Nutritional Profile of Annona Muricata and Ageratum Conyzoides Based Herbal Teas for the Management of a Precancerous Lesion: Atypical Hyperplasia of the Mammary Gland

Introduction

Hyperplasia, a term for an abnormally large volume of a tissue or organ due to an increase in the number of its cells [1], is characterized by the proliferation of epithelial cells of a specific tissue. It becomes atypical when the proliferating cells are abnormal or in an unusual way. When this abnormal proliferation attacks the cells that line the ducts or lobules of the breast it is called Atypical Mammary Hyperplasia (AMH) [2]. The prevalence of AMH is increasing every year and it is one of the leading breast disorders in women of childbearing age [3]. AMH is considered a potential precancerous lesion that can progress to breast cancer if left undiagnosed and untreated [4,5]. Breast cancer is a heterogeneous disease in which normal cells of the mammary gland adopt a malignant phenotype and proliferate indefinitely and uncontrollably, destroying breast tissue [6]. According to a report by the World Health Organization (WHO) and the International Agency for Research on Cancer (IARC), approximately 22 million people will be annually affected by cancer by 2030: 8 million more than in 2012 [7]. In 2021, cancer was the leading cause of death worldwide, accounting for about 10 million deaths: the most common in terms of new cases being breast cancer with 2.26 million cases [8]. In Cameroon, the figures are even more alarming and breast cancer is the most common in terms of prevalence and mortality. According to several studies, 70-80% of patients are diagnosed at advanced stages and nearly 2000 women die from it each year [9]. In addition to the delay in diagnosis, other risk factors for cancer include gender, age, family history, reproductive risk factors and obesity [10]. Obesity is particularly characterized by an abundance of adipose tissue where oestrogen synthesis takes place, and it plays a major role in both abnormal cell proliferation in the mammary gland and in the uterus, and that can progress to malignancy and metastasis [11]. There is an association between high fat, protein and carbohydrate diets and the increased risk of breast cancer through increased oestrogen production, hyperinsulinaemia and insulin resistance. High fat and protein diets play a role in the increase in oestrogen production by adipose tissues [12,13].

Polyphenols are good in slowing down cell proliferation; they are particularly good in the development of blood vessels and in inactivating carcinogenic products through their antioxidant activities. Plants, taken in form of herbal tea, are used by about 80% of the world’s population for their curative virtues [14,15]. They can help female populations to reduce the risk of developing gynaecological cancers. Annona muricata and Ageratum conyzoides are two plants from the Annonaceae and Asteraceae families respectively, which have shown convincing results in previous studies. Nutritionally, Annona muricata leaves are rich in protein (25 ± 0.06 g/100g), lipid (21.22 ± 1.01 g/100g), carbohydrate (16.62 ± 0.09 g/100g), Potassium (363.05 mg/kg), Calcium (11183.50 mg/kg), Sodium (694.86 mg/kg), Magnesium (9619 mg/kg) and Iron (139.50 mg/kg) [16,17]. Ageratum Conyzoides leaves contain crude protein (24.53 ± 0.104), lipid (3.78 ± 0.069), carbohydrate (36.81 ± 0.006), Sodium (88.50 ± 0.346), Potassium (139.10 ± 0.006), Calcium (220.60 ± 0.173), Magnesium (110.13 ± 0.115), Iron (22.73 ± 0.289), Zinc (43.50 ± 0.173) and Phosphorus (380.13 ± 0.173) [18]. The rich nutritional composition of these plants is responsible for their medicinal effects, including antibacterial, antiproliferative and even anticancer activities. Referring to their antiproliferative and anticancer effects, Annona muricata has shown convincing results on breast cells with 98% efficacy in previous studies [19]. Adebayo, et al. [20] demonstrated that the in vitro anticancer activity of aqueous extracts of Ageratum Conyzoides is more effective on lung, uterine, breast and liver cells. Based on these results, a combination of the two plants in the form of formulas proved to have more interesting properties leading to promising conclusions on slowing down AMH (Publication in progress). The main objective of this work was to evaluate the nutritional properties of formulations made by combining Annona muricata and Ageratum Conyzoides.

Materials and Methods

Plant Material

Annona muricata and Ageratum Conyzoides were collected in Yaoundé, Centre region in October and November 2019 between 5:30 and 6:30 am. They were identified by comparison at the National Herbarium of Cameroon against the numbers 6575 SRFK (YA) and 18681/ SRF/Cam (YA) respectively. The collected plants were weighed and spread out for 24 hours on large trays in a room (20-21°C) for wilting.

Preparation of Extracts

The wilted leaves were dried at 40°C in a ventilated oven for 24 hours. Once dry, they were milled using a blender and then sieved using a 150 μm mesh size sieve. The obtained powders were infused in boiling water in the ratios of 1/5 and 1/6 g/mL for Annona muricata and Ageratum Conyzoides respectively for 2 hours, stirring every 30 minutes. The solution was filtered, and the residue was rinsed twice. The obtained solution was freeze-dried, and a mixing plan used in industrial research and development studies [21,22] helped to have five combinations of the freeze-dried extracts in the proportions 100/0; 75/25; 50/50; 25/75 and 0/100 corresponding to C100Rh; C75Rh; C50Rh; C25Rh and C0Rh respectively (C for Annona muricata and Rh for Ageratum Conyzoides).

Crude Protein Content

Crude protein content was determined according to the method described by Devani and al (1989) [23]: it is a spectrophotometric method based on the conversion of total organic nitrogen to ammonium sulphate.

Carbohydrate Content

The determination of soluble carbohydrates was carried out using the Chronolab assay kit based on the oxidation of glucose to gluconic acid catalysed by glucose oxidase (GOD). The hydrogen peroxide (H2O2) produced is detached using a chromogenic oxygen acceptor, phenol ampirone in the presence of peroxidase (POD).

Lipid Content

Total lipids were extracted with Soxhlet according to the Russian method as described by Bourely, (1982) [24] based on the differential solubility of lipids in organic solvents (hexane) at high temperatures about 12 hours.

Mineral Content

The minerals Ca, Na, Mg and K were analysed according to the method described by Horwitz, (2000) [25] based on the separation of the minerals from the sample matrix by wet digestion of the organic matter. The separated minerals were diluted in acid and their content determined by atomic absorption spectrophotometry.

Statistical Analysis

Statistical analyses were performed using IBM SPSS 20.0 software for Windows. The data were analysed by 1-way ANOVA and Tukey’s multiple range test was used to determine the differences among samples. Significant levels were defined as probabilities of 0.05 or less. The obtained results were presented as mean ± standard error of the mean (SEM). Excel 2016 software was used for graphical representations.

Results and Discussion

Crude Protein, Fat and Soluble Carbohydrate Content

Table 1 shows the macronutrient content of the different combinations (formulations). Protein content ranged from 25.07 g/100g DM to 12.01 g/100g DM: fat content from 22.42 g/100g DM to 20.65 g/100g DM and carbohydrate content from 0.63 g/100g DM to 0.23 g/100g DM with statistical significant difference (p< 0.05) between the protein content of the different herbal tea combinations. Ageratum Conyzoides leaf tea C0Rh had the lowest protein content (12.01 g/100 g DM) compared to Annona muricata leaf tea C100Rh (25.07 g/100 g DM) and the combinations C75Rh, C50Rh, C25Rh of 17.65 g/100 g; 17.30 g/100 g and 16.42 g/100 g DM respectively. A significant difference (p< 0.05) was also observed between the lipid content of the different combinations. Ageratum Conyzoides leaf tea C0Rh had the highest lipid content (22.41 g/100 g DM) compared to Annona muricata leaf tea C100Rh (21.57 g/100g DM). Of the three combinations, the lipid content of the C50Rh herbal tea combination was the highest (22.42 g/100g DM) followed by the C75Rh and C25Rh formula. Ageratum Conyzoides leaf tea C0Rh had the lowest soluble carbohydrate content 0.23 g/100 g DM, followed by C75Rh, C100Rh, C25Rhand C50Rh.

Table 1: Protein, lipid, and soluble carbohydrate contents (g/100g DM) of herbal tea formulations based on Ageratum Conyzoides and Annona muricata leaves.

Note: Means assigned with different letters in the same rows are significantly different (p<0.05). C100Rh: 100% Annona muricata leaf powder; C75Rh: 75% Annona muricata leaf powder and 25% Ageratum conyzoides leaf powder; C50Rh: 50% Annona muricata leaf powder and 50% Ageratum conyzoides leaf powder; C25Rh: 25% Annona muricata leaf powder and 75% Ageratum conyzoides leaf powder; C0Rh: 100% Ageratum conyzoides leaf powder.

The protein content of A. muricata in the present study was approximately equal to 25 ± 0.06 g/100g DM, same value obtained by Usunobun and al. (2012) [26] in Nigeria; higher than 24.3 ± 0.1 g/100g DM and 15.74 ± 1.01 g/100g DM obtained by Rosemary and al. (2017) [27] and Ogbonna and al. (2019) [28] respectively. The protein content of A. conyzoides was lower than 14.73 g/100g DM and 15.67 g/100g DM respectively obtained by Agbafor and al. (2015) [29] and Agunbiande and al. (2012) [30]. These differences could be explained by various parameters such as the age of the plant before harvesting, the application of chemical or natural fertilizers on the harvesting land, the nature of the soil [31], and the different treatments carried out on the plant material such as drying. The combination of 75% A. muricata and 25% A. conyzoides tea had the highest protein content after the 100% A. muricata formula at 17.65 g/100 g DM. According to the Agence Nationale de Sécurité Sanitaire de l’Alimentation de l’Environnement et du Travail (ANSES) the average daily protein intake of a woman is 0.9 g/kg body weight. This means that a woman weighing 60 kg should have a protein intake of 54 g per day. The average protein content of 100 g of herbal tea dry matter (average of the five contents obtained) is 17.69 g/100g. Therefore, consumption of 100g of herbal tea per day provides 5.08 g of protein, thereby contributing approximately 10% of the protein RDA for a 60 kg woman. The protein intake of the tea (depending on the quantity) can be subtracted from the food intake. A high protein intake can lead to a rapid proliferation of tumour cells due to the stimulation of growth hormone (GH), which itself promotes the synthesis of IGF 1, another factor in the proliferation of tumour cells [32]. A lipid-rich diet has a higher energy density than one rich in other macronutrients. The role of lipids has been discussed in the development of overweight and obesity. When fatty acids are involved in energy production, the polyunsaturated fatty acids are oxidised, leaving the saturated fatty acids to accumulate in adipose tissue, where they trigger abundant oestrogen production [32].

As breast cancer prevention is a major public health objective, the quantification of lipids in our herbal tea samples was of paramount importance. The lipid content of A. muricata C100Rh tea (21.57 g/100g DM) was slightly higher than 21.22 ± 1.01 g/100 g DM obtained by Usunobun et al. (2015) [33] in Nigeria. That of A. conyzoides C0Rh tea (22.41 g/100g DM) was significantly higher than 5.67 g/100 g DM obtained by Agubiande, et al. (2012) [30]. The combination of 75% A. muricata and 25% A. conyzoides C75Rh had an average lipid content of 21.13 g/100 g DM. ANSES recommends an average daily lipid intake of 1 g/kg body weight for a woman. This means that a 60 kg woman should consume 60 g of fat per day. The average fat content of 100g dry mass is 21.63g, contributing 36% of a 60kg woman’s requirement. The fat intake from the tea (depending on the amount) can be subtracted from the food intake as too much fat can lead to increased fatty tissue in the body which is a source of abundant oestrogen production and thus increases the risk of developing cancer of the primary oestrogen targets [34]. Carbohydrates, when consumed in large quantities and in absence of balanced caloric expenditure, can be considered as risk factors for several diseases such as cancer. The combination of 50% A. muricata and 50% A. conyzoides (C50Rh) showed the highest soluble carbohydrate content 0.63g/100g DM followed by the combination of 25% A. muricata and 75% A. conyzoides (C75Rh) 0.61g/100g DM which is not very advantageous in this case. The soluble carbohydrate content of the combination of 75% A. muricata and 25% A. conyzoides (C75Rh) was 0.33g/100g DM, which would be an advantage for the management of people at risk.

Mineral Contents (Na, Ca, K, Mg)

Table 2 shows the mineral (Na, K, Mg and Ca) content of the herbal tea formulations based on Ageratum conyzoides and Annona muricata leaf powder. Minerals have various roles in cell proliferation. Of the three intermediate combinations, the tea combination with 75% Annona muricata and 25% Ageratum conyzoides (C75Rh) had the highest Na content (63.03 mg/100 g DM), followed by C50Rh (56.53 mg/100g DM) and C25Rh (48.46 mg/100g DM). This C75Rh combination also had the highest K content (467.09 mg/100 g DM). Sodium is frequently ingested in food in the form of naturally occurring sodium chloride (table salt). It is stimulated by growth factors and ensures the alkalinisation of the intracellular environment, a process essential for the activation of DNA synthesis and thus the initiation of the mitotic cycle. Potassium is nutritionally important for pH regulation and the proper functioning of carbohydrate and protein metabolism. Most people with cancer generally have too much sodium, not enough potassium, and a pH of 4, which leads to tissue damage and weakening of organs [35]. The potassium content of the tea would regulate the pH of the body by filling the potassium deficiency in people at risk. The combination of 75% Annona muricata and 25% Ageratum conyzoides herbal tea had the lowest calcium content of 18.19 mg/100 g DM; calcium is known to be a macro element required for hormone release [36]. Hypercalcaemia would lead to the deposition of calcium salts in the ducts of the breast, exposing the woman to precancerous connections [37].

Table 2: Content of mineral elements (Na, K, Mg and Ca) (mg/100g DM).

Note: Means assigned with different letters in the same rows are significantly different (p<0.05). C100Rh: 100% Annona muricata leaf powder; C75Rh: 75% Annona muricata leaf powder and 25% Ageratum conyzoides leaf powder; C50Rh: 50% Annona muricata leaf powder and 50% Ageratum conyzoides leaf powder; C25Rh: 25% Annona muricata leaf powder and 75% Ageratum conyzoides leaf powder; C0Rh: 100% Ageratum conyzoides leaf powder.

This low Ca content would be an asset as it would solve the problems of mammary calcifications, since high concentrations of calcium salts puts the individual at risk of developing benign tumours. The Magnesium content of the combination of Annona muricata and Ageratum Conyzoides herbal tea was 27.53 mg/100 g. Magnesium is a mineral required in hormone metabolism. It is also important for the release and action of insulin [38]. The higher the amount of magnesium in women at increased risk of cancer, the more it will lead to hyperinsulinemia, a factor that promotes the development of cancerous disease. According to WHO, the recommended daily intake of sodium is not more than 2000 mg/day [39]. Consumption of 100 g of C75Rh herbal tea meets 3.15% of the daily sodium requirement. For potassium, the European Food Safety Authority (EFSA) recommends an intake of 3500 mg/day for adult women (EFSA, 2016). C75Rh herbal tea meets 13.34% of the daily potassium requirement. The intake of potassium will promote the proper functioning of carbohydrate and protein metabolism. As for magnesium, the recommended intake for adults is 6 mg/kg/day [40] and the lowest magnesium content was that of the C25Rh formulation (4.87mg/100g DM). Finally, for calcium, the recommended nutritional intake for adults is 900 mg, for women over 55 and the elderly the requirement is 1200 mg/day. The highest calcium content was found in C50Rh (570.56 mg/100g DM) and the lowest in C75Rh (18.19 mg/100g DM). Calcium is known to be a macro element necessary for the release of hormones. The higher its content in the body, the more calcium salts will be deposited in the ducts of the breast exposing the woman to precancerous connections, the C75Rh formulation with a content of 18.19 mg/100g DM would be an asset for women at risk.

Conclusion

The objective of this study was to study the nutritional profile of herbal teas made from leaf powder of Annona muricata and Ageratum Conyzoides leaves combined in various proportions for the treatment of atypical hyperplasia. It was found that of the five combinations, the one with 75% Annona muricata and 25% Ageratum Conyzoides presented a better nutritional profile because of its low nutrient content (proteins and carbohydrates) and medium mineral content (Na, K and Ca). These results suggest a real asset for the dietary management of young women at risk of developing breast cancer or not. Furthermore, due to the high content of phenolic compounds in the C75Rh combination, its antiproliferative activity on mammary precancerous lesions induced in Wistar rats was studied and it showed an inhibitory effect on the disordered proliferation of mammary gland cells. As this work is in the process of being published, there are questions about the nature of the bioactive compounds present in the tea and mainly responsible for its antiproliferative activity.

Acknowledgments

The authors are thankful to all those who contributed to this work. Special thanks to Pr. Fokom Raymond, Mr. Jean Pierre and Mr. Jean Paul for the supply of some reagents.

Conflict of Interest

The authors declare no conflicts of interest for this study.

Data Availability

The datas analyzed during the current study are available from the corresponding author upon reasonable request.

Author’s Contribution

Fokou Elie, Mbacham Wilfred and Mercy Tah-Monunde conceived the study, edited and reviewed the manuscript; Mono Anne Sophie prepared the proposal and conducted laboratory analysis under the guidance of Djouhou Michelle and drafted the manuscript. Djouhou Michelle and Maptououm Laure were involved in data analysis and reviewing of the manuscript. All authors read and approved the final manuscript.

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Comparison of Two Indexes of Adherence to Mediterranean Diet in Adolescents: A Cross Sectional Pilot Study

Introduction

The traditional Mediterranean Diet (MD) refers to the dietary pattern of the Mediterranean basin defined by prevalent consumption of foods of vegetable origin, regular but moderate intake of dairy products, low to moderate intake of fish and poultry, and very low intake of red meat. Other fundamental characteristics are seasonality, biodiversity, and the use of local and traditional food products; moreover, MD has also qualitative cultural and lifestyle elements, such as frugality, sobriety, conviviality, typical recipes, physical activity and adequate rest [1,2]. The MD has been identified a healthy dietary pattern for the prevention of non-communicable diseases, being associated with a better health status and a prolonged lifespan [3-6]. In evaluating the relationships between diet and health, a particular interest has developed to approximate the overall adherence to certain dietary patterns.

The assessment of MD adherence is made possible by using specific indexes (or scoring systems), suitable for adults [7], as well as for children and adolescents [8]. However, measuring the level of adherence to MD is not immediate: a single method does not exist, as the definition of MD is influenced by socio-cultural, religious, and economic factors [7,9,10]. There are several methods assessing different elements, i.e. the intake of various nutrients/foods and/or specific food habits, to derive a score which may be used to categorize population groups. Differences between the various MD scores are mostly determined by the selection of the items and by the different cut-off points or scoring criteria adopted (mainly medians and tertiles) [7]. The first and most extensively used index is the Mediterranean Dietary Score (MDS), proposed by Trichopoulou et al. in 1995 and revised in 2003 [11,12], suitable for adults and elderly. Other indexes have been later proposed and adapted, each of them being appropriate for a specific population and underlying some specific aspects of the MD [9,13].

In recent years, a gradual withdrawal of MD fundamentals has been found especially in Mediterranean countries [14-18], and also in Italy [19,20]. This downward trend has been observed mainly for the younger age groups [21] and, as early eating habits influence adulthood health status [22], it is very important to focus the attention on these population groups. Adolescence is defined as the broad age range from 10–24 years [23]. To the best of our knowledge, the MD adherence indexes adopted for adolescents are: the Mediterranean Diet Quality Index in children and adolescents (KIDMED) [24], which is the most widely used scoring system; adapted versions of the original MDS [11,12], based on dietary records; and MD Scores, based on Food Frequency Questionnaires (FFQs), which represent a commonly used, reproducible and valid approach for dietary assessment [25]. The aim of this paper is to compare two different indexes to assess the adherence to MD in a sample of adolescents: the KIDMED index [24] and the Mediterranean Diet Serving Score (MDSS), based on a FFQ and suitable for adults as well as for adolescents [26]. This work represents only one aspect of a broader and more articulated research that has as its purpose to evaluate if and to what extentthe encounter between different cultures, in the context of globalization present in modern society,may have influenced lifestyle, eating habits, adherence to MD and nutritional status of adolescents of different cultures and geographical proveniences [27,28].

Materials and Methods

Participants and Study Design

The study sample was recruited in Youth Aggregation Centers, located in Rome, from October 2020 to March 2023 and included free-living adolescents. Before starting the data collection, the objective of the research and the intention to publish the results in scientific papers were described in detail. Participation in the study was fully voluntary and anonymous and volunteers could with-draw from the study at any time and for any reason. After obtaining informed consent, qualified interviewers administered questionnaires in a “face to face” assisted modality, on one occasion. They were designed to obtain information about lifestyle, eating habits, food consumption and adherence to MD; all the data were handled according to the European General Data Protection Regulation – GDPR 679/2016. In the case of minors, both releases were signed by parents or by whoever had parental responsibility. As the assessment did not involve invasive procedures or induce changes in dietary patterns, the approval by the Ethics Committee was not necessary.

Lifestyle and Eating Habits

Information about demographic factors and social aspects (age, sex, country of origin, education and/or occupation, living arrangement), leisure time activity (type and dedicated time) eating habits (frequency of meal consumption, away-from-home eating, water, softdrinks, and alcohol consumption, eating differently from family members or roommates) were collected through a specifically designed questionnaire.

Food Consumption

Participants completed a qualitative FFQ related to the previous 12 months. The frequencies were expressed as “never”, or as number of servings/day or servings/week or servings/month or servings/ year.

Adherence to MD

Two validated score systems were selected: the KIDMED index [24,29], easy tool suitable for our sample, and the MDSS [26], based on the frequency of consumption of foods and food groups, in absence of information on nutrients and quantities in grams of foods. It was calculated based on the food items of the FFQ, which in a later time have been gathered into 13 items, according to the MD pyramid [1]. The KIDMED Questionnaire: The KIDMED questionnaire could be self-administered or conducted by trained interviewers and consists of 16 dichotomous (positive/negative) items, where the 4 questions denoting a negative connotation to the MD are scored with -1, and the 12 questions denoting a positive connotation are scored with +1 (Table 1). The score obtained summing the single answers let to classify the adherence to MD into three levels: low (≤ 3), medium (4–7), high (≥ 8) [24,29].

Table 1: KIDMED test to assess the Mediterranean diet quality [24].

Note: KIDMED – Mediterranean Diet Quality Index in children and adolescents.

The Mediterranean Dietary Serving Score (MDSS): The MDSS is based on the latest update of the MD pyramid [1] and consists of 14 items for adults and 13 items for adolescents. Individuals whose intake is within the number of recommended servings are awarded a score of 3, 2, or 1 point for recommendations expressed in times/ meal, times/day, or times/week, respectively. A score of 0 is given when the number of servings/meal, week or day is higher or lower than recommended (Table 2). The MDSS total score ranges between 0 and 24 points for adults and elderly, and between 0 and 23 for adolescents (because fermented beverages consumption is not considered for this age group). The sum of the scores of each item let to classify the adherence to MD into three levels: low (0-7), medium (8-15), high (16-23) [26]. The MDSS is comparable with the MDS created by Trichopoulou [11,12].

Table 2: According with the new MD pyramid [1].

Note: ** Main meals: breakfast, lunch and dinner.
a. Bread, breakfast cereals, rice and pasta.
b. Olive oil used on salads or bread or for frying.
c. Milk, yoghurt, cheese, ice-cream.
d. Poultry.
e. Pork, beef, or lamb.
f. Sugar, candies, pastries, sweetened fruit juices, and soft drinks.
g. Wine and beer.

Statistical Analysis

Statistical analysis was performed by the MedCalc software version 20.106 for Windows, setting the significance at p < 0.05. Continuous variables were presented as means and standard deviations (sd), while categorical variables as frequencies. Means were compared through the Student’s t test, after checking the normal distribution by using the Shapiro–Wilk test, while differences between categorical variables through the Pearson chi-square test.

Results

Data were collected on 223 volunteers (130 males, mean age 17.2±3.8 years and 93 females, mean age 16.4±3.3 years, P=ns) (data not shown). Table 3 reports the frequency of meal consumption. There were no statistically significant differences between males and females. In our sample, 31.5% of males and 39.8% of females sometimes skipped breakfast, due to insufficient time or appetite and to the will to sleep longer; 13.1% of males and 5.4% of females never did breakfast. There was also a percentage of volunteers not having lunch or dinner every day (12.6% and 10.3% respectively). The tendency of eating between meals was once a day for 36.0% of volunteers (34.1% of males and 38.7% of females), twice a day for 35.6% of them (38.8% of males and 31.2% of females) and more than twice a day for 14.0% of them (10.0% of males and 19.3% of females). Table 4 reports the MD adherence by both the KIDMED index and the MDSS. A mean KIDMED score (5.9±2.5 for males and 5.1±2.8 for females) indicates a medium adherence, without significant differences between males and females. 50.8% of males and 49.5% of females had a medium adherence to MD; a great percentage of the sample (27.4%) had a high adherence, mainly represented by male volunteers (30.7%).

Table 3: Frequency of Meal Consumption by Sex.

Note: Categorical variables are presented as percentages. Statistical analysis: Chi square test; ns=not significant.

Table 4: Adherence to Mediterranean Diet (MD) by KIDMED Index and MDSS by Sex.

Note: Continuous variables are presented as mean ± sd and categorical variables as percentages. Statistical analysis: Student’s T-Test and Chi square test; ns=not significant.

The mean MDSS score was 5.4±2.5 for males and 4.4±2.2 for females, without significant differences (P=ns), indicating a low MD adherence. Indeed, 83.9% of total sample had a low adherence according to the MDSS (79.2% of males and 90.3% of females) and no one had high adherence. There were statistically significant differences in MDSS categories between sexes (P=0.026). The adherence to MD was deepened by considering the responses to each single question of the KIDMED questionnaire, shown in Table 5, and the single items of the MDSS, reported in Table 6. Regarding the KIDMED index (Table 5), among the behaviors with a negative connotation with respect to the MD, those that prevailed were, for 43.9% of the sample, the consumption of baked goods or pastries for breakfast (by 44.6% of males and 43.0% of females) and skipping breakfast due to lack of time, a desire to sleep longer in the morning and a lack of appetite (by 43.8% of males and 44.1% of females), without significant differences between sexes. Among the behaviors with a positive connotation with respect to the MD, the use of olive oil was the most widespread among all the participants in the study (97.3% of the total sample, 96.2% of males and 98.9% of females, stated that they use it regularly at home), followed by the consumption of pasta or rice almost every day (80.7% of the total sample, 89.2% of males and 68.8% of females, P=0.000). The less followed behavior with a positive connotation with respect to the MD was the daily consumption of two yoghurts and/or some cheese (40 g) (by 21.5% of the total sample, 24.6% of males and 17.2% of females).

Table 5: Percentage of “yes” answers to each single question of the KIDMED questionnaire.

Note: Data presented as percentages are referred to “yes” answers. Statistical analysis: Chi square test; ns=not significant; 1. “Yes” answers with a positive score (+1); 2. “Yes” answers with a negative score (−1).

Table 6: Percentage of volunteers Within and Outside Recommendations According to MDSS.

Note: *According with the new MD pyramid [1]. Statistical analysis: Chi square test.

Regarding fruit and vegetables consumption, about the 40.0% of the total sample had a second fruit every day, or fresh or cooked vegetables more than once a day. The deepening of single items of the MDSS is described in Table 6, which shows the population distribution according to MDSS cut-off points. Regarding the total sample, the highest adherence to the recommendations was identified for potatoes (75.3%), legumes (53.4%) and red meat (46.6%), whereas the lowest was for vegetables (0.4%), olive oil (2.7%) and sweets (4.9%). Males complied better with all the recommendations except for fruit, white meat, and sweets. Most of sample was below the recommendation for vegetables (99.6%), olive oil (96.9%) and fruit (91.0%), and above the recommended values for sweets (95.1%), red meat (53.4%) and white meat (44.8%). There were not significant differences between sexes (P=ns). In Table 7 is summarized the adherence to MD according to the two different indexes. There are some aspects not included in MDSS, such as going to fast-food (hamburger) restaurant or skipping breakfast, whereas KIDMED questionnaire doesn’t include consumption of potatoes, eggs, white meat, and red meat. Higher differences were observed for those foods which have to be consumed in every main meal, i.e. fruit, vegetables, cereals and olive oil.

Table 7: Comparison Between KIDMED index and MDSS.

Note: *According with the new MD pyramid [1]. Categorical variables are presented as percentages. Statistical analysis: Chi square test.

According to the KIDMED questionnaire, 73.1% of volunteers consumed 1 serving/day of fruit (including fruit juice) and 40.8% of them 2 servings/day (vs 8.5% of the sample being within recommendation according to MDSS); 65.9% consumed 1 serving/day of vegetables and 40.4% more than 1 serving/day (vs 0.4% of the sample being within recommendation according to MDSS); 80.7% consumed ≥ 5 servings/week of cereals and 60.5% consumed cereals or grains for breakfast (vs 35.9% of the sample being within recommendation according to MDSS); 97.3% used olive oil at home (vs 2.7% of the sample being within recommendation according to MDSS). Similar percentages were found for legumes (59.6% of volunteers within recommendation according to KIDMED vs 53.4% of volunteers within recommendation according to MDSS), even if the number of portions considered was different between the two tools. Only in case of fish, the methodologies can be compared both for servings/week and for the percentages of volunteers within recommendation (50.2% according to KIDMED vs 43.9% according to MDSS). In Table 8 is reported the distribution of volunteers according to both indexes. Only 26.5% of the sample had the same level of MD adherence in both indexes (47 volunteers having low adherence and 12 volunteers having a medium adherence), while the remaining 73.5% of volunteers was differently distributed among the three levels of MD adherence (P=0.000).

Table 8: Distribution of volunteers according to both indexes.

Note: Statistical analysis: Chi square test.

Discussion

The aim of this paper is to compare two different methodologies used to assess the adherence to MD in a sample of adolescents: the KIDMED index [24] and the MDSS [26]. Our results describe a sample of adolescents with a mean KIDMED score indicating a medium adherence to MD and a mean MDSS score indicating a low adherence. Most of volunteers are differently distributed among the three levels of MD adherence evaluated with the two indexes. According to KIDMED questionnaire, the 27.4% of our sample has a high MD adherence and 50.2% a medium adherence. A systematic review [30] conducted on participants 2–17 years old, shows that the KIDMED questionnaire is the method most often used, also for the Italian adolescents. In the study conducted by Bonaccorsi, et al. [31] on 314 volunteers aged 6-14 years old, high adherence is found in 24.8%, medium adherence in 56.4% and low adherence in 18.8% of the sample.

A cross-sectional study, conducted on adolescents of 11–16 years, reveals that 9.1% of the sample has high adherence, 61.0% medium adherence and 29.9% had low adherence to MD, measured by KIDMED [32]. The results of another cross-sectional study [16] carried out on students of 11-14 years, show that most subjects (60%) have a medium adherence to MD, and 12% has high adherence, according to KIDMED. Recently, in the DIMENU cross-sectional study carried out on participants aged 14-17, a medium adherence to MD assessed by the KIDMED score is reported in the 60.87% of the sample [33]. Archero, et al. [34] in a cross-sectional study on volunteers aged 6-16 years, highlight high adherence in 19.6%, medium adherence in 63.7% and low adherence in 16.7% of the sample, measured by KIDMED. The results obtained in our research according to MDSS show that most of the sample has low adherence (83.9%), while the remaining percentage has medium adherence. To the best of our knowledge, in Italy there are no studies on adolescents in which MDSS has been used. In the ARIANNA cross-sectional survey, this tool is used for participants aged ≥17 years, but results are not yet available [35].

Some literature studies compare different methodologies to assess the MD adherence; anyway, it is important to underline that the KIDMED score was not included in these studies. Milá Villaroel, et al. [36], basing on the analysis of 10 indexes of MD adherence reliability and founding a moderate correlation among them, conclude that a consensus on the components included in the MD indexes should be reached. Hernández Ruiz et al. [37] identifying 22 different indexes, confirm the findings of Milá Villaroel, et al. [36]: although all these indexes are related to the Mediterranean dietary pattern, they very differently evaluate the dietary characteristics of the MD. Differences encountered are based on the considered items, as they can be adapted to the population´s dietary habits, and whether their consumption is valued positively or negatively; the scoring scheme and finally, the cut-off points of each index and the contribution of each component to the overall score.

In a systematic review [9], 28 MD adherence scores are analyzed, many of them developed in Mediterranean countries and applied to the general population. Indeed, measuring food consumption and eating habits is far from being simple. Dietary pattern can be defined using a general description, dietary pyramids, a priori scoring systems (diet index), a posteriori dietary pattern formation, or by quantifying food and nutrient content [38,39]. The indexes are usually based on data acquired within a 24-hour quantitative intake recall, dietary records or FFQs. FFQ is one of the most used approaches for dietary assessment, also in adolescents [40], showing in addition good reproducibility and validity for MD assessment [25]. This method has been chosen in our study, in place of dietary records or 24-hour recall. Indeed, capturing detailed information about all foods and beverages would be difficult to carry out, due to the linguistic difficulties of some volunteers hailing from different countries. In our study, a qualitative FFQ was used to calculate MDSS, based on the latest MD recommendations. The MDSS classifies consumption frequency in servings per meal, day, or week and, considering the upper and lower recommended limits for each food group (when available), it penalizes individuals both when they do not reach the recommended intake and when they exceed it. Moreover, total score is more influenced by meeting, or not, the recommendations of the foods at the base of the pyramid than those at its apex [26].

Most indexes of MD adherence consider 9 items in comparison to the 14 included in the MDSS, which differentiates between the consumption of fresh fruit and dried fruit/nuts, cereals, and potatoes and red and white meats and introduces two new items to assess the intake of eggs and sweets. This permits a more accurate diagnosis of adherence to MD [41]. Even if FFQs are very useful to determine the dietary intake at the same time are time-consuming for volunteers and require complex data management and processing. To overcome this issue, numerous short indexes have been developed, among which the KIDMED index [24]. It has been used for more than a decade but, based on the scientific evidence, some authors suggests some changes to the original version of the KIDMED questionnaire; these changes are represented by adding the term ‘whole-grain’ to the eighth and the ninth questions of the questionnaire, due to the recognized importance of the whole grain cereals and by deleting ‘or fruit juice’ from the first question of the questionnaire [42]. Other authors [43] update the 2019 KIDMED questionnaire producing a revised version, named KIDMED 2.0, adapting the instrument to the real MD. The original version of the questionnaire is however the one mainly used and, consequently, to compare literature data on adolescents, is the only questionnaire allowed.

However, the KIDMED questionnaire considers different aspects respect to MDSS: a lack of uniformity in the components of the diet between the two measures and, even within the same food group, the number of servings differs markedly among the indexes. Anyway, it is important to underline that, in our study, the answers relating to food consumption are congruent between the two methodologies, because they were immediately checked in the presence of the subject to avoid inconsistent reporting. The first obvious discrepancy between the two methodologies is that in the MDSS only fruit consumption is mentioned (1-2 servings/main meal, i.e. 3-6 servings/day), while in KIDMED fruit is associated with fruit juice consumption. According to the KIDMED questionnaire, 73.1% of volunteers consumed 1 serving/day of fruit (including fruit juice) and 40.8% consumed 2 servings/day, differently from the 8.5% of the sample within fruit recommendation according to MDSS: some volunteers answered positively to the first question of KIDMED as they consumed fruit juice and not fruit, data confirmed by the FFQ.

The WHO recommends eating at least 400 g (approximatively 5 servings) of fruit and vegetables per day [44], but their levels in adolescent diets are often low [45], as confirmed also by our results, keeping in mind that the first question of the KIDMED questionnaire considers, besides fruit, also fruit juice which, unlike whole fruit, is low in fiber and rich in sugar [46]. Moreover, the MDSS approach gives greater importance to foods (fruit, vegetables, olive oil, cereals) that should be consumed during the 3 main meals (breakfast, lunch, dinner). Nonetheless, 44.9% of the sample does not consume breakfast every day, hence, does not assume fruit for breakfast. There is also a percentage of volunteers not consuming lunch or dinner every day (respectively 13.5% and 11.2%), with effect on consumption of those food which are at the base of the MD pyramid. Moreover, those who regularly have breakfast do not eat fruit during this meal, as confirmed by the lifestyle questionnaire. The same trend is observed for vegetables, olive oil and cereals (bread, breakfast cereals, rice, and pasta) which, according to the MD pyramid [1] and consequently to the MDSS [26], should be consumed in the 3 main meals.

According to the MD pyramid [1] these food categories can also be found throughout the day; for this reason, we have considered the daily servings obtained by FFQ (fruit 3-6 servings/day; vegetables ≥ 6 servings/day; cereals 3-6 servings/day; olive oil servings/ day). Moreover, volunteers that skip breakfast answered “No” to the 3 KIDMED questions related to breakfast (Has cereals or grains for breakfast; Uses dairy product for breakfast; Has commercially baked goods or pastries for breakfast) and consequently, the score of these questions was “zero”. To better explain, in case of the consumption of commercially baked goods or pastries for breakfast, our volunteers are not virtuous, but they just skip breakfast, and consequently they are not assigned a negative score, even if skipping breakfast is an incorrect dietary behavior. The KIDMED questions should be rephrased, also because skipping breakfast is a very common dietary habit in children and adolescents, to which the KIDMED is addressed. It has been shown that children and adolescents who ate breakfast regularly were more likely to adhere to the MD than those who did not eat breakfast [47].

Rosi and coworkers [48] reported only 14% of participants were breakfast skippers, while in our sample this percentage is 44.9%. The percentage of breakfast skippers is like the results produced by Nardone and colleagues [45] showing 4 out of 10 adolescents skipped breakfast, while a recent systematic review on 286,804 children and adolescents living in 33 countries [49] reported extremely wide variability, ranging from 0.7% to 74.7% of prevalence of breakfast skippers, according to the definition of breakfast skipping used. reported above, another discrepancy between the two measures is related to olive oil, because the KIDMED questionnaire registers only the domestic consumption, without specifying the number of servings, while MDSS considers as recommended one serving per main meal (at the base of the pyramid with fruit, vegetables, and cereals). In our research, this difference is well highlighted by the percentages of adherent volunteers to olive oil consumption which are deeply dissimilar (97.3% according to KIDMED and 2.7% according to MDSS).

The two indexes consider a different number of servings for legumes and nuts. For legumes, the KIDMED index indicates an intake equal to or higher than 1 serving a week, while MDSS considers a double frequency of consumption per week. Anyway, the percentage of adherent volunteers is similar in our sample. In case of nuts, this difference is more evident: the KIDMED index indicates an intake of 2-3 servings a week, while MDSS considers a daily frequency of consumption (1-2 servings). For this reason, the percentage of adherent volunteers according to KIDMED questionnaire was more than double compared to the MDSS (35.4% vs 15.2%). Fish was the only item, which is perfectly comparable between the two methodologies, as the frequency of consumption is more than 2 servings/week, and moreover, the percentages of adherent volunteers are similar (50.2% according to KIDMED and 43.9% according to MDSS). Noteworthy differences occur for dairy products and sweets, which make the questionnaires not-comparable.

Finally, there are some components not included in one or the other questionnaire: potatoes, white and red meat consumption is not included in the KIDMED questionnaire, while the habit to go to fast-food restaurant and to skip breakfast are not included in MDSS, which considers only food consumption and not dietary habits. For these reasons, it is not possible to compare the two indexes, for these items. The strength of this study is that it is an in-person-administered survey: the administration of questionnaires occurred during face-to-face interviews conducted on one occasion, thus optimizing data quality, and allowing to verify immediately the consistency of answers related to food consumption. This kind of procedure inevitably imply the involvement of a small sample of volunteers, differently from online surveys which allow to reach a larger number of respondents; anyway, they don’t give a chance to directly check the answers in the presence of the subjects to avoid bias and inconsistent reporting. The use of internationally validated questionnaires, suitable for adolescents, is another strength of the study. A limitation could be the current unequal number of males and females but, as we didn’t investigate sex related parameters and didn’t use sex specific questionnaires, in our opinion this item could be negligible for the purpose of the present research.

Conclusion

In understanding the relationship between MD and health, the evaluation of MD adherence is fundamental. Considering our sample of adolescents, we decided to apply the KIDMED questionnaire which is surely the most easy and suitable methods for younger population groups. Anyway, as also speculated in this paper, it does not give an exhaustive and real picture of adherence to MD, reason for which it has been reformulated, even if the original version is still the most used. Having available data on frequencies of consumption from FFQ, we established to calculate the MDSS and compare it to KIDMED to evaluate the correspondence of information obtained with the two different methods. As there are differences between methods, it is fundamental to recommend striving for agreement on the number food groups to consider, how they are measured and the dietary behaviors and lifestyle factors to include, as well as the methodology for assigning points in MD scoring systems. This approach will let comparisons across cultures and geographic regions since the topic is of great importance. Indeed, the lack of adherence of younger age groups to healthy dietary patterns, such as the MD, underlines the need to develop and apply nutritional education programs.

Author Contributions

All the authors conceptualized and designed the current research, performed the observational study and data analysis, wrote, and critically reviewed the manuscript; they have read and agreed to the published version of the manuscript.

Funding

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This research did not receive any specific grant from funding agencies in the public, commercial, or not -for-profit sectors.

Acknowledgments

The authors gratefully thank the Youth Aggregation Centers for their availability and all the volunteers who provided time for interviews. Moreover, a special acknowledgment goes to Dr Sara Mestici; with her collaborative attitude, she effectively contributes to the provision of the results.

Declarations of Competing Interest

None.

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A Comprehensive Review on the Effect of Exercise on Healthy Life in the Improvement of Quality of Life

Introduction

While heart diseases are defined as a concept that describes various diseases that may occur in the heart or blood vessels and pose a risk for a healthy life, cardiovascular system diseases are also known as very risky problems in terms of general health. Although there are many causes of cardiovascular system diseases, especially in recent years, it has been observed that overweight and obesity have extremely negative effects on heart health. Clinical studies show that overweight and obesity are associated with cardiovascular risk. These studies, conducted mostly in middle and older age groups, reveal a clear consensus that overweight and obesity increase the risk of death from cardiovascular diseases in the elderly, and that healthy weight control is essential for a long and quality life, especially heart diseases (Elagizi, et al. [1,2]). A large body of research has demonstrated that regular exercise behavior can improve cardiovascular health and reduce the risk of heart disease and stroke. For example, studies found that regular exercise can lower blood pressure, reduce LDL cholesterol levels, and even improve overall heart function. In addition to cardiovascular health benefits, regular exercise behavior has been found to be an effective tool for weight management (White M.P. et al. 2019). Some studies by (Jakicic et al. [3,4]) and (Petridou, et al. [5]) found that regular exercise can lead to significant weight loss and even prevent weight regain after diet-induced weight loss. According to the results of numerous studies on healthy living, it has been found that regular exercise behavior has a positive effect on mental health, which is of great importance in the process of sustainable healthy living and healthy aging.

There are many studies highlighting the effect mechanisms of physical activity on psychological wellness during the healthy aging process. One possible mechanism for the positive effect of exercise on mental health is the release of endorphins, naturally occurring chemicals in the body that can improve mood and reduce pain (Craft & Landers, [6,7]). Exercise can also increase blood flow to the brain, which can reduce the risk of cognitive decline and help to support mental and psychological health (Hassmen, Koivula, & Uutela, [8]); Gard et al. 2014). Regular exercise has been shown to reduce symptoms of depression and anxiety (Craft, Landers, et al.1998; Mandolesi et al. [9]), improve mood, increase self-confidence and self-esteem, and reduce stress (Malm, Jakobsson,Isaksson, [10,11]). In addition, regular exercise has been found to have a protective effect against the onset of mental health disorders such as stress, depression and anxiety, which negatively affect the healthy living process (Bond, et al. [12-14]). In addition to physical and mental health benefits, exercise has also been linked to improvements in overall quality of life. A study by D’Aurea et al. (2022) found that regular exercise can improve sleep quality, reduce stress levels, and increase overall happiness. Overall, the evidence suggests that exercise can have numerous positive effects on both individual and societal levels of sustainable quality of life. By promoting physical and mental health, improving overall quality of life, and promoting sustainable living practices, exercise can be an important tool for achieving sustainable development goals.

Discussion

Healthy aging is is a multifaceted concept that covers physical, mental and socio-psychological health. In addition to treatment measures that play an important role in reducing aging, Regular exercise behavior, together with healthy nutrition, can contribute to a healthy life and healthy aging process (Dominguez, et al. [15,16]). The effect of exercise on healthy life in the process of sustainable quality of life has been extensively researched in recent years. Studies aiming to examine the effect of a sedentary lifestyle on chronic disease and early death prove that regular physical activity is effective in preventing various chronic diseases and early death (Anderson & Durstine, [17]). Many studies show that there is a linear relationship between physical activity and health status, and that regular physical activity habits and increases in fitness lead to improvements in health status. Therefore, the importance of regular physical activity should be emphasized to reduce the risk of disease and premature death associated with physical inactivity. A study by (Warburton et al. [18]) found that regular exercise can improve cardiovascular health and reduce the risk of chronic diseases such as diabetes and obesity.

Similarly, a study by (Booth, et al. [19]) found that exercise can improve mental health and reduce the risk of depression and anxiety. In addition to physical health benefits, exercise has been found to have numerous positive effects on mental health and well-being. A study by (D’Aurea, et al. [20]) found that regular exercise can improve sleep quality, reduce stress levels, and increase overall happiness. Similarly, a study by (Craft & Perna [21]) found that exercise can reduce symptoms of depression and anxiety and even improve cognitive function. Also, a study by (Gill, et al. [22]) found that regular exercise can improve the neurological functions, including improving memory and attention and it has been found to have a positive effect on mental health by enhancing cognitive functions [23-25].

Conclusion

Healthy aging should be considered as a multidimensional concept that includes physical, mental and socio-emotional health. In addition to medical treatments that play an important role in slowing down the aging process, regular physical activity and healthy nutrition are known to make significant contributions to a healthy life. As scientific methods focus on healthy aging and the analysis and synthesis of aging modules, many new scientific findings will be revealed on this subject. There are various clinical studies in the field of healthy aging and sustainable healthy living, focusing on the solutions offered by anti-aging, maturation and aging, and ensuring the improvement that the individual can develop against the negative effects of aging, and active research has been carried out on this subject in recent years. A healthy lifestyle and healthy aging process are of great importance. Regular physical activity and exercise are associated with many different health benefits across all age groups, including older adults.

Not only the physical effects of exercise, but also the psychological effects play an important role in the process of a sustainable healthy life. Research has shown that exercise has positive effects on the tracking system, musculoskeletal system, balance, and mobility in older adults. It has also been determined that regular exercise improves mental health and alleviates psychological problems related to stressful life. As a result, it is known that regular physical activity habits contribute to increasing a healthy lifestyle. I am an individual at all ages, but especially regular exercise and healthy nutrition should be encouraged to support the healthy aging of the elderly and increase their freedom of life. In this respect, it is extremely important to introduce regular exercise habits into daily life in order to support possible treatment applications in the sustainable healthy living process and to enable the healthy aging process.

Acknowledgment

We would like to express our special thanks to Evangelia STAVROPOULOU for her very successful contribution to the literature research process and unique academic support in the publication during the process of this review article.

Conflict of Interest

The author certifies that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.

Funding

The author certifies that there is no funding from any financial organization regarding the material discussed in the manuscript or contributions.

Author Contributions

All the authors read and approved the final version of the manuscript.

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Advances in Understanding and Management of Atypical Fibroxanthoma

Introduction

Atypical Fibroxanthoma (AFX) is an infrequent, yet diagnostically challenging cutaneous malignancy characterised by spindle to epithelioid cells [4]. Its clinical behaviour ranges from indolent to locally aggressive, necessitating prompt and accurate diagnosis for optimal management [5]. While histopathological assessment remains fundamental, recent molecular studies have shed light on the underlying genetic alterations driving AFX tumorigenesis [6].

Epidemiology

AFX represents approximately 1% of all cutaneous tumours, predominantly affecting elderly individuals with a predilection for sun-exposed areas [7]. Its incidence is rising, likely due to increased awareness and improved diagnostic techniques. Epidemiological studies have also highlighted associations with immunosuppression and previous radiation exposure [8].

Pathogenesis

Chronic sun exposure and immunosuppression are implicated in AFX pathogenesis [9]. Molecular studies have identified genetic alterations, including TP53 mutations, chromosomal abnormalities, PDGFRB rearrangements, and CDKN2A loss, contributing to dysregulated cell proliferation and tumour growth [10,11]. Additionally, dysregulation of signalling pathways such as the PI3K/AKT/mTOR pathway has been implicated in AFX pathogenesis [12].

Clinical Presentation

AFX typically manifests as a solitary, rapidly growing nodule or plaque on sun- damaged skin, with variable clinical features such as erythema, ulceration, and bleeding [13]. Rarely, regional lymph node involvement or distant metastasis may occur. Clinicians should be vigilant in recognising AFX, particularly in patients with a history of chronic sun exposure or immunosuppression [14].

Histopathological Characteristics

Histologically, AFX exhibits a dermal proliferation of spindle to epithelioid cells with marked cytological atypia and brisk stromal inflammation [15]. Immunohistochemical staining aids in diagnosis, with positive staining for vimentin and CD10 [16]. Additional markers, including p16, p53, and Ki-67, may also be used to characterise AFX and assess its proliferative activity [17].

Molecular Pathology

Genetic alterations, including TP53 mutations, chromosomal abnormalities, PDGFRB rearrangements, and CDKN2A loss, have been identified in AFX [18,19]. These molecular findings provide insights into the underlying mechanisms of tumorigenesis and may offer potential therapeutic targets in the future. Furthermore, gene expression profiling studies have revealed distinct molecular signatures associated with AFX subtypes, which may have prognostic implications and guide personalised treatment approaches [20].

Differential Diagnosis

AFX can mimic other spindle cell tumours, necessitating careful histopathological evaluation and immunohistochemical staining to differentiate it from its mimics, such as dermatofibrosarcoma protuberans, spindle cell squamous cell carcinoma, and leiomyosarcoma [21]. Ancillary techniques such as fluorescence in situ hybridisation (FISH) and next- generation sequencing (NGS) may also be utilised to aid in the differential diagnosis of AFX [22].

Treatment Options

Surgical excision with clear margins remains the mainstay of treatment for AFX, with Mohs micrographic surgery offering precise tumour removal and preservation of surrounding healthy tissue [23]. Adjuvant therapies, including radiation therapy and topical imiquimod, may be considered in select cases, particularly in unresectable or recurrent lesions [24]. Emerging targeted therapies, such as tyrosine kinase inhibitors and immunotherapy, hold promise for the management of advanced or metastatic AFX and warrant further investigation in clinical trials [25].

Prognosis

AFX generally carries a favourable prognosis, with low rates of metastasis and disease- specific mortality [26]. However, local recurrence rates vary depending on factors such as tumour size, depth of invasion, and adequacy of surgical excision. Long-term follow-up is recommended to monitor for recurrence and assess treatment outcomes [27].

Conclusion

Atypical Fibroxanthoma is a rare but clinically significant cutaneous malignancy characterised by spindle to epithelioid cells [28]. Recent advancements in molecular pathology have improved our understanding of its pathogenesis and may facilitate the development of targeted therapeutic strategies [29]. Further research is warranted to elucidate the molecular mechanisms underlying AFX tumorigenesis and to optimise treatment approaches for this challenging neoplasm.

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Dystocia in Local Goat in Southern Ethiopia

Introduction

Dystocia, which translates to “difficult labor,” is a Greek word. According to Waage and Wangensteen, (2013), (Ahmed [1,2]) dystocia commonly happens when stage one labor does not progress to stage two labor or when thirty minutes pass before stage two labor or parturition begins. Stage one of parturition (labor) is defined as the preparation for fetal expulsion and includes decreased appetite, preparing a birthing area, isolation from the herd, restlessness, and leading up to the early uterine contractions, which become stronger and more frequent toward the end of this stage Majeed and Taha [3]. Fetal expulsion or delivery of the neonate is stage two of parturition. The third stage of parturition follows and ends with the expulsion of the fetal membranes, which usually takes place within 4-6 hours Ismail [4]. In small ruminants, dystocia can be caused by either maternal or fetal factors. It occurs because of fetal-maternal disproportion, multiple fetuses within the pelvic canal, fetal mal-positioning, incomplete dilation of the cervix, and uterine inertia (Brounts, et al. [5-7]).

Dystocia because of fetal-maternal disproportion occurs when a heavy male breed is mated with a small female breed Hafez, et al. [8,9]. In this condition, the birth of a fetus does not occur through the birth canal because of the giant size of the fetus or newborn and the small size of the birth canal Tripathi and Mehta [9]. Fetal causes of dystocia mainly include malposition, fetal emphysema, simultaneous presentation of twins and monsters, and a higher incidence of fetal dystocia. Maternal causes of dystocia include incomplete vulva and cervical dilatation (ring womb), a narrow pelvis, uterine inertia, and uterine torsion (Kumar et al., 2015; Noakes, et al., 2009). In small ruminants, in most cases in which assistance is needed, manual correction and vaginal delivery are achieved. It is important to emphasize here the idea that extensive attempts at manual delivery of the fetus are associated with poorer outcomes for both dam and newborn survival. Ideally, in cases where there is complete dilation of the cervix, a decision should be made if the size of the fetus allows its passage through the birth canal without causing extensive damage using traction Ismail [4].

Because of the small diameter of the pelvic canal, only limited manual manipulation of the fetus to relieve dystocia is possible; thus, a cesarean section is often necessary. Furthermore, the diameter of the birth canal of small ruminants is small, making manual manipulation of the fetus very difficult. Cesarean section was reported to be an effective method for the treatment of most types of dystocia and was safe for dams as well as fetuses, especially when performed as early as possible after the onset of labor. Cesarean section is the only treatment for fetal-maternal dystocia, followed by systemic administration of antibiotics and non-steroidal anti-inflammatory drugs. Single- layer cesarean section closure must be done in two layers despite single-layer closure, as it confirms zero leakage after checking uterine leakage. Cesarean or c-section is less required in sheep and goats than in cattle because of the lesser frequency of fetal-maternal disproportion as the basis of dystocia, i.e., only 1 out of 5 compared with 1 out of 2 in cattle Al-Timimi [10]; Kachiwal 2000; Bhattacharyya, et al. [11].

Also, failure of or incomplete dilatation of the cervix is a relatively frequent cause of cesarean section. More rare indications are fetal retention, usually in cases of multiple births, irreducible uterine torsion, and fetal malformation (Waage and Wangensteen, 2013). Indications for cesarean section in small ruminants include inadequate dilatation of the cervix, irreducible malpresentation, fetopelvic disparity, fetal abnormalities, and abnormalities of the dam’s genital tract Brounts, et al. [5]. When vaginal birth fails, some might consider euthanasia an alternative to cesarean section. Of interest to the sheep farmer, apart from animal welfare aspects, is the economic outcome after cesarean section, which depends on the costs of surgery and the outcomes of the ewe and her offspring. Short-term effects concerning complications survival and subsequent performance must be taken into account. Thus, to assess the effect of the cesarean section, cases should be monitored for a considerable period after surgery (Waage and Wangensteen, 2013). Fetal deaths are frequently observed when ewes undergo emergency cesarean delivery (Waage and Wangensteen, 2013). Early recognition and interference in cases of dystocia will result in a satisfactory outcome both for the dam and the neonate Ismail [4]. Generally, the outcome and success rates are much higher if surgery is performed early when the fetus is alive or freshly dead. Survival of both the dam and newborn was significantly affected by the length of time delay between the start of labor and the time of presentation for surgical intervention.

Prolonged labor before surgery impaired prognosis. So, early intervention by performing cesarean section can result in the delivery of live lambs and kids and much healthier dams (Waage and Wangensteen, 2013, Ismail [4]). Factors related to surgical skills will likely affect the prognosis of cesarean cases. The risk of postoperative complications, including infections, is likely to increase with increasing surgery time Scott, 1989. It has been reported that the most common complication following surgery was retained placenta, which was more likely to occur in ewes that received prolonged assistance before surgery. Moreover, it was reported that vaginal tears and secondary uterine inertia (hypocalcemia) are common in non-surviving sheep and goats undergoing surgery for dystocia. The most common complications reported are acute fatal peritonitis associated with uterine tear during surgery, retained fetal membranes, and acute metritis. Vaginal or uterine tears are common in cases with delayed surgical intervention, excessive pre-operative manipulation, or mishandling Ismail [4].

Case History and Clinical Examination

A local goat, aged about 4 years and 40 kg bodyweight with a history of partial one foreleg out from vulva 24 hours ago was presented at a veterinary clinic in Gesuba town. The animal has been suffering from this condition for over the past three hours. The owner said that they kept different goats in the home together. the goat was injured before pregnancy in her pelvic area and the was bone fractured. On clinical examination, body temperature was 40⁰C with normal respiration and tachycardia and there was no injury and hemorrhage over limb. Animal dehydration status is normal, checked by skin fold test or skin turgor test, but the animal was restless. The fetus was alive as checked or confirmed by their reflexes. The presentation, posture, and position of the fetus were normal but did not come out from the pelvic canal due to oversize and problems in the birth canal. Depending on the diagnosis and clinical examinations, the case was decided to be treated as an emergency surgical manipulation by a cesarean section to deliver a kid.

Surgical Correction and Treatment

After proper restraining, the goat was placed on the right lateral recumbency exposing the left flank. the hairs on the left para-lumbar fossa were clipped by using a surgical blade and 0.05 mg/kg xylazine intramuscularly for mild sedation. next, the surgical site was scrubbed with Iodine tincture as an antiseptic and 10% lidocaine for the inverted L-block on the left para-lumbar fossa as a local anesthetic agent. About 20 cm long vertical incision was placed on the left para- lumbar fossa (skin), approximately 10-12 cm below the transverse process with the help of a surgical blade. Then, the approaches were continued using a combination of blunt and sharp dissection over subcutaneous tissues and abdominal muscle layer to approach the abdominal cavity (Figure 1). Finally, the uterine horn of the gravid uterus was grabbed and exteriorized gently to avoid perforation, and placed holding suture was on the uterine horn to hold it outside the abdominal cavity.

Figure 1

The incision in the uterus was performed on the gravid horn and removed the fetus from the uterine horn by grasping the posterior limb of the fetus with the intact umbilical cord. The fetus was detached from the umbilical cord and nostrils were cleared from fluid to facilitate proper respiration. In addition, bleeding during the procedure was managed by applying sterile gauze using different straight and curved hemostatic forceps on the site. Single layer closure has been done by using Chromic Catgut with Cushing suture pattern and perforation has been checked by uterus leaking technique. surprisingly due to the 1.5 mm suture gap, there was no leakage found from the uterus and the animal has been recovered quite efficiently. The uterus was washed to remove blood clots and foreign particles with normal saline. Pen stirp 1ml/25 kg was dispensed in the uterus and in the abdominal cavity to prevent secondary bacterial infection. Simple interrupted suture on the muscles layer and simple continuous suture on a subcutaneous layer with chromic catgut. A simple interrupted suture pattern was applied on the skin with silk suture material (Figure 2).

Figure 2

Postoperative Follow-Up and Results

Post-operatively, tetracycline wound spray was also applied over the wound area. Besides, the goat was kept on antibiotic Pen stirp 1ml/25 kg was given for five days intramuscularly to prevent secondary infection. Furthermore, the goat owner was also advised to closely monitor and allow the goat in a favorable place and supply with good nutrition to facilitate wound healing. The complete healing was recorded on the 15th day post-operation without complications. Moreover, after one month of follow-up, the goat and its lamb were in good health (Figure 3).

Figure 3

Results and Discussion

Small ruminants are often more likely to experience dystocia than larger ruminants, and cervical obstruction or failure to dilate the cervix is a major contributing factor (Adams and Nairn, 1983; Brounts et al. [5]. Cervical ripening is a multi-factorial process that results from collagen being broken down by enzymes, inflammation, and hormone modulation. Animals may have cervical dilatation failure whenever there are changes to the abovementioned systems (Gahalot et al., 2017). The exact etiology of the condition is not known but several predisposing factors like hypocalcemia, hypophosphatemia (Al-Sultan and Majeed, 1996), and hormonal or mineral imbalances have been reported (Braun, 1997). In literature, the main indication for cesarean section in goats is incomplete dilatation of the cervix is true for present cases Brounts et al. [5]. Dystocia during first delivery and probably due to the narrow pelvis of the dam may be because of breeding the animals at a young age and/or poor state of nutritional management during gestation. Feto-pelvic disproportion has been reported as one of the main causes of dystocia in cattle (Dhaliwal, 1979) and ewes Brounts et al. [5].

Dystocia is life-threatening for both the dam and fetuses which needs immediate intervention. Proper diagnosis of causes of dystocia is very important to adopt accurate obstetrical operations. Radiography could be a tool to diagnose different causes of dystocia in small animals when other methods fail to do so Ahmed, et al. [12]. Survival of both the dam and newborn is significantly affected by the length of time laps between the commencement of labor and the time of presentation for cesarean section (Sharma et al., 2014). Several workers reported that cesarean section becomes necessary in goats to relieve dystocia due to relatively oversized fetus and narrow pelvis of the dam when manual obstetrical operations are difficult for vaginal delivery Roberts [13], Tibary, 2004; Hussain [14]. Generally, the success rate of cesarean section is higher if performed early when the fetus is alive or freshly dead Hussain [14]. The present case report describes the survival of dams following a cesarean section of a live fetus. Postoperatively after a few hours the animal was completely active and there were no signs of internal or external bleeding.

Post-operative care and antibiotic treatment to avoid any secondary bacterial infection and facilitate the wound healing process Majeed, et.al [4,15]. Dystocia due to fetal-maternal disproportion in small ruminants is a clinical case due to different sized mix breed herding at the same place that has been successfully managed by cesarean followed by using broad-spectrum antibiotic i.e., from group Tetracycline and non-steroidal anti-inflammatory drug i.e., Flunixin Meglumine therapy and the same was depicted Ahmed et.al [1,15]. If cesarean does not occur in time, it will be fatal for the dam and fetus’s life as it may result in the death of the fetus and ultimately mummification and maceration steep toward the low body condition score of animals resulting in the death of goat by systemic infection Ahmed et.al [1,4,16] (Kachiwal, 2000). This type of dystocia mainly occurs due to the sexual matting of large and small breeds and the same has happened in this case. For the prevention of this sort of dystocia, it is necessary to keep the same type of breeds in a herd instead of mixed breeds herd at the same place [17-21].

Conclusion

Dystocia due to fetal-maternal disproportion is common in different size mix breed herds at the same place. This can be overcome by separating flocks of the same size breed. In small ruminants, the digital manipulation of the fetus is often manageable. Prolonged and delayed attempts for vaginal delivery affect the outcome of the case severely. Therefore, cases of dystocia due to incomplete dilatation of the cervix in doe can be managed successfully by surgical intervention without any postoperative complication.

Declarations

Competing Interests

All authors declare no competing conflicts of interest.

Data Availability Statement

The data used in the current study are referenced in the article.

Funding Statement

The current study was conducted without the support of funding sources.

Consent for Publication

Not applicable.

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Climate and Its Relation to Greenhouse Gases

Introduction

Almost all human activities generate garbage or waste that is released into the environment, among them are waste that goes into the atmosphere and deteriorates air quality, balance and climate stability. Regarding climate instability, a few years ago it only caught the attention of scientists, but nowadays it is very common for us to find in the vox populi, in newspapers, magazines, newscasts and electronic media, information that hurricanes are increasingly more intense, that the rainy and drought seasons are increasingly irregular and that the summers are increasingly hotter and the winters colder and more unstable, attributing the responsibility for these events to the so-called climate change, climatic instability caused largely by the enormous amounts of greenhouse gases (GHG) that are produced and released into the environment and the atmosphere. But what is climate change? In short, climate change has been defined as any change in the climate over time as a result of natural variability or human activities. These changes can occur throughout the year both on land as well as air and sea. In order to understand how climate change occurs and what causes it, it is necessary to understand mainly the phenomenon known as the greenhouse effect, which is partially responsible for the variation in the planet’s temperature, along with two other factors, the Sun and the distance from our planet. him. Among the gases that make up the atmosphere (mainly nitrogen and oxygen), those that have the greatest impact on the temperature imbalance are the so-called GHGs.

A GHG is one that has the property and capacity to absorb radiation, returning it to the Earth’s surface, thereby causing an increase in temperature, particularly where these gases are concentrated and in general and gradually throughout the planet, producing the phenomenon known as the greenhouse; This result is a phenomenon produced by these gases that are found in the atmosphere and that retain part of the solar energy reflected by the ground, preventing it from going out into outer space, absorbing it and transforming it into an internal molecular movement that produces the elevation of the ambient temperature (CEPSA [1]). In November 1997, at the convention on climate change, organized by the United Nations (UN) in the city of Kyoto, Japan, what is known as the Kyoto Protocol was agreed upon, which had the objective of reducing emissions of these gases to 5.2% below normal levels. from 1995 depending on the gas being treated; In that document, the main GHGs were established: carbon dioxide (CO2), ozone (O3), methane (CH4), nitrous oxide (N2O), Hydrofluorocarbons (HFCs), perfluorocarbons (PFCs) and sulfur hexafluoride (SF6) and although Water vapor was not included in this list, it is the one that produces the most greenhouse effect, due to its ability to retain the heat emanating from the earth’s surface. Currently, the activities carried out by human beings over the years have generated enormous quantities of GHG, concentrating these gases in large population centers, where industrial, agricultural and livestock activity is very high.

These gas concentrations cause variations in those areas the atmospheric composition influencing and altering sooner or later this climatic composition. Certain GHGs arise naturally, but are influenced directly or indirectly by human activities while others are entirely man-made; those that arise naturally are: water vapor (H2O), carbon dioxide (CO2), ozone (O3), methane (CH4) and nitrous oxide (N2O) and those that are completely artificially produced for the benefit and/or comfort of the living things are: chlorofluorocarbons (CFCs), hydrofluorocarbons (HFCs) and hydrochlorofluorocarbons (HCFCs) (collectively called halocarbons), and fully fluorinated species, such as sulfur hexafluoride (SF6). As mentioned, water vapor produced naturally or as a result of some human activity is the gas that most contributes to the greenhouse effect and is the one most directly linked to the climate; This is because the evaporation of water depends mainly on the high temperature of the surface and because this vapor passes through the atmosphere in very rapid cycles and with a duration in the atmosphere of approximately eight days; According to the Intergovernmental Panel on Climate Change (IPCC), water vapor, fog and haze are responsible for between 36% and 70% of the greenhouse effect, which together with the byproducts of burning Fossil fuels are mainly responsible for global warming. Furthermore, this warming causes a feedback loop, that is, the higher the temperature, the more water vapor is produced when it evaporates, which in turn generates a greater greenhouse effect, which in turn raises the temperature and consequently greater water vapor and so on.

Carbon dioxide (CO2) is a byproduct of cellular respiration and the burning of fossil fuels; ozone (O3) in the stratosphere with the passage of time and due to the effect of other gases has caused the thinning of the ozone layer, causing the passage of harmful ultraviolet (UV) rays over time and in the troposphere where there are A higher concentration of this gas contributes to the generation of the greenhouse effect; Methane (CH4) is a gas produced as a result of the digestion of food consumed mainly by cattle and cattle. The emissions and concentrations of other GHGs depend directly on the quantities of these chemicals produced at the expense of demand for the benefit and comfort of people. Regarding their elimination process, all GHGs depend to some extent on the climate and are largely eliminated through chemical or photochemical reactions within the atmosphere. Only CO2, which is the most abundant and most researched GHG, is eliminated. through temporary reservoirs or storage deposits, such as the atmosphere itself, terrestrial plants, soil, water, ice and ocean sediments (Henry Oswaldo Benavides Ballesteros H O, León Aristizabal G E [2]). In the last two centuries, 2.4 billion tons of CO2 have been produced and expelled into the atmosphere, which is the equivalent of what 522,000 million cars would emit annually to this day, massive quantities of the main GHG that in turn represents the soul of human civilization but also in these times its nightmare. According to scientists, there is very little time left for this extreme climate situation of high temperatures and historic high cooling to be irreversible and that if nothing is done to reduce this type of gas emissions, it is certain that the climate will worsen, as well as its consequences (Howe Verhovek S [3]).

Measuring the impact that GHGs have produced to date on the environment and climate is a complex task; however, various ways have been proposed to do so, including the Living Planet Index (LPI) and the Environmental Sustainability Index (ESI), of all these ways of measuring it, the best known is through the Ecological Footprint proposed by the Canadian Ecologist William Rees and Mathis Wackernagel in 1996. The Ecological Footprint is an environmental indicator of an integrative nature of the impact exerted by a certain community (country, region or city) on its environment, both the necessary resources and the waste generated for the maintenance of the community’s production and consumption model are considered (Rees and Wackernagel, 2000). In Mexico, based on the National Inventory of GHG Emissions, it was calculated that in 2018 873 million tons of these gases were emitted. The panorama was extremely worrying if we added that emissions increased by 45% more than the estimated in 1990 (Secretariat of the Environment and Natural Resources SEMARNAT [4]). In this case, the sector that emitted the most and continues to emit the most emissions and waste of these gases is the producer of fuel for all types of transportation, because it mainly includes the use of fossil fuels, and is also responsible for the generation of Electrical energy is another highly polluting sector, especially this second sector since it is responsible for producing around 70% of GHG emissions, due to the excessive use of fossil fuels to produce the energy necessary to supply human consumption.

Other industrial processes such as the production of cement, glass, steel, paper, food and beverages also emit a large amount of GHG into the atmosphere; The total emissions from this type of industries represent 9%. On the other hand, agricultural activities generate two types of greenhouse gases methane (CH4) that add up to 8% of the emissions from these activities and nitrous oxide (N20). These gases are generated by fermentation in the digestive system of livestock or directly from manure and its handling, as well as from crops and decomposing soil, since the microorganisms present in it promote their production and release into the atmosphere. The waste generated in homes also emits GHG, representing approximately 12% of the total production in Mexico. On the other hand, municipal and industrial wastewater, as well as livestock, emit methane (CH4) and nitrous oxide (N2O) when as well as the burning of hazardous waste such as corrosive, reactive, explosive, toxic, flammable or infectious biological materials (CRETIB), especially outdoors. The correct management of this waste and the adequate treatment of wastewater have undoubted benefits, however, despite everything, they generate secondary products, among which are GHGs. GHG emissions due to these situations and others such as the disposal of solid waste in inadequate garbage dumps increased 96% between 1990 and 2002. The most serious thing about this situation is that despite the improvement of infrastructure and the acquisition of more and better inputs to correctly carry out these activities, emissions continue to increase, estimating that by 2025 they could increase by 140%.

Regarding the search for comfort and benefit of modern societies, certain GHGs have been produced such as trifluoromethane (CHF3), also known as fluoroform, which is used in the manufacture of silicon chips and as a fire suppressant, it is the most abundant hydrofluorocarbon (HFC) gas, remains in the atmosphere for 260 years and traps heat 11,700 times longer than carbon dioxide (CO2), hexafluoroethane (C2F6), used in the creation of semiconductors, remains in the atmosphere up to 10,000 years, this longevity, together with its ability to retain heat 9,200 times more than carbon dioxide CO2, has sparked the interest of the IPCC to follow closely, sulfur hexafluoride (SF6), which is an inert gas widely used in the electronics industry as an insulator; The IPCC considers it the most powerful GHG in the world, with a capacity to trap heat 22,200 times more than carbon dioxide (CO2) and trichlorofluoromethane (CFC-11), this refrigerant causes several negative effects on the environment, in addition Retaining heat 4,600 times longer than carbon dioxide (CO2), it reduces the ozone layer (O3) faster than any other refrigerant, without forgetting the environmental impact of chlorine. The list could expand as scientists study the phenomenon more; is the case of sulfuryl fluoride (SO2F2), used as a fumigant against termites, its capacity as a GHG has been announced in March by scientists from the Massachusetts Institute of Technology (MIT) and it has a useful life of 40 years and is capable of trapping heat 4,800 times more than CO2, although in the atmosphere it is only found in 1.5 parts per billion, this amount increases by 5% per year according to a recent article published in the Journal of Geophysical Research (ECODES (s/f) [5]).

Natural Elimination of the Main and Largest GHG (Carbon Dioxide, CO2)

Forests, jungles and mainly plankton and algal forests of the seas and oceans participate in the flow of carbon by capturing it because it is necessary for them to carry out photosynthesis and subsequent storage in their biomass, which is why they become sinks. coal; However, when vegetation is removed and burned to convert the land into pastures or crop fields or its reproduction is reduced (as in the case of plankton and kelp), the vegetation is eliminated and a large part of the stored carbon is quickly converted into CO2 and released back into the atmosphere or it is not absorbed and it is then that they inversely become a source of CO2 emissions, thereby increasing pollution by generation and emissions of GHGs into the atmosphere. An alarming fact is that, until the end of the 20th century, 87.7% of global CO2 emissions were generated by the United States alone, which emitted 30.3% of the total, Europe generated 27.7%, the Soviet Union 13.7% and the region of China and India 12.2%. South and Central America contributed just 3.8% of the world total; Currently, these figures have been increasing alarmingly due in some cases to the improvement in purchasing power, which is why more electrical household appliances are purchased and consequently greater consumption of electrical energy or the purchase of more automotive units, to the creation and urbanization of new and large population centers generated a greater carbon footprint and, on the other hand, political indifference to face the commitment accepted when signing the wide variety of global agreements regarding the reduction of GHGs and the lack of maintenance of equipment. responsible for incinerating and/or treating wastewater correctly, as well as the lack of vigilance or corruption when it comes to monitoring the correct application of the rules for soil change and livestock management and feeding (European Parliament News [6]).

Effects of Climate Change

The degradation, pollution, alteration and imbalance of the gases that make up the atmosphere generates adverse effects and consequences on the climate, causing disorders and alterations on ecosystems in general, all elements of the habitat and biosphere environment have complex relationships and interactions among themselves. Therefore, altering some causes changes in the remaining elements, sometimes imperceptible and other times very obvious. Vegetation, for example, can only reproduce and grow successfully only within a specific range of temperatures and respond to certain amounts of rain and humidity. Animals, for their part, also need certain ranges of temperature and rainfall. Throughout history the Earth has had changes in the climate that have even caused the extinction of species, however, when the change in climate does not occur gradually and is accelerated like what is currently happening, they are magnified, accelerated and They generate impacts linked together. Currently, due to climate change, for example, more intense hurricanes occur in greater numbers, causing flooding and destruction in coastal populations, an increase in global temperature and of the oceans and seas, causing glaciers to melt more quickly and increasing the level of the sea and consequently change in the pH of the water and affectation of marine flora and fauna, in other places intensifying and prolonging droughts and their consequences, these are only some consequences of climate change without considering all the effects produced and their consequences.

While drought seasons intensify in Africa, at the same time in other parts of the world there are reports of more intense rains and storms, as well as frost and/or snowfall; In recent years, atypical meteorological phenomena have occurred that have to do with the warming of the seas and that have caused greater loss of human life and property. A recent study shows that in recent years an increasing number of intense hurricanes have been recorded, that is, those that were commonly category 3 have become category 4 or 5 on the Saffir- Simpson Scale, characterized by winds higher than 250 kilometers per hour and destruction of housing infrastructure in general, flooding of population centers near the coast causing the population to be evacuated due to the mortal risk of remaining in their homes. Regarding rainfall, there is a tendency towards greater precipitation where the rain was constant and regular, while where the rains depend on the meteorological phenomena that occur in the Pacific Ocean, the trend seems to be in the opposite direction, generating uncontrollability. in the agricultural and fishing season cycles with the economic and social consequences that such climatic alterations produced in the communities that depend directly and indirectly on these activities and the products generated. As a consequence of global warming, changes have been observed in regions as distant as the poles, since in just under 40 years, the accelerated disappearance of the Arctic ice sheet has been observed, with close to 20% of its surface loss, which would be equivalent Half of the Mexican territory has thawed.

At the other pole, on the Antarctic continent, in 2002 the Larsen B platform fractured, causing an ice surface of 3,240 square kilometers to detach and melt (United Nations [7]). These thaws have not only affected the Arctic and Antarctic, directly altering not only the habitat of polar fauna, but also that of the seas and oceans; On the other hand, the cold areas with perpetual ice found in the high areas of mountains and volcanoes are also affected by the same consequences of global warming, for example, the glaciers of the Swiss Alps have lost more than a third of its surface and at least half of its mass between the year 1850 to the present and scientists have calculated that, by the year 2050, 75% of the glaciers in the Alps could disappear. In Mexico there are also glaciers and they are mainly found in the high areas of Iztaccíhuatl, Popocatépetl and Pico de Orizaba and although their reductions have not been so great, they are very important, for example, the glaciers of Iztaccíhuatl have suffered reductions of up to 55% Between 1960 to date, in Pico de Orizaba and Popocatépetl, the trend has been similar, but in the latter the reduction has been more accelerated due to the volcanic activity that has occurred in recent years. Experts have estimated that if current rates of shrinkage of Mexican glaciers continue, they may disappear completely in less than 30 years. During the 20th century and as a result of the melting of the polar caps and glaciers that flow into the seas and ocean, its level rose at an average speed of 1 to 2 millimeters per year, which is equivalent to a total rise of 10 to 20 centimeters in the century, this increase might seem minimal to us, but in fact it is worrying for many countries since there are numerous cities located in low coastal areas that can be flooded or are already in that process and many are even below sea level , as is the case of Amsterdam where the risk is greater, whose altitude is 4 meters below sea level.

This implies that many millions of people are susceptible in the near future to suffering more serious consequences than they already have from flooding due to rising levels, cities such as Cairo, Lagos, Maputo, Bangkok, Dhaka, Jakarta, Bombay, Shanghai, Copenhagen, London, Los Angeles, New York, Buenos Aires or Santiago de Chile, among others, could have serious repercussions in the immediate future due to overflows and/or floods of the oceans and seas. Likewise, biodiversity in many regions is already suffering severe and critical adverse changes due to the effects of climate change, many species of flora have migrated or have disappeared or are in the process, likewise species of fauna or flora that do not They can migrate, either due to movement, physiological or behavioral limitations, they could disappear. Austrian scientists found that some species of animals and plants from alpine areas have moved up to four meters per decade from their original areas to higher places in the mountains or volcanoes they inhabit, this is explained and is due to the increase in temperature, since the lower parts of the mountains and volcanoes have increased their temperature and the alpine species that depend to survive on low temperatures present in the higher parts have migrated to increasingly higher altitudes to find suitable habitats to survive; Changes in temperature also have effects on the feeding strategies of some species that depend on ice shelves to hunt their prey. For example, the population of Adelie Penguins in Antarctica went from 1990 to 2004 from a population of 320 pairs with offspring down to just 54 (and continuing to decline), in a place where the average temperature has increased by almost 5.5°C in fifty years.

The polar bears of Hudson Bay, on the southern limits of North America, do not fully meet their nutritional needs, since the period in which they hunt seals annually has been reduced or they have been migrating to other colder regions, which previous because the ice shelves from which they hunt fracture at least three weeks earlier than usually occurred, in addition to the already described examples of polar bears and penguins, these changes will also surely harm seals, walruses and other marine mammals. Coral reefs have been suffering from so-called coral bleaching due to global warming; This condition is due to the fact that a coral can be made up of one or many tiny polyps, small circles that cover its surface and through which they feed. Within these polyps, the corals shelter certain microscopic unicellular algae from which they obtain nutrients. and to which they offer protection and waste that they use as food, when the sea temperature increases or the sea water is contaminated, sediments are deposited on the corals and the algae are detached from the polyps, which leads to the loss of their color. and allows us to observe its white skeleton built of calcium carbonate beneath the tissue; The loss of coral reefs is not only important because of their tourist attraction, but because extraordinarily diverse ecosystems are consolidated around them. In the Indian Ocean, for example, 46% of corals have bleached, while in the Pacific, coral mortality has been recorded that fluctuated between 50% and 70%.

In early 2007, the world’s attention was directed to the IPCC, which presented a preview of the Fourth Assessment Report of the Intergovernmental Panel on Climate Change, stating that if the necessary policies are not adopted to minimize the causes of climate change , mainly due to the emission of GHGs, in the year 2100 the global concentration of carbon dioxide (CO2) will be between 540 and 970 parts per million, currently it is almost 380 and in pre-industrial times it was 280 parts per million. This evaluation report predicts that, for the last decade of the 21st century, the temperature could increase between 1.8 and 4°C and with it the sea level could rise from 18 to 59 centimeters, causing serious and extensive flooding, mainly in core areas. coastal populations; A temperature increase of this magnitude can be accompanied by drought conditions in some places and intense rains in others. In the case of turtles and other species of reptiles, such as crocodiles and alligators, the sex ratio could be altered as a result of the increase in temperature, since in this group of animal’s temperatures is key to defining the sex of the offspring. Between two and three degrees of difference are enough to define whether the calf is female or male. This change in sexual proportions alters the structure of populations, since competition for a mate increases and many individuals may be left without a partner to reproduce. The volume of plankton can also be further affected and reduced and because it is found at the beginning of the food chain of all marine ecosystems, it can affect the global productivity of the oceans and sea, and with it, the global production of products. fisheries for human consumption.

The human population will also have to face and suffer from future changes, for example, an increase in heat-related deaths and illnesses and a decrease in deaths caused by winter cold have been predicted in some temperate regions; Strong changes in temperature also have effects on the geographical distribution of disease-transmitting animals or vectors, such as mosquitoes, ticks, bedbugs, fleas, cockroaches, rats, mice and others. Some of them, such as those that transmit dengue and malaria, live in warm areas, so if global temperature increases and areas with warm climates expand, they could move to latitudes that were previously cold, expanding transmission areas. of malaria and dengue, diseases that annually kill a large number of people in the world. Also, the quality and availability of water, one of the most important resources for humanity, could be affected by the increase in global temperature. The increase in temperature reduces the concentration of oxygen dissolved in water, which is essential for survival of aquatic organisms, added to this, the available water reserves will be greatly reduced in many of the areas where it is currently insufficient due to evaporation processes. The impacts described above come from mathematical models that, although they try to take into account most of the variables that currently act on the climate, are unable to accurately predict what will happen in the future, that is, although they are likely to occur in the way it has been predicted can also be done differently and with another magnitude. The impacts on the environment generated by climate change can also affect society in different aspects, including quality of life and health. a complex system of pipes that descend 750 meters. underground, at that depth the water combined with CO2 meets a terrestrial layer of porous basalt that traps the CO2 creating dots in the cream-colored rock.

This capture technology so far only represents 0.1% of the extraction of this gas. On the other hand, in Arizona a team of engineering researchers is experimenting with a mechanical tree that will be able to do the function and work of a thousand natural trees in the near future in terms of capturing and storing this gas. In another part of the planet, in Australia, an oceanography researcher works with a marine garden made up of kelp and wakame, which are a type of algae that grows in shallow oceans and can reach great heights and are full of health benefits. health, this researcher tries to find and understand the way in which these algae can store billions of tons of CO2; This with the intention of finding immediate alternatives to reduce the concentration of CO2 in the atmosphere, which for thousands of years remained below 280 ppm until the Industrial Revolution began to raise it, currently reaching 420 ppm. Another project is to plant genetically treated trees to grow quickly, last for many years and not require much water to do so. These forests would be natural carbon sinks so they would store the carbon they capture for many years. The company CLIMEWOKS uses another technological alternative that consists of capturing carbon directly from the air, for this it has built giant fans that would direct the air from the atmosphere through filters towards liquid and solid solvents to which the CO2 adheres to trap it and then These would be buried underground, despite using geothermal energy for their operation, the main problem is the high cost that their massive implementation would imply.

The TURECEK Company develops modules that work with solar energy to trap CO2 and store it in fissures in the earth. To do so, it builds units that are the size of a small tent that contain a fan that directs the air towards a panel made of polymer. special that separates CO2 from other gases and then deposits the polymer in large earthly concavities, On the other hand, for some years now, plants have been cultivated, harvested and burned to obtain energy (biomass), which absorb carbon when growing, trap and store the CO2 that is released during this conversion, which must be purified and trapped in large limestone filters before burying this substrate in the subsoil, its handling requires great care to avoid leaks; Another option is carbon mineralization, stones such as basalt can react and adhere to carbon in the atmosphere and then deposit those sediments in large caves. Using smart agriculture can also help sequester carbon, rather than tilling entire fields, managed tillage keeps it in the soil, equally cover crops between seasons also reduce the release of carbon from cropland. Despite the use of science and technology to reduce and trap GHG emissions and in particular CO2, it is urgent that humanity and industry do two things at the same time, reduce their current and future emissions and reverse the effects. From what has already been issued, under this position there is great optimism among scientists when it comes to technology, but not so when it comes to considering the political disposition and its representatives.

Conclusion

Climate change caused by the large amount of GHG that currently exists in the planet’s atmosphere is so severe, broad and critical that it is not possible to solve it in a short or medium time, nor by means of a technology and/or strategy, to This requires the sum and symbiosis of several, as well as the political availability of the rulers and the population in general. The hopeful thing is that there are currently several technologies, some still in development, that can be applied to solve the problem of climate change; the big problem for their application continues to be their high costs. For now, changing the way we behave, modifying the way we produce and consume energy is essential to find significant global results. Promote policies and actions to have less waste and more intelligent use of our renewable and non-renewable resources, improve the efficient use of energy and fuel savings for the vehicles that are produced, the increase in the use of wind energy and solar energy, the use of biofuels from organic waste and the efficient and effective protection of terrestrial and maritime forests (algae and corals), are also effective actions to mainly reduce CO2 (the main GHG) and other gases that trap heat on the planet due to the greenhouse effect they cause. However, the global demand to stop, control and reduce greenhouse gas emissions is fundamental, little progress has been achieved, look back, support and promote the work, projects and progress achieved so far with the use of science and technology regarding the capture, storage or elimination of GHGs, mainly CO2, is possibly the way to more quickly confront, control and/ or reduce the climate imbalance produced by global warming and cooling; Although hopes are placed on the massive development of technology, we must not forget that practically none of them are harmless, each one to a lesser or greater extent can also cause pollution, which is why it is necessary to continue working to find their application with the highest level of social, energy and anti-pollution responsibility.

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Generalized Doppler Effect in Wave Interaction with Uneven Moving Boundaries of Mobile Environments

Introduction

In medical diagnostics and measurement technology, devices are widely used that work using the Doppler Effect – changing the frequency of signals reflected from moving objects [1-3]. This effect is widely used in the technology of remote measurements of the speed of moving objects of any nature, especially in the case of unavailability of direct measurements. It is known [4-6] that the Doppler Effect underlies modern theories about the Universe [7]. Numerous varieties of the Doppler Effect are known and experimentally recorded [8-10], including: anomalous [11-13], reverse [14-16], double [17], angular [18-20], rotational [21-23] , multimode [4], in chiral [24-26], nonlinear [27], metamedia [19] media, in acousto-optics, magnetooptics, plasmonics, etc. [28]. These varieties of the Doppler Effect significantly expand the possibilities of its use in various fields of wave technology in wide frequency ranges (optical, microwave) of waves of various physical natures (elastic, electromagnetic, etc.). At the same time, it should be noted that in practice the Doppler Effect can provide relatively high accuracy only in relatively short periods of time when measuring instruments are operating, when the speed of moving and studied objects can be considered constant during the measurement period. In this case, the conditions for uniform relative movement of the radiation source and wave receiver or a wave reflected from a uniformly moving interface between the media are met. At the same time, it is known that in the general case this condition is not met and the accuracy of measurements due to the failure to meet the conditions for the Doppler Effect is low, and conclusions based on these measurements may be erroneous.

In a number of measurement technology problems, for example, in radar problems, there is always uneven displacement or movement of interacting objects, which is called the micro-Doppler effect [10]. Under these conditions, the characteristics of the effect change over time [8,29] and taking into account the unevenness of movement becomes an important task in practice. To describe and analyze the Doppler Effect, fairly clear geometric models are usually used [1,2,26], which make it possible to analyze relatively simple situations. At the same time, to solve a wider and more complex range of problems, a less developed wave theory can be used, used in particular in [4], to study the multimode Doppler Effect in a waveguide structure in [4]. The problem of wave reflection from moving interfaces between media was considered by many authors [6] without taking into account all the parameters of the adjacent media, as an analysis of the significantly influencing characteristics of the effect shows. In particular, the influence of the movement of media adjacent to the boundary was not considered. This may be due to the fact that usually the effect of the movement of the media themselves was previously considered an effect of second order of smallness due to the relatively low speed of this movement. The analysis shows that the movement of media leads to entrainment of the wave process by these media, the emergence of non-reciprocity of the parameters of wave-guiding media for forward and backward waves, to a change in the parameters of wave structures and to a qualitative change in the physical properties of these structures.

This paper examines the features of the reflection of waves from an unevenly moving interface in the general case of moving media, the influence of the uneven movement of media adjacent to the interface on the features of changes in the frequency of elastic waves reflected and passing through the interface, for which these effects are more pronounced than for electromagnetic waves due to the low speed of these waves. It has been established that, in addition to the Doppler effect, when the boundary moves unevenly, an additional time-no stationary frequency shift is observed, determined by the magnitude of its acceleration, similar to the effect previously considered by Ritz [30]. In analytical form, relations are obtained for finding the frequencies and coefficients of reflection and transmission of waves during their interaction with non-uniformly moving boundaries of media in the general case, taking into account the movement of media adjacent to the boundary. It has been established that the frequency shift of reflected waves from an accelerated moving interface can be observed in the absence of the classical Doppler Effect and, under certain conditions, exceed the frequency shift observed due to the Doppler Effect, which determines the need to revise a number of results based on the Doppler Effect.

Simulation

Let us consider the reflection of waves from a non-uniformly moving interface between two media, each of which, in the general case, can also move. Let the flat interface between two media move along the 0x axis (Figure 1). At a constant speed of movement, its coordinate is described by the dependence x(t)=ut. In this case, the well-known Doppler Effect is observed [4]. With uniformly accelerated motion of the boundary in the same direction, the change in the coordinates of the interface is described by the function:

where a is acceleration. Note that the dependence of the change in the frequency of the reflected wave on the magnitude of the acceleration for the problem of accelerated approach of the source and receiver of radiation was considered by Ritz, who discovered its difference from the Doppler Effect [31-33]. In the case of accelerated motion of the boundary, the effect of changing the frequency of the wave reflected from it (and differing from the Doppler Effect) can be called the Ritz effect. The possibility of a more general case of uneven movement of the interface between media, when the coordinate of the interface between media is described by the function x (t ) = ∫u (t )dt , where u (t ) is the speed variable in time, requires a separate description. For elastic waves, physical processes are expressed more clearly due to the comparability of wave speeds and the speeds of movement of media and interfaces observed under normal conditions. For electromagnetic waves, in particular light waves, these effects, due to the high speed of light, will appear over long periods of time or when waves propagate in slow-moving media and structures. Let there be an acoustic medium to the left of the boundary (Figure 1), characterized by the density ρ₁ and speed of propagation of waves in the medium c₁ , and a medium with similar parameters, respectively ρ₂ , c₂ ,to the right. Without dwelling on the question of the practical feasibility of the model, for generality we assume that each of these media can move along the 0x axis with speeds υ₁ and υ₂. In this case, each of the media carries away the wave process with its movement. Then the speed of propagation of waves along the axis (forward waves) in the first medium is equal to c₁₀ = c₁ +υ₁ , and the speed of propagation of waves in the opposite direction (reverse waves) is equal to c_1R= c₁ −υ₁ . In the second semi-infinite medium, waves (passing through the interface between the media) propagate waves only in the direction of the 0x axis with speed c_2T = c₂ +υ₂ . There are no waves in the opposite direction.

Figure 1

The pressure functions of the incident p10, reflected p10 and transmitted p2T waves through the interface must satisfy the wave equations in the first and second media, and must also satisfy the boundary conditions on the moving interface (1), which can be represented in the form of relations [6,31]:

The choice of different frequencies for direct ω, ω₁ , ω₂ , backward and transmitted waves in (2) is determined by the Doppler effect and allows us to obtain an analytical solution for calculating the parameters of direct and reflected waves that satisfies the wave equations in both media and the boundary conditions at the moving interface between the media. Note that the change in the frequency of the transmitted wave depending on the frequency of the incident wave has not been studied previously [2,3,26], perhaps for the simple reason that this change in frequency occurs only when the parameters of the first and second media differ, that is. for a case that was not considered.

Main Results

Substituting the desired solutions in the form of functions (3) into the first boundary condition (2), taking into account (1), gives the relation:

Taking into account the movement of media, relation (4) can be represented as:

Relation (4) relates the frequencies of the reflected wave 1 ω and the wave that passed through the interface between the media 2 ω , with the frequency of the incident wave. In previous studies [1-3], they were limited to analyzing the case of a constant speed of movement of the interface between media (Doppler Effect) (u = const ) , in which the change in the frequency of reflected waves does not change with time. This approximation is mainly used in practice.

In this case, the magnitude of the acceleration is a = 0 . This approximation can also be used for uneven motion during periods of time when the condition: at <<с is satisfied, which, however, is violated over time. Over time, the speed change parameter increases and the main effect of frequency change associated with acceleration becomes. This effect also manifests itself in the general case for variable acceleration. When the relation at >c is satisfied, this effect becomes more significant than the frequency shift in accordance with the Doppler effect.

Note that the Doppler Effect is considered for the reflected wave. Therefore, it is necessary to fulfill the condition  when the wave catches up with the interface between the media when the first medium and the boundary are moving or, if the first medium is stationary at 1ñ > u . Generalization to the case of accelerated motion (Ritz effect) leads to the need to fulfill the relation  (the wave catches up with the interface between the media escaping with acceleration). If  the wave in the first wave moves at the speed of the interface, the effect of wave reflection is not observed. Under the condition 1 1 ñ =υ , the speed of movement of the medium reaches the speed of wave propagation. In this case, there is no reflected wave and the frequency of the reflected wave (3)1 1 ñ →υ tends to 1 ω →0 . Thus, from relation (3) it follows that the change in the frequency of reflected waves includes a time-constant component (depending on the speed of movement of the interface – this is the Doppler Effect), and a time-variable component (at constant acceleration – this is the Ritz effect). In this case, a change in the frequency of the reflected wave is observed over a limited period of time, equal to constant acceleration , and the frequency of the reflected waves decreases 1 ω →0 over time until the moment when the speed of movement of the interface, which increases due to acceleration, reaches the speed of propagation of the incident wave.

The speed of movement of the interface between the media at this moment in time reaches the speed of the wave. During this time, the interface between the media shifts by a distance equal to

Discussion

In the simplest known case of a uniformly moving interface between moving media (a=0, u≠0), from equation (4) the following relations follow:

If the media, as in commonly considered configurations, are immobile, then υ1=0, υ2=0 and general relations (3) turn into the known relations of the Doppler Effect [1,2]:

The second of these relations shows that a change in the frequency of a wave that has passed through the interface between media is observed only if these media have different wave propagation speeds c₁ (≠) c₂ . If the medium on both sides of the interface is the same, then the frequency of the transmitted wave does not change 2 ω =ω . In this case, if the interface between the media moves in the direction of propagation of the incident wave (velocities u and c1 coincide in direction, that is, the interface “runs away” from the wave), then the frequency of the reflected wave is less than the frequency of the wave. In the more general case of uneven motion of the interface between media ( a ≠ 0 ), the dependence of the relative change in the frequency of reflected and transmitted waves through an accelerated moving interface (at u/c=0.1 and u/c=0.2) on the parameter at is described by the relation (4 ) and is shown in Figure 2. The frequency shift due to the Doppler Effect depends only on speed and does not depend on time. The frequency shift occurring due to the accelerated movement of the interface becomes decisive after a time interval t~u/a even at a small acceleration a, when it reaches the value at~u. Accounting for both boundary conditions (2) gives a system of equations for determining the amplitudes of the reflected and transmitted waves, which takes the form:

Figure 2

Hence, the reflection coefficient R and the transmission coefficient T are determined by the relations:

from which it follows that the coefficients R and T depend only on the parameters of the media, the velocities of the waves in these media and on the velocities of the media themselves (Figure 3). It is possible to ensure the absence of a reflected wave by fulfilling the following relation between the velocities and parameters of the media:  . If the media are stationary, then υ₁ = υ₂ = 0 the reflection coefficient is determined by the relation:

If the second medium is stationary ( υ₂ = 0 ), then the matching condition and the absence of a reflected wave can be achieved by moving the first medium with a speed. Thus, the movement of the media can provide the condition for matching the parameters of the media, in which there is no reflection from the interface between the media.

Figure 3

Conclusion

The stationary change in the frequency of waves, observed when they are reflected from uniformly moving objects (Doppler effect) or interfaces between media, depends not only on the speed of movement of the interfaces, but also on the parameters of the adjacent media. The effect of changing the frequency of the reflected signal due to accelerated movement leads to a significant distortion of the results of measuring the speed of moving objects during high, even short-term accelerated movements of objects or when measuring the speed of distant objects even at low accelerations, when the parameter of the product of acceleration and time becomes comparable to the speed of the object. This shows the need to adjust the settings of multiple Doppler sensors. Coordination of the media, which makes it possible to ensure the absence of a reflected wave, can be ensured both by selecting the parameters of the adjacent media and by the movement of these media. When reflected from non-uniformly moving objects or media interfaces, an additional frequency change occurs, the magnitude of which depends on time and on the acceleration of motion. This frequency shift can also exist in those moments when the Doppler Effect is absent (the speed of the interface movement u=0).

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Prevalence of Suffering from Partner Violence in Women

Introduction

The Royal Spanish Academy defines “woman” as “a person of the female sex,” “who has reached adulthood” and/or “who has the qualities considered feminine par excellence.” [1]; Sex” is the biological, anatomical and genetic characteristic with which a person is born [2] and “gender” is the dynamic, psychological and cultural construct to which one is rooted, susceptible to transformation and variable according to the sociodemographic conditions of each region. of the world [3]; without necessarily existing a dependency between the two [4]. The archetype of the word comes from an uncertain etymological root of the Latin “mulier” or “Mulieris”, and has had different interpretations, in Rome, during the 1st century BC, as something “soft or watery”; at the beginning of the 20th century in Europe, as “weaker and more tender”; America, as “soft and delicate”; In the 21st century, it begins to be understood as “being feminine”, but without complete attachment to Latin [5]. Women have fought to change the concept in an erroneous way, such as the “weaker sex” [6], with “enlightened feminism” [7] women have shown to have outstanding intellectual, strategic, and artistic capacity [8], with the arrival of the Industrial Revolution women have acquired an economic independence never seen before [9]. In Mexico, there are traditional ideas that the function of women is reproductive and domestic [10], with the decree of women’s right to vote, [11] the presentation of voluntary motherhood, the end of sexual violence and the right to free choice sexual, [12] the creation of norms for Equality between Women and Men, [13] it has been possible to modify that notion [14].

However, the growing wave of violence that women currently face, inside and outside their home, must be considered [15]; acts of multi-causal origin to regain the physical, economic, sexual, and psychological submission of women [16]. The World Health Organization refers to a Public Health problem in women, called violence within the couple. In 2018, 2.7 out of every 10 women between 15 and 49 years old experienced physical, sexual or both violence at the hands of your romantic partner at some point in your life [17]; This act being a behavior of the partner or ex-partner that causes physical, sexual, psychological and economic harm to the woman [18], of multi-causal origin [19], with repercussions on the quality of life of the woman and those around her, mainly her children [20]. However, culture and social environment for women have an important role, considering religion, ideology, customs, and traditions [21]. The most common prevalence and characteristics of intimate partner violence against women have been identified [22]. Some studies refer to the age of 15 years or less, sexual violence prevails [23], physical violence in young adults [24], and psychological violence at older ages [25]. Predominant in married [26], separated, and divorced women [27], with low or no schooling [28], correlated with unemployment, low or no monthly income and little opportunity for the victim to become independent [29]. A history of having experienced some type of domestic violence increases the risk of exposure to a new violent episode [30]; and especially if the victim’s mother also suffered some type of abuse [31].

The use and abuse of addictive substances is also closely related [32]. Referring as the most frequent violence to psychological violence, followed by physical and sexual violence [33]. Various instruments have been used to measure violence against women, such as the Index of Spouse Abuse (ISA) [34]; the Partner Violence Screen (PVS) [35], the Abuse Assessment Screen (AAS) [36]. However, due to the small population studied and the few items they cover, their uses have been limited [37]. The Woman Abuse Screening Tool (WAST) instrument has been the most appropriate questionnaire for screening intimate partner violence against women [38], presenting added items to leave eight items on sexual violence [39], evaluating whether there is confrontation with the couple [40]. This instrument is practical, flexible, easy to understand and use in non-established contexts of violence [41], with a Cronbach’s alpha of 91.5% [42]. In Mexico, in 2019, psychological violence is identified as the most common exercised by the current partner [43]. In 2022, in a Family Medicine Unit of the Mexican Social Security Institute (IMSS), the emotional dependence between the victim and their perpetrator is reported as a risk factor [44]. Also implementing in the IMSS the virtual help module [45], with a gender violence detection system through the resolution of a variant of the WAST instrument [46]. Therefore, the present objective is to identify the prevalence of female patients aged 20-59 years from the U.M.F. 20 “Vallejo” who are at risk of suffering from intimate partner violence.

Materials and Methods

This is an observational, cross-sectional, descriptive study, carried out with prior informed consent in 1,043 women between 20 and 59 years of age, users of the U.M.F. No. 20 “Vallejo”, first level care unit of the Mexican Social Security Institute, in both care shifts, within the period from March 2023 to February 2024.

Method

If the patient, with prior informed consent, agreed to participate in the study according to the following inclusion criteria: women between 20 and 59 years of age who were beneficiaries of the Family Medicine Unit No. 20 Vallejo, of the IMSS in any care shift, who were in a heterosexual romantic relationship currently or within the previous year 2022. Exclusion criteria: illiterate women; who came to the consultation accompanied by their partner or children; who presented severe cognitive impairment or with mental health disorders and those who found it difficult or impossible to sign the informed consent or complete the instrument. And elimination criteria: female patients who did not sign the informed consent due to forgetfulness, error or own desire and those who decided to withdraw from the study even after providing personal information, signing the consent or answering the instrument.

Variables

In the questionnaire used as the first part, sociodemographic and employment data were collected from the women surveyed, identifying the following variables: age, which was defined as the number of years of existence of the woman expressed as the period from her birth to the current moment, with interval measurement scale categorized as 20-24 years of age, 25-29, 30-34, 35-39, 40-44, respectively.; civil status defined as the legal status of the person in the family and society, determining her capacity to exercise certain rights and obligations, with a nominal measurement scale categorized as single, married, common law, divorced, separated, widowed.; schooling defined as the highest level of studies carried out by a student within an educational establishment and that has already been completed, with an ordinal measurement scale categorized as preschool, primary, secondary, technical baccalaureate, high school, university, postgraduate or master’s degree.; occupation defined as the job, job or trade performed by an economically active person, with a nominal scale categorized as legislator or manager, professional, technician, office worker, service or sales employee, farmer or fisherwoman, artisan, machinery operator or assembler, merchant, cleaning worker, security guard, armed forces, unemployed.; average monthly income defined as different human groups into which a given society is stratified, based on their related social and economic conditions, based on their similar social and economic conditions, with an ordinal scale categorized as A/B: Upper class ($51,100/month), C+: Lower upper class ($27,800/month), C: Upper middle class ($19,900/month), D+: Lower middle class ($12,300/month), D: Upper lower class ($8,900/ month), E: Lower class ($5,400/month).

The variable that describes Intimate Partner Violence against women, defined as any deliberate act or omission that causes psychological, physical, patrimonial, economic, sexual harm or death to the woman, simply due to her gender condition and that occurs within of an intimate relationship, current or past, with an ordinal scale categorized as a suspicious case if the score is 15 points or more and a negative case if the score is 14 points or less. sing the WAST questionnaire as the second part of the questionnaire to measure this last variable, containing 8 Likert-type questions, from 1 to 3 where 1 is the least intense option or “never”, 2 is “sometimes” or “some difficulty”.” and 3 “many times; obtaining a minimum score of 8 and a maximum of 24. The cut-off points are: 14 or less, negative case; 15 or more points indicates a suspicious case. The time given to patients to complete the test was approximately 5 to 10 minutes.

Statistics

To calculate the sample size, statistical data from the U.M.F. No. 20 “Vallejo” on the number of female users between 20 and 59 years of age, within the year 2022; determining a total of 45,964 women. Subsequently, using the formula of finite populations, with a Confidence Interval of 95%, a maximum accepted estimation error of 3%, assuming a probability of success of 50%, it was established that it was necessary to survey the number of 1,043 women, to carry out the study. Descriptive statistics were used for the variables age, marital status, education, occupation, and monthly income. For the variable intimate partner violence against women, indicated as a suspicious case if a score of 15 points or more is obtained and a negative case if a score of 14 points or less is obtained, according to the WAST instrument and age by age group, a contingency table using the Pearson Chisquare test, adding in this contingency table demographic variables (education, occupation, monthly income, occupation) in bar graphs, considering a value p < 0.05, as statistically significant, using the IBM SPSS statistical package version 26.0 for the analysis.

Results

In the present study, 1043 women from the Family Medicine Unit Number 20 were analyzed, predominating the age group of 25 to 29 years, 19.2% (n=200), followed by 50 to 54 years, 13.2% (n=138) and in last place the group of 20 to 24 years old 8.8% (n=92); respectively; marital status with the same frequency in Married and Single 31.3% (n= 326) and lastly divorced 5.1% (n=53); Schooling predominated bachelor’s degree 31.4% (n=327) followed by high school 23.9% (n=249), lastly preschool 0.5% (n=5); occupation predominantly unemployed 27.0% (n= 282), followed by professional 24.5% (n= 256); monthly income predominated lower lower class 41.4% (n=432) and lastly the upper upper class 2.0% (n=21) (Table 1). It was reported in the outcome of women by age group from 20 to 59 years according to the WAST instrument as a method to detect intimate partner violence in the U.M.F. 20 Vallejo 20.7% (n= 216) of suspicious cases and 79.3% (n= 827) of negative cases, with p value=0.000. Suspected cases predominated in the age groups of 55 to 59 years, 50 to 54 years and 45 to 49 years, with 24.5% (n= 53), 18.5% (n= 40) and 12.5% (n= 27); respectively. Negative cases predominated in the age groups of 25 to 29 years, 40 to 44 years, and 30 to 34 years, with 20.6% (n= 170), 14.0% (n= 116) and 12.3% (n= 102); respectively (Table 2).

Table 1: Demographic characteristics of the of the female population between 20 and 59 years old at risk of suffering intimate partner violence from the U.M.F. 20 Vallejo.

Note: A/B $51.100/ month; C+ $ 27.800/month; C $ 199800/month; D+ $ 12.300/month; D $ 8.900/month; E $ 5.400/month.

Table 2: Contingency table of woman aged 20-59 years according to the WAST instrument as a method for detecting intimate partner violence at the U.M.F. 20 Vallejo.

Note: *p< 0.05 Pearson Chi-square test

Suspected cases in women in the age group of 20 to 59 years by education according to the WAST instrument as a method to detect intimate partner violence at the U.M.F. 20, primary education predominated 8.8% (n= 19) in the age group 55 to 59 years and bachelor’s degree 6.02% (n=13) in the age group 30 to 34 years, 95% CI (0.000 – 0.003), p =0.000 (Figure 1). The negative cases predominated in the bachelor’s degree 11.85% (n= 98) in the age group 25 to 29 years and high school 5.80% (n= 48) in the age group 20 to 24 years, 95% CI (0.000 – 0.003), p =0.000 (Figure 2). Suspected cases in women in the age group of 20 to 59 years by occupation, reported to service employees 6.02% (n= 13), followed by unemployed 5.09% (n= 11) in the age group 55 to 59 years and professional 5.09% (n=11) in the age group 25 to 29 years; 95% CI (0.000 – 0.003), p =0.000 (Figure 3). Negative cases predominated in professional occupation 8.46% (n= 70) in the age group 25 to 29 years, 95% CI (0.000 – 0.003), p =0.000. Suspected cases in women in the age group of 20 to 59 years by marital status, predominance separated in first place 7.87% (n= 17), followed by widowed 6.02% (n=13) in age group 55 to 59 years; single 7.41% (n=16) in age group 25 to 29 years, 95% CI (0.000 – 0.003), p =0.000 (Figure 4).

The negative cases were dominated by single marital status 14.03% (n= 116) and 7.50% (n=62) in the age group 20 to 29 years and 20 to 24 years; respectively, 95% CI (0.000 – 0.003), p =0.000 (Figure 5). The behavior of suspected cases in women in the age group of 20 to 59 years by monthly income, reports monthly income E: Low lower class with first place 12.50% (n= 27) in age group 55 to 59 years and 7.87 % (n= 17) in age group 50 to 54 years, 95% CI (0.000 – 0.003), p =0.000 (Figure 6). Negative cases also predominated E: the lower lower class 7.50% (n= 62) in the age group 50 to 54 years and second place 7.13% (n= 59) in the age group 20 to 24 years; respectively, 95% CI (0.037 – 0.051), p=0.037.

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Discussion

It is important to consider the social context of the present study in relation to the place where it was carried out as a method to detect intimate partner violence in women in suspected cases where significant differences were reported in separated marital status, primary schooling and age from 55 to 59 years. with 24.5% (n=53), unlike a study carried out on women in Brazil where post-traumatic stress disorders due to sexual assault were studied, which contrasts with age (24.4 years), education (secondary) and marital status [47] (single); without significant difference for education and marital status., another study from Brazil reports physical violence against women where married marital status, occupation as a housewife and ages between 20 and 40 years predominate [48]. Similar psychological dimensions involved in the WAST instrument of our study were developed in a study from Chile with gender violence in university students, reporting psychological violence through emotional blackmail and threats with a prevalence of 28% and 30.4%, respectively. Also highlighting that higher education is not a protective factor against gender violence [49], however there are mechanisms such as reporting to respond and preventive actions with care workshops [50].

In Spain, a study carried out on childhood victimization reported an increase of 4.7 times the risk of presenting a risk of victimization in the partner (p=0.002) and 3.1 times the risk of presenting it in women vs. men (p=0.016). [51] In Colombia, it was determined that those who seek social support for gender violence are separated-divorced people [52]. In Ecuador, the poverty factor has a risk of presenting violence against women, being consistent with our study, with reporting of E: low lower class in suspected cases as a method to detect intimate partner violence in women with 12.50% (n= 27), predominantly [53].

Conclusion

In the present study, it was possible to identify a prevalence of 20.7% in women aged 20 to 59 years in the context of the U.M.F. 20 “Vallejo” who are at risk of suffering intimate partner violence, with specific characteristics in certain age groups, education, occupation and monthly income, this corresponds to continuing with prevention actions in social, psychological and clinical aspects on the risk of suffer intimate partner violence to avoid recurrence and improve the quality of life of users.

Gratefulness

To the management staff of the OOAD DF Norte, director of the UMF 20, to the medical staff who contributed as a work team to make this article a reality for the benefit of IMSS users.

Ethical and Legal Aspects

Data was included in a registry approved by the ethics and health research committee.

Conflict of Interests

The researchers of this article declare that there is no financial interest or conflict of interest.

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Evaluation of the Impact of a Novel Nutraceutical Product through the Membrane Lipidomic Analysis in Aging Dogs with Canine Cognitive Dysfunction

Introduction

The population of aging dogs has grown significantly over the last decades. There are more than 30 million senior and geriatric dogs over the age of seven years in the USA and over 15 million in Europe [1]. Dogs live much longer due to the excellent care level and preventive examinations [2]. However, prolonged life expectancy frequently accompanies age-related diseases such as the Canine Cognitive Dysfunction (CCD) [3,4]. The CCD estimated prevalence ranges from 14% to over 60% and increases as dogs age, even if small breeds are at higher risk because of their greater lifespan [5,6]. Canine cognitive dysfunction is a neurodegenerative syndrome of older dogs characterized by a gradual but progressive cognitive decline [7-9]. Clinical signs are a continuing cognitive impairment and changes in daily routine, including disorientation, altered social interactions, change in sleepwake cycle, aimless wandering, barking, house soiling, alterations in activity level, and anxiety level changes [10,11]. The first approach to diagnose CCD is a clinical evaluation aimed at excluding any systemic or neurologic disease that might mimic CCD symptoms [12]. Diagnostics may require physical and neurologic examinations, bloodwork, thoracic radiographs, and abdominal ultrasound, followed in some cases by a brain MRI and cerebrospinal fluid analysis to evaluate for structural changes such as brain atrophy or inflammation, infection, and neoplasia [13]. The establishment of an accurate diagnosis is often reached with the help of validated screening questionnaires that are related to the owner’s perception, sound tools to rate different stages of CCD and to distinguish this disease from a simple decrease in psychomotor activity caused by general aging of the organism [14- 18].

Canine cognitive dysfunction resembles human Alzheimer’s disease (AD) [19,20], sharing many similarities in the neuropathological abnormalities associated with the disease process [21-24]. Among the pathological brain alterations commonly witnessed in both humans and dogs with cognitive dysfunction, there are cerebrovascular diseases, neuronal mitochondrial dysfunction, oxidative brain damage, impaired neuronal glucose metabolism, astrocyte dysfunction, microglial activation, glutamate-mediated excitotoxic neuronal damage, and abnormal deposition of beta-amyloid plaques [6,8,21,25-27]. Moreover, amyloid beta deposits in dogs have DNA fragments that are highly conserved with those of their human counterpart [28,29]. A-beta amyloid plaques seem to have an essential role in the neuronal death cascade. However, there might be differences between dogs and humans in the persistence of plaques and their distribution, suggesting additional pathogenetic mechanisms in the canine species, such as failure of myelin and synapsis homeostasis and activation of brain damage through immunological mechanisms [24,30,31].

Other common characteristics of CCD and human AD are a loss in synapsis and low plasma and phospholipid precursors levels in the brain [32,33]. In order to hinder cognitive decline and neurodegeneration, the formation of new synapsis, namely synaptogenesis, should be stimulated. This process dramatically relies on the availability of phospholipid precursors to the brain, particularly phosphocholine, an intermediate synthesizing phosphatidylcholine (PC), representing the major component of neuronal brain membranes. In addition, it provides the phosphocholine moiety needed to synthesize sphingomyelin, the other major choline-containing brain phospholipid. Phosphatidylethanolamine synthesis also utilizes ethanolamine instead of choline. At the same time, phosphatidylserine, the third major structural phosphatide, is produced by exchanging a serine molecule for the choline in PC or the ethanolamine in phosphatidylethanolamine [32,34-36]. Phosphatidylcholine synthesis occurs thanks to 3 precursors: docosahexaenoic acid (DHA), uridine, and choline, which reach the brain entirely or predominantly from circulation.

Since the enzymes that synthesize PC act on all of them and have low affinities for these substrates, the blood levels of the three precursors can influence the overall PC synthesis rate [37,38]. The literature demonstrates that brain PC synthesis rapidly increased in rodents fed with a diet supplemented with choline, uridine, and DHA [37,38], while positive outcomes resulted from two clinical trials performed on patients with early AD receiving these three compounds [39,40]. The assumption that the clinical effects of these elements are mediated by an enhanced synaptogenesis is consistent with the observation that the characteristic impairment of connectivity between the varied areas of AD brain was also ameliorated [41]. Therapeutic strategies that may slow the progression and improve signs of CCD include drugs, functional foods, and nutritional supplementation oriented to reduce damage caused by oxidative stress, correct metabolic changes associated with cognitive decline, reduce inflammation, and ameliorate mitochondrial function, neuronal health, and signaling [42].

Strictly adhering to a treatment protocol combining drug treatment, dietary supplementation, and environmental enrichment results in satisfactory clinical outcomes [8]. Dietary supplements, more adequately known as nutraceuticals, have shown a sharp increase in veterinary medicine in recent years [43]. In modern medicine, the administration of nutraceuticals in the prevention and treatment of animal diseases is as joint as it is in humans due to their safety profile, cost-effectiveness, and ease of finding [44]. Nutraceuticals are undefined compounds used to improve the state of health, prevent malignant processes, and control symptoms of different diseases. Their therapeutic components are derived mainly from plants, animals, and marine sources [45] and typically contain the required amounts of vitamins, lipids, proteins, and minerals [46]. Nutraceuticals vary according to their mechanism of action, ranging from an anti-cancer to an antioxidant or anti-inflammatory activity [47]. There is a reasonable body of literature confirming the positive effects of nutraceutical supplementation on neuronal damage and cognition in rat models, dogs, and humans with cognitive impairment [48-55]. However, research needs more objective and unbiased methods to detect the actual effect of nutraceutical treatments.

In 2020, Prasinou et al. disclosed a novel molecular approach helpful in assessing nutritional treatments in health and canine disease conditions [56]. These authors selected a cluster of ten fatty acids representative of cell membranes’ structural and functional properties and quantitatively analyzed them from erythrocyte membrane glycerophospholipids of healthy dogs. These ten fatty acids belonging to saturated (SFA), monounsaturated (MUFA), and polyunsaturated (PUFA) fatty acids families were extracted from red blood cells (RBC) because the RBC composition of fatty acids is informative of fatty acid levels present in various tissues such as muscles, liver, and in tissues not withdrawable during life like retina or brain [57,58]. Lipidomics, the science that studies metabolic pathways through lipids composition [59], has already been widely applied in human research [60-62] but has been scarcely studied in veterinary medicine [63].

Prasinou et al. described the healthy dog lipidomic membrane profile, creating a benchmark for evaluating metabolic and nutritional status in physiological and pathological conditions [56,64]. This study aims to assess whether Neurogen Pet Ultra® dietary supplementation results in significant molecular changes by a quantitative analysis of the lipidomic profile in aging dogs affected by CCD compared to a cohort of senior healthy dogs. Neurogen Pet Ultra® is a nutraceutical product addressed to geriatric dogs and indicated for neuronal protection and neurotransmission regeneration by means of activation of synaptic membranes. It provides the dogs with choline, uridine, DHA, and various antioxidant and anti-inflammatory compounds.

Materials and Methods

Animals

Twenty-four dogs with signs compatible with CCD were enrolled in this prospective study between January 2019 and January 2020. Dogs with symptoms consistent with cognitive dysfunction were identified through clinical signs and examined by a neurologist (FC). Diagnosis requested the employment of a questionnaire, clinical evaluation, and confirmed in selected cases with advanced diagnostic imaging (brain MRI). The recruited subjects were eligible for the study if they were aged over 108 months in the case of small/medium size dogs or over 84 months in the case of medium/large size dogs. The dogs differed in breed, sex, and body weight (see Tables 1-3). Epileptic dogs and dogs with metabolic or behavioral disorders were equally included in the study, as well as animals already receiving treatments for CCD. Moreover, dog owners must be willing to administer the dietary supplementation accordingly. Exclusion criteria comprised dogs with other concurrent documented diseases that might have signs similar to CCD, such as structural brain lesions (i.e., neoplasia, vascular disease, others); subjects already treated with different nutraceuticals for geriatric diseases; patients in need of further medications due to subsequent medical conditions; dogs presenting adverse events following the dietary supplementation under investigation; animals reporting a deterioration in the clinical status during the observation period. The study protocol did not require any evaluation concerning the type of diet the owners feed their dogs, albeit dietary regimens were registered.

Study Design

Each dog received Neurogen Pet Ultra® (NBF Lanes, Milan, Italy) at the dosage of ½ tablet per 5 kgs live weight once a day for 60 days. Follow-up also included the following 60 days in which Neurogen Pet Ultra® was not administered. Table 4 lists the product components. Animals were not subjected to additional supplementation or change in their daily diet over the observation period. After a first medical examination at study entry (T0), the experimental subjects underwent follow-up visits on day 30 (T30), day 60 (T60), day 90 (T90), and day 120 (T120), when the investigators recorded clinical data. Blood samples were harvested at days 0, 60, and 120 for the lipidomic profile assessment.A control group of 8 healthy elderly dogs, whose fatty acid- based membrane lipidome profile was already studied, has been extrapolated from the Prasinou et al. article [56]. The research was conducted according to the directive 2010/63/EU, article 1 (paragraph 5f); the present study did not imply any form of animal suffering or health risk, since it focused on the administration of a natural substance. However, compliance with the decree ensured a further safeguard for the patient’s health. All dogs enrolled in this study were owned and subjected to regular veterinary checks.

Membrane Lipidome Analysis

Red blood cells (RBC) were isolated from 1 mL of fresh EDTA- treated whole blood sample and used for performing the lipidome analysis as previously described [56,64]. The fatty acid-based membrane lipidome analysis consists of 8 steps, as summarized below and in Figure 1. The process starts with separating erythrocytes from plasma, followed by the washings and centrifugation for membrane isolation that produces a pellet that is suspended in water to extract lipids. The organic layer produced is then separated and evaporated under a vacuum to dryness. A thin layer chromatography (TLC) using chloroform/methanol/water is performed to determine the purity of the phospholipid fraction to avoid plasma contaminants that could interfere with the fatty acid quality and quantity. The phospholipid extract is transesterified at room temperature for 10 min to obtain the fatty acid methyl esters (FAMEs) derived from the fatty acid residues present in membrane glycerophospholipids. The final steps include the extraction of the FAME, their evaporation under vacuum, and the FAME extract’s gas chromatography (GC) analysis. The FAME quantification is performed using CG analysis, as already described [56,64].

Figure 1

The values of the cluster of ten fatty acids representative of the primary fatty acid moieties of the cell membrane are expressed in μg/ mL. Each quantity is reported as a percentage relative to the cluster as the sum of the quantities (100%) that corresponds to > 97% of the peaks found in the GC runs. The fatty acid quantities identified are also helpful for calculating the corresponding lipid indexes by performing mathematical operations of sums and ratios. The cluster of the selected ten fatty acids, including lipid indexes, are detailed in Table 5. The cohort of fatty acids includes palmitic (C16:0) and stearic (C18:0) acids as SFAs; palmitoleic (C16:1), oleic (9c, C18:1), and vaccenic (11c, C18:1) acids as MUFAs; linoleic (LA, C18:2), dihomo-gamma- linolenic (DGLA; C20:3), and arachidonic (AA, C20:4) acids as PUFA omega-6; and eicosapentaenoic (EPA, C20:5) and docosahexaenoic (DHA, C22:6) acids as PUFA omega-3.

These values are also reported as total fatty acid contents (total SFAs, total MUFAs, total PUFAs), and lipid indexes calculated as follows: SFA/MUFA ratio; omega-6/omega-3 ratio; PUFA balance = [(%EPA + %DHA) / Total PUFA] × 100; Unsaturation Index (UI) = (%MUFA × 1) + (%LA × 2) + (%DGLA × 3) + (%ARA × 4) + (%EPA × 5) + (%DHA × 6); Peroxidation Index (PI) = (%MUFA × 0.025) + (%LA × 1) + (%DGLA × 2) + (%ARA × 4) + (%EPA × 6) + (%DHA × 8). As previously published, the GC method can successfully separate all ten fatty acids without overlapping other peaks, particularly the positional and geometrical isomers of the unsaturated fatty acids [56,64].

Statistical Analysis

A statistical analysis was performed using the software GraphPad Prism V.6.01. All data were evaluated using standard descriptive statistics and reported as the mean ± SD or as median and range (minimum- maximum), based on their distribution. Normality was checked using the D’Agostino Pearson test. Variables sequentially collected were compared using repeated measure one-way analysis of variance or a Friedman test according to their distribution, and a post hoc test (Holm-Sidak test or Dunn test) was performed. According to distribution, the data obtained from both groups were compared using an unpaired t-test or Mann-Whitney test. A p-value < 0.05 was considered significant.

Results

Twenty-four elderly dogs were diagnosed with CCD and enrolled in this study between January 2019 and January 2020. In comparison, information on the 8 healthy old dogs staged as a control group originated from a previously published paper [56,64]. Out of 24 sick dogs, only 12 remained in the study until the end of the follow-up (T120), so these last 12 dogs acted as the treatment group to evaluate the effect of Neurogen Pet Ultra®. The distribution of the 32 dogs’ characteristics at baseline is detailed in Table 1, Table 2, and Table 3; the three groups are largely comparable. The dogs belonged to different breeds, including mixed breeds, both male and female, in some cases sterilized, with varying body weights (range 4,2-40), but all aged over 102 months (range 102-216). The first finding of this study, which was crucial for the subsequent evaluation of the nutraceutical treatment, comes from the comparison between the erythrocyte membrane fatty acids of healthy aging dogs and those of aging dogs affected by CCD at study entry (T0). The quantitative GC analysis of the cluster of ten fatty acids representative of the primary fatty acid moieties present in the cell membrane (see Table 5) displayed meaningful changes in CCD subjects. The distribution of modifications to the lipidomic profile arising from the onset of CCD is reported in Table 6.

Table 1: Demographic characteristics of old dogs with canine cognitive dysfunction treated with dietary supplementation (Neurogen Pet Ultra ®) that completed the follow-up period.

Table 2: Demographic characteristics of old dogs with canine cognitive dysfunction treated with dietary supplementation (Neurogen Pet Ultra®) that did not complete the follow-up period.

Table 3: Demographic characteristics of senior healthy dogs.

Table 4: Composition for 1 tablet of Neurogen Pet Ultra® (1250 mg).

Table 5: List of fatty acids selected for this research, including fatty acid families and lipid indexes.

Note: Total saturated fatty acids (SFAs) = % C16:0 + % C18:0.
Total monounsaturated fatty acids (MUFAs) = % 9c, C16:1 + % 9c, C18:1 + % 11c, C18:1.
Polyunsaturated fatty acids (PUFAs) omega-6 = %LA + %DGLA + %ARA.
Polyunsaturated fatty acids (PUFAs) omega-3 = %EPA + %DHA.
Total PUFAs = %LA + %DGLA + %ARA + %EPA + %DHA.
SFA/MUFA = (% C16:0 + % C18:0) / (% C16:1 + % 9c, C18:1 + % 11c, C18:1).
Omega-6/omega-3 ratio = (%LA + %DGLA + %ARA) / (%EPA + %DHA).
PUFA balance = [(%EPA + %DHA) / Total PUFA] × 100.
UI = (%MUFA × 1) + (%LA × 2) + (%DGLA × 3) + (%ARA × 4) + (%EPA × 5) + (%DHA × 6).
PI = (%MUFA × 0.025) + (%LA × 1) + (%DGLA × 2) + (%ARA × 4) + (%EPA × 6) + (%DHA × 8).

Table 6: Mean values of the ten fatty acids derived from the erythrocyte membrane glycerophospholipids of the 8 healthy dogs, and all the 24 dogs affected by canine cognitive dysfunction at Time Zero.

Note: *Values are expressed as μg/mL.
SD = standard deviation.

Specifically, CCD dogs showed a statistically significant increase of stearic acid (p = 0.0001) and total SFAs (p = 0.0011); an increase of total MUFAs (p = 0.0194); a significant decrease of ARA (p = 0.0011), PUFAs omega-6 (p = 0.0006) and total PUFAs (p = 0.0009); a significant decrease of unsaturation (p = 0.0016) and peroxidation (p = 0.0019) index value (UI and PI, respectively). On the other hand, there were no differences between healthy and CCD dogs regarding the levels of palmitic acid, palmitoleic acid, oleic acid, vaccenic acid, LA, DGLA, EPA and DHA, PUFA omega-3, SFA/MUFA ratio, omega-6/ omega-3 ratio, and PUFA balance. Among the 24 CCD dogs, only 12 subjects have gone through the whole follow-up period till day 120 (T120). However, the fatty acid levels at time zero (T0) of this limited cohort of dogs show the same values and the same significant differences compared to healthy dogs (see Table 7), except for DGLA, whose increment reaches the significant level only in the smaller group of dogs that completed the study. Therefore, any DGLA modification after the nutraceutical treatment was not considered valid.

Table 7: Mean values of the ten fatty acids derived from the erythrocyte membrane glycerophospholipids of the 8 healthy dogs, and the 12 dogs with canine cognitive dysfunction that completed the study at Time Zero.

Note: *Values are expressed as μg/mL.
SD = standard deviation.

The second finding that fulfills this study’s main purpose relates to assessing the effect of Neurogen Pet Ultra® in aging dogs affected by CCD. The beneficial effect of the dietary supplementation has been validated using a statistical comparison between the levels of fatty acids under investigation (expressed as μg/mL) in the treatment group at the end of the study period (T 120) and the levels of fatty acids in the control group. Upon completion of the follow-up period (60 days of dietary supplementation plus a further 60 days of observation), the 12 experimental CCD dogs expressed overlapping lipidomic profiles with that of the 8 healthy dogs, as described in Table 8. The unbalance in the fatty acids content following CCD development seems to normalize because of the supplementation with Neurogen Pet Ultra®. Out of 8 parameters that were significantly changed in the treatment group compared to the control group at T0, 7 are within the “normal” range at T120: stearic acid, total SFAs, ARA, PUFAs omega-6, total PUFAs, UI, and PI.

Table 8: Mean values of the ten fatty acids derived from the erythrocyte membrane glycerophospholipids of the 8 healthy dogs and the 12 dogs with canine cognitive dysfunction that completed the study at Time 120.

Note: *Values are expressed as μg/mL.
SD= standard deviation.

Total MUFA values represent the unique parameter that did not restore to normal at the end of the study. At the same time, oleic acid increase (p = 0.02) is the only discrepancy induced by the nutraceutical supplementation at 120 days. The final statistical analysis concerns the comparison of amounts of the ten fatty acids (including fatty acid families and lipid indexes) within the treatment group at T0, T60, and T120. Provided that the occurrence of CCD implies a remarkable disparity of the erythrocyte membrane lipidome in aging dogs, noticeable modifications of this imbalance were observed after Neurogen Pet Ultra® supplementation (see Table 9). Indeed, stearic acid decreased significantly at T 120 (p = 0,0087) as well as for total SFAs (p = 0,052), even though this parameter featured a significant decrease already at T60; DHA and PUFA omega-3 values were both declined after the whole follow-up period (p = 0,0038 and p = 0,0278, respectively); SFA/MUFA ratio decreased significantly (p = 0,0023) as well as for PUFA balance values (p = 0,0087) at T120, while omega-6/ omega-3 ratio underwent a substantial increase (p = 0,0087) at the end of the study.

Table 9: Changes in fatty acid values of the treatment group resulting from Neurogen Pet Ultra® dietary supplementation at day 0, 60, and 120.

Note: *Values are expressed as μg/mL.
SD = standard deviation.

Discussion

Canine cognitive dysfunction is one of the most common neurodegenerative conditions affecting senior dogs [3,23]. With aging dog populations, the prevalence of CCD is increasing [5,65]. However, the pathogenesis of this debilitating illness is still not fully understood, and a multifactorial origin is postulated [9,23,66]. The incidence of CCD does not differ between breeds, nor are there specific differences in clinical signs comparing various breeds [17], so it remains a fact that aging is the most significant risk factor for canine neurodegeneration [4,7]. Although investigators studied many therapies, there are currently no effective pharmaceutical cures for CCD, and the treatment options available are very few, most of them carriers of possible adverse events [67]. As with most canine health and behavior disorders, CCD can be most successfully managed when identified as soon as a dog reaches the clinical threshold for the disease. Therefore, it is imperative to rely on early recognition and intervention [67]. In addition, the treatment of cognitive impairment needs a multimodal approach that includes drugs, balanced diets, supplementations, and environmental and behavioral modification, and it should focus on prevention, maintaining brain health, slowing down cognitive decline, and improving clinical signs [3,67-70]. There is increasing evidence that nutraceutical remedies act similarly to medicines with fewer side effects, which is why nutraceuticals became complementary to drugs, often reducing the dosage of conventional medication or extending its effectiveness [46,71,72]. Not surprisingly, various investigations into nutraceuticals aimed to ameliorate canine cognitive decline have been disclosed in recent years [73-76].

Neurogen Pet Ultra® is a unique nutraceutical product formulated with active ingredients selected on the basis of clinical evidence. It provides geriatric dogs with choline, uridine, and DHA, nutritional precursors needed for synthesizing PC that is essential for membrane phospholipids and for the growth of dendritic processes [32,34-38]. Omega-3 fatty acids supplementation improves membrane physiology and clear improvements in neuronal membrane permeability [76,77]. This product encloses polyphenols and Vitamin E, an antioxidant mix that can hinder the degenerative action of free radicals and counteract oxidative stress by slowing cellular aging [68,78,79]. Neurogen Pet Ultra® also provides animals with curcumin that sharply contrasts neuroinflammation, thus delaying neuronal degeneration [52], and supportive herbal extracts [74].

To evaluate the efficacy of a treatment, investigators routinely rely on validated questionnaires such as CADES (CAnine DEmentia Scale) [14]. These screening tools are a scale rating that monitors the clinical course of the symptomatology during the follow-up period. Limitations to these methods are attributed mainly to pet owners’ perceptions that cannot be unbiased due to their emotional solid involvement; for example, it is common for owners to underestimate their pet’s body condition score and many other clinical or behavioral signs. Several authors have already evaluated using the CADES questionnaire combined with novel biomarkers to predict CCD more readily in elderly dogs [80-82]. Furthermore, recent studies developed and validated a new methodology to support the rapid diagnosis of disease and to objectively assess treatments’ effect in sick dogs using as a benchmark the lipidomic RBC membrane profile of healthy dogs [56,64].

The present study assessed the benefits of Neurogen Pet Ultra® daily administration in elderly dogs with cognitive dysfunction using quantitative analysis of the cluster of fatty acids previously studied by Prasinou and other authors in a subgroup of healthy aging dogs [56]. The first important outcome of this prospective observational study regards the marked changes in the lipidomic profile observed when comparing senior healthy and CCD dogs. In animal models and humans, increased or decreased fatty acid levels are linked to various diseases [64,83,84]. Changes in lipid composition in the case of human AD have already been published [85]. Nevertheless, this cutting-edge evidence is relevant since it comes specifically from research on dogs with cognitive dysfunction. Interestingly, although aging is associated with significant changes in human lipid content [85], the previous analysis on the RBC membrane lipidome of 68 healthy dogs did not show relevant correlations between fatty acid values and age. In fact, the only significant correlation was found with EPA [56]. On the contrary, the development of CCD in this experimental cohort generated significant changes in the abundance of 8 lipid parameters out of 20 analyzed. The overall fatty acid distribution reported in Table 6 points out that the healthy senior dog erythrocyte membrane lipidome is made up almost exclusively of PUFA omega-6 and total SFAs (about 90% of the total RBC fatty acids) followed by total MUFAs, similar to what occurs in other mammalian species [86]. Instead, omega-3 fatty acids account for a minimal concentration, underlining the difference with human lipidome [60,63,86].

Canine cognitive dysfunction promoted a marked and significant increase in SFAs (from 36,59 μg/mL to 43,27 μg/mL; p = 0,0011), mainly driven by stearic acid (p = 0,0001). In humans, SFAs can be regarded as the most harmful type of fatty acids because they can induce the development of atherosclerosis through lipid mediators. High levels of SFAs might be a trigger for insulin resistance and inflammation [87]. Moreover, patients affected by different diseases show a significantly higher proportion of SFAs, being these fatty acids capable of promoting inflammatory signaling by stimulating TLR2 and TLR4 (Toll-like receptors 2 and 4) [88]. Therefore, it can be assumed that an increased concentration of saturated acids in aging dogs is proof of pathological cognitive dysfunction; however, it is also a driving factor for the occurrence of this disease that should be counteracted, possibly with dietary supplementation.

The MUFA pathways, along with SFA and PUFA ones, participate in the specific fatty acid distribution of healthy dog erythrocyte membranes, so it is essential to underline that total MUFAs increased in the RBC membrane of the experimental cohort of CCD dogs (from 11,31 μg/mL to 13,05 μg/mL; p = 0.0194), contributing to the unbalance of fatty acids content. Increased stearic acid levels probably promoted a growth in the monounsaturated acid levels. Polyunsaturated fatty acids represent the other great family of fatty acids fundamental to membrane homeostasis. Omega-3 and omega-6 PUFAs are essential fatty acids for eukaryotic cells, so they are taken from the diet and then enzymatically processed to provide long-chain PUFAs. Feeding dogs with enough omega-6 fatty acids is essential to maintain a good health status. In particular, linoleic acid is necessary to ensure its metabolic transformations to DGLA and arachidonic acid, predominant in dogs, because they are leading structural and functional components of cell membranes [56]. On that basis, it is worthwhile to highlight that CCD dogs here experienced a remarkable reduction in total PUFA content with respect to healthy senior dogs (p = 0,0009), with a highly significant alteration also for omega-6 fatty acids that decreased from a mean value of 50,01 μg/mL to 40,82 μg/mL (p = 0,0006). The evidence that cognitive dysfunction promotes an alteration of the lipidomic profile in aging dogs may relate to the marked decrease of arachidonic acid that drops from 34,07 μg/mL to 26,71 μg/mL (p = 0,0011) even if the mean values of linolenic acid, its precursor, did not significantly change between healthy and CCD dogs. The partial inhibition of this metabolic step that leads to ARA synthesis could indicate a condition of unbalance of the membrane lipids caused by canine cognitive dysfunction.

In this observational study, CCD was even accompanied by a significant decrease in both unsaturation and peroxidation indexes (p = 0,0016; p = 0,0019, respectively). These parameters have already been used for objectively evaluating the homeostasis of membranes in terms of fluidity and oxidative damage [89]. Lipid rafts from the frontal cortex of early AD patients exhibit unusually low unsaturation and peroxidation indexes compared to healthy controls [90]. By setting the two indexes as parameters that mirror an optimal environment for maintaining membrane functions in veterinary medicine, it seems clear that perturbations in the mean values of these indexes indicate a membrane alteration triggered by the disease. Proceeding with the discussion of the results, Table 8 shows the central node of this research, namely the actual clinical effect of Neurogen Pet Ultra® dietary supplementation in CCD aging dogs measured through quantitative analysis of erythrocyte membrane fatty acids. The efficacy of this formulation mirrors the “normalization” of almost all the parameters following the period of treatment with the nutraceutical product. By comparing the fatty acids content between the 8 healthy dogs of the control group and the 12 CCD dogs who achieved the last day of the follow-up period, it was gratifying to see that all SFAs and PUFAs, whether they were checked individually or per family, did not differ in quantitative terms. The unique parameter that remained modified after the treatment was related to the MUFA family (p = 0,02). However, since PUFAs and SFAs are the fatty acid families that mainly contribute to the formation of RBC membranes, it is possible to claim that Neurogen Pet Ultra® is capable of supporting the resetting of the appropriate distribution of fatty acids in the erythrocyte membrane, thus ameliorating its structure and functions. The mean values related to two ratios between the families (SFA/MUFA; omega-6/omega-3) and three indexes (UI; PI; PUFA balance) of the experimental cohort after the treatment period also resulted within the ranges expressed by healthy subjects.

Even the length of the follow-up period is worthy of note: the dogs that completed the study were treated with nutraceutical supplementation for 60 consecutive days; after that, they were observed for an additional 60 days before performing the final molecular-based testing. This protocol was designed precisely for estimating any improvement even after discontinued treatment, thus measuring its benefits over time. Finally, the ability of Neurogen Pet Ultra® to remodel the RBC membrane has been further corroborated by a separate analysis within the treatment group. The fatty acid proportion was evaluated and compared at study entry, after two months, and at the end of the study. Bearing in mind that the starting point was a situation where CCD dogs displayed a fatty acid biodistribution out of balance, the lipidome profile underwent adjustments after 60 days of nutraceutical administration, followed by two additional months of observation. The significant decrease of stearic acid and total SFAs (p = 0,0087 and p = 0,0052, respectively) is desirable given that this fatty acid family is more damaging to the central nervous system. In addition, it should be noted that the reduced amount of SFA family was already significant at day 60.

The last point that requires careful consideration is the change in PUFA omega-3 content. Both DHA and omega-3 family decreased noticeably after dietary supplementation (p = 0,0038 and p = 0,184) despite DHA being integrated. Because of the previous omega-6 reduction triggered by cognitive dysfunction, it can be argued that this omega-3 fatty acid rearrangement is beneficial. Since dogs’ omega-6/ omega-3 ratio is incredibly high and crucial for maintaining specific metabolic effects, this ratio can be restored even through omega-3 lowering. It certainly needs to be emphasized that the SFA/MUFA ratio and PUFA balance went to substantial changes (p values = 0,0023; 0,0087); therefore, these modified lipid indexes stand for an active remodeling process of the RBC membrane structure induced by Neurogen Pet Ultra® intake. We are conscious that this study has some limitations since it has been conducted on a restricted number of patients, while information on the control group was gathered previously. Despite the modern molecular-based methodology proposed, we also acknowledge that a traditional clinical evaluation of the nutraceutical effects is needed to reinforce the results.

The outcomes herein reported fully meet the primary endpoint by providing unbiased evidence of Neurogen Pet Ultra® benefits in the case of CCD. Nevertheless, the implications the study offers in terms of new scientific knowledge are much broader, as we were able to deliver more profound insights into the mechanism of CCD occurrence. Our findings validate the use of membrane lipidome analysis for an early, fast, and accurate diagnosis of canine cognitive dysfunction, in addition to a reliable method for monitoring different therapies. We advocate setting up further studies with this scientific approach to thoroughly investigate any added advantage of dietary supplementation with Neurogen Pet Ultra® in preventing and treating canine neurodegeneration.

Conclusion

The present study objectively discloses the effect of Neurogen Pet Ultra® dietary supplementation in aging dogs affected by canine cognitive dysfunction. One of the strengths of this research is the molecular- based approach performed to demonstrate the nutraceutical effect. Using a tested protocol, the authors examined and quantified a cluster of ten fatty acids in the erythrocyte membrane of a cohort of diseased dogs, being erythrocyte fatty acids composition informative of the brain tissue. The outcomes displayed that Neurogen Pet Ultra® supplementation can significantly adjust the fatty acid alterations caused by cognitive dysfunction in senior dogs, thus supporting the remodeling of the cell membrane structure. Resetting the levels of most lipid components is fundamental to maintaining the functional properties of cell membranes, essential for canine health.

Acknowledgment

The authors thank Giuseppe Pappini and Carlo Maria Colombo for NBF Lanes financial support.

Conflicts of Interest

Two co-authors of the present study, Prof. Alessandro Gramenzi and Dr. Giulia Pignataro, are NBF consultants.

Funding

This research was funded by the NBF Lanes Srl, Corso di Porta Vittoria 14, 20122 Milano.

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Assessment of Imaging Performance of Fluorine-18 and Gallium-68 on Mediso NanoScan PC Small-Animal PET/CT Scanner

Introduction

Tracers labeled with positron-emitting radioactive nuclides such as ¹¹C, ¹⁵O, ¹³N, and ¹⁸F are widely used in positron emission tomography (PET) (Liu, et al. [1]). The most commonly used radiopharmaceutical is 2-[¹⁸F]fluoro-2-deoxy-D-glucose ([¹⁸F]FDG). It is mainly used for diagnosis and staging of various cancer types (Liu, et al. [1]). Interest in other radioisotopes such as Gallium-68 (⁶⁸Ga), Cupper-62 (62Cu), Rubidium-82 (82Rb), Iodine-124 (124I) and Zirconium-89 (89Zr) have grown recently (Attarwala, et al. [2]). These radioisotopes have distinctive physical properties, such as long half-lives, unique decay modes, or mode of complexation. Gallium-68 (⁶⁸Ga) is increasingly being used in radiolabeling of small molecules such as peptides and antibodies (Attarwala, et al. [2,3]). It has also been used to label peptide agents showing promising results particularly in imaging neuroendocrine tumors (Jalilian [3]). ⁶⁸Ga is readily available by simple elution of ⁶⁸Ge/⁶⁸Ga generators. Additionally, ⁶⁸Ga is easily labelled using commercially available labelling kits (Attarwala et al., Conti et al., Cañadas et al., [2,4,5]). However, its relatively large positron range causes significant blurring of ⁶⁸Ga-PET images without positron range correction. This effect is very clearly seen in small-animal PET imaging where blurring on a milli-meter scale can clearly effect image quality (Cañadas, et al. [5]).

While the positron range in itself is a fundamental physical aspect of the slowing-down processes of highly energetic positrons, it is possible to correct for the blurring effect in the reconstructed images. This can be achieved by incorporating a deconvolution model based on the knowledge of the spatial distribution of the blurring effect of the positron range in the reconstruction algorithm (Rahmim et al., Ruangma et al., Disselhorst et al., [6-8]). Liu and Laforest have shown that the incorporation of point spread function modelling increases quantitative accuracy of small lesions for long-range positron emitters (Liu, et al. [1]). The imaging performance of a variety of small-animal PET imaging systems and a range of radioisotopes (tracers), including ¹⁸F, ⁶⁸Ga, ⁶⁴Cu, ⁸⁹Zr, ¹¹C, ¹²⁴I, ⁸⁹Rb have been evaluated. These investigations have demonstrated the affect of positron range on image quality and image resolution in particular (Alanazi, Szanda et al., Nagy et al., Gaitanis et al., Presotto et al., Ferguson et al., Teuho et al., and Liu et al. [1,2,5, 8-16]). This study aims to evaluate the performance of a small-animal Mediso nanoScan PET/CT system using 18F and 68Ga positron emitter radioisotopes. The study aims to evaluate the effects of choice of reconstruction algorithm on image quality. It also aims to assess the effect of application of CT based attenuation and scatter corrections and Monte Carlo simulation based positron range correction supported by the Tera-Tomo™ multi-GPU based reconstruction algorithm (Peter Major, et al. [10,17]). The image quality evaluation study is performed according to the NEMA NU 4-2008 standards protocols.

Materials and Methods

NanoScan PET/CT Scanner and Radioisotopes

In this study, a small animal Mediso-nanoScan PET/CT scanner (Hungary) was used to evaluate the image quality and spatial resolution of phantom images acquired using ¹⁸F and ⁶⁸Ga radioisotopes. The nanoScan PET/CT is a compact multimodality small-animal scanner developed and manufactured by Mediso Medical Imaging Systems (Hangary). The system combines large axial and trans-axial fields of view of the PET ring and high-performance CT system with Tera-Tomo 3D PET iterative reconstruction algorithm. The Tera-Tom properietry 3D iterative reconstruction applies deep Monte Carlo based physical modelling of particle-level interaction from positron emission to detection. It incorporates advanced corrections for energy, time, dead-time and random events and positron range with CT based attenuation and scatter corrections (Peter Major, et al. [10,17]). A comparison between image quality and spatial resolution of ⁶⁸Ga and ¹⁸F radioisotopes was performed.

¹⁸F was selected as a reference radioisotope as it is the most commonly used PET tracer and is required for most NEMA standard assessments. Most important physical characteristics of both radioisotopes are summarized in Table 1. It is important to remember that ⁶⁸Ga typically emits extra photons (1.08 MeV) at a low branching ratio (0.03) and produces a very small number of single events. Despite being small compared to those caused by annihilation photons, these high-energy photons contribute to the image’s creation and cannot be ignored (Cañadas, et al. [5]). King Faisal Specialist Hospital and Research Centre produces both radioisotopes in-house in its Cyclotron facility: ¹⁸F is obtained from a CS-30 cyclotron (Cyclotron Co. Ltd, Obninsk, Russia) as [¹⁸F]FDG; and ⁶⁸Ga is obtained from a 68Ge/⁶⁸Ga generator as [⁶⁸Ga]GaCl3 (eluted with 3 mL of 0.5 mol/L hydrochloric acid).

Table 1: The most important physical characteristics of ¹⁸F and ⁶⁸Ga (Cañadas, et al. [2,5]).

Evaluation of Spatial Resolution

Spatial resolution is a critical measure to assess imaging performance of PET systems (Gong, et al. [18]). According to the NEMA NU 4-800 standard on performance measurement of small animal positron emission tomography systems, a 22Na point source should be used to measure the spatial resolution of small animal PET scanners (Szanda, et al. [10]). In this investigation, two test objectives were used for spatial resolution assessment of the nanoScan PET/CT scanner. The first test object is made of a small acrylic sphere (< 1 mm radius) glued on a thin acrylic plate. The sphere was injected with 2 MBq and 0.8 MBq for ¹⁸F and ⁶⁸Ga, respectively to create a small spherical point source. The sphere and plate were positioned in air at 3 radial positions (0, 10, and 15 mm from the axis of the scanner). In the second test, 75 mm long glass capillary tubes with 1.0 mm inner diameter and 0.3 mm wall thickness were injected with 2.8 MBq of ¹⁸F and ⁶⁸Ga radioisotopes each. The capillary tubes was positioned in air at 3 radial positions (0, 10, and 15 mm from the central axis of the scanner). Mediso proprietary Nucline acquisition software was used to perform the scans. It provides graphical user interface (GUI) for setting up and starting acquisitions, operating mechanical functions, starting PET reconstruction, visualizing results, and other operational functions.

List-mode data was acquired in 1–5 coincidence mode, with an energy window of 400–600 keV and coincidence window of 5 ns. Acquired data was normalized to correct for different detection efficiencies and then and re-binned into a set of 0.3 mm bin-sized 2D sinograms using the Single-Slice Rebinning (SSRB) method (Daube-Witherspoon ME, et al. [19]) with a maximum ring difference of 16. Sinograms data was then reconstructed using a Filtered-Back Projection (2D-FBP) algorithm with a Median filter (Hounsfield GN [20]). Ordered-Subset Expectation Maximization (2D-OSEM) algorithm with 4 subsets, and 8 iterations (Hudson HM, et al. [21]), and the Tera-Tomo 3D-OSEM reconstruction algorithm with 8 iterations. In all three reconstruction algorithms (2D-FBP, 2D-OSEM, 3D-OSEM), randoms, attenuation, and scatter corrections were applied. However, positron range correction was only applied in the TeraTomo 3D-OSEM reconstruction algorithm. Image spatial resolution was reported as the Full Width at Half-Maximum (FWHM) and Full Width at Tenth-Maximum (FWTM) of tangential and radial profiles obtained across the activity point and line sources.

Image Quality Evaluation

The NEMA NU-4 compatible image-quality phantom (part no. PH- 60-00-42; Mediso Ltd.) was used for image-quality evaluation. The phantom comprises of three compartments as illustrated in Figure 1. The first compartment is homogeneous block (30 mm diameter and 30 mm length) used to calculate the system’s Signal-to-Noise Ratio (SNR). The second compartment consists of two cylinders filled with air and water respectively (length 15 mm; outer diameter 10 mm; wall thickness 1 mm) used to estimate Scatter Fraction (SF), and Spill- Over Ratio (SOR). The third compartment is a solid PMMA region (30 mm in diameter and 20 mm in length) with 5 holes drilled through with different diameters 1, 2, 3, 4, and 5 mm, used to assess Recovery Coefficient (RC). The image quality phantom was filled with a total volume of 25 mL activity for each study. To ensure equivalent number of positron decays, we used 20 minutes acquisition time for both radioisotopes studies, and injected the phantom with 3.7 MBq of 18F and 4.17 MBq of ⁶⁸Ga.

Figure 1

The phantom was positioned on the scanner bed and centered manually. The acquired data were reconstructed with the Tera-Tomo 3D-OSEM algorithm with a 0.3 mm voxel size and 8 iterations settings. Random, attenuation, scatter, positron range and dead-time corrections, and normalization were applied. A cylindrical Volume of Interest (VOI_uniform) with dimensions (22.5 mm-diameter and 10 mm-long) was drawn over the center of the phantom to determine image uniformity. In this VOI, the mean, minimum, and maximum pixel values, as well as the Standard Deviation percentage (% STD), were calculated. Line profile of pixel values along the activity rods were obtained. Recovery Coefficient (RC) curves were obtained by mean pixel value over a 10-mm volume of interest at the center of the active rods (VOI_rods). Recovery Coefficient (RC) for each rod size is obtained by dividing average pixel values of the line profile measured along the rods by mean pixel values obtained from the uniform region VOI_uniform as stated in equation (1) below.

The STD percentage of the RC (%STDRC) is calculated using the STD and mean values of the line profiles and uniform region’s VOI as stated in equation (2) below.

To assess accuracy of scatter correction for both radioisotopes, Spill-Over Ratio (SOR) is obtained by dividing average activity concentration in each of the two cold air and water cylinders by the average activity concentration in the uniform region as defined in equation (3) below. The average activity concentration in each of the two cylinders is obtained as the mean pixel values of 4 mm diameter and 7.5 mm long Volumes of Interest (VOI) calculated at the center of each cylinder.

The STD percentage of SOR (% STD_SOR) is calculated using the STD and mean pixel values of air and water cold cylinders VOI_air/water and uniform region’s VOI_uniform as defined in equation (4) below.

Results

Spatial Resolution

For the glass capillaries tubes positioned at radial positions (0, 10, and 15 mm from the axis of the scanner), obtained FWHM (FWTM) values were 2.2 (4.8), 1.5 (3.4), and 0.9 (1.6) mm for ¹⁸F radioisotope reconstructed using FBP, 2D-OSEM, and 3D-OSEM algorithms respectively. Corresponding values using the spherical point sources (Acrylic spheres) were 2.3 (5.2), 1.9 (4.1), 1.3 (2.4) mm for ¹⁸F, and 3.4 (7.1), 3.3 (6.0), 1.3 (2.3) mm for ⁶⁸Ga. FWHM and FWTM values obtained for various radial positions and two different setups are shown in Figures 2-5. FWHM to FWTM ratio (FWHM/FWTM) values for glass capillaries tubes’ pixel value profiles using FBP, 2D-OSEM, and 3D-OSEM reconstruction algorithms were 0.45, 0.44, and 0.56 for ¹⁸F; and 0.43, 0.46, and 0.54 for ⁶⁸Ga respectively. Corresponding values obtained using the spherical point sources were 0.44, 0.46, and 0.54 for ¹⁸F and 0.48, 0.55, and 0.56 for ⁶⁸Ga respectively.

Figure 2

Figure 3

Figure 4

Figure 5

Image Quality

Figure 6 below shows circular ROIs drawn on axial images of the three compartments of the image quality phantom reconstructed using the Tera Tom 3D-OSEM reconstruction algorithm. Obtained ROI pixel values were used to calculate Recovery Coefficient (RC) and Spill-Over Ratio (SOR) values for ¹⁸F and ⁶⁸Ga filled phantoms.

According to Teuho et al., Recovery Coefficient (RC) of uniformly distributed radioactivity in a phantom, should be approximately be equal to 1 (Teuho, et al. [15]). Using the Tera Tom 3D-OSEM reconstruction algorithm with all previously mentioned corrections applied, RC values ranged from 0.08 to 1.3 for ¹⁸F and from 0.09 to 1.16 for ⁶⁸Ga as illustrated in Figure 7. RC values for ⁶⁸Ga were generally lower than those obtained for ¹⁸F for all rods sizes. However, difference between RC values for ¹⁸F and ⁶⁸Ga for the same rod sizes were statistically insignificant. Image noise obtained as the standard deviation (% STD) of pixel values in the uniform region of the phantom was 10.1 % for ¹⁸F and 11.6 % for ⁶⁸Ga (Table 2). Spill-Over Ratio (SOR) values in air were (0.11 and 0.23); and (0.2 and 0.37) in water for ¹⁸F and ⁶⁸Ga, respectively (Table 3). As would be expected, the spill-in effects are higher for ⁶⁸Ga than ¹⁸F images. It is also noticeable that the spill-in effects are generally higher for water filled cylinders than for air-filled cylinders for both radioisotopes (¹⁸F and ⁶⁸Ga).

Table 2: Statistical Uniformity parameters in terms of mean, maximum and minimum values of activity concentration and % STD obtained in uniform Volume of Interest (VOI_uniform).

Table 3: Spill-Over Ratio (SOR) and percentage Standard Deviation (% STD) for cold water- and air- cylinders for both ¹⁸F and ⁶⁸Ga filled phantoms.

Figure 6

Figure 7

Discussion

In this study a comparison between image quality and spatial resolution performance of a Mediso NanoScan PET/CT system using ¹⁸F and ⁶⁸Ga radioisotopes is reported. Our results indicate that physical properties of a radioisotope significantly influence the performance of small animal PET/CT scanner’s image quality parameters such as RC, SOR, and spatial resolution. Spherical Acrylic point sources and cylindrical line sources of each radioisotopes were used to estimate radial and tangential resolution for both ¹⁸F and ⁶⁸Ga reconstructed images. Variation of spatial resolution with radial position in the axial field of view is also investigated for both radioisotopes according to NEMA NU 4 protocols. FWHM and FWTM of the line profiles were used to compare spatial resolution performance of the scanner using the 2D-FBP, 2D-OSEM, and the Tera-Tomo 3D-OSEM reconstruction algorithms. Tera-Tomo 3D-OSEM algorithm produced the highest resolution images followed by the 2D-OSEM iterative reconstruction algorithm.

There was a significant variation in spatial resolution values between measurements performed using Acrylic spheres and glass capillary tubes. This could be attributed to the partial volume effect on the spherical point sources in comparison to the cylindrical capillary tubes line sources. This may also be attributed to the large difference in the density of glass tube walls (2.2 g/cm3) and the sphere’s acrylic walls (<1.2 g/cm3), as wall thickness and density effects positron range and radioactivity spill-out significantly. As to be expected, spatial resolution was poorer for ⁶⁸Ga than ¹⁸F for images reconstructed using the 2D-FBP and 2D-OSEM reconstruction algorithms (Figures 2-5). This is mainly due to the effect of positron range on the spatial resolution of PET images (Bai, et al. [22]). However, there was no significant difference in spatial resolution for ⁶⁸Ga than ¹⁸F for images reconstructed using the Tera-Tomo 3D-OSEM reconstruction algorithm, which can be attributed to the effectiveness of the positron range correction applied in this reconstruction algorithm.

Considering that an ideal FWTM/FWHM ratio for a Gaussian distribution profile is 1.82, and should be ≤ 1.9, our resolution estimation results have met this requirement for all image reconstruction algorithms performed using the glass capillary tubes in contrast to earlier reported results by Cañadas, et al. [5,8], where an important deviation from a Gaussian profile (ratio ~1.82) was observed for ⁶⁸Ga. However, an apparent deviation from the ideal Gaussian profile for both radioisotopes was observed when spherical point sources was used. This again could be attributed to the inherent partial volume effect of the spherical shape of the point source and the lower density of the Acrylic point source wall. The possibility of formation of micro air bubbles in the small sphere may also cause significant partial volume effect resulting in the over estimation of FWHM of point spread function of the point source. Furthermore, a gradual deterioration in the spatial resolution was observed, especially for 2D-FBP and 2D-OSEM reconstruction methods, with increased distance from the center of the axial FOV, which agrees with the results of Attarwala [2]. Employing the Tera-Tomo 3D-OSEM reconstruction algorithm has produced significantly improved spatial resolution values for both radioisotopes with almost identical spatial resolution values for ⁶⁸Ga and ¹⁸F. These results agree with those reported earlier by Dahle, et al. [9,23], who both showed that spatial resolution values were greatly enhanced by using the 3D-OSEM reconstruction algorithm. A NEMA NU-4 compatible image-quality phantom was used to evaluate image-quality parameters for ¹⁸F and ⁶⁸Ga imaging (Teuho, et al. [15]). Images with comparable injected activities were acquired using 30 minutes acquisitions and reconstructed using the Tera-Tomo 3D-OSEM algorithm. Our results have shown that there was no significant variation in noise levels expressed as % STD for the two radioisotopes. We did not investigate the effect of choice of image reconstruction algorithm on image quality parameters such image noise, contrast recovery and spill-over ratio due to technical issues and time constraints.

However, it is well established that choice of reconstruction algorithm and reconstruction parameters including number of iteration, subset and choice of filter will affect image quality parameters as reported by Disselhorst et al. and others (Disselhorst, et al. [8]). It is has been reported that ⁶⁸Ga will have lower contrast recovery expressed as RC due to the relatively high positron range (Attarwala, et al. [2,15]). However, our results have shown insignificant reduction in estimated RC using ⁶⁸Ga as compared to ¹⁸F. This better than expected result is probably due to implementation of the positron range correction technique on the Tear Tomo 3D-OSEM algorithm. Furthermore, the RC values for both ¹⁸F and ⁶⁸Ga have exceeded 1 for rods with diameters 4 and 5 mm (Figure 6). This could be attributed to the distinct hot object edge artifacts; in which the hot rods’ activity was underestimated inside and overstated at the edges (Ferguson, et al. [14]). However, our results differ from those of Ferguson et al., who suggested that RC values exceed one for only short-range positron emitters such as 64Cu and ¹⁸F (Ferguson, et al. [14]), while our results have shown RC values exceeding 1 for a relatively long positron range emitter ⁶⁸Ga.

As stated by Teuho et al., Spill-Over Ratio (SOR) in a model system should be equal to 0, as there is no actual radioactivity in cold regions (Teuho, et al. [15]). Low SOR values however indicate how well the scatter and positron range correction work for the system (Attarwala, et al. [2]). Our results have shown that SOR in both air and water was significantly higher for ⁶⁸Ga (0.23 and 0.37, respectively) than for ¹⁸F (0.11 and 0.2, respectively). This variation is in agreement with previous published results (Teuho, et al. [2,5,8,15]). Furthermore, SOR values were significantly higher in water than in air for both radioisotopes. This is probably due to the huge difference in positron range in water and air (positron range in air is 1.66 m and 6.69 m for ¹⁸F and ⁶⁸Ga respectively). Positron range effects can be ignored in SOR estimation in air but not for water or equivalent tissues (Disselhorst, et al. [8,24]).

Conclusion

Our study provided a comparison of image quality indictors including spatial resolution between ¹⁸F and ⁶⁸Ga radioisotopes on a small-animal scanner (Mediso NanoScan PET/CT). This allowed the investigation of the effect of positron range on image quality and spatial resolution. It also allowed the evaluation of the positron range correction technique implemented on the Tera-Tomo 3D-OSEM reconstruction algorithm. Significant reduction in spatial resolution was observed when using ⁶⁸Ga with 2D-FBP and 2D-OSEM reconstruction algorithms. However, employing the Tera-Tomo 3D-OSEM reconstruction algorithm with positron range correction has resulted in identical and improved spatial resolution parameters for both radioisotopes. There was no significant difference in image noise (% STD) for ¹⁸F and ⁶⁸Ga images performed using the Tera-Tomo 3D-OSEM reconstruction algorithm. Insignificant reduction in estimated RC using ⁶⁸Ga as compared to ¹⁸F was observed. This indicates the effectiveness of the positron range correction technique implemented on the Tera-Tomo 3D-OSEM algorithm. Spill-Over Ratio (SOR) values measured in both air and water cold cylinders were significantly higher for ⁶⁸Ga than for ¹⁸F. SOR values were significantly higher in water than in air for both radioisotopes indicating that positron range effects can be ignored in air but not for water or equivalent tissues in quantification studies.

Conflict of Interest

The authors declare that there is no conflict of interest regarding the publication of this paper.

Funding Statement

The authors express their thanks and appreciation to King Faisal Specialist Hospital and Research Centre (KFSH&RC) for providing access to facilities and radioisotopes including the Mediso-nanoScan PET/CT scanner (RAC # 2210033).

Contributions

All authors contribute, read, and approved the final manuscript

Author’s Statement

This submission complies with ethical guidelines and all authors contributed to this manuscript.

Acknowledgments

The authors express their thanks and appreciation to King Faisal Specialist Hospital and Research Centre (KFSH&RC) for providing access to facilities and radioisotopes including the Mediso-nanoScan PET/CT scanner, and to Mr. Y. AlMalki for his technical assistance. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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