Open Access Journals on Medical Research

Tuberculosis in South Asia

Introduction

Tuberculosis (TB) is a potentially serious infectious disease that mainly affects the lungs though it can spread to other parts of the body one’s brain or spine. The bacteria are spread when an infected person coughs or sneezes. It can also spread through saliva (by kissing or sharing drinks etc.). A type of bacteria called Mycobacterium tuberculosis is the cause of the disease. Tuberculosis (TB) remains formidable threat to global health despite concerted efforts during past decades by the global health community. From 1990 to 2015 the death rate has decreased by 47 percent [1]. Among 22 countries which had high TB burden, 16 countries have achieved millennium development goals for TB. A target has been set by the Global Health Community for the post 2015, ENO-TB strategy of reduction of 25 percent in occurrence and a reduction of 75 percent in mortality between 2015 and 2025 and a mortality reduction of 95 percent and 90 percent reduction in occurrence by the year 2035 [1]. As per the news item published in the daily, “The Times of India” dated 16th February 2020, The Health Minister of India, Dr. Harsh Vardhan informed that the country has even more ambitious target of eliminating the disease from the country by the year, 2025 and said that the mission Indradhanush is already operational for the purpose.
Although the elimination of a disease like TB is a very complex problem but by the time our understanding and knowledge about infectious diseases, global connections, resources and range of intercession have also increased. In order to increase the rate of TB elimination we must increase the research to maximum level in high burden countries like India, Pakistan, Nepal, Bangladesh, Afghanistan, Maldives, Bhutan and Sri Lanka. The WHO South-East Asia (SEA) is habitat for 26% of the world’s population with 44% heavy load of TB. An estimated 4.4 million community got sick with TB and estimated 6,38,000 passed away due to the disease which is over half of the global TB deaths [2]. The coming into view of rifampicin resistant or multidrug resistant tuberculosis (MDR) is one of the most important challenges to the control of tuberculosis pandemic. The main reason for drug resistant tuberculosis is either the transmission of already resistant strain of Mycobacterium tuberculosis or the suboptimal cure of susceptible strains. It is no surprise therefore that having such a high burden of TB disease, this region has an estimated number of 184336 MDR cases among the total recorded TB cases which brings it on third position in the list of Global MDR burden region [2]. Most of the MDR cases in this field remain untouched due to considerable gap between the agreement and useful putting into effect [3]. As such, there is an imperative need to improve and strengthen research capacity in these high burden countries.

Treatment

In our fight against drug resistant TB, a great achievement is the recent licensing of bedaquiline and delamanid [4-5]. Effective treatment depends upon accurate diagnosis. In the region, the Gene X port scale up has significantly increased detection of MDR TB [6]. Until there is an improvement in molecular drug susceptibility testing for drugs other than rifampicin, phenotype drug susceptibility testing will remain essential to treat MDR TB cases properly. Although the population is very large (1.8 billion), the ratio of DST laboratories to population remains as low as 0.2 labs per 5M population. It is estimated that the cost of treatment of a single MDR patient is about 500 times the cost of a drug susceptible patient. Therefore investment in strong MDR TB control is very cost effective if it successfully checks MDR transmission. Apart from this, the new shorter MDR TB treatment regimen from Bangladesh is now adopted and recommended by WHO and it should also be implemented region wide.

Infection Control and their Prevention

The huge number of latently infected individuals throughout the world presents an extensive challenge to eradication efforts. Various models created for control strategies suggested that TB suppression is not possible without get to grips with latent TB. A rough estimate was that about 1/3rd of the world population was suffering from latent TB. But according to a recent study the risk of infection per year estimates around one fourth of the total world population which counts near 1.7B [7]. In the last decade, Isoniazid preventive therapy (IPT) in endemic countries has received serious consideration. Shorter regimens must be developed for its large scale feasibility also the regimens appropriate in the area of high isoniazid resistance. The 12 doses given 3 months rifapentine isoniazid regimine is a step in the right direction but more must be done to value carefully worked designs for IPT scaleup beyond those currently being undertaken in people living with HIV. South Asia is also vulnerable to natural disasters and political un stability. Contingency measures are to be kept in place for prevention of decease spread in case a disaster visits. The 2015 earthquake in Nepal and lack of proper delivery system of healthcare measures is a grim reminder in this regard.

Research Priorities

The discovery of an effective vaccine to treat TB seems an unachievable goal in the near future. While efforts must be kept on for finding a vaccine, we should find a way to eradicate TB without using any vaccine. In large urban cities and remote rural areas of south Asian region research priorities must aim at packaged intrusions. Research capacity should be increased by broadening the network of good clinical practice (GCP) compliant, clinical trial sites to spread up novel regimen evaluations, improving laboratory capacity for bacterial culture and testing of drug susceptibility. Networking research wideness the region to share and scale up best experiences, enthralling governments and fund providers in research plans to make certain move and political promise to keep giving money (Figure 1).

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Figure 1: Isoniazid.

Conclusion

There is an golden chance for the scientific fraternity to embark on a path of new research to find real cure for the huge majority of TB afflicted population in the South Asian region. South Asia has patients with a large number of innovative scientists who can enable it to seize the opportunity and lead global TB eradication programs by representing feasibility in cities with high population density and remotest reaches of the Himalayas. Political will and leadership with a vision are the two attributes which are essential to facilitate the loftily programme of TB elimination. The malady cannot be overcome by a single intervention, instead continuous innovations are required. This is the time that researchers in the region join hands, work with cohesion, develop an unified agendum to ensure successful research for TB eradication. The sooner, the better.

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Open Access Journals on Medical Research

Pfizer/Biontech Post Vaccine Acute St-Elevation Myocardial Infarction: A Case

Introduction

Corona-type viruses have been known for years. In the 2019/2020 period, a new type of SARS-Corona virus-2 (SARS-CoV- 2=COVID-19) started to spread all over the world. Several vaccines have been approved against COVID-19. Each of these sought to respond to the pandemic by providing individual protection against COVID-19. One of these vaccines is the messenger ribonucleic acid (mRNA) COVID-19 (Pfizer/BioNTech) vaccine that is based on new gene-based technology. It was recommended that the vaccine be administered in two doses, with a 21-day interval between them. Based on current knowledge, COVID-19 mRNA vaccines have a high efficacy rate of approximately 95%. Current research data indicate that the odds of contracting COVID-19 infection are approximately 95% lower in fully vaccinated persons than in unvaccinated persons. Its effectiveness in preventing severe COVID-19 disease (for example, hospitalization) is about 85%. It is not yet known how long the protection of this vaccine will last. The Pfizer/BioNTech vaccine is approved for people 12 years of age and older. However, some unexpected side effects may occur after vaccinations for COVID-19 infection, the future of which is still unknown. After one of the mRNA vaccines, local and general reactions can be seen as a result of the interaction of the vaccine with the body. These reactions are generally expected to disappear within 2 days after vaccination [1].
Although rare cases of myocarditis and pericarditis have been reported among cardiac side effects after the mRNA covid-19 vaccine, no significant heart attack was observed in vaccinated patients. It is thought that other medical conditions that develop during or after vaccination may not always be related to the vaccine, since large-scale vaccination is performed [2]. Diagnosing postvaccine Acute Myocardial İnfarction (AMI) can be difficult. Because muscle pain at the injection site may cause ischemic symptoms to be overlooked and delayed admission. As far as we can search the literature, we know of two cases of acute myocardial infarction in the United States that started less than 24 hours after the first dose of the COVID-19 vaccine [3]. In our case, we aimed to emphasize acute myocardial infarction and other possible side effects that developed within hours after the second dose of Pfizer/BioNTech COVID-19 vaccine, in the light of the literature.

Case Report

A 39-year-old male patient said he had shoulder, arm, and chest pain lasting more than three days after receiving the first dose of the Pfizer/BioNTech COVID-19 vaccine. There was a relief after taking paracetamol and nonsteroidal anti-inflammatory tablets for his pain. He attributed all these pains to possible post-vaccine reasons. After 21 days, the patient applied to the emergency service with complaints of discomfort and tightness in the chest, which started 1.5-2 hours after the second dose of vaccine, continued for more than 10 minutes. Also, the patient could not describe the nature of the pain exactly. He stated that the pain was not like in the first dose, but was suppressive, restless, starting from the scaphoid and spreading to the jaw and neck. The patient had both vaccines done on the left arm. The patient did not have the fever or respiratory distress at the time of admission and did not report any drug-vaccine allergy. There was no cardiac history in his own and first-degree relatives. In his examination; general condition was good. Vital signs are blood pressure; arterial 130/70mmHg, temperature: 36.5 °C, oxygen saturation parameter (SpO2): 99%, heart rate: 70/minute. When the heart was listened to, there was no additional sound and no murmur. Peripheral pulses were palpable. Other system examinations were normal. Electrocardiography (ECG), complete blood count, biochemistry, and cardiac biomarkers were requested from the patient. Polymerase Chain Reaction (PCR) was requested for COVID-19 in the nasopharyngeal aspirate in the emergency department, the test result was negative.
In the ECG taken in the fifth minute of the patient’s admission to the emergency department; ST-segment elevation in inferior leads (II, III, and aVF) and reciprocal ST-segment depression in I and aVL were detected (Figure 1). The patient’s cardiac troponin T value at admission; 8.83pg/dl (range: 0-14pg/ml), 5097pg/dl at the 6th hour and 2689pg/dl at the 24th hour. ECG monitoring, 300 mg acetylsalicylic acid, 300 mg clopidogrel orally, 5000 IU heparin intravenously were administered to the patient with the diagnosis of acute inferior myocardial infarction. Emergency coronary angiography showed globular thrombus and occluded proximal segment of the Right Coronary Artery (RCA) with TIMI 0 flow (Figure 2). RCA emergency Percutaneous Coronary İntervention (PCI) was performed. Dilated with balloon and stent placed. After coronary angiography, a good outcome was detected with the TIMI III flow (Figures 3 & 4). The patient did not have chest pain after percutaneous coronary intervention. On the fifth day of hospitalization, the patient had minimal hypokinesia of the right inferior wall and was discharged with a cured left ventricular ejection fraction of 60-65%.

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Figure 1: ST elevation in lead II, III and AVF (blue arrow) and ST depression in I and AVL (red arrow).

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Figure 2: Critical stenosis of middle segment of RCA-black arrow.

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Figure 3: Balloon and stent inserted to middle segment of right coronary artery-black arrow.

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Figure 4: Balloon and stent inserted to middle segment of right coronary artery-black arrow.

Discussion

Although vaccines dedicated to COVID-19 disease have been approved as safe, both the disease and the vaccine are relatively new [4-6]. This requires that even very rare events be widely shared and discussed with the medical community. We report a case of AMI occurring within two hours of the second dose of the COVID-19 vaccine. The angiographic features of the case suggested acute thrombotic events as the underlying mechanism. Although it is not possible to establish a causal relationship between vaccination and cardiac events, it is important to remember that the mechanisms of thrombotic events in COVID-19 are still not fully understood [7]. Vaccines are considered the most effective drugs in public health. Side effects may also occur due to some reactions after vaccination. It is often difficult to say whether the reactions are caused by the vaccine itself or by other factors causing AMI. Excipients used in vaccines; it is used to improve stability, increase solubility and improve absorption. However, these substances contribute significantly to the development of IgE-mediated anaphylactic reactions during vaccination [8].
Kounis syndrome is an allergic reaction to various substances, including excipients, resulting in acute coronary syndrome [9]. This could be a possible explanation for the AMI clinic post- COVID-19 vaccine. It may also occur through prothrombotic immune thrombocytopenia, which is similar to heparin-induced thrombocytopenia, leads to vaccine-induced thrombotic events [10]. Although there is limited data on the risk of thrombotic events with vaccines currently in use in the United States, there have been recent reports of thrombotic events after COVID-19 vaccines in Europe. No acute myocardial infarction was reported in the original study of vaccines used in Covid-19. They also did not present a significant difference in thrombotic events between the vaccinated and placebo groups. However, cases have started to be presented after these vaccines, rarely. Boivin et al. reported a 96-year-old female case who had AMI one hour after the Moderna COVID-19 vaccine, and one case of AMI in a 63-year-old healthy man two days after the AstraZeneca COVID-19 vaccine [11]. Maadarani AstraZeneca presented a case of AMI after the COVID-19 vaccine [12]. Tajstra on the other hand, reported an 86-year-old male case of AMI after Pfizer/BioNTech Covid-19 vaccine [13]. Finally, Jonathan presented two cases of AMI, one male, and one female, after the Moderna vaccine [3]. Our case can be seen as the second case in the literature after Pfizer/BioNTech Covid-19 vaccine. The Vaccine Adverse Event Reporting System (VAERS) [14], the National Vaccine Safety passive monitoring system, reported that side effects occurred after receiving the second dose of 76% of mRNA vaccines made so far. According to the Centers for Disease Control and Prevention (CDC) case definitions, The median interval from vaccination to symptom onset was 2 days (range = 0-40 days). Some of the symptoms were seen within 7 days of vaccination in 92% of patients. Acute clinical courses were generally mild [15]. About 78% of Suspected Adverse Events (AEs) were reported in individuals younger than 60 years of age. In clinical trials of mRNA vaccines, it has been noted that people younger than 60 years tend to experience more reactogenic AEs than those aged 60 and over. In general, younger individuals have more active immune responses and may experience more AEs to vaccines. This is part of the body’s natural response to build immunity to COVID-19 infection. Approximately 66% of AEs have been reported in women.
Of the 7.5 million doses of Pfizer-BioNTech and Moderna mRNA vaccine administered in total, 0.12% were reported as suspicious and 0.005% as severe AEs. The most commonly reported serious side effects are anaphylaxis and other serious allergic reactions. Also asthma exacerbation, difficulty breathing, fast heart rate, increase or decrease in blood pressure, chest discomfort and pain, pericarditis or myocarditis, syncope, numbness of limb, weakness or pain, vision changes, increase in liver enzymes, thyroid gland dysfunction, muscle injury, joint pain, seizures, tinnitus, infections, and blood clots. Most individuals who develop severe AE are reported to have recovered. A higher incidence of heart attacks and strokes was not observed in locally vaccinated persons. It’s also important to note that heart attacks and strokes can occur naturally, regardless of whether people are vaccinated or not. It is argued that the benefits of COVID-19 vaccines continue to outweigh the known risks in the pandemic [2].

Conclusion

As the COVID-19 vaccine campaign moves into a new phase worldwide and vaccines become available to the general public, there will be more data on safety and potential side effects. Further data on nationwide myocardial infarction or other thrombotic events before and after vaccination initiation should be carefully considered and interpreted before commenting on any possible causal relationship.

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Journals on Microbiology Research

Comparison of the Reaction Time Between Motorcycle Road Racer and Motorcyclists

Introduction

Motorbike speed is one of the most popular sports in the world, which requires excellent physical and mental performance from the racers. However, the importance of applying the Theory of Sports Training in the sport is little explored, especially in the amateur categories. The lack of information occurs due to the little involvement of Sports Medicine and Sports Sciences in the different categories of motorbike [1]. It is recognized that this sport is extremely challenging and dangerous [2,3]. The motorbike racers is exposed to extreme conditions and high physical demands, on the other hand, in motorsports, the victories depend, not only on the individual physical capacity of the athlete but on a set of factors, such as riding technique, mental effort, equipment efficiency and resistance, race strategy and team competence [4].
Even though physical fitness is not the main determinant for win in Motor Sports, it is still observed that training and racing require athletic skills and physical demands from the driver, similar to athletes in other sports [5]. In this sense, Ebben, et al. [6] point out that the aerobic resistance of racer drivers is similar to that of boxing, basketball, football, athletics, among others. Reaction Time (RT) is the time interval between the generation of visual, audible or tactile stimuli and motor action. This time revolves around values that correspond to milliseconds and depends on factors such as age, physical conditioning, cognitive level, emotional state and gender [7]. Although Reaction Time is related to genetic conditions, it is a sensory quality that can be trained. High-performance athletes can achieve a decrease of approximately 15% in reaction time due to years of training [8]. For the athlete to react to a stimulus, there is a latency period (just before muscle contraction begins), which is shorter or slower due to the quality of the processing of the reference by the central nervous system [7]. It is observed that the RT of elite athletes, is faster when compared to beginners or untrained [9].
According to Tønnessen, et al. [10], in a study comparing the reaction time of 1,319 world champions in the 100-meter dash category, it was observed that the RT of male athletes (0.166 ± 0.030 seconds) was significantly lower than that of female athletes (0.176 ± 0.034 seconds). There was no relationship between RT and height. The best RT was obtained in the age group between 26 and 29 years old in males (0.150 ± 0.017 seconds) and athletes over 30 years old (0.153 ± 0.020 seconds). According to Sparrow, et al. [11], the type of training, testing and the modality practiced by the athlete can influence RT. Gélat, et al. [12] point out that the emotional and attention state interferes with RT, which can be faster, in motivating conditions and/or when the individual remains more attentive and slower, in situations of demotivation or depression. The neurosensory response is extremely important for Motor Sports racers, such as motorcycle race. Being that during races the racers receives different visual, auditory, tactile stimuli and the respective reception, transfer, processing and transfer of this information, in the shortest possible time, are essential for the performance during the competitions.

Objective

To compare reaction time of Track Day racers with motorcyclists.

Method

Sample Characteristics

The accessibility sample consisted of 10 volunteers, who were divided into two groups with five individuals each: G1) Amateur Track Day racers 29.4 + 2.6 years old and G2) Motorcyclists, without experience in motorcycle competitions, with an average age of 22 + 1.4 years.

Inclusion Criteria

• Motorbike racers: participation in at least six Track Day races between 2018 and 2019; male; physically active; age over 18 years; signing the Free and Informed Consent Form (ICF).
• Motorcyclists: National Driver’s License Category A; use of the motorcycle as a means of transportation at least four times a week between the years 2018 and 2019; male; physically active; age over 18 years; signing the Free and Informed Consent Form (ICF).

Exclusion Criteria

Any type of injury that makes bodily movements impossible. Consumption of substances with caffeine two hours from the tests. Do not accept to participate in the research.

Ethical Procedures

The research project was submitted to and approved by the Research Ethics Committee of the School of Physical Education of Jundiaí (Jundiaí, São Paulo/Brazil) Protocol Number 3.401.727 and all volunteers signed the IC to participate in the study.

Experimental Procedures

To evaluate the Reaction Time, FITLIGHT® system was used, a device that allows the random activation of up to eight LEDs, wirelessly, according to the programming of the respective lighting time and activation time determined by the researcher, with the registration of the number of hits and reaction time in milliseconds. The equipment allows identifying, besides the Reaction Time, other variables such as several correct answers in the tasks as well as the total activity time. The equipment’s different schedules have the potential to quantify Reaction Time, as well as to develop specific exercises or activities that simulate real movements during the specific movement of various sports.
The study volunteers underwent two tests performed sequentially to identify the Reaction Time: T1) specific test performed on a Honda CBR 600cc motorcycle, supported on a specific easel, where each rider and motorcyclist remained in the riding position responding to luminous signs of the LEDs that were positioned laterally (left and right side) at the height of the volunteer’s eyes and two others on the ground beside the front tire, during the test the objective of turning the handlebar left or right or directing the left hand or right as the LEDs are being turned on randomly; T2) non-specific test, with the led’s positioned in front of each individual on a bench, the objective being to direct the right or left hand to each random lighting of the led for the respective Reaction Time record. In the two tests, the respective positions of the subjects were standardized. Each test has a total of 30 random LEDs, with an estimated time to complete each task of around thirty-five seconds. The variables obtained in each task were: average Reaction Time (in milliseconds), the number of hits and errors, that is, if the volunteer was able to respond to the stimulus in the time that the LEDs remained on (0.500 ms), as well as the total test run time.

Data Analysis

For comparison between moments, Student’s t-test paired for variables with normal distribution was used, with Bonferroni’s post-test adopting a 5% significance level for all analyzes. Using the Graph Pad software. The data were also presented descriptively in graphical and tabular form.

Results

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Figure 1: Comparison of Reaction Time in the specific test.

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Figure 2: Comparison of Reaction Time in the general test.

Graphs 1-6 show the results obtained in the present study. It can be seen that in the specific test the Track Day racers performed better than the motorcyclists concerning the following variables: reaction time, number of hits and total time to perform the task. However, in test 2 considered as a non-specific evaluation of the TR, there was no statistical difference in the variables studied, except in the total time to perform the task, which was significantly less in favor of the Track Day racers (Graphs 1-6).

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Figure 3: Comparison of the number of hits in the number of hits in the specific test.

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Figure 4: Comparison of the number of hits in the general test.

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Figure 5: Comparison of average total time to complete the specific test.

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Figure 6: Comparison of average total time to complete the general test.

Discussion

The identification of the reaction time levels of a sports modality must follow its characteristics and physiological influences in its execution. For Backman, et al. [13], in Motor Sports, the effect of driving the vehicle on the locomotor system and the muscular effort in its entirety during practice should be considered. In this sense, it can be inferred that in the motorbike the dynamic component of the modality is characterized by the great demand for body displacement during the practice, that is, use of the muscles of the trunk, abdomen and upper limbs for driving, acceleration, clutch control and motorcycle braking and use of the lower limbs together with the pelvic girdle muscles for positioning in curves, braking and downshifting. The static component is also observed, concerning the core and shoulder girdle muscles, and the modality is considered to have a vigorous level of physical effort, due to the high level of dehydration, above-average heart rate and high probability of falling and risk of collisions. In this sense, familiarity with the equipment during the specific test may be one of the reasons for the better performance of the racers compared to motorcyclists. However, Track Day racers did not train Reaction Time in their weekly exercise routine. Inferring that the best results obtained in the specific test are related to the practice of the sport, according to the principle of specificity [14].
Remaud, et al. [15] highlighted the demands for attention associated with postural control during Reaction Time and considered that both the focus of attention and the difficulty of a postural task are potential factors that influence RT. The authors evaluated thirteen young people in two tests that consisted of being as quiet as possible on a force platform in different postural conditions, while simultaneously performing a simple TR task. The difficulty of the postural task was handled by various combinations of three support bases (feet together, apart and single leg) and two visual conditions (eyes open and closed). Participants were instructed to focus on balance or performance on RT, depending on the test session. Participants responded more quickly in all dualtask conditions by focusing on TR performance than on balance. The modified attention allocation index indicated that the participants’ ability to modulate their allocation of attentional resources to respond positively to instruction was more pronounced in the most challenging postural condition. This can explain in our study, the better performance of Track Day racers in the task performed in the riding position and on a motorcycle of a similar model to the one that is usually used in their routines.
These results indicate that Track Day racers concerning the reactions on the motorcycle stand out from the racers, as expected due to the experience on the tracks and familiarity with the model of the motorcycle that was used as a reference for carrying out the tests. A similar result was observed in the study by Van Leeween, et al. [16], where racers drivers were better in all measures of reaction time compared to drivers. On the other hand, in the nonspecific test, the performance of the two groups of motorcyclists was equivalent, but with a noticeable difference in terms of the test execution time.

Practical Applications

For a motorcyclist to participate in the Track Day, the racers don’t need to have experience in racing tracks and/or to have taken a Riding Course and a motorcycle-specific preparation for competitions is also not necessary. Due to these facilities, many motorcyclists, even without the proper preparation to enter a racetrack, are attracted to participate in these races, in some cases with rented competition motorcycles. Our data showed that, for example, the Reaction Time of racers experienced in this type of event is higher than that of motorcyclists who, although experienced, never participated in Track Day. In this sense, the results of this study indicate that it is not enough for the rider alone have experience of daily use of motorcycles in traffic, as the specificity of the riding position seems to significantly interfere in the Reaction Time test. In this sense, it is suggested that motorcyclists who have never participated in Track Day should take courses and training before starting the practice. On the other hand, we suggest that the specific test protocol used in the present study to evaluate the Reaction Time can be used for the evaluation and/or training of motorbike racers.

Conclusion

It was observed that the Track Day racers presented the best reaction time, number of hits and time of execution of the task than the racers in the specific test. However, in the non-specific reaction time test, there was no significant difference between the volunteers studied, except in the time of the test, where the Track Day racers were faster. These data contribute to reinforce the importance of specificity in motorsports and to the establishment of benchmarks concerning the reaction time of motorbike racers.

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Open Access Journals on Behavioral Medicine journals

Evaluation of Myocardial Infarction in Pigs by Coronary Spectral CT Angiography

Introduction

Acute Myocardial Infarction (MI) is one of the major causes of morbidity and mortality in western countries [1]. Noninvasive imaging for MI detection, patient risk stratification and treatment monitoring is needed especially when Electrocardiography (ECG) and myocardial enzyme with no clinical clue. Multidetector CT has been used for heart morphology and coronary stenosis evaluation for many decades [2,3]. However, even mild coronary stenosis could not always exclude vessel-specific myocardial ischemia [4]. In addition, additional contrast material and radiation exposure limit the use of CT perfusion to evaluate myocardium [5]. In clinical practice, comprehensive evaluation of coronary artery stenosis and myocardial perfusion in one examination is desirable [6]. The advent of dual energy spectral CT has sparked renewed interest in clinical applications for many organs [7-10]. The strength of monochromatic energy image in improving image quality at optimal keV and increasing Contrast to Noise Ratio (CNR) at high keV was outlined; [11,12] the iodine density image was highlighted for the capability to quantify Iodine Concentration (IC) [11,13]. Consequently, our study aims to evaluate the myocardial infarction by Coronary Spectral CT Angiography (S-CCTA) based on the advantages mentioned above to discuss its strength, potential and limitations. The purpose of the current study was in three folds:
1. To investigate whether S-CCTA could assess MI
2. To define the best parameters of S-CCTA for MI delineation
3. To discuss the correlation between myocardial IC and cardiomyocyte apoptosis.

Methods

Experimental Protocol

Twelve Chinese mini pigs (5 males; weight, 20.23 ±1.23 kg; age, 6.0±0.7 months) underwent Percutaneous Coronary Intervention (PCI) to produce acute ischemia/reperfusion MI model by balloon dilatation. In compliance with the NIH guidelines for the use of laboratory animal, these mini pigs received human care. After premedication with ketamine (20 mg/kg), Xylazine Hydrochloride (1.5mg/kg) and atropine (0.02mg/kg), the pigs were intubated, anesthetized and mechanically ventilated with an admixture of 2.5%-3.5% sevoflurane and 100% oxygen. Baseline heart rate, ECG, and weight of animals were acquired before modeling. Before PCI, heparin was administered at an initial dose of 10,000IU intravenously, followed by additional 4,000IU/h to maintain anticoagulation throughout the PCI procedure [14]. The angioplasty balloon was positioned in the Left Anterior Descending Artery (LAD) just distal to the first diagonal artery. Acute MI was confirmed by ECG. After 90-minutes occlusion, the angioplasty balloon was drawn back.

Spectral CT

After 4±1 days of MI model establishment, the pigs underwent S-CCTA. Induced anesthesia was the same as above during PCI. Anesthesia was maintained by intravenous disoprofol (5mg/ kg/h). Respirator was used to help and control the breathing. All pigs underwent S-CCTA on a single-source dual energy spectral CT (Discovery CT750 HD CT Freedom Edition scanner, GE Healthcare, USA). The parameters were as following: Gemstone spectral imaging mode with fast peak tube voltage switching between 80 and 140 kVp during a single rotation, axial plane with 64 × 0.625 mm collimation, 350-msec gantry rotation time, 175-msec x-ray exposure time. All pigs received 1.5 ml/kg contrast material (Ultravist 370, Bayer Schering Pharma, Germany) followed by 30 ml saline at a flow rate of 3.0ml/s. Bolus tracking with a Region of Interest (ROI) was placed in ascending aorta and was used to synchronize the arrival of contrast material to start the image acquisition (trigger threshold of 120 HU). The radiation dose was recorded.

S-CCTA Images Processing

The monochromatic S-CCTA images were reconstructed in standard short axial plane with slice thickness of 2.00 mm. The density of infarction area (referred to the myocardium in tan or white color on TTC stain mentioned later), remote myocardium (myocardium in an unaffected coronary artery territory, usually the inferior wall) and the noise were measured. Identical ROIs on S-CCTA images of different energy were adopted by adjusting monochromatic energy from 40keV to 140 keV at a 5keV interval. The optimal keV was chosen based on the CNR, signal to noise ratio (SNR) and noise.

CNR = (HUremote – HUinfarct) / noise
SNR = HUinfarct / noise.

Where H Uremote represented the mean CT value of remote myocardium, and HUinfarct indicated the mean CT value of infarction. The noise was derived from the standard deviation of CT value in the remote myocardium.
In addition, the mean CT value of infarction area, risk area (the adjacent segments of infarction) and remote myocardium was recorded respectively to observe the density change from 40keV to 140 keV at a 5keV interval (spectral curve).
The optimal keV and iodine density images of S-CCTA were reconstructed in short axis. Thereafter, the CT value and IC were measured in the infarction region, risk area and remote myocardium of each pig. To maintain the consistency of the size, shape and position of ROIs among different CT images, the ROIs were automatically copied by the software and adjusted slightly by hand if necessary. Images were assessed in consensus by two experienced readers (A and B, with 5 and 11 years of experience in CCTA, respectively). For infarction observation, the CNR and SNR were calculated as mentioned above. Finally, objects with noreflow phenomenon at late enhancement imaging were recorded. The IC in no-reflow region (persistent hypo-enhancement on late enhancement images), infarction and remote myocardium was measured on S-CCTA.

Histopathology

At the conclusion of radiologic examinations, the animals were euthanized with overdose vecuronium bromide. The hearts were sliced into short axises of about 4mm and incubated in the triphenyl tetrazolium chloride (TTC) (1mg/100ml) at a temperature of 37℃ by water bath. Remote normal myocardium was delineated as the living tissue and is colored with red, while the infarcted tissue is colored in pale tan. With consideration of S-CCTA images and gross specimen, serial cutting sections in the no-reflow region, infarction and remote myocardium were used for immunofluorescent staining. Terminal deoxynucleotidyl transferase-mediated dUTP Nick-End Labeling (TUNEL) stain of myocardium was used to observe cardiomyocyte apoptosis. The TUNEL positive nuclei were counted by image analysis system “Image-Pro Plus Version 6.0”. The mean values of positive nuclei count/area were recorded [15].

Infarcted Segment Evaluation of S-CCTA and TTC Stain

MI of all the 17 segments according to standardized myocardial segmentation was evaluated by the two points scoring systems. (15) S-CCTA image analysis was performed by using dedicate visual evaluation on optimal energy images. For S-CCTA, score 0 indicated no hypo-perfusion, score 1 represented hypo-perfusion observed. For TTC stain, myocardium in tan color was classified as infarction (score 1) and viable myocardium in red color was regard as score 0 (Figure 1) [16].

biomedres-openaccess-journal-bjstr

Figure 1: Both S-CCTA
A. TTC Stain
B. Showed myocardial infarction (the 7th and 8th segments).

Statistical Analysis

The differences among or between groups were compared by using one-way Analysis of Variance (ANOVA) and Least Significant Difference (LSD) test or t test. The spectral curve of different regions was fitted by the best regression model on curve estimation provided by PASW. Accordingly, the value of slope was derived from the preferred curve. For comparing among different regions, the value of slope underwent logarithmic transformation [17]. The categorical inter-method agreement between S-CCTA and TTC stain was calculated by using the Cohen κ [16]. Receiver Operating Characteristic (ROC) curve was used to investigate the ability of S-CCTA on differentiating infarcted myocardial segments taking the TTC stain as gold standard. The correlation between IC and cardiomyocyte apoptosis of no-reflow region, infarction and remote myocardium was tested by Pearson correlation analysis. P<0.05 was considered statistically significant.

Results

Acute MI models were performed in 12 pigs. However, 2 of them died of ventricular fibrillation soon after PCI, 1 of them died during the S-CCTA examination. Finally, 9 Chinese mini-pigs (6 females; weight, 20.17±1.35kg; age, 5.3±0.6months) were included in current study. The heart rate during S-CCTA was 87±6 per minute. The radiation dose of S-CCTA was about 18.45mGy (CTDI), 193.70- 258.27 mGy.cm (DLP).

Spectral Curve

As the monochromatic energy increase from 40keV to 140keV, the CT value steadily decreased in remote myocardium, risk area and infarction (396.08~58.01HU, 61.30~344.62HU, and 86.80~36.90HU, respectively) (Figure 2). Significant differences of CT value among three regions at 40keV were observed (ps≤0.001). The exponential regression model was optimal for the spectral curve after comparison by curve estimation.16 The logarithmic transformed slopes of remote myocardium, risk area and infarct myocardium were 5.06±0.26, 4.71±0.27 and 2.78±1.03 respectively. Significant difference was detected among them (ps<0.001) (Figure 2).

biomedres-openaccess-journal-bjstr

Figure 2: The spectral curve of the mean attenuation density in the remote myocardium, risk area and infarction myocardium.

CT Value or IC Differences

The CT value or IC difference among infarction, risk area and remote myocardium was shown in Table 1 (ps<0.05).

biomedres-openaccess-journal-bjstr

Table 1: The CT Value, IC or slope of spectral curve difference among Infarction, Risk Area and Remote Myocardium.

Optimal Kev Setting for Differentiation

The scatterplot showed that 70keV ~75keV were the optimal monochromatic energies to observe infarction region on S-CCTA with higher CNR, SNR and lower noise (Figure 3). 70keV was chosen as the optimal monochromatic energy in our study for the following measurement and comparison. The 70 keV multiplanar reconstruction images could be overlaid with iodine density images; it could provide intuitionistic observation for the location and margin of MI (Figure 4).

biomedres-openaccess-journal-bjstr

Figure 3: The CNR, SNR and noise change from 40keV to 140keV.

biomedres-openaccess-journal-bjstr

Figure 4: A 7-month female pig with acute MI. The anterior and anterior septal hypo-perfusion was demonstrated in A. High voltage B. 70keV C. Iodine density D. 70keV overlay with iodine density images. D depicted the hypo-perfusion more clearly.

Diagnostic Ability of S-CCTA to Differentiate Infarct Segments

biomedres-openaccess-journal-bjstr

Figure 5: ROC curve of CT value obtained by S-CCTA to distinguish infarct segments from normal myocardial segments confirmed by TTC stain.

Categorical inter-method agreement between S-CCTA and TTC stain was almost perfect (κ= 0.821, 95%CI=0.729~0.913, p<0.001). 53 of 153 segments (34.64%) were considered as infarction on S-CCTA, compared with 64 segments (41.83 %) that were confirmed as infarcted on TTC stain (Table 2). ROC curve showed high diagnostic accuracy of S-CCTA to differentiate myocardial infarct segments (sensitivity, 0.813; specificity, 0.989; positive predictive value, 0.981; negative predictive value, 0.880 and accuracy, 0.901, p<0.001) (Figure 5).

biomedres-openaccess-journal-bjstr

Table 2: Inter-method agreement between S-CCTA and TTC stain.

Correlation Between IC and Apoptosis

The ICs of no-reflow region, infarction and remote myocardium were (1043±282) ug/cm3, (1867±344) ug/cm3, and (3507±331) ug/cm3 respectively. There were significant differences among them (p<0.001). The mean number of apoptosis cells in no-reflow region, infarction and remote myocardium were (2661±231)/mm2, (2270±241)/mm2 and (74±41) /mm2 respectively (Figure 6). There was significant difference among them (p<0.001). A significant inverse correlation was found between IC and cardiomyocyte apoptosis (r2=0.879, p<0.001).

biomedres-openaccess-journal-bjstr

Figure 6: Fused images of TUNEL stain and DAPI stain.
A. There were few normal cells (blue cell: indicated cells with normal double-stranded DNA) but a lot of cardiomyocyte apoptosis in the no-reflow region
B. There were abundant of apoptosis cells and normal cells in the infarction area.
C. A large number of normal cells but few apoptosis cells were detected in the remote myocardium.

Discussion

S-CCTA for Myocardial Infarction Assessment

The spectral CT may improve its ability to differentiate myocardial infarction from remote myocardium. This may be due to three following factors: its high CNR of higher photon energy (70keV) images, its higher photon energy (140keV) images and its ability to alleviate of beam hardening artifacts [8,11]. Previous studies have similar results with our study no matter for the myocardial perfusion or delayed enhancement [18,19] The 70keV images was selected as optimal monochromatic image to evaluate acute myocardial infarction due to with high CNR, SNR and low noise. And previous study also confirmed that The CT value of 70keV monochromatic image was similar to those of 120kVp CT images with lower noise [12,20].

IC and Cardiomyocyte Apoptosis

TUNEL stain aims to detect apoptotic cell and necrotic cell [21,22]. Apoptosis, necrosis and, possibly, autophagy determined the ultimate number of viable cardiomyocytes following MI [23,24]. Persistent ischemia without reperfusion eventually causes cardiomyocytes to die by a necrotic pathway. While, following myocardial ischemia/reperfusion, apoptosis is one of the major pathways that lead to the process of cell death [25]. In the current study, we correlated the IC of myocardium with TUNEL stain to investigate the capability of spectral CT to assess MI. The results showed that both the IC calculated on S-CCTA and cardiomyocyte apoptosis measured by TUNEL stain could differentiate no-reflow region from infarction or remote myocardium. Furthermore, the myocardial IC on S-CCTA correlated with the TUNEL stain of apoptosis cell in acute MI mini-pig models. Cardiomyocyte is permanent cell, no cardiomyocyte apoptosis detected in the normal heart. Previous study confirmed this point [26]. However, few apoptotic cells were found in the remote myocardium in our study which is probably caused by the overall ischemia situation. Significant difference of the number of cardiomyocyte apoptosis in no-reflow region and infarction area was detected.
No-reflow phenomenon reflects severe reperfusion injury. Ischemia/reperfusion injury (no-reflow phenomenon) initiates a wide and complex array of inflammatory responses that aggravate local injury [27-29]. It explains why apoptotic and necrotic cells was much more prominent in no-reflow region compared with infarction area without no-reflow phenomenon. The IC calculated on S-CCTA images reflected the myocardial perfusion and distribution of blood flow. Following ischemia/reperfusion, the higher the myocardial perfusion, the fewer the apoptotic and necrotic cells induced. This may be responsible for that the IC had a negative correlation with the TUNEL stain of apoptosis cell in acute MI. The iodine quantification on S-CCTA may add valuable information for risk stratification in the future.

Limitations

There were some limitations in the current study. First, the number of pigs enrolled was relatively small and with similar habitus, further study is necessary to determine whether our results are applicable to patients with larger and various figures. Second, this model only investigates early hypo perfused myocardial infarctions and the results cannot be extrapolated to later time points of infarct healing. Finally, for the mandatory use of prospective triggering and high heart rate of the pigs, we did not conduct the analysis including coronary artery stenosis and stress myocardial perfusion assessment.

Conclusion

S-CCTA could assess MI by CT value on 70keV images, IC and spectral curve. In addition, IC calculated on S-CCTA may indirectly reflect myocardial damage which could potentially add valuable information for risk stratification in the future.

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Evaluation of Myocardial Infarction in Pigs by Coronary Spectral CT Angiography

Introduction

Acute Myocardial Infarction (MI) is one of the major causes of morbidity and mortality in western countries [1]. Noninvasive imaging for MI detection, patient risk stratification and treatment monitoring is needed especially when Electrocardiography (ECG) and myocardial enzyme with no clinical clue. Multidetector CT has been used for heart morphology and coronary stenosis evaluation for many decades [2,3]. However, even mild coronary stenosis could not always exclude vessel-specific myocardial ischemia [4]. In addition, additional contrast material and radiation exposure limit the use of CT perfusion to evaluate myocardium [5]. In clinical practice, comprehensive evaluation of coronary artery stenosis and myocardial perfusion in one examination is desirable [6]. The advent of dual energy spectral CT has sparked renewed interest in clinical applications for many organs [7-10]. The strength of monochromatic energy image in improving image quality at optimal keV and increasing Contrast to Noise Ratio (CNR) at high keV was outlined; [11,12] the iodine density image was highlighted for the capability to quantify Iodine Concentration (IC) [11,13]. Consequently, our study aims to evaluate the myocardial infarction by Coronary Spectral CT Angiography (S-CCTA) based on the advantages mentioned above to discuss its strength, potential and limitations. The purpose of the current study was in three folds:
1. To investigate whether S-CCTA could assess MI
2. To define the best parameters of S-CCTA for MI delineation
3. To discuss the correlation between myocardial IC and cardiomyocyte apoptosis.

Methods

Experimental Protocol

Twelve Chinese mini pigs (5 males; weight, 20.23 ±1.23 kg; age, 6.0±0.7 months) underwent Percutaneous Coronary Intervention (PCI) to produce acute ischemia/reperfusion MI model by balloon dilatation. In compliance with the NIH guidelines for the use of laboratory animal, these mini pigs received human care. After premedication with ketamine (20 mg/kg), Xylazine Hydrochloride (1.5mg/kg) and atropine (0.02mg/kg), the pigs were intubated, anesthetized and mechanically ventilated with an admixture of 2.5%-3.5% sevoflurane and 100% oxygen. Baseline heart rate, ECG, and weight of animals were acquired before modeling. Before PCI, heparin was administered at an initial dose of 10,000IU intravenously, followed by additional 4,000IU/h to maintain anticoagulation throughout the PCI procedure [14]. The angioplasty balloon was positioned in the Left Anterior Descending Artery (LAD) just distal to the first diagonal artery. Acute MI was confirmed by ECG. After 90-minutes occlusion, the angioplasty balloon was drawn back.

Spectral CT

After 4±1 days of MI model establishment, the pigs underwent S-CCTA. Induced anesthesia was the same as above during PCI. Anesthesia was maintained by intravenous disoprofol (5mg/ kg/h). Respirator was used to help and control the breathing. All pigs underwent S-CCTA on a single-source dual energy spectral CT (Discovery CT750 HD CT Freedom Edition scanner, GE Healthcare, USA). The parameters were as following: Gemstone spectral imaging mode with fast peak tube voltage switching between 80 and 140 kVp during a single rotation, axial plane with 64 × 0.625 mm collimation, 350-msec gantry rotation time, 175-msec x-ray exposure time. All pigs received 1.5 ml/kg contrast material (Ultravist 370, Bayer Schering Pharma, Germany) followed by 30 ml saline at a flow rate of 3.0ml/s. Bolus tracking with a Region of Interest (ROI) was placed in ascending aorta and was used to synchronize the arrival of contrast material to start the image acquisition (trigger threshold of 120 HU). The radiation dose was recorded.

S-CCTA Images Processing

The monochromatic S-CCTA images were reconstructed in standard short axial plane with slice thickness of 2.00 mm. The density of infarction area (referred to the myocardium in tan or white color on TTC stain mentioned later), remote myocardium (myocardium in an unaffected coronary artery territory, usually the inferior wall) and the noise were measured. Identical ROIs on S-CCTA images of different energy were adopted by adjusting monochromatic energy from 40keV to 140 keV at a 5keV interval. The optimal keV was chosen based on the CNR, signal to noise ratio (SNR) and noise.

CNR = (HUremote – HUinfarct) / noise
SNR = HUinfarct / noise.

Where H Uremote represented the mean CT value of remote myocardium, and HUinfarct indicated the mean CT value of infarction. The noise was derived from the standard deviation of CT value in the remote myocardium.
In addition, the mean CT value of infarction area, risk area (the adjacent segments of infarction) and remote myocardium was recorded respectively to observe the density change from 40keV to 140 keV at a 5keV interval (spectral curve).
The optimal keV and iodine density images of S-CCTA were reconstructed in short axis. Thereafter, the CT value and IC were measured in the infarction region, risk area and remote myocardium of each pig. To maintain the consistency of the size, shape and position of ROIs among different CT images, the ROIs were automatically copied by the software and adjusted slightly by hand if necessary. Images were assessed in consensus by two experienced readers (A and B, with 5 and 11 years of experience in CCTA, respectively). For infarction observation, the CNR and SNR were calculated as mentioned above. Finally, objects with noreflow phenomenon at late enhancement imaging were recorded. The IC in no-reflow region (persistent hypo-enhancement on late enhancement images), infarction and remote myocardium was measured on S-CCTA.

Histopathology

At the conclusion of radiologic examinations, the animals were euthanized with overdose vecuronium bromide. The hearts were sliced into short axises of about 4mm and incubated in the triphenyl tetrazolium chloride (TTC) (1mg/100ml) at a temperature of 37℃ by water bath. Remote normal myocardium was delineated as the living tissue and is colored with red, while the infarcted tissue is colored in pale tan. With consideration of S-CCTA images and gross specimen, serial cutting sections in the no-reflow region, infarction and remote myocardium were used for immunofluorescent staining. Terminal deoxynucleotidyl transferase-mediated dUTP Nick-End Labeling (TUNEL) stain of myocardium was used to observe cardiomyocyte apoptosis. The TUNEL positive nuclei were counted by image analysis system “Image-Pro Plus Version 6.0”. The mean values of positive nuclei count/area were recorded [15].

Infarcted Segment Evaluation of S-CCTA and TTC Stain

MI of all the 17 segments according to standardized myocardial segmentation was evaluated by the two points scoring systems. (15) S-CCTA image analysis was performed by using dedicate visual evaluation on optimal energy images. For S-CCTA, score 0 indicated no hypo-perfusion, score 1 represented hypo-perfusion observed. For TTC stain, myocardium in tan color was classified as infarction (score 1) and viable myocardium in red color was regard as score 0 (Figure 1) [16].

biomedres-openaccess-journal-bjstr

Figure 1: Both S-CCTA
A. TTC Stain
B. Showed myocardial infarction (the 7th and 8th segments).

Statistical Analysis

The differences among or between groups were compared by using one-way Analysis of Variance (ANOVA) and Least Significant Difference (LSD) test or t test. The spectral curve of different regions was fitted by the best regression model on curve estimation provided by PASW. Accordingly, the value of slope was derived from the preferred curve. For comparing among different regions, the value of slope underwent logarithmic transformation [17]. The categorical inter-method agreement between S-CCTA and TTC stain was calculated by using the Cohen κ [16]. Receiver Operating Characteristic (ROC) curve was used to investigate the ability of S-CCTA on differentiating infarcted myocardial segments taking the TTC stain as gold standard. The correlation between IC and cardiomyocyte apoptosis of no-reflow region, infarction and remote myocardium was tested by Pearson correlation analysis. P<0.05 was considered statistically significant.

Results

Acute MI models were performed in 12 pigs. However, 2 of them died of ventricular fibrillation soon after PCI, 1 of them died during the S-CCTA examination. Finally, 9 Chinese mini-pigs (6 females; weight, 20.17±1.35kg; age, 5.3±0.6months) were included in current study. The heart rate during S-CCTA was 87±6 per minute. The radiation dose of S-CCTA was about 18.45mGy (CTDI), 193.70- 258.27 mGy.cm (DLP).

Spectral Curve

As the monochromatic energy increase from 40keV to 140keV, the CT value steadily decreased in remote myocardium, risk area and infarction (396.08~58.01HU, 61.30~344.62HU, and 86.80~36.90HU, respectively) (Figure 2). Significant differences of CT value among three regions at 40keV were observed (ps≤0.001). The exponential regression model was optimal for the spectral curve after comparison by curve estimation.16 The logarithmic transformed slopes of remote myocardium, risk area and infarct myocardium were 5.06±0.26, 4.71±0.27 and 2.78±1.03 respectively. Significant difference was detected among them (ps<0.001) (Figure 2).

biomedres-openaccess-journal-bjstr

Figure 2: The spectral curve of the mean attenuation density in the remote myocardium, risk area and infarction myocardium.

CT Value or IC Differences

The CT value or IC difference among infarction, risk area and remote myocardium was shown in Table 1 (ps<0.05).

biomedres-openaccess-journal-bjstr

Table 1: The CT Value, IC or slope of spectral curve difference among Infarction, Risk Area and Remote Myocardium.

Optimal Kev Setting for Differentiation

The scatterplot showed that 70keV ~75keV were the optimal monochromatic energies to observe infarction region on S-CCTA with higher CNR, SNR and lower noise (Figure 3). 70keV was chosen as the optimal monochromatic energy in our study for the following measurement and comparison. The 70 keV multiplanar reconstruction images could be overlaid with iodine density images; it could provide intuitionistic observation for the location and margin of MI (Figure 4).

biomedres-openaccess-journal-bjstr

Figure 3: The CNR, SNR and noise change from 40keV to 140keV.

biomedres-openaccess-journal-bjstr

Figure 4: A 7-month female pig with acute MI. The anterior and anterior septal hypo-perfusion was demonstrated in A. High voltage B. 70keV C. Iodine density D. 70keV overlay with iodine density images. D depicted the hypo-perfusion more clearly.

Diagnostic Ability of S-CCTA to Differentiate Infarct Segments

biomedres-openaccess-journal-bjstr

Figure 5: ROC curve of CT value obtained by S-CCTA to distinguish infarct segments from normal myocardial segments confirmed by TTC stain.

Categorical inter-method agreement between S-CCTA and TTC stain was almost perfect (κ= 0.821, 95%CI=0.729~0.913, p<0.001). 53 of 153 segments (34.64%) were considered as infarction on S-CCTA, compared with 64 segments (41.83 %) that were confirmed as infarcted on TTC stain (Table 2). ROC curve showed high diagnostic accuracy of S-CCTA to differentiate myocardial infarct segments (sensitivity, 0.813; specificity, 0.989; positive predictive value, 0.981; negative predictive value, 0.880 and accuracy, 0.901, p<0.001) (Figure 5).

biomedres-openaccess-journal-bjstr

Table 2: Inter-method agreement between S-CCTA and TTC stain.

Correlation Between IC and Apoptosis

The ICs of no-reflow region, infarction and remote myocardium were (1043±282) ug/cm3, (1867±344) ug/cm3, and (3507±331) ug/cm3 respectively. There were significant differences among them (p<0.001). The mean number of apoptosis cells in no-reflow region, infarction and remote myocardium were (2661±231)/mm2, (2270±241)/mm2 and (74±41) /mm2 respectively (Figure 6). There was significant difference among them (p<0.001). A significant inverse correlation was found between IC and cardiomyocyte apoptosis (r2=0.879, p<0.001).

biomedres-openaccess-journal-bjstr

Figure 6: Fused images of TUNEL stain and DAPI stain.
A. There were few normal cells (blue cell: indicated cells with normal double-stranded DNA) but a lot of cardiomyocyte apoptosis in the no-reflow region
B. There were abundant of apoptosis cells and normal cells in the infarction area.
C. A large number of normal cells but few apoptosis cells were detected in the remote myocardium.

Discussion

S-CCTA for Myocardial Infarction Assessment

The spectral CT may improve its ability to differentiate myocardial infarction from remote myocardium. This may be due to three following factors: its high CNR of higher photon energy (70keV) images, its higher photon energy (140keV) images and its ability to alleviate of beam hardening artifacts [8,11]. Previous studies have similar results with our study no matter for the myocardial perfusion or delayed enhancement [18,19] The 70keV images was selected as optimal monochromatic image to evaluate acute myocardial infarction due to with high CNR, SNR and low noise. And previous study also confirmed that The CT value of 70keV monochromatic image was similar to those of 120kVp CT images with lower noise [12,20].

IC and Cardiomyocyte Apoptosis

TUNEL stain aims to detect apoptotic cell and necrotic cell [21,22]. Apoptosis, necrosis and, possibly, autophagy determined the ultimate number of viable cardiomyocytes following MI [23,24]. Persistent ischemia without reperfusion eventually causes cardiomyocytes to die by a necrotic pathway. While, following myocardial ischemia/reperfusion, apoptosis is one of the major pathways that lead to the process of cell death [25]. In the current study, we correlated the IC of myocardium with TUNEL stain to investigate the capability of spectral CT to assess MI. The results showed that both the IC calculated on S-CCTA and cardiomyocyte apoptosis measured by TUNEL stain could differentiate no-reflow region from infarction or remote myocardium. Furthermore, the myocardial IC on S-CCTA correlated with the TUNEL stain of apoptosis cell in acute MI mini-pig models. Cardiomyocyte is permanent cell, no cardiomyocyte apoptosis detected in the normal heart. Previous study confirmed this point [26]. However, few apoptotic cells were found in the remote myocardium in our study which is probably caused by the overall ischemia situation. Significant difference of the number of cardiomyocyte apoptosis in no-reflow region and infarction area was detected.
No-reflow phenomenon reflects severe reperfusion injury. Ischemia/reperfusion injury (no-reflow phenomenon) initiates a wide and complex array of inflammatory responses that aggravate local injury [27-29]. It explains why apoptotic and necrotic cells was much more prominent in no-reflow region compared with infarction area without no-reflow phenomenon. The IC calculated on S-CCTA images reflected the myocardial perfusion and distribution of blood flow. Following ischemia/reperfusion, the higher the myocardial perfusion, the fewer the apoptotic and necrotic cells induced. This may be responsible for that the IC had a negative correlation with the TUNEL stain of apoptosis cell in acute MI. The iodine quantification on S-CCTA may add valuable information for risk stratification in the future.

Limitations

There were some limitations in the current study. First, the number of pigs enrolled was relatively small and with similar habitus, further study is necessary to determine whether our results are applicable to patients with larger and various figures. Second, this model only investigates early hypo perfused myocardial infarctions and the results cannot be extrapolated to later time points of infarct healing. Finally, for the mandatory use of prospective triggering and high heart rate of the pigs, we did not conduct the analysis including coronary artery stenosis and stress myocardial perfusion assessment.

Conclusion

S-CCTA could assess MI by CT value on 70keV images, IC and spectral curve. In addition, IC calculated on S-CCTA may indirectly reflect myocardial damage which could potentially add valuable information for risk stratification in the future.

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Open Access Journals on Medical Research

A Method for Automatic Control of the Process of Dynamic Settling of Oil Emulsion

Introduction

There is a known method for regulating the oil-water interface level using a capacitive sensor with an impact on the flow rate of discharged (drainage) water [1,2] The drawback of this method is that over time, asphalt-resinous substances and high-melting point oil paraffins accumulate on the surface of the sensing element, leading to a change in the gravity of the sensing element and, consequently, to a significant decrease in the accuracy of regulation. We know of a method and a system for automatic control of the oil-water interface level in the process of dynamic settling of oil emulsion [3], where the differential hydrostatic pressure between two specified points located in the upper and lower parts of the settler is measured. However, it has a significant accuracy error due to the expansion of the differential pressure gauge scale.

Problem Statement

Development of a more accurate and reliable method for measuring and regulating the oil and water interface level in settlers of a thermochemical treatment unit, in which dynamic settling of emulsified water droplets in an oil environment is carried out [4].

Solution

This goal is achieved by first taking samples of the upper pressure gauge and determining in laboratory conditions the density of water and oil, as well as the content of water and asphaltresinous substances in the intermediate emulsion layer (IEL) formed on the surface of the water cushion between the water and oil layers. Then, taking into account the measured values, the water cushion level is determined from the following formula:

where are the water cushion level in the settler, the height of lower pressure gauge and the distance between the gauges, respectively, cm; is the water level (water-IEL interface level) between the gauges, cm; are the density of water, oil and oil emulsion (OE), respectively, kg/cm3; is the differential hydrostatic pressure between the lower and the upper gauges, kg/cm2; are fractional content of water and asphaltenes, respectively.
Figure 1 shows a schematic diagram that interprets the operation of the method for automatic control of the process of dynamic settling of OE, which consists in regulating the water cushion level in the TCOTU settler. The method is implemented as follows. Via pipeline 1, OE with increased water concentration enters settler 2. The settled oil with a small (residual) water concentration is removed from the settler via pipeline 3. Pressure gauges 4 and 5 installed at points corresponding to the permissible maximum and minimum water cushion (WC) level, according to the TCOTU master production record, with differential pressure gauge 6 measure the differential hydrostatic pressure (DHP) between points 4 and 5. The output of DHP 6 is connected to the input of control and display unit (CDU) 7. In order to improve the accuracy of DHP measurement, gauge 5 is connected through the separation vessel 8 with the negative chamber of the differential pressure gauge in CDU 7, the values of , determined in laboratory conditions, are introduced. Sampling is carried out at point 9. In the CDU, the WC level is calculated from formulas (1)-(3).

biomedres-openaccess-journal-bjstr

Figure 1: Schematic diagram of automatic control of the process of dynamic settling of OE.

The obtained value from the CIB output goes to WC level regulator 10 connected to actuating mechanism (AM) 11 installed in the drainage water discharge line. In regulator 9, the signal received from unit 7 is compared with the regulator’s setpoint and, if the bias is upward, the flow rate of drainage water increases and vice versa.
Thus, the oil and water interface level (WC) is automatically determined by the differential hydrostatic pressure measured between points 4 and 5, taking into account the quality indicators of OE – .

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The Application of Nanobody in Non-invasive Imaging of Cancers

Introduction

Imaging is a useful and essential tool for making the correct clinical decisions for many diseases, including cancer. Many different imaging modalities have been developed ranging from conventional microscopy methods, aimed at single cells and multiphoton intravital microscopy, to non-invasive methods at the organismal level, such as single-photon emission computed tomography (SPECT), positron emission tomography (PET), magnetic resonance imaging, computed tomography (CT), bioluminescence and ultrasound imaging. The ability to image biological processes of a living animal and to diagnose signs of disease, have always been desirable goals. With the ongoing development of targeted therapies, it has become more and more important to visualize the presence tumor antigens and immune infiltrates to predict responsiveness [1].
There are several factors to be necessarily considered for designing a specific imaging agent. Once a tracer is injected into the blood stream, it must penetrate the tissue and then bind to its target. A tracer may accumulate in a tissue without binding specifically to its target. Furthermore, immunohistochemistry is needed to perform to confirm specificity and characterize the sensitivity of a tracer. Molecular imaging with labeled antibodies, extensively with labeled monoclonal Abs (mAbs), has been intensely explored, due to their particular characteristics such as high affinity and high specificity, and considered one of the best biomolecules applied for detection and targeting purposes. This can be useful for research, diagnostics, and therapeutic applications [2]. The application of antibodies in molecular imaging can help to overcome the challenge of specificity. Antibodies exist for many cell-surfaceavailable markers. Antibodies can detect cancer-specific markers and identify components of the tumor Extracellular Matrix (ECM) or tumor-infiltrating immune cells. Using radiolabeled antibodies and antibody fragments as imaging agents can be able to visualize and track location, movement and quantity of the target molecule, thereby showing insight into its dynamics.
However, antibodies’ difficult tissue penetration and longer serum half-life are strong obstacles in creating high-contrast images and cancer detection. The optimal non-invasive imaging agent would be able to penetrate tissues to allow rapid imaging after injection and show high specificity and sensitivity. The patient’s radiation exposure time should be minimized. Single domain Abs or commonly named nanobodies (Nbs), produced mainly in camelids such as llamas, alpacas, or camels, are only 15- kDa small size and improve the penetrability when compared with the performance of conventional mAbs (150 kDa) [3]. Moreover, Nbs own the characteristic of rapid renal clearance, avoiding toxicity effects [4]. One of the main advantages of obtaining Nbs by recombinant technology is that several tags can be fused in their tertiary structure such as His-tag or even fluorescent labels like the green fluorescent protein (GFP) [5]. Considering these characteristics, Nbs are particularly suited for targeting tumors and non-invasive imaging. Thus, Nbs form quite suitable candidates, ensuring minimal non-target retention to create a high tumor-tobackground ratio (T/B) shortly after administration.

Nanobody

Nbs, the single domain antigen-binding fragments obtained mainly from the Camellidae such as llamas, alpacas, or camels. Normally, IgGs are formed from four polypeptidechains comprising two light chains (L) and two heavy chains (H). These host animals have the ability to produce immunoglobulins which only contain the heavy chain (HcAb) and completely lack the light chain. The heavy chain is structured into two constant regions (CH2 and CH3), a long hinge region, and the Ag-binding domain VHH [6]. Specifically, VHH is formed from different regions, ones that are more conserved (FR) and others that are responsible for the specific recognition of the Ag, called complementary determining regions (CDRs) [7]. Nbs present three CDRs instead of six occurring in conventional Abs [8,9]. The one called CDR3, usually longer than the VH domains of mAbs, being the region that shows best degree of recognition [4] (Figure1). Nbs have numerous attractive advantages over conventional monoclonal antibodies (mAbs) [5-7] include small size (15 kDa), high stability, high solubility and specificity, ease of genetic design and excellent tissue penetration in vivo.

biomedres-openaccess-journal-bjstr

Figure 1: The comparison between mAb and HcAb.

The folding of CDR3 loop and the hydrophilic content of the framework-2 region keeps Nbs high solubility in aqueous solutions and lack of aggregation [10]. High thermal stability keeps Nbs full binding capacities for 1 week at 37℃ [11], and even completely reversible after long incubation periods at 90°C [12]. High tolerance against extreme pHs makes Nanobody great stability between pH 7.4 and [10,13] as well as in the presence of proteases [14]. The optimal biophysical and biochemical properties allow Nbs to be used for diagnostic purposes.

Recognition of Hidden Epitopes

Crystallographic studies of Nbs have revealed that in most cases the Ag-binding surface is clefts and cavities [15]. The lack of variable light chain (VL) is balanced with a VHH region that shows an extended CDR1 and a more exposed CDR3. These structural changes allow Nbs to bind planar surfaces and cavities, and also possibly bind the protruding loops or clefts [16]. Therefore, this feature of Nbs and their smaller size explain the ability of Nanobody to bind and neutralize targets that are notoriously difficult to hit with conventional Abs.

The Development and Production of Nbs

Obtaining libraries that contain the required genetic information is critical to produce Nbs with high specificity and affinity properties. At present, there are mainly three technologies for Agspecific Nbs’ preparation including immune, naïve, or synthetic libraries [9]. Immune libraries are the most common option for the development of Nbs, which requires an active immunization of Camelidae animals. Once the specific sequence is amplified from the extraction of mRNA from isolated lymphocytes and inserted in a cloning vector, the screening process is performed to isolate the most suitable Nbs by taking advantage of phage display technology, or using other methods like cell surface display and so on (Figure 2) [9]. However, phage display selection is the most commonly used strategy for this sort of screening, which is relatively fast to produce Nbs and has low cost, compared with the conventional polyclonal and monoclonal Abs.Nanobody selection based on naïve libraries takes advantage of the natural immunological diversity of the host animal without immunization [3,17]. Clearly, in any case, the success of this process depends on the amount of blood samples collected and it should be taken into account that only high specificity Abs can be obtained.

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Figure 2: The development of Nbs from immune libraries.

Other strategies from semisynthetic/ synthetic libraries are based mainly on randomly varying the corresponding CDR sequences to generate higher degree of diversity than when the protocol performed depends on naïve libraries. Therefore, these sorts of libraries are considered a promising alternative to the conventional method including immunization of animals. Regarding of Nanobody production, a wide range of different expression models can be used including organisms such as bacteria, yeast, fungi, insect cells, mammalian cells, or even plant hosts [18]. The most widely used expression system is Escherichia coli, which expresses proteins in different cellular compartments. The main advantage of working with this expression host is that it enables the production of soluble functional Nbs and that requires cheap protocols. Conversely, the yields are not very high compared with organisms such as yeast or fungi. Another usual way to produce Nbs uses mammalian cells.
This is the most suitable choice when Nbs are produced for therapeutic purposes, although their cost, long time requirements, and complex handling do not make them the first option. Other possible methods include the use of yeast and fungi, which have already been successfully applied, but the production process is still complex. Moreover, the fact that Nbs can be expressed in different organisms is an advantage with respect to conventional mAb production since it allows insertion of customized tags, production at low cost, and high production scale [2]. Unlike the mAbs production, which requires sophisticated machinery only found in eukaryotic systems and uses very large mammalian cell cultures and long screening and purification steps, leading to very expensive production costs, Nbs are a good alternative to solve the problem of mAb production costs. Nbs can be easily expressed in microbial systems such as bacteria, yeasts, fungi 9 and rapidly screening from display libraries. Moreover, using sequencing technologies, it is particularly easier for high-throughput screenings. All of these production and selection advantages result in lower manufacturing prices.

Introduction to Molecular Imaging Technologies

The focus on the diagnosis of tumor imaging is just critical, as the tumor’s antigen profiles obtained by visual imaging are essential to maximize therapeutic efficacy. A variety of imaging modalities are utilized in cancer diagnosis, and molecular imaging techniques have shown potential in improving existing techniques [1]. Mainly, there are two imaging techniques mentioned frequently, including nuclear imaging technique and the optical imaging technique. The nuclear techniques of PET and SPECT comprise the majority of molecular imaging studies due to the advantages of their high sensitivity, quantitative output, and clinical relevance. For tracking, Nbs are tagged with a positron-emitting nuclide (e.g., 18F, 68Ga, 89Zr) for PET, and gamma-emitting nuclides (e.g., 99mTc) are used for SPECT [1]. The optical imaging techniques, including ultrasound, quantum dots, and magnetic resonance imaging (MRI), have also been studied with Nbs. Nbs tagged with fluorescent dyes, offers the advantages of simplicity, flexibility, cost effectiveness, and safety, although the technique has weaker penetration.
Ultrasound imaging utilizes reflected sound waves from tissues, and Nbs have been tagged to contrast agents, microbubbles, and nanobubbles. Even though it is a comparatively safer technique, its applications are currently limited to systemic vasculature [19]. Quantum dots are fluorescent nanocrystals that have recently demonstrated tumor imaging potential for their superior stability, adaptable properties, and multiplex detection. However their low biocompatibility limited their current implementation. Nanobodyconjugated quantum dots targeting epidermal growth factor receptor vIII (EGFRvIII) [20], carcinoembryonic antigen (CEA) [21], and cytotoxic T lymphocyteantigen-4 (CTLA-4) 22 have achieved enhanced targeting with minimal toxicity in vivo [20,22]. MRI is a more expensive technique that utilizes strong magnetic fields to generate higher resolution images. Nbs coated magnetoliposomes [23], super paramagnetic nanoparticles [24], and fluorescent streptavidin [25] has paired with the technology for detecting ovarian tumors.

Imaging Cancer Biomarkers Against by Nbs

Currently, Nbs against cancer biomarkers, such as human epidermal growth factor receptor type 2 (HER2) are in clinical testing [26-28]. HER2, an oncogene that encodes a transmembrane tyrosine kinase receptor, is used as a classifier of invasive breast cancer and a major therapeutic target. HER2 is over expressed in 15-20% of patients with breast cancer [29-30]. Based on the success of a phase I clinical trial of a 68Ga-HER2 nanobody that could detect primary and metastatic tumors without adverse effects [31], the phase II clinical trial was performed. Notably, the HER2-CAIX combination synergistically enhanced the T/B ratio and could also detect lung metastases [32]. Nbs targeting other cancer biomarkers, such as a sepidermal growth factor receptor hepatocyte growth factor [33], carcinoembryonic antigen [34] and HER [35]. have been developed, radiolabeled and used in mouse models. Notably, vascular cell adhesion molecule-1 (VCAM-1) is a marker associated with metastasis and immune evasion, and anti- VCAM-1 nanobody microbubbles have been used for ultrasound imaging of murine carcinomas [19].
Additionally, 89Zr-HER3 [35], 18F-HER2 [36], and 68Ga-NOTACD20 [37] Nbs, 99mTc-EGFR [38] 99mTc-EGFR-cartilage oligomeric matrix protein (COMP) [39], 99mTc-dipeptidyl-peptidase-like protein 6 (DPP6) [40], 99mTc-mesothelin [41], and 131I-HER2 [42] nanobody probes have also demonstrated high T/B ratios. Additionally, anti-EGFR nanobody probes have been utilized in dual-isotope SPECT [43] and optical imaging 44, with an enhanced T/B ratio vs. mAb-based probes [44,45]. Other studies have assessed nanobody probes targeting immune checkpoints (ICP) CTLA-4 and programmed death ligand 1 (PDL1) [45] for nuclear imaging with high T/B ratios [46,47] have demonstrated success in various tumor models. An anti-human PD-L1 nanobody was developed for non-invasively imaging [48], which can detect PD-L1 in melanoma and breast tumors and showed high signal-to-noise ratios in tumors. Compared with immunohistochemistry, Wholebody noninvasive imaging of PD-L1, is likely to be more informative, which can provide visualization, localization and quantification of its expression throughout the body.
The studies published recently about a 99mTc-labeled anti- PD-L1 nanobody at an early phase I, showed that no drug-related adverse events were observed. Tumor images with good signalto- background ratios were obtained 2 h post injection and signal was mainly detected in the kidneys, spleen, liver and bone marrow [49]. Overall, Nbs have proved to be excellent imaging agents to assess the presence or absence of important cancer biomarkers on metastatic lesions and primary tumors according to the results shown from several preclinical [37,50,51] and early clinical imaging studies [31,52].

Outlook

While we have focused mainly on image a range of infectious diseases, Nbs, possessing the own advantageous physicochemical properties, such as the high tolerance of Nbs against extreme pHs, high temperatures and high concentrations of organic solvents have opened a wide range of applications for the detection of small molecule. Their nano-size enables enhanced tumor penetration and access to hidden and/or intracellular epitopes, their stability and manufacturing ease are favorable for large-scale production, and their superior paratope diversity allows an extensive arsenal for tumor antigen targeting. Nbs owning high sequence similarity with human VH domains 52 possess low immunogenicity and are appropriate for human administration. Combined with their size, structure, low agglutination, coupling efficiency, tissue penetrability and rapid renal clearance and no side effects, Nbs are a real desirable for imaging purposes. Nbs can overcome some of the limitations that first-generation Abs showed.
Using nanobody-based imaging probes has shown improved visualization compared to traditional mAb-based probes. For high affinity Nbs’ development, considering about the animal welfare, semisynthetic/synthetic libraries have been used for producing high affinity Nbs instead of the immune antibody library. However, there are still more requirement of rational and faster panning methods are applied to ensure the production of Nbs with the feature of high affinity and selectivity. With several Nbs having advanced to the clinic, and with FDA approval of one nanobodybased drug, in addition to imaging applications of Nbs, we forecast that, Nbs will be the leading actor to being developed as many innovative and high potential molecules for cancer immuneimaging and immunotherapy in the near future.

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Journals on Biomedical Research

Ultrasonic Frame of Reference for the Downward Displacement of the Posterior and Anterior Leaflets of the Tricuspid Valve in Children

Introduction

In downward displacement of the tricuspid valve [1], displacement of the septal and posterior valves is most common; however, the anterior tricuspid valve can also be moved downward [2]. During ultrasound, the root of the mitral valve in the apical fourchamber view of Ebstein malformation can be used as a reference structure for the downward movement deformity of the tricuspid septal valve [3]: however, there is a lack of reference structures for the downward movement deformity of the posterior and anterior tricuspid valves. Thus, the aim of this study was to evaluate the tricuspid annulus and inferior margin of the coronary sinus were as reference structures for posterior tricuspid valve downward movement. In addition, we aimed to evaluate two-chamber and four-chamber view tricuspid annulus as reference structures for anterior valve downward movement malformation, as well as exploring the reference structure for evaluating the degree of posterior and anterior tricuspid valve downward displacement.

Methods

Selection and Description of Patients

From May 2005 to April 2019, all children with tricuspid valve downward displacement diagnosed by echocardiography in our hospital were selected as study participants. Exclude children with unclear diagnosis. Of them, 18 were male and 24 female patients, aged between 7 months and 15.9 years, with a median age of 3 years and 10 months. Of the 42 total patients selected, 40 also exhibited atrial septal defect. Among them, two cases were further complicated with ventricular septal defect, one with pulmonary valve stenosis, one with ventricular septal defect and pulmonary valve stenosis, and one with single atrium and pulmonary valve stenosis. Two cases were not complicated with any congenital heart disease deformities. These complications are summarized in Table 1.

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Table 1: Clinical Information.

Note: ASD: atrial septal defect; VSD: ventricular septal defect; PS: stenosis of pulmonary valve; SA: single atrium

Research Methods

Research Methods

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Note: RV right ventricle; RA right atrium

Figure 1: The apical four-chamber view showing that the arrow points to the front of the tricuspid valve annulus. A is the attachment point of the anterior tricuspid valve on the annulus. In the picture, the anterior tricuspid valve is attached to the anterior part of the tricuspid annulus, and the position is normal.

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Table 2: Number of patients with different leaflets moving.

Note: SV: Septal valve; PV: Posterior valve; AV: Anterior valve.

Ethical approval was granted by the medical ethical committee of The First Affiliated Hospital of Gannan Medical University with the following reference number: LLSC-202105401. All study participants provided oral informed consent. The tricuspid septal and anterior valves were displayed by echocardiography in the apical four-chamber section, using the ultrasonic examination instrument Philips EPIQ7C and GE VIVID7 (Philips, Amsterdam, Netherlands), with a probe frequency of 3–8 MHz. The descending degree of the tricuspid septal valve was evaluated according to the position of the anterior mitral valve attached to the intracardiac septum, and the size of the atriated right ventricle and right ventricular cavity was observed. The apical four-chamber probe was then rotated about 45° clockwise to make the left atrium, left ventricle, and interventricular septum disappear gradually, exposing the right atrium, right ventricle, and right ventricular posterior wall. The shape, activity and position of the anterior valve of the anterior wall of the right ventricle and the posterior valve of the posterior wall of the right ventricle were observed, the tricuspid annulus and coronary sinus were displayed, and the distance between the attachment point of the posterior tricuspid valve, the inferior edge of the tricuspid annulus, and the inferior edge of the coronary sinus was measured. The apical four-chamber and right cardiac two-chamber views were used to evaluate the downward movement of the anterior tricuspid valve using the tricuspid annulus as the reference structure (Figure 1). The location of tricuspid regurgitation was revealed by color doppler ultrasound (Table 2).

Results

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Note: ATV the anterior tricuspid leaflet; STV the septal tricuspid leaflet; RV right ventricle; RA right atrium; LV left ventricle; LA left atrium

Figure 2: The apical four-chamber view showing that the position of the septal leaflet of the tricuspid valve is significantly lower than the root of the mitral valve during systole.

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Note: ATV the anterior tricuspid leaflet; STV the septal tricuspid leaflet; RV right ventricle; RA right atrium; LV left ventricle; LA left atrium.

Figure 3: The apical four-chamber view showing that the position of the septal leaflet of tricuspid valve is significantly lower than the root of the mitral valve during diastole.

In 42 patients with Ebstein malformation, the septal and posterior valves moved downward simultaneously in 39 patients; the simple septal valve moved downward in one case; the posterior and anterior valve moved downward at the same time in one case; and the septal, posterior, and anterior valves moved downward simultaneously in one case. Aside from two patients with tricuspid septal and posterior valve downward movement and partial slight downward movement of anterior valve, ultrasound was consistent with the results of operation. Ultrasound showed downward displacement of the tricuspid septal valve in 41 patients (Figures 2 & 3). Aside from two patients in which downward movement of the tricuspid septal valve was so severe that the distance between the tricuspid valve and the root of the mitral valve could not be measured, the attachment point of the tricuspid septal valve was 2.22 ±1.11 cm from the root of the mitral valve in 39 patients.

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Note: PTV posterior tricuspid leaflet; ATV anterior tricuspid leafle;ARV atrialized right ventricle ;RV right ventricle ;RA right atrium

Figure 4: Apical right heart two-chamber view showing that the posterior tricuspid leaflet moves down from the tricuspid annulus and lower edge of the coronary sinus to the apex. The position of the anterior tricuspid leaflet is normal, and the leaflet is elongated.

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Note: PTV posterior tricuspid leaflet; ATV anterior tricuspid leaflet; ARV atrialized right ventricle; RV right ventricle; RA right atrium

Figure 5: Right ventricular inflow tract view showing that the posterior tricuspid leaflet moves down from lower edge of the coronary sinus to the apex. The position of the anterior tricuspid leaflet is normal, and the leaflet is elongated.

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Note: ATV the anterior tricuspid leaflet; STV the septal tricuspid leaflet; RV right ventricle; RA right atrium; LV left ventricle; LA left atrium

Figure 6: The apical four-chamber view showing that the anterior tricuspid leaflet moves down significantly, and the position of the root of the septal tricuspid leaflet is normal.

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Note: PTV posterior tricuspid leaflet; ATV anterior tricuspid leaflet; ARV atrialized right ventricle; RV right ventricle; RA right atrium

Figure 7: Apical right heart two-chamber view showing downward motility of the anterior tricuspid leaflet and the posterior tricuspid leaflet positions.

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Table 3: Distance of downward movement of different valves.

Note: SV: Septal valve; PV: Posterior valve; AV: Anterior valve.

Ultrasound also showed downward movement of the posterior tricuspid valve in 41 patients with reference to the tricuspid annulus or the inferior edge of the coronary sinus in the view of the inflow tract of the right ventricle and the right ventricle (Figures 4 & 5). Aside from the severe downward movement of the posterior tricuspid valve reaching the cardiac apex that could not be measured in three patients, the distance between the root of the posterior tricuspid valve and the inferior edge of the tricuspid annulus or inferior margin of coronary sinus was 2.71 ±1.08 cm in 38 patients. In one patient, downward movement of the anterior and posterior tricuspid valves, was confirmed by both operation and ultrasound. Ultrasound showed that the position of the anterior tricuspid valve had moved downward in the apical four-chamber section (Figure 6), which was 2.2 cm away from the tricuspid annulus, and that the anterior tricuspid valve was attached to the anterior wall of the right ventricle. The two-chamber view of the apical right heart showed the downward movement of the posterior tricuspid valve (Figure 7), and the distance from the tricuspid annulus was 1.3 cm (Table 3).

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Note: ATV the anterior tricuspid leaflet; STV the septal tricuspid leaflet; RV right ventricle; RA right atrium

Figure 8: Color Doppler showing that the orifice position of the tricuspid regurgitation (blue shunt) has downward direction, and that the direction of the TR flow has an anterolateral bias in patients with downward displacement of the anterior leaflet in the apical four-chamber view.

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Note: ATV the anterior tricuspid leaflet; STV the septal tricuspid leaflet; RV right ventricle; RA right atrium; LV left ventricle; LA left atrium

Figure 9: Color Doppler in the apical four-chamber view showing that the orifice position of the tricuspid regurgitation (blue shunt) has significant downward motility.

The right atrium was significantly enlarged, the atriated right ventricle was located on the anterolateral side, and the anterior lobe of the tricuspid valve was not obviously lengthened. The apical four-chamber section showed that the tricuspid septal valve was in the exact position. Color Doppler confirmed that the position and direction of the tricuspid regurgitation orifice had shifted to the anterolateral side of the anterior tricuspid valve (Figure 8). Color Doppler ultrasound showed that the position of the tricuspid regurgitation orifice was significantly decreased in 42 patients (Figure 9).

Discussion

In the apical four-chamber ultrasound section, the downward movement of the anterior tricuspid valve shows that the septal valve of the tricuspid valve moves away from the root of the mitral valve, but the reference structure of the posterior and anterior tricuspid valve is rarely reported. All the three valvular lobes of tricuspid valve are attached to tricuspid annulus, while the tricuspid annulus is a cardiac fibrous scaffold structure composed of dense connective tissue. Ultrasound shows hyperechoic light band, and the coronary sinus is located above the tricuspid annulus [4]. The posterior tricuspid valve is distant from the tricuspid annulus in the two-chamber view of the apical right heart [5]. In this study, in 40 children with posterior tricuspid valve displacement, the downward movement of the tricuspid valve was evaluated by ultrasound in the view of the apical right cardiac chamber and the inflow tract of the right ventricle, with the tricuspid annulus and the inferior edge of the coronary sinus as reference structures. Results were confirmed by operation. It is suggested that the downward displacement of the posterior tricuspid valve can be well evaluated by taking the tricuspid annulus and the inferior edge of the coronary sinus as reference structures in the two- chamber view of the right portion of the apical heart.
Downward movement of the anterior tricuspid valve is rare. When the anterior tricuspid valve moves downward, the anterior tricuspid valve is distant from the tricuspid annulus. Ultrasound shows that the tricuspid annulus has a strong echo band, which can show the downward movement of the anterior tricuspid valve. Of the two children with anterior tricuspid valve displacement in this study, one case showed that the anterior tricuspid valve moved away from the tricuspid annulus in the apical four-chamber and two-chamber views of the right heart. The ultrasonic diagnosis of the downward displacement of the anterior tricuspid valve was consistent with the results of the operation. In the other case, the downward movement of the septal and posterior tricuspid valves was diagnosed by ultrasound, and it was found that in addition to the downward movement of the septal and posterior tricuspid valves, there was also a slight downward movement of the anterior valve, which may have resulted from the large area and three-dimensional structure of the anterior tricuspid valve. Among them, part of the slight downward movement of the structure was not related to the change of hemodynamics. The position of the tricuspid annulus attached to the anterior tricuspid valve is sometimes difficult to display. Ultrasound can be expected to show a strong echo light band between the right atrium and the right ventricle from multiple angles, such as the apical four-chamber section, the right cardiac two-chamber section and the right ventricular inflow tract. Detailed attention is required to observe whether the anterior tricuspid valve is attached to the anterior position of the tricuspid annulus. In this study, the ultrasound of one patient with downward displacement of the anterior tricuspid valve showed that there was a hyperechoic light mass in the anterior tricuspid valve attached to the anterior wall of the right ventricle on the apical four-chamber section, which may have been caused by the myocardial echo contrast of the implantation of the root of the anterior tricuspid valve into the anterior wall of the right ventricle. It is suggested that the implantation a of strong echo light mass at the root of the anterior tricuspid valve into the anterior wall of the right ventricle may have been a sign of the downward movement of the anterior tricuspid valve in the apical four-chamber section.
The area of the anterior tricuspid valve was the largest among the three valves, and it was semicircular, accounting for 2/3 of the function. The hemodynamic changes of the downward movement of the anterior tricuspid valve were significantly greater than those of the other two valves, the right atrium was significantly enlarged, the atrial right ventricular wall had become thinner, and cardiac function was poor. Obvious enlargement of the right atrium must sometimes be distinguished from the right atrial aneurysm when the tricuspid annulus and the anterior tricuspid valve move downward at the same time. When the anterior tricuspid valve moved downward in this study, the position of the tricuspid annulus was normal, and only the anterior tricuspid valve moved downwards. The strong echo light mass at the root of the anterior tricuspid valve was implanted into the anterior wall of the right ventricle, and color Doppler showed that the direction of tricuspid regurgitation shifted to the anterolateral direction. This may also have been a sign of downward movement of the anterior tricuspid valve.
The downward movement of the tricuspid valve is more common with downward movement of the tricuspid septal valve, posterior valve, and the lengthy and increased amplitude of the anterior tricuspid valve [6,7]. It is rare that the position of anterior and posterior tricuspid valve is downward, and the position of septal valve is normal, but no matter which valve moves downward, the position of tricuspid annulus remains unchanged, and the position of tricuspid opening remains far away from tricuspid annulus. In this paper, 42 patients with tricuspid regurgitation were shown by color doppler ultrasonography, which suggested that the downward position of tricuspid regurgitation was an important sign in the diagnosis of tricuspid regurgitation.

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Journals on Medical Microbiology

Cognition and Omega-3 Fatty Acids: A Narrative Review of the Literature

Introduction

Today we know quite well that a healthy lifestyle that includes physical activity, healthy eating or moderating toxic habits such as alcohol and tobacco are associated with a lower risk of suffering from some diseases such as cardiovascular diseases, some types of neoplasms, and neurodegenerative diseases in general, and particularly cognitive impairment or dementia. The dietary factor is perhaps the most complex, and much research is aimed at elucidating which foods are associated with this beneficial effect and why. To this respect, one of the most implicated actors in this positive effect has shown to be polyunsaturated fatty acids, especially those belonging to the omega-3 group. Fatty acids are biomolecules consisting of a linear hydrocarbon chain of variable length, with a carboxyl group (-COOH) at one end and a methyl group (-CH3) at the other. The carbon atoms in the chain are joined by single or double covalent bonds. The absence of double bonds defines the acid as saturated, while the presence of one double bond in the chain defines it as monounsaturated acid, and the presence of multiple double bonds as polyunsaturated. Polyunsaturated fatty acids are known by their acronym PUFAs (Poly Unsaturated Fatty Acids). Omega-3 fatty acids (ω-3), together with omega-6 fatty acids (ω-6), make up the group of so-called essential fatty acids, which owe their name to the fact that they are essential for the body since the body is not capable of producing them on its own and must acquire them from foods that contain them. Whether a fatty acid is referred to as omega-3 or omega-6 is established by the location of the first double bond from the methyl-terminal end. In omega-3s, the double bond is at carbon 3 [C3-C4] and can also be identified as n-3. In omega-6, the double bond is at carbon 6 (C6-C7) and is also known as n-6.
Excessive amounts of omega-6 polyunsaturated fatty acids (PUFA), marked by an increased dietary high omega-6/omega-3 ratio, as is increasingly common in current Western diets, are currently speculated to promote the pathogenesis of many diseases, including cardiovascular, cancer, inflammatory and autoimmune diseases [1]. In contrast, omega-3 fatty acids have been shown to play an important role in altering blood lipid profiles and membrane lipid composition and affecting eicosanoid biosynthesis, cell signaling cascades and gene expression, which positively influences health status. This effect seems to have been proven in cardiovascular diseases [atrial fibrillation, atherosclerosis, thrombosis, inflammation, and heart disease, among others], diabetes, cancer, depression, or autoimmune diseases (e.g., rheumatoid arthritis). Its beneficial influence on brain function in the diet of pregnant and lactating women has also been studied [2,3]. The first evidence of this beneficial effect was provided by epidemiological studies which revealed that the traditional Greenlandic diet, rich in marine mammals and fish, reduced the incidence of cardiovascular disease in both the Inuit population and in the Danish people who immigrated to these latitudes, belonging to a different ethnic group [4]. There is currently a tendency in today’s diets to over-consume omega-6 in relation to omega-3 due to the high consumption of vegetable oils by Western society, which means that this ratio can be as high as 20:1, very different from the current recommendations which advise that the omega-6/omega-3 acid ratio should be approximately 4:1, as was the case until the beginning of the 20th century. We could hypothesize that diets rich in omega-3 polyunsaturated fatty acids would be beneficial for the functioning of neural structures, since one out of three fatty acids in the central nervous system are long-chain polyunsaturated fatty acids, and it could be thought that an inadequate balance between these (ω-6/ω-3) would lead to neuropsychological alterations [5].
Omega-3 PUFAs originate mainly from the marine environment or the vegetal kingdom and include α-linolenic acid (ALA; 18:3 ω-3), stearidonic acid (SDA; 18: 4 ω-3), eicosapentaenoic acid (EPA; 20:5 ω-3), docosapentaenoic acid (DPA; 22:5 ω-3) and docosahexaenoic acid (DHA; 22:6 ω-3). Some plant seeds, such as flax, chia and canola seeds, are good sources of ALA, which serves as precursor for the synthesis of other long-chain PUFAs (see Figure 1) in the human body such as DHA or EPA. However, the production of longchain ω-3 PUFA from ALA is very limited, with conversion rates of around 5% through this metabolism. Therefore, the best sources of PUFA ω-3 are those of marine origin. Long-chain ω-3s, such as EPA and DHA, are found in the lipids of fatty fish; in the fat tissue of marine mammals; in algae and marine fungi; as well as small crustaceans that are part of krill. The bioavailability of ω-3 PUFAs is also influenced by the organic structure of the ingested form from the diet (see Figure 2), whether ethyl ester (EE), triacylglycerol (TAG) or phospholipids (PL). The most common form of lipids in nature is TAG, which has the best bioavailability compared with EE. PLs are rare in nature and data on their bioavailability are limited and inconclusive [4]. For these reasons, most supplements are made from fish oils or other marine organisms such as krill, as the bioavailability of these products is higher than with vegetal origin products, in addition to the higher proportion of long-chain omega-3 PUFA (DHA or EPA) of higher biological value [6].

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Figure 1: Metabolism of ω-3 PUFAs.

The ω-3 PUFAs appear to exert a very important action on neuron membranes, especially in the synaptic regions of neurons (and to a greater extent, in areas of grey matter), where they accumulate in greater proportion and are essential components of the phospholipid membrane, so their importance is vital for the stability of the dynamic structure and functional activity of neurons, as they can alter the fluidity of the lipid membrane (displacing cholesterol from it) and promote synaptic plasticity, which is essential for learning, memory and other cognitive processes. They also act as sources of communication for second messengers between neurons, enhance the coupling of G-proteins involved in many signal transduction pathways and are involved in direct lipid-related transcription functions. DHA, one of the most important and final products of their metabolism (see Figure 1), constitutes more than 90% of the ω-3 and 10% to 20% of the total lipids in the brain. It is mainly incorporated into phosphatidylethanolamine, phosphatidylserine and, in smaller amounts, into phosphatidylcholine in synaptic terminals, mitochondria and endoplasmic reticulum. In fact, DHA is able to modulate cellular properties and physiological processes such as membrane fluidity, neurotransmitter release, gene expression, myelination, neuroinflammation and neuronal growth [6].

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Figure 2: Structure of lipids.

The purpose of this review is to examine the current evidence on the relationship between dietary omega-3 supplementation and improved cognition or prevention of cognition-related diseases such as Alzheimer’s disease (AD). To this end, we will discuss the evidence both for and against, in preclinical experiments as well as in clinical studies in humans, including normal ageing, subjective memory or cognitive complaints, mild cognitive impairment, and even AD.

Preclinical Data

The potential effect of omega-3 fatty acids on cognition has been studied in multiple experimental preclinical studies in animals, both in models of normal ageing and in models of AD. The most common model of normal animal ageing is the canine model. Dogs are capable of developing age-related cognitive decline similar to that found in humans and their diet is similar to ours [7]. They can therefore be used to study the potential effects of nutritional supplements in controlled settings. Most studies use nutritional supplements that combine different types of omega-3s, as well as amino acids and antioxidants. The results obtained in these studies support the hypothesis that these supplements would have a beneficial effect on cognition and learning in older animals [7-9], although there are some studies that do not find these benefits [10].
AD models are usually conducted in rodents. These often consist of transgenic strains with mutations that predispose to developing the disease and have been used extensively in AD treatment research. Most studies of omega-3 supplementation in these animal models have shown benefits on delayed cognitive impairment [11], cognitive decline, behavioral symptoms [12], and have even shown to reduce beta-amyloid deposits [12]. An interesting study used transgenic mice for amyloid precursor protein (APP)-animal model for AD-, compared with others that carry the same pathogenic mutation together as well as another mutation that induces the passage of endogenous omega-6 to omega-3, obtaining that the latter showed a lower progression of cognitive and behavioral symptoms compared to the former, displaying the potential protective effect of omega-3 versus omega-6 [13]. This beneficial effect of omega-3s is also observed in other studies in a murine model of epilepsy [14]. In contrast, other studies have not found this beneficial effect in transgenic mice, although they do find it in normal mice [15].

Clinical Evidence

Normal Ageing

We have found and reviewed 13 randomized trials examining the effects of omega-3 dietary supplements in healthy elderly subjects, looking at their cognitive performance. Most used DHA supplements, sometimes combined with EPA, with a daily dose ranging from 350 mg to 3,000 mg of DHA obtained from fish oil in most cases, and in one case from krill [16]. Cognitive performance was measured using a cognitive assessment protocol that typically includes tests of memory, attention, working memory, verbal fluency, and processing speed [17]. The results of most of them showed significant differences when assessing the cognitive effects of omega-3s [16,18-23]. One of the studies used the measurement of P300 evoked potentials as the primary endpoint, resulting in an improvement in the omega-3 treated group [24].
In other studies, on the contrary, no significant differences were found in the parameters evaluated, although most of them used lower daily doses (200-300 mg DHA), compared to the previous studies [25-27], or otherwise the sample size was small [28], which could explain this difference in results. The exception is the study by Danthiir et al. which used high doses (1,720 mg DHA) and only obtained a slight tendency towards improvement in some of the evaluated endpoints [29]. In addition to the above, mention could be made of the Spanish WAHA study, which did not examine the direct effect of omega-3 supplements but studied the effect of a dietary supplement with walnuts in a population-based cohort. Although there were no significant results in cognitive variables after two years of intervention, improvements in functional networks mediated by functional magnetic resonance imaging (fMRI) during working memory tasks were shown [30].

Subjective Memory Complaints

Studies related to subjective cognitive, or memory complaints are scarce, perhaps because the concept is more difficult to categorize or define than normal ageing or the “classic” mild cognitive impairment or dementia. By subjective memory complaints (SMC) we mean individuals who present a subjective perception of poor cognitive performance in general [and memory in particular] but which present a neuropsychological examination within the normal range. It has been established that this altered perception of one’s own cognition could be caused by the onset of a very incipient cognitive impairment which eventually cannot be detected in neuropsychological tests, or by a poor estimation of one’s own abilities (meta-cognition) due to executive dysfunction [31].
The main study in this subgroup of subjects is the MAPT study which randomized French elderly people to multi-domain intervention groups [cognitive stimulation, physical activity, and nutrition], to omega-3 supplementation or both interventions versus placebo. This study found no improvement in patients who underwent intervention [32]. Anyway, a subgroup analysis subsequently found that omega-3 supplementation would be partially beneficial in those subjects who had low baseline omega-3 levels [33]. Another ambitious trial in this population subset is the PONDER study, for which no results have yet been obtained [34]. Otherwise, some studies have found objective improvements on cognitive performance or in regional blood flow measured by fMRI in the posterior cingulum [35-37].
The study by Yurko-Mauro et al. in patients with memory complaints who met criteria for “age-related cognitive decline” has been considered as positive. This is a randomized, doubleblind, placebo-controlled clinical trial in which 900 mg per day of DHA (n=242) or placebo (n=243) was administered to individuals with memory complaints of average age 70 years for a period of 6 months, showing statistically significant differences in favor of DHA administration in validated cognitive tests frequently used for the assessment of memory and learning ability. The authors concluded that DHA supplementation at a dose of 900 mg/day improved memory and learning capacity in individuals with SMC [35]. In addition, there is a meta-analysis evaluating the results of 15 clinical studies, most of them observational, which concluded that DHA/EPA supplementation has a beneficial effect on memory in adult individuals. The review concludes that episodic memory tests of adults with SMC were significantly improved (p<0.004) with DHA/EPA supplementation. Furthermore, and regardless of cognitive status at baseline, DHA/EPA supplementation [at doses >1 g per day] was able to improve episodic memory (p<0.04). Changes in semantic and working memory from baseline were significant with DHA, but no differences between groups were detected [38].

Mild Cognitive Impairment

Mild cognitive impairment [(MCI) is probably the main risk factor for developing dementia, and specifically amnestic MCI (the most prevalent entity) is the most important risk factor for the development of AD. For this reason, preventive treatments have become a relevant source of study in patients with MCI, as they could have a potential role in the prevention of AD. Currently, there is no drug approved for use in MCI, but some strategies such as cognitive stimulation, physical exercise, and dietary recommendations, including omega-3 PUFAs, are under study. Randomized clinical trials that have studied the effect of omega-3 PUFA in patients with MCI have been reviewed. Most studies showed improvements in scores on working memory tests, as well as episodic memory [39- 41], although there are others that evaluated test scores (FSIQ -Full Scale Intelligence Quotient- and WAIS -Wechsler Adult Intelligence Scale), or even depressive symptom scales (GDS -Geriatric Depression Scale) [42,43], which concluded with positive results in favor of omega-3 supplementation. All the mentioned studies above followed patients for 6 months to one year, while studies with shorter follow-up did not seem to obtain statistically significant differences [44].
Additionally, there have been several published meta-analyses and systematic reviews on the effect of omega-3s in patients with MCI. The overall conclusion of all of them points to the beneficial effect of omega-3 PUFA in MCI patients [45,46]. The most recent meta-analysis of 25 studies (n=787) indicated that omega-3s appear to have no effect on overall cognitive function (Hedge’s g= 0.02; 95% confidence interval= -0.12 to 0.154), although it may have a beneficial effect on memory (Hedge’s g=0.31; p=0.003; z=2.945) [46]. Another meta-analysis by Zhang et al. analysed all studies in which the MMSE (Mini Mental State Examination) was used for the assessment of these patients treated with DHA/EPA supplements and concluded that the treatment seems to statistically decrease the rate of cognitive decline in terms of MMSE score (WMD=0.15 (0.05-0.25); p=0.003), so the hypothesis that omega-3 could help to prevent global cognitive decline -in addition to memory- in elderly people with MCI seems to be supported [47].
Neuroimaging research has looked at the effect that omega-3 PUFA may have on the brain in patients with MCI. One of these studies used fMRI to study regional blood flow. It appeared that treatment with omega-3 for 26 weeks is able to increase blood flow in posterior cortical areas, typically affected in MCI [48]. Another study analysed the evolution of brain volume measured by MRI in patients with MCI with results showing that, after treatment with DHA supplementation for one year, the hippocampal volume measured by brain MRI was larger in treated patients than in those receiving placebo [49].

Alzheimer´s Disease

There have been numerous studies on omega-3 PUFA in AD with overall slightly favorable results. In this context, multiple randomized, double-blind, placebo-controlled trials have been conducted, some of which have had great scientific impact. So far, no treatment has been found that is able to significantly improve AD, so most studies are trying to elucidate whether treatments are able to reduce the degree of disease progression. One of the first studies that attempted to address the effect of omega-3s on the progression of AD was the Omega AD study. In this study, patients were treated with high-dose DHA and EPA (1.7 g DHA + 0.6 g EPA) versus placebo, with omega-3 supplementation in both groups continuing for a further period from 6 months to 1 year. The main publication derived from the research showed a tendency for the treatment group to have less disease progression at the cognitive level [measured by MMSE and ADAS-Cog -Alzheimer´s Disease Assessment Scale-Cognitive], although significant differences were only obtained in the mildest AD subgroup [50]. In this trial, a better outcome in patients with higher plasma levels of omega-3 was found [51] and also in those with higher homocysteine levels, which the authors relate to a hypothetical synergistic effect with B vitamins [52]. The same study attempted to assess the efficacy on behavioral symptoms without improvement, with the exception of depressive symptoms, a finding that has been observed in other settings of cognitive impairment [53]. This beneficial effect of omega-3s has been replicated in other clinical trials using similar doses of DHA for 6-12 months with improvements in cognitive scales such as the ADAS-Cog [54–56].
On the other hand, some studies have found no significant differences in terms of cognitive assessment [44]. The most relevant is that of Quinn et al., concluding that treatment with DHA (2 g/day) for 18 months did not produce a relevant cognitive effect as measured by MMSE and subscales of the ADAS-Cog [57]. Studies related to some complex nutrient formulations with high doses of omega-3 PUFA in their composition [1200 mg DHA + 300 mg EPA per 125 ml] are worth mentioning. Some studies with these products have shown beneficial effects of daily treatment. The primary endpoint of the trial was the change in memory as measured by the NTB [Neuropsychological Test Battery] Z-score at 24 weeks of treatment. The study showed statistically significant differences in NTB score in favor of the treated arm [p=0.023; Cohen’s d=0.21; 95% confidence interval (-0.06 – 0.9)) [58,59].

Conclusion

Although evidence is not conclusive, several investigations support that supplementation with omega-3 PUFA, especially DHA, may have beneficial effects on cognition in healthy older adults with SMC and even in patients with MCI or AD. These effects should generally appear at high daily doses [800-900 mg and above] and over long periods of time (6 months or longer). This protective effect has not been shown in all studies, so it is possible that the results may be influenced by other variables; in this sense, it is important to mention that ensuring adequate long-term adherence to treatment [at least 6-12 months] may be important to establish a possible objective benefit. Anyway, more soundly designed interventional clinical trials are needed to ascertain relevant issues concerning dose and supplementation duration. It has been found that plasma omega-3 values or homocysteine levels may be related with these discrepancies among different studies, with subjects with lower plasma omega-3 and homocysteine levels benefiting more from this effect. In any case, these supplements appear to have no relevant adverse effects and may have other added benefits such as reduced cardiovascular risk. Moreover, their indication is not at odds with other recommendations such as cognitive stimulation and physical exercise, so that, altogether, these measures can be medically recommended.

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Open Access Journals on Life sciences

A Review on the End-of-Life Care

Background to the Study

There is a huge distinction between death occurrences and believes in the past and that of the present. The level of death occurrence in the present is increasing compared to the past, and it is influenced by population increase, globalization, global warming, internet penetration, and various discoveries. According to Carlet et al (2004), death is frequently a private, religious or spiritual event with a group of family members and friends gathered around the victim. However, as death becomes a topic of public concern and more technical procedures are introduced to avoid or facilitate dying, the picture of death is changing [1]. End-of-life care for families and their patients has been a hot topic for the past two to three decades. This could be in consequence of a variety of factors, such as the shifting away from a solely medical perspective and toward a more holistic approach to caring. [2], for example, said that the early 1970s saw a transition away from a strictly biomedical survival perspective and toward a dual perspective. This includes focusing care on saving lives and enhancing medical identities, as well as taking care of family members and developing numerous social identities. Supporting life procedures and modern technology introduced to the treatment process contribute to hospitalized patients’ disease becoming more severe [3].
Most significantly, in today’s healthcare environment, families and their patients expect to be included in the treatment process as well as in the decision-making process [4]. The majority of the literature fails to define end-of-life or end-of-life care precisely. However, several authors have attempted to provide a succinct definition of this phrase. These two descriptions appear to be general and ambiguous. It seems to be difficult to establish a precise or comprehensive description of end-of-life care. End-of-life care in the intensive care unit (ICU) was detailed in detail by Alhalaiqa, et al. [5]. Alhalaiqa, et al. [5] pointed out that while delivering endof- life care, there are no set processes or stages to follow, making it difficult to assess the effectiveness of any end-of-life programs. However, talking about death and end-of-life care, as well as having open dialogues about these topics, may contribute to the development of guidelines for healthcare professionals to provide high-quality end-of-life care [6]. End-of-life care is also implemented differently depending on a variety of criteria, including the patients’ conditions and the departments. Cardiopulmonary resuscitation of victims and their relatives in acute settings is the focus of this study.

Literature Review

Stages of the Crisis at a Duration of Unexpected Death

There are four stages of crisis during a sudden death. They are recognized and listed in order of difficulty from the least challenging to the most challenging crisis. If any of these stages are not addressed, a person may progress from one to a more challenging one.
First stage: The presence of a threat that could harm patients’ family members is included in the first stage. This threat may cause stress or tension among the relatives. This stage may entail family members’ fears that something dangerous has occurred to their loved one in the case of cardiopulmonary resuscitation (CPR). The routine coping mechanism, according to Wright (1996), may be able to settle this stage. Wright also stressed the necessity of previous experience in minimizing the effect of this stage. Surprisingly, many studies revealed that most of family members who have previously watched family-witnessed resuscitation (FWR) wish to be present again.
Second Stage: Failure to treat persons in this stage may result in their illness worsening and sending them to the second stage, according to Wright (1996). The failure of previous experience to tell people how to deal with this new situation may have resulted in the second stage. Feelings of helplessness and hopelessness may intensify throughout this stage. People at this stage, according to Wright [7,8], try to find alternatives. Wright revealed that relatives can utilize the trial-and-error method to figure out the most acceptable response to the situation. Allowing FWR was supposed to increase death acceptance and assist bereaved families [9]. This would hasten the grief process and make it easier to accept death as a natural result of CPR.
Third Stage: Intense activity, disorder, and disarray characterize the third stage; old fears resurface, preoccupation with trivia, and make-or-break action are all present. This stage, according to Wright, might be overcome by changing the focus. Wagner. Several studies have found that family members prefer to attend their loved one’s CPR because it allows them to acquire information more rapidly. This would also lessen family tensions and reduce the likelihood of inappropriate behavior.
Fourth Stage: The patient’s relatives may be unable to cope with the situation in the fourth stage, and they may retreat and feel gloomy. Wright recommended taking various actions to manage this stage, including addressing the relatives’ comfort requirements, listening to them without passing judgment, creating a quiet environment, and taking a break from the problem-solving effort.
These interventions were suggested in the literature as a way to satisfy the requirements of the patients’ relatives during CPR. The necessity of treating families as soon as possible would be highlighted if healthcare providers considered these stages and recognized each one. This prevents families from progressing to a more mature level. Individuals do not, however, necessarily progress through all of these stages. Some people may skip the early stages and proceed straight to the advanced stages, while others may adjust to abrupt death and recover swiftly without experiencing any of the preceding stages. The determinants of grief and the elements that affect bereaved individuals during sudden death are discussed in the next section.

Causal Factors of Grieving

In general, there was no systematic study of the elements that would affect families’ reactions and feelings during and after CPR, especially if the CPR resulted in death, in the literature. It might be worthwhile to go over these points again. Several elements were thought to play a role in creating the powerful feelings, reactions, and grief experienced by people who died suddenly. Six elements, according to Wright (1996), have a significant part in deciding how families grieve when a cherished person dies suddenly. These factors include:

a. Manner of Death

The patient’s condition at the time of death should be known to healthcare personnel. It was generally assumed that natural (normal) death would cause less grief than unnatural (abnormal) death. The natural deaths are usually unavoidable, while the unnatural deaths are usually avoidable. The avoidable deaths are mostly caused by either human or mechanical faults. Parkes, who worked as a psychiatrist, noted that all of the bereaved people he had visited had experienced extremely traumatic kinds of bereavement. It has been stated that healthcare professionals who deal with CPR in consequence of injuries and accidents are more rigorous about allowing family members to participate in CPR. The majority of these health care providers raised serious worries about the presentation’s potential adverse effects on family members.
Helmer, et al. (2000) revealed that comparing trauma patients to those with medical disorders is problematic. It would be more difficult to blame others if the death was caused by natural causes such as disease, according to Wright (1996). Family members, on the other hand, may blame healthcare experts, health organizations, culture, or society, as well as themselves for not being able to do more than they did. The sadness of losing a loved one will be magnified if he or she was far away from home or family members, according to Wright (1996). This demonstrates the importance of family members being present during CPR.

b. The Identity of that Person (victim)

The depth of the grieving process is influenced by the patient’s status in the family [10]. Wright (1996), on the other hand, cautioned against making assumptions based on this characteristic. For instance, if a breadwinner dies unexpectedly, such person will be mourned for a long time because of the significant position that the person occupied in the family. Furthermore, the loss of one of the parents would have an impact on other susceptible family members’ feelings of stability and safety.

c. Manner of Attachment

The loss of a key individual is usually the most distressing. This can involve the death of someone with whom one has had a close relationship. As a result, the stronger the link between the grieving individual and the deceased person, the more severe the adverse effects on the departed relatives are expected. This, however, is not a rule. A father or a mother, for example, may have various amounts of attachment to their sons. To determine the strength of this relationship, a series of questions should be asked. This includes questions like, “What does this loss represent?” and “What security or safety aspects might be jeopardized in consequence of this loss?” Wright (1996) noted that the provision of answers to the following questions would aid in determining the bereaved person’s level of vulnerability. Another essential aspect of the attachment’s nature is the security and safety difficulties that arise in consequence of the relationship (Wright, 1996). For example, losing a father who is the family’s sole source of money might increase tension among family members.

d. Past Precursor

Wright (1996) emphasizes the importance of previous crisis experience, particularly abrupt death, in coping with the new crisis. When people have a positive experience, Wright says, it helps them cope with the new incident, and vice versa. To put it another way, what one person considers a loss may not be considered a loss by another. It’s possible that being exposed to the crisis will help people build skills and experience in dealing with similar situations in the future. It was discovered that family members with prior FWR experience were more confident in their attitudes during CPR. Furthermore, those who had been exposed to a comparable situation or had died suddenly were more cooperative with medical personnel performing CPR. They also provide assistance and comfort to the other families.

e. Individual Characteristics

Personal qualities have a vital influence in determining how people react when they are dying or receiving CPR. People react to abrupt death in different ways, according to Wright (1996). Wright also discovered that the personality of a patient’s relatives has an impact on how they search for a healthy grieving resolution. Emotional and physical well-being, according to Walsh and Crumbie (2007), provides people with more resources to cope with losses. As a result, healthcare providers should be prepared to cope with people who have a wide range of mental and physical resources for coping with loss, as well as diverse levels of social support. It appears that assessing people’s personalities or evaluating the differences between patients’ families is challenging during CPR. It appears critical to emphasize that not only personal traits influence people’s reactions, but also other elements such as family preparation, healthcare preparation, and environmental preparation. Family members should, for example, be accompanied by trained staff during CPR [11], and they should be given a suitable location to sit and observe the treatment [11].

f. Social, Cultural and Religious Factors

When people lose a loved one, social, cultural, and religious variables may have a big impact on their feelings and reactions [12]. The availability of social assistance would help to mitigate the crises’ impact on patients’ families. In a survey done by Al-hassan and Hweidi (2004), relatives of Jordanian patients rated their need for assistance as the lowest. Jordanians, it was revealed, rely on other family and friends for the majority of their support. Jordanian people in critical care units were assessed in this study, which was conducted in Jordan. However, the goal of this study was to look at the needs of critically sick patients’ families, not their needs at the time of death.
Some religions and cultures, according to Wright (1996), assist the bereaved by providing support and confirmation of their worth. Some religions, such as Christianity, are commonly thought to be helpful and supportive in the event of a sudden death, according to Wright. Jordanians’ opinions and behavior on matters such as health and death are heavily influenced by religion and culture. People in Jordan, for example, rely on their relatives for financial and psychological support rather than relying on other resources such as hospital professionals. As a result, Jordanian patients frequently have a significant number of visitors. The task of healthcare professionals is made more difficult by the fact that they must deal with a huge number of visitors.
In conclusion, these six criteria appear to be critical to consider when devising any remedy to lessen the harshness of the grieving process. Understanding these characteristics will aid healthcare providers in identifying the issues that patients may need to address in order to begin the grieving process. The following discussion looks at a suitable plan for treating bereaved family members appropriately.

Dealing with Bereaved Relatives at the Time of Sudden Death and During CPR

In the work of literatures, there is no mention of FWR in relation to end-of-life care. According to Kubler-Ross, grievers go through five stages (1969). Denial or a sense of isolation may be felt by grievers at first. This means that grievers may say things like “don’t say that” or “no, he didn’t die” to indicate their disbelief. Wright (1996) suggested a number of methods for coping with this emotion, including finding a polite way to inform family members of the bad news. According to Davidhizar and Newman-Eiger, nurses, on the other hand, should comprehend the value of denial (1998). They claimed that denial is one of the safest strategies to deal with the unfathomable.
Second, grievers may have a sense of rage. They might start saying things like ‘why me?’ or ‘why now?’ as a form of protest. Kubler-Ross warned that grievers might start blaming the healthcare experts for the lack of justice. Nurses and other professionals may take this personally (Wright, 1996). Bereaved people may begin haggling with healthcare providers. They might try to put off the inevitable. Healthcare practitioners, according to Wright (1996), should accept this and endeavor to reach an agreement with grievers to prove the death. Bereaved people may experience depression. When grievers can no longer deny or relocate, they enter this stage.
Grievers may experience feelings of sadness and crying at this stage as they begin to recognize reality. Withdrawal, stillness, and helplessness may be observed by bereaved people. As grievers reach the acceptance stage, they stop striving to ignore or avert the unavoidable death. Grievers begin to comprehend the concept of death at this point, and they begin to relax and feel at ease. Research by Brysiewicz, et al. [13] employed a semi-structured interviews to examine the ED healthcare personnel’ capability to handle the situation of sudden death. The study reacted to findings of previous studies that was conducted by Brysiewicz [14]. A model was created to give healthcare personnel guidance on how to cope with unexpectedly bereaved families before, during, and after death. This approach instructs healthcare providers on how to cope with family members while performing CPR. This would also make it easier for families to accept their loved one’s death. Before the happiness of death, the first half of this paradigm includes instructions for dealing with bereaved families. This comprises implementing two ways to improve the department’s performance, as well as enhancing the department’s culture and guaranteeing enough resources. The model’s second section contains suggestions for coping with bereaved relatives after they have died. This involves making the caring process more efficient. Three ways are expected to do this. Proximity, sensitive communication, and sensitive deathtelling are the three.
The model’s final piece includes advice for dealing with bereaved families when a loved one has passed away. This entails giving family members the best possible support. It was recommended that two approaches be taken. Assisting and supporting medical professionals, as well as assisting mourning families, are among these responsibilities. Once this paradigm was established, ED professionals’ capacity to communicate with families at the time of a loved one’s death was stated to improve [13]. Data from a prior study was used to develop this model. The use of qualitative design allows researchers to gain valuable insight into the perspectives and recommendations of families and healthcare providers. However, because of the small size of the original study’s sample and the fact that the data was taken from a single institution, the findings are limited in their generalizability. This model was implemented in ED. The current study, on the other hand, adopts a different approach. Regarding the effects as influenced by technology, the majority of studies and reviews focused on FWR in emergency departments, according to a review of the literature. The current study, on the other hand, is concerned with the views of healthcare workers and family members in adult critical care settings. As a result, it appears that some light should be shed on nature and the characteristics of the critical care environment. In terms of the rate of CPR, patient conditions, and work environment, Demir [15] highlighted that there are certain distinctions between the ED and critical care units. Critical care units (CCUs) are specialized units for patients with lifethreatening illnesses [16]. During the care of a critically ill patient, healthcare workers are expected to face numerous physiological and psychological problems.
Furthermore, critical care specialists are increasingly expected to provide psychological and emotional support to the families of critically sick patients [17]. Critical care workers, according to Offord [18], are expected to deal with dying patients and bereaved relatives more than experts in other departments [19]. The conditions of patients in critical care units differ from those in other departments. In critical care settings, CPR is a regular technique. Hadders [20] found that ICU clinicians are frequently unsure regarding the resuscitation results of their patients. According to Hadders, individuals who receive CPR either survive or recover completely. After CPR, most survivors rely on machines and technology to keep them alive. According to Benner, et al. [17], the necessity of providing psychological and emotional care for patients and their relatives in critical care settings is undervalued. More than anything else, this was intended to result from a focus on the patient’s biological demands. It should be noted, however, that the presence of a family member in one of the critical care units will upset established family roles and will frequently throw a family into disarray [21].
Moving forward, a number of studies have demonstrated the necessity of assisting critically ill patients’ families and include them in patient care [22]. The critical care environment differs from other hospital departments in that it typically contains skilled healthcare staff as well as advanced technology [16]. In these situations, multiple machines and monitors must be present surrounding each patient. Professionals encounter a number of obstacles in this context. Medical experts had to learn how to use all of the new machines and technologies to begin with [16]. Dealing with technology should not prevent healthcare workers from considering other patients’ and family members’ psychological needs [16]. Sundin-Huard (2005) noted that critical care personnel are frequently obsessed with their patients’ immediate physical and technological needs [23]. To ensure patient survival, technology should be employed to provide maximal patient benefit while also considering the needs of other patients and their families [16].
Mosenthal, et al. [10] noted that people are increasingly seeking death dignity without unnecessary using life-prolonging gadgets, but they equally value high technology’s promise of cure and spectacular lifesaving measures. As a result, it appears that providing technical assistance to critically sick patients and their families, as well as describing the role of each machine in the patient’s environment is enhanced. In a critical care setting, Hadders [20] explained how critically ill patients and their families felt about being reliant on technology. FWR has been reported to be more acceptable in the ED than in critical care settings in the literature. According to Bennun [16], critical care specialists place a greater emphasis on technology than on providing psychosocial treatment to patients and their families.
Because critical care specialists are so focused on technology, they overlook other parts of care, such as family-centered care [24]. This was also assumed to be the reason why critical care workers resisted allowing family members to participate in treatments like CPR [25]. The current investigation takes place in a critical care setting. The majority of the literature, however, is based on evidence from ED settings. As a result, it appears that the findings of these investigations must be taken into account. However, the mind should be awakened to consider the contrasts between the emergency department and critical care settings.

Empirical Review

Mcmahon-Parkes et al. (2009) examined the opinions of patients who survived CPR and those who were not resuscitated. Patients were indifferent about compromising confidentiality in consequence of FWR, according to the researchers. Redley, et al. [26] noted that the ethical principles surrounding FWR should be further discussed. The importance of a qualitative approach in studying FWR would provide a broader perspective on these principles and their impact on healthcare professionals’ and families’ perceptions. Several questions must be answered, such as “who is the person that will be authorized to witness CPR?’ How many people should be present while CPR is performed?’ How about the other family members? Who will look after them? Would additional relatives be willing to stay outside the resuscitation room with you?” All of these questions should be addressed with consideration for the culture of the responders.
A qualitative approach would reveal more information about what family members wish to accomplish while in the resuscitation chamber. Allowing FWR, for example, could affect the public’s trust in the medical profession, according to Rosenczweig [27]. However, the manner in which this would occur was not specified. Other topics are expected to be examined more if a qualitative approach is used. Fulbrook, et al. [11] stressed the importance of recognizing the differences in healthcare systems between countries, which can have an impact on outcomes. In addition, it was identified that the role of self-assurance in accepting or rejecting FWR be investigated [28-30]. They found that nurses’ opinions toward FWR are unaffected by previous experience with the procedure. This finding differs from that of other studies, which found a link between a lack of experience with FWR and unfavorable or doubtful attitudes [31,32]. Fulbrook, et al. [11] advocated for more research into the aspects that influence how people make decisions about the FWR. Many studies about FWR were evaluated by Redley, et al. [26]. They claimed that FWR might infringe on a patient’s privacy. Because there is a scarcity of information about ethical principles from the perspective of patients, these principles may be questioned.
Ardley, et al. [9,33-36] discovered that the majority of the research used quantitative designs. These findings are consistent with other studies that analyzed numerous empirical investigations on FWR. However, it is agreed that this is insufficient rationale for choosing a qualitative approach. As a result, other flaws in using a quantitative technique to examine FWR should be identified. In general, quantitative research is thought to be reductionist [37]. This means that using a quantitative approach will leave some variables unaddressed. As a result, using a qualitative method should provide a more holistic view of the subject under inquiry [38,39]. Fulbrook, et al. [11] investigated the opinions of European Nurses that are saddled with the responsibilities of critical care concerning FWR. Furthermore, some researchers acknowledged the influence of culture and religion on people’s opinions toward FWR [40-42]. These topics, in contrast require further investigation and debate. To do this, healthcare professionals and the general public must be encouraged to engage in open debate and free discussion on FWR [43,44]. Most of the above problems could be explained by using a qualitative design.

Specific Literature Review on FWR and Research Gaps

In the study of Axelsson, et al. [45], six studies were undertaken in the United States, four in the United Kingdom, one in Australia, and one in Sweden. Alhalaiqaa, et al. [46] identified that only two studies were conducted from a Western point of view, and that all these studies may not have considered the views of emergency situation professionals. Also, none of them were conducted in Africa, and none of them supported FWR. However, cultural differences are expected to emerge inside Western countries as well [47]. Fulbrook, et al. [11] examined the attitudes of critical care nurses concerning FWR in Europe. In their studies, they noticed that there were certain differences between British nurses and nurses from other European countries. Walker [36] noted that the global movement to study FWR should focus on cultural differences not only between nations, but also inside the local and national healthcare systems.
Davidson, et al. [23] also noted that the impact of spiritual and religious beliefs on patients’ healthcare decisions has not been extensively investigated in the works of literature. This also implies that the impact of religion on people’s opinions toward FWR was not sufficiently examined in literature. According to MacKenzie, et al. [48], highly religious and spiritual people believe in prayer and divine intervention to promote health, but they also seek healing and care from healthcare experts. According to Davidson, et al. [23], the severity of the illness has an impact on the patients’ motivation to care spiritually and religiously. One of the most critical situations is CPR. Furthermore, Ong, et al. [40] conducted four studies in Turkey, and two studies in Singapore. Five of the studies looked at healthcare workers. Similarly, in all of these investigations, the majority of healthcare professionals were opposed to FWR. In a unique Asian study, roughly 73 percent of family members preferred FWR and believed it would help them cope with their bereavement [28,47]. It’s crucial to note that all the six research employed a survey research design.
This may impede your ability to achieve a wider understanding of the issue. This could also explain why there isn’t much evidence about the impact of religion and culture on FWR in this research. More crucially, the majority of these research either used a survey that had already been used in a Western study or created their own survey questionnaire based on existing literature. This may limit the ability to attribute negative sentiments about FWR to specific cultural or religious factors. Four of the six studies listed above were conducted in Turkey. The majority of Turks are Muslims, as is well known. Turkey has closer connections with Arab countries. Some of this research suggested that religion and culture play a role in influencing healthcare professionals’ opinions toward FWR. However, none of this research looked into these difficulties in depth.
For example, Badir, et al. [41] and Demir [42] suggested that cultural and theological factors could explain the disparity in attitudes between Turkish healthcare workers and their Western counterparts. However, the word “cultural differences” is used here in a broad sense without specifying what kind of cultural differences are being discussed. Nurses and doctors are likely to be opposed to FWR because they are afraid of being harmed by family members, particularly if the patient dyes [42,48]. Demir suggested that further research be done on the impact of cultural problems on people’s attitudes toward FWR. Nigeria and Turkey do share significant parallels, particularly in terms of the majority of their populations being Muslims.
There are, nevertheless, some distinctions between the two countries. In Turkey, for example, the general system is secular. At this time, the Turkish people follow the Western countries in that they distinguish between religion and other parts of life. In Nigeria, however, the situation is somewhat different. The Nigerian people incorporate religion into every area of their lives, though Nigeria’s medical industry is not regulated based on the derivatives of Islamic religion. But there are some believes that shapes the mentality of Nigerian Muslims; for instance, they believe that life is a divine trust and Islam does not allow a person to die voluntarily [7,49]. This implies that in Muslim countries, orders like “do not resuscitate” (DNR) are controversial.
Sharp and Frederick (1989) observed that since 1998, all US acute and chronic care hospitals have been required to establish policies that affirm the patient’s right to determine DNR orders. The United Kingdom and Australia have issued similar policies [50]. In Muslim countries, the situation is considerably different. The DNR order is incompatible with Islam’s principles [51,31,32]. Stopping supportive therapies when a patient is terminally or seriously sick is a contentious issue [46,52]. It may be claimed that family members would request to observe CPR in order to ensure that their loved one receives the finest possible treatment and that everything possible is done for them. All of the foregoing arguments illustrate that Western and Nigerian cultures have some cultural differences. It was also emphasized that there is a need to considering culture and religion while making healthcare decisions. An examination of the literature also suggests that other factors may influence people’s perceptions toward FWR are education, training, and experience [53-60].
In the literature, the social dimension has also gotten a lot of attention. FWR implementation demands enough resources as well as a set budget. As a result, the economic component must be considered when starting any FWR endeavor [61-70]. Furthermore, this study will be place in critical care facilities with high-tech environs. It is important to think about how modern technology will affect healthcare practitioners and their families. To take into account all of the preceding aspects, in addition to the fact that this study is unique in Nigeria, a conceptual or theoretical framework is required to better explain the function of all of these factors in forming people’s opinions toward FWR. While looking for nursing theories, it was discovered that Leininger’s cultural care theory might address all of these concerns. Furthermore, applying this theory is supposed to demonstrate the distinctions between Western and Nigerian cultures. This would aid in obtaining the advantages of the existing literature. At the same time, this hypothesis will not overlook Nigerian’s unique characteristics [71- 73].

Conclusion

Despite the fact that much has been published on FWR, it remains a difficult, debatable, and diverse topic. The findings of the earlier studies showed that there are overlapping ideas and perceptions about the predicted outcomes of allowing FWR. The bulk of these research revealed that family members would desire to attend CPR for their loved ones. The majority of patients thought FWR was convenient and would help the resuscitated patient. Nonetheless, a few patients raised concerns about the impact of this presence on the competence of health personnel, and also on the impact on the resuscitated patient. In researches that concerned the examination of health professionals’ views and opinions about FWR, the results were usually mixed. Several researches have shown that FWR has significant benefits for family members, patients, and health care workers. However, numerous studies have identified concerns about this presentation, such as the psychological impact on family members, the additional stress that this presence may cause for health professionals, and the potential for legal action in consequence of this presence.

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