Optimization of the adult zebrafish ECG method for assessment of drug-induced QTc prolongation

IntroductionRecent studies have shown the utility of adult zebrafish ECG (electrocardiogram) in assessing drug-induced QTc prolongation. While the method has significant advantages over current ECG animal models including ethical issues, low compound requirement and expense, adoption of the method into drug discovery programs has been hampered by specific limitations. The limitations include the inability to determine the exact dose of test compound administered, and potential effects due to variables such as flow rate of oral perfusion and immobilization method. We describe a refined method for the reproducible recording of the adult zebrafish ECG and illustrate its application in investigating drug-induced QTc prolongation using the histamine receptor antagonist Terfenadine as a test drug.MethodWe chose to perform parenteral administration of test drug instead of perfusion on the basis of mg per kg body weight of adult zebrafish. Acclimatization and immobilization methods were optimized to avoid ECG artifacts due to sudden environmental changes. We further modified the formula for QT correction and ensured reproducible recording of stable ECGs. Various concentrations of Terfenadine were used and the resultant proarrhythmic effects were analyzed as compared to the baseline and untreated controls.ResultsNormal, stable and reproducible ECGs were recorded in all zebrafish. Terfenadine at the rate of 0.1 mg/kg body weight was found to be the NOAEL. We found an excellent correlation between known QTc effects in humans and those observed in adult zebrafish at all concentrations. All Terfenadine-induced proarrhythmic effects observed in zebrafish were dose and time dependent.DiscussionWe report a refined method for reproducible recording of stable zebrafish ECGs to facilitate its routine application in preclinical investigation of QTc-prolonging drugs with reliable estimation of NOAEL. Our study is of relevance to the development and use of alternate animal models in drug discovery.     

Histomorphometric and sympathetic innervation of the human internal thoracic artery

INTRODUCTION:

Internal thoracic artery (ITA) is an established arterial graft for the coronary artery by‐pass surgery. Special micro‐anatomical features of the ITA wall may protect it from age related pathological changes. One of the complications seen after coronary artery bypass grafting is vasospasm. Sympathetic nerves may be involved in vasospasm.

OBJECTIVE:

To ascertain the sympathetic innervation of the internal thoracic artery and to assess the effect of aging on this artery by histomorphometry.

METHOD:

Fifty‐four human internal thoracic artery samples were collected from 27 cadavers (19 male and 8 female) with ages of 19 to 83 years. Samples were divided into three age groups: G1, 19–40 years; G2, 41–60 years; G3, ≥61 years. Sections (thickness 5 µm) of each sample were taken and stained with hematoxylin–eosin and Verhoeff–Van Gieson stains. Five of fifty‐four samples were processed for tyrosine hydroxylase immunostaining.

RESULTS:

The thickness of the tunica intima was found to be constant in all age groups, whereas the thickness of the tunica media decreased in proportion to age. Verhoeff–Van Gieson staining showed numerous elastic laminae in the tunica media. Significant differences (p<0.0001) in the number of elastic laminae were found between G1 with G2 cadavers, between G2 and G3 cadavers and between G3 and G1 cadavers. Tyrosine hydroxylase immunostaining demonstrated sympathetic fibers, located mainly in the tunica adventitia and the adventitia–media border. The sympathetic nerve fiber area and sympathetic index were found to be 0.0016 mm² and 0.012, respectively.

DISCUSSION:

Histology of the ITA showed features of the elastic artery. This may be associated with lower incidence of Atherosclerosis or intimal hyperplasia in ITA samples even in elderly cases. Low sympathetic index (0.012) of ITA may be associated with fewer incidences of sympathetic nervous systems problems (vasospasm) of the ITA.

CONCLUSION:

Sympathetic nerve fibers are present in the adventitia of the internal thoracic artery. This is an elastic artery, although anatomically it is considered to be medium‐sized. The sympathetic index may be used for analysis of sympathetic nerve fiber‐related problems of the internal thoracic artery.     

Histomorphometric and sympathetic innervation of the human superficial temporal artery

Context:

Following microvascular surgeries, stenosis and spasm of the arterial graft or the recipient vessel are serious complications which are often caused by intimal hyperplasia and perivascular nerves, respectively.

Aims:

The purpose of this study was to understand the characteristics of arterial wall and sympathetic innervation of the human superficial temporal artery (STA) and also, the effect of aging on STA.

Methods and Materials:

Fifty-two fresh human STA (frontal branch) samples were obtained from 26 cadavers (19 males and 7 females) between the ages of 19 and 83 years. Samples were divided into three age groups: G1, 19-40 years; G2, 41-60 years; G3, over 61 years. 5μm-thin sections of each sample were taken and stained with haematoxylin-eosin, Verhoff''s and tyrosine hydroxylase (TH) immunostaining.

Results:

The well-defined internal elastic lamina (IEL) was observed in all samples of STA, whereas external elastic lamina (EEL) was not prominent in almost all cases or absent in few cases. This might be the important factor in the process of intimal and medial hyperplasia in the frontal branch of STA. Notably, intimal thickening appeared from second decade of life. Sympathetic fibres are located mainly in tunica adventitia and outer media. Mean adventitial and sympathetic areas were found to be 0.080 and 0.010mm², respectively.

Statistical analysis used:

One-way ANOVA followed by Tukey HSD post hoc test by using the SPSS 11.5 software.

Conclusions:

STA is prone to age related pathological changes. Sympathetic index may be used for analysis of sympathetic fibre-related problems (vasospasm, migraine) of the STA.     

Automation of immunohistochemical evaluation in breast cancer using image analysis

AIM: To automate breast cancer diagnosis and to study the inter-observer and intra-observer variations in the manual evaluations.METHODS: Breast tissue specimens from sixty cases were stained separately for estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER-2/neu). All cases were assessed by manual grading as well as image analysis. The manual grading was performed by an experienced expert pathologist. To study inter-observer and intra-observer variations, we obtained readings from another pathologist as the second observer from a different laboratory who has a little less experience than the first observer. We also took a second reading from the second observer to study intra-observer variations. Image analysis was carried out using in-house developed software (TissueQuant). A comparison of the results from image analysis and manual scoring of ER, PR and HER-2/neu was also carried out.RESULTS: The performance of the automated analysis in the case of ER, PR and HER-2/neu expressions was compared with the manual evaluations. The performance of the automated system was found to correlate well with the manual evaluations. The inter-observer variations were measured using Spearman correlation coefficient r and 95% confidence interval. In the case of ER expression, Spearman correlation r = 0.53, in the case of PR expression, r = 0.63, and in the case of HER-2/neu expression, r = 0.68. Similarly, intra-observer variations were also measured. In the case of ER, PR and HER-2/neu expressions, r = 0.46, 0.66 and 0.70, respectively.CONCLUSION: The automation of breast cancer diagnosis from immunohistochemically stained specimens is very useful for providing objective and repeatable evaluations.     

Are health care workers following preventive practices in the COVID-19 pandemic properly? - A cross-sectional survey from India

Background and aimsHealth care workers (HCWs) are at increased risk of getting infected with Coronavirus disease 2019 (COVID-19) and suboptimal preventive practices have been identified as an important risk factor in this regard. This study was done to evaluate the preventive practices being followed by health care workers and identify reasons for suboptimal compliance.MethodsA cross-sectional survey was done in HCWs belonging to various occupational roles and socio-cultural backgrounds across India through online platforms and telephonic interviews from July 30, 2020 to August 30, 2020. A scientifically designed and pre-validated questionnaire with good validity (CVR = 0.87, S-CVI/Av = 0.978) and internal consistency (Cronbach’s alpha coefficient = 0.85) was used.ResultsThe responses of 956 participants were analysed. Various suboptimal practices like touching outer surface of masks, lack of social distancing in cafeteria and duty rooms, inability to wash hands for adequate duration and properly follow steps of hand hygiene, inability to don and doff PPE properly, carrying PPE to duty rooms before completely doffing, use of personal mobile phones during duty and improper sleep were identified. Lack of knowledge, long duty hours, shortage of PPE, high patient workload, and casual attitude regarding own safety were identified as important barriers. Resident doctors and paramedical staff in the age group 18–30 years reported lower adherence.ConclusionsSuboptimal compliance in preventive practices like handling PPE, distancing in cafeteria/duty rooms and hand hygiene is not uncommon in HCWs. Certain barriers are identified which should be addressed to ensure adequate safety of HCWs against COVID-19.     

Geriatric Mental Healthcare Training: A Mini-Fellowship Approach to Interprofessional Assessment and Management of Geriatric Mental Health Issues

IntroductionThe population in United States aged 65 and older has rapidly grown and is projected to grow faster than any other segment of the population. Despite this demographic shift, the nation's geriatric workforce is shrinking.AimThe primary goal of the fellowship was to form a learning collaborative that would help trainees in medicine, nursing, social work, pharmacy and occupational/physical therapy understand the roles of each discipline involved in the provision of geriatric mental healthcare and to enhance basic knowledge of common geriatric syndromes.MethodsFaculty from the University of Pittsburgh developed a format for the mini-fellowship. Trainees from five disciplines were recruited for participation in the mini-fellowship. This was offered annually over four-year period, hosted by the John A. Hartford Foundation Centers of Excellence in Geriatric Psychiatry at the University of Pittsburgh and University of California at San Diego.ResultsEighty-one participants across five schools of the health sciences completed the mini-fellowship. Feedback was positive: most participants appreciated learning from other team members, endorsed appreciation of the contributions of other disciplines to patient care, and reported improved understanding of three major geriatric syndromes.ConclusionConducting an interdisciplinary mini-fellowship in geriatric mental health was feasible and well received by trainees. The fellowship enabled better appreciation for the provision of geriatric mental health care within the context of an interprofessional team. However, decanal and faculty leadership across the schools needs to place greater emphasis on the importance of interprofessional team-based learning and to free up time for such activity.     

CD36 genetic polymorphism and salivary cues are associated with oleic acid sensitivity and dietary fat intake

There is a lack of research on the combined effects of genetic variations (specifically CD36 SNPs—rs1761667 and rs1527483), dietary food habits (vegetarian or not), and the salivary environment on obesity and taste sensitivity, especially in the Indian population. The current study aims to better understand the relationship between impaired taste perception, fat consumption, higher BMI and obesity development by examining the combined association between CD36 SNPs, oleic acid (OA) detection threshold, and food habits among Indian participants. Furthermore, the relationship between oral fatty acid (FAs) sensitivity and taste physiology factors linked to inflammation and salivary proteins was considered. Participants with the minor allele (AA/AG) of CD36 (in both rs1527483 and rs1761667) consumed more fat, particularly saturated FAs (p = 0.0351). Salivary lipopolysaccharide, which causes inflammation, was significantly greater in non-vegetarians with a higher BMI (p < 0.05), and it exhibited a negative correlation (r = −0.232 and p < 0.05) with Ki67 gene expression, a marker for taste progenitor cells. A positive correlation (r = 0.474, p = 0.04) between TLR4 mRNA levels and the OA detection threshold was also observed. Participants with BMI > 25 kg/m² had substantially higher TNF-α and IL-6 receptor mRNA expression levels, but there were no significant differences between the vegetarian and non-vegetarian groups. However, salivary CA-VI, which has a buffering capability on the oral environment, was lower in non-vegetarian adults with BMI >25. Thus, it was shown that non-vegetarians with overweight and obesity in India were in at-risk groups for the CD36 SNP (AA/AG at rs1761667 and rs1527483) and had higher levels of inflammatory markers, which exacerbated alterations in food behaviour and physiological changes, indicating their relevance in the development of obesity.