Computational protocol: Plant-based foods containing cell wall polysaccharides rich in specific active monosaccharides protect against myocardial injury in rat myocardial infarction models

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Protocol publication

[…] The animal experiments were conducted according to the guidelines for the animal care and use of laboratory animal protocols approved by the Institutional Animal Care and Research Advisory Committee of Catholic University of Daegu. Myocardial injury was generated through LAD coronary artery ligation in male Sprague-Dawley rats (250–300 g, 8 weeks) (Samtaco Inc., Osan, Korea) as previously described. The rats were anaesthetized with intramuscular injections of ketamine (100 mg/kg) and xylazine (5 mg/kg), intubated, and ventilated with air. The heart was exposed by a left thoracic incision, and the LAD artery was ligated approximately 5 mm down from the aortic origin by passing a 5–0 Prolene suture (BV-1, Ethicon) around the LAD artery and then double-knotting the suture. Whether the LAD artery was occluded or not was confirmed by observing LV wall alterations via the development of a pale colour. Subsequently, IR or ischaemia-only experiments were performed as follows. In the SISR experiments, the hearts were ligated for 30 minutes and then reperfused for 3 hours. In the SILR experiments, the hearts were ligated for 30 minutes and then reperfused for 7 days. In the LI experiments, the heart remained ligated for up to 30 days. IS was assessed through 2,3,5-triphenyltetrazolium chloride (TTC) (Sigma-Aldrich) or haematoxylin & eosin (H&E) staining for the SISR and LI experiments, respectively. In the case of TTC staining, the LAD was religated. Then, 1 ml of 1.0% Evans blue (Sigma-Aldrich) was infused through the jugular vein; subsequently, the AAR was defined as the area that was not infiltrated by the dye. The heart was excised into 4 pieces approximately 3-mm thick, and the pieces were stained in 1 ml of 1.5% TTC at 23 °C for 5 minutes. Subsequently, IA was defined as the area that was not stained by TTC. BZ was defined as the region in which the IA is excluded from the AAR. AAR and IA were determined by computerized planimetry using ImageJ software (NIH, v1.47), and IS was defined as a percentage of the IA relative to the AAR. In the case of H&E staining, the pieces were fixed, sectioned on 5-μm thick slides, and stained with H&E. The IA was identified by viewing the sections under a microscope, and the IS was calculated as a percentage of the IA relative to the LV area. To assess the average infarcted LV wall thickness, five evenly spaced radians were passed through the infarct with the centre of the LV cavity as a reference, and the average infarcted LV wall thickness was calculated. To determine the average non-infarcted LV wall thickness, the septal LV wall thickness was measured in a similar manner. To quantify the infarct expansion, the expansion index (EI), which accounts for LV dilation and infarct wall thinning, was employed, for which LV cavity area was also determined by computerized planimetry. For the oral sample administration, rats were randomly assigned to sham, control, or sample-treated groups. In the sample-treated group, the rats received the samples supplemented at various doses with a standard chow diet for 3 days prior to occlusion. For the tail-vein injection sample administration, the rats received the samples once 1 hour prior to occlusion. In the control group, the rats received the standard chow diet only. In the sham group, the experimental procedures were the same as those in the control group but without ligation.Assessments of IS, apoptotic cells, LV wall thickness, collagen content, and expansion index were performed as previously described. The MDA levels in the AAR were measured as previously described. Detailed procedures are provided in . [...] The sample size in the MI experiments was pre-determined using a power analysis (G*Power 3.1.7 program) as previously described. The data are expressed as the mean ± SEM. All of the statistical analyses were performed in SPSS (IBM Corporation, v19). For all of the data, Shapiro–Wilk and Levene statistics were applied a priori to verify the normality and homogeneity of variances, respectively. The data that met the assumptions for a one-way ANOVA were analysed using ANOVA with post hoc tests. Non-normal datasets were analysed by the Kruskal–Wallis test using a post hoc pairwise comparison of mean ranks. Between two groups, an unpaired two-sided Student’s t-test was used. Differences between the control and sample-treated groups were considered statistically significant at P < 0.05. […]

Pipeline specifications

Software tools ImageJ, G*Power, SPSS
Applications Miscellaneous, Microscopic phenotype analysis
Diseases Coronary Disease, Heart Failure, Cardiomyopathies, Myocardial Infarction
Chemicals Adenosine Triphosphate, Arabinose, Monosaccharides, Xylose