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The primary endpoint of the in vivo ischaemia-reperfusion injury model was myocardial infarct size as a percentage of AAR (IS/AAR%). The AAR is defined as the region of myocardium subject to ischaemia. Infarct size and AAR were determined by histological staining of ex vivo heart preparations (summarised in Figure 3.9).

The area of myocardial infarction was determined by TTC staining whereby TTC serves as an enzymatic substrate for intracellular dehydrogenases in living cells causing it to change colour from an off-white precipitate to an intense red colour. Dead cells no longer contain these dehydrogenase enzymes meaning that TTC remains an off-white colour in areas of infarction. The colour of TTC stained tissues allows accurate distinction between the living and dead regions of myocardium (see Figure 3.9). This TTC-based staining method has been validated in numerous previous studies against classical microscopic histological indicators of cell death (reviewed by Kloner, 1993; Black and Rodger, 1996). The area of myocardium deemed ‘at-risk’ was delineated by perfusion of Evans blue dye following re-occlusion of the LAD suture which results in blue staining of the area of myocardium that was perfused during the ischaemic event; this staining method is summarised in Figure 3.9.

i

Histological staining method

Hearts were extracted from mice anaesthetised with ketamine (100 mg/kg [Vetlar, Bioniche Animal Health, Canada]), xylazine (20 mg/kg [Rompun, Bayer, UK]) and atropine (0.6 mg/kg [Sigma-Aldrich, UK]). The chest was rapidly opened and the heart extracted by cutting the aorta immediately before the aortic loop. The aorta was cannulated onto a 21 gauge (G) standard metal perfusion cannula to allow retrograde perfusion of the heart (Figure 3.9 A).

The heart was perfused manually taking care not to exert excessive pressure that may damage the coronary system. The heart was first washed by perfusion of saline (1 ml) to remove blood from the coronary system (Figure 3.9 B). The area of infarction was then delineated by perfusion of TTC (7 ml of 1% TTC in phosphate-buffered saline [PBS], pre-warmed to 37°C) (Figure 3.9 C). The LAD suture from the surgical procedure was securely re-occluded and Evans blue dye (1.5 ml 0.5% Evans blue made in distilled water) then perfused under running water to delineate the AAR (Figure 3.9 D).

Figure 3.9: Histological staining method for myocardial infarction and area-at-risk

A) Hearts were extracted and the aorta cannulated for manual perfusion. B) Residual blood was removed by perfusion of saline. C) Staining for infarction was by TTC perfusion at 37ºC where living tissue stains bright red and dead tissue remains off-white in colour. D) The AAR was delineated by perfusion of Evans blue dye following re-occlusion of the LAD ligating suture whereby the non-AAR stains blue and the AAR remains unstained by Evans blue.

ii

Processing of stained hearts

Hearts were immediately stored at -20°C for 1–2 days and then manually sliced using a sharp scalpel to give 5 transverse slices from the apex of the heart with the uppermost slice at the approximate level of LAD occlusion (Figure 3.10). Heart slices were washed in saline and fixed in 10% formalin for 90-120 minutes at room temperature. The right ventricle was dissected from each heart slice and the resulting left ventricle slices were imaged in a custom-made acrylic block spaced by 0.5 mm using an Epson scanner (Epson Perfection V100 Photo, Epson, UK) at 1200 dpi, 0%, 15% and 100% brightness.

B. C. D.

A.

Figure 3.10: Processing method of preparing histologically stained hearts for imaging A) Hearts were rapidly sliced using a sharp scalpel to take five transverse slices from the apex of the heart to the approximate level of the LAD suture. B) After briefly washing and formalin fixation for 90-120 minutes, the right ventricle (RV) was removed from each heart slice and discarded. C) Left ventricle (LV) slices were imaged using a scanner – example of mid-heart slice with area of infarction (off-white colour) and AAR (all of the area not stained blue) indicated.

iii

Quantification of infarct size and area-at-risk

Infarct size and AAR were quantified by planimetry using ImageJ software (version 1.45s, National Institutes of Health, USA). Raw images (at 15% brightness) were assessed by manual thresholds to identify the total left ventricular area (LV area), area of Evans blue staining and area of infarction. Myocardial AAR as a percentage of left ventricle area (AAR/LV%) was calculated as: (LV area – Evans blue area) / (LV area). Infarct size as a percentage of AAR (IS/AAR%) was calculated as: (Infarct area) / (AAR/LV%). This method is summarised in in Figure 3.11 and the precise details and validation of this method is provided in chapter 4.

Figure 3.11: Planimetry method for quantification of infarct size and area-at-risk

A) Raw heart images were prepared by removal of the background image. B) AAR was quantified by manual threshold of the green channel image and the number of pixels of Evans blue staining recorded. C) Infarct area was quantified by manual threshold of the red channel image and the number of infarct pixels (off-white) recorded. Total LV area was quantified by manual threshold to record the number of pixels. AAR was calculated as (LV area – Evans blue area) / (LV area).

B. C. A. Formalin fixation _Infarct Area-at-risk LV RV C. A. B. Raw image RGB scan at 15% brightness

Evans blue threshold Red channel image with manual threshold applied

Infarct threshold

Green channel image with manual threshold applied

Hearts were excluded from the dataset if the myocardial AAR was outside the predefined inclusion range of 40–75% AAR/LV. No statistically significant differences were observed in AAR/LV% between groups within experiments presented. Mean AAR/LV% for each group are reported alongside the results for each experiment.

Statistical analysis: All percentage areas are reported as mean ± standard error of

mean (SEM). Note that regression analysis values for slopes are given as the regression parameter ± standard deviation, as detailed below. Statistical analysis was completed using GraphPad Prism® version 5.0 (GraphPad Software, USA) or subsidiary GraphPad StatMate software version 2.0 (GraphPad Software, USA), as detailed below. Details of statistical tests and sample sizes are provided alongside all results.

Comparison of means: Sample means were compared using an appropriate statistical test determined by the number of groups and type of data being compared. Where two independent groups were compared, data were analysed using an unpaired t-test. Where more than two independent groups were compared, data were analysed by one- way ANOVA and Bonferroni test comparing relevant columns of data as indicated. In cases where two dependent groups were compared, data were analysed using a paired t-test since measurements were made of the same parameter by two different methods. Paired t-tests were only appropriate for the comparison of histological and cardiac MRI endpoints where the use of paired t-tests is specifically reported in the text. For all these statistical analyses, the statistical significance level was set at 5% (α=0.05) and statistical significance reported where the computed P-value was less than 0.05. Significance was reported using standard significance coding: *P<0.05, **P<0.01 and ***P<0.001. No statistical significance was reported where P>0.05 (indicated as ‘NS’). Regression: Where appropriate, regression analysis of myocardial infarct size and AAR was undertaken using the Deming (Model II) regression method. Standard regression analysis was not appropriate in this case since it assumes that only the Y variable is subject to error, whereas in this case both measurement of AAR (X variable) and infarct size (Y variable) are subject to a similar degree of error. Deming regression analysis was therefore undertaken where the error of both the X and Y variables was assumed to be approximately equal. This regression analysis was used to define the relationship between infarct size and AAR and reported as: y=mx+c where m is the slope and c is the intercept when x=0. Regression slope is reported as mean ± standard deviation (SD) and Y intercept reported for when x=0.

Regression values were reported as r2 and the relationship determined for each group compared by evaluating the slopes. Where the slopes of the two groups were not

statistically significant, the elevation of the lines (intercepts) was compared. Statistical significance was set at 5% (α=0.05) and significance was reported when the computed P-value was less than 0.05 using standard significance coding.

Statistical power: Statistical power calculations were performed to determine the approximate sample sizes required for a prospective study or to retrospectively evaluate statistical power to ensure that sufficient sample sizes were investigated. For all tests, a statistical power of 80% was deemed the minimal acceptable level of power in accordance with published guidelines (Cohen, 1988; Townend, 2002). All power calculations were performed using GraphPad StatMate software version 2.0 (GraphPad Software, USA) according to the statistical power equations published by Cohen (1988). Where no statistical significance was observed, the statistical power of the analysis was assessed using the observed sample sizes and standard deviations to determine the difference between groups that could be detected with 80% statistical power.

Sample size calculations for prospective studies were estimated based on 5% significance level (α=0.05) and 80% statistical power (β=0.2) and the required sample size estimated based on the predicted effect size. Specific details of power and sample size calculations for the assessment of statistical power are provided alongside results.

3.3. Ischaemia-reperfusion in vivo non-recovery model

A non-recovery in vivo model of ischaemia-reperfusion was also undertaken to permit assessment of myocardial infarct size following short durations of reperfusion. The surgical protocol was as described above (see 3.2.1) with minor modifications.

3.3.1. Non-recovery anaesthetics

It was not possible to undertake non-recovery surgery using isoflurane anaesthesia within our laboratory due to the constraints of ventilation systems and apparatus. A standard non-recovery injectable anaesthetic cocktail was therefore used for all non- recovery in vivo procedures undertaken. Mice were anaesthetised with an injectable anaesthetic cocktail containing ketamine (100 mg/kg [Vetlar, Bioniche Animal Health, Canada]), xylazine (20 mg/kg [Rompun, Bayer, UK]) and atropine (0.6 mg/kg [Sigma- Aldrich, UK]) administered as a single intraperitoneal bolus. Anaesthetic depth was monitored and maintained by additional anaesthetic doses. Ventilation stroke volume was 200 µl and stroke rate was 120 strokes/minute supplied with oxygen (1.5 L/minute).