Poster R14 - Quantifying Intramyocardial Blood Volume using µ-SPECT Imaging as a Measure of Coronary Microvascular Function in Mice

Introduction: Coronary microvascular disease (CMVD) affects the small blood vessels of the heart and accounts for 30-50% of ischemic heart disease. Progress in the field requires new quantitative methods to assess the coronary microvasculature in preclinical models. We are developing µ-SPECT imaging methods to measure coronary microvascular function in mice. We have previously reported the feasibility of measuring intramyocardial blood volume (IMBV) using red blood cells labeled with Technetium-99m pyrophosphate. The relative increase in IMBV seen under hyperemic or stress conditions reflects coronary microvascular function or vasodilatory capacity. Here, we report our experience using these methods to measure change in IMBV in several mouse models of cardiovascular disease including high fat diet, aging, and hypercholesterolemia.

Materials and

Methods: Male and female C57BL/6 mice aged 12 to 42 weeks (n=29) were treated with one or more cardiovascular risk factors including a 45% high fat diet for six months, aged greater than 9 months, and hypercholesterolemia using Apolipoprotein E knockout (ApoE-/-) mice. As adeno-associated virus (AAV) mediated overexpression is an effective way to evaluate gene expression in mouse models, we also assessed the effects of AAV-mediated green fluorescent protein (GFP) overexpression on IMBV. We injected mice with pyrophosphate , followed by Tc99m-pertechnatate for in vivo red blood cell labelling. We then acquired µ-SPECT images under hyperemic (stress) conditions (2.5% isoflurane) and basal rest conditions (1.25% isoflurane). In a subset of mice, we compared 3% to 2.5% isoflurane to evaluate hyperemic conditions. We quantified myocardial activity concentration using an in-house developed MATLAB software. The percent change in activity concentration between rest and stress conditions is the change in IMBV or △IMBV = (IMBV[stress] –IMBV[rest])/ IMBV[rest] and reflects coronary microvascular function. Outliers were removed based on Grubbs method with alpha significance of 0.1. For statistics, we performed Student's T test for direct comparisons between male and females and used ANOVA with multiple comparisons. p< 0.05 was considered significant.

Results, Conclusions, and Discussions: We present an updated protocol for quantifying △IMBV using µ-SPECT. In general, IMBV was quantifiable in >90% of mice, and outliers were often due to mouse motion or poor image quality. We compared males (n=3) and females (n=4) each in the high fat diet and aged cohorts and found no significant sex differences (18% vs 19% , p=0.79 and 17% vs 19%, p=0.74, respectively). We found no significant difference for IMBV between 2.5% and 3% isoflurane (p =0.25), therefore, 2.5% isoflurane was used throughout. Cardiovascular disease risk factors generally lowered △IMBV (Figure). High fat diet had a 0.75-fold reduction on △IMBV (23% ± 3.4 vs 17% ± 2.4, p=0.03). Aging had a similar reduction in △IMBV 0.73-fold (17% ± 2.7, p=0.01). ApoE-/- induces hypercholesterolemia, another well-established cardiovascular risk factor. ApoE-/- mice fed a high fat diet had a 0.48-fold reduction in △IMBV (11% ± 2.3, p< 0.01), and ApoE-/- mice aged to 10 months had a 0.54-fold decrease in △IMBV (12% ± 2.6, p< 0.01). AAV-GFP did not significantly reduce △IMBV (18% ± 5.6, p=0.13) suggesting a mild but not statistically significant decrease in microvascular function with GFP overexpression.

µ-SPECT-based imaging methods using commercially available tracers offer novel methods to quantify coronary microvascular function in mice. We saw no sex-based difference in △IMBV in our cohorts with one cardiovascular risk factor, consistent with human cardiac perfusion imaging PET data. Mouse models of cardiovascular disease using established diet induced obesity, aging, and hypercholesterolemic risk factors reduced △IMBV, and exposure to two cardiovascular risk factors had additive effect on △IMBV. AAV-GFP had a mild and non-significant effect on △IMBV. △IMBV may be helpful in evaluating mouse models of CMVD and assessing the effects on different interventions. Additional studies are needed to understand the test-retest performance of △IMBV, and the interaction between imaging sensitivity and cardiovascular disease.

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