# Calculation of Myocardial Percentage Collagen, Mast Cell Count and Presence of Arteriosclerosis in Dogs With Myxomatous Mitral Valve Disease and Control Dogs–A Pilot Study

## TL;DR

This pilot study tested methods to measure heart tissue changes in dogs with a heart valve disease and found no significant differences compared to healthy dogs.

## Contribution

The study established a feasible method to calculate myocardial collagen percentage and determined the sample size needed for future research.

## Key findings

- No significant difference in myocardial collagen percentage was found between dogs with and without MMVD.
- A sample size of at least 26 dogs per group is needed for future studies to detect potential differences.
- No significant association was found between collagen percentage and mast cell count in MMVD cases.

## Abstract

This study aimed to (1) determine the feasibility of calculating percentage myocardial collagen (PMC) in formalin‐fixed, paraffin‐embedded canine tissue samples, (2) explore relationships between PMC, mast cell (MC) count and myocardial arteriosclerosis/arteriolosclerosis (MA) and (3) calculate the sample size required to compare PMC between dogs with and without myxomatous mitral valve disease (MMVD).

Histological sections were prepared from retrospective samples of formalin‐fixed, wax‐embedded ventricular myocardial tissue from 15 dogs with, and seven dogs without, MMVD. Sections from each sample were stained with Masson's trichrome, haematoxylin and eosin (H&E) or toluidine blue. In each Masson's trichrome‐stained section, digital images of 12 random fields were captured and the total image area and total collagen area were measured using computer‐assisted morphometric analysis. Mean collagen area, mean total image area and mean PMC ([mean collagen area/mean total area] × 100) were calculated per sample. MCs were counted manually in toluidine blue‐stained sections and the mean MC count was calculated from 10 fields. MA was identified by visual inspection of transversely cut vessels in each H&E‐stained section.

No significant difference was detected between MMVD and control dogs in PMC (p = 0.063), MC (p = 0.476) or MA (p = 0.172). A sample size calculation suggests that data from at least 26 MMVD dogs and 26 controls are required to detect a difference in PMC. No significant association was detected between PMC and MC count (p = 0.606) in cases of MMVD.

The methods described are feasible and the sample size for a definitive study has been estimated.

This pilot study explored the feasibility of calculating myocardial percentage collagen (PMC), mast cell (MC) count and assessing arteriosclerosis in canine myocardial tissue. No significant difference in PMC, MC count or arteriosclerosis was detected between dogs with and without myxomatous mitral valve disease (MMVD). A sample size calculation suggests that at least 26 dogs per group are needed for future studies to determine whether a true difference in myocardial fibrosis exists.