This study used 21 male Wistar rats, which are commonly used in swimming-based cardiovascular experiments. Animals were stratified into three age cohorts: juvenile (3–4 months), adult (12–13 months), and geriatric (18–19 months), with 7 rats per cohort. These age cutoffs were defined based on established rodent cardiac aging literature. Juvenile rats (1–3 months correspond to young adults with preserved cardiomyocyte proliferative capacity and minimal baseline fibrosis. Adult rats represent mature animals at peak contractile performance with established extracellular matrix homeostasis but declining regenerative potential. Geriatric rats exhibit age-related fibrotic accumulation, increased myocardial stiffness, and reduced adaptive reserve. All animals underwent the same 6-week resistance-loaded swimming protocol. No sedentary control group was included, as the primary objective was to characterize age-dependent responses to a standardized overload stimulus rather than to compare exercise versus rest within each age group. Animals were housed in a conventional laboratory facility at 22±2°C with relative humidity maintained within the accepted mammalian laboratory range, under a 12:12-hour reversed light-dark cycle with the dark phase scheduled from 07:00 to 19:00 to match exercise timing. Standard pellet chow and water were provided "ad libitum", and animals were acclimatized for 7 days before the intervention. Experimental conduct and husbandry were aligned with the Guide for the Care and Use of Laboratory Animals and with ARRIVE 2.0 reporting principles. 11,12 Randomization to age cohorts was based on date of birth. No animals were excluded from the analysis, and no adverse events requiring early termination occurred during the study period.

​

Aquatic adaptation and resistance-loaded swimming

To reduce non-exercise stress, rats were acclimatized to shallow warm water during the week preceding training. Swimming was then performed individually in a cylindrical tank filled to a depth sufficient to prevent tail support, with water maintained at 30-32°C. Rats swam for 60 minutes daily, 7 days per week, for 6 weeks. A lead weight equal to 4% of each rat's body weight was attached to the proximal tail at the start of each session and adjusted weekly after weighing. The selected water temperature and duration were based on established rat swimming protocols. In contrast, the external load was chosen to create a resistance-dominant stimulus rather than free-swimming endurance alone. 6-8,13-15

 

Tissue collection and histological processing

Twenty-four hours after the final training session, rats were deeply anesthetized with intraperitoneal sodium pentobarbital (60 mg/kg) and euthanized by exsanguination followed by thoracotomy. Hearts were rapidly excised, rinsed in cold phosphate-buffered saline, blotted dry, weighed, and fixed in 10% neutral buffered formalin for 24 to 48 hours.

​

Transverse left ventricular sections were obtained at three standardized levels: basal, mid-ventricular, and apical. Sections were paraffin-embedded, cut, and stained with hematoxylin-eosin (H&E) for cellular injury and vascular congestion, and with Masson trichrome for collagen deposition.

​

Microscopic examination was performed systematically across four left ventricular regions: the anterior wall, lateral wall, posterior wall, and interventricular septum. This systematic approach ensured that regional variation in injury burden was captured without post hoc selection bias.

​

Histopathological assessment

Two blinded observers evaluated all histological slides. Discrepancies in scoring were resolved by consensus review with a third observer.

​

For fibrosis quantification, five non-overlapping fields per region (20 fields per heart) were captured from Masson trichrome-stained sections at 200× magnification using a digital microscope camera. Collagen area fraction (percentage of blue-stained area relative to total myocardial tissue area) was calculated using ImageJ software with a standardized color thresholding protocol.

​

Cardiomyocyte injury was evaluated on H&E-stained sections and categorized semiquantitatively as: Grade 0 (none), Grade 1 (mild, scattered hypereosinophilic myocytes), Grade 2 (moderate, multifocal necrosis with inflammatory infiltrate), or Grade 3 (severe, confluent areas of necrosis). Vascular congestion was graded semiquantitatively as Grade 0 (absent), Grade 1 (mild), Grade 2 (moderate), or Grade 3 (severe) based on the degree of capillary and venous dilatation.

​

Statistical analysis

Data are presented as mean ± standard deviation (SD). Semiquantitative injury and congestion grades were compared using the Kruskal-Wallis test with Dunn's post-hoc correction. A two-sided p-value <0.05 was considered statistically significant. All analyses were performed using GraphPad Prism software (version 9.0).