Enhancing Cardiac Health: Lipoxin A4’s Role in Diabetes-Induced Cardiac Dysfunction | Cardiovascular Diabetology
Animals and Experimental Design
Table of Contents
Mice were used following protocols approved by the Alfred Medical Research and Education Precinct Animal Ethics Committee (E/1755/2017/B). The study aligned with National Health and Medical Research Council Australia guidelines. All mice were housed in the AMREP Animal Centre on a standard mouse chow diet with unlimited access to water. An age-matched group of 6-week-old ApoE−/− male mice (C57BL/6 background) was divided into diabetic and non-diabetic (ND) groups at the study’s start.
Diabetic mice received daily intraperitoneal (i.p.) injections of streptozotocin (STZ) for five days (55 mg/kg/day). The ND group received a vehicle control. Blood glucose levels and body weight were monitored weekly. Each group was further split into two subgroups, receiving either a treatment vehicle or LXA4 (5 µg/kg) via i.p. injection twice weekly from week 11 to week 16.
At the study’s end, diabetic status was confirmed through HbA1c levels (≥10%) and glucose levels (≥25 mmol/L). Mice with glucose levels ≤26 mmol/L but HbA1c levels ≥10% were also included.
Echocardiography
Left ventricular (LV) function was assessed one week before the endpoint using echocardiography. Mice were anesthetized with a cocktail of ketamine, xylazine, and atropine. Four imaging modes were used: B-mode, M-mode, pulsed wave Doppler, and tissue Doppler, employing a Vevo2100 imaging system. An experienced investigator validated the analyzed images.
Histology
The upper LV portion was embedded in paraffin, sectioned at 4 µm, and scanned for analysis. Picrosirius red staining determined myocardial fibrosis, while hematoxylin and eosin (H&E) staining assessed cardiomyocyte size. Collagen deposition percentages were calculated using ImageJ software.
The middle LV portion was frozen and sectioned at 6 µm. Immunofluorescence staining identified macrophages in the myocardium, utilizing specific antibodies. DAPI was used for nuclei staining, and autofluorescence was reduced with Sudan Black.
Gene Expression
RNA was extracted from the LV using a GenElute™ kit. cDNA was synthesized with a High-Capacity cDNA Reverse Transcription Kit. Quantitative real-time PCR (qRT-PCR) measured the expression of relevant genes using SYBR® Green Mix and mouse-specific primers. Comparative analysis used the 2−ΔΔCt method, normalizing against β-actin.
Western Blotting
LV tissue was extracted with RIPA buffer. Protein concentration was determined with a BCA protein assay kit. Proteins were loaded onto gels, transferred to membranes, and exposed to specific primary antibodies overnight. After secondary antibody treatment, images were captured and quantified.
Statistical Analysis
Data are presented as mean ± SEM. Experimental groups (ND + vehicle, ND + LXA4, diabetic + vehicle, diabetic + LXA4) were compared using two-way ANOVA with Fisher’s LSD post hoc test. A significance level of p < 0.05 was set for analysis.