CardiLect: A combined cross-species lectin histochemistry protocol for the automated analysis of cardiac remodelling

Abstract

Background: Cardiac remodelling, a crucial aspect of heart failure, is commonly investigated in preclinical models by quantifying cardiomyocyte cross-sectional area (CSA) and microvascular density (MVD) via histological methods, such as immunohistochemistry. To achieve this, optimized protocols are needed, and the species specificity is dependent on the antibody used. Lectin histochemistry offers several advantages compared to antibody-based immunohistochemistry, including as cost-effectiveness and cross-species applicability. Direct comparisons between the two methods are lacking from the literature.

Methods and results: In this study, we compared antibody- and lectin-based methods for the histological assessment of cardiomyocyte CSA (with the use of anti-laminin and wheat germ agglutinin [WGA]) and microvascular density (utilizing anti-CD31 and isolectin B4 [ILB4]) using different embedding and antigen/carbohydrate retrieval techniques. Here, we describe a detailed, easy-to-use combined lectin histochemistry protocol (WGA and ILB4, 'CardiLect' protocol) for the histological assessment of cardiac remodelling. The lectin-based approach has been evaluated on a cross-species basis, and its efficacy has been demonstrated in zebrafish, rodents, large animals and human samples. We provide an ImageJ script ('CardiLect Analyser') for automated image analysis, validated in a preclinical heart failure model by correlating histological parameters with echocardiographic findings. CSA showed a significant positive correlation with left ventricular (LV) mass (P = 0.0098, r_S = 0.7545) and significant negative correlation with markers of systolic function, such as ejection fraction (EF) (P = 0.0402, r_S = -0.6364). Microvascular density showed significant negative correlation with LV mass (P = 0.0055, r_S = -0.7622) and significant positive correlation with EF (P = 0.0106, r_S = 0.7203).

Conclusions: The described combined lectin histochemistry protocol with the provided ImageJ script is an easy-to-use, cost-effective, cross-species approach for the histological assessment of cardiac remodelling.

Keywords: Cardiac remodelling; Echocardiography; Heart failure; Immunohistochemistry; Lectin; Transverse aortic constriction.

Figure 1

Embedding and antigen retrieval methods affect laminin staining in rat heart. Hearts from control and TAC subjected rats were cut frozen or paraffin embedded and subjected to citrate, Tris or proteinase K‐based antigen retrieval. Sections were then stained for laminin in 1:50, 1:100 or 1:200 dilution. Representative images are shown. Scale bars = 100 μm. FFPE, formalin fixed paraffin embedded; TAC, transverse aortic constriction.

Figure 2

Embedding and antigen retrieval methods affect WGA staining in rat heart. Hearts from control and TAC subjected rats were cut frozen or paraffin embedded and subjected to citrate, Tris or proteinase K‐based antigen retrieval. Sections were then stained with WGA lectin in 1:50, 1:100 or 1:200 dilution. Representative images are shown. Scale bars = 100 μm. FFPE, formalin fixed paraffin embedded; TAC, transverse aortic constriction.

Figure 3

Embedding and antigen retrieval methods affect CD31 staining in rat heart. Hearts from control and TAC subjected rats were paraffin embedded and subjected to citrate, Tris or proteinase K‐based antigen retrieval. Sections were then stained for CD31 in 1:50, 1:100 or 1:200 dilution. Representative images are shown. Scale bars = 100 μm. FFPE, formalin fixed paraffin embedded; TAC, transverse aortic constriction.

Figure 4

Embedding and antigen retrieval methods affect ILB4 staining in rat heart. Hearts from control and TAC subjected rats were paraffin embedded and subjected to citrate, Tris or proteinase K‐based antigen retrieval. Sections were then stained with ILB4 lectin in 1:50, 1:100 or 1:200 dilution. Representative images are shown. Scale bars = 100 μm. FFPE, formalin fixed paraffin embedded; TAC, transverse aortic constriction.

Figure 5

Representative images of combined lectin histochemistry in rats and mice subjected to either sham or TAC surgery. Scale bars = 100 μm. TAC, transverse aortic constriction.

Figure 6

The CardiLect Analyser automatically measure the cross‐sectional area and count the capillary density. The data are saved to a csv file and can be further analysed statistically. The flowchart illustrates the functioning of the script step‐by‐step. Refer to the Methods section for further details.

Figure 7

Correlation between echocardiographic parameters of LV remodelling and histological parameters measured by CardiLect Analyser after lectin histochemistry. (A) Rats were subjected to sham or TAC surgery. Echocardiography and lectin histochemistry were carried out after 14–16 weeks of follow‐up. (B) Representative M‐mode echocardiographic images of sham and TAC‐operated animals after follow‐up. (C) Echocardiographic parameters of LV remodelling showing increased LV mass and decreased systolic function after TAC surgery. *P < 0.05, unpaired t‐test (n = 4 for sham, n = 7 for TAC groups; n represents the number of animals). (D) Cardiomyocyte cross‐sectional area (CSA) and microvascular density (MVD), histological parameters of LV remodelling, were significantly altered after TAC surgery, as measured by CardiLect Analyser after lectin histochemistry. (E,F) CSA and MVD, as measured by CardiLect Analyser, significantly correlate with echocardiographic parameters of LV remodelling (P values represents statistical significance of Spearman's correlation test). CSA, cross‐sectional area; EF, ejection fraction; FS, fractional shortening; LV, left ventricle; LVRi, left ventricular remodelling index; MVD, microvascular density; TAC, transverse aortic constriction.

Figure 8

Representative images of combined lectin histochemistry in cardiac tissues of hamsters, landrace pigs, zebrafish embryo and humans. Scale bars = 100 μm, unless specified otherwise.