Long-term histological effects of high-dose prednisolone administration on the mitral valve in normal Beagle dogs

Abstract

Background: In recent years, left ventricular hypertrophy and cardiac dysfunction have been reported in human and canine patients with hypercortisolism and in dogs treated experimentally with high-dose prednisolone. However, to our knowledge, there have been no reports on the effects of hyperglucocorticism (HGC) on the mitral valve (MV).

Aim: This study aimed to compare the MV in dogs treated with high-dose prednisolone with that in healthy dogs to investigate the effects of HGC on the MV.

Methods: We investigated the effects of HGC on the MV by comparing samples obtained from high-dose glucocorticoid (GC)-treated (P) and healthy (C) dogs. The P group included healthy Beagle dogs (n = 6) treated with prednisolone (2 mg/kg, bid, po) for 84 days and the C group included healthy Beagle dogs (n = 6) euthanized for unrelated reasons. The anterior and posterior mitral leaflets (AML and PML, respectively) from both groups were harvested and stained with hematoxylin-eosin, Alcian blue, and Masson trichome. Additionally, adiponectin (ADN) and GC receptor immunohistochemistry were performed. Histological evaluation was performed in the atrialis, spongiosa, fibrosa, and all layers of the proximal, middle, and distal regions of the AML and PML.

Results: The proportion of the spongiosa layer thickness to the total thickness was higher in the P than in the C group (proximal and middle AML). However, the proportion of the fibrosa layer thickness to the total thickness was lower in the P than in the C group (middle PML). Areas of acidic sulfated mucosubstance deposition were smaller in the fibrosa layer and all layers (middle AML), while those of collagen deposition were smaller in the spongiosa and total layers (proximal and middle AML), in the P than in the C group. Additionally, ADN expression in the spongiosa layer was higher in the P than in the C group (middle AML).

Conclusion: These findings suggest that long-term administration of synthetic GCs induces histological changes in the MV. These changes may lead to MV dysfunction in dogs with HGC.

Keywords: Anterior mitral leaflet; Canine; Cushing syndrome; Hyperglucocorticism; Posterior mitral leaflet.

Fig. 1.. Image showing each layer (atrialis, spongiosa, and fibrosa layer) used for measurement in a HE-stained sample in the proximal of the AML. (A) Atrialis layer; (B) spongiosa layer; and (C) fibrosa layer. Scale bar: 100 μm.

Fig. 2.. HE-stained sample images of the MV (AML and PML) in the control and prednisolone groups. C group: Control group; P group: Prednisolone group; blue bar: atrialis layer; yellow bar: spongiosa layer; green bar: fibrosa layer; *: in univariable linear regression, the percentage (%) of the total layer occupied by the spongiosa layer was higher in group P than in group C at the proximal (p = 0.018) and middle AML (p = 0.011). In contrast, the percentage of the fibrosa layer thickness to the total layer thickness (%) was lower in group P than in group C in the middle PML region (p = 0.047) (Table 2); †: in univariable linear regression, cell counts in the fibrosa layer were lower in group P than in group C at the proximal AML (p = 0.033), in the spongiosa layer (p = 0.046), and overall (p = 0.049) at the middle AML region (Table 4). Scale bar: 100 μm.

Fig. 3.. AB-stained sample images of the MV (AML and PML) in the control and prednisolone groups. The light blue stained areas indicate the presence of acidic sulfated mucosubstances. C group: Control group; P group: Prednisolone group; blue bar: atrialis layer; yellow bar: spongiosa layer; green bar: fibrosa layer; *: in univariable linear regression, the percentage (%) of the positively stained area was lower in group P than in group C in the fibrosa layer of the middle AML (p = 0.031) and the total layer of the middle AML (p = 0.023) (Table 6). Scale bar: 100 μm.

Fig. 4.. MT-stained sample images of the MV (AML and PML) in the control and prednisolone groups. The blue-stained areas indicate the presence of collagen-rich deposits. C group: Control group; P group: Prednisolone group; blue bar: atrialis layer; yellow bar: spongiosa layer; green bar: fibrosa layer; *: univariable linear regression analysis of the percentage of the positively stained area (%) was lower in group P than in group C in the spongiosa layer and total layers of the proximal AML (p = 0.032 and 0.004, respectively); in the total layer of the middle of the AML (p = 0.004); in the fibrosa layer and total layers of the proximal PML (p = 0.006 and 0.004, respectively); in the fibrosa layer and total layers in the middle PML (p = 0.009 and 0.002, respectively); and in the fibrosa layer and total layers in the distal of the PML (p = 0.041, 0.034) (Table 8). Scale bar: 100 μm.

Fig. 5.. ADN-immunohistochemistry images of the MV (AML and PML) in the control and prednisolone groups. Arrowheads show ADN-positive areas in the cytoplasm of mature adipocytes in the spongiosa layer. C group: Control group; P group: Prednisolone group; blue bar: atrialis layer; yellow bar: spongiosa layer; green bar: fibrosa layer; *: in univariable linear regression the percentage of the ADN-positive areas in the spongiosa layer was higher in group P than in group C in the middle of the AML (p = 0.016) (Table 9). Scale bar: 100 μm.

Fig. 6.. GCR immunohistochemistry images of the MV (AML and PML) in the control and prednisolone groups. Arrowheads show GCR-positive nuclei in the spongiosa (mainly expressed in adipocytes and cardiomyocytes) and fibrosa layer. C group: Control group; P group: Prednisolone group; blue bar: atrialis layer; yellow bar: spongiosa layer; green bar: fibrosa layer; *: univariable linear regression analysis of the percentage of GCR-positive nuclei (%) was lower in group P than in group C in the fibrosa layer in the proximal AML (p = 0.039); in the spongiosa layer in the distal AML (p = 0.034); in the fibrosa and total layers in the proximal PML (p = 0.031 and 0.031, respectively) (Table 12). GCR was expressed in the spongiosa and fibrosa layers of the AML and PML in groups C and P. Scale bar: 100 μm.