Morphological characterisation of portal myofibroblasts and hepatic stellate cells in the normal dog liver

Abstract

Background: Hepatic fibrosis is a common outcome of hepatic injury in both man and dog. Activated fibroblasts which develop myofibroblastic characteristics play an essential role in hepatic fibrogenesis, and are comprised of three subpopulations: 1) portal or septal myofibroblasts, 2) interface myofibroblasts and 3) the perisinusoidally located hepatic stellate cells (HSC). The present study was performed to investigate the immunohistochemical characteristics of canine portal myofibroblasts (MF) and HSC in the normal unaffected liver as a basis for further studies on fibrogenesis in canine liver disease.

Results: In the formalin-fixed and paraffin embedded normal canine liver vimentin showed staining of hepatic fibroblasts, probably including MF in portal areas and around hepatic veins; however, HSC were in general negative. Desmin proved to react with both portal MF and HSC. A unique feature of these HSC was the positive immunostaining for alpha-smooth muscle actin (alpha-SMA) and muscle-specific actin clone HHF35 (HHF35), also portal MF stained positive with these antibodies. Synaptophysin and glial fibrillary acidic protein (GFAP) were consistently negative in the normal canine liver. In a frozen chronic hepatitis case (with expected activated hepatic MF and HSC), HSC were negative to synaptophysin, GFAP and NCAM. Transmission electron microscopy (TEM) immunogold labelling for alpha-SMA and HHF35 recognized the positive cells as HSC situated in the space of Disse.

Conclusion: In the normal formalin-fixed and paraffin embedded canine liver hepatic portal MF and HSC can be identified by alpha-SMA, HHF35 and to a lesser extent desmin immunostaining. These antibodies can thus be used in further studies on hepatic fibrosis. Synaptophysin, GFAP and NCAM do not seem suitable for marking of canine HSC. The positivity of HSC for alpha-SMA and HHF35 in the normal canine liver may eventually reflect a more active regulation of hepatic sinusoidal flow by these HSC compared to other species.

Figure 1

Vimentin. Normal canine liver, stained with vimentin antibody. a) In the portal area, smooth muscle cells of portal vasculature, most spindle-shaped stromal cells and neural cells (arrows) are positive. b) HSC were generally negative, although some individual positive cells are present (arrows).

Figure 2

Desmin. Normal canine liver, stained with desmin antibody. a) A HSC (right) is positive in the perinuclear cytoplasm, weakly extending into a cytoplasmic process; also a negative vitamin A-storing HSC (arrow) is present. b) In the portal area, a moderate to strong staining is present in the smooth muscle cells of the arterial tunica muscularis and in the perivenular smooth muscle cells of the portal vein. HSC are weakly positive in the perinuclear cytoplasm.

Figure 3

α-SMA. Normal canine liver, stained with α-SMA antibody. a) Portal areas show positivity around the bile ducts, in the arterial tunica media, and in the wall of the portal veins. There is slightly irregular moderate staining in the perisinusoidal spaces throughout the parenchyma. b) HSC stain positive, producing a thin irregular positive band lining the sinusoids. c) HSC stain positive. A positive cell containing one large vacuole (arrow-head is placed in vacuole) and a dislocated nucleus is seen. d) In the portal area there is strong positivity around the bile ducts and in the arterial tunica media, and moderate positivity in the wall of the portal veins, while endothelial cells remain negative (horizontal arrow). A portal MF with moderate positivity (vertical arrow) is present.

Figure 4

HHF35 (muscle-specific actin, clone HHF35). Normal canine liver, stained with HHF35 antibody. a) HSC stain positive. Cells with few, small vacuoles stain positive (arrowhead). b) HSC stain positive. A vitamin A-storing HSC is negative (arrow).

Figure 5

TEM of α-SMA. Immunogold labelling for α-SMA. The HSC is located in Disse's space between the endothelial cell and the hepatocyte. It has subendothelial cytoplasmic extensions and a prominent large lipid vacuole. The positive signal is present in the extensions (arrows). E = endothelial cell, HSC = hepatic stellate cell, L = lipid vacuole, S = sinusoid.

Figure 6

TEM of HHF35 (muscle-specific actin, clone HHF35). Immunogold labelling for HHF35. The positive signal (arrows) is present in the subendothelial cellular extensions of the hepatic stellate cell situated in Disse's space. E = endothelial cell, H = hepatocyte, MV = hepatocytic microvilli, S = sinusoid.