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

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 (α-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 α-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 α-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 α-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

Comparison of different methods to obtain and store liver biopsies for molecular and histological research

BACKGROUND: To minimize the necessary number of biopsies for molecular and histological research we evaluated different sampling techniques, fixation methods, and storage procedures for canine liver tissue. For addressing the aim, three biopsy techniques (wedge biopsy, Menghini, True-cut), four storage methods for retrieval of RNA (snap freezing, RNAlater, Boonfix, RLT-buffer), two RNA isolation procedures (Trizol and RNAeasy), and three different fixation protocols for histological studies (10% buffered formalin, RNAlater, Boonfix) were compared. Histological evaluation was based on hematoxylin-eosin (HE) and reticulin (fibrogenesis) staining, and rubeanic acid and rhodanine stains for copper. Immunohistochemical evaluation was performed for cytokeratin-7 (K-7), multidrug resistance binding protein-2 (MRP-2) and Hepar-1. RESULTS: RNA quality was best guaranteed by the combination of a Menghini biopsy with NaCl, followed by RNAlater preservation and RNAeasy mini kit extraction. These results were confirmed by quantitative RT-PCR testing. Reliable histological assessment for copper proved only possible in formalin fixed liver tissue. Short formalin fixation (1-4 hrs) improved immunohistochemical reactivity and preservation of good morphology in small liver biopsies. CONCLUSION: At least two biopsies (RNAlater and formalin) are needed. Since human and canine liver diseases are highly comparable, it is conceivable that the protocols described here can be easily translated into the human biomedical field

Regenerative and fibrotic pathways in canine hepatic portosystemic shunt and portal vein hypoplasia, new models for clinical hepatocyte growth factor treatment

BACKGROUND: We analyzed two spontaneous dog diseases characterized by subnormal portal perfusion and reduced liver growth: (i) congenital portosystemic shunts (CPSS) without fibrosis and (ii) primary portal vein hypoplasia (PPVH), a disease associated with fibrosis. These pathologies, that lack inflammation or cholestasis, may represent simplified models to study liver growth and fibrosis. To investigate the possible use of those models for hepatocyte growth factor (HGF) treatment, we studied the functionality of HGF signaling in CPSS and PPVH dogs and compared this to aged-matched healthy controls. RESULTS: We used quantitative real-time polymerase chain reaction (Q-PCR) to analyze the mRNA expression of HGF, transforming growth factor β1 (TGF-β1), and relevant mediators in liver biopsies from cases with CPSS or PPVH, in comparison with healthy control dogs. CPSS and PPVH were associated with a decrease in mRNA expression of HGF and of MET proto-oncogene (c-MET). Western blot analysis confirmed the Q-PCR results and showed that intracellular signaling components (protein kinase B/Akt, ERK1/2, and STAT3) were functional. The TGF-β1 mRNA levels were unchanged in CPSS whereas there was a 2-fold increase in PPVH indicating an active TGF-β1 pathway, consistent with the observation of fibrosis seen in PPVH. Western blots on TGF-β1 and phosphorylated Smad2 confirmed an activated pro-fibrotic pathway in PPVH. Furthermore, Q-PCR showed an increase in the amount of collagen I present in PPVH compared to CPSS and control, which was confirmed by Western blot analysis. CONCLUSION: The pathophysiological differences between CPSS and PPVH can adequately be explained by the Q-PCR measurements and Western blots. Although c-MET levels were reduced, downstream signaling seemed to be functional and provides a rational for HGF-supplementation in controlled studies with CPSS and PPVH. Furthermore both diseases may serve as simplified models for comparison with more complex chronic inflammatory diseases and cirrhosis

Effects of dental probing on occlusal surfaces - A scanning electron microscopy evaluation

The aim of this clinical-morphological study was to investigate the effects of dental probing on occlusal surfaces by scanning electron microscopy (SEM). Twenty sound occlusal surfaces of third molars and 20 teeth with initial carious lesions of 17- to 26-year-old patients (n = 18) were involved. Ten molars of each group were probed with a sharp dental probe (No. 23) before extraction; the other molars served as negative controls. After extraction of the teeth, the crowns were separated and prepared for the SEM study. Probing-related surface defects, enlargements and break-offs of occlusal pits and fissures were observed on all occlusal surfaces with initial carious lesions and on 2 sound surfaces, respectively. No traumatic defects whatsoever were visible on unprobed occlusal surfaces. This investigation confirms findings of light-microscopic studies that using a sharp dental probe for occlusal caries detection causes enamel defects. Therefore, dental probing should be considered as an inappropriate procedure and should be replaced by a meticulous visual inspection. Critical views of tactile caries detection methods with a sharp dental probe as a diagnostic tool seem to be inevitable in undergraduate and postgraduate dental education programmes. Copyright (c) 2007 S. Karger AG, Basel

The establishment and characterization of the first canine hepatocellular carcinoma cell line, which resembles human oncogenic expression patterns

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most worldwide frequent primary carcinomas resulting in the death of many cirrhotic patients. Unfortunately, the molecular mechanisms of this cancer are not well understood; therefore, we need a good model system to study HCC. The dog is recognized as a promising model for human medical research, namely compared with rodents. The objective of this study was to establish and characterize a spontaneous canine tumor cell line as a potential model for studies on HCC. RESULTS: Histomorphological, biochemical, molecular biological and quantitative assays were performed to characterize the canine HCC cell line that originated from a dog with a spontaneous liver tumor. Morphological investigations provided strong evidence for the hepatocytic and neoplastic nature of the cell line, while biochemical assays showed that they produced liver-specific enzymes. PCR analysis confirmed expression of ceruloplasmin, alpha-fetoprotein and serum albumin. Quantitative RT-PCR showed that the canine HCC cell line resembles human HCC based on the measurements of expression profiles of genes involved in cell proliferation and apoptosis. CONCLUSIONS: We have developed a novel, spontaneous tumor liver cell line of canine origin that has many characteristics of human HCC. Therefore, the canine HCC cell line might be an excellent model for comparative studies on the molecular pathogenesis of HCC