Guy C. M. Grinwis

The Dog as an Animal Model for Intervertebral Disc Degeneration

Study Design. Prospective observational and analytic study. Objective. To investigate whether spontaneous intervertebral disc degeneration (IVDD) occurring in both chondrodystrophic (CD) and nonchondrodystrophic dogs (NCD) can be used as a valid translational model for human IVDD research. Summary of Background Data. Different animal models are used in IVDD research, but in most of these models IVDD is induced manually or chemically rather than occurring spontaneously. Methods. A total of 184 intervertebral discs (IVDs) from 19 dogs of different breeds were used. The extent of IVDD was evaluated by macroscopic grading, histopathology, glycosaminoglycan content, and matrix metalloproteinase 2 activity. Canine data were compared with human IVD data acquired in this study or from the literature. Results. Gross pathology of IVDD in both dog types (CD and NCD) and humans showed many similarities, but the cartilaginous endplates were significantly thicker and the subchondral cortices significantly thinner in humans than in dogs. Notochordal cells were still present in the IVDs of adult NCD but were not seen in the CD breeds or in humans. Signs of degeneration were seen in young dogs of CD breeds (<1 year of age), whereas this was only seen in older dogs of NCD breeds (5–7 years of age). The relative glycosaminoglycan content and metalloproteinase 2 activity in canine IVDD were similar to those in humans: metalloproteinase 2 activity increased and glycosaminoglycan content decreased with increasing severity of IVDD. Conclusion. IVDD is similar in humans and dogs. Both CD and NCD breeds may therefore serve as models of spontaneous IVDD for human research. However, as with all animal models, it is important to recognize interspecies differences and, indeed, the intraspecies differences between CD and NCD breeds (early vs. late onset of IVDD, respectively) to develop an optimal canine model of human IVDD.

Intervertebral disc degeneration in the dog. Part 2: Chondrodystrophic and non-chondrodystrophic breeds

Dogs can be grouped into two distinct types of breed based on the predisposition to chondrodystrophy, namely, non-chondrodystrophic (NCD) and chondrodystrophic (CD). In addition to a different process of endochondral ossification, NCD and CD breeds have different characteristics of intravertebral disc (IVD) degeneration and IVD degenerative diseases. The anatomy, physiology, histopathology, and biochemical and biomechanical characteristics of the healthy and degenerated IVD are discussed in the first part of this two-part review. This second part describes the similarities and differences in the histopathological and biochemical characteristics of IVD degeneration in CD and NCD canine breeds and discusses relevant aetiological factors of IVD degeneration.

Estrogenic effects in fish in The Netherlands: some preliminary results

Recently, a large-scale field study in The Netherlands has focused on the effects of estrogenic contaminants on feral fish populations. The freshwater bream (Abramis brama) and the estuarine flounder (Platichthys flesus) were sampled at a large number of locations in the spring and autumn of 1999. Concentrations of the yolk protein vitellogenin (VTG) in blood plasma of male flounders were small at most sites. At two sites, however, moderately elevated concentrations were found in autumn. Both sites were situated in the same industrial harbour zone also receiving effluent from sewage treatment works. At many sites VTG levels in male bream were significantly greater than at the control site. The greatest concentrations were observed in individuals collected from a small stream, close to the discharge of a relatively large municipal waste water treatment plant. This was also the only site where considerable intersex occurred; 37% of male bream exhibited ovotestes. Ovotestis was not observed in any of the male flounder captured. The results from The Netherlands are briefly discussed and compared with the well-known case studies in the UK.

Aquatic toxicology:: opportunities for enhancement through histopathology

This paper briefly reviews the application of histopathology as aninstrument or endpoint in toxicity studies in fish. For long this has been applied rather occasionally in (regulatory) toxicology, and was mainly of interest in fundamental studies and limited carcinogenicity experiments. However, nowadays there are various incentives that ask for the application of pathology, such as field monitoring of pollution effects, the wish for optimal use and lower species of laboratory animals, the availability of modern histology techniques, and insight and interest in mechanistic data. This is timely illustrated by the current broad interest in endocrine disrupting pollutants-a threat mainly in the aquatic environment-where histopathological organ and tissue changes in intact sentinel fish species provide pivotal diagnostic and mechanistic features.

Toxicity of TCDD in European flounder (Platichthys flesus) with emphasis on histopathology and cytochrome P450 1A induction in several organ systems

The present study is part of a series of experiments, set up to elucidate the impact of aquatic pollution on fish health in the marine and estuarine environment. In the Dutch coastal and estuarine waters, European flounder (Platichthys flesus) showed a relatively high prevalence of (pre)neoplastic liver lesions and lymphocystis virus disease. The hypothesis of a causal relationship between pollution and these diseases was supported by semi-field experiments. Therefore a series of laboratory experiments was performed to further substantiate causality and identify the xenobiotics that may play a major role in the field. Polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) are important environmental pollutants. They are highly persistent, highly lipophilic, and have shown to induce several toxic effects in mammalian and non-mammalian species at relatively low concentrations. This report describes a study in which European flounder were orally exposed to the most toxic PCDD congener, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or to harbor sludge extract under controlled laboratory conditions. The effects on several organs (liver, gills, gastro-intestinal tract, thyroid gland, gonads, spleen and mesonephros) were examined microscopically. Induction and localization of cytochrome P4501A (CYP1A) immunoreactivity, and effects on hepatocyte-proliferation were visualized immunohistochemically. Effects on thymus size were examined by morphometric analysis. Oral exposure of flounder to 0.0125 or 0.3125 µg TCDD/kg bw, or to 0.3125 µg TEQ/kg bw of a harbor sludge extract, weekly for 8 weeks, induced a significant increase in CYP1A immunoreactivity in hepatocytes. Single administration of higher doses (20, 100 and 500 µg/kg bw) of TCDD also induced a significant increase CYP1A immunoreactivity in the endothelium in all organs examined, and in the epithelium of the digestive tract, liver, and mesonephros. Remarkably, strong immunoreactivity was noted in a distinct cell population of the hematopoietic tissue in the mesonephros and spleen, which has not been described in fish previously. Moreover, oral exposure to 20 µgTCDD/kg bw resulted in an increased mitotic activity, and an increased hepatosomatic index was found after exposure to 500 µgTCDD/kg bw. In the thymus only a trend in size reduction was noted, again in the highest dose group. Nevertheless, no marked pathology was detected even in fish exposed to a single dose of 500 µg TCDD/kg body weight. The present experiments show that, under the actual experimental conditions, European flounder is relatively insensitive to the toxic effects of TCDD. However, we assume that exposure to TCDD (and related substances) may promote the development of tumors in the field.

Short-term toxicity of bis(tri-n-butyltin)oxide in flounder (Platichthys flesus) : Pathology and immune function

Abstract The present study is part of a project that focuses on the relationship between environmental pollution and fish diseases. Field studies in various polluted coastal areas in Europe and the United States of America clearly indicate a relationship between pollution and the increase in prevalence of tumours and infectious diseases in fish. Research under controlled laboratory conditions is necessary to prove causal links between specific xenobiotics and disease prevalence. One of the chemicals of interest in the myriad of xenobiotics found in polluted waters and sediments is the organotin compound tributyltin (TBT), originating mainly from antifouling paints used on the hulls of ships. This report describes a study in which flounders (Platichthys flesus) were exposed to bis(tri-n-butyltin)oxide (TBTO) in the water under controlled laboratory conditions. The effects on several organs (gills, skin, eye, liver, mesonephros, ovary/testis, spleen, and gastrointestinal tract) were examined using histopathology, and morphometric analysis of the thymus was performed to assess the target organ(s) for TBTO in this fish species. Also the function of the non-specific and specific resistance was studied using ex vivo/in vitro immune function tests. Exposure of flounder to TBTO, in concentrations which were in the same order of magnitude as maximum TBT levels measured in the field (experiment: 17.3 μg TBT/l; field: 7.2 μg TBT/l), caused mortality after 7–12 days, resulted in gill lesions, and induced significant reduction of the non-specific resistance. A significant decrease of the relative thymus volume, but no marked effects on the specific immune system were noted after exposure to TBTO.

Intervertebral disc degeneration in the dog. Part 1: Anatomy and physiology of the intervertebral disc and characteristics of intervertebral disc degeneration

Intervertebral disc (IVD) degeneration is common in dogs and can give rise to a number of diseases, such as IVD herniation, cervical spondylomyelopathy, and degenerative lumbosacral stenosis. Although there have been many reports and reviews on the clinical aspects of canine IVD disease, few reports have discussed and reviewed the process of IVD degeneration. In this first part of a two-part review, the anatomy, physiology, histopathology, and biochemical and biomechanical characteristics of the healthy and degenerated IVD are described. In Part 2, the aspects of IVD degeneration in chondrodystrophic and non-chondrodystrophic dog breeds are discussed in depth.

Intervertebral disc disease in dogs – Part 2: Comparison of clinical, magnetic resonance imaging, and histological findings in 74 surgically treated dogs

The relationship between intervertebral disc (IVD) disease and IVD degeneration remains unclear. The aim of the present study was to compare the clinical severity of IVD herniation (IVDH), determined with a neurological grading system, with findings of magnetic resonance imaging (MRI) and histology using grading systems for IVD degeneration in chondrodystrophic (CD; n=37) and non-chondrodystrophic (NCD; n=37) dogs. This study is the second part of a two-part investigation, where the first part involved the development and validation of a histological grading scheme for classification of canine IVD degeneration. IVD degeneration graded on MRI correlated significantly with IVD degeneration graded on histology, but not with pre-operative clinical signs. Hansen type 1 hernias were more common in the cervical and thoracolumbar segments and Hansen type 2 hernias were more common in the lumbosacral segment. Type 1 hernias occurred more often in CD dogs than in NCD dogs, and CD dogs were clinically more severely affected than NCD dogs. The grade of IVD degeneration on MRI was higher in CD dogs than in NCD dogs, but there was no difference between dogs with type 1 and type 2 hernias. No significant differences in histological grade were found between CD and NCD dogs or between type 1 and type 2 hernias. It was possible to conclude that IVD degeneration did not correlate with the neurological severity of IVDH. The extent of degeneration identified on MRI correlated with degeneration seen histologically. Although the MRI grading system reflected the severity of IVD degenerative changes as confirmed by histopathology, it appeared less useful in predicting the clinical implications.

Gene expression profiling of early intervertebral disc degeneration reveals a down-regulation of canonical Wnt signaling and caveolin-1 expression: implications for development of regenerative strategies

IntroductionEarly degeneration of the intervertebral disc (IVD) involves a change in cellular differentiation from notochordal cells (NCs) in the nucleus pulposus (NP) to chondrocyte-like cells (CLCs). The purpose of this study was to investigate the gene expression profiles involved in this process using NP tissue from non-chondrodystrophic and chondrodystrophic dogs, a species with naturally occurring IVD degeneration.MethodsDual channel DNA microarrays were used to compare 1) healthy NP tissue containing only NCs (NC-rich), 2) NP tissue with a mixed population of NCs and CLCs (Mixed), and 3) NP tissue containing solely CLCs (CLC-rich) in both non-chondrodystrophic and chondrodystrophic dogs. Based on previous reports and the findings of the microarray analyses, canonical Wnt signaling was further evaluated using qPCR of relevant Wnt target genes. We hypothesized that caveolin-1, a regulator of Wnt signaling that showed significant changes in gene expression in the microarray analyses, played a significant role in early IVD degeneration. Caveolin-1 expression was investigated in IVD tissue sections and in cultured NCs. To investigate the significance of Caveolin-1 in IVD health and degeneration, the NP of 3-month-old Caveolin-1 knock-out mice was histopathologically evaluated and compared with the NP of wild-type mice of the same age.ResultsEarly IVD degeneration involved significant changes in numerous pathways, including Wnt/β-catenin signaling. With regard to Wnt/β-catenin signaling, axin2 gene expression was significantly higher in chondrodystrophic dogs compared with non-chondrodystrophic dogs. IVD degeneration involved significant down-regulation of axin2 gene expression. IVD degeneration involved significant down-regulation in Caveolin-1 gene and protein expression. NCs showed abundant caveolin-1 expression in vivo and in vitro, whereas CLCs did not. The NP of wild-type mice was rich in viable NCs, whereas the NP of Caveolin-1 knock-out mice contained chondroid-like matrix with mainly apoptotic, small, rounded cells.ConclusionsEarly IVD degeneration involves down-regulation of canonical Wnt signaling and Caveolin-1 expression, which appears to be essential to the physiology and preservation of NCs. Therefore, Caveolin-1 may be regarded an exciting target for developing strategies for IVD regeneration.

Canonical Wnt signaling in the notochordal cell is upregulated in early intervertebral disk degeneration

The notochordal cell (NC) of the nucleus pulposus (NP) is considered a potential NP progenitor cell, and early intervertebral disk (IVD) degeneration involves replacement of NCs by chondrocyte‐like cells (CLCs). Wnt/β‐catenin signaling plays a crucial role in maintaining the notochordal fate during embryogenesis, but is also involved in tissue degeneration and regeneration. The canine species, which can be subdivided into non‐chondrodystrophic and chondrodystrophic breeds, is characterized by differential maintenance of the NC: in non‐chondrodystrophic dogs, the NC remains the predominant cell type during the majority of life, with IVD degeneration only occurring at old age; conversely, in chondrodystrophic dogs the NC is lost early in life, with concurrent degeneration of all IVDs. This study investigated Wnt/β‐catenin signaling in the healthy, NC‐rich NP and early degenerated, CLC‐rich NP of both breed types by immunohistochemistry of β‐catenin and relative gene expression of brachyury and cytokeratin 8 (notochordal markers) and Wnt targets axin2, cyclin D1, and c‐myc. Both NCs and CLCs showed nuclear and cytoplasmic β‐catenin protein expression and axin2 gene expression, but β‐catenin signal intensity and Wnt target gene expression were higher in the CLC‐rich NP. Primary NCs in monolayer culture (normoxic conditions) showed Wnt/β‐catenin signaling comparable to the in vivo situation, with increased cyclin D1 and c‐myc gene expression. In conclusion, Wnt/β‐catenin signaling activity in the NC within the NC‐rich NP and in culture supports the role of this cell as a potential progenitor cell; increased Wnt/β‐catenin signaling activity in early IVD degeneration may be a reflection of its dual role.