The establishment of potential cerebrospinal fluid biomarkers for canine degenerative myelopathy

Canine degenerative myelopathy(DM) is a late onset neurodegenerative disease that primarily affects German Shepherd dog (GSD), though a number of other specific breeds are also affected. The underlying cause of the disorder remains elusive, though recent advances have implicated a mutation of superoxide dismutase 1(Sod1) in the aetiology, also implying DM is a potential orthologue of human amyotrophic lateral sclerosis. The identification of the Sod1 mutation raises the index of suspicion for an individual animal, however it is not specifically diagnostic as a proportion of dogs homozygous for the Sod1 mutation do not develop DM. Therefore, there is a clinical need for the development of specific biomarker(s) for DM to support genetic test. The aim of this study was to establish potential biomarkers for DM by exploring canine cerebrospinal fluid (CSF). A dual strategy was adopted;1) Evaluation of potential ALS biomarkers in DM CSF, 2) Identification of novel biomarker(s) in DM CSF. The cases selected in this project had a presumptive diagnosis of DM and were homozygous for Sod1 mutation. Preliminary characterisation by Western blot and mass spectrometry identified four protein candidates in DM CSF, comprised of cystatin C, transthyretin (dimeric and monomeric TTR), haptoglobin and clusterin. Since the validity of these putative biomarkers may be influenced by pre-analytical variables that may arise from the clinical environment, we therefore assessed the impact of three potential sample handling practices on these four proteins. The results from these experiments demonstrate that dimeric TTR and clusterin were affected by sample handling conditions. Therefore, an appropriate protocol for CSF sample handling was established. Western blot analyses indicated that clusterin is the most viable biomarker candidate for DM. Clusterin was significantly elevated in DM CSF when compared to a range of neurological conditions. The second potential candidate for DM biomarker is TTR, which is potentially reduced, an observation similar to those found in ALS CSF. The relationship of these proteins in the pathogenic mechanisms that underpin DM is unclear. However, based on observations on ALS, it is reasonable to speculate that their alterations are associated with a toxic gain of function of the mutant SOD1 protein. The successful characterisation of clusterin and TTR in DM CSF may therefore represent components of a panel of emerging biomarkers that may combine to distinguish DM in the clinic and provide further insights into the disease mechanisms

In vitro evidence consistent with an interaction between wild‐type and mutant SOD1 protein associated with canine degenerative myelopathy

Canine degenerative myelopathy (DM) is a progressive neurological disorder that may be considered to be a large animal model for specific forms of the fatal human disease, familial amyotrophic lateral sclerosis (fALS). DM is associated with a c118G>A mutation of the superoxide dismutase 1 (Sod1) gene, and a significant proportion of cases are inherited in an autosomal recessive manner in contrast to the largely, but not exclusively, dominant mode of inheritance in fALS. The consensus view is that these Sod1/SOD1 mutations result in a toxic gain of function but the mechanisms remain unclear. Here we used an in vitro neuroblastoma cell line transfection system to monitor wild-type and mutant forms of SOD1 fusion proteins containing either a Cherry or an enhanced green fluorescent protein (EGFP) tag. These fusion proteins retained SOD1 enzymatic activity on a native gel assay system. We demonstrate that SOD1 aggregate density is significantly higher in DM transfectants compared to wild-type. In addition, we show by co-immunoprecipitation and confocal microscopy, evidence for a potential interaction between wild-type and mutant forms of SOD1 in co-transfected cells. While in vitro studies have shown SOD1 heterodimer formation in fALS models, this is the first report for DM SOD1. Therefore, despite for the majority of cases there is a difference in the mode of inheritance between fALS and DM, a similar interaction between wild-type and mutant SOD1 forms can occur. Clarifying the role of SOD1 in DM may also be of benefit to understanding the role of SOD1 in fALS

A retrospective study of vertebral fracture and luxation in dogs presented to University Veterinary Hospital, Universiti Putra Malaysia in 2015 to 2017

To date, there is data paucity on canine vertebral fracture and luxation (VFL) in the local setting. Therefore, it was hypothesized that the geographical location and stray dog population would influence VFL cases in the University Veterinary Hospital, Universiti Putra Malaysia (UVH, UPM). This study aimed to describe the incidence and characteristics of VFL in dogs presented to UVH, UPM. Medical records, including radiographic images of 74 dogs between 2015 to 2017, were reviewed. VFL dominated the spinal cord disease in dogs at 49% (n = 36/74), exceeding intervertebral disc disease (IVDD) and acute noncompressive nucleus pulposus extrusion (ANNPE). Half of VFL cases were contributed by intact male, small breed dogs aged more than one-year-old, with 52% (n = 11/19) of cases caused by vehicular accidents. Almost two-thirds (n = 21/36) of dogs with VFL were outdoor or stray dogs, and the Th3-L3 region was the most susceptible (52%, n = 19/36) for VFL. More than 70% (n = 25/36) of the patients had unstable fractures, highly associated with severity. In conclusion, the occurrence of VFL in UVH, UPM is three times higher than reported in western countries and most likely contributed by a large number of outdoor and stray dogs

Canine vertebral screw and rod fixation system in dogs

Bone plates and screws are often recommended to fix vertebral fracture and luxation in dogs although several complications had been reported. The canine vertebral screw and rod fixation (CVSRF) system, a device tailored for the canine spine, is a modified system from the human pedicle screw. This study aimed to determine the optimal corridor implantation of CVSRF and to investigate the potential trauma to the vertebrae and spinal cord in medium-sized dogs. Two screws of 16 mm and 20 mm and rods of 40 mm and 45 mm in length were inserted into the pedicles of L1 and L2 in six dogs. Safe implantation angles for 16 mm screw were 52.67° ± 10.40ºand 58.59° ± 7.72ºat L1 and L2, respectively. The angle of the 20 mm screw at L1 was recorded at 56.03°±5.34ºand 55.67° ± 2.89ºat L2. No gross and histological lesion was found on the spinal cord and vertebrae although minimal microfractures of the vertebrae were observed histologically. Findings from this study suggest that CVSRF is feasible for medium-sized dogs using 16 mm screws, however, a long-term study is required to determine the stability and durability of the system

Comparison of bone and articular cartilage changes in osteoarthritis: a micro-computed tomography and histological study of surgically and chemically induced osteoarthritic rabbit models

Background: Osteoarthritis (OA) is a multifaceted condition that affects both the subchondral bones and the articular cartilage. Animal models are widely used as an effective supplement and simulation for human OA studies in investigating disease mechanisms and pathophysiology. This study is aimed to evaluate the temporal changes of bone and cartilage in surgically and chemically induced osteoarthritis using micro-computed tomography and histology. Methods: Thirty rabbits underwent either anterior cruciate ligament transection (ACLT) procedure or injected intraarticularly with monosodium iodoacetate (MIA, 8 mg) at the right knee joint. The subchondral bones were scanned via micro-CT, and articular cartilage was assessed histologically at 4-, 8- and 12-week post-induction. Results: Based on bone micro-architecture parameters, the surgically induced group revealed bone remodelling processes, indicated by increase bone volume, thickening of trabeculae, reduced trabecular separation and reduced porosity. On the other hand, the chemically induced group showed active bone resorption processes depicted by decrease bone volume, thinning of trabeculae, increased separation of trabecular and increased porosity consistently until week 12. Histologically, the chemically induced group showed more severe articular cartilage damage compared to the surgically induced group. Conclusions: It can be concluded that in the ACLT group, subchondral bone remodelling precedes articular cartilage damage and vice versa in the MIA group. The findings revealed distinct pathogenic pathways for both induction methods, providing insight into tailored therapeutic strategies, as well as disease progression and treatment outcomes monitoring

Synovial fluid proteome profile of surgical versus chemical induced osteoarthritis in rabbits

Background: Animal models are significant for understanding human osteoarthritis (OA). This study compared the synovial fluid proteomics changes in surgical and chemical induced OA models. Methods: Thirty rabbits either had anterior cruciate ligament transection (ACLT) procedure or injected intra-articularly with monosodium iodoacetate (MIA, 8 mg) into the right knee. The joints were anatomically assessed, and the synovial fluid proteins analyzed using two-dimensional polyacrylamide gel electrophoresis (2DGE) and MALDI TOF/TOF mass spectrometry analysis at 4, 8 and 12 weeks. The proteins' upregulation and downregulation were compared with control healthy knees. Results: Seven proteins (histidine-rich glycoprotein, beta-actin-like protein 2 isoform X1, retinol-binding protein-4, alpha-1-antiproteinase, gelsolin isoform, serotransferrin, immunoglobulin kappa-b4 chain-C-region) were significantly expressed by the surgical induction. They characterized cellular process (27%), organization of cellular components or biogenesis (27%), localization (27%) and biological regulation (18%), which related to synovitis, increased cellularity, and subsequently cartilage damage. Three proteins (apolipoprotein I-IV precursor, serpin peptidase inhibitor and haptoglobin precursor) were significantly modified by the chemical induction. They characterized stimulus responses (23%), immune responses (15%), biological regulations (15%), metabolism (15%), organization of cellular components or biogenesis (8%), cellular process (8%), biological adhesions (8%) and localization (8%), which related to chondrocytes glycolysis/death, neovascularization, subchondral bone necrosis/collapse and inflammation. Conclusions: The surgical induced OA model showed a wider range of protein changes, which were most upregulated at week 12. The biological process proteins expressions showed the chemical induced joints had slower OA progression compared to surgical induced joints. The chemical induced OA joints showed early inflammatory changes, which later decreased