Abstract
Abstract
Cranial cruciate ligament (CrCL) disease/rupture causes pain and osteoarthritis (OA) in dogs. α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-2 and kainate (KA)-1 glutamate receptors (GluR) and the excitatory amino acid transporter-1 (EAAT-1) and EAAT-3 are expressed in joint tissues from OA patients and rodent arthritis models and represent potential therapeutic targets.
Objectives
To evaluate glutamate signalling in canine diseased and normal CrCL and meniscus by immunohistochemistry (IHC).
Methods
Surgical waste (CrCL, n=5 and medial meniscus, n=3) were obtained from canines with CrCL disease (RCVS ethics approval:2017/14/Alves) and normal analogous tissues (n=2). IHC optimization was performed for rabbit polyclonal (AMPA-2:ab52176, KA-1:ab67402, EAAT-1:ab416) and monoclonal (EAAT-3:ab124802) antibodies from Abcam. IHC was optimised over antibody dilutions from 1:100 to 1:5000 alongside equivalent IgG isotype controls (ab37415 and ab172730) and negative controls (TBS/Tween buffer without primary antibodies). IHC staining was compared in diseased and normal tissues and disclosed with 3,3’-Diaminobenzidine (DAB).
Results
Specific immunostaining was observed for all primary antibodies, at concentrations between 2.0×10−4 mg/mL to 1.0×10−2 mg/mL, depending on the tissue and primary antibody. All GluR and transporters were expressed in the cellular membrane, in the normal and diseased CrCL and meniscus. Healthy CrCL showed a well-organized microstructure, with normal positively labelled ligamentocytes, whereas diseased CrCL microstructure was disrupted, with many positively stained fibroblastic cells in the epiligamentous region and evident neovascularization, indicative of ongoing repair. The normal and diseased meniscal tissues showed similar chondrocytes-like cells labelling and microstructure. Negative controls demonstrated no labelling.
Conclusions
GluR and transporters expression is altered in canine diseased CrCLs, implicating glutamate signalling in this pathology. Since AMPA/KA GluR antagonists alleviate joint degeneration in post-traumatic OA in rodent models, they may be useful for the treatment of CrCL disease in dogs, as well as translated to other veterinary and human orthopaedic diseases.
