Cranial cruciate ligament (CrCL) disease/rupture is a highly prevalent orthopaedic disease in dogs and common cause of pain, lameness, and secondary joint osteoarthritis (OA). Previous experiments investigating the role of glutamate receptors (GluR) in arthritic degeneration and pain revealed that OA biomarkers assessing early bone turnover and inflammation, including osteoprotegerin (OPG) and the receptor activator of nuclear factor kappa-B ligand (RANKL) are more likely to be influenced by glutamate signalling. Moreover, interleukin-6 (IL-6) has a complex and potentially bi directional (beneficial and detrimental) effect, and it is a critical mediator of arthritic pain, OA progression and joint destruction. 1) to recruit dogs undergoing CrCL disease/rupture surgery and obtain discarded synovial fluid (SF) and serum/plasma (ethics approval, RCVS:2017/14/Alves); 2) to quantify the biomarkers listed above in the SF and serum/plasma by enzyme linked immunosorbent assay (ELISA); 3) to assess radiographic OA at the time of surgery and correlate it with the biomarkers and clinical findings. Abnova, Abcam and AMSBIO ELISA kits were tested using a validation protocol relating the standard curve to a dilution series of SF and serum/plasma (1× to 1/50×), with and without SF hyaluronidase treatment to evaluate linearity, specificity and optimal dilutions. Validated ELISA kits were used to measure [IL-6], glutamate [glu], [RANKL] and [OPG] in SF and serum/plasma. For each dog, CrCL disease pre-operative lameness scores were graded as: (1) mild, (2) moderate (easily visible), (3) marked (encumbered), (4) non-weightbearing lameness. Blinded OA scoring was performed on radiographs [15–60, normal-severe OA].Abstract
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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. To evaluate glutamate signalling in canine diseased and normal CrCL and meniscus by immunohistochemistry (IHC). 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).Abstract
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Cranial cruciate ligament (CrCL) disease in dogs causes pain and osteoarthritis (OA) and surgical treatment does not prevent OA progression. Glutamate receptor (GluR) antagonists alleviate pain and degeneration in rodent models of OA, but it is unknown whether they are a suitable treatment for dogs. Understanding GluR signalling in CrCL disease may lead to novel therapeutics in both veterinary and human medicine. To determine whether age, breed, sex, weight, and therapeutic(s) influence lameness and pre-operative radiographic OA scoring in dogs with CrCL disease and whether GluRs are expressed, in this disease. Surgical waste (CrCL and medial meniscus), clinical data, stifle radiographs, lameness scores (1–4, mild-unloading limb) were obtained with full informed consent (RCVS ethics approval, ref: 2017/14/Alves). OA scoring was performed on radiographs [VCOT, 2017, 30(6):377–384, 15–60, normal-severe OA], and α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-2 and kainate (KA)-1 GluR expression compared in diseased Abstract
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1. Measurements have been made of the relative calcification of different types of bone in tibia of the rabbit at the ages of six weeks, three and a half months and seven months by comparing their absorption of x-rays. 2. Calcified cartilage is between 8 and 10 per cet more highly calcified than periosteal and endosteal bone and about 20 per cent more highly calcified than bone formed immediately adjacent to cartilage. 3. Young and adult bones have a framework of approximately the same strength; that is, calcified cartilage, bone adjacent to cartilage and the interstitial areas of periosteal and endosteal bone have each approximately the same degree of calcification at all ages. 4. Adult rabbit bone approaches uniform calcification throughout, equal to the calcification of the interstitial areas of periosteal and endosteal bone. Evidence for this is the replacement of the lowly calcified epiphysial bone by osteones of higher calcification.
1. The detailed anatomy and calcification of the upper half of the tibia in rabbits varying in age from six weeks to twelve months has been studied. 2. The structure of the bone varies at different levels, but a section taken from the same level in the tibia from animals of the same age presents a reasonably constant picture. 3. It has been shown that this variation in structure at different levels is directly related to a difference between the axis of growth and the bone axis. This difference is a result of the unique shape of the tibia. 4. Autoradiographic studies confirm the localised concentration of radioactive strontium in areas of active bone formation where uptake is rapid. 5. The long retention of radioactive strontium in the skeleton (that is, the slow turnover) is a result of the slowness of resorption of bone (endosteal, periosteal or Haversian) in the cortex. Not only is the process slow but it is extremely localised. 6. The significance of these anatomical and physiological characteristics in relation to radiation injury is discussed.
