Abstract
Platelet concentrates are widely used in mammalian regenerative medicine to improve tissue healing. Chelonians (Testudines) would benefit from the application of thrombocyte preparations to regenerate damaged tissues, since traumatic injuries are leading causes of morbidity and mortality for both wild-living and domesticated animals. The aim of this study was to establish a protocol that optimized the recovery of the thrombocytes from blood samples and to show the efficacy of thrombocyte-enriched plasma in chelonians. Peripheral blood samples were obtained from Testudo spp. (n = 12) and Trachemys scripta elegans (n = 10). Blood cells were fractionated by sodium diatrizoate-sodium polysucrose density gradient using a two-step centrifugation protocol. Thrombocytes and leukocytes were isolated and resuspended to obtain thrombocyte-leucocyte rich plasma (TLRP). The mean recovery of leukocytes and thrombocytes was 48.9% (±4.0 SEM, n = 22) of the whole blood cell content. No statistically significant difference was observed between blood samples collected from different turtle species. The ability of TLRP to form a gel was evaluated by adding variable concentrations of calcium gluconate at room temperature and at 37°C. A reliable and consistent clotting of the TLRP was obtained in glass tubes and dishes by adding 5-20% v/v of a 100 mg/ml solution of calcium gluconate. Furthermore, in order to test the clinical efficacy of TLRP, a preliminary evaluation was performed on four turtles (Testudo spp.) with traumatic injuries. In all the four animals, a successful clinical outcome was observed. The results demonstrated that a thrombocyte-enriched plasma, comparable to mammalian platelet rich plasma, can be prepared from chelonian blood samples. Furthermore, although the low number of cases presented does not allow definitive conclusions from a clinical point of view, their outcome suggests that TLRP application could be further investigated to improve the healing process of both soft and hard tissue injuries in chelonians.
Highlights
Chelonians have become increasingly popular pets in recent years and internal medicine and surgery of turtles currently involve a growing number of veterinary practitioners
The method here proposed resulted in a better separation of leukocytes and thrombocytes on an Hystopaque layer when compared with the methods described in the literature for both Testudo spp. and Trachemys scripta elegans [19]
When centrifugation on Histopaque layer was perfomed at 150xg for 20 minutes, leukocytes and thrombocytes formed an evenly dispersed opaque cloud clearly separated from the erythrocyte pellet, as visible in Fig 2B
Summary
Chelonians have become increasingly popular pets in recent years and internal medicine and surgery of turtles currently involve a growing number of veterinary practitioners. Tortoises are included in appendix ii of the Convention on International Trade of Endangered Species (C.I.T.E.S., Washington D.C. USA, 1973). Traumatic injuries are one of the most common problems which veterinary practitioners must face, for turtles living in the wild and in captivity. Head and different soft tissue injuries can be severely debilitating, carapace fractures are frustrating. They may cause long-lasting disability and sufference, since they usually require a long time to heal and to obtain good anatomical and functional recovery [1,2,3]. Current treatment for fracture stabilization based on the use of non-organic materials such as screws, epoxy resins, pins, and/or wires, can induce adverse reactions which delay the healing process [1, 4]
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