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Abstract
Introduction Superficial digital flexor tendon (SDFT) damage is one of the most common orthopaedic injuries in horses. The SDFT is adapted as an energy storing tendon to aid high-speed locomotion, however repetitive cyclical loading can result in core tendon temperatures reaching 48°C.2 Such high temperatures may induce cell damage via apoptotic pathways and loss of cell viability, predisposing the tendon to permanent damage.1 However the effect of physiologically relevant oxygen conditions on tenocyte stress pathways has not been investigated. We hypothesised that hypoxic culture condition will attenuate hyperthermia induced stress in equine SDFT tenocytes compared to cultures in atmospheric (20.9%) oxygen levels
Methods Equine SDFT cells were cultured in DMEM containing 10% foetal bovine serum at 37 °C in both 20% and 8% oxygen conditions at >75% confluency, before transiently inducing hyperthermia by elevating the temperature to 45 °C for 10 min. Expression of heat shock protein-25 (HSP-25), HSP-72 (protective stress pathways) and Daxx (Death domain-associated protein) was assayed by western blot and Annexin A1 (apoptosis associated protein) by immunocytochemistry at 1, 2, 4 and 8 h post heating. The viability of the cells was assessed using AlamarBlue® (Promega, UK).
Results HSP-25 and HSP-72 expression increased over time post heat-shock by 2- and 10-fold respectively (Figure 1), whereas Daxx expression remained unchanged (data not shown) Annexin A1 homodimer (nuclear isoform) had constitutive higher expression in 20% oxygen compared to 8% regardless of temperature conditions (Figure 1). The monomeric form of Annexin A1 was of low abundance in both groups and was not affected by heating (data not shown). Tenocyte proliferation was initially decreased after heating at 24 h but the cells showed recovery by 72 h. Recovery rates were delayed in 20% oxygen up to 48 h post heating. (Figure 2).
Western blot analysis of tenocyte lysates following an episode of hyperthermia
Tenocyte proliferation following hyperthermia
Discussion The induction of HSP-25 and HSP-75 in response to hyperthermia suggests a protective mechanism exists in tenocytes to reduce cell damage. The response is particularly acute under physiological oxygen levels (8%) where the reduced expression of Annexin A1 homodimer and the lack of Daxx induction further suggests that tenocytes are resistant to a short episode hyperthermia. In addition the recovery of proliferation was delayed under 20% oxygen. However, whether repeat bouts of hyperthermia in vivo alleviate the resistance of tenocyte to recover needs further investigation.
References 1 Patterson-Kane JC, Firth EC. Vet J. 2009;181(2):79–89
2 Wilson AM, Goodship AE. J Biomech. 1994;27(7):899–905