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Abstract
Introduction Injuries of forelimb superficial digital flexor tendon (SDFT) are a common injury in athletic horses (Patterson-Kane et al., 2009). During high speed locomotion the core temperature of SDFT has been recorded to rise up to 48 oC, as a result of energy loss within the hysteresis of the stretch-recoil loop (Wilson and Goodship, 1994), which has been hypothesised as a contributing factor to cell death and ultimately tendon damage. To further understand the mechanism of hyperthermia induced damage we investigated the induction of apoptosis via stress proteins associated with hyperthermia.
Methods Equine SDFT (from horses aged 7 ± 1.5 years) were obtained from an abattoir. Only tendons with no history of injury were used. The tensile region was dissected aseptically and cut into 20 mm x 2 mm x 2 mm explants using a multiple blade cutting apparatus (Dudhia et al., 2007). After overnight culture in 8% O2, 5% CO2 and 87% N2 (DMEM with no serum) hyperthermia was induced by heating the media to 45 oC and holding for 10 min before cooling to 37 oC. Media temperature was monitored with 10KΩ thermistor sensors (Omega, UK) (Figure 1). Samples were collected from both control (37 oC) and the heated group up to 8 h after heating. Explants were immediately fixed in 4% PBS buffered formalin and processed for immunohistochemistry using specific antibodies: Connexin-32, Annexin V, Heat shock protein 25 (HSP25), Matrix metalloproteinase 1 (MMP1), ADAMts4.
Real-time temperature monitoring of the media. The set temperature was reached in the media and explants (data not shown) within 37 min although cooling had a slightly extended toe region
Results Connexin-32, Annexin V and HSP25 were expressed significantly higher by 2 h and remained elevated up to 8 h compared to 0 h and the equivalent control time point in heated explants (Figure 2). Although Annexin V expression was significantly higher than the control group it remained relatively stable up to 8 h. MMP1 was transiently and significantly increased at 2 and 4 h but declined by 8 h, whereas there was no significant change in the expression of ADAMts4 post-heating.
Discussion A transient episode of hyperthermia induced early apoptotic signals (Annexin V and HSP25) in SDFT explants. The upregulation of Connexin-32 suggests increased formation of gap junctions which may facilitate an increase in transfer of apoptotic signals between cells and orchestrate the spread of apoptosis to neighbouring tenocytes.
Protein levels in SDFT explants. Immunohistochemistry images were semi-quantitatively analysed using WCIF ImageJ software. The integrated density value (IDV) was normalised to the 0 h control group
References Dudhia J, et al. Aging Cell. 2007;6(4):547–556
Patterson-Kane JC, EC Firth. Vet J. 2009;181(2):79–89
Wilson AM, Goodship AE. J Biomech. 1994;27(7):899–905