A Study On The Time-dependent Expression Of Caspase-6 During Repair Of Skin Contusion In Rats

A study on the time-dependent expression ofcaspase-6 during repair of skin contusion in ratsObjectiveTo investigate the time-dependent expression of caspase-6 during repair of skin contusion in rats.Methods1,Establishment of animal model and groupingA total of 30 healthy, adult rats, weighting 220-250g were divided into 10 groups. An acute contusion model was used to stroken the rats'right gastrocnemius muscles after 0.2% intraperitoneal injection. The skin specimens were taken from the contusion sites after the animals were sacrificed by cervical dislocation under anaesthetization at 3h, 6h, 12h, 1d, 3d, 5d, 7d, 10d, 14d(3rats in each group)postcontusion. The remaining 3rats were used as controls. Then the specimens were embeded with OCT, and rapidly frozenin.2,Immunohistochemical detection of caspase-6Caspase-6 was detected by immunohistochemical SP method. Briefly, after serum incubation, sections were incubated with polyclonal antibodies against caspase-6 at 4℃overnight at a dilution of 1: 100. Rabbit anti-goat IgG-biotiny incubated at RT for 20 minutes. The slides were incubated with SP at RT for 20minutes. Visualization of antibody binding was the DAB method. Finally, the sections were counterstained with hematoxylin, dehydrated and mounted. H. E staining of the sections was routinely conducted.3,Analysis of caspase-6 by Western blotWhole cell extract proteins and then the proteins was transferred to PVDF membrane, blocked in 5% nonfat dry milk and 0.1% Tween-20. Membrane was probed with rabbit-anti-goat secondary antibody. Immunoreactive bands were visualized by ECL.Results1,Macroscopic findingsAt 3h postcontusion, hemorrhage and cutaneous edema were obviously observed in contused skin. The degree of hemorrhage and cutaneous edema were relieved at 6h. At 1d postcontusion, cutaneous edema was always exist in contused skin, but hemorrhage was lessened. At 3d postcontusion, cutaneous edema was almost entire absorbed. Buffchange was seen in contused skin at 5d. From 7d to 14d, the color in contused skin was the same as the uncontused skin.2,Histological changes of contusionAt 3h postcontusion, cuticular layer and dermis layer was separated, polymorphonuclear cells(PMNs) appeared at the margin and peripheral zone of the contusion and markedly increased in number from 6h after injury. A large number of PMNs, together with some inflammatory mononuclear cells, accumulated in the contusion at 1d. The number of total infiltrating cells increased remarkably at 3d postcontusion and reduced thereafter. At 5d postcontusion, collagen fibrils increased. From 7d to 14d, the inflammatory cells were almost replaced by collagen fibrils.3,The expressions of caspase-6 in contused skinIn the control specimens, caspase-6 was detected in the epidermis, hair follicle, sebaceous gland. In the contusion specimens, at 3h postcontusion of dermis layer, caspase-6 was detected in many fibroblastic cells(FBCs) and gradually increased at 6h, peaked from 12h to 3d and from 5d posttrauma, the positive cells were decreased, at 14d postcontusion, weak expression of caspase-6 was detected in FBCs. At 3h postcontusion of hypodermis, weak expression of caspase-6 was detected in cytoplasm of PMNs that infiltrated into the injured area. From 6h to 12h postcontusion, the expression ofcaspase-6 was prominently increased. At 1d postcontusion, almost all of the mononuclear cells(MNCs) and PMNs were caspase-6 positive. At 3d postcontusion, immunoreactivity of caspase-6 was mainly detected in FBCs. From 5d to 7d postcontusion, the weak immunostaining of caspase-6 was detected in the cytoplasm of FBCs. At 10d and 14d postcontusion, the positive cells were gradually decreased. No immunostaining of caspase-6 was seen in control sections.The ratio of caspase-6 positive cells elevated(25.78%±1.38%) from 3h, and then increased gradually from 6h to ld and peaked at 3d after contusion(54.58%±5.64%). Thereafter, its level decreased at 5d(28.39%±4.50%), and then kept steadily. The positive cells decreased largely at 10d(20.43%±2.36%), and was low at 14d(11.31%±2.19%).By statistical analysis of variance, there were significant differences in the comparision of the caspase-6 positive cell ratio among all the neighbor time groups(P＜0.01).4,Western blot data analysisThe obvious activation bands of 10kDa were noticed at 1d, 3d and 5d after contusion.ConclusionThe results suggest that caspase-6 may play a major role in the injury-induced inflammatory response. Furthermore, caspase-6 may be used as a marker for the contusion age determination, as caspase-6 was time-dependently expressed in PMNs, MNCs and FBCs during skin repair in rats.