A Study On The Time-dependent Expression Of Caspase-8 During The Skin Incised Wound Healing In Mice

IntroductionSkin wound healing process may be arbitrarily categorized into three phases, the inflammation, the proliferation and the remodeling phases. Emigration of white blood cells, principally neutrophils and macrophages is an important defence mechanism. They can engulf and digest foreign particulate matter such as bacteria and the debris of dead cells.Programmed cell death, or apoptosis, is a physiological process of cellular autodestruction. Apoptosis plays a critical role in development, maintenance of homeostasis and host defense in multicellular organisms. Dysregulation of this process is implicated in various diseases ranging from cancer and autoimmune disorders to neurodegenerative disease and ischemic injuries.The central component of apoptosis is a protease called caspase. The caspase family consists of 14 members, caspase - 1 to caspase — 14. They are both cysteine protease, which use cysteine as the nucleophilic group for substrate cleavage, and aspases, which cleave the peptide bond C - terminal to as-partic acid residues. Consequently, caspase often function in cascades. An upstream caspase, called initiator caspase ( caspase — 8 , - 9 ) , inactivated by its interaction with the caspase adapter(s). This activation represents a key regulatory step in apoptosis. Once activated, the cleaved initiator caspase activates one or more downsream caspase, called effector caspase, caspase - 3 , - 6 and - 7. The activated effector caspase then degrade various cellular proteins, leading to apoptotic cell death.Studies showed apoptosis occurs when cellular amount decrease. It is no doubt that caspase - 8, an initiator of caspase family, plays an important roleduring skin wound healing.To investigate whether caspase - 8 is expressed in the polymorphonuclear cells (PMNs) , mononuclear cells (MNCs) and fibroblastic cells (FBCs) during the skin incised wound healing and the applicability of using caspase - 8 to estimate the skin wound age, immunohistochemical and Western blotting studies on the expression of caspase - 8 were performed on skin incised wound at different post - traumatic intervals in mice.Materials and MethodsA total of 33 eight -week - old male mice, each weighted 35 g -40g. A 1. 5 - cm - long incision was made with a bistoury in the skin layer on the central dorsum under sterile technique after the mouse was anesthetized by aether. Then each mouse was fed with sterilized food and distilled water in individual cage to prevent infection. The 2cm x 2cm specimens were taken from the wounded sites after the mice were executed by cervical dislocation at Oh, 3h, 6h, 12h, Id, 3d, 5d, 7d, lOd and 14d ( 3 mice in each group ) post -wounding. The remaining 3 mice were used as controls.1. ImmunohistostainingFor immunohistostaining, the materials were fixed in 10% formalin for 12 hours then embedded in paraffin. After being cut into 5(xm thick, the paraffin sections were deparaffinized in xylene and rehydrated in graded alcohol. Endogenous peroxidase activity was quenched for 20 minutes at room temperature in PBS containing 3% hydrogen peroxide. After being washed with PBS, sections were subjected to antigen retrieval by incubating in antigen retrieval solution at 96°C for 3 minutes in a microwave oven. The sections were washed in PBS and incubated for 20 minutes with a blocking solution consisting of 10% normal goat serum in PBS at room temperature. The rabbit polyclonal caspase - 8 antibody ( Neo Markers) was applied overnight at 4°C at a concentration of 1 : 200. Then the sections were washed with PBS and incubated with goat anti - rabbit immu-noglobulins at room temperature for 20 minutes. After being washed with PBS, sections were incubated with Streptavidin - Peroxidase ( S - P) at room tempera-ture for 20 minutes. The peroxidase reaction was developed with 3,3'- diamino-benzidine (DAB) to visualize the specific antigen. Nuclei were routinely counter — stained with hematoxylin.As immunohistochemical controls, some sections were reacted with PBS in place of the primary antibody. No false positive reaction was detected in the sections. Hematoxylin and eosin (HE) staining was also performed in each group.The cytoplasm of caspase - 8 positive cells was brown. The data analysis were performed by SPSS 10. 0 for Windows with a significant level of P <0. 05.2. Western blot Analysis of caspase -8Whole cell extract proteins were loaded at lOOjxg/lane on 12% [w/v] SDS -PAGE. Proteins were transferred to PVDF membranes, blocked in 5% nonfat dry milk and 0. 1% Tween —20. Membranes were probed with polyclonal antibody against caspase -8 (Neo Markers). Immunoreactive bands were visualized by DAB for detecting the band of cleaved caspase — 8.Results1. Morphological changes of the skin incised woundAt 3h post - injury, PMNs appeared at the peripheral zone of the wound and increased markedly and peaked at 6h. Many MNCs together with some PMNs accumulated in the wound at Id. From 3d to 5d, the early fibro — prolifer-ative phase, the cells were almost composed of MNCs and FBCs. From 7d to 14d post - injury the epidermal cells proliferated and covered the wound cavity completely.2. Immunohistochemical detection of caspase -82. 1 Immunostaining of the uninjured skin in the control group In the uninjured specimens, caspase - 8 immunoreactivity was detected in the epidermis, hair follicles, sebaceous glands and sweat glands by immunostaining.2. 2 Immunostaining of the injured skin in the experimental groups The ratio of caspase -8 positive cells was low in the sections aged 3h(4. 05 ± 0. 26) % . From 6h post - injury, the ratio increased remarkably and peaked at3d (54.75 ±5.30)% post - injury and decreased thereafter. At 14d post - injury , the ratio was (21. 58 ± 3. 52 ) % .By statistical analysis of variance, there were significant differences in the comparison of the caspase - 8 positive cell ratios between the neighbor time groups (P <0.05) except the 0 hour group.3. Detection of cleaved caspase - 8 by Western blotting The obvious bandsof 44/42 kD were noticed at Id, 3d, 5d and 7d post -injury, indicating that caspase - 8 was activated.DiscussionCleavage of caspase —8 (FLICE/MACH) is the first step in the cascade of apoptotic events induced by Fas. Many studies showed that caspase - 8 is associated with pathogenesis of many diseases and injuries in human being, such as cancer, neurodegenerative disease, parasitosis, trauma and so on. However, there is no study on the possible role of caspase — 8 in the skin wound healing heretofore.This study applied immunohistochemical and Western blotting techniques to detect the expression of caspase - 8 during the skin incised wound healing. The results showed that caspase - 8 expressed in the epidermis, hair follicles, sebaceous glands and sweat glands in the normal skin. The caspase - 8 positive MNCs and FBCs were detected in the skin incised wound region. The expression of caspase - 8 in the two categories of cells during the skin incised wound healing was time - dependent. The present results suggested that caspase - 8 may play an important role in inducing apoptosis of the MNCs and FBCs during the skin incised wound healing in mice. Furthermore, caspase -8 may be used as a marker for estimating the wound age since the expression of caspase - 8 in the MNCs and FBCs during the skin incised wound healing was time - dependent.No definite expression of caspase - 8 was detected in the PMNs throughout the whole skin incised wound healing process in the present study. An immunohistochemical study on Fas and Fas ligand in skin wound healing indicated neither Fas nor Fas ligand staining was observed in the PMNs throughout the woundhealing process as reported previously and apoptosis of PMNs occurred, suggesting that other pathway(s) which induced the apoptosis of PMNs can not be excluded.Conclusion1. This study showed that caspase - 8 expressed in the epidermis, hair follicles, sebaceous glands and sweat glands in the mice skin for the first time.2. The experiment indicated firstly that the caspase —8 positive MNCs and FBCs were detected in the skin incised wound region. The expression of caspase- 8 in the two categories of cells during the skin incised wound healing was time- dependent. No definite expression of caspase - 8 was detected in PMNs during the skin incised wound healing in mice.3. The results suggested that caspase - 8 may be used as a marker for estimating the skin incised wound age.