| IntroductionIt is well established that skin wound healing is a continuous and complex process which can be roughly divided into three phases; the early acute exudative , fibro - proliferative and tissue remodeling phases. In these phases, many kinds of inflammatory cells and cytokines are involved in to remove the wounded and necrotic tissue and then to remodel the newly regenerated skin tissue.NO is an active multifunctional diffused signaling molecule which is synthesized by NOS(nitric oxide synthase). Three NOS isoforms have been identified, which are encoded by different genes located on different chromosome. Two enzymes isoforms are constitutively expressed ( endothelial and neural cNOS) , whereas another isoform is an inducible enzyme (iNOS). It is suggested that NO plays a pivotal role in the process of inflammatory phase, cell proliferation, differentiation , apoptosis and angiogenesis, matrix deposition and tissue remodeling during skin wound healing. We suppose that the expressions of NOS iso-forms - iNOS and eNOS reflect the outcome of NO and its functions.To confirm the biological functions of NOS and explore its applicability to skin wound age estimation, immunohistochemical investigation on the expressions of two isoforms of NOS were performed on mice cutaneous incised wound at different post - traumatic intervals in the present study.Materials and MethodsA total of 36 healthy adult mice, each weights 35 to 40g. A 1. 5 - cm -long incision was made with a scalpel in the skin layer on the central dorsum under sterile technique. After wounding, each mouse was individually housed in acage and given sterilized chow and redistilled water to prevent bacterial infection. 1. Ocm x 1.5cm specimens were taken from the wounded sites after the animals were sacrificed by cervical dislocation under anaesthetization at 0h,1h,3h, 6h,12h,1d,3d,5d,7d,l0d,14d(3mice in each group) post wounding. The remaining 3 mice were used as controls. Then the specimens were fixed, dehydrated, cleared, followed by embedding in paraffin, 5|xm consequently paraffin sections were made for the following procedures.Immunohistochemical SP method was used to detect the expressions of iNOS and eNOS, after inhibition of endogenous peroxidase activity (3% hydrogen peroxide PBS solution for 20 min) , antigen retrieval was then performed by heating in a microwave oven in 10mM citrate buffer for 10 min. After serum incubation, sections were incubated with rabbit polyclonal antibodies against iNOS and eNOS at 4°C for one night at a dilution of 1 -.400 respectively, and another lOmin at RT to enhance incubation. Then the sections were incubated by biotinylated goat an-ti - rabbit second antibody at room temperature for 20 min, followed by being incubated with SP reagent at room temperature for 20 min. DAB was used as the chromogen for visualization. Finally, the sections were counterstained with he-matoxylin, dehydrated and mounted.Positive cells of iNOS and eNOS, including polymorphonuclear cells (PMNs) , mononuclear cells (MNCs) , fibroblastic cells (FBCs) were identified and counted in 10 randomly selected fields in the three sections of each groups under microscope at a 400 - fold magnification. The ratio of iNOS - and eNOS -positive cells to total cells were calculated and expressed as (x ± SD).The data were analyzed using SPSS for Windows 11.0 with a significant level of P<0.01.ResultsThe expressions of iNOS and eNOS in skin wound healing: in the control specimens, iNOS was detected in the epidermis, hair follicle, sebaceous gland, and it was negative in the endothelial cells of the vessels although eNOS was seen in the endothelial cells with low density. Expressions of iNOS and eNOSwere detected in polymorphonuclear cells (PMNs) in the wound site 3h post injury. In the wounded specimens aged from 6h to 24h after injury, iNOS and eNOS were identified in a large number of infiltrating PMNs and part of mononu-clear cells. Afterwards, the MNCs and FBCs accounted for the most part of the iNOS - and eNOS - positive cells. Especially dynamic expression was observed in the endothelial cells of neonatal blood vessels in granulation tissue.The ratios of iNOS - and eNOS - positive cells and data analysis: The ratio of iNOS - positive cells was low in the wounded specimens aged lh post - injury (9.25% ±1.44%), and then increased gradually and peaked on day 1 after wounding (86.65% ±4.46%). Thereafter, its level of expression kept steadily. Then maximized again on day 10 (68.87% ±2.65% ) and decreased in the specimens on day 14 (36. 10% ±3. 61% ). The ratio of eNOS - positive cell was low from lh to 3h post wounding (12. 34% ±3. 44% ) and maximized on day 3 after wounding (90. 25% ±3. 25% ) , in the next 5 days, its ratio was stable and then decreased gradually until day 14 after injury (28. 67% ±3. 60%).By statistical analysis of variance, except Oh to lh group, there are significant differences in the comparision of the iNOS and eNOS positive cell ratios between all the neighbor time groups (P <0.01) , among which the days 1 and 10 group of iNOS and day 3 group of eNOS after wounding have the significant difference in the comparison of the positive cell ratios with all the other time groups ( P < 0.01). Except the days 5 and 7 of iNOS group, the day 7 of eNOS groups, the other groups have significant differences between the neighbor groups.DiscussionAt present, more and more studies have confirmed that NO plays a pivotal role in the process of inflammatory phase, cell proliferation and differentiation, cell apoptosis and angiogenesis, matrix deposition and remodeling during skin wound healing. So in the process of skin wound healing, the dynamic expressions of iNOS and eNOS which synthesize NO reflect the outcome of NO and itsbiological functions. Furthermore, in the iNOS and eNOS knockout mice the li-fespan of wound healing prolongs, which explain the important roles of isoforms of NOS. The expressions of iNOS and eNOS were studied in the present study to invetigate the biological functions of NO during skin incised wound healing. Hitherto, no related studies have been reported on expressions of isoforms of NOS in cutaneous wound in situ by immunohistochemical techniques.In the present study, expressions of iNOS and eNOS were observed in poly-morphonuclear cells, mononuclear cells and fibroblastic cells in the wound site and peripheral zone of the incised skin wound during healing process in mice. Both of the two NOS isoforms were also detected in the dermis and endothelial cells of the capillaries, which suggest NO participates in all the events of wound healing, including affecting inflammatory phase of wound healing, regulating blood flow in dermis, adjusting cell proliferation and differentiation and apopto-sis, forming granulation tissue and neonatal vessel, and matrix deposition and tissue remodeling. After calculating the ratios of iNOS - and eNOS - positive cells, the ratio of iNOS - positive cells peaked on day 1 post - injury, which was conformed to the consequences of previous studies that the infiltrating cell on day 1 is mostly monocytes and all of them are iNOS - positive. In addition, from the day 5 after wound, iNOS was positive in the endothelial cells of vessels in neonatal granulation tissue, and eNOS was seen in the vessel in dermis and hypo-dermis, especially in the neonatal capillary in granulation tissue. It is suggested that the isoforms of NOS have significant roles in the forming of vessel.The results suggest that iNOS and eNOS play important roles in all the e-vents during skin wound healing. At the same time, the ratios of iNOS - and eNOS - positive cells including polymorphonuclear cells, mononuclear cells and fibroblastic cells were time dependent, which suggested that iNOS and eNOS can be used as a marker for wound age estimation.Conclussion1. In the control specimens, iNOS was detected in the epidermis, hair follicle , sebaceous gland, and eNOS was also seen in the endothelial cells of the... |
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