The Changes Of T Lymphocyte Subsets In The Peripheral Blood And Injured Site Of SD Rats After Acute Spinal Cord Injury

Background: According to the traditional view the autoimmune response willaggravate the secondary damage and against nerve regeneration after central nervoussystem injury. In recent years, Israel Schwartz research team found that the autoimmuneresponse after central nervous system injury also play protective and promote role forthe nerve regeneration, and proposed the protective autoimmune theory in central nervessystem injury. We think that the reasons lead to academia debate possible relative to thecomplexity of the local immune microenvironment after spinal cord injury(SCI). AfterSCI, T lymphocytes in the peripheral blood and damage local parts includes CD3~+,CD4~+, CD8~+T cells, and Treg cell subsets are all involved in the autoimmune response,some of T cell subpopulation have protective potential role, and others likely havedamage potential for nerves, and overall final results depend on the balance amongthese different T cell subsets after SCI.Objective: To investigate the dynamic changes of the proportion of T lymphocytesubsets in SD rats peripheral blood, injured part before and after SCI.Methods:1. By using New York university impactor system, the SD rat moderate spinal corddamage model was established and the effect was evaluated.2. By using flow cytometry and staining with fluorescent antibodies, anti-rat CD3-FITC,CD8-PE, CD4-PE, CD25-PERCP, and FOXP3-APC, the proportion of T lymphocytesubsets in the peripheral blood in rats before and after SCI were detected.3. Local tissue lymphocytes were isolated from infiltrated lymphocytes in fresh spinalcord by different density Percoll separation liquid, and the CD8, CD4T cell subsetsand Treg cells were detected by flow cytometry.4. By using histoimmunefluorescent staining techniques the local infiltrated Tlymphocyte subsets(CD4~+,Treg cells) in spinal cord were verified and analysed beforeand after SCI.Results:1. SD rats T9spinal cord were impacted by New York university impactor system (10 g*25mm). SD rats both two lower limbs lost activity and resulted in the paralytics.Pathological examincation showed significantly difference. The SD rat moderate SCImodel was successfully established.2. By using flow cytometry for detection of T cell subsets, before and after SCI, theCD3~+CD8~+cells, Treg/CD3~+CD4~+proportions in SD rat peripheral blood had nochanges and significant difference (p>0.05), at the1st,3rd,5th, and7thday after SCI.The CD3~+T cell, CD3~+CD4~+T cells proportion and the CD3~+CD4~+/CD3~+CD8~+ratio was significantly lower than the normal SD rats and have significant difference (p<0.05). At the2ndweek after SCI, each of these T cells subsets all recovered topreoperative level. Compared with the two time points, have no significant difference (p>0.05).3. We detect the local infiltrated T lymphocytes subsets in injured spinal cord by flowcytometry. T cells cannot be detected in health SD rat spinal cord. The ratio of CD4~+cell in local injury part has significant difference between the3rdday respectively withthe1stweek,the3rdweek after SCI(p<0.05). The ratio of CD4~+cell in local injury parthas significant difference between the1stweek and the3rdweek after SCI(p<0.05). Theratio of CD8~+cell in local part has significant difference between the3rddayrespectively with the1stweek, the3rdweek after SCI(p<0.05). The ratio of Treg/CD4~+cell in local part has significant difference between the2ndweek respectively with the3rdday, the1stweek and the3rdweek after SCI(p<0.05). There is no significantdifference between the rest of the time points (p>0.05).4. Useing immunohistochemistry fluorescent staining technique to analysis and verifylocal infiltrated T lymphocyte subsets(CD4~+,Treg) in injured spinal cord. CD4+celland Treg cell in normal SD rat spinal cord is undetectable. Except between the1stweek,the2ndand the3rdweek. The amount of CD4~+cells have no significant difference(p>0.05). All of the remain have significant difference(p<0.05). Except between the1stweek and the3rdweek. The amount of Treg cells have no significant difference (p>0.05).All of the remain have significant difference (p<0.05). Between the1stday and the2ndweek after SCI, among the3rdday, the1stweek and the3rdweek, the Treg/CD4~+ratiohave no significant difference(p>0.05). All of the remain have significant difference0(p<0.05).Conclusion：1. It is a ideal and reliable method that establish SD rats moderate SCImodels by New York university impactor system.2. In the peripheral blood of SD rats after SCI,the major change is the proportion of CD3~+lymphocytes, CD3~+CD4+lymphocytes and the ratio of CD3~+CD4~+/CD3~+CD8~+down.3. In the peripheralblood of SD rats after SCI, the proportion of CD3~+CD8~+lymphocytes and the ratio ofTreg/CD3~+CD4~+have no change.4.We detect the local infiltrated lymphocytes in thefresh injured spinal cord by By flow cytometry. T cells cannot be detected in healthSD rat spinal cord, and compare with at the1st,3rd,7th,14thand21thday after SCI. Alarge number of T lymphocytes appear in injured part are significantly different.Relative in peripheral blood, the main T cells in the injured part is CD3~+CD4~+cellsand their ratio up, the proportion of CD3~+CD8~+and the ratio of Treg/CD3~+CD4+down.5. CD4~+T and Treg cell infiltrating in normal SD rats spinal cord is undetectableby immunohistochemistry fluorescent staining technique. CD4~+T and Treg cells and theTreg/CD4~+ratio rise obviously at the1st,3rd,7th,14thand21thday after injury and haveobvious difference to normal SD rats. At the2ndweek after SCI, the amount ofCD4~+,Treg cells rise to the peak, then start to down. The Treg/CD4~+ratio have twopeaks at the1stday and the2ndweek after SCI.