The Role Of T Lymphocytes In Mice Spinal Cord Injury

Neuroinflammation is one of the most important mechanisms in secondaryinjury of SCI. Mammals have both innate and adaptive immunity. Innateimmunity activates adaptive immunity through antigen presentation andcytokine release. And it is also the effective system of adaptive immunity.Research shows that T lymphocytes can regulate the response of innateimmunity, which is of great significance in the lethality of mouse hepatitis virus(MHV) infection. The immune response in SCI is somewhat similar to that inother parts of the body. But it has its own distinctions. The function of Tlymphocytes in the early stage of SCI is still unknown. In our study, asthymicmice, thymectomic mice and CD4+T lymphocytes depleted mice were used tocreate models of mice lack T lymphocytes or CD4+T lymphocytes, in order tostudy their functions in SCI.This research can be divided into the three following parts:PartⅠ [Objective] To investigate the role of T lymphocytes in spinal cord injury, weused BALB/c mice and BALB/c-nude-/-mice (athymic nude mice with BALB/cbackground) which have no T lymphocytes in blood circulation, and comparedthe functional and histological recovery of the two groups.[Methods] Sever spinal cord compression model was made at T8spinal cordsegment in6to8week-old BALB/c and BALB/c-nude-/-mice. We used BassoMouse Scale (BMS) to evaluate the locomotor recovery of the two groups ofmice after SCI. GFAP was used to delineate the lesion area, and NeuN to markthe survived neurons around the lesion. Degenerated myelin was distinguishedby Luxol fast blue dyeing.[Result] BMS results showed that the athymic mice had a significantly betterfunctional recovery than the control group. Histologically, the lesion areasdelineated by GFAP staining were smaller in the athymic mice（0.33±0.04mm2）compared to the BALB/c mice（0.57±0.04mm2） at7dpi. NeuN stainingshowed that the counts of neuron decreased sharply within800μm areas rostralor caudal to the lesion centers. However there were no significant differencesbetween the two groups. Luxol fast blue dyeing showed that athymic mice hadless pathological lesion in white matter.PartⅡ[Objective] To explore the gene expression of different cytokines, IL-1β、IL-6、IL-4、IL-10、MCP-1and MIP-1, in the injured spinal cord during the earlystage of mouse spinal cord injury. Compare the possible expression differencesbetween BALB/c and BALB/c-nude-/-mice.[Methods] Still we used the sever compression model of SCI at T8spinalcord segment.5mm spinal cord tissue centered with the injury site wascollected at different time points. Total RNA was extracted using TRIzol reagent according to the manufacturer’s instruction. cDNAs were reverse transcribedwith Oligo (dT) as primer. And qPCR was used to quantify the expression ofdifferent cytokines.[Results] At the early stage of mouse spinal cord injury, pro-inflammatorycytokines IL-1β, IL-6, MCP-1and MIP-1increased in no time, peaked at6hours post injury, decreased thereafter, and restored to sham level during24hours to3days post injury. However, IL-4, known as a main anti-inflammatorycytokine, slowly decreased at the early stage of spinal cord injury, reached thelowest level at3days post injury, then began to increase. IL-10, anotheranti-inflammatory cytokine, had a little peak at6hours post injury. Theexpressions of these6different cytokines in BALB/c mice and in athymic micehad no significant differences.Part Ⅲ[Objective] To verify the results of PartⅠ, neonatal thymectomy was used toremove mature T lymphocytes of mice. And peritoneal injection of CD4monoclonal antibody was used to deplete T lymphocytes in the circulation.[Methods] Thymuses of neonatal mice from birth to day3were removedunder microscope. And the mice then were raised up. Flow cytometry assay wasused to assure the effectiveness of the surgery. At different time points after SCI,Locomotor function was evaluated according to BMS.To deplete the CD4+T lymphocytes, we peritoneally injected CD4monoclonal antibody into BALB/c mice,200μl every time each mouse (thecontrol group received the same amount of sterile1M PBS),1time every3days.Flow cytometry assay was used to assure the effectiveness of the antibody. Afterthe third time of injection, compression SCI models were performed. BMS wasused to evaluate the locomotor recovery. [Results] The BMS scores of the thymectomic mice at different time pointpost SCI had no differences compared to normal control group. And thefunctional recovery of CD4+T lymphocyte depleted group and the PBS injectioncontrol group had no significant difference either.ConclusionsAccording to the studies above, we could conclude:1. Athymic mice recovered better than BALB/c mice, both functionally andhistologically after SCI, while the thymectomic mice and CD4+Tlymphoyte depleted mice had no better recovery. This implies that in micewith BALB/c background, T lymphocyte plays an insignificant role inearly stage of spinal cord injury. And there are other reasons which canexplain why athymic mice recovered better.2. During the early stage of spinal cord injury, pro-inflammatory cytokines inthe injury site were sharply upregulated. While anti-inflammatorycytokines either decreased or didn’t change. This makes the microenvironment of the injured spinal cord become pro-inflammatory, leads tothe secondary inflammatory injury.