Experimental Study On Mechanism Of Immunosuppressant FK506 To Accelerate Regeneration Of Peripheral Nerves

Objective1. To monitor the change of T lymphocyte subsets of peripheral blood and the deposition of IgG and the expression of MHC II in local area of lesioned nerve after injury and during regeneration of sciatic nerves of rats.2. To study the effect of FK506 on the change of above-mentioned immunological indexes and to explore the relationship between the immunosuppression of FK506 and the regeneration of peripheral nerves.Methods1. The models of transection and anastomosis in situ of sciatic nerves were made in SD rats with microscope: Anesthetize a rat and immobilizate it in prone position. Make a longitudinal incision about 2cm long on the posterior surface of right buttock and thigh and expose the sciatic nerve. Transect the nerve at about 1cm below greater sciatic foramen and then anastomose it in situ with 3 ⁴ interrupted stitches of 10/0 monofilament nylon silk. Close the incision.2. Five male or female adult SD rats constituted the normal control group. A segment about 8mm long (4mm long proximally and distally from the stoma) of right sciatic nerve of each rat was cut off and was made into cryo-slices l0μm thick. Detect IgG by indirect immunofluorescent staining and detect MHC II by indirect immunoenzymatic staining to investigate the deposition of IgG and expression of MHC II in normal nerve tissue. Then draw 2³ml blood from the heart. Detect T lymphocyte subsets of peripheral blood by direct immunofluorescent FCM (flow cytometry) and get their normal values.3. Sixty male or female adult SD rats weighing about 200²50g received the surgery of transection and anastomosis in situ of sciatic nerves and then were allocated randomly into FK506 group, CsA group and placebogroup. Each group had 20 rats and received FK506 1mg/kg ?d-1, CsA l0mg/kg ?d-1 or correspondent volume of normal saline by infusing these solutions into stomach, respectively. Five rats were taken from each group 1, 2, 3 and 4 weeks postoperatively and were drawn materials to detect IgG, MHCII and T lymphocyte subsets. The difference between pre- and postoperation and between different groups of these indexes were assessed.4. The same models as above were made in fifteen male or female adult SD rats weighing 200²50g. These rats were allocated randomly into three groups, each having five, and received FK506 1mg/kg ?d-1, CsA l0mg/kg ?d-1 or correspondent volume of normal saline by infusing these solutions into stomach, respectively. Have a walking test and get the footprints to calculate SFI every half month postoperatively. Materials were drawn three months postoperative to make histological examination by myelin staining and HE staining. The thickness of myelin and the diameter of axis-cylinders were measured and the number of myelinated nerve fibers was counted to evaluate the regeneration of the lesioned nerves.Results1. All rats survived well. No death and no infection of the incisions appeared. The third, fourth and fifth toes of right lower limbs in three rats lost. The stoma were seen smooth without laceration while drawing materials.2. Immunohistochemical staining of IgG and MHC II of normal control group demonstrated light staining of normal nerve tissues. The result of T lymphocyte subsets(%): CD3+ 59.89 ?6.23, CD3+CD4+ 39.71 ?5.35, CD3+CD8+20.45 + 1.77, CD3+CD4⁺/CD3+CD8+1.94 ?.20.3. By contrast with normal contrast group, the immunohistochemical staining of IgG and MHC II of placebo group demonstrated stronger staining on epineurium and scattered staining of nerve tissue, and the proportion of CD3+CD4+ and the ratio of CD4"7CD8+ increased, especially two weeks postoperative.4. By contrast with placebo group, FK506 group and CsA group have lighterimmunohistochemical staining. The proportion of each T lymphocyte subset reduced and the ratio of CD4+/CD8+ decreased during the whole period of administration.5. The data of dynamic SFI revealed that in contrast to placebo group and CsA group, FK506 group g...