| ObjectiveThe purpose of this experimental study is based on the model that cut both sides of the sciatic nerve of SD rats, make the end to end anastomosis of epineurium immediately, and treate by using neurotropin (NTP) early after the operation. Comparing with the control group to observe the changes of morphous of L4-6 spinal cord anterior horn motoneuron and Nissl's body, check the expression of Bcl-2 and Bax by immunohistochemical staining, counts of apoptosis cell with TUNEL staining and observe the morphous of nerve fiber on anastomotic stoma, then to discuss that the protection of NTP for the anterior motoneuron of spinal cord which correspond to the sciatic nerve injuried, and approve that if NTP can accelerate the peripheral nerve to regenerate. It provides some experimental gists of which using NTP early after injured peripheral nerve in order to accelerate the nerve regeneration, and discusses the possible mechanism of action on it primarily.MethodsOne hundred and fourteen healthy male SD rats that everyone had eight weeks old and approximate 200-250 gram were divided randomly into four groups: Normal control group(group A)with 6 rats, without anything to deal with; Experiment group with 108 rats, all of them had end to end anastomosis of epineurium instantly after cut both sides of sciatic nerve. According to the measures of postoperative drug treatment, experiment group was divided into three groups. Group B was not treated, 36 rats. Group C was treated with NTP after operation, 36 rats. Group D was treated with normal saline (NS) after operation, 36 rats. Then, go one step further, divided the SD rats of group B, group C and group D into six subgroups according to the time of them were sacrificed: 1std,4thd,9thd,14thd,21std and 28thd, and every subgroup included 6 rats. About 1 hour after the operation, group C were treated with NTP 1.2NU(1ml)/kg·d, group D treaded with NS 1ml/kg·d, both of two groups had drugs through intraperitoneal injection with regular time and dose, about one time one day until they were sacrificed. Group B had no drug treatment. After the rats were died, fixated their spinal cord segments(L4-6), and then stained with HE, Nissl's body, Bcl-2 and Bax immunohistochemical methods, Tunel methods. Fixated nerve around sciatic nerve anastomotic stoma and staining with silver to observe the changes of regeneration of nerve fiber.Results1 Rats behave after the operation Compared with forward the operation, all rats of experiment group appeared, dysphoria, anorexia, lost weight on differ grade. During the observation after operation, both of hind limbs of over half rats of group B and group D had appeared, lameness, dropsy, self-mutilation, and ulcer to bone. Compared group C with group B and group D, the rats had appeared later, lesser extent, and they recovered earlier. The motor function of experiment group all had appeared restoration with different levels about 4 weeks later. Especially, about 3 weeks later, group C could crawl and stand in short time with both hind limbs, had better effect than group B and group D.2 HE staining of spinal cord Under light microscope, normal control group: the karyon of the motoneuron is roundness, blue dyed, stated in the centre of motoneuron, nucleolus clearly, Nissl's body in cytoplast obviously, and uniformly distributed. Experimental group: the karyon of the motoneuron is shift, Nissl's body condense and decrease, the amount of nerve cells decrease. The motoneuron quantity had statistical significance differences from 14d-subgroups(P<0.05). Compared group C to group B and group D, the motoneuron quantity decrease slower. Experimental group had little of apoptosis cells, as karyons dense and stained, showed blue-black, endochylema was rosiness, the body of them was shrink.3 Staining of Nissl's body in spinal cord Under light microscope, normal control group: the Nissl's body could see clearly in neurons, was triangle or oval bit. Experimental group: Nissl's body quantity had decreased, dissolved or disappeared, however the group C was better than group B and group D. With the observational time going by, they got to be better gradually.4 Bcl-2 and Bax immunohistochemical staining of spinal cord Under light microscope, Normal control group had not found positive cells. Experimental group could find them. The intensity of immunohistochemical expression was analyzed by average optical density of immunohistochemical analyze system of Hpias-1000 pathological graph analytical system. The highest expression of Bcl-2 and Bax all reached in the 9d-subgroups of experimental group. The expression of Bcl-2 had differences in group B, group C and group D. Hereinto the expression of Bcl-2 in the 9d-subgroup,14d-subgroup and 21d-subgroup had statistical significance differences(P<0.05), however Bax not. The value of Bcl-2/Bax of Group C was higher than group B and group D.5 TUNEL staining of spinal cord Under light microscope, no positive cells were found in normal control group, 1d-subgroup of experimental group had little of positive cells; then the quantity of positive cells of 9d-subgroup began to have statistical significant differences(P<0.05); the highest expression of positive cells reached in 14d-subgroup of experimental group, but the quantity of positive cells of group C were less than group B and group D, and there was nosignificant difference between group B and group D. The positive cells of experimental group would decreased along with the observational time going by. The apoptosis speed, form 9thd to 14thd, was the maximum, and the curve slope of arcuation was the slopest.6 Nerve fiber staining Observed the sections of nerve fiber silver staining of experimental group: in 1d-subgroup, 4d-subgroup and 9d-subgroup, there was not nerve fiber passing through anastomotic stoma, and nerve fiber arranged in disorder. The little nerve fiber of 14d-subgroup begun to pass through anastomotic stoma. In group C, some nerve fiber had passed through the anastomotic stoma in 21d-subgroup, and more nerve fiber in 28d-subgroup, but arranged also in disorder. In group B and group D, the state in 28d-subgroup was the same as in 21d-subgroup of group C, and there was not obviously difference between group B and group D.Conclusions1 Cutting the sciatic nerves of SD rats will induce the death of spinal cord anterior horn motoneuron, and the main mode of death is apoptosis.2 After made the operation of end to end anastomosis of epineurium for abscised sciatic nerve, applied NTP early, not only protected spinal cord anterior horn motoneuron to excess apoptosis, but also could promote repair and regenerate following the sciatic nerve injuried.3 After made the operation of end to end anastomosis of epineurium for abscised sciatic nerve, the mechanism on decreasing the apoptosis of spinal cord anterior horn motoneuron by early application of NTP, neurotropin has relation with the value of Bcl-2/Bax heighten. |
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