Study On Spinal Motor Neuron Protection By Cntf After Operation On Different Stages In Rats With Sciatic Nerve Injury

ObjectiveThe experimental study in rats were designed to simulate the patients with sciatic nerve injury who delayed their medical visit. The sciatic nerves were transected and repaired after a delay of different stages. Ciliary neurotrophic factor (CNTF) was used during and/or after surgery. The rats were dissected on the 3rd day after CNTF treatment, and the Ca2+ concentrations of spinal tissue were detected and compared with those in control group. The other rats were dissected 4 weeks after CNTF treatment, and the apoptotic cells were counted with TUNEL staining; the morphological changes of L4-6 spinal neurons were examined; silver staining of nerve fibers in nerve anastomosis area were observed. The results were analyzed to explore the effects of ciliary neurotrophic factor (CNTF) on motor neurons protection of spinal cord segment with traumatic sciatic nerve injury and promotion to regeneration of peripheral nerve. The study provides the experimental basis for application of medication after peripheral nerve injury on different stages. Experimental methods192 healthy male SD rats (8weeks old), weighing 200-250 g , were divided into 4 groups: control group(group A, n=12); Experimental group, sciatic nerve were transected and the outer membrane were repaired(group B,n=60);Treated by CNTF after surgery(group C, n=60); Treated by saline after surgery(group D, n=60). Group B, C, and D were divided into 5 subgroups separately by the stages of immediately, 3d, 7d, 14d, 21d after sciatic nerve injuries(n=12). Group A was as controls, Group B, C, and D were repaired on different time points. CNTF 200 ng/kg·d was injected during operation and CNTF 100 ng/kg?d at amputated sciatic nerves after operation in group C. NS 100 ng/kg?d was injected into amputated sciatic nerves 1h after operation in group D. There was no medication treatment in the other groups. 6 rats were sacrificed randomly on 3d after injury in group B, C and D separately. Ca²⁺ concentrations in spinal cord segment L4-6 were detected. 6 rats were sacrificed randomly in 28d subgroups of group B, C and D separately. HE staining and TUNEL staining were made in spinal cord segment L4-6, and silver staining in anastomotic sciatic nerves.Result1. General index of rats1.1 Behavior after surgery There were dysphoria, disappitite and weight loss in different degrees in group B, C and D. Muscular atrophy in hind limbs, edema, cannibalism, erosion deep into bone in more than half of the rats in group B and C. The rats in group C began to eat more and earlier than those in group B and D, with lighter symptoms and earlier recovery. There were different degrees of recovery in motor function of both hind limbs in 21d subgroups of group B, C and D. The rats in group C could crawl with both hind limbs and stand for a short time after 1-2w with better motor recovery.1.2 HE staining of spinal cordUnder light microscope: Neurons were round, light-blue colored, located in the center, with homogeneous distribution of nissl bodies in group A; the nuclears were not in the center, with nissl bodies concentrated, reduced, and the cell numbers decreased in of group B, C and D. The numbers of motor nerve cells decreased more in all 7d subgroups than those in other time point subgroups, with lower reduce occurred in C.2. Index in 3d acute stage2.1 Ca²⁺ concentration detectionCa²⁺ concentration in 1cm spinal cord segment was detected by atomic absorption spectrometer on 3d after injury. By the formula: Concentration of Ca²⁺ = Ca²⁺ ion neurons concentration/quality after dried×dilution multiple. Concentration of Ca²⁺ was lower in group C than those in group B and D. T≥t0.01(10)(3.169)with statistical significance.3 Index in 28d chronic stage3.1 TUNEL staining of spinal cord Under light microscope: the positive expression of apoptotic cell was in the nuclear, TUNEL staining apoptotic cells were characterized with the nuclear chromosome condensed, cell broken and apoptosis bodies formed. There was no positive expression of apoptosis cells in group A but in all subgroups of group B, C and D. There were significantly different in positive apoptotic cell numbers in all 7d subgroups than those in other subgroups(P<0.05),with the lowest in 7d subgroups. There were no significant differences between subgroups in group B and D.3.2 Nerve fiber stainingBy nerve fiber silver staining, there were some nerve fibers across the anastomosis area seen in immediately, 3, 14d, 21d subgroups; There were more nerve fibers across the anastomosis area in 7d subgroup, with less normal arrangement; There were less nerve fibers across the anastomosis area in 7d subgroups of group B and D than those of group C. There were no significant differences in microscopic changes between B and D.Conclusion1. That the sciatic nerves are transected and repaired at different stages may induce apoptosis of spinal cord anterior horn motor neurons.2. After the sciatic nerves were transected and repaired in rats, early use of CNTF may decrease Ca²⁺ concentration in spinal tissue and keep ion balance in and out of cells in the acute stage. This may decrease apoptosis of spinal motor neurons and keep regeneration of peripheral nerve axon.3. CNTF may reduce apoptosis when sciatic nerves of rats are transected and repaired within 21days.4. CNTF may promote axon regeneration of sciatic nerves when sciatic nerves of rats are transected and repaired within 21days.