The Effect Of Expression Of Gfap In Spinal Cord By Cntf After Operation On Rats With Sciatic Nerve Injury

Objective In this study, patients with sciatic nerve injury in simulated clinical nerve anastomosis in time after the repair operation and application of ciliary neurotrophic factor,through the comparison with the control group,observed the performance and HE staining,the average absorbance of immunohistochemical staining of glial fibrillary acidic protein (GFAP), apoptosis motor neuron marked by Terminal–deoxynucleotidyl transferase mediated nick end labeling of corresponding spinal segment of sciatic nerve in the1st,3rd,7th, 14th, 21st and 28th day after the operation, to investigate the effects of ciliary neurotrophic factor on the expression of glia fibrillary acidic protein in the corresponding segment spinal anterior horn astrocytes after anastomosing apocoptic sciatic nerve in rats, the protection of astrocytes(AS) to motor neuron, providing some experimental evidence for medication after repair peripheral nerve injury operation.Methods 1. Model preparation and group:The male 8 weeks 114 SD rats, body weight 200～250g, were divided into control group(n=6), Model group(n=36), Saline group(n=36), Medicine group(n=36). All rats were anaesthetized with volume fraction of 10% chloral hydrate (3ml/kg) intraperitoneal injection except control group, exposed bilateral sciatic nerve, cutting the sciatic nerve in the hole about 0.5cm from the infrapiriformis foramen, epineurial suture under the operating microscope with 10-0 nylon medical suture line, 4-pin at each end, 90-degree angle, give antibiotics to prevent infection in surgery area. Give medicine group a intramuscular injection with CNTF100 ng / kg every day, saline group equivalent normal saline. Taking 1 rat of control group, 6 rats of model group, 6 rats of normal saline group and 6 rats of medicine group, open the chest and exposed heart after intraperitoneal anesthesia volume fraction 10% chloral hydrate (3ml/kg) in the1st, 3rd, 7th, 14th and 28th day after the operation. Then penetrate aorta through apical with thick needle, fixed needle, blood through cut the right atrial appendage with eye scissors, inject warm saline 200ml quickly, then inject 4% paraformaldehyde 200ml after the blood flow became transparent, keep dripping for 1h with 200ml PBS. As the same time, the eyeball of rat became semitransparent,spitted white foam,tongue fell, muscle vibratted, stiff limbs and tails twitch, the color of liver became yellow. L4-6 segment spinal was tracked retrograde along the exposed sciatic nerve, then cut out it and putted into 4% paraformaldehyde for 24h, paraffin embedding finally. Coronal full-thickness 4～5μm biopsy,select two pieces of each rat each staining, expand slices in 45℃water bath, scoop up with slide glass soaked in polylysine.2. Examination method: HE staining,GFAP immunohistochemical staining, apoptosis motor neurons marked by Tunel.3. Deal with results: The data was show as x±s, carried on statistics analysis with SPSS13.0 statistical software, difference had statistical significance when P<0.05.Results 1. The performance of rats after operation: there are varying degrees of irritability, loss of appetite, weight loss in the operated rats. Anastomosis during the observation, model and Saline group of rats, almost dual-hindlimb muscle atrophy appears, peripheral edema, since the food, such as bone erosion depth of the performance of the medicine group and the model and saline groups compared to the general back and eat rats, and less of these symptoms, there were also earlier recovery. Three groups of rats around in the post-3w double hindlimb motor function of the varying degrees of recovery, which recovery of medicine group than the model and saline group were better in about 1～2w can be expressed as two-hind crawling, standing a short period of time. 2. HE staining of spinal cord: We can see motor neurons with larger cell body was purple block, had many synapses under the microscope, a big round or oval nucleus and prominent nucleoli with clear membrane, less chromatin in nucleus. There are many scattered small nuclei blue-violet, they were glial cell nucleus, the largest round or oval nucleus and less chromatin one was astrocyte. Nucleus in anterior horn motor neurons of control group were lightly blue stained in the central of cell body and manifest, nissl body was manifest and uniform distribution. The number of motor neurons of operated rats reduced, nuclear deviated, nissl bodies were reduced and lightly stained, emerged vacuole cell, glial cells increased around the neurons. The number of neurons started decreasing from the 7th day, especially the 14th day. The number of medicine group neurons was more than other two group each time point from the 3rd day (P<0.05).3. Immunohistochemistry staining: It was positive staining that GFAP of astrocytes showed clear brown under the microscope. There was not obviously expression in the control group, but gradually increased in others from the1st day, reached the peak in the 14th day,difference had statistical significance compared with the model group and the saline group(P<0.05), high expression of GFAP of medicine group continued to the 28th day, but the high expression of GFAP of model and saline group just continued to the 14th day, restores normally basically in the 28th day.4. TUNEL apoptosis detection: Apoptotic cells of neurons in the spinal cord anterior horn under the microscope, the features of apoptotic cells labeled by TUNEL were that cells shrinked,chromatin was dense patchy uptake, nucleus were pyknosis,nuclear fragmentated, formed apoptosis bodies. There were not positive cells in control group, the operated rats emerged TUNEL-positive cells in the 1st day, reached the peak in the 14th day, reduced significantly in the 21st day. The apoptotic cells number of medicine group reduced significantly than other two group each time point from the 3rd day (P<0.05).Conclusions 1. Anastomosing apocoptic sciatic nerve of rats can induce motor neuron apoptosis in spinal cord anterior horn. 2. GFAP expression of the corresponding spinal segment was promoted through applying CNTF after anastomosing apocoptic sciatic nerve. 3. Glial cells increased can decreased the number of apoptosis motor neurons, in other words, protect damaged motor neuron cells. 4. Applying CNTF can decrease the number of apoptosis motor neurons after anastomosing apocoptic sciatic nerve.