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Purpose of this study is to simulate clinical patients with sciatic nerve injury in the after timely surgical nerve anastomosis repair and ciliary neurotrophic factor (Ciliary Neurotrophic Factor, CNTF), compared with the normal control group, the rats were observed postoperative performance, HE staining, surgery after 1,3,7,14,21,28 d sciatic nerve to the spinal cord stage of immunohistochemical staining of glial fibrillary acidic protein (Glial fibrillary acidic protein, GFAP) mean absorbance deoxynucleotidyl transferase-mediated gap end labeling (Terminal-deoxynucleotidyl transferase mediated nick end labeling, Tunel) labeled spinal cord apoptosis motor neuron cell count, to explore the impact of CNTF GFAP expression of the corresponding segments of the spinal cord after peripheral nerve injury, astrocytes (Astrocytes , AS) protection of motor neurons, provide some experimental basis for clinical peripheral nerve injury, surgical repair after medication. Method 1 model preparation and grouping: 114 Weight 200 ~ 250g 8-week-old male SD rats were randomly divided into control group (n = 6), model group (n = 36), saline group (n = 36) , drug group (n = 36), in addition to the control group, all rats with the volume fraction of 10% chloral hydrate (3ml/kg) anesthetized by intraperitoneal injection exposed sciatic nerves from the piriform hole about 0.5cm cut off the sciatic nerve, 10-0 medical surgical microscope nylon suture line epineurium anastomosis, end 4-pin, needle away from the angle of 90 degrees, given antibiotics to prevent surgery area infected. Drug after rats daily CNTF100 ng / kg, saline group given an intramuscular injection of normal saline was given an intramuscular injection. 1,3,7,14,21,28 d after randomly taken from a control group of rats, take the model group, saline group and drug group of six rats, with a volume fraction of 10% chloral hydrate (3ml / kg) intraperitoneal anesthesia thoracotomy exposed the heart, aortic apical penetration with a thick needle, fixed needle with ophthalmic scissors cut right atrial appendage bloodletting, rapid injection of warm saline 200 ml of blood to be outflow becomes clear, rapid injection in PBS containing 4% paraformaldehyde fixative 200ml, then 200 ml of the same fixative maintain infusion of about 1h after drawn. Perfused rat eyeball becomes visible when fall outside the white translucent, foaming at the mouth, tongue, muscles trembling, twitching limbs stiff, the tail stiff straight, the liver becomes pale yellow color. Exposed on both sides of the sciatic nerve, take out along the sciatic nerve retrograde tracing connected segments L4-6 spinal cord. The spinal cord was immersed in PBS containing 4% paraformaldehyde fixative. The specimens are immersed in fixative 24h after paraffin embedding. Coronavirus layer slice, each rat of each stain selected two sheet thickness 4 ~ 5μm, Expand 45 ° C water bath, with a poly-L-lysine treated slides fish. 2. Detection method: HE staining, GFAP immune staining staining Tunel marked apoptosis of motor neurons. Result processing: The data obtained are x ± s statistical analysis in Excel database finishing with SPSS13.0 statistical package, the same point in time between the two groups compared using analysis of variance, P lt; 0.05 for difference statistically significant. Results 1. Postsurgical performance: postoperative rats are different degrees of irritability, loss of appetite, weight loss. Anastomosis observation period, the model group, saline group, almost all of the posterior limbs of rats muscle atrophy, peripheral edema, since addicted performance drugs rats eating early and more, the low level of the above symptoms, rapid recovery. Model group, saline rats have varying degrees of recovery motor function of hind limbs in about three weeks, the drug group, about 1 to 2 weeks has been restored. 2 HE staining of the spinal cord: light microscope, visible sports neuron cell body was purple lump, synapses more, the center is a large round or oval nuclei, prominent nucleoli, clear nuclear membrane, nuclear chromatin. More dispersed around the neurons blue purple smaller nuclei, glial cell nucleus. The nucleus, were round or oval, chromatin lesser astrocytes. Horn motor neurons in the nucleus in the control group round, blue lightly stained, the cell body is located in the central, prominent nucleoli, intracytoplasmic Nissl bodies, evenly distributed. The postoperative rat motor neuron cells decrease in the number of, fine nuclear partial bit Nissl bodies decreased lightly stained, and vacuoles cells, glial cells around neurons increased reactive. After 7d number of neurons significantly reduced 14d number of neurons decreased the most. Drug group at each time point from 3d the neuron counts significantly more than the model group and the saline group (P lt; 0.05). 3. Spinal cord anterior horn immunohistochemical staining: GFAP immunohistochemistry showed the yellow-brown staining in the cytoplasm in the light microscope, astrocytes stained positive. Control rats no significant positive expression of the postoperative rats the 1d began GFAP expression gradually increased and reached the peak at 14d when compared with the model group and the saline group, the difference was statistically significant (P lt; 0.05), the drug group GFAP high expression continued until the 28d, the model group, saline group GFAP highly expressed only lasts until the 14d, to 28d when returned to normal. Spinal front corner TUNEL apoptosis staining: the light microscope, anterior horn motor neurons of apoptotic cells positive expression in the nucleus, TUNEL labeled apoptotic cells characterized as cell shrinkage, the nuclear chromatin uptake dense punctate pyknosis, nuclear fragmentation, formation of apoptotic bodies (Apoptsis body). No positive control group cells after 1d rats have TUNEL-positive cells, 14d of apoptosis peak on day 21 significantly reduced. Drug group from 3d apoptotic cell count compared with the model group, saline group was significantly reduced (P lt; 0.05). Conclusion 1 can induce apoptosis in the anterior horn motor neurons after sciatic nerve transection anastomosis. Sciatic nerve transection anastomosis application of CNTF can promote the corresponding spinal segment GFAP rat sciatic nerve transection anastomosis application of CNTF can promote GFAP expression of the corresponding spinal segment. 3. Sciatic nerve transection anastomosis, reactive hyperplasia of astrocytes can reduce the number of motor neuron apoptosis, damaged motor neuron cells have a protective effect. 4. Sciatic nerve transection anastomosis application CNTF can reduce the number of motor neuron apoptosis.
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