| IntroductionIt is a hot spot that the recovery of neural function after spinal cord injuries . The transplantation of neural stem cells is helpful to the recovery of the spinal cord. But now we concentrate on the neural stem cells in the striatum corpora and cort , not on the spinal cord derived neural stem cells. We investigate the culture and transplantation of spinal cord derived neural stem cells and effect to the spinal cord functional recovery.Material and Methods1 Experimental animals and groups;The experiments were conducted on 160 female Sprague - Dawley rats weighing 200 - 250g obtained from China Medical University. Animals were divided randomly into a injury group, a vacant group, a transplant group and a sham operation group.2 The spinal cord injury model of mice: One hundred and twenty S - D rats were performed under anesthesia induced by sodium pentobarbital ,2% (25mg/ kg). Fixation and sterilization,dorsal laminectomy was performed at the T10 -T12. In 120 animals,a traumatic injury was produced in exposed spinal cord by traverse with microscissors. The paraspinal muscles and subcutaneous tissues were subsequently closed with a No. 3 -0 absorbable suture. Immediate complete paraplegia was observed in every case and persisted throughout the entire studyperiod. The control group underwent injury but not to transplant. A vacant group underwent not to injury. The sham operation group underwent dorsal laminecto-my not to injury spinal cord. In these rats, we perceived no postoperative deficits.3 Neural stem cells isolating culture: In early experiment,30 rats ,include 15 female rats and 15 male rats, lived together. When we found vaginal secretions, it is EOd. At the E10. 5d, rats were anesthetized , sterilized, opened abdominal cavity, exposed two sides uterus. We clamped the capitular head of uterus , sheared comu uteri, put it into culture dish. The uterine wall and birth membranes were dissected away,The ependymal cell of central canal of spinal cord were disassociated,cleaned in Hanks balanced salt solution,dissected with scalpel, filtered with 200 wells screen. Blood vessel and connective tissue and tissue pieces were removed. The cells were collected in tubules, boasted into one cell suspention. The cells were counted about 1 x lOVml , built in 50ml culture flask. Cultivate condition;6ml DMEM/F12 + 120ulB27 + ^ulbFGF^?^ ,5 % CO2. The cells were passaged to the fifth generation, assessmented by nestin.4 Cellular transplant: On the second day, rats in the cellular transplantation group were anaesthetized, injected cells about 5 x 10 into iritermedius of spinal cord. Room temperature 30t, we massage the bladder till independence bladder, intramuscular gentamycin lOOOOu/d to prevent infection. 80 rats in the control group and sham operation group were injected Sodium Chloride,the vacant group not deal with.5 Ethology evaluation: The rats were tested through tiltboard and Tarlov score after transplantation 1 week,2weeks,4weeks. Animals stand still 5 second on the tiltboard . We write down the degree of the tiltboard. Tarlov score: animals were tested for movements of the hind limb . no autonomy movement scored 0;only hip and knee joint non reflex movement scored 1;hip and knee and ankle joint movement scored 2;animals can support body quality and mis match walking scored 3;animals can go concordantly and interphalangeal joint movement scored 4;normal gait scored 5.6 Histology;Id, 3d, 1 week, 2 weeks, 4 weeks after the transplantation, animals were anesthetized and perfused. The spinal cord was removed en bloc,fixed by 4 % PFA. It is made to paraffin section and processed for immunohisto-chemical and hematoxylin and eosin studies.7 Electron microscope;Animals were lavaged with 50ml PBS,200ml 8g/L PFA,25g/L glutaral,fixed with osmic acid,dehydrated,embed,stained with ura-nyl acetate and citric acid alum, observed through electron microscope.8 Statistics analysis;The date was analyzed by using SPSS, Student t - test. P <0.01, significant deviation.Results1 HE stain;In the vacant and sham operation group, histological studies showed the features of the normal spinal cord. In the control group, histology showed atrophy, glial scar, capsular space in the spinal cord. In the transplantation group, histology showed no atrophy in the spinal cord.2 Immunohistochemical stain: Neural stem cells survived well in vivo in the transplantation group. CD95 and P75 express obviously diminished in the transplantation group.3 Electron microscope: Apoptosis cells is obviously diminished in the transplantation group than the control group.4 Ethology evaluation;tiltboard test after transplantation, 1 week,the control group 18.5 ±0.76°, the transplantation group 38. 3 ±0. 84°;2 weeks, the control group 19.7 ±0.66°, the transplantation group 39.4 ±0.78°;4 weeks, the control group 22. 3 ±0.69°, the transplantation group 45.0 ±0. 81°;the vacant group and sham group is normal. Values between the control group and the transplantation group were considered statistically significant different, P = 0. 0022, P <0. 01. Tarlov score, 1 week, the control group 3. 32 ±0. 34, the transplantation group 4. 37 ±0.45;2 weeks, the control group 3.41 ± 0.43, the transplantation group 4.45 ± 0.38;4 weeks, the control group 3.45 ±0.48, the transplantation group 4.63 ±0.47,the vacant and sham operation group was normal. P =0.0017, P <0.01.DiscussionThe recovery of SCI is a medical puzzle problem ever since a long time ago. NSCs is applied for recovery of the spinal cord. The transplantation of NSCs is a new strategy for treatment. The spinal cord comes from the tail of embryo medullary tube. The archaeneurocoele developed central tunnel. It has many nerve cell clump. It is made of large neuron, middle neuron,small neuron. Motoneu-ron on the ventricornu is the largest neuron in the spinal cord. Its dendron extend as long as lOOOum. The axis of motoneuron extend to the skeletal muscle. The spinal cord derived neural stem cells and brain derived neural stem cells have some different characteristics.There are many NSCs on the ependyma of spinal cord. The NSCs can proliferated by self replication on the growth period and satisfy the demand of forming many cells. The part of daughter cell differentiated into neuron glial cell. These cells are in quiescent condition on adult period. They are hyperplasia postinju-ry. Spinal cord derived NSCs was isolated from E14. 5d embryo. It goes down to the fifth generation. The NSCs transplanted into cervical cord of rats. The neural precursor cell divided and grew after 5 weeks. The neuron erupted tubercle and inserted in the spinal cord and formed tubercle structure. The tubercle length of spinal cord derived neural stem cells is more longer than that of the brain derived neural stem cells . These feature are fit for the structure and function in vivo.The neural stem cells colony from E10.5d embryon spinal cord go down to the future generation,immunoreaction masculine. At 1 week, values between the control group and the transplantation group were considered statistically different, P <0. 01. The function of spinal cord was partial recovery. At 4 weeks, values between the control group and the transplantation group were considered statistically significant different, P<0. 01. Neural stem cells survived well in injuried spinal cord. A clear statistically different expression after transplantation. The number of apoptosis is obviously diminished in the transplantation group in TUNEL. The author think spinal cord derived neural stem cells aresuitable for repairing the function of spinal cord.ConclusionsThe author think spinal cord derived neural stem cells are suitable for repairing the function of spinal cord.
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