| BackgroundAcute spinal cord injury includes primary injury and secondary injury. Primary injury refers to the injured due to the vertebrae of the shift, dislocation or intervertebral disc protruding into the spinal canal compression of spinal cord or fracture fragments of spinal cord injury caused by spinal cord compression and impact, resulting in spinal cord laceration and contusion and laceration and shearing injury, primary injury is immediately generated by the external force and irreversible damage. Secondary injury refers to by the primary injury initiated a series of chemical and biological factors involved in the complex process, resulting in damage to the spinal cord to further increase, the spinal cord damage sometimes even more than the primary injury. The pathological mechanism of secondary injury is very complex, involving many factors, and these factors influence each other, which results in the initial lesions around the integrity of tissue self destructive lesions. At present the research on secondary spinal cord injury mainly concentrated in the following aspects: changes of ischemia and hypoxia, microcirculation injury, spinal cord ischemia after delayed hypoperfusion, inflammatory reaction, free radical reaction and lipid peroxidation and so on.Spinal cord injury (SCI) is a kind of severe central nervous system (CNS) injury. Epidemiological studies show that this injury is more common in young men adults, most often occurs in young people aged between 16-30,40.4% in traffic accident and 27.9% in high fall injury. Spinal cord injury will seriously affect the quality of life of patients and reduce their life expectancy. The SCI is showing a high incidence rate, high disability rate, high cost and low age that the characteristics of "three high and one low" in clinical. A lot of useful exploration for the treatment of SCI. But so far, the effect is not ideal.With the study of pathophysiological mechanism of SCI, researcher have found that the excitatory amino acid (EAAs) plays an important role in spinal cord injury. Excitatory amino acid (EAAs) including glutamic acid (Glu) and aspartic acid (Asp), they are the important excitatory neurotransmitters in the central nervous system. In the normal circumstances of the nerve endings, when depolarization of nerve endings, the Ca2+-dependent way continuously release EAAs, on specific membrane receptors, causing the change of a series of second messenger in cell, and itself will be degradated by the internal enzyme and uptake from the synaptic rapidly eliminated. EAAs in the role of neurotoxin in the secondary spinal cord injury is mainly manifested in two aspects:one is the swelling of nerve cells in acute permeability in a few hours after the injury caused by EAAs receptor hyperactivity mediated by Na+ and Cl", internal flow and water flow characteristics of passive; the other is delayed injury occurred in a few hours to a few days after the injury due to excessive excitement caused by NMDAR nerve cells, with the Ca2+ internal flow caused by intracellular Ca2+overload, the biological oxidation of mitochondrial function will be inhibited and decreased ATP generation, accelerate the degradation of protein and phospholipase, promote the dissolution of directly caused the degeneration and necrosis of nerve cells; on the other hand, intracellular calcium overload. Free radicals are produced, causing oxidative stress. Free radicals are chemically active, strong oxidation, serious damage to the normal structure of cell membrane, activation of cysteine and aspartic acid protease family (caspases), caused by a series of enzyme cascade, eventually leading to the occurrence of apoptosis.Axonal demyelination also plays an important role in spinal cord injury. In the central nervous system, myelin is an important structure that oligodendrocyte formation by wrapping axons, axonal myelination, conduction velocity increased by nearly 100 times, to complete the exchange of information and intricate. At the same time, axonal myelination, myelinated nerve fibers maintain saltatory conduction, saving a lot of energy. Moreover, intact myelin can limit glutamate and CD8+T cells on axon attack. In recent years, the research of the spinal cord injury of myelin and oligodendrocytes in nerve repair has been more highly focused. Therefore, to seek an effective method for promoting tissue regeneration of myelin is the key to the treatment of spinal cord injury. However, there is no effective method to promote myelination.At present, NMDAR antagonists have been widely used in neuron injury caused by CNS damage. The NMDAR and ion channel blockers such as MK-801 and ketamine, benzene Gua pyridine phencyclidine prevent Ca2+ enters the cell, to avoid intracellular Ca2+ overload and excited toxicity. Therefore, NMDAR has been used as a target for the treatment of CNS damage, and there is a similar mechanism of toxicity in acute SCI. On spinal cord injury application NMDAR antagonist agent (CPP) intrathecal injection can effectively alleviate nerve function and tissue damage, antagonistic Na+, Ca2+ influx can alleviate the damage of nerve cells, and to gain time for other treatment. Memantine is a new type of NMD A (N-methy-D-aspastate) receptor antagonist with low moderate to moderate affinity, voltage dependent and non competitive.1- amino -3,5 two methyl, molecular formula for C12H21N, HC1, its relative molecular mass is 215.76, the pure white or white powder, soluble in water. It was first used in the treatment of moderate to severe Alzheimer’s disease (AD), now it has been used in the treatment of other diseases. Memantine as NMDAR blocker, can effectively block the binding of glutamate and ion channel receptors, reduce glutamate (Glu) pathological concentration increased resulted in neuronal damage. Memantine in NMDAR binding sites and Mg2+ overlap, but unlike Mg2+, its affinity with the NMDAR is low, the binding to the receptor dissociation. In addition, memantine with voltage dependent, normal physiological activities, due to the postsynaptic membrane of strong depolarization of the dissociation from the receptor, but in the longer duration of moderate degree of depolarization, such as damage has occurred in the memantine will continue to stay in the receptor, to play a closed role.Oligodendrocytes precursor cells (OPCs) is a kind of neural epithelium cells derived from the neural tube of early embryonic development, which is widely distributed in the central nervous system of animals. Oligodendrocyte precursor cells as a mature oligodendrocytes (OL) progenitor cells was less, in a variety of factors under the action of migration and differentiation into mature oligodendrocytes, participate in the formation of myelination of axons in the central nervous system. Compared with other sources of stem cells, OPCs transplantation is an ideal cell source for the treatment of spinal cord injury.In this study, the memantine combined with OPCs transplantation for the treatment of spinal cord injury in rats, to investigate the memantine block glutamate excitatory poisoning, reduce NMDAR excessive activation of oxidative stress produced by damage to the spinal cord; and on the other hand, memantine can by blocking glutamate on transplantation of OPCS injury, reduce the transplantation of OPCS death and promote cell transplantation survival and differentiation, and make the function of the myelin sheath regeneration better. This will provide a new idea for the treatment of spinal cord injury.Objective1、Investigate the effect of memantine on spinal cord injury and recovery of motor function of rats and related mechanism.2、Memantine combined with OPCs transplantation for the treatment of spinal cord injury in rats, to observe rat motor function recovery, to explore the status of oxidative stress in spinal cord injury and changes of OPCs survival and differentiation and myelination of axons regeneration.Methods1、80 healthy adult male SD rats, using clamping method to establish the animal model of T12 spinal cord injury in rats, and randomly assigned for sham operation group (group A), model group (group B), memantine group (Group C) and MK-801 group (D group), with 20 rats in each group. Preoperative and postoperative 24h,72h, 5d,7d of BBB scores, and xanthine oxidation method and thiobarbituric acid method was used to detect the damage of spinal cord tissue homogenate by superoxide dismutase (SOD) and malondialdehyde (MDA) levels; HE staining, Nissl staining observation of spinal cord injury group fabric of pathological changes and TUNEL assay was used to detect the neuronal cell apoptosis.2、Primary culture to obtain a sufficient number of OPCs, and the use of immune fluorescence labeled OPCs specific antibody O4 for identification. Healthy adult male 100 SD rats, using clamping method to establish the animal model of T12 spinal cord injury in rats, and were randomly divided into model group (group A), PBS group (group B), memantine group (Group C), OPCS group (Group D), MEM+ OPCS group (E group),20 rats in each group. In the memantine injected after the completion of the 1 d,3 d,5 d,7 d, five rats from each group, SOD and MDA were detected.7 days after the models were established, the OPCs transplantation was performed in situ. BBB scores were observed every week after cell transplantation, and the motor function of the hind limbs was observed. After 8 weeks of transplantation, the survival and differentiation of OPCs and the regeneration of myelin sheath were observed by immunofluorescence double labeling.Results1、The establishment of spinal cord injury modelThe moment when clamping against rat spinal cord, rat tail appeared transient continuous spastic swing, the lower limbs transient continuity back shrinkage flutter like, then the lower limbs flaccid paralysis. The ischemic area and edema were observed in the spinal cord. Experimental animal model of spinal cord injury was successfully established.2、The culture and identification of the precursor cells (OPCs) of the humanOne day after primary culture mixed glial cells adhered to the wall, day 2 visible cells into a group of adherent growth, a small round, oval or polygonal,8-9 days visible obvious cell stratification and the lower layer is confluent, covered with a culture astrocytes in the bottom of the bottle. The upper layer is closely attached to OPCs in the astrocyte surface, OPCs somata showed small round, refraction strong, part of the cells out 1,2 processes. A large number of OPCs were isolated by the method of oscillating separation and differential adhesion, and OPCs was detected by immunocytochemistry. Most of the cells were positive for 04 antibody.3、Motor function scoreIn the first part of the experiment, the memantine intervention group and MK-801 treatment group and model group compared to rat BBB score was significantly increased, differences at each time point has statistical significance (P< 0.05), but memantine intervention group and MK-801 treatment group at each time point BBB evaluation phase had no significant difference (P>0.05). In the second part of the experiment, each experimental group rat BBB score are prolonging, the score gradually increased, the intervention group with memantine, OPCS transplant group and combined treatment group compared with model group and PBS group in each time, the difference is statistically significant (P< 0.05).4、The SOD, MDA detectionIn the first part of the experiment, the postoperative day 1 injury in rats SOD values than those in the sham operation group was significantly lower, and the difference is statistically significant (P< 0.05); after the adoption of memantine and MK-801 intervention 3 d,5 d,7 d, SOD level gradually increased, compared with the model group, and the difference is significant (P<0.05), but on the seventh day, the intervention group with memantine and MK-801 treatment group and sham operation group compared to the change of SOD and no significant difference. Memantine intervention group and MK-801 intervention group at each time point was no significant difference between SOD values (P>0.05). Postoperative memantine intervention group, MK-801 intervention group MDA value gradually decreased, compared with the model group, the MDA content had significant difference (P< 0.05). In the second part of the experiment, and the level of SOD in the model group and PBS in the intervention group with the prolongation of time SOD value gradually decreased; In the memantine group, OPCS group and combined group, the level of SOD with the extension of time, value of SOD increased gradually, compared with the model group and PBS group in each time point, the difference is statistically significant (P< 0.05). Postoperative MDA in Memantine group, OPCS group and combined group value gradually decreased. Compared with the model group and PBS group, the MDA content have significant difference (P<0.05).5、HE and Nissl stainingHE staining and Nissl staining found that the sham operation group at each time point spinal cord tissue structure without obvious abnormalities; within the gray matter of the spinal cord tissues of model group showed a large amount of hemorrhage, the white matter structure disorder, and there are empty, gray white matter junction is not clear, nerve fibers loosely arranged, the swelling of nerve cells, interstitial inflammatory cell infiltration; memantine group within the spinal cord tissue also visible small hemorrhage, mild swelling of cells, a few inflammatory cells infiltration; MK-801 group of spinal cord tissue in neural cells appeared mild swelling and nerve fibers in mild osteoporosis. Nissl staining was seen in model group, neurons and glial cells Nissl body pale staining, number significantly reduced or even disappear, memantine and MK-801 group Nissl body structure rules, the number compared with the sham operation group decreased slightly.6、TUNEL cell apoptosis detectionTUNEL assay it was found that the first day after operation the injury group rat neural cell apoptosis rate than those in the sham operation group was significantly higher, difference was statistically significant (P<0.05); then the memantine group and MK-801 group of nerve cell apoptosis rate decreased,3 d,5 d,7 d compared with the model group, the difference has statistical significance (P<0.05), memantine group and MK-801 group at each time point, the nerve cell apoptosis rate differences had no statistical significance (P>0.05)7、The survival and differentiation of OPCs were observed by immunofluorescence double labeling.CC1 was a marker of mature oligodendrocyte cells, under the microscope, red light was stimulated, and NG2 was OPCs specific marker, the green light was stimulated under the microscope. Memantine group, OPCS group and combined group can see obvious red and green signal, combined group, the positive signal is more obvious than other group.8、The regeneration of myelin sheath was observed by immunofluorescence double labeling.Under the microscope, MBP was the specific marker of myelin sheath, the yellow light was stimulated, and NFM was the axon specific marker, the green light was stimulated under the microscope. The experimental group can see remyelination of the fluorescence signal and memantine group, OPCS group and the combined group of myelin regeneration more significantly than that of model group.9、statistical analysisConclusionsMemantine can block excessive activation of NMDAR function, reduce the injury of rat spinal cord after oxidative stress reaction, reduce the apoptosis of nerve cells and spinal cord injury, promote the recovery of motor function of rats with spinal cord injury. Memantine combined with oligodendrocyte precursor cells (OPCS) transplantation for treatment of spinal cord injury rats, can effectively promote the transplantation of oligodendrocyte cell survival and differentiation, can effectively promote remyelination. |
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