Study Of The Motor Capacity, Pathologic Changes And Caspase-3 Expression After Spinal Cord Injury In Rats

Spinal cord injury is a kind of serious diseases that endangers people's health.There was a reported incidence rate of 20-40 per million population due to traffic accidents,falling, violence,physicl exersise and so on. Among the resons above, traffic accident was a contributing factor in 48% of injuries .Recently there is an ascedning trend in the incidence rate.The death of neuronal cells from primary and secondary injuries results in complete loss of the sensory, voluntary motor,and autonomic nervous system below the site of injury.Individuals with trauma to the spinal cord often suffer from paraplegia or quadriplegia.And the high expenses on treatment and care bring a heavy burden to the relatives of the patiens and the society. As evidenced by the poor neurologic recovery after most spinal cord injuries and the paucity of pharmacologic treatments currently available, it should be obvious to even the most casual observer that our present understanding of these pathophysiologic processes and how to manipulate them is fairly rudimentary.Allen studied SCI since the beginging of last century. A lot of researches at present demonstrate that apoptosis may play an important role in the secondary injury, it is not only in neuron but also in astrocyte,oligodendrocyte,microglia,etc.Our experiments are aimed to study the numerical and morphological changes of neurons and astrocytes during the apoptosis. The majority of neurons in the spinal cord are interneurons in the ascending and descending tracts which plays an important role in maintaining functions of motor ,sensoryand autonomic nervous.Giving limited ability of self repair and regeneration of neurons after SCI,our research on neurons after SCI is with great significance. In the early hours after SCI, astrocytes become "reactive", which can provide physical support for neurons and induce regeneration of nerves for the repair of injured spinal cord. Several days after the injury,reactive astrocytes may turn into formative ones which can result in the formation of glial scars .The scars can prevent nerves from regeneration mechanically and chemically. It is an effective way in the treatment of SCI making good use of reactive astrocyte and inhibiting the formation of glial scars. It is generally considered that the apoptotic cell death is closely related to the activation of caspases among which suggests that caspase-3 is just the corresponding member in mammalian animals, caspase-3 is the pivotal enzyme in the mammalian apoptotic cell death .Thereafter,it is called " death protease". A recent study demonstrated that spinal cord injury rapidly increased the activation of caspase-3. Harmful factors and protective measures can be probed by the investigation of the expression of caspase-3 after SCI which can provides bases for the use of caspase-3 inhibitor in the treatment of SCI.We studied the changes of motor capacity, pathological processes, neurons, astrocytes andcaspase-3 p20 after SCI with CBS scales, HE staining and immunohistochemistry methods to investigate the cellular and molecular mechanisms related in secondary apoptosis in SCI.The main results of our research and discussion are as follows:1 .Establishment of spinal cord weight-drop-injury modelIt is quite important to establish an ideal spinal cord injury model for the study on the SCI. Using Allen's method,we set up a spinal cord drop injury model in rats with self-made strike mechanism .After the successful establishment of the model,we observed significant differences between the injury and control groups .the spinal cord neurological function was normal in the control group while in the injury group muscular tension decreased in the hind limbs and reflexes decreased with different extent or even disappeared below the injury level. This model is quite scientific for studies on SCI with the following advantages:easy to put into practice; inducing severe paraplegia highly repetitive; similar with clinical SCI in the pathological changes and function hindrance;with intact spinal dura mater which can avoidthe infiltration of extraneous substances into the injury area. Therefore, it is a idealer model.2. Establishment of scales for motor capacityIt is of great importance to evaluate the motor capacity to demonstrate the extent of spinal cord injury and recovery by the animals' activities.We applied combined behavioral scales to score the motor activities after SCI and got the results that there were significant difference between the scores of each items and the total score.We observed worse recovery of toe spread and contact place responses which represents fine muscular activites and better recovery of incline plane and open-field locomotion which represents unelaborate muscular activites.Given the differences between the recovery of muscular functions, further studies should be done to investigate the mechanisms and the protective measures.3. Study of pathological processes after SCI with HE stainingWe study the pathological processes after SCI with HE staining of spinal cord slides. We observed there existed hemorrage,swelling cells and infiltration of inflammatory cells in the central gray matter of injury areas during 12h-ld.On the 3 day,we observed necrosis focuses with acidophilic granules .On the 5 day, cystoid changes occurred in the gray matter. During 7-14 day , the number of the neurons dramatically decreased and the formation of cavitation was seen which suggests that cavitation is formed after necrosis and organization in the injury area.4. The changes of astrocytes after SCIThe activation of astrocyts and fibrosis is one of processes involved in the phathological changes. We stained astrocytes through immunohistochemical reactions of reagents with the GEAR There was an increasing trend in the number and areas of GFAP-positive cells after SCI. There was significant difference between the injury groups and control groups on Id and 3d which suggests that astrocytes is a state of cellular proliferation after SCI.5.The changes of neurons after SCIIt is a quite important study on neurons in SCI since it is known that the self repair and regeneration is so limited. We stained the neurons through immunohistochemical reactions of reagents with the NSE and received the results that the number of neurons was significantlydecreased in 12h after injuries.The numbers of neurons are decreased by 55.41 %, 68.73 % and 80.13 % on the 12h> Id and 3d after SCI and there was no significant difference between the numbers of neurons during 3d and 14d. It shows that the number of neurons is decreased with large scales in the early stage(during 12h) after SCI and it doesn't change significantly after 3d. This suggests that SCI brings great damage to neurons and neurons are with so restricted capacity in repair and regeneration. So the protective measures should be taken in the early stage after SCI6 The changes of the expression of cleaved caspase-3 after SCIIn order to investigate the cellular mechanism involved in the neuronal apoptosis after SCI,we surveyed injured spinal cord for the cleaved caspase-3 with immunohistochemical methods and found that the cleaved caspase-3 was present and upregulated immediately after SCI and reached its peak during 24—72h while it was absent in the control group. It suggestes that the activity of caspase-3 was enhanced in the traumatic spinal cord which plays an important role in the cell apoptosis.The study above is quite fundamental on SCI and reaches the results that the weight-dropping model is is a very much idealer one; the scores of CBS and various items of CBS are dramatically decreased after SCI; the number of neurons is decreased in the early hours in the penumbra area; the astrocytes' proliferation in the early stage is a protective factor while glial scars can prevent axons from regeneration mechanically and chemically in the late stage; the rule of changes of caspase-3 after SCI: the number of caspase-3-positive cells was increased and reached its peak during 24—72h.Generally speaking, we investigated unclarified pathological changes and cellular and molecular mechnisms in SCI ,thus provided experimental basis and ideas for designing new measures and medicines for the treatement of SCI. So it is reasonable that our results are with great significance in theory and appliance.