| The functional restoration after peripheral nerve injury is a vital aspect in traumatic surgery. The functional status of neurons in spinal cord affects the functional restoration directly. Several aspects regarding neuron functionality are of key interest in the field of traumatic surgery. These include whether peripheral nerve injury can cause apoptosis of the corresponding neurons; as an important secondary messenger, whether Ca2+ takes part in the apoptosis process; and the pathway of the apoptosis. This experiment is designed to explore whether motoneurons undergo apoptosis following peripheral nerve injury, the role of Ca2+ in this process, and the corresponding signal transduction pathway involved. We used the whole-cell patch-clamp techniques, in situ hybridization, and immunohistochemistry to study the apoptosis of neuron after peripheral nerve injury, the changes in L-type calcium channel on the nerve cell membrane, the changes of the free calcium ions in nerve cell, and the ultrastructure of nerve cells.The study result will help to improve the functional restoration after peripheral nerve injury.The adult wister rats were divided into pseudo-operation group(disposed the brachial plexus nerve root, but not cutted it off), control group(brachial plexus nerve root amputating wound group) and experimental Group(brachial plexus nerve root amputating wound + verapamil group). We first established the animal model of brachial plexus nerve root amputating wound, then applied the calcium ions blocker on the animal model, and observed the changes of motoneurons in spinal cord after nerve injury.1. Detect the L-type calcium channel of motoneuron using the whole-cell patch-clamp recording techniques. Under the microscope, the C5～T1 spinal cord and dura mater of spinal cord were taken out, immediately followed by dissociation of the nerve cells of spinal cord anterior horn under sterile condition. The isolated nerve cells were then cultured in appropriate medium for 24 to 48 hours. Nerve cells with diameter between 50 to 70 micrometer were selected, and L-type calcium channel on the nerve cell membranes were studied. 2.Monitor the changes in intra-cellular Ca2+ concentration using spectrofluorometer.3. Detect motoneruron apoptosis using the TUNEL method. 4. Observe the ultrastructure of spinal cord anterior horn cells with transmission electron microscope.5. Determine the transcription level of CaM with in situ hybridization.6. Oberve the C-fos gene expression in spinal cord 7. Perform neuron countingSummary of the study results: 1.Following the peripheral nerve injury, the electrical current of L-type calcium channel on the nerve cell membrane changes with time. In the control group, the ion channels on the nerve cell membrane opened (and the electrical currant increased) immediately after peripheral nerve injury. The Electric current decreased slightly 24 hours later, and maintained at relatively stable level for one week. About two weeks after nerve injury, the electrical current dropped back to normal level. On the contrary, the ion channels on the nerve cell membrane maintained at the normal level throughout the experimental procedure in the experimental group. 2. The Ca2+ concentration within motoneuron increased immediately after the peripheral nerve injury in control group and experimental group. The increase in Ca2+ continued and reached its peak by 48 hours, then gradually decrease. One week after nerve injury, the Ca2+ level in the experimental group dropped back to normal level, however, the Ca2+ level in control group remained to be slightly higher than that in pseudo-operation group. There is some difference between control group and experimental group, and between control group and pseudo-operation group.3. No apoptosis in motoneurons were observed within 48 hours post- nerve injury. Apoptotic cells in both control group and the experimental group were found after 48 hours. The number of apoptotic cells reached the peak in one week, then began to decrease... |
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