| The treatment of spinal cord injury is a problem that puzzled the orthopaedic scientists. The extent of initial damage to the spinal cord produced by a spinal cord injury depends on factors such as the mechanism of injury, force of the impact, cord displacement, acceleration, and kinetic energy of compression. The primary injury is followed by secondary injury mechanisms that are associated with physiological, biochemical, immunological, and cellular changes in the injured cord. Spinal cord compression and displacement from bone fragments, ligaments, or hematoma within the spinal canal may contribute to the initial damage by stimulating this secondary injury cascade. Mitigation of this secondary injury process by spinal cord decompression may be an important factor in the preservation of neurological function. Retrospective studies have demonstrated that surgical decompression increases neurological recovery. But there is no consensus regarding the most effective time-frame for intervention. Following the developing studies of spinal cord injury, scientists find out that the functional recovery depends on the preservation of neurons and the regeneration of axon. Neurotrophic factors, such as glial cell line-derived neurotrophic factor, can't cross the blood-brain barrier. Gene therapy is a new method following spinal cord injury. GDNF, a member of the transformation growth factor-βsuperfamily, is known to be the most active neurotrophic factor for motoneurons. The adenoviral vector is easy to be produced and is proven to mediate successful expression of target gene. In the previous gene therapy studies, the volum of the intraspinal injection changed from 0.5ul to 6ul. Whether there is a relationship between the volum of the intraspinal injection and the extent of spinal cord injury has not been inspected. The first objective of these series studies is to investigate the relationship between sustained spinal cord compression and the extent of spinal cord injury with histological examination, regional spinal cord blood flow, somatosensory evoked potentials and functional assessments. The second objective of these series studies is to investigate the relationship between the volum of intraspinal injection and the extent of spinal cord injury, and then find out the safe intraspinal injection volum. The third objective of these series studies is to assess the effect of adenovirus-mediated GDNF transfer in vivo on recovery of motor function following moderate spinal cord injury with safe intraspinal injection volum. Part one: Relationship between sustained spinal cord compression and neuronal functional recovery following spinal cord compression injury in dogs. Objective: The objective of this study is to investigate the relationship between sustained spinal cord compression and the extent of spinal cord injury with histological examination, regional spinal cord blood flow, somatosensory evoked potentials and functional assessments. Methods: Twenty-four dogs underwent sustained spinal cord compression for 30 or 180 minutes. The cords were compressed with the loading device of a hydraulic piston. When somatosensory evoked potentials declined by 50%, dynamic compression loading was no longer increased but was sustained. Sustained compression was continued for 30 minutes in 12 dogs and for 180 minutes in the other 12 dogs. Somatosensory evoked potentials were regularly monitored throughout the duration of the sustained compression. After the designed time, the compression was discontinued and somatosensory evoked potentials were monitored at the end of the dynamic loading, 60 minutes, 120 minutes, 180 minutes and 28 days after the decompression. Regional blood flow were monitored during the period of sustained compression as well as 180 minutes and 28 days after the decompression. Functional motor recovery was judged throughout a 28-day period after the injury with a modification of the standard Talov system. Thelesion and damage to the tissue were assessed with histological analysis 28 days after the experiment. Results: At the end of the dynamic loading and before sustained compression there was a decline in the amplitude of the somatosensory evoked potentials to 20% of the baseline. After decompression, somatosensory evoked potentials recovered to 42.8%±5.7% in 30-minute group, then recovered slowly. Somatosensory evoked potentials recovered to 48.3%±7.7% in 30-minute group 28 days after the decompression. In contrast, decompression did not result in recovery of somatosensory evoked potentials in 180-minute group (p<0.05). Regional blood flow decreased during the period of sustained compression. The 30-minute group had significant better regional blood flow than the 180-minute group after the decompression and 28 days after the decompression (p<0.05). Regional blood flow recovered gradually after the decompression. Regional blood flow recovered so close to the baseline 28 days after the decompression,but the baseline has a significant better regional blood flow (p<0.05). Functional motor test demonstrated a rapid recovery of hindlimb motor function in 30-minute group. Nearly, all the dogs in 30-minute group could walk smoothly. In contrast, five of the dogs in 180-mintue group were capable of weight-bearing. The 30-minute group had significant better Talov motor scores at all time-points (p<0.05). The sustained compression injury produced cavitation of the central region of the spinal cord parenchyma at the T13 epicenter, leaving a rim of intact white matter. The 30-minute group had a smaller lesion volume than the 180-minute group did (p<0.05). Conclusion: 1. Sustained spinal cord compression is an important factor in the secondary injury process. A longer duration of compression injury is associated with reduced electrophysiological recovery, increased pathological changes, and significantly greater functional impairment. 2. The result underscore the importance of timely decompression to improve long-termfunction recovery after spinal cord injury. Part two: Relationship between the volum and drug of the intraspinal injection and the extent of spinal cord injury in normal rats. Objective: The objective of this study is to investigate the relationship between the volum and drug of intraspinal injection and the extent of spinal cord injury, and then find out the safe intraspinal injection volum for the following studies. Methods: Ninety-six adult Sprague-Dawley rats were divided into twelve groups: NS-2ul group, NS-4ul group, NS-6ul group, NS-10ul group, Buffer-2ul group, Buffer-4ul group, Buffer-6ul group, Buffer-10ul group, Ad-LacZ-2ul group, Ad-LacZ-4ul group, Ad-LacZ-6ul group, Ad-LacZ-10ul group. According to the principle of the division, Saline or Virus Buffer or Ad-LacZ (1×109 pfu/ml) was injected into the dorsal column of the spinal cord at T13 level immediately after the laminectomy was performed. Six adult Sprague-Dawley rats were in the control group that only performed the laminectomy. The BBB Locomotor Rating Scale was used to evaluate the hindlimb functional improvement of treated animals with spinal cord contusion. In this study, the behavioral analysis was performed at 1, 2, 3, 4, 5, 6, 7 days and 2, 3, 4 weeks after the surgery. Histological analysis was processed 1, 2, 4 weeks after the surgery with HE stain and Toluidine blue stain. X-Gal staining was performed to demonstrate the adenovirus-mediated expression of the LacZ transgene in vivo. The reservation rate of functional residual tissue was measured to assess the extent of spinal cord injury 4 weeks after the injection. Results: The volum of 2, 4ul intraspinal injection caused short-time motor deficits. The hindlimb motor function recovered within 1 week, and no deficit was found. The volum of 6ul intraspinal injection caused long-time motor deficits. The hindlimb motor function of most rats recovered within 2 weeks, and partial slight functional deficit was found. The volum of 10ul intraspinal injection caused severe motor deficits. Moderate functional deficit was found in most rats. Statistically significant differences in BBB scoresamong different volum groups and control group (p<0.05). Statistically significant differences in BBB scores among Ad-LacZ group, NS group, and Buffer group (p<0.05). There was no statistically significant differences in BBB scores among Ad-LacZ-2ul group, Ad-LacZ-4ul group and control group (p>0.05). The extent of tissue damage increased following the increasing intraspinal injection volum. The 2ul groups had smaller lesion volumes than the 10ul groups. Different drugs that injected caused different tissue damage. NS group and Buffer group had better functional residual tissue than the Ad-LacZ group did (p<0.05). The distribution of the reporter gene LacZ was shown by X-Gal staining in Ad-LacZ 2ul, 4ul, 6ul and 10ul group. The gene LacZ could be found 4 weeks after the intraspinal injection. Conclusion: 1. The intraspinal injection result in functional deficit and tissue damage. The extent of tissue damage increased following the increasing intraspinal injection volum. 2. Different drugs that injected caused different tissue damage. The intraspinal injection of Ad-LacZ results in more tissue damage. The volum of 2, 4ul is relatively the safe volum for injection. Part three: The effect of adenovirus-mediated GDNF transfer in vivo on recovery of motor function following moderate spinal cord injury in rats. Objective: The objective of this study is to assess the effect of adenovirus-mediated GDNF transfer in vivo on recovery of motor function following moderate spinal cord injury with safe intraspinal injection volum. Methods: The SCI model of acute posterior compression of spinal cord was established according to the method of Nystrom in sixty adult Sprague-Dawley rats at T13 level. Spinal cord compression injuries were produced in rats by applying weight of 35g for 5 minutes to a metal plate 2.5×5.0 mm in size, placed on the exposed midthoracic dura covering the spinal cord. The models were divided into three groups: control group, Ad-LacZ group and Ad-GDNF group. After the surgery, 2ul of 1×109 pfu/ml Ad-LacZ or Ad-GDNF was injected into the dorsal column of the spinal cord. Thecontrol group didn't do the injection. RT-PCR analysis was performed for control group, Ad-LacZ group and Ad-GDNF group rats 1, 2, 4 weeks after the injection. The behavioral analysis was performed at 1, 2, 3, 4, 5, 6, 7 days and 2, 3, 4 weeks after the surgery. Histological analysis was processed 3, 5 days and 1, 2, 4 weeks after the surgery with HE stain and Toluidine blue stain. The reservation rate of functional residual tissue was measured to assess the extent of spinal cord injury 4 weeks after the injection. Immunohistochemistry stain for GDNF was performed 4 weeks after the intraspinal injection. X-Gal staining was performed to demonstrate the adenovirus-mediated expression of the LacZ transgene in vivo. Results: GDNF mRNA expression could be detected in rats in control group, Ad-LacZ group and Ad-GDNF group 1 week after the injection and it also could be detected in rats receiving Ad-GDNF 4 weeks after the injection. The hindlimb motor function recovered slowly within the first week, and it recovered rapidly within the next one week. The Ad-GDNF group had a better BBB score 2 weeks after the injection (p<0.05). After two weeks the hindlimb motor function recovered gradually, but the speed was slow down. The hindlimb motor function of the rats in Ad-GDNF group and control group recovered 4 weeks after the injection. There was no significant difference between Ad-GDNF group and control group (p>0.05). Swelling was so significant 5 days after the surgery. There were caves in the gray matter and there were severe tissue damages in the white matte too 4 weeks after the surgery.. Ad-GDNF group had a better functional residual tissue than the Ad-LacZ group and control group did (p<0.05). In contrast to the Ad-LacZ group and control group, GDNF-positive neurons could be observed in Ad-GDNF group. There were faint GDNF immunostaining in Ad-LacZ group and control group. Conclusion: 1. The result demonstrate that 2ul of the adenovirus-mediated GDNF transfer in vivo could improve the motor function in rats with moderate spinal cord injury, and it can shorten the recovering period. 2. One week to two weeks after the injection is the key period of...
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