Motor Neuron Degeneration And Protective Strategy

Motor neuron degeneration is a pathology event in many nervous system disorders. As a consequence, the loss of motor function often persist for the life of the patient and influence in diverse ways, not only the patient, but also family members and society at large. There is two types of motor neuron degeneration: acute and chronic. Acute traumatic spinal cord injury (SCI) results in a devastating loss of neurological function, Especially motor function, chronic selective motor neuron degeneration is the character of motor neuron disease especially amyotrophic lateral sclerosis(ALS). The pathophysiology of SCI includes a primary mechanical injury and a delayed secondary injury. The cell death from the mechanical injury is predominated by necrosis, secondary injury events trigger a continuum of necrotic and apoptotic cell death mechanisms. These secondary events include vascular abnormalities, ischemia-reperfusion, glutamate excitotoxicity, oxidative stress and inflammatory response. Amyotrophic lateral sclerosis (ALS) is an adult-onset, progressive and fatal neurodegenerative disease that causes motoneuron degeneration, skeletal muscle atrophy, paralysis and death. Ninety percent of the cases are sporadic and 10% are familial. For a long time, studies of this disease were limited to pathological descriptions of autopsy specimens. Direct experimental study on the mechanism of motoneuron degeneration was not possible because suitable models for the disease were lacking. A breakthrough occurred in 1993 when mutations in the Cu,Zn superoxide dismutase (SOD1) gene were discovered to cause approximately 25% of familial ALS. By expressing the disease-causing mutant SOD1 in different systems, numerous in-vitro and in-vivo models have been created to address the mechanism and the treatment of the disease. The etiology of SALS, which accounts for the majority of all ALS cases, remains to be resolved, and several pathogenic mechanisms may be involved: 1) excitotoxicity, 2)oxidative stress, 3) autoimmunity, 4) mitochondria dysfunction, 5) over phosphorylation of neurofilament , 6) deficiency of neurotrophic factors. et al.Model systems such as experimental animals and cell cultures are often used to understand the pathogenesis of motor neuron degeneration. cell culture model include primary motor neuron culture, NSC34 cell line and organotypic spinal cord or brain slice culture. The organotypic culture of brain slice and spinal cord slice can survive for a long time with intact form maintaining the synapse of neuron-neuron and neuron-glial cells. So compared with motor neuron culture, organotypic culture is more suitable to study chronic motor neuron degeneration.for study acute and chronic motor neuron degeneration, different animal model developed by different injury factor. Depress and axotomy can induce acute motor neuron injury. Because G93A mouse can mimic the chronic progressive feature of ALS and parts of its pathologic and biochemical changes (inclusion formation; neurofilament aggregation; motor neuron apoptosis; free radicals and glutamate increase). The study on ALS models is mainly focused on SOD1 transgenic mice models.To investigate the pathogenesis of acute and chronic motor neuron degeneration and the potential protective agents, we design this expriment. First, we develop an organotypic spinal cord and brain slice culture model. Second, according to our observation, there is a motor neuron loss after dissection, it can mimic acute motor neuron degeneration by serious spinal cord injury. In this model, we study whether phaseⅡenzyme inducer have neuroprotection on acute motor neurons injury. Third, based on the chronic excitotoxicity pathogenesis, using brain slice culture and spinal cord slice culture respectively, add threo-hydroxyaspartate (THA) to culture medium, we established the culture model for ALS. Study whether phaseⅡenzyme inducer, ceftriaxone, and IGF-1 have neuroprotection on chronic motor neurons degeneration.At last, we want to establish transgenic mouse model of familial amyotrophic lateral sclerosis and identified the genotype of the first filial generation. PartⅠThe methord of organotypic spinal cord culture model.Objective: Establish the organotypic spinal cord culture model to oberserved motor neuron survival in vitro.Methods: Organotypic spinal cord cultures were prepared using lumbar spinal cord slices from 7-day-old rat. Ventral motor neurons survival was evaluated by culture morphology and by monoclonal antibody SMI-32, a nonphosphorylated neurofilament marker. The lumber spinal cords of 1-day-old rats, 1-month-old rats and 2-month-old rats were dissection at 30μm, stained with SMI-32.Results: We found the motor neuron were loss within 1 week culture, after 1 week culture, the system is stable. The loss of motor neuron due to dissection.Conclusions: We developed a methord of organotypic culture model in vitro, SMI-32 is a perfect marker for motor neurons. This system is very suitable for studying pathogenesis and neuroprotection of ALS.PartⅡThe neuroprotective potential of phaseⅡenzyme inducer on motor neuron survival in traumatic spinal cord injury in vitroObjective: PhaseⅡenzyme inducer is a kind of compound which can promote the expression of antioxidative enzymes through Nrf2 activation. Recently, it has been reported that these compound show neuroprotective effect via to combat oxidative stress. Whether phaseⅡenzyme inducer has neuroprotective effect on traumatic spinal cord injury, it is unclear. Here, we want to identified the effect of phaseⅡenzyme inducer on traumatic spinal cord injury in an organotypic spinal cord culture system.Methods: Organotypic spinal cord cultures were prepared using lumbar spinal cord slices from 7-day-old rat.when culture, slices were respectively treated with t-BHQ, D3T, CPDT to achieve a final concentration of 30μmol/L(DMSO for drug solubilizer)in the culture medium. No drugs were added to the control group, while 0.1% DMSO was added as drug solubilizer control. Each group consisted 10-15 slices. After 1week culture, the cultures were fixed with 4% paraformaldehyde. Ventral motor neurons survival was evaluated by monoclonal antibody SMI-32, a nonphosphorylated neurofilament marker, immunohistochemistry staining compared with controls. slices for RT-PCR were take out after 48h culture, Glutamate concentrations in culture mediums at culture 48 hours were also measured.Results: we found the motor neuron loss within 1 week culture, after 1 week culture, the system is stable. The loss of motor neuron due to dissection can mimic severe traumatic spinal cord injury. Moreover, Glutamate is increased when culture 48 hours, decreased after 1 week culture, and there is no significant change between 1 and 4 weeks culture. Furthermore, we found phaseⅡenzyme inducer: t-BHQ, D3T, CPDT can promote motor neuron survival after dissection and Nrf2 and HO-1mRNA overexpression after CPDT treatment.Conclusions: in this study, those results demonstrated that glutamate is possible involvement in motor neuron death after traumatic spinal cord injury and Nrf2/HO-1 pathway show neuroprotective potential on motor neuron survival in traumatic spinal cord injury in vitro.PartⅢNeuroprotective potential of phaseⅡenzyme inducer on THA-induced motor neuron death in vitroObjective: in this study, we want to investigate the role of phaseⅡenzyme inducer on THA-induced motor neuron death.Methods:The organotypic spinal cord cultures were prepared from 7 day old rat pup lumbar spinal cords. Lumbar spinal cords were collected under sterile conditions, sectioned transversely at 350μm intervals. Slices were placed on the inserts. After 1 week in culture, the THA (Threo-hydroxyaspartate) group, treated with THA alone to achieve a final concentration of 100μmol/L. For the phaseⅡenzyme inducer48h +THA group, tissues were treated with phaseⅡenzyme inducer to achieve a final concentration of 15,30μmol/L, 48 hour later, the same concentration phaseⅡenzyme inducer and 100μmol/L THA were added to the medium. For the phaseⅡenzyme inducer +THA group, tissues were treated with phaseⅡenzyme inducer to achieve a final concentration of 15,30μmol/L and 100μ mol/L THA were added simultaneously, 0.1%DMSO was added to the drug solubilizer control, no drugs added to the control group. After treament for 3 weeks in culture, tissue were harvested.Results: In this experiment we found 100 micromol/L THA resulted in a significant decrease of motor neurons in the anteror horn of the spinal cord after treament with THA for 3 weeks. At the same time, the extrcellular glutamate level is nearly 2-fold elevated.PhaseⅡenzyme inducer can prevent THA-induced motor neuron death, increase motor neuron suvival. phaseⅡenzyme inducer also show mitochronial protection. The glutamate and GLT-1 expression were not affected after treatment with CPDT for 3 weeks,but Nrf2 activation and antioxidative enzyme HO-1 overexpression contribute to the neuroprotective effect of phaseⅡenzyme inducer.Conclusions: motor neurons in such cultures are vulnerable to exictotoxicity. phaseⅡenzyme inducer can protect motor neuron from THA-induced motor neuron death, moreover, it appear to reduce the loss of motor neuron by Nrf2 activation and antioxidative enzyme HO-1 overexpression not due to effect on glutamate transport.PartⅣNeuroprotective potential of CPDT on THA-induced cortical motor neuron death in an Organotypic Culture ModelObjective: Based on the chronic excitotoxicity pathogenesis in ALS, using brain slice culture to observe the effect of threo-hydroxyaspartate (THA), the inhibitor of glutamate transport, on cortical motor neurons. And test the role of CPDT in this model.Methods: The frontal- parietal lobe from 1-day-old rat was sliced into 350μm-thick sections. 100μmol/L THA were added into the culture medium after cultured for 2 weeks. explants were treated with CPDT to achieve a final concentration of 15,30μM, 48 hour later, the same concentration CPDT and 100μM THA were added to the medium simultaneously. 0.1%DMSO was added to the drug solubilizer control, no drugs added to the control group.Results: Chronic THA treatment of the slices resulted in pyramidal cell death, treatment of the slices with CPDT significantly protected the CMNs against THA toxicity. CPDT treatment for 48 hours, Nrf2 translocated to necleus. By western blot analysis, Nrf2 level increased in nuclei after CPDT treatment for 48 hours.HO-1 protein by CPDT added to culture medium. Morever,HO-1 protein is kept increasing after treatment with CPDT for 3 weeks.Conclusions: This study demonstrates that the inhibitor of glutamate transport, THA, could produce a loss of pyramidal cells. CPDT can protect motor neuron from THA-induced motor neuron death, moreover, it appear to reduce the loss of motor neuron by Nrf2 activation and antioxidative enzyme overexpression.PartⅤNeuroprotective potential of ceftriaxone on motor neuron suvival of ratObjective: study the role of ceftriaxone on THA-induced motor neuron injury.Methods: The organotypic spinal cord and brain cultures were prepared from 7-day-old rat pup lumbar spinal cords and 1-day-old brain tissue. They were collected under sterile conditions, sectioned transversely at 350μm intervals. Slices were placed on the inserts. After 1 week in culture of spinal cord slice and 2 weeks of brain slice, the THA group, treated with THA alone to achieve a final concentration of 100μmol/L. for the ceftriaxone +THA group, tissues were co-treated with ceftriaxone to achieve a final concentration of 100μm, and simultaneously with 100μm THA. 0.1%DMSO was added to the drug solubilizer control. no drugs added to the control group. After 3 weeks treatment, tissue were harvested, the number of motor neurons were oberserved by immunohistochemistryResults: THA induced selective motor neuron death. ceftriaxone can prevent motor neuron death, increase motor neuron suvival.Conclusion: ceftriaxone have neuroprotective potential on motor neuron death.βantibiotic may provide beneficial effect for Amyotrophic lateral sclerosis (ALS) treatment.PartⅥNeuroprotective potential of IGF-1 on motor neuron suvival of ratObjective: study the role of IGF-1 on THA-induced motor neuron injury.Methods: The organotypic spinal cord and brain cultures were prepared from 7-day-old rat pup lumbar spinal cords and 1-day-old brain tissue. They were collected under sterile conditions, sectioned transversely at 350μm intervals. Slices were placed on the inserts. After 1 week in culture for spinal cord slice and 2 weeks for brain slice, the THA group, treated with THA alone to achieve a final concentration of 100μmol/L. for the IGF-1 +THA group, tissues were co-treated with IGF-1 to achieve a final concentration of 100μM, and simultaneously with 100μM THA. no drugs added to the control group. After treatment for 3 weeks, tissue were harvested, the number of motor neurons were oberserved by immunohistochemistry.Results: THA induced selective motor neuron death. IGF-1 can prevent motor neuron death, increase motor neuron suvival.Conclusion: IGF-1 have neuroprotective potential on motor neuron death. IGF-1 may provide beneficial effect for Amyotrophic lateral sclerosis (ALS) treatment.PartⅦToxic effect of hmSOD1 on motor neuron in transgenic mouseObjective: establish transgenic mouse models of familial amyotrophic lateral sclerosis (FALS) and identified the genotype of the first filial generation. And observed the behavior change at different stage of G93A mouse.Methods: Mated B6SJLSOD1G93A1Gur/J with B6SJLF1/J to produce the filial generation. The genomic DNA was obtained from tail tissue or tain blood. Amplified the hmSOD1 gene fragment by PCR. The PCR produce was purified for gene sequence analysis. We also observed the behavior change at different stage of G93A mouse.Results: 40 mice were born and the positivity rate for hmSOD1 gene were 42.5%. the G93A mice show abnormal splaying or hindlimb shaking at 15 weeks, and weakness and paralysis in both hindlimbs, at last inability toright itself within 20s when placed on its back, we consider it reach to endpoint.Conclusion: PCR can identified the genotype of the filial generation, hmSOD1 induce motor function loss. It is a classic animal model to study ALS.PartⅧFormation of a spinal cord and muscle organotypic co-culture system in vitroObjective: To develop a spinal cord slice and muscle co-culture system in vitro.Methods: The organotypic spinal cord and skeleton muscle were prepared from 7-day-old rat. They were collected under sterile conditions, sectioned transversely at 350μm intervals. Slices were placed on the inserts. After 3 weeks treatment, tissue were harvested, the number of motor neurons were oberserved by SMI-32 immunostaining. Neuromuscular junctions of muscle labeled withα-bungarotoxin-conjugated rhodamine and A cetylchelin esterase. We also observed the ultrastructure of muscles in co-culture tissue.Results: positive-Neuromuscular junctions were labeled withα-bungarotoxin-conjugated rhodamine and A cetylchelin esterase, Ventral horn motor neurons (VMN) can build junction with muscle in vitro. The number of motor neurons in spinal cord and muscle co-culture is more than spinal cord-only culture.Conclusion: These results suggested that Spinal cord and muscle co-culture is a good model for study the interaction between motor neurons and muscle. Spinal cord and muscle co-culture can improve motor neuron survival than spinal cord slice culture only. It maybe due to neurotrophic factor secreted to the medium by muscle.