The Establishment Of Culture Model For Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a common progressive neurodegenerative disease in central nervous system. It is characterized by selective degeneration of motor neurons in brain and spinal cord, which clinical features are delayed onset, chronic progression, weakness and atrophy of limbs'muscles, the occurrence of pathologic reflex. ALS is a lethal disease, with most patients died of respiratory failure 3-5 years later. From its stigmata of episode and heredity, ALS can be divided into familial amyotrophic lateral sclerosis (FALS) and sporadic amyotrophic lateral sclerosis (SALS). FALS is less than 10% of the total ALS patients, while SALS is more than 90%. FALS is mainly caused by the mutation of copper/zinc superoxide dismutase (Cu/Zn SOD) gene, whose site is at 21q22.1～22.2. This suggests that FALS should be a kind of genetic 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. At present, the study on ALS models is mainly focused on SOD1 transgenic mice models and culture models in vitro. The virtue of transgenic models is that it may mimic the chronic progressive feature and parts of its pathologic and biochemical changes (inclusion formation; neurofilament aggregation; motor neuron apoptosis; free radicals and glutamate increase). The flaw is that it comes from the feature of FALS (SOD1 gene mutation). It is a model of FALS, it cannot represent the SALS which is the predominance in ALS. Besides, transgenic technique is too complex and expensive to develop. Most laboratories cannot master the technique, which limit the use of the model. Traditional motor neuron culture is another model. Its virtue is little interference. We can observe motor neurons'form and function change directly and developmentally. The defect is its operation is too complex. And the survival time of motor neuron in culture is too short, which doesn't fit to study the progressive degenerative disease. Purified motor neuron culture cannot mimic the physiological enviroment in vivo such as the interaction between neurons and astrocytes. So purified motor neuron culture is neither an ideal model to study ALS. 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 ALS. Based on the chronic excitotoxicity pathogenesis, using brain slice culture and spinal cord slice culture respectively, we established the culture model for ALS. And explored the protective function of glial cell derived neurotrophic factor (GDNF) in this model. There are four parts in my article. The first part: we established the technique of rat motor cortex culture and compared the cultured cortex with those live in vivo. The second part: based on the chronic excitotoxicity pathogenesis, using threo-hydroxyaspartate (THA), the inhibitor of glutamate transporter, we established the model of motor-cortical pyramidal cells injured. The third: using threo-hydroxyaspartate (THA), the inhibitor of glutamate transport, we established the organotypic spinal cord culture model of motor neurons injured while interneurons preserved. The forth part: we observed the protective function of GDNF to motor neuron in our model. Part ⅠRat brain slice culture and the identification of motor cortical pyramidal cells with immunohistochemistry staining Objective: Develop a method of organotypic culture of rat brain slice. Pyramidal cells in motor cortex were evaluated by immunohistochemistry staining monoclonal antibody SMI-32, a nonphosphorylated neurofilamentmarker. Compare the cultured brain slice with those live in vivo. Establish the culture technique of brain slice which can mimic the physiological enviroment in vivo. Methods: Brain slice cultures were prepared using 1-day-old rat pups. Dissociated the intact brain and cut into two parts (left and right) along the midline under sterile conditions. The frontal-parietal lobe were sliced transversely into 350μm-thick sections with a McIIwain tissue chopper. Sections were then transferred into cold (4℃) sterile GBSS and gently separated. The first three slices from frontal lobe were abandoned. The second three slices were carefully placed on the surface of membrane insert with three slices per insert. These inserts were placed in 35 mm(6 wells) culture plates containing 1 ml of culture medium and kept in a 5% CO2 /95% air incubator at 37℃. Culture medium was changed twice weekly. Fixed the slices at the time of 2 weeks, 3 weeks, 4 weeks and 5 weeks respectively. Pyramidal cells were evaluated by nonphosphorylated neurofilament H (SMI-32) immunohistochemistry staining with 200×lens. Assay the level of lactate dehydrogenase (LDH) in culture mediums. Rat motor cortexes were stained with HE, and pyramidal cells were counted. Results: The brain slices can survive in culture for more than 1 month with excellent organotypic cellular organization and more than 85% of the slices can survive in the culture system. After SMI-32 immunohistochemistry staining, in the deep cortex (the V layer), there is a platoon of SMI32-positive cells paralleling with the cortical surface. The SMI32-positive cells were pyramidal or oval with long dendrites extending to the cortical surface. The diameter was between 25μm and 50μm. The number of pyramidal cells maintained stable after cultured for 2 weeks. The level of LDH in culture medium at different culture time had no significant differences. The six layers appeared distinctly in motor cortex in vivo after HE staining. The pyramidal cells in layer V were deep blue, with triangle shape. The number of pyramidal cells had no differences compared with those age-matched in vivo. Conclusions: In this study, brain slice culture was successfullyestablished. Pyramidal cells in layer V can be identified by SMI-32 immunohistochemistry staining. Cultured brain slice had high survival rate and long survival time with intact form. The number of pyramidal cells did not decrease with culture time and had no significant differences with those in vivo. To some extent this organotypic culture system can mimic the neurons survival environment in vivo, which provides an effective method to study diseases about upper motor neuron, such as amyotrophic lateral sclerosis. Part ⅡThe chronic injures of glutamate transporter inhibitors to motor cortical pyramidal cells Objective: 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. Establish the brain slice culture model of upper motor neurons degeneration, which is mediated by glutamate. Methods: The frontal-parietal lobe from 1-day-old rat was sliced into 350μm-thick sections. Various concentrations of THA(50μmol/L,100μmol/L ,500μmol/L) were respectively added into the culture medium after cultured for 2 weeks. At different culture times (2 weeks, 3 weeks, 5 weeks), cortical pyramidal cells were evaluated by monoclonal antibody SMI-32, a nonphosphorylated neurofilament marker. Ultrastructural alternations of the neuropathology were examined by electron transmission microscopy. Glutamate concentrations in culture mediums at different times were also measured. Results: The results showed that the brain slices in control group could maintain excellent organotypic cellular organization and a stable population of motor cortical pyramidal cells. THA could produce a slow dose-dependent loss of pyramidal cells and an increase of glutamate concentration. Pyramidal cells appeared mild to middle degree of vacuolar degeneration. Mitochondria swelled and cristae broke, appearing vacuole state.Conclusions: The inhibitor of glutamate transport, THA, could produce a slow dose-dependent loss of pyramidal cells and an increase of glutamate concentration. Ultrastructural changes showed mitochondria swelling and vacuolar degeneration. These changes were similar to the neuropathologic manifestation of ALS. It suggested that the brain slice culture model of upper motor neurons degeneration, which is mediated by THA, was an ideal model to study ALS. It provided an effective method to investigate the mechanism of ALS and to explore new neuroprotective treatment. Part ⅢThe chronic injures of glutamate transporter inhibitors to motor neurons in organotypic spinal cord culture Objective: Based on the chronic excitotoxicity pathogenesis in ALS, using spinal cord slice culture to observe the effect of threo-hydroxyaspartate (THA), the inhibitor of glutamate transport, on spinal motor neurons. Establish the organotypic spinal cord culture model of lower motor neurons degenerated while interneurons preserved, which is mediated by glutamate. Methods: Organotypic spinal cord cultures were prepared using lumbar spinal cord slices from 8-day-old rat. Various concentrations of THA(50μmol/L,100μmol/L ,500μmol/L) were continuously added into the culture medium after cultured seven days. Ventral α-motor neurons survival was evaluated by culture morphology and by monoclonal antibody SMI-32, a nonphosphorylated neurofilament marker, immunohistochemistry staining compared with controls. Dorsal interneurons were identified and counted by calretinin immunohistochemistry staining. Ultrastructural alternations of the neuropathology were examined by electron transmission microscopy. Glutamate concentrations in culture mediums at different times were also measured. Results: The results showed that the spinal cord explants in control group could maintain excellent organotypic cellular organization and a stable population of ventral α-motor neurons. THA could produce a slowdose-dependent loss ofα-motor neurons and an increase of glutamate concentration in culture medium. Low concentration of THA(50μmol/L ) caused only slight toxic to the cultured spinal cord slice. 100μmol/M THA resulted in significantly decrease of α-motor neuron and ultrastructural alternations after cultured 4 weeks while the inter-neurons in the dorsal horn were less affected. These changes were similar to changes in ALS. High concentration of THA(500μmol/L) produced severe toxicity that lead to significantly decrease of both motor neurons in ventral horn and interneurons in dorsal horn. Conclusions: Treatment with THA (100μmol/L) could cause selective α-motor neuron death with interneurons in dorsal horn little damaged, which could mimic the feature of pathological changes in ALS. Chronic inhibition of glutamate transport with THA could provide an effective organotypic culture model for studying pathogenesis and neuroprotection of ALS. Part ⅣThe protective effects of GDNF on ALS culture model Objective: Use brain slice culture and spinal cord culture to investigate the protective effects of glial cell derived neurotrophic factor (GDNF) on injured upper and lower motor neurons which is mediated by glutamate. Provide theoretic basis for treating ALS. Methods: Organotypic brain slice cultures and spinal cord cultures were made of 1-day-old rats and 1-day-old respectively. After 2 weeks'brain slice culture or 1 week's spinal cord culture, THA/MS ( THA concentration 100μM) containing various concentrations of GDNF ( 1 ng/ml,5 ng/ml,50 ng/ml) were continuously added into the culture medium. Culture medium was changed twice weekly. 3 weeks later, slices were taken out. SMI-32 immunohistochemistry stained to observe the changes of cortical pyramidal cells and spinal motor neurons, and compared with THA/MS group. Results: GDNF resulted in a dose-dependent protective...