Huperzine A Protects Motor Neurons In An Organotypic Spinal Cord Culture Model Of Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease, selectively involving in the upper and lower motor neurons. The disease is characterized by progressive muscle weakness and the patient's death is usually caused by respiratory failure, occurring 3-5 years after the first appearance of symptoms. Several mechanisms for the pathogenesis of ALS have been proposed: 1) mutations of the copper/zinc superoxide dismutase (Cu/Zn SOD) gene; 2) glutamate excitotoxicity; 3) high energy demands coupled with mitochondrial dysfunction; 4) oxidative stress ; 5)formation of autoantibodies, et al. Glutamate excitotoxicity is one of important mechanisms for the pathogenesis of sporadic amyotrophic lateral sclerosis(SALS). When the function of glutamate transporters is significantly inhibited, excessive extracelluar accumulation of glutamate surrounding the synaptic cleft may cause exicitotoxic damage to the postsynaptic neruons and other surrounding tissues through activation of glutamate receptors.It is very important for the cell or tissue culture in vitro especially the organotypic spinal cord culture for the study of ALS. Organotypic cultures of postnatal spinal cord slices have the advantages of maintaining intact morphology and local synaptic connection in culture and may maintain for three months, which is essential for modeling the chronic damage occurring in ALS. We developed a model of organotypic spinal cord culture, as a model of ALS in vitro, based on the technique of organotypic spinal cord culture. The cultures are treated with threohydroxyaspartate(THA) to selectively inhibit glutamate transporters, which raise the extracellular glutamte concentration and mimick the chronic damage to glutamate transporters that have been observed in ALS patients. It is a very good model to be used to study some medicines which can protect against motor neurons damage and provide apossibility for these medicines to be used by ALS patients.It is very difficult to cure ALS. ALS therapies include some approaches: antiglutamate> neurotrophic factors> free radical scavengers ^ channel blockers and cell and gene therapies, et al. Riluzole, the first therapeutic drug of motor neuron disease approved by FDA , is believed to exert its effects by reducing release of glutamate into the synaptic cleft, but the protection of riluzole is limited and it can only prolong the patient's life about several months and can't cure ALS.Huperzine A (Hup A), a Lycopodium alkaloid isolated from the Chinese herb Huperzia serrata Trev, is a potently reversible and selective acetylcholinesterase inhibitor. However, previous studies revealed importantly that huperzine A had exhibited broad neuroprotection against glutamate excitotoxicity > oxidative stress and apoptosis, et al. So , it is very useful to study the neuroprotection of Huperzine A.Basing on glutamate excitotoxicity ,and using the technique of organotypic spinal cord culture, we made the organotypic spinal cord culture model of ALS in vitro. After 1 week culture in vitro, tissues were co-treated with different concentrations of huperzine A to be used to study the protection of huperzine A against motor neurons damage. Our study included four parts. In the first part, we established the technique of organotypic spinal cord culture and studied whether treatment of organotypic spinal cord cultures with huperzine A alone was neurotoxic to motor nuerons. In the second part, We developed a model of organotypic spinal cord culture, basing on glutamate excitotoxicity and using the technique of organotypic spinal cord culture. The cultures are treated with threohydroxyaspartate(THA) to selectively inhibit glutamate transporters. Tissues were co-treated with different concentrations of huperzine A and THA to be used to study the protection of huperzine A against motor neurons damage at different levels. In the third part, we studied the protection of different concentrations of riluzole against motor neurons damage with the model of organotypic spinal cord culture and compared the protection of huperzine A and riluzole. In the fourth part, we discussed themechanisms of the protection of huperzine A against motor neurons damage.Part I A study on establishment of organotypic spinal cord culture and neurotoxicity of huperzine A to motor neuronsObjective: To establish the technique of organotypic spinal cord culture and identify a motor neurons in the ventral horn. To study whether treatment of organotypic spinal cord cultures with huperzine A alone was neurotoxic to motor nuerons through observing morphology of cultures and numbering a motor neurons in the ventral horn . To prepare for the study of protection of huperzine A against motor neurons damage.Methods: Organotypic spinal cord cultures were prepared using lumber spinal cord slices from 8-day-old rat pups. Neunatal rat pups were decapitated , and the spinal cords were rapidly harvested . Lumber spinal cords were collected under sterile condition and sectioned transversely at 350-jim intervals with a Mcllwain tissue chopper. Sections were then transferred to sterile Gey's balanced salt solution and gently separated at room temperature .Cultures were carefully placed on the surface of Millipore Millicell—CM insert(five slices per membrane).Such tissue grew optimally at an air/fluid interface, so it was important to remove any excess medium on the membrane surface around cultures.The membranes were placed in 35-um culture wells containing lml incubation medium.Cultures were incubated at 37°C in a 5% CO2/ 95% humidified enviroment.Culture medium, along with any added pharmacological agents, was changed twice weekly. In all experiments, cultures were allowed an 8-day recovery period after initial preparation. The cultures were divided into two groups:?normal control group? (2)100 u mol-L'1 Hup A group. To observe the growth of cultures with inverted microscope every day and measure the transverse diameters and anteroposterior diameters of the spinal cord cultures every week . After 4 weeks culture in vitro, the spinal cord cultures were taken out and a motor neurons in the ventral horn were identified with SMI-32 immuohistochemicaistaining.Results: The spinal cord cultures of normal control group had excellentorganotyopic cellular organization. The cultures became bigger than ever. A light halo could be found around the culture. The dorsal horn was darker than the ventral horn .There were many typical tightly packed small cells in the dorsal horn.The ventral horn was brighter than the dorsal horn. The cells in the ventral horn were big and sporadic. The spinal cord cultures of 100 u mol-L"1 Hup A group have the same organotyopic .organization as those of normal control group. The transverse diameters and anteroposterior diameters of the spinal cord cultures of the two groups had no statistic difference . a motor neurons in the two groups could be detected easily with SMI-32 immuohistochemical staining in the ventral horn , which were >25 u m in diameter with long slim axons that spread through the culture to make synaptic connections. A stable population of a motor neurons (about 5-15 a motor neurons) was found in each side of the ventral horn. The numbers of a motor neurons of two groups had no statistic difference after 4 weeks culture in vitro.Conclusions: In this study, the technique of organotypic spinal cord culture was successfully established, a motor neurons in the ventral horn could be identified with SMI-32 immunohistochemical staining easily. The cultures maintained stable number of a motor neurons and good culture morphology. Cultures were treated with high concentration of huperzine A After 4 weeks culture in vitro. The numbers of a motor neurons and culture morphology of two groups have no statistic difference. So ,we could think that huperzine A was not neurotixic to motor neurons. This part can be a good base to study the protection of huperzine A against motor neurons damage.Part II The establishment of the organotypic spinal cord culture model of amyotrophic lateral sclerosis and the study on protection of huperzine A against motor neurons damageObjective: There is increasing evidence that glutamate-mediated excitotoxicity plays an important role in the pathogenesis of ALS. The aim of this part is to develop a model for selective motor neuron death in organotyic spinal cord cultures to mimic the feature of ALS which is selective motorneuron loss. Cultures were co-treated with different concentrations of huperzine A and THA to be used to study the protection of huperzine A against motor neurons damage from various angles.Methods: Organotypic spinal cord cultures were prepared using lumbar spinal cord slices from 8-day-old rat pups. The cultures were divided into 5 groups:(Dnormal control group; ?model group; (3)1 u mol-L"1 Hup A group; (4)10 u mol-L"1 Hup A group; (5)100 u mol-L"1 Hup A group. Culture medium, along with THA and Hup A, was changed twice weekly. In all experiments ,cultures were allowed an 8-day recovery period after initial preparation. To observe the growth of cultures with inverted microscope every day. After 4 weeks culture in vitro, the spinal cord cultures were taken out and culture medium was collected and a motor neurons were identified in the ventral horn. The level of lactate dehydrogenase (LDH) in culture medium was assayed and the structure change of cultures was observed with transmission electron microscope.Results: The spinal cord cultures of 100 u mol-L'1 Hup A group had better growth and had the similar change with those of normal control group. The cultures of 1 and 10 u mol-L'1 Hup A groups had bad growth and had the similar change with those of model group. After 4 weeks culutre in vitro, the number of a motor neurons decreased (PO.05) and the LDH in culture medium of model group increased (PO.05) vs normal control group; the number of a motor neurons increased (P<0.05) and the LDH in culture medium of 10011 mol-L-1 HupA group decreased ( PO.05 )v.s model group, a motor neurons in the cultures of model group contained a lot of cytoplasmic vacuoles .However, a - motor neurons in 100 u mol-L"1 Hup A group cultures contained few cytoplasmic vacuoles.Conclusions: Treatment with 100 y mol-L"1 THA, a selective glutamate transporter inhibitor, could cause selective a motor neurons damage in ventral horn which could provided an effective organotypic culture model for studying pathogenesis of ALS and neuroprotection of some medicines. When cultures were co-treated with different concentrations of huperzine A and THA,1 u mol-L"1 and. 10 u- mol-L"1 Hup A couldn't resist the effects of THA. However , 10014 mol-L"1 huperzine A could protect the motor nuerons against THA. The 100 u mol-L"1 Hup A was found to reduce a motor neuron death and the release of LDH and improve the condition of neuron growth on spinal cord cultures, so Hup A was shown to have protection against a motor neurons damage.PartlTJ The comparison of protection against a motor neurons damage between huperzine A and riluzoleObjective: Riluzole reportedly acts to inhibit presynaptic glutamate release and is the only FDA approved drug which was used in the treatment of motor neuron disease, but the protection of riluzole is limited and it can only prolong the patient's life about several months. In this part, based on the model of organotypic spinal cord culture, culutres were co-treated with different concentrations of riluzole and THA to study the protection of riluzole against motor neuron damage and to compare the protection between huperzine A and riluzole.Methods: Organotypic spinal cord cultures were prepared using lumbar spinal cord slices from 8-day-old rat pups. The cultures were divided into 4 groups :(Dmodel group ; (2)1 u mol-L"1 riluzole group; (3)10 u mol-L"1 riluzole group; (4)100 li mol-L"1 riluzole group. To number the a motor neurons in the ventral horn with SMI-32 immuohistochemical staining and to compare the protection between huperzine A and riluzole.Results: The number of a motor neurons in cultures of 1 u mol-L"1 riluzole group is 10.1+4.0 per organotypic culture, and have no statistic difference compared with model group(P > 0.05). the number of a motor neurons in cultures of 10 u mol-L"1 riluzole group is 12.2+4.5 per organotypic culture, and have no statistic difference compared with model group(P > 0.05). The number of a motor neurons in cultures of 100 u mol-L"1 riluzole group is 16.3+4.3 per organotypic culture, and have statistic difference compared with model group (PO.05); and have no statistic difference compared with 100 u mol-L'1 Hup A group(P > 0.05).Conclusions: When tissues were co-treated with different concentrations of riluzole and THA, 1 u mol-I/'and 10 u mol-L'1 riluzole couldn't resist the effects of THA. However, 100 u mol-L-1 riluzole could protect the a motor nuerons against THA.The numbers of a motor neurons in cultures of 100 u mol-L"1 riluzole group and 100 u mol-L'1 huperzine A group had no statistic difference. So, it was very important for us to study the protection of huperzine A against motor neurons damage.PartIV The study on the mechanisms of the protection of huperzine A against motor neurons damageObjective: Glutamate excitotoxicity and oxidative stress are associated with pathogenesis of ALS. Previous studies showed that huperzine A protected neuronal-like cells through anti-oxidative stress and anti-glutamate excitotoxicity. In this part, we studied the mechanisms of protection of huperzine A against motor neurons damage.Methods: Organotypic spinal cord cultures were prepared using lumbar spinal cord slices from 8-day-old rat pups. The cultures were divided into 3 groups:? normal control group? (2) model group; (3) 100 u mol-L*1 Hup A group. Culture medium, along with THA and Hup A ,was changed twice weekly. In all experiments ,cultures were allowed an 8-day recovery period after initial preparation.The culture medium of the second ,third and fourth week was collected. Glutamate concentration and the level of MDA in samples of culture medium at weekly intervals were assayed.Results: A persistent elevation of glutamate concentration in the culture medium didn't be found in culture medium of normal control group. Chronic inhibition of glutamate transport with THA produced a persistent elevation of glutamate concentration in the culture meidum of model group, which were 2.4 ± 0.8 u mol-L'1 ^ 2.7 ± 0.5 u mol-L"1 > 4.5 + 0.6 u mol-L'! respectively. Glutamate concentration in the culture meidum of 100 u mol-L"1 Hup A group also had persistent elevation ,which were 2.3 + 0.9 u mol-L"1 ?? 2.5+ 0.6 u mol-L^and 4.2 +1.0 u mol-L"1 respectively. Glutamate concentration in the culture meidum of lOOumol-L'1 Hup A group and model group had no...