Mechanisms Involved In Lipopolysaccharide-Mediated Motor Neuron Injury

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease, selectively involving in the upper and lower motor neurons. Until now the exact mechanisms underlying ALS are still less clear. Several hypotheses to explain motor neuron degeneration have been proposed, including mitochondrial dysfunction, protein aggregate formation, excitotoxicity, axonal transport malfunction, oxidative damage, lack of growth factors, and inflammation. There exist complex interactions among these different pathways, which contribute to the development of ALS. Recently, increasing evidences have showed that motor neuron death is non-cell-autonomous, glial cells and inflammation playing a key role in the pathogenesis of ALS.It has been reported that numerous activated microglia, reactive astrocytes, dendritic cells, cytokines and chemokines are present in the spinal cord of ALS patients and mice. In addition, in the mutant SOD1G93A transgenic mice, inflammatory responses are present early in disease before any evidence of dysfunction. Analyses of serum, skin and muscle from ALS patients indicate widespread inflammatory responses. All these data suggest that inflammation is an important event in the pathogenesis of ALS and may mediate motor neuron injury.Inflammatory reactions are composed of innate immune response and acquired immune response. It has been paid more attention to the significant role of innate immunity in the neurodegenerative diseases. Components of the innate defense system in the CNS include microglia and astrocytes, which are strongly activated in most neurodegenerative diseases and produce a variety of inflammatory mediators and other injury response factors. Inflammation is of dual roles. It is designed to remove or inactivate noxious agents and to promote healing, whereas the unregulated or inappropriate responses might be neurotoxic and trigger neurodegeneration. At present, there are still many problems about the precise role of inflammation in ALS and the mechanisms involved, which deserve to be studied.Oxygen free radicals are generated over the course of inflammatory processes, which could lead to the formation of oxidative stress. One abundant source of oxygen free radicals in the CNS is the respiratory burst system of activated glial cells. In the inflammatory processes, NADPH oxidase is activated, releasing large numbers of superoxide. Superoxide is the precursor of hydrogen peroxide, hypochlorous acid, hydroxyl radical species, and can further form peroxynitrite by reacting with nitric oxide (NO). In addition, NADPH oxidase-derived reactive oxygen species (ROS) can mediate proinflammatory gene expression and lead to the production of inflammatory cytokines. NADPH oxidase is therefore considered as a key factor involved in oxidative stress and inflammatory processes, which are both implicated in the pathogenesis of ALS. So we speculated that NADPH oxidase may mediate the toxicity to motor neurons.Besides ROS, reactive nitrogen species (RNS) are another type of free radicals, in which NO is the most important. During the process of inflammation, the expression or enzyme activity of nitric oxide synthase (NOS) in neurons and glia is elevated, generating a large amount of NO. Study on the role of NOS and NO in the pathogenesis of ALS has been the focus of research field. NO, acting as an important biological messenger, has multiple roles. It is a fundamental signaling device regulating virtually every critical cellular function, as well as a potent mediator of cellular damage in a wide range of conditions. In view of its complicated effects, it is necessary to explore the role of NO in inflammation-mediated motor neuron injury.Organotypic spinal cord slice cultures preserve an in vivo horizontal architecture and are therefore expected to reproduce cellular interactions under inflammatory conditions in the central nervous system more precisely than dissociated cell cultures. Lipopolysaccharide (LPS), which is a strong activator of innate immunity, can lead to the release of a variety of inflammatory cytokines through toll-like receptor (TLR). In the present study, we used organotypic spinal cord slice cultures to develop an in vitro model of inflammation-mediated motor neuron impairment by adding LPS to the culture medium, and further examined the mechanisms involved. Our study includes three parts. In the first part, using LPS to induce inflammatory reactions in the spinal cord slice cultures, we established an in vitro model that damage is relatively specific for motor neurons. Next, we explored the reasons responsible for the vulnerability of motor neuron. In the second part, based on the in vitro model, we investigated the changes in NADPH oxidase by RT-PCR and immunoblot and verified that NADPH oxidase is indispensable for inflammation-mediated motor neuron injury from different directions. In the third part, we confirmed that the enzyme activity of inducible NOS (iNOS) and neuronal NOS (nNOS) is elevated, as well as an over-production of NO. However, NOS selective or non-selective inhibitor could not protect motor neurons from damage, indicating that NO may not play a crucial role in mediating motor neuron injury in this model.PartⅠStudy on lipopolysaccharide-induced motor neuron injury and mechanisms responsible for its vulnerabilityObjective: The study was aimed to investigate whether LPS can induce inflammation in the organotypic spinal cord slice cultures and to establish an in vitro model that motor neurons are selectively impaired. In addition, we explored the reasons accounting for the vulnerability of motor neurons.Methods: Organotypic spinal cord cultures were prepared using lumbar spinal cord slices from 7-day-old rat. After 1 week in culture, the explants were treated with medium alone or LPS with different concentrations (1, 10, 30, 60μg/ml). At different culture times, motor neurons in the ventral horn and interneurons in the dorsal horn were counted based on SMI-32 and calretinin immunohistochemical staining, respectively. The ultrastructure of explants were observed using electromicrography. Lactate dehydrogenase (LDH) in the culture medium was measured by colorimetry. The changes in mRNA expression of TNF-αand IL-1βafter LPS exposure were examined using RT-PCR. 30μg/ml LPS group was chose for the following drug-treated experiments. Spinal cord slices, after 1 week in culture, were treated with medium alone, 30μg/ml LPS or combination of 10μmol/L BAPTA-AM and 30μg/ml LPS for 2 weeks. At the end of treatment, motor neurons were evaluated by immunostaining with SMI-32.Results:⑴The spinal cord explants in the control group preserved excellent organotypic cellular organization with a stable population of motor neurons. The number of motor neurons showed no significant difference in 1μg/ml LPS group compared to the control one at different culture time (P>0.05). After exposure to 10μg/ml LPS, the number of motor neurons gradually decreased with increasing time, and the effect reached statistical significance at 3 weeks of treatment (P<0.05). The decrease in the number of motor neurons was significant at 3 or 2 weeks after application of 30 and 60μg/ml LPS, respectively (P<0.05, P<0.01).⑵After treatment with 1, 10, and 30μg/ml LPS for 1 to 3 weeks, or treatment with 60μg/ml LPS for 1 to 2 weeks, the number of interneurons did not change significantly (P>0.05). Three weeks after exposure to 60μg/ml LPS, the number of calretinin-positive neurons in the dorsal horns significantly decreased compared to the control group (P<0.05).⑶Compared with the control group, the amount of LDH significantly increased in the group treated with 10μg/ml LPS for 3 weeks (P<0.05). There was a significant increase in LDH release after explants were treated with 30 or 60μg/ml LPS for 2 weeks (P<0.05).⑷The ultrastructure of neurons and glial cells in the control group was normal. After explants exposed to 30μg/ml LPS for 2 weeks, the ultrastructure of motor neurons showed extensive vacuolation and swollen mitochondria, whereas interneurons were relatively normal. After treatment with LPS, the soma of microglia enlarged evidently, and numerous lipid droplets and membranous bodies were observed within cytoplasm.⑸After treatment with 30 or 60μg/ml LPS for 1 day, the message level of TNF-αwas significantly elevated (P<0.05, P<0.01). Similarly, the mRNA level of IL-1βwas obviously higher in the explants treated with 30 or 60μg/ml LPS for 3 days than in normal controls (P<0.01).⑹The number of surviving motor neurons was significantly increased in the group with concomitant application of BAPTA-AM and LPS compared to the LPS group (P<0.01). Many motor neurons preserved better morphology with smooth and thick neurites in cotreated cultures.Conclusions: LPS could induce inflammatory responses in the organotypic spinal cord slice cultures, and lead to the demise of motor neurons and elevation of LDH release in a dose- and time- dependent manner. Exposure of slice cultures to 30μg/ml LPS for 2 weeks caused significant decrease in the number of motor neurons, whereas the survival of interneurons showed little change, which is similar to the pathology of ALS and can be applied to studies on the pathogenesis of ALS. Motor neurons lacked the expression of calretinin and BAPTA-AM, an intracellular calcium chelator, ameliorated motor neuron injury, indicating that the low capacity of calcium buffering may partially account for the vulnerability of motor neurons. This in vitro model provides a useful way to investigate the pathogenesis underlying ALS and to develop effective therapeutic strategies.PartⅡNADPH oxidase involved in lipopolysaccharide-induced motor neuron injuryObjective: Based on the in vitro model that motor neurons are selectively impaired in the organotypic spinal cord slice cultures, the present study was aimed to determine the changes in NADPH oxidase and clarify the key mechanisms implicated in motor neuron injury under inflammatory conditions.Methods: Organotypic spinal cord cultures were prepared using lumbar spinal cord slices from 7-day-old rats. After 1 week in culture, the explants were treated with medium alone, 30μg/ml LPS or combination of apocynin (0.5 or 1 mmol/L) with LPS (30μg/ml) for 2 weeks. The media were changed twice a week along with the test compounds. At the end of the treatment, the slices were immunostained with SMI-32 for motor neurons. The changes in mRNA expression of NADPH oxidase subunits gp91phox and p47phox after LPS and apocynin treatment were examined by RT-PCR. The protein level of p47phox in cytosolic and membranous components was further determined using immunoblot. LDH efflux to the culture medium and malondialdehyde (MDA) in the explants were measured using corresponding detection kits.Results:⑴Compared with the control group, the mRNA expression of gp91phox obviously enhanced in the explants exposed to 30 or 60μg/ml LPS for 3 days (P<0.05). Similarly, the mRNA level of gp91phox in the slices treated with 30μg/ml LPS was signinficantly higher than that in the normal controls (P<0.01). There was no notable difference in the p47phox message level between the control and LPS group at different time points (P>0.05).⑵The protein level of p47phox in the membrane was significantly increased in the slices exposed to LPS for 2 weeks than in the controls (P<0.01), and it was notably decreased after apocynin treatment (P<0.01).⑶The number of motor neurons was significantly increased in the group with concomitant application of apocynin and LPS compared to the LPS group (P<0.01). Many motor neurons had well-preserved morphology and long neurites.⑷Compared with the control group, the amount of LDH was significantly elevated in the group treated with 30μg/ml LPS for 2 weeks (P<0.05). LDH level was significantly decreased in the group cotreated with apocynin and LPS compared to the LPS group (P<0.05).⑸Exposure of slice cultures to LPS enhanced malondialdehyde production, significantly higher than that in the control (P<0.01). Combined application of apocynin with LPS abolished LPS-induced increase in malondialdehyde level (P<0.05, P<0.01).Conclusions: LPS could induce p47phox to translocate from cytoplasm to the plasma membrane, indicating that NADPH oxidase became activated. Apocynin inhibited NADPH oxidase activation by reducing LPS-induced p47phox translocation to the cell membrane, and afforded a significant protective effect against inflammation-mediated cytotoxicity. At the same time, the beneficial effects of apocynin on slice cultures were further supported by analyses of LDH and MDA. These findings suggest that NADPH oxidase may play a critical role in motor neuron damage caused by LPS-induced inflammation, which could lead to development of strategies of treatment for ALS.PartⅢEffect of nitric oxide on motor neuron damage induced by lipopolysaccharideObjective: Based on the in vitro model that motor neurons are selectively impaired in the organotypic spinal cord slice cultures, to investigate the changes in NO production as well as message expression and enzyme activity of NOS after LPS exposure. By using NOS selective or non-selective inhibitor, to further explore the precise role of NO in LPS-induced motor neuron injury.Methods: Organotypic spinal cord cultures were prepared using lumbar spinal cord slices from 7-day-old rat. After 1 week in culture, the explants were treated with medium alone or LPS with different concentrations (30 or 60μg/ml). Three days or two weeks after LPS exposure, the slices were collected for analyses of NOS message expression and enzyme activity. In addition, after treatment with LPS for 3 days, 1 week and 2 weeks, the nitrite level in the medium, which reflects NO production, was detected by the Griess reaction. 30μg/ml LPS group was chose for the drug-treated experiments. Spinal cord slices were treated with medium alone, LPS, combination of 1400W and LPS, combination of AG and LPS, combination of 7-NI and LPS or combination of L-NAME and LPS. NO production was measured after treatment for 3 days, 1 week or 2 weeks. And motor neuron survival was evaluated by SMI-32 immunohistochemical staining.Results:⑴Compared with the control group, iNOS mRNA expression markedly enhanced in the explants treated with 30 or 60μg/ml LPS for 3 days (P<0.01), whereas nNOS mRNA level showed no significant change (P>0.05). Two weeks after exposure to 30μg/ml LPS, iNOS mRNA level was significantly increased compared to the normal controls (P<0.01), whereas there was no obvious change in nNOS message expression (P>0.05).⑵Enzyme activities of total NOS, iNOS and nNOS were significantly higher in the slices treated with 30μg/ml LPS for 3 days than in the controls (P<0.01). Total NOS activity was significantly elevated in the slices treated with 30μg/ml LPS for 2 weeks compared to the control group (P<0.05).⑶NO level in the culture medium was very low in the control group at different time points. Three days or one week after 30μg/ml LPS treatment, NO production was obviously increased compared to the controls (P<0.01). Two weeks after LPS exposure, the level of NO tapered, but was still higher than that in the control group (P<0.01).⑷Combined application of 1400W or AG with LPS obviously lowered LPS-induced increase in nitrite levels (P<0.01), but they showed no significant beneficial effect on motor neuron impairment caused by LPS (P>0.05).⑸Combined application of 7-NI with LPS significantly decreased LPS-induced NO elevation (P<0.01), but it afforded no significant neuroprotective effect against cytotoxicity caused by LPS (P>0.05).⑹Combined application of L-NAME with LPS abolished LPS-induced NO increase (P<0.01), but it showed no significant neuroprotective effect on motor neuron survival (P>0.05).Conclusions: NOS activity and NO production obviously increased in the organotypic spinal cord slice cultures after LPS exposure. However, results obtained from NOS selective or non-selective inhibitor do not support that NO is of crucial effect on motor neuron injury in this model, and suggest that simply suppressing NO production could not improve motor neuron survival obviously. In view of its complicated effects, regulating the production of NO derived from different NOS at different periods of diseases may be a more promising therapeutic strategy.