Study On Immune Factors And Neuroglia Cells In Rat Models Of Delayed Neuropathological Sequelae After CO Poisoning

Carbon monoxide (CO) is an odorless, colorless, toxic gas and stable product of incomplete hydrocarbon combustion. CO poisoning is the most common accidental poisoning. It is a poison agent with high incidence rate and mortality rate. It can be seen in the situation of fire disaster, excessive leakage of vehicle's end gas, and accidents happened in industry or in the use of stove for heating, cooking and bathing in our daily life. Suicide with CO also can be heard sometimes. After a period of"latent phase"(days to weeks) post treatment, 3%~30% patients may appear delayed neuropsychologic sequelae (DNS) which is characterized by dementia, psychiatric symptoms and extrapyramidal symptoms. At present, the mechanism of DNS is still not clear.Recently, immunologic mechanism of DNS is given more and more reconstruction. Traditional points of view presume that central nervous system (CNS) is an immunological-privileged site. Nowadays, studies have showed that CNS is not an absolute immunological-privileged site and the permeability of blood brain barrier (BBB) may be changed in some definite condition. It can make immune factors permeate to brain, and meanwhile in CNS there are antigen presenting cells (APC) and immunocytes that release many kinds of immune factors. These immune factors can make APC be activated and present antigens. After that progress, antigens will be cleared. When cerebral ischemia reperfusion, there will be a lot of free radicals in brains of patients with CO poisoning. Free radicals may increase the lipid peroxidation of cellular membrane. Some products of lipid peroxidation make cerebral tissue protein denature and bring out antigen materials. Antigen materials or lipid peroxidation itself can stimulate immune reaction of CNS. The visible"latent phase"may be the result of the process of immunocytes activation in CNS immune reaction after brain injury.Neuroglia cells are obbligato part in nervous system and play an important role on physiological function of CNS. Their functions mainly include support, give nutrition, protect and repair neurons. When brain tissue damaged, there are not only injury of neurons, but also alteration of neuroglia cells. They respond to brain damage by generation, deformation and secreting cytokines. Neuroglia cells play a double role on CNS injury. Sometimes they protect neurons, and sometimes they damage neurons. Mammals'nervous system maturates at embryonic period, and neural stem cells (NSCs) can differentiate to neurons, astrocytes and oligodendrocytes during the process. There are few NSCs in CNS of adult mammals and they always gather in subventricular zone (SVZ) and subgranular zone (SGZ). It is reported that after brain injured NSCs could repair brain tissue damage by generation, immigration and differentiation. There may be some regularity on neuroglia cells and NSCs in brains of patients with DNS after CO poisoning. The effect of hyperbaric oxygen (HBO) treatment is predominant for DNS. It is suggested that HBO may improve patients'clinical symptoms by effecting neuroglia cells.On account of above hypothesis, expression of immune factors such as major histocompatibility complexⅡ(MHCⅡ) and interferonγ(IFN-γ) in brains of rat models was observed on this study by setting up rat model of DNS after CO poisoning and the mechanism of DNS was to be discussed. We observed variation of astrocytes, oligodendrocytes, microglias, NSCs and neurons on brains of these models and the effect on variation of these cells after HBO treatment. The mechanism of DNS and the HBO therapeutic mechanism were to be further discussed.The rat models of DNS after CO poisoning were established by the method of acute static inhalation of CO. the pathological change of cerebral cortex and hippocampus area was acquired by technique of histopathology. The expression of MHCⅡand IFN-γ, the variation of neuroglia cells and NSCs and the effect of HBO treatment on these cells were observed by immunohistochemistry staining.The main results of present work are as follows:(1) Changes of pathomorphism: there were extensive pathological changes and obvious neuron degeneration and necrosis on cerebral cortex, hippocampus and dentate gyrus on H-E staining preparations of DNS group. Cortex of apical lobe was damaged most seriously. We found neuroglia cells generate at the site of neuron degeneration and necrosis. Hippocampal pyramidal cell layer got thinner. Apparent neuron degeneration and necrosis could be seen in CA1 region of hippocampus. These pathological changes that peaked on the 7th day could be found in all the groups of DNS. Ultramicrostructure showed that the size of neurons was normal, nuclear membrane of neuron was integrity, nucleolus was clear and cell organs were complete in control group. But in DNS group neurons got augmente, bioblast became swelling, cell organs got disaggregated and the density of chromatin became thick and inhomogeneous.(2) Characteristc of immunohistochemistry staining of MHCⅡand IFN-γ: there was little expression of MHCⅡand IFN-γin control group, but in hippocampus and cortex area of DNS groups, the expression of these two proteins was distinct. The expression of MHCⅡwas enhanced in the 3rd day, peaked in the 7th day(P<0.05), decreased in the 10th day and hardly found in the 20th day. The expression of IFN-γwas peaked in the 3rd day(P<0.05), a little decreased in the 7th day and barely noticed in the 10th day and the 20th day.(3) Variation of neuroglia cells and NSCs: the result of immunohistochemistry staining of nestin,GFAP,OX-42 and RIP showed that compared to control group expression of nestin and RIP was decreased(P<0.05) and expression of GFAP and OX-42 was increased(P<0.05) in DNS group. The shape of GFAP+ cells and OX-42+ cells was changed and these cells were assumed at active state. In HBO group expression of nestin and RIP was decreased (P<0.05)and expression of GFAP and OX-42 was increased(P<0.05)compared to control group.But compered to DNS group expression of nestin and RIP was increased (P<0.05)and expression of GFAP and OX-42 was decreased(P<0.05).In a word, this study demonstrated that the immune factors MHCⅡand IFN-γwere expressed in rat models of DNS after CO poisoning. Referred to the site of appearance, they always lay in the region where neuron degeneration and necrosis occurred. And referred to the time course, their expression fastigium had a time interval. It is also showed in this study that there were variations on shape and quantity of neuroglia cells. The site and time course of microglias'active state were coincident with MHCⅡexpression and the shape of MHCⅡ+ cells was similar with neuroglia cells. At the base of these findings, we can make our conclusion that the following mechanism may contribute to the onset of DNS. After acute CO poisoning, brain tissues were affected by many injury factors and neurons and neuroglia cells got damaged. Impaired neurons and neuroglia cells released cytokines such as IFN-γ, and because of these cytokines resting microglias were activated to become APC and expressed MHCⅡ. APC presented antigens that conjugated with MHCⅡto T lymphocyte. After that immune reaction course, brain tissues were damaged and caused secondary damage. As a result, delayed neuron necrosis occurred in this area, decreased NSCs cut down brain tissue repair simultaneously and DNS was observed in patients. On the foundation of above study, the following research must be emphasized on finding the direct evidence of immune reaction including immunogen, immune effector cells and exploring its mechanism.