Study On Expressions Of VCAM-1, IFN-γ And CD4～+ T Lymphocyte In Rat Models Of Delayed Neuropathological Sequelae After CO Poisoning And Developments After HBO Treatment

Most patients of acute carbon monoxide (CO) poisoning can be recovered after prompt and valid therapy, but 10%～30% of them will develop delayed neuropsychologic sequelae (DNS) which is characterized by dementia, psychiatric symptoms and extrapyramidal symptoms after a period of"latent phase"(days to weeks). Clinically, DNS is one of the most serious sequelae after acute CO poisoning. Patients with DNS have many symptoms, such as cognitive handicap, dysmnesy and extrapyramidal injury that make great influences on their living, their families and the society. Furthermore, there has been no effective treatment for DNS up to now. Therefore, it becomes one of the clinical researching emphasis concerning pathological changes and mechanism of DNS and how to prevent DNS beforehand.Many patients have no obvious symptoms because of the obvious latent phase during the period of CO poisoning and DNS. So it can be concluded that there are possibly delayed pathological changes that induce immunological injury in the brain.Due to blood brain barrier (BBB), brain tissues are isolated from blood system and become a partial immunological privileged site where there should be no lymphocytes and immune factors in normal situation.Vascular cell adhesion molecule-l (VCAM-1) can conjugate with T lymphocytes and mononuclear macrophage of blood system and can make them be activated and immigrate to tissues at last.CD4~+ T lymphocytes mainly participate in cell immunity and delayed type hypersensitivity (DTH) and induce cell degeneration and necrosis by releasing cytokines to damage the target cell with antigens indirectly.Interferon-γ(IFN-γ) is the main lymphocyte factor released in the effector phase of immunological response by CD4~+ T lymphocytes. It can activate the process of angtigen presentation, reinforce the ability of sterilization and induce the vascular endothelial cell to activate the express of adhesion molecule. So VCAM-1 is prerequisite for the system cell immunity in brain tissues, CD4~+ T lymphocyte is the main body of DTH, and IFN-γis the direct effect factor of the immunological response.In order to investigate the immunological reaction in rat brains with DNS after acute CO poisoning, DNS models of rats after acute CO poisoning which should be consistent with the clinic symptoms of DNS patients are established. Immunohistochemistry staining is used to detect the express of VCAM-1 and IFN-γand the infiltration of CD4~+ T lymphocyte and to explore their time regularity and distribution feature. Moreover, hyperbaric oxygen (HBO) is used to treat DNS and changes of these indexes mentioned above are discussed. On the other hand, H-E staining is applied to observe the morphologic changes of neurons in rat model and to reveal the pathological features of DNS.The main results of present work are as follows:1.The characteristic of morphologic changes of neurons in rat models with DNS after CO poisoningAfter acute CO poisoning, there are many degenerated and necrotic neurons in cerebral cortex and hippocampus. The main phenomenon was as follows: the cell body of neurons crenulated, the spacing appeared around the cell, the cytoplasm showed characteristic of philo-eosine, the nucleolus was anachromasis and dissymmetry, especially karyopyknosis and the cytoplasm condensed with the nucleus showing vague circumscription.The spacing around blood vessels and cells enlarged and the fibrous tissues got sparse. The generation of the neuroglia cells happened at the site of neuronal degeneration and necrosis. There were nucleated cells inside lumen of blood vessel in brains. Cortex of apical lobe and temporal lobe were damaged most seriously. Hippocampal pyramidal cell layer got thinner, pyramidal cells at the both sides of the cell layer were almost disappeared, and the middle pyramidal cells atrophied and degenerated. The most serious damage can be seen in CA1 and CA4 region of hippocampus.These pathological changes that peaked at the 7th day can be found in all the groups of CO poisoning. The number of degenerated and necrotic neurons at the 7th day had significant statistical difference (P<0.01) compared with what at other poisoning groups. Moreover, there were also statistical differences (P<0.05) between HBO group and the poisoning groups at the corresponding time.2. The time regularity and distribution feature of the expression and infiltration of VCAM-1, IFN-γ, CD4~+ T lymphocytes(1)Endothelial cells of the cerebral capillary vessels and degenerated and necrotic neurons mainly expressed VCAM-1. Expression of VCAM-1 reached the peak at the 1st day and possessed significant statistical difference (P<0.01) compared with which of other poisoning groups. The expression decreased after the treatment of HBO and possessed significant statistical difference (P<0.05) compared with which of other poisoning groups at the corresponding time.(2)The CD4~+ T lymphocytes initiated infiltration at the 1st day and reached the peak at the 7th day (P<0.01). The infiltrated CD4~+ T lymphocytes with such brown shapes as round, ellipse or triangle in a diameter of micrometers to decade micrometers were distributed around the blood vessels of apical lobe and hippocampus. The infiltration decreased in HBO group (P<0.05).(3)IFN-γwas expressed mainly by the neurons and partially by neuroglia cells. The expression reached the peak at the 3rd day (P<0.01). The positive cells with such brown shapes as triangle, polygon or irregularity suffused the whole region of the cerebral cortex and hippocampus. It was found that the IFN-γ+ cells always lay in the region of neuronal degeneration and necrosis after application of hematoxylin. Expression of IFN-γdecreased in HBO group (P<0.05).According to the experimental results mentioned above, the neuronal degeneration and necrosis in the region of cerebral cortex and hippocampus were duo to hypoxia after acute CO poisoning. VCAM-1 was expressed greatly by those neurons and endothelial cells of the cerebral capillary vessels. CD4~+ T lymphocytes in the hematological system were induced to pass through BBB, infiltrate into the brain tissues, release lymphocyte factor IFN-γ, and participate in cell immunity and DTH. Finally, the neuron damage in the region of cerebral cortex and hippocampus became worse.At the same time, neurons and neuroglia cells expressed IFN-γafter CO poisoning. IFN-γcould induce microglia to express major histocompatibility complex-Ⅱ(MHC-Ⅱ), activate CD4~+ T lymphocyte, and make continuous immunological reaction in the brains. HBO treatment had the advantages to decrease expression of VCAM-1 and IFN-γas well as decrease the infiltration of the CD4~+ T lymphocyte, and to lighten the damages of brain tissues from immunological reactions.Collectively, our work has demonstrated that immunological reactions participate in DNS after acute CO poisoning, and that HBO treatment lighten the brain damage through restraining the immunological reactions. Therefore, HBO is helpful to prevent DNS beforehand.