| Because of the existence of Blood Brain Barrier (BBB), CNS is considered the immune privilege site, moreover, the inflammatory infection and immune response in it has been a focus of attention and controversy. Identification of Toll-like receptors (TLRs) promotes comprehension of the mechanisms of inflammation reaction in the brain and draws much attention. Toll-like receptor4(TLR4) is the first toll-related protein found by humans, and it is widely distributed in tissues and cells in the organism. Studies have shown the expression of TLR4on the glia in the CNS of mammalian, and confirmed that glia exert its function through the binding of TLR4and its ligand in the inflammatory process. Currently, systematic data for the relationship between TLR4and inflammatory and immune response in poultry is still lacking. Therefore, adopting4day-old chicks as the experimental objects, this research aims to explore the influence of Lipopolysaccharide (LPS) and Salmonella administration on the tissue structure and ultrastructure of BBB in the brain and also the distribution and expression levels of TLR4to lay a sound foundation for revelation of mechanisms of the innate immunity. The main contents are as follows:1The effects of LPS and Salmonella adminstration on brain tissue structureThis research is performed to explore the influences of bacteria and their products from periphery on the changes in brain structure through intraperitoneal injection of LPS and Salmonella of2day-old chicks. Results:Comparing with the control group, with extension of time, cerebellar pia mater vascular dilation, congestion, swelling, thickening of pia mater gradually, and Purkinje cell layer of micro vascular hyalinization were found after LPS stimulation. Comparing with the control group, with the time of LPS administration increasing, there were increasing inflammatory cells beside the ventricular in the cerebrum. After Salmonella administration, cerebellar pia mater hyperemia, inflammatory cell infiltration, vascular obstruction and vascular invasion cell inflammation around the microvessel and late visible meningeal congestion, edema, hemorrhage neuron necrosis and vacuolization are found. Hyperemia and swelling of microvessel under pia mater, thickening of the pia mater, Inflammatory substances leaking, inflammatory cells increasing and tissue necrosis after LPS stimulus compared with the control group. Hyperemia and hemangiectasis under pia mater, thickening of the pia mater and hemangiectasis, inflammatory cells increasing and tissue necrosis after salmonella stimulus compared with the control group. These results suggest us that, from the perspective of morphology, peripheral injection of LPS and Salmonella cause pathological changes of brain tissue CNS to some extent.2The effect of salmonella administration on the ultrastructure of BBB in the brainThis experiment is preformed to investigate the effects of microbial infection on the structure of BBB in chickens ’ brain in the electron microscope. The research shows an intact structure of microvascular wall around nerve fiber network in the control group and dissolved microvascular endothelial cell nucleus, changes of the endothelial cytoskeleton and cell membrane expansion at12h after Salmonella adminsstration. The results also shows degradation of nerve fiber net around the microvascular wall and degradation of cellular processes and neuronal axons and dendrites at72h after Salmonella adminsstration. It suggests that microbial peripheral infection affect its functions and also the homeostasis of the micro-environment in the CNS through the structural changes of BBB.3Distribution patterns of TLR4in the brain of two-day chickenImmunohistochemistry was used to look into the distribution characteristics of TLR4in chicken’ brain. Results:TLR4was constitutively expressed in the brain chicken of developmental stages of chicken. Its expression focuses mainly on cerebral pia mater, microvessels and neurons and cerebellar pia mater, Purkinje cells and medullary neurons, and also the microvascular wall with relative weak positive signal. The results realize us that, TLR4was expressed in normal conditions and may be involved in the recognition and response to exogenous stimuli in the chicken CNS. At the same time, recognition and initiate of innate immunity may depend not only on the pia mater and the circulation of blood, may also depend on the immune related factors from the neurons in parenchyma. 4The influences of LPS and Salmonella stimulation on the TLR4expression level in the brainThe expression level of TLR4has been detected at different time points after peripheral administration of LPS and salmonella on2day-old chickens at Oh,12h,36h and72h after collecting the brain tissue samples through the method of double antibody sandwich ELISA, respectively. Results:After LPS and salmonella stimulation, the expression level of TLR4in the cerebrum decreases first and increases then with the stimulus and is down to the lowest at36h. The TLR4expression in the cerebellum is opposite, after LPS stimulation, it increases first and decreases then, reaching the maximum at12h. This conclusion suggests the time of response between cerebrum and cerebellum after pathogenic peripheral stimulation be different, and the mechanism of identification of TLR4for pathogens and its expression which were induced to also be different.The expression level of Iba-1has been detected at different time points after peripheral administration of LPS and salmonella through the method of double antibody sandwich ELISA. Results:In the cerebrum, after LPS stimulation, the expression level of Iba-1increases and reaches the peak at12h after infection, the Iba-1expression increases and reaches the peak at36h after salmonella infection, in the cerebrum. It is opposite in the cerebellum, after LPS stimulation, it decreases after infection of both LPS and salmonella. The results suggest that LPS and salmonella both can activate microglia in the cerebrum.The expression level of E-selectin has been detected at different time points after peripheral administration of LPS and salmonella through the method of double antibody sandwich ELISA. Results:In the cerebrum, the expression level of E-selectin decreases and reaches the lowest point at72h after LPS infection, the Iba-1expression decreases and reaches the lowest point at36h after salmonella infection. In the cerebrum, after LPS stimulation, the E-selectin expression has been a waving trend and it increases after salmonella infection.The expression level of calprotectin has been detected at different time points after peripheral administration of LPS and salmonella through the method of double antibody sandwich ELISA. Results:In the cerebrum, the expression level of E-selectin increases first and decreases then and it is significantly higher at12h and lower at72h after infection of LPS and salmonella compared with control group. The expression level of E-selectin is opposite in the cerebellum, it decreases and reaches the lowest point at72h after LPS infection and it decreases first and decreases then and reaches the maximum at3h after salmonella infection. The results indicate that cerebrum maybe the key part of inflammation in the central nervous system. |
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