The Effect Of PirB On Neuronal Synaptic Loss During Chronic Brain Inflammation

Objective This project was designed to investigate the changes of expression of paired-immunoglobulin-like receptor B (PirB) in neurons and activation of brain astroglial cells, the changes of synaptophysin (SYN) expression, as well as the changes of animals behavior; Further to investigate the effect of PirB immune-mediated control on neuronal synaptic loss and learning-memory deficits during chronic brain inflammatory induced by lipopolysaccharide (LPS).Methods In our study, normal adult SD rats were randomly divided into experimental and control groups. Rats were injected stereotaxically with LPS (10μg in4μl PBS)into the right lateral hippocampus,Vehicle(phosphate-buffer-ed saline, PBS) was used as control. After the animals survived for30days, we examined the response of astroglial cells, using glial fibrillary acidic protein (GFAP) as astrocyte marker. Expression of PirB and synaptophysin in the hippocampus of different treatment was also investigated, using immunohistochemistry. Their number and immunosignal intensity were evaluated with quantitative image analysis, and the Western blot technique was also used to quantitative analysis. The changes of neuronal structure was examined in the rat brain by Golgi staining. Double labeling with immunofluorescence was used to detect whether PirB was expressed not only by neurons but also by glial cells. To study the histochemical relationship of PirB with synapses, PirB and SYN double staining was performed. Spatial learning and memory abilities in rat were tested by Morris water maze.Results In both the PBS-injected control cases and the LPS-treated ones, GFAP-positive astrocytes were distributed in the piriform and entorhinal cortex, throughout the hippocampal formation, and subcortical white matter such as in the corpus callosum. Astrocytes with the morphological features of resting cells, with smaller cell bodies and thinner processes, were detected in the cortex and in the hippocampal formation of the control animals. After the endotoxin treatment, GFAP-positive astrocytes was significantly activated in the rat brain, especially in the hippocampal formation、the piriform and entorhinal cortex,These immunolabeled astrocytes appeared hypertrophic, with a relatively large cell bodies and very thick processes. Relative to the PBS-treated control group, the number and average OD values of GFAP positive astrocytes were significantly increased (P<0.01) in the experimental groups with respect to control groups in the CA3field of the hippocampus and DG at30days after LPS ih injections.In the PBS-treated controls, PirB neuron-like cells were mainly observed in the cerebral cortical5, with round/ovale or pyramidal shape. PirB staining consisted of brownish reaction products in the membrane of the cells. At30days after intrahippocampal LPS injection,Some large size PirB neuron-like cells and some small size PirB astrocytes-like cells were observed in the cerebral cortical4and5, and pyramidal cell layer in CA1-CA3area, as well as granular cell layer of dentate gyrus of the hippocampal formation,PirB positive reaction products located mainly in the somata and processes of the neurons. Quantitative analysis and statistical evaluation documented in the analyzed structures in each group highly significant increases (P<0.005) of the number of cells and their immunostaining intensity after LPS treatment with respect to the PBS-treated controls,.Immunoblot detected that the levels of the PirB protein in the hippocampi and cortex ipsilatcral to the injection, were dramatically elevated in the LPS-treatcd relative to PBS-treated animals surviving30days (P<0.001).The increased PirB labeling was localized to astrocytes and neurons,which co-expressed MAP-2. We also carried out double labeling for PirB and the microglia marker CD11b, but did not detect double labeling.Presynaptic protein SYN immunostaining is present mainly in the stratum oriens, stratum radiatum, molecular layer in CA1area, neuropil of CA3sectors of the Ammon’s horn and the non-cellular layers of dentate gyrus, where appears be puncta and discrete. A reduced synaptophysin immunolabeling was seen across the hippocampus and dentate gyrus in LPS-injected brains relative to controls, which was confirmed by densitometric analysis over two representative areas, CA1area of the hippocampus and the hilar regions of the dentate gyrus (P<0.05). Double labeling of SYN and PirB in CA3area of the ipsilateral hippocampus from an LPS-treated animal. There exists fine terminal-like PirB reactivity in the mossy fiber field, which seemingly overlaps with strong SYN reactivity at the mossy fiber terminals. SYN reactive products localized in the CA3pyramidal neuronal somata.Western blot analysis detected that the levels of the SYN protein in the hippocampi and cortex ipsilateral to the injection, were dramatically decreased in the LPS-treated relative to PBS-treated animals surviving30days, that reach statistic significance (P<0.05).The dendritic lengths in the basal dendrites and the density of dendritic spines of pyramidal neurons in the CA3region of hippocampus were dramatically decreased in the LPS-injected animals in comparison to those of PBS-treated controls by Golgi staining, that reach the statistic significance (P <0.05)The escape latency in LPS group increased significantly and was statistical significance (P<0.05) by the4th training day of the maze trials relative to PBS treated group.The swim paths recorded in the5th day trials indicated that:the PBS-treated rats swam mainly within the quadrant where the platform was located, or within the adjacent two quadrants after the platform was removed. In contrast, LPS-treated rats swam mostly along the side of the pool, with the swim paths distributed in all pool quadrants largely in a random manner. Quantitative analysis showed that the LPS-treated rats spent significantly less time in the target quadrant relative to PBS controls in the5th day of maze test (P<0.01).Conclusion LPS can induce activation of astrocytes and upregulation of PirB expression in the activated astrocytes, reduction of synaptophysin (SYN) expression, as well as impairment of learning and memory in rats. The dendritic lengths in the basal dendrites and the density of dendritic spines of pyramidal neurons in the CA3region of hippocampus were dramatically decreased in the LPS-injected animals in comparison to those of vehicle-injected group by Golgi staining. Double labeling of SYN and PirB showed that there exists fine terminal-like PirB reactivity overlaps with strong SYN reactivity at the mossy fiber terminals in CA3area of hippocampus. These results suggest that PirB may be involved in immune-mediated control of the synaptic plastic changes and deficits in learning and memory during inflammatory brain diseases.