The Experimental Study On The Role Of Meningeal Cells In Sensation Of Immune Signals

Meningeal connective tissue, which shields the whole brain, is subdivided into three different compartments: dura mater, arachnoid and pia mater, forming a thin and tight membrane texture. Functional investigation in neuroscience has generally been predicated on the assumption that the brain parenchyma is structurally and functionally separated from meninges. It seemed that the meninges were merely protective membranes that do not serve any physiological function in adult life. In 1990s, Frederic et al challenged this prevailing view by using immunohistochemical methods to specifically mark the meningeal cells and astrocytes. The results were surprising. It showed us first time a cellular network that courses through all layers of meninges, intimately connects to the glial networks. This conception of meningeo-glial network calls for a reconsideration of the role of meninges and appears to reflect the possibility of meninges' involvement in physiological or pathological processes in CNS.Meninges are formed by kinds of cells, which facilitates their complicated functions. It is recognized that meninges are actively involved in neuronal cells differentiation, cerebellum maturation and enzyme-barrier constitution. However, we know little about the role of meninges playing in sensing peripheral inflammatory insults and regulating immunoreaction of CNS. Most evidence is acquired accidentally by investigating brain parenchyma, hi the preceding experiments, central or peripheral injection of immunal stimulators induced evident increase of pro-inflammation cytokines(e.g.IL-1β, IL-6) immunoreactivity and up-regulation of c-fos, c-jun in many sites of parenchyma, but it was surprising that meninges' responses were prior to other sites. All of these results indicate the possible involvement of meninges in CNS immunoreaction, simultaneously they bring up new questions: which cells in meninges participate in the neuroimmunomodulation? Can they sense and transmit immune signals individually? Do they interact with glials and neurons?The identity of meningeal cells remains unclear nowadays, but more research inclined to treat meningeal cells as a special kind of fibroblast-like cells. So, the present study included three sets of experiments which were made on animal models and cultured cells respectively, aiming to investigate the role of meninges in sensation and regulation of immune signals in CNS. The first part of study was to observe the expression of MAPK molecules in meninges and morphological changes of astrocytes beneath pia mater after intraperitoneal injection of LPS. Then in the second set of experiment, we established a culture system of meningeal cells and investigated effects of different culture medium or immune stimulators on meningeal cells. In the last experiment, we evaluated the role of MAPK pathways in LPS-induced expression of inflammatory cytokine receptors by meningeal cells.Based on the immunoreaction of meningeal cells, the president study showed the function of meninges from a new aspect by using cell culture, immunohistochemistry method in light microscope level and Western blot analysis. The results primarily proved the ability of meningeal cells of sensing and transmitting immune signals.The main results are as follows:1. After intraperitoneal injection of LPS for half an hour, we observed the expression of phospho-ERK1/2 molecules by meningeal cells. The positive cells limited in certain regions: part of meninges overlaid on fissure, temporal and ventral brain, meninges covered cerebella and spinal leptomeninges. Cells in meninges exhibited dense-stained nucleus, while cells of spinal meninges showed stained nucleus and processes projecting into parenchyma. These results indicated that meningeal cells tend to be activated by peripheral stimuli and react differently according to their location.2. After intraperitoneal injection of LPS for 0.5h, 2h and 4h respectively, we detected the changes of astrocytes that were just beneath the meninges by using immunohistochemistry methods with GFAP marker. At 0.5h, the immunoreactivity for GFAP of glial-limiting membranes was considerably significant, with swollen astrocytic body and shortened processes, while few astrocytes in parenchyma were densely marked. At 2h, reaction of astrocytes forming glia limitans decreased, compared to the enhanced reaction in parenchyma. Then at 4h, although most of astrocytes in the brain were activated, cells in glial-limiting membranes showed shrinked bodies and elongate processes. These results suggested that after sensation of peripheral stimuli the reaction of glial-limiting membranes, formed by meningeal cells and astrocytes, is evident and prior to parenchyma. It is possible that meningo-astrocytic cross-talk plays an important role in inflammatory reaction in CNS.3. After cultured meningeal cells in different condition, we detected the difference of purity and cellular property of different culture system. When cultured in...