The Cellular Bases Of Brain Lateralization Affecting Neuroimmunoendocrine

Background & Objective:Brain lateralization is a universal phenomenon, ascertained by anatomical, neurochemical and functional data, brain lateralization is involved in the regulation of physiological systems including the immune system. Two sides of the brain are differently involved in the modulation of immune responses as demonstrated by lesional and behavioral approaches. In the physiological condition the right neocortex depresses immune functions, whereas the left neocortex enhances immune functions. Brain lateralization has effect on the immune system through hypothalamic-pituitary-adrenal (HPA) axis. Animal studies have provided compelling evidences that IL-1β , IL-6 and TNF- α had immunosuppressive roles in the CNS. The former research of our laboratory also demonstrated that there was a strong correlation between the levels of IL-1β and IL-6 in brain cortial tissue homogenates and behavioral lateralization, and cytokine asymmetries had a strong correlation with the direction and the intensity of behavior lateralization. In the CNS, the neuroglia is the primary source for cytokines and is believed to be immunocompetent defense cell, it plays an important role in neuroimmunoendocrine networks . This research has established glial cells cultures from brains of right and left cortex, hope that could be found what role of the glial cells (which are the main element of the cortex ) performed in brain lateralization. The purpose of this report is to study the quantity of the neuroglia between the right and left cortex and the cytokines secreting ability of the glial cells, analyze their roles in the relationship between the immune system and the brain lateralization. Methods:Cultures were prepared from the brains of 1-2-day-old Balb/c mice, the animals were killed by decapitation, whole brain was cleared from adhering meninges and blood vessels and mechanically dissociated, washed with Hank's solution, the left and right cerebral cortices wereseparated and plated into different tubes. The fibroblasts were removed by allowing the cells to adhere to the surface of the plate for 30 min, after turn up the plate, the cells which can not stick are glial cells. The cells solutions were divided into two groups: group one were breed on the small glass slides for preparing the immunohistochemical stain which detect the GFAP and CNPase expression; group two for cytokines detection. Then re-suspended cells in 6ml l:l(v/v) Dulbecco's modified Eagle's medium and F12 which containing 20% (v/v) fetal calf serum(FCS),100ug/ml streptomycin, 50unit/ml penicillin, 25ug/ml gentamycin and 2.5ug/ml amphoteicin B and adjusted the cells at lX106/ml in group two(n=13), cells were plated at 24-well plates, these primary cultures were maintained in a 5% CO2 humidified incubator at 37°C for 10-15 days. After confluented, glial cells cultured on 24-well plates were washed twice and replenished with fresh medium without FCS, LPS were added in a final concentration of lOug/ml, control experiments were also performed. Samples of culture medium were harvested at 24 hour and different time point respectively after LPS stimulation, and cytokine levels were determined by ELISA kits. The cells solution for determining the quantity of glial cells were prepared with the same method in another group. Statistical analysis was performed by Student's T test, and data are expressed as means ± standard error mean and the significance level was defined at PO.05. Results:1. The quantity of the left and the right glial cells of Balb/c mice (n=30) were 1.596+0.558X106 and 1.486 + 0.658 X106 respectively, there were no differences in two groups (P=0.122).2. There were many GFAP and CNPase positive cells in the left and right cerebral cortex glial cells, GFAP positive cells were much more than CNPase positive cells, and there existed some differences in morphology and distribution between two kinds of the cells. There were no significance difference in GFAP and CNPase expression between the left and the right sides.3. IL-1 3 , IL-6 and TNF- a level in supernatants of the left and the right cerebral cortex glial cells culture: IL-1 3 level in right cortex was higher than that of the left but had no statistic meaning (P=0.152) in normal group; the right and the left's IL-1 3 levels were all higher after LPS stimulation and had significance difference ( P=0.022 and P=0.007 respectively). IL-1 3 level in right cortex was higher than that of the left (P=0.043). IL-6 level in normalgroup was higher in right cortex than that of in left cortex, the difference had no significance(P=0.232); In LPS treated group, IL-6 level was much higher than that of the normal group^=0.002 and P—0.000 respectively), and IL-6 level in right cortex was higher than that of the left (P=0.012). TNF- a level in right cortex was higher than that of the left but had no statistic meaning (P=0.262) in normal group; the right and the left's TNF- a levels were all higher after LPS stimulation and only the right sides increased significantly (/M).O43). TNF- a level in right cortex was higher than that of the left (/M).038).4. Kinetic studies showed different patterns of cytokine IL-ip, IL-6 and TNF-a production after incubation with LPS. IL-ip and TNF-a were the first two kinds of cytokines to had been produced, followed by IL-6. IL-lp started to increase at 3-6 hour, then increased steadily; TNF-a started to increase at 3 hour, which increased and reached maximal level at 6 hour and 12 hour points, but couldn't keep that level thereafter. Then TNF-a level decreased, at 24 hour and 48 hour, TNF-a was back to the basic level. After about 12 hour of stimulation, the production of IL-6 also started to increase, at 24 hour, it increased steady and maintained this trend, the maximal level at 72 hour. These results indicated that the time courses of LPS-induced IL-6, IL-lp and TNF-a releasing were difference, under experimental conditions the non-stimulated glial cells produced few quantities of cytokines. In the kinetic studies, IL-1 p and the IL-6 levels in the right were higher than those of in the left.Conclusion:1. There is no significant difference in quantity of the glial cells between the left and the right cortex, indicating that the brain lateralization may correspond to the glial cells secretion ability rather than their quantity.2. There are no differences in GFAP and CNPase expression between the left and the rightcerebral cortex glial cells.3. There is a significant difference in IL-1J3 level which released by the right cortex stimulated by LPS, the level of IL-1P are higher in the right than that of in the left.4. The right cortex is found to be more sensitive to the elevation of IL-6 and TNF-a induced byLPS, the levels of IL-6 and TNF-a released by glial cells are higher in the right than those of in the left, it can be proposed that the higher expression of IL-6 and TNF-a in the right cortex glial cells may contribute to the immunosuppression of the right cortex.5. Kinetic studies of these three cytokines further indicate that these cytokines might be interactive as indicated by the difference in time course of their expression, with the IL-ip being the earliest and IL-6 being the latest. At the whole time course, IL-1J3 and IL-6 levels in the right are higher than those of in the left.