| BackgroundBrain edema is a common critical disease, which can cause intracranial hypertension, and then reduce cerebral blood flow and cause secondary ischemic brain injury or hernia. Hypertonic saline has been widely used in the treatment of patients with brain edema resulting from cerebral infarction, hemorrhage or traumatic brain injury. The common used sodium concentration of hypertonic saline is3%,5%,7.5%,10%and23.4%. Recently several studies suggest the relative superiority of hypertonic saline to mannitol. Both animal and human studies have demonstrated the ability of hypertonic saline to lower intracranial hypertension when used as both continuous infusions and intermittent, bolus infusions, and it has low incidence of side effects.However, little was known as of the action of hypertonic saline besides its osmotic effect in the treatment of brain edema. The possible mechanisms of action of hypertonic saline have osmotic effect, increasing cerebral perfusion pressure, modulating the inflammatory response, and restoring the neuronal membrane Na+, K+-ATPase activity. Zeng HK et al reported that10%sodium chloride (NaCl) can down-regulate expression of aquaporin4(AQP4) in perivascular astrocytes in a rat cerebral ischemic edema model. Our previous study confirmed that in addition to its osmotic force,3%NaCl exerted anti-edema effects possibly through inhibiting the expression of AQP4in bacterial meningitis induced by E.coli in rabbits. Lipopolysaccharide (LPS) is the main component of the cell wall and pathogenic component of E.coli, Gram-negative bacterium. LPS is closely related to the occurrence of cerebral edema. Investigating the effect of3%NaCl on up-regulation of brain Inflammatory cytokines and AQP4during LPS induced mice brain edema would provide clues to reveal the non osmotic effect mechanism of3%NaCl on brain edema in bacterial meningitis.Objective(1) By investigating the effect of3% NaCl on brain Inflammatory cytokines and AQP4expression in cerebral edema induced by LPS to demonstrate the role and mechanism of3% NaCl on AQP4expression in LPS induced mice brain edema.(2) To investigate the role of PKC in mediating AQP4expression downregulation induced by3% NaCl in the primary astrocytes.Chapter Ⅰ The Building of Lipopolysaccharide-induced Mouse Brain Edema Model.1.1Methods(1) KunMing mice were randomized into control group, LPS group. LPS group was given LPS as a single injection of10mg/kg,20mg/kg or40mg/kg i.p.. Control group received0.9%NaCl as control. The mice were sacrificed at6h or12h after injection of LPS. Brains were quickly removed and brain water content was measured.(2) KunMing mice were randomized into control group, LPS group. LPS group was given LPS as a single injection of10mg/kg i.p. Control group received0.9%NaCl as control. The mice were sacrificed at8h or12h after injection of LPS. Brain water content, Interleukin-1beta (IL-1β) or tumor necrosis factor alfa (TNFα) mRNA and protein, immunoglobulin G (IgG) protein in brain tissues were performed respectively.All values were presented as mean±S.E.M. Differences in various groups were determined by one-way analysis of variance. A value of p<0.05was considered statistically significant.1.2Results(1) The brain water content was significantly increased in LPS-treated mice at6h,12h following LPS injection when compared with corresponding controls (p<0.05). There are no differences between LPS groups (10mg/kg,20mg/kg or40mg/kg). (2) The brain water content was significantly increased in LPS-treated mice at8h following LPS injection when compared with corresponding controls (p<0.05), IgG in brain tissues increased in LPS-treated mice at8h,12h following LPS injection (p<0.05). There is no difference between LPS groups.(3) IL-1β and TNFa mRNA or protein in brain tissues increased at8h,12h following LPS injection, compared with corresponding controls (p<0.01, p<0.05). There is no difference between IL-1β or TNFa groups.1.3Conclusion(1) Intraperitoneal injection of LPS (10mg/kg) for6hours can induce brain edema. Increasing the time or dose of LPS don’t accelerate brain edema, which indicates that intraperitoneal injection of Intraperitoneal injection of LPS (10mg/kg) for6hours can induce a stable model of brain edema.(2) Intraperitoneal injection of LPS (10mg/kg) can induce up-regulated expression of IL-1β and TNFα, disruption of the blood-brain barrier.Chapter Ⅱ Effects and Mechanisms of3%Sodium Chloride on Lipopolysaccharide-induced Mouse Brain Edema2.1MethodsKunMing mice were randomized into control group, LPS group and HS group. LPS group was given LPS (Sigma) as a single injection of lOmg/kg i.p.. HS group was given3%NaCl as a single injection of20ml/kg i.v. through the tail vein at6h following injection of LPS, and control group received0.9%NaCl as control. The mice were sacrificed at8h or12h after injection of LPS. Measurements of brain water content, interleukin-1beta (IL-1β), tumor necrosis factor alfa (TNFa), immunoglobulin G (IgG), AQP4mRNA and protein in brain tissues were performed. All values were presented as mean±.E.M. Differences in various groups were determined by one-way analysis of variance. A value ofp<0.05was considered statistically significant.2.2Results(1) Treatment with3%NaCl for2h or6h the brain water content was significantly decreased (p<0.05). There is no difference between HS groups(2) Treatment with3%NaCl for2h the IgG in brain tissues didn’t significantly change (p>0.05); Treatment with3%NaCl for6h the IgG in brain tissues was significantly decreased (p<0.05)(3) Treatment with3%NaCl for2h the IL-1βmRNA in brain tissues was significantly decreased (p<0.01); the IL-1βprotein in brain tissues didn’t significantly change (p>0.05). Treatment with3%NaCl for6h IL-1βmRNA and protein was significantly decreased (p<0.01).The Variation of TNF-a mRNA and protein were consistent with IL-1β.(4) AQP4mRNA and protein in brain tissues were significantly increased in LPS-treated mice at8h,12h following LPS injection, compared with corresponding controls (P<0.01). Treatment with3%NaCl for2h the AQP-4mRNA in brain tissues was significantly decreased (p<0.01); the AQP-4protein in brain tissues didn’t significantly change (p>0.05). Treatment with3%NaCl for6h AQP-4mRNA and protein was significantly decreased (p<0.01, p<0.05).2.3Conclusion(1)3%NaCl can ameliorate LPS-induced cerebral edema, reduced expression of inflammatory cytokines in vivo.(2)3%NaCl can inhibit the expression of AQP-4, and down-regulating the expression of proinflammatory cytokines (IL-1β and TNFa) may be one of mechanisms of inhibiting the expression of AQP-4by3%NaCl.ChapterⅢ Effects of PKC on inhibition of the expression of AQP4induced by3%NaCl 3.1Methods(1) KunMing mice were randomized into control group, LPS group and HS group. LPS group was given LPS (Sigma) as a single injection of10mg/kg i.p.. HS group was given3%NaCl as a single injection of20ml/kg i.v. through the tail vein at6h following injection of LPS, and control group received0.9%NaCl as control. The mice were sacrificed at8h or12h after injection of LPS. PKCa in the membrane structure of brain tissue were performed.(2) The fourth-generation of primary cultured astrocytes were randomly divided into control group, IL-1β group, IL-1β+HS group. Measurements of cell viability, AQP4mRNA and protein in astrocytes were performed.(3) The fourth-generation of primary cultured astrocytes were randomly divided into control group, IL-1β group, IL-1β+HS group, IL-1β+CalphostinC group and IL-1β(3+CalphostinC+HS group. Measurements of AQP4mRNA and protein in astrocytes were performed. All values were presented as mean±.E.M.Differences in various groups were determined by one-way analysis of variance. A value of p<0.05was considered statistically significant.3.2Results(1) Treatment with3%NaCl for6h, the optical density of protein kinase C alpha significantly increased, compared with corresponding controls or LPS group (P<0.05).(2) There are no statistical differences between cell viability in control group, IL-1β group, IL-1β+HS group (P>0.05).(3) IL-1β significantly increased the AQP4mRNA expression and membrane protein level in IL-1β-treated astrocytes (p<0.01).(4)3%NaCl significantly decreased AQP4mRNA and membrane protein level in the IL-1β+3%NaCl-treated astrocytes (p<0.01).(5) Pretreatment with Calphostin C, a specific inhibitor of PKC, attenuated the decrease of AQP4mRNA and protein in the primary astrocytes induced by3%NaCl. 3.3Conclusion(1)3%NaCl can block the increase of AQP4mRNA and protein in the primary astrocytes induced by IL-1β.(2)3%NaCl can block the increase of AQP4expression in the primary astrocytes induced by IL-1β through activation of PKC.Conclusion(1) Intraperitoneal injection of LPS (10mg/kg) for6hours can induce brain edema and set up a stable model of brain edema; LPS can induce up-regulated expression of IL-1β and TNFa, disruption of the blood-brain barrier.(2)3%NaCl can ameliorate LPS-induced cerebral edema, and reduce expression of inflammatory cytokines in vivo.(3)3%NaCl can inhibit the expression of AQP-4, and down-regulating the expression of proinflammatory cytokines (IL-1β and TNFa) may be one of mechanisms of inhibiting the expression of AQP-4by3%NaCl.(4)3%NaCl can blocke the increase of AQP4mRNA and protein in the primary astrocytes induced by IL-1β. Activation of PKC may be one of mechanisms of inhibiting the expression of AQP-4by3%NaCl. |
PDF Full Text Request