BackgroundHypertonic saline (HS) has been successfully used clinically for treatment of various forms of cerebral edema. Up-regulated expression of Na-K-Cl Cotransporter 1 (NKCC1) and inflammatory mediators such as tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β) has been demonstrated to be closely associated with the pathogenesis of cerebral edema resulting from a variety of brain injuries. This study aimed to explore if alleviation of cerebral edema by 10% HS might be effected through down-regulation of inflammatory mediator expression in the microglia, and thus result in decreased NKCC1 expression in astrocytes in the cerebral cortex bordering the ischemic core.MethodsThe Sprague-Dawley (SD) rats that underwent right-sided middle cerebral artery occlusion (MCAO) were used for assessment of NKCC1, TNF-α and IL-1β expression using Western blotting, double immunofluorescence and real time RT-PCR, and the model also was used for evaluation of brain water content (BWC) and infarct size. SB203580 and SP600125, specific inhibitors of the p38 and JNK signaling pathways, were used to treat primary microglia cultures to determine whether the two signaling pathways were required for the inhibition of HS on microglia expressing and secreting TNF-α and IL-1β using Western blotting, double immunofluorescence and enzyme-linked immunosorbent assay (ELISA). The effect of TNF-α and IL-1β on NKCC1 expression in primary astrocyte cultures was determined. In addition, the direct inhibitory effect of HS on NKCC1 expression in primary astrocytes was also investigated by Western blotting, double immunofluorescence and real time RT-PCR.ResultsBWC and infarct size decreased significantly after 10% HS treatment. TNF-α and IL-1β immunoexpression in microglia was noticeably decreased. Concomitantly, NKCC1 expression in astrocytes was down-regulated. TNF-α and IL-1β released from the primary microglia subjected to hypoxic exposure and treatment with 100 mM HS were decreased. NKCC1 expression in primary astrocytes was concurrently and progressively down-regulated with decreasing concentration of exogenous TNF-α and IL-1β. Additionally, 100 mM HS directly inhibited NKCC1 up-regulation in astrocytes under hypoxic condition.ConclusionsThe results suggest that 10% HS alleviates cerebral edema through inhibition of the NKCC1 Cotransporter, which is mediated by attenuation of TNF-α and IL-1β stimulation on NKCC1.

Hypoxic exposure in the perinatal period causes periventricular white matter damage (PWMD), a condition associated with myelination abnormalities. Under hypoxic conditions, glial cells were activated and released a large number of inflammatory mediators in the PWM in neonatal brain, which may result in oligodendrocyte (OL) loss and axonal injury. This study aims to determine if astrocytes are activated and generate proinflammatory cytokines that may be coupled with the oligodendroglial loss and hypomyelination observed in hypoxic PWMD. Twenty-four 1-day-old Wistar rats were exposed to hypoxia for 2 h. The rats were then allowed to recover under normoxic conditions for 7 or 28 days before being killed. Another group of 24 rats kept outside the chamber was used as age-matched controls. Upregulated expression of TNF-α and IL-1β was observed in astrocytes in the PWM of P7 hypoxic rats by double immunofluorescence, western blotting and real time RT-PCR. This was linked to apoptosis and enhanced expression of TNF-R1 and IL-1R1 in APC+ OLs. PLP expression was decreased significantly in the PWM of P28d hypoxic rats. The proportion of myelinated axons was markedly reduced by electron microscopy (EM) and the average g-ratios were higher in P28d hypoxic rats. Upregulated expression of TNF-α and IL-1β in primary cultured astrocytes as well as their corresponding receptors in primary culture APC+ oligodendrocytes were detected under hypoxic conditions. Our results suggest that following a hypoxic insult, astrocytes in the PWM of neonatal rats produce inflammatory cytokines such as TNF-α and IL-1β, which induce apoptosis of OLs via their corresponding receptors associated with them. This results in hypomyelination in the PWM of hypoxic rats.

Background: Increased permeability of pulmonary capillary is a common consequence of sepsis that leads to acute lung injury. In this connection, ulinastatin, a urinary trypsin inhibitor (UTI), is used clinically to mitigate pulmonary edema caused by sepsis. However, the underlying mechanism of UTI in alleviating sepsis-associated pulmonary edema remains to be fully elucidated. As tight junctions (TJs) between the pulmonary microvascular endothelial cells (PMVECs) play a pivotal role in the permeability of pulmonary capillary, this study investigated the effect of UTI on expression of junctional proteins in PMVECs during sepsis.Methods: Male adult Sprague Dawley rats were subjected to cecal ligation and puncture (CLP) and divided into sham, CLP, and UTI+CLP groups. UTI was administered every 8 h for 3 days before CLP. At 48 h after surgery, Evans blue (EB) was administered to evaluate the pulmonary vascular leakage. Histological staining was used for evaluation of lung injury score. Using immunofluorescence staining and Western blot, the expression of junctional proteins (occludin, claudin-5, and ZO-1) in pulmonary endothelia was assessed. In vitro, PMVECs were divided into control, lipopolysaccharide (LPS), and UTI+LPS groups for examination of expression of junctional proteins and TNF-α as well as inhibitor of NF-κB (IκB), p38 mitogen-activated protein kinases (p38 MAPKs), c-Jun N-terminal kinases (JNKs), and extracellular signal-regulated kinases (ERKs) signaling pathways. Additionally, the expression of various junctional proteins was determined in PMVECs of control, LPS, and TNF-α receptor antagonist-LPS groups. PMVECs were also treated with TNF-α and TNF-α receptor antagonist and the expression of various junctional proteins was assessed.Results: Compared with the CLP group, UTI markedly decreased EB leakage and lung injury score. The expression of occludin, claudin-5, and ZO-1 was decreased in both CLP rats and LPS-treated PMVECs, but it was reversed by UTI and TNF-α receptor Ulinastatin Protects Septic Pulmonary Endothelia antagonist. TNF-α expression was vigorously elevated in the lung of CLP rats and in LPS-challenged PMVECs, which were suppressed by UTI. In addition, TNF-α also reduced occludin, claudin-5, and ZO-1 expression in PMVECs, but these effects of TNF-α were antagonized by pretreatment with TNF-α receptor antagonist. Furthermore, UTI inhibited LPS-induced activation of NF-κB and mitogen-activated protein kinases (MAPKs) pathways in PMVECs.Conclusion: UTI effectively protects TJs and helps to attenuate the permeability of pulmonary capillary endothelial cells during sepsis through inhibiting NF-κB and MAPKs signal pathways and TNF-α expression.