Study On The Role And Mechanism Of Hyperbaric Oxygen In Early Brain Injury After Experimental Subarachnoid Hemorrhage

Aneurysmal Subarachnoid hemorrhage(SAH) is a devastating disease frequently leading to death or neurological deficits in the world, the occurrence of cerebral vasospasm after SAH used to be considered as the most important contributor to high rates of mortality and morbidity, however, a randomized, blinded clinical trial using an endothelin antagonist, clazosentan, has found that clazosentan does not significantly improve mortality or clinical outcome, even though angiographic vasospasm is reversed. This result indicates that other pathophysiological factors, which are independent of angiographic vasospasm, can contribute to the outcome of patients. Up to now, there has been a growing body of evidence indicating that the inflammation accompanying SAH may play an important role in devastating neurological outcomes. In addition, the term "early brain injury" has recently been used to describe the mechanisms of acute neurologic deterioration after SAH, including cell death, cerebral edema, and neuronal dysfunction within the first 72h of the onset. Great evidences for the purpose of highlighting a strong contribution of inflammation to EBI after SAH has been found in animal and clinical studies, so treatment of inflammation within the first 72h has also been considered as a major target in the management of patients surviving cerebral aneurysm rupture.Toll-like receptors (TLRs) are a family of pattern recognition receptors which can detect microbes and trigger a signaling cascade that culminates in the transcription of pro-inflammatory and immunomodulatory factors involved in innate and adaptive immunity. Of the 12 TLRs identified in mouse, toll-like receptors 4 (TLR4) has been demonstrated to play an important role in initiating the inflammation related to stroke, Alzheimer’s disease, Huntington’s disease and Parkinson’s disease. A growing number of studies have shown that the activation of the TLR4 and nuclear factor-kappa B (NF-κB) signaling pathway is involved in inflammatory processes in early brain damage after SAH. It has also been reported that suppression of TLR4/NF-κB signaling pathway can down-regulate the gene expression of many inflammatory mediators, such as tumor necrosis factor-α(TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), intercellular adhesion molecule-1 (ICAM-1), improve neurologic results and alleviate brain injury after SAH. Therefore, TLR4/NF-κB signaling pathway may be a therapeutic target for EBI after SAH.As a nondrug and noninvasive treatment, hyperbaric oxygen (HBO) has been widely used in clinical practice for many diseases and has led to satisfactory outcomes, especially in the areas of acute carbon monoxide poisoning, cerebral ischemia, decompression sickness. Even though investigators have demonstrated that HBO can be used as an adjunct treatment of SAH to counter cerebral vasospasm because of its potent anti-inflammatory and anti-oxidant properties, the evidence of physiologic and molecular mechanisms and efficacy of HBO therapy in reducing inflammatory cytokines in the early brain injury period after SAH is still limited.Recently, a series of studies has shown that, by interfering with the TLR/NF-κB signaling pathway, HBO therapy can attenuate inflammation and exert a protective effect in experimental animal models of spinal cord injury (SCI) and multiple organ failures. These provide a brilliant prospect for the further study of HBO on treatment of EBI and its anti-inflammation mechanism after SAH. The experimental SAH model was established according to the prechiasmatic injection method, and the present study attempted to provide a novel mechanism that HBO could inhibit the activation of TLR4 induced by SAH and suppress TLR4/NF-κB signaling pathway.Part 1 Neuroprotective effect of hyperbaric oxygen in experimental Subarachnoid hemorrhage model of early brain injuryObjective:we established an animal model that can effectively simulate the early brain injury induced by SAH, for providing reliable experimental conditions to study the neuroprotective effect of hyperbaric oxygen in experimental SAH model.Methods:A total of 150 Sprague-Dawley male rats were randomly assigned to 5 groups (n=30 for each group):(1) the sham group (SH); (2) the SH+2.8 atmospheres absolute (ATA) HBO group; (3) the SAH group; (4) the SAH+2.0ATA HBO group; (5) the SAH+2.8ATA HBO group. The experimental Subarachnoid hemorrhage model of early brain injury was established according to the prechiasmatic injection method. After flushing CO2 from a small hyperbaric chamber with 100% 02, rats in the SH +2.8ATA HBO and SAH+2.8ATA HBO groups were placed inside for a 90min exposure to HBO (100% 02) at 2.8ATA. The chamber pressure was increased to 2.8ATA in 15 min and then gradually decreased to normal pressure (20% 02) in 15 mins at the end of the session. In the SAH+2.0ATA HBO group, the animals were treated with 2.0ATAHBO using the same method. Animals in each of these three groups received five sessions of HBO treatment in total, which started 12 h after SAH induction and was given once daily in the first day and twice on the second and third day. At the planed time point, the animals were anesthetized and decapitated for different assays. In each group,5 rats were manipulated for detecting brain water content, and five were used for blood-brain barrier (BBB) permeability. In order to check the effects of HBO treatment on neurological deficit after SAH,15 of the rats in each group were subdivided into three subgroups (n= 5 per group) corresponding to the following post-operative time points:24h,48h and 72h to evaluate Neurological functions.Results:Compared with SAH group, the mortality of SAH+2.0ATA group and SAH+2.8ATA group at 24h after SAH significantly increased, and the mortality of SAH+2.8ATA was higher than that in SAH+2.0ATA, even though there was no statistical difference. Neurologic scores in the SAH group were significantly lower than that in the sham group at different time points (P<0.01), while Treatment with HBO both at 2.8ATA and 2.0ATA dramatically raised neurological scores compared with the SAH group at 24h,48h and 72h (P<0.05) even though the point level calculated in these two groups was significantly lower than the control value of sham group (P< 0.05). A significant increase in water content was detected in the cerebral hemispheres at 48h after SAH (P<0.01). However, no difference in water content in cerebellum or brain stem was noted (P>0.05). The mean value of brain water content in cerebral was evidently decreased by HBO administration of both 2.0ATA and 2.8ATA as compared with SAH group (P<0.05). As compared to animals of the SH group, rats in SAH group demonstrated a significant increase in the amount of Evans blue dye extravasation at 48h (P<0.01) and the levels of extravasated dye were significantly reduced by 2.0ATA and 2.8ATA HBO treatment compared to SAH group (P< 0.05).There was no significant difference in any measured parameters between the SH and the SH+2.8ATA HBO groups (P>0.05). Although there was no significant difference in brain water content and neurologic score between the SAH+2.0ATA HBO and the SAH+2.8ATA HBO groups (P>0.05), high dose of HBO administration have led to amelioration in BBB permeability with statistical differences at 48h after SAH (P<0.05), indicating that there may be a reduced BBB opening in response to the high dosage of HBO treatment.Conclusion:1. This study confirmed that the rat prechiasmatic injection model could lead to obvious neurological dysfunction, cerebral edema and elevation of BBB permeability at 48h after SAH, and this model could be used as an ideal animal model in this study to investigate the changes of early brain injury after SAH.2. When rats of SAH were early intervened by HBO, it may increase the mortality rate of rats within 24 hours, but it would improve neurological dysfunction after 24 hours. HBO reduced cerebral edema and elevation of BBB permeability at 48h after SAH, and the effect of treatment for reducing BBB permeability in SAH+2.8ATA group is better than that in SAH+2.0ATA groupPart 2 The influence of hyperbaric oxygen in TLR4/NF-κB signaling pathway after experimental subarachnoid hemorrhage in ratsObjective:This study was undertaken to investigate the influence of HBO administration on the TLR4/NF-κB signaling pathway and the expression of cytokines in cortical specimen of rats at the early stage of SAH.Methods:The group of experimental animal, modeling and hyperbaric oxygen treatment were the same with the former part. In each group, fifteen rats were taken for molecular biological and biochemical experiments, and another five rats were used for immunohistochemistry studies. The time of sacrifice was determined according to the different parameters measured:Real time polymerase chain reaction (RT-PCR) analysis:48h after SAH; Enzyme-linked immunosorbent assay (ELISA):48h after SAH; Western blot analysis:24h,48h and 72h after SAH; Immunohistochemistry:48h after SAH.Results:Western blot showed that TLR-4 and NF-κB were expressed at a low level in cortex in the SH and SH+2.8ATA groups. The protein expression did not differ significantly between these two groups(P> 0.05). However, the protein levels of TLR-4 and NF-κB were increased significantly in rats subjected to SAH (P<0.01). Moreover, TLR-4 and NF-κB protein expression reached peak values at 48h after SAH. Treatment with HBO decreased these cytokine levels after SAH at 24h,48h and 72h when compared with those of SAH group,The mRNA levels of TLR-4 and NF-κB were significantly elevated after SAH when compared with the sham group. Treatment with HBO led to a significant decrease in the mRNA level of TLR-4 and NF-κB (P<0.05) at 48h after SAH. Although these data showed that the average mRNA and protein levels were still higher in dosage of 2.0ATA HBO administration, there was no significant statistical difference in any measured parameters between the SAH+2.0ATA HBO and SAH+2.8ATA HBO treatment groups (P>0.05). ICAM-1 activity was inhibited by HBO administration groups (P<0.05), and 2.8ATA HBO administration could not inhibit the activity of ICAM-1 obviously by comparison with that of the SAH+2.0ATA HBO group (P> 0.05). The concentrations of mediators were present at low levels in the sham groups, and the expression of TNF-α, IL-1β and IL-6 did not differ significantly between the sham and sham+2.8ATA HBO groups (P> 0.05). The levels of the three pro-inflammatory cytokines in cortical were dramatically increased after SAH at 48h (P< 0.01). However, the expression of mediators in rats subjected to SAH were attenuated significantly after treatment of HBO both at the dosages of 2.0ATA and 2.8ATA (P< 0.05). The dosage of 2.8ATA HBO administration to rats of SAH could not suppress the expression of TNF-α, IL-1β and IL-6 obviously than those in the SAH+2.0ATA HBO group (P>0.05). The results of the immunohistochemistry experiment revealed a significant increase of TLR4 and NF-κB positive cells in the cortex of the SAH group (P<0.01). These proteins’immunoreactivity was mainly observed in neurons and a little in glia cells. Similar to the Western blot, HBO administration (both 2.0ATA and 2.8 ATA) reduced the number of TLR4 and NF-κB positive cells remarkably in the cortical of rats at 48h after blood injection (P< 0.05), but the 2.8ATA HBO administration group could not suppress the number of positive cells obviously more than the SAH+2.0ATA HBO group (P>0.05).Conclusion:1. This study has shown that the activation of the TLR4/NF-κB signaling pathway and downstream inflammatory cytokines are involved in inflammatory processes in early brain injury after SAH.2. Our study showed that treatment with HBO significantly decreased the expressions of TLR4, NF-κB and the downstream inflammatory agents, such as TNF-α, IL-6, IL-1β and ICAM-1, indicating that HBO treatment may result in abatement of the development of EBI after SAH, possibly through suppression of TLR4/NF-κB signaling pathway.3. The effect of treatment for reducing expression of TLR4/NF-κB signaling pathway in SAH+2.8ATA group is not better than that in SAH+2.0ATA group.