| Hypoxic-ischemic encephalopathy(HIE)is a common but severe complication of asphyxia that occurs during the perinatal period with a high rate of morbidity and mortality(Shah,2004;Tsuji,2004).HIE is also the major recognized perinatal cause of neurological morbidity in full-term newborns(Triulzi,2006)and can result in mental impairment, seizures and permanent motor deficits,such as cerebral palsy(Fawke, 2002).At present,there are no effective means of repairing the hypoxic-ischemic brain damage(HIBD)once damage has occurred. Therefore,it is necessary that a suitable therapy for HIBD is developed. Increasing knowledge of the renewal of neural stem cells within the central nervous system(CNS)has shed new light on severe HIBD(Gage, 2000).It was found that NSCs were capable of being activated to proliferation and recruited migration the affected areas.However,the capacity for recruiting endogenous NSCs is limited(Parent 2002;Goings 2004).Thus,other strategies used to modify endogenous neurogenesis after ischemic brain injury have been described(Felling,2006).Hyperbaric oxygen(HBO)treatment is one potential therapy that has been used for the treatment of neonatal diseases including HIE for many years(Rudge,1993;Calvert,2002;Calvert,2003).Recent studies have shown that HBO is neuroprotective by promoting cell proliferation after brain injury(Gutsaeva,2006).We have also found that HBO therapy can protect the NSCs and stimulate cell proliferation in the hippocampal dentate gyrus(DG)areas and subventricular zone(SVZ).These observations have prompted us to ask the following questions:â…°)are the cells that are stimulated by HBO proliferating NSCs?â…±)are proliferating NSCs capable of migrating to the affected areas and differentiating into mature neurons?â…²)is the neuroprotective effect induced by HBO therapy associated with the proliferation of NSCs? If this is the case, perhaps activation of endogenous NSCs(that reside the brain throughout life)could be induced by the application of HBO in HIBD rats.Previous studies have shown that the therapeutic window is associated with the therapeutic effects,and HBO therapy is highly efficient in reducing infarct volume and promoting the recovery of neurobehavioral function within 6 hours after brain damage in an adult model of middle cerebral artery occlusion(MCAO)[4,5].The neonatal brain,however,is in the developmental stage,so it is quite different from the adult brain in both anatomic structure and metabolism.Thus,it is unlikely that the therapeutic window for HBO therapy in the adult can be reliably applied to neonates.Few reports have discussed the therapeutic window of HBO therapy for neonatal HIBD and the 6-hour therapeutic window of HBO therapy as determined from the adult model of MCAO dramatically limits the application of HBO therapy in HIE.Therefore,it would be of value to find the appropriate therapeutic window of HBO therapy for neonatal rats after HIBD.Wnt signaling is implicated in the control of cell growth and differentiation during CNS development(Yu,2006).It can regulate the proliferation and differentiation of NSC,thus promote the development of nerve system.Wnt-3,as the starting protein of Wnt signaling pathway,is closely associated with the proliferation of NSCs(Roelink,1990;Lie, 2005),β-Catenin is an essential component of the canonical Wnt signaling system that controls decisive steps in development and plays an important role in signal transduction(Korinedk,1998;Zechner,2003).Is the proliferation of NSCs is correlated with the Wnt signaling pathway? It will provide theory basis for the application of HBO therapy in HIBD.Partâ… hyperbaric oxygen promoting the proliferation, migration and differentiation in neonatal rats after hypoxie-ischemic brain damageObjective:To examine whether HBO promotes neural stem cells (NSCs)to proliferate and differentiate in perinatal hypoxic-ischemic(HI) rats. Methods:Seven-day-old rat pups were subjected to unilateral carotid artery ligation followed by 2 hours of hypoxia(8%O2).HBO was administered(2 ATA(atmosphaera absolutus),once daily for 7 days) within 3 hours after HI.BrdU/nestin immunofluorescence was examined in the subventricular zone(SVZ)and dentate gyrus(DG).Nestin protein was detected by Western blot analysis at various time points(between 6 hours and 14 days)after HI.BrdU/doublecortin(DCX)immunofluorescence was examined 7 and 14 days after HI.BrdU/β-tubulin, BrdU/GFAP,BrdU/O4 immunofluorescence and myelin basic protein (MBP)immunohistochemistry were examined in the injured brain 28 days after HI.Pathological changes of the brain tissue were also detected 28 days after HI.Results:In HI rats treated with HBO,proliferation of endogenous NSCs was observed in the SVZ and DG.Cell numbers peaked 7 days after HI and proliferating NSCs migrated to the cerebral cortex at 14 d after HI.Twenty-eight days after HI,an increase in newly generated neurons,oligodendrocytes and MBP was observed in the HBO group compared to the untreated and HI-treated rats.Conclusions:This study suggests that HBO treatment may promote neurogenesis of the endogenous NSCs in perinatal HI rats,contributing to repair of the injured brain. Partâ…¡Therapeutic window of hyperbaric oxygen therapy for hypoxic-ischemic brain damage in newborn ratsObjective:We propose the hypothesis that HBO treatment is effective in the treatment of HIBD regardless of when it is started.Methods:Seven-day-old Sprague-Dawley rats underwent left carotid ligation followed by 2 hours of hypoxic stress(8%O2 at 33℃). Hyperbaric oxygen therapy was administered 3,6,12,24,and 72 hours after HIBD.5-bromo-2'-deoxyurindine and 5-bromo-2'- deoxyuridine /nestin were detected by immunofluorescence and nestin was detected by western blotting analysis 10 d after HIBD.T-maze forced alternation,the foot-fault test,and the radial arm maze were conducted at P 22 d,P 30 d, and P 34 d(14 d after HIBD).Thereafter,cerebral morphology was examined by Nissl-staining 28 d after HIBD.Results:There were remarkable increases in the proliferation of neural stem cells in the HBO-treated group,3,6,12,and 24 hours after HIBD,as compared with the HIBD group.The HBO-treated group,3,6, and 12 hours after HIBD,performed better in the behavioral test and had less neural loss in the hippocampal CA1 region as compared with the HIBD group.Conclusions:The therapeutic window for effective HBO treatment could be delayed up to 12 hours after HIBD.The mechanism resulting in the neuroprotective effects of HBO may involve stimulation of endogenous neural stem cells to proliferate and differentiate,which then leads to recovery from brain damage.Partâ…¢Hyperbaric oxygen inducing the neurogenesis after neonatal hypoxic hschemic injury via Wnt signalingObjective:Hyperbaric oxygen(HBO)treatment promotes the proliferation of NSCs in neonatal HI rats.The Wnt signaling pathway was associated with neurogenesis.Thereby this study examined whether HBO treatment after neonatal hypoxic ischemic injury could stimulate the proliferation of NSCs via the Wnt signaling.Methods:Seven-day-old Sprague-Dawley rats underwent left carotid ligation followed by 2 hours of hypoxic stress(8%O2 at 33℃). Hyperbaric oxygen therapy was administered within 3 hrs after hypoxia-ischemia(HI)daily for continuous 7 days.The nestin / Wnt-3 and nestin/β-catenin protein in subventricular zone(SVZ)of the ischemic brain were double-stained by immunofluorescence and analyzed by confocal scanning microscopy at 6 h,24 h,3 d,5 d,7 d,14 d after HI. The whole cell Wnt-3,Nestin,β-catenin and nuclearβ-catenin protein were examined by western-blots at 6 h,24 h,3 d,5 d,7 d,14 d after HI. Linear correlation was used to analyze the correlation between Wnt-3 and nestin protein,β-catenin and nestin protein respectively.Results:In the normal control group,less Wnt-3 protein were observed in the membrane and lessβ-catenin in the cytoplasm of the NSCs.An initial increase of nestin protein was observed after HBO therapy at 24 h after HI and it reached a higher level at 3 d,5 d,7 d after HI and it decreased 14 d after HI.The Wnt-3 protein was observed in the membrane of the NSCs,it got an initial increase at 6 h after HI and it rose to a higher level at 3d,5d and 7 d after HI,then it declined at 14 d after HI.β-catenin protein is observed an initial increase at 24 h after HI as compared with the control group,then the translocation ofβ-catenin to the nuclear was also detected at 3 d after HI and it reached a higher level at 5 d and 7 d after HI.The increase of nestin protein is closely correlated with Wnt-3 andβ-catenin protein(P＜0.05)respectively.Conclusion:The HBO treatment is capable of stimulating the proliferation of NSCs in neonatal rats after HI and the proliferation is correlated with the increase of Wnt-3 protein and the activation ofβ-cateninThe main contents and conclusions of the research were summarized as follows:1.Hyperbaric oxygen therapy could promote the endogenous NSCs to proliferate,migrate and differentiate,thus restore the hypoxic ischemic brain damage. 2.The therapeutic window of HBO therapy for hypoxic ischemic brain damage in neonatal rats could be prolonged to 12 h after HIBD.3.The proliferation of NSCs in neonatal hypoxic ischemic rats after HBO is correlated with the increase of Wnt-3 protein and activation ofβ-catenin,that is,HBO therapy promote the endogenous NSCs to proliferate via the Wnt signaling pathway. |
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