The Role And Mechanism Of Nerve Adhesion Molecule NB-3 In The Development Of High Altitude Cerebral Edema

Our country is the one with the largest plateau,longest border line and most resident in high altitude.However,environment in high altitude is rather special,which is characterized with decreased oxygen content,low temperature,decreased humidity,increased temperature difference and strong ultraviolet radiation.Of these factors,hypoxia is regarded as the primary cause of high altitude related illness.Brain is the most sensitive organ to hypoxia and shows the least resistant to hypoxia.When oxygen supply is in shortage,metabolism,function and morphology of the brain gets changed.Sometimes cerebral edema and cerebral hemia could be induced.Brain injury induced by hypoxia is a dynamic,multi-factor inter-active,progressive process involving endothelial cell injury,vasogenic edema and cytotoxic edema.Hypoxia is accompanied with dysfunction of gastrointestine,inflammation and increased risk of infection.Research shows that mountaineers with inflammation-inducing disease in high altitude were more vulnerable to acute mountain sickness(AMS),and the lake louise score is positive correlated to the frequency of infectious symptoms.It is also reported that pro-inflammation cytokines in the plasma of AMS patients got up-regulated.However,relationship of inflammation and high altitude cerebral edema are seldom investigated.Blood-brain barrier(BBB)lays key role in maintaining the stability of the micro-environment of central nervous system.BBB consists of endothelial cell,pericyte,endfoot of astrocyte and basement membrane.The barrier function is mainly maintained by specialized endothelial cells.These specialized endothelial cells are tightly connected together by tight junction proteins,thus to inhibit molecules to cross the barrier through the intercellular pathway.What's more,these specialized endothelial cells express a series of specific transporters and have an extreme low rate of endocytosis and pinocytosis,thus to limit molecules crossing the barrier through the transcellular pathway.Meanwhile,pericyte,astrocyte and vascular basement membrane crosstalk with these specialized endothelial cells through various pathways in the formation and maintenance of BBB.Cell adhesion molecule plays key role in mediating adhesion and interactions between various cells and extracellular matrix.NB-3 is a memberof immunoglobulin superfamily.Through homophilic and heterophilic interactions,NB-3 plays roles inneural stem cell differention,projection of axons,neuron survival and apoptosis,axon regeneration and so on.What's more,NB-3 also plays roles in autism,attention deficit hyperactivity disorder,intellectual disability,schizophrenia,depression,anxiety,apositia and bipolar disorder.Previous investigations of our laboratory showed that loss of NB-3 aggravated brain injury induced by cerebral ischemia.At the same time,NB-3 knock-out mice showed hypoxia intolerance.So we focused on the function and mechanism of NB-3 on high altitude cerebral edema in this study.PurposeMore and more research and clinic investigations show that inflammation plays key role in AMS and HAPE.While the relationship of inflammation and HACE is seldom investigated.So we investigated the function of inflammation in the occurrence of HACE.The core event of HACE is the disruption of BBB,which is a multi-cellular structure.Cell adhesion molecules play key roles in mediating interactions between cells and extracellular matrix.It is reported that NB-3 play important roles in neural injury,especially in hypoxic injury.Then we wonder if NB-3also play important roles in HACE,and investigate the probable underlying mechanism.ContentsThis work contains two parts: firstly,we investigated how inflammation affects the occurrence of HACE considering the role of inflammation plays in AMS.Secondly,based on the mouse model of HACE,we investigated the role NB-3 plays in HACE and the mechanism how NB-3 affects the progression of HACE.Specific research diagram is shown as follows:Hypoxia augments LPS induced inflammation and triggers HACE in mice Loss of NB-3 aggravates the progression of HACE MethodsBrain water content were evaluated with dry-wet weight method and magnetic resonance imaging(MRI);BBB permeability were evaluated by checking evens blue and Ig G leakage;specific gene transcription were evaluated by real-time PCR;specific cytokine content in serum were evaluated by ELISA;withimmunofluorescence,activation of microglia and astocyte,location of NB-3,translocation of tight junctions and capillary expansion were evaluated;blood supply were evaluated by laser doppler;specific protein expression were detected by western blot;ultrastructure were observed by transmission electron microscope(TEM);morphological changes were evaluated by HE staining and Nissl staining and Thy-1-YFP transgene mice,cognitive and motor function were evaluated by morris water maze and rota-rod test.ResultsPart one: hypoxia augments LPS induced inflammation and induces HACE1.Hypobaric hypoxia up-regulated the expression of pro-inflammatory cytokines both in serum and brain parenchyma.Mice were subjected to hypobaric hypoxia exposure for different duration.As time of exposure prolongs,IL-1? and IL-6 got up-regulated in serum.TNF-? in brain parenchyma got up-regulated after 12 hour hypoxia exposure.After 24 hours exposure of hypoxia,IL-1?,IL-6 and TNF-? got up-regulated in brain parenchyma.2.Hypobaric hypoxia augmented LPS induced inflammation and induced cerebral edema.Mice were subjected to LPS injection of different concentrations.LPS of low concentration(<5 mg/kg)had no influence on brain water content.However,when these mice were subjected to hypoxia exposure thereafter,cerebral edema was induced.Investigation to the content of pro-inflammatory cytokines in serum and brain parenchyma showed that hypoxia exposure significantly augmented LPS-induced IL-1?,IL-6 and TNF-? up-regulation.3.Combination of hypobaric hypoxia and LPS injection induced activation of microglia and HIF signaling pathway.Activation of microglia and astrocyte were evaluated.We found that LPS injection induced microglia activation with no effect on astrocyte.At the same time,HIF-1?,HIF-2? and VEGF up-regulated significantly in group treated with hypobaric hypoxia exposure and LPS injection.4.Combination of hypobaric hypoxia and LPS injection induced BBB leakage.Permeability of BBB showed no change in group treated with hypobaric hypoxia or LPS injection.When these two treatments were combined,BBB leakage was induced.TEM results showed tight junction opening;Western blot results showed down-regulation of Occludin and VE-Cadherin.5.Combination of hypobaric hypoxia and LPS injection induced neural injury.No injury was induced in group treated with hypobaric hypoxia or LPS injection.When these two treatments were combined,lightly stained cytoplasma and darkly stained cell nucleus were observed in HE staining sections;Nissl bodies became less and lightly stained in Nissl staining sections;projection neurons became less and axons got shrunk in thy-1-YFP transgene mice.6.Combination of hypobaric hypoxia and LPS injection induced cognitive and motor dysfunction.Morris water maze and Rota-rod test were applied to evaluate the cognitive and motor function.No dysfunction was observed in group treated with hypobaric hypoxia exposure or LPS injection.When these two treatments were combined,latency to find the platform prolonged;platform entries,pathlength and time in the target quadrant decreased;latency and rotation mice falling off the rolling rod decreased significantly.Part two: Loss of NB-3 aggravates the progression of HACE1.Neural adhesion molecule NB-3 expresses in CNS and located in neuron.Protein of different organs were extracted.Western blot results showed that NB-3expressed in brain with different abundance.Immunofluorescence results showed that NB-3 located in neuron but not in endothelia cell,pericyte,microglia and astrocyte.2.Hypobaric hypoxia decreases the expression of NB-3.Mice were subjected to hypoxia exposure for different duration,western blot results showed that hypoxia exposure induced down-regulation of NB-3.As hypoxia exposure prolonged,down-regulation of NB-3 got more distinct.3.Loss of NB-3 aggravated the progression of HACE.When HACE was induced,compared with WT mice,brain water content increased more severe in NB-3 knock-out mice.Disruption of BBB was more severe in NB-3 knock-out mice.More evens blue and Ig G leaked from blood into brain parenchyma.4.Loss of NB-3 aggravated systemic and neural inflammation in HACE.Pro-inflammatory cytokines in serum and brain parenchyma were evaluated.When HACE was induced,compared with WT mice,IL-1?,IL-6,TNF-? up-regulated more evident in NB-3 knock-out mice in serum and brain parenchyma.Activation of microglia was more universal in NB-3 knock-out mice when HACE was induced.5.Loss of NB-3 aggravated the opening of BBB in HACE.Opening of tight junction,basement membrane degradation,mitochondria swelling were more severe in NB-3 knock-out mice when HACE was induced.Western blot results showed severer down-regulation of Occludin and Claudin-5.Immunofluorescence results showed severer translocation of Occludin and Claudin-5.6.Loss of NB-3 aggravated blood vessel expansion in HACEWe labeled blood vessels and capillaries and measured the diameter of these blood vessels and capillaries.Results showed that expansion of both blood vessels and capillaries were more severer in NB-3 knock-out mice when HACE was induced.Blood supply increased significantly in NB-3 knock-out mice when HACE was induced.7.Loss of NB-3 aggravated neural injury in HACE.When HACE was induced,lightly stained cytoplasma and darkly stained nucleus were more universal in NB-3 knock-out mice in HE staining sections;decrease of nissl bodies was more severe,nissl bodies became more light-stained in NB-3knock-out mice in Nissl staining sections;loss of projection neurons and axon shrunk became more severer in Thy-1-YFP mice8.Loss of NB-3 aggravated cognitive and motor dysfunction in HACE.When HACE was induced,latency to the platform prolonged more significantly,platform entries,pathlength and time in target quadrant decreased more significantly,latency and rotation mice falling off the rolling rod decreased more significantly in NB-3 knock-out mice.9.NB-3 regulated BBB permeability via VEGF signaling pathway in HACE.When HACE was induced,NB-3 was induced to express in the end-foot of astrocyte.Expression and activation of MMP-9 and VEGF showed no difference between WT and NB-3 knock-out mice.However,down-regulation of VEGFR2 was more significantly in WT mice compared with NB-3 knock-out mice.At the same time,Notch1 signaling pathway was activated in WT mice but not in NB-3 knock-out mice.ConclusionIn our experiments,we proved that high altitude hypobarbic hypoxia could augment LPS induced inflammation,thus to increase BBB permeability and induce neural injury,and result in the occurrence of HACE eventually.NB-3 got down-regulated when HACE evolved.Loss of NB-3 aggravated a series of pathophysiologic changes including severer inflammation,increased hydrostatic pressure,severer BBB disruption and severer cognitive and motor dysfunction.With these results,we get more understanding to the occurrence and progression of HACE.At the same time,new insights into prevention and treatment to HACE are offered.