Notch1Mediates Postnatal Neurogenesis In Hippocampus Enhanced By Intermittent Hypoxia

Intermittent hypoxia (IH) is generally defined as repeated episodes of hypoxiainterspersed with normoxic periods. It has been found that chronic and moderatehypoxia exposure are able to promote the tolerance to hypoxia, and have preventive ortherapeutic effects on various diseases. In addition, IH can reduce the injury of centralnervous system, prevent or cure a variaty of nervous diseases, enhance the functionsof central nervous system, and promote the expression of Spine-associatedRap-specific GTPase (SPAR) and the ability of learning and memory in mice.Active adult neurogenesis is spatially restricted under normal conditions to twospecific neurogenic brain regions, the subgranular zone (SGZ) in the dentate gyrusof the hippocampus and the subventricular zone (SVZ) of the lateral ventricles. Onehallmark of adult neurogenesis is its sensitivity to physiological and pathologicalstimuli at almost every stage, from proliferation of neural precursors to development,maturation, integration, and survival of newborn neurons. Physiological stimuliinclude learning and physical exercise; Phathological simuli include stroke andepilepsy.It has been found that there is a close relationship between hypoxia andneurogenesis. At first, embryonic and adult neurogenesis take place under moderatehypoxia. Secondly, moderate hypoxia exists during the process of neurogenesisinduced by various physiological and pathological stimuli. Thirdly, in vitro studiesshow that moderate hypoxia can promote the NSCs proliferation and differentiation todopaminergic neurons.Notch proteins are cell surface transmembrane receptors that mediate multipleimportant cellular functions through cell–cell interactions, including organogenesis,self-renewal of stem cells, decisions of cell fate, differentiation and death. In theprocess of neural development, Notch1maintains the undifferentiated state of NSCsand regulates the NSCs self-renewal and differentiation. Recent studies showed that Notch1mediated the maintenance of undifferentiated state of NSCs and signalingtransduction in hypoxia. In vitro studies showed that hypoxia inhibits thedifferentiation of NSCs and musle stem cells through Notch1pathway, and activatesthe transcription of Notch1pathway and expression of Notch1target genes. Inaddition, there is crosstalk between Notch1pathway and HIF-1pathway which isgenerally accepted as the canonical pathway stimulates by hypoxia: HIF-1α andNotch ICD form a point of convergence between the two signaling mechanisms,leading to stabilization of Notch ICD, recruitment of HIF-1α to Notch-responsivepromoters, and activation of Notch downstream genes (Hey and Hes). These studiesimplied that Notch1may play a significant role in various biological processesinduced by hypoxia.Our objectives include3aspects: to investigate whether hypoxic environmentcould induce hypoxic niche in the brain of alive animals; to determine whether IHcould promote hippocampal neurogenesis, including proliferation and differentiationof NSCs, survival and migration of newborn neurons, and the development ofdendrite spines; to detect whether Notch1is involved in the neurogenesis promoted b yIH.1. PO2measure ment in brain of alive animals: At first, we detected the spatialdistribution of PO2in Brain. In the experiments, we chose two lines across the coronalslides of brain: A line (AP,0mm; LR,1.3mm; D,0.0～-9.0mm) and B line(AP,3.6mm; LR,2.0mm; D,0.0～-6.0mm). Before the measurement, we drilled one holeat the starting point of A line (AP,0mm; LR,1.3mm; D,0.0mm) and B line (AP,3.6mm; LR,2.0mm; D,0.0mm). Then, implant the oxygen probe into the brain a longA line or B line. The interval between each measurement point is0.1mm. The maps ofPO2distribution in brain is drawn according to the data. Next, we detected thedynamic changes of PO2in various cerebral areas, including cortex, lateral ventricles,hippocampus, and the third ventricles, thalamus and striatum. In the lateral ventricles,we continuously monitored the PO2for1h. In the other areas, we monitored for10-13min. The respiratory rate was recorded every10min. At last, we measured PO2in thebrain of alive animals on plateau. In order to detect the effect of hypoxia on the PO2inbrain, we selected various cerebral area, including cortex, lateral ventricle, and DG ofhippocampus, to monitor the changes of cerebral PO2in hypoxic environment. In theexperiment, we tested the cerebral PO2at sea level,2000m and3000m altitude.We found that the distribution ofPO2in various cerebral regions were not only spatially heterogeneous, but also temporally heterogeneous-----the trends of PO2dynamic changes were different in different cerebral regions. The PO2levels inparenchyma are stable and maintain at2mmHg, while PO2levels in ventricles areabout45-50mmHg and in a dynamic state. In addition, PO2levels in DG ofhippocampus are dynamic and about6-8mmHg. In addition, It was shown that PO2inventricles and DG of hippocampus decreased with the reduced PO2in environment,which indicated that low PO2level in environment can induce the hypoxic niche inbrain. However, PO2in cortex can not change with the PO2in environment.2. Detection of the neurogenesis in hippocampus after IH and the effect ofNotch1: The WT and Notch1+/-mice2days after the birth were treated with IH(2000m,4h/day for4weeks). In the detection of NSCs proliferation, the mice wereinjected with Brdu (100mg/kg once,6times,2h intervals)0or14days2h after IHtreatment, and were perfused immediately2h after the last Brdu injection. In thedetection of differentiation and migration of newborn neurons, mice were injectedwith Brdu (50mg/kg once per day for3days)0,14and28days after IH treatment,and perfused after the last Brdu injection. In the detection of the development of spine,Nothch1+/-mouse were mated with Thy1positive mice to acquire WT/Thy1positiveand Notch1+/-/Thy1positive mouse. Then, the above two genenotypes were treatedwith IH and perfused. The brains of above perfused mouse were acquired and used forfrozen sections and immunohistochemisty. Microscopy images were taken andanalyzed with using Image-Pro-Plus Image Processing and Analysis Software.The results showed the phenomena below. Firstly, IH promotes the NSCsproliferation in DG of hippocampus, which can continue for at least2weeks. Next,IH is able to increase the number of newborn neurons in DG, but does not affect thedifferentiation of NSCs. Thirdly, the migration of newborn neurons in DG is enhancedby IH. At last, IH prolongs the length of apical and basal dentritic spines and enhancesthe density of apical dentritic spines, but does not affect the density of basal dentriticspines. However, the above effects of IH on the neurogenesis in DG are all inhibitedin Notch1+/-mice, which indicated that Notch1is involved in the enhancedneurogenesis in DG of hippocampus.3. The detection of Notch1pathway after IH: WT and Notch1+/-mice weretreated with IH (2000m altidude for4h per day,4weeks). Total protein inhippocampus was extracted and used for Western Blot. The expression of NICD,Hes1and Hes5were detected in WT and Notch1+/-mouse after Normoxia or IH treatment. Data were analyzed with ImageJ software.It was shown that Notch1-Hes1pathway is activated by IH in WT mice, insteadof that in Notch1+/-mice. The above molecular mechanism might explain theenhanced neurogenesis by IH in WT mice, but not in Notch1+/-mice. In addition,results indicated that Hes5may be not involved in neurogenesis in DG promoted byIH.In conclusion,1. Hypoxia in vitro reduces the PO2in vivo and induces thehypoxic niches in brain of alive animals.2. Intermittent hypoxia (IH) enhances theneurogenesis in DG of hippocampus.3.Notch1is involved in the IH-enhancedneurogenesis in hippocampus at multiple stages.4. IH can activate the Notch1signaling pathway.It has been found that postnatal neurogenesis contributes to olfaction, learning,memory, and mood regulation. The present study provides a novel method to inducepostnatal neurogenesis at multiple stages, which may ultimately lead to newtherapeutic strategies to enhance functional neurogenesis for treatment of mentaldisorders, degenerative neurological disorders, and injury repair. With the development of railway and air transport, there are millions of peopletraveling into the plateau region at present. However, the recent studies have found thatmore than half of people, who ascend to5,000m, will develop acute mountain sickness(AMS). The Lake Louise Consensus Group defi ned AMS as the presence of a headachein an unacclimatised person who has recently arrived at an altitude above2500m plus thepresence of one or more of the following symptoms: gastrointestinal disorders (such as,anorexia, nausea, or vomiting); insomnia; dizziness; lassitude; or fatigue. HACE isdiagnosed clinically in individuals who have recently arrived at high altitude, most ofwhom will have symptoms of AMS or HAPE. HACE is characterised by varying degreesof confusion, ataxia of gait, disturbances of consciousness, and psychiatric changes thatcan progress to deep coma. Many chemical materials are involved in the development ofHACE, including nitric oxide (NO), adenosine, oxygen radicals, VEGF and so on. All theabove chemicals will induce the damages of the vascular basement and the vasogenicendema. The treatments of AMS and HACE includes fast decrease of the altitude, oxygensupply, administration of dexamethasone and acetazolamide, and hyperbaric chambertreatment.Heat strock proteins (HSPs) are the products of highly conservative genes, and canbe induced by series of stresses. HSPs include various families. HSP70is the mostobviously increased protein in the heat stress, the mechanisms of which is most clearlyunderstood. HSP70is a kind of molecular chaperones, able to stabilize the proteinconformation and assist to protein folding, transportation, and release. In addition, HSP70can maintain the cellular homeostasis and resist the apoptosis. Recently, studies havefound that hypoxia can induce the expression of HSP70. It is also increasingly concernedthat HSP70is able to act as a marker of hypoxic stress. The most important one is that the induction of HSP70could protect various organs from injuries caused byischemia/hypoxia. Studies have found that HSP70could reduce the damages of lungs andlivers induced by hypoxia, and preserve the brain and heart from the ischemic injuries.Geranylgeranylacetone (GGA) is a kind of retinoid materials, and induces theexpression of HSP70in various organs. Studies have shown that GGA is able to reducethe injuries of organs in digestive system, including atrophic gastritis, pancreatitis,oxidative injuries of colonic epithelial cells. In addition, GGA can induce HSP70toprotect chondrocyte, retina, cochlea, lungs, kidneys and cardiac muscle cells from stressinjuries. Recent studies have found that GGA also can reduce the injuries caused byischemia/hypoxia in central nervous system. Researchers administrated GGA1h beforethe middle cerebral artery occlusion (MACO), and found that GGA could obviouslyinduce the expression of HSP70in neurons and glias in the border of infarction zone, andreduce the infarction zone of brain.Objectives1. The first objective is to confirm the protective effects of GGA on acute hypoxicinjuries and cerebral damages, and to search the novel drugs preventing acute hypoxicinjuries.2. The second objective is to identify the mechanism of prevention of injuries andcerebral damages induced by acute hypoxia after administration of GGA, and to providenovel idea to prevent injuries caused by acute hypoxia in efficient and unharmful way.Methods1. The measurement of survival rate and time in lethally acute hypoxia:GGA as an emulsion with0.0056%α-tocopherol and2%gum Arabic was givenintraperitoneally at a dose of1,000mg/kg. The dose volume was10mL/kg. Mice in thecontrol group were given the same dose of vehicle (2%gum Arabic in0.0056%α-tocopherol). In the studies of lethally acute hypoxic tolerance, mice in GGA or controlgroup were administered with1,000mg/kg GGA or vehicle intraperitoneally1h beforethe onset of the hypoxic exposure. Mice were acclimatized in the chambers (model:DYC-DWI; Guizhou Fenglei, China) in room air for30min to1h before experiments.The altitude of10,000m at a velocity of approximately50m/s is selected for lethallyacute hypoxia, under which untreated mice died in a relatively uniform time period.Survival under lethally acute hypobaric hypoxia was examined in control mice and mice pretreated with GGA (1,000mg/kg). The above two groups were then exposed tosimulated acute hypoxia of10,000m at a velocity of50m/s for15min. The time ofdeath was measured after achieving the altitude and was defined as the time of the lastbreath by an investigator who was blindfolded. The survival rate was defined as the ratio(the number of surviving mice/the number of total mice).2. The pathological detection of cerebral injuries caused by sublethally acute hypoxia:The altitude of8,300m at a velocity of approximately10–20m/s was selected forsublethally acute hypobaric hypoxia, which, when administered via either anenvironmental chamber or a ventilator, was severe enough to induce typical hypoxicorgan injuries yet allowed the mice to survive so that hypoxia-induced pathologicalchanges could be analyzed. After the exposure to sublethally acute hypoxia for6h,animals were immediately anesthetized and were then perfused. The whole brain wasremoved and postfixed. For histological analysis with Nissl s staining, consecutivecoronal sections were prepared using frozen sectioning technique (model: E, Thermo,USA). The cortex above the hippocampus and dorsal hippocampus were examined, andneural damage was evaluated in each hemisphere.3. The detection of HSP70in brain before and after the sublethally acute hypoxicexposure:After exposure to1,7, or1h normoxia+6h sublethally acute hypoxia, animalswere immediately anesthetized. The cortex and hippocampus were removed and groundto extract the total protein. The protein (100μg) was subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis and electroblotted onto nitrocellulose membrane. Themembrane was blocked, washed, and probed with mouse monoclonal antibody of HSP70(1:1,000, Calbiochem). The membrane was washed and incubated with antimouse IgGhorseradish peroxidase conjugate (1:1,000) for2h at room temperature. The membranewas then incubated with chemiluminescent substrate (Santa Cruz) and the bands weredeveloped using X-ray films (Kodak, Rochester, NY, USA).4. The detection of NOS activity before and after the sublethally acute hypoxic exposure:NO synthases (NOS) comprise constitutive (neuronal NOS (nNOS) and endothelialNOS (eNOS)) and inducible forms(iNOS). Total NOS (TNOS) and iNOS activity ofmice exposed to1,7, or1h normoxia+6h sublethally acute hypoxia were determinedusing a Jiancheng kit (Jiancheng Ltd., Nanjing, China) following the manufacturer sinstructions. 5. Statistical analysis:Comparisons among groups were made with a one-way analysis of variancefollowed by unpaired Student s t tests with the Bonferroni correction. Comparisonsbetween control and mice pretreated with1,000mg/kg GGA or normoxic and hypoxicmice were made with unpaired Student s t test. A P value of <0.05was consideredsignificant.Results1. GGA pretreatment renders mice more resistant to acute hypoxia:Mice exposed to simulated acute hypoxia of10,000m at a velocity of about50m/sin a hypoxia chamber were monitored for survival for15min. Two thirds of the controlmice died within3min (Fig.1; average survival time of4.28±4.29min, n=15), and thesurvival rate decreased to6.67%15min after the mice were exposed to simulated acutehypoxia of10,000m. Surprisingly, the mice pretreated with GGA (1,000mg/kg)1hbefore exposure to acute hypoxia survived more than5min (average survival time of9.55±3.12, n=16, P<0.005vs. controls) and the survival rate significantly improved byabout three times (the survival rate increased to18.75%) as compared to the control mice.Thus, these data suggest that GGA pretreatment renders mice more resistant to acutehypoxia.2. GGA pretreatment reduces cerebral injuries caused by acute hypoxia:The mice were exposed to simulated acute hypoxia of8,300m (sublethal hypoxia)at a velocity of about10–20m/s for6h. The neurons in the cortex of mice exposed tohypoxia were shrunken and slightly scattered. However,1,000mg/kg GGA pretreatment1h before sublethally acute hypoxic exposure obviously reduced the shrinking of neuronsin the cortex. In hippocampal CA2and CA3subfields of mice exposed to sublethallyacute hypoxia, the neurons were significantly shrunken, irregularly arranged, and weaklystained.. Pretreatment of1,000mg/kg GGA significantly attenuated the deterioration ofthe neurons, which were less shrunken, arranged more regularly, and stained moreintensively. These results indicated that GGA pretreatment reduces the cortex andhippocampal injuries caused by acute hypoxia.3. GGA pretreatment induces the HSP70expression in cortex and hippocampus:We found that the expression of HSP70was significantly elevated in both cortexes(Fig.3a,c) and in the hippocampus (Fig.3b,d) with1h normoxic exposure after1,000 mg/kg GGA administration. The above data indicated that GGA induced the expressionof HSP70, which might be involved in the protective role of GGA against acute hypoxicinjury.4. GGA pretreatment inhibit the TNOS and iNOS activity in cortex and hippocampus:We found that the appreciably increased activity of iNOS induced by sublethallyacute hypobaric hypoxia at8,300m was significantly reduced tothat of control values by1,000mg/kg GGA pretreatment in the cortex. Similarly,1,000mg/kg GGA pretreatment also reduced the slightly increased activity of iNOS or TNOSfollowing sublethally acute hypoxia,in the cortex or the hippocampus. The results showedthat GGA pretreatment inhibit the TNOS and iNOS activity in cortex and hippocampus.Conclusions1. GGA pretreatment markedly increased acute hypoxic tolerance and attenuated acutehypoxic damage to the brain compared to the control mice.2. We show that the preinduced HSP70and inhibited iNOS may be the primaryunderlying mechanism of improved acute hypobaric hypoxic tolerance. The findings ofthis study will help in the design and development of novel therapeutic strategies to useGGA as drug for promoting acclimatization to high altitude and preventing HACE.