An Experimental Study Of Chronic Hypoxia Effects On Myelinogenesis And Brain Function In Adult Mice

Chronic hypoxia exposure is currently recognized as one of the risk factors for the health of adults.There are quite a lot clinical diseases,such as CMS,COPD and OSA,et.al,which can make patients in a state of chronic hypoxia to a certain extent.Long term chronic hypoxia can cause temporary or permanent damage to multiple organs,including central nervous system,cardiovascular system,blood system,lung,kidney,etc.Physiological compensation and even disease changes will appear under long-term chronic hypoxia.Among them,brain is the most important and sensitive organ to hypoxia.During and after exposure to chronic hypoxia,brain function defects are mainly manifested as abnormal movement,impaired learning and memory ability,attention disorder,etc.However,how chronic hypoxia leads to brain function defects in adults is still unclear.In recent years,researchers have used DTI sequence scanning technology to detect and analyze the integrity of central nervous system nerve fiber tracts in different brain regions of patients with OSAHS and CMS.As a result of extensive damage of white matter,it was closely associated with the impairment of the integrity of fibers in the CNS,and atrophy of white matter has been revealed as the main lesion.White matter is an important component of the central nervous system,which accounting for approximatelly 60%of the brain weight.It is mainly composed of a large number of axons and myelin sheaths surrounded by oligodendrocytes（OLs）.Myelin sheath is an important structure of vertebrate central nervous system,which can nourish and protect neurons.Myelin sheath is isolated on the axons of neurons and has the characteristics of selective and segmental distribution.There are a large number of voltage-gated sodium channels（Nav）in the gap between the myelinated segments,so-called as node of Ranvier.In the CNS and PNS,Ranvier nodes are essential infrastructures for the transmission and propagation of action potential on individual myelinated axons between the adjacent nodes.In different vertebrate species,the starting point of myelin development is different,and it is generally believed that the development of myelin begins at the 14th week of embryo in human.Myelin formation accelerated three months before birth and continued to form after birth.Myelinated OLs mainly come from the differentiation and development of oligodendrocyte precursor cells（OPCs）.Recent studies have found that OPCs distributes uniformly in the central nervous system of adult animals and maintains a relatively constant cell density.This feature may be relevant to the sensory acquisition of the environment,or directly linked to the motor skill learning in adult mice.Some researches even reported that the myelination of adult vertebrate is conducive to the consolidation of memory.Moreover,studies have shown that although the development of myelin begins in the embryo and reaches the peak before adulthood,there are still a large number of new myelin sheath formation after adulthood and continue to the old age.What is the myelination of CNS in adult animals for?Does chronic hypoxia interfere with myelination and cause neurological dysfunction in adult animals?Is there a direct relationship between hypomyelination and neurological dysfunction?Can promoting myelination improves the dysfunction caused by chronic hypoxia?In view of the above problems,we mainly carried out the following four aspects of experimental researches:（1）NG2-Cre ER^TM;Tau-m GFP model was established to observe the new myelin sheath of CNS in adult mice.（2）The hypoxia model of adult mice was established to observe the effects of chronic hypoxia on myelingenesis and motor function in adult mice;PLP-Cre ER^TM;m T/m G mouse model was used to observe the effect of chronic hypoxia on pre-exiting myelin in adult mice.（3）Knockout Olig2 specifically in OPCs to mimic hypomyelination and explore whether the myelin sheath deficits of the CNS is harmful to the motor coordination function of adult vertebrates.（4）Explore whether the drug-based treatment strategy can effectively promote myelination and improve functional defects,and promoting myelination may be a potential therapeutic strategy against chronic hypoxia.During our researches,we mainly use immunofluorescent staining to explore histological changes of myelin;at the same time,behavioral test,such as open field test and beam walking test,were used to record and analysis neurofunctions.Main results were listed below:1.Myelination is active in the central nervous system of adult miceThe formation of myelin in mice begins at birth,and peaks at 4-week afterbirth.For adults,there have already been so much of myelin in the central nervous system.Therefore,it is difficult to observe the new myelin sheath with traditional methods.In order to observe the myelination of CNS in adult mice and visualize the new myelin.We used NG2-Cre ER^TM;Tau-m GFP reporter mice,which were induced by tamoxifen at the age of 6months and then taken histology after 4 months.The myelination of CNS was observed by immunofluorescence staining.As the result we have found,myelination in motor cortex（M1 and M2）,sensory cortex（S1）,corpus callosum（CC）,striatum,anterior commissure（AC）and hippocampus of adult mice are crazy active.By calculating the density of new myelin,we found that the density of new myelin in different brain regions of CNS is different,more myelin was formed in CC and AC.2.Effects of chronic hypoxia on myelination of CNS and motor coordination in adult miceAfter 4 days of hypoxic preconditioning,the 4-month-old NG2-Cre ER^TM;Tau-m GFP reporter mice were divided into two groups in a random manner,the experient group mice were exposed to 10%oxygen to establish a chronic hypoxia model,while the control group mice maintained in a normoxic condition（21%oxygen）.Then,after the treatment of hypoxia lasting for 4 weeks,tissue samples were obtained.the brains were collected for examining m GFP positive new myelin.It is evident that the m GFP-positive new myelin and oligodendrocytes were greatly decreased in the corpus callosum and motor cortex of the hypoxic mice as compared to the normoxic controls.The number of OPCs had no significant changes,suggesting that the decrease of myelination induced by chronic hypoxia may be due to the differentiation disorder of OPCs.To understand if pre-existing myelin is sensitive to hypoxia in the brains,we used the PLP-Cre ER^TM;m T/m G line to label pre-exiting myelin,4-month-old mice was induced before hypoxia exposure,after 4 weeks,it was found that the myelin sheath had no significant changes after chronic hypoxia.By using behavior test,such as the open filed test and the modified beam walking test,we have found that chronic hypoxia could not impair general activities but induce motor coordination deficits in adult mice.The staining results of NF200 and Neu N suggested that the number of neurons and axons in the central nervous system did not change significantly after chronic hypoxia.3.Inhibited myelination can lead to motor coordination dysfunction in adult miceBy hybridizing NG2-Cre ER^TM;Tau-m GFP mice with Olig2fl/fl mice,we obtained a gene knockout mouse model(NG2-Cre ER^TM;Tau-m GFP;Olig2 fl/fl)that can specifically knock out the transcription factor Olig2 in oligodendrocyte cell line.The results of immunofluorescence staining showed that the myelin formation in corpus callosum,cortex and hippocampus of Olig2 knockout adult mice were inhibited,which was consistent with the myelin lesions of chronic hypoxia mice.These results suggest that Olig2 knockout mouse model can effectively simulate the changes of myelin sheath induced by chronic hypoxia,which was helpful to understand the role of myelination in motor coordination function of adult mice.The results of beam walking test indicated that Olig2 knockout adult mice had motor coordination dysfunction.Because Olig2 specific knockout mice only inhibit myelination,but do not affect neurons and other cells,suggesting that myelinogenesis was necessary for motor coordination in adults.4.Enhancing myelination by clemastine treatment improve chronic hypoxia induced motor coordination function deficitSince myelinogenesis is necessary for the motor coordination function in adults,we next wanted to explore whether enhancing oligodendrocyte differentiation in adults could improve chronic hypoxia associated functional deficits.Clemastine is a muscarinic receptor antagonist approved by FDA for the treatment of allergy,which can promote myelination in vivo.By using the treatment of Clemastine,strikingly,we found clemastine treatment greatly increased m GFP positive newly-formed myelin in motor cortex and corpus callosum,which indicating the drug treatment can enhance myelinogenesis in chronic hypoxic brains.The results of beam walking test indicated that enhancing myelination can rescue motor coordination of hypoxia adult mice,which suggesting myelination enhancing therapeutics may be a promising strategy to prevent hypoxia induced neuro-functional impairments in adultsIn this study,we mapped myelinogenesis in adult CNS and found that myelinogenesis was active throughout the whole brain,especially in motor cortex and corpus callosum.Intermittent chronic hypoxia inhibited new myelin generation,along with impaired motor coordination function without altering pre-existing myelin in adult mice.To mimic the inhibited myelingoenesis in hypoxic brains,we used Olig2 conditional knockout mice,since Olig2 has been reported to be a positive regulator of OPC differentiation.Our results indicate that inhibiting myelinogenesis by Olig2 deletion can directly cause motor coordination dysfunction.Clemastine is an FDA-approved muscarinic receptor antagonist that can promote myelinogenesis in vivo.Our results showed that clemastine treatment enhanced myelination during chronic hypoxia and improved neurological functions in hypoxic adult mice.Taken together,our data indicated that myelination in adult brain is active,chronic hypoxia inhibits myelinogenesis and causes brain functional deficits and enhancing myelinogenesis may be a potential strategy to improve chronic hypoxia induced neurofunctional deficits.