Effects Of Hypoxia On Some Proteins Relating To Neuroprotection In The Brain

Hypoxic diseases are common in clinical work involved in respiration, neuron,heart and so on.Because of its consumption of oxygen in large quantity and not enough energy in store,the brain is sensitive to hypoxia and damaged easily.Therefore,protecting brain against hypoxia is a crucial topic in neuronal research.Under hypoxia,some changes take place in the brain including deficiency of ATP production,acidosis,disorder of ion transportation of cellular merabrane,alteration of membrane potential,which result in a decease of neurotransmitter synthesis and changes of receptor expression.The abnormalities of these functional proteins affect the synaptic transmission and lead to the dysfunction and structural changes in neuron cells.Amino acid neurotransmitters are important transmitters in the brain.The balance of excitatory amino acids and inhibitory amino acids is needed for brain cells to maintain their normal functions.The former is represented with glutamic acid and aspartate mediating most part of rapid excitatory synaptic transmission,while the latter is represented with r-aminobutyric acid(GABA) and glycine mediating most part of rapid inhibitory synaptic transmission. Recently,researchers have found that the inhibitory amino acids play a more important role in protecting brain tissues from hypoxia/ischemia and other stress.Furthermore,inhibitory amino acids show various effects on different developmental neurons.But so far,the expression of GABA and glycine receptors in different age neurons and effects of inhibitory amino acids on the two receptors in hypoxia are unclearAnother transmitter system in the brain is acetylcholine.It participates in the information transformation of movement and sensation in the body.The choline acetyltransferase(CHAT)is a rate-limiting enzyme in acetylcholine (Ach)synthesis and its abnormality is correlated to Alzheimer's disease(AD) .Hypoxia is a one cause of AD.But which link the hypoxia involved in AD remains to be further studied.In the first part of the recent study,rat cortex neurons were cultured under hypoxia.The inhibitory amino acids GABA,glycine and taurine were added into the medium respectively,then the GABA_ARα1 and GlyRα1 were measured.Through the experiment,the effects of inhibitory amino acids on expression of the two receptors could be studied in vitro.In addition,many research reports have indicated that DOR(δ-opioid receptor)can protect brain from hypoxia/ischemia.In the second and third part of the recent study,transgenic DOR mice were used to study the expression GABA_ARα1 and ChAT in cortex neurons under hypoxia.These results can help us to know the effects of hypoxia and transgenic DOR on GABA_ARα1 and ChAT in vivo.PARTⅠEffects of inhibitory amino acids on GABA_A and glycine receptor expression in cortical neurons during development and under hypoxiaObjective:To study effects of inhibitory amino acids on expression of GABA_A and glycine receptors in cortical neurons during development and under hypoxia.Methods:Primary neuron cultures were prepared from the cerebral cortex of pregnant rats(E17-18)and exposed to hypoxia(1%oxygen).The inhibitory amino acids including gamma-aminobutyric acid(GABA),aminoaceic acid (glycine)and 2-aminoethanesulfonic acid(taurine)were separately added to culture medium with deferent final concentrations(10-2000μM)at DIV 4 and 20 respectively.DADLE,DOR(δ-opioid receptor)agonist,was applied to DIV-4 and DIV-20 dishes.The density of GABA_ARα1 and GlyRα1 protein was measured by Western blot.Results:(1)GABA_ARα1 and GlyRα1 underwent differential changes in expression during neuron development in-vitro.GABA_ARα1 density was very low in the immature(DIV4)cortical neurons but increased with increasing days in culture.In contrast,GlyRα1 density in early development time had reached to high level already.(2)In hypoxic environment,the density of GABA_ARα1 and GlyRα1 increased in immature neurons but decreased in mature(DIV20)neurons.(3)In normoxia,GABA and glycine tended to decrease GABA_ARα1 density in immature neurons,but increased the density in mature neurons.(4)Under hypoxia,GABA,glycine and taurine all decreased GABA_ARα1 and GlyRα1 density in immature neurons.While in mature neurons,they withstood the reduction of GABA_ARα1 and GlyRα1 expression induced by hypoxia.(5)Activation of DOR increased GABA_ARα1 and GlyRα1 expression in both immature and mature neurons under normoxia and in mature neurons exposed to hypoxia.Conclusions:In immature neurons,inhibitory amino acids decreased GABA_ARα1 and GlyRα1 expression under hypoxia or normoxia owing to their cellular toxicity.But in mature neurons,the inhibitory amino acids withstood the reduction of GABA_ARα1 and GlyRα1 expression induced by hypoxia. Meanwhile,they protected cortical neurons and up-regulated the expression of GABA_ARα1 and GlyRα1 in normoxia.The activation of DOR also brought about cellular protection and enhanced the expression of these two receptors. PARTⅡEffects of chronic hypoxia on choline acetyltransferase (CHAT)in DOR transgenic mouse brainObjective:to investigate the expression of choline acetyltransferase(ChAT) in transgenic mouse neurons and study the protection ofδ-opioid receptors (DOR)for neurons in vivo under chronic hypoxia.Methods:The tDOR(transgenicδ-opioid receptor)and wild type mice, including adult(P30,postnatal 30)and later stage of development(P20, postnatal 20),were used in this experiment.The brain tissues of these mice were harvested after hypoxic insult or in normoxia for 1 day,3 days,5 days and 7 days respectively.The density of ChAT was measured with Western blot in different brain regions like cerebral cortex,subcortex,hippocampus,brainstem and cerebellum.Results:In tDOR mice,under normoxia,the density of ChAT was decreased compaired with wild type mice in 4 brain regions,i.e.,cortex,subcortex, hippocampus and brainstem.A 7-day hypoxia exposure led to a significant decrease of ChAT density among the wild type mice in the cortex, hippocampus and brainstem.In contrast,among the tDOR mice,the same hypoxia induced an increase of ChAT expression in these brain regions,Conclusions:These results suggested that ChAT expression in wild type mouse brain decreased after chronic hypoxia.While in transgenic mice,the high expression of DOR withstood ChAT decrease caused by chronic hypoxia, which showed the protection of DOR for the cholinergic neurons in chronic hypoxia.PARTⅢEffects of hypoxia on expression of GABA_A receptors in cortical neurons of transgenic miceObjective:To investigate the effects of hypoxia and tDOR(transgenicδ-opioid receptor)on expression of GABA_A receptor in mouse cortical neurons and explore the protection of DOR for neurons under hypoxia in vivo.Methods:The tDOR and wild type mice were exposed to hypoxia or normoxia. The brain tissues of these mice were harvested after hypoxic insult or in normoxia for 1day,3 days,5 days and 7 days respectively.The GABA_ARα1 level in cerebral cortex was measured with Western blot.Results:In normoxia,the density of GABA_ARα1 in tDOR mouse cortical tissues was lower than that in wild type mouse.This difference was more significant after chronic hypoxia(P＜0.05).In addition,the hypoxia insult during 1-7 days couldn't result in apparent changes of GABA_ARα1 expression in wild type mice.Conclusions:The expression of GABA_ARα1 in transgenic mouse brain was lower than that in wild type mouse.Chronic hypoxia further down-regulated this expression of cortical neurons in tDOR mice.The DOR inhibited the expression of GABA_ARα1 and showed its protection for mouse brain.The hypoxia for 7 days couldn't produce any significant change in GABA_ARα1 expression among wild type mice.