Inhibition Of HIF-1α Affects The Expression Of GLUT1 And GLUT3 And The Repair Of White Matter Injury In Brain Of Cerebral Palsy Model Mice

BackgroundCerebral Palsy(CP)is a non-progressive and permanent neurodevelopmental disorder syndrome caused by various risk factors.It happens within one month before or after birth,and is the main cause of disability in children.There are 3.3 sick kids per 1,000 newborns,additionally some symptoms will become more obvious as children grow older.Up to now,there is no method to cure cerebral palsy once and for all,but supportive treatment and drug therapy can alleviate cerebral palsy symptoms to a certain extent and improve patients’ motor ability and living ability.Researchers have neverstoppedstudying a lot of pathomechanism research on cerebral palsy.It is confirmed that cerebral ischemia-hypoxia are the main causes of cerebral palsy.The periventricular leukomalacia(PVL)induced byischemia-hypoxiais characterized asthe stagnation of oligodendrocyte precursor cell(OPC)maturation and differentiation,which results in myelin sheath damage and nerve conduction interruption.Therefore,intervention measures topromote differentiation and maturation of OPC myelin sheath regeneration through improving the neurological development environment in CP patients’ brain,has become a novel point in cerebral palsy in recent years.Hypoxia inducible factor(HIFs)is an important component of hypoxia transcription reaction.OPC is very sensitive to the decrease of concentration of oxygen in brain under ischemia-hypoxia conditions.HIF-1a is highly expressed in cells,which latterly transferred to nucleus together with HIF-1β to regulate gene transcription of pathways related to cell survival so as to stimulate the adaptation of brain and cells which are exposed to hypoxia injury.In addition,HIF-la can induce and activate the expression of various metabolic enzymes and transporters.Glucose is the main source of brain energy metabolism,but it cannot freely pass through cell membrane.Glucose transporters(GLUTs)are required to transport across the membrane to provide oxygen and nutrients for nerve cells.Previous studies have found that HIF-la may giverise to GLUTs upstream gene family SLC2A to encode GLUT subtype 1 in CNS endothelial cells and astrocytes meanwhileGLUTsubtype 3 in neurons during ischemia and hypoxia,thus inducing accelerated compensation of glycolysis in the brain and optimizing energy production.However,it is not clear the relationship between HIF-1α promoting OPC and encoding GLUTs.Therefore,we assumed that HIF-la may promote OPC differentiation and myelin regeneration after CP by inducing the expression level of GLUT1 and GLUT3.After inhibiting HIF-1α,the down-regulation of GLUT1 and GLUT3 expessions may not be able to promote the rehabilitation of white matter injury.PurposeIn this experiment,we aimed to imitate the clinical symptoms and nervous system changes in CP brain by establishing a CP model mouse underischemia-hypoxia conditions combined with LPS infection.Injecting Oltipraz to inhibit HIF-la protein expressionlevel to explore the relationship and mechanism between OPC differentiation,OL maturation,myelin sheath regeneration and GLUTs content in CP mice brain,and that to provide feasible ideas for further research and clinical treatment.Methods(1)Cerebral Palsy Model and Oltipraz TreatmentAccording to the neurological characteristics,newborn mice were selected to establish CP models to simulate human pathogenesis.Postnatal day 6(PND)were anesthetized with a self-made anesthetic tool,and then placed on the ice for deep anesthesia.After fixing the four limbs,the mice were cleaned the skin with iodophor.A midline ventral incision was made in the anterior neck.Under a stereomicroscope,the bifurcation of the right common carotid artery was approached by gently retracting the omohyoid and sternocleidomastoid muscles.The fascia around the carotid sheath was removed,as well the proximal internal branch isolated from the nearby vagus nerve nerve and sympathetic ganglia with surgical operation draw hook.The internal carotid artery was then cauterized using anelectronic cauterizing tip.Following cauterization,the skin incision was sutured,and the animal was kept warm until fully awake and then returned to the dam.One hour after ligation,the animal is placed in a sealed chamber infused with nitrogen until level of 8.0%O2 is reached,then the pup was exposed to 90 min in the hypoxia environment.After hypoxia exposure,the mouse recovered for 30 min and then returned to the dam again.For creation of brain injury combining infection/inflammation with hypoxia/ischemia,the animal wasalso injected intraperitoneally with lipopolysaccharide(LPS,1 mg/kg).Within 12 hour post-cerebral palsy,the model mice of cerebral palsy were randonmly divided into four groups:(a)saline;(b)one low-dose Oltipraz(35 mg/kg);(c)one middle-dose Oltipraz(75 mg/kg);(d)one high-dose Oltipraz(150 mg/kg).The newborn mice received intraperitoneal injection ofHIF-la inhibitor Oltipraz with a minimum tolerable dose of 0.2ml.(2)Behavior TestsHorizontal Grid test18cm ×35cm metal grid was used to test the grip strength of mice.Tin paper and tape were used tightly to seal off the edges of the metal mesh to prevent mice from escaping from the test.PND35 Mice were set on the grid and shook gently three or four times to make them hold on to the wire,and then turn the grid upside down.The investigator used a stopwatch to quantitate the latency to fall off the wire lid.Pole-climbing TestA plastic ball with a diameter of 3 cm was stuck to the top of a wood pole 60 cm long and 1 cm thick.Two layers of gauze were wrapped around the wood pole to prevent slippage.The PND35 mice were placed on the top of the ball and three data indicators were recorded:(a)the time of climbing the upper half of the rod length;(b)the time of climbing down tothe second half of the rod length;(c)time for mice to climb the whole length of the rod.Scoring record,3 points in 3 seconds to complete one of the above actions;2 points in 6 seconds;1 point in more than 6 seconds.(3)In order to evaluate the white matter damage,the expression level of myelin basic protein(MBP),mature oligodendrocyte(CC1)and oligodendrocyte precursor cell(Olig2)in mouse brain were examined by immunofluorescence(IF)technique.(4)Western blot was used to detect the expression levels of HIF-1α,GLUT1 and GLUT3 protein in different brain regions and different groups.Results(1)PND30 CP model mice showed large area infarction of cerebral hemisphere on ligation side.H&E staining results identically showed cell swelling,inflammatory infiltration and cell death in mice brain.(2)PND30 CP mice showed decreased grip with limb disharmony(p<0.05).(3)MBP density decreased in the brain tissue of PND 14 and PND21CP mice,CC1 expression decreased in different brain regions(p<0.05)but Olig2 expression level remained the same,indicating decrease of myelin sheath and OL quantity and OPC differentiation retardation.(4)The expression of HIF-la in cortex,hippocampus and basal ganglia(p<0.05)region of PND 14 CP mice increased,while the expression of CP mice in PND21 and PND28 decreased in accordance with the expression of group.The expression of GLUT1 and GLUTS increased(p<0.05),indicating that PND14 may be the critical point of CP change.(5)Western blot showed that the expression of MBP,GLUT1 and GLUT3 in PND14 decreased(p<0.05)after inhibiting HIF-1α by injecting oltipraz,but the expression of Olig2 protein remained unchanged indicating that inhibition of HIF-la leads to myelin sheath reduction and low glucose transport efficiency.Conclusion(1)The myelin sheath density and OLs quantity in nervous system decreased in CP model mice,leading to OPC differentiation and maturation barrier and white matter damage.(2)The inhibition of HIF-la led to the decreased expression of GLUT1 and GLUT3,the poor effeciency of glucose energy supply rate and that aggravation of white matter damaged,which means that HIF-la may play an important role in neuroprotective effect in the brain.