Effects Of GABA_A Receptor Regulators On Axonal Growth And Motor Function After Cerebral Ischemia

Objective:Stroke is one of the major causes of death and disability around the world.Ischemic stroke is the most common type,which is caused by the blood limitation in some brain area.As an effective treatment for stroke,thrombolytic therapy must be taken within a few hours after it onset.Compared with the acute stage,the repair stage provides a wide time window lasting from days to months for stroke recovery.Brain tissue recovery and plasticity in stroke recovery,such as neurogenesis,axonal growth and remyelination,are closely related to behavioural recovery after stroke.To indicate a key therapeutic target may improve self-repairing ability including neurogenesis,axonal growth,remyelination,which benefits behavioral recovery at the same time.GABA_A receptors play a key function in the development of brain and its plasticity.Therefore,we would like to regulate GABA_A receptors by their agonist muscimol and antagonist L655,708,to study whether the regulation of GABAergic inhibition effects the axonal regeneration,oligodendrogenesis,remyelination and functional recovery in the sustained focal cerebral ischemia.Methods:1.AnimalsThe Wistar male rats were randomly divided into 3 groups:sham-operated rats(SHAM;n=14),endothelin-1(ET-1)induced focal ischemic rats(ISC;n=16),ischemic rats treated with GABA_A receptor agonist muscimol(ISC+Muscimol;n=17).In part two,were randomly divided into 3groups:sham-operated rats(SHAM;n=14),rats subjected to focal cerebral ischemia by ET-1(ISC;n=14)and ischemic rats treated with?5 subunit containing GABA_A receptor inverse agonist L655,708(ISC+L655,708;n=15).2.ET-1 stroke modelAccording to the rat brain stereoscopic anatomy atlas,3 sites received ET-1 micro-injection in left cerebral cortex and striatum.Each stereotaxic coordinate injected 2?l ET-1(0.5?g/?l).Sham-operated rats received the same volume of normal saline as with ET-1 injection.3.In vivo muscimol administrationThe cannula connecting with muscimol(10mM)containing pump was implanted at the cortical surface)under anesthesia at the 7 day ater stroke.4.In vivo L655,708 administrationL655,708 was dissolved in dimethyl sulfoxide(DMSO).The drug was given by intraperitoneal injection once a day at a dose of 1 mg/kg for 2 weeks.5.Anterograde tracing of corticospinal tract(CST)Biotinylated Dextran Amine was injected into right motor cortex to label CST fibers at 14 day after stroke.6.Behavior analysisAt 4/5 week after stroke,tapered/ledged beam-walking test,cylinder test and sticky label test were used to evaluate the sensorimotor function of rats impaired limbs.7.Tissue PreparationAfter the behavioral tests,there were 84 rats left in total,14 rats in each group.Eignteenrats(average 3 from each group)were for western blot analysis.Other rats were perfused with 4%paraformaldehyde(PFA)for immunobistochemical analysis.8.Western blot analysisThe following primary antibodies were used:rabbit polyclonal anti-NogoR,rabbit polyclonal anti-NogoA,rabbit polyclonal ROCK,rabbit monoclonal anti-RhoA,mouse monoclonal anti-MBP,rabbit polyclonal anti-PSD-95,rabbit polyclonal anti-vGlut1 to detected expressions in perilesional cortex or denervated spinal cord.9.Immunohistochemical analysisThe following primary antibodies were used:rabbit polyclonal anti-NogoR,rabbit polyclonal anti-NogoA,rabbit polyclonal ROCK,rabbit monoclonal anti-RhoA,rabbit polyclonal anti-GFAP,rabbit monoclonal anti-Iba-1,rabbit polyclonal anti-NG2,mouse monoclonal anti-MBP,rabbit polyclonal anti-vGlut1,rabbit polyclonal anti-PSD-95 for immunofluorescence staining.10.Statistical analysisStatistical analysis was conducted with SPSS software vision 17.All data are reported as mean±SD.The repeated measures ANOVA or one-way ANOVA was used to analyze the data.P<0.05 was considered a statistically significant difference.Results:1.Survival of animalsThere were 62 rats received ET-1 injection in total and 2 rats died in the operation.Four rat in was excluded for its inability to complete behavioral tests.2.Muscimol has no effect on the growth of crossing CSTAt the 35 day after ischemic injure,the length of crossing midline fibers in ISC group significantly increased(P<0.01).There was no significant difference between ISC group and ISC+Muscimol group(P>0.05).3.Muscimol has no effect on the expression of axonal growth inhibitors after strokeThere were no significant difference between ISC group and ISC+Muscimol group on the number of NogoA-,NogoR-,RhoA-,and ROCK-positive cells and the dose of the axonal growth inhibitors in the perilesional cortex(P>0.05).4.Partially restoration of synaptic markers by muscimol treatmentMuscimol only significantly elevated the expression of PSD-95(P<0.01)and had no benefit to relieve the vGlut-1 reduction(P>0.05).5.Partially improved behavioral performance by drug treatmentIn the tapered/ledged beam-walking test,The slip ratio of paretic limbs were decreased after 2weeks muscimol treatment in recovery stage(P<0.01).In the cylinder test,there was no significant difference between ISC group and ISC+mucsimol group(P>0.05).In the stickly label teat,after treatment of muscimol,the time taken to contact tapes significant decreased compared with ISC group(P<0.01),but the time to remove tapes had no different with ISC group(P>0.05).6.Infarct volumesThere was no significant difference in the infarct volume between the experimental groups.7.L655,708 increases the growth of crossing CSTThere was a significant increase in the length of crossing midline fibers in the ISC group(P<0.01).In the L655,708 treatment group,there was a significant increase in the total length of the midline crossing fibers(P<0.01).8.L655,708 decreases the expression of axonal growth inhibitors after strokeTheir levels were found to be significantly increased after stroke compared with the SHAM group(P<0.01).In addition,L655,708 significantly decreased the number of NogoA-,NogoR-,RhoA-and ROCK-positive cells and their expressions in the peri-infarct cortex(P<0.01).9.Restoration of synaptic markers by L655,708 treatmentL655,708 treatment elevated the expressions of both vGlut-1 and PSD-95 as compared with the untreated ISC group(P<0.01).10.Promoting regeneration of astrocytes,microglia,oligodendrocyte precursor cells andmyelination of oligodendrocyte by L655,708 treatment.L655,708 treatment elevated the numbers of these positive cells further(P<0.01).And,the protein expression of MBP could be elevated by L655,708 treatment(P<0.01).11.Improved behavioral outcomes by drug treatment L655,708 treatment for 2 weeks decreased the impaired forelimb and hind-limb slips compared with the untreated ISC group in the beam-walking test(P<0.01).There was a significant improvement in the forelimb function after the stroke evoked by L655,708 treatment in the cylinder test(P<0.05).The time for impaired forelimbs needed to contact adhesive tape and remove them significantly increased after stroke.The effect of L655,708 treatment was more significant compared with the untreated ISC group(P<0.01).Conclusion:Our study proves that activating GABA_A receptors by muscimol during chronic repair phase can slightly promote the recovery of motor and sensory function after stroke.However,the activation of GABA_A receptor does not promote axon regeneration and improve the inhibitory environment of axon growth in the cortex around the infarcted area.L655,708 antagonizes GABA_A receptors and promotes axonal regeneration,oligodendrocyte proliferation,myelination and motor function recovery of ischemic rats by inhibiting NogoA/NogoR and RhoA/ROCK signaling pathways.Therefore,muscarinol can not be used as a therapeutic drug for brain protection and brain plasticity after ischemia.L655,708 has the advantage of less side effects,which makes it possible to be used as a drug to promote brain tissue repair and behavioral recovery in acute and convalescent stage after ischemia in the future.