Objective:To explore the role of P2Y1Receptor(P2Y1R)-activated astrocytes in delayed encephalopathy after acute carbon monoxide poisoning(DEACMP).Methods:SD rats were randomly divided into Control,DEACMP,DEACMP+DMSO,and DEACMP+MRS2179 groups.The latter three groups were exposed to static CO inhalation to establish the DEACMP model,while the Control group inhaled air.The rats in DEACMP+MRS2179 group were injec-ted with 3 μl of P2Y1R antagonist MRS2179 with concentration of 1 mmol/L via lateral ventricle,while the DEACMP+DMSO group an equal volume of DMSO.At 14 and 21 day after DEACMP model establishment,the spatial learning and memory ability of rats were observed by Morris water maze,the injury of pyramidal cells in the CA1 area of the hippo-campus was observed by HE staining.The protein levels of glial fibrillary acidic protein(GFAP)and P2Y1receptor(P2Y1R)in the hippocampal tissues of rats were detected by immunohistochemistry and Western blot.The expressions of interleukin-1β(IL-1β),interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)in the hippocampal tissues of rats were detected by ELISA.Results:In comparison with the Control group,the escape latency was found to be signifi-cantly prolonged in the DEACMP group and the DEACMP+DMSO group.Furthermore,the expression levels of GFAP,P2Y1R,IL-1β,IL-6,and TNF-α were significantly increased in the pyramidal cells of the CA1 region of the hippocam-pal tissue(P<0.05).In contrast,the escape latency was significantly reduced and the expression levels of GFAP,P2Y1R,IL-1β,IL-6,and TNF-α were significantly decreased in the DEACMP+MRS2179 group when compared with the DEACMP+DMSO group(P<0.05).Conclusion:P2Y1R activates astrocytes to release inflammatory factors lead-ing to the onset of cognitive impairment in DEACMP,and inhibition of P2Y1R reduces the expression of inflammatory factors and thus improves cognitive function in DEACMP.

Objective:To explore the repair mechanism of bone marrow stromal cells(BMSCs)transfected with miR-21 in rat sciatic nerve injuries.Methods:In vitro,rat BMSCs were transfected with miR-21 agonist(agomir-21)or blocker(antagomir-21)and their respective empty vectors,respectively,and divided into Control group,agomir-21 group,agomir-NC group,antagomir-21 group and antagomir-NC group.A 12 mm rat sciatic nerve injury model contai-ning decellularized nerve area was prepared,and the cells in each group were labeled with Hoechst33342 for postopera-tive implantation of decellularized nerve area.At 14 d after operation,the recovery of neuromotor function was evaluated by footprint test and the wet weight ratio of gastrocnemius.Immunofluorescence staining was applied to detect the expression of neurofilament protein 200(NF200),myelin basic protein(MBP),S100 calcium binding protein β(S100β)and glial fibrillary acidic protein(GFAP)within the regenerating nerve.Results:Compared with Control group,the sciatic nerve function index(SFI)and the wet weight ratio of gastrocnemius were significantly increased in agomir-21 group(P<0.05);and the density of regenerated nerve fibers and the expression of myelin marker proteins were increased,and the differentiation rate of BMSCs into Schwann cell-like cells(SCLCs)was elevated significantly(P<0.05).Conclusion:miR-21 significantly promotes peripheral nerve regeneration by enhancing the differentiation of BMSCs into SCLCs in vivo.

Objective:To explore the effect of melatonin on mitophagy in the brain tissue of rats with early subarach-noid hemorrhage(SAH).Methods:The rat model of SAH was established by internal carotid artery puncture and trea-ted with melatonin by intraperitoneal injection.At 24 hours after the operation,the neurological function of rats was de-tected by mNSS score and balance beam test.The contents of reactive oxygen species(ROS),malondialdehyde(MDA),and glutathione(GSH)and in brain tissue were detected by commercial reagent kits.The expression changes of PINK1/Parkin,microtubule-associated protein1A/1B-light chain3(LC3)and p62 in brain tissue were detected by Western blot.Results:Melatonin can significantly improve the neurological function of SAH rats,reduce the con-tents of MDA(P<0.05)and ROS(P<0.05),and increase the content of GSH(P<0.05).Meanwhile,Western blot detection indicated that melatonin could increase the expression of PINK1 and Parkin(P<0.05),increase the ratio of LC3-II/LC3-I(P<0.05),and reduce the expression of p62(P<0.05).Conclusion:Melatonin alleviates early brain injury in rats with subarachnoid hemorrhage through PINK1/Parkin-mediated mitophagy.

Objective:To explore the role of the expression of peroxisome proliferator-activated receptor β/δ(PPARβ/δ)in the anti-inflammatory mechanism of acupuncture(AC)against cerebral ischemia-reperfusion injury(CIRI)in rat hippocampal tissue.Methods:A total of 60 SD rats were randomly divided into Sham group,CIRI group,CIRI+AC group and CIRI+AC+PPARβ/δsiRNA group.The CIRI rat model was prepared by the method of middle cerebral artery occlusion,and acupuncture treatment was used for intervention.The neurological function of rats was assessed by the modified neurological severity score(mNSS)method after 72 hours of intervention.The brain in-farction volume ratio was observed by 2,3,5-triphenyltetrazolium chloride(TTC)staining method.The pathological morphological changes of brain tissue were observed by hematoxylin-eosin(HE)staining.The expression levels of PPARβ/δ,IL-1β,and IL-10 mRNA in the hippocampal region were detected by RT-qPCR.Results:Compared with the Sham group,the mNSS scores and the brain infarction volume ratio in all modeling groups were significantly in-creased.Compared with the CIRI group,the mNSS scores and the brain infarction volume ratio in the CIRI+AC group were significantly reduced.The expression levels of PPARβ/δ and IL-10 in the hippocampus were significantly upregu-lated,while the expression level of IL-1β was significantly downregulated(P<0.01),with scattered inflammatory cell infiltration observed.Compared with the CIRI+AC group,there was no significant difference in the mNSS scores and the brain infarction volume ratio in the CIRI+AC+PPARβ/δsiRNA group.The expression level of PPARβ/δ in the hippocampus was significantly downregulated(P<0.01),while no significant difference was found in the expression level of IL-10 and IL-1β mRNA,with a small amount of inflammatory cell infiltration observed.Conclusion:Acupunc-ture ameliorates the recovery of neurological function and the ratio of cerebral infarction volume in rats with CIRI,and its mechanism may be associated with the upregulation of PPARβ/δ expression,thereby reducing inflammatory respon-ses and exerting neuroprotective effects.

Objective:To anatomically and phenotypically characterize the insular cortex(IC)-nucleus tractus soli-tarii(NTS)neural pathway.Methods:Adult male Sprague-Dawley rats were divided into three experimental cohorts for neural circuit tracing.Anterograde labeling was achieved by injecting anterograde self-complementary adeno-associated viruses(scAAVs)into the IC.Retrograde tracing involved NTS injections of either retrograde scAAVs or FluoroGold(FG),combined with immunofluorescence histochemical staining to identify IC-originating projection neurons.For postsynaptic neurochemical phenotype characterization,IC was injected with AAV2/1-CaMKII-Cre,while a mixture of AAV2/9-Syn-DIO-mCherry and AAV2/9-VGAT1-EGFP was injected into the NTS.The rats were allowed to survive for one week following scAAVs or FG injection or four weeks after recombinase-dependent systems injection.Then the rats were sacrificed,and serial brain sections were prepared for immunofluorescence histochemical staining(brain section containing FG)and subsequent fluorescence/confocal microscopic analysis.Results:(1)Anterograde viral tracing re-vealed dense axonal terminals from the IC projecting to the medial subnucleus of the NTS,while retrograde tracing re-vealed that IC neurons projecting to the NTS were predominantly localized within the dysgranular layer;(2)IC-NTS projection neurons were exclusive glutamatergic(100%,n=3);(3)NTS neurons receiving IC inputs were mainly lo-calized in the medial subnucleus,and were predominantly GABAergic(79.8±3.2%,n=3).Conclusion:The pres-ent results indicate that a descending pathway from excitatory neurons of the IC terminates onto inhibitory neurons of the NTS,which might represent a potential neuromodulatory target for visceral pain disorders.

Objective:To explore the expression and ultrastructure distribution of cofilin and its potential mecha-nisms in postsynaptic mitochondrial anchoring and synaptic plasticity in the pre-B?tzinger complex(pre-B?tC)in rats.Methods:Using neurokinin 1 receptor(NK1R)as a morphological marker of the pre-B?tC,we examined the expres-sion and ultrastructure distribution of cofilin in pre-B?tC neurons of normoxic(NOR)and acute intermittent hypoxic(AIH)rats by combining Western blot,RT-qPCR,immunofluorescence,and immunoelectron microscopy.Results:Cofilin was expressed in the nuclei,somata and dendrites of NK1R-positive neurons in the pre-B?tC and its expression decreased after AIH intervention in this region detected.The results of immunoelectron microscopy showed that cofilin was expressed in the dendritic spines and postsynaptic filamentous or flocculent structures of pre-B?tC neurons.A total of 317 synapses were detected in pre-B?tC(NOR:122;AIH:195).In the NOR group,65.6％of the postsynaptic had cofilin expression,while in the AIH group,the proportion decreased to 47.2％.Combining with the role of cofilin in the regulation of actin severing,it is suggested that the severing effect of cofilin on postsynaptic actin is weakened af-ter AIH intervention,which may further enhance the postsynaptic mitochondrial anchoring.Conclusion:Cofilin,an ac-tin binding protein,plays a crucial role in regulating shearing and depolymerization of actin.Decreased cofilin expres-sion induced by AIH suggests an enhanced actin polymerization,which may be involved in the postsynaptic anchoring of mitochondria and the regulation of synaptic plasticity in the pre-B?tC.

Objective:To investigate the effects of melatonin(Mel)on inflammatory damage in the retina of rats with oxygen-induced retinopathy(OIR)and the molecular mechanisms.Methods:Healthy neonatal SD rats were di-vided into the sham group(Sham),the model group(OIR),and the melatonin treatment group(OIR+Mel).The OIR model was induced by alternating 50%/10%oxygen concentration exposure for 14 d.The OIR+Mel group was in-jected intraperitoneally with 10 mg-kg-1 melatonin.Hematoxylin-eosin(HE)staining was used to observe the morpho-logical changes in the retinal tissue;immunohistochemical staining was used to detect the expression of retinal cleaved-caspase-1 and IL-1β proteins;and immunofluorescence staining was used to detect the expression of cGAS-STING-NL-RP3 signaling molecules and gasdermin(GSDMD)in the microglia of the retina.Results:HE staining results showed that compared with the Sham group,the retinal cells in the OIR group were disorganized and the thickness of the inner retina was significantly thinner,and the retinal cells in the OIR+Mel group were more neatly arranged compared with those in the OIR group(P<0.05).Immunohistochemical staining results showed that the number of cleaved-caspase-1+and IL-1β+cells in the retina of rats in the OIR group increased significantly compared with that in the Sham group,and the number of cleaved-caspase-1+and IL-1β+cells in the retina of rats in the OIR+Mel group decreased signifi-cantly compared with that of the OIR group(P<0.05).Immunofluorescence staining results showed that the number of cGAS+,STING+and NLRP3+cells in the retina of rats in the OIR group increased significantly compared with that in the Sham group,and the number of cGAS+,STING+and NLRP3+cells in the retina of rats in the OIR+Mel group de-creased significantly compared with that in the OIR group(P<0.05);The number of Iba-1+/N-GSDMD+cells in-creased significantly in the OIR group compared with the Sham group,whereas the number of Iba-1+/N-GSDMD+cells in the OIR+Mel group was significantly less than that in the OIR group,but still more than that in the Sham group(P<0.05).Conclusion:Mel inhibits the pyroptosis of retinal microglia,thus attenuates retinal inflammatory injury in OIR rats,and its mechanism may be related to the cGAS-STING-NLRP3 signaling pathway.

Objective:To explore the efferent projections of glutamatergic neurons in the basal forebrain(BF)and the function of its projections to lateral habenula(LHb)under isoflurane anesthesia.Methods:Channelrhodopsin-as-sisted circuit mapping(CRACM)was used to explore the efferent projections of BF glutamatergic neurons in cre driver mice lines.On this basis,we optogenetically activated the BF glutamatergic projections to LHb to explore its function in wake-anesthesia regulation of isoflurane anesthesia.Results:The glutamatergic projections of BF mainly project to LHb,optogenetic activation of BF glutamatergic projections to LHb could significantly prolonged the time of loss of righ-ting reflex(LORR)and shortened the time of recovery of righting reflex(RORR).Optical stimulation of this projection reduced the burst suppression ratio(BSR)under 1.0%isoflurane anesthesia.When the mice were anesthetized by 0.8%isoflurane,a significant decrease of δ power percentage was observed,while the power percentage of β bands and γ bands increased.Conclusion:The glutamatergic neurons of BF mainly project to LHb,activation of this projection can not only prolong the induction time but also shorten the emergence time.Furthermore,this projection also exerts a pro-arousal effect under the maintenance phase of isoflurane anesthesia.

Objective:To investigate the effects of acupuncture intervention on the expression of circRNA_017109 and miR-138-5p in the hippocampal region of rats with cerebral ischemia-reperfusion injury(CIRI),and to explore the potential mechanism of acupuncture in treating CIRI.Methods:Forty-five Sprague-Dawley(SD)rats were divided into sham group,middle cerebral artery occlusion/reperfusion(MCAO/R)group,and acupuncture(MCAO/R+AC)group.The MCAO/R and MCAO/R+AC groups were subjected to the modified Longa suture method to establish the MCAO/R model.The MCAO/R+AC group received acupuncture at the"Dazhui","Renzhong",and"Baihui"acu-points.Neurological impairment was assessed via the modified neurological severity score(mNSS).while cerebral infarct volume was assessed through TTC staining.Histological alterations in brain tissue were examined via HE stai-ning,and the expression of circRNA_017109 and miR-138-5p in the hippocampal area were determined using RT-qPCR.Results:The mNSS scores and cerebral infarct volume ratios in the model groups were markedly higher than those in the sham group(P<0.01),but these were significantly reduced in MCAO/R+AC group compared to the MCAO/R group(P<0.01).HE staining showed severe necrosis and inflammation in the MCAO/R group's hippocam-pus,while the MCAO/R+AC group had minimal necrosis and scattered inflammation.RT-qPCR results indicated that the expression of circRNA_017109 and miR-138-5p was downregulated in the MCAO/R group,while MCAO/R+AC group upregulated the expression of circRNA_017109 and miR-138-5p(P<0.05,P<0.01).Conclusion:Acupunc-ture can improve neurological deficits,reduce cerebral infarction volume,and alleviate inflammatory cell infiltration in CIRI rats by upregulating the expression of circRNA_017109 and miR-138-5p,thereby exerting neuroprotective effects.

Objective:To explore the effects of adenosine on learning and memory in rats thromboembolic stroke(TS)and its mechanism based on PI3K/Akt/CREB signaling pathway.Methods:36 male SD rats were randomly di-vided into sham group(Sham),TS group,TS+adenosine(TS+Ade)group and TS+adenosine+Akt inhibitor(TS+Ade+MK-2206)group.Preparation of rat TS model used autologous thrombus intravascular formation method.Mor-ris water maze(MWM)test was used to observe the learning and memory ability of rats.The morphological changes of hippocampal neurons were observed by Nissl staining.The expression of glutamate transporter 1(GLT-1)and glial fi-brillary acidic protein(GFAP)in CA1 region was determined by immunofluorescence.The protein expressions of PI3K,p-PI3K,Akt,p-Akt,CREB and p-CREB were determined by Western blot.Results:Through the rat TS mod-el,adenosine was able to improve the learning and memory ability,improve the hippocampal neuron cell morphological abpormality and vacuolation,nucleolar condensation and other phenomena in rats.Immunofluorescence results showed that GLT-1 and GFAP were co-localized,and adenosine could significantly enhance the fluorescence intensity of GFAP and GLT-1.Western blot results showed that adenosine can enhance the expression of p-PI3K,p-Akt,and p-CREB proteins,but the use of Akt inhibitors can to some extent inhibit the phosphorylation of PI3K,Akt,and CREB.Conclusion:Adenosine may enhance the expression of GLT-1 in astrocytes and increase the clearance of glutamate through activation of PI3K/Akt/CREB signaling pathway,thereby reducing the excitotoxicity and improving recognition and memory function.However,this effect is partially blocked by Akt inhibitors.