TRPC6 Knockout Alleviates Renal Fibrosis through PI3K/AKT/GSK3B Pathway

Objective Renal fibrosis is the ultimate pathway of various forms of acute and chronic kidney damage.Notably,the knockout of transient receptor potential channel 6(TRPC6)has shown promise in alleviating renal fibrosis.However,the regulatory impact of TRPC6 on renal fibrosis remains unclear.Methods In vivo,TRPC6 knockout(TRPC6−/−)mice and age-matched 129 SvEv(WT)mice underwent unilateral renal ischemia-reperfusion(uIR)injury surgery on the left renal pedicle or sham operation.Kidneys and serum were collected on days 7,14,21,and 28 after euthanasia.In vitro,primary tubular epithelial cells(PTECs)were isolated from TRPC6−/−and WT mice,followed by treatment with transforming growth factorβ1(TGFβ1)for 72 h.The anti-fibrotic effect of TRPC6−/−and the underlying mechanisms were assessed through hematoxylin-eosin staining,Masson staining,immunostaining,qRT-PCR,and Western blotting.Results Increased TRPC6 expression was observed in uIR mice and PTECs treated with TGFβ1.TRPC6−/−alleviated renal fibrosis by reducing the expression of fibrotic markers(Col-1,α-SMA,and vimentin),as well as decreasing the apoptosis and inflammation of PTECs during fibrotic progression both in vivo and in vitro.Additionally,we found that the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(AKT)/glycogen synthase kinase 3 beta(GSK3β)signaling pathway,a pivotal player in renal fibrosis,was down-regulated following TRPC6 deletion.Conclusion These results suggest that the ablation of TRPC6 may mitigate renal fibrosis by inhibiting the apoptosis and inflammation of PTECs through down-regulation of the PI3K/AKT/GSK3βpathway.Targeting TRPC6 could be a novel therapeutic strategy for preventing chronic kidney disease.

Effect of the combination of high-frequency repetitive magnetic stimulation and neurotropin on injured sciatic nerve regeneration in rats

Repetitive magnetic stimulation is effective for treating posttraumatic neuropathies following spinal or axonal injury.Neurotropin is a potential treatment for nerve injuries like demyelinating diseases.This study sought to observe the effects of high-frequency repetitive magnetic stimulation,neurotropin and their combined use in the treatment of peripheral nerve injury in 32 adult male Sprague-Dawley rats.To create a sciatic nerve injury model,a 10 mm-nerve segment of the left sciatic nerve was cut and rotated through 180°and each end restored continuously with interrupted sutures.The rats were randomly divided into four groups.The control group received only a reversed autograft in the left sciatic nerve with no treatment.In the high-frequency repetitive magnetic stimulation group,peripheral high-frequency repetitive magnetic stimulation treatment(20 Hz,20 min/d)was delivered for 10 consecutive days after auto-grafting.In the neurotropin group,neurotropin therapy(0.96 NU/kg per day)was administrated for 10 consecutive days after surgery.In the combined group,the combination of peripheral high-frequency repetitive magnetic stimulation(20 Hz,20 min/d)and neurotropin(0.96 NU/kg per day)was given for 10 consecutive days after the operation.The Basso-Beattie-Bresnahan locomotor rating scale was used to assess the behavioral recovery of the injured nerve.The sciatic functional index was used to evaluate the recovery of motor functions.Toluidine blue staining was performed to determine the number of myelinated fibers in the distal and proximal grafts.Immunohistochemistry staining was used to detect the length of axons marked by neurofilament 200.Our results reveal that the Basso-Beattie-Bresnahan locomotor rating scale scores,sciatic functional index,the number of myelinated fibers in distal and proximal grafts were higher and axon lengths were longer in the high-frequency repetitive magnetic stimulation,neurotropin and combined groups compared with the control group.These measures were not significantly different among the high-frequency repetitive magnetic stimulation,neurotropin and combined groups.Therefore,our results suggest that peripheral high-frequency repetitive magnetic stimulation or neurotropin can promote the repair of injured sciatic nerves,but their combined use seems to offer no significant advantage.This study was approved by the Animal Ethics Committee of the Affiliated Changzhou No.2 People’s Hospital of Nanjing Medical University,China on December 23,2014(approval No.2014keyan002-01).

Hydrogen sulfide enhances adult neurogenesis in a mouse model of Parkinson's disease

Hydrogen sulfide(H_2S) is regarded to be a protectant against diseases of the central nervous system and cardiovascular system.However, the mechanism by which H_2S elicits neuroprotective effects in the progression of Parkinson's disease(PD) remains unclear.To investigate the role of H_2S in delaying the pathological process of PD, we used the most common sodium hydrosulfide(Na HS) as an H_2S donor and established a mouse model of PD induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/probenecid(MPTP/p) in the present study.Our results show that H_2S reduced neuronal loss during the progression of PD.Notably, we found that H_2S exhibited protective effects on dopaminergic neurons.Excitingly, H_2S also increased the proliferation of neural stem cells in the subventricular zone.Next, we evaluated whether the neuroprotective effects of H_2S on dopaminergic neurons in PD are dependent on adult nerve regeneration by treating primary adult neural stem cells cultured ex vivo with 1-methyl-4-phenylpyridine.Our results show that H_2S could prevent nerve injury induced by 1-methyl-4-phenylpyridine, promote the growth of neurospheres, and promote neurogenesis by regulating Akt/glycogen synthase kinase-3β/β-catenin pathways in adult neural stem cells.These findings confirm that H_2S can increase neurogenesis in an adult mouse model of PD by regulating the Akt/glycogen synthase kinase-3β/β-catenin signaling pathway.This study was approved by the Animal Care and Use Committee of Nanjing Medical University, China(IACUC Approval No.1601153-3).

Preventive and Therapeutic Potential of Vitamin C in Mental Disorders

In this review,we summarize the involvement of vitamin C in mental disorders by presenting available evidence on its pharmacological effects in animal models as well as in clinical studies.Vitamin C,especially its reduced form,has gained interest for its multiple functions in various tissues and organs,including central nervous system（CNS）.Vitamin C protects the neuron against oxidative stress,alleviates inflammation,regulates the neurotransmission,affects neuronal development and controls epigenetic function.All of these processes are closely associated with psychopathology.In the past few decades,scientists have revealed that the deficiency of vitamin C may lead to motor deficit,cognitive impairment and aberrant behaviors,whereas supplement of vitamin C has a potential preventive and therapeutic effect on mental illness,such as major depressive disorder（MDD）,schizophrenia,anxiety and Alzheimer＇s disease（AD）.Although several studies support a possible role of vitamin C against mental disorders,more researches are essential to accelerate the knowledge and investigate the mechanism in this field.

Identification and Function of Acid-sensing Ion Channels in RAW 264.7 Macrophage Cells

Activation of acid-sensing ion channels （ASICs） plays an important role in neuroinflammation. Macrophage recruitment to the sites of inflammation is an essential step in host defense. ASIC1 and ASIC3 have been reported to mediate the endocytosis and maturation of bone marrow derived macrophages. However, the expression and inflammation-related functions of ASICs in RAW 264.7 cells, another common macrophage, are still elusive. In the present study, we first demonstrated the presence of ASIC 1, ASIC2a and ASIC3 in RAW 264.7 macrophage cell line by using reverse transcriptase polymerase chain reaction （RT-PCR）, Western blotting and immunofluorescence experiments. The non-specific ASICs inhibitor amiloride and specific homomeric ASICla blocker PcTxl reduced the production of iNOS and COX-2 by LPS-induced activating RAW 264.7 cells. Furthermore, not only amiloride but also PcTxl inhibited the migration and LPS-induced apoptosis of RAW 264.7 cells. Taken together, our findings suggest that ASICs promote the inflammatory response and apoptosis of RAW 264.7 cells, and ASICs may serve as a potential novel target for immunological disease therapy.

ROCK Inhibition with Fasudil Promotes Early Functional Recovery of Spinal Cord Injury in Rats by Enhancing Microglia Phagocytosis

Emerging evidence indicates that microglia activation plays an important role in spinal cord injury（SCI） caused by trauma. Studies have found that inhibiting the Rho/Rho-associated protein kinase（ROCK） signaling pathway can reduce inflammatory cytokine production by microglia. In this study, Western blotting was conducted to detect ROCK2 expression after the SCI; the ROCK Activity Assay kit was used for assay of ROCK pathway activity; microglia morphology was examined using the CD11 b antibody; electron microscopy was used to detect microglia phagocytosis; TUNEL was used to detect tissue cell apoptosis; myelin staining was performed using an antibody against myelin basic protein（MBP）; behavioral outcomes were evaluated according to the methods of Basso, Beattie, and Bresnahan（BBB）. We observed an increase in ROCK activity and microglial activation after SCI. The microglia became larger and rounder and contained myelin-like substances. Furthermore, treatment with fasudil inhibited neuronal cells apoptosis, alleviated demyelination and the formation of cavities, and improved motor recovery. The experimental evidence reveals that the ROCK inhibitor fasudil can regulate microglial activation, promote cell phagocytosis, and improve the SCI microenvironment to promote SCI repair. Thus, fasudil may be useful for the treatment of SCI.

Brain-derived neurotrophic factor and neural plasticity in a rat model of spinal cord transection

The present study employed a rat model of T10 spinal cord transection. Western blot analyses revealed increased brain-dedved neurotrophic factor （BDNF） expression in spinal cord segments caudal to the transection site following injection of replication incompetent herpes simplex virus vector （HSV-BDNF） into the subarachnoid space. In addition, hindlimb locomotor functions were improved. In contrast, BDNF levels decreased following treatment with replication defective herpes simplex virus vector construct small interference BDNF （HSV-siBDNF）. Moreover, hindlimb locomotor functions gradually worsened. Compared with the replication incompetent herpes simplex virus vector control group, extracellular signal regulated kinasel/2 expression increased in the HSV-BDNF group on days 14 and 28 after spinal cord transection, but expression was reduced in the HSV-siBDNF group. These results suggested that BDNF plays an important role in neural plasticity via extracellular signal regulated kinasel/2 signaling pathway in a rat model of adult spina cord transection.

Inhibition of ciliary neurotrophic factor in a rat model of transected spinal cord

Ciliary neurotrophic factor （CNTF） dramatically increases following spinal cord injury and participates in the repair process, although some studies have shown that CNTF plays a role in promoting glial scar formation following spinal cord injury. The antibody closure model can be used to inhibit CNTF expression following spinal cord injury, thereby furthering the understanding of the role of CNTF in spinal cord injury repair. In the present experiment, spinal catheters were placed in the vertebral canal of spinal cord transected rats, and CNTF antibodies were injected following fixation of the paraspinal muscle catheter. At 24 hours after a single CNTF antibody injection, CNTF expression decreased in the thoracic and lumbar spinal cord and recovered to normal levels by 48 72 hours. CNTF antibody treatment can effectively block endogenous CNTF expression in the thoracic and lumbar spinal cord during an interval of less than 24 hours in transected rats.

Dimethyl sulfide,a metabolite of the marine microorganism,protects SH-SY5Y cells against 6-hydroxydopamine and MPP~＋-induced apoptosis

Dimethyl sulfide(DMS)has been historically recognized as a metabolite of the marine microorganism or a disgusting component for the smell of halitosis patients.In our recent study,DMS has been identified as a cytoprotectant that protects against oxidative-stress induced cell death and aging.We found that at near-physiological concentrations,DMS reduced reactive oxygen species(ROS)in cultured PC12 cells and alleviated oxidative stress.The radical-scavenging capacity of DMS at near-physiological concentration was equivalent to endogenous methionine(Met)-centered antioxidant defense.Methionine sulfoxidereductase A(MsrA),the key antioxidant enzyme in Met-centered defense,bound to DMS and promoted its antioxidant capacity via facilitating the reaction of DMS with ROS through a sulfonium intermediate at residues Cys72,Tyr103,Glu115,followed by the release of dimethyl sulfoxide(DMSO).MTT assay and trypan blue test indicated that supplement of DMS exhibited cytoprotection against 6-hydroxydopamine and MPP+induced cell apoptosis.Furthermore,Msr A knockdown abolished the cytoprotective effect of DMS at near-physiological concentrations.The present study reveals new insight into the potential therapeutic value of DMS in Parkinson disease.

Epigenetic regulation of SARS-Cov-2 spike protein modulates cellular viability and nascent RNA transcription in neuronal cells

SARS-CoV-2 has been demonstrated to be highly susceptible to neuron-and glial-like cells,1 but there is no consensus regarding how the virus affects neurons following neuroinvasion.It is therefore unknown if and how SARS-CoV-2 maintains the homeostasis of neuronal cells.Herein,we revealed that the receptor-binding domain(RBD)of the SARS-CoV-2 spike protein acted as an extracellular remodeling component to undermine epigenetic regulation,perturbnascentRNA(nsRNA)transcription,promote mitochondrial biogenesis,and increase cellular viability in SH-SY5Y cells.

The Rho-associated kinase inhibitors Y27632 and fasudil promote microglial migration in the spinal cord via the ERK signaling pathway

Rho-associated kinase（ROCK） is a key regulatory protein involved in inflammatory secretion in microglia in the central nervous system.Our previous studies showed that ROCK inhibition enhances phagocytic activity in microglia through the extracellular signal-regulated kinase（ERK） signaling pathway,but its effect on microglial migration was unknown.Therefore,in this study,we investigated the effects of the ROCK inhibitors Y27632 and fasudil on the migratory activity of primary cultured microglia isolated from the spinal cord,and we examined the underlying mechanisms.The microglia were treated with Y27632,fasudil and/or the ERK inhibitor U0126.Cellular morphology was observed by immunofluorescence.Transwell chambers were used to assess cell migration.ERK levels were measured by incell western blot assay.Y27632 and fasudil increased microglial migration,and the microglia were irregularly shaped and had many small processes.These inhibitors also upregulated the levels of phosphorylated ERK protein.The ERK inhibitor U0126 suppressed these effects of Y27632 and fasudil.These findings suggest that the ROCK inhibitors Y27632 and fasudil promote microglial migration in the spinal cord through the ERK signaling pathway.

HOXA11-AS aggravates microglia-induced neuroinflammation after traumatic brain injury

Long noncoding RNAs(lnc RNAs)participate in many pathophysiological processes after traumatic brain injury by mediating neuroinflammation and apoptosis.Homeobox A11 antisense RNA(HOXA11-AS)is a member of the lnc RNA family that has been reported to participate in many inflammatory reactions;however,its role in traumatic brain injury remains unclear.In this study,we established rat models of traumatic brain injury using a weight-drop hitting device and injected LV-HOXA11-AS into the right lateral ventricle 2 weeks before modeling.The results revealed that overexpression of HOXA11-AS aggravated neurological deficits in traumatic brain injury rats,increased brain edema and apoptosis,promoted the secretion of proinflammatory factors interleukin-1β,interleukin-6,and tumor necrosis factorα,and promoted the activation of astrocytes and microglia.Microglia were treated with 100 ng/m L lipopolysaccharide for 24 hours to establish in vitro cell models,and then transfected with pc DNA-HOXA11-AS,mi R-124-3 p mimic,or sh-MDK.The results revealed that HOXA11-AS inhibited mi R-124-3 p expression and boosted MDK expression and TLR4-nuclear factor-κB pathway activation.Furthermore,lipopolysaccharide enhanced potent microglia-induced inflammatory responses in astrocytes.Forced overexpression of mi R-124-3 p or downregulating MDK repressed microglial activation and the inflammatory response of astrocytes.However,the mi R-124-3 p-mediated anti-inflammatory effects were reversed by HOXA11-AS.These findings suggest that HOXA11-AS can aggravate neuroinflammation after traumatic brain injury by modulating the mi R-124-3 p-MDK axis.This study was approved by the Animal Protection and Use Committee of Southwest Medical University(approval No.SMU-2019-042)on February 4,2019.

Ligustrazine Attenuates Hyperhomocysteinemia-induced Alzheimer-like Pathologies in Rats

Ligustrazine,an alkaloid extracted from the traditional Chinese herbal medicine Ligusticum Chuanxiong Hort,has been clinically applied to treat the cerebrovascular diseases.Hyperhomocystcincmia(Hhcy)is an independent risk factor for Alzheimer's disease(AD).Memory deficits can be caused by Hhcy via pathologies of Aβ-like tau and amyloid-β(Aβ)in the hippocampus.Here,we investigated whether homocysteine(Hey)can induce Aβ-like pathologies and the effects of ligustrazine on these pathologies.The Hey rat model was constructed by 14-day Hey injection via vena caudalis,and rats were treated with daily intragastric administration of ligustrazine at the same time.We found that the pathologies of tau and Aβ were induced by Hey in the hippocampus,while the Hcy-induced tau hyperphosphorylation and Aβ accumulation could be markedly attenuated by simultaneous ligustrazine treatment.Our data demonstrate that ligustrazine may be used as a promising neuroprotective agent to treat the Hcy-induced Aβ-like pathologies.

Astrocyte transplantation for repairing the injured spinal cord

Spinal cord injury(SCI)leads to permanent deficits in neural function without effective therapies,which places a substantial burden on families and society.Astrocytes,the major glia supporting the normal function of neurons in the spinal cord,become active and form glial scars after SCI,which has long been regarded as a barrier for axon regeneration.However,recent progress has indicated the beneficial role of astrocytes in spinal repair.During the past three decades,astrocyte transplantation for SCI treatment has gained increasing attention.In this review,we first summarize the progress of using rodent astrocytes as the primary step for spinal repair.Rodent astrocytes can survive well,migrate extensively,and mature in spinal injury;they can also inhibit host reactive glial scar formation,stimulate host axon regeneration,and promote motor,sensory,respiratory,and autonomic functional recovery.Then,we review the progress in spinal repair by using human astrocytes of various origins,including the fetal brain,fetal spinal cord,and pluripotent stem cells.Finally,we introduce some key questions that merit further research in the future,including rapid generation of large amounts of human astrocytes with high purity,identification of the right origins of astrocytes to maximize neural function improvement while minimizing side effects,testing human astrocyte transplantation in chronic SCI,and verification of the long-term efficacy and safety in large animal models.

Ankaflavin ameliorates steatotic liver ischemiareperfusion injury in mice

BACKGROUND： It is well-known that steatotic liver is more susceptible to ischemia-reperfusion （I/R） injury during liver transplantation, liver resection and other liver surgeries. The increasing incidence of non-alcoholic fatty liver disease （NAFLD） decreases the availability of liver donors. Although steatotic liver is now accepted as a source of liver for trans- plantation, NAFLD exacerbates the liver injury after liver surgery. The present study was to investigate the protective role of ankaflavin in steatotic liver I/R injury.

Updated Understanding of the Glial-Vascular Unit in Central Nervous System Disorders

The concept of the glial-vascular unit(GVU)was raised recently to emphasize the close associations between brain cells and cerebral vessels,and their coordinated reactions to diverse neurological insults from a“glio-centric”view.GVU is a multicellular structure composed of glial cells,perivascular cells,and perivascular space.Each component is closely linked,collectively forming the GVU.The central roles of glial and perivascular cells and their multi-level interconnections in the GVU under normal conditions and in central nervous system(CNS)disorders have not been elucidated in detail.Here,we comprehensively review the intensive interactions between glial cells and perivascular cells in the niche of perivascular space,which take part in the modulation of cerebral blood flow and angiogenesis,formation of the blood-brain barrier,and clearance of neurotoxic wastes.Next,we discuss dysfunctions of the GVU in various neurological diseases,including ischemic stroke,spinal cord injury,Alzheimer’s disease,and major depression disorder.In addition,we highlight the possible therapies targeting the GVU,which may have potential clinical applications.

Nerve Growth Factor Inhibits Gd^(3+)-sensitive Calcium Influx and Reduces Chemical Anoxic Neuronal Death

To investigate whether glutamate and voltage-gated calcium channels-independent calcium influx exists during acute anoxic neuronal damage and its possible relationship to neuronal protective function of NGF. In in vitro model of acute anoxia, hippocampal cultures from newborn rats were exposed to 3 mmol/L KCN. Changes of intracellular Ca^2＋ concentration （[Ca^2＋]i） were monitored by con-focal imaging and cell viability was assayed by PI and cFDA staining. The results showed that after treatment with primary hippocampal cultures with 3 mmol/L KCN for 15 min, [Ca^2＋]i was significantly increased 6.27-fold compared to pre-anoxia level and 73.3% of the cells died. When combination of 20 μmol/L MK-801 （glutamate receptor antagonist）, 40 μmol/L CNQX （AMPA receptor antagonist） and 5 μmol/L nimodipine （voltage-gated calcium channel antagonist） （hereafter denoted as MCN） were administrated to hippocampal cultures, levels of [Ca^2＋]i and cell death rate induced by KCN were partially reduced by 35.9% and 47.5% respectively. However, Gd^3＋ （10 μmol/L） almost completely blocked KCN-mediated [Ca^2＋]i elevation by 81.9% and reduced neuronal death by 88.8% in the presence of MCN. It is noteworthy that NGF, used in combination with MCN, inhibited KCN-induced [Ca^2＋]i increase by 77.4% and reduced cell death by 87.1%. Only PLC in- hibitor U73122 （10 μmol/L） abolished NGF effects. It is concluded that Gd^3＋-sensitive calcium influx, which is NMDA （glutamate receptor） and voltage-gated calcium channels-independent, is responsible for acute anoxic neuronal death. NGF can inhibit Gd^3＋-sensitive calcium influx and reduce anoxic neuronal death through activating PLC pathway.

Digoxin Ameliorates Glymphatic Transport and Cognitive Impairment in a Mouse Model of Chronic Cerebral Hypoperfusion

The glymphatic system plays a pivotal role in maintaining cerebral homeostasis.Chronic cerebral hypoperfusion,arising from small vessel disease or carotid stenosis,results in cerebrometabolic disturbances ultimately manifesting in white matter injury and cognitive dysfunction.However,whether the glymphatic system serves as a potential therapeutic target for white matter injury and cognitive decline during hypoperfusion remains unknown.Here,we established a mouse model of chronic cerebral hypoperfusion via bilateral common carotid artery stenosis.We found that the hypoperfusion model was associated with significant white matter injury and initial cognitive impairment in conjunction with impaired glym・phatic system function.The glymphatic dysfunction was associated with altered cerebral perfusion and loss of aquaporin 4 polarization.Treatment of digoxin rescued changes in glymphatic transport,white matter structure,and cognitive function.Suppression of glymphatic functions by treatment with the AQP4 inhibitor TGN-020 abolished this protective effect of digoxin from hypoperfusion injury.Our research yields new insight into the relationship between hemodynamics,glymphatic transport,white matter injury,and cognitive changes after chronic cerebral hypoperfusion.

Needle Sensation and Personality Factors Influence Therapeutic Effect of Acupuncture for Treating Bell＇s Palsy： A Secondary Analysis of a Multicenter Randomized Controlled Trial

Background： It has not been solved what kind of needle sensation might influence outcomes of acupuncture treatment. Effects of personality factors on the therapeutic effect of acupuncture have not been investigated. This study aimed to find the effects of the traits of personality on the objective outcome when different acupuncture techniques were used in treating patients with Bell＇s palsy. Methods： We performed a secondary analysis of a prospective multicenter randomized controlled trial of acupuncture for Bell＇s palsy. Patients were randomly assigned to the de qi and control groups, respectively. The primary outcome was facial nerve function at month 6. The intensity of each needle sensation was rated by a visual analog scale. Psychosocial thctors were assessed by the pretreatment mediator questionnaire： 16 Personality Factor Questionnaire （16PF） was used tbr assessing personality factors and digit cancellation test for assessing attention. Results： After 6 months, patients in the de qi group had better facial function （adjusted odds ratio [OR]： 4.16, 95% confidence interval [CI]： 2.23-7.78）. Path analysis showed that intensity of needle sensation of fullness had direct effect on House-Brackmann （HB） score at month 6. In de qi group, the low HB score on day I （OR： 0.13, 95% （_7： 0.03-0.45） and the low Social Boldness score （OR： 0.63, 95% CI： 0.41-0.97） in 16PF were associated with better facial function. In control group, low HB score on day 1 （OR： 0.25, 95% CI： 0.13-0.50）, low Vigilance score （OR： 0.66, 95% CI： 0.50-0.88）, and high Tension score （OR： 1.41,95% CI： 1.12 1.77） in 16PF were related to better facial function. Conclusions： The needle sensation of fullness could predict better facial function and personality traits might influence outcomes of acupuncture treatment. Both of them should be considered seriously in acupuncture treatment and research.

Multiple Mild Stimulations Reduce Membrane Distribution of CX3CR1 Promoted by Annexin al in Microglia to Attenuate Excessive Dendritic Spine Pruning and Cognitive Deficits Caused by a Transient Ischemic Attack in Mice

A transient ischemic attack(TIA)can cause reversible and delayed impairment of cognition,but the specific mechanisms arestill unclear.Annexin al(ANXA1)is a phospholipid-binding protein.Here,we confirmed that cognition and hippocampal synapses were impaired in TIA-treated mice,and this could be rescued by multiple mild stimulations(MMS).TIA promoted the interaction of ANXAl and CX3CR1,increased the membrane distribution of CX3CR1 in microglila,and thus enhanced the CX3CR1 and CX3CL1 interaction.These phenomena induced by TIA could be reversed by MMS.Meanwhile,the CX3CR1 membrane distribution and CX3CR1-CX3CL1 interaction were upregulated in primary cultured microglia overexpressing ANXAl,and the spine density was significantly reduced in co-cultured microglia overexpressing ANXAl and neurons.Moreover,ANXAl overexpression in microglia abolished the protection of MMS after TIA.Collectively,our study provides a potential strategy for treating the delayed synaptic injury caused by TIA.