Neuronal autophagy aggravates microglial inflammatory injury by downregulating CX3CL1/fractalkine after ischemic stroke

Ischemic stroke often induces excessive neuronal autophagy, resulting in brain damage; meanwhile, inflammatory responses stimulated by ischemia exacerbate neural injury. However, interactions between neuronal autophagy and microglial inflammation following ischemic stroke are poorly understood. CX3CL1/fractalkine, a membrane-bound chemokine expressed on neurons, can suppress microglial inflammation by binding to its receptor CX3CR1 on microglia. In the present study, to investigate whether autophagy could alter CX3CL1 expression on neurons and consequently change microglial inflammatory activity, middle cerebral artery occlusion(MCAO) was established in Sprague-Dawley rats to model ischemic stroke, and tissues from the ischemic penumbra were obtained to evaluate autophagy level and microglial inflammatory activity. MCAO rats were administered 3-methyladenine(autophagy inhibitor) or Tat-Beclin 1(autophagy inducer). Western blot assays were conducted to quantify expression of Beclin-1, nuclear factor kappa Bp65(NF-κB), light chain 3B(LC3B), and CX3CL1 in ischemic penumbra. Moreover, immunofluorescence staining was performed to quantify numbers of LC3B-, CX3CL1-, and Iba-1-positive cells in ischemic penumbra. In addition, enzyme linked immunosorbent assays were utilized to analyze concentrations of tumor necrosis factor alpha(TNF-α), interleukin 6(IL-6), interleukin 1 beta(IL-1β), and prostaglandin E2(PGE2). A dry/wet weight method was used to detect brain water content, while 2,3,5,-triphenyltetrazolium chloride staining was utilized to measure infarct volume. The results demonstrated that autophagy signaling(Beclin-1 and LC3B expression) in penumbra was prominently activated by MCAO, while CX3CL1 expression on autophagic neurons was significantly reduced and microglial inflammation was markedly activated. However, after inhibition of autophagy signaling with 3-methyladenine, CX3CL1 expression on neurons was obviously increased, whereas Iba-1 and NF-κB expression was downregulated; TNF-α, IL-6, IL-1β, and PGE2 levels were decreased; and cerebral edema was obviously mitigated. In contrast, after treatment with the autophagy inducer Tat-Beclin 1, CX3CL1 expression on neurons was further reduced; Iba-1 and NF-κB expression was increased; TNF-α, IL-6, IL-1β, and PGE2 levels were enhanced; and cerebral edema was aggravated. Our study suggests that ischemia-induced neuronal autophagy facilitates microglial inflammatory injury after ischemic stroke, and the efficacy of this process may be associated with downregulated CX3CL1 expression on autophagic neurons.

Enriched environment boosts the post-stroke recovery of neurological function by promoting autophagy

Autophagy is crucial for maintaining cellular homeostasis,and can be activated after ischemic stroke.It also participates in nerve injury and repair.The purpose of this study was to investigate whether an enriched environment has neuroprotective effects through affecting autophagy.A Sprague-Dawley rat model of transient ischemic stroke was prepared by occlusion of the middle cerebral artery followed by reperfusion.One week after surgery,these rats were raised in either a standard environment or an enriched environment for 4 successive weeks.The enriched environment increased Beclin-1 expression and the LC3-II/LC3-I ratio in the autophagy/lysosomal pathway in the penumbra of middle cerebral artery-occluded rats.Enriched environment-induced elevations in autophagic activity were mainly observed in neurons.Enriched environment treatment also promoted the fusion of autophagosomes with lysosomes,enhanced the lysosomal activities of lysosomal-associated membrane protein 1,cathepsin B,and cathepsin D,and reduced the expression of ubiquitin and p62.After 4 weeks of enriched environment treatment,neurological deficits and neuronal death caused by middle cerebral artery occlusion/reperfusion were significantly alleviated,and infarct volume was significantly reduced.These findings suggest that neuronal autophagy is likely the neuroprotective mechanism by which an enriched environment promotes recovery from ischemic stroke.This study was approved by the Animal Ethics Committee of the Kunming University of Science and Technology,China(approval No.5301002013855)on March 1,2019.

Dynamic changes in neuronal autophagy and apoptosis in the ischemic penumbra following permanent ischemic stroke

The temporal dynamics of neuronal autophagy and apoptosis in the ischemic penumbra following stroke remains unclear.Therefore,in this study,we investigated the dynamic changes in autophagy and apoptosis in the penumbra to provide insight into potential therapeutic targets for stroke.An adult Sprague-Dawley rat model of permanent ischemic stroke was prepared by middle cerebral artery occlusion.Neuronal autophagy and apoptosis in the penumbra post-ischemia were evaluated by western blot assay and immunofluorescence staining with antibodies against LC3-Ⅱ and cleaved caspase-3,respectively.Levels of both LC3-Ⅱ and cleaved caspase-3 in the penumbra gradually increased within 5 hours post-ischemia.Thereafter,levels of both proteins declined,especially LC3-Ⅱ.The cerebral infarct volume increased slowly 1–4 hours after ischemia,but subsequently increased rapidly until 5 hours after ischemia.The severity of the neurological deficit was positively correlated with infarct volume.LC3-Ⅱ and cleaved caspase-3 levels were high in the penumbra within 5 hours after ischemia,and after that,levels of these proteins decreased at different rates.LC3-Ⅱ levels were reduced to a very low level,but cleaved caspase-3 levels remained high 72 hours after ischemia.These results indicate that there are temporal differences in the activation status of the autophagic and apoptotic pathways.This suggests that therapeutic targeting of these pathways should take into consideration their unique temporal dynamics.

Autophagy Elicits Neuroprotection at the Subacute Phase of Transient Cerebral Ischaemia but Has Few Effects on Neurological Outcomes After Permanent Ischaemic Stroke in Rats

Objective Autophagy was prominently activated by cerebral ischaemia.This study was to investigate the exact role of autophagy in ischaemic stroke.Methods Two rat models of transient middle cerebral artery occlusion(tMCAO)and permanent MCAO(pMCAO)were prepared.The brain tissues in the penumbra were obtained to observe the dynamic variations of autophagy activity with Beclin1 and LC3 antibodies by Western blotting.At the characteristic time points,when autophagy activity was markedly elevated or reduced,the autophagy activation signaling was intervened with rapamycin and 3-methyladenine,respectively.Thereafter,key proteins in the autopahgic/lysosomal pathway were detected with the antibodies of LC3,p62,ubiquitin,LAMP-1 and cathepsin B.Meanwhile,TTC staining,neurological score and immunofluorescence were performed to evaluate brain infarct volume,neurological deficit and neuron survival,respectively.Results Both Beclin1 and LC3 expression levels were remarkably altered at 6 h,12 h,2 days and 7 days after tMCAO.Interestingly,the dynamic changes of autophagy activity following pMCAO were identical to those after tMCAO.Neither autophagy induction nor autophagy inhibition was able to ameliorate the pMCAO-induced neurological injury due to lysosomal dysfunction,as indicated by low levels of LAMP-1 and cathepsin B,accompanied with the accumulation of LC3-II,ubiquitin and insoluble p62.Comparatively,autophagy induction elicited overt neuroprotection at 2 and 7 days after tMCAO,and this neuroprotection might be elicited by the enhancement of autophagy flux.Conclusion Our study suggests that autophagy confers neuroprotection at the subacute phase of tMCAO but has few effects on neurological outcomes after pMCAO.