The role of autophagic and lysosomal pathways in ischemic brain injury******

Autophagy is involved in neural cell death after cerebral ischemia. Our previous studies showed that rapamycin-induced autophagy decreased the rate of apoptosis, but the rate of apoptosis was in-creased after the autophagy inhibitor, 3-methyladenine, was used. In this study, a suture-occluded method was performed to generate a rat model of brain ischemia. Under a transmission electron microscope, autophagic bodies and autophagy lysosomes were markedly accumulated in neurons at 4 hours post brain ischemic injury, with their numbers gradually reducing over time. Western blotting demonstrated that protein levels of light chain 3-II and cathepsin B were significantly in-creased within 4 hours of ischemic injury, but these levels were not persistently upregulated over time. Confocal microscopy showed that autophagy was mainly found in neurons with positive light chain 3 signal. Injection of rapamycin via tail vein promoted the occurrence of autophagy in rat brain tissue after cerebral ischemia and elevated light chain 3 and cathepsin B expression. However, in-jection of 3-methyladenine significantly diminished light chain 3-II and cathepsin B expression. Results verified that autophagic and lysosomal activity is increased in ischemic neurons. Abnormal components in cells can be eliminated through upregulating cell autophagy or inhibiting autophagy after ischemic brain injury, resulting in a dynamic balance of substances in cells. Moreover, drugs that interfere with autophagy may be potential therapies for the treatment of brain injury.     

Excessive Autophagy Contributes to Neuron Death in Cerebral Ischemia

Aims: To determine the extent to which autophagy contributes to neuronal death in cerebral hypoxia and ischemia. Methods: We performed immunocytochemistry, western blot, cell viability assay, and electron microscopy to analyze autophagy activities in vitro and in vivo. Results: In both primary cortical neurons and SH‐SY5Y cells exposed to oxygen and glucose deprivation (OGD)for 6 h and reperfusion (RP) for 24, 48, and 72 h, respectively, an increase of autophagy was observed as determined by the increased ratio of LC3‐II to LC3‐I and Beclin‐1 (BECN1) expression. Using Fluoro‐Jade C and monodansylcadaverine double‐staining, and electron microscopy we found the increment in autophagy after OGD/RP was accompanied by increased autophagic cell death, and this increased cell death was inhibited by the specific autophagy inhibitor, 3‐methyladenine. The presence of large autolysosomes and numerous autophagosomes in cortical neurons were confirmed by electron microscopy. Autophagy activities were increased dramatically in the ischemic brains 3–7 days postinjury from a rat model of neonatal cerebral hypoxia/ischemia as shown by increased punctate LC3 staining and BECN1 expression. Conclusion: Excessive activation of autophagy contributes to neuronal death in cerebral ischemia.     

A combination of four active compounds alleviates cerebral ischemia–reperfusion injury in correlation with inhibition of autophagy and modulation of AMPK/mTOR and JNK pathways

SMXZF is a combination of Rb1, Rg1, schizandrin, and DT‐13 (6:9:5:4) derived from Sheng‐mai San, a widely used Chinese traditional medicine for the treatment of cardiovascular and cerebral diseases. The present study explores the inhibitory effects and signaling pathways of SMXZF on autophagy induced by cerebral ischemia–reperfusion injury. Male C57BL/6 mice were subjected to ischemia–reperfusion insult by right middle cerebral artery occlusion (MCAO) for 1 hr with subsequent 24 hr reperfusion. Three doses of SMXZF (4.5, 9, and 18 mg/kg) were administered intraperitoneally (i.p.) after ischemia for 1 hr. An autophagic inhibitor, 3‐methyladenine (3‐MA; 300 μg/kg), was administered i.p. 20 min before ischemia as a positive drug. We found that SMXZF significantly increased cerebral blood flow and reduced the infarct volume, brain water content, and the neurological deficits in a dose‐dependent manner. Similar to the positive control, SMXZF at 18 mg/kg also significantly inhibited autophagosome formation. Immunofluorescence staining and Western blotting demonstrated that SMXZF could significantly decrease the expression levels of beclin1 and microtubule‐associated protein 1 light chain 3. SMXZF also remarkably inhibited the phosphorylation of adenosine monophosphate‐activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) as well as the expression of c‐Jun N‐terminal kinase (JNK) and its phosphorylation induced by 24 hr reperfusion. Finally, we demonstrated that the optimal administration time of SMXZF was at the early period of reperfusion. This study reveals that SMXZF displays neuroprotective effect against focal ischemia–reperfusion injury, possibly associated with autophagy inactivation through AMPK/mTOR and JNK pathways. © 2014 Wiley Periodicals, Inc.     

Autophagy in acute brain injury: feast, famine, or folly?

In the central nervous system, increased autophagy has now been reported after traumatic brain and spinal cord injury, cerebral ischemia, intracerebral hemorrhage, and seizures. This increase in autophagy could be physiologic, converting damaged or dysfunctional proteins, lipids, and/or organelles to their amino acid and fatty acid components for recycling. On the other hand, this increase in autophagy could be supraphysiologic, perhaps consuming and eliminating functional proteins, lipids, and/or organelles as well. Whether an increase in autophagy is beneficial (feast) or detrimental (famine) in brain likely depends on both the burden of intracellular substrate targeted for autophagy and the capacity of the cell's autophagic machinery. Of course, increased autophagy observed after brain injury could also simply be an epiphenomenon (folly). These divergent possibilities have clear ramifications for designing therapeutic strategies targeting autophagy after acute brain injury and are the subject of this review. This article is part of a Special Issue entitled "Autophagy and protein degradation in neurological diseases."     

Neuronal autophagy in cerebral ischemia

Autophagy has evolved as a conserved process for the bulk degradation and recycling of cytosolic components, such as long-lived proteins and organelles. In neurons, autophagy is important for homeostasis and protein quality control and is maintained at relatively low levels under normal conditions, while it is upregulated in response to pathophysiological conditions, such as cerebral ischemic injury. However, the role of autophagy is more complex. It depends on age or brain maturity, region, severity of insult, and the stage of ischemia. Whether autophagy plays a beneficial or a detrimental role in cerebral ischemia depends on various pathological conditions. In this review, we elucidate the role of neuronal autophagy in cerebral ischemia.     

Peripheral administration of fetuin-A attenuates early cerebral ischemic injury in rats

Cerebral ischemia-elicited inflammatory responses are driven by inflammatory mediators produced both by central (e.g., neurons and microglia) and infiltrating peripheral immune cells (e.g., macrophage/monocyte), and contribute to the evolution of tissue injury. A ubiquitous molecule, spermine, is released from injured cells, and counter-regulates release of various proinflammatory cytokines. However, the spermine-mediated anti-inflammatory activities are dependent on the availability of fetuin-A, a liver-derived negative acute-phase protein. Using an animal model of focal cerebral ischemia (i.e., permanent middle cerebral artery occlusion, MCAo), we found that levels of fetuin-A in the ischemic brain tissue were elevated in a time-dependent manner, starting between 2 and 6 h, peaking around 24 to 48 h, and returning to baseline 72 h after MCAo. When administered peripherally, exogenous fetuin-A gained entry across the BBB into the ischemic brain tissue, and dose dependently reduced brain infarct volume at 24 h after MCAo. Meanwhile, fetuin-A effectively attenuated (i) ischemia-induced HMGB1 depletion from the ischemic core; (ii) activation of centrally (e.g., microglia) and peripherally derived immune cells (e.g., macrophage/monocytes); and (iii) TNF production in ischemic brain tissue. Taken together, these experimental data suggest that fetuin-A protects against early cerebral ischemic injury partly by attenuating the brain inflammatory response.     

短期禁食诱导自噬对大鼠脑缺血损伤的保护作用

目的探讨短期禁食对大鼠脑局灶性缺血再灌注损伤的保护作用及机制。方法 57只成年雄性SD大鼠随机分为五组。假手术组(S组)、缺血再灌注组(I/R组)、短期禁食组(Fasting组)、自噬抑制剂3-甲基腺嘌呤组(3-MA组)和溶剂对照组(vehicle组)。I/R组采用右侧大脑中动脉阻闭120 min后再灌注72 h。Fasting组大鼠禁食但不禁水48 h后给予脑缺血再灌注损伤。3-MA组将3-MA 30μg溶于生理盐水10μl,于短期禁食前30 min行侧脑室注射给药,24 h后第二次侧脑室注射给药,共两次。脑缺血损伤检测指标为神经行为学评分和脑梗死容积。自噬的检测采用Westernblot测定微管相关蛋白1轻链3-II(LC3-II)的表达。结果再灌注后72 h,与I/R组比较,Fasting组可改善神经行为学评分,缩小脑梗死容积。Fasting组LC3-II的蛋白表达显著高于I/R组和S组。与Fasting组比较,3-MA组神经行为学评分降低,脑梗死容积扩大,脑损伤程度3-MA组与I/R组差异无统计学意义,表明抑制短期禁食所诱导的自噬,短期禁食的脑保护作用消失。结论短期禁食通过诱导自噬的形成减轻其后发生的大鼠局灶性脑缺血再灌注损伤。     

Autophagy: novel insights into therapeutic target of electroacupuncture against cerebral ischemia/reperfusion injury

Electroacupuncture is known as an effective adjuvant therapy in ischemic cerebrovascular disease. However, its underlying mechanisms remain unclear. Studies suggest that autophagy, which is essential for cell survival and cell death, is involved in cerebral ischemia reperfusion injury and might be modulate by electroacupuncture therapy in key ways. This paper aims to provide novel insights into a therapeutic target of electroacupuncture against cerebral ischemia/reperfusion injury from the perspective of autophagy. Here we review recent studies on electroacupuncture regulation of autophagy-related markers such as UNC-51-like kinase-1 complex, Beclin1, microtubule-associated protein-1 light chain 3, p62, and autophagosomes for treating cerebral ischemia/reperfusion injury. The results of these studies show that electroacupuncture may affect the initiation of autophagy, vesicle nucleation, expansion and maturation of autophagosomes, as well as fusion and degradation of autophagolysosomes. Moreover, studies indicate that electroacupuncture probably modulates autophagy by activating the mammalian target of the rapamycin signaling pathway.This review thus indicates that autophagy is a therapeutic target of electroacupuncture treatment against ischemic cerebrovascular diseases.     

The Akt/glycogen synthase kinase-3β pathway participates in the neuroprotective effect of interleukin-4 against cerebral ischemia/reperfusion injury

Interleukin-4(IL-4) has a protective effect against cerebral ischemia/reperfusion injury. Animal experiments have shown that IL-4 improves the short-and long-term prognosis of neurological function. The Akt(also called protein kinase B, PKB)/glycogen synthase kinase-3β(Akt/GSK-3β) signaling pathway is involved in oxidative stress, the inflammatory response, apoptosis, and autophagy. However, it is not yet clear whether the Akt/GSK-3β pathway participates in the neuroprotective effect of IL-4 against cerebral ischemia/reperfusion injury. In the present study, we established a cerebral ischemia/reperfusion mouse model by middle cerebral artery occlusion for 60 minutes followed by a 24-hour reperfusion. An IL-4/anti-IL-4 complex(10 μg) was intraperitoneally administered 30 minutes before surgery. We found that administration of IL-4 significantly alleviated the neurological deficits, oxidative stress, cell apoptosis, and autophagy and reduced infarct volume of the mice with cerebral ischemia/reperfusion injury 24 hours after reperfusion. Simultaneously, IL-4 activated Akt/GSK-3β signaling pathway. However, an Akt inhibitor LY294002, which was injected at 15 nmol/kg via the tail vein, attenuated the protective effects of IL-4. These findings indicate that IL-4 has a protective effect on cerebral ischemia/reperfusion injury by mitigating oxidative stress, reducing apoptosis, and inhibiting excessive autophagy, and that this mechanism may be related to activation of the Akt/GSK-3β pathway. This animal study was approved by the Animal Ethics Committee of Renmin Hospital of Wuhan University, China(approval No. WDRY2017-K037) on March 9, 2017.     

p53 induction contributes to excitotoxic neuronal death in rat striatum through apoptotic and autophagic mechanisms

The present study sought to investigate mechanisms by which p53 induction contributes to excitotoxic neuronal injury. Rats were intrastriatally administered the N‐methyl‐d ‐aspartate (NMDA) receptor agonist quinolinic acid (QA), the changes in the expression of p53 and its target genes involved in apoptosis and autophagy, including p53‐upregulated modulator of apoptosis (PUMA), Bax, Bcl‐2, damage‐regulated autophagy modulator (DRAM) and other autophagic proteins including microtubule‐associated protein 1 light chain 3 (LC3) and beclin 1 were assessed. The contribution of p53‐mediated autophagy activation to apoptotic death of striatal neurons was assessed with co‐administration of the nuclear factor‐kappaB (NF‐κB) inhibitor SN50, the p53 inhibitor Pifithrin‐alpha (PFT‐α) or the autophagy inhibitor 3‐methyladenine (3‐MA). The increased formation of autophagosomes and secondary lysosomes were observed with transmission electron microscope after excitotoxin exposure. QA induced increases in the expression of p53, PUMA, Bax and a decrease in Bcl‐2. These changes were significantly attenuated by pre‐treatment with SN50, PFT‐α or 3‐MA. SN50, PFT‐α or 3‐MA also reversed QA‐induced upregulation of DRAM, the ratio of LC3‐II/LC3‐I and beclin 1 protein levels in the striatum. QA‐induced internucleosomal DNA fragmentation and loss of striatal neurons were robustly inhibited by SN50, PFT‐α or 3‐MA. These results suggest that overstimulation of NMDA receptors can induce NF‐κB‐dependent expression of p53. p53 participates in excitotoxic neuronal death probably through both apoptotic and autophagic mechanisms.     

Severe global cerebral ischemia-induced programmed necrosis of hippocampal CA1 neurons in rat is prevented by 3-methyladenine: a widely used inhibitor of autophagy

The true programmed mechanisms of delayed neuronal death induced by global cerebral ischemia/reperfusion injury remain incompletely characterized. Autophagic cell death and programmed necrosis are 2 kinds of programmed cell death distinct from apoptosis. Here, we studied the death mechanisms of hippocampal cornu ammonis 1 neuronal death after a 20-minute severe global ischemia/reperfusion injury in young adult rats and the effects of 3-methyladenine (3-MA), a widely used inhibitor of autophagy. The morphological changes detected by electron microscopy, together with the activation of autophagy, transferase-mediated UTP nick end-labeling-positive neurons, and delayed death, demonstrated that cornu ammonis 1 neuronal death induced in this paradigm was programmed necrosis. No significant activation of caspase-3 after injury was detected by Western blot and immunohistochemistry. Treatment with 3-MA provided time-dependent protection against cornu ammonis 1 neuronal death at 7 days of reperfusion when it was administered before ischemia; administration 60 minutes after reperfusion was not beneficial. The redistribution of the lysosomal enzyme cathepsin B after injury was inhibited by 3-MA administered before ischemia, suggesting that this might be another important mechanism for the protective effect of 3-MA in ischemic neuronal injury.     

Puerarin protects rat brain against ischemia/reperfusion injury by suppressing autophagy via the AMPK-mT OR-ULK1 signaling pathway

Puerarin suppresses autophagy to alleviate cerebral ischemia/reperfusion injury, and accumulating evidence indicates that the AMPKm TOR signaling pathway regulates the activation of the autophagy pathway through the coordinated phosphorylation of ULK1. In this study, we investigated the mechanisms underlying the neuroprotective effect of puerarin and its role in modulating autophagy via the AMPK-m TOR-ULK1 signaling pathway in the rat middle cerebral artery occlusion model of cerebral ischemia/reperfusion injury. Rats were intraperitoneally injected with puerarin, 50 or 100 mg/kg, daily for 7 days. Then, 30 minutes after the final administration, rats were subjected to transient middle cerebral artery occlusion for 90 minutes. Then, after 24 hours of reperfusion, the Longa score and infarct volume were evaluated in each group. Autophagosome formation was observed by transmission electron microscopy. LC3, Beclin-1 p62, AMPK, m TOR and ULK1 protein expression levels were examined by immunofluorescence and western blot assay. Puerarin substantially reduced the Longa score and infarct volume, and it lessened autophagosome formation in the hippocampal CA1 area following cerebral ischemia/reperfusion injury in a dose-dependent manner. Pretreatment with puerarin(50 or 100 mg/kg) reduced Beclin-1 expression and the LC3-II/LC3-I ratio, as well as p-AMPK and p S317-ULK1 levels. In comparison, it increased p62 expression. Furthermore, puerarin at 100 mg/kg dramatically increased the levels of p-m TOR and p S757-ULK1 in the hippocampus on the ischemic side. Our findings suggest that puerarin alleviates autophagy by activating the APMK-m TOR-ULK1 signaling pathway. Thus, puerarin might have therapeutic potential for treating cerebral ischemia/reperfusion injury.     

Splicing factor NSSR1 reduces neuronal injury after mouse transient global cerebral ischemia

This study focuses on the function of NSSR1, a splicing factor, in neuronal injury in the ischemic mouse brain using the transient global cerebral ischemic mouse model and the cultured cells treated with oxygen‐glucose deprivation (OGD). The results showed that the cerebral ischemia triggers the expression of NSSR1 in hippocampal astrocytes, predominantly the dephosphorylated NSSR1 proteins, and the Exon3 inclusive NCAM‐L1 variant and the Exon4 inclusive CREB variant. While in the hippocampus of astrocyte‐specific NSSR1 conditional knockdown (cKD) mice, where cerebral ischemia no longer triggers NSSR1 expression in astrocytes, the expression of Exon3 inclusive NCAM‐L1 variant and Exon4 inclusive CREB variant were no longer triggered as well. In addition, the injury of hippocampal neurons was more severe in astrocyte‐specific NSSR1 cKD mice compared with in wild‐type mice after brain ischemia. Of note, the culture media harvested from the astrocytes with overexpression of NSSR1 or the Exon3 inclusive NCAM‐L1 variant, or Exon4 inclusive CREB variant were all able to reduce the neuronal injury induced by OGD. The results provide the evidence demonstrating that: (1) Splicing factor NSSR1 is a new factor involved in reducing ischemic injury. (2) Ischemia induces NSSR1 expression in astrocytes, not in neurons. (3) NSSR1‐mediated pathway in astrocytes is required for reducing ischemic neuronal injury. (4) NCAM‐L1 and CREB are probably mediators in NSSR1‐mediated pathway. In conclusion, our results suggest for the first time that NSSR1 may provide a novel mechanism for reducing neuronal injury after ischemia, probably through regulation on alternative splicing of NCAM‐L1 and CREB in astrocytes. GLIA 2015;63:826–845     

大鼠局灶性脑缺血再灌注损伤后自噬的初步研究

目的：研究大鼠局灶性脑缺血再灌注损伤后自噬溶酶体相关蛋白随损伤时间表达的规律,检测自噬体、溶酶体的变化。方法：采用线栓法建立大鼠大脑中动脉缺血再灌注损伤模型,免疫组织化学法检测损伤周边区（皮质）自噬溶酶体相关蛋白Beclinl、cathepsinB的表达,透射电镜观察脑缺血再灌注损伤后神经细胞内自噬体的形成和溶酶体的激活。结果：免疫组织化学检测显示,脑缺血再灌注损伤后4hBeclinl阳性细胞增加（P〈0．01）,24h达高峰,5d仍有较多阳性细胞（P〈0．01）。脑缺血再灌注损伤后2hcathepsinB阳性细胞增加（P〈0．01）,12h达高峰,5d仍有较多阳性细胞（P〈0．01）。透射电镜显示,脑缺血再灌注损伤后2h即可见到损伤周边区神经细胞内自噬体形成、溶酶体增多,12-24h最为明显,持续到5d。脑缺血再灌注损伤后表现为自噬溶酶体相关蛋白Beclin1、cathepsinB阳性细胞数随损伤时间而变化,以及缺血半暗带内自噬体的形成和溶酶体的激活。结果：大鼠局灶性脑缺血再灌注损伤能激活自噬溶酶体途径。     

Inhibition of cysteine cathepsin B and L activation in astrocytes contributes to neuroprotection against cerebral ischemia via blocking the tBid‐mitochondrial apoptotic signaling pathway

The roles of cathepsins in the ischemic astrocytic injury remain unclear. Here, we test the hypothesis that activation of cathepsin B and L contributes to the ischemic astrocyte injury via the tBid‐mitochondrial apoptotic signaling pathways. In the rat models of pMCAO, CA‐074Me or Clik148, a selective inhibitor of cathepsin B or cathepsin L, reduced the infarct volume, improved the neurological deficits and increased the MAP₂ and GFAP levels. In OGD‐induced astrocyte injury, CA‐074Me or Clik148 decreased the LDH leakage and increased the GFAP levels. In the ischemic cortex or OGD‐induced astrocytes injury, Clik148 or CA‐074Me reversed pMCAO or OGD‐induced increase in active cathepsin L or cathepsin B at 3 h or 6 h, increase in tBid, reduction in mitochondrial cytochrome‐c (Cyt‐c) and increase in cytoplastic Cyt‐c and active caspase‐3 at 12–24 h of the late stage of pMCAO or OGD. CA‐074Me or Clik148 also reduced cytosolic and mitochondrial tBid, increased mitochondrial Cyt‐c and decreased cytoplastic Cyt‐c and active caspase‐3 at 6 h of the early stage of Bid activation. CA‐074Me or Clik148 blocked the pMCAO‐induced release of cathepsin B or L from the lysosomes into the cytoplasm and activation of caspase‐3 in ischemic astrocytes at 12 h after ischemia. Concurrent inhibition of cathepsin B and cathepsin L provided better protection on the OGD‐induced astrocytic apoptosis than obtained with separate use of each inhibitor. These results suggest that inhibition of the cysteine cathepsin B and cathepsin L activation in ischemic astrocytes contributes to neuroprotection via blocking the tBid‐mitochondrial apoptotic signaling pathway. GLIA 2014;62:855–880     

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.     

Protective role of autophagy in neonatal hypoxia-ischemia induced brain injury

Autophagy, an intracellular bulk degradation process of cellular constituents, plays a key role in cell homeostasis and can be induced by stresses, such as nutrient depletion, closed head injury or focal cerebral ischemia. This study focuses on the role of autophagy in neonatal hypoxia-ischemia (HI). Enhanced beclin 1 expression, a Bcl-2-interacting protein required for autophagy, has been used as a marker of autophagy. Beclin 1 was significantly increased at short times after HI, both in the hippocampus and in the cerebral cortex. Beclin 1-positive cells were found in the injured but not in the contralateral side and co-localized with MAP2 but not with GFAP or ED1, indicating that the protein is over-expressed in neurons. Beclin 1-positive cells were also TUNEL-positive. 3-Methyladenine and wortmannin, that inhibit autophagy, significantly reduced beclin 1 expression and switched the mechanism of the cell death mode from apoptosis to necrosis. Conversely, rapamycin, that increases autophagy, augmented beclin 1 expression, reduced necrotic cell death, and decreased brain injury. A prophylactic treatment with simvastatin or hypoxic preconditioning also increased beclin 1 expression. Taken together, these data indicate that autophagy is increased in neuronal cells after neonatal hypoxia-ischemia and suggest that over-activation of autophagic pathways represents a potential protective mechanism in the early stage of the brain injury.     

Spermidine preconditioning ameliorates laurate-induced brain injury by maintaining mitochondrial stability

Ischemic precondition plays a protective effect during cerebral ischemia. This effect partly depends on the autophagic activity. However, whether the activity of autophagy can exert the protective effects after cerebral ischemia is unclear. In this study, rats were treated with spermidine, an activator of autophagy, and injected with sodium laurate via the internal carotid artery to stimulate cerebral small vessel disease (CSVD). The effects of the spermidine precondition on brain injury were evaluated by behavioural test, histology assay, ultrastructure observation, and autophagic-related signals. Furthermore, the mitochondria of brain tissue were isolated, and mitDNA were extracted. The stability of mitDNA was analyzed by quantitative real-time PCR. Results showed that the penetrating artery of the striatum was damaged. This damage was accompanied by neural inflammation characterized by an increase in Fluoro-Jade C (FJC)-positive cells after sodium laurate injection. Spermidine pretreatment decreased the deletion of mitDNA and the autophagy hyperactivity induced by the laurate injection. Likewise, spermidine reduced the neurological deficit and FJC reactivation of striatum at 48 h after laurate injection. These results suggested sodium laurate injection through the internal carotid artery can induce the pathological features of CSVD characterized by the damage of penetrating artery, neurological deficit, mitochondrial impairment, and autophagic hyperactivity. Pretreatment with spermidine can ameliorate these outcomes. Further study indicated that the protective effect of the spermidine precondition is associated with the maintenance of mitochondrial stability and proper autophagy activity.     

肢体缺血预处理对脑缺血再灌注损伤大鼠自噬的影响

目的探讨肢体缺血预处理对脑缺血再灌注损伤大鼠自噬的影响。方法将60只Wistar大鼠随机分为假手术组(Sham组)、缺血再灌注组(I/R组)、肢体缺血预处理组(LIPC组)、3-甲基嘌呤组(3-MA组),每组15只。制作脑缺血再灌注、肢体缺血预处理及3-MA干预大鼠模型,在脑缺血2 h再灌注24 h后进行神经功能缺陷评分和脑梗死体积测定,HE染色观察细胞形态学改变,Western Bloting法检测自噬相关蛋白Beclin-1、Cathepsin B的表达。结果与I/R组比较,LIPC组神经功能缺陷评分降低(P<0.05),脑梗体积明显减小(P<0.05),细胞损伤、坏死减轻(P<0.05),Beclin-1、Cathepsin B的蛋白表达明显减弱(P<0.05)。结论 LIPC对缺血再灌注损伤大脑具有保护作用,其机制可能与减弱自噬水平有关。