Study On The Mechanism Of Autophagy During Spinal Cord Ischemia - Reperfusion Injury

Objective:(1) To elucidate the role and mechanism of autophagy in reperfusion of ischemic spinal cord, by applying the model of spinal cord ischemic reperfusion injury in vivo and neurons oxygen-glucose-deprivation/reoxygenation(OGD/R) injury in vitro.(2) To further investigate the role and function of autophagy during neurons oxygen-glucose-deprivation/reoxygenation(OGD/R) injury in vitro by applying JNK inhibitor SP600125.(3) To study the influence of ischemic preconditioning on autophagy, and investigate the neuroprotection of ischemic preconditioning. Methods:Cell experiments in vitro: Cortical neurons were isolated from Sprague-Dawley rats using the method described by Takei with modifications and identified by immunofluorescence.The cortical neurons were randomly assigned to one of four groups:control group: The neurons in the blank control group were cultured in glucose-containing Dulbecco’s modified Eagle’s medium(DMEM) at 37°C in an incubator containing 5% CO2., Experimental group(OGD/R group, further divided into four groups depended on the difference reoxygenation time points: group A: 0.5 hours, group B: 2 hours, group C: 6 hours, group D: 12 hours): the neurons were washed with glucose-free phosphate buffered saline(PBS) to remove the primary medium, and then incubated in 2 ml of glucose-free DMEM. Cells were placed in an anaerobic chamber aerated with an aerobic gas mix(95% N2 and 5% CO2) at 37°C for 30 min to reduce the oxygen content to less than 1%. The chamber was sealed, and the medium was replaced with glucose-containing DMEM. The neurons were immediately transferred to an incubator containing 5% CO2 and cultured for 0.5, 2, 6 and 12 h at 37°C., JNK inhibitor treatment group: Cells in the JNK inhibitor treatment group were maintained in glucose-containing DMEM in an incubator containing 5% CO2 at 37°C. When the neurons reached maturity, the medium was supplemented with 10 μmol/L SP600125(Sigma-Aldrich, St. Louis, MO, USA) dissolved in dimethyl sulfoxide(DMSO). After 0.5 h, the medium was replaced with normal DMEM for a further culture of 0.5, 2, 6 and 12 h., and JNK inhibitor pretreatment + OGD/R group(Specific groups are exactly the same as Group II):the neurons were maintained in medium supplemented with 10 μmol/L SP600125 dissolved in DMSO 0.5 h prior to the OGD/R(Chen et al. 2011); experimental procedures and subgrouping were performed as described for the experimental group. Neuronal viability assessment by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT) assay. Transmission electron microscopy(TEM) was applied to investigate ultrastructural changes in mitochondrial, endoplasmic reticulum, and the formation of autophagosome or phagophores in each group. The activation of LC3I/II、Beclin-1、Bcl-2 and p-Bcl-2(ser70)was detected by Westernblot. the interactions between Beclin-1 and Bcl-2 was performed by coimmunoprecipitation analysis. The localization of LC3 and Beclin-1 were analyzed by immunofluorescence.Animal experimental in vivo: Fifty Sprague-Dawley rats were randomly assigned to one of the four groups: GroupⅠ: sham-operation group(Sham group, n = 5) the peritoneum was dissected under the same anesthesia protocol and the abdominal aorta was exposed but not occluded, GroupⅡ: Ischemic reperfusion group(I/R group, n = 20, further divided into four groups depended on the difference reperfusion time points: group A: 3 hours, group B: 6 hours, group C: 12 hours, group D: 24 hours):After a 3- to 4-cm medial incision, the abdominal aorta was exposed at the level of the left renal artery. Spinal cord ischemia was induced by introducing an infra-renal aortic cross-clamp for 0.5 h followed by 3, 6, 12, or 24 h reperfusion before animal sacrifice and analysis., GroupⅢ: Ischemic Preconditioning group(IPC group, n = 5):Prior to the 0.5 h of ischemia, rats in the IPC group underwent three cycles of 5 min of ischemia followed by 5 min of reperfusion, Group Ⅳ: Ischemic Preconditioning+ Ischemic reperfusion group(IPC+I/R group, n=20, Specific groups are exactly the same as Group II):After IPC, rats were then subjected to I/R as described. Changes in spinal cord morphology were observed by Hematoxylinand eosin(HE) stain. To assess the viability of neurons at different time points during reperfusion, spinal cord sections were stained with an anti-neuronal nuclei(Neu N) monoclonal antibody. Spinal cord sections were prepared for transmission electron microscopy(TEM) to investigate ultrastructural changes in mitochondrial, endoplasmic reticulum, and the formation of autophagosome or phagophores. The activation of LC3I/II 、 Beclin-1 、 Bcl-2 and p-Bcl-2( ser70) was detected by Westernblot. the interactions between Beclin-1 and Bcl-2 was performed by coimmunoprecipitation analysis. The localization of LC3 and Beclin-1 were analyzed by immunohistochemistry. Results:Cell experimental in vitro:Ⅰ) Identification of cortical neurons : Cultured cells isolated from Sprague-Dawley rats stained positive for MAP-2 by immunofluorescence, confirming their identity as cortical neurons with a purity > 95%.Ⅱ)Neuronal viability assessment by the MTT assay: Neuronal viability was slightly lower in the JNK inhibitor treated group than in the blank control group, reflecting the effect of the JNK inhibitor on neurons(P > 0.05). Neuronal viability showed a tendency to decrease gradually with increasing reoxygenation time in the experimental group and the JNK inhibitor pretreatment + OGD/R group, with a more notable reduction in the experimental group(P < 0.05). Neuronal viability decreased significantly in the experimental group at 6 and 12 h of reoxygenation, with relative survival rates of 29% and 13%, respectively, while these values were 56% and 41% in the JNK inhibitor pretreatment + OGD/R group. These results indicated that OGD/R caused significant damage to neurons and pretreatment with JNK inhibitors suppressed this effect.Ⅲ)Electron microscopic observation: The neuronal soma was plump and showed normal morphology in the control group, with intact nuclear and plasma membranes and normal aggregation of organelles including mitochondria and endoplasmic reticulum. No damage such as fragment-like dispersion or swelling was observed. A large number of crescent-shaped or goblet-like phagophores were observed in the experimental group after 0.5 h of reoxygenation; most of them had double- or multiple-layer membrane structures and contained cytoplasmic components. After 2 h of reoxygenation, autophagosomes with double- or multiple-layer membrane structures containing cytoplasmic components or cell fractions were observed; organelles such as mitochondria and endoplasmic reticulum showed normal morphology, without mitochondrial swelling, crista disorder or reduction, endoplasmic reticulum swelling or extension. After 6 h of reoxygenation, autophagic vacuoles fused with lysosomes forming autolysosomes with single-layer membrane structures characteristic of the preliminary binding of autophagosomes to lysosomes, and the gradual degradation of cytoplasmic components or cell fractions within the autolysosome was observed. In addition, a large number of phagophores appeared in the plasma of neurons. After 12 h of reoxygenation, severe nuclear pyknosis and chromatin margination were observed, and the condensation of the cytoplasm induced aggregation of organelles(mitochondria and endoplasmic reticulum). Hydrolyzation of the contents of autolysosomes resulted in the appearance of vacuoles, and a large number of new autophagosomes containing mitochondria, endoplasmic reticulum and ribosomes were observed. The neuronal soma was not plump in the JNK inhibitor treatment group and the nuclear membrane and plasma membrane showed slight shrinking; however, no cellular damage was observed. The appearance of autophagosomes and autolysosomes showed at 0.5h, 2h, and decreased at 6h and did not increase at 12 h when compared with the OGD/R group.Ⅳ)Immunofluorescence and western blot detection of LC3 expression: LC3 expression was barely detectable in the blank control and JNK inhibitor treatment groups. In the experimental group, LC3 positive neurons increased gradually at 0.5 and 2h of reoxygenation, and LC3 expression increased significantly in the cytoplasm of neurons. LC3 expression decreased slightly at 6h of reoxygenation, but rapidly increased at 12 h. The JNK inhibitor pretreatment + OGD/R group showed a similar pattern of increase-decrease of LC3 expression at 0.5, 2 and 6h of reoxygenation, with a more intense staining pattern than that of the experimental group; however, unlike the experimental group, the number of LC3 positive neurons did not increased in this group after 12 h. Western blot analysis confirmed the increase-decrease pattern of LC3 II expression in the experimental group and the JNK inhibitor pretreatment + OGD/R group at 0.5, 2 and 6h of reoxygenation, as well as the higher level of expression in the JNK inhibitor pretreatment + OGD/R group. LC3 II expression increased rapidly at 12 h in the experimental group whereas it showed a slight increase in the JNK inhibitor treatment group(P >0.05).Ⅴ)Immunofluorescence and western blot detection of Beclin-1 expression: Beclin-1 plays an important role in autophagosome formation and it is therefore considered as a marker for the activation of autophagosomes. Immunofluorescence analysis showed a gradual increase in the number of Beclin-1 positive neurons and staining intensity in correlation with reoxygenation time in the experimental group. However, in the JNK inhibitor pretreatment + OGD/R group, Beclin-1 expression did not increase until after 12 h of reoxygenation. Western blot analysis confirmed that Beclin-1 expression increased gradually in the experimental group, and this increase was suppressed by JNK inhibitor treatment, with the inhibitory effect decreasing gradually until 12 h of reoxygenation.Ⅵ) Determination of JNK activity and Bcl-2 expression: The level of phosphorylated JNK increased gradually with reoxygenation time in the experimental group, and this effect was suppressed in the JNK inhibitor pretreatment + OGD/R group, which showed a slight increase in phosphorylated JNK at 12 h of reoxygenation(P < 0.05). In a similar pattern, the level of phosphorylated Bcl-2(ser70) increased gradually in the experimental group and showed a slight increase in the JNK inhibitor pretreatment + OGD/R group after 12 h of reoxygenation(P < 0.05).Ⅶ)Interaction between Beclin-1 and Bcl-2: Our results showed the gradual dissociation of the Beclin-1/Bcl-2 complex during reoxygenation in the experimental group. On the other hand, a relatively stable Beclin-1/Bcl-2 complex was observed in the JNK inhibitor pretreatment + OGD/R group, with slight complex dissociation detected after 12 h of reoxygenation.Animal experimental in vivo:Ⅰ)The result of HE staining: In the sham group, the spinal cord exhibited a clear boundary between the gray and white matter and showed little evidence of congestion, edema, or neuronal death. Furthermore, blood vessels and the central canal were of normal morphology. Compared with sham group and IPC group, spinal cord in I/R group exhibited edema in group A, and patchy hemorrhages, and swollen neurons were shown in group B, C and D with longer reperfusion times. However, spinal cord in IPC+ I/R group shown edema and punctate hemorrhage only in group D.Ⅱ)The result of immunohistochemical staining with the Neu N antibody: In the sham group, the mean total number of Neu N-positive neurons per section of anterior spinal cord was 375±16, compared to 209±6 after 3h, 164±4 after 6h, 112±3 after 12 h, and 75±2 after 24 h of reperfusion in the I/R group. In the IPC group, the mean total number of Neu N-positive neurons was 370±13 per section, not significantly different from the sham group. In the IPC+I/R group, neuronal numbers were 356±11 after 3h, 348±12 after 6h, 344±11 after 12 h, and 337±10 after 24 h of reperfusion. The number of surviving spinal neurons was less than the sham group only at 24 h reperfusion and was significantly higher than the I/R group at each reperfusion time point.Ⅲ)The result of Transmission electron microscope: In the sham group, spinal neurons exhibited normally shaped nuclei, numerous mitochondria, and an extensive endoplasmic reticulum but few autophagosomal vacuoles. Spinal cord neurons in sections from I/R group rats contained many more vesicles compared to sham-treated rats, indicative of accelerated autophagy.The vacuoles observed after 3h reperfusion were relatively small and resembled nascent autophagosomes or phagophores in the early stage of engulfment. Moreover, nascent autophagosomes were isolated from the rest of the cellular contents by an ER-like membrane also encircling cytoplasm, mitochondria, and even peroxisomes. The organelles such as mitochondria and endoplasmic reticulum showed normal morphology, without mitochondrial swelling, crista disorder or reduction, endoplasmic reticulum swelling or extension. After 6h of reperfusion, autophagosomes with double- or multiple-layer membrane structures containing cytoplasmic components or cell fractions were observed. In contrast, spinal neurons in the IR group after 12 h reperfusion exhibited numerous double-membrane autophagosomal vacuoles containing cargo, such as membranous structures and fragments of cytoplasmic organelles, as well as secondary lysosomes at various stages of digestion. Larger autophagosomes containing mitochondria and rough ER were also observed, as well as swollen mitochondria and dark(activated) lysosomes. We also obtained images during the fusion of autophagosomes with lysosomes after 12 h reperfusion. After 24 h reperfusion, autophagosomes and autolysosomes were still found, while lysosomal structures containing heterogeneously electron dense materials were detected with increasing frequency. Alternatively, numerous autophagic vacuoles(AVs) with the characteristic morphological features of autophagosomes were less detected in IPC+I/R group rats.Ⅳ)The result of Western blot analysis: In Sham and IPC groups, the expressions of LC3-II, Beclin-1 and p-Bcl-2(ser70) were very low. In I/R group and IPC+ I/R group, the expressions of LC3-II、Beclin-1 and p-Bcl-2(ser70) continued to increase at 3h, 6h, 12 h and 24 h after reperfusion. In the IPC+I/R group, the post-ischemic increase in LC3-II, Beclin-1 and p-Bcl-2(ser70) expressions were significantly lower than in I/R group rats at all reperfusion time points and was significantly higher than sham group rats only at 24 h after reperfusion.Ⅴ)The result of coimmunoprecipitation analysis: The expression of the Beclin-1/Bcl-2 complex did not change significantly until 3h after reperfusion in I/R group and then decreased significantly over the next 21 h of reperfusion as indicated by co-immunoprecipitation of Beclin-1. In the IPC+I/R group, however, co-immunoprecipitation of Beclin-1 decreased only after 24 h of reperfusion.Ⅵ)The result of immunostaining analysis: In sham group rats, only weak cytoplasmic Beclin-1 and LC3 II immunostaining was observed, while both cytoplasmic staining intensity and the number of Beclin-1-positive and LC3II-positive neurons increased significantly with reperfusion time in I/R rats. In I/R group, Mild cytoplasmic staining of Beclin-1 and were observed after 3 h and 6h of reperfusion but staining intensity increased significantly between 12 h and 24 h. In the IPC+I/R group, Beclin-1 and LC3 II immunostaining resembled that in sham rats at 3h, 6h, and 12 h. Only after 24 h of reperfusion were a greater number of(mildly stained) Beclin-1-positive and LC3II-positive neurons observed compared to sham-operated rats. In the IPC+I/R group, the cytoplasmic staining of LC3 II and Beclin-1 was significantly lower than in I/R group rats at all reperfusion time points and was significantly higher than sham group rats. Conclusion:(1) The level of p-Bcl-2(Ser70) is increased in reperfusion of ischemic spinal cord, which leads to the decrease in Beclin-1/Bcl-2 complex, the activity of Beclin-1, and the autophagic cell death.(2) IPC significantly inhibited the phosphorylation of Bcl-2, decreased the activity of Beclin-1, and then maintained the Beclin-1/Bcl-2 complex, inhibited autophagic cell death in reperfusion of ischemic spinal cord.(3) During oxygen-glucose-deprivation/reoxygenation(OGD/R) injury, the autophagic flux activated at the initial phase of reoxygenation(0.5h and 2 h) in our study may have a protective function in neurons, while the autophagy triggered at the late phase of reoxygenation(12h) may be associated with cell death.(4) The activity of autophagic cell death was induced by the increase of p-Bcl-2(Ser70), decrease in Beclin-1/Bcl-2 complex, and the activity of Beclin-1.(5) The regulation of the JNK/Beclin-1/Bcl-2 signaling pathway may be one of the mechanisms underlying the OGD/R-induced autophagic cell death of neurons.