Binxiao Su

Limb remote ischemic preconditioning protects the spinal cord from ischemia-reperfusion injury: a newly identified nonneuronal but reactive oxygen species-dependent pathway.

Background:It remains to be established whether spinal cord ischemic tolerance can be induced by limb remote ischemic preconditioning (RIPC), and the mechanisms underlying the neuroprotective effects of RIPC on the spinal cord need to be clarified. Methods:Spinal cord ischemia was studied in New Zealand White rabbits. In experiment 1, all rabbits were subjected to 20-min spinal cord ischemia by aortic occlusion. Thirty minutes before ischemia, rabbits were subjected to sham intervention or RIPC achieved by bilateral femoral artery occlusion (10 min ischemia/10 min reperfusion, two cycles). Dimethylthiourea (500 mg/kg, intravenously), a hydroxyl radical scavenger, or vehicle was given 1 h before RIPC. Antioxidant enzyme activity was measured along with spinal cord histology and neurologic function. In experiment 2, rabbits were subjected to spinal cord ischemia, with or without RIPC. In addition, rabbits were pretreated with various doses of hexamethonium. Results:RIPC improved neurologic function and reduced histologic damage. This was associated with increased endogenous antioxidant activity. Dimethylthiourea inhibited the protective effects of RIPC. In contrast, there was no effect of hexamethonium on the protective effect of RIPC. Conclusions:An initial oxidative stress acts as a trigger to upregulate antioxidant enzyme activity, rather than the neural pathway, and plays an important role in the formation of the tolerance against spinal cord ischemia by limb RIPC.

SEVOFLURANE PRECONDITIONING INDUCES RAPID ISCHEMIC TOLERANCE AGAINST SPINAL CORD ISCHEMIA/ REPERFUSION THROUGH ACTIVATION OF EXTRACELLULAR SIGNAL-REGULATED KINASE IN RABBITS

BACKGROUND: The protective effect of sevoflurane preconditioning against spinal cord ischemia/reperfusion (I/R) is unclear. We designed this study to investigate whether sevoflurane preconditioning could induce rapid ischemic tolerance to the spinal cord in a rabbit model of transient spinal cord ischemia and how the role of extracellular signal-regulated kinase (ERK) is involved. METHODS: To test whether preconditioning with sevoflurane induces rapid ischemic tolerance, New Zealand White male rabbits were randomly assigned to three groups. Animals in the Sev group received preconditioning with 3.7% sevoflurane (1.0 minimum alveolar anesthetic concentration) in 96% oxygen for 30 min, whereas animals in the O2 group serving as controls inhaled only 96% oxygen for 30 min. The Sham group received the same anesthesia and surgical preparation but no preconditioning or spinal cord I/R. To evaluate the role of ERK activation in sevoflurane preconditioning, rabbits were randomly assigned to four groups. U0126, an ERK inhibitor, was administered IV 20 min before the beginning of preconditioning in the U0126 + O2 and U0126 + Sev groups. Dimethylsulfoxide was administered IV at the same time in the vehicle + O2 and vehicle + Sev groups. At 1 h after preconditioning, the animals were subjected to spinal cord I/R induced by infrarenal aorta occlusion. All animals were assessed at 48 h after reperfusion with modified Tarlov criteria, and the spinal cord segments (L5) were harvested for histopathological examination, TUNEL staining, and Western blot of phosphor-ERK1/2. RESULTS: The animals in the Sev group had higher neurological scores and more normal motor neurons than those in the O2 group (P < 0.01 for each comparison). Compared with vehicle + Sev group, the U0126 + Sev group had worse neurological outcomes, fewer viable neurons, more apoptotic neurons, and significantly decreased ERK1/2 phosphorylation (P ≤ 0.01 for each comparison). There were no significant differences in the outcomes among vehicle + O2, U0126 + O2, and U0126 + Sev groups. CONCLUSIONS: This study demonstrates that sevoflurane preconditioning induces rapid tolerance to spinal cord I/R in rabbits, and the tolerance is possibly mediated through the activation of ERK. These data suggest that sevoflurane preconditioning might provide a new practical method for protecting perioperative spinal cord I/R.

Cannabinoid 1 receptor mediation of spinal cord ischemic tolerance induced by limb remote ischemia preconditioning in rats.

OBJECTIVE The aim of this study was to examine the influence of endogenous cannabinoids on neuroprotection of the spinal cord afforded by limb remote ischemic preconditioning. METHODS In experiment 1 (RIPC group), 3 cycles of limb remote ischemic preconditioning within different episodes (2, 3, or 5 minutes) were induced before spinal cord ischemia in rats (N = 5, n = 8). In experiment 2, animals were pretreated intravenously by the vehicles, cannabinoid 1 (AM251, 1 mg/kg) or cannabinoid 2 (AM630, 1 mg/kg) receptor antagonist 15 minutes before remote ischemic preconditioning, or else they were subjected to a sham operation. Thirty minutes after the pretreatment, spinal cord ischemia was induced (N = 8, n = 8). In experiment 3, the arachidonylethanolamide and 2-arachidonoylglycerol contents in the spinal cord after remote ischemic preconditioning and spinal cord ischemia were detected in rats (N = 2, n = 12). Spinal cord ischemia was induced by 12 minutes of thoracic aorta occlusion in rats. Neurologic function was assessed 24 and 48 hours after reperfusion. Histopathologic examination was performed and the number of normal neurons in anterior spinal cord were counted. RESULTS In experiment 1, 3 cycles of limb remote ischemic preconditioning (3 minutes of ischemia/3 minutes of reperfusion) induced ischemic tolerance on the spinal cords of the rats. The RIPC group showed a significant reduction in motor deficit index (P < .01) as well as an increase in the number of normal neurons (P < .01). In experiment 2, the cannabinoid 1 receptor antagonist AM251 pretreatment abolished the protective effects of remote preconditioning. In experiment 3, arachidonylethanolamide content in spinal cord was elevated by remote ischemic preconditioning in rats. CONCLUSION These results indicated that endogenous cannabinoids, through acting on cannabinoid 1 receptors, were involved in the neuroprotective phenomenon on spinal cords of limb remote ischemic preconditioning.

Activation of orexin signal in basal forebrain facilitates the emergence from sevoflurane anesthesia in rat.

Orexinergic system may play an important role in the regulation of anesthesia-arousal. However, which region or which pathway mediated the effect of orexins was still unclear. In current study, we investigated whether activation of orexin signals in basal forebrain (BF) may alter electroencephalographic activity, induction and emergence time to sevoflurane anesthesia in rats. Either orexin-A or orexin-B was injected into the BF while measuring electroencephalogram (EEG) under 1.0 minimum alveolar concentration (2.4%) sevoflurane anesthesia. The induction and emergence time of sevoflurane anesthesia were measured respectively after an injection of orexin receptor agonist (orexin-A or orexin-B) or antagonist (SB-334867A) into the BF also. We found that the administration of orexin-A (30, 100 pmol) and orexin-B (100 pmol) changed the burst and suppression patterns to arousal EEG in rat under sevoflurane anesthesia. Comparing with orexin-B, injection of lower dose of orexin-A induced more arousal EEG. Intrabasalis microinjection of orexin-A shorted the emergence time, whereas intrabasalis microinjection of SB-334867A (5 microg, 20 microg) delayed the emergence time to sevoflurane anesthesia, without changing anesthetic induction. These findings indicate that the orexin signals in basal forebrain, a middle region of the cholinergic ventral ascending arousal system, plays a crucial role in the anesthesia-arousal regulation.

TREK1 activation mediates spinal cord ischemic tolerance induced by isoflurane preconditioning in rats.

The aim of this study is to examine the role of one of the two-pore (2P) domain K(+) channels, TREK (TWIK-related K(+) channels, TREK)-1, mediated neuroprotection on spinal cord afforded by isoflurane preconditioning. In Experiment 1, male Sprague-Dawley rats were randomly assigned to control (Con) group, an isoflurane preconditioning (Iso) group, and sham group. Twenty-four hours after the last pretreatment, spinal cord ischemia was induced in Con and Iso groups. Neurobehavioral testing and histopathologic examination were performed after reperfusion. In Experiment 2, the expression of the TREK1 in the spinal cord was assessed by immunohistochemistry, Western blot and real-time polymerase chain reaction. In Experiment 3, Amiloride, a blocker of stretch-sensitive channels, was administered intraperitoneally immediately prior to each isoflurane preconditioning. Iso group showed a significant reductions in motor deficit index as well as increases in the number of normal neurons compared with the Con group. The expression of TREK1 protein and the level of mRNA after ischemia were higher in the rats of the Iso group than those in the Con group. Amiloride pretreatment abolished the protective effects of Iso preconditioning. These finding indicate that isoflurane preconditioning had a neuroprotective effect against spinal cord ischemia reperfusion injury. These effects may be mediated through the TREK1 pathway.

Ischemic postconditioning protects the spinal cord from ischemia–reperfusion injury via modulation of redox signaling

BACKGROUND It is well known that ischemic postconditioning reduces ischemic-reperfusion injury, but the underlying mechanism is not fully understood. The current study investigated the role of reactive oxygen species-mediated upregulation of endogenous antioxidant enzymes in the generation of a protective effect induced by ischemic postconditioning against spinal cord reperfusion injury in the rabbit. METHODS New Zealand White rabbits were randomly allocated to sham, ischemia-reperfusion, and postconditioning groups (3 cycles of 30 seconds of reperfusion and 30 seconds of occlusion during the onset of reperfusion). Spinal cord ischemia was induced by clamping the infrarenal abdominal aorta for 20 minutes in the ischemia-reperfusion and postconditioning groups. Forty-eight hours after reperfusion, the neurologic status of the lower limbs was assessed. Blood samples were collected for analysis of serum neuron-specific enolase levels, and the lumbar spinal cord segments (L5-7) were harvested for histopathologic and antioxidant enzyme activities and mRNA analysis with or without administration of N-2-mercaptopropionylglycine (an effective oxygen free radical scavenger) given at different reperfusion times. RESULTS Continuous administration of N-2-mercaptopropionylglycine for 13 minutes, starting at 10 minutes before (but not 10 minutes after) the beginning of reperfusion, attenuated the neuroprotective effect of postconditioning against spinal cord ischemia and reversed the increase in activity of the antioxidant enzymes superoxide dismutase and catalase in spinal cord tissue subjected to ischemic postconditioning. CONCLUSIONS The results indicate that reactive oxygen species-triggered upregulation of endogenous antioxidant enzyme activities may be involved in the mechanism of neuroprotection of ischemic postconditioning.

GAPDH/Siah1 cascade is involved in traumatic spinal cord injury and could be attenuated by sivelestat sodium

The glyceraldehyde-3-phosphate dehydrogenase (GAPDH)/Siah1 signaling pathway has been recognized as a sensor of nitric oxide (NO). It is associated with a variety of injurious conditions, suggesting its therapeutic potential for spinal cord injury (SCI). Sivelestat sodium (SIV), a neutrophil elastase (NE) inhibitor initially used to treat acute lung injury, has been known to protect against compression-induced and ischemic SCI. However, little is known about the relationship between the GAPDH/Siah1 cascade and SIV. Thus, we aimed to assess the role of GAPDH/Siah1 cascade in traumatic SCI and its possible link with SIV. Rats were assigned to four groups: sham group, SCI group, 5-mg/kg SIV group, and 10-mg/kg SIV. The traumatic SCI was induced by dropping a 10-g impactor from a height of 25mm on the dorsal surface of T9 and T10. SIV was injected intraperitoneally immediately after surgery. Our results showed that the nuclear translocation of GAPDH was induced together with the nuclear translocation of Siah1 and the formation of the GAPDH/Siah1 complex in the spinal cord after traumatic SCI. However, the activation of the GAPDH/Siah1 cascade was attenuated by treatment with SIV. We also found that SIV suppressed apoptosis, NE and inducible nitric oxide synthase (iNOS) protein expressions, the number of NE and iNOS immunostained cells, the production of interleukin (IL)-1β and tumor necrosis factor-alpha (TNF-α), and the activation of nuclear factor kappa light-chain enhancer of activated B cells (NF-κB) signaling in the spinal cord. The behavioral tests showed that SIV promoted functional recovery after traumatic SCI as reflected in the sustained increase in the Basso-Beattie-Bresnahan (BBB) scores throughout the observation period. In conclusion, our results reveal GAPDH/Siah1 as a novel signaling pathway during the progression of SCI, which can be blocked by SIV.

Electroacupuncture pretreatment attenuates spinal cord ischemia-reperfusion injury via inhibition of high-mobility group box 1 production in a LXA4 receptor-dependent manner.

Paraplegia caused by spinal cord ischemia is a severe complication following surgeries in the thoracic aneurysm. HMGB1 has been recognized as a key mediator in spinal inflammatory response after spinal cord injury. Electroacupuncture (EA) pretreatment could provide neuroprotection against cerebral ischemic injury through inhibition of HMGB1 release. Therefore, the present study aims to test the hypothesis that EA pretreatment protects against spinal cord ischemia-reperfusion (I/R) injury via inhibition of HMGB1 release. Animals were pre-treated with EA stimulations 30min daily for 4 successive days, followed by 20-min spinal cord ischemia induced by using a balloon catheter placed into the aorta. We found that spinal I/R significantly increased mRNA and cytosolic protein levels of HMGB1 after reperfusion in the spinal cord. The EA-pretreated animals displayed better motor performance after reperfusion along with the decrease of apoptosis, HMGB1, TNF-α and IL-1β expressions in the spinal cord, whereas these effects by EA pretreatment was reversed by rHMGB1 administration. Furthermore, EA pretreatment attenuated the down-regulation of LXA4 receptor (ALX) expression induced by I/R injury, while the decrease of HMGB1 release in EA-pretreated rats was reversed by the combined BOC-2 (an inhibitor of LXA4 receptor) treatment. In conclusion, EA pretreatment may promote spinal I/R injury through the inhibition of HMGB1 release in a LXA4 receptor-dependent manner. Our data may represent a new therapeutic technique for treating spinal cord ischemia-reperfusion injury.

Inhibiting a spinal cord signaling pathway protects against ischemia injury in rats

Objective The aim of the study was to examine whether the cannabinoid agonist WIN55212‐2 could attenuate ischemic spinal cord injury (SCI) in rats through inhibition of GAPDH/Siah1 signaling. Methods Male Sprague‐Dawley rats were distributed randomly into 5 groups: (1) sham group that received no aortic occlusion and injected intraperitoneally (i.p.) with vehicle control after reperfusion; (2) control group that received a 12‐minute aortic occlusion and injected i.p. with vehicle control after reperfusion; (3) WIN55212‐2 group (WIN) that received the aortic occlusion and injected i.p. with 1 mg/kg of WIN55212‐2 after reperfusion; and (4) WIN55212‐2 plus AM251 group and (5) WIN55212‐2 plus AM630 group that received the same surgical operation as the WIN group, except that 1 mg/kg of AM251 or AM630 was injected i.p. 30 min before each dose of WIN55212‐2 injection, respectively. Neurologic function was assessed 48 hours after reperfusion. Histopathologic examination was performed to determine the number of normal neurons in anterior spinal cord. Protein expression of active caspase‐3, total caspase‐3, glyceraldehyde 3‐phosphate dehydrogenase (GAPDH), inducible nitric oxide synthase (iNOS), nuclear factor kappa light chain enhancer of activated B cells (NF‐&kgr;B), Siah1, tumor necrosis factor &agr;, and interleukin 1&bgr; were determined with Western blot and enzyme‐linked immunosorbent assay; coimmunoprecipitation assays were also used to determine GAPDH/Siah1 complexing. Finally, terminal deoxynucleotidyl transferase dUTP nick end labeling staining was used to determine neuronal apoptosis in the lumbar spinal cord. Results The nuclear translocation of GAPDH and Siah1 in the spinal cord was initiated after ischemic spinal cord injury (SCI) along with the increased formation of GAPDH/Siah1 complexes. However, the activation of GAPDH/Siah1 was blocked by WIN. In addition, the treatment of WIN55212‐2 promoted neuronal survival in the spinal cord, reduced apoptosis and inflammation, and improved neurologic scores. Furthermore, these beneficial effects of WIN55212‐2 were abolished by the combined treatment of the CB2 antagonist AM630, but not the CB1 antagonist AM251. Conclusions Our findings reveal GAPDH/Siah1 signaling cascades as a novel therapeutic target for ischemic SCI and identify WIN55212‐2 with the potential to treat ischemic SCI by targeting this pathway. Graphical abstract Figure. No Caption available.

The synthetic cannabinoid WIN55212-2 ameliorates traumatic spinal cord injury via inhibition of GAPDH/Siah1 in a CB2-receptor dependent manner

The essential role of GAPDH/Siah1 signaling pathway in the pathogenesis of various injurious conditions such as traumatic spinal cord injury (SCI) has been gradually recognized. However, the drugs targeting this signaling pathway are still lacking. The endocannabinoid system, including its receptors (CB1 and CB2), act as neuroprotective and immunomodulatory modulators in SCI. WIN55212-2, an agonist for CB1 and CB2 receptors, has been demonstrated with anti-inflammatory and anti-apoptotic effects in multiple neurological diseases. Therefore, the present study aimed to investigate whether WIN55212-2 could promote functional recovery after traumatic SCI via inhibition of the GAPDH/Siah1 signaling. The traumatic SCI was induced by dropping a 10-g impactor from 25mm on the dorsal surface of T9 and T10. Our results showed that WIN55212-2 alleviated the activation of GAPDH/Siah1 signaling pathway after SCI, as indicated by the reduction in GAPDH nuclear expression, GAPDH-Siah1 complex formation and iNOS protein expression. Furthermore, WIN55212-2 reduced apoptosis, production of IL-1β and TNF-α and activation of NF-κB signaling in the spinal cord after SCI. The behavioral tests showed that WIN55212-2 improved the functional recovery after traumatic SCI as indicated by sustained increase in the locomotor scores. However, these neuroprotective effects of WIN55212-2 were blocked in the presence of the combined treatment of AM630 (an antagonist of CB2) rather than AM251 (an antagonist of CB1). In conclusion, our study indicates that, WIN55212-2 improves the functional recovery after SCI via inhibition of GAPDH/Siah1 cascades in a CB2 receptor dependent manner, indicative of its therapeutic potential for traumatic SCI or other traumatic conditions.