Spinal Ischemia-reperfusion Injury Research Articles

Retraction Note: Neuroprotective effect of Buyang Huanwu Decoction on spinal ischemia-reperfusion injury in rats is linked with inhibition of cyclin-dependent kinase 5

Retraction Note: Neuroprotective effect of Buyang Huanwu Decoction on spinal ischemia-reperfusion injury in rats is linked with inhibition of cyclin-dependent kinase 5

Simulated microgravity-cultured mesenchymal stem cells improve recovery following spinal cord ischemia in rats.

Spinal cord ischemia is a potential complication of thoracoabdominal aortic surgery that may induce irreversible motor disability. We investigated the therapeutic efficacy of simulated microgravity-cultured mesenchymal stem cell (MSC) injection following spinal cord ischemia-reperfusion injury. Sprague-Dawley rats were divided into sham, phosphate-buffered saline (PBS), normal gravity-cultured MSC (MSC-1 G), and simulated microgravity-cultured MSC (MSC-MG) groups. Spinal cord ischemia was induced by transient balloon occlusion of the thoracic aorta, which was followed immediately by PBS or MSC injection into the left carotid artery. Hindlimb motor function was evaluated by the Basso-Beattie-Bresnahan (BBB) scale. Spinal cords were removed 1, 3, or 7 days post-injury for immunohistochemical staining and Western blot analysis. One day post-injury, a few infiltrating inflammatory cells and small vacuoles were observed without significant group differences, followed over several days by progressive spinal cord degeneration. Glial fibrillary acidic protein (GFAP)-positive (reactive) astrocyte numbers were increased in all three groups, and brain-derived neurotrophic factor (BDNF) was colocalized with GFAP-positive cells in spinal ventral horn. Animals in the MSC-MG group demonstrated greater BDNF-positive astrocyte numbers, reduced caspase-3-positive cell numbers, and superior motor recovery. Microgravity-cultured MSC-based therapy may improve functional recovery following spinal ischemia-reperfusion injury by promoting astrocytic BDNF release, thereby preventing apoptosis.

Effects of ligustrazine on autophagy-related proteins after spinal cord ischemia-reperfusion injury

Objective To investigate the effect of ligustrazine on autophagy-related proteins Beclin l, LC3 and P62 after spinal cord ischemia-reperfusion injury. Methods A total of 48 SD rats were randomly divided into sham operation group, model group, ligustrazine group and 3-MA group. The rats were intraperitoneally injected with ligustrazine injection 0.16 mg/kg in the Ligustrazine group, the rats were intraperitoneally injected with 3-methyladenine injection 0.015 mg/kg in the inhibitor group, and the rats were intraperitoneally injected with normal saline of equal volume in the sham operation group and model group. Spinal cord ischemia-reperfusion model was established in all groups except sham-operated group after administration. After molding behavioral scores were scored after 3 and 6 hours of ischemia, and the expression of Beclin l, LC3 and P62 was detected by immunohis-tochemistry. Results After 3 and 6 hours, compared with the model group, the behavioral score (3 h: 2.33 ± 0.58 vs. 0.67 ± 0.58, 6 h: 3.33 ± 0.58 vs. 1.33 ± 0.58) of the rats in ligustrazine group significantly increased (P<0.05). Compared with the model group, the expression of Beclinl (3 h: 348.00×104 ± 0.27×104vs. 659.00×104 ± 0.11×104; 6 h: 38.00×104 ± 0.19×104vs. 557.00×104 ± 0.26×104), LC3 (3 h: 357.00×104 ± 0.48×104vs. 686.00×104 ± 0.33×104; 6 h: 334.00×104 ± 0.51×104vs. 673.00×104 ± 0.22×104), P62 (3 h: 357.00×104 ± 0.48×104vs. 830.00×104 ± 0.48×104; 6 h: 315.00×104 ± 0.12×104vs. 591.00×104 ± 0.36×104) in ligustrazine group were significantly decreased (P<0.05). Conclusions The ligustrazine may regulate autophagy in two directions and protect nerve cells. Key words: Chuanxiongzine; Spinal cord ischemia; Reperfusion injury; Beclin-1; Rats

Role of ERK signaling pathway in intrathecal dexmedetomidine-induced reduction of spinal cord ischemia-reperfusion injury in rats

Objective To evaluate the role of extracellular signal-regulated kinase (ERK) signaling pathway in intrathecal dexmedetomidine-induced reduction of spinal cord ischemia-reperfusion (I/R) injury in rats. Methods Eighty clean-grade male Sprague-Dawley rats, aged 9-10 weeks, weighing 300-350 g, were divided into 4 groups (n=20 each) using a random number table method: sham operation group (group S), spinal cord I/R group (group I/R), dexmedetomidine group (group D), and dexmedetomidine plus ERK signaling pathway blocker PD98059 group (group P). Spinal cord ischemia was produced by cross-clamping of the abdominal aorta distal to the left renal artery for 25 min followed by reperfusion to establish the model of spinal cord I/R injury.Dexmedetomidine 1 μg/kg was intrathecally injected at 20 min before establishing the model in D and P groups, PD98059 2 mg/kg was given via the tail vein at the same time in group P, and the equal volume of normal saline was given instead in S and I/R groups.Five rats were selected at 6, 8, 10 and 12 h of reperfusion, and the modified Basso, Beattie, Bresnahan (BBB) scale was used to assess the hindlimb locomotor function.Five rats were sacrificed after assessing the locomotor function at 6 h of reperfusion, and the L3-5 segments of the spinal cord were taken for determination of cell apoptosis (by TUNEL) and expression of phosphorylated ERK (p-ERK) (by Western blot). The apoptosis index was calculated. Results Compared with group S, the BBB scores were significantly decreased at each time point of reperfusion, the apoptosis index was increased, and the expression of p-ERK was up-regulated in the other three groups (P 0.05). Compared with group D, the BBB scores were significantly decreased at each time point of reperfusion, the apoptosis index was increased, and the expression of p-ERK was down-regulated in group P (P<0.05). Conclusion The mechanism by which intrathecal dexmedetomidine reduces spinal cord I/R injury is related to activating ERK signaling pathway in rats. Key words: Dexmedetomidine; Reperfusion injury; Spinal cord; Extracellular signal-regulated MAP kinases

The effects of barnidipine on an experimental ischemia reperfusion model of spinal cord injury and comparison with methyl prednisolone.

OBJECTIVE:Increased intracellular calcium concentration plays an important role in the secondary mechanism of spinal cord injury. In the presenting experimental study, we aimed to evaluate the healing effect of barnidipine, which has a high affinity for L-type calcium channels, on acute spinal cord injury and to compare its effects with those of methylprednisolone.METHODS:A total of 32 Spraque Dawley albino adult female rats were divided into 4 groups; group 1: sham-operated (n=8), group 2: only ischemia (n=6), group 3: barnidipine-treated (n=8), and group 4: methylprednisolone-treated (n=6). An ischemia-reperfusion model was created by clipping the abdominal aorta in the rats. Motor examination was performed 1 hour after the surgical procedure and before sacrification. Immediately following the second motor examination, rats were sacrificed and tissue samples were taken for histopathological examination and for testing of tissue malondialdehyde (MDA) levels.RESULTS:A significant correlation of motor examination was found between the sham-operated and barnidipine-treated groups and the sham-operated and only ischemia groups at the 1st and 24th hour (p<0.008). There was no significant difference between the only ischemia and barnidipine-treated groups and only ischemia and methylprednisolone-treated groups (p>0.008). Light microscopic examination of the sham-operated group revealed findings consistent with normal spinal cord structure. In group 2, 3, and 4, light microscopic examination revealed polymorphonuclear leukocyte infiltration and a small amount of axonal swelling. There was no significant correlation between the ischemia and barnidipine-treated groups and the barnidipine and methylprednisolone groups in terms of MDA levels (p>0.008).CONCLUSION:A single dose of barnidipine (10 mg/kg) and methylprednisolone are not effective and not sufficient to prevent spinal ischemia-reperfusion injury in rats.

Isoflurane Impairs Motor Function Recovery by Increasing Neuroapoptosis and Degeneration During Spinal Ischemia-Reperfusion Injury in Rats.

Spinal cord ischemia (SCI) leads to variable degrees of neurologic deficit in patients undergoing major cardiovascular surgery. The effect of intraoperative neuroprotection against SCI and the subsequent ischemia-reperfusion injury is still limited. Because isoflurane is a commonly used anesthetic agent during major operation, and its neuroprotective and neurotoxicity effects have both been discussed, this study aimed to investigate the effect of isoflurane on the spinal cord's functional recovery in a rat model of cord ischemia. Rats were randomly anesthetized by parenteral anesthetic (Zoletil) and isoflurane (0% and 1.5% v/v in oxygen). Cord ischemia was induced by cross-clamping of thoracic aorta at the level of T5, and cord perfusion was resumed after 25 minutes. The motor function was assessed independently up to 48 hours after reperfusion. Spinal cords were harvested and analyzed for molecular and histologic changes. The locomotor rating scale was significantly reduced in rats that received isoflurane treatment during SCI at 12 to 48 hours after reperfusion. Isoflurane enhanced the expression of heme oxygenase-1, glial fibrillary acidic protein, cleaved caspase-3, and Iba-1 in the spinal cord. Increased apoptotic cells and the presence of axonal damage were also observed in the histologic sections. Our results demonstrate that the administration of inhaled isoflurane in spinal cord ischemia-reperfusion injury impairs the recovery of motor function. This response is associated with the neuronal apoptosis and degeneration. This study highlights the potential adverse effect of isoflurane on the functional recovery of ischemic spinal cord during major aortic surgery.

Ischemia and reperfusion injury of the spinal cord: experimental strategies to examine postischemic paraplegia

Thoracoabdominal aortic replacement, necessary in case of injuries, aneurysms and dissections, shows a high complication rate as a consequence of the perioperative ischemia / reperfusion-sequence (I/R). Clamping above and below the lesion leads to the spinal cord suffering from ischemia. Clamping times of less than 30 minutes show only a small risk of neurological deficit, while longer periods increase paraplegia rates disproportionately. The subsequent reperfusion as a second hit causes additional damage to the spinal cord. Up to 30 % of all patients who require being treated in the thoracoabdominal part of the aorta suffer from paraplegia within the first 24 postoperative hours (Kahn et al., 2012).

The protective effect of hypoxic-preconditioned BMSCs on spinal cord tissue after ischemia reperfusion injury in rats

Objective To investigate the protective effect of hypoxic-preconditioned bone marrow mesenchymal stem cells (BMSCs) on spinal cord tissue after ischemia reperfusion injury. Methods Healthy adult Sprague Dawley (SD) rats weighing 200~250 grams (g) were randomly divided into 3 groups with 6 animals in each group: The sham group received simple surgical manipulation without ischemia/reperfusion treatment; The spinal cord ischemia/reperfusion group (Control group) only received spinal cord ischemia/reperfusion surgery. The hypoxic preconditioned BMSC transplantation group (HP-MSCs group) was injected with hypoxic preconditioned BMSCs 2 days before ischemia/reperfusion. The control group, HP-MSCs group received spinal cord ischemia/reperfusion for 10 min and observed for 48 h. The permeability of the blood-spinal cord barrier was examined with Evans blue (EB), and the histomorphology changes were observed with hematoxylin and eosin (HE) staining. Results EB red fluorescence was significantly weakened in the HP-MSCs group than that in the Control group (P<0.05=, and more intact motor neurons were found in the lumbar spinal cords in the HP-MSCs group than that in the Control group(P<0.05=. Conclusions The hypoxic-preconditioned BMSCs could effectively attenuate spinal cord ischemia reperfusion injury, it may be associated with protective effect of the blood-spinal cord barrier integrity. Key words: Anoxia; Hematopoietic stem cell transplantation; Mesenchymal stem cell transplantation; Spinal cord ischemia/SU; Reperfusion injury/SU

The neuroprotective mechanism of puerarin in the treatment of acute spinal ischemia–reperfusion injury is linked to cyclin-dependent kinase 5

Puerarin is shown to exert a variety of pharmacological effects including neuroprotective properties. However, mechanisms of the action are not fully understood. This study was designed to explore the mechanism of puerarin in treatment of acute spinal ischemia–reperfusion injury in rats. Acute spinal ischemia–reperfusion injury was conducted by aortic occlusion in twenty-eight male Sprague-Dawley rats, weighting 230–250g. The animals were randomly divided into four groups. In the animals with puerarin treatment, 50mg/kg of puerarin was injected intraperitoneally after reperfusion, and followed by the same dose of injection every 24h for 2 days. In the animals with roscovitine pre-treatment, 30mg/kg roscovitine was intravenously administrated 60min before spinal ischemia started. After spinal ischemia for 60min followed by 48h of reperfusion, the motor function, spinal infarction volume, apoptosis indices and the activities of Cdk5 and p25 were examined. Acute spinal ischemia–reperfusion resulted in an injury of the spines associated with motor deficit, elevation of Cdk5 and p25 activities, and increase in the spinal apoptosis number and spinal infarction volume. Puerarin improved motor function associated with the decreased apoptosis number, spinal infarction volume, and Cdk5 and p25 activities. The present study indicated that reduction of spinal injury was associated with inhibition of Cdk5 and p25, and that inhibition of Cdk5 and p25 was one of the neuroprotective mechanisms in the puerarin treatment of acute ischemia/reperfusion-induced spinal injury in rats.

Effects of simvastatin on heat hock protein 70 after spinal cord ischemia reperfusion injury

Objective To investigate the protective effect of simvastatin on spinal cord ischemia/reperfusion injury through the observation of the changes of heat shock protein 70 (Hsp70).Methods 24healthy adult Tibet mini-pigs of either sex,weighted 20-30 kg,were randomly divided into three groups (n =8),including sham operation group (group C),ischemia/reperfusion group (group I/R) and simvastatin experimental group (group Sim).In group I/R and Sim,the models of spinal cord ischemia/reperfusion were established by the occlusion of thoracic aorta and subclavian artery (70 min).The accessory hemiazygos vein was isolated and the distal end was ligated.In group I/R,1000 ml lactated ringer solution were pumped into the vein and the rate was 860 ml/h during the ischemia time.In group Sim,1000 ml simvastatin solution were pumped into the vein and the dose was 0.25 mg/kg during the ischemia time.Feed the swines of the experimental group simvastatin tablets 80 mg/d,orally for 30 days.After the operation,the motor function scores were recorded at 6,24,48 h (see Taylor motor function scoring system).All eight swines of each group were killed at 30 days after reperfusion,taking the L2-L5 and sacral spinal cord as quickly as possible.The hematoxylin and eosin (HE) staining method and the NISSLE staining method were adopted to observe the morphological changes of neurons and the immunohistochemical method was adopted to detect the changes of Hsp70.Results In group Sim,the 24,48 h motor function scores (3.2 ± 0.3,3.6 ± 0.2 respectively) after operation were significantly higher than that in group I/R (2.2 ±0.4,2.8 ± 0.4 respectively) (P ＜ 0.05).Compared with group I/R,the morphology of neurons in group Sim had a slight damage and higher expression changes of Hsp70 can be found in group Sim (P ＜ 0.05).Conclusion Simvastatin has a significant protective effect on the spinal cord ischemia reperfusion injury and the potential mechanism may be related to the up-regulation of Hsp70. Key words: Simvastatin; Spinal cord; Ischemia/reperfusion injury; Heat shock protein 70; Protection

Adenovirus-mediated gene transfer of rat heme oxygenase-1 against rat spinal cord ischemia-reperfusion injury

Objective To study the protective effect of adenovirus-mediated heme oxygenase-1 (HO-1) transfection on the spinal cord ischemia-reperfusion injury (IRI) in rats.Methods SD rats were randomly divided into sham operation group (group A),ischemia reperfusion group (group B) and AdV-rHO-1 group (group C),The rats in groups B and C were given spinal cord ischemia for 30 min.Physiological saline,AdV-EGFP,and AdV-rHO-1 were injected in groups A,B and C,respectively.Hind legs nerve function grading,apoptotic cells,immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR) were used to evaluate the degree of spinal cord injury.Results The hind legs nerve function grading (2.83 ± 0.75) in group C was obviously better than in group B (1.5 ± 0.55) after reperfusion for 2 days (P ＜ 0.01).The histopathologic changes in group C were obviously milder than those in group B.Immunohistochemistry and RT-PCR indicated that transgene was already expressed in group C.The level of HO-1 in group C (35.85 ±6.59) was higher than that in group A (7.24 ± 1.15) or group B (8.21 ± 3.05) (P ＜ 0.01).Conclusion Adenovirus-mediated gene transfection of rHO-1 has significantly protective effect on spinal cord IRI. Key words: Gene transfection ; Heme oxygenase-1 ; Adenovirus ; Spinal cord ischemia; Reperfusion injury

Effect of tacolimus postconditioning on serine-threonine protein kinase signaling pathway via regulating generation of reactive oxygen species after spinal cord ischemia-reperfusion injury

Objective To explore the influence of tacrolimus postconditioning (TP) on serine-threonine protein kinase (Akt) signaling pathway afier spinal cord ischemia-reperfusion injury (SCIRI)and the role of reactive oxygen species (ROS) in the mechanism.Methods The rat model of SCIRI was prepared on the basis of Taira' s method.Eighty male SD rats were randomly divided into 4 groups:sham operation (SO) group,ischemia-reperfusion (IR) group,TP group,and TP plus N-Acetylcysteine (TP +N) group.IR group underwent reperfusion for 20 min after spinal cord ischemia.TP group experienced a single injection of tacrolimus (0.5 mg/kg) through the left common carotid artery right at the onset of reperfusion,with remaining procedures the same as IR group.TP + N group was injected through the left common carotid artery with N-Acetylcysteine (150 mg/kg) 5 min before spinal cord ischemia,with remai-ning procedures the same as TP group.SO group received exclusively femoral artery catheterization.Fluo-rospectrophotometry was employed to detect the level of ROS in injured spinal cord segments,and immuno-blotting was used to determine the expression of phosphorylated Akt (p-Akt) at 15 min after reperfusion.Flow cytometry was applied to assess apoptosis of neural cells at 24 h after reperfusion.Nissl's staining was utilized to observe pathological changes of injured spinal cord segments at 7th day after reperfusion.Re-sults The ROS levels in spinal cord tissue were 49.0 ± 3.5,145.3±11.7,79.0±6.3 and12.8 ± 0.9in SO group,IR group,TP group,and TP + N group at 15 rain after reperfusion,respectively.The ROSlevel in TP group was significantly lower than that in IR group at 15 min after reperfusion (P ＜0.01),and that in TP + N group was significantly lower than the other three groups (P ＜ 0.01).The p-Akt expression in spinal cord tissue was 0.54 ± 0.06,0.34 ± 0.03,0.42 ± 0.07,and 0.28 ± 0.05 in SO group,IR group,TP group,and TP + N group at 15 min after reperfusion respectively.The p-Akt expression was sig-nificantly lower in IR group than in SO group (P ＜ 0.01),higher in TP group than in IR group (P ＜0.05),and lower in TP + N group than in SO group (P ＜0.01) or in TP group (P ＜0.01).The percentages of apoptotic cells in spinal cord tissue were (7.5 ± 1.8) ％,(31.4 ± 5.5) ％,(22.3-± 4.0) ％,and (33.3 ± 3.1) ％ in SO group,IR group,TP group,and TP + N group at 15 min after reperfusion respectively.The percentage of apoptotic cells was significantly higher in IR or TP + N group than in TP group (P ＜0.01),while there was no significant difference between IR group and TP + N group (P ＞0.05).The spinal cord sections revealed that pathological changes could hardly be observed in SO group,and mild in TP group but serious in IR and TP + N groups.Conclusion TP produces protective effect on ischemic spinal cord via positive regulation of Akt signaling pathway,and ROS,acting as a signaling molecule,plays an indispensable role in initiating the mechanism. Key words: Spinal cord ischemia; Tacrolimus ; Reactive oxygen species ; Serine-threonine protein kinase

Effects of drug volume on the model for regional drug protection against spinal cord ischemiareperfusion injury in rabbits

Objective To compare the effects of different drug volume on the model for regional aorta-infused drug protection against spinal cord ischemia-reperfusion injury in rabbits.Methods Spinal cord ischemia model was induced in New Zealand white rabbits by infrarenal abdominal aorta occlusion with a balloon catheter for 30 min.Another polycarbonate catheter was inserted into the abdominal aorta with the tip close to the site of occlusion via the femoral artery.During the ischemic period,nothing (V0 group) or different volume (1.5,3.0 or 6.0 ml/kg) of normal saline containing methylene blue (V1.5,V3 and V6 groups) was consecutively infused into the occluded aortic segments via the polycarbonate catheter at different rate of 3.0,6.0,or 12.0 ml/(kg·h).The bilateral common iliac arteries were clamped simultaneouly to prevent drug solution leakage.Thirty min later,blood flow was regained for reperfusion.The change of mean aortic pressure distal to the occluded site was measured throughout the operation.Neurological function was assessed according to the modified Tarlov scales at the moment of palinesthesia and 6,24 and 48 h after reperfusion.Lumbar segements of the spinal cord (L4-5) were harvested at 48 h after reperfusion for histopathologic evaluation,and the total number of normal motor neurons in the anterior horn was counted.Results The abdominal aorta was not completely occluded in V6 group.In V3 and V6 groups,the distal aortic pressure during ischemia was higher than in V0 and V1.5 groups with (41.0 ± 5.5) and (49.4 ± 4.5) vs.(17.9±2.6) and (22.5±1.7) mmHg [1 mmHg=0.133kPa,P＜0.05],while the difference value between ischemia and the first 10 min of reperfusion was lower than in V0 and V1.5 groups with (44.9±8.9)and (32.3±9.4) vs.(62.5±6.3) and (61.8±4.5) mmHg (P＜0.05).Tarlovscores were higher in V6 group than in V0 group at all intervals (P ＜ 0.05).The number of normal motor neurons was greater in V6 group than in V0 group with 10 (5.0-12.0) vs.0 (0-4.5) (P＜0.05).There was no significant difference between other groups and V0 group (P ＞ 0.05).Conclusion Drug volume lower than 3 ml/kg can ensure the model stability of spinal cord injury when infused into the occluded aortic segments during the ischemic period.The optimal volume was 1.5 ml/kg. Key words: Spinal cord ischemia; Reperfusion injury; Arterial infusions; Models,animal

Modification of the minimally invasive model for regional drug protection against spinal cord ischemia-reperfusion injury in rabbits

Objective To establish a minimally invasive model for regional aorta-infused drug protection against spinal cord ischemia-reperfusion injury in rabbits.Methods Spinal cord ischemia was induced in New Zealand rabbits by infrarenal abdominal aorta occlusion with different balloon catheters or an artery clamp for 30 min.During ischemia,normal saline or lidocaine 40 mg/kg was consecutively infused into the occluded aortic segments via a polycarbonate catheter through the femoral artery.Thereafter,blood flow was regained for reperfusion.Neurological function was assessed according to Tarlov scales postoperatively.L4-5 segements of the spinal cord were harvested at 48 h after reperfusion for histopathologic evaluation,and the total number of normal motor neurons in the anterior horn was counted.Results The 4F SwanGanz balloon catheter combined with a polycarbonate catheter was appropriate for modeling.Tarlov scores,the incidence of paraplegia (87.5％ in normal saline groups; 25.0％ vs.12.5％ in lidocaine groups) and the number of normal motor neurons [0 (0-1.75) vs.1 (0-2.00) in normal saline groups ; 8.5 (2.25-12.00) vs.9.0 (4.75-10.75) in lidocaine groups] were similar between balloon occlusion and aorta clamping while the same solution was infused (P ＞ 0.05).Conclusion The 4F Swan-Ganz balloon catheter combined with a polycarbonate catheter can be used to establish the minimally invasive model for regional drug protection against spinal cord ischemia-reperfusion instead of conventional aorta clamping in rabbits. Key words: Spinal cord ischemia; Reperfusion injury ; Aorta occlusion ; Models,animal

Protective effect of tacrolimus postconditioning on oxidative stress injury provoked by spinal cord ischemia-reperfusion in rats

To investigate whether protective effect of tacrolimus postconditioning on rats' spinal cord ischemia-reperfusion injury is mediated by up-regulation of activity of endogenous antioxidant enzymes and down-regulation of production of oxygen free radicals. Ninety male Sprague-Dawley rats were randomly divided into ischemia-reperfusion (IR) group, tacrolimus postconditioning (TP) group and sham operation (SO) group. The model of spinal cord ischemia was prepared by means of catheterization through femoral artery and balloon dilatation. IR group underwent reperfusion 20 min after spinal cord ischemia. TP group experienced a single injection of tacrolimus (0.5 mg/kg) through the left common carotid artery at the onset of reperfusion. SO group received femoral artery catheterization only. Fluoro spectro photometry was employed to detect the level of reactive oxygen species (ROS) in injured spinal cord segment at 15 minutes after reperfusion. The content of malondialdehyde (MDA) and activity of antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-PX) were determined at 15 minutes, 1, 6, and 24 hours after reperfusion respectively. BBB scale was conducted to evaluate hindlimb motor function at 14 days after reperfusion. The level of ROS in TP group was significantly lower than that in IR group at 15 minutes after reperfusion. The activity of SOD was significantly higher in TP group than in IR group at all observational time points, while the activities of CAT and GSH-PX were significantly higher in TP group than in IR group at 1 and 6 hours after reperfusion. The content of MDA in TP group was significantly less than that in IR group at all observational time points. The motor function score of TP group was significantly superior to that of IR group at 14 days after reperfusion. Tacrolimus post conditioning can improve activity of endogenous antioxidant enzymes, decrease production of oxygen free radicals, suppress lipid peroxidation, and thereby promote functional recovery after spinal cord ischemia-reperfusion injury in rats.

Effects of adenosine preconditioning on expression of B lymphocytes/leukemia-2 and bcl-2 associated X protein following the spinal cord ischemia-reperfusion injury of rats

Objective To study the expression levels of B lymphocytes/leukemia-2 (bcl-2) and bcl-2 associated X protein (bax) following ischemia-reperfusion injury and their action mechanism on apoptosis for investigating the protection of spinal cord I-R injury by means of adenosine preconditioning.Methods The experimental model was established in rats through cross-clamping of abdominal aorta inferior to the left renal artery.Twenty-seven rats were randomly divided into three groups:group A (n =9),sham group; group B (n =9),I-R group ; and group C (n =9),administration with adenosine group.Spinal cord function was assessed by the Modified Tarlov Scale at 12,24 and 48 h after reperfusion.At 48 h after reperfusion,histologic analysis was done by hematoxylin and eosin (HE) and Nissl staining,ultrastructures were observed under transmission electron microscopy,and the expression levels of bcl-2 and bax pro tein and mRNA were detected by using Western blotting and real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) respectively.Results HE staining and Nissl staining results showed:group B showed extensive spinal cord injury such as cell edema and dissolution of Nissl substance when compared to group C; the number of apoptotic cells in group C was less than that in group B.Western blotting and RT-qPCR results showed adenosine could reduce bax expression and increase the bcl-2 expression.Conclusion Adenosine can reduce spinal cord I-R and improve neurological function. Key words: Spinal ischemia; Reperfusion injury; Apoptosis ; B leukemia/lymphocytes-2 ; bcl-2 associated X protein

Effect of hypoxic preconditioning on anti-inflammatory responses of bone marrow mesenchymal stem cells in rats: in vitro and in vivo experiments

Objective To investigate the effect of hypoxic preconditioning on anti-inflammatory responses of bone marrow mesenchymal stem cells (BMSCs) in rats through in vitro and in vivo experiments.Methods In vitro experiment The isolated rat BMSCs were cultured by whole bone marrow adherence method.The cells at passage 3 were seeded in 24-well plates at a density of 1 × 106 cells/ml and randomly divided into 5 groups (n =8 wells each) using a random number table:control group (group C),normoxia-incubated group (group N),hypoxic preconditioning group (group H),hypoxia preconditioning + STAT3 inhibitor Stattic group (group HS) and hypoxia preconditioning + anti-IL-10 monoclonal antibody group (group HA).In group C,BMSCs were incubated in DMEM culture medium.In group N,BMSCs were exposed to21％ O2-74％ N2-5.0％ CO2 for48 h.In group H,BMSCs were exposed to 0.5％ O2-94.5％ N2-5.0％ CO2 for 24 h followed by 24 h exposure to normoxia.In HS and HA groups,500 μg/ml Stattic and 100 μg/rnl anti-IL-10 monoclonal antibody were added to the culture medium before hypoxia preconditioning,respectively.The expression of phosphorylated STAT3 (p-STAT3) and IL-10 was determined by Western blot.In vivo experiment Healthy male Sprague-Dawley rats,weighing 300-350 g,in which intrathecal catheters were successfully implanted without complications,underwent spinal cord ischemia by occlusion of the thoracic aorta combined with controlled hypotension.Three hundred rats with spinal cord I/R injury were randomly divided into C,N,H,HS and HA groups (n =60 each) using a random number table.Immediately after onset of reperfusion,DMEM medium 300 μl was injected intrathecally in group C,and BMSC suspension 300 μl (1 × 106 cells/ml) was injected intrathecally in N,H,HS and HA groups.Neurological function was scored before ischemia and at 4,12,24 and 48 h of reperfusion (T0-,).The animals were then sacrificed and the lumbar segment of spinal cord was removed for detection of the content of IL-10,TNF-α,IL-1β,IL-6,monocyte chemotactic protein-1 (MCP-1),and macrophage inflammatory protein-1 α (MIP-1 α) (by ELISA) and the number of activated microglia (by immuno-histochemistry).Results Compared with C and N groups,the expression of pSTATβ and IL-10 was significantly up-regulated,the neurological function score and IL-10 content were increased,the content of TNF-α,IL-1β,IL-6,MCP-1 and MIP-1α and the number of activated microglia were decreased in group H (P ＜ 0.05).Compared with group H,the expression of p-STAT3 and IL-10 in group HS and expression of IL-10 in group HA was significantly down-regulated,and the neurological function score and IL-10 content were decreased,and the content of TNF-α,IL-13,IL-6,MCP-1 and MIP-1α and the number of activated microglia were increased in HS and HA groups (P ＜ 0.05).Conclusion Hypoxic preconditioning can enhance anti-inflammatory effects of BMSCs,thus increasing BMSCs-induced reduction of spinal cord ischemia-reperfusion injury in rats. Key words: Cell hypoxia; Ischemica preconditioning; Mesenchymal stem cells transplantation ; Spinal cord ; Inflammation

RETRACTED ARTICLE: Neuroprotective effect of Buyang Huanwu Decoction on spinal ischemia-reperfusion injury in rats is linked with inhibition of cyclin-dependent kinase 5

BackgroundBuyang Huanwu Decoction (BYHWD), a traditional Chinese medicine formula, has been shown to exert a variety of pharmacological effects including neuroprotective properties. However, the mechanism of neuroprotection is not fully understood. This study was designed to explore the mechanism of BYHWD in the treatment of spinal ischemia-reperfusion injury in rats.MethodsTwenty-eight male Sprague–Dawley rats, weighting 250–280 g, were used, and were randomly divided into four groups with 7 animals in each: sham operation group (Control), spinal ischemia with saline (SI + Saline), spinal ischemia with BYHWD (SI + BYHWD), and spinal ischemia with roscovitine (SI + R). After 60 minutes of spinal ischemia followed by 72 hours of reperfusion, motor function of hind limbs, spinal ischemic infarction volume, the number of apoptotic cells, and cyclin-dependent kinase 5 (Cdk5) were examined.ResultIschemia-reperfusion resulted in injury of the spines, while BYHWD significantly improved spinal function. The spinal infarction volume, number of apoptotic cells, and Cdk5 were decreased by administration of BYHWD. The similar improvements were seen with the pre-treatment of roscovitine.ConclusionsBYHWD prevented the ischemia-reperfusion-induced spinal injury in rats. The protective function of BYHWD was, in part, linked with inhibition of Cdk5.

Comparison of neuroprotective effect of two postconditioning methods on spinal cord ischemia-reperfusion injury in rats

Objective To compare neuroprotective effect of mechanical postconditioning (MP) vs.tacrolimus postconditioning (TP) on spinal cord ischemia-reperfusion (IR) injury in rats.Methods Forty-five male SD rats were randomly divided into IR group,MP group and TP group.The model of spinal cord ischemia was established by means of catheterization through femoral artery and balloon dilatation.IR group underwent 20-min reperfusion after spinal cord ischemia.MP group received 3 cycles of 30-s reperfusion/30-s ischemia at the onset of reperfusion.TP group experienced a single injection of tacrolimus (0.5 mg/kg) through the left common carotid artery at the onset of reperfusion.Tarlov scale was conducted to evaluate hindlimb motor function,and Western blotting was applied to determine the expression of Caspase-3 in spinal cord at 1st,3rd,and 7th day after reperfusion.Results The motor function scores of both MP and TP groups were significantly higher than those of IR group with 1.75 ±0.23 and 1.62 ±0.41 vs.0.33±0.47 at 1stday,2.670.35 and 2.46±0.33 vs.1.53 ±0.44 at 3rd day,and 3.06±0.27 and 3.09 ±0.22 vs.2.54 ±0.31 at 7th day respectively after reperfusion (P ＜0.05).There were no significant differences in the motor function scores between MP and TP groups at all observational time points (P ＞ 0.05),though the score in TP group was higher than that in MP group at 7th day after reperfusion.The expression of Caspase-3 in spinal cord of both MP and TP groups was significantly lower than that of IR group with 0.26 ± 0.03 and 0.27 ± 0.02 vs.0.32 ± 0.06 at 1 st day,0.29 ± 0.04 and 0.30 ± 0.07 vs.0.40 ±0.08 at 3rd day,0.28 ±0.07 and 0.28 ±0.03 vs.0.39 ±0.10 at 7th day respectively after reperfusion (P ＜ 0.05).There were no significant differences in the Caspase-3 expression between MP and TP group at all observational time points (P ＞ 0.05).Conclusion Tacrolimus postconditioning possesses neuroprotective effect comparative to classic mechanical postconditioning,and both postconditioning methods can alleviate spinal cord ischemia-reperfusion injury in rats. Key words: Spinal cord ischemia; Reperfusion injury; Tacrolimus postconditioning

Role of CXCR4-FAK signaling pathway in migration and adhesion of hypoxia-preconditioned bone marrow mesenchymal stem cells towards damaged tissues resulting from spinal cord ischemia-reperfusion injury in rats

Objective To investigate the role of CXC chemokine receptor 4-focal adhesion kinase (CXCR4-FAK) signaling pathway in migration and adhesion of hypoxia-preconditioned bone marrow mesenchymal stem cells (BMSCs) towards damaged tissues resulting from spinal cord ischemia-reperfusion (I/R) injury in rats.Methods Part I Rat BMSCs transfected with recombinant adenovirus-mediated green fluorescent protein gene 3 were seeded in 24-well plates and randomly divided into 5 groups (n =18 wells each):control group (group C),normoxia-incubated group (group N),HP group (group H),HP + CXCR4 antagonist AMD3100 group (group HA) and HP + FAK inhibitor FAK-related nonkinase group (group HF).In group C,BMSCs were incubated in DMEM culture medium.In group N,BMSCs were exposed to 21％ O2-74％ N2-5％ CO2 for 36 h.In group H,BMSCs were exposed to 0.5％O2-94.5％ N2-5.0％CO2 for 24 h followed by 12 h exposure to normoxia.In groups HA and HF,5 μg/ml AMD3100 and 10 μg/ml FAK-related nonkinase were added to the culture medium before HP,respectively.The expression of stromal derived factor-1α (SDF-1α),CXCR4 and phosphorylated FAK (p-FAK) in BMSCs was determined by Western blot.The migratory capability and adhesive ability of BMSCs were measured by Transwell invasive assay and fibronectin adhesive assay,respectively.Part Ⅱ Two hundred and sixteen male Sprague-Dawley rats weighing 300-350 g were used and 210 out of the 216 rats underwent spinal cord ischemia by occlusion of the thoracic aorta combined with controlled hypotension.Thirty-six rats were chosen and sacrificed before spinal cord I/R and at 12 h and 1,3,5 and 7 days of reperfusion (T0-5) and the lumbar segment of spinal cord was removed for detection of the content of SDF-1α.The left 180 rats with spinal cord I/R were randomly divided into 5 groups (n =36 each).IT DMEM medium 300 μl was injected in group C and BMSC suspension 300 μl (1 × 106/ml) was injected in groups N,H,HA and HF immediately after onset of reperfusion.Neurological function was scored at T0-5.The animals were then sacrificed and the lumbar segment of spinal cord was removed for detection of the degree of BMSC aggregation.Results There was no significant difference in the expression of SDF-1α,CXCR4 and p-FAK,migratory capability and adhesive ability of BMSCs,neurological function scores and degree of BMSC aggregation between groups C and N (P ＞ 0.05).Compared with group N,the expression of SDF-1α,CXCR4 and p-FAK was significantly up-regulated,and migratory capability and adhesive ability of BMSCs,neurological function scores and the degree of BMSC aggregation were increased in group H,while no significant change was found in the expression of p-FAK,migratory capability and adhesive ability of BMSCs,neurological function scores and degree of BMSC aggregation in HA and HF groups (P ＞ 0.05).Compared with group H,the expression of p-FAK was down-regulated and the migratory capability and adhesive ability of BMSCs,neurological function scores and degree of BMSC aggregation were decreased in groups HA and HF (P ＜0.05).The content of SDF-1α was significantly higher at T2,3 than at T0.Conclusion HP can promote migration and adhesion of BMSCs towards damaged tissues resulting from spinal cord I/R injury through CXCR4-FAK signaling pathway in rats; thus CXCR4-FAK signal pathway provides the protective effect on spinal cord. Key words: Receptors, CXCR4; Focal adhesion protein-tyrosine kinases; Mesenchymal stem cell transplantation ; Cell Hypoxia ; Ischemic preconditioning ; Cell movement; Cell adhesion; Spinal cord ; Reperfusion injury