Key genes expressed in different stages of spinal cord ischemia/reperfusion injury ischemia/reperfusion injury

The temporal expression of microRNA after spinal cord ischemia/reperfusion injury is not yet fully understood.In the present study,we established a model of spinal cord ischemia in Sprague-Dawley rats by clamping the abdominal aorta for 90 minutes,before allowing reperfusion for 24 or 48 hours.A sham-operated group underwent surgery but the aorta was not clamped.The damaged spinal cord was removed for hematoxylin-eosin staining and RNA extraction.Neuronal degeneration and tissue edema were the most severe in the 24-hour reperfusion group,and milder in the 48-hour reperfusion group.RNA amplification,labeling,and hybridization were used to obtain the microRNA expression profiles of each group.Bioinformatics analysis confirmed four differentially expressed microRNAs(miR-22-3p,miR-743b-3p,miR-201-5p and miR-144-5p) and their common target genes(Tmem69 and CxcllO).Compared with the sham group,miR-22-3p was continuously upregulated in all three ischemia groups but was highest in the group with no reperfusion,whereas miR-743b-3p,miR-201-5p and miR-144-5p were downregulated in the three ischemia groups.We have successfully identified the key genes expressed at different stages of spinal cord ischemia/reperfusion injury,which provide a reference for future investigations into the mechanism of spinal cord injury.     

Ginsenoside Rd inhibits apoptosis following spinal cord ischemia/reperfusion injury

Ginsenoside Rd has a clear neuroprotective effect against ischemic stroke. We aimed to verify the neuroprotective effect of ginsenoside Rd in spinal cord ischemia/reperfusion injury and explore its anti-apoptotic mechanisms. We established a spinal cord ischemia/reperfusion injury model in rats through the occlusion of the abdominal aorta below the level of the renal artery for 1 hour. Successfully established models were injected intraperitoneally with 6.25, 12.5, 25 or 50 mg/kg per day ginsenoside Rd. Spinal cord morphology was observed at 1, 3, 5 and 7 days after spinal cord ischemia/reperfusion injury. Intraperitoneal injection of ginsenoside Rd in ischemia/reperfusion injury rats not only improved hindlimb motor function and the morphology of motor neurons in the anterior horn of the spinal cord, but it also reduced neuronal apoptosis. The optimal dose of ginsenoside Rd was 25 mg/kg per day and the optimal time point was 5 days after ischemia/ reperfusion. Immunohistochemistry and western blot analysis showed ginsenoside Rd dose-de- pendently inhibited expression of pro-apoptotic Caspase 3 and down-regulated the expression of the apoptotic proteins ASK1 and JNK in the spinal cord of rats with spinal cord ischemia/reper- fusion injury. These findings indicate that ginsenoside Rd exerts neuroprotective effects against spinal cord ischemia/reperfusion injury and the underlying mechanisms are achieved through the inhibition of ASK1-JNK pathway and the down-regulation of Caspase 3 expression.     

Aldehyde dehydrogenase 2 overexpression inhibits neuronal apoptosis after spinal cord ischemia/reperfusion injury

Aldehyde dehydrogenase 2(ALDH_2)is an important factor in inhibiting oxidative stress and has been shown to protect against renal ischemia/reperfusion injury.Therefore,we hypothesized that ALDH_2 could reduce spinal cord ischemia/reperfusion injury.Spinal cord ischemia/reperfusion injury was induced in rats using the modified Zivin’s method of clamping the abdominal aorta.After successful model establishment,the agonist group was administered a daily consumption of 2.5%alcohol.At 7 days post-surgery,the Basso,Beattie,and Bresnahan score significantly increased in the agonist group compared with the spinal cord ischemia/reperfusion injury group.ALDH_2expression also significantly increased and the number of apoptotic cells significantly decreased in the agonist group than in the spinal cord ischemia/reperfusion injury group.Correlation analysis revealed that ALDH_2 expression negatively correlated with the percentage of TUNEL-positive cells(r=-0.485,P0.01).In summary,increased ALDH_2 expression protected the rat spinal cord against ischemia/reperfusion injury by inhibiting apoptosis.     

丹参酮对大鼠脊髓缺血再灌注损伤NMDAR1蛋白表达的影响

BACKGROUND： Tanshinone has been previously shown to be involved in the prevention and treatment of cerebral ischemia/reperfusion injury. In addition, excitatory amino acid-mediated neu- rotoxicity may induce neuronal damage following spinal cord ischemia/reperfusion injury.
OBJECTIVE： To explore the interventional effect of tanshinone on N-methyl-D-aspartate receptor 1 （NMDAR1） protein expression in a rat model of spinal cord ischemia/reperfusion injury.
DESIGN, TIME AND SETTING： A randomized molecular biology experiment was conducted at the Traumatology ＆ Orthopedics Laboratory of Fujian Hospital of Traditional Chinese Medicine （Key Laboratory of State Administration of Traditional Chinese Medicine） between September 2007 and May 2008. MATERIALS： A total of 88 Sprague Dawley rats were randomly divided into a sham operation （n = 8）, model （n = 40）, and tanshinone （n = 40） groups. Thirty minutes after ischemia, rats in the model and tanshinone groups were observed at hour 0.5, 1, 4, 8, and 12 following perfusion, with eight rats for each time point. METHODS： Abdominal aorta occlusion was performed along the right renal arterial root using a Scoville-Lewis clamp to induce spinal cord ischemia. Blood flow was recovered 30 minutes following occlusion to establish models of spinal cord ischemia/reperfusion injury. Abdominal aorta occlusion was not performed in the sham operation group. An intraperitoneal injection of tanshinone ⅡA sulfonic sodium solution （0.2 L/g） was administered to rats in the tanshinone group, preoperatively. In addition, rats in the sham operation and model groups were treated with an intraperitoneal injection of the same concentration of saline, preoperatively.
MAIN OUTCOME MEASURES： NMDAR1 protein expression in the anterior horn of the spinal cord, accumulative absorbance, average absorbance, and area of positive cells were detected in the three groups through immunohistochemistry.
RESULTS： All 88 rats were included in the final analysis. （1） NMDAR1 protein expression increased following 30-minute ischemia/1-hour reperfusion injury to the spinal cord, and reached a peak 4 hours after reperfusion. （2） Accumulative absorbance and average absorbance of NMDAR1, as well as area of positive cells in the model group, were significantly greater than the sham operation group at each time point （P 〈 0.05）. However, values in the tanshinone group were significantly less than the model group （P 〈 0.05）.
CONCLUSION： NMDAR1 protein expression was rapidly increased following spinal cord ischemia/reperfusion injury and reached a peak 4 hours following reperfusion. In addition, tanshinone downregulated NMDAR1 protein expression in the anterior horn of the spinal cord.     

Oxidative phosphorylated neurofilament protein M protects spinal cord against ischemia/reperfusion injury

Previous studies have shown that neurofilament protein M expression is upregulated in the early stage of spinal cord ischemia/reperfusion injury, indicating that this protein may play a role in the injury process. In the present study, we compared protein expression in spinal cord tissue of rabbits after 25 minutes of ischemia followed by 0, 12, 24, or 48 hours of reperfusion with that of sham operated rabbits, using proteomic two-dimensional gel electrophoresis and mass spec- trometry. In addition, the nerve repair-related neurofilament protein M with the unregulated expression was detected with immunohistochemistry and western blot analysis. Two-dimen- sional gel electrophoresis and mass spectrometry showed that, compared with the sham group, upregulation of protein expression was most significant in the spinal cords of rabbits that had undergone ischemia and 24 hours of reperfusion. Immunohistochemical analysis revealed that neurofilament protein M was located in the membrane and cytoplasm of neuronal soma and axons at each time point after injury. Western blot analysis showed that neurofilament protein M expression increased with reperfusion time until it peaked at 24 hours and returned to baseline level after 48 hours. Furthermore, neurofilament protein M is phosphorylated under oxidative stress, and expression changes were parallel for the phosphorylated and non-phosphorylated forms. Neurofilament protein M plays an important role in spinal cord ischemia/reperfusion injury, and its functions are achieved through oxidative phosphorylation.     

Stress protein expression in early phase spinal cord ischemia/reperfusion injury*

Spinal cord ischemia/reperfusion injury is a stress injury to the spinal cord. Our previous studies using differential proteomics identified 21 differentially expressed proteins (n > 2) in rabbits with spinal cord ischemia/reperfusion injury. Of these proteins, stress-related proteins included protein disulfide isomerase A3, stress-induced-phosphoprotein 1 and heat shock cognate protein 70. In this study, we established New Zealand rabbit models of spinal cord ischemia/reperfusion injury by abdominal aorta occlusion. Results demonstrated that hind limb function initially improved after spinal cord ischemia/reperfusion injury, but then deteriorated. The pathological morphology of the spinal cord became aggravated, but lessened 24 hours after reperfusion. However, the numbers of motor neurons and interneurons in the spinal cord gradually decreased. The expression of protein disulfide isomerase A3, stress-induced-phosphoprotein 1 and heat shock cognate protein 70 was induced by ischemia/reperfusion injury. The expression of these proteins increased within 12 hours after reperfusion, and then decreased, reached a minimum at 24 hours, but subsequently increased again to similar levels seen at 6-12 hours, showing a characterization of induction-inhibition-induction. These three proteins were expressed only in cytoplasm but not in the nuclei. Moreover, the expression was higher in interneurons than in motor neurons, and the survival rate of interneurons was greater than that of motor neurons. It is assumed that the expression of stress-related proteins exhibited a protective effect on neurons.     

Differential protein expression in spinal cord tissue of a rabbit model of spinal cord ischemia/reperfusion injury

New Zealand rabbits were randomly divided into an ischemia group(occlusion of the abdominal aorta for 60 minutes),an ischemia-reperfusion group(occlusion of the abdominal aorta for 60 minutes followed by 48 hours of reperfusion) and a sham-surgery group.Two-dimensional gel electrophoresis detected 49 differentially expressed proteins in spinal cord tissue from the ischemia and ischemia/reperfusion groups and 23 of them were identified by mass spectrometry.In the ischemia group,the expression of eight proteins was up regulated,and that of the remaining four proteins was down regulated.In the ischemia/reperfusion group,the expression of four proteins was up regulated,and that of two proteins was down regulated.In the sham-surgery group,only one protein was detected.In the ischemia and ischemia/reperfusion groups,four proteins overlapped between groups with the same differential expression,including three that were up regulated and one down regulated.These proteins were related to energy metabolism,cell defense,inflammatory mechanism and cell signaling.     

异丙酚及缺血预处理联合对主动脉阻断致脊髓损伤的保护

目的 探讨缺血预处理(ischemic preconditioning,IPC)和异丙酚（Propofol）预处理对兔主动脉阻断所致脊髓损伤的防治作用其可能作用机制。 方法 本实验有二处理因素：缺血预处理及异丙酚预处理。采用2×2析因实验设计,分四个实验组：缺血再灌注损伤组（A组,空白组）、缺血预处理组（B组）、异丙酚组（C组）及缺血预处理和异丙酚联合预处理组（D组）。雄性日本大白兔32只,随机分为4组,每组8只。各组阻断腹主动脉40min,再灌注7d。B组IPC5min,再灌注30min后阻断腹主动脉40min,再灌注7d;C组静注异丙酚5mg/kg 10min后阻断腹主动脉40min,再灌注7d;D组IPC5min,,再灌注20min时静注异丙酚5mg/kg,再灌注30min时阻断腹主动脉血流40分钟,再灌注7天。分别测定阻断前10min（C-10）、开放前即刻（C40）、再灌注60min（R60）及7d（R7d）血清MDA、SOD;观察术后后肢神经功能;脊髓病理学观察;脊髓凋亡神经元。 结果 ①缺血再灌注后B、C、D组MDA值明显高于C-10值及A组相应时点值（p<0.05）, SOD值变化同MDA变化相反（p<0.05）; B组缺血后各时点MDA值明显低于C组(P<0.05), D组缺血后各时点MDA值明显低于B、C组(P<0.05),SOD值变化同MDA变化相反（p<0.05）。②B、C、D组凋亡细胞数明显少于A组(P<0.05); B组明显少于C组(P<0.05),D组明显少于B、C组(P<0.05)。③B、C、D组瘫痪发生率明显低于A组(P<0.05),B组瘫痪发生率明显低于C组(P<0.05),D组瘫痪发生率明显低于B、C组(P<0.05);B、C、D组后肢神经功能评分明显高于A组(P<0.05), B组后肢神经功能评分高于C组(P<0.05),D后肢神经功能评分高于B、C组(P<0.05)。④ D组脊髓病理变化明显轻于A、B、C组(P<0.05)。结论 缺血预处理和异丙酚预处理对兔主动脉阻断所致脊髓损伤都有良好的防治作用;缺血预处理对脊髓缺血再灌注损伤的作用明显好于异丙酚;缺血预处理和异丙酚联合应用对脊髓缺血再灌注损伤有更加良好的防治作用,表现出一定的交互作用。缺血预处理和异丙酚联合预处理对兔主动脉阻断所致脊髓损伤其机制可能与其抗氧化反应作用有关。     

MicroRNA-101a-3p mimic ameliorates spinal cord ischemia/reperfusion injury

miR-101a-3p is expressed in a variety of organs and tissues and plays a regulatory role in many diseases,but its role in spinal cord ischemia/reperfusion injury remains unclear.In this study,we established a rat model of spinal cord ischemia/reperfusion injury by clamping the aortic arch for 14 minutes followed by reperfusion for 24 hours.Results showed that miR-101a-3p expression in L4-L6 spinal cord was greatly decreased,whereas MYCN expression was greatly increased.Dual-luciferase reporter assay results showed that miR-101a-3p targeted MYCN.MYCN immunoreactivity,which was primarily colocalized with neurons in L4-L6 spinal tissue,greatly increased after spinal cord ischemia/reperfusion injury.However,intrathecal injection of an miR-101a-3p mimic within 24 hours before injury decreased MYCN,p53,caspase-9 and interleukin-1βexpression,reduced p53 immunoreactivity,reduced the number of MYCN/NeuN-positive cells and the number of necrotic cells in L4-L6 spinal tissue,and increased Tarlov scores.These findings suggest that the miR-101a-3p mimic improved spinal ischemia/reperfusion injury-induced nerve cell apoptosis and inflammation by inhibiting MYCN and the p53 signaling pathway.Therefore,miR-101a-3p mimic therapy may be a potential treatment option for spinal ischemia/reperfusion injury.     

MicroRNA miR-133b is essential for functional recovery after spinal cord injury in adult zebrafish

MicroRNAs (miRNAs) play important roles during development and also in adult organisms by regulating the expression of multiple target genes. Here, we studied the function of miR-133b during zebrafish spinal cord regeneration and show upregulation of miR-133b expression in regenerating neurons of the brainstem after transection of the spinal cord. miR-133b has been shown to promote tissue regeneration in other tissue, but its ability to do so in the nervous system has yet to be tested. Inhibition of miR-133b expression by antisense morpholino (MO) application resulted in impaired locomotor recovery and reduced regeneration of axons from neurons in the nucleus of the medial longitudinal fascicle, superior reticular formation and intermediate reticular formation. miR-133b targets the small GTPase RhoA, which is an inhibitor of axonal growth, as well as other neurite outgrowth-related molecules. Our results indicate that miR-133b is an important determinant in spinal cord regeneration of adult zebrafish through reduction in RhoA protein levels by direct interaction with its mRNA. While RhoA has been studied as a therapeutic target in spinal cord injury, this is the first demonstration of endogenous regulation of RhoA by a microRNA that is required for spinal cord regeneration in zebrafish. The ability of miR-133b to suppress molecules that inhibit axon regrowth may underlie the capacity for adult zebrafish to recover locomotor function after spinal cord injury.     

Expression profiles of microRNAs after focal cerebral ischemia/reperfusion injury in rats

Rat models of focal cerebral ischemia/reperfusion injury were established by occlusion of the middle cerebral artery.Microarray analysis showed that 24 hours after cerebral ischemia,there were nine up-regulated and 27 down-regulated microRNA genes in cortical tissue.Bioinformatic analysis showed that bcl-2 was the target gene of microRNA-384-5p and microRNA-494,and caspase-3 was the target gene of microRNA-129,microRNA-320 and microRNA-326.Real-time PCR and western blot analyses showed that 24 hours after cerebral ischemia,bcl-2 mRNA and protein levels in brain tissue were significantly decreased,while caspase-3 mRNA and protein levels were significantly increased.This suggests that following cerebral ischemia,differentially expressed microRNA-384-5p,microRNA-494,microRNA-320,microRNA-129 and microRNA-326 can regulate bcl-2 and caspase-3 expression in brain tissue.     

脊髓缺血性损害时检测血糖、丙二醛、神经源性胞浆酶的意义

目的 观察家兔脊髓缺血性损害时血糖、丙二醛(MDA)、神经源性胞浆酶的变化,探讨其临床意义。方法 16只家兔随机分为二组:假手术组和缺血再灌注组,缺血再灌注组分缺血40min和再灌注后1h两时点采血。分别检测各组血糖、MDA、血清肌酸激酶(CK)、肌酸激酶脑型同工酶(CK-BB)、乳酸脱氢酶(LDH)及天门冬氨酸氨基转移酶(AST)等的含量。结果 缺血期血糖、CK、CK-BB及MDA均增高。再灌注后血糖下降,MDA持续增高,CK、CK-BB较缺血期进一步升高。结论 脊髓缺血再灌注损伤时CK、CK-BB明显增高,实时检测CK-BB的变化可反映脊髓损伤的程度。     

丹参酮IIa拮抗脊髓缺血再灌注损伤：干预谷氨酸释放的上游因素

目的：探讨丹参酮调控谷氨酸转运体功能对脊髓缺血再灌注损伤的作用。方法：88只SD 大鼠结扎腹主动脉,制作脊髓缺血再灌注损伤模型。按随机数字表法将动物分为假手术组（n=8）,模型组（n=40）和丹参酮组（n=40）。分别在脊髓缺血再灌注损伤后0.5h、1h、4h、8h、12h相应时点检测各组谷氨酸转运体功能和Na+-K+−ATP酶活性。结果： ①大鼠脊髓组织谷氨酸转运体功能及Na+-K+−ATP酶活性在脊髓缺血再灌注损伤后0.5h后开始下降,4h后降到最低点,其后活性逐渐恢复,但12h后仍未及正常水平。②丹参酮组各观测点脊髓组织谷氨酸转运体功能及Na+-K+−ATP酶活性均较其它两组高。结论：①大鼠脊髓缺血再灌注损伤时脊髓谷氨酸转运体功能和Na+-K+−ATP酶活性均下降;②丹参酮可能通过保护脊髓谷氨酸转运体功能和Na+-K+−ATP酶活性的下降,从而减轻大鼠脊髓缺血再灌注损伤。     

Blood flow and vascular permeability during motor dysfunction in a rabbit model of spinal cord ischemia.

BACKGROUND AND PURPOSE: Delayed deterioration of neurological function after central nervous system ischemia is a well-documented clinical problem. The purpose of our study was to elucidate the role of spinal cord blood flow and spinal cord-blood barrier integrity in the evolution of delayed neurological deterioration after transient spinal cord ischemia in rabbits. METHODS: Anesthetized rabbits were subjected to lumbar spinal cord ischemia (25 minutes) and variable periods of reperfusion (30 minutes to 48 hours after ischemia). Regional spinal cord blood flow was monitored by carbon-14-labeled iodoantipyrine autoradiography; vascular permeability was assessed by quantitative microhistofluorescence of Evans blue-albumin in frozen sections of spinal cord. Hindlimb motor function was assessed by standard scoring system and tissue edema by wet/dry weight method. RESULTS: Hindlimb motor function indicated complete paralysis during ischemia and partial gradual recovery upon reperfusion (up to 8 hours), followed by progressive deterioration to severe deficits over 48 hours. Severe vascular permeability disruption was noticed early (30 minutes) after reperfusion, but almost complete recovery reestablished at 8 hours was followed by a secondary progressive increase in vascular permeability. Blood flow was reduced by 20-30% (p less than 0.01) 4 hours after ischemia in the gray matter, but hyperemia (200-300%, p less than 0.01) was observed 12-24 hours after ischemia. Spinal cord water content increased by 5.7% (p less than 0.05) 24 hours after ischemia. CONCLUSIONS: This study demonstrates that delayed neurological and motor deterioration after spinal cord ischemia is associated with severe progressive breakdown of spinal cord-blood barrier integrity that develops late (hours) after the injury. Our data suggest that no ischemic insult in early or late reperfusion is associated with delayed motor deterioration.     

Propofol application combined with ischemic preconditioning for aortic occlusion-induced spinal cord injury

BACKGROUND:Propofol combined with ischemic preconditioning(IPC)could prevent spinal ischemia/reperfusion injury,However,the effect of this combination remains poorly understood.OBJECTIVE:To measure neuroprotection of IPC in combination with propofol in a rabbit model of aorta occlusion-induced spinal injury.DESIGN,TIME AND SETTING:A randomized,controlled,animal experiment was performed at the Laboratory of Anesthesiology,Wuhan University and Central Laboratory of the First Clinical Medical College,China Three Gorges University,from October 2006 to April 2008.MATERIALS:Propofol was purchased from AstraZeneca,UK; malondialdehyde(MDA)and superoxide dismutase(SOD)kits were purchased from Nanjing Jiancheng Bioengineering Institute,China.METHODS:A total of 32 male,Japanese White rabbits,were randomly assigned to model,IPC,propofol,and combination groups,with eight rabbits in each group,using 2 × 2 factorial experimental design.Spinal ischemia/reperfusion injury was induced by abdominal aorta occlusion for 40 minutes and reperfusion for 7 days.The IPC group was subjected to IPC treatment for 30 minutes prior to occlusion; the propofol group was treated with propofol for 10minutes before occlusion; and the combination group underwent IPC treatment for 30 minutes and propofol for 10 minutes prior to occlusion.MAIN OUTCOME MEASURES:Serum MDA levels and SOD activity were detected 35 minutes prior to occlusion,immediately after reperfusion,and 60 minutes and 7 days after reperfusion,respectively.Rabbit hind limb nerve function and spinal pathological changes following injury were observed,and spinal neuronal apoptosis was determined using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling method.RESULTS:Serum MDA levels,spinal neuronal apoptosis,and palsy incidence following injury were greatest in the propofol group,followed by the IPC and combination groups(P < 0.01),while SOD activity and hind limb neurological scores were greatest in the combination group,followed by the IPC and propofol groups.Spinal cord injury in the combination group was slight(P < 0.01).CONCLUSION:IPC and propofol treatment resulted in a synergistic effect for treating spinal ischemia/reperfusion injury.Combined application was superior to IPC or propofol treatment,suggesting that the protection of spinal cord injury may relate with anti-peroxidation.     

Hyperlipidemia affects neuronal nitric oxide synthase expression in brains of focal cerebral ischemia rat model★

BACKGROUND: Hyperlipidemia, a risk factor for ischemic cerebrovascular disease, may mediate production of neuronal nitric oxide synthase (nNOS) to induce increased nitric oxide levels, resulting in brain neuronal injury. OBJECTIVE: To investigate effects of hyperlipidemia on brain nNOS expression, and to verify changes in infarct volume and pathology during reperfusion, as well as neuronal injury following ischemia/reperfusion in a rat model of focal cerebral ischemia. DESIGN, TIME AND SETTING: Complete, randomized grouping experiment was performed at the Laboratory of Physiology, Shanxi Medical University from March 2005 to March 2006. MATERIALS: A total of 144 eight-week-old, male, Wistar rats, weighing 160-180 g, were selected. A rat model of middle cerebral artery occlusion was established by suture method after 4 weeks of formulated diet. Nitric oxide kit and rabbit anti-rat nNOS kit were respectively purchased from Nanjing Jiancheng Bioengineering Institute, China and Wuhan Boster Biological Technology, Ltd., China. METHODS: The rats were equally and randomly divided into high-fat diet and a normal diet groups. Rats in the high-fat diet group were fed a high-fat diet, consisting of 10% egg yolk powder, 5% pork fat, and 0.5% pig bile salt combined with standard chow to create hyperlipidemia. Rats in the normal diet group were fed a standard rat chow. A total of 72 rats in both groups were randomly divided into 6 subgroups: sham-operated, 4-hour ischemia, 4-hour ischemia/2-hour reperfusion, 4-hour ischemia/4-hour reperfusion, 4-hour ischemia/6-hour reperfusion, and 4-hour ischemia/12-hour reperfusion, with 12 rats in each subgroup. MAIN OUTCOME MEASURES: nNOS expression was measured by immunohistochemistry, and pathomorphology changes were detected by hematoxylin-eosin staining. Infarct volume and nitric oxide levels were respectively measured using 2, 3, 5-triphenyltetrazolium chloride (TTC) and immunohistochemistry. RESULTS: In the ischemic region, pathology changes were significant in the 4-hour ischemia/4-hour, 4-hour ischemia/6-hour reperfusion, and 4-hour ischemia/12-hour reperfusion subgroups fed on a high-fat diet compared to the same groups fed on a normal diet. In each ischemia subgroup, nNOS expression in brain tissues was higher than in the sham-operated subgroups fed on either the high-fat diet or normal diet (P < 0.01). At each ischemia/reperfusion time point, rats fed on a high-fat diet expressed higher levels of nNOS compared to rats fed on the normal diet (P < 0.05). When tissue was stained with TTC, a white infarction area was detected in the ischemic hemisphere, demonstrating that the infarct volume gradually increased with prolonged reperfusion time in each ischemia subgroup. At each ischemia/reperfusion time point, the infarct volume was larger in rats fed on a high-fat diet compared to those fed on a normal diet. CONCLUSION: nNOS expression was greater in hyperlipidemia rats following ischemia/reperfusion. Cerebral ischemia/reperfusion injury is aggravated with prolonged reperfusion time. Key Words: focal cerebral ischemia; hyperlipidemia; ischemia/reperfusion injury; neuronal nitric oxide synthase     

Protective effects of human umbilical cord mesenchymal stem cell vein transplantation against spinal cord ischemia/reperfusion injury in rats

BACKGROUND: The majority of studies addressing spinal cord ischemia/reperfusion injury (SCIRI) have focused on drugs, proteins, cytokines, and various surgical techniques. A recent study reports that human umbilical cord mesenchymal stem cell (hUCMSC) transplantation achieves good therapeutic effects, but the mechanisms underlying nerve protection remain poorly understood.OBJECTIVE: To observe survival of transplanted hUCMSCs in SCIRI rat models and the influence on motor function in the hind limbs, to determine interleukin-8 expression and cellular apoptosis in spinal cord tissues, and to verify the hypothesis that hUCMSC transplantation exhibits protective effects on SCIRI.DESIGN, TIME AND SETTING: A randomized, controlled, animal experiment was performed at the Laboratory of the Department of Orthopedics in the First Affiliated Hospital of Soochow University,China between January 2007 and December 2008.MATERIALS: hUCMSCs were harvested from umbilical cord blood of healthy pregnant women after parturition in the Obstetrical Department of the First Affiliated Hospital of Soochow University, China. Rabbit anti-human BrdU monoclonal antibody was provided by DAKO, USA. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) Kit and enzyme-linked immunosorbent assay (ELISA) Kit were purchased by Wuhan Boster, China. METHODS: A total of 72 healthy, Wistar, adult rats were randomly assigned to three groups: sham-surgery, model, and transplantation, with 24 rats in each group. SCIRI was induced in the model and transplantation groups via the abdominal aorta block method. The inf rarenal abdominal aorta was not blocked in the sham-surgery group. Prior to abdominal aorta occlusion, 0.2-0.3 mL bromodeoxyuridine (BrdU)-labeled hUCMSCs suspension (cell concentration 5 × 10~3/μL) was injected through the great saphenous vein of the hind limb, and an equal volume of physiological saline was administered to the model and sham-surgery groups.MAIN OUTCOME MEASURES: Pathological observation of rat spinal cord tissues was performed by hematoxylin-eosin staining at 6, 24, and 48 hours post-surgery. Immunohistochemistry was applied to determine hUCMSCs survival in the spinal cord. The amount of cellular apoptosis and interleukin-8 expression in spinal cord tissues was assayed utilizing the TUNEL and ELISA methods, respectively. Motor function in the hind limbs was evaluated according to Jacob's score. RESULTS: Numerous BrdU-positive cells were observed in spinal cord tissues from the transplantation group. The number of apoptotic cells and interleukin-8 levels significantly decreased in the transplantation group (P < 0.05), pathological injury was significantly ameliorated, and motor function scores significantly increased (P < 0.05) compared with the model group. CONCLUSION: Via vein transplantation, hUCMSCs were shown to reach and survive in the injury area. Results suggested that the transplanted hUCMSCs contributed to significantly improved pathological changes in the injured spinal cord, as well as motor function, following SCIRI. The protective mechanism correlated with inhibition of cellular apoptosis and reduced production of inflammatory mediators.     

Bone marrow mesenchymal stem cells repair spinal cord ischemia/reperfusion injury by promoting axonal growth and anti-autophagy

Bone marrow mesenchymal stem cells can differentiate into neurons and astrocytes after trans- plantation in the spinal cord of rats with ischemia/reperfusion injury. Although bone marrow mesenchymal stem cells are known to protect against spinal cord ischemia/reperfusion injury through anti-apoptotic effects, the precise mechanisms remain unclear. In the present study, bone marrow mesenchymal stem cells were cultured and proliferated, then transplanted into rats with ischemia/reperfusion injury via retro-orbital injection. Immunohistochemistry and immunofluorescence with subsequent quantification revealed that the expression of the axonal regeneration marker, growth associated protein-43, and the neuronal marker, microtubule-as- sociated protein 2, significantly increased in rats with bone marrow mesenchymal stem cell transplantation compared with those in rats with spinal cord ischemia/reperfusion injury. Fur- thermore, the expression of the autophagy marker, microtubule-associated protein light chain 3B, and Beclin 1, was significantly reduced in rats with the bone marrow mesenchymal stem cell transplantation compared with those in rats with spinal cord ischemia/reperfusion injury. Western blot analysis showed that the expression of growth associated protein-43 and neuro- filament-H increased but light chain 3B and Beclin 1 decreased in rats with the bone marrow mesenchymal stem cell transplantation. Our results therefore suggest that bone marrow mes- enchymal stem cell transplantation promotes neurite growth and regeneration and prevents autophagy. These responses may likely be mechanisms underlying the protective effect of bone marrow mesenchymal stem cells against spinal cord ischemia/reperfusion injury.