Pharmacological preconditioning ameliorates neurological injury in a model of spinal cord ischemia

BACKGROUND: Pharmacological openers of mitochondrial ATP-sensitive potassium (mitoKATP) channels have been shown to mimic ischemic preconditioning (IPC) in both the brain and myocardium. We hypothesized that similar endogenous mechanisms exist in the spinal cord and that diazoxide, a potent mitoKATP opener, could reduce neurologic injury after aortic cross-clamping in a model of spinal cord ischemia. METHODS: The infra-renal aorta was cross-clamped in 45 male New Zealand white rabbits for 20 minutes. Control animals received no pretreatment. Diazoxide-treated animals were dosed (5 mg/kg) 15 minutes before cross-clamp. A third group underwent 5 minutes of IPC 30 minutes before cross-clamp. Two groups received KATP antagonists, 5-hydroxydecanoic acid (5-HD, 20 mg/kg) or glibenclamide (1.0 mg/kg), before diazoxide administration. Systemic hypotension was induced in a final group with excess isoflurane. Tarlov Scoring was used to assess neurologic function at 24 and 48 hours, after which, the spinal cords were procured for histopathological analysis. RESULTS: Tarlov scoring demonstrated marked improvement in the Diazoxide group compared with control at 24 hours (p < 0.02) and 48 hours (p < 0.009). Moreover, no further neurologic injury occurred in this group at 7 days. IPC-treated animals showed neurologic improvement but were not significantly different from controls. Further, administration of glibenclamide was effective in antagonizing diazoxide's protective effect. CONCLUSIONS: Administration of diazoxide resulted in significant improvement in neurologic outcome in this model. This protective effect improved outcome at both early and late time points. Further, the antagonistic effect of glibenclamide implicates diazoxide's ATP-dependent potassium channel agonism as the mechanism of protection. Overall, this study suggests that diazoxide may be useful in the prevention of neurologic injury after thoracic aneurysm surgery.     

Experimental study of the protection of ischemic preconditioning to spinal cord ischemia.

BACKGROUND: Since the advent of ischemic preconditioning in myocardium, more and more attention has been paid to ischemic preconditioning in the central nervous system (CNS). This study was designed to evaluate the protective effect of ischemic preconditioning on spinal cord ischemia. METHODS: Interventional neuroradiological techniques were used to induce spinal cord ischemia in a rabbit model. Hydrogen electrode technique was used to determine the regional blood flow of the spinal cord. Catecholamines and their metabolites were measured by high performance liquid chromatography (HPLA). Spinal cord evoked potentials were recorded to show spinal cord neurofunction. RESULTS: After 5 minutes ischemic preconditioning with 20 minutes reperfusion, the regional spinal cord blood flow (rSCBF) was increased, as may be seen by the slight increase of catecholamine, especially NE. This is in positive proportion to the cAMP and indicates the enhancement of the metabolic activities of the spinal cord. After 30 minutes of irreversible ischemia, the great increase in catecholamine caused vascular spasm, endotheliocyte fissure, multiple hemorrhagic suffusion, and necrosis, which would injure the spinal cord as a result. The slight increase of the rSCBF and the maintenance of the rSCBF after irreversible ischemia may enhance the protection of ischemic preconditioning to the spinal cord neurofunction, which was proved by spinal cord evoked potentials (SCEPs). CONCLUSIONS: Our study showed that 5 minutes of ischemic preconditioning can increase the rSCBF, enhance the tolerance of the spinal cord to irreversible ischemia, and protect the neurofunction of the spinal cord. The biological mechanism of the protective effect of ischemic preconditioning to spinal cord ischemia should be further studied.     

Ischemic preconditioning reduces neurologic injury in a rat model of spinal cord ischemia.

BACKGROUND: Ischemic preconditioning (IPC) is an endogenous cellular protective mechanism whereby brief, noninjurious periods of ischemia render a tissue more resistant to a subsequent, more prolonged ischemic insult. We hypothesized that IPC of the spinal cord would reduce neurologic injury after experimental aortic occlusion in rats and that this improved neurologic benefit could be induced acutely after a short reperfusion interval separating the IPC and the ischemic insult. METHODS: Forty male Sprague-Dawley rats under general anesthesia were randomly assigned to one of two groups. The IPC group (n = 20) had 3 minutes of aortic occlusion to induce spinal cord ischemia 30 minutes of reperfusion, and 12 minutes of ischemia, whereas the controls (n = 20) had only 12 minutes of ischemia. Neurologic function was evaluated 24 and 48 hours later. Some animals from these groups were perfusion-fixed for hematoxylin and eosin staining of the spinal cord for histologic evaluation. RESULTS: Survival was significantly better at 48 hours in the IPC group. Sensory and motor neurologic function were significantly different between groups at 24 and 48 hours. Histologic evaluation at 48 hours showed severe neurologic damage in rats with poor neurologic test scores. CONCLUSIONS: Ischemic preconditioning reduces neurologic injury and improves survival in a rat model of spinal cord ischemia. The protective benefit of IPC is acutely invoked after a 30-minute reperfusion interval between the preconditioning and the ischemic event.     

肢体缺血预处理对兔颈髓慢性缺血后再灌注损伤的影响

目的：探讨肢体缺血预处理对兔颈髓慢性缺血后再灌注损伤的影响及其机制。方法：建立兔颈髓慢性压迫损伤模型，随机分为实验组和对照组。实验组在脊髓减压再灌注前给予肢体缺血预处理，对照组不做预处理。两组兔在减压前和减压后2h、8h、24h，7d、21d分别进行后肢神经功能评分。在减压后即刻、0．5h、1h和以上时间点分别进行脊髓血流测定。处死动物取脊髓(C1～T1)进行组织病理学检查。结果：实验组于再灌注8h后神经功能评分明显好于对照组。实验组脊髓组织丙二醛含量于再灌注8h后显著低于对照组。两组再灌注后脊髓血流均显著下降，实验组优于对照组。病理学检查示对照组神经元呈缺血改变，白质水肿、脱髓鞘；实验组病理改变轻微。实验组热休克蛋白70表达阳性，对照组为阴性。结论：肢体缺血预处理对脊髓慢性缺血后的再灌注损伤具有明显保护作用。     

缺血预适应对缺血脊髓保护作用的实验研究

目的：探讨缺血预适应对缺血脊髓的保护作用及其可能机制。方法：48只成年大耳白兔随机分为两组，每组24只，建立脊髓缺血模型。缺血预适应组（IPC组）采用腹主动脉阻断5min，开放15min的预适应方案后，再阻断40min后开放。对照组常规阻断腹主动脉40min后开放。分别于阻断前、阻断40min后、开放后2、8、24和72h，测定脑脊液超氧化物歧化酶（SOD）、脊髓脂质过氧化物酶（LPO）及脊髓组织含水量，并行双后肢神经功能评分。结果：IPC组各时相脑脊液SOD活性及神经功能评分显著优于对照组（P＜0．05），脊髓LPO含量及组织含水量明显低于对照组（P＜0．05）。结论：缺血预适应通过调动与增强脊髓组织内源性抗损害机制对缺血脊髓发挥保护作用。     

The effect of thalidomide on spinal cord ischemia/reperfusion injury in a rabbit model

STUDY DESIGN: Randomized study. OBJECTIVES: To evaluate the effects of thalidomide on spinal cord ischemia/reperfusion injury via reduced TNF-alpha production. SETTING: Animal experimental laboratory, Clinical Research Institute of Seoul National University Hospital, Seoul, Korea. METHODS: Spinal cord ischemia was induced in rabbits by occluding the infrarenal aorta. Rabbits in group N did not undergo ischemic insult, but rabbits in groups C (the untreated group), THA, and THB underwent ischemic insult for 15 min. The THA and THB groups received thalidomide (20 mg/kg) intraperitoneally (i.p.) before ischemia, but only the THB group received thalidomide (i.p., 20 mg/kg) after 24 and 48 h of reperfusion. After evaluating neurologic functions at 1.5 h, 3, and 5 days of reperfusion, rabbits were killed for histopathologic examination and Western blot analysis of TNF-alpha. RESULTS: The THA and THB groups showed significantly less neurologic dysfunction than the C group at 1.5 h, 3, and 5 days of reperfusion. The number of normal spinal motor neurons in ventral gray matter was higher in THA and THB than in C, but no difference was observed between THA and THB. Western blot analysis showed a significantly higher level of TNF-alpha in C than in THA and THB at 1.5 h of reperfusion, but no difference was observed between C, THA, or THB at 3 or 5 days of reperfusion. CONCLUSION: Thalidomide treatment before ischemic insult reduces early phase ischemia/reperfusion injury of the spinal cord in rabbits.     

远程缺血预处理对脊髓缺血再灌注损伤保护作用的研究进展

近年来,需要术中阻断胸腹主动脉的外科手术大大增加,由此引起的脊髓缺血性损伤会导致脊髓功能障碍,甚至截瘫[1];脊髓慢性压迫性疾病如颈椎病、椎管狭窄症、后纵韧带骨化症等在行减压手术后可发生脊髓损伤,即脊髓缺血再灌注损伤（spinal cord ischemic reperfusion injury,SCII）。对脊髓损伤目前尚缺乏确切有效的治疗方法,预防脊髓损伤和阻止脊髓继发性损伤是目前研究的重点。远程缺血预处理（remote ischemic preconditioning,RIPC）是指一个器官或组织经短暂缺血再灌注处理后通过释放生化信使到循环中或激活神经通路而对远隔器官产生保护作用。1997年,Matsuyama等[2]首先报道了脊髓RIPC对脊髓缺血再灌注损伤的保护作用,目前已成为脊髓缺血再灌注损伤研究的热点。现就RIPC对脊髓缺血再灌注损伤保护作用的研究进展综述如下。     

缺血预处理对缺血脊髓微循环影响的实验研究

目的 探讨缺血预处理（IPC）对缺血损伤脊髓的保护作用及其相关机制.方法 48只健康新西兰大白兔随机分为缺血预处理组（IPC组）和缺血组,应用腹主动脉夹闭法建立脊髓缺血模型.观察两组脊髓组织缺血前、缺血40min及再灌注后2、8、24、72 h内皮素-1（ET-1）、血栓素A2（TXA2）与前列环素（PGI2）含量,并观察脊髓组织病理形态学改变及双后肢神经功能评分.结果 IPC组各时间点ET-1含量、TXB2/6-keto-PGF1α比值均显著低于缺血组（P＜0.05）.两个时间点上IPC组后肢神经功能评分明显高于缺血组（P＜0.001、0.01）,病理学累积评分明显低于缺血组（P＜0.001）.结论 IPC对主动脉阻断所致脊髓缺血再灌注损伤有良好的保护作用,抗“无再灌流”作用可能是其保护作用的重要机制之一.     

Effect of ischemic pretreatment on heat shock protein 72, neurologic outcome, and histopathologic outcome in a rabbit model of spinal cord ischemia

OBJECTIVE: In the present study, we investigated the effect of ischemic pretreatment on heat shock protein 72 concentration and neurologic and histopathologic outcome after transient spinal cord ischemia. METHODS: In 28 New Zealand White rabbits, an aortic occlusion device was placed infrarenally. The animals were randomly assigned to 2 groups: ischemic pretreatment (n = 14 animals) and control (n = 14 animals). The duration of ischemic pretreatment was 6 minutes. After 24 hours, the aorta was occluded for 26 minutes in both groups of animals. Neurologic function was assessed 24 and 48 hours after the definite ischemic insult. At 48 hours, the animals were killed for histopathologic evaluation of the spinal cord. In a separate set of animals, heat shock protein 72 levels were determined in the lumbar spinal cord after both a 6- and 10-minute ischemic period, with the use of a Western blot analysis. RESULTS: No significant difference in neurologic outcome between the groups was observed at 24 and 48 hours. The incidence of paraplegia and severe paresis at 48 hours was 79% in the control group and 92% in the ischemic pretreatment group. There was no difference in histopathologic scores between the groups. Heat shock protein 72 could be clearly detected 1 and 2 days after 6- or 10-minute periods of spinal cord ischemia. CONCLUSIONS: In the present rabbit study, ischemic pretreatment could not induce tolerance against a moderately severe spinal cord ischemic insult, despite increased heat shock protein 72 levels after the preconditioning stimulus.     

Brain and spinal cord preconditioning for the protection against ischemic injury

Recent studies have suggested that the brain preconditioning could induce tolerance to ischemia in humans. It has been believed that newly synthesized proteins are required for the acquisition of delayed tolerance in the brain and spinal cord. However, the mechanism other than the synthesis of neuroprotective proteins may also play a pivotal role. Preconditioning may reprogram the response to ischemic injury as seen during hibernation. Preconditioning with hyperbaric oxygen, volatile anesthetics, and xenon seems to be the focus of the attention from the standpoint of the clinical setting. Strong neuroprotection by the preconditioning with isoflurane and xenon is reported in animal experiments and may change the traditional idea of neuroprotection by anesthetics. The discovery that erythropoietin exerts neuroprotective properties has opened new therapeutic avenues. Erythropoietin is induced in the brain by hypoxic preconditioning and by the pharmacological preconditioning. In addition, the intravenous administration of erythropoietin has been shown to be safe and beneficial for acute stroke in humans. Therefore, erythropoietin is now one of the most promising neuroprotective agents. The research in the brain and spinal cord preconditioning will contribute to the elucidation of the mechanism of ischemic injury and to the establishment of new therapies for neuroprotection.     

Two episodes of remote ischemia preconditioning improve motor and sensory function of hind limbs after spinal cord ischemic injury

Objectives: To investigate the effect of one and two remote ischemia preconditioning episodes (1-RIPC or 2-RIPC, respectively) on neuro-protection after spinal cord ischemic injury (SCI) in rats.Design: Experimental animal study.Setting: College of Medicine, King Khalid University, Abha, KSA.Interventions: Male rats (n = 10/group) were divided into control, sham, SCIRI, 1-RIPC + SCIRI, and 2-RIPC + SCIRI. SCI was induced by aortic ligation for 45 min and each RIPC episode was induced by 3 cycles of 10 min ischemia/10 min perfusion. The two preconditioning procedures were separated by 24 h.Outcome measures: after 48 h of RIPC procedure, Tarlov’s test, withdrawal from the painful stimulus and placing/stepping reflex (SPR) were used to evaluate the hind limbs neurological function. SC homogenates were used to measure various biochemical parameters.Results: Motor and sensory function of hind limbs were significantly improved and levels of MDA, AOPPs, PGE2, TNF-α, and IL-6, as well as the activity of SOD, was significantly decreased in SC tissue in either 1 or 2 episodes of RIPC intervention. Concomitantly, levels of total nitrate/nitrite and eNOS activity were significantly increased in both groups. Interestingly, except for activity of SOD, eNOS and levels of nitrate/nitrite, the improvements in all neurological biochemical endpoint were more profound in 2-RIPC + SCIRI compared with 1-RIPC + SCIRI.Conclusion: applying two preconditioning episodes of 3 cycles of 10 min ischemia/10 min perfusion, separated by 24 h, boost the neuro-protection effect of RIPC maneuver in rats after ischemic induced SCI in rats.     

Flumazenil mimics whereas midazolam abolishes ischemic preconditioning in a rabbit heart model of ischemia-reperfusion

BACKGROUND: The goal of the current study was to assess the effects of flumazenil, a benzodiazepine receptor antagonist, in limiting infarct size and in reducing hydroxyl free radical production. METHODS: After intravenous salicylate (100 mg/kg) administration, rabbits were subjected to 40 min of regional myocardial ischemia and 2 h of reperfusion. In one group, flumazenil (0.05 mg/kg) and, in another, midazolam (0.05 mg/kg) was administered 15 min before 40 min of ischemia. Ischemic preconditioning (IP) was elicited by 5 min of ischemia followed by 10 min of reperfusion (before the 40-min ischemia period). In two other groups, midazolam was added to flumazenil and IP. Infarct size was determined using triphenyl tetrazolium chloride staining. The authors quantified the hydroxyl-mediated conversion of salicylate to its 2,3- and 2,5-dihydroxybenzoate derivatives during reperfusion by high-performance liquid chromatography coupled with electrochemical detection. Results are expressed as mean +/- SEM. RESULTS: Flumazenil, like IP, significantly decreased infarct size (23 +/- 4 and 22 +/- 5%, respectively, vs. 57 +/- 6% in control group; P < 0.01). Midazolam inhibited the effects of flumazenil and IP. Flumazenil and IP significantly limited the increase in the normalized concentrations of 2,3- and 2,5-dihydroxybenzoic acids. With midazolam, however, the increase was comparable to that of the control group. 5-Hydroxydecanoate, a selective mitochondrial adenosine triphosphate-sensitive K channel blocker, given with flumazenil, abolished the protection obtained with the latter. CONCLUSIONS: Flumazenil mimics preconditioning to decrease infarct size and hydroxyl radical production during reperfusion. Midazolam, however, abolishes these effects. Blockade of benzodiazepine receptors is upstream to the mitochondrial adenosine triphosphate-sensitive K channels in the preconditioning cascade.     

快速交换球囊扩张导管技术制备兔急性脊髓压迫损伤模型

目的 :探讨快速交换球囊扩张导管技术制备兔单纯脊髓压迫损伤模型的可行性。方法 :将24只新西兰大白兔随机分为3组,麻醉成功后备皮,以T10为中心取背部正中切口,长约4cm,显露T8~T11,假手术组(A组)行T8一侧椎板切除,不置入球囊;对照组(B组)将T8一侧椎板切除后置入球囊达T10水平,不扩张球囊;实验组(C组)行T8一侧椎板切除后,将球囊通过T8置入达T10水平,将球囊快速充起,占据椎管前后径约30%,术中采用CT平扫确定球囊位置并记录球囊扩张达到占位程度时的压强值并维持此压强值压迫48h。术前和术后48h行体感诱发电位(SSEP)检查并记录图形和数值变化,采用改良Tarlov评分记录各组术后48h动物后肢活动情况。同时记录各组兔术后1d、2d体重下降值和饮食量。术后2d各组随机选取5只实验兔取出以T10为中心脊髓组织进行病理学检查,每组剩余3只均以T8为中心上下约0.5cm范围进行拆线,B、C组将球囊内压力减为负压后将球囊轻轻抽出,最后缝合切口,继续观察并记录术后4d、7d、14d时体重下降值及饮食量。结果:C组造模球囊扩张达到椎管前后径30%占位程度时球囊内压强为67.23±22.34k Pa,95%置信区间为(48.55~85.91)k Pa。造模术后C组SSEP波幅显著性降低,与A、B组术后及C组术前比较均有统计学差异(P0.05),A组与B组比较差异无统计学意义(P0.05)。造模后48h改良Tarlov评分A组(6.00±0.00分)与B组(5.88±0.35分)比较差异无统计学意义(P0.05),A、B组与C组(1.13±0.35分)比较差异均有统计学意义(P0.05)。术后1d、2d、4d、7d、14d时3组兔体重下降值及饮食量均无统计学差异(P0.05),术后14d时体重恢复至正常体重,术后7d时饮食恢复正常。结论:快速交换球囊扩张导管技术制备兔单纯脊髓压迫损伤模型可模拟临床急性脊髓压迫损伤的状态。     

Heat stress increases the effectiveness of early ischemic preconditioning in spinal cord protection.

OBJECTIVE: This study was aimed to test the hypothesis that the combination of heat stress and early ischemic preconditioning (IP) applied before aortic occlusion would be protective against spinal cord ischemic injury. METHODS: Thirty Whister-Albino rats were randomly divided into three groups. In group 1 (n=10), aorta was clamped just distal to the left renal artery and above the iliac bifurcation for 45 min. Group 2 (n=10) had 5 min of transient aortic occlusion and 30 min later underwent an additional 45 min. In group 3 (n=10), animals were heated to 41 degrees C and maintained at this temperature for 15 min. Twenty-four hours later, this hyperthermia pretreated group underwent the same early IP model of aortic occlusion. Neurologic status was assessed on postoperative 24 and 48 h by using the 15-point neurologic performance scale. Spinal cords were harvested for histopathological grading (1-4) and evaluated for the presence of heat shock protein-ubiquitin staining. RESULTS: At 24 and 48 h, the mean neurologic performance scores of the group 1 were found to be significantly lower than those of groups 2 and 3. Although the neurologic assessment of rats performed on the 24h did not reveal statistically significant difference between groups 2 and 3 (P=0.069); on 48 h, the mean neurologic scores of the group 3 were significantly higher than those of group 2 (P=0.005). At 48 h, a delayed neurologic deterioration was seen in groups 1 and 2 when compared to the results obtained at 24h. Histologic evaluation correlated well with the neurologic outcome with the least cellular damage in group 3. There were six rats with ubiquitin expression and this was detected only in animals pretreated with sublethal heat stress. CONCLUSIONS: An early IP model with a short reperfusion interval does not give the minimal required time for the HSPs expression and is associated with a delayed neurologic deterioration. Neuroprotection provided by heat stress combined with an early IP model lasts up to 48 h and heat shock protein-ubiquitin induction may be responsible in this phenomenon.