MCI-186 reduces oxidative cellular damage and increases DNA repair function in the rabbit spinal cord after transient ischemia

Background

Paraplegia is a serious complication of operations on the thoracic and thoracoabdominal aorta. To investigate the mechanism by which motor neurons are damaged during these operations, we have reported a rabbit model of spinal cord ischemia. We also tested whether a free radical scavenger MCI-186 that is useful for treating ischemic damage in the brain can protect against ischemic spinal cord damage.

Methods

Fifteen minutes of ischemia was induced, then MCI-186 or vehicle was injected intravenously. Cell damage was analyzed by observing the function of the lower limbs and by counting the number of motor neurons. To investigate the mechanism by which MCI-186 prevents ischemic spinal cord damage, we observed the immunoreactivity of 8-hydroxy-2'-deoxyguanosine as an oxidative DNA damage marker and redox effector as a DNA repair marker.

Results

In sham control, 8-hydroxy-2'-deoxyguanosine was not observed, and the nuclear expression of redox effector was observed. In vehicle injection group (group I), the nuclear expression of 8-hydroxy-2'-deoxyguanosine was observed at 1 and 2 days after reperfusion. The nuclear expression of redox effector was observed at 8 hours and 1 day, and disappeared at 2 days after transient ischemia. In MCI-186 injection group (group M), the nuclear expression of 8-hydroxy-2'-deoxyguanosine was not observed, and redox effector was observed at 8 hours and 1 and 2 days.

Conclusions

These results suggest that redox effector decreased in motor neurons after transient ischemia and this reduction preceded oxidative DNA damage. MCI-186 works as a radical scavenger and reduced oxidative DNA damage, so redox effector did not disappear. MCI-186 could be a strong candidate for a use as a therapeutic agent in the treatment of ischemic spinal cord injury.     

亚低温对脊髓损伤后神经细胞凋亡的影响

目的观察大鼠脊髓损伤(SCI)细胞凋亡现象及亚低温对细胞凋亡的影响。方法大鼠SCI后分别于8h、24h、7d取材，采用常规病理HE染色和末端脱氧核苷酸转移酶(TdT)介导的dutp缺口末端标记技术(TNEUL)，研究亚低温对大鼠SCI后神经细胞凋亡的影响。结果SCI后常温组8h灰质区出现较多凋亡细胞，24h时白质和灰质内均有凋亡细胞分布，7d后凋亡细胞多见于白质；亚低温组凋亡细胞明显减少(P＜0．05)。结论亚低温明显减少SCI后细胞凋亡的发生，从病理上为亚低温脊髓保护提供了可靠的依据。     

Effects of adenosine monophosphate-activated kinase in the ventral horn of rabbit spinal cord after transient ischemia

Abstract

Objective

To investigate the effect compound C, an adenosine monophosphate-activated kinase (AMPK) inhibitor, has on motor neurons of rabbit spinal cord after ischemia/reperfusion.

Design

Compound C (30 mg/kg) was administered intraperitoneally to rabbits 30 minutes before ischemia and the animals were sacrificed at 15 minutes after ischemia/reperfusion to measure lactate levels and at 72 hours after ischemia/reperfusion for morphological study.

Results

The administration of compound C did not produce any significant changes in physiological parameters such as pH, arterial blood gas (PaCO₂ and PaO₂), and blood glucose in rabbit either at 10 minutes before ischemia or at 10 minutes after reperfusion. However, the administration of compound C did significantly ameliorate lactate acidosis at 15 minutes after reperfusion. In addition, the administration of compound C significantly improved the neurological scores of the rabbits and reduced the neuronal death seen in the ventral horn of their spinal cords at 72 hours after ischemia/reperfusion.

Conclusions

Inhibition of AMPK can ameliorate the ischemia-induced neuronal death in the spinal cord via the reduction of early lactate acidosis.     

Effects of adenosine monophosphate-activated kinase in the ventral horn of rabbit spinal cord after transient ischemia

Objective

To investigate the effect compound C, an adenosine monophosphate-activated kinase (AMPK) inhibitor, has on motor neurons of rabbit spinal cord after ischemia/reperfusion.

Design

Compound C (30 mg/kg) was administered intraperitoneally to rabbits 30 minutes before ischemia and the animals were sacrificed at 15 minutes after ischemia/reperfusion to measure lactate levels and at 72 hours after ischemia/reperfusion for morphological study.

Results

The administration of compound C did not produce any significant changes in physiological parameters such as pH, arterial blood gas (PaCO₂ and PaO₂), and blood glucose in rabbit either at 10 minutes before ischemia or at 10 minutes after reperfusion. However, the administration of compound C did significantly ameliorate lactate acidosis at 15 minutes after reperfusion. In addition, the administration of compound C significantly improved the neurological scores of the rabbits and reduced the neuronal death seen in the ventral horn of their spinal cords at 72 hours after ischemia/reperfusion.

Conclusions

Inhibition of AMPK can ameliorate the ischemia-induced neuronal death in the spinal cord via the reduction of early lactate acidosis.     

Role of free radical scavenger in protection of spinal cord during ischemia

Previous work in our laboratory established an experimental model for the production of paraplegia in the anesthetized normothermic adult mongrel dog. The current study involves 24 animals divided into two equal groups: Group 1 served as control, and Group 2 received treatment with scavenger agent. Vascular occlusive clamps were placed on the thoracic aorta proximal to the left subclavian artery, on the left subclavian artery at its origin, and on the distal thoracic aorta at the diaphragm for 30 minutes. In Group 1,200 ml of normal saline solution (37 degrees C) was perfused into the occluded aortic segment at the rate of 0.33 ml per kilogram of body weight per minute. In Group 2, 90% dimethyl sulfoxide (DMSO) in a dose of 0.1 gm/kg in normal saline solution (37 degrees C) for a total volume of 200 ml, was likewise injected into the occluded aortic segment at the same infusion rate. Animals were observed for evidence of paresis in the postoperative period. Microscopic analysis revealed evidence of ischemic myelopathy in the control group but none in the treated group. Under the conditions of this experiment, we conclude that the scavenger agent DMSO has a highly protective effect on the spinal cord during ischemic insult.     

胸椎管狭窄症术后脊髓缺血再灌注损伤的临床分析

目的 探讨胸椎管狭窄症患者手术后出现脊髓功能受损的原因,总结脊髓手术后缺血再灌注损伤(spinal cord ischemic reperfusion injury,SCII)[1]的预处理和早期治疗方法.方法 回顾性分析我科2年内收治的32例胸椎管狭窄症患者术后脊髓恢复情况,出现脊髓损伤患者的临床资料及处理方法,并对其预后进行客观评估.结果 2年内在收治并手术的胸椎管狭窄症患者中,手术减压前给予1克甲基强的松龙预防.5例患者于手术后出现不同程度的脊髓功能受损,即刻给予大剂量甲基强的松龙冲击治疗、脱水药及神经营养药,1例患者症状改善不理想,2例患者症状部分改善,生活可自理,2例患者基本恢复正常.结论 胸椎管狭窄症患者手术后出现的脊髓功能受损可能是脊髓缺血再灌注损伤引起,再灌注损伤在胸椎管狭窄症患者中较多见,但出现严重监床症状的少见,诊断有一定的困难.妥善的处理可望改善患者的生存质量.     

MCI-186 (edaravone), a free radical scavenger, attenuates hepatic warm ischemia–reperfusion injury in rats

Hepatic warm ischemia-reperfusion injury (IRI) during hepatectomy and liver transplantation is a major cause of liver dysfunction in which the pathologic role of free radicals is a major concern. To assess the effect of MCI-186 (edaravone) on hepatic IRI, male Wistar rats were subjected to partial hepatic ischemia for 60 min after pretreatment with vehicle (group C) or MCI-186 (group M), or after both MCI-186 pretreatment and additional administration of MCI-186 12 h after reperfusion (group MX). Groups M and MX showed significantly lower levels of serum alanine aminotransferase and hepatic lipid peroxidation than group C, and also significantly lower expression levels of mRNA for cytokines, chemokines and intercellular adhesion molecule-1. There were fewer tissue monocytes and neutrophils in groups M and MX than in group C. These effects were more marked in group MX than in group M. Our findings suggest that treatment with MCI-186 attenuates hepatic IRI in this rat in vivo model.     

Changes of blood flow,oxygen tension,action potential and vascular permeability induced by arterial ischemia or venous congestion on the spinal cord in canine model

It is generally considered that the genesis of myelopathy associated with the degenerative conditions of the spine may result from both mechanical compression and circulatory disturbance. Many references about spinal cord tissue ischemic damage can be found in the literature, but not detailed studies about spinal cord microvasculature damage related to congestion or blood permeability. This study investigates the effect of ischemia and congestion on the spinal cord using an in vivo model. The aorta was clamped as an ischemia model of the spinal cord and the inferior vena cava was clamped as a congestion model at the 6th costal level for 30 min using forceps transpleurally. Measurements of blood flow, partial oxygen pressure, and conduction velocity in the spinal cord were repeated over a period of 1 h after release of clamping. Finally, we examined the status of blood‐spinal cord barrier under fluorescence and transmission electron microscope. Immediately after clamping of the inferior vena cava, the central venous pressure increased by about four times. Blood flow, oxygen tension and action potential were more severely affected by the aorta clamping; but this ischemic model did not show any changes of blood permeability in the spinal cord. The intramedullar edema was more easily produced by venous congestion than by arterial ischemia. In conclusions, venous congestion may be a preceding and essential factor of circulatory disturbance in the compressed spinal cord inducing myelopathy. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:139–146, 2012     

Effects of intrathecal bupivacaine in conjunction with hypothermia on neuronal protection against transient spinal cord ischemia in rats

BACKGROUND: Excitotoxic neuronal injury from ischemia may be reduced by local anesthetics. We investigated the neuroprotective effects of intrathecally administered bupivacaine and hypothermia in a rat model of transient spinal cord ischemia. METHODS: PE-10 intrathecal catheter-implanted male Sprague-Dawley rats were randomly assigned to one of four groups: normothermia (NT) and hypothermia (HT) groups (given 15 microl of normal saline) and bupivacaine (B) and bupivacaine-hypothermia (BHT) groups (given 15 mul of 0.5% bupivacaine). Transient spinal cord ischemia was induced by inflation of a 2F Fogarty catheter placed in the aortic arch for 12 min. The rectal temperature was maintained at 37.0 +/- 0.5 degrees C for the NT and B groups, and at 34.5 +/- 0.5 degrees C for the HT and BHT groups. Motor and sensory deficit scores were assessed 2 and 24 h after reperfusion. Lumbar spinal cords were harvested for histopathology and immunoreactivity of heat shock protein 70 (HSP70). RESULTS: After reperfusion, the motor and sensory deficit scores of the NT group were significantly higher than those of the HT (P < 0.05) and BHT (P < 0.001) groups. Significant differences were evident in the motor and sensory deficit scores between the HT and BHT groups at 24 h (P < 0.05). Neuronal cell death and immunoreactivity of HSP70 were frequently observed in the NT and BT groups, but not in the HT and BHT groups. CONCLUSIONS: These results collectively suggest that intrathecal bupivacaine does not provide neuroprotection during normothermic transient spinal cord ischemia in rats, but enhances the neuroprotective effects of hypothermia.     

胚胎脊髓不同移植方法对损伤后大鼠脊髓轴索病理的影响

目的研究胚胎脊髓不同移植方法对损伤后大鼠脊髓轴索病理的影响。方法将成年大鼠40只均分为四组,A组：单纯脊髓挫伤和半切洞组;B组：脊髓挫伤和半切洞＋胚胎脊髓移植组;C组：脊髓挫伤和半切洞＋胚胎脊髓移植＋损伤区上下神经根吻合组;D组：脊髓挫伤和半切洞＋胚胎脊髓移植＋带蒂大网膜移植组。移植后6周,应用行为学检查,观察大鼠功能恢复情况,用光、电镜检查计算轴索和观察轴索病理变化,采用计算机图像分析技术,进行定量分析。结果脊髓损伤后6周A组轴索丢失最严重,各组轴索都有不同程度的减少,其减少程度顺序是A组〉B组〉C组〉D组。D组与其它三组比较有显著差异性。残留的轴索数目与神经功能的改善相平行。结论大鼠胚胎脊髓和大网膜移植后对损伤的大鼠脊髓轴索有保护作用并能促进大鼠后肢神经功能的恢复。     

Beneficial effect of the oxygen free radical scavenger amifostine (WR-2721) on spinal cord ischemia/reperfusion injury in rabbits

Background  

Paraplegia is the most devastating complication of thoracic or thoraco-abdominal aortic surgery. During these operations, an ischemia-reperfusion process is inevitable and the produced radical oxygen species cause severe oxidative stress for the spinal cord. In this study we examined the influence of Amifostine, a triphosphate free oxygen scavenger, on oxidative stress of spinal cord ischemia-reperfusion in rabbits.     

Effect of puerarin on neural function and histopathological damages after transient spinal cord ischemia in rabbits

Ischemicspinalcordinjuryremainsadisastrouscomplicationofthedescendingandthoracoabdominalaortaeafteroperation .Thereportedincidenceofparaplegiarangesfrom 4 %to 33% .1Therefore ,agreatnumberofeffortshavebeenfocusedonsolvingthisproblem ,whichincludehypothermia ,cerebrospinalfluiddrainage ,temporarybypassandpharmacologicalagents .2 Thecellularandmolecularmechanismsofischemicspinalcordinjuryhavenotbeentotallyelucidated .Butvariousstudieshavesuggestedthatfreeradicalproduction ,calciumaccumulationand…     

MCI-186 prevents spinal cord damage and affects enzyme levels of nitric oxide synthase and Cu/Zn superoxide dismutase after transient ischemia in rabbits

OBJECTIVE: The mechanism of spinal cord injury is believed to be related to the vulnerability of spinal motor neuron cells against ischemia. We tested whether MCI-186, which is useful for treating ischemic damage in the brain, can protect against ischemic spinal cord damage. METHODS: After induction of ischemia, MCI-186 or vehicle was injected intravenously. Cell damage was analyzed by observing the function of the lower limbs and by counting the number of motor neurons. To investigate the mechanism by which MCI-186 prevents ischemic spinal cord damage, we observed the immunoreactivity of Cu/Zn superoxide dismutase, neuronal nitric oxide synthase, and endothelial nitric oxide synthase. RESULTS: MCI-186 eased the functional deficits and increased the number of motor neurons after ischemia. The induction of neuronal nitric oxide synthase was significantly reduced by the treatment with MCI-186. Furthermore, the increase in the induction of endothelial nitric oxide synthase and Cu/Zn superoxide dismutase was more pronounced. CONCLUSION: These results indicate that MCI-186 may protect motor neurons from ischemic injury by reducing neuronal nitric oxide synthase and increasing endothelial nitric oxide synthase. MCI-186 may be a strong candidate for use as a therapeutic agent in the treatment of ischemic spinal cord injury.     

Aprotinin reduces injury of the spinal cord in transient ischemia

Objective: The protective effect of aprotinin, which is a protease inhibitor, was assessed in a rabbit spinal cord ischemia model. Design: Randomized, controlled, prospective study. Setting: University research laboratory. Subjects: New Zealand white rabbits (36) of both sexes. Methods: In 24 animals, ischemia was induced with midline laparotomy and clamping the aorta just distal to left renal artery and proximal to aortic bifurcation for 20 min. Aprotinin was given 30 000 KIU as a short intravenous injection after anesthesia, and was followed by 10 000 KIU/h by continuous infusion in group 1 (n=12). Similar volume of saline solution was used in control group of animals (group 2, n=12). Group 3 of animals (sham group, n=12) were anesthetized and subjected to laparotomy without aortic occlusion. Physiological parameters and somatosensory evoked-potentials (SEP) were monitored in animals before ischemia, during ischemia and in the first 60 min of reperfusion. Their neurological outcome was clinically evaluated up to 48 h postischemia. Their motor function was scored, and the intergroup differences were compared. The animals were sacrificed after two days of postischemia. Their spinal cord, abdominal aorta, and its branches were processed for histopathological examination. Results: In group 3, SEP amplitudes did not change during the procedures, and all animals recovered without neurologic deficits. At the end of ischemic period, the average amplitude was reduced to 53±7% of the baseline in all ischemic animals. This was followed by a gradual return to 89±8 and 81±13% of the initial amplitude after 60 min of reperfusion in group 1 and group 2 correspondingly (P>0.05). The average motor function score was significantly higher in group 1 than group 2 at 24 and 48 h after the ischemic insult (P<0.05). Histological observations were clearly correlated with the neurological findings. Conclusion: The results suggest that aprotinin reduces spinal cord injury and preserves neurologic function in transient spinal cord ischemia in rabbits.     

大鼠钳夹式急性脊髓损伤模型的制备与评价

[目的]建立并评估大鼠钳夹式脊髓损伤模型,为研究急性脊髓损伤的机制及后续治疗提供基础。[方法]40只成年S-D大鼠,随机分为脊髓损伤组和假手术组,脊髓损伤组行T10水平的椎板切除术,并用一动脉瘤夹瞬间释放,造成脊髓的急性挫伤,假手术组大鼠则仅行T10水平的椎板切除,术后进行行为学评价,血清学检测及病理学检查。[结果]脊髓损伤组大鼠术后运动功能评分(BBB评分)低于假手术组大鼠,术后4 h血清TNF-α和IL-6的含量高于假手术组,且两组差异具有统计学意义,病理学检查脊髓损伤组有脊髓实质结构破坏、空洞及瘢痕形成,假手术组未见明显异常。[结论]本研究建立的大鼠急性脊髓损伤模型,能够反映脊髓损伤的病理生理过程,并具有较好的重复性及稳定性,可用于脊髓损伤的研究。     

多节段脊髓平面损伤后大鼠神经源性膀胱模型的制备

目的研制一种具有临床相似性、可调控性、可重复性的脊髓损伤后神经源性膀胱尿道功能障碍动物模型的制模方法。方法采用改良Allen法,将10g重的柯氏针从20cm高以自由落体落下,撞击在动物脊柱背侧的垫片上,造成一定程度的脊髓损伤。结果在脊髓休克期,骶上脊髓损伤(胸腰段)组和骶髓损伤组动物平均每次挤压膀胱排尿量差异无统计学意义(P>0.05),在脊髓休克期后,两组之间差异有统计学意义(P<0.001)。结论改良Allen法具有临床相似性等优点,能为脊髓损伤后神经源性膀胱尿道功能障碍的研究提供理想的动物模型。     

Experimental bypass surgery between the spinal cord and caudal nerve roots for spinal cord injuries

Spinal cord injuries often cause permanent neurological deficits and are still considered as inaccessible to efficient therapy. Injured spinal cord axons are unable to spontaneously regenerate in adult mammalians. Re-establishing functional activity especially in the lower limbs by reinnervating the caudal infra-lesional territories could represent an attractive therapeutic strategy. For several years, we have studied and developed surgical bypasses using peripheral nerve grafts bridging the supra-lesional rostral spinal cord to the caudal infra-lesional lumbar roots. Main objectives were: 1- to overcome the spinal cord lesion and the consecutive glial barrier blocking the axonal regeneration; 2- to find and bring an alternative source of regenerating axons; 3- to guide those axons toward precisely definite targets (for example, lower limb muscles). We report here the results of our experimental research, which led us from animal experimental models (rodents, primates) to the first human experimentation. Limitations of the method (especially technical pitfalls) are numerous. However, we have obtained encouraging results in our attempts to "repair" the motor pathway. Functional recovery with strong evidence of centrifugal axonal regeneration from the spinal cord to the periphery has been observed. Regarding the sensory pathway, we have found evidence of centripetal axonal regeneration from the periphery toward the spinal cord. Further studies are obviously advocated, but our experimental model of spinal cord - nerve roots bypasses may be integrated in future "repair" strategies of both motor and sensory pathways following spinal cord injury.     

The prevention of iatrogenic spinal cord injury utilizing the evoked spinal cord potential

Summary The evoked spinal cord potential elicited by direct stimulation of the cord has been used clinically to monitor cord function in the course of operations on the spine. The technique used allows measurement of a relatively large amplitude of potential, which is fairly stable against anaesthetics and related drugs, by means of a simple recording system and is sensitive enough to indicate cord damage. Continuous monitoring can easily be carried out. We have encountered no complications when using this method on 99 patients.

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Résumé Le potentiel évoqué provoqué par la stimulation directe de la moelle épinière a été utilisé en clinique pour contrôler la fonction de la moelle lors des interventions sur le rachis. Cette technique permet de mesurer une assez grande amplitude de potentiel, qui est relativement stable à l'égard des anesthésiques et d'autres drogues de même type, grâce à un système simple d'enregistrement; il est suffisamment sensible pour détecter des altérations de la moelle. Une surveillance continue peut aisément être effectuée. Aucun incident n'a été rencontré chez 99 malades lors de l'utilisation de cette méthode.