AR-A014418 as a glycogen synthase kinase-3 inhibitor: anti-apoptotic and therapeutic potential in experimental spinal cord injury.

OBJECTIVES: We aimed to investigate the effects of AR-A014418, a strong inhibitor specific to GSK-3beta, on neuronal apoptosis and neuroprotection in the traumatic SCI model. MATERIALS AND METHODS: In this study, three groups were generated from 36 Wistar rats; (1) control, (2) spinal cord trauma group created by clip compression technique after laminectomy, and (3) AR-A014418 (4mg/kg, i.p., DMSO) treatment group after laminectomy and spinal cord trauma. The TUNEL assay for apoptosis detection, immunohistochemical staining for bax and TGF-beta were applied in spinal cord tissues. For light microscopic examination, necrotic, and apoptotic cells were counted, and PMNL counting was applied to detect inflammation. Functional recovery was tested by field locomotor test in the 3rd and 7th days following surgery. RESULTS: In the trauma group, diffuse hemorrhage, cavitation, necrosis and edematous regions, degeneration in motor neurons and leukocyte infiltration were observed in gray matter. In the AR-A014418-treated groups, healthy cells were observed in more places compared to the trauma groups, however, cavitation, hemorrhagic, and edematous areas were seen in gray matter. In the AR-A014418-treatment groups, the number of apoptotic cells in the 3rd and 7th days (respectively; p<0.05, p<0.01), were significantly decreased compared to the trauma groups, as were the levels of bax (p<0.01) and TGF-beta 1 immunoreactivity. Results of the locomotor test were significantly increased in the treatment group (p<0.001) as compared to the trauma group. CONCLUSIONS: In this experimental spinal cord trauma model study neural apoptosis was significantly triggered in secondary damage developed after trauma, however, neurological healing was expedited by preventing mitochondrial apoptosis and reducing the inflammation by the potent inhibitor AR-A014418, which is GSK-3beta selective.

Extract EGb 761 pretreatment limits ubiquitin positive aggregates in rabbit spinal cord neurons after ischemia-reperfusion.

1. Ubiquitin immunohistochemistry was used for investigation of time dependent changes of ubiquitin in the nerve cells reacting to ischemic/reperfusion damage. In the rabbit spinal cord ischemia model a period of 30 min ischemia followed by 24 and 72 h of reperfusion caused neuronal degeneration selectively in the ventral horn motor neurons as well as interneurons of the intermediate zone. 2. Ubiquitin aggregates were accumulated in the neurons of lamina IX and the neurons of intermediate zone destined to die 72 h after 30 min of the spinal cord ischemia. 3. The activation of ubiquitin hydrolytic system is related to a defective homeostasis and could trigger different degenerative processes. Having in mind this, we used EGb 761 to rescue the motor neurons and interneurons against ischemia/reperfusion damage. Our results show that after 30 min of ischemia and 24 or 72 h of reperfusion with EGb 761 pre-treatment for 7 days the vulnerable neurons in the intermediate zone and lamina IX exhibit marked elevation of ubiquitin-positive granules in the cytoplasm, dendrites and nuclei. Abnormal protein aggregates have not been observed in these cells. 4. The rabbits were completely paraplegic after 30 min of ischemia and 24 or 72 h of reperfusion. However, after 7 days EGb 761 pre-treatment, 30 min of ischemia and 24 or 72 h of reperfusion the animals did not show paraplegia. 5. Evaluated ubiquitin-positive neurons of the L(5)-L(6) segments showed significant decrease in number and significant increase of density after 30 min of ischemia followed by 24 h and mainly 72 h of reperfusion. Ubiquitin immunohistochemistry confirmed the protective effect of EGb 761 against ischemia/reperfusion damage in the rabbit spinal cord.

Preoperative stress conditioning prevents paralysis after experimental aortic surgery: increased heat shock protein content is associated with ischemic tolerance of the spinal cord.

BACKGROUND: All forms of surgical therapy are stressful and injurious. The problems of paralysis, renal dysfunction, and colonic ischemia associated with aortic occlusion are due to acute ischemia-reperfusion injury at the cellular level. Acute-anterior spinal cord ischemia is the most devastating outcome of these iatrogenic-ischemic events. The majority of surgical procedures are performed electively and therefore provide an opportunity to preoperatively condition the patient to minimize these ischemia-related morbidities. OBJECTIVES: We sought to determine whether acute spinal cord injury associated with aortic occlusion can be prevented by induction of the cellular stress response by means of preoperative administration of whole-body hyperthermia or stannous chloride. METHODS: The study consisted of an experimental rabbit model of infrarenal aortic occlusion for 20 minutes at normothermic body temperature. RESULTS: Control rabbits experienced an 88% (7/8) incidence of paralysis after spinal cord ischemia induced by 20 minutes of aortic occlusion, whereas animals treated preoperatively with either whole-body hyperthermia (0/9) or stannous chloride (0/4) never became paralyzed (P <.001 for control vs treated groups). Ischemic protection of the spinal cord was associated with increased content of stress proteins within tissues of pretreated animals. CONCLUSION: Prior induction of the heat shock response in the whole animal will increase the content of stress proteins within the spinal cord and other tissues and result in the prevention of hind-limb paralysis associated with aortic occlusion. We have designated the preoperative induction of the cellular stress response for the prevention of ischemic tissue injury stress conditioning. We suggest that stress-conditioning protocols represent the opportunity to practice preventative medicine at the molecular level.

Time course studies on the effectiveness of tetrodotoxin in reducing consequences of spinal cord contusion.

Focal injection of the sodium channel blocker tetrodotoxin (TTX) into the injury site at either 5 or 15 min after a standardized thoracic contusion spinal cord injury (SCI) reduces white matter pathology and loss of axons in the first 24 hr after injury. Focal injection of TTX at 15 min after SCI also reduces chronic white matter loss and hindlimb functional deficits. We have now tested the hypothesis that the reduction in chronic deficits with TTX treatment is associated with long-term preservation of axons after SCI and compared both acute (24 hr) and chronic (6 weeks) effects of TTX administered at 15 min prior to and 5 min or 4 hr after SCI. Our results indicate a significant reduction of acute white matter pathology in rats treated with TTX at 15 min before and 5 min after injury but no effect when treatment was delayed until 4 hr after contusion. Compared with injury controls, groups treated with TTX at 5 min and 4 hr after injury did not show a significant deficit reduction, nor was there a significant sparing of white matter at 6 weeks compared with injury controls. In contrast, the group treated with TTX at 15 min before SCI demonstrated significantly reduced hindlimb functional deficits beginning at 1 week after injury and throughout the 6 weeks of the study. This was associated with a significantly higher axon density in the ventromedial white matter at 6 weeks. The results demonstrate that blockade of sodium channels preserves axons from loss after SCI and points to the importance of time of administration of such drugs for therapeutic effectiveness.

Third place winner of the Conrad Jobst Award in the gold medal paper competition. Prevention of spinal cord dysfunction in a new model of spinal cord ischemia.

Paraplegia or paraparesis caused by temporary cross-clamping of the aorta is a devastating sequela in patients after surgery of the thoracoabdominal aorta. No effective clinical method is available to protect the spinal cord from ischemic reperfusion injury. A small animal (rat) model of spinal cord ischemia is established to better understand the pathophysiological events and to evaluate potential treatments. Eighty-one male Sprague-Dawley rats weighing 300 g to 350 g were used for model development (45) and treatment evaluation (36). The heparinized and anesthetized rat was supported by a respirator following tracheostomy. The thoracic aorta was cannulated via the left carotid artery for post-clamping intra-aortic treatment solution administration. After thoracotomy, the aorta was freed and temporarily clamped just distal to the left subclavian artery and just proximal to the diaphragm for different time intervals: 0, 5, 10, 15, 20, 25, 30, 35, and 40 minutes (five animals per group). The motor function of the lower extremities postoperatively showed consistent impairment after 30 minutes clamping (5/5 rats were paralyzed), and this time interval was used for treatment evaluation. For each treatment, six animals per group were used, and direct local intra-aortic infusion of physiologic solution (2 mL) at different temperatures with or without buffer substances was given immediately after double cross-clamp to protect the ischemic spinal cord. Arterial blood (2 mL) was infused in the control group. The data indicate that the addition of HCO3-(20 mM) to the hypothermic (15 degrees C) solution offered complete protection of the spinal cord from ischemic injury.(ABSTRACT TRUNCATED AT 250 WORDS)

Graft replacement of the descending thoracic aorta: results of "open" distal anastomosis.

With the advent of rapid autotransfusion, we began to repair aneurysms of the descending thoracic and thoracoabdominal aorta by using an "open" technique, in which a single cross-clamp is placed proximal to the aneurysm to exsanguinate the lower body. To determine whether open distal anastomosis effectively protects against spinal cord injury, we studied 71 consecutive patients (50 men, 21 women) who underwent this procedure beginning in April 1989. The patients ranged in age from 31 to 83 years (mean, 63.3 years). Most patients were hypertensive (n = 61; 86%) and symptomatic (n = 54; 76%). Most had been diagnosed with medial degeneration (n = 45; 63.4%) or aortic dissection (n = 16; 22.5%). Five patients (7.0%) were admitted with aortic rupture. We replaced the entire descending thoracic aorta in 31 (43.7%), the thoracoabdominal aorta in 21 (29.6%), and a segment of the descending thoracic aorta in 19 (26.7%). The average distal ischemic time was 22.4 minutes (range, 11 to 42 minutes). The amount of blood returned through the autotransfusion device averaged 2,099 mL. Eight patients (11.3%) died within 30 days (multiple organ failure, 3; hemorrhage, 2; coexisting ischemic heart disease, 3). Spinal cord dysfunction occurred in 6 patients (8.5%) (lower extremity paraparesis, 4; paraplegia, 2). Renal insufficiency requiring dialysis occurred in 4 patients (5.6%). We believe that the low incidence of spinal cord injury and renal insufficiency in this series may have resulted from the free draining of the intercostal and lumbar arteries during aortic occlusion, which decreases cerebrospinal fluid and central venous pressures.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of sodium nitroprusside on spinal cord perfusion and paraplegia during aortic cross-clamping.

To evaluate the effects of sodium nitroprusside (SNP) on hemodynamics, cerebrospinal fluid dynamics, and neurological outcome after 30 minutes of thoracic aortic occlusion, we monitored proximal and distal blood pressure, cerebrospinal fluid pressure, spinal cord blood flow, and somatosensory evoked potentials. In group 1 (n = 6), no attempts were made to control proximal hypertension, whereas in group 2 (n = 6), proximal blood pressure was controlled with intravenous infusion of SNP. There was no significant difference in proximal or distal blood pressure or cerebrospinal fluid pressure between the two groups at baseline. During the crossclamp interval, the mean proximal aortic pressure rose from 108 +/- 21 to 146 +/- 14 mm Hg (p less than 0.001) in the control group, whereas the mean blood pressure in the SNP group was maintained at 99.8 +/- 12 mm Hg (p = not significant compared with baseline blood pressure). Mean distal aortic pressure decreased from systemic values to 23 +/- 7 mm Hg in control animals and to 11 +/- 5 mm Hg in the SNP group (p less than 0.005). In the latter group, cerebrospinal fluid pressure increased significantly from 10.6 +/- 1.9 to 20.1 +/- 5.5 mm Hg (p less than 0.005). In animals receiving SNP, spinal cord blood flow was decreased in the lower spinal cord segments and increased in the upper cord segments. When compared with controls, this difference did not reach significance.(ABSTRACT TRUNCATED AT 250 WORDS)