Tempol, a superoxide dismutase mimetic, inhibits Wallerian degeneration following spinal cord injury by preventing glutathione depletion and aldose reductase activation.

Spinal cord contusion injury results in Wallerian degeneration of spinal cord axonal tracts, which are necessary for locomotor function. Axonal swelling and loss of axonal density at the contusion site, characteristic of Wallerian degeneration, commence within hours of injury. Tempol, a superoxide dismutase mimetic, was previously shown to reduce the loss of spinal cord white matter and improve locomotor function in an experimental model of spinal cord contusion, suggesting that tempol treatment might inhibit Wallerian degeneration of spinal cord axons. Here, we report that tempol partially inhibits Wallerian degeneration, resulting in improved locomotor recovery. We previously reported that Wallerian degeneration is reduced by inhibitors of aldose reductase (AR), which converts glucose to sorbitol in the polyol pathway. We observed that tempol inhibited sorbitol production in the injured spinal cord to the same extent as the AR inhibitor, sorbinil. Tempol also prevented post-contusion upregulation of AR (AKR1B10) protein expression within degenerating axons, as previously observed for AR inhibitors. Additionally, we hypothesized that tempol inhibits axonal degeneration by preventing loss of the glutathione pool due to polyol pathway activity. Consistent with our hypothesis, tempol treatment resulted in greater glutathione content in the injured spinal cord, which was correlated with increased expression and activity of γ-glutamylcysteine ligase (γGCL; EC 6.3.2.2), the rate-limiting enzyme for glutathione synthesis. Administration of the γGCL inhibitor buthionine sulfoximine abolished all observed effects of tempol administration. Together, these results support a pathological role for polyol pathway activation in glutathione depletion, resulting in Wallerian degeneration after spinal cord injury. Interestingly, methylprednisolone, oxandrolone, and clenbuterol, which are known to spare axonal tracts after spinal cord injury, were equally effective in inhibiting polyol pathway activation. These results suggest that prevention of AR activation is a common target of many disparate post-SCI interventions.

Role of the Polyol Pathway in Locomotor Recovery and Wallerian Degeneration after Spinal Cord Contusion Injury.

Spinal cord contusion injury leads to Wallerian degeneration of axonal tracts, resulting in irreversible paralysis. Contusion injury causes perfusion loss by thrombosis and vasospasm, resulting in spinal cord ischemia. In several tissues, including heart and brain, ischemia activates polyol pathway enzymes-aldose reductase (AR) and sorbitol dehydrogenase (SDH)-that convert glucose to sorbitol and fructose in reactions, causing oxidative stress and tissue loss. We sought to determine whether activation of this pathway, which has been termed glucotoxicity, contributes to tissue loss after spinal cord contusion injury. We tested individual treatments with AR inhibitors (sorbinil or ARI-809), SDH inhibitor (CP-470711), superoxide dismutase mimetic (tempol), or combined sorbinil and tempol. Each treatment significantly increased locomotor recovery and reduced loss of spinal cord tissue in a standard model of spinal cord contusion in rats. Tissue levels of sorbitol and axonal AR (AKR1B10) expression were increased after contusion injury, consistent with activation of the polyol pathway. Sorbinil treatment inhibited the above changes and also decreased axonal swelling and loss, characteristic of Wallerian degeneration. Treatment with tempol induced recovery of locomotor function that was similar in magnitude, but non-additive to sorbinil, suggesting a shared mechanism of action by reactive oxygen species (ROS). Exogenous induction of hyperglycemia further increased injury-induced axonal swelling, consistent with glucotoxicity. Unexpectedly, contusion increased spinal cord levels of glucose, the primary polyol pathway substrate. These results support roles for spinal glucose elevation and tissue glucotoxicity by the polyol pathway after spinal cord contusion injury that results in ROS-mediated axonal degeneration.

Therapeutic target for external beam x-irradiation in experimental spinal cord injury.

OBJECTIVE: X-irradiation has been shown to be beneficial to recovery from spinal cord injury (SCI); however, the optimal therapeutic target has not been defined. Experiments were designed to determine the optimal target volume within the injured spinal cord for improving functional recovery and sparing tissue with stereotactic x-irradiation. METHODS: SCI was produced in rats at the T10 level. A 20-Gy dose of radiation was delivered with a single, 4-mm-diameter, circular radiation beam centered either on the injury epicenter or 4 or 8 mm caudal or rostral to the injury epicenter. Locomotor function was determined for 6 weeks with the Basso, Beattie, and Bresnahan locomotor scale and tissue sparing by histological analysis of transverse sections along the spinal cords. RESULTS: X-irradiation of spinal cord segments at 4 mm, but not 8 mm, caudal or rostral to the contusion epicenter resulted in increases in locomotor recovery. Consistently, significant tissue sparing also occurred with x-irradiation centered at those sites, although irradiation centered 4 mm rostral to the epicenter led to tissue sparing along the greatest length of the spinal cord. Interestingly, regression analysis of these variables demonstrated that the quantitative relationship between the amount of tissue spared and the improvement in locomotion recovery was greatest in a region several millimeters rostral to the injury epicenter. CONCLUSIONS: These results indicate that x-irradiation in a region rostral to the injury epicenter is optimal for recovery from SCI. This minimal target should be attractive for therapeutic application since it allows a greatly reduced target volume so that uninjured tissue is not needlessly irradiated.

Stem cell therapy and curcumin synergistically enhance recovery from spinal cord injury.

Acute traumatic spinal cord injury (SCI) is marked by the enhanced production of local cytokines and pro-inflammatory substances that induce gliosis and prevent reinnervation. The transplantation of stem cells is a promising treatment strategy for SCI. In order to facilitate functional recovery, we employed stem cell therapy alone or in combination with curcumin, a naturally-occurring anti-inflammatory component of turmeric (Curcuma longa), which potently inhibits NF-κB. Spinal cord contusion following laminectomy (T9-10) was performed using a weight drop apparatus (10 g over a 12.5 or 25 mm distance, representing moderate or severe SCI, respectively) in Sprague-Dawley rats. Neural stem cells (NSC) were isolated from subventricular zone (SVZ) and transplanted at the site of injury with or without curcumin treatment. Functional recovery was assessed by BBB score and body weight gain measured up to 6 weeks following SCI. At the conclusion of the study, the mass of soleus muscle was correlated with BBB score and body weight. Stem cell therapy improved recovery from moderate SCI, however, it had a limited effect on recovery after severe SCI. Curcumin stimulated NSC proliferation in vitro, and in combination with stem cell therapy, induced profound recovery from severe SCI as evidenced by improved functional locomotor recovery, increased body weight, and soleus muscle mass. These findings demonstrate that curcumin in conjunction with stem cell therapy synergistically improves recovery from severe SCI. Furthermore, our results indicate that the effect of curcumin extends beyond its known anti-inflammatory properties to the regulation of stem cell proliferation.

Perfusion imaging of spinal cord contusion: injury-induced blockade and partial reversal by ß2-agonist treatment in rats.

OBJECT: Traumatic injury to the spinal cord results in considerable delayed tissue loss. The authors investigated the extent to which ischemia occurs following contusion-induced spinal cord injury and whether ischemia exacerbates tissue damage that leads to the loss of locomotor function. They also determined if ischemia is reversed with ß2-adrenoceptor agonist treatment, which has been established to be neuroprotective following contusion injury. METHODS: The extent and role of circulation loss in spinal cord injury was determined in an established experimental model of contusion injury. The spinal cord dura mater of Wistar rats was exposed by performing a laminectomy at T-8 to T-11. Laser Doppler perfusion imaging was then used to measure microcirculation in the exposed spinal cord. After imaging, a moderately severe contusion injury was produced using a weight-drop device unto the exposed dura at T-10. Perfusion imaging was again performed, scans were quantitated, and integrated intensities were compared. RESULTS: Postinjury imaging revealed an 18%-27% reduction in perfusion in regions rostral and caudal to the injury site, and a 68% reduction was observed at the contusion epicenter. These perfusion losses persisted for at least 48 hours. At 24 hours after injury, some rats were intraperitoneally injected with 2 mg/kg of the ß2-adrenoceptor agonist clenbuterol, which has been shown to promote the partial recovery of locomotor function and spare spinal cord tissue when administered within 2 days after contusion injury. Clenbuterol injection caused a gradual increase in perfusion, which was detectable at 30 minutes postinjection and continued over time, resulting in an 127% overall increase in perfusion at the epicenter 24 hours after treatment. CONCLUSIONS: These results suggest that the occurrence of chronic perfusion loss after contusion contributes to delayed damage and tissue loss. In contrast, ß2-adrenoceptor agonist treatment may exert neuroprotection by restoring perfusion, thereby preventing ischemic neurodegeneration. The ability of laser Doppler imaging to measure the loss of perfusion and its restoration upon treatment suggests that it may have clinical utility in the assessment and treatment of spinal cord injury.

Recovery from spinal cord injury using naturally occurring antiinflammatory compound curcumin: laboratory investigation.

OBJECT: Spinal cord injury (SCI) is a debilitating disease. Primary SCI results from direct injury to the spinal cord, whereas secondary injury is a side effect from subsequent edema and ischemia followed by activation of proinflammatory cytokines. These cytokines activate the prosurvival molecule nuclear factor-κB and generate obstacles in spinal cord reinnervation due to gliosis. Curcumin longa is an active compound found in turmeric, which acts as an antiinflammatory agent primarily by inhibiting nuclear factor-κB. Here, the authors study the effect of curcumin on SCI recovery. METHODS: Fourteen female Sprague-Dawley rats underwent T9-10 laminectomy and spinal cord contusion using a weight-drop apparatus. Within 30 minutes after contusion and weekly thereafter, curcumin (60 mg/kg/ml body weight in dimethyl sulfoxide) or dimethyl sulfoxide (1 ml/kg body weight) was administered via percutaneous epidural injection at the injury site. Spinal cord injury recovery was assessed weekly by scoring hindlimb motor function. Animals were killed 6 weeks postcontusion for histopathological analysis of spinal cords and soleus muscle weight evaluation. RESULTS: Curcumin-treated rats had improved motor function compared with controls starting from Week 1. Body weight gain significantly improved, correlating with improved Basso-Beattie-Bresnahan scores. Soleus muscle weight was greater in curcumin-treated rats than controls. Histopathological analysis validated these results with increased neural element mass with less gliosis at the contusion site in curcumin-treated rats than controls. CONCLUSIONS: Epidural administration of curcumin resulted in improved recovery from SCI. This occurred with no adverse effects noted in experimental animals. Therefore, curcumin treatment may translate into a novel therapy for humans with SCI.

Partial functional recovery after complete spinal cord transection by combined chondroitinase and clenbuterol treatment.

Spinal cord injury not only disrupts axonal tracts but also causes gliotic, fibrotic, and Schwannotic scarring with resulting deposition of chondroitin sulfate proteoglycans (CSPGs) which prevent axonal reconnection and recovery of locomotor function. Here, we determined whether recovery of locomotor function could be promoted after complete transection, by degrading CSPGs enzymatically within the injury site with chondroitinase ABC (chABC) together with treatment with the beta(2)-adrenoceptor agonist, clenbuterol, a neuroprotective agent which can promote regrowth of lower motoneurons. Partial recovery of locomotor function was observed 8-12 weeks postinjury only after combined chABC and clenbuterol treatment. The recovery of locomotor function coincided with the presence of axons caudal to the injury site arising from neurons of the reticular, vestibular, and red nuclei also only with combined chABC and clenbuterol treatment. Axons myelinated by Schwann cells were most prominent in the transection site in the combined treatment group. Clenbuterol treatment activated cAMP response element binding protein within retrogradely traced neurons which has been associated with axonal regrowth. ChABC treatment decreased scarring due to both CSPG and collagen deposition as well as the gap between intact regions of the spinal cord. ChABC also increased numbers of phagocytic cells which remove myelin debris as well as populations of astrocytes thereby aiding blood-spinal cord barrier reformation. Together the results suggest that chABC and clenbuterol can act synergistically to promote recovery of locomotor function.

Improved functional recovery with oxandrolone after spinal cord injury in rats.

At present, only the corticosteroid, methylprednisolone, is used for acute spinal cord injury to improve function. However, improvements are modest, and are associated with myopathy and immunosuppression so that alternative treatments are needed. Oxandrolone is an androgenic steroid with potential neuroprotective properties that is used to prevent muscle loss and is not immunosuppressive. Oxandrolone increased locomotor recovery concomitant with reduced loss of cord tissue in a standard weight drop model of spinal cord contusion injury indicating oxandrolone as a possible alternative to methylprednisolone. Oxandrolone also increased axonal sprouting within the ventral horns distal to the injury consistent with formation of relay circuits mediating locomotor recovery.

Differential skeletal muscle gene expression after upper or lower motor neuron transection.

Causes of disuse atrophy include loss of upper motor neurons, which occurs in spinal cord injury (SCI) or lower motor neurons (denervation). Whereas denervation quickly results in muscle fibrillations, SCI causes delayed onset of muscle spasticity. To compare the influence of denervation or SCI on muscle atrophy and atrophy-related gene expression, male rats had transection of either the spinal cord or sciatic nerve and were sacrificed 3, 7, or 14 days later. Rates of atrophy increased gradually over the first week after denervation and then were constant. In contrast, atrophy after SCI peaked at 1 week, then declined sharply. The greater atrophy after SCI compared to denervation was preceded by high levels of ubiquitin ligase genes, MAFbx and MuRF1, which then also markedly declined. After denervation, however, expression of these genes remained elevated at lower levels throughout the 2-week time course. Interestingly, expression of the muscle growth factor, IGF-1 was increased at 3 days after denervation when fibrillation also peaks compared to SCI. Expression of IGF-1R, GADD45, myogenin, and Runx1 were also initially increased after denervation or SCI, with later declines in expression levels which correlated less well with rates of atrophy. Thus, there were significant time-dependent differences in muscle atrophy and MAFbx, MuRF1, and IGF-1 expression following SCI or denervation which may result from distinct temporal patterns of spontaneous muscle contractile activity due to injury to upper versus lower motor neurons.

Stereotactic radiosurgery improves locomotor recovery after spinal cord injury in rats.

OBJECTIVE: Currently, because of the precision of stereotactic radiosurgery, radiation can now be delivered by techniques that shape the radiation beam to the tissue target for a variety of clinical applications. This avoids unnecessary and potentially damaging irradiation of surrounding tissues inherent in conventional irradiation, so that irradiation of the minimum volume of tissue necessary for optimal therapeutic benefit can be achieved. Although conventional x-irradiation has been shown to improve recovery from spinal cord injury in animals, the efficacy of targeted irradiation of the injured spinal cord has not been demonstrated previously. The purpose of these studies was to determine whether stereotactic x-irradiation of the injured spinal cord can enhance locomotor function and spare spinal cord tissue after contusion injury in a standard experimental model of spinal cord injury. METHODS: Contusion injury was produced in rats at the level of T10 with a weight-drop device, and doses of x-irradiation were delivered 2 hours after injury via a Novalis, 6-MeV linear accelerator shaped beam radiosurgery system (BrainLAB USA, Westchester, IL) in 4 sequential fractions, with beam angles 60 to 70 degrees apart, at a rate of 6.4 Gy/minute. The target volume was a 4 x 15-mm cylinder along the axis of the spinal cord, with the isocenter positioned at the contusion epicenter. Locomotor function was determined for 6 weeks after injury with the 21-point Basso, Beattie, and Bresnahan (BBB) locomotor scale and tissue sparing in histological sections of the spinal cord. RESULTS: Locomotor function recovered progressively during the 6-week postinjury observation period. BBB scores were significantly greater in the 10-Gy x-irradiated group compared with controls (9.4 versus 7.3; P < 0.05), indicating hind limb weight support or dorsal stepping in contrast to hind limb joint mobility without weight bearing. Doses in the range of 2 to 10 Gy increased BBB scores progressively, whereas greater doses of 15 to 25 Gy were associated with lower BBB scores. The extent of locomotor recovery after treatment with x-irradiation correlated with measurements of spared spinal cord tissue at the contusion epicenter. CONCLUSION: These results suggest a beneficial role for stereotactic radiosurgery in a rat model of acute spinal cord contusion injury and raise hopes for human treatment strategies. Additional animal studies are needed to further define potential benefits.

Inhibition of x-irradiation-enhanced locomotor recovery after spinal cord injury by hyperbaric oxygen or the antioxidant nitroxide tempol.

OBJECT: Hyperbaric oxygen (HBO), the nitroxide antioxidant tempol, and x-irradiation have been used to promote locomotor recovery in experimental models of spinal cord injury. The authors used x-irradiation of the injury site together with either HBO or tempol to determine whether combined therapy offers greater benefit to rats. METHODS: Contusion injury was produced with a weight-drop device in rats at the T-10 level, and recovery was determined using the 21-point Basso-Beattie-Bresnahan (BBB) locomotor scale. Locomotor function recovered progressively during the 6-week postinjury observation period and was significantly greater after x-irradiation (20 Gy) of the injury site or treatment with tempol (275 mg/kg intraperitoneally) than in untreated rats (final BBB Scores 10.6 [x-irradiation treated] and 9.1 [tempol treated] compared with 6.4 [untreated], p < 0.05). Recovery was not significantly improved by HBO (2 atm for 1 hour [BBB Score 8.2, p > 0.05]). Interestingly, the improved recovery of locomotor function after x-irradiation, in contrast with antiproliferative radiotherapy for neoplasia, was inhibited when used together with either HBO or tempol (BBB Scores 8.2 and 8.3, respectively). The ability of tempol to block enhanced locomotor recovery by x-irradiation was accompanied by prevention of alopecia at the irradiation site. The extent of locomotor recovery following treatment with tempol, HBO, and x-irradiation correlated with measurements of spared spinal cord tissue at the contusion epicenter. CONCLUSIONS: These results suggest that these treatments, when used alone, can activate neuroprotective mechanisms but, in combination, may result in neurotoxicity.

iNOS expression in rat aorta is increased after spinal cord transection: a possible cause of orthostatic hypotension in man.

Orthostatic hypotension commonly occurs in persons with spinal cord injury (SCI), limiting rehabilitation and independence. Findings of increased production of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) after exposure to simulated microgravity suggest that increased iNOS expression contributes to OH in persons with SCI. To test this possibility, male Wistar rats underwent surgical transection of the spinal cord (T10) or sham-SCI surgery followed by euthanasia 3, 7 or 14 days later. Expression in thoracic aortic of inducible (iNOS), endothelial (eNOS) and neuronal (nNOS) NOS was then determined. In SCI rats, expression of iNOS mRNA was decreased at 3 days, had returned to normal levels of expression at 7 days and was increased at 14 days post-SCI (1.8-fold). In contrast, levels of eNOS mRNA were increased at 3 days (1.4-fold), then declined over time reaching levels by day 14 that were reduced compared to sham-SCI (0.23-fold). There were no significant effects of SCI on nNOS expression. These findings suggest a possible role for increased iNOS expression in the pathogenesis of OH in persons with SCI.

Beta2-adrenoreceptor agonist-enhanced recovery of locomotor function after spinal cord injury is glutathione dependent.

The beta2-adrenoreceptor agonist, clenbuterol, has been shown to spare spinal cord tissue and enhance locomotor recovery in an experimental model of spinal cord contusion injury. A likely mechanism of neurodegeneration following spinal cord injury involves generation of toxic levels of reactive oxygen species (ROS), e.g., O2-*, H2O2 and OH*, which overwhelm endogenous antioxidants. Agents, such as clenbuterol, that oppose neurodegeneration and improve recovery of locomotor function may possibly act by improving redox status. Consistent with reduced oxidative stress by beta2-agonist treatment following injury, prior blockade of synthesis of the antioxidant tripeptide, glutathione, with buthionine sulfoximine completely inhibited the ability of clenbuterol to enhance locomotor recovery and spare spinal cord tissue. Moreover, at 8 h postinjury, clenbuterol caused an increase in glutathione reductase activity, an indicator of cellular redox status, at the injury site that was also blocked by buthionine sulfoximine. Although clenbuterol improved locomotor recovery only when administered within a therapeutic window of several days postinjury, the accumulation of protein carbonyls in the spinal cord at 1 week postinjury, a consequence of ongoing ROS-mediated neurodegeneration, was also decreased by clenbuterol in a glutathione-dependent manner. Together, these results suggest that activation of beta2-adrenoreceptors during the acute phase of injury stimulates glutathione-dependent antioxidative processes, that lead to reduced oxidative damage and greater locomotor function as the injury evolves during the subacute and chronic phases.

Clenbuterol retards loss of motor function in motor neuron degeneration mice.

Motor neuron degeneration (mnd) mice exhibit lysosomal accumulation of lipofuscin-like material that is associated with progressive loss of motor function and strength. Motor dysfunction scores at 8.5-9 months of age were highly correlated with the occurrence of abnormal spinal motor neurons with eccentric nuclei, although the total numbers of motor neurons were not significantly reduced. Nuclear eccentricity is a characteristic of the axon reaction that results from injury and subsequent compensatory axonal sprouting indicating axonal/synaptic dysfunction in mnd motor neurons. Treatment with clenbuterol, a beta(2)-adrenoceptor agonist that can enhance regeneration of motor neuron axons, opposed the development of motor deficits in parallel with a reduced proportion of motor neurons with eccentric nuclei consistent with improved synaptic function. Clenbuterol also opposed decreases in grip strength and muscle mass suggesting beta(2)-agonist treatment as a potential therapeutic modality for lipofuscinoses.

Tempol, a nitroxide antioxidant, improves locomotor and histological outcomes after spinal cord contusion in rats.

We have determined whether the nitroxide antioxidant, tempol, can oppose tissue loss and improve recovery of locomotor function following contusion injury of the spinal cord. Contusion injury was produced in rats at the level of T10 with a weight-drop device and locomotor recovery was determined with the 21-point Basso, Beattie and Bresnahan (BBB) scale. Locomotor function recovered progressively during the 6-week postinjury observation period and was significantly greater in tempol-treated (275 mg/kg i.p., 20 min postinjury) compared to vehicle-treated rats (final BBB scores: 9.1 versus 6.4). Similarly enhanced locomotor recovery was observed with doses of tempol in the range of 138-550, but not 69 mg/kg, and with injection at 48 h postinjury indicating a therapeutic time-window of at least several days. The extent of recovery correlated with measurements of sparing of spinal cord white matter in a region several millimeters in length extending rostrally from the contusion epicenter. In contrast, loss of gray matter was unaffected by tempol treatment. Since tempol acts by scavenging reactive oxygen species (ROS) such as superoxide and hydroxyl radicals, the improved locomotor recovery and spared spinal cord tissue following contusion injury provides evidence of a direct role of ROS-mediated neurodegeneration in spinal cord injury.