Search

Migrating activity of reactive astrocytes induced after spinal cord injury (SCI) controls glial scar formation by limiting inflammatory responses around the injury area, and, therefore, can be beneficial for regenerative responses of spinal axons. Recently, we found that cell division cycle 2 (cdc2) activity in primary astrocytes facilitated neurite outgrowth of co-cultured neurons. Here, we investigated the effects of cdc2 activity on regenerative processes in vivo after SCI. Administration of the cdc2 inhibitor purvalanol A restricted compaction of the injury cavity and astrocyte infiltration into the cavity. After SCI, regenerative responses of anterogradely labeled corticospinal tract (CST) axons were attenuated by purvalanol A treatment. Using the polymeric tube that was implanted into the spinal cord as a nerve guide conduit, we found that purvalanol A treatments reduced astrocyte migration into the tube graft and, in parallel, retarded the extension of spinal axons into the tube. These results suggest that astrocytes with cdc2 activity may play a permissive role in mediating regrowth of spinal axons after lesion.

Spinal cord injury is a major medical problem worldwide. Unfortunately, we still do not have suitable therapeutic agents for the treatment of spinal cord injury and prevention of its devastating consequences. Scientists and physicians are baffled by the challenges of controlling progressive neurodegeneration in spinal cord injury, which has not been healed with any currently-available treatments. Although extensive work has been carried out to better understand the pathophysiology of spinal cord injury, our current understanding of the repair mechanisms of secondary injury processes is still meager. Several investigators reported the crucial role played by various proteases after spinal cord injury. Understanding the beneficial and harmful roles these proteases play after spinal cord injury will allow scientists to plan and design appropriate treatment strategies to improve functional recovery after spinal cord injury. This review will focus on various proteases such as matrix metalloproteinases, cysteine proteases, and serine proteases and their inhibitors in the context of spinal cord injury.

Inducible nitric oxide synthase (iNOS) is a key mediator of inflammation and oxidative stress produced during pathological conditions, including neurodegenerative diseases and central nervous system (CNS) injury. iNOS is responsible for the formation of high levels of nitric oxide (NO). The production of highly reactive and cytotoxic NO species, such as peroxynitrite, plays an important role in secondary tissue damage. We have previously demonstrated that acute administration of iNOS antisense oligonucleotides (ASOs) 3 h after moderate contusive spinal cord injury (SCI) potently inhibits iNOS-mediated increases in NO levels, leading to reduced blood-spinal cord barrier permeability, decreased neutrophil accumulation, and less neuronal cell death. In the current study we investigated if iNOS ASOs could also provide long-term (10-week) histological and behavioral improvements after moderate thoracic T8 contusive SCI. Adult rats were randomly assigned to three groups (n=10/group): SCI alone, SCI and mixed base control oligonucleotides (MBOs), or SCI and iNOS ASOs (200 nM). Oligonucleotides were administered by spinal superfusion 3 h after injury. Behavioral analysis (Basso-Beattie-Bresnahan [BBB] score and subscore) was employed weekly for 10 weeks post-SCI. Although animals treated with iNOS ASOs demonstrated no significant differences in BBB scores compared to controls, subscore analysis revealed a significant improvement in foot positioning, trunk stability, and tail clearance. Histologically, while no gross improvement in preserved white and gray matter was observed, greater numbers of surviving neurons were present adjacent to the lesion site in iNOS ASO-treated animals than controls. These results support the effectiveness of targeting iNOS acutely as a therapeutic approach after SCI.

The inhibitory environment of the injured spinal cord is an obstacle to functional recovery and axonal regeneration in adult mammals. We had previously shown that injection of adeno-associated virus (AAV) encoding the L1 cell adhesion molecule (AAV-L1) at the time of acute thoracic compression injury of adult mice promotes locomotor recovery, which is associated with ameliorated astrogliosis and improved axonal regeneration in the lumbar spinal cord. In the present study, we investigated whether delayed injection of AAV-L1, chondroitinase ABC (Chase), or the combination of the two agents into the mouse spinal cord 3 weeks after injury would also lead to improved recovery. The Basso Mouse Scale showed enhanced locomotor recovery 12 weeks after application of the agents in all treatment groups compared to the control group that was injected with AAV encoding green fluorescent protein (AAV-GFP). Investigation of hindlimb function using single-frame motion analysis revealed, however, that L1 overexpression, but not injection of Chase, improved voluntary movements without body weight support, whereas injection of Chase, but not L1 overexpression, enhanced body weight support during stepping. Mice with the combined application of AAV-L1 and Chase showed improvement in both parameters. Enhanced motor recovery after combined application correlated with increased densities of cholinergic and GABAergic terminals at motoneuronal cell bodies, and of lamina-specific glutamatergic sensory afferents 15 weeks after injury, indicating enhanced synaptic rearrangements in the lumbar spinal cord below the lesion site. These findings suggest that L1 overexpression combined with Chase application may contribute to the treatment of sub-chronic spinal cord injury.

Cardiovascular diseases are one of the principal causes of death and disability in people with spinal cord injury (SCI). The present study was designed to investigate if acute treatment with FPTIII (an inhibitor of Ras-GTPase farnesylation) or MG132 (an inhibitor of ubiquitin-proteasome pathway [UPS]) or administration of angiotensin-(1-7), also known as Ang-(1-7), (a known inhibitor of cardiac NF-kB) would be cardioprotective. The weight drop technique produced a consistent contusive injury of the spinal cord at the T13 segment. Hearts were isolated from rats either 6 months (SCI-6) or 12 months (SCI-12) after SCI. Hearts were perfused for 30 min and then subjected to 30 min ischemia followed by 30 min reperfusion (I/R). Recovery of cardiac function after I/R was measured as left ventricular developed pressure (P(max)) and coronary flow (CF). Drugs were given during perfusion before hearts were exposed to ischemia and reperfusion. Percent recovery (%R) in P(max) and CF in hearts from control animals were 48±6 and 50±5, respectively, whereas none of the hearts from animals with SCI recovered after 30 min of ischemia. Treatment with FPTIII, MG 132, or Ang-(1-7) before ischemia for 30 min led to significant recovery of heart function following ischemia in SCI-6 but not in SCI-12 animals. Thus we have shown that acute treatments with FPTIII, MG132, or Ang-(1-7) improve cardiac recovery following ischemic insult in animals with SCI and may represent novel therapeutic agents for preventing ischemia-induced cardiac dysfunction in patients with SCI.

We tested the hypothesis that a selective phosphodiesterase type 4 inhibitor (PDE4-I; IC486051) would attenuate early inflammatory and oxidative processes following spinal cord injury (SCI) when delivered during the first 3 days after injury. Rats receiving a moderately severe thoracic-clip-compression SCI were treated with the PDE4-I (0.5, 1.0, and 3.0 mg/kg IV) in bolus doses from 2-60 h post-injury. Doses at 0.5 mg/kg and 1.0 mg/kg significantly decreased myeloperoxidase (MPO) enzymatic activity (neutrophils), expression of a neutrophil-associated protein and of ED-1 (macrophages), and estimates of lipid peroxidation in cord lesion homogenates at 24 h and 72 h post-injury by 25-40%. The 3.0 mg/kg dose had small or no effects on these measures. The PDE4-I treatment (0.5 or 1.0 mg/kg) reduced expression of the oxidative enzymes gp91(phox), inducible nitric oxide synthase, and cyclooxygenase-2, and diminished free radical generation by up to 40%. Treatment with 0.5 mg/kg PDE4-I improved motor function (as assessed by the Basso-Beattie-Bresnahan scale) significantly from 4-8 weeks after SCI (average difference 1.3 points). Mechanical allodynia elicited from the hindpaw decreased by up to 25%. The PDE4-I treatment also increased white matter volume near the lesion at 8 weeks after SCI. In conclusion, the PDE4-I reduced key markers of oxidative stress and leukocyte infiltration, producing cellular protection, locomotor improvements, and a reduction in neuropathic pain. Early inhibition of PDE4 is neuroprotective after SCI when given acutely and briefly at sufficient doses.

Despite the diversity of cells available for transplantation into sites of spinal cord injury (SCI), and the known ability of transplanted cells to integrate into host tissue, functional improvement associated with cellular transplantation has been limited. One factor potentially limiting the efficacy of transplanted cells is poor cell survival. Recently we demonstrated rapid and early death of Schwann cells (SCs) within the first 24 h after transplantation, by both necrosis and apoptosis, which results in fewer than 20% of the cells surviving beyond 1 week. To enhance SC transplant survival, in vitro and in vivo models to rapidly screen compounds for their ability to promote SC survival are needed. The current study utilized in vitro models of apoptosis and necrosis, and based on withdrawal of serum and mitogens and the application of hydrogen peroxide, we screened several inhibitors of apoptosis and necrosis. Of the compounds tested, the calpain inhibitor MDL28170 enhanced SC survival both in vitro in response to oxidative stress induced by application of H2O2, and in vivo following delayed transplantation into the moderately contused spinal cord. The results support the use of calpain inhibitors as a promising new treatment for promoting the survival of transplanted cells. They also suggest that in vitro assays for cell survival may be useful for establishing new compounds that can then be tested in vivo for their ability to promote transplanted SC survival.

Growth-inhibitory chondroitin sulfate proteoglycans (CSPG) are a primary target for therapeutic strategies after spinal cord injury because of their contribution to the inhibitory nature of glial scar tissue, a major barrier to successful axonal regeneration. Chondroitinase ABC (ChABC) digestion of CSPGs promotes axonal regeneration beyond a lesion site with subsequent functional improvement. ChABC also has been shown to promote sprouting of spared fibers but it is not clear if functional recovery results from such plasticity. Here we sought to better understand the roles rostral or caudal sprouting may play in ChABC-mediated functional improvement. To achieve this, ChABC or vehicle was injected rostral or caudal to a unilateral C5 injury. When injected rostral to a hemisection, ChABC promoted significant sprouting of 5HT+ fibers into dorsal and ventral horns. When ChABC was injected into tissue caudal to a hemisection, no additional sprouting was observed. When injected caudal to a hemicontusion injury, ChABC promoted sprouting of 5HT+ fibers into the ventral horn but not the dorsal horn. None of this sprouting resulted in a change in the synaptic component synapsin, nor did it impact performance in behavioral tests assessing motor function. These data suggest that ChABC-mediated sprouting of spared fibers does not necessarily translate into functional recovery.

Abstract Physical training in experimental animals can improve locomotor activity via the regulation of spinal neural circuitry or peripheral nerve regeneration. Here we investigated the effects of treadmill training (TMT) on regenerative responses of the corticospinal tract (CST) after contusive spinal cord injury (SCI). One week after injury of the low thoracic spinal cord, rats were given TMT or sedentary treatment for 1-4 weeks. Anterograde tracing of descending CST axons revealed that TMT enhanced collateral arborization of CST axons surrounding the injury cavity and promoted extension into the caudal spinal cord. The number of oligodendrocytes in the vicinity of the injury cavity was significantly increased at 2 or 4 weeks after TMT compared to sedentary controls. The data further showed that TMT increased phosphorylation of Erk1/2 in the motor cortex as well as the spinal cord injury area, and inhibition of Erk1/2 activity by administration of the MEK1 inhibitors PD98059 and U0126 reduced collateral outgrowth of descending CST axons in TMT animals. TMT for 2-4 weeks significantly improved behavioral scores as assessed by the Basso-Beattie-Bresnahan scale, as well as on motor function and gridwalk testing. Our data imply that Erk1/2 may be an important mediator for transmitting signals from the injury site to the cell body, and further suggest that activation of the Erk1/2 signaling pathway may be involved in enhanced outgrowth of CST axons after TMT.

Formation of a glial scar is one of the major obstacles to axonal growth after injury to the adult CNS. In this study, we have addressed this issue by focusing on reactive astrocytes in a mouse model of spinal cord injury (SCI). First, we attempted to identify profile changes in the expression of astrocytic gliosis 10 days after injury by using gliosis-specific microdissection, genome-wide microarray, and MetaCore(trade mark) pathway analysis. This systematic data processing revealed many intriguing activated pathways. However, considering that proliferation/mitosis is one of the most prominent features of reactive astrocytes, we focused on the functional role of the Ras-MEK-ERK signaling cascades in reactive astrocytes. SCI-induced proliferation of reactive astrocytes in the lesion is in accordance with the increase in the expression and phosphorylation of MEK-ERK. Second, to reduce reactive gliosis after SCI, liposomes containing the interferon-beta (IFN-beta) gene were administered locally 30 min after injury. At 14 days after this treatment, GFAP-positive intensity and MEK-ERK phosphorylation at the lesion were reduced. In the animals receiving the IFN-beta gene, significant recovery of neurobehavior and parameters of electrophysiology following SCI was revealed by assessments of rotarod performance and improvements in the Basso Mouse Scale for locomotion and cortical motor-evoked potentials. SCI resulted in the degeneration of biotinylated dextran amine-labeled descending corticospinal tract axons, but the IFN-beta gene delivery induced regrowth of a large number of corticospinal tract axons. These results suggest that liposome-mediated IFN-beta gene delivery inhibits glial scar formation after SCI and promotes functional recovery.

Although calpain (calcium-activated cysteine protease) inhibition represents a rational therapeutic target for spinal cord injury (SCI), few studies have reported improved functional outcomes with post-injury administration of calpain inhibitors. This reflects the weak potency and limited aqueous solubility of current calpain inhibitors. Previously, we demonstrated that intraspinal microinjection of the calpain inhibitor MDL28170 resulted in greater inhibition of calpain activity as compared to systemic administration of the same compound. In the present study, we evaluated the ability of intraspinal MDL28170 microinjection to spare spinal tissue and locomotor dysfunction following SCI. Contusion SCI was produced in female Long-Evans rats using the Infinite Horizon impactor at the 200-kdyn force setting. Open-field locomotion was evaluated until 6 weeks post-injury. Histological assessment of tissue sparing was performed at 6 weeks after SCI. The results demonstrate that MDL28170, administered with a single post-injury intraspinal microinjection (50 nmoles), significantly improves both locomotor function and pathological outcome measures following SCI.

Matrix metalloproteinase-9 (MMP-9) activation plays an important role in blood-brain barrier (BBB) dysfunction after central nervous system injury. Oxidative stress is also implicated in the pathogenesis after cerebral ischemia and spinal cord injury (SCI), but the relationship between MMP-9 activation and oxidative stress after SCI has not yet been clarified. We examined MMP-9 expression after SCI using copper/zinc-superoxide dismutase (SOD1) transgenic (Tg) rats. Our results show that MMP-9 activity significantly increased after SCI in both SOD1 Tg rats and their wild-type (Wt) littermates, although the increase was less in the SOD1 Tg rats. This pattern of MMP-9 expression was further confirmed by immunostaining and Western blot analysis. In situ zymography showed that gelatinolytic activity increased after SCI in the Wt rats, while the increase was less in the Tg rats. Evans blue extravasation increased in both the Wt and Tg rats, but was less in the SOD1 Tg rats. Inhibitor studies showed that, with an intrathecal injection of SB-3CT (a selective MMP-2/MMP-9 inhibitor), the MMP activity, Evans blue extravasation, and apoptotic cell death decreased after SCI. We conclude that increased oxidative stress after SCI leads to MMP-9 upregulation, BBB disruption, and apoptosis, and that overexpression of SOD1 in Tg rats decreases oxidative stress and further attenuates MMP-9 mediated BBB disruption.

We conducted a study of whether treatment with glial cell line-derived neurotrophic factor (GDNF) initiated at 2 or 4 weeks after spinal-root avulsion could promote survival and regulate neuronal nitric oxide synthase (nNOS) expression in adult rat spinal motoneurons. By 6 weeks after root avulsion, the treatment given at 2 weeks not only increased motoneuron survival (86.1% vs. 27.9%), but also reversed the atrophy of injured motoneurons and increased their somatic size (101.3% vs. 52.9%) in comparison to the untreated control group of animals. All surviving motoneurons in the GDNF-treated group showed immunoreactivity for choline acetyltransferase. In contrast, GDNF treatment at 4 weeks post-injury failed to promote motoneuron survival (33.1% vs. 27.9%) at 6 weeks compared to the control group. Both the 2- and 4-week post-injury treatments downregulated nNOS expression. This finding suggests that injured adult motoneurons die shortly (a few weeks in the rat) after root avulsion injury, but can be saved from degeneration by treatment within the proper time frame after injury, which in the case of GDNF treatment in rats, appears to be within 2 weeks of the avulsion injury of the spinal root. These findings provide useful information for choosing the best time frame for the potential clinical treatment of brachial plexus avulsion.

Rho is an important signaling target to promote repair following spinal cord injury (SCI). Myelin-derived inhibitory proteins, as well as other classes of known growth inhibitory proteins, block regeneration by signaling activation of Rho. Here, we review the molecular mechanisms of Rho activation after SCI and explain known Rho signaling antagonists. We review the data on use of Rho antagonists to promote axon regeneration, neuroprotection, and functional recovery after SCI. We report on efforts to translate the nonclincal studies on rodents to clinical trials in patients with acute SCI.

Acute traumatic spinal cord injury (SCI) results in a devastating loss of neurological function below the level of injury and adversely affects multiple systems within the body. The pathobiology of SCI involves a primary mechanical insult to the spinal cord and activation of a delayed secondary cascade of events, which ultimately causes progressive degeneration of the spinal cord. Whereas cell death from the mechanical injury is predominated by necrosis, secondary injury events trigger a continuum of necrotic and apoptotic cell death mechanisms. These secondary events include vascular abnormalities, ischemia-reperfusion, glutamate excitotoxicity and disturbances in ionic homeostasis, oxidative cell injury, and a robust inflammatory response. No gold standard therapy for SCI has been established, although clinical trials with methylprednisolone (NASCIS II and III) and GM-1 ganglioside (Maryland and Sygen) have demonstrated modest, albeit potentially important therapeutic benefits. In light of the overwhelming impact of SCI on the individual, other therapeutic interventions are urgently needed. A number of promising pharmacological therapies are currently under investigation for neuroprotective abilities in animal models of SCI. These include the sodium (Na+) channel blocker riluzole, the tetracycline derivative minocycline, the fusogen copolymer polyethylene glycol (PEG), and the tissue-protective hormone erythropoietin (EPO). Moreover, clinical trials investigating the putative neuroprotective and neuroregenerative properties ascribed to the Rho pathway antagonist, Cethrin (BioAxone Therapeutic, Inc.), and implantation of activated autologous macrophages (ProCord; Proneuron Biotechnologies) in patients with thoracic and cervical SCI are now underway. We anticipate that these studies will harken an era of renewed interest in translational clinical trials. Ultimately, due to the multi-factorial pathophysiology of traumatic SCI, effective therapies will require combined approaches.

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.

Traumatic spinal cord injury (SCI) results in widespread neuronal cell death. Recent studies have suggested that activated calpain mediates neuronal cell death in the central nervous system. We conducted a study to determine whether calpain mediates neuronal cell death in the motor neurons of the spinal cord after SCI, and whether postinjury administration of the calpain inhibitors N-acetyl- Leu-Leu-Met-CHO (ALLM) and calpain inhibitor III (CI III) (MDL28170) reduces the motor disturbances in rats with a model of SCI. Adult male Wistar rats were subjected to SCI by application of a 20-g weight impactor probe to the spinal cord at T12 for 20 min. The rats were divided into three groups according to whether they were injected intravenously with 0.05-2.5 mg/kg ALLM, 10 mg/kg CI III, or 0.1% DMSO as a control every 24 h for 1 week after SCI. Calpain was activated in the spinal cord at 8 h, 24 h, and 5 days after SCI, and administration of ALLM inhibited its activation. ALLM, as compared to the DMSO vehicle alone, also significantly reduced the number of motor neurons in spinal-cord lesions that were positively labeled at 24 h after SCI with the terminal deoxynucleotidyl transferase-uridine nucleotide end-labeling (TUNEL) technique. Additionally, both the inclined plane test and footprint analysis showed markedly better motor activity after 4 weeks in rats injected with ALLM or CI III than in rats given vehicle only. These results suggest that activation of calpain plays a critical role in the neuronal cell death that follows SCI, and that calpain inhibitors may have benefit in treating the motor disturbances that follow SCI.

Antioxidant enzymes including superoxide dismutase (SOD) may play a role in the mechanism by which cells counteract the deleterious effects of reactive oxygen species (ROS) after spinal cord injury (SCI). Cu/Zn and MnSOD are especially potent scavengers of superoxide anion and likely serve important cytoprotective roles against cellular damage. We investigated expression of SOD after SCI to address its role during the early stages of injury. MnSOD activity was increased 4 h after SCI and persisted at elevated levels up to 24-48 h; by contrast, Cu/ZnSOD activity was not changed. RT-PCR and Western blot analyses showed increased levels of MnSOD mRNA and protein, respectively, by 4 h and reached maximum levels by 24-48 h. Double immunostaining revealed that MnSOD protein was localized within neurons and oligodendrocytes. Tumor necrosis factor-alpha (TNF-alpha) was administered locally into uninjured spinal cords to examine potential mechanisms for MnSOD induction after injury. TNF-alpha administered exogenously increased MnSOD expression in uninjured spinal cords. Western blot and immunostaining also revealed that a transcription factor, NF-kappaB, was activated and translocated into the nuclei of neurons and oligodendrocytes. By contrast, administration of neutralizing antibody against TNF-alpha into injured spinal cords attenuated the increase in MnSOD expression and activation of NF-kappaB. Double immunostaining revealed that MnSOD was co-localized with NF-kappaB in neurons and oligodendrocytes after SCI. These results suggest that TNF-alpha may be an inducer of NF-kappaB activation and MnSOD expression after SCI and that MnSOD expression induced by TNF-alpha is likely mediated through activation of NF-kappaB.

While the average age for patients admitted with spinal cord injury is 32 years, patients under the age of 16 account for 5% of spinal cord injured persons. For these younger patients, an increased mortality up to 24 h post-injury has been reported, however, survivors may regain more function than their adult counterparts, suggesting that age may play a role in injury tolerance. While the use of growth factors as a therapy for spinal cord injury is well researched, the response of the developing cord to secondary injury has not been thoroughly investigated. Following spinal cord injury, Ca(2+) influx can activate enzymes such as calpain, a Ca(2+)-dependent protease, which plays a role in the pathogenesis of spinal cord injury in rats. The present investigation revealed that following spinal cord injury, calpain upregulation was significantly less (15.3%) in the 21-day-old rats than in either 45-day-old (70%) or 90-day-old (99.6%) rats, as shown by Western blot and in situ immunofluorescent studies. Expression of the endogenous calpain inhibitor, calpastatin, was significantly higher in juvenile rats than adult rats. Juvenile rats with spinal cord injury also showed a reduced Bax:Bcl-2 ratio (4:1 vs. 6:1), reduced caspase-3 staining, reduced myelin loss (3% vs. 18%), and less neuronal DNA damage, as compared to older rats. These results suggest that increased calpastatin levels found in juvenile rats muted calpain activity and neuronal apoptosis, following spinal cord injury.

Heme oxygenase-1 (HO-1) has been shown to alter vascular function in part by attenuating inflammation. We induced HO-1 in blood vessels in the spinal cord by systemic administration of hemin. Twenty-four hours later, immediately prior to euthanasia, fluorescence conjugated Lycopersicon esculentum (tomato) lectin was given intravenously to label the vasculature. HO-1 was induced in blood vessels, particularly in the white matter, as evidenced by the immunolocalization of HO-1 in lectin positive vessels. Western blots confirmed the hemin-mediated induction of HO-1 in the uninjured spinal cord. We next examined the extent to which treatment with hemin or vehicle, 24 h prior to a moderate contusion injury, influenced early vascular dysfunction in the injured cord. All animals were euthanized 24 h after injury. Luciferase, a marker of barrier integrity, was given intravenously 30 min prior to euthanasia. The spinal cord was either prepared for quantification of luciferase activity or fixed by vascular perfusion and prepared for the immunolocalization of neutrophils. There was a significant attenuation of barrier permeability to luciferase and a significant reduction in the number of neutrophils in hemin treated animals as compared to the vehicle treated group. Together, these findings demonstrate that vascular induction of HO-1 modulates barrier function and neutrophil infiltration and suggest that this protein may be useful for limiting the early vascular dysfunction and inflammation that occurs in the acutely injured spinal cord., (Copyright 2004 Mary Ann Liebert, Inc.)

Olfactory ensheathing cells (OECs) were transplanted in adult rats after photochemical injury of the spinal cord. Rats received either 180,000 OECs suspended in DMEM or DMEM alone. Locomotor ability scored by the BBB-scale, pain sensibility, and motor and somatosensory evoked potentials were evaluated during the first 14 days post-surgery. At 3, 7, and 14 days, 5 rats per day of both groups were perfused and transverse sections from proximal, lesioned and distal spinal cord blocks were stained for COX-2, VEGF, GFAP and lectin. The BBB-score and the amplitude of motor and somatosensory evoked potentials were significantly higher in OEC- than in DMEM-injected animals throughout follow-up, whereas the withdrawal latency to heat noxious stimulus was lower in OEC- than in DMEM-injected rats. The area of preserved spinal cord and the levels of COX-2 and VEGF staining were significantly higher in OEC- than in DMEM-injected rats. GFAP- but no LEC-positive cells expressed COX-2 staining in OEC-transplanted rats. The density of blood vessels was also significantly increased in OEC- with respect to DMEM-injected rats. Our results show that OECs promote functional and morphological preservation of the spinal cord after photochemical injury, increasing neoangiogenesis and up-regulation of COX-2 and VEGF expression in astrocytes., (Copyright 2004 Mary Ann Liebert, Inc.)

Calpains (calcium-activated cysteine proteases) are strongly implicated in the secondary damage that follows contusion injury to the spinal cord. Calpains are activated within a few minutes following injury and their elevated activity persists for 24 h, thereby providing a reasonable window of opportunity for postinjury inhibition. Previous studies demonstrated decreased axonal damage and neurofilament proteolysis with postinjury intravenous administration of relatively low concentrations of the calpain inhibitors leupeptin, E-64-D, and calpeptin. We sought to determine if conditions under which calpain inhibitors were administered in previous studies resulted in effective calpain inhibition, and to identify conditions that result in significant calpain inhibition following spinal cord injury. Contusive spinal cord injury was produced in female Long-Evans rats using the NYU impactor at the 12.5-25-mm height setting. The results demonstrate that intravenous administration of 1 mg/kg E-64-D or 250 micro g/kg calpeptin does not inhibit total calpain activity in the rat spinal cord, measured using a BODIPY-FL labeled casein assay. Intravenous administration of MDL28170 (20 mg/kg) resulted in mild calpain inhibition and a modest decrease in the proteolysis of calpain substrates alpha-spectrin and MAP2. Intraspinal microinjection of 50 nmoles/19 micro g MDL28170, either 30 min prior to or 20 min following contusion injury, resulted in a more robust inhibition of total calpain activity and greater attenuation of alpha-spectrin breakdown and MAP2 proteolysis. The decreased proteolysis persisted 24 h postinjury. Together, the results demonstrate that direct microinjection of the calpain inhibitor MDL28170 is more effective than intravenous infusion in reducing calpain activity and decreasing the injury-induced proteolysis of calpain substrates.

The aim of this study was to observe the time course of NO production and NOS expression in the spinal cord following acute traumatic injury. Rat spinal cord was injured by extradural static weight-compression, which resulted in an incomplete transverse spinal cord lesion with paralysis of the lower extremities. Using this model, measurement of NO by microdialysis and Griess reaction and histological and immunohistochemical examinations using polyclonal antibodies to nNOS and iNOS were performed from immediately to 14 days after injury. In injured cord, the amount of NO markedly increased immediately after injury and gradually decreased between 1 and 12 h after injury. A second wave of increase in NO level was observed at 24 h and 3 days after injury. Histologically, hematomas and necrotic changes were observed after injury and demyelination of nerve fibers increased with time in the compressed segment. Immunohistochemically, the number of cells with expression of nNOS was increased immediately to 12 h after injury. Expression of iNOS was observed from 12 h to 3 days after injury. These findings suggested that the initial maximal increase of NO production might be caused mainly by nNOS and that the second wave of increase in NO might be due mainly to iNOS.

Free radical-mediated mechanisms of cellular damage have been implicated in the early stages of spinal cord injury (SCI). Manganese superoxide dismutase (MnSOD) is a potent scavenger of superoxide radicals and likely serves an important cytoprotective role in preventing cellular damage after SCI. We have evaluated the expression of MnSOD to address its role during the early events of SCI using a well-established rat contusion model. Northern analysis showed a rapid induction of MnSOD mRNA between 2 and 6 h post injury. Observed time-dependent increases in MnSOD message was maximal 6 h post injury over that of MnSOD mRNA levels induced by laminectomy alone. Immunoblot and immunohistochemical analysis demonstrated increased expression of MnSOD protein 24 h after SCI with localization primarily within neurons. Interestingly, laminectomy alone also caused an induction of MnSOD gene and protein expression. To evaluate one potential mechanism of MnSOD induction, we microinjected the naive spinal cord with IL-1beta, which caused a similar fold induction of MnSOD mRNA levels by 6 h as observed with SCI, thus implicating it as a potential inducer of MnSOD during SCI. In summary, these results demonstrate that this potent cytoprotective antioxidant enzyme is rapidly and significantly induced as a consequence of SCI.

Nitric oxide (NO) has been shown to play an important role in the pathophysiology of traumatic brain injury (TBI) and cerebral ischemia. However, its contribution to the pathogenesis of traumatic spinal cord injury (SCI) remains to be clarified. This study determined the time course of constitutive and inducible nitric oxide synthases (cNOS and iNOS, respectively) after SCI. Rats underwent moderate SCI at T10 using the NYU impactor device and were allowed to survive for 3, 6, or 24 h and 3 days after SCI (n = 5 in each group). For the determination of enzymatic activities, spinal cords were dissected into five segments, including levels rostral and caudal (remote) to the injury site. Other rats were perfusion fixed for the immunohistochemical localization of iNOS protein levels. cNOS activity was significantly decreased at 3 and 6 h within the traumatized T10 segment and at 3, 6, and 24 h at the rostral (T9) level (p < 0.05). Rostral (T8) and caudal (T11, T12) to the injury site cNOS activity was also decreased at 3 h after injury (p < 0.05). However, cNOS activity returned to control levels within 6 h at T8, T11 and T12 and at one day at T10 and T9 segments. iNOS enzymatic activity was elevated at all time points tested (p < 0.05), with the most robust increase observed at 24 h. Immunostaining for iNOS at 24 h revealed that a significant cellular source of iNOS protein appeared to be invading polymorphonuclear leukocytes (PMNLs). To assess the functional consequences of iNOS inhibition, aminoguanidine treatment was initiated 5 min after SCI and rats tested using the BBB open field locomotor score. Treated rats demonstrated significantly improved hindlimb function up to 7 weeks after SCI. Histopathological analysis of contusion volume showed that aminoguanidine treatment decreased lesion volume by 37% (p < 0.05). In conclusion, these results indicate that (1) cNOS and iNOS activities are regionally and temporally affected after moderate SCI, (2) the early accumulation of PMNLs are a potentially significant source of NO-induced cytotoxic products, and (3) acute aminoguanidine treatment significantly improves functional and histopathological outcome after SCI.

Tissue damage and neurological dysfunction after spinal cord injury may result, in part, from delayed or secondary mechanisms that appear to involve several endogenous factors. Among them, neutrophils are known to play important roles in the pathomechanisms of the secondary injury, that is, neutrophils are activated by an interaction with the endothelial cells, migrate into the damaged tissue and release several kinds of proteases or oxygen radicals. In the present study, we examined heme oxygenase-1 expression in the damaged spinal cord. The administration of an inhibitor of heme oxygenase-1 in vivo produced a delayed recovery of motor function after spinal cord injury, suggesting that heme oxygenase-1 may play roles as an endogenous anti-inflammatory enzyme and protective gene in the damaged and inflammatory tissue. We found that many neutrophils expressing heme oxygenase-1 mRNA and protein were recruited into the damaged spinal cord with extensive hemorrhages during early stage of spinal cord injury. In an in vitro study, neutrophils incubated with proinflammatory cytokines, such as interleukin-1, 6 or interferon-gamma, expressed heme oxygenase-1 mRNA and protein. Based on these findings we conclude that the activated neutrophils can express heme oxygenase-1 in the injured spinal cord tissue, perhaps expecting modulatory and neuroprotective actions in the inflammatory response to spinal cord injury.

Secondary tissue damage after spinal cord injury (SCI) may be due to inflammatory mediators. After SCI, the nuclear factor-kappaB (NF-kappaB) transcription factor can activate many pro-inflammatory genes, one of which is inducible nitric oxide synthase (iNOS). iNOS catalyzes the synthesis of nitric oxide (NO), a key inflammatory mediator, which in turn reacts with superoxide to generate peroxynitrite. Peroxynitrite is a strong oxidant that can damage cellular enzymes, membranes, and subcellular organelles through the nitration of tyrosine residues on proteins. The presence of nitrotyrosine (NT) is an indirect chemical indicator of toxic NO and peroxynitrite-induced cellular damage. Using a New York University (NYU) impactor to induce SCI in adult rats, we examined the temporal and cellular expression of iNOS and NT. We observed a progressive increase in iNOS expression in the injured cord starting at day 1 with maximal expression occurring at day 7, as determined by Western blot analysis. iNOS expression corresponded temporally to an increase in iNOS enzyme activity after SCI. In parallel with the progressive increase in iNOS activity, NT expression also increased with time after SCI. The iNOS and NT immunoreactivity was localized in neurons, astrocytes, endothelial cells and ependymal cells at the epicenter and adjacent to the region of spinal cord impact and injury. Results from the present study suggest that increased iNOS and peroxynitrite anion, as reflected by the progressive accumulation of NT in the injured impacted spinal cord, may contribute to the secondary injury process after SCI.

Although superoxide dismutase (SOD) has been reported to promote functional recovery in ischemic spinal cord injury, it presents many difficulties in practical use primarily due to its short half-life in vivo and low tissue affinity. In this study, we investigated the effects of a new type of SOD, a lecithinized superoxide dismutase (PC-SOD), on motor disturbances, spinal cord edema, levels of myeloperoxidase (MPO), and spinal cord blood flow (SCBF) after spinal cord injury (SCI) in rats. PC-SOD is reported to show a delayed plasma disappearance in vivo in rats and has a higher affinity for vascular endothelium cells, neutrophils, and other cells than unmodified SOD. PC-SOD (4000 units/kg), unmodified SOD (4000 units/kg), or vehicle was injected intravenously 30 min after SCI. Four hours after SCI, SOD activities in spinal cord tissue and plasma were significantly higher in the PC-SOD group than in the unmodified SOD group. In the PC-SOD-treated rats, motor function was significantly better than in the other 2 groups of rats. PC-SOD significantly suppressed MPO activity, an indicator of neutrophils infiltration, in the spinal cord, at 4, 8, and 24 h after SCI, and spinal cord edema at 24 h after SCI. Moreover, the decrease of SCBF after SCI was less marked in the PC-SOD group. The present results suggest that lecithinization can improve the drug delivery of SOD to the spinal cord and PC-SOD may be an alternative pharmacological treatment for SCI.

An increase in pressure up to 15 atm was used to condense the cellular membrane of cells in culture thereby eliciting a mechanical-like trauma. This trauma is similar to a compression-like spinal cord injury or brain injury. The cells used in this study were ROC-1 oligodendroglia, N1E-115 neuroblastoma, and human umbilical vein endothelial (HUVE) cells. Total fatty acid (FA) release and release of lactate dehydrogenase (LDH) into the extracellular medium were used as indices of cellular trauma. Pressure-induced FA release, dependent on pressure and pressure duration, occurred with all cell types. The level of pressure needed to cause the greatest increase in FA levels was 10 atm for ROC-1 cells (3 min duration), 15 atm for N1E-115 cells (3 min duration), and 15 atm for HUVE cells (10 min duration). With each cell type, the released FA were reacylated or metabolized between 10 and 30 min of recovery. Following a 12- to 24-h recovery period, N1E-115 and HUVE cells release more FA, indicating that the initial perturbation of the membrane was not fully reversible. LDH levels were significantly increased in both the N1E-115 and HUVE cultures following 24 h of recovery. This efflux of LDH indicates irreversible membrane damage, suggesting that the trauma may be irreversible at longer recovery times.

High-dose methylprednisolone (MP) given to patients within 8 h of traumatic spinal cord improved neural function at 6 and 12 months, suggesting a probable secondary injury process that may be amenable to therapeutic intervention. Vascular injury plays an important role in the secondary injury process of CNS trauma. We have examined the effect of MP on vascular changes, including tissue edema, vascular permeability, and polymorphonuclear (PMN) cell infiltration in a rat model of spinal cord impact injury. MP significantly reduced extravasation of fluorescein isothiocyanate dextran (FITC-D), a macromolecular tracer, by 64.3% and 50.7% with trauma forces of 20 and 40 g-cm, respectively, when MP was administered IV immediately after trauma at a bolus of 165 mg/kg, with a subsequent continuous MP infusion at 31.5 mg/kg/h for 23 h. MP reduced the water content in the 40 g-cm traumatic cord lesion to 73.0% compared to the traumatic control (74.3%, p < 0.001) at the same schedule of large dose 24-h infusion. The same doses of MP showed a trend to decrease the extent of neutrophil infiltration as determined by myeloperoxidase (MPO) activity, but the change was not significant. MP had little effect in decreasing FITC-D extravasation and cord edema when given at a lower dose (bolus of 30 mg/kg with continued infusion of 1.3 mg/kg/h for 23 h). MP did not reduce extravasation of FITC-D and edema when administered IV as one bolus injection at high (165 mg/kg) or low (30 mg/kg) doses.(ABSTRACT TRUNCATED AT 250 WORDS)

The excessive generation of free radicals is thought to be one of the major mechanisms leading to tissue injury in various pathological conditions, including ischemia, inflammation, and trauma. Conversion of xanthine dehydrogenase (XDH) to xanthine oxidase (XO) contributes to the formation of superoxide, an oxygen radical. We measured XDH and XO activity using a newly developed fluorometric assay in an experimental spinal cord injury model in rats. XO activity increased by more than 100% 4 h after spinal cord trauma. Total (XDH + XO) activity also increased by 96% during the same period. Allopurinol, an inhibitor of XO (100 mg/kg/day x 2 days, i.p.), completely inhibited plasma and spinal cord XO activity but did not affect posttraumatic edema determined by water content or polymorphonuclear (PMN) cell infiltration reflected by myeloperoxidase (MPO) activity in traumatized spinal cord. These results indicate that XDH conversion to XO may not be the major mechanism of oxygen radical formation in the pathogenesis of vasogenic edema or inflammatory response in this experimental spinal cord injury model in rats.