Alexander G. Rabchevsky

Experimental Modeling of Spinal Cord Injury: Characterization of a Force-Defined Injury Device

We examined the ability of a novel spinal cord injury (SCI) device to produce graded morphological and behavioral changes in the adult rat following an injury at thoracic level 10 (T10). The injury device uses force applied to the tissue as the control variable rather than tissue displacement. This has the advantage of eliminating errors that may arise from tissue movement prior to injury. Three different injury severities, defined by the amount of force applied to the exposed spinal cord at T10 (100, 150, and 200 kdyn), were evaluated at two different survival times (7 and 42 d). Unbiased stereology was employed to evaluate morphological differences following the injury. Quantitative behavioral assessment employed the Basso, Beattie, and Bresnahan locomotive rating scale. There was a significant force-related decline in locomotive ability following the injury. Animals subjected to a 200-kdyn injury performed significantly worse than animals subjected to a 100- and 150-kdyn injury. The locomotor ability at different days post injury significantly correlated with the amount of force applied to the spinal cord. Statistical analysis revealed several significant force-related morphological differences following the injury. The greatest loss of white and gray matter occurred at the site of injury impact and extended in both a rostral and caudal direction. Animals subjected to the greatest force (200 kdyn) displayed the least amount of spared tissue at both survival times indicative of the most severe injury. The amount of spared tissue significantly correlated with the locomotor ability. This novel rodent model of SCI provides a significant improvement over existing devices for SCI by reducing variability with a constant preset force to define the injury.

Mitochondrial permeability transition in CNS trauma: Cause or effect of neuronal cell death?

Experimental traumatic brain injury (TBI) and spinal cord injury (SCI) result in a rapid and significant necrosis of neuronal tissue at the site of injury. In the ensuing hours and days, secondary injury exacerbates the primary damage, resulting in significant neurologic dysfunction. It is believed that alterations in excitatory amino acids (EAA), increased reactive oxygen species (ROS), and the disruption of Ca2+ homeostasis are major factors contributing to the ensuing neuropathology. Mitochondria serve as the powerhouse of the cell by maintaining ratios of ATP:ADP that thermodynamically favor the hydrolysis of ATP to ADP + Pi, yet a byproduct of this process is the generation of ROS. Proton‐pumping by components of the electron transport system (ETS) generates a membrane potential (ΔΨ) that can then be used to phosphorylate ADP or sequester Ca2+ out of the cytosol into the mitochondrial matrix. This allows mitochondria to act as cellular Ca2+ sinks and to be in phase with changes in cytosolic Ca2+ levels. Under extreme loads of Ca2+, however, opening of the mitochondrial permeability transition pore (mPTP) results in the extrusion of mitochondrial Ca2+ and other high‐ and low‐molecular weight components. This catastrophic event discharges ΔΨ and uncouples the ETS from ATP production. Cyclosporin A (CsA), a potent immunosuppressive drug, inhibits mitochondrial permeability transition (mPT) by binding to matrix cyclophilin D and blocking its binding to the adenine nucleotide translocator. Peripherally administered CsA attenuates mitochondrial dysfunction and neuronal damage in an experimental rodent model of TBI, in a dose‐dependent manner. The underlying mechanism of neuroprotection afforded by CsA is most likely via interaction with the mPTP because the immunosuppressant FK506, which has no effect on the mPT, was not neuroprotective. When CsA was administrated after experimental SCI at the same dosage and regimen used TBI paradigms, however, it had no beneficial neuroprotective effects. This review takes a comprehensive and critical look at the evidence supporting the role for mPT in central nervous system (CNS) trauma and highlights the differential responses of CNS mitochondria to mPT induction and the implications this has for therapeutically targeting the mPT in TBI and SCI.

Role of peroxynitrite in secondary oxidative damage after spinal cord injury

Peroxynitrite (PON, ONOO−), formed by nitric oxide synthase‐generated nitric oxide radical (˙NO) and superoxide radical (O2˙−), is a crucial player in post‐traumatic oxidative damage. In the present study, we determined the spatial and temporal characteristics of PON‐derived oxidative damage after a moderate contusion injury in rats. Our results showed that 3‐nitrotyrosine (3‐NT), a specific marker for PON, rapidly accumulated at early time points (1 and 3 h) and a significant increase compared with sham rats was sustained to 1 week after injury. Additionally, there was a coincident and maintained increase in the levels of protein oxidation‐related protein carbonyl and lipid peroxidation‐derived 4‐hydroxynonenal (4‐HNE). The peak increases of 3‐NT and 4‐HNE were observed at 24 h post‐injury. In our immunohistochemical results, the co‐localization of 3‐NT and 4‐HNE results indicates that PON is involved in lipid peroxidative as well as protein nitrative damage. One of the consequences of oxidative damage is an exacerbation of intracellular calcium overload, which activates the cysteine protease calpain leading to the degradation of several cellular targets including cytoskeletal protein (α‐spectrin). Western blot analysis of α‐spectrin breakdown products showed that the 145‐kDa fragments of α‐spectrin, which are specifically generated by calpain, were significantly increased as soon as 1 h following injury although the peak increase did not occur until 72 h post‐injury. The later activation of calpain is most likely linked to PON‐mediated secondary oxidative impairment of calcium homeostasis. Scavengers of PON, or its derived free radical species, may provide an improved antioxidant neuroprotective approach for the treatment of post‐traumatic oxidative damage in the injured spinal cord.

Dose-response curve and optimal dosing regimen of cyclosporin A after traumatic brain injury in rats

Acute neuropathology following experimental traumatic brain injury results in the rapid necrosis of cortical tissue at the site of injury. This primary injury is exacerbated in the ensuing hours and days via the progression of secondary injury mechanism(s) leading to significant neurological dysfunction. Recent evidence from our laboratory demonstrates that the immunosuppressant cyclosporin A significantly ameliorates cortical damage following traumatic brain injury. The present study extends the previous findings utilizing a unilateral controlled cortical impact model of traumatic brain injury in order to establish a dose-response curve and optimal dosing regimen of cyclosporin A. Following injury to adult rats, cyclosporin A was administrated at various dosages and the therapy was initiated at different times post-injury. In addition to examining the effect of cyclosporin A on the acute disruption of the blood-brain barrier following controlled cortical impact, we also assessed the efficacy of cyclosporin A to reduce tissue damage utilizing the fluid percussion model of traumatic brain injury. The findings demonstrate that the neuroprotection afforded by cyclosporin A is dose-dependent and that a therapeutic window exists up to 24h post-injury. Furthermore, the optimal cyclosporin dosage and regimen markedly reduces disruption of the blood-brain barrier acutely following a cortical contusion injury, and similarly affords significant neuroprotection following fluid percussion injury. These findings clearly suggest that the mechanisms responsible for tissue necrosis following traumatic brain injury are amenable to pharmacological intervention.

Basic fibroblast growth factor (bFGF) enhances functional recovery following severe spinal cord injury to the rat

We have recently demonstrated that following a moderate contusion spinal cord injury (SCI) to rats, subsequent administration of basic fibroblast growth factor (bFGF) significantly enhances functional recovery and tissue sparing. To further characterize the effects of bFGF, we evaluated its efficacy after a more severe contusion injury at T(10) using the NYU impactor. Immediately after SCI, osmotic minipumps were implanted into the lateral ventricle and lumbar thecal sac to deliver bFGF at 3 or 6 microg per day versus control vehicle for 1 week. Animals were behaviorally tested for 6 weeks before histological assessment of tissue sparing through the injured segment and glial reactivity distal to the lesion. Compared to moderate SCI, all rats had more prolonged and sustained functional deficits 6 weeks after severe contusion. Subjects treated with bFGF had pronounced recovery of hindlimb movements from 2 to 6 weeks compared to controls, manifested in significantly higher behavioral scores. Only marginal tissue sparing was seen rostral to the injury in bFGF-treated spinal cords versus controls. Optical density measurements of astrocyte and microglial cell immunoreactivity in bFGF-treated spinal cords showed that after 6 weeks they approximated controls, although astrocyte immunoreactivity remained higher in controls rostrally. In summary, intrathecal infusion of bFGF following severe SCI significantly restores gross hindlimb motor function that is not correlated with significant tissue sparing. In light of previous evidence that pharmacological intervention with bFGF after moderate SCI enhances tissue preservation, the current findings indicate that yet undefined mechanisms contribute to the enhanced functional recovery following bFGF treatment.

Cyclosporin A Treatment Following Spinal Cord Injury to the Rat: Behavioral Effects and Stereological Assessment of Tissue Sparing

The immunosuppressant drug cyclosporin A (CsA) has significant neuroprotective properties following CNS injury. In the present study, we assessed the efficacy of CsA therapy following a moderate spinal cord injury (SCI). Adult female rats were injured with the NYU impactor from a height of 12.5 mm, and CsA or vehicle therapy was initiated 15 min after the injury. All animals were behaviorally tested with the BBB locomotor rating scale prior to morphological assessment of changes in the spinal cord. CsA therapy failed to significantly improve the behavioral recovery following the injury. Using a unique stereological approach to assess tissue damage, it was determined that CsA did not alter the amount of spared tissue. The possible neuroprotective effects of CsA, observed in other models of CNS injury, do not appear to influence SCI pathology, perhaps reflecting both anatomical and physiological differences between these distinct regions of the CNS.

Efficacy of methylprednisolone therapy for the injured rat spinal cord

Currently the synthetic glucocorticosteroid methylprednisolone sodium succinate (MPSS) is the standard therapy after acute spinal cord injury (SCI) in humans based on reported neurological improvements. The mechanisms for its beneficial actions are not entirely clear, but experimental evidence suggests MPSS affords some degree of neuroprotection. As many studies with rat models of SCI have been unable to demonstrate improved behavioral outcome or tissue sparing after MPSS treatment, we chose to stereologically assess whether it alters lesion volume and tissue sparing over time, as well as long‐term behavioral recovery. Adult rats subjected to contusion SCI with the NYU impactor were administered either MPSS or saline for 24 hr beginning 5 min post injury. Over time the lesion dimensions were extremely dynamic, such that by 6 weeks post injury the volumes were reduced to a third of those seen after the first week. MPSS marginally reduced lesion volumes across time vs. controls, but the amount of spared gray and white matter remained unaltered between the two groups. Behavioral results further showed that MPSS failed to improve recovery of hind‐limb function. These findings add to the emerging scrutiny of MPSS as the standard therapy for acute SCI, as well as indicate the existence of a therapeutic window for tissue sparing restricted to the first several days after this type of SCI in rats. Equally important, our results caution the use of lesion volume dimensions or percent tissue sparing at the epicenter as indicators of therapeutic efficacy because neither reflects the actual amount of tissue sparing.

Intrinsic Differences in Brain and Spinal Cord Mitochondria: Implication for Therapeutic Interventions

It is well known that regions of the CNS differentially respond to insults. After brain injury, cyclosporine A reduces damage but is ineffective following spinal cord injury. We address this disparity by assessing several parameters of mitochondrial physiology in the normal neocortex and spinal cord. In situ measurements of O  2– · production, lipid peroxidation, and mitochondrial DNA oxidation revealed significantly higher levels in spinal cord vs. neocortical neurons. Real‐time PCR demonstrated differences in mitochondrial transcripts coupled with decreases in complex I enzyme activity and respiration in spinal cord mitochondria. The threshold for calcium‐induced mitochondrial permeability transition was substantially reduced in spinal cord vs. neocortex and modulated by lipid peroxidation. These intrinsic differences may provide a pivotal target for strategies to ameliorate neuronal damage following injury, and this imbalance in oxidative stress may contribute to the susceptibility of spinal cord motor neurons in neuropathologies such as amyotrophic lateral sclerosis. J. Comp. Neurol. 474:524–534, 2004.

Genetic Manipulation of Intraspinal Plasticity after Spinal Cord Injury Alters the Severity of Autonomic Dysreflexia

Severe spinal cord injuries above mid-thoracic levels can lead to a potentially life-threatening hypertensive condition termed autonomic dysreflexia, which is often triggered by painful distension of pelvic viscera (bladder or bowel) and consequent sensory fiber activation, including nociceptive C-fibers. Interruption of tonically active medullo-spinal pathways after injury causes disinhibition of thoracolumbar sympathetic preganglionic neurons, and intraspinal sprouting of nerve growth factor (NGF)-responsive primary afferent fibers is thought to contribute to their hyperactivity. We investigated spinal levels that are critical for eliciting autonomic dysreflexia using a model of noxious colorectal distension (CRD) after complete spinal transection at the fourth thoracic segment in rats. Post-traumatic sprouting of calcitonin gene-related peptide (CGRP)-immunoreactive primary afferent fibers was selectively altered at specific spinal levels caudal to the injury with bilateral microinjections of adenovirus encoding the growth-promoting NGF or growth-inhibitory semaphorin 3A (Sema3a) compared with control green fluorescent protein (GFP). Two weeks later, cardio-physiological responses to CRD were assessed among treatment groups before histological analysis of afferent fiber density at the injection sites. Dysreflexic hypertension was significantly higher with NGF overexpression in lumbosacral segments compared with GFP, whereas similar overexpression of Sema3a significantly reduced noxious CRD-evoked hypertension. Quantitative analysis of CGRP immunostaining in the spinal dorsal horns showed a significant correlation between the extent of fiber sprouting into the spinal segments injected and the severity of autonomic dysreflexia. These results demonstrate that site-directed genetic manipulation of axon guidance molecules after complete spinal cord injury can alter endogenous circuitry to modulate plasticity-induced autonomic pathophysiology.

Segmental organization of spinal reflexes mediating autonomic dysreflexia after spinal cord injury

Spinal cord injuries above mid-thoracic levels can lead to a potentially life-threatening hypertensive condition termed autonomic dysreflexia that is often triggered by distension of pelvic viscera (bladder or bowel). This syndrome is characterized by episodic hypertension due to sudden, massive discharge of sympathetic preganglionic neurons in the thoracolumbar spinal cord. This hypertension is usually accompanied by bradycardia, particularly if the injury is caudal to the 2nd to 4th thoracic spinal segments. The development of autonomic dysreflexia is correlated with aberrant sprouting of peptidergic afferent fibers into the spinal cord below the injury. In particular, sprouting of nerve growth factor-responsive afferent fibers has been shown to have a major influence on dysreflexia, perhaps by amplifying the activation of disinhibited sympathetic neurons. Using a model of noxious bowel distension after complete thoracic spinal transection at the 4th thoracic segment in rats, we selectively altered C-fiber sprouting, at specified spinal levels caudal to the injury, with microinjections of adenovirus encoding the growth-promoting nerve growth factor or the growth-inhibitory semaphorin 3A. This was followed by assessment of physiological responses to colorectal distension and subsequent histology. Additionally, anterograde tract tracers were injected into the lumbosacral region to compare the extent of labeled propriospinal rostral projections in uninjured cords to those in cords after complete 4th thoracic transection. In summary, overexpression of chemorepulsive semaphorin 3A impeded C-fiber sprouting in lumbosacral segments and mitigated hypertensive autonomic dysreflexia, whereas the opposite results were obtained with nerve growth factor overexpression. Furthermore, compared to naïve rats, there were significantly more labeled lumbosacral propriospinal projections rostrally after thoracic injury. Collectively, our findings suggest that distension of pelvic viscera increases the excitation of expanded afferent terminals in the disinhibited lumbosacral spinal cord. This, in turn, triggers excitation and sprouting of local propriospinal neurons to relay visceral sensory stimuli and amplify the activation of sympathetic preganglionic neurons in the thoracolumbar cord, to enhance transmission in the spinal viscero-sympathetic reflex pathway. These responses are manifested as autonomic dysreflexia.