Sensory Axon Regeneration: A Review from an in vivo Imaging Perspective.

Injured primary sensory axons fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. Re-entry is prevented at the dorsal root entry zone (DREZ), the CNS-PNS interface. Why axons stop or turn around at the DREZ has generally been attributed to growth-repellent molecules associated with astrocytes and oligodendrocytes/myelin. The available evidence challenges the contention that these inhibitory molecules are the critical determinant of regeneration failure. Recent imaging studies that directly monitored axons arriving at the DREZ in living animals raise the intriguing possibility that axons stop primarily because they are stabilized by forming presynaptic terminals on non-neuronal cells that are neither astrocytes nor oligodendrocytes. These observations revitalized the idea raised many years ago but virtually forgotten, that axons stop by forming synapses at the DREZ.

In vivo imaging of dorsal root regeneration: rapid immobilization and presynaptic differentiation at the CNS/PNS border.

Dorsal root (DR) axons regenerate in the PNS but turn around or stop at the dorsal root entry zone (DREZ), the entrance into the CNS. Earlier studies that relied on conventional tracing techniques or postmortem analyses attributed the regeneration failure to growth inhibitors and lack of intrinsic growth potential. Here, we report the first in vivo imaging study of DR regeneration. Fluorescently labeled, large-diameter DR axons in thy1-YFPH mice elongated through a DR crush site, but not a transection site, and grew along the root at >1.5 mm/d with little variability. Surprisingly, they rarely turned around at the DREZ upon encountering astrocytes, but penetrated deeper into the CNS territory, where they rapidly stalled and then remained completely immobile or stable, even after conditioning lesions that enhanced growth along the root. Stalled axon tips and adjacent shafts were intensely immunolabeled with synapse markers. Ultrastructural analysis targeted to the DREZ enriched with recently arrived axons additionally revealed abundant axonal profiles exhibiting presynaptic features such as synaptic vesicles aggregated at active zones, but not postsynaptic features. These data suggest that axons are neither repelled nor continuously inhibited at the DREZ by growth-inhibitory molecules but are rapidly stabilized as they invade the CNS territory of the DREZ, forming presynaptic terminal endings on non-neuronal cells. Our work introduces a new experimental paradigm to the investigation of DR regeneration and may help to induce significant regeneration after spinal root injuries.

Concussive brain trauma in the mouse results in acute cognitive deficits and sustained impairment of axonal function.

Concussive brain injury (CBI) accounts for approximately 75% of all brain-injured people in the United States each year and is particularly prevalent in contact sports. Concussion is the mildest form of diffuse traumatic brain injury (TBI) and results in transient cognitive dysfunction, the neuropathologic basis for which is traumatic axonal injury (TAI). To evaluate the structural and functional changes associated with concussion-induced cognitive deficits, adult mice were subjected to an impact on the intact skull over the midline suture that resulted in a brief apneic period and loss of the righting reflex. Closed head injury also resulted in an increase in the wet weight:dry weight ratio in the cortex suggestive of edema in the first 24 h, and the appearance of Fluoro-Jade-B-labeled degenerating neurons in the cortex and dentate gyrus of the hippocampus within the first 3 days post-injury. Compared to sham-injured mice, brain-injured mice exhibited significant deficits in spatial acquisition and working memory as measured using the Morris water maze over the first 3 days (p<0.001), but not after the fourth day post-injury. At 1 and 3 days post-injury, intra-axonal accumulation of amyloid precursor protein in the corpus callosum and cingulum was accompanied by neurofilament dephosphorylation, impaired transport of Fluoro-Gold and synaptophysin, and deficits in axonal conductance. Importantly, deficits in retrograde transport and in action potential of myelinated axons continued to be observed until 14 days post-injury, at which time axonal degeneration was apparent. These data suggest that despite recovery from acute cognitive deficits, concussive brain trauma leads to axonal degeneration and a sustained perturbation of axonal function.

Distinct muscarinic acetylcholine receptor subtypes contribute to stability and growth, but not compensatory plasticity, of neuromuscular synapses.

Muscarinic acetylcholine receptors (mAChRs) modulate synaptic function, but whether they influence synaptic structure remains unknown. At neuromuscular junctions (NMJs), mAChRs have been implicated in compensatory sprouting of axon terminals in paralyzed or denervated muscles. Here we used pharmacological and genetic inhibition and localization studies of mAChR subtypes at mouse NMJs to demonstrate their roles in synaptic stability and growth but not in compensatory sprouting. M(2) mAChRs were present solely in motor neurons, whereas M(1), M(3), and M(5) mAChRs were associated with Schwann cells and/or muscle fibers. Blockade of all five mAChR subtypes with atropine evoked pronounced effects, including terminal sprouting, terminal withdrawal, and muscle fiber atrophy. In contrast, methoctramine, an M(2/4)-preferring antagonist, induced terminal sprouting and terminal withdrawal, but no muscle fiber atrophy. Consistent with this observation, M(2)(-/-) but no other mAChR mutant mice exhibited spontaneous sprouting accompanied by extensive loss of parental terminal arbors. Terminal sprouting, however, seemed not to be the causative defect because partial loss of terminal branches was common even in the M(2)(-/-) NMJs without sprouting. Moreover, compensatory sprouting after paralysis or partial denervation was normal in mice deficient in M(2) or other mAChR subtypes. We also found that many NMJs of M(5)(-/-) mice were exceptionally small and reduced in proportion to the size of parental muscle fibers. These findings show that axon terminals are unstable without M(2) and that muscle fiber growth is defective without M(5). Subtype-specific muscarinic signaling provides a novel means for coordinating activity-dependent development and maintenance of the tripartite synapse.

Neuromuscular transmission failure and muscle fatigue in ankle muscles of the adult rat after spinal cord injury.

Current evidence suggests that significant morphological changes occur in nerve-muscle connections caudal to spinal cord injury (SCI). To determine whether neuromuscular junction (NMJ) function is compromised after SCI, we investigated the contribution of NMJ failure to hindlimb muscle fatigue in control and spinalized adult rats. Repetitive supramaximal nerve stimulation was applied to two muscle-nerve preparations: medial gastrocnemius (MG)-tibial and tibialis anterior (TA)-peroneal. NMJ transmission failure was evident in control and SCI animals after repetitive stimulation. At 2 wk post-SCI, NMJ transmission failure was greater in SCI animals compared with controls, but the difference was not significant (P = 0.205 for the MG and P = 0.053 for the TA). At 6 wk post-SCI, there was a significant but small difference in NMJ transmission failure for the TA between control and spinal animals. These results demonstrate that, although there may be a mild decrement in NMJ function, NMJ transmission remains largely intact for supramaximal nerve stimulation.

Pathology of blast-related brain injury.

Blasts are responsible for about two-thirds of the combat injuries in Operation Iraqi Freedom and Operation Enduring Freedom, which include at least 1,200 traumatic brain injuries. Blasts inflict damage to the brain directly and by causing injuries to other organs, resulting in air emboli, hypoxia, and shock. Direct injuries to the brain result from rapid shifts in air pressure (primary blast injury), from impacts with munitions fragments and other objects propelled by the explosion (secondary blast injury), and from collisions with objects and rapid acceleration of individuals propelled by the explosion (tertiary blast injury). Tertiary injury can occur from a building or other structure collapsing and from an individual being thrown by the blast wind. The pathological consequences of secondary and tertiary blast injuries are very likely to be similar to those of other types of mechanical trauma seen in civilian life. The damage attributable to the specific effects of a blast, however, has received little study, although it has been assumed to include the focal and diffuse lesions characteristic of closed head injuries. Available clinical studies of blast injuries show focal damage similar to that found in other types of closed head injuries but have not determined whether diffuse axonal injury also occurs. In this article, we will try to reach a better understanding of the specific pathology of blast-related brain injury by reviewing the available experimental studies and the autopsy reports of victims of terrorist attacks and military casualties dating back to World War I.

Fibrillation potentials following spinal cord injury: improvement with neurotrophins and exercise.

Fibrillation potentials and positive sharp waves (spontaneous potentials) are the electrophysiological hallmark of denervated skeletal muscle, and their detection by intramuscular electromyography (EMG) is the clinical gold standard for diagnosing denervated skeletal muscle. Surprisingly, spontaneous potentials have been described following human and experimental spinal cord injury (SCI) in muscles innervated by spinal cord segments distal to the level of direct spinal injury. To determine whether electrophysiological abnormalities are improved by two therapeutic interventions for experimental SCI, neurotrophic factors and exercise training, we studied four representative hindlimb muscles in adult domestic short-hair cats following complete transection of the spinal cord at T11-T12. In untreated cats, electrophysiological abnormalities persisted unchanged for 12 weeks postinjury, the longest duration studied. In contrast, fibrillations and positive sharp waves largely resolved in animals that underwent weight-supported treadmill training or received grafts containing fibroblasts genetically modified to express brain-derived neurotrophic factor and neurotrophin-3. These findings suggest that neurotrophins and activity play an important role in the poorly understood phenomenon of fibrillations distal to SCI.

Paralysis elicited by spinal cord injury evokes selective disassembly of neuromuscular synapses with and without terminal sprouting in ankle flexors of the adult rat.

Neuromuscular junctions (NMJs) innervated by motor neurons below spinal cord injury (SCI) have been reported to remain intact despite the interruption of supraspinal pathways and the resultant loss of activity. Here we report notably heterogeneous NMJ responses to SCI that include overt synapse disassembly. Complete transection of the thoracic spinal cord of adult rats evoked massive sprouting of nerve terminals in a subset of NMJs in ankle flexors, extensor digitorum longus, and tibialis anterior. Many of these synapses were extensively disassembled 2 weeks after spinal transection but by 2 months had reestablished synaptic organization despite continuous sprouting of their nerve terminals. In contrast, uniform and persistent loss of acetylcholine receptors (AChRs) was evident in another subset of NMJs in the same flexors, which apparently lacked terminal sprouting and largely maintained terminal arbors. Other synapses in the flexors, and almost all the synapses in the ankle extensors, medial gastrocnemius, and soleus, remained intact, with little pre- or postsynaptic alteration. Additional deafferentation of the transected animals did not alter the incidence or regional distribution of either type of the unstable synapses, whereas cycling exercise diminished their incidence. The muscle- and synapse-specific responses of NMJs therefore reflected differential sensitivity of the NMJs to inactivity rather than to differences in residual activity. These observations demonstrate the existence of multiple subpopulations of NMJs that differ distinctly in pre- and postsynaptic vulnerability to the loss of activity and highlight the anatomical instability of NMJs caudal to SCI, which may influence motor deficit and recovery after SCI.

Neural stem cells may be uniquely suited for combined gene therapy and cell replacement: Evidence from engraftment of Neurotrophin-3-expressing stem cells in hypoxic-ischemic brain injury.

Previously, we reported that, when clonal neural stem cells (NSCs) were transplanted into brains of postnatal mice subjected to unilateral hypoxic-ischemic (HI) injury (optimally 3-7 days following infarction), donor-derived cells homed preferentially (from even distant locations) to and integrated extensively within the large ischemic areas that spanned the hemisphere. A subpopulation of NSCs and host cells, particularly in the penumbra, "shifted" their differentiation towards neurons and oligodendrocytes, the cell types typically damaged following asphyxia and least likely to regenerate spontaneously and in sufficient quantity in the "post-developmental" CNS. That no neurons and few oligodendrocytes were generated from the NSCs in intact postnatal cortex suggested that novel signals are transiently elaborated following HI to which NSCs might respond. The proportion of "replacement" neurons was approximately 5%. Neurotrophin-3 (NT-3) is known to play a role in inducing neuronal differentiation during development and perhaps following injury. We demonstrated that NSCs express functional TrkC receptors. Furthermore, the donor cells continued to express a foreign reporter transgene robustly within the damaged brain. Therefore, it appeared feasible that neuronal differentiation of exogenous NSCs (as well as endogenous progenitors) might be enhanced if donor NSCs were engineered prior to transplantation to (over)express a bioactive gene such as NT-3. A subclone of NSCs transduced with a retrovirus encoding NT-3 (yielding >90% neurons in vitro) was implanted into unilaterally asphyxiated postnatal day 7 mouse brain (emulating one of the common causes of cerebral palsy). The subclone expressed NT-3 efficiently in vivo. The proportion of NSC-derived neurons increased to approximately 20% in the infarction cavity and >80% in the penumbra. The neurons variously differentiated further into cholinergic, GABAergic, or glutamatergic subtypes, appropriate to the cortex. Donor-derived glia were rare, and astroglial scarring was blunted. NT-3 likely functioned not only on donor cells in an autocrine/paracrine fashion but also on host cells to enhance neuronal differentiation of both. Taken together, these observations suggest (1) the feasibility of taking a fundamental biological response to injury and augmenting it for repair purposes and (2) the potential use of migratory NSCs in some degenerative conditions for simultaneous combined gene therapy and cell replacement during the same procedure in the same recipient using the same cell (a unique property of cells with stem-like attributes).

Immunosuppression with either cyclosporine a or FK506 supports survival of transplanted fibroblasts and promotes growth of host axons into the transplant after spinal cord injury.

Fibroblasts that have been genetically modified to secrete neurotrophins can stimulate axonal regeneration, rescue injured neurons, and improve function when grafted into a spinal cord injury site. These grafts are usually allografts that require immunosuppression to prevent rejection. In this study, we compared the effects of two immunophilin-ligands (cyclosporine A [CsA] and FK506) that are used clinically to prevent transplant rejection on protection of grafted fibroblasts. As there are risks associated with prolonged immunosuppression, we compared the effects of 2 or 8 weeks of administration of these drugs, in combination with our standard methylprednisolone protocol, in animals that survived for 8 weeks, to determine whether a shorter course of immunosuppression would be effective. Outcome measures included fibroblast survival, infiltration of activated macrophages and microglia into the graft, final lesion size, and growth of host axons into the graft. The graft consisted of a Vitrogen matrix into which fibroblasts were suspended; the graft was placed into a C3/C4 lateral funiculus lesion. The fibroblasts were isolated from a transgenic strain of Fischer rats that produce the marker alkaline phosphatase (Fb/AP). This enabled us to track the grafted fibroblasts and to evaluate the extent of their survival. The grafted matrix filled the lesion cavity. The density of fibroblasts within the matrix differed according to treatment. Fibroblast survival was most robust in animals that received 8 weeks of immunophilin-ligand treatment. FK506 supported greater Fb/AP survival than CsA. ED-1 immunostaining for activated microglia and macrophages showed an inverse correlation between AP immunoreactivity and the density of immune cells within the graft. Thus, prolonged administration of either FK506 or CsA was necessary for maximal fibroblast survival and for limiting the macrophage invasion of the graft. None of the FK506 or CsA protocols modified the size of the lesion, indicating that these immunophilin-ligands had little effect on secondary enlargement of the lesion and therefore little neuroprotective effect. Because immunophilin-ligands have been shown to be neurotrophic, we used RT-97 immunostaining for neurofilaments and calcitonin gene related protein (CGRP) staining for dorsal root axons to visualize axons that grew into the graft. Some axons grew into the matrix even in the absence of immunophilin-ligand treatment, suggesting that the Vitrogen matrix itself is permissive, but all of the immunophilin-ligand protocols were much more effective in eliciting axonal growth. Growth of axons into the transplants was equally increased by drug treatment for 2 or 8 weeks. Thus, both treatments improved fibroblast survival, diminished immune cell invasion, and promoted axonal growth, and a 2-week course of treatment with either immunophilin-ligand was as effective as 8 weeks in stimulating axonal growth.

Ex vivo evaluation of ADC values within spinal cord white matter tracts.

BACKGROUND AND PURPOSE: Our purpose was to evaluate the effect of fixative on apparent diffusion coefficient (ADC) values and anisotropy within spinal cord white matter. As glutaraldehyde (GL) better preserves axonal ultrastructure as compared with paraformaldehyde (PF), we hypothesize that spinal cord white matter fixed with GL will have increased anisotropic water diffusion as compared with specimens fixed with PF. METHODS: Eleven rats were perfusion-fixed with either 4% PF or a combination of 2.5% GL and 4% PF. Diffusion-weighted imaging of the ex vivo spinal cord was performed using a 9.4T magnet with b values up to 3100 s/mm(2). In-plane resolution was 39 mum x 39 mum, and section thickness was 500 mum. RESULTS: Overall, animals fixed with a combination of GL and PF (GL-PF) showed a greater increase in longitudinal ADC (lADC) as compared to those fixed with PF only, without differences in transverse ADC (tADC). As a consequence of the increased lADC, overall anisotropic diffusion increased in those animals fixed with GL-PF, as measured with an anisotropy index (AI = tADC/lADC). Evaluation of specific tracts demonstrated that lADC for animals fixed with GL-PF were significantly elevated in the rubrospinal, vestibulospinal, and reticulospinal tracts as compared with animals fixed with PF only. CONCLUSION: Using a fixative of GL-PL results in increased anisotropy (decreased AI values) in spinal cord white matter tracts, as compared with PF fixation only, largely owing to increases in the lADC values. This finding may be due to better fixation of intra-axonal cytoskeletal proteins that results when GL is combined with PF and sheds further light on underlying sources of anisotropic water diffusion in CNS white matter.

Fate of immortalized human neuronal progenitor cells transplanted in rat spinal cord.

BACKGROUND: Replacement of neurons and glia by transplantation has been proposed as a therapy for neurodegenerative diseases, including amyotrophic lateral sclerosis. This strategy requires using human motor neuronal progenitor cells or xenografts of animal cells, but there is little evidence that xenografted neuronal cells can survive in spinal cord despite immunosuppression. OBJECTIVE: To clarify the mechanisms responsible for the death of xenografted neurons in spinal cord. METHODS: Cells from an immortalized, neuronally committed, human embryonic spinal cord-derived cell line (HSP1) that expresses motor neuronal properties in vitro were transplanted into adult rat spinal cord. The rats were killed at intervals up to 8 weeks and serial sections through the graft sites were processed for immunofluorescence using primary antibodies against human nuclear and mitochondrial antigens, microtubule-associated protein 2, TUJ1, CD5, natural killer cells, and activated microglia-macrophages, caspase-3 and caspase-9. RESULTS: Grafted cells did not migrate and underwent partial differentiation along a neuronal pathway. They were rejected after 4 weeks despite cyclosporine immunosuppression. Cells died by apoptosis via the cytochrome c/caspase-9/caspase-3 pathway. The host response included natural killer cells and activated microglia-macrophages but few T cells. CONCLUSIONS: Intraspinal neuronal xenotransplantation failed because of apoptotic cell death. Neither T cells nor the spinal cord environment, which favors gliogenesis, are likely to have been responsible, but natural killer cells may have been involved.

Alginate encapsulated BDNF-producing fibroblast grafts permit recovery of function after spinal cord injury in the absence of immune suppression.

Encapsulation of cells has the potential to provide a protective barrier against host immune cell interactions after grafting. Previously we have shown that alginate encapsulated BDNF-producing fibroblasts (Fb/BDNF) survived for one month in culture, made bioactive neurotrophins, survived transplantation into the injured spinal cord in the absence of immune suppression, and provided a permissive environment for host axon growth. We extend these studies by examining the effects of grafting encapsulated Fb/BDNF into a subtotal cervical hemisection on recovery of forelimb and hindlimb function and axonal growth in the absence of immune suppression. Grafting of encapsulated Fb/BDNF resulted in partial recovery of forelimb usage in a test of vertical exploration and of hindlimb function while crossing a horizontal rope. Recovery was significantly greater compared to animals that received unencapsulated Fb/BDNF without immune suppression, but similar to that of immune suppressed animals receiving unencapsulated Fb/BDNF. Immunocytochemical examination revealed neurofilament (RT-97), 5-HT, CGRP and GAP-43 containing axons surrounding encapsulated Fb/BDNF within the injury site, indicating axonal growth. BDA labeling however showed no evidence of regeneration of rubrospinal axons in recipients of encapsulated Fb/BDNF, presumably because the amounts of BDNF available from the encapsulated grafts are substantially less than those provided by the much larger numbers of Fb/BDNF grafted in a gelfoam matrix in the presence of immune suppression. These results suggest that plasticity elicited by the BDNF released from the encapsulated cells contributed to reorganization that led to behavioral recovery in these animals and that the behavioral recovery could proceed in the absence of rubrospinal tract regeneration. Alginate encapsulation is therefore a feasible strategy for delivery of therapeutic products produced by non-autologous engineered fibroblasts and provides an environment suitable for recovery of lost function in the injured spinal cord.

Apparent diffusion coefficients in spinal cord transplants and surrounding white matter correlate with degree of axonal dieback after injury in rats.

BACKGROUND AND PURPOSE: Abnormal apparent diffusion coefficient (ADC) values in injured spinal cord white matter and fibroblast transplants have been shown to correspond with qualitative histologic findings of axonal loss or regeneration. We proposed that ADC values would correlate with quantitative axonal tracing in the transected rubrospinal tract (RST). METHODS: Eleven rats received right-sided lateral funiculus lesions at C3-4 (disrupting the RST) and transplantation of fibroblasts that were unmodified or modified to secrete brain-derived neurotrophic factor (BDNF). Behavioral tests measured hindlimb function at 1, 2, 4, 6, 8, 10, and 12 weeks after injury. At 12 weeks after injury, the antegrade axon tracer biotinylated dextran amine was stereotactically injected into the red nucleus to label the injured RST axons. Animals were sacrificed 2 weeks later. Diffusion-weighted MR imaging of the excised, fixed spinal cord specimens was then performed at 9.4 T. RESULTS: In white matter surrounding transplants, ADC values transverse to axons were elevated and ADC values longitudinal to axons were decreased. These ADC values were more abnormal closer to the transplant, and this correlated with decreases in numbers of labeled RST axons. ADC values in BDNF-expressing fibroblast transplants were significantly lower than those in unmodified fibroblast transplants, and these lower values correlated with decreased axonal dieback. Behaviorally, all animals showed partial recovery, but animals with BDNF-expressing fibroblast transplants had slightly improved hindlimb function compared to those with unmodified fibroblast transplants. CONCLUSION: ADC values may be able to evaluate graft function after spinal cord injury by demonstrating the degree of axonal dieback and preservation.

Abnormal spontaneous potentials in distal muscles in animal models of spinal cord injury.

Spontaneous potentials in skeletal muscle distal to human spinal cord injury (SCI) have been reported in the literature. Two animal models of SCI were studied for the presence of similar potentials. Six rats and two cats with surgical transections of the thoracic spinal cord were followed for 4-6 weeks with serial electromyography. As a control for the effects of anesthesia and serial testing, three intact rats were anesthetized and tested weekly for 4 weeks. In rats with spinal cord transection, spontaneous potentials emerged 4-7 days after surgery and persisted for the duration of the study (28-32 days). Spontaneous potentials were absent in controls at all timepoints. In cats, spontaneous potentials were observed 8 days postinjury and gradually diminished, starting at 2 weeks. Spontaneous potentials therefore occur after SCI in animals as well as in humans. The utilization of animal models will facilitate the understanding of alterations that occur distal to spinal cord lesions and affect the function of lower motor neurons, leading to peripheral denervation after SCI.

Towards a definition of recovery of function.

In this review we consider recovery of function after spinal cord injury, and, in particular, recovery improved following intraspinal cellular transplants. Some recovery occurs spontaneously and this can be especially dramatic in neonates, supporting the notion that developing and adult spinal cord respond differently to injury. Recovery can be improved in both neonates and adults by appropriate cellular transplants into the injury site. We describe several functional tests used in animals with spinal lesions and transplants. We compare the effects of transplants of fetal tissue and genetically modified fibroblasts into neonatal and adult injury sites on recovery of motor and sensorimotor function. Fetal tissue transplants support greater recovery and elicit more regeneration in neonates than in adults. Transplants of fibroblasts modified to produce neurotrophic factors however support both recovery and axonal growth even in adults. The contribution of the transplant to recovery is shown by the loss of function that follows a second lesion just rostral to the original lesion/transplant site. The effect of the re-lesion indicates that the recovery is mediated by the presence of the transplant but the way in which transplants act to promote recovery may include a number of mechanisms, including regeneration and sprouting, neuroprotection, and modifications of organization of spared CNS structures.

Neurotrophic effects on dorsal root regeneration into the spinal cord.

Dorsal root ganglion neurons exhibit a robust and generally successful regenerative response following injury of their peripheral processes. Regeneration fails, however, after section of their central processes in the dorsal roots or dorsal columns. Experiments characterizing the attenuated response of these neurons to injury, and the inhibition of regeneration exerted by astrocytes and oligodendrocytes within the dorsal root entry zone and spinal cord, have contributed important insights into the failure of regeneration after injury to the central nervous system (CNS). Interventions that have enhanced the metabolic response of injured dorsal root ganglion neurons, and altered the inhospitable environment, have increased sensory afferent regeneration and recovery. There is reason to expect that these strategies will help to develop clinically applicable treatments of CNS injuries.

Repair of chronic spinal cord injury.

Advances in medical and rehabilitative care now allow the 10-12,000 individuals who suffer spinal cord injuries each year in the United States to lead productive lives of nearly normal life expectancy, so that the numbers of those with chronic injuries will approximate 300,000 at the end of the next decade. This signals an urgent need for new treatments that will improve repair and recovery after longstanding injuries. In the present report we consider the characteristics of the chronically injured spinal cord that make it an even more challenging setting in which to elicit regeneration than the acutely injured spinal cord and review the treatments that have been designed to enhance axon growth. When applied in the first 2 weeks after experimental spinal cord injury, transplants, usually in combination with supplementary neurotrophic factors, and possibly modifications of the inhibitory central nervous system environment, have produced limited long-distance axon regeneration and behavioral recovery. When applied to injuries older than 4 weeks, the same treatments have almost invariably failed to overcome the obstacles posed by the neurons' diminished capacity for regeneration and by the increasing hostility to growth of the terrain at and beyond the injury site. Novel treatments that have stimulated regeneration after acute injuries have not yet been applied to chronic injuries. A therapeutic strategy that combines rehabilitation training and pharmacological modulation of neurotransmitters appears to be a particularly promising approach to increasing recovery after longstanding injury. Identifying patients with no hope of useful recovery in the early days after injury will allow these treatments to be administered as early as possible.

Ex vivo MR determined apparent diffusion coefficients correlate with motor recovery mediated by intraspinal transplants of fibroblasts genetically modified to express BDNF.

The purpose of this study was to determine whether apparent diffusion coefficients (ADCs) in ex vivo spinal cord white matter, calculated from diffusion weighted MR (DWI) images, correlate with axonal growth and behavioral recovery following subtotal hemisection and transplantation of fibroblasts genetically modified to express brain derived neurotrophic factor (BDNF). These genetically modified fibroblasts have been shown to promote axonal growth, diminish retrograde degenerative changes in axotomized Red nucleus neurons, and are associated with behavioral recovery. Since changes in ADC appear to reflect damage to axons and myelin sheaths, which conventional MR techniques do not identify, partial repair mediated by BDNF-secreting fibroblasts should be detected with ADC measures. Accordingly, we transplanted unmodified fibroblasts (Fb-UM) or fibroblasts modified to secrete BDNF (Fb-BDNF) into cervical subtotal hemisection cavities in adult rats. Rats with Fb-BDNF transplants showed significantly greater behavioral recovery over 12 weeks, as measured by tests of forelimb exploration and open field locomotor activity. Lesion sizes and transplant survival did not differ between the two groups, but immunocytochemical examination showed substantial growth of axons into the Fb-BDNF grafts and little growth into the Fb-UM grafts. Fixed spinal cords were imaged in a 9.4-T magnet. ADCs perpendicular (tADC) and parallel (lADC) to the long axis of the cord were measured in the dorsal lateral white matter, rostral and caudal to the transplant. tADC values and anisotropy index (AI = tADC/lADC) were elevated in both transplant types, indicating white matter damage, but were closer to normal in rats with Fb-BDNF, consistent with known neuroprotection and axonal growth elicited by BDNF. Closer to normal tADC and AI values correlated with improved behavioral recovery. These findings suggest that high-resolution imaging with measurement of tADC and lADC can provide a measure of functionally significant repair that may otherwise go undetected with conventional MR techniques.

Patient selection for clinical trials: the reliability of the early spinal cord injury examination.

Patients with incomplete spinal cord injuries can spontaneously recover motor function. Because of this, phase I and II trials of invasive interventions for acute spinal cord injury will likely involve neurologically complete injuries. It is therefore important to reliably identify complete injuries as early as possible. We examined the reliability of the early examination in motor complete spinal cord injuries by retrospectively analyzing the stability of baseline neurological status determined within 2 days of injury in 103 subjects. Baseline neurological status was compared to neurological status at follow-up, preferably within one week (101 of 103 subjects). When available (n = 68), neurological status at 1 year or later was also compared. Overall, 6.2% (5/81) of motor complete, sensory complete (ASIA A) subjects converted to motor complete, sensory incomplete status (ASIA B) between the initial and follow-up assessments; however, none exhibited motor recovery (ASIA C or D). At initial follow-up, 9.3% (4/43) of ASIA A subjects with factors affecting examination reliability were reclassified as ASIA B injuries compared to 2.6% (1/38) of ASIA A subjects without such factors. At year 1 or later, 6.7% (2/30) of ASIA A subjects without factors affecting exam reliability, converted to ASIA B status. None developed volitional motor function below the zone of injury. For subjects with factors affecting exam reliability, 17.4% (4/23) of ASIA A subjects converted to incomplete status and 13.0% (3/23) regained some motor function by one year or later (ASIA C or D). These data suggest that it is possible to identify within 48 h of injury, a subset of patients with a negligible chance for motor recovery who would be suitable candidates for future clinical trials of invasive treatments.