Spinal axon regeneration induced by elevation of cyclic AMP.

Myelin inhibitors, including MAG, are major impediments to CNS regeneration. However, CNS axons of DRGs regenerate if the peripheral branch of these neurons is lesioned first. We show that 1 day post-peripheral-lesion, DRG-cAMP levels triple and MAG/myelin no longer inhibit growth, an effect that is PKA dependent. By 1 week post-lesion, DRG-cAMP returns to control, but growth on MAG/myelin improves and is now PKA independent. Inhibiting PKA in vivo blocks the post-lesion growth on MAG/myelin at 1 day and attenuates it at 1 week. Alone, injection of db-cAMP into the DRG mimics completely a conditioning lesion as DRGs grow on MAG/myelin, initially, in a PKA-dependent manner that becomes PKA independent. Importantly, DRG injection of db-cAMP results in extensive regeneration of dorsal column axons lesioned 1 week later. These results may be relevant to developing therapies for spinal cord injury.

Delayed transplantation with exogenous neurotrophin administration enhances plasticity of corticofugal projections after spinal cord injury.

Functional deficits following spinal cord injury (SCI) result from a disruption of corticofugal projections at the lesion site. Not only direct regeneration of the severed axons but also anatomical re-organization of spared corticofugal pathways can reestablish connections between the supraspinal and spinal motor centers. We have previously shown that delayed transplantation of fetal spinal cord tissue and neurotrophin administration by two weeks after SCI supported recovery of forelimb function in adult rats. The current study determined whether the same intervention enhances plasticity of corticofugal fibers at the midbrain and spinal cord level. Anterograde tracing of the left corticorubral fibers revealed that the animals with transplants and neurotrophins (BDNF or NT-3) increased the extent of the traced fibers crossing to the right red nucleus (RN), of which the axons are spared by a right cervical overhemisection lesion. More neurons in the left motor cortex were recruited by the treatment to establish connections with the right RN. The right corticorubral projections also increased the density of midline crossing fibers to the axotomized left RN in response to transplants and neurotrophins. Transplants plus NT-3, but not BDNF, significantly increased the amount of spared corticospinal fibers in the left dorsolateral funiculus at the spinal level both rostral and caudal to the lesion. These results suggest that corticofugal projections retain the capacity until at least two weeks after injury to undergo extensive reorganization along the entire neuraxis in response to transplants and neurotrophins. Targeting anatomical plasticity of corticofugal projections may be a promising strategy to enhance functional recovery following incomplete SCI.

Labeling of dendritic spines with the carbocyanine dye DiI for confocal microscopic imaging in lightly fixed cortical slices.

Visualization of dendritic spines is an important tool for researches on structural synaptic plasticity. Fluorescent labeling of the dendrites and spines followed by confocal microscopy permits imaging a large population of dendritic spines with a higher resolution. We sought to establish an optimal protocol to label neurons in cortical slices with the carbocyanine dye DiI for confocal microscopic imaging of dendritic spines. DiI finely labeled dendrites and spines in slices prefixed (by cardiac perfusion) with 1.5% paraformaldehyde to the similar extent that could be achieved in live preparation. In contrast, fixation with 4% paraformaldehyde severely compromised dye diffusion. Confocal microscopy showed that structural integrity of dendrites and spines was preserved much better in lightly (1.5%) fixed slices than those prepared without fixation. Quantitative measurement revealed that spine density was lower in live slices than that counted in lightly fixed slices, suggesting that fixation is necessary for an adequate evaluation of spine density. The quality of confocal microscopic images obtained from lightly fixed slices allowed us to observe distinctive morphologies such as branched spines and dendritic filopodium, which may be indicative of structural changes at synapses. This method will thus be useful for studying structural synaptic plasticity.

Degradation of chondroitin sulfate proteoglycans potentiates transplant-mediated axonal remodeling and functional recovery after spinal cord injury in adult rats.

Transplantation of growth-permissive cells or tissues was used to bridge a lesion cavity and induce axonal growth in experimental spinal cord injury (SCI). Axonal interactions between host and transplant may be affected by upregulation of inhibitory chondroitin sulfate proteoglycans (CSPGs) following various transplantation strategies. The extent of axonal growth and functional recovery after transplantation of embryonic spinal cord tissue decreases in adult compared to neonatal host. We hypothesized that CSPGs contribute to the decrease in the extent to which transplant supports axonal remodeling and functional recovery. Expression of CSPGs increased after overhemisection SCI in adult rats but not in neonates. Embryonic spinal cord transplant was surrounded by CSPGs deposited in host cord, and the interface between host and transplant seemed to contain a large amount of CSPGs. Intrathecally delivered chondroitinase ABC (C'ase) improved recovery of distal forelimb usage and skilled motor behavior after C4 overhemisection injury and transplantation in adults. This behavioral recovery was accompanied by an increased amount of raphespinal axons growing into the transplant, and raphespinal innervation to the cervical motor region was promoted by C'ase plus transplant. Moreover, C'ase increased the number of transplanted neurons that grew axons to the host cervical enlargement, suggesting that degradation of CSPGs supports remodeling not only of host axons but also axons from transplanted neurons. Our results suggest that CSPGs constitute an inhibitory barrier to prevent axonal interactions between host and transplant in adults, and degradation of the inhibitory barrier can potentiate transplant-mediated axonal remodeling and functional recovery after SCI.

Delayed intervention with transplants and neurotrophic factors supports recovery of forelimb function after cervical spinal cord injury in adult rats.

The adult central nervous system is capable of considerable anatomical reorganization and functional recovery after injury. Functional outcomes, however, vary greatly, depending upon size and location of injury, type and timing of intervention, and type of recovery and plasticity evaluated. The present study was undertaken to assess the recovery of skilled and unskilled forelimb function in adult rats after a C5/C6 spinal cord over-hemisection and delayed intervention with fetal spinal cord transplants and neurotrophins. Recovery of forelimb function was evaluated during both target reaching (a skilled behavior) and vertical exploration (an unskilled behavior). Anatomical tracing and immunohistochemistry were used to assess the growth of descending raphespinal, corticospinal, and rubrospinal fibers at the injury site, tracts that normally confer forelimb function. Delayed intervention with transplants and either brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NT-3) restored skilled left forelimb reaching to pre-injury levels. Animals showed recovery of normal reaching movements rather than compensation with abnormal movements. Transplants and NT-3 also improved right forelimb use during an unskilled vertical exploration, but not skilled right reaching. Intervention with fetal transplant tissue supported the growth of descending serotonergic, corticospinal, and rubrospinal fibers into the transplant at the lesion site. The addition of neurotrophins, however, did not significantly increase axonal growth at the lesion site. These studies suggest that the recovery of skilled and unskilled forelimb use is possible after a large cervical spinal cord injury following delayed intervention with fetal spinal cord and neurotrophins. Plasticity of both spared and axotomized descending pathways likely contributes to the functional recovery observed.

Transplants and neurotrophic factors increase regeneration and recovery of function after spinal cord injury.

Earlier studies suggested that while after spinal cord lesions and transplants at birth, the transplants serve both as a bridge and as a relay to restore supraspinal input caudal to the injury (Bregman, 1994), after injury in the adult the spinal cord transplants serve as a relay, but not as a bridge. We show here, that after complete spinal cord transection in adult rats, delayed spinal cord transplants and exogenous neurotrophic factors, the transplants can also serve as a bridge to restore supraspinal input (Fig. 9). We demonstrate here that when the delivery of transplants and neurotrophins are delayed until 2 weeks after spinal cord transection, the amount of axonal growth and the amount of recovery of function are dramatically increased. Under these conditions, both supraspinal and propriospinal projections to the host spinal cord caudal to the transection are reestablished. The growth of supraspinal axons across the transplant and back into the host spinal cord caudal to the lesion was dependent upon the presence of exogenous neurotrophic support. Without the neurotrophins, only propriospinal axons were able to re-establish connections across the transplant. Studies using peripheral nerve or Schwann cell grafts have shown that some anatomical connectivity can be restored across the injury site, particularly under the influence of neurotrophins (Xu et al., 1995a,b; Cheng et al., 1996; Ye and Houle, 1997). Without neurotrophin treatment, brainstem axons do not enter [figure: see text] the graft (Xu et al., 1995a,b; Cheng et al., 1996; Ye and Houle, 1997). Similarly, cells genetically modified to secrete neurotrophins and transplanted into the spinal cord influence the axonal growth of specific populations of spinally projecting neurons (Tuszynski et al., 1996, 1997; Grill et al., 1997; Blesch and Tuszynski, 1997). Taken together, these studies support a role for neurotrophic factors in the repair of the mature CNS. The regrowth of supraspinal and propriospinal input across the transection site was associated with consistent improvements in hindlimb locomotor function. Animals performed alternating and reciprocal hindlimb stepping with plantar foot contact to the treadmill or stair during ascension. Furthermore, they acquired hindlimb weight support and demonstrated appropriate postural control for balance and equilibrium of all four limbs. After spinal cord injury in the adult, the circuitry underlying rhythmic alternating stepping movements is still present within the spinal cord caudal to the lesion, but is now devoid of supraspinal control. We show here that restoring even relatively small amounts of input allows supraspinal neurons to access the spinal cord circuitry. Removing the re-established supraspinal input after recovery (by retransection rostral to the transplant) abolished the recovery and abolished the serotonergic fibers within the transplant and spinal cord caudal to the transplant. This suggests that at least some of the recovery observed is due to re-establishing supraspinal input across the transplant, rather than a diffuse influence of the transplant on motor recovery. It is unlikely, however, that the greater recovery of function in animals that received delayed transplant and neurotrophins is due solely to the restoration of supraspinal input. Recent work by Ribotta et al. (2000) suggests that segmental plasticity within the spinal cord contributes to weight support and bilateral foot placement after spinal cord transection. This recovery of function occurs after transplants of fetal raphe cells into the adult spinal cord transected at T11. Recovery of function appears to require innervation of the L1-L2 segments with serotonergic fibers, and importantly, animals require external stimulation (tail pinch) to elicit the behavior. In the current study, animals with transection only did not develop stepping overground or on the treadmill without tail pinch, although the transplant and neurotrophin-treated groups did so without external stimuli. Therefore both reorganization of the segmental circuitry and partial restoration of supraspinal input presumably interact to yield the improvements in motor function observed. It is unlikely that the recovery of skilled forelimb movement observed can be mediated solely by reorganization of segmental spinal cord circuitry. We suggest that the restoration of supraspinal input contributes to the recovery observed. It is likely that after CNS injury, reorganization occurs both within the spinal cord and at supraspinal levels, and together contribute to the recovery of automatic and skilled forelimb function and of locomotion. In summary, the therapeutic intervention of tissue transplantation and exogenous neurotrophin support leads to improvements in supraspinal and propriospinal input across the transplant into the host caudal cord and a concomitant improvement in locomotor function. Paradoxically, delaying these interventions for several weeks after a spinal cord transection leads to dramatic improvements in recovery of function and a concomitant restoration of supraspinal input into the host caudal spinal cord. These findings suggest that opportunity for intervention after spinal cord injury may be far greater than originally envisioned, and that CNS neurons with long-standing injuries may be able to re-initiate growth leading to improvement in motor function.

Delayed rehabilitation with task-specific therapies improves forelimb function after a cervical spinal cord injury.

PURPOSE: The effect of activity based therapies on restoring forelimb function in rats was evaluated when initiated one month after a cervical spinal cord injury. METHODS: Adult rats received a unilateral over-hemisection of the spinal cord at C4/5, which interrupts the right side of the spinal cord and the dorsal columns bilaterally, resulting in severe impairments in forelimb function with greater impairment on the right side. One month after injury rats were housed in enriched housing and received daily training in reaching, gridwalk, and CatWalk. A subset of rats received rolipram for 10 days to promote axonal plasticity. Rats were tested weekly for six weeks for reaching, elevated gridwalk, CatWalk, and forelimb use during vertical exploration. RESULTS: Rats exposed to enriched housing and daily training significantly increased the number of left reaches and pellets grasped and eaten, reduced the number of right forelimb errors on the gridwalk, increased right forelimb use during vertical exploration, recovered more normal step cycles, and reduced their hindlimb base of support on the CatWalk compared to rats in standard cages without daily training. CONCLUSIONS: Delayed rehabilitation with enriched housing and daily forelimb training significantly improved skilled, sensorimotor, and automatic forelimb function together after cervical spinal cord injury.

Activity-based therapies to promote forelimb use after a cervical spinal cord injury.

Significant interest exists in strategies for improving forelimb function following spinal cord injury. We investigated the effect of enriched housing combined with skilled training on the recovery of skilled and automatic forelimb function after a cervical spinal cord injury in adult rats. All animals were pretrained in skilled reaching, gridwalk crossing, and overground locomotion. Some received a cervical over-hemisection lesion at C4-5, interrupting the right side of the spinal cord and dorsal columns bilaterally, and were housed in standard housing alone or enriched environments with daily training. A subset of animals received rolipram to promote neuronal plasticity. Animals were tested weekly for 4 weeks to measure reaching, errors on the gridwalk, locomotion, and vertical exploration. Biotinylated dextran amine was injected into the cortex to label the corticospinal tract. Enriched environments/daily training significantly increased the number and success of left reaches compared to standard housing. Animals also made fewer errors on the gridwalk, a measure of coordinated forelimb function. However, there were no significant improvements in forelimb use during vertical exploration or locomotion. Likewise, rolipram did not improve any of the behaviors tested. Both enriched housing and rolipram increased plasticity of the corticospinal tract rostral to the lesion. These studies indicate that skilled training after a cervical spinal cord injury improves recovery of skilled forelimb use (reaching) and coordinated limb function (gridwalk) but does not improve automatic forelimb function (locomotion and vertical exploration). These studies suggest that rehabilitating forelimb function after spinal cord injury will require separate strategies for descending and segmental pathways.

Modulation of dendritic spine remodeling in the motor cortex following spinal cord injury: effects of environmental enrichment and combinatorial treatment with transplants and neurotrophin-3.

Incomplete spinal cord injury (SCI) elicits structural plasticity of the spared motor system, including the motor cortex, which may underlie some of the spontaneous recovery of motor function seen after injury. Promoting structural plasticity may become an important component of future strategies to improve functional outcomes. We have recently observed dynamic changes in the density and morphology of dendritic spines in the motor cortex following SCI. The present study sought to test whether SCI-induced changes in spine density and morphology could be modulated by potential strategies to enhance functional recovery. We examined the effects of enriched environment, transplants, and neurotrophin-3 on the plasticity of synaptic structures in the motor cortex following SCI. Housing rats in an enriched environment increased spine density in the motor cortex regardless of injury. SCI led to a more slender and elongated spine morphology. Enriched housing mitigated the SCI-induced morphological alterations, suggesting that the environmental modification facilitates maturation of synaptic structures. Transplantation of embryonic spinal cord tissue and delivery of neurotrophin-3 at the injury site further increased spine density when combined with enriched housing. This combinatorial treatment completely abolished the injury-induced changes, restoring a preinjury pattern of spine morphology. These results demonstrated that remodeling of dendritic spines in the motor cortex after SCI can be modulated by enriched housing, and the combinatorial treatment with embryonic transplants and neurotrophin-3 can potentiate the effects of enriched housing. We suggest that synaptic remodeling processes in the motor cortex can be targeted for an intervention to enhance functional recovery after SCI.

Remodeling of synaptic structures in the motor cortex following spinal cord injury.

After spinal cord injury (SCI), structural reorganization occurs at multiple levels of the motor system including the motor cortex, and this remodeling may underlie recovery of motor function. The present study determined whether SCI leads to a remodeling of synaptic structures in the motor cortex. Dendritic spines in the rat motor cortex were visualized by confocal microscopy in fixed slices, and their density and morphology were analyzed after an overhemisection injury at C4 level. Spine density decreased at 7 days and partially recovered by 28 days. Spine head diameter significantly increased in a layer-specific manner. SCI led to a higher proportion of longer spines especially at 28 days, resulting in a roughly 10% increase in mean spine length. In addition, filopodium-like long dendritic protrusions were more frequently observed after SCI, suggesting an increase in synaptogenic events. This spine remodeling was accompanied by increased expression of polysialylated neural cell adhesion molecule, which attenuates adhesion between the pre- and postsynaptic membranes, in the motor cortex from as early as 3 days to 2 weeks after injury, suggesting a decrease in synaptic adhesion during the remodeling process. These results demonstrate time-dependent changes in spine density and morphology in the motor cortex following SCI. This synaptic remodeling seems to proceed with a time scale ranging from days to weeks. Elongation of dendritic spines may indicate a more immature and modifiable pattern of synaptic connectivity in the motor cortex being reorganized following SCI.