Weight-supported Stepping Research Articles

Exercise therapy guides cortical reorganization after midthoracic spinal contusion to enhance control of lower thoracic muscles, supporting functional recovery

Postural control is critical for locomotion, allowing for gait changes, obstacle avoidance and navigation of rough terrain. A major problem after spinal cord injury (SCI) is regaining the control of balance to prevent falls and further injury. While the circuits for locomotor pattern generation reside in the spinal cord, postural control consists of multiple, complex networks that interact at the spinal, brainstem and cortical levels. After complete SCI, cortical reorganization establishes novel control of trunk musculature that is required for weight-supported stepping. In this study, we examined the impact of exercise therapy on cortical reorganization in the more clinically relevant models of both moderate and severe midthoracic contusion injury in the rat. Results demonstrate that both spontaneous recovery and therapy induced recovery of weight-supported stepping utilize cortical reorganization. Moreover, exercise therapy further improves outcome by enhancing cortical control of lower thoracic muscles enabling improvements in interlimb coordination associated with improved balance that increases weight-supported stepping. The outcome of this study suggest that cortical control of posture is key to functional improvement in locomotion. This information can be used to improve the timing and type of therapy after SCI by considering changes along the entire neural axis.

Unintentional Effects from Housing Enhancement Resulting in Functional Improvement in Spinal Cord-Injured Mice.

It is well established that both positive and negative housing conditions of laboratory animals can affect behavioral, biochemical, and physiological responses. Housing enhancements have been shown to have beneficial effects on locomotor outcomes in rodents with spinal cord injury (SCI). Subsequent to an unplanned housing enhancement of the addition of a balcony to home cages by animal care personnel at a research facility, a retrospective analysis of multiple SCI studies was performed to determine whether outcomes differed before (four studies, N = 28) and after (four studies, N = 23) the addition of the balcony. Locomotor and morphological differences were compared after a mild-moderate T9 spinal cord contusion injury in wild-type mice. Post-injury assessments of locomotor function for 6 weeks included Basso Mouse Scale (BMS) and treadmill kinematic assessments (week 6). Balcony-housed mice showed greater improvements not only in basic locomotor functions (weight-supported stepping, balance) compared to those in standard housing, but also surpassed mice in standard housing without the balcony in higher-order locomotor recovery outcomes, including BMS late-stage recovery measures (paw, tail, and trunk indices). Additionally, balcony-housed mice had overall higher BMS scores, consistently attained more BMS subscores, and had better treadmill track width and stride length compared to those with no balcony. The housing enhancement of a balcony led to unforeseen consequences and unexpected higher recovery outcomes compared to mice in standard housing. This retrospective study highlights the importance of housing conditions in the key outcomes of locomotor recovery after incomplete contusive SCIs in mice.

Combining neuroprotective agents: effect of riluzole and magnesium in a rat model of thoracic spinal cord injury

Background ContextDamage to the spinal cord can result in irreversible impairments or complete loss of motor, sensory, and autonomic functions. Riluzole and magnesium have been widely investigated as neuroprotective agents in animal models of spinal cord injury. As these drugs protect the injured spinal cord through different mechanisms, we aimed to investigate if their neuroprotective efficacy could be cumulative. PurposeThis study aimed to investigate the neuroprotective efficacy of combined administration of riluzole and magnesium chloride in a contusive model of thoracic spinal cord injury. Study DesignAn in vivo experiment was set using female Wistar Han rats that underwent a thoracic spinal cord contusion (T8) using a weight drop method. An hour after injury, animals were randomly distributed to receive (1) saline, (2) riluzole (2.50 mg/kg), (3) magnesium chloride (24.18 mg/kg) in a polyethylene glycol formulation, or (4) a combined treatment (riluzole and magnesium). Subsequent treatments were given in four intraperitoneal injections (spaced 12 hours apart). MethodsThe Basso, Beattie, and Bresnahan locomotor rating scale, an activity box test, and a swimming test were used to evaluate behavioral recovery over a 4-week period. Histologic analysis of the spinal cords was performed to measure the extent and volume of the lesion, axonal preservation, serotonergic and glutamatergic fiber sparing, motor neuron survival, and inflammation. ResultsOur results show that only the riluzole treatment significantly improved behavioral recovery up to 4 weeks after injury when compared with saline controls (6.2±1.8), with animals achieving weight-supported stepping (9.1±1.2). Riluzole also promoted tissue sparing with significant differences achieved from 200 to 600 µm (caudally to the lesion epicenter), and reduced lesion volume, with animals presenting a significantly smaller lesion (3.23±0.26 mm3) when compared with the saline-treated group (4.74±0.80 mm3), representing a 32% decrease in lesion volume. Riluzole treatment induced significant axonal preservation, as well as serotonergic fiber sparing, caudally to the injury epicenter. ConclusionsOur results suggest that the combined treatment, although simultaneously targeting two excitotoxic-related mechanisms, did not further improve behavioral and histologic outcome when compared with riluzole given alone.

A combination therapy of neural and glial restricted precursor cells and chronic quipazine treatment paired with passive cycling promotes quipazine-induced stepping in adult spinalized rats

IntroductionIn order to develop optimal treatments to promote recovery from complete spinal cord injury (SCI), we examined the combination of: (1) a cellular graft of neural and glial restricted precursor (NRP/GRP) cells, (2) passive exercise, and (3) chronic quipazine treatment on behavioral outcomes and compared them with the individual treatment elements. NRP/GRP cells were transplanted at the time of spinalization.MethodsDaily passive exercise began 1 week after injury to give sufficient time for the animals to recover. Chronic quipazine administration began 2 weeks after spinalization to allow for sufficient receptor upregulation permitting the expression of its behavioral effects. Behavioral measures consisted of the Basso, Beattie, and Bresnahan (BBB) locomotor score and percent of weight-supported steps and hops on a treadmill.ResultsRats displayed an increased response to quipazine (BBB ≥ 9) beginning at 8 weeks post-injury in all the animals that received the combination therapy. This increase in BBB score was persistent through the end of the study (12 weeks post-injury).ConclusionUnlike the individual treatment groups which never achieved weight support, the combination therapy animals were able to perform uncoordinated weight-supported stepping without a body weight support system while on a moving treadmill (6.5 m per minute) and were capable of supporting their own weight in stance during open field locomotion testing. No regeneration of descending serotonergic projections into and through the lesion cavity was observed. Furthermore, these results are a testament to the capacity of the lumbar spinal cord, when properly stimulated, to sustain functioning locomotor circuitry following complete SCI.

Role of cortical reorganization on the effect of 5-HT pharmacotherapy for spinal cord injury

Cortical reorganization or expansion of the intact cortical regions into the deafferented cortex after complete spinal transection in neonatally spinalized rats was shown to be essential for increases in weight-supported stepping at adulthood. The novel somatotopic organization identified in these animals can be induced by exercise or spinal transplants that bridge the site of injury. However, the role of cortical reorganization in increased weight-supported (WS) stepping after pharmacotherapy is unknown. For the neonatally spinalized rat model, the 5-HT2C receptor agonist 1-(m-chlorophenyl)-piperazine hydrochloride (mCPP) increases the number of WS steps taken when administered to adult rats spinalized as neonates (mCPP+) though not all animals showed this effect (mCPP−). Since no differences in the behavior of the animals off-drug has been demonstrated, it is unclear why acute administration of 5-HT affects only a subset of animals. One possibility is that differences in cortical organization between mCPP+ and mCPP− may contribute to the differences in the functional effect of mCPP. To test this, we recorded from single neurons in the deafferented hindlimb sensorimotor cortex during passive sensory stimulation of the cutaneous surface of the forepaws and during active sensorimotor stimulation of the forepaws while the animals locomoted on a motorized treadmill. Our results show that neurons recorded from mCPP+ animals increased their responsiveness to both passive and active stimulation off-drug in comparison to neurons from mCPP− animals. These data suggest that differences in the cortical organization of mCPP+ compared to mCPP− animals may be at least partially responsible for the effect of a 5-HT2C receptor agonist on functional outcome.

Tail nerve electrical stimulation combined with scar ablation and neural transplantation promotes locomotor recovery in rats with chronically contused spinal cord

To date, few treatment strategies applying cellular transplantation to the chronically injured spinal cord have yielded significant functional improvement in animal experiments. Here we report that significant improvement of locomotor function was achieved in rats with chronic spinal cord injury (SCI) by the application of combination treatments with tail nerve electrical stimulation (TANES), which can activate the central pattern generator, inducing active weight-supported stepping. Contusion injury (25mm) to spinal cord T10 was produced by using the NYU impactor device in female, adult Long–Evans rats. Rats in 2 of 4 groups with SCI received basic treatments (scar ablation followed by transplantation of lamina propria of olfactory mucosa and cultured olfactory ensheathing cells into the lesion cavity) 6weeks after SCI. Rats both with and without basic treatments were subjected to TANES one week after secondary surgery or 7weeks after SCI. Sixteen weeks after secondary surgery or 22weeks after SCI rats in two groups receiving TANES significantly improved their functional recovery compared with those without TANES, when evaluated with BBB open field rating scale (p<0.01). Among them, however, rats with basic treatments performed better than those without basic treatments. TANES may contribute to the activity-dependent plasticity below the injury level, which is critical for functional recovery. Additionally, TANES may promote axonal regeneration, including those from supraspinal level. Since TANES demonstrated considerable potential for achieving improvement of functional recovery in rat model, it would suggest a new strategy for chronic SCI.

Acetyl-l-carnitine treatment following spinal cord injury improves mitochondrial function correlated with remarkable tissue sparing and functional recovery

We have recently documented that treatment with the alternative biofuel, acetyl-l-carnitine (ALC, 300 mg/kg), as late as 1 h after T10 contusion spinal cord injury (SCI), significantly maintained mitochondrial function 24 h after injury. Here we report that after more severe contusion SCI centered on the L1/L2 segments that are postulated to contain lamina X neurons critical for locomotion (the “central pattern generator”), ALC treatment resulted in significant improvements in acute mitochondrial bioenergetics and long-term hind limb function. Although control-injured rats were only able to achieve slight movements of hind limb joints, ALC-treated animals produced consistent weight-supported plantar steps 1 month after injury. Such landmark behavioral improvements were significantly correlated with increased tissue sparing of both gray and white matter proximal to the injury, as well as preservation of choline acetyltransferase (ChAT)-positive neurons in lamina X rostral to the injury site. These findings signify that functional improvements with ALC treatment are mediated, in part, by preserved locomotor circuitry rostral to upper lumbar contusion SCI. Based on beneficial effects of ALC on mitochondrial bioenergetics after injury, our collective evidence demonstrate that preventing mitochondrial dysfunction acutely “promotes” neuroprotection that may be associated with the milestone recovery of plantar, weight-supported stepping.

Bone Loss following Spinal Cord Injury in a Rat Model

The current study was undertaken to follow the time course of bone loss in the proximal tibia of rats over several weeks following thoracic contusion spinal cord injury (SCI) of varying severity. It was hypothesized that bone loss would be more pronounced in the more severely injured animals, and that hindlimb weight bearing would help prevent bone loss. Twenty-six female Sprague-Dawley rats (200-225 g, 6-7 weeks old) received standard thoracic (T9) injuries at energies of 6.25, 12.5, 25, or 50 g-cm. The rats were scored weekly for hindlimb function during locomotion. At 0, 2 or 3, and 8 weeks, high-resolution micro-CT images of each right tibia were obtained. Mechanical indentation testing was done to measure the compressive strength of the cancellous bone structure. The 6.25 g-cm group showed near normal locomotion, the 12.5 and 25 g-cm groups showed the ability to frequently or occasionally generate weight-supported plantar steps, respectively, and the 50 g-cm group showed only movement without weight-supported plantar stepping. The 6.25, 12.5 and 25 g-cm groups remained at the same level of bone volume fraction (cancBV/TV=0.24±0.07), while the 50 g-cm group experienced severe bone loss (67%), resulting in significantly lower (p<0.05) bone volume fraction (cancBV/TV=0.11±0.05) at 8 weeks. Proximal tibia cancellous bone strength was reduced by approximately 50% in these severely injured rats. Instead of a linear proportionality between injury severity and bone loss, there appears to be a distinct functional threshold, marked by occasional weight-supported stepping, above which bone loss does not occur.

Spontaneous development of full weight-supported stepping after complete spinal cord transection in the neonatal opossum, Monodelphis domestica.

Spinal cord trauma in the adult nervous system usually results in permanent loss of function below the injury level. The immature spinal cord has greater capacity for repair and can develop considerable functionality by adulthood. This study used the marsupial laboratory opossum Monodelphis domestica, which is born at a very early stage of neural development. Complete spinal cord transection was made in the lower-thoracic region of pups at postnatal-day 7 (P7) or P28, and the animals grew to adulthood. Injury at P7 resulted in a dense neuronal tissue bridge that connected the two ends of the cord; retrograde neuronal labelling indicated that supraspinal and propriospinal innervation spanned the injury site. This repair was associated with pronounced behavioural recovery, coordinated gait and an ability to use hindlimbs when swimming. Injury at P28 resulted in a cyst-like cavity encased in scar tissue forming at the injury site. Using retrograde labelling, no labelled brainstem or propriospinal neurons were found above the lesion, indicating that detectable neuronal connectivity had not spanned the injury site. However, these animals could use their hindlimbs to take weight-supporting steps but could not use their hindlimbs when swimming. White matter, demonstrated by Luxol Fast Blue staining, was present in the injury site of P7- but not P28-injured animals. Overall, these studies demonstrated that provided spinal injury occurs early in development, regrowth of supraspinal innervation is possible. This repair appears to lead to improved functional outcomes. At older ages, even without detectable axonal growth spanning the injury site, substantial development of locomotion was still possible. This outcome is discussed in conjunction with preliminary findings of differences in the local propriospinal circuits following spinal cord injury (demonstrated with fluororuby labelling), which may underlie the weight bearing locomotion observed in the apparent absence of axons bridging the lesion site in P28-injured Monodelphis.

Functional role of exercise-induced cortical organization of sensorimotor cortex after spinal transection

Spinal cord transection silences neuronal activity in the deafferented cortex to cutaneous stimulation of the body and untreated animals show no improvement in functional outcome (weight-supported stepping) with time after lesion. However, adult rats spinalized since neonates that receive exercise therapy exhibit greater functional recovery and exhibit more cortical reorganization. This suggests that the change in the somatotopic organization of the cortex may be functionally relevant. To address this issue, we chronically implanted arrays of microwire electrodes into the infragranular layers of the hindlimb somatosensory cortex of adult rats neonatally transected at T8/T9 that received exercise training (spinalized rats) and of normal adult rats. Multiple, single neuron activity was recorded during passive sensory stimulation, when the animals were anesthetized, and during active sensorimotor stimulation during treadmill-induced locomotion when the animal was awake and free to move. Our results demonstrate that cortical neurons recorded from the spinalized rats that received exercise 1) had higher spontaneous firing rates, 2) were more likely to respond to both sensory and sensorimotor stimulations of the forelimbs, and also 3) responded with more spikes per stimulus than those recorded from normal rats, suggesting expansion of the forelimb map into the hindlimb map. During treadmill locomotion the activity of neurons recorded from neonatally spinalized rats was greater during weight-supported steps on the treadmill compared with the neuronal activity during nonweight supported steps. We hypothesize that this increased activity is related to the ability of the animal to take weight supported steps and that, therefore, these changes in cortical organization after spinal cord injury are relevant for functional recovery.

Tail nerve electrical stimulation induces body weight-supported stepping in rats with spinal cord injury

Walking or stepping has been considered the result from the activation of the central pattern generator (CPG). In most patients with spinal cord injury (SCI) the CPG is undamaged. To date, there are no noninvasive approaches for activating the CPG. Recently we developed a noninvasive technique, tail nerve electrical stimulation (TANES), which can induce positive hind limb movement of SCI rats. The purpose of this study is to introduce the novel technique and examine the effect of TANES on CPG activation. A 25 mm contusion injury was produced at spinal cord T10 of female, adult Long-Evans rats by using the NYU impactor device. Rats received TANES (∼40 mA at 4 kHz) 7 weeks after injury. During TANES all injured rats demonstrated active body weight-supported stepping of hind limbs with left–right alternation and occasional front–hind coordination, resulting in significant, temporary increase in BBB scores ( p < 0.01). However, there is no response to TANES from rats with L2 transection, consistent with other reports that the CPG may be located at L1–2. S1 transection negatively implies the key role of TANES in CPG activation. The TANES not only renders paralyzed rats with a technique-induced ability to walk via activating CPG, but also is likely to be used for locomotor training. It has more beneficial effects for physical training over other training paradigms including treadmill training and invasive functional electrical stimulation. Therefore the TANES may have considerable potential for achieving improvement of functional recovery in animal models and a similar method may be suggested for human study.

Exercise Induces Cortical Plasticity after Neonatal Spinal Cord Injury in the Rat

Exercise-induced cortical plasticity is associated with improved functional outcome after brain or nerve injury. Exercise also improves functional outcomes after spinal cord injury, but its effects on cortical plasticity are not known. The goal of this investigation was to study the effect of moderate exercise (treadmill locomotion, 3 min/d, 5 d/week) on the somatotopic organization of forelimb and hindlimb somatosensory cortex (SI) after neonatal thoracic transection. We used adult rats spinalized as neonates because some of these animals develop weight-supported stepping, and, therefore, the relationship between cortical plasticity and stepping could also be examined. Acute, single-neuron mapping was used to determine the percentage of cortical cells responding to cutaneous forelimb stimulation in normal, spinalized, and exercised spinalized rats. Multiple single-neuron recording from arrays of chronically implanted microwires examined the magnitude of response of these cells in normal and exercised spinalized rats. Our results show that exercise not only increased the percentage of responding cells in the hindlimb SI but also increased the magnitude of the response of these cells. This increase in response magnitude was correlated with behavioral outcome measures. In the forelimb SI, neonatal transection reduced the percentage of responding cells to forelimb stimulation, but exercise reversed this loss. This restoration in the percentage of responding cells after exercise was accompanied by an increase in their response magnitude. Therefore, the increase in responsiveness of hindlimb SI to forelimb stimulation after neonatal transection and exercise may be due, in part, to the effect of exercise on the forelimb SI.

MDP (Muramyl Dipeptide) Limits Anatomical Damage and Neurological Dysfunction in Rat with Contusive Spinal Cord Injury (SCI) (101.16)

Abstract MDP is the minimum structural unit responsible for the immunoadjuvancy of bacterial cell wall peptidoglycans. Many properties of MDP manifest potential therapeutic efficacy for SCI. These include stimulation of phagocytosis of immunocompetent cells, thereby facilitating myelin removal with minimal beneficial proinflammatory cytokine alterations,reduced glutamate release,increased intra- and extra-cellular Mg2+, thereby blocking NMDA receptors, andsuppressor cytokine expression, such as IL-10. The current experiment delivered MDP by injection, 6 hours post T8 moderate contusive spinal cord injury in adult Fischer rats. At 2 wk after injury, histological examination revealed reductions in lesion volume associated with deposits of chondroitin sulfate proteoglycan and more myelinated axons. The activated (ED1+) macrophage/microglia count was higher in the more limited injury site of MDP animals. Concurrently, most were Mac1+, indicating active phagocytosis. Furthermore, MDP treated animals displayed fewer ED1+ cells rostral and caudal to the lesion compared with controls. Behaviorally, MDP animals displayed frequent co-ordination with weight-supported steps (BBB, 13.7 ± 1.6) compared with the occasional stepping in controls (BBB, 10 ± 0.6; n=6, p&amp;lt;0.05). Thus, MDP appears to be a novel therapeutic agent to treat acute SCI. Supported by CRF, Hollfelder Foundation, and Miami Project

Role of the 5-HT 2C receptor in improving weight-supported stepping in adult rats spinalized as neonates

Loss of descending serotonergic (5-HT) projections after spinal cord injury (SCI) contributes to motor deficits and upregulation of receptors on partially denervated serotonergic targets in the spinal cord. Serotonergic agonists acting on these upregulated receptors are potential therapeutic agents that could ameliorate motor deficits. However, modification of 5-HT receptors following complete spinal cord injury results in different effects by 5-HT 2C receptor agonists and antagonists. For example, administration of 5-HT 2C receptor agonists suppresses locomotor activity in normal animals, but enhances it in spinalized animals. In addition, administration of 5-HT 2C receptor agonists does not induce activity-dependent hindlimb tremors in normal animals, but does induce them in spinalized animals. We therefore extended our previous work with the 5-HT 2C receptor agonist 1-( m-chlorophenyl)-piperazine hydrochloride (mCPP), which enhances weight-supported stepping when administered to adult rats spinalized as neonates, to identify the optimal dose for improved weight-supported stepping with minimal side effects. In order to determine whether mCPP enhances weight-supported stepping after SCI is through activation of the 5-HT 2C receptor, we performed the following experiments. We determined that stimulation of the 5-HT 1A receptor did not contribute to this improvement in weight-support. We reversed the increase in mCPP-induced weight-supported stepping with SB 206,553, a 5-HT 2C receptor antagonist. We also provide evidence for denervation-induced upregulation of 5-HT 2C receptors in the injured spinal cord. Since mCPP does not have the behavioral toxicity associated with non-selective 5-HT 2 receptor agonists, targeting the 5-HT 2C receptor may have clinical relevance for the treatment of SCI.

Robotic Gait Analysis of Bipedal Treadmill Stepping by Spinal Contused Rats: Characterization of Intrinsic Recovery and Comparison with BBB

There is a critical need to develop objective, quantitative techniques to assess motor function after spinal cord injury. Here, we assess the ability of a recently developed robotic device (the "rat stepper") to characterize locomotor impairment following contusion injury in rats. In particular, we analyzed how the kinematic features of hindlimb movement during bipedal, weight-supported treadmill stepping change following contusion, and whether these changes correlate with the recovery of open field locomotion. Female, Sprague-Dawley rats (n=29, 8 weeks of age) received mid thoracic contusion injuries of differing severities (11 mild, nine moderate, nine severe, and four sham). In a first experiment, 16 of the animals were evaluated weekly for 12 weeks using the robotic stepping device. In a second experiment, 17 of the animals were evaluated every other day for 4 weeks. The contused animals recovered open field locomotion based on the Basso, Beattie, and Bresnahan Scale (BBB) analysis, with most of the recovery occurring by 4 weeks post-injury. Analysis of 14 robotic measures of stepping revealed that several measures improved significantly during the same 4 weeks: swing velocity, step height, step length, hindlimb coordination, and the ability to support body weight. These measures were also significantly correlated with the BBB score. The number of steps taken during testing was not directly related to intrinsic recovery or correlated to the BBB score. These results suggest that it is the quality of weight-supported steps, rather than the quantity, that best reflects locomotor recovery after contusion injury, and that the quality of these steps is determined by the integrity of extensor, flexor, and bilateral coordination pathways. Thus, by measuring only a few weight-supported steps with motion capture, a sensitive, valid measure of locomotor recovery following contusion injury can be obtained across a broad range of impairment levels.

Locomotor performance of the rat after neonatal repairing of spinal cord injuries: quantitative assessment and electromyographic study.

In spinal cord injuries, various attempts have been made to reconstruct neural connections once disrupted. To improve current procedures and develop therapeutic methodologies it appears important to compare these reconstructive attempts via a standardized quantification of any ensuing functional recovery with parallel correlations to any potentially repaired neural connections. We have reported previously a quantitative assessment of neural connections across the graft site of rats whose spinal cord segments were neonatally replaced with embryonic spinal cord segments or a peripheral nerve section under comparable conditions. Using this same experimental model the present study assessed locomotor performance quantitatively using an open field locomotor scale at various postoperative intervals from day 0 to 5 weeks postinjury. To examine hind-forelimb coordination in further detail, electromyography was employed to record simultaneously from all four limbs during locomotion. Half of the rats whose spinal cord segments were repaired by replacement with embryonic homologous structures acquired virtually normal locomotor function, with a delay of five days compared with that of sham-operated rats. Detailed analysis revealed an abnormality in ankle joint movement and the stability of trunk during locomotion. Electromyography revealed that the pattern of locomotion in these rats was similar to controls. Grafted segments joined with the host spinal cord without gliosis at the host-graft interface. The remaining rats with an embryonic tissue graft showed various grades of hind-forelimb coordination. Gliosis and cavity were observed at the host-graft interface. The rats whose spinal cord was repaired by periphral nerve graft lacked hind-forelimb coordination despite the achievement of weight-supported steps. It appears likely that the grade of locomotor performance depends on quantity and quality of reestablished neural connections across the graft.

Axonal Regeneration and Functional Recovery after Complete Spinal Cord Transection in Rats by Delayed Treatment with Transplants and Neurotrophins

Little axonal regeneration occurs after spinal cord injury in adult mammals. Regrowth of mature CNS axons can be induced, however, by altering the intrinsic capacity of the neurons for growth or by providing a permissive environment at the injury site. Fetal spinal cord transplants and neurotrophins were used to influence axonal regeneration in the adult rat after complete spinal cord transection at a midthoracic level. Transplants were placed into the lesion cavity either immediately after transection (acute injury) or after a 2-4 week delay (delayed or chronic transplants), and either vehicle or neurotrophic factors were administered exogenously via an implanted minipump. Host axons grew into the transplant in all groups. Surprisingly, regeneration from supraspinal pathways and recovery of motor function were dramatically increased when transplants and neurotrophins were delayed until 2-4 weeks after transection rather than applied acutely. Axonal growth back into the spinal cord below the lesion and transplants was seen only in the presence of neurotrophic factors. Furthermore, the restoration of anatomical connections across the injury site was associated with recovery of function with animals exhibiting plantar foot placement and weight-supported stepping. These findings suggest that the opportunity for intervention after spinal cord injury may be greater than originally envisioned and that CNS neurons with long-standing injuries can reinitiate growth, leading to improvement in motor function.

The Serotonergic 5-HT2C Agonist m-Chlorophenylpiperazine Increases Weight-Supported Locomotion without Development of Tolerance in Rats with Spinal Transections

The direct serotonergic agonist, m-chlorophenylpiperazine (m-CPP), displays high efficacy at 5-HT2C receptors. Systemic administration of m-CPP increased dramatically the percentage of weight-supported steps made on a treadmill by rats with complete midthoracic spinal transections. The improvement in motor function occurred in rats with grafts of fetal spinal cord into the site of transection (transplant rats) and in spinal rats without grafts (spinal rats). m-CPP produced a therapeutic action with its first administration and after 14 single daily injections. In contrast, the serotonin and norepinephrine reuptake inhibitor, chlorimipramine (CMI), failed to enhance weight support during 21 days of treatment. The results imply that stimulating directly 5-HT2C receptors restores postural support after spinal injury. Thus, 5-HT2C agonists are candidates for treating spinal patients chronically without the development of tolerance.

Direct Agonists for Serotonin Receptors Enhance Locomotor Function in Rats that Received Neural Transplants after Neonatal Spinal Transection

We analyzed whether acute treatment with serotonergic agonists would improve motor function in rats with transected spinal cords (spinal rats) and in rats that received transplants of fetal spinal cord into the transection site (transplant rats). Neonates received midthoracic spinal transections within 48 hr of birth; transplant rats received fetal (embryonic day 14) spinal cord grafts at the time of transection. At 3 weeks, rats began 1-2 months of training in treadmill locomotion. Rats in the transplant group developed better weight-supported stepping than spinal rats. Systemic administration of two directly acting agonists for serotonergic 5-HT(2) receptor subtypes, quipazine and (+/-)-1-[2, 5]-dimethoxy-4-iodophenyl-2-aminopropane), further increased weight-supported stepping in transplant rats. The improvement was dose-dependent and greatest in rats with poor to moderate baseline weight support. In contrast, indirectly acting serotonergic agonists, which block reuptake of 5-HT (sertraline) or release 5-HT and block its reuptake (D-fenfluramine), failed to enhance motor function. Neither direct nor indirect agonists significantly improved locomotion in spinal rats as a group, despite equivalent upregulation of 5-HT(2) receptors in the lumbar ventral horn of lesioned rats with and without transplants. The distribution of immunoreactive serotonergic fibers within and caudal to the transplant did not appear to correspond to restoration of motor function. Our results confirm our previous demonstration that transplants improve motor performance in spinal rats. Additional stimulation with agonists at subtypes of 5-HT receptors produces a beneficial interaction with transplants that further improves motor competence.

Fetal Transplants Alter the Development of Function after Spinal Cord Transection in Newborn Rats

Pieces of fetal spinal tissue were transplanted into the site of complete midthoracic spinal transections in neonatal rat pups (transplant rats). The development of locomotion in these animals was compared with that of unoperated control rats and rats that received spinal transections alone (spinal rats). Reflex, treadmill and overground locomotion, staircase descent, and horizontal ladder crossing for a water reward were tested in control, spinal, and transplant rats from 3 weeks to adulthood. All tests were readily performed by control animals. Most spinal rats were unable to make many linked weight-supported steps on these tasks. Transplant rats were variable in their locomotor capabilities, but a subset of rats were able to demonstrate coordinated and adaptable locomotion on these tasks. Some transplant rats performed better on more challenging tasks, suggesting that motor strategies for these tasks used different information, perhaps from descending systems. Transplanted tissue survived, and in most cases there was immunocytochemical staining of serotonergic fibers passing into and caudal to the transplant, supporting the conclusion that descending systems grew through the transplanted tissue. Integration with the host tissue was often poor, suggesting that nonspecific or trophic effects of the transplant might also contribute to the development of locomotor function. Therefore several mechanisms may contribute to the repair of injured spinal cord provided by transplants that permit the development of useful locomotion.