Neonatal midthoracic spinal cord injury disrupts the development of postural reflexes and hindlimb locomotion. The recovery of rhythmical alternating movements, such as locomotion, is enhanced in injured animals receiving fetal spinal cord transplants. Neonatal cervical spinal cord injury disrupts not only locomotion but also skilled forelimb movement. The aims of this study were to determine the consequences of cervical spinal cord injury on forelimb motor function and to determine whether transplants of fetal spinal cord support normal development of skilled forelimb use after this injury. Three-day-old rats received a cervical spinal cord lesion at C3, with or without a transplant of fetal cervical spinal cord (embryonic day 14); unoperated pups served as controls. Animals were examined daily during the first month of life using a behavioral protocol that assessed reflexes, postural reactions, and forelimb motor skills. They also were trained and tested as adults to assess performance in goal-directed reaching tasks. The onset of postural reflexes was delayed in the lesion-only group, and goal-directed reaching and associated postural adjustments failed to develop. The transplant group developed reflex responses and skilled forelimb activity that resembled normal movement patterns. Transplant animals developed both target reaching and accompanying postural adjustments. Target reaching requires integration of segmental, intersegmental, and supraspinal input to propriospinal and motor neurons over many spinal cord levels. Transplants may support the reestablishment of input onto these neurons, permitting the development of skilled forelimb activity after neonatal cervical spinal cord injury. The neuroanatomical reorganization of descending and propriospinal input was examined in the companion paper (Diener and Bregman, 1998).
Cervical spinal cord injury at birth permanently disrupts forelimb function in goal-directed reaching. Transplants of fetal spinal cord tissue permit the development of skilled forelimb use and associated postural adjustments (Diener and Bregman, 1998, companion article). The aim of this study was to determine whether transplants of fetal spinal cord tissue support the remodeling of supraspinal and segmental pathways that may underlie recovery of postural reflexes and forelimb movements. Although brainstem-spinal and segmental projections to the cervical spinal cord are present at birth, skilled forelimb reaching has not yet developed. Three-day-old rats received a cervical spinal cord overhemisection with or without transplantation of fetal spinal cord tissue (embryonic day 14); unoperated pups served as normal controls. Neuroanatomical tracing techniques were used to examine the organization of CNS pathways that may influence target-directed reaching. In animals with hemisections only, corticospinal, brainstem-spinal, and dorsal root projections within the spinal cord were decreased in number and extent. In contrast, animals receiving hemisections plus transplants exhibited growth of these projections throughout the transplant and over long distances within the host spinal cord caudal to the transplant. Raphespinal axons were apposed to numerous propriospinal neurons in control and transplant animals; these associations were greatly reduced in the lesion-only animals. These observations suggest that after neonatal cervical spinal cord injury, embryonic transplants support axonal growth of CNS pathways and specifically supraspinal input to propriospinal neurons. We suggest that after neonatal spinal injury in the rat, the transplant-mediated reestablishment of supraspinal input to spinal circuitry is the mechanism underlying the development of target-directed reaching and associated postural adjustments.
We are using neural tissue transplantation after spinal cord injury to identify the rules which determine the response of young neurons to injury, to identify the mechanisms underlying anatomical plasticity and recovery of function following spinal cord injury, and to determine the conditions which change during development, leading to the more restricted growth capacity of mature neurons following injury. Spinal cord lesions at birth interrupt different pathways at different relative stages in their development. Neural tissue transplants modify the response of the immature central nervous system neurons to injury. In the current studies, we have used neuroanatomical and behavioral methods to compare the response of the late-developing corticospinal pathway with that of brainstem-spinal pathways which are intermediate in their development and that of the relatively mature dorsal root pathway. We find that both late-developing and regenerating neuronal populations contribute to the transplant-induced anatomical plasticity, and suggest that this anatomical plasticity underlies the transplant-mediated sparing and recovery of function.
We have studied the locomotor development of kittens that received complete low thoracic spinal cord transections and embryonic spinal cord transplants as newborns. Embryonic spinal cord (E21-E26) transplanted into the site of a transection integrated well with the host spinal cord and promoted the development of overground locomotion. Spinalized kittens with transplants were first distinguished from spinalized kittens during the 2nd and 3rd postnatal weeks when kittens with transplants positioned their hindlimbs underneath their bodies which promoted support of the hindquarters. By postnatal Week 6, kittens with transplants exhibited overground locomotion characterized by full weight support and moderate balance control. By 20 weeks of age, as many as 96% of the step cycles showed full weight support and as few as 2% of the step cycles were interrupted by a fall. Most kittens also showed coordination between the forelimbs and the hindlimbs. They differed from normal in the precocious onset of reflex stepping and in the less precise interlimb coordination and more precarious balance during overground locomotion. The overground locomotor performance of kittens with transplant greatly exceeded that of spinal kittens without transplants since few spinalized kittens showed any full-weight-supported step cycles and none showed coordination between the forelimbs and the hindlimbs. In the absence of a transplant, no fibers could grow across the lesion site. In the presence of a transplant, fibers grew across the lesion site and established anatomical connectivity with the host. Host segmental systems identified by the presence of calcitonin gene-related peptide- and substance P-immunoreactive fibers were found throughout the transplants. Descending host systems of supraspinal origin were identified by serotonin- and dopamine β-hydroxylase-immunoreactive fibers throughout the transplants. The growth of supraspinal axons into the transplant, and in one case into the caudal host spinal cord, provided a possible anatomical basis for the development of coordinated overground locomotion.
OBJECTIVES/GOALS: Almost 8 million Americans live with disability caused by stroke. However, recent advances in stroke rehabilitation are costly and lack resemblance to activities of daily living. The goal of this study was to develop a rehabilitation platform to increase stroke patients paretic limb use using inexpensive virtual reality and exoskeleton devices. METHODS/STUDY POPULATION: We conducted a feasibility study with 2 hemiplegic stroke participants. They reached for targets in a virtual reality environment using both hands. They completed 162 reaches divided into 3 blocks. Following baseline, we used an exoskeleton to provide 50% arm weight compensation to the paretic limb and used wrist weights to provide 50% arm weight resistance to the non-paretic limb. We removed the exoskeleton and wrist weights during the retention block. We used electromyography to approximate muscle activity in the biceps brachii. Relative contribution (RC) was calculated as the displacement of the paretic arm divided by the sum of displacements for both arms. Muscle contribution (MC) was calculated as the root mean square of paretic arm muscle activity divided by the sum of activity for both arms. RESULTS/ANTICIPATED RESULTS: During baseline, RC of the impaired limb was 44% and 48%, and MC of the impaired bicep was 43% and 35% in two mild to moderately impaired patients (Fugl-Meyer Upper Extremity scores of 43 and 37, respectively). During loading, RC increased by 5.6% and 1.1% and MC decreased by 8.3% and 11.8%. These data suggest hemiplegic stroke participants alter limb coordination when our device normalizes muscular output asymmetries between limbs. Importantly, these results closely match data from our previous work in 12 healthy controls, where we found a 2% increase in RC is significantly predicted by a 11% decrease in MC. By collecting more data on stroke patients, we will quantify this tradeoff between coordination and muscle activity modulation, allowing us to optimize the exoskeleton mechanics to maximize paretic limb use. DISCUSSION/SIGNIFICANCE: We demonstrate our platform is well tolerated by mild to moderately impaired stroke patients; this feasibility study forms the basis for low cost at-home technologies for stroke rehabilitation. With further development, clinicians can use our platform to fine-tune the level of limb constraint based on the individual needs of the patient.
Functional recovery after spinal cord injury (SCI) may result in part from axon outgrowth and related plasticity through coordinated changes at the molecular level. We employed microarray analysis to identify a subset of genes the expression patterns of which were temporally coregulated and correlated to functional recovery after SCI. Steady-state mRNA levels of this synchronously regulated gene cluster were depressed in both ventral and dorsal horn neurons within 24 h after injury, followed by strong re-induction during the following 2 wk, which paralleled functional recovery. The identified cluster includes neuritin, attractin, microtubule-associated protein 1a, and myelin oligodendrocyte protein genes. Transcriptional and protein regulation of this novel gene cluster was also evaluated in spinal cord tissue and in single neurons and was shown to play a role in axonal plasticity. Finally, in vitro transfection experiments in primary dorsal root ganglion cells showed that cluster members act synergistically to drive neurite outgrowth.
This study was undertaken to determine the locomotor capability of kittens whose spinal cords were transected at birth. The postnatal development of reflex and goal-directed locomotion was examined during the first 5 postnatal months in kittens that received low thoracic spinal cord transections as newborns. Some spinal kittens developed aberrant quadrupedal forms of locomotion. The onset of quadrupedal locomotion, however, was delayed by 2-3 months compared to the normal kitten (42) and deteriorated by 5 months of age. Qualitative and quantitative analyses demonstrated that the quadrupedal locomotion was abnormal. Although some step cycles were characterized by full weight support, the typical hindlimb step cycle of the best performing cat showed inadequate weight support and balance. No spinal cat was able to coordinate the hindlimbs with the forelimbs during overground locomotion on a runaway or during quadrupedal locomotion on a treadmill. Neuroanatomical tracing with WGA-HRP and immunocytochemical techniques showed no axonal regeneration or growth into or across the lesion sites. The aberrant form of quadrupedal locomotion developed without descending input to the caudal spinal cord. The variability in performance among animals suggested that compensatory strategies were important factors in the spinal kitten's achievement of quadrupedal locomotion. Hindlimb weight-supported stepping during quadrupedal locomotion in some animals underscored the capacity of the isolated caudal spinal cord to generate both rhythmical stepping movements and weight support. The maintenance of developmentally immature, but functional, hindlimb postures suggested that the development of the isolated caudal spinal cord was arrested in the absence of descending input.
Neural tissue transplantation techniques have been used to study neuronal-target interactions during development and to study CNS repair and recovery of function after injury. We describe methods of neural tissue transplantation after spinal cord injury in newborn and adult mammals, with an emphasis on preparation of embryonic spinal cord tissue for transplantation and subsequent analysis of host-transplant interactions.
It is often assumed that the response of the immature nervous system to injury is more robust and exhibits greater anatomical reorganization and greater recovery of function than in the adult. In the present experiments the extent of recovery of function after spinal cord injury at birth or at maturity was assessed. We used a series of quantitative tests of motor behavior to measure reflex responses and triggered movements and to examine different components of locomotion. Rats received a midthoracic "over-hemisection" at birth or as adults. The neonatal operates were allowed to mature and the adult operates were allowed to recover. The animals were trained to walk on a treadmill and to cross runways of varying difficulty. The animals were tested for reflex responses and triggered movements, videotaped while crossing the runways, and footprinted while walking on the treadmill. The adult operates had greater deficits in the reflex responses than the neonatal operates. The adult operates lost the contact placing response and had a decreased hopping response in the ipsilateral limb, while these responses were not impaired in the neonatal operates. Although the contact placing response in the neonatal operates was spared, a greater stimulus was necessary to induce the response than in control animals. In contrast, the neonatal operates had greater deficits in locomotion. Footprint analysis revealed that the animals' base of support was significantly greater after the neonatal injury than after the adult injury, and deficits in limb rotation were larger in the neonatal operates than in the adult operates. Both groups crossed the grid with a similar number of steps but the adult operates made significantly more errors with the hindlimb ipsilateral to the lesion than the contralateral one, while the neonatal operates made an equivalent number of errors with both limbs. The neonatal operates took longer to execute the climb test and used a different movement pattern than the adult operates. The neonatal operates had a different locomotor pattern than the adult operates. Despite greater recovery of reflex responses after spinal cord injury at birth, the pattern of locomotion exhibits greater deficits when compared with the same lesion in the adult. Just as the anatomical consequences of injury to the developing nervous system are not uniform, similarly, the behavioral consequences are also not uniform. Spinal cord injury before the mature pattern of locomotion has developed results in a different motor strategy than after injury in the adult. The observation that locomotor patterns and motor strategies differ between newborn and adult operates suggests that the mechanisms underlying the recovery also must differ.
