Karim Fouad

Combining Schwann Cell Bridges and Olfactory-Ensheathing Glia Grafts with Chondroitinase Promotes Locomotor Recovery after Complete Transection of the Spinal Cord

Numerous obstacles to successful regeneration of injured axons in the adult mammalian spinal cord exist. Consequently, a treatment strategy inducing axonal regeneration and significant functional recovery after spinal cord injury has to overcome these obstacles. The current study attempted to address multiple impediments to regeneration by using a combinatory strategy after complete spinal cord transection in adult rats: (1) to reduce inhibitory cues in the glial scar (chondroitinase ABC), (2) to provide a growth-supportive substrate for axonal regeneration [Schwann cells (SCs)], and (3) to enable regenerated axons to exit the bridge to re-enter the spinal cord (olfactory ensheathing glia). The combination of SC bridge, olfactory ensheathing glia, and chondroitinase ABC provided significant benefit compared with grafts only or the untreated group. Significant improvements were observed in the Basso, Beattie, and Bresnahan score and in forelimb/hindlimb coupling. This recovery was accompanied by increased numbers of both myelinated axons in the SC bridge and serotonergic fibers that grew through the bridge and into the caudal spinal cord. Although prominent descending tracts such as the corticospinal and reticulospinal tracts did not successfully regenerate through the bridge, it appeared that other populations of regenerated fibers were the driving force for the observed recovery; there was a significant correlation between numbers of myelinated fibers in the bridge and improved coupling of forelimb and hindlimb as well as open-field locomotion. Our study tests how proven experimental treatments interact in a well-established animal model, thus providing needed direction for the development of future combinatory treatment regimens.

Nogo-A Antibody Improves Regeneration and Locomotion of Spinal Cord-Injured Rats

Spinal cord trauma leads to loss of motor, sensory and autonomic functions below the lesion. Recovery is very restricted, due in part to neurite growth inhibitory myelin proteins, in particular Nogo‐A. Two neutralizing antibodies against Nogo‐A were used to study recovery and axonal regeneration after spinal cord lesions. Three months old Lewis rats were tested in sensory‐motor tasks (open field locomotion, crossing of ladder rungs and narrow beams, the CatWalk® runway, reactions to heat and von Frey hairs). A T‐shaped lesion was made at T8, and an intrathecal catheter delivered highly purified anti‐Nogo‐A monoclonal IgGs or unspecific IgGs for 2 weeks. A better outcome in motor behavior was obtained as early as two weeks after lesion in the animals receiving the Nogo‐A antibodies. Withdrawal responses to heat and mechanical stimuli were not different between the groups. Histology showed enhanced regeneration of corticospinal axons in the anti‐Nogo‐A antibody groups. fMRI revealed significant cortical responses to stimulation of the hindpaw exclusively in anti‐Nogo‐A animals. These results demonstrate that neutralization of the neurite growth inhibitor Nogo‐A by intrathecal antibodies leads to enhanced regeneration and reorganization of the injured CNS, resulting in improved recovery of compromised functions in the absence of dysfunctions. Ann Neurol 2005

Neuronal coordination of arm and leg movements during human locomotion

We aimed to study the neuronal coordination of lower and upper limb muscles. We therefore evaluated the effect of small leg displacements during gait on leg and arm muscle electromyographic (EMG) activity in walking humans. During walking on a split‐belt treadmill (velocity 3.5 km/h), short accelerations or decelerations were randomly applied to the right belt during the mid or end stance phase. Alternatively, trains of electrical stimuli were delivered to the right distal tibial nerve. The EMG activity of the tibialis anterior (TA), gastrocnemius medialis (GM), deltoideus (Delt), triceps (Tric) and biceps brachii (Bic) of both sides was analysed. For comparison, impulses were also applied during standing and sitting. The displacements were followed by specific patterns of right leg and bilateral arm muscle EMG responses. Most arm muscle responses appeared with a short latency (65–80 ms) and were larger in Delt and Tric than in Bic. They were strongest when deceleration impulses were released during mid stance, associated with a right compensatory TA response. A similar response pattern in arm muscles was obtained following tibial nerve stimulation. The arm muscle responses were small or absent when stimuli were applied during standing or sitting. The arm muscle responses correlated more closely with the compensatory TA than with the compensatory GM responses. The amplitude of the responses in most arm muscles correlated closely with the background EMG activity of the respective arm muscle. The observations suggest the existence of a task‐dependent, flexible neuronal coupling between lower and upper limb muscles. The stronger impact of leg flexors in this interlimb coordination indicates that the neuronal control of leg flexor and extensor muscles is differentially interconnected during locomotion. The results are compatible with the assumption that the proximal arm muscle responses are associated with the swinging of the arms during gait, as a residual function of quadrupedal locomotion.

Reorganization of descending motor tracts in the rat spinal cord

Following lesion of the central nervous system (CNS), reinnervation of denervated areas may occur via two distinct processes: regeneration of the lesioned fibres or/and sprouting from adjacent intact fibres into the deafferented zone. Both regeneration and axonal sprouting are very limited in the fully mature CNS of higher vertebrates, but can be enhanced by neutralizing the neurite outgrowth inhibitory protein Nogo‐A. This study takes advantage of the distinct spinal projection pattern of two descending tracts, the corticospinal tract (CST) and the rubrospinal tract (RST), to investigate if re‐innervation of denervated targets can occur by sprouting of anatomically separate, undamaged tracts in the adult rat spinal cord. The CST was transected bilaterally at its entry into the pyramidal decussation. Anatomical studies of the RST in IN‐1 antibody‐treated rats showed a reorganization of the RST projection pattern after neutralization of the myelin associated neurite growth inhibitor Nogo‐A. The terminal arborizations of the rubrospinal fibres, which are normally restricted to the intermediate layers of the spinal cord, invaded the ventral horn but not the dorsal horn of the cervical spinal cord. Moreover, new close appositions were observed, in the ventral horn, onto motoneurons normally receiving CST projections. Red nucleus microstimulation experiments confirmed the reorganization of the RST system. These observations indicate that mature descending motor tracts are capable of significant intraspinal reorganization following lesion and suggests the expression of cues guiding and/or stabilizing newly formed sprouts in the adult, denervated spinal cord.

Spontaneous locomotor recovery in spinal cord injured rats is accompanied by anatomical plasticity of reticulospinal fibers

Although injured axons in mammalian spinal cords do not regenerate, some recovery of locomotor function following incomplete injury can be observed in patients and animal models. Following a lateral hemisection injury of the thoracic spinal cord, rats spontaneously recover weight‐bearing stepping in the hind limb ipsilateral to the injury. The mechanisms behind this recovery are not completely understood. Plasticity in the reticulospinal tract (RtST), the tract responsible for the initiation of walking, has not been studied. In this study, rats received lateral thoracic hemisection of the spinal cord, and RtST projections were compared in two groups of rats, one early in recovery (7 days) and the other at a time point when weight‐bearing stepping was fully regained (42 days). We found that this recovery occurs in parallel with increased numbers of collaterals of spared RtST fibers entering the intermediate lamina below the injury at L2. Sprouting of injured RtST fibers above the lesion was not found. In conclusion, our study suggests that sprouting of spared RtST fibers might be involved in the recovery of locomotion following incomplete spinal cord injury.

Functional switch between motor tracts in the presence of the mAb IN-1 in the adult rat

Fine finger and hand movements in humans, monkeys, and rats are under the direct control of the corticospinal tract (CST). CST lesions lead to severe, long-term deficits of precision movements. We transected completely both CSTs in adult rats and treated the animals for 2 weeks with an antibody that neutralized the central nervous system neurite growth inhibitory protein Nogo-A (mAb IN-1). Anatomical studies of the rubrospinal tracts showed that the number of collaterals innervating the cervical spinal cord doubled in the mAb IN-1- but not in the control antibody-treated animals. Precision movements of the forelimb and fingers were severely impaired in the controls, but almost completely recovered in the mAb IN-1-treated rats. Low threshold microstimulation of the motor cortex induced a rapid forelimb electromyography response that was mediated by the red nucleus in the mAb IN-1 animals but not in the controls. These findings demonstrate an unexpectedly high capacity of the adult central nervous system motor system to sprout and reorganize in a targeted and functionally meaningful way.

Regenerating corticospinal fibers in the Marmoset (Callitrix jacchus) after spinal cord lesion and treatment with the anti‐Nogo‐A antibody IN‐1

Neutralizing the myelin‐associated growth inhibitor Nogo‐A in adult spinal cord‐injured rats can promote regeneration of injured and compensatory sprouting of uninjured axons. Nogo‐A is present in humans, making its neutralization a possible novel treatment option for paraplegic patients. In this study we examined the effects of an extensively used anti‐Nogo‐A antibody (mAb IN‐1) on the regenerative capabilities of lesioned corticospinal tract (CST) axons in a primate, the Marmoset monkey. Unilateral thoracic lesions of the CST were performed in six adult Marmosets, followed by the application of mAb IN‐1 into the cerebrospinal fluid circulation by a graft of hybridoma cells. A unilateral injection of biotin dextran amine into the motor cortex was performed to analyse sprouting and regeneration of the lesioned axons. In the control antibody‐treated animal CST fibers stopped rostral to the lesion site and often showed retraction bulbs. In contrast, in four out of five mAb IN‐1‐treated animals fine labeled neurites had grown into, through and around the lesion site. Thus, this study provides first anatomical evidence that in primates, the neutralization of the myelin‐associated inhibitor Nogo‐A results in increased regenerative sprouting and growth of lesioned spinal cord axons.

Enhancement and Resetting of Locomotor Activity by Muscle Afferentsa

Abstract: The generation of the normal motor pattern for walking in mammals requires feedback from muscle proprioceptors. Two characteristics of the motor pattern particularly dependent on proprioceptive signals are (1) the magnitude of activity in knee and ankle extensor muscles and (2) the duration of extensor bursts during stance. Sensory regulation of these characteristics ensures that the level of activity in extensor muscles during stance is appropriate for the load carried by the leg and that the swing phase is not initiated when a leg is loaded. Many different groups of afferents from flexor and extensor muscles can influence the locomotor pattern. Most attention has focused on the action of group I afferents from ankle extensors. Electrical stimulation of these afferents during extension increases the duration and the magnitude of extensor activity. The prolongation of extensor activity depends in part on excitation of the extensor half‐center by group Ib afferents from Golgi tendon organs. The enhancement of the magnitude of extensor bursts is produced primarily via disynaptic and polysynaptic pathways opened only during locomotion. The involvement of the proprioceptive signals in the generation of locomotor activity means that the gains in reflex pathways must be constantly calibrated according to the biomechanical properties of the locomotor system. Alteration of these properties by weakening ankle extensor muscles has recently been found to produce compensatory changes in proprioceptive influences on the locomotor pattern.

Red nucleus projections to distinct motor neuron pools in the rat spinal cord

Despite being one of the more extensively investigated descending pathways of the rat spinal cord, the termination pattern and postsynaptic targets of the rubrospinal tract (RST) still present some unresolved issues. In addition to locomotor functions, the RST is implicated in the control of limb movements such as reaching and grasping. Although a strong RST projection onto interneurons of intermediate Rexeds laminae V and VI have been described through the entire length of the rat spinal cord, the existence of direct rubro‐motoneuronal connections have not been demonstrated. In the present study, anterograde tracing of the rat RST with biotinylated dextran amine (BDA) was combined with injections of cholera toxin β‐subunit (CTβ) into selected groups of forelimb muscles to analyze in detail the rubral projections to the forelimb areas of the cervical spinal cord. The double‐staining procedure suggested a direct projection from the RST to specific populations of motoneurons. Three populations of forelimb muscles were distinguished, i.e., paw, “distal” muscles; forearm, “intermediate” muscles; and upper arm, “proximal” muscles. A somatotopic distribution of the corresponding motor neuron pools was present in the spinal cord segments C4‐Th1. Rubrospinal axons were seen in close apposition to the distal and intermediate muscle motoneurons, but were consistently absent in the most ventrally situated motor column projecting to proximal muscles. Microstimulation of the red nucleus resulted in electromyographic responses with shorter latency in the distal forelimb muscles than in proximal muscles. These experiments support a specific, preferential role of the RST in distal forelimb muscle control. J. Comp. Neurol. 448:349–359, 2002.

Restoring walking after spinal cord injury.

One of the most obvious deficits following a spinal cord injury is the difficulty in walking, forcing many patients to use wheelchairs for locomotion. Over the past decade considerable effort has been directed at promoting the recovery of walking and to find effective treatments for spinal cord injury. Advances in our knowledge of the neuronal control of walking have led to the development of a promising rehabilitative strategy in patients with partial spinal cord injury, namely treadmill training with partial weight support. The current focus is on developing more efficient training protocols and automating the training to reduce the physical demand for the therapists. Mechanisms underlying training-induced improvements in walking have been revealed to some extent in animal studies. Another strategy for improving the walking in spinal cord injured patients is the use of functional electric stimulation of nerves and muscles to assist stepping movements. This field has advanced significantly over the past decade as a result of developments in computer technology and the miniaturization of electronics. Finally, basic research on animals with damaged spinal cords has focused on enhancing walking and other motor functions by promoting growth and regeneration of damaged axons. Numerous important findings have been reported yielding optimism that techniques for repairing the injured spinal cord will be developed in the near future. However, at present no strategy involving direct treatment of the injured spinal cord has been established for routine use in spinal cord injured patients. It now seems likely that any successful protocol in humans will require a combination of a treatment to promote re-establishing functional connections to neuronal networks in the spinal cord and specialized rehabilitation training to shape the motor patterns generated by these networks for specific behavioral tasks.