Increased expression of glutamate decarboxylase (GAD(67)) in feline lumbar spinal cord after complete thoracic spinal cord transection

To determine changes in gamma-aminobutyric acid (GABA) in the spinal cord in response to a complete transection, we examined the cellular and tissue changes of the two forms of GABA synthetic enzyme glutamate decarboxylase (GAD(65) and GAD(67)). In situ hybridization, immunohistochemistry, and Western blot analyses show that spinal cord transection between thoracic segments 12 and 13 results in an increase of GAD(67), but not GAD(65), protein and mRNA in the lumbar spinal cord. This increase occurs mainly in the dorsal horn and persists for at least 12 months. In addition, there was relatively high GAD(67)-immunoreactivity around the central canal, with dorsolateral GAD(67)-immunoreactive fibers extending toward the ependyma and into the central canal in the transected animals. We suggest that an increase in GAD(67) leads to increased GABA production in spinal neurons below the injury site, resulting in altered inhibition and trophic support during posttrauma recovery and adaptation. Increased GABA synthesis around the central canal, in the vicinity of ependymal cells, may represent part of a regenerative process in the mammalian spinal cord, reminiscent of that observed in lower vertebrates.     

Axonal regeneration after spinal cord transection and reconstruction

Following complete transection of the spinal cord, cats were separated into 2 groups to undergo: (i) surgical reconstruction of the disconnected cord using a neuroactive agent mixed into a collagen matrix bridge and omental transposition and (ii) cord transection-only. After 90 days, animals were killed and the brain and spinal cord were removed for immunohistochemistry. Two weeks prior to sacrifice, spinal cord blood flows were measured and the retrograde axonal tracer Fluoro-Gold was injected below the transection site. Gross inspection of the spinal cords at autopsy showed excellent integration and continuity of the collagen matrix bridge with the proximal-distal stumps in the surgical reconstruction group. In the transection-only group, the proximal-distal stumps were connected by a fibrotic, often tapered in the middle, tissue bridge. Results show that omental transposition in the surgical reconstruction group increased spinal cord blood flow by 58% when compared to transection-only animals. Fluoro-Gold was found in mesencephalic and brainstem catecholaminergic and cholinergic neurons known to send axons to the spinal cord. Immunohistochemical staining with antibodies against catecholamine synthesizing enzymes tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DBH) showed that surgical reconstruction treated cat cords but not transection-only, developed dense bundles of dopaminergic and noradrenergic fibers which were present in the collagen matrix bridge and in the distal spinal cord. Extension of these catecholaminergic fibers in surgical reconstruction treated cats showed maximal outgrowth of 90 mm below the transection site when the neuroactive agent 4-aminopyridine was mixed into the collagen matrix. In addition, the synaptogenic marker synaptophysin (SYN) was observed on preganglionic sympathetic neurons in association with dopaminergic- and noradrenergic-containing varicosities distal to the collagen matrix bridge, an indication that neo-synaptic contacts may have been made on these previously denervated neurons. No TH, DBH or SYN was observed below the transection site in transection-only cats. These findings indicate that surgical reconstruction treated cords can develop dense supraspinal fiber outgrowth across a treated collagen matrix bridge fed by an omental blood supply and that these fibers may have made neo-synaptic contacts with appropriate distal spinal cord target tissue.     

Metabolism of rat skeletal muscle after spinal cord transection

We investigated the energy metabolism of the gastrocnemius muscle of the rat after spinal cord transection, using in vivo (31)P magnetic resonance spectroscopy (MRS). Spectra were obtained at rest and during exercise and recovery before, and at different time-points after, spinal cord transection. At rest, the adenosine triphosphate (ATP) level was not altered and the intracellular pH became permanently more alkaline. In electrically stimulated muscle, cord transection caused a greater phosphocreatine depletion than in control animals, and the maximum rate of oxidative ATP synthesis was significantly diminished; at days 30 and 60 after transection, an intracellular acidification was observed at the end of exercise. These effects indicate that, as in humans, spinal cord transection in rats leads to a decrease in mitochondrial oxidative metabolism and probably to an increase in anaerobic metabolism. This experimental model may prove useful for evaluating various approaches to improve muscle function in paraplegia.     

Cell death in Clarke's column after spinal cord transection

The death of embryonic central nervous system (CNS) neurons deprived of a target is well established. In adult rats, similar cell death of corticospinal and rubrospinal motor neurons occurs as a delayed response to spinal cord transection. We document the loss of neurons in Clarke's column, secondary ascending spinocerebellar neurons in adult rats, after complete spinal cord transection at T-9. Twenty-five weeks after spinal cord transection, horseradish peroxidase (HRP) studies showed a dramatic loss of labeled cells in rats with transected spinal cords as compared to matched control rats. Cresyl echt violet-stained sections failed to support the hypothesis that unlabeled cells persist in a shrunken, inactive state; instead we found far fewer identifiable neurons in Clarke's column. Although we saw little gliosis in the area of cell loss, gliosis was evident in the adjacent corticospinal tract which was severed in the original surgical injury. Amputation of the right hind limb resulted in a paradoxical increase in labeled Clarke's column cells on the right. Total cells stained with cresyl echt violet in amputated animals were not different from right to left. The increase in labeled cells on the amputated side may have been caused by an increase in metabolic activity of these deafferentated neurons which resulted in more effective axoplasmic transport of the HRP label.     

Retrograde transport in corticospinal neurons after spinal cord transection

Complete spinal cord transection at T-6/T-7 in rats caused a decrease in the number of surviving corticospinal neurons. Cell death began 5 and 10 weeks after cord injury. The number of surviving cells decreased progressively for at least 25 weeks after injury. Surviving cells were identified by their ability to transport horseradish peroxidase (HRP) retrograde from a T-1/T-2 insertion site to cortical cell somas. Therapy aimed at promoting corticospinal tract regeneration must be started early after spinal cord injury.     

Axonal regrowth after spinal cord transection in adult zebrafish

Using axonal tracers, we characterized the neurons projecting from the brain to the spinal cord as well as the terminal fields of ascending spinal projections in the brain of adult zebrafish with unlesioned or transected spinal cords. Twenty distinct brain nuclei were found to project to the spinal cord. These nuclei were similar to those found in the closely related goldfish, except that additionally the parvocellular preoptic nucleus, the medial octavolateralis nucleus, and the nucleus tangentialis, but not the facial lobe, projected to the spinal cord in zebrafish. Terminal fields of axons, visualized by anterograde tracing, were seen in the telencephalon, the diencephalon, the torus semicircularis, the optic tectum, the eminentia granularis, and throughout the ventral brainstem in unlesioned animals. Following spinal cord transection at a level approximately 3.5 mm caudal to the brainstem/spinal cord transition zone, neurons in most brain nuclei grew axons beyond the transection site into the distal spinal cord to the level of retrograde tracer application within 6 weeks. However, the individually identifiable Mauthner cells were never seen to do so up to 15 weeks after spinal cord transection. Nearly all neurons survived axotomy, and the vast majority of axons that had grown beyond the transection site belonged to previously axotomized neurons as shown by double tracing. Terminal fields were not re-established in the torus semicircularis and the eminentia granularis following spinal cord transection. J Comp Neurol 377:577–595, 1997. © 1997 Wiley-Liss, Inc.     

Effects of sacrocaudal spinal cord transection and transplantation of fetal spinal tissue on withdrawal reflexes of the tail

Reflex responses to electrocutaneous stimulation of the tail were characterized in awake cats, before and after transection of the spinal cord at sacrocaudal levels S3-Ca1. Consistent with effects of spinal transection at higher levels, postoperative cutaneous reflexes were initially depressed, and the tail was flaccid. Recovery ensued over the course of 70-90 days after sacrocaudal transection. Preoperative and chronic postlesion reflexes elicited by electrocutaneous stimulation were graded in amplitude as a function of stimulus intensity. Chronic postlesion testing of electrocutaneous reflexes revealed greater than normal peak amplitudes, peak latencies, total amplitudes (power), and durations, particularly for higher stimulus intensities. Thus, sacrocaudal transection produced effects representative of the spastic syndrome. In contrast, exaggerated reflex responsivity did not develop for a group of cats that received transplants of fetal spinal cord tissue within sacrocaudal transection cavities at the time of injury, in conjunction with long-term immunosuppression by cyclosporine. We conclude that gray matter replacement and potential neuroprotective actions of the grafts and/or immunosuppression prevent development of the spastic syndrome. This argues that the spastic syndrome does not result entirely from interruption of long spinal pathways.     

Motor axonal regeneration after partial and complete spinal cord transection

We subjected rats to either partial midcervical or complete upper thoracic spinal cord transections and examined whether combinatorial treatments support motor axonal regeneration into and beyond the lesion. Subjects received cAMP injections into brainstem reticular motor neurons to stimulate their endogenous growth state, bone marrow stromal cell grafts in lesion sites to provide permissive matrices for axonal growth, and brain-derived neurotrophic factor gradients beyond the lesion to stimulate distal growth of motor axons. Findings were compared with several control groups. Combinatorial treatment generated motor axon regeneration beyond both C5 hemisection and T3 complete transection sites. Yet despite formation of synapses with neurons below the lesion, motor outcomes worsened after partial cervical lesions and spasticity worsened after complete transection. These findings highlight the complexity of spinal cord repair and the need for additional control and shaping of axonal regeneration.     

Loss of neurons in the red nucleus after spinal cord transection

Red nucleus neurons, particularly those of the caudal one-half of the nucleus, die or severely atrophy following complete spinal cord transection at T9. The size of residual horseradish peroxidase-labeled cells was smaller at 10 and 15 weeks, but those survivors which could be labeled at 25 weeks were normal in size. Hematoxylin and eosin-stained sections of the red nucleus at 52 weeks postoperative showed loss of cells from all size groups.     

Postcordotomy spontaneous dysesthesias in macaques: recurrence after spinal cord transection

Monkeys chronically attack the hypoalgesic hindlimb after thoracic contralateral anterolateral cordotomy or hemisection. This compulsive behavior could be induced by innocuous stimulation in the hypoalgesic region, and it also appeared to occur spontaneously. The postcordotomy spontaneous compulsive self-directed behavior was studied in 4 macaques after subsequent upper lumbar crush spinal transection. Despite the paraplegia and bilateral analgesia/anesthesia, this spontaneous abnormal behavior continued to be directed to the same hindlimb as before transection, but not to the opposite hindlimb. Hence, it is concluded that the recurring syndrome originated from the initial contralateral cordotomy. The rationale for the presumption of postcordotomy spontaneous dysesthesias is presented, and the experimental results are offered in refutation of alternative interpretive hypotheses. In conjunction with previous findings, these results lead to the argument that postcordotomy dysesthesias are caused by a neuropathological compensatory response to partial deafferentation of brain somatosensory neurons.     

Catecholamine fiber regeneration across a collagen bioimplant after spinal cord transection

A cell-free bovine derived collagen matrix was used to study potential axonal regeneration in transected rat spinal cord. Rats were initially subjected to a 200 g/cm force acceleration injury at T₁₀ and 10 days later, the spinal cord was totally transected at the injury site. Controls had their spinal cord stumps juxtaposed end-to-end following transection. Experimental rats had 3–4 mm of spinal cord tissue trimmed from the proximo-distal stumps. The semi-fluid collagen material was implanted to bridge the proximo-distal ends and after several hours, the collagen graft polymerized to a firm gel. Rats were observed for 90 days. After 90 days, animals were evaluated using somatosensory evoked potentials, local spinal cord blood flow, and Catecholamine histofluorescence in and around the site of transection. Results suggest that the collagen bioimplant can support the development of anastomotic blood vessels with the cord as well as provide a non-hostile environment to regenerating spinal cord axons.     

Morphometric analysis of effects of spinal cord transection

After spinal cord transection in the rat, endothelial reactivity to horseradish peroxidase (HRP) was quantified at the ultrastructural level. The ratio of the area of labeled endothelial vesicles to area of endothelium (pinocytotic index) was established using morphometric techniques. From 3 h through 7 days the pinocytotic index was significantly elevated as far as 10 mm distal to the transection site. Correlating these findings with a previously established blood vessel classification model, the tracer uptake was identified as both a pinocytotic and vesicular transport mechanism. The latter was associated with an acute response lasting as long as 1 day after transection. Thereafter, the barrier appeared to reestablish its selectivity and only pinocytotic uptake of HRP was apparent.     

Passive exercise and fetal spinal cord transplant both help to restore motoneuronal properties after spinal cord transection in rats

Spinal cord transection influences the properties of motoneurons and muscles below the lesion, but the effects of interventions that conserve muscle mass of the paralyzed limbs on these motoneuronal changes are unknown. We examined the electrophysiological properties of rat lumbar motoneurons following spinal cord transection, and the effects of two interventions shown previously to significantly attenuate the associated hindlimb muscle atrophy. Adult rats receiving a complete thoracic spinal cord transection (T-10) were divided into three groups receiving: (1) no further treatment; (2) passive cycling exercise for 5 days/week; or (3) acute transplantation of fetal spinal cord tissue. Intracellular recording of motoneurons was carried out 4-5 weeks following transection. Transection led to a significant change in the rhythmic firing patterns of motoneurons in response to injected currents, as well as a decrease in the resting membrane potential and spike trigger level. Transplants of fetal tissue and cycling exercise each attenuated these changes, the latter having a stronger effect on maintenance of motoneuron properties, coinciding with the reported maintenance of structural and biochemical features of hindlimb muscles. The mechanisms by which these distinct treatments affect motoneuron properties remain to be uncovered, but these changes in motoneuron excitability are consistent with influences on ion conductances at or near the initial segment. The results may support a therapeutic role for passive limb manipulation and transplant of stem cells in slowing the deleterious responses of motoneurons to spinal cord injury, such that they remain more viable for subsequent alternative strategies.     

Recovery of the hopping response after complete spinal cord transection in the cat

The hopping response reappears in the hind limb of the cat after complete spinal cord transection at postoperative day X̄ = 11.6. This response is hypermetric and slow compared with that in the normal cat and only forward, medial, and lateral hopping can be elicited. Anatomical and physiological changes in the spinal cord which may account for the reappearance of the hopping response are discussed.