Andrew R. Blight

Myelination in the Absence of Galactocerebroside and Sulfatide: Normal Structure with Abnormal Function and Regional Instability

The vertebrate nervous system is characterized by ensheathment of axons with myelin, a multilamellar membrane greatly enriched in the galactolipid galactocerebroside (GalC) and its sulfated derivative sulfatide. We have generated mice lacking the enzyme UDP-galactose:ceramide galactosyltransferase (CGT), which is required for GalC synthesis. CGT-deficient mice do not synthesize GalC or sulfatide but surprisingly form myelin containing glucocerebroside, a lipid not previously identified in myelin. Microscopic and morphometric analyses revealed myelin of normal ultrastructural appearance, except for slightly thinner sheaths in the ventral region of the spinal cord. Nevertheless, these mice exhibit severe generalized tremoring and mild ataxia, and electrophysiological analysis showed conduction deficits consistent with reduced insulative capacity of the myelin sheath. Moreover, with age, CGT-deficient mice develop progressive hindlimb paralysis and extensive vacuolation of the ventral region of the spinal cord. These results indicate that GalC and sulfatide play important roles in myelin function and stability.

Extracellular calcium ionic activity in experimental spinal cord contusion.

Abstract Entry of calcium ions (Ca2+) into axons has been hypothesized by several investigators to play a major role in myelopathy of spinal trauma. Although some evidence supports this hypothesis, a rapid and large influx of Ca2+ into cells prior to development of necrotic changes has not been demonstrated in spinal injury. Because large influxes of Ca2+ into cells are manifested by decreases in extracellular Ca2+, we measured extracellular Ca2+ activity in contused cat spinal cords, using ion-selective microelectrodes and relating the Ca2+ changes to local blood flow and evoked potentials. Within 5 min of 400 g·cm contusion, we found decreases of spinal extracellular Ca2+ from normal pre-injury levels (1.0–1.3 mM) to The initial extracellular Ca2+ fall preceded the onset of significant blood flow changes in spinal cord white matter. Thus, the Ca2+ change was related to the trauma and not to subsequent blood flow alterations. Evoked potentials often recovered transiently at 1–2 h after injury, suggesting that some axons were not immediately destroyed by the contusion and their subsequent survival or function was influenced by factors other than the initial Ca2+ derangement. Neither blood flow changes nor action potential conduction recovery depended on specific levels of extracellular Ca2+. In conclusion, our data support the hypothesis of Ca2+-mediated injury of spinal tracts but additional work is required to define the role of Ca2+ in the later phases of spinal injury.

Central axons in injured cat spinal cord recover electrophysiological function following remyelination by Schwann cells

Axonal morphometry of the lesion site was studied at 3 months after standardized weight-drop contusion injury of the thoracic spinal cord in adult cats. From a sample of 25 injured animals, 12 examples were found in which all surviving axons in the dorsal column were remyelinated by Schwann cells, at the level of the lesion. The dorsolateral tracts were also peripherally myelinated in 6 of these cases, and there was no central myelination in complete transverse sections through the lesion in four animals. In these cases, Schwann cell myelination was prevalent for several millimeters on either side of the lesion center. The extent of Schwann cell invasion correlated with the intensity of injury, measured by overall axon loss. Cortical somatosensory evoked potentials (CSEP) were recorded from all animals before and at intervals for 12 weeks after injury. CSEP to hindlimb (tibial nerve) stimulation were lost immediately at injury but some recovery took place during the first month. The extent of CSEP recovery correlated negatively but weakly with overall axon loss. Clear SEP were recorded at 3 months post-injury in 3 of the animals in which the dorsal columns were remyelinated by Schwann cells; in one of these, the dorsolateral funiculi were also peripherally myelinated. In another, oligodendrocyte myelination was absent from the entire transverse section of the lesion site. Thus, abnormal remyelination by cells of the peripheral nervous system, which is known to occur in a variety of central demyelinating conditions, is capable of restoring effective action potential conduction in mammalian spinal cord sensory tracts.

Effect of 4-aminopyridine on axonal conduction-block in chronic spinal cord injury

The spinal cords of 18 anesthetized cats were injured by standardized contusion. The animals were maintained for 4-16 months, then the thoracic spinal cord was isolated in vitro at 25 degrees C. Microelectrode recordings were made from single axons conducting through the lesion in ventral and lateral tracts. On warming the tissue, action potential conduction was found to block at temperatures below 36 degrees C in 29% of 129 axons tested. Of 17 axons in which it was possible to demonstrate a block below physiological temperature, apply 0.1-1 mM 4-AP, wait for 10 min and test conduction again, 7 showed increases in blocking temperature and 4 of these restored to conduction above 37 degrees C. The other 10 fibers showed no improvement in blocking temperature. 4-AP also increased the spontaneous activity of axons. It was concluded that 4-AP may eventually be useful in chronic spinal cord injuries, because it improves safety factor in some axons and increases excitability in others, which may compensate to some extent for the reduction in density of projections through the lesion.

Augmentation by 4-aminopyridine of vestibulospinal free fall responses in chronic spinal-injured cats

This study examines the effect of the potassium channel blocker 4-aminopyridine (4-AP) on free fall responses (FFR) in the hindlimb muscles of chronically spinal injured cats. The thoracic spinal cord of 7 adult female cats was injured by a standardized contusion method. At 3-7 months post-injury the FFR in 6 hindlimb muscles was recorded electromyographically in each animal, under ketamine sedation. The normal short-latency response to a sudden drop was severely attenuated in all injured animals and practically undetectable in 2 cases. Within 15 min following intravenous administration of 1 mg/kg 4-AP, there was profound augmentation of the amplitude of the FFR and a tendency toward normalization of latency in all animals, though the normal amplitude range was not attained. The same 4-AP dose produced a relatively small increase of FFR amplitude in only 2 of 4 normal, uninjured animals tested. The data are consistent with previous observations that low doses of 4-AP restore conduction in some critically demyelinated axons, and provide support for the hypothesis that conduction block in surviving axons is responsible for a proportion of the dysfunction in chronic spinal injury. Augmentation of FFR in injured animals may also result partly from increased transmitter release in both spinal cord and periphery, due to the presynaptic effects of 4-AP.

Delayed application of direct current electric fields in experimental spinal cord injuries

The cutaneus trunci muscle (CTM) reflex of guinea pigs depends on an ascending afferent pathway within the ventrolateral funiculus of the thoracic spinal cord. The expression of this reflex is a phasic contraction of back skin in response to tactile stimulation, which is permanently eliminated by transection of the ventrolateral funiculus. It was shown previously that when a polarized (rostrally negative), weak (300-400 μV/mm) DC electric field is placed across a lateral hemisection of the spinal cord in adult guinea pigs at the time of injury, approximately 13% recover the reflex, while sham-treated animals remain unchanged. In this study, a similar approach was used, except that three months were allowed to pass between the time of hemisection and experimental treatment. No recovery of the CTM reflex was observed in 13 animals with rostrally negative fields, 8 of which were followed for at least 9 months, 5 for 3 months; or in 11 animals with caudally negative applied fields, 8 of which were followed for 9 months and 3 for 3 months.

Axonal Morphometric Correlates of Evoked Potentials in Experimental Spinal Cord Injury

Cortical somatosensory-evoked potentials recorded at a succession of intervals after contusion injury of the cat spinal cord were compared with the numbers of axons surviving in transverse sections of the lesion at 3 months postinjury. In a sample of 25 animals, with injuries varying from ca.87% to 99% loss of myelinated axons in the white matter, there was a significant correlation between chronic axonal survival and the amplitude of the SEP recorded from 30 min to 1 day postinjury. There was little or no correlation between axon numbers and SEPs recorded at 3 months, within this narrow but crucial range of injury intensity. Robust, relatively normal waveforms could be obtained from animals with as few as 2% of the original axon population surviving at the lesion site, and from animals in which all the surviving axons in the dorsal columns were restricted to the outer 100–200 um of tissue. In other cases, repeatable SEPs could not be recorded from animals with >5% survival of axons. SEPs were lost immediately after contusion injury, but partially recovered within the first 4 hours in ca.30% of cases. There was a secondary severe reduction of SEP amplitude within the first day in most examples of early recovery. Most of the final recovery of chronic SEP amplitude occurred within 2–4 weeks.