Faculty Bibliography

Previous attempts to generate myelin in the myelin-deficient rat spinal cord by transplanting mouse glia were not successful. In order to determine whether this result was due to graft rejection or to interspecies mismatch of cellular or molecular components at the axoglial junction, we have repeated the experiment in cyclosporine-treated rats. Our results show that in the immunosuppressed hosts, foetal glial xenografts form an abundance of myelin within the dorsal columns at or near the injection site about two weeks after the operation. In some cases, myelination extends virtually across the entire width of the dorsal columns. Ultrastructurally, the myelin sheaths are normal in all respects, including the presence of the 'radial component'. The lateral edges of the myelin lamellae form typical paranodal axoglial junctions, some displaying periodic 'transverse bands'. We infer that previous mouse to rat xenograft failures reflect host immune response rather than mismatch of heterologous junctional components. We also compared foetal, early post-natal and adult xenografts. Foetal donor cells, containing an abundance of precursors but virtually no mature oligodendrocytes, are more effective than neonatal donor cells in forming myelin, and after adult grafts, we found no myelin formation. Thus, in xenografts, as in allografts, foetal precursor cells are far more suitable than glia from mature donors in generating significant amounts of myelin

Nodal and paranodal regions of myelinated sciatic nerve fibres from diabetic (db/db) mice were examined in freeze fracture replicas. In some fibres, the axolemma was found to display abnormalities in the paranodal region. These include shallow, undifferentiated junctional indentations, thinning of the indentations with widening of the non-junctional grooves between them, particle clusters within the non-junctional grooves, and patches in which axolemmal E-face particles are distributed randomly rather than in the form of linear strings within grooves. Nodal structure, in contrast, is hardly affected. Nodal E-face and P-face particle densities in db/db axons are not significantly different from those in age-matched controls, although we found a few examples in which the E-face density fell slightly below the normal range. Occasional fibres showing evidence of paranodal or segmental demyelination were also seen. The results support paranodal pathology as a potential basis for reduced nerve conduction velocity in diabetic nerves but provide no evidence for significant changes in nodal structure or in nodal Na channel density in sciatic nerve fibres of the db/db mouse

Minced and triturated fragments from the spinal cord of normal rat fetuses (15-18 days gestation) labeled with the fluorescent dye fast blue (FB) were successfully transplanted into juvenile myelin-deficient rat spinal cord under direct observation. Clusters of myelinated fibers were found subsequently in the recipient spinal cord, and, by fluorescence microscopy, clusters of FB-labeled cells were found at corresponding sites. The results indicate that the surgical approach used is suitable for transplantation of tissue fragments into a defined region of juvenile rat spinal cord, that FB can be used to locate the transplanted cells subsequently, and that FB does not interfere with maturation of the donor glia or with myelin formation

Structurally normal myelin sheaths develop in the spinal cord of juvenile myelin-deficient rats (mdr) 11 days after transplantation of normal fetal spinal cord fragments or cultured cells that do not yet express galactocerebroside. Cultures result in more extensive myelin formation, and in both cases the myelin that forms is located primarily at or near the site of transplantation. Myelin formation also occurs after transplantation of postnatal donor tissue, but the extent diminishes with donor age, and none was seen after transplantation of adult donor tissue over the two-week period studied. Injection of killed tissue, tissue derived from mouse donors or an extract of myelin also did not lead to myelin formation. The results imply that myelin formed in the host following transplantation was generated by oligodendrocytes newly differentiated from donor precursor cells rather than by donor oligodendrocytes that were already mature at the time of transplantation or by host oligodendrocytes that took up components of the injected material. We conclude that exogenous fetal glial cell precursors are able to survive, differentiate and form myelin in the environment of the juvenile mdr spinal cord

The time course of the physiological changes accompanying Wallerian degeneration in the frog is markedly prolonged in comparison with that in mammals. Following transection of frog sciatic nerve, stimulation of the distal segment results in muscle contraction of normal amplitude through day 4, after which tension and EMG signals decline rapidly to levels that are undetectable by day 7. The compound action potential in the severed nerve continues for a much longer period of time, however, persisting as long as approximately 6 weeks. The amplitude remains at normal levels for nearly 3 weeks, after which it declines progressively, approaching zero at approximately 6 weeks. Conduction velocity remains normal in both alpha- and beta-fibers for approximately 3 weeks; thereafter, velocity in the alpha-fibers declines to approximately 71% of normal, and beta-fiber conduction is no longer detectable. Contraction could be elicited by direct stimulation of the muscle during the entire 6-week period of the study. The failure of nerve-mediated muscle activity in the face of persistent nerve conduction could be the result of either a defect in the most distal portions of the nerve fibers or a defect in neuromuscular transmission. These results suggest that EMG evaluation may be more sensitive than nerve conduction studies for clinical assessment of axonopathy at early stages

We have identified three examples of female Wistar rats exhibiting the tremor and seizures characteristic of the X-linked myelin deficiency (md) mutation, which is ordinarily seen only in males. Cytogenetic study of two of these animals has shown them to have 41 chromosomes instead of the normal 42. The missing chromosome was identified as an X chromosome by G-banding analysis. These animals thus have an XO genotype comparable to that in Turner's syndrome. Anatomically, one of the animals, which was studied in detail, showed no abnormality of the uterus, and the ovaries, although somewhat smaller than normal, were histologically indistinguishable from those in a normal female rat. No evidence of endocardial fibroelastosis was detected, nor was there any anomaly of the aorta. The myelin deficiency in the central nervous system was comparable to that in hemizygous mutant male rats. XO monosomy in the Wistar rat thus has little effect on phenotype and is more comparable to that in mice than to Turner's syndrome in man. The myelin-deficient rat is useful for studies of X-chromosome monosomy since XO females can readily be identified by the neurological syndrome characteristic of the md mutation

Evidence is reviewed that the paranodal axoglial junction plays important roles in the differentiation and function of myelinated axons. In myelin-deficient axons, ion flux across the axolemma is greater than that in myelinated fibers because a larger proportion of the axolemma is active during continuous, as opposed to saltatory, conduction. In addition, older myelin-deficient rats that have developed spontaneous seizures display small foci of node-like E-face particle accumulations in CNS axons as well as more diffuse regions of increased particle density and number. Assuming that the E-face particles represent sodium channels, such regions could underlie high sodium current density during activity, low threshold for excitation, and increased extracellular potassium accumulation. Depending on the degree of spontaneous channel opening, they could also represent sites of spontaneous generation of activity. The appearance of seizures and their gradual increase in frequency and severity could represent an increase in the number of such regions. In addition, diminution in the dimensions of the extracellular space during maturation would result in increased extracellular resistance, which, together with increasing axonal diameter, would tend to increase the likelihood of ephaptic interaction among neighboring axons as well as the likelihood of extracellular potassium rises to levels that could cause spontaneous activity

We recorded somatosensory evoked potentials SEPs), extracellular K+ ionic activity ([K+]e), and K+ clearance rates in the spinal cords of 14 myelin-deficient mutant rats and 16 normal male littermates at 16-41 days after birth. Tested under pentobarbital anesthesia (25 mg/kg ip) and hypothermic conditions (32-34 degrees C), myelin-deficient rats had longer cortical SEP latencies (67 +/- 20 ms) compared to those in normal siblings (48 +/- 15 ms; P less than 0.05). Mean baseline [K+]e levels were 2.6 +/- 0.5 mM in myelin-deficient rats and 2.6 +/- 0.8 mM in normal siblings. Clearance times of KCl solutions injected into the spinal cord were biphasic and exponential. The mean initial and secondary exponential half-times were 1.0 +/- 0.5 and 2.7 +/- 1.7 min for myelin-deficient rats and 0.8 +/- 0.4 and 3.8 +/- 3.2 min for normal siblings. Repetitive sciatic nerve stimulation (2-20 Hz, 2- to 6-s trains) produced 1-3 mM transient [K+]e rises in thoracic and lumbar cords of myelin-deficient rats. The [K+]e rises were largest in the dorsal spinal cord at 200-500 microns depth. The normal siblings had smaller or no stimulus-induced [K+]e rises. In myelin-deficient rats, injection of 1 mM 4-aminopyridine (4-AP) solution into the thoracic spinal cord completely suppressed the stimulus-induced [K+]e and markedly increased spinal and cortical SEP amplitudes for several hours. In the normal siblings, the 4-AP injections transiently blocked spinal conduction for 20-30 min but thereafter enhanced cortical SEP amplitudes for 2-3 h. We conclude that sciatic nerve stimulation produces spinal cord [K+]e rises in myelin-deficient rat larger than those in the normal siblings, that the [K+]e transients represent increased K+ release rather than impaired K+ clearance, and that the K+ ions come from 4-AP blockable sources

Previous studies of the myelin-deficient rat spinal cord have suggested that astrocytes may play a role in preventing the formation of myelin in this mutant, or causing its breakdown. Comparison of mutant and normal littermate spinal cords shows a marked hypertrophy of astrocytes in the mutant in both gray matter and fiber tract regions. Nevertheless, when normal fetal spinal cord fragments are transplanted into mutant host spinal cord, clusters of normal-looking myelin sheaths develop with no sign of attack by host astrocytes

The myelin-deficient mutant rat develops generalized tonic seizures and dies during the fourth postnatal week. Surgical constriction of the lower thoracic spinal cord, performed either after the seizures have appeared or before, eliminates the seizures, or delays their onset, and prolongs the life of the animals. These observations support the view that the seizures in these animals can be triggered by abnormal activity originating in the myelin-deficient spinal cord and can be blocked by preventing that activity from ascending to higher levels. Similar seizures and 'paroxysmal' phenomena occur in other myelin-deficient conditions including multiple sclerosis