Demyelination and Schwann cell responses adjacent to injury epicenter cavities following chronic human spinal cord injury.

The natural history of post-traumatic demyelination and myelin repair in the human spinal cord is largely unknown and has remained a matter of speculation. A wealth of experimental studies indicate that mild to moderate contusive injuries to the mammalian spinal cord evolve into a cavity with a preserved rim of white matter in which a population of segmentally demyelinated axons persists. It is believed that such injured axons have abnormal conduction properties. Theoretically, such axons might show improved function if myelin repair occurred. Schwann cells can remyelinate axons affected by multiple sclerosis, but little evidence exists that such repair can occur spontaneously following traumatic human SCI. Therefore, it is important to determine if chronic demyelination is present following human spinal cord injury. There are no previous reports that have conclusively demonstrated demyelination in the human spinal cord following traumatic spinal cord injury using immunohistochemical techniques. Immunohistochemical methods were used to study the distribution of peripheral and central myelin proteins as well as axonal neurofilament at the injury epicenter in 13 postmortem chronically injured human spinal cords 1-22 years following injury. Of these seven could be assessed by our methods. We found that some axonal demyelination can be detected even a decade following human SCI and indirect evidence that invading Schwann cells contributed to restoration of myelin sheaths around some spinal axons.

The effect of surgically implanted bullet fragments on the spinal cord in a rabbit model.

BACKGROUND: Whether or not to remove bullets or bullet fragments from the spinal column of a neurologically intact patient has been a subject of continual debate. The controversy is due in part to a lack of information about the long-term effects of bullet fragments on spinal cord tissue. Although many studies have demonstrated the toxic effects of metal fragments on brain tissue, to our knowledge no one has evaluated the effects of the metals contained in commercially available bullets on spinal cord tissue. METHODS: Copper, aluminum, and lead fragments from three commercially available bullet cartridges were implanted in intradural and extradural locations in seventeen New Zealand White rabbits. At an average of 9.8 months, the metal content of specimens of blood, cerebrospinal fluid, and liver were determined. The spinal cords were harvested and examined histologically. RESULTS: There was a significant increase in the copper level of blood from the rabbits with an implanted copper fragment compared with that of the control animals (p = 0.007). Concentrations of copper and lead were not elevated, compared with the control values, in the serum or liver. Histological examination of the spinal cords revealed major destruction of both the axons and the myelin of the dorsal column adjacent to the intradural copper fragments. Intradural fragments of lead caused similar destruction of myelin and axons in the dorsal column, but to a lesser degree. Minimal spinal cord or meningeal histological changes were noted around the aluminum intradural fragments, and no pathological changes were found near any fragments placed in an extradural location. CONCLUSIONS: The results of this study show that certain metals contained in commercially available bullets can cause varying degrees of neural destruction independent of the initial mechanical injury caused by implantation. Of the three metals tested, copper fragments consistently caused a substantial localized area of neural injury within the spinal cord. CLINICAL RELEVANCE: In our study, copper fragments caused local neural toxicity involving as much as 10% of the spinal cord area, suggesting that there may be a scientific basis for removal of copper fragments lodged in the spinal cord, even in the absence of a neurological deficit.

Embryonic cord transplants in peripheral nerve restore skeletal muscle function.

The rapid atrophy of skeletal muscle after denervation severely compromises efforts to restore muscle function. We have transplanted embryonic day 14-15 (E14-E15) ventral spinal cord cells into adult Fischer rat tibial nerve stump to provide neurons for reinnervation. Our aim was to evaluate medial gastrocnemius reinnervation physiologically because this transplant strategy will only be effective if the reinnervated muscle contracts, generates sufficient force to induce joint movement, and is fatigue resistant enough to shorten repeatedly. Twelve weeks posttransplantation, brief duration electrical stimuli applied to the transplants induced medial gastrocnemius contractions that were strong enough to produce ankle movement in 4 of 12 rats (33%). The force of these four "low-threshold" reinnervated muscles and control muscles declined only gradually during five hours of intermittent, supramaximal stimulation and without depression of EMG potential area, which is strong evidence of functional neuromuscular junctions and fatigue resistant muscles. Sectioning of the medial gastrocnemius nerves confirmed that these contractions were innervation dependent. Weakness in low-threshold reinnervated muscles (8% control force) related to incomplete reinnervation, reductions in muscle fiber size, specific tension, and/or the presence of nonfunctional neuromuscular junctions. Muscle reinnervation achieved using this novel transplantation strategy may salvage completely denervated muscle and may provide the potential to evoke limb movement when injury or disease precludes or delays peripheral axon regeneration.

Density dependent regulation of human Schwann cell proliferation.

Cessation of division is prerequisite for Schwann cell differentiation but regulation of this critical function is poorly understood. Heregulin/forskolin-induced growth of human Schwann cells (HSCs) in vitro was found to be strongly regulated by cell density and thus could model some aspects of negative growth-regulation in vivo. To better understand this phenomenon, the production of an autocrine growth-inhibitor and the role of contact-inhibition were investigated. The possible involvement of two membrane proteins, contactinhibin (CI) and peripheral myelin protein 22 (PMP22) in regulating growth was studied. Thymidine-labeling of HSCs on collagen-coated dishes was inhibited at cell densities less than one tenth of the density at maximal growth-inhibition. Medium from high density cultures did not inhibit the thymidine-labeling of HSCs at low density, a result that argues against the production of a soluble inhibitor. The expression of CI and PMP22 in nerve and HSCs, and the effect of a function-blocking antibody to CI on HSC growth, were determined. CI was detected in fresh nerve by western blotting, and could easily be detected by immunocytochemistry in cultured HSCs by five days and for several weeks thereafter. Twenty-four hour treatment with anti-CI antibody did not increase the thymidine-labeling of HSCs at any density but a significant increase in HSC number was observed in cultures treated with anti-CI for 20 days. This increase was not related to decreased cell death. PMP22, unlike other myelin proteins, was not down-regulated after nerve dissociation and by seven days nearly all HSCs were PMP22 positive. These results provide evidence for a contact-mediated mechanism of growth-regulation in HSCs and suggest that CI is involved in this mechanism.

The changes in human spinal sympathetic preganglionic neurons after spinal cord injury.

We have applied conventional histochemical, immunocytochemical and morphometric techniques to study the changes within the human spinal sympathetic preganglionic neurons (SPNs) after spinal cord injury. SPNs are localized within the intermediolateral nucleus (IML) of the lateral horn at the thoraco-lumbar level of the spinal cord and are the major contributors to central cardiovascular control. SPNs in different thoracic segments in the normal spinal cord were similar in soma size. SPNs in the IML were also identified using immunoreactivity to choline acetyltransferase. Soma area of SPNs was 400.7+15 microm2 and 409.9+/-22 microm2 at the upper thoracic (T3) and middle thoracic (T7) segments, respectively. In the spinal cord obtained from a person who survived for 2 weeks following a spinal cord injury at T5, we found a significant decrease in soma area of the SPNs in the segments below the site of injury: soma area of SPNs at T8 was 272.9+/-11 microm2. At T1 the soma area was 418+/-19 microm2. In the spinal cord obtained from a person who survived 23 years after cord injury at T3, the soma area of SPNs above (T1) and below (T7) the site of injury was similar (416.2+/-19 and 425.0+/-20 microm2 respectively). The findings demonstrate that the SPNs in spinal segments caudal to the level of the lesion undergo a significant decrease of their size 2 weeks after spinal cord injury resulting in complete transection of the spinal cord. The impaired cardiovascular control after spinal cord injury may be accounted for, in part, by the described changes of the SPNs. The SPNs in spinal segments caudal to the injury were of normal size in the case studied 23 years after the injury, suggesting that the atrophy observed at 2 weeks is transient. More studies are necessary to establish the precise time course of these morphological changes in the spinal preganglionic neurons.

Long-Term Culture of Lobster Central Ganglia: Expression of Foreign Genes in Identified Neurons.

The ventral nerve cords of lobsters (Homarus americanus) can be cultured in vitro for at least 7 weeks. Over this period, neurons maintain their normal electrophysiological features and continue, among other measures of neuronal health, to synthesize RNA and proteins. One application of this culture system is demonstrated: the manipulation of gene expression in identified neurons. After intracellular injection of complementary RNA (cRNA) encoding green fluorescent protein (GFP), the amount of protein product measured by fluorescent confocal microscopy increases for 4 days and then decreases to background by day 10. Thus, translation of the injected message must have increased for 4 days before declining. Moreover, after injection of cRNA encoding {beta}-galactosidase, the levels of enzyme activity were measured using a fluorogenic substrate, revealing a peak of {beta}-galactosidase activity at 6 to 9 days; this activity was still detectable for at least 10 days after injection. Therefore, either GFP or {beta}-galactosidase can be used as an injectable marker, allowing in vivo quantitation of expression in individual cells over time. We measured long-lasting expression of these proteins after a single injection, suggesting that it may be possible to manipulate the levels of expression of any gene of interest.

Schwann cells genetically modified to secrete human BDNF promote enhanced axonal regrowth across transected adult rat spinal cord.

The infusion of BDNF and NT-3 into Schwann cell (SC) grafts promotes regeneration of brainstem neurones into the grafts placed in adult rat spinal cord transected at T8 (Xu et al., 1995b). Here, we compared normal SCs with SCs genetically modified to secrete human BDNF, grafted as trails 5 mm long in the cord distal to a transection site and also deposited in the transection site, for their ability to stimulate supraspinal axonal regeneration beyond the injury. SCs were infected with the replication-deficient retroviral vector pL(hBDNF)RNL encoding the human preproBDNF cDNA. The amounts of BDNF secreted (as detected by ELISA) were 23 and 5 ng/24 h per 106 cells for infected and normal SCs, respectively. Biological activity of the secreted BDNF was confirmed by retinal ganglion cell bioassay. The adult rat spinal cord was transected at T8. The use of Hoechst prelabelled SCs demonstrated that trails were maintained for a month. In controls, no SCs were grafted. One month after grafting, axons were present in SC trails. More 5-HT-positive and some DbetaH-positive fibres were observed in the infected vs. normal SC trails. When Fast Blue was injected 5 mm below the transection site (at the end of the trail), as many as 135 retrogradely labelled neurones could be found in the brainstem, mostly in the reticular and raphe nuclei (normal SCs, up to 22, mostly in vestibular nuclei). Numerous neurones were labelled in the ventral hypothalamus (normal SCs, 0). Also, following Fast Blue injection, a mean of 138 labelled cells was present in dorsal root ganglia (normal SCs, 46) and spinal cord (39 vs. 32) rostral to the transection. No labelled spinal neurones rostral to the transection were seen when SCs were not transplanted. Thus, the transplantation of SCs secreting increased amounts of BDNF improved the regenerative response across a transection site in the thoracic cord. Moreover, the enhanced regeneration observed with infected SCs may be specific as the largest response was from neurones known to express trkB.

Human sympathetic preganglionic neurons and motoneurons retrogradely labelled with DiI.

The retrograde tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) was used to label sympathetic preganglionic neurons (SPN) and motoneurons (MN) in postmortem human spinal cord. Seven months after microinjection of DiI into the ventral part of spinal thoracic segments T4 and T8, DiI-labelled neurons were identified and analyzed. Cryostat sections of spinal cord were prepared for light microscopy, while vibratome sections were analyzed using confocal microscopy. The majority of retrogradely labelled SPNs were located within the intermediolateral nucleus, with a few labelled dendrites having a mediolateral orientation. SPNs were also located within the nucleus intercalatus, around the central canal and in the lateral funiculus. Cell bodies of retrogradely labelled IML neurons were oval, kite- or spindle-shaped. The soma area of SPNs in T4 was approximately 422.9 +/- 20.9 microm2 with a median diameter of 14 +/- 0.6 microm. MNs in the ventral horn were round or oval in shape and often appeared with a few labelled neurites. The soma area of the MNs in T4 was approximately 842.3 +/- 35.1 microm2, with a median diameter of 18.3 +/- 0.1 microm. The mean values for MN soma area and diameter measurements were significantly greater compared to SPNs. However, no difference was observed between MNs in different segments or between SPNs in the same segments. No retrogradely labelled cells were observed within the dorsal horn. These findings indicate that DiI is a useful method for studying fixed human central nervous system tissue.

High-resolution diffusion-weighted MR of fresh and fixed cat spinal cords: evaluation of diffusion coefficients and anisotropy.

PURPOSE: To use high-resolution diffusion-weighted and calculated apparent diffusion coefficient (ADC) MR imaging to determine whether fixation and storage influence diffusion anisotropy in white matter tracts of cat spinal cord specimens. METHODS: Four cat cord specimens were imaged using a diffusion-weighted spin-echo sequence. Diffusion encoding was applied in the section-select axis (parallel to white matter tracts) and in the read axis (perpendicular to white matter tracts). Five sets of axial diffusion-weighted images were acquired with b values ranging from 0 to 800 s/mm2 and used to obtain calculated ADC images and to determine diffusion coefficients in different regions of the white matter tracts. RESULTS: After cord fixation, a decrease in T2 relaxation and spin density in the white matter caused the signal intensity to appear similar on diffusion-weighted images when the diffusion-probing gradient was applied along both the section-select and read axes. On the calculated ADC images, however, distinct differences in signal intensities were seen in the section-select and read axes. CONCLUSION: Although there is little difference in signal intensity in the white matter tracts on diffusion-weighted images when diffusion encoding is applied in the section-select or read axis in the fixed specimens, calculated ADC images confirm that diffusion anisotropy is maintained. Therefore, calculated ADC images may be helpful in the evaluation of fixed spinal cord specimens.

Influence of IN-1 antibody and acidic FGF-fibrin glue on the response of injured corticospinal tract axons to human Schwann cell grafts.

Two strategies have been shown by others to improve CST regeneration following thoracic spinal cord injury: 1) the administration of a monoclonal antibody, IN-1, raised against a myelin-associated, neurite growth inhibitory protein, and 2) the delivery of acidic fibroblast growth factor (aFGF) in fibrin glue in association with peripheral nerve grafts. Because autologous transplantation of human Schwann cells (SCs) is a potential strategy for CNS repair, we evaluated the ability of these two molecular agents to induce CST regeneration into human SC grafts placed to span a midthoracic spinal cord transection in the adult nude rat, a xenograft tolerant strain. IN-1 or control (HRP) antibodies were delivered to the injury/graft region by encapsulated hybridoma cells ("IN-1 ravioli") or daily infusion of hybridoma culture supernatant; aFGF-fibrin glue was placed in the same region in other animals. Anterograde tracing from the motor cortex using the dextran amine tracers, Fluororuby (FR) and biotinylated dextran amine (BDA), was performed. Thirty-five days after grafting, the CST response was evaluated qualitatively by looking for regenerated CST fibers in or beyond grafts and quantitatively by constructing camera lucida composites to determine the sprouting index (SI), the position of the maximum termination density (MTD) rostral to the GFAP-defined host/graft interface, and the longitudinal spread (LS) of bulbous end terminals. The latter two measures provided information about axonal die-back. In control animals (graft only), the CST did not enter the SC graft and underwent axonal die-back [SI = 1.4 +/- 0.1, MTD = 2.0 +/- 0.2, LS = 1.3 +/- 0.3, (n = 3)]. Results of IN-1 delivery from ravioli did not differ from controls, but injections of IN-1-containing supernatant resulted in a significant degree of sprouting but did not prevent axonal die-back [SI = 1.9 +/- 0.1, MTD = 1.5 +/- 0.2, LS = 1.1 +/- 0.1, (n = 7)] and traced fibers did not enter grafts. Acidic FGF dramatically reduced axonal die-back and caused sprouting [SI = 2.0 +/- 0.1 (n = 5), MTD = 0.5 +/- 0.04 (n = 6), LS = 0.4 +/- 0.1 (n = 6)]. Some traced fibers entered SC grafts and in 2/6 cases entered the distal interface. We conclude that 1) human SC grafts alone do not support the regeneration of injured CST fibers and do not prevent die-back, 2) grafts plus IN-1 antibody-containing supernatant support some sprouting but die-back continues, and 3) grafts plus aFGF-fibrin glue support regeneration of some fibers into the grafts and reduce die-back.

The astroglial response to Wallerian degeneration after spinal cord injury in humans.

We describe the changes exhibited by astrocytes in areas of Wallerian degeneration after spinal cord injury in humans using glial fibrillary acidic protein immunohistochemistry correlated to standard histology at time points ranging from 8 days to 23 years after injury. Astrocytes were slow to react; a slight increase in immunoreactivity was observed at 4 months. Over time they began to lose immunoreactivity in both the somata and the processes as the debris from the degenerative process was cleared. By 1 year after injury the staining intensity had decreased to levels which were lower than in normal areas of the cord. This hypointense staining persisted for at least 23 years after injury. These findings are significantly different from those observed in animal studies and emphasize the need for additional pathological studies of human spinal cord injury.

The ability of human Schwann cell grafts to promote regeneration in the transected nude rat spinal cord.

Advances in the purification and expansion of Schwann cells (SCs) from adult human peripheral nerve, together with biomaterials development, have made the construction of unique grafts with defined properties possible. We have utilized PAN/PVC guidance channels to form solid human SC grafts which can be transplanted either with or without the channel. We studied the ability of grafts placed with and without channels to support regeneration and to influence functional recovery; characteristics of the graft and host/graft interface were also compared. The T9-T10 spinal cord of nude rats was resected and a graft was placed across the gap; methylprednisolone was delivered acutely to decrease secondary injury. Channels minimized the immigration of connective tissue into grafts but contributed to some necrotic tissue loss, especially in the distal spinal cord. Grafts without channels contained more myelinated axons (x = 2129 +/- 785) vs (x = 1442 +/- 514) and were larger in cross-sectional area ( x = 1.53 +/- 0.24 mm2) vs (x = 0.95 +/- 0.86 mm2). The interfaces formed between the host spinal cord and the grafts placed without channels were highly interdigitated and resembled CNS-PNS transition zones; chondroitin sulfate proteoglycans was deposited there. Whereas several neuronal populations including propriospinal, sensory, motoneuronal, and brainstem neurons regenerated into human SC grafts, only propriospinal and sensory neurons were observed to reenter the host spinal cord. Using combinations of anterograde and retrograde tracers, we observed regeneration of propriospinal neurons up to 2.6 mm beyond grafts. We estimate that 1% of the fibers that enter grafts reenter the host spinal cord by 45 days after grafting. Following retrograde tracing from the distal spinal cord, more labeled neurons were unexpectedly found in the region of the dextran amine anterograde tracer injection site where a marked inflammatory reaction had occurred. Animals with bridging grafts obtained modestly higher scores during open field [(x = 8.2 +/- 0.35) vs (x = 6.8 +/- 0.42), P = 0.02] and inclined plane testing (x = 38.6 +/- 0. 542) vs (x = 36.3 +/- 0.53), P = 0.006] than animals with similar grafts in distally capped channels. In summary, this study showed that in the nude rat given methylprednisolone in combination with human SC grafts, some regenerative growth occurred beyond the graft and a modest improvement in function was observed.

Improved method for harvesting human Schwann cells from mature peripheral nerve and expansion in vitro.

The use of cellular prostheses containing large populations of Schwann cells (SC) has been proposed as a future therapeutic approach in the repair of neural tissue. We have sought to define an efficient protocol for the harvest and expansion of human SC from mature human peripheral nerve. We evaluated SC proliferation occurring within fresh explants and studied the relationship between certain parameters (cell yield, purity, and rate of SC proliferation) and the conditions of maintenance of nerve explants prior to dissociation. In addition, we studied SC proliferation after dissociation in a variety of conditions. We observed that SC within explants divide at a low rate during the first 3 weeks following explantation; this proliferation falls to near zero during the fourth week. The cell yield, SC purity, and proliferation rate following dissociation were all increased when nerve explants were exposed to heregulin/ forskolin for 2 weeks prior to dissociation. Electron microscopic analysis showed that heregulin/forskolin exerted trophic effects on SC within explants. Following dissociation, SC growth in heregulin/forskolin-containing medium was more rapid on laminin or collagen than on poly-L-lysine. These results provide new insights into human SC biology and suggest several procedural improvements for harvesting and expanding these cells. The new method we describe shortens our previous procedure by 4-6 weeks and provides a 30-50-fold increase in the number of SC obtained relative to the earlier procedure.

Identification of Gas6 as a growth factor for human Schwann cells.

Schwann cells are one of the principal components of the peripheral nervous system. They play a crucial role in nerve regeneration and can be used clinically in the repair of injured nerves. We have established serum-free, defined culture conditions that rapidly expand adult human Schwann cells without fibroblast growth. We find that Gas6, a ligand for the Axl and Rse/Tyro3 receptor protein tyrosine kinase family, stimulates human Schwann cell growth, increasing both cell number and thymidine incorporation. Gas6 has synergistic effects with the other known human Schwann cell mitogens, heregulin/glial growth factor and forskolin. Addition of Gas6 causes phosphorylation of Axl and Rse/Tyro3 simultaneously and results in ERK-2 activation. A combination of Gas6 with heregulin and forskolin, on a defined background, supports maximal Schwann cell proliferation, while preserving the typical Schwann cell morphology and expression of the Schwann cell markers S-100, glial fibrillary acidic protein, and low-affinity nerve growth factor receptor. Gas6 mRNA is present in both spinal motor neurons and large neurons of the dorsal root ganglia, and neural injury has been reported to upregulate Rse/Axl in the schwann cell. This is the first demonstration of a potentially important biological role for the human Gas6/Rse-Axl system.

Performance of hair breeds and prolific wool breeds of sheep in southern Illinois: wool production and fleece quality.

The objective of this study was to compare weight and quality of fleeces of different F1 ewe types produced from breeds with a broad range of fleece types. Weights of 629 fleeces produced during 1988 through 1991 from F1 ewes that were daughters of Suffolk and Targhee dams and Finnsheep, Combo-6, Booroola Merino, St. Croix, and Barbados sires were recorded. Staple length was measured on the mid-side of each ewe present in 1991. Fleeces shorn in 1991 were sent to a wool marketing organization, and staple length, wool grade, and clean fleece yield were subjectively estimated (n = 220). Mid-side fleece samples were collected from no more than two randomly selected ewes from each subclass (breed of dam-breed of sire-age of ewe) in 1991 (n = 78) and sent to a wool laboratory where fiber diameter, yield, and percentage of colored, med, and kemp fibers were objectively determined. Ewes from Targhee dams produced fleeces with greater weight, greater fiber length, smaller fiber diameter, lower yield, and fewer colored fibers than ewes from Suffolk dams (all differences significant, P < .01). Booroola Merino-sired ewes produced heavier (P < .01) fleeces than did Finnsheep- and Combo-6-sired ewes (4.13 and 3.09 kg, respectively), and in turn, Finnsheep- and Combo-6-sired ewes produced heavier (P < .01) fleeces than did ewes sired by hair breed rams (3.09 and 1.70 kg, respectively). Among hair breed-sired ewes, St. Croixsired ewes produced heavier (P < .01) fleeces than did Barbados-sired ewes (1.88 and 1.52 kg, respectively). Fleeces produced by Booroola Merino-sired ewes had smaller (P < .01) fiber diameter than all sire breed groups except Combo-6-sired ewes, and fleeces produced by St. Croix-sired ewes had greater (P < .01) fiber diameter than all other sire breed groups. Lab scoured yield was greater (P < .01) for fleeces from ewes from hair breed than for fleeces from ewes from wool breed sires (74.2 vs 66.1%). Proportions of undesirable fibers (med, kemp, and colored) were 20 to 600 times greater (P < .01) in fleeces of ewes from hair breed sires than in fleeces of ewes from wool breed sires. In general, F1 ewes from Booroola Merino sires produced the heaviest, highest quality fleeces, and ewes from the hair breed sires of St. Croix and Barbados produced the lightest, lowest quality fleeces. Ewes from Finnsheep and Combo-6 sires produced fleeces that were more similar to the fleeces of ewes from Booroola Merino sires than to the fleeces of ewes from the hair breed sires.

Clinical syndromes associated with disproportionate weakness of the upper versus the lower extremities after cervical spinal cord injury.

Patients with cervical spinal cord injuries who present with weakness or paralysis of the hands and arms with relative preservation of lower extremity strengths are often categorized as having two clinical syndromes, cruciate paralysis and acute central cervical spinal cord injury. The explanation for the pathophysiological findings of the dissociated strength in the upper versus the lower extremities has relied on the assumption that there is a localized injury within a somatotopically organized corticospinal tract. This article summarizes the evidence that there is no somatotopic organization within the corticospinal tract in the medulla or cervical spinal cord in primates. An alternative hypothesis for these two syndromes is presented and is based on evidence that has demonstrated that the corticospinal tract in primates is critical for hand function but not for locomotion. Other prevailing theories are reviewed. Thus, we propose that a syndrome consisting of relatively greater hand and arm weakness compared with leg weakness can occur after an injury to the corticospinal tracts in the medulla or the cervical cord. The proposed mechanism, based on the function of the corticospinal tract, unifies a spectrum of injuries of the lower medulla and cervical spinal cord, which produce similar clinical syndromes (cruciate paralysis and acute central cervical spinal cord injury).

Human Schwann cells in vitro. I. Failure to differentiate and support neuronal health under co-culture conditions that promote full function of rodent cells.

Schwann cells (SCs) play critical roles in regeneration after injury to the peripheral nervous system and can also induce axonal regeneration in the central nervous system. Transplantation of purified SCs into sites of neural injury in rodents has confirmed the remarkable ability of these cells to promote axonal regrowth, suggesting that human application of SC transplantation could be valuable. In this report, we have compared the functional capacities of SCs derived from adult human and rodent nerves by of SCs derived from adult human and rodent nerves by maintaining SCs from these two sources in culture with sensory neurons. We noted that techniques commonly in use for maintaining pure rat SC populations are not sufficient to sustain populations of human SCs free of fibroblasts. In these co-cultures, human SCs express a limited profile of characteristic behaviors and they proliferate more slowly than rat SCs in response to axonal contact. Slow SC proliferation, relative to that of contaminating fibroblasts, leads to a high proportion of fibroblasts in the cultures. After 3 to 4 weeks of co-culture with neurons, human SCs express extracellular matrix molecules, but only partially ensheathe axons, whereas rat SCs differentiate, form basal lamina, and ensheathe or myelinate axons. Co-culture of sensory neurons with human (but not rat) SC preparations (or conditioned medium therefrom) leads to a progressive neuronal atrophy characterized by shrinking neuronal cell bodies and a decrease in the density of the neurite network in the culture dish. As the divergent effects of human and rat SCs on neuronal health were also observed in co-cultures with human sensory neurons, these effects reflect differences between the rat and human-derived SC populations, rather than a species mismatch between SCs and neurons. The marked differences in behavior observed between rat and human SCs derived by the same methods requires further exploration if human-derived SCs are to be considered in the treatment of disease. In a companion article we report experiments that define culture conditions more effective in promoting human SC function in vitro.

Human Schwann cells in vitro. II. Myelination of sensory axons following extensive purification and heregulin-induced expansion.

Co-culture conditions are well established in which Schwann cells (SCs) derived from immature or adult rats proliferate and form myelin in response to contact with sensory axons. In a companion article, we report that populations of adult-derived human Schwann cells (HASCs) fail to function under these co-culture conditions. Furthermore, we report progressive atrophy of neurons in co-cultures containing populations of either human fibroblasts or HASCs (which contain both SCs and fibroblasts). Two factors that might account for the insufficiency of the co-culture system to support HASC differentiation are the failure of many HASCs to proliferate and the influence of contaminating fibroblasts. To minimize fibroblast contamination of neuron-HASC co-cultures, we used fluorescence-activated cell sorting to highly purify HASC populations (to more than 99.8%). To stimulate expansion of the HASC population, a mitogenic mixture of heregulin (HRG beta 1 amino acid residues 177-244; 10 nM), cholera toxin (100 ng/mL), and forskolin (1 microM) was used. When these purified and expanded HASCs were co-cultured with embryo-derived rat sensory neurons, neuronal shrinkage did not occur and after 4 to 6 weeks some myelin segments were seen in living co-cultures. This myelin was positively identified as human by immunostaining with a monoclonal antibody specific to the human peripheral myelin protein P0 (antibody 592). Although this is the first reported observation of myelination by HASCs in tissue culture, it should be noted that myelination occurred more slowly and in much less abundance than in comparable cultures containing adult rat-derived SCs. We anticipate that further refinements of the HASC co-culture system that enhance myelin formation will provide insights into important aspects of human SC biology and provide new opportunities for studies of human peripheral neuropathies.