Fine structure of nerve fibers and growth cones of isolated sympathetic neurons in culture.

The leading tips of elongating nerve fibers are enlarged into "growth cones" which are seen in tissue culture to continually undergo changes in conformation and to foster numerous transitory slender extensions (filopodia) and/or a veillike ruffling sheet. After explantation of 1-day-old rat superior cervical ganglia (as pieces or as individual neurons), nerve fibers and tips were photographed during growth and through the initial stages of aldehyde fixation and then relocated after embedding in plastic. Electron microscopy of serially sectioned tips revealed the following. The moving parts of the cone, the peripheral flange and filopodia, contained a distinctive apparently filamentous feltwork from which all organelles except membranous structures were excluded; microtubules were notably absent from these areas. The cone interior contained varied forms of agranular endoplasmic reticulum, vacuoles, vesicles, coated vesicles, mitochondria, microtubules, and occasional neurofilaments and polysomes. Dense-cored vesicles and lysosomal structures were also present and appeared to be formed locally, at least in part from reticulum. The possible roles of the various forms of agranular membranous components are discussed and it is suggested that structures involved in both the assembly and degradation of membrane are present in the cone. The content of these growing tips resembles that in sensory neuron growth cones studied by others.

Movements of the Schwann cell nucleus implicate progression of the inner (axon-related) Schwann cell process during myelination.

Although it has been known for several decades that peripheral myelin is formed from an extended, spiraled, and compacted sheet of Schwann cell (SC) plasma membrane, the mechanism by which this unique spiraling is accomplished remains unknown. We have studied the movements of SC nuclei before, during, and subsequent to myelin formation (over periods of 24-72 h) to determine if this nuclear motion (noted in earlier reports) would provide useful insights into the mechanism of myelinogenesis. We used rodent sensory neuron and SC cultures in which initiation of myelinogenesis is relatively synchronized and bright field conditions that allowed resolution of the axon, compact myelin, and position of the SC nucleus. Observed areas were subsequently examined by electron microscopy (EM); eight myelinating SCs with known nuclear movement history were subjected to detailed EM analysis. We observed that, prefatory to myelination, SCs extended along the length of larger axons, apparently competing with adjacent SCs for axonal surface contact. This lengthening preceded the deposition of compact myelin. SC nuclear circumnavigation of the axon was found to attend early myelin sheath formation. This movement was rarely greater than 0.25 turns per 3 h; on the average, more nuclear motion was seen in relation to internodes that formed during observation (0.8 +/- 0.1 turns/24 h) than in relation to those that had begun to form before observation (0.3 +/- 0.1 turns/24 h). Nuclear circumnavigation generally proceeded in one direction, could be in similar or opposite direction in neighboring myelinating SCs on the same axon, and was not proportional to the number of major dense lines within the myelin sheath. A critical finding was that, in all eight cases examined, the overall direction of nuclear movement was the same as that of the inner end of the spiraling SC process, and thus opposite the direction of the outer end of the spiral. We conclude that the correspondence of the direction of nuclear rotation and inner end of the spiraling cytoplasmic lip implicates active progression of the inner lip over the axonal surface to form the membranous spiral of myelin, the nuclear motion resulting from towing by the advancing adaxonal lip. This interpretation fits with finding basal lamina and macular adhering junctions associated with the external lip of SC cytoplasm; these attributes would imply anchorage rather than movement of this region of the SC.

Evidence that contact with connective tissue matrix is required for normal interaction between Schwann cells and nerve fibers.

Explants of fetal rat sensory ganglia, cultured under conditions allowing axon and Schwann cell outgrowth in the absence of fibroblasts, occasionally develop nerve fascicles that are partially suspended in culture medium above the collagen substrate. In these suspended regions, fascicles are abnormal in that Schwann cells are decreased in number, are confined to occasional clusters along the fascicle, provide ensheathment for only a few axons at the fascicle periphery, and do not form myelin. When these fascicles are presented with a substrate of reconstituted rat-tail collagen, Schwann cell numbers increase, ensheathment of small nerve fibers occurs normally, and larger axons are myelinated. We conclude that, for normal development, Schwann cells require contact with extracellular matrix as well as axons. The Schwann cell abnormalities in suspended fascicles are similar to those observed in nerve roots of dystrophic mice.

Cytological studies of organotypic cultures of rat dorsal root ganglia following X-irradiation in vitro. I. Changes in neurons and satellite cells.

Long-term organotypic cultures of rat dorsal root ganglia were exposed to a single 40 kR dose of 184 kvp X-rays and studied in the living and fixed states by light or electron microscopy at 1-14 day intervals thereafter. Within the first 4 days following irradiation, over 30% of the neurons display chromatolytic reactions (eccentric nuclei, peripheral dispersal of Nissl substance, central granular zone) as well as abnormal nucleolar changes and dissociation of ribosomes from endoplasmic reticulum cisternae. Some satellite cells undergo retraction or acute degeneration, leaving only basement membrane to cover the neuron in these areas. 8 days after irradiation, neurons also exhibit (a) areas in which ribosomes are substantially reduced, (b) regions of cytoplasmic sequestration, (c) extensive vacuolization of granular endoplasmic reticulum and Golgi complex, and (d) diversely altered mitochondria (including the presence of ribosome-like particles or association with abnormal glycogen and lipid deposits). Nucleolar components become altered or reoriented and may form abnormal projections and ringlike configurations. Sizeable areas of the neuronal soma are now denuded of satellite cells; underlying these areas, nerve processes are found abnormally invaginated into the neuronal cytoplasm. By the 14th day following irradiation, most neurons display marked degenerative changes including extensive regions of ribosome depletion, sequestration, vacuolization, autolysis, and, in some areas, swirls of filaments, myelin figures, and heterogeneous dense bodies. These observations demonstrate that X-irradiation produces profound cytopathological changes in nervous tissue isolated from the host and that many of these changes resemble the effects of radiation on nervous tissue in vivo.

Cytological studies of organotypic cultures of rat dorsal root ganglia following X-irradiation in vitro. II. Changes in Schwann cells, myelin sheaths, and nerve fibers.

Under suitable conditions rat dorsal root ganglia differentiate and myelinate in culture, providing an organotypic model of the ganglion (8). Mature cultures of this type were irradiated with a 40 kR dose of 184 kvp X-rays and, after daily observation in the living state, were fixed for light and electron microscopy. Within 24 hr after irradiation, numerous Schwann cells investing unmyelinated axons acutely degenerate. The axons thus denuded display little change. Conversely, few ultrastructural changes develop in Schwann cells investing myelinated axons until after the 4th day. During the 4-14 day period, these Schwann cells and their related myelin sheaths undergo progressive deterioration. Associated axons decrease in diameter but are usually maintained. Myelin deterioration begins as a nodal lengthening and then progresses along two different routes. In intact Schwann cells, fragmentation of myelin begins in a pattern reminiscent of Wallerian degeneration, but its slow breakdown thereafter suggests metabolic disturbances in these Schwann cells. The second pattern of myelin deterioration, occurring after complete degeneration of the related Schwann cell, involves unusual configurational changes in the myelin lamellae. Atypical repeating periods are formed by systematic splitting of lamellae at each major dense line with further splitting at the intraperiod line (Type I) or by splitting in the region of every other intraperiod line (Type II); some sheaths display a compact, wavy, inner zone and an abnormally widened lamellar spacing peripherally (Type III). Extensive blebbing of myelin remnants characterizes the final stages of this extracellular myelin degradation. These observations provide the first description of ultrastructural changes produced by ionizing radiation in nerve fascicles in vitro.

Morphological changes in the neuritic growth cone and target neuron during synaptic junction development in culture.

Our object was to characterize the morphological changes occurring in pre- and postsynaptic elements during their initial contact and subsequent maturation into typical synaptic profiles. Neurons from superior cervical ganglia (SCG) of perinatal rats were freed of their supporting cells and established as isolated cells in culture. To these were added explants of embryonic rat thoracic spinal cord to allow interaction between outgrowing cord neurites and the isolated autonomic neurons. Time of initial contact was assessed by light microscopy; at timed intervals thereafter, cultures were fixed for electron microscopy. Upon contact, growth cone filopodia became extensively applied to the SCG neuronal plasmalemma and manifested numerous punctate regions in which the apposing plasma membranes were separated by only 7-10 nm. The Golgi apparatus of the target neuron hypertrophied, and its production of coated vesicles increased. Similar vesicles were seen in continuity with the SCG plasmalemma near the close contact site; their apparent contribution of a region of postsynaptic membrane with undercoating was considered to be the first definitive sign of synapse formation. Tracer work with peroxidase and ferritin confirmed that the traffic of coated vesicles within the neuronal soma is largely from Golgi region to somal surface. Subsequent to the appearance of postsynaptic density, the form and content of the growth cone was altered by the loss of filopodia and the appearance of synaptic vesicles which gradually became clustered opposite the postsynaptic density. As the synapse matured, synaptic vesicles increased in number, cleft width and content increased, presynaptic density appeared, branched membranous reticulum became greatly diminished, and most lysosomal structures disappeared. Coated vesicles continued to be associated with the postsynaptic membrane at all stages of maturation. The incorporation of Golgi-derived vesicles into discrete regions of the cell membrane could provide the mechanism for confining specific characteristics of the neuronal membrane to the synaptic region.

Comparison of nerve cell and nerve cell plus Schwann cell cultures, with particular emphasis on basal lamina and collagen formation.

The availability of cultures of normal cells (NCs) and Schwann cells (SCs) with and without fibroblasts has allowed us to investigate the sources of endoneurial and perineurial constituents of peripheral nerve. NCs cultured alone, devoid of ensheathment but healthy in appearance, lack basal lamina and extracellular fibrils. In contrast, when SCs accompany NCs, basal lamina and extracellular fibrils are consistently visible around SCs in outgrowth areas formed de novo in culture. These fibrils average 18 nm in diameter, exhibit a repeating banding pattern, and are trypsin-resistant and collagenase-sensitive. Collagen synthesis is also indicated by the incorporation of [14C]proline into peptide-bound hydroxy-proline in NC + SC or SC cultures. That the [14C]hydroxyproline polypeptides formed in NC + SC cultures are collagenous was determined in part by pepsin digestion-ammonium sulfate precipitation-polyacrylamide gel electrophoresis techniques; the 14C-polypeptides migrate to the positions of alpha 1 (I), alpha 2, alpha 1 (III), and alpha B chains of type I, type III, and A-B collagens. Also formed are thin, ruthenium red-preserved strands interconnecting basal laminae. SC ensheathment of axons is similar to that found in the animal; one SC is related to a number of unmyelinated axons or a single myelinated axon. This proclivity to ensheathe and myelinate axons indicates that SC function is not lost during the preparative procedures or after lengthy isolation in culture and provides the most reliable means for SC identification. Perineurial ensheathment and macrophages are lacking in NC + SC culture preparations divested of fibroblasts. We conclude that SCs do not form perineurium or the larger diameter collagen fibrils typical of endoneurium but that in combination with neurons they generate biochemically detectable collagens and morphologically visible basal lamina and thin collagenous fibrils.

Specific asparagine-linked oligosaccharides are not required for certain neuron-neuron and neuron-Schwann cell interactions.

To determine whether specific asparagine-linked (N-linked) oligosaccharides present in cell surface glycoproteins are required for cell-cell interactions within the peripheral nervous system, we have used castanospermine to inhibit maturation of N-linked sugars in cell cultures of neurons or neurons plus Schwann cells. Maximally 10-15% of the N-linked oligosaccharides on neuronal proteins have normal structure when cells are cultured in the presence of 250 micrograms/ml castanospermine; the remaining oligosaccharides are present as immature carbohydrate chains not normally found in these glycoproteins. Although cultures were treated for 2 wk with castanospermine, cells always remained viable and appeared healthy. We have analyzed several biological responses of embryonic dorsal root ganglion neurons, with or without added purified populations of Schwann cells, in the presence of castanospermine. We have observed that a normal complement of mature, N-linked sugars are not required for neurite outgrowth, neuron-Schwann cell adhesion, neuron-induced Schwann cell proliferation, or ensheathment of neurites by Schwann cells. Treatment of neuronal cultures with castanospermine increases the propensity of neurites to fasciculate. Extracellular matrix deposition by Schwann cells and myelination of neurons by Schwann cells are greatly diminished in the presence of castanospermine as assayed by electron microscopy and immunocytochemistry, suggesting that specific N-linked oligosaccharides are required for the expression of these cellular functions.

Differentiation of axon-related Schwann cells in vitro. I. Ascorbic acid regulates basal lamina assembly and myelin formation.

Rat Schwann cells cultured with dorsal root ganglion neurons in a serum-free defined medium fail to ensheathe or myelinate axons or assemble basal laminae. Replacement of defined medium with medium that contains human placental serum (HPS) and chick embryo extract (EE) results in both basal lamina and myelin formation. In the present study, the individual effects of HPS and EE on basal lamina assembly and on myelin formation by Schwann cells cultured with neurons have been examined. Some batches of HPS were unable to promote myelin formation in the absence of EE, as assessed by quantitative evaluation of cultures stained with Sudan black; such HPS also failed to promote basal lamina assembly, as assessed by immunofluorescence using antibodies against laminin, type IV collagen, and heparan sulfate proteoglycan. The addition of EE or L-ascorbic acid with such HPS led to the formation of large quantities of myelin and to the assembly of basal laminae. Pretreatment of EE with ascorbic acid oxidase abolished the EE activity, whereas trypsin did not. Other batches of HPS were found to promote both basal lamina and myelin formation in the absence of either EE or ascorbic acid. Ascorbic acid oxidase treatment or dialysis of these batches of HPS abolished their ability to promote Schwann cell differentiation, whereas the subsequent addition of ascorbic acid restored that ability. Ascorbic acid in the absence of serum was relatively ineffective in promoting either basal lamina or myelin formation. Fetal bovine serum was as effective as HPS in allowing ascorbic acid (and several analogs but not other reducing agents) to manifest its ability to promote Schwann cell differentiation. We suggest that ascorbic acid promotes Schwann cell myelin formation by enabling the Schwann cell to assemble a basal lamina, which is required for complete differentiation.

A light and electron microscope study of long-term organized cultures of rat dorsal root ganglia.

Dorsal root ganglia from fetal rats were explanted on collagen-coated coverslips and carried in Maximow double-coverslip assemblies for periods up to 3 months. These cultured ganglia were studied in the living state, in stained whole mounts, and in sections after OsO(4) fixation and Epon embedment. From the central cluster of nerve cell bodies, neurites emerge to form a rich network of fascicles which often reach the edge of the carrying coverslip. The neurons resemble their in vivo counterparts in nuclear and cytoplasmic content and organization; e.g., they appear as "light" or "dark" cells, depending on the amount of cytoplasmic neurofilaments. Satellite cells form a complete investment around the neuronal soma and are themselves everywhere covered by basement membrane. The neuron-satellite cell boundary is complicated by spinelike processes arising from the neuronal soma. Neuron size, myelinated fiber diameter, and internode length in the cultures do not reach the larger of the values known for ganglion and peripheral nerve in situ (30). Unmyelinated and myelinated nerve fibers and associated Schwann cells and endoneurial and perineurial components are organized into typical fascicles. The relationship of the Schwann cell and its single myelinated fiber or numerous unmyelinated fibers and the properties of myelin, such as lamellar spacing, mesaxons, Schmidt-Lanterman clefts, nodes of Ranvier, and protuberances, mimic the in vivo pattern. It is concluded that cultivation of fetal rat dorsal root ganglia by this technique fosters maturation and long-term maintenance of all the elements that comprise this cellular community in vivo (except vascular components) and, furthermore, allows these various components to relate faithfully to one another to produce an organotypic model of sensory ganglion tissue.

Perineurium originates from fibroblasts: demonstration in vitro with a retroviral marker.

A cellular sheath, the perineurium, forms a protective barrier around fascicles of nerve fibers throughout the peripheral nervous system. In a study to determine the cellular origin of perineurium, a culture system was used in which perineurium forms after purified populations of sensory neurons, Schwann cells, and fibroblasts are recombined. Before recombination, the Schwann cells or the fibroblasts were labeled by infection with a defective recombinant retrovirus whose gene product, beta-galactosidase, is histochemically detectable in the progeny of infected cells. Perineurial cells were labeled when fibroblasts had been infected but not when Schwann cells had been infected. Thus, perineurium arises from fibroblasts in vitro and, by implication, in vivo as well.

Designing cell- and gene-based regeneration strategies to repair the injured spinal cord.

There is an array of new and promising strategies being developed to improve function after spinal cord injury (SCI). The targeting of a diversity of deleterious processes within the tissue after SCI will necessitate a multi-factorial intervention, such as the combination of cell- and gene-based approaches. To ensure proper development and design of these experiments, many issues need to be addressed. It is the purpose of this review to consider the strategies involved in testing the efficacy of these new combinations to improve axonal regeneration. For cell-based therapy, issues are choosing a SCI model, the time of cell implantation, placement of cells and their subsequent migration, fluid versus solid grafts, use of agents to prevent immune rejection, and tracking of implanted cells. Grafting is also discussed in view of improving function, reducing secondary damage, bridging the injured spinal cord, supporting axonal regrowth, replacing lost neurons, facilitating myelination, and promoting axonal growth from the implant into the cord. The choice of a gene delivery system, gene-based therapies in vivo to provide chemoattractant and guidance cues, altering the intrinsic regenerative capacity of neurons, enhancing endogenous non-neuronal cell functions, and targeting the synthesis of growth inhibitory molecules are also discussed, as well as combining ex vivo gene and cell therapies.

Motor enrichment sustains hindlimb movement recovered after spinal cord injury and glial transplantation.

PURPOSE: This study investigated whether enrichment improves hindlimb movement following complete spinal cord transection and transplantation of olfactory ensheathing glia (OEG), with or without a Schwann cell (SC) bridge. METHODS: Motor activity was encouraged through provision of motor enrichment housing (MEH); a multi-level cage containing ramps, textured surfaces and rewards. Hindlimb joint movement was assessed weekly for 22 weeks starting one week post-surgery, comparing rats housed in MEH to those in basic housing (BH). Transganglionic tracer was injected into the crushed right sciatic nerve three days prior to sacrifice, allowing sensory axons in the dorsal columns to be visualized by immunolabeling. Serotonergic axons and glial cells expressing low affinity nerve growth factor receptor were identified by immunolabeling. RESULTS: All rats, having received transplants, recovered some hindlimb movement. Rats housed in BH progressively lost recovered hindlimb function whereas recovered hindlimb movements were sustained in most rats in MEH. In rats transplanted with SCs and OEG, effects of MEH were first significant 14 weeks after injury. In rats transplanted with OEG, a trend was seen from 14 weeks after injury, but this did not reach significance. In all rats, traced sensory axons died back from sites of transplantation and did not regenerate rostrally. Further, in no rat were serotonergic axons observed regenerating into, around or beyond transplants. CONCLUSIONS: Transection and transplantation of SC/OEG or OEG induced recovery of hindlimb function. This recovered hindlimb movement was sustained in rats housed in MEH but was progressively lost in rats housed in BH. Because benefits of MEH were not observed until 14 weeks after injury, long-term assessment of behavior is recommended. BH conditions are not conducive to maintenance of recovered hindlimb function, and MEH should be used in studies of recovery of function following spinal cord injury.

Survival, integration, and axon growth support of glia transplanted into the chronically contused spinal cord.

Due to an ever-growing population of individuals with chronic spinal cord injury, there is a need for experimental models to translate efficacious regenerative and reparative acute therapies to chronic injury application. The present study assessed the ability of fluid grafts of either Schwann cells (SCs) or olfactory ensheathing glia (OEG) to facilitate the growth of supraspinal and afferent axons and promote restitution of hind limb function after transplantation into a 2-month-old, moderate, thoracic (T8) contusion in the rat. The use of cultured glial cells, transduced with lentiviral vectors encoding enhanced green fluorescent protein (EGFP), permitted long-term tracking of the cells following spinal cord transplantation to examine their survival, migration, and axonal association. At 3 months following grafting of 2 million SCs or OEG in 6 microl of DMEM/F12 medium into the injury site, stereological quantification of the three-dimensional reconstructed spinal cords revealed that an average of 17.1 +/- 6.8% of the SCs and 2.3 +/- 1.4% of the OEG survived from the number transplanted. In the OEG grafted spinal cord, a limited number of glia were unable to prevent central cavitation and were found in patches around the cavity rim. The transplanted SCs, however, formed a substantive graft within the injury site capable of supporting the ingrowth of numerous, densely packed neurofilament-positive axons. The SC grafts were able to support growth of both ascending calcitonin gene-related peptide (CGRP)-positive and supraspinal serotonergic axons and, although no biotinylated dextran amine (BDA)-traced corticospinal axons were present within the center of the grafts, the SC transplants significantly increased corticospinal axon numbers immediately rostral to the injury-graft site compared with injury-only controls. Moreover, SC grafted animals demonstrated modest, though significant, improvements in open field locomotion and exhibited less foot position errors (base of support and foot rotation). Whereas these results demonstrate that SC grafts survive, support axon growth, and can improve functional outcome after chronic contusive spinal cord injury, further development of OEG grafting procedures in this model and putative combination strategies with SC grafts need to be further explored to produce substantial improvements in axon growth and function.

Comparison of iNOS inhibition by antisense and pharmacological inhibitors after spinal cord injury.

Inducible nitric oxide synthase (iNOS) is a key mediator of inflammation during pathological conditions. We examined, through the use of selective iNOS inhibitors, the role of iNOS in specific pathophysiological processes after spinal cord injury (SCI), including astrogliosis, blood-spinal cord barrier (BSCB) permeability, polymorphonuclear leukocyte infiltration, and neuronal cell death. Administration of iNOS antisense oligonucleotides (ASOs) (intraspinally at 3 h) or the pharmacological inhibitors, N-[3(Aminomethyl) benzyl] acetamidine (1400 W) (i.v./i.p. 3 and 9 h) or aminoguanidine (i.p. at 3 and 9 h) after moderate contusive injury decreased the number of iNOS immunoreactive cells at the injury site by 65.6% (iNOS ASOs), 62.1% (1400 W), or 59% (aminoguanidine) 24 h postinjury. iNOS activity was reduced 81.8% (iNOS ASOs), 56.7% (1400 W), or 67.9% (aminoguanidine) at this time. All iNOS inhibitors reduced the degree of BSCB disruption (plasma leakage of rat immunoglobulins), with iNOS ASO inhibition being more effective (reduced by 58%). Neutrophil accumulation within the injury site was significantly reduced by iNOS ASOs and 1400 W by 78.8% and 20.9%, respectively. Increased astrogliosis was diminished with iNOS ASOs but enhanced following aminoguanidine. Detection of necrotic and apoptotic neuronal cell death by propidium iodide and an FITC-conjugated Annexin V antibody showed that iNOS inhibition could significantly retard neuronal cell death rostral and caudal to the injury site. These novel findings indicate that acute inhibition of iNOS is beneficial in reducing several pathophysiological processes after SCI. Furthermore, we demonstrate that the antisense inhibition of iNOS is more efficacious than currently available pharmacological agents.

Spontaneous axonal regeneration in rodent spinal cord after ischemic injury.

Here we present evidence for spontaneous and long-lasting regeneration of CNS axons after spinal cord lesions in adult rats. The length of 200 kD neurofilament (NF)-immunolabeled axons was estimated after photochemically induced ischemic spinal cord lesions using a stereological tool. The total length of all NF-immunolabeled axons within the lesion cavities was increased 6- to 10-fold at 5, 10, and 15 wk post-lesion compared with 1 wk post-surgery. In ultrastructural studies we found the putatively regenerating axons within the lesion to be associated either with oligodendrocytes or Schwann cells, while other fibers were unmyelinated. Immunohistochemistry demonstrated that some of the regenerated fibers were tyrosine hydroxylase- or serotonin-immunoreactive, indicating a central origin. These findings suggest that there is a considerable amount of spontaneous regeneration after spinal cord lesions in rodents and that the fibers remain several months after injury. The findings of tyrosine hydroxylase- and serotonin-immunoreactivity in the axons suggest that descending central fibers contribute to this endogenous repair of ischemic spinal cord injury.

Bridging areas of injury in the spinal cord.

There is a devastating loss of function when substantial numbers of axons are interrupted by injury to the spinal cord. This loss may be eventually reversed by providing bridging prostheses that will enable axons to regrow across the injury site and enter the spinal cord beyond. This review addresses the bridging strategies that are being developed in a number of spinal cord lesion models: complete and partial transection and cavities arising from contusion. Bridges containing peripheral nerve, Schwann cells, olfactory ensheathing glia, fetal tissue, stem cells/neuronal precursor cells, and macrophages are being evaluated as is the administration of neurotrophic factors, administered by infusion or secreted by genetically engineered cells. Biomaterials may be an important factor in developing successful strategies. Due to the complexity of the sequelae following spinal cord injury, no one strategy will be effective. The compelling question today is: What combinations of the strategies discussed, or new ones, along with an initial neuroprotective treatment, will substantially improve outcome after spinal cord injury?

Implantation of cultured sensory neurons and Schwann cells into lesioned neonatal rat spinal cord. I. Methods for preparing implants from dissociated cells.

Our goal was to devise methods of implanting defined populations of the cellular constituents of peripheral nerve into regions of spinal cord injury. This objective derived from the knowledge that the cellular environment of peripheral nerve is known to be supportive of axon regeneration from both central and peripheral neurons. Two of the constituents of the peripheral nerve environment known to influence axonal growth are the Schwann cell and extracellular matrix (particularly basal lamina), both of which can be obtained in culture. We describe here large-scale methods of establishing purified populations of rat sensory neurons to which purified populations of Schwann cells were added. These essentially monolayer preparations were then scrolled and cut into lengths of proper shape and size to provide implants for sites of spinal cord injury in newborn rats. We also describe methods enabling the addition of leptomeningeal components to the implants; this addition contributes a proliferating population of vascular endothelial cells (identified by immunostaining) to the otherwise vasculature-free neuron/Schwann cell implant. Light and electron microscopic observations were made to characterize the implants. When the implant was ready for use, it contained Schwann cells that were differentiated, i.e., had begun to ensheathe axons and form basal lamina. The use of a medium containing human plasma to foster endothelial cell growth led to increased neurite fasciculation and Schwann cell migratory activity in the outgrowth, particularly when the neurons and Schwann cells were cultured on leptomeninges. The second paper in this series reports the deportment of these implants and their influence on corticospinal tract growth after placement into regions of dorsal column injury in neonatal rats (Kuhlengel et al., J. Comp. Neurol 293:74-91, 1990).

Implantation of cultured sensory neurons and Schwann cells into lesioned neonatal rat spinal cord. II. Implant characteristics and examination of corticospinal tract growth.

The purpose of this study was to test the effectiveness of implants derived from peripheral neural tissue to serve as bridges following interruption of the developing corticospinal tract (CST). Implants prepared from purified populations of cultured dorsal root ganglion neurons (DRGNs) and Schwann cells (SCs) (Kuhlengel et al., J. Comp. Neurol. 293:63-73, 1990) were placed into thoracolumbar regions of neonatal rat spinal cord from which a 2-mm length of dorsal columns had been removed by suction. These cords were examined by a number of techniques 10 days to 6 months later. The implants, recognizable by their DRGN content, filled the vacated dorsal columns and survived the longest periods examined. The most effective method to maintain implant position was dorsal placement of collagen-coated Nitex filter. Implants were inserted either at the time of lesioning or 5 days later. The implant survival rate was better (72% vs. 50%) and meningeal scarring was less with immediate implantation, but delayed implantation resulted in better implant-cord fusion and the implant better filled the lesion cavity. DRGN/SC implants became well vascularized without leptomeningeal cells; this may explain why implant survival was not improved with leptomeningeal cell addition. Particularly well-differentiated implants (full extracellular matrix production and myelination) did not fuse as well with cord as did those less well differentiated. The addition of nerve growth factor to the Nitex filter collagen coating led to improved survival of DRGNs in implants. Electron microscopy showed that astrocytes populated the implant-cord junction region and migrated into implants. Typical SCs related to nonmyelinated and myelinated axons were present in implants. Close proximity of astrocytes and central myelin to SCs and peripheral myelin demonstrated good implant integration with cord. Clusters of SCs, astrocytes, and axons, all enclosed within a common basal lamina, were observed in implants. Immunostaining for GFAP and laminin confirmed our microscopy findings that SCs did not migrate from implant into host but that astrocytes left host tissue to enter implants. Neuroanatomical tracing of CST neurons with HRP-WGA showed that labeled fibers were not present in the implant but were fasciculated just beneath in gray matter. These fibers remained clustered in gray matter underneath the ventral dorsal columns caudal to the lesion. In lesioned but not implanted rats, labeled fibers were only diffusely distributed in gray matter. Delayed implantation led to more variation in fasciculation compared with immediate implantation.(ABSTRACT TRUNCATED AT 400 WORDS)

Axonal regeneration into Schwann cell-seeded guidance channels grafted into transected adult rat spinal cord.

Schwann cells (SC) have been shown to promote regeneration in both the peripheral and central nervous systems. In this study we tested the ability of SC to enhance axonal regeneration in adult rat spinal cord by grafting SC-seeded guidance channels into transected cords. SC were purified in culture from adult inbred rat sciatic nerves, suspended in Matrigel, and seeded into semipermeable PAN/PVC channels (2.6 mm I.D. x 10 mm long) at a final density of 120 x 10(6) cells/ml. Channels filled with Matrigel alone served as controls. Adult isologous rat spinal cords were transected at the T8 level, and segments T9-T11 were removed. The rostral stump was inserted 1 mm into channels with capped distal ends. One month after grafting, a vascularized tissue cable was present within the channel in all animals. In SC-seeded channels (n = 14), a mean of 501 myelinated axons was found in the cable, and many axons extended 9-10 mm. Electron microscopy revealed typical SC ensheathment and myelination of axons with four times more unmyelinated than myelinated axons. Control channels (n = 8) contained fewer myelinated axons (mean = 71). When SC were prelabeled in culture with a nuclear dye, labeled nuclei were observed at 30 days, confirming SC survival. Astrocytes identified by glial fibrillary acidic protein staining did not migrate far into the cable, and prelabeled SC did not enter the cord. Lack of immunostaining for serotonin and dopamine beta-hydroxylase indicated that supraspinal axons did not regenerate into the cable. Fast Blue injections into the middle of the cable (n = 3) marked spinal cord interneurons (mean = 306) as far as nine segments rostral (25 mm, C7) extending axons into the graft; fewer dorsal root ganglion neurons were retrogradely labeled. In conclusion, purified populations of SC transplanted within channels promote both propriospinal and sensory axonal regeneration in the adult rat thoracic spinal cord.