Retrograde labeling of regenerated electromotor neurons with HRP in a teleost fish,Sternarchus albifrons: Relation to cell death

Summary Back-labeling of regenerated electromotor neurons in the teleost Sternarchus albifrons was performed to test the hypothesis that, in regenerated spinal cord, incorrectly located electromotor neurons are eliminated because their axons do not reach the correct target area (electric organ). In each cross section examined, all of the regenerated electromotor neurons ipsilateral to the implantation site were labeled with horseradish peroxidase, including those ectopic cells located at the edge of the cord, which are later eliminated by selective cell death. Retrograde labeling of these ectopic neurons demonstrates that their axons do extend into the correct target area (the regenerated electric organ). Thus total misdirection of the axons cannot be the cause of their subsequent cell death. We conclude that selective neuronal death in this system does not reflect the absence of axonal projection to the correct target area.A preliminary report on this work has been presented in Soc. Neurosci. Abstracts 10:48 (1984)     

Regeneration of spinal electrocyte fibers in Sternarchus albifrons: Development of axon-schwann cell relationships and nodes of Ranvier

Summary The electrocyte fibers in the gymnotid Sternarchus albifrons are highly differentiated myelinated axons which exhibit several types of nodes of Ranvier and characteristically short internode lengths. In the present study, regeneration of the electrocyte fibers following removal of the tail was examined by electron microscopy. By 36 days following extirpation, the regenerating electrocyte axons exhibit Type I nodes of Ranvier, with a normal morphology, and Type II nodes of Ranvier with a large nonmyelinated gap and a polypoid elaboration of the axon surface. Moreover, in the regenerating axons the internode length diameter ratios are quite small. Thus, relatively normal axon-Schwann cell relations and a relatively normal differentiation of the axon surface are achieved during regeneration of the Sternarchus electrocyte fibers.Supported in part by the Medical Research Service, Veterans Administration and by grants from the National Institutes of Health and the Paralyzed Veterans of America     

Neuronal differentiation in vitro from precursor cells of regenerating spinal cord of the adult teleost Apteronotus albifrons

This study documents neuronal differentiation in vitro from undifferentiated precursor cells of caudalmost regenerating spinal cord of the teleost Apteronotus albifrons. At 11 days in vitro, cells from the caudalmost tip of the regenerating cord are flat and polygonal in shape, lack neuronal processes and do not stain with antibody against neuron-specific filaments. At 15 days in vitro, some of the caudalmost cells have developed short, neurite-like processes; at 18 days in vitro, some cells react positively with antibody against neuron-specific filaments. At 26 days in vitro, many of the caudalmost cells have long branching neurites and react positively with anti-neurofilament antibody. Addition of insulin-like growth factor-I to the medium accelerates the process of neuronal differentiation from the caudalmost precursor cells in vitro. The source of these precursor cells is ultimately cells of the ependymal layer of adult spinal cord. Further investigation of the factors that control production and differentiation of these cells will be important in defining the developmental potential possible for vertebrate spinal cord cells and may aid in creating an optimal environment for regeneration of axons within mammalian spinal cord.     

Myelinated nerve cell perikaryon in mouse spinal cord

Summary In the thoracic cord (posterior horn region) of a wild mouse, we have observed a small nerve cell soma completely enveloped by a myelin sheath. The number of myelin lamellae varied between 7 and 12. In one place, the existence of an inner mesoperikaryon could also be shown. The significance of this fortuitous finding has not yet been explained.

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Zusammenfassung Im Thorakalmark (Hinterhornbereich) einer Wildmaus wurde ein kleines Nervenzellperikaryon beobachtet, das vollständig von einer Markscheide umhüllt war. Die Zahl der Markscheidenlamellen variierte zwischen 7 und 12. An einer Stelle konnte ein sogenanntes inneres Mesoperikaryon nachgewiesen werden. Die Bedeutung dieses zufällig erhobenen Befundes ist vorerst noch offen.

     

Ependyma and meninges of the spinal cord of the mouse

Summary In addition to ependymal epithelial cells, numerous tanycytes are found along the entire central canal of the mouse. These tanycytes are arranged in clusters in the cervical, thoracic and lumbar segments of the spinal cord. In the conus medullaris, tanycytes separate and ensheath bundles of myelinated and unmyelinated axons; their processes take part in the formation of the stratum marginale gliae. In the caudal part of the spinal cord, the ventral wall of the central canal is thin and some areas are reduced to a single-cell thickness. In this region, ependymal cells participate directly in the formation of the stratum marginale gliae.The meninges consist of the intima piae, the pia mater, the arachnoid, a subdural neurothelium and the dura mater. The subarachnoid space appears occluded and opens only around the spinal roots. In the vicinity of the spinal ganglia, the dura mater, the subdural neurothelium and the arachnoid form a cellular reticulum.     

Presynaptic and postsynaptic organelles of synapses formed in cultures of previously dissociated mouse spinal cord

Summary This study describes some of the ultrastructural features of presynaptic and postsynaptic organelles at synapses developed in cultures of previously dissociated mouse spinal cord cells. Particular attention was paid to the agranular reticulum which is well developed at many presynaptic and postsynaptic sites, either in the form of simple tubules or cisternae, or more complex networks and often closely associated with mitochondria. In addition, the disposition of microtubules at and close to synaptic specializations is described. These and other features of synaptic zones, such as granular vesicles in presynaptic sites, are discussed in relation to cultures developed on feeder layers and synapses in vivo, and in relations to possible degenerative and regenerative events in the cell cultures.     

Neurogenesis during caudal spinal cord regeneration in adult newts

 After tail amputation in urodele amphibians, dramatic changes appear in the spinal cord rostral to the amputation level. Transection induces a proliferation response in cells lining the ependymal canal, giving rise to an ependymal tube in which neurogenesis occurs. Using the thymidine analog bromodeoxyuridine (BrdU) in short- and long-term labeling of cells undergoing DNA synthesis (S phase of the cell cycle), specific cell markers, and cell cultures, we show that neurons derive from the proliferative ependymal layer of the ependymal tube. Received: 30 November 1998 / Accepted: 22 December 1998     

Generation of electromotor neurons in Sternarchus albifrons: differences between normally growing and regenerating spinal cord

This study examines the regulation of the number of electromotor neurons during postnatal growth of the spinal cord in the gymnotiform teleost Sternarchus albifrons. It specifically asks whether a large overproduction of electromotor neurons and a wave of cell death, similar to those occurring during spinal cord regeneration in this species, play a role in the on-going growth at the caudal tip of the normal spinal cord. Neurons are produced from ependymal precursors at the caudal end of the spinal cord during both normal growth in the adult and regeneration of the spinal cord in this species. Previous studies have demonstrated that during spinal cord regeneration after amputation of the tail in Sternarchus, there is an initial massive (up to fivefold) overproduction of electromotor neurons, followed by a wave of cell death which reduces the number of these neurons to the normal level. In the present study, transverse sections through the caudalmost spinal segment of normal adult Sternarchus were examined. Proceeding rostrally from the caudal tip of the cord, the number of electromotor neurons increases monotonically to reach the normal number at a site 4-5 mm rostral to the caudal tip. Neither a massive overproduction of electromotor neurons nor a wave of neuronal death are observed during on-going growth of the normal spinal cord. The mechanisms by which the neuronal number is modulated are therefore different in the on-going normal growth of spinal cord versus regeneration of spinal cord in this species.     

Reissner's fiber and the wall of the central canal in the lumbo-sacral region of the bovine spinal cord

Summary Reissner's fiber (RF) of the subcommissural organ (SCO), the central canal and its bordering structures, and the filum terminale were investigated in the bovine spinal cord by use of transmission electron microscopy, histochemical methods and light-microscopic immunocytochemistry. The primary antisera were raised against the bovine RF, or the SCO proper. Comparative immunocytochemical studies were also performed on the lumbo-sacral region of the rat, rabbit, dog and pig.At all levels of the bovine spinal cord, RF was strongly immunoreactive with both antisera. From cervical to upper sacral levels of the bovine spinal cord there was an increasing number of ependymal cells immunostainable with both antisera. The free surface of the central canal was covered by a layer of immunoreactive material. At sacral levels small subependymal immunoreactive cells were observed. From all these structures sharing the same immunoreactivity, only RF was stained by the paraldehyde-fuchsin and periodicacid-Schiff methods.At the ultrastructural level, ependymal cells with numerous protrusions extending into the central canal were seen in the lower lumbar segments, whereas cells displaying signs of secretory activity were principally found in the ependyma of the upper sacral levels. A few cerebrospinal fluid-contacting neurons were observed at all levels of the spinal cord; they were immunostained with an anti-tubulin serum.The lumbo-sacral segments of the dog, rat and rabbit, either fixed by vascular perfusion or in the same manner as the bovine material, did not show any immunoreactive structure other than RF.The possibilities that the immunoreactive ependymal cells might play a secretory or an absorptive role, or be the result of post-mortem events, are discussed.Supported by Grant I/38259 from the Stiftung Volkswagenwerk, Federal Republic of Germany, and Grant RS-82-18 from the Dirección de Investigaciones, Universidad Austral de ChileThe authors wish to thank Dr. Enrique Romeny from the Valdivia abattoir for kindly providing the bovine spinal cords     

Protective effect of leptin-mediated caveolin-1 expression on neurons after spinal cord injury

Spinal cord injury (SCI) causes long-term disability and has no effective clinical treatment. After SCI, extracellular adenosine triphosphate (ATP) leads to an influx of extracellular Ca²⁺, and this Ca²⁺ overload causes neuronal toxicosis and apoptosis. The biological functions of leptin have been widely investigated in the central nervous system. In this study, we discovered that the administration of leptin could improve locomotor recovery following SCI. The aim of this study was to determine the neuroprotective mechanism of leptin in vivo and in vitro. The neuronal apoptosis and Ca²⁺ imaging signal induced by ATP were suppressed by leptin, due to elevated caveolin-1 expression. In vivo two-photon observations revealed that leptin reduced the neuronal Ca²⁺ imaging signal in the exposed spinal cords of live Thy1-YFP mice. In conclusion, leptin promotes locomotor functional recovery and suppresses neuronal impairment after SCI, suggesting that leptin has a promising clinical therapeutic value for treatment of SCI.     

Up-regulation of neural stem cell markers suggests the occurrence of dedifferentiation in regenerating spinal cord

Following tail amputation in urodele amphibians, an ependymal tube, that resembles a developing neural tube, forms from ependymal cells that migrate from the cord stump and elongates by cell proliferation. Expression of the keratin pair 8 and 18 has been observed in the developing urodele nervous system and is maintained in the ependymal cells of the mature cord. We show here that expression of these keratins is not unique to urodeles, but is also observed in the radial glia of the human spinal cord, suggesting that these proteins might play a role both in neural development and regeneration. Analysis of their expression in the regenerating spinal cord following tail amputation shows that their expression, as well as that of glial fibrillary acidic protein (GFAP), is maintained in the ependymal tube during regeneration, though differences in their levels of expression are observed along the anteroposterior axis and appear to be related to the progression of morphogenesis. In addition, we show that following tail amputation the ependymal tube expresses the neural stem cell markers nestin and vimentin, which are undetectable in normal urodele spinal cord. This up-regulation of neural stem cell markers shows that the ependymal cells undergo a phenotypic change. Whereas maintenance of keratin and GFAP expression in the adult ependyma may reflect a higher plasticity of these cells in adult urodeles than in other vertebrates, re-expression of markers of early neural development suggests the occurrence of a dedifferentiation process in the spinal cord in response to injury.Edited by J. Campos-Ortega     

Scanning electron-microscopic study of Reissner's fiber in the caudal spinal cord of the cat and rabbit

Summary The caudal portion of Reissner's fiber was examined by scanning electron microscopy (SEM) in the spinal cord of the cat and rabbit. In some preparations of both species the fiber displayed in the sinus terminalis of the central canal either stump-like terminations or structural modifications such as knot-like swellings and convolutions. In the same area homogeneous material could also be found, which obviously originated from the disintegrating fiber.     

Radiography used to measure internal spinal cord deformation in an in vivo rat model

Little is known about the internal mechanics of the in vivo spinal cord during injury. The objective of this study was to develop a method of tracking internal and surface deformation of in vivo rat spinal cord during compression using radiography. Since neural tissue is radio-translucent, radio-opaque markers were injected into the spinal cord.Two tantalum beads (260 µm) were injected into the cord (dorsal and ventral) at C5 of nine anesthetized rats. Four beads were glued to the lateral surface of the cord, caudal and cranial to the injection site. A compression plate was displaced 0.5 mm, 2 mm, and 3 mm into the spinal cord and lateral X-ray images were taken before, during, and after each compression for measuring bead displacements. Potential bead migration was monitored for by comparing displacements of the internal and glued surface beads.Dorsal beads moved significantly more than ventral beads with a range in averages of 0.57–0.71 mm and 0.31–0.35 mm respectively. Bead displacements during 0.5 mm compressions were significantly lower than 2 mm and 3 mm compressions. There was no statistically significant migration of the internal beads.The results indicate the merit of this technique for measuring in vivo spinal cord deformation. The pattern of bead displacements illustrates the complex internal and surface deformations of the spinal cord during transverse compression. This information is needed for validating physical and finite element spinal cord surrogates and to define relationships between loading parameters, internal cord deformation, and biological and functional outcomes.     

Basis of progesterone protection in spinal cord neurodegeneration

Progesterone neuroprotection has been reported in experimental brain, peripheral nerve and spinal cord injury. To investigate for a similar role in neurodegeneration, we studied progesterone effects in the Wobbler mouse, a mutant presenting severe motoneuron degeneration and astrogliosis of the spinal cord. Implant of a single progesterone pellet (20 mg) during 15 days produced substantial changes in Wobbler mice spinal cord. Morphologically, motoneurons of untreated Wobbler mice showed severe vacuolation of intracellular organelles including mitochondria. In contrast, neuropathology was less pronounced in Wobbler mice receiving progesterone, together with a reduction of vacuolated cells and preservation of mitochondrial ultrastructure. Determination of mRNAs for the 3 and β1 subunits of neuronal Na, K-ATPase, showed that mRNA levels in untreated mice were significantly reduced, whereas progesterone therapy re-established the expression of both subunits. Additionally, progesterone treatment of Wobbler mice attenuated the aberrant expression of the growth-associated protein (GAP-43) mRNA which otherwise occurred in motoneurons of untreated animals. The hormone, however, was without effect on astrocytosis of Wobbler mice, determined by glial fibrillary acidic protein (GFAP)-immunostaining. Lastly, progesterone treatment of Wobbler mice enhanced grip strength and prolonged survival at the end of the 15-day observation period. Recovery of morphology and molecular motoneuron parameters of Wobbler mice receiving progesterone, suggest a new and important role for this hormone in the prevention of spinal cord neurodegenerative disorders.     

Fine structure of growth cones in the upper dorsal horn of the adult primate spinal cord in the course of reactive synapto-neogenesis

Summary Following transganglionic degenerative atrophy of primary afferent terminals induced by a crush-injury of the sciatic nerve, a regenerative process takes places in the upper dorsal horn of the lumbar spinal cord in the primate Macacus rhesus. Axonal growth cones are characterized by cisterns of axoplasmic reticulum; filopodia emanating from growth cones are electron-optically translucent sheet-like expansions, often containing growth-cone vesicles. Axoplasmic reticulum appears also in preterminal portions of regenerating axons. Dendritic growth cones contain a fine, filamentous matrix; electron-dense membrane specializations can be seen in well-defined areas of their surfaces. Immature synapses are formed between filopodia of axonal growth cones and dendritic growth cones. Electron-microscopic structures of this unique CNS regeneration are similar to those seen in the course of embryonic development of the spinal cord.     

Avian pancreatic polypeptide (APP) immunoreactive neurons in the spinal cord and spinal trigeminal nucleus

Using indirect immunofluorescence technique, avian pancreatic polypeptide (APP) immunoreactive cell bodies and fibres have been observed in the superficial laminae of the dorsal horn of the spinal cord and of the spinal trigeminal nucleus. Fibres were also seen in the ventral horns, in low numbers at the cervical and thoracic levels and in high numbers at the lower lumbar and upper sacral levels. Neither total cord transection, nor dorsal rhizotomy, nor capsaicin treatment seemed to affect the APP systems described above. The present findings suggest that an APP-like peptide may be involved in processing of sensory information at the level of the first relay station.     

Length-tension properties of ankle muscles in chronic human spinal cord injury

Contracture, or loss of range of motion (ROM) of a joint, is a common clinical problem in individuals with spinal cord injury (SCI). In order to measure the possible contribution of changes in muscle length to the loss of ankle ROM, the active force vs. angle curves for the tibialis anterior (TA) and gastrocnemiussoleus (GS) were measured in 20 participants, 10 with SCI, and 10 gender and age matched, neurologically intact (NI) individuals. Electrical stimuli were applied to the TA and GS motor nerves at incremented angles of the entire ROM of the ankle and the resulting ankle and knee torques were measured using a multi-axis load cell. The muscle forces of the TA and GS were calculated from the torque measurements using estimates of their respective moment arms and the resulting forces were plotted against joint angle. The force–angle relation for the GS at the ankle (GSA) was significantly shifted into plantar flexion in SCI subjects, compared to NI controls (t-test, p<0.001). Similar results were obtained based upon the GS knee (GSK) force–angle measurements (p<0.05). Conversely, no significant shift in the force–angle relation was found for the TA (p=0.138). Differences in the passive ROM were consistent with the force–angle changes. The ROM in the dorsiflexion direction was significantly smaller in SCI subjects compared to NI controls (p<0.05) while the plantar flexion ROM was not significantly different (p=0.114). Based upon these results, we concluded that muscle shortening is an important component of contracture in SCI.     

Synaptic profiles in the outgrowth from chick spinal cord in vitro

Summary Synaptic profiles have been identified in the outgrowth from chick embryo spinal cord maintained in vitro for short periods. Profiles corresponding to types that may be excitatory and inhibitory in the intact central nervous system have been found. Their presence outside expiants, and in occasional relation to glial cells, suggests that neurites themselves may possess a generalised capacity for synapse formation under appropriate circumstances, rather than be limited to specific targets.     

Changes in progenitor populations and ongoing neurogenesis in the regenerating chick spinal cord

The chick spinal cord can regenerate following injury until advanced developmental stages. It is conceivable that changes in stem/progenitor cell plasticity contribute to the loss of this capacity, which occurs around E13. We investigated the contribution of proliferation, phenotypic changes in radial glia progenitors, and neurogenesis to spinal cord regeneration. There was no early up-regulation of markers of gliogenic radial glia after injury either at E11 or E15. In contrast, increased proliferation in the grey matter and up-regulation of transitin expression following injury at E11, but not E15, suggested high levels of plasticity within the E11 spinal cord progenitor population that are lost by later stages. Changes in neural progenitors with development were also supported by a higher neurosphere forming ability at E11 than at E15. Co-labelling with doublecortin and neuron-specific markers and BrdU in spinal cord sections and dissociated cells showed that neurogenesis is an ongoing process in E11 chick spinal cords. This neurogenesis appeared to be complete by E15. Our findings demonstrate that the regeneration-competent chick spinal cord is less mature and more plastic than previously believed, which may contribute to its favourable response to injury, and suggest a role for neurogenesis in maintaining regenerative capacity.     

Synenkephalin in bovine and human spinal cord

Summary Synenkephalin, which comprises 70 residues at the aminoterminal of proenkephalin, was studied with immunocytochemical methods in the human and bovine spinal cord. Immunoreactive fibers had the same general distribution as methionine-enkephalin, but not as leucine-enkephalin fibers. They were found in all spinal layers and were most numerous in lamina II (outer zone) and V–VI (lateral portion). Synenkephalin immunoreactivity was overall less dense than that of the enkephalins. These results suggest that proenkephalin is the precursor protein also in enkephalinergic neurons of the human spinal cord.