Cell death of asynaptic neurons in regenerating spinal cord

The weakly electric fish Sternarchus albifrons possesses a unique class of asynaptic neurons, the electromotor neurons, whose axons constitute the electric organ. The cell bodies of origin of the electrocyte axons are located in the spinal cord. Both spinal cord and electromotor neurons ( electrocytes ) regenerate after amputation of the tail. Sternarchus spinal cords which have regenerated for 1 or more years show a progression in number of perikarya of electromotor neurons along the rostro-caudal axis. The most recently regenerated region of the cord is at the caudal end, which consists of a tube of ependyma. Progressing rostrally along regenerated spinal cord from the caudal end, numerous cells are generated and large numbers of electromotor neurons differentiate. The maximum number of electromotor neurons per transverse section of regenerated cord is five times higher than in normal mature cord. Rostral to this, the number of electromotor neurons decreases gradually to the normal number near the transition zone (the border with unregenerated cord). As the more rostral regenerated cord has presumably had a longer period of regeneration, we conclude that excess numbers of electromotor neurons are generated initially, and that subsequently the number of these neurons is decreased by cell death. This conclusion is supported by the fact that younger regenerates (2-4 months) have larger-than-normal numbers of perikarya of electromotor neurons extending up to the transition zone (Anderson and Waxman , 1981). No evidence of migration or depletion of electromotor neurons from unregenerated cord rostral to the amputation has been observed. Since the axons of the electromotor neurons in Sternarchus do not normally form any synapses, this study provides evidence that factors other than synaptic competition must be responsible for determining cell death during regeneration of these spinal neurons.     

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.     

Morphology of regenerated spinal cord in Sternarchus albifrons

Summary The tail of the gymnotid Sternarchus albifrons, including the spinal cord, regenerates following amputation. Regenerated spinal cord shows a rostro-caudal gradient of differentiation. Cross sections of the most distal regenerated cord show radially enlarged ependymal cells, relatively undifferentiated cells, and numerous blood vessels. More anterior sections contain well differentiated electromotor neurons, glial cells, and myelinated axons. The number of electromotor-neuron cell bodies in cross sections of regenerated spinal cord is three to six times the number in nonregenerated cord. Distinct tracts of axons, easily identifiable in normal cord, are not distinguishable in cross sections of regenerated cord. Some reorganization of the spinal cord also appears to take place anterior to the site of transection.Individual electromotor neurons in the regenerated spinal cord have morphologies largely similar to those of normal electrocytes, i.e., cell bodies are rounded, lack dendrites, have synapses characterized by gap junctions with presynaptic axons, and lack an unmyelinated initial segment. The presence of electromotor neurons with normal morphology in regenerated spinal cord correlates with the re-establishment of relatively normal electrocyte axonSchwann cell relationships in the regenerating electric organ of this sternarchid.Supported in part by the Medical Research Service, Veterans Administration and by a grant from the National Institutes of Health. We also thank the Paralyzed Veterans of America for their support. We thank Mary E. Smith and Susan Cameron for excellent technical support     

Keratin 8 of simple epithelia is expressed in glia of the goldfish nervous system

The intermediate filament protein composition in glial cells of goldfish optic nerve differs from that found in glial cells of the goldfish spinal cord and brain. Brain and spinal cord glial cells contain glial fibrillary acidic protein (GFAP), whereas glial cells in the optic nerve contain ON3. The ON3 protein of the goldfish optic nerve was recently identified as the goldfish equivalent to the mammalian type II keratin 8 protein. In addition to the ON3 protein, the goldfish optic nerve also contains a 48-kDa protein. Immunoblotting experiments suggest that this protein is equivalent to the mammalian type I keratin 18 protein, which typically pairs with keratin 8 to form filaments. We show that these proteins are not specific to the optic nerve. The ON3 and 48-kDa proteins of the goldfish optic nerve share common antigenic properties with the predominant keratin pair expressed in the goldfish liver. These proteins are also expressed at low levels in the goldfish brain and spinal cord. In addition RNase protection assays and Northern blots indicate that the mRNA for the ON3 protein in optic nerve is identical to the message found in other goldfish tissues. The expression of ON3 was also examined in cultured glial cells from goldfish spinal cord and optic nerve and cultured fibroblast cells. Analysis of intermediate filament protein expression in cultured glial cells taken from goldfish spinal cord demonstrated the absence of GFAP in these cells and the expression of ON3. This protein was also the predominant intermediate filament protein of cultured optic nerve glial cells and fibroblasts. The differences in the expression of intermediate filament proteins in mammals and lower vertebrates are discussed. In addition, we discuss how the expression of a simple epithelial keratin pair in glial cells of the goldfish optic nerve may be associated with this system's capacity for continuous growth and regeneration.     

Survey of Intermediate Filament Proteins in Optic Nerve and Spinal Cord: Evidence for Differential Expression

The distribution of intermediate filament proteins in optic nerve and spinal cord from rat, hamster, goldfish, frog, and newt were analyzed by two-dimensional gel electrophoresis. General as well as specific monoclonal and polyclonal antibodies were reacted against putative intermediate filament proteins. In vitro incubations of excised optic nerve in the presence of [35S]methionine distinguished between neuronal and nonneuronal intermediate filament proteins. The proteins of the intermediate filament complex in the two tissues for rat and hamster were similar. The typical neurofilament triplet and glial fibrillary acidic protein (GFAP) were observed. Vimentin was more concentrated in the optic nerve than in the spinal cord. The goldfish, newt, and frog contained neurofilament proteins in the 145-150K range and in the 70-85K range. In addition, predominant neurofilament proteins in the 58-62K molecular-weight range were found in all three species. In contrast to mammalian species, the goldfish, newt, and frog displayed extensive heterogeneity between optic nerve and spinal cord in the expression of both neuronal and nonneuronal intermediate filament proteins. The distinctive presence of low-molecular-weight intermediate filament proteins and their high concentration in the optic nerve and spinal cord of these nonmammalian vertebrates is discussed in terms of neuronal development and regeneration.     

Formation of the peripheral nervous system during tail regeneration in urodele amphibians: ultrastructural and immunohistochemical studies of the origin of the cells.

In the regenerating newt tail, epimorphic regeneration--which recapitulates morphologically normal embryonic development--proceeds along a rostrocaudal differentiation gradient. Innervation of the new myomeres results from the spinal roots of segments rostral to the amputation plane and from ventral roots emerging from the lateroventral region of the regenerating spinal cord, in which motor neurons are differentiating. Electron microscopy and an indirect immunofluorescence study with anti-glial fibrillary acid protein (GFAP) confirm that the ventrolateral part of the regenerated ependymal tube gives rise to cells of the ventral root sheath and the spinal ganglia. Anti-GFAP and anti-neurofilament antibodies showed that ependymoglial cells and Schwann cells may play a role in neuronal pathfinding by helping guide and stabilize pioneering axons as they extend toward the myomeres. The carbohydrate epitope NC-1 is expressed in the spinal cord, in sheath cells of the spinal ganglia and in the non-myelin-forming Schwann cells of the peripheral nervous system. L1, a Ca++ independent neural cell adhesion molecule, was detected in the axonal compartments of the regenerating spinal cord, on immature and/or non-myelin-forming Schwann cells within the peripheral nervous system (PNS), and on nerve fibers within the regenerate. These immunohistochemical observations collectively support the hypothesis that Schwann cells already present in the blastema could be involved in organizing neural pathways.     

Cloning of Multiple Forms of Goldfish Vimentin: Differential Expression in CNS

Abstract: In efforts to determine the primary structure of intermediate filament proteins in the goldfish visual pathway, we isolated clones from a retinal λgt11 cDNA expression library that represent goldfish vimentin. We show that there are at least two forms of goldfish vimentin, designated as vimentin α and vimentin β. RNase protection assays indicate that vimentin α mRNA is expressed in low amounts in retina, optic nerve, and brain and in higher amounts in spinal cord. In contrast, vimentin β mRNA is expressed in low amounts in retina, optic nerve, brain, and spinal cord and in very high amounts in eye lens. Immunohistochemical studies show that in the optic nerve, vimentin α is mainly restricted to blood vessels, meninges, and septa. Light staining is observed with this antibody in an astrocytic glial pattern throughout the optic nerve. Two-dimensional gel analysis shows that all of these goldfish vimentins are low abundant components of optic nerve cytoskeletal preparations.     

Inhibition of gecko GSK-3β promotes elongation of neurites and oligodendrocyte processes but decreases the proliferation of blastemal cells

GSK-3β signaling is involved in regulation of both neuronal and glial cell functions, and interference of the signaling affects central nervous system (CNS) development and regeneration. Thus, GSK-3β was proposed to be an important therapeutic target for promoting functional recovery of adult CNS injuries. To further clarify the regulatory function of the kinase on the CNS regeneration, we characterized gecko GSK-3β and determined the effects of GSK-3β inactivation on the neuronal and glial cell lines, as well as on the gecko tail (including spinal cord) regeneration. Gecko GSK-3β shares 91.7-96.7% identity with those of other vertebrates, and presented higher expression abundance in brain and spinal cord. The kinase strongly colocalized with the oligodendrocytes while less colocalized with neurons in the spinal cord. Phosphorylated GSK-3β (pGSK-3β) levels decreased gradually during the normally regenerating spinal cord ranging from L13 to the 6th caudal vertebra. Lithium injection increased the pGSK-3β levels of the corresponding spinal cord segments, and in vitro experiments on neurons and oligodendrocyte cell line revealed that the elevation of pGSK-3β promoted elongation of neurites and oligodendrocyte processes. In the normally regenerate tails, pGSK-3β kept stable in 2 weeks, whereas decreased at 4 weeks. Injection of lithium led to the elevation of pGSK-3β levels time-dependently, however destructed the regeneration of the tail including spinal cord. Bromodeoxyuridine (BrdU) staining demonstrated that inactivation of GSK-3β decreased the proliferation of blastemal cells. Our results suggested that species-specific regulation of GSK-3β was indispensable for the complete regeneration of CNS.     

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)     

Differences in growth of neurons from normal and regenerated teleost spinal cord in vitro

Summary Explants and dissociated cells from normal adult spinal cord and regenerating cord of the teleostApteronotus albifrons were grown in vitro for periods of 8 to 12 wk. During this time the neurons showed extensive neurite outgrowth. Neurite outgrowth from tissue explants and dissociated cells of regenerated spinal cord starts sooner and is more profuse than that from normal (unregenerated) cord. Neurite outgrowth is maximized by using adhesive substrata and a high density of explants or dissociated cells. Inasmuch asApteronotus does regenerate its spinal cord naturally after injury, whereas mammals do not, this culture system will be useful to study factors that control (permit) regeneration of spinal neurons in this adult vertebrate.     

Immunohistochemical analysis of nervous system regeneration in chimeric individuals of Dorvillea bermudensis (Polychaeta, Dorvilleidae)

In regeneration experiments, 0.5% of the two- or five-segmented fragments of the polychaete Dorvillea bermudensis were found unexpectedly transplanted: two fragments of each that were lying close together during the initial period, fused and regenerated a chimeric individual. Of the three theoretical possibilities (i.e. fusion of (i). two posterior ends; (ii). one anterior and one posterior end; (iii). or two anterior ends) only the last two were realized. The similarly oriented fragments regenerated a normal animal while anterior-anterior fused ones produced two heads or a double head. Whether the ventral cords of the fragments are located vis-à-vis or adjacent, influences the course of regeneration as well. Immunohistochemical methods (anti-acetylated alpha-tubulin) in conjunction with confocal laser scanning microscopy were used to investigate the wiring pattern of the nervous systems of the grafts. In all cases, at least two supraesophageal ganglia were formed and palps, antennae and nuchal organs were innervated by the correct nerves but, in special cases, were innervated vice versa from the other brain. From these results it can be concluded that fusion of a regenerating connective with another connective results in formation of a new brain, irrespective of whether it belongs to the same nerve cord or not.     

58,000 Dalton Intermediate Filament Proteins of Neuronal and Nonneuronal Origin in the Goldfish Visual Pathway

A group of proteins in the goldfish optic nerve with a molecular weight of 58K daltons was analyzed by two-dimensional gel electrophoresis. Results show that the proteins are differentially phosphorylated and found exclusively in a cytoskeletal-enriched fraction. The proteins from this fraction can be reconstituted into typical intermediate filament structures, as shown by electron microscopy. Two components which are of neuronal origin are transported within the slow phase of transport. The 58K proteins are the most abundant proteins in the optic nerve, and they are distinct from actin and tubulin. It was concluded that they are intermediate filament proteins. Cytoskeletal preparations of rat spinal cord, rat optic nerve, and goldfish optic nerve were compared by one-dimensional gel electrophoresis. The rat spinal cord contains glial fibrillary acidic protein (GFAP), and the rat optic nerve contains vimentin and GFAP, in addition to the neurofilament triplet. A typical mammalian neurofilament triplet is not detected in the goldfish optic nerve, while the major cytoskeletal constituent is a 58K band which coelectrophoreses with vimentin in the rat optic nerve by one-dimensional gel electrophoresis.     

Spinal cord regeneration in a tail autotomizing urodele

Adult urodele amphibians possess extensive regenerative abilities, including lens, jaws, limbs, and tails. In this study, we examined the cellular events and time course of spinal cord regeneration in a species, Plethodon cinereus, that has the ability to autotomize its tail as an antipredator strategy. We propose that this species may have enhanced regenerative abilities as further coadaptations with this antipredator strategy. We examined the expression of nestin, vimentin, and glial fibrillary acidic protein (GFAP) after autotomy as markers of neural precursor cells and astroglia; we also traced the appearance of new neurons using 5‐bromo‐2′‐deoxyuridine/neuronal nuclei (BrdU/NeuN) double labeling. As expected, the regenerating ependymal tube was a major source of new neurons; however, the spinal cord cranial to the plane of autotomy showed significant mitotic activity, more extensive than what is reported for other urodeles that cannot autotomize their tails. In addition, this species shows upregulation of nestin, vimentin, and GFAP within days after tail autotomy; further, this expression is upregulated within the spinal cord cranial to the plane of autotomy, not just within the extending ependymal tube, as reported in other urodeles. We suggest that enhanced survival of the spinal cord cranial to autotomy allows this portion to participate in the enhanced recovery and regeneration of the spinal cord. J. Morphol. 2011. © 2011 Wiley Periodicals, Inc.     

The molecular cloning of glial fibrillary acidic protein in <Emphasis Type="Italic">Gekko japonicus</Emphasis> and its expression changes after spinal cord transection

The glial fibrillary acidic protein (GFAP) is an astrocyte-specific member of the class III intermediate filament proteins. It is generally used as a specific marker of astrocytes in the central nervous system (CNS). We isolated a GFAP cDNA from the brain and spinal cord cDNA library of Gekko japonicus, and prepared polyclonal antibodies against gecko GFAP to provide useful tools for further immunochemistry studies. Both the real-time quantitative PCR and western blot results revealed that the expression of GFAP in the spinal cord after transection increased, reaching its maximum level after 3 days, and then gradually decreased over the rest of the 2 weeks of the experiment. Immunohistochemical analyses demonstrated that the increase in GFAP-positive labeling was restricted to the white matter rather than the gray matter. In particular, a slight increase in the number of GFAP positive star-shaped astrocytes was detected in the ventral and lateral regions of the white matter. Our results indicate that reactive astrogliosis in the gecko spinal cord took place primarily in the white matter during a short time interval, suggesting that the specific astrogliosis evaluated by GFAP expression might be advantageous in spinal cord regeneration.     

Monoclonal antibody to rat brain actin antigenically enhanced with HVJ (Sendai Virus) M protein

Summary Monoclonal antibody to rat brain actin was easily produced using HVJ (Sendai Virus) M protein to enhance the antigenicity of the actin. This monoclonal antibody was determined to be IgM with a kappa light chain. By immunoblot analysis the antibody was also shown to react with rat brain actin but not with HVJ M protein on nitrocellulose sheets. Utilizing the antibody, neuronal cytoplasm in the cerebral cortex, the anterior and posterior horns in the spinal cord, the spinal ganglion and astrocytes showed positive immunohistochemical staining by light microscopy. However, Purkinje cells showed variable staining, some staining intensely, while others were negative. All of neurons in specific anatomical locations showed always positive staining but variable intensities. Vascular walls were stained only faintly. By electron microscopy, neuronal cytoplasm showed diffuse positive staining. Other areas showed a positive reaction, including dendrites, the postsynaptic densities, and a few capillary endothelial cells and arterial smooth muscle cells. The results suggest that the HVJ M protein was effective for producing monoclonal antibody to brain actin, and that the antibody could be utilized for the immunohistochemical study of neuronal elements in both normal and pathological conditions.     

Monoclonal antibody to rat brain actin antigenically enhanced with HVJ (Sendai virus) M protein

Monoclonal antibody to rat brain actin was easily produced using HVJ (Sendai Virus) M protein to enhance the antigenicity of the actin. This monoclonal antibody was determined to be IgM with a kappa light chain. By immunoblot analysis the antibody was also shown to react with rat brain actin but not with HVJ M protein on nitrocellulose sheets. Utilizing the antibody, neuronal cytoplasm in the cerebral cortex, the anterior and posterior horns in the spinal cord, the spinal ganglion and astrocytes showed positive immunohistochemical staining by light microscopy. However, Purkinje cells showed variable staining, some staining intensely, while others were negative. All of neurons in specific anatomical locations showed always positive staining but variable intensities. Vascular walls were stained only faintly. By electron microscopy, neuronal cytoplasm showed diffuse positive staining. Other areas showed a positive reaction, including dendrites, the postsynaptic densities, and a few capillary endothelial cells and arterial smooth muscle cells. The results suggest that the HVJ M protein was effective for producing monoclonal antibody to brain actin, and that the antibody could be utilized for the immunohistochemical study of neuronal elements in both normal and pathological conditions.     

Fas and FasL Expression in the Spinal Cord Following Cord Hemisection in the Monkey

The changes of endogenous Fas/FasL in injured spinal cord, mostly in primates, are not well known. In this study, we investigated the temporal changes in the expression of Fas and FasL and explored their possible roles in the ventral horn of the spinal cord and associated precentral gyrus following T(11) spinal cord hemisection in the adult rhesus monkey. A significant functional improvement was seen with the time going on in monkeys subjected to cord hemisection. Apoptotic cells were also seen in the ventral horn of injured spinal cord with TUNEL staining, and a marked increase presents at 7 days post operation (dpo). Simultaneously, the number of Fas and FasL immunoreactive neurons in the spinal cords caudal and rostral to injury site and their intracellular optical density (OD) in the ipsilateral side of injury site at 7 dpo increased significantly more than that of control group and contralateral sides. This was followed by a decrease and returned to normal level at 60 dpo. No positive neurons were observed in precentral gyrus. The present results may provide some insights to understand the role of Fas/FasL in the spinal cord but not motor cortex with neuronal apoptosis and neuroplasticity in monkeys subjected to hemisection spinal cord injury.     

The Expression of Nerve Growth Factor Receptor on Schwann Cells and the Effect of These Cells on Regeneration of Axons in Traumatically Injured Human Spinal Cord

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Anatomical specificity of regenerated muscle sensory afferents in the spinal cord of the bullfrog

The specificity of central projections made by regenerated muscle sensory fibers in the brachial spinal cord was studied with anatomical tracing methods. Sensory fibers were interrupted by freezing dorsal roots in postmetamorphic bullfrogs. After several months, regenerated sensory fibers were labeled with horseradish peroxidase applied to the triceps brachii muscle nerve, and their arborizations within the spinal cord were reconstructed from serial cross sections. Most of the regenerated projections from triceps muscle sensory afferents ended in or near their normal terminal field. A few branched and appeared to terminate more dorsally than normal, however, sometimes within the region where cutaneous afferents normally terminate. In contrast to the normal pathway followed by muscle afferents within the spinal cord, many regenerated afferents grew along the circumference of the spinal cord, just under the pial surface, and then turned abruptly toward the midline and into their appropriate terminal region. This suggests that regenerating afferents may actively seek out their appropriate targets and are not simply passively guided to them.