Proximal Axon Stump Research Articles

Stressed axons craving for glial sugar: links to regeneration?

Stressed axons craving for glial sugar: links to regeneration?

Traumatology of the optic nerve and contribution of crystallins to axonal regeneration

Within a few decades, the repair of long neuronal pathways such as spinal cord tracts, the optic nerve or intracerebral tracts has gone from being strongly contested to being recognized as a potential clinical challenge. Cut axonal stumps within the optic nerve were originally thought to retract and become irreversibly necrotic within the injury zone. Optic nerve astrocytes were assumed to form a gliotic scar and remodelling of the extracellular matrix to result in a forbidden environment for re-growth of axons. Retrograde signals to the ganglion cell bodies were considered to prevent anabolism, thus also initiating apoptotic death and gliotic repair within the retina. However, increasing evidence suggests the reversibility of these regressive processes, as shown by the analysis of molecular events at the site of injury and within ganglion cells. We review optic nerve repair from the perspective of the proximal axon stump being a major player in determining the successful formation of a growth cone. The axonal stump and consequently the prospective growth cone, communicates with astrocytes, microglial cells and the extracellular matrix via a panoply of molecular tools. We initially highlight these aspects on the basis of recent data from numerous laboratories. Then, we examine the mechanisms by which an injury-induced growth cone can sense its surroundings within the area distal to the injury. Based on requirements for successful axonal elongation within the optic nerve, we explore the models employed to instigate successful growth cone formation by ganglion cell stimulation and optic nerve remodelling, which in turn accelerate growth. Ultimately, with regard to the proteomics of regenerating retinal tissue, we discuss the discovery of isoforms of crystallins, with crystallin beta-b2 (crybb2) being clearly upregulated in the regenerating retina. Crystallins are produced and used to promote the elongation of growth cones. In vivo and in vitro, crystallins beta and gamma additionally promote the growth of axons by enhancing the production of ciliary neurotrophic factor (CNTF), indicating that they also act on astrocytes to promote axonal regrowth synergistically. These are the first data showing that axonal regeneration is related to crybb2 movement within neurons and to additional stimulation of CNTF. We demonstrate that neuronal crystallins constitute a novel class of neurite-promoting factors that probably operate through an autocrine and paracrine mechanism and that they can be used in neurodegenerative diseases. Thus, the post-injury fate of neurons cannot be seen merely as inevitable but, instead, must be regarded as a challenge to shape conditions for initiating growth cone formation to repair the damaged optic nerve.

Rapid, effective, and long‐lasting behavioral recovery produced by microsutures, methylene blue, and polyethylene glycol after completely cutting rat sciatic nerves

Behavioral function lost in mammals (including humans) after peripheral nerve severance is slowly (weeks to years) and often poorly restored by 1-2-mm/day, nonspecifically directed outgrowths from proximal axonal stumps. To survive, proximal stumps must quickly repair (seal) plasmalemmal damage. We report that, after complete cut- or crush-severance of rat sciatic nerves, morphological continuity, action potential conduction, and behavioral functions can be consistently (>98% of trials), rapidly (minutes to days), dramatically (70-85% recovery), and chronically restored and some Wallerian degeneration prevented. We assess axoplasmic and axolemmal continuity by intra-axonal dye diffusion and action potential conduction across the lesion site and amount of behavioral recovery by Sciatic Functional Index and Foot Fault tests. We apply well-specified sequences of solutions containing FDA-approved chemicals. First, severed axonal ends are opened and resealing is prevented by hypotonic Ca²⁺-free saline containing antioxidants (especially methylene blue) that inhibit plasmalemmal sealing in sciatic nerves in vivo, ex vivo, and in rat B104 hippocampal cells in vitro. Second, a hypotonic solution of polyethylene glycol (PEG) is applied to open closely apposed (by microsutures, if cut) axonal ends to induce their membranes to flow rapidly into each other (PEG-fusion), consistent with data showing that PEG rapidly seals (PEG-seals) transected neurites of B104 cells, independently of any known endogenous sealing mechanism. Third, Ca²⁺-containing isotonic saline is applied to induce sealing of any remaining plasmalemmal holes by Ca²⁺-induced accumulation and fusion of vesicles. These and other data suggest that PEG-sealing is neuroprotective, and our PEG-fusion protocols that repair cut- and crush-severed rat nerves might rapidly translate to clinical procedures.

Accumulation of the inhibitory receptor EphA4 may prevent regeneration of corticospinal tract axons following lesion

Abstract We have examined the expression of Eph receptors and their ephrin ligands in adult rat spinal cord before and after lesion. Neurons in adult motor cortex express EphA4 mRNA, but the protein is undetectable in uninjured corticospinal tract. In contrast, after dorsal column hemisection EphA4 protein accumulates in proximal axon stumps. One of the ligands for EphA4, ephrinB2, is normally present in the grey matter flanking the corticospinal tract but after injury is markedly up-regulated in astrocytes in the glial scar. The result is that, after a lesion, corticospinal tract axons bear high levels of EphA4 and are surrounded to front and sides by a continuous basket of cognate inhibitory ephrin ligand. We suggest that a combination of EphA4 accumulation in the injured axons and up-regulation of ephrinB2 in the surrounding astrocytes leads to retraction of corticospinal axons and inhibition of their regeneration in the weeks after a spinal lesion.

Regulation of the expression and phosphorylation of microtubule-associated protein 1B during regeneration of adult dorsal root ganglion neurons

Microtubule-associated protein 1B is a major constituent of the neuronal cytoskeleton during the early stages of development. This protein and its phosphorylated isoform, microtubule-associated protein 1B-P, defined by the monoclonal antibody 1B-P [Boyne L. J. et al. (1995) J. Neurosci. Res. 40, 439–450], are present in growing axons and concentrated in the distal end near the growth cone. In most regions of the central nervous system, microtubule-associated protein 1B and microtubule-associated protein 1B-P are developmentally down-regulated. They remain, however, at relatively high levels in the adult peripheral nervous system, where microtubule-associated protein 1B-P is localized exclusively in axons. The aim of this study was to examine the levels of microtubule-associated protein 1B and its phosphorylated isoform during regenerative growth of peripheral axons. Following transection and re-apposition of the sciatic nerve at midthigh, the levels of total microtubule-associated protein 1B, microtubule-associated protein 1B-P and microtubule-associated protein 1B messenger RNA were analysed in dorsal root ganglion neurons and sciatic nerve axons using western blots and RNase protection assays. After the lesion, there was a small decrease in the levels of microtubule-associated protein 1B and its messenger RNA in dorsal root ganglion neurons. The proximal axonal stump showed a similar decrease in the levels of microtubule-associated protein 1B 30 days after lesion and returned to normal 60–90 days post-lesion. In the distal stump of the sciatic nerve, the levels of microtubule-associated protein 1B increased dramatically and rapidly between three and 14 days, but the protein was localized mainly in activated Schwann cells and myelin-like structures, and not in axons [Ma D. et al. (1999) Brain Res. 823, 141–153]. With the regeneration of axons into the distal stump, an intense expression of microtubule-associated protein 1B was observed in these axons. Microtubule-associated protein 1B-P, however, disappeared from the degenerated distal axonal stump as early as three days post-operation, and was absent in the regenerating axons and in Schwann cells between three and 14 days. The levels of microtubule-associated protein 1B-P recovered slowly and did not reach the normal levels even after 90 days post-operation. In contrast to the response following transection, the levels of microtubule-associated protein 1B and microtubule-associated protein 1B-P were much less affected after nerve crush. We propose that the relatively high levels of microtubule-associated protein 1B and its messenger RNA in adult dorsal root ganglions support peripheral neuron regeneration. The presence of microtubule-associated protein 1B in the regenerating axons suggests that microtubule-associated protein 1B is involved in axonal growth during peripheral nerve regeneration. However, the phosphorylated microtubule-associated protein 1B-P isoform, associated with growing axons during development, is not present in the regenerating axons after transection, presumably because of changes in the activities of kinases and phosphatases associated with the injury. These observations underscore the difference between axonal development and regeneration and the importance of injury-related effects that occur locally.

Neurosurgery of the Peripheral Nervous System: Injuries, Degeneration, and Regeneration of the Peripheral Nerves

Increased knowledge is shedding new light on our understanding of the biologic processes involved in nerve regeneration and degeneration. Regeneration is a multistep event that seems to be controlled by neurotrophic factors. Following traumatic, agerelated, or disease-related nerve injuries, these molecules can be expressed and released by target cells, Schwann cells, neuron ensheathing glial cells, or neural somata themselves. The success of nerve regeneration depends on the survival of axotomized neurons, the efficacy of axonal outgrowth from the neurons, and the specificity of peripheral target reinnervation by the regenerating axons. Specific molecules help the injured neurons to survive. Trophic factors, such as nerve growth factors, are taken from either the terminal target tissue or cells such as Schwann cells by the axon and transported retrogradely to the neuronal cell body where they support gene regulation and promote survival of the neuron. A peripheral nerve, regardless of the mechanism of the lesion (e.g., compression, laceration, transection), responds to injury in a predictable way. After peripheral nerve sectioning, changes occur at the level of the proximal and distal axon stumps, their parent neuronal cell bodies, and peripheral nerve targets. Changes in the neuronal cell body consist of chromatolysis, swelling, and displacement of the Nissl substance to the periphery. During chromatolysis, the lipids and proteins necessary for axonal regeneration are synthesized and transferred along the proximal axon stump by so-called “axonal transport.” Axonal elongation begins with emerging axonal sprouts. In the distal axon stump, wallerian degeneration occurs. The axon and myelin degenerate and their debris is removed by phagocytic cells. Empty endoneurial tubes remain within the perineurium and epineurium of the distal nerve stump. Then, in such tubes, the residual Schwann cells proliferate, forming the Bungner’s bands that are ready to receive regenerating axons from the proximal nerve stump. Here, the emerging sprouts, in the appropriate environment, can grow across the area of damage and enter and regenerate along the endoneurial tubes in the distal nerve stump. Chemotactic factors are believed to guide the growing axonal sprouts to reinnervate their original peripheral targets. Only when regenerating axons reach a diameter of two microns do the Schwann cells start remyelination. If the regenerating axons do not reach a distal endoneurial tube or the tube has been replaced by fibrosis, they will form a local tangle or neuroma. A peripheral nerve lesion also leads to pathologic changes in the end organs. For example, by 3 weeks after nerve injury, the denervated muscle undergoes fibrosis. The fibrosis will gradually replace the muscle fibers and after 2 years often only scar tissue is present. Therefore, the muscle should be reinnervated within 18 months after nerve injury to eventually provide a functional outcome. Proximal nerve lesions are therefore, more at risk for poor outcome because of the longer time required for the regenerated axons to reach the target muscles. Turning our attention to the principal kinds of lesions that occur in the peripheral nerves and referring to the classification of Seddon, the following types of nerve injuries can be outlined: Address reprint requests to: Eduardo Fernandez, M.D., Department of Neurosurgery, Catholic University School of Medicine, 00168 Rome, Italy.

A time-dependent loss of retrograde transport ability in distally axotomized rubrospinal neurons.

Studies on the effect of axotomy on adult intrinsic central projection neurons have generally assumed that the severed proximal axonal stumps were still capable of retrogradely transporting tracer at varying times after injury. Failure of transport was interpreted as neuronal death, which is at odds with current understanding that central projection neurons survived distal axotomy. We used lumbar spinal cord-projecting rubrospinal neurons of the rat as a model to evaluate the ability of injured neurons to transport tracer retrogradely at different times after distal axotomy. We examined only the caudal part of the red nucleus, since rubrospinal neurons are concentrated here. In control animals, tracer applied to the rubrospinal tract at the T10 vertebral level labeled ventrolateral rubral neurons, while C3 application marked all rubral neurons. From 3 days after a T10 axotomy and tracer application, most ventrolateral neurons were no longer labeled by another tracer application at the C3 vertebral level via an axonal cut. The phenomenon was not caused by tracer toxicity, since a T10 tractotomy without tracer application also prevented these axotomized neurons from being labeled when treated similarly. Thus, neuronal retrograde transport capability was seriously retarded 3 days after a distal axotomy. Loss of retrograde transport may merely suggest that a mechanism no longer in service has been switched off, or perhaps it may insulate injured neurons from the effect of lesion site-derived factors. Using this property, we were able to localize cervical spinal cord-projecting rubrospinal neurons in the caudal red nucleus.(ABSTRACT TRUNCATED AT 250 WORDS)

Neurofilament sidearm proteolysis is a prominent early effect of axotomy in lamprey giant central neurons.

In the accompanying paper, it was shown that axotomy of lamprey spinal axons induces the rapid formation of condensed neurofilamentous masses in the proximal axon stump near the lesion. In this study, we used immunocytochemical and Western blot analysis to characterize these masses further and to determine the time course of their formation and dispersal. We show that monoclonal antibodies specific to the "rod" domain of lamprey neurofilament protein strongly stain such masses in tissue sections without staining other axonal neurofilaments. Antibodies specific for the neurofilament "sidearm" domain fail to recognize neurofilamentous masses but stain other axonal neurofilaments. Western blots of spinal cord segments from the lesion site were compared to unlesioned cord and to samples of cord distant from the lesion. We found that a neurofilament rod-specific antibody identified breakdown products of the same size as the rod domain in samples from the lesion site, but not elsewhere. Other lesion-specific neurofilament breakdown products were recognized by a sidearm-specific antibody. This lesion-specific pattern of neurofilament proteolysis was visible at 1 day postlesion and was still present 3 weeks later. Immunocytochemistry showed masses of rod-staining neurofilaments in axon stumps by 12 hours postlesion that remained for 1-2 weeks postaxotomy; these dispersed with the onset of regeneration. Such neurofilament rod staining was also prominent in distal axon stumps undergoing Wallerian degeneration. We conclude that axotomy induces neurofilament sidearm proteolysis near the lesion, permitting antibody access to the rod domain. We suggest that sidearm loss causes the high packing density of neurofilaments within neurofilamentous masses near the lesion site and that neurofilament sidearm proteolysis can be used to distinguish degenerative from regenerative changes in lesioned lamprey axons.

Nerve fibre regeneration after traumatic lesions of the CNS; progress and problems

Four distinct phases can be distinguished in the regenerative response of a lesioned CNS axon: sprouting of the proximal axon stump, elongation, target recognition, and formation of appropriate synapses. These processes can be observed in such a way only in lower vertebrates, in particular, in the optic system. In these species and systems, both, the guidance mechanisms leading regenerating fibres to their former target areas, and the mechanisms responsible for specific synapse formation are retained throughout life. As during development, guidance crucially depends on the presence of favourable substrate molecules, and on chemotropic signals (Dodd & Jessel 1989; Tessier-Lavigne et al .1988; Harris 1989). The cell biological mechanisms responsible for target recognition including the arrest of long-distance growth, the initiation of side branch formation and terminal arborization, and the selection of specific post-synaptic partners (cell type; soma, proximal or distal dendrite, spines, axons) remain unknown up to now.

Involvement of neurofilaments in the radial growth of axons

The control of radial growth of axons is of functional importance because caliber is a principal determinant of conduction velocity in myelinated nerve fibers. Neurofilaments, the major cytoskeletal protein in myelinated nerves, appear to be intrinsic determinants of caliber. Evidence supporting this derives first from the linear relationship between neurofilament content and axonal diameter. Further, following distal axonal injury in a peripheral nerve, caliber is reduced in the proximal axonal stumps. This reduction in caliber is itself due to selective suppression of neurofilament gene expression, thereby leading to lower levels of newly synthesized neurofilament subunits transported into the axon and a consequent decrease in axonal neurofilament content. To demonstrate directly the physiological consequence of altering normal neurofilament accumulation, we have elevated neurofilament expression by introducing additional genes into transgenic mice. The clear result is that increases in NF-L content alone are not sufficient to increase axonal caliber. To test the consequence of disruption of normal filament accumulation, we have identified dominant assembly-disrupting mutants in NF-L and NF-M and have used these to produce transgenic animals in which neurofilament assembly should be disrupted.

Neurotoxicity of MDMA and Related Compounds: Anatomic Studiesa

The cytotoxic effects of amphetamine derivatives were studied by immunocytochemistry to identify the cellular compartments affected by these drugs, to obtain morphologic evidence of neuronal degeneration, and to assess the potential for regeneration. The substituted amphetamines, MDA, MDMA, PCA, and fenfluramine, all release serotonin and cause acute depletion of 5-HT from most axon terminals in forebrain. (1) Unequivocal signs of axon degeneration were seen at 36-48 hour survivals: 5-HT axons exhibited increased caliber, huge, swollen varicosities, fragmentation, and dilated proximal axon stumps. (2) Fine 5-HT axon terminals were persistently lost after drug administration, while beaded axons and raphe cell bodies were spared. These two types of 5-HT axons, which arise from separate raphe nuclei and form distinct ascending projections, are differentially vulnerable to psychotropic drugs. (3) From 2-8 months after treatment, there was progressive serotonergic re-innervation of neocortex along a fronto-occipital gradient. Longitudinal 5-HT axons grew into layers I and VI from rostral to caudal, before sprouting into middle cortical layers; this bilaminar pattern of growth simulates perinatal development of 5-HT innervation. This study demonstrates differential vulnerability of 5-HT projections, evidence for axonal degeneration, and sprouting of 5-HT axons leading to re-innervation of forebrain. While the sprouting axons are anatomically similar to the type that was damaged, it is not known whether a normal pattern of innervation is re-established.

Regeneration of nerve fibres in the anterior medullary velum of neonatal and weanling rats

The responses to injury of myelinated central axons in neonatal (5-day) and weanling (30-day) rats were compared, using the trochlear nerve fibres in the anterior medullary velum (AMV) as an experimental system. In intact animals, fibres of the trochlear nerve decussate completely in the AMV, to enter the contralateral nerve. Sprouting from proximal axon stumps was seen within 36 h after injury in silver-stained, whole mounts of mid-sagittally lesioned AMVs from weanlings. Subsequent growth produced a tangled axon mass which persisted until 15-20 days post-lesion (d.p.l.) but then began to degenerate. By 50 d.p.l., the majority of surviving axons looped within the AMV to enter the ipsilateral trochlear nerve; few axons remained growing randomly within the AMV. Axonal debris persisted within the velum 50 d.p.l. In neonatal rats, lesioned trochlear axons initially developed axon sprouts, but then degenerated with rapid clearance of debris, leaving the AMV devoid of both axons and degeneration products by 6 d.p.l. From 8 d.p.l., the velum became repopulated by axons which either grew randomly or looped to enter the ipsilateral peripheral nerve. Both patterns of axon growth were still observed 50 d.p.l. In both experimental groups, horseradish peroxidase injection to the right superior oblique muscle 40 d.p.l. indicated that at least some of the looping fibres observed within the lesioned AMVs originated in the ipsilateral trochlear nucleus. The origin of those axons growing randomly within the AMVs of neonatally-lesioned animals is unknown. The results are discussed with reference to current ideas concerning regeneration-inhibiting elements in the CNS, and regeneration-promoting factors in the PNS.

Regeneration of the neurohemal terminals for identified cerebral neurosecretory cells in an insect

The axons of specific neurosecretory cells, L-NSC III, in the brain of the tobacco hornworm, Manduca sexta, were transected during larval-pupal development to study the effects of this type of lesion on these peptidergic neurons and to begin to identify factors that may regulate their regeneration and growth. The two somata of these bilaterally paired neurons produce the prothoracicotropic hormone and are located in the pars intercerebralis. Their axons exit from the contralateral brain lobe via a retrocerebral nerve and pass through the corpus cardiacum before terminating at the glandular corpus allatum. At the corpus allatum, the L-NSC III axons arborize to form the terminal neurohemal organ for prothoracicotropic hormone release. The retrocerebral nerve was severed either in vitro followed by brain transplantation or in situ; in either protocol, the distal axon segments and corpus allatum were removed. The ability of the injured L-NSC III axons to regenerate was assessed immunocytologically by using a monoclonal antibody against the prothoracicotropic hormone. In both treatments, the proximal axon stumps exhibited regenerative growth as early as 1 day after axotomy, and, by the third day, neurites had extended. By the fifth day, the regenerating axons had branched to form terminal varicosities similar to those of a normal neurohemal organ. The regenerated neurohemal structure appeared to be functional, because larvae that had been bilaterally axotomized were able to metamorphose to pupae, a process requiring temporally precise periods of prothoracicotropic hormone release. In addition to the regeneration of the terminal axon structures, several other responses to axotomy and retrocerebral organ excision occurred. These included an apparent accumulation of prothoracicotropic hormone in the axons and regenerating neurohemal-like structure, sprouting of ectopic neurites from the axotomized somata, and a change in shape of the cell bodies from spherical to avoid.

De novo formation of axon-like processes from axotomized retinal ganglion cells which exhibit long distance growth in a peripheral nerve graft in adult hamsters

Damaged axons in the central nervous system of the adult mammal can stimulated to regenerate extensively into a peripheral nerve graft. It was generally believed that the new axonal sprouts which extend into the graft arose from the injured proximal axonal stumps. However, when retinal ganglion cells of the adult hamster were axotomized by crushing the optic nerve and the proximal axonal stump was not in direct apposition to the graft, a new axon-like process could be seen to be emitted from either the cell soma or dendrite and extended in the graft for at least 1–2 cm. This axon-like process was distinct from the original injured axon which could still be seen to course towwards the optic disc in the retina. Evidently, even a fully differentiated central nervous system neuron of the adult mammal retains a great degree of morphological plasticity so that if the original axon is discouraged to regrow after injury, other parts of the neuron can act as favourable sites for the sprouting of a new axon-like process.

The pattern of dendritic sprouting and retraction induced by axotomy of lamprey central neurons

We have investigated some of the factors controlling the distribution of axonal and dendritic sprouting following axotomy of a subset of Muller giant interneurons (anterior bulbar cells or ABCs) in the hindbrain of the larval sea lamprey (Petromyzon marinus). Sprouts originated from different sites in the cell depending on the distance of the axonal lesion from the soma. When the axon was cut close to the soma (within 500 microns), the dendritic tips sprouted profusely, whereas the proximal axon stump showed few sprouts and frequently disappeared entirely. Axotomy further from the soma (1000-1400 microns) resulted in less sprouting from the dendrites and more from the axon stump, with the total amount of dendritic plus axonal sprouting remaining constant. Axotomy at sites distant from the soma (1 cm or more) did not result in dendritic sprouting. No sprouts were ever observed emerging from the soma proper or from the axon stump except at the lesion site. Neuritic sprouts from dendrites and axon were similar in their gross morphology. Sprouts resembled axons rather than dendrites whatever their sites of origin; they followed linear, rostrocaudally oriented paths in the "basal plate" region of the hindbrain. Dendritic and axonal sprouts grew both rostrally and caudally within the brain. Either "close" or "distant" axotomy resulted in the retraction of the dendritic tree and of both dendritic and axonal sprouts by several months postaxotomy. Reaxotomy close to the soma 30 d after a distant axotomy accelerated the onset of this evoked dendritic retraction. Reaxotomy close to the soma also induced sprouting significantly sooner than did close axotomy alone. These results suggest that axotomy close to the soma causes axonal regeneration to be shunted into ectopic locations at the dendritic tips. The emerging sprouts then follow guidance cues appropriate for regenerating ABC axons.

Dendritic amputation redistributes sprouting evoked by axotomy in lamprey central neurons

In the previous paper (Hall and Cohen, 1988), we showed that axotomy of anterior bulbar cells (ABCs) in the hindbrain of the larval lamprey results in the sprouting of axonlike neurites from either the end of the proximal axon stump, the dendritic tips, or both, depending on the site of axotomy. Here we show that, unlike axotomy, dendritic amputation (dendrotomy) does not by itself induce sprouting from ABCs. However, dendrotomy does induce sprouting from dendrites in the immediate vicinity of the dendritic lesion in cells that have been previously axotomized. We found that dendrotomy acts primarily to rearrange the distribution of sprouts induced by axotomy rather than serving as an additional stimulus to neurite outgrowth. We propose that (1) dendritic sprouting in ABCs occurs because the dendritic tips become attractive sites for sprout initiation when they are either directly injured (as with dendrotomy) or are situated relatively close to the site of injury (as with axotomy close to the soma), and (2) the axon stump, dendritic stumps, and uninjured dendritic tips of the cell compete to initiate a limited total amount of sprouting induced by axotomy. The probability that a given locus will support sprouting is determined both by its proximity to the nearest lesion site and by whether there are other attractive potential sprouting sites in the cell.

Survival and subsequent regeneration of olfactory neurons after a distal axonal lesion.

If an axonal lesion is made close enough to the cell body, injured olfactory neurons degenerate and are replaced by new nerve cells arising from undifferentiated mucosal basal cells. Therefore, under these conditions neural regeneration occurs through a process similar to neuronal development. The use of the long garfish olfactory nerve has revealed that neuronal death is not an inevitable consequence of an axonal injury and that the extent of cell death depends on the distance between the site of injury and the perikaryon. A lesion located up to 40 mm from the cell body induces the death of all mature neurons. Between 60 and 100 mm an increasing proportion of neurons survive the injury (from 60 to more than 90%) and are able to regenerate their distal segment. During regeneration, two populations of growing axons have been characterized. The fastest growing fibres (5.8 mm per day) correspond to the small population of neurons which were already growing at the time of the lesion and are able to survive an injury at any distance along the nerve. The majority of the regenerating fibres grow at 0.8 mm per day and corresponds to the damaged mature neurons. Elongation velocities were not affected by the distance from injury to cell body or by the mode of neural repair (development or regeneration). The initial delay between the injury and the beginning of elongation increases linearly with distance at a rate of 1 day cm-1 and is independent of the elongation velocity of the growing neurons. This indicates that the mechanism responsible for the beginning of axonal growth is initiated at or near the cell body and involves the entire axonal stump and not only the area surrounding the crush site. The ability of nerve cells to survive an injury may depend on the length of the axonal stump remaining attached to the cell body and on the level of protein synthesis at the time of the crush. From preliminary results it can be hypothesized that the length of the initial delay is determined by a reorganization of the cytoskeletal elements in the proximal axonal stump.

Changes in neurofilament gene expression occur after axotomy of dorsal root ganglion neurons: an in situ hybridization study.

Neurofilaments (NFs) are predominant elements in large myelinated axons, where they are thought to serve the important function of maintaining axonal caliber. Previous studies have shown that changes in NF synthesis and axonal transport occur after axonal injury in rat dorsal root ganglion (DRG) cells. The resulting reduction in the NF supply to DRG axons is thought to be largely responsible for the observed decrease in axonal diameter in the proximal axonal stump after an injury. In the present study, we test the hypothesis that a change in NF gene expression precedes the changes in synthesis and transport of NF proteins. To address this hypothesis, the levels of mRNA encoding the 68-kilodalton (kd) neurofilament protein (NF68) in adult rat DRG neurons were assessed at different times after peripheral axotomy using in situ hybridization. For these studies we used a 35S-labeled cDNA probe to NF68. The levels of NF68 mRNA in sensory neurons located in ipsilateral fourth and fifth lumbar DRG at 1, 7, and 14 days after sciatic nerve crush were compared to those in normal DRG neurons using quantitative autoradiography. In large DRG neurons (greater than 1000 micron 2), the levels of NF68 mRNA were significantly reduced relative to normal at 1, 7, and 14 days after axotomy. Medium-sized cells (601-1000 micron 2) exhibited a reduction only at 14 days postinjury, and small-sized cells were not significantly affected. These findings indicate that larger DRG neurons which give rise to large myelinated sensory axons exhibit a change in NF gene expression after axonal injury. The observed changes in NF68 mRNA levels temporally precede changes in NF synthesis and transport in injured DRG cells. Thus, a change in NF gene expression may be an important component of an effective regenerative response and a critical step at which axonal caliber is regulated in injured neurons.

Neurofilament gene expression: a major determinant of axonal caliber.

Within the wide spectrum of axonal diameters occurring in mammalian nerve fibers, each class of neurons has a relatively restricted range of axonal calibers. The control of caliber has functional significance because diameter is the principal determinant of conduction velocity in myelinated nerve fibers. Previous observations support the hypothesis that neurofilaments (NF) are major intrinsic determinants of axonal caliber in large myelinated nerve fibers. Following interruption of axons (axotomy) by crushing or cutting a peripheral nerve, caliber is reduced in the proximal axonal stumps, which extend from the cell bodies to the site of axotomy. (The distal axonal stumps, which are disconnected from the cell bodies, degenerate and are replaced by the outgrowth of regenerating axonal sprouts arising from the proximal stump). This reduction in axonal caliber in the proximal stumps is associated with a selective diminution in the amount of NF protein undergoing slow axonal transport in these axons, with a decrease in axonal NF content, and with reduced conduction velocity. The present report demonstrates that changes in axonal caliber after axotomy correlate with a selective alteration in NF gene expression. Hybridization with specific cDNAs was used to measure levels of mRNA encoding the 68-kDa neurofilament protein (NF68), beta-tubulin, and actin in lumbar sensory neurons of rat at various times after crushing the sciatic nerve. Between 4 and 42 days after axotomy by nerve crush, the levels of NF68 mRNA were reduced 2- to 3-fold. At the same times, the levels of tubulin and actin mRNAs were increased several-fold. These findings support the hypothesis that the expression of a single set of neuron-specific genes (encoding NF) directly determines axonal caliber, a feature of neuronal morphology with important consequences for physiology and behavior.

Differential time course of the response to axotomy induced by cut or crush in the leech AP cell

The time course of the reaction to axotomy in the leech AP cell was determined by measuring the duration of the spontaneous spikes at different times after the operation. The axotomy performed by section of the segmental roots containing the AP axon induced an increase of the spike duration, which persisted over 30 days. A different time course was found when the axotomy was performed by nerve crush: the changes in duration of the spontaneous spikes, which occurred during the early 2 weeks, were significantly reduced afterwards. Dye staining of some cells axotomized by crushing revealed that the reversion of the changes, which had been set up by axotomy, was in some cases concomitant with the reconnection between proximal and distal axon stumps. The section of a single axonal branch was never sufficient to affect the membrane properties of the AP cells. It is concluded that the changes observed in axotomized AP cells are not produced by simple axonal injury and that the maintainance of normal properties in the somatic membrane requires the presence of at least part of the distal axon arborization.