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PubMed1992

Passive electrical properties of motoneurons in aged cats following axotomy.

The objective of this study was to determine whether the aging process influences the changes in the electrophysiological properties of motoneurons that occur as a consequence of axotomy. Accordingly, using intracellular recording and stimulating techniques, the basic electrical properties of control (unaxotomized) and axotomized spinal cord motoneurons of aged cats were determined. Compared with control motoneurons, axotomized motoneurons exhibited increases in input resistance (Rin), membrane time constant (tau b) and the equalizing time constant (tau c). While the electrotonic length (L) remained unchanged, axotomy induced a decrease in the total cell capacitance (Ccell). The post-axotomy reduction of Ccell indicates that the motoneuron surface area was reduced and the increased membrane time constant indicates that there was an increase in membrane resistivity (Rm). The post-axotomy conservation of L accompanied by an increase in Rm suggests that aged axotomized motoneurons undergo geometrical changes. Furthermore, calculations based on cable theory suggest that the diameter of the equivalent cylinder (d) decreased following axotomy, whereas the equivalent cylinder length (l) remained unaffected. It is concluded that axotomy produces significant alterations in the soma-dendritic portion of aged spinal motoneurons, as indicated by the changes found in their passive electrophysiological properties, and that the pattern of the response that occurs in axotomized motoneurons of adult cats is also present in axotomized motoneurons of aged animals.

Aging↗
PubMed1996

Vasoactive intestinal peptide expression in enteric neurons is upregulated by both colchicine and axotomy.

Axotomy is known to induce changes in neuropeptide expression in several types of neurons. Colchicine blocks the axonal transport and may mimic axotomy. The effects of colchicine-treatment and axotomy (local nerve crush by clamping of the gut) on enteric neurons expressing vasoactive intestinal peptide, neuropeptide Y and nitric oxide synthase were studied in rat small intestine by immunocytochemistry and in situ hybridization. Colchicine treatment significantly increased the number of submucous and myenteric neurons expressing vasoactive intestinal peptide and its mRNA. In contrast, an increase in the number of neuropeptide Y or nitric oxide synthase expressing neurons could not be detected. Axotomy markedly increased the number of myenteric vasoactive intestinal peptide-immunoreactive neurons in the segment located orally to the lesion, but not in the segment anally to the lesion, whereas that of nitric oxide synthase and neuropeptide Y expressing neurons was not affected. Double immunostaining revealed that the myenteric neurons containing nitric oxide synthase were induced by colchicine and axotomy to express vasoactive intestinal peptide. The present data indicate that colchicine and axotomy may induce marked changes in the neuropeptide expression of enteric neurons.

Animals↗
PubMed1997

Differential effects of axotomy on the in vivo synthesis of the stress-inducible and constitutive 70-kDa heat-shock proteins in rat dorsal root ganglia.

The purpose of this study was to the test the hypothesis that heat-shock protein expression is upregulated (or induced) in dorsal root ganglia (DRG) following axotomy. To test this hypothesis, DRG or sciatic nerve (SN) proteins were pulse-labelled in vivo with [35S]methionine and the metabolic synthesis of two major 70-kDa heat-shock proteins, the constitutive species (hsc70) and stress-inducible species (hsp68), were analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and fluorography. Results showed that DRG hsp68 expression was absent (or barely detectable) under normal (sham-axotomy) conditions. However, following long-range axotomy (35 mm from DRG), there was a delayed (> 12 h post-axotomy) and transient upregulation of DRG hsp68 metabolic synthesis. Control studies demonstrated that, although DRG hsp68 was upregulated, hsp68 was not induced in SN regions proximal to the crush site. In contrast to DRG hsp68 expression, there was abundant DRG hsc70 synthesis under normal conditions that did not significantly change following axotomy. These results suggest that a specific stress protein response is induced in DRG following axotomy.

Animals↗
PubMed1993

Spatiotemporal distribution of Ca2+ following axotomy and throughout the recovery process of cultured Aplysia neurons.

This study investigates the alterations in the spatiotemporal distribution pattern of the free intracellular Ca2+ concentration ([Ca2+]i) during axotomy and throughout the recovery process of cultured Aplysia neurons, and correlates these alterations with changes in the neurons input resistance and trans-membrane potential. For the experiments, the axons were transected while imaging the changes in [Ca2+]i with fura-2, and monitoring the neurons' resting potential and input resistance (Ri) with an intracellular microelectrode inserted into the cell body. The alterations in the spatiotemporal distribution pattern of [Ca2+]i were essentially the same in the proximal and the distal segments, and occurred in two distinct steps: concomitantly with the rupturing of the axolemma, as evidenced by membrane depolarization and a decrease in the input resistance, [Ca2+]i increased from resting levels of 0.05-0.1 microM to 1-1.5 microM along the entire axon. This is followed by a slower process in which a [Ca2+]i front propagates at a rate of 11-16 microns/s from the point of transection towards the intact ends, elevating [Ca2+]i to 3-18 microM. Following the resealing of the cut end 0.5-2 min post-axotomy, [Ca2+]i recovers in a typical pattern of a retreating front, travelling from the intact ends towards the cut regions. The [Ca2+]i recovers to the control level 7-10 min post-axotomy. In Ca(2+)-free artificial sea water (2.5 mM EGTA) axotomy does not lead to increased [Ca2+]i and a membrane seal is not formed over the cut end. Upon reperfusion with normal artificial sea water, [Ca2+]i is elevated at the tip of the cut axon and a membrane seal is formed. This experiment, together with the observations that injections of Ca2+, Mg2+ and Na+ into intact axons do not induce the release of Ca2+ from intracellular stores, indicates that Ca2+ influx through voltage gated Ca2+ channels and through the cut end are the primary sources of [Ca2+]i following axotomy. However, examination of the spatiotemporal distribution pattern of [Ca2+]i following axotomy and during the recovery process indicates that diffusion is not the dominating process in shaping the [Ca2+]i gradients. Other Ca2+ regulatory mechanisms seem to be very effective in limiting these gradients, thus enabling the neuron to survive the injury.

Animals↗
PubMed1994

Effect of peripheral axotomy on expression of neuropeptide Y receptor mRNA in rat lumbar dorsal root ganglia.

Using in situ hybridization, the expression of the mRNA for a neuropeptide Y (NPY) receptor, was studied in lumbar (L) 4 and 5 dorsal root ganglia (DRGs) of normal rats and at various intervals after unilateral sciatic nerve transection. Twenty percent of all normal DRG neurons were NPY receptor mRNA-positive, and the majority of these neurons were of the small type, with only a few labelled medium-sized and large neurons. In L5 normal ganglia NPY receptor mRNA colocalized with substance P, calcitonin gene-related peptide and galanin mRNAs in small neurons, but not in medium-sized or large neurons containing these peptides. NPY receptor mRNA was not observed in somatostatin or nitric oxide synthase mRNA-positive neurons. Sciatic nerve transection induced a marked decrease in NPY receptor mRNA levels. However, in parallel there was a transient increase in the number of NPY receptor mRNA-positive small neuron profiles, but the intensity of labelling was mostly very low, although a few strongly labelled, small neuron profiles were also encountered. In addition, axotomy caused a marked increase in the number of NPY receptor mRNA-positive large neuron profiles in the ipsilateral DRGs, and they constituted 15-20% of counted DRG neuron profiles and 45-65% of counted large neuron profiles, 7-28 days after axotomy. In L5 DRGs, ipsilateral to the axotomy, NPY receptor mRNA colocalized with NPY mRNA in many large and some medium-sized neuron profiles, with galanin mRNA in some small, medium-sized and large neuron profiles and with vasoactive intestinal polypeptide mRNA in some small and medium-sized neuron profiles and a few large profiles. Occasionally, NPY receptor mRNA was observed in nitric oxide synthase mRNA-positive small neurons. In the dorsal horn, NPY receptor mRNA-positive small neurons were concentrated in lamina II at L4 and L5 levels, and were scattered in deeper laminae. No marked changes were observed ipsilateral to the axotomy. No NPY receptor mRNA-positive cells were found in the normal rat gracile nucleus, or in this nucleus after axotomy. These results show that a NPY receptor may be a prejunctional receptor in primary afferent neurons and play a role in the modulation of somatosensory information, both in normal and lesioned primary afferent DRG cells. However, axotomy induced a distinct shift in NPY receptor mRNA expression from small to large neurons, indicating that sensitivity to NPY is switched from one modality to another.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1984

Effects of denervation and axotomy on nervous system-specific protein, ornithine decarboxylase, and other enzyme activities in the superior cervical sympathetic ganglion of the rat.

The time courses of changes of three enolase isozymes (alpha alpha, alpha gamma, and gamma gamma), S-100 protein, 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), ornithine decarboxylase (ODC), beta-galactosidase, and glucose-6-phosphate dehydrogenase (G6PDH) were examined from 1 to 14 days after cutting of the preganglionic nerve (denervation) or the postganglionic nerve (axotomy) of the superior cervical sympathetic ganglion (SCG) of the rat. The wet weight and protein content in the axotomized SCG increased continuously, to nearly twice those of the denervated SCG for 1-2 weeks after the operations. Among enolase isozymes in the SCG, neuron-specific gamma gamma-enolase decreased rapidly after denervation and stayed at a low level for 2 weeks, whereas the isozyme remained almost unchanged after axotomy. On the contrary, ganglionic alpha alpha-enolase and the alpha gamma-hybrid form increased remarkably to reach a maximum at the second day after axotomy, and remained above control for 1 to 2 weeks; these two enolase isozymes showed little change after denervation. Denervation caused a much larger increase than did axotomy in the ganglionic S-100 protein, an astrocyte-specific protein, during the first week after the operation, while the protein content decreased after 2 weeks of either denervation or axotomy. CNPase, a myelin-associated enzyme, rose suddenly 2 days after axotomy, and remained at a rather high level compared with the denervated ganglion, which showed little variation.(ABSTRACT TRUNCATED AT 250 WORDS)

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1988

Electrophysiology of degenerating neurones in the vagal motor nucleus of the guinea-pig following axotomy.

1. The electrophysiological properties of motoneurones in the dorsal motor nucleus of the vagus in the guinea-pig were studied at different times following cervical vagotomy. The results were compared both to normal neurones and to results obtained at the same time from intact neurones located in the contralateral nucleus. 2. The input resistances of axotomized neurones are significantly higher than those of normal neurones (66 +/- 29 compared to 45 +/- 17 M omega). This difference was seen during the first month following axotomy without any sign of a time-dependent process. On the other hand, no change in resting potential was observed. 3. Significant reduction in action potential amplitude was observed 1 month after axotomy (from 97.8 +/- 8 to 87 +/- 7 mV) and was followed by slow recovery lasting more than 1 year. Neither the Na+ conductance nor the voltage-dependent K+ conductance responsible for the fast rise and fall of the action potential, respectively, were affected by axotomy. 4. One month after axotomy the action potential duration in axotomized neurones was found to be shorter than that of normal neurones (0.9 +/- 0.1 ms compared to 1.1 +/- 0.04 ms). We show that this decrease in duration reflects a reduction in the depolarizing hump on the falling phase of the action potential, which is known to express the Ca2+ conductance activated during the action potential. A slow recovery of the spike duration was observed, although an age-dependent reduction in duration was also observed in neurones in the contralateral nucleus. 5. Two K+ conductances, the Ca2+-dependent and the A type, decrease 1 month after axotomy and follow a similar time course of recovery to that of the reduction in action potential duration and amplitude. 6. The firing pattern of axotomized neurones undergoes profound alteration, manifested as an increase in firing duration as a response to a rectangular current pulse. Examination of these alterations reveals that the reduction in both K+ conductances is responsible for the observed changes. 7. The results are discussed within the framework of the degenerative response known to take place in the nucleus following axotomy. We hypothesize that the observed phenomena reflect an increase in intracellular Ca2+ concentration which, in turn, inactivates the Ca2+ and K+ conductances. Furthermore this rise in intracellular Ca2+ may eventually be responsible for cell death.

Action Potentials↗
PubMed1988

The effects of axotomy on electrophysiological properties of B cells of bullfrog sympathetic ganglia conditioned by a previous lesion.

In bullfrog sympathetic B cells, axotomy decreases the amplitude and decay time of membrane afterhyperpolarization (AHP) and increases action potential (AP) duration. A second (test) axotomy, 7 days after an initial (conditioning) axotomy, did not amplify these changes. No recovery of AHP amplitude or AP duration occurred by 56 days post-axotomy, but AHP decay time recovered 21 days earlier than after test axotomy alone. Conditioning, previously shown to accelerate regeneration, speeds the return to normal of those membrane properties previously shown to recover after axotomy.

Animals↗
PubMed1993

Alterations in membrane potential after axotomy at different distances from the soma of an identified neuron and the effect of depolarization on neurite outgrowth and calcium channel expression.

1. Intracellular recordings were made from the soma of an identified neuron B5 within the buccal ganglion of the mollusc, Helisoma trivolvis, during axotomy induced by crushing or cutting the esophageal nerve. Axotomy was associated with a rapid depolarization and occasionally a burst of action potentials (injury discharge). The magnitude of the membrane depolarization in the soma in response to axotomy decayed exponentially when the distance between the soma and site of injury was increased. Input resistance measurements taken during axotomy showed that a barrier to current flow formed rapidly and gradually recovered within 2 h. A barrier to the diffusion of intracellularly injected carboxyfluorescein formed at the site of injury within 15 min of axotomy. 2. To examine the effect of chronic depolarization on neurite outgrowth, the extracellular potassium ion concentration [K+]o was manipulated. The membrane potential of neurons B5 exhibited a 51.8 mV/decade potassium dependence between 20 and 150 mM [K+]o. The initiation of neurite outgrowth from axons crushed 800 microns from the soma and bathed in different concentrations of [K+]o was examined by fluorescence microscopy after filling neurons with Lucifer yellow. We compared the percentage of axons with sprouts 9 and 24 h after organ culture in saline containing [K+]o ranging from 0.1 to 50 mM. Sprouting occurred from 33% of neurons B5 in normal saline (1.7 mM [K+]o) after 9 h and from 100% of neurons after 24 h. No sprouting was observed from neurons B5 9 or 24 h after axotomy when bathed in saline containing reduced or elevated concentrations of [K+]o. 3. To examine the effects of chronic depolarizatin on neurite outgrowth over several days, neurons B5 were axotomized close to the soma and maintained in organ culture in Liebovitz medium (defined medium or medium conditioned with central ganglia). Neurite outgrowth was ranked from 0 to 5 after filling neurons with Lucifer yellow, and our analysis indicated that a small increase in neurite outgrowth occurred in medium supplemented with 10 mM potassium. 4. Elevated potassium did not trigger neurite outgrowth from isolated neurons B5 in cell culture within defined medium, but whole-cell patch-clamp analysis revealed that chronic depolarization associated with elevated potassium altered the expression of calcium currents. Low-voltage-activated (LVA) and high-voltage-activated (HVA) calcium currents were detected in acutely isolated neurons B5.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1988

Spontaneous activity in afferent and efferent fibers after chronic axotomy: response to potassium channel blockade.

Distally propagating spontaneous impulses in acutely and chronically cut rat saphenous nerve were examined to determine (1) the origin(s) of the activity, (2) the fiber types involved, and (3) whether the activity was affected by potassium channel blockade. Under deep pentobarbital anesthesia, six male Sprague-Dawley rats underwent L3 cauda equina section, then unilateral saphenous axotomy. The nerve was then dissected into 30-50 microfilaments and surveyed for spontaneous activity using a modification of the microfilament recording method. Afterward, the nerve was cut back, and a potassium channel blocking agent (gallamine) was administered. The axonal activity was once again surveyed in the same fashion. Twenty-eight rats underwent unilateral saphenous axotomy 1-8 weeks prior to similar recordings, and the neuroma was excised just before microfilament dissection. Spontaneous discharges in these preparations originated from three foci: (1) antidromic activity from in-continuity dorsal root ganglia (DRG), (2) orthodromic activity from sympathetic neurons, and (3) antidromic activation of dichotomizing afferent axons in the peripheral nerve. There was significantly more antidromic activity from DRG in rats with prior axotomies than in control animals (t = 2.38; p less than 0.025), and gallamine produced a significant increase in DRG activity in the chronically lesioned nerve (t = 2.43; p less than 0.005), but not in acutely lesioned controls. However, most of the spontaneous activity in these preparations was from sympathetic efferents. This activity was decreased significantly by chronic axotomy (t = 2.635; p less than 0.01), and it was not affected by potassium channel blockade with gallamine. In two microfilaments, spontaneous antidromic action potentials were observed in conjunction with a clear receptive field on blood vessels in the nearby fascia. Both of these presumably dichotomized axons were found in acutely cut nerve, thus were not the result of retrograde sprouting from a neuroma. It was concluded that (1) chronic axotomy of sensory afferents produced ectopic activity in their respective DRG, (2) gallamine administration increased spontaneous activity from DRG in chronically axotomized rats, (3) ongoing sympathetic efferent activity in rat saphenous nerve was decreased by distal axotomy for up to 8 weeks, and (4) rare branched sensory afferents occasionally exhibit spontaneous activity.

Adrenergic Fibers↗
PubMed1991

Axotomy-like changes in cat motoneuron electrical properties elicited by botulinum toxin depend on the complete elimination of neuromuscular transmission.

The electrical properties of cat medial gastrocnemius (MG) spinal motoneurons were studied 14-21 d following injection of type A botulinum toxin (BTX) into the MG muscle. Treated MG muscles were atrophic, displayed pronounced fibrillation activity, and were markedly but not completely paralyzed. MG motoneuron electrical properties from animals with the highest MG muscle-twitch forces (greater than 20 gm) appeared normal, while motoneuron properties from animals with the lowest MG muscle-twitch forces (less than 10 gm) exhibited axotomy-like changes, though these changes were less pronounced than after axotomy itself. No changes in the axonal conduction velocity were observed, however. Motoneuron connectivity with MG muscle fibers was determined following intracellular stimulation of MG motoneurons by averaging EMG signals from 3 or 4 pairs of recording electrodes inserted into the BTX-treated MG muscles. Normal electrical properties were observed among motoneurons in which detectable EMG activity linked to the intracellular stimulation pulse was observed. The level of this connectivity, however, indicated that a relatively small number of muscle fibers were activated by individual motoneuron action potentials. Axotomy-like changes of electrical properties were observed in MG motoneurons that could not be associated with detectable EMG activity in the BTX-treated MG muscle following repeated trials of intracellular stimulation. These results indicate that the existence of effective neuromuscular transmission at a small number of motor terminals is sufficient to prevent the appearance of axotomy-like changes in motoneuron electrical properties, and that the absence of such transmission at all motor terminals is associated with the appearance of axotomy-like changes. The results suggest that the effects of axotomy itself on motoneuron properties may be based upon the loss or elimination of a potent interaction between muscle and motoneurons normally mediated by neuromuscular transmission.

Animals↗
PubMed1988

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.

Animals↗
PubMed1996

Trophic effects of androgen: receptor expression and the survival of laryngeal motor neurons after axotomy.

To determine whether changes in androgen receptor (AR) expression are associated with trophic actions of androgens, we have examined the laryngeal motor nucleus (N. IX-X) of Xenopus laevis 1 and 5 months after section of the laryngeal nerve. In situ hybridization was used to recognize cells expressing mRNA for the Xenopus AR and bromodeoxyuridine to assess cell proliferation. In addition, the total number of cells was determined in untreated and dihydrotestosterone (DHT)-treated animals after 5 months of axotomy. After 1 month of axotomy, the number of AR mRNA-expressing cells in N. IX-X is 1.8-fold higher than in the intact side. Androgen upregulates expression of AR mRNA in N. IX-X on both the intact and the axotomized sides, suggesting that the increase is independent of contact with muscle. Neither the axotomy- nor the androgen-induced increase in number of cells expressing AR mRNA is attributable to cell proliferation. Five months after axotomy, both the total number of cells and the number of AR mRNA-expressing cells are severely decreased in the axotomized N. IX-X. DHT treatment mitigates the cell loss in N. IX-X induced by prolonged axotomy; the effect includes maintenance of AR mRNA-expressing cells. Gonadally intact males have more cells in the axotomized N. IX-X than castrated animals, suggesting that androgen acts at physiological levels as a trophic hormone. Axotomy-induced upregulation of AR expression may facilitate the trophic actions of androgens.

Age Factors↗
PubMed1999

Influence of the axotomy to cell body distance in rat rubrospinal and spinal motoneurons: differential regulation of GAP-43, tubulins, and neurofilament-M.

Axotomized motoneurons regenerate their axons regardless of whether axotomy occurs proximally or distally from their cell bodies. In contrast, regeneration of rubrospinal axons into peripheral nerve grafts has been detected after cervical but not after thoracic injury of the rubrospinal tract. By using in situ hybridization (ISH) combined with reliable retrograde tracing methods, we compared regeneration-associated gene expression after proximal and distal axotomy in spinal motoneurons versus rubrospinal neurons. Regardless of whether they were axotomized at the iliac crest (proximal) or popliteal fossa (distal), sciatic motoneurons underwent highly pronounced changes in ISH signals for Growth Associated Protein 43 (GAP-43) (10-20x increase) and neurofilament M (60-85% decrease). In contrast, tubulin ISH signals substantially increased only after proximal axotomy (3-5x increase). To compare these changes in gene expression with those of axotomized rubrospinal neurons, the rubrospinal tract was transected at the cervical (proximal) or thoracic (distal) levels of the spinal cord. Cervically axotomized rubrospinal neurons showed three- to fivefold increases in ISH signals for GAP-43 and tubulins (only transient) and a 75% decrease for neurofilament-M. In sharp contrast, thoracic axotomy had only marginal effects. After implantation of peripheral nerve transplants into the spinal cord injury sites, retrograde labeling with the sensitive retrograde tracer Fluoro-Gold identified regenerating rubrospinal neurons only after cervical axotomy. Furthermore, rubrospinal neurons specifically regenerating into the transplants were hypertrophied and expressed high levels of GAP-43 and tubulins. Taken together, these data support the concept that, even if central nervous system (CNS) axons are presented with a permissive/supportive environment, appropriate cell body responses to injury are a prerequisite for CNS axonal regeneration.

Animals↗
PubMed1998

Differential regulation of levels of nicotinic receptor subunit transcripts in adult sympathetic neurons after axotomy.

Axotomy of adult peripheral neurons produces decreases in the levels of transcripts for a number of proteins involved in synaptic transmission. For example, tyrosine hydroxylase and neuropeptide Y mRNA decrease in axotomized sympathetic neurons in the superior cervical ganglion (SCG). In the present study, the effects of axotomy on the expression of nicotinic receptor subunit transcripts were examined in the SCG and the results were compared to those produced by deafferentation and explantation. Normally, neurons in the SCG express five different nicotinic subunits: alpha3, alpha5, alpha7, beta2, and beta4. Forty-eight hours after axotomy in vivo or explantation, dramatic decreases in these transcripts were seen, except for beta2, which increased. In contrast, deafferentation of the SCG had negligible effects on any of these transcripts. Both leukemia inhibitory factor (LIF) and nerve growth factor (NGF) have been shown to play a role in the decrease in neuropeptide Y mRNA expression after axotomy. In the cases of these nicotinic receptor transcripts, however, similar decreases were seen in wild-type and LIF knockout animals. Furthermore, administration of an antiserum to NGF in intact animals produced no changes in transcript levels. On the other hand, providing exogenous NGF to axotomized SCG in vivo or in explant cultures partially prevented the decreases in the transcripts for alpha3, alpha5, alpha7, and beta4. These data indicate that axotomy produces dramatic decreases in the expression of several nicotinic receptor subunit transcripts, and that the molecular signals underlying these changes differ from those previously shown to mediate the decrease in neuropeptide Y expression.

Animals↗
PubMed2003

Critical calpain-dependent ultrastructural alterations underlie the transformation of an axonal segment into a growth cone after axotomy of cultured Aplysia neurons.

The transformation of a stable axonal segment into a motile growth cone is a critical step in the regeneration of amputated axons. In earlier studies we found that axotomy of cultured Aplysia neurons leads to a transient and local elevation of the free intracellular Ca2+ concentration, resulting in calpain activation, localized proteolysis of submembranal spectrin, and, eventually, growth cone formation. Moreover, inhibition of calpain by calpeptin prior to axotomy inhibits growth cone formation. Here we investigated the mechanisms by which calpain activation participates in the transformation of an axonal segment into a growth cone. To that end we compared the ultrastructural alterations induced by axotomy performed under control conditions with those caused by axotomy performed in the presence of calpeptin, using cultured Aplysia neurons as a model. We identified the critical calpain-dependent cytoarchitectural alterations that underlie the formation of a growth cone after axotomy. Calpain-dependent processes lead to restructuring of the neurofilaments and microtubules to form an altered cytoskeletal region 50-150 microm proximal to the tip of the transected axon in which vesicles accumulate. The dense pool of vesicles forms in close proximity to a segment of the plasma membrane along which the spectrin membrane skeleton has been proteolyzed by calpain. We suggest that the rearrangement of the cytoskeleton forms a transient cellular compartment that traps transported vesicles and serves as a locus for microtubule polymerization. We propose that this cytoskeletal configuration facilitates the fusion of vesicles with the plasma membrane, promoting the extension of the growth cone's lamellipodium. The growth process is further supported by the radial polymerization of microtubules from the growth cone's center.

Animals↗
PubMed2002

FK506 increases peripheral nerve regeneration after chronic axotomy but not after chronic schwann cell denervation.

Poor functional recovery after peripheral nerve injury is attributable, at least in part, to chronic motoneuron axotomy and chronic Schwann cell (SC) denervation. While FK506 has been shown to accelerate the rate of nerve regeneration following a sciatic nerve crush or immediate nerve repair, for clinical application, it is important to determine whether the drug is effective after chronic nerve injuries. Two models were employed in the same adult rats using cross-sutures: chronic axotomy and chronic denervation of SCs. For chronic axotomy, a chronically (2 months) injured proximal tibial (TIB) was sutured to a freshly cut common peroneal (CP) nerve. For chronic denervation, a chronically (2 months) injured distal CP nerve was sutured to a freshly cut TIB nerve. Rats were given subcutaneous injections of FK506 or saline (5 mg/kg/day) for 3 weeks. In the chronic axotomy model, FK506 doubled the number of regenerated motoneurons identified by retrograde labeling (from 205 to 414 TIB motoneurons) and increased the numbers of myelinated axons (from 57 to 93 per 1000 microm2) and their myelin sheath thicknesses (from 0.42 to 0.78 microm) in the distal nerve stump. In contrast, after chronic denervation, FK506 did not improve the reduced capacity of SCs to support axonal regeneration. Taken together, the results suggest that FK506 acts directly on the neuron (as opposed to the denervated distal nerve stump) to accelerate and promote axonal regeneration of neurons whose regenerative capacity is significantly reduced by chronic axotomy.

Animals↗
PubMed2005

The effects of delayed nerve repair on neuronal survival and axonal regeneration after seventh cervical spinal nerve axotomy in adult rats.

It has been proposed clinically that delayed surgery after traumatic brachial plexus injury may adversely affect functional outcome. In the present experimental study the neuroprotective and growth-promoting effects of early and delayed nerve grafting following proximal seventh cervical spinal nerve (C7) axotomy were examined. The ventral branch of C7 spinal nerve was transected and axons projecting out of the proximal nerve stump were labelled with Fast Blue (FB). At the same time, the biceps brachii muscle was denervated by transecting the musculocutaneous nerve at its origin. Neuronal survival and muscle atrophy were then assessed at 1, 4, 8 and 16 weeks after permanent axotomy. In the experimental groups, a peripheral nerve graft was interposed between the transected C7 spinal nerve and the distal stump of the musculocutaneous nerve at 1 week [early nerve repair (ENR)] or 8 weeks [delayed nerve repair (DNR)] after axotomy. Sixteen weeks after nerve repair had been performed, a second tracer Fluoro-Ruby (FR) was applied distal to the graft to assess the efficacy of axonal regeneration. Counts of FB-labelled neurons revealed that axotomy did not induce any significant cell loss at 4 weeks, but 15% of motoneurons and 32% of sensory neurons died at 8 weeks after injury. At 16 weeks, the amount of cell loss in spinal cord and dorsal root ganglion (DRG) reached 29 and 50%, respectively. Both ENR and DNR prevented retrograde degeneration of spinal motoneurons and counteracted muscle atrophy, but failed to rescue sensory neurons. Due to substantial cell loss at 8 weeks, the number of FR-labelled neurons after DNR was significantly lower when compared to ENR. However, the proportion of regenerating neurons among surviving motoneurons and DRG neurons remained relatively constant indicating that neurons retained their regenerative capacity after prolonged axotomy. The results demonstrate that DNR could protect spinal motoneurons and reduce muscle atrophy, but had little effect on sensory DRG neurons. However, the efficacy of neuroprotection and axonal regeneration will be significantly affected by the amount of cell loss already presented at the time of nerve repair.

Animals↗