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PubMed1994

Type III sodium channel mRNA is expressed in embryonic but not adult spinal sensory neurons, and is reexpressed following axotomy.

1. In situ hybridization with subtype-specific probes was used to ask whether there is a change in the types of sodium channels that are expressed in dorsal root ganglion (DRG) neurons after axotomy. 2. Types I and II sodium channel mRNA are expressed at moderate-to-high levels in control DRG neurons of adult rat, but type III sodium channel mRNA is not detectable. 3. When adult rat DRG neurons are examined by in situ hybridization 7-9 days following axotomy, type III sodium channel mRNA is expressed at moderate-to-high levels, in addition to types I and II mRNA that are present at relatively high levels. 4. To determine whether the expression of type III sodium channel mRNA following axotomy represents up-regulation of a gene that had been expressed at earlier developmental stages, we also studied DRG neurons from embryonic (E17) rats. In these embryonic DRG neurons, type I sodium channel mRNA is expressed at low levels, type II mRNA at high levels, and type III at high levels. 5. These results demonstrate altered expression of sodium channel mRNA in DRG neurons following axotomy, and suggest that in at least some DRG neurons, there is a de-differentiation after axotomy that includes a reversion to an embryonic mode of sodium channel expression. Different channel characteristics, as well as an altered spatial distribution of sodium channels, may contribute to the electrophysiological changes that are observed in axotomized neurons.

Animals↗
PubMed1995

Axotomy induces a transient and localized elevation of the free intracellular calcium concentration to the millimolar range.

1. Axonal transection triggers a cascade of pathological processes that frequently lead to the degeneration of the injured neuron. It is generally believed that the degenerative process is triggered by an overwhelming influx of calcium through the cut end of the axon. 2. Theoretical considerations and indirect observations suggest that axotomy is followed by an increase in the free intracellular calcium concentration ([Ca2+]i) to the millimolar level. In contrast, only relatively modest and transient elevation in [Ca2+]i to the micromolar level was revealed by recent fura-2 studies. 3. In the current study we used the low-affinity Ca2+ indicator mag-fura-2 to reexamine the spatiotemporal distribution pattern of Ca2+ after axotomy and to map the free intracellular Mg2+ concentration gradients. 4. We report that axotomy elevates [Ca2+]i well beyond the "physiological" range of calcium concentrations, to levels > 1 mM near the tip of the cut axon and to hundreds of micromolars along the axon further away from the cut end. Nevertheless, [Ca2+]i recovers to the control levels within 2-3 min after the resealing of the cut end. 5. A comparison of the behavior of fura-2 and mag-fura-2 in the cytosol of the axotomized neurons reveals that the determination of [Ca2+]i by fura-2 largely underestimates the actual intracellular Ca2+ concentrations. 6. Experiments in which one branch of a bifurcated axon was transected revealed that the elevation in [Ca2+]i is confined to the transected axonal branch and does not spread beyond the bifurcation point. 7. After axotomy, the intracellular Mg2+ concentration equilibrates rapidly with the external concentration and then recovers at a rate somewhat slower than that of [Ca2+]i. 8. To the best of our knowledge, this study is the first direct demonstration that axotomy elevates [Ca2+]i to the millimolar range and that neurons are able to recover from these extreme calcium concentrations.

Animals↗
PubMed1996

Axotomy induces retraction of the dendritic arbor of adult rat rubrospinal neurons.

The effect of distal axonal injury on the soma-dendritic morphology of intrinsic central neurons was examined using adult rat lumbar spinal cord-projecting rubrospinal neurons as a model. The soma-dendritic morphology was revealed using an improved Golgi-aldehyde method. Impregnated neurons were reconstructed in the two-dimensional plane for analysis. Four weeks after axotomy, neurons had reduced soma sizes and remained multipolar in shape. Some dendrites were found to end not far from their cell bodies. In addition, no long dendrite was identified following axotomy. Sholl's analysis [The Organization of the Cerebral Cortex. London, Methuen, [1956] revealed that axotomized neurons had fewer dendritic branches than control neurons. Total dendritic length was also reduced. Subsequent analyses showed that the average number of dendritic trunks was not altered however the mean number of terminal branches per dendritic trunk was reduced. The dendritic membrane of the normal neurons was usually smooth with occasional short protuberances on the proximal dendrites and spines on the distal dendrites, which did not change after axotomy. In control neurons, we identified an elaborate type of dendritic structure named dendritic appendage aggregates. These aggregates were located preferentially on terminal dendrites and were classified into three categories according to their complexity. The incidence of occurrence for these aggregates decreased following distal axotomy. These phenomena indicate that rat lumbar spinal cord-projecting rubrospinal neurons retract their distal dendrites in response to distal axotomy. The observed anatomic restructuring following axonal injury is likely to be accompanied by an alteration of afferents which normally synapse on distal dendrites.

Animals↗
PubMed1994

Nitric oxide synthase in motor neurons after axotomy.

Nitric oxide synthase (NOS), an enzyme involved in synthesis of nitric oxide (NO), has been localized in many diverse cell types. In the CNS and PNS, discrete neuron cell groups express NOS constitutively. Recent evidence indicates that NOS is inducible in neurons normally not expressing NOS. After transection of peripheral nerves, NOS expression was significantly up-regulated in the axotomized sensory ganglion cells, whereas in the corresponding motor neurons NOS was not induced unless axon regeneration was prevented and ensuing neuron death became massive. Studies on axotomy-induced NOS have been limited largely to spinal nerves, with only one reported in the vagus nerve. The aim of this study was to determine whether NOS induction in motor neurons of the brainstem after axotomy is regulated in a manner similar to that of the spinal cord. By NADPH-diaphorase histochemistry and NOS immunocytochemistry, the status of NOS in neurons of the hypoglossal nucleus, dorsal motor nucleus of the vagus, and motor nucleus of the facial nerve was examined 2 weeks after unilateral transection of the respective cranial nerves, and the results were compared with those of spinal motor neurons after transection of the sciatic nerve. NOS, undetectable in neurons of the three cranial motor nuclei of sham-operated animals, was observed in about 30-50% of neurons in the cranial motor nuclei ipsilateral to axotomy, but it was not detected in spinal motor neurons after axotomy. NOS localized in axotomized cranial motor neurons was unrelated to NOS of macrophages or endothelial cells. There was no appreciable cell loss from axotomy at this period except in the dorsal motor nucleus of the vagus, where some loss was observed. The results indicate that there is a fundamental difference in the regulation of NOS expression between motor neurons of the cranial and spinal nerves. The possible role of NOS/NO acting as cytoprotective or cytotoxic agent on injured motor neurons is discussed. Motor neurons of cranial and spinal nerves may serve as a useful model to further define the roles of NOS/NO in neurons, especially after traumatic injury.

Amino Acid Oxidoreductases↗
PubMed1991

The effect of age on motor neurone death following axotomy in the mouse.

The ability of mouse motor neurones to survive axotomy during the first month of life was studied. The motor neurones that lie in the dorsolateral columns of spinal segments C7 and C8 and supply the flexor muscles of the forepaw were axotomized by cutting and removing part of the median and ulnar nerves above the elbow. The number and position of cell bodies with axons in these nerves were confirmed by retrograde labelling of the cut axons with horseradish peroxidase. The ability of these neurones to survive axotomy varies with the age of the animal at the time of axotomy. When the axons are sectioned within the first four postnatal days, 80-90% of the cell bodies will die, more than half of this death occurring in less than one week after axotomy. If the animals are one week old at the time the nerves are cut, a significantly smaller number (50%) die (P = 0.013), and the time-course of death is different, with eight to ten days elapsing before half the death has occurred. 40% of the neurones will die if sectioned at two weeks of age, and it is not until four weeks of age that more than 90% of the cells can survive axotomy. We conclude, therefore, that the kinetics of motor neurone death, as well as the final extent of neuronal loss, are affected by the age at which the animal is axotomized.

Aging↗
PubMed2005

[Protective effects of transplantation of hCNTF-transfected cell line on axotomy-induced degeneration of retinal ganglion cells].

OBJECTIVE: To investigate the protective effects of transplantation of cell line stably expressing and secreting human ciliary neurotrophic factor (hCNTF) on the degeneration of retinal ganglion cells (RGCs) after optic nerve transection. METHODS: Plasmid encoding hCNTF was transfected into human lung fibroblast (HLF) cell line, then the stably transfected clones were selected with methopterin. In adult SD rats, RGCs were labeled with retrograde axonal tracer fluorogold (FG) injected to their targets including dorsal lateral geniculate nuclei (dLGN) and superior colliculi (SC). Seven days later, the optic nerve was transected alone or in combination with transplantation of HLF cells. Five, 14, 17, 21 and 28 days after axotomy, the retinas were mounted and examined under fluorescence microscope to observe the RGCs. RESULTS: Compared to the controls, the density of RGC in axotomy group decreased by 67.44% and 82.73% on the 14th and 28th day, respectively. In the eyes with hCNTF-transfected HLF cells transplantation, RGC density was higher than that of the axotomy group on the 5th, 17th, 21st day after axotomy (P < 0.05). On the 5th day, the morphology of RGC in the hCNTF group remained the same as the controls, whereas the morphology of RGC in the axotomy alone group began to change. CONCLUSIONS: hCNTF administered at the time of optic nerve transection can protect RGC from degeneration, increasing the numbers of surviving RGCs and delaying the death of RGCs.

Animals↗
PubMed1982

Neurogenesis in the vomeronasal epithelium of adult garter snakes. 2. Reconstitution of the bipolar neuron layer following experimental vomeronasal axotomy.

Postnatal neurogenesis and reconstitution of the neuronal layer of the vomeronasal epithelium have been demonstrated in adult garter snakes following vomeronasal axotomy. Two weeks following axotomy the vomeronasal epithelium was depleted of its bipolar layer but the basal, undifferentiated cells were actively proliferating. In subsequent weeks the undifferentiated cell layer continued to increase its cell population through mitosis and began to occupy the neuron-depleted zone of the receptor cell column. Four weeks following axotomy the denervated epithelium contained an expanded Ud cell layer which occupied the basal one-half to two-thirds of the receptor cell column. The cells at the base of the expanded Ud cell layer were morphologically similar to Ud cells in a normal epithelium whereas the cells at the apex of the columns resembled normal differentiating neurons. A few necrotic cells could still be detected within the apical, cell-depleted zone. By the eighth post-operative week the receptor cell column was fully occupied with cells formed as a result of Ud cell proliferation. The most apical cells, 6-10 cells deep, were morphologically similar to normal bipolar neurons with a dendritic process reaching the lumen of the VN organ. The remaining cells were morphologically similar to normal differentiating or Ud cells. Sixteen weeks following axotomy a larger portion of cells in the receptor cell column were fully differentiated bipolar neurons. The Ud cell population was reduced and, as in the normal epithelium, occupied only the basal portion of each receptor cell column. The regenerated neurons of the VNO were capable of synthesizing and transporting macromolecules to the telencephalon as demonstrated by autoradiography following intraepithelial injections of [3H]proline. Newly formed axons terminated in the accessory olfactory bulb within 8 weeks following axotomy. These results support the view that the basal Ud cells were the source of neurons in the regenerating vomeronasal organ and demonstrate a dynamic process of neuronal proliferation, differentiation and maturation in the denervated vomeronasal epithelia of adult garter snakes.

Afferent Pathways↗
PubMed1995

Contributing factors to poor functional recovery after delayed nerve repair: prolonged axotomy.

The contribution of prolonged motoneuron axotomy to the poor functional recovery after delayed nerve repair was determined by means of a nerve cross-anastomosis paradigm in the rat. The tibial nerve was axotomized up to 12 months before it was cross-sutured to the distal stump of the freshly cut common peroneal nerve to innervate the freshly denervated tibialis anterior muscle. Three to 17 months later, muscle and motor unit (MU) forces were measured to quantify the number of axons that had successfully regenerated and reinnervated the muscle. The extent of axonal branching was estimated by the innervation ratio (IR) (i.e., the number of muscle fibers innervated by each axon), which was obtained directly by counting muscle fibers in a single glycogen-depleted MU in each muscle and indirectly by calculation. The total number of MUs in each muscle significantly decreased with progression of axotomy and was only 35% of the control when axotomy was prolonged more than 3 months. Concurrently, MU force and IR increased exponentially, with a mean increase of threefold when axotomy was more than 3 months, which largely compensated for the reduction in the number of axons that reinnervated the muscle. Consequently, muscles reinnervated by tibial motor axons that had been axotomized up to 12 months produced as much force as those reinnervated by freshly axotomized tibial motor axons. Muscle weight, size, and muscle fiber size were similar to those after immediate nerve suture. Although prolonged axotomy does not compromise the number of muscle fibers innervated by each axon, it does reduce the capacity of motor axons to regenerate and thus is an important contributing factor to the poor functional recovery in delayed nerve repair.

Anastomosis, Surgical↗
PubMed1993

Phenotypic plasticity in adult sympathetic ganglia in vivo: effects of deafferentation and axotomy on the expression of vasoactive intestinal peptide.

The expression of neurotransmitters/neuromodulators in sympathetic neurons is regulated by anterograde and retrograde mechanisms. We have examined the role of such mechanisms in the regulation of the neuropeptide vasoactive intestinal peptide (VIP). The adult rat superior cervical ganglion (SCG) contains low levels of peptide-like immunoreactivity (IR) and mRNA for VIP. Some VIP-IR nerve processes, but only a few VIP-IR cell bodies, are detectable. Previous evidence demonstrates, however, that after the SCG is placed in organ culture for 48 hr, the level of VIP-IR and VIP mRNA and the number of VIP-IR cell bodies and fibers increase considerably. Two of the possible causes for these changes in peptide expression in sympathetic neurons are deafferentation and axotomy, both of which occur when the SCG is placed in culture. To determine the importance of deafferentation, the preganglionic cervical sympathetic trunk was cut and the ganglion left in situ. Forty-eight hours later, VIP-IR increased twofold. A corresponding increase in the number of VIP-IR nerve processes was seen, but there was no detectable change in the number of VIP-IR cell bodies. The content of VIP/PHI mRNA also increased by 1.8-fold. The effect of axotomy on VIP-IR was examined by cutting the postganglionic internal and external carotid nerves and leaving the ganglion in situ. Forty-eight hours later, the level of VIP-IR increased 22-fold, many immunostained neurons were found, and the content of VIP mRNA increased over fivefold. After either deafferentation or axotomy, changes in VIP-IR were accompanied by comparable changes in the related molecule peptide histidine isoleucine amide (PHI)-IR. Neuropeptide Y-IR, on the other hand, decreased after deafferentation and increased only twofold after axotomy. The results indicate plasticity in the expression of VIP- and PHI-IR in adult sympathetic neurons in vivo, and suggest that the changes previously seen in organ culture were primarily a response to axotomy.

Animals↗
PubMed1997

Calcium/calmodulin-dependent protein kinase II expression in motor neurons: effect of axotomy.

Although Ca2+/calmodulin-dependent (CaM) protein kinase II isoforms are present in the nervous system in high amounts, many aspects of in vivo expression, localization, and function remain unexplored. During development, CaM kinase IIalpha and IIbeta are differentially expressed. Here, we examined CaM kinase II isoforms in Sprague-Dawley rat sciatic motor neurons before and after axotomy. We cut the L4-5 spinal nerves unilaterally and exposed the proximal nerve stumps to a fluoroprobe, to retrogradely label the neurons of origin. Anti-CaM kinase IIbeta antibody showed immunoreactivity in motor neurons, which decreased to low levels by 4 days after axotomy. We found a similar response by in situ hybridization with riboprobes. The decrease in expression of mRNA and protein was confined to fluorescent motor neurons. For CaM kinase IIalpha, in situ hybridization showed that the mRNA was in sciatic motor neurons, with a density unaffected by axotomy. However, these neurons were also enlarged, suggesting an up-regulation of expression. Northern blots confirmed an mRNA increase. We were unable to find CaM kinase IIalpha immunoreactivity before or after axotomy in sciatic motor neuron cell bodies, suggesting that CaM kinase IIalpha is in the axons or dendrites, or otherwise unavailable to the antibody. Using rats with crush lesions, we radiolabeled axonal proteins being synthesized in the cell body and used two-dimensional polyacrylamide gel electrophoresis with Western blots to identify CaM kinase IIalpha as a component of slow axonal transport. This differential regulation and expression of kinase isoforms suggests separate and unique intracellular roles. Because we find CaM kinase IIbeta down-regulates during axonal regrowth, its role in these neurons may be related to synaptic transmission. CaM kinase IIalpha appears to support axonal regrowth.

Animals↗
PubMed2000

Response of abducens internuclear neurons to axotomy in the adult cat.

The highly specific projection of abducens internuclear neurons on the medial rectus motoneurons of the oculomotor nucleus constitutes an optimal model for investigating the effects of axotomy in the central nervous system. We have analyzed the morphological changes induced by this lesion on both the cell bodies and the transected axons of abducens internuclear neurons in the adult cat. Axotomy was performed by the transection of the medial longitudinal fascicle. Cell counts of Nissl-stained material and calretinin-immunostained abducens internuclear neurons revealed no cell death by 3 months postaxotomy. Ultrastructural examination of these cells at 6, 14, 24, and 90 days postaxotomy showed normal cytological features. However, the surface membrane of axotomized neurons appeared contacted by very few synaptic boutons compared to controls. This change was quantified by measuring the percentage of synaptic coverage of the cell bodies and the linear density of boutons. Both parameters decreased significantly after axotomy, with the lowest values at 90 days postlesion ( approximately 70% reduction). We also explored axonal regrowth and the possibility of reinnervation of a new target by means of anterograde labeling with biocytin. At all time intervals analyzed, labeled axons were observed to be interrupted at the caudal limit of the lesion; in no case did they cross the scar tissue to reach the distal part of the tract. Nonetheless, a conspicuous axonal sprouting was present at the caudal aspect of the lesion site. Structures suggestive of axonal growth were found, such as large terminal clubs, from which short filopodium-like branches frequently emerged. Similar findings were obtained after parvalbumin and calretinin immunostaining. At the electron microscopy level, biocytin-labeled boutons originating from the sprouts appeared surrounded by either extracellular space, which was extremely dilated at the lesion site, or by glial processes. The great majority of labeled boutons examined were, thus, devoid of neuronal contact, indicating absence of reinnervation of a new target. Altogether, these data indicate that abducens internuclear neurons survive axotomy in the adult cat and show some form of axonal regrowth, even in the absence of target connection.

Abducens Nerve↗
PubMed2002

Involvement of cyclin-dependent kinases in axotomy-induced retinal ganglion cell death.

We have tested the role of cyclin-dependent kinases (CDKs) in the type 3B death of axotomized retinal ganglion cells, by injecting intraocularly olomoucine, roscovitine, or butyrolactone I. Each of these inhibits CDK1, CDK2, and CDK5; CDK1 and CDK2 are involved in cell proliferation, whereas CDK5 is involved in neuronal differentiation. The inhibitors partially protected ganglion cells against the effects of axotomy. These agents may affect the ganglion cells directly, because CDK1, its regulatory subunit cyclin B1, and CDK5 were identified immunohistochemically in the perikarya of ganglion cells, and this was confirmed for CDK1 and CDK5 in Western blots of the ganglion cell layer. These blots showed an axotomy-induced phosphorylation of CDK5 occurring remarkably quickly (within 6 hours of axotomy) but little if any change in the phosphorylation state of CDK1. In addition, we studied the expression of proliferation markers, including proliferating cell nuclear antigen (PCNA) and the synthesis of DNA, by immunohistochemical and autoradiographic methods. Normal or axotomized ganglion cells did not express PCNA and did not synthesize DNA. Although we cannot exclude the possibility that axotomized ganglion cells may leave their quiescent state, our data show that they did not progress beyond the G1 phase of the cell cycle. Finally, in contrast to inhibitors of CDKs, cell cycle blockers with different targets than CDKs did not protect ganglion cells. Globally, our results suggest that axotomy-induced death of ganglion cells involves the activation of CDK1, CDK2, or CDK5 (most probably CDK5) but not the full cell cycle machinery.

Animals↗
PubMed2002

Alterations to neuronal polarity following permanent axotomy: a quantitative analysis of changes to MAP2a/b and GAP-43 distributions in axotomized motoneurons in the adult cat.

Following axotomy, morphologically unusual, distal processes (UDPs) emerge from motoneuron dendrites. These processes contain an axonal protein, growth-associated protein 43 (GAP-43) but lack immunostaining for the dendritic protein microtubule-associated protein 2a/b (MAP2a/b). Thus, it appears that neuronal polarity alters following axotomy. Our goal was to describe this change in neuronal polarity on a more detailed and quantitative level. We asked two questions: Following axotomy, where in the entire neuron does the immunoreactivity for MAP2a/b and GAP-43 change and do these changes reflect a transformation of dendrite to axon or growth from terminal dendrites? Using intracellular labeling and immunocytochemistry, changes in MAP2a/b and GAP-43 immunoreactivity were also found in processes with a morphology typical of terminal branches of intact motoneurons (called simple distal processes [SDPs]), as well as UDPs. Trajectories (the path from the soma to a single terminus) with UDPs and SDPs were longer than trajectories without these processes, and trajectories with UDPs were the longest. Trajectories without UDPs or SDPs were similar in length to trajectories from intact motoneurons. The distance from the soma to the point where MAP2a/b immunoreactivity became absent in trajectories with UDPs or SDPs was similar to the length of trajectories from intact motoneurons. Thus, following axotomy, two morphologically distinct types of axon-like processes emerge from dendrites. The formation of these processes does not involve a transformation of the original dendrite, but rather growth at the ends of dendrites.

Animals↗
PubMed2002

Primary motor neurons fail to up-regulate voltage-gated sodium channel Na(v)1.3/brain type III following axotomy resulting from spinal cord injury.

Epilepsy occurs in a small proportion of patients with spinal cord injury (SCI), but whether it is due to concomitant traumatic head injury or to changes in cortical motor neurons secondary to axotomy within the spinal cord is not known. Na(v)1.3/brain type III sodium channel expression is up-regulated following peripheral axotomy of dorsal root ganglion (DRG) and facial motor neurons, but, to date, Na(v)1.3 expression has not been examined in upper (cortical) motor neurons following axotomy associated with SCI. In the present study, we examine Na(v)1.3 expression in upper motor neurons within rat primary motor cortex following midthoracic (T9) dorsal column transection, which severs the axons of those cells. Axotomized pyramidal cells were identified by retrograde transport of fluorogold. Immunolabeled cells were confined to layer V of the primary motor cortex and exhibited low levels of Na(v)1.3 staining. After axotomy, no significant changes were detected in Na(v)1.3 density or distribution in injured or uninjured cells, compared with control brains, in contrast to up-regulation of Na(v)1.3 in ipsilateral DRG neurons after sciatic nerve transection. These results do not preclude a role for voltage-gated sodium channels in post-SCI epilepsy but suggest that up-regulated expression of Na(v)1.3 channel is not involved.

Animals↗
PubMed2002

Changes in visual response properties of cat retinal ganglion cells within two weeks after axotomy.

After optic nerve transection beta cells of cat retinal ganglion cells (RGCs) suffer from rapid cell death from 3 to 7 days, whereas alpha cells gradual cell death until 14 days. Here we report electrophysiological properties of Y- (morphological alpha) and X- (morphological beta) cells at 5 and 14 days after axotomy in comparison with those of intact Y- and X-cells. Most of the axotomized RGCs revealed characteristic visual response properties that enable us to classify them into Y- or X-cells. Physiological sampling ratio of X-cells sharply decreased from day 5 to 14 after axotomy, corresponding to the previous morphological results. As compared with intact RGCs, axotomized RGCs of both Y- and X-type revealed the following abnormalities: smaller receptive field centers, weaker visual responses and lower spontaneous activities. Intracellular injections of Lucifer yellow into axotomized and intact RGCs at eccentricities 0-6 mm from the area centralis revealed no sign of shrinkage in dendritic field size of either alpha or beta cells on day 5 and day 14 after axotomy, revealing that observed smaller receptive field centers of axotomized RGCs on day 5 were not due to the change of dendritic field sizes. These results suggest that the major events occurring shortly after axotomy are significant loss of synaptic inputs from afferent neurons in the retina and/or changes of membrane properties of axotomized RGCs. These events can also explain lower spontaneous activities and weaker visual responses of axotomized RGCs.

Action Potentials↗
PubMed2003

No overlap of sensitivity to capsaicin and expression of galanin in rat dorsal root ganglion neurons after axotomy.

The neuropeptide galanin is known to have an antinociceptive effect under neuropathic conditions. After axotomy, galanin is upregulated in sensory neurons, presumably in the capsaicin-sensitive ones. Here, the sensitivity to capsaicin and the expression of galanin were simultaneously examined by double-staining in individual, dissociated rat dorsal root ganglion neurons (1) after axotomy of the sciatic nerve for up to 14 days and (2) in culture for up to 4 days without prior nerve injury. Ten days after axotomy, the proportion of capsaicin-sensitive neurons had decreased by 36 percentage points (from 63% to 27%), whereas the proportion of galaninergic neurons had increased by 33 percentage points (from 3% to 36%). These changes were also observed in neurons kept in culture, where the regulation was attenuated by the addition of nerve growth factor (NGF) or glial cell line-derived neurotrophic factor (GDNF) to the medium. After axotomy, galaninergic neurons had a soma size-distribution profile similar to the capsaicin-sensitive neurons, but there was no colocalization of capsaicin sensitivity and galanin expression in individual neurons. In culture, some neurons showed colocalization after 30 h and 48 h, but not after 6 h or 96 h. We conclude that the upregulation of galanin in an individual neuron is preceded by downregulation of its capsaicin sensitivity both in NGF-dependent peptidergic and in GDNF-dependent non-peptidergic neurons, indicating a change in phenotype.

Animals↗
PubMed2003

Primary sensory neuronal rescue with systemic acetyl-L-carnitine following peripheral axotomy. A dose-response analysis.

The loss of a large proportion of primary sensory neurons after peripheral nerve axotomy is well documented. As a consequence of this loss, the innervation density attained on completion of regeneration will never be normal, regardless of how well the individual surviving neurons regenerate. Acetyl-L-carnitine (ALCAR), an endogenous peptide in man, has been demonstrated to protect sensory neurons, thereby avoiding loss after peripheral nerve injury. In this study we examined the dose-response effect of ALCAR on the primary sensory neurons in the rat dorsal root ganglia (DRG) 2 weeks after sciatic nerve axotomy. Six groups of adult rats (n=5) underwent unilateral sciatic nerve axotomy, without repair, followed by 2 weeks systemic treatment with one of five doses of ALCAR (range 0.5-50 mg/kg/day), or normal saline. L4 and L5 dorsal root ganglia were then harvested bilaterally and sensory neuronal cell counts obtained using the optical disector technique. ALCAR eliminated neuronal loss at higher doses (50 and 10 mg/kg/day), while lower doses did result in loss (12% at 5 mg/kg/day, p<0.05; 19% at 1 mg/kg/day, p<0.001; 23% at 0.5 mg/kg/day, p<0.001) compared to contralateral control ganglia. Treatment with normal saline resulted in a 25% (p<0.001) loss, demonstrating no protective effect in accordance with previous studies.ALCAR preserves the sensory neuronal cell population after axotomy in a dose-responsive manner and as such, has potential for improving the clinical outcome following peripheral nerve trauma when doses in excess of 10 mg/kg/day are employed.

Acetylcarnitine↗
PubMed2004

Expression of axotomy-inducible and apoptosis-related genes in sensory nerves of rats with experimental diabetes.

In diabetes, peripheral nerves suffer deficient neurotrophic support-a situation which resembles axotomy. This raises the question: does inappropriate establishment of an axotomised neuronal phenotype contribute to diabetic neuropathy, and in extremis, does this provoke apoptosis? We hybridized reverse-transcribed RNA, from the dorsal root ganglia (DRG) of 8-week streptozotocin (STZ)-induced diabetic rats, to Affymetrix Rat Genome U34A chips and scanned the array for expression of (a) genes that are upregulated by axotomy, (b) proapoptotic and (c) anti-apoptotic genes. Expression of the axotomy-responsive genes coding for growth-associated protein 43 (GAP-43), galanin, neuropeptide Y (NPY), pre-pro-vasoactive intestinal polypeptide (pre-pro-VIP), neuronal nitric oxide synthase (nNOS), protease nexin 1, heat-shock protein 27 (HSP 27) and myosin light chain kinase II (MLCK II) was unaffected in ganglia from diabetic rats compared to controls; thus, no axotomised phenotype was established. The expression of the majority of proapoptotic genes in the DRG was also unaltered (bax, bad, bid, bok, c-Jun, p38, TNFR1, caspase 3 and NOS2). Similarly there was no change in expression of the majority of antiapoptotic genes (bcl2, bcl-xL, bcl-w, NfkappaB). These alterations in gene expression make it clear that neither axotomy nor apoptotic phenotypes are established in neurones in this model of diabetes.

Animals↗