Effects Of Axotomy Research Articles

Efectos de la axotomía en neuronas nociceptivas de la sanguijuela

It has been described several similarities between mammalian and invertebrate nociceptors. Lateral N neuron, in the segmental ganglion of the leech Hirudo medicinalis, behave like polimodal nociceptors in mammals, meanwhile medial N cells is similar to mechano nociceptors (Pastor, Soria, Belmonte 1996). The effects of axotomy in sensory neurons in leeches are poorly understood. We have focused on the electrophysiological properties of leech nociceptive neurons after axotomy. In N lateral neurons, axotomy induced an increase in the total membrane capacitance, membrane time constant, decrease in the steady state/maximum hyperpolarization rate, increment in the time constant of voltage relaxation of inward rectifier, increase in action potential duration, dV/dtmax, dV/dtmin, and maximum amplitude and duration of after hyperpolarization; firing threshold (always in not axotomized) and an increment in adaptation spike frequency. In N medial cells, axotomy produced a slight depolarization and a decrease in Rinput, a rise in the time constant of voltage relaxation of inward rectifier, a decrement of action potential maximum amplitude, dV/dtmax, dV/dtmin, and after hyperpolarization maximum amplitude, with an increase in action potential and after hyperpolarization duration. Finally, firing threshold increased both in axotomized and not axotomized after axotomy. Axotomy not only induced changes in injured cells, but modified neurons whose axons did not run into the cutting nerves. axotomy induces different changes in N lateral and N medial nociceptive cells, and it is suggested some kind of communication between axotomized and not axotomized cells

Monocyte chemoattractant protein (MCP)-1 is rapidly expressed by sympathetic ganglion neurons following axonal injury.

EDI-immunoreactive macrophages, absent from the superior cervical ganglia (SCG) of normal rats, appear in these ganglia within 48h after postganglionic axotomy. Further, resident macrophages show changes after axotomy. Since chemokines function as chemoattractants and activators of leukocytes, the effects of axotomy on chemokine expression in the SCG were examined. Within 6 h after nerve transection, increases were seen in mRNA levels for monocyte chemoattractant protein (MCP)-1. MCP-1 mRNA was concentrated in a population of neurons, while MCP-1 protein was localized to endothelial cells. This axotomy-induced neuronal MCP-1 expression may trigger the infiltration and/or activation of macrophages in SCG after injury.

Axotomy- and autotomy-induced changes in the excitability of rat dorsal root ganglion neurons.

The spontaneous, ectopic activity in sensory nerves that is induced by peripheral nerve injury is thought to contribute to the generation of "neuropathic" pain in humans. To examine the cellular mechanisms that underlie this activity, neurons in rat L(4)-L(5) dorsal root ganglion (DRG) were first grouped as "large," "medium," or "small" on the basis of their size (input capacitance) and action potential (AP) shape. A fourth group of cells that exhibited a pronounced afterdepolarization (ADP) were defined as AD-cells. Whole cell recording was used to compare the properties of control neurons with those dissociated from rats in which the sciatic nerve had been sectioned ("axotomy" group) and with neurons from rats that exhibited self-mutilatory behavior in response to sciatic nerve section ("autotomy" group). Increases in excitability in all types of DRG neuron were seen within 2-7 wk of axotomy. Resting membrane potential (RMP) and the amplitude and duration of the afterhyperpolarization (AHP) that followed the AP were unaffected. Effects of axotomy were greatest in the small, putative nociceptive cells and least in the large cells. Moderate changes were seen in the medium and AD-cells. Compared to control neurons, axotomized neurons exhibited a higher frequency of evoked AP discharge in response to 500-ms depolarizing current injections; i.e., "gain" was increased and accommodation was decreased. The minimum current required to discharge an AP (rheobase) was reduced. There were significant increases in spike width in small cells and significant increases in spike height in small, medium, and AD-cells. The electrophysiological changes promoted by axotomy were intensified in animals that exhibited autotomy; spike height, and spike width were significantly greater than control for all cell types. Under our experimental conditions, spontaneous activity was never encountered in neurons dissociated from animals that exhibited autotomy. Thus changes in the electrical properties of cell bodies alone may not entirely account for injury-induced spontaneous activity in sensory nerves. The onset of autotomy coincided with alterations in the excitability of large, putative nonnociceptive, neurons. Thus large cells from the autotomy group were much more excitable than those from the axotomy group, whereas small cells from the autotomy group were only slightly more excitable. This is consistent with the hypothesis that the onset of autotomy is associated with changes in the properties of myelinated fibers. Changes in Ca2+ and K+ channel conductances that contribute to axotomy- and autotomy-induced changes in excitability are addressed in the accompanying paper.

Nicotinic acetylcholine receptor subunit proteins ?7 and ?4 decrease in the superior cervical ganglion after axotomy

Synaptic transmission in the superior cervical ganglion (SCG) is mediated by nicotinic acetylcholine receptors (nAChR). After transection of the postganglionic nerves of the SCG in the adult rat, the transcript levels of four of the five nAChR subunits present in the ganglion, alpha3, alpha5, alpha7, and beta4, decrease dramatically. In the present study, the effect of axotomy on nAChR subunit expression was examined at the protein level, focusing on the alpha7 and beta4 subunits. Immunohistochemistry with monoclonal antibody mAb306 (for the alpha7 subunit) and polyclonal antibody 4886 (for the beta4 subunit) showed that immunoreactivities for both alpha7 and beta4 subunits were concentrated in neurons in the intact ganglion. Results from double staining with antibodies to these subunits and to tyrosine hydroxylase, the enzyme that catalyzes the rate-limiting step in the biosynthesis of the sympathetic neurotransmitter norepinephrine, demonstrated that most neurons in the SCG express both the alpha7 and beta4 subunits. Three days after axotomy, the number of immunolabeled neurons and the intensity of the immunostaining per labeled neuron were decreased for both subunits. Decreases in subunit levels were also observed by Western blot analysis. Observing changes in these subunits over time after surgery revealed that, while the protein level of the alpha7 subunit recovered substantially within 2 weeks after the lesion, that of the beta4 subunit stayed low. These data demonstrate that decreases in nicotinic receptor subunits are among the changes in proteins that occur in axotomized sympathetic neurons, and suggest that these decreases may contribute to the depression in ganglionic synaptic transmission observed in axotomized ganglia.

Effect of Axotomy on Expression of NPY, Galanin, and NPY Y1 and Y2 Receptors in Dorsal Root Ganglia and the Superior Cervical Ganglion Studied with Double-Labeling in Situ Hybridization and Immunohistochemistry

Using double-labeling techniques for both in situ hybridization and immunohistochemistry some peptides and peptide receptors were studied quantitatively in a sensory and a sympathetic ganglion after axotomy. In the lumbar 5 dorsal root ganglion (DRG) normally no neuropeptide Y- and only a few galanin-positive cell bodies are seen. Following complete transection of the sciatic nerve around 60% of all neuropeptide Y (NPY) neuron profiles (NPs) were galanin positive (+) and 33–44% of all galanin NPs were NPY+. A good agreement between immunohistochemistry and in situ hybridization was observed for NPY and galanin. NPY Y1- and Y2-receptor (R) mRNAs were found in around 40% of all NPY mRNA+ NPs, and more than half of the Y1-R mRNA+ NPs and two-thirds of the Y2-R mRNA+ NPs were NPY+. In addition, more than one-third of the galanin mRNA-containing NPs showed colocalization with NPY receptor mRNAs and up to 70% of the Y2-R mRNA+ NPs also expressed galanin mRNA. In the control superior cervical ganglion (SCG) 10% of the NPY+ NPs were Y2-R mRNA+, and 85% of the Y2-R+ NPs were NPY mRNA+, and the corresponding percentages after axotomy were around 35 and 45%, respectively. Following axotomy of the carotid nerves around half of all NPY+ NPs were galanin+, and conversely around 50% of all galanin NPs were NPY+ at the mRNA level, whereas much lower percentages (15 and 9%, respectively) were observed with immunohistochemistry. These results demonstrate that double-labeling procedures are valid tools to quantitatively evaluate coexistence situations in sensory and sympathetic ganglia, showing a high degree of coexistence for NPY and galanin in axotomized neurons both in the lumbar 5 DRG and in the SCG. However, the immunohistochemical analysis in the SCG demonstrated much lower numbers of peptide-positive neurons than seen with in situ hybridization, suggesting that the latter technique is more sensitive. The fact that a considerable number of neurons express NPY together with Y1- and/or Y2-Rs indicates that both receptors may act as autoreceptors, the Y1-R presumably at the level of the cell body and the Y2-R on nerve terminals in the dorsal horn and/or the periphery. The present results also show that in both sensory and sympathetic neurons there is a strong upregulation of the Y2-R after nerve injury, suggesting a possible role in trophic and regenerative events.

Androgen receptor mRNA regulation in adult male and female hamster facial motoneurons: effects of axotomy and exogenous androgens.

Testosterone propionate (TP) administration at the time of facial nerve injury in the adult hamster augments the regenerative properties of the injured facial motoneurons (FMN), with the androgen receptor (AR) playing a key role in mediating the actions of TP on facial nerve regeneration. The purpose of the present study was to determine the effects of axotomy on AR mRNA expression in FMN. This was accomplished using in situ hybridization in conjunction with a (35)S-labeled AR riboprobe. Gonadally intact adult male and gonadectomized (gdx) adult female hamsters were subjected to a right facial nerve axotomy, with the left side serving as internal, unoperated control. Half the animals were subcutaneously implanted with a 10-mm TP Silastic capsule, and the other half were sham-implanted. An additional group of nonaxotomized, gonadally intact males was also included. Postaxotomy survival times were 1, 4, and 7 days. At 1 postoperative day 1, there were no effects of axotomy on AR mRNA levels. By postoperative days 4 and 7, axotomy caused a significant decrease in AR mRNA levels in FMN of gonadally intact males, relative to either the contralateral control FMN of the same animals or FMN from the group of gonadally intact males that were not subjected to facial nerve axotomy. There were no significant differences between AR mRNA levels in contralateral control FMN and FMN from the gonadally intact group of nonaxotomized males. TP administration at the time of axotomy had no effect on AR mRNA levels in either the axotomized or contrala(teral control FMN of gonadally intact males, relative to the nonaxotomized, gonadally intact male group. Corroborating our previous work, AR mRNA levels were reduced in the contralateral control FMN of gdx females, relative to the nonaxotomized, gonadally intact male group, with axotomy having no additional effects. The data are discussed in a mechanistic framework suggesting how TP acts to augment facial nerve regeneration.

Factors regulating AMPA-type glutamate receptor subunit changes induced by sciatic nerve injury in rats.

Excitatory glutamatergic neurotransmission at Ia afferent-motoneuron synapses is enhanced shortly after physically severing or blocking impulse propagation of the afferent and/or motoneuron axons. We considered the possibility that these synaptic changes occur because of alterations in the number or properties of motoneuron alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) receptors. Therefore, we quantitatively analyzed glutamate receptor (GluR)1, GluR2/3, and GluR4 AMPA subunit immunoreactivity (ir) in motoneurons 3, 7, or 14 days after axotomy or continuous tetrodotoxin (TTX) block of the sciatic nerve. GluR1-ir remained low in experimental and control motoneurons with either treatment and at any date. However, there was a large reduction of GluR2/3-ir (peak at 7 days >60% reduced) and a smaller, but statistically significant, reduction of GluR4-ir (around 10% reduction at days 3, 7, and 14) in axotomized motoneurons. TTX sciatic blockade did not affect AMPA subunit immunostainings. Axonal injury or interruption of the trophic interaction between muscle and spinal cord, but not activity disruption, appears therefore more likely responsible for altering AMPA subunit immunoreactivity in motoneurons. These findings also suggest that synaptic plasticity induced by axotomy or TTX block, although similar in the first week, could be related to different mechanisms. The effects of axotomy or TTX block on motoneuron expression of the metabotropic glutamate receptor mGluR1a were also studied. mGluR1a-ir was also strongly decreased after axotomy but not after TTX treatment. The time course of the known stripping of synapses from the cell somas of axotomized motoneurons was studied by using synaptophysin antibodies and compared with AMPA and mGluR1a receptor changes. Coverage by synaptophysin-ir boutons was only clearly decreased 14 days post axotomy and not at shorter intervals or after TTX block.

Experimental studies of agents with potential neuroprotective properties

Acta Ophthalmologica ScandinavicaVolume 77, Issue S229 p. 27-28 Free Access Experimental studies of agents with potential neuroprotective properties Larry A. Wheeler, Larry A. Wheeler Allergan, Inc., Department of Biological Sciences, 2525 Dupont Drive, Irvine, CA 92612, USASearch for more papers by this author Larry A. Wheeler, Larry A. Wheeler Allergan, Inc., Department of Biological Sciences, 2525 Dupont Drive, Irvine, CA 92612, USASearch for more papers by this author First published: 13 November 2014 https://doi.org/10.1111/j.1600-0420.1999.tb01133.xAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. References Quigley HA, Nickells RW, Kerrigan LA et al. (1995): Retinal ganglion cell death in experimental glaucoma and after axotomy occurs by apoptosis. Invest Ophth Vis Sci 36: 774– 786. Kerrigan LA, Zack DJ, Quigley HA, Smith SD & Pease ME (1997): TUNEL-positive ganglion cells in human open-angle Glaucoma. Arch Ophthamol 115: 1031– 1035. Nickells RW (1996): Retinal ganglion cell death in glaucoma: The how, the why and the maybe. J Glaucoma 5: 345– 356. Vorwerk CK, Lipton SA, Zurakowski D et al. (1996): Chronic low-dose glutamate is toxic to retinal ganglion cells. Invest Ophth Vis Sci 37(8): 1618– 1624. Dryer EB, Zurakowski D, Schumer RA, Podos SM & Lipton SA (1996): Elevated glutamate in the vitreous body of humans and monkeys with glaucoma. Arch Ophthalmol 114: 299– 305. Duvdevani R, Rosner M, Belkin M, Sautter J, Sabel BA & Schwartz M (1990): Graded crush of the rat optic nerve as a brain injury model: combining electrophysiological and behavioral outcome. Restor Neurol Neurosci 2: 31– 38. Yoles E & Schwartz M (1998): Elevation of intraocular glutamate levels in rats with partial lesion of the optic nerve. Arch Ophthalmol 116: 906– 910. Garcia-Valenzuela E, Shareef S, Walsh J & Sharma SC (1995): Programmed cell death of retinal ganglion cells during experimental glaucoma. Exp Eye Res 61(1): 33– 44. Yoles E, Wheeler LA & Schwartz M (1999): Aipha-2-adrenoreceptor agonists are neuroprotective in a rat model of optic nerve degeneration. Invest Ophthal V15 Sci; in press. Hicks D, Heidinger V, Mohand-Said S, Sahel J & Dreyfus H (1998): Growth factors and gangliosides as neuroprotective agents in excitotoxicity and ischemia. Gen Pharmacol 30(30): 265– 273. Sievers J, Hausmann B, Unsicker K & Berry M (1987): Fibroblast growth factors promote the survival or adult rat retinal cells after transection of the optic nerve. 76: 157– 162. Peinado-Ramon P, Salvador M, Villegas-Perez MP & Vidat-Sanz M (1996): Effects of axotomy and intraocular administration of NT-4, NT-3, and brain-derived neurotrophic factor on the survival of adult rat retinal ganglion cells. Invest Ophthalmol Vis Sci 37: 489– 500. Zhang C, Takahasi K, Lam TT & Tso MO (1994): Effects of basic fibroblast growth factor in retinal ischemia. Invest Ophthalmol Vis Sci 35(8): 3163– 3168. Wen R, Cheng T, Li Y, Cao W & Steinberg RH (1996): Alpha 2-adrenergic agonists induce basic fibroblast growth factor expression in photoreceptors in vivo and ameliorate light damage. J Neurosci 16(19): 5986– 5992. Peng M, Yiwen L, Zhijun L, Liu C, Latties A & Wen R (1998): Alpha-2-adrenergic agonists selectively activate extracellular signal-regulated kinases in Mueller cells in vivo. Invest Ophthal Vis Sci 39: 721– 1726. Hasson D, Lai RK, Chun T & Wheeler LA (1997): Functional protection of rat retina ischemic injury by brimonidine, Soc Neuroscience 23: 72– 74. Chao DT & Korsmeyer SJ (1998): Bcl-2 family: regulators of cell death. Ann Rev Immunol 16: 395– 419. Volume77, IssueS229December 1999Pages 27-28 ReferencesRelatedInformation

Differential regulation of cytoskeletal gene expression in hamster facial motoneurons: Effects of axotomy and testosterone treatment

Differential regulation of cytoskeletal gene expression in hamster facial motoneurons: Effects of axotomy and testosterone treatment

Protection of axotomized ganglion cells by salicylic acid

Neuronal survival is influenced by the redox environment, and it has been shown that antioxidants protect developing neurons from the effects of axotomy. Here, we show that the intraocular injection of salicylic acid (SA) reduces the number of dying axotomized ganglion cells in the chick embryo. The antioxidant properties of SA are probably responsible for its protective effects, whose U-shaped dose-dependency matches that of several other antioxidants. We conclude that SA protects axotomized neurons by maintaining the redox status near an optimal set-point.

Differential regulation of cytoskeletal gene expression in hamster facial motoneurons: Effects of axotomy and testosterone treatment

We have previously demonstrated that systemic administration of testosterone increases the rate of axonal regeneration following facial nerve crush in adult male hamsters. In the present study, the molecular mechanisms by which androgens could enhance axonal regeneration were examined at a cellular level. Specifically, the following question was addressed using quantitative in situ hybridization with cDNA probes complementary to betaII, and alpha1 tubulin mRNAs: Does exogenous testosterone augment axotomy-induced changes in tubulin mRNA expression in hamster facial motoneurons (FMN)? Castrated adult male hamsters were subjected to right facial nerve severance, with the left side serving as internal control. One-half of the animals received testosterone replacement in the form of subcutaneously implanted silastic capsules containing crystalline testosterone propionate, and the other half were implanted with blank capsules immediately following the axotomy. Postoperative survival times from 2-14 days were examined. Axotomy alone resulted in a significant increase in the levels of both betaII and alpha1 tubulin mRNAs in facial motor neurons between 2-14 days after injury. Administration of testosterone selectively augmented the axotomy-induced increases in betaII-tubulin, but not alpha1 tubulin, mRNA, levels at 7 and 14 days post axotomy. These results demonstrating an effect of testosterone in altering the neuronal cytoskeletal response to axotomy suggest that testosterone may enhance the regenerative properties of motor neurons via molecular mechanisms that involve selective alterations of the neuronal cytoskeleton.

Effects of axotomy on the expression and ultrastructural localization of N-cadherin and neural cell adhesion molecule in the quail ciliary ganglion: an in vivo model of neuroplasticity

Postganglionic nerve crush of the avian ciliary ganglion induces detachment of preganglionic terminals from the soma of the injured ciliary neurons, followed by reattachment at about the same time that the postganglionic axons regenerate to their targets. In order to determine the role played by cell adhesion molecules in this response, we have studied injury-induced changes in the amount and distribution of N-cadherin and neural cell adhesion molecule, together with modifications in the expression of their messenger RNAs. Both N-cadherin and neural cell adhesion molecule immunoreactivities associated with postsynaptic specializations decreased between one and three days following postganglionic nerve crush, preceding the detachment of the preganglionic boutons. Immunoreactivities subsequently increased between 13 and 20 days, in parallel with restoration of synaptic contacts on the ganglion cells and the progressive reinnervation of the peripheral targets. In contrast to the rapid decrease in immunoreactivity, the messenger RNA levels of N-cadherin and neural cell adhesion molecule both increased after crush, and remained elevated throughout the 20-day period of the experiment. These results are consistent with roles for N-cadherin and neural cell adhesion molecule in the maintenance of synaptic contacts. The rapid regulation of these proteins in injury-induced synaptic plasticity occurs at the post-transcriptional level, whereas longer term regulation associated with the re-establishment of synapses may be promoted by the increased levels of gene expression.

Ribosomal RNA transcriptional activation and processing in hamster facial motoneurons: Effects of axotomy with or without exposure to testosterone

A key step in the ability of neurons to survive injury and successfully regenerate involves ribosomal RNA production. Testosterone propionate (TP), augments facial nerve regeneration in the adult hamster. TP modulates the nucleolar reaction in injured facial motoneurons, such that mature ribosome levels increase more rapidly and in greater magnitude than with injury only. In this study, molecular and electron microscopic stereologic approaches were used to determine the effects of axotomy and steroid treatment on ribosomal transcription and processing in facial motoneurons. Castrated adult male hamsters were subjected to right facial nerve transection at the stylomastoid foramen. Half the animals were subcutaneously implanted with one Silastic TP capsule, with the remainder sham implanted. For the in situ hybridization experiments, postoperative survival times were 0.5, 2, or 6 hours. In situ hybridization with a ribosomal DNA probe specific to the external transcribed spacer region located at the 5' end of the ribosomal gene was accomplished. Transcriptional activation of the rRNA gene occurred rapidly, within 2 hours, after injury only. Unexpectedly, TP treatment did not alter the time course or magnitude of rRNA transcriptional activity. For the electron microscope experiments, the postoperative time of 12 hours was selected. Stereologic analysis of 3 nucleolar subcomponents, fibrillar centers (site of rRNA transcription), nucleolonema (site of rRNA processing), and granular material (site of preribosome storage), was accomplished. TP decreased the nucleolonemal strands and the granular material, relative to injury only. These results suggest that, although rRNA transcription is rapidly activated by axotomy, rRNA processing is temporarily stalled. TP does not affect the early, axotomy-induced transcriptional activation of the ribosomal gene, but may, instead, prevent the subsequent disruption in rRNA processing. An hypothesis for the molecular mechanism by which steroids augment the regenerative capabilities of injured facial motoneurons is presented.

The Expression of P2X3Purinoreceptors in Sensory Neurons: Effects of Axotomy and Glial-Derived Neurotrophic Factor

We have studied the distribution and regulation of the P2X3receptor (a ligand-gated ion channel activated by ATP) in adult dorsal root ganglion (DRG) neurons using a polyclonal antibody. P2X3receptor immunoreactivity was normally present in about 35% of L4/5 DRG neurons, virtually all small in diameter. In the dorsal horn, P2X3receptor expression was restricted to the terminals of sensory neurons terminating in lamina IIinner. P2X3receptors were expressed in approximately equal numbers of sensory neurons projecting to skin and viscera but in very few of those innervating skeletal muscle. P2X3receptors were found mostly in sensory neurons that bind the lectin IB4. After sciatic nerve axotomy, P2X3receptor expression dropped by more than 50% in L4/5 DRG. Glial cell line-derived neurotrophic factor (GDNF), delivered intrathecally, completely reversed axotomy-induced down-regulation of the P2X3receptor. We conclude that P2X3receptors are normally expressed in nociceptive primary sensory neurons, predominantly the nonpeptidergic nociceptors. P2X3receptors are down-regulated following peripheral nerve injury and their expression can be regulated by GDNF.

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.

Differential effects of facial nerve transection on heat shock protein 70 expression in the developing and adult hamster facial nucleus.

In this study, the effects of axotomy on heat shock protein 70 (hsp70) protein levels were analyzed by immunoblotting with a hsp70 antibody that recognized constitutive and inducible forms of the protein. The right facial nerve of postnatal day 4 (neonate) or 100 (adult) hamsters was transected at its exit from the stylomastoid foramen, with the left nerve serving as internal control. Postoperative survival times were 2, 6, 12, and 24 h, with 3 animals per time point. Tissue punches containing individual facial nuclear groups were collected and homogenized. Approximately 10 microg of total protein was loaded onto paired gels and electrophoretically separated. Immunoblots of one gel from each pair were prepared, with the other gel stained in 0.2% Coomassie blue and used for verification of equality of lane loading. The results indicate that hsp70 protein levels increase in the adult, but decrease in the neonate, after axotomy. It is concluded that a stress response to cellular damage is an initial component of the classically described "axon reaction" and that hsp70 plays a role in mediating motoneuron survival after peripheral nerve transection.

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.

Effect of permanent axotomy on number and volume of dorsal root ganglion cell bodies

The effects of axotomy on the size and number of rat dorsal root ganglion cells was studied using stereological methods. Twenty adult Wistar rats were axotomized by transection of the right fifth lumbar spinal nerve approximately 7 mm distal to the fifth lumbar dorsal root ganglion (DRG-L5). The corresponding ganglia from the nonaxotomized side served as controls. The DRG-L5 were removed for study 4, 8, 15, and 45 days after axotomy. The number of neurons in each DRG-L5 was determined from estimates of the numerical density, NV, made with disectors and estimates of the volume of the ganglion using the Cavalieri principle. The mean cell body volume was determined with the vertical planar rotator method. There was a progressive loss of nerve cells during the postoperative period. There was a loss of 6% (not significant) after 4 days, 19% (not significant) after 8 days, 22% (2P < 0.05) after 15 days, and 35% (2P < 0.005) after 45 days. The relative reduction in cell number 45 days after axotomy was larger for B-cells (43%) than for A-cells (15%). The mean nerve cell body volume for the entire DRG-L5 cell population was reduced by 33% (2P < 0.005) 4 days after axotomy and remained so throughout the experimental period. The distribution of the individual cell volumes in the ganglia appeared to be uniformly shifted to lower values. It is concluded that permanent axotomy of the fifth lumbar spinal nerve results in a substantial loss of dorsal root ganglion cells and is well-suited as a model for studying the potential protective effects of neurotrophic factors using modern stereological techniques.

The effects of axotomy and preganglionic denervation on the expression of pituitary adenylate cyclase activating peptide (PACAP), galanin and PACAP type 1 receptors in the rat superior cervical ganglion

The effects of axotomy, chemical sympathectomy and preganglionic denervation on the expression of the neuropeptides, pituitary adenylate cyclase-activating peptide (PACAP), galanin (GAL), and the PACAP type 1 receptor in the rat superior cervical ganglion (SCG) were investigated by immunocytochemistry, in situ hybridization and receptor autoradiography. An antibody recognizing the rat vesicular acetylcholine transporter (VAChT) was used for the detection of preganglionic cholinergic fibers. In the normal SCG, PACAP-immunoreactivity (-IR) was present in numerous, basket-forming, preganglionic nerve fibers, while very few SCG neurons expressed PACAP. GAL-IR was restricted to occasional neurons, and a few nerve fibers, most of which were, in addition, PACAP-IR. PACAP type 1 receptors were expressed in all nerve cell bodies. Axotomy resulted in a rapid and prominent upregulation of PACAP in a large number of nerve cell bodies. There was a large increase also in GAL expression in many nerve cell bodies. In contrast, there was a marked decline in PACAP type 1 receptor expression. Chemical sympathectomy by administration of the catcholaminergic neurotoxin, 6-hydroxydopamine (6-OHDA), gave rise to similar changes. Preganglionic denervation led to the disappearance of PACAP- and VAChT-IR baskets and to the upregulation of PACAP and GAL expression in neurons located close to the entrance of the sympathetic chain, whereas PACAP type 1 receptor expression was not affected. PACAP and GAL were coexpressed in most neurons after axotomy and chemical sympathectomy. Taken together, these results indicate that disruption of target contact and/or the infliction of an injury to the axons of the sympathetic neurons, rather than the preganglionic output, regulates the expression of PACAP, GAL and the PACAP type 1 receptor.

Axotomy increases the excitability of dorsal root ganglion cells with unmyelinated axons.

Axotomy increases the excitability of dorsal root ganglion cells with unmyelinated axons. J. Neurophysiol. 78: 2790-2794, 1997. To better understand the neuronal mechanism of neuropathic pain, the effect of axotomy on the excitability of dorsal root ganglion (DRG) cells with unmyelinated axons (C cells) was investigated. Whole cell patch-clamp recordings were performed on intact DRG cells with intact axons or with axons transected 7-12 days earlier. C cells were identified by 1) soma size, 2) action potential morphology, 3) conduction velocity, and 4) in some cases, injection of Fast Blue into the injured nerve fibers. Axotomy reduced (more negative) action potential threshold but did not significantly change resting membrane potential, action potential duration, or maximal depolarization rate. Axotomy significantly increased the peak sodium current measured under voltage-clamp conditions. In Fast Blue-labeled (injured) cells, the tetrodotoxin (TTX)-sensitive current was enhanced while the TTX-resistant current was reduced. These results suggest that axotomy increased the excitability of C cells, possibly because of a preferential increase in expression of TTX-sensitive sodium currents.