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PubMed1990

A comparison of peripheral and central axotomy effects on neurofilament and tubulin gene expression in rat dorsal root ganglion neurons.

The expression of major cytoskeletal protein mRNAs was studied in adult rat dorsal root ganglion (DRG) neurons after crushing either their central or peripheral branch axons. mRNA levels in DRG neurons were examined by quantitative in situ hybridization with radiolabeled cDNA probes specific for the low-molecular-weight neurofilament protein (NF-L) and beta-tubulin. The large-sized (greater than 1000 microns 2) neurons which give rise to myelinated axons in lumbar ganglia (L4 and L5) were studied 1 d through 8 weeks after either dorsal root or sciatic nerve crush. NF-L and beta-tubulin mRNA levels in axotomized DRG neurons were compared to those in contralateral control DRG neurons, as well as to those in normal (completely untreated) DRG cells. In the case of NF-L mRNA, changes were observed after central as well as peripheral branch axotomy and the time course and magnitude of changes were similar after both types of axotomy. NF-L mRNA levels initially decreased (first 2 weeks after crush) and then began to return towards control levels at longer survival times. Similar, but less pronounced, changes in NF-L mRNA levels also occurred in contralateral DRG neurons (which were uninjured); the changes in contralateral neurons were not simply a result of surgical stress since no changes in NF-L mRNA levels were observed in sham-operated DRG neurons. In the case of tubulin mRNA, changes were observed after central as well as peripheral branch axotomy by in situ hybridization, but the time course and magnitude of changes were different after each type of axotomy.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Administration of testosterone attenuates neuronal loss following axotomy in the brain-stem motor nuclei of female rats.

This study was undertaken to elucidate whether (1) administration of testosterone to female rats attenuates axotomy-induced neuronal loss; (2) the efficacy of testosterone treatment is related to the age of animals, the dosage given, and the time and duration of the treatment; (3) neurons which project or terminate aberrantly can survive; and (4) the trophic actions of testosterone on neuronal survival and axonal outgrowth are operated under the same mechanisms. The hypoglossal and facial nerves were transected unilaterally at 3 and 6 weeks of age. In order to establish the dose-response curve, testosterone propionate (TP) at doses of 0.5, 1.0, 2.0, or 5.0 mg was injected subcutaneously twice weekly during the first 4 postaxotomy weeks, and once weekly thereafter for an additional 6 weeks. Neuronal numbers in the hypoglossal and facial motor nuclei were counted 10-12 weeks after axotomy in serial paraffin sections stained with cresyl violet. To determine the time course of TP effect, neuronal numbers were counted at 1, 4, 12, and 20 weeks after axotomy. In addition, neuronal loss 12 weeks after axotomy in rats treated with TP for the first 3 postaxotomy weeks only was compared with that in rats withheld TP treatment until the 5th postaxotomy week. To determine axonal projections and terminations of the surviving neurons, HRP retrograde tracing technique was used. Results indicated that TP treatment significantly attenuated neuronal loss in prepubertal and young adult female rats in a dose- and time-dependent manner. Only doses which elevated serum testosterone to levels comparable to or surpassing normal male levels were effective.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

Axotomy-induced parallel increases in electrical and dye coupling between identified neurons of Helisoma.

The electrically coupled buccal ganglion neurons 4R and 4L of the snail, Helisoma, display predictable plasticity. The strength of the electronic synapse between them increases significantly following axotomy. Synaptic strength was assayed by measurements of electrical coupling coefficients and by assessment of dye coupling (passage of dye into the uninjected neuron) following injection of Lucifer Yellow CH into one neuron. Within 3 to 5 days, axotomy induced an increase in electrical coupling coefficients between neurons 4R and 4L from 0.54 +/- 0.11 (n = 13) in normal preparations to 0.72 +/- 0.14 (n = 24). A parallel axotomy-induced increase in the probability of dye coupling occurred. Only 27% (n = 27) of normal neuron 4 pairs were dye coupled, compared with 87% (n = 15) of axotomized neuronal pairs. Irrespective of treatment, electrical and dye measurements in the same neuron 4 pairs showed a consistent correlation between the magnitude of the electrical coupling coefficients and the probability of detectable dye coupling. No dye coupling was observed in neuronal pairs with electrical coupling coefficients less than 0.50. Dye coupling always occurred when coupling coefficients were greater than 0.70. Sixty-seven percent of neuronal pairs with intermediate coupling (0.50 to 0.70) coefficients displayed dye coupling. The results show that axotomy evokes a predictable enhancement of communication at an "identified" electronic synapse and suggests that electrical and dye coupling are mediated by similar mechanisms.

Animals↗
PubMed1995

Axotomy-induced axonal degeneration is mediated by calcium influx through ion-specific channels.

We examined the role of extracellular calcium entry, the possible involvement of axonal calcium channels, and the potential protective effect of calcium channel and calpain antagonists in axotomy-induced axonal degeneration using murine dorsal root ganglia in cell culture. We found that calcium entry is both necessary and sufficient to induce axonal degeneration after axotomy, and may be inhibited by cobalt, manganese, dihydropyridines, and bepridil. Tetrodotoxin and omega-conotoxin are ineffective in preventing axonal degeneration. The activation of calpains also appears to be necessary and sufficient for axonal degeneration to proceed, and can be blocked with membrane-permeant leupeptin analogs and the oxirane aloxistatin. Although other calcium-activated events may occur, it appears that inhibition of calpain is sufficient to preserve the axon at the light microscope level, and to prevent axonal cytoskeleton degradation as detected by immunofluorescent staining. Our results suggest that axonal degeneration after axotomy involves the following sequence of events: (1) a lag-period after axotomy prior to the onset of axonal degeneration, (2) entry of calcium into the axon through an intact axolemma via a calcium-specific ion transport mechanism, (3) activation of calcium-dependent effector molecules such as calpains, (4) degradation of the axonal cytoskeleton. The details of the second step require further elucidation, and are of particular interest because this step is a potential target for therapies directed towards peripheral neuropathies.

Animals↗
PubMed1993

Treatment of the adult retina with microglia-suppressing factors retards axotomy-induced neuronal degradation and enhances axonal regeneration in vivo and in vitro.

To monitor the cascade of events initiated by injury of adult neurons, and to explore whether and how neighboring microglial cells contribute to the degradation of lesioned neurons, axotomy-induced ganglion cell degeneration was investigated in adult rats. Suppression of macrophage and microglia activity during the weeks following transection of the optic nerve was performed with the immunoglobulin-derived tripeptide Thr-Lys-Pro, which is a macrophage inhibitory factor (MIF) and retards the activity of cells of monocytic origin. Single or repeated injection of MIF into the vitreous body during and after transection of the optic nerve resulted in significant retardation of axotomy-induced ganglion cell degradation in the retina as detected by specific labeling with the retrogradely transported fluorescent dye 4Di-10ASP. MIF specifically altered the morphology of labeled microglial cells from a ramified to an oval, less ramified shape, indicating that these cells were targets of its activity. Injection of the tetrapeptide macrophage stimulating factor, also known as tuftsin (Thr-Lys-Pro-Arg), revealed effects opposite to those described for the MIF: it increased the number of labeled microglial cells and enhanced the devastating effects of axotomy on ganglion cells. The viability of rescued ganglion cells in retinas treated with the various drugs was assessed both in vivo and in vitro. (1) Intravitreal injection of MIF to prevent degradation of neurons combined with transplantation of autologous peripheral nerve grafts, which facilitate regrowth of the transected neurites, revealed that significantly more ganglion cells contributed to axonal regeneration (17.1%) than in untreated controls (9.5%). (2) Explantation of retinas that were pretreated with MIF in situ revealed higher incidence of axonal outgrowth in organ cultures than untreated control explants or retinas treated with either the basic fibroblast growth factor or brain-derived neurotrophic factor. The present results demonstrate that axotomy initializes a cascade of microglia-mediated autodestructive retinal responses, which culminate in degradation of "sick," but obviously viable neurons. We postulate that the retinal microglial system has a key role in recognizing and eliminating severed neurons.

Amino Acid Sequence↗
PubMed1997

Electron microscopic study of sprouting dendrites in the ciliary ganglia of cat and monkey (Macaca fascicularis) following pre- and post-ganglionic axotomy.

The present paper reports the ultrastructure of dendritic sprouting and formation of associated synapses in the ciliary ganglion of cat and monkey induced by pre- and post-ganglionic axotomy. In both series of experiments, sprouting dendrites were observed mostly at 3-5 days postoperatively; such profiles were identified by their dense packing of mitochondria and glycogen-like granules. In longitudinal section, such profiles appeared as expanded extensions from the normal-looking dendritic trunks. None were observed to arise directly from the neuronal soma. After preganglionic nerve section, the cross-sectional diameters of such profiles measured 2.2 +/- 1.0 microm (range: 0.9-6.2 microm) in cat and 2.4 +/- 0.7 microm (range: 0.9-5.5 microm) in monkey. After postganglionic nerve section, the cross-sectional diameters of such profiles measured 2.1 +/- 0.7 microm (range: 0.8-4.5 microm) in cat and 2.8 +/- 1.4 microm (range: 1.1-7.0 microm) in monkey. After preganglionic axotomy, in both cat and monkey, the axon terminals began to degenerate at 3 days postoperatively and disappeared by 5 days postoperatively. However, at later postoperative survival periods, the axon terminals reappeared and were observed to make synaptic contacts with the sprouting dendrites. Some of the sprouting dendrites were observed to degenerate, some as early as 3 days postoperatively; such profiles did not appear to have any synapse on them. After postganglionic axotomy, such sprouting dendritic profiles were also observed to make synaptic contacts with axon terminals; some were only closely associated with profiles filled with synaptic vesicles. The results thus suggest that through the formation of new synapses, sprouting of dendrites may have a role to play in neuronal survival after axotomy.

Animals↗
PubMed1997

Androgen mitigates axotomy-induced decreases in calbindin expression in motor neurons.

Androgens can rescue axotomized motor neurons from cell death. Here we examine a possible mechanism for this trophic action in juvenile Xenopus laevis: regulation of a calcium-binding protein, calbindin, after axotomy. Western analysis revealed that a monoclonal antibody to calbindin D specifically recognizes a single approximately 28 kDa band in X. laevis CNS and rat cerebellum. Retrograde transport of peroxidase combined with immunohistochemistry demonstrated that somata, axons, and synaptic terminals of laryngeal motor neurons in nucleus (N.) IX-X of X. laevis are calbindin-positive. The number of calbindin-positive cells was compared in the intact and axotomized sides of N.IX-X of gonadectomized males that were either hormonally untreated or DHT-treated for 1 month. Although axotomy decreased the number of calbindin-positive cells by 86% in hormonally untreated males, the decrease was only 56% in DHT-treated animals. Compared with hormonally untreated animals, the number of calbindin-labeled cells in N.IX-X of DHT-treated males was increased in both the intact (14%) and axotomized sides (75%). We conclude that axotomy decreases and that DHT enhances calbindin immunoreactivity in N.IX-X. Axotomy-induced decrease in calbindin immunoreactivity precedes cell loss in N.IX-X and may impair the capacity of motor neurons to regulate cytoplasmic calcium. Androgen-mediated maintenance of calbindin expression is thus a candidate cellular mechanism for trophic maintenance of hormone target neurons.

Animals↗
PubMed1999

Effects of aging and axotomy on the expression of neurotrophin receptors in primary sensory neurons.

Aging is accompanied by declined sensory perception, paralleled by widespread dystrophic and degenerative changes in both central and peripheral sensory pathways. Several lines of evidence indicate that neurotrophic interactions are of importance for a maintained plasticity in the adult and aging nervous system, and that changes in the expression of neurotrophins and/or their receptors may underpin senile neurodegeneration. We have here examined the expression of neurotrophin receptor (p75NTR, trkA, trkB, and trkC) mRNA and protein in intact and axotomized primary sensory neurons of young adult (3 months) and aged (30 months) rats. To examine possible differences among primary sensory neuron populations, we have studied trigeminal ganglia (TG) as well as cervical and lumbar dorsal root ganglia (DRG). In intact aged rats, a decrease in trk (A/B/C) mRNA labeling densities and protein-like immunoreactivities was observed. The decrease was most pronounced in lumbar DRG. In contrast, a small, not statistically significant, increase of p75NTR expression was observed in aged DRG neuron profiles. After axotomy, a down-regulation of mRNA and protein levels was observed for all neurotrophin receptors (p75NTR, trkA, trkB and trkC) in both young adult and aged rats. Consistent with the higher expression levels of neurotrophin receptors in unlesioned young adult primary sensory neurons, the relative effect of axotomy was more pronounced in the young adult than aged rats. Although a decrease in mean cell profile cross-sectional areas was found during aging and after axotomy, the characteristic distribution of neurotrophin receptor expression in different populations of NRG neurons was conserved. The present findings suggest an attenuation of neurotrophic signaling in primary sensory neurons with advancing age and that the expression of p75NTR and trks is regulated differently during aging. A similar dissociation of p75NTR and trk regulation has previously been reported in other neuronal systems during aging, suggesting that there may be a common underlying mechanism. Decreased access to ligands, disturbed axon function and systemic changes in androgen/estrogen levels are discussed as inducing and/or contributing factors.

Aging↗
PubMed1999

Peripheral axotomy slows motoneuron degeneration in a transgenic mouse line expressing mutant SOD1 G93A.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder that involves motoneuron degeneration, paralysis, and death. Mutations in Cu, Zn superoxide dismutase (SOD1) are one cause of this disease. It has been a puzzle as to why mutations in SOD1, an enzyme expressed in many neuronal types, selectively kill motoneurons. To begin to explore the factors that determine this selectivity, we carried out peripheral axotomy in mice expressing a mutant SOD1 (G93A mice) and controls (nontransgenic mice and mice expressing wild-type human SOD1). Axotomy in controls induced a predicted axonal atrophy and a small degree of axon loss. The axonal atrophy led to an increase in the number of small axons and a decrease in the number of large axons. In contrast to the controls, axotomy in G93A mice reduced the extent of axon degeneration at the end stage of the disease, leading to an increase in the number of surviving motor axons. Interestingly, all of the increased surviving axons were in the axon group with diameters smaller than 4.5 microm. This result suggests an apparent threshold of vulnerability that is correlated with axon size.

Amino Acid Substitution↗
PubMed1999

Changes in expression of voltage-gated potassium channels in dorsal root ganglion neurons following axotomy.

Several families of voltage-gated potassium channels (Kv), including a spectrum of subtypes, are involved in regulating and modifying the integration and transmission of electrical signals in the nervous system. However, the specific patterns of Kv expression in normal or injured dorsal root ganglion (DRG) neurons have not been studied. Previous studies have examined the expression of voltage-gated sodium channels in DRG neurons, and also the selective up- and downregulation of several of these channels following axonal injury to the DRG neurons. In the present study, we used immunocytochemical methods to investigate the expression of Kv channels (Kv1.1, 1.2, 1.3, 1.4, 1.5, 1.6, and 2.1) in DRG cells cultured from control and axotomized adult rats. Kv1.2 and 2.1 immunoreactivity in DRG neurons showed large decreases following axotomy, whereas Kv1.1 and 1.3 showed smaller decreases. Kv1.4 and 1.6 immunostaining were not altered by axotomy, and Kv1.5 immunoreactivity was low in both control and axotomized DRG neurons. These results provide molecular correlates for the expression of multiple K+ currents in normal DRG neurons and indicate that, in relation to changes in sodium channel expression, there are decreases in specific potassium channels following axotomy in these cells. The alterations in K+ and Na+ channel expression following axonal injury may lead to changes in electrical excitability of the DRG neurons, and might contribute to chronic pain syndromes.

Animals↗
PubMed1999

Localization and regulation of basic fibroblast growth factor (FGF-2) and FGF receptor-1 in rat superior cervical ganglion after axotomy.

In response to peripheral nerve lesion, synthesis of basic fibroblast growth factor (FGF-2) increases in sensory ganglia and motoneurons. Here, we investigated the axotomy-induced regulation of FGF-2 and FGF receptor-1 (FGFR-1) expression in the autonomic nervous system using the sympathetic superior cervical ganglion of the adult rat as a model. Transcripts for both proteins were detected by ribonuclease protection assay. Western blotting indicated the presence of all three FGF-2 isoforms (18, 21, and 23 kD) in the superior cervical ganglion. Immunohistochemical analysis revealed FGF-2 localization in nuclei of satellite cells surrounding postganglionic perikarya. After transection of the carotid nerves, the number of FGF-2-immunoreactive glial cells increased. FGF-2 mRNA was up-regulated within 6 h and remained elevated for 3 weeks. The 18-, 21-, and 23-kD isoforms were all increased 7 days after axotomy. FGFR-1 immunoreactivity was observed in neuronal and nonneuronal nuclei in the normal rat superior cervical ganglion. In contrast to FGF-2, expression of FGFR-1 was unchanged in ganglia after axotomy. Taken together, the present results suggest that FGF-2 participates in neuron-glial interactions of sympathetic ganglia and may be involved in sympathetic neuron survival or nerve regeneration after nerve lesion.

Animals↗
PubMed2002

Ultrastructure of retinal ganglion cell death after axotomy in chick embryos.

Axotomy often leads to neuronal death, which occurs after a particularly short delay in immature animals. Tectal lesions were made in embryonic day (E) 12 chick embryos, thereby axotomizing the retinal ganglion cells of the contralateral eye, which then died within 3 days. We here describe the ultrastructural changes in the axotomized ganglion cells. The main changes were nuclear invagination and type 3B (cytoplasmic type) cell death characterized by dilation of the perinuclear space, endoplasmic reticulum, and Golgi apparatus. However, nuclear invagination was never seen in type 3B dying cells. All the axotomy-induced retinal ganglion cell death appears to have been of type 3B; apoptosis was not induced by axotomy, as was confirmed by additional light microscopic experiments showing that it did not increase the frequency of apoptotic markers revealed by terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (the TUNEL method) labeling and immunoreactivity for activated caspase-3. However, the latter methods did show small numbers of apoptotic cells dying naturally even in control retinas. After the death of the axotomized ganglion cells, they were phagocytosed mainly in Müller processes. The present findings open up the chick tectal lesion model as a system for analyzing type 3B neuronal death in vivo.

Animals↗
PubMed2005

Effects of systemically administered NT-3 on sensory neuron loss and nestin expression following axotomy.

Previous work has shown that administration of the neurotrophin NT-3 intrathecally or to the proximal stump can prevent axotomy-induced sensory neuron loss and that NT-3 can stimulate sensory neuron differentiation in vitro. We have examined the effect of axotomy and systemic NT-3 administration on neuronal loss, apoptosis (defined by morphology and activated caspase-3 immunoreactivity), and nestin expression (a protein expressed by neuronal precursor cells) in dorsal root ganglia (DRG) following axotomy of the adult rat sciatic nerve. Systemic administration of 1.25 or 5 mg of NT-3 over 1 month had no effect on the incidence of apoptotic neurons but prevented the overall loss of neurons seen at 4 weeks in vehicle-treated animals. Nestin-immunoreactive neurons began to appear 2 weeks after sciatic transection in untreated animals and steadily increased in incidence over the next 6 weeks. NT-3 administration increased the number of nestin-immunoreactive neurons at 1 month by two- to threefold. Nestin-IR neurons had a mean diameter of 20.78 +/- 2.5 microm and expressed the neuronal markers neurofilament 200, betaIII-tubulin, protein gene product 9.5, growth associated protein 43, trkA, and calcitonin gene-related peptide. Our results suggest that the presence of nestin in DRG neurons after nerve injury is due to recent differentiation and that exogenous NT-3 may prevent neuron loss by stimulating this process, rather than preventing neuron death.

Animals↗
PubMed2004

Axotomy-dependent and -independent synapse elimination in organ cultures of Wld(s) mutant mouse skeletal muscle.

Progressive "dying back" neurodegenerative diseases are debilitating due to loss of connectivity after nerve terminal and axonal withdrawal, which impairs peripheral nerve function and leads ultimately to neuronal cell death. The mutant mouse (Wallerian degeneration slow; Wld(s)) provides an accessible model system to understand orthograde and retrograde degeneration, because in these mice axotomy induces slow, progressive withdrawal of nerve terminals from motor endplates. Axon degeneration itself is about 10 times slower than in wild-type mice. We describe an organ culture paradigm that permits direct observation of the progressive changes in morphology of neuromuscular junctions in Wld(s) mutant mice. Normal nerve terminal and motor endplate morphology were maintained at most Wld(s) neuromuscular junctions for up to 72 hr in vitro. At others, synaptic boutons were removed from postsynaptic junctional folds in piecemeal fashion, as observed in adults in vivo. By contrast, nerve terminals degenerated rapidly and synchronously in wild-type muscle cultures, resembling Wallerian degeneration in vivo. These observations confirm that in Wld(s) mice, axotomy triggers a mechanism of nerve-terminal withdrawal that seems qualitatively different from that in wild-type animals. The piecemeal dismantling of presynaptic terminals resembles that occurring during neonatal synapse elimination. Organ cultures of neonatal Wld(s) muscle maintained for 1-2 days in vitro also showed no evidence of synaptic terminal degeneration, but elimination of polyneuronal innervation progressed in vitro at approximately the same rate as in vivo. Taken together, the data suggest that both natural and axotomy-induced forms of synapse withdrawal may be accessible to continuous observation and analysis, in organ-cultures of Wld(S) mouse muscles. This offers several advantages over repeated visualization of synaptic remodeling that has thus far been possible only in vivo.

Animals↗
PubMed2002

Down-regulation of glutamate-induced conductances of retinal horizontal cells after ganglion cell axotomy.

After a complete optic nerve section (ONS), retinal neurons may display retrograde transneuronal modifications in synaptic structure and function related to the retinal disconnection from the brain. The molecular and physiological basis of these changes is not yet fully understood. Immunoreactivity for calbindin was used to specifically immunolabel the horizontal cells (HC) in order to study any morphologic changes in the outer plexiform layer (OPL) after axotomy-induced degeneration of retinal ganglion cells (RGC) in the rabbit retina. Glutamate-gated conductance expressed by HC enzymatically dissociated from the rabbit retina were studied at 12 and 21 days after ONS by using the whole-cell voltage-clamp technique. The amplitudes of glutamate-induced currents on HC were significantly reduced 3 weeks after axotomy. However, no morphologic changes within the OPL were detected coincident with the progressive loss of glutamatergic responses; similarly, HC dissociated from the axotomized retinal tissue did not differ in morphology or appearance from control retinas. The main finding in this study is that the HC experiment a retrograde transneuronal down-regulation of their ionotropic glutamate-induced conductance following axotomy-induced degeneration of RGC.

Animals↗
PubMed1998

Streptozotocin-induced diabetes mellitus causes changes in primary sensory neuronal cytoskeletal mRNA levels that mimic those caused by axotomy.

Dorsal root ganglion (DRG) sensory neurons are particularly vulnerable to diabetes mellitus. There is evidence that the disease decreases both circulating and retrogradely transported neurotrophic factors that are essential to the normal maintenance and function of these cells. A substantive loss of trophic support should cause DRG neurons to respond as though they were axotomized and, like an axotomy, cause significant changes in cytoskeletal gene expression within these cells. Such changes might contribute to the deficits in sensory neuronal function that characterize diabetic neuropathy. The current study used quantitative in situ hybridization to test the hypothesis that streptozotocin-induced diabetes, like an axotomy, increases class III beta-tubulin gene expression and decreases neurofilament 68-kDa gene expression in lumbar DRG neurons. In animals that had been diabetic for 8 weeks with mean blood glucose levels of 340 mg/dl, lumbar DRG class III beta-tubulin mRNA mean steady-state levels were twofold higher than those in age-matched nondiabetic controls. Moreover, in the same animals, diabetes decreased lumbar DRG 68-kDa neurofilament mRNA mean steady-state levels by more than half. These data show that diabetes causes changes in primary sensory neuronal cytoskeletal gene expression that mimic those caused by axotomy. Moreover, they support the idea that a loss of neurotrophic support contributes to the pathogenesis of diabetic neuropathy.

Animals↗
PubMed2000

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.

Animals↗
PubMed2002

Expression of c-Jun N-terminal kinases after axotomy in the dorsal motor nucleus of the vagus nerve and the hypoglossal nucleus.

c-Jun N-terminal kinases (JNKs)/stress-activated protein kinases (SAPKs) are a subgroup of mitogen-activated protein kinases (MAPKs), and important mediators of signal transduction from the cell surface to the nucleus. JNK phosphorylates the transcription factor c-Jun. c-Jun is one of the earliest and most consistent markers for neurons that respond to nerve-fiber transection. To elucidate the c-Jun metabolism after axotomy, we investigated the expression of JNKs mRNA and of JNKs and c-Jun proteins following vagus and hypoglossal nerve transection. We found that JNK 1, JNK 2 and JNK 3 mRNA were positive in the cytoplasm of neuronal and glial cells. JNK 1 and JNK 2 protein were distributed mainly in the cytoplasm of neurons and glial cells, while only JNK 3 immunoreactivity was observed intensely in the nuclei of neuronal cells. Activated JNK was also observed intensely in the nuclei of neuronal cells. In sham-operated animals, the cytoplasm of a few glial cells showed moderate immunoreactivity for activated JNK, while after axotomy the cytoplasm of all perineuronal microglial cells were stained intensely. Up-regulated c-Jun and phosphorylated c-Jun immunoreactivities were found in the nuclei of neuronal cells in the severed side of both the dorsal motor and hypoglossal nuclei. These results indicate that the principal activity of JNKs in neurons is contributed largely by JNK 3 under both normal and axotomized conditions, and that JNKs play an important role in signal transduction of perineuronal microglial cells after axotomy.

Animals↗