Effects Of Peripheral Axotomy Research Articles

Transcript expression of vesicular glutamate transporters in lumbar dorsal root ganglia and the spinal cord of mice – Effects of peripheral axotomy or hindpaw inflammation

Using specific riboprobes, we characterized the expression of vesicular glutamate transporter (VGLUT)1–VGLUT3 transcripts in lumbar 4–5 (L4–5) dorsal root ganglions (DRGs) and the thoracolumbar to lumbosacral spinal cord in male BALB/c mice after a 1- or 3-day hindpaw inflammation, or a 7-day sciatic nerve axotomy. Sham animals were also included. In sham and contralateral L4–5 DRGs of injured mice, VGLUT1-, VGLUT2- and VGLUT3 mRNAs were expressed in ∼45%, ∼69% or ∼17% of neuron profiles (NPs), respectively. VGLUT1 was expressed in large and medium-sized NPs, VGLUT2 in NPs of all sizes, and VGLUT3 in small and medium-sized NPs. In the spinal cord, VGLUT1 was restricted to a number of NPs at thoracolumbar and lumbar segments, in what appears to be the dorsal nucleus of Clarke, and in mid laminae III–IV. In contrast, VGLUT2 was present in numerous NPs at all analyzed spinal segments, except the lateral aspects of the ventral horns, especially at the lumbar enlargement, where it was virtually absent. VGLUT3 was detected in a discrete number of NPs in laminae III–IV of the dorsal horn. Axotomy resulted in a moderate decrease in the number of DRG NPs expressing VGLUT3, whereas VGLUT1 and VGLUT2 were unaffected. Likewise, the percentage of NPs expressing VGLUT transcripts remained unaltered after hindpaw inflammation, both in DRGs and the spinal cord. Altogether, these results confirm previous descriptions on VGLUTs expression in adult mice DRGs, with the exception of VGLUT1, whose protein expression was detected in a lower percentage of mouse DRG NPs. A detailed account on the location of neurons expressing VGLUTs transcripts in the adult mouse spinal cord is also presented. Finally, the lack of change in the number of neurons expressing VGLUT1 and VGLUT2 transcripts after axotomy, as compared to data on protein expression, suggests translational rather than transcriptional regulation of VGLUTs after injury.

Effect of Peripheral Axotomy on Gene Expression of NIDD in Rat Neural Tissues

Peripheral nerve lesion-induced production of neuronal nitric oxide synthase (nNOS) was implicated to influence a range of postaxotomy processes necessary for neuronal survival and nerve regeneration (Zochodne et al., Neuroscience, 91:1515-1527, 1999; Keilhoff et al., Journal of Chemical Neuroanatomy, 24:181-187, 2002, Nitric Oxide, 10:101-111, 2004). Protein-protein interactions represent an important mechanism in the control of NOS spatial distribution or activity (Alderton et al., Biochemical Journal, 357:593-615, 2001; Dedio et al., FASEB Journal, 15:79-89, 2001; Zimmermann et al., Proceedings of the National Academy of Sciences, 99:17167-17172, 2002). As one of the nNOS-binding proteins, nNOS-interacting DHHC domain-containing protein with dendritic mRNA (NIDD) has recently been identified to increase nNOS enzyme activity by targeting nNOS to the synaptic plasma membrane in a postsynaptic density protein 95/discs-large/zona occlusens-1 domain dependent manner (Saitoh et al., Journal of Biological Chemistry, 279:29461-29468, 2004). In this paper, we established a rat model with peripheral axotomy to investigate the gene expression patterns of NIDD in neural tissues using TaqMan quantitative real-time polymerase chain reaction and in situ hybridization combined with immunofluorescence. It revealed that NIDD mRNA was upregulated after sciatic nerve transection with the similar expressing styles as that of the nNOS in the injured nerves, corresponding dorsal root ganglia, and lumbar spinal cord. These findings imply that NIDD may be involved in the different pathological conditions including nerve regeneration, neuron loss or survival, and even pain process, possibly via regulating the enzyme nNOS activity.

Effect of peripheral axotomy on pain-related behavior and dorsal root ganglion neurons excitability in NPY transgenic rats

In order to clarify the physiologic role of NPY in sensory processing, we obtained intracellular recordings of DRG neurons from wild type (WT) and NPY overexpressing transgenic rats (NPY-TG) before and after injury. We investigated medium and large diameter DRG neurons since upregulation of NPY peptide following the nerve injury occurs primarily in those cells. Neurons were classified as Aα/β and Aδ using conduction velocity and action potential duration. Prior to the injury, Aα/β neurons of NPY-TG rats conducted more slowly and had a more brief AHP than similar cells from the WT group. Aδ neurons at baseline conducted faster in TG animals compared to WT. Ligation of the 5th lumbar spinal nerve (SNL) produced certain changes in Aα/β cells that were evident only in the TG group. These include increased refractory period, increased input resistance, AHP prolongation and a depolarizing shift in threshold for AP initiation. The expected injury-induced CV slowing was not seen in NPY-TG Aα/β cells. In the Aδ cell group, injury produced a depolarizing shift in the resting membrane potential, an increase in AP duration and decrease in AHP and refractory period duration only in WT rats, while NPY-TG cells lacked these injury-induced changes. Behavior tests showed diminished sensory response to nerve injury in NPY-TG rats, i.e., shorter duration of enhanced pain-related behavior and attenuation of contralateral effect. In conclusion, our observations suggest that NPY overexpression leads to reduced neuronal activity following nerve injury in a cell-specific manner.

Morphine-induced in vivo release of spinal cholecystokinin is mediated by delta-opioid receptors--effect of peripheral axotomy.

Morphine and other opioid agonists induce spinal in vivo release of cholecystokinin (CCK), a neuropeptide with anti-opioid properties. However, so far the opioid receptor subtype responsible for this effect has not been determined. In the present in vivo microdialysis study, the morphine-induced release of cholecystokinin-like immunoreactivity (CCK-LI) in the dorsal horn was completely blocked by the delta-opioid antagonist naltrindole (10 microM in the perfusion fluid). Neither the mu-opioid receptor antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr amide (CTOP; 10 microM in the perfusion fluid), nor the kappa-opioid receptor antagonist nor-binaltorphimine (nor-BNI); 10 microM in the perfusion fluid) had any significant effect in this respect. In addition, systemic administration of the delta-opioid receptor agonist BW373U86 (1 mg/kg, s.c.) and spinal administration of the delta(2)-opioid receptor agonist, Tyr-D-Ala-Phe-Glu-Val-Val-Gly amide ([D-Ala(2)] deltorphin II) (1 microM in the perfusion fluid) induced a significant increase of the CCK-LI level. The effect of BW373U86 on spinal CCK-LI release was completely blocked by spinal administration of naltrindole. The mu-opioid receptor agonist [D-ala(2)-N-Me-Phe(4)-Gly(5)-ol]-enkephalin (DAMGO) (1 microM in the perfusion fluid or 1 mg/kg, s.c.) failed to alter the CCK-LI level. Peripheral nerve lesions have previously been shown to down-regulate mu- and delta-opioid receptors in the dorsal horn, to increase the gene-expression of CCK and CCK-receptor mRNA in dorsal root ganglion neurons and to alter the potassium-induced spinal CCK-LI release. After complete sciatic nerve transection, administration of the two selective delta-opioid receptor agonists induced a significant release of CCK-LI, which was comparable to controls. In contrast, neither systemic nor spinal administration of morphine and DAMGO altered the spinal CCK-LI release in axotomized animals. The present data indicate that the delta-opioid receptor mediates morphine-induced CCK-LI release in the spinal cord.

Effects of peripheral axotomy on cholecystokinin neurotransmission in the rat spinal cord.

Because cholecystokinin (CCK) acts as a "functional" endogenous opioid antagonist, it has been proposed that changes in central CCKergic neurotransmission might account for the relative resistance of neuropathic pain to the analgesic action of morphine. This hypothesis was addressed by measuring CCK-related parameters 2 weeks after unilateral sciatic nerve section in rats. As expected, significant decreases (-25-38%) in the tissue concentrations and in vitro release of both substance P and calcitonin gene-related peptide were noted in the dorsal quadrant of the lumbar spinal cord on the lesioned side. In contrast, the tissue levels and in vitro release of CCK were unchanged in the same area in lesioned rats. Measurements in dorsal root ganglia at L4-L6 levels revealed no significant changes in proCCK mRNA after the lesion. However, sciatic nerve section was associated with a marked ipsilateral increase in both CCK-B receptor mRNA levels in these ganglia (+70%) and the autoradiographic labeling of CCK-B receptors by [3H]pBC 264 (+160%) in the superficial layers of the lumbar dorsal horn. Up-regulation of CCK-B receptors rather than CCK synthesis and release probably contributes to increased spinal CCKergic neurotransmission in neuropathic pain.

Effects of peripheral axotomy on neuropeptides and nitric oxide synthase in dorsal root ganglia and spinal cord of the guinea pig: an immunohistochemical study

The effect of axotomy (3, 10 and 21 days) on the expression of some neuronal markers was analysed in dorsal root ganglia and spinal cord of guinea-pigs using immunohistochemistry. Three weeks following injury, substance P-like immunoreactivity (-LI) was slightly reduced in the DRGs of the ipsilateral side, whereas a marked increase in neuropeptide Y(NPY)-LI could be detected ipsilaterally and a smaller increase contralaterally. NPY-LI was mainly expressed in small, but also some medium-sized and large neuron profiles after axotomy. Galanin-LI showed a moderate bilateral increase. No significant changes could be observed in DRGs for calcitonin gene-related peptide (CGRP)-, vasoactive intestinal polypeptide-, peptide histidine isoleucine- or nitric oxide synthase-LIs. In the ventral horn CGRP-LI was slightly increased bilaterally in motoneurons, most pronounced on the injured side. Autotomy behaviour was seen in seven of the nine animals in the twenty-one day group. The present results demonstrate that also in guinea-pigs several peptides undergo distinct changes in their expression after peripheral nerve injury. However, in contrast to rats and monkeys, galanin-LI is only moderately increased in guinea-pigs. Neuropeptide Y showed a dramatic increase mainly in small neurons, in contrast to the upregulation in large neurons in the rat. Thus, distinct species differences exist with regard to the cellular response to nerve injury.

Neuropeptide Y and galanin binding sites in rat and monkey lumbar dorsal root ganglia and spinal cord and effect of peripheral axotomy.

Using monoiodinated peptide YY (PYY) and galanin as radioligands, and neuropeptide Y (NPY) fragments, the distribution of NPY binding sites and its subtypes Y1 and Y2, and of galanin binding sites, was investigated in rat and monkey lumbar (L) 4 and L5 dorsal root ganglia (DRG) and spinal cord before and after a unilateral sciatic nerve cut, ligation or crush. Receptor autoradiography revealed that [125I]PYY bound to some DRG neurons and a few nerve fibres in normal rat DRG, and most of these neurons were small. NPY binding sites were observed in laminae I-IV and X of the rat dorsal horn and in the lateral spinal nucleus, with the highest density in laminae I-II. [125I]PYY binding was most strongly attenuated by NPY13-36, a Y2 agonist, and partially inhibited by [Leu31,Pro34]NPY, a Y1 agonist, in both rat DRG and the dorsal horn of the spinal cord. These findings suggest that Y2 receptors are the main NPY receptors in rat DRG and dorsal horn, but also that Y1 receptors exist. After sciatic nerve cut, PYY binding markedly increased in nerve fibres and neurons in DRG, especially in large neuron profiles, and in laminae III-IV of the dorsal horn, as well as in nerve fibres in dorsal roots and the sciatic nerve. Incubation with NPY13-36 completely abolished PYY binding, which was also reduced by [Leu31,Pro34] NPY. However, the increase in PYY binding seen in laminae I-IV of the ipsilateral dorsal horn after axotomy was not observed after coincubation with [Leu31,Pro34] NPY. NPY binding sites were seen in a few neurons in monkey DRG and in laminae I-II, X and IX of the monkey spinal cord. The intensity of PYY binding in laminae I-II of the dorsal horn was decreased after axotomy. Galanin receptor binding sites were not observed in rat DRG, but were observed in the superficial dorsal horn of the spinal cord, mainly in laminae I-II. Axotomy had no effect on galanin binding in rat DRG and dorsal horn. However, galanin receptor binding was observed in many neurons in monkey L4 and L5 DRG and in laminae I-IV and X of monkey L4 and L5 spinal cord, with the highest intensity in laminae I-II. No marked effect of axotomy was observed on the distribution and intensity of galanin binding in monkey DRG or spinal cord.(ABSTRACT TRUNCATED AT 400 WORDS)

Secretory pathways of neuropeptides in rat lumbar dorsal root ganglion neurons and effects of peripheral axotomy.

Using immunocytochemistry combined with confocal and electron microscopy, the secretory pathways related to substance P (SP), calcitonin gene-related peptide (CGRP), galanin (GAL), and neuropeptide Y (NPY) were investigated in neurons in rat lumbar (L) 4 and L5 dorsal root ganglia (DRGs) before and after peripheral axotomy. All four peptides were processed through the regulated secretory pathway in many small neurons in normal DRGs, and CGRP through this pathway also in some large neurons. In many small neurons, two neuropeptides could be sorted into the same or separate large dense-core vesicles (LDCVs). The LDCVs had a significantly larger diameter in small as compared to large DRG neurons. Fourteen days after sciatic nerve cut, the levels of SP- and CGRP-like immunoreactivities (-LIs) and the number of LDCVs containing these peptides were markedly reduced, but SP- and CGRP-LIs were still seen in the regulated pathway. GAL-LI was markedly increased in many small neurons and some large neurons and NPY-LI mainly in large neurons. Both peptides were particularly abundant in the Golgi region. In small neurons, the number of LDCVs containing GAL- or NPY-LI was increased, but did not appear to reach the numbers containing SP- or CGRP-LI in normal DRG neurons. After axotomy, CGRP-LI and GAL-LI were often in separate LDCVs. One type of NPY-positive large neurons showed budding off of LDCVs after axotomy, but also some "scattered" labeling in the cytoplasm. In the second type, NPY-LI was mainly found in multivesicular bodies. In several myelinated nerve fibers a "diffuse" distribution of NPY was seen together with some LDCVs containing NPY-LI. In contrast, in unmyelinated nerve fibers, NPY-, GAL-, SP-, and CGRP-LIs were always observed in LDCVs. Thus, both in normal and axotomized DRG neurons, peptides are processed through the regulated pathway. However, in some large neurons, NPY is, in addition, secreted through the constitutive pathway, perhaps as a consequence of limited sorting mechanisms for NPY, i.e., the plasticity of the secretory mechanisms does not match the rate of peptide synthesis after axotomy.

Ultrastructural studies on peptides in the dorsal horn of the rat spinal cord—IV. Effects of peripheral axotomy with special reference to neuropeptide Y and vasoactive intestinal polypeptide/peptide histidine isoleucine

Using immunofluorescence histochemistry and pre- and post-embedding immunoelectron microscopy the rat lumbar dorsal horn was analysed in normal rats and 14 days after unilateral transection of the sciatic nerve. A marked increase in neuropeptide Y-like immunoreactivity was observed in the ipsilateral, superficial dorsal horn, especially in laminae III and IV, of the lumbar 4-5 spinal cord segments after peripheral axotomy. In the ipsilateral lamina II two types of neuropeptide Y-immunoreactive, presumably primary afferent terminals could be identified at the ultrastructural level. The first type contained many large dense-core vesicles (100-155 nm in diameter), whereas a second, more common type had only a few and smaller large dense-core vesicles (80-100 nm in diameter), plus synaptic vesicles of varying diameter (50-85 nm), large empty vesicles and tubular structures. Only occasionally were neuropeptide Y-positive terminals in lamina II involved in the formation of axonal labyrinths. In the ipsilateral lamina III, the number of neuropeptide Y-positive nerve terminals markedly increased after axotomy, with a moderate increase in lamina IV. These neuropeptide Y-positive terminals were morphologically similar to the second type of neuropeptide Y-positive terminal in lamina II, i.e. contained many synaptic vesicles (45-50 nm in diameter), a few small large dense-core vesicles (80-100 nm in diameter), electron-dense granular matrix and a few tubular structures. Fusion of synaptic vesicles with the plasma membrane was often observed at these synapses. These terminals frequently formed glomeruli but were not involved in axonal labyrinths. With regard to local neurons, neuropeptide Y-like immunoreactivity was observed in many dendrite-like profiles mostly making synaptic contacts with neuropeptide Y-negative dendrites and only rarely contacting the central terminal of the glomeruli. Neuropeptide Y-positive nerve endings were mainly seen in lamina I and the outer third of lamina II. After peripheral axotomy the number of vasoactive intestinal polypeptide/peptide histidine isoleucine immunoreactive terminals was increased in laminae I and II. They contained many large dense-core vesicles (100-120 nm in diameter), and some of them were positive for vasoactive intestinal polypeptide/peptide histidine isoleucine. Morphologically, the terminals were characterized by a granular matrix, tubular structures, empty vesicles, reduction in synaptic vesicles and absence of postsynaptic densities. Vasoactive intestinal polypeptide/peptide histidine isoleucine-like immunoreactivities were often found in association with labyrinth formation.(ABSTRACT TRUNCATED AT 400 WORDS)

Ultrastructural studies on peptides in the dorsal horn of the rat spinal cord--III. Effects of peripheral axotomy with special reference to galanin.

In this study co-localization of galanin- with calcitonin gene-related peptide (CGRP)-like immunoreactivity was examined in dorsal root ganglion neurons 14 days after sciatic nerve cut using a laser scanning confocal microscope. CGRP- and galanin-like immunoreactivities were also analysed in the dorsal horn of the spinal cord of these animals with immunofluorescence microscopy. The ultrastructural changes in galanin-immunoreactive, presumably primary afferent terminals in the superficial dorsal horn, were studied as well as the relationship between galanin-, substance P- and CGRP-like immunoreactivities in primary afferent terminals. Local galanin-positive neurons in lamina II were also analysed after peripheral axotomy. Under the confocal microscope, CGRP-like immunoreactivity was located in the perinuclear region, probably the Golgi complex, and in dot-like structures, probably representing large dense-core vesicles, in normal dorsal root ganglion neurons. However, after peripheral axotomy CGRP was mainly detected in dot-like structures. Only a slight decrease in percentage of CGRP neurons in dorsal root ganglion was seen after axotomy, and about 84% of the galanin-positive neurons contained CGRP. The field of galanin-positive nerve fibres in the superficial lumbar (L)4 and L5 dorsal horn expanded and the intensity of staining for CGRP was reduced in these regions 14 days after sciatic nerve cut. Using pre-embedding immunoelectron microscopy, several morphological changes were observed in galanin-positive terminals in laminae I and II ipsilateral to the lesion. Most importantly, the most frequently occurring type of galanin-positive terminals (type 1) showed distinct changes with a granular matrix, many immunoreactive, peripherally located large dense-core vesicles, empty large vesicles and synaptic vesicles which were displaced from the presynaptic zone. Other galanin-positive terminals underwent even more pronounced morphological changes, including extensive vesiculolysis, also of large dense-core vesicles, filamentous degeneration or formation of axonal labyrinths. An increased number of galanin-positive nerve terminals was observed in lamina III of the ipsilateral dorsal horn after axotomy. They did not form glomeruli and contained few large dense-core vesicles. Post-embedding immunocytochemistry combined with quantitative analysis revealed that significant changes occurred in a proportion of terminals also with regard to peptide content in large dense-core vesicles after axotomy. Thus, the percentage of galanin-positive large dense-core vesicles increased in several cases and that of substance P- and CGRP-immunoreactive ones decreased.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of peripheral axotomy on presynaptic axon terminals with GABA‐like immunoreactivity

The facial nerve was unilaterally crushed at its exit from the stylomastoid foramen in three 3-month old male rats. After 10 days survival, before the regenerating axons had reinnervated their target muscles, the facial nucleus was examined to determine central patterns of response in material prepared to demonstrate the presence of GABA-like immunoreactivity with postembedding procedures using gold-labeled secondary antibody. The uninjured nucleus served as a control. In both control and injured nuclei, the GABAergic terminals synapse with all parts of the motor neurons, except the axon, and exhibit diverse morphologies. GABAergic axon terminals vary in their size and in the electron density of their axoplasm and the majority of the terminals contain pleomorphic vesicle profiles that display a range in their packing density and size. In both control and injured facial nuclei, only approximately 40% of the axon terminal profiles with pleomorphic vesicles exhibit GABA immunoreactivity. A morphometric analysis of the synaptic vesicle profiles in the GABA-positive terminals reveals that following axotomy there is no change in the mean number of synaptic vesicle profiles per GABAergic terminal profile. However, the mean size of the synaptic vesicle profiles in these terminals shows an axotomy-induced 50% increase, without change in the shapes of the enlarged vesicle profiles. Also, the numerical density of gold particles associated with the GABA-positive terminals is consistently greater in the injured than the control axon terminals. In the control animals quantitative analysis of the relative distribution of all axon terminal profiles in the neuropil categorized by the shape of their vesicle profiles as round, pleomorphic, or flat is 57:37:6. Ten days after axotomy the ratio of these categories in the injured nucleus has shifted to 35:60:5. This study demonstrates that the functional state of a postsynaptic target can influence the morphology of vesicle profiles in presynaptic elements as well as patterns of its afferent input.

Effect of Peripheral Axotomy on Expression of Neuropeptide Y Receptor mRNA in Rat Lumbar Dorsal Root Ganglia

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

Nitric oxide synthase-like immunoreactivity in lumbar dorsal root ganglia and spinal cord of rat and monkey and effect of peripheral axotomy.

With the immunofluorescence technique, nitric oxide synthase (NOS)-like immunoreactivity (LI) was found in a few medium-sized and small sensory neurons in lumbar (L) 4 and L5 dorsal root ganglia (DRG) of normal rat, and in most of these neurons, NOS-LI coexisted with calcitonin gene-related peptide and sometimes with substance P and galanin. NOS-immunoreactive nerve fibers, terminals and small neurons were also located in the dorsal horn of the segments 4 and 5 of the rat lumbar spinal cord with the highest density in inner lamina II. Many NOS-positive neurons and fibers were seen in the area around the central canal. A sparse network of NOS-immunoreactive nerve fibers was found in the ventral horn. After unilateral sciatic nerve cut in the rat, the number of NOS-positive neurons increased in the ipsilateral L4 and L5 DRGs, mainly in medium and small neurons, but also in some large neurons and very small neurons. NOS-LI could now also be seen in the ipsilateral dorsal roots, and in an increased number of fibers and terminals in both outer and inner lamina II of the ipsilateral dorsal horn. The number of NOS-immunoreactive neurons in lamina II of the ipsilateral dorsal horn was reduced. In the monkey L4 and L5 DRGs, many small neurons were NOS-immunoreactive, but only a few weakly stained nerve fibers and terminals were found in laminae I-IV of the dorsal horn at L4 and L5 lumbar levels. A few NOS-positive neurons were present in lamina X. The number of NOS-immunoreactive neurons was somewhat reduced in DRGs 14 days after peripheral axotomy, but no certain effect was seen in the dorsal horn. These results, together with earlier in situ hybridization studies, demonstrate that axotomy in rat induces a marked upregulation of NOS synthesis in primary sensory neurons, thus suggesting a role for NO in lesioned sensory neurons. In contrast, no such effect was recorded in monkey, perhaps indicating distinct species differences.

Expression of the class III beta-tubulin gene during axonal regeneration of rat dorsal root ganglion neurons.

The effect of peripheral axotomy on the expression of the class III beta-tubulin gene in adult dorsal root ganglion (DRG) neurons was examined. Of the 5 isotypic classes of beta-tubulin expressed in the mammalian nervous system, only the class III beta-tubulin is neuron specific. While information about the expression of several of the tubulin genes during neuronal development and regeneration has become available recently, very little is known about the expression of beta III-tubulin during axonal regeneration. To explore this issue, we examined axotomy-induced changes in beta III-tubulin mRNA levels in adult rat lumbar dorsal root ganglion (DRG) neurons at different times (1-28 days) after unilateral sciatic nerve crush using northern blotting of total RNA and quantitative in situ hybridization. These studies showed an initial decrease in beta III-tubulin mRNA levels in axotomized DRG neurons as compared to contralateral controls at 1 day after injury followed by robust increases in beta III-tubulin mRNA levels relative to contralateral controls from 1 to 4 weeks after injury. We postulate that beta III-tubulin may play an essential role in axonal growth because of its unique neuron-specific pattern of expression and its substantial increase in neurons that have been stimulated to regrow their axons.

Cytochrome oxidase activity in vagal and glossopharyngeal visceral sensory neurons of the rat: effect of peripheral axotomy

Cytochrome oxidase (CO) activity, an endogenous metabolic marker, was examined in visceral sensory neurons of the rat nodose and petrosal ganglia by using enzyme histochemistry. In the normal nodose and petrosal ganglia, nerve cells showed various degrees of staining intensity. The population of darkly stained neurons in the nodose ganglion was higher than in the petrosal ganglion. Axotomy of the peripheral axons of these bipolar sensory neurons was used to study potential changes in ganglionic cellular metabolism associated with loss of afferent inputs and/or injury. Peripheral axotomy had a significant effect on CO activity in the nodose ganglion. By 3 days after axotomy, darkly stained neurons decreased in number and lightly stained neurons, which were not observed in the normal ganglion, appeared in the nodose ganglion. At 7 days after axotomy, the average population of these lightly stained neurons increased to 29% in the nodose ganglion. Subsequently, the population decreased so that at 14 days and 21 days, 19% and 7% respectively of neurons were stained lightly. Even at 28 days after axotomy, the lightly stained neurons were still observed. In the petrosal ganglion, no remarkable change was observed at any stage after axotomy. These results suggest that metabolic activity decreases in some nodose neurons after peripheral nerve section.

Increased neuropeptide Y (NPY)-like immunoreactivity in rat sensory neurons following peripheral axotomy

The effects of peripheral axotomy (sciatic nerve transection) on the presence and distribution of neuropeptide Y (NPY) in rat dorsal root ganglion (DRG) and spinal grey matter were examined using immunocytochemistry. In normal rats and on the sham-operated side of experimental rats, NPY-like immunoreactivity (NPYir) was observed in all laminae of the lumbar spinal cord, with an especially dense concentration of immunostained axons and axonal varicosities in laminae I-II of the dorsal horn. There was no detectable NPYir in L4-L5 DRG cells from normal rats or from the sham-operated side of experimental rats. At 14 days after axotomy, there was a large ipsilateral increase in the density of NPYir axons and varicosities in the lumbar spinal cord on the side of the nerve injury; this was especially apparent in laminae III-V. In the same rats, NPYir was observed in many small, medium, and large neurons in the L4-L5 DRGs on the side of the severed nerve.