Peripheral axotomy affects nicotinamide adenine dinucleotide phosphate diaphorase and nitric oxide synthases in the spinal cord of the rabbit

Using nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry and nitric oxide synthase (NOS) immunocytochemistry combined with radioassay of calcium-dependent NOS activity, we examined the occurrence of NADPHd staining and NOS immunoreactivity (NOS-IR) in the dorsal root ganglia (DRG) neurons, dorsal root afferents, and axons projecting via gracile fascicle to gracile nucleus 14 days after unilateral sciatic nerve transection in the rabbit. Mild to moderate NADPHd staining and NOS-IR appeared in a large number of small and medium-sized to large neurons in the ipsilateral L4-L6 DRG, accompanied by enhanced NOS-IR of thick myelinated fibers in the ipsilateral L4-L6 dorsal roots. A noticeable increase in the density of punctate NADPHd staining occurred throughout laminae I-IV in the ipsilateral medial part of the dorsal horn in L4-L6 segments. Concurrently, a statistically significant decrease in the number of small NADPHd-exhibiting neurons in laminae I-II and, in contrast to this, a statistically significant increase of medium-sized to large NADPHd-stained somata in the ipsilateral laminae III-VI of L4-L6 segments were found. A detailed compartmentalization of L4-L6 segments into gray and white matter regions disclosed substantially increased catalytic NOS activity and inducible NOS mRNA levels in the dorsal horn and dorsal column ipsilaterally to the peripheral injury. A noticeable increase in the number of thick myelinated NADPHd-exhibiting and NOS-IR axons was noted in the ipsilateral gracile fascicle, terminating in dense, punctate NADPHd staining in the neuropil of the gracile nucleus. These observations indicate that the de novo-synthesized NOS in the lesioned primary afferent neurons resulting after sciatic nerve transection may be involved in an increase in NADPHd staining and NOS-IR in the ipsilateral dorsal roots and dorsal horn of L4-L6 segments, whence NOS could be supplied to ascending axons of the gracile fascicle.     

Glutamate uptake is attenuated in spinal deep dorsal and ventral horn in the rat spinal nerve ligation model

Alteration of glutamatergic (GLU) neurotransmission within the spinal cord contributes to hyperalgesic and allodynic responses following nerve injury. In particular, changes in expression and efficacy of glutamate transporters have been reported. Excitatory, pain transmitting primary afferent neurons utilizing glutamate as an excitatory neurotransmitter project to both superficial (I-II) and deep (III-V) laminae of the dorsal horn. These experiments were designed to examine changes in glutamate uptake occurring concomitantly within the spinal deep dorsal and ventral horn in situ after experimentally induced neuropathic pain. In vivo voltammetry, using microelectrode arrays configured for enzyme-based detection of GLU were employed. Sprague-Dawley rats had either sham surgery or tight ligation of L5 and L6 spinal nerves (SNL). Four to six weeks later, the L4-L6 spinal cord of chloral hydrate-anesthetized animals was exposed, and ceramic-based glutamate microelectrodes equipped with glass micropipettes 50 microm from the recording surfaces were placed stereotaxically at sites within the spinal cord. Pressure ejection of GLU into the ipsilateral L5-L6 spinal cord resulted in a 72% reduction of GLU uptake in SNL rats compared to sham controls in the ipsilateral L5-L6 deep dorsal horn and a 96% reduction in the ventral horn. In contrast, in the same animals, the contralateral L5-L6 or the ipsilateral L4 spinal cord showed no change in glutamate uptake. The data suggest that spinal nerve ligation produced attenuated glutamate uptake activity extending into the deep dorsal and ventral horn. The study suggests that plasticity related to spinal nerve injury produces widespread alteration in glutamate transporter function that may contribute to the pathophysiology of neuropathic pain.     

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. © 1993 Wiley-Liss, Inc.     

Neuronal nitric oxide synthase is upregulated in a subset of primary sensory afferents after nerve injury which are necessary for analgesia from alpha2-adrenoceptor stimulation

alpha2-Adrenoceptor (AR) agonists increase in analgesic potency and efficacy after peripheral nerve injury, and their effects are blocked by neuronal nitric oxide synthase (nNOS) inhibitors and M4 muscarinic receptor antagonists only after injury. We tested whether nNOS and M4 muscarinic receptors are co-expressed in the spinal cord, and whether destruction of a subset of sensory afferents which are essential to alpha2-AR analgesia would also destroy nNOS and M4 receptor expression. Male Sprague-Dawley rats underwent left L5 and L6 spinal nerve ligation. Lumbar spinal cord was removed and immunostained for M4 muscarinic receptors and nNOS alone and for co-expression. Others received intrathecal injection of saporin linked to an antibody to the neurotrophin receptor p75(NTR), which eliminates cells expressing this receptor and the analgesic effects of alpha2-AR agonists. nNOS staining of fibers in the superficial dorsal horn was dramatically increased after spinal nerve ligation, and this was abolished by saporin linked anti-p75(NTR) treatment. In contrast, nNOS staining in dorsal horn neurons was unaltered by these manipulations. M4 receptors were present on neurons in the dorsal horn, some of which co-expressed nNOS, but their pattern of expression was not altered by these manipulations. Peripheral nerve injury increases nNOS expression in fibers in the superficial dorsal horn, some of which likely express p75(NTR), and alpha2-AR agonists may reduce injury-induced sensitization by activation of nNOS in these fibers In contrast, changes in nNOS and M4 receptor location on spinal cord neurons are not responsible for increased analgesic potency of alpha2-AR agonists after nerve injury.     

Distribution of NADPH-d and nNOS-IR in the thoracolumbar and sacrococcygeal spinal cord of the guinea pig

The distribution of NADPH-d staining and neuronal nitric oxide synthase (nNOS)-immunoreactivity in the spinal cord of the guinea pig was studied to evaluate the potential role of nitric oxide in lumbosacral afferent and spinal autonomic pathways and to compare the distribution of these two markers to that observed in other species. NADPH-d staining and nNOS-immunoreactivity were present in neurons and fibers in the superficial dorsal horn, dorsal commissure and in neurons around the central canal in all levels of the spinal cord examined. Sympathetic preganglionic neurons in the thoracic and rostral lumbar segments identified by choline acetyl transferase (ChAT) immunoreactivity exhibited prominent NADPH-d staining and nNOS-immunoreactivity; whereas the ChAT-immunoreactive parasympathetic preganglionic neurons in the sacral segments were not stained. The most prominent NADPH-d staining in the sacral segments occurred in fibers extending from Lissauer's tract through laminae I along the lateral edge of the dorsal horn to the region of the sacral parasympathetic nucleus (lateral collateral pathway of Lissauer). These fibers were prominent in the S1-S3 segments but not in adjacent (L5-L7 and Cx1) or thoracolumbar segments. These NADPH-d fibers were, for the most part, not nNOS-immunoreactive, but did overlap with a prominent fiber bundle containing vasoactive intestinal polypeptide immunoreactivity in the sacral spinal cord. These results indicate that nitric oxide may function as a transmitter in thoracolumbar sympathetic preganglionic neurons, but not in sacral parasympathetic preganglionic neurons. Although the functional significance of the NADPH-d positive, nNOS-negative fiber bundle on the lateral edge of the sacral dorsal horn remains to be determined, this fiber tract may represent, in part, visceral afferent projections to the sacral parasympathetic nucleus.     

Restoration of substance P and calcitonin gene-related peptide in dorsal root ganglia and dorsal horn after neonatal sciatic nerve lesion

Dorsal root ganglion (DRG) neurons decrease their substance P (SP) synthesis after peripheral nerve lesions. Levels in the dorsal horn also decline but return to normal if regeneration is successful. In adults, when regeneration is prevented, recovery of SP in the dorsal horn is slow and incomplete, whereas in newborns, recovery is rapid and complete even though retrograde cell death of DRG neurons is greater than in adults. We have examined the mechanisms that might account for the rapid and complete recovery of SP and calcitonin-gene related peptide (CGRP) in the dorsal horn after peripheral nerve injury in newborns. Peptides were compared in the L4 and L5 DRG and spinal cord segments of normal rats and in rats surviving 6 days to 4 months after sciatic nerve section/ligation within 24 hours of birth. Sciatic nerve section/ligation produced 50% neuron death in L4 and L5 DRGs, but immunocytochemical methods showed that both SP-immunoreactivity (-IR) and CGRP-IR recovered completely in dorsal horn. Radioimmunoassay confirmed that recovery of SP was not an artefact due to shrinkage. β-Preprotachykinin (PPT)-mRNA hybridization and SP-IR were observed mostly in small neurons; α-CGRP-mRNA-hybridized and CGRP-IR neurons were more heterogeneous. The percentage of DRG neurons that contained SP (～ 25%) or CGRP (～ 50%) was the same in normal newborn and adult rats. Neither selective cell survival nor change in neuron phenotype was likely to contribute to the recovery seen in the dorsal horn, and DRG neurons ipsilateral to the lesion exhibited the same level of hybridized β-PPT-mRNA and α-CGRP-mRNA as intact DRG neurons. Because neither the constitutive level of expression of the genes nor peptide levels increased above those observed in intact DRG neurons, these mechanisms were also not responsible. Axotomized DRG neurons, however, contributed to recovery. Recovery was also due to sprouting by neurons in intact DRGs rostral and caudal to L4 and L5. © 1993 Wiley-Liss, Inc.     

Changes in galanin immunoreactivity in rat lumbosacral spinal cord and dorsal root ganglia after spinal cord injury

Alterations in the expression of the neuropeptide galanin were examined in micturition reflex pathways 6 weeks after complete spinal cord transection (T8). In control animals, galanin expression was present in specific regions of the gray matter in the rostral lumbar and caudal lumbosacral spinal cord, including: (1) the dorsal commissure; (2) the superficial dorsal horn; (3) the regions of the intermediolateral cell column (L1-L2) and the sacral parasympathetic nucleus (L6-S1); and (4) the lateral collateral pathway in lumbosacral spinal segments. Densitometry analysis demonstrated significant increases (P < or = 0.001) in galanin immunoreactivity (IR) in these regions of the S1 spinal cord after spinal cord injury (SCI). Changes in galanin-IR were not observed at the L4-L6 segments except for an increase in galanin-IR in the dorsal commissure in the L4 segment. In contrast, decreases in galanin-IR were observed in the L1 segment. The number of galanin-IR cells increased (P < or = 0.001) in the L1 and S1 dorsal root ganglia (DRG) after SCI. In all DRG examined (L1, L2, L6, and S1), the percentage of bladder afferent cells expressing galanin-IR significantly increased (4-19-fold) after chronic SCI. In contrast, galanin expression in nerve fibers in the urinary bladder detrusor and urothelium was decreased or eliminated after SCI. Expression of the neurotrophic factors nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) was altered in the spinal cord after SCI. A significant increase in BDNF expression was present in spinal cord segments after SCI. In contrast, NGF expression was only increased in the spinal segments adjacent and rostral to the transection site (T7-T8), whereas spinal segments (T13-L1; L6-S1), distal to the transection site exhibited decreased NGF expression. Changes in galanin expression in micturition pathways after SCI may be mediated by changing neurotrophic factor expression, particularly BDNF. These changes may contribute to urinary bladder dysfunction after SCI.     

L1 CAM expression in the superficial dorsal horn is derived from the dorsal root ganglion

The cell adhesion molecule L1 is highly expressed on embryonic axons and may play a role in axonal outgrowth and fasciculation. Generally only low levels of L1 are found in adult spinal cord except for intense labeling in Lissauer's tract, in laminae I-II, and on dorsolateral funicular axons. In this study we determine the source of L1 immunoreactivity in the dorsal spinal cord, the presence of L1 expression on sprouting axons, and the effect of exercise on L1 expression. We determined the source of L1 immunoreactivity in the superficial dorsal horn by performing acute unilateral rhizotomies (T12-L4) in adult rats. This resulted in a marked decrease in L1 expression in Lissauer's tract and laminae I-II on the deafferented side. The peptidergic and nonpeptidergic small-diameter primary afferent markers, calcitonin gene-related peptide (CGRP) and the lectin IB4 respectively, closely correlated with L1 expression and also decreased dramatically after rhizotomy. Considering its developmental role, we asked whether L1 was expressed on sprouting axons following chronic rhizotomy. L1 and CGRP, but not IB4, were detected on sprouting axons. Lastly, we investigated the effect of exercise on L1 expression by giving animals with chronic rhizotomies free access to an exercise wheel. After extensive exercise, L1, CGRP, and IB4 expression levels were unchanged compared with those of sedentary chronic animals. Combined, these data demonstrate that the dorsal root ganglia is a major source of L1-positive axons in the superficial dorsal horn, that both L1 and CGRP identify sprouting axons following rhizotomy, and that exercise does not upregulate L1 expression.     

Simultaneous identification of unmyelinated and myelinated primary somatic afferents by co-injection of isolectin B4 and Cholera toxin subunit B into the sciatic nerve of the rat

Several studies have used the transganglionic tracers cholera toxin subunit B (CTb) and either Bandeiraea simplicifolia isolectin B4 (IB4) or wheat-germ agglutinin (WGA) to label myelinated and unmyelinated afferent fibres respectively. In this study, we aim to determine whether co-injection of CTb and either IB4 or WGA into the sciatic nerve of rat will selectively label myelinated and unmyelinated simultaneously. A double immunofluorescence approach was used to detect these tracers in dorsal root ganglia (DRGs) and afferent fibre terminals in the spinal cord. CTb- and IB4-labelled neurons were seen mainly in L4 and L5 DRGs, with CTb labelling detected primarily in large sized neurons and IB4 staining seen mainly in smaller cells. Only a minority of CTb labelled DRG neuron profiles (5.1%) were also labelled with IB4. In the spinal cord, IB4-labelling was largely confined to lamina II of spinal segments L3-L5, whereas CTb-labelled terminals were seen in all laminae but sparse in lamina II. Confocal microscopy showed no evidence for colocalisation of CTb and IB4 labelling in any terminals in laminae I-III. Although the central distribution of CTb labelling in laminae I and II inner-IV had the same rostro-caudal and medio-lateral coverage as IB4 labelling in spinal segments L3-L5, CTb labelling in ventral laminae (of putative proprioceptor afferents) extended between T12 and S1. Similar patterns of central labelling were found when CTb and WGA were injected together. We therefore concluded that this co-injection approach provides a reliable method to identify both myelinated and unmyelinated somatic primary afferents simultaneously.     

Plastic changes and nitric oxide synthase induction in neurons which innervate the regenerated tail of the lizard Gekko gecko: II. The response of dorsal root ganglion cells to tail amputation and regeneration

The lizard tail regenerates after amputation, which severs the spinal cord and spinal nerves. Dorsal root ganglia (DRGs) do not regenerate in the regrowing tail, which is innervated by DRGs rostral to the amputation. With Nissl staining, NADPH-diaphorase histochemistry and nitric oxide synthase (NOS) immunohistochemistry, we investigated NOS expression and its relationship with structural changes in DRG neurons of caudotomized lizards. First, by horseradish peroxidase retrograde tracing we here provided evidence that the sensory innervation of the regenerated tail derives only from the three pairs of DRGs rostral to the amputation plane. These ganglia were then analyzed in control animals with original intact tail, at 5, 15 and 30 days after caudotomy, and at 8 months in lizards with mature regenerates. Caudotomy elicited in DRG neurons marked hypertrophy that persisted after tail regeneration. In control ganglia, most neurons were lightly NADPH-diaphorase-positive, a few were unstained or intensely stained. Tail transection elicited marked staining up-regulation, and an increase in the proportion of intensely positive neurons. The staining intensity peaked in DRG neurons at 15 days and was still significantly increased in respect to controls several months after complete tail regeneration. NOS immunoreactivity in DRGs matched the histochemical findings. NADPH-diaphorase positivity was also enhanced in the dorsal horn superficial laminae of the corresponding spinal segments. We demonstrate that transection of the lizard spinal nerves, provoked by tail loss, elicits in the axotomized primary sensory neurons marked NOS enhancement, which accompanies axon elongation in the regrowing tail and persists after the end of this process.     

Development of tolerance to the antinociceptive effect of systemic morphine at the lumbar spinal cord level: a c-Fos study in the rat

The development of tolerance to the antinociceptive effects of morphine was investigated in rats using carrageenin-induced spinal c-Fos expression. We took advantage of this technique to especially study, at the cellular level, in freely moving animals, the development of tolerance based on the visualization of dorsal horn spinal cord neurons which play a major role in nociceptive processes. Two hours after intraplantar injection of carrageenin (6 mg/150 μl of saline), c-Fos-like immunoreactivity (FLI) was observed predominantly in the superficial and deep laminae of the dorsal horn in segments L4 and L5 of the spinal cord. In naive rats, acute intravenous morphine (3 mg/kg, i.v.) reduced the number of superficial and deep FLI neurons; 49% and 59% reduction respectively (p<0.0001 for both). In morphine-pretreated rats (daily administration of subcutaneous morphine: 1, 3, 5, 10, 20 or 40 mg/kg once a day for 4 days), antinociceptive tolerance tested on day 5 by acute morphine (3 mg/kg, i.v.) was manifest in those groups pretreated with the highest doses of morphine (10, 20 or 40 mg/kg). From regression analysis, it appeared that tolerance to the antinociceptive effect of morphine developed progressively as a function of the chronic morphine dose used on neurons involved in spinal nociceptive processes (superficial and deep dorsal horn neurons). Similarly, in rats pretreated with 10 mg/kg of morphine over 1, 2, 3 or 4 days, tolerance progressively developed, for both spinal neuronal populations, as a function of the duration of the pretreatment. These results are discussed in the context of the several possible sites of action of morphine.     

Transneuronal labeling of spinal interneurons and sympathetic preganglionic neurons after pseudorabies virus injections in the rat medial gastrocnemius muscle.

The distribution of retrogradely and transneuronally labeled neurons was studied in CNS of rats 4 days after injections of the Bartha strain of pseudorabies virus (PRV) into the medial gastrocnemius (MG) muscle. Tissue sections were processed for immunohistochemical detection of PRV. Retrogradely labeled cells were identified in the ipsilateral MG motor column in the caudal L4 and the L5 spinal segments. In order to evaluate the efficacy of PRV retrograde cell body labeling, the number of PRV retrogradely labeled neurons in the MG motor column was compared to the number labeled with two conventional retrograde cell body markers--Fluoro-Gold and cholera toxin-HRP. A ratio of 1:3 representing medium-sized (less than 30 microns) versus large neurons (greater than 30 microns) was found in the Fluoro-Gold dye experiments; a 1:2 ratio was seen in the PRV experiments. In contrast, when cholera toxin-HRP was used as a retrograde marker, mainly large neurons were labeled; the medium-to-large cell body ratio was 1:10 suggesting cholera toxin-HRP may have a greater affinity for the terminals of alpha-motoneurons as opposed to gamma-motoneurons. Transneuronally labeled cells were identified in the L1-L6 spinal gray matter, intermediolateral cell column (T11-L2), lateral spinal nucleus and medial part of lamina VII in C4 and C5 spinal segments, brainstem (caudal raphe nuclei, rostral ventrolateral medulla, A5 cell group, paralemniscal nucleus, locus coeruleus, subcoeruleus nucleus, red nucleus) and paraventricular hypothalamic nucleus. In the L5 spinal cord, transneuronally labeled neurons were seen in the ipsilateral spinal laminae I and II and bilaterally in spinal laminae IV-VIII, and X. Similar results were obtained in rats that had chronic unilateral L3-L6 dorsal rhizotomies indicating most of the labeling was due to retrograde transneuronal cell body labeling. In order to determine whether PRV was transported into the spinal cord by the dorsal root axons, the ipsilateral dorsal root ganglia (DRGs) were examined for PRV immunoreactivity; none was found. However, using the polymerase chain reaction, viral DNA was shown to be present in the ipsilateral DRGs indicating that some of spinal cord cell body labeling may have resulted from anterograde transneuronal labeling, as well.     

Ureteral ligation induces Fos expression in the dorsal horn

To describe a sympathetic afferent circuit, the left ureter was ligated in anesthetized rats for 1.5–2 h followed by immunocytochemical processing to localize expression of either the immediate early gene (IEG) c-fos or Krox-24 in the spinal cord or dorsal root ganglia (DRG). No IEG expression was detected in DRG. Both Fos and Krox-24 expression was found in the dorsal horn. More Fos immunocytochemically stained cells were found in the dorsal hom both ipsi- and contralateral to the ligated ureter at spinal segments T₁₀–T₁₃ after ureteral ligation than after either sham ligation or anesthesia control procedures. More Fos stained cells were in the dorsal horn ipsilateral to the ligated ureter than on the contralateral side. The Fos staining patterns in the dorsal horn of ligated and sham-ligated animals were similar with most labeled cells in dorsomedial portions of laminae I and II. In contrast, the Fos staining pattern in the dorsal horn in anesthetized animals (unoperated controls) was noticeably different from operated animals with the most Fos cells in the ventrolateral part of laminae I-II. These results indicate that (1) Fos immunocytochemistry may be useful for tracing sympathetic afferent pathways, (2) the sensory pathway activated by ureteral ligation enters the spinal cord at lower thoracic levels, where renal and upper ureteral afferents are terminating, and (3) some of this sympathetic afferent pathway is located contralateral to the stimulated kidney. Neurons activated by ureteral ligation in the contralateral dorsal horn may mediate reno-renal reflexes.     

Expression of nitric oxide synthase in the spinal cord in C57BL/6J mice with congenital muscular dystrophy

Autoradiography with the nitric oxide synthase (NOS) inhibitor ((3)H)nitro-L-arginine ([(3)H]L-NNA) was used to quantify NOS in cervical and lumbar spinal cord in normal and dystrophic mice. A single homogeneous population of binding sites was seen in all subregions of the gray matter in normal mice and in the superficial dorsal horn in dystrophic mice. However, in dystrophic mice, two populations were revealed in the deeper dorsal, intermediate, and ventral subregions. Pronounced immunoreactivity for neuronal NOS (nNOS), and weak immunoreactivity for endothelial NOS (eNOS), were revealed in all subregions in normal and dystrophic mice. Inducible NOS (iNOS) immunoreactivity was negligible in normal mice but intense in the deeper dorsal, intermediate, and ventral subregions in dystrophic mice. The higher affinity ((3)H)L-NNA binding site colocalized with nNOS and the lower affinity site with iNOS. It is suggested that expression of iNOS is associated with the pathological changes occurring in congenital muscular dystrophy.     

Increased uptake and transport of cholera toxin B-subunit in dorsal root ganglion neurons after peripheral axotomy: Possible implications for sensory sprouting

In the present study we show that, in contrast to the rat, injection of cholera toxin B-subunit (CTB) into the intact sciatic nerve of Macaca mulatta monkey gives rise to labelling of a sparse network of fibers in laminae I–II of spinal cord and of some mainly small dorsal root ganglion (DRG) neurons. Twenty days after sciatic nerve cut, the percentage of CTB-positive lumbar 5 (L5) DRG neuron profiles increased from 11% to 73% of all profiles. In the spinal cord, a marked increase in CTB labelling was seen in laminae I, II, and the dorsal part of lamina III. In the rat L5 DRGs, 18 days after sciatic nerve cut, the percentage of CTB- and CTB conjugated to horseradish peroxidase (HRP)-labelled neuron profiles increased from 45% to 81%, and from 54% to 87% of all neuron profiles, respectively. Cell size measurements in the rat showed that most of the CTB-positive neuron profiles were small in size after axotomy, whereas most were large in intact DRGs. In the rat spinal dorsal horn, a dense network of CTB-positive fibers covered the whole dorsal horn on the axotomized side, whereas CTB-labelled fibers were mainly seen in laminae III and deeper laminae on the contralateral side. A marked increase in CTB-positive fibers was also seen in the gracile nucleus. The present study shows that in both monkey and rat DRGs, a subpopulation of mainly small neurons acquires the capacity to take up CTB/CTB-HRP after axotomy, a capacity normally not associated with these DRG neurons. These neurons may transganglionically transport CTB and CTB-HRP. Thus, after peripheral axotomy, CTB and CTB-HRP are markers not only for large but also for small DRG neurons and, thus, possibly also for both myelinated and unmyelinated primary afferents in the spinal dorsal horn. These findings may lead to a reevaluation of the concept of sprouting, considered to take place in the dorsal horn after peripheral nerve injury. J. Comp. Neurol. 404:143–158, 1999. © 1999 Wiley-Liss, Inc.     

The regional distribution of nitric oxide synthase activity in the spinal cord of the dog

The aim of this study was to examine the distribution of calcium-dependent nitric oxide synthase activity (cNOS) in the white and gray matter in cervical, thoracic, lumbar and sacral segments of the spinal cord and cauda equina of the dog. The enzyme's activity, measured by the conversion of [3H]arginine to [3H]citrulline revealed considerable region-dependent differences along the rostrocaudal axis of the spinal cord in general and in cervical (C1, C2, C4, C6 and C8) and lumbar (L1-L3, L4-L7) segments in particular. In the non-compartmentalized spinal cord, the cNOS activity was lowest in the thoracic and highest in the sacral segments. No significant differences were noted in the gray matter regions (dorsal horn, intermediate zone and ventral horn) and the white matter columns (dorsal, lateral and ventral) in the upper cervical segments (C1-C4), except for a significant increase in the ventral horn of C4 segment. In C6 segment, the enzyme's activity displayed significant differences in the intermediate zone, ventral and lateral columns. Surprisingly, extremely high cNOS activity was noted in the dorsal horn and dorsal column of the lowest cervical segment. Comparing the enzyme's activity in upper and lower lumbar segments of the spinal cord, cNOS activity prevailed in L4-L7 segments in the dorsal horn and in all the above mentioned white matter columns.     

Anatomical evidence that the uninjured adjacent L4 nerve plays a significant role in the development of peripheral neuropathic pain after L5 spinal nerve ligation in rats

Rats develop hyperalgesia and allodynia in the hind paw after L5 spinal nerve ligation. Phosphorylated extracellular regulated kinase (pERK) was used as a pain marker to investigate the potential role of adjacent uninjured L4 nerve in the development of heat hyperalgesia after L5 nerve injury. Left L5 nerve was ligated and sectioned in rats. Three days later, rats were randomly assigned to five groups; each had both hind paws immersed in water at different temperatures (no heat, 37, 42, 47, and 52°C) under sevoflurane anesthesia for 2 minutes. Five minutes after stimulation the rats were sacrificed and sections of L3–L6 spinal segments were stained immunocytochemically with pERK antibody. pERK immunoreactivity, which is not detectable in the normal spinal cord, was discernible in neurons (not glia) of the superficial dorsal horn after noxious heat stimuli. pERK‐positive neurons clearly overlapped in laminae I–II with normal unmyelinated and thin myelinated afferents labeled with calcitonin gene‐related peptide and isolectin B4, and injured unmyelinated afferents labeled with vasoactive intestinal polypeptide. There was a linear increase in pERK immunoreactivity on both sides with an increase in temperature. Importantly, the number of positive pERK neurons was significantly higher in the ipsilateral side of L4 spinal segment, which receives innervation from uninjured L4 nerve, compared with the contralateral control side, which receives both uninjured L4 and L5 spinal nerves. The data demonstrate that the uninjured L4 nerve plays an important role in the development of heat hyperalgesia at the spinal cord level after L5 nerve injury. J. Comp. Neurol. 523:1731–1747, 2015. © 2015 Wiley Periodicals, Inc.     

Nitric oxide synthase expression and cell changes in dorsal root ganglia and spinal dorsal horn of developing and adult Rana esculenta indicate a role of nitric oxide in limb metamorphosis

Metamorphosis of amphibians requires reconfiguration of sensory and locomotor neural networks. In view of such plastic changes and implications of nitric oxide (NO) in neural developmental shaping, we examined via histochemistry and immunohistochemistry its synthetic enzyme nitric oxide synthase (NOS) in dorsal root ganglia (DRGs) and dorsal horn of the developing and adult frog Rana esculenta. In limb DRGs, NOS positivity was first and selectively detected just before limb bud appearance, increased during metamorphosis, and was then down-regulated. In adulthood, NOS was expressed in some DRG neurons at all segmental levels. Similar features were detected in the dorsal horn neuropil. In limb DRGs, cell counts in Nissl-stained sections revealed a twofold increase of differentiated neurons during metamorphosis and an additional twofold increase in adulthood. Perikaryal sizes in limb DRGs did not vary during metamorphosis but increased and were more heterogeneous in the adult frog, probably reflecting adaptation to body size. NOS and cell changes during metamorphosis were much less marked in DRGs at other levels. Carbocyanine tracing documented selective labeling of NOS-expressing hindlimb DRG neurons from the spinal nerve at the time of initiation of hindlimb movements. The findings show that, in limb DRG neurons, NOS parallels cell differentiation and limb development during metamorphosis. The data also provide evidence of NOS expression in DRG cells innervating the hindlimbs when sensorimotor circuits become functionally mature. This study indicates a key role of NO production in the maturation of sensory functions that subserves in amphibians the transition from swimming to tetrapod locomotion.     

Immune cell involvement in dorsal root ganglia and spinal cord after chronic constriction or transection of the rat sciatic nerve

Chronic constriction injury (CCI) of the sciatic nerve in rodents produces mechanical and thermal hyperalgesia and is a common model of neuropathic pain. Here we compare the inflammatory responses in L4/5 dorsal root ganglia (DRGs) and spinal segments after CCI with those after transection and ligation at the same site. Expression of ATF3 after one week implied that 75% of sensory and 100% of motor neurones had been axotomized after CCI. Macrophage invasion of DRGs and microglial and astrocytic activation in the spinal cord were qualitatively similar but quantitatively distinct between the lesions. The macrophage and glial reactions around neurone somata in DRGs and ventral horn were slightly greater after transection than CCI while, in the dorsal horn, microglial activation (using markers OX-42(for CD11b) and ED1(for CD68)) was greater after CCI. In DRGs, macrophages positive for OX-42(CD11b), CD4, MHC II and ED1(CD68) more frequently formed perineuronal rings beneath the glial sheath of ATF3+ medium to large neurone somata after CCI. There were more invading MHC II+ macrophages lacking OX-42(CD11b)/CD4/ED1(CD68) after transection. MHC I was expressed in DRGs and in spinal sciatic territories to a similar extent after both lesions. CD8+ T-lymphocytes aggregated to a greater extent both in DRGs and the dorsal horn after CCI, but in the ventral horn after transection. This occurred mainly by migration, additional T-cells being recruited only after CCI. Some of these were probably CD4+. It appears that inflammation of the peripheral nerve trunk after CCI triggers an adaptive immune response not seen after axotomy.     

Cholecystokinin-like immunoreactivity in the rat spinal cord: Effects of thoracic transection

A study of cholecystokinin-like immunoreactivity in the lumbar (L1-L5) spinal cord segments of rats was realised 24-48 hours after complete thoracic transection (T6-T8). A comparison was made with corresponding spinal cord segments from control and sham-operated animals. The immunocytochemical study with light microscopy showed cholecystokinin-like immunoreactive cell bodies in laminae VII and X at L1-L5, caudal to the transection. In addition, the immunoreactivity was greatly enhanced in bundles of the dorsolateral funiculus compared to sham-operated animals. Our results suggest that part of cholecystokinin-like cell bodies of laminae VII and X send projections to supraspinal sites. Some of these supraspinal projections would go through the dorsolateral funiculus. In the lumbar dorsal horn of operated animals, the immunoreactivity was greatly enhanced in lamina I, while it was slightly decreased in lamina II, compared to control animals. Using electron microscopy, in lamina I, the immunoreactivity localized in different neurites was generally very intense. Moreover, axon terminals showed swelling: their mean size was 0.8-1.8 microns (0.5-1.2 in control animals). This result suggests that some cholecystokinin-like neurons also project to lamina I of rostral cervical segments. In lamina II, numerous degenerating axons were observed (24 hours after thoracic spinal transection). This would suggest that part of descending cholecystokinin-like projections terminate in lamina II.