Deafferentation-induced expansion of saphenous terminal field labelling in the adult rat dorsal horn following pronase injection of the sciatic nerve

We have examined the effect of the degeneration of sciatic nerve afferents on the distribution of saphenous terminals in the adult rat dorsal horn. Deafferentation was produced by injection into the sciatic nerve of pronase, a combination of proteolytic enzymes, which causes death of ganglion cells and degeneration of their terminal fields. The saphenous terminal fields were labelled by exposing the cut nerve to a combination of horseradish peroxidase (HRP) and wheat germ agglutinin-horseradish peroxidase (WGA-HRP). Terminals were mainly found in the superficial dorsal horn, indicating that small-diameter afferents were heavily labelled. In one group of control animals, the normal sciatic and normal saphenous terminal fields were shown to be bilaterally symmetrical. In the experimental group, the initial injection of one sciatic nerve with pronase was followed 4 months later by bilateral HRP/WGA-HRP labelling of both saphenous nerves. In each animal, the terminal field of the saphenous nerve on the lesioned side was expanded in the medial, lateral, and caudal directions. Medially and laterally, the expanded terminal field overlapped more of the sciatic territory than normal; caudally, saphenous terminals were found in the rostral portion of the L5 segment, in an area normally filled by sciatic terminals and devoid of saphenous terminals. The expansion resulted in a total saphenous area 26% larger than the control side. Electron microscopy demonstrated that the label in both the normal and expanded territories was primarily contained in axons and terminals, with minor transneuronal labelling. Labelled terminals in the expanded areas were both simple terminals with round, clear vesicles, and glomerular terminals with multiple synaptic contacts; these terminal types resemble those previously described for primary afferents in the superficial dorsal horn. Although the preexistence of "silent" synaptic terminals in the expanded areas cannot be disproven, the data support the hypothesis that primary afferents in the adult have the potential to sprout and establish synapses when the conditions of the deafferentation are favorable.     

The sprouting of saphenous nerve terminals in the spinal cord following early postnatal sciatic nerve section in the rat

Transganglionic labelling of the saphenous nerve in rats with WGA-HRP revealed the central distribution of its terminals in the lumbar dorsal horn. The terminal field was clearly defined and consistent in rats aged between day 6 and day 90. If, however, the sciatic nerve was sectioned on day 1 of postnatal life, the saphenous terminal field expanded to occupy twice the normal area (measured between the L2 and L4 boundaries). The spread was caudal, medial, and lateral into areas normally occupied by sciatic nerve terminals. This sprouting was very weak if the sciatic nerve was sectioned later in postnatal life, on day 5, and nonexistent if sectioning took place on day 10. Crushing the sciatic nerve on day 1 also triggered the effect but the spread of the terminal field was less than that produced by section of the sciatic nerve. There was no evidence of sprouting from the contralateral intact sciatic nerve. The results demonstrate the necessity of intact afferent input during a critical period of neonatal life in order to maintain the precise somatotopic termination pattern of dorsal root afferents.     

Neuropeptide Y and Galanin Binding Sites in Rat and Monkev Lumbar Dorsal Root Ganalia 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 1–11. [¹²⁵I]NPY binding was most strongly attenuated by NPY_13–36, 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 NPY_13–36 completely abolished PYY binding, which was also reduced by [Leu,³¹ 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. The present results indicate that after axotomy the synthesis of NPY receptors is increased in rat DRG neurons, especially in large neurons, and is transported to the laminae I–IV of the ipsilateral dorsal horn and into the sciatic nerve. No such up-regulation of the NPY receptor occurred in monkey DRG after axotomy. The Y2 receptor seems to be the main NPY receptor in DRG and the dorsal horn of the rat and monkey spinal cord, but Y1 receptors also exist. The increase in NPY binding sites in laminae I–IV of the dorsal horn after axotomy partly represents Y1 receptors. In contrast to the rat, galanin binding sites could be identified in monkey lumbar DRG. No effect of axotomy on the distribution of galanin binding sites in rat or monkey DRG and dorsal horn was detected, suggesting their presence on local dorsal horn neurons (or central afferents).     

The somatotopic organization of primary afferent terminals in the superficial laminae of the dorsal horn of the rat spinal cord

Transganglionic transport of wheatgerm agglutinin conjugated horse-radish peroxidase (WGA-HRP) was used to reveal the central distribution of terminals of primary afferent fibers from peripheral nerves innervating the hind leg of the rat. In separate experiments the sizes and locations of cutaneous peripheral receptive fields were determined by electrophysiological recording techniques for each of the nerves that had been labeled with WGA-HRP. By using digital image analysis, the sizes and positions of the peripheral receptive fields were correlated with the areas of superficial dorsal horn occupied by terminals of primary afferents from each of these receptive fields. Data were obtained from the posterior cutaneous nerve of the thigh, lateral sural, sural, saphenous, superficial peroneal, and tibial nerves. The subdivisions of the sciatic nerve, the sural, lateral sural, superficial peroneal, and tibial nerves each projected to a separate and distinct region of the superficial dorsal horn and collectively formed a "U"-shaped zone of terminal labeling extending from lumbar spinal segments L2 to the caudal portions of L5. The gap in the "U" extended from L2 to the L3-4 boundary and was occupied by terminals from the saphenous nerve. Collectively, all primary afferents supplying the hindlimb occupied the medial 3/4 of the superficial dorsal horn with terminals from the tibial nerve lying most medially and occupying the largest of all the terminal fields. Afferents from the superficial peroneal lay in a zone between the medially situated tibial zone and the more laterally placed sural zone. Afferents from the posterior cutaneous nerve were located most caudally and laterally. Terminal fields from the posterior cutaneous and saphenous nerves differed from the others in having split representations caused presumably by their proximity to the mid-axial line of the limb. Comparisons between the peripheral and the central representations of each nerve revealed that 1 mm2 of surface area of the superficial dorsal horn serves approximately 600-900 mm2 of hairy skin and roughly 300 mm2 of glabrous skin. The vast majority of terminal labeling observed in the dorsal horn was found in the marginal layer and substantia gelatinosa, suggesting that small diameter afferents have an orderly somatotopic arrangement in which each portion of the skin surface is innervated by afferent fibers that terminate in preferred localities within the dorsal horn.     

Expansion of spinal cord primary sensory afferent projection following combined sciatic nerve resection and saphenous nerve crush: a horseradish peroxidase study in the adult rat

Transganglionic transport of horseradish peroxidase was used to study the potential for collateral sprouting of saphenous nerve afferent fibers in the lumbar dorsal horn of the adult rat following (1) combined unilateral saphenous nerve crush and ipsilateral sciatic nerve resection, (2) unilateral saphenous nerve crush, and (3) unilateral sciatic nerve resection. The saphenous nerve on the nonlesioned contralateral side served as control. Eight weeks after the lesion(s) the animals were subjected to bilateral application of horseradish peroxidase to the saphenous nerves. The distribution of the ensuing labeling in the superficial dorsal horn was subsequently mapped. Combined saphenous nerve crush and sciatic nerve resection resulted in expansion of the saphenous nerve projection area in the dorsal horn when compared to the nonlesioned control side (mean = 13%, P less than 0.05). No expansion of the saphenous nerve projection was found following isolated saphenous nerve crush or sciatic nerve resection, respectively (P greater than 0.05). The findings indicate that in the adult rat, central processes of primary sensory neurons which are regenerating their peripheral processes can extend collateral sprouts into adjacent projection areas in the superficial dorsal horn subjected to previous deafferentation by peripheral nerve resection.     

Functional Connections Formed by Saphenous Nerve Terminal Sprouts in the Dorsal Horn Following Neonatal Sciatic Nerve Section

The rostrocaudal distribution of saphenous nerve inputs into the lumbar dorsal horn from L2 to L6 has been investigated in urethane anaesthetized rats whose left sciatic nerve was cut and ligated at birth. In normal cord, electrical stimulation of the saphenous nerve evoked dorsal horn spikes in L2 to caudal L4. Few or no spikes were evoked in L5. After neonatal sciatic nerve section, saphenous nerve stimulation evoked spikes throughout segments L2 to L6. Dorsal horn cell receptive fields were also altered following neonatal sciatic nerve section. A somatotopic map of the lumbar enlargement in normal rats was constructed from the receptive fields (RFs) of adjacent dorsal horn cells. Cells with RFs in the saphenous skin region were concentrated in L3 and rostral L4 and very few were found in L5. After neonatal sciatic nerve section, however, a substantial number of cells with low threshold saphenous skin RFs were also found in caudal L4 and throughout L5. These results show that the central saphenous nerve terminal sprouts that grow into the sciatic terminal region following neonatal sciatic nerve section (Fitzgerald, 1985, J. Comp. Neurol., 240, 414-422; Fitzgerald et al., 1990, J. Comp. Neurol., 300, 370-385) form functional connections. This results in dorsal horn cells that are not normally influenced by saphenous nerve inputs developing substantial low threshold RFs in saphenous nerve skin regions.     

Neonatal Sciatic Nerve Section Results in a Rearrangement of the Central Terminals of Saphenous and Axotomized Sciatic Nerve Afferents in the Dorsal Horn of the Spinal Cord of the Adult Rat

Previous studies have shown that following neonatal peripheral nerve injury, adjacent intact myelinated and unmyelinated primary afferents sprout into the central denervated terminal area. The present study investigates this in more detail and goes further, to study the fate of the central terminals of the surviving axotomized primary afferent neurons. Bulk labelling of the sciatic and saphenous nerves with horseradish peroxidase conjugated to choleragenoid (B-HRP), to label the A fibres, or wheatgerm agglutinin (WGA-HRP), to label C fibres were employed to investigate the central consequences of sciatic nerve section and ligation on the day of birth, in adult rats. Bulk labelling of the axotomized sciatic or intact saphenous nerve with either tracer and comparison with contralateral controls revealed alterations to the terminal field. The intact saphenous nerve terminal field expanded caudally from mid L4 to the L4-L5 boundary when labelled with WGA-HRP and to the sacral cord when labelled with B-HRP. Labelling the axotomized sciatic nerve with either tracer revealed little change in the overall somatotopic organization of central terminals, although labelling was less intense compared to control nerves and more variable with WGA-HRP. Invasion of the substantia gelatinosa (SG) by axotomized A fibres was observed in segments L3-5, into the area occupied by axotomized C fibres. This area was also invaded by intact saphenous A fibres in the L4–5 segments. These results demonstrate that following neonatal nerve section: (i) axotomized primary afferents are able to retain a ‘normal’ somatotopic map in the rostrocaudal plane; (ii) both A and C fibres from adjacent intact nerves sprout into the denervated territory, but A fibres sprout further caudally; (iii) axotomized A fibres and invading intact A fibres both sprout dorsally into denervated SG. As a result, there is considerable overlap between nerve territories in denervated spinal cord, suggesting that competition for laminar termination sites exists between A and C fibres and also between axotomized and intact primary afferents.     

坐骨神经非冻结性冷损伤后背根神经节细胞的凋亡

目的观察大鼠坐骨神经在非冻结性低温作用后,腰段脊髓L4-L6背根神经节(dorsal root ganglion,DRG)凋亡神经元的数量以及形态学改变,探讨周围神经非冻结性冷损伤致DRG神经元凋亡的情况。方法雄性Wistar大鼠33只,随机分为1周组、2周组、3周组,每组11只。每只大鼠取任意一侧坐骨神经为实验侧,给予冷损伤(4℃,2h),对照侧坐骨神经同样方法暴露,不给予低温处理。根据坐骨神经和DRG的病理变化,分别取两侧的L4、L5、L6背根神经节于低温结束后1周、2周、3周采用流式细胞仪(Annexin/PI法)和Tunel法对DRG神经元凋亡进行定量和定性检测。结果流式细胞仪(Annexin/PI法)定量测定结果显示,实验侧凋亡率明显高于对照侧。Tunel法检测发现受冷侧的DRG出现典型的Tunel染色阳性的早期凋亡细胞,半定量测定显示实验侧DRG神经元凋亡率明显高于对照侧。两种方法均显示受冷后1周开始凋亡细胞增多,2周时到达高峰,3周时略下降。结论非冻结性低温可以造成坐骨神经对应的L4、L5、L6DRG神经元出现凋亡,凋亡的高峰出现在冷损伤后2周,以早期凋亡为主。凋亡可能是坐骨神经冷损伤的机制之一。     

Do central terminals of intact myelinated primary afferents sprout into the superficial dorsal horn of rat spinal cord after injury to a neighboring peripheral nerve?

In order to investigate whether normal myelinated primary afferent axons sprout into the territories of adjacent injured peripheral nerve fibers in the superficial dorsal horn of the spinal cord, adult rats underwent either sectioning of the saphenous or femoral nerves on one side, or else unilateral denervation of the skin of the posterior thigh. Two weeks later cholera toxin B subunit (CTb), which is normally transported selectively by myelinated somatic primary afferents, was injected into the ipsilateral (intact) sciatic nerve. The relationship between CTb, vasoactive intestinal peptide (VIP), and binding of Bandeiraea simplicifolia isolectin B4 (IB4) was then examined in the ipsilateral dorsal horn of the second to fifth lumbar spinal segments (L2-L5). Sectioning of the femoral or saphenous nerves resulted in a reduction of IB4 binding in laminae I-II in the medial third of the dorsal horn of L2, L3, and the upper part of L4. VIP-immunoreactivity was upregulated in exactly the same regions in which IB4-binding was reduced. These correspond to the areas that were previously innervated by unmyelinated afferents in the sectioned nerves. CTb-labeling was detected in regions known to receive input from myelinated sciatic afferents: lamina I and a band extending from the inner part of lamina II (IIi) to lamina V in the L3-5 segments, and the deepest part of the dorsal horn in L2. Importantly, no CTb-labeling was detected in the outer part of lamina II (IIo) in the denervated areas. Sectioning of branches of the posterior cutaneous nerve of the thigh resulted in a reduction of IB4-binding and upregulation of VIP-immunoreactivity in the lateral part of the superficial dorsal horn of caudal L4 and L5. Again, CTb-immunoreactivity showed the normal sciatic pattern in L4-L5, with no labeling detected in lamina IIo in the denervated region. These results do not support the suggestion that the central terminals of intact myelinated afferents sprout into regions of lamina II occupied by adjacent nerves that have been axotomized peripherally.     

Expression of c-Jun as a Response to Dorsal Root and Peripheral Nerve Section in Damaged and Adjacent Intact Primary Sensory Neurons in the Rat

It was previously shown that the immediate early gene, c-jun , was highly expressed over long periods, in both peripheral sensory and motor neurons following axon damage or block of axoplasmic transport. Here we have examined the question of whether the expression of c-Jun protein is related to axon injury per se or to the process of axon growth. We have examined dorsal root ganglion (DRG) cells subjected to different manipulations which are associated with varying degrees of regrowth, as follows: (i) after peripheral nerve section, where it appears that all damaged neurons make some regenerative effort. 1 – 24 days after sciatic nerve section and ligation most cells in L4/L5 DRG were c-Jun-positive; (ii) after section of the central processes of the DRG cells, which then showed a slow and limited regrowth of their axons towards, but not into, the spinal cord. This resulted in a variable, but significant, expression of c-Jun in a small number of DRG cells; (iii) in intact sensory neurons that were offered the opportunity to sprout into adjacent denervated peripheral tissue. The sciatic nerve was ligated and the response of cells in the L3 ganglia (many of which project to the saphenous nerve) was measured. A small but significant number of cells were c-Jun-positive; (iv) in intact sensory neurons that were offered the opportunity to sprout centrally into partialy denervated neuropil of the spinal cord. We examined neurons in the L3 DRG after rhizotomy of the adjacent L4/L5 dorsal roots. Previous work suggests that sensory neurons show at best a very limited growth under these conditions. No significant increase was seen in c-Jun expression in these cases. These results suggest that c-Jun expression is closely correlated with growth and regeneration, and not simply a consequence of neuronal injury.     

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.     

Increase of preprotachykinin mRNA and substance P immunoreactivity in spared dorsal root ganglion neurons following partial sciatic nerve injury

Complete sciatic nerve injury reduces substance P (SP) expression in primary sensory neurons of the L4 and L5 dorsal root ganglia (DRG), due to loss of target-derived nerve growth factor (NGF). Partial nerve injury spares a proportion of DRG neurons, whose axons lie in the partially degenerating nerve, and are exposed to elevated NGF levels from Schwann and other endoneurial cells involved in Wallerian degeneration. To test the hypothesis that SP is elevated in spared DRG neurons following partial nerve injury, we compared the effects of complete sciatic nerve transection (CSNT) with those of two types of partial injury, partial sciatic nerve transection (PSNT) and chronic constriction injury (CCI). As expected, a CSNT profoundly decreased SP expression at 4 and 14 days postinjury, but after PSNT and CCI the levels of preprotachykinin (PPT) mRNA, assessed by in situ hybridization, and the SP immunoreactivity (SP-IR) of the L4 and L5 DRGs did not decrease, nor did dorsal horn SP-IR decrease. Using retrograde labelling with fluorogold to identify spared DRG neurons, we found that the proportion of these neurons expressing SP-IR 14 days after injury was much higher than in neurons of normal DRGs. Further, the highest levels of SP-IR in individual neurons were detected in ipsilateral L4 and L5 DRG neurons after PSNT and CCI. We conclude that partial sciatic nerve injury elevates SP levels in spared DRG neurons. This phenomenon might be involved in the development of neuropathic pain, which commonly follows partial nerve injury.     

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.     

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.     

Effect of nerve section on the spinal distribution of neighboring nerves

The spinal cord distribution of axonal terminals of peripheral nerves that innervate the skin of the upper medial thigh was examined in rats using transganglionic transport of horseradish peroxidase (HRP) and wheat-germ agglutinin-conjugated HRP (WGA-HRP). Chronic transection of the sciatic nerve or both the sciatic and saphenous nerves did not alter this distribution. Therefore, long-distance sprouting of intact 'thigh nerve' afferents in the dorsal horn is apparently not the mechanism whereby spinal dorsal horn neurons deafferented by sciatic and saphenous neurectomy, gain novel receptive fields in the cutaneous distribution of neighbouring intact nerves of the thigh.     

Sciatic nerve transection produces death of dorsal root ganglion cells and reversible loss of substance P in spinal cord

Sciatic nerve section has been shown to reduce substance P (SP) in the dorsal horn of the spinal cord, but the mechanism which underlies the reduction is not understood. Whether SP levels subsequently recover as they do after dorsal rhizotomy has also been unknown. To test the hypothesis that transganglionic degeneration of primary afferents contributes to the reduction of SP, we have studied the changes in SP which result from section of the cat sciatic nerve and determined the extent of dorsal root ganglion (DRG) cell death. Sciatic nerve section resulted in DRG cell death, but the amount was variable and not seen in all animals. Reduction in dorsal horn and DRG SP was seen in all animals, and in the spinal cord it was followed by recovery. These sequelae resemble the changes which follow dorsal rhizotomy. After sciatic nerve section, the reduction in dorsal horn SP is small than after rhizotomy, the recovery more complete, and both the reduction and the recovery proceed more slowly. Evidence is presented that similar mechanisms may contribute to depletion of intraspinal SP after sciatic nerve section and after dorsal rhizotomy. The mechanisms contributing to recovery of spinal cord SP after sciatic nerve section may resemble known mechanisms of recovery that occur when the lesion is central.     

A-fiber sprouting in spinal cord dorsal horn is attenuated by proximal nerve stump encapsulation

Peripheral nerve transection in the rat alters the spinal cord dorsal horn central projections from both small and large DRG neurons. Injured neurons with C-fibers exhibit transganglionic degeneration of their terminations within lamina II of the spinal cord dorsal horn, while peripheral nerve injury of medium to large neurons induces collateral sprouting of myelinated A-fibers from lamina I and III/IV into lamina II in rats, cats, and primates. To date, it is not known what sequelae are responsible for the collateral sprouting of A-fibers after peripheral nerve injury, although target-derived factors are thought to play an important role. To determine whether target-derived factors are necessary for changes in A-fiber laminar terminations in rat spinal cord dorsal horn, we unilaterally transected the sciatic nerve and ensheathed the proximal nerve stump in a silicone cap. Three days before sacrifice of rat, the injured sciatic nerve was injected with cholera toxin beta-subunit conjugated to horseradish peroxidase (betaHRP) that effectively labels both peripheral and central A-fiber axons. The effect of the ligature, axotomy, and silicone cap treatment was evaluated by analyzing the extent of betaHRP-, Substance P-(SP-), and isolectin B4- (IB4-) immunoreactive (ir) fibers in the somatotopically appropriate spinal cord dorsal horn regions. In all animals, 2-5 weeks after nerve transection (treated or otherwise), IB4- and SP-ir is absent from lamina II. Animals without nerve cap treatment exhibited robust fiber sprouting into lamina II at 2 weeks. In sharp contrast, animals treated with silicone caps did not exhibit betaHRP-ir fibers in lamina II at 2 weeks. This observation was extended up to 5 weeks postinjury. These results suggest that axotomy-induced expansion of betaHRP-ir primary afferent central terminations in the spinal cord dorsal horn is dependent on factors produced in the injury site milieu. While our understanding of local repair mechanisms of injured peripheral nerves is incomplete, it is clear that the time-dependent production of growth factors in the nerve injury microenvironment favor nerve fiber outgrowth, both peripherally and centrally.     

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.     

Changes in the somatotopic organization of the cat lumbar spinal cord following peripheral nerve transection and regeneration

Glass microelectrodes were used to record the activity of neurones in the left dorsal horn of the L6 segment of the spinal cord of normal cats and cats in which the left sciatic and saphenous nerves had been cut 1 or 9 months previously. In the normal animals the receptive fields of L6 dorsal horn neurones excited by tactile stimulation of the leg were somatotopically organized, with neurones in the medial and central dorsal horn having receptive fields on the distal parts of the leg, particularly the toes, and neurones in the lateral dorsal horn having receptive fields on the proximal parts of the leg, buttock and lower back. This somatotopy has been shown before. One month after nerve section no cells responded to tactile stimulation of the distal leg and cells in the medial and central parts of the dorsal horn now had receptive fields on the proximal leg, buttock and back. There did not appear to be any somatotopic organization of these new receptive fields. Lateral dorsal horn neurones had normal receptive fields. Nine months after nerve section neurones in the medial and central parts of the lumbar dorsal horn had receptive fields on the distal leg but they showed several abnormal features and there was no evidence of a return of the somatotopic organization seen in normal animals. Lateral dorsal horn cells still had normal receptive fields.