Neurofilament phosphorylation in axons and perikarya: immunofluorescence study of the rat spinal cord and dorsal root ganglia with monoclonal antibodies

Rat dorsal root ganglia and spinal cord were stained with 12 monoclonal antibodies reacting with phosphorylated epitopes of two neurofilament proteins (NF 150K and NF 200K). Three monoclonal antibodies were axon-specific in both locations; neuronal perikarya were not stained. Nine monoclonal antibodies stained a subpopulation of neurofilament-positive sensory neurons, as indicated by double labeling experiments with polyclonal antibodies reacting with phosphorylated and dephosphorylated forms of the neurofilament protein triplet. Of these nine antibodies, two stained motor neuron perikarya in the spinal cord, while the remaining seven antibodies were axon-specific in this location. Subpopulations of stained and unstained motor neurons were not observed. With all 12 antibodies, the staining pattern in the lumbar dorsal root ganglia and spinal cord remained unchanged following sciatic nerve crush and ligature. The findings suggest that, in the neurofilament, some phosphorylated epitopes are axon specific, while other phosphorylated epitopes are present in both axons and perikarya. Furthermore, they suggest that differences exist between neuronal populations as to the presence of phosphorylated epitopes in perikaryal neurofilaments. It remains to be seen whether phosphorylation events in perikarya and axons have similar or different effects on neurofilament structure and function.     

Changes of chemoarchitectural organization of the rat spinal cord following ventral and dorsal root transection.

Time-related changes in the distribution of chemical messengers in the rat spinal cord following the transection of dorsal and ventral roots were observed by using immunohistochemistry for the following antigens: microtubule-associated protein 2 (MAP2), calcitonin gene-related peptide (CGRP), substance P (SP), galanin (Gal), Met-enkephalin (Enk), neuropeptide Y (NPY), and serotonin (5-HT). To investigate dendrocytoarchitectonic organizational changes, morphometric analyses were performed on both the gray and the white matter of tissue samples stained with MAP2 antiserum. A significant reduction in the area of gray matter on the lesioned side was seen from 1 to 24 weeks postoperation, and progressive changes in the shape of the gray matter were also observed. CGRP-immunoreactive fibers were reduced in number in the posterior horn after root transection, except in the lateral part of lamina I. In contrast, CGRP immunoreactivity in the anterior horn cells of the ipsilateral side was increased early after transection, but later it progressively decreased. Root transection also caused significant reduction in the number of SP-immunoreactive fibers in the posterior horn, but no changes were seen in the anterior horn. Gal immunoreactivity was also affected by root transection, and it changed in a similar way to CGRP immunoreactivity. 5-HT-immunoreactive fibers were increased in the posterior horn after transection, and later decreased. In the anterior horn, there were no changes in the intensity or distribution pattern of 5-HT-immunoreactive nerve fibers following root transection. Enk and NPY immunoreactivity in the anterior and posterior horns was not affected by root transection up to 24 weeks postoperative. These results show that spinal root transection caused significant changes in the chemoarchitectural organization of nerve fibers containing certain types of chemical messengers, such as CGRP, SP, Gal, and 5-HT, in addition to altering dendritic geometry in the spinal cord.     

Neurofilament sidearm proteolysis is a prominent early effect of axotomy in lamprey giant central neurons

In the accompanying paper, it was shown that axotomy of lamprey spinal axons induces the rapid formation of condensed neurofilamentous masses in the proximal axon stump near the lesion. In this study, we used immunocytochemical and Western blot analysis to characterize these masses further and to determine the time course of their formation and dispersal. We show that monoclonal antibodies specific to the “rod” domain of lamprey neurofilament protein strongly stain such masses in tissue sections without staining other axonal neurofilaments. Antibodies specific for the neurofilament “sidearm” domain fail to recognize neurofilamentous masses but stain other axonal neurofilaments. Western blots of spinal cord segments from the lesion site were compared to unlesioned cord and to samples of cord distant from the lesion. We found that a neurofilament rodrspecific antibody identified breakdown products of the same size as the rod domain in samples from the lesion site, but not elsewhere. Other lesion-specific neurofilament breakdown products were recognized by a sidearm-specific antibody. This lesion-specific pattern of neurofilament proteolysis was visible at 1 day postlesion and was still present 3 weeks later. Immunocytochemistry showed masses of rod-staining neurofilaments in axon stumps by 12 hours postlesion that remained for 1–2 weeks postaxotomy; these dispersed with the onset of regeneration. Such neurofilament rod staining was also prominent in distal axon stumps undergoing Wallerian degeneration. We conclude that axotomy induces neurofilament sidearm proteolysis near the lesion, permitting antibody access to the rod domain. We suggest that sidearm loss causes the high packing density of neurofilaments within neurofilamentous masses near the lesion site and that neurofilament sidearm proteolysis can be used to distinguish degenerative from regenerative changes in lesioned lamprey axons. © 1995 Wiley-Liss, Inc.     

Inflammation of rat dorsal root ganglia below a mid-thoracic spinal transection

Macrophages and T-lymphocytes invade the spinal cord in and around a lesion and spinal microglia are converted into macrophages. After spinal transection at T8 in rats, T-lymphocyte and major histocompatibility complex II+ (MHC II+) macrophage numbers were increased within dorsal root ganglia (DRGs) below the lesion. Inflammation was greater in DRGs closer to the site of transection. After 8 weeks, MHC II+cell density had fallen by 30% but T-lymphocyte numbers were undiminished. In lumbosacral DRGs, inflammation preceded inflammation within the spinal cord. The responses in distant DRGs are hard to reconcile with the limited damage to sensory neurons produced by the lesion. Inflammation of DRGs after spinal injury may contribute to hyper-reflexia and pain.     

Regulation of aberrant neurofilament phosphorylation in neuronal perikarya. II. Correlation with continued axonal elongation following axotomy.

Neurofilaments (NF) are normally poorly phosphorylated in neuronal perikarya and highly phosphorylated in axons. Aberrant NF phosphorylation in the neuronal perikaryon has been demonstrated in a number of human and experimental disorders. In this study, we have asked whether expression of these phosphorylated NF (pNF) epitopes is dependent upon continued axonal regeneration following nerve transection (axotomy). This hypothesis was tested using the neurotoxic chemical acrylamide (AC) which is known to inhibit axonal regeneration following systemic administration. First, we examined whether AC acts at the level of the neuronal perikaryon to inhibit axonal elongation. Systemic, high dose intraperitoneal (IP) AC administration totalling 150 mg/kg (75 mg/kg x 2) did not impair the axotomy-induced reordering of slow axonal transport in the neuronal perikaryon. Next, we studied the ability of AC to directly prevent nerve outgrowth at the growing tips of axons. Subperineurial injection of AC (0.1 M), which in preliminary studies was found not to produce nerve fiber damage, markedly reduced the extent of nerve outgrowth when injected proximal to a nerve crush; this was shown by a reduction in the extent of radiolabeling and number of axonal sprouts in the distal stump seven days following nerve crush. Using this protocol, a 67% decrease in the number of neuronal perikarya in the L4 and L5 dorsal root ganglia demonstrating immunoreactivity to antibody 07-05 (directed against pNF epitopes) was observed in AC-injected compared to contralateral saline-injected crushed nerves. Taken together, the results indicate that inhibition of axonal regeneration in the distal stump by AC reduces aberrant NF phosphorylation in the neuronal perikaryon following axotomy.     

Neurofilament antibodies and spiral ganglion neurons of the mammalian cochlea

The spiral ganglia of the cat, gerbil, mouse, rat, and human were immunohistochemically stained with various monoclonal neurofilament antibodies. Three antibodies to the 200-kD neurofilament protein (R-3, Dr?ger et al., '84; ICN anti-200, clone NE14, Debus et al., '83; RT-97, Wood and Anderton, '81) labeled the somata of type II spiral ganglion neurons but not those of type I ganglion neurons. In the extreme base of the cochlea of cats, mice and rats, there was intense labeling of a few (less than 0.5% of the total ganglion population) large neurons resembling type I ganglion neurons. Several other neurofilament antibodies (Amersham anti-68, Amersham and ICN anti-160, and SMI-32) did not specifically label type II ganglion neurons but instead labeled all neurons of the spiral ganglion. These two patterns of labeling prompted us to investigate the cause for this difference. Because antibodies against the 200-kD neurofilament protein preferentially labeled type II neurons and because 200-kD neurofilament is highly phosphorylated, we treated cochlear tissue with alkaline phosphatase in order to remove phosphate groups. This treatment eliminated the intense labeling of type II neurons with R-3, ICN anti-200, and RT-97, but had no effect on the intense labeling of ganglion cell bodies observed with the other neurofilament antibodies tested. This evidence suggests that labeling occurs because of the cytoplasmic presence of phosphorylated 200-kD neurofilament protein in type II ganglion neurons. Populations of neurons may thus differ in their neurofilament epitopes and monoclonal antibodies can be used to mark such differences.     

Changes in cholinergic and opioid receptors in the rat spinal cord,dorsal root and sciatic nerve after ventral and dorsal root lesion

Summary Changes in the distribution of³H-quinuclidinylbenzilate (³ H-QNB),³ H-acetylcholine (³ H-ACh) and³ H-alpha-bungarotoxin (alpha-BTx) binding sites were studied with the use of quantitative in vitro autoradiography in the L4–L6 segments of rats 7 days after ventral L4–L6-rhizotomies and 24 hours after ligation of the dorsal roots L4–L6. The changes in the binding sites of these ligands and of³ H-etorphine binding sites were also studied in the dorsal roots of the rats operated with dorsal root ligation and in the sciatic nerves (around a ligature) in the rats operated with ventral rhizotomy. After ventral rhizotomy³ H-QNB binding sites in the ipsilateral motor neuron area were decreased by about 25% from 100±5 to 73±5 fmol/mg wet weight. After dorsal root ligation³ H-QNB binding sites in the ipsilateral posterior horn were reduced by about 30% from 91±5 to 64±7 fmol/mg wet weight. No significant changes in the binding of the other cholinergic ligands in the spinal cords were observed after the operations. In the dorsal root³ H-alpha-Btx and³ H-etorphine binding sites were higher on the distal side of the ligation (3.5±0.8 and 14±4 fmol/mg wet weight, respectively) than on the proximal side (0.7±0.5 and 2.4±1.2 fmol/mg wet weight, respectively).The same level of³ H-ACh (total, muscarinic and nicotinic) binding was observed on both sides of the ligation. In the sciatic nerve³ H-QNB and total, muscarinic and nicotinic ACh binding sites were higher on the proximal side of the ligation than on the distal side. Except for a small emergence of muscarinic-ACh binding distally to the ligation there were no changes in the number of binding sites in the sciatic nerve after the ventral rhizotomy.Muscarinic antagonist binding sites are probably located on the perikarya of the motor neurons and presynaptically on the primary afferents in the posterior horn and in the dorsal root. Cholinergic agonist binding sites in the spinal cord seem less sensitive to axonal damage than antagonist binding sites. Cholinergic and opioid receptors in peripheral nerves are transported in both anterograde and retrograde directions and their origin seems to be the dorsal root ganglion.     

Anatomical correlates of locomotor recovery following dorsal and ventral lesions of the rat spinal cord

The present study was designed to relate functional locomotor outcome to the anatomical extent and localization of lesions in the rat spinal cord. We performed dorsal and ventral lesions of different severity in 36 adult rats. Lesion depth, spared total white matter, and spared ventrolateral funiculus were compared to the locomotor outcome, assessed by the BBB open-field locomotor score and the grid walk test. The results showed that the preservation of a small number of fibers in the ventral or lateral funiculus was related to stepping abilities and overground locomotion, whereas comparable tissue preservation in the dorsal funiculus resulted in complete paraplegia. The strongest relation to locomotor function was between the BBB score and the lesion depth as well as the BBB score and the spared white matter tissue in the region of the reticulospinal tract. Locomotion on the grid walk required sparing in the ventrolateral funiculus and additional sparing of the dorsolateral and dorsal funiculus, where the cortico- and rubrospinal tracts are located.     

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.     

Adult neurogenesis in primate and rodent spinal cord: comparing a cervical dorsal rhizotomy with a dorsal column transection

Neurogenesis has not been shown in the primate spinal cord and the conditions for its induction following spinal injury are not known. In the first part of this study, we report neurogenesis in the cervical spinal dorsal horn in adult monkeys 6-8 weeks after receiving a well-defined cervical dorsal rhizotomy (DRL). 5-bromo-2-deoxyuridine (BrdU) was administered 2-4 weeks following the lesion. Cells colabeled with BrdU and five different neuronal markers were observed in the peri-lesion dorsal horn 4-5 weeks after BrdU injection. Those colabeled with BrdU and neuron-specific nuclear protein, and BrdU and glial fibrillary acidic protein were quantified in the dorsal horn peri-lesion region, and the ipsi- and contralateral sides were compared. A significantly greater number of BrdU/neuron-specific nuclear protein- and BrdU/glial fibrillary acidic protein-colabeled cells were found on the lesion side (P<0.01). These findings led us to hypothesize that neurogenesis can occur within the spinal cord following injury, when the injury does not involve direct trauma to the cord and glial scar formation. This was tested in rats. Neurogenesis and astrocytic proliferation were compared between animals receiving a DRL and those receiving a dorsal column lesion. In DRL rats, neurogenesis was observed in the peri-lesion dorsal horn. In dorsal column lesion rats, no neurogenesis was observed but astrocytic activation was intense. The rat data support our hypothesis and findings in the monkey, and show that the response is not primate specific. The possibility that new neurons contribute to recovery following DRL now needs further investigation.     

Prepro-VIP and preprotachykinin mRNAs in the rat dorsal root ganglion cells following peripheral axotomy

Using in situ hybridization histochemistry, we examined the expression of prepro-vasoactive intestinal polypeptide (VIP) mRNAs and preprotachykinin (PPT) mRNAs which coded for substance P (SP) in the rat dorsal root ganglion (DRG) following spinal nerve transection. VIP mRNAs increased dramatically in the DRG neurons after transection of the peripheral branch of the spinal nerve (sciatic nerve), whereas PPT mRNAs showed a gradual decrease for a few weeks. Dorsal rhizotomy or axotomy of the central branch of DRG cells had little influence on VIP-mRNAs and no effect on PPT mRNA expression. These results demonstrated an activation of VIP biosynthesis in the DRG neurons due to axotomy of the peripheral branch, which was opposite to the reaction of PPT mRNA to the same treatment.     

Increased brain-derived neurotrophic factor immunoreactivity in rat dorsal root ganglia and spinal cord following peripheral inflammation

Our recent study showed that peripheral inflammation induced an increased expression of brain-derived neurotrophic factor (BDNF) mRNA which was mediated by nerve growth factor (NGF) in the dorsal root ganglion (DRG). In the present study, we evaluated the change of BDNF immunoreactivity in the DRG and spinal cord following peripheral inflammation by means of immunohistochemistry. Significant increases in the percentage of BDNF-immunoreactive (IR) neuron profiles in the L5 DRG and marked elevation in the expression of BDNF-IR terminals in the spinal dorsal horn were observed following peripheral tissue inflammation produced by an intraplantar injection of Freund's adjuvant into the rat paws. These findings suggest that peripheral tissue inflammation induces an increased BDNF synthesis in the DRG and an elevated anterograde transport of BDNF to the spinal dorsal horn. The functional role of this increased BDNF was discussed briefly.     

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.     

Differentiation and migration of astrocytes in the spinal cord following dorsal root injury in the adult rat

Nerve fibre degeneration in the spinal cord is accompanied by astroglial proliferation. It is not known whether these cells proliferate in situ or are recruited from specific regions harbouring astroglial precursors. We found cells expressing nestin, characteristic of astroglial precursors, at the dorsal surface of the spinal cord on the operated side from 30 h after dorsal root injury. Nestin-expressing cells dispersed to deeper areas of the dorsal funiculus and dorsal horn on the operated side during the first few days after injury. Injection of bromodeoxyuridine (BrdU) 2 h before the end of the experiment, at 30 h after injury, revealed numerous BrdU-labelled, nestin-positive cells in the dorsal superficial region. In animals surviving 20 h after BrdU injection at 28 h postlesion, cells double-labelled with BrdU and nestin were also found in deeper areas. Labeling with BrdU 2 h before perfusion showed proliferation of microglia and radial astrocytes in the ventral and lateral funiculi on both sides of the spinal cord 30 h after injury. Nestin-positive cells coexpressed the calcium-binding protein Mts1, a marker for white matter astrocytes, in the dorsal funiculus, and were positive for glial fibrillary acidic protein (GFAP), but negative for Mts1 in the dorsal horn. One week after injury the level of nestin expression decreased and was undetectable after 3 months. Taken together, our data indicate that after dorsal root injury newly formed astrocytes in the degenerating white and grey matter first appear at the dorsal surface of the spinal cord from where some of them subsequently migrate ventrally, and differentiate into white- or grey-matter astrocytes.     

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.     

Bismuth-induced neuronal cell death in rat dorsal root ganglion: a stereological study

The goal of the present study has been to quantify the morphological changes in myelinated nerve roots and dorsal root ganglion (DRG) cells in rats exposed to bismuth subnitrate. Male Wistar rats (n =30) were divided into three groups of ten animals. The ten animals in each group were given intraperitoneal injections of one of the following: (1) 500 mg/kg bismuth subnitrate, (2) 1,000 mg/kg bismuth subnitrate, or (3) saline. The mean total cell number of B-cells in the DRG was significantly smaller in the two treated groups, 18% (2P <0.001) and 23% (2P <0.001), respectively, than it was in the control group. In addition, there was an 11% reduction in the number of A-cells (2P =0.039) in rats exposed to the highest concentration of bismuth. Bismuth did not affect the total number or mean cross sectional area of axons and myelin sheaths of the myelinated nerve fibers in the ventral or dorsal nerve root of the DRG. This is the first study to investigate pathological changes of the peripheral nervous system after bismuth intoxication.     

Somatosensory corticospinal tract axons sprout within the cervical cord following a dorsal root/dorsal column spinal injury in the rat

The corticospinal tract (CST) is the major descending pathway controlling voluntary hand function in primates, and though less dominant, it mediates voluntary paw movements in rats. As with primates, the CST in rats originates from multiple (albeit fewer) cortical sites, and functionally different motor and somatosensory subcomponents terminate in different regions of the spinal gray matter. We recently reported in monkeys that following a combined cervical dorsal root/dorsal column lesion (DRL/DCL), both motor and S1 CSTs sprout well beyond their normal terminal range. The S1 CST sprouting response is particularly dramatic, indicating an important, if poorly understood, somatosensory role in the recovery process. As rats are used extensively to model spinal cord injury, we asked if the S1 CST response is conserved in rodents. Rats were divided into sham controls, and two groups surviving post-lesion for ~6 and 10 weeks. A DRL/DCL was made to partially deafferent one paw. Behavioral testing showed a post-lesion deficit and recovery over several weeks. Three weeks prior to ending the experiment, S1 cortex was mapped electrophysiologically, for tracer injection placement to determine S1 CST termination patterns within the cord. Synaptogenesis was also assessed for labeled S1 CST terminals within the dorsal horn. Our findings show that the affected S1 CST sprouts well beyond its normal range in response to a DRL/DCL, much as it does in macaque monkeys. This, along with evidence for increased synaptogenesis post-lesion, indicates that CST terminal sprouting following a central sensory lesion, is a robust and conserved response.     

Time course of substance P expression in dorsal root ganglia following complete spinal nerve transection

Recent evidence suggests that substance P (SP) is up-regulated in primary sensory neurons following axotomy and that this change occurs in larger neurons that do not usually produce SP. If this is so, then the up-regulation may allow normally neighboring, uninjured, and nonnociceptive dorsal root ganglion (DRG) neurons to become effective in activating pain pathways. By using immunohistochemistry, we performed a unilateral L5 spinal nerve transection on male Wistar rats and measured SP expression in ipsilateral L4 and L5 DRGs and contralateral L5 DRGs at 1-14 days postoperatively (dpo) and in control and sham-operated rats. In normal and sham-operated DRGs, SP was detectable almost exclusively in small neurons (< or =800 microm2). After surgery, the mean size of SP-positive neurons from the axotomized L5 ganglia was greater at 2, 4, 7, and 14 dpo. Among large neurons (>800 microm2) from the axotomized L5, the percentage of SP-positive neurons increased at 2, 4, 7, and 14 dpo. Among small neurons from the axotomized L5, the percentage of SP-positive neurons was increased at 1 and 3 dpo but was decreased at 7 and 14 dpo. Thus, SP expression is affected by axonal damage, and the time course of the expression is different between large and small DRG neurons. These data support a role for SP-producing, large DRG neurons in persistent sensory changes resulting from nerve injury.