Differential distribution of the tetrodotoxin-sensitive rPN4/NaCh6/Scn8a sodium channel in the nervous system

Voltage-gated sodium channels underlie the generation of action potentials in excitable cells. Various sodium channel isoforms have been cloned, functionally expressed and distinguished on the basis of their biophysical properties or differential sensitivity to tetrodotoxin (TTX). In the present study, we have investigated the immunolocalization of the TTX-sensitive sodium channel, rPN4/NaCh6/Scn8a, in discrete areas of the rat nervous system. Thus, in na?ve animals, PN4 was abundantly expressed in brain, spinal cord, dorsal root ganglia (DRG) and peripheral nerve. The presence of PN4 at the nodes of Ranvier in the sciatic nerve suggests the importance of this sodium channel in peripheral nerve conduction. In addition, the pattern of PN4 immunolabeling was determined in DRG, spinal cord and sciatic nerve in rats subjected to chronic constriction nerve injury (CCI).     

Regulation of TRPV2 by axotomy in sympathetic, but not sensory neurons

Neuropathic pain results from traumatic or disease-related insults to the nervous system. Mechanisms that have been postulated to underlie peripheral neuropathy commonly implicate afferent neurons that have been damaged but still project centrally to the spinal cord, and/or intact neurons that interact with degenerating distal portions of the injured neurons. One pain state that is observed following peripheral nerve injury in the rat is thermal hyperalgesia. The noxious heat-gated ion channel TRPV1 may be responsible for this increased sensitivity, as it is up-regulated in L4 dorsal root ganglion (DRG) neurons following L5 spinal nerve lesion (SpNL). The TRPV1 homologue TRPV2 (or VRL-1) is another member of the TRPV subfamily of TRP ion channels. TRPV2 is a nonselective cation channel activated by high noxious temperatures (>52 degrees C) and is present in a subset of medium- to large-diameter DRG neurons. To establish whether TRPV2 is endogenous to the spinal cord, we examined its expression in the dorsal horn following rhizotomy. We found no significant decrease in TRPV2 immunoreactivity, suggesting that TRPV2 is endogenous to the spinal cord. In order to determine whether TRPV2, like TRPV1, is regulated by peripheral axotomy, we performed L5 SpNL and characterized TRPV2 distribution in the DRG, spinal cord, brainstem, and sympathetic ganglia. Our results show that peripheral axotomy did not regulate TRPV2 in the DRG, spinal cord, or brainstem; however, TRPV2 was up-regulated in sympathetic postganglionic neurons following injury, suggesting a potential role for TRPV2 in sympathetically mediated neuropathic pain.     

Distribution of paclitaxel within the nervous system of the rat after repeated intravenous administration

The distribution of paclitaxel (Taxol) within the central and peripheral nervous system after repeated administration of this antineoplastic agent is still largely unknown. In this study we determined for the first time paclitaxel tissue concentration in the brain, spinal cord, dorsal root ganglia (DRG) and sciatic nerve using an experimental paradigm in the rat which reproduces the features of paclitaxel peripheral neurotoxicity in humans. Pathological confirmation of the onset of paclitaxel-induced peripheral neurotoxicity was performed. In order to achieve reliable results even with low concentrations of paclitaxel, a newly reported analytical method (high-performance liquid chromatography with tandem mass spectrometry) was used. We demonstrated that paclitaxel has easy access to the DRG, where it accumulates, while the lowest concentrations of the drug were measured in the brain. The intermediate concentrations of paclitaxel observed in the sciatic nerve and spinal cord may be due to paclitaxel transport along the centrifugal and centripetal branches of the DRG neuron axons.     

Dynamic changes in glypican-1 expression in dorsal root ganglion neurons after peripheral and central axonal injury

Glypican-1, a glycosyl phosphatidyl inositol (GPI)-anchored heparan sulphate proteoglycan expressed in the developing and mature cells of the central nervous system, acts as a coreceptor for diverse ligands, including slit axonal guidance proteins, fibroblast growth factors and laminin. We have examined its expression in primary sensory dorsal root ganglion (DRG) neurons and spinal cord after axonal injury. In noninjured rats, glypican-1 mRNA and protein are constitutively expressed at low levels in lumbar DRGs. Sciatic nerve transection results in a two-fold increase in mRNA and protein expression. High glypican-1 expression persists until the injured axons reinnervate their peripheral targets, as in the case of a crushed nerve. Injury to the central axons of DRG neurons by either a dorsal column injury or a dorsal root transection also up-regulates glypican-1, a feature that differs from most DRG axonal injury-induced genes, whose regulation changes only after peripheral and not central axonal injury. After axonal injury, the cellular localization of glypican-1 changes from a nuclear pattern restricted to neurons in noninjured DRGs, to the cytoplasm and membrane of injured neurons, as well as neighbouring non-neuronal cells. Sciatic nerve transection also leads to an accumulation of glypican-1 in the proximal nerve segment of injured axons. Glypican-1 is coexpressed with robo 2 and its up-regulation after axonal injury may contribute to an altered sensitivity to axonal growth or guidance cues.     

Axonal injury-dependent induction of the peripheral benzodiazepine receptor in small-diameter adult rat primary sensory neurons

The peripheral benzodiazepine receptor (PBR), a benzodiazepine but not γ‐aminobutyric acid‐binding mitochondrial membrane protein, has roles in steroid production, energy metabolism, cell survival and growth. PBR expression in the nervous system has been reported in non‐neuronal glial and immune cells. We now show expression of both PBR mRNA and protein, and the appearance of binding of a synthetic ligand, [³H]PK11195, in dorsal root ganglion (DRG) neurons following injury to the sciatic nerve. In naïve animals, PBR mRNA, protein expression and ligand binding are undetectable in the DRG. Three days after sciatic nerve transection, however, PBR mRNA begins to be expressed in injured neurons, and 4 weeks after the injury, expression and ligand binding are present in 35% of L4 DRG neurons. PBR ligand binding also appears after injury in the superficial dorsal horn of the spinal cord. The PBR expression in the DRG is restricted to small and medium‐sized neurons and returns to naïve levels if the injured peripheral axons are allowed to regrow and reinnervate targets. No non‐neuronal PBR expression is detected, unlike its putative endogenous ligand the diazepam binding inhibitor (DBI), which is expressed only in non‐neuronal cells, including the satellite cells that surround DRG neurons. DBI expression does not change with sciatic nerve transection. PBR acting on small‐calibre neurons could play a role in the adaptive survival and growth responses of these cells to injury of their axons.     

Attempts to facilitate dorsal column axonal regeneration in a neonatal spinal environment

The response to injury of ascending collaterals of dorsal root axons within the dorsal column (DC) was studied after neonatal spinal overhemisection (OH) made at different levels of the spinal cord. The transganglionic tracer, cholera toxin conjugated to horseradish peroxidase, and the anterograde tracer, biotinylated dextran amine, were used to label dorsal root ganglion cells with peripheral axons contributing to the sciatic nerve. There was no indication of a regenerative attempt by DC axons at acute survival times (3 days and later) after cervical injury, replicating previous work done at chronic survival periods (Lahr and Stelzner [1990] J. Comp. Neurol. 293:377–398). There was also no evidence of DC regeneration after lumbar OH injury even though immunohistochemical studies using the oligodendrocyte markers Rip and myelin basic protein showed few oligodendrocytes in the gracile fasciculus at lumbar levels at birth. Therefore, the lack of myelin in the dorsal funiculus at lumbar levels does not enhance the growth of neonatally axotomized DC axons. In addition, DC axons did not regenerate when presented with fetal spinal tissue implanted into thoracic OH lesions, even though positive control experiments showed that segmental dorsal root axons containing calcition gene-related peptide and corticospinal axons grew into these implants, replicating previous work of others. When a thoracic OH lesion, with or without a fetal spinal implant, was combined with sciatic nerve injury to attempt to stimulate an intracellular regenerative response of DRG neurons, again, no evidence of DC axonal regeneration was detected. Quantitative studies of the L4 and L5 dorsal root ganglia (DRG) showed that OH injury did not result in DRG neuronal loss. However, sciatic nerve injury did result in significant post-axotomy retrograde cell loss of DRG neurons, even in groups receiving thoracic embryonic spinal implants, and is one explanation for the minimal effect of sciatic nerve injury on DC regeneration. Although fetal tissue did not appear to rescue a significant number of DRG neurons, the quantitative analysis showed an enlargement of the largest class of DRG neuron, the class that contributes to the DC projection, in all groups receiving fetal tissue implants. This apparent trophic effect did not affect DC regeneration or neuronal survival after peripheral axotomy. Further studies are needed to determine why DC axons do not regenerate in a neonatal spinal environment or within fetal tissue implants, especially because previous work by others in both the developing and adult spinal cord shows that dorsal root axons will grow within the same type of fetal spinal implant. © 1996 Wiley-Liss, Inc.     

Novel sites of aldose reductase immunolocalization in normal and streptozotocin-diabetic rats

Glucose metabolism by aldose reductase (AR) is implicated in the pathogenesis of many diabetic complications, including neuropathy. We have re-evaluated the distribution of AR in the sciatic nerve and dorsal root ganglion (DRG) of normal rats, expanded these observations to describe the location of AR in the spinal cord and footpad skin, and investigated whether diabetes alters the distribution of AR. In sciatic nerve, AR was restricted to cytoplasm of myelinated Schwann cells and endothelial cells of epineurial, but not endoneurial, blood vessels. AR immunoreactivity (IR) was present in satellite cells in the DRG. In skin, AR-IR was detected in vascular endothelial cells, Schwann cells of myelinated fibers, and axons of perivascular sympathetic nerves. AR was localized selectively to oligodendrocytes of the white matter of spinal cord. The distribution of AR-IR in sciatic nerve, DRG, skin, and spinal cord was not altered by up to 12 weeks of streptozotocin-induced diabetes. Identification of perineuronal satellite cells, oligodendrocytes, and perivascular sympathetic nerves as AR-expressing cells reveals them as cellular sites with the potential to contribute to diabetic neuropathy.     

Spinal cord microglial cells and DRG satellite cells rapidly respond to transection of the rat sciatic nerve

Transection of the rat sciatic nerve induces retrograde changes in the dorsal root ganglia (DRG) neurons and in the motoneurons in the ventral grey matter of the lumbar L4-L6 spinal cord segments. In the ipsilateral dorsal grey matter and in the ipsilateral nucleus gracilis, transganglionic changes occur in the terminal fields of the centrally projecting axons of injured DRG neurons. As revealed by immunocytochemistry, the neuronal reactions were associated with a rapid proliferation and activation of microglial cells in the lumbar spinal cord as well as in the nucleus gracilis. Reactive microglial cells were detected as early as 24 h after sciatic axotomy. The microglial reaction had a maximum around day 7 postlesion and disappeared around 6 weeks after axotomy. In addition to light microscopy, activated, perineuronal microglia were identified by immuno-electron microscopy in the ventral grey matter. In the DRG, satellite cells constitutively expressed major histocompatibility complex (MHC) class II antigens. Sciatic axotomy led to a proliferation of satellite cells and an increased expression of MHC class II molecules in particular. This satellite cell reaction started 24 h after axotomy and continued to increase gradually until about 6 weeks after the lesion. Resident macrophages, detected in the DRG interstitial tissue by their expression of monocyte/macrophage markers, also reacted to sciatic axotomy. Our data suggest that (1) sciatic axotomy leads to a rapid microglial reaction in both the ventral and dorsal grey matter of the lumbar spinal cord and in the ipsilateral nucleus gracilis; (2) the immunophenotype of activated microglia following sciatic axotomy is comparable with that observed after axotomy of cranial nerves, e.g. the facial nerve; (3) satellite cells in DRG constitutively express MHC class II molecules; and (4) sciatic axotomy leads to a rapid activation of satellite cells and interstitial macrophages in the axotomized DRG.     

Focal and generalized peripheral nerve dysfunction in spinal cord-injured patients.

SUMMARY: The present study was undertaken to quantitate the incidence and clinical patterns of peripheral nerve dysfunction distal to the level of injury in patients with spinal cord injury (SCI). Through retrospective analysis, SCI patients were identified after referral for neurophysiologic investigation of new neuropathic symptoms. In total, peripheral nerve or nerve root lesions developed in 34 SCI patients, most commonly within the first year after SCI. Carpal tunnel syndrome was the most common upper-limb neuropathy (34%); sciatic neuropathy was the most common lower-limb abnormality (8.5%). A significant proportion of SCI patients had neurophysiological evidence of generalized peripheral nerve dysfunction, specifically axonal neuropathy (18%). Tetraplegic patients developed more frequent peripheral nerve lesions than paraplegics. Although most SCI patients presented within 4 years of their original injury, in a more chronic population of SCI patients that developed neuropathy 5 years after injury, 60% had evidence of coexistent syrinx formation. Maintenance of peripheral nerve function is a critical issue in all acute SCI and rehabilitation units, particularly in the context of spinal cord neuronal regeneration projects.     

Satellite-cell-derived nerve growth factor and neurotrophin-3 are involved in noradrenergic sprouting in the dorsal root ganglia following peripheral nerve injury in the rat

Injury to a peripheral nerve induces in the dorsal root ganglia (DRG) sprouting of sympathetic and peptidergic terminals around large-diameter sensory neurons that project in the damaged nerve. This pathological change may be implicated in the chronic pain syndromes seen in some patients with peripheral nerve injury. The mechanisms underlying the sprouting are not known. Using in situ hybridization and immunohistochemical techniques, we have now found that nerve growth factor (NGF) and neurotrophin-3 (NT3) synthesis is upregulated in satellite cells surrounding neurons in lesioned DRG as early as 48 h after nerve injury. This response lasts for at least 2 months. Quantitative analysis showed that the levels of mRNAs for NT3 and NGF increased in ipsilateral but not contralateral DRG after nerve injury. Noradrenergic sprouting around the axotomized neurons was associated with p75-immunoreactive satellite cells. Further, antibodies specific to NGF or NT3, delivered by an osmotic mini-pump to the DRG via the lesioned L5 spinal nerve, significantly reduced noradrenergic sprouting. These results implicate satellite cell-derived neurotrophins in the induction of sympathetic sprouting following peripheral nerve injury.     

Presence of spinal B7.2 (CD86) but not B7.1 (CD80) co-stimulatory molecules following peripheral nerve injury: role of nondestructive immunity in neuropathic pain

Previous work in our laboratory demonstrated spinal neuroimmune activation and leukocyte trafficking into the central nervous system (CNS) parenchyma in a rat model of neuropathic pain. Recent studies suggest that co-stimulatory molecules B7.1 (CD80) and B7.2 (CD86) play a differential role in the effect of beneficial versus deleterious CNS autoimmune responses. In the present study, we determined the lumbar spinal expression of the co-stimulatory molecules B7.1 and B7.2 in a rat model of neuropathy. We observed intense B7.2 microglial immunoreactivity in the lumbar spinal cord following the injury but no expression of B7.1. These data suggest a role of protective CNS autoimmunity following peripheral nerve injury.     

Expression of HGF and cMet in the peripheral nervous system of adult rats following sciatic nerve injury

Hepatocyte growth factor (HGF) exhibits neurotrophic properties on different types of neuron, including motor, sensory and parasympathetic neurons. We demonstrate that sciatic nerve ligation induces an increase of the HGF receptor, c-met, mRNA in the distal segment of the sciatic nerve to the ligation site and a delayed elevation in the proximal segment. Immunohistochemical analysis revealed co-localization of cMet and GFAP and indicates that Schwann cells express cMet in the sciatic nerve after injury. HGF mRNA was detected in the spinal cord and DRG, and nerve injury did not alter the expression. These data demonstrate that the expression of HGF and cMet in the peripheral nervous system shows the unique pattern of regulation following nerve injury.     

Pericellular Griffonia simplicifolia I isolectin B4-binding ring structures in the dorsal root ganglia following peripheral nerve injury in rats

Patients with a peripheral nerve injury often suffer from persistent chronic pain, but the underlying mechanism remains largely unknown. The persistent nature of the pain suggests injury-induced profound structural changes along the sensory pathways. In the present study, using the plant Griffonia simplicifolia I isolectin B4 (IB4) as a marker for nonpeptidergic small sensory neurons, we sought to examine whether these neurons sprout in the dorsal root ganglia (DRG) in response to peripheral nerve injury. The lumbar 5 (L5) spinal nerve was transected, and rats were allowed to survive for varying lengths of time before IB4 histology was performed. We found that a subpopulation of IB4-positive sensory neurons sprouted robustly after spinal nerve injury. Twelve weeks after spinal nerve injury, the IB4-positive ring structures became dramatic and encircled both large and small neurons in the DRG. The aberrant sprouting of small sensory neurons was also demonstrated by retrograde labeling. The processes of satellite cells surrounding large sensory neurons also became IB4 positive, and 87.8% of perineuronal IB4-positive ring structures intermingled and/or coexpressed with glial fibrillary acidic protein-positive satellite cells. Thus, the sprouting axons of IB4-positive neurons were intermingled with IB4-positive satellite cells, forming perineuronal ring structures surrounding large-diameter neurons. Ultrastructural examinations further confirmed that IB4-positive nerve terminals were entangled with satellite cells and IB4-negative unmyelinated sprouting fibers around sensory neurons. These studies have provided the first evidence that a subpopulation of IB4-binding small sensory neurons sprouts and forms perineuronal ring structures together with IB4-positive satellite cells in response to nerve injury. The significance of the sprouting of IB4-positive neurons remains to be determined.     

Upregulation of brain-derived neurotrophic factor in the sensory pathway by selective motor nerve injury in adult rats

Selective motor nerve injury by lumbar 5 ventral root transection (L5 VRT) induces neuropathic pain, but the underlying mechanisms remain unknown. Previously, increased expression and secretion of brain-derived neurotrophic factor (BDNF) had been implicated in injury-induced neuropathic pain in the sensory system. In this study, as a step to examine potential roles of BDNF in L5 VRT-induced neuropathic pain, we investigated BDNF gene and protein expression in adult rats with L5 VRT. L5 VRT induced a dramatic upregulation of BDNF mRNA in intact sensory neurons in the ipsilateral L5 dorsal root ganglia (DRG), in non-neuronal cells in the ipsilateral sciatic nerve, and in motoneurons in the ipsilateral spinal cord. L5 VRT also induced de novo synthesis of BDNF mRNA in spinal dorsal horn neurons and in glial cells in the white matter of the ipsilateral spinal cord. Consistent with the mRNA expression pattern, BDNF protein was also mainly upregulated in all populations of sensory neurons in the ipsilateral L5 DRG and in spinal neurons and glia. Quantitative analysis by ELISA showed that the BDNF content in the DRG and sciatic nerve peaked on day 1 and remained elevated 14 days after L5 VRT. These results suggest that increased BDNF expression in intact primary sensory neurons and spinal cord may be an important factor in the induction of neuropathic pain without axotomy of sensory neurons.     

Bortezomib-induced peripheral neurotoxicity: a neurophysiological and pathological study in the rat

Bortezomib is a new proteasome inhibitor with a high antitumor activity, but also with a potentially severe peripheral neurotoxicity. To establish a preclinical model and to characterize the changes induced on the peripheral nerves, dorsal root ganglia (DRG) and spinal cord, bortezomib was administered to Wistar rats (0.08, 0.15, 0.20, 0.30 mg/kg/day twice [2q7d] or three times [3q7d] weekly for a total of 4 weeks). At baseline, on days 14, 21 and 28 after the beginning the treatment period and during a 4-week follow-up period sensory nerve conduction velocity (SNCV) was determined in the tail of each animal. Sciatic nerve, DRG and spinal cord specimens were processed for light and electron microscope observations and morphometry. At the maximum tolerated dose bortezomib induced a significant reduction in SNCV, with a complete recovery at the end of the follow-up period. Sciatic nerve examination and morphometric determinations demonstrated mild to moderate pathological changes, involving predominantly the Schwann cells and myelin, although axonal degeneration was also observed. Bortezomib-induced changes were also observed in DRG and they were represented by satellite cell intracytoplasmatic vacuolization due to mitochondrial and endoplasmic reticulum damage, closely resembling the changes observed in sciatic nerve Schwann cells. Only rarely did the cytoplasm of DRG neurons has a dark appearance and clear vacuoles occurring in the cytoplasm. Spinal cord was morphologically normal. This model is relevant to the neuropathy induced by bortezomib in the treatment of human malignancies and it could be useful in increasing our knowledge regarding the mechanisms underlying bortezomib neurotoxicity.     

Regulation of neuronal and glial galectin-1 expression by peripheral and central axotomy of rat primary afferent neurons

Galectin-1 (Gal1) is an endogenously-expressed protein important for the embryonic development of the full complement of primary sensory neurons and their synaptic connections in the spinal cord. Gal1 also promotes axonal regeneration following peripheral nerve injury, but the regulation of Gal1 by axotomy in primary afferent neurons has not yet been examined. Here, we show by immunohistochemistry and in situ hybridization that Gal1 expression is differentially regulated by peripheral nerve injury and by dorsal rhizotomy. Following peripheral nerve injury, the proportion of Gal1-positive DRG neurons was increased. An increase in the proportion of large-diameter DRG neurons immunopositive for Gal1 was paralleled by an increase in the depth of immunoreactivity in the dorsal horn, where Gal1-positive terminals are normally restricted to laminae I and II. Dorsal rhizotomy did not affect the proportions of neurons containing Gal1 mRNA or protein, but did deplete the ipsilateral dorsal horn of Gal1 immunoreactivity, indicating that it is transported centrally by dorsal root axons. Dorsal rhizotomy also resulted in an increase in Gal1 mRNA the nerve peripheral to the PNS-CNS interface (likely within Schwann cells and/or macrophages), and to a lesser extent within deafferented spinal cord regions undergoing Wallerian degeneration. This latter increase was notable in the dorsal columns and along the prior trajectories of myelinated afferents into the deeper dorsal horn. These results show that neuronal and glial expressions of Gal1 are tightly correlated with regenerative success. Thus, the differential expression pattern of Gal1 following peripheral axotomy and dorsal rhizotomy suggests that endogenous Gal1 may be a factor important to the regenerative response of injured axons.