A comparative histological analysis of two models of nerve root avulsion injury in the adult rat.

AIMS: This study has investigated the reliability of the artificial surgical model dorsal root rhizotomy (DRR), to the surgical tearing of the roots, avulsion, that occurs clinically. Root avulsion of the limb nerves is common in high-impact motor vehicle accidents and results in paraesthesia, paralysis and intractable pain. Limited treatment options are largely due to a lack of basic research on underlying mechanisms, and few animal models. We assess this limitation by histologically assessing the spatial and temporal injury profile of dorsal root avulsion (DRA) and DRR within the spinal cord. METHODS: Rats underwent DRR, DRA or sham surgery to the L3-L6 dorsal roots unilaterally. At 1, 2, 14, and 28 days post injury, immunohistochemical density staining was used to characterize the progression of spinal cord trauma. Neuronal (NeuN) and vascular degeneration (RECA-1), inflammatory infiltrate (ED1, anti-neutrophil), gliosis (Iba1, GFAP) and apoptosis (TUNEL) were assessed. RESULTS: Unilateral DRA produced a prolonged and bilateral glial and inflammatory response, and vascular degeneration compared to transient and unilateral effects after DRR. Transsynaptic neurodegeneration after DRA was greater than after DRR, and progressed across 28 days coinciding with gliosis and macrophage infiltration. CONCLUSIONS: Rhizotomy leads to a milder representation of the spinal cord trauma that occurs after 'true' avulsion injury. We recommend DRA be used in the future to more reliably model clinical avulsion injury. Avulsion is an injury with a chronic profile of degenerative and inflammatory progression, and this theoretically provides a window of clinical therapeutic opportunity in treatment of secondary trauma progression.

Differential effects of riluzole on subpopulations of adult rat dorsal root ganglion neurons in vitro.

Riluzole is clinically approved for the treatment of motoneuron disease. We have previously shown that this drug is neuroprotective for both sensory neurons and motoneurons and promotes neurite outgrowth [Bergerot A, Shortland PJ, Anand P, Hunt SP, Carlstedt T (2004) Exp Neurol 187(2):359-366; Shortland PJ, Leinster VH, White W, Robson LG (2006) Eur J Neurosci 24:3343-3353]. This study explored the effects of exogenous administration of 0.1 muM riluzole on the neurite growth of specific subpopulations of adult rat dorsal root ganglion (DRG) neurons in vitro. Neuronal branching and neurite length were measured in calcitonin gene related peptide (CGRP), Griffonia simplicifolia Isolectin B4 (IB4), N52 and parvalbumin positive neuronal subpopulations. Riluzole was found to enhance neurite branching in both CGRP and IB4 positive neurons compared to vehicle treated cultures. However, neurite length was only significantly increased in CGRP positive neurons in riluzole treated cultures. The results suggest that riluzole affects specific subpopulations of sensory neurons in vitro and that its effects may be mediated through activation of neurotrophic factor receptors, since neurite outgrowth could be blocked by the administration of K252a (at 10 nM). Riluzole may offer a new pharmacological approach to promote sensory regeneration following small fibre neuropathies.

Reg-2 expression in dorsal root ganglion neurons after adjuvant-induced monoarthritis.

Reg-2 is a secreted protein that is expressed de novo in motoneurons, sympathetic neurons, and dorsal root ganglion (DRG) neurons after nerve injury and which can act as a Schwann cell mitogen. We now show that Reg-2 is also upregulated by DRG neurons in inflammation with a very unusual expression pattern. In a rat model of monoarthritis, Reg-2 immunoreactivity was detected in DRG neurons at 1 day, peaked at 3 days (in 11.6% of DRG neurons), and was still present at 10 days (in 5%). Expression was almost exclusively in the population of DRG neurons that expresses the purinoceptor P2X(3) and binding sites for the lectin Griffonia simplicifolia IB4, and which is known to respond to glial cell line-derived neurotrophic factor (GDNF). Immunoreactivity was present in DRG cell bodies and central terminals in the dorsal horn of the spinal cord. In contrast, very little expression was seen in the nerve growth factor (NGF) responsive and substance P expressing population. However intrathecal delivery of GDNF did not induce Reg-2 expression, but leukemia inhibitory factor (LIF) had a dramatic effect, inducing Reg-2 immunoreactivity in 39% of DRG neurons and 62% of P2X(3) cells. Changes in inflammation have previously been observed predominantly in the neuropeptide expressing, NGF responsive, DRG neurons. Our results show that changes also take place in the IB4 population, possibly driven by members of the LIF family of neuropoietic cytokines. In addition, the presence of Reg-2 in central axon terminals implicates Reg-2 as a possible modulator of second order dorsal horn cells.

Neurochemical characterization of neuronal populations expressing protein kinase C gamma isoform in the spinal cord and gracile nucleus of the rat.

Protein kinase C gamma (PKCgamma) is widely distributed throughout the CNS and is thought to play a role in long term hyper-excitability in nociceptive neurones. Here, we provide the first report of PKCgamma cells in the dorsal column nuclei of the adult rat. Retrograde labeling of PKCgamma cells from the thalamus with choleragenoid revealed that 25% of the PKCgamma positive gracile cells projected to the thalamus. Further, we have characterized the distribution of PKCgamma within gracile nucleus in terms of colocalization with various neurotransmitter receptors or enzymes and calcium binding proteins, and compared this with PKCgamma colocalization in cells of laminae I-III of the spinal cord. We show that approximately 90% of the PKCgamma cells in the gracile nucleus and 60% in the dorsal horn were immuno-positive for the AMPA receptor subunit glutamate 2/3 (GluR2/3). Little coexpression was seen with neurokinin 1 receptor, nitric oxide synthase (NOS) and the AMPA receptor subunit GluR1, markers of distinct neuronal subpopulations. In the spinal cord, a quarter of PKCgamma cells expressed calbindin, but very few cells did so in the gracile nucleus. Electrical stimulation at c-fiber strength of the normal or injured sciatic nerve was used to induce c-fos as a marker of postsynaptic activation in the spinal cord and gracile nucleus. Quantitative analysis of the number of PKCgamma positive gracile cells that expressed also c-fos increased from none to 24% after injury, indicating an alteration in the sensory activation pattern in these neurones after injury. C-fos was not induced in inner lamina II following c-fiber electrical stimulation of the intact or axotomized sciatic nerve, indicating no such plasticity at the spinal cord level. As dorsal column nuclei cells may contribute to allodynia after peripheral nerve injury, pharmacological modulation of PKCgamma activity may therefore be a possible way to ameliorate neuropathic pain after peripheral nerve injury.

Spinal cord compression and dorsal root injury cause up-regulation of activating transcription factor-3 in large-diameter dorsal root ganglion neurons.

Spinal cord injury causes damage to ascending and descending tracts, as well as to local circuits, but relatively little is known about the effect of such injury on sensory neurons located within adjoining ganglia. We have therefore used immunocytochemistry for activating transcription factor-3 (ATF3), a sensitive marker of axonal damage, in order to examine the effects of spinal cord injury in rats on dorsal root ganglion (DRG) neurons. A 50-g static compression injury applied to the dorsal surface of the T12 thoracic spinal cord led to an up-regulation of ATF3 that was maximal at 1 day and affected 12-14% of DRG neurons in ganglia caudal to the injury (T13-L3). A similar response was seen after a T12 hemisection that transected the dorsal columns except that compression injury, but not hemisection, also evoked ATF3 expression in ganglia just rostral to the injury (T10, T11). ATF3 was up-regulated exclusively in DRG neurons that were of large diameter and immunoreactive for heavy neurofilament. Small-diameter cells, including the population that binds the lectin Grifffonia simplicifolia IB4, did not express ATF3 immunoreactivity. A similar pattern of ATF3 expression was induced by dorsal rhizotomy. The data show for the first time that ATF3 is up-regulated after spinal cord and dorsal root injury, but that this up-regulation is confined to the large-diameter cell population.

Delayed administration of NGF reverses nerve injury induced central alterations of primary afferents.

We have examined whether delayed exogenous NGF administered to an axotomised peripheral nerve reverses the increased transganglionic choleragenoid (CTB) labelling in lamina II. Two, four, eight or 18 weeks after bilateral sciatic nerve section, NGF was applied unilaterally for an additional 2-week period to the transected nerve stump. The transganglionic choleragenoid labelling and substance P (SP) expression were determined and compared to the contralateral axotomised side in the spinal cord dorsal horn. Delayed NGF administration reversed the transganglionic choleragenoid labelling in lamina II when administered 2 or 18 weeks after the sciatic nerve lesion, but not at 4 or 8 weeks. There was also a clear recovery of SP on the axotomised, NGF-treated side 2 or 18 weeks after the sciatic nerve lesion, but not at the intermediate survival times. At the longer survival time, however, there was a recovery of SP regardless of NGF treatment. These results suggest that there is a critical window as to when NGF administration can be effective in reversing axotomy-induced changes in the spinal cord.

Axotomy results in major changes in BDNF expression by dorsal root ganglion cells: BDNF expression in large trkB and trkC cells, in pericellular baskets, and in projections to deep dorsal horn and dorsal column nuclei.

Brain derived neurotrophic factor (BDNF) is normally expressed by a small number of predominantly trkA-expressing dorsal root ganglion cells. Using immunocytochemistry and in situ hybridization, we have examined the effect of sciatic nerve section on the expression of BDNF in the adult rat. Following axotomy there was a long lasting (4-week) increase in BDNF mRNA and protein in large-diameter, trkB- and trkC-expressing dorsal root ganglion cells. By 2 days postaxotomy, expression of BDNF mRNA had increased from 2% of trkB cells to 50%, and from 18% of trkC cells to 56%. In contrast, BDNF expression in most trkA cells was unchanged, although was increased in the small population of medium- and large-sized trkA cells. Following axotomy, BDNF-immunoreactive terminals appeared in the central axonal projections of large-diameter cells, including the deep dorsal horn and gracile nucleus. Neuropeptide Y was also upregulated following axotomy and was coexpressed with BDNF in the cell bodies and central terminals of the large cells. Ultrastructural analysis in lamina IV of the spinal cord revealed that BDNF terminals in these central projections establish synaptic contacts. Immunoreactivity at 4 weeks was also observed in pericellular baskets that contained calcitonin gene-related peptide (CGRP) and surrounded trkA- and trkB-expressing cells in L4 and L5 lumbar ganglia. These baskets are likely to arise from local, highly immunoreactive, BDNF/CGRP/trkA-expressing cells. Our results identify several novel targets for BDNF and imply that it acts locally in both autocrine and paracrine modes, as well as centrally in a synaptic mode, to modulate the response of somatosensory pathways in nerve injury.

Transganglionic transport of the lectin soybean agglutinin (Glycine max) following injection into the sciatic nerve of the adult rat.

The lectin soybean agglutinin (SBA) from Glycine max binds to small-sized dorsal root ganglion cells and their central terminals in the superficial dorsal horn of the spinal cord. Here we investigated the ability of SBA and SBA conjugated to horseradish peroxidase (SBA-HRP) to trace thin calibre afferents into the spinal cord from a peripheral nerve. Following injection into the sciatic nerve, labelled cells in the dorsal root ganglion were predominantly small-sized but some medium-sized cells were also labelled. Colocalization studies of transported SBA with various neuronal markers showed that all neurons that transported SBA-HRP showed SBA binding, indicating high uptake specificity for the conjugate. 15% were immunoreactive for RT97 indicating that some axons were myelinated, and 54% also expressed binding sites for isolectin B4 from Griffonia simplicifolia, a selective marker for a subpopulation of unmyelinated afferents. With regard to neurotransmitter content, 43% of the SBA cells contained calcitonin gene-related peptide, 33% contained substance P and 2.5% somatostatin. In addition, 3% contained carbonic anhydrase. Centrally, injection of SBA in the sciatic nerve resulted in labelled terminals in somatotopically appropriate regions of laminae I-II of the dorsal horn, and in the gracile nucleus. A few neurons in the dorsal horn were labelled indicating that some transneuronal transport of SBA had occurred. The results show that SBA can be used as a transganglionic tracer to label fine calibre primary afferents that project to laminae I-II of the spinal cord and the gracile nucleus. It appears to label more afferents than isolectin B4, including also a subpopulation of myelinated afferents.

Peripheral and central predictors of whisker afferent morphology in the rat brainstem.

Prior studies suggest that whisker afferents have but one central projection pattern, despite their association with differing peripheral receptors that predict central morphology in other systems. Target factors in barrelettes are thought to dictate afferent projection patterns; yet, barrelettes differ in their size, shape and development. We tested the hypothesis that whisker afferents have differing morphologies that are predicted by peripheral and central factors. Branching patterns and collaterals of 78 Neurobiotin-stained afferents were compared in rats. Fibers from one whisker had precisely somatotopic projections but highly varied morphologies. For the entire sample, analysis of variance revealed significant intrafiber variance in collateral number and arbor shape that was attributed to the target subnucleus. Significant interfiber variance did not reflect response adaptation rate, direction sensitivity, whisker row origin or parent fiber bifurcation in the trigeminal root. Instead, we found the following. 1) Mandibular fibers had more elongated arbors than maxillary axons. In subnuclei interpolaris and principalis, mandibular fibers had larger arbors with more boutons/collateral than maxillary axons; in oralis and interpolaris, mandibular fibers had fewer collaterals than those of the maxillary division. 2) Upper lip whisker axons had more boutons than those from the B-D row in all subnuclei. 3) Rostral whisker are afferents had larger arbors and more boutons than those from middle or caudal arcs due to significant arc effects in interpolaris and oralis. Thus, whisker afferents are not structurally uniform, and some morphological features are predictable. Intrafiber variance is attributed to the central target; interfiber variance reflects maxillary versus mandibular origin, upper lip origin and whisker rostrocaudal arc.

Central projections of identified trigeminal primary afferents after molar pulp deafferentation in adult rats.

It is known that removal of the tooth pulp from mandibular molar teeth in adult rats alters the mechanoreceptive field properties of many low-threshold mechanoreceptive neurons in the trigeminal brainstem nuclear complex. The present study investigates one possible way that such deafferentation-induced receptive field changes could occur: altered central projections of uninjured trigeminal low-threshold mechanoreceptive primary afferent fibers. Intra-axonal injection of horseradish peroxidase (n = 22) or neurobiotin (n = 44) into characterized fibers was performed ipsilateral to, and 10-32 days after, removal of the coronal pulp from the left mandibular molars in adult rats. Collaterals were reconstructed, quantified, and compared by means of multivariate analyses of variance to equivalent fibers stained in normal adult rats. Stained mechanosensitive fibers from experimental animals were rapidly conducting and responded to light mechanical stimulation of one vibrissa, one tooth, oral mucosa, facial hairy skin, or guard hairs. Their central projections were indistinguishable from those of control axons in all four trigeminal subnuclei. The numbers of collaterals, areas subtended by collateral arbors, numbers of boutons per collateral, and arbor circularity did not differ from those of control afferents. Collateral somatotopy was also unaffected. These data suggest that following pulpotomy, the central collaterals of uninjured trigeminal afferents display normal morphologies and maintain normal somatotopy. Changes in the morphology of low-threshold primary afferents cannot account for the changes that occur in the receptive field properties of trigeminal brainstem neurons after pulp deafferentation.

Trigeminal structure-function relationships: a reevaluation based on long-range staining of a large sample of brainstem a beta fibers.

Prior studies suggest that some classes of thickly myelinated (A beta) afferents have distinct morphologies in the trigeminal (V) brainstem complex, and that single fibers have collaterals with different shapes in the four V subnuclei. However, these conclusions are based upon relatively few and incompletely stained fibers and limited statistical rigor. In the present study, 104 fibers were stained more completely with neurobiotin in rats to provide within-fiber intersubnucleus comparisons, and between-fiber intrasubnucleus comparisons, of collaterals associated with a vibrissa, guard hairs, hairy skin, glabrous skin, or oral structures. Collaterals from all functional categories had similar qualitative features and were distributed somatotopically in the transverse plane according to known maps. Fiber categories were not disproportionately represented at particular sites along the brainstem's rostrocaudal axis, although most fibers adhered to an onion-leaf topography in caudalis. Surprisingly few structure-function relationships were revealed by multivariate analysis of variance and post hoc group comparisons, as follows: Arbors were larger in caudalis than in any other subnucleus; collaterals were most numerous in interpolaris; vibrissa afferents had more collaterals than oral and guard hair afferents; and oral fibers had larger arbors than vibrissa or guard hair afferents in subnucleus oralis. Peripheral receptor association and response adaptation rate failed to predict arbor shapes and terminal bouton numbers in any V subnucleus. These data confirm that the locations of V primary afferent arbors are predicted by their receptive fields. However, collateral number and morphology are predicted only to a very limited extent by the V subnucleus and peripheral receptor affiliation--a conclusion that contrasts with those of most prior studies of somatosensory primary afferents.