Sharon Averill

Immunocytochemical Localization of trkA Receptors in Chemically Identified Subgroups of Adult Rat Sensory Neurons

Immunocytochemistry has been used to examine the location of trkA, the high‐affinity receptor for nerve growth factor, in adult rat dorsal root ganglia, trigeminal ganglia and spinal cord. TrkA immunoreactivity was observed in small and medium sized ganglion cells and in the dorsal horn of the spinal cord. In lumbar L4 and L5 ganglia trkA‐immunoreactive cells constitute 40% of dorsal root ganglion cells and range in size from 15 to 45 μm in diameter. Double labelling using markers for various dorsal root ganglion subpopulations revealed that virtually all (92%) trkA‐immunoreactive cells express calcitonin gene‐related peptide (CGRP) immunoreactivity. In contrast only 4 and 13% of trkA‐immunoreactive cells are labelled by the monoclonal antibody LA4 or the lectin Griffonia simplicifolia IB4, markers for small non‐peptide‐containing cells. Eighteen percent of trkA‐immunoreactive cells belong to the ‘large light’subpopulation, identified by their strong immunostaining by the neurofilament antibody RT97. TrkA immunoreactivity in the dorsal horn is heaviest in laminae I and II outer, has a similar distribution to CGRP, and is depleted by dorsal rhizotomy. Our results show that trkA‐expressing cells in dorsal root ganglia correspond almost exactly with the CGRP, peptide‐producing population. The receptor is present not only on cell bodies but also on central terminals. Non‐peptide‐containing small cells, which constitute 30% of dorsal root ganglion cells, are not trkA‐immunoreactive and therefore most probably are functionally independent of nerve growth factor.

Postnatal Changes in the Expression of the trkA High-affinity NGF Receptor in Primary Sensory Neurons

In development ∼70–80% of dorsal root ganglion (DRG) cells are dependent on nerve growth factor (NGF) for their survival, while in the adult only some 40% of DRG cells express the high‐affinity NGF receptor, trkA. This discrepancy suggests that trkA expression, and therefore neurotrophin sensitivity, may alter as the animal matures. We have tested this possibility by counting the number of L4/5 DRG neurons showing immunoreactivity for trkA in rats from the day of birth to postnatal day 14. We also examined changes in p75 and IB4 labelling. On the day of birth, 71% of DRG cells were found to express trkA. However, this percentage gradually fell with age and reached adult levels at postnatal day 14. The expression of p75 did not parallel that of trkA, remaining relatively constant at between 45 and 50% of cells from birth to postnatal day 14. Over the same period there was a marked increase in the proportion of cells which bind the lectin IB4 from 9 (day of birth) to 40% (day 14). Since in the adult the 1B4 population consists of small cells which mostly do not express trkA, this finding suggests that the postnatal down‐regulation of trkA occurs in this population. Consistent with this suggestion are the results of double labelling for trkA and IB4, which confirmed that at times intermediate between birth and postnatal day 14 there was a high degree of coexpression between these markers (which is absent in the adult). This result also suggests that the down‐regulation of trkA is unlikely to be directly responsible for the emerging IB4 binding.

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.

NGF and GDNF ameliorate the increase in ATF3 expression which occurs in dorsal root ganglion cells in response to peripheral nerve injury

Activating transcription factor‐3 (ATF3) is a member of the ATF/CREB transcription factor superfamily and is induced in dorsal root ganglion (DRG) cells after nerve injury. In order to study the regulation of ATF3, we have examined the effect of nerve growth factor (NGF) and glial cell line‐derived neurotrophic factor (GDNF) on ATF3 expression. In untreated rats, sciatic nerve transection induced ATF3 immunoreactivity in 82% of L4 DRG cells at 14 days after axotomy. Intrathecal delivery of NGF or GDNF for 2 weeks commencing immediately after injury reduced the ATF3 expression to 35 and 23% of DRG cells, respectively. Cell size analysis indicated that NGF had protected a population of mainly small‐ to medium‐sized cells, but that the GDNF had protected a population of both small and large cells. This effect was confirmed by double labelling for P2X3, CGRP and 200 kDa neurofilament, markers for small peptide‐poor cells, peptide‐rich cells and large cells, respectively. Thus GDNF reduced the percentage of ATF3‐immunoreactive P2X3 cells from 70 to 4%, and the percentage of ATF3‐immunoreactive neurofilament cells from 63 to 24%. NGF was less effective than GDNF in reducing ATF3 expression in these cell types, but reduced the percentage of ATF3‐immunoreactive CGRP cells from 10% to < 1%. These results show that ATF3 expression in specific populations of DRG cells can be modulated by exogenous supplementation of specific trophic factors, and suggest that ATF3 expression may normally be induced by the loss of target‐derived NGF and GDNF.

Nerve growth factor modulates the activation status and fast axonal transport of ERK 1/2 in adult nociceptive neurones

Mature dorsal root ganglion cells respond to neurotrophins, and the intracellular signalling pathways activated by neurotrophins have been characterized in vitro. We have now used immunocytochemistry and Western blots to examine the expression and activation of extracellular signal-regulated protein kinase-1/2 (ERK) in rat dorsal root ganglion cells in vivo, using antisera to total (tERK) and phosphorylated (pERK) forms. This has revealed a number of novel findings. tERK immunoreactivity is present in most dorsal root ganglion cells but is expressed most strongly in small (nociceptive) cells and, surprisingly, is absent in a population of large cells that expressed trkB or trkC but mainly lack p75(NTR) immunoreactivity. In contrast pERK is prominent in a few trkA cells and in satellite glial cells, and is further increased by NGF treatment. tERK and pERK both undergo fast anterograde and retrograde axonal transport, indicated by accumulation at a sciatic nerve ligature, and NGF reduces the level of retrograde pERK transport.

Erythropoietin and carbamylated erythropoietin are neuroprotective following spinal cord hemisection in the rat

The cytokine erythropoietin (EPO) has been shown to be neuroprotective in a variety of models of central and peripheral nervous system injury. Derivatives of EPO that lack its erythropoietic effects have recently been developed, and the initial reports suggest that they have a neuroprotective potential comparable to that of EPO. One such derivative is carbamylated EPO (CEPO). In the current study we compared the effects of treatment with EPO and CEPO on some of the early neurodegenerative events that occur following spinal cord injury (SCI) induced by hemisection. Adult male Wistar rats received a unilateral hemisection of the spinal cord. Thirty minutes and 24 h following injury, animals received an intraperitoneal injection of saline, EPO (40 µg/kg) or CEPO (40 µg/kg). Results indicated that 3 days post‐injury, both CEPO and EPO decreased to a similar extent the size of the lesion compared with control animals. Both compounds also decreased the number of terminal transferase‐mediated dUTP nick‐end labelling (TUNEL)‐labelled apopotic nuclei around the lesion site, as well as the number of axons expressing the injury marker β‐amyloid precursor protein. EPO and CEPO also increased Schwann cell infiltration into the lesion site, although neither compound had any effect on macrophage infiltration either within the lesion site itself or in the surrounding intact tissue. In addition, immunohistochemistry showed an increased expression of both the EPO receptor and the β common receptor subunit, the components of the receptor complex proposed to mediate the neuroprotective effects of EPO and CEPO in neurons near the site of the injury. The results show that not only does CEPO have an efficacy comparable to that of EPO in its neuroprotective potential following injury, but also that changes in the receptors for these compounds following SCI may underlie their neuroprotective efficacy.

ATF3 expression in L4 dorsal root ganglion neurons after L5 spinal nerve transection

Activating transcription factor 3 (ATF3) is a widely used marker of damaged primary sensory neurons that is induced in essentially all dorsal root ganglion (DRG) neurons by spinal nerve axotomy. Whether such injuries induce its expression in neurons of adjacent DRGs remains unknown. Following L5 spinal nerve ligation, experimental but not sham‐operated rats develop thermal and mechanical hypersensitivity. In the L4 DRG, 11–12% of neurons were ATF3 positive by 1 day post‐surgery, and numbers remain unchanged at 2 weeks. Importantly, sham exposure of the L5 spinal nerve produced a nearly identical number of ATF3‐positive neurons in the L4 DRG and also a substantial increase in the L5 DRG, with a similar time‐course to experimental animals. There was no correlation between behaviour and magnitude of ATF3 expression. Co‐localization studies with the DRG injury markers galanin, neuropeptide Y and nitric oxide synthase (NOS) showed that approximately 75, 50 and 25%, respectively, of L4 ATF3‐positive neurons co‐expressed these markers after L5 transection or sham surgery. Additionally, increases in galanin and NOS were seen in ATF3‐negative neurons in L4. Our results strongly suggest that the surgical exposure of spinal nerves induces ATF3 in the L4–5 DRG, irrespective of whether the L5 nerve is subsequently cut. This probably reflects minor damage to the neurons or their axons but nevertheless is sufficient to induce phenotypic plasticity. Caution is therefore warranted when interpreting the phenotypic plasticity of DRG neurons in adjacent ganglia in the absence of positive evidence that they are not damaged.

Transplantation of olfactory ensheathing cells fails to promote significant axonal regeneration from dorsal roots into the rat cervical cord

The olfactory ensheathing cell (OEC) is a class of glial cell that has been reported to support regeneration in the central nervous system after various types of lesions, including rhizotomy of spinal dorsal roots at thoracic, lumbar and sacral levels. We have therefore carried out a detailed anatomical analysis to assess the efficacy of dorsal horn OEC transplants at promoting regeneration of primary afferents across the dorsal root entry zone (DREZ) at the cervical level in the adult rat. OECs were cultured from adult rat olfactory bulb and immunopurified (90% purity). Regeneration by large diameter afferents and by both peptidergic and non-peptidergic small diameter afferents was assessed using respectively cholera toxin B (CTB) labelling and immunocytochemistry for calcitonin gene-related peptide (CGRP) and the purinoceptor P2X3. Following an extensive (C3-T3) rhizotomy, CGRP and P2X3 immunoreactive axons regenerated across the rhizotomy site as far as the DREZ but there was no evidence of regeneration across the DREZ, except through sites where the OEC transplant was directly grafted into the DREZ. No evidence of regeneration into the dorsal horn by CTB-labelled axons was obtained. In addition, there was little sign of sprouting by intact axons in the vicinity of OEC transplant sites. In contrast to these results in vivo, cocultures of OECs and adult dorsal root ganglion cells showed that OECs stimulate extensive neurite outgrowth. The failure of the OECs to promote regeneration in vivo following cervical rhizotomy is therefore most likely due to factors in the environment of the graft site and/or the method of transplantation.

TrkA immunoreactive neurones in the rat spinal cord

We report the presence in rat spinal cord of a novel neuronal system expressing tyrosine kinase receptor (trkA), the high affinity receptor for nerve growth factor (NGF). TrkA immunoreactive cell bodies were observed in the intermediate grey matter of the spinal cord and were classified into three main groups: central canal cells located dorsolateral to the aqueduct, partition cells located between lamina X, and the lateral border of the intermediate grey, and a morphologically heterogeneous group which included large cells located near the lateral border. In situ hybridization confirmed that cells in all these areas express trkA mRNA. Combined immunofluorescence and retrograde Fluoro‐Gold labelling was used to further characterise the projections and neurotransmitter profile of the trkA cells. Although often located in the vicinity of preganglionic cell groups, trkA immunoreactive cells are not themselves preganglionic. Rather, the central canal and partition cells belong to a neurochemically complex cholinergic propriospinal system. Many partition cells coexpress trkA, choline acetyltransferase (ChAT), the low affinity neurotrophin receptor, p75, and nicotinamide adenine dinucleotide phosphate‐diaphorase (NADPH‐d). In contrast, trkA immunoreactive central canal cells express ChAT, but do not express p75 and only a subpopulation express NADPH‐d. The large trkA immunoreactive cells located on the lateral border do not express ChAT. TrkA immunoreactive fibres were also present and were located in the dorsal horn, in the dorsal columns, and in a bundle ventral to the aqueduct. However, double labelling revealed that the trkA immunoreactive fibres are not intrinsic but are primary afferent in origin and coexpress p75.

The characteristics of neuronal injury in a static compression model of spinal cord injury in adult rats

Studies of spinal cord injury using contusion (impact) injury paradigms have shown that neuronal death is an acute event that is largely over within 24 h. However, much less is known about cell death following compression injury, despite compression being a key component of natural spinal injuries. We have therefore used neuronal nuclei (NeuN) immunostaining to examine the spatiotemporal pattern of neuronal loss after static compression injury in adult rats. 3D reconstruction was used to reveal the full effect of the injury. Neuronal loss at the injury epicentre, assessed by NeuN immunostaining, amounted to 44% at 1 day but increased to 73% at 3 days and 81% at 1 month. Neuronal loss was also seen 5 mm rostral and caudal to the epicentre, but was not significant until 3 days. NeuN loss was greatest in the ventral horns and in the intermediate grey matter, with the lateral dorsal horns relatively spared. Cystic cavities formed after injury, but were not evident until 4 weeks and were small in size. In contrast to the slow profile of neuronal loss, the compression injury also evoked a transient expression of activating transcription factor‐3 (ATF3) and activated c‐Jun in neurons. ATF3 expression peaked at 3 days and declined at 7 days. Our spatiotemporal analysis of compression injury shows that neuronal loss is much more protracted than in contusion injury, and highlights the potential for neuroprotective strategies. This study is also the first indication of ATF3 involvement in spinal cord injury.