Time course changes to structural, mechanical and material properties of bone in rats after complete spinal cord injury.

OBJECTIVE: Characterise the spatiotemporal trabecular and cortical bone responses to complete spinal cord injury (SCI) in young rats. METHODS: 8-week-old male Wistar rats received T9-transection SCI and were euthanised 2-, 6-, 10- or 16-weeks post-surgery. Outcome measures were assessed using micro-computed tomography, mechanical testing, serum markers and Fourier-transform infrared spectroscopy. RESULTS: The trabecular and cortical bone responses to SCI are site-specific. Metaphyseal trabecular BV/TV was 59% lower, characterised by fewer and thinner trabeculae at 2-weeks post-SCI, while epiphyseal BV/TV was 23% lower with maintained connectivity. At later-time points, metaphyseal BV/TV remained unchanged, while epiphyseal BV/TV increased. The total area of metaphyseal and mid-diaphyseal cortical bone were lower from 2-weeks and between 6- and 10-weeks post-SCI, respectively. This suggested that SCI-induced bone changes observed in the rat model were not solely attributable to bone loss, but also to suppressed bone growth. No tissue mineral density differences were observed at any time-point, suggesting that decreased whole-bone mechanical properties were primarily the result of changes to the spatial distribution of bone. CONCLUSION: Young SCI rat trabecular bone changes resemble those observed clinically in adult and paediatric SCI, while cortical bone changes resemble paediatric SCI only.

Human olfactory mesenchymal stromal cell transplants promote remyelination and earlier improvement in gait co-ordination after spinal cord injury.

Autologous cell transplantation is a promising strategy for repair of the injured spinal cord. Here we have studied the repair potential of mesenchymal stromal cells isolated from the human olfactory mucosa after transplantation into a rodent model of incomplete spinal cord injury. Investigation of peripheral type remyelination at the injury site using immunocytochemistry for P0, showed a more extensive distribution in transplanted compared with control animals. In addition to the typical distribution in the dorsal columns (common to all animals), in transplanted animals only, P0 immunolabelling was consistently detected in white matter lateral and ventral to the injury site. Transplanted animals also showed reduced cavitation. Several functional outcome measures including end-point electrophysiological testing of dorsal column conduction and weekly behavioural testing of BBB, weight bearing and pain, showed no difference between transplanted and control animals. However, gait analysis revealed an earlier recovery of co-ordination between forelimb and hindlimb stepping in transplanted animals. This improvement in gait may be associated with the enhanced myelination in ventral and lateral white matter, where fibre tracts important for locomotion reside. Autologous transplantation of mesenchymal stromal cells from the olfactory mucosa may therefore be therapeutically beneficial in the treatment of spinal cord injury. GLIA 2017 GLIA 2017;65:639-656.

Sulfatase-mediated manipulation of the astrocyte-Schwann cell interface.

Schwann cell (SC) transplantation following spinal cord injury (SCI) may have therapeutic potential. Functional recovery is limited however, due to poor SC interactions with host astrocytes and the induction of astrogliosis. Olfactory ensheathing cells (OECs) are closely related to SCs, but intermix more readily with astrocytes in culture and induce less astrogliosis. We previously demonstrated that OECs express higher levels of sulfatases, enzymes that remove 6-O-sulfate groups from heparan sulphate proteoglycans, than SCs and that RNAi knockdown of sulfatase prevented OEC-astrocyte mixing in vitro. As human OECs are difficult to culture in large numbers we have genetically engineered SCs using lentiviral vectors to express sulfatase 1 and 2 (SC-S1S2) and assessed their ability to interact with astrocytes. We demonstrate that SC-S1S2s have increased integrin-dependent motility in the presence of astrocytes via modulation of NRG and FGF receptor-linked PI3K/AKT intracellular signaling and do not form boundaries with astrocytes in culture. SC-astrocyte mixing is dependent on local NRG concentration and we propose that sulfatase enzymes influence the bioavailability of NRG ligand and thus influence SC behavior. We further demonstrate that injection of sulfatase expressing SCs into spinal cord white matter results in less glial reactivity than control SC injections comparable to that of OEC injections. Our data indicate that sulfatase-mediated modification of the extracellular matrix can influence glial interactions with astrocytes, and that SCs engineered to express sulfatase may be more OEC-like in character. This approach may be beneficial for cell transplant-mediated spinal cord repair. GLIA 2016 GLIA 2017;65:19-33.

Inhibitory Interneurons That Express GFP in the PrP-GFP Mouse Spinal Cord Are Morphologically Heterogeneous, Innervated by Several Classes of Primary Afferent and Include Lamina I Projection Neurons among Their Postsynaptic Targets.

The superficial dorsal horn of the spinal cord contains numerous inhibitory interneurons, which regulate the transmission of information perceived as touch, pain, or itch. Despite the importance of these cells, our understanding of their roles in the neuronal circuitry is limited by the difficulty in identifying functional populations. One group that has been identified and characterized consists of cells in the mouse that express green fluorescent protein (GFP) under control of the prion protein (PrP) promoter. Previous reports suggested that PrP-GFP cells belonged to a single morphological class (central cells), received inputs exclusively from unmyelinated primary afferents, and had axons that remained in lamina II. However, we recently reported that the PrP-GFP cells expressed neuronal nitric oxide synthase (nNOS) and/or galanin, and it has been shown that nNOS-expressing cells are more diverse in their morphology and synaptic connections. We therefore used a combined electrophysiological, pharmacological, and anatomical approach to reexamine the PrP-GFP cells. We provide evidence that they are morphologically diverse (corresponding to "unclassified" cells) and receive synaptic input from a variety of primary afferents, with convergence onto individual cells. We also show that their axons project into adjacent laminae and that they target putative projection neurons in lamina I. This indicates that the neuronal circuitry involving PrP-GFP cells is more complex than previously recognized, and suggests that they are likely to have several distinct roles in regulating the flow of somatosensory information through the dorsal horn.

A putative relay circuit providing low-threshold mechanoreceptive input to lamina I projection neurons via vertical cells in lamina II of the rat dorsal horn.

BACKGROUND: Lamina I projection neurons respond to painful stimuli, and some are also activated by touch or hair movement. Neuropathic pain resulting from peripheral nerve damage is often associated with tactile allodynia (touch-evoked pain), and this may result from increased responsiveness of lamina I projection neurons to non-noxious mechanical stimuli. It is thought that polysynaptic pathways involving excitatory interneurons can transmit tactile inputs to lamina I projection neurons, but that these are normally suppressed by inhibitory interneurons. Vertical cells in lamina II provide a potential route through which tactile stimuli can activate lamina I projection neurons, since their dendrites extend into the region where tactile afferents terminate, while their axons can innervate the projection cells. The aim of this study was to determine whether vertical cell dendrites were contacted by the central terminals of low-threshold mechanoreceptive primary afferents. RESULTS: We initially demonstrated contacts between dendritic spines of vertical cells that had been recorded in spinal cord slices and axonal boutons containing the vesicular glutamate transporter 1 (VGLUT1), which is expressed by myelinated low-threshold mechanoreceptive afferents. To confirm that the VGLUT1 boutons included primary afferents, we then examined vertical cells recorded in rats that had received injections of cholera toxin B subunit (CTb) into the sciatic nerve. We found that over half of the VGLUT1 boutons contacting the vertical cells were CTb-immunoreactive, indicating that they were of primary afferent origin. CONCLUSIONS: These results show that vertical cell dendritic spines are frequently contacted by the central terminals of myelinated low-threshold mechanoreceptive afferents. Since dendritic spines are associated with excitatory synapses, it is likely that most of these contacts were synaptic. Vertical cells in lamina II are therefore a potential route through which tactile afferents can activate lamina I projection neurons, and this pathway could play a role in tactile allodynia.

Developing and Investigating a Nanovibration Intervention for the Prevention/Reversal of Bone Loss Following Spinal Cord Injury.

Osteoporosis disrupts the fine-tuned balance between bone formation and resorption, leading to reductions in bone quantity and quality and ultimately increasing fracture risk. Prevention and treatment of osteoporotic fractures is essential for reductions in mortality, morbidity, and the economic burden, particularly considering the aging global population. Extreme bone loss that mimics time-accelerated osteoporosis develops in the paralyzed limbs following complete spinal cord injury (SCI). In vitro nanoscale vibration (1 kHz, 30 or 90 nm amplitude) has been shown to drive differentiation of mesenchymal stem cells toward osteoblast-like phenotypes, enhancing osteogenesis and inhibiting osteoclastogenesis simultaneously. Here, we develop and characterize a wearable device designed to deliver and monitor continuous nanoamplitude vibration to the hindlimb long bones of rats with complete SCI. We investigate whether a clinically feasible dose of nanovibration (two 2 h/day, 5 days/week for 6 weeks) is effective at reversing the established SCI-induced osteoporosis. Laser interferometry and finite element analysis confirmed transmission of nanovibration into the bone, and microcomputed tomography and serum bone formation and resorption markers assessed effectiveness. The intervention did not reverse SCI-induced osteoporosis. However, serum analysis indicated an elevated concentration of the bone formation marker procollagen type 1 N-terminal propeptide (P1NP) in rats receiving 40 nm amplitude nanovibration, suggesting increased synthesis of type 1 collagen, the major organic component of bone. Therefore, enhanced doses of nanovibrational stimulus may yet prove beneficial in attenuating/reversing osteoporosis, particularly in less severe forms of osteoporosis.

A comparative study of glial and non-neural cell properties for transplant-mediated repair of the injured spinal cord.

Cell transplantation is one strategy for encouraging regeneration after spinal cord injury and a range of cell types have been investigated for their repair potential. However, variations in study design complicate determination of which cells are most effective. In this study we have carried out a direct comparison of the regenerative and integrative properties of several cell preparations following transplantation into the lesioned rat spinal cord. Transplants included: (i) purified olfactory ensheathing cells (OECs) and (ii) fibroblast-like cells, from olfactory bulb (OBFB-L), (iii) a 50:50 mixture of (i) and (ii) (OEC/OBFB-L), (iv) dissociated nasal mucosa (OM), (v) purified peripheral nerve Schwann cells (SCs), (vi) peripheral nerve fibroblasts, and (vii) skin fibroblasts (SF). All transplants supported axonal regeneration: OECs and SCs promoted the greatest regeneration while OBFB-like cells were least efficient and mixed cell populations were less effective than purified populations. Tract-tracing experiments demonstrated that none of the cell types promoted regeneration beyond the lesion. Although all cell types prevented cavity formation, the extent of astrocytic hypertrophy [GFAP immunoreactivity (IR) at the transplant/lesion site] differed markedly. OECs and SCs were associated with the least GFAP-IR, fibroblasts and fibroblast-like cells resulted in greater GFAP-IR while hypertrophy surrounding transplants of OM was most extensive. These differences in host-transplant reactivity were confirmed by transplanting cells into normal spinal cord where the cellular interaction is not complicated by injury. Thus, purified glial cells have advantages for transplant-mediated repair, combining maximal support for axonal regeneration with a minimal astrocytic reaction around the transplant site.

Neurochemical characterisation of lamina II inhibitory interneurons that express GFP in the PrP-GFP mouse.

BACKGROUND: Inhibitory interneurons in the superficial dorsal horn play important roles in modulating sensory transmission, and these roles are thought to be performed by distinct functional populations. We have identified 4 non-overlapping classes among the inhibitory interneurons in the rat, defined by the presence of galanin, neuropeptide Y, neuronal nitric oxide synthase (nNOS) and parvalbumin. The somatostatin receptor sst2A is expressed by ~50% of the inhibitory interneurons in this region, and is particularly associated with nNOS- and galanin-expressing cells. The main aim of the present study was to test whether a genetically-defined population of inhibitory interneurons, those expressing green fluorescent protein (GFP) in the PrP-GFP mouse, belonged to one or more of the neurochemical classes identified in the rat. RESULTS: The expression of sst2A and its relation to other neurochemical markers in the mouse was similar to that in the rat, except that a significant number of cells co-expressed nNOS and galanin. The PrP-GFP cells were entirely contained within the set of inhibitory interneurons that possessed sst2A receptors, and virtually all expressed nNOS and/or galanin. GFP was present in ~3-4% of neurons in the superficial dorsal horn, corresponding to ~16% of the inhibitory interneurons in this region. Consistent with their sst2A-immunoreactivity, all of the GFP cells were hyperpolarised by somatostatin, and this was prevented by administration of a selective sst2 receptor antagonist or a blocker of G-protein-coupled inwardly rectifying K+ channels. CONCLUSIONS: These findings support the view that neurochemistry provides a valuable way of classifying inhibitory interneurons in the superficial laminae. Together with previous evidence that the PrP-GFP cells form a relatively homogeneous population in terms of their physiological properties, they suggest that these neurons have specific roles in processing sensory information in the dorsal horn.

Grpr expression defines a population of superficial dorsal horn vertical cells that have a role in both itch and pain.

ABSTRACT: Neurons in the superficial dorsal horn that express the gastrin-releasing peptide receptor (GRPR) are strongly implicated in spinal itch pathways. However, a recent study reported that many of these correspond to vertical cells, a population of interneurons that are believed to transmit nociceptive information. In this study, we have used a GRPR CreERT2 mouse line to identify and target cells that possess Grpr mRNA. We find that the GRPR cells are highly concentrated in lamina I and the outer part of lamina II, that they are all glutamatergic, and that they account for ∼15% of the excitatory neurons in the superficial dorsal horn. We had previously identified 6 neurochemically distinct excitatory interneuron populations in this region based on neuropeptide expression and the GRPR cells are largely separate from these, although they show some overlap with cells that express substance P. Anatomical analysis revealed that the GRPR neurons are indeed vertical cells, and that their axons target each other, as well as arborising in regions that contain projection neurons: lamina I, the lateral spinal nucleus, and the lateral part of lamina V. Surprisingly, given the proposed role of GRPR cells in itch, we found that most of the cells received monosynaptic input from Trpv1-expressing (nociceptive) afferents, that the majority responded to noxious and pruritic stimuli, and that chemogenetically activating them resulted in pain-related and itch-related behaviours. Together, these findings suggest that the GRPR cells are involved in spinal cord circuits that underlie both pain and itch.

Neuropeptide Y-expressing dorsal horn inhibitory interneurons gate spinal pain and itch signalling.

Somatosensory information is processed by a complex network of interneurons in the spinal dorsal horn. It has been reported that inhibitory interneurons that express neuropeptide Y (NPY), either permanently or during development, suppress mechanical itch, with no effect on pain. Here, we investigate the role of interneurons that continue to express NPY (NPY-INs) in the adult mouse spinal cord. We find that chemogenetic activation of NPY-INs reduces behaviours associated with acute pain and pruritogen-evoked itch, whereas silencing them causes exaggerated itch responses that depend on cells expressing the gastrin-releasing peptide receptor. As predicted by our previous studies, silencing of another population of inhibitory interneurons (those expressing dynorphin) also increases itch, but to a lesser extent. Importantly, NPY-IN activation also reduces behavioural signs of inflammatory and neuropathic pain. These results demonstrate that NPY-INs gate pain and itch transmission at the spinal level, and therefore represent a potential treatment target for pathological pain and itch.

Transplant-mediated repair properties of rat olfactory mucosal OM-I and OM-II sphere-forming cells.

Olfactory mucosa is a source of cells for transplant-mediated repair of spinal cord injury (SCI) and is currently being assessed in clinical trials. We previously reported that olfactory mucosa can generate two types of sphere-forming cells with stem cell-like properties. Here we have assessed the repair potential of these cells in a rodent SCI model. Sphere-forming cells transplanted into a dorsal column injury integrated with the host spinal cord, filling the injury cavity, but showed no evidence of differentiation in vivo. Moreover, transplants supported robust axonal regeneration, particularly when suspensions of smaller spheres, rather than large aggregates, were transplanted. However, tract-tracing of dorsal column fibers showed that regenerating axons did not extend beyond the transplant. These observations show that undifferentiated olfactory spheres, though capable of supporting axonal regeneration, do not show any advantage over olfactory ensheathing cells isolated from adult olfactory tissue. In addition, olfactory spheres induced a greater astrocytic hypertrophy at the injury site than previously observed for purified olfactory ensheathing cells.

Spatiotemporal responses of trabecular and cortical bone to complete spinal cord injury in skeletally mature rats.

Objective: Characterise the spatiotemporal responses of trabecular and cortical bone to complete spinal cord injury (SCI) in the skeletally mature rat in the acute (4-week) period following injury. Methods: The spinal cord of 5-month old male rats was transected at the T9 level. Outcome measures were assessed using micro-computed tomography, three-point bending and serum markers at 1-, 2-, and 4-weeks post-transection. Comparison was made with time-0 and sham animals. Results: Lower levels of circulating serum bone formation markers and higher bone resorption markers suggested uncoupled bone turnover as early at 1-week post-transection. Micro-computed tomography showed metaphyseal and epiphyseal trabecular bone loss was observed only at 4-weeks post-transection. The bone loss was site-specific with a more severe reduction in trabecular BV/TV observed in the metaphyseal (50%) relative to epiphyseal (19%) region. Metaphyseal trabecular bone exhibited a 54% reduction in connectivity density while the epiphyseal trabecular bone was unaffected. Cortical bone deficits were not seen over the time periods examined. Conclusions: The study demonstrates that the skeletally mature spinal cord transected rat model replicates the biphasic pattern of osteoporotic changes observed in the human SCI population, providing a relevant model for testing the efficacy of interventions against SCI-induced osteoporosis.

Evidence against AMPA receptor-lacking glutamatergic synapses in the superficial dorsal horn of the rat spinal cord.

Pure NMDA receptor (NMDAr)-mediated EPSCs, thought to correspond to "silent" glutamatergic synapses that lack AMPA receptors (AMPArs), have been observed in superficial spinal dorsal horn of neonatal but not adult rats. Recent anatomical studies suggest that AMPArs are present at virtually all glutamatergic synapses in this region in adults. We used antigen retrieval to examine colocalization of AMPArs and PSD-95 (a marker for glutamatergic synapses) in laminae I-II of neonatal and adult rats. We found a high degree of colocalization in all cases, which suggests that AMPArs are present in the great majority of glutamatergic synapses even in neonatal animals. We therefore reexamined evidence for silent synapses by performing blind whole-cell recordings from superficial dorsal horn neurons in slices from neonatal or adult rats, with focal stimulation to activate glutamatergic synapses. On some occasions in both neonatal (10 of 109, 9%) and adult (9 of 77, 12%) slices, NMDAr-mediated EPSCs were observed when the holding potential was raised to +50 mV at a stimulus strength that had failed to evoke AMPAr-mediated EPSCs. However, in all cases tested, AMPAr-mediated EPSCs were then observed when the cell was returned to -70 mV; this and other properties of the EPSCs suggest that they do not represent genuine silent synapses. When compared with previous findings, our results indicate that the appearance of silent synapses depends on experimental protocol. This suggests that pure NMDAr-mediated EPSCs seen in previous studies do not correspond to AMPAr-lacking synapses but result from another mechanism, for example, loss of labile AMPArs from recently formed synapses.

Olfactory mucosa for transplant-mediated repair: a complex tissue for a complex injury?

Damage to the brain and spinal cord leads to permanent functional disability because of the very limited capacity of the central nervous system (CNS) for repair. Transplantation of cells into regions of CNS damage represents one approach to enhancing this repair. At present, the ideal cell type for transplant-mediated repair has not been identified but autologous transplantation would be advantageous. Olfactory tissue, in part because of its capacity for regeneration, has emerged as a promising source of cells and several clinical centers are using olfactory cells or tissues in the treatment of CNS damage. Until now, the olfactory ensheathing cell, a specialized glial cell of the olfactory system has been the main focus of attention. Transplants of this cell have been shown to have a neuroprotective function, support axonal regeneration, and remyelinate demyelinated axons. However, the olfactory mucosa is a heterogeneous tissue, composed of a variety of cells supporting both its normal function and its regenerative capacity. It is therefore possible that it contains several cell types that could participate in CNS repair including putative stem cells as well as glia. Here we review the cellular composition of the olfactory tissue and the evidence that equivalent cell types exist in both rodent and human olfactory mucosa suggesting that it is potentially a rich source of autologous cells for transplant-mediated repair of the CNS.

Identification of nonepithelial multipotent cells in the embryonic olfactory mucosa.

Olfactory mucosal (OM) tissue, a potential source of stem cells, is currently being assessed in the clinic as a candidate tissue for transplant-mediated repair of spinal cord injury. We examined the ability of embryonic rat OM tissue to generate stem cells using culture conditions known to promote neural stem cell proliferation. Primary spheres formed that proliferated and exhibited two main morphologies: (a) CNS neurosphere-like (OM-I) and (b) small, tight spheroid-like (OM-II). The OM-I spheres expressed the neural stem cell marker nestin but also markers of peripheral glia, neurons, and connective tissue. Further studies demonstrated the presence of multipotential mesenchymal-like stem cells within OM-I spheres that differentiated into bone, adipose, and smooth muscle cells. In contrast, the OM-II spheres contained mainly cytokeratin-expressing cells. Immunolabeling of rat olfactory tissue with Stro-1, CD90, and CD105 showed the presence of multipotent mesenchymal cells in the lamina propria, whereas cytokeratin was expressed by the epithelial cells of the olfactory epithelium. In addition, a comparable pattern of immunoreactivity was detected in human tissue using Stro-1 and cytokeratin, suggesting the presence of similar cells in this tissue. The identification of a nonepithelial multipotent cell in the OM may explain the varied reports on olfactory stem cell differentiation capacity in vitro and in vivo and illustrates the cellular complexity of this tissue as a potential source of stem cells for transplantation and translation to the clinic.

A novel poly-ε-lysine based implant, Proliferate®, for promotion of CNS repair following spinal cord injury.

The limited regenerative capacity of the CNS poses formidable challenges to the repair of spinal cord injury (SCI). Two key barriers to repair are (i) the physical gap left by the injury, and (ii) the inhibitory milieu surrounding the injury, the glial scar. Biomaterial implantation into the injury site can fill the cavity, provide a substrate for cell migration, and potentially attenuate the glial scar. We investigated the biological viability of a biocompatible and biodegradable poly-ε-lysine based biomaterial, Proliferate®, in low and high cross-linked forms and when coated with IKVAV peptide, for SCI implantation. We demonstrate altered astrocyte morphology and nestin expression on Proliferate® compared to conventional glass cell coverslips suggesting a less reactive phenotype. Moreover Proliferate® supported myelination in vitro, with myelination observed sooner on IKVAV-coated constructs compared with uncoated Proliferate®, and delayed overall compared with maintenance on glass coverslips. For in vivo implantation, parallel-aligned channels were fabricated into Proliferate® to provide cell guidance cues. Extensive vascularisation and cellular infiltration were observed in constructs implanted in vivo, along with an astrocyte border and microglial response. Axonal ingrowth was observed at the construct border and inside implants in intact channels. We conclude that Proliferate® is a promising biomaterial for implantation following SCI.

Global and site-specific analysis of bone in a rat model of spinal cord injury-induced osteoporosis.

Micro-Computed Tomography bone analysis is the gold standard method for assessing trabecular and cortical bone microarchitecture in small animal bones. This technique reports morphometric parameters as averages over selected volumes of interest (VOIs). This study proposes the introduction of an additional global 2D morphometric step into the analysis process, that provides a survey of the underlying morphometric variation present throughout both trabecular and cortical bone. The visualisation of these morphometric distributions provides a systematic approach to VOI selection that provides rationale and adds confidence to subsequent 3D morphometric analysis. To test the applicability and value of this methodological addition it was applied to the distal femur of a rat model of spinal cord injury (SCI)-induced osteoporosis. The 2D morphometric variation of both trabecular and cortical bone was quantified as a function of bone length. SCI-induced osteoporosis was localised in i) trabecular bone, where metaphyseal bone was more severely affected than epiphyseal bone, and there was a significant reduction in Distal Femoral Trabecular Extent, a new parameter defined here that quantifies how far trabecular bone penetrates in to the marrow cavity, ii) cortical bone, where diaphyseal bone underwent significant lowering of both cortical area and thickness, while distal-metaphyseal bone did not. Theses site-specific changes were validated, further elucidated and compared with follow-up conventional 3D analysis. The techniques applied here are equally applicable to other long bones (tibia, humerus, radius, ulna), other types of imaging modality and other types of experimental design including the effects of rehabilitation, aging, loading, gene knockout and pharmacological intervention.

Morphological and functional properties distinguish the substance P and gastrin-releasing peptide subsets of excitatory interneuron in the spinal cord dorsal horn.

Excitatory interneurons account for the majority of neurons in the superficial dorsal horn, but despite their presumed contribution to pain and itch, there is still limited information about their organisation and function. We recently identified 2 populations of excitatory interneuron defined by expression of gastrin-releasing peptide (GRP) or substance P (SP). Here, we demonstrate that these cells show major differences in their morphological, electrophysiological, and pharmacological properties. Based on their somatodendritic morphology and firing patterns, we propose that the SP cells correspond to radial cells, which generally show delayed firing. By contrast, most GRP cells show transient or single-spike firing, and many are likely to correspond to the so-called transient central cells. Unlike the SP cells, few of the GRP cells had long propriospinal projections, suggesting that they are involved primarily in local processing. The 2 populations also differed in responses to neuromodulators, with most SP cells, but few GRP cells, responding to noradrenaline and 5-HT; the converse was true for responses to the µ-opioid agonist DAMGO. Although a recent study suggested that GRP cells are innervated by nociceptors and are strongly activated by noxious stimuli, we found that very few GRP cells receive direct synaptic input from TRPV1-expressing afferents, and that they seldom phosphorylate extracellular signal-regulated kinases in response to noxious stimuli. These findings indicate that the SP and GRP cells differentially process somatosensory information.

Upregulation of substance P in low-threshold myelinated afferents is not required for tactile allodynia in the chronic constriction injury and spinal nerve ligation models.

It has been proposed that substance P and calcitonin gene-related peptide (CGRP) are upregulated in low-threshold myelinated primary afferents after certain types of nerve injury, and that release of substance P from these afferents contributes to the resulting tactile allodynia. To test this hypothesis, we looked for neuropeptides in Abeta primary afferent terminals in the ipsilateral gracile nucleus and spinal dorsal horn in three nerve injury models: sciatic nerve transection (SNT), spinal nerve ligation (SNL), and chronic constriction injury (CCI). We also looked for evidence of neurokinin 1 (NK1) receptor internalization in the dorsal horn after electrical stimulation of Abeta afferents. We found no evidence of either substance P or CGRP expression in injured Abeta terminals in the spinal cord in any of the models. Although substance P was not detected in terminals of injured afferents in the gracile nucleus, CGRP was expressed in between 32 and 68% of these terminals, with a significantly higher proportion in the SNL and CCI models, compared with SNT. In addition, we did not detect any Abeta-evoked NK1 receptor internalization in neurons from laminas I, III, or IV of the dorsal horn in the CCI or SNL models. These results do not support the proposal that substance P is present at significant levels in the terminals of injured Abeta primary afferents in neuropathic models. They also suggest that any release of substance P from injured Abeta afferents is unlikely to activate NK1 receptors in the dorsal horn or contribute to neuropathic pain.

FGF/heparin differentially regulates Schwann cell and olfactory ensheathing cell interactions with astrocytes: a role in astrocytosis.

After injury, the CNS undergoes an astrocyte stress response characterized by reactive astrocytosis/proliferation, boundary formation, and increased glial fibrillary acidic protein (GFAP) and chondroitin sulfate proteoglycan (CSPG) expression. Previously, we showed that in vitro astrocytes exhibit this stress response when in contact with Schwann cells but not olfactory ensheathing cells (OECs). In this study, we confirm this finding in vivo by demonstrating that astrocytes mingle with OECs but not Schwann cells after injection into normal spinal cord. We show that Schwann cell-conditioned media (SCM) induces proliferation in monocultures of astrocytes and increases CSPG expression in a fibroblast growth factor receptor 1 (FGFR1)-independent manner. However, SCM added to OEC/astrocyte cocultures induces reactive astrocytosis and boundary formation, which, although sensitive to FGFR1 inhibition, was not induced by FGF2 alone. Addition of heparin to OEC/astrocyte cultures induces boundary formation, whereas heparinase or chlorate treatment of Schwann cell/astrocyte cultures reduces it, suggesting that heparan sulfate proteoglycans (HSPGs) are modulating this activity. In vivo, FGF2 and FGFR1 immunoreactivity was increased over grafted OECs and Schwann cells compared with the surrounding tissue, and HSPG immunoreactivity is increased over reactive astrocytes bordering the Schwann cell graft. These data suggest that components of the astrocyte stress response, including boundary formation, astrocyte hypertrophy, and GFAP expression, are mediated by an FGF family member, whereas proliferation and CSPG expression are not. Furthermore, after cell transplantation, HSPGs may be important for mediating the stress response in astrocytes via FGF2. Identification of factors secreted by Schwann cells that induce this negative response in astrocytes would further our ability to manipulate the inhibitory environment induced after injury to promote regeneration.