John S. Riddell

Selective loss of spinal GABAergic or glycinergic neurons is not necessary for development of thermal hyperalgesia in the chronic constriction injury model of neuropathic pain.

&NA; GABA and glycine are inhibitory neurotransmitters used by many neurons in the spinal dorsal horn, and intrathecal administration of GABAA and glycine receptor antagonists produces behavioural signs of allodynia, suggesting that these transmitters have an important role in spinal pain mechanisms. Several studies have described a substantial loss of GABA‐immunoreactive neurons from the dorsal horn in nerve injury models, and it has been suggested that this may be associated with a loss of inhibition, which contributes to the behavioural signs of neuropathic pain. We have carried out a quantitative stereological analysis of the proportions of neurons in laminae I, II and III of the rat dorsal horn that show GABA‐ and/or glycine‐immunoreactivity 2 weeks after nerve ligation in the chronic constriction injury (CCI) model, as well as in sham‐operated and naïve animals. At this time, rats that had undergone CCI showed a significant reduction in the latency of withdrawal of the ipsilateral hindpaw to a radiant heat stimulus, suggesting that thermal hyperalgesia had developed. However, we did not observe any change in the proportion of neurons in laminae I–III of the ipsilateral dorsal horn that showed GABA‐ or glycine‐immunoreactivity compared to the contralateral side in these animals, and these proportions did not differ significantly from those seen in sham‐operated or naïve animals. In addition, we did not see any evidence for alterations of GABA‐ or glycine‐immunostaining in the neuropil of laminae I–III in the animals that had undergone CCI. Our results suggest that significant loss of GABAergic or glycinergic neurons is not necessary for the development of thermal hyperalgesia in the CCI model of neuropathic pain.

Populations of inhibitory and excitatory interneurons in lamina II of the adult rat spinal dorsal horn revealed by a combined electrophysiological and anatomical approach.

&NA; Lamina II contains a large number of interneurons involved in modulation and transmission of somatosensory (including nociceptive) information. However, its neuronal circuitry is poorly understood due to the difficulty of identifying functional populations of interneurons. This information is important for understanding nociceptive processing and for identifying changes that underlie chronic pain. In this study, we compared morphology, neurotransmitter content, electrophysiological and pharmacological properties for 61 lamina II neurons recorded in slices from adult rat spinal cord. Morphology was related to transmitter content, since islet cells were GABAergic, while radial and most vertical cells were glutamatergic. However, there was considerable diversity among the remaining cells, some of which could not be classified morphologically. Transmitter phenotype was related to firing pattern, since most (18/22) excitatory cells, but few (2/23) inhibitory cells had delayed, gap or reluctant patterns, which are associated with A‐type potassium (IA) currents. Somatostatin was identified in axons of 14/24 excitatory neurons. These had variable morphology, but most of those tested showed delayed‐firing. Excitatory interneurons are therefore likely to contribute to pain states associated with synaptic plasticity involving IA currents. Although noradrenaline and serotonin evoked outward currents in both inhibitory and excitatory cells, somatostatin produced these currents only in inhibitory neurons, suggesting that its pro‐nociceptive effects are mediated by disinhibition. Our results demonstrate that certain distinctive populations of inhibitory and excitatory interneuron can be recognised in lamina II. Combining this approach with identification of other neurochemical markers should allow further clarification of neuronal circuitry in the superficial dorsal horn.

Lack of evidence for significant neuronal loss in laminae I–III of the spinal dorsal horn of the rat in the chronic constriction injury model

&NA; Peripheral nerve injury leads to structural and functional changes in the spinal dorsal horn, and these are thought to be involved in the development of neuropathic pain. In the chronic constriction injury (CCI) model, abnormal ‘dark’ neurons and apoptotic nuclei have been observed in laminae I–III of the dorsal horn in the territory innervated by the injured sciatic nerve. These findings have been taken as evidence that there is significant neuronal death in this model, and it has been suggested that loss of inhibition resulting from death of GABAergic inhibitory interneurons contributes to the neuropathic pain. However, loss of neurons from the dorsal horn has not been directly demonstrated in neuropathic models, even though this issue is of considerable importance for our understanding of the mechanisms that underlie neuropathic pain. In this study, we have looked for evidence of neuronal death by using a stereological method (the optical disector) with NeuN‐immunostaining, and examining spinal cords of naïve rats, and of rats that had undergone CCI or sham operations. All of the CCI animals showed clear signs of thermal hyperalgesia. However, the numbers of neurons in laminae I–III of the ipsilateral dorsal horn in these animals did not differ significantly from those on the contralateral side, nor from those of sham‐operated or naïve animals. These results do not, therefore, support the suggestion that there is significant neuronal death in the dorsal horn in this model.

Olfactory ensheathing cells (OECs) and the treatment of CNS injury: advantages and possible caveats

One of the main research strategies to improve treatment for spinal cord injury involves the use of cell transplantation. This review looks at the advantages and possible caveats of using glial cells from the olfactory system in transplant‐mediated repair. These glial cells, termed olfactory ensheathing cells (OECs), ensheath the axons of the olfactory receptor neurons. The primary olfactory system is an unusual tissue in that it can support neurogenesis throughout life. In addition, newly generated olfactory receptor neurons are able to grow into the CNS environment of the olfactory bulb tissue and reform synapses. It is thought that this unique regenerative property depends in part on the presence of OECs. OECs share some of the properties of both astrocytes and Schwann cells but appear to have advantages over these and other glial cells for CNS repair. In particular, OECs are less likely to induce hypertrophy of CNS astrocytes. As well as remyelinating demyelinated axons, OEC grafts appear to promote the restoration of functions lost following a spinal cord lesion. However, much of the evidence for this is based on behavioural tests, and the mechanisms that underlie their potential benefits in transplant‐mediated repair remain to be clarified.

Olfactory ensheathing cell transplantation as a strategy for spinal cord repair--what can it achieve?

Restoring function to the injured spinal cord represents one of the most formidable challenges in regenerative medicine. Glial cell transplantation is widely considered to be one of the most promising therapeutic strategies, and several differentiated glial cell types—in particular, Schwann cells and olfactory ensheathing cells (OECs)—have been proposed as transplant candidates. In this Review, we analyze evidence from animal studies for improved functional recovery following transplantation of OECs into spinal cord injuries, and examine the mechanisms by which repair might be achieved. Data obtained using various injury models support the view that OEC transplants can promote functional recovery, but accumulating anatomical evidence indicates that although axons regenerate within a transplant, they do not cross the lesion or reconnect with neurons on the opposite side to any significant extent. Consequently, it is possible that neuroprotection and promotion of sprouting from intact fibers are the main mechanisms that contribute to functional recovery. We conclude that for the foreseeable future the clinical benefits of OEC transplants alone are likely to be modest. The future potential of cell transplantation strategies will probably depend on the success with which the transplants can be combined with other, synergistic, therapies to achieve significant regeneration of axons and re-establish functionally useful connections across a spinal cord injury.

Olfactory ensheathing cell grafts have minimal influence on regeneration at the dorsal root entry zone following rhizotomy

The effectiveness of grafts of olfactory ensheathing cells (OECs) as a means of promoting functional reconnection of regenerating primary afferent fibers was investigated following dorsal root injury. Adult rats were subjected to dorsal root section and reanastomosis and at the same operation a suspension of purified OECs was injected at the dorsal root entry zone and/or into the sectioned dorsal root. Regeneration of dorsal root fibers was then assessed after a survival period ranging from 1 to 6 months. In 11 animals, electrophysiology was used to look for evidence of functional reconnection of regenerating dorsal root fibers. However, electrical stimulation of lesioned dorsal roots failed to evoke detectable cord dorsum or field potentials within the spinal cord of any of the animals examined, indicating that reconnection of regenerating fibers with spinal cord neurones had not occurred. In a further 11 rats, immunocytochemical labeling and biotin dextran tracing of afferent fibers in the lesioned roots was used to determine whether regenerating fibers were able to grow into the spinal cord in the presence of an OEC graft. Although a few afferent fibers could be seen to extend for a limited distance into the spinal cord, similar minimal in‐growth was seen in control animals that had not been injected with OECs. We therefore conclude that OEC grafts are of little or no advantage in promoting the in‐growth of regenerating afferent fibers at the dorsal root entry zone following rhizotomy.

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.

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. STEM CELLS 2009;27:2196–2208

Interneurones mediating presynaptic inhibition of group II muscle afferents in the cat spinal cord.

1. To investigate whether dorsal horn interneurones with input from group II muscle afferents induce depolarization of sensory fibres, simultaneous recordings were made from single interneurones in the sacral segments and from sacral dorsal root filaments using the spike‐triggered averaging technique. 2. The spike potentials of eighteen out of thirty‐eight interneurones tested were followed by dorsal root potentials (DRPs). The DRPs occurred at latencies of 2 and 6‐8 ms. Interneurones evoking DRPs at latencies of up to 2 ms are considered likely to be last‐order interneurones in pathways of presynaptic inhibition, while those inducing DRPs at longer latencies are considered likely to be first‐order interneurones. The former were activated by peripheral afferents with somewhat longer latencies than the latter. However, all interneurones were co‐activated by group II muscle and cutaneous afferents, indicating that the depolarization of group II muscle afferents, which these afferents induce, may be mediated by the same interneurones. 3. DRPs evoked by electrical stimulation of peripheral nerves were recorded from both sacral and midlumbar dorsal root filaments. The amplitudes of these DRPs were closely related to the potency with which group II afferents of various nerves activate dorsal horn interneurones in the sacral and midlumbar segments and group II afferents contributed to them more effectively than group I afferents. The second stimulus in a train was more effective than the first, while a third stimulus had little additional effect, indicating that the interneurones involved are relatively easily activated. 4. Intraspinal stimuli applied within the dorsal horn, at the sites where the largest field potentials of group II origin were recorded, evoked distinct DRPs. However, the location of the first‐ and last‐order interneurones in pathways of primary afferent depolarization (PAD) could not be differentiated by this approach because the same stimuli induced positive potentials, which masked the onset of DRPs and precluded localization of the sites from which DRPs might be evoked monosynaptically.