A characterization of white matter pathology following spinal cord compression injury in the rat.

Our laboratory has previously described the characteristics of neuronal injury in a rat compression model of spinal cord injury (SCI), focussing on the impact of this injury on the gray matter. However, white matter damage is known to play a critical role in functional outcome following injury. Therefore, in the present study, we used immunohistochemistry and electron microscopy to examine the alterations to the white matter that are initiated by compression SCI applied at T12 vertebral level. A significant loss of axonal and dendritic cytoskeletal proteins was observed at the injury epicenter within 1day of injury. This was accompanied by axonal dysfunction, as demonstrated by the accumulation of ß-amyloid precursor protein (ß-APP), with a peak at 3days post-SCI. A similar, acute loss of cytoskeletal proteins was observed up to 5mm away from the injury epicenter and was particularly evident rostral to the lesion site, whereas ß-APP accumulation was prominent in tracts proximal to the injury. Early myelin loss was confirmed by myelin basic protein (MBP) immunostaining and by electron microscopy, which also highlighted the infiltration of inflammatory and red blood cells. However, 6weeks after injury, areas of new Schwann cell and oligodendrocyte myelination were observed. This study demonstrates that substantial white matter damage occurs following compression SCI in the rat. Moreover, the loss of cytoskeletal proteins and accumulation of ß-APP up to 5mm away from the lesion site within 1day of injury indicates the rapid manner in which the axonal damage extends in the rostro-caudal axis. This is likely due to both Wallerian degeneration and spread of secondary cell death, with the latter affecting axons both proximal and distal to the injury.

Regenerative potential of silk conduits in repair of peripheral nerve injury in adult rats.

Various attempts have been made to develop artificial conduits for nerve repair, but with limited success. We describe here conduits made from Bombyx mori regenerated silk protein, and containing luminal fibres of Spidrex(®), a silk-based biomaterial with properties similar to those of spider silk. Assessment in vitro demonstrated that Spidrex(®) fibres support neurite outgrowth. For evaluation in vivo, silk conduits 10 mm in length and containing 0, 100, 200 or 300 luminal Spidrex(®) fibres, were implanted to bridge an 8 mm gap in the rat sciatic nerve. At 4 weeks, conduits containing 200 luminal Spidrex(®) fibres (PN200) supported 62% and 59% as much axon growth as autologous nerve graft controls at mid-conduit and distal nerve respectively. Furthermore, Spidrex(®) conduits displayed similar Schwann cell support and macrophage response to controls. At 12 weeks, animals implanted with PN200 conduits showed similar numbers of myelinated axons (81%) to controls, similar gastrocnemius muscle innervation, and similar hindpaw stance assessed by Catwalk footprint analysis. Plantar skin innervation was 73% of that of controls. PN200 Spidrex(®) conduits were also effective at bridging longer (11 and 13 mm) gaps. Our results show that Spidrex(®) conduits promote excellent axonal regeneration and function recovery, and may have potential for clinical application.

The effect of mechanical strain or hypoxia on cell death in subpopulations of rat dorsal root ganglion neurons in vitro.

Spinal nerves and their associated dorsal root ganglion (DRG) cells can be subject to mechanical deformation and hypoxia associated with pathology such as disc herniation, spinal stenosis and spine trauma. There is very limited information on the response of adult DRG neurons to such stressors. In this study we used an in vitro approach to examine the response of adult DRG cells to (a) mechanical, hypoxic, and combined injuries; and (b) to compare the effects on injury on nociceptive and non-nociceptive neurons, as well as on non-neuronal cells. Mechanical injury (20% tensile strain) led to significant neuronal cell death (assessed by ethidium homodimer-1 labelling), which was proportional to strain duration (5 min, 1 h, 6 h or 18 h). Hypoxia (2% O(2) for 24 h) also promoted death of DRG neurons, and was further enhanced when mechanical strain and hypoxia were combined. Both mechanical strain and hypoxia significantly decreased the maximum neurite length. Conversely, death of non-neuronal cells was only increased by hypoxia and not by mechanical strain. Total cell death in response to mechanical injury or hypoxia was similar in both non-nociceptive (neurofilament, NF-200 immunoreactive) and nociceptive (calcitonin gene-related peptide, CGRP immunoreactive) neurons, but apoptosis (assessed by activated caspase-3 immunostaining) was significantly higher in CGRP than NF-200 neurons. Surprisingly, cell death of non-peptidergic nociceptors (identified by Griffonia simplicifolia IB4 lectin binding) was already high in control cultures, and was not increased further by either mechanical stretch or hypoxia. These results provide detailed information on the response of adult DRG subpopulations to hypoxia and mechanical strain, and describe in vitro models that could be useful for screening potential neuroprotective agents.

Activation transcription factor-3 activation and the development of spinal cord degeneration in a rat model of amyotrophic lateral sclerosis.

It has been reported that an early activation of glial fibrillary acid protein (GFAP) in astroglial cells occurs simultaneously in peripheral nerves and spinal cord from the G93A SOD1 mouse model of amyotrophic lateral sclerosis (ALS), an invariably fatal neurodegenerative disorder. In ALS, the contribute to the pathological process of different cell types varies according to the disease stage, with a florid immune response in spinal cord at end stage disease. In this study, we have mapped in different anatomical sites the process of disease-induced functional perturbation from a pre-symptomatic stage using a marker of cellular distress expressed in neurons and glial cells, the activating transcription factor 3 (ATF-3), and applied large-scale gene expression analysis to define the pattern or transcriptional changes occurring in spinal cord from the G93A SOD1 rat model of ALS in parallel with ATF-3 neuronal activation. From the disease onset onward, transgenic lumbar spinal cord displayed ATF-3 transcriptional regulation and motor cells immunostaining in association with the over-expression of genes promoting cell growth, the functional integrity of cell organelles and involved in the modulation of immune responses. While spinal cord from the pre-symptomatic rat showed no detectable ATF-3 transcriptional regulation, ATF-3 activation was appreciated in large size neurofilament-rich, small size non-peptidergic and parvalbumin-positive neurons within the dorsal root ganglia (DRG), and in ventral roots Schwann cells alongside macrophages infiltration. This pattern of peripheral ATF-3 activation remained detectable throughout the disease process. In the G93A SOD1 rat model of ALS, signs of roots and nerves subtle distress preceded overt clinical-pathological changes, involving both glial cells and neurons that function as receptors of peripheral sensory stimuli from the muscle. In addition, factors previously described to be linked to ATF-3 activation under various experimental conditions of stress, become switched on in spinal cord from the end-stage transgenic rat model of ALS.

The neuroprotective effects of fibronectin mats and fibronectin peptides following spinal cord injury in the rat.

We have shown previously that mats made from the glycoprotein fibronectin are permissive for axonal growth when implanted into the injured spinal cord. Recent evidence has indicated that fibronectin and its peptides also have neuroprotective effects in the CNS. We have therefore examined the neuroprotective effects of fibronectin applied to a spinal cord injury site. Adult rats with fibronectin mats implanted into a spinal cord lesion cavity had decreased apoptosis in the intact adjoining spinal cord tissue at 1 and 3 days post-injury compared to rats that had gelfoam implanted into the lesion cavity. Rats with fibronectin mat implants also showed enhanced hindlimb locomotor performance for the first 3 weeks post-surgery compared to control animals. To further examine the neuroprotective potential of fibronectin following spinal cord injury, we examined the effects of placing fibronectin mats over the site of a spinal cord hemisection or of delivering a solution derived from a dissolved fibronectin mat. The effects of these treatments were compared with control animals and animals that were treated with a fibronectin peptide (PRARIY) that has been shown to decrease secondary damage in a rodent model of cerebral ischemia. Results showed that both types of fibronectin mat treatment resulted in decreased lesion size, apoptosis, and axonal damage within the first week post-injury compared to control animals and were comparable in their neuroprotective efficacy to treatment with the fibronectin peptide. The results of the current study indicate that fibronectin based biomaterials have neuroprotective effects following spinal cord injury, in addition to their previously reported ability to promote axonal regeneration.

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.

The impact of neurotrophin-3 on the dorsal root transitional zone following injury.

STUDY DESIGN: Morphological and Stereological assessment of the dorsal root transitional zone (DRTZ) following complete crush injury, using light microscopy (LM) and transmission electron microscopy (TEM). OBJECTIVES: To assess the effect of exogenous neurotrophin-3 (NT-3) on the response of glial cells and axons to dorsal root damage. SETTING: Department of Anatomy, University College Cork, Ireland and Department of Physiology, UMDS, University of London, UK. METHODS: Cervical roots (C6-8) from rats which had undergone dorsal root crush axotomy 1 week earlier, in the presence (n=3) and absence (n=3) of NT-3, were processed for LM and TEM. RESULTS: Unmyelinated axon number and size was greater in the DRTZ proximal (Central Nervous System; CNS) and distal (Peripheral Nervous System; PNS) compartments of NT-3-treated tissue. NT-3 was associated with a reduced astrocytic response, an increase in the proportion of oligodendrocytic tissue and a possible inhibition or delay of microglial activation. Disrupted-myelin volume in the DRTZ PNS and CNS compartments of treated tissue was lower, than in control tissue. In the PNS compartment, NT-3 treatment increased phagocyte and blood vessel numbers. It decreased myelinating activity, as sheath thickness was significantly lower and may also account for the noted lower Schwann cell and organelle volume in the test group. CONCLUSIONS: Our observations suggest that NT-3 interacts with non-neuronal tissue to facilitate the regenerative effort of damaged axons. This may be as a consequence of a direct action or indirectly mediated by modulation of non-neuronal responses to injury.

A combination of intravenous and dietary docosahexaenoic acid significantly improves outcome after spinal cord injury.

Previous studies have shown that omega-3 polyunsaturated fatty acids such as alpha-linolenic acid and docosahexaenoic acid (DHA) are neuroprotective in models of spinal cord injury (SCI) in rodents. However, the mechanism of action underlying these effects has not been elucidated, and the optimum treatment regime remains to be defined. We have therefore carried out a detailed analysis of the effects of DHA in adult rats subject to thoracic compression SCI. Saline or DHA (250 nmol/kg) was administered intravenously (i.v.) 30 min after compression. After injury, the saline group received a standard control diet for 1 or 6 weeks, whereas DHA-injected animals received either a control or a DHA-enriched diet (400 mg/kg/day) for 1 or 6 weeks. Other groups received a DHA-enriched diet only for 1 week following injury, or received acute DHA (250 nmol/kg; i.v.) treatment delayed up to 3 h after injury. We also assessed oxidative stress and the inflammatory reaction at the injury site, neuronal and oligodendrocyte survival and axonal damage and the locomotor recovery. At 24 h, lipid peroxidation, protein oxidation, RNA/DNA oxidation and the induction of cyclooxygenase-2 were all significantly reduced by i.v. DHA administration. At 1 week and 6 weeks, macrophage recruitment was reduced and neuronal and oligodendrocyte survival was substantially increased. Axonal injury was reduced at 6 weeks. Locomotor recovery was improved from day 4, and sustained up to 6 weeks. Rats treated with a DHA-enriched diet in addition to the acute DHA injection were not significantly different from the acute DHA-treated animals at 1 week, but at 6 weeks showed additional improvements in both functional and histological outcomes. DHA treatment was ineffective if the acute injection was delayed until 3 h post-injury, or if the DHA was administered for 1 week solely by diet. Our results in a clinically relevant model of SCI show that significant neuroprotection can be obtained by combining an initial acute i.v. injection of DHA with a sustained dietary supplementation. Given that the safety and tolerability of preparations enriched in omega-3 fatty acids is already well-documented, such a combined DHA treatment regime deserves consideration as a very promising approach to SCI management.

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 microg/kg) or CEPO (40 microg/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 beta-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 beta 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.

Costorage of BDNF and neuropeptides within individual dense-core vesicles in central and peripheral neurons.

Some central and peripheral neurons synthesize brain-derived neurotrophic factor (BDNF), and, after anterograde transport, release it at synapses. By immunocytochemistry, we examined, in rat and mouse, the subcellular localization of BDNF and BDNF/peptide coexistence, under normal conditions or after intrathecal infusion of nerve growth factor. In dorsal root ganglion neurons and afferent terminals, and in the parabrachial projection to amygdala, we show that BDNF is costored in individual dense-core vesicles (DCVs) with the neuropeptides calcitonin gene-related peptide (CGRP) and substance P. At both locations, nerve endings costoring all three peptides were fairly rare. Remarkably however, costorage occurred in a stoichiometric ratio of 0.7 BDNF:1 CGRP:1 substance P, and DCVs contained 31 (spinal cord) -36 (amygdala) times the amount of BDNF detected in agranular vesicles. This is the first direct demonstration in peripheral and central neurons from two different mammals, that a growth factor is selectively packaged together with neuropeptide transmitters within individual DCVs. It provides structural bases for differential release upon stimulation, and has important implications for understanding BDNF transmitter function.

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.

International spinal research trust research strategy. III: A discussion document.

STUDY DESIGN: Discussion document. OBJECTIVES/METHODS: To review the Research Strategy of the International Spinal Research Trust (ISRT), which identifies key areas of basic and clinical research that are likely to be beneficial in developing potential treatments for spinal cord injury for funding. This strategy is intended to both guide the programme of research towards areas of priority and stimulate discussion of the different avenues of research. This latest document has been developed to take into account the scientific progress in the 6 years since publication of the previous Research Strategy. RESULTS/DISCUSSION: The latest scientific developments in research designed to repair the spinal cord and restore function following injury and how they might impact on spinal cord injury research are highlighted.

Immunohistochemical co-localization of transient receptor potential vanilloid (TRPV)1 and sensory neuropeptides in the guinea-pig respiratory system.

Electrophysiological studies within the lung have documented the presence of heterogenous groups of afferent fibers composed of Adelta and C-fibers and studies of somatosensory nerves within the skin reveal a complex pattern of distribution of sensory neuropeptides and transient receptor potential vanilloid (TRPV)1 positive nerves. However, the anatomical location of these different subpopulations of nerves within the lung has not been extensively studied. In the present study we have demonstrated that TRPV1 axons represented only a small proportion of the total number of PGP9.5 staining nerves within guinea-pig tracheal epithelium and only half the number of TRPV1 axons was immunopositive for substance P. In contrast, most TRPV1 positive neurones found within guinea-pig intrapulmonary airways were found to co-localize with sensory neuropeptides substance P and calcitonin gene-related peptide within and beneath the epithelium, around blood vessels, within airway smooth muscle and alveoli, indicative of heterogeneity of TRPV1 positive axons throughout the airways. However, in the smooth muscle layer of the trachea there was evidence of substance P and calcitonin gene-related peptide containing nerves that did not stain for TRPV1. We also demonstrated a complete loss of TRVP1 positive axons in the trachea and intrapulmonary airways and associated loss of bronchoconstriction induced by capsaicin, in animals chronically treated with capsaicin. However, some neuropeptide immunoreactive axons remained in the smooth muscle layer of capsaicin-treated animals which could represent the small subset of neuropeptide containing fibers which do not co-localize with TRPV1. We have provided evidence of heterogeneity of TRPV1 positive nerve fibers, including fibers characterized by lack of co-localization with neuropeptides in various regions of the airways and the existence of neuropeptide containing fibers that were not TRPV1 positive in guinea-pigs.

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.

Characterization of non-neuronal elements within fibronectin mats implanted into the damaged adult rat spinal cord.

Previous studies have shown that mats made from fibronectin (FN) integrate well into spinal cord lesion sites and support extensive axonal growth. Using immunohistochemistry, we have investigated the non-neuronal factors that contribute to these properties. Extensive vascularization was observed in FN mats by 1 week along with heavy macrophage infiltration by 3 days post-implantation. By 1 week post-implantation, laminin tubules had formed and were associated with axons and p75 immunoreactive Schwann cells. By 4 weeks post-implantation, most axons were associated with Schwann cell derived myelin. Few oligodendrocytes were present within the mat, even with an increase in the number of oligodendrocyte precursors around the implant site by 7 days post-implantation. Astrocyte proliferation also occurred in the intact tissue, with a prominent glial scar forming around the implant within 4 weeks. However, by 2 months post-implantation astrocytes were present in the FN implant site and were intermingled with the axons. Axonal ingrowth and integration of the FN mats is probably due to the ability of FN mats to support and organize infiltration of Schwann cells and deposition of laminin. At later time points, myelinated axons remain in the implant site, even after other elements (e.g. macrophages and laminin) have disappeared. Both of these properties are likely to be important in the design of biomaterial bridges for CNS regeneration.

Identification of the sites of expression of triple A syndrome mRNA in the rat using in situ hybridisation.

Triple A syndrome is characterised by achalasia, alacrima, adrenocorticotropin-resistant adrenal insufficiency and a variable and progressive neurological phenotype. It is caused by mutations in a gene that is normally referred to as the triple A syndrome gene (AAAS) and which has recently been shown to encode a nuclear pore protein named ALADIN (alacrima, achalasia, adrenal insufficiency neurologic disorder). In this study we performed in situ hybridisation with radioactive oligonucleotide probes in the adult and developing rat and present the first detailed map of AAAS mRNA expression. Consistent with a role for AAAS in adrenal function, we detected high levels of its mRNA in the adrenal cortex. On the other hand hepatocytes, enteric smooth muscle and fibroblasts had relatively little or no detectable AAAS mRNA. In both the peripheral and central nervous systems, AAAS mRNA was abundantly expressed. Neurons in sensory and sympathetic ganglia expressed high levels. CNS expression was highest in neurons of the cerebral cortex, cerebellum, hippocampus, motor-associated nuclei of the brainstem including cranial nerve nuclei, and ventral horn of the spinal cord. Although neuronal expression of AAAS mRNA was striking, non-neuronal cells including those of the circumventricular organs and fibrous astrocytes also expressed AAAS mRNA. Within the developing embryo, the highest levels of expression were found in neural tissues. These findings indicate a widespread but not ubiquitous or uniform expression of AAAS mRNA in the rat. Robust expression in neural systems associated with cognitive, motor and sensory functions is consistent with the myriad of symptoms experienced by patients with triple A syndrome.

Hypoxia-induced apoptosis in adult rat dorsal root ganglion neurons in vitro.

Following spinal root injury, dorsal root ganglia suffer mechanical trauma and compromised blood supply. Little is known about the consequences for neuronal survival. Here we used cyanide treatment in vitro to examine effects of moderate hypoxia on adult rat dorsal root ganglion cells identified by GAP-43 immunostaining. 400 microM-4 mM cyanide caused sustained increases in intracellular Ca2+. Cyanide at 2 mM led to a significant increase in apoptosis, detected using TUNEL labelling and confirmed by ultrastructural analysis, and a further increase when cultures were left overnight in fresh medium. Our study shows that dorsal root ganglion neurons die by apoptosis following hypoxia and that cell death increases over time. Cyanide response provides a simple assay for testing neuroprotective agents and examining underlying mechanisms.

Differential pH and capsaicin responses of Griffonia simplicifolia IB4 (IB4)-positive and IB4-negative small sensory neurons.

Protons play a key role in nociception caused by inflammation and ischaemia, but little is known about the relative sensitivities of different dorsal root ganglion (DRG) neurons. We have therefore examined the responses in vitro of rat DRG cells classified according to whether or not they bind Griffonia simplicifolia IB4 (IB4), a lectin which is widely used to distinguish between two major populations of small diameter neurons. Under voltage-clamp conditions, proton-activated inward currents were found in approximately 90% of small DRG neurons and showed one of three waveforms: transient, sustained or mixed. The majority of IB4-positive (IB4+) neurons (63%) gave rise to sustained inward currents that were sensitive to capsazepine. In contrast, the most prevalent waveform in small IB4-negative (IB4-) neurons (69%) was a mixed response containing transient and sustained components. The transient component was inhibited by amiloride whilst the sustained component showed a variable sensitivity to capsazepine. We also found that more IB4+ cells responded to capsaicin and, on average, gave rise to a larger magnitude of response than small IB4- neurons, consistent with their higher prevalence and greater amplitude of vanilloid receptor 1 (TRPV1)-like acid responses. The increase in intracellular Ca(2+) induced by capsaicin was also slightly greater in IB4+ neurons and in these cells its magnitude correlated with the level of TRPV1 immunoreactivity. Our data suggest that acid-sensing ion channels (ASICs) and TRPV1 are the major acid-sensitive receptors in small IB4- neurons, whilst TRPV1 is the predominant one in IB4+ neurons. Because ASIC-like responses were approximately 10-fold more sensitive to changes in H(+) than TRPV1-like responses, we speculate that small IB4- rather than IB4+ neurons play an essential role in sensing acid. Our results also highlight differences in capsaicin responses between IB4+ and IB4- small neurons and reveal the close link between capsaicin responses and levels of TRPV1 expression.

The effects of treatment with antibodies to transforming growth factor beta1 and beta2 following spinal cord damage in the adult rat.

We recently showed axonal ingrowth into fibronectin (FN) mats implanted into the spinal cord. However, little axonal growth was found from FN mats into intact spinal cord. Previous research has shown that this is due in part to astrocytosis around an area of CNS damage. Antibodies to transforming growth factor beta (TGFbeta) can diminish this astrocytosis. TGFbeta also has effects on macrophages and Schwann cells, both of which infiltrate the spinal cord following damage. We examined the axonal, Schwann cell, and macrophage infiltration into FN mats as well as the level of astrocytosis and chondroitin sulfate proteoglycan NG2 around FN implants incubated in TGFbeta antibodies and implanted into a lesion cavity in the spinal cord. We also examined the effects of applying TGFbeta antibodies to a spinal cord hemisection site. Anti-TGFbeta1 within FN mats resulted in extensive cavitation, with the area of damage being larger than the original lesion. Cavitation was also seen following application of anti-TGFbeta1 to a spinal cord hemisection site. No cavitation was seen following saline, non-immune IgG or anti-TGFbeta2 treatment. However, anti-TGFbeta2 treatment did result in diminished axonal growth and Schwann cell and macrophage infiltration. Around the implant site, anti-TGFbeta2 treatment resulted in a reduction in the level of astrocytosis but had not effect on levels of NG2. Similar effects were seen following anti-TGFbeta2 application to spinal cord hemisection sites. The results suggest that anti-TGFbeta1 exacerbates secondary damage by preventing the anti-inflammatory effect of endogenous TGFbeta1. Anti-TGFbeta2 did not enhance axonal regeneration in this model but did slightly reduce astrocytosis.

Protective role of vanilloid receptor type 1 in HCl-induced gastric mucosal lesions in rats.

BACKGROUND: Effects of vanilloid-receptor agonists and antagonists on HCl-induced gastric lesions in rats were investigated to elucidate the role of vanilloid receptor type 1 (VR1) in gastric mucosal defense mechanisms. METHODS: Gastric lesions in rats were evaluated after intragastric administration of 0.6 N HCl. The localization of VR1 in the stomach was investigated immunohistochemically. RESULTS: Intragastric administration of capsaicin inhibited the formation of gastric lesions in a dose-dependent manner (0.1-2.5 mg/kg). The functional VR1 antagonists ruthenium red and capsazepine markedly aggravated HCl-induced gastric lesions in rats. The gastroprotective effect of capsaicin was attenuated by ruthenium red or capsazepine. It is reported that resiniferatoxin, [6]-gingerol and lafutidine are compounds that activate VR1 and/or capsaicin-sensitive afferent neurons. These compounds significantly inhibited the formation of HCl-induced gastric lesions, and their gastroprotective effects were inhibited by treatment with ruthenium red. The immunohistochemical studies revealed that nerve fibers expressing VR1 exist along gastric glands in the mucosa, around blood vessels in the submucosa, in the myenteric plexus, and in the smooth muscle layers, especially the circular muscle layer. CONCLUSION: The results of this study suggest that VR1 plays a protective role in the gastric defensive mechanism in rats.