Novel Use of Dual Anti-Inflammatory Therapy to Overcome Drug Resistance and Improve Functional Recovery Following Spinal Cord Injury

Over 1.2 million Americans are currently living with a traumatic spinal cord injury (SCI). Despite the need for effective therapies, there are currently no proven effective treatments that can improve recovery of function in SCI patients. Many therapeutic compounds have shown promise in preclinical models of SCI, but all of these have fallen short in clinical trials. P-glycoprotein (Pgp) is an active transporter expressed on capillary endothelial cell membranes at the blood-spinal cord barrier (BSCB). Pgp limits passive diffusion of blood-borne drugs into the CNS, by actively extruding drugs from the endothelial cell membrane. Pgp can become pathologically up-regulated, thus greatly impeding therapeutic drug delivery (‘multidrug resistance’). Importantly, many drugs that have been evaluated for the treatment of SCI are Pgp substrates. We hypothesized that Pgp-mediated drug resistance diminishes the delivery and efficacy of neuroprotective drugs following SCI. We observed a progressive, spatial spread of Pgp overexpression within the injured spinal cord. To assess Pgp function, we examined spinal cord uptake of systemically-delivered riluzole, a drug that is currently being evaluated in clinical trials as an SCI intervention. Blood-to-spinal cord riluzole penetration was reduced following SCI in wild-type but not Pgp-null rats, highlighting a critical role for Pgp in mediating spinal cord drug resistance after injury. Others have shown that pro-inflammatory signaling drives Pgp up-regulation in cancer and epilepsy. We have detected inflammation in both acutely- and chronically-injured spinal cord tissue. We therefore evaluated the ability of the dual COX-/5-LOX inhibitor licofelone to attenuate Pgp-mediated drug resistance following SCI. Licofelone treatment both reduced spinal cord Pgp levels and enhanced spinal cord riluzole bioavailability following SCI. Thus, we propose that licofelone may offer a new combinatorial treatment strategy to enhance spinal cord drug delivery following SCI. Additionally, we assessed the ability of licofelone, riluzole, or both to enhance recovery of locomotor function following SCI. We found that licofelone treatment conferred a significant improvement in hindlimb function that was sustained through the end of the study. In contrast, riluzole did not improve functional outcome. We therefore conclude that licofelone holds promise as a potential neuroprotective intervention for SCI.

TRPV1 Channels Contribute to Behavioral Hypersensitivity and Spontaneous Activity in Nociceptors After Spinal Cord Injury

A majority of persons who have sustained spinal cord injury (SCI) develop chronic pain. While most investigators have assumed that the critical mechanisms underlying neuropathic pain after SCI are restricted to the central nervous system (CNS), recent studies showed that contusive SCI results in a large increase in spontaneous activity in primary nociceptors, which is correlated significantly with mechanical allodynia and thermal hyperalgesia. Upregulation of ion channel transient receptor vanilloid 1 (TRPV1) has been observed in the dorsal horn of the spinal cord after SCI, and reduction of SCI-induced hyperalgesia by a TRPV1 antagonist has been claimed. However, the possibility that SCI enhances TRPV1 expression and function in nociceptors has not been tested. I produced contusive SCI at thoracic level T10 in adult, male rats and harvested lumbar (L4/L5) dorsal root ganglia (DRG) from sham-treated and SCI rats 3 days and 1 month after injury, as well as from age-matched naive control rats. Whole-cell patch clamp recordings were made from small (soma diameter <30 >μm) DRG neurons 18 hours after dissociation. Capsaicin-induced currents were significantly increased 1 month, but not 3 days, after SCI compared to neurons from control animals. In addition, Ca2+ transients imaged during capsaicin application were significantly greater 1 month after SCI. Western blot experiments indicated that expression of TRPV1 protein in DRG is also increased 1 month after SCI. A major role for TRPV1 channels in pain-related behavior was indicated by the ability of a specific TRPV1 antagonist, AMG9810, to reverse SCI-induced hypersensitivity of hindlimb withdrawal responses to heat and mechanical stimuli. Similar reversal of behavioral hypersensitivity was induced by intrathecal delivery of oligodeoxynucleotides antisense to TRPV1, which knocked down TRPV1 protein and reduced capsaicin-evoked currents. TRPV1 knockdown also decreased the incidence of spontaneous activity in dissociated nociceptors after SCI. Limited activation of TRPV1 was found to induce prolonged repetitive firing without accommodation or desensitization, and this effect was enhanced by SCI. These data suggest that SCI enhances TRPV1 expression and function in primary nociceptors, increasing the excitability and spontaneous activity of these neurons, thus contributing to chronic pain after SCI.

External validation study of a clinical prediction rule for ambulation outcomes after traumatic spinal cord injury

The main objective of this study was to determine the external validity of a clinical prediction rule developed by the European Multicenter Study on Human Spinal Cord Injury (EM-SCI) to predict the ambulation outcomes 12 months after traumatic spinal cord injury. Data from the North American Clinical Trials Network (NACTN) data registry with approximately 500 SCI cases were used for this validity study. The predictive accuracy of the EM-SCI prognostic model was evaluated using calibration and discrimination based on 231 NACTN cases. The area under the receiver-operating-characteristics curve (ROC) curve was 0.927 (95% CI 0.894 – 0.959) for the EM-SCI model when applied to NACTN population. This is lower than the AUC of 0.956 (95% CI 0.936 – 0.976) reported for the EM-SCI population, but suggests that the EM-SCI clinical prediction rule distinguished well between those patients in the NACTN population who were able to achieve independent ambulation and those who did not achieve independent ambulation. The calibration curve suggests that higher the prediction score is, the better the probability of walking with the best prediction for AIS D patients. In conclusion, the EM-SCI clinical prediction rule was determined to be generalizable to the adult NACTN SCI population.^

A new feature extraction method for signal classification applied to cat spinal cord signals

In the spinal cord of the anesthetized cat, spontaneous cord dorsum potentials (CDPs) appear synchronously along the lumbo-sacral segments. These CDPs have different shapes and magnitudes. Previous work has indicated that some CDPs appear to be specially associated with the activation of spinal pathways that lead to primary afferent depolarization and presynaptic inhibition. Visual detection and classification of these CDPs provides relevant information on the functional organization of the neural networks involved in the control of sensory information and allows the characterization of the changes produced by acute nerve and spinal lesions. We now present a novel feature extraction approach for signal classification, applied to CDP detection. The method is based on an intuitive procedure. We first remove by convolution the noise from the CDPs recorded in each given spinal segment. Then, we assign a coefficient for each main local maximum of the signal using its amplitude and distance to the most important maximum of the signal. These coefficients will be the input for the subsequent classification algorithm. In particular, we employ gradient boosting classification trees. This combination of approaches allows a faster and more accurate discrimination of CDPs than is obtained by other methods.

A computational model coupling mechanics and electrophysiology in spinal cord injury

Traumatic brain injury and spinal cord injury have recently been put under the spotlight as major causes of death and disability in the developed world. Despite the important ongoing experimental and modeling campaigns aimed at understanding the mechanics of tissue and cell damage typically observed in such events, the differenti- ated roles of strain, stress and their corresponding loading rates on the damage level itself remain unclear. More specif- ically, the direct relations between brain and spinal cord tis- sue or cell damage, and electrophysiological functions are still to be unraveled. Whereas mechanical modeling efforts are focusing mainly on stress distribution and mechanistic- based damage criteria, simulated function-based damage cri- teria are still missing. Here, we propose a new multiscale model of myelinated axon associating electrophysiological impairment to structural damage as a function of strain and strain rate. This multiscale approach provides a new framework for damage evaluation directly relating neuron mechanics and electrophysiological properties, thus provid- ing a link between mechanical trauma and subsequent func- tional deficits.

A data-globe and immersive virtual reality environment for upper limb rehabilitation after spinal cord injury

While a number of virtual data-gloves have been used in stroke, there is little evidence about their use in spinal cord injury (SCI). A pilot clinical experience with nine SCI subjects was performed comparing two groups: one carried out a virtual rehabilitation training based on the use of a data glove, CyberTouch combined with traditional rehabilitation, during 30 minutes a day twice a week along two weeks; while the other made only conventional rehabilitation. Furthermore, two functional indexes were developed in order to assess the patient’s performance of the sessions: normalized trajectory lengths and repeatability. While differences between groups were not statistically significant, the data-glove group seemed to obtain better results in the muscle balance and functional parameters, and in the dexterity, coordination and fine grip tests. Related to the indexes that we implemented, normalized trajectory lengths and repeatability, every patient showed an improvement in at least one of the indexes, either along Y-axis trajectory or Z-axis trajectory. This study might be a step in investigating new ways of treatments and objective measures in order to obtain more accurate data about the patient’s evolution, allowing the clinicians to develop rehabilitation treatments, adapted to the abilities and needs of the patients.

A computational model coupling mechanics and electrophysiology in spinal cord injury

Traumatic brain injury and spinal cord injury have recently been put under the spotlight as major causes of death and disability in the developed world. Despite the important ongoing experimental and modeling campaigns aimed at understanding the mechanics of tissue and cell damage typically observed in such events, the differentiated roles of strain, stress and their corresponding loading rates on the damage level itself remain unclear. More specifically, the direct relations between brain and spinal cord tissue or cell damage, and electrophysiological functions are still to be unraveled. Whereas mechanical modeling efforts are focusing mainly on stress distribution and mechanistic-based damage criteria, simulated function-based damage criteria are still missing. Here, we propose a new multiscale model of myelinated axon associating electrophysiological impairment to structural damage as a function of strain and strain rate. This multiscale approach provides a new framework for damage evaluation directly relating neuron mechanics and electrophysiological properties, thus providing a link between mechanical trauma and subsequent functional deficits

Signal changes in the spinal cord of the rat after injection of formalin into the hindpaw: Characterization using functional magnetic resonance imaging

Changes in metabolism and local circulation occur in the spinal cord during peripheral noxious stimulation. Evidence is presented that this stimulation also causes signal intensity alterations in functional magnetic resonance images of the spinal cord during formalin-induced pain. These results indicate the potential of functional magnetic resonance imaging in assessing noninvasively the extent and intensity of spinal cord excitation in this well characterized pain model. Therefore, the aim of this study was to establish functional magnetic resonance imaging as a noninvasive method to characterize temporal changes in the spinal cord after a single injection of 50 μl of formalin subcutaneously into the hindpaw of the anesthetized rat. This challenge produced a biphasic licking activity in the freely moving conscious animal. Images of the spinal cord were acquired within 2 min, enabling monitoring of the site and the temporal evolution of the signal changes during the development of formalin-induced hyperalgesia without the need of any surgical procedure. The time course of changes in the spinal cord functional image in the isoflurane-anesthetized animal was similar to that obtained from behavioral experiments. Also, comparable physiological data, control experiments, and the inhibition of a response through application of the local anesthetic agent lidocaine indicate that the signal changes observed after formalin injection were specifically related to excitability changes in the relevant segments of the lumbar spinal cord. This approach could be useful to characterize different models of pain and hyperalgesia and, more importantly, to evaluate effects of analgesic drugs.

Reelin controls position of autonomic neurons in the spinal cord

Mutation of the reeler gene (Reln) disrupts neuronal migration in several brain regions and gives rise to functional deficits such as ataxic gait and trembling in the reeler mutant mouse. Thus, the Reln product, reelin, is thought to control cell–cell interactions critical for cell positioning in the brain. Although an abundance of reelin transcript is found in the embryonic spinal cord [Ikeda, Y. & Terashima, T. (1997) Dev. Dyn. 210, 157–172; Schiffmann, S. N., Bernier, B. & Goffinet, A. M. (1997) Eur. J. Neurosci. 9, 1055–1071], it is generally thought that neuronal migration in the spinal cord is not affected by reelin. Here, however, we show that migration of sympathetic preganglionic neurons in the spinal cord is affected by reelin. This study thus indicates that reelin affects neuronal migration outside of the brain. Moreover, the relationship between reelin and migrating preganglionic neurons suggests that reelin acts as a barrier to neuronal migration.

Agmatine reverses pain induced by inflammation, neuropathy, and spinal cord injury

Antagonists of glutamate receptors of the N-methyl-d-aspartate subclass (NMDAR) or inhibitors of nitric oxide synthase (NOS) prevent nervous system plasticity. Inflammatory and neuropathic pain rely on plasticity, presenting a clinical opportunity for the use of NMDAR antagonists and NOS inhibitors in chronic pain. Agmatine (AG), an endogenous neuromodulator present in brain and spinal cord, has both NMDAR antagonist and NOS inhibitor activities. We report here that AG, exogenously administered to rodents, decreased hyperalgesia accompanying inflammation, normalized the mechanical hypersensitivity (allodynia/hyperalgesia) produced by chemical or mechanical nerve injury, and reduced autotomy-like behavior and lesion size after excitotoxic spinal cord injury. AG produced these effects in the absence of antinociceptive effects in acute pain tests. Endogenous AG also was detected in rodent lumbosacral spinal cord in concentrations similar to those previously detected in brain. The evidence suggests a unique antiplasticity and neuroprotective role for AG in processes underlying persistent pain and neuronal injury.

Segment-specific pattern of sympathetic preganglionic projections in the chicken embryo spinal cord is altered by retinoids

Sympathetic preganglionic neurons exhibit segment-specific projections. Preganglionic neurons located in rostral spinal segments project rostrally within the sympathetic chain, those located in caudal spinal segments project caudally, and those in midthoracic segments project either rostrally or caudally in segmentally graded proportions. Moreover, rostrally and caudally projecting preganglionic neurons are skewed toward the rostral and caudal regions, respectively, of each midthoracic segment. The mechanisms that establish these segment-specific projections are unknown. Here we show that experimental manipulation of retinoid signaling in the chicken embryo alters the segment-specific pattern of sympathetic preganglionic projections and that this effect is mediated by the somitic mesoderm. Application of exogenous retinoic acid to a single rostral thoracic somite decreases the number of rostrally projecting preganglionic neurons at that level. Conversely, disrupting endogenous synthesis of retinoic acid in a single caudal thoracic somite increases the number of rostrally projecting preganglionic neurons at that level. The number of caudally projecting neurons does not change in either case, indicating that the effect is specific for rostrally projecting preganglionic neurons. These results indicate that the sizes of the rostrally and caudally projecting populations may be independently regulated by different factors. Opposing gradients of such factors along the longitudinal axis of the thoracic region of the embryo could be sufficient, in combination, to determine the segment-specific identity of preganglionic projections.

Preferential synaptic relationships between substance P-immunoreactive boutons and neurokinin 1 receptor sites in the rat spinal cord

Substance P plays an important role in the transmission of pain-related information in the dorsal horn of the spinal cord. Recent immunocytochemical studies have shown a mismatch between the distribution of substance P and its receptor in the superficial laminae of the dorsal horn. Because such a mismatch was not observed by using classical radioligand binding studies, we decided to investigate further the issue of the relationship between substance P and its receptor by using an antibody raised against a portion of the carboxyl terminal of the neurokinin 1 receptor and a bispecific monoclonal antibodies against substance P and horseradish peroxidase. Light microscopy revealed a good correlation between the distributions of substance P and the neurokinin 1 receptor, both being localized with highest densities in lamina I and outer lamina II of the spinal dorsal horn. An ultrastructural double-labeling study, combining preembedding immunogold with enzyme-based immunocytochemistry, showed that most neurokinin 1 receptor immunoreactive dendrites were apposed by substance P containing boutons. A detailed quantitative analysis revealed that neurokinin 1 receptor immunoreactive dendrites received more appositions and synapses from substance P immunoreactive terminals than those not expressing the neurokinin 1 receptor. Such preferential innervation by substance P occurred in all superficial dorsal horn laminae even though neurokinin 1 receptor immunoreactive dendrites were a minority of the total number of dendritic profiles in the above laminae. These results suggest that, contrary to the belief that neuropeptides act in a diffuse manner at a considerable distance from their sites of release, substance P should act on profiles expressing the neurokinin 1 receptor at a short distance from its site of release.

Cloning of the cDNA encoding the urotensin II precursor in frog and human reveals intense expression of the urotensin II gene in motoneurons of the spinal cord

Urotensin II (UII) is a cyclic peptide initially isolated from the caudal neurosecretory system of teleost fish. Subsequently, UII has been characterized from a frog brain extract, indicating that a gene encoding a UII precursor is also present in the genome of a tetrapod. Here, we report the characterization of the cDNAs encoding frog and human UII precursors and the localization of the corresponding mRNAs. In both frog and human, the UII sequence is located at the C-terminal position of the precursor. Human UII is composed of only 11 amino acid residues, while fish and frog UII possess 12 and 13 amino acid residues, respectively. The cyclic region of UII, which is responsible for the biological activity of the peptide, has been fully conserved from fish to human. Northern blot and dot blot analysis revealed that UII precursor mRNAs are found predominantly in the frog and human spinal cord. In situ hybridization studies showed that the UII precursor gene is actively expressed in motoneurons. The present study demonstrates that UII, which has long been regarded as a peptide exclusively produced by the urophysis of teleost fish, is actually present in the brain of amphibians and mammals. The fact that evolutionary pressure has acted to conserve fully the biologically active sequence of UII suggests that the peptide may exert important physiological functions in humans.

Tonic nicotinic modulation of serotoninergic transmission in the spinal cord

The spinal serotoninergic projection from the raphe magnus has been shown to modulate nociceptive inputs, and activation of this projection mediates nicotine-elicited analgesia. Here, we investigate the interactions between cholinergic and serotoninergic systems in the spinal cord, by conducting serotonin [5-hydroxytryptamine (5-HT)] efflux experiments on mouse spinal slices. At least three spinal populations of nicotinic receptors are distinguished that affect 5-HT release. The first could be directly located on serotoninergic terminals, is insensitive to nanomolar concentrations of methyllicaconitine (MLA), and may be subjected to a basal (not maximal) cholinergic tone. The second is tonically and maximally activated by endogenous acetylcholine, insensitive to nanomolar concentrations of MLA, and present on inhibitory neurons. The last is also present on inhibitory neurons but is sensitive to nanomolar concentrations of MLA and not tonically activated by acetylcholine. Multiple nicotinic acetylcholine receptor populations thus differentially exert tonic or not tonic control on 5-HT transmission in the spinal cord. These receptors may be major targets for nicotine effects on antinociception. In addition, the presence of a tonic nicotinic modulation of 5-HT release indicates that endogenous acetylcholine plays a role in the physiological regulation of descending 5-HT pathways to the spinal cord.