A new ketogenic formulation improves functional outcome and reduces tissue loss following traumatic brain injury in adult mice.

Rationale: Traumatic brain injury (TBI) leads to neurological impairment, with no satisfactory treatments available. Classical ketogenic diets (KD), which reduce reliance on carbohydrates and provide ketones as fuel, have neuroprotective potential, but their high fat content reduces compliance, and experimental evidence suggests they protect juvenile brain against TBI, but not adult brain, which would strongly limit their applicability in TBI. Methods: We designed a new-KD with a fat to carbohydrate plus protein ratio of 2:1, containing medium chain triglycerides (MCT), docosahexaenoic acid (DHA), low glycaemic index carbohydrates, fibres and the ketogenic amino acid leucine, and evaluated its neuroprotective potential in adult TBI. Adult male C57BL6 mice were injured by controlled cortical impact (CCI) and assessed for 70 days, during which they received a control diet or the new-KD. Results: The new-KD, that markedly increased plasma Beta-hydroxybutyrate (ß-HB), significantly attenuated sensorimotor deficits and corrected spatial memory deficit. The lesion size, perilesional inflammation and oxidation were markedly reduced. Oligodendrocyte loss appeared to be significantly reduced. TBI activated the mTOR pathway and the new-KD enhanced this increase and increased histone acetylation and methylation. Conclusion: The behavioural improvement and tissue protection provide proof of principle that this new formulation has therapeutic potential in adult TBI.

Serotonin Expression in the Song Circuitry of Adult Male Zebra Finches.

Serotonin is an important neurotransmitter of the brain, but its role in song control remains to be fully demonstrated. Using male zebra finches (Taeniopygia guttata) that have song learning and production capabilities, we analysed the serotonin expression levels in the song nuclei and adjacent areas (peri-song nuclei) using immunohistochemistry. Key song nuclei were identified using combinations of Hoechst, choline acetyltransferase, and a neurofilament (NN18) marker in reference to the ZEBrA atlas. Mean serotonin expression was highest in interfacial nucleus (Nif) and lower in the other song nuclei in the following order (in order of highest first): interfacial nucleus (Nif) > Area X > dorsomedial part of the intercollicular nucelus (DM) > robust nucleus of the archistriatum (RA) > lateral magnocellular nucleus of the anterior neostriatum (LMAN) > ventral respiratory group (VRG) > dorsolateral nucleus of the medial thalamus (DLM) > the nucleus HVC (proper name) > tracheosyringeal motor nucleus (nXIIts). However, the mean serotonin expression (in order of highest first) in the peri-song nuclei regions was: peri-DM > peri-nXIIts > supra-peri-HVC > peri-RA > peri-DLM > peri-Area X > infra-peri-HVC > peri-VRG > peri-LMAN > peri-Nif. Interestingly, serotoninergic fibers immunostained for serotonin or the serotonin transporter can be found as a basket-like peri-neuronal structure surrounding cholinergic cell bodies, and appear to form contacts onto dopaminergic neurones. In summary, serotonin fibers are present at discrete song nuclei, and peri-song nuclei regions, which suggest serotonin may have a direct and/or modulatory role in song control.

A Single Injection of Docosahexaenoic Acid Induces a Pro-Resolving Lipid Mediator Profile in the Injured Tissue and a Long-Lasting Reduction in Neurological Deficit after Traumatic Brain Injury in Mice.

Traumatic brain injury (TBI) can lead to life-changing neurological deficits, which reflect the fast-evolving secondary injury post-trauma. There is a need for acute protective interventions, and the aim of this study was to explore in an experimental TBI model the neuroprotective potential of a single bolus of a neuroactive omega-3 fatty acid, docosahexaenoic acid (DHA), administered in a time window feasible for emergency services. Adult mice received a controlled cortical impact injury (CCI) and neurological impairment was assessed with the modified Neurological Severity Score (mNSS) up to 28 days post-injury. DHA (500 nmol/kg) or saline were injected intravenously at 30 min post-injury. The lipid mediator profile was assessed in the injured hemisphere at 3 h post-CCI. After completion of behavioral tests and lesion assessment using magnetic resonance imaging, over 7 days or 28 days post-TBI, the tissue was analyzed by immunohistochemistry. The single DHA bolus significantly reduced the injury-induced neurological deficit and increased pro-resolving mediators in the injured brain. DHA significantly reduced lesion size, the microglia and astrocytic reaction, and oxidation, and decreased the accumulation of beta-amyloid precursor protein (APP), indicating a reduced axonal injury at 7 days post-TBI. DHA reduced the neurofilament light levels in plasma at 28 days. Therefore, an acute single bolus of DHA post-TBI, in a time window relevant for acute emergency intervention, can induce a long-lasting and significant improvement in neurological outcome, and this is accompanied by a marked upregulation of neuroprotective mediators, including the DHA-derived resolvins and protectins.

Docosahexaenoic acid reduces microglia phagocytic activity via miR-124 and induces neuroprotection in rodent models of spinal cord contusion injury.

Microglia are activated after spinal cord injury (SCI), but their phagocytic mechanisms and link to neuroprotection remain incompletely characterized. Docosahexaenoic acid (DHA) has been shown to have significant neuroprotective effects after hemisection and compression SCI and can directly affect microglia in these injury models. In rodent contusion SCI, we demonstrate that DHA (500 nmol/kg) administered acutely post-injury confers neuroprotection and enhances locomotor recovery, and also exerts a complex modulation of the microglial response to injury. In rodents, at 7 days after SCI, the level of phagocytosed myelin within Iba1-positive or P2Y12-positive cells was significantly lower after DHA treatment, and this occurred in parallel with an increase in intracellular miR-124 expression. Furthermore, intraspinal administration of a miR-124 inhibitor significantly reduced the DHA-induced decrease in myelin phagocytosis in mice at 7 days post-SCI. In rat spinal primary microglia cultures, DHA reduced the phagocytic response to myelin, which was associated with an increase in miR-124, but not miR-155. A similar response was observed in a microglia cell line (BV2) treated with DHA, and the effect was blocked by a miR-124 inhibitor. Furthermore, the phagocytic response of BV2 cells to stressed neurones was also reduced in the presence of DHA. In peripheral monocyte-derived macrophages, the expression of the M1, but not the M0 or M2 phenotype, was reduced by DHA, but the phagocytic activation was not altered. These findings show that DHA induces neuroprotection in contusion injury. Furthermore, the improved outcome is via a miR-124-dependent reduction in the phagocytic response of microglia.

Brain Phospholipid Precursors Administered Post-Injury Reduce Tissue Damage and Improve Neurological Outcome in Experimental Traumatic Brain Injury.

Traumatic brain injury (TBI) leads to cellular loss, destabilization of membranes, disruption of synapses and altered brain connectivity, and increased risk of neurodegenerative disease. A significant and long-lasting decrease in phospholipids (PLs), essential membrane constituents, has recently been reported in plasma and brain tissue, in human and experimental TBI. We hypothesized that supporting PL synthesis post-injury could improve outcome post-TBI. We tested this hypothesis using a multi-nutrient combination designed to support the biosynthesis of PLs and available for clinical use. The multi-nutrient, Fortasyn® Connect (FC), contains polyunsaturated omega-3 fatty acids, choline, uridine, vitamins, cofactors required for PL biosynthesis, and has been shown to have significant beneficial effects in early Alzheimer's disease. Male C57BL/6 mice received a controlled cortical impact injury and then were fed a control diet or a diet enriched with FC for 70 days. FC led to a significantly improved sensorimotor outcome and cognition, reduced lesion size and oligodendrocyte loss, and it restored myelin. It reversed the loss of the synaptic protein synaptophysin and decreased levels of the axon growth inhibitor, Nogo-A, thus creating a permissive environment. It decreased microglia activation and the rise in ß-amyloid precursor protein and restored the depressed neurogenesis. The effects of this medical multi-nutrient suggest that support of PL biosynthesis post-TBI, a new treatment paradigm, has significant therapeutic potential in this neurological condition for which there is no satisfactory treatment. The multi-nutrient tested has been used in dementia patients and is safe and well tolerated, which would enable rapid clinical exploration in TBI.

Galectin-3 released in response to traumatic brain injury acts as an alarmin orchestrating brain immune response and promoting neurodegeneration.

Traumatic brain injury (TBI) is currently a major cause of morbidity and poor quality of life in Western society, with an estimate of 2.5 million people affected per year in Europe, indicating the need for advances in TBI treatment. Within the first 24 h after TBI, several inflammatory response factors become upregulated, including the lectin galectin-3. In this study, using a controlled cortical impact (CCI) model of head injury, we show a large increase in the expression of galectin-3 in microglia and also an increase in the released form of galectin-3 in the cerebrospinal fluid (CSF) 24 h after head injury. We report that galectin-3 can bind to TLR-4, and that administration of a neutralizing antibody against galectin-3 decreases the expression of IL-1ß, IL-6, TNFα and NOS2 and promotes neuroprotection in the cortical and hippocampal cell populations after head injury. Long-term analysis demonstrated a significant neuroprotection in the cortical region in the galectin-3 knockout animals in response to TBI. These results suggest that following head trauma, released galectin-3 may act as an alarmin, binding, among other proteins, to TLR-4 and promoting inflammation and neuronal loss. Taking all together, galectin-3 emerges as a clinically relevant target for TBI therapy.

A Single Dose of Docosahexaenoic Acid Increases the Functional Recovery Promoted by Rehabilitation after Cervical Spinal Cord Injury in the Rat.

Task-specific rehabilitation has been shown to promote functional recovery after acute spinal cord injury (SCI). Recently, the omega-3 polyunsaturated fatty acid, docosahexaenoic acid (DHA), has been shown to promote neuroplasticity after SCI. Here, we investigated whether the combination of a single bolus of DHA with rehabilitation can enhance the effect of DHA or rehabilitation therapy in adult injured spinal cord. We found enhanced functional improvement with DHA in combination with rehabilitation compared with either treatment alone in a rat cervical lateral hemisection SCI model. This behavioral improvement correlated with a significant sprouting of uninjured corticospinal and serotonergic fibers. We also observed that the greatest increase in the synaptic vesicle protein, synaptophysin, and the synaptic active zone protein, Bassoon, occurred in animals that received both DHA and rehabilitation. In summary, the functional, anatomical, and synaptic plasticity induced by task-specific rehabilitation can be further enhanced by DHA treatment. This study shows the potential beneficial effects of DHA combined with rehabilitation for the treatment of patients with SCI.

A nutrient combination designed to enhance synapse formation and function improves outcome in experimental spinal cord injury.

Spinal cord injury leads to major neurological impairment for which there is currently no effective treatment. Recent clinical trials have demonstrated the efficacy of Fortasyn® Connect in Alzheimer's disease. Fortasyn® Connect is a specific multi-nutrient combination containing DHA, EPA, choline, uridine monophosphate, phospholipids, and various vitamins. We examined the effect of Fortasyn® Connect in a rat compression model of spinal cord injury. For 4 or 9 weeks following the injury, rats were fed either a control diet or a diet enriched with low, medium, or high doses of Fortasyn® Connect. The medium-dose Fortasyn® Connect-enriched diet showed significant efficacy in locomotor recovery after 9 weeks of supplementation, along with protection of spinal cord tissue (increased neuronal and oligodendrocyte survival, decreased microglial activation, and preserved axonal integrity). Rats fed the high-dose Fortasyn® Connect-enriched diet for 4 weeks showed a much greater enhancement of locomotor recovery, with a faster onset, than rats fed the medium dose. Bladder function recovered quicker in these rats than in rats fed the control diet. Their spinal cord tissues showed a smaller lesion, reduced neuronal and oligodendrocyte loss, decreased neuroinflammatory response, reduced astrocytosis and levels of inhibitory chondroitin sulphate proteoglycans, and better preservation of serotonergic axons than those of rats fed the control diet. These results suggest that this multi-nutrient preparation has a marked therapeutic potential in spinal cord injury, and raise the possibility that this original approach could be used to support spinal cord injured patients.

A Single Bolus of Docosahexaenoic Acid Promotes Neuroplastic Changes in the Innervation of Spinal Cord Interneurons and Motor Neurons and Improves Functional Recovery after Spinal Cord Injury.

Docosahexaenoic acid (DHA) is an ω-3 polyunsaturated fatty acid that is essential in brain development and has structural and signaling roles. Acute DHA administration is neuroprotective and promotes functional recovery in animal models of adult spinal cord injury (SCI). However, the mechanisms underlying this recovery have not been fully characterized. Here we investigated the effects of an acute intravenous bolus of DHA delivered after SCI and characterized DHA-induced neuroplasticity within the adult injured spinal cord. We found robust sprouting of uninjured corticospinal and serotonergic fibers in a rat cervical hemisection SCI model. A mouse pyramidotomy model was used to confirm that this robust sprouting was not species or injury model specific. Furthermore, we demonstrated that corticospinal fibers sprouting to the denervated side of the cord following pyramidotomy contact V2a interneurons. We also demonstrated increased serotonin fibers and synaptophysin in direct contact with motor neurons. DHA also increased synaptophysin in rat cortical cell cultures. A reduction in phosphatase and tensin homolog (PTEN) has been shown to be involved in axonal regeneration and synaptic plasticity. We showed that DHA significantly upregulates miR-21 and downregulates PTEN in corticospinal neurons. Downregulation of PTEN and upregulation of phosphorylated AKT by DHA were also seen in primary cortical neuron cultures and were accompanied by increased neurite outgrowth. In summary, acute DHA induces anatomical and synaptic plasticity in adult injured spinal cord. This study shows that DHA has therapeutic potential in cervical SCI and provides evidence that DHA could exert its beneficial effects in SCI via enhancement of neuroplasticity. SIGNIFICANCE STATEMENT: In this study, we show that an acute intravenous injection of docosahexaenoic acid (DHA) 30 min after spinal cord injury induces neuroplasticity. We found robust sprouting of uninjured corticospinal and serotonergic fibers in a rat hemisection spinal cord injury model. A mouse pyramidotomy model was used to confirm that the robust sprouting involved V2a interneurons. We show that DHA significantly upregulates miR-21 and phosphorylated AKT, and downregulates phosphatase and tensin homolog (PTEN), which is involved in suppressing anatomical plasticity, in corticospinal neurons and in primary cortical neuron cultures. We conclude that acute DHA can induce anatomical and synaptic plasticity. This provides direct evidence that DHA could exert its beneficial effects in spinal cord injury via neuroplasticity enhancement.

Laminin promotes vascular network formation in 3D in vitro collagen scaffolds by regulating VEGF uptake.

Angiogenesis is an essential neovascularisation process, which if recapitulated in 3D in vitro, will provide better understanding of endothelial cell (EC) behaviour. Various cell types and growth factors are involved, with vascular endothelial growth factor (VEGF) and its receptors VEGFR1 and VEGFR2 key components. We were able to control the aggregation pattern of ECs in 3D collagen hydrogels, by varying the matrix composition and/or having a source of cells signalling angiogenic proteins. These aggregation patterns reflect the different developmental pathways that ECs take to form different sized tubular structures. Cultures with added laminin and thus increased expression of α6 integrin showed a significant increase (p<0.05) in VEGFR2 positive ECs and increased VEGF uptake. This resulted in the end-to-end network aggregation of ECs. In cultures without laminin and therefore low α6 integrin expression, VEGFR2 levels and VEGF uptake were significantly lower (p<0.05). These ECs formed contiguous sheets, analogous to the 'wrapping' pathway in development. We have identified a key linkage between integrin expression on ECs and their uptake of VEGF, regulated by VEGFR2, resulting in different aggregation patterns in 3D.

Docosahexaenoic acid attenuates the early inflammatory response following spinal cord injury in mice: in-vivo and in-vitro studies.

BACKGROUND: Two families of polyunsaturated fatty acid (PUFA), omega-3 (ω-3) and omega-6 (ω-6), are required for physiological functions. The long chain ω-3 PUFAs, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), have significant biological effects. In particular, DHA is a major component of cell membranes in the brain. It is also involved in neurotransmission. Spinal cord injury (SCI) is a highly devastating pathology that can lead to catastrophic dysfunction, with a significant reduction in the quality of life. Previous studies have shown that EPA and DHA can exert neuroprotective effects in SCI in mice and rats. The aim of this study was to analyze the mechanism of action of ω-3 PUFAs, such as DHA, in a mouse model of SCI, with a focus on the early pathophysiological processes. METHODS: In this study, SCI was induced in mice by the application of an aneurysm clip onto the dura mater via a four-level T5 to T8 laminectomy. Thirty minutes after compression, animals received a tail vein injection of DHA at a dose of 250 nmol/kg. All animals were killed at 24 h after SCI, to evaluate various parameters implicated in the spread of the injury. RESULTS: Our results in this in-vivo study clearly demonstrate that DHA treatment reduces key factors associated with spinal cord trauma. Treatment with DHA significantly reduced: (1) the degree of spinal cord inflammation and tissue injury, (2) pro-inflammatory cytokine expression (TNF-α), (3) nitrotyrosine formation, (4) glial fibrillary acidic protein (GFAP) expression, and (5) apoptosis (Fas-L, Bax, and Bcl-2 expression). Moreover, DHA significantly improved the recovery of limb function.Furthermore, in this study we evaluated the effect of oxidative stress on dorsal root ganglion (DRG) cells using a well-characterized in-vitro model. Treatment with DHA ameliorated the effects of oxidative stress on neurite length and branching. CONCLUSIONS: Our results, in vivo and in vitro, clearly demonstrate that DHA treatment reduces the development of inflammation and tissue injury associated with spinal cord trauma.

Omega-3 fatty acids and traumatic neurological injury: from neuroprotection to neuroplasticity?

Omega-3 polyunsaturated fatty acids (PUFAs) are compounds that have a structural role in the nervous system and are essential for neurodevelopment. Results obtained with docosahexaenoic acid and eicosapentaenoic acid show therapeutic potential in neurotrauma. Traumatic brain injury (TBI) and spinal cord injury (SCI) can lead to major disability and have a significant socioeconomic cost. Thus, there is an unmet need for acute neuroprotection and for treatments that promote neuroregeneration. Acute administration of omega-3 PUFAs after injury and dietary exposure before or after injury improve neurological outcomes in experimental SCI and TBI. The mechanisms involved include decreased neuroinflammation and oxidative stress, neurotrophic support, and activation of cell survival pathways. This review raises questions that must be addressed before successful clinical translation.

Lentiviral mediated expression of a NGF-soluble Nogo receptor 1 fusion protein promotes axonal regeneration.

Nogo receptor 1 (NgR1) mediates the inhibitory effects of several myelin-associated inhibitors (MAIs) on axonal regeneration in the central nervous system. A truncated soluble NgR1 (sNgR) has been reported to act as a decoy receptor to block the actions of MAIs. In this study, we fused the sNgR to nerve growth factor (NGF) and used NGF as a carrier to deliver sNgR to the intercellular space to neutralize MAIs. NGF in NGF-sNgR remained biologically active and induced sprouting of calcitonin gene related peptide containing axons when expressed in the spinal cord using a lentiviral vector (LV). Secreted NGF-sNgR promoted neurite outgrowth of dissociated dorsal root ganglion neurons on myelin protein substrate. In a rat dorsal column transection model, regenerating sensory axons were found to grow into the lesion cavity in animals injected with LV/NGF-sNgR, while in animals injected with LV/GFP or LV/NGF-GFP few sensory axons entered the lesion cavity. The results indicate that NGF-sNgR fusion protein can reduce the inhibition of MAIs and facilitate sensory axon regeneration. The fusion constructs may be modified to target other molecules to promote axonal regeneration and the concept may also be adapted to develop gene therapy strategies to treat other disorders.

Activation of neuronal transient receptor potential vanilloid 1 channel underlies 20-hydroxyeicosatetraenoic acid-induced vasoactivity: role for protein kinase A.

A rise in intraluminal pressure triggers vasoconstriction in resistance arteries, which is associated with local generation of the vasoconstrictor 20-hydroxyeicosatetraenoic acid (20-HETE). Importantly, dysregulation of 20-HETE synthesis and activity has been implicated in several cardiovascular disease states, including ischemic disease, hypertension, and stroke; however, the exact molecular pathways involved in mediating 20-HETE bioactivity are uncertain. We investigated whether 20-HETE activates the transient receptor potential vanilloid 1 (TRPV1) and thereby regulates vascular function and blood pressure. We demonstrate that 20-HETE causes dose-dependent increases in blood pressure, coronary perfusion pressure (isolated Langendorff), and pressure-induced constriction of resistance arteries (perfusion myography) that is substantially attenuated in TRPV1 knockout mice and by treatment with the neurokinin 1 receptor antagonist RP67580. Furthermore, we show that both channel activation (via patch-clamping of dorsal root ganglion neurons) and vessel constriction are enhanced under inflammatory conditions, and our findings indicate a predominant role for protein kinase A-mediated sensitization of TRPV1 in these phenomena. Finally, we identify a prominence of these pathway in males compared with females, an effect we relate to reduced protein kinase A-induced phosphorylation of TRPV1. 20-HETE-induced activation of TRPV1, in part, mediates pressure-induced myogenic constriction and underlies 20-HETE-induced elevations in blood pressure and coronary resistance. Our findings identify a novel vasoconstrictor 20-HETE/TRPV1 pathway that may offer potential for therapeutic targeting in cardiovascular diseases associated with elevated 20-HETE implicated in dysregulated organ blood flow, such as stroke or hypertension.

Improved outcome after spinal cord compression injury in mice treated with docosahexaenoic acid.

In this study we have characterised the locomotor recovery, and temporal profile of cell loss, in a novel thoracic compression spinal cord injury (SCI) in the mouse. We have also shown that treatment with docosahexaenoic acid (DHA) is neuroprotective in this model of SCI, strengthening the growing literature demonstrating that omega-3 polyunsaturated fatty acids are neuroprotective after SCI. Compression SCI in C57BL/6 mice was produced by placing a 10 g weight for 5 min onto a 2 mm × 1.5 mm platform applied to the dura at vertebral level T12. Mice partly recovered from complete hindlimb paralysis and by 28 days post-surgery had plateaued at an average BMS locomotor score of 4.2, equivalent to weight support with plantar stepping. During the same period, neuronal loss at the epicentre increased from 26% of ventral horn neurons by day 1, to 68% by day 28. Delayed loss of oligodendrocytes was also seen (e.g. 84% by day 28 in the dorsal columns) and microglia/macrophage activation was maximal at 7 days. In contrast, axonal damage, judged by a decrease in the non-phosphorylated form of 200 kD neurofilament, was an early event, as the loss was seen by day 1 and did not change markedly over time. Mice that received an intravenous (i.v.) injection of 500 nmol/kg DHA 30 min after SCI, showed improved locomotor recovery and, at 28 day survival, reduced neuronal, oligodendrocyte and neurofilament loss, and reduced microglia/macrophage activation. For some of these indices of SCI, enrichment of the diet with 400 mg/kg/day DHA led to further improvement. However, dietary DHA supplementation, without the initial i.v. injection, was ineffective.

Transgenic mice with high endogenous omega-3 fatty acids are protected from spinal cord injury.

Omega-3 polyunsaturated fatty acids have been shown to have therapeutic potential in a variety of neurological disorders, including acute traumatic injury of the spinal cord. We addressed the question whether the neuroprotective effect of these compounds after spinal cord injury could also be seen when their level is raised in tissues prophylactically, prior to injury. In this study we used transgenic fat-1 mice to examine whether enriching spinal cord tissue in endogenous omega-3 polyunsaturated fatty acids has an effect on the outcome after compression spinal cord injury. The results demonstrate that after thoracic compression spinal cord injury, fat-1 mice display better locomotor recovery compared with the wild-type mice on a high omega-6 diet (high omega-6 polyunsaturated fatty acids in tissues), and wild-type mice on a normal diet (controls). This is associated with a significant increase in neuronal and oligodendrocyte survival and a decrease in non-phosphorylated neurofilament loss. The protection from spinal cord injury in fat-1 mice was also correlated with a reduction in microglia/macrophage activation and in pro-inflammatory mediators. In vitro experiments in dorsal root ganglia primary sensory neurons further demonstrated that a fat-1 tissue background confers robust neuroprotection against a combined mechanical stretch and hypoxic injury. In conclusion, our studies support the hypothesis that a raised omega-3 polyunsaturated fatty acid level and an altered tissue omega-6/omega-3 ratio prior to injury leads to a much improved outcome after spinal cord injury.

Limiting spinal cord injury by pharmacological intervention.

The direct primary mechanical trauma to neurons, glia and blood vessels that occurs with spinal cord injury (SCI) is followed by a complex cascade of biochemical and cellular changes which serve to increase the size of the injury site and the extent of cellular and axonal loss. The aim of neuroprotective strategies in SCI is to limit the extent of this secondary cell loss by inhibiting key components of the evolving injury cascade. In this review we will briefly outline the pathophysiological events that occur in SCI, and then review the wide range of neuroprotective agents that have been evaluated in preclinical SCI models. Agents will be considered under the following categories: antioxidants, erythropoietin and derivatives, lipids, riluzole, opioid antagonists, hormones, anti-inflammatory agents, statins, calpain inhibitors, hypothermia, and emerging strategies. Several clinical trials of neuroprotective agents have already taken place and have generally had disappointing results. In attempting to identify promising new treatments, we will therefore highlight agents with (1) low known risks or established clinical use, (2) behavioral data gained in clinically relevant animal models, (3) efficacy when administered after the injury, and (4) robust effects seen in more than one laboratory and/or more than one model of SCI.

The spatiotemporal localization of JAM-C following sciatic nerve crush in adult rats.

JAM-C is a junctional adhesion molecule, enriched at tight junctions on endothelial and epithelial cells, and also localized to Schwann cells at junctions between adjoining myelin end loops. The role of JAM-C following peripheral nerve injury (PNI) is currently unknown. We examined the localization of JAM-C after sciatic nerve crush injury in adult rats. JAM-C immunoreactivity was present in paranodes and incisures in sham surgery control nerve, but distal to the crush injury significantly decreased at three and 14 days. JAM-C was re-expressed at 28 days and, by 56 days, was significantly increased in the distal nerve compared to controls. In a 7-mm length of sciatic nerve sampled distal to the crush site, the densities of JAM-C immunoreactive paranodes increased in the distal direction. Conversely, the densities of JAM-C immunoreactive incisures were highest immediately distal to the crush site and decreased in the more distal direction. Further analysis revealed a strong correlation between JAM-C localization and remyelination. Fifty-six days after crush injury, greater densities of JAM-C paranodes were seen compared to the nodal marker jacalin, suggesting that paranodal JAM-C precedes node formation. Our data are the first to demonstrate a potential role of JAM-C in remyelination after PNI.

Docosahexaenoic acid, but not eicosapentaenoic acid, reduces the early inflammatory response following compression spinal cord injury in the rat.

Docosahexaenoic acid (DHA, 22 : 6) and eicosapentaenoic acid (EPA, 20 : 5) are omega-3 polyunsaturated fatty acids (n-3 PUFAs) with distinct anti-inflammatory properties. Both have neuroprotective effects acutely following spinal cord injury (SCI). We examined the effect of intravenous DHA and EPA on early inflammatory events after SCI. Saline, DHA or EPA (both 250 nmol/kg) were administered 30 min after T12 compression SCI, to female Sprague-Dawley rats. DHA significantly reduced the number of neutrophils to some areas of the injured epicentre at 4 h and 24 h. DHA also reduced C-reactive protein plasma levels, whereas EPA did not significantly reduce neutrophils or C-reactive protein. Laminectomy and SCI elicited a sustained inflammatory response in the liver, which was not reversed by the PUFAs. The chemokine KC/GRO/CINC and the cytokine IL-6 provide gradients for chemotaxis of neutrophils to the epicentre. At 4 h after injury, there was a significant increase in IL-6, KC/GRO/CINC, IL-1ß and tumour necrosis factor-α in the epicentre, with a return to baseline at 24 h. Neither DHA nor EPA returned their levels to control values. These results indicate that the acute neuroprotective effects of n-3 PUFAs in rat compression SCI may be only partly attributed to reduction of some of the early inflammatory events occurring after injury.

20-Hydroxyeicosatetraenoic acid (20-HETE) is a novel activator of transient receptor potential vanilloid 1 (TRPV1) channel.

TRPV1 is a member of the transient receptor potential ion channel family and is gated by capsaicin, the pungent component of chili pepper. It is expressed predominantly in small diameter peripheral nerve fibers and is activated by noxious temperatures >42 °C. 20-Hydroxyeicosatetraenoic acid (20-HETE) is a cytochrome P-450 4A/4F-derived metabolite of the membrane phospholipid arachidonic acid. It is a powerful vasoconstrictor and has structural similarities with other TRPV1 agonists, e.g. the hydroperoxyeicosatetraenoic acid 12-HPETE, and we hypothesized that it may be an endogenous ligand for TRPV1 in sensory neurons innervating the vasculature. Here, we demonstrate that 20-HETE both activates and sensitizes mouse and human TRPV1, in a kinase-dependent manner, involving the residue Ser(502) in heterologously expressed hTRPV1, at physiologically relevant concentrations.