Wenlong Huang

TRPV1, but not P2X3, requires cholesterol for its function and membrane expression in rat nociceptors

We examined the importance of membrane cholesterol for the function and expression of TRPV1 (vanilloid receptor subtype 1) and P2X3 in adult rat dorsal root ganglion (DRG) neurons. Cholesterol, an essential component of lipid rafts, was depleted using methyl β‐cyclodextrin (MCD). We found that MCD significantly reduced TRPV1‐mediated capsaicin‐ and proton‐activated currents. By contrast, inward currents activated by α,β‐methylene ATP (α,β‐meATP), a non‐hydrolysable ATP analogue, were not altered. Immunoreactivity for TRPV1, but not P2X3, in the plasma membrane was markedly reduced by MCD. A reduction of TRPV1 protein in membrane fractions was found following cholesterol depletion. Our data show that the level of cholesterol determines the activity and the amount of membrane TRPV1, suggesting that TRPV1 might be localized in cholesterol‐rich microdomains in nociceptors. The differential dependence on the membrane cholesterol of TRPV1 and P2X3 may have physiological significance in nociception during inflammation.

TRPC4 in Rat Dorsal Root Ganglion Neurons Is Increased after Nerve Injury and Is Necessary for Neurite Outgrowth

Canonical transient receptor potential (TRPC) receptors are Ca2+-permeable cation channels that have a variety of physiological functions and may be involved in neuronal development and plasticity. We investigated the expression profile of TRPC channels in adult rat dorsal root ganglia (DRG) after nerve injury and examined the role of TRPC4 in neurite outgrowth in cultured DRG neurons. Sciatic nerve transection and microinjection of dibutyryl cAMP were employed to induce axonal regeneration in vivo. TRPC4 mRNA was significantly increased whereas TRPC1, TRPC3, TRPC6, and TRPC7 remained unaltered after nerve injury or dibutyryl-cAMP microinjection. The increases in TRPC4 transcript and protein were transient with maximal levels reached at 2 or 7 days, respectively. In addition, TRPC4 transcript in ND7/23 and NDC cells, hybrid cell lines derived from neonatal DRG and neuroblastoma, was substantially increased on differentiation, characterized by neurite outgrowth. In adult DRG, TRPC4 immunoreactivity was found in small and large neurons, and nerve injury increased the number of TRPC4-immunoreactive cells, particularly in large neurons. TRPC4 immunoreactivity was present in growth cones at various stages of DRG neurite outgrowth in vitro. Suppression of TRPC4 by a specific small interfering RNA or antisense significantly reduced the length of neurites in cultured DRG neurons. Expression of short hairpin RNA significantly down-regulated TRPC4 protein level and shortened neurite lengths in differentiated ND7/23 cells. The reduction in neurite lengths in ND7/23 cells was rescued by overexpression of human TRPC4. Our results suggest that TRPC4 contributes to axonal regeneration after nerve injury.

Docosahexaenoic Acid Prevents White Matter Damage after Spinal Cord Injury

We have previously shown that the omega-3 fatty acid docosahexaenoic acid (DHA) significantly improves several histological and behavioral measures after spinal cord injury (SCI). White matter damage plays a key role in neurological outcome following SCI. Therefore, we examined the effects of the acute intravenous (IV) administration of DHA (250u2009nmol/kg) 30u2009min after thoracic compression SCI in rats, alone or in combination with a DHA-enriched diet (400u2009mg/kg/d, administered for 6 weeks post-injury), on white matter pathology. By 1 week post-injury, the acute IV DHA injection led to significantly reduced axonal dysfunction, as indicated by accumulation of β-amyloid precursor protein (-55% compared to vehicle-injected controls) in the dorsal columns. The loss of cytoskeletal proteins following SCI was also significantly reduced. There were 43% and 73% more axons immunoreactive for non-phosphorylated 200-kD neurofilament in the ventral white matter and ventrolateral white matter, respectively, in animals receiving DHA injections than vehicle-injected rats. The acute DHA treatment also led to a significant improvement in microtubule-associated protein-2 immunoreactivity. By 6 weeks, damage to myelin and serotonergic fibers was also reduced. For some of the parameters measured, the combination of DHA injection and DHA-enriched diet led to greater neuroprotection than DHA injection alone. These findings demonstrate the therapeutic potential of DHA in SCI, and clearly indicate that this fatty acid confers significant protection to the white matter.

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 24u2003h. 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 1u2003day but increased to 73% at 3u2003days and 81% at 1u2003month. Neuronal loss was also seen 5u2003mm rostral and caudal to the epicentre, but was not significant until 3u2003days. 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 4u2003weeks 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 3u2003days and declined at 7u2003days. 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.

Improved Outcome after Peripheral Nerve Injury in Mice with Increased Levels of Endogenous Omega-3 Polyunsaturated Fatty Acids

Functional recovery after a peripheral nerve injury (PNI) is often poor. There is a need for therapies that protect neurons against injury and enhance regeneration. Omega-3 polyunsaturated fatty acids (PUFAs) have been shown to have therapeutic potential in a variety of neurological disorders, including acute traumatic injury. The objective of this study was to assess the neuroprotective and pro-regenerative potential of ω-3 PUFAs in PNI. We investigated this in mice that express the fat-1 gene encoding for ω-3 fatty acid desaturase, which leads to an increase in endogenous ω-3 PUFAs and a concomitant decrease in ω-6 PUFAs. Dorsal root ganglion (DRG) neurons from wild-type or fat-1 mice were subjected to a mechanical strain or hypoxic injury, and cell death was assessed using ethidium homodimer-1 labeling. The fat-1 background appears to confer robust neuroprotection against both injuries. We then examined the early functional and morphological changes in wild-type and fat-1 mice after a sciatic nerve crush. An accelerated functional recovery 7 d after injury was seen in fat-1 mice when assessed using von Frey filaments and the sciatic nerve functional index. These observations were also mapped to changes in injury-related markers. The injury-induced expression of ATF-3 was decreased in the DRG of fat-1 mice, whereas the axons detected 6 mm distal to the crush were increased. Fat-1 animals also had some protection against muscle atrophy after injury. In conclusion, both in vitro and in vivo experiments support the idea that a higher endogenous ω-3 PUFA could lead to beneficial effects after a PNI.

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.

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 1u2003day 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.

The acute administration of eicosapentaenoic acid is neuroprotective after spinal cord compression injury in rats

The aim of the present study was to investigate the effects of treatment with eicosapentaenoic acid (EPA) after spinal cord compression injury in adult rats. Saline or EPA (250 nmol/kg) was administered intravenously 30 min after compression injury. Locomotor recovery was assessed daily using the BBB open-field locomotor score. One week after injury, animals were sacrificed and the spinal cord tissue containing the compression epicenter, and the adjacent rostral and caudal segments, was immunostained using specific markers for neurons, oligodendrocytes, axonal injury, and macrophages/microglia. Administration of EPA resulted in decreased axonal injury and increased neuronal and oligodendrocyte survival, in the lesion epicenter and adjacent tissue. The behavioural assessment mirrored the neuroprotective effects and showed a significantly improved functional recovery in animals treated with EPA compared to the saline-treated controls over the 7-day period. These observations suggest that EPA has neuroprotective properties when administered after spinal cord trauma.

Schwann cell-specific JAM-C-deficient mice reveal novel expression and functions for JAM-C in peripheral nerves

Junctional adhesion molecule‐C (JAM‐C) is an adhesion molecule expressed at junctions between adjacent endothelial and epithelial cells and implicated in multiple inflammatory and vascular responses. In addition, we recently reported on the expression of JAM‐C in Schwann cells (SCs) and its importance for the integrity and function of peripheral nerves. To investigate the role of JAM‐C in neuronal functions further, mice with a specific deletion of JAM‐C in SCs (JAM‐C SC KO) were generated. Compared to wild‐type (WT) controls, JAM‐C SC KO mice showed electrophysiological defects, muscular weakness, and hypersensitivity to mechanical stimuli. In addressing the underlying cause of these defects, nerves from JAM‐C SC KO mice were found to have morphological defects in the paranodal region, exhibiting increased nodal length as compared to WTs. The study also reports on previously undetected expressions of JAM‐C, namely on perineural cells, and in line with nociception defects of the JAM‐C SC KO animals, on finely myelinated sensory nerve fibers. Collectively, the generation and characterization of JAM‐C SC KO mice has provided unequivocal evidence for the involvement of SC JAM‐C in the fine organization of peripheral nerves and in modulating multiple neuronal responses.—Colom, B., Poitelon, Y., Huang, W., Woodfin, A., Averill, S., Del Carro, U., Zambroni, D., Brain, S. D., Perretti, M., Ahluwalia, A., Priestley, J. V., Chavakis, T., Imhof, B. A., Feltri, M. L., Nourshargh, S. Schwann cell‐specific JAM‐C‐deficient mice reveal novel expression and functions for JAM‐C in peripheral nerves. FASEB J. 26, 1064–1076 (2012). www.fasebj.org

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.