Could an endoneurial endothelial crosstalk between Wnt/β-catenin and Sonic Hedgehog pathways underlie the early disruption of the infra-orbital blood-nerve barrier following chronic constriction injury?

BackgroundBlood–nerve barrier disruption is pivotal in the development of neuroinflammation, peripheral sensitization, and neuropathic pain after peripheral nerve injury. Activation of toll-like receptor 4 and inactivation of Sonic Hedgehog signaling pathways within the endoneurial endothelial cells are key events, resulting in the infiltration of harmful molecules and immunocytes within the nerve parenchyma. However, we showed in a previous study that preemptive inactivation of toll-like receptor 4 signaling or sustained activation of Sonic Hedgehog signaling did not prevent the local alterations observed following peripheral nerve injury, suggesting the implication of another signaling pathway.MethodsUsing a classical neuropathic pain model, the infraorbital nerve chronic constriction injury (IoN-CCI), we investigated the role of the Wnt/β-catenin pathway in chronic constriction injury-mediated blood–nerve barrier disruption and in its interactions with the toll-like receptor 4 and Sonic Hedgehog pathways. In the IoN-CCI model versus control, mRNA expression levels and/or immunochemical detection of major Wnt/Sonic Hedgehog pathway (Frizzled-7, vascular endothelial-cadherin, Patched-1 and Gli-1) and/or tight junction proteins (Claudin-1, Claudin-5, and Occludin) readouts were assessed. Vascular permeability was assessed by sodium fluorescein extravasation.ResultsIoN-CCI induced early alterations in the vascular endothelial-cadherin/β-catenin/Frizzled-7 complex, shown to participate in local blood–nerve barrier disruption via a β-catenin-dependent tight junction protein downregulation. Wnt pathway also mediated a crosstalk between toll-like receptor 4 and Sonic Hedgehog signaling within endoneurial endothelial cells. Nevertheless, preemptive inhibition of Wnt/β-catenin signaling before IoN-CCI could not prevent the downregulation of key Sonic Hedgehog pathway readouts or the disruption of the infraorbital blood–nerve barrier, suggesting that Sonic Hedgehog pathway inhibition observed following IoN-CCI is an independent event responsible for blood–nerve barrier disruption.ConclusionA crosstalk between Wnt/β-catenin- and Sonic Hedgehog-mediated signaling pathways within endoneurial endothelial cells could mediate the chronic disruption of the blood–nerve barrier following IoN-CCI, resulting in increased irreversible endoneurial vascular permeability and neuropathic pain development

Freezing of Enkephalinergic Functions by Multiple Noxious Foci: A Source of Pain Sensitization?

BACKGROUND:The functional significance of proenkephalin systems in processing pain remains an open question and indeed is puzzling. For example, a noxious mechanical stimulus does not alter the release of Met-enkephalin-like material (MELM) from segments of the spinal cord related to the stimulated area of the body, but does increase its release from other segments. METHODOLOGY/PRINCIPAL FINDINGS:Here we show that, in the rat, a noxious mechanical stimulus applied to either the right or the left hind paw elicits a marked increase of MELM release during perifusion of either the whole spinal cord or the cervico-trigeminal area. However, these stimulatory effects were not additive and indeed, disappeared completely when the right and left paws were stimulated simultaneously. CONCLUSION/SIGNIFICANCE:We have concluded that in addition to the concept of a diffuse control of the transmission of nociceptive signals through the dorsal horn, there is a diffuse control of the modulation of this transmission. The "freezing" of Met-enkephalinergic functions represents a potential source of central sensitization in the spinal cord, notably in clinical situations involving multiple painful foci, e.g. cancer with metastases, poly-traumatism or rheumatoid arthritis

Early alterations of Hedgehog signaling pathway in vascular endothelial cells after peripheral nerve injury elicit blood-nerve barrier disruption, nerve inflammation, and neuropathic pain development

Changes in the nerve's microenvironment and local inflammation resulting from peripheral nerve injury participate in nerve sensitization and neuropathic pain development. Taking part in these early changes, disruption of the blood–nerve barrier (BNB) allows for infiltration of immunocytes and promotes the neuroinflammation. However, molecular mechanisms engaged in vascular endothelial cells (VEC) dysfunction and BNB alterations remain unclear. In vivo, BNB permeability was assessed following chronic constriction injury (CCI) of the rat sciatic nerve (ScN) and differential expression of markers of VEC functional state, inflammation, and intracellular signaling was followed from 3 hours to 2 months postinjury. Several mechanisms potentially involved in functional alterations of VEC were evaluated in vitro using human VEC (hCMEC/D3), then confronted to in vivo physiopathological conditions. CCI of the ScN led to a rapid disruption of endoneurial vascular barrier that was correlated to a decreased production of endothelial tight-junction proteins and an early and sustained alteration of Hedgehog (Hh) signaling pathway. In vitro, activation of Toll-like receptor 4 in VEC downregulated the components of Hh pathway and altered the endothelial functional state. Inhibition of Hh signaling in the ScN of naive rats mimicked the biochemical and functional alterations observed after CCI and was, on its own, sufficient to evoke local neuroinflammation and sustained mechanical allodynia. Alteration of the Hh signaling pathway in VEC associated with peripheral nerve injury, is involved in BNB disruption and local inflammation, and could thus participate in the early changes leading to the peripheral nerve sensitization and, ultimately, neuropathic pain development

Mycolactone displays anti-inflammatory effects on the nervous system

International audienceBackground.Mycolactone is a macrolide produced by the skin pathogen Mycobacterium ulcerans, with cytotoxic, analgesic and immunomodulatory properties. The latter were recently shown to result from mycolactone blocking the Sec61-dependent production of pro-inflammatory mediators by immune cells. Here we investigated whether mycolactone similarly affects the inflammatory responses of the nervous cell subsets involved in pain perception, transmission and maintenance. We also investigated the effects of mycolactone on the neuroinflammation that is associated with chronic pain in vivo.Methodology/ Principle findings.Sensory neurons, Schwann cells and microglia were isolated from mice for ex vivo assessment of mycolactone cytotoxicity and immunomodulatory activity by measuring the production of proalgesic cytokines and chemokines. In all cell types studied, prolonged (>48h) exposure to mycolactone induced significant cell death at concentrations >10 ng/ml. Within the first 24h treatment, nanomolar concentrations of mycolactone efficiently suppressed the cell production of pro-inflammatory mediators, without affecting their viability. Notably, mycolactone also prevented the pro-inflammatory polarization of cortical microglia. Since these cells critically contribute to neuroinflammation, we next tested if mycolactone impacts this pathogenic process in vivo. We used a rat model of neuropathic pain induced by chronic constriction of the sciatic nerve. Here, mycolactone was injected daily for 3 days in the spinal canal, to ensure its proper delivery to spinal cord. While this treatment failed to prevent injury-induced neuroinflammation, it decreased significantly the local production of inflammatory cytokines without inducing detectable cytotoxicity.Conclusion/ Significance.The present study provides in vitro and in vivo evidence that mycolactone suppresses the inflammatory responses of sensory neurons, Schwann cells and microglia, without affecting the cell viability. Together with previous studies using peripheral blood leukocytes, our work implies that mycolactone-mediated analgesia may, at least partially, be explained by its anti-inflammatory properties

Anti-inflammatory effects of mycolactone on primary DRG and Schwann cells.

<p>Production of CCL-2 (A), IL-6 (B) and TNF-α (C) by mouse dorsal root ganglion (DRG) cells exposed to subtoxic doses of mycolactone (ML) or equivalent volume of vehicle (DMSO) for 30 min prior to 16 h of stimulation with 1 μg/ml LPS. Controls (NA) are vehicle-treated, non-activated cells. (D) IL-6 production by Schwann cells (SCs) exposed to ML or vehicle (DMSO) for 30 min prior to stimulation with 1 μg/m LPS + 20 ng/ml IFN-γ or not (NA). (E) Flow cytometry analysis of TLR4 surface expression by SCs exposed to increasing doses of ML for 16 h. Data are mean values of OD or MFI ± SEM of triplicates, and are representative of two independent experiments.</p

Intrathecal injection of mycolactone triggers a decrease of pro-inflammatory cytokines in spinal cord of Sham rats.

<p>(A) Experimental procedure: chronic constriction injury (CCI) was induced in rats by partial ligation of the sciatic nerve. At day 2 (D2) post-operation, rats were treated daily by intrathecal injections of mycolactone (ML) or vehicle (Veh) during 3 days and were sacrificed at D5. Sham-operated rats were submitted to the same procedure without CCI. Ipsilateral DRGs from lumbar regions 4 to 6 (L4-L6) and ipsilateral dorsal horn of the spinal cord were collected. (B-G) Expression levels of the pro-inflammatory mediators Il-1β, TIMP-1, IL-6, IFN-γ and IL-2 in the spinal cord (SpC) or in the dorsal root ganglion (DRG), 5 days post CCI or Sham treatment, in Vehicle or ML injected rats. Mean fold changes ± SEM compared to sham treated rats injected with vehicle (n = 6–9). Statistics: Mann whitney, * p<0.05, ** p<0.01, *** p<0.001. (H) Colocalization of Dapi and TUNEL stainings in the ipsilateral region of spinal cord slices from rats injected with DMSO vehicle (left) or ML (middle) daily during three days via intrathecal route. TUNEL positive controls are spinal cord slices from rats injected with vehicle, treated with DNase before staining. Scale bar = 50μm.</p

Cytotoxicity of mycolactone on PNS and CNS cell subsets.

<p>(A-B) Cytotoxicity of mycolactone (ML) on dorsal root ganglion (DRG) neurons, identified by β-III tubulin staining. (A) Proportion of TUNEL⁺ DRG neurons, relative to the total number of DRG neurons, after 24 h of exposure to increasing doses of ML. Red lines indicate mean percentages. Data are from two independent experiments, with at least 10 acquisition fields per dose, and 200 cells per dose. Statistics: Mann whitney, * p<0.05, ** p<0.01, *** p<0.001. (B) Viability of DRG neurons after 24 or 48 h of exposure to ML expressed as percentages relative to vehicle-treated controls. Data are from two independent experiments, with at least 10 acquisition fields per dose, and 200 cells per dose (C-F) Cell viability, as assessed by MTT reduction, of primary mouse Schwann cells incubated with ML or vehicle for 48 h (C), primary cortical astrocytes for 72 h (D), cortical neurons for 72 h (E) and microglia for 48 h (F). IC₅₀ indicates the concentration of ML leading to 50% cell death, compared to vehicle-treated controls. Data are mean percentages ± SD of triplicates, and are representative of three independent experiments.</p

Mycolactone suppresses the production of pro-inflammatory mediators by activated microglia.

<p>IL-6 (A) and TNF-α (B) production by primary mouse cortical microglia exposed to mycolactone (ML) or vehicle for 16 h, prior to a 8 h activation with 100 ng/ml LPS. (C) Flow cytometry analysis of intracellular NOS-2 in primary cortical microglia pre-treated with ML for 30 min, prior to 16 h activation with 100 ng/ml LPS + 20 ng/ml IFN-γ, in presence of ML. (D) Flow cytometry analysis of surface expression of TLR4 (black) and IFN-γ receptor (CD119, gray) in microglia exposed to ML for 16 h. Data are means IL-6 or TNF-α levels ± SEM (A-B), mean fluorescence intensity ± SEM (C) and mean percentage of suppression compared to vehicle (D) of duplicates, and are representative of two independent experiments.</p