Neuroprotective effects of 3α-DIOL corrective treatment against PAC-induced alterations in sciatic nerves and intraplantar skin.

<p>(<b>A</b>-<b>C</b>) Chartbars show NF200 (<b>A</b>) or CNPase (<b>C</b>) expression level in sciatic nerve sections or IENF density in intraplantar skin sections (<b>B</b>) dissected from (VEH_crem + VEH_hpc)-, (PAC + VEH_hpc)-, (VEH_crem + 3α-DIOL)- and (PAC + 3α-DIOL)-treated rats. (<b>A</b>) Each value is expressed as mean (+ SEM) of actual counts for NF200-positive fibers detected in sciatic nerve sections. (<b>B</b>) Each value is expressed as mean (+ SEM) of IENF density (counts for PGP9.5-positive terminals) detected in intraplantar skin sections. (<b>C</b>) Each value is expressed as percent (+ SEM) of CNPase-positive cells bodies detected in sciatic nerve sections of control (VEH_crem + VEH_hpc)-treated rats. n=8 per group. *** <i>p</i><0.001 vs (VEH_crem + VEH_hpc), ### <i>p</i><0.001 vs (PAC + VEH_hpc).</p

Comparison of IENF density, NF-200 or CNPase expression in vehicle- and PAC-treated rat nerves.

<p>(<b>A</b>) Comparative analysis of NF200-immunostaining density (actual counts for NF200-positive fibers) detected in sciatic nerve sections dissected from vehicle- and PAC-treated rats. (<b>B</b>) Comparative analysis of IENF density (counts for PGP9.5-positive terminals) measured in vehicle- and PAC-treated rat intraplantar skin sections. (<b>C</b>) Comparative analysis of the numbers of CNPase-positive Schwann cell bodies detected in sciatic nerve sections dissected from vehicle- and PAC-treated rats. Each value is expressed as percent (+ SEM) of CNPase-positive cells bodies detected in sciatic nerve sections of control (vehicle-treated) rats. n=8 per group. *** <i>p</i><0.001.</p

Photomicrographs of sagittal sections of sciatic nerves (A,B,E,F) or hind paw intraplantar skins (C,D) dissected from vehicle (A,C,E)- and PAC (B,D,F)-treated rats.

<p>Nerve sections were labeled with the monoclonal NF200 antibody (<b>A</b>,<b>B</b>) or with the monoclonal anti-CNPase (<b>E</b>,<b>F</b>) revealed with Alexa-488-conjugated donkey anti-mouse. (<b>C</b>,<b>D</b>) Intraplantar skin sections were labeled with the polyclonal anti-PGP9.5 revealed with FITC-conjugated goat anti-rabbit. White arrows indicated intraepidermal nerve fibers. Scale bar=50µm.</p

PAC effects on rat sciatic nerve action potential (NPA) peak amplitude and conduction velocity.

<p>(<b>A</b>) Mean traces of biphasic NAPs recorded from animals treated with vehicle (<i>black curve</i>) or PAC (<i>grey curve</i>). The nerve was stimulated with a supra-maximal pulse potential (1 V) as indicated by the protocol trace (<i>lower trace</i>). The arrows 1-3 indicate the beginning of the stimulation artifact (1) and the onset of NAP in control (2) and PAC (3) conditions giving a calculated NAP CV_latency in this case of 39.6 and 23.2 m/s for control and PAC, respectively. Note the reduction of NAP peak amplitude in PAC condition (1.2 mV versus 2.9 mV in controls). (<b>B</b>,<b>C</b>) NAP conduction velocity (<b>B</b>) and peak amplitude (<b>C</b>) histograms of statistical data obtained for vehicle- and PAC-treated rats (n=8 for each condition). ** <i>p</i><0.01, *** <i>p</i><0.005.</p

Effects of 3α-DIOL (4 mg/kg/2 days) corrective (A-E) or prophylactic (F-J) treatment on PAC-induced neuropathic pain symptoms.

<p>(<b>A</b>-<b>C</b>,<b>F</b>-<b>H</b>) Action of 3α-DIOL against PAC-induced mechanical allodynia (<b>A</b>,<b>F</b>) and hyperalgesia (<b>B</b>,<b>C</b>,<b>G</b>,<b>H</b>). Chartbars show the mean + SEM of the percentages of paw withdrawal responses to mechanical stimulation by von Frey filament 4 g (<b>A</b>,<b>F</b>), 15 g (<b>B</b>,<b>G</b>) or 26 g (<b>C</b>,<b>H</b>) (n=8 per group). (<b>D</b>,<b>I</b>) Effect of 3α-DIOL against PAC-evoked cold-allodynia. (<b>E</b>,<b>J</b>) 3α-DIOL effects on the heat thermal nociceptive thresholds of vehicle- and PAC-treated rats. Each bar represents the mean + SEM of 6 observations in each of 8 rats. Non-parametric Mann-Whitney <i>U</i> test was used for the analysis of the von Frey test results and two-way repeated measures ANOVAs followed by Newman-Keuls <i>post </i><i>hoc</i> comparisons were used for acetone and Plantar Tests. Statistical differences between controls and each treatment group at each testing day are shown. * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.005. * vs (VEH_crem + VEH_hpc), # vs (PAC + VEH_hpc).</p

Dose- and injection frequency-dependent effects of corrective 3α-DIOL treatment on the mechanical nociceptive thresholds of control- and PAC-treated rats.

<p>Corrective treatment every 2- (<b>A</b>) or 4-days (<b>B</b>) consisted in starting 3α-DIOL (2 or 4 mg/kg) or VEH_hpc i.p. administrations 2 days after the end of PAC treatment. Threshold values represent responses to 26 g von Frey filament (% ± SEM). Non-parametric Mann-Whitney <i>U</i> test was used. Statistical differences between controls and each treatment group at each testing day are shown. n=6 per group; * <i>p</i><0.05. * compared to VEH_crem+VEH_hpc; # compared to PAC+VEH_hpc.</p

Photomicrographs of sagittal sections of sciatic nerves (A-D,I-L) or intraplantar skins (E-H) dissected from (VEH_crem + VEH_hpc)(A,E,I)-, (PAC + VEH_hpc)(B,F,J)-, (VEH_crem + 3α-DIOL)(C,G,K)- or (PAC + 3α-DIOL)(D,H,L)-treated rats.

<p>Nerve sections were labeled with the monoclonal NF200 antibody (<b>A</b>-<b>D</b>) or with the monoclonal anti-CNPase (<b>I</b>-<b>L</b>) revealed with Alexa-488-conjugated donkey anti-mouse. (<b>E</b>-<b>H</b>) Intraplantar skin sections were labeled with the polyclonal anti-PGP9.5 revealed with FITC-conjugated goat anti-rabbit. White arrows indicated intraepidermal nerve fibers. Scale bar, 50 µm.</p

Curative effects of 3α-DIOL against PAC-induced reduction of sciatic nerve action potential parameters (CV_latency: A; CV_peak: B and peak amplitude: C).

<p>Histograms of normalized CV_latency and CV_peak show their reduction by PAC and recovery by 3α-DIOL treatment (<b>A</b>,<b>B</b>). Mean CV values were calculated as % of the mean CV obtained from vehicle-treated rats. (<b>C</b>) Recovery from PAC-induced NAP peak amplitude decrease by 3α-DIOL treatment. Each value is the mean (+SEM) of NAP peak amplitude obtained from 8 rat sciatic nerves per each group investigated. ** <i>p</i><0.01, *** <i>p</i><0.005. * vs (VEH_crem + VEH_hpc), # vs (PAC + VEH_hpc).</p

Effect of PAC treatment on the rat mechanical (A-C) and thermal (D, E) nociceptive threshold.

<p>(<b>A</b>-<b>C</b>) Time-course of mechanical allodynia (<b>A</b>) and hyperalgesia (<b>B</b>,<b>C</b>) induced by PAC treatment. Graphs show the mean + SEM of the percentages of paw withdrawal responses to mechanical stimulation by von Frey filament 4 g (<b>A</b>), 15 g (<b>B</b>) or 26 g (<b>C</b>) (n=8 per group). (<b>D</b>,<b>E</b>) Effects of PAC treatment on rat cold (<b>D</b>) or heat (<b>E</b>) thermal nociceptive threshold assessed by acetone (<b>D</b>) or Plantar (<b>E</b>) tests. Each point represents the mean + SEM of 6 observations in each of 8 rats. Non-parametric Mann-Whitney <i>U</i> test was used for the analysis of the von Frey test results and one-way repeated measures ANOVAs followed by Newman-Keuls <i>post </i><i>hoc</i> comparisons were used for acetone and Plantar tests. Statistical differences between control and paclitaxel group on each testing day are shown. * <i>p</i><0.05, *** <i>p</i><0.005. </p

Xanthurenic Acid Binds to Neuronal G-Protein-Coupled Receptors That Secondarily Activate Cationic Channels in the Cell Line NCB-20

<div><p>Xanthurenic acid (XA) is a metabolite of the tryptophan oxidation pathway through kynurenine and 3-hydroxykynurenine. XA was until now considered as a detoxification compound and dead-end product reducing accumulation of reactive radical species. Apart from a specific role for XA in the signaling cascade resulting in gamete maturation in mosquitoes, nothing was known about its functions in other species including mammals. Based upon XA distribution, transport, accumulation and release in the rat brain, we have recently suggested that XA may potentially be involved in neurotransmission/neuromodulation, assuming that neurons presumably express specific XA receptors. Recently, it has been shown that XA could act as a positive allosteric ligand for class II metabotropic glutamate receptors. This finding reinforces the proposed signaling role of XA in brain. Our present results provide several lines of evidence in favor of the existence of specific receptors for XA in the brain. First, binding experiments combined with autoradiography and time-course analysis led to the characterization of XA binding sites in the rat brain. Second, specific kinetic and pharmacological properties exhibited by these binding sites are in favor of G-protein-coupled receptors (GPCR). Finally, in patch-clamp and calcium imaging experiments using NCB-20 cells that do not express glutamate-induced calcium signals, XA elicited specific responses involving activation of cationic channels and increases in intracellular Ca²⁺ concentration. Altogether, these results suggest that XA, acting through a GPCR-induced cationic channel modulatory mechanism, may exert excitatory functions in various brain neuronal pathways.</p> </div