Immunofluorescent staining of TRPV1 and analysis of its expression pattern in DRG neurons at varying time points after the establishment of DMA.

<p><b>A–E</b>, representative photographs of TRPV1 staining in DRG of different groups. <b>F</b>, illustration of the percentage of TRPV1-IR neurons over total neurons in vehicle, DMA 7 d, 14 d, 21 d and 28 d group (n = 4–6). <b>G</b>, histogram for size distribution of TRPV1-IR neurons. Note the decreased distribution of TRPV1 in neurons with cross-sectional area larger than 400–500 and increased distribution of this protein within the range of 100–400 µm² (n = 4–6). <b>H</b>, comparison of the optical density due to TRPV1 immunostaining in small-sized (100–500 µm²) and medium-sized (500–1200 µm²) DRG neurons of different groups (n = 4–6). *<i>P</i><0.05, **<i>P</i><0.01 <i>vs</i>. vehicle control. Scale bar  = 100 µm.</p

Percentage of NF200-, IB4- and CGRP-IR neurons in vehicle-treated and DMA 14 d groups (n = 4–6).

<p>*<i>P</i><0.05, **<i>P</i><0.01 <i>vs</i>. vehicle group.</p

DM and DMA rat model induced by STZ injection.

<p>Time course of blood glucose concentration (<b>A</b>), body weight (<b>B</b>) and paw withdrawal threshold (<b>C</b>) alterations after STZ or citrate buffer (vehicle) treatment. Data are presented as meanS.E.M. (n = 30), **<i>P</i><0.01 <i>vs</i>. vehicle control.</p

Immunofluorescent staining of TRPV1 in plantar skin of hind paw with the progression of DMA.

<p>A–E, typical photograsph of TRPV1 immunoreactivities in vehicle and DMA model rats at varied time points. A′–C′, the magnified pictures from the rectangle areas in A–C. F, optical density analysis of TRPV1 immunoreactivities in epidermis and dermis displaying strong enhancement on DMA 7 d and 14 d. Sc, Ep and De are the abbreviation of stratum corneum, epidermis and dermis, respectively. Scale bar  = 20 µm</p

Single intrathecal application of TRPV1 antagonists alleviates mechanical allodynia and thermal hyperalgesia.

<p><b>A</b> and <b>B</b>, single application of RR and CPZ at varing doses caused differential inhibition of mechanical sensitivity of DMA rats. <b>C</b>, single application of RR and CPZ at high dose caused significant inhibition of thermal hyperalgesia in DM rats. *<i>P</i><0.05, **<i>P</i><0.01 <i>vs</i>. vehicle control of the same time. ^#<i>P</i><0.05, ^##<i>P</i><0.01 <i>vs</i>. drugs of middle dose.</p

Immunofluorescent staining of TRPV1 in spinal dorsal horn (SDH) with the progression of DMA.

<p><b>A–E</b>, representative photographs of TRPV1 staining in SDH in vehicle, DMA 7 d, 14 d, 21 d and 28 d groups. <b>F</b>, optical density analysis of TRPV1 staining in SDH showing the increased TRPV1-IR neurons on DMA 7 d and 14 d group (n = 4–6). Scale bar  = 100 µm.</p

Double labeling rate of TRPV1 immunoreactivity with that of NF200, CGRP or IB4 in DRG neurons (n = 4–6).

<p>*<i>P</i><0.05 <i>vs</i>. vehicle group.</p

Multiple intrathecal applications of TRPV1 antagonists cumulatively antagonize mechanical allodynia.

<p><b>A</b> and <b>B</b>, long-term effects of multiple applications of ruthenium red and capsazepine for consecutive 7 days (n = 6). <b>C</b> and <b>D</b>, reconstruction of the decay response diagram from <b>A</b> and <b>B</b> after the withdrawal of RR (<b>A</b>) and CPZ (<b>B</b>) (n = 6).The value in longitudinal axis was normalized by the thresholds value on DMA 20 d. The linear regressions were performed to calculate the decay rates and to evaluate the half decay times (‘Decay₅₀’), and the results are shown above the plots. *<i>P</i><0.05, **<i>P</i><0.01 <i>vs</i>. vehicle control of the same time. ^#<i>P</i><0.05, ^##<i>P</i><0.01 <i>vs</i>. drugs of middle dose.</p

The paired-pulse responses were affected by GABApentin and NASPM in different ways.

<p>(A-B) Bath application of GABApentin (50 μm) or NASPM (50 μM) has no effect on the ratio and amplitude of the paired-pulse depression (PPD) in rats with sham surgery. (C) GABApentin increased the ratio of the PPD and inhibited the amplitude of the first eEPSC in rats with CMI. (D) NASPM decreased the amplitude of the paired responses but did not affect the paired-pulse ratio in CMI rats. (F) Summarized results for the effects of GABApentin and NASPM on the paired-pulse responses in rats with sham or CMI treatment. #, NASPM vs. baseline, <i>p</i> > 0.05; ǂ, GABApentin vs. baseline, <i>p</i> > 0.05; *, GABApentin vs. baseline, <i>p</i> < 0.05; **, NASPM <i>vs</i>. baseline, <i>p</i> < 0.01.</p

Fluorescent images showing that Fos-ir neurons were highly expressed in the NTS of rats with CMI.

<p>(A) A sample slice showing Fos-expression in the NTS. (B) A magnified image of the rectangle in (A). (C-D) The synaptophysin-ir terminals and NeuN-ir neurons shown within the same area as (B). (E) The merged image from (B-D) showing that synaptophysin-ir terminals made close connection to the NeuN-ir neurons, especially to those with Fos-immunoreactivity. 10n, dorsal motor nucleus of vagus; SolC, commissural part of the NTS; SolL, lateral part of the NTS; SolM, medial part of the NTS; st, solitary tract. Scale bars: 500 μm (A) and 20 μm (B-E).</p