DRG ATF3, Iba1 immunohistochemistry and sciatic nerve myelin staining following intrathecal vector injection.

<p>A) Expression of ATF3 in the L5 DRGs in rats treated with spinal nerve ligation (SNL, 2 weeks), si-Luc or si-TOR AAV5 vectors (4 weeks). B) Myelin staining of the sciatic nerve from rats treated with si-Luc, si-TOR or si-TOR' vectors (4 weeks). C) Expression of IbaI in the L5 DRGs in a naïve rat or rats received si-Luc, si-TOR or GFP-only vectors. A cervical DRG from a rat treated with si-TOR vector was also shown. Scale bar, 100 µm.</p

Expression of mTOR in lumbar DRGs following intrathecal vector injection.

<p>A) Schematic drawing of the AAV vector that encodes sh-RNA. The sequences of the two active and one control siRNA (si-Luc) are shown. B) Representative Western blots showing levels of mTOR and beta-tubulin in naïve rats or at two week following vector administration in the lumbar DRGs (L4 and L5 DRGs from both sides were pooled). C) Histogram represents the mean mTOR levels in respect to the naive group. The data are presented as mean ± SEM of 4–6 rats per group. D) Representative Western blots showing levels of mTOR and beta-tubulin at five week following vector administration in the lumbar DRGs (L4 and L5 DRGs from both sides were pooled). E) Histogram represents the mean mTOR levels in respect to the control group (si-Luc). The data are presented as mean ± SEM of 4 rats per group. F, G and H) Confocal images of mTOR (red), GFP (green) and NeuN (blue) in L5 DRG at two week following F) si-Luc, G) si-TOR and H) si-TOR' vector administration. Scale bar, 100 µm. PGK, phosphoglycerate kinase promoter; ITR, inverted terminal repeat; U6, U6 promoter.</p

Expression of GFP in different populations of neurons in the lumbar DRG.

<p>A,B C and D) GFP (green) was co-labeled with A) NF200, B) TRPV1, C) CGRP and D) IB4 (red) at two week following vector administration. Arrowheads indicate co-localization of GFP with the respective cell marker. E) Bar graph represents percentage of GFP-positive neurons co-labeled with the aforementioned markers. The data are presented as mean ± SEM of 3 rats. Scale bar, 100 µm.</p

Weight gain and nociceptive behaviors following the siRNA vectors.

<p>A) Daily weight gain of rats after receiving no vector (control), si-Luc or si-TOR vector. The mean weight gain in a two week period following vector injection is shown. B) Thermal and C) mechanical thresholds for naïve rats and rats treated with GFP, si-Luc or si-TOR vector for two weeks. D) Total number of flinches in phase I (1–9 min) and II (10–60 min) following intraplantar formalin (2.5%, 50 µL) injection. Rats received si-Luc or si-TOR vector two weeks before the formalin tests. E) Mechanical thresholds of the ipsilateral paw following unilateral L5 and L6 spinal nerve ligation. Rats received si-Luc or si-TOR vector two weeks before the SNL surgery. The data are presented as mean ± SEM of 6–8 rats per group.</p

Expression of GFP and NeuN in rat DRG following intrathecal vector injection.

<p>A,B and C) GFP (green) and NeuN (red) were co-labeled in A) lumbar, B) thoracic and C) cervical DRG at 2 week-post vector injection. D) Bar graph represents size distribution of DRG neurons with and without GFP labeling. A total of 3781 neurons from the L4, L5 and L6 DRGs of 3 animals were included. Scale bar, 250 µm.</p

Expression of mTOR in L5 DRG following intrathecal vector administration.

<p>A) Confocal images of mTOR (red) co-labeled with NF200, CGRP, TRPV1 or IB4 (green) in L5 DRG 2 weeks following the si-Luc or si-TOR vector injection. Double labeled neurons are indicated with asterisks. B) Bar graph represents percentage of mTOR-positive neurons co-labeled with the aforementioned markers. The data are presented as mean ± SEM of 3–4 rats except TRPV1 represents a single rat. C) GFP (green) and mTOR (red) in L5 DRG at 1 week following the si-Luc vector and 1, 2 and 5 weeks following the si-TOR vector injection. Scale bars, A, 50 µm and C, 100 µm.</p