4-(4- chlorophenyl)24-hydroxy-a,a-diphenyl-1-piperidinepentanenitrile hydrochloride (bx513)  (Tocris)

Journal: Anesthesiology

Article Title: Chemokine (C-C motif) Receptor 5 Is an Important Pathological Regulator in the Development and Maintenance of Neuropathic Pain

doi: 10.1097/aln.0000000000000190

Figure Lengend Snippet: Fig. 1. Expression levels of chemokine (C-C motif) ligand 3 (CCL3), chemokine (C-C motif) receptor 1 (CCR1), and chemokine (C-C motif) receptor 5 (CCR5) messenger RNA (mRNA) in the spinal cord after injury to the L5 spinal nerve. Total RNA extracted from the rat spinal cord was subjected to real-time quantitative reverse transcription polymerase chain reaction analysis for CCL3 (A), CCR5 (B), and CCR1 (C) mRNA expression after peripheral nerve injury. Bar graphs show the average fold increase in the level of mRNA expression in spinal cord sagittal hemisections compared with the mean expression level of mRNA in naive (day 0) animals. Each measurement was normalized to glyceraldehyde-3-phosphate dehydrogenase content. Data are the means ± SEM of five individual animals (*P < 0.05; vs. the naive spinal cord, one-way ANOVA post hoc Tukey test). CD11b/c-positive cells sorted from day 1 spinal cord (6,000 cells from each sample) were also subjected to real-time quantitative reverse transcrip- tion polymerase chain reaction analysis of CCL3 transcript (D). Data are the means ± SEM of nine individual animals (*P < 0.05, paired t test). Fluorescent in situ hybridization for CCL3 mRNA was performed using ViewRNA assay to visualize the transcript localization in day 1 spinal cord tissue (E–N). Spotted fluorescent signal of Fast Red represents CCL3 mRNA localization (E, F, I, J) and corresponding transmitted light images (G, H, K, L) are shown. High-magnification images of E with G and F with H indicated by the white square region in G and H are represented in M and N. Scale bars indicate 100 μm (E–L) and 20 μm (M and N).

Article Snippet: For examining the effect of CCR1 or CCR5 antagonists on rCCL3-induced tactile allodynia, preinjection (30 min before intrathecal injection of rCCL3) of the selective CCR1 antagonist BX513 hydrochloride (0.01, 0.1, and 1 pmol/10 μl; Tocris Bioscience, Bristol, United Kingdom) or the selective CCR5 antagonist maraviroc (Selzentry, Celsentri) (1, 10, and 100 pmol/10 μl; Sellek, Houston, TX) was performed.

Techniques: Expressing, Reverse Transcription, Polymerase Chain Reaction, In Situ Hybridization

Journal: Anesthesiology

Article Title: Chemokine (C-C motif) Receptor 5 Is an Important Pathological Regulator in the Development and Maintenance of Neuropathic Pain

doi: 10.1097/aln.0000000000000190

Figure Lengend Snippet: Fig. 4. Effect of chemokine (C-C motif) ligand 3 (CCL3)–neutralizing antibody, chemokine (C-C motif) receptor 1 (CCR1), and chemokine (C-C motif) receptor 5 (CCR5) antagonists on peripheral nerve injury–induced tactile allodynia. The CCL3-neutralizing antibody (NAb) or mouse immunoglobulin G 2 for isotype control (mIgG2, 20 ng in 10 μl in an injection, n = 6 for each group) was intrathecally injected once daily during the indicated time to rats with L5 spinal nerve transection (A). The CCR5 antagonist maraviroc (B, vehicle, 1, 10, and 100 pmol in 10 μl phosphate buffered saline, n = 6 for each) and the CCR1 antagonist BX513 hydrochloride (C, vehicle and 1 pmol in 10 μl phosphate buffered saline, n = 6 and 7) were intrathecally injected once daily at the indicated time to rats with L5 spinal nerve transection. The paw-withdrawal thresholds of the hind paw in response to tactile stimulation in rats were examined at the indicated time points after peripheral nerve injury. Each data point represents the mean ± SEM of the paw-withdrawal thresholds (*P < 0.05; vs. mIgG2 or vehicle group [ipsilateral side] at each time point by multiple comparison by Bonferroni test after a repeated-measure two-way ANOVA). L5 spinal cord segments from 3-day postoperative rats with intrathecal administration of neutralizing antibody or isotype IgG (D, E, H, and I), and 7-day postoperative rats with maraviroc or vehicle were subjected to immunohistochemistry using OX-42 antibody (F, G, J, and K). Scale bar indicates 100 μm.

Article Snippet: For examining the effect of CCR1 or CCR5 antagonists on rCCL3-induced tactile allodynia, preinjection (30 min before intrathecal injection of rCCL3) of the selective CCR1 antagonist BX513 hydrochloride (0.01, 0.1, and 1 pmol/10 μl; Tocris Bioscience, Bristol, United Kingdom) or the selective CCR5 antagonist maraviroc (Selzentry, Celsentri) (1, 10, and 100 pmol/10 μl; Sellek, Houston, TX) was performed.

Techniques: Control, Injection, Saline, Comparison, Immunohistochemistry