Article Figures & Data
Figures
- Figure 1.
Nicotine alleviates mechanical allodynia and thermal hyperalgesia in SNL rats in a α7-nAChR-dependent manner. A, The timeline shows nicotine, MLA and saline injection date, and the surgery and tissue harvest date. B, C, Intraperitoneal injection of nicotine significantly reduced mechanical allodynia (B) and thermal hyperalgesia (C) from 14 to 28 d after surgery compared with that of intraperitoneal injection of saline and MLA (1 mg/kg) reversed the analgesic effect (n = 6–8/group). *p < 0.05, **p < 0.01, versus the sham group; #p < 0.05, ##p < 0.01, versus the SNL plus saline group; ^^p < 0.01, versus the SNL plus nicotine (1 mg/kg) group.
- Figure 2.
Nicotine inhibits L5 spinal nerve regeneration in the SNL model. A, Schematic of an SNL model and the cut site of the L5 spinal nerve. The red arrow shows the injection site of Dil; Dil was injected just after the L5 nerve transection. B–E, Microscope images demonstrating the regenerated L5 nerve (arrows) distal to the injury site observed on day 14 in saline group and on day 28 in the saline group, nicotine group, and nicotine plus MLA group. F–H, Representative images of L5 DRG showing Dil transported from the hindpaw on day 28 in the saline group (F), nicotine group (G), and nicotine plus MLA group (H). I–K, Double immunofluorescence labeling of Dil with CGRP (I), IB4 (J), and NF200 (K) in the L5 dorsal horn. Scale bar, 50 μm. L–N, Representative images of L5 spinal nerve proximal to the injury site showing Dil transported from the hindpaw on day 28 in the saline group (L), nicotine group (M), and nicotine plus MLA group (N). Scale bar, 100 μm.
- Figure 3.
Nicotine reduces GAP43 expression in DRG and spinal nerve after L5 nerve ligation. A–D, Representative immunofluorescence images of L5 DRG showing GAP43 (red) and neuronal marker PGP9.5 (green) expression in the sham group (A), SNL + saline group (B), SNL + nicotine group (C), and SNL + nicotine + MLA group (D). E, Immunofluorescence intensity analysis shows that the immunoreactivity of GAP43 in the L5 DRG was reduced at postoperative day 28 in the nicotine group and reversed in the nicotine plus MLA group. n = 8 sections from 3 rats per group. F, Representative Western blots and quantification of GAP43 in the above four groups. G–J, Representative immunofluorescence images of L5 spinal nerve showing GAP43 expression in the sham group (G), SNL + saline group (H), SNL + nicotine group (I), and SNL + nicotine + MLA group (J). K, Immunofluorescence intensity analysis shows that the immunoreactivity of GAP43 in the L5 spinal was reduced at postoperative day 28 in the nicotine group and reversed in nicotine plus MLA group. n = 9 sections from 3 rats per group. L, Representative Western blots and quantification of GAP43 in L5 spinal nerve in the above four groups. n = 3. *Compared with the sham group; #compared with the SNL group. Scale bar, 50 μm.
- Figure 4.
Nicotine alleviates pain behaviors and reduced GAP43 expression in the nerve crush model. A, B, Nicotine injection alleviated mechanical allodynia (A) and thermal hyperalgesia (B) in comparison with vehicle (saline solution) injection and MLA reversed this effect. n = 7/group. C, D, DRG sections from cellular regions (C) or axonal regions (D) stained for GAP43 from crush + saline group, crush + nicotine group, and crush + nicotine + MLA group. Scale bar, 100 μm. E, F, Summary data of GAP43 intensity from cellular (E) and axonal (F) regions. **p < 0.01, versus the crush plus saline group; ##p < 0.01, versus the crush plus nicotine group; ^^p < 0.01, versus the SNL plus nicotine (1 mg/kg) group.
- Figure 5.
Effect of nicotine on functional measurement of regeneration. A, The schematic and representative recording trace showing in vivo recording setup of CAPs 10 d after tibial crush injury in the saline injection, nicotine injection, and nicotine plus MLA injection groups. The CAPs were measured in response to stimulation distal to the injury site (S2) and then proximal to the crush site (S1). B, The schematic and representative recording trace showing EMG recording of gastrocnemius setup 10 d after tibial crush injury in saline injection, nicotine injection, and nicotine plus MLA injection groups. The EMG was measured in response to stimulation proximal to the crush site (S1) and distal to the injury site (S2). C, D, The ratios of early component of CAPs (C) and EMG (D) evoked with distal and proximal stimuli were measured in the above three groups, respectively. A ratio value of 1 would be expected for a normal nerve, and 0 would be expected for no regeneration of nerve. N = 6 rats/group. *p < 0.05.
- Figure 6.
Nicotine increases PTEN expression in DRG nociceptive neurons. A–D, Immunostaining shows PTEN in PGP9.5-positive neurons in sham plus saline, crush plus saline, crush plus nicotine, and crush plus nicotine plus MLA groups. Scale bar, 50 μm. E, The percentage of PTEN-positive neurons was reduced in the crush plus saline group and reversed in the crush plus nicotine groups. n = 3. *p < 0.05, versus the sham plus saline group; #p < 0.05, versus the crush plus saline group; ^p < 0.05, versus the crush plus nicotine group. F, Western blot shows the effect of nicotine on PTEN expression. n = 3. *p < 0.05, versus the sham plus saline group. G–I, Double immunofluorescence staining of PTEN with nociceptive cell markers TRPV1, IB4, and CGRP. n = 3. Scale bar, 50 μm.
- Figure 7.
Nicotine administration reduces microphage density in DRG. A–D, Immunostaining of IBA1 in the sham plus saline, crush plus saline, crush plus nicotine, and crush plus nicotine plus MLA groups in DRG. Scale bar, 50 μm. E, The IBA1 intensity shows nicotine reduced microphage density and was reversed by MLA. n = 6. ***p < 0.001, versus the sham plus saline group; ###p < 0.001, versus the crush plus saline group; ^^p < 0.01, versus the crush plus nicotine group. F, Western blot shows the effect of nicotine on IBA1 expression. n = 3. G–J, Immunostaining of IBA1 in the sham plus saline, crush plus saline, crush plus nicotine, and crush plus nicotine plus MLA groups in nerves. Scale bar, 50 μm. K, The IBA1 intensity analysis. n = 6. ***p < 0.001, versus the sham plus saline group; ###p < 0.001, versus the crush plus saline group; ^^^p < 0.001, versus the crush plus nicotine group.
- Figure 8.
The PTEN inhibitor SF1670 negates analgesic effects of nicotine. A, Diagram showing the nicotine, SF1670, and I3C injection dates, the surgery and tissue harvest dates, and the behavioral test timeline. B, C, Intraperitoneal injection of SF1670 significantly reduced mechanical allodynia (B) and thermal hyperalgesia (C) from 4 to 7 d after surgery compared with that of intraperitoneal injection of DMSO (n = 6–8/group). *p < 0.05, **p < 0.01, versus the nicotine plus SF1670 group. D–I, DRG sections from cellular regions (D–F) or axonal regions (G–I) stained for GAP43 from nicotine + DMSO group, nicotine + SF1670 group, and nicotine + I3C group. Scale bar, 100 μm. J–L, DRG sections from cellular regions stained for IBA1. M, N, Summary data of GAP43 intensity from cellular (M) and axonal (N) regions. O, Summary data of GAP43 intensity from cellular regions ***p < 0.001, versus the nicotine plus DMSO group; ##p < 0.01, versus the nicotine plus SF1670 group.
In this issue
