Neuropeptide Y acts at Y1 receptors in the rostral ventral medulla to inhibit neuropathic pain

Abstract

Brain microinjection studies in the rat using local anesthetics suggest that the rostral ventral medulla (RVM) contributes to the facilitation of neuropathic pain. However, these studies were restricted to a single model of neuropathic pain (the spinal nerve ligation model) and to just two stimulus modalities (non-noxious tactile stimulus and heat). Also, few neurotransmitter systems have been shown to modulate descending facilitation. After either partial sciatic nerve ligation (PSNL) or spared nerve injury (SNI), we found that unilateral or bilateral microinjection of lidocaine into the RVM reduced not only mechanical allodynia (decreased threshold to von Frey hairs and/or an automated device) and mechanical hyperalgesia (increased paw lifting in response to a noxious pin), but also cold hypersensitivity (increased lifting in response to the hindpaw application of a drop of acetone). Application of a drop of water did not elicit paw withdrawal, indicating that the acetone test is indeed a measure of cold hypersensitivity. We found significant neuropeptide Y Y1-like immunoreactivity within, and lateral to, the midline RVM. Intra-RVM injection of neuropeptide Y (NPY) dose-dependently inhibited the mechanical and cold hypersensitivity associated with PSNL or SNI, an effect that could be blocked by the Y1 receptor antagonist BIBO 3304. We conclude that medullary facilitation spans multiple behavioral signs of allodynia and hyperalgesia in multiple models of neuropathic pain. Furthermore, NPY inhibits behavioral signs of neuropathic pain, possibly by acting at Y1 receptors in the RVM.

Figure 1. Microinjection sites within the RVM

Photomicrographs showing placement of A) midline single cannula tract (bregma – 10.04 mm) and B) bilateral cannula tracts (bregma – 10.80 mm) within the RVM in brain sections counterstained with cresyl violet. Section thickness = 40 μm. Brightfield photomicrographs of Y1 receptor immunoreactivity in the RVM at C) bregma – 10.08 mm and D) bregma – 10.80 mm. Scale bars = 200 μm for C, D. The boxed areas in C and D represent areas of higher magnification as shown in E and F, respectively. Scale bars = 100 μm for E, F.

Figure 2. Intra-RVM lidocaine decreases spared nerve injury (SNI)-induced allodynia

Bilateral microinjection of 2% lidocaine (A–C) or unilateral microinjection of 4% lidocaine (D–F) into the RVM. Lidocaine reversed the drop in mechanical threshold as assessed with von Frey hairs [A,D, P<0.01], and a machine-mounted filament (B, MMF, P<0.01). Lidocaine also reversed cold allodynia (C,F) assessed as the duration of response to a drop of acetone. Lidocaine did not change responses at the paw contralateral to SNI. n=6–15. Error bars = SEM. ★P<0.01, saline vs lidocaine at the ipsilateral hindpaw, Mann-Whitney U test subsequent to Kruskal Wallis (A) or post-hoc Bonferroni subsequent to ANOVA (B–C).

Figure 3. Intra-RVM NPY decreases SNI -induced allodynia

Unilateral microinjection of NPY into the midline RVM dose-dependently reduced mechanical allodynia (von Frey, Panels A–B; machine-mounted filament, Panels C–D), and cold allodynia (Panel E–F). The line graphs in panels A, C, E illustrate time course data collected from the paw ipsilateral to SNI. The histograms in panels B, D, F summarize the data obtained at the 15′ time point from the ipsilateral and contralateral sides. Numbers of animals in each group are in parenthesis. Error bars = SEM. ★P<0.05 vs saline, Mann-Whitney U test subsequent to Kruskal Wallis (A–B) or post-hoc Bonferroni subsequent to ANOVA (C–F).

Figure 4. Intra-RVM lidocaine decreases partial sciatic ligation (PSNL)-induced allodynia and hyperalgesia

Unilateral microinjection of lidocaine into the midline RVM reduced mechanical allodynia (VF, Panels A–B), mechanical hyperalgesia (Panels C–D), and cold allodynia (Panel E–F). The line graphs in panels A, C, E illustrate time course data. The histograms in panels B, D, F summarize the change in behavior from baseline (t=0) to post injection (mean of t=15–90) at the ipsilateral and contralateral sides. Lidocaine did not changes responses at the paw contralateral to PSNL. Numbers of animals in each group are in parenthesis. Error bars = SEM. ★P<0.05 vs saline, Mann-Whitney U test subsequent to Kruskal Wallis (A–B) or post-hoc Bonferroni subsequent to repeated-measures ANOVA (C–F).

Fig 5. Intra-RVM NPY decreases PSNL -induced allodynia and hyperalgesia

Unilateral microinjection of NPY into the midline RVM reduced mechanical allodynia (Panels A–B) mechanical hyperalgesia (Panels C–D), and cold allodynia (Panel E–F). The line graphs in panels A, C, E illustrate time course data collected from the paw ipsilateral to SNI. The histograms in panels B, D, F summarize the change in behavior from baseline (t=0) to post injection (mean of t=10–30) at the ipsilateral and contralateral sides. NPY did not changes responses at the paw contralateral to SNI. Numbers of animals in each group are in parenthesis. Error bars = SEM. ★P<0.05 vs saline, Mann-Whitney U test subsequent to Kruskal Wallis (A–B) or post-hoc Bonferroni subsequent to repeated-measures ANOVA (C–F).

Figure 6. Intra-RVM NPY dose-dependently decreases PSNL-induced allodynia and hyperalgesia

The line graphs in panels A and C illustrate time course data collected from the paw ipsilateral to SNI. The histograms in panels B and D summarize the change in von Frey threshold from baseline (t=0) to post injection (mean of timepoints 10–30) at the ipsilateral and contralateral sides. Error bars = SEM. Numbers of animals in each group are in parenthesis. ★P<0.05, Mann-Whitney U test subsequent to Kruskal Wallis (A–B) or post-hoc Bonferroni subsequent to repeated-measures ANOVA (C–D).

Figure 7. Intra-RVM delivery of the selective Y1 receptor antagonist BIBO3304 reverses the effects of NPY in the PSNL model

When administered 15 min before NPY (1 μg), the unilateral microinjection BIBO (1 μg) but not saline prevented the anti-allodynic (Panels A–B) and anti-hyperalgesic (Panels C–D) effects of NPY at the hindpaw ipsilateral to nerve injury. BIBO had no effect when administered alone. Numbers of animals in each group are in parenthesis. Error bars = SEM. ★ P<0.05 vs saline, *P<0.05 vs NPY alone, Mann-Whitney U test subsequent to Kruskal Wallis or post-hoc Bonferroni subsequent to ANOVA.

Figure 8. The acetone test is a behavioral measure of cold hypersensitivity

Time spent lifting the ipsilateral paw over a 60-s observation period in response to a drop of acetone (A), water (B), or to its immersion in 1-cm deep ice water (C) after sham (n=3) or SNI surgery (n=7). In contrast to acetone, a drop of water did not evoke a robust response. However, ice water produced a greater response in SNI rats as compared to Sham rats; no difference was observed at the contralateral paw. These data indicate that the response to acetone reflects cold hypersensitivity rather than mechanical allodynia. Error bars = SEM.

Figure 9. Intra-RVM delivery of NPY does not affect behaviour in animals with sham surgery

Behavior was evaluated in the ipsilateral and contralateral paws (relative to sham surgery) before and after microinjection of NPY (5 μg bilateral) into the RVM at t = 0. NPY did not change the withdrawal response to innocuous mechanical stimuli (Panels A–B), a noxious mechanical stimulus (Panel C), or a cold stimulus (Panel D). n=6.