Noradrenergic neurons in the locus coeruleus contribute to neuropathic pain

Abstract

Current theories of neuropathic hypersensitivity include an imbalance of supraspinal inhibition and facilitation. Our overall hypothesis is that the locus coeruleus (LC), classically interpreted as a source of pain inhibition, may paradoxically result in facilitation after tibial and common peroneal nerve transection (spared sural nerve injury--SNI). We first tested the hypothesis that non-noxious tactile hind paw stimulation of the spared sural innervation territory increases neuronal activity in the LC in male rats. We observed a bilateral increase in the stimulus-evoked expression of transcription factors Fos and phosphorylated CREB (pCREB) in LC after SNI but not sham surgery; these markers of neuronal activity correlated with the intensity of tactile allodynia. We next tested the hypothesis that noradrenergic neurons contribute to the development of neuropathic pain. To selectively destroy these neurons, we delivered antidopamine-beta-hydroxylase saporin (anti-DbetaH-saporin) into the i.c.v. space 2 weeks before SNI. We found that anti-DbetaH-saporin, but not an IgG-saporin control, reduced behavioral signs of tactile allodynia, mechanical hyperalgesia, and cold allodynia from 3 to 28 days. after SNI. Our final experiment tested the hypothesis that the LC contributes to the maintenance of neuropathic pain. We performed SNI, waited 2 weeks for maximal allodynia and hyperalgesia to develop, and then administered the local anesthetic lidocaine (4%) directly into the LC parenchyma. Lidocaine reduced all behavioral signs of neuropathic pain in a reversible manner, suggesting that the LC contributes to pain facilitation. We conclude that, in addition to its well-known inhibition of acute and inflammatory pain, the LC facilitates the development and maintenance of neuropathic pain in the SNI model. Further studies are needed to determine the facilitatory pathways emanating from the LC.

Figure 1. Stimulus-induced Fos expression in the LC after SNI

Panel A: Tactile allodynia (drop in von Frey threshold), mechanical hyperalgesia (increased response to pin prick), and cold allodynia (increased response to topical acetone) in rats with spared nerve injury (SNI) or sham surgery. There was no difference in hypersensitivity among SNI rats that were later assigned to the unstimulated (US) or stimulated with non-noxious stroking of the footpad (S) condition. Panel B: The total number of Fos-immunoreactive (Fos-ir) profiles in the LC (both sides) in sham and SNI rats 2 wk following SNI. Innocuous tactile footpad stimulation increased Fos in SNI rats. Panels C, E, G, I: The number of Fos-ir profiles in 4 distinct coronal divisions throughout the rostro-caudal extent of the LC. The effect of footpad stimulation in SNI rats reached statistical significance in the -9.90 to -10.15 mm division. Panels D, F, H, J: Representative photomicrographs of stimulated Fos-ir at each coronal division of the LC in an SNI rat. CG=central gray; DMTg=dorsomedial tegmental area; g7=facial nerve; Me5=mesencephalic 5^th nucleus; mlf=medial longitudinal fasciculus; scp=superior cerebellar peduncle; SGe=supragenual nucleus. Magnification = 3X. Data is expressed as mean ± SEM. n=3-12. ★p < 0.05.

Figure 2. Fos expression in the LC after Innocuous paw stimulation and/or SNI

Representative photomicrographs of Fos expression in the LC (~ -9.90 mm from bregma) following footpad stimulation to a sham (A), or SNI rat (B), or following no stimulation of a sham (C) or SNI rat (D). Magnification = 10X.

Figure 3. Stimulus-induced pCREB expression in the LC after SNI

Panel A: The total number of pCREB-immunoreactive (pCREB-ir) profiles in the LC (both sides) in sham and SNI rats 2 wk following SNI. The representative photomicrographs (-9.90 mm from bregma) from a sham (B) and an SNI rat (C) illustrate that innocuous tactile footpad stimulation in the setting of nerve injury increased pCREB-ir. Panel D: Quantification of pCREB-ir following footpad stimulation to sham and SNI rats within each of 4 coronal divisions of the LC. Magnification = 15X. n=5-12. ★p < 0.05.

Figure 4. Pontine DβH immunoreactivity following anti-DβH-saporin

Photomicrographs depicting low (2X, Panels A-B) and high (10X, Panels C-H) power images from a representative saporin IgG control (A, C, E, G) or an anti-DβH-saporin-treated rat (B, D, F, H). Anti-DβH-saporin almost completely eliminated DβH-immunoreactivity (DβH-ir) in the LC (C vs D) but neither A5 (E vs F) nor A7 nuclei (G vs H).

Figure 5. SNI-induced allodynia and hyperagesia after anti-DβH-saporin

Line graphs in the left panels (A, C, E, G) illustrate time course data collected from the paw ipsilateral to SNI. Histograms at the right (B, D, F, H) summarize the change in behaviour (mean of day 3-28) at the ipsilateral and contralateral sides. Mechanical threshold was assessed with both von Frey hairs (A, B) and an automated machine-mounted probe (MMP, C, D). Mechanical hyperalgesia was assessed as paw withdrawal duration to a blunt pin (Pin Prick, E, F). Cold allodynia was assessed as paw withdrawal duration to topical plantar acetone (G, H). Anti-DβH-saporin reduced all behavioral signs of neuropathic pain [★p’s < 0.05, Mann-Whitney U test subsequent to Kruskal Wallis (A, B) or post-hoc Bonferroni subsequent to ANOVA (C-H)]. Values represent mean ± SEM. n=4.

Figure 6. SNI-induced allodynia and hyperalgesia after intra-LC lidocaine

Line graphs illustrating time course data for mechanical threshold assessed with either von Frey hairs (A), or an automated machine-mounted probe (MMP, B), a noxious mechanical blunt pin (prick) stimulus (C), or a cold (topical acetone) stimulus (D). When delivered 2 wk after SNI, bilateral microinjection of lidocaine (4%), but not saline, reduced all signs of neuropathic pain at the paw ipsilateral to SNI. Data is expressed as mean ± SEM. n=3-6. ★p < 0.01.

Figure 7. Microinjection sites within the LC

The Nissl section on the right illustrates a representative deposit of India ink within and above the LC (mapped to be approximately -0.8 mm from interaural). Landmarks are provided on the left. mcp=middle cerebellar peduncle; Me5=mesencephalic 5 nucleus; Mo=motor 5 nucleus; s5=sensory root of the 5^th nucleus; Tz=nucleus of the trapezoid body.