Glial activation in the rostroventromedial medulla promotes descending facilitation to mediate inflammatory hypersensitivity

Abstract

Substantial evidence shows that activation of glial cells in the spinal cord may promote central sensitization and pain. Descending facilitation from the rostroventromedial medulla (RVM) is a critical component in the maintenance of chronic pain states, although the precise mechanisms through which facilitation maintains pain are unclear. Here, we investigated the possibility that glial activation in the RVM could promote descending facilitation from the RVM in states of inflammatory pain. Peripheral inflammation was induced with carrageenan injected into the hindpaws of male Sprague-Dawley rats, and behavioral responses to noxious thermal and light tactile stimuli were determined. Microinjection of the glial inhibitors minocycline or fluorocitrate, or of the p38 mitogen-activated protein kinase (MAPK) inhibitor SB 203580, produced a significant and time-related attenuation of behavioral hypersensitivity resulting from hindpaw inflammation. Carrageenan-induced inflammation increased immunolabeling for microglia and astrocytes in the RVM, as well as for phosphorylated p38 MAPK. Phosphorylated p38 MAPK was found in microglia and neurons of the RVM. Inflammation-induced microglial and astrocytic activation in the RVM were attenuated by RVM microinjection of the glial inhibitors. The data show that inflammatory pain is associated with glial activation in the RVM that probably participates in driving descending pain facilitation. These findings reveal a novel site of glial modulation of inflammatory pain.

Fig. 1

A representative photomicrograph of a medullary section at the level of the rostroventromedial medulla (RVM) counterstained with Nissl stain. The microinjection sites in the RVM were visualized with the microinjection of India ink to verify proper cannula placement. Scale bar: 100 μm.

Fig. 2

Medullary sections that include the rostroventromedial medulla (RVM) obtained from rats treated with saline (A and C) or subjected to carrageenan-induced inflammation (B and D) and immunolabeled for OX-42 (microglia; A and B) and glial fibrillary acidic protein (GFAP) (astrocytes; C and D). Rats with inflammation indicate an intensification in labeling for OX-42 (B) and GFAP (D) along with changes in morphology indicative of activation of glia. Scale bar: 100 μm. Insets show magnified microglia cells (A and C) and astrocytes (B and D). Scale bar: 25 μm. (E) All RVM images are representative of multiple sections examined between −10.8 and −11.4 mm from bregma (Paxinos & Watson, 1998). The drawing represents the RVM location, including the nucleus raphe magnus and gigantocellularis pars alpha (green area). The shaded rectangle represents the location of the photomicrograph.

Fig. 3

Microinjection of the microglia inhibitor minocycline (25 μg) into the rostroventromedial medulla (RVM) produced a time-dependent attenuation of carrageenan-induced thermal hyperalgesia (A) and tactile allodynia (B). *Significant (P < 0.05) increases in responses relative to the post-carrageenan values. BL, mean baseline responses; Carr., mean post-carrageenan responses. (C) Paw volumes (mL) were measured 30 min after microinjection into the RVM of minocycline or vehicle in rats treated with carrageenan or saline. No significant difference was observed after the microinjection of minocycline into the RVM.

Fig. 4

Medullary sections obtained from rats treated with hindpaw injections of saline (A) or subjected to carrageenan-induced inflammation and receiving microinjections of vehicle (B) or 25 μg of minocycline (C) in the rostroventromedial medulla (RVM) were labeled with OX-42 for immunofluorescent visualization of microglia. Microinjection of minocycline into the RVM 3 h after hindpaw injection of carrageenan produced a reduction in immunofluorescence intensity for OX-42 along with morphological changes suggesting reduced microglial activation. Scale bar: 100 μm. (D) The top blot shows examples of the bands immunoreactive against anti-ionized calcium-binding adaptor molecule 1 (Iba1). The bottom blot shows bands immunoreactive against anti-tubulin after stripping and reprobing of the same membrane. The bar graph shows the mean levels of Iba1 normalized to tubulin. Carrageenan administration produced significantly increased levels of Iba1 as compared with saline-treated animals. Protein levels of Iba1 from animals treated with minocycline in the RVM were not significantly different from those in saline-treated animals. *P < 0.05, relative to saline/saline.

Fig. 5

Microinjection of the astrocyte inhibitor fluorocitrate (1 μg) into the rostroventromedial medulla (RVM) produced a time-dependent attenuation of carrageenan-induced thermal hyperalgesia (A) and tactile allodynia (B). *Significant (P < 0.05) increases in responses relative to the post-carrageenan values. BL, mean baseline responses; Carr., mean post-carrageenan responses. (C) Paw volumes (mL) were measured 60 min after microinjection into the RVM of fluorocitrate or vehicle in rats treated with carrageenan or saline.

Fig. 6

Medullary sections obtained from rats treated with hindpaw injections of saline (A) or subjected to carrageenan-induced inflammation and receiving microinjections of vehicle (B) or 1 μg of fluorocitrate (C) in the rostroventromedial medulla (RVM) were labeled with glial fibrillary acidic protein (GFAP) for immunofluorescent visualization of astrocytes. Microinjection of fluorocitrate into the RVM 3 h after hindpaw injection of carrageenan produced a reduction in immunofluorescence intensity for GFAP along with morphological changes suggesting reduced astrocyte activation. Scale bar: 100 μm. (D) The top blot shows examples of the bands immunoreactive against anti-GFAP. The bottom blot shows the bands immunoreactive against anti-tubulin after stripping and reprobing of the same membrane. The bar graph shows the mean levels of GFAP normalized to tubulin. Carrageenan administration produced significantly increased protein levels of GFAP as compared with saline-treated animals. Protein levels of GFAP from animals treated with fluorocitrate in the RVM were not significantly different from those in saline-treated animals. *P < 0.05, relative to saline/saline.

Fig. 7

Medullary sections were obtained from saline-treated control rats (A, C and E) and rats with carrageenan-induced inflammation (B, D and F) 3 h after injection. Sections were immunolabeled for phosphorylated p38 mitogen-activated protein kinase (MAPK) (red), and co-labeled (green) with OX-42 (microglia; A and B), glial fibrillary acidic protein (GFAP) (astrocytes; C and D) and neuronal nuclei (NeuN) (neurons; E and F). The label for phosphorylated p38 MAPK is co-localized with microglia and neurons, but not with astrocytes. Additionally, there is a visible increase in expression of microglia co-expressing phosphorylated p38 MAPK after carrageenan-induced inflammation, whereas there is no observable change in neurons expressing this marker. Scale bar: 100 μm. White arrows indicate examples of co-localization.

Fig. 8

Microinjection of the p38 mitogen-activated protein kinase (MAPK) inhibitor SB 203580 (10 μg) into the rostroventromedial medulla (RVM) produced a time-dependent attenuation of carrageenan-induced thermal hyperalgesia (A) and tactile allodynia (B). *Significant (P < 0.05) increases in responses relative to the post-carrageenan values. BL, mean baseline responses; Carr., mean post-carrageenan responses. *P = 0.05 as compared with saline. Medullary sections obtained from rats treated with hindpaw injections of saline (C) or with carrageenan-induced inflammation and receiving microinjections of vehicle (D) or 10 μg of SB 203580 (E) in the RVM were labeled with OX-42 for immunofluorescent visualization of microglia. Microinjection of SB 203580 into the RVM 3 h after hindpaw injection of carrageenan produced a reduction in immunofluorescence intensity for OX-42 along with morphological changes suggesting reduced microglial activation. Scale bar: 100 μm.