Glial activation in the collagenase model of nociception associated with osteoarthritis

Abstract

Background Experimental osteoarthritis entails neuropathic-like changes in dorsal root ganglia (DRG) neurons. Since glial activation has emerged as a key player in nociception, being reported in numerous models of neuropathic pain, we aimed at evaluating if glial cell activation may also occur in the DRG and spinal cord of rats with osteoarthritis induced by intra-articular injection of collagenase. Methods Osteoarthritis was induced by two injections, separated by three days, of 500 U of type II collagenase into the knee joint of rats. Movement-induced nociception was evaluated by the Knee-Bend and CatWalk tests during the following six weeks. Glial fibrillary acidic protein (GFAP) expression in satellite glial cells of the DRG was assessed by immunofluorescence and Western Blot analysis; the pattern of GFAP and activating transcription factor-3 (ATF-3) expression was also compared through double immunofluorescence analysis. GFAP expression in astrocytes and IBA-1 expression in microglia of the L3-L5 spinal cord segments was assessed by immunohistochemistry and Western Blot analysis. The effect of the intrathecal administration of fluorocitrate, an inhibitor of glial activation, on movement-induced nociception was evaluated six weeks after the first collagenase injection. Results GFAP expression in satellite glial cells of collagenase-injected animals was significantly increased six weeks after osteoarthritis induction. Double immunofluorescence showed GFAP upregulation in satellite glial cells surrounding ATF-3-positive neurons. In the spinal cord of collagenase-injected animals, an ipsilateral upregulation of GFAP and IBA-1 was also observed. The inhibition of glial activation with fluorocitrate decreased movement- and loading-induced nociception. Conclusion Collagenase-induced knee osteoarthritis leads to the development of nociception associated with movement of the affected joint and to the activation of glial cells in both the DRG and the spinal cord. Inhibition of glial cell activation by fluorocitrate decreases these osteoarthritis-associated nociceptive behaviours. These results suggest that glial cell activation may play a role in the development of chronic pain in this experimental model of osteoarthritis.

Keywords: animal model; astrocytes; fluorocitrate; microglia; satellite glial cells.

Figure 1.

Nociceptive behaviour. Nociception associated with movement and loading on the joint was evaluated by the Knee-Bend (a) and CatWalk (b) tests in saline-injected control and collagenase-injected OA animals. Knee-Bend score (a) is presented as the difference between ipsilateral and contralateral scores. CatWalk data (b) are expressed as the percentage of total ipsilateral paw print intensity (%TIPPI). Mean ± SEM, two-way ANOVA followed by Bonferroni post-hoc test for comparisons between groups at each time point. ***p < 0.001, comparisons between the control and the collagenase group.

Figure 2.

GFAP expression in the DRG. Representative images of GFAP labelling in SGCs of L4 DRG, six weeks after the first saline (a) or collagenase (b) injection; Scale bar: 50 µm. (c) Immunofluorescence analysis of GFAP expression in L3, L4 and L5 ipsilateral DRG of saline- and collagenase-injected rats, six weeks after injection; GFAP expression significantly increased at six weeks of OA. Data are presented as the percentage of GFAP-encircled neurons in L3, L4 and L5 DRG. (d) Western Blot analysis of GFAP levels in L3–L5 DRG of control and collagenase-injected rats, six weeks after injection. Results are presented as the ipsilateral/contralateral ratio of GFAP levels (GFAP/GAPDH values). Mean ± SEM. Mann–Whitney test, **p < 0.01, for comparisons between control and collagenase-injected animals.

Figure 3.

ATF-3 and GFAP double labelling. Representative images of double immunofluorescence labelling for ATF-3 and GFAP in L4 ipsilateral DRG of saline-injected (a) and collagenase-injected (b) rats, six weeks after injection. Scale bar: 50 µm. (c) The percentage of GFAP-encircled neurons within the ATF-3 positive population increased in collagenase-injected animals in comparison with control animals. Mean ± SEM. Mann–Whitney test; **p < 0.01.

Figure 4.

GFAP expression in the spinal cord. Representative images of GFAP labelling in the SC, six weeks after the first saline (a) or collagenase (b) injection. Representative images of GFAP labelling in the ipsilateral (c, e) and contralateral (d, f) dorsal horn, six weeks after the first saline (c, d) or collagenase (e, f) injection; Scale bar: 50 µm. (g) Densitometric quantification of GFAP labelling in laminae I-III of the dorsal horn of control and collagenase-injected rats six weeks after injection, presented as the ipsilateral/contralateral ratio. (h) Quantitative WB analysis of GFAP expression in the spinal cord of control and collagenase-injected rats six weeks after injection, presented as the ipsilateral/contralateral ratio of GFAP levels (GFAP/GAPDH). Mean ± SEM. Mann–Whitney test; **p < 0.01.

Figure 5.

Iba-1 expression in the Spinal Cord. Representative images of Iba-1 labelling in the SC, six weeks after the first saline (a) or collagenase (b) injection. Representative images of Iba-1 labelling in the ipsilateral (c, e) and contralateral (d, f) dorsal horn, six weeks after the first saline (c, d) or collagenase (e, f) injection; Scale bar: 50 µm. (g) Densitometric quantification of Iba-1 labelling in laminae I–III of the dorsal horn of control and collagenase-injected rats six weeks after injection, presented as the ipsilateral/contralateral ratio. (h) Quantitative WB analysis of Iba-1 expression in the spinal cord of control and collagenase-injected rats six weeks after injection, presented as the ipsilateral/contralateral ratio of Iba-1 levels (Iba-1/GAPDH). Mean ± SEM. Mann–Whitney test; **p < 0.01.

Figure 6.

Fluorocitrate administration. The effect of fluorocitrate administration (30 µL, 0.1 nM, intrathecal) on movement- and loading-induced nociception was assessed by the Knee-Bend (a) and the CatWalk (b) tests six weeks after injection of collagenase. Nociception was assessed on day 0, before the first collagenase injection, and before fluorocitrate administration for determination of baseline values (open circles); the effect of fluorocitrate or vehicle was determined 1, 2, 3, 4 and 6 h after administration (filled symbols). Mean ± SEM. Repeated measures ANOVA followed by Dunnett’s post-hoc test; *p < 0.05, **p < 0.01, ***p < 0.001.