Mechanical hypersensitivity, sympathetic sprouting, and glial activation are attenuated by local injection of corticosteroid near the lumbar ganglion in a rat model of neuropathic pain

Abstract

Background and objectives: Inflammatory responses in the lumbar dorsal root ganglion (DRG) play a key role in pathologic pain states. Systemic administration of a common anti-inflammatory corticosteroid, triamcinolone acetonide (TA), reduces sympathetic sprouting, mechanical pain behavior, spontaneous bursting activity, and cytokine and nerve growth factor production in the DRG. We hypothesized that systemic TA effects are primarily due to local effects on the DRG.

Methods: Male Sprague-Dawley rats were divided into 4 groups: SNL (tight ligation and transection of spinal nerves) and normal with and without a single dose of TA injectable suspension slowly injected onto the surface of DRG and surrounding region at the time of SNL or sham surgery. Mechanical threshold was tested on postoperative days 1, 3, 5, and 7. Immunohistochemical staining examined tyrosine hydroxylase and glial fibrillary acidic protein in DRG and CD11B antibody (OX-42) in spinal cord.

Results: Local TA treatment attenuated mechanical sensitivity, reduced sympathetic sprouting in the DRG, and decreased satellite glia activation in the DRG and microglia activation in the spinal cord after SNL.

Conclusions: A single injection of corticosteroid in the vicinity of the axotomized DRG can mimic many effects of systemic TA, mitigating behavioral and cellular abnormalities induced by spinal nerve ligation. This provides a further rationale for the use of localized steroid injections clinically and provides further support for the idea that localized inflammation at the level of the DRG is an important component of the spinal nerve ligation model, commonly classified as neuropathic pain model.

Figure 1

Effect of local triamcinolone acetonide (TA) injection on mechanical pain behavior induced by spinal nerve ligation (SNL). Baseline withdrawal threshold to mechanical stimulation of the hind paws was measured in the ipsilateral paw on three measurements taken over 5 days before the surgery, and the average is plotted as the postoperative day 0 value. *, Days on which there was a significant difference between group SNL+TA and group SNL rats (one-way ANOVA). The ipsilateral reduction in threshold caused by SNL alone was significantly different from the normal group on all postoperative days; for clarity no symbols are added to indicate this.

Figure 2

Local TA injection reduced sympathetic basket formation in the DRG after SNL on postoperative day (POD) 5 and POD 7. a – c, examples of sympathetic fiber basket formation in dorsal root ganglion sections stained for tyrosine hydroxylase on POD 5. White arrows indicate examples of neurons surrounded by basket formation. Black arrows indicate sympathetic fibers innervating vascular processes. (a) Example from a SNL rat. The inset shows a magnified view of neurons surrounded by basket formation. (b) Example from a SNL+TA rat. (c) Example from a Normal+TA rat. Scale bar = 50 μm. Graphical summary for POD 5 (left) and POD 7 (right). Upper graphs give the total density of basket formations. In the lower graphs, DRG neurons were classified by diameter as small (<30 μm), medium (30-50 μm), or large (>50 μm). *, significantly different from respective SNL group.

Figure 3

Effect of local TA injection on glial activation. TOP: Sections of DRG stained for GFAP (red) and NeuN (Green). GFAP was barely detectable in normal rat DRG (f). Following spinal nerve injury, the expression of GFAP was dramatically increased on POD 1 (a), and remained very high on POD 3 (b). Applying TA locally to the axotomized DRG starting at the time of nerve injury reduced the nerve-injury induced GFAP expression at these time points (c, POD 1; d, POD 3). GFAP was barely detectable in examples from a Normal+TA rat at POD 1 (e) or later time points (not shown; see Fig. 4). Scale bar=50μm. BOTTOM: microglial activation on POD3 as measured by OX-42(CD11B antibody, a microglia marker) staining (red) in spinal cord. Panels on the top and bottom show dorsal horn portions of the spinal cord, the sides contra- and ipsi-lateral to nerve injury, respectively. Scale bar=100 μm. SNL caused increased OX-42 staining ipsilaterally (a) and, to a lesser degree, contralaterally (b). Reduced staining was seen in SNL animals that received a TA injection (c, d). Low levels of staining were observed in normal animals (e, f); or in normal animals treated with TA (not shown).

Figure 4

Quantitative summary of glial activation data exemplified in Figs. 2 and 3. Top: Time course of effect of local TA injection on satellite glia activation in the DRG, measured as percent of neurons surrounded by GFAP staining. *, Days on which there was a significant difference between SNL+TA and SNL rats (two-way ANOVA). Both SNL and SNL+TA groups were significantly different (p<0.001) from the Normal+TA group at all postoperative time points. Bottom: Summary of relative intensity of staining for the microglia marker OX 42, examined on POD 3 in spinal cord. For the normal group, ipsilateral and contralateral data were combined to a single value. * Significantly different from SNL group. # Significantly different from Normal group