Chemical and mechanical nerve root insults induce differential behavioral sensitivity and glial activation that are enhanced in combination

Abstract

Both chemical irritation and mechanical compression affect radicular pain from disc herniation. However, relative effects of these insults on pain symptoms are unclear. This study investigated chemical and mechanical contributions for painful cervical nerve root injury. Accordingly, the C7 nerve root separately underwent chromic gut exposure, 10gf compression, or their combination. Mechanical allodynia was assessed, and glial reactivity in the C7 spinal cord tissue was assayed at days 1 and 7 by immunohistochemistry using GFAP and OX-42 as markers of astrocytes and microglia, respectively. Both chromic gut irritation and 10gf compression produced ipsilateral increases in allodynia over sham (p<0.048); combining the two insults significantly (p<0.027) increased ipsilateral allodynia compared to either insult alone. Behavioral hypersensitivity was also produced in the contralateral forepaw for all injuries, but only the combined insult was significantly increased over sham (p<0.031). Astrocytic activation was significantly increased over normal (p<0.001) in the ipsilateral dorsal horn at 1 day after either compression or the combined injury. By day 7, GFAP-reactivity was further increased for the combined injury compared to day 1 (p<0.001). In contrast, spinal OX-42 staining was generally variable, with only mild activation at day 1. By day 7 after the combined injury, there were significant (p<0.003) bilateral increases in OX-42 staining over normal. Spinal astrocytic and microglial reactivity follow different patterns after chemical root irritation, compression, and a combined insult. The combination of transient compression and chemical irritation produces sustained bilateral hypersensitivity, sustained ipsilateral spinal astrocytic activation and late onset bilateral spinal microglial activation.

Fig. 1

Average mechanical allodynia in the ipsilateral forepaw for sham (○), chromic exposure (○), 10gf compression (□) and chromic+10gf compression (●). Allodynia is measured by the number of paw withdrawals for stimulation with each von Frey filament: (A) 1.4g, (B) 2g, and (C) 4g. Chromic exposure produced a slight increase in mechanical allodynia that was significant over sham (p<0.041) on day 1 for testing with the 2g and 4g filaments and on day 3 for testing with all filaments. By day 5 chromic exposure was not significantly different than sham. Following 10gf compression, ipsilateral allodynia was sustained, displaying significant elevation over sham on day 7 (p<0.047). Chromic+10gf compression produced significant increases in ipsilateral allodynia over chromic exposure (p<0.01), 10gf compression (p<0.033) and sham (p<0.043). Asterisks indicate significant difference between chromic+10gf compression and sham (*), chromic exposure (**) and 10gf compression (***). Pound sign (#) indicates significant difference between 10gf compression and sham, and delta (δ) indicates significant difference between chromic exposure and sham.

Fig. 2

Average mechanical allodynia in the contralateral forepaw for sham (○), chromic exposure (○), 10gf compression (□) and chromic+10gf compression (●) for (A) 1.4 g, (B) 2g, and (C) 4g von Frey filaments. Chromic exposure and 10gf compression produced slight increases in allodynia over sham. However, chromic+10gf compression produced increased contralateral allodynia compared to sham, chromic exposure and 10gf compression, for testing with the (A) 1.4g, (B) 2g, and (C) 4g filaments. Asterisks indicate significant differences between chromic+10gf compression and sham (*), chromic exposure (**) and 10gf compression (***). Pound sign (#) indicates significant difference between 10gf compression and sham.

Fig. 3

Representative ipsilateral (B, D, F, H, J, L) and contralateral (C, E, G, I, K, M) C7 spinal cord sections stained against GFAP after chromic exposure (B, C, H, I), 10gf compression (D, E, J, K) or chromic+10gf compression (F, G, L, M) on day 1 (B–G) or day 7 (H–M) following injury. A matching normal naive sample (A) was assigned baseline levels of staining. Scale bar shown in (A) is 100μm and applies to all panels.

Fig. 4

Automated densitometry results quantifying percentage of positive pixels reactive for GFAP staining, relative to normal levels at day 1 and day 7 following chromic exposure, 10gf compression, chromic+10gf compression and sham. Significant (# p<0.001) elevations over normal were detected for ipsilateral 10gf compression and chromic+10gf compression at day 1. Ipsilateral 10gf compression was also significantly elevated over sham, chromic exposure, and contralateral expression at day 1 (* p<0.022). All injury groups were elevated in the ipsilateral dorsal horn over normal at day 7 (# p<0.044) with both 10gf compression and chromic+10gf compression also elevated over contralateral expression (* p<0.003). Ipsilateral GFAP staining at day 7 for chromic+10gf compression was significantly greater than the expression at day 1 in the ipsilateral dorsal horn, the expression at day 7 following chromic exposure and the expression at day 7 following sham (* p<0.001).

Fig. 5

Representative ipsilateral (B, D, F, H, J, L) and contralateral (C, E, G, I, K, M) C7 spinal cord sections stained against OX-42 after chromic exposure (B, C, H, I), 10gf compression (D, E, J, K) or chromic+10gf compression (F, G, L, M) on day 1 (B–G) or day 7 (H–M) following injury. A matching normal naive sample (A) was assigned baseline levels of staining. Scale bar shown in (A) is 100μm and applies to all panels.

Fig. 6

Automated densitometry results quantifying relative OX-42 staining at day 1 and day 7 following chromic exposure, 10gf compression, chromic+10gf compression and sham. At day 1, reactivity in the bilateral dorsal horns for all injuries was not significantly different from sham or normal. By day 7, ipsilateral OX-42 staining following 10gf compression or chromic+10gf compression was significantly increased compared to day 1 (* p<0.001). These day 7 increases were also significant compared to normal (# p<0.001), sham (* p<0.001), and day 7 chromic exposure (* p<0.004). Contralateral increases were also significant compared to normal following chromic+10gf compression (# p=0.003) and day 1 chromic+10gf compression (* p=0.008).