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Release of interleukin 1ß from spinal microglia in the rat isolated dorsal horn Mounting evidence supports a role for inflammatory mediators released by activated spinal microglia in the maintenance of persistent pain states (Watkins & Maier, 2003). We evaluated whether activation of microglial cells in the dorsal horn released quantifiable levels of the proinflammatory cytokine Interleukin1 β (IL1 b ). We used an in vitro rat dorsal horn preparation (Malcangio & Bowery, 1996) to chemically stimulate microglial by lipopolysaccharide (LPS) . We then examined the occurrence of microglial activation in the dorsal horn by quantification of phosphorylated p38 MAP kinase (p-p38) using immunohistochemistry (IHC). Lumbar spinal cord from male Wistar rats were excised and dorsal slices with dorsal roots attached continuously superfused with oxygenated Krebs’ solution (1ml/min). Before and after stimulation three fractions were collected to assess basal and recovery IL1β levels respectively. Chemical stimulation of microglia with LPS (10μg/ml; Sigma) was applied for 8 min and superfusates assayed for IL1β content by ELISA. LPS stimulation of IL1 b release was also examined in the presence of either Tetrodotoxin (300nM; TTX; Tocris), the glial inhibitor Fluorocitrate (100μM; Sigma), the p38MAPK inhibitor SB203580 (10μM; Tocris) or the Caspase-1 inhibitor Z-VAD-FMK (10μM; Sigma). At the end of these experiments slices were prepared for IHC. Following LPS application the levels of IL1 b in dorsal horn superfusates were increased to 574%±141 above basal levels (4.6±1.1pg/8ml fraction, n=5, P<0.05). In non-stimulated dorsal horn slices the mean p-p38 immunoreactivity (IR) was 31.2±2.6 fluorescence per 100μm2 (n=6). Following LPS superfusion p-p38 IR was increased to 93.2±9.2 fluorescence per 200μm2 (n= 6, P<0.001). In the presence of Fluorocitrate, SB203580 and Z-VAD-FMK, but not TTX, no significant increase in IL1β release or p38 phosphorylation occurred above basal levels following LPS activation of microglia (n=5-7). IL1 b can be readily released in the dorsal horn following activation of microglia by LPS, via a p38 MAPK and Caspase-1 dependent mechanism. We suggest that this microglial cell-derived IL1 b may modulate central neuronal plasticity following peripheral nerve injury and spinal cord injury.
Malcangio, M. & Bowery, N.G. (1996). Pain, 66, 351 - 358. |
