Abstract
ObjectiveBacterial lipopolysaccharide (LPS) may contribute to the manifestation of inflammatory pain within structures of the afferent somatosensory system. LPS can induce a state of refractoriness to its own effects termed LPS tolerance. We employed primary neuro-glial cultures from rat dorsal root ganglia (DRG) and the superficial dorsal horn (SDH) of the spinal cord, mainly including the substantia gelatinosa to establish and characterize a model of LPS tolerance within these structures.MethodsTolerance was induced by pre-treatment of both cultures with 1 µg/ml LPS for 18 h, followed by a short-term stimulation with a higher LPS dose (10 µg/ml for 2 h). Cultures treated with solvent were used as controls. Cells from DRG or SDH were investigated by means of RT-PCR (expression of inflammatory genes) and immunocytochemistry (translocation of inflammatory transcription factors into nuclei of cells from both cultures). Supernatants from both cultures were assayed for tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) by highly sensitive bioassays.ResultsAt the mRNA-level, pre-treatment with 1 µg/ml LPS caused reduced expression of TNF-α and enhanced IL-10/TNF-α expression ratios in both cultures upon subsequent stimulation with 10 µg/ml LPS, i.e. LPS tolerance. SDH cultures further showed reduced release of TNF-α into the supernatants and attenuated TNF-α immunoreactivity in microglial cells. In the state of LPS tolerance macrophages from DRG and microglial cells from SDH showed reduced LPS-induced nuclear translocation of the inflammatory transcription factors NFκB and NF-IL6. Nuclear immunoreactivity of the IL-6-activated transcription factor STAT3 was further reduced in neurons from DRG and astrocytes from SDH in LPS tolerant cultures.ConclusionA state of LPS tolerance can be induced in primary cultures from the afferent somatosensory system, which is characterized by a down-regulation of pro-inflammatory mediators. Thus, this model can be applied to study the effects of LPS tolerance at the cellular level, for example possible modifications of neuronal reactivity patterns upon inflammatory stimulation.
Highlights
Nociception is the sensory response of an organism to potentially harmful stimuli
In both investigated neuro-glial structures we observed a highly significant reduction of LPS10-induced expression of tumor necrosis factor-α (TNF-α) after pre-treatment with LPS1. This result is in line with studies on peripheral macrophages demonstrating that a drastic reduction of TNF-α induction is a key read-out for endotoxin tolerance under in vivo or in vitro conditions [9–12, 1 3
The operation of such mechanisms in our dorsal root ganglia (DRG) and superficial dorsal horn (SDH) primary cultures is indicated by a tendency for increased expression of IL-10 in the LPS1/LPS10 compared to the PBS/LPS10 group (Figs. 1 and 2)
Summary
Nociception is the sensory response of an organism to potentially harmful stimuli. It protects the organism from injury and tissue damage [1]. Peripheral nociceptive nerve endings detect painful stimuli, e.g. strong heat, intense cold, mechanical and chemical stimuli. Their cell bodies are located in the dorsal root ganglia (DRG) [2]. From DRG neuronal information is forwarded via axons to the superficial dorsal horn (SDH) of the spinal cord including laminae I and II termed substantia gelatinosa [3]. DRG and SDH represent principle transfer stations
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