Sensitization of primary cultures from rat dorsal root ganglia with lipopolysaccharide (LPS) requires a robust inflammatory response.

OBJECTIVE: We investigated whether it is possible to induce a state of "LPS-sensitization" in neurons of primary cultures from rat dorsal root ganglia by pre-treatment with ultra-low doses of LPS. METHODS: DRG primary cultures were pre-treated with low to ultra-low doses of LPS (0.001-0.1 µg/ml) for 18 h, followed by a short-term stimulation with a higher LPS-dose (10 µg/ml for 2 h). TNF-α in the supernatants was measured as a sensitive read out. Using the fura-2 340/380 nm ratio imaging technique, we further investigated the capsaicin-evoked Ca2+-signals in neurons from DRG, which were pre-treated with a wide range of LPS-doses. RESULTS: Release of TNF-α evoked by stimulation with 10 µg/ml LPS into the supernatant was not significantly modified by pre-exposure to low to ultra-low LPS-doses. Capsaicin-evoked Ca2+-signals were significantly enhanced by pre-treatment with LPS doses being above a certain threshold. CONCLUSION: Ultra-low doses of LPS, which per se do not evoke a detectable inflammatory response, are not sufficient to sensitize neurons (Ca2+-responses) and glial elements (TNF-α-responses) of the primary afferent somatosensory system.

Gabapentinoids Suppress Lipopolysaccharide-Induced Interleukin-6 Production in Primary Cell Cultures of the Rat Spinal Dorsal Horn.

INTRODUCTION: Gabapentin and pregabalin are drugs to treat neuropathic pain. Several studies highlighted effects on presynaptic terminals of nociceptors. Via binding to α2δ subunits of voltage-gated calcium channels, gabapentinoids modulate the synaptic transmission of nociceptive information. However, recent studies revealed further properties of these substances. Treatment with gabapentin or pregabalin in animal models of neuropathic pain resulted not only in reduced symptoms of hyperalgesia but also in an attenuated activation of glial cells and decreased production of pro-inflammatory mediators in the spinal dorsal horn. METHODS: In the present study, we aimed to investigate the impact of gabapentinoids on the inflammatory response of spinal dorsal horn cells, applying the established model of neuro-glial primary cell cultures of the superficial dorsal horn (SDH). We studied effects of gabapentin and pregabalin on lipopolysaccharide (LPS)-induced cytokine release (bioassays), expression of inflammatory marker genes (RT-qPCR), activation of transcription factors (immunocytochemistry), and Ca2+ responses of SDH neurons to stimulation with substance P and glutamate (Ca2+-imaging). RESULTS: We detected an attenuated LPS-induced expression and release of interleukin-6 by SDH cultures in the presence of gabapentinoids. In addition, a significant main effect of drug treatment was observed for mRNA expression of microsomal prostaglandin E synthase 1 and the inhibitor of nuclear factor kappa B. Nuclear translocation of inflammatory transcription factors in glial cells was not significantly affected by gabapentinoid treatment. Moreover, both substances did not modulate neuronal responses upon stimulation with substance P or glutamate. CONCLUSION: Our results provide evidence for anti-inflammatory capacities of gabapentinoids on the acute inflammatory response of SDH primary cultures upon LPS stimulation. Such effects may contribute to the pain-relieving effects of gabapentinoids.

Systemic Lipopolysaccharide Challenge Induces Inflammatory Changes in Rat Dorsal Root Ganglia: An Ex Vivo Study.

Inflammatory processes within the peripheral nervous system (PNS) are associated with symptoms of hyperalgesia and allodynia. Pro-inflammatory mediators, such as cytokines or prostaglandins, modulate the excitability of nociceptive neurons, called peripheral sensitization. Here, we aimed to examine if previously reported effects of in vitro stimulation with lipopolysaccharide (LPS) on primary cell cultures of dorsal root ganglia (DRG) reflect changes in a model of LPS-induced systemic inflammation in vivo. Male rats were intraperitoneally injected with LPS (100 µg/kg) or saline. Effects of systemic inflammation on expression of inflammatory mediators, neuronal Ca2+ responses, and activation of inflammatory transcription factors in DRG were assessed. Systemic inflammation was accompanied by an enhanced expression of pro-inflammatory cytokines and cyclooxygenase-2 in lumbar DRG. In DRG primary cultures obtained from LPS-treated rats enhanced neuronal capsaicin-responses were detectable. Moreover, we found an increased activation of inflammatory transcription factors in cultured macrophages and neurons after an in vivo LPS challenge compared to saline controls. Overall, our study emphasizes the role of inflammatory processes in the PNS that may be involved in sickness-behavior-associated hyperalgesia induced by systemic LPS treatment. Moreover, we present DRG primary cultures as tools to study inflammatory processes on a cellular level, not only in vitro but also ex vivo.

Manifestation of lipopolysaccharide-induced tolerance in neuro-glial primary cultures of the rat afferent somatosensory system.

OBJECTIVE: Bacterial 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. METHODS: Tolerance 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. RESULTS: At 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. CONCLUSION: A 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.

Primary culture of the rat spinal dorsal horn: a tool to investigate the effects of inflammatory stimulation on the afferent somatosensory system.

One maladaptive consequence of inflammatory stimulation of the afferent somatosensory system is the manifestation of inflammatory pain. We established and characterized a neuroglial primary culture of the rat superficial dorsal horn (SDH) of the spinal cord to test responses of this structure to neurochemical, somatosensory, or inflammatory stimulation. Primary cultures of the rat SDH consist of neurons (43%), oligodendrocytes (35%), astrocytes (13%), and microglial cells (9%). Neurons of the SDH responded to cooling (7%), heating (18%), glutamate (80%), substance P (43%), prostaglandin E2 (8%), and KCl (100%) with transient increases in the intracellular calcium [Ca2+]i. Short-term stimulation of SDH primary cultures with LPS (10 µg/ml, 2 h) caused increased expression of pro-inflammatory cytokines, inflammatory transcription factors, and inducible enzymes responsible for inflammatory prostaglandin E2 synthesis. At the protein level, increased concentrations of tumor necrosis factor-α (TNFα) and interleukin-6 (IL-6) were measured in the supernatants of LPS-stimulated SDH cultures and enhanced TNFα and IL-6 immunoreactivity was observed specifically in microglial cells. LPS-exposed microglial cells further showed increased nuclear immunoreactivity for the inflammatory transcription factors NFκB, NF-IL6, and pCREB, indicative of their activation. The short-term exposure to LPS further caused a reduction in the strength of substance P as opposed to glutamate-evoked Ca2+-signals in SDH neurons. However, long-term stimulation with a low dose of LPS (0.01 µg/ml, 24 h) resulted in a significant enhancement of glutamate-induced Ca2+ transients in SDH neurons, while substance P-evoked Ca2+ signals were not influenced. Our data suggest a critical role for microglial cells in the initiation of inflammatory processes within the SDH of the spinal cord, which are accompanied by a modulation of neuronal responses.

Neuroinflammation in Primary Cultures of the Rat Spinal Dorsal Horn Is Attenuated in the Presence of Adipose Tissue-Derived Medicinal Signalling Cells (AdMSCs) in a Co-cultivation Model.

Neuroinflammation within the superficial dorsal horn (SDH) of the spinal cord induces inflammatory pain with symptoms of hyperalgesia and allodynia. Glial activation and production of inflammatory mediators (e.g. cytokines) is associated with modulation of nociceptive signalling. In this context, medicinal signalling cells, e.g. obtained from adipose tissue (AdMSCs), gained attention due to their capacity to modulate the inflammatory response in several diseases, e.g. spinal cord injury. We applied the recently established mixed neuroglial primary cell culture of the rat SDH to investigate effects of AdMSCs on the inflammatory response of SDH cells. Following establishment of a co-cultivation system, we performed specific bioassays for tumour necrosis factor alpha (TNFα) and interleukin (IL)-6, RT-qPCR and immunocytochemistry to detect changes in cytokine production and glial activation upon inflammatory stimulation with lipopolysaccharide (LPS). LPS-induced expression and release of pro-inflammatory cytokines (TNFα, IL-6) by SDH cells was significantly attenuated in the presence of AdMSCs. Further evidence for anti-inflammatory capacities of AdMSCs derived from a blunted LPS-induced TNFα/IL-10 expression ratio and suppressed nuclear translocation of the inflammatory transcription factor nuclear factor kappa B (NFκB) in SDH microglial cells. Expression of IL-10, transforming growth factor beta (TGF-ß) and TNFα-stimulated gene-6 (TSG-6) was detected in AdMSCs, which are putative candidates for anti-inflammatory capacities of these cells. We present a novel co-cultivation system of AdMSCs with neuroglial primary cultures of the SDH to investigate immunomodulatory effects of AdMSCs at a cellular level.

Effects of gabapentinoids on responses of primary cultures from rat dorsal root ganglia to inflammatory or somatosensory stimuli.

Background Gabapentinoids are known to reduce neuropathic pain. The aim of this experimental study was to investigate whether gabapentinoids exert anti-inflammatory and/or anti-nociceptive effects at the cellular level using primary cultures of rat dorsal root ganglia (DRG). Methods Cells from rat DRG were cultured in the presence of gabapentin or pregabalin, and we tested the effects of subsequent stimulation with lipopolysaccharide (LPS) on the expression of genes (real-time polymerase chain reaction) and production of tumor necrosis factor-α (TNFα) and interleukin-6 (IL-6) by specific bioassays. Using Ca2+ imaging, we further investigated in neurons the effects of gabapentinoids upon stimulation with the TRPV-1 agonist capsaicin. Results There is a small influence of gabapentinoids on the inflammatory response to LPS stimulation, namely, a significantly reduced expression of IL-6. Pregabalin and gabapentin further seem to exert a moderate inhibitory influence on capsaicin-induced Ca2+ signals in DRG neurons. Conclusions Although the single inhibitory effects of gabapentinoids on inflammatory and nociceptive responses are moderate, a combination of both effects might provide an explanation for the proposed function of these substances as an adjuvant for the reduction of neuropathic pain.