Dataset Information

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

ABSTRACT:

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.

SUBMITTER: Nurnberger F

PROVIDER: S-EPMC8800878 | biostudies-literature | 2022 Feb

REPOSITORIES: biostudies-literature

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Publications

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

Nürnberger Franz F Leisengang Stephan S Ott Daniela D Murgott Jolanta J Gerstberger Rüdiger R Rummel Christoph C Roth Joachim J

Inflammation research : official journal of the European Histamine Research Society ... [et al.] 20211223 2

<h4>Objective</h4>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.<h4>Methods</h4>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 im ...[more]

PMID: 34940887

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Project description:BackgroundATP-sensitive potassium (KATP) channels in neurons mediate neuroprotection, they regulate membrane excitability, and they control neurotransmitter release. Because loss of DRG neuronal KATP currents is involved in the pathophysiology of pain after peripheral nerve injury, we characterized the distribution of the KATP channel subunits in rat DRG, and determined their alterations by painful axotomy using RT-PCR, immunohistochemistry and electron microscopy.ResultsPCR demonstrated Kir6.1, Kir6.2, SUR1 and SUR2 transcripts in control DRG neurons. Protein expression for all but Kir6.1 was confirmed by Western blots and immunohistochemistry. Immunostaining of these subunits was identified by fluorescent and confocal microscopy in plasmalemmal and nuclear membranes, in the cytosol, along the peripheral fibers, and in satellite glial cells. Kir6.2 co-localized with SUR1 subunits. Kir6.2, SUR1, and SUR2 subunits were identified in neuronal subpopulations, categorized by positive or negative NF200 or CGRP staining. KATP current recorded in excised patches was blocked by glybenclamide, but preincubation with antibody against SUR1 abolished this blocking effect of glybenclamide, confirming that the antibody targets the SUR1 protein in the neuronal plasmalemmal membrane. In the myelinated nerve fibers we observed anti-SUR1 immunostaining in regularly spaced funneled-shaped structures. These structures were identified by electron microscopy as Schmidt-Lanterman incisures (SLI) formed by the Schwann cells. Immunostaining against SUR1 and Kir6.2 colocalized with anti-Caspr at paranodal sites.DRG excised from rats made hyperalgesic by spinal nerve ligation exhibited similar staining against Kir6.2, SUR1 or SUR2 as DRG from controls, but showed decreased prevalence of SUR1 immunofluorescent NF200 positive neurons. In DRG and dorsal roots proximal to axotomy SLI were smaller and showed decreased SUR1 immunofluorescence.ConclusionsWe identified Kir6.2/SUR1 and Kir6.2/SUR2 KATP channels in rat DRG neuronal somata, peripheral nerve fibers, and glial satellite and Schwann cells, in both normal state and after painful nerve injury. This is the first report of KATP channels in paranodal sites adjacent to nodes of Ranvier and in the SLI of the Schwann cells. After painful axotomy KATP channels are downregulated in large, myelinated somata and also in SLI, which are also of smaller size compared to controls.Because KATP channels may have diverse functional roles in neurons and glia, further studies are needed to explore the potential of KATP channels as targets of therapies against neuropathic pain and neurodegeneration.

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Dataset Information

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

Objective

Methods

Results

Conclusion

Publications

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

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