Primary culture of the rat spinal dorsal horn: a tool to investigate the effects of inflammatory stimulation on the affe
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NEUROSCIENCE
Primary culture of the rat spinal dorsal horn: a tool to investigate the effects of inflammatory stimulation on the afferent somatosensory system Stephan Leisengang 1,2,3 & Franz Nürnberger 1 & Daniela Ott 1 & Jolanta Murgott 1 & Rüdiger Gerstberger 1 & Christoph Rummel 1,2,3 & Joachim Roth 1,2,3 Received: 22 April 2020 / Revised: 28 August 2020 / Accepted: 14 October 2020 # The Author(s) 2020
Abstract 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. Keywords Somatosensory system . Substantia gelatinosa . Primary culture . Inflammation . Neuronal and glial responses
Introduction A commentary to this article is available online at https://doi.org/10.1007/ s00424-020-02479-x * Joachim Roth [email protected] 1
Department of Veterinary Physiology and Biochemistry, Justus-Liebig-University Giessen, Frankfurter Strasse 100, 35392 Giessen, Germany
2
Center for Mind, Brain and Behavior – CMBB, Philipps-University Marburg, Marburg, Germany
3
Center for Mind, Brain and Behavior – CMBB, Justus-Liebig-University of Giessen, Giessen, Germany
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