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.
Highlights
A commentary to this article is available online at https://doi.org/10.1007/ s00424-020-02479-xSomatosensory information from peripheral nociceptors and thermosensors is transmitted by afferent axons of dorsal root ganglia (DRG) to neurons in the superficial dorsal horn (SDH) of the spinal cord, including laminae I and II termed substantia gelatinosa [1]
Thermal responsiveness of primary SDH neurons was investigated by changing the temperature of Ca2+ imaging buffer from 37 °C to either 25 °C or 45 °C
We further investigated the neuronal responses to glutamate (10 μM, 180 s) and substance P (1 μM, 180 s), two important neurotransmitters within the dorsal horn of the spinal cord involved in the transmission of painful stimuli
Summary
Somatosensory information from peripheral nociceptors and thermosensors is transmitted by afferent axons of dorsal root ganglia (DRG) to neurons in the superficial dorsal horn (SDH) of the spinal cord, including laminae I and II termed substantia gelatinosa [1]. Most of the primary afferent neurons are nociceptors or thermoreceptors, especially those with thin myelinated or unmyelinated fibers [12]. These neurons employ glutamate as the principle transmitter to carry noxious or thermal information to the second order neurons located within the SDH [1]. For the synaptic transmission from nociceptive fibers to SDH neurons, neuropeptides including substance P (SP) were identified [13] and the neurokinin 1 receptor for SP is present in projection neurons in lamina I of the spinal cord [14]. Ca2+ imaging is an appropriate tool to characterize the responsiveness of SDH neurons to the relevant transmitters (glutamate and SP) involved in the transmission of nociceptive signals into the spinal cord
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