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

2020 ◽  
Vol 472 (12) ◽  
pp. 1769-1782 ◽  
Author(s):  
Stephan Leisengang ◽  
Franz Nürnberger ◽  
Daniela Ott ◽  
Jolanta Murgott ◽  
Rüdiger Gerstberger ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Transcription Factors ◽  
Substance P ◽  
Primary Culture ◽  
Primary Cultures ◽  
Somatosensory System ◽  
Microglial Cells ◽  
Prostaglandin E ◽  
Short Term ◽  
Stimulation Of

AbstractOne 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.

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

2021 ◽  
Vol 70 (4) ◽  
pp. 429-444
Author(s):  
Franz Nürnberger ◽  
Stephan Leisengang ◽  
Daniela Ott ◽  
Jolanta Murgott ◽  
Rüdiger Gerstberger ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Nuclear Translocation ◽  
Mrna Level ◽  
Primary Cultures ◽  
Somatosensory System ◽  
Cellular Level ◽  
Microglial Cells ◽  
Substantia Gelatinosa ◽  
Tnf Α ◽  
Pre Treatment

Abstract 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.

Use of NK1 receptor antagonists in the exploration of physiological functions of substance P and neurokinin A

1995 ◽  
Vol 73 (7) ◽  
pp. 903-907 ◽  
Author(s):  
M. Qtsuka ◽  
K. Yoshioka ◽  
M. Yanagisawa ◽  
H. Suzuki ◽  
F.-Y. Zhao ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Electrical Stimulation ◽  
Substance P ◽  
Guinea Pig ◽  
Saphenous Nerve ◽  
Ventral Root ◽  
Neonatal Rat ◽  
Neurokinin A ◽  
Receptor Antagonists ◽  
Stimulation Of

Tachykinin NK1 receptor antagonists were used to explore the physiological functions of substance P (SP) and neurokinin A (NKA). Pharmacological profiles of three NK1 receptor antagonists, GR71251, GR82334, and RP 67580, were examined in the isolated spinal cord preparation of the neonatal rat. These tachykinin receptor antagonists exhibited considerable specificities and antagonized the actions of both SP and NKA to induce the depolarization of ventral roots. Electrical stimulation of the saphenous nerve with C-fiber strength evoked a depolarization lasting about 30 s of the ipsilateral L3 ventral root. This response, which is referred to as saphenous-nerve-evoked slow ventral root potential (VRP), was depressed by these NK1 receptor antagonists. In contrast, the saphenous-nerve-evoked slow VRP was potentiated by application of a mixture of peptidase inhibitors, including thiorphan, actinonin, and captopril in the presence of naloxone, but not after further addition of GR71251. Likewise, in the isolated coeliac ganglion of the guinea pig, electrical stimulation of the mesenteric nerves evoked in some ganglionic cells slow excitatory postsynaptic potentials (EPSPs), which were depressed by GR71251 and potentiated by peptidase inhibitors. These results further support the notion that SP and NKA serve as neurotransmitters producing slow EPSPs in the neonatal rat spinal cord and guinea pig prevertebral ganglia.Key words: substance P, neurokinin A, neurotransmitter, tachykinin antagonist, spinal cord.

Myocardial ischemia induces the release of substance P from cardiac afferent neurons in rat thoracic spinal cord

2004 ◽  
Vol 286 (5) ◽  
pp. H1654-H1664 ◽  
Author(s):  
Fang Hua ◽  
Brian A. Ricketts ◽  
Angela Reifsteck ◽  
Jeffrey L. Ardell ◽  
Carole A. Williams
Keyword(s):  
Spinal Cord ◽  
Coronary Artery ◽  
Substance P ◽  
Coronary Artery Occlusion ◽  
Artery Occlusion ◽  
Afferent Neurons ◽  
Thoracic Spinal Cord ◽  
Thoracic Spinal ◽  
Cardiac Afferents ◽  
Stimulation Of

Antibody-coated microprobes were inserted into the thoracic (T3–4) spinal cord in urethane-anesthetized Sprague-Dawley rats to detect the differences in the release of immunoreactive substance P-like (irSP) substances in response to differential activation of cardiac nociceptive sensory neurons (CNAN). CNAN were stimulated either by intrapericardial infusion of an inflammatory ischemic exudate solution (IES) containing algogenic substances (i.e., 10 mM each of adenosine, bradykinin, prostaglandin E2, and 5-hydroxytryptamine), or by transient occlusion of the left anterior descending coronary artery (CoAO). There was widespread basal release of irSP from the thoracic spinal cord. Stimulation of the CNAN by IES did not alter the pattern of release of irSP. Conversely, CoAO augmented the release of irSP from T3–4 spinal segments from laminae I–VII. This CoAO-induced irSP release was eliminated after thoracic dorsal rhizotomy. These results indicate that heterogeneous activation of cardiac afferents, as with focal coronary artery occlusion, represents an optimum input for activation of the cardiac neuronal hierarchy and for the resultant perception of angina. Excessive stimulation of cardiac nociceptive afferent neurons elicited during regional coronary artery occlusion involves the release of SP in the thoracic spinal cord and suggests that local spinal cord release of SP may be involved in the neural signaling of angina.

Substance P and endogenous opioids: how and where they could play a role in cluster headache

Cephalalgia ◽  
1983 ◽  
Vol 3 (1_suppl) ◽  
pp. 143-145 ◽  
Author(s):  
Federigo Sicuteri ◽  
Liborio Rainò ◽  
Pierangelo Geppetti
Keyword(s):  
Spinal Cord ◽  
Substance P ◽  
Cluster Headache ◽  
Endogenous Opioids ◽  
P Release ◽  
Antidromic Stimulation ◽  
Étude Clinique ◽  
Substance P Release ◽  
Midollo Spinale ◽  
Stimulation Of

Substance P appears to be involved in the transmission of pain signals from the periphery to the spinal cord and brain stem. Substance P containing neurons are responsible for the neurogenic vasodilation identical to that obtained by substance P release evoked by antidromic stimulation of these fibres. Both endogenous opioids and somatostatin inhibit the release of substance P from central and peripheral endings. Present pharmacological investigations conclude that morphine and somatostatin are as effective as ergotamine in reducing the pain of CH. All three drugs are significantly more active than placebo. Somatostatin and opiates could act by inhibiting the release of substance P. La Substance P (SP) représente le probable transmitteur des sensations douloureuses de la périphérie à la moelle épinière et au tronc encéphalique. Les neurones contenants la SP semblent être aussi responsables de la vasodilatation neurogenique identique à celle qui peut être obtenue par le “release” de SP, induit par la stimolation antidromique de ces fibres. Soit ces opiaces endogènes que la somatostatine (SR1F) inhibent le “release” de SP par les terminaisons nerveuses céntrales et périphériques. Notre étude clinique parvient à la conclusion que la morphine et le SRIF ont une efficacité semblabe à celle de l'ergotamine en rediure la douleur pendant l'attaque de céphalée “en chapelet”, et que tous les troi medicaments sont significativement actifs plus que le placebo. Le SRIF et les opiacés pourraient agir en inhibant le “release” de SP par les terminaissons neuronales céntrales et périphériques. La sostanza P viene indicata come il probabile trasmettitore delle sensazioni dolorifiche dalla periferia al midollo spinale ed al tronco encefalico. I neuroni contenenti SP sembrano inoltre essere responsabili della vasodilatazione neurogenica identica a quella ottenibile dal “release” di SP indotto dalla stimoalzione antidromica di queste fibre. Sia gli oppiacei endogeni che la Somatostatina inibiscono il “release” di sostanza P dai terminali nervosi centrali e periferici. Dalla presente ricerca emerge che la morfina ed il SRIF hanno una efficacia simile a quella dell'ergotamina nel ridurre il dolore nell'attacco di CH, e che tutti e tre i farmaci sono significativamente più attivi rispetto al placebo. SRIF ed oppiacei potrebbero agire inibendo il release di SP dalle terminazioni nervose sia a livello centrale che periferico.

Depression of Nociceptive Sensory Activity in the Rat Spinal Cord Due to the Intrathecal Administration of Drugs: Effect of Diazepam

Neurosurgery ◽  
1984 ◽  
Vol 15 (6) ◽  
pp. 917-920 ◽  
Author(s):  
Ilmar Jurna
Keyword(s):  
Spinal Cord ◽  
Substance P ◽  
Nerve Fibers ◽  
Afferent Nerve ◽  
Spinal Reflexes ◽  
Sensory Response ◽  
Motor Responses ◽  
Afferent Nerve Fibers ◽  
C Fiber ◽  
Stimulation Of

Abstract The intrathecal (i.t.) administration of morphine inhibits nociceptive motor responses and activity in ascending axons evoked by stimulation of nociceptive afferent nerve fibers (nociceptive sensory response) in the rat. The i.t. administration of cholecystokinin octapeptide and ceruletide inhibits nociceptive motor responses, but does not affect ascending nociceptive activity. This shows that drug-induced depression of nociceptive motor responses is not always associated with depression of the nociceptive sensory response of the spinal cord. The microiontophoretic application of substance P excites single dorsal horn neurons that respond to noxious stimulation, whereas the i.t. administration of substance P inhibits both nociceptive motor and sensory responses. Thus, the results obtained from the i.t. administration of a drug may differ from those obtained from its application to single spinal neurons. Diazepam inhibits spinal reflexes and may reduce pain sensation in humans. To assess whether a spinal action is involved in the pain-relieving effect of diazepam, experiments were carried out on spinalized rats in which activity evoked by the stimulation of nociceptive and nonnociceptive afferent nerve fibers of the sural nerve was recorded from single ascending axons below the site of spinal cord transection. Diazepam, 20 ųg i.t., reduced activity evoked by afferent A delta and C fiber stimulation and by stimulation of afferent A beta fibers. The depressant effect caused by diazepam, 2 mg/kg i.v., on C fiber-evoked ascending activity was reduced by the i.t. injection of the benzodiazepine antagonist, Ro 15-1788 (40 ųg), an imidazodiazepine. It is concluded that the depression by diazepam of C fiber-evoked ascending activity contributes to pain relief caused by the drug.

Loss of adrenergic regulation of cAMP production in the FRTL-5 cell line

1986 ◽  
Vol 111 (1) ◽  
pp. 54-61 ◽  
Author(s):  
Maria Luisa Brandi ◽  
Carlo M. Rotella ◽  
Roberto Zonefrati ◽  
Roberto Toccafondi ◽  
Salvatore M. Aloj
Keyword(s):  
Primary Culture ◽  
Cell Line ◽  
Adrenergic Receptors ◽  
Primary Cultures ◽  
Human Thyroid ◽  
Camp Production ◽  
Thyroid Cells ◽  
Adrenergic Regulation ◽  
Rat Thyroid ◽  
Stimulation Of

Abstract. Rat thyroid cells in primary culture augment cAMP production when challenged with β-adrenergic agonists; at 10−5m the potency is isoproterenol > nor-epinephrine > epinephrine. In analogy with human thyroid cells, rat thyroid primary cultures display α-adrenergic-stimulated cGMP production which inhibits TSH and norepinephrine stimulation of cAMP. Adrenergic regulation of cyclic nucelotide production is lost in the cloned thyroid cell line of rat origin known as FRTL-5. Also the potentiating effect of phentolamine on TSH stimulation of cAMP production in thyroid primary cultures becomes an inhibitory one in the FRTL-5 cells. Neither 'soluble factors' nor contamination of other cell populations could account for the different behaviour of the primary culture and the cell line toward adrenergic regulation. The reported activation by norepinephrine of iodide efflux in FRTL-5 cells rules out the loss of specific adrenergic receptors in the FRTL-5 cells. It is proposed that the cloning of FRTL-5 cells from primary cultures causes an 'alteration' in the coupling of adrenergic receptors to the adenylate cyclase system. This alteration does not affect those mechansism of message transduction that do not involve cAMP as the signal.

Electrical Stimulation of the Anterior Cingulate Cortex Reduces Responses of Rat Dorsal Horn Neurons to Mechanical Stimuli

2005 ◽  
Vol 94 (1) ◽  
pp. 845-851 ◽  
Author(s):  
Arun K. Senapati ◽  
Stacey C. Lagraize ◽  
Paula J. Huntington ◽  
Hilary D. Wilson ◽  
Perry N. Fuchs ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Dorsal Horn ◽  
Anterior Cingulate ◽  
Spinal Cord Dorsal Horn ◽  
Mechanical Stimuli ◽  
Short Term ◽  
Dorsal Horn Neurons ◽  
Spinal Cord Dorsal ◽  
Stimulation Of

The anterior cingulate cortex (ACC) is involved in the affective and motivational aspect of pain perception. Behavioral studies show a decreased avoidance behavior to noxious stimuli without change in mechanical threshold after stimulation of the ACC. However, as part of the neural circuitry of behavioral reflexes, there is no evidence showing that ACC stimulation alters dorsal horn neuronal responses. We hypothesize that ACC stimulation has two phases: a short-term phase in which stimulation elicits antinociception and a long-term phase that follows stimulation to change the affective response to noxious input. To begin testing this hypothesis, the purpose of this study was to examine the response of spinal cord dorsal horn neurons during stimulation of the ACC. Fifty-eight wide dynamic range spinal cord dorsal horn neurons from adult Sprague-Dawley rats were recorded in response to graded mechanical stimuli (brush, pressure, and pinch) at their respective receptive fields, while simultaneous stepwise electrical stimulations (300 Hz, 0.1 ms, at 10, 20, and 30 V) were applied in the ACC. The responses to brush at control, 10, 20, and 30 V, and recovery were 14.2 ± 1.4, 12.3 ± 1.2, 10.9 ± 1.2, 10.3 ± 1.1, and 14.1 ± 1.4 spikes/s, respectively. The responses to pressure at control, 10, 20, and 30 V, and recovery were 39.8 ± 4.7, 25.6 ± 3.0, 25.0 ± 3.0, 21.6 ± 2.4, and 34.2 ± 3.7 spikes/s, respectively. The responses to pinch at control, 10, 20, and 30 V, and recovery were 40.7 ± 3.8, 30.6 ± 3.1, 27.8 ± 2.8, 27.2 ± 3.2, and 37.4 ± 3.9 spikes/s, respectively. We conclude that electrical stimulation of the ACC induces significant inhibition of the responses of spinal cord dorsal horn neurons to noxious mechanical stimuli. The stimulation-induced inhibition begins to recover as soon as the stimulation is terminated. These results suggest differential short-term and long-term modulatory effects of the ACC stimulation on nociceptive circuits.

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