Dorsal Horn Neurons Research Articles

Inhibition of synaptic transmission by anandamide precursor 20:4-NAPE is mediated by TRPV1 receptors under inflammatory conditions.

Transient receptor potential ion channel, vanilloid subfamily, type 1 (TRPV1) cation channel, and cannabinoid receptor 1 (CB1) are essential in the modulation of nociceptive signaling in the spinal cord dorsal horn that underlies different pathological pain states. TRPV1 and CB1 receptors share the endogenous agonist anandamide (AEA), produced from N-arachidonoylphosphatidylethanolamine (20:4-NAPE). We investigated the effect of the anandamide precursor 20:4-NAPE on synaptic activity in naive and inflammatory conditions. Patch-clamp recordings of miniature excitatory postsynaptic currents (mEPSCs) from superficial dorsal horn neurons in rat acute spinal cord slices were used. Peripheral inflammation was induced by subcutaneous injection of carrageenan. Under naive conditions, mEPSCs frequency (0.96 ± 0.11 Hz) was significantly decreased after 20 μM 20:4-NAPE application (55.3 ± 7.4%). This 20:4-NAPE-induced inhibition was blocked by anandamide-synthesizing enzyme N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD) inhibitor LEI-401. In addition, the inhibition was prevented by the CB1 receptor antagonist PF 514273 (0.2 μM) but not by the TRPV1 receptor antagonist SB 366791 (10 μM). Under inflammatory conditions, 20:4-NAPE (20 μM) also exhibited a significant inhibitory effect (74.5 ± 8.9%) on the mEPSCs frequency that was prevented by the TRPV1 receptor antagonist SB 366791 but not by PF 514273 application. Our results show that 20:4-NAPE application has a significant modulatory effect on spinal cord nociceptive signaling that is mediated by both TRPV1 and CB1 presynaptic receptors, whereas peripheral inflammation changes the underlying mechanism. The switch between TRPV1 and CB1 receptor activation by the AEA precursor 20:4-NAPE during inflammation may play an important role in nociceptive processing, hence the development of pathological pain.

481-P: 10kHz SCS Restores Mechanical and Thermal Neural Processing in a Rodent Model of Painful Diabetic Neuropathy

A recent randomized controlled trial of 10 kHz spinal cord stimulation (SCS) in patients with painful diabetic neuropathy (PDN) has demonstrated significant and durable improvements in neurologic status, particularly sensory restoration of the foot (Petersen et al 2021, Argoff et al 2023). In order to gain further mechanistic insight into the improvements in sensory processing by 10kHz SCS, we analyzed the receptive field (RF) size and responses of spinal dorsal horn (DH) neurons to mechanical and thermal stimuli of the paw in a PDN rat model. For PDN-model, Rats were given a single effective dose of streptozotocin (STZ), while Naïve animals were injected with 0.9% saline. Four groups were studied: Naïve, STZ, STZ+ShamSCS, and STZ+10kHzSCS. Rats were anesthetized with urethane and a laminectomy was performed for DH neurons recording. We analyzed RF size and the response of DH neurons to vibrotactile stimulation and thermal sensation. Both the Naïve and STZ+10kHzSCS groups had significantly smaller RFs than the STZ Control and STZ+ShamSCS groups. In response to a 1 Hz vibration frequency, Naïve and STZ+10kHzSCS groups had higher peak power spectral density (PSD) of DH firing than STZ+ShamSCS, and PSD for the STZ+10kHzSCS group was statistically higher than for the STZ Control group. Response of DH neurons showed no significant differences between groups during the thermal stimulation, but the post-stimulus firing rates for the STZ+10kHzSCS group were significantly higher compared to STZ Control and STZ+ShamSCS. These data suggest that 10 kHz SCS amplified the DH response to large fiber (vibration) and small fiber (warm) signals and normalized the receptive field of the paw (acuity) in PDN rats. In diabetes, where peripheral sensory compromise from chronic hyperglycemia can lead to loss of protective sensation, such ‘tuned amplification’ as provided by 10 kHz SCS in the central nervous system may compensate for the reduced functionality seen in the peripheral sensory systems. Disclosure K.Lee: Employee; Nevro Corp. D.Lee: Employee; Nevro Corp. D.Wang: Employee; Nevro Corp. Z.B.Kagan: Employee; Nevro Corp. K.Bradley: Employee; Nevro Corp.

Impaired Ventrolateral Periaqueductal Gray-Ventral Tegmental area Pathway Contributes to Chronic Pain-Induced Depression-Like Behavior in Mice.

Chronic pain conditions within clinical populations are correlated with a high incidence of depression, and researchers have reported their high rate of comorbidity. Clinically, chronic pain worsens the prevalence of depression, and depression increases the risk of chronic pain. Individuals suffering from chronic pain and depression respond poorly to available medications, and the mechanisms underlying the comorbidity of chronic pain and depression remain unknown. We used spinal nerve ligation (SNL) in a mouse model to induce comorbid pain and depression. We combined behavioral tests, electrophysiological recordings, pharmacological manipulation, and chemogenetic approaches to investigate the neurocircuitry mechanisms of comorbid pain and depression. SNL elicited tactile hypersensitivity and depression-like behavior, accompanied by increased and decreased glutamatergic transmission in dorsal horn neurons and midbrain ventrolateral periaqueductal gray (vlPAG) neurons, respectively. Intrathecal injection of lidocaine, a sodium channel blocker, and gabapentin ameliorated SNL-induced tactile hypersensitivity and neuroplastic changes in the dorsal horn but not depression-like behavior and neuroplastic alterations in the vlPAG. Pharmacological lesion of vlPAG glutamatergic neurons induced tactile hypersensitivity and depression-like behavior. Chemogenetic activation of the vlPAG-rostral ventromedial medulla (RVM) pathway ameliorated SNL-induced tactile hypersensitivity but not SNL-elicited depression-like behavior. However, chemogenetic activation of the vlPAG-ventral tegmental area (VTA) pathway alleviated SNL-produced depression-like behavior but not SNL-induced tactile hypersensitivity. Our study demonstrated that the underlying mechanisms of comorbidity in which the vlPAG acts as a gating hub for transferring pain to depression. Tactile hypersensitivity could be attributed to dysfunction of the vlPAG-RVM pathway, while impairment ofthe vlPAG-VTA pathway contributed to depression-like behavior.

Descending Facilitation of Nociceptive Transmission From the Rostral Ventromedial Medulla Contributes to Hyperalgesia in Mice with Sickle Cell Disease

Sickle cell disease (SCD) is an inherited blood disorder that is associated with acute episodic and chronic pain. Mice with SCD have robust hyperalgesia mediated, in part, by sensitization of spinal dorsal horn neurons. However, underlying mechanisms are not fully understood. Since the rostral ventromedial medulla (RVM) is a major component of descending circuitry that modulates nociceptive transmission in the spinal cord, we examined if the RVM contributes to hyperalgesia in mice with SCD. Injection of lidocaine, but not vehicle, into the RVM eliminated mechanical and heat hyperalgesia in sickle (HbSS-BERK) mice without altering mechanical and heat sensitivity in naïve C57B mice. These data indicate that the RVM contributes to the maintenance of hyperalgesia in mice with SCD. In electrophysiological studies, we determined the changes in response properties of RVM neurons that might contribute to hyperalgesia in sickle mice. Recordings were made from single ON, OFF, and Neutral cells in the RVM of sickle and control (HbAA-BERK) mice. Spontaneous activity and responses of ON, OFF and Neutral cells evoked by heat (50 °C) and mechanical (26 g) stimuli applied to the hind paw were compared between sickle and control mice. Although there were no differences in the proportions of functionally-identified neurons or spontaneous activity between sickle and control mice, evoked responses of ON cells to heat and mechanical stimuli were increased approximately 3-fold in sickle mice as compared to control mice. Thus, the RVM contributes to hyperalgesia in sickle mice via a specific ON cell-dependent descending facilitation of nociceptive transmission.

Spinal neuromodulation mitigates myocardial ischemia-induced sympathoexcitation by suppressing the intermediolateral nucleus hyperactivity and spinal neural synchrony.

Myocardial ischemia disrupts the cardio-spinal neural network that controls the cardiac sympathetic preganglionic neurons, leading to sympathoexcitation and ventricular tachyarrhythmias (VTs). Spinal cord stimulation (SCS) is capable of suppressing the sympathoexcitation caused by myocardial ischemia. However, how SCS modulates the spinal neural network is not fully known. In this pre-clinical study, we investigated the impact of SCS on the spinal neural network in mitigating myocardial ischemia-induced sympathoexcitation and arrhythmogenicity. Ten Yorkshire pigs with left circumflex coronary artery (LCX) occlusion-induced chronic myocardial infarction (MI) were anesthetized and underwent laminectomy and a sternotomy at 4-5 weeks post-MI. The activation recovery interval (ARI) and dispersion of repolarization (DOR) were analyzed to evaluate the extent of sympathoexcitation and arrhythmogenicity during the left anterior descending coronary artery (LAD) ischemia. Extracellular in vivo and in situ spinal dorsal horn (DH) and intermediolateral column (IML) neural recordings were performed using a multichannel microelectrode array inserted at the T2-T3 segment of the spinal cord. SCS was performed for 30 min at 1 kHz, 0.03 ms, 90% motor threshold. LAD ischemia was induced pre- and 1 min post-SCS to investigate how SCS modulates spinal neural network processing of myocardial ischemia. DH and IML neural interactions, including neuronal synchrony as well as cardiac sympathoexcitation and arrhythmogenicity markers were evaluated during myocardial ischemia pre- vs. post-SCS. ARI shortening in the ischemic region and global DOR augmentation due to LAD ischemia was mitigated by SCS. Neural firing response of ischemia-sensitive neurons during LAD ischemia and reperfusion was blunted by SCS. Further, SCS showed a similar effect in suppressing the firing response of IML and DH neurons during LAD ischemia. SCS exhibited a similar suppressive impact on the mechanical, nociceptive and multimodal ischemia sensitive neurons. The LAD ischemia and reperfusion-induced augmentation in neuronal synchrony between DH-DH and DH-IML pairs of neurons were mitigated by the SCS. These results suggest that SCS is decreasing the sympathoexcitation and arrhythmogenicity by suppressing the interactions between the spinal DH and IML neurons and activity of IML preganglionic sympathetic neurons.

Comparison of intrathecal dexmedetomidine and intrathecal fentanyl as an adjuvant to bupivacaine during spinal anaesthesia for lower limb orthopedic surgery

Background: In this study, we have compared the addition of fentanyl 25 mcg and dexmedetomidine 5 mcg to 15 mg of 0.5% hyperbaric bupivacaine for spinal anesthesia separately for patient undergoing lower limb orthopedic surgery. Dexmedetomidine is an α-2 adrenoreceptor agonist and it can prolong the motor and sensory block for long spinal anesthesia. It act by binding to presynaptic C-fiber and postsynaptic dorsal horn neurons. The analgesic action is a result of depression of release of C-fiber transmitters and hyperpolarization of postsynaptic dorsal horn neurons. Aims and Objectives: The present study compares the onset, duration of sensory and motor block, postoperative analgesia, hemodynamic changes, and adverse effects of intrathecal dexmedetomidine and intrathecal fentanyl as an adjuvant to bupivacaine during spinal anesthesia for lower limb orthopedic surgery. Materials and Methods: Patient was randomly grouped by close-envelope technique into the three equal group of 30 in each group. Total 90 patients were including in this study. The blind nature of the study was maintained and the study drug is given according as, Group-1: 15 mg of 0.5% hyperbaric bupivacaine. (Control group), Group-2: 15 mg of 0.5% hyperbaric bupivacaine with 25 mcg of fentanyl, and Group-3: 15 mg of 0.5% hyperbaric bupivacaine with 5 mcg dexmedetomidine for spinal anesthesia. Results: Patients in dexmedetomidine Group-3 had a significantly longer sensory (160±18.5 min) and motor block (242±22 min) time as compared to patients in fentanyl and control group (P<0.001). The time to first request of analgesic in the post-operative period was also longer in dexmedetomidine group (245±3.6 min) when compared to bupivacaine and fentanyl in which it was 125±1.0 min and 220±2.5 min, respectively (P<0.001). Conclusion: We concluded that intrathecal dexmedetomidine with bupivacaine for spinal anesthesia is associated with prolong motor and sensory block then fentanyl 25 μg with bupivacaine and bupivacaine alone.

Electroacupuncture inhibits dendritic spine remodeling through the srGAP3-Rac1 signaling pathway in rats with SNL

Previous studies have shown that peripheral nerve injury can lead to abnormal dendritic spine remodeling in spinal dorsal horn neurons. Inhibition of abnormal dendritic spine remodeling can relieve neuropathic pain. Electroacupuncture (EA) has a beneficial effect on the treatment of neuropathic pain, but the specific mechanism remains unclear. Evidence has shown that slit-robo GTPase activating protein 3 (srGAP3) and Rho GTPase (Rac1) play very important roles in dendritic spine remodeling. Here, we used srGAP3 siRNA and Rac1 activator CN04 to confirm the relationship between SrGAP3 and Rac1 and their roles in improving neuropathic pain with EA. Spinal nerve ligation (SNL) was used as the experimental model, and thermal withdrawal latency (TWL), mechanical withdrawal threshold (MWT), Western blotting, immunohistochemistry and Golgi-Cox staining were used to examine changes in behavioral performance, protein expression and dendritic spines. More dendritic spines and higher expression levels of srGAP3 were found in the initial phase of neuropathic pain. During the maintenance phase, dendritic spines were more mature, which was consistent with lower expression levels of srGAP3 and higher expression levels of Rac1-GTP. EA during the maintenance phase reduced the density and maturity of dendritic spines of rats with SNL, increased the levels of srGAP3 and reduced the levels of Rac1-GTP, while srGAP3 siRNA and CN04 reversed the therapeutic effects of EA. These results suggest that dendritic spines have different manifestations in different stages of neuropathic pain and that EA may inhibit the abnormal dendritic spine remodeling by regulating the srGAP3/Rac1 signaling pathway to alleviate neuropathic pain.

An electrophysiological method for evaluation of topical antipruritic drugs on itch-related neuronal activities in the spinal cord in hairless mice

To evaluate the effects of antipruritic drugs, it is important to determine whether the neural responses induced by physiological itch stimuli are suppressed. Although there are several behavioral assessments for topical antipruritic drugs applied to the skin, there are few established methods at neuronal levels using in vivo electrophysiological recordings for predicting local efficacy of antipruritic drugs for cutaneous application. To establish an assessment of topical antipruritic drugs applied to skin using in vivo extracellular recording from neurons in the superficial dorsal horn, we examined the relationships between itch-related biting behavior and spinal neuronal responses elicited by intradermal injection of pruritogen serotonin (5-HT) in hairless mice. The efficacy of topical occlusive application of local anesthetics was also evaluated by an in vivo electrophysiological method. 5-HT significantly increased the firing frequency in spinal neurons. The spinal firing frequency time course was similar to that of the biting behavior after the 5-HT injections. The 5-HT-induced spinal responses were significantly decreased by topical occlusive application of lidocaine or a Nav 1.7 channel blocker to the calf. The intradermal 5-HT injection-induced spinal neuronal responses appeared to be suppressed by topical occlusive application of lidocaine or a Nav1.7 channel blocker. The electrophysiological method for evaluating topical antipruritic drugs may be beneficial in assessing local effects on the skin.

Intrathecal administration of botulinum toxin type a antagonizes neuropathic pain by countering increased vesicular nucleotide transporter expression in the spinal cord of chronic constriction injury of the sciatic nerve rats

Botulinum toxin type A (BoNT/A) induces direct analgesic effects in neuropathic pain by inhibiting the release of substance P, calcitonin gene-related peptide (CGRP) and glutamate. Vesicular nucleotide transporter (VNUT) was responsible for the storage and release of ATP in vivo, and one of the mechanisms underlying neuropathic pain is VNUT-dependent release of extracellular ATP from dorsal horn neurons. However, the analgesic effect of BoNT/A by affecting the expression of VNUT remained largely unknown. Thus, in this study, we aimed to elucidate the antinociceptive potency and analgesic mechanism of BoNT/A in chronic constriction injury of the sciatic nerve (CCI) induced neuropathic pain. Our results showed that a single intrathecal injection of 0.1 U BoNT/A seven days after CCI surgery produced significant analgesic activity and decreased the expression of VNUT in the spinal cord of CCI rats. Similarly, BoNT/A inhibited the CCI-induced increase in ATP content in the rat spinal cord. Overexpression of VNUT in the spinal cord of CCI-induced rats markedly reversed the antinociceptive effect of BoNT/A. Furthermore, 33 U/mL BoNT/A dramatically reduced the expression of VNUT in pheochromocytoma (PC12) cells but overexpressing SNAP-25 increased VNUT expression in PC12 cells. Our current study is the first to demonstrate that BoNT/A is involved in neuropathic pain by regulating the expression of VNUT in the spinal cord in rats.

Metformin inhibits spontaneous excitatory postsynaptic currents in spinal dorsal cord neurons from paclitaxel-treated rats.

Cancer patients treated with paclitaxel often develop chemotherapy-induced peripheral neuropathy, which has not been effectively treated with drugs. The anti-diabetic drug metformin is effective in the treatment of neuropathic pain. The aim of this study was to elucidate effect of metformin on paclitaxel-induced neuropathic pain and spinal synaptic transmission. Electrophysiological experiments on rat spinal slices were performed in vitro and mechanical allodynia quantified in vitro. The present data demonstrated that intraperitoneal injection of paclitaxel produced mechanical allodynia and potentiated spinal synaptic transmission. Intrathecal injection of metformin significantly reversed the established mechanical allodynia induced by paclitaxel in rats. Either spinal or systemic administration of metformin significantly inhibited the increased frequency of spontaneous excitatory postsynaptic currents (sEPSCs) in spinal dorsal horn neurons from paclitaxel-treated rats. We found that 1 h incubation of metformin also reduced the frequency rather than the amplitude of sEPSCs in the spinal slices from paclitaxel-treated rats. These results suggested that metformin was able to depress the potentiated spinal synaptic transmission, which may contribute to alleviating the paclitaxel-induced neuropathic pain.

Downregulation of ciRNA-Kat6b in dorsal spinal horn is required for neuropathic pain by regulating Kcnk1 in miRNA-26a-dependent manner.

Nerve injury-induced maladaptive changes in gene expression in the spinal neurons are essential for neuropathic pain genesis. Circular RNAs (ciRNA) are emerging as key regulators of gene expression. Here, we identified a nervous-system-tissues-specific ciRNA-Kat6 with conservation in humans and mice. We aimed to investigate whether and how spinal dorsal horn ciRNA-Kat6b participates in neuropathic pain. Unilateral sciatic nerve chronic constrictive injury (CCI) surgery was used to prepare the neuropathic pain model. The differentially expressed ciRNAs were obtained by RNA-Sequencing. The identification of nervous-system-tissues specificity of ciRNA-Kat6b and the measurement of ciRNA-Kat6b and microRNA-26a (miRNA-26a) expression level were carried out by quantitative RT-PCR. The ciRNA-Kat6b that targets miRNA-26a and miRNA-26a that targets Kcnk1 were predicted by bioinformatics analysis and verified by in vitro luciferase reports test and in vivo experiments including Western-blot, immunofluorescence, and RNA-RNA immunoprecipitation. The correlation between neuropathic pain and ciRNA-Kat6b, miRNA-26a, or Kcnk1 was examined by the hypersensitivity response to heat and mechanical stimulus. Peripheral nerve injury downregulated ciRNA-Kat6b in the dorsal spinal horn of male mice. Rescuing this downregulation blocked nerve injury-induced increase of miRNA-26a, reversed the miRNA-26a-triggered decrease of potassium channel Kcnk1, a key neuropathic pain player, in the dorsal horn, and alleviates CCI-induced pain hypersensitivities. On the contrary, mimicking this downregulation increased the miRNA-26a level and decreased Kcnk1 in the spinal cord, resulting in neuropathic pain-like syndrome in naïve mice. Mechanistically, the downregulation of ciRNA-Kat6b reduced the accounts of miRNA-26a binding to ciRNA-Kat6b, and elevated the binding accounts of miRNA-26a to the 3' untranslated region of Kcnk1 mRNA and degeneration of Kcnk1 mRNA, triggering in the reduction of KCNK1 protein in the dorsal horn of neuropathic pain mice. The ciRNA-Kat6b/miRNA-26a/Kcnk1 pathway in dorsal horn neurons regulates the development and maintenance of neuropathic pain, ciRNA-Kat6b may be a potential new target for analgesic and treatment strategies.

Effects of Post Trauma Morphine On Dorsal Horn Neuron Excitability: Studies using cFOS and RNAscope

Previous research has shown that a 5-day course of morphine enhances nociceptive sensitivity and allodynia when given 10 days after chronic constriction injury (CCI) as measured by the Von Frey test, a test where the hindpaw is poked with force (measured in grams) calibrated filament. This increased sensitivity to touch suggests that post trauma morphine makes second order sensory dorsal horn neurons more excitable. Therefore, it is hypothesized that during morphine enhanced allodynia, dorsal horn neurons will be more excitable to nociceptive stimulus. It was found that morphine enhanced allodynia causes more excitable neurons across a larger spatial range of the spinal cord, both rostral-caudal and dorsal-ventral along the dorsal horn. Mechanisms for the increased excitability of the dorsal horn are proposed and explored. These findings add to a robust literature which has detailed the paradoxical pain amplifying effects of morphine. Further, this study predicts that hyperexcitability of pain and touch pathways may occur as a clinically unintended side effect of morphine when administered to treat ongoing neuropathic pain. Lay Summary When working on the body, opioids take effect on various cells- including cells of the nervous system known as Glia. These glial cells produce various inflammatory responses that are typically known to decrease inflammation in the body. However, prior literature has revealed that following an activated state, these cells enter a “primed” form. If a second immunological activation occurs during this primed state, glial cells have been shown to release proteins that actually potentiate the state of pain. This study examines this hypothesis through the context of peripheral nerve injury (modeling injury, surgery, or trauma), followed by an administration of morphine. Using advanced imaging techniques, we are able to visualize exactly what modulators are inducing this potentiated pain response, as well as where in the nervous system these cells are found. Morphine has a potent effect on various aspects of the nervous system which may be alleviated by other treatment routes that focus specifically on the cell type and location that release the molecules that initiate the healing process. By unveiling the details of this mechanism, we can better understand how to treat patients following injury or surgery without inducing a higher pain response that can be caused by a short-term administration of morphine. To see the complete thesis, please visit https://scholar.colorado.edu/concern/undergraduate_honors_theses/0k225c53t.

A phosphodeficient a3 glycine receptor mutation alters synaptic glycine and GABA release in mouse spinal dorsal horn neurons

A phosphodeficient a3 glycine receptor mutation alters synaptic glycine and GABA release in mouse spinal dorsal horn neurons

Spinal neuromodulation by intrathecal oxytocin administration mitigates the myocardial ischemia induced sympathoexcitation by suppressing the ischemia-related spinal neural hyperactivity

Background: Myocardial ischemia can lead to fatal ventricular arrhythmias by disrupting the cardiospinal neural network which controls the sympathoexcitation. Spinal neuromodulation techniques could decrease myocardial ischemia-induced sympathoexcitation by modulating the spinal neural network. Hypothesis: We hypothesize that spinal oxytocin (OXT) administration decreases myocardial ischemia-induced sympathoexcitation by suppressing spinal neural hyperactivity in the dorsal horn (DH) and intermediolateral nucleus regions (IML). Materials and Methods: Anesthetized pigs underwent laminectomy to expose the spinal T2 section and a sternotomy to expose the heart for performing left anterior descending coronary artery ischemia (LAD) with and without intrathecal oxytocin (90ug) treatment. Electrocardiograms were recorded using a 56-channel mesh electrode to evaluate the activation recovery interval (ARI) and dispersion of repolarization (DOR) which are the markers for sympathoexcitation and arrhythmogenecity. Extracellular in-vivo spinal neural recordings were done by using a 64-channel microelectrode array inserted at the T2 segment of the spinal cord to record the activity of DH and IML neurons. Oxytocin was administered intrathecally at T1-T3. Spinal neural activity, ARI, and DOR were evaluated during myocardial ischemia with vs. without intrathecal oxytocin administration. Results: Sixteen Yorkshire pigs entered into this study. In 13 animals (LAD:7, LAD+OXT:6) ARI and DOR were evaluated during long LAD ischemia (1hr). ARI shortening due to LAD was mitigated by OXT (LAD:-33 ± 4 ms; LAD+OXT: -15 ± 1 ms, P=0.001). OXT decreased the LAD-induced augmented DOR (LAD: 2194± 310 ms2; LAD+OXT: 1099± 305 ms2, P=0.029). We have further evaluated the effect of OXT on short LAD ischemia (5 mins) and the impact of intrathecal OXT agonist on the spinal cord neural processing of myocardial ischemia in 3 pigs. Out of 88 recorded neurons, LAD increased the firing rate of more neurons than the LAD+OXT (LAD: increased 52, decreased 12 neurons; LAD+OXT: increased 16, decreased 14 neurons, P=0.007). The LAD-induced spinal cord hyperactivity was mitigated after intrathecal OXT administration (LAD:1.57 ± 0.52 Hz to LAD+OXT: 0.02 ± 0.22 Hz, P=0.0001). OXT also mitigated the reperfusion-induced spinal neuronal hyperactivity (LAD:2.16 ± 0.59 Hz to LAD+OXT: -0.36 ± 0.28 Hz, P<0.0001). OXT showed a similar effect in reducing the LAD-induced sympathoexcitation in these three animals (ARI shortening: LAD: -69 ± 14 ms; LAD+OXT: -38 ± 8 ms, P=0.035). Conclusion: Myocardial ischemia induces sympathoexcitation by increasing the spinal neural activity. OXT mitigates the adverse effect of myocardial ischemia by suppressing spinal neural hyperactivity. Funding: NIH (R01) HL136836 (Dr.Mahajan) and University of Pittsburgh Medical Center Competitive Medical Research Fund (Dr.Salavatian). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

STING controls opioid-induced itch and chronic itch via spinal tank-binding kinase 1-dependent type I interferon response in mice

BackgroundPatients receiving epidural or intrathecal opioids administration for neuraxial analgesia frequently suffer from an irritating itch. STING (stimulator of interferon genes), an innate immune modulator, is strongly implicated in pain pathogenesis via neuron-immune modulation. Given that pain and itch share some common neurocircuits, we evaluate the therapeutic potential of STING agonists in opioid-induced itch and chronic itch.MethodsOpioids (morphine, fentanyl and sufentanil) were intrathecally injected to induce acute itch. Chronic itch was induced by dry skin and contact dermatitis. Opioids analgesic effect, itch-induced scratching behavior, spinal expression of STING, phosphorylation of TBK1 (tank-binding kinase 1), IRF3 (interferon regulatory factor-3) and ERK (extracellular signal-regulated kinase), as well as production of IFN-α and IFN-β were examined. STING agonists (DMXAA and ADU-S100), TBK1 inhibitor, recombinant IFN-α and IFN-β elucidated the mechanism and treatment of itch. Whole-brain functional connectivity was evaluated using resting-state fMRI.ResultsWe report the primary expression of STING protein by the spinal dorsal horn neurons. Intraperitoneal injection of DMXAA dose-dependently reduces morphine-induced scratch bouts, without impairing morphine antinociception. Simultaneously, DMXAA alleviates fentanyl- and sufentanil-induced itching-like behavior, and chronic scratching behavior caused by dry skin and contact dermatitis. Furthermore, DMXAA drastically increases spinal phosphorylation of TBK1 and IRF3 following morphine exposure, dry skin and contact dermatitis. DMXAA-induced anti-pruritus effects and spinal productions of IFN-α and IFN-β are compensated by intrathecal delivery of the TBK1 inhibitor. Also, ADU-S100, recombinant IFN-α and IFN-β exhibits remarkable attenuation in scratching behaviors after morphine injection and dermatitis. Recombinant IFN-α inhibits morphine-induced spinal phosphorylation of ERK. Finally, DMXAA prevents dermatitis-induced the increase of cerebral functional connectivity between regions of interests such as primary somatosensory cortex, piriform cortex, retrosplenial cortex, colliculus and ventral thalamus.ConclusionsSTING activation confers protection against opioid-induced itch and chronic itch through spinal up-regulation of TBK1-IRF3-type I interferon cascades in mice, suggesting that STING agonists are promising candidates in translational development for pruritus relief.

Inflammation and subsequent nociceptor sensitization in the bone marrow are involved in an animal model of osteoarthritis pain

AimsThis study aimed to determine whether pathological changes in the bone marrow cause Osteoarthritis (OA) pain based on magnetic resonance imaging (MRI), immunohistochemistry, and electrophysiology. Main methodsAdjuvant-induced arthritis (AIA) was achieved by injecting 150 μL of complete Freund's adjuvant into the right knee joints of male Sprague-Dawley rats. AIA rats were compared with saline-injected rats. Key findingsAIA significantly induced mechanical hyperalgesia and spontaneous pain in the right hind paw 1–14 days after induction. Intratibial injection of 50 μL of 1 % lidocaine significantly suppressed AIA-induced mechanical hyperalgesia (p = 0.0001) and spontaneous pain (p = 0.0006) 3 days after induction. In T2-weighted MRI, AIA induced high-signal intensity within the proximal tibial metaphysis, and the mean T2 values in this area significantly increased on days 3 (p = 0.0043) and 14 (p = 0.0012) after induction. AIA induced intraosseous edema and significantly increased the number of intraosseous granulocytes on days 3 (p < 0.0001) and 14 (p < 0.0001) after induction. The electrophysiological study on days 3–7 after induction showed significantly increased spontaneous firing rates (p = 0.0166) and evoked responses to cutaneous stimuli (brush, p < 0.0001; pinching, p = 0.0359) in the right hind paw plantar surface and intratibial stimuli (p = 0.0002) in wide-dynamic-range neurons of the spinal dorsal horn. SignificanceIntraosseous changes caused by OA induce hypersensitivity in the sensory afferents innervating bone marrow may be involved in OA pain. Novel bone marrow-targeted therapies could be beneficial for treating OA pain.

NMDA Receptors at Primary Afferent-Excitatory Neuron Synapses Differentially Sustain Chemotherapy- and Nerve Trauma-Induced Chronic Pain.

The spinal dorsal horn contains vesicular glutamate transporter-2 (VGluT2)-expressing excitatory neurons and vesicular GABA transporter (VGAT)-expressing inhibitory neurons, which normally have different roles in nociceptive transmission. Spinal glutamate NMDAR hyperactivity is a crucial mechanism of chronic neuropathic pain. However, it is unclear how NMDARs regulate primary afferent input to spinal excitatory and inhibitory neurons in neuropathic pain. Also, the functional significance of presynaptic NMDARs in neuropathic pain has not been defined explicitly. Here we showed that paclitaxel treatment or spared nerve injury (SNI) similarly increased the NMDAR-mediated mEPSC frequency and dorsal root-evoked EPSCs in VGluT2 dorsal horn neurons in male and female mice. By contrast, neither paclitaxel nor SNI had any effect on mEPSCs or evoked EPSCs in VGAT neurons. In mice with conditional Grin1 (gene encoding GluN1) KO in primary sensory neurons (Grin1-cKO), paclitaxel treatment failed to induce pain hypersensitivity. Unexpectedly, SNI still caused long-lasting pain hypersensitivity in Grin1-cKO mice. SNI increased the amplitude of puff NMDA currents in VGluT2 neurons and caused similar depolarizing shifts in GABA reversal potentials in WT and Grin1-cKO mice. Concordantly, spinal Grin1 knockdown diminished SNI-induced pain hypersensitivity. Thus, presynaptic NMDARs preferentially amplify primary afferent input to spinal excitatory neurons in neuropathic pain. Although presynaptic NMDARs are required for chemotherapy-induced pain hypersensitivity, postsynaptic NMDARs in spinal excitatory neurons play a dominant role in traumatic nerve injury-induced chronic pain. Our findings reveal the divergent synaptic connectivity and functional significance of spinal presynaptic and postsynaptic NMDARs in regulating cell type-specific nociceptive input in neuropathic pain with different etiologies.SIGNIFICANCE STATEMENT Spinal excitatory neurons relay input from nociceptors, whereas inhibitory neurons repress spinal nociceptive transmission. Chronic nerve pain is associated with aberrant NMDAR activity in the spinal dorsal horn. This study demonstrates, for the first time, that chemotherapy and traumatic nerve injury preferentially enhance the NMDAR activity at primary afferent-excitatory neuron synapses but have no effect on primary afferent input to spinal inhibitory neurons. NMDARs in primary sensory neurons are essential for chemotherapy-induced chronic pain, whereas nerve trauma causes pain hypersensitivity predominantly via postsynaptic NMDARs in spinal excitatory neurons. Thus, presynaptic and postsynaptic NMDARs at primary afferent-excitatory neuron synapses are differentially engaged in chemotherapy- and nerve injury-induced chronic pain and could be targeted respectively for treating these painful conditions.

DHX9/DNA-tandem repeat-dependent downregulation of ciRNA-Fmn1 in the dorsal horn is required for neuropathic pain.

Circular RNAs (ciRNAs) are emerging as new players in the regulation of gene expression. However, how ciRNAs are involved in neuropathic pain is poorly understood. Here, we identify the nervous-tissue-specific ciRNA-Fmn1 and report that changes in ciRNA-Fmn1 expression in spinal cord dorsal horn neurons play a key role in neuropathic pain after nerve injury. ciRNA-Fmn1 was significantly downregulated in ipsilateral dorsal horn neurons after peripheral nerve injury, at least in part because of a decrease in DNA helicase 9 (DHX9), which regulates production of ciRNA-Fmn1 by binding to DNA-tandem repeats. Blocking ciRNA-Fmn1 downregulation reversed nerve-injury-induced reductions in both the binding of ciRNA-Fmn1 to the ubiquitin ligase UBR5 and the level of ubiquitination of albumin (ALB), thereby abrogating the nerve-injury-induced increase of ALB expression in the dorsal horn and attenuating the associated pain hypersensitivities. Conversely, mimicking downregulation of ciRNA-Fmn1 in naïve mice reduced the UBR5-controlled ubiquitination of ALB, leading to increased expression of ALB in the dorsal horn and induction of neuropathic-pain-like behaviors in naïve mice. Thus, ciRNA-Fmn1 downregulation caused by changes in binding of DHX9 to DNA-tandem repeats contributes to the genesis of neuropathic pain by negatively modulating UBR5-controlled ALB expression in the dorsal horn.

In Vivo Measurements Reveal that Both Low- and High-frequency Spinal Cord Stimulation Heterogeneously Modulate Superficial Dorsal Horn Neurons

Current sub-perception spinal cord stimulation (SCS) is characterized by the use of high-frequency pulses to achieve paresthesia-free analgesic effects. High-frequency SCS demonstrates distinctive properties from paresthesia-based SCS, such as a longer time course to response, implying the existence of alternative mechanism(s) of action beyond gate control theory. We quantified the responses to SCS of single neurons within the superficial dorsal horn (SDH), a structure in close proximity to SCS electrodes, to investigate the mechanisms underlying high-frequency SCS in 62 urethane-anesthetized male rats. Sciatic nerve stimulation was delivered to isolate lumbar SDH neurons with evoked C-fiber activity. The evoked C-fiber activity before and after the application of SCS was compared to quantify the effects of SCS across stimulation intensity and stimulation duration at three different stimulation frequencies. We observed heterogeneous responses of SDH neurons which depended primarily on the type of unit. Low-threshold units with spontaneous activity, putatively inhibitory interneurons, tended to be facilitated by SCS while the other unit types were suppressed. The effects of SCS were more prominent with increased stimulation duration from 30 s to 30 m across frequencies. Our results highlight the importance of inhibitory interneurons in modulating local circuits of the SDH and the importance of local circuit contributions to the analgesic mechanisms of SCS.

Characterisation of NPFF-expressing neurons in the superficial dorsal horn of the mouse spinal cord

Excitatory interneurons in the superficial dorsal horn (SDH) are heterogeneous, and include a class known as vertical cells, which convey information to lamina I projection neurons. We recently used pro-NPFF antibody to reveal a discrete population of excitatory interneurons that express neuropeptide FF (NPFF). Here, we generated a new mouse line (NPFFCre) in which Cre is knocked into the Npff locus, and used Cre-dependent viruses and reporter mice to characterise NPFF cell properties. Both viral and reporter strategies labelled many cells in the SDH, and captured most pro-NPFF-immunoreactive neurons (75–80%). However, the majority of labelled cells lacked pro-NPFF, and we found considerable overlap with a population of neurons that express the gastrin-releasing peptide receptor (GRPR). Morphological reconstruction revealed that most pro-NPFF-containing neurons were vertical cells, but these differed from GRPR neurons (which are also vertical cells) in having a far higher dendritic spine density. Electrophysiological recording showed that NPFF cells also differed from GRPR cells in having a higher frequency of miniature EPSCs, being more electrically excitable and responding to a NPY Y1 receptor agonist. Together, these findings indicate that there are at least two distinct classes of vertical cells, which may have differing roles in somatosensory processing.