Physiology education in China: the current situation and changes over the past 3 decades.

OBJECTIVE: As an experimental biological science, physiology has been taught as an integral component of medical curricula for a long time in China. The teaching effectiveness of physiology courses will directly affect students' learning of other medical disciplines. The purpose of this study is to investigate the current situation and changes in physiology teaching over 30 years in Chinese medical schools. METHODS: National survey was conducted online on the platform SoJump via WeChat and the web. The head of the physiology department in medical school was asked to indicate the information of physiology education from three periods: 1991-2000, 2001-2010, and 2011-2020. The responses of 80 leaders of the Department of Physiology from mainland Chinese medical schools were included in the study for analysis. RESULTS: The survey showed that the class hours, both of theory and practice, had been decreased. During the past 20 years, the total number of physiology teachers, the number of physiology teachers who had been educated in medical schools, and the number of technicians had been reduced, whereas teachers with doctor's degrees had been increased. In addition to traditional didactic teaching, new teaching approaches, including problem-based learning/case-based learning/team-based learning, integrated curriculum and formative evaluation systems, had been employed, mostly for more than 5 years, in some medical schools. CONCLUSION: The present study has provided historical data regarding the current status of physiology education in China and that in the past thirty years by showing that physiology education in China has developed quickly,even it faces many challenges.

Impaired Lactate Release in Dorsal CA1 Astrocytes Contributed to Nociceptive Sensitization and Comorbid Memory Deficits in Rodents.

BACKGROUND: Memory deficits are a common comorbid disorder in patients suffering from neuropathic pain. The mechanisms underlying the comorbidities remain elusive. The hypothesis of this study was that impaired lactate release from dysfunctional astrocytes in dorsal hippocampal CA1 contributed to memory deficits. METHODS: A spared nerve injury model was established to induce both pain and memory deficits in rats and mice of both sexes. von Frey tests, novel object recognition, and conditioned place preference tests were applied to evaluate the behaviors. Whole-cell recording, fiber photometry, Western blotting, and immunohistochemistry combined with intracranial injections were used to explore the underlying mechanisms. RESULTS: Animals with spared sciatic nerve injury that had displayed nociception sensitization or memory deficit comorbidities demonstrated a reduction in the intrinsic excitability of pyramidal neurons, accompanied by reduced Ca2+ activation in astrocytes (ΔF/F, sham: 6 ± 2%; comorbidity: 2 ± 0.4%) and a decrease in the expression of glial fibrillary acidic protein and lactate levels in the dorsal CA1. Exogenous lactate supply or increasing endogenous lactate release by chemogenetic activation of astrocytes alleviated this comorbidity by enhancing the cell excitability (129 ± 4 vs. 88 ± 10 for 3.5 mM lactate) and potentiating N-methyl-d-aspartate receptor-mediated excitatory postsynaptic potentials of pyramidal neurons. In contrast, inhibition of lactate synthesis, blocking lactate transporters, or chemogenetic inhibition of astrocytes resulted in comorbidity-like behaviors in naive animals. Notably, ß2-adrenergic receptors in astrocytes but not neurons were downregulated in dorsal CA1 after spared nerve injury. Microinjection of a ß2 receptor agonist into dorsal CA1 or activation of the noradrenergic projections onto the hippocampus from the locus coeruleus alleviated the comorbidity, possibly by increasing lactate release. CONCLUSIONS: Impaired lactate release from dysfunctional astrocytes, which could be rescued by activation of the locus coeruleus, led to nociception and memory deficits after peripheral nerve injury.

Deactivation of dorsal CA1 pyramidal neurons projecting to medial prefrontal cortex contributes to neuropathic pain and short-term memory impairment.

ABSTRACT: Neuropathic pain after peripheral nerve injury is a multidimensional experience that includes sensory, affective, and cognitive components that interact with one another. Hypoexcitation of the medial prefrontal cortex (mPFC) was observed in mice with peripheral nerve injury, but the changes in neural inputs onto the mPFC have not been completely explored. Here, we report that the neural terminals from the dorsal hippocampus CA1 (dCA1) form excitatory connection with layer 5 pyramidal neurons in the prelimbic area (PrL) of the mPFC. Spared nerve injury (SNI) induced a reduction in the intrinsic excitability of dCA1 pyramidal neurons innervating the PrL and impairment in excitatory synaptic transmission onto dCA1 pyramidal cells. Specifically, activating the neural circuit from dCA1 to mPFC alleviated neuropathic pain behaviors and improved novel object recognition ability in SNI mice, whereas deactivating this pathway in naïve animals recapitulated tactile allodynia and memory deficits. These results indicated that hypoactivity in dCA1 pyramidal cells after SNI in turn deactivated layer 5 pyramidal neurons in PrL and ultimately caused pain hypersensitivity and memory deficits.

Activation of Beta-adrenergic Receptors Upregulates the Signal-to-Noise Ratio of Auditory Input in the Medial Prefrontal Cortex and Mediates Auditory Fear Conditioning.

Norepinephrine (NE) is involved in auditory fear conditioning (AFC) in posttraumatic stress disorder (PTSD). However, it is still unclear how it acts on neurons. We aimed to investigate whether the activation of the ß-adrenergic receptor (ß-AR) improves AFC by sensitization of the prelimbic (PL) cortex at the animal, cellular, and molecular levels. In vivo single-cell electrophysiological recording was used to characterize the changes in neurons in the PL cortex after AFC. Then, PL neurons were locally administrated by the ß-AR agonist isoproterenol (ISO), the GABAaR agonist muscimol, or intervened by optogenetic method, respectively. Western blotting and immunohistochemistry were finally used to assess molecular changes. Noise and low-frequency tones induced similar AFC. The expression of ß-ARs in PL cortex neurons was upregulated after fear conditioning. Microinjection of muscimol into the PL cortex blocked the conformation of AFC, whereas ISO injection facilitated AFC. Moreover, PL neurons can be distinguished into two types, with type I but not type II neurons responding to conditioned sound and being regulated by ß-ARs. Our results showed that ß-ARs in the PL cortex regulate conditional fear learning by activating type I PL neurons.

MiR-184-5p represses neuropathic pain by regulating CCL1/CCR8 signaling interplay in the spinal cord in diabetic mice.

BACKGROUND: Diabetic neuropathic pain (DNP) is a serious complication for diabetic patients involving nervous system. MicroRNAs (miRNAs) are small-noncoding RNAs which are dysregulated in neuropathic pain, and might be critical molecules for pain treatment. Our previous study has shown miR-184-5p was significantly downregulated in DNP. Therefore, the mechanism of miR-184-5p in DNP was investigated in this study. METHODS: A DNP model was established through streptozotocin (STZ). The pharmacological tools were injected intrathecally, and pain behavior was evaluated by paw withdrawal mechanical thresholds (PWMTs). Bioinformatics analysis, Dual-luciferase reporter assay and fluorescence-in-situ-hybridization (FISH) were used to seek and confirm the potential target genes of miR-184-5p. The expression of relative genes and proteins was analyzed by quantitative reverse transcriptase real-time PCR (qPCR) and western blotting. RESULTS: MiR-184-5p expression was down-regulated in spinal dorsal on days 7 and 14 after STZ, while intrathecal administration of miR-184-5p agomir attenuates neuropathic pain induced by DNP and intrathecal miR-184-5p antagomir induces pain behaviors in naïve mice. Chemokine CC motif ligand 1 (CCL1) was found to be a potential target of miR-184-5p and the protein expression of CCL1 and the mRNA expression of CCR8 were up-regulated in spinal dorsal on days 7 and 14 after STZ. The luciferase reporter assay and FISH demonstrated that CCL1 is a direct target of miR-184-5p. MiR-184-5p overexpression attenuated the expression of CCL1/CCR8 in DNP; intrathecal miR-184-5p antagomir increased the expression of CCL1/CCR8 in spinal dorsal of naïve mice. CONCLUSION: This research illustrates that miR-184-5p alleviates DNP through the inhibition of CCL1/CCR8 signaling expression.

Activation of the bile acid receptors TGR5 and FXR in the spinal dorsal horn alleviates neuropathic pain.

AIMS: Beyond digestion, bile acids have been recognized as signaling molecules with broad paracrine and endocrine functions by activating plasma membrane receptor (Takeda G protein-coupled receptor 5, TGR5) and the nuclear farnesoid X receptor (FXR). The present study investigated the role of bile acids in alleviating neuropathic pain by activating TGR5 and FXR. METHOD: Neuropathic pain was induced by spared nerve injury (SNI) of the sciatic nerve. TGR5 or FXR agonist was injected intrathecally. Pain hypersensitivity was measured with Von Frey test. The amount of bile acids was detected using a bile acid assay kit. Western blotting and immunohistochemistry were used to assess molecular changes. RESULTS: We found that bile acids were downregulated, whereas the expression of cytochrome P450 cholesterol 7ahydroxylase (CYP7A1), a rate-limiting enzyme for bile acid synthesis, was upregulated exclusively in microglia in the spinal dorsal horn after SNI. Furthermore, the expression of the bile acid receptors TGR5 and FXR was increased in glial cells and GABAergic neurons in the spinal dorsal horn on day 7 after SNI. Intrathecal injection of either TGR5 or FXR agonist on day 7 after SNI alleviated the established mechanical allodynia in mice, and the effects were blocked by TGR5 or FXR antagonist. Bile acid receptor agonists inhibited the activation of glial cells and ERK pathway in the spinal dorsal horn. All of the above effects of TGR5 or FXR agonists on mechanical allodynia, on the activation of glial cells, and on ERK pathway were abolished by intrathecal injection of the GABAA receptor antagonist bicuculline. CONCLUSION: These results suggest that activation of TGR5 or FXR counteracts mechanical allodynia. The effect was mediated by potentiating function of GABAA receptors, which then inhibited the activation of glial cells and neuronal sensitization in the spinal dorsal horn.

Glucocorticoid regulation of lactate release from spinal astrocytes contributes to the induction of spinal LTP of C-fiber-evoked field potentials and the development of mechanical allodynia.

High-frequency stimulation (HFS) of the sciatic nerve leads to long-term potentiation (LTP) at C-fiber synapse and long-lasting pain hypersensitivity. The underlying mechanisms, however, are still unclear. In the present study, we investigated the involvement of astrocytes derived l-lactate in the spinal dorsal horn subsequent to glucocorticoid (GC) secretion into the plasma in this process using Sprague-Dawley rats and Aldh1L1-CreERT2 mice of either sex. We found that HFS increased l-lactate and monocarboxylate transporters 1/2 (MCT1/2) in the spinal dorsal horn. Inhibition of glycogenolysis or blocking lactate transport prevented the induction of spinal LTP following HFS. Furthermore, Chemogenetical inhibition of dorsal horn astrocytes, which were activated by HFS, prevented spinal LTP, alleviated the mechanical allodynia and the decreased the level l-lactate and GFAP expression in the dorsal horn following HFS. In contrast, Chemogenetics activation of dorsal horn astrocytes in naïve rats induced spinal LTP as well as mechanical allodynia, and increased GFAP expression and l-lactate. Application of l-lactate directly to the spinal cord of naïve rats induced spinal LTP, mechanical allodynia, and increased spinal expression of p-ERK. Importantly, HFS increased GC in the plasma and glucocorticoid receptor (GR) expression in spinal astrocytes, adrenalectomy or knocking down of GR in astrocytes by using Cre-Loxp system blocked the mechanical allodynia, prevented the spinal LTP and the enhancement of lactate after HFS. These results show that lactate released from spinal astrocytes following glucocorticoid release into the plasma enhance synaptic transmission at the C-fiber synapse and underlie pain chronicity.

MiR-106b-5p Attenuates Neuropathic Pain by Regulating the P2X4 Receptor in the Spinal Cord in Mice.

The P2X4 receptor (P2X4R) can be upregulated after nerve injury, and its mediated spinal microglial activation makes a critical contribution to pathologically enhanced pain processing in the dorsal horn. Although some studies have partly clarified the mechanism underlying altered P2X4R expression, the specific mechanism is not well understood. MicroRNAs (miRNAs) are small noncoding RNAs which control gene expression by binding with their target mRNAs. Thus, in the present study, we investigated whether miRNA is involved in the pathogenesis of neuropathic pain by regulating P2X4R. Our results showed that P2X4R was upregulated in the spinal dorsal horn of mice following spared nerve injury (SNI), and 69 miRNAs (46 upregulated and 23 downregulated miRNAs) were differentially expressed (fold change > 2.0, P < 0.05). P2X4R was found to be a major target of miR-106b-5p (one of the downregulated miRNAs) using bioinformatics technology; quantitative real-time PCR analysis confirmed the change in expression of miR-106b-5p, and dual-luciferase reporter assays confirmed the correlation between them. Fluorescence in situ hybridization was used to show cell co-localization of P2X4R and miR-106b-5p in the spinal dorsal horn. Transfection with miR-106b-5p mimic into BV2 cells reversed the upregulation of P2X4R induced by lipopolysaccharide (LPS). Moreover, miR-106b-5p overexpression significantly attenuated neuropathic pain induced by SNI, with decreased expression of P2X4R mRNA and protein in the spinal dorsal horn; intrathecal miR-106b-5p antagomir induced pain behaviors, and increased expression of P2X4R in the spinal dorsal horn of naïve mice. These data suggest that miR-106b-5p can serve as an important regulator of neuropathic pain development by targeting P2X4R.

Activation of the dorsal, but not the ventral, hippocampus relieves neuropathic pain in rodents.

ABSTRACT: Accumulating evidence suggests hippocampal impairment under the chronic pain phenotype. However, it is unknown whether neuropathic behaviors are related to dysfunction of the hippocampal circuitry. Here, we enhanced hippocampal activity by pharmacological, optogenetic, and chemogenetic techniques to determine hippocampal influence on neuropathic pain behaviors. We found that excitation of the dorsal (DH), but not the ventral (VH) hippocampus induces analgesia in 2 rodent models of neuropathic pain (SNI and SNL) and in rats and mice. Optogenetic and pharmacological manipulations of DH neurons demonstrated that DH-induced analgesia was mediated by N-Methyl-D-aspartate and µ-opioid receptors. In addition to analgesia, optogenetic stimulation of the DH in SNI mice also resulted in enhanced real-time conditioned place preference for the chamber where the DH was activated, a finding consistent with pain relief. Similar manipulations in the VH were ineffective. Using chemo-functional magnetic resonance imaging (fMRI), where awake resting-state fMRI was combined with viral vector-mediated chemogenetic activation (PSAM/PSEM89s) of DH neurons, we demonstrated changes of functional connectivity between the DH and thalamus and somatosensory regions that tracked the extent of relief from tactile allodynia. Moreover, we examined hippocampal functional connectivity in humans and observe differential reorganization of its anterior and posterior subdivisions between subacute and chronic back pain. Altogether, these results imply that downregulation of the DH circuitry during chronic neuropathic pain aggravates pain-related behaviors. Conversely, activation of the DH reverses pain-related behaviors through local excitatory and opioidergic mechanisms affecting DH functional connectivity. Thus, this study exhibits a novel causal role for the DH but not the VH in controlling neuropathic pain-related behaviors.

Liver X Receptor α in Sciatic Nerve Exerts an Alleviating Effect on Neuropathic Pain Behaviors Induced by Crush Injury.

Peripheral nerve injury often leads to neuropathic pain. In the present study, we assessed the role of liver x receptor alpha (LXRα), an oxysterol regulated nuclear transcription factor that promotes reverse cholesterol transport and alternative (M2) macrophage activation, in the development of neuropathic pain. We found that compared to WT mice, in LXRα knockout mice the development of mechanical allodynia following sciatic nerve crush was accelerated and the duration was prolonged. Furthermore, the expression of M1-like macrophage marker iNOS and M1-like macrophages inducer hydrogen peroxide (H2O2) was increased, whereas expression of M2 macrophage marker arginase-1 (Arg-1) and interleukin-10 (IL-10) was reduced in the sciatic nerve of LXRα knockout mice. Moreover, peri-sciatic administration of LXRs agonist GW3965, immediately after the nerve crush, into wild type mice, suppressed the mechanical allodynia induced by crush injury. GW3965 also suppressed the expression of iNOS and production of H2O2 in the injured nerve and enhanced the expression of IL-10 and Arg-1. Importantly, peri-sciatic administration of IL-10 neutralization antibody prevented the alleviating effect of GW3965 on mechanical allodynia. Altogether, these results indicates that the lack of LXRα in the sciatic nerve results in an augmented inflammatory profile of macrophages, which ultimately speed up the development of neuropathic pain and dampen its recovery following nerve injury. Activation of LXRα by its agonist might rebalance the neuroprotective and neurotoxic macrophage phenotypes, and thus alleviate the neuropathic pain behavior.

Adaptive alterations in the mesoaccumbal network after peripheral nerve injury.

ABSTRACT: The nucleus accumbens (NAc) and the ventral tegmental area (VTA) are critical hubs in the brain circuitry controlling chronic pain. Yet, how these 2 regions interact to shape the chronic pain state is poorly understood. Our studies show that in mice, spared nerve injury (SNI) induced alterations in the functional connectome of D2-receptor expressing spiny projection neurons in the core region of the NAc-enhancing connections with prelimbic cortex and weakening them with basolateral amygdala. These changes, which were attributable in part to SNI-induced suppression of VTA dopaminergic signaling, were adaptive because mimicking them chemogenetically alleviated the anxiety and social withdrawal accompanying injury. By contrast, chemogenetic enhancement of activity in VTA dopaminergic neurons projecting to the medial shell of the NAc selectively suppressed tactile allodynia in SNI mice. These results suggest that SNI induces regionally specific alterations in VTA dopaminergic signaling in the NAc to promote environmental reengagement after injury. However, countervailing, homeostatic mechanisms limit these adaptive changes, potentially leading to the chronic pain state.

Normalization of magnesium deficiency attenuated mechanical allodynia, depressive-like behaviors, and memory deficits associated with cyclophosphamide-induced cystitis by inhibiting TNF-α/NF-κB signaling in female rats.

BACKGROUND: Bladder-related pain symptoms in patients with bladder pain syndrome/interstitial cystitis (BPS/IC) are often accompanied by depression and memory deficits. Magnesium deficiency contributes to neuroinflammation and is associated with pain, depression, and memory deficits. Neuroinflammation is involved in the mechanical allodynia of cyclophosphamide (CYP)-induced cystitis. Magnesium-L-Threonate (L-TAMS) supplementation can attenuate neuroinflammation. This study aimed to determine whether and how L-TAMS influences mechanical allodynia and accompanying depressive symptoms and memory deficits in CYP-induced cystitis. METHODS: Injection of CYP (50 mg/kg, intraperitoneally, every 3 days for 3 doses) was used to establish a rat model of BPS/IC. L-TAMS was administered in drinking water (604 mg·kg-1·day-1). Mechanical allodynia in the lower abdomen was assessed with von Frey filaments using the up-down method. Forced swim test (FST) and sucrose preference test (SPT) were used to measure depressive-like behaviors. Novel object recognition test (NORT) was used to detect short-term memory function. Concentrations of Mg2+ in serum and cerebrospinal fluid (CSF) were measured by calmagite chronometry. Western blot and immunofluorescence staining measured the expression of tumor necrosis factor-α/nuclear factor-κB (TNF-α/NF-κB), interleukin-1ß (IL-1ß), and N-methyl-D-aspartate receptor type 2B subunit (NR2B) of the N-methyl-D-aspartate receptor in the L6-S1 spinal dorsal horn (SDH) and hippocampus. RESULTS: Free Mg2+ was reduced in the serum and CSF of the CYP-induced cystitis rats on days 8, 12, and 20 after the first CYP injection. Magnesium deficiency in the serum and CSF correlated with the mechanical withdrawal threshold, depressive-like behaviors, and short-term memory deficits (STMD). Oral application of L-TAMS prevented magnesium deficiency and attenuated mechanical allodynia (n = 14) and normalized depressive-like behaviors (n = 10) and STMD (n = 10). The upregulation of TNF-α/NF-κB signaling and IL-1ß in the L6-S1 SDH or hippocampus was reversed by L-TAMS. The change in NR2B expression in the SDH and hippocampus in the cystitis model was normalized by L-TAMS. CONCLUSIONS: Normalization of magnesium deficiency by L-TAMS attenuated mechanical allodynia, depressive-like behaviors, and STMD in the CYP-induced cystitis model via inhibition of TNF-α/NF-κВ signaling and normalization of NR2B expression. Our study provides evidence that L-TAMS may have therapeutic value for treating pain and comorbid depression or memory deficits in BPS/IC patients.

Nicotinamide adenine dinucleotide phosphate oxidase 2-derived reactive oxygen species contribute to long-term potentiation of C-fiber-evoked field potentials in spinal dorsal horn and persistent mirror-image pain following high-frequency stimulus of the sciatic nerve.

High-frequency stimulation (HFS) of the sciatic nerve has been reported to produce long-term potentiation (LTP) and long-lasting pain hypersensitivity in rats. However, the central underlying mechanism remains unclear. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) belongs to a group of electron-transporting transmembrane enzymes that produce reactive oxygen species (ROS). Here, we found that NOX2 was upregulated in the lumbar spinal dorsal horn after HFS of the left sciatic nerve, which induced bilateral pain and spinal LTP in both male and female rats. Blocking NOX2 with blocking peptide or shRNA prevented the development of bilateral mechanical allodynia, the induction of spinal LTP, and the phosphorylation of N-methyl-d-aspartate (NMDA) receptor 2B (GluN2B) and nuclear factor kappa-B (NF-κB) p65 after HFS. Moreover, NOX2 shRNA reduced the frequency and amplitude of both spontaneous excitatory postsynaptic currents and miniature excitatory postsynaptic currents in laminar II neurons. Furthermore, 8-hydroxyguanine (8-OHG), an oxidative stress marker, was increased in the spinal dorsal horn. Spinal application of ROS scavenger, Phenyl-N-tert-butylnitrone (PBN), depressed the already established spinal LTP. Spinal application of H2O2, one ROS, induced LTP and bilateral mechanical allodynia, increased the frequency and amplitude of spontaneous excitatory postsynaptic currents in laminar II neurons, and phosphorylated GluN2B and p65 in the dorsal horn. This study provided electrophysiological and behavioral evidence that NOX2-derived ROS in the spinal cord contributed to persistent mirror-image pain by enhancing the synaptic transmission, which was mediated by increasing presynaptic glutamate release and activation of NMDA receptor and NF-κB in the spinal dorsal horn.

Activation of liver x receptors prevents the spinal LTP induced by skin/muscle retraction in the thigh via SIRT1/NF-Κb pathway.

It has been reported that skin/muscle incision and retraction (SMIR) in the thigh, produces mechanical allodynia in the hind paw, far from the site of incision/retraction. The mechanical allodynia lasts about 22 days, indicating chronic post-operative pain develops. The precise mechanisms, however, are largely unclear. In the current study, we further found that SMIR surgery induced LTP of c-fiber evoked field potentials that lasted at least 4 h. The mRNA and protein level of tumor necrosis factor-alpha (TNFα) and acetylated nuclear factor-kappaB p65 (ac-NF-κB p65) in the lumbar spinal dorsal horn was gradually increased during LTP development, while pretreatment with either TNFα neutralization antibody or NF-κB inhibitor PDTC completely prevented the induction of LTP. Moreover, the expression of Silent information regulator 1 (SIRT1) in the lumbar spinal dorsal horn was decreased and activation of SIRT1 by SRT1720 also prevented the induction of LTP. Importantly, the spinal expression of Liver X receptors (LXRs) was increased, both at mRNA and protein level following SMIR. Application of LXRs agonist T0901317 to the spinal dorsal horn prevented LTP induction following SMIR. Mechanistically, T0901317 enhanced the expression of SIRT1 and decreased the expression of ac-NF-κB p65 and TNFα. Spinal application of SIRT1 antagonist EX-527, 30 min before T0901317 administration, completely blocked the inhibiting effect of T0901317 on LTP, and on expression of ac-NF-κB p65 and TNFα. These results indicated that activation of LXRs prevented SMIR-induced LTP by inhibiting NF-κB/TNFα pathway via increasing SIRT1 expression.

Rostral ventromedial medulla-mediated descending facilitation following P2X7 receptor activation is involved in the development of chronic post-operative pain.

Chronic postsurgical pain (CPSP) remains a medical problem. Whether the descending modulation of nociceptive transmission from the rostral ventromedial medulla (RVM) plays a role in CPSP induced by skin/muscle incision and retraction (SMIR) in the thigh is still unknown. In this study, we found that SMIR surgery, which induced either bilateral or unilateral mechanical allodynia, activated microglia, and up-regulated interleukin-1ß (IL-1ß), an important cytokine, and 8-hydroxyguanine, an oxidative stress marker in the RVM. In addition, the release of 5-hydroxytryptamine (5-HT) was increased in the ipsilateral and contralateral RVM in rats with either bilateral or unilateral pain following SMIR. The 5-HT level increase, 5-HT3 receptor (5-HT3R) up-regulation, and microglia activation were found bilaterally in SMIR rats with bilateral pain, but only ipsilaterally in SMIR rats with unilateral pain. The intrathecal injection of the 5-HT3R antagonist Y25130 prevented the development of CPSP and the activation of spinal microglia induced by SMIR. Furthermore, P2X7 receptor (P2X7R) was up-regulated in microglia in the RVM. The microinjection of the P2X7R antagonist brilliant blue G (BBG, a non-competitive P2X7R antagonist) into the RVM prevented the development of mechanical allodynia, inhibited the activation of microglia, and decreased the expression of IL-1ß and 8-hydroxyguanine in the RVM following SMIR. Importantly, BBG injected into the RVM also decreased the activation of microglia and the level of 5-HT in the lumbar 3 (L3) spinal cord. The microinjection of the P2X7R agonist BzATP, the NADPH oxidase activator phorbol-12-myristate-13-acetate, or IL-1ß into the RVM induced bilateral mechanical allodynia, microglia activation, and 5-HT release in the L3 spinal dorsal horn. Taken together, P2X7R activation in microglia in the RVM following SMIR might be responsible for the development of CPSP via activating descending serotonergic pathway. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Withdrawal from spinal application of remifentanil induces long-term potentiation of c-fiber-evoked field potentials by activation of Src family kinases in spinal microglia.

It is well known that remifentanil, a widely used intravenous anesthesia drug, can paradoxically induce hyperalgesia. The underlying mechanisms are still not clear despite the wide investigations. The present study demonstrated that withdrawal from spinal application of remifentanil could dose-dependently induce long term potentiation (LTP) of C-fiber evoked field potentials. Remifentanil withdrawal could activate Src family kinases (SFKs) in microglia, and upregulate the expression of tumor necrosis factor alpha (TNFα) in spinal dorsal horn. Furthermore, pretreatment with either microglia inhibitor Minocycline, SFKs inhibitor PP2 or TNF αneutralization antibody could block remifentanil withdrawal induced spinal LTP, whereas supplement of recombinant rat TNFα to the spinal cord could reverse the inhibitory effect of Minocycline or PP2 on remifentanil withdrawal induced LTP. Our results suggested that TNFαrelease following SFKs activation in microglia is involved in the induction of LTP induced by remifentanil withdrawal.

The role of P2X7R/ERK signaling in dorsal root ganglia satellite glial cells in the development of chronic postsurgical pain induced by skin/muscle incision and retraction (SMIR).

The mechanisms of chronic postsurgical pain remain to be elucidated. We reported here that skin/muscle incision and retraction (SMIR), a rat model of postsurgical pain, phosphorylated the extracellular regulated protein kinases (ERK) signaling components c-Raf, MEK (ERK kinase) and ERK1/2 in lumbar 3 dorsal root ganglion (L3 DRG) in rats. Intrathecal injection of ERK specific inhibitor SCH772984 suppressed the mechanical allodynia induced by SMIR. Furthermore, SMIR upregulated tumor necrosis factor alpha (TNFα) in L3 DRG, which could be inhibited by SCH772984. Intrathecal injection of TNF antagonist Etanercept could also inhibit the mechanical allodynia and the increased ERK phosphorylation in L3 DRG induced by SMIR. In addition, immunofluorescent data showed that P2X7R was located exclusively in GFAP labeled satellite glial cells and was highly colocalized with p-ERK1/2 following SMIR. Pretreatment with P2X7R antagonist Brilliant Blue G (BBG) could also block the mechanical allodynia, inhibited the phosphorylation of c-Raf, MEK, ERK1/2, and decrease the expression of TNF-α. Finally, intrathecal injection of BzATP produced mechanical allodynia and induced ERK phosphorylation in satellite glial cells in L3 DRG. Thus, P2X7R activation in satellite glial cells in L3 DRG, leading to a positive feedback between ERK pathway activation and TNF-α production, is suggested to be involved in the induction of chronic postsurgical pain following SMIR.

Liver X Receptor α Is Involved in Counteracting Mechanical Allodynia by Inhibiting Neuroinflammation in the Spinal Dorsal Horn.

BACKGROUND: Liver X receptors, including α and ß isoforms, are ligand-activated transcription factors. Whether liver X receptor α plays a role in neuropathic pain is unknown. METHODS: A spared nerve injury model was established in adult male rats and mice. Von Frey tests were performed to evaluate the neuropathic pain behavior; Western blot and immunohistochemistry were performed to understand the underlying mechanisms. RESULTS: Intrathecal injection of a specific liver X receptor agonist T0901317 or GW3965 could either prevent the development of mechanical allodynia or alleviate the established mechanical allodynia, both in rats and wild-type mice. GW3965 could inhibit the activation of glial cells and the expression of tumor necrosis factor-α (mean ± SD: 196 ± 48 vs. 119 ± 57; n = 6; P < 0.01) and interleukin 1ß (mean ± SD: 215 ± 69 vs. 158 ± 74; n = 6; P < 0.01) and increase the expression of interleukin 10 in the spinal dorsal horn. All of the above effects of GW3965 could be abolished by liver X receptor α mutation. Moreover, more glial cells were activated, and more interleukin 1ß was released in the spinal dorsal horn in liver X receptor α knockout mice than in wild-type mice after spared nerve injury. Aminoglutethimide, a neurosteroid synthesis inhibitor, blocked the inhibitory effect of T0901317 on mechanical allodynia, on the activation of glial cells, and on the expression of cytokines. CONCLUSIONS: Activation of liver X receptor α inhibits mechanical allodynia by inhibiting the activation of glial cells and rebalancing cytokines in the spinal dorsal horn via neurosteroids.

Oral Application of Magnesium-L-Threonate Attenuates Vincristine-induced Allodynia and Hyperalgesia by Normalization of Tumor Necrosis Factor-α/Nuclear Factor-κB Signaling.

BACKGROUND: Antineoplastic agents, including vincristine, often induce neuropathic pain and magnesium deficiency clinically, but the causal link between them has not been determined. No drug is available for treating this form of neuropathic pain. METHODS: Injection of vincristine (0.1 mg · kg · day, intraperitoneally, for 10 days) was used to induce nociceptive sensitization, which was accessed with von Frey hairs and the plantar tester in adult male Sprague-Dawley rats. Magnesium-L- threonate was administered through drinking water (604 mg · kg · day). Extracellular and intracellular free Mg were measured by Calmagite chromometry and flow cytometry. Molecular biologic and electrophysiologic experiments were performed to expose the underlying mechanisms. RESULTS: Vincristine injection induced allodynia and hyperalgesia (n = 12), activated tumor necrosis factor-α/nuclear factor-κB signaling, and reduced free Mg in cerebrospinal fluid by 21.7 ± 6.3% (mean ± SD; n = 13) and in dorsal root ganglion neurons by 27 ± 6% (n = 11). Reducing Mg activated tumor necrosis factor-α/nuclear factor-κB signaling in cultured dorsal root ganglion neurons. Oral application of magnesium-L-threonate prevented magnesium deficiency and attenuated both activation of tumor necrosis factor-α/nuclear factor-κB signaling and nociceptive sensitization (n = 12). Mechanistically, vincristine induced long-term potentiation at C-fiber synapses, up-regulated N-methyl-D-aspartate receptor type 2B subunit of N-methyl-D-aspartate receptor, and led to peptidergic C-fiber sprouting in spinal dorsal horn (n = 6 each). The vincristine-induced pathologic plasticity was blocked by intrathecal injection of nuclear factor-κB inhibitor (n = 6), mimicked by tumor necrosis factor-α, and substantially prevented by oral magnesium-L-threonate (n = 5). CONCLUSIONS: Vincristine may activate tumor necrosis factor-α/nuclear factor-κB pathway by reduction of intracellular magnesium, leading to spinal pathologic plasticity and nociceptive sensitization. Oral magnesium-L-threonate that prevents the magnesium deficiency is a novel approach to prevent neuropathic pain induced by chemotherapy.