Search

Single-cell transcriptomics analysis is an advanced technology that can describe the intracellular transcriptome in complex tissues. It profiles and analyses datasets by single-cell RNA sequencing. Neurodegenerative diseases are identified by the abnormal apoptosis of neurons in the brain with few or no effective therapy strategies at present, which has been a growing healthcare concern and brought a great burden to society. The transcriptome of individual cells provides deep insights into previously unforeseen cellular heterogeneity and gene expression differences in neurodegenerative disorders. It detects multiple cell subsets and functional changes during pathological progression, which deepens the understanding of the molecular underpinnings and cellular basis of neurodegenerative diseases. Furthermore, the transcriptome analysis of immune cells shows the regulation of immune response. Different subtypes of immune cells and their interaction are found to contribute to disease progression. This finding enables the discovery of novel targets and biomarkers for early diagnosis. In this review, we emphasize the principles of the technology, and its recent progress in the study of cellular heterogeneity and immune mechanisms in neurodegenerative diseases. The application of single-cell transcriptomics analysis in neurodegenerative disorders would help explore the pathogenesis of these diseases and develop novel therapeutic methods.

Gastric cancer (GC) causes no symptoms at early stages. However, with the progression, GC causes symptoms mimicking normal gastrointestinal issues, such as irritable bowel syndrome (IBS), gastroesophageal reflux disease (GERD), gastritis, or peptic ulcers (PU). CircRNA circSOBP has been characterized as a critical regulator in prostate cancer. The present study aimed to study its involvement in GC.Plasma samples were collected from GC patients (Decreased circSOBP RNA accumulation was observed in GC tissues compared to normal tissue samples. Decreased plasma circSOBP accumulation was only observed in GC patients, but not other patients, compared to HCs. With plasma circSOBP as a biomarker, GC patients were separated from other patients and HCs. Patients with high plasma or tissue levels of circSOBP showed better survival conditions. In addition, plasma and tissue circSOBP levels were only closely correlated with GC patients' tumor metastasis, but not other clinical factors.Decreased circSOBP accumulation may be applied in clinical practice to improve the diagnosis and prognosis of GC.

OBJECTIVE To investigate the effect and potential mechanism of quercetin on inflammation, oxidative stress, apoptosis, and mitochondrial structure and function in H9C2 cells. MATERIALS AND METHODS H9C2 cells were obtained from the Shanghai Institutes for Biological Sciences, Chinese Academy of Science, and randomly divided into six groups: control, model, PVT1 overexpression (OV), quercetin, OV + quercetin, and NAC groups. The CCK-8 assay was performed to examine cell proliferation. Flow cytometry was used to examine cell apoptosis, cell membrane potential, and ROS levels. The expression of endothelial nitric oxide synthase (eNOS), malondialdehyde (MDA), and superoxide dismutase (SOD) was measured by ELISA and a Biochemical kit. Western blotting was used to determine the levels of p-DRP1 (s637), MFN2, NF-kB, p-NF-kB, IkB, and p-IkB. IL-6, IL-10, TNF-α, and IL-1β mRNA expression was examined by RT-PCR. Electron microscopy was used to observe the structure of mitochondria in H9C2 cells. RESULTS MDA, p-NF-κB, p-IKB, IL-6, IL-1β, and TNF-α expression levels, and the cell apoptosis rate were significantly higher in the model group than in the control group (P

Reperfusion of blood flow during ischemic myocardium resuscitation induces ischemia/reperfusion (I/R) injury. Oxidative stress has been identified as a major cause in this process. Quercetin (QCT) is a member of the flavonoid family that exerts antioxidant effects. The aim of this study was to investigate the preventive effects of QCT on I/R injury and its underlying mechanism. To this end, H9c2 cardiomyocytes were treated with different concentrations of QCT (10, 20, and 40 μM) and subsequently subjected to oxygen-glucose deprivation/reperfusion (OGD/R) administration. The results indicated that OGD/R-induced oxidative stress, apoptosis, and mitochondrial dysfunction in H9c2 cardiomyocytes were aggravated following 40 μM QCT treatment and alleviated following the administration of 10 and 20 μM QCT prior to OGD/R treatment. In addition, OGD/R treatment inactivated ERK1/2 signaling activation. The effect was mitigated using 10 and 20 μM QCT prior to OGD/R treatment. In conclusion, these results suggested that low concentrations of QCT might alleviate I/R injury by suppressing oxidative stress and improving mitochondrial function through the regulation of ERK1/2-DRP1 signaling, providing a potential candidate for I/R injury prevention.

BACKGROUND: Multiorgan injury has been implicated in patients with coronavirus disease 2019 (COVID-19). We aim to assess the impact of organ injury (OI) on prognosis according to the number of affected organs at admission. METHODS: This is a retrospective cohort study of patients with confirmed COVID-19 in Wuhan Third Hospital & Tongren Hospital of Wuhan University from February 17 to March 22, 2020. We classified the patients according to the presence and number of damaged organs (heart, liver, and kidney). RESULTS: The percentage of patients with no, one, two, or three organs affected was 59.75%, 30.46%, 8.07%, and 1.72%, respectively. With the increasing number of OI, there is a tendency of gradual increase regarding the white blood cell counts, neutrophil counts, levels of C-reactive protein, lactate dehydrogenase, D-dimer, and fibrinogen as well as the incidence of most complications. In a Cox regression model, individuals with OI, old age, and an abnormal level of C-reactive protein were at a higher risk of death compared with those without. Patients with three organ injuries had the highest mortality rate [57.9%; hazard ratio (HR) with 95% confidence interval (CI) vs. patients without OI: 22.31 (10.42-47.77), those with two (23.6%; HR 8.68, 95% CI 4.58-16.48), one (8.6%; HR 3.1, 95% CI 1.7-5.7), or no OI (2.6%); P < 0.001]. CONCLUSION: The increasing number of OI was associated with a high risk of mortality in COVID-19 infection. This article is protected by copyright. All rights reserved.

This retrospective study was designed to explore whether neutrophil to lymphocyte ratio (NLR) is a prognostic factor in patients with coronavirus disease 2019 (COVID‐19). A cohort of patients with COVID‐19 admitted to the Tongren Hospital of Wuhan University from 11 January 2020 to 3 March 2020 was retrospectively analyzed. Patients with hematologic malignancy were excluded. The NLR was calculated by dividing the neutrophil count by the lymphocyte count. NLR values were measured at the time of admission. The primary outcome was all‐cause in‐hospital mortality. A multivariate logistic analysis was performed. A total of 1004 patients with COVID‐19 were included in this study. The mortality rate was 4.0% (40 cases). The median age of nonsurvivors (68 years) was significantly older than survivors (62 years). Male sex was more predominant in nonsurvival group (27; 67.5%) than in the survival group (466; 48.3%). NLR value of nonsurvival group (median: 49.06; interquartile range [IQR]: 25.71‐69.70) was higher than that of survival group (median: 4.11; IQR: 2.44‐8.12; P, Highlights NLR is a significant prognostic biomarker of outcomes in critically ill patients with COVID‐19.COVID‐19 is more likely to infected those elder men with chronic comorbidities.NLR may also help in the early identification of older patients at higher risk of COVID‐19.Close monitoring and timely intervention are needed for elderly patients with COVID‐19.

Systemic lupus erythematosus (SLE) is a multi-organ disease of unknown etiology in which the normal immune responses are directed against the body's own healthy tissues. Patients with SLE often suffer from chronic pain. Currently, no animal studies have been reported about the mechanisms underlying pain in SLE. In this study, the development of chronic pain in MRL lupus-prone (MRL/lpr) mice, a well-established lupus mouse model, was characterized for the first time. We found that female MRL/lpr mice developed thermal hyperalgesia at the age of 13 weeks, and mechanical allodynia at the age of 16 weeks. MRL/lpr mice with chronic pain had activation of microglia and astrocytes, over-expression of macrophage colony-stimulating factor-1 (CSF-1) and interleukin-1 beta (IL-1β), as well as suppression of glial glutamate transport function in the spinal cord. Intrathecal injection of either the CSF-1 blocker or IL-1 inhibitor attenuated thermal hyperalgesia in MRL/lpr mice. We provide evidence that the suppressed activity of glial glutamate transporters in the spinal dorsal horn in MRL/lpr mice is caused by activation of the CSF-1 and IL-1β signaling pathways. Our findings suggest that targeting the CSF-1 and IL-1β signaling pathways or the glial glutamate transporter in the spinal cord is an effective approach for the management of chronic pain caused by SLE.

Patients receiving paclitaxel for cancer treatment often develop an acute pain syndrome (paclitaxel-associated acute pain syndrome, P-APS), which occurs immediately after paclitaxel treatment. Mechanisms underlying P-APS remain largely unknown. We recently reported that rodents receiving paclitaxel develop acute pain and activation of spinal microglial toll like receptor 4 (TLR4) by paclitaxel penetrating into the spinal cord is a critical event in the genesis of P-APS. Our current study dissected cellular and molecular mechanisms underlying the P-APS. We demonstrated that bath-perfusion of paclitaxel, at a concentration similar to that found in the cerebral spinal fluid in animals receiving i.v. paclitaxel (2 mg/kg), resulted in increased calcium activity in microglia instantly, and in astrocytes with 6 min delay. TLR4 activation in microglia by paclitaxel caused microglia to rapidly release interleukin-1β (IL-1β) but not tumor necrosis factor α, IL-6, or interferon-γ. IL-1β release from microglia depended on capthepsin B. IL-1β acted on astrocytes, leading to elevated calcium activity and suppressed glutamate uptake. IL-1β also acted on neurons to increase presynaptic glutamate release and postsynaptic AMPA receptor activity in the spinal dorsal horn. Knockout of IL-1 receptors prevented the development of acute pain induced by paclitaxel in mice. Our study indicates that IL-1β is a crucial molecule used by microglia to alter functions in astrocytes and neurons upon activation of TLR4 in the genesis of P-APS, and targeting the signaling pathways regulating the production and function of IL-1β from microglia is a potential avenue for the development of analgesics for the treatment of P-APS.

This study by Stockstill et al. demonstrates that bortezomib-induced neuropathic pain is driven by S1P receptor 1 (S1PR1) activation in spinal cord astrocytes. Disrupting spinal astrocyte S1PR1 signaling attenuates neuropathic pain by reducing neuroinflammation and presynaptic glutamate release., The development of chemotherapy-induced painful peripheral neuropathy is a major dose-limiting side effect of many chemotherapeutics, including bortezomib, but the mechanisms remain poorly understood. We now report that bortezomib causes the dysregulation of de novo sphingolipid metabolism in the spinal cord dorsal horn to increase the levels of sphingosine-1-phosphate (S1P) receptor 1 (S1PR1) ligands, S1P and dihydro-S1P. Accordingly, genetic and pharmacological disruption of S1PR1 with multiple S1PR1 antagonists, including FTY720, blocked and reversed neuropathic pain. Mice with astrocyte-specific alterations of S1pr1 did not develop neuropathic pain and lost their ability to respond to S1PR1 inhibition, strongly implicating astrocytes as a primary cellular substrate for S1PR1 activity. At the molecular level, S1PR1 engaged astrocyte-driven neuroinflammation and altered glutamatergic homeostasis, processes blocked by S1PR1 antagonism. Our findings establish S1PR1 as a target for therapeutic intervention and provide insight into cellular and molecular pathways. As FTY720 also shows promising anticancer potential and is FDA approved, rapid clinical translation of our findings is anticipated.

Excessive activation of glutamate receptors in spinal dorsal horn neurons is a key mechanism leading to abnormal neuronal activation in pathological pain conditions. Previous studies have shown that activation of glutamate receptors in the spinal dorsal horn is enhanced by impaired glial glutamate transporter functions and proinflammatory cytokines including interleukin-1 beta (IL-1β). In this study, we for the first time revealed that spinal glial glutamate transporter activities in the neuropathic animals are attenuated by endogenous IL-1β. Specifically, we demonstrated that nerve injury results in an increased expression of IL-1β and activation of PKC in the spinal dorsal horn as well as suppression of glial glutamate uptake activities. We provided evidence that the nerve-injury induced suppression of glial glutamate uptake is at least in part ascribed to endogenous IL-1β and activation of PKC in the spinal dorsal horn. IL-1β reduces glial glutamate transporter activities through enhancing the endocytosis of both GLT-1 and GLAST glial glutamate transporters. The IL-1β induced trafficking of glial glutamate transporters is through the calcium/PKC signaling pathway, and the dynamin-dependent endocytosis, which is dependent on the integrity of actin filaments. The signaling pathway regulating glial glutamate transporters revealed in this study provides novel targets to attenuate aberrant activation of glutamate receptors in the spinal dorsal horn, which could ultimately help the development of analgesics.

Excessive activation of glutamate receptors and overproduction of proinflammatory cytokines, including interleukin-1β (IL-1β) in the spinal dorsal horn, are key mechanisms underlying the development and maintenance of neuropathic pain. In this study, we investigated the mechanisms by which endogenous IL-1β alters glutamatergic synaptic transmission in the spinal dorsal horn in rats with neuropathic pain induced by ligation of the L5 spinal nerve. We demonstrated that endogenous IL-1β in neuropathic rats enhances glutamate release from the primary afferent terminals and non-NMDA glutamate receptor activities in postsynaptic neurons in the spinal dorsal horn. Myeloid differentiation primary response protein 88 (MyD88) is a mediator used by IL-1β to enhance non-NMDA glutamate receptor activities in postsynaptic neurons in the spinal dorsal horn. Presynaptic NMDA receptors are effector receptors used by the endogenous IL-1β to enhance glutamate release from the primary afferents in neuropathic rats. This is further supported by the fact that NMDA currents recorded from small neurons in the dorsal root ganglion of normal rats are potentiated by exogenous IL-1β. Furthermore, we provided evidence that functional coupling between IL-1β receptors and presynaptic NMDA receptors at the primary afferent terminals is mediated by the neutral sphingomyelinase/ceramide signaling pathway. Hence, functional coupling between IL-1β receptors and presynaptic NMDA receptors at the primary afferent terminals is a crucial mechanism leading to enhanced glutamate release and activation of non-NMDA receptors in the spinal dorsal horn neurons in neuropathic pain conditions. Interruption of such functional coupling could be an effective approach for the treatment of neuropathic pain.

RATIONALE Paclitaxel, an antitumor agent for the treatment of several types of cancers, has recently been reported to cause impaired cognitive function and neuropathic pain in humans. To assess the effects of paclitaxel on the central nervous system, a sensitive and accurate method is required to quantify paclitaxel concentrations in plasma and brain tissue obtained from rodents receiving paclitaxel. METHODS The biological samples were prepared by liquid-liquid extraction and separated by a 3.5 min reversed-phase liquid chromatography (RPLC) method using a BDS Hypersil C8 column under isocratic conditions. Paclitaxel was quantified using multiple reaction monitoring (MRM) with a triple quadrupole tandem mass spectrometer working in the positive electrospray ionization (ESI+) mode. A stable isotope labeled analogue of paclitaxel was used as the internal standard (IS). RESULTS The method was validated to be precise and accurate within the dynamic range of 0.5-100 ng/mL based on 100 μL plasma and 1.5–300 ng/g based on 33 mg of brain tissue in homogenate. This method was applied to samples from 2 mg/kg intravenously dosed rats. The plasma concentrations were observed to be 26.62 ± 8.93 ng/mL and brain concentrations 11.08 ± 4.18 ng/g when measured 4 h post-dose. CONCLUSIONS This rapid LC/MS/MS method was validated to be sensitive, specific, precise and accurate for the quantification of paclitaxel in rat plasma and brain tissue homogenate. Application of the method to study samples provided sufficient proof of blood–brain barrier penetration of paclitaxel, allowing further investigation of its influence on the central nervous system. Copyright © 2013 John Wiley & Sons, Ltd.

Activation of N-methyl-D-aspartate (NMDA) receptors (NMDARs) is a crucial mechanism underlying the development and maintenance of pain. Traditionally, the role of NMDARs in the pathogenesis of pain is ascribed to their activation and signalling cascades in postsynaptic neurons. In this study, we determined if presynaptic NMDARs in the primary afferent central terminals play a role in synaptic plasticity of the spinal first sensory synapse in a rat model of neuropathic pain induced by spinal nerve ligation. Excitatory postsynaptic currents (EPSCs) were recorded from superficial dorsal horn neurons of spinal slices taken from young adult rats. We showed that increased glutamate release from the primary afferents contributed to the enhanced amplitudes of EPSCs evoked by input from the primary afferents in neuropathic rats. Endogenous activation of presynaptic NMDARs increased glutamate release from the primary afferents in neuropathic rats. Presynaptic NMDARs in neuropathic rats were mainly composed of NR2B receptors. The action of presynaptic NMDARs in neuropathic rats was enhanced by exogenous D-serine and/or NMDA and dependent on activation of protein kinase C. In contrast, glutamate release from the primary afferents in sham-operated rats was not regulated by presynaptic NMDARs. We demonstrated that the lack of NMDAR-mediated regulation of glutamate release in sham-operated rats was not attributable to low extracellular levels of the NMDAR agonist and/or coagonist (D-serine), but rather was due to the insufficient function and/or number of presynaptic NMDARs. This was supported by an increase of NR2B receptor protein expression in both the dorsal root ganglion and spinal dorsal horn ipsilateral to the injury site in neuropathic rats. Hence, suppression of the presynaptic NMDAR activity in the primary sensory afferents is an effective approach to attenuate the enhanced glutamatergic response in the spinal first sensory synapse induced by peripheral nerve injury, and presynaptic NMDARs might be a novel target for the development of analgesics.

Emerging studies have shown that pharmacological activation of adenosine monophosphate-activated protein kinase (AMPK) produces potent analgesic effects in different animal pain models. Currently, the spinal molecular and synaptic mechanism by which AMPK regulates the pain signaling system remains unclear. To address this issue, we utilized the Cre-LoxP system to conditionally knockout the AMPKα1 gene in the nervous system of mice. We demonstrated that AMPKα1 is imperative for maintaining normal nociception, and mice deficient for AMPKα1 exhibit mechanical allodynia. This is concomitantly associated with increased glutamatergic synaptic activities in neurons located in the superficial spinal dorsal horn, which results from the increased glutamate release from presynaptic terminals and function of ligand-gated glutamate receptors at the postsynaptic neurons. Additionally, AMPKα1 knockout mice have increased activities of extracellular signal-regulated kinases (ERK) and p38 mitogen-activated protein kinases (p38), as well as elevated levels of interleukin-1β (IL-1β), reactive oxygen species (ROS), and heme oxygenase 1 (HO-1) in the spinal dorsal horn. Systemic administration of a non-specific ROS scavenger (phenyl-N-tert-butylnitrone, PBN) or a HO-1 activator (Cobalt protoporphyrin IX, CoPP) attenuated allodynia in AMPKα1 knockout mice. Bath-perfusion of the ROS scavenger or HO-1 activator effectively attenuated the increased ROS levels and glutamatergic synaptic activities in the spinal dorsal horn. Our findings suggest that ROS are the key down-stream signaling molecules mediating the behavioral hypersensitivity in AMPKα1 knockout mice. Thus, targeting AMPKα1 may represent an effective approach for the treatment of pathological pain conditions associated with neuroinflammation at the spinal dorsal horn.

Ca2+-calmodulin (CaM) dependent protein kinase II (CaMKII) is an important intracellular signal transduction pathway. CaMKII is rich in the primary sensory neurons and specifically presents in the small- and medium-sized neurons. It remains unclear about the modulation on the excitability of primary sensory neurons by Ca2+–CaM–CaMKII pathway. By current clamp recording, we found that the excitability of capsaicin-sensitive small and medium trigeminal ganglion (TG) neurons was significantly reduced by a CaM specific antagonist (W-7) and a CaMKII antagonist (KN-93). The inhibition is represented as the reduction of numbers of action potential (AP), decrease of the amplitude of AP, increase of threshold, and prolongation of duration of AP. Consistently, by voltage clamp recording, we found that both voltage-gated sodium channels (VGSCs) and voltage-gated potassium channels (VGPCs) were inhibited by W-7 and KN-93 in the order of total sodium (Na+) current (INa-T) > sustained potassium (K+) current (IK) > A-type K+ current (IA). In addition, AIP (a selective CaMKII peptide inhibitor) and KN-93 caused a similar inhibition of INa-T and IK. Those evidences show that the excitability of capsaicin sensitive small and medium TG neurons can be regulated by Ca2+–CaM–CaMKII pathway through modulating VGSCs and VGPCs. Considering the specific distribution of CaMKII and its susceptibility to many analgesic stimuli, Ca2+–CaM–CaMKII pathway may play an important role in the peripheral sensory transduction, especially in nociception.

Background: Neuroinflammation and dysfunctional glial glutamate transporters (GTs) in the spinal dorsal horn are implicated in the genesis of neuropathic pain. The authors determined whether adenosine monophosphate–activated protein kinase (AMPK) in the spinal dorsal horn regulates these processes in rodents with neuropathic pain. Methods: Hind paw withdrawal responses to radiant heat and mechanical stimuli were used to assess nociceptive behaviors. Spinal markers related to neuroinflammation and glial GTs were determined by Western blotting. AMPK activities were manipulated pharmacologically and genetically. Regulation of glial GTs was determined by measuring protein expression and activities of glial GTs. Results: AMPK activities were reduced in the spinal dorsal horn of rats (n = 5) with thermal hyperalgesia induced by nerve injury, which were accompanied with the activation of astrocytes, increased production of interleukin-1β and activities of glycogen synthase kinase 3β, and suppressed protein expression of glial glutamate transporter-1. Thermal hyperalgesia was reversed by spinal activation of AMPK in neuropathic rats (n = 10) and induced by inhibiting spinal AMPK in naive rats (n = 7 to 8). Spinal AMPKα knockdown (n = 6) and AMPKα1 conditional knockout (n = 6) induced thermal hyperalgesia and mechanical allodynia. These genetic alterations mimicked the changes of molecular markers induced by nerve injury. Pharmacological activation of AMPK enhanced glial GT activity in mice with neuropathic pain (n = 8) and attenuated glial glutamate transporter-1 internalization induced by interleukin-1β (n = 4). Conclusions: These findings suggest that enhancing spinal AMPK activities could be an effective approach for the treatment of neuropathic pain.

Toll like receptor 4 (TLR4) is an innate immune pattern recognition receptor, expressed predominantly on microglia in the CNS. Activation of spinal TLR4 plays a critical role in the genesis of pathological pain induced by nerve injury, bone cancer, and tissue inflammation. Currently, it remains unknown how synaptic activities in the spinal dorsal horn are regulated by TLR4 receptors. Through recording GABAergic currents in neurons and glial glutamate transporter currents in astrocytes in rodent spinal slices, we determined whether and how TLR4 modulates GABAergic synaptic activities in the superficial spinal dorsal horn. We found that activation of TLR4 by lipopolysaccharide (LPS) reduces GABAergic synaptic activities through both presynaptic and postsynaptic mechanisms. Specifically, LPS causes the release of IL-1β from microglia. IL-1β in turn suppresses GABA receptor activities at the postsynaptic site through activating protein kinase C (PKC) in neurons. GABA synthesis at the presynaptic site is reduced upon activation of TLR4. Glial glutamate transporter activities are suppressed by IL-1β and PKC activation induced by LPS. The suppression of glial glutamate transporter activities leads to a deficiency of glutamine supply, which results in an attenuation of the glutamate-glutamine cycle-dependent GABA synthesis. These findings shed light on understanding synaptic plasticity induced by activation of TLR4 under neuroinflammation and identify GABA receptors, glial glutamate transporters, IL-1β and PKC as therapeutic targets to abrogate abnormal neuronal activities following activation of TLR4 in pathological pain conditions.

Paclitaxel is a chemotherapeutic agent widely used for treating carcinomas. Patients receiving paclitaxel often develop neuropathic pain and have a reduced quality of life which hinders the use of this life-saving drug. In this study, we determined the role of GABA transporters in the genesis of paclitaxel-induced neuropathic pain using behavioral tests, electrophysiology, and biochemical techniques. We found that tonic GABA receptor activities in the spinal dorsal horn were reduced in rats with neuropathic pain induced by paclitaxel. In normal controls, tonic GABA receptor activities were mainly controlled by the GABA transporter GAT-1 but not GAT-3. In the spinal dorsal horn, GAT-1 was expressed at presynaptic terminals and astrocytes while GAT-3 was only expressed in astrocytes. In rats with paclitaxel-induced neuropathic pain, the protein expression of GAT-1 was increased while GAT-3 was decreased. This was concurrently associated with an increase in global GABA uptake. The paclitaxel-induced attenuation of GABAergic tonic inhibition was ameliorated by blocking GAT-1 but not GAT-3 transporters. Paclitaxel-induced neuropathic pain was significantly attenuated by the intrathecal injection of a GAT-1 inhibitor. These findings suggest that targeting GAT-1 transporters for reversing disinhibition in the spinal dorsal horn may be a useful approach for treating paclitaxel-induced neuropathic pain. Patients receiving paclitaxel for cancer therapy often develop neuropathic pain and have a reduced quality of life. In this study, we demonstrated that animals treated with paclitaxel develop neuropathic pain, have enhancements of GABA transporter-1 protein expression and global GABA uptake, as well as suppression of GABAergic tonic inhibition in the spinal dorsal horn. Pharmacological inhibition of GABA transporter-1 ameliorates the paclitaxel-induced suppression of GABAergic tonic inhibition and neuropathic pain. Thus, targeting GAT-1 transporters for reversing GABAergic disinhibition in the spinal dorsal horn could be a useful approach for treating paclitaxel-induced neuropathic pain.

Ranolazine attenuates cardiac arrhythmic activity associated with hypoxia and hydrogen peroxide (H2O2) by inhibition of late sodium current (late INa). The mechanism of ranolazine's action on Na channels was investigated using whole-cell and single-channel recording from guinea pig isolated ventricular myocytes. Hypoxia increased whole-cell late INa from -0.48 ± 0.02 to -3.99 ± 0.07 pA/pF. Ranolazine at 3 and 9 μmol/L reduced the hypoxia-induced late INa by 16% ± 3% and 55% ± 3%, respectively. Hypoxia increased the mean open probability and open time of Na-channel late openings from 0.016 ± 0.001 to 0.064 ± 0.007 milliseconds and from 0.693 ± 0.043 to 1.081 ± 0.098 milliseconds, respectively. Ranolazine at 3 and 9 μmol/L attenuated the hypoxia-induced increase of open probability by 19% ± 7% and 61% ± 1%, and increase of open time by 26% ± 19% and 74 ± 21%, respectively. H2O2 increased the mean open probability and open time of Na-channel late openings from 0.013 ± 0.002 to 0.107 ± 0.015 milliseconds and from 0.689 ± 0.075 to 1.487 ± 0.072 milliseconds, respectively. Ranolazine at 3 and 6 μmol/L reduced the H2O2-induced increase of mean open probability by 60% ± 7% and 95% ± 2%, and the increase of mean open time by 31% ± 21% and 82% ± 8%. In conclusion, the inhibition by ranolazine of hypoxia- and H2O2-stimulated late INa is due to reduction of both the open probability and open time of Na-channel late openings.

Excessive activation of glutamate receptors in spinal dorsal horn neurons is a key mechanism leading to abnormal neuronal activation in pathological pain conditions. Previous studies have shown that activation of glutamate receptors in the spinal dorsal horn is enhanced by impaired glial glutamate transporter functions and pro-inflammatory cytokines including interleukin-1 beta (IL-1β). In this study, we for the first time revealed that spinal glial glutamate transporter activities in the neuropathic animals are attenuated by endogenous IL-1β. Specifically, we demonstrated that nerve injury results in an increased expression of IL-1β and activation of PKC in the spinal dorsal horn as well as suppression of glial glutamate uptake activities. We provided evidence that the nerve-injury induced suppression of glial glutamate uptake is at least in part ascribed to endogenous IL-1β and activation of PKC in the spinal dorsal horn. IL-1β reduces glial glutamate transporter activities through enhancing the endocytosis of both GLT-1 and GLAST glial glutamate transporters. The IL-1β induced trafficking of glial glutamate transporters is through the calcium/PKC signaling pathway, and the dynamin-dependent endocytosis, which is dependent on the integrity of actin filaments. The signaling pathway regulating glial glutamate transporters revealed in this study provides novel targets to attenuate aberrant activation of glutamate receptors in the spinal dorsal horn, which could ultimately help the development of analgesics.

Paclitaxel, an antitumor agent for the treatment of several types of cancers, has recently been reported to cause impaired cognitive function and neuropathic pain in humans. To assess the effects of paclitaxel on the central nervous system, a sensitive and accurate method is required to quantify paclitaxel concentrations in plasma and brain tissue obtained from rodents receiving paclitaxel.The biological samples were prepared by liquid-liquid extraction and separated by a 3.5 min reversed-phase liquid chromatography (RPLC) method using a BDS Hypersil C8 column under isocratic conditions. Paclitaxel was quantified using multiple reaction monitoring (MRM) with a triple quadrupole tandem mass spectrometer working in the positive electrospray ionization (ESI+) mode. A stable isotope labeled analogue of paclitaxel was used as the internal standard (IS).The method was validated to be precise and accurate within the dynamic range of 0.5-100 ng/mL based on 100 μL plasma and 1.5-300 ng/g based on 33 mg of brain tissue in homogenate. This method was applied to samples from 2 mg/kg intravenously dosed rats. The plasma concentrations were observed to be 26.62 ± 8.93 ng/mL and brain concentrations 11.08 ± 4.18 ng/g when measured 4 h post-dose.This rapid LC/MS/MS method was validated to be sensitive, specific, precise and accurate for the quantification of paclitaxel in rat plasma and brain tissue homogenate. Application of the method to study samples provided sufficient proof of blood-brain barrier penetration of paclitaxel, allowing further investigation of its influence on the central nervous system.

Activation of N-methyl-d-aspartate (NMDA) receptors (NMDARs) is a crucial mechanism underlying the development and maintenance of pain. Traditionally, the role of NMDARs in the pathogenesis of pain is ascribed to their activation and signalling cascades in postsynaptic neurons. In this study, we determined if presynaptic NMDARs in the primary afferent central terminals play a role in synaptic plasticity of the spinal first sensory synapse in a rat model of neuropathic pain induced by spinal nerve ligation. Excitatory postsynaptic currents (EPSCs) were recorded from superficial dorsal horn neurons of spinal slices taken from young adult rats. We showed that increased glutamate release from the primary afferents contributed to the enhanced amplitudes of EPSCs evoked by input from the primary afferents in neuropathic rats. Endogenous activation of presynaptic NMDARs increased glutamate release from the primary afferents in neuropathic rats. Presynaptic NMDARs in neuropathic rats were mainly composed of NR2B receptors. The action of presynaptic NMDARs in neuropathic rats was enhanced by exogenous d-serine and/or NMDA and dependent on activation of protein kinase C. In contrast, glutamate release from the primary afferents in sham-operated rats was not regulated by presynaptic NMDARs. We demonstrated that the lack of NMDAR-mediated regulation of glutamate release in sham-operated rats was not attributable to low extracellular levels of the NMDAR agonist and/or coagonist (d-serine), but rather was due to the insufficient function and/or number of presynaptic NMDARs. This was supported by an increase of NR2B receptor protein expression in both the dorsal root ganglion and spinal dorsal horn ipsilateral to the injury site in neuropathic rats. Hence, suppression of the presynaptic NMDAR activity in the primary sensory afferents is an effective approach to attenuate the enhanced glutamatergic response in the spinal first sensory synapse induced by peripheral nerve injury, and presynaptic NMDARs might be a novel target for the development of analgesics.

J. Neurochem. (2012) 121, 526–536. Abstract Decreased GABAergic synaptic strength (‘disinhibition’) in the spinal dorsal horn is a crucial mechanism contributing to the development and maintenance of pathological pain. However, mechanisms leading to disinhibition in the spinal dorsal horn remain elusive. We investigated the role of glial glutamate transporters (GLT-1 and GLAST) and glutamine synthetase in maintaining GABAergic synaptic activity in the spinal dorsal horn. Electrically evoked GABAergic inhibitory post-synaptic currents (eIPSCs), spontaneous IPSCs (sIPSCs) and miniature IPSCs were recorded in superficial spinal dorsal horn neurons of spinal slices from young adult rats. We used (2S,3S)-3-[3-[4-(trifluoromethyl)benzoylamino]benzyloxy]aspartate (TFB-TBOA), to block both GLT-1 and GLAST and dihydrokainic acid to block only GLT-1. We found that blockade of both GLAST and GLT-1 and blockade of only GLT-1 in the spinal dorsal horn decreased the amplitude of GABAergic eIPSCs, as well as both the amplitude and frequency of GABAergic sIPSCs or miniature IPSCs. Pharmacological inhibition of glial glutamine synthetase had similar effects on both GABAergic eIPSCs and sIPSCs. We provided evidence demonstrating that the reduction in GABAergic strength induced by the inhibition of glial glutamate transporters is due to insufficient GABA synthesis through the glutamate–glutamine cycle between astrocytes and neurons. Thus, our results indicate that deficient glial glutamate transporters and glutamine synthetase significantly attenuate GABAergic synaptic strength in the spinal dorsal horn, which may be a crucial synaptic mechanism underlying glial–neuronal interactions caused by dysfunctional astrocytes in pathological pain conditions.

Ca(2+)-calmodulin (CaM) dependent protein kinase II (CaMKII) is an important intracellular signal transduction pathway. CaMKII is rich in the primary sensory neurons and specifically presents in the small- and medium-sized neurons. It remains unclear about the modulation on the excitability of primary sensory neurons by Ca(2+)-CaM-CaMKII pathway. By current clamp recording, we found that the excitability of capsaicin-sensitive small and medium trigeminal ganglion (TG) neurons was significantly reduced by a CaM specific antagonist (W-7) and a CaMKII antagonist (KN-93). The inhibition is represented as the reduction of numbers of action potential (AP), decrease of the amplitude of AP, increase of threshold, and prolongation of duration of AP. Consistently, by voltage clamp recording, we found that both voltage-gated sodium channels (VGSCs) and voltage-gated potassium channels (VGPCs) were inhibited by W-7 and KN-93 in the order of total sodium (Na(+)) current (INa-T)sustained potassium (K(+)) current (IK)A-type K(+) current (IA). In addition, AIP (a selective CaMKII peptide inhibitor) and KN-93 caused a similar inhibition of INa-T and IK. Those evidences show that the excitability of capsaicin sensitive small and medium TG neurons can be regulated by Ca(2+)-CaM-CaMKII pathway through modulating VGSCs and VGPCs. Considering the specific distribution of CaMKII and its susceptibility to many analgesic stimuli, Ca(2+)-CaM-CaMKII pathway may play an important role in the peripheral sensory transduction, especially in nociception.

The systematic designing idea of optical simulation software for simulating and analyzing the beam propagation in the optical waveguides is described in the paper. Based on the theory of Finite-difference beam propagation method for calculation, the feasibility analysis and detailed module partitions with their respective functions of systematic design and implementation of optical simulation software on usual PC-based platform are discussed. With the systematic analysis and implementation, various types of waveguides can be edited, data of field distribution can be calculated, and visual resulting graphics can be displayed in the software system. Then the simulation of beam propagation through optical devices can be realized based on the systematic implementation.

Paclitaxel, a powerful anti-neoplastic drug, often causes pathological pain, which significantly reduces the quality of life in patients. Paclitaxel-induced pain includes pain that occurs immediately after paclitaxel treatment (paclitaxel-associated acute pain syndrome, P-APS) and pain that persists for weeks to years after cessation of paclitaxel treatment (paclitaxel induced chronic neuropathic pain). Mechanisms underlying P-APS remain unknown. In this study, we found that paclitaxel causes acute pain in rodents in a dose-dependent manner. The paclitaxel-induced acute pain occurs within 2 hrs after a single intravenous injection of paclitaxel. This is accompanied by low levels of paclitaxel penetrating into the cerebral spinal fluid and spinal dorsal horn. We demonstrated that an intrathecal injection of paclitaxel induces mechanical allodynia in a dose-dependent manner. Paclitaxel causes activation of toll like receptor 4 (TLR4) in the spinal dorsal horn and dorsal root ganglions. Through activating TLR4, paclitaxel increases glutamatergic synaptic activities and reduces glial glutamate transporter activities in the dorsal horn. Activations of TLR4 are necessary in the genesis of paclitaxel-induced acute pain. The cellular and molecular signaling pathways revealed in this study could provide rationales for the development of analgesics and management strategies for P-APS in patients.