Evaluating Oral Probiotic Supplements as Complementary Treatment in Advanced Lung Cancer Patients Receiving ICIs: A Prospective Real-World Study.

OBJECTIVE: To evaluate the effectiveness of oral probiotic supplements in patients undergoing immune checkpoint inhibitors (ICIs) for the treatment of advanced lung cancer. METHODS: This prospective real-world study enrolled patients with advanced lung cancer who were receiving ICIs as part of their treatment. The patients were divided into 2 groups: Group OPS received oral probiotic supplements along with ICIs, while Group C did not. The primary endpoint was progression-free survival (PFS). The secondary outcome measure was the objective response rate (ORR). RESULTS: A total of 253 patients were included in the study, with 71 patients in Group OPS and 182 patients in the control group (Group C). No significant differences were observed in the median PFS between the 2 groups for all patients. However, for small cell lung cancer (SCLC) patients, the median PFS was significantly better in the Group OPS compared to the Group C (11.1 months vs 7.0 months, P = .049). No significant differences were observed in median PFS for the non-small cell lung cancer (NSCLC) cohort between the 2 groups, but a trend towards better median PFS in Group OPS was noticed (16.5 months vs 12.3 months, P = .56). The ORR for the entire cohort was 58.0%. CONCLUSION: Oral probiotics supplements in combination with ICIs included regimen may improve the outcome in patients with advanced SCLC. The above points should be proved by further study.

This study examined whether the addition of oral probiotic supplements to ICIs could enhance the treatment of advanced lung cancer. A total of 253 patients with advanced lung cancer were involved in the study, with some receiving probiotics in combination with ICIs and others not. The findings revealed that patients with SCLC who took probiotics had significantly better PFS compared to those who did not. Additionally, there was a tendency towards enhanced PFS in NSCLC patients who received probiotics. In conclusion, the study indicates that incorporating oral probiotics with ICIs may lead to better outcomes for patients with advanced SCLC, although further research is necessary to validate these results.This real world study explores whether oral probiotic supplements along with immune checkpoint inhibitors (ICIs) can help treat advanced lung cancer. The study included 253 patients with advanced lung cancer receiving ICIs treatment, part of them taking probiotics along with ICIs. The results showed that patients with small cell lung cancer (SCLC) who took probiotics had better progression-free survival (PFS) compared to those who didn't. There was also a trend towards better PFS in non-small cell lung cancer (NSCLC) patients who took probiotics. Overall, the study suggests that taking oral probiotics along with ICIs may improve outcomes for patients with advanced SCLC, but more research is needed to confirm these findings.

Effect of sympathetic sprouting on the excitability of dorsal root ganglion neurons and afferents in a rat model of neuropathic pain.

Increased sympathetic nerve excitability has been reported to aggravate a variety of chronic pain conditions, and an increase in the number of sympathetic nerve fibers in the dorsal root ganglion (DRG) has been found in neuropathic pain (NP) models. However, the mechanism of the neurotransmitter norepinephrine (NE) released by sympathetic nerve fiber endings on the excitability of DRG neurons is still controversial, and the adrenergic receptor subtypes involved in this biological process are also controversial. In our study, we have two objectives: (1) To determine the effect of the neurotransmitter NE on the excitability of different neurons in DRG; (2) To determine which adrenergic receptors are involved in the excitability of DRG neurons by NE released by sprouting sympathetic fibers. In this experiment, a unique field potential recording method of spinal cord dorsal horn was innovatively adopted, which can be used for electrophysiological study in vivo. The results showed that： Forty days after SNI, patch clamp and field potential recording methods confirmed that NE enhanced the excitability of ipsilateral DRG large neurons, and then our in vivo electrophysiological results showed that the α2 receptor blocker Yohimbine could block the excitatory effect of NE on A-fiber and the inhibitory effect on C-fiber, while the α2A-adrenergic receptor agonist guanfacine (100 µM) had the same biological effect as NE. Finally, we concluded that NE from sympathetic fiber endings is involved in the regulation of pain signaling by acting on α2A-adrenergic receptors in DRG.

Mussel-Inspired Durable TiO2/PDA-Based Superhydrophobic Paper with Excellent Self-Cleaning, High Chemical Stability, and Efficient Oil/Water Separation Properties.

Oceanic oil spill and the discharge of industrial oily wastewaters can cause significant threats to the ecological environment and human health. Herein, we design a durable TiO2/PDA-based superhydrophobic paper for efficient oil/water separation. Bioinspired from mussel adhesive proteins, the mechanical durability of the as-prepared superhydrophobic paper is enhanced by the deposition of polydopamine (PDA) onto cellulosic fibers via self-polymerization of dopamine. The TiO2/PDA-based superhydrophobic paper shows a high water contact angle of 168.2° and an oil contact angle of ∼0°, exhibiting excellent superhydrophobicity and superoleophilicity. Furthermore, the as-prepared superhydrophobic paper possesses excellent chemical stability, thermal stability, and mechanical durability in terms of being immersed in corrosive solutions and solvents and boiling water and being subjected to the sandpaper abrasion test, respectively. More importantly, the separation efficiency of the TiO2/PDA-based superhydrophobic paper for an oil/water mixture is 97.2%, and it maintains a separation efficiency above 94.3% even after 15 cyclic separation processes. Furthermore, the separation efficiency for water-in-oil emulsions is higher than 93.7% after 15 cyclic separation tests, showing its excellent recyclable stability for water-in-oil emulsions. Therefore, the rationally designed TiO2/PDA-based superhydrophobic paper shows great potential in the practical applications of self-cleaning, antifouling, and oil/water separation.

Wrinkled Titanium Carbide (MXene) with Surface Charge Polarizations through Chemical Etching for Superior Electromagnetic Interference Shielding.

Despite the fact that the high conductivity of two-dimensional laminated transition metal carbides/nitrides (MXenes) contributes to the outstanding electromagnetic interference (EMI) shielding by the reflection of electromagnetic waves (EWs), it is difficulty to improve EMI shielding by pursuing higher conductivity due to the limitation of intrinsic properties. Here, we achieve superior EMI shielding by introducing the absorption of EWs in MXenes with micro-sized wrinkles which are induced by abundant Ti vacancies under chemical etching. The shielding effectiveness is up to 107 dB at a thickness of 20 µm. Combining with atomic-scale structure observation and the first-principles calculations, it is concluded that the promotion of EMI shielding originates from the resonant absorption of formed electric dipoles induced by the asymmetrical distribution of charge densities near Ti vacancies. Our results could open a new vista for developing two-dimensional EMI shielding materials.

BDNF promotes activation of astrocytes and microglia contributing to neuroinflammation and mechanical allodynia in cyclophosphamide-induced cystitis.

BACKGROUND: Patients with interstitial cystitis/bladder pain syndrome (IC/BPS) often grieve over a low quality of life brought about by chronic pain. In our previous studies, we determined that neuroinflammation of the spinal dorsal horn (SDH) was associated with mechanisms of interstitial cystitis. Moreover, it has been shown that brain-derived neurotrophic factor (BDNF) participates in the regulation of neuroinflammation and pathological pain through BDNF-TrkB signaling; however, whether it plays a role in cyclophosphamide (CYP)-induced cystitis remains unclear. This study aimed to confirm whether BDNF-TrkB signaling modulates neuroinflammation and mechanical allodynia in CYP-induced cystitis and determine how it occurs. METHODS: Systemic intraperitoneal injection of CYP was performed to establish a rat cystitis model. BDNF-TrkB signaling was modulated by intraperitoneal injection of the TrkB receptor antagonist, ANA-12, or intrathecal injection of exogenous BDNF. Mechanical allodynia in the suprapubic region was assessed using the von Frey filaments test. The expression of BDNF, TrkB, p-TrkB, Iba1, GFAP, p-p38, p-JNK, IL-1ß, and TNF-α in the L6-S1 SDH was measured by Western blotting and immunofluorescence analysis. RESULTS: BDNF-TrkB signaling was upregulated significantly in the SDH after CYP was injected. Similarly, the expressions of Iba1, GFAP, p-p38, p-JNK, IL-1ß, and TNF-α in the SDH were all upregulated. Treatment with ANA-12 could attenuate mechanical allodynia, restrain activation of astrocytes and microglia and alleviate neuroinflammation. Besides, the intrathecal injection of exogenous BDNF further decreased the mechanical withdrawal threshold, promoted activation of astrocytes and microglia, and increased the release of TNF-α and IL-1ß in the SDH of our CYP-induced cystitis model. CONCLUSIONS: In our CYP-induced cystitis model, BDNF promoted the activation of astrocytes and microglia to release TNF-α and IL-1ß, aggravating neuroinflammation and leading to mechanical allodynia through BDNF-TrkB-p38/JNK signaling.

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.

Impact of Dwell Time of Retrievable IVC Filters on IVC Lumen Diameter: A Series of 36 Cases.

BACKGROUND: Inferior vena cava (IVC) filters are effective in preventing pulmonary embolism in patients at risk. This study aimed to investigate whether the dwell time of retrievable IVC filters have impact on IVC lumen diameter. METHODS: The clinical data of 36 patients treated with retrievable IVC filters from January 2016 to November 2018 were retrospectively collected. A total of 33 filters were successfully removed. At times of filter placement and removal, the IVC lumen diameter (at upper, middle, and lower levels of the filter), distance between the filter upper end and the right renal vein opening, and degree of filter tilt were measured. RESULTS: IVC filters were placed because of deep vein thrombosis in the lower limbs after fractures in 26 patients. The median dwell time of the IVC filters was 18 days. From the time of filter placement to that of removal, the IVC diameter decreased significantly at the middle (28.07 ± 5.92 vs. 25.73 ± 7.33 mm, P = 0.002) and lower levels (27.48 ± 4.73 vs. 26.36 ± 4.72 mm, P = 0.003) of the filters. No significant difference was noticed in the IVC diameter at the upper levels of the filters (27.78 ± 6.43 vs. 27.11 ± 6.63 mm, P = 0.082). Positive correlation was noticed between filter dwell time and IVC diameter changes at the upper (r = 0.381, P = 0.029) and middle (r = 0.555, P = 0.001) levels of the filters. No significant change was noticed in the distance from the filter upper end to the right renal vein opening and the degree of filter tilt. CONCLUSIONS: Retrievable IVC filters are associated with IVC stenosis. The severity of IVC stenosis is positively correlated with the dwell time of filters.

Upregulation of tumor necrosis factor-alpha in the anterior cingulate cortex contributes to neuropathic pain and pain-associated aversion.

Injury associated pain involves subjective perception and emotional experience. The anterior cingulate cortex (ACC) is a key area involved in the affective component of pain processing. However, the neuroimmune mechanisms underlying enhanced ACC excitability following peripheral nerve injury are still not fully understood. Our previous work has shown that tumor necrosis factor-alpha (TNF-α) overexpression leads to peripheral afferent hyperexcitability and synaptic transmission potentiation in spinal cord. Here, we aimed to reveal the potential role of ACC TNF-α in ACC hyperexcitability and neuropathic pain. c-Fos, a widely used neuronal activity marker, was induced especially in contralateral ACC early [postoperative (PO) 1 h] and later (PO day 7 and 10) during the development of neuropathic pain. Spared nerve injury (SNI) elevated TNF-α level in contralateral ACC from PO day 5 to 14, delayed relative to decreased ipsilateral paw withdrawal threshold apparent from PO day 1 to 14. Microinjection of anti-TNF-α antibody into the ACC completely eliminated c-Fos overexpression and greatly attenuated pain aversion and mechanical allodynia induced by SNI, suggesting an important role of ACC TNF-α in the pain aversiveness and pain maintenance. Furthermore, modulating ACC pyramidal neurons via a Gi-coupled human M4 muscarinic receptor (hM4Di) or a Gq-coupled human M3 muscarinic receptor (hM3Dq), a type of designer receptors exclusively activated by designer drugs (DREADD), greatly changed the ACC TNF-α level and the mechanical paw withdrawal threshold. The positive interactions between TNF-α and ACC neurons might modulate the cytokine microenvironment thus contribute to the neuropathic pain.

Differential regulation of GSK-3ß in spinal dorsal horn and in hippocampus mediated by interleukin-1beta contributes to pain hypersensitivity and memory deficits following peripheral nerve injury.

Accumulating evidence shows that inhibition of glycogen synthase kinase-3beta (GSK-3ß) ameliorates cognitive impairments caused by a diverse array of diseases. Our previous work showed that spared nerve injury (SNI) that induces neuropathic pain causes short-term memory deficits. Here, we reported that GSK-3ß activity was enhanced in hippocampus and reduced in spinal dorsal horn following SNI, and the changes persisted for at least 45 days. Repetitive applications of selective GSK-3ß inhibitors (SB216763, 5 mg/kg, intraperitoneally, three times or AR-A014418, 400 ng/kg, intrathecally, seven times) prevented short-term memory deficits but did not affect neuropathic pain induced by SNI. Surprisingly, we found that the repetitive SB216763 or AR-A014418 induced a persistent pain hypersensitivity in sham animals. Mechanistically, both ß-catenin and brain-derived neurotrophic factor (BDNF) were upregulated in spinal dorsal horn but downregulated in hippocampus following SNI. Injections of SB216763 prevented the BDNF downregulation in hippocampus but enhanced its upregulation in spinal dorsal horn in SNI rats. In sham rats, SB216763 upregulated both ß-catenin and BDNF in spinal dorsal horn but affect neither of them in hippocampus. Finally, intravenous injection of interleukin-1beta that induces pain hypersensitivity and memory deficits mimicked the SNI-induced the differential regulation of GSK-3ß/ß-catenin/BDNF in spinal dorsal horn and in hippocampus. Accordingly, the prolonged opposite changes of GSK-3ß activity in hippocampus and in spinal dorsal horn induced by SNI may contribute to memory deficits and neuropathic pain by differential regulation of BDNF in the two regions. GSK-3ß inhibitors that treat cognitive disorders may result in a long-lasting pain hypersensitivity.

Neuregulin-1-ErbB signaling promotes microglia activation contributing to mechanical allodynia of cyclophosphamide-induced cystitis.

AIMS: Central sensitization playsimportant roles in cyclophosphamide (CYP)-induced cystitis. In addition, as a visceral pain, CYP-induced chronic pain shares common pathophysiological mechanisms with neuropathic pain. Previous studies demonstrated that neuregulin-1 (Nrg1)-ErbB signaling contributes to neuropathic pain, but whether and how this signaling influences mechanical allodynia in CYP-induced cystitis is unclear. This study aimed to determine whether and how Nrg1-ErbB signaling modulates mechanical allodynia in a CYP-induced cystitis rat model. METHODS: Systemic injection with CYP was used to establish a rat model of bladder pain syndrome/interstitial cystitis (BPS/IC). An irreversible ErbB family receptor inhibitor, PD168393, and exogenous Nrg1 were intrathecally injected to modulate Nrg1-ErbB signaling. Mechanical allodynia in the lower abdomen was assessed with von-Frey filaments using the up-down method. Western blot analysis and immunofluorescence staining were used to measure the expression of Nrg1-ErbB signaling, Iba-1, p-p38, and IL-1ß in the L6-S1 spinal dorsal horn (SDH). RESULTS: We observed upregulation of Nrg1-ErbB signaling as well as overexpression of the microglia activation markers Iba-1 and p-p38 and the proinflammatory factor, interleukin-1ß (IL-1ß), in the SDH of the cystitis group. Further, treatment with PD168393 attenuated mechanical allodynia in CYP-induced cystitis and inhibited microglia activation, leading to decreased production of IL-1ß. The inhibitor PD168393 reversed the algesic effect of exogenous Nrg1 on the cystitis model. CONCLUSIONS: Nrg1-ErbB signaling may promote microglia activation, contributing to mechanical allodynia of CYP-induced cystitis. Our study showed that modulation of Nrg1-ErbB signaling may have therapeutic value for treating pain symptoms in BPS/IC.

TNF-α Differentially Regulates Synaptic Plasticity in the Hippocampus and Spinal Cord by Microglia-Dependent Mechanisms after Peripheral Nerve Injury.

Clinical studies show that chronic pain is accompanied by memory deficits and reduction in hippocampal volume. Experimental studies show that spared nerve injury (SNI) of the sciatic nerve induces long-term potentiation (LTP) at C-fiber synapses in spinal dorsal horn, but impairs LTP in the hippocampus. The opposite changes may contribute to neuropathic pain and memory deficits, respectively. However, the cellular and molecular mechanisms underlying the functional synaptic changes are unclear. Here, we show that the dendrite lengths and spine densities are reduced significantly in hippocampal CA1 pyramidal neurons, but increased in spinal neurokinin-1-positive neurons in mice after SNI, indicating that the excitatory synaptic connectivity is reduced in hippocampus but enhanced in spinal dorsal horn in this neuropathic pain model. Mechanistically, tumor necrosis factor-alpha (TNF-α) is upregulated in bilateral hippocampus and in ipsilateral spinal dorsal horn, whereas brain-derived neurotrophic factor (BDNF) is decreased in the hippocampus but increased in the ipsilateral spinal dorsal horn after SNI. Importantly, the SNI-induced opposite changes in synaptic connectivity and BDNF expression are prevented by genetic deletion of TNF receptor 1 in vivo and are mimicked by TNF-α in cultured slices. Furthermore, SNI activated microglia in both spinal dorsal horn and hippocampus; pharmacological inhibition or genetic ablation of microglia prevented the region-dependent synaptic changes, neuropathic pain, and memory deficits induced by SNI. The data suggest that neuropathic pain involves different structural synaptic alterations in spinal and hippocampal neurons that are mediated by overproduction of TNF-α and microglial activation and may underlie chronic pain and memory deficits. SIGNIFICANCE STATEMENT: Chronic pain is often accompanied by memory deficits. Previous studies have shown that peripheral nerve injury produces both neuropathic pain and memory deficits and induces long-term potentiation (LTP) at C-fiber synapses in spinal dorsal horn (SDH) but inhibits LTP in hippocampus. The opposite changes in synaptic plasticity may contribute to chronic pain and memory deficits, respectively. However, the structural and molecular bases of these alterations of synaptic plasticity are unclear. Here, we show that the complexity of excitatory synaptic connectivity and brain-derived neurotrophic factor (BDNF) expression are enhanced in SDH but reduced in the hippocampus in neuropathic pain and the opposite changes depend on tumor necrosis factor-alpha/tumor necrosis factor receptor 1 signaling and microglial activation. The region-dependent synaptic alterations may underlie chronic neuropathic pain and memory deficits induced by peripheral nerve injury.

Early CALP2 expression and microglial activation are potential inducers of spinal IL-6 up-regulation and bilateral pain following motor nerve injury.

Previous work from our laboratory showed that motor nerve injury by lumbar 5 ventral root transection (L5-VRT) led to interleukin-6 (IL-6) over-expression in bilateral spinal cord, and that intrathecal administration of IL-6 neutralizing antibody delayed the induction of mechanical allodynia in bilateral hind paws. However, early events and upstream mechanisms underlying spinal IL-6 expression following L5-VRT require elucidation. The model of L5-VRT was used to induce neuropathic pain, which was assessed with von Frey hairs and the plantar tester in adult male Sprague-Dawley rats. Calpain-2 (CALP2, a calcium-dependent protease) knockdown or over-expression and microglia depletion were conducted intrathecally. Western blots and immunohistochemistry were performed to explore the possible mechanisms. Here, we provide the first evidence that both IL-6 and CALP2 levels are increased in lumbar spinal cord within 30 min following L5-VRT. IL-6 and CALP2 co-localized in both spinal dorsal horn (SDH) and spinal ventral horn. Post-operative (PO) increase in CALP2 in ipsilateral SDH was evident at 10 min PO, preceding increased IL-6 at 20 min PO. Knockdown of spinal CALP2 by intrathecal CALP2-shRNA administration prevented VRT-induced IL-6 overproduction in ipsilateral spinal cord and alleviated bilateral mechanical allodynia. Spinal microglia activation also played a role in early IL-6 up-regulation. Macrophage/microglia markers ED1/Iba1 were increased at 30 min PO, while glial fibrillary acidic protein (astrocyte) and CNPase (oligodendrocyte) markers were not. Increased Iba1 was detected as early as 20 min PO and peaked at 3 days. Morphology changed from a small soma with fine processes in resting cells to an activated ameboid shape. Depletion of microglia using Mac-1-saporin partially prevented IL-6 up-regulation and attenuated VRT-induced bilateral mechanical allodynia. Taken together, our findings provide evidence that increased spinal cord CALP2 and microglia cell activation may have early causative roles in IL-6 over-expression following motor nerve injury. Agents that inhibit CALP2 and/or microglia activation may therefore prove valuable for treating neuropathic pain.

Mechanisms for therapeutic effect of bulleyaconitine A on chronic pain.

Bulleyaconitine A, a diterpenoid alkaloid isolated from Aconitum bulleyanum plants, has been used for the treatment of chronic pain in China since 1985. Clinical studies show that the oral administration of bulleyaconitine A is effective for treating different kinds of chronic pain, including back pain, joint pain, and neuropathic pain with minimal side effect in human patients. The experimental studies have revealed that bulleyaconitine A at therapeutic doses potently inhibits the peripheral sensitization and central sensitization that underlie chronic pain and has no effect on acute pain. Bulleyaconitine A preferably blocks tetrodotoxin-sensitive voltage-gated sodium channels in dorsal root ganglion neurons by inhibition of protein kinase C, and the effect is around 600 times more potent in neuropathic animals than in naïve ones. Bulleyaconitine A at 5 nM inhibits the hypersensitivity of dorsal root ganglion neurons in neuropathic rats but has no effect on excitability of dorsal root ganglion neurons in sham group. Bulleyaconitine A inhibits long-term potentiation at C-fiber synapses in spinal dorsal horn, a synaptic model of pathological pain, preferably in neuropathic pain rats over naïve rats. The following mechanisms may underlie the selective effect of bulleyaconitine A on chronic pain. (1) In neuropathic conditions, protein kinase C and voltage-gated sodium channels in dorsal root ganglion neurons are upregulated, which enhances bulleyaconitine A's effect. (2) Bulleyaconitine A use-dependently blocks voltage-gated sodium channels and therefore inhibits the ectopic discharges that are important for neuropathic pain. (3) Bulleyaconitine A is shown to inhibit neuropathic pain by the modulation of spinal microglia, which are involved in the chronic pain but not in acute (nociceptive) pain. Moreover, bulleyaconitine A facilitates the anesthetic effect of morphine and inhibits morphine tolerance in rats. Together, bulleyaconitine A is able to inhibit chronic pain by targeting at multiple molecules. Further clinical and experimental studies are needed for evaluating the efficacy of bulleyaconitine A in different forms of chronic pain in patients and for exploring the underlying mechanisms.

Heterosynaptic long-term potentiation from the anterior cingulate cortex to spinal cord in adult rats.

Spinal nociceptive transmission receives biphasic modulation from supraspinal structures. Recent studies demonstrate that the anterior cingulate cortex facilitates spinal excitatory synaptic transmission and nociceptive reflex. However, whether the top-down descending facilitation can cause long-term synaptic changes in spinal cord remains unclear. In the present study, we recorded C-fiber-evoked field potentials in spinal dorsal horn and found that the anterior cingulate cortex stimulation caused enhancement of C-fiber-mediated responses. The enhancement lasted for more than a few hours. Spinal application of N-methyl-D-aspartate (NMDA) receptor antagonist D-AP5 abolished this enhancement, suggesting that the activation of the NMDA receptor is required. Furthermore, spinal application of methysergide, a serotonin receptor antagonist, also blocked the anterior cingulate cortex-induced spinal long-term potentiation. Our results suggest that the anterior cingulate cortex stimulation can produce heterosynaptic form of long-term potentiation at the spinal cord dorsal horn, and this novel form of long-term potentiation may contribute to top-down long-term facilitation in chronic pain conditions.

Bulleyaconitine A attenuates hyperexcitability of dorsal root ganglion neurons induced by spared nerve injury: The role of preferably blocking Nav1.7 and Nav1.3 channels.

Background Oral administration of Bulleyaconitine A, an extracted diterpenoid alkaloid from Aconitum bulleyanum plants, is effective for treating chronic pain in rats and in human patients, but the underlying mechanisms are poorly understood. Results As the hyperexcitability of dorsal root ganglion neurons resulting from the upregulation of voltage-gated sodium (Nav) channels has been proved critical for development of chronic pain, we tested the effects of Bulleyaconitine A on Nav channels in rat spared nerve injury model of neuropathic pain. We found that Bulleyaconitine A at 5 nM increased the threshold of action potentials and reduced the firing rate of dorsal root ganglion neurons in spared nerve injury rats but not in sham rats. Bulleyaconitine A preferably blocked tetrodotoxin-sensitive Nav channels over tetrodotoxin-resistant ones in dorsal root ganglion neurons of spared nerve injury rats. Bulleyaconitine A was more potent for blocking Nav1.3 and Nav1.7 than Nav1.8 in cell lines. The half maximal inhibitory concentration (IC50) values for resting Nav1.3, Nav1.7, and Nav1.8 were 995.6 ± 139.1 nM, 125.7 ± 18.6 nM, and 151.2 ± 15.4 µM, respectively, which were much higher than those for inactivated Nav1.3 (20.3 ± 3.4 pM), Nav1.7 (132.9 ± 25.5 pM), and Nav1.8 (18.0 ± 2.5 µM). The most profound use-dependent blocking effect of Bulleyaconitine A was observed on Nav1.7, less on Nav1.3, and least on Nav1.8 at IC50 concentrations. Bulleyaconitine A facilitated the inactivation of Nav channels in each subtype. Conclusions Preferably blocking tetrodotoxin-sensitive Nav1.7 and Nav1.3 in dorsal root ganglion neurons may contribute to Bulleyaconitine A's antineuropathic pain effect.

Improving the Sensitivity of Solid-Phase Microextraction by Reducing the Volume of Off-Line Elution Solvent.

Solid-phase microextraction (SPME) coupled with liquid chromatograph (LC) is widely used to detect polar and ionic organic compounds, including various pharmaceuticals and endogenous bioactive compounds. In this study, a small-sized insert tube for use in the commercial autosampler vial was designed for eluting the extracted analytes from SPME fibers for LC analysis. By using this custom-made insert tube as an alternative to the commercial insert tube, the volume of the elution solvent was reduced by four-fifths. Even though smaller fractions of the analytes were eluted from the fiber coatings, the analyte concentrations in the elution solutions were substantially increased by using the custom-made insert tube. Therefore, larger amounts of the analytes could be injected to LC and higher signal-to-noise ratios could be achieved, even at smaller injection volumes. Since the elution in the custom-made insert tube was nonexhaustive, four strategies were developed to figure out the extracted amounts in the fiber coatings. In combination with the sampling-rate calibration method, these strategies were successfully used to determine the concentrations of fluoxetine in living tilapias. This study provides a simple but effect way for improving the analytical sensitivity when coupling SPME with LC.

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.

Upregulation of N-type calcium channels in the soma of uninjured dorsal root ganglion neurons contributes to neuropathic pain by increasing neuronal excitability following peripheral nerve injury.

N-type voltage-gated calcium (Cav2.2) channels are expressed in the central terminals of dorsal root ganglion (DRG) neurons, and are critical for neurotransmitter release. Cav2.2 channels are also expressed in the soma of DRG neurons, where their function remains largely unknown. Here, we showed that Cav2.2 was upregulated in the soma of uninjured L4 DRG neurons, but downregulated in those of injured L5 DRG neurons following L5 spinal nerve ligation (L5-SNL). Local application of specific Cav2.2 blockers (ω-conotoxin GVIA, 1-100 µM or ZC88, 10-1000 µM) onto L4 and 6 DRGs on the operated side, but not the contralateral side, dose-dependently reversed mechanical allodynia induced by L5-SNL. Patch clamp recordings revealed that both ω-conotoxin GVIA (1 µM) and ZC88 (10 µM) depressed hyperexcitability in L4 but not in L5 DRG neurons of L5-SNL rats. Consistent with this, knockdown of Cav2.2 in L4 DRG neurons with AAV-Cav2.2 shRNA substantially prevented L5-SNL-induced mechanical allodynia and hyperexcitability of L4 DRG neurons. Furthermore, in L5-SNL rats, interleukin-1 beta (IL-1ß) and IL-10 were upregulated in L4 DRGs and L5 DRGs, respectively. Intrathecal injection of IL-1ß induced mechanical allodynia and Cav2.2 upregulation in bilateral L4-6 DRGs of naïve rats, whereas injection of IL-10 substantially prevented mechanical allodynia and Cav2.2 upregulation in L4 DRGs in L5-SNL rats. Finally, in cultured DRG neurons, Cav2.2 was dose-dependently upregulated by IL-1ß and downregulated by IL-10. These data indicate that the upregulation of Cav2.2 in uninjured DRG neurons via IL-1ß over-production contributes to neuropathic pain by increasing neuronal excitability following peripheral nerve injury.

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.

Src-family kinases activation in spinal microglia contributes to central sensitization and chronic pain after lumbar disc herniation.

Background Lumbar disc herniation is a major cause of radicular pain, but the underlying mechanisms remain largely unknown. Spinal activation of src-family kinases are involved in the development of chronic pain from nerve injury, inflammation, and cancer. In the present study, the role of src-family kinases activation in lumbar disc herniation-induced radicular pain was investigated. Results Lumbar disc herniation was induced by implantation of autologous nucleus pulposus, harvest from tail, in lumbar 4/5 spinal nerve roots of rat. Behavior test and electrophysiologic data showed that nucleus pulposus implantation induced persistent mechanical allodynia and thermal hyperalgesia and increased efficiency of synaptic transmission in spinal dorsal horn which underlies central sensitization of pain sensation. Western blotting and immunohistochemistry staining revealed that the expression of phosphorylated src-family kinases was upregulated mainly in spinal microglia of rats with nucleus pulposus. Intrathecal delivery of src-family kinases inhibitor PP2 alleviated pain behaviors, decreased efficiency of spinal synaptic transmission, and reduced phosphorylated src-family kinases expression. Furthermore, we found that the expression of ionized calcium-binding adapter molecule 1 (marker of microglia), tumor necrosis factor-α, interleukin 1 -ß in spinal dorsal horn was increased in rats with nucleus pulposus. Therapeutic effect of PP2 may be related to its capacity in reducing the expression of these factors. Conclusions These findings suggested that central sensitization was involved in radicular pain from lumbar disc herniation; src-family kinases-mediated inflammatory response may be responsible for central sensitization and chronic pain after lumbar disc herniation.