Rab8a/SNARE complex activation promotes vesicle anchoring and transport in spinal astrocytes to drive neuropathic pain.

Neuropathic pain (NPP) remains a clinically challenging condition, driven by the activation of spinal astrocytes and the complex release of inflammatory mediators. This study aimed to examine the roles of Rab8a and SNARE complex proteins in activated astrocytes to uncover the underlying mechanisms of NPP. The research was conducted using a rat model with chronic constriction injury (CCI) of the sciatic nerve and primary astrocytes treated with lipopolysaccharide. Enhanced expression of Rab8a was noted specifically in spinal dorsal horn astrocytes through immunofluorescence. Electron microscopy observations showed increased vesicular transport and exocytic activity in activated astrocytes, which was corroborated by elevated levels of inflammatory cytokines such as interleukin (IL)-1ß and tumor necrosis factor (TNF)-α detected through quantitative PCR. Western blot analyses confirmed significant upregulation of Rab8a, VAMP2, and Syntaxin16 in these cells. Furthermore, the application of botulinum neurotoxin type A (BONT/A) reduced the levels of vesicle transport-associated proteins, inhibiting vesicular transport in activated astrocytes. These findings suggest that the Rab8a/SNARE pathway in astrocytes enhances vesicle transport and anchoring, increasing the secretion of bioactive molecules that may play a crucial role in the pathophysiology of NPP. Inhibiting this pathway with BONT/A offers a novel therapeutic target for managing NPP, highlighting its potential utility in clinical interventions.

Neutrophil extracellular traps as a unique target in the treatment of inflammatory pain.

Pain is a widespread motivation for seeking healthcare and stands as a substantial global public health concern. Despite comprehensive investigations into the mechanisms of pain sensitization induced by inflammation, efficacious treatments options remain scarce. Neutrophil extracellular traps (NETs) have been associated with the progression and tissue damage of diverse inflammatory diseases. This study aims to explore the impact of NETs on the progression of inflammatory pain and explore potential therapeutic approaches. Initially, we observed neutrophil infiltration and the formation of NETs in the left hind paw of mice with inflammatory pain induced by complete Freund's adjuvant (CFA). Furthermore, we employed the peptidyl arginine deiminase 4 (PAD4) inhibitor Cl-amidine (diluted at 50 mg/kg in saline, administered via tail vein injection once daily for three days) to impede NETs formation and administered DNase1 (diluted at 10 mg/kg in saline, once daily for three days) to break down NETs. We investigated the pathological importance of peripheral NETs formation in inflammatory pain and its influence on the activation of spinal dorsal horn microglia. The findings indicate that neutrophils infiltrating locally generate NETs, leading to an increased release of inflammatory mediators that worsen peripheral inflammatory reactions. Consequently, this results in the transmission of more harmful peripheral stimuli to the spinal cord, triggering microglial activation and NF-κB phosphorylation, thereby escalating neuroinflammation and fostering pain sensitization. Suppression of peripheral NETs can mitigate peripheral inflammation in mice with inflammatory pain, reverse mechanical and thermal hypersensitivity by suppressing microglial activation in the spinal cord, ultimately diminishing inflammatory pain. In conclusion, these discoveries propose that obstructing or intervening with NETs introduces a novel therapeutic avenue for addressing inflammatory pain.

Ulinastatin inhibits microglia activation in spinal cord via P2Y12 receptor in a rat neuropathic pain model.

Ulinastatin, a broad spectrum of serine protease inhibitor, has been found to alleviate neuropathic pain (NPP). However, its mechanism is not completely clear. Here, a sciatic nerve ligation rat model and BV2 microglial cells were used to investigate the effect of Ulinastatin on the activation of microglia and P2Y12 receptors in vivo and in vitro. Levels of P2Y12 receptor and NF-κB (P65) expression in the dorsal horn of the lumbar enlargement region of the spinal cord and BV2 cells were assessed by immunohistochemistry and double-label immunofluorescence assays. Levels of IL-1ß and TNF-α in cell culture medium and cerebrospinal fluid (CSF) were examined by ELISA. The results showed that Ulinastatin reduced the release of inflammatory IL-1ß and TNF-α by inhibiting the activation of spinal microglia. Ulinastatin down-regulated P2Y12 receptor and NF-κB (P65) expression in the spinal microglia of the chronic constrictive injury model. The results indicated that Ulinastatin may attenuate the activation of spinal microglia after peripheral nerve injury by inhibiting the activation of P2Y12 receptor signal pathway in microglia. NF-kB may play a key role in the mechanism of Ulinastatin.

Propofol downregulates the activity of glutamatergic neurons in the basal forebrain via affecting intrinsic membrane properties and postsynaptic GABAARs.

Propofol anesthesia rapidly causes loss of consciousness, while the neural mechanism underlying this phenomenon is still unclear. Glutamatergic neurons in the basal forebrain play an important role in initiation and maintenance of wakefulness. Here, we selectively recorded the activity of glutamatergic neurons in vGlut-2-Cre mice. Propofol induced outward currents in a concentration-dependent manner. Bath application of propofol generated membrane hyperpolarization and suppressed the firing rates in these neurons. Propofol-induced stable outward currents persisted after blockade of the action potentials, implying a direct postsynaptic effect of propofol. Furthermore, propofol selectively increased the GABAergic inhibitory synaptic inputs via affecting the GABAARs, but did not affect the glutamatergic transmissions. Together, propofol inhibits the excitability of the glutamatergic neurons via direct influencing the membrane intrinsic properties and the inhibitory synaptic transmission. This inhibitory effect might provide a novel mechanism for the propofol-induced anesthesia.

Circular RNA SFMBT2 Inhibits the Proliferation and Metastasis of Glioma Cells Through Mir-182-5p/Mtss1 Pathway.

Glioma is a common type of tumor in human central nervous system, and it is characterized with high mobility and mortality. The prognosis of patients with advanced glioma remains poor. Thus, it is necessary to develop novel therapeutic approaches for the treatment of this disease. Circular RNAs are a group of noncoding RNAs which have been detected in eukaryotic cells. They are tissue-specific and characterized with a more stable structure compared with linear RNAs. Recently, studies have revealed that certain circular RNAs are involved in biological processes such as gene regulation; however, the functions of most circular RNAs remain unknown and require further investigation. Furthermore, circular RNAs can act as "sponges" of its target microRNA, consequently suppressing their activity. Additionally, impaired expression of circular RNAs is reported in different diseases including cancer. In our study, low expression of circular RNA Scm like with 4 Mbt domains 2 was detected in glioma samples. Furthermore, reduced circRNA Scm like with 4 Mbt domains 2 expression was observed in human glioma cell lines compared to normal astrocyte cells. Additionally, overexpression of circRNA Scm like with 4 Mbt domains 2 suppressed the growth and metastasis of glioma cells in vitro. Moreover, microRNA-182-5p could be a downstream molecule of circRNA Scm like with 4 Mbt domains 2. The influenced of microRNA-182-5p-induced proliferation, migration, and invasion of glioma cells could be abrogated by overexpressed circRNA Scm like with 4 Mbt domains 2. In addition, metastasis suppressor 1 was predicted as a novel target of microRNA-182-5p, and its expression was restored by circRNA Scm like with 4 Mbt domains 2. In summary, our findings provided novel insight into the roles of circRNA Scm like with 4 Mbt domains 2 in glioma. More importantly, circRNA Scm like with 4 Mbt domains 2/microRNA-182-5p/metastasis suppressor 1 axis could be a putative therapeutic target for the treatment of patients with glioma.

Medication Adherence and Associated Factors for Children With Tic Disorders in Western China: A Cross-Sectional Survey.

Background: Adherence to treatment remains important for successful tic disorder (TDs) management, but no studies had previously been carried out to assess adherence or the factors that affect it in children with TDs. This study therefore aimed to explore adherence to prescribed medication among children with tic disorders and to examine the associated factors. Methods: This was a cross-sectional study in western China, where children with tic disorders were recruited consecutively in 2018. We used the eight-item Morisky Medication Adherence Scale (MMAS-8) to assess adherence. We included sociodemographic data, disease status, medication status, and family conditions as independent variables and used an ordinal logistic regression model to examine the factors affecting medication adherence. Results: A total of 204 patients were included, with a response rate of 96.2%. The majority of participants (77.5%) were male, aged 7.69 ± 2.58 years. In total, 37.7% were newly diagnosed, 46.1% were review patients, and 16.2% were recurrent patients. Only 40.7% of patients showed high adherence (MMAS-8 score, 8). Decreasing quality of life (adjusted odds ratio 0.974) and living in non-rural areas (adjusted odds ratio 2.361) were significant independent determinants of non-adherence. Conclusion: The results suggest that primary healthcare providers in pediatric clinics should focus on medication adherence counseling for children with tic disorders who have a lower quality of life and live in non-rural areas.

A combined treatment with erythrocyte lysis solution and Sudan Black B reduces tissue autofluorescence in double-labeling immunofluorescence.

The autofluorescence of animal tissues complicates the results obtained using fluorescence microscopy. Many techniques have been used to reduce autofluorescence; however, all these techniques have the disadvantage of reducing the intensity of immunofluorescence staining. We observed the features of autofluorescence in formalin-fixed paraffin-embedded (FFPE) vascularized liver and kidney sections and assessed the effects of an intravascular treatment with erythrocyte lysis solution (ELS) before a routine perfusion with normal saline (NS) and Sudan Black B (SBB) treatment after antigen retrieval on reducing autofluorescence reduction and the visualization of antigens to establish an optimal method for reducing autofluorescence. Erythrocytes exhibited bright autofluorescence in FFPE liver and kidney sections, which altered the results of actin and destrin immunofluorescence staining. The SBB treatment significantly reduced background autofluorescence and exerted a moderate effect on reducing the autofluorescence of erythrocytes, and the intravascular ELS treatment eliminated erythrocyte autofluorescence in FFPE liver and kidney sections. A combined treatment with ELS and SBB further reduced autofluorescence but did not decrease actin and destrin immunofluorescence staining in double-labeled FFPE liver and kidney sections. In conclusion, the application of an intravascular ELS treatment before the NS perfusion combined with an SBB treatment after antigen retrieval is a simple and efficient strategy for reducing autofluorescence in FFPE vascularized tissues and can be broadly used in fluorescence microscopy analyses.

Inhibition of GSK3 and MEK induced cancer stem cell generation via the Wnt and MEK signaling pathways.

Cancer stem cells (CSCs) are considered to be tumor­initiating cells, responsible for tumor invasive growth and dissemination to distant organ sites. Typically, radiation treatment and chemotherapy should target CSCs. However, current research investigating CSCs is impeded by the difficulty of isolating pure CSCs and maintaining them in vitro. In the present study, the synergistic inhibition of glycogen synthase kinase 3 and mitogen­activated protein kinase kinase using small molecules, CHIR99021 and PD184352, efficiently generated CSCs from immortalized human mammary epithelial cells (HMLEs) and resulted in the acquisition of mesenchymal traits and the expression of epithelial­mesenchymal transition markers. The cell proliferation, invasion and migration of HMLE cells were significantly promoted by CHIR99021 and PD184352 (P<0.05). Furthermore, the cell cycle was shifted from the G0/G1 phase to the G2/M phase, and the apoptotic rate was suppressed in HMLE cells following treatment with CHIR99021 and PD184352. Compared with control group, the stimulated cells exhibited an increased ability to form mammospheres and regenerate a tumor. In addition to these properties, the induced cells also exhibited notable chemotherapy resistance. In vivo, the treatment of cells with CHIR99021 and PD184352 promoted the growth of HMLE­engrafted tumor types. These results provide a practical strategy for the generation of CSCs using small molecules in vitro, which provides a cell resource that may be used for drug screening. Additionally, the present results additionally highlighted the synergistic functions of Wnt and mitogen­activated protein kinase kinase signaling pathways in tumorigenesis.

Ulinastatin attenuates neuropathic pain via the ATP/P2Y2 receptor pathway in rat models.

Ulinastatin, a serine protease inhibitor, which has anti-inflammatory properties and neuroprotective effects, is used to treat acute inflammatory disorders. Recent evidence indicates that administration of ulinastatin alleviates pain in rat model of neuropathic pain (NPP). However, its effect on NPP and the underlying mechanism requires further study. In this study, we evaluated the role of intrathecal administration of ulinastatin in rats with sciatic nerve ligation and observed the effect of ulinastatin on the ATP/P2Y2 receptor pathway. We performed mechanical and thermal sensitivity measurements, immunohistochemistry and double-label immunofluoresence studies to evaluate P2Y2 receptor and adenosine 5'-monophosphate-activated protein kinase (AMPK) expression in the dorsal horn of the lumbar enlargement region of the spinal cord, and a luciferase assay for the detection of ATP levels in the cerebrospinal fluid. The results showed that ulinastatin prevented the development of mechanical allodynia and thermal hypersensitivity in the rat sciatic nerve ligation model. Ulinastatin reduced the level of extracellular ATP, down-regulated P2Y2 receptor and AMPK expression in the spinal dorsal horn of the chronic constrictive injury model. We found that increased expression of P2Y2 receptor in microglia was likely involved in the activation of microglia after nerve injury, and ulinastatin inhibited the abnormal microglia activation in the dorsal horn after nerve injury. These findings demonstrated that ulinastatin might be a potential and effective drug for the treatment of NPP via the suppression of the ATP/P2Y2 receptor pathway.

A preliminary study on the role of the complement regulatory protein, cluster of differentiation 55, in mice with diabetic neuropathic pain.

The aim of this study was to investigate the role of the complement regulatory protein cluster of differentiation 55 (CD55) in the pathogenesis of diabetic neuropathic pain (DNP). Healthy adult male C57BL/6J mice were intraperitoneally injected with streptozotocin (STZ) in order to induce DNP. Peripheral blood glucose and protein, and the mRNA expression levels of C3 and CD55 in the spinal cord were determined. In addition, the behaviors of these mice were observed. The results showed that STZ­treated mice displayed the clinical manifestations of diabetes mellitus, and that their peripheral blood glucose was markedly increased. On the 21st and 28th days following the STZ injection, the mechanical pain threshold and thermal pain threshold of the mice were dramatically reduced (P<0.05). |Additionally, 14 days post­STZ injection, the mRNA expression of C3 in the spinal cord was significantly increased, which continued for 28 days. On the 21st and 28th days, the number of C3 positive cells in the spinal cord was markedly increased. Seven days after the STZ injection, the number of cells positive for CD55 was markedly reduced in the spinal dorsal horn and subsequently remained at a low level. The mRNA expression of CD55 also was significantly reduced (P<0.05) and remained so for 28 days. The reduction in the expression levels of CD55 occurred earlier than the changes in the expression of C3, suggesting that the downregulation of CD55 expression precedes, and has an important role regarding, the activation of C3 in the occurrence and development of DNP.

Early high volume lung lavage for acute severe smoke inhalation injury in dogs.

The aim of the present study was to investigate the safety and short­term results of early high volume lung lavage in the treatment of acute severe smoke inhalation injuries in dogs. A high­volume normal saline complex solution lavage in the left lung was performed 1 h subsequent to bilateral pulmonary acute severe sawdust smoke inhalation injury in dogs. Lavage of the right lung was conducted following an interval of 30 min or 4 h. The perfluorodecalin lavage was performed in dogs with unilateral pulmonary acute severe sawdust smoke inhalation injury. The present study identified that lavage with an interval of 4 h between two lungs was safer compared with a 30­min interval. Following lavage, the increase in the levels of free radical metabolites and inflammatory mediators in the lung homogenate was reduced. Acute severe smoke inhalation injury in one lung evidently caused a secondary injury to the other lung in the dogs. Perfluorodecalin lavage did not achieve the same effect in cleansing the lungs as the normal saline, but a greater comprehensive short­term outcome was obtained. These observations demonstrated that early high­volume lung lavage following severe smoke inhalation injury could relieve primary injuries and secondary local and general inflammatory reactions in dogs. An improved comprehensive short­term outcome was obtained in the perfluorodecalin­lavaged dogs.

Abnormal activation of complement C3 in the spinal dorsal horn is closely associated with progression of neuropathic pain.

The aim of the present study was to investigate the role of complement activation in the pathogenesis of neuropathic pain (NPP) induced by peripheral nerve injury. We modified a classical chronic constriction injury (CCI) model (mCCI), and verified its reliability in rats. Furthermore, reverse transcription-PCR and immunohistochemistry were conducted to investigate complement activation in the spinal dorsal horn and the effect of a complement inhibitor, cobra venom factor (CVF), on the behavior of the mCCI model rats. We found that rats in the mCCI group presented a better general condition, without signs of autophagy of the toes. Moreover, mCCI induced a significant increase (+40%) in the expression of component 3 (C3) mRNA in the spinal dorsal horn, which was associated with hyperalgesia. Correlation analysis showed a negative correlation between the mechanical pain threshold and the expression of C3 in the spinal cord. Administration of CVF reduced the occurrence of hyperalgesia in mCCI rats and nearly reversed the hyperalgesia. In addition, the mCCI rats exhibited significantly less spinal superoxide dismutase activity and significantly greater levels of maleic dialdehyde compared to the sham-operated rats. Transmission electron micrographs revealed mitochondrial swelling, cell membrane damage, and cristae fragmentation in the neurons of the spinal dorsal horn 14 days after mCCI. Mitochondrial swelling was attenuated in mCCI rats receiving CVF. The findings demonstrated that abnormal complement activation occurred in the dorsal horn of the spinal cord in rats with NPP, and C3 in the spinal dorsal horn could play an important role in the cascade reaction of complements that are involved in the development of hyperalgesia.