TRPC通道阻断剂SKF-96365对蜜蜂毒肽诱发初级感觉细胞内向电流和胞内钙增高的作用(英文)

目的蜜蜂毒肽是蜜蜂粗毒中的主要物质。外周皮下注射蜜蜂毒肽可导致持续性自发痛和痛觉过敏。本研究旨在研究瞬时受体电势C(transient receptor potential canonical,TRPC)通道在蜜蜂毒肽诱致的初级感觉神经元活化中的介导作用。方法运用全细胞膜片钳和激光共聚焦测钙技术,检测TRPC通道抑制剂SKF-96365对蜜蜂毒肽诱致的急性分离大鼠背根神经节细胞胞内钙和内向电流升高的影响。结果电压钳记录的91个背根神经节细胞中,蜜蜂毒肽可诱发43.9%(40/91)的细胞产生内向电流,而不同浓度的SKF-96365(1,5,10μmol/L)均明显抑制了背根神经节细胞的内向电流,且呈剂量相关性。应用激光共聚焦钙成像技术记录的210个背根神经节细胞中,67.6%的细胞对蜜蜂毒肽敏感,产生胞内钙离子浓度的升高,而SKF-96365能抑制这种胞内钙浓度的升高,抑制率为46.5%。结论 SKF-96365能够抑制蜜蜂毒肽引起的背根神经节中小神经元的活化,提示TRPC通道介导了蜜蜂毒肽对初级感觉神经元的激活作用。     

背根神经节参与疼痛机制研究进展

背根神经节神经元是躯干、四肢痛觉的初级传入神经元,具有传输和调节机体感觉、接受和传导伤害性感受的功能,在疼痛机制中发挥重要作用。临床以背根神经节为靶点的介入治疗和神经调节治疗可以缓解疼痛。本文拟就背根神经节的钠离子通道、胶质细胞和炎症反应在疼痛机制中的研究进展进行综述。     

抑制容量性钙内流对U87侵袭及迁移力的影响

目的探讨抑制容量性钙内流(CCE)对胶质瘤U87细胞侵袭及迁移力的影响。方法采用免疫印迹(Western blot)检测基质相互作用分子1(STIM1)RNA干涉(RNAi)后U87细胞STIM1表达变化;活细胞钙成像检测CCE抑制剂(SKF-96365)处理及STIM1 RNAi后U87细胞CCE的改变;细胞划痕和Transwell侵袭实验检测CCE对U87细胞侵袭及迁移力的影响。结果 SKF-96365处理及STIM1RNAi均能明显抑制U87细胞的CCE(P0.05);与对照组比较,抑制CCE后U87细胞迁移及侵袭能力明显降低(P0.05)。结论抑制CCE可有效抑制U87细胞迁移及侵袭力,其机制可能与调控细胞内钙稳态相关。     

由感觉和交感节神经元组成的中枢外反射弧的形态学研究

用免疫细胞化学和辣根过氧化物酶（HRP）逆行追踪法研究了感觉神经节和交感神经节中P物质（SP）样免疫反应物的分布及两种神经节之间的联系。在所有背根节出现大量SP免疫阳性神经元胞体，但在胸交感干节和腹腔-前肠系膜神经节仅有大量的SP阳性纤维和终末，而无阳性胞体。分组切断与腹腔-前肠系膜神经节相联系的神经支后，该节内的SP免疫反应物仅在切断与背根节的联系后明显减少或消失。将HRP注入腹腔神经节，在同侧C8～L3背根节出现大量标记细胞。以上结果表明．在感节中的SP纤维和终末来自背根节初级传入神经元的轴突侧支，提示由初级传人神经元与交感节主细胞在中枢外直接形成了短反射环路。     

嗅鞘细胞对培养脊髓神经元生长状态的影响

目的研究嗅鞘细胞(OECs)对体外培养脊髓背根神经节神经元生长状态的影响。方法取新生大鼠脊髓背根神经节细胞与嗅鞘细胞共培养,在显微镜下观察神经元生长发育情况,染色后进行细胞计数,并测定细胞活性。结果共培养组细胞密度明显高于对照组,神经元胞体大而饱满,突起较长,细胞活性较高。结论嗅鞘细胞可明显促进体外培养脊髓背根神经节神经元的生长,提高细胞活性。     

脑神经肽对人胚神经细胞生长发育及对小鼠急性缺血脑组织的影响

目的和方法应用体外培养的人胚背根神经节和大脑皮层神经元，研究了神经肽对神经组织和神经细胞生长发育的影响，还通过小鼠的急性脑缺血模型研究神经肽对急性脑缺血的作用。结果发现神经肽可使背根神经节突起的长度和密度增加，皮层神经元存活数增加，神经元分化率增高，胞体增大，突起延长，细胞生长加快。超微结构观察显示神经肽可增强细胞内的合成代谢和细胞间连接。脑神经肽可使小鼠脑组织水肿较轻神经元无明显空泡，细胞超微结构改变较轻微，脑组织LDH活性高于对照组。结论神经肽对神经组织具有营养作用，并对急性脑缺血也具有保护作用。     

大鼠背根神经节细胞的纯化培养

目的:在NB1培养基中建立体外胚胎大鼠背根神经节(DRG)细胞的纯化培养体系。方法:取胎鼠的背根神经节,用胰蛋白酶消化法分离成单细胞,通过差速贴壁法进行背根神经节神经元的分离纯化,在NB1中培养,用活细胞计数和神经元特异性的烯醇化酶(NSE)免疫组织化学染色相结合判定培养神经元的纯度。结果:纯化培养神经元生长状态良好,培养4天时神经元纯度为91％左右。结论:采用差速贴壁法可获得高纯度的神经元,可作为神经科学研究的重要工具。     

凝血酶对原代培养海马神经元游离钙浓度的影响

目的研究凝血酶对原代培养海马神经元内游离Ca^2 水平的影响。方法采用原代培养大鼠海马神经元方法，用钙离子指示剂Fura-2双波长荧光检测凝血酶对海马神经元内游离钙浓度的影响。结果(1～40)U／ml凝血酶可使海马神经元内游离Ca^2 水平显著升高，且呈剂量依赖性。凝血酶受体激活肽可明显升高细胞内游离[Ca^2 ]i。当胞外Ca^2 为1．3mmol／L时，40U／ml凝血酶可使海马神经元游离[Ca^2 ]i明显增加；而当胞外Ca^2 为0．0mmol／L时，40U／ml凝血酶不影响海马神经元游离[Ca^2 ]i。预先加入MK-801可显著降低凝血酶的升钙作用。结论凝血酶使神经细胞内游离Ca^2 浓度异常升高的作用机制可能是通过激活凝血酶受体，继而激活NMDA受体门控的Ca^2 通道介导胞外Ca^2 大量内流。     

背根神经节神经元胞体膜可能存在NMDA自身受体

应用经酶和机械消化分离的大鼠背根神经节（ＤＲＧ）神经元进行实验。首先运用全细胞膜片箝技术找到ＮＭＤＡ可引起一内向电流的神经元,此内向电流可被甘氨酸明显增强,并被ＮＭＤＡ受体的特异性拮抗剂ＡＰＶ完全阻断。随后,把经电生理技术证实膜上存在ＮＭＤＡ受体的这一ＤＲＧ神经元用微吸管转移到载玻片上,进行谷氨酸神经递质免疫组织化学检测。１０／１８的ＤＲＧ细胞呈免疫反应阳性,结果为ＤＲＧ神经元膜上存在ＮＭＤＡ自身受体提供了新证据。     

电压门控钠通道、钙通道与抗癎治疗

电压门控钠通道介导再生内向电流,这些电流负责脑神经元动作电位的初始去极化.抗痫药以及许多抑制和癫痫发作有关的异常神经元兴奋的有前景的化合物,大多是通过钠通道完成电压和频率依赖的离子电流的抑制.电压激活钙通道在控制神经元兴奋性、神经保护作用、癫癎波的发放和癎性发作的维持及传播中起重要作用.     

Inhibition of P2X receptor-mediated inward current by protein kinase C in small-diameter dorsal root ganglion neurons of adult rats

Objective

The aim of the present study is to verify the ATP-induced varied responses in isolated dorsal root ganglion (DRG) neurons of the adult rat, and investigate the modulatory effects of specific P2X receptor agonist β, γ-me-ATP and protein kinase C (PKC) on P2X receptor-mediated inward current in DRG neurons.

Methods

Whole cell patch-clamp was employed to record the currents on acutely isolated DRG neurons in the adult rats.

Results

β, γ-me-ATP, similar as ATP, evoked 2 distinct subtypes of P2X receptor-mediated inward currents in a dose-dependent manner in DRG neurons. Activation of PKC by phorbol 12,13-dibutyrate (PDBu) significantly inhibited both subtypes of inward currents mediated by P2X receptors in a dose-dependent manner.

Conclusion

Activation of PKC negatively modulated the P2X receptor-mediated currents in rat DRG neurons, which may be of benefit to preventing the over-excitation of nociceptor under inflammatory or neuropathic conditions.     

Effects of pentobarbital anesthesia on nociceptive processing in the medial and lateral pain pathways in rats

Objective

To investigate the effects of pentobarbital anesthesia on nociceptive processing in the medial and lateral pain pathways.

Methods

Laser stimulation was employed to evoke nociceptive responses in rats under awake or anesthetic conditions. Pain-related neuronal activities were simultaneously recorded from the primary somatosensory cortex (SI), ventral posterolateral thalamus (VPL), anterior cingulate cortex (ACC), and medial dorsal thalamus (MD) with 4 eight-wire microelectrode arrays.

Results

Compared with the awake state, pentobarbital anesthesia significantly suppressed the neural activities induced by noxious laser stimulation. Meanwhile, the pain-evoked changes in the neuronal correlations between cortex and thalamus were suppressed in both medial and lateral pain pathways. In addition, the spontaneous firing rates in all the 4 areas were altered (including inhibition and excitation) under the condition of anesthesia.

Conclusion

The nociceptive processing in the brain can be dramatically changed by anesthesia, which indicates that there are considerable differences in the brain activities between awake and anesthetized states. It is better to employ awake animals for recording neural activity when investigating the sensory coding mechanisms, especially pain coding, in order to obtain data that precisely reflect the physiological state.     

Competitive Testing of Health Behavior Theories: How Do Benefits,Barriers, Subjective Norm,and Intention Influence Mammography Behavior?

Background

Competitive hypothesis testing may explain differences in predictive power across multiple health behavior theories.

Purpose

We tested competing hypotheses of the Health Belief Model (HBM) and Theory of Reasoned Action (TRA) to quantify pathways linking subjective norm, benefits, barriers, intention, and mammography behavior.

Methods

We analyzed longitudinal surveys of women veterans randomized to the control group of a mammography intervention trial (n?=?704). We compared direct, partial mediation, and full mediation models with Satorra–Bentler χ ² difference testing.

Results

Barriers had a direct and indirect negative effect on mammography behavior; intention only partially mediated barriers. Benefits had little to no effect on behavior and intention; however, it was negatively correlated with barriers. Subjective norm directly affected behavior and indirectly affected intention through barriers.

Conclusions

Our results provide empiric support for different assertions of HBM and TRA. Future interventions should test whether building subjective norm and reducing negative attitudes increases regular mammography.     

Initial bradykinin triggers calcium-induced calcium release in C6 glioma cells and its significance

Objective

To investigate the underlying mechanism for the selective modulation of the permeability of blood-tumor barrier (BTB) by small dose of bradykinin (BK).

Methods

C6 glioma cells were treated with BK, and changes of intracellular nitric oxide (NO) and intracellular calcium level were measured with fluorescent spectrophotometer.

Results

The initial application of BK easily triggered extracellular calcium influx, which resulted in intracellular calcium store release in C6 glioma cells. The above mechanism was also named ryanodine mediated calcium induced calcium release (CICR). We also detected a long-lasting intracellular NO elevation in C6 glioma cells upon BK treatment. Further study showed that ryanodine mediated CICR contributed greatly to the secondary NO elevation induced by BK treatment.

Conclusion

These results suggested that BK triggered CICR in C6 glioma cells and the associated NO generation might be the underlying mechanism for the selective modulation of BTB permeability by BK.     

TRPC6 Inhibited NMDA Current in Cultured Hippocampal Neurons

N-methyl-d-aspartate (NMDA) receptors play a key role in excitatory synaptic transmission, plasticity and neural development, and they also mediate excitotoxicity that is involved in both acute neuronal damage and chronic neurodegenerative diseases. Regulation of the NMDA channel activity is critical for the pathological processes of these diseases. The canonical transient receptor potential channels (TRPCs) are Ca²⁺-permeable nonselective cation channels with various physiological functions, including promoting neuronal survival. Here, we reported that TRPC6, a member of the TRPC family, inhibited the NMDA-induced current in primary cultured hippocampal neurons. Overexpression of TRPC6 or application of 1-oleoyl-2-acetyl-sn-glycerol, a compound known to activate TRPCs, inhibited the NMDA-induced current in these neurons assayed by the whole-cell patch-clamp recording. Consistently, downregulation of TRPC6 or application of SKF96365, a compound known to inhibit TRPCs, enhanced this current. The peak amplitude of the NMDA current in the neurons isolated from TRPC6 transgenic mice was greatly suppressed than that in the neurons isolated from the wild-type littermates. Furthermore, TRPC6 might activate calcineurin to inhibit the activity of NMDA receptors in cultured hippocampal neurons. Together, these results suggested that TRPC6 might be a novel negative modulator of NMDA receptors.     

Recombinant human erythropoietin counteracts cisplatin-induced visceral hyperalgesia

Objective

Cisplatin exerts its cytotoxic effect through distinct DNA lesions, leading to peripheral neuropathy. The risk of sensory neuropathy is a common problem during cancer treatment with cisplatin, leading to somatic hyperalgesia. Yet, data focussing on cisplatin-induced impairment of the autonomic nervous system are limited. The present study was aimed to investigate the effect of recombinant human erythropoietin (rhEPO) on cisplatin-induced visceral hyperalgesia.

Methods

C57BL/6 mice were treated either with cisplatin (2 mg/kg, once per week) or with cisplatin (2 mg/kg, once per week) plus rhEPO (40 μg/kg, 3 times per week) for 8 weeks. Controls were treated with saline. To quantify the visceromotor response (VMR) at week 9, standardized electrodes were implanted into the external oblique musculature for electromyographic recordings. After that, animals were decapitated and dorsal root ganglia (DRG) was removed for transmission electron microscopy studies.

Results

Cisplatin-treated mice showed a significant increase of VMR compared to the controls [(7080 ± 969) vs (2864 ± 279); P < 0.001], while rhEPO dramatically counteracted this effect [(2962 ± 336) vs (7080 ± 969); P < 0.001)]. Transmission electron microscopy revealed cisplatin-induced structural lesions of nuclear membrane in DRG cells, which could be ameliorated by rhEPO.

Conclusion

Erythropoietin can significantly ameliorate the cisplatin-induced visceral hyperplasia and DRG nuclear membrane structure damage in mice, indicating a neuroprotective role of erythropoietin.     

Ensheathing cells utilize dynamic tiling of neuronal somas in development and injury as early as neuronal differentiation

Background

Glial cell ensheathment of specific components of neuronal circuits is essential for nervous system function. Although ensheathment of axonal segments of differentiated neurons has been investigated, ensheathment of neuronal cell somas, especially during early development when neurons are extending processes and progenitor populations are expanding, is still largely unknown.

Methods

To address this, we used time-lapse imaging in zebrafish during the initial formation of the dorsal root ganglia (DRG).

Results

Our results show that DRG neurons are ensheathed throughout their entire lifespan by a progenitor population. These ensheathing cells dynamically remodel during development to ensure axons can extend away from the neuronal cell soma into the CNS and out to the skin. As a population, ensheathing cells tile each DRG neuron to ensure neurons are tightly encased. In development and in experimental cell ablation paradigms, the oval shape of DRG neurons dynamically changes during partial unensheathment. During longer extended unensheathment neuronal soma shifting is observed. We further show the intimate relationship of these ensheathing cells with the neurons leads to immediate and choreographed responses to distal axonal damage to the neuron.

Conclusion

We propose that the ensheathing cells dynamically contribute to the shape and position of neurons in the DRG by their remodeling activity during development and are primed to dynamically respond to injury of the neuron.

     

Inhibition of phosphorylated STAT3 by cucurbitacin I enhances chemoradiosensitivity in medulloblastoma-derived cancer stem cells

Introduction

CD133 (PROM1) is a potential marker for cancer stem cells (CSCs), including those found in brain tumors. Recently, medulloblastoma (MB)-derived CD133-positive cells were found to have CSC-like properties and were proposed to be important contributors to tumorigenicity, cancer progression, and chemoradioresistance. However, the biomolecular pathways and therapeutic targets specific to MB-derived CSCs remain unresolved.

Materials and methods

In the present study, we isolated CD133⁺ cells from MB cell lines and determined that they showed increased tumorigenicity, radioresistance, and higher expression of both embryonic stem cell-related and drug resistance-related genes compared to CD133^? cells. Bioinformatics analysis suggested that the STAT3 pathway might be important in MB and CD133⁺ cells. To evaluate the effects of inhibiting the STAT3 pathway, MB-derived CD133^+/? cells were treated with the potent STAT3 inhibitor, cucurbitacin I. Treatment with cucurbitacin I significantly suppressed the CSC-like properties and stemness gene signature of MB-derived CD133⁺ cells. Furthermore, cucurbitacin I treatment increased the apoptotic sensitivity of MB-derived CD133⁺ cells to radiation and chemotherapeutic drugs. Notably, cucurbitacin I demonstrated synergistic effects with ionizing radiation to inhibit tumorigenicity in MB-CD133⁺-inoculated mice.

Results

These results indicate that the STAT3 pathway plays a key role in mediating CSC properties in MB-derived CD133⁺ cells. Targeting STAT3 with cucurbitacin I may therefore represent a novel therapeutic approach for treating malignant brain tumors.     

Endogenous Inhibition of the Nociceptive Flexion Reflex (NFR) and Pain Ratings During the Menstrual Cycle in Healthy Women

Background

The menstrual cycle influences pain, with symptoms often increasing during the premenstrual (late-luteal) phase. Deficiencies in endogenous inhibition of afferent nociception at the spinal level might contribute to menstrual phase-related changes in pain.

Purpose

This study assessed whether conditioned pain modulation (CPM) of spinal nociception differs between mid-follicular and late-luteal phases.

Methods

CPM was evoked by a blood pressure cuff affixed to the right forearm and inflated to induce ischemia in 41 healthy women during both menstrual phases. Suprathreshold electric stimuli were delivered to the left sural nerve to evoke pain and the nociceptive flexion reflex (NFR) before, during, and after forearm ischemia.

Results

Forearm ischemia produced CPM of electrocutaneous pain and NFR, but inhibition did not differ across mid-follicular and late-luteal phases.

Conclusions

Mechanisms contributing to changes in experimental pain across mid-follicular and late-luteal phases in healthy women are not due to deficits in CPM of spinal nociception.