Rhodiola rosea extract protects human cortical neurons against glutamate and hydrogen peroxide-induced cell death through reduction in the accumulation of intracellular calcium

The aim of this study was to investigate the neuroprotective effects of a titolated extract from Rhodiola rosea L. (RrE) and of salidroside (Sa), one of the major biologically active compounds extracted from this medicinal plant, against oxidative stressor hydrogen peroxide (H₂O₂) and glutamate (GLU)‐induced cell apoptosis in a human cortical cell line (HCN 1‐A) maintained in culture. The results obtained indicate that exposure of differentiated HCN 1‐A neurons to GLU or H₂O₂ resulted in concentration‐dependent cell death. A 24 h pre‐treatment with RrE significantly increased cell survival and significantly prevented the plasma membrane damage and the morphological disruption caused by GLU or H₂O₂, indicating that neurons treated with RrE were protected from the neurotoxicity induced by the oxidative stressor used. In addition, RrE significantly reduced H₂O₂ or GLU‐induced elevation of intracellular free Ca²⁺ concentration. The results obtained have also shown that Sa caused similar effects in all experimental models used; however, the potency of the action was lower than that of the extract containing corresponding quantities of Sa. These findings indicate that RrE has a neuroprotective effect in cortical neurons and suggest that the antioxidant activity of the RrE, due to the structural features of the synergic active principles they contain, may be responsible for its ability to stabilize cellular Ca²⁺ homeostasis. Copyright © 2011 John Wiley & Sons, Ltd.     

Elevated expression of chloride intracellular channel 1 is correlated with poor prognosis in human gliomas

Background

Chloride intracellular channel 1 (CLIC1) is expressed ubiquitously in human tissues and is involved in the regulation of cell cycle, cell proliferation and differentiation. Recent studies have shown that CLIC1 is highly expressed in several human malignant tumors. However, its roles in human gliomas are still unclear. The aim of this study was to investigate the clinicopathological significance and prognostic value of CLIC1 expression in human gliomas.

Methods

CLIC1 expression in human gliomas and nonneoplastic brain tissues was measured by real-time quantitative RT-PCR assay and immunohistochemistry. Its association with clinicopathological factors or prognosis in patients with gliomas was statistically analyzed.

Results

The expression of CLIC1 at both mRNA and protein levels was significantly increased in high-grade (Grade III~IV) glioma tissues compared with that in low-grade (Grade I~II) and nonneoplastic brain tissues, and was up-regulated with ascending tumor World Health Organization (WHO) grades. The elevated expression of CLIC1 protein was also significantly correlated with low Karnofsky performance score (KPS) (P=0.008). Moreover, both univariate and multivariate analysis shown that high CLIC1 expression was significantly associated with poor prognosis in patients with gliomas (P<0.001 and P=0.01, respectively). In particular, the elevated CLIC1 expression also correlated with shorter overall survival in different glioma subgroups stratified according to the WHO grading.

Conclusions

Our data provide the first evidence that CLIC1 expression might play an important role in the regulation of aggressiveness in human gliomas. The elevated expression of CLIC1 might represent a valuable prognostic marker for this disease.     

Characterization of transient receptor potential vanilloid channel 4 (TRPV4) in human corneal endothelial cells

The transient receptor potential vanilloid 4 (TRPV4) is a Ca²⁺-and Mg²⁺ permeable cation channel that might be a cellular osmosensor since it is activated upon hypotonic cell swelling. TRPV4 is also thermosensitive and responds to moderate heat (from 24 to 27 °C) as well as to phorbol esters (4α-PDD) and several endogenous substances including arachidonic acid (AA), the endocannabinoids anandamide and 2-AG, and cytochrome P-450 metabolites of AA, such as epoxyeicosatrienoic acids. The resulting Ca²⁺ influx occurring in response to swelling induces regulatory volume decrease (RVD) behavior. As regulation of cell volume is essential for corneal endothelial function, we determined whether human corneal endothelial cells have functional TRPV4 channel activity. RT-PCR identified TRPV4 gene expression in the HCEC-12 cell line as well as two clonal daughter cell lines (HCEC-H9C1, HCEC-B4G12). [Ca²⁺]_i transients were monitored in fura-2 loaded cells. Nonselective cation channel currents were recorded in the whole-cell mode of the planar patch-clamp technique. TRPV4 mRNA was found in HCEC-12 and the clonal daughter cell lines. TRPV4 channel agonists (4α-PDD and GSK1016790A; both 5 μmol/l) as well as moderate heat (<40 °C) elicited [Ca²⁺]_i transients. Hypotonicity increased [Ca²⁺]_i and nonselective cation channel currents in HCEC-12 cells. There is functional TRPV4 expression in HCEC-12 and in its clonal daughter cell lines based on Ca²⁺ transients and underlying currents induced by known activators of this channel.     

Human anterior lens capsule epithelial cells contraction

Purpose: Human anterior lens epithelial cells, attached to surgically isolated capsules, were found to contract upon stimulation. The purpose of this study was to characterize these contractions, which create gaps between cells, and to assess the underlying physiological mechanisms and their possible association with cataract formation. Methods: Lens capsules obtained during cataract surgery were stained with fluorescent dye Fura‐2. Its fluorescence, upon excitation at 360 and 380 nm, was imaged to monitor changes in cell morphology and cytosolic free Ca²⁺concentrations ([Ca²⁺]_i) in response to pharmacological stimulation by acetylcholine (ACh) and to mechanical stimulation by flow of saline or direct contact. Results: Epithelial cells contracted in approximately a third of preparations when stimulated by either ACh application, fluid movement or direct mechanical contact. Contractions started either before or at best simultaneously with the rise in [Ca²⁺]_i. Contractions also occurred when there was hardly any change in [Ca²⁺]_i upon application of physiological saline alone. The probability of contractions occurring did not differ significantly among cortical, nuclear and combined cortical + nuclear cataract. Conclusions: This study provides the evidence that contractions of the anterior lens epithelial cells take place in significant portion of human lens anterior capsule postoperative preparations after non‐specific stimulation. Contractions are at least partially independent of changes in [Ca²⁺]_i. They can be mechanically induced, are localized and reversible and have a fast response and did not differ among different types of cataract. Physiological and clinical significance of this phenomenon remains to be elucidated.     

多电极阵列记录技术在大鼠视网膜生理研究中的应用

目的 采用多电极阵列（MEA）技术探索记录Sprague-Dawley（SD）大鼠视网膜电生理特征的方法。方法 实验研究。采用成年清洁级雄性SD大鼠6只。处死后急性分离出视网膜神经感觉层,将其转移到MEA生物芯片上,使神经节细胞一面贴于电极阵列,用充氧的Ringer′s液孵育15 min后,记录不同光照刺激或电刺激模式下大鼠视网膜电生理特征。所有数据经Shapiro-Wilk检验呈正态分布,以x±s进行描述。结果 MEA能够记录到SD大鼠视网膜典型的光刺激诱发场电位改变,这种光诱发场电位的幅值随刺激光亮度增加先增大后稳定。暗适应后给予高光亮度（256 cd/m2）刺激时,长时程光照后（6～10 s）,可记录到一个明显的撤光相关场电位改变,表现为一个负向偏折。根据光诱发神经节细胞的放电模式与光刺激的关系,可将神经节细胞分为ON、OFF及ON/OFF型。电刺激诱发神经节细胞的放电主要集中在刺激后的50 ms内。结论 MEA技术可以在保证视网膜神经回路完整的情况下,从多个方面（感觉细胞、双极细胞、神经节细胞3个水平）对视网膜的功能做出客观的评定。     

肾移植后排斥与感染中细胞免疫功能的监测

背景：Cylex ImmuKnow检测方法是惟一得到FDA认可的检测移植受者细胞免疫功能的方法, 它直接反映细胞免疫的功能,具有灵敏度高、特异性强,结果量化等优点。 目的：通过对肾移植后患者监测细胞免疫功能iATP值(intracellular adenosine triphosphatei),分析细胞免疫功能与肾移植术后排斥或感染的关系。 方法：140例肾移植受者采用ImmuKnowTM-Cylex®方法检测细胞免疫功能,收集数据结合患者临床状态分为排斥组18例,感染组35例与稳定组87例,同时收集61份健康人群血样检测细胞免疫功能数据作对照。分析细胞免疫功能与肾移植患者移植后发生排斥与感染的关系。 结果与结论：细胞免疫功能iATP值检测结果显示,感染组患者有71.4%(n=25)分布在低免疫区,同比对照组、稳定组与排斥组比例明显增高(P < 0.05)。感染组iATP值显著低于其他3组。说明感染与细胞免疫功能低下两者具有明显的相关性,细胞免疫功能测定对监测肾移植后感染具有显著意义。提示ImmuKnowTM-Cylex®方法测定细胞免疫功能为肾移植后并发感染的诊断与治疗提供可靠客观依据,但与肾移植后排斥反应的发生未见明显的相关性,有待于大样本的实验证实。     

骨髓间充质干细胞向神经元样细胞分化前后基因及Ca2+浓度的变化

背景：单唾液酸四己糖神经节苷脂(Monosialotetrahexosylganglioside,GM1)在神经细胞的生长发育、分化、再生和细胞内外信息传递等多种生理过程中发挥重要作用。 目的：探讨GM1对骨髓间充质干细胞按照Woodbury经典诱导方案将其诱导分化为神经元样细胞前后基因方面和细胞内游离Ca2+浓度的变化。 方法：分离纯化SD大鼠骨髓间充质干细胞进行传代培养,传至第5代时,待细胞融汇成致密单层后,加入50 mmol/L神经节苷脂设为GM1组,预培养24 h后按照Woodbury经典诱导方案进行诱导,设置对照组,采用免疫组化和Realtime PCR技术分别检测诱导前后生长相关蛋白43、神经元特异性烯醇化酶、神经丝蛋白和神经巢蛋白的蛋白和mRNA表达的变化,采用激光扫描共聚焦显微镜检测诱导前后细胞内游离钙离子浓度的变化。 结果与结论：①加入GM1组诱导分化后较对照组生长相关蛋白43、神经元特异性烯醇化酶、神经丝蛋白和神经巢蛋白表达水平增高(P < 0.05),GM1能够促骨髓间充质干细胞诱导分化为神经元样细胞。②更换诱导液后,两组细胞内荧光强度逐渐增加,到100 s 达高峰值,其后逐渐减弱,但20 min 时细胞荧光强度仍高于诱导前,加入GM1组,细胞内游离Ca2+浓度较对照组有所增加(P < 0.05)。说明GM1能够促进细胞内Ca2+浓度增加,游离Ca2+在诱导过程中可能有促进作用。③诱导后生长相关蛋白43、神经元特异性烯醇化酶、神经丝蛋白和神经巢蛋白基因表达改变不显著,说明Woodbury经典诱导方案可能为转录后水平即蛋白水平的调控。     

【In Press】 Is Marcus Gunn jaw winking a primitive reflex? I. Rat neuroanatomy

AIM: To investigate a possible trigeminal proprioceptive-oculomotor neural pathway and explore possible synaptic connections between neurons in this pathway. Attempt to bring a new insight to mechanism of Marcus Gunn Syndrome (MGS). METHODS: Anterograde and retrograde tract tracing combined with immunofluorescent stain in rats. Intracellular injection of tracer into electrophysiologically identified rat mesencephalic trigeminal nucleus (Vme) neurons and tracing axon trajectory. RESULTS: Following injections of anterograde tracers into the Vme, labeled terminals were observed ipsilateral in oculomotor and trochlear nuclei (III/IV), as well as in their premotor neurons in interstitial nucleus of Cajal and Darkschewitsch nucleus (INC/DN). Combining with Choline Acetyltransferase (ChAT) immunofluorescent stain, it showed that Vme projecting terminals contact upon ChAT positive III/IV motoneurons under confocal microscope. By retrograde labeling premotor neurons of the III, it showed that Vme neuronal terminals contact with retrogradely labeled pre-oculomotor neurons in the INC/DN. Axons of intracellularly labeled Vme neurons that respond to electric stimuli of the masseter nerve traveled into the ipsilateral III. CONCLUSIONS: There may exist a trigeminal proprioceptive-oculomotor system neural circuit in the rat, which is related to vertical-torsional eye movements. Possible association of this pathway with MGS etiology was discussed.