AmphotericinB在全细胞穿孔膜片钳技术中的应用研究

目的：为研究海德氏突触的电生理特性,建立用amphotericinB穿孔的膜片钳技术。方法：本文应用两性霉素B（amphotericinB）在小鼠脑干的calyx细胞上进行穿孔膜片钳技术的研究。结果：应用amphotericinB进行穿孔后,通道电流衰减现象显著变慢。AmphotericinB的最适浓度为400tc／ml。结论：本研究摸索出了一种稳定的穿孔膜片钳全细胞记录技术,可以更有效,更真实的反应神经元通道电流的电生理特性。为穿孔膜片钳技术在听觉信息传导和调控研究中的应用提供了基础资料。     

海马脑片盲法膜片钳全细胞记录技术

本文较为详细地介绍了海马脑片盲法膜片钳全细胞记录技术,对其关键步骤和需要注意的问题进行了重点说明,同时对CA1区锥体神经元突触活动的特点,电压门控性Ca^2 通道以及谷氨酸（glutamate,Glu)γ－氨基丁酸（GABA）受体通道电流性质等进行了观察和分析,实验结果为采用海马脑片盲法膜片钳全细胞记录技术研究海马神经元离子通道动力学性质和中枢神经系统药物对突触活动的影响提供了可靠的依据。     

利用全细胞膜片钳技术记录大鼠脑片NMDA电流的研究*

目的: 采用全细胞膜片钳技术记录大鼠脑片实验中NMDA电流,并介绍诱发NMDA电流的自制刺激电极制作方法。方法: 在大鼠目标脑区快速取材并获取活性良好的脑片,分别通过含受体激动剂NMDA的孵育液灌流和电刺激诱发NMDA电流两种方式进行膜片钳记录;利用针灸针自制刺激电极。结果: 通过记录到大鼠脑片神经元的EPSC和AP可判断神经元的状态,比较两种方法诱导的NMDA电流幅度,即直接灌流受体激动剂NMDA(282.0±24.3) pA和自制刺激电极诱发(261.4±40.1)pA,二者电流幅度无明显差异(P＞0.05,n=4);自制刺激电极与进口刺激电极诱发的NMDA电流幅度分别为(267.2±36.5)pA vs (239.2±41.0)pA,二者电流幅度无明显差异(P＞0.05,n=4),证明自制刺激电极成功。结论: 大鼠脑片实验中,通过直接灌流激动剂与电刺激两种方式均可诱导NMDA电流,自制刺激电极为在脑片上记录诱发电流提供了一种经济、可靠的实验手段,便于各实验室应用。     

A need to provide explanations for observed biological effects of radiofrequency exposure

Abstract

Although there is scientific consensus that radiofrequency (RF) exposure at high intensity can cause thermal effects, including well-established adverse health effects, there is still considerable controversy on whether low-intensity RF exposure can cause biological effects, especially adverse health effects. The objective of this paper is to describe several reported “non-thermal” effects that were later shown to be due to a weak thermal effect or an experimental artifact by properly conducted and thorough follow-on scientific research. First, the multiple factors that can cause different RF energy absorption in biological tissues are reviewed and second, several examples of experimental artifacts in published papers are described to demonstrate the importance of paying attention to dosimetry and temperature control. For example, isolated nerve response studies show that when temperature of the RF-exposed tissues is controlled, effects disappeared. During RF exposure, conductive electrodes routinely used in physiological studies have been shown to cause field intensification at the tips or contacts of the electrodes with biological tissue; thus, the RF exposure at the site of measurement could be much higher than the incident field. In some in vitro studies, a lack of temperature uniformity in RF-exposed cell cultures and rate of heating explain changes originally reported to be due to low-level RF exposure. In other studies, detailed dosimetry studies have identified artifacts that explain the reasons why so-called “non-thermal” effects were mistakenly reported. Researchers should look for explanations for their own findings, and not expect others to figure out what was the reason for their observed effects.     

New head exposure system for use in human provocation studies with EEG recording during GSM900- and UMTS-like exposure

A new head exposure system for double blinded human provocation studies, which requires EEG recording during exposure with GSM900- and UMTS-like signals has been developed and dosimetrically evaluated. The system uses planar patch antennas fixed at 65 mm distance from the subject's head by a special headset, which provides minimum impairment of the test subjects and ensures an almost constant position of the antennas with respect to the head, even in case of head movements. Compared to exposure concepts operating small antennas in close proximity to the head, the concept of planar antennas at a certain distance from the head produces a much more homogeneous SAR distribution in the temporal and parietal lobe of the brain. At the same time the resulting uncertainty of exposure due to variations in head size, variations of the dielectric properties of tissues and unavoidable small changes of the antenna's position with respect to the head, is reduced to the order of approximately 3 dB, which is a significant improvement to comparable head exposure systems reported in literature in the past. To avoid electromagnetic interference on the EEG recording caused by the incident RF-field an appropriate double-shielded filter circuit has been developed. Furthermore, the effect of the presence of the sintered Ag/AgCl EEG electrodes and electrode wires on the SAR distribution inside the head has been investigated and was found to be minimal if the electrode wires are arranged orthogonal to the incident electric field vector. EEG electrode arrangement parallel to the incident field vector, however, might cause drastic changes in the SAR distribution inside the head.     

Characterization of mechanosensitive channels in Escherichia coli cytoplasmic membrane by whole-cell patch clamp recording

Whole-cell patch clamp recordings were done on giant protoplasts of Escherichia coli. The pressure sensitivity of the protoplasts was studied. Two different unit conductance mechanosensitive channels, 1100 ± 25 pS and 350 ± 14 pS in 400 mm symmetric KCl solution, were observed upon either applying positive pressure to the interior of the cells or down shocking the cells osmotically. The 1100 pS conductance channel discriminated poorly among the monovalent ions tested and it was permeable to Ca²⁺ and glutamate^?. Both of the two channels were sensitive to the osmotic gradient across the membrane; the unit conductances of the channels remained constant while the mean current of the cell was increased by increasing the osmotic gradient. Both of the channels were voltage sensitive. Voltage-ramp results showed that the pressure sensitivity of protoplasts was voltage dependent: there were more channels active upon depolarization than hyperpolarization. The mech anosensitive channels were reversibly blocked by gadolinium ion. Also they could reversibly be inhibited by protons. Mutations in two of the potassium efflux systems, KefB and KefC, did not affect the channel activity, while a null mutation in the gene for KefA changed the channel activity significantly. This indicates a potential modulation of these channels by KefA.     

Cylindrical waveguide applicator for in vitro exposure of cell culture samples to 1.9-GHz radiofrequency fields

An applicator for in vitro cell culture exposure was developed based on a circularly polarized, cylindrical waveguide for the 1.9-GHz frequency band used by Personal Communications Services (PCS) in Canada. The applicator consists of two coaxial Petri dishes that sit on the open end of the cylindrical waveguide. The inner 60-mm Petri dish contains the cell culture while the outer 150-mm dish contains coolant water, which is circulated from a pump. A dosimetric evaluation was made using thermometric and E-field probe techniques. The latter allowed the entire inner dish to be scanned to determine the range of specific absorption rates (SARs) pertinent to the expected position of the cells. A representative SAR rate (SAR per unit of input power) of 8.6 +/- 2.1 W/kg/W (95th percentile) was determined 1 mm from the bottom, for a 10 ml sample volume of standard medium. Evaluation of the cooling system demonstrated that following an initial 0.3 degrees C temperature increase, a constant temperature was maintained for 24 h when the waveguide was energized to achieve an average sample SAR of 10 W/kg. These properties enable both acute and sub-acute in vitro bio-effect studies to be performed on a variety of cell culture samples.     

Space efficient system for whole-body exposure of unrestrained rats to 900 MHz electromagnetic fields

The aim of this study was to design, implement and analyze a space-efficient setup for the whole-body exposure of unrestrained Wistar rats to radiofrequency (RF) electromagnetic fields at 900 MHz. The setup was used for 2 years in a cocarcinogenesis study and part of it for 5 weeks in a central nervous system (CNS) study. Up to 216 rats could be placed in separate cages in nine different exposure chambers on three racks requiring only 9 m2 of floor area (24 rats per m2). Chambers were radial transmission lines (RTL), where the rats could freely move in their cages where food and drinking water was provided ad libitum except during RF exposure periods. Dosimetrical analysis was based on FDTD computations with heterogeneous rat models and was validated with calorimetrical measurements carried out with homogeneous phantoms. The estimated whole-body average specific absorption rates (SAR) of rats were 0 (sham), 0.4, and 1.3 W/kg in the cocarcinogenesis study and 0 (sham), 0.27, and 2.7 W/kg in the CNS study with an estimated uncertainty of 3 dB (K = 2). The instantaneous and lifetime variations of whole-body average SAR due to the movement of rats were estimated to be 2.3 and 1.3 dB (K = 1), respectively.     

Reevaluation and improved design of the TEM cell in vitro exposure unit for replication studies

The transverse electromagnetic (TEM) cell system developed by Litovitz et al. and utilized by Penafiel et al. for the exposure of cells in T25 flasks at 835 MHz has been reevaluated for the purpose of replicating the studies published by Penafiel. The original setup has been reconstructed as closely as possible, with improvements enabling blinded exposures, forced cooling and better repeatable positioning of the flasks, as well as tight exposure and environmental parameter control. The signal unit can simulate the original signal but also enables various other exposure schemes. The setup has been evaluated for four T25 flasks filled with 5 and 10 ml of cell medium by experimental and numerical means. Comparing E field, SAR and temperature measurements resulted in good agreement: <0.4 dB (4.5%) for E field and 0.48 dB (10.5%) for SAR. The overall average SAR within the medium is 6.0 W/kg at 1 W input power with a standard deviation of less than 52%. The temperature increase was determined to be 0.13 degrees C/(W/kg). This can be reduced to 0.045 degrees C/(W/kg) by applying active air flow cooling. The comparison of SAR values from temperature measurements with the corresponding simulated values resulted in excellent agreement. These results do not correspond to the previous study reporting an average SAR within the medium of 2.5 W/kg at an input power of 0.96 W.     

Isolation and patch clamp measurements of xylem contact cells for the study of their role in the exchange between apoplast and symplast of leaves

Because of their importance for nutrition, a method was developed to patch xylem contact cells in leaves of Vicia faba and maize. Since the lignification of older cells was a major obstacle for isolating protoplasts which could be patched, only young leaves (fourth fully developed leaf) were used. An important step in the isolation of these cells was the infiltration of the leaves and their exposure to enzymes for several hours, allowing mesophyll cells to be removed whilst having most of the xylem contact cells attached to the xylem. Channel activity in cell-attached mode or in excised patches could only be observed if an internal coating of sigmacote was used to block diffusion ions out of the pipette glass. Two different types of K+ channels were identified by measuring the reversal potential at different concentrations of KCl. One channel (SC) had a symmetric IV curve with a high probability of remaining open, irrespective of membrane potential; the other channel was an inward rectifier. The symmetical channel could be blocked weakly by Na+ but it was permeable to .     

Determination of the induced ELF electric field distribution in a two layer in vitro system simulating biological cells in nutrient solution

In-vitro studies of biological effects of electromagnetic fields are often conducted with cultured cells either in suspension or grown in a monolayer. In the former case, the exposed medium can be assumed to be homogeneous; however, eventually the cells settle to the bottom of the container forming a two layer system with different dielectric and conductive properties. In the present work the effect of this separation on the electric field distribution is calculated and experimentally measured at selected positions for a commonly used exposure configuration. The settled cell suspension is modeled by a well-defined two layer system placed in a rectangular container with the base of the container parallel to the direction of the magnetic field. Theoretical calculations based on numerical techniques are done for various two layer systems with different conductivities in each layer. The agreement between the theoretical calculations and the experimental measurements is within ± 1.5 mV/m, or 10% of the maximum induced field when the conductivity of the lower layer is ten times that of the upper layer. This result is well within experimental error. When the thickness of one of the layers is small compared to the thickness of the other layer, it is found that the electric field distribution is essentially that of the homogeneous case. The latter situation corresponds to a typical cell exposure condition. © 1993 Wiley-Liss, Inc.     

Evaluation of genotoxic effects in human leukocytes after in vitro exposure to 1950 MHz UMTS radiofrequency field

In the present study the third generation wireless technology of the Universal Mobile Telecommunication System (UMTS) signal was investigated for the induction of genotoxic effects in human leukocytes. Peripheral blood from six healthy donors was used and, for each donor, intermittent exposures (6 min RF on, 2 h RF off) at the frequency of 1950 MHz were conducted at a specific absorption rate of 2.2 W/kg. The exposures were performed in a transverse electro magnetic (TEM) cell hosted in an incubator under strictly controlled conditions of temperature and dosimetry. Following long duration intermittent RF exposures (from 24 to 68 h) in different stages of the cell cycle, micronucleus formation was evaluated by applying the cytokinesis block micronucleus assay, which also provides information on cell division kinetics. Primary DNA damage (strand breaks/alkali labile sites) was also investigated following 24 h of intermittent RF exposures, by applying the alkaline single cell gel electrophoresis (SCG)/comet assay. Positive controls were included by treating cell cultures with Mitomycin-C and methylmethanesulfonate for micronucleus and comet assays, respectively. The results obtained indicate that intermittent exposures of human lymphocytes in different stages of cell cycle do not induce either an increase in micronucleated cells, or change in cell cycle kinetics; moreover, 24 h intermittent exposures also fail to affect DNA structure of human leukocytes soon after the exposures, likely indicating that repairable DNA damage was not induced.     

Stimulated rapid expression in vitro for early detection of in vivo T-cell receptor mutations induced by radiation exposure

The T-cell receptor (TCR) mutation assay for in vivo somatic mutations is a sensitive indicator of exposure to ionizing radiation. However, this assay cannot be immediately applied after radiation exposure because expression of a mutant phenotype may require as long as several months. In the present study, we eliminate this time lag by stimulating lymphocytes with a mitogen that can accelerate the turnover of TCR protein expression in T-cells. When lymphocytes obtained from healthy donors were irradiated with various doses of X-rays and cultured with human interleukin-2 after phytohemagglutinin (PHA) pulse stimulation, the mutant frequency (MF) of CD4⁺ T-cells increased dose dependently during the first 7 days, then decreased rapidly due to the growth disadvantage of mutant cells. This suggests that PHA stimulation can shorten the expression time of a mutant phenotype to within a week after radiation exposure. The relationship between radiation dose and TCR MF on the seventh day was best fitted by a linear-quadratic dose–response model. We applied this improved TCR mutation assay to gynecological cancer patients who received 5 days of localized radiotherapy, totaling about 10 Gy. The in vivo TCR MF in the patients did not change within a week after radiotherapy, whereas the in vitro TCR MF of PHA-stimulated lymphocytes from the same patients significantly increased 7 days after initiating culture. The estimated mean radiation dose to the peripheral blood lymphocytes of the cancer patients was about 0.9 Gy, based on the in vitro linear-quadratic dose–response curve. This estimated dose was close to that described in a previous report on unstable-type chromosome aberrations from cervical cancer patients after receiving the same course of radiotherapy. On the basis of these findings, we propose that the improved TCR mutation assay is a useful biological dosimeter for recent radiation exposure.     

Plasmalemma patch clamp experiments in plant root cells: procedure for fast isolation of protoplasts with minimal exposure to cell wall degrading enzymes

Summary A convenient and rapid isolation procedure for root cell protoplasts suitable for patch clamp experiments, was developed for root cells of tomato (Lycopersicon esculentum) andPlantago species, grown on hydroculture. The procedure is based on a minimal exposure of cells to cell wall degrading enzyme mixtures. After an incubation period of 30 min in a cell wall degrading enzyme mixture all free floating cells were discarded. Subsequently the root material was rinsed and a second group of cells, still present inside the tissue, was freed by application of mechanical pressure. The newly released protoplasts were filtered and collected on the glass bottom of a patch clamp dish. The bathing medium was rinsed extensively removing cellulose fibrils and protoplasts not attached to the glass. Removal of these cellulose fibrils significantly improved the seal success ratio. The isolated protoplasts were suitable for patch clamp experiments in the cell-attached patch, the whole cell and the isolated patch configuration.Abbreviations BSA bovine serum albumin - BTP bis-tris propane - CAP cell-attached patch - OOP outside out patch - PEG polyethylene glycol - WC whole cell     

Evaluation of genotoxic effects in human peripheral blood leukocytes following an acute in vitro exposure to 900 MHz radiofrequency fields

Human peripheral blood leukocytes from healthy volunteers have been employed to investigate the induction of genotoxic effects following 2 h exposure to 900 MHz radiofrequency radiation. The GSM signal has been studied at specific absorption rates (SAR) of 0.3 and 1 W/kg. The exposures were carried out in a waveguide system under strictly controlled conditions of both dosimetry and temperature. The same temperature conditions (37.0 +/- 0.1 degrees C) were realized in a second waveguide, employed to perform sham exposures. The induction of DNA damage was evaluated in leukocytes by applying the alkaline single cell gel electrophoresis (SCGE)/comet assay, while structural chromosome aberrations and sister chromatid exchanges were evaluated in lymphocytes stimulated with phytohemagglutinin. Alterations in kinetics of cell proliferation were determined by calculating the mitotic index. Positive controls were also provided by using methyl methanesulfonate (MMS) for comet assay and mitomycin-C (MMC), for chromosome aberration, or sister chromatid exchange tests. No statistically significant differences were detected in exposed samples in comparison with sham exposed ones for all the parameters investigated. On the contrary, the positive controls gave a statistically significant increase in DNA damage in all cases, as expected. Thus the results obtained in our experimental conditions do not support the hypothesis that 900 MHz radiofrequency field exposure induces DNA damage in human peripheral blood leukocytes in this range of SAR.     

Evaluation of environmental and biological monitoring methods for toluene exposure assessment in paint industry

The aim of this study was to assess the exposure to Toluene in paint industry and to evaluate the environmental and biological monitoring techniques for the assessment of occupational exposure to this aromatic hydrocarbon. In this study, personal active and passive air sampling for toluene measurements, blood and urine sampling respectively for B-Tol and HA or U-Tol analyses for eight workers from two paint and thinner production factories were collected during four successive working days. Correlations were analyzed between biological indicators and environmental toluene exposure levels.The concentration of Toluene measured in air samples ranged from 0.2 to 414.0 ppm (mean = 59.8 ppm), with high variability of atmospheric levels between activities and between days. No significant difference was found between airborne toluene concentrations measured by the two sampling methods. The correlation between air concentrations sampled by the diffusive sampling method and the biomarkers was the best for HA (r = 0.902, p < 0.01), followed by B-Tol (r = 0.820; p < 0.01), o-Cr (r = 0.691; p < 0.01) and U-Tol (r = 0.607; p < 0.05). The correlation was better between air concentrations and urinary metabolites HA and o-Cr for exposure levels higher than 50 ppm (r = 0.931; p < 0.01), and lower than 300 ppm (r = 0.827; p < 0.01), respectively.According to our results, workers in the studied industries are highly exposed to Toluene. Given the high correlation found between toluene concentrations in samples taken on dosimeters and those actively sampled on charcoal tubes, it may be assumed that both sampling methods are valuable. Despite the influencing factors, HA was found to be a reliable biological indicator for the monitoring of occupational exposure to toluene for high exposure levels. However, B-Tol seems to be an interesting alternative, since it is more specific and showed the best correlations with airborne toluene levels.     

Tuning,coupling and matching of a resonant cavity in microwave exposure system for biological objects

A new microwave exposure system for biological experiments with well-defined exposure conditions and improved control of the exposure parameters consisting of variable frequency power source, coaxial to waveguide transition, matching network and single-mode resonant cavity with movable shorting plunger was fabricated and characterized. The introduction of a biological sample into a resonant cavity has a large impact on its field configuration and port impedance. As such, the properties, geometry and position of the biological sample become a part of the electrical properties of the microwave circuit. With that change, the electrical properties of the resonant cavity, such as impedance, quality factor and resonant frequency, also change. In this study, an appropriate coupling system with effective power transfer and an algorithm to tuning and coupling of resonant cavity in resonance before and after the introduction of biological sample have been proposed. This procedure will lead to a known dose distribution within the biological sample and allow a better comparison with other studies. Coupling of the electromagnetic energy into a resonant cavity was experimentally investigated. Graphical representation of cavity impedance in case of undercoupled, critically coupled and overcoupled cavity has been presented. Critical coupling of an empty resonant cavity has been accomplished at voltage standing wave ratio (VSWR) 1.01, at resonance frequencies 900 and 947.5 MHz. Critical coupling with the introduction of a biological sample has been accomplished at VSWR ≤ 1.07 for frequency bandwidth 1 MHz and VSWR ≤ 1.5 for frequency bandwidth up to 5 MHz with central frequency 947.5 MHz.     

Development of a higher power intermediate‐frequency magnetic field exposure system for in vitro studies

In a previous article we developed an in vitro 23 kHz magnetic field (MF) exposure system that generated an MF of 532 µT_rms. Using this system, the biological effects of 23 kHz MFs on cell functions have been reported. To further clarify the biological effect of intermediate‐frequency (IF) MFs and investigate the dose–response relationship in cell lines, an exposure system that generates stronger MFs is required. To meet this requirement, we developed a 6.25 mT_rms MF exposure system for in vitro study. This level is 1000 times the reference level for the general public in the ICNIRP guidelines. This system provides an MF of 6.25 mT_rms at 23 kHz with a uniformity within ±5%. To verify that in vitro experimental conditions are maintained, we examined the temperature, environmental MF, and MF leakage for a sham exposure system. In addition, we examined the harmonics, coil shape, and heat generated in the medium by the high‐strength MF. As a result, it was confirmed that this system can be used to evaluate the biological effects of IF MFs. This article presents the design and successful construction of the in vitro exposure system. Bioelectromagnetics 31:156–163, 2010. © 2009 Wiley‐Liss, Inc.