Relationship between Locomotor Activity and Monoamines Following Single and Double Transient Forebrain Ischemia in Gerbils

The relationship between locomotor activity and monoamine levels in gerbils after single and/or double forebrain ischemic insult was studied. Locomotor hyperactivity was observed after the first ischemic episode, but the gerbils failed to show hyperactivity after the second ischemic episode induced one week later. The monoamine levels were determined in order to clarify the biochemical basis of post-ischemic locomotor hyperactivity. Norepinephrine increased in response to first ischemic episode but remained at normal levels after the second episode of ischemia. Metabolites of dopamine and serotonin increased after both the first and second ischemic insults, which indicates that these monoamines do not play significant roles in post-ischemic locomotor activity. Therefore, increases in norepinephrine after first ischemic insult may play a role in increasing locomotor activity during the period following such an episode.     

Sine Wave Extremely Low Frequency Magnetic Fields Protect Chick Embryos Against UV-Induced Death

Four-day-old chicken embryos were exposed to extremely low frequency (ELF) magnetic fields (MF) prior to UV exposure (75 min, predominantly UV-C, 0.4 mW/cm²) to investigate possible MF-mediated protection against lethal effects of UV. The UV exposure typically resulted in a 20% survival rate (as judged by beating hearts) in sham-exposed embryos 3 h postexposure. In contrast, exposure to a 50 (10, 50, or 100 µT) or 60 Hz (10 µT) vertical MF caused a significant increase in survival rate, observed only 30 min after UV exposure. No difference in protection levels was seen between these exposure intensities. A horizontal 50 Hz MF (10, 50, or 100 µT) did not result in the general protection against UV-induced death observed for vertical fields, suggesting that the size of the induced electric field (which differs between horizontal and vertical exposure) is important for the MF-induced protection. To explore the molecular mechanisms involved in this effect, immunoblotting experiments with an antibody against the inducible form of hsp70 were performed. These showed that application of MF (50 Hz, 200 µT, 1 h) induced hsp70 expression in human K562 cells.     

Effects of Extremely Low-Frequency Magnetic Field on Growth and Differentiation of Human Mesenchymal Stem Cells

Human Mesenchymal Stem Cells (hMSCs) were exposed to a developed extremely low-frequency (ELF) magnetic fields (50?Hz ,20?mT ELF) system to evaluate whether exposure to (ELF) magnetic fields affects growth, metabolism, and differentiation of hMSCs. MTT method was used to determine the growth and metabolism of hMSCs following exposure to ELF magnetic fields. Na⁺/K⁺ concentration and osmolality of extracelluar were measured after exposured culture. Alkaline phosphatase (ALP) assay and Calcium assay, ALP staining, and Alizarin red staining were performed to evaluate the osteogenic differentiation of hMSCs under the ELF magnetic field exposure. In these experiments, the cells were exposed to ELF for up to 23 days. The results showed that exposure to ELF magnetic field could inhibit the growth and metabolism of hMSC, but have no significant effect on differentiation of hMSCs. These results suggested that ELF magnetic field may influence the early development of hMSCs related adult cells.     

The Free Radical Scavenger, α-Lipoic Acid, Protects Against Cerebral Ischemia-Reperfusion Injury in Gerbils

-Lipoic acid (thioctic acid) was tested for its neuroprotective activity in a Mongolian gerbil model of forebrain ischemia/reperfusion. Adult gerbils were treated for 7 days with two intraperitoneal injections per day of -lipoic acid (20 mg/kg), vehicle or saline and on the 7th day the animals were subjected to 5 min of forebrain ischemia. Ischemic injury was assessed by monitoring the increases in locomotor activity and from the extent of damage to the CA1 hippocampal pyramidal cell layer after 5 days of recovery. By both criteria, -lipoic acid was neuroprotective against ischemia/reperfusion evoked cerebral injury.     

Ornithine Decarboxylase Activity and Edema Formation in Cerebral Ischemia of Conscious Gerbils

Abstract: General anesthetic agents often affect the biochemical and physiologic changes triggered by cerebral ischemia. This study examined the regional activities of ornithine decarboxylase (ODC) in gerbils subjected to 5 min of bilateral carotid occlusion without anesthesia. At 2, 4, and 6 h of reperfusion, significant ODC activity was observed in both the cortex and the hippocampus. Pretreatment with α-difluoromethylornithine (DFMO) significantly blocked the ODC activity at 2, 4, and 6 h. Significant edema formation was found at 2, 4, and 6 h. At 2 h, edema formation was unaffected by administration of DFMO. However, DFMO treatment reduced later edema formation at 4 and 6 h. These results demonstrate that ODC activity and edema formation are delayed in gerbils after the induction of transient ischemia even with the removal of anesthetic agents and their potentially protective effects. These findings suggest that ODC activity and its induction of delayed cerebral edema are specific to cerebral ischemia and not to an anesthetic effect. DFMO treatment reduced both the ODC activity and edema formation, indicating a role for polyamines in postischemic edema formation.     

弱极低频磁场对Actin骨架组装效率的频率窗口效应初探

前期研究发现,50 Hz弱磁场辐照能明显降低细胞的微丝含量和组装效率,对actin骨架形态也有明显影响.电磁生物学效应是否与辐照场频率相关,一直受到研究者的关注.单体球状肌动蛋白(G-actin)是带电结构,电磁场频率会影响其振荡频率并对微丝聚合效率产生影响.本文从细胞骨架形态和蛋白质两层次,采用免疫荧光技术考察0.4 m T,在35~140 Hz范围内5个频率的极低频磁场(ELF-MF)对FL细胞中纤维状肌动蛋白(F-actin)含量的影响,并采用荧光共振能量转移技术(FRET)验证效应最明显的频率对离体G-actin组装效率的干扰程度.结果显示,相比假辐照组,细胞中F-actin含量在50 Hz辐照组下降了(34.66±3.14)%,110 Hz次之,而另外3组(35、70和140 Hz)无显著性差异.同时利用FRET方法验证,在50 Hz磁场辐照下,离体环境中G-actin组装成F-actin的效率较假辐照组、35和70 Hz组显著降低.经初步分析,G-actin在弱ELF-MF中受到以洛伦兹力和感生电场力的合力为主的相关电磁力干扰,致使组装效率下降,且由于工频磁场周期与微丝组装周期的特殊相干性,在50 Hz频率附近可能存在一个外磁场干扰actin骨架组装的频率窗口.     

Extremely Low Frequency Magnetic Fields Suppress the Reduction of Germination Rate of Arabidopsis thaliana Seeds Kept in Saturated Humidity

The effects of an extremely low frequency magnetic field (ELFMF) on the germination of plant seeds were examined. The decrease in the germination activity of the seeds of Arabidopsis thaliana WS kept in saturated humidity and high temperature (37°C) was suppressed by the exposure to a 400 mT ELFMF.     

Behavioral Differences of the Insect Morimus funereus (Coleoptera,Cerambycidae) Exposed to an Extremely Low Frequency Magnetic Field

Changes in the behavior of Morimus funereus individuals were investigated as early manifestations of the contact of a living system with a changed environment primarily established via the nervous system. These experiments were aimed at revealing possible behavioral differences of a laboratory population of cerambycid beetle M. funereus in an “open field” before and after exposure to an extremely low frequency magnetic field (ELF‐MF, 50 Hz, 2 mT). The experimental groups were divided into several activity categories and exposed to ELF‐MF. The results showed that the activity increased in the groups with medium and low motor activity, but decreased in highly active individuals. High individual variability was found in the experimental groups, as well as differences in motor activities between the sexes, both before and after exposure to ELF‐MF. According to preliminary results, we assume the changes of activity in both sexes after exposure to ELF‐MF. The results showed a tendency toward locomotor activity decrease, the affect being more pronounced in females. As opposed to this type of activity, stereotypic activity of males was increased after the exposure, whereas females maintained the expected tendency of decrease. However, we did not obtain statistically significant differences because of a high individual variability and a low total number of individuals in the experiment (N = 28). Only a detailed analysis of the locomotor activity at 1‐min intervals showed some statistically significant differences in behavior between the sexes.     

Effects of Extremely Low Frequency Magnetic Fields on Blood Coagulation in Mice: An Initial Study

OF1 mice were chronically exposed to a 50‐Hz sinusoidal East–West magnetic field 15 µT (rms), in order to evaluate the blood coagulation variations related to the effect of this nonionizing radiation. Mating and pregnancy of ancestors (first generation), and birth, lactation, and development of second‐generation female mice until adulthood took place in the experimental field. A global blood coagulation study of both control and exposed 14‐ to 15‐week‐old and 50‐ to 52‐week‐old, second‐generation females was carried out. Plasma calcium content was determined by atomic absorption spectrophotometry. Different steps of blood coagulation were studied by thromboelastography (TEG) in whole blood (WB), platelet‐rich plasma (PRP), and platelet‐poor plasma (PPP). A significant decrease (approximately 34.5%) of calcium concentration was detected with aging; however, no change was induced by medium‐term or long‐term exposure to extremely low frequency magnetic field (ELF‐MF). Medium‐term exposure could not be related to noticeable changes in global coagulation. However, a great deterioration of fibrin clot formation in mature exposed female mice was detected as a result of the long‐term exposure that was strengthened by aging. These deficiencies seemed to be compensated by the discrete, although statistically not significant, decrease of platelet counts and the significant decrease of blood cells' mean corpuscular volume associated to ELF‐MF exposure of 50‐Hz, 15 µT. Consequently, whole blood TEG values of mature exposed female mice were similar to those from the young control group. In view of the obtained results, further studies on variations associated with ELF‐MF exposure in different coagulation parameters will be necessary.     

Characterization of Levels of Extremely Low Frequency Magnetic Fields Emitted From Portable Hand‐Held Fans

This study aims to assess the levels of extremely low frequency magnetic fields (ELF‐MF) emitted from portable hand‐held fans (HHFs) and their principal frequency and to identify factors influencing these levels. We collected a total of eleven models of HHF and monitored the ELF‐MF as a function of fan speed and distance from the fan. EMDEX II was used to monitor the ELF‐MF. An SMP2 EMF‐meter equipped with a P400 field probe was used to determine the levels of ELF‐MF and the frequency spectrum. Ten of the fans, excluding only one bladeless‐fan model, emitted a high level of ELF‐MF near the source of the HHF direct‐current motor. The maximum measured level of ELF‐MF ranged from 14.07 to 218.7 µT. All measurements of the ELF‐MF taken within 10 cm from the HHFs showed values higher than 1.0 µT. ELF‐MF levels were found to decrease markedly with distance, regardless of the HHF product. The level of ELF‐MF rose noticeably with increased fan speed. The speed of and distance from the HHF significantly influenced the level of ELF‐MF. All principal frequencies ranged from 1 to 300 Hz, which falls in the typical range of ELF. Bioelectromagnetics. 2019;40:569–577. © 2019 Bioelectromagnetics Society.     

Pre- and Posttreatment With Edaravone Protects CA1 Hippocampus and Enhances Neurogenesis in the Subgranular Zone of Dentate Gyrus After Transient Global Cerebral Ischemia in Rats

Edaravone is clinically used for treatment of patients with acute cerebral infarction. However, the effect of double application of edaravone on neurogenesis in the hippocampus following ischemia remains unknown. In the present study, we explored whether pre- and posttreatment of edaravone had any effect on neural stem/progenitor cells (NSPCs) in the subgranular zone of hippocampus in a rat model of transient global cerebral ischemia and elucidated the potential mechanism of its effects. Male Sprague-Dawley rats were divided into three groups: sham-operated (n = 15), control (n = 15), and edaravone-treated (n = 15) groups. Newly generated cells were labeled by 5-bromo-2-deoxyuridine. Immunohistochemistry was used to detect neurogenesis. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling was used to detect cell apoptosis. Reactive oxygen species (ROS) were detected by 2,7-dichlorofluorescien diacetate assay in NSPCs in vitro. Hypoxia-inducible factor-1α (HIF-1α) and cleaved caspase-3 proteins were quantified by western blot analysis. Treatment with edaravone significantly increased the number of NSPCs and newly generated neurons in the subgranular zone (p < .05). Treatment with edaravone also decreased apoptosis of NSPCs (p < .01). Furthermore, treatment with edaravone significantly decreased ROS generation and inhibited HIF-1α and cleaved caspase-3 protein expressions. These findings indicate that pre- and posttreatment with edaravone enhances neurogenesis by protecting NSPCs from apoptosis in the hippocampus, which is probably mediated by decreasing ROS generation and inhibiting protein expressions of HIF-1α and cleaved caspase-3 after cerebral ischemia.     

HTEA对全脑缺血/再灌注大鼠脑血流及海马细胞凋亡的影响

目的:研究上胸段硬膜外阻滞(high thoracic epidural anesthesia,HTEA)对大鼠全脑缺血再灌注损伤(global cerebral ischemia,GCI)再灌注期间脑血流及凋亡相关蛋白Bcl-2和Bax的影响。方法:本研究选用成年雄性Wistar大鼠成功进行T4-5间隙硬膜外置管,并建立四血管阻断的全脑缺血模型进行15 min的全脑缺血。根据通过硬膜外导管输注药物不同,随机分为四组:假手术组(Sham,0.9%生理盐水)、假手术-硬膜外组(Sham-HTEA,0.25%布比卡因)、全脑缺血组(GCI,0.9%生理盐水)和硬膜外组(HTEA,0.25%布比卡因)。给药时间从缺血前15 min开始以20μL/h的速度持续输注至再灌注24 h。缺血前至再灌注2 h观察平均动脉压(MAP)和心率(HR),激光多普勒血流仪监测脑血流(cerebral blood flow,CBF),Western-blot检测再灌注24 h海马凋亡蛋白Bcl-2和Bax含量。结果:HTEA组与GCI组相比,缺血期间及再灌注2 h内的MAP和HR无统计学差异,而与Sham组相比,GCI组的MAP缺血时升高,而再灌注时降低(P0.05);再灌注10 min的高灌注期HTEA组CBF明显低于GCI组(123.1%±35.2%vs 177.5%±32.4%,P0.01),再灌注60 min至120 min的低灌注期的大部分时间点HTEA组CBF均高于GCI组(P0.05);再灌注24 h海马组织Bax/Bcl-2比例明显降低(P0.01)。结论:0.25%的布比卡因20μL·h-1连续上胸段硬膜外阻滞可以维持血流动力学稳定,且可改善大鼠全脑缺血再灌注后低灌注期的脑血流量,并减少再灌注24 h海马Bax/Bcl-2比例。     

骨髓间质干细胞移植对大鼠脑缺血再灌注凋亡蛋白Caspase-3的影响

目的研究静脉移植骨髓间充质干细胞（MSCs）对脑缺血再灌注模型大鼠神经功能及凋亡相关蛋白caspase-3的影响。方法体外培养及扩增MSCs后,用绿色荧光染料羟基荧光素二醋酸盐琥珀酰亚胺脂（CFSE）标记,通过静脉途径移植给大脑中动脉缺血2 h再灌注的SD大鼠,按不同时间点取材,荧光显微镜观察BMSCs在脑内的分布,免疫组织化学染色及RT-PCR检测大鼠脑内caspase-3蛋白表达情况。结果移植组在移植后第6天神经功能明显好于对照组（P〈0.05）。移植组移植后3、12、24、48、72 h caspase 3免疫组化阳性目标面密度分别为（1.34±0.31）%、（3.98±0.67）%、（5.58±0.92）%、（4.65±0.69）%、（3.51±0.63）%,对照组分别为（2.09±0.19）%、（5.23±0.30）%、（6.89±0.57）%、（5.93±0.56）%、（4.39±0.57）%,移植组和对照组比较均（P〈0.05）。6h及7 d移植组caspase 3阳性目标面密度分别为（2.81±0.35）%、（1.64±0.29）%,与对照组（3.92±0.44）%,（2.29±0.21）%比较差异显著（P〈0.01）。移植组相应时间点caspase-3的表达明显低于对照组（P〈0.05,P〈0.01）;移植组大鼠缺血侧皮层的caspase-3 mRNA相对量明显低于对照组（P〈0.01）。结论经静脉注射骨髓间充质干细胞可明显改善神经功能。其可能通过下调caspase-3表达方式对脑缺血再灌注损伤起保护作用。     

Interaction of Microwaves and a Temporally Incoherent Magnetic Field on Single and Double DNA Strand Breaks in Rat Brain Cells

The effect of a temporally incoherent magnetic field noise on microwave-induced DNA single and double strand breaks in rat brain cells was investigated. Four treatment groups of rats were studied: microwave-exposure (continuous-wave 2450-MHz microwaves, power density 1 mW/cm², average whole-body specific absorption rate of 0.6 W/kg), noise-exposure (45 mG), microwave + noise-exposure, and sham-exposure. Animals were exposed to these conditions for 2h. DNA single- and double-strand breaks in brain cells of these animals were assayed 4h later using a microgel electrophoresis assay. Results show that brain cells of microwave-exposed rats had significantly higher levels of DNA single- and double-strand breaks when compared with sham-exposed animals. Exposure to noise alone did not significantly affect the levels (i.e., they were similar to those of the sham-exposed rats). However, simultaneous noise exposure blocked microwave-induced increases in DNA strand breaks. These data indicate that simultaneous exposure to a temporally incoherent magnetic field could block microwave-induced DNA damage in brain cells of the rat.     

Free Fatty Acids, Neutral Glycerides, and Phosphoglycerides in Transient Focal Cerebral Ischemia

Abstract: Cerebral ischemia is known to cause an increase in levels of free fatty acids (FFAs) and diacylglycerols (DGs), although the mechanism(s) leading to these changes is not well understood. In this study, we examined FFA and DG levels along with those of other lipids in rats during and after transient focal cerebral ischemia induced by temporary occlusion of the right middle cerebral artery (MCA) and both common carotid arteries. During the duration of ischemia (15–60 min), there was a time-dependent increase (two- to 10-fold) in FFA levels in the right MCA cortex, whereas levels of DG and other lipids were not altered appreciably. FFA levels in right MCA cortex returned to near control values after reperfusion. However, following a 60-min ischemic insult, there was a second phase of FFA level increase that was evident after 16 h. The FFAs accumulated during the ischemia period were different from those after reperfusion, suggesting differences in mechanisms for their release. During the second phase of FFA release, there were increases in levels of DGs and triacylglycerols (TGs) with unusually high proportions of 20:4(n-6) and 22:6(n-3). The increases in FFA, DG, and TG levels were marked by a decrease in content of phosphoglycerides (PGs). It is interesting that the increases in levels of FFAs and neutral glycerides accounted only for 10% of the total PGs depleted. The lipid changes during this reperfusion period correlated well with the development of cortical infarct. Because FFAs are potent inhibitors of mitochondrial respiratory function, the time-dependent FFA accumulation during the ischemia period may be an important determinant for the extent of ischemia-induced injury after reperfusion.     

Mitochondria and Neuronal Death/Survival Signaling Pathways in Cerebral Ischemia

Apoptotic cell death pathways have been implicated in acute brain injuries, including cerebral ischemia, brain trauma, and spinal cord injury, and in chronic neurodegenerative diseases. Experimental ischemia and reperfusion models, such as transient focal/global ischemia in rodents, have been thoroughly studied and suggest the involvement of mitochondria and the cell survival/death signaling pathways in cell death/survival cascades. Recent studies have implicated mitochondria-dependent apoptosis involving pro- and anti-apoptotic protein binding, the release of cytochrome c and second mitochondria-derived activator of caspase, the activation of downstream caspases-9 and –3, and DNA fragmentation. Reactive oxygen species are known to be significantly generated in the mitochondrial electron transport chain in the dysfunctional mitochondria during reperfusion after ischemia, and are also implicated in the survival signaling pathway that involves phosphatidylinositol-3-kinase (PI3-K), Akt, and downstream signaling molecules, like Bad, 14-3-3, and the proline-rich Akt substrate (PRAS), and their bindings. Further studies of these survival pathways may provide novel therapeutic strategies for clinical stroke.Special issue dedicated to Lawrence F. Eng.     

中药复方防治脑缺血的研究进展

脑缺血是危害人类健康的主要疾病之一,其在老年人中发病率较高,且具高死亡率、高致残率和高复发率的特点,给社会和家 庭带来了巨大负担。目前,基于脑缺血损伤的不同机制,针对性开发出多种抗脑缺血药物,但往往疗效不甚理想。而中药复方因其多途径、 多靶点的作用方式,在预防和治疗脑缺血及脑缺血再灌注损伤中有其独特优势,临床应用前景看好。分类综述传统中药复方、现代复方 制剂及临床经验方对脑缺血的治疗作用研究进展。     

Recovery from Edema and of Protein Synthesis Differs Between the Cortex and Caudate Following Transient Focal Cerebral Ischemia in Rats

Postischemic recovery from brain edema and of protein synthesis was examined following 1 h of middle cerebral artery (MCA) occlusion in rats. Recovery from brain edema and of protein synthesis showed a good correlation until 7 days after reperfusion in each area (cerebral cortex or lateral caudate) in the occluded MCA side. However, regional differences in the above types of recovery in the cortex and in the lateral caudate were found for the first time in this experiment. A profound inhibition of protein synthesis and formation of brain edema began sooner in the lateral caudate than in the cortex and continued long after reperfusion. Grades of cerebral blood flow during ischemia and the early period of reperfusion were almost the same in the two regions. Therefore, the regional differences in the above recoveries may not be due to the difference in the blood flow during ischemia and reperfusion, but may be partly attributable to the imbalance of excitatory and inhibitory innervation in the above two areas of the brain, may be due to a distinctive response to ischemic stress, and may be caused also by the potentiative effect of free arachidonate on the excitotoxic mechanism.     

Evaluation of Radiofrequency and Extremely Low‐Frequency Field Levels at Children's Playground Sites in Greece From 2013 to 2018

From 2013 to 2018, in‐situ measurements of radiofrequency (RF) electromagnetic fields (EMF) and extremely low‐frequency (ELF) electric and magnetic fields in 317 existing and under‐construction children's playground facilities, in 16 municipalities all over Greece, were carried out by the Greek Atomic Energy Commission (EEAE). These measurements were conducted following legislative framework established in 2009, which requires that compliance with the established exposure limits for EMFs should be verified in playground areas. The results are presented by the value of the electric field (E) and exposure ratio (Λ) for the RF EMF, as well as the value of the electric field (E) and magnetic flux density (B) for the ELF electric and magnetic fields. Statistical analysis tools were applied on measurement data and conclusions have been made, taking into consideration: (i) environment type (urban/suburban), and (ii) vicinity to any transmitting installations. Measurement results correspond to the typical EMF background levels for each environment type. Concerning the environment type, RF EMF, and ELF electric/magnetic field measurements reveal no differentiation between urban and suburban environments. Bioelectromagnetics. 2019;40:602–605. © 2019 Bioelectromagnetics Society.     

Stereological and Flow Cytometry Characterization of Leukocyte Subpopulations in Models of Transient or Permanent Cerebral Ischemia

Microglia activation, as well as extravasation of haematogenous macrophages and neutrophils, is believed to play a pivotal role in brain injury after stroke. These myeloid cell subpopulations can display different phenotypes and functions and need to be distinguished and characterized to study their regulation and contribution to tissue damage. This protocol provides two different methodologies for brain immune cell characterization: a precise stereological approach and a flow cytometric analysis. The stereological approach is based on the optical fractionator method, which calculates the total number of cells in an area of interest (infarcted brain) estimated by a systematic random sampling. The second characterization approach provides a simple way to isolate brain leukocyte suspensions and to characterize them by flow cytometry, allowing for the characterization of microglia, infiltrated monocytes and neutrophils of the ischemic tissue. In addition, it also details a cerebral ischemia model in mice that exclusively affects brain cortex, generating highly reproducible infarcts with a low rate of mortality, and the procedure for histological brain processing to characterize infarct volume by the Cavalieri method.