Effects of millimeter-wave radiation on monolayer cell cultures. II. Scanning and transmission electron microscopy

Both thermal and athermal effects of millimeter-wave radiation on BHK-21/C13 cells were sought using scanning and transmission electron microscopy in conjunction with an in vitro technique that allows direct exposure of monolayer cultures to high average power densities. Culture dishes were irradiated by placing them on the open end of an E- or U-band wave guide. This technique exposes different regions of the cell monolayer lying along the longer axis of the wave guide aperture to varying power densities ranging from zero at each edge to twice the average power density at the center. Cell ultrastructure was unaffected by microwave radiation for 1 hour (41.8 or 74.0 GHz, average power densitites = 320 or 450 mW/cm², respectively) with or without cooling by rapid recirculation of the culture medium. Temperature in recirculated cultures was held at 37.2 °C, and that in noncooled cultures never exceeded 42 °C during irradiation at either power density. In contrast, cell morphology was affected by microwave exposure whenever irradiation conditions were altered so that the temperature of the monolayer reached or exceeded 44.5 °C. Ultrastructural alterations included breakage of cell processes, progressive detachment of cells from the substrate, increased clumping of heterochromatin in the nuclei, and the appearance of large empty vesicles in the cytoplasm. Such morphological changes resulted from either application of higher average power densities or irradiation at the power densities described above at a higher ambient temperature (>38.5°C).     

Analysis of temperature effect on the germination of New York lettuce seeds

When New York lettuce seeds were imbibed below 20°C in thedark, full germination was observed, whereas none occurred above35°C even under red-light irradiation. Partial treatmentwith high temperature during imbibition revealed the involvementof a thermo-labile process in the germination. This processwas completely inhibited above 30°C in the dark, but re-activatedby the following incubation below 20°C. The longer the seedswere preincubated above 30°C, the longer the period below20°C required for re-activation. The results suggest thatthe thermo-labile process is controlled by a thermo-labile factor.The factor was inactivated slowly at 25°C and rapidly above30°C to a form which could be re-activated below 20°Cbut was irreversibly inactivated when the seeds were imbibedat temperatures above 45°C for more than 40 hr. The escapereaction of the phytochrome system proceeded even at 35°Cwhereas no germination was observed at that temperature. Thus,die germination of lettuce seeds is regulated not only by thephytochrome system but also by the thermolabile factor. (Received August 11, 1975; )     

The effect of 2450 MHz microwave radiation on histamine secretion by rat peritoneal mast cells

Isolated rat peritoneal mast cells actively secrete histamine in response to reaginic or chemical stimulation. Mast cells were irradiated in a waveguide microwave exposure chamber at 2450 MHz with power absorptions of 8.2 and 41.0 mW/g for periods up to 3 h. These levels of microwave absorption caused no change in the morphological characteristics or viability of the cells. Irradiated mast cells were stimulated with compound 48/80, a potent, noncytotoxic histamine releasing agent. The dose response curves showed that neither prior nor simultaneous irradiation of mast cells at 37°C affected 48/80-induced secretion. However, microwave power absorptions of 41.0 mW/g inhibited secretion at 44.0°C. Precise measurements of the effect of heat on secretion indicated that this level of inhibition could have been produced by a radiation induced increase in cell temperature between 0.4 and 0.9°C above ambient levels. Alternatively, the heat stress produced at 44°C may have sensitized the cells to the electromagnetic effects of the microwave radiation. Rat peritoneal mast cells can therefore be useful as a model for the study of functioning secretory cells during microwave irradiation and can also be used to monitor the synergistic effects of cell heating during in vitro exposure.     

Inhibition of cortical neural networks using infrared laser

The aim of the present study is to optimize parameters for inhibiting neuronal activity safely and investigating thermal inhibition of rat cortex neural networks in vitro by continuous infrared (IR) laser. Rat cortex neurons were cultured on multi‐electrode arrays until neural networks were formed with spontaneous neural activity. Neurons were then irradiated to inhibit the activity of the networks using different powers of 1550 nm IR laser light. A finite element heating model, calibrated by the open glass pipette method, was used to calculate temperature increases at different laser irradiation intensities. A damage signal ratio (DSR) was evaluated to avoid excessive heating that may damage cells. The DSR predicted that cortex neurons should be safe at temperatures up to 49.6°C for 30 seconds, but experiments suggested that cortex neurons should not be exposed to temperatures over 46°C for 30 seconds. Neural response experiments showed that the inhibition of neural activity is temperature dependent. The normal neural activity could be inhibited safely with an inhibition degree up to 80% and induced epileptiform activity could be suppressed. These results show that continuous IR laser radiations provide a possible way to safely inhibit the neural network activity.      

Local tumor hyperthermia using a computer-controlled microwave system

A minicomputer-based system was designed to control the microwave (2.45-GHz) power to four local hyperthermia applicators. Errors in temperature measurement, due to electromagnetic field interactions with small thermocouple probes, are minimized by sampling the temperature only when the microwave power is off. The programmable controller can regulate the temperature in tumors in 0.1 °C increments from 30 to 60 °C. This technique reduces temperature differences throughout the tumor at steady state to less than 0.4 °C and prevents skin burns.     

In vivo regional levels of PGE and thromboxane in mouse brain: Effect of decapitation,focused microwave fixation,and indomethacin

A focused microwave fixation technique was tested for use in determining basal PGE and thromboxane B₂ levels of mouse brain. Focused microwave irradiation (3.5 Kw/0.4 sec) to the head of C3H mice produced basal values of PGE and TXB₂ which were five-fold less than those in animals killed by decapitation. Indomethacin (10 mg/kg) pretreatment blocked the decapitation rise in PGE and TXB₂ levels and gave values similar to focused microwave irradiation. Indomethacin pretreatment combined with microwave fixation did not reduce PG levels more tham microwave treatment alone. When microwave fixation was used, there was no difference in regional (cerebral cortex, whole cerebellum, midbrain, hypothalamus) levels of either PGE or TXB₂. However, PGE levels were significantly higher than TXB₂ in all regions. After decapitation there was a greater increase in TXB₂ than PGE. The cerebellum produced less PGE and TXB₂ after decapitation compared to the other regions. Our results confirm the usefulness of the focused microwave irradiation technique for examining $\text{in vivo}$ basal prostaglandin levels in mouse brain.     

Fluorescence depolarization studies of the phase transition in multilamellar phospholipid vesicles exposed to 1.0-GHz microwave radiation

The phase transition in multilamellar dimyristoylphosphatidylcholine (DMPC) vesicles was studied during exposure to continuous wave 1.0-GHz microwave radiation. Fluorescence depolarization measurements using a lipid-seeking molecular probe, diphenylhexatriene (DPH). were performed as a function of temperature. Semilog plots of microviscosity versus temperature illustrate the phase transition which shows a 5°C shift when the vesicles are treated with chloroform as a positive control. No shift of the phase transition was found during exposure to microwave radiation at specific absorption rates between 1 and 30 W/kg. Samples were exposed in a rectangular transmission line (TEM cell), and specific absorption rates were calculated from electrical measurements of incident, reflected, and transmitted power. Samples were exposed to increasing intensities of radiation, while the temperature was maintained at either 23.5 or 25.5 °C; these temperatures represented the two ends of the phase transition region for these vesicles. No statistically significant difference was found between exposed and control samples. These results are in contrast to those of others using laser Raman spectroscopy to measure the phase transition in similar multilamellar vesicles exposed to microwave radiation.     

Studies on microwave and blood-brain barrier interaction

This investigation was aimed at correlating changes of blood-brain-barrier permeability with the quantity and distribution of absorbed microwave energy inside the brain of adult Wistar rats anesthetized by sodium pentobarbital. Through use of thermographic methods and a direct-contact applicator at the animal's head, the pattern of absorbed microwave energy was determined. Indwelling catheters were placed in the femoral vein and in the left external carotid artery. Evans blue and sodium fluorescein in isotonic saline were used as visual indicators of barrier permeation. Exposure to pulsed 2,450-MHz radiation for 20 min at average power densities of 0.5, 1, 5, 20, 145 or 1,000 mW/cm², which resulted in average specific absorption rates (SARs) of 0.04, 0.08, 0.4, 1.6, 11.5 or 80.0 mW/g in the brain, did not produce staining, except in the pineal body, the pituitary gland, and the choroid plexus — regions that normally are highly permeable. Except for these regions, staining was also absent in the brains of sham-exposed animals. The rectal temperature, as monitored by a copper-constantan thermocouple, showed a maximum increase of less than 0.75°C from a mean pre-exposure temperature of 36.6°C. The highest brain temperature recorded in a similar group of animals using a thickfilm carbon thermistor was less than 41.0°C.     

Exacerbation of Brain Pathology After Partial Restraint in Hypertensive Rats Following SiO2 Nanoparticles Exposure at High Ambient Temperature

This investigation examines the possibility that exposure to silica dust of hypertensive individuals may exacerbate brain pathology and sensory motor dysfunction at high environmental temperature. Hypertension was produced in rats (200–250 g) by two-kidney one clip (2K1C) method, and in these animals, SiO₂ nanoparticles (NPs; 50 to 60 nm) were administered at 50 mg/kg, i.p. daily for 1 week. On the 8th day, these rats were subjected to partial restraint in a Perspex box for 4 h either at room temperature (21 °C) or at 33 °C in a biological oxygen demand incubator (wind velocity, 2.6 cm/s; relative humidity, 65 to 67 %). In these animals, behavioral functions, blood–brain barrier (BBB) permeability to Evans blue albumin (EBA) and radioiodine (^[131]-Iodine), brain water content and neuronal injuries were determined. Hypertensive rats subjected to 4 h restraint at room temperature did not exhibit BBB dysfunction, brain edema, neural injury, or alterations in rotarod or inclined plane angle performances. However, when these hypertensive rats were subjected to restraint at 33 °C, breakdown of the cortical BBB (EBA, +38 %; radioiodine, +56 %), brain water (+0.88 %), neuronal damages (+18 %), and behavioral impairment were exacerbated. Interestingly, SiO₂ exposure to these rats further exacerbated BBB breakdown (EBA, 280 %; radioiodine, 350 %), brain edema (4 %), and neural injury (30 %) after identical restraint depending on the ambient temperature. SiO₂ treatment also induced brain pathology and alteration in behavioral functions in normotensive rats after restraint at high temperature. These observations clearly show that hypertension significantly enhances restraint-induced brain pathology, and behavioral anomalies particularly at high ambient temperature and SiO₂ intoxication further exacerbated these brain pathologies and cognitive dysfunctions.     

Characteristics of microwave evoked body movements in mice

Microwave evoked body movements were studied in mice. A resonant cavity was used to provide head and neck exposure of the mouse to pulsed and gated continuous wave (CW) 1.25 GHz microwaves. No difference in response to pulsed and gated CW stimuli of equal average power was found. The incidence of the microwave evoked body movements increased proportionally with specific absorption (dose) when the whole-body average specific absorption rate was at a constant level (7300 W/kg). Under a constant average specific absorption rate, the response incidence reached a plateau at 0.9 kJ/kg. For doses higher than 0.9 kJ/kg, response incidence was proportional to the specific absorption rate and reached a plateau at 900 W/kg. Body movements could be evoked by a single microwave pulse. The lowest whole-body specific absorption (SA) tested was 0.18 kJ/kg, and the corresponding brain SA was 0.29 kJ/kg. Bulk heating potentials of these SAs were less than 0.1 °C. For doses higher than 0.9 kJ/kg, the response incidence was also proportional to subcutaneous temperature increment and subcutaneous heating rate. The extrapolated absolute thresholds (0% incidence) were 1.21 °C temperature increment and 0.24 °C/s heating rate. Due to high subcutaneous heating rates, these microwaves must be perceived by the mouse as an intense thermal sensation but not a pain sensation because the temperature increment was well below the threshold for thermal pain. Results of the present study should be considered in promulgation of personnel protection guideline against high peak power but low average power microwaves. © 1994 Wiley-Liss, Inc.     

Resection of meningiomas with implantable microwave coagulation

Implantable microwave coagulation was used to perform resection on 62 patients that had intracranial meningiomas. When 20–60 W microwave power was applied for 15 s, the temperature at the center of the tumor tissue was 43–63°C; 30 mm from the center, the temperature was under 40°C. Histological changes in the center of the tumor showed coagulative necrosis, diminished nuclei, and obliterated blood vessels. The changes at 10–20 mm from the center of the tumor showed coagulative necrosis and degeneration and, 30–50 mm from the center of the tumor, showed normal cell morphology after microwave coagulation. The thermal field in brain tumor has an effective diameter of about 40 mm. No side effects on the normal brain tissues were observed. The amount of blood loss during the operation was minimal while the meningioma was coagulated, especially when the meningioma was located at the skull base or in the parasagittal or cerebral convexity region. After microwave coagulation, the entire tumor could easily be removed. Among the 62 surgically treated cases, gross total tumor excision was 85%. No postoperative complications occurred after microwave coagulation, and there was no operative mortality in the series. We believe that this new technique has the advantage of simplicity, less blood loss, and smooth postoperative procedures. Hemostatic effects during the operation are satisfactory, and blood transfusion can be reduced by 50–60%. © 1996 Wiley-Liss, Inc.     

Effect of ionic strength on the inactivation of micro-organisms by microwave irradiation

Microwave irradiation at 2450 MHz inactivated the cells of Escherichia coli, Staphylococcus aureus and Candida albicans suspended in a phosphate buffer. The rate of cell inactivation was proportional to that of the increase in temperature accompanied by microwave irradiation. The inactivation rates of E. coli and C. albicans were affected by addition of NaCl and KCl, but not by sucrose. The maximal inactivation effect was exerted at concentrations of 0.5-1.0 mol l-1, and the end-point temperature was the highest at the same salt concentrations. Correlation of both the electroconductivity and di-electric loss of ionic solutions with the heating by microwave irradiation was discussed.     

Studies on temperature-dependent ultraviolet light-sensitive mutants of bacteriophage T4: the structural gene for T4 endonuclease V.

Mutants of bacteriophage T4 which exhibit increased sensitivity to ultraviolet radiation specifically at high temperature were isolated after mutagenesis with hydroxylamine. At 42 °C the mutants are twice as sensitive to ultraviolet light as T4D, whereas at 30 °C they exhibit survival curves almost identical to that of the wild-type strain. Complementation tests revealed that the mutants possess temperature-sensitive mutations in the v gene.Evidence is presented to show that T4 endonuclease V produced by the mutants is more thermolabile than the enzyme of the wild-type. (1) Extracts of cells infected with the mutants were capable of excising pyrimidine dimers from ultraviolet irradiated T4 DNA at 30 °C, but no selective release of dimers was induced at 42 °C. (2) Endonuclease V produced by the mutant was inactivated more rapidly than was the enzyme from T4D-infected cells when the purified enzymes were incubated in a buffer at 42 °C. From these results it is evident that the v gene is the structural gene for T4 endonuclease V, which plays an essential role in the excision-repair of ultraviolet light-damaged DNA.The time of action of the repair endonuclease was determined by using the mutant. Survival of a temperature-sensitive v mutant, exposed to ultraviolet light, increased when infected cells were incubated at 30 °C for at least ten minutes and then transferred to 42 °C. It appears that repair of DNA proceeds during an early stage of phage development.     

Effects of millimeter-wave radiation on monolayer cell cultures. III. A search for frequency-specific athermal biological effects on protein synthesis

A method recently developed in this laboratory has been used to directly expose BHK-21/C13 cells to high levels of microwave radiation without significant microwave-induced heating (? 0.1 °C). Monolayer cultures were grown on microwave-transparent polystyrene coverslips, placed on the open end of a wave guide, and maintained at 37.2 °C during irradiation at frequencies in both the E- and U-bands (average power densities 292 and 177 mW/cm², respectively). Effects of microwave radiation were assessed at 0.1 GHz increments in the ranges of 38–48 GHz and 65–75 GHz. Protein synthesis was measured in quadruplicate cultures that were allowed to incorporate labeled methionine during the 15-minute period of microwave irradiation. Autoradiographs of each monolayer culture were scanned along the region corresponding to the longer axis of the wave guide aperture using a microdensitometer to quantify incorporation. Since microwave power incident on the cells was previously shown to vary along this axis according to a cosine² relationship from zero at each edge of the wave guide to twice the average power density at the center of the wave guide, this technique should reveal biological effects that might only be manifested in narrow amplitude domains or “power windows.” Observations of protein synthesis in monolayer cultures irradiated at 202 closely spaced frequencies in the E- and U-bands failed to reveal changes associated with microwave exposure. Thus no evidence was obtained in support of the existence of frequency-specific athermal biological effects of microwaves. In addition, no support was found for the existence of amplitude-specific “power windows”.     

Effects of microwave exposure and temperature on survival of mice infected with Streptococcus pneumoniae

Female CD-1 mice were injected with an LD₅₀ dose of Streptococcus pneumoniae and then exposed to 2.45 GHz (CW) microwave radiation at an incident power density of 10 mW/cm² (SAR = 6.8 W/kg), 4 h/d for 5 d at ambient temperatures of 19 °C, 22 °C, 25 °C, 28 °C, 31 °C, 34 °C, 37 °C and 40 °C. Four groups of 25 animals were exposed at each temperature with an equal number of animals concurrently sham-exposed. Survival was observed for a 10-d period after infection. Survival of the sham-exposed animals increased as ambient temperature increased from 19 °C–34 °C. At ambient temperatures at or above 37 °C the heat induced in the body exceeded the thermoregulatory capacity of the animals and deaths from hyperthermia occurred. Survival of the microwave-exposed animals was significantly greater than the shams (～20%) at each ambient temperature below 34 °C. Based on an analysis of the data it appears that the hyperthermia induced by microwave exposure may be more effective in increasing survival in infected mice than hyperthermia produced by conventional methods (ie, high ambient temperature). Microwave radiation may be beneficial to infected animals at low and moderate ambient temperatures, but it is detrimental when combined with high ambient temperatures.     

Acetylcholinesterase activity of the polychaete Nereis diversicolor: effects of temperature and salinity

In order to estimate the potential use of the mean wholebody acetylcholinesterase (AChE) activity from the ragworm Nereis diversicolor for the biological assessment of pollution by anticholinesterase agents in estuarine areas, we measured the effects of the main abiotic factors (i.e. temperature and salinity) on AChE activity. We report here that AChE activity tends to decrease in individuals sampled in tanks at a salinity of 30‰ as temperature increases. No tendencies in the evolution of AChE activity were observed in individuals sampled in tanks at a salinity of 15‰. In contrast, salinity seems to have a greater effect on AChE activity than temperature. At a temperature of 12°C, a salinity of 30‰ provokes a significant transient increase of AChE 2 days after the beginning of the maintenance period compared with a salinity of 15‰. The effects are short-term stress effects. We noticed only a transient increase of AChE activity between 2 days for individuals maintained in tanks at temperature of 20°C and salinity of 15 and 30‰, respectively, and 8 days for individuals maintained in tanks at salinity of 30‰ and at a temperature of 12°C after the beginning of the maintenance period, confirming the more pronounced effect of salinity over temperature.     

Temperature-dependent Activation of Neurons by Continuous Near-infrared Laser

Optical control of neuronal activity has a number of advantages over electrical methods and can be conveniently applied to intact individual neurons in vivo. In this study, we demonstrated an experimental approach in which a focused continuous near-infrared (CNI) laser beam was used to activate single rat hippocampal neurons by transiently elevating the local temperature. Reversible changes in the amplitude and kinetics of neuronal voltage-gated Na and K channel currents were recorded following irradiation with a single-mode 980 nm CNI-laser. Using single-channel recordings under controlled temperatures as a means of calibration, it was estimated that temperature at the neuron rose by 14°C in 500 ms. Computer simulation confirmed that small temperature changes of about 5°C were sufficient to produce significant changes in neuronal excitability. The method should be broadly applicable to studies of neuronal activity under physiological conditions, in particular studies of temperature-sensing neurons expressing thermoTRP channels.     

Genome shuffling improves production of the low-temperature alkalophilic lipase by Acinetobacter johnsonii

The production of a low-temperature alkalophilic lipase from Acinetobacter johnsonii was improved using genome shuffling. The starting populations, obtained by UV irradiation and diethyl sulfate mutagenesis, were subjected to recursive protoplast fusion. The optimal conditions for protoplast formation and regeneration were 0.15 mg lysozyme/ml for 45 min at 37°C. The protoplasts were inactivated under UV for 20 min or heated at 60°C for 60 min and a fusant probability of ~98% was observed. The positive colonies were created by fusing the inactivated protoplasts. After two rounds of genome shuffling, one strain, F22, with a lipase activity of 7 U/ml was obtained.     

Synthesis and screening for acetylcholinesterase inhibitor activity of some novel 2-butyl-1,3-diaza-spiro[4,4]non-1-en-4-ones: derivatives of irbesartan key intermediate

The association of bioactive nucleus with other pharmacological agents is hoped to improve the efficacy of the treatment by combining the effects of different pharmacological mechanisms of action. Keeping this in view, a series of 2-butyl-1,3-diaza-spiro[4,4]non-1-en-4-one derivatives have been synthesized by interaction of 2-butyl-1,3-diaza-spiro[4,4]non-1-en-4-one with different bioactive aralkyl halides in presence of powdered potassium carbonate by two different methods viz., conventional and microwave irradiation. The yields under conventional and microwave irradiation methods were in the range of 60-65% and 80-90%, respectively. The structure elucidation of the new compounds has been carried out with the help of elemental analysis and spectral data. All the synthesized compounds have been screened for their efficacy as acetylcholinesterase (AChE) inhibitor. AChE inhibitory activity study was carried out by using Ellman colorimetric assay with neostigmine as a reference standard against targets from different species, such as pure electric eel AChE, human serum AChE, and rat brain AChE. Among the compounds synthesized, compounds 5a, 5b, 5j showed good inhibition against AChE.     

On the Use of Microwave Radiation Energy for Brain Tissue Fixation

Abstract: Focused microwave irradiation (MWR) is an increasingly accepted method of sacrifice of laboratory animals such as the mouse or rat. By fixing the brain within a fraction of a second with heat inactivation, the investigation of fast neurochemical events may be obtained. Even though the technique is widely utilized, its application is inconsistent. This report illustrates some of the requirements necessary for the proper application of MWR for the sacrifice of animals, particularly those related to the length of time MWR is applied and the efficiency with which generated MWR power is coupled to the brain tissue. Studies were performed on the mouse, using either a 2.5 KW or 6.3 KW generator with a focused, closed system waveguide at time intervals of 350 or 500 ms or 1.4 s. During each of these intervals MWR was varied so that core brain temperatures for all groups were held between 83 and 95°C. In contrast with reported studies that used full animal restraint, all animals were minimally restrained for less than 1 s before sacrifice. Tissue content of cyclic AMP, an index of neuronal activity grossly affected by subtle changes in the activity of adenylate cyclase and/or phosphodiesterases, was monitored. No differences in tissue cyclic AMP content in any of 12 brain regions were detected after MWR, either at 350 or 500 ms. A substantial increase in cyclic AMP content occurred in 8 of 12 brain regions examined following microwave irradiation for 1.4 s. On the basis of these experiments, accurate determination of cyclic AMP in rodent brain requires that the maximum time interval of MWR exposure should not exceed 500 ms.