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Brain temperature measurements in rats: a comparison of microwave and ambient temperature exposures.

In an effort to understand microwave heating better, regional brain and core temperatures of rats exposed to microwave radiation (2450 MHz) or elevated air temperatures were measured in two studies. In general, we have found no substantial evidence for temperature differentials, or "hot spots," in the brain of these animals. In the first study, after a 30-min exposure, no temperature differences between brain regions either after microwave or ambient air exposure were found. However, a highly significant correlation between brain and core temperatures was found and this correlation was the same for both microwave and ambient air heating. In the second study, time-temperature profiles were measured in rats exposed to either 30 mW/cm2 or 36.2 degrees C. In this study, the 30-min exposure period was divided into seven intervals and the change in temperature during each period was analyzed. Only the cortex showed significantly different heating rates between the air heating and microwave heating; however, this difference disappeared after the initial 5 min of exposure.

Air↗

Some behavioral effects of short-term exposure of rats to 2.45 GHz microwave radiation.

Rats were tested for neurobehavioral alterations immediately after exposure to 2.45-GHz (CW) microwave radiation at 10 mW/cm2 for 7 h. Behavioral tests used were locomotor activity, startle to an acoustic stimulus and acquisition and retention of a shock-motivated passive avoidance task. Both horizontal and vertical components of locomotor activity were assessed in 5-min epochs for a period of 30 min using photoelectric detectors. Microwave-exposed animals exhibited less activity than sham-exposed animals. This was most evident during the last 10-15 min of the 30-min test session. Twenty identical acoustical stimuli (8 KHz, 110 dB) were delivered to each rat at 40-s intervals. The microwave-exposed animals were less responsive to the stimuli than sham-exposed animals. Microwave exposure had no effect on the retention of a passive avoidance procedure when tested at 1 week after training. Both the locomotor activity and acoustic startle data demonstrate that, under the conditions of this experiment, microwave exposure may alter responsiveness of rats to novel environmental conditions or stimuli.

Animals↗

Japanese encephalitis virus (JEV): potentiation of lethality in mice by microwave radiation.

The expression of Japanese Encephalitis Virus (JEV) lethality in mice requires entry of the virus into the central nervous system. This entry is presumably through the capillary endothelial cells (CEC), because entry between CECs is inhibited by bands of circumferential tight-junctions. A viremic stage occurs during the first 4 to 5 days after JEV administration in mice, and both microwave radiation (2.45-GHz, continuous wave, 10-min exposure) and hypercarbia were employed to increase CEC permeability to JEV. Exposure to microwaves at power densities of 10-50 mW/cm2 resulted in a dose-dependent increase in JEV-induced lethality. Mice did not become tolerant or sensitized to microwave potentiation of JEV-induced mortality because 4 daily exposures at 10 or 50 mW/cm2 (SARS, approximately 24-98 W/kg) did not alter the lethality pattern to subsequent microwave radiation of JEV-exposed animals. Similarly, hypercarbia (5, 10, and 20% CO2) was observed to produce a dose-dependent increase in JEV-induced lethality. Both microwave radiation and hypercarbia are thought to promote pinocytosis in CNS capillary endothelial cells. This may be one mechanism by which they enhance JEV-induced lethality in adult Swiss-Cox mice.

Animals↗

Opioid receptor subtypes that mediate a microwave-induced decrease in central cholinergic activity in the rat.

We performed experiments to investigate subtypes of opioid receptors in the brain involved in the effect of acute (45 min) pulsed microwave exposure (2,450-MHz, 2-microseconds pulses, 500 pps, average power density 1 mW/cm2, peak-power density, 1 W/cm2, average whole body SAR 0.6 W/kg) on cholinergic activity in the rat brain. Rats were pretreated by microinjection of specific antagonists of mu, delta, and kappa opioid-receptors into the lateral cerebroventricle before exposure to microwaves. The data showed that all three subtypes of opioid receptors are involved in the microwave-induced decrease in cholinergic activity in the hippocampus. However, the microwave-induced decrease in cholinergic activity in the frontal cortex was not significantly affected by any of the drug treatments, confirming our previous conclusion that the effect of microwaves on the frontal cortex is not mediated by endogenous opioids.

Animals↗

Increased sensitivity of the non-human primate eye to microwave radiation following ophthalmic drug pretreatment.

Previous studies in our laboratory have established that pulsed microwaves at 2.45 GHz and 10 mW/cm2 are associated with production of corneal endothelial lesions and with disruption of the blood-aqueous barrier in the non-human primate eye. In the study reported here we examined ocular damage in monkeys (M. mulatta and M. fascicularis) following topical treatment with one of two ophthalmic drugs (timolol maleate and pilocarpine) that preceded exposure to pulsed microwaves. Anesthetized monkeys were sham exposed or exposed to pulsed, 2.45 GHz microwaves (10 microseconds, 100 pps) at average power densities of 0.2, 1, 5, 10, or 15 mW/cm2 4 h a day for 3 consecutive days (respective SARs were 0.052, 0.26, 1.3, 2.6, and 3.9 W/kg). Immediately before microwave exposure, one or both eyes were treated topically with one drop of 0.5% timolol maleate or of 2% pilocarpine. Following administration of a drug, we observed a significant reduction in the power-density threshold (from 10 to 1 mW/cm2) for induction of corneal endothelial lesions and for increased vascular permeability of the iris. Diagnostic procedures (in vivo specular microscopy and fluorescein iris angiography) were performed following each exposure protocol. In addition, increased vascular permeability was confirmed with horseradish peroxidase tracer techniques. Although we did not measure intraocular temperatures in experimental animals, the results suggest that a mechanism other than significant heating of the eye is involved. Our data indicate that pulsed microwaves at an average SAR of 0.26 W/kg, if administered after pretreatment with ophthalmic drugs, can produce significant ocular effects in the anesthetized primate.

Administration, Topical↗

The effect of pulsed microwaves on passive electrical properties and interspike intervals of snail neurons.

The effects of pulsed microwaves (2.45 GHz, 10 microseconds, 100 pps, SAR: 81.5 kW/kg peak, 81.5 W/kg average) on membrane input resistance and action potential (AP) interval statistics were studied in spontaneously active ganglion neurons of land snails (Helix aspersa), at strictly constant temperature (20.8 +/- .07 degrees C worst case). Statistical comparison with sham-irradiated neurons revealed a significant increase in the mean input resistance of neurons exposed to pulsed microwaves (P < or = .05). Pulsed microwaves had no visible effect on mean AP firing rate; this observation was confirmed by analysis of interspike intervals (ISIs). Using an integrator model for spontaneously active neurons, we found the net input current to be more variable in neurons exposed to pulsed microwaves. The mean input current was not affected. The standard deviation of ISIs and the autocorrelation of the input current were marginally affected, but these changes were not consistent across neurons. Although the observed effects were less obvious than those reported in other studies, they represent evidence of a direct interaction between neurons and pulsed microwaves, in the absence of macroscopic temperature changes. The data do not suggest a single, specific mechanism for such interaction.

Animals↗

Microwave irradiation as a form of fixation for light and electron microscopy.

Microwave irradiation was used to fix a wide range of surgical and autopsy specimens as well as tissue from freshly killed rats. Although there appeared to be varying optimum temperatures of fixation for different tissues, from a practical standpoint, the heating to 58 degrees C of tissues submerged in normal saline resulted in fixation of a quality comparable with that produced by conventional fixation to 10 per cent formalin. Microwave irradiation applied to tissues which had up to 1 h prior immersion in 10 per cent formalin also produced excellent preservation of cytomorphology, the time required for microwave fixation being no more than 150 s. This form of rapid heat fixation had no deleterious effects on special stains, sectioned as well as control fixed blocks and produced less shrinkage artefact than conventional formalin fixation. Immunocytochemical staining for more stable cytoplasmic antigens revealed no significant difference between microwave fixation compared with formalin fixation. The more labile membrane associated antigens of lymphocytes, however, were not preserved by either method of fixation. Microwave fixation was also found to be applicable to electron microscopy. Tissue samples immersed in 2.5 per cent glutaraldehyde and fixed in 90 s by irradiation to 50 degrees C showed excellent preservation of ultrastructural morphology.

Animals↗

Limiting racemization and aspartimide formation in microwave-enhanced Fmoc solid phase peptide synthesis.

Microwave energy represents an efficient manner to accelerate both the deprotection and coupling reactions in 9-fluorenylmethyloxycarbonyl (Fmoc) solid phase peptide synthesis (SPPS). Typical SPPS side reactions including racemization and aspartimide formation can occur with microwave energy but can easily be controlled by routine use of optimized methods. Cysteine, histidine, and aspartic acid were susceptible to racemization during microwave SPPS of a model 20mer peptide containing all 20 natural amino acids. Lowering the microwave coupling temperature from 80 degrees C to 50 degrees C limited racemization of histidine and cysteine. Additionally, coupling of both histidine and cysteine can be performed conventionally while the rest of the peptide is synthesized using microwave without any deleterious effect, as racemization during the coupling reaction was limited to the activated ester state of the amino acids up to 80 degrees C. Use of the hindered amine, collidine, in the coupling reaction also minimized formation of D-cysteine. Aspartimide formation and subsequent racemization of aspartic acid was reduced by the addition of HOBt to the deprotection solution and/or use of piperazine in place of piperidine.

Amino Acid Sequence↗

Fourier-transform EPR at high-field/high-frequency (3.4 T/95 GHz) using broadband stochastic microwave excitation.

Stochastic excitation with a full-width-half-maximum bandwidth of 250 MHz was used to perform Fourier-transform (FT) high-field/high-frequency electron paramagnetic resonance (EPR) at 3.4T/95 GHz (W-band). Thereby, the required microwave peak power is reduced by a factor of tau(p)/T1 as compared to equivalent pulsed FT EPR in which the spin system with spin-lattice relaxation time T1 is excited by a single microwave pulse of length tau(p). Stochastic EPR is particularly interesting under high-field/high-frequency conditions, because the limited output power of mm microwave sources, amplifiers, and mixers makes pulse FT EPR in that frequency domain impossible, at least for the near future. On the other hand, FT spectroscopy offers several advantages compared to field-swept magnetic resonance methods, as is demonstrated by its success in NMR and X-band EPR. In this paper we describe a novel stochastic W-band microwave bridge including a bimodal induction mode transmission resonator that serves for decoupling the microwave excitation and signal detection. We report first EPR measurements and discuss experimental difficulties as well as achieved sensitivity. Moreover, we discuss future improvements and the possibility for an application of stochastic W-band FT EPR to transient signals such as those of photoexcited radical pairs in photosynthetic reaction centers.

Fourier Analysis↗

Problems and artifacts of microwave accelerated procedures in neurohistotechnology and resolutions.

Microwaving artifacts in histoprocessing and staining arise from the acceleration of diffusional and reactive processes. Because such accelerations provide the advantages of microwaving, and because microwave ovens cannot distinguish desirable from undesirable accelerations, artifacts are inevitable. Such microwaving problems can be categorized as follows: histoprocessing and staining reagents may be lost or altered; staining targets may move away from their in vivo sites, may be totally lost from the specimen, or may be altered; physical characteristics such as permeability of specimens or embedding resins may be changed; and staining processes themselves are sometimes different at elevated temperatures. The most general tips for detecting and/or avoiding such problems are to monitor and control the temperature of the reagents and the specimen, to standardize the procedures, and to observe the specimen and reagents carefully during microwaving when a new procedure is being introduced to the laboratory.

Animals↗

Microwave treatment enhances the immunostaining of amyloid deposits in both the transmissible and non-transmissible brain amyloidoses.

The immunolocalization of amyloid deposits containing either protease-resistant prion protein (PrP) or amyloid beta protein (A beta) in the brains of patients with transmissible or non-transmissible cerebral amyloidoses has been greatly facilitated by the pretreatment of tissue sections with concentrated formic acid. We have investigated whether microwave processing of formalin-fixed tissue sections would obviate the need for formic acid pretreatment. Exposure of brain sections to microwaves, even for periods as brief as 1 sec, greatly enhanced the immunostaining of PrP and A beta amyloid deposits in both the transmissible and non-transmissible brain amyloidoses. Microwaving for 1 min yielded staining intensities similar to that following pretreatment with concentrated formic acid for 10 min. Moreover, the combination of formic acid pretreatment and microwave processing resulted in an even more intense staining of amyloid deposits. Microwave processing, which is easy to perform and comparatively inexpensive, makes exposure to the potentially toxic fumes of formic acid unnecessary.

Alzheimer Disease↗

Enhancement of the immunocytochemical detection of antigens by microwave irradiation. Benefits and limitations analysed in isolated plant nuclei and Drosophila embryos in toto.

Performing the antibody reaction under controlled heating through microwave irradiation results in significant improvements in the immunovisualization of antigens, such as shortening the times of incubation, lowering the antibody titres used and allowing the detection of difficult, inaccessible antigens. In addition to investigate the basis of the enhancement, we have extended to more intact samples such as isolated plant nuclei and in toto Drosophila embryos the results previously reported only with tissue sections. A drop of heterologous anti-nucleolin antibody covering isolated nuclei from onion root meristems spread on a glass slide was microwave irradiated, resulting in clear immunofluorescent labelling of the nucleoli. This result was never previously obtained in the absence of microwave treatment, even using the complicated procedure previously reported for the homologous identification of this nuclear protein. Using the much larger and to some extent impermeable Drosophila embryos, we were able to show that the incubation time and concentration of the anti-myosin antibody can be strongly reduced by performing the reaction at 45 degrees C under microwave irradiation. The controlled increase in temperature is the main factor responsible for these improvements; the importance of maintaining an adequate mixing of the samples is also emphasized. The proper implementation of these two experimental conditions will require the introduction of appropriate mixing accessories and temperature measuring probes for samples of small volume in current microwave laboratory ovens.

Animals↗

Potency of microwave irradiation during fixation for electron microscopy.

Liver, skeletal muscle, peripheral nerves, pancreas, thyroid and adrenal cortex were prepared for electron microscopy employing microwave energy either during prefixation with glutaraldehyde or instead of prefixation. Microwave irradiation in the presence of glutaraldehyde in Na/K-phosphate or Na-cacodylate containing CaCl2 and MgCl2 led to distinct appearance of membranes, mainly plasma membrane, and membranes of SER, Golgi complex and mitochondria in liver, pancreas and muscle. The area of high quality fixation, however, was limited to the periphery of samples. On the other hand, SER was dilated in cells of the adrenal cortex, and RER markedly vacuolated in thyroid follicular cells. Microwave irradiation in the presence of Na/K-phosphate and subsequent osmication resulted in preservation of the ultrastructure in similar quality as was obtained by osmication without previous immersion in glutaraldehyde. However, the preservation of SER and Golgi complex in liver and pancreas, and of mitochondria in muscle was greatly improved. Small myelin sheaths remained intact whereas large ones showed focal disintegration. We consider that enhancement of fixation by microwave energy may greatly improve preservation of membranes in some tissues. Successful fixation depends on the use of glutaraldehyde during microwave irradiation, the type of buffer, the addition of ions to increase stabilization, the exposure time to heat, and on postosmication.

Adrenal Cortex↗

Microwave fixation provides excellent preservation of tissue, cells and antigens for light and electron microscopy.

It was demonstrated that microwave energy used simultaneously in combination with low concentrations of glutaraldehyde (0.05%) and formaldehyde (2.0%) rapidly preserved light microscopic histology and excellent fine structural details, as well as a variety of cytoplasmic and membrane-bound antigens. Specimen blocks up to 1 cm3 can be fixed in as brief a time as 26 ms using a specially designed microwave device (ultrafast microwave fixation method). The fast microwave fixation method, using a commercially available device, was successfully used to preserve granule-bound rat mast cell chymase which was subsequently detected by a postembedding immunogold procedure. Control of the following parameters is important to the microwave fixation method: (1) specimens with one dimension less than 1 cm; (2) irradiation temperatures lower than 50 degrees C; (3) irradiation times less than 50 s; (4) immediate replacement of the postirradiation solution with cold storage buffer; (5) fixing the specimen within 15 min after it is removed from its blood supply.

Animals↗

The use of microwave irradiation with low formalin concentrations to enhance the conversion of dopamine into norsalsolinol in rat brain: a pilot study.

The fixation of the neurotransmitter dopamine in the central nervous system by perfusion with formalin solutions seems to take place mainly via the formalin-induced condensation product norsalsolinol. In the present investigation the influence of microwave irradiation of the formalin-induced condensation of dopamine was studied in vitro and in vivo by making use of different, relatively low, formalin concentrations. It appeared that in vitro and in vivo the dopamine conversion was complete with 4% formalin and no influence of microwaves was noted. However, by making use of much lower formalin concentrations (0.2% and 0.4%) the condensation of dopamine was strongly augmented, in vitro (200%) and in vivo (at least 500%) using microwave techniques. There was a considerable loss in non-microwaved tissue (30%) after perfusion in vivo. This was lower (10%) in microwaved tissue. In experiments with perfused brain tissue which allowed a more complete calculation, a loss was found. This might be caused by a strong binding of dopamine and/or norsalsolinol to tissue components or to side reactions that could not be traced by the present experimental techniques.

Animals↗

Microwave fixation: its potential for routine techniques, histochemistry, immunocytochemistry and electron microscopy.

Human tissues, both biopsy and postmortem, and tissues from rodents were fixed by microwaves at various temperatures and compared against formaldehyde-fixed material. Conventional stains, including trichromes, worked well. Red cells were lysed, but white cells were fixed, thus permitting diagnoses of various inflammatory states. Malignant cells were equally well-preserved by the two methods. Histochemical investigations of mucosubstances, lipids and various hydrolases showed no significant difference between the two techniques. Some neurological stains, however, were not as good following microwave treatment. Immunocytochemical localization of IgA, IgM and IgG showed no significant difference after microwave fixation compared to that in tissues fixed with formaldehyde. Microwave fixation did not lead to a greater tissue shrinkage than that obtained with formaldehyde fixation. Both were significantly less than that following treatment with phosphate-buffered saline alone. Electron microscopy gave results which were interpretable, but with damage resembling early postmortem change. Microwave fixation is complete in approximately 1-2 min. The mechanism of fixation appears to be due to denaturation associated with disulphide bond formation and a decrease in solubility of proteins.

Animals↗

Effect of microwave heating on the migration of dioctyladipate and acetyltributylcitrate plasticizers from food-grade PVC and PVDC/PVC films into olive oil and water.

Migration of dioctyladipate (DOA) and acetyltributylcitrate (ATBC) plasticizers from plasticized polyvinylchloride (PVC) and polyvinylidene chloride (PVDC)/PVC (Saran) films into both olive oil and distilled water during microwave heating has been studied. The plasticizer migrating into olive oil and water was determined using an indirect GC method after saponification of the ester-type plasticizer (DOA or ATBC) and subsequent collection of the alcohol component of the ester, namely: 2-ethyl-1-hexanol and 1-butanol, respectively. Migration was dependent on heating time, microwave power setting, the nature of the food simulant and the initial concentration of the plasticizer in the film. Migration of DOA into olive oil reached equilibrium after heating for 10 min at full power (604.6 mg DOA/l). Migration into distilled water was 74.1 mg/l after 8 min of microwave cooking at full power. The amount of ATBC migrating into olive oil reached equilibrium after heating for 10 min at full power (73.9 mg ATBC/l). Migration into distilled water was 4.1 mg/l after heating at full power for 8 min. Control samples containing olive oil gave DOA migration values which were significantly higher than the upper limit for global migration (60 mg/l) set by the European Community. It is proposed that PVC should not be used in direct contact with food in the microwave oven, while Saran may be used with caution in microwave heating and reheating applications, avoiding its direct contact with high fat foodstuffs.

Adipates↗

Extracellular calcium and microwave enhancement of membrane conductance in snail neurons.

Microwave irradiation has been shown to decrease the input resistance of snail neurons. In this study, we examined the role of extracellular calcium in triggering the microwave-induced enhancement of membrane conductance. Two sets of experiments were conducted. In the first set, nerve cells were superfused using Ringer solution with added Cd2+ (0.9 mM) which is a known blocker of calcium channels. In the second set, cells were superfused with low Ca2+ (0.7 mM) Ringer solution. Microwave irradiation was conducted at 2,450 MHz for 30 min with a specific absorption rate of 13 mW/g. It was found that 7 mM to 0.7 mM lowering of Ca2+ in bathing solution as well as blocking of calcium channels in neuronal membrane by means of Cd2+ did not influence the fall in membrane resistance induced by microwave radiation. In fact, the observed changed in membrane resistance in these experiments were nearly equal to those observed for neurons superfused by normal Ringer's. Thus, these results rule out the possible contribution of external Ca2+ in the observed microwave effect. Experiments with high Ca2+ solution also support this conclusion.

Animals↗