Microwave inactivation — a technique with promise and pitfalls

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Determination of brain tissue substance concentrations Microwave irradiation is a particularly useful technique for the in vivo study of rapidly metabolized heat-stable substances such as acetylcholine, 3,-aminobutyric acid (GABA), cyclic AMP, cyclic GMP, and certain components of the glycolytic pathway 1~,8,1=,14.The use of this technique considerably changes estimates of normal levels of acetylcholine and choline in brain. The rapid inactivation of cholinesterase activity results in much higher levels of acetylcholine than would R. H. Lenox, P. V. Brown and J. L. Meyorhoff otherwise be found. Studies comparing brain values of intermediary metabolites, e.g. creatinephosphate, ATP, ADP, pyruvate, glucose, For many studies ofmetabolic changes occurring in brain tissue, it is imperative to be able etc., show that whole brain values to halt metabolism throughout the brain in a matter o f milliseconds. Some techniques, obtained using a very rapid freezing such as decapitation into liquid nitrogen, can take more than a minute to freeze all o f the technique, i.e. freeze blowing (approx. brain; and the end result is a very hard block. The technique described in this paper, 0.5 s), are quite comparable with values microwave inactivation, provides a very fast method o f halting metabolism and still obtained with newer microwave inactivation techniques with inactivation times in allows for easy dissection o f the tissue. the range of 0.4-2.0 s7. Determination of Levels of a number of chemicals in the but do not permit regional dissection. brain levels of cyclic AMP is particularly brain are subject to rapid post-mortem Moreover, enzyme systems may recover limited by rapid changes occurring during changes. In order to avoid these artifac- during thawing, thus producing additional sacrifice. The concentration of cyclic AMP in the cerebellum increases over tual changes and assess accurately levels artifactual change. Microwave heating, on the other hand, fivefold within 90 s of decapitation. Using of these metabolites in vivo, it is necessary to inactivate the enzymes involved in their provides a promising approach based newer microwave systems, levels of whole metabolism as rapidly as possible during upon the principle that most enzymes are brain cyclic AMP are similar to values the sacrifice procedure. Conventional heat-labile and denature irreversibly at obtained after freeze-blowing. Thus, using methods have employed immersion or temperatures in the range of 55-90"C. a rapid freezing technique for comparison, decapitation into liquid nitrogen and/or Stavinoha demonstrated that the use of microwave enzyme inactivation appears Freon-12 to stop brain enzyme activity. high-intensity microwave irradiation has to be at least as efficient in most cases, Freezing methods depend on the thermal the capability of sacrificing an animal while offering the advantage of permitting conduction properties of the tissue and and simultaneously inactivating brain regional dissection. thus may require up to 90 s for complete enzymes in milliseconds. This technique enzyme inactivation in all brain regionP ~. reduces the artifacts seen in decapitation Evolution of microwave inactivation More rapid freezing methods, such as the or conventional freezing methods, while systems The microwave inactivation systems freeze-blowing technique, have decreased permitting accurate regional dissection of have changed considerably since their first the time of inactivation considerably, the brain at room temperature L12'1'.

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Microwave exposure time (s) Fig. I. Determination o f adequate duration o f microwave exposure to stabilize levels o f cyclic AMP. Brain regions o f animals exposed to different duration o f microwave were dissected immediately following exposure. Regions from one hemisphere remained at room temperature while those from the other hemispheres were immediately heated at 90°C for 10 min. In order to demonstrate stable tissue levels of cyclic A M P for prolonged dissection at room temperature, we selected as our criterion for adequate duration o f exposure that exposure time at which the levels o f cyclic A M P in both the 90°C heated and the corresponding room temperature regions were statistically equivalent, and when both curves were approaching an asymptote. Thus adequate exposure at these microwave parameters was determined to be 3 s for the cortex, 4 s for the cerebellum and 5 s for the hypothalamus.

© Elsevicr/North-Hollaod Biomedical Press 1979

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introduction early in this decade. At that time animals were exposed to microwave irradiation in systems that resembled commercial microwave heating ovens. This meant that animals were placed in an oven cavity and were exposed to whole body irradiation for 30 s duration at a power level of approximately 1.25 kW and frequency of 2450MHz. The required exposure duration of 30s, however, was too long to avoid anoxic metabolic artifacts. It soon became evident that it would be preferable to focus the microwave energy into the head of the animal, thereby increasing the efficiency of energy delivered to the brain in order to achieve the required temperature rise of 40-50"C in less time. Our laboratory and others explored ways of inserting only the head of an animal into a modified waveguide chamber, thus limiting exposure. Animals were placed in a plexiglass tube with the tube then inserted into the waveguide. This development, along with the availability of highpowered microwave sources (2.5-7 kW) permitted decreased exposure times as short as 1-5 s for the rat and 150 msto 1 s in the mouse. Factors affecting the reliability of the methodology The minimum power and efficiency required of a microwave system depends upon both the post-mortem activity and the heat lability of the enzyme system to be inactivated. It appears that choline acetyltransferase is more labile to heat denaturation than is acetylcholinesterase, which begins losing activity at 56°C, completely losing its activity at approximately 70°C. In the inactivation of cyclic AMP metabolism, adenylate cyclase appears more labile to microwave inactivation than phosphodiesterase e. Metabolic systems with lower postmortem enzymatic activity and greater heat lability will have less artifactual change in substrate concentration during the inactivation period. For example, cyclic AMP can increase 5-10-fold within 90 s during the post-mortem period, while G A B A has been reported to increase by a maximum of 50% over 10 minutes. Moreover, we have observed microwave inactivation of the metabolic enzymes to be more rapid for the G A B A system than for the cyclic AMP system. Hence the power or efficiency requirements of a microwave inactivation system might he less if G A B A were the only substrate to be measured.

Non-uniform enzyme inactivation It is important to be aware of the limitations of the microwave techniques presently available. In evaluating various microwave inactivation systems for the determination of regional levels of cyclic AMP and G A B A in brain, it became apparent that enzyme inactivation throughout the brain was non-uniform*~. Butcher and Butcher observed this non-uniformity of heating using histochemical techniques for acetylcholinesterase and sodium N A D H diapborase in rat brain s. It is clear from our studies that different regions of the rat brain require different durations of microwave exposure in order to stabilize cyclic AMP levels*. This suggested a gradient of enzyme inactivation within the rat brain (Fig. 1). The precise regional heating and enzyme inactivation patterns of the brain depend upon the pattern of absorption of microwave energy and the thermal diffusion properties of the tissue. The microwave field pattern generated in the brain is affected by the nature and configuration of the dielectric materials making up the head (i.e. brain, bone, muscle, skin). The non-uniform distribution of dielectric throughout the rat's head tends to produce a non-uniform field within the brain. Another issue of concern was the orientation and mobility of the animal relative to the microwave field within the waveguide chamber. The configuration of

the microwave energy absorbed is also dependent on the orientation of the dielectrics in relation to the polarization of the incident microwave field at the time of exposure. Hence, movement of the animal's head within a waveguide chamber during exposure alters the configuration of 'the microwave field generated within the brain, and the resulting pattern of enzyme inactivation across brain regions. Thus, the mobility of the animal within the field creates significant problems with regard to reproducibility from animal to animal. Conventional freezing methods have the theoretical advantage of reproducibility in that the liquid nitrogen, given a sufficient volume, can be considered independent of the object being introduced. Therefore, the freezing or inactivation pattern as shown by Swaab 1~, and others, is dependent only upon the type and amount of materials composing the object, and their distribution. This is in contrast with microwave inactivation which is dependent upon the dynamic interaction between the field (the inactivating medium) and the orientation of the rat's head during exposure. This close interaction in the microwave system between the load (the rat's head) and the inactivating medium (the microwave field) leads to the requirement, in most cases, to immobilize an animal during, or prior to, sacrifice. This requirement is shared by most sacrifice methods, but it should be noted that forced immobilization may introduce

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108 artifacts of its own, since it is a potent stressor. Moreover, we have found that immobilization blocks the elevations in cerebellar cGMP induced by cold exposure or apomorphine injectionlk

TINS - April 1979

microwave exposure results in significant distortion of the field. Using a thermistor probe thrust into the brain seconds after exposure, we measured temperatures in the range of 90-100°C for exposures adequate to stop the metabolism of cyclic AMP throughout the brain. Butcher and Butcher have similarly examined temperature in the brain, finding temperatures in the range of 78 ° adequate for inactivation of acetylcholinesterase. Stavinoha and colleagues have also used thermography to estimate temperature gradients contained in mouse brains during microwave inactivation is.

Quality of the equipment We have also found it necessary to control microwave power source parameters in order to achieve reproducible enzyme inactivation. The net power absorbed by a given load (animal being sacrificed) is determined by the difference between the incident and reflected power. The incident power may vary from exposure to exposure with fluctuations in Artifacts of microwave systems line voltage, resolution capacity of the Since large amounts of energy are being timing circuit, and with the age of the deposited into the brain over very short output tube, causing variations in the periods of time, changes in tissue range of 15-20%. Electronic modifica- morphology and possible subsequent tions have been designed to control artifacts might be expected. Diffusion of precisely the intensity and duration of the dopamine from regions of high concentraincident power~. The reflected power can tion to contiguous regions of low concenbe minimized by precise impedance trations have been shown to occur during matching of the load to the power source. irradiationL This possibility has been The quality of this tuning depends upon the raised with regard to acetylcholine and frequency of the microwave output as well histamine as well. Histological changes as upon the size, configuration, and following microwave irradiation include dielectric characteristics, of the load within vacuolization, a marked decrease in the waveguide system. Because the dielec- staining for myelin and axon sheaths ~, as tric characteristics vary between animals, well as changes in cell bodiesa.L our equipment has the capability of in-line impedance matching each animal with an appropriate tuner, and of monitoring the incident and reflected power with a strip chart recorder (Fig. 2). A wide frequency match is used to accommodate changes in dielectric characteristics of the animal during heating which would otherwise result in excessive amounts of reflected power. Periodic checks are made on the O frequency spectrum of the power source 90 and the accuracy of the timing circuit. Adequate inactivation The non-uniformity of the microwave field generated within the brain makes it essential that the duration of microwave exposure be sufficient to inactivate enzymes in the least heated regions. However, optimal microwave exposure requires not only an irreversible inactivation of enzyme systems and heat stability of the suhstrates measured, but also preservation of the structural integrity of the brain tissue. Non-uniform microwave field concentrations produce 'hot spots', where brain tissue may vacuolize. Efforts to measure temperatures in the brain during microwave exposure have been limited by the technology available. The use of in situ thermistors during the

In addition, there is a question as to whether alternative synthetic/degradative routes might be activated during microwave inactivation such as was suggested in the case of cyclic GMP or histamine. Kimura and Murad have demonstrated that significant amounts of cyclic GMP can be formed non-enzymatically from GTP when guanylate cyclasc reaction mixtures are heated at a pH near neutrality. This raises the possibility that d e n o v o synthesis of cyclic GMP during the heating process could occur with sufficiently prolonged inactivation times. Domino and colleagues have observed increased levels of histamine following microwave ~nactivation which could not be accounted for by blockade of histamine catabolism or microwave-induced histidine decarboxylation. They suggested the possibility of histamine formation secondary to microwave-induced conversion of peptido histamines. Current state of microwave technology Despite its limitations, microwave inactivation has broad utility in the neurosciences when the technique is carefully employed. Any laboratory using the technique should document the reliability of the power source being used. It is important to verify that each animal being

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CONTROL (sacrificed by decapitation) Fig. 3. Coronal and sagittal sections o f rat brain folio wing exposure at 2450 or 986 MHz after rotation of the rat to one o f four angular positions (0, 90, 180, or 270 degrees) within the waveguide. An exposure time was deliberately selected to produce a gradient o f enzyme inactivation in the brains o f rats exposed to either frequency. This gradient appears to be independent o f the rotation o f the animal at 986, while markedly angle-dependent at 2450 MHz.

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5. Lenox, R. H., Gandhi, O. P., Meyerhoff, J. L. sacrificed is coupled to the microwave milliseconds. If we are to be able to and Grove, H.M. (1976) IEEE Trans. field with the same efficiency, thereby correlate such events with neurochemical Microwave Theory Tech. 24, 58--61. estimating the net power received by each changes in rapidly turning over meta6. Lenox, R. H., Meyerhoff, J. L., Gandhi, O. P. animal. With the microwave waveguide bolites, it will be necessary to apply and Wray, H.L. (1977) J. Cyclic Nucieotide applicators presently in use, the investi- increasing amounts of power for shorter Research 3, 367-379. 7. Lust, W. D.,Passonneau, J. V.andVeech, R. L. gator must determine the configuration of periods of time. In order to accomplish (1973) Science 181,280--282. heating patterns within the brain by this most effectively it will be necessary to 8. Medina, M. A., Jones, D. J., Stavinoha, W. B. assessing the pattern of regional enzyme apply the microwave energy as uniformly and Ross, D.H. (1975) J. Neurochem. 24, inactivation versus duration of exposure as possible throughout the brain. The 223-227. for each region of interest. It is critical that requirement for increased amounts of 9. Meyerhoff, J.L., Balcom, G.J. and Lenox, R. H. (1978) Brain Res. 152, 161-169. a time course of inactivation be power incident to the animal is technodocumented for each enzyme involved in logically achievable, but generation of a 10. Meyerhoff, J. L., Gandhi, O. P., Jacobi, J. H. and Lenox, R. H. (1979) IEEE Trans. Microthe metabolism of the substrate of uniform field within the brain during a wave Theory Tech. (in press). interest, since irreversible inactivation of waveguide exposure requires further 11. Meyerhoff, J. L, Lenox, R.H., Kant, G.J., different enzymes may require differing research. Studies of the parameters Mougey, E. H., Pennington,L. L. and Sessions, G. R. (1978) Society for Neuroscience Abs. 4, amounts of heat energy deposition. Since n e c e s s a r y to attain this goal are in 1111. the fields generated within the animal are progress. Microwave irradiation will conalso orientation-dependent in current tinue to be very useful to the neuro- 12. Schmidt, M.J., Schmidt, D. E. and Robison, G. A. (1971) Science 173, 1142-1143. microwave systems, there is a persisting sciences, but variables inherent in the 13. Stavinoha, W. B. (1978) In: D. Jenden (ed.), requirement to immobilize the animal to a technology must be recognized and Cholinergic Mechanisms and Psychopharmaco. logy, Plenum Press, New York, pp. 169-179. standard orientation vis-a-vis the polariza- controlled wherever possible. 14. Stavinoha,W., Pepelko, B. and Smith,P. (1970) tion of the microwave E field. These kinds Pharmacologist 12, 257. of parameters must be worked out for Reading list 1. Balcom, G.J., Lenox, R.H. and Meyerhoff, 15. Swaab, D.F. (1971) J. Neurochem. 18, each inactivation unit and for each species 2085-2092. J. L. (1975) J. Neurochem. 24, 609-613. of animal to be studied. Once these 2. Brown, P.V., Lenox, R.H. and Meyerhoff, conditions have been clearly designated, J.L. (1978) IEEE Trans. Biomed. Eng. 25, R. H. Lenox and P. V. Brown are members of the the investigator can use the microwave 205-208. Department of Psychiatry, Neuroscience Research technique to advantage. 3. Butcher, L. L. and Butcher, S. G. (1976) Life Unit, and J. L. Meyerhoff is Chief of the NeuroendoSci. 19, 1079-1088. crinology and Neurochemistry Branch, Department 4. Jones, D. J., Medina, M. A., Ross, D. H. and of Medical Neurosciences, Division of NeuroFuture of microwave inactivation systems Stavinoha, W.B. (1974) Life Sci. 14, psychiatry, Walter Reed Army Medical Center, Microwave inactivation technology 1577-1585. Washington, DC 20012, U.S.A. continues to develop; attempts are being made to overcome some of the limitations of the techniques noted above. The energy t c a I t| I't, distribution and accompanying heat pattern within a load exposed in a microwave field are dependent not only upon the shape, dielectric properties, and its orientation with respect to the field, but also upon the frequency of the microwave field. Our laboratory has studied the pattern of energy, deposition at different frequencies using cytochemical techniques to identify patterns of enzyme Present clinical education leaves medical students to develop their diagnostic expertise inactivation. We previously observed that through experience alone. However, recent studies have s h o w n that the attainment o f this heat deposition as well as pattern of expertise can be accelerated by the teaching o f appropriate information and decisions enzyme inactivation in the rat brain at theory. John Balla feels that students can, and should, be actively taught to convert 2450 MHz with two different applicator clerical, inductive data collection into an expert technique which follows specific leads in designs was non-uniform and markedly a deductive process. affected by rotation of the rat head. Using Over the last few years the process of Kleinmuntza was one of the first to a waveguide applicator designed for clinical diagnosis has been the subject of study the diagnostic process, and he felt exposure at 986 MHz, recent studies in increasing scrutiny. The realization that that the highly structured nature of the our laboratory have shown non- the diagnostic process lends itself to clinical data which was presented to the uniformity of heat deposition in the brain, clinical analysis has important practical as neurologist made it particularly amenable but have also demonstrated that the well as theoretical implications. From the to analysis. He was able to study a number pattern of energy absorption at 986 MHz practical point of view, we may be able to of neurologists using a technique similar is independent of orientation of the ask if teaching methods could be to the game of '20 questions'. A n 'actor', animal within the waveguide (Fig. 3) 2. improved and whether computer techno- who would have to be an expert Thus, the use of this frequency with rats logy has a place in clinical diagnosis. From neurologist, would think of a specific might alleviate the requirement of rigor- the theoretical side, we can expect diagnosis. The 'examiner' would then ask ous immobilization of the subject during significant advances in diagnostic skills questions to which 'yes' or 'no' answers sacrifice. once we have a better understanding of only were given. Kleinmuntz was able to Neurophysiological events occur in the diagnostic process itself. show that the expert 'examiner' tended to

The neurological diagnostic process

(~ El~vler/Nonh~HollandBiomedicalPress1979