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Study of the use of the microwave magnetic field for the rapid inactivation of brain enzymes.

A new model of a microwave device was developed with a power of 10 kW at 2450 MHz. In order to accomplish even distribution of heating with minimum trauma and with a maximum certainty about enzyme inactivation, a modified magnetic field distribution was utilized rather than the conventional electric field. An integrated tuning system was used to increase efficiency and distribution of microwave energy absorption. This increased the ability of the instrument to properly inactivate the enzymes in the brain of both mice and large rats. In general, the time of irradiation for the rat was 600 to 900 msec and for the mice, 100 to 330 msec. The animal chambers used were designed so as not to impair breathing or too severely restrict movement. The effects of these improvements on microwave irradiation were confirmed by 1) observation of brain appearance, 2) effects on succinic dehydrogenase and cholinesterase activity, 3) measurement of regional temperatures in the animal's brain, 4) thermograms of the brain, 5) electron microscopic examination of brain tissue and 6) measurement of endogenous acetylcholine and catecholamines.

The technique of microwave irradiation (MWR) for the inactivation of brain enzymes of animals has been widely used since its introduction in 1970 (1). It has gained widespread acceptance because it is a sacrifice method which makes possible sampling and measurement of many enzymatically destroyed brain neurochemicals by inactivating the enzymes, lessening the influence of postmortem changes (2,3).
Discussion of the method and its application has been the subject of several reviews (4-6) and scientific reports . Many of these results have been recently summarized in the Proceedings of the Symposium on "Drug Effects on Rapidly Metabolized Compounds in the CNS: Rapid Tissue Fixation with MWR (1981)" which was held at a Satellite Symposium of the 8th International Congress of Pharmacology in Tokyo, 1981 (10).
However, there is still misunderstanding about the application of microwave fixation not because of doubt about the usefulness of the technique, but because of some unsuccessful applications. These errors seem to occur primarily because of the nonuniformity of heating which results in incorrect sampling and assay of levels of endogenous compounds (29, 31). A primary reason for these problems most likely lies in the use of inadequately controlled microwave devices. Use of various simple microwave devices which do not have adequate control and adjustment mechanisms has given rise to a multitude of problems related to the uniformity of heating, residual enzyme activity, coupling efficiency of the device, stress-induced changes in brain chemistry and compound translocations associated with tissue disruption. Heating brain tissue by microwave energy makes it possible to rapidly heat the brain and inactivate enzymes; however, it does not necessarily provide uniform distribution of the heat or the ability to repeat the same distribution in subsequent use. (6,10,32).
To organize a system to obtain proper distribution of tissue heating, a certainty of enzyme inactivation with minimum trauma and retention of valid labile compound concentration, the following criteria should be addressed: The instrument: 1. Must provide a wide distribution of power in the brain. 2. Must have adequate power and coupling efficiency to heat the brain rapidly. 3. Should have a rapid rise time, that is, the time required for full power to be delivered to the tissue should be a small segment of the firing interval. 4. Must be readily tuneable so that the power distribution can be optimized for variation in animals.
The animal: 1. Should be comfortable in the container.
2. Should not be unduly restrained. 3. Should not be detained in the container or apparatus for more than a few seconds before sacrifice.
The tissue: 1. Upon dissection, following microwave heating, the brain should appear hardened and intact with a uniform, grey appearance with no apparent bloody areas. 2. Must have been heated over a wide area with the adequacy of the distribution reflected by: a. A relatively even temperature distribution, as measured immediately following heating. b. A widespread enzyme inactivation which occurs very early during power application: that is, most heating occurs by primary power deposition and not conduction from heated to non-heated tissue. 3. Concentration of enzymically labile compounds whose action is rapidly terminated by enzyme action should yield different values following microwave irradiation sacrifice and sacrifice by decapitation.
In order to satisfy these criteria for successful inactivation of brain enzymes in rat and mouse, an instrument has been developed for applying the magnetic field (H-field) with a tuning system.
In this paper, we describe the NJE2603 system with a 10 kW input operating at a frequency of 2450 MHz and discuss its Appearance of the rat brain following microwave irradiation in H-magnetic (H) and E-electric (E) field modes: The gross appearance of the brain following MWR in H-field and E-field is shown in Figs. 3 and 4.
Most available and currently used microwave units utilize the E-field with a power output of 3-5 kW. This power was used to evaluate the heating effect in the H-field as compared with that of the E-field.
Comparison of the tissue appearance in dorsal, (Fig. 3A and 4A) ventral ( Fig. 3B and 4B) and sagittal ( Fig. 3C and 4C) views shows significant differences in uniformity of heating between the fields; the appearance of the tissue irradiated in the H-field mode was much more uniform than that of the tissue exposed to the primarily E-field mode. Rapid and uniform coagulation of blood is important for limiting contamination of compounds between blood and tissues. We did not specifically study this problem, but most analytical techniques for studying     5. Thermogram of sagittal, frontal and horizontal sections of the rat and the mouse brain following MWR. Rats were exposed at 6 kW for 600 msec and mice at 6 kW for 120 msec. The photos show the energy distribution and heat conduction in the brain following MWR utilizing the magnetic field. The position and extent of the brain is outlined in dotted lines superimposed on the thermogram. The temperature color code is red (A) for the highest temperature with lower temperatures being successively lettered and colored tan, green and blue, the lowest temperature.
The mark "Ar" in the pictures indicate artifacts from the edge of a frame supporting the tissue. compared with those obtained from the decapitated tissue sample. Both light and electron microscopic examinations were performed to visualize the interface between the striatum and cortex, the shape of the nerve cell, the general tissue structure, and the preservation of the synapse and synaptic vesicles. Histochemical examination on tissue heated by a 5 kW device has been reported (21, 22).
In Fig. 8, electron micrographs of cortex sections following MWR and decapitation are presented. There was no significant structural destruction in sections prepared either way, although slight vacuolation was seen in the tissue following MWR. Synapses and synaptic vesicles were well defined in both sections. Mitochondria can be seen along    reported for the rat following 900 msec irradiation in this paper show that significant regional differences in norepinephrine, DOPAC and dopamine do occur when compared with decapitation sacrifice. The evaluation of stress to animals of being placed in the chamber was carefully performed by the method of Blank (23). No effect of pressure induced changes on levels of the assayed compounds was found between the two sacrifice procedures.
Examination by electron microscopy of the microwave heat fixed tissue was done to evaluate the possibility of structural damage caused by the heat exposure. It was concluded that the changes of compound levels were not the result of tissue disruption following rapid heating of the brain by irradiation. Histochemical fluorescence of brain catecholamines is diminished following microwave irradiation because there is a temperaturedependent decrease of formaldehyde indirect fluorescence in brain tissue that has been heated. Use of glyoxylic acid in the procedure resulted in an increase in fluorescence to a level comparable to unheated tissue (21, 22). Evaluation of microwave irradiation using the glyoxylic acid method demonstrated that proper microwave heating preserves the integrity of the tissue with no discernable diffusion of the catecholamines.
The use of this instrument to rapidly sacrifice and denature enzymes in the central nervous system of small animals improves the accuracy and precision of the data since the degradation and synthesis of compounds can be stopped during a precisely known time interval. The technique is also relatively atraumatic, with unconsciousness occurring within approximately the first 10 percent of the exposure period. The use of rapid microwave sacrifice should result in more comparable concentrations of enzymatically labile compounds in the brain being reported between laboratories. This improved accuracy should lessen the need for so many laboratories to perform repetitive experiments.
In summary: The ability of a microwave enzyme inactivating instrument to heat and inactivate enzymes in the brain without causing tissue disruption was studied. Following this, the effect of rapid inactivation on levels of labile compounds in regions of the brain was evaluated.
Except for epine-