Effects of respiratory gases on cytochrome a in intact cerebral cortex: Is there a critical Po2?

Brain Research, 108 (1976) 143-154

143

© ElsevierScientificPublishingCompany,Amsterdam- Printed in The Netherlands

EFFECTS OF RESPIRATORY GASES ON CYTOCHROME A IN INTACT CEREBRAL CORTEX: IS THERE A CRITICAL Po2?

MYRON ROSENTHAL, JOSEPH C. LAMANNA, FRANS F. Jt~BSIS, AND JOSEPH E. LEVASSEUR, HERMES A. KONTOS AND JOHN L. PATTERSON

Department of Physiology and Pharmacology, Duke University, Durham, N.C. 27710 and Medical College of Virginia, Virginia Commonwealth University, Health Sciences Division, Richmond, Va. 23298 (U.S.A.) (Accepted October 8th, 1975)

SUMMARY

Changes in the redox level of cytochrome a and in the amount of oxygenated hemoglobin were measured by dual wavelength reflectance spectrophotometry in the intact cerebral cortex of cats (cerveau isol6 preparation) and in unanesthetized rabbits with chronically implanted cranial windows. Increases in inspired oxygen were accompanied by an increase in the oxidation level of cytochrome a and an increase in the amount of oxygenated hemoglobin in the optical field. These changes were larger in the presence of 5 ~o COz. Reduction of the inspired oxygen concentration produced a decrease in the oxidation/reduction ratio of cytochrome a and a disoxygenation o f hemoglobin. The presence of CO2 at these lower oxygen levels diminished the reduction of cytochrome a and the disoxygenation of hemoglobin. These data indicate that, in the resting subject, the reduction levels of cytochrome a are well above the low values seen in isolated mitochondria. They also indicate that the blood supply to the cerebral cortex is regulated at a level of slight hypoxia.

INTRODUCTION

Functional and metabolic activity of the brain are intimately connected with the supply and consumption of oxygen from the circulation. Much is known of the basic nature of the intracellular oxygen utilization processes (see e.g. refs. 11 and 12) but the fundamental concept of the Po2 level required for normal cellular metabolism remains open. Measurements of changes of blood flow and oxygen consumption for the brain have been approached by means of arterio-venous (A-V) differences or with oxygen polarographic techniques and these have been correlated with the functional

144 or pathological state of the system15,16,~. For example, Gurdjian et al. 8 found that when dogs inspire less than 11 ~,,i oxygen, cerebral lactic acid concentration increases and EEG slowing occurs. Davies and Bronk 6 found with surface electrodes that oxygen tension fell linearly during local vascular occlusion and reasoned that the cortex consumes oxygen at an undiminished rate until the tissue Poz reaches a lower than normal level (below 5 mm Hg). It is relatively difficult to determine the first changes of oxygen utilization with these techniques. It seemed therefore appropriate to determine the adequacy of the 02 supply by monitoring the steady-state oxidationreduction level of cytochrome a and the oxygenation state of hemoglobin during the administration of various respiratory gas mixtures. The classic studies of Chance and Williams 5 on isolated mitochondria from rat liver showed that cytochromes a and a3 are highly oxidized under all conditions of respiratory chain function except during hypoxia and anoxia. Thus, cytochrome a is no more than about 1~ reduced during slow rates of O2 utilization when the ADP (adenosine diphosphate) concentration is low and rate limiting. In this condition (the 'resting metabolism' or 'State 4'), tight coupling between O2 uptake and oxidative phosphorylation produces a slow rate of both processes because of a lack of high energy phosphate ( ~ P) acceptor. When more ADP is made available, the rate of O2 uptake increases and cytochrome a becomes slightly more reduced (3-4~) whereas cytochrome a3 remains practically totally oxidized (1 ~ reduced). Only when 02 becomes limiting do increased reduction levels of these cytochromes occur with concommitant loss of the rate of 02 utilization and the rate of oxidative phosphorylation. The first decreases in the rate of oxidative metabolism are subtle when the reduction levels of cytochromes a and a3 first increase, yet they do take place 11,12. Thus, these two members of the respiratory chain provide very sensitive indices to adequacy of 02 provision. Recent development of a technique for dual wavelength monitoring of changes in the steady state reduction level of cytochrome a allows us to study the adequacy of 02 provision in the intact, normally circulated cerebral cortex. In addition, redox levels of cytochromes b and c and the oxygenation state of hemoglobin can be monitored and fluorometry provides measurements on intramitochondrial reduced nicotinamide adenine dinucleotide (NADH) as previously described la. Monitoring of cytochrome a3 has as yet not been succesfully accomplished because of the interference of the very large Soret-region band of hemoglobin. In this article we describe the first results of administration of various levels of 02 to awake, unanesthetized rabbits with chronic cranial windows and to cats with acute brain stem transections. METHODS

Biological preparation

The chronic experiments were repeated at least 3 times on 8 awake rabbits provided with cranial windows and the acute experiments were performed on 5 cats with bilateral midcoUicular brain stem transections (cerveau isol6 preparation). The cranial windows were manufactured, according to the procedure of Levasseur

145 et al. TM from high optical quality Plexiglas polished to produced a clear, scratch free

surface. Rabbits were used because of their docility and the ease with which they can be handled for experimentation in the awake state. To provide the windows, the rabbits were anesthetized with sodium pentobarbital (i.v., 45 mg/kg) intubated and artificially respired. At this point, the skin and muscle overlying the calvaria was retracted and a hole drilled in the exposed bone. The dura was then removed generally along with the thin arachnoid membrane and the acrylic window was set into a bone opening and pressed in position on the bone flange. Dental acrylic was then applied at the interface between bone and window and over previously embedded stainless steel screws. Tissue adaptation to the implant was virtually complete within two weeks after surgery but the rabbits used in this study had post surgical delay times varying from 4 weeks to 5 years. During the experiments, the rabbits were wrapped in a towel and placed in a plaster-of-paris body cast form-fitted to the animal in a resting position. A plastic mask was placed over the animal's head with an opening on top for the window exposure. Air and other gas mixtures were blown into the front of the mask and allowed to diffuse out the back end. Rabbits generally stayed quiet under the microscope optics of the spectrophotometric monitoring system for an hour or more (except at very low inspired 02 levels) although an adaptor was utilized to maintain a constant distance between the microscope epi-illumination system and the cortical surface. Cats were prepared under ether anesthetic. They were provided with a tracheal and a femoral vein cannula, a portion of the calvaria was removed and an electrolytic brain stem transection was made at the mid-collicular level (A-P 0.0). At this point, anesthesia was withdrawn and the cats were allowed to recover from the surgery for approximately 4 h before the experiments were performed. The cats were then paralyzed with gallamine triethiodide (Flaxedil), and respired with a positive pressure ventilator. Changes in gas mixtures were made by altering the input to the respirator. Optical signals were recorded from exposed areas along the suprasylvian gyri avoiding major bloodvessels as much as possible. Instrumentation

Reflectance spectrophotometry was performed with a modification of the dual wavelength technique of Chance 1. Monochromatic beams of 3-4 nm bandwidth alternately illuminate a 3-ram area of the cerebral cortex through a microscope epiillumination system (Leitz Ultropak) for 'dark field' reflected light. A single tungsten filament lamp functions as the light source for the two monochromators (Bausch and Lomb High Intensity). The light beams are presented alternately at a frequency of 60 Hz; modulation was by means of slotted discs driven by a synchronous motor. The beams enter light conducting fibers mounted at the monochromator exit slits. The fibers are randomly combined into a single bundle which delivers light at the filament plane of the epi-illumination system. Light scattered back from the tissue enters the objective lens (3.8 ×) and its intensity is measured by an end window photomultiplier tube (EMI 9698B) mounted in the barrel of the microscope. Use of

146 the dark field system assures a minimal and negligible contribution of light specutarly reflected from the pial surface. A negative feedback circuit maintains constancy of the signal of the 'reference' wavelength by altering the high voltage supply to the photomultiplier dynodes. This voltage is kept constant during the next half cycle when the 'sample' wavelength illuminates the cortex. Thus, the system is stabilized for optical density changes occurring equally at both wavelengths. The current pulses from the photomultiplier are amplified, substracted and the difference displayed on a chart recorder. In addition, the feedback induced variations in the high voltage are also recorded and provide continuous monitoring of the amount of hemoglobin in the optical field since vascular changes are the main source of optical density variations in the tissue. The output of a photomultiplier tube responds logarithmically to dynode voltage changes. Thus, changes in the high voltage supplying the photomultiplier after feedback control to constant output are directly related to optical density changes and thus to the hemoglobin concentration. To monitor the hemoglobin (Hb) oxygenation state, a wavelength isosbestic for Hb and HbO2 is chosen as a reference and one with maximal difference is chosen for the measuring or 'sample' beam. For measurement of the redox changes of cytochrome a, the 'sample' beam is set at or near the absorption maximum of the respiratory chain component and a wavelength for reference is selected which undergoes an equal optical density change for Hb-HbO2 shifts as the 'sample' wavelength. Hemoglobin spectra were obtained from washed red blood cells by standard transillumination spectrophotometry; disoxygenation was produced by equilibration with N2. For lack of an existing term, such a reference wavelength is referred to as 'equibestic'. In practice the equibestic wavelength is chosen close to a hemoglobin isobestic point in order to avoid possible effects of large changes which may have different wavelength characteristics within the bandwidth of the two monochromatic beams. This safe-guard often requires selection of a 'sample' wavelength that does not quite coincide with the absorption maximum of the cytochrome under study. Thus, the cytochrome signal may be somewhat attenuated. The feedback signal generated for an equibestic wavelength is generally indicative of the total amount of Hb in the field and provides a satisfactory means of monitoring vascular changes. Nevertheless, we prefer to limit specific statements concerning Hb to measurements utilizing isosbestic points. This technique of dual wavelength reflectance spectrophotometry could be complicated by a number of different factors, mainly: (1) inadequate separation of cytochrome and Hb spectral changes; (2) interference by wavelength dependent scattering properties, especially during increased neuronal activity when small cytochrome changes might possibly be expected; and (3) effects of very large Hb concentration and oxygenation changes. For assessment of the basic point raised in the first question, verification of the correct interpretation of the signals assigned to cytochrome a and to Hb from dual wavelength data was made by the performance of a series of measurements at different wavelengths in the region of the absorption band in question. Such 'action spectra' have validated the dual wavelength techniquO a.

147 Further corroborative measurements with two reference wavelengths were made occasionally (triple wavelength spectrophotometry). The two reference wavelengths, one shorter and one longer than the 'sample' wavelength, were chosen so that the sum of the optical changes during Hb-HbO2 transitions was equal to the change at the sample wavelength. This method eliminates, to a maximal degree possible, wavelength-dependent, scattering artifacts. In no instance was this latter control found to be required to follow the cytochrome a or hemoglobin effects. The third possible interference is negated by measurements of total ischemia and re-establishment of blood flow in cat cerebral cortex 24. In those studies, it was observed that the kinetics of cytochrome a and Hb are different and change independently of each other during and after repetitive ischemic episodes. Finally, it is of some importance to note that contrary to intuition the optical measurements for Hb during oxygenation changes are smaller on an absolute scale than those for cytochrome a (see Figs. 2 and 3). This is probably related to the fact that the lowest oxygen levels of respiratory gases employed still provided better than 90 ~o saturation of Hb. Thus, the present dual wavelength technique is sufficient and adequate to make the measurements required for the objectives of this study. Changes in the redox level of cytochrome a are presented in two ways. Most often, these changes are expressed as a per cent of full scale light levels with zero being no light and 100~ being the 'rest' level signal. 'Rest' is defined as the level found in the unstressed, unmanipulated preparation before any experimental procedures have been undertaken. The cerveau isol6 cat preparations typically exhibit sleep ECoG patterns during this phase while the rabbit showed a pattern progressively more reminiscent of the sleeping state. In a few cases, however, changes are expressed as a per cent of the total labile or 'reducible' cytochrome a signal. The total signal amplitude was determined by monitoring the change from peak oxidation level of the cytochrome produced by inspiration of 95 ~o O~ in 5 ~o COs with the redox level of cytochrome a produced by terminal Nz breathing. This was done only in the cat preparations that did not show trauma effects of repeated manipulations of the inspired gases. Changes in the blood volume were expressed also as a per cent of full scale in most cases although some examples based on the per cent of total blood volume were obtained. Hemoglobin oxygenation changes are expressed as percentages of full scale light levels. RESULTS

When the inspired gas mixture was switched from normal room air to one containing higher levels of 02, an oxidation of cytochrome a was always seen together with an increase in total oxygenated Hb in the field. In contrast, administration of gas mixtures with lower oxygen tensions produced a reduction of cytochrome a and a disoxygenation of hemoglobin in both cats and rabbits. Fig. 1 shows the records of a typical experiment in the rabbit. In Fig. 1A, responses of cytochrome a to the change from room air to 50 ~ oxygen with 5 ~o COs and 45 ~o N~ and from room air

148

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to 8 % O2 in 92 % N2 are shown in the top and bottom traces, respectively. Fig. I B represents the changes in hemoglobin oxygenation with similar gas mixture alterations. These data were taken consecutively and illustrate how peak values were acquired to produced the graphs shown in subsequent figures. All 8 rabbits were monitored several times at different sites under the window and each gave qualitatively similar responses, as did the cats during acute experiments. The optical changes due to hemoglobin oxygenation are smaller than those of cytochrome a and, in addition, the hemoglobin changes exhibit very different kinetics. Apparently two different effects are present: increased 02 delivery at higher respiratory 02 levels and some vasoregulation on plasma Po2 in the absence of Pcoz changes in the inspired air. When 5 % CO2 plus 20 % 02 is administered after respiration on room air, larger Hb changes accompany an increased level of cytochrome a oxidation (see below). The changes in cytochrome a redox state produced by a number of 02 mixtures with and without 5 % CO2 are given in Fig. 2. These data were taken at a single recording site with the total measurement time lasting approximately 2 h. The other rabbits and the cats were qualitatively similar although quantitative differences are present between animals mostly with regard to changes in Po2 close to room air levels. The changes for each rabbit, however, could be duplicated from day to day and measurements at different locations provided practically superimposable curves for each rabbit. It was consistently found that at each 02 level, a mixture of 5 % CO2 increased the level of cytochrome a oxidation but an apparent saturation of cytochrome a occurs at the higher levels of 02 admininistration. Below 8 % 02 with 5 % COz, the rabbits became restless and studies of further decreases in O2 were precluded by their movements. S i ~ c a n t l y , the restless behavior occured earlier and at higher Po2

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levels in the absence of CO2. In that case, 12 ~o 02 was the lowest Po2 at which measurements could be made. Calibration of the total amount of reducible cytochrome a requires a period of complete anoxia. In view of the restlessness of the unparalyzed rabbit and the possible danger to these valuable animals, such absolute calibrations were made only in the acute cat experiments. In these, oxidation of cytochrome a by 100 ~o 09. from the room air level equalled approximately 20% of the total signal between 100 ~o 09. and terminal anoxia. Since the curve of cytochrome a oxidation does not yet show a completion of the effect at 100 % 09.. it can only be stated that cytochrome a is more than 20 ~o reduced under normal respiratory conditions. Preliminary monitoring by dual-wavelength reflection spectrophotometry of signals presumed to be cytochromes b (564-586 nm) and c (550-586 nm) and of N A D H by fluorometry showed the same effects, i.e., an increased level of oxidation of these respiratory chain components with 100 % 09. and increased reduction levels at 09 values below 20 %. Fig. 3 shows changes in the oxygenation state of hemoglobin produced by alterations in the inspired gas mixtures. These results were recorded in the same rabbit as shown in Fig. 2. Again, a saturation effect occurs at higher 09. levels with an increase in oxygenation accompanying the inclusion of 5 % CO9. at each 09. level. Nearly identical curves were obtained in all rabbits each tested at least 3 times on different days and in the cats. Note that the level for disoxygenated H b at 8 ~o 09.

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is not the final equilibrated value for the rabbits but rather represents the point at which the animals became restless. Decreases of end expiratory CO2 were produced by increasing the ventilation rate in paralyzed cats. The effects of this procedure on cortical cytochrome a, blood volume and hemoglobin are displayed in Figs. 4 and 5. Fig. 4 illustrates that decreased expired CO2 is accompanied by an increased reduction level of cytochrome a together with a decrease in blood volume. Re-establishment of 'rest' CO2 levels corresponds with a return of the cytochrome a signal and blood volume to baseline. The onset of cytochrome a recovery, however, lags slightly the onset of the return 5 9 0 nm

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Fig. 4. The effect of hyperventilation on cytochrome a and blood volume. Decreased end-expired CO2 produced in this manner consistently inere,ased cytoehrome a reduction levels and decreased the volume of blood within the optical field.

151

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of COg and blood volume. Similarly in Fig. 5, decreased COg is accompanied by the blood volume decrease and a disoxygenation of the hemoglobin in the optical field• When ventilation rate is decreased, expired COg levels returned to baseline. The increased end expired COg is accompanied by an increase in blood volume which is later followed by an increase in the oxygenation state of hemoglobin. The hemoglobin disoxygenation with hyperventilation, however, was considerably smaller than that produced by anoxia caused by stopping ventilation. In the latter case, a 1-min period without respiration produced a decrease in the hemoglobin oxygenation state approximately 2.5 times that produced by hyperventilation. DISCUSSION

Chance and Williams 5 demonstrated that the velocity of oxygen consumption in mitochondria is independent of oxygen concentration when the supply of oxygen is in excess. They found that only when the concentration of oxygen becomes limiting does cytochrome a become significantly reduced and does ATP synthesis become diminished. The question is, 'at what values is oxygen limiting in the cerebral cortex', i.e. what is the critical Po27 The concept of 'critical Po2', although presently ill-defined, has been used frequently as a designation of that tissue Pog level below which oxygen consumption declines, implying insufficiency of oxidative energy provision. Since such a limit suffers immediately from the difficulty of establishing a first decrease, a decrease of a given fraction is used as a more fruitful goal. From a biochemical point of view, a 50 % inhibition of activity, related to the Km of enzyme reactions, has been proposed as a better endpointa,12, 22. However, this parameter does not carry sufficient information concerning the functional effects of the lack of oxygen. The present data indicate that a sharp boundary does not exist but that a continuum between Og levels, cytochrome oxidation state and high energy provision (and Og utilization) and perhaps functional activity produces a highly complex, mutually dependent system• Further investigations aimed especially at clarifying the functional parameters in this complex are presently underway 17. On the basis of early determination of the oxygen affinity of reduced cyto-

152 chrome a3, J6bsis 1l calculated an upper limit value of 5 mm Po2 as critical (detined as a 10°/~oloss in oxygen uptake) for ascites tumor cells, liver cells and isolated liver mitochondria. Chance et al. 3 have recently determined critical Po2 levels considerably below l mm Hg. In order to relate this seemingly low mitochondrial oxygen requirement to intact, functioning cerebral cortex, however, we must take into account the ventilation and perfusion factors which determine the delivery of oxygen to the cells. Grote e t al. 7 found in dogs that when arterial Po2 was lowered to values below 60 mm Hg and venous Po2 below 34 mm Hg critical conditions for the brain oxygen supply were produced. Chance et al. 2 fluorometrically recorded increased N A D H reduction levels in rat brain when the animal was switched from room air to 100 °/ .J,, N2 or to 3 % 02 in N2. Also, Chance e t al. 4 found that decreasing inspired 02 to 40 mm Hg produced a NAD reduction of only 35 % of that reduced by anoxia at which pressure less than 10% of the hemoglobin remained oxygenated. In the present case we have observed a continuous adjustment of cytochrome a reduction to much smaller changes in Po2. Davies and Bronk 6 calculated that the cortex should function normally until a limiting level of less than 5 mm Hg Po2 is reached. In another experiment, however, they found normoxic Po2 values varying from 2 mm to about 10 mm and concluded that the cortex is normally near a state of oxygen insufficiency. Their data were derived from surface electrodes and therefore are relevant to the optical surface measurements of the present experiments. However, the optical data are probably derived from an estimated 1 mm depth and would therefore contain information from deeper layers than the 02 surface electrode studies. Our data suggest that the blood supply to the cerebral cortex is regulated at a level of slight hypoxia. Because of the very gradual decrease in rate of oxidative metabolism with increases in respiratory chain reduction levels, the so-called 'cushion effect', these small changes would be difficult to identify with A-V difference techniques. In the Chance and Williams scheme for isolated mitochondria in vitro 5, cytochromes a and aa are practically completely oxidized under both resting (state 4) and actively phosphorylating (state 3) conditions because of high affinity of the reduced forms of these cytochromes for oxygen. Recent work on the redox response of cytochrome a ÷ a3 of isolated mitochondria to changes in 02 concentrations supports this basic concept 22. This scheme is open to question, however, in certain intact tissues that seem to have in common a high proportion of active transport activity. Ramirez 23 showed that cytochrome aa in heart muscle responds with a transinet oxidation to a contraction. More recently, Muraoka and Slater 21 noted a larger than expected reduction level of cytochrome aa and also found that the transition from resting to active metabolism (ADP addition) produced transient oxidations. Hersey and J6bsis 1° reported that all cytochromes including a and a3 are reduced when the intact gastric mucosa is stimulated to secrete acid by histamine and this was shown to be under conditions of adequate oxygen supply 9. Similarly, midgut preparations from silkworms exhibit high reduction levels of the respiratory chain components which vary with the rate of potassium transporP 9,2°. In addition,

153 in intact cat cerebral cortex, J6bsis et ai. 14 reported with reflection spectrophotometry that cytochromes b, c and a are transiently oxidized during evoked potential activity and in spreading cortical depression. These investigators estimated from terminal anoxic transients and maximum oxidation levels produced by spreading depression that cytochrome a is least 20 % reduced under 'resting' conditions. Data presented here confirm that cytochrome a is, at rest, not nearly at the low reduction levels seen in isolated mitochondria. Increasing inspired oxygen levels produces an oxidation of cytochrome a while the cytochrome has a sharp reductive response to any lowering of inspired oxygen. Under normal respiratory condition, this cytochrome is at least 20% reduced instead of the 1-4% observed in isolated mitochondria. It may be that in the intact cerebral cortex, cytochrome a needs a much higher concentration of oxygen to b e highly oxidized than has been generally accepted. Kinetic analysis of the turnover of the respiratory chain shows that the higher reduction levels observed must necessarily be accompanied by a (slight) decrease in 02 uptake rates 11. If this is true, a critical evaluation and possible redefinition of the critical Po2 may be in order. If not, important differences exist between respiratory chain function in the intact cortex as compared to isolated mitochondria. ACKNOWLEDGEMENTS We wish to thank Mr. Ronald Overaker for his assistance in the design and development of the optical instrumentation used in this study, and Mr. David Martel for assistance in making many of the measurements presented. This work was supported by PHS Grants NS-10384, NS-06233 and MH-12333 (to M.R., J.C.L. and F.F.J.) and by SCOR Grant HL-14251 from the N H L I and by a contact from the U.S. Army Research and Development Command DAMD1774-C4021 (to J.E.L., H.A.K. and J.L.P.). Dr. Patterson is a recipient of a Research Career Award from the National Heart and Lung Institute. Dr. Kontos is occupant of the Virginia Heart Association Chair of Cardiovascular Research.

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154 7 GROTE, J., KREUSCHER,H., SCHUBERT,R., AND RUSS, H. J., In 6th Europ. Cbng. Microcirculath~, Karger, Basel, 1970, pp. 294-297. 8 GURDJIAN, E., WEBSTER, J., AND STONE, W., Cerebral constituents in relation to blood gases, Amer. J, Physiol., 156 (1940) 149-157. 9 HERSEY,S. J., AND HIGH, W. L., Effect of unstirred layers on oxygenation of frog gastric mucosa, Amer. J. Physiol., 223 (1972) 903-909. 10 HERSEY,S. J., AND JOBSIS, F. F., Redox changes in the respiratory chain related to acid secretion by the intact gastric mucosa, Biochem. biophys. Res. Commun., 36 (1969) 243-250. 11 J6BSIS, F. F., Basic processes in cellular respiration. In Handbook of Physiology, Respiration 1, Amer. Physiol. Soc., Washington D.C., 1964, pp. 63-124. 12 JSBsls, F. F., Oxidative metabolism at low Pos, Fed. Proc., 31 (1972) 1404-1413. 13 J6BSls, F. F., O'CONNOR, M. J., VITALE, k., AND VREMAN,O., Intracellular redox changes in functioning cerebral cortex. I. Metabolic effects of epileptiform activity, J. Neurophysiol., 34 (1971) 735-749. 14 J6BSlS, F. F., ROSENTHAL,M., LAMANNA, J., LOTHMAN, E., CORDINGLEY,G., AND SOMJEN, G., Metabolic activity in epileptic seizures. In D. INGVARAND N. LASSEN(Eds.), Benson Symposium on the Working Brain, Munksgaard, Copenhagen, 1975, pp. 185-196. 15 KETY, S. S., Blood flow and metabolism of the human brain in health and desease. In K. A. C. ELLIOTT, I. H. PAGE AND J. H. QUASTEL(Eds.), Neurochemistry, Thomas, Springfield, Ill., 1962, pp. 113-127. 16 KETY, S. S., AND SCHMIDT,C. F., The nitrous oxide method for the flow in man: theory, procedure and normal values, Jr. clin. Invest., 27 (1949) 476-483. 17 LAMANNA, J. C., WATKINS, G., AND ROSENTHAL, M., Relationship of inspired oxygen, redox level of cytochrome a and ECoG cats, Physiologist, 18 (1975) 284. 18 LEVASSEUR,J. E., WEI, E. P., RAPER, A. J., KONTOS, H. A., AND PATTERSON,J. L., Detailed description of a cranial window technique for acute and chronic experiments, Stroke, 6 (1975) 308-317. 19 MANDEL, L. J., MOFFETT, n., AND JOBSIS, F'. F., Redox state of respiratory chain enzymes and potassium transport in silkwork midgut, Fed. Proc., 33 (1974) 1258. 20 MANDEL,L. J., MOFFETT, n. F., AND JOBSIS,F. F., Redox state of respiratory chain enzymes and potassium transport in silkworm midgut, Biochem. biophys. Acta (Amst.), 408 (1975) 123-134. 21 MURAOKA,S., AND SLATER,E. C., The redox state of respiratory chain components in rat liver mitochondria. II. The 'crossover' on the transition from state 3 to state 4, Biochim. biophys. Acta (Amst.), 180 (1969) 227-236. 22 OSHINO, N., SUGANO, T., OSHINO, R., AND CHANCE, B., Mitochondrial function under hypoxic conditions: The steady states of cytochrome a + aa and their relation to mitochondrial energy states, Biochim. biophys. Acta (Amst.), 368 (1974) 298-310. 23 RAMIREZ, J., Oxidation-reduction changes of cytochromes following stimulation of amphibian cardiac muscle, J. Physiol. (Lond.), 147 (1959) 14-32. 24 ROSENTHAL, i . , MAP,TEL, D., LAMANNA, J. C., AND J6BS[S, F. F., In situ studies of oxidative energy metabolism during transient cortical ischemia in cats, Exp. NeuroL, O0 (1975) 000-000. 25 SCHMIDT,C. F., Cerebral blood supply and cerebral oxidative metabolism. In F. DICKENS AND E. NElL (Eds.), Oxygen in the Animal Organism, Pergamon Press, New York, 1964, pp. 433-442.