Impedance characteristics of cortical and subcortical structures: Evaluation of regional specificity in hypercapnea and hypothermia

EXPERIMENTAL

NEUROLOGY

11,

190-216

impedance Characteristics Structures: Evaluation in Hypercapnea W. R.

ADEY,

Departments of Anatomy of California, Los Long

R. T.

(1965)

of Cortical of Regional and KADO,

and Subcortical Specificity

Hypothermia AND

D. 0.

WALTER]

and Physiology, and Brain Research Angeles, and Veterans Administration Beach and Los Angeles, California Received

August

17,

Institute, University Hospitals,

1964

Measurements of electrical impedance have been made in focal volumes of approximately 1.0 mma of allocortical and subcortical tissue in the cat, with microvolt signals at 1000 cycle/set. The technique allows simultaneous recording of the relative magnitude of resistive and reactive components. Measurements were made with chronically implanted electrodes, and also in acute preparations, either immobilized with gallamine triethiodide or with an upper cervical spinal transection. Qualitative differences were noted in amygdaloid impedance changes during behavioral responses characterized by similar cortical EEG records, as in paradoxical sleep and behavioral arousal. Brief alerting stimuli reduced resistance and increased capacitance in the amygdala, hippocampus and midbrain reticular formation. These responses lasted 6G120 set, and differed in relative size of resistive and reactive shifts in the various structures. Inhalation of 7% carbon dioxide in air produced only resistive shifts in the amygda!a, but caused substantial shifts in both resistance and reactance in the hippocampus and midbrain reticular formation, amounting to 2-40/o of baseline values. However, equally large impedance responses to physiological stimuli occurred with just detectable increases in endogenous carbon dioxide production. The effects of hypothermia in the range 28-21 C on cerebral impedance, carbon dioxide excretion and blood pressure were studied in shivering and immobilized preparations. In shivering animals, resistance and reactance showed only minor perturbations in hippocampus and midbrain reticular formation until the core temperature fell to approximately 25 C. Both resistance and reactance rose sharply in the range 25-21 C, and lagged in return to baseline during temperature recovery. In immobilized animals, impedance shifts followed the general contour r This study was assisted by Grant MH-03708 from the National Institutes of Health, and by Grant AF-AFOSR 246-63 from the U. S. Air Force Office of Scientific Research. Histological preparations were made by Miss Cora Rucker and Miss Arlene Koithan. Figures and data were prepared for publication by Miss Hiroho Kowto. 190

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of falling temperature, and closely paralleled the carbon dioxide excretion, both in the phase of falling temperature, and in the recovery phase when carbon dioxide excretion continued to drop below control levels. The characteristic excursions of resistive and capacitive impedance induced by hypothermia had no primary relationship to systemic blood pressure, which remained unaltered during their development, and which could also vary substantially without modifying the course or direction of impedance shifts. Temporary cardiac arrest in hypothermia was associated with impedance shifts an order of magnitude larger than those in physiological manipulations. The evidence suggests that impedance responses reflect changes in intrinsic characteristics of cerebral tissue, rather than relating in a direct fashion to cerebral blood flow or blood pressure. In a system with intraneuronal, intraneuroglial and extracellular compartments, carbon dioxide may exert a regulatory function on the selective exchange of sodium and chloride ions between neuronal elements and vascular capillaries, perhaps within the neuroglial compartment. The relative contributions of extracellular and neuroglial compartments to the observed conductance, and their possible relationship to substantial differences between impedance phenomena in physiological responses and in terminal asphyxia, are discussed. Introduction

Measurements of cerebral impedance with microvolt signals at 1000 cycle/set have indicated the feasibility of detecting impedance changes in restricted volumes of cerebral tissue relating to states of alertness, sleep, anesthesia, and epileptic discharges, and in relation to the acquisition of a learned habit (1, 2). Although these measurements have revealed empirically both brief and enduring modifications in the conductance characteristics of cerebral tissue, the nature of these changes, and their exact location within the complexly interrelated tissue compartments has remained obscure. At this stage, therefore, identification of the qualitative nature of the underlying mechanisms is of considerable importance. It is unlikely that, in the gamut of shifting cerebral functions from altered attention to terminal asphyxia and death, a single simple mechanism, such as ionic redistribution between tissue compartments, would necessarily explain all observed changes. Establishment of the essential independence of the more rapid physiologically induced impedance transients from such factors as blood pressure and blood flow, for example, would not necessarily preclude these factors as partially or indirectly causal in certain long term impedance changes. From the point of view of a tricompartmental model having intraneuronal, intraneuroglial and extracellular divisions, the low membrane resistance of neuroglia cells (12) may offer a preferential pathway for

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the applied measuring currents. On the other hand, recent models of the impedance characteristics of cerebral tissue (19, 20) have suggested that the neuroglial tissue cannot be equated unreservedly with the extracellular space in its contribution to the observed impedance. In particular, the nature of the reactive component remains obscure, particularly if its frequency-dependent characteristics are viewed as relating to the time constants of either neuronal or neuroglial cellular membranes, or to a combination of both. In this study, we have utilized a modification of our initial technique which allows simultaneous examination of a quadrature, or “reactive” component of the impedance, as well as the “resistive” measure on which our previous studies were based. We have examined the characteristics of the rapid spontaneous fluctuations in impedance baseline revealed by this method in such regions as the midbrain reticular formation, amygdala and hippocampus. We have found regional differences in these apparently spontaneous impedance signals, but they are not discussed in detail here. Careful evaluation has revealed that they may arise in part from exceedingly small (but partially coherent) amounts of energy at the impedance signal frequency of 1000 cycle/set, and may originate in electrophysiological tissue generators, particularly in the midbrain and pontine reticular formations, and geniculate bodies. On the other hand, this impedance measuring technique has reliably revealed with great sensitivity the amount and direction of slower changes induced in the resistive and reactive components by such manipulations as transient alterations in blood carbon dioxide levels and induction of hypothermia. Particular attention has been directed to possible relationships between these impedance changes and systemic blood pressure. Methods In a total of twenty-three cats, three different experimental preparations have been used. Five of these animals were preparations with electrodes chronically implanted bilaterally in the dorsal hippocampus, the amygdala and the rostra1 midbrain reticular formation. Thirteen other animals were prepared surgically under ether anesthesia, with identical electrode placements, and subsequently immobilized with Flaxedil (gallamine triethiodide) . Local anesthesia with procaine hydrochloride was maintained in all pressure points around the face and in tracheotomy and cannulation incisions. A third series of five cats were also prepared acutely under ether anesthesia, and a cervical transection performed at Cl. These

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animals were also maintained with artificial respiration and local anesthesia. Histological examination was made of all electrode placements. Systemic blood pressure was recorded by cannulation of the femoral artery. In one chronic preparation, an electromagnetic flowmeter probe designed to fit the common carotid artery (diameter 1.5 mm) was implanted. Body temperature was recorded with a rectal thermistor electrode, and in the acute preparations a thermistor sensor was also placed in contact with the unopened dura mater and sealed in place with bone wax. Continuous monitoring of alveolar carbon dioxide levels was performed with a Godart Capnograph, which permitted analysis of carbon dioxide levels in each breath. Hypothermia was induced either in a chamber containing cannisters of dry ice, or by rapid evaporation of acetone from the skin surface. All physiological parameters were recorded on a Grass sixchannel polygraph. Impedance measurements involved an extension of techniques described elsewhere (1, 2). Stereotaxic placement of stainless steel coaxial electrodes permitted examination of impedance characteristics in focal volumes of cerebral tissue, estimated at less than 1.0 mm3 in previous studies. All measurements were made at 1000 cycle/set, with a sinusoidal signal of approximately 20 uV RMS applied to the coaxial electrode. The tissue path formed one leg of a Wheatstone bridge. Bridge balance was secured through adjustable resistive and capacitive elements arranged in parallel in an adjoining leg. Unbalance signals were amplified with a low noise differential amplifier, having 1.0 kilocycle/set input filters with a _t 7.5 cycle/set bandpass, and with a decremental response of 6 db per octave around the center frequency. The residual signal was examined with a pulse sampling technique, using two O.l-msec square wave gates having a fixed phase relation to the sine wave bridge signal. The pulse positions were set initially by introducing in separate operations a resistive and reactive unbalance at either the bridge or the animal. The resistive gating pulse was positioned so that the integrated output of the gate remained unaltered when a reactive unbalance was introduced. Conversely, in positioning the reactive gating pulse, after rebalancing for a complex impedance null, resistive unbalance was introduced, and the position of the reactive pulse adjusted until no change in output of the reactive gate occurred. The gating pulses were then 90’ apart, and had appropriate phase relations to the bridge excitation signal for mutually exclusive recording of the resistive and reactive components, and elimination of reactive effects of associated circuitry. This square wave sampling system

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thus produced two trains of amplitude modulated pulses, which were subsequently integrated to produce a graphic output with an adjustable bandwidth from dc to 30 cycle/set. Both were simultaneously recorded from a single electrode, and displayed with other physiological data. Sensitivity could be adjusted by increasing the integration time of the output of the gating system, thus effectively narrowing the bandwidth of the readout. This was acceptable for baselinereadings, and allowed differential sensitivity substantially in excessof 0.1 per cent. Results

GENERALFEATURESOF

IMPEDANCE RESPONSES TOPHYSIOLOGICAL

STIMULI

Our previous observations with a system monitoring only the ‘kesistive” component had revealed rapid transient impedance changes to a variety of environmental stimuli ( 1) . These findings have been confirmed in the present study, and related to simultaneouseffects in “capacitive” readouts (Fig. 1). Repeated loud noisesproduced transient falls of about 250 ohms RESPONSES

T O VARIOUS

STIMULI

IN THE

H,PPOCAMP”S

FIG. 1. Simultaneous records of hippocampal resistive impedance (R. D. HIPP. RES.), expressed in kilohms, and reactive impedance (R. D. HIPP. CAP.), expressed in kilopicofarads. These tracings show effects of auditory, visual and somatic stimuli. A small transient increase in expired CO, accompanies each stimulus.

in dorsal hippocampal resistive impedance, of the order of 2.0 per cent of the baseline value. Simultaneous capacitive measurementsindicated transient increasesof 200 picofarad; or about JO per cent of the baseline value. This aspect of an inverse relationship between the resistive and capacitive componentshas been found to typify hippocampal responsesto

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a variety of stimuli, but is by no meansa universal phenomenon in cerebral tissue, as will be discussedbelow. Figure 1 also shows the effects of brief painful squeezesto the paw. They were of substantially greater amplitude than those to noise, but responsesto both the auditory and painful stimuli showed adaptation in repeated presentations. Carbon dioxide levels monitored in the expired air showeda small increasefollowing each stimulus, and these also adapted to sequential presentations of both noxious and auditory stimuli. INTERRELATIONS OF RESISTIVE AND CAPACITIVE COMPONENTS IN AMYGDALOID RECORDS IN CHANGING STATES OF CONSCIOUSNESS

Changing states of consciousnessfrom wakefulness to drowsinessand sleep have been shown to produce slow baseline shifts in the resistive impedance component (1). We have related these slow shifts in amygdaloid impedance records to characteristic features of the EEG in states of sleep and arousal (Fig. 2), with evidence that the impedance records provide a clear differential monitor of differing behavioral states in which the EEG may exhibit basically similar amplitude and frequency patterns. During sleep (Fig. 2A), epochs of high amplitude rhythmic spindle activity were recorded in visual cortex (L.V.C.) and septum (L. SEPTUM), and in lesssustained amounts in hippocampal leads (L.M.H. and R.M.H.). These epochs were irregularly interrupted by low amplitude records, lasting up to several minutes, and characterized by behavioral aspectsof ‘Lparadoxical” sleep, including eye movements. Resistive amygdaloid impedancemeasurements[L. AMYG. (R) ] showeda slow rise and return to baseline coincident with the paradoxical episodes,whereas the capacitive lead [L. AMYG. (C) ] fell slightly over the same time course as the resistive shift. Brief behavioral arousal from sleep (Fig. 2B) also produced a low amplitude EEG record for almost a minute with gradual recrudescenceof high amplitude spindles in visual cortical (L.V.C.) and septal (L. SEPTUM) leads as the animal returned to sleep. The amygdaloid impedance records were strikingly different from those in paradoxical sleep, however, and exhibited the general features of arousal phenomena described above in the hippocampus. There was a prolonged slow fall in the resistive component [L. AMYG (R) ] over a period of about 5 min, with gradual return to baseline level over the same period. There was a slight simultaneousincreasein the capacitive component, which maximized at the sametime as the resistive shift.

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196 REGIONAL

DIFFERENCES

IN IMPEDANCE

RESPONSES TO HYPERCAPNEA

Detection of both qualitative and quantitative differences in impedance changes evoked in different cortical and subcortical regions by identical peripheral stimuli, or by a singleglobal shift in the cerebral milieu interne, would assistmaterially in assigningsignificance to a local functional frame in the production of the evoked changes. Hypercapnea induced by inIMPEDANCE

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-

100 SEC

8. AROUSAL

FIG. 2. Resistive (L. AMYG. (R)) and reactive (L. AMYG. (C)j amygdaloid impedance changes in sleep (A) and behavioral arousal (B). Recurrent episodes of paradoxical sleep, characterized by low amplitude EEG records, showed a rise in resistive impedance and a slight decrease in capacitance. Arousal records, however, showed reduced resistance and slightly increased capacitance. Abbreviations: L.M.H. and R.M.H., left and right hippocampus; L.V.C., left visual cortex.

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halation of 7 per cent carbon dioxide in air has clearly disclosed evidence of regional differences in responsiveness to cerebral hypercapnea. Amygdaloid Impedance Responses to Inhalation of Carbon Dioxide. Equilibration of the expired air with the increased carbon dioxide level (7%) in the stimulating gas mixture occurred after 8-10 breaths over a period of 30 set (Fig. 3). The equilibrium condition was maintained for 2-3 min, and, after removal of the test gas source from the respirator system, baseline levels were restored after 7-10 min. The amygdaloid resistive impedance component (AMYG. RES.) fell progressively throughout the exposure to raised carbon dioxide (Fig. 3A), A 129

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FIG. 3. Effects of inhalation of 7 per cent CO, in air on amygdaloid impedance in two subjects. In each case, amygdaloid resistance (AMYG. RES.) fell, but there was little effect in the reactive component (AMYG. CAP.). Other abbreviations: L. MB. RF., left midbrain reticular formation; R. D. HIPP., right dorsal hippocampus.

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declining almost 3 per cent of the baseline value shortly after completion of an exposure of 3 min, and returning to baseline levels after a further 3 min. By contrast, the capacitive impedance lead (AMYG. CAP.) showed a minimal decrease, with a similar time course to the resistive lead. EEG records from the rostra1 midbrain reticular formation (L. MB. RF.) showed an increased amplitude in the first half of the exposure, with a progressive decline thereafter. Dorsal hippocampal EEG records (R. D. HIPP.) decreased sharply in amplitude in the initial phase of hypercapnea, and remained below the amplitude of control records for the duration of administration of CO- and for more than 10 min thereafter. A no7

CEREBRAL IMPEDANCE IN THE RETICULAR L. MB R F RES

RESPONSES FORMATION

TO

HYPERCAPNEA 4/29/w

FIG. 4. Changes in resistive and reactive impedance (L. MB. R. F. RES. and L MB. R. F. CAP.) in the rostra1 midbrain reticular formation during inha!ation of 7 per cent CO, in air in two acute immobilized preparations. Resistance fell and capacitance increased in both cases. Other abbreviations: R. AMYG., right amygdala; R. D. HIPP., right dorsal hippocampus.

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These findings in an immobilized animal were repeated in another preparation (Fig. 3B) with essentially identical reults. The substantial decrease in the amygdaloid resistive lead was without a comparable shift in the capacitive lead, which showed only a slight decrease that maximized 3 min after completion of the CO2 exposure, with return to baseline levels 6 min later. Effects of Hypercapnea on Midbrain Reticular Impedance. In striking contrast to the effects of raised systemic carbon dioxide levels in the amygdala, both resistive (L. MB. RF. RES.) and capacitive (L. MB. RF. CAP.) midbrain impedance readings were substantially modified (Fig. 4A). Capacitance was increased over a time course which essentially mir-

FIG. 5. Hypercapnea induced by inhalation of 7 per cent CO, in air, showing effects on dorsal hippocampal impedance (R. D. HIPP. RES. and R. D. HIPP. CAP.). This stimulus produced a substantially greater change in both resistance and reactance in this animal than an identical stimulus to the midbrain reticular formation (Fig. 4) or amygdala (Fig. 3). Other abbreviations as in Fig. 4.

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rored the course of the simultaneous decrease in the resistive component, Return to baseline impedance levels occurred after approximately 4 min. These records from the same acute immobilized preparation as in Fig. 3A also showed decreased amplitude of the hippocampal EEG (R. D. HIPP.) , but no major modification of the amygdaloid EEG (R. AMYG.). Similar tests in another acute immobilized preparation (Fig. 4B), from which the amygdaloid records of Fig. 3B were obtained, showed larger but qualitatively similar shifts in both resistive and capacitive midbrain

FIG. 6. Repeated exposure to brief episodes of hypercapnea, with impedance changes at successively deeper levels in the hippocampus (see text). LittIe change was discerned with the electrode tip located near the alvear surface (A). Records B, C, D and E were obtained in successive steps 300, 200, 100 and 2OOt4 respectively below the previous CO, challenge. Maximal changes occurred in the dendritic layer of the pyramidal cells.

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impedance parameters, but the return to baseline levels was much slower than in the preparation of Fig. 4A. Effects of Hypercupnea on Hippocampal Impedance. Susceptibility of hippocampal tissue to raised carbon dioxide in acute experiments varied substantially in different experiments and appeared to depend in considerable degree on the length of preliminary ether anesthesia and any associated hyperventilation, and on minor variations in electrode placements in relation to the laminar configuration of the hippocampal pyramidal cells. Figure 5 shows the effects of 2.5 min exposure to 7 per cent carbon dioxide in a sensitive preparation. As in the reticular formation, the resistive impedance parameter fell sharply at the same time as the capacitive parameter increased. Peak values of the shifts were beyond available pen excursions, but in both parameters showed higher percentage shifts from baseline impedance than any encountered in rostra1 midbrain reticular leads. On the basis of available data, this differential susceptibility to hypercapnea of both resistive and capacitive measures in hippocampal tissue appeared characteristic of this tissue. Preliminary exploration of certain caudal brainstem areas has indicated a similar susceptibility and is under further investigation. Laminar Exploration of Hippocampal Responses to Recurrent Episodes of Hypercapnea. Further evidence of qualitative focal differences in responseto brief epochsof raised carbon dioxide tension was sought with a coaxial electrode advanced vertically through the dorsal hippocampus. At each level, inhalation of 7 per cent carbon dioxide in air for 40 set achieved an equilibrium with alveolar air (Fig. 6). The initial hypercapnic episode (A), with the recording electrode located in alvear tissue above the level of the pyramidal cell bodies, was not followed by a definite impedance change. At a site 300 p deeper, with the coaxial dipole now spanning a substantial part of the pyramidal dendritic field, resistive impedance increased and the capacitance fell (B). At a point 100 p deeper than B, a further exposure elicited an even greater drop in capacitance and a similar increase in resistance (C) . These responsesdisappeared abruptly 100 u below C at D, and further exploration at three further levels 200 p a part failed to produce responsesto hypercapnea. The regional responsivenessthus defined is in anatomical agreement with the location of the bodies and dendritic layers of the pyramidal cells. However, it should be emphasized that this observation can be interpreted only as indicative of a qualitative difference in impedance shifts in closely adjacent regions, since the incremental advance of the electrode through

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the tissues was in each instance associated with a rapid baseline drift, presumably due to cellular disruption or some disturbance at the interface between the tissue and the electrode. This declined exponentially to a stable baseline over a period of 30-60 min. Serial examination as performed in Fig. 6 required a much more rapid advancement of the electrode, and it will be noted that the direction of resistive and capacitive shifts prior to baseline stabilization were in the opposite sense to those seen in the hippocampus after stabilization (Fig. 5). Similar reversals in direction of impedance changes induced by hyperventilation have been seen in the implanted human hippocampus, reported elsewhere (18). EFFECTS

OF

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EXCRETION

ON

AND

BLOOD

IMMOBILIZED

CEREBRAL PRESSURE

IMPEDANCE, IN

SHIVERING

CARBON

DIOXIDE

AND

PREPARATIONS

The effects of hypothermia on reticular and hippocampal impedance was tested in animals with chronically implanted electrodes and in acute preparations with surgery performed under ether anesthesia and subsequently immobilized either with Flaxedil or by transection of the spinal cord at Cl. It was hoped in this way to determine whether impedance shifts with falling temperature have a simple relationship to the temperature gradient, or might have a nonlinear relationship, arising perhaps from local metabolic factors, or from the complex interplay of such parameters as blood pressure and blood flow, carbon dioxide tension, or the effects of circulating metabolites of extracerebral origin. Impedance Efiects in Midbrain Reticular Formation During Hypothermia. In chronic animals allowed to shiver freely during the induction of hypothermia, cortical and subcortical impedance leads showed polyphasic changes. Resistive and capacitive measurements (Fig. 7) in general, shifted oppositely, but were not strictly mirror images. In the rostra1 midbrain reticular formation (Fig. 7), the resistive impedance showed minor perturbations until the core temperature dropped to 25 C. The resistance then rose sharply from 16.4 to 17.1 kilohms, as the temperature continued to drop to a minimum of 21.3 C. Recovery of body temperature followed an essentially linear course over a period of 2 hours, but was accompanied by a sharp fall in resistance from 17.1 to 15.7 kilohms in the first half hour of the recovery cycle, Resistance remained at this level until the experiment was terminated. Capacitance contours showed a substantial early rise as temperatures fell from 38 to 30 C. In the range from 2 to 25 C, both resistance and capacitance remained constant. As

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the temperature continued to fall to 21 C, the capacitance fell sharply, mirroring the sudden simultaneous rise in resistance, and exhibiting a similar rebound and overshoot in the temperature recovery cycle. The capacitance also exhibited a hysteresis, persisting at a level substantially above the value of the precooling baseline. Hypothermic Changes in Hippocampd Tissue. In chronic preparations, as hippocampal impedance followed the same general configuration hypothermic records in the reticular formation. In a typical example IMPEDANCE RETICULAR

CHANGES FORMATION

IN THE IN HYPOTHERMIA

during hypothermic FIG. 7. Typical configuration of midbrain impedance shifts episode in a cat with chronically implanted electrodes, with sudden appearance ot rapid resistive and capacitive changes at about 25 C, and an overshoot in both resistance and reactance during temperature recovery.

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(Fig. 8), an initial small resistive rise as the temperature fell from 38 to 28 C was followed by a much larger rise from 10.1 to 10.9 kilohms as the temperature fell to 21 C. Recovery of temperature was accompanied by a sharp initial fall with a small overshoot, and perturbations around the precooling values during the remainder of the experiment. Modifications in capacitance were proportionately larger than in the reticular formation. A fall in temperature from 37.8 C to 26 C raised the capacitance from 8.3 to 9.5 kilopicofarads. In the range from 25 to 21 C, capaciHIPPOCAMPAL IMPEDANCE CHANGES IN HYPOTHERMIA IN CHRONIC PREPARATION

FIG. 8. Effects of resistive and reactive maximum changes in about 27 C. Overshoot than in the midbrain.

hypothermia in chronically implanted cat on hippocampal components of impedance. As in the midbrain (Fig. 7), the both factors occurred rapidly after body temperature fell to in impedance values during temperature recovery was larger

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tance fell to 8.9 kilopicofarads, but was followed by a large and sustained rise to 10.1 kilopicofarads, remaining at this level for about 2 hours during the recovery cycle, and falling precipitously towards the original value as the body temperature rose above 34 C. Eflects of Cardiac Awest During Hypothermia on Hippocampal Impedance. Much attention has been directed by van Harreveld and his col-

leagues (10, 11) to the effects of circulatory arrest on cerebral impedance. In the present study, cardiac arrest occurred following a period of arhythmia with body temperatures below 21 C. It was thus possible to compare HIPPOCAMPAL WITH CARDIAC

FIG. 9. A brief episode contrast with the relatively phase, resistance increased the cardiac arrest.

IMPEDANCE ARREST

IN

HYPOTHERMIA

of cardiac arrest during hypothermia below 22 C. By small impedance responses in the hypothermic induction and capacitance fell by an order of magnitude during

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impedance changes in this period of extreme disruption of the milieu interne with preceding effects in essentially normal physiological responses (Fig. 9). In a chronic preparation, hippocampal resistance rose from 37.0 to 39.0 kilohms during the period of initial temperature drop from 38 to 23 C. At the onset of a period of cardiac arrest, beginning at a body temperature of 21 C and lasting approximately 2 min, resistance rose sharply to 65 kilohms. Reestablishment of normal cardiac action reversed this change over a period of 10 min. Capacitance readings showed a slow and relatively small decline in the greater part of the initial temperature drop from 35 to 21 C. By contrast, cardiac arrest resulted in an immediate fall from 3.9 to 1.8 kilopicofarads, with resumption of the prearrest value within 15 min. It may be emphasized that these dramatic effects of cardiac arrest are almost an order of magnitude larger than the impedance changes arising from physiological manipulations, such as peripheral sensory stimuli, or HIPPOCAMPAL

IMPEDANCE

IN

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IN

ACUTE

PREPARATION

0 37 z 35 2 2 33 t:

31

t- 29 27

4:oo

4:30

5:oo

5:30

6:OO

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7:oo

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FIG. 10. Relationship of hippocampal impedance excretion in immobilized animal. It will be noted run a closely parallel course.

plot that

during hypothermia CO, and impedance

to CO, factors

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accompanying a learned performance or from inhalation of carbon dioxideenriched mixtures, as described above. It is also obvious that, despite their magnitude, they were reversible when the cardiac arrest was brief, and the metabolic levels substantially reduced by hypothermia. In many other instances where the cardiac arrest proceeded to a fatal termination in the absence of hypothermia, these changes were sustained, with a decline of the capacitance after 10-15 min to approximately one-third of its value prior to cardiac arrest. In the later cases it was regularly observed that a latent interval of 90-150 set preceded the appearance of the large and rapidly developing impedance shift. In this interval a small fall in resistance and rise in capacitance was usually noted. Relationship Between Hippocampal Impedance Changes and Expired Carbon Dioxide Levels During Hypothermia. In an animal immobilized with gallamine triethiodide, lowering the rectal temperature from 36.3 to 27.5 C resulted in a fall in capacitance that was closely paralleled by the fall in expired carbon dioxide (Fig. 10). An encdphale isole’ preparation subjected to a more profound thermal drop in rectal temperature from 36.6 to 24.6 C exhibited a similar relationship to exhaled carbon dioxide levels, both in the period of induction of hypothermia, and in the recovery phase when carbon dioxide excretion continued to drop substantially below control levels (Fig. 11). Cortical surface temperature was also plotted in this animal, and fell only to 26.2 C, and began to climb again 20 min earlier than the rectal temperature. This differential between cerebral and visceral temperature gradients allowed the observation that both tissue capacitance and carbon dioxide levels followed the contour for rectal, rather than cerebral temperature. Moreover, nonshivering preparations, with either cervical sections or immobilized with curarizing agents, did not show the delayed impedance changes characteristic of the shivering animals (Figs. 8 and 9) during the period of falling temperature. These differentials in impedance changes between shivering and nonshivering animals will be discussed below. Relations Between Blood Pressure and Impedance During Hypothermia. Although the impedance measuring technique used here has not revealed rhythmic changes synchronous with vascular pulsation, it would be of considerable importance to determine possible long-term relationship between baseline impedance values and such vascular factors as blood pressure. It was found that femoral arterial blood pressure showed only minor perturbations during induction of major degrees of hypothermia, and that

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‘33NV113VdV3

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concomitant hippocampal impedance changes with a typical hypothermic configuration occurred prior to the late onset of substantial changes in blood pressure. Typical changes in these parameters in an encdpphale isole’ preparation are shown in Fig. 12. Blood pressure remained between 70 and 8.5 mm during recovery from the cervical transection under ether HIPPOCAMPAL

IMPEDANCE

CHANGES

IN

HYPOTHERMIA

KAW-3X,

T/6,

FIG. 12. Changes in blood pressure in cat with cervical cord transection during hypothermia, plotted concurrently with core temperature and resistive and reactive impedance factors. No relationship was noted between blood pressure and the typical impedance changes.

anesthesia, and during induction of hypothermia with a fall in rectal temperature from 38 to 26.5 C. During this induction phase, hippocampal resistive impedance rose from 13.2 to 19.7 kilohms. Capacitance rose sharply from 6.5 to 11.2 kilopicofarads during recovery from surgery, and then decreased to 9.2 kilopicofarads as rectal temperature was lowered from 38 to 26.5 C. As the temperature was raised again to 38 C, both resistance and

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capacitance returned to their initial levels, but during this recovery period, blood pressure rose temporarily from 82 to 120 mm. In summary, the characteristic excursions of resistive and capacitive impedance induced by a brief hypothermic episode appear to have no primary relationship to systemic blood pressure, which remains unaltered during the development of the main impedance changes, and which may also vary substantially without modifying the course or direction of these shifts. Discussion

Manipulation of arterial carbon dioxide concentration will produce changes in the tonus of cerebral vessels, increased arterial pCOz lowering tonus, increasing rate of circulation and diminishing the arteriovenous oxygen gradient. The present study of hypercapnea with 7 per cent carbon dioxide has indicated several complex aspects of interrelationship between cerebral carbon dioxide levels and impedance values, and emphasizes the probability that the impedance responses reflect changes in intrinsic characteristics of cerebral tissue, rather than relating in a direct fashion to cerebral blood flow or blood pressure. The evidence rests firstly on the finding that the qualitative nature of the impedance response, as it involves relative shifts in resistive and reactive components, changes sharply between different brain regions. The purely resistive shift so induced in the amygdala of the cat, and confirmed with implanted electrodes in man (18), has no counterpart in the substantial simultaneous changes in both resistive and reactive components detected in the reticular formation, and even more obviously in the hippocampus. An identical stimulus to three such regions, inducing presumably identical changes in vascular reflexes, would scarcely be expected to produce such different effects. Such differential effects in the reactive component would also appear to exclude the tissue-electrode interface (6) as the sole or even the major site of these changes. Moreover, evidence was found that responses to hypercapnea were also qualitatively dependent on the time that had elapsed after insertion of the electrode into a particular region, a finding also confirmed in man (18). This suggests that functional organization of the tissue, including its peremptory but temporary disruption by insertion of the electrode, with concomitant release of intracellular contents, is a more important factor in the nature of the impedance responses than mere changes in local vascular reflex patterns. This view is supported by findings in hypothermia that changes in end-tidal carbon dioxide levels provided closer

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parallels to the observed impedance changes than systemic blood pressure, both during induction of hypothermia, and in the recovery phase when carbon dioxide excretion continued to drop substantially below control levels (Fig. 11). It is thus important to assess the role of carbon dioxide in the compartmental divisions of cerebral tissue. Evaluated in the frame of the tricompartmental system discussed above, with intraneuronal, intraneuroglial and extracellular compartments, metabolically produced carbon dioxide may undergo conversion to carbonic acid in the presence of carbonic anhydrase, and not diffuse directly into the blood as molecular carbon dioxide (26). Intervention of the neuroglia cells between neuronal elements and the vascular capillaries has suggested to Tschirgi that this reaction might take place within the neuroglial compartment, with selective exchange of hydrogen and bicarbonate ions so formed with sodium and chloride ions drawn from the plasma. This notion of a regulatory action by carbon dioxide, and its site of action in neuroglial tissue, has been studied in the horizontal and Muller cells of the fish retina by Svaetechin et al. (23). Ammonia depolarized and carbon dioxide hyperpolarized these cells, and these substances were therefore characterized as critical metabolic regulators, with carbon dioxide contributing hydrogen ions to metabolic systems. Tsukada (27) has drawn attention to the transient but substantial increase in free ammonia in the brain of the mouse in avoidance behavior. The present study has indicated that very small changes in endogenous carbon dioxide production in response to physiological stimuli (Fig. 1) are associated with impedance shifts as large as those seen with much larger changes in blood carbon dioxide levels, induced by high concentrations of inspired carbon dioxide. This supports the view that these impedance responses occur. in a buffer compartment, isolated in considerable degree from the full effects of altered blood carbon dioxide levels. This and previous studies ( 1, 2) have consistently indicated impedance shifts towards lower resistance and increased reactance in response to alerting stimuli. Conversely, states of decreasing alertness, including sleep and anesthesia, were associated with increased resistance and decreased reactance. These findings have posed a paradox, since at least some of these conditions characterized by opposite shifts in states of consciousness, such as alerting and sleep, are also associated with raised systemic carbon dioxide levels. Moreover, essentially identical impedance shifts have been produced in the hippocampus of man ( 18) by hyperventilation and by

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hypercapnea, with decreased resistance and increased reactance in both cases, although the hypercapnic response in man was more complex than that described here in the cat. These seemingly paradoxical shifts may find an explanation in studies of the effects of carbon dioxide on electrical conductance of sea water ( 17), which have shown that its highest conductance is at pH7. Increasing alkalinity decreases conductivity, apparently due to increasing relative numbers of carbonate over bicarbonate ions. On the other hand, increasing acidity also decreases conductivity, apparently from a quite different mechanism involving preponderance of molecular carbon dioxide over carbonic acid. The present study, and the effects noted by Park et al. (17), direct attention to the need for careful control of carbon dioxide metabolism in measurement of neuroglial membrane resistance. High values noted in perfused preparations in the absence of a controlled carbon dioxide environment (13, 16) may require further assessment. In turn, we may consider the interaction of carbon dioxide with ionic fluxes between intracellular and extracellular compartments, and associated movements of fluid. Van Harreveld (9) has emphasized the presence of a substantial extracellular space in normal cerebral tissue rapidly fixed by his technique, and the progressive loss of this space after an interval of 2-8 min, with an apparent transfer of fluid to now swollen neuroglia cells. Our findings support the view that a cataclysmic change in tissue ionic distribution associated with terminal asphyxia or cardiac arrest is delayed for approximately 2 min, and is associated with rising resistance and falling capacitance, which transcend by an order of magnitude the physiological impedance responses (Fig. 10). Swollen neuroglial elements are characteristic of certain types of cerebral edema (14), although it has recently been emphasized that cerebral edema may be associated with an intraneuroglial accumulation of fluid in some pathological states of cerebral gray matter, and in other states, with extracellular accumulation of fluid in white matter (7, 24, 25). The ionic basis of impedance shifts in spreading depression, a condition associated with cortical swelling (8) has been attributed by Ranck (21) to depolarization of astrocytes by an increased external potassium ion concentration and a decreased potassium ion conductance, a condition which he considers analogous to the “anomalous rectification” in skeletal muscle described by Adrian and Freygang (3). However, measurement of fluid and ionic displacement in cerebral

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edema induced by triethyl tin acetate (22) has indicated accumulation of sodium and chloride ions in a neuroglial volume increased in size at the expense of extracellular space, with no evidence of displacement of potassium ions. Extracellular space estimated by their technique would occupy about 1.5 per cent of the cerebral volume. Our findings in hypercapnea may be compatible with the results of Reed et al. (22) in cerebral edema, whereas the much larger impedance shifts associated with terminal asphyxia, cardiac arrest, or spreading depression, and particularly our observation of a rapid and gross terminal decline in the reactive impedance component, suggest a substantially different mechanism. Here, an even greater distortion of physiological relationships, involving membrane transport mechanisms responsible for normal intracellular potassium levels, may lead to its accumulation in an extracellular compartment, with still further effects as suggested by Ranck. The present study has confirmed and extended the observations of Collewijn and Schade (5) on the effects of hypothermia on cerebral impedance. Our use of a more profound hypothermia down to 21 C has enabled detection of regional differences between the midbrain reticular formation and the hippocampus (Figs. 7 and 8), with a proportionately larger overshoot in the hippocampal reactive component during the recovery phase from 21 to 34 C. This relative insusceptibility of the reticular formation to hypothermia parallels the findings of Massopust et al. (15) of a similar resistance to obliteration of the EEG in the reticular formation during hypothermia. In the present study, the return of resistance and reactance to baseline values in both reticular formation and hippocampus lagged considerably behind the temperature recovery from 21 C. This was not observed by Collewijn and Schad6 (5) and apparently relates to the deeper hypothermia in the present study. Chang (4) has emphasized the susceptibility of dendritic components of cortical evoked potentials to temperatures from 21 to 28 C, and the absence of comparable effects in the axons of subjacent white matter. Our findings suggest that it may be necessary to take account of other tissue compartments, such as the neuroglial and extracellular, in the full evaluation of this hypothermic effect, if the observed impedance shifts have a substantial basis in these non-neural compartments. The occurrence of altered dendritic phenomena and sustained impedance shifts in the same hypothermic range suggests that they may entail an important functional interrelationship between neuroglial and dendritic elements, with modula-

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tion of dendritic activity by the enclosing neuroglial envelopes through direct control of metabolic transport mechanisms, or through adjustments in the ionic environment of the extracellular space (2, 13, 16). The complexity of environmental factors in determining hypothermic impedance shifts are clearly seen in this study. In immobilized animals and following cervical transection, impedance changes closely followed the initial fall in temperature, as noted previously by Collewijn and SchadC (5). By contrast, shivering preparations with chronically implanted electrodes maintained their impedance around baseline levels with core temperatures as low as 25 C, at which point both resistance and reactance changed sharply. Blood pressure measurements did not reveal any clear relationship to this sharp transition, nor were there definitive relationships between impedance and blood pressure in other aspects of a hypothermic cycle (Fig. 12). It would appear that these differences between shivering and nonshivering preparations may relate to a circulating metabolite. Thus, although we may seek an explanation of these impedance variations in the simple mechanisms of sodium, potassium and chloride ion exchanges, these ionic shifts may themselves be controlled by other, more complex factors. Certainly, the notion that carbon dioxide, for example, may be the ultimate arbiter of these effects should be treated with caution. We have noted the occurrence of large impedance responses to sensory stimuli, yet with only minor shifts in tidal carbon dioxide levels, and the relative inefficiency of inhaled carbon dioxide in promoting comparable impedance responses. This invites the notion that tissue compartments which underlie the impedance response, presumably neuroglial and extracellular, have an inherent capacity to isolate the neuron from the immediate impact of circulatory metabolites, and as discussed above, are only indirectly influenced in their conductance characteristics by such mechanical factors as blood presure. Such a functional frame renders all the more important elucidation of their functional relationship with the citadel of the neuron which they so intimately enclose, particularly since this study suggests that, in the intricacies of such neuronal-neuroglial relationships, regional differences may determine the very qualitative nature of the impedance response. References 1.

ADEY,

tissue

W.

R., R. T., KADO, and J. Didio. 1962. Impedance of animals using microvolt signals. Ezptl. Neural.

measurements 5: 47-66.

in brain

IMPEDANCE

IN

HYPERCAPNEA

AND

HYPOTHERMIA

215

2. ADEY, W. R., R. T. KADO, J. Dmro, and W. J. SCHINDLER. 1963. Impedance changes in cerebral tissue accompanying a learned discriminative performance in the cat. Exptl. Neurol. 7: 2.59-281. 3. ADRIAN, R. H., and W. H. FREYGANG.1962. The potassium and chloride conductance of frog muscle membrane. J. Physio. London 163: 61-103. 4. CHANG, H. T. 1959. The evoked potentials, pp. 299-314. In “Handbook of Physiology, Section 1, Neurophysiology,” Volume 1, J. Field, H. W. Magoun, V. E. Hall (eds.), Am. Physiol, Sot., Washington (Ref. p. 302). 5. COLLEWIJN, H., and J. P. SCHAD~. 1962. Cerebral impedance changes in hypothermia. Arch. Intern. Physiol. Biochem. 70: 200-210. 6. GESTELAND, R. C., B. HOWLAND, J. Y. LETTVIN, and W. H. PITTS. 1959. Comments on microelectrodes. Proc. Inst. Radio Eng. 47: 1856-1861. 7. GONATAS, N. K., H. M. ZIMMERMAN, and S. LEVINE. 1963. Ultrastructure of inflammation with edema in the rat brain. Am. J. Path. 42: 45.5-469. 8. HARREVELD, A. VAN. 1957. Changes in volume of cortical neuronal elements during asphyxiation. Am. J. Physiol. 191: 233-242. 9. HARREVELD,A. VAN. 1964. Extracellular space in central nervous tissue. Federetion

Proc.

23: 304.

10. HARREVELD,A. VAN, and culatory arrest. Am. J. 11. HARRE~ELD, A. VAN, and cortex after circulatory Physiol.

S. OCHS. 1956. Cerebral impedance changes after cirPhysiol. 187: 180-192. J. P. SCHAD%. 1960.

Chloride movement in cerebral arrest and during spreading depression. J. Cell. Camp.

54: 65-84.

12. HILD, W., and I. TASAKI. 1962. Morphological and physiological properties of neurons and glial cells in tissue culture. J. Neurophysiol. 26: 277-304. 13. KUFFLER, S. W., and D. D. POTTER. 1964. Glia in the leech central nervous system: physiological properties and neuron-glia relationship. J. Neurophysiol. 27: 290-320. 14. LUSE, S. A., and B. HARRIS. 1961. Brain ultrastructure in hydration and dehydration. Arch. Neural. 4: 139-152. 15. MASSOPUST, L. C., M. S. ALBIN, H. W. BARNES, R. MEDER, and H. W. KRETCHMER. Cortical and subcortical responses to hypothermia. Exptl. Neural. 9: 249-261. 16. NICHOLLS, J. G., and S. W. KU~ER. 1964. Extracellular space as a pathway for exchange between blood and neurons in the central nervous system of the leech: ionic composition of glial cells and neurons. J. Neurophysiol. 27: 645-671. 17. PARK, K., P. K. WEIL, and A. BRADSHAW. 1964. Effect of carbon dioxide on the electrical conductance of sea water. Natare 201: 1283-1284. 18. PORTER, R., W. R. ADEY, and R. T. KADO. 1964. Measurement of electrical impedance in the human brain. Some preliminary observations. Neurology, 14: 1002-1012. 19. RANCK, J. B. 1963a. Specific impedance of rabbit cerebral cortex. Erptl. Neural. 7: 144-152. 20. RANCK, J. B. 1963b. Analysis of specific impedance of rabbit cerebral cortex. Ex#tl. Neural. 7: 153-174. 21. RANCK, J. B. 1964. Specific impedance of cerebral cortex during spreading

216

ADEY,

KADO, AND WALTER

depression, and an analysis of neuronal, neuroglial and interstitial contributions. Exptl. Neural. 9: 1-16. 22. REED, D. J., D. M. WOODBURY,and R. L. HOLTZER. 1964. Brain edema, electrolytes and skeletal muscle. Arch. Neural. 10: 604-616. 23. SVAETECHIN, G., R. FATEHCHAND, M. LAUFER, P. WITKOWSKI, K. NEGISHI, and A. SELVIN DE TESTA. 1963. Interaction glia-neuronal: Su dependencia metabolica. Una nueva teoria acerca de1 sistema nerviosa. Acta Cientifica Venezolana supp1. 1, 14: 135-153. 24. TORACK, R. M., R. D. TERRY, and H. M. ZIMMERMAN. 1959a. The fine structure of cerebral fluid accumulation. 1. Swelling secondary to cold injury. Am J. Pathol. 35: 1135-1147. 25. TORACK, R. M., R. D. TERRY, and H. M. ZIMMERMAN. 1959b. The fine structure of cerebral fluid accumulation. 11. Swelling produced by triethyl tin poisoning and its comparison with that in the human brain. Am. J. Pathol. 36: 273-287. 26. TSCHIRGI, R. D. 1958. The blood-brain barrier, pp. 130-138. In: “Biology of Neuroglia” W. F. Windle led.]. Thomas, Springfield, Illinois. 27. TSUKADA, Y. 1964. Amino-acid metabolism and its relations to brain functions. “Proceedings of the Conference on Neurological Basis of Behavior, National Science Foundation, Honolulu, 1964.” (In press).