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Reprinted from Excerpta Medica International Congress Series No. 399 Anaesthesiology.

Proceedings of the VI World Congress of Anaesthesiology, Mexico City, April 24-30-1976. Excerpta Medica Amsterdam (ISBN 90 219 0327 0)

Oxygen and microarea related to anesthesia 487

initial value and at 25 by more than 200%. The maximal increase of the perfusion rate is reached at 300% of the initial value. This seems to be the highest compensatory capability of capillary perfusion. Studies of diffusion parameters and its influence by anesthetic drugs are possible with a model developed with the giant neurons of marine gastropodes (Aplysia californensis). This model has been developed recently in our laboratory for studies of oxygen transfer across the cell membrane, especially of facilitated diffusion when the membrane characteristics are changed by pharmacologic agents (Fig. 6). When an oxygen measuring electrode is driven through the cell membrane, the oxygen partial pressure drops to 1/3 inside the membrane in relation to outside. Inside the cell, there is little change in the oxygen partial pressure; at the other side of the cell, we observe again the same oxygen partial pressure gradients. The cells are put into a chamber with a thin layer of saline above the cells and a saline reservoir. The cells are supplied with O2 by a constant gas flow across the surface of the saline (Fig. 6). The oxygen partial pressure on the outside of the cell membrane (extracellular space) can be changed by changing the oxygen concentration in the supplying gas. At low extracellular PO2 values, the oxygen partial pressure drop across the membrane only amounts to 10%-15%. This means that the cell membrane has a large regulative capacity for the oxygen supply of the cell interior with its oxygen metabolizing organelles.
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Fig. 6. Simplified diagram of the experimental set-up for studies on giant neurons in marine gastropodes concerning oxygen diffusion problems across the cell membrane. REFERENCES Bicher, H.I. and Knisely, M.H. (19713): J. appl. Physiol., 28, 387. Cater, B.P. and Silver, I.A. (1961): In: Reference Electrodes. Editors: B.J.G. Ires and J.G. Janz. Academic Press, London. Erdmann, W. and Kunke, S. (1973): In: Oxygen Transport to Tissue. Editors: H.I. Bicher and B.S. Bruele. Plenum Publishing Co., New York. Erdmann, W., Kunke, S. and Krell, W. (1973): In: Oxygen Supply. Editor: M.L. Kessler. Urban and Schwarzenberg, Munich.

Modern methodology in the study of micropbysiologic functions


HAIM I. BICHER
Department of Radiation Medicine, Roswell Park Memorial Institute, Buffalo, N.Y., U.S.A.

Knowledge of physiological functions in intact tissues at the cellular level is of great import clinically, notably in anesthesiology and neonatology. To the basic researcher the ability to objectively determine parameters such as PO2 and ionic concentrations is invaluable. In recent years advances in the development of chemical microsensors have enhanced the precision and reliability of these determinations. The sensors, having a tip diameter of 1-5/am, can be placed in tissue without disturbing microcirculation, and are capable of detecting changes in the molecular composition of extra- or intracellular fluids. The first ultramicroelectrode of this type was developed by Cater and Silver (1961): for TPO2 determinations. This was a stainless steel needle of tip diameter 1 /~m which was electroplated with a noble metal (usually platinum). The probe was insulated with Araldite 985E, and then coated with a nitrocellulose membrane. This was a major breakthrough in that the small tip diameter virtually assured the elimination of stirring artifacts and minimized microcirculatory damage. The nitrocellulose membrane was effective in helping to maintain a reasonable degree of precision in the calibration of these electrodes. However, these probes still suffered from major drawbacks. They were subject to protein deposition, poisoning by sulfhydryl compounds, and the plating was susceptible to formation of small micropores which acted like small galvanic cells. Consequently, their calibration for absolute POz measurement in tissue was very difficult. A further development by Silver (1965) served to substantially eradicate the aforementioned problems. The probe consists of a platinum wire attached by means of silver paint onto a copper wire, and then electropolished to a tip as small as 0.5/2m in diameter. This tip is very thinly coated (but not completely to the end) with glass which is part of a capillary tube that is fused onto the platinum and which ensheathes the length of the copper wire. This basic probe is coated with successive layers of collodion, electrolyte solution, and DPS, the final tip diameter being 3-5/~m. This electrode is based on tbe Clark (1956) principle, and can be calibrated very accurately for absolute TPO2 measurements due to the size of the tip relative to its membrane covering. A major shortcoming of this oxygen ultramicroelectrode is its fragility, and it can only be used in soft tissues or for surface measurements. Bicher and Knisely (1970) successfully used a platinum on glass probe which has several advantages. It can be ground to a very small tip and has the capability of serving as either an open tip, or with certain modifications, as an internal reference (for intracellular recordings) oxygen ultramicroelectrode. The oxygen cathode basically consists of a very finely drawn (1 ~m or less) glass pipette which has a thin fdm overcoating of platinum. Each basic probe is then coated with 2 layers of an oxygen impervious resin, Seran F 310, leaving an exposed length of tip of approximately 2 om in diameter. A major problem encountered with this electrode is the questionable insulating quality of the outer membrane resins which may result in drift and unreliable calibrations. Erdmann et al. (1973b) developed a multielectrode in order to gain a 3-dimensional picture of PO2 in tissue. Based upon the gold-in-glass electrode developed by Erdmann (1971), the multielectrode consists of 6 such electrodes equidistant surrounding a central

Microphysiologic functions 489 one and affixed to it with silver print. The silver print also serves well as an indifferent Ag-AgCl-electrode when connected to another conducting wire. All tips are covered by a special plastic membrane. Erdmanns group has also developed a system for measurement of PO2 in the fetal scalp (Erdmann et al., 1973a). This probe, which simultaneously measures ECG, is composed of a basic gold in glass Erdmann (1971) electrode fixed in a 2 mm spring-loaded steel cannula. To this 02-sensing portion is affixed another steel cannula which serves as the ECG probe. The 2 bent tips are brought together with a ring and thus pressed into the scalp. An indifferent Ag-AgC1 clamp electrode is fixed somewhere in the tissue of the vagina. This system is not as yet refined, but has great potential for the monitoring and control of the very critical function of feto-maternal oxygen exchange. Whalen et al. (1967) described a recessed type of microelectrode. It consists of a molten metal-filled glass micropipette in which the metal does not quite reach the tip of the pipette. Gold may be electroplated onto the metal to adjust the depth of the recess. The recess is filled with collodion, which serves as a diffusion barrier. This probe may have a tip diameter as small as 2 ,um, and with an external reference electrode may be used to measure intracellular PO2. The collodion, while possibly lengthening the response time, makes the electrode relatively immune to poisoning by protein or sulfhydryl compounds. Some disadvantages are the difficulty of construction, the fragility of the probe, and the difficulties encountered in recording the small currents which are generated. TRANSCUTANEOUS AND BLOOD O2 ELECTRODES The efforts to continuously record blood PO2 with catheter electrodes have met with varying degrees of success, and problems such as stability, stirring artifacts, and maintenance of physiological integrity have yet to be completely overcome. Bicher et al. (1973b) took a major step in eradicating problems of drift, calibration, miniaturization, and ease of production with a clinically useful intraarterial catheter electrode system. The electrode consists of an Ag-AgC1 plated copper anode and an Ag-plated copper cathode exposed to an electrolvte chamber enclosed by a membrane which is pervious to O2 but semipervious to water. The system is completed by a polarizing cell and a current amplifier with a digital ammeter which may be calibrated to read PO2. The electrode is easily fitted through a 20-gauge cannula. The cannula is complimented by a delivery head which not only permits completion of the electrode circuit, but also allows access to the cannula for blood sampling and recording of arterial pressure. Laboratory and clinical tests have shown this electrode system very effective and accurate. However, some problems still exist relating to mass production. Harris and Nugent (1973) based their blood probe on an electrode developed by International Biophysics Corporation (Irvine, Calif.) in which the anode and cathode are separated, with only the cathode placed in the blood stream and the anode topically attached. The gold cathode, the tip of which is coated with Hydron, enters an Argyle catheter through a side hole. This does not significantly interfere with taking blood samples, measuring blood pressure, etc. Some problems encountered with this electrode are the slow response time and a somewhat large discrepancy in values when comparisons are made with a gas analyzer. Huch et al (1973) have developed a catheter electrode based upon the Clark principle. It consists of a 15/Jm platinum wire welded onto a 100/Jm platinum wire and then inserted into a glass capillary which is fused around the wire. This is then attached with araldite to a long silver tube which serves as the anode and which carries the means of attachment to the catheter at one end and a thread for screwing on a Teflon cap at the

490 H.I. Bieher other end. The cap feature of the probe guarantees small size and prevention of loss of electrode parts in the blood vessel. The sensing portion of the probe is covered with 12/am membranes of Teflon and cuprophane, minimizing stirring artifact and response time. Questions arise as to the electrodes insulation and the affinity for platelets to the active site. A fascinating method of determining arterial PO2 has been developed by Lubbers et al. (1973). The method rests on the principle that PO2 measured at the skin surface is an indication of the local blood POz when factors such as blood flow, blood composition and O2 consumption of the skin are taken into account. The basic skin surface PO2 probe consists of a flat, glass insulated 3-wire platinum cathode and a silver chloride reference electrode mounted in lucite. Also constructed according to the Clark principle, the electrode is covered by membranes of cuprophane and Teflon, and is surrounded by an electrolyte chamber which holds 0.2 M KC1. These are held in place by a Teflon O-ring. Minimizing the influence of blood flow and skin respiration by inducing hyperemia and decreasing respiration by means of drugs has produced less than satisfying results. More successful has been the addition of a small heating coil to the electrode which serves as a better hyperemic agent. The heating, however, produces other physiological complications which must be considered. The accurate determination of perfusion efficiency, then, is the major obstacle in the development and widespread clinical use of this method for a correct transcutaneous measurement of arterial blood PO2. pH AND ION MICROELECTRODES Other microelectrodes are now available to measure K+, Na+, Cl-, pH, etc. An antimony electrode has long been used in the measurement of pH, but investigators have found that the electrode potential is linear with increasing pH only to pH 7.0. Consequently, these electrodes require frequent calibration. Bicher and Ohki (1971) successfully used an anti~nony pH microelectrode, the design of which follows the same general lines as that of the oxygen ultramicroelectrode reported earlier by Bicher and Knisely (1970). Basically it consists of a very finely drawn (1 /am or less tip diameter) glass micropipette which has a thin trim overcoating of antimony. It is then coated with 2 layers of an insulating epoxy resin leaving an exposed tip of approximately 2/am in length. A microcalomel electrode inserted into the same cell serves as a reference. The results obtained in the squid giant axon were very satisfactory. However, if this design is to be used successfully in mammalian cells, it should be modified by falling the micropipette with KC1 and using that as an internal reference. Designs for glass pH microelectrodes have been developed, most notably by Hinke (1967), and Thomas (1970). The Thomas electrode consists of a pyrex glass micropipette drawn to a fine point into which is inserted and fused a second pipette made of pH sensitive glass. The tip of the pH sensitive glass pipette is recessed in the tip of the pyrex glass pipette and the electrode is filled with KC1 electrolyte. The Hinke-type electrode also consists of a pH sensitive glass micropipette inside of a pyrex glass pipette, the major difference being that the tip of the pH sensitive micropipette is not recessed, but extrudes from the pyrex glass pipette. A silver/silver chloride electrode is inserted into the electrode stem which is filled with 0.1 N HC1. The Hinke microelectrode then, has an exposed tip and its response time is instantaneous. This is an advantage over the Thomas microelectrode in which the recessed tip may cause a response time of up to several minutes. The Hinke microelectrode has a major drawback in that its long sensing length (50/am) limits it to use in large cells only. The Thomas electrode, on the other hand, has a sensing length of 1-2/am. Development of potassium, chloride and other ion-selective microelectrodes, most

Microphysiologic functions 491 notably by Walker (1971) and Silver (1973) are based upon the same pattern as the glass pH electrodes, but the glass used is K-sensitive or Cl-sensitive, etc. One of the many expansions of the realm of scientific knowledge which the described methodology has facilitated is in the area of brain tissue response to hypoxia, a field in which Bichers group has been prominent (Bicher, 1973). The availability of oxygen throughout the brain is far from homogeneous, with oxygen tensions varying 30 or 40 mm Hg within a distance of a few/Jm (Bicher et at., 1970, 1971, 1973a; Bicher and Knisely, 1970). However, the PO2 at a given microarea of cerebral tissue is remarkably constant, and can be changed only through major alterations in blood supply or the composition of respiratory gases (Bicher and Knisely, 1970; Bicher et at., 1973a; Silver, 1965). Bichers group has defined the different processes arrived at maintaining the constant brain cell oxygen microenvironment as "oxygen autoregulatory mechanisms. Experimental investigations in the brain cortex and spinal cord of cats with an ultramlcrooxygen electrode (Bicher and Knisely, 1970; Bicher et al.. 1970, 1973a) have produced the following results: (1)Tissue PO2 during a short period of anoxia was initially decreased, but then leveled out for a certain period of time at a relatively constant plateau, even when arterial PO2 values were under 20 mm Hg. (2) As arterial PO2 tension increases from depressed levels near zero. tissue tension responds; but on returning to normal tissue concentrations, oxygen tension overshoots the pre-existing normal level. The reoxygenation time (period of time needed to reach the prenitrogen 02 levels after reverting the respiratory mixture) is short and very reproducible (Bicher et at.. 1971). (3) There is a remarkable increase in cerebral blood flow paralleling the period of decreased TPO2. Measurement of local electrical activity and tissue oxygen tension with the same microelectrode indicates a significant reduction in electrical activity when tissue tension is in the "plateau region. Measurement of loca! electrical activity and tissue oxygen tension with the same microelectrode indicates a significant reduction in electrical activity when tissue tension is in the plateau region. Therefore there are 4 criteria that are used to identify the oxygen autoregulation mechanism in brain tissue, following a short period of anoxic anoxia: (1)short "reoxygenation time; (2)increase in cerebral blood flow: (3)presence of an overshoot; (4) presence of a period of electrical silence paralleling the period of TPO2 depression. In earlier theoretical investigations based on the above experimental data (Reneau et at., 1970), they demonstrated that the overshoot phenomena could be explained as a time delay in the flow rate vs arterial PO~ curve (steady state), i.e., hyperemia for a short period of time after arterial blood had returned to normal conditions. Later, based on the assumption that decreased electrical activity during reduced tissue oxygen tension was an indication of reduced oxygen utilization, they inferred that the plateau region of tissue PO~, in spite of decreasing arterial tension, could be explained as a reduction in cerebral oxygen consmnption. These experiments and theoretical reasoning led to the assumption that there is in brain tissue an oxygen sensor, able to regulate flow and neuronal activity according to TPO~ changes (Bicher, 1973: Bicher et al., 1971, 1973a). When interpreting anoxic anoxaa experiments, they assumed then that the decreased availability of oxygen in the arterial blood is only reflected in brain tissue after the transport delay is overcome. At this point, tissue PO~ begins to fall and the next defense mechanism is started, namely the increase in arterial blood flow. As this mechanism fails, because by that time arterial POz is nearing zero, the electrical activity of the brain is stopped in a very sharp way. This shut off point is very reproducible at a given microarea of brain tissue and usually occurs when the given POz in that area is reduced by about 30% (Bicher et al., 1973a).

492 H.L Bicher The assumption is then made that brain electrical activity is related to tissue oxygen consumption and electrical silence means a sharp decrease in oxygen utilization. Two facts in the results presented tend to support this assumption: (1) the PO2 level in cortex stabilizes when neuronal firing stops; (2)computer simulations match the actual PO2 values almost exactly when the consumption term is made equal to zero (R = 0), instead of being kept at a constant (Bicher et al., 1973~a). If the electrical silence is a compensatory mechanism then there are 2 possibilities for its function: (1)neurons are very sensitive to their O2 atmosphere, and a slight drop is enough to silence them, or (2) this is an inhibitory reflex, in which case a tissue PO2 receptor should be theorized. Opitz and Schneider (1950) have assumed that such a receptor exists in tissue and predicted that it is possibly located at the venous end of the capillaries; if it exists, it should have the capability of both increasing blood flow and the reflex inhibition of synaptic or neuronal potentials. Under hypoglycemic conditions, the regulatory mechanisms described hitherto are markedly altered. The electrical activity is only sporadic, and does not change much with a decrease in oxygen supply. Blood flow compensation occurs very early, paralleling in time the changes in arterial PO=. The delay mechanism is absent (Bicher et al., 1973a). Conclusions are that under those circumstances, the main compensation mechanism is blood flow. REFERENCES Bicher, H.I. (1973): In: Oxygen Transport to Tissue, p. 205. Editors: H.I. Bicher and D.F. Bruley. Plenum Press, New York, N.Y. Bicher, H.I., Bruley, D.F., Reneau, D.D. and Knisely, M.H. (1971): .l. Physiol. (Lond.), 217, 689. Bicher, H.I. and Knisely, M.H. (1970): J. appl. Physiol., 28, 387. Bicher, H.I. and Ohki, S. (1971): Biochirn. biophys. Acta (Amst.), 75, 849. Bicher, H.I., Reneau, D.D., Bruley, D.F. and Knisely, M.H. (1973a): Amer. J. Physiol., 224, 275. Bicher, H.I., Reneau, D.D. and Knisely, M.H. (1970): In: Blood Oxygenation, p. 201. Editor: D. Hershey. Plenum Press, New York, N.Y. Bicher, H.I., Rubin, J.W. and Adams, R.J. (1973b): In: Oxygen Transport to Tissue, p. 107. Editors: H.I. Bicher and D.F. Bruley. Plenum Press, New York, N.Y. Cater, D. and Silver, I. (1961): In: Reference Electrode, Chapter II. Editor: J. Janz. Academic Press, London~New York. Clark, L.C. (1956): Trans. Amer. Soc. artif, intern. Org., 2, 41. Erdmann, W. (1971): A Quickly Produced Ultramicro Electrode for Oxygen, H2-clearance and Action Potential Measurement. Mount Sinai Prize of Anesthesiology. Mount Sinai School of Medicine. Erdmann, W., Kunke, S., Heidenreich, J., Dempsey, W., Gunther, H. and Schafer, H. (1973a): In: Oxygen Transport to Tissue, p. 1129. Editors: H.I. Bicher and D.F. Bruley. Plenum Press, New York, N.Y. Erdmann, W., Kunke, S. and Krell, W. (1973b): In: Oxygen Supply, p. 169. Editors: M. Kessler, D.F. Bruley, L.C. Clark, D.W. Lubbers, I.A. Silver and J. Strauss. Urban and Schwarzenberg, Munich-Berlin. Harris, T.R. and Nugent, M. (1973): In: Oxygen Transport to Tissue, p. 1109. Editors: H.I. Bicher and D.F. Bruley. Plenum Press, New York, N.Y. Hinke, J.A.M. (1967): In: Glass Electrodes for Hydrogen and Other Cations. Editor: G. Eisenman. Marcel Dekker, New York, N.Y. Huch, A., Lubbers, D.W. and Huch, R. (1973): In: Oxygen Transport to Tissue, p. 1113. Editors: H.I. Bicher and D.F. Bruley. Plenum Press, New York, N.Y.

Microphysiologic functions 493


Lubbers, D.W., Huch, R. and Huch, A. (1973): In: Oxygen Transport to Tissue. p. 115. Editors: H.I. Bicher and D.F. Bruley. Plenum Press, New York, N.Y. Opitz, W. and Schneider, M. (1950): Ergebn. Physiol.. 46, 126. Reneau, D.D., Bicher, H.I., Bruley, D.F. and Knisely, M.H. (1970): In: Blood Oxygenation, p. 175. Editor: D. Hershey. Plenum Press, New York, N.Y. Silver, I.A. (1965): Med. Electron. Biol. Eng., 3, 377. Silver, I.A. (1973): In: Oxygen Transport to Tissue. p. 223. Editors: H.I. Bicher and D.F. Bruley. Plenum Press, New York, N.Y. Thomas, R.C. (1970): J. Phvsiol. (Lond.), 210. 82. Whalen, W.J., Riley, J. and Nair, P. (1967): J. appl. Physiol., 23. 798. Walker, J.L. (1971): Analy~. Chem.. 43, 89.

Pathogenesis and therapy of ischemic-anoxic brain damage


EDWIN M. NEMOTO Department of Anesthesiology, Critical Care Medicine Program, University of Pittsburgh School of Medicine, Pittsburgh, Pa., U.S.A.

Anesthesiologists constantly work with patients in the ever-present danger of cerebral ischemic-anoxic insults in the operating room as a result of the direct effect of anesthetics which may cause myocardial depression, increased myocardial irritability, ventricular fibrillation and arrhythmias or for any of a number of other reasons. They may also be faced with a patient in prolonged coma or brain death following extended periods on cardiopulmonary bypass. In addition, more anesthesiologists appear to be involved in the care and treatment of the critically ill patient. Although much has been learned about the pathogenesis and therapy of ischemic-anoxic brain damage the exact pathologic mechanisms involved, definitive criteria for the application of appropriate therapy and, indeed, clear-cut proof of the efficacy of presently used therapies remain to be elucidated. Therefore, it is appropriate that in this Neuroscience section ofModern Methods in Experimental Medicine we discuss the problems, methods and our present knowledge on the pathogenesis and therapy of postischemic (PI) encephalopathy. Among the questions which remain to be answered in this field of research are: (a) the tolerance of the brain to ischemic-anoxia; (b) the time course and magnitude of pathophysiological and biochemical processes which add or result in the development of PI encephalopathies; (c) the definitive criteria for application of the appropriate therapy of proven efficacy; (d) the variables which may provide a clue for prognosis and diagnosis; and (e)the correlation between EEG, neurologic deficit examination and brain histopathology in prognosis, diagnosis, and evaluation of efficacy of therapeutic procedures. The problems involved in studying the pathogenesis and clinically feasible therapies for ischemic anoxic brain damage are obviated by reports in the literature claiming that the tolerance of the brain to ischemic anoxia is in some reports 4 minutes (Wolin and Massopust, 1972) and at the other extreme 60 minutes (Hossman et al., 1973). The specific problems in this field of research are as follows: (1) the precise duration and the completeness of the ischemic-anoxic insult; (2)the morbidity and mortality associated with methods of inducing global brain ischemia requiring radical surgical procedures; (3) the sensitivity of the variables monitored PI in evaluating the severity of neurologic damage (i.e. brain biochemistry, gross neurologic deficit examination, psychological testing, or other physiological variables - cerebral blood flow, intracranial pressure, brain tissue PO2); (4) the standardization and adequacy of PI intensive care to avoid premature death as a result of complications; (5)the inherent probable correlation between neu~ologic deficit and duration of ischemia, which adds to the difficulty of precisely deter:nining the threshold of ischemic-anoxic brain damage (Fig. 1); (6)the evaluation of therapeutic efficacy in terms of physiological and biochemical variables rather than zlinically relevant ultimate neurologic outcome; and (7) the use of a variety of animal ;pecies which probably adds to data variability. Restricting our discussion to global brain ischemia, a variety of methods have been ased in past studies for producing global brain ischemia. The methods used as well as their ~hortcomings are as follows: (1)aortic and venae cava clamping (radical surgical prozedures making PI intensive care difficult and adds to morbidity and mortality) (Snyder et