0080_REPRINTED FROM_ EXCERPTA MEDICA INTERNATIONAL CONGRESS SERIES NO 399 ANAESTHESIOLOGY.pdf

8/14/2019 0080_REPRINTED FROM_ EXCERPTA MEDICA INTERNATIONAL CONGRESS SERIES NO 399 ANAESTHESIOLOGY.pdf

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Reprinted fromExcerpta Med ica International Congress Series No. 399 Anaesthesiology.Proceedings of the VI Wo rld Congress of Anaesthesiology,Mex ico City, April 24-30-1976.Excerpta Medica Amsterdam(ISBN 90 219 0327 0)


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Oxygen and microarea related to anesthesia 48 7initial value and at 25 by more than 200 . The maximal increase of the perfusion rate isreached at 300 of the initial value. This seems to be the highest compensatory capabilityof capillary perfusion.Studies of diffusion parameters and its influence by anesthetic drugs are possible w itha model developed with the giant neurons of marine gastropodes Aplysia californensis).This mod el has been developed recently in our laboratory for studies of oxygen transferacross the cell membrane, especially of facilitated diffusion when the membrane charac-teristics are changed by pharmacologic agents (F ig. 6).When an oxygen measuring electrode is driven through the cell memb rane, the oxygenpartial pressure drops to 1/3 inside the m embrane in relation to outside. Inside the cell,there is little change in the oxygen partial pressure; at the other side of the cell, weobserve again the same oxygen partial pressure gradients. The cells are put into a chamb erwith a thin layer of saline above the cells and a saline reservoir. The cells are supplied withO2 by a constant gas flow across the surface of the saline (Fig. 6). The oxygen partialpressure on the outside of the cell mem brane (extracellular space) can be changed bychanging the oxygen concentration in the supplying gas. At low extracellular PO2 values,the oxygen partial pressure drop across the memb rane only amounts to 10 -15 . Thismeans that the cell memb rane has a large regulative capacity for the oxygen supply of thecell interior with its oxygen metabolizing organe lles.

G A S M t X T U R E

B I N O C U L A RM I C R O S C O P E

N O R M A L S A L I N ER E S E R V O I R

Fig. 6. Simplified diagram of the experimental set-up for studies on giant neurons inma rine gastropodes concerning oxygen di f fus ion problems across the ce ll membrane .

REFERENCESBicher, 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. Bicherand 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.


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odern methodology in the study ofmicropbysiologic functionsH A I M I B I C H E R

Department of Radiation Medicine, Roswell ParkMemorial Institute, Buffalo, N.Y., U.S.A.

Knowledge of physiological functions in intact tissues at the cellular level is of greatimport clinically, notably in anesthesiology and neonatology. To the basic researcher theability to objectively determine parameters such as PO2 and ionic concentrations isinvaluable. In recent years advances in the development of chemical microsensors haveenhanced the precision and reliability of these determinations. The sensors, having a tipdiameter of 1-5/am, can be placed in tissue without disturbing microcirculation, and arecapable 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 waselectroplated with a noble metal (usually platinum). The probe was insulated with Aral-dite 985E, and then coated with a nitrocellulose membrane. This was a major break-through in that the small tip diameter virtually assured the elimination of stirring artifactsand minimized microcirculatory damage. The nitrocellulose membrane was effective inhelping 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 proteindeposition, poisoning by sulfhydryl compounds, and the plating was susceptible toformation of small micropores which acted like small galvanic cells. Consequently, theircalibration for absolute POz measurement in tissue was very difficult.A further development by Silver (1965) served to substantially eradicate the afore-mentioned problems. The probe consists of a platinum wire attached by means of silverpaint 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 acapillary tube that is fused onto the platinum and w hich ensheathes the length of the copperwire. 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 tothe size of the tip relative to its membrane covering. A major shortcoming of this oxygenultramicroelectrode is its fragility, and it can only be used in soft tissues or for surfacemeasurements.Bicher and Knisely (1970) successfully used a platinum on glass probe which hasseveral advantages. It can be g round to a v ery small tip and has the capability of serving aseither an open tip, or with certain modifications, as an internal reference (for intracellu-lar recordings) oxygen ultramicroelectrode. The oxygen cathode basically consists of avery finely drawn (1 ~m or less) glass pipette which has a thin fdm overcoating ofplatinum. 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. Amajor problem encountered with this electrode is the questionable insulating quality ofthe outer membrane resins which may result in drift and unreliable calibrations.

Erdmann et al. (1973b) developed a multielectrode in order to gain a 3-dimensionalpicture 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


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Microphysiologic functions 489one and affixed to it with silver print. The silver print also serves well as an indifferentAg-AgCl-electrode when connected to another conducting wire. All tips are covered by aspecial plastic membrane.

Erdmanns group has also developed a system for measurement of PO2 in the fetalscalp (Erdmann et al., 1973a). This probe, which simultaneously measures ECG, is com-posed of a basic gold in glass Erdmann (1971) electrode fixed in a 2 mm spring-loadedsteel cannula. To this 02-sensing portion is affixed another steel cannula which serves asthe ECG probe. The 2 bent tips are brought together with a ring and thus pressed into thescalp. 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 controlof the very critical function of feto-maternal oxygen exchange.Whalen et al. (1967) described a recessed type of microelectrode. It consists of amolten metal-filled glass micropipette in which the metal does not quite reach the tip ofthe 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 mayhave a tip diameter as small as 2 ,um, and with an external reference electrode may beused to measure intracellular PO2. The collodion, while possibly lengthening the responsetime, makes the electrode relatively immune to poisoning by protein or sulfhydryl com-pounds. 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 ELECTRODESThe efforts to continuously record blood PO2 with catheter electrodes have met with

varying degrees of success, and problems such as stability, stirring artifacts, and main-tenance 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 catheterelectrode system. The electrode consists of an Ag-AgC1 plated copper anode and anAg-plated copper cathode exposed to an electrolvte chamber enclosed by a membranewhich is pervious to O2 but semipervious to water. The system is completed by a polari-zing cell and a current amplifier with a digital ammeter which may be calibrated to readPO2. The electrode is easily fitted through a 20-gauge cannula. The cannula is compli-mented 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 pres-sure. Laboratory and clinical tests have shown this electrode system very effective andaccurate. However, some problems still exist relating to mass production.Harris and Nugent (1973) based their blood probe on an electrode developed byInternational Biophysics Corporation (Irvine, Calif.) in which the anode and cathode areseparated, with only the cathode placed in the blood stream and the anode topicallyattached. The gold cathode, the tip of which is coated with Hydron, enters an Argylecatheter through a side hole. This does not significantly interfere with taking bloodsamples, measuring blood pressure, etc. Some problems encountered with this electrodeare the slow response time and a somewhat large discrepancy in values when comparisonsare 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 theninserted into a glass capillary which is fused around the wire. This is then attached witharaldite to a long silver tube which serves as the anode and which carries the means ofattachment to the catheter at one end and a thread for screwing on a Teflon cap at the


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490 H.I. Bieherother end. The cap feature of the probe guarantees small size and prevention of loss ofelectrode parts in the blood vessel. The sensing portion of the probe is covered with12/am membranes of Teflon and cuprophane, minimizing stirring artifact and responsetime. Questions arise as to the electrodes insulation and the affinity for platelets to theactive 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 anindication of the local blood POz when factors such as blood flow, blood compositionand O2 consumption of the skin are taken into account. The basic skin surface PO2 probeconsists of a flat, glass insulated 3-wire platinum cathode and a silver chloride referenceelectrode mounted in lucite. Also constructed according to the Clark principle, the elec-trode is covered by membranes of cuprophane and Teflon, and is surrounded by an elec-trolyte chamber which holds 0.2 M KC1. These are held in place by a Teflon O-ring. Mini-mizing the influence of blood flow and skin respiration by inducing hyperemia and de-creasing respiration by means of drug s has produced less than satisfying results. More suc-cessful has been the addition of a small heating coil to the electrode which serves as a bet-ter hyperemic agent. The heating, however, produces other physiological complicationswhich must be considered. The accurate determination of perfusion efficiency, then, isthe major obstacle in the development and widespread clinical use of this method for acorrect transcutaneous measurement of arterial blood PO2.

pH AND ION MICROELECTRODESOther microelectrodes are now available to measure K ,Na+, Cl-, pH, etc. An anti-

mony electrode has long been used in the measurement of pH, but investigators havefound that the electrode potential is linear with increasing pH only to pH 7.0. Conse-quently, these electrodes require frequent calibration. Bicher and Ohki (1971) success-fully used an anti~nony pH microelectrode, the design of which follows the same generallines 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) glassmicropipette which has a thin trim overcoating of antimony. It is then coated with2 layers of an insulating epoxy resin leaving an exposed tip of approximately 2/am inlength. A microcalomel electrode inserted into the same cell serves as a reference. Theresults obtained in the squid giant axon were very satisfactory. However, if this design isto be used successfully in mammalian cells, it should be modified by falling the micro-pipette 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 micropipettedrawn to a fine point into which is inserted and fused a second pipette made of pH sensi-tive glass. The tip of the pH sensitive glass pipette is recessed in the tip of the pyrex glasspipette and the electrode is filled with KC1 electrolyte. The Hinke-type electrode alsoconsists of a pH sensitive glass micropipette inside of a pyrex glass pipette, the major dif-ference being that the tip of the pH sensitive micropipette is not recessed, but extrudesfrom the pyrex glass pipette. A silver/silver chloride electrode is inserted into the elec-trode stem which is filled with 0.1 N HC1. The Hinke microelectrode then, has an exposedtip and its response time is instantaneous. This is an advantage over the Thomas micro-electrode 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 sensinglength of 1-2/am.Development of potassium, chloride and other ion-selective microelectrodes, most


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Microphysiologic functions 493Lubbers, 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 Oxygena-tion, 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): Ana ly~. Chem.. 43, 89.


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Pathoge nesis and therapy of ischem ic ano xic brain dam ageEDWIN 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 cerebralischemic-anoxic insults in the operating room as a result of the direct effect of anes-thetics which may cause myocardial depression, increased myocardial irritability, ven-tricular fibrillation and arrhythmias or for any of a number of other reasons. Theymay also be faced with a patient in prolonged coma or brain death following extendedperiods on cardiopulmonary bypass. In addition, more anesthesiologists appear to beinvolved in the care and treatment of the critically ill patient. Although much has beenlearned about the pathogenesis and therapy of ischemic-anoxic brain damage the exactpathologic mechanisms involved, definitive criteria for the application of appropriatetherapy and, indeed, clear-cut proof of the efficacy of presently used therapies remainto be elucidated. Therefore, it is appropriate that in this Neuroscience section ofModernMethods in Experimental Medicine we discuss the problems, methods and our presentknowledge on the pathogenesis and therapy of postischemic (PI) encephalopathy.

Among the questions which remain to be answered in this field of research are: (a) thetolerance of the brain to ischemic-anoxia; (b) the time course and magnitude of patho-physiological and biochemical processes which add or result in the development of PIencephalopathies; (c) the definitive criteria for application of the appropriate therapy ofproven efficacy; (d) the variables which may provide a clue for prognosis and diagnosis;and (e)the correlation between EEG, neurologic deficit examination and brain histo-pathology in prognosis, diagnosis, and evaluation of efficacy of therapeutic procedures.

The problems involved in studying the pathogenesis and clinically feasible therapiesfor ischemic anoxic brain damage are obviated by reports in the literature claiming thatthe tolerance of the brain to ischemic anoxia is in some reports 4 minutes (Wolin andMassopust, 1972) and at the other extreme 60 minutes (Hossman et al., 1973). Thespecific problems in this field of research are as follows: (1) the precise duration and thecompleteness of the ischemic-anoxic insult; (2)the morbidity and mortality associatedwith methods of inducing global brain ischemia requiring radical surgical procedures;(3) the sensitivity of the variables monitored PI in evaluating the severity of neurologicdamage (i.e. brain biochemistry, gross neurologic deficit examination, psychologicaltesting, or other physiological variables - cerebral blood flow, intracranial pressure, braintissue PO2); (4) the standardization and adequacy of PI intensive care to avoid prematuredeath 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 oftherapeutic efficacy in terms of physiological and biochemical variables rather thanzlinically relevant ultimate neurologic outcome; and (7) the use of a variety of animal;pecies w hich probably adds to data variability.

Restricting our discussion to global brain ischemia, a variety of methods have beenased in past studies for producing global brain ischemia. The m ethods used as w ell as their~hortcomings are as follows: (1)aortic and venae cava clamping (radical surgical pro-zedures making PI intensive care difficult and adds to morbidity and mortality) (Snyder et

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0080_REPRINTED FROM_ EXCERPTA MEDICA INTERNATIONAL CONGRESS SERIES NO 399 ANAESTHESIOLOGY.pdf