Pflfigers Archiv

Pfl/igers Arch. 378, 47-53 (1978)

EuropeanJournal

of Physiology 9 by Springer-Verlag1978

Time Course of Changes of Extracellular H + and K + Activities During and After Direct Electrical Stimulation of the Brain Cortex* Rudolf Urbanics**, Elfriede Leniger-Follert, and Dietrich W. Ltibbers Max-Planck-Institut fiir Systemphysiologie,Rheinlanddamm 201, D-4600 Dortmund 1, Federal Republic of Germany

Abstract. The kinetics of H + and K + activities were recorded during and after direct electrical activation of the brain cortex (cat). H + activity was measured with H+-sensitive glass microelectrodes (tip diameters of 1 - 4 gin) and K + activity was registered with doublebarrelled ion-sensitive microelectrodes (tip diameters of 1 - 3 gm). It could be shown that extracellular H + activity initially decreased for a few seconds and increased only after the 7. s. M a x i m u m acidosis was always noticed after stimulation ended. Alkalotic as well as acidotic changes were the higher the stronger the stimulation parameters were. K + activity increased very rapidly after stimulation began, reached its maxim u m when stimulation ended and then decreased to its initial value with an undershoot. It is concluded that the functional hyperemia of microflow could be triggered by the rapid increase in K + activity, whereas the initial alkalotic change of extracellular p H means that H + activity does not play a role in the first phase of this kind of hyperemia. The alkalotic shift is interpreted to be caused by the washout of CO 2 due to the rapid increase in microflow. In the further course, H + activity obviously contributes to the maintenance of functional hyperemia. In this later period K + activity is always below the control value. Key words: Extracellular H + activity - K + Activity Brain cortex - Microflow regulation - Direct electrical activation.

Send offprintrequests to E. Leniger-Follert at the above address. * Parts of the results were presented at "47. Tagung der Deutschen Physiologischen Gesellschaft" September 13-18, 1976 in Regensburg and at "13. Dortmunder Arbeitsgespr/ich"March 11-12, 1977 ** Present address: Experimental Research Dept., Semmelweis Medical School, Budapest, Hungary

Introduction It has been known for several years that regional cerebral blood flow is adapted to the metabolic demand of the brain cortex (for reviews see Betz [2]; Liibbers [17]; Purves [22]). In studies published recently, Leniger-Follert and Ltibbers [12,13] investigated the time course of the increase in flow produced by direct electrical stimulation. It was found that the capillary flow in the brain cortex increased in the activated tissue immediately when electrical stimulation began. Since the local tissue oxygen partial pressures (Po2) increased concomitantly with the capillary flow, functional hyperemia could not be caused by local hypoxia under these experimental conditions. Therefore, another mechanism was suggested to trigger the increase in microflow. Betz et al. [4, 5] and Kuschinsky et al. [9] deduced from microapplication studies in the pial vessels using artificial liquor that local extracellular hydrogen ions and potassium ions play a role in the metabolic regulation of brain circulation. So far, little information has been available about direct measurements of extracellular p H in the brain cortex. Most p H measurements were done either in the cerebral spinal fluid or in the vicinity of the pial vessels. Extracellular K + activity was directly recorded immediately around the pial vessels to study the influence of potassium ions on the diameters of the pial vessels. The purpose of the majority of the measurements of K + activity within the cortex was to investigate the behaviour of K + during indirect stimulation, epilepsia, anoxia and ischemia. Lewis et al. [16] and Prince et al. [21] already examined the changes of K + activity during direct electrical stimulation of the brain cortex. The present study was undertaken to investigate the kinetics of both local extracellular H + and K + activities during and after direct stimulation by direct microelectrode measurements within the gray matter of the brain. We examined the question of

0031-6768/78/0378/0047/$01.40

48

Pflfigers Arch. 378 (1978)

whether these ions are involved in the increase of capillary flow (microflow) and contribute to the maintenance of functional hyperemia under these conditions.

Methods Experimental animals were 15 adult cats (body weight o f 2 . 0 3.0 kg). After administration of 0.2 ml of Bellafolin the animals were anesthetized with Nembutal (pentobarbital 35mg/kg). Some cats breathed spontaneously, some were paralyzed with Flaxedil (gallaminetriethiodide 1 5 - 20 mg/kg) and mechanically ventilated. Endtidal CO2 concentrations recorded with an Uras analyzer were in the range of 3 . 5 - 4 vol%. Arterial blood pressure was continuously recorded in the abdominal aorta with a Statham element and was in the normal range. Rectal temperature was stabilized at 38 ~ C. Arterial pO2, pCO2 and p H were analyzed at intervals throughout the experiments. ArterialpO 2 a m o u n t e d to 94.6 _+ 9.6 Torr and arterial p H to 7.37 _+ 0.06 a n d p C O 2 to 27 + 4 Torr. After trepanation of the calvarium on one side, a piece of the dura mater was removed for inserting the microelectrode. To minimize brain movements of the animals ventilated mechanically, a p n e u m o t h o r a x was set at both sides a n d the liquor was sucked off. Measurements of extracellular potassium ions were done by double-barrelled liquid ion exchanger microelectrodes (tip diameter 1 - 3 pm) according to Lux and Neber [19]. The reference channel was filled with 150 mmo] NaC1 and served to register the DC-potentials. The other barrel contained the ion exchanger ( C o m i n g No. 477317) below 100 mmoI KCI_ A separate Ag/AgCI surface electrode placed on an agar bridge was used as a second reference electrode located about 1 - 2 m m distant from the microelectrode. The signals of both barrels were led to a high input impedance amplifier (1013 f2) with capacitance neutralization and registered against the surface reference. The output of both amplifier channels were led to the second stage of the differential amplifier and so, the corrected K + potential could be recorded additionally (Fig. 1 a). Thus, disturbances to the signal of the K + electrode caused by DC-changes in the stimulated area were avoided [11, 26]. Local extracellular H + activity was measured with H +-sensitive microelectrodes according to Saito et al. [24]. A n H +-sensitive tip was fused in lead glass. The electrode was filled with a buffer solution (1 part standard acetate buffer, p H 4.62, 2 parts saturated KC1 solution (cf. Saito et al. [24])).An internal reference electrode of Ag/AgC1 was inserted into the buffer. The head of the electrode was insulated by pitch and teflone. The H +-sensitive tip was 1 - 4 p m in diameter and about 2 0 - 80 g m long. The response time for 90 % was 1 - 3 s, the reaction time was less than I s. The sensitivity of the electrodes was 58 - 63 m V / p H unit at 37 ~ C. The drift was up to 1 m V / p H unit a n d hour. The resistance was about 1 0 - 4 0 GO. In some experiments (4 cats) we used an Ingold Ag/AgCl-electrode via an agar bridge as an external reference electrode. The reference electrode was positioned on the surface of the brain about 1 - 11/2 m m from the inserting point o f the measuring electrode. In another experiment, we used a micropipette filled with 1 5 0 m m o l NaC1 as the second reference electrode that was located at the same depth but about 100 g m distant from the measuring electrode. The signal was recorded by a digital electrometer with high input impedance (Keithley 616). In those five experiments the p H signal was not corrected for D C disturbances. In another two experiments where K + and H + activities were measured simultaneously, the reference barrel of the K + electrode served also as the reference electrode for the measurements of H + activity (Fig. 1 a). In these cases, the pH signal was corrected for D C potentials. In another two cats an H+-sensitive glass electrode was used where a thin silver film sputtered on the glass shaft served as the reference

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~mV/pH b Fig. 1 a and b. Circuit diagrams showing the arrangement for measurements o f K § and H + activities, a Simultaneous measurement o f K + and H + activity; h Measurement of H + activity using a p H microelectrode with reference electrode sputtered onto its shaft

(Fig. I b). In these cases, the possible influence of D C potentials was eliminated owing to the close neighborhood of the sputtered reference to the sensitive gtass electrode. We did not see any principal differences in the time course o f the H + activity under either condition. So we may assume that, under our experimental conditions, the influence of D C potentials on the time course of the pH signal is negligible. The electrodes were calibrated with buffer solutions at 37 ~ C (pH 6.83; 7.15; 7.33) before and after measurement in the tissue. The H +- and K+-sensitive electrodes were m o u n t e d on a counter-balanced holder which followed the movements of the brain caused by ventilation and blood pressure. The electrodes were inserted about 5 0 0 - 1 5 0 0 p m into the gyrus suprasylvius. In one experiment microflow and H § activity, and in another microflow and K + activity were recorded simultaneously. Microflow was continuously measured by local hydrogen clearance according to Liibbers and Stosseck [18] with a multiwire surface element (outer diameter 1.6 mm) including four measuring sites as developed by Leniger-Follert and Liibbers [13]. The H § or K § electrode was inserted about i m m distant from the border of the multiwire surface element. Direct electrical stimulation o f the brain cortex was achieved by two silver wires which were placed on the surface of the brain at a distance of about 5 ram. Frequency, amplitude, and time of stimulation were varied. Stimulation time was 1 - 2 0 s, amplitude 5 - 20 V, frequency 5 - 4 0 Hz, and pulse duration 1 ms, in one case 0.2 ms. To protect the brain from loss of heat and drying up, the surface of the brain was covered by warmed paraffin oil after the electrodes were inserted.

R. Urbanics et al. : H + and K § Activities as Regulatory Factors for Microflow

Tests of the Method M. Purves has suggested (personal communication) that the pH electrodes may change their sensitivity or position of the mV/pH curve on the pH axis, respectively, when inserted into the tissue. We checked this possible disturbance as follows. A basin (diameter 1 6 18 mm) formed of dental cement was placed over the skull hole and filled with thermoequilibrated buffer solution which immediately contacted the brain. The pH of the buffer solution was chosen so as to closely correspond to the pH actually measured in the brain by the precalibrated pH microelectrode. The electrode was changed from the tissue directly into the buffer solution, In two control experiments the electrode showed the same potential readings as in the tissue. So we think that inserting the electrode into the tissue does not distinctly change the sensitivity and position in our measurements. According to H. Caspers (personal communication), highfrequency signals could disturb the measurements with ion-selective electrodes owing to the rectifier effect of the border zones. We tested this possible effect on the signals of the K + electrodes by generating high frequencies of0.11 - 115 MHz and a voltage of 60 inV. We could not see any effect although the generator was in a Faraday's cage where the electrodes were mounted in the cat brain.

49

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The behaviour of local p H was investigated in 9 cats during and after direct electrical stimulation. Figure 2 shows the dependence of changes in H + activity on the stimulation parameters recorded at one measuring point. Time of stimulation varied between i and 10 s (upper part) and frequency between 20 and 40 Hz in this case (lower part). With the beginning of direct electrical stimulation, an initial alkalotic shift occurred which was followed by an acidotic change. The acidotic change was observed at the earliest at about the 7. s. Maximum acidosis occurred some time after stimulation ended. The extent of the alkalotic change and the following acidosis were higher as the time of stimulation became longer or the frequency higher. Furthermore, the maximal changes occurred later and the recovery period was longer, as the stimulation parameters became stronger. In some cases, H § activity returned to control values with an undershoot. Similar results were obtained when the amplitude of stimulation was changed. In a total of 8 series (6 cats) voltages varied between 5 and 20V. The stimulation time was varied in 5 series (4 cats) and frequency in 5 series (4 cats). Before stimulation the control level of p H was 7.17 with a standard deviation, 6, of + 0.069 at 9 measuring points. The m a x i m u m acidosis noticed after stimulation was 6.8 at 20V, 10Hz, pulse duration 1 ms and duration of stimulation 5 s. As we did not use a stronger stimulation amplitude, we do not know whether or not this acidosis was a ceiling level. Figure 3 shows the simultaneous measurement of H § activity, K § activity and DC-changes. As described above an initial alkalotic shift occurred followed by a longer acidotic change. K + activity increased immedi-

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ately after direct stimulation began and reached its m a x i m u m when stimulation ended. K + activity then decreased to its initial level with an undershoot. The D C potential changed in negative direction. Figure 4 shows the dependence of the extent of K § increase on the stimulation parameters. The increase was the higher the higher voltage, frequency and time of stimulation were. Control values of K + activity before stimulation were 3.47 + 0.45mmol in 10 cases measured in 8 cats. In a total of 11 series (6 cats) the voltage

50

Pfltigers Arch. 378 (1978) 5S

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was varied between 5 - 2 0 V (5, 10, 15, 20V). The m a x i m u m K + value reached with m a x i m u m voltage was about 9 - 1 0 mmol. In 7 series (6 cats) the stimulation time varied between 1 and 20 s. In a few cases, a ceiling (plateau)was reached before stimulation ended that did not change when the stimulation time was prolonged. The ceiling value increased when the amplitude increased. Frequency was varied in 6 series (5 cats) between 5-- 20 Hz. Figure 5 shows an example for simultaneous measurement of microflow and K + activity. After stimulation began, K + increased immediately and microflow changed within i - 2 s at the three measuring sites. When the stimulation time lasted for-2 or 5 s, hyperemia and K + changes were distinctly stronger. Figure 6 shows an example for simultaneous measurements o f microflow, p H and DC. In this registra-

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tion, microflow was recorded at two sites. The temporal relationship between the variables in response to the electrical stimulation can be seen clearly.

Discussion

A. Methodological Problems To avoid serious damage to the tissue at the site where the microelectrode was inserted, we used small K § and H + needle electrodes with tip diameters of about 1 4 gm. The sensitive lengths of the p H electrodes varied between 20 and 100gin. In spite of such small tip diameters, insertion of the microelectrode may slightly alter the brain tissue at the site of measurement as substantiated by t h e histological examination after insertion of Po2 needle electrodes of the same tip

R. Urbanics et al. : H + and K + Activities as Regulatory Factors for Microflow

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diameters (Seidl, Leniger-Follert, Wrabetz, Lfibbers, unpublished results). However, small capillary hemorrhages have not been observed at the site of measurement when such small tip diameters were used, so that disturbance of the microcirculation and therefore changes of the blood/brain D C potentials can be excluded in our experiments. The problem of measuring absolute p H values in vivo is a very difficult one. As described in the methodological part, we made some efforts to exclude possible artefacts. We think that the measurements o f p H that were corrected for D C potentials are reliable with regard to the absolute values. The results of the p H measurements were expressed in absolute values as shown in the different figures. In the 5 experiments where correction was not done, the p H values may' have been falsified by D C changes. However, the main purpose of this investigation was to examine the time course of the behaviour of H § and K + related to the time course of microflow. As we did not see any principal differences in the time course of H § under either condition we assume that the time course of the p H changes was correctly measured. The time course of p H changes during and after stimulation was .independent of the control level of p H before stimulation, i.e., it was the same even if the control level was more acidotic than the mean value of about 7.17. Moreover, the response time of the electrodes used was fairly short. The response time of the K § electrodes was between 5 and 20ms [19], whereas that of the p H electrodes was about 1 - 3 s for the 90 ~ value. The reaction time of the p H electrodes was below 1 s.

B. Discussion of the Results The aim of the present study was to investigate whether the extracellular H + and K + ions play a role in

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triggering and/or maintenance of functional hyperemia of microflow during and after direct electrical stimulation of the brain cortex. In previous investigations Leniger-Follert and Lfibbers [14,15] had shown that the increase in metabolism occurred within 0.1 s and that microflow increased within 1 - 2 s after the beginning of direct activation of the cortex [12,13]. M a x i m u m hyperemia always occurred some time after stimulation ended and depended on the strength of the stimulation parameters. The present microflow measurements performed with the same method confirmed the earlier findings. Our results clearly showed that H § activity did not increase initially after the beginning of stimulation but decreased for a few seconds. This initial alkalotic shift is interpreted as a washout of CO2 due to the rapid increase in microflow. At about the 7. s, H + activity distinctly increased and showed an acidotic m a x i m u m some time after stimulation ended. As H § activity did not increase initially it could not be the trigger for the rapid increase in microflow. However, simultaneous measurements of microflow and H § activity showed that extracellular H + activity and microflow run similarly in the later period, i. e., m a x i m u m and return to the initial value show the same time course (see Fig. 6). We therefore conclude that H + activity contributes to the maintenance of functional hyperemia. In contrast to H + activity, K + activity increased rapidly within some milliseconds after the beginning of stimulation and preceded the increase in microflow. It reached its m a x i m u m when stimulation ended and then decreased whereas microflow had not yet reached its m a x i m u m at that time. Microflow was still increased when K + activity showed an undershoot below the control level. We conclude from the time course of the behaviour of K § that the rapid increase in extracellular K § activity could be one of the mechanisms that trigger

52

the increase in microflow and that, after the end of stimulation, K § activity does not contribute to the functional hyperemia. Recently Betz and Heuser [3], Heuser et al. [7] confirmed our results. They used glass microelectrodes with tips of 8 - 10 gm in diameter and found that, under the same conditions of electrical stimulation of the brain cortex, pH showed at first an alkaline shift followed by a distinct increase. They also reported that K § activity increased immediately and showed that Ca z+ activity changed as well under the same conditions. Ca z+ activity decreased initially at about the same time when K § activity increased. Since perfusion experiments with artificial liquor showed that decrease in Ca 2 § activity and increase in K + activity in the surrounding of pial vessels cause dilatation of the pial arterioles (Betz et al. [5]; Kuschinsky et al. [9]) we may assume that changes in K § and Ca 2 § activities provide the initial trigger signals for the vessels immediately after the beginning of direct activations. In the later phase of hyperemia, other factors, such as H § activity, adenosine (Rubio et al. [23]; Wahl and Kuschinsky [27]) and PQ (Hilton and Chir [8]) and possible unknown factors could be responsible for the maintenance of functional hyperemia. Our results confirm the assumption of Astrup et al. [1] that the pH hypothesis of the metabolic control within the brain is probably not quite valid. This hypothesis [6,10, 20, 25] suggested that the extracellular pH is the main regulator of the vessels during changes of metabolism. Our findings have clearly shown that H § activity cannot change the microflow in the initial phase during activation and therefore cannot adapt the flow to the initial metabolic demand. Acknowledgements. We want to thank Dr. A. Lehmenk/ihler, Priv.Doz. Dr. H. Acker and Prof. Dr. M. Kessler for their advice in preparing the K + electrodes, D. Sylvester for constructing the amplifier for K + measurements and C. Danz for skillful technical assistance.

References 1. Astrup, J., Heuser, D.0 Lassen, N. A., Nilsson, B., Norberg, R., Siesj6, B. K. : Evidence against H + and K + as the main factors in regulation of cerebral blood flow during epileptic discharges, acute hyperemia, amphetamine intoxication and hypoglycemia. 9 A micro-electrode study. In: Ionic actions on vascular smooth muscle (E. Betz, ed.), pp. 110-115. Berlin-Heidelberg-New York: Springer 1976 2. Betz, E. : Cerebral blood flow: its measurement and regulation. Physiol. Roy. 52, 595-630 (1972) 3. Betz, E., Heuser, D. : Effects of electrical stimulation on vascular walls and perivascular tissue of the arterial brain vessels. In: Functional hyperemia. Mechanisms of metabolic regulation of microcirculation in different organs. Arzneimittelforsch. (Drug Res.) 27, 1516 (1977)

Pfliigers Arch. 378 (1978) 4. Betz, E., EnzenroB, H. G., Vlahov, V.: Interaction of H + and Ca 2 + in the regulation of local pial vascular resistance. Pfliigers Arch. 343, 7 9 - 8 8 (1973) 5. Betz, E., Brandt, H., Czornai, M. : Ionic control of pial arterial resistance. In: Blood flow and metabolism in the brain (M. Harper, B. Jennett, D. Miller, J. Rowan, eds.), pp. 9.12-9.14. London: Churchill-Livingstone 1975 6. Gotoh, F., Tazaki, Y., Meyer, J. S. : Transport of gases through brain and their extravascular vasomotor action. Exp. Neurol. 4, 48 (1961) 7. Heuser, D., Astrup, J., Lassen, N. A., Nilsson, B., Norberg, K., Siesj6, B. K.: Are H + and K + factors for the adjustment of cerebral blood flow to changes in functional state. A microelectrode study. In : Cerebral function, metabolism and circulation (D. H. Ingvar, N. A. Lassen, eds.), pp. 216-217. Copenhagen: Munksgaard 1977 8. Hilton, S. M., Chir, B.: A new candidate for mediator of functional vasodilatation in skeletal muscle. Circ. Res., Suppl. I, 28/29, 1-70--I-72 (1971) 9. Kuschinsky, W., Wahl, M., Bosse, O., Thurau, K. : Perivascnlar potassium and pH as determinants of local piai arterial diameter in cats. A microapplication study. Circ. Res. 31,240-247 (1972) 10. Lassen, N. A. : Brain extracellular pH: The main factor controlling cerebral blood flow. Scand. J. Clin. Lab. Invest. 22, 2 4 7 252 (1968) 11. Lehmenktihler, A., Speckmann, E.-J., Caspers, H.: Cortical spreading depression in relation to potassium activity, oxygen tension, local flow and carbon dioxide tension. In: Ion and enzyme electrodes in biology and medicine (M. Kessler, L. C. Clark, Jr., D. W. Ltibbers, I. A. Silver, W. Simon, eds.), pp. 311 - 315. Mtinchen-Berlin-Wien: Urban & Schwarzenberg 1976 12. Leniger-Follert, E., Ltibbers, D. W. : Response ofmicroflow and local tissue PO2 of brain cortex to direct electrical stimulation. Pfltigers Arch. 359, R 75 (1975) 13. Leniger-Follert, E., Ltibbers, D. W.: Behavior ofmicroflow and local pO 2 of the brain cortex during and after direct electrical stimulation. A contribution to the problem of metabolic regulation of microcirculation in the brain. PfliJgers Arch. 366, 3 9 - 4 4 (1976) 14. Leniger-Follert, E., Liibbers, D. W. : Microflow, local tissuepO2 and metabolism of the brain cortex during and after direct electrical stimulation. In: Functional hyperemia. Mechanisms of metabolic regulation of microcirculation in different organs. Arzneimittelforsch. (Drug Res.) 27, 1517 (1977) 15. Leniger-Follert, E., Urbanics, R., Harbig, K., Liibbers, D. W. : The behavior of local pH and NADH-fluorescence during and after direct activation of the brain cortex. In: Cerebral function, metabolism and circulation (D. H. Ingvar, N. A. Lassen, eds.), pp. 214-215. Copenhagen: Munksgaard 1977 16. Lewis, D. V., Schuette, W. H. : NADH fluorescence, [K+]0 and oxygen consumption in cat cerebral cortex during direct cortical stimulation. Brain Res. 110, 523-535 (1976) 17. Ltibbers, D. W. : Physiologic der Gehirndurchblutung. In: Der Hirnkreislauf (H. G/inshirt, ed.), pp. 212-260. Stuttgart: G. Thieme 1972 18. Lfibbers, D. W., Stosseck, K.: Quantitative Bestimmung der lokalen Durchblutung durch elektrochemisch im Gewebe erzeugten Wasserstoff. Naturwissenschaften 57, -311 -- 312 (1970) 19. Lux, H. D., Neher, E. : The equilibration time course of K + in cat cortex. Exp. Brain Res. 17, 190--205 (1973) 20. McDowall, D. G., Harper, A. M. : CBF and CSF pH in the monkey during prolonged hypocapnia. Scand. J. Lab. Clin. Invest., Suppl. 102, VIII E (1968) 21. Prince, D. A., Lux, H. D., Neher, E.: Measurements of extracellular potassium activity in cat cortex. Brain Res. 50, 489-495 (1973)

R. Urbanics et al. : H + and K + Activities as Regulatory Factors for Microflow 22. Purves, M. J.: The physiology of the cerebral circulation. Cambridge: University Press 1972 23. Rubio, R., Berne, R. M., Bockman, E. L., Curnish, R. R.: Relationship between adenosine concentration and oxygen supply in rat brain. Am. J. Physiol. 228, 1896-1902 (1975) 24. Saito, Y., BanmgSxtl, H., Liibbers, D. W. : The RF-sputtering technique as a method for manufacturing needle-shaped pH microelectrodes. In: Ion and enzyme electrodes in biology and medicine (M. Kessler, L. C. Clark, Jr., D. W. Liibbers, I. A. Silver, W. Simon, eds.), pp. 103-109. MSnchen-Berlin-Wien: Urban & Schwarzenberg 1976

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25. SkinhCe, E. : Regulation of cerebral blood flow as a single function of the interstitial pH. Acta Neurol. Scan& 42, 6 0 4 - 607 (1966) 26. Speckmann, E. J., Caspers, H. : Messung des Sauerstoffdruekes mit Platinmikroelektroden im Zentralnervensystem. Pfltigers Arch. 318, 7 8 - 8 4 (1970) 27. Wahl, M., Kuschinsky, W. : The dilatatory action of adenosine on pial arteries of cats and its inhibition by theophylline. Pfl/.igers Arch. 362, 5 5 - 5 9 (1976)

Received September 12, 1978