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Transport of gases through brain and their extravascular vasomotor action
EXPERIMENTAL
4, 48-58
NEUROLOGY
Transport
of
(1961)
Gases
Extravascular FUMIO Department
Neurology,
Brain
Vasomotor
GOTOH, YOSHIAKI of
Through
Wayne Detroit, Received
TAZAKI,
Their
Action
AND JOHN S. MEYERS
State University Michigan May
and
College
of
Medicine,
5, 1961
The rate of transport of oxygen and nitrogen through living cat brain was compared with that through dead brain and effects of extravascular gases on cerebral vasomotor action were investigated. For these purposes gases were directly supplied to cerebral cortex using a plastic tent over the exposed cortex while brain-oxygen tension and cortical blood flow were recorded by the polarographic and thermistor methods, respectively. Transport of oxygen and nitrogen through living brain tissue was far faster than through dead brain. Capillary blood flow played an important role in the transport of gases. Direct external application of oxygen to cerebral cortex by plastic tent caused cerebral vasoconstriction. Carbon dioxide gas applied directly and from without to cerebral cortex resulted in dilation of cerebral vessels and this action was inhibited by previous intravenous injection of large amounts of acetazoleamide.
Introduction The rates at which gasesand especially oxygen diffuse through different tissuesare important in the application and interpretation of physiological measurementsof tissue blood flow and metabolism. Knowledge of the diffusion rate of oxygen through tissue is particularly important when tissue-oxygen tensionsare measuredby the polarographic technique using the oxygen electrode (2, 3, 5). This method requires that a constant, small amount of oxygen be utilized by the electrode, hence the polarographic current is proportional not only to tissue-oxygen tension but also to the diffusion coefficient of oxygen. In 1919, Krogh (7) systematically investigated the rate of diffusion of 1 This work was supported by grants from the National Institute of Neurological Diseases and Blindness and the National Heart Institute of the U. S. Public Health Service. Doctors Gotoh and Tazaki are Fellows of the Michigan Heart Association. Mr. Peter Miller provided technical assistance. 48
GAS
TRANSPORT
THROUGH
BRAIN
49
gases through animal tissues. Since then, diffusion coefficients of gases in various tissues, such as collagen, skin, muscle, nerve, and brain, have been reported (4, 13, 14). In none of these studies were measurements made with living tissue in situ. As far as we are aware, there is no knowledge available concerning transport of gases through living brain. The purpose of this communication is to compare the rate of transport of oxygen through living brain with that of dead brain, and furthermore, to determine whether gases diffusing from outside the brain to vessels cause vasoconstriction or vasodilation. For these purposes, a new technique of recording the rate of transport of oxygen and nitrogen in living tissue has been developed. Using this, it was shown that rate of transport of gases in living brain is far faster than that in dead brain and that gases may exert vasomotor influences from the extravascular as well as from the endovascular side. The observation is of fundamental importance in considering how local cerebral circulation is controlled and maintained to meet local circulatory requirements (9). Methods Experiments were performed on five cats. The weight of the animals ranged from 2.5 and 3.5 kg. After anesthesia by intraperitoneal injection of Nembutal (25 mg/kg body weight), a tracheostomy was performed, a tracheal cannula was inserted and connected to a Harvard variable speed respirator pump which maintained pulmonary respiratory exchange constant. The pial surface of the cerebral cortex was exposed unilaterally by removing the overlying skull and dura mater. The cortical oxygen availability from several small, discrete areas of cortex was recorded simultaneously by the polarographic method. Opentip polarographic platinum electrodes, 250~ in diameter, insulated with Teflon except for their tips, were thrust into the cerebral cortex to a depth of 1 to 4 mm. Silver-silver chloride reference electrodes were placed under the dura mater in the temporal region. A polarizing potential of 0.7 volts was supplied between the reference and platinum electrodes by means of a potentiometer and a battery. The platinum electrode was connected to the negative pole of the potentiometer through a resistance of 33 kQ. The output from either side of this resistance was connected to the input of an Offner polygraph (type R) and recorded by ink-writing oscillographs after amplification by d-c amplifiers. Cortical blood flow was recorded by means of a thermistor placed
50
GOTOH,
TAZAKI,
AND
MEYER
on the cortex. The thermistor was wired into one arm of a Wheatstone bridge, the output of which was connected to the input of one channel of the polygraph. Femoral blood pressure was recorded by the use of a polyethylene catheter connected to a Statham pressure transducer. “Alveolar carbon dioxide concentration”2 was continuously monitored by means of a Liston-Becker infrared gas analyzer to confirm the consistency of gas exchange of the lung during these experiments. The exposed cerebral cortex was completely covered with a tent made of double sheets of thick, Saran polymer plastic. Two lengths of polyethylene tubing, one for the gas inlet and the other for the gas outlet, were sealed into the transparent tent. This system permitted rapid and direct supply of various kinds of gas mixtures to the cortex through the exposed pial surface independently of systemic blood gases which were maintained constant. When oxygen (100%) was supplied into the tent at constant gas flow, it diffused into the cortex or capillary blood through the pial surface and reached the oxygen electrodes. The rate of increase of oxygen available to the electrode was a function of the rate of transport of oxygen through brain tissue. Thus, it is possible to estimate the rate of transport of oxygen through living brain tissue by continuously recording the oxygen available to the electrodes inserted at several, measured depths of the cortex. Other readings were made 30 to 60 min after death caused by Nembutal in massive dosage. Results
Transport of Oxygen and Nitrogen in Living Brain and Dead Brain. Oxygen and nitrogen (both, 100%) were supplied to the cortex through the Saran tent before and after the animals were sacrificed by the intravenous injection of 300 mg of Nembutal per kilogram with circulatory and respiratory arrest. Respiration was kept constant by the artificial respirator. Polarographic electrodes were inserted 2 mm in depth. Gas flow was kept constant at the rate of 250 ml per minute, which proved to have no direct cooling effect on cortex (Fig. 1). Application of oxygen to the living brain from without, by the tent, produced a prompt rise in oxygen available to the cortex. After switching from oxygen to air, the oxygen availability fell rapidly and returned to 2 Gas sample was drawn through a catheter placed in the trachea the terminal bronchioles to avoid aspiration of mucus.
rather
than
in
GAS
TRANSPORT
THROUGH
51
BRAIN
its original level. After sacrifice with circulatory arrest, however, these changes were far slower in spite of the same rate and external administration of the gas flow and the use of same animals in identically the same way except that circulation had ceased. Nitrogen (100%) by the tent caused a rapid fall of oxygen availability of the cortex, although the change was slightly slower than the oxygen effect (Fig. 1). The oxygen level was promptly restored after supply of air to the tent. In dead brain, these changes were extremely slow and
OXYGEN
N I TROGEN
EPG
7
N2 AIR
Z 3 6
t AIR
NZ AIR EPG
1
FIG. 1. Rate of transport of oxygen and nitrogen in living and dead cat brain. Direct application of oxygen (100%) to exposed living brain by tent caused a prompt rise in oxygen availability of cortex (EPG). After sacrifice, this change was delayed and far slower. Nitrogen (lOO’j&) by tent caused an instant fall of oxygen. In dead brain, this change was extremely slow and less remarkable.
less remarkable. These data prove that transport of oxygen and nitrogen through living brain tissue is far faster than that through dead brain. Influence of Extravascular Gases on Cerebral Vessels. Effects of extravascular oxygen and carbon dioxide on cerebral cortical blood flow (T) were also investigated (Fig. 2). Gases were supplied to the cortical surface by the plastic tent at the same rate and by the same method as described previously. Application of oxygen (100%) through the tent to the living brain caused an instant and progressive decrease of cortical blood flow. Oxygen available to platinum electrodes placed at various depths in
52
GOTOH,
TAZAKI,
the brain also was increased, but the depth of the electrode tip and current. In ‘the living animal, the trode and capillary seemed to be than to the depth of the electrode.
AND
MEYER
there was poor relationship between the rate of increase of polarographic distance between the tip of the elecmore related to the rate of increase In other words, transport of oxygen
t 35%COz
t AIR
65202
t
B
FIG. 2. Influence of extravascular oxygen and carbon dioxide on cerebra1 cortical blood flow (T) and oxygen availabilities (EPG 1: 3 mm in depth; EPG 2: 2 mm). Application of oxygen (100%) by tent to living cat brain caused an instant, progressive decrease of cortical blood flow and increase in oxygen availabilities. Note faster response in EPG 1 in spite of deeper position. Gas mixture (35% CO, + 65% 0,) by tent increased cortical blood flow and oxygen availabilities. In dead brain, no change in cortical temperature (T) was seen, and increases in oxygen availabilities were slower and less marked.
through living brain tissue depends not only on diffusion but also on capillary blood flow since gases diffuse from tissue into the circulation and are carried more rapidly than by simple diffusion. The latter factor seemed to play a considerable part in transport of gases through living brain tissue. When air flow through the tent was resumed, the cortical
GAS
blood flow tissues fell When a applied to
TRANSPORT
THROUGH
53
BRAIN
promptly returned to resting levels as oxygen available to the slowly. gas mixture of carbon dioxide (35%) in oxygen (6.5 ‘$6) was the living cortex through the tent, increases in both cortical
A T EPG-I +
3s%cO2*65%02
AIR
‘30SEC’ .
B
METAZOLAMIDE) w
FIG. 3. Effect of acetazoleamide on vasomotor action of extravascular carbon dioxide. A, effect of carbon dioxide by tent before acetazoleamide injection. Note a rise in cortical blood flow (T). B, effect of carbon dioxide by tent after acetazoleamide injection (500 mg). Vasodilator action of carbon dioxide was inhibited.
blood flow and cortical oxygen availabilities resulted without significant change in blood pressure. When the air flow was resumed, cortical blood flow and oxygen availabilities gradually returned to resting levels. Changes of cortical blood flow by the extravascular application of oxygen
54
GOTOH,
TAZAKI,
AND
MEYER
and carbon dioxide must be attributable to a vasomotor action of the gases, since there was no significant change in blood pressure. In the dead brain, cortical temperature was not altered by changes in oxygen or carbon dioxide content of the tent. This excluded the possibility of temperature artifact by the flow of gases through the tent. Increases in oxygen availability in dead brain by application of oxygen or mixtures of carbon dioxide and oxygen were slower and less remarkable than in living brain, which is further evidence of participation of circulation in transport of gases through living brain tissue. Effect of Acetazoleamid-e on Extravascular Vasomotor Action of Carbon Dioxide. Previous studies (8, 9) have shown that large amounts of acetazoleamide inhibit the vasomotor action of endovascular carbon dioxide. In the present series of experiments, in order to determine whether or not acetazoleamide also inhibits the vasomotor action of application of extravascular carbon dioxide; effects of extravascular carbon dioxide (35% ) in oxygen (657%) by tent on cortical blood flow and oxygen availability were studied before and after intravenous injection . of 500 mg of acetazoleamide. Figure 3A illustrates the effect of extravascular carbon dioxide before acetazoleamide injection and Fig. 3B shows the same after acetazoleamide injection. After acetazoleamide injection, extravascularly applied carbon dioxide did not increase cortical blood flow. Rise in oxygen availability was due to the oxygen present in the gas mixtures. After acetazoleamide injection, carbon dioxide ceased to have vasodilator action, which was present before injection. However, oxygen may still have caused its usual vasoconstrictor effect which was formerly overriden by the carbon dioxide present in the mixture. Cerebral vessels of the animals treated with large amounts of acetazoleamide no longer repond to either endovascular or extravascular carbon dioxide. Discussion
Since Davies and Brink (3) first applied polarographic methods to measuring oxygen tension in animal tissues, many attempts have been made to calibrate the polarographic electrode in vitro. Roseman, Goodwin, and McCulloch ( 12) compared the polarographic current passed through the brain with the current which flowed when the same electrodes were immersed in stirred and unstirred solutions equilibrated with room air. They concluded that the oxygen tension of brain, if calculated from the unstirred solutions as a standard, was many atmospheres, whereas if
GAS
TRANSPORT
THROUGH
BRAIN
55
calculated from stirred solution, it was the more reasonable value of less than 30 mm Hg. Montgomery and Horwitz (10) measured oxygen tension in intact human skin. They calibrated the electrode by inserting it into excised, dead skin immersed in solutions of several known oxygen tensions. Their normal values of oxygen tensions in human skin were 87 mm Hg, which seemed to be unusually high. None of these calibrations has been theoretically justified, since diffusion coefficients for living tissue are not yet known. According to Kolthoff and Lingane (6), one dimensional polarographic current can be expressed by the following equation: it = nFyCA(D/t)s where it = electrode current in amperes at the time t, n = number of electrons used per molecule of oxygen electrolyzed, Fy = the Faraday, C = initial uniform concentration of oxygen, A = area of the platinum surface in cm2, D = diffusion coefficient of oxygen in cm2/sec. According to this equation, the diffusion coefficient for oxygen in tissue is an important factor, as well as the oxygen concentration of the tissue. Clark and associates (2) developed a membrane-covered polarographic electrode for measuring blood oxygen tension. As far as flowing blood is concerned, the concentration gradient of oxygen does not extend beyond the membrane of this electrode; therefore, calibration of the electrode can be performed in stirred solutions of known oxygen tensions. Recently, Thews (13) analyzed oxygen diffusion in the brain theoretically, and calculated the pOz of the gray matter to be 17 mm Hg at the least supplied place. This value agrees fairly well with our findings in viva (9)) which have shown that normal values of cortical tissue ~0s varied between 10 and 20 mm Hg (10-35, with large electrode), depending on respiratory exchange, cortical blood flow, cortical metabolism, and pH. Zero oxygen tension determined in stirred saline correspondswell with oxygen available, determined at the cerebral cortex following apnea or prolonged nitrogen breathing. However, validity of this procedure of calibration for measuring brain tissue oxygen tension has not yet been determined. The present results of our experiments show evidence for participation of capillary circulation in transport of oxygen through living brain tissue.
56
GOTOH,
TAZAKI,
AND
MEYER
This transport of oxygen is far faster than that expected from diffusion alone. Thus, there are “convection currents” or continuously and rapidly moving currents of oxygen moving through the tissues which are generated by the rapid capillary circulation. Thus, the oxygen concentration gradient does not reach beyond the membrane of the polarographic electrode placed on the pial surface since the capillary circulation has a “stirring effect.” Therefore, as long as circulation is maintained, the calibration in stirred solutions of known oxygen tensions seems to be valid for measuring oxygen tension of brain tissues using the membrane-covered oxygen electrode. Under such conditions, “tissue oxygen tension” measured by the polarographic method should be defined as the oxygen available to the tissue on which the electrode is placed. Calibrations of tissue ~02 in mm Hg [for example, 20 mm Hg) should be interpreted as “the oxygen available to the tissue beneath the electrode is equivalent to stirred saline equilibrated at 20 mm Hg pOz at the same temperature.” The objections to calibrations of tissue or blood by stirred solutions as described above can be overcome either by the use of a membrane permitting slow diffusion of oxygen or minimizing the size of the platinum cathode within the membrane. The first method ensures that movement of oxygen across tissues always exceeds the movement of oxygen within the electrode and hence the gradient of oxygen tension does not extend beyond the membrane. The second method has proven ideal in our hands. We now use a platinum cathode within the membrane less than 100 mu in diameter and only its cross-sectional area is exposed. Such a small cathode consumes so little oxygen that the oxygen gradient does not extend beyond the membrane. Since the electrical output is small greater amplification is needed. This electrode is extremely stable, gives a rapid response time (in seconds) and permits calibration of tissue ~02 in absolute units of mm Hg from stirred or unstirred solutions or gases. Hence tissue calibrations are unaffected by tissue “convection currents” derived from blood flow. Zero readings are the same in viva as in vitro whether achieved by ischemia, nitrogen breathing, or death. Stirring and flow changes do not effect the electrodes within physiological ranges. It has long been assumedthat extravascular carbon dioxide in brain tissuesproduced by cerebral metabolism could dilate cerebral vessels. Our previous studies (9) proved that increased tissue carbon dioxide resulting from increasedcerebral metabolism dilated cerebral vesselsand maintained local circulatory homeostasis. This provided indirect evidence of vaso-
GAS TRANSPORT
THROUGH
BRAIN
57
motor action of extravascular carbon dioxide. The present experiment gives direct evidence for a vasodilator action of extravascular carbon dioxide diffusing through tissue as occurs from cerebral metabolism. It is still obscure whether or not extravascular carbon dioxide must diffuse into the endovascular space to be effective. We suspected previously that it must act directly on smooth muscle of the walls of small arteries (9). Acetazoleamide inhibits the vasomotor action of both endovascular and extravascular carbon dioxide. Therefore, inhibition of vasomotor action of carbon-dioxide inhalation is not due to the disturbance of delivery of carbon dioxide resulting from carbonic anhydrase inhibition in red cells. The mechanism of inhibition must be due to inhibition of carbonic anhydrase at the target point, presumably the cerebral vessel wall itself. Carbonic anhydrase enhances the reaction from CO2 + Hz0 + H+ + HCOB- ; therefore, the final factor responsible for cerebral vasomotor action must be either Hi- or HC03-. However, after acetazoleamide injection, cerebral vessels do not respond to HC03(NaHC03) (9). Hence, H+ seems to be responsible; however, the fact that H+ is less potent a vasodilator than CO, itself remains to be explained. The reason for the more potent effect of CO2 is that the latter passes more readily than H+ through the cell membrane of smooth muscle and lowers the intracellular pH more rapidly (1, 10). It is, thus, concluded that the final factor responsible for cerebral vasomotor activity is a change in intracellular H+ of smooth muscle fibers of cerebral arterioles. References 1. 2. 3.
4. 5.
6.
7.
P. C., Studies of the internal pH of large muscle and nerve fibres. J. Physiol. London 142: 22-62, 1958. CLARK, L. C., R. WOLF, D. GRANCER, and Z. TAYLOR, Continuous recording of blood oxygen tensions by polarography. J. Appl. Physiol. 6: 189-193, 1953. DAVIS, P. W., and F. BRINK, JR., Microelectrodes for measuring local oxygen tension in animal tissues. Rev. Sci. Instr. 13: 524-533, 1942. FENN, W. O., The carbon dioxide dissociation curve of nerve and muscle. Am J. Physiol. 65: 207-223, 1928. GOTOH, F., and J. S. MEYER, A combined electrode for recording absolute tensions of oxygen and carbon dioxide from small areas of tissue. Electroenceph. Clin. Neurophysiol. 13: 119-122, 1961. “Polarography.” New York, Interscience KOLTHOFF, I. M., and J. J. LINGANE, Publishers, 1941. KROGH, A., The rate of diffusion of gases through animal tissues, with some remarks on the coefficient of invasion. J. Physiol. London 52: 391-403, 1919. CALDWELL,
58
GOTOH,
TAZAKI,
AND
MEYER
8. MEYER, J. S., and F. GOTOH, Metabolic and electroencephalographic effects of hyperventilation. A.M.A. Arch. Neurol. 3: 539-552, 1960. 9. MEYER, J. S., and F. GOTOH, Interaction of cerebral hemodynamics and metabolism. Neurology (Suppl.) 11: 46-65, 1961. 10. MEYER, J. S., F. GOTOH, and Y. TAZAKI, CO, narcosis. An experimental study. Neurology 11: 524-537, 1961. 11. MONTGOMERY, H., and 0. HORWITZ, Oxygen tension of tissues by the polarographic method. I. Introduction: oxygen tension and blood flow of the skin of human extremities. /. Clin. Invest. 99: 1120-1130, 1950. 12. ROSEMAN, E., C. W. GOODWIN,and W. S. MCCULLOCH, Rapid changes in cerebral oxygen tension induced by altering the oxygenation and circulation of the blood. J. Neurophysiol. 9: 33-40, 1946. 13. THEWS, G., Die Sauerstoff diffusion im Gehirn. Arch. ges. Physiol., Pf&iiger’s 271: 197-226, 1960. 14. WRIGHT, C. I., The diffusion of carbon dioxide in tissues. J. Gen. Physiol. 17: 657-676,
1934.
4, 48-58
NEUROLOGY
Transport
of
(1961)
Gases
Extravascular FUMIO Department
Neurology,
Brain
Vasomotor
GOTOH, YOSHIAKI of
Through
Wayne Detroit, Received
TAZAKI,
Their
Action
AND JOHN S. MEYERS
State University Michigan May
and
College
of
Medicine,
5, 1961
The rate of transport of oxygen and nitrogen through living cat brain was compared with that through dead brain and effects of extravascular gases on cerebral vasomotor action were investigated. For these purposes gases were directly supplied to cerebral cortex using a plastic tent over the exposed cortex while brain-oxygen tension and cortical blood flow were recorded by the polarographic and thermistor methods, respectively. Transport of oxygen and nitrogen through living brain tissue was far faster than through dead brain. Capillary blood flow played an important role in the transport of gases. Direct external application of oxygen to cerebral cortex by plastic tent caused cerebral vasoconstriction. Carbon dioxide gas applied directly and from without to cerebral cortex resulted in dilation of cerebral vessels and this action was inhibited by previous intravenous injection of large amounts of acetazoleamide.
Introduction The rates at which gasesand especially oxygen diffuse through different tissuesare important in the application and interpretation of physiological measurementsof tissue blood flow and metabolism. Knowledge of the diffusion rate of oxygen through tissue is particularly important when tissue-oxygen tensionsare measuredby the polarographic technique using the oxygen electrode (2, 3, 5). This method requires that a constant, small amount of oxygen be utilized by the electrode, hence the polarographic current is proportional not only to tissue-oxygen tension but also to the diffusion coefficient of oxygen. In 1919, Krogh (7) systematically investigated the rate of diffusion of 1 This work was supported by grants from the National Institute of Neurological Diseases and Blindness and the National Heart Institute of the U. S. Public Health Service. Doctors Gotoh and Tazaki are Fellows of the Michigan Heart Association. Mr. Peter Miller provided technical assistance. 48
GAS
TRANSPORT
THROUGH
BRAIN
49
gases through animal tissues. Since then, diffusion coefficients of gases in various tissues, such as collagen, skin, muscle, nerve, and brain, have been reported (4, 13, 14). In none of these studies were measurements made with living tissue in situ. As far as we are aware, there is no knowledge available concerning transport of gases through living brain. The purpose of this communication is to compare the rate of transport of oxygen through living brain with that of dead brain, and furthermore, to determine whether gases diffusing from outside the brain to vessels cause vasoconstriction or vasodilation. For these purposes, a new technique of recording the rate of transport of oxygen and nitrogen in living tissue has been developed. Using this, it was shown that rate of transport of gases in living brain is far faster than that in dead brain and that gases may exert vasomotor influences from the extravascular as well as from the endovascular side. The observation is of fundamental importance in considering how local cerebral circulation is controlled and maintained to meet local circulatory requirements (9). Methods Experiments were performed on five cats. The weight of the animals ranged from 2.5 and 3.5 kg. After anesthesia by intraperitoneal injection of Nembutal (25 mg/kg body weight), a tracheostomy was performed, a tracheal cannula was inserted and connected to a Harvard variable speed respirator pump which maintained pulmonary respiratory exchange constant. The pial surface of the cerebral cortex was exposed unilaterally by removing the overlying skull and dura mater. The cortical oxygen availability from several small, discrete areas of cortex was recorded simultaneously by the polarographic method. Opentip polarographic platinum electrodes, 250~ in diameter, insulated with Teflon except for their tips, were thrust into the cerebral cortex to a depth of 1 to 4 mm. Silver-silver chloride reference electrodes were placed under the dura mater in the temporal region. A polarizing potential of 0.7 volts was supplied between the reference and platinum electrodes by means of a potentiometer and a battery. The platinum electrode was connected to the negative pole of the potentiometer through a resistance of 33 kQ. The output from either side of this resistance was connected to the input of an Offner polygraph (type R) and recorded by ink-writing oscillographs after amplification by d-c amplifiers. Cortical blood flow was recorded by means of a thermistor placed
50
GOTOH,
TAZAKI,
AND
MEYER
on the cortex. The thermistor was wired into one arm of a Wheatstone bridge, the output of which was connected to the input of one channel of the polygraph. Femoral blood pressure was recorded by the use of a polyethylene catheter connected to a Statham pressure transducer. “Alveolar carbon dioxide concentration”2 was continuously monitored by means of a Liston-Becker infrared gas analyzer to confirm the consistency of gas exchange of the lung during these experiments. The exposed cerebral cortex was completely covered with a tent made of double sheets of thick, Saran polymer plastic. Two lengths of polyethylene tubing, one for the gas inlet and the other for the gas outlet, were sealed into the transparent tent. This system permitted rapid and direct supply of various kinds of gas mixtures to the cortex through the exposed pial surface independently of systemic blood gases which were maintained constant. When oxygen (100%) was supplied into the tent at constant gas flow, it diffused into the cortex or capillary blood through the pial surface and reached the oxygen electrodes. The rate of increase of oxygen available to the electrode was a function of the rate of transport of oxygen through brain tissue. Thus, it is possible to estimate the rate of transport of oxygen through living brain tissue by continuously recording the oxygen available to the electrodes inserted at several, measured depths of the cortex. Other readings were made 30 to 60 min after death caused by Nembutal in massive dosage. Results
Transport of Oxygen and Nitrogen in Living Brain and Dead Brain. Oxygen and nitrogen (both, 100%) were supplied to the cortex through the Saran tent before and after the animals were sacrificed by the intravenous injection of 300 mg of Nembutal per kilogram with circulatory and respiratory arrest. Respiration was kept constant by the artificial respirator. Polarographic electrodes were inserted 2 mm in depth. Gas flow was kept constant at the rate of 250 ml per minute, which proved to have no direct cooling effect on cortex (Fig. 1). Application of oxygen to the living brain from without, by the tent, produced a prompt rise in oxygen available to the cortex. After switching from oxygen to air, the oxygen availability fell rapidly and returned to 2 Gas sample was drawn through a catheter placed in the trachea the terminal bronchioles to avoid aspiration of mucus.
rather
than
in
GAS
TRANSPORT
THROUGH
51
BRAIN
its original level. After sacrifice with circulatory arrest, however, these changes were far slower in spite of the same rate and external administration of the gas flow and the use of same animals in identically the same way except that circulation had ceased. Nitrogen (100%) by the tent caused a rapid fall of oxygen availability of the cortex, although the change was slightly slower than the oxygen effect (Fig. 1). The oxygen level was promptly restored after supply of air to the tent. In dead brain, these changes were extremely slow and
OXYGEN
N I TROGEN
EPG
7
N2 AIR
Z 3 6
t AIR
NZ AIR EPG
1
FIG. 1. Rate of transport of oxygen and nitrogen in living and dead cat brain. Direct application of oxygen (100%) to exposed living brain by tent caused a prompt rise in oxygen availability of cortex (EPG). After sacrifice, this change was delayed and far slower. Nitrogen (lOO’j&) by tent caused an instant fall of oxygen. In dead brain, this change was extremely slow and less remarkable.
less remarkable. These data prove that transport of oxygen and nitrogen through living brain tissue is far faster than that through dead brain. Influence of Extravascular Gases on Cerebral Vessels. Effects of extravascular oxygen and carbon dioxide on cerebral cortical blood flow (T) were also investigated (Fig. 2). Gases were supplied to the cortical surface by the plastic tent at the same rate and by the same method as described previously. Application of oxygen (100%) through the tent to the living brain caused an instant and progressive decrease of cortical blood flow. Oxygen available to platinum electrodes placed at various depths in
52
GOTOH,
TAZAKI,
the brain also was increased, but the depth of the electrode tip and current. In ‘the living animal, the trode and capillary seemed to be than to the depth of the electrode.
AND
MEYER
there was poor relationship between the rate of increase of polarographic distance between the tip of the elecmore related to the rate of increase In other words, transport of oxygen
t 35%COz
t AIR
65202
t
B
FIG. 2. Influence of extravascular oxygen and carbon dioxide on cerebra1 cortical blood flow (T) and oxygen availabilities (EPG 1: 3 mm in depth; EPG 2: 2 mm). Application of oxygen (100%) by tent to living cat brain caused an instant, progressive decrease of cortical blood flow and increase in oxygen availabilities. Note faster response in EPG 1 in spite of deeper position. Gas mixture (35% CO, + 65% 0,) by tent increased cortical blood flow and oxygen availabilities. In dead brain, no change in cortical temperature (T) was seen, and increases in oxygen availabilities were slower and less marked.
through living brain tissue depends not only on diffusion but also on capillary blood flow since gases diffuse from tissue into the circulation and are carried more rapidly than by simple diffusion. The latter factor seemed to play a considerable part in transport of gases through living brain tissue. When air flow through the tent was resumed, the cortical
GAS
blood flow tissues fell When a applied to
TRANSPORT
THROUGH
53
BRAIN
promptly returned to resting levels as oxygen available to the slowly. gas mixture of carbon dioxide (35%) in oxygen (6.5 ‘$6) was the living cortex through the tent, increases in both cortical
A T EPG-I +
3s%cO2*65%02
AIR
‘30SEC’ .
B
METAZOLAMIDE) w
FIG. 3. Effect of acetazoleamide on vasomotor action of extravascular carbon dioxide. A, effect of carbon dioxide by tent before acetazoleamide injection. Note a rise in cortical blood flow (T). B, effect of carbon dioxide by tent after acetazoleamide injection (500 mg). Vasodilator action of carbon dioxide was inhibited.
blood flow and cortical oxygen availabilities resulted without significant change in blood pressure. When the air flow was resumed, cortical blood flow and oxygen availabilities gradually returned to resting levels. Changes of cortical blood flow by the extravascular application of oxygen
54
GOTOH,
TAZAKI,
AND
MEYER
and carbon dioxide must be attributable to a vasomotor action of the gases, since there was no significant change in blood pressure. In the dead brain, cortical temperature was not altered by changes in oxygen or carbon dioxide content of the tent. This excluded the possibility of temperature artifact by the flow of gases through the tent. Increases in oxygen availability in dead brain by application of oxygen or mixtures of carbon dioxide and oxygen were slower and less remarkable than in living brain, which is further evidence of participation of circulation in transport of gases through living brain tissue. Effect of Acetazoleamid-e on Extravascular Vasomotor Action of Carbon Dioxide. Previous studies (8, 9) have shown that large amounts of acetazoleamide inhibit the vasomotor action of endovascular carbon dioxide. In the present series of experiments, in order to determine whether or not acetazoleamide also inhibits the vasomotor action of application of extravascular carbon dioxide; effects of extravascular carbon dioxide (35% ) in oxygen (657%) by tent on cortical blood flow and oxygen availability were studied before and after intravenous injection . of 500 mg of acetazoleamide. Figure 3A illustrates the effect of extravascular carbon dioxide before acetazoleamide injection and Fig. 3B shows the same after acetazoleamide injection. After acetazoleamide injection, extravascularly applied carbon dioxide did not increase cortical blood flow. Rise in oxygen availability was due to the oxygen present in the gas mixtures. After acetazoleamide injection, carbon dioxide ceased to have vasodilator action, which was present before injection. However, oxygen may still have caused its usual vasoconstrictor effect which was formerly overriden by the carbon dioxide present in the mixture. Cerebral vessels of the animals treated with large amounts of acetazoleamide no longer repond to either endovascular or extravascular carbon dioxide. Discussion
Since Davies and Brink (3) first applied polarographic methods to measuring oxygen tension in animal tissues, many attempts have been made to calibrate the polarographic electrode in vitro. Roseman, Goodwin, and McCulloch ( 12) compared the polarographic current passed through the brain with the current which flowed when the same electrodes were immersed in stirred and unstirred solutions equilibrated with room air. They concluded that the oxygen tension of brain, if calculated from the unstirred solutions as a standard, was many atmospheres, whereas if
GAS
TRANSPORT
THROUGH
BRAIN
55
calculated from stirred solution, it was the more reasonable value of less than 30 mm Hg. Montgomery and Horwitz (10) measured oxygen tension in intact human skin. They calibrated the electrode by inserting it into excised, dead skin immersed in solutions of several known oxygen tensions. Their normal values of oxygen tensions in human skin were 87 mm Hg, which seemed to be unusually high. None of these calibrations has been theoretically justified, since diffusion coefficients for living tissue are not yet known. According to Kolthoff and Lingane (6), one dimensional polarographic current can be expressed by the following equation: it = nFyCA(D/t)s where it = electrode current in amperes at the time t, n = number of electrons used per molecule of oxygen electrolyzed, Fy = the Faraday, C = initial uniform concentration of oxygen, A = area of the platinum surface in cm2, D = diffusion coefficient of oxygen in cm2/sec. According to this equation, the diffusion coefficient for oxygen in tissue is an important factor, as well as the oxygen concentration of the tissue. Clark and associates (2) developed a membrane-covered polarographic electrode for measuring blood oxygen tension. As far as flowing blood is concerned, the concentration gradient of oxygen does not extend beyond the membrane of this electrode; therefore, calibration of the electrode can be performed in stirred solutions of known oxygen tensions. Recently, Thews (13) analyzed oxygen diffusion in the brain theoretically, and calculated the pOz of the gray matter to be 17 mm Hg at the least supplied place. This value agrees fairly well with our findings in viva (9)) which have shown that normal values of cortical tissue ~0s varied between 10 and 20 mm Hg (10-35, with large electrode), depending on respiratory exchange, cortical blood flow, cortical metabolism, and pH. Zero oxygen tension determined in stirred saline correspondswell with oxygen available, determined at the cerebral cortex following apnea or prolonged nitrogen breathing. However, validity of this procedure of calibration for measuring brain tissue oxygen tension has not yet been determined. The present results of our experiments show evidence for participation of capillary circulation in transport of oxygen through living brain tissue.
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This transport of oxygen is far faster than that expected from diffusion alone. Thus, there are “convection currents” or continuously and rapidly moving currents of oxygen moving through the tissues which are generated by the rapid capillary circulation. Thus, the oxygen concentration gradient does not reach beyond the membrane of the polarographic electrode placed on the pial surface since the capillary circulation has a “stirring effect.” Therefore, as long as circulation is maintained, the calibration in stirred solutions of known oxygen tensions seems to be valid for measuring oxygen tension of brain tissues using the membrane-covered oxygen electrode. Under such conditions, “tissue oxygen tension” measured by the polarographic method should be defined as the oxygen available to the tissue on which the electrode is placed. Calibrations of tissue ~02 in mm Hg [for example, 20 mm Hg) should be interpreted as “the oxygen available to the tissue beneath the electrode is equivalent to stirred saline equilibrated at 20 mm Hg pOz at the same temperature.” The objections to calibrations of tissue or blood by stirred solutions as described above can be overcome either by the use of a membrane permitting slow diffusion of oxygen or minimizing the size of the platinum cathode within the membrane. The first method ensures that movement of oxygen across tissues always exceeds the movement of oxygen within the electrode and hence the gradient of oxygen tension does not extend beyond the membrane. The second method has proven ideal in our hands. We now use a platinum cathode within the membrane less than 100 mu in diameter and only its cross-sectional area is exposed. Such a small cathode consumes so little oxygen that the oxygen gradient does not extend beyond the membrane. Since the electrical output is small greater amplification is needed. This electrode is extremely stable, gives a rapid response time (in seconds) and permits calibration of tissue ~02 in absolute units of mm Hg from stirred or unstirred solutions or gases. Hence tissue calibrations are unaffected by tissue “convection currents” derived from blood flow. Zero readings are the same in viva as in vitro whether achieved by ischemia, nitrogen breathing, or death. Stirring and flow changes do not effect the electrodes within physiological ranges. It has long been assumedthat extravascular carbon dioxide in brain tissuesproduced by cerebral metabolism could dilate cerebral vessels. Our previous studies (9) proved that increased tissue carbon dioxide resulting from increasedcerebral metabolism dilated cerebral vesselsand maintained local circulatory homeostasis. This provided indirect evidence of vaso-
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motor action of extravascular carbon dioxide. The present experiment gives direct evidence for a vasodilator action of extravascular carbon dioxide diffusing through tissue as occurs from cerebral metabolism. It is still obscure whether or not extravascular carbon dioxide must diffuse into the endovascular space to be effective. We suspected previously that it must act directly on smooth muscle of the walls of small arteries (9). Acetazoleamide inhibits the vasomotor action of both endovascular and extravascular carbon dioxide. Therefore, inhibition of vasomotor action of carbon-dioxide inhalation is not due to the disturbance of delivery of carbon dioxide resulting from carbonic anhydrase inhibition in red cells. The mechanism of inhibition must be due to inhibition of carbonic anhydrase at the target point, presumably the cerebral vessel wall itself. Carbonic anhydrase enhances the reaction from CO2 + Hz0 + H+ + HCOB- ; therefore, the final factor responsible for cerebral vasomotor action must be either Hi- or HC03-. However, after acetazoleamide injection, cerebral vessels do not respond to HC03(NaHC03) (9). Hence, H+ seems to be responsible; however, the fact that H+ is less potent a vasodilator than CO, itself remains to be explained. The reason for the more potent effect of CO2 is that the latter passes more readily than H+ through the cell membrane of smooth muscle and lowers the intracellular pH more rapidly (1, 10). It is, thus, concluded that the final factor responsible for cerebral vasomotor activity is a change in intracellular H+ of smooth muscle fibers of cerebral arterioles. References 1. 2. 3.
4. 5.
6.
7.
P. C., Studies of the internal pH of large muscle and nerve fibres. J. Physiol. London 142: 22-62, 1958. CLARK, L. C., R. WOLF, D. GRANCER, and Z. TAYLOR, Continuous recording of blood oxygen tensions by polarography. J. Appl. Physiol. 6: 189-193, 1953. DAVIS, P. W., and F. BRINK, JR., Microelectrodes for measuring local oxygen tension in animal tissues. Rev. Sci. Instr. 13: 524-533, 1942. FENN, W. O., The carbon dioxide dissociation curve of nerve and muscle. Am J. Physiol. 65: 207-223, 1928. GOTOH, F., and J. S. MEYER, A combined electrode for recording absolute tensions of oxygen and carbon dioxide from small areas of tissue. Electroenceph. Clin. Neurophysiol. 13: 119-122, 1961. “Polarography.” New York, Interscience KOLTHOFF, I. M., and J. J. LINGANE, Publishers, 1941. KROGH, A., The rate of diffusion of gases through animal tissues, with some remarks on the coefficient of invasion. J. Physiol. London 52: 391-403, 1919. CALDWELL,
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8. MEYER, J. S., and F. GOTOH, Metabolic and electroencephalographic effects of hyperventilation. A.M.A. Arch. Neurol. 3: 539-552, 1960. 9. MEYER, J. S., and F. GOTOH, Interaction of cerebral hemodynamics and metabolism. Neurology (Suppl.) 11: 46-65, 1961. 10. MEYER, J. S., F. GOTOH, and Y. TAZAKI, CO, narcosis. An experimental study. Neurology 11: 524-537, 1961. 11. MONTGOMERY, H., and 0. HORWITZ, Oxygen tension of tissues by the polarographic method. I. Introduction: oxygen tension and blood flow of the skin of human extremities. /. Clin. Invest. 99: 1120-1130, 1950. 12. ROSEMAN, E., C. W. GOODWIN,and W. S. MCCULLOCH, Rapid changes in cerebral oxygen tension induced by altering the oxygenation and circulation of the blood. J. Neurophysiol. 9: 33-40, 1946. 13. THEWS, G., Die Sauerstoff diffusion im Gehirn. Arch. ges. Physiol., Pf&iiger’s 271: 197-226, 1960. 14. WRIGHT, C. I., The diffusion of carbon dioxide in tissues. J. Gen. Physiol. 17: 657-676,
1934.