J. Phy8iol. (1967), 188, pp. 13-23 13With 4 text-figuresPrinted in Great Britain

MEAN CARBON DIOXIDE TENSION IN THE BRAIN AFTERCARBONIC ANHYDRASE INHIBITION

BY J. BRZEZINSKI,* A. KJALLQUIST AND B. K. SIESJOFrom the Department of Neurosurgery, University of Lund,

and the Neurosurgical Research Laboratory, the Hospital of Lund,Sweden

(Received 18 March 1966)

SUMMARY

1. Measurements of the C02 tensions in arterial blood, in blood from thesuperior sagittal sinus, in cisternal cerebrospinal fluid, and on the surfaceof the cerebral cortex were made in spontaneously breathing anaesthetizedcats and rats after inhibition of carbonic anhydrase with acetazolamide indoses of 50-150 mg/kg.

2. After an acetazolamide dose of 50 mg/kg in cats there was a meanincrease in the c.s.f. C02 tension of 8 mm Hg; after a dose of 100 mg/kgthe corresponding mean increase was 13 mm Hg.

3. Since the CO2 tension measured in venous blood was only moderatelyinfluenced by the acetazolamide, the normal Pco2 difference betweenvenous blood and c.s.f. was markedly reduced. The apparent arterial C02tension, i.e. the C02 tension measured in vitro, always changed to the sameextent as the c.s.f. C02 tension.

4. The findings were confirmed by measurements of the C02 tension onthe surface of the cerebral cortex, and by measurements of the blood andc.s.f. C02 tensions in the rat.

5. It is concluded that the mean tissue C02 tension of the brain can beestimated from the measured arterial C02 tension, even under conditionsof carbonic anhydrase inhibition.

INTRODUCTION

The carbonic anhydrase inhibitor acetazolamide (Diamox) has markedeffects on the central nervous system. It increases the threshold forexperimentally induced convulsions (Woodbury & Karler, 1960), reducesthe rate of c.s.f. formation (Tschirgi, Frost & Taylor, 1954; Wistrand,Nechay & Maren, 1961; Oppelt, Patlak & Rall, 1964), and alters the

* On leave of absence from the Department of Neurosurgery, University of Lodz, Poland.

J. BRZEZINSKI AND OTHERS

normal concentration ratios for chloride and bicarbonate ions betweenblood plasma and c.s.f. (Davson & Luck, 1957; Maren & Robinson, 1960;Schwab, 1962). Brain tissues have a high concentration of carbonieanhydrase which is chiefly in the choroid plexus and the glia cells (Ashby,Garzoli & Schuster, 1952; Giacobini, 1962). The localization of the enzymesuggests that it is involved in active transport mechanisms, but its exact.role in the brain is unknown, and studies of the bicarbonate concentrationin the brain after acetazolamide (Koch & Woodbury, 1960) have failed toreveal any significant influence on tissue acid-base parameters.The main difficulty in studies of tissue acid-base parameters after

acetazolamide lies in the assessment of the true in vivo tissue CO2 tensions.It is well recognized that carbonic anhydrase inhibition leads to a disequi-librium in the CO2 buffer system (Carter & Clark, 1958; Mithoefer, 1959;Cain & Otis, 1960a, b; Wistrand et al. 1961), and that the CO2 tensionsmeasured in arterial and venous blood samples do not represent the truein vivo values. The true mean CO2 tensions of tissues have been approachedby the subcutaneous gas pocket technique (Mithoefer & Davis, 1958) or bycalculation (Cain, 1959), but the results cannot be used to predict themean CO2 tension of a tissue like the brain.The development of a CO2 electrode for measurements on the surface

of tissues (Siesj6, 1961, 1965) has made it possible to study CO2 tensiongradients in the brain. It has been found that the CO2 tension measuredon the surface of the brain, and the c.s.f. CO2 tension, agree with the meantissue CO2 tension calculated from a tissue model using diffusion equations(Ponten & Siesj6, 1966). The present paper gives an account of measure-ments of the tissue surface and c.s.f. CO2 tensions after carbonic anhydraseinhibition and the relations between these tensions and the CO2 tensionsmeasured in samples of arterial and cerebral venous blood. The resultsshow that there is an agreement between the tissue surface and the c.s.f.CO2 tensions even after carbonic anydrase inhibition, and they indicatethat the mean tissue CO2 tension can be either estimated from the CO.tension measured in c.s.f. or calculated from the CO2 tension measuredin samples of arterial blood.

METHODSThe experiments were carried out on twenty-three cats and forty-eight rats. The cats

were anaesthetized with sodium phenobarbitone (initial dose 100 mg/kg body weightyor sodium pentobarbitone (25 mg/kg), while the rats were anaesthetized with sodiumphenobarbitone (150 mg/kg), the initial doses being supplemented with small doses repeatedas required. There was no difference between the results obtained with the two anaesthetics.All animals were tracheotomized and breathed air spontaneously. In all animals one femoralartery was cannulated for sampling of arterial blood, and for blood pressure recording with a

capacitive electromanometer (Elema, Stockholm). If the mean blood pressure fell below

14

BRAIN PCO2 AFTER ACETAZOLAMIDE 1590 mm Hg no further measurements of the CO2 tensions were made. In all cats one femoralvein was cannulated for injections. The rectal temperature of each animal was measuredwith an electrothermometer (Elektrolaboratoriet, Copenhagen).

SamplingAll blood samples were collected in heparinized glass capillaries (40-60 ,t1. capacity).

Arterial blood was obtained from the femoral artery, and venous blood from the superiorsagittal sinus, exposed through a burr hole. Venous blood and c.s.f. were sampled in capil-laries drawn out to a fine point at one end. The c.s.f. samples were obtained from the cisternamagna after exposure of the atlanto-occipital membrane. The sampling of c.s.f. in the ratsrequired the application of gentle suction to the end of the capillary. Arterial samples wereusually taken both before and after the c.s.f. samples, and a second set of samples was takenfrom the animal whenever possible. Manipulation of the atlanto-occipital membrane some-times gave rise to a slight hyperpnoea, indicated by a lowering of the arterial CO2 tensionby 1-4 mm Hg. If the arterial CO2 tension taken before and after the c.s.f. sample differedby 10% or more of the first sample, the result of the set of measurements was discarded.If the difference was smaller the result was accepted but the c.s.f. value was referred to theCO2 tension in the preceding arterial sample to avoid any effect due to manipulation of themembrane. Blood from the superior sagittal sinus could be obtained without suction in bothspecies.

AnalysesThe pH and the PCO2 values were measured within 1 min after withdrawal using a micro -

pH electrode (Radiometer, Copenhagen) and a micro-PC02 electrode (Eschweiler and Co.,Kiel). Both electrodes were maintained at 37-5 + 0-.1 C by means of water circulating from-a water-bath. The factors used when correcting for differences in temperature between theanimals and the electrodes were, for blood samples, 0-015 pH units/' C for pH and 4-6 %/0 Cfor PC02, while the corresponding factor for c.s.f. PCO2 was 3-0 %/0 C (Pont6n & Siesj6, 1966).The range of animal temperatures was 36 0-39.00 C.The tissue CO2 tension was measured only in cats. The tissue CO2 electrode was placed

in a burr hole over the parietal cortex. It was calibrated and used as previously described~Pont6n & Siesjo, 1966).

The over-all accuracy of the measurements of the arterial and the cerebral venous CO2tension was estimated to + 2 % (coefficient of variation). For the c.s.f. and the tissue CO2tensions the corresponding accuracy has been estimated to + 3 % (Ponten & Siesjo, 1966).The CO2 tensions measured in arterial and venous blood from acetazolamide-treated

-animals reflect an equilibrium in the CO2 buffer system which is not attained in the capil-laries in vivo (see Discussion). For that reason the terms arterial and venous C02 tension willbe used only for control conditions in which no inhibitor was administered, while the word-apparent will be used to characterize the arterial and venous CO2 tensions in animals givenacetazolamide. It was assumed that the c.s.f. and the tissue C02 electrode came into equili-brium with the tissue after acetazolamide.

Experimental variablesThe carbonic anhydrase inhibitor acetazolamide (Diamox, American Cyanamide Co.,

New York) was given as a 10% solution of the sodium salt in distilled water (1 g/10 ml.).In the cat, 50 mg/kg was given intravenously in the course of 10-15 sec, either as a singledose or repeated once or twice at intervals of 1 hr. Arterial, venous and c.s.f. CO2 tensionswere measured successfully in fifteen cats while the remaining eight cats were used formeasurements of the CO2 tension on the surface of the brain. In eight cats of the first groupcontrol values were obtained before acetazolamide was administered, giving a total ofeighteen complete analyses. Corresponding PCO2 values after acetazolamide were obtained

J. BRZEZINSKI AND OTHERSin all the fifteen cats with a total of eighty-nine complete analyses. A complete analysisimplied that arterial and venous blood as well as c.s.f. could be sampled successively withoutany further delay than that caused by the measurement of the preceding sample.The rats were given acetazolamide in doses of 50 mg/kg; some received a single dose,

others received a second dose after an interval of 1 hr. The animals were anaesthetized andprepared 1 or 2 hr respectively after the last dose.

Control experiments were carried out by injecting three cats with an equimolar dose of2-acetamino-1,3,4-thiadiazole-5 (N-t-butyl)-sulphonamide (CL 13850, American CyanamideCo., New York), a similar compound which is devoid of carbonic anhydrase inhibitor activity(Wistrand et al. 1961). To each mole of CL 13850, 1-5 moles of NaOH was added. An equalamount of base was thus injected with the two drugs.

RESULTS

Experiments on cats. Measurements of the c.s.f. and the cortical surfaceCO2 tensions in cats anaesthetized with barbiturates indicate that themean tissue CO2 tension exceeds the arithmetic mean of the arterial andthe cerebral venous CO2 tensions by about 0-5 mm Hg. Since the arterio-venous PCO2 difference at an arterial PCO2 of 40 mm Hg is close to 11 mmHg it follows that the mean tissue CO2 tension is about 6-0 mm Hg higherthan the arterial and about 5 mm Hg lower than the venous CO2 tension(Ponten & Siesjo, 1966).These general results were confirmed in the present experiments. Thus,

measurements of the blood and the c.s.f. CO2 tensions before the injectionof acetazolamide showed that the mean difference between the c.s.f. andthe arterial CO2 tensions was 5-6 mm Hg while the corresponding venous-c.s.f. difference was 5-7 mm Hg (see Table 1 below). These relations werenot significantly altered by the intravenous injection of the control sub-stance CL 13850 in three cats. The only effects which could be referred tothe injection were transient variations in the arterial CO2 tension. Thus,arterial samples drawn within 1 min after the injection often showed adecrease in the CO2 tension. This decrease was often followed by an in-creased CO2 tension for 15-30 min. Similar effects were seen after injectingNaOH in an equal amount.

Injection of acetazolamide in a dose of 50 mg/kg was invariably followedby an increase in the c.s.f. CO2 tension and in the apparent arterial CO2tension (Fig. 1). The changes measured in cerebral venous blood wereeither variable or insignificant. The absolute increase in the c.s.f. CO2tension varied from 3-5 to 13 mm Hg with a mean of 8 mm Hg.

If a second acetazolamide dose of 50 mg/kg was given there was usuallya further increase in the c.s.f. and in the apparent arterial CO2 tensions sothat the mean total increase in the c.s.f. CO2 tensions after 100 mg/kgwas 13 mm Hg. The effects seen upon increasing the dose to 150 mg/kgwere inconsistent.Although there was a consistent increase in the c.s.f. CO2 tensions after

16

BRAIN Pco2 AFTER ACETAZOLAMIDEan acetazolamide dose of 50 mg/kg, and usually a larger increase after100 mg/kg, the magnitude of the changes varied too widely to allow thec.s.f. CO2 tension to be predicted from the dose administered. This isclearly illustrated by Fig. 2, which shows an experiment in which threedoses of Diamox of 50 mg/kg each were given to a cat with subsequentmeasurement of the CO2 tensions. Here, the maximal observed increase

50 r-

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he

2v 30

20

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:-'.-

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aDiamox 50 mg/kg

a Fem. art.O Sup. sag. sin.* C.s.f.

I ~~~~I I

0 1 2Time (hr)

3 4 5

Fig. 1. The relation between the CO2 tensions in arterial blood (triangles), in bloodfrom the superior sagittal sinus (squares) and in cisternal c.s.f. (filled circles).Cat, pentobarbitone anaesthesia and spontaneous respiration. Acetazolamide50 mg/kg was given at zero time.

60 r-

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Fig. 2. The relation between the CO2 tensions in arterial blood (triangles), incerebral venous blood (squares) and in c.s.f. (filled circles). Cat, pentobarbitoneanaesthesia and spontaneous respiration. Three doses of acetazolamide of 50 mg/kg each were given with an interval of 1 hr (D).

17a-A^

I I

2 Physiol. I88

i A

J. BRZEZINSKI AND OTHERS

in the c.s.f. CO2 tension was only 3 mm Hg. However, there was a con-stant relation between the c.s.f. CO2 tension and the apparent arterial CO2tension, irrespective of the dose or the time lag between drug administra-tion and analysis (Fig. 1 and 2). Figure 3, which relates the arterial CO2 to

70

60 - o°00

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0 80o%

0~~~~~

0~~~~~

40 O40, - i ° X

30 40 50 60Art. Pco2 (mm Hg)

Fig. 3. The relation between the C02 tensions measured in arterial blood and inc.s.f. samples in uninjected control cats (filled circles; eighteen observations) or incats injected I.v. with 50-150 mg/kg of acetazolamide (unfilled circles; eighty-nineobservations).

the corresponding c.s.f. CO2 tensions, shows all control analyses and allanalyses obtained after acetazolamide. Table 1 shows the c.s.f.-arterial,and the venous-c.s.f. Pco, differences obtained in the whole material, aswell as in the groups given different doses of acetazolamide. Statisticalanalysis confirmed that the c.s.f.-arterial PCO differences after acetazol-amide did not significantly differ from those of the uninjected controlgroup (P > 0 1). However, the venous-c.s.f. PCO2 differences after acetazol-amide were significantly lower than in the control group (P < 0001).

18

BRAIN Poo AFTER ACETAZOLAMIDE 19This means that the usual relation between the arterial, the venous, andthe c.s.f. CO2 tensions, in which the c.s.f. CO2 tensions exceed the meancapillary CO2 tensions by about 0-5 mm Hg (Pont6n & Siesj6, 1966), is notmaintained after carbonic anhydrase inhibition. However, the apparentarterial CO2 tension in animals which have been given acetazolamide canbe used to calculate the c.s.f. CO2 tension, exactly as can be done in theanimals which had not received the drug.

TABLE 1. The relation between the arterial, the cerebral venous, and the c.s.f. CO2 tensionsafter acetazolamide in cats under pentobarbitone or phenobarbitone anaesthesia. The valuesgiven are the means + S.E.

C.s.f. PCO2 VenousNo. Arterial C.s.f. Venous minus Pco2

Type of of PCo2 Pco2 Pco2 arterial minusexpt. expt. (mm Hg) (mm Hg) (mm Hg) pco2 c.s.f. Pco2

Control 18 36-6+0-7 42-5+0-6 48-2+0-9 5-6+0-3 5-7+0-3(uninjected)

Acetazolamide50 mg/kg 24 37-6+0-5 44-5+0-5 43-7+0-8 6-9+0-4 -0-8+0-5100 mg/kg 38 45-2+0-5 50-4+0-7 51-6+0-7 5-2+0-3 1-2+0-4150 mg/kg 27 44-8+1-2 51-2+1-2 52-6+1-4 6-4+0-3 1-4+0-4

All animals in- 89 43-0+0-6 49-0+0-6 49-7+0-7 6-0+0-3 0-7+0-3jected withacetazolamide

TABLE 2. Acid-base parameters in arterial blood and cisternal c.s.f. inrats after acetazolamide (means and standard errors)

Act. Stand.No Arterial C.s.f. HCO3- HC03-

Type of of PCO2 pco2 APco2 (m- (m-expt. expt. pH (mm Hg) (mm Hg) (mm Hg) moles/l.) moles/l.)

Controls 21 7-42+0-01 40-9+0-8 46-1+1-1 5-2+0-7 27-1+0-5 26-6 +0-6uninjectedCL 13850 5 7-42+ 0-01 43-5+1-3 48-6+1-1 5-1+1-2 27-7+0-7 25-6+0-4Acetazolamide

50 mg/kg 8 7-28+0-01 48-9+2-0 55-1+2-2 6-4+1-0 22-4+ 0-8 20-7+0-5100 mg/kg 8 7-27 + 0-01 44-9+1-5 50-1+1-6 5-2+0-9 19-2+0-6 18-3+0-6

Measurements were made of the pH and the CO2 tension in arterial blood, and of the CO2tension of the c.s.f. APCo2 is the difference between the CO2 tensions in c.s.f. and in aterialblood. The number of experiments given for the uninjected controls (21) refers to the PCO,measurements. The pH and the bicarbonate figures are the means of only ten measurements.

Measurements of the CO2 tension on the surface of the cerebral cortexwere performed as control experiments in five cats. The results of theseexperiments were similar to those obtained on the c.s.f. Thus, there was asimilar increase in the cortical surface PCO2 as in the c.s.f. Pco2, and therelation between the arterial and the tissue surface CO2 tensions aftervarious doses of acetazolamide was similar to the relation between thearterial and the c.s.f. CO2 tensions. The results are exemplified by Fig. 4.

2-2

J. BRZEZINSKI AND OTHERS

In this experiment the absolute increase in the tissue surface CO2 tensionwas 9 mm Hg, and the Pco. difference between the apparent arterial andcortical surface CO2 tensions remained fairly constant around 7 mm Hg.Rat experimenrt8. Table 2 contains the mean values for the parameters

studied. The difference in CO2 tension between the arterial blood and c.s.f.was 5-2 + 0 7 mm Hg in the uninjected control group and 5-1 + 1F2 mm Hgin the group injected with the control substance 13850. These differences

60

A Fem. art.o Sup. sag. sin.* C.s.f.

00-8°50 ko' t_

X40__A

tlD D D

30 1 l I-1 0 1 2 3 4 5 6 7

Time (hr)

Fig. 4. The relation between the C00 tensions in arterial (triangles) and in cere-bral venous (squares) blood, and the C02 tension measured on the surface of thetissue with a C02 electrode (half-filled circles). Cat, pentobarbital anaesthesia,spontaneous respiration. At zero time, acetazolamide 50 mg/kg was given i.v.

are slightly lower thanthe corresponding differences in the cat (Pont6n &Siesj6, 1966, and Table 1 above), a fact which probably reflects thedifficulty of sampling c.s.f. in rats. Control experiments showed that thearteriovenous Pco2 difference in uninjected rats was similar to that in thecat (11-0 + 0-8 mm Hg at a mean arterial Pco2 of 39-9 mm Hg, n = 6).In both the rats given an acetazolamide dose of 50 mg/kg, and in those

receiving 100 mg/kg, there were clear signs of an acidosis. Thus, boththe pH values and the plasma standard bicarbonate concentrationsafter acetazolamide were significantly lower than in the control group(P < 0-001). However, although both the apparent arterial and the c.s.f.CO2 tensions were increased after acetazolamide, especially after 50 mg/kg(P < 0-001), there was a proportional increase of the two and conse-quently no change in the relation between them. These findings confirmthe results obtained in the cats.

20

BRAIN Pco0 AFTER ACETAZOLAMIDE

DISCUSSION

It is well established that inhibition of carbonic anhydrase leads to adisequilibrium in the CO2 buffer system in the body, i.e. that the hydrationof carbon dioxide or the dehydration of carbonic acid will no longer becomplete during the passage of the circulating blood through the vascularbeds of the peripheral tissue or the lungs. This disequilibrium results in acontinuous increase in the CO2 tension of blood as it flows from the lungsand approaches the tissues, and a CO2 tension of the emerging venous bloodhigher than that of blood in main peripheral veins. When arterial andvenous blood samples are drawn and analysed for their acid-base para-meters there will be time for a complete equilibrium and, consequently,the measured CO2 tensions will deviate from those existing in vivo.The disequilibrium existing in vivo wil complicate the estimation of the

mean tissue CO2 tensions in the peripheral tissues. After acetazolamide theCO2 tensions measured in arterial samples with in vitro methods mayexceed the alveolar tensions by as much as 20 mm Hg, while the samemethods underestimate the in vivo venous CO2 tensions by about 4 mm Hg(Carter & Clark, 1958; Cain, 1959; Wistrand et al. 1961). However, thesefigures will be of limited value for the assessment of the mean tissue CO2tensions since the information needed concerns the gradients of CO2tension in the tissue capillaries.The related difficulties have made it necessary to approach the tissue

tensions after carbonic anhydrase inhibition with other methods. By meansof indirect calculations it has been estimated that the tissue CO2 tensionsincrease by 10-33 mm Hg after a conventional dose of Diamox (Cain,1959). A more direct estimate of 9 mm Hg was obtained by using the sub-cutaneous gas pocket technique in rats (Mithoefer & Davis, 1958). Theparadoxical effects of a decreased alveolar CO2 tension and an increasedtissue CO2 tension were confirmed by Meyer & Gotoh (1961), who reportedthat the brain tissue CO2 tension often increased more than 20 mm Hgafter a 500 mg dose of Diamox to a curarized monkey. However, theirmethods do not allow further quantitative conclusions.The present experiments have shown that inhibition of carbonic

anhydrase with acetazolamide causes an increase in the mean tissue CO2tension in the brain, as indicated by the c.s.f. CO2 tension, and by the CO2tension measured on the surface of the tissue. The most important findingis that the resulting tissue CO2 tension has a constant relation to theapparent arterial CO2 tension, i.e. to the CO2 tension measured with invitro methods. Moreover, the difference between the mean tissue CO2tension and the apparent arterial CO2 tension (about 6 mm Hg) was equalto the corresponding difference in the uninjected animal at the same arterial

21

J. BRZEZINSKI AND OTHERS

Pco0. This relation allows the mean tissue C02 tension to be calculated byadding 6 mm Hg to the arterial C02 tension measured with conventionalmethods on arterial blood samples.The conclusions drawn from the present experiments regarding the

tissue C02 tension are based on the assumption that both the C02 tensionmeasured in cisternal c.s.f. and the C02 tension measured on the surface ofthe tissue with a C02 electrode, reflect the mean tissue C02 tension. Thereis ample evidence that this is normally the case. Thus both these experi-mental C02 tensions agree with the mean tissue C02 tension calculatedfrom the diffusion equations applied to the Krogh (1918-19) diffusioncylinder (Pont6n & Siesjo, 1966). There is no simple way to derive atheoretical mean tissue C02 tension to compare with the experimentallyobtained ones when the carbonic anhydrase has been inhibited. This ismainly due to the difficulty of assessing the kinetics of the enzyme reac-tions in vivo (cf. Roughton, 1943-44), and clearly such a calculation wouldhave to be based on rather tenuous assumptions with regard to thelongitudinal and radial tension gradients within the tissue. However, thereseems to be no reason to suspect the validity of the c.s.f. as a tonometer, orthat a tissue C02 electrode does not reflect a mean tissue C02 tension inthe underlying tissue, also under conditions of carbonic anhydraseinhibition.

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CAiN, S. M. (1959). Respiratory effects of carbonic anydrase inhibition. Dissertation. Gaines-ville: University of Florida.

CAN, S. M. & OTis, A. B. (1960a). Effect of carbonic anhydrase inhibition on mixed venousC02 tension in anaesthetized dogs. J. appl. Physiol. 15, 390-392.

CAN, S. M. & OTis, A. B. (1960b). C02 retention in anaesthetized dogs after inhibition ofcarbonic anhydrase. Proc. Soc. exp. Biol. Med. 103, 439-441.

CARTEmR, E. T. & CLARK, R. T., Jr. (1958). Respiratory effects of carbonic anhydraseinhibition in the trained anaesthetized dog. J. appl. Physiol. 13, 42-46.

DAvsoN, H. & LuCK, C. P. (1957). The effect of acetazolamide on the chemical compositionof the aqueous humour and cerebrospinal fluid of some mammalian species and on therate of turnover of 24Na in these fluids. J. Phy8iol. 137, 273-293.

GiACOBINi, E. (1962). A cytochemical study of the localization of carbonic anhydrase inthe nervous system. J. Neurochem. 9, 169-177.

KOCH, A. & WOODBIuRY, D. M. (1960). Carbonic anhydrase inhibition and brain electrolytecomposition. Am. J. Physiol. 198, 434-440.

KROGH, A. (1918-19). The number and distribution of capillaries in muscles with calcula-tion of the oxygen pressure head necessary for supplying the tissue. J. Phy8iol. 52,409-415.

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BRAIN PCO2 AFTER ACETAZOLAMIDE 23MITHOEFER, J. C. (1959). Inhibition of carbonic anhydrase: its effect on carbon dioxide

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8cand. 67, 129-140.ROUGHTON, F. J. W. (1943-44). Some recent work on the chemistry of carbon dioxide

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TSCHIRGI, R. D., FROST, R. W. & TAYLOR, J. L. (1954). Inhibition of CSF formation by acarbonic anhydrase inhibitor, 2-acetylamino-1,3,4-thiadiazole-5-sulfonamide (Diamox).Proc. Soc. exp. Biol. Med. 87, 373-376.

WISTRAND, P. NECHAY, B. R. & MAREN, T. M. (1961). Effects of carbonic anhydraseinhibition on cerebrospinal and intraocular fluids in the dog. Acta pharmac. tox. 17,315-336.

WOODBURY, D. M. & KARLER, R. (1960). The role of carbon dioxide in the nervous system.Anaesthesiology 21, 686-703.