The Measurement of Cerebral Blood Flow in the Rat

Transcription

1 The Measurement of Cerebral Blood Flow in the Rat BY AKIRA MATSUMOTO, M.D., RICHARD NAMON, YOHICHI UTSUNOMIYA, M.D., KYUYA KOGURE, M.D., PERITZ SCHEINBERG, M.D., AND OSCAR M. REINMUTH, M.D. Abstract: The Measurement of Cerebral Blood Flow in the Rat Cerebral blood flow (CBF) was determined in the rat under 70% nitrous oxide anesthesia and pentobarbital anesthesia. The application of the Fick principle technique of Kety et al. was modified utilizing l Xe infused intravenously steadily for 0 seconds, at which time the animal was decapitated and the head frozen in liquid nitrogen. A prior femoral artery to femoral vein shunt was led through a polyethylene catheter of 0. ml volume. This catheter passed as a coil in a Nal crystal well-counter with the arterial Xe concentration curve recorded by a ratemeter-recorder system. The results of the hemispheric blood flow (HBF) were: under 70% nitrous oxide anesthesia in normocapnia (Paco, 8 mm Hg), 8 ± ml/00 gm per minute; with hypocapnia (Paco, 0 mm Hg), 0 ± ml/00 gm per minute; with hypercapnia (Paco, mm Hg), 87 ± 0 ml/00 gm per minute; and with pentobarbital anesthesia (Paco, 8 mm Hg), ± 8 ml/00 gm per minute. Additional Key Words regional cerebral blood flow nitrous oxide pentobarbital hypercapnia hypocapnia Xe Downloaded from by on December, 08 Introduction D A great deal of interest has been attached to the study of metabolic functions in the rat brain. In many instances these metabolic data depend in part for their interpretation on knowledge of overall cerebral metabolic rate and of cerebral blood flow (CBF). Consequently, it is desirable to have methods for measurement of CBF in small animals. The usual modification of the Fick technique and of external detector washout method have made CBF measurement in the rat less than satisfactory, since there is considerable technical difficulty in serial sampling of blood from the sagittal sinus of small animals and because of the problems posed by the small size of the brain. Recently we established a method which modifies the Fick principle used by Kety, Landau and Reivich utilizing Xe to measure CBF in the rat, the details of which are described here. Methods The experiments were performed utilizing male Wistar rats weighing 0 to 0 gm. Anesthesia was induced with ether, and mg per kilogram of body weight of tubocurarine From the Cerebral Vascular Disease Research Center, Department of Neurology, University of Miami School of Medicine, P.O. Box 087, Biscayne Annex, Miami, Florida. Dr. Matsumoto is presently in the Department of Neurological Surgery, Okayama University Medical School, -- Shikata-cho, Okayama City, 700 Japan. This work was supported by the National Institute of Neurological Diseases and Stroke Research Grant NS and the Meyer Gold Research Fund. Reprint requests to Dr. Reinmuth. chloride was injected intraperitoneally. Tracheostomy was performed immediately thereafter and an endotracheal tube was inserted to maintain artificial ventilation with 70% nitrous oxide and 0% oxygen. Rectal temperature was controlled at 7 C with an electric lamp. The right femoral artery was cannulated with a PE-0 polyethylene tube (O.D. 0.08"), and a PE-0 polyethylene tube (O.D. 0.0") was cannulated through the right jugular vein into the superior vena cava as close as possible to the right atrium. The artery was used for continuous monitoring of arterial blood pressure and for anaerobic sampling of arterial blood. The vein was used for infusion of the radioactive tracer. A small coil (volume: 0. ml, total length: cm) of PE-0 tubing was placed between the left femoral artery and the right femoral vein as an arteriovenous shunt. This was inserted into a well scintillation counter (Harshaw Type SW-- W, " diameter Nal crystal) and was used for continuously recording the concentration of the radioactive tracers in the arterial blood by a ratemeter (Nuclear-Chicago, Model 87 Dual Channel Ratemeter) and recorder (Hewlett-Packard, Model 700B-9- Strip Chart Recorder). The animal was fixed in the prone position on an operating stand to which a decapitator was attached. A midsagittal incision was made over the cranial vault and the skin was reflected. A small bottomless plastic cup was sutured in the wound over the cranial vault for eventual freezing (fig. ). The animals were maintained with Paco, values of to 0 mm Hg, Pao, values of 9 to 0 mm Hg and blood pressure of over 00 mm Hg. After a steady state period of at least 0 minutes, Xe physiological saline solution containing 00 to 00 nc\ per milliliter of Xe (Mallinckrodt, Xeneisol, 0.0 to.7 mci per milliliter) was infused steadily at a constant speed (0. ml per minute) with an infusion pump (Braun Apparatebau, Type 80 Infusion Pump, West Germany). I.V. infusion was chosen over inhalation, since for a fixed dose of l Xe the I.V. delivery provided the 0 Stroke. Vol., November-December 97

2 MEASUREMENT OF CBF I N THE RAT Experimental arrangement for rat CBF. The picture illustrates the rat fixed in a headholder with the mechanical decapitator in place, the animal respirator to tracheal cannula, the microinfusion pump for l"xe solution infusion, the arteriovenous shunt leading to a wellcounter, and the ratemeter-recording devices. Downloaded from by on December, 08 highest arterial IM Xe concentration as well as a linear change in concentration. Thirty seconds later, decapitation was performed. Simultaneously the head was frozen with liquid nitrogen to trap xenon in the brain tissue, since the temperature of liquid nitrogen ( C) is lower than the melting point of xenon ( l.9 C). The brain was chiseled out in the frozen state, and divided into right and left hemispheres. Each hemisphere was put into a separate test tube and measured for radioactivity using the same wellcounter which was used for measuring the arterial concentration of xenon. After determining the weight of each hemisphere, hemispheric blood flow (HBF) was calculated according to the formula of Kety:'- Ci(T) = Xki J? T Ca-e- K " T - t) dt where Ci(T) is the concentration of xenon in the brain tissue at time T (0 seconds in these studies), X is the blood-brain partition coefficient for xenon, Ki is the rate of blood flow per unit weight of brain tissue multiplied by the reciprocal of the partition coefficient for that tissue, and Ca is the concentration of xenon in arterial blood. Flow was calculated from a hand integration of the arterial "'Xe time-concentration curve, using an ingenious simplification of computation devised by Goldman and Sapirstein. For the curve integration, no correction was made for the smearing factor of the A-V shunt coil. Since l u Xe of any given arterial concentration arrived in the brain and in the A-V shunt coil at a different time, this time difference was measured and the recorded arterial curve shifted accordingly. This correction varied from three to four seconds. To determine the value of X, rats were used and divided into two groups of six rats. Following the same procedure as outlined above, MXe solution (0 to 0 iic\ per milliliter) was infused intravenously at a constant speed of 0. ml per minute. In one group of animals after the arterial Slrokt, Vol., Nonmbtr-Dtcimbtr 97 curve of xenon had reached a plateau maintained for one minute, and five or six minutes after xenon injection had begun, arterial blood was sampled and decapitation was performed. In the other group arterial blood sampling and decapitation were performed ten minutes after starting infusion of the xenon solution. The X value was calculated from the ratio of the radioactivity in a unit of brain tissue to a unit of blood. The size of a rat's brain is small and contains small relative amounts of white matter, and we did not separate gray matter and white matter in the hemispheres. After a regular steady state period of 0 minutes, either hypocapnia or hypercapnia was induced. Hypocapnia was induced by increasing the tidal volume and Paco, was kept around 0 mm Hg. Hypercapnia was induced by adding CO, gas to the inspired gas and Paco, was adjusted to 0 mm Hg as nearly as possible. In both cases, the ventilation rate was kept constant. HBF determination was performed five minutes after the beginning of either hypocapnia or hypercapnia. To assess the effect of pentobarbital anesthesia, the animals were tracheostomized, cannulated and fixed in the prone position on an operating stand under 70% nitrous oxide anesthesia. Pentobarbital (0 mg per kilogram) was then injected intraperitoneally. At the same time inspired gas was switched to 70% nitrogen instead of 70% nitrous oxide. Then the animals were kept at a steady state (and at Paco, of to 0 mm Hg and Pao, of 9 to 0 mm Hg) for a period of 0 minutes until HBF determinations were performed. Results The values of A, which were determined from the rat hemispheres, are shown in table. These values are very close to unity which is often assumed to be the A value. We utilized unity as the value of X to simplify the calculation of HBF. The results of the HBF in the rat in various conditions are as shown in tables and. In normocapnia under 70% nitrous oxide anesthesia HBF was 8 ± ml/00 gm per minute (mean ± SD); with hypocapnia the Paco, was about 0 mm Hg and HBF was 0 ± ml/00 gm per minute; with hypercapnia the Paco, was mm Hg and HBF was 87 ± 0 ml/00 gm per minute. With pentobarbital anesthesia the Paco, was 8 mm Hg and HBF was ± 8 ml/00 gm per minute. Discussion A great variety of methods for determining CBF have been developed since Kety and Schmidt" established the nitrous oxide method. External detection and the use of radionuclide tracers allow not only CBF of the whole brain, but also regional CBF to be determined in both man and the experimental animal. Every method has some disadvantage, and there is as yet no perfect method for all purposes. There are technical difficulties especially in determining CBF in small animals like the rat. Nevertheless, several investigators have already reported CBF values in the rat brain utilizing different methods (table ). The particular importance of adapting CBF determinations to the rat as an experimental animal

3 MATSUMOTO, NAMON, UTSUNOMIYA, KOGURE, SCHEINBERG, REINMUTH TABLE Parfition Coefficient (\) of m Xe in the Rat Brain Hemispheres at Different Times of Equilibrium Animal Pocoj Pao; BP Hb X of hemispheres no. ph (gm%) R side Lside Group. - minutes decapitation Mean SD SE Group. J 0 minutes decapitation Mean SD SE R = right, L = left. Downloaded from by on December, 08 depends upon the frequency with which this animal has served as a model for a variety of neurochemical, physiological and pathological changes in the brain. In many experimental states a knowledge of the effects of a given manipulation of the model on the CBF would be valuable or even critical to know. Not only the small size of the animal but the peculiarities of its brain arterial supply and venous drainage as well as the location of the brain in the cranium pose serious difficulties in translating methods useful in other animals to rats or smaller animals. Moreover, there are many experimental states in which the assumptions of the Kety-Schmidt technique (and its radionuclide-related techniques) cannot be made. As examples, local or regional brain disease may violate the assumption of homogeneous perfusion, and changing physiological states associated with an acute stress may violate the requirement of a steady state in regard to several variables. One method may be perfectly suitable for one kind of experiment, while completely invalid for another. One of the most difficult and most important of the requirements is that of the steady state, and a method demanding relatively short time intervals during which a steady state must be produced (particularly in regard to blood pressure, Pao,, Paco,, intracranial pressure, and distribution of cerebral edema or ischemia) would have great utility. Regional clearance of hydrogen, measured at the tip of a hydrogen electrode, allows quantitative estimates of CBF at the electrode tip. 7 This does not allow estimates of larger regions as of average hemispheric flow. Utilization of thermocouples 8 or thermistors allows monitoring of directional changes, but cannot be readily calibrated to derive quantitative flow and, like the hydrogen electrode, may produce local tissue damage on insertion. Aukland 9 studied the effect of electrode damage and concluded it to be minimal, and Haining et al. 7 agreed in a study of the rat brain. Our own experience with fine thermistor probes indicates that although gross damage to the tissue by the probe is readily recognized by the poor response characteristics in the measurements, minimal injury is not unusual and must be a factor in disturbing the local physiological state of the tissue, not only potentially altering flow characteristics but, more importantly, disturbing the biochemical state of the regional tissue. Radionuclide-labeled microspheres 0 pose a problem in achieving and maintaining uniform suspension during injection and also require simultaneous measurement of the cardiac output. These limitations may be serious in studies assessing changing experimental conditions or in which biochemical measurements are to be compared. A similar objection may be raised to the indicatorfractionation technique of Goldman and Sapirstein using u C-antipyrine. Both of these methods have the sometimes-advantage of allowing mean values in dissectable regions of the brain. Sokoloff and Reivich et al. - reported regional CBF values by an autoradiographical measurement of u C-deoxyglucose accumulation in the brain. Under physiological conditions, and perhaps in some Stroke, Vol.. November-December 97

4 MEASUREMENT OF CBF IN THE RAT TABLE Experimental Data of Hemispheric Blood Flow Under 70% N,O Anesthesia Downloaded from by on December, 08 Animal no. Paco* 7. Normocapnia Mean 7.7 SD 0.8 SE 0.. Hypocapnia 7 Mean SD SE Hypercapnia Mean.7 SD. SE. ph Poo? pathophysiological states, local glucose metabolism correlated closely with local blood flow. Whether this relationship holds in other pathological states, e.g., infarction, is doubtful. This reservation and the necessity of this method for a steady state of approximately 0 minutes during flow measurement would prevent application to many experimental situations. The method has other extremely important advantages aside from these, referable to assessing alterations and distribution of metabolism relative to the functional organization of the brain. Recently Eklof and Siesjo et al. " 8 have actively investigated the measurement of CBF in the rat. They discussed two methods. The first was modified from BP 70 ] Hb (Bm%) R HBF(rnl/00gm/rnin) L Kety, Landau, and Reivich, using several kinds of radioisotopes and directly counting the radioactivity in the brain tissues. The second was a Kety-Schmidt method using Xe gas inhalation and calculating CBF from its arterial and venous desaturation curves. They pointed out that with all indicators the Landau- Reivich method showed low CBF values in hypercapnic conditions compared to values calculated from the A-V difference of oxygen and carbon dioxide. They showed variation of CBF values depending upon the decapitation timing in the Landau-Reivich method. Consequently, they concluded that the Kety-Schmidt method was preferable and suggested that the Landau-Reivich method would be effective only if U C- Slroke, Vol., November-December 97

5 MATSUMOTO, NAMON, UTSUNOMIYA, KOGURE, SCHEINBERG, REINMUTH TABLE Experimental Data of Hemispheric Blood Flow Under Pentobarbital Anesthesia Animal no Mean SD SE Pacoi PH Pao» BP Hb (gm%) R HBF(rnl/ 00 grn/min) L ethanol was used as a tracer, and decapitation was done 0 seconds after starting the radioisotopic injection in the rat. The Kety-Schmidt method is preferred, since one can get the value of the cerebral metabolic rate of oxygen utilization (CMR0 ) and that of CBF at the same time, and there are fewer hypothetical assumptions in the formula for the CBF calculation than in other methods. But in the Kety-Schmidt method as modified by Eklof and Siesjo, blood transfusion was necessary to prevent blood pressure decreases while sampling blood for the determination of CBF. It might be difficult to be certain that this method can be performed under uniform physiological conditions. Eklof and his co-workers observed that C-ethanol, by virtue of its faster blood-to-brain equilibration, is a preferable indicator. In their method, as in that presently described, the bloodradionuclide concentration curve must be accurately assessed. Direct arterial sampling is necessary for measuring the 0 activity of C-ethanol a technical Downloaded from by on December, 08 TABLE Cerebral Blood Flow Studies in the Rat Author Method Anesthesia Leatherman et al., 97 8 Haining et al., 98 7 Pannier et al., 97 0 Goldman et al., 97 Eklof et al., 97 ' Ekiof et al., 97 Gjedde et al., 97 8 Present study Thermocouple Hydrogen clearance Ce microspheres C-antipyrine indicator fractionation technique Xe inhalation desaturation, Kety-Schmidt I C-antipyrine tissue direct count C-ethanol tissue direct count H-water tissue direct count Xe tissue direct count Xe inhalation desaturation, Kety-Schmidt Xe tissue direct count A-V shunt Unanesthetized and pentobarbital Awake Pentobarbital Awake 70% N O 70% N O 70% N O 70% N O 70% N O 70% N,O 70% N O Pentobarbital 7 0 CBF (ml/ 00 Paco- gm/min) Area of (mean ± SE) brain ± ± 0. ± 0. ±.0 ± 0. ± 0. *.0 ± 0.7 ± 0.9 ± 0.7 ±.0 ± 0.8 ±.0 ±.0 ± 0. ± 0. ±. ± =t = ± 00 ± = = 9 =*= 0 ± ± 0 ± 0 ± 90 ± 7 ± ± 98 ± 0 ± 8 ± 87 ± ± Thermocoup Frontal cortex Whole brain Whole brain Hemisphere Stroke, Vol.. November-December 97

6 MEASUREMENT OF CBF IN THE RAT Downloaded from by on December, 08 problem of surprising magnitude because of the need to relate the timing of the samples to the brain-arrival time of blood of comparable concentration. Since they showed that a second source of error related to assumptions of homogeneous perfusion requires utilization of short infusion times (recommended 0 seconds), such sampling time errors add to the difficulty of the method. Use of Xe allows direct measurement of the blood concentration in an artificial A-V shunt, by virtue of the y activity of the radionuclide, and the time error between shunt and brain can be directly assessed by external detection of radionuclide peak between shunt and brain. Gjedde et al. 7 ' 8 have recently improved the method of Eklof by cannulation of the rat jugular bulb and this application of the Kety-Schmidt technique, using integrated blood samples, appears to be the most satisfactory method for experimental states in which a uniform, stable alteration in CBF may be assessed for the whole brain. It must be observed that the assumption that the lateral sinus-jugular bulb blood of the rat is representative of mixed cerebral venous total outflow has never been established for the rat, nor for any animal other than monkey or man, and in those species only incompletely. This assumption is essential for estimation of CBF by the Kety- Schmidt technique and is probably true for the rat based on indirect evidence (e.g., derived flow values vary appropriately with changes in Paco ). Since there are no published values for the partition coefficient (X) determined in the rat brain for Xe, we evaluated this in a way suitable for the particular application we had in mind for this method. That is, our biochemical determinations were mean values for particular gross segments of whole brain representing indeterminate mixtures of gray and white matter, and it is for such whole brain mixtures of comparable kind that we wish to derive flow values. Similarly, our arterial Xe concentration was achieved by slow ramp intravenous injection of Xe saline solutions which resulted in a very much more rapid arterial plateau than that achieved by breathing a fixed concentration of gaseous Xe. In our determination of X, arterial plateau occurred prior to five minutes in all animals, judged by the continuously recorded A-V shunt coil values. In the second group of animals, the infusion time was doubled to ten minutes, and the derived values shown in table indicate essentially identical results. Hemoglobin values in these animals varied from to 7 gm % and in this range the variation in X related to hemoglobin differences is slight and can be disregarded, although variations beyond these concentrations should not be ignored. 9 In the application that we have for this method, animals with hemoglobin outside these ranges would not be appropriate, but should one study, for example, hypovolemic hypotension produced by phlebotomy, different X values would need to be determined for measurements made on extremes of hemoglobin concentrations. For purposes of validation of any method of cerebral circulation, no ideal reference standard exists, a problem which has always bedeviled investigators interested in the cerebral circulation. Direct methods, such as the bubble-flowmeter, are not adaptable to the vascular tree of lower order mammals, for example. The next best approach is to use the best validated method used by others. For the rat as well as for most species, this is a Kety-Schmidt method, and yet application of this approach is still in a developmental stage methodologically for the rat (as has been already noted). A third approach is to use a physiological stimulus whose quantitative effect has been found to be relatively predictable and reproducible in many species and test a new method's ability to assess expected changes from that stimulus. It is this approach that has been chosen here. In using a Paco, change to produce changes in CBF, it seemed reasonable to choose values that lie at the extremes of variations that might occur under physiological stress, and not employ extremes which result in indeterminate secondary effects or which are beyond those attainable by usual physiological situations. For Paco, this range probably extends from to 0 mm Hg to 0 to 70 mm Hg. This range includes the "straightline" portion of the curves published by many investigators for CBF versus Paco, relationships in a variety of animals. 0 " To choose greater extremes would be expected to lead to incorrect assumptions regarding the slope of that response curve inside narrower limits of variation, since it is well known that the slope of the curve changes beyond the extremes of mm Hg to 70 mm Hg at both the high and the low range. As noted in table, the summarized results of others are in most instances derived from values of Paco, beyond these limits at one end or the other of the range. The presently described method is applicable to measurements in any desired region of the brain which can be physically separated by dissection in the ultracold (liquid nitrogen temperature) brain. This includes separation of hemispheres and of brain stem. It also includes separation of parts of the brain that can be achieved by scraping into smaller fractions, so that a sample of one or another portion of the cortex may be taken. Because of the brittle nature of the brain at - 9 C, sharp dissection of central nuclei, for example, is difficult or impossible. The use of xenon as the tracer imposes the requirement of maintaining the very cold state until the brain material can be counted for radioactivity. Kety-Schmidt methods that are dependent on venous outflow tracer curves are useless in experimental states in which regional flow changes are known to be present. A further utility of this method is that it is much less dependent on steady-state assumptions than the Stroke, Vol., November-December 797

7 MATSUMOTO, NAMON, UTSUNOMIYA, KOGURE, SCHEINBERG, REINMUTH Downloaded from by on December, 08 Kety-Schmidt method. Not only is the time during which constancy of cerebral perfusion must be assumed much shorter (0 seconds), but the effect of changes of Paco,, pressure, shifting intracranial pressure, etc., is less since in this method the variable that most requires being "steady" is only the arterial perfusion of the brain area to be studied, and is not dependent on shifts in venous drainage which are known to occur readily in jugular vein blood under a variety of otherwise unimportant stresses that may occur in experimental models. Prior application of methods similar to the present one has involved methods other than decapitation to kill the animal at the conclusion of the tracer infusion time usually an intravenous bolus of KC. If one follows the intra-arterial pressure curve at such a time, pressure falls to zero over 0 or more seconds, although ECG evidence of rhythmic heart beat may disappear in three to five seconds. Uncertainty as to the nature of some continuing arterial perfusion of the brain over time intervals of more than one or two seconds (out of a total of 0 seconds) introduces a variable of unacceptable magnitude in the calculation and, since it is indeterminate, not correctable. In this and similar methods we consider decapitation with precisely determined timing the only acceptable method. The selection of a 0-second infusion time was first made on empiric grounds. This relates to the use of an intravenous ramp injection of radionuclide. The rate of infusion is limited by volume considerations, and the time of arterial equilibrium is fixed by rate of infusion and rate of blood to alveolar outflow. In our application the arterial curve rises steeply and fairly linearly for 0 to 0 seconds and then begins an asymptotic relationship to a plateau of final arterial concentration which, as noted, was usually achieved between three and four minutes. The nature of the method is such that large increments of arterial activity in successive increments of time are desirable, and this was best achieved in the first 0 seconds. Eklof has completely described additional theoretical reasons why a 0-second time interval is preferable and more accurate than longer time intervals. The use of an A-V shunt for recording the arterial Xe concentration avoids the difficulties of serial blood sampling under rigorous closed system requirements and with attendant problems in syringe and system dead space correction. For study of circumstances in which a uniform alteration of CBF can be assessed, this method does not allow derivation of a value for CMRO. Such a determination would not be especially meaningful, of course, in experimental states with known regional alterations in brain flow, but might be very useful in other situations. To obtain this value for use with the present method, a sample of sagittal sinus or jugular blood and an arterial sample would need to be separately taken, presumably before or during the final 0 seconds of flow determination, for analysis of oxygen content and determination of arteriovenous oxygen difference. The determination of biochemical changes in the brain used for regional flow assessment is readily carried out after brain radioactivity counting with one single limitation, that is, that transcalvarial freezing of the sample can begin only at the instant of decapitation. Certain kinds of biochemical alterations, especially in parts of the brain deeper than to mm from the surface, may occur before freezing arrests these postmortem artifacts. ' Nonetheless biochemical, neurotransmitter, neurophysiological and related determinations can be assessed along with regional blood flow in precisely the same anatomical location, in the same animal and under the identical experimental conditions utilizing this method. No other published method applicable to the rat, of which we have knowledge, has this capability. Summary A method of quantitatively assessing cerebral blood flow in the rat is described utilizing Xe infused as a solution intravenously. After 0 seconds of Xe infusion the animal is decapitated and the brain frozen by exposure of the dissected calvarium to liquid nitrogen, followed by freezing of the whole head. The actual time-concentration change in arterial blood is directly measured by passing an external arteriovenous shunt through a gamma well-counter. CBF is measured in any or all desired parts of the brain dissected in the cold state, and flow calculated according to a formula derived by Kety. The method is shown to produce values of CBF that vary quantitatively in the expected fashion for changes of Paco, and in states of nitrous oxide and pentobarbital anesthesia, as well as being closely comparable in magnitude to values obtained by others utilizing standard Kety-Schmidt methods. The method has the important advantage of being accomplished in a short time interval of 0 seconds (allowing short-time steady-state requirements), of assessing either regional, hemispheric or whole brain flow, and of allowing measurement of any other metabolic, biochemical or pharmacological variable in the very same tissue, subject only to limitations which are discussed. It is adaptable to other radionuclide tracers with modifications. The calculations can be derived easily without resorting to a computer, and the method employs equipment likely to be readily available in a laboratory where y radionuclides are used, without major alterations in equipment. References. Kety SS, Landau WM, Freygang WH Jr, et ah Estimation of regional circulation in the brain by uptake of an inert gas. Fed Proc :8, 9 Sfrole, Vol., November-December 97

8 MEASUREMENT OF CBF IN THE RAT Downloaded from by on December, 08 0,.. Landau WM, Freygang WH Jr, Rowland LP, et ah The local circulation of the living brain; values in the unanesthetized and anesthetized cat. Trans Amer Neurol Assoc 80:- 9, 9 Reivich M, Jehle J, Sokoloff L, et ah Measurement of regional cerebral blood flow with antipyrine- C in awake cats. J Appl Physiol 7:9-00, 99 Kety SS: Measurement of local blood flow by the exchange of an inert, diffusible substance. In Bruner HD (ed): Methods in Medical Research. Chicago, Yearbook, p 8-, 90 Goldman H, Sapirstein LA: Brain blood flow in the conscious and anesthetized rat. Amer J Physiol :-, 97 Kety SS, Schmidt CF: The nitrous oxide method for the quantitative determination of cerebral blood flow in man: Theory, procedure, and normal values. J Clin Invest 7:7-8, 98 Haining JL, Turner MD, Pantall RM: Measurement of local cerebral blood flow in the unanesthetized rat using a hydrogen clearance method. Circulation Research :-, 98 Leatherman NE, Bean JN: Continuous recording of regional changes in brain blood flow by a probe. J Appl Physiol :8-88, 97 Aukland K: Hydrogen polarography in measurement of local blood flow; theoretical and empirical basis. Acta Neurol Scand (suppl) :-, 9 Pannier JL, Leusen I: Circulation to the brain of the rat during acute and prolonged respiratory changes in the acid-base balance. Pflu'gers Arch 8:7-9, 97 Sokoloff L, Reivich M, Shapiro H, et al: Local glucose uptake in the brain in vivo, (abstract) Stroke : (Mar-Apr) 97 Reivich M: Blood flow metabolism couple in brain. In Plum F (ed): Brain Dysfunction in Metabolic Disorders. New York, Raven Press, p -0, 97 EkISf B, Holmin T, Johannsson H, et al: Cerebral blood flow and cerebral metabolic rate for oxygen in rats with portacaval anastomosis. Acta Physiol Scand 90:7-, 97. Eklof B, Siesjo BK: Cerebral blood flow in ischemia caused by carotid artery ligation in the rat. Acta Physiol Scand 87:9-77, 97. Eklof B, Lassen NA, Nilsson L, et al: Blood flow and metabolic rate for oxygen in the cerebral cortex of the rat. Acta Physiol Scand 88:87-89, 97. Eklof B, Lassen NA, Nilsson L, et ah Regional cerebral blood flow in the rat measured by the tissue sampling technique; a critical evaluation using four indicators C-antipyrine, "Cethanol, H-water and Xe. Acta Physiol Scand 9:-0, Gjedde A, Caronna J, Hindfeldt B, et ah Whole brain blood flow and oxygen metabolism in the rat. Society for Neuroscience, Program of the Fourth Annual Meeting, St. Louis, Gjedde A, Caronna J, Hindfeldt B, et ah Whole brain blood flow and oxygen metabolism in the rat. Read at the Cerebrovascular Clinical Research Center Workshop, Miami, Florida, February 7-8, Conn HL Jr: Equilibrium distribution of radioxenon in tissue: Xenon-hemoglobin association curve. J Appl Physiol :0-070, 9 0. Harper AM, Glass HI: Effect of alteration in the arterial carbon dioxide tension on the blood flow through the cerebral cortex at normal and low arterial blood pressures. J Neurol Neurosurg Psychiat 8:9-, 9. Reivich M: Arterial Pco, and cerebral hemodynamics. Amer J Physiol 0:-, 9. James IM, Millar RA, Purves MJ: Observations on the extrinsic neural control of cerebral blood flow in the baboon. Circulation Research :77-9, 99. Kogure K, Busto R, Scheinberg P, et ah Dynamics of cerebral metabolism during moderate hypercapnia. J Neurochem :7-78, 97. Swaab DF: Pitfalls in the use of rapid freezing for stopping brain and spinal cord metabolism in rat and mouse. J Neurochem 8:08-09, 97 Stroke, Vol., November-December 97 7