Abstract
The effects of equipotent doses of halothane (1.05%) versus isoflurane (1.38%) anesthesia on CMRglc were determined autoradiographically using the 2-p4C]deoxyglucose technique in the rat. Eight anatomi cally standardized coronal sections were selected and dig itized from the autoradiographs. Mean CMRglc was de termined for hemispheric, neocortical, and subcortical re gions at each anatomic level, and a neocortical! subcortical CMRglc ratio was calculated. In addition, the current CMRglc autoradiographs, as well as previous CBF autoradiographs obtained under identical experimental conditions were examined to characterize and compare flow/metabolism relationships for the two anesthetics. For this analysis, CBF was determined in 80 selected anatomic areas, and the values from each area were plot ted against CMRglc values obtained from identical areas. In all major regions, mean CMRglc was greater with halo thane than with isoflurane. The neocortical/subcortical ratio, reflecting the pattern of CMRglc distribution, was Employing the e4C]iodoantipyrine (e4C]IAP) au toradiographic method, we recently demonstrated that while 1.0 MAC (minimum alveolar concentra tion) halothane and isoflurane had similar effects on hemispheric (whole brain) CBP in the rat, markedly different flow distribution patterns within that hemi sphere were produced by each agent (Hansen et aI., 1988). In particular, halothane produced a dispro portionate increase in neocortical blood flow (rela tive to flow in subcortical structures), while flow
Summary:
The effects of equipotent doses of halothane (1.05%) versus isoflurane (1.38%) anesthesia on CMRglc were determined autoradiographically using the 2-p4C]deoxyglucose technique in the rat. Eight anatomi cally standardized coronal sections were selected and dig itized from the autoradiographs. Mean CMRglc was de termined for hemispheric, neocortical, and subcortical re gions at each anatomic level, and a neocortical! subcortical CMRglc ratio was calculated. In addition, the current CMRglc autoradiographs, as well as previous CBF autoradiographs obtained under identical experimental conditions were examined to characterize and compare flow/metabolism relationships for the two anesthetics. For this analysis, CBF was determined in 80 selected anatomic areas, and the values from each area were plot ted against CMRglc values obtained from identical areas. In all major regions, mean CMRglc was greater with halo thane than with isoflurane. The neocortical/subcortical ratio, reflecting the pattern of CMRglc distribution, was Employing the e4C]iodoantipyrine (e4C]IAP) au toradiographic method, we recently demonstrated that while 1.0 MAC (minimum alveolar concentra tion) halothane and isoflurane had similar effects on hemispheric (whole brain) CBP in the rat, markedly different flow distribution patterns within that hemi sphere were produced by each agent (Hansen et aI., 1988) . In particular, halothane produced a dispro portionate increase in neocortical blood flow (rela tive to flow in subcortical structures), while flow also greater during halothane anesthesia. This suggests that isoflurane has a disproportionate effect on neocorti cal metabolism resembling patterns previously seen for CBF. Analysis of CBF versus CMRglc plots for each an esthetic group showed two parallel lines with nearly iden tical slopes, but different Y intercepts. We conclude that the distribution of CMRglc observed during 1 MAC (min imum alveolar concentration) halothane and isoflurane anesthesia parallels the distribution of CBF. This finding supports the conclusion that flow-metabolism coupling is intact during halothane and isoflurane anesthesia, and that drug induced changes in cerebral metabolism may play an important role in determining the CBF response to that drug. Furthermore, there is evidence that, at a given level of CMRglc, isoflurane may have greater va sodilating capabilities than halothane. Key Words: Anes thetics, volatile: halothane, isoflurane-Autora diography-Brain blood flow-Cerebral metabolism. distribution patterns seen with isoflurane were much more uniform. The reasons for these differing effects of volatile agents on regional flow distribu tion remain unknown-but it is almost certainly not a simple matter of the drugs having different intrin sic vasodilating potencies. If this were the case, it would be difficult to explain the unique flow distri bution patterns seen for each agent.
Summary:
A more likely explanation for the differences in regional CBP during halothane versus isoflurane anesthesia may be their differing effects on cerebral metabolism. Isoflurane is a more potent metabolic depressant than is halothane (perhaps in keeping with its more pronounced ability to reduce cerebral electrical activity) (Cucchiara et aI., 1974; Todd and Drummond, 1984; Maekawa et aI., 1986) . In the conscious state, CBP and metabolism are interre-lated ("coupled"), such that increases (decreases) in metabolism (CMR) are accompanied by increases (decreases) in CBF (Olesen, 197 1; Sokoloff, 1981) . However, the role of metabolic changes in control ling blood flow during anesthesia is unclear. It has long been believed that volatile agents "uncouple" flow and metabolism (i.e., increase CBF, decrease CMR) (Kuramoto et aI., 1979; Lassen and Shapiro, 1981) . In spite of this belief, recent work suggests that this may not be true. Drummond et aI. (1986) , working in rabbits, showed that when differences in the metabolic responses to halothane and isoflurane were removed (by barbiturate loading), both agents had identical effects on CBF. This would suggest that "coupling" may play an important role in de fining the CBF response to these agents under nor mal circumstances. In other words, the lower me tabolism associated with isoflurane may act, via a coupled vasoconstriction, to attenuate the indepen dent vasodilating effect of the drug, with the result being only small changes in CBF. By contrast, since halothane has a smaller effect on CMR, less indi rect, metabolically mediated vasoconstriction would be expected, direct vasodilation would be "unopposed," and the observed flows would be higher.
Summary:
To further examine the possible role of metabo lism in defining global and regional CBF responses to the volatile anesthetics, we performed a compar ative examination of the effects of 1 MAC halothane versus isoflurane on local CMRglc using the 2-rt4C]deoxyglucose autoradiographic method (Sokoloff et aI., 1977) . Regional CMRgl C values were then compared with regional [14C]IAP autora diographic CBF data obtained under identical cir cumstances in an earlier experiment (Hansen et aI., 1988) . To better provide evidence confirming the presence or absence of a coupled relationship be tween flow and metabolism, both current CMR data and our earlier CBF data were reexamined, and a structure by structure comparison of CBF and CMRglc was performed.
Summary:
Based on these studies, we believe that a strong case can be made that coupling between flow and metabolism persists during 1 MAC concentrations of halothane and isoflurane, and that differences in the cerebral metabolic response to these drugs plays an important role in the flow patterns seen.
METHODS
This study was approved by the Institutional Animal Care and Vse Committee. Male Sprague-Dawley rats weighing between 310 and 390 g (Biolabs; St. Paul, MN) were fasted for 6 h. Rats were randomly anesthetized with 1.5% halothane or 2.0% isoflurane in 33% 021
METHODS
J Cereb Blood Flow Metab, Vol. 9, No.3, /989 balance N2. A tracheostomy was performed, and the an imal connected to a small animal ventilator set to a tidal volume of 2.5 ml and a respiratory rate of 60/min. Fem oral arterial and venous catheters were inserted via cut down and all wound sites were infiltrated with 2.0% lidocaine. The arterial catheter was connected to a trans ducer for blood pressure monitoring, while the venous catheter was used for drug and fluid administration. Hep arin (200 LV.) and D-tubocurarine (2 mg) were adminis tered intravenously to each rat.
METHODS
Total surgical preparation time was 35 min (measured from the time of anesthesia induction). Immediately fol lowing completion of surgery, the inspired anesthetic agent concentration (measured with Puritan Bennetl Datex Model 222 Anesthetic Agent Analyzer) was re duced to either 1 MAC halothane (1.05%) or 1 MAC iso flurane (1.38%) in 33% 02lbalance N2 (White et aI., 1974) . Rats remained anesthetized an additional 35 min prior to initiation of CMRglc determination. During this time, the ventilator was adjusted to maintain normoxia (Pa02 = 110-140 mm Hg) and normocarbia (Pac02 = 38-42 mm Hg). Body temperature was monitored via a rectal probe and maintained within the range of 36.8°-37 .2°C by surface heating or cooling. Mean arterial blood pressure (MABP) was continuously monitored and maintained within the range of 90 to 100 mm Hg by infusion of whole donor rat blood.
CMR g lc determination
Thirty-five minutes after the end of surgery, animals were given 2 ml whole blood. Final MABP and arterial blood gas values were recorded and 100 fLCi/kg of 2-[14C]deoxyglucose (2-[14C]DG, specific activity 50-56 mCilmmol, New England Nuclear, Boston, MA) was in fused intravenously at a constant rate over 30 s via an infusion pump. At defined time intervals over the next 45 min, fourteen lOO-fLl arterial blood samples were col lected. Samples were immediately centrifuged and the plasma saved for later determination of 14C activity and glucose concentration. The animals were then decapi tated, the brains rapidly removed and frozen immediately in 2-methylbutane (-40°C). Twenty microliters of each timed arterial plasma sample was placed on chromatog raphy paper and dried for 24 h, then eluted 24 h in 1 ml water and 9 ml liquid scintillation cocktail (Ready-Solv HPIb, Beckman, Fullerton, CA). Radioactivity was de termined by liquid scintillation counting (Searle, Model 6880, Des Plaines, IL) using an external quench correc tion. Plasma glucose values were determined by the glu cose oxidase method (Beckman Glucose Analyzer-2, Ful lerton, CA). Animals in which subsequent plasma glucose values deviated more than 10% from baseline levels were excluded from the study.
Autoradiography and image analysis
Brains from eight halothane and eight isoflurane anesthetized animals were analyzed. Frozen brains were cut in 20 fLm thick serial coronal sections on a cryostat ( -20°C). Quadruplicate sections taken at 240 fLm inter vals were mounted on glass slides, dried for 5 min on a hot plate (50°C), and exposed to Kodak SB-5 autoradio graphic film for 10 days in an X-ray cassette along with seven e4C]methylmethacrylate standards (Amersham, Arlington Hts, IL). Autoradiographic images of eight standardized brain sections were chosen from each ani mal for further analysis, based upon anatomical land-marks (see Hansen et aI., 1988) . These images were con verted to digitized optical density images on a scanning microdensitometer system (Eikonix-Kodak, Bedford, MA.) with a camera aperture of 100 fLm. Optical densi ties from these autoradiographic images, standard radio activity values derived from 1 4C standards, and timed ar terial blood radioactivity values were entered into a Dig ital Micro-Vax computer system. Recalculation of CMRglc values from brain images employed the equations developed by Sokoloff et ai. (1977) (lumped constant = 0.483), with mathematical operations performed using a quantitative glucose utilization program (A Toga, Wash ington Univ., St. Louis, MO).
Data analysis
Two different analytical approaches were used. The first was very similar to that used for examination of re gional CB F in our earlier experiment (Hansen et aI., 1988) . Individual digitized CMRglc images were pseudo color enhanced and displayed on a cathode ray screen. Using an operator-controlled cursor, regions of interest were circumscribed and mean CMRglc for each region recorded, along with the area of each region (in pixel units). The three primary regions of interest in each sec tion were hemisphere, neocortex, and subcortex, where subcortex was defined as the total hemispheric area mi nus the area of the neocortical mantle (see Hansen et aI., 1988) . Ventricular spaces were subtracted prior to calcu lations. Olfactory cortex is a limbic structure and was therefore included in subcortex (Hornykiewcz and Liv ingston, 1978) . The neocortical and subcortical CMRglc values from each section were then used to calculate a neocorticaVsubcortical CMR il lc ratio. "Whole brain" val ues for hemispheric, neocortical, and subcortical CMRglc as well as the neocorticaVsubcortical ratio were deter mined as area weighted averages of values from each of the eight sections in each rat. A two-way analysis of vari ance (ANOV A) (section level versus anesthetic with sec tion level as a repeated variable) was then used to inde pendently compare values for each region at each ana tomic level as well as for the brain as a whole.
Data analysis
In addition to these large areas, ten anatomical struc tures were outlined and CMRglc for each determined (these structures are listed in Table 2 ). These CMRglc values were compared between anesthetic groups by an unpaired Student's t test.
Data analysis
The second analysis compared CMRglc values mea sured within 80 anatomically defined areas (representing 49 anatomic structures) to CBF values in corresponding areas, which were measured by reexamining digitized e4C]lAP autoradiographs obtained in our previous CBF study (Hansen et aI., 1988) . Specifically, CBF and CMRg1c images were pseudo-color enhanced and dis played on a cathode ray screen. Using an operator controlled cursor, ten 500 square pixel unit areas were consistently identified on each of the same eight anatom ically standardized brain sections from each animal and CBF and CMRglc values were determined from each area. These areas were selected to sample as many anatomi cally diverse areas as possible. Individual CBF and CM Rglc values for each area were then averaged over all animals within an anesthetic group. Mean CMRglc values for each of 80 areas within an anesthetic group were then plotted versus corresponding mean CBF values from the identical regions. Best fit lines were determined for both groups from these CBF and CMRg1c data points using a linear, least squares fit. Slopes and Y intercepts for each anesthetic group were compared using a regression anal ysis with indicator variables (Neter et aI., 1983) . All CBF and CMRglc values are reported as mean ± standard de viation with significance assumed when p < 0.05.
RESULTS
Physiologic values determined immediately prior to CMRglc determination are reported in Table 1 , along with similar data from CBF animals. No sig nificant intergroup differences were noted with re spect to Pao2, Paco2, pH, MABP, or temperature. Larger amounts of infused blood were required to maintain MABP in the CMRglc groups because of larger volumes of blood samples drawn. Table 2 summarizes our "whole brain" CMRglc results for the three major tissue regions, as well as values from ten anatomical regions. Average hemi spheric, neocortical, and subcortical CMR g lc values were lower in the isoflurane group (p < .001 for each region), as was the neocorticaVsubcortical ra tio (p < .00 1). Local CMR g lc was also lower in the isoflurane group for all local anatomical structures. Anatomically comparable data for CBF is also listed in Table 2 .
RESULTS
Mean CBF values from the 80 selected white and gray matter structures ranged from 59 to 195 ml 100g -I min -I during halothane and isoflurane an esthesia; corresponding mean CMRglc values from the same structures ranged from 24 to 98 j.Lmol 100g-1 min -I. Regression analysis of CBF versus CMRglc values plotted on a structure by structure basis for each anesthetic group is shown in Fig. 1 Hansen et al. (1988) .
DISCUSSION
In the nonanesthetized state, cerebral blood flow and metabolism are tightly coupled, such that in creases or decreases in metabolism are accompa nied by corresponding changes in blood flow (Ole sen, 197 1; Sokoloff, 198 1; Kuschinsky et aI., 1981) . Using the [14C]IAP and 2-deoxyglucose (2-DG) techniques to measure CBF and CMRg1c' respec tively, Greenberg et ai. (1979) have demonstrated identical spatial distribution of flow and metabolism following vibrissal stimulation in the rat. Similarly, Silver (1978) has demonstrated rapid increases in microvascular flow following neuronal stimulation. These and many other studies indicate that normal flow is indeed regulated (in part) by changes in func tional and metabolic activity. Previous studies have examined CBF and CMR g lc at various minimum alveolar concentration (MAC) levels of halothane and isoflurane anesthesia (Kuramoto et aI., 1979; Todd and Drummond, 1984; Maekawa et aI., 1986) . These studies have shown that at increasing MAC levels, CBF increases while CMRglc is either unchanged or (more commonly) decreased. Based on these observations, some au thors have concluded that the anesthetic agents studied caused an "uncoupling" of flow and metab olism.
DISCUSSION
In our current study, we have noted differences in the CMRglc effects of halothane and isoflurane, not just in terms of their absolute values, but also in terms of the spatial distribution patterns. Specifi cally we observed that not only was regional CMRglc lower with 1 MAC isoflurane than with 1 MAC halothane, but also that the ratio of neocorti cal to subcortical CMRglc was lower with isoflurane (Table 2 ). This observation suggests that isoflurane has a proportionately greater effect on neocortical CMRglc (relative to its overall CMRglc impact) than does halothane. Of greater importance, however, is that this CMRglc distribution reflects a pattern also seen for CBF under similar anesthetic conditions (Hansen et aI., 1988) , with isoflurane-anesthetized rats having lower neocortical flows and lower neo cortical/subcortical ratios than animals given halo thane. In other words, anesthetic-related flow pat terns are paralleled by patterns in metabolism. While this alone is evidence suggesting a relation ship between these two variables, we have also demonstrated a linear relationship between CBF and CMRglc over a wide range of flow and CMRglc values during 1 MAC anesthesia with both agents; brain areas with high CMRglc values had higher measured flows, while areas with low CMRglc val ues had low CBF. This is identical to the linear relationship observed in awake animals-a relation ship that is considered strong evidence for the ex istence of a coupled relationship between these two parameters. In short, these two findings suggest that flow and metabolism are, in fact, "coupled" during 1 MAC doses of both of these anesthetics. They also indirectly suggest that differences in me tabolism produced by these agents may influence the changes in CBF produced by these drugs. This postulate could be better examined by recording both flow and metabolism in the same animal, before and after some intervention that is known to increase CMRglc, e.g., a seizure. If CBF and CMRglc are indeed "coupled," one would expect to see parallel increases in both values. Unfortunately, this demonstration is more difficult to carry out. The auto radiographic methods used in the current study are not compatible with such repeated mea sures, and the usual methods for recording "whole brain" CBF and CMR [e.g., 133Xe washout com bined with arteriovenous (A-V) 02/glucose differences] are confounded by the fact that the cal culation of CMR is dependent on the measurement of CBF (which makes a linear regression compari son of CBF versus CMR suspect). However, the work of Kuramoto et aI., (1979) using volumetri cally determined CBF, identified parallel increases in CBF and CMR02 during peripheral stimulation in dogs breathing I MAC halothane-again evidence supporting our contention that flow and metabolism remain tightly coupled during the administration of clinically relevant concentrations of volatile anes thetics.
DISCUSSION
This conclusion has a number of important impli cations. Studies which have examined neocortical CBF have shown halothane to have greater effects than isoflurane (Todd and Drummond, 1984; Eintrei et aI., 1985; Scheller et aI., 1986) . Based on these observations, it has been suggested that halothane has greater direct vasodilating capabilities than iso flurane (Shapiro, 1986) . However, comparison of CBF during halothane versus isoflurane anesthesia when metabolic differences are minimized (barbit urate loading) indicates that this may not be true (Drummond et aI., 1986) . This question can also be examined in our current study. As can be seen from the plots of CBF versus CMRglc in Fig. 1 , the line for isoflurane is above that for halothane. This sug gests that, at a given value of CMRglc, isoflurane anesthetized animals may have greater flows than animals given halothane. These observations sug gest that despite a greater overall depression of CMRglc during isoflurane compared to halothane anesthesia, the potentially greater vasodilating ca pabilities of isoflurane may account for equal hemi spheric flow values previously reported for the two agents (Theye and Michenfelder, 1968; Cucchiara et aI., 1974; Stullken et aI., 1977) .
DISCUSSION
In the interpretation of our results, methodologi cal considerations must be evaluated. First, the se lection of large brain areas for our initial determi nation of CBF and CMRglc was somewhat different from the approach used by others (which is more similar to the multiple spot samples used in our sec ond analysis). Furthermore, area weighted CMRglc values from eight standardized brain sections were taken to reflect whole brain values. However the brain sections chosen for analysis were based on subcortical anatomical structures within these sec tions, not the uniformity of anteroposterior distance between sections. Examination of the anatomical location of our selected sections shows a relative bias toward centrally located sections at the ex pense of more rostral and caudal sections. In the second analysis, eighty 500 square pixel unit areas, representing 49 independent anatomical brain struc tures, were consistently selected for CBF and CMRglc determination from identical anatomical sites over eight autoradiographic brain sections. These areas were selected to represent a wide range of CMRglc values, yet a sampling bias with relative exclusion of very low CMRglc values (white matter) is apparent. It is possible, although unlikely, that the inclusion of a greater number of these areas would alter our results.
DISCUSSION
Second, animals in the flow and metabolism stud ies were anesthetized for somewhat different peri ods of time prior to the initiation of CBF or CMRglc determinations (1 h 30 min versus 1 h 10 min re spectively, measured from the time of induction). Due to the differences in time required to measure CBF versus CMRglc (45 s vs 45 min, respectively), an identical anesthetic exposure period for the two studies was difficult to establish. However, the ma jority of 2-DG utilization occurs early in the 45 min measurement period (Sokoloff et aI., 1977) . There fore, the anesthetic equilibration period in the me tabolism study was chosen such that the midpoint of the CMRglc determination would closely approx imate the 1 h 30 min total anesthetic exposure time allowed in the previous CBF study. As a result, animals in the CBF study groups versus those in the CMRglc study were exposed to the anesthetics stud ied for slightly different time periods. Since adap tation to anesthetic-produced changes in flow and metabolism occurs over several hours, the errors related to these small time differences would be ex pected to be minimal (Warner et al., 1985) .
DISCUSSION
In summary, the relationship between CBF and CMRglc in a variety of anatomic brain structures during 1 MAC halothane and isoflurane anesthesia was best represented by two parallel lines with equal slopes, but different Y intercepts. This, com bined with the similar CBF and CMRglc distribution patterns seen with a given agent, is strong evidence in support of the persistence of intact flow metabolism coupling during 1 MAC anesthesia. In addition, at any given level of CMRglc, isoflurane anesthetized rats had greater CBF values. Whether this indicates that isoflurane is a more potent direct vasodilator, or whether other factors that influence CBF are being differentially altered by these drugs (e.g., neuronal input) remains to be determined. These observations may have further applications in experiments in which the effects of inhalational anesthetics are studied in the presence of an equally established level of CMR, such as barbiturate pre treatment. These findings also suggest, as was pre viously noted by Scheller et al. (1987) and Drum mond et al. (1986) , that the effects of an anesthetic agent on CBF may depend upon the preexisting metabolic state of the brain and that metabolic ac tivity must be taken into consideration when draw ing any conclusions about the mechanisms by which volatile agents alter flow.

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