Effects of Xenon on Cerebral Blood Flow and Cerebral Glucose Utilization in Rats

After obtaining approval from the Institutional Animal Care Committee (Regierungspraesidium Karlsruhe, Germany), the experiments were performed on 48 male Sprague-Dawley rats weighing 316 ± 26 g (Charles River Deutschland, Sulzfeld, Germany). Animals were kept under temperature-controlled environmental conditions on a 14:10 light:dark cycle, were fed standard diet (Altromin C 1000; Altromin, Lage, Germany), and were allowed free access to food and water until starting the experiments.

Rats were anesthetized by inhalation of isoflurane (1 MAC) in oxygen–nitrous oxide (fraction of inspired oxygen [Fio²]= 0.4). Anesthesia was maintained breathing the same gas mixture spontaneously via  a nose cone. Cannulation of right femoral artery and vein was performed using polyethylene catheters (PE-50; Labokron, Sinsheim, Germany). Oxygen saturation was monitored using pulse oximetry (Nonin 8600V Pulse Oximeter, Nonin Medical Inc., Plymouth, MN). After wound closure, animals were placed in tunnels (Braintree Scientific, Boston, MA) for recovery breathing nitrogen– oxygen (Fio²= 0.3) in a gas-sealed head chamber. Gas within the head chamber was exchanged by a ventilation circuit with a flow of 200 ml/min (Cicero; Draeger AG, Lübeck, Germany). The gas concentrations were monitored by mass spectrometry (Xenotec 2000, Draeger AG). Mean arterial blood pressure and heart rate were registered continuously by a quartz pressure transducer (Hewlett-Packard, Palo Alto, CA). Arterial blood gases were checked in a pH–blood gas analyzer (AVL Gas Check 939; AVL, Graz, Austria). Rectal temperature was controlled between 37°C and 37.5°C with a heating lamp.

After a recovery period of 60 min, all rats were placed in a tunnel where they breathed spontaneously. According to applied gas mixture and the duration of gas administration, rats were randomly assigned to various groups (fig. 1).

Steady state conditions were assumed after 45 min of xenon equilibration within the rat brain because the equilibration time of xenon is 2 min for grey matter and more than 30 min in white matter. 15 

With each treatment, six rats were used for the autoradiographic determination of local CBF and six rats for the measurement of local CGU according to previous descriptions. 17,18In the short inhalation groups, only local CBF experiments could be performed. Comparable local CGU experiments using deoxyglucose were not possible because of the long experimental time (45 min) of this type of experiment.

For the measurement of local CGU, 125 μCi/kg body weight of 2-[1-¹⁴C]deoxy-d-glucose (specific activity, 50–56 mCi/mmol; New England Nuclear, Dreieich, Germany) were injected as a pulse via  the femoral venous catheter within 20 s after a period of 30-min steady state inhalation of the respective gas concentration. Timed arterial blood samples of 80 μl were collected through the arterial catheter at 15, 30, and 45 s and at 1, 2, 3, 5, 7.5, 10, 15, 25, 35, and 45 min. The blood samples were immediately centrifuged and stored on ice until assays for plasma 2-[1-¹⁴C]deoxy-d-glucose and glucose concentrations were performed. Immediately after the final arterial blood sample was collected, the animal was decapitated, and the brain was rapidly removed and frozen in isopentane chilled to −60°C.

For the measurement of local CBF, after various inhalation periods in the respective groups, 100 μCi/kg body weight of 4-iodo[N-methyl-¹⁴C]antipyrine (specific activity, 54 mCi/mmol; Amersham-Buchler, Braunschweig, Germany) dissolved in 1 ml of saline was infused continuously at a progressively increasing infusion rate for 1 min via  the femoral venous catheter. The progressively increasing infusion rate, a modification of the method described previously, 17was chosen to minimize equilibration of rapidly perfused tissues with arterial blood during the period of measurement. During the 1-min infusion period, 14–20 timed blood samples were collected in drops from the free-flowing arterial catheter directly onto filter paper disks (1.3 cm in diameter) that had been prepared in small plastic beakers and weighed. The samples were weighed, and radioactivity was estimated with a liquid scintillation counter (TriCarb 4000 series; Canberra Packard, Frankfurt, Germany) after extraction of the radioactive compound with ethanol. After the 1-min infusion and sampling, the animal was decapitated. The brain was removed as quickly as possible and frozen in isopentane chilled to −60°C. In both the 2-[1-¹⁴C]deoxy-d-glucose and 4-iodo[N -methyl-¹⁴C]antipyrine experiments, the frozen brains were coated with chilled embedding medium (Lipshaw, Detroit, MI), stored at −80°C in plastic bags, sectioned in 20-μm sections at −20°C in a cryostat, and autoradiographed along with precalibrated [¹⁴C]methyl methacrylate standards.

Local tissue concentrations of [¹⁴C] were determined from the autoradiographs by densitometric analysis. Local CGU and CBF were calculated from the local concentrations of [¹⁴C] and the time courses of the plasma [¹⁴C]deoxyglucose and iodo[¹⁴C]antipyrine concentrations, including corrections for the lag and washout in the arterial catheter. The washout correction rate constant was 100/min, and the brain–blood partition coefficient for iodo[¹⁴C]antipyrine was 0.9 in our rats.

Autoradiographic images were converted to digitized optical density images by an image processing system (MCID; Imaging Research, St. Catharines, Canada). For measurements of separate brain structures, an ellipsoid cursor was used and adjusted to the size of the individual region. For both measurement of mean CBF and mean CGU, the area and optical density of the whole coronal sections were analyzed from sections of the whole brain selected every 200 μm. As a result, the area-weighted means of all measured sections throughout the rat brain were obtained.

Two kinds of analysis were performed: (1) for testing of the effects of different concentrations of xenon on glucose utilization during steady state conditions, glucose utilization and physiologic variables were compared between a control group and two groups of steady state inhalation of either 30 or 70% xenon; (2) for testing the effects of 70% xenon inhalation on CBF, values of CBF and physiologic variables were compared between a control group (no inhalation of xenon) and three groups of 2 min, 5 min, and steady state (45 min) inhalation of 70% xenon.

Data were evaluated by analysis of variance, and differences between the experimental groups were investigated by t  tests for multiple comparisons with Bonferroni correction. 19Data are presented as mean ± SD. The level of statistical significance was set at 0.05. The overall relation between local CGU and local CBF in the examined structures of the brain (fig. 2) was assessed by the least-squares fit of the data to y = ax + b, where x is the mean local CGU in a given region, and y is the mean local CBF in that same area. Contrasts of slopes of the local CBF–CGU regression lines were tested by common t  test statistics with Bonferroni correction for multiple comparisons. Because of the limitations of this kind of analysis, an additional, more rigorous statistical approach using log-transformed data was applied, examining the relation of local CBF and local CGU by a repeated measure of the analysis of variance according to McCulloch et al.  20and Ford et al.  21For this analysis, a computer software package (BMDP2v; BMDP Statistical Software Inc., Los Angeles, CA) considering interanimal variability and enabling the detection of heterogeneities in the relation between local CGU and CBF was used.

Steady state inhalation of xenon did not result in changes of the physiologic variables at any concentration tested (table 1). During inhalation of 70% xenon for 2 and 5 min, changes of arterial carbon dioxide tension, plasma glucose levels, and heart rate were found.

Mean CBF values during 30 or 70% steady state inhalation of xenon were not different from conscious control values (fig. 3). Local CBF remained unchanged in all structures tested during steady state inhalation of 30 and 70% xenon (table 2).

During 2- and 5-min inhalation of 70% xenon, however, mean CBF increased by 48 and 37% compared with conscious controls, respectively (fig. 3). Analysis of the various loci (table 2) showed that this increase in mean CBF was caused by a large increase in local CBF in all cerebral cortical structures (pyriform, frontal, sensory motor, parietal, cingulate, auditory, and visual cortex). In contrast to these local CBF increases, local CBF was decreased after 2-min (by 32%) or 5-min inhalation (by 36%) in the cerebellar cortex during 70% xenon inhalation.

Mean CGU did not change during 30% (54 ± 5 μmol · 100 g⁻¹· min⁻¹) or 70% xenon inhalation (49 ± 2 μmol · 100 g⁻¹· min⁻¹) compared with the conscious control group (54 ± 5 μmol · 100 g⁻¹· min⁻¹). Values of local CGU are shown in table 3. Compared with controls, inhalation of 30 and 70% xenon induced significant decreases in local CGU in 7 and 18 of the 40 examined brain regions, respectively.

The relation between local CGU and CBF at steady state exposure to xenon is plotted in figure 2. Close correlations were found between local CGU and CBF for control conditions (r = 0.90;P < 0.05), 30% xenon (r = 0.94;P < 0.05), and 70% xenon (r = 0.89;P < 0.05). As revealed from the analysis of log-transformed data previously mentioned, 20the slopes of the regression lines were not significantly different between controls (1.6 ml/μmol;P > 0.05) and 30% xenon (1.8 ml/μmol;P > 0.05), but both were different from 70% xenon (2.4 ml/μmol;P ≤ 0.001).

The data obtained show that steady state inhalation of 30 or 70% xenon did not result in changes of mean or local CBF or mean CGU, whereas local CGU was decreased in 7 by 30% xenon and 18 of the 40 brain structures by 70% xenon. In contrast to these moderate effects at steady state, mean CBF was considerably increased during the first 5 min of inhalation of 70% xenon. This increase was caused by an increase in local CBF in most cortical brain structures. The correlation between local CBF and local GCU that existed during control conditions was maintained during steady state inhalation of both 30 and 70% xenon, although at an increased slope at 70% xenon.

The most important result of this investigation is that steady state inhalation of 70% xenon does not result in an increase in CBF as had been expected on the basis of several previous results. 11,12However, in these previous studies, short inhalation periods of less than 5 min were used. Because at that time xenon was only used for xenon computed tomography during short exposure times and in low concentrations because of its high costs, long-term effects of xenon were of little interest. The situation has now changed. Xenon anesthesia is in phase III clinical studies, and the costs can be reduced by recycling techniques. With the intended use of xenon in high concentrations for anesthesia, interest in the effects of long exposure times of xenon on CBF is growing. In concert with our results of steady state inhalation, a recently published study in pigs used a ventilation period of 30 min and failed to show any effect of various doses of xenon in propofol-sedated pigs on regional blood flow measured by the sagittal sinus outflow technique. 22Moreover, the time dependency of the xenon effect on CBF is known from a previous investigation. 13Hartmann et al.  13reported that an adaptation of xenon-induced initial increases in CBF exists. Using the intra-arterial xenon-133 method for the measurement of local CBF in baboons, the adaptation of an initially increased CBF after the first 4 min occurred during the continuous inhalation of 35% xenon for 45 min to the baseline CBF. Our study using autoradiographic CBF determination now might further support the report of a reversal of initial xenon-induced increase of CBF because after steady state inhalation of 70% xenon for a 45-min period, no differences in mean CBF and mean CGU were detected.

We were unable to detect the origin of the cortical increases in CBF. The cause of these changes remains unknown. Several possibilities exist that might explain the effects, such as an increase in arterial carbon dioxide partial pressure (Paco²), neuro-excitation, or dilation of cerebral vessels by xenon. Each of these possibilities are considered in the following paragraphs, although other possibilities cannot be excluded.

First, an increased Paco² must be considered as a cause of the increased CBF during the first minutes of the inhalation because Paco²was increased by 4 mmHg (70% xenon, 2 min) and 5 mmHg (70% xenon, 5 min). Such an increase in Paco²should induce a general increase in CBF by 10–15% according to previous studies of our group. 23Therefore, the general increase in CBF by 28 and 17% as measured in the present study during 2- and 5-min 70% xenon inhalation could be well explained by the increase in Paco^2.However, the increase in Paco²cannot be the cause of large increase in neocortical CBF by 97 and 63% as found in the present study. With respect to the specific cortical effects, this interpretation is consistent with previous results of Junck et al.  11During 1- and 2-min inhalation of 80% xenon, the investigators found a small increase in Paco²of less than 2 mmHg but a large increase in neocortical local CBF by 75 and 96%, respectively.

Second, neuro-excitation could be the cause of the observed increase in CBF during short-term inhalation of 70% xenon. The increase in CBF could be either secondary to an increase in brain activity at the onset of xenon inhalation or directly caused by an activation of specific neuronal receptors by xenon. An increase in brain activity could be concomitant to an enhanced innervation of respiratory muscles to meet the increased respiratory work during the inhalation of xenon because 70% xenon has a 4.6-fold higher specific weight than air. 24The increased CBF would then reflect the enhanced activity of neurons that activate the efferent tracts to respiratory muscles. An activation of specific receptors by xenon appears possible because xenon induces inhibition of excitatory N -methyl-d-aspartate receptors in contrast to the activation of inhibitory γ-aminobutyric acid type A receptors by the majority of other general anesthetics. 25Direct neuro-excitatory effects of xenon should be reflected in changes in local CGU in brain regions where these receptors are localized. However, because the [¹⁴C]deoxyglucose method requires stable experimental conditions over 45 min, local CGU could not be measured in the short-term inhalation groups during the present investigation. Therefore, neuro-excitation could not be demonstrated or excluded as a cause for the increase in local CBF observed in the present study during the first minutes of 70% xenon inhalation.

Third, the most likely cause of the observed initial increase in CBF in the short-term inhalation groups is a direct vasodilator action of xenon that wanes within the next 45 min. During 2-min inhalation of 70% xenon, local CBF was doubled in neocortical brain structures (frontal, sensory motor, parietal, auditory cortex) from 137 ± 8 to 277 ± 27 ml · 100 g⁻¹· min⁻¹. After 45 min, CBF had returned to preinhalation levels. An initial marked increase in CBF that showed a tendency to decrease with time has also been described for nitrous oxide 26and halothane. 27–29During 1 h of 70% inhalation of nitrous oxide in goats, Pelligrino et al.  26observed an increase in CBF that was mainly restricted to cortical brain structures. The maximum increase of 65% compared with controls was found at 15 min, and an increase of 43% was still found after 60 min. 26As to halothane, CBF was nearly doubled in goats within 4 min after start of 1% halothane anesthesia and returned to preanesthetic levels after 2.5 h. 28Likewise, 5 min after start of inhalation in dogs, 1.33% halothane induced an increase in CBF by 79%. During continuous inhalation, CBF then decreased at a rate of 8.7% per hour. 29Compared with the effects of nitrous oxide and halothane, the dilating effects of xenon were restricted to cortical regions, less prominent for the mean CBF (maximum increase of 48%) and below detection after 45 min. The cause of the observed initial increases in CBF by xenon, nitrous oxide, and halothane are unclear as well as the mechanism of adaptation of CBF during longer inhalation periods.

Steady state inhalation of xenon produced a metabolic depression pattern only present at the higher concentration (70%) and located mainly in the cerebral cortex. A predominant suppression of cortical metabolism has already been described for the halogenated ethers isoflurane, sevoflurane, and desflurane. 30–32At a concentration sufficient to induce anesthesia (1 MAC), these anesthetics induce a reduction of mean CGU in conscious rats by 43, 34, and 52%, respectively. In contrast, we were not able to demonstrate a reduction of mean CGU even by high concentrations of xenon. This may be caused by the failure to attain a xenon concentration of 1 MAC at atmospheric pressure because a pressure of 1.61 ± 0.17 atm is necessary to obtain 1 MAC xenon concentration in rats. 5In addition, because of the rather small number of experimental animals used for the experiment, it cannot be ruled out that a small reduction of mean CGU might have occurred that could not be detected statistically.

The physiologic relation between the cerebral blood of cerebral glucose metabolism to blood flow is varied by other volatile anesthetics as previously shown. 31,33–35However, the shift of the relation to higher levels (2.4 ml/μmol) is in the level of 1 MAC isoflurane and sevoflurane (2.5 to 2.6 and 2.3 ml/μmol) 36but less than the one of desflurane at 1 MAC (3.5 ml/μmol). 34Based on minor effects of 0.5 MAC xenon on the cerebral metabolism, this suggests the presence of a homogenous flow increase in the majority of structures even if the increases in mean and local CBF did not reach the level of significance.

In conclusion, during the first minutes of xenon inhalation, mean CBF is increased by a maximum of approximately 50%. The cause of the transient initial vasodilation might be either a direct vascular action of xenon or an indirect effect mediated by a xenon-induced metabolic stimulation. During steady state inhalation of 70% xenon, changes of cerebral metabolism and CBF were not detected. Because of local changes in blood flow and metabolism, the coupling of local CBF to local CGU is reset but shifted to a higher level as already known from other inhalational anesthetics.