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Journal of Cerebral Blood Flow & Metabolism (2007) 27, 588–596 & 2007 ISCBFM All rights reserved 0271-678X/07 $30.00

Isoflurane strongly affects the diffusion of intracellular metabolites, as shown by 1H nuclear magnetic resonance spectroscopy of the monkey brain Julien Valette1, Martine Guillermier2, Laurent Besret2, Philippe Hantraye1,2, Gilles Bloch1 and Vincent Lebon1 Commissariat a` l’E´nergie Atomique, Service Hospitalier Fre´de´ric Joliot, Orsay, France; 2Unite´ de Recherche Associe´e 2210, Commissariat a` l’E´nergie Atomique—Centre National de la Recherche Scientifique, Orsay, France


Isoflurane is a volatile anesthetic commonly used for animal studies. In particular, diffusion nuclear magnetic resonance (NMR) spectroscopy is frequently performed under isoflurane anesthesia. However, isoflurane is known to affect the phase transition of lipid bilayer, possibly resulting in increased permeability to metabolites. Resulting decreased restriction may affect metabolite apparent diffusion coefficient (ADC). In the present work, the effect of isoflurane dose on metabolite ADC is evaluated using diffusion tensor spectroscopy in the monkey brain. For the five detected intracellular metabolites, the ADC exhibits a significant increase when isoflurane dose varies from 1% to 2%: 13%68% for myo-inositol, 14%613% for total N-acetyl-aspartate, 20%618% for glutamate, 27%67% for total creatine and 53%617% for total choline. Detailed analysis of ADC changes experienced by the five different metabolites argues in favor of facilitated metabolite exchange between subcellular structures at high isoflurane dose. This work strongly supports the idea of metabolite diffusion in vivo being significantly restricted in subcellular structures at long diffusion time, and provides new insights for interpreting ADC values as measured by diffusion NMR spectroscopy. Journal of Cerebral Blood Flow & Metabolism (2007) 27, 588–596. doi:10.1038/sj.jcbfm.9600353; published online 21 June 2006 Keywords: brain; diffusion; monkey; NMR spectroscopy; isoflurane

Introduction Owing to its unique ability to probe the intracellular space, diffusion-weighted (DW) nuclear magnetic resonance (NMR) spectroscopy has proven to be a valuable tool to explore brain cells in vivo under normal or pathological conditions (Posse et al, 1993; Merboldt et al, 1993; Wick et al, 1995; van der Toorn et al, 1996; Dijkhuizen et al, 1999; Pfeuffer et al, 2000; Abe et al, 2000; de Graaf et al, 2001; Dreher et al, 2001; Harada et al, 2002; Valette et al, 2005a; Ellegood et al, 2005). The apparent diffusion coefficient (ADC) of metabolites reflects several biophysical parameters such as viscosity, cell swelCorrespondence: Dr V Lebon, Commissariat a` l’E´nergie Atomique, Service Hospitalier Fre´de´ric Joliot, 4 place du Ge´ne´ral Leclerc, Orsay 91401, France. E-mail: [email protected] Received 25 January 2006; revised 11 April 2006; accepted 3 May 2006; published online 21 June 2006

ling, restriction in subcellular structures, cytoplasmic streaming, etc. However, the relative contribution of each parameter to NMR-measured ADC remains unclear (for a review see Nicolay et al, 2001), limiting data interpretation. Halogenated volatile anesthetics such as isoflurane or halothane are commonly used for DWNMR spectroscopy of the brain (Merboldt et al, 1993; Wick et al, 1995; van der Toorn et al, 1996; Dijkhuizen et al, 1999; Pfeuffer et al, 2000; Abe et al, 2000; de Graaf et al, 2001; Dreher et al, 2001). These anesthetics constitute a rather homogenous class of drugs, which interact similarly with biological membranes. In particular, there is a correlation (described by the Meyer-Overton theory) between lipid solubility of inhaled anesthetics and the minimal alveolar concentration, suggesting that anesthesia occurs when a given number of inhalational anesthetic molecules dissolve in lipid cell membranes. Although the exact mechanism provok-

Isoflurane strongly affects metabolite ADC in the brain J Valette et al 589

ing anesthesia is still debated, it has been proven that the presence of volatile anesthetic in lipid bilayers induces changes in membrane fluidity, viscosity and permeability (Trudell et al, 1973; Qin et al, 1995). Under normal physiological conditions and in the absence of halogenated anesthetics, brain metabolites hardly cross lipid bilayers. For this reason, perturbation of membrane properties induced by halogenated volatile anesthetics may potentially affect the diffusion of brain metabolites. Surprisingly, the effect of volatile anesthetic on intracellular metabolites diffusion has never been evaluated. However, any potential effect may reveal critical for comparative studies. It could also give an insight into the controversial mechanism by which halogenated anesthetics operate. Finally, the potential perturbing effect of volatile anesthetics on membrane permeability may bring out the importance of restriction phenomenon in the process of intracellular metabolite diffusion. In the present study, macaque monkeys were anesthetized with two different doses of isoflurane. The effect of isoflurane dose on brain metabolite diffusivity was assessed by diffusion tensor spectroscopy (DTS) for improved measurement accuracy and reproducibility (Valette et al, 2005b; Ellegood et al, 2006). Experiments show that isoflurane significantly increases the diffusion of all detected metabolites: N-acetyl-aspartate, creatine, choline, glutamate and myo-inositol. Comparison of ADC changes experienced by the five metabolites strongly argues in favor of isoflurane partly releasing subcellular restriction on metabolite diffusion, this interpretation being consistent with previously reported DW-magnetic resonance spectroscopy (MRS) studies.

Materials and methods Animal Handling Five experiments were performed on two healthy macaque monkeys (Macaca fascicularis, body weight B7 kg). Procedures were in accordance with the recommendations of the European Community (86/609) and the French National Committee (87/848). Primary anesthesia was induced by a small intramuscular (i.m.) ketamine–xylazine injection (B1 mL). Animals were intubated and ventilated with a 55:45 mixture of oxygen and air, and a variable dose of isoflurane (CHF2-O-CHClCF3). The body temperature was maintained at physiological temperature by circulation of warm water. Physiological parameters were monitored using an MR compatible Maglife system (Schiller Me´dical SA, Wissembourg, France). The animal was held in the sphinx position and the head was positioned in a stereotaxic frame with a bite-bar and ear rods. Isoflurane dose was first set to 1% (the minimal dose to maintain anesthesia) and DW-MRS was performed under

this conditions (B1-h acquisition). Isoflurane dose was then set to 2% and no acquisition was performed for 30 to 45 mins to allow for physiological parameters stabilization, essentially decreased blood pressure: systolic/diastolic pressure dropped from 6276/2674 to 4873/157 5 mm Hg (mean7s.d.) for the five experiments. Another complete DW-MRS acquisition was then performed. Note that other physiological parameters remained relatively unaltered when varying isoflurane dose: body temperature slightly changed from 37.11C71.01C to 36.21C70.91C, breathing frequency increased from 970 min1 to 107 1 min1, expired CO2 decreased from 3273 to 2972 mm Hg. Heart rate was 9078 min1 under 1% isoflurane. Owing to the use of a pulse oximeter, decreased blood pressure associated with high anesthetic dose did not allow to monitor heart rate under 2% isoflurane, except for one single experiment where heart rate proved to remain stable. A randomized temporal sequence of anesthetic dose might a priori have limited bias; however, little is known on the clearance of isoflurane in the monkey brain. In this context, starting the experiment with a 1-h session at 2% isoflurane may have biased the following measurement at 1% isoflurane. Therefore, all experiments began at 1% isoflurane. To make sure the measured changes in ADC were associated with isoflurane increase and not with a possible adaptation to prolonged anesthesia, an additional experiment was performed in which the isoflurane dose was maintained at 1% (two complete DW-MRS acquisitions were successively acquired under 1% isoflurane).

Nuclear Magnetic Resonance Acquisition Experiments were performed on a whole-body 3 Tesla system (Bruker, Ettlingen, Germany) equipped with a gradient coil reaching 44 mT/m in 400 ms. A surface coil was used for radiofrequency emission and detection. Anatomical localization was achieved with a T1-weighted image. An 18  18  18 mm3 (5.8 mL) voxel was positioned in the fronto-parietal lobe, including grey and white matter (Figure 1). Shimming was performed with Fastmap (Bruker, Ettlingen, Germany) (Gruetter, 1993), leading to B5 Hz linewidth as measured on water. The DW sequence

Figure 1 Voxel position in the monkey brain displayed on a scout T1-image. Journal of Cerebral Blood Flow & Metabolism (2007) 27, 588–596

Isoflurane strongly affects metabolite ADC in the brain J Valette et al 590

was built from a STEAM (Stimulated Echo Acquisition Mode) scheme with TE = 21 ms, TR = 2500 ms and mixing time TM = 110 ms, corresponding to diffusion gradient length d = 8 ms and diffusion gradient separation D = 121 ms, as described elsewhere (Valette et al, 2005a). Water suppression was performed with an optimized 8-pulses VAPOR scheme (Tkac et al, 1999). Improved localization was obtained with outer volume suppression consisting in home made dual-band hyperbolic secant pulses applied at three different power levels (pulse duration = 3 ms, spectral width = 12 kHz). To correct for small movement artifact, individual scan zero-order phasing was performed (Posse et al, 1993; Valette et al, 2005a). Diffusion tensor spectroscopy was implemented to minimize measurement variability: the trace ADC, which can be derived from DTS, is rotationally invariant and thus insensitive to possible variations of head orientation relative to the gradient coil. The complete determination of the diffusion tensor requires diffusion measurement along six directions. For maximizing the diffusion gradient strength Gd these directions were XZ, XZ, YZ, Y-Z, XY, XY. After the acquisition of a first spectrum with Gd = 0 (number of transients NT = 256), the entire set of six directions was entirely acquired at b = 1000 s/mm2 in the order given above, then the six directions were again entirely acquired at b = 2000 s/mm2. Note that using a larger number of b values is not theoretically required since the logarithm of metabolite signal attenuation is essentially linear in this range of b values for the diffusion time used here (Pfeuffer et al, 2000; Valette et al, 2005a). The acquisition of more b values would improve measurement accuracy, but of course at the expense of time resolution. For each diffusion weight, 128 transients were collected. Before the acquisition of DW spectra, a macromolecule spectrum was acquired using the metabolitenulling method (TI/TR = 365/1000 ms, NT = 1024) (Behar et al, 1994). For each experiment, spectra processing first consisted in subtracting macro-molecules from all DW spectra. The resulting spectra were analyzed with LCModel (Provencher, 1993). The basis-set was made of total N-acetyl aspartate (tNAA including NAA and NAAG), total creatine (tCr including creatine Cr and phosphocreatine PCr), total choline (tCho including phosphorylcholine PC and glycerophosphorylcholine GPC), glutamate (Glu), myo-inositol (Ins), glutamine, lactate, aspartate, taurine and glucose (Valette et al, 2005a). Signal attenuation along each direction as determined by LCModel was then automatically processed with home-made routines to estimate log-linear best fit and to calculate the diffusion tensor D for tNAA, Glu, tCr, tCho and Ins. The trace (i.e. rotationally invariant) ADCtrace = trace(D)/3 was then derived. At the beginning and at the end of each ADC measurement, control spectra (no diffusion gradients, NT = 32) were acquired to assess signal stability on anesthesia. Additionally, in one experiment, a macro-molecule spectrum was acquired at the beginning and at the end of NMR measurement for independent evaluation of macro-molecule stability under isoflurane anesthesia. Journal of Cerebral Blood Flow & Metabolism (2007) 27, 588–596

Results Effect of Isoflurane Dose

As shown by control spectra displayed in Figure 2, the concentration of all detected metabolites remained stable during the entire experiment. The only metabolite exhibiting variation was lactate, showing a significant increase when changing isoflurane dose from 1% to 2%. Similarly the macro-molecule spectrum remained stable under isoflurane anesthesia (1% and 2%). A stack-plot of diffusion weighted signal attenuation (along the XZ direction) is presented in Figure 3. Values of ADCtrace measured under 1% isoflurane and 2% isoflurane are reported in Table 1, revealing an average ADCtrace increase of 25%78% for the five dose varying experiments. Increases ranged from 13% for Ins to 53% for tCho, as displayed in Figure 4. For each metabolite, ADCtrace variation was evaluated by a Student’s paired t-test for each set of two successive dose-varying experiments. Statistical significance (which was set to P < 0.05) was achieved for all detected metabolites. Water diffusivity was also measured for three experiments, demonstrating a moderate (3%) but significant increase when changing isoflurane dose from 1% to 2% (Table 1). Experiment at Constant 1% Isoflurane Dose

The additional experiment in which isoflurane dose was maintained at 1% during the 2 DW measure-

Figure 2 Typical control spectra (2 Hz line-broadening) acquired at the beginning of the 1% isoflurane dose experiment and at the end of the 2% isoflurane dose experiment. The difference spectrum demonstrates excellent stability throughout the experiment. Note the lactate increase induced by isoflurane anesthesia at 1.33 ppm.

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reflects measurement noise ( + 6% for tNAA, 6% for tCr, + 6% for Glu, 8% for tCho and 11% for Ins).

Discussion Measurement Reliability

Figure 3 Stack-plot of DW-spectra collected along the XZ direction for one experiment under 1% isoflurane (0.5 Hz line broadening). Note the small lactate signal at 1.33 ppm.

Table 1 ADCtrace measured under 1% and 2% isoflurane, and corresponding ADC increase (mean7s.d., N = 5 except for water N = 3) ADCtrace (mm2/ms) ADCtrace (mm2/ms) 1% isoflurane 2% isoflurane tNAA tCr Glu tCho Ins Water

0.13670.006 0.10670.016 0.22370.026 0.09470.010 0.19270.019 0.68070.016

0.15570.021 0.13570.022 0.26370.029 0.14470.022 0.21770.030 0.69770.011

ADC increase

14713% (P < 0.05) 2777% (P < 0.001) 20718% (P < 0.03) 53717% (P < 0.002) 1378% (P < 0.02) 370% (P < 0.05)

80% 70% 60% 50% 40% 30% 20% 10% 0% tNAA






Figure 4 Apparent diffusion coefficient increase measured for brain metabolites between 1% and 2% isoflurane dose (average of diffusivity increase for each set of two successive experiments, N = 5 except for water N = 3).

ments did not show any significant variation in ADCtrace. On average, the five detected metabolites exhibited a 2% change between the two subsequent measurements of ADCtrace, which likely

Diffusion tensor spectroscopy allowed excellent measurement reproducibility as compared with conventional single direction ADC measurement: typical s.d. on ADCtrace is B10%, as compared with an B25% standard deviation on single direction ADC measured under similar experimental conditions (Valette et al, 2005a), demonstrating the superiority of DTS. This difference can be ascribed to anisotropic diffusion of brain metabolites in tissues (Valette et al, 2005b; Ellegood et al, 2006) causing increased inter-experiment variability with single direction ADC measurement, while rotationally invariant ADCtrace as derived from DTS experiment is not sensitive to anisotropy. For each detected metabolite, ADCtrace is very close to the single direction ADC value reported in the same volume of interest (Valette et al, 2005a), confirming significant differences among metabolite diffusion coefficients, ranging from B0.1 mm2/ms for tCho to B0.2 mm2/ms for Glu. Comparison with rodent data acquired with a similar NMR sequence (Pfeuffer et al, 2000) reveals differences in ADC values. For the five detected metabolites, ADC values as reported by Pfeuffer et al, at 2% isoflurane are 21 to 55% lower than ADC values as measured in our monkey study. This might be partly explained by the lower b values and the lower B0 used in our study (Valette et al, 2005a). Possible interspecies differences might also contribute to the discrepancy between the rodent and the primate measurements. The ADC values measured in the monkey brain for the five metabolites present a higher dispersion than the one reported in the rat brain (Pfeuffer et al, 2000; Dreher et al, 2001). However, our monkey study is in agreement with human studies reporting rather dispersed ADCs for the three most observed metabolites tCho, tCr and tNAA (Posse et al, 1993; Ellegood et al, 2005). Moreover, it should be pointed out that glutamate and myo-inositol ADC have never been measured in primates (including humans) except by our group, so that comparative values are still lacking for these two metabolites. Prolonged 1% isoflurane anesthesia had no significant effect on ADCtrace, as demonstrated by the additional experiment in which two subsequent measurements were performed at 1% isoflurane (2% ADCtrace decrease as compared with 25%78% increase when changing isoflurane dose from 1% to 2%). In addition to metabolite and macro-molecule signal stability, this demonstrates that diffusivity changes between 1% and 2% isoflurane cannot be ascribed to systematic bias such as coil Journal of Cerebral Blood Flow & Metabolism (2007) 27, 588–596

Isoflurane strongly affects metabolite ADC in the brain J Valette et al 592

de-tuning, variation of metabolite concentration, baseline variation induced by the isoflurane dose or brain adaptation to prolonged anesthesia. Only lactate level increased with isoflurane dose, but this cannot affect diffusion measurement since lactate resonates B90 Hz away from the closest detected metabolite (tNAA). Lactate accumulation is likely to result from isoflurane inhibiting mitochondrial respiration, as it has already been reported (Brabec et al, 1984). It has been argued that changes in magnetic susceptibility as caused by blood oxygenation level dependent (BOLD) effect might bias the effective b value (i.e. b value including microscopic background gradients), resulting in artifactual ADC variations (Zhong et al, 1991). However, our data tend to show that ADC measurements are not affected by this effect in our experimental conditions. First, changes in magnetic susceptibility may potentially result in measurable linewidth changes on 1H spectra. In the present work, the absence of significant linewidth change can be visually assessed in Figure 2, which demonstrates lineshape stability during the entire course of the experiment. Measuring the full-width at half-maximum (FWHM) on spectra at b = 0 under 1% and 2% isoflurane quantitatively demonstrates that metabolite linewidth exhibits no significant change (FWHM change is 3%76% for tCr and 5%77% for tNAA, N = 5), suggesting the absence of significant susceptibility change. Consequently, the effective b values must be identical under 1% and 2% isoflurane. The fact that no significant BOLD effect could be observed might be explained by the high oxygen content (B65%) of the inhalated gas that guarantees blood to remain highly saturated in oxygen regardless of the effect of isoflurane on oxidative metabolism. Finally, even considering the improbable case where susceptibility changes could induce significant variation of the effective b values without significant linewidth variation, the effect on ADC should be similar for all molecules. This is clearly not consistent with the range of ADC variation observed in the present study (from 3% for water to 53% for tCho), ruling out the possibility of any significant bias associated with blood susceptibility changes between both isoflurane doses.

Effect of Isoflurane on Metabolite Diffusion

The effect of isoflurane on metabolite diffusivity appears to be dramatic. Since isoflurane impairs energy metabolism, a decreased diffusivity would be expected if energy-dependent cytoplasmic streaming was to contribute significantly to the ADC (Agutter et al, 1995; Wheatley, 1998; Nicolay et al, 2001). In contrast, metabolite diffusivities exhibit strong increases with isoflurane dose, suggesting that another phenomenon dominates the diffusion process at the time scale of DW-NMR. Journal of Cerebral Blood Flow & Metabolism (2007) 27, 588–596

Importance of Restriction Effects

Although the free, unrestricted intracellular diffusion coefficient is expected to differ between detected metabolites because of their various sizes, there are some experimental evidences that brain metabolite ADC in vivo do not directly depend on the free diffusion coefficient (Nicolay et al, 2001; Pfeuffer et al, 2000). Theoretical models (e.g. Tanner and Stejskal, 1968) show that the ADC is relatively insensitive to the free diffusion coefficient for intracellular metabolites experiencing significant restriction, although the importance of such restriction effects for brain metabolites has never been clearly assessed in vivo. Given the high membrane content of the intracellular space (106 mm2 intracellular membrane surface for 1000 mm3 intracellular volume (Agutter et al, 1995)) and the apparent limited contribution of cytoplasmic streaming for the observed brain metabolites, metabolites must encounter several barriers during the long diffusion time used for NMR measurements (Nicolay et al, 2001), so that the mean distance traveled during the diffusion time must be dominated by the geometry of the diffusion space rather than by free diffusion. For this reason, NMR-measured ADC will be primarily interpreted in terms of restriction effects in the following discussion, and consequently ADC changes will be regarded as changes in metabolite restriction.

Alteration of Membrane Restriction by Isoflurane

Under normal physiological conditions and in the absence of halogenated anesthetics, lipid bilayers are almost totally impermeable to brain metabolites, requiring active transportation by specialized transporters. Restriction effects are therefore expected to be very strong in that case. The effect of volatile anesthetics on membranes permeability might explain the partial release of metabolite restriction, that is, the facilitated exchange of metabolites through the membranes, ultimately resulting in an increased ADC as observed in our study. Indeed, in vitro experiments have established that lipid bilayers that are predominantly in gel phase under normal physiological conditions, are affected by volatile anesthetics: halogenated gas decrease the transition temperature from the gel phase to the liquid-crystalline phase (Trudell et al, 1973), so that the transition temperature may decrease down to physiological temperature (Tsukamoto et al, 1992). Knowing that transmembrane permeability is maximal at the transition temperature (Antonov et al, 1980; Clerc and Thompson, 1995), this mechanism might explain how isoflurane increases membrane permeability. This interpretation is supported by the finding that isoflurane makes lipid bilayers partly permeable to charged molecules that do not cross biological membranes under normal physiological

Isoflurane strongly affects metabolite ADC in the brain J Valette et al 593

conditions (Qin et al, 1995). Our experimental finding that water diffusion is substantially less affected by isoflurane than metabolite diffusion also supports this interpretation: indeed biological membranes are already largely permeable to water molecules. Isoflurane-induced changes in membrane permeability can occur either at the cell membrane level or at subcellular organelle level. In the first case, intracellular metabolites would be released in the extracellular space where diffusivity is expected to be higher. Global ADC would increase consequently. However, metabolites are unlikely to be significantly released in the extracellular space, even at 2% isoflurane dose. In the pioneer work of Pfeuffer et al (2000) performed in the rat brain under 2% isoflurane at very high b values, none of the five metabolites measured in the present study exhibited any detectable extracellular component. This suggests that isoflurane has little effect on cell membrane permeability in vivo, so that metabolites remain purely intracellular. In this context increased permeability might be explained by facilitated exchange between subcellular compartments such as mitochondria, nucleus, endoplasmic reticulum, lysosome, Golgi apparatus, etc. Arguments favoring this interpretation can be inferred from the comparison of ADC changes experienced by the five different metabolites. Negative Correlation between Apparent Diffusion Coefficient and Apparent Diffusion Coefficient Change

As discussed previously, the ADC of intracellular metabolites must primarily reflect metabolite restriction, which may differ due to specific compartmentation. In this context, increasing organelle permeability should have a stronger effect on heavily restricted metabolites. In other words, metabolites having a low ADC such as tCr or tCho (Table 1) should experience a stronger ADC increase under 2% isoflurane as compared with metabolites having a high ADC (Glu or Ins). As shown in Figure 5, this prediction is verified by our data exhibiting a negative correlation between ADC and ADC change on isoflurane. This observation argues in favor of isoflurane releasing subcellular restriction, leading to homogenized intracellular metabolite diffusion.

Figure 5 Negative correlation between ADC and ADC increase.

thesia. For studies performed with halogenated volatile anesthetics, ADC coefficient of variation is V ¼ 6%  3% (eight reported studies: Merboldt et al, 1993; Wick et al, 1995; van der Toorn et al, 1996; Dijkhuizen et al, 1999; Pfeuffer et al, 2000; Abe et al, 2000; de Graaf et al, 2001; Dreher et al, 2001). Studies performed with other—or without—anesthetics reveal a significantly higher coefficient of variation V ¼ 13%  4% (four reported studies: Posse et al, 1993; Harada et al, 2002; Valette et al, 2005a; Ellegood et al, 2005). This literature review includes all brain DW-MRS studies currently published to our knowledge. It clearly shows that ADC coefficient of variation is significantly reduced when halogenated volatile anesthetics are used. This trend is confirmed by the present report where two different doses of isoflurane were used: under high isoflurane dose, the coefficient of variation was V ¼ 7%, as compared with V ¼ 19% under lower isoflurane dose. The finding of metabolite ADCs converging under the action of isoflurane brings new arguments in favor of isoflurane homogenizing intracellular diffusion. The interpretation of isoflurane partly releasing metabolite restriction argues in favor of metabolite diffusion being significantly restricted in subcellular structures, so that the range of diffusivity measured for various metabolites (from 0.09470.010 mm2/ms for tCho to 0.2237 0.026 mm2/ms for glutamate under 1% isoflurane) could be largely explained by differences in restriction under normal conditions.

Convergence of Metabolite ADCs under the Action of Isoflurane

Another way to test the hypothesis of isoflurane homogenizing diffusion within the intracellular space is to calculate ADC coefficient of variation among metabolites: V ¼ s:d:ðADCÞ=meanðADCÞ. Performing the calculation for tNAA, tCr and tCho allows to re-visit all DW-MRS studies reported in the literature to test the hypothesis of a reduced coefficient of variation under halogenated anes-

Specific Effect of Isoflurane on Subcellular Membranes

Although the study of membrane permeability goes beyond the scope of this work, an overview of currently available knowledge on this matter may provide possible explanation for subcellular membranes behaving differently from plasmic membranes in the presence of high isoflurane doses. Journal of Cerebral Blood Flow & Metabolism (2007) 27, 588–596

Isoflurane strongly affects metabolite ADC in the brain J Valette et al 594

Indeed, organelle membranes present important structural differences with cell membranes: for example, mitochondria or microsome membranes contain much less cholesterol (B5%) than cell membranes (B30%) (Shechter, 1990; Romsicki and Sharom, 1997). The fact that cholesterol affects the phase transition of biological membranes (Shechter, 1990; Gaus et al, 2003) might provide a possible explanation for organelle and cell membranes being differently sensitive to isoflurane, knowing that membrane permeability is maximal at the phase transition while it is lower when membranes are in pure gel or pure liquid-crystalline phase (Antonov et al, 1980; Clerc and Thompson, 1995).

Choline Compounds

As compared with the four other detected metabolites, the diffusion of tCho exhibits two striking differences: the ADC of choline compounds appears significantly low (0.09470.010 mm2/ms for tCho versus 0.16470.053 mm2/ms for the other metabolites) and much more sensitive to isoflurane (B50% increase when increasing isoflurane from 1% to 2% versus B20% for other metabolites). Considering the relationship between diffusion and restriction as previously discussed, the diffusion of tCho suggests that, under normal conditions, a large fraction of tCho pool is confined in a highly hindered space. Owing to molecular crowding and diffusional obstructions, this fraction is likely to have a relatively long rotational correlation time (LopezBeltran et al, 1996; Garcia-Perez et al, 1999), resulting in a shorter relaxation time T2. In this case, the highly restricted pool of tCho would be mainly detectable at short echo time, while longer echo times would only detect the less restricted pool of tCho. Once again, this interpretation can be tested by comparing all diffusion measurements of tCho reported up to now: literature review reveals that studies measuring a relatively smaller ADC for tCho were performed at shorter echo-time than studies reporting high ADC for tCho (TE = 62750 ms (Posse et al, 1993; Wick et al, 1995; van der Toorn et al, 1996; Pfeuffer et al, 2000; Abe et al, 2000; de Graaf et al, 2001; Valette et al, 2005a) versus TE = 1437 23 ms (Merboldt et al, 1993; Dijkhuizen et al, 1999; Dreher et al, 2001; Harada et al, 2002; Ellegood et al, 2005)). The relationship between echo-time and ADC for tCho is particularly striking when limiting comparison to studies that were performed without halogenated anesthetics. In this case, a maximal fraction of tCho is supposed to be confined in hindered space, and a gap is expected between tCho ADC and other metabolites. As shown in Figure 6, there is a remarkable positive correlation (R2 = 0.89) between echo-time and normalized ADC for tCho (i.e. ADC for tCho divided by the average ADC of tNAA, tCr and tCho) (Posse et al, 1993; Harada et al, 2002; Valette et al, 2005a; Ellegood et al, 2005). This Journal of Cerebral Blood Flow & Metabolism (2007) 27, 588–596

Figure 6 Normalized ADC of tCho (i.e. tCho ADC divided by the average of tNAA, tCr and tCho ADCs) as a function of echo-time TE. Data are taken from former studies performed without halogenated anesthetics (Posse et al, 1993; Harada et al, 2002; Valette et al, 2005a; Ellegood et al, 2005).

shows that long TE ‘filter’ the signal of a slowdiffusing tCho fraction. This observation supports the idea that a fraction of tCho is heavily restricted in subcellular structures. A possible reason for this could be that choline compounds are potentially present within the lipid bilayer, because PC and GPC are involved in membrane maintenance and degradation pathways.

Conclusion This study shows that the diffusivity of intracellular metabolites tNAA, tCho, tCr, Glu and Ins is significantly increased by high isoflurane dose in the monkey brain. Detailed analysis of diffusion changes experienced by the five detected metabolites shows that ADC and ADC changes are negatively correlated and that metabolite ADC converges under the action of isoflurane. This finding is further supported by literature review demonstrating that metabolite ADCs measured under halogenated volatile anesthetics exhibit a significantly smaller coefficient of variation than ADC measured with other anesthetics. Altogether these observations strongly support the idea of isoflurane partly releasing metabolite restriction at subcellular level. While metabolites are compartmentalized in various organelles under normal conditions, restriction in these compartments is attenuated at high isoflurane dose. In the limit of high membrane permeability, metabolites tend to diffuse in the same minimally hindered diffusion space, and metabolite ADCs increase and tend to converge. The particular diffusion of tCho, which ADC is lower than other metabolites and more sensitive to isoflurane, likely reflects the existence of a highly restricted large pool. Future studies using DW-NMR spectroscopy to probe subcellular structures should take the effect

Isoflurane strongly affects metabolite ADC in the brain J Valette et al

of volatile anesthetics into account to preserve the variety of diffusion spaces. This work argues in favor of restriction by membranes being of critical importance on the diffusion process of metabolites at the time-scale of in vivo DW-NMR spectroscopy (B100 ms), while the free diffusion coefficient, and consequently temperature and viscosity, may be of secondary importance (at least within a reasonable range). It suggests that, under normal physiological conditions, organelles can be considered as relatively independent compartments regarding their metabolite content. In the case of mitochondria, this observation is consistent with the recent observation by 13C NMR that mitochondrial and cytosolic glutamate/a-ketoglutarate pools are not in fast equilibrium (Garcia-Martin et al, 2002), suggesting that the exchange through the mitochondrial membrane could be the limiting step. Finally, the loss of clear subcellular compartmentation under the action of volatile anesthetics may have physiological implications. For example, it is generally accepted that mitochondrial/cytosolic compartmentation is required for efficient mitochondrial function (Stryer, 1981; Sonnewald et al, 2004). In this context, an increase in mitochondrial membrane permeability could be a possible mechanism by which isoflurane inhibits oxidative metabolism.

Acknowledgements The authors thank Dr Denis Le Bihan for helpful and supporting discussions.

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