Quantification of Cerebral Glc Under Pentobarbital Anesthesia
Due to low pentobarbital concentration and rapid Eth consumption in the rat brain, these two compounds are almost invisible in the in vivo 1
H spectra of brain acquired in the present study; in contrast, the PG solvent provides intense proton signals across a wide range of chemical shift (2
). In the present study, we found that the presence of PG signal significantly influences quantification of lactate (Lac), Glc and glutathione, which have low concentrations (~1 mM) and which resonances partially overlap with the PG resonances in the rat brain under deep pentobarbital anesthesia (High-PB). The concentrations of those metabolites were overestimated by the LCModel fitting if the PG resonance peaks were not considered and corrected in LCModel fitting. One example of Glc quantification shown in suggests a very large error (> 2-fold) in quantifying [Glc] under the High-PB condition if the PG contribution was not taken into consideration.
Another challenge faced by the quantification of absolute metabolite concentration is how to choose an internal concentration reference using a physiologically stable metabolite. The common practice is to use [tCr] as a standard reference, which was assumed as a constant of 8 mM under all the anesthesia conditions in this study. However, this quantification approach can become problematic if [tCr] changes under various anesthesia conditions. We have tested this question using the results from the present study and found no statistical differences between the tCr peak (3.01 ppm) integral measured under the light isoflurane anesthesia and that under Low-PB, as well as High-PB, condition (P
> 0.05; n
= 8, unpaired two-tail t
test). This is evident from the following quantification results:
is the integral of tCr resonance peak quantified by the Varian software package. The results indicate that [tCr] remains constant under a wide range of physiologic conditions and, thus, the tCr resonance peak provides a reliable and stable internal reference for in vivo proton MRS quantification of other brain metabolites.
Alterations in Cerebral Metabolites Under Deep Anesthesia
The cerebral metabolism is tightly correlated to brain function and neuronal activity (26
). Generally, increased brain activities require higher brain energy demands, resulting in a decrease of Glc concentration and possibly an increase of Lac concentration in the brain (19
). In addition, an increase in brain activity also leads to increased neurotransmitter cycling rates (24
), which may ultimately change the concentrations of neurotransmitters such as glutamate/glutamine and γ-aminobutyric acid (28
). For instance, the transient increases in the concentrations of lactate and glutamate, accompanied with the tendency of concentration decrease in Glc, have been observed in the human visual cortex during sustained visual stimulation (19
). It was also reported that there are significant changes in a number of brain metabolites and neurotransmitters, including the ratio of PCr versus Cr, γ-aminobutyric acid, glutamine, and glutamate, as well as neurotransmission cycling rate at near-freezing body temperature in hibernating mammals (31
). In the present study, the results based on the LCModel fitting of each animal proton MRS data ( and ) reveal that besides a significant increase of PG concentration when the pentobarbital dose was increased, three compounds, γ-aminobutyric acid, taurine, and Glc, were found to be significantly decreased (P
< 0.05, unpaired two-tail t
test) at the isoelectric state as compared to other two anesthesia states (isoflurane and Low-PB). The concentrations of γ-aminobutyric acid and Glc are relatively low in the brain, resulting in low signal-to-noise ratios and large LCModel fitting errors, indicated by the high values of Cramer-Rao lower bounds, in particular, under the isoelectric condition (). To reduce the uncertainty of spectral fitting results, the spectra were summed from all rat measurements, respectively, for the three anesthesia conditions (isoflurane, Low-PB, High-PB), and each summed spectrum from each of the three groups was fitted with LCModel again. When doing so, only the Glc and PG changes were found to be statistically significant and had the same trends as those found in individual-animal analysis, as shown in and .
Interestingly, there were no statistically significant changes in the concentrations of Lac and neurotransmitters such as glutamate/glutamine while brain activity was significantly depressed, especially at the isoelectric state, in which the spontaneous EEG activity is completely suppressed. Therefore, one should be cautious to interpret the concentration changes associated with brain bioenergetic changes. On the other hand, the cerebral metabolic rates (e.g., CMRglc) may provide a more informative measure to evaluate brain activity and energy changes in the anesthetized rat model.
Alterations in CMRglc Under Various Anesthesia Conditions and Neurometabolic Coupling
The brain Glc concentration, [Gi], gradually decreased from the isoflurane to High-PB anesthesia state only if the PG resonance peaks between 3.1 and 4.0 ppm were accounted for in the LCModel fitting (see and ). [Gi] showed an increase under deep anesthesia condition if the contaminations from the PG resonance peaks were not corrected in LCModel fitting. This quantification error could result in an overestimated [Gi] () and consequently to an underestimated CMRglc value at the isoelectric condition leading to a large decrease of 89% in the measured CMRglc as compared to that measured under the isoflurane anesthesia condition (). In contrast, the percentage of CMRglc decrease became 37% after the correction of PG contribution.
The observation of reduction in the brain Glc concentration when the brain EEG activity was suppressed under the isoelectric condition in the present study seemingly contradicts with other brain activation studies showing a decreased brain Glc concentration when the brain activity level was increased by brain stimulation (19
). However, this apparent discrepancy can be explained by the blood plasma Glc concentration change, which was substantially decreased under the isoelectric condition in the present study (). Since the brain Glc concentration is tightly coupled to the plasma Glc concentration and is regulated by the Glc transportation across the blood-brain barrier according to Eqs. 1
, a significant reduction in plasma Glc level could lead to a large decrease in brain Glc concentration, as observed in the present study.
Several lines of evidence support our observation of brain Glc reduction under the isoelectric condition. First, it has been shown that a concentration gradient between the plasma and brain Glc concentrations was preserved under the deep pentobarbital anesthesia condition, showing a much larger [G0
] than that of [Gi
] with a [G0
] ratio about 3 to 4 (24
). The measured [G0
] ratios under isoflurane (4.9 ± 1.0), Low-PB (4.3 ± 1.0), and High-PB (4.3 ± 1.6) conditions in the present study are in line with this range. Second, we have applied the quantitative relationship between steady-state [G0
] and [Gi
] as reported in the literature (24
) to estimate the values of [Gi
] based on the [G0
] values measured in the present study, and the estimated [Gi
] values were 2.3 mM, 1.3 mM, and 0.7 mM under the isoflurane, Low-PB, and High-PB anesthesia condition, respectively, which are in good agreement with our LCModel fitting results with PG correction ( and ). Third, the measured ratios of CMRglc
were from 0.3 to 0.4 for the isoflurane and Low-PB anesthesia conditions. They are similar to the value of CMRglc
= 0.38, measured under α-chloralose anesthesia conditions (35
One important finding from the present study is that the CMRglc
value remained 63% under the isoelectric condition compared to the isoflurane anesthesia condition (). This result suggests a significant amount of brain energy in the absence of brain spike activity; and this “housekeeping” energy is essential for maintaining cellular integrity in the brain. Note that the CMRglc
measurement in the present study reflects the total metabolic rate, which includes oxidative and nonoxidative Glc metabolism in both neuronal and glial cells. The reduction in total (or oxidative) CMRglc
in the rat brain by deep pentobarbital anesthesia was reported to be 40 ~ 64% compared to nitrous oxide analgesia, light α-chloralose anesthesia, or the awake conditions (24
). This metabolic rate reduction is consistent with other measurements showing a 61% reduction in the cerebral metabolic rate of oxygen (38
) and a 48% reduction in the cerebral metabolic rate of ATP under the isoelectric condition compared to the light isoflurane condition (11
However, one should be cautious when quoting the “housekeeping” energy as the percentage of the total brain energy budget since the value of total brain energy depends on the brain state referred. In the present study, the reference brain state was based on the light isoflurane anesthesia condition, in which the Glc metabolic rate has been significantly suppressed compared to an awake condition. For instance, cerebral metabolic rate of oxygen was reduced 35% in the dog brain under anesthesia with 2% end-tidal isoflurane concentration compared to an awake state (39
). Similarly, CMRglc
was found to reduce 41% in the rat cortical regions covering the motor, visual, auditory, and somatosensory cortices under anesthesia with 1.5% end-tidal isoflurane concentration (averaged CMRglc
= 0.53 µmol/g/min) compared to an awake state (averaged CMRglc
= 0.90 µmol/g/min) (40
). To consider this reduction (assuming approximately 41%) effect in the cerebral metabolic rates between the isoflurane and awake conditions, the “housekeeping” brain energy left under the isoelectric state determined by CMRglc
measurement in our study became ~37% in comparison with that in the awake brain. In the absolute quantification scale, the CMRglc
value in the rat under the isoelectric condition was estimated to be 0.33 µmol/g/min, which was in an excellent agreement with the values of oxidative CMRglc
measured by in vivo 13
C MRS under the isoelectric condition with High-PB (24
) and the autoradiographic result of total CMRglc
from 0.26 to 0.33 µmol/g/min in the sensory cortices (41
). It has been suggested that a substantial portion of this Glc metabolic activity under the isoelectric condition may be attributed from the glial cell (24
). Moreover, there is a decent agreement (in both absolute and relative scales) between the total (oxidative plus nonoxidative) CMRglc
values estimated by this study and by autoradiographic measurements under the isoelectric condition (40
) and the oxidative CMRglc
values measured by in vivo 13
C MRS (24
) and other indirect measurements (11
). This similarity suggests that a significant amount of “housekeeping” brain energy is produced by oxidative Glc metabolism.
The relatively small portion of “housekeeping” brain energy estimated in this study is consistent with the notion that the majority of brain energy is used to support brain activity and neuronal signaling in a resting and awake brain (11
). Moreover, CMRglc
is tightly correlated to SEI under varied anesthesia conditions (). This result indicates a tight neurometabolic coupling across a wide range of brain physiologic conditions, although SEI only provides a relative measure of spontaneous brain activity level.
This coupling relation can be quantified by a linear approximation as described by Eq. 3
In Eq. 
and , the CMRglc
values were relative to the isoelectric CMRglc
value (= 0.33 mol/g/min), which was estimated in the present study and reported in the literature (24
It is interesting to note that if [Gi] was quantified without using PG correction in the LCModel fitting, it could lead to an overestimated [Gi], ultimately, an underestimated CMRglc under the isoelectric condition with a large reduction of 89% compared to the isoflurane anesthesia condition (). This could result in an unreasonably smaller “housekeeping” energy of ~6% compared to the awake condition. These comparisons indicate again the importance of correcting PG contaminations for reliable quantification of the brain Glc concentration after the application of pentobarbital injection solution.
PG as a Biomarker Reflecting Anesthesia Depth, Brain Activity, and Bioenergetics
The PG concentration can approach up to 6 mM in the rat brain under the High-PB anesthesia condition ( and ). This unique solvent provides an intense, stable, well-resolved resonance peak at 1.13 ppm, which can be robustly detected by in vivo proton MRS and readily quantified by using LCModel fitting or manual integration (). Therefore, the brain PG concentration can be conveniently measured and quantified in a clinic MRI/MRS scanner.
Our results clearly indicate that the brain PG concentration is tightly correlated to the spontaneous EEG activity level quantified by SEI, as well as to the cerebral metabolic activity level quantified by relative CMRglc (). These findings lead to the conclusion that the PG resonance peak at 1.13 ppm closely reflects the pentobarbital concentration in the brain, and it provides a reliable and quantitative biomarker to link the levels of both EEG and brain metabolic activities, as well as anesthesia depth with varied pentobarbital dosage.