In vivo proton MRS to quantify anesthetic effects of pentobarbital on cerebral metabolism and brain activity in rat.

Sodium pentobarbital, one type of barbiturate, has been popularly used in clinic for treatment of seizures and preoperative sedation. It is also a common agent used for general anesthesia in clinical and animal research. Pentobarbital rapidly distributes to all tissues, with a higher concentration in brain, liver, and kidneys due to high lipid solubility. It can enhance the γ-aminobutyric acid receptor-coupled response, thereby directly depressing neuronal excitability and resulting in an anesthesia effect in the brain and vanishing consciousness (see a recent review article, Alkire et al. (1), and references therein). The suppression of neuronal activity is also accompanied by significant depression of cerebral metabolic activity due to the inhibition of nicotinamide adenine dinucleotide (NADH) oxidation in the respiratory chain, and a deep pentobarbital anesthesia can induce a complete suppression of brain electroencephalography (EEG) activity thereby reaching an isoelectric state. Therefore, the delivery of pentobarbital into a living body can lead to a variety of brain physiologic changes, including EEG activity, cerebral metabolites/metabolic rates, and cerebral blood flow, and all these changes are expected to be related to the pentobarbital concentration reached inside the brain. It is thus essential to seek a robust, reliable tool to noninvasively assess the brain pentobarbital concentration in vivo and, ultimately, to study the quantitative relationship between pentobarbital anesthesia depth and physiologic changes. In vivo proton (1H) magnetic resonance spectroscopy (MRS) could provide a suitable approach for fulfilling this task. Nevertheless, the detection sensitivity of in vivo proton MRS is still limited to directly measuring the brain pentobarbital due to a very low concentration under normal anesthesia conditions. For instance, a large dose of 70 mg pentobarbital per kg of body weight could induce a maximum brain pentobarbital concentration of approximately 300 µmol per kg brain tissue if one assumes that pentobarbital distributes uniformly in the body and it is not metabolized. Such a low concentration is barely detectable by in vivo proton MRS if the sampling time is inadequately long. However, to enhance the solubility of pentobarbital, the commercial available pentobarbital injection solution usually contains large amounts of water, ethanol (Eth) and propylene glycol (PG). It has been demonstrated that the 1H signal of Eth is not detectable in the rat brain with the pentobarbital anesthetic, presumably because of rapid metabolism of Eth in a living body (2). In contrast, the PG signal can be reliably detected by in vivo proton MRS (2–5). It is interesting to note that both pentobarbital and PG are chemically stable in the brain and primarily metabolized in the liver in which pentobarbital is oxidized and PG is converted to L-lactate (6). Therefore, the measure of the cerebral PG concentration could provide an indirect, sensitive index reflecting the pentobarbital concentration in the brain. In the present study, we used the localized in vivo proton MRS and EEG to quantitatively study the relationships among the alterations in cerebral metabolites/metabolic rates and EEG activity levels in the rat brain under varied anesthesia depths, which were achieved by the application of isoflurane and two different doses of sodium pentobarbital. This study aims to address the following questions: What is the implication of NMR signals of PG on quantification of brain glucose (Glc) concentration? Do the brain metabolite concentrations change with varied anesthesia depth? What is the quantitative relationship of neurometabolic coupling between brain EEG activity and cerebral metabolic rate? Can the PG signal measured by in vivo proton MRS provide a unique biomarker for monitoring anesthesia depth and cerebral metabolic rate changes caused by pentobarbital?