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In Response

Xie, Zhongcong MD, PhD; Wang, Hui MD, PhD

doi: 10.1213/ANE.0000000000001177
Letters to the Editor: Letter to the Editor
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Department of Anesthesia, Critical Care, and Pain Medicine, Massachusetts General Hospital and Harvard Medical School, Charlestown, Massachusetts, zxie@mgh.harvard.edu

Department of Anesthesia, Beijing Chaoyang Hospital, Capital Medical University, Beijing, China

We would like to thank Drs. Kaufman and Jensen1 for their detailed and constructive comments on our recent article in which we showed that 2-deoxy-D-glucose enhances anesthetic effects in mice.2 The last experiment described in the article used magnetic resonance spectroscopy (MRS) to detect the effects of anesthetic isoflurane on adenosine triphosphate (ATP) levels in the mouse brain. Drs. Kaufman and Jensen request clarification of some aspects of those experiments and challenge the data derived from the MRS studies. We agree with some of their concerns and provide the following clarifications and explanations.

First, with respect to questions about methodology related to the 31P-MRS studies, the studies were conducted in the Capital Medical University in Beijing, P. R. China, and were performed with a Bruker Clinscan 7.0 Tesla MR system (Bruker-Biospin, Billerica, MA), operating at a proton frequency of 300 MHz and a 31P frequency of 121 MHz. Single-pulse 31P spectra were obtained with a surface coil applied to the mouse skull via a 2-second repetition time, spectral bandwidth of 10 kHz, 128 acquisitions, and a 90-degree flip angle. Peak areas were calculated with Siemens Syngo 2004 B software (Bruker-Biospin, Billerica, MA).

Second, regarding concerns that the spectra shown in Figure 6, B and C, “do not resemble typical 31P-MRS spectra,” have “very low signal-to-noise ratios and baselines that fluctuate wildly,” and “do not appear to conform to source data peaks or baselines,” the authors acknowledge that the MRS studies reported in the article may suffer from technical limitations and that the data presented in Figure 6, B and C, are questionable and may not be considered without further validation. Part of this may be the result of experimental conditions. Most studies use anesthetized animals as controls and gather spectra data over a long period of time (30–60 minutes), whereas our control mice were awake, restrained, and with neither anesthesia nor previous acclimation to restraint during the imaging studies, and spectra were gathered over 4 to 10 minutes, whereas spectra were gathered in the anesthetized mice during the transition from general anesthesia to consciousness (i.e., emergence). This protocol might have produced restraint stress in the control mice, undermining the quality of the spectra and confounding the interpretation of the spectroscopy studies. Thus, the data obtained from MRS studies remain preliminary and are not of sufficient quality to be analyzed. Nevertheless, the main finding in the manuscript is that 2-deoxy-D-glucose enhances anesthetic effects in mice and, although the behavioral findings would be stronger if properly performed MRS studies had provided additional support, this conclusion remains valid without the MRS data.

Third, Drs. Kaufman and Jensen point out that the phosphocreatine (PCr)/β-ATP ratios presented in Table 2 appear low (0.18 and 0.77 for control and anesthesia mice, respectively) and are inconsistent with those reported in the literature (approximately 1.7).3,4 Our low values could be a measurement error or simply reflect differences in experimental conditions noted previously. Indeed, there is precedent for PCr/β-ATP ratios in this range in the literature in certain conditions, although not in brain—e.g., prostate carcinoma in rats (0.86 for hormone-sensitive tumor and 0.26 for hormone-resistant tumor, respectively)5 and human prostate adenocarcinoma in mice6 under pentobarbital5 and ketamine-xylazine6 anesthesia, respectively. Moreover, the use of an entirely different method of measuring the ATP levels in vitro yielded results similar to the MRS experiments.

Zhongcong Xie, MD, PhD

Department of Anesthesia, Critical Care, and Pain Medicine

Massachusetts General Hospital

and Harvard Medical School

Charlestown, Massachusetts

zxie@mgh.harvard.edu

Hui Wang, MD, PhD

Department of Anesthesia

Beijing Chaoyang Hospital

Capital Medical University

Beijing, China

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REFERENCES

1. Kaufman MJ, Jensen JEN. Regarding “2-deoxy-D-glucose enhances anesthetic effects in mice.” Anesth Analg. 2016;122:1224–5
2. Wang H, Xu Z, Wu A, Dong Y, Zhang Y, Yue Y, Xie Z. 2-Deoxy-D-glucose enhances anesthetic effects in mice. Anesth Analg. 2015;120:312–9
3. Bresnen A, Duong TQ. Brain high-energy phosphates and creatine kinase synthesis rate under graded isoflurane anesthesia: an in vivo (31) P magnetization transfer study at 11.7 tesla. Magn Reson Med. 2015;73:726–30
4. in ‘t Zandt HJ, Renema WK, Streijger F, Jost C, Klomp DW, Oerlemans F, Van der Zee CE, Wieringa B, Heerschap A. Cerebral creatine kinase deficiency influences metabolite levels and morphology in the mouse brain: a quantitative in vivo 1H and 31P magnetic resonance study. J Neurochem. 2004;90:1321–30
5. Vigneron DB, Hricak H, James TL, Jajodia PB, Nunes L, Narayan P. Androgen sensitivity of rat prostate carcinoma studied by 31P NMR spectroscopy, 1H MR imaging, and 23Na MR imaging. Magn Reson Med. 1989;11:152–60
6. Kurhanewicz J, Dahiya R, Macdonald JM, Jajodia P, Chang LH, James TL, Narayan P. Phosphorus metabolite characterization of human prostatic adenocarcinoma in a nude mouse model by 31P magnetic resonance spectroscopy and high pressure liquid chromatography. NMR Biomed. 1992;5:185–92
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