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. Author manuscript; available in PMC: 2025 Feb 1.
Published in final edited form as: Magn Reson Med. 2023 Oct 17;91(2):681–686. doi: 10.1002/mrm.29888

Subcutaneous deuterated-substrate administration in mice: An alternative to tail vein infusion

Kyu-Ho Song 1, Xia Ge 1, John A Engelbach 1, Liu Lin Thio 2, Jeffrey J Neil 1,2, Joseph JH Ackerman 1,3,4,5, Joel R Garbow 1,5
PMCID: PMC10966607  NIHMSID: NIHMS1971108  PMID: 37849055

Abstract

Purpose

Tail-vein catheterization and subsequent in-magnet infusion is a common route of administration of deuterium-labeled substrates in small-animal deuterium MR studies. With mice, because of the tail vein’s small diameter, this procedure is challenging. It requires considerable personnel training and practice, is prone to failure, and may preclude serial studies. Motivated by the need for an alternative, the time courses for common small-molecule deuterated substrates and downstream metabolites in brain following subcutaneous (sc) infusion were determined in mice and are presented herein.

Methods

Three deuterium-labeled substrates – [6,6-2H2]glucose, [2H3]acetate, and [3,4,4,4-2H4]beta-hydroxybutyrate – and 2H2O (D2O) were administered to mice in-magnet via sc catheter. Brain time courses of the substrates and downstream metabolites (and HOD) were determined via single-voxel deuterium MR spectroscopy.

Results

Subcutaneous catheter placement and substrate administration was readily accomplished with limited personnel training. Substrates reached pseudo steady state in brain within ~ 30–40 minutes of bolus infusion. Time constants characterizing the appearance in brain of deuterated substrates or HOD following D2O administration were similar (~15 min).

Conclusion

Administration of deuterated substrates via sc catheter for in vivo DMRS experiments with mice is robust, requires limited personnel training, and enables substantial dosing. It is suitable for metabolic studies where pseudo-steady-state substrate administration/ accumulation is sufficient. It is particularly advantageous for serial longitudinal studies over an extended period, as it avoids inevitable damage to the tail vein following multiple catheterizations.

Keywords: Deuterium, MR Spectroscopy, Mice, Subcutaneous, Brain

1. INTRODUCTION

Deuterium (2H) magnetic resonance spectroscopy (DMRS) and imaging (DMRI) of 2H-labeled substrates and their subsequent downstream metabolites is an emerging arena of metabolic imaging (1–28). With small-animal models, tail-vein catheterization is a common in-magnet route of substrate administration. With mice, a ubiquitous laboratory rodent model, the small diameter of the tail vein makes placing and maintaining a tail-vein catheter challenging. Indeed, the training of personnel to perform mouse tail-vein catheterization can require many practice sessions and some individuals are never able to successfully perform the procedure with an acceptable success rate. Further, in studies requiring repeated administration of substrates to individual mice over periods spanning multiple days or weeks, damage to the tail vein incurred following a very limited number of catheter placements (e.g., two or three) is often unavoidable, even with skilled personnel. This, obviously, limits the feasibility of extended longitudinal studies with individual mice, thus negating a great strength of noninvasive imaging.

Our laboratory is pursuing longitudinal serial DMRS studies of individual mice over multi-week periods of time. Faced with the above-described challenge, we considered alternative routes of in-magnet administration of 2H-labeled substrates and explored the feasibility and delivery characteristics of in-magnet administration of 2H-labeled substrates via a subcutaneous (sc) catheter. The sc administration of fluids, known in the medical literature as clysis, has been employed in various forms for nearly two centuries in both people (29–31) and animals (32,33).

2. METHODS

All procedures involving mice were approved by the Washington University Institutional Animal Care and Use Committee (IACUC). Experiments employed young wild-type hybrid mice (mean age 27 days postpartum) from an ongoing study that were the progeny of mating C57BL/6J females with asymptomatic 129S6/SvEvTac males heterozygous for a targeted deletion of the first exon in the α1 subunit of the neuronal, voltage-gated sodium channel (34). Mice were anesthetized with isoflurane, and a sc catheter was placed in the midline of the upper back, just below the neck region. Mice were placed in the scanner magnet (Agilent/Varian 11.74T DirectDrive™ system) and monitored for maintenance of normal body temperature and respiration. Single-voxel localized magnetic field shimming used a 1H volume coil and typically achieved a water 1H linewidth (full width at half maximum intensity) of 40 – 50 Hz with symmetrical lineshape. Single-voxel DMRS data were collected using a two-turn, 2.1-cm diameter surface coil and the SPECIAL pulse sequence (35) with outer-volume suppression (36). Nominal voxel dimensions were 3 × 6 × 5 mm3, and the voxel was completely contained within the brains of all subjects. DMRS data were acquired in 5-minute time blocks, two time-blocks prior to substrate administration and 14 time-blocks following substrate administration. Other data acquisition and analysis details are as described previously (8). Each 2H-labeled substrate was administered individually at a 1-molar concentration in 0.9% saline solution at a dose of 25 μL/g-body-weight (e.g., 500 μL in a 20g mouse). In separate experiments, pure (55.5 M) D2O saline was administered subcutaneously at the same volumetric dose.

An advantage of in-magnet substrate administration is that pre-substrate-injection data acquisition yields the natural-abundance semi-heavy water (HOD) signal amplitude, providing a convenient deuterium concentration reference (8). With knowledge of substrate and metabolite relaxation time constants and labeling stoichiometry, 2H resonance amplitudes can be converted to aqueous molar concentrations.

The initial ~30 minutes following sc “bolus” (~40 sec) administration of 2H-labeled substrate or D2O saline were characterized by a near exponential rise in brain concentration of 2H-labeled substrate or HOD. Exponential modeling of this time-course data yielded time constants characterizing the kinetics of 2H-labeled substrate/HOD appearance in brain following bolus sc administration.

3. RESULTS

Magnetization saturation corrections employed relaxation T1 values determined in vivo by other laboratories (3, 23, 28). Stoichiometry related label loss corrections followed published reports (6, 23).

Figure 1 shows Fourier-transformed (frequency-domain) DMRS data summed over the first 70 minutes following sc administration of Glc, Act, and βHB substrates for representative individual mice. Time-domain data for these same mice were modeled as the sum of exponentially decaying sinusoids using Bayesian analysis methods developed in our laboratory (37–39) – estimating resonance frequencies, decay-rate constants, and amplitudes – and the frequency-domain representation is shown below the actual data. Figure 2 shows averaged substrate and metabolite concentration time courses (Glc, n = 7; Act, n = 6; βHB, n = 6) following sc administration. Uptake of subcutaneously administered water is of interest as a freely diffusible, small-molecule reference species that is, obviously, present in abundance in aqueous solutions containing the substrate of interest. Figure 3 shows the HOD time course following D2O infusion (n = 6).

Figure 1.

Figure 1.

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Top panels: Exemplary mouse brain single-voxel (SPECIAL (35) with outer-volume suppression (36); 3 × 6 × 5 mm3 nominal dimensions) 2H MR spectra for data summed over the first 70 minutes following sc administration of, from left to right, Glc, Act, and βHB substrates. Each substrate was administered as a ~40-sec bolus of a 1-M solution (in saline), at a dose of 25μL/g-body-weight. A 10-Hz line broadening exponential apodization function was applied to the time-domain data prior to Fourier transformation. The intensities observed in these MR spectra are resonance amplitudes and not metabolite concentrations. Bottom panels: Frequency-domain representation of the Bayesian-estimated time-domain model (37–39) of the data shown in the top panel.

Figure 2.

Figure 2.

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Mean (± SEM) substrate, and downstream HOD and metabolite concentration time courses, following sc administration of, from left to right, Glc (n = 7), Act (n = 6), and βHB (n = 6) as 1-M substrate solutions (in saline) at a dose of 25μL/g-body-weight. As per Methods, using the known natural-abundance HOD concentration, substrate and metabolite T1 and T2 relaxation time constants, and labeling stoichiometry, 2H resonance amplitudes were converted to aqueous molar concentrations. Bottom panels: substrate and downstream metabolite time courses. Top panels: downstream HOD time courses.

Figure 3.

Figure 3.

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Mean (± SEM) HOD time course following sc administration of pure D2O (55.5 M) saline at a dose of 25μL/g-body-weight (n = 6).

Exponential time constants [min], mean:SD, for the appearance of 2H-labeled substrates or HOD in brain following sc bolus administration were Glc 16:6, Act 12:6, βHB 16:5, and HOD 16:4.

4. DISCUSSION

Subcutaneous administration of 2H-labeled substrates is convenient, robust, and provides relatively rapid, yet controlled, release of the administered dose. Distinct from mouse tail-vein catheterization, sc catheterization requires minimal personnel training or skill. In our hands, no adverse events following sc administration have been observed, and it is likely that higher doses could be tolerated. Likewise, serial administration of multiple sc doses would be expected to maintain pseudo-steady-state brain concentrations over extended observation periods. While we have not explored the appearance of 2H-labeled substrates in other tissues/organs following sc administration, we hypothesize similar, if not more rapid, kinetics given the vascular delivery challenge posed by the blood-brain-barrier.

Time constants for the appearance in brain of the 2H-labeled substrates examined herein (Glc, Act, βHB), and HOD following D2O saline administration, were remarkably similar. The time constants characterizing the appearance in brain of these 2H-labeled substrates were sufficiently short that the kinetics for the appearance of downstream metabolites were also observable. Substrates and HOD showed a pseudo-exponential increase in brain concentration over the initial ~30 minutes post sc administration and then reached a pseudo steady state for ~25 minutes, decreasing late in the time course. This contrasts with the kinetics following intravenous (tail vein) bolus administration in which, for example, the mouse/rat brain concentration of [6,6-2H2]glucose reaches a maximum ~10 minutes following infusion but then drops to ~50% of its maximum value ~20 minutes later(13,26). Because 2H MRS in vivo requires that deuterated substrates are delivered at substantially high pharmacologic doses, the subsequent metabolic kinetics of [6,6-2H2]glucose or other labeled substrates to downstream products is expected to be relatively independent of delivery method.

As expected of normal brain metabolism, Glc, Act, and βHB administration resulted principally in the appearance of the downstream “metabolite” Glx via oxidative respiration/tricarboxylic acid (TCA) cycle. This was particularly evident following administration of βHB. Only very low levels of Lac were detected following Glc administration and essentially none during Act and βHB administration. As noted earlier, the mice employed in this demonstration were quite young, scanned ~ 1-week postweaning, mean age 27 days postpartum. The brains of young rodents, similar to human newborns and infants, show increased capacity to remove and oxidize ketone bodies compared to adults (Hawkins, Williamson, and Krebs, 1971, Biochem. J. 122:13–18; Robinson and Dermot, 1980, Physiol. Rev. 60:143–187; Kraus, Sehlenker, and Schwedesky, 1974, Hoppe-Seyker’s Z. Physiol. Chem. 355:164–170).

5. CONCLUSIONS

Compared with tail-vein catheterization, administration of deuterated substrates via sc catheter for in vivo DMRS experiments with mice is robust, requires limited personnel training, and enables substantial dosing. It is particularly advantageous for serial longitudinal studies over an extended period, as it avoids inevitable damage to the tail vein following multiple catheterizations. This protocol can be extended to other laboratory animals and other MR-active labels in addition to 2H (e.g., 1H, 13C, 15N, 19F, 31P). Despite its many procedural advantages, sc substrate administration as described herein is not suitable for metabolic kinetic studies requiring tight control of substrate bolus arrival and/or tight control of blood substrate levels. Nevertheless, it will be suitable for numerous metabolic studies in which pseudo-steady-state substrate administration/appearance is sufficient.

ACKNOWLEDGMENTS

The authors are pleased to acknowledge support from the Dravet Syndrome Foundation and JAM for Dravet, the Washington University Intellectual and Developmental Disabilities Center (P50 HD103525), the Alvin J Siteman Cancer Center (P30 CA091842) Small-Animal Cancer Imaging Shared Resource, and the Mallinckrodt Institute of Radiology and its Small-Animal MR Facility. We thank Professor Jennifer Kearney, Northwestern University, for supplying heterozygous mutant 129S6/SvEvTac male breeder mice.

REFERENCES

  • 1.Batsios G, Taglang C, Tran M, Stevers N, Barger C, Gillespie AM, Ronen SM, Costello JF, Viswanath P. Deuterium Metabolic Imaging Reports on TERT Expression and Early Response to Therapy in Cancer. Clin Cancer Res 2022;28(16):3526–3536. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Cember ATJ, Wilson NE, Rich LJ, Bagga P, Nanga RPR, Swago S, Swain A, Thakuri D, Elliot M, Schnall MD, Detre JA, Reddy R. Integrating (1)H MRS and deuterium labeled glucose for mapping the dynamics of neural metabolism in humans. Neuroimage 2022;251:118977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.De Feyter HM, Behar KL, Corbin ZA, Fulbright RK, Brown PB, McIntyre S, Nixon TW, Rothman DL, de Graaf RA. Deuterium metabolic imaging (DMI) for MRI-based 3D mapping of metabolism in vivo. Sci Adv 2018;4(8):eaat7314. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.De Feyter HM, de Graaf RA. Deuterium metabolic imaging - Back to the future. J Magn Reson 2021;326:106932. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.de Graaf RA, Hendriks AD, Klomp DWJ, Kumaragamage C, Welting D, Arteaga de Castro CS, Brown PB, McIntyre S, Nixon TW, Prompers JJ, De Feyter HM. On the magnetic field dependence of deuterium metabolic imaging. NMR Biomed 2020;33(3):e4235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.de Graaf RA, Thomas MA, Behar KL, De Feyter HM. Characterization of Kinetic Isotope Effects and Label Loss in Deuterium-Based Isotopic Labeling Studies. ACS Chem Neurosci 2021;12(1):234–243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Ding H, Haroon A, Wan S, Niendorf T, Adeleke S. Old Discovery Leading to New Era: Metabolic Imaging of Cancer with Deuterium MRI. Magnetochemistry 2022;9:6. [Google Scholar]
  • 8.Ge X, Song K-H, Engelbach JA, Yuan L, Gao F, Dahiya S, Rich KM, Ackerman JJ, Garbow JR. Distinguishing Tumor Admixed in a Radiation Necrosis (RN) Background: 1H and 2H MR With a Novel Mouse Brain-Tumor/RN Model. Front Oncol 2022;12:885480. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Hesse F, Somai V, Kreis F, Bulat F, Wright AJ, Brindle KM. Monitoring tumor cell death in murine tumor models using deuterium magnetic resonance spectroscopy and spectroscopic imaging. Proc Natl Acad Sci U S A 2021;118(12). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Kaggie JD, Khan AS, Matys T, Schulte RF, Locke MJ, Grimmer A, Frary A, Menih IH, Latimer E, Graves MJ, McLean MA, Gallagher FA. Deuterium metabolic imaging and hyperpolarized (13)C-MRI of the normal human brain at clinical field strength reveals differential cerebral metabolism. Neuroimage 2022;257:119284. [DOI] [PubMed] [Google Scholar]
  • 11.Kreis F, Wright AJ, Hesse F, Fala M, Hu DE, Brindle KM. Measuring Tumor Glycolytic Flux in Vivo by Using Fast Deuterium MRI. Radiology 2020;294(2):289–296. [DOI] [PubMed] [Google Scholar]
  • 12.Liu Y, De Feyter HM, Fulbright RK, McIntyre S, Nixon TW, de Graaf RA. Interleaved fluid-attenuated inversion recovery (FLAIR) MRI and deuterium metabolic imaging (DMI) on human brain in vivo. Magn Reson Med 2022;88(1):28–37. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Lu M, Zhu XH, Zhang Y, Mateescu G, Chen W. Quantitative assessment of brain glucose metabolic rates using in vivo deuterium magnetic resonance spectroscopy. J Cereb Blood Flow Metab 2017;37(11):3518–3530. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Mahar R, Chang MC, Merritt ME. Measuring NQO1 Bioactivation Using [(2)H(7)]Glucose. Cancers (Basel) 2021;13(16). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Mahar R, Donabedian PL, Merritt ME. HDO production from [(2)H(7)]glucose Quantitatively Identifies Warburg Metabolism. Sci Rep 2020;10(1):8885. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Mahar R, Zeng H, Giacalone A, Ragavan M, Mareci TH, Merritt ME. Deuterated water imaging of the rat brain following metabolism of [(2) H(7) ]glucose. Magn Reson Med 2021;85(6):3049–3059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Markovic S, Roussel T, Agemy L, Sasson K, Preise D, Scherz A, Frydman L. Deuterium MRSI characterizations of glucose metabolism in orthotopic pancreatic cancer mouse models. NMR Biomed 2021;34(9):e4569. [DOI] [PubMed] [Google Scholar]
  • 18.Peters DC, Markovic S, Bao Q, Preise D, Sasson K, Agemy L, Scherz A, Frydman L. Improving deuterium metabolic imaging (DMI) signal-to-noise ratio by spectroscopic multi-echo bSSFP: A pancreatic cancer investigation. Magn Reson Med 2021;86(5):2604–2617. [DOI] [PubMed] [Google Scholar]
  • 19.Polvoy I, Qin H, Flavell RR, Gordon J, Viswanath P, Sriram R, Ohliger MA, Wilson DM. Deuterium Metabolic Imaging-Rediscovery of a Spectroscopic Tool. Metabolites 2021;11(9). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Ruhm L, Avdievich N, Ziegs T, Nagel AM, De Feyter HM, de Graaf RA, Henning A. Deuterium metabolic imaging in the human brain at 9.4 Tesla with high spatial and temporal resolution. Neuroimage 2021;244:118639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Seres Roig E, De Feyter HM, Nixon TW, Ruhm L, Nikulin AV, Scheffler K, Avdievich NI, Henning A, de Graaf RA. Deuterium metabolic imaging of the human brain in vivo at 7 T. Magn Reson Med 2023;89(1):29–39. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Simoes RV, Henriques RN, Cardoso BM, Fernandes FF, Carvalho T, Shemesh N. Glucose fluxes in glycolytic and oxidative pathways detected in vivo by deuterium magnetic resonance spectroscopy reflect proliferation in mouse glioblastoma. Neuroimage Clin 2022;33:102932. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Soni ND, Swain A, Jacobs P, Juul H, Armbruster R, Nanga RPR, Nath K, Wiers C, Detre J, Reddy R. In vivo assessment of beta-hydroxybutyrate metabolism in mouse brain using deuterium ((2) H) MRS. Magn Reson Med 2023;90(1):259–269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Straathof M, Meerwaldt AE, De Feyter HM, de Graaf RA, Dijkhuizen RM. Deuterium Metabolic Imaging of the Healthy and Diseased Brain. Neuroscience 2021;474:94–99. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Taglang C, Batsios G, Mukherjee J, Tran M, Gillespie AM, Hong D, Ronen SM, Artee Luchman H, Pieper RO, Viswanath P. Deuterium magnetic resonance spectroscopy enables noninvasive metabolic imaging of tumor burden and response to therapy in low-grade gliomas. Neuro Oncol 2022;24(7):1101–1112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Veltien A, van Asten J, Ravichandran N, de Graaf RA, De Feyter HM, Oosterwijk E, Heerschap A. Simultaneous Recording of the Uptake and Conversion of Glucose and Choline in Tumors by Deuterium Metabolic Imaging. Cancers (Basel) 2021;13(16). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.von Morze C, Engelbach JA, Blazey T, Quirk JD, Reed GD, Ippolito JE, Garbow JR. Comparison of hyperpolarized (13) C and non-hyperpolarized deuterium MRI approaches for imaging cerebral glucose metabolism at 4.7 T. Magn Reson Med 2021;85(4):1795–1804. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Wang T, Zhu XH, Li H, Zhang Y, Zhu W, Wiesner HM, Chen W. Noninvasive assessment of myocardial energy metabolism and dynamics using in vivo deuterium MRS imaging. Magn Reson Med 2021;86(6):2899–2909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Barua P, Bhowmick BK. Hypodermoclysis--a victim of historical prejudice. Age Ageing 2005;34(3):215–217. [DOI] [PubMed] [Google Scholar]
  • 30.Cullingworth CJ. Subcutaneous Injection of Mercury. Lancet 1874;103:653 – 654. [Google Scholar]
  • 31.Lawrence G Tools of the Trade, the Hypodermice Syringe. Lancet 2002;359:1074. [Google Scholar]
  • 32.Danowski TS, Winkler AW, Elkinton JR. Biochemical and Hemodynamic Changes Following the Subcutaneous Injection of Glucose Solution. J Clin Invest 1947;26(5):887–891. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Havlena J, Werboff J. Postnatal Effects of Control Fluids Administered to Gravid Rats. Psychological Reports 1963;12(1):127 – 131. [Google Scholar]
  • 34.Hawkins NA, Calhoun JD, Huffman AM, Kearney JA. Gene expression profiling in a mouse model of Dravet syndrome. Exp Neurol 2019;311:247–256. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Mlynarik V, Gambarota G, Frenkel H, Gruetter R. Localized short-echo-time proton MR spectroscopy with full signal-intensity acquisition. Magn Reson Med 2006;56(5):965–970. [DOI] [PubMed] [Google Scholar]
  • 36.Felmlee JP, Ehman RL. Spatial presaturation: a method for suppressing flow artifacts and improving depiction of vascular anatomy in MR imaging. Radiology 1987;164(2):559–564. [DOI] [PubMed] [Google Scholar]
  • 37.Bretthorst GL. Bayesian Analysis V. Amplitude Estimation, Multiple Well-Separated Sinusoids. Journal of Magnetic Resonance 1992;98:501–523. [Google Scholar]
  • 38.Bretthorst GL, Hung CC, d’Avignon DA, Ackerman JJH. Bayesian Analysis of Time-Domain Magnetic Resonance Signals. J Magn Reson 1988;79:369–376. [Google Scholar]
  • 39.Quirk JD, Bretthorst GL, Garbow JR, Ackerman JJH. Magnetic Resonance Data Modeling: The Bayesian Analysis Toolbox. Concepts Magn Reson Part A 2019;47A:e21467. [Google Scholar]

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