Talia Harris1, Assad Azar1, Gal Sapir1, Ayelet Gamliel1, Atara Nardi-Schreiber1, Jacob Sosna1, J. Moshe Gomori1, and Rachel Katz-Brull1
1Hadassah-Hebrew University Medical Center, Jerusalem, Israel
Synopsis
Translating the hyperpolarized signal observed in the
brain in vivo to cerebral metabolic rates is not straightforward, as the
observed signals reflect also the influx of metabolites produced in the body,
the cerebral blood volume and flow, and the rate of transport across the blood
brain barrier. We introduce a robust method to study rapid metabolism of
hyperpolarized substrates ex vivo in viable rat brain slices and
demonstrate its ability to characterize rates of LDH and PDH activities.
Despite variations in these measured rates, we saw that the Lactate to
Bicarbonate ratio is highly reproducible across all samples.
Introduction
The development of the dissolution dynamic nuclear
polarization (dDNP) methodology by Ardenkjaer-Larsen et al.1 that can enhance the liquid
state 13C NMR signal by four orders of magnitude has enabled a
re-examination of cerebral MRS using hyperpolarized 13C-labeled
substrates in vivo 2-10.
The metabolic phenotype observed in these studies may be convoluted by significant
influx of metabolic products from other organs to the brain during the
hyperpolarization acquisition window and by transport of metabolites across the
blood brain barrier (BBB). For these reasons we were interested in developing a
system to investigate the intrinsic metabolic properties of brain tissues using
hyperpolarized substrates without the convoluting effects present in vivo.
Previously developed hyperpolarized perfusion-injection bioreactors11,12 cannot be used to study brain metabolism as
growing cultured neuronal cells to the cell densities necessary for such
experiments is difficult and, further, these cultured cells poorly represent the
cellular heterogeneity and the complexity of the brain tissue in vivo13. We have adapted a perfusion
system designed for maintenance of viability in ex vivo brain slices thus
preserving the in vivo architecture of the brain and allowing
investigation of metabolism therein. In such a system the metabolic rates
observed will not be affected by influx of metabolites from the periphery or
the BBB transport 14-18.
The system allows rapid and repeated injections of hyperpolarized solutions. Using
this perfusion-injection system we have characterized the cerebral metabolism
of [1-13C]pyruvate in real time. Material and Methods
13C NMR spectra were acquired using a 5.8T
high-resolution spectrometer (RS2D). Dissolution dynamic
nuclear polarization (dDNP) was performed using a spin polarizer (HyperSense). 13C spectra were acquired with a 10-20° nutation angle and repetition
time of 5 s. All experiments were performed on 500µm slices of the entire rat
cerebrum perfused with artificial cerebral spinal fluid (aCSF) at 36°C. Kinetic data were fit assuming
constant metabolic rate and T1 heterogeneity as described previously19. An additional time shift factor was introduced to
account for sample settling. Results
Upon injection of hyperpolarized [1-13C]pyruvate,
we were able to clearly observe the appearance [1-13C]lactate and [13C]bicarbonate.
(Figure 1) In some experiments a very small signal of [1-13C]alanine
was observed as well (Figure 1B).
In all 16 experiments we have observed the production
of hyperpolarized lactate, however, only in 13 experiments there was sufficient
signal-to-noise ratio to characterize the dynamics of the [1-13C]lactate
signal. For 8 of these 13 experiments there was also sufficient signal-to-noise
to characterize the dynamics of the [13C]bicarbonate signal. For the
remaining 5 experiments the [13C]bicarbonate signal could be
observed but not analyzed kinetically. A typical experimental time course in
which both [1-13C]lactate and [13C]bicarbonate signals
could be fit to the kinetic model described above is shown in Figure 2. Additionally,
the longitudinal relaxation constants for the different species were fit,
resulting in T1,pyruvate=55±4s (n=13), T1,lactate=27±4s
(n=13) and T1,bicarbonate=15±6s (n=8).
Assuming 0.5 mL of hyperpolarized solution in the
coil, while the remaining volume of approximately 0.875 mL is occupied by brain
slices metabolic rates in the range of 1.3 – 8.3 nmol/s were observed for the
production of lactate, and rates in the range of 0.42 – 0.76 nmol/s were
observed for the production of bicarbonate.
Despite the variation in the metabolic rates, we
observed a strong correlation between the signals of [1-13C]lactate
and [13C]bicarbonate. Plotting the lactate-to-pyruvate ratio versus
the bicarbonate-to-pyruvate ratio for a summed spectra spanning 20 – 65 s after
injection of hyperpolarized pyruvate for all of the 13 experiments for which
bicarbonate signal could be observed, it can be appreciated that the [1-13C]lactate
signal is ≈14
fold higher than that of [13C]bicarbonate (Figure 3).Discussion
We have established the ability of our system to
characterize the enzyme activities of lactate dehydrogenase and pyruvate
dehydrogenase complex in ex vivo brain slices using hyperpolarized [1-13C]pyruvate.
Although there was variation in the absolute rates of lactate and bicarbonate
production, presumably due to differences in the amount of viable brain tissue
present in the probe, the ratio of [1-13C]lactate to [13C]bicarbonate
production is highly reproducible and appears to be an inherent property of the
perfused brain tissue slices in this model system. Conclusion
We have demonstrated a system to characterize the
metabolism of brain tissue ex vivo using hyperpolarized substrates without
the convoluting effects that complicate such interpretation in vivo.
Further work is under way to study the effect of different conditions and
therapeutic treatments on metabolic rates in order to better understand
cerebral metabolism as well as to aid in planning and interpretation of such hyperpolarized
studies in vivo. Acknowledgements
This study was supported by the
European Research Council (ERC award number 338040 to RKB).References
- Ardenkjaer-Larsen, J. H. et al. Increase in signal-to-noise ratio of > 10,000 times in liquid-state NMR. Proc. Natl. Acad. Sci. U S A 100, 10158-10163 (2003)
- Mishkovsky, M. & Comment, A. Hyperpolarized MRS: New tool to study real-time brain function and metabolism. Anal. Biochem.(2016)
- Chavarria, L., Romero-Gimenez, J., Monteagudo, E., Lope-Piedrafita, S. & Cordoba, J. Real-time assessment of 13C metabolism reveals an early lactate increase in the brain of rats with acute liver failure. NMR Biomed. 28, 17-23, (2015).
- Josan, S. et al. Effects of isoflurane anesthesia on hyperpolarized 13C metabolic measurements in rat brain. Magn. Reson. Med. 70, 1117-1124,(2013).
- Butt, S. A. et al. Imaging cerebral 2-ketoisocaproate metabolism with hyperpolarized 13C magnetic resonance spectroscopic imaging. J. Cereb. Blood. Flow Metab. 32, 1508-1514, (2012).
- Mayer, D. et al. Dynamic and high-resolution metabolic imaging of hyperpolarized [1-13C]-pyruvate in the rat brain using a high-performance gradient insert. Magn. Reson. Med. 65, 1228-1233, (2011).
- Marjańska, M. et al. In vivo 13C spectroscopy in the rat brain using hyperpolarized [1-13C]pyruvate and [2-13C]pyruvate. J. Magn. Reson. 206, 210-218, (2010).
- Hurd, R. E. et al. Metabolic imaging in the anesthetized rat brain using hyperpolarized [1-13C] pyruvate and [1-13C] ethyl pyruvate. Magn. Reson. Med. 63, 1137-1143, (2010).
- Hurd, R. E. et al. Cerebral dynamics and metabolism of hyperpolarized [1-13C]pyruvate using time-resolved MR spectroscopic imaging. J. Cereb. Blood Flow Metab. 30, 1734-1741, (2010)
- DeVience, S. J. et al. Metabolic imaging of energy metabolism in traumatic brain injury using hyperpolarized [1-13C] pyruvate. Sci. Reports 7 (2017)
- Keshari, K. R. et al. Hyperpolarized C-13 spectroscopy and an NMR-compatible bioreactor system for the investigation of real-time cellular metabolism. Magn. Reson. Med. 63, 322-329, (2010).
- Keshari, K. R. et al. Hyperpolarized (13)C spectroscopy and an NMR-compatible bioreactor system for the investigation of real-time cellular metabolism. Magn Reson Med 63, 322-329, (2010).
- Harris, T., Eliyahu, G., Frydman, L. & Degani, H. Kinetics of hyperpolarized C-13(1)-pyruvate transport and metabolism in living human breast cancer cells. Proc. Natl. Acad. Sci. U.S.A 106, 18131-18136, (2009)
- Katz-Brull, R., Koudinov, A. R. & Degani, H. Direct detection of brain acetylcholine synthesis by magnetic resonance spectroscopy. Brain Res. 1048, 202-210 (2005)
- Lipton, P. & Whittingham, T. S. in Brain Slices (ed Raymond Dingledine) 113-153 (Springer US, 1984)
- Okada, Y. & Lipton, P. in Handbook of neurochemistry and molecular neurobiology: Brain energetics. Integration of molecular and cellular processes (eds Abel Lajtha, Gary E. Gibson, & Gerald A. Dienel) 17-39 (Springer US, 2007)
- Pardridge, W. M. Brain metabolism: a perspective from the blood-brain barrier. Physiol. Rev. 63, 1481-1535 (1983)
- Hertz, L. Metabolic studies in brain slices - past, present, and future. Front. Pharmacol. 3, (2012).
- Allouche-Arnon,
H. et al. Quantification of rate
constants for successive enzymatic reactions with DNP hyperpolarized MR. NMR Biomed 27, 656-662 (2014).