Synopsis

Keywords: Neurodegeneration, Hyperpolarized MR (Non-Gas)

It is unclear how serum lactate perturbations influence the quantification of cerebral blood flow (CBF) and metabolism using proton perfusion imaging and hyperpolarized ¹³C MRI. Low dosage L-lactate injection increased CBF and cerebral LDH flux, whilst high dosage lactate decreased CBF and maintained the increased LDH flux, relative to saline administration. Lactate administration did not alter apparent oxidative metabolism. This suggests the efficacy of probing oxidative metabolism with ¹³C MRI when alterations in serum lactate are present (e.g. in pathology). But highlights the requirement for serum lactate measurement prior to interpretation of ¹³C metabolic data.

Introduction

Hyperpolarized [1-¹³C]pyruvate MRI can directly observe [1-¹³C]lactate and ¹³C bicarbonate formation, thus probing real-time lactate dehydrogenase (LDH) and pyruvate dehydrogenase (PDH) flux in-vivo¹. Combining hyperpolarized ¹³C MRI in tandem with proton MR perfusion imaging can probe cerebral blood flow (CBF)² and metabolic changes³ present in pathology. However, it is unknown how perturbations in serum lactate, a signalling molecule⁴ and substrate for oxidative brain metabolism⁵, which is altered in pathology⁶ and ageing⁷, influence apparent cerebral perfusion and metabolism. This study aimed to characterise CBF and ¹³C metabolic responses to varying concentrations of L-lactate in the anesthetized rodent brain.

Methods

Protocol
Sprague Dawley rats were imaged using a 60:40% O₂:N₂O protocol. 10 animals were imaged once without a pre-scan intervention of saline or lactate to estimate cerebral perfusion.Using a cross-over design, 3 animals were imaged three times, receiving a 0.5mL intra-peritoneal injection, prior to imaging, of either saline or sodium L-lactate at 0.5 (half dose) and 1g/kg (full dose). Animals were anesthetised with 2.5% isoflurane (2% isoflurane during scanning). Serum lactate was measured prior to injection and following scanning.

Hyperpolarization
Approximately 30muL of 40mg [1-¹³C]pyruvic acid (Merck, New Jersey, USA) was mixed with trityl radical, OX063 (15mM) (Oxford Instruments, Abingdon, UK) and gadolinium (3L, 1:50 dilution in water) (Dotarem, Guerbet, France) and was hyperpolarized in a prototype hyperpolarizer system at 3.35 T for approximately 1 hour as previously described⁸. Samples were dissolved using sodium hydroxide buffer (approximately 4.5 mL) and 1 mL of final solution was injected over 4 seconds with a 200µL saline chase.

MRI
Animals were scanned using a 2-channel ¹³C receive coil using a ¹H/¹³C volume coil for radiofrequency transmission (Rapid Biomedical, Rimpar). A pulse-acquire spectroscopic slab was placed through the brain of each animal (gauss excitation pulse, excitation pulse width = 100ms, receive bandwidth = 5kHz, TR = 1s, TE = 0.3ms, slice thickness = 20mm, FA = 15° , time points = 240). Animals were transferred to a second custom made cradle using a 4-channel ¹H phased array head coil (Rapid Biomedical, Rimpar), for perfusion assessment (200µL Dotarem with 200µL saline chase, single slice 2D Gradient Echo, slice through mid-brain,) and anatomical T1-weighted imaging pre- and post-gadolinium bolus.

Post-processing
Spectra were processed using jMRUI (v5.1) using [1-¹³C]pyruvate, [1-¹³C]lactate, ¹³C bicarbonate, [1-¹³C]alanine, and [1-¹³C]pyruvate-hydrate as basis functions summed over the time ¹³C signal was present. [1-¹³C]lactate:[1-¹³C]pyruvate and ¹³C bicarbonate:[1-¹³C]pyruvate ratios were calculated.Cerebral perfusion data were post-processed using model-free deconvolution. A region of interest was drawn in supplying vessels to generate CBF, Cerebral Blood Volume (CBV), Time to peak, and Mean Transit Time maps. Measurement reproducibility was assessed by comparison of ROI’s drawn by five independent observers.

Statistical analysis
All statistical analysis was performed in R (4.0.5). Intra-class correlation coefficient (ICC) was calculated for inter-observer reliability of perfusion measurements. A Kruskal-Wallis test was used to assess variance between groups using paired post-hoc testing with a Bonferroni correction. Statistical significance was accepted at P<0.05

Results

Cerebral perfusion reproducibility.
CBF reproducibility was (0.96 [0.92-0.99], CBV (0.98 [0.97-0.99], TTP (0.98 [0.96-0.99]), and MTT (0.99 [0.98-0.99].

Lactate modulates cerebral perfusion in the anesthetised rodent brain
CBF increased between saline and half-dose lactate (112±5 vs 127±2 mL/100g/min) and decreased between saline and full dose lactate (112±5 vs 90±5 mL/100g/min) and between half dose and full dose lactate (127±2 vs 90±5 mL/100g/min) (Fig 2. A). CBV remained consistent between conditions (P=0.6). High dose lactate increased MTT relative to saline injection (0.62±0.04 s-1 vs0.39±0.05) and half dose lactate (0.62±0.04 vs 0.49±0.07 s^-1) (Fig 2. C). TTP remained consistent between conditions (P=0.2). Measurements of lactate exchange, but not PDH flux, vary with lactate dose. The [1-¹³C]lactate:[1-¹³C]pyruvate ratio increased following half dose lactate relative to saline (0.59±0.15 vs 0.22±0.05). This was maintained at high dose lactate (0.62±0.04 vs 0.59±0.15) (Fig 3.A) End-scan serum lactate concentrations were greater following half dose (2.6±0.8 vs 1.3±0.2 mmolL^-1) and full dose lactate relative to saline (6±2 vs 1.3±0.2 mmolL^-1) and at full dose relative to half dose (6±2 mmolL^-1 vs 2.6±0.8 mmolL^-1) (Fig 3. C).¹³Cbicarbonate:[1-¹³C]pyruvate ratio was consistent between conditions (P=0.1 Fig 3.B).

Discussion

Consistent with the modulatory role of lactate on CBF, half dose lactate administration increased CBF, whereas high dose lactate decreased CBF relative to half dose lactate and saline conditions.¹³Clactate:¹³Cpyruvate ratio, reflecting the cerebral ¹²C lactate pool size and LDH flux, increased similarly following half and full dose lactate administration, relative to saline. ¹³C bicarbonate:¹³C pyruvate ratio, reflecting PDH flux and oxidative metabolism, was unresponsive to L-lactate administration and associated CBF response. This may reflect PDH saturation from the ¹³C pyruvate bolus or altered brain oxygen extraction fraction following CBF changes. These data may indicate the feasibility of probing oxidative metabolism using ¹³C MRI when serum lactate is perturbed due to pathology⁶ or therapeutic intervention⁹. Further, apparent alterations in LDH flux may reflect serum lactate changes as opposed to an inherent cellular pathology. Therefore, multi-variate analysis including serum metabolites are needed to elucidate clinical pathology.

Conclusion

These data demonstrate preservation of cerebral oxidative metabolism (¹³C bicarbonate:¹³C-pyruvate) in the healthy rodent brain during experimental modulation of CBF and apparent cerebral LDH flux via L-lactate administration.

Acknowledgements

No acknowledgement found.