The effects of acute Methylene Blue administration on cerebral blood flow and metabolism in humans and rats

Methylene Blue (MB) is a brain-penetrating drug with putative neuroprotective, antioxidant and metabolic enhancing effects. In vitro studies suggest that MB enhances mitochondrial complexes activity. However, no study has directly assessed the metabolic effects of MB in the human brain. We used in vivo neuroimaging to measure the effect of MB on cerebral blood flow (CBF) and brain metabolism in humans and in rats. Two doses of MB (0.5 and 1 mg/kg in humans; 2 and 4 mg/kg in rats; iv) induced reductions in global cerebral blood flow (CBF) in humans (F(1.74, 12.17)5.82, p = 0.02) and rats (F(1,5)26.04, p = 0.0038). Human cerebral metabolic rate of oxygen (CMRO2) was also significantly reduced (F(1.26, 8.84)8.01, p = 0.016), as was the rat cerebral metabolic rate of glucose (CMRglu) (t = 2.6(16) p = 0.018). This was contrary to our hypothesis that MB will increase CBF and energy metrics. Nevertheless, our results were reproducible across species and dose dependent. One possible explanation is that the concentrations used, although clinically relevant, reflect MB’s hormetic effects, i.e., higher concentrations produce inhibitory rather than augmentation effects on metabolism. Additionally, here we used healthy volunteers and healthy rats with normal cerebral metabolism where MB’s ability to enhance cerebral metabolism might be limited.

CBF data were acquired using 3D pCASL to determine changes in regional resting perfusion.The sequence included background suppression for optimum reduction of the static tissue signal, which increased sensitivity to the labelled arterial blood signal.The labelling RF pulse had a duration of 1.8s and a postlabelling delay of 2.025s.Four control-label pairs were collected, each within ∼45 s, to derive a mean perfusion weighted difference image.After the postlabeling delay, images were acquired using a multishot, segmented 3D fast spin echo stack-of-spiral sequence (8-arms) with an effective spatial resolution of 2 × 2 × 3 mm.A proton density (PD) image was also acquired using the same acquisition parameters to enable the computation of quantitative CBF maps 1 .
CBF maps were produced from ASL and PD images according to methods recommended by the ASL consensus paper 2 .Raw PD and T1w images were co-registered, and transformations were applied to CBF maps which were also normalised to MNI space using SPM12 software 3 .Normalised CBF maps were smoothed using an 8-mm FWHM kernel.Global CBF values in the grey matter were extracted with the Functional Software Library suite (FSL; 4 ) and were compared across conditions using non-parametric tests (Wilcoxon signed ranks test).Voxel-wise comparisons of the CBF maps across treatments was performed using permutation tests, with and without global CBF covariance.
Voxel-wise R2' and OEF values were estimated from the ASE data, according to Blockley and Stone 5,6 .Field maps were also acquired to correct for macroscopic magnetic field inhomogeneities 5 .

Computation of CMRO2
CMRO2 calculations were carried out, using the method outlined by 7 , which is based on the Fick's principle 8 .Briefly, CMRO2 was calculated via the equations below: Where CRBC is the oxygen carrying capacity of blood and is a product of the mean corpuscular haemoglobin concentration (MCHC) and the amount of oxygen that can be carried by 1 g of pure haemoglobin, which is a known practical quantity of 1.34 mL/g.Ya is the oxygenation level of arterial blood, which we assumed to be the reasonable value of 0.98 for healthy subjects.Therefore, we adapted Equation (1) to calculate CMRO2 for each participant as shown below in Equation (2).Hct was measured along with MCHC and other blood parameters on each visit, as well as before and after each treatment.CBF and OEF were obtained from the ASL and ASE scans, as outlined above.Our MRIderived CBF units were mL blood/100g tissue/min, therefore the values we obtained for CMRO2 had units of mL O2/100g tissue/min.This was converted to the more universally used CMRO2 units which are μmol O2/100 g tissue/min by converting mL O2 to μmol O2, as 1 mol of gas corresponds to 22.4 L.

Rat study Animal information and drug administration
All animal experiments were conducted in accordance with the Home Office Animals Scientific Procedures Act, UK, 1986 (Project licence P023CC39A) and were approved by the King's College London ethical review committee.Adult (10-16 weeks old), male Sprague-Dawley rats, Charles River, UK) were grouphoused at 21 ± 1 °C in a 12-h light:dark cycle and with ad libitum access to standard rat chow and drinking water.Animals were housed for a minimum of one week prior to any experimental procedures.Animals were assigned to either dose of MB, or to vehicle treatment, using simple randomisation.MB was the same as used for human study, diluted in 5% glucose before intravenous administration.All rats received a relevant dose of MB in 1ml/kg over 5 minutes (i.e.infusion rate 0.2ml/min).The reporting of this study complies with the Animal Research Reporting in vivo experiments (ARRIVE) guidelines 9 .

MRI protocol and analysis
Rats were initially anaesthetised with 2.5-3% isoflurane in an 80:20 mix of air to medical oxygen for cannulation of the tail and femoral veins.Isoflurane was discontinued (while air and oxygen continued to be administered) and anaesthesia switched to α-chloralose by injecting a bolus of 65 mg/kg via the tail vein cannula before infusion at a rate of 30 mg/kg/hr.Body temperature, respiration rate and pulse oximetry (SA Instruments, Inc; Stony Brook, NY) were monitored throughout the experiment.Data were acquired using a 9.4 T Bruker Biospec MR scanner with 86 mm volume, four channel array receiver, and arterial spin labelling coil.The scanning protocol consisted of a structural scan, followed by 10 minute BOLD protocol, 12 minutes CBF protocol, iv MB administration and 5 minutes later, repetition of BOLD and CBF protocols.BOLD response was measured using a GE-EPI sequence with the following parameters: TR 1000 ms, TE 18 ms, 90∘ , 24 slices, 64 x 64 matrix with voxels 0.35 x 0.35 x 0.8 mm).Cerebral blood flow (CBF) measurements were derived from the pairs of tagged and control images using the continuous arterial spin-labelling (CASL) method with the following parameters: TR 4000 ms, TE 20 ms, 90∘ , 96 x 96 matrix, single 1 mm slice, 8 seconds per volume.
During the scanning we also applied somatosensory, non-noxious electrical stimulation to the right forepaw via a plantar TENS pad and dorsal platinum needle with the following stimulation parameters: 200mV, 3 Hz, 0.4 ms, 2 mA 10 .We alternated periods of rest and stimulation as follows: for BOLD scans, 10 epochs of 40 seconds with no stimulation and 20 seconds with stimulation, resulting in total of 600 brain volumes (400 resting, 200 stimulated), and for CBF scans it was ten epochs of 48 seconds with no stimulation and 24 seconds with stimulation, resulting in single slice CBF maps with 60 volumes at rest and 30 stimulated (sCBF) volumes.This 22 minute protocol was repeated five minutes after administration of either 2 mg/kg (n=3) or 4 mg/kg (n=4) iv MB (1 mL/kg).Additionally, six rats underwent BOLD protocol only, and were administered 0.5 mg/kg MB.
The structural image was corrected for inhomogeneous signal intensity using N4BiasFieldCorrection 11 , and normalised to a rat template image 12 with antsRegistration 13 .BOLD volumes were aligned to the first volume with AFNI's 3dVolReg 14,15 .The mean image across time was calculated from the motion-corrected volume which was subsequently registered to the structural image.Finally, the motion-corrected volume was warped to the template image by concatenating the transformations from BOLD-to-structural and structural-to-template.A general linear model was fit to the boxcar model from the forepaw stimulation paradigm convolved with a haemodynamic response function and the realignment parameters from motion correction for each subject.Random effects were analysed with a full factorial analysis of dose (2 or 4 mg/kg) and MB status (before or after MB).Contrasts positively associated with stimulation were generated with one-sided t-tests which were statistically assessed at the group level with a two-tailed factorial design and the pTFCE toolbox 16 for SPM12 3 .
CASL single slice images were corrected for motion artefacts by registering the timeseries to the mean volume, before absolute CBF images were calculated using qi-asl from Quantitative Imaging Tools 17 .Mean CBF time courses were extracted from the whole slice (whilst avoiding its edges), and two bilateral ellipsoid ROIs in the somatosensory cortex using Jim8 software (Xinapse Systems, UK).

In vivo autoradiography protocol and analysis
Rats in the autoradiography experiment (n=32) were anaesthetised with isoflurane in order to cannulate the femoral vein and artery.Animals for the conscious 2DG cohort (n=18) were then restrained by wrapping their torso and lower limbs with a loose-fitting plaster cast (3M), and once this had hardened, the anaesthesia was terminated.The rats were monitored for absence of stress visually and by measuring their plasma glucose periodically, ensuring it is within normal physiological values.Animals from anaesthetised 2DG cohort remained under a low dose of isoflurane (1-1.2% in air/oxygen 80/20).At 90 minutes from discontinuing anaesthesia (in conscious animals), or ca.120 minutes after starting the anaesthesia, all rats were intravenously administered 2 mg/kg MB or vehicle.After 30 minutes, 14 C-2-DG (Perkin Elmer, USA) was infused over 30 seconds intravenously and 14 timed arterial blood samples collected as previously described (Littlewood et al., 2006).After the final blood sample at 45 min post-14 C-2-DG, the animals were decapitated and the brains rapidly removed, frozen in cold isopentane (~-40ºC) and stored at -80ºC.Quantification of plasma glucose and 14 C was carried out using the blood glucose analyser (YSI 2300) and scintillation counter (Beckman Coulter LS 6500), respectively.Brains were cryosectioned at 20 µm and exposed to x ray film (Kodak Biomax MR-2) alongside calibrated 14 C standards (American Radiolabelled Chemicals) for 7 days, after which they were developed in Optimax 2010 x-ray film processor.Brain glucose utilisation (GU) quantification was carried out by calculating µmol/100g/min from the optical densities in films, and from plasma glucose and 14 C, according to the methodology originally described by Sokoloff et al. 18 using MCID software (Interfocus, UK).Six large ellipsoid ROIs across an entire section at approximately 1, -3, -6 and -8 mm from Bregma 19 were averaged to estimate whole brain glucose utilisation.

Statistics
All statistical analyses (human and rat experiments) were performed with Graphpad Prism 8 (version 8.1.1)and all reported p-values have been adjusted for multiple comparisons using the Holm-Sidak test for multiple comparisons.All data was tested for normality using the D'Agostino & Pearson test, outliers were tested using the Grubbs method (α=0.05).
For the human data, determination of statistical significance was carried out using a repeated measured oneway analysis of variance (ANOVA) with a Geisser-Greenhouse correction, or in case of incomplete data sets, due to outliers, a mixed analysis model was used.
For the rat CBF data (Figure 3), 2 and 4 mg/kg MB doses rats were combined and a paired t test used to ascertain the differences between pre and post-MB scans.Also, one sample t test was used to demonstrate significance of percent changes from baseline (Figure 5a).For each rat, CBF data was collected from both the left and right hemispheres and then averaged.For the conscious autoradiography (Figure 4), Mann Whitney t-tests were used to determine statistical significance; those were corrected for multiple comparisons using Holm Sidak method.
Additionally, to compute dose-related trends, a simple linear regression analysis for trend was also carried out to determine any significant dose-dependent effects for the relevant human and animal data.
Unless otherwise mentioned, all error bars in the figures are standard error of mean (SEM).The data shown in Table 1 are mean ± standard deviation (SD).
All image analyses were conducted by the operators blinded to the identity of the group or drug dose.