INTRODUCTION

Disruptions in oxidative metabolism may occur in neurological diseases, such as Multiple Sclerosis (MS)^1,2, Alzheimer's Disease³, Parkinson’s disease⁴. Non-invasive examination of oxidative metabolism and the multiple physiological parameters involved in this process, such as oxygenation levels, dynamics of the blood flow, and mitochondrial status, is crucial to further understand the role of these parameters in neurological diseases. We are implementing a novel multimodal imaging technique combining Near-infrared Spectroscopy (NIRS) and MRI to study simultaneously, and non-invasively, physiological alterations in the brain of mouse models. Using this technique, we are monitoring oxygenation (S_tO₂) and mitochondrial capacity focusing on the key enzyme Cytochrome C Oxidase (CCO), while providing information on the process of blood delivery (CBF) and O₂ consumption (CMRO₂) throughout the brain of the cuprizone mouse model. This is a well-known model for the study of demyelination and spontaneous remyelination, which is also useful to study human MS⁵.

METHODS

Twenty C57BL/6J male mice (8-week-old) were separated into Control (n = 9) and Cuprizone (n = 11) groups. Control mice received normal mouse chow, while cuprizone mice received a diet of ground chow mixed with cuprizone⁶. After 5 weeks, cuprizone diet was terminated, and the Cuprizone group was given normal diet for 4 additional weeks, for recovery. NIRS-MRI imaging were performed for both groups prior to the cuprizone diet initiation (Week 0), at the end of the cuprizone exposure (Week 5) and after cuprizone cessation (Week 9). Statistical analysis was performed comparing CPZ and CTRL groups over 3 different time points using mixed factorial ANOVA with Bonferroni post-hoc test. During imaging, the mice were spontaneously ventilated with a gas mixture of 70% N₂ and 30% O₂ in addition to 2% isoflurane. A 9.4T MRI with a 35mm volume coil was used to quantify Magnetization Transfer Ratio (MTR) as a marker for de/remyelination. A single axial slice was acquired using a spin-echo sequence with the following parameters: TR/TE=2500/15 ms, FOV=12.8x12.8 mm, voxel resolution=100x100x1500 µm³. 40 MT pulses of Gauss shape were applied. The MT pulse B₁ strength was 10.0 µT with 15 ms duration, 1ms interpulse delay, 182.7 Hz bandwidth, and an offset frequency of 1500 Hz. An identical reference magnitude image, M₀, was collected with no MT pulse. In total, two images (with and without MT pulse) were collected over a period of 12 min, and MTR maps were generated. ROI were selected within the corpus callosum and cerebral cortex. For perfusion measurement, the same slice was acquired using a CASL-HASTE sequence with the following parameters: TR/TE=3000/13.5 ms, FOV=25.6×25.6 mm, matrix size=128×128 pixels, slice thickness=1.5 mm, 16 averages. Four perfusion images were collected per measurement: 2 control images and 2 tagged images, to correct for magnetization transfer. Following these images, a T₁ map was obtained in the same location using a RARE-VTR sequence where effective TE=20 ms, TR=100, 500, 1000, 3000, and 7500 ms. Together, the four perfusion images and the T₁ map were collected over a period of 14 min. CBF was calculated on a voxel-by-voxel basis⁷. In addition, a two-dimensional T₂-weighted RARE sequence was acquired with TR/TE=3000/32 ms, FOV=25.6x25.6 mm, matrix size=256x256, voxel resolution=100x100 µm², slice thickness=0.5 mm, 10 averages, and acquisition time=16 min. We measured the concentration of hemoglobin and S_tO₂ in mouse cortex, in addition to the concentration and redox state of CCO using a custom-built broadband NIRS device and in-house developed processing algorithms. CMRO₂ was calculated based on the modified Fick principle⁸.

RESULTS

Cuprizone mice showed reduced OEF (31.8%, p≤0.05) and CMRO₂ (47.2%, p≤0.001) that were resolved after cessation of cuprizone exposure, in addition to a decrease in hemoglobin concentration (28.42%, p≤0.05), an increase in tissue oxygenation (5.7%, p≤0.05) but no change in CBF. The oxidized state of CCO increased (36.9%, p≤0.01) significantly in cuprizone mice while the reduced state decreased (34.4%, p≤0.05). The total amount of the enzyme was not affected by the cuprizone diet, however, it decreased at week 9 both in control (23.1%, p≤0.01) and cuprizone (28.8%, p≤0.01) groups. A reduced value of MTR at week 5 was observed in the cuprizone group both in cerebral cortex (3.2%, p≤0.05) and corpus callosum (5.1%, p≤0.001). T₂-weighted images of cuprizone mice showed a strong gray-white matter contrast which decreased at week 5 and increased back after recovery.

DISCUSSION

Higher levels of oxidized CCO were found in cuprizone mice, which could be an adaptation in response to an impairment in the electron transport chain. The abnormal redox state of CCO led to lower O₂ extraction fraction, lower O₂ consumption rate by the tissue, and thus a higher O₂ availability (S_tO₂) in cortical microvessels. These metabolic changes were associated with the impaired myelination occurring in the gray matter (GM) as well as the white matter of the cuprizone mouse model.

CONCLUSION

We were able to detect significant metabolic alterations in the GM of cuprizone mice using novel NIRS-MRI multimodality system. Demyelination and possible mitochondrial involvement were supported by reduced MTR, increased redox state of CCO, and reduced CMRO₂. The novel multimodal imaging technique applied here shows promise for noninvasively assessing parameters associated with oxidative metabolism in both mouse models of neurological disease and for translation to study oxidative metabolism in human brain.

Acknowledgements

This work was supported by an NIH R21 grant, Canada Foundation for Innovation (CFI), Natural Sciences and Engineering Research Council (NSERC), Discovery grant, and the Biomedical Engineering Graduate Program (BMEG) at U of C.