Cancer Therapy & Oncology

International Journal
ISSN: 2473-554X

Review Article Cancer Ther Oncol Int J

Volume 22 Issue 1 - July 2022
DOI: 10.19080/CTOIJ.2022.22.556076
Copyright © All rights are reserved by Tyrus Omondi Swaya

900 MHz Microimaging and Proton MRS of

Transgenic Mice Brain in vivo Theranostic
Monitoring: MRS data correlation with Human
Neurodegenerative Disease Burden for Translational
Medicine
Rakesh Sharma1,2
1
Department of Nanobiotechnology COE and NHMFL, Florida State University, Tallahassee, FL 32304
and
2
NMR Lab, Shriners Children Hospital, Massachusetts General Hospital, Harvard Medical School, Boston, MA
Email: rksz2009@gmail.com and rsharma14@mgh.harvard.edu; Phone: 1-646-856-1592 and
011-91-9548336632
Submission: June 15, 2022; Published: July 28, 2022
*Corresponding author: Rakesh Sharma, NMR Lab, Shriners Children Hospital, Massachusetts General Hospital, Harvard Medical School, Boston,
MA

Abstract

With available only one high resolution MRI imager at ultrahigh 21 Tesla or 900 MHz magnetic field, intact mice brain microimaging is becoming
state of art in visualizing brain structural details to evaluate the neurological disease burden and therapeutic monitoring in pre-clinical trials.
High resolution and high signal noise ratio within short scan time are benefits of ultrahigh magnetic field MRI. One of the major applications
of ultrahigh magnetic field is precise localization of CNS anatomical landmarks and micron-level visualization of brain structure changes to
visualize them with validation process. In brain, a hippocampus structure called ‘dentate gyrus’ is made of granule cells, and it is widely accepted
as possible MRI visible structure using different MRI contrast mechanisms.

The granule cells of dentate gyrus are representative site of drug or neuropharmaceutical compounds induced morphological changes.
Coregistration of MRI visible neuroanatomical regions with NMR visible spectroscopic peaks offers Magnetic Resonance Spectroscopic Imaging
(MRSI) and neurostaining identification further provide finer details of hippocampus structures. Recently, other applications of functional MR
microimaging and nanoparticle based newer methods are emerging as potential molecular imaging tools. Ultrahigh Magnetic field Nuclear
Magnetic Resonance (NMR) microscopy with ultrahigh resolution MR spectroscopy is emerging as spectroscopic imaging tool to study metabolic
events and protein structural-functional characterization in the small animals. The present study introduces only one available 900 MHz
NMR imager-spectrometer with detailed chronological review of previous and possible theranosis potentials in animal brain microimaging
and association of NMR visible peaks from hippocampus with possible neurodegenerative diseases using high MR signal intensity and high
resolution spectra. Ultrahigh field 900 MHz NMR spectrometer was converted to 900 MHz microscopic MR imager to generate both images and
spectra. The imager was suitable research tool for brain microscopy, brain spectroscopy, protein structural characterization and drug therapeutic
monitoring in theranosis. Other applications of functional imaging and nanoparticle based newer methods are emerging as potential molecular
imaging tools.

Keywords: Theranostic Magnetic Resonance Monitoring; Magnetic Resonance Microscopy; Brain metabolism; Animal models; Transgenic mice;
Nonhuman primates; Alzheimer’s disease; Huntington’s disease; Parkinson’s disease; Cerebral ischemia; Multiple sclerosis

Introduction
human diseases. To test and monitor the new drug action on gene,
Over last three decades, transgenic mice brain models have
transgenic animal models are used to study pathochemical effects
been in use for translational medicine (translation of experimental
by in vivo theranostic monitoring as research tool of translational
drug testing in clinical phase trial). Transgenic animals offer
medicine. With technical developments, magnetic resonance
‘service at wish’ to develop experimental neurodegenerative
imaging and NMR spectroscopy have taken shape as ‘Theranostic
disease with known knock-out gene characteristics and pre-
MR Monitoring’ means changes in NMR microimaging of tissue
programed metabolic or genetic disorders mimicking with

Cancer Ther Oncol Int J 22(1): CTOIJ.MS.ID.556076 (2022) 001

 Cancer Therapy & Oncology International Journal
pathochemical composition and microscopy of metabolite
distribution appearing as ‘real time molecular maps’ with
dynamic ‘NMR peak fingerprints’ used in preclinical evaluation
in neuro medicine. Idea is to observe the patho-physiochemical
signals changed parallel with disease prognosis or post-drug
effects as ‘fingerprint of test drug induced brain function’. If
disease sensitive NMR signal changes or test drug action specific
representative pre-clinical signal changes exactly match with
human disease behavior, it may also suggest better therapy and
diagnosis (theranosis) in disease management. It is translatable
approach so called ‘translational research’.
Now noninvasive in vivo magnetic resonance spectroscopy
with imaging (MRSI) is a routine technique to study the drug
actions on brain metabolism used in translational medicine.
Present time combined multimodal complementary techniques
such as fluorescence microscopy, bioluminescence with
noninvasive MR microscopy and MRSI are also emerging for better
pathophysiochemical molecular understanding and real-time
theranostic monitoring in neurobiology and translational clinical
research. However, still challenges are microimaging artifact
minimization, tissue disease sensitive or drug chemosensitive
representative metabolites identification as ‘theranostic
monitoring fingerprints’. Ultrahigh magnetic fields up to 7T-70T or
300 MHz - 3000 MHz, ultrafast imaging and robust softwares are
under investigation as neuroscience and/or drug discovery tools
[1-7]. Till date, ultrahigh field scanners at 11.7T, 14T, 16.4T, 17 T,
21.1T, 25 T spectrometers provided significant and necessary new
gains in resolution and sensitivity for animal model experiments
[1-7].
The present review highlights the development of present-time
ultrahigh magnetic field 21 Tesla proton MR microimaging and
MRS studies of brain in small sized bird, transgenic mice animals
featuring in vivo MR spectral ultrahigh resolution or chemical shift
dispersion and sensitivity of endogenous neurochemicals with
microscopic studies on brain MR slices for possible indications
in neurodysfunction and neurodegenerative diseases [1-6].
Recently, major achievement was made in functional ultrafast
MR imaging (fMRI) of small animal brain behavior studies [7].
The cerebral microimaging and MRS of transgenic mice models
predict the real time ‘theranostic monitoring’ of tissue behavior
in human brain disease prognosis and therapeutic intervention.
Mice neurogenetics exhibits very close homology of monkeys and
humans [7].
In recent past, MRSI studies achieved spatial localization with
the use of a point-resolved spectroscopy (PRESS) or stimulated
echo acquisition mode (STEAM) [8] and chemical shift CHESS [9-
11] sequences. Initially, small transgenic animal size, poor spatial
localization and long scan times were limitations on high field 21.1
T MRI use [1,3-7]. With technical advances in MR spectroscopy,
uniform homogeneous ultra-high magnetic field up to 25 tesla and
ultrafast EPI imaging techniques have made possible noninvasive
metabolic rapid theranostic screening (signatures) of eighteen
How to cite this article: Rakesh S. 900 MHz Microimaging and Proton MRS of Transgenic Mice Brain in Vivo Theranostic Monitoring: MRS Data
002 Correlation with Human Neurodegenerative Disease Burden for Translational Medicine. Canc Therapy & Oncol Int J. 2022; 22(1): 556077.
DOI: 10.19080/CTOIJ.2022.22.556077
neurometabolites as “neurochemical profile” [12-18] to evaluate
mice CNS cell metabolic dysfunction in disease and theranostic
NMR monitoring [19-30].
Present review discusses the chronology of high field magnet
technology and MRS technique development. Author shares his
opinion on possibility of ultrahigh 21 tesla MR applications of
tissue composition and metabolite measurement by combined
MRI and MRS of transgenic animal brain models translatable
to human brain disorders “as possible ultra high field 21 Tesla
MRS theranostic monitoring applications” to characterize
neurochemical and metabolic status of brain diseases in the light
of previous studies [1-7,10, 31].
Technical developments in NMR microimaging and
MR Spectroscopy
Major developments were design of ultrawide 138 mm bore
superconducting magnet to achieve ultrahigh magnetic field
for high SNR resolution with temperature control, continuous
anesthesia flow, least inhomogeneity and fast imaging pulse
sequences to reconstruct images in less time [1-7].
Chronology of Ultra-High Field Magnet Technology
Over years of efforts, superconducting magnet was developed
and ultrahigh magnetic field was achieved operable at 900
MHz [3-7]. Author summarized major technical developments
in the construction of high temperature superconducting
magnet and imaging pulse sequences [31]. Development of
ultrawide bore and ultrahigh 900 MHz magnetic field for water-
fat imaging technology with neurochemical spectroscopy was
major achievement. A decade ago, 21.1 Tesla magnetic field or
900 MHz imaging gantry system was made of Nb3Sn and NbTi
conductors in a set of epoxy impregnated long solenoids plus
compensation coils for uniformity at National High Magnetic Field
Lab, Tallahassee (Figure 1). The 900 MHz with 138 mm wide bore
NMR spectrometer magnet provided an increased field strength,
higher spectrometer frequency, ultrawide bore and economically
feasible metabolic imager [32-34].
Design of Radiofrequency animal birdcage high
temperature sensitive coil
Other major development was design of microimaging Rf
birdcage coils and imaging probes. Such advanced technology made
possible to keep small animal for long 6 hours under isofluorane
anesthesia as shown in Figure 2 [1]. In vivo MR microscopy with
spectroscopy at 900 MHz magnetic field was another achievement
to get better SNR, sharp peaks of neurochemicals and good water
suppression [6-7].
Transgenic animals and anesthesia
After transgenic animal resource developed in NIH, presently,
transgenic mice serve as animal models of desired experimental
disease gene specific controlled interventions as breakthrough in
translational theranosis.
 Cancer Therapy & Oncology International Journal

Figure 1: The vertical bore (left) magnet design of superconducting gantry operable at ultrahigh 900 mHz magnetic field (on right)
is shown. It is made of several layers of TXI CryoProbe walls directly in contact of helium and liquid nitrogen. In center, hole is to
place receiver-transmitter Rf insert coil to place samples or animal [30].

Figure 2: illustrates a 21 T MR spectrometer, Rf insert with animal heart and tuning console (panel on top). The animal can be
placed in insert and kept alive by supply of oxygen and anesthesia. Experimental setup for localized proton MRS of mice brain in
vivo at 21.1 T. Signal excitation is given by homogeneous RF coil and signal received by decoupled quadrature surface coil placed
on top of the mice head. The animal holder and bed keep the mice warm show in panels . A home-made stereotaxic palate holder
with an adjustable nose cone is connected to a respirator for artificial ventilation, a pressure sensor, and a rectal thermometer
(panel at bottom). See references [2,33].

How to cite this article: Rakesh S. 900 MHz Microimaging and Proton MRS of Transgenic Mice Brain in Vivo Theranostic Monitoring: MRS Data
003 Correlation with Human Neurodegenerative Disease Burden for Translational Medicine. Canc Therapy & Oncol Int J. 2022; 22(1): 556077.
DOI: 10.19080/CTOIJ.2022.22.556077
 Cancer Therapy & Oncology International Journal
What are major obstacles in ultrahigh magnetic field MR
Spectroscopic Imaging?
i. The major obstacle in animal MRI and MRS studies is
the need of safe long-time anesthesia to minimize stressful drug
effects or undesired artifacts due to animal movements. Protocols
for anesthesia vary for different animals at different imaging
sites. Protocols also depend on the initial access to the awake
animal, total examination time and actual scientific question of
investigation. At our laboratory, follow-up MRS measurements
need interventional techniques such as intravenous injections,
spontaneous breathing of an anesthetic gas via a mask, or
endotracheal intubation with artificial ventilation as standard
procedures to provide transgenic animal safety, recovery rate, the
ability to perform adjustments during the experiment up to several
hours [3]. Intubation is extremely valuable for investigations of
small young and aged animals, severely affected animals, and rare
mutants with unknown pathophysiology as shown in Figure 2.
ii. After initiation of anesthesia, animals are placed in a
prone or supine position on a commercially available or custom-
built bed (animal slider) of 21Tesla NMR imager (see Figure 2).
To fix animal brain inside gantry, stereotaxic device is used to
immobilize the animal head. Continuous noninvasive monitoring
of basic life functions such as breathing amplitude and frequency,
blood oxygenation level, and rectal temperature is difficult under
long-time anesthesia to ensure adequate survival rates. Other
additional devices are permanent catheters or electrodes fixed
for electrophysiological recordings may be necessary for special
applications.
iii. Cost of transgenic mice husbandry, possible mice
transportation and keeping them alive during imaging is also
critical issue.
iv. Long time animal brain exposure effects of ultrahigh
magnetic field are an inconclusive, uninvestigated and unknown
concern if high magnetic field exposure can damage the brain
or central nervous system territories or induce neuronal
dysfunctional except manageable mild sensory effects such as
vertigo, metallic taste, and magnetophosphenes. The single piece
of 21 Tesla MR imager till date is under observation for any
untoward event or known neuronal dysfunction after first NASA
report on high field hazards on muscle exposed to SAR >8 W/kg
[1]. Moreover, several advantages are now known.
Advantages of ultrahigh resolution MRI microimaging
Recently, several advantages of ultrahigh magnetic field have
been reported to achieve high resolution microimaging, high SNR,
multicontrast, high spatial resolution, diffusion tensor imaging
(DTI), flow studies etc [30]. However, inhomogeneity also linearly
grows with high magnet field strength. Disadvantages of ultrahigh
magnet field strength are not reported yet as safe technique or it
remains to establish the disadvantages of body exposure to high
magnetic fields.
How to cite this article: Rakesh S. 900 MHz Microimaging and Proton MRS of Transgenic Mice Brain in Vivo Theranostic Monitoring: MRS Data
004 Correlation with Human Neurodegenerative Disease Burden for Translational Medicine. Canc Therapy & Oncol Int J. 2022; 22(1): 556077.
DOI: 10.19080/CTOIJ.2022.22.556077
Application of ultra-high-resolution MRI of mice brain:
Notable examples are following:
i. Mice brain atlas in localization of site-specific damage:
Alcoholic encephalopathy; 2. Transgenic knock-out mice brain
and effect of alcohol, colchicine etc;
ii. Stereotactic spectroscopic image slices and cubes
to visualize test drug induced morphometric changes with
possible pathochemical variation or distribution of metabolite
concentrations;
iii. Altered peak shapes as proportionate neuron
dysfunction or neurodisorder;
iv. Transgenic mice models of neurogenetics for
investigations of the genetic control sensitive metabolites actively
participating in cellular metabolism and physiological processes
[33].
Localized Proton 21 Tesla MRS of Transgenic animal
brain in vivo
Experimental setup of 21.1 Tesla MR scanner
The majority of transgenic animal MRS studies employ a
surface coil for signal reception either in transmit/receive mode
or in combination with a homogeneous radiofrequency (RF)
excitation coil. It enhances SNR x 300 times advantage from very
small 1 - 8 mm3 mm<cube> volumes-of-interest (VOI) close to the
surface coil [1]. It may also suffer from a fading signal for VOIs at
increasing distance from the coil. The coils are set perpendicular
to the static magnetic field. These coils are geometrically and/or
actively decoupled. For best SNR quality is obtained by placing
the head of the animal (partly) within a single-looped circular
or elliptic surface coil. Actively decoupled, slightly bent coils
similar to the shape of the head are shown in Figure 3 for MRS of
mice brain at 21.1 T magnetic field. Now phased array coils are
available with more SNR for 21.1 Tesla magnetic field strengths/
frequencies used for transgenic animal research. Structural
Validation of brain structures: The mouse brains (n = 3) were
perfusion-fixed for MRI microscopy. The dentate gyrus of dorsal
hippocampus in both left and right sides show collateral control.
The susceptibility matching eliminates both extraneous proton
signal and coil loading (see Figure 5).
Mice brain atlas: The axial, coronal and sagittal planes in 2D
slices reconstruct the 3D brain atlas by volume rendering. The
main features at ultrahigh resolution are: distinct gray and white
matter tissues and neuron proton density in different structures
(shown in Figure 5).
Why 21.1 Tesla MRS better? Routine animal MRI systems
function at higher 9.4 T, 11.7 T, 21.1 T magnetic field strengths
with smaller bore sizes 35 mm - 135 mm and stronger gradient
systems. Human clinical MRI systems have wide 2.5 feet bore
magnet. The high magnetic fields (2.35 T vs. 9.4 T and 21.1 Tesla)
have merits for the resolution enhancement of individual (spin-
 Cancer Therapy & Oncology International Journal

coupled) metabolite resonances [35-39]. At 2.35 T, proton spectra
of myo-inositol (Ins) and creatine (Cr) mixture have a superior
peak intensity ratio due to apparent singlet resonance of Ins with
better relative detectability. At 9.4 T, proton spectra of a mixture
of N-acetylaspartate (NAA), glutamate (Glu), and glutamine
(Gln) demonstrate the improved separation of the Glu (2.35
ppm) and Gln multiplets (2.45 ppm) with better detectability. At
21.1 T proton spectra of neurometabolite mixture demonstrate
the sharp narrow NMR peaks with improved separation of less
abundant multiplets better over 9.4 T spectrum (Figure 4). Larger
chemical shift dispersion can be achieved by advanced shimming
techniques such as FASTMAP [40] with strong shim currents.

Figure 3: On left, localized proton MRS (STEAM) of metabolite solutions at a low field strength (2.35 T, TR/TE = 10,000/10 ms)
and a high field strength (9.4 T, TR/TE = 15,000/10 ms). (on Top) for 1:1 mixture of myo-inositol (Ins) and creatine (Cr) and (bottom)
2:2:1 mixture of N-acetylaspartate (NAA), glutamate (Glu), and glutamine (Gln). While Ins is easily detectable at low fields due to
its apparent singlet resonance with a much better separation of Glu and Gln is achieved at high fields. On right, localized proton
MRS of metabolite at ultrahigh field strength (21.1 T, TR/TE = 2000/2 ms) show 1:1 mixture of myo-inositol (Ins) and creatine (Cr)
and (bottom) 2:2:1 mixture of N-acetylaspartate (NAA), glutamate (Glu), and glutamine (Gln). On left, localization of voxel is shown
in mice brain hippocampus. Source: NHMFL data base.

Ultrahigh 21.1 Tesla magnetic field strength offers high Signal-
to-noise (SNR) with spatial high resolution (0.05 ppm or 1.5 Hz or
15 micrometers) at short echo time MR spectra acquisitions from
small VOIs or very clear cerebral smaller sub-regions such as the
transgenic mice hippocampus.
Localization technique
Without spatial localization, in vivo MRS spectral peaks suffer
from systematic problems. For localization, surface coils are
placed directly on the brain or skull of mice [12,13,41,42]. These
studies suffer from sensitivity profile of the surface coil though.
Smaller assigned VOI and signal-noise contamination
from surrounding tissue pose problem. Two decades ago, MR
spectroscopy was interpreted in terms of resonance intensity
ratios or peak area ratio as comparable concentrations of
metabolites in brain without water suppression. Such studies
faced obstacles but advent of single-voxel localized MRS [8] and
chemical shift imaging [9-11] together simplified the imaging
and localized spectroscopy together and technique was termed
‘magnetic resonance spectroscopic imaging’ (MRSI). Actual
localization of assigned VOI or reduced field-of-view for MRSI
was achieved with the use of a PRESS or STEAM sequence as
shown in Figure 3. With technical advance for proton MRS, water
suppression mode visualized weaker metabolite peaks.
gap (PRESS selects....) tissue volume at TE=145-270
ms to get high SNR (sharp 2D or 3D metabolite choline;
creatine; N-acetylasparatate; lactate; and lipid peaks) free
from susceptibility of brain lipids and sinuses to define tissue
heterogeneity. The choline peak comes from choline-rich
membrane synthesis and turnover. Creatine comes from cellular

How to cite this article: Rakesh S. 900 MHz Microimaging and Proton MRS of Transgenic Mice Brain in Vivo Theranostic Monitoring: MRS Data
005 Correlation with Human Neurodegenerative Disease Burden for Translational Medicine. Canc Therapy & Oncol Int J. 2022; 22(1): 556077.
DOI: 10.19080/CTOIJ.2022.22.556077
 Cancer Therapy & Oncology International Journal

energetics. N-acetylaspartate is a neuronal marker. Lactate tumor has decreased N-acetylaspartate and increased choline and
reflects anaerobic metabolism. Lipids come from neuronal cell variable levels of creatine. Peaks of lactate and lipids show high
breakdown by necrosis. The normal brain has N-acetylaspartate in regions of necrosis, metastatic and brain tumors. Large voxels
approximately twice the intensity of choline and creatine. Brain contain a mixture of tumor, necrosis, and normal brain tissue.

Figure 4a: (a) A explorative in vivo coronal image of the finch brain acquired using a 2D fat-suppressed fast spin-echo sequence
at a resolution of 100x100x500 μm in 5.5 min; (b) The adult male zebra finch bird; (c) A single 2D section taken from a 3D GRE
dataset acquired at 21.1 T from a fixed finch thalamus at 40-μm isotropic resolution; (d) Segmentation of song nuclei (Area
X, Fascii, robust nucleus of the arcopallium; RA and dorsal part of the medial thalamus; DLM) using 3D images so that these
neuroanatomical areas can be compared quantitatively between treatment groups; (e) Test of MR localized spectroscopy in brain
phantom solution performed at UWB 900. (f)The voxel volume was 8 μL, TR/TE = 4000/ 19 ms, accumulation time was 18 min.
Permission from NHMFL database as NSF user [5].

Figure 4b: Generic localization sequences are shown for in vivo proton MRS using spin echoes (PRESS) or stimulated echoes
(STEAM) and Multislice Multiple Echo (MSME) sequence. Image and spectrum reconstruction is sillustrated for MR spectroscopic
imaging (see panel at bottom).

How to cite this article: Rakesh S. 900 MHz Microimaging and Proton MRS of Transgenic Mice Brain in Vivo Theranostic Monitoring: MRS Data
006 Correlation with Human Neurodegenerative Disease Burden for Translational Medicine. Canc Therapy & Oncol Int J. 2022; 22(1): 556077.
DOI: 10.19080/CTOIJ.2022.22.556077
 Cancer Therapy & Oncology International Journal

Figure 5: (on top) A metabolic sketch is illustrated showing normal MRI-visible metabolic products formed during metabolism in
normal mice brain with a representative spectrum obtained at 21.1 Tesla MR spectroscopy.
(on middle panel)) MRI-visible NMR spectroscopy peaks in different normal brain structures are shown at 21.1 Tesla MR
spectroscopic imaging. On right, coronal images A to D and 3D MRI images E to F by color coded segmentation with validation are
shown by MR microscopy [51]. Notice the corresponding high resolution NMR peaks on left and accurate delineation of different
brain structures A to D on top at right. Corresponding metabolite cocentrations (in mM) are shown from different brain structures
shown in Table at bottom. Reproduced from NHMFL database.

How to cite this article: Rakesh S. 900 MHz Microimaging and Proton MRS of Transgenic Mice Brain in Vivo Theranostic Monitoring: MRS Data
007 Correlation with Human Neurodegenerative Disease Burden for Translational Medicine. Canc Therapy & Oncol Int J. 2022; 22(1): 556077.
DOI: 10.19080/CTOIJ.2022.22.556077
 Cancer Therapy & Oncology International Journal

Chemical shift stimulated (CHESS) method is a water-selective times partly counter balance the enhanced spin polarization and
RF excitation pulse followed by a spoiler gradient pulse [9-11]. compromise the achievable gain in SNR per unit time. It is done by
Moreover, these techniques have advantages and drawbacks choice of TE Aand TR
of MRS versus MRSI and PRESS versus STEAM. Conceptually,
MRS visible Metabolites at 21.1 Tesla or 900 MHz: What
high-quality proton MR spectra measure the in vivo metabolite
we predict from MRS?
concentrations (as neurochemical insight of long-term disease
prognosis). Figure 4 shows generic localization pulse sequences Many cerebral metabolites have been identified in localized
for in vivo proton MRS using spin echoes (PRESS) or stimulated proton MR spectra of animal brain in vivo after post-processing and
echoes (STEAM). curve fitting by NMR-2 and Topspin softwares [43]. Under normal
physiological conditions the chemical shift in frequency range
Two decades ago, MR spectroscopy was interpreted in terms
between 1 and 4 ppm comprises resonances of unsaturated lipids
of resonance intensity ratios or peak area ratio as comparable
(Lp) at -0.2-1.0 ppm, lactate (Lac) at 1.33 ppm, pyruvate (Pyu) at
concentrations of metabolites in brain without water suppression.
1.35 ppm as mitochondrial stress markers, alanine (Ala) at 1.4
Multivoxel 1-H MRSI ppm, acetate (Ace) at 1.8 ppm, N-acetylaspartate (NAA) at 2.0 ppm,
N-acetylaspartylglutamate (NAAG) at 2.1 ppm, γ-aminobutyric
Three-dimensional 1-H MRSI data with CHESS selective water
acid (GABA) at 1.5 ppm as neurodegeneration markers, Glu, Gln,
suppression and conventional volume selection radiofrequency
aspartate (Asp) at 2.2-2.8 ppm; Cr, phosphocreatine (PCr) at 3.0
pulses suppress lipids but compromise the quality of the MRS
ppm, choline-containing compounds (Cho) at 3.2 ppm as energy
data obtained. Alternative radiofrequency pulses with spatially
metabolic markers; taurine (Tau) at 3.4 ppm, Myo-Ins and scyllo-
selective saturation bands improve the spatial and frequency
inositol at 3.5 ppm, glucose (Glc) at 3.8 ppm, as neuronal and glial
selection. Sharp transition band applied parallel to selective
markers; glycine, glutathione, phosphoethanolamine, vitamin C,
volume edges make cubic shape or oblique orientation to anatomic
and threonine in downfield 4-6 ppm; as well as macromolecules
volume. The multislice and multiple echo time (MSME) techniques
(MM) and (un)-saturated lipids in upfield -0.2 – 1.0 ppm as
provide spatial localization with lipid suppression by spatial
oxidative stress markers see (Figure 5). Under pathological
2 2 2
first order X,Y,Z and second order XY,ZX,XY,Z2 and ....Z<square>
and X,square>-Y<square> field-based shimming and frequency
conditions or after drug administration additional metabolites

Z and X Y
selective pulses, inversion recovery, and spatial saturation
may become detectable including ethanol, methanol, glycerol,
acetone, propan-1,2-dyol, guanidinoacetate, pyruvate, succinate
pulses. However, brain sinuses or cavities show susceptibility
(Suc) and several aromatic compounds with resonances in proton
artifacts. In multislice, slice gaps avoid the crosstalk and two
MR spectrum beyond 5 ppm up field.
acquisitions cover complete mice brain. Other method is echo
planar spectroscopic imaging(EPSI) with oscillating gradients in Often NAA and NAAG (tNAA), Cr and PCr (tCr), and Cho
one spatial dimension or spiral sampling in a plane from complete are considered as “major three” brain metabolites. These are
brain volume coverage. Hybrid PRESS-echo planar spectroscopic abundant in relatively high concentrations with singlet nature
imaging technique with spatially selective saturation bands are of their methyl resonances. Lactate (Lac) doublet resonance
used to mask large k-space free from susceptibility artifacts. signal is detected at small spin echo times with low SNR. Doublet
resonance signal from Lac is detected flipped down at high TE
For spectroscopic imaging techniques, AFFIRMATIVE
when its concentration increases above normal physiological
and QUADCON may produce metabolite images in 3D spatial
levels. Short echo times facilitate the direct observation of
resolution with sharp NMR peaks from each pixel coordinate for
additional resonances from Ins, Glu, Gln, and Glc at low fields in
quantitative studies of regional brain metabolism in anesthetized
rodents and monkey brain, and Tau at high fields in mice brain.
mice at ultrahigh 21.1 T magnetic fields see (Figures 3 & 4). For
These metabolites predict physiological processes in specific
a reasonable SNR in 15 minutes, the minimum VOI = 1×1×1
cell type see (Figure 5). Disease-related alterations of respective
mm<cube>...... or 1 μL is chosen for high-field proton MRS
metabolite levels may reflect specific changes in intracellular

nm3
protocols. Even smaller structures such as pure white matter in
the mice corpus callosum need VOI of 250×100×500 m<cube>.....
to achieve high SNR × 300 gain. So, it requires unrealistic longer
measuring times. It is possible that ultrahigh magnetic fields can
solve this limitation, because presently available magnets are
already close to the technical limit of about 23.5 T. Hopefully, new
Bruker Mini 135 gradient coil may provide peak gradient strength
of >450 mT/m using GREAT60 amplifiers possibly for primate
and rodent model MRI studies beyond proton probes. Moreover,
field-dependent increases in T1 and decreases in T2 relaxation
metabolism and tissue composition. Metabolite peak pattern
also characterizes the cerebral energy status in the glial cell
population, or the neuroaxonal integrity and function.
Briefly, NAA has been demonstrated to be predominantly
localized within neurons [44] and is commonly considered as
a marker for vital neuroaxonal tissue. The energy metabolites
Cr and PCr are linked to oxidative phosphorylation and
mitochondrial function and are constituents of both neuronal
and glial cells [45]. A pathological loss of neurons as suggested

How to cite this article: Rakesh S. 900 MHz Microimaging and Proton MRS of Transgenic Mice Brain in Vivo Theranostic Monitoring: MRS Data
008 Correlation with Human Neurodegenerative Disease Burden for Translational Medicine. Canc Therapy & Oncol Int J. 2022; 22(1): 556077.
DOI: 10.19080/CTOIJ.2022.22.556077
 Cancer Therapy & Oncology International Journal

by reduced NAA therefore causes a similar reduction of tCr unless simultaneous metabolite concentration changes.
compensated for by increased glial cell density (e.g., due to reactive
iii. Use an internal (e.g., brain tissue water) or external
astrogliosis). Elevated concentrations of Lac indicate enhanced
reference to measure concentration of metabolites. Similar
nonoxidative glucose consumption, not only in brain cells but
with humans, cerebral metabolite concentrations may be
also due to the anaerobic metabolism of infiltrating macrophages
measured using surface coils or phase array coils comparable to
(e.g., during neurodegeneration). The Cho resonance comprises
homogeneous RF coils [48].
phosphocholine as a precursor for membrane synthesis as well
as glycerophosphocholine as the corresponding membrane iv. Spectral evaluation in objective manner is best by fully
degradation product. Accordingly, elevated Cho concentrations automatic and without user interference.
may either arise from enhanced membrane turnover (e.g., during
v. Available software packages such as LCModel estimate
development or in brain tumors) or from accumulation of myelin
the metabolite concentrations as well as measures of reliability
breakdown products (e.g., during active demyelination). A
(e.g., Cramer–Rao lower bounds) [49].
diminished Cho level has been described in Pelizaeus–Merzbacher
disease due to hypomyelination [46]. Finally, Ins arises from vi. Follow the general conditions of MRS quantitation
free myo-inositol, which is the most important non-nitrogenous similar to human studies.
organic osmolyte in the brain with very high concentrations in
Present state-of-art
astrocytes [47]. Thus, elevated Ins concentrations are most often
interpreted as increased cellular density of astrocytes (e.g., due to State-of-art localized proton MRS of animal brain in vivo
astrocytosis or in glial tumors). requires the consideration of a number of technical components:
(i) the design of an appropriate experimental setup; (ii) the
The MRS-detectable metabolites predict physiological or
optimization of a short echo time MRS or MRSI localization
biochemical events many neurodegenerative tissue features
technique based on PRESS or STEAM; (iii) a calibration method for
may be defined under physiological and pathological conditions.
quantification of metabolite concentrations; (iv) an objective and
These are discussed in detail for animals [13-30]. Many possible
reliable spectral evaluation method; and (v) a sound interpretation
metabolic pathways are described in the disease models in
of quantitative MRS findings strongly depends on a thorough
subsequent sections see (Figure 5).
understanding of the disease specific physiological functions of
Quantification different brain structures and correlation of biochemical-MRS
visible metabolites.
MRS peaks translate into metabolite concentration at
minimum T1 and T2 effects during data acquisition using Tip® and High Field MR Microimaging and Spectroscopy in
Paravision software. For it, spectra are first deconvoluted, zero- Disease Mice Models
filled, apopdized, Fourier-transformed, zero-order rephasing,
The following description offers a guideline to investigators
frequency corrected, baseline corrected, NAA peak assigned,
for study design on feasibility or possible metabolites’ theranostic
curve fitted and peak area calculated against caliberated internal
outcome from 21.1 Tesla MR scanner to predict the disease
standard suppressed water peak using AQSES, NMR-2 or SID
prognosis and therapeutic monitoring closer with reports in
softwares [4]. MRS peak or metabolite concentration alterations
animals reviewed recently [50].
reflect the pathological contrast. Metabolite T1 and T2 relaxation
constants (FWHM or peak width at half hight) are usually altered Mice models
in a disease condition [1,2]. Three following approaches reduce
The first proton MRS studies of mice brain in vivo were
the time-consuming measurements of the actual relaxation times
performed on exposed brain without spatial localization [10].
for corrections of T1- and T2-weighted spectra on 21.1 Tesla MRS
Superficial skin, muscle, and fat, investigations of the mice brain
Scanner.
metabolism needed the fully localized acquisition techniques
i. Choose suitable acquisition parameters such as long for the analysis of genetic mice models. Initially, in last century
repetition times TR= 5xT1 and short echo times TE<T2 so called focus was on inborn errors of metabolism by comparing cerebral
‘paravision multislice multiecho MSME’ pulse sequence. Long metabolite profile of mutant mice. Further examples refer to a
TR cause excessive measuring times for single-voxel MRS for mice model of histidinemia [41] and scrapie [42], transgenic mice
investigating brain metabolites. overexpressing the human ornithine decarboxylase gene [43],
and a mice model of Duchenne muscular dystrophy [44]. Present
ii. Avoid the use of relative peak heights or intensities
availability of genetically modified animals from NIH source paved
(areas) of individual resonances. Metabolite ratios (e.g., relative
the development of mice brain MRS since last decade. Figure 6
to tCr) can be sufficient to identify a neurochemical disturbance.
shows the fully localized proton MRS of the normal and ischemic
Many neuropathologies involve alterations in cellular density and
mice brain with high Lac peak at 1.33 ppm at low TE [51].
therefore affect the tCr level. Peak ratio measurement can miss

How to cite this article: Rakesh S. 900 MHz Microimaging and Proton MRS of Transgenic Mice Brain in Vivo Theranostic Monitoring: MRS Data
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DOI: 10.19080/CTOIJ.2022.22.556077
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Figure 6: Feasibility of localized proton MRS of mice hippocampus in vivo (4.7 T, PRESS, TR/TE = 2500/20 ms, 2×2×2 mm3,
128 accumulations). This early application corresponds to the normal and ischemic hemisphere of a C57BL/6J mice 3.5 h after
occlusion of the middle cerebral artery. The accumulation of Lac is accompanied by decreases of NAA, tCr, and Cho (arrows).

Normal Mice Brain Physiology best due to the enhanced chemical shift dispersion with increased
linewidths larger than coupling constants see (Figure 5). For
Localized proton MR spectra of normal mice hippocampus are
example, resolution of the weakly coupled doublet resonances for
depicted in Figure 7 at two different field strengths. The readily
Lac (7 Hz, centered at 1.33 ppm) and Ala (7 Hz, centered at 1.48
accessible metabolites are tNAA, tCr, Cho, Ins, Glc, Tau, and Glu. At
ppm) is easily achieved as shown in Figure 7. Lactate is important
low field (2.35 T) detection of Ins and Glc is better because their
neurochemical showing doublet alongwith methyl and methylene
strongly spin-coupled multiplets merge into apparent singlet
resonances of mobile lipids and/or macromolecules. Table 1
resonances but identification of Tau and Glu (separated from Gln)
summarizes the linewidths of water proton resonances for mice
is better at high field (9.4 T). At 21 Tesla, spectral resolution is
brain in vivo in experimental settings.
Table 1: Proton MRS linewidths of mice brain in vivo for different magnetic field strengths (in Tesla) and volumes-of-interest (VOI).
Field/T VOI/mm3 Linewidth/Hz
2.35 4.0×3.0×4.0 7–9
4.7 3.4×1.7×1.7 18
4.7 6.0×3.5×3.0 11
7 2.0×2.0×2.2 13–17
9.4 2.0×1.5×2.5 10–14
9.4 1.5×1.5×1.5 12–20
9.4 2.5×2.5×2.5 20
11.7 2.0×2.0×2.0 20-25
21.1 1.0×1.0×1.0 25-35

The proton resonance frequency ranges from 7 Hz (2.35 T) a better SNR. This SNR gain offers the use of small VOIs to assess
to 20Hz (9.4 T) depending on field strength and size of the VOI, metabolic evaluation of different mice brain structures such as
regional differences in different brain areas of mice strain. At 900 hippocampus (see comparison in Figure 9).
MHz (21.1 T), enhanced proton spin polarization is translated into

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Figure 7: Localized proton MRS of mice (C57BL/6) brain hippocampus in vivo (NMRI) is shown at different field strengths. In
comparison to low field acquisitions (2.35 T, STEAM, TR/TE = 6000/20 ms, 4.0×3.0×4.0 mm3, 512 accumulations) the use of a high
field (9.4 T and 21.1 T, STEAM, TR/TE = 6000/10 ms) shows lower number of accumulations (32 accumulations) or a much smaller
volume (2.0×1.2×2.2 mm3 and 1.0× 1×1 mm3 covering the hippocampus) at still reduced measuring time (128 accumulations).
Metabolite resonances refer to tNAA, Glu, tCr, Cho, Ins, Tau, Glc, and MM from VOI [7].

How neurochemical in vivo MRS proton resonances indicate different pathochemical-neurophysiology functions
of brain in different tissues? How much metabolites can answer?
Table 2: MRS-PRESS (TR=2500 ms, TE=24 ms, 7T) of ethyl nitrosourea induced and GL261 mice model also showed similar metabolite changes.

Disease Model Transgenic Mice NMR Metabolites Reference

High NAA, and Glx
NMRI,BALB/c, C57BL/6,CBA, CBA/
Brain Maturation Disorders Low Cho, Tau in striatum, Cerebrum, hippocampus, 53,57,58
BL6,C57BL/6, 129/SvJ,FVB/N mice
Cerebellum, thalamus
Cerebral Ischemia C57BL/6,BALB/c, NMRI mice Low NAA, High Lac 63,69,70
Low NAA/tCr and high Cho/tCr
Multiple Sclerosis 129/SvJ,FVB/N, CBA/6, C57BL/6 71,72,73
in whole brain, hippocampus, CSF
Low NAA in substantia nigra, pars compacta & striatum,
Parkinson Disease C57BL/6 mice 74,75,76
High Glx and GABA in striatum
High Cho, Glx levels and
77,78,79,
Huntington Disease N171-82Q, R6/2 mice
Low NAA/tCr ratio in Striatum, Cortex, Cerebellum, brain 80,81
stem
Alzheimer Disease B6/SJL, APP-PS1 mice LowNAA/tCr in cortex, Glu/tCr in brain stem 85,86,87, 88

How to cite this article: Rakesh S. 900 MHz Microimaging and Proton MRS of Transgenic Mice Brain in Vivo Theranostic Monitoring: MRS Data
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DOI: 10.19080/CTOIJ.2022.22.556077
 Cancer Therapy & Oncology International Journal

OxiPhos,Energy Metabolism (GAMT−/−) mice Low tCr in whole brain 30

Amylotrophic Lateral Syn- G93A, Mecp2−/y High Glx/tCr, Low Glx/Ins, High NAA/tCr ratios in brain
94
drome, Ritts Syndrome gene model stem, cortex, cerebellum, striatum
Canvan Disease C57BL/6 model High NAA in hippocampus 95
Low NAA/tCr ratio and
Down Syndrome Ts65Dn model 96
High Ins/tCr ratios in cerebellum
HIV Encephalitis HIV-1 model Low NAA/tCr in whole brain 97
Prion Disease C57BL/6, C506M3 ++ Ins/tCr ratio in whole brain 98
Glioma GL261 model Low NAA, Cr/Cr+PCr, High Lac 99,100

mm3 mm<cube>,
Figure 8: Mice brain in vivo (C57BL/6-SJL) during maturation. Localized proton MRS (9.4 T, PRESS, TR/TE = 2000/130 ms, 2×2×2
512 accumulations) of the thalamus as a function of postnatal age (days 5–21) reveals spectral alterations such as
3
an increase of NAA and decrease of Tau. At 21.1 T, PRESS, TR/TE = 2000/130 ms, 1 × 1 ×1 mm<cube>, 512 accumulations from
thalamus of 21 days mice shows more metabolite peaks. Modified from reference [59].
mm
In case of no brain biopsy or autopsy, a gross evaluation of maturation disorder. Autocorrected T2 relaxation times reflect the
in vivo MRS visible metabolites suggests a possibility of brain true changes in metabolite concentrations. NAA, Cho, Tau, and Glx
metabolic alteration see (Figure 5) and its association with MRI concentration changes during mice brain maturation were similar
visible brain anatomic changes shown in Table 2 for the following to humans. NAA/tC in transgenic mice brain remained same
diseases. between 4 and 24 months but reduced in maturation disorder see
(Figures 8) [53]. In adult mice, regional cerebral concentration
Brain maturation disorders
differences show metabolite ratios to tCr by proton MRSI [54-56]
Proton MRS monitors the metabolic alterations during brain or, altered absolute metabolite concentrations by quantitative
maturation in the mice thalamus, olfactory bulb, and cerebellum proton MRS [57,58]. Localized proton MR spectra of mice cerebral
[52]. Figure 8 shows serial proton MR spectra from the thalamus cortex, hippocampus, striatum, and cerebellum showed many
for postnatal days 5–21 that reveal increased NAA and Glx specific metabolite changes during embryonic stage see (Figure
signals as well as decreased Cho and Tau resonances with age or 9) [59-61].

How to cite this article: Rakesh S. 900 MHz Microimaging and Proton MRS of Transgenic Mice Brain in Vivo Theranostic Monitoring: MRS Data
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DOI: 10.19080/CTOIJ.2022.22.556077
 Cancer Therapy & Oncology International Journal

Figure 9: Regional metabolite profiles of mice brain in vivo (C57BL/6) as detected by localized proton MRS (9.4 T, STEAM, TR/
TE = 5000/2 ms) of the cerebral cortex (2.5×1.2×3.0 mm<cube>, 320 accumulations), hippocampus (2.0×1.2×2.2 mm<cube>, 320

mm3
accumulations), striatum (1.7×2.0×3.0 mm3, 160 accumulations), and cerebellum (2.0×1.7×1.8 mm3, 320 accumulations). See
reference [60].
mm3

mm3
Figure 10: Cerebral ischemia in different mice strains. Localized proton MRS (2.35 T, STEAM, TR/TE = 6000/20 ms, 4×3×4
mm<cube>....., 512 accumulations) of the brain in vivo and 1 h postmortem (same animal) for a (top) NMRI, (middle) BALB/c, and
(bottom) C57BL/6 mice. Apart from decreased Glc and increased Lac concentrations postmortem, the specific vulnerability of the
C57BL/6 strain to ischemia is demonstrated by severely reduced levels of tNAA, tCr, and Cho (arrows). See reference [63].

How to cite this article: Rakesh S. 900 MHz Microimaging and Proton MRS of Transgenic Mice Brain in Vivo Theranostic Monitoring: MRS Data
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 Cancer Therapy & Oncology International Journal
Different transgenic mice strains may show subtle and distinct
metabolite level differences in NMRI, BALB/c, and C57BL/6 mice
[62], CBA, CBA/BL6, and C57BL/6 mice [63], and 129/SvJ, FVB/N,
and C57BL/6 mice [64]. These mice with different metabolic
endophenotypes serve for genetic engineering mice models. As an
example, Figure 8 compares in vivo and early postmortem proton
MR spectra of the brain of NMRI, BALB/c, and C57BL/6 mice.
MRI volumetry is another indicator. The C57BL/6 strain
showed largest ventricular spaces of different structures by proton
21 T MRI [65]. Spectroscopy showed elevated concentrations
of tNAA, tCr, Cho, Glc, and Lac relative to BALB/c mice and
elevated Glc concentrations relative to NMRI mice. Morphological
differences and metabolic heterogeneity were reported for the
129/SvJ, FVB/N, and C57BL/6 mice strains [63]. MRI volumetry
revealed significant alterations in cerebellar and hippocampal
volume as well as differences in the thickness of the motor cortex
[65]. The metabolite ratios to tCr yielded the highest tCr level and
lowest NAA/tCr ratio in the C57BL/6 strain [66]. In CBA, CBA/
BL6, and C57BL/6 mice striatum concentration showed small
differences for Lac, NAAG, Glu, Gln, Ins, and phosphoethanolamine
[66,67]. At 21.1 T, metabolite peaks of GABA, MM, Ala were visible
[68].
In vivo metabolite concentrations in mice brain change
as a function of age, brain region, and mice strain matched
with concentration values determined by in vitro proton NMR
spectroscopy of brain tissue extracts qualitatively similar to the
metabolite profiles of other mammal brains.
Cerebral ischemia
Cerebral ischemia is a condition where the brain or parts of
it do not receive sufficient blood flow or insufficient oxygen and
glucose to maintain normal physiological functions. Blockage
or obstruction of the supplying arterial vessels causes oxygen
insufficiency. Severe or prolonged brain ischemia results acute
stroke, unconsciousness, tissue damage, or death.
In a transgenic C57BL/6 mice model of focal cerebral
ischemia, Figure 10 demonstrates a pronounced increase of
Lac in the affected brain hemisphere and a distinct decrease of
NAA 2 h after occlusion of the middle cerebral artery. The lack of
oxygen supply causes a failure of the energy metabolism which
involves a switch from oxidative phosphorylation to nonoxidative
glucose consumption. This process utilizes local glygocen
stores to produce Lac. The reduced Cho and tCr may be due to
dilution of all metabolites or vasogenic edema which widens the
extracellular space by influx of water from the capillary bed [69].
These responses match with global ischemia in C57BL/6 mice 1 h
after cardiac arrest. Noteworthy, only Glc and Lac concentrations
increased without any change in tNAA, tCr, and Cho in BALB/c
and NMRI mice [63]. The enhanced vulnerability of C57BL/6
mice to cerebral ischemia seems strain-dependent differences of
the cerebral energy metabolism and/or the formation of edema
[70]. The 21.1 tesla MR spectroscopy of cerebral ischemia brain
How to cite this article: Rakesh S. 900 MHz Microimaging and Proton MRS of Transgenic Mice Brain in Vivo Theranostic Monitoring: MRS Data
0014 Correlation with Human Neurodegenerative Disease Burden for Translational Medicine. Canc Therapy & Oncol Int J. 2022; 22(1): 556077.
DOI: 10.19080/CTOIJ.2022.22.556077
structures from different mice strains may further answer specific
pathophysiological processes and neurochemical alterations.
Multiple sclerosis
In humans, proton MRS with brain multiple sclerosis lesions
is routine indicating inflammation, demyelination, and axonal
damage. A large variety of animal models ranging from monkeys
to mice mimic the pathologic conditions of multiple sclerosis.
The mice model of multiple sclerosis is experimental allergic
encephalomyelitis (EAE) showing inflammatory demyelination
in the spinal cord. Most in vivo MRI studies indicate white
matter and gray matter disturbances with tissue metabolism or
metabolite composition using MRS. Theiler’s murine encephalitis
virus infection model of demyelinating disease serves animal
model in C57BL/6J mice [71]. At 3–7 days after intracerebral
virus injection serial T1-weighted MRI and proton MRS (spectra
not shown) revealed periventricular and parahippocampal
hypointense lesions with decreasing NAA/tCr and Cho/tCr ratios.
Normalization of the MRI hypointensities 45 days after injection
showed normal NAA/tCr and Cho/tCr ratios, suggesting active
regeneration.
NMRI, BALB/c, and C57BL/6 mice [62], CBA, CBA/BL6, and
C57BL/6 mice [63], and 129/SvJ, FVB/N, and C57BL/6 mice
models represent reversible demyelination after feeding toxin
cuprizone to transgenic mice models of multiple sclerosis. These
reversed pathophysiologic mechanisms may answer correction
of demyelination and/or axonal damage to some extent [72].
Mechanisms regulating disease progression and tissue repair in
multiple sclerosis need high resolution proton MRS in vivo and
histopathologic assessments [73].
Parkinson’s disease
Parkinson’s disease is a chronic motor system disorder
from a progressive loss of dopamine-producing cells in the
substantia nigra. The reduced level or even lack of dopamine
leads to a generalized slowness of movements (bradykinesia),
muscular rigidity, and tremor which is most noticeable during
rest. Degeneration of nigrostriatal dopaminergic neurons or
Parkinson’s disease was induced by 1-methyl-4-phenyl-1,2,3,6-
tetrahydropyridine (MPTP) intoxication [74,75]. Quantitative
proton MRSI in 2 days and 6 days post-MPTP injected mice
demonstrated a significantly decreased NAA concentration in
the substantia nigra, pars compacta and striatum, regions most
affected in human Parkinson disease. More interestingly, MPTP-
intoxicated mice after receiving immune cells (recovered from
copaxone-vaccinated animals) presented similar NAA levels as
found in sham-treated animals, whereas adoptive transfer of
immune cells from ovalbumin-immunized animals to MPTP-
treated mice showed reduced NAA levels and degenerated
dopaminergic neurons with their projections [75]. Another
proton MRS study reported increased striatal Lac/tCr ratios
within 2 h after MPTP injection and return to basal levels after 7
h of injection [76]. Pre-treatments with deprenyl, a monoamine
 Cancer Therapy & Oncology International Journal

oxidase B inhibitor, or with GBR-12909, a dopamine uptake
inhibitor, attenuated both the increases in striatal Lac/tCr ratios
and the subsequent loss of dopaminergic nerve terminals in
MPTP-treated mice [76]. The ultra-high field 21.1 T proton MRS
has potentials to detect metabolic alterations better in Parkinson’s
disease and monitor disease progression and treatment.
Huntington’s disease
Huntington’s disease is a progressive neurodegenerative
disorder due to a genetic defect of a cytosine–adenosine–
guanosine (CAG) sequence responsible to code an expanded
polyglutamine huntingtin protein in the tract. Several transgenic
mice models NMRI, BALB/c, C57BL/6, CBA, CBA/BL6, 129/SvJ,
FVB/N have been developed that differ in CAG repeat length.
For example, N171-82Q mice with 82 CAG repeats exhibit a
progressive neurological phenotype at about 3 months of age
with a 2D NMR pattern [77]. Mice of the R6/1 line with 111–121
CAG repeats showed progressive symptoms by 4–5 months of age
and therefore serve as an adult-onset model [78,79]. The more
intensively studied R6/2 mice with 150 CAG repeats showed first
behavioral symptoms at about 1–2 months and were considered
as ideal model of juvenile-onset Huntington’s disease [80,81].
The proton MRS assessed the neurochemical profile of the
striatum during disease progression. Longitudinal measurements
of relative metabolite ratios in the juvenile R6/2 mice model
showed a nonlinear decrease in striatal NAA levels starting at
about 6 weeks of age coincident with the onset of symptoms.
Histopathology demonstrated that the in vivo NAA/tCr reduction
was due to neuronal dysfunction [80]. R6/1 mice model showed
decreased striatal NAA levels without any change in Cho and
tCr levels by proton MRS studies [81]. R6/2 mice model showed
elevated NAA [77]. As demonstrated in Figure 11, the severity of
the NAA reduction strongly correlated with the CAG repeat length
as well as with the levels of gene expression and protein context
[77]. Behavioral testing and proton MRS have been used to assess
therapeutic effects of various drugs in transgenic mice models
of Huntington’s disease. Daily administration of CGS21680,
an A2A adenosine receptor-selective agonist, improved the
motor performance, prevented a reduction of brain weight, and
normalized the elevated blood glucose levels in the R6/2 mice. It
also significantly reduced the increased Cho levels in the striatum,
but decreased NAA/tCr ratio [80].

mm3
Figure 11: Transgenic mice model of Huntington’s disease. Localized proton MRS (4.7 T, PRESS, TR/TE = 2000/136 ms,
6.0×3.5×3.0 mm3, 256 accumulations) of the striatum of a N171-82Q mice with 82 cytosine–adenosine–guanosine (CAG) repeats
(on left). A YAC72 mice with 72 CAG repeats (low expression level), and a wild-type (WT) YAC mice shows NAA/tCr ratio decreases
with increasing length of CAG repeats (on right). See reference [78].

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DOI: 10.19080/CTOIJ.2022.22.556077
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Neuroprotective effects of creatine were observed in N171-
82Q and R6/2 animals. Dietary creatine supplementation showed
increased brain tCr concentrations and delayed the decreases
in NAA by proton MR spectroscopy to improve survival, to slow
down the development of brain atrophy, to reduce the formation
of intranuclear inclusions, and delayed the development of
hyperglycemia. These results suggest a correction of metabolic
dysfunction in Huntington’s disease therapeutic method to
strengthen the energy metabolism in order to delay the pathologic
process [81]. The neurochemical profile in the striatum of R6/2
mice is distinct transgenic mice animal model of Huntington’s
disease different from other non-transgenic primates and rodents
[82,83]. The 21.1 T proton MRS may answer these differences and
may open additional insights into progression and treatment of
Huntington’s disease.
Alzheimer’s disease
Alzheimer’s disease is neurodegenerative disorder among
seniors with cognitive function loss evident with amyloid plaques
and neurofibrillary tangles in the central nervous system.
Proton in vivo MRSI offers theranostic monitoring in evaluation
of Alzheimer’s disease and new test drug discovery [3]. The
neurochemical abnormalities of Alzheimer’s disease have been
studied by proton MRS in different transgenic mice models [4,84].
Efforts were made to study 1H-13C NMR spectroscopy to study
metabolomics [85]. In first mice study on mutant forms of human
presenilin 2 (PS2) and APP (PS2APP) expression confirmed
severe amyloidosis in the neocortex and hippocampus with in
vivo proton MRS showing stable NAA/tCr, Glu/tCr and Ins/tCr
ratio ratios in first 12-month-old animals but decreased NAA/
tCr and Glu/tCr ratios in 24-month-old animals [86]. A second
mice study on mutant human presenilin 1 (PS1) and amyloid-β
precursor protein (APP-PS1) confirmed the amyloid plaques with
in vivo proton MRS showing reduced biomarkers NAA/tCr and
Glu/tCr ratios initially in 3-month-old animals and reduced NAA/
tCr and Glu/tCr in 23-month-old animals [86]. In contrast to said
studies, elevated Ins/tCr ratio is shown in Figure 12 to suggest
that plaque load in APP-PS1 mice is genotype dependent [87].

Figure 12: Transgenic mice model of Alzheimer’s disease. Localized proton MRS (9.4 T, LASER, TR/TE = 3000/28 ms, 18 μl, 192
accumulations) of the brain of a mice (B6/SJL) which coexpresses mutated human presenilin 1 and amyloid-β precursor protein
(APP-PS1) at 16, 20, and 23 months of age. When scaled to tCr (dotted line) and in comparison to an age-matched wild-type (WT)
animal, the old APP-PS1 mice exhibits decreased NAA/tCr and Glu/tCr ratios as well as an increased Ins/tCr ratio (arrows). See
reference. [114].

A third transgenic mice study on mutant forms of human
amyloid precursor protein (APPTg2576) expression confirmed
β-amyloid plaques in cortex with proton MRS in vivo metabolite
ratio of NAA/tCr, Cho/tCr, Glx/tCr, and Tau/tCr intensities ratios
during 10-19 months [88]. In contrast, wild-type mice in vitro
NMR spectroscopy showed decreased NAA/tCr and Glu/tCr
ratios and an increased Tau/tCr ratio, normal Ins/tCr ratio [89].
Proton MRS visible metabolites in human Alzheimer’s disease do
resemble with transgenic APP-PS1 neurochemical profile.

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DOI: 10.19080/CTOIJ.2022.22.556077
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Oxidative Phosphorylation and Energy metabolism
In vivo MRS studies of transgenic mice knockout model of
guanidinoacetate methyltransferase (GAMT) deficiency (creatine
deficiency) with altered high-energy phosphoryl transfer
metabolism showed reduced brain tCr levels [30]. Similar to
human ‘creatine deficiency” patients [89,90], Figure 13 [30]
shows combined in vivo and in vitro phosphorus and proton MRS
of brain and muscle in a longitudinal study of biosynthesis and
intracellular transport of creatine and creatine supplementation
in GAMT-deficient mice that creatine uptake was faster in skeletal
muscle than in brain.

mm3
Figure 13: Transgenic mice model of guanidinoacetate methyltransferase (GAMT) deficiency. Localized proton MRS (7.0 T,
STEAM, TR/TE = 5000/10 ms, 3×3×3 mm3, 64 accumulations) of the brain of a guanidinoacetate methyltransferase-deficient
(GAMT−/−) mice demonstrates a dramatic loss in tCr (arrows) relative to a wild-type (WT) animal. See reference [30].

Creatine kinase (CK) enzyme indicates status of oxidative
phosphorylation in muscle and brain. Transgenic mice
lacking cytosolic creatine kinase isoenzyme (B-CK−/−) or the
mitochondrial ubiquitous isoenzyme (UbCKmit−/−) showed
normal PCr and adenosine triphosphate (ATP) as well as tCr
and NAA in brain by phosphorus and proton MRS [91]. Double
knockout (CK−/−) mice showed 20-30% tCr+PCr with increased
NAA concentrations and normal high-energy phosphates in all
brain regions [91]. This elevated NAA levels reflected the changed
neuron cell size, neuronal volume, cell type and cerebral osmolite
distribution means genetic control in disturbed oxidative
phosphorylation or muscle physiology [91]. The potential of 21.1
T MRS in knockout mice is awaited.
Transgenic mice models of ALS, Rett Syndrome
Familial amytrophic lateral sclerosis (ALS) neurodegenerative
disease or motor neuron loss in transgenic mice (G93A human
superoxide dismutase mutation) was characterized by increased
Glx/tCr ratios but normal NAA/tCr and Cho/tCr ratios within 100
days and motor loss, premature death in a month [92]. Creatine
supplementation increased longevity and motor performance
confirmed by increased Glx/tCr ratio in proton MRS at 80 days
of age [93]. Rett syndrome is an X-linked neurologic disorder
(mutation in methyl-CpG-binding protein 2 (MECP2) gene) [94].
Proton MRS in Mecp2−/y gene knock out mice showed decreased
Glx/Ins ratio with increased NAA/tCr ratio is shown in Figure 14.
The potential of 21.1 T MRS in knockout mice is awaited.
Transgenic mice model of Canvan’s Disease
A proton MRS study of a Canvan disease knockout Cmice
revealed increase of the NAA/tCr ratio in the thalamus of
homozygous mice relative to wild-type and heterozygous animals
[95]. Canavan disease is leukodystrophy with mental retardation,
abnormal muscle tone, and abnormal head size appearance due
to aspartoacyclase deficiency genetic defect (NAA is not broken
down) showed increased cellular NAA by proton MRS of affected
children and impairs the normal function of the nervous system.
High-resolution proton NMR spectroscopy of brain extracts
showed increased NAA. The potential of 21.1 T MRS in knockout
mice is awaited.
Transgenic mice model of Down Syndrome
The Ts65Dn mice model of Down syndrome shows mental
retardation, abnormal cell division due to segmental trisomy 21
characterized by three (instead of two) copies of chromosome
21 and decreased NAA/tCr and elevated Ins/tCr ratios but

How to cite this article: Rakesh S. 900 MHz Microimaging and Proton MRS of Transgenic Mice Brain in Vivo Theranostic Monitoring: MRS Data
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DOI: 10.19080/CTOIJ.2022.22.556077
 Cancer Therapy & Oncology International Journal

inconclusive Ins resonance by proton MRS study similar with and Ts65Dn mice but the NAA/tCr and Cho/tCr ratios remained
metabolites in patients shown in Figure 15. Subsequently, one unaltered [96]. The potential of 21.1 T MRS in knockout mice is
month lithium treatment decreased Ins/tCr ratio in patients awaited.

mm 3
Figure 14: Transgenic mice model of Rett syndrome. Localized proton MRS (4.7 T, PRESS, TR/TE = 1500/16 ms, 3.5×3.5×3.5
mm3, 256 accumulations) of the brain of a Mecp2−/y mice shows decreased Ins and increased Tau resonances (arrows) relative to
a control. Metabolite ratios with the sum of all metabolite signals reveal increased NAA/sum and decreased Glx/sum and Ins/sum
ratios in Mecp2−/y mice (arrows). See reference [94].

Figure 15: Transgenic mice model of Down syndrome. Localized proton MRS (4.7 T, STEAM, TR/TE = 1500/30 ms, 5×3×5 mm3,
mm3
512 accumulations) of the brain of a Ts65Dn mice reveals a decreased NAA/tCr and increased Ins/tCr ratio (arrows) in comparison
to a control [96].

How to cite this article: Rakesh S. 900 MHz Microimaging and Proton MRS of Transgenic Mice Brain in Vivo Theranostic Monitoring: MRS Data
0018 Correlation with Human Neurodegenerative Disease Burden for Translational Medicine. Canc Therapy & Oncol Int J. 2022; 22(1): 556077.
DOI: 10.19080/CTOIJ.2022.22.556077
 Cancer Therapy & Oncology International Journal

Figure 16: (on left row) Cerebrospinal fluid (CSF) filled ventricular spaces have been highlighted in color. D2 knockout mouse
brain shows large ventricles compared to the D2 (+/+) mouse brain (middle) following ethyl alcohol exposure. The control mouse
brain displays smaller ventricles than either of treatment groups. It suggests that D2 receptor modulate EtOH induced toxicity. (on
right row) colchicine treatment showed shrunk hippocampus or atrophy (presentation at NHMFL open house, 2006).

HIV encephalitis models C57BL/6, BSE strain 6PB1 or C506M3 scrapie strains of
prion diseases with increased Ins/tCr ratio and ataxia in 20 days
The human immunodeficiency virus (HIV) lead to the
of preclinical stage [98] followed by a decreased NAA/tCr ratio at
acquired immune deficiency syndrome (AIDS) causing incapable
advanced stages. Murine ME7 model also showed reduced NAA/
WBC, opportunistic infections and tumors. HIV-1 encephalitis
tCr by in vivo MRS with early behavioral deficits and pathological
mice model (human HIV-infected monocyte-derived macrophages
alterations [98].
injected into the subcortex of immunodeficient mice) showed
focal giant cell encephalitis, reactive astrocytes, microgliosis, and Glioma
neuronal dropout 7 days after injection with reduced NAA without
A MRS imaging (nonlocalized 2D J and correlation spectroscopy)
Cho and tCr altered concentrations in injected hippocampus
of glioma using spin-echo correlation spectroscopy (SECSY) pulse
suggesting edema or enlarged cerebrospinal fluid (CSF) spaces,
sequence at 4.7 T revealed low creatine+phosphocreatine/choline
neuronal dysfunction regions and metabolic deficit [97]. The
ratio, low NAA, high lactate peaks. By MRS-PRESS (TR=2500 ms,
potential of 21.1 T MRS in knockout mice is awaited.
TE=24 ms, 7T) of ethyl nitrosourea induced and GL261 mice
Prion diseases model also showed similar metabolite changes [99]. In other
study, MRS(LASER, TR=3 sec, TE=60 ms, 7T) showed low NAA,
The infectious cell membrane protein misfolding and altered
low creatine in transgenic mice model [100].
three-dimensional configuration causes the Prion diseases
(human Creutzfeldt–Jakob disease, scrapie in sheep and goats, Future Attempts Functional 21.1 Tesla MRI Imaging
and the bovine spongiform encephalopathy (BSE) or “mad cow Of Transgenic Animals
disease”). It is transmissible spongiform encephalopathies or
At low magnetic field, transgenic mice fMRI experiments
extensive vacuole formation in the central nervous system. Proton
showed promise to evaluate the brain functions and
MRS showed altered cerebral Ins and tCr metabolites in mice

How to cite this article: Rakesh S. 900 MHz Microimaging and Proton MRS of Transgenic Mice Brain in Vivo Theranostic Monitoring: MRS Data
0019 Correlation with Human Neurodegenerative Disease Burden for Translational Medicine. Canc Therapy & Oncol Int J. 2022; 22(1): 556077.
DOI: 10.19080/CTOIJ.2022.22.556077
 Cancer Therapy & Oncology International Journal
neuroactivation events [5]. As of today, the possibility of 21 Tesla
fMRI experiments is unknown or not reported while 25 Tesla NMR
magnet systems are available. There is rapidly growing interest
in making radiological imaging techniques sensitive to specific
molecules or biological processes. The new awareness of animal
rights, molecular genetics and available mice models of human
diseases envisions the future of micro-imaging in development of
molecular biology. Nuclear imaging techniques, optical imaging
techniques, and MRI techniques all are used in routine over years
to understand the pathophysiology of disease and new targets
for An example of ethanol and colchicine treatment on D2 (+/+)
mice hippocampus size by 21 Tesla MRI is shown in Figure 15 [8].
therapeutic intervention as new tools of ‘theranostic monitoring’.
Molecular in vivo MR imaging of the brain offers significant
challenges due to the problem of delivering agents through the
blood-brain barrier, expansive cost, keeping small animal intact
for long time undisturbed physiology in magnet during real-time
monitoring. Transgenic mice engineering will solve partly these
problems such as producing a large number of amyloid plaques
and smart MRI visible nanoparticle accumulation. New strategy
of coupling MRI contrast agents to peptides or antibodies that
recognize specific targets across blood-brain barrier, is rapidly
fast-growing area in molecular imaging. Another very promising
area for molecular imaging is to monitor cell migration in vivo.
New cell tracking methods will be developed to label a specific
cell population, either in vitro or in vivo, with smart MRI visible
contrast agents and then to follow the movement of these cells
in the animal or track the effected cell metabolites at micrometer
scale. Typically, nanometer-sized iron-oxide-based contrast
agents are used for endocytosis to get these particles into cell
and tracking metabolic events. The advantage of these iron
oxide particles is that nanoparticle loaded single cells can cause
sufficient contrast to change detectable by MRSI. In near future,
recent advances in 21 Tesla MRSI-based cell tracking will answer
unknown facts to study neurodegenerative and neurodysfunction
diseases of the brain.
Present State of Art
Primates and rodents offer a close similarity to humans in
terms of anatomical, physiological, immunological, and functional
characteristics of the central nervous system. Monkey models are
ideal for theranostic trials of specific neurological disorders (e.g.,
multiple sclerosis) and psychiatric syndromes (e.g., stress and
depression). Apart from transgenic mice, gerbils, squirrels, guinea
pigs, and rabbits are also widely used as major experimental
ex vivo or in vitro animal models for human brain diseases.
However, primates, rodents and monkeys are not suitable for
138 mm ultrawide MRSI. With concern of animal rights and
quickly available transgenic mice with NIH/NIBIB database,
now transgenic mice techniques only show promise to explore
genetic insight of cognitive and psychiatric diseases by simple
proton MRSI in vivo. Continuing progress in ultrahigh-field 21.1-
30 Tesla MR microscopy, in vivo MRSI and NIH/NIBIB database
How to cite this article: Rakesh S. 900 MHz Microimaging and Proton MRS of Transgenic Mice Brain in Vivo Theranostic Monitoring: MRS Data
0020 Correlation with Human Neurodegenerative Disease Burden for Translational Medicine. Canc Therapy & Oncol Int J. 2022; 22(1): 556077.
DOI: 10.19080/CTOIJ.2022.22.556077
on genetically modified mice, it is likely that in vivo experiments
on transgenic species will be preferred. Author hopes that the
animal NMR spectroscopy studies discussed in this review will
help scientists to find new applications for transgenic animal
species in translational medicine to understand human diseases
including other organ diseases. High NMR resolution and high
SNR without noise, altered concentrations of disease or tissue
specific neurometabolites to correlate them with role of brain
physiological functions in brain diseases still remain challenges
and inconclusive. More MRI technical advances and better spectral
resolution techniques supported with bench experiments will
investigate true representative neurometabolite variations during
disease development and prognosis [103-110]. In this direction,
university of Geneva and Bruker Biospin team developed and
tested superconducting coil to generate magnetic field of 23.5 - 25
Tesla towards attempts to 30 -35 Tesla by using 1-micron YBCO
copper oxide superconductor wrapped 140-meter 3 mm steel
tape around cylinder coil cooled with liquid helium temperature
of −269°C (4.2 K) with capability of microimaging. In present time,
both NMR and MR microscopy techniques are used in chemical
and pharmaceutical industries while biomedical and medical
applications are very limited. With above attempts, there is ample
opportunity of MR imaging with spectroscopy use in theranosis,
drug discovery, metobolomics, structural protein chemistry and
food analysis.
Conclusion
Localized proton ultrahigh in vivo MRS and MRSI (imager
up to 21.1 Tesla magnetic field) of animal brain has emerged as
theranostic monitoring means “disease detection and confirming
disease diagnosis and testing the drug effect real-time monitoring”
based on structural and metabolic distribution in whole brain for
both preclinical research and basic transgenic animal neuroscience.
The cerebral metabolism in transgenic mice models of human
brain diseases is better explained with proton MR spectral
localization methods to identify metabolite NMR signal intensities
and measuring absolute metabolite concentrations. Localized
proton MRS still today is choice to evaluate neurochemical
profiles in transgenic mice, rats, and nonhuman primate animal
models of neurodegenerative diseases. Genetically modified mice
(transgenic mice technology) models available at NIH resource
center have replaced experiments on nonhuman primates
because of close similarity of mice brain genetics with human
genetics associated with diseases. Noninvasive MRSI studies are
new theranostic tools of molecular biology and neurochemical
genetics in preclinical neurotranslational research and medicine.
Altered neurometabolite profiles ( MR visible neurochemicals)
indicate the intraneuron metabolism or brain cell composition to
predict pathophysiological changes in neurological and psychiatric
diseases as potential guide to discover or search effective drug or
novel therapeutic intervention in ischemia, hypoxia, Perkinson
disease, multiple sclerosis, Rett disease, Alzheimer disease,
oxidative stress diseases, Huntington disease, Down’s syndrome,
 Cancer Therapy & Oncology International Journal
HIV and Canvan disease. Ultrahigh field magnetic resonance
spectroscopy technology will serve better in theranosis and new
drug testing.
Acknowledgment
Author acknowledges the National Science Foundation
sponsored Users’ Facility Program opportunity offered by Director
at National High Magnetic Field Laboratory, Tallahassee, FL to
initiate biomedical imaging program at department of Chemical
and Biomedical Engineering, FAMU-FSU College of Engineering,
Florida State University, Tallahassee, FL during years 2004-2010.
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How to cite this article: Rakesh S. 900 MHz Microimaging and Proton MRS of Transgenic Mice Brain in Vivo Theranostic Monitoring: MRS Data
0023 Correlation with Human Neurodegenerative Disease Burden for Translational Medicine. Canc Therapy & Oncol Int J. 2022; 22(1): 556077.
DOI: 10.19080/CTOIJ.2022.22.556077
 Cancer Therapy & Oncology International Journal

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How to cite this article: Rakesh S. 900 MHz Microimaging and Proton MRS of Transgenic Mice Brain in Vivo Theranostic Monitoring: MRS Data
0024 Correlation with Human Neurodegenerative Disease Burden for Translational Medicine. Canc Therapy & Oncol Int J. 2022; 22(1): 556077.
DOI: 10.19080/CTOIJ.2022.22.556077