Non-invasive Clinical Imaging of Cerebral Metabolism Following Brain Injury Using 13C Magnetic Resonance Spectroscopy
Phase: NA
Conditions: Traumatic Brain Injury
Sponsor: Loma Linda University

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Non-Invasive Clinical Imaging of Cerebral Metabolism Following 
Traumatic Brain Injury Using 13C Magnetic Resonance Spectroscopy 
NCT02745210 
2-9-2019 

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Background  
Despite the decline in fatal TBI incidence in recent years, TBI morbidity remains a public health 
challenge and is the leading cause of disability in the United States (1,2,4). Residual impairments 
have been estimated to occur in 67% to 100% of TBI survivors, with cognitive impairments being 
the most disabling sequelae. (3,4) In addition, TBI has emerged as one of the most significant health 
risks to military personnel related to the wars in Iraq and Afghanistan (3) and an important medical 
concern due to potential long-term physical, cognitive and functional disabilities (5). To combat 
these effects, new research is needed to identify mechanisms of injury that will lead to potential 
targets for therapeutic interventions that improve neurological outcome.   
 
One promising area of research is the cerebral metabolic dysfunction following TBI. Studies of 
post-traumatic cerebral metabolism have shown that CMRglc decreases for a period of days, 
weeks or months after injury with the duration and degree of hypometabolism correlating to level of 
consciousness and a strong predictor of long-term neurological outcome (6,7,8). However, specific 
changes in intermediary carbohydrate metabolic pathways have not yet been identified. In addition, 
the role of astrocyte metabolism in the post-injury metabolism has not been studied. This study 
uses in vivo 13C MRS at 3T, a novel method in the clinical study of TBI, to non-invasively study the 
metabolic fate and flux of glucose (metabolized in both neurons and astrocytes) and acetate 
(metabolized in astrocytes) through metabolic pathways during the hypometabolic period. In 
addition, we will study the association of decreased cerebral perfusion with changes in glucose and 
acetate metabolism following TBI, as changes in cerebral perfusion could profoundly affect 
cerebral metabolism by limiting substrate availability.  
 
Cellular Mechanisms Affecting Cerebral Metabolism 
While, mitochondrial dysfunction has been proposed as a key factor contributing to the hypometabolic 
response following injury, the down regulation and/or inhibition of enzymes or cofactors associated 
with intermediary metabolism may also decrease glucose metabolism following TBI. For example, 
increased production of reactive oxygen species (ROS) such as superoxide radical, hydrogen 
peroxide, and hydroxyl radical following TBI (9,10) could inactivate pyruvate dehydrogenase (PDH), due 
to its sensitivity to ROS (11,12).  Impaired PDH activity after TBI would limit the flux of substrates into 
the TCA cycle, decreasing oxidative metabolism and increasing the flux of substrate through 
anaplerotic pathways as a way of supplying needed TCA cycle intermediates to affected cells.  
 
Astrocyte Metabolism and Intracellular Metabolic Compartmentation Following TBI  
Still unclear is the role of metabolic coupling between astrocytes and neurons following TBI. It has 
long been recognized that astrocytes play a pivotal role in meeting the energy requirements of 
neurons through various metabolic pathways, including the glutamate-glutamine cycle which links the 
exchange of neurotransmitter glutamate (Glu) and glutamine (Gln) between glutamatergic neurons 
and astrocytes (13). Moreover, the net synthesis of Glu in neurons requires a flux of TCA cycle 
intermediates, notably Gln, from astrocytes (14) as neurons lack the capacity to generate TCA cycle 
intermediates. The net synthesis of TCA cycle intermediates, Glu and Gln depends upon the entry of 
pyruvate via an anaplerotic pathway into the TCA cycle. In the brain this is exclusively achieved by 
pyruvate carboxylase (PC), an astrocyte specific enzyme (15,16). Numerous in vitro studies have 
shown that astrocytes supply TCA cycle substrates to neurons during periods of glucose and/or 
oxygen deprivation (17,18,19), suggesting that astrocytes may play an even greater nutritional role for 
neurons in the injured state. Given the essential role of neuron-astrocyte metabolic coupling in normal 
brain, a greater appreciation of the effect of TBI on metabolic coupling is an important and necessary 
contribution to understanding of metabolic dysfunction following TBI. Moreover, the identification of 
novel metabolic therapies that target astrocytes may prove important in limiting neurological injury. In 

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this study, we will use [1, 2 13C2] acetate as an astrocyte specific metabolic substrate together with [1-
13C] glucose to study injury-induced changes in neuron-astrocyte metabolic coupling. 
 
Decreased Cerebral Perfusion Following TBI  
Decreased cerebral perfusion following TBI is associated with reductions in the utilization of 
glucose which could profoundly affect cerebral glucose metabolism by limiting substrate 
availability. In this study, we will use MR dynamic susceptibility contrast perfusion weighted 
imaging (DSC-PWI) to measure changes in cerebral perfusion in the hypometabolic period 
following TBI. DSC-PWI can provide a measure of tissue perfusion similar to results found using 
more traditional methods of CBF determination, with the advantage of relative higher spatial 
resolution, the absence of need for radioactive isotopes, and can be combined with other MRI 
studies in one evaluation.  
 
Tracking the Metabolic Fate of Glucose: 13C-MR spectroscopy 
In order to determine how glucose is being metabolized, 13C MRS offers an attractive, novel method 
to study alterations in multiple enzyme complexes following a TBI. The primary advantage of this 
technique arises from its ability to distinguish 13C incorporation into multiple metabolites as well as 
into the specific carbon positions within the same metabolite, resulting in a detailed analysis of the 
metabolic fate of 13C label (20,21,22). The relative 13C enrichment at each carbon position and the ratios 
between isotopomers of Glu and Gln gives additional information regarding enzyme usage and 
metabolic compartmentation (23,24,25,26). The acquisition of 13C spectra over time enables the dynamic 
measurement and calculation of in vivo rates of flux through metabolic pathways, an important 
technique to study in vivo cerebral metabolism. To our knowledge, the use of 13C glucose together 
with 13C acetate to measure metabolic flux simultaneously has not been performed in either the 
normal or injured human brain. In this study, we will use of [1-13C] glucose together with [1, 2 13C2] 
acetate to measure changes in both neuronal and astrocyte metabolism in both TBI and control 
subjects.   
 
Significance 
Detailed knowledge of the metabolic alterations following TBI will provide a significant advancement 
to our understanding of the hypometabolic response to TBI, which is key information for the future 
development and testing of novel therapeutic interventions that by-pass or compensate for the 
metabolic dysfunction. 
 
Study Objectives 
The goals of this inter-departmental study are: 1) the clinical application of in vivo 13C MRS at 3T to 
identify specific metabolic alterations following TBI and 2) determine if changes in cerebral perfusion 
effect metabolism by limiting metabolic substrate flow into the brain following TBI.  
 
Hypothesis 
We hypothesize that following TBI, metabolic pathways are altered causing an incomplete oxidative 
of glucose in neurons and astrocytes resulting in a decrease in cerebral metabolism.  
 
Research Design and Methods 
Imaging Protocol Overview 
All control and TBI subjects will undergo an identical MRS/MRI protocol and neurological exam. 
MRS/MRI will be performed on a 3T Siemens TIM Trio system equipped with a second 
radiofrequency channel (Siemens Medical Solutions, Erlangen, Germany) using a specially designed 
and manufactured 1H-13C dual tuned volume head coil (Advanced Imaging Research, Cleveland, 

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OH). Sagittal 3D T2 weighted (T2WI) and 3D T1 weighted (T1WI) MR images, reformatted in axial 
and coronal planes, will be obtained for image co-registration and segmentation. The 2D DSC-PWI 
sequence will be used to determine cerebral blood flow (CBF), cerebral blood volume (CBV), mean 
transit time (MTT), and the cerebral metabolic rate of oxygen (CMRO2). Detailed sequence 
description and image processing methods are explained below. 
 
TABLE 1. Imaging Parameters for 3.0T  Siemens TIM Trio Scanner 
Pulse 
Sequence 
Plane 
TR 
(ms) 
TE 
(ms) 
Slice 
Thickness 
(mm) 
FOV 
(mm2) 
Time 
(min) 
NA 
Note 
T2 SPC 
3D 
sagittal 
3200 
458 
1 
290x232 
4:30 
1 
matrix = 320x319x320, iPAT=2; 
ETL=139; BW=744Hz/px  
T1  
MP-
RAGE 
3D 
sagittal 
1950 
2.26 
1 
256x224 
3:54 
1 
FA=90º, matrix = 256x256x246, 
BW=200Hz/px, iPAT=2 
DSC-PWI 2D 
axial 
1700 
40 
5 
(20% gap) 
220x220 
1:30 
1 
Matrix=128x100, measures = 
50, FA=90º, fat saturation, 
BW=1860Hz/px, Echo spacing 
=.72 
ISIS-
DEPT 13C 
MRS 
2D 
axial 
2500 
 
 
 
160x200 
60:00 24 Waltz16 1H decoupling, FA=45º, 
outer volume suppression, 
200s rectangular excitation 
pulse, voxel size = 7x5x6xcm3 
TOTAL IMAGING TIME:  70 minutes plus shimming time 
 
In vivo 13C MRS  
[1-13C] glucose and [1, 2-13C2] acetate infusion protocol 
To reduce the complicating effects of endogenous glucose or the conversion of acetate to glucose in 
the liver, both control and TBI subjects will undergo a hyperinsulemic euglycemic glucose clamp via 
insulin infusion (60mU . m-2 . min-1) in conjunction with a variable infusion of 20% dextrose solution. 
Following the insulin clamp, 30 ml of 100% isotopically enriched [1-13C] glucose and [1, 2-13C2] 
acetate (Cambridge Isotope Laboratories, Andover, MA, USA) in a 20% w/v solution will be infused 
for 10 minutes followed by a constant infusion (3mg . kg-1 . min-1) of 30% isotopically enriched [1-13C] 
glucose and [1, 2-13C2] acetate. This infusion protocol results in normoglycemic plasma glucose levels 
of ~ 5.3 mmol/L(28), which will be maintained for the duration of the 13C MRS experiment. Blood 
samples (0.5ml) will be drawn in duplicate at 5-minute intervals for the immediate measurement of 
plasma glucose level using an Accu-Chek glucose meter and for measuring plasma glucose and 
acetate isotopic enrichment by high-resolution 1H and 13C NMR spectroscopy. 
 
ISIS-DEPT 13C MRS sequence  
Using the T2WI images, a 7 x 5 x 6 cm3 volume covering portions of the fronto-parietal lobe will be 
positioned, avoiding areas of obvious contusion. Direct detection, localized in vivo 13C MRS spectra 
will be acquired using a gradient-spoiled semi-adiabatic distortionless enhanced polarization transfer 
(DEPT) sequence (TR = 2.5 sec, interpulse delay = 3.8 ms, 45º flip angle, 128 acquisitions and 
WALTZ-16 decoupling applied during the acquisition) with three-dimensional image selected in vivo 
spectroscopy (ISIS) localization and outer volume saturation (27,28). Spectra will be post-processed off-
line using NUTS data processing software (Acorn NMR Inc., Livermore, CA). All relevant peaks will 
be identified and assigned by comparison to previously published values (21,22,23), integrated, and the 
relative amount of 13C incorporation into Glu and Gln will be calculated. To calculate the various flux 

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parameters, a two-compartment model of flow from [1-13C] glucose and [1, 2 13C2] acetate will be 
implemented in CWave software using modifications of previously published equations (27). 
 13C MRS voxel tissue composition  
In vivo MRS does not have the spatial resolution or the sensitivity to eliminate partial volume 
effects of different tissue types. Using an automatic MRS voxel segmentation program developed in 
our laboratory incorporating routines from MATLAB (Version 7.0.4, MathWorks, Natick, MA, USA) 
and SPM5 (The Wellcome Department of Imaging Neuroscience, Institute of Neurology, University 
College London, London, England), the relative gray matter and white matter tissue components will 
be calculated and applied as correction to factors to the 13C enrichment curves.  
 
Plasma NMR spectroscopy 
The peripheral metabolism of infused 13C labeled substrates is a confounding factor when measuring 
metabolism in the brain. Acetate metabolism is known to occur in the liver, resulting in the synthesis 
of glucose by gluconeogenesis (30). In the brain, 13C labeled glucose produced in the liver would lead 
to isotopomer labeling patterns similar to the metabolism of [1, 2-13C2] acetate resulting in a potential 
source of error in our experiments. As a result, the fractional 13C enrichment of all plasma glucose 
isotopomers will be calculated by 13C and 1H high resolution NMR using a Bruker 600 MHz 
spectrometer to correct brain isotopomer enrichment levels for the contribution of peripheral 
metabolism. 
 
DSC-Perfusion Weighted imaging 
A 2D single shot gradient echo echo-planar imaging (EPI) sequence will be used to obtain DSC_PWI 
using the following parameters: TR/TE = 1700/40 ms, FA = 90º, slice thickness = 5mm, matrix size = 
1282, and 50 measures. Gadolinium-DPTA (Gd-DTPA) contrast (0.1 mmol/kg) will be administered 
intravenously at the completion of the 5th scan. The resultant images are transferred to an offline 
program where CBV, CBF and MTT maps will be generated for each slice. To calculate CMRO2, an 
arterial input function is chosen from the M1 section of the middle cerebral artery for each subject. A 
least squares exponential fitting is used to minimize secondary circulation; a singular value 
decomposition deconvolution is then used to obtain estimates of CBF and CBV.  
 
To determine the effect of substrate limitation on glucose metabolism the position of the MRS voxel 
will be overlaid on CBF, CBV, and CMRO2 maps using an automatic image overlay program 
developed in our laboratory incorporating routines from MATLAB (Version 7.0.4, MathWorks, Natick, 
MA, USA).  The relative CBF (ml/100g/min), CBV (ml/100g), and CMRO2 (ml/100g/min) within the 
region of the MRS voxel will then be compared to the final 13C Glu and Gln enrichment pools to 
determine if substrate limitation correlates with metabolite enrichment. 
 
Clinical Chart Review 
Additional Clinical Information including: age, gender, demographic data, medical history, cause of 
brain injury, GCS score (initial, admission, and lowest post-resuscitation), Abbreviated Injury Score 
(AIS), pupillary reaction at admission, presence of associated injuries, laboratory (BUN, creatinine, 
alcohol and drug screening) and radiographic data, number of days after insult, length of patient’s 
unconsciousness, length of post-traumatic amnesia (PTA), evidence of hypoxia, duration of 
ventilatory support, time to follow commands, medication regimen, and duration of stay in the ICU will 
be recorded from the patients’ chart.  
 
Neurological Assessment 
Neurological assessment will consist of a standardized examination of neurologic function as listed in 
Table 2 by a neurologist or intensivist on the day of admission and the day of the MRS/MRI study.   

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Table 2:  Neurologic examination  
1. GCS score (initial, admission, post-
resuscitation) 
7. Motor tone and strength 
2. Abbreviated Injury Score (AIS) 
8. Deep tendon reflexes 
3. Length of unconsciousness 
9. Sensory function (touch, pain, temperature) 
4. Duration of PTA 
10. Cerebellar and basal ganglia function and gait 
5. Time until able to follow commands 
11. GOS score 
6. Cranial nerve examination 
12. Length of stay in ICU 
 
Enrollment 
Subjects with severe brain trauma will be recruited from admissions to the medical, surgical, or 
neurological services of LLUMC, which is a Level 1 Trauma Center verified by the American College 
of Surgeons.  
 
TBI Patient enrollment 
Only patients that are hemodynamically stable will be targeted for enrollment and the MRS/MRI 
studies will be ordered only after scheduled repairs for traumatic injuries have taken place. TBI 
participants will undergo the studies depending on clinical status as determined by the intensivist, Dr. 
Dorotta, attending neurosurgeon or neurologist. Sedation and mechanical ventilation will be continued 
and monitored for all patients during transport and during the studies by Critical Care nursing and 
respiratory therapy staff according to hospital policies. Once a potential candidate has been identified, 
Dr. Bartnik Olson will interview the candidate and/or family members, screen for inclusion/exclusion 
criteria, and enroll the candidate by obtaining the properly signed consent. The specific inclusion and 
exclusion criteria are: 
 
TBI Patient Inclusion Criteria:  
 Patients will be at least 18 years of age without gender or ethnic restrictions. 
 Severe accidental TBI defined as the lowest post-resuscitation GCS < 8 prior to administration 
of sedatives or paralytics. 
 Eligibility for MRI per routine screening checklist. 
 
TBI Patient Exclusion Criteria: 
 History of neurosurgical intervention, excluding the placement of ventriculostomy shunt 
 History of a prior known brain injury with associated loss of consciousness. 
 History of a known neurological disorder prior to qualifying injury. 
 History of psychiatric disorder. 
 History of diabetes or current unstable serum glucose level. 
 Renal insufficiency or known history of kidney disease. 
 Known contraindication to MRI such as, pacemaker, pregnancy, and/or other non-MR 
compatible implanted device. 
 
Ten (10) severe TBI patients will have the MRS/MRI study between 3 and 7 days post-injury to target 
the hypometabolic period.  
 
Control Subject enrollment 
Five (5) age/sex matched normal volunteers will be recruited as control subjects during the first year 
of the study.  Control subjects will be recruited from Loma Linda University and/or Medical Center 

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staff, student or Resident populations. The specific inclusion and exclusion criteria for the control 
subjects are: 
 
Control Subject Inclusion Criteria:  
 At least 18 years of age without gender or ethnic restrictions. 
 Eligibility for MRI per routine screening checklist. 
 
Control Subject Exclusion Criteria: 
 MRI Department staff or subordinate of project Investigator. 
 History of neurosurgical intervention, excluding the placement of ventriculostomy shunt. 
 History of a prior known brain injury with associated loss of consciousness. 
 History of a known neurological disorder. 
 History of psychiatric disorder. 
 History of diabetes or current unstable serum glucose level. 
 Renal insufficiency or known history of kidney disease. 
 Previous allergic reaction to gadolinium MR contrast. 
 Known contraindication to MRI such as, pacemaker, pregnancy, and/or other non-MR 
compatible implanted device. 
 
Personnel 
In addition to the principal investigator and co-investigators, research assistants or student may 
perform chart review and data base entry. 
 
Statistical Analysis 
The data analysis will emphasize descriptive and graphical statistics. Descriptive statistics used to 
detect statistically significant differences between control and TBI subjects will include means and 
associated 95% confidence intervals, one-way ANOVA with Scheffe post-hoc analysis, and Pearson 
correlation coefficients to identify possible correlations between metabolic changes and/or perfusion 
changes with neurological status.  
  
Security 
The study principal investigator will keep all information obtained from the study in a locked filling 
cabinet and password protected database.  A study number will replace control and subject names 
and any PHI will be removed. 
 
 
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