A Magnetic Resonance Approach to Neurodegeneration
by
Yin-Ching Iris Chen
B.S. Electrical Engineering
National Taiwan University, Taipei, Taiwan (1989)
M.S. Biomedical Engineering
National Yang-Ming Medical College, Taipei, Taiwan (1991)
SUBMITTED TO THE HARVARD-MIT DIVISION OF HEALTH SCIENCES AND
TECHNOLOGY
AND THE DEPARTMENT OF NUCLEAR ENGINEERING
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF
Doctor of Philosophy
in
Radiological Sciences
at the
Massachusetts Institute of Technology
May, 1997
© 1996 Yin-Ching Iris Chen
All rights reserved
The author hereby grants to MIT permission to reproduce and to distribute publicly paper and electronic
copies of this thesis document in whole or in part.
Signature of Author ............................ .......... ;.-.... /. ...... ..
Harvard-MIT Div'on of Health Sciens and Technology, and
Department of Nyclear Engineering, May, 1997
Certified
A pproved
by
by
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............................................
by
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* ** * * * .
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Professor David Cory
Reader
.Professor
,,
A ccepted
........................
Srrofessor Bruce G. Jenkins
Thesis Supervisor
..............................
~rlflRu1IC·PR Rnsen
ider
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. .......
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M. Flint Beal
Reader
-.. . ....................................
Srofessor Jeffrey P. Friedberg
/ hair an, Departmental Committee on Graduate Students
JUL 10 19971 Si3nrv
1
ABSTRACT
A Magnetic Resonance Approach to Neurodegeneration
by
Yin-Ching Iris Chen
Submitted to the Harvard-MIT division of Health Sciences and Technology
and MIT Department of Nuclear Engineering
on May, 1997 in partial fulfillment of the requirements for the
degree of Doctor of Philosophy in Radiological Sciences
The primary objectives of this thesis were to investigate and verify the
utility of magnetic resonance techniques to probe neurodegenerative
processes. We focused primarily on Parkinson's disease (PD). We
demonstrated how various factors could alter the accuracy in estimating the
absolute metabolite concentration by using computer simulation. We found
that it would be difficult to obtain accurate relaxation times T, and T 2 due
to the problems of spectral overlap , macromolecular contamination and Jcoupling effects. In estimating the metabolite concentration, we found that
the errors propagated from the T 2 errors are more critical than the T,
errors. In human study, we found that PD patients' striatal lactate level
was elevated 59% than the normal controls (p < 0.004). We also found a
significant lactate asymmetry in the 2 striata, which is significant higher in
the PD group (64.51% increase, p < 0.05) in the "big side". However, we
did not have similar finding in the animals of PD model. However, the
neuronal marker N-acetylaspartate (NAA) in these animals showed a
significant decrease in the striatum ipsilateral to the lesioning (9.61%, p <
0.0003). To assess the neuronal activity by using pharmacological MRI
(phMRI), we used D-amphetamine and CFT as our specific dopaminergic
ligands. We have shown that 1) the BOLD signal change delineated to the
blood pCO z and global blood pressure changes, 2) phMRI response is
regionally specific to those brain areas with high dopaminergic
innervation, 3) phMRI response correlates the "C-CFT binding via PET,
4) phMRI time course correlates the dopamine release assessed by
microdialysis. We tested the phMRI technique on 3 groups of animals. We
showed the phMRI responses were symmetric in the normal control rats,
unilateral ablated in the unilateral dopaminergic denervated striatum, and
restored right at the graft site in the dopaminergic cell transplanted rats.
In conclusion, the data presented in this thesis demonstrate the ability of
using MR technique to explore the neurodegenerative processes and could
elucidate treatment strategies and benefit the patients suffering from
neurodegenerative diseases.
Thesis Supervisor: Bruce Jenkins, Ph.D.; Department of Radiology, Massachusetts
general Hospital, Harvard Medical School.
Acknowledgment
I would like to express my sincere gratitude to Dr. Bruce Jenkins. In
the last five years, he has been a wise and inspirative mentor and
has become a true friend. As been a foreign student in this country
with both language and culture barrier, I thank his great patience
and support to guide and assist me through the tough graduate
school journey.
I would also like to deliver my hearty gratitude to many of the
scientists for their assistance in my work in addition to many
I would like to thank Rick Matthews for
memorable experiences.
neurochemical
and
neurosurgical
knowledgeable
providing
information in animal model of rats. Without him, lots of the animal
work involved in this thesis could not possibly be done. I also like to
deliver my sincere appreciation to Dr. John Keltner. John has been a
great friend who has been encouraging me to explore new scientific
frontiers and sharing me the most up to date scientific information
without a little bit of selfishness. I also like to deliver my thanks to
Dr. Wendy Galpern and Dr. Anna-Liisa Brownell for their excellent
expertise in animal neuro-transplantation and positron tomographic
imaging.
I am also grateful to Dr. Bruce Rosen for 6 years of
excellent education and mentorship within the MGH NMR laboratory,
Harvard-MIT Division of Health Sciences and Technology, as well as
in the Nuclear Engineering Department.
I would like to express my great appreciation to my friends and
family. My deepest gratitude goes to Dr. Beverly Yu. Without her, I
won't be able to come to study at the great school MIT and won't be
able to come to the dreamland laboratory in MGH. She was not only
a great mentor for my master thesis, she also provides a family like
hearty and dearing support.
I thank my parents, most of all, who have provides me with unfailing
support through this entire experience.
Dedication
I dedicate
this
thesis to my
loving parents
Chun-Yu Chen & Mei-Chun Lin
for their
encouragement and wisdom to guide
the travails of my young life.
I also dedicate this
thesis to
Wei-Chun
my beloved
Chang
for his hearty support
in the past, now, and future.
me
through
husband
TABLE OF CONTENTS
CHAPTER 1 BACKGROUND -- WHY MR COULD BE USEFUL FOR
.....................................
STUDY OF NEURODEGENERATION
11
REVIEW OF THE BASAL GANGLIA SYSTEM ................................................................................ 12
12
The neuronal netw ork ...........................................................................................................
.........
The basal ganglialnetwork......................................
................. 13
POSSIBLE HYPOTHESES INVOLVED IN BASAL GANGLIA DEGENERATION ............................................
18
Mitochondrialenergy impairment............................................ .....................................
18
Free Radical .......................................................................................................................
20
Iron accumulation................. ................................................................
21
WHAT MR CAN DO FOR THE STUDY OF NEURODEGENERATION ....................................................... 22
Is 'H Magnetic resonancespectroscopy (MRS) useful to investigate the mitochondrialenergy stress
theory?.......... ...................................................................................................................
23
What canfunctional MR technique do to investigate the neurotransmissionsystem? ...................
25
27
REFERENCES ..........................................................................................................................
CHAPTER 2 ERROR ANALYSIS OF THE CEREBRAL METABOLITE
CONCENTRATIONS IN
..................
1H
MAGNETIC RESONANCE SPECTROSCOPY
................................. ..
INTRODUCTION ..................................
33
.............................
................................................................................ 33
METHOD TO ESTIMATE THE CEREBRAL METABOLITE CONCENTRATIONS FROM 'H MR SPECTROSCOPY...
SPECTRAL QUALITY ...........................................................................................
Spectral Overlap..................................
..............
.
............
.....
38
....................................... 38
Signal to Noise Ratio (SNR)............................................................................................
PROBLEMS WITH RELAXATION RATE MEASUREMENTS ...................................................................
.............
40
41
41
Methods to measure the relaxation times Ti and T2 ......................................
M ethods ..........................................................................
35
........ .. 44
Comp uter Sim ulation .......................................................................................................... 44
Phantom and in vivo spectra..................................................................
.............
..... 48
RESULTS AND DISCUSSIONS .....................................................................................................
Spectral sim ulation.........................................................
49
49
T2 measurement errors ............................................................................................... 58
Absolute concentration measurement errors .................................................................
........ 63
.......
...
...... ........................................ 67
REFERENCES ..............................................
...
............................................. 69
..
CONCLUSIONS .................................
CHAPTER 3 1H MAGNETIC RESONANCE SPECTROSCOPIC STUDIES
.. 72
IN PARKINSON'S DISEASE PATIENTS ............................
..... 72
INTRODUCTION AND BACKGROUND ............................................................................
.....................
............
M ETHODS ..................................................................
77
.................
............................
.
.. ...
.. .
RESULTS ..................................................................
78
Average resultfrom the individualspectra ................................................................................
Asymmetric of the lactate levels in the two striataof the subject...................
....................... 80
84
A spartateElevation..............................................................................................................
D ISCUSSION......
75
................................................................................................................. 87
Morphology and SNR considerations...............................
.................................... 87
TherapeuticImplication................................................................................................... 90
A sym metric Lactate levels .............................................................................
CONCLUSIONS ...........................................
................. 91
........................................................................ 92
REFERENCES ........................................................................................................................... 92
CHAPTER 4 AN ANIMAL MODEL OF PARKINSON'S DISEASE USING
ANATOMICAL
--6-HYDROXYDOPAMINE LESIONS IN RATS
AND METABOLITE MAGNETIC RESONANCE IMAGING
97
.........................................................
STUDIES ............
..... 97
INTRODUCTION AND BACKGROUND .................................................................................
M ETHO DS ...........
.........................
.....................
............................................................... 99
...................... 99
Animals .........................................................
MR Studies...................................................................................................
.................... 100
............. 103
DataAnalysis..........................................................................
R ESULTS:.............................................................................................................................
103
..................
03
M etabolite images ..............................................................................................................
106
Water images.................
..................................................
D ISCUSSION .................... .....................
CONCLUSIONS ..................................
.............................................................................. 108
...................................
................................................
APPENDIX-BEHAVIORAL TEST....................................
............................................
REFERENCE ..............................................................................................................
111
112
.... 113
CHAPTER 5 PHARMACOLOGICAL MRI (phMRI)....................119
....... .
INTRODUCTION ........................
M ETH O DS .............................
... .......................
.....................
Animal preparation..........
.........................
.. ...........
119
.............................
.. ..........................................
122
................
122
...............................................................
MR M easurements ..................................................................................
...................... 122
124
PET M easurements .........................................................................................................
Microdialysis.................
Data Analysis
....................................................
........
....................................
125
..................
125
.............
127
RESULTS ................................................................................................................................
.. 144
D ISCUSSION ..............................................................................................................
C ONCLUSIONS ..........................................................................
........................................
148
RE FERENCES .........................................................................................................................
CHAPTER 6 THESIS SUMMARY
148
AND FUTURE WORK...............152
1
TECHNICAL CONCERNS IN USING H-MRS TO EXPLORE THE STRIATAL METABOLITES ........................
154
1H-MRS IN PD PATIENTS AND ANIMAL MODEL OF PD................................................................156
VALIDATION OF USING PHARMACOLOGICAL MRI (phMRI) TO ASSESS THE DOPAMINERGIC NEURONAL
ACTIVITY ..............................................................................................................................
C ONCLUSIONS ..........................................
........................................................................
REFERENCES..................................................................................................................
160
163
...... 164
LIST OF TABLES
Table 1-1
Common 'H-MRS detectable neurochemicals ....................... 24
Table 2-1
Common 'H-MRS detectable neurochemicals ..................
. 37
Table 2-2 List of major spectral overlap groups in 'H-MRS ................... 44
46
Table 2-3
Spectral elements used in Lorentzian line simulation ................
Table 3-1
Clinical characteristics of the PD patients in our study .............. 77
Table 3-2 Cerebral metabolite levels measured from PD patients and controls ... 78
Table 3-3 Correlation of the PD lac/NAA ratio with clinical parameters ....... 83
Table 3-4 Cerebral metabolite levels: big versus small groups ..................
83
LIST OF FIGURES
Fig 1-1
Basal ganglia circuit I ................................................
15
Fig 1-2
Basal ganglia circuit II ...........................................................
18
Fig 2-1
Computer synthesized spectra, AR2* = 10Hz ..................................
50
Fig 2-2
Computer synthesized spectra, AR2* = 50Hz .................................
53
Fig 2-3
NAA signal intensity as a function of echo time TE ............................ 60
Fig 2-4 Creatine and choline signal intensity as a function of echo time TE ....... 61
Fig 2-5
Stack spectra with different TE: phantom study ............................
62
Fig 2-6
Stack spectra with different TE: human study ................................
62
65
Fig 2-7 NAA concentration error as function of errors in TI and T2 .......... .
66
Fig 2-8
NAA concentration error as function of TR ..................................
Fig 2-9
In vivo
Fig 3-1
Striatal spectra from PD patients and normal controls ......................
79
Fig 3-2 Distribution of the individual Lad/NAA in PD patients and controls ......
81
Asymmetric lactate spectra ......................................................
82
Fig 3-3
Fig 3-4
Lac/NAA ratio: big versus small groups .....................................
Fig 3-5
Spectra with elevated aspartate peaks .....................................
Fig 3-6
Criterion to screen striatal spectra ...................................
Fig 4-1
68
spectra from 1.5T and 4.7T ........................................
84
. 86
90
T2 and diffusion weighted images in 6-OHDA lesioned rats ................ 105
Fig 4-2
rCBV and rCBF maps in 6-OHDA lesioned rats .............................
105
Fig 4-3
NAA image from 6-OHDA lesioned rats .....................................
107
Fig 5-1
phMRI responses versus blood pressure and pCO2 changes .............
128
Fig 5-2
Regional specificity in phMRI .....................................
129
Fig 5-3
Partial volume averaging in the parietal cortex ..............................
130
Fig 5-4
Blood pressure and pCO2 changes in all animals..........................
132
Fig 5-5
phMRI time courses with microdialysis data ...................................
133
Fig 5-6
phMRI activity: good to bad activation maps ...............................
135
Fig 5-7
phMRI time courses in the unilaterally lesioned rats ......................
136
Fig 5-8 rCBV and rCBF maps in the unilaterally lesioned rats .....................
137
Behavior versus phMRI and dopamine release .................................
138
Fig 5-9
Fig 5-10 phMRI and PET images .....................................
Fig 5-11 Asymmetry in lesioned only and neuron transplanted rats .................
Fig 5-12 Correlations: behavior versus PET and phMRI ................................
141
142
143
useful
Why
---
Background
Chapter 1
of
study
for
could
MR be
neurodegeneration?
In this thesis, we demonstrate the use of magnetic resonance (MR) as
diseases of the basal ganglia.
a tool to investigate neurodegenerative
In
order
answer
to
resonance
is
a
neurodegenerative
mechanisms
the
useful
of whether
question
we
processes,
of neurodegeneration
investigation
the
for
tool
or not
by
start
defining
magnetic
of
the
possible
and then explain MR techniques
The overall outline is:
may be employed to verify the hypotheses.
* review of the basal ganglia system,
* review
of hypotheses
concerning
the
mechanisms
of basal
ganglia degeneration,
* demonstrations
of MR to investigate
process.
11
the
neurodegenerative
Review of the basal ganglia system:
and the neural
anatomy
neuronal
ganglia function.
mechanisms
the
ganglia, including
of the basal
This section is a brief review
basal
in the
involved
circuits
In addition, this section will discuss the possible
in disorders
involved
of the
basal
such
ganglia
as
Parkinson's disease (PD) and Huntington's disease (HD).
The
The
neuronal
network:
question of how the brain works has. never been fully answered.
However, scientists have found their ways to explore
mechanisms of brain function.
brain.
The brain is comprised of hundreds of
Groups of neurons
billions of neurons.
the possible
compose suborgans
of the
How the individual neurons talk to each other has never been
easily answered.
rely on stereotyped
In short, neurons
signals and chemical signals to communicate.
neurons
play
and
integrate
out the
electrical
The interconnections of
Two types
functions.
of
membrane potential signals are used by neurons: localized potentials
The localized potentials are only effective in a
and action potentials.
very short
and the neurons
distance
potentials to transmit
bridge for
have to rely on the action
signals over long distances (via axons).
individual neurons to talk to each other is primarily
neurotransmitters
and their corresponding receptors.
the action potentials at the axonal terminals
neurotransmitters from the presynaptic neurons.
neurotransmitters to their corresponding
electrical
Different
on
potentials
types
of
neurons
via
The arrival of
the release
of
The binding of the
receptors can modulate the
postsynaptic
the
causes
The
possess
neuronal
different
membrane.
kinds
of
neurotransmitters and receptors, which may potentiate or inhibit the
12
action
of the
formation
neurons
postsynaptic
on the
potentials
(excitatory post synaptic potential (EPSP) or inhibitory post synaptic
potential
Based
(IPSP)).
and inhibitory
modulated
and transmitted.
ganglia
The basal
network system.
signals can be
neuronal
complicated
of this
is a submodule
Although the complete mechanism of how the basal
ganglia function is yet to be determined,
mapped
the
network:
ganglia
basal
between
balance
the neuronal
interactions,
excitatory
The
precise
on the
out some
neurotransmission
scientists have successfully
Based
routes.
on these
neurotransmission routes, numerous hypotheses have been proposed
to reveal the role basal ganglia may be playing
ganglia may
The basal
act as a self-regulated
in brain
function.
current-regulation
center to modulate the cortical activities. Further elaboration
of this
current regulation theory (described below) may be applied to help
solve the etiologic mysteries
such as Parkinson's
disorders,
(HD), hemiballismus,
of some of the basal ganglia relative
disease
athetosis, etc.
(PD) , Huntington's
disease
Let's take a closer look of the
basal ganglia.
Terminology of basal ganglia subunits:
The traditional term of "basal ganglia" refers to the deep gray matter
buried inside the cerebrum: caudate nucleus (CA), putamen,
(internal
/ external) globus pallidus (GPi/GPe), subthalamic nucleus (STN), and
nigra (SN).
substantia
Because the caudate
nucleus
and putamen
share the same embryological origin, these two organs are generally
referred
as the neostriatum
accumbens,
or striatum.
which is the fusion part between
13
However,
caudate
the
nucleus
nucleus and
putamen, is generally referred to as part of the limbic system but not
The putamen
striatum.
and globus pallidus comprise the lenticular
nucleus because of their lens like shapes.
The combination
of the
lenticular nucleus and the neostriatum is called the corpus striatum.
Basal ganglia circuit theory:
The
basically
striata
C-shaped
form
courses
along
the
lateral
ventricles and receive a broad range of signals from various cortical
Although the striatum is relatively small in volume compared
areas.
to the cerebral
cortex, the topographical
innervation
pattern
from
and to the cortical areas has been mapped out by immunohistologic
afferent neurotransmitter
techniques. Generally, the primary
striatum
is glutamate.
input primarily
its
have
major
The caudate nucleus receives
from the prefrontal
role
primarily receives the glutamatergic
glutamatergic
cortex and thus is thought to
functions;
in cognitive
of the
while
the
putamen
input from the somatosensory
and motor cortex and thus its involvement
in motor functions. The
excitatory cortico-striatal glutamatergic neurons set the basic tone of
the striatal neuronal firing rate. The striatum then serves as a relay
or
modulation
glutamatergic
neurotransmitters,
suborgans
reach
center
which
can
input
signal
by
or
interactions
modulate
with
the
other
such as dopamine, serotonin, etc., through several
of basal ganglia.
thalamus
integrate
The signals from basal ganglia finally
and are relayed
back to the cerebral
cortex
via
glutamate.
In order to understand
how the basal ganglia are involved in motor
activity, we may simplify the complicated
cortical route as shown in figure
14
cortical-striatal-thalamic-
1-1. As shown in the figure, both
and STN receive
the striatum
excitatory
input from
glutamatergic
cortex and they also share the GPi as the main output node to the
(GABA, inhibitory)
thalamus
cortex (glutamate, excitatory).
to inhibit
fast latency
which then relays the signal back to
The cortical-STN-GPi route is of short
unwanted
motions
and thus
The cortical-striatal-pallidal-thalamic
posture.
latency with a tendency
to inhibit the activity
maintain
the
is of
long
route
in GPi and thus to
disinhibit the desired motor activity. [1]
The two parallel
Figure 1-1
globus pallidus.
disynaptic
from
cortex to
The solid arrow indicates an excitatory pathway and
pathway.
GPe: globus pallidus
SNr: substantia
nigra pars reticulata,
the open arrow indicates an inhibitory
external,
pathways
GPi: globus pallidus internal,
STN: subthalamic nucleus.
The long latency
pathways:
segment
circuit
can be further
divided
into
2 parallel
the direct versus the indirect pathway ( figure 1-2).
has
its
particular
neurotransmitters
such
as
Each
GABA,
substance P (SP), enkephalin, etc., as specified in figure 1-2 and they
15
are
inhibitory
mainly
layout, the direct pathway
neuropathway
to
According
substances.
kind
this
is essentially
of
the positive
feedback route while the indirect pathway is the negative feedback
on the
Based
route.
while
pathway
hypokinetic
theory,
can be the result of an overactive
disorders, such as Parkinsonism
'indirect'
circuit
ganglial
basal
disorders,
hyperkinetic
like Huntington's
'indirect'
disease and hemiballismus, could be due to an underactive
pathway. [2,3,4,5] This simplified circuit theory provides a plausible
regarding
movement
therapeutic
strategies.
mechanism
valuable
disorders
also
and
has
Subthalamotomy
(VA/VL) have been performed
been
Pallidotomy
performed in PD patients to improve parkinsonism. [6,7]
and thalamotomy
provides
in PD patients
to
reduce some of the side effects caused by the dopamine replacement
therapy
( "wearing-off" and "on-off" effects) and the occurrence
of
dyskinesias caused by levodopa.
However, some of the clinical findings from the stereotaxic
in Parkinson's disease have contradicted this theory.
thalamotomy,
For example,
a
which might be expected to increase motor disability
according to the circuit theory, does not worsen movement
trials. [8]
surgery
in clinical
This may only suggest that the basal ganglia function is far
more complicated
then the simple circuit theory presented and the
etiology of the neuronal
degeneration
in the striatum
is far from
understood.
The nigro-striatal
role
in
neurons
dopaminergic
modulation
of the
main
seem to play an important
afferent
glutamatergic
signal.
Dopamine can either reduce the presynaptic neuronal activity (via D2
autorecepters),
enhance
the postsynaptic
16
neuronal
activity (via Dl
or inhibit the postsynaptic
receptors, mainly on the direct pathway),
neuronal activity (via D2 receptors, mainly on the indirect pathway).
The pathologic finding in idiopathic Parkinsonism
tremendous
compacta.
amount
of cell
The evidence
loss
in
the
from
is that there is a
substantia
the
postmortem
pars
nigra
studies
on
the
patients with idiopathic Parkinson's disease showed that the onset of
the parkinsonism did not show up approximately to 80-90% neuronal
loss in the SNc. [9,10,11]
An animal model of parkinsonism
uses the selective dopaminergic
OHDA) to interrupt
also demonstrated
neurotoxin
cells die and how the insufficient
striatum
Several
causes the dysfunction
hypotheses
dopamine
have been proposed
some of the most popular hypotheses.
17
concentration
of the striatum
these neuronal degeneration processes.
pathway
(6-
in rats
The reasons why the nigral
[12]
similar results.
6-hydroxydopamine
dopaminergic
the nigro-striatal
which
are
in an attempt
in the
still unclear.
to explain
The next section will discuss
Figure
1-2
pathways.
indicates
Basal ganglia motor circuit
-- direct
versus
indirect
Solid arrow indicates excitatory pathway and open arrow
inhibitory
pathway
The direct
pathway.
is a positive
feedback loop and the indirect pathway is a negative feedback loop.
DA: dopamine, Enk: enkephaline, Glu: glutamate, GPe: globus pallidus external,
GPi: globus
pallidus
SNc: substantia
internal,
substantia nigra pars reticulata, STN: subthalamus
nigra
pars
compacta,
SNr:
nucleus, SP: substance P.
Possible etiologic hypotheses of basal ganglia depeneration
Mitochondrial
energy
This hypothesis
postulates
impairment;
that
impairment
of the
mitochondrial
electron transport chain either directly or indirectly causes the cell
The evidence for this was discovered
death in the substantia nigra.
accidentally in humans who had injected the street drug 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine
18
(MPTP),
which was a contaminant
The drug abusers who received MPTP
of synthetic heroin analogue.
that
on MPTP discovered
studies
Further
Parkinson's
disease.
this compound
has the
idiopathic
resembling
symptoms
developed
potential to destroy the dopaminergic neurons in the substantia nigra
methyl-4-phenylpyridinium
transport
and an increase
in the conversion
expected and were observed
can
mitochondrial
in the
a decrease
Consequently,
chain.
MPP'
ion (MPP') is.
I (NADH dehydrogenase)
complex
animal
but its metabolite
MPTP is not neurotoxic
Actually,
species.
other
and some
primates,
pars compacta (SNc) in human,
1-
inhibit
electron
in the NAD+/NADH ratio
to lactate are
rate of pyruvate
in cell culture studies
by Vyas el. al.
[13]
The key enzyme required to oxidize MPTP is the monoamine oxidase
subtype
B (MAO-B), which is located on the astrocytes.
substance MPTP +, and then
oxidized by MAO-B to the intermediate
subsequently
oxidized
This
to MPP'.
MPTP is
metabolite
MPP + is then
released from the glial cell and freely diffuses into the extracellular
space.
Since MPP + is a substrate of the dopamine reuptake protein, it
to the substantia
can be transferred
reuptake
MPP ÷
pathway.
mitochondrial
matrix
is
finally
the dopamine
inside
accumulated
to inhibit the action of complex
causes the nigral cells to die. [14,15]
findings, researchers
nigra through
have proposed
the
I and thus
Inspired by the MPTP/MPP +
that the neuronal degeneration
in idiopathic parkinsonism may be caused by some environmental
or
endogenous toxins which may induce similar damage as MPP + does in
the
mitochondrial
electric
transport
chain
through the MAO-B/dopamine reuptake pathway.
involved in idiopathic parkinsonism,
19
in the
SNc, possibly
If MAO-B is really
using MAO-B inhibitors may be
able to postpone the neuronal degenerative process. Clinical trials to
treat
PD patients
selective
L-dopa
long-lasting
and
hypothesis
the
may
parkinsonism
be
with
conjugating
deprenyl,
the
to
56%
up
showed
inhibitor,
MAO-B
score. [16,17] This clinical evidence
in the disability
improvement
supported
with
that
the
to
related
of
etiology
stress.
energy
mitochondrial
However, other studies showed little effect.
idiopathic
the
The latter finding may
be more consistent with the observation that by the time someone is
with PD, 80 - 90% of the nigral dopamine
diagnosed
are
neurons
already dead.
Free
Radicals:
section, MPP'
As mentioned in the mitochondrial energy impairment
the activity of NADH dehydrogenase
will interrupt
transport chain.
However, some of the researchers
in the electron
suggest that the
mechanism of toxicity of MPP + is not to cause the interruption
of the
electron transport but to generate oxygen radicals (superoxide) while
the electron transport is blocked at the stage of complex I. This free
radical theory
is supported
by the evidence
of reduced
levels
of
glutathione and other antioxdants in substantia nigra and other brain
areas
after
provided
by
studies
oxidants can provide
MPP + toxicity. [20,21]
no detectable
mismatch
[18,19]
the MPTP neurotoxicity.
increase
between
showing
free
radical
neuroprotection
Further
evidence
scavengers
and
in animal
is
anti-
model of MPTP /
However, further studies showed that there is
in lipid peroxidation
the
degree
20
of
[22] and
inhibition
in
there
the
is a
NADH
and
dehydrogenate
[23]
formation.
superoxide
of
rate
the
Nonetheless, all of the evidence suggests that free radical formation
cause of neurotoxin
may not be the primary
the
by
done
damage
the
accelerate
of MPP' but it ma y
mitochondrial
of
cessation
respiration.
accumulation:
Iron
an abnormal
reported
important
and
increase
neuronal
enzymatic
dysfunction.
catalysis
of the
iron
Therefore,
the
[24] and the disturbance
development
may lead to neuronal
homeostasis
Iron is an
in iron concentration.
trace element for electron transport,
have
PD patients
of the
brains
of post-mortem
studies
Several
finding of high nigral iron concentrations in PD patients may suggest
of PD. Several groups have
that iron is involved in the pathogenesis
suggested
that iron may
free radical
prompt
and
formation
also
catalyze lipid peroxidation, which may subsequently lead to neuronal
death.
However, from post-mortem
studies with human brains, the
abnormal nigral iron levels were only found in the severely affected
but
PD patients
histochemical
model
affected
not in mildly
And
[25]
studies in rats with 6-OHDA lesions, a typical animal
of PD which
is able
to induce
SNc/VTA
There
areas.
contralateral side. [26]
neuronal
increase
SNc/VTA area, showed a substantial
ipsilateral
PD patients.
were
degeneration
in Fe staining
no
changes
in
in the
in
the
These data suggested that the high iron level
in SNc may be a secondary effect of the neurodegenerative
process.
Although up to now how and why the iron accumulates in the SNc is
still unclear, Oestreicher
proposed
3 possible mechanisms:
1) iron
may be associated with the infiltrating reactive glial cells; 2) loss of
21
the dopaminergic neurons in the SNc may cause disregulation
of the
nigral iron homeostasis; 3) 6-OHDA lesions lead to the dysfunction of
the
blood-brain-barrier.
What MR can do for the study of neurodepeneration
As we discussed in the previous
hypotheses
section, different
have
been proposed in an attempt to explain the neuronal degeneration
PD.
in
Among the 3 most popular hypotheses, we found that the energy
impairment theory may be the most plausible one in that it has the
evidence
appropriate
will search
for
in PD patients
by
The first part of this thesis
most direct evidence.
to support
this hypothesis
using magnetic resonance spectroscopy (MRS) to investigate cerebral
metabolites such as the energy impairment marker of lactate and the
marker
neuronal
In addition
of N-acetylaspartate.
to the patient
group, a well characterized animal model for PD, the specific selective
lesioning in the nigro-striatal
dopaminergic neurons by the toxin 6-
(6-OHDA), provides us a further
hydroxy-dopamine
opportunity
investigate the hypothesis that insufficient dopaminergic
in the
striatum
accumulation
changes.
can induce
theory
striatal
neuronal
can also be investigated
stress.
to
innervation
The iron
by monitoring
AR2*
We therefore propose to use MR as a tool to investigate the
question of whether or not in these 6-OHDA lesioned animals there is
any disturbance of the striatal metabolite levels, or any change in the
hemodynamic properties.
using functional
MRI (fMRI) to observe
transmitter-receptor
response.
Furthermore, we will test the possibility of
and
activity
the
the coupling between
corresponding
We will propose to use pharmacological
22
the
neuronal
stimulation
to
make this neuronal coupling visible and thus we will propose to call
this novel technique as pharmacological MRI (phMRI).
Before we can go further in to the experimental
set up, we have to
answer several questions:
Is 'H
Magnetic
investigate
the
The individual
experience
resonance
spectroscopy
mitochondrial
'H nucleus
under
a slightly different
momentum,
a steady
magnetic
called chemical
nuclei of different molecular structures
useful
stress
theory?
magnetic
to
field
will
field due to the shielding
electrons.
effect set up by the surrounding
magnetic
energy
(MRS)
This perturbation
shift, enables
of the
us to detect
via MR spectroscopy.
There
are a number of cerebral molecules which are 'H magnetic resonance
visible [27,28]. Table 1-1 shows a partial list and the properties
these
molecules.
Among
these
MR visible
prominent one is N-Acetylaspartate
molecules,
the
of
most
(NAA), which is believed to be
the marker of normal neurons because it is found almost exclusively
in neurons but not glial cells [29,30]. The spectral peak labeled Cr at
3.03 ppm is composed of both Cr and PCr. When energy impairment
occurs, one expects hydrolysis
intensity
same
would not change
for both molecules.
internal
standard
of PCr to Cr and the total resonance
assuming
Creatine
when absolute
molecules is necessary.
23
the relaxation
is thus
or relative
usually
times are the
taken
quantification
as an
of other
Compound
Structure
Relevance to Neurodegeneration
(Chemical Shifts and Approximate
Human Brain Concentration)
N-acetylaspartate (NAA)
2.023 ppm (CH 3)
2.52 ppm (3CH) 2.70 ppm q3'CH)
=8mM
Neuronal Marker
Higher Concentration in Grey Matter
Compared to White Matter
Excitatory neurotransmitter
-
Aspartate (asp)
2.69 ppm (1CH)
ppm (@'CH)
2.82 ppm
2.82
=2 mM
C--CH2-CH--C
IO
NHI
$'CH)
May possess excitotoxic potential
NH2
Also composed of other trimethylamines
CHa
Choline / Phosphocholine (CHO)
3.23ppm (CH) 3
- 1.6 mM
Creatine /Phosphocreatine (Cr/PCr)
3.95 ppm (CH)
HO-CH2 -CH
H3
2
I
May represent lipid breakdown products
and/or gliosis
CHa
C--CH2 -N-C-N
CHa NH
Hs
Peak represents sum of total creatine.
Supplies phosphate for conversion of
ADP to ATP in creatine kinase reaction.
=7.69mM
g-aminobutyric acid (GABA)
1.91 ppm (3CIH)
2.31 ppm (roCH) 3.02 ppm (CH22
S3 mM
NH-
H 2 -CH
2
H2
2
H
O
CH 2 H
4H
o-D-Glucose
3.2-4.1 ppm ([2-6]CH)
5.32 ppm ([1] CH)
=0-3 mM
cH
/1
H
IO
OH
OH
Inhibitory Neurotransmitter
Large fraction of basal ganglia neurons
are GABAergic
Fuel for the brain - most ND conditions
show hypo-metabolism of glucose
Detectable in 2D studies in vivo but
quantitation at low field strengths nearly
impossible except potentially peak at
5.32 ppm.
Major excitatory neurotransmitter in brain.
Potential Excitotoxin.
Quantifiable at high fields
Source of glutamate for neurotransmission
Quantitation problems as hard as for
glutamate.
OH
OH
Myo-inositol ([mI)
.54 ppm ([1,3]CHH
I
H
OHC
I
OH
Cl-I)
1.33 ppm (13
'0.5 mM
Table 1-1.
"
0
Precursor of inositol polyphosphate (IP 3 )
intracellular messengers.
I
H
H
Lactate
Possible osmoregulator.
I
I
<- CH 3
End product of anaerobic glycolysis
Barely detectable in normal brain.
Common neurochemicals observable in proton MRS
24
Table
1-1
factors
indicates
of the
that
cerebral
MRS would allow
status
following events relevant
during
us to evaluate
physiological
to neurodegeneration.
amount of NAA (2.023ppm), one can determine
the neurons
under
neurotoxic challenge.
acid amount (1.33
stress the neurons are experiencing.
serve
as an index
neuronal
to support
degeneration
the
energy
stimulation
may
be
of
the lactic
of energy
impairment
can
theory
in
Furthermore,
by
(Glx) level, one can investigate
the possibility that the vulnerability
dopaminergic
the survivability
the degree
earlier.
or
By monitoring the
This lactate measurement
as we mention
monitoring the glutamate/glutamine
challenge
By monitoring
ppm), one can investigate
many
of the striatal
caused
by
neurons
glutamate
after
toxicity.
Overall, although MRS possesses some technical difficulties related to
the
insensitivity
of MR in general,
spatial, temporal,
scope
of this
such as the limitation
and chemical resolutions,
text
to explain,
it still
which are
provides
us
in the
beyond
an
the
excellent
opportunity to investigate cerebral events at the molecular level.
In
this thesis, we will test the hypothesis that PD may be the result of
energy impairment
in the electron transport
chain. And we would
like to apply the MRS technique to PD patients to investigate whether
there is any abnormality in the cerebral lactate levels.
Furthermore,
we would like to investigate any other abnormality of the 1H spectra
possibly found in this PD patient group.
What can functional
neurotransmission
MR
technique do
to investigate
the
system?
Neuronal activity is coupled with changes in regional cerebral blood
flow (rCBF) [31], regional
cerebral
25
volume
(rCBV) [32,33], blood
oxygenation
[32,34], and metabolism
emission tomography
(PET) studies
change in oxygen consumption
Results of positron
[35,36].
indicate
there
during neuronal
is no or little
activity
[32,34,36].
Based on the rationale that changes in rCBF will be 2-4 times larger
than changes in rCBV [37], the venous oxygenation will increase from
base line level 30% to 75% due to neuronal
oxygen consumption
[32,34,36].
these
changes
hemodynamic
activity
with constant
that
Kwong et. al. demonstrated
are
detectable
using
by
magnetic
resonance techniques without exogenous contrast agents [38]. In the
baseline condition, deoxyhemoglobin,
a magnetic
field gradient
sets up
which is paramagnetic,
at the interface
of the vessels
and
surrounding tissues. This inhomogeneous field is manifested
effect
the magnetic
and will decrease
While in the activation
oxyhemoglobin
state, the increase
(fMRI).
of
of paramagnetic
The concept of detecting the oxygenation
as the BOLD effect
MR is also known
by using
investigate
intensity.
causes an increase in the MR signal intensity due
to a reduction in T2* effect.
Oxygenation
as a T2*
in the concentration
and a decrease in the concentration
deoxyhemoglobin
changes
signal
resonance
the
[39]
Level Dependent)
the brain
functions
has
been
widely
(Blood
applied
and is thus called functional
to
MRI
Although fMRI has been proved to be powerful to map out
neuronal systems, such as language[40,41], the visual system [42,43],
auditory
ability
system
to
[44], etc., the question
observe
neurotransmitter
the
in
vivo
of whether
neuronal
level is yet to be determined.
fMRI has the
activity
at
the
Spatial, temporal
resolution and the signal to noise ratio (SNR) are the main issue for
MR to go down to the in vivo
activation.
molecular level to follow the neuronal
However, since neuronal activity is coupled with rCBF and
26
rCBV, there
is a possibility
of investigating
activity by following the corresponding
the
neurotransmitter
this thesis, we propose to investigate the hemodynamic
the
pharmacological
application
system
neurotransmission
dopaminergic
We
stimulation.
of functional
to
propose
MRI as pharmacological
in
properties
of
response
to
name
this
new
MRI or phMRI.
With the aid of phMRI technique, we may monitor the remaining
the dopaminergic
in the elderly brain to evaluate
innervation
In
responses.
hemodynamic
of
the
possibility of progressive PD before any symptom shows up.
Based on the background
the ability
regarding
to use MR to study
basal ganglia degeneration
Parkinsonism,
and
in the following
chapters this thesis will discuss:
* Development
of
appropriate
techniques
MR
to
study
neurodegeneration
* Human studies of PD using MRS
* Animal studies of PD models
using MRS and hemodynamics
measurements.
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Chapter 2
Error Analysis
Concentrations in
Metabolite
Magnetic
of the Cerebral
Resonance
1H
Spectroscopy
Introduction
Over the past decade
has developed
metabolites
1H
Magnetic Resonance spectroscopy ( 1 H MRS)
tool for investigation
into a powerful
in vivo and in vitro. Comparisons
using
of cerebral
both relative
amplitudes among the metabolite peaks [1,2] as well as the absolute
metabolite concentrations
neurochemical
challenges.
[3,4,5] have been proposed to monitor the
changes during cerebral pathologies
However,
the
absolute
different
groups
are
metabolite
not always
or physiological
concentrations
consistent.
For
reported
from
instance,
the NAA concentration could be as low as 4 p4 mol/g [6] or
33
as high as 17 .t mol/g [8] and the creatine values have been reported
7.7 p. mol/g
between
measurement
or
relative
[6] and 10 pt mol/g
absolute
of an internal
existence
should
reference
constant
remain
signals
metabolite
during
pathophysiological conditions.
standards.
internal
standard
reliable.
standards
However,
and
Creatine [7,8] and unsuppressed water
used
as the
in the invariance
of the
peaks
spectral
uncertainty
have
investigators
such as MnC12-doped
comparison
[9] water
also has some difficulties,
signal amplitude
or trimethylsilylethanol
such as the distortion
This
beside the head.
concentration
Nonetheless,
standard
if the accurate
sample is placed
absolute
is achievable, it is highly desirable
of the
(BI field
due to the possible field inhomogeneity
and static field B0 ) when the external
less
external
using
proposed
(TSE) [10,11] with known T, and T2 values and concentrations.
approach
the
amount
physiological
makes the relative concentration
Several
for
it is
is
whose
peak
various
are two of the most popular
internal
estimate,
concentration
metabolite
whether
comparison,
metabolite
of an accurate
One criterion
[8].
metabolite
because it would
allow one to:
1) make inter-subject comparisons,
2) make
physiologic
parameter
estimations,
such
as
CMRglucose calculations.
In this chapter, we will discuss the possible factors which may alter
the
accuracy
of the metabolite
amounts
spectroscopy.
34
estimated
from
1H MR
Method to Estimate the Cerebral Metabolite Concentrations
from 1 H MR Spectroscopv:
different
In principle,
magnetic
different
chemical
groups
slightly
may experience
fields due to shielding
by electrons
and
thus
express themselves at different MR spectral positions called chemical
The chemical shift is invariant
shifts.
in ppm but is
as expressed
proportional to the magnetic field strength as expressed
in Hz. Like
fingerprints,
most of the chemical groups have their own chemical
shift values
and
are
represented
specific chemical shift locations (ppm or Hz).
at the
centered
as resonances
Depending upon the
scalar coupling effects among different chemical groups of the same
molecule, the lines may be split into doublet, triplet,
order
multiplicities.
observable
identified
cerebral
and
Over the past several
spectral
assigned
peaks
to their
from
or even higher
decades,
1H
corresponding
MRS
most of the
have
chemical
been
groups.
Table 2-1 is a list of most of the major cerebral metabolites
are
1H
MRS detectable.
which
From basic magnetic resonance spectroscopy
theory, the signal intensity of a singlet spectral peak from a spinecho experiment should follow the expression shown in equation 2-1:
SIAa = MoA(1- exp(-
TR
T1Aa
) )exp(-
TE
)
(2-1)
T2Aa
where
SIAa = signal intensity from the spectral peak 'a' of molecule A
MoA = proton density of molecule A
35
c NAa X [A]
(2-2)
NAa = number of protons in this spectral group 'a' of molecule
A,
[A]
= concentration of molecule A.
And
SIAa
SIBb
SI
MoA(1-exp(MoB(1-exp(-
TR
TAa
TR
) )exp(-
NoB[B](1- exp(-
where,
a and
b are
the
T2Aa
TR
T1Aa
TIBb
)
(2-3)
TE)
) )exp(-
T1Bb
NoA[A](1- exp(-
TE
T2Bb
) )exp(-
TE
)
T2Aa
(2-4)
)
) )exp(T2Bb
2 spectral
molecules A and B.
36
peaks
from
the
2 different
Compound
(Chemical Shifts and Approximate
Human Brain Concentration)
Structure
N-acetylaspartate (NAA)
2.023 ppm (CH)
2.70 ppm O'CH)
2.52 ppm(CH)
= 8mM
O0 C--
Relevance to Neurodegeneration
PO
Hz2
Neuronal Marker
NH
Higher Concentration in Grey Matter
Compared to White Matter
C=O
Excitatory neurotransmitter
Aspartate (asp)
2.69 ppm(~CH)
2.82 ppm (13'CH)
=2 mM
C-CH
2 -CH&G- "
0
H•
0
Choline / Phosphocholine (CHO)
3.23ppm (CH3
= 1.6 mM
H
Cratin3.03
/ Phosphocreatine (Cr/PCr)
O
CH 3
Also composed of other trimethylamines
H3
May represent lipid breakdown products
and/or gliosis
HH
C -- CH -N--C- NH 3
,H 3iH
3.95 ppm (CHO)
=7.6mM
g-aminobutyric acid (GABA)
1.91 ppm (1CH2 )
2.31 ppm (tcCH 2) 3.02 ppm (TCH 2)
S3 mM
May possess excitotoxic potential
NH -CH
2 -CH
2-CH 2-C
Peak represents sum of total creatine.
Supplies phosphate for conversion of
ADP to ATP in creatine kinase reaction.
O
20
CH 20H
a-D-Glucose
3.2-4.1 ppm ([2-6]CH)
5.32 ppm ([1] CH)
-0-3 mM
H
H
Glutamate (Glu)
2.06 ppm 'CH) 2.3.76 ppm (CH)
ppm
6-2.10
I
OH
0
Major excitatory neurotransmitter in brain.
Potential Excitotoxin.
Quantifiable at high fields
Nl 2
0-
Source of glutamate for neurotransmission
0
0
Quantitation problems as hard as for
glutamate.
C
H
N+H3
'CH) 3.76
Glutamine (Gln)
2.14 ppm q3CH2)
2.46 ppm (ICH2) 3.78 ppm (aCH)
-3mM
-H
-CH2
OC-CH 2 CH 2 CH -COH
Myo-inositol (ml)
3.54 ppm ([1,3]CH)
5 mM
Detectable in 2D studies in vivo but
quantitation at low field strengths nearly
impossible except potentially peak at
5.32 ppm.
OH
I
H
- 6 - 10 mM
Fuel for the brain - most ND conditions
show hypo-metabolism of glucose
H
I
OH
Inhibitory Neurotransmitter
Large fraction of basal ganglia neurons
are GABAergic
OH
Possible osmoregulator.
H
OH
L/ i
Precursor of inositol polyphosphate (IP3)
OH/ J
NH\H
H I
I
I
intracellular messengers.
H
OH
H
Lactate
1.33 ppm CH3)
-0.5 mM
OH
H
End product of anaerobic glycolysis
Barely detectable in normal brain.
Table 2-1 Common neurochemicals observable in proton MRS
37
In theory, if the molar concentration
the absolute concentration
of chemical A is known, then
of chemical B can be estimated
with the
knowledge of T, and T2 values of both of the spectral groups a and b:
(1- exp(-
TR
) )exp(-
TE
)
[B] = SIBb A]NAa
T1Aa
T2Aa
TE)
)exp()
(l-exp(NBb
SIAa
TlBb
T2Bb
studies,
spectral
For in vivo cerebral
(2-5)
the molar levels
of cellular
water and creatine are relatively stable and are usually taken as the
internal spectral standards, to map out the amounts of other cerebral
However,
metabolites.
measurements
feasible when
absolute
of
according
metabolite
equation
the
are
only
signal intensities
In the following sections,
discuss the possible errors which may propagate
concentration
(2-5),
concentrations
of the spectral
the measurements
and T I/ T 2 values are accurate.
to
we will
into the absolute
measurements.
Spectral quality:
Spectral
Overlap
The spectral peaks can be expressed as Lorentzian lines centered
their corresponding
chemical shifts (ppm or Hz).
at
The line width of
the Lorentzian line is proportional to the relaxation rate R2* (R2* =
1/ T2*, Av 1/2 = 1/HT 2*) which is composed of several factors:
R 2=
R2 + XR
n
2n
(2-6)
38
relaxation rate of this particular
where R2 is the intrinsic spin-spin
field and R2ns are some external
molecule in the magnetic
factors
which can accelerate the relaxation rate, such as the inhomogeneity
In the practical world, there are
of the magnetic field, flow rate, etc.
several
factors
which may
lead to magnetic
shim of the magnetic field caused by sudden
such as an imperfect
changes of the magnetic
susceptibility
two
compartments
different
tissue
brain-CSF or brain-air
boundary,
field inhomogeneity,
Xm close to the interface
(e.g.,
grey-white
sinus interface,
etc.).
of
matter
The bigger
the relaxation rate is, the broader the Lorentzian line width will be.
The typical line width of 1H MR cerebral spectra can as low as 2 Hz
However, since the chemical shift
in small single voxel experiments.
distance
(Hz) between
strength
of magnetic
2 chemical
field, the
1H
groups
is proportional
to the
spectra from a low field clinical
magnet (0.5 ~ 1.5 T) may not allow one to resolve one individual
spectral peak from the adjacent ones at a given broad spectral
line
Prior work using 2D COSY experiments has demonstrated
that
width.
there
are many
overlapping
1H chemical
shifts
resolved without the use of the 2D technique
composition may be even more complicated
peaks
are J-coupled
and split to various
undergo phase modulation
to
the
choice
The spectral
if some of the spectral
multiplicities
which can
We will
General speaking, one can reduce the
spectral line width with a better
care
[12].
as a function of echo time (TE).
discuss this phenomenon later.
with
which cannot be
shim of the magnetic
of the
imaging
voxel
field , and
cover
as
few
heterogeneous tissues as possible, and to keep the voxel as far away
39
such as sinuses with huge magnetic
from interfaces
susceptibility
differences as is possible.
Signal to Noise
Ratio (SNR)
The concentrations
of cerebral
are
metabolites
millimolar range while the cellular water/lipid
To detect these concentration
molar range.
bigger
Therefore,
than
water/lipid
signal
the
suppression
cerebral metabolite signals visible.
from
the
in
are in the
in the
differences
1H
such
huge
cerebral
metabolites.
has to be applied to make the
However, imperfect
suppression can still lead to some problems.
present
contents
in the
the signal from water or lipid will be thousands
MRS measurement,
of times
generally
quantities
water/lipid
As water and lipid are
compared
to
cerebral
the
metabolites, reduction of the signal intensity to only one tenth of the
original
water/lipid
amount
still leaves
As most of the receiver bits
much bigger than the metabolite signal.
would be used
to digitize the massive
would not be enough dynamic range
signals of cerebral
ratio
(SNR) of the
metabolite signals.
metabolites.
metabolites
Furthermore,
or lipid signal
the water
water/lipid
to digitize
signals,
there
the tiny spectral
In other words, the signal to noise
will be
too
small
as the shoulders
to
detect
the
of the water/lipid
peaks may extend over quite a wide range of chemical shift with
non-negligible
amplitude,
this may lead to problems
shift and phase distortion of the metabolite peaks.
SNR the
1H
analysis
will be and the more
MR spectrum
metabolite concentration
has, the more
measurements
40
is.
So, the lower the
complicated
questionable
the
like baseline
the spectral
accuracy
of the
Problems with relaxation rate measurements:
Methods
The
to
measure
the
relaxation
longitudinal
disturbed magnetization
value is traditionally
by a series
measured
several
the
fast
how
equilibrium.
The T,
recovery
of inversion
variable inversion recovery
waiting
time (T 2) is a function of echo
The spin-spin relaxation
through
Ti and T 2 ---
describes
will return to thermal
experiments
by series of spin-echo
time TE and can be measured
stepping
times
(TI)
time
stepping through
experiments
times (TI).
relaxation
TE values.
Equations
(2-7)
and
(2-8)
describe the mathematical expressions of these measurements:
SI = Mo(1 -2exp(-
TE
TR
TR
)
) )exp())(1 - exp(T1
TI
T2
(2-7)
= (TE2 - TE2)
In(SI1 - SI2)
(2-8)
However, there are a number
of possible problems
involved in the
relaxation time measurements:
Partial volume averages and heterogeneous environments:
Due to the limitation
through
resolutions,
plane
heterogeneous
resolution,
of spatial
structures
spectral
the
Different types
white matter(WM),
may
voxel
and may be contaminated
adjacent to but outside the voxel.
gray matter(GM),
for both in plane
and
cover
by structures
of tissues (e.g.,
and cerebrospinal
fluid (CSF))
have different metabolite compositions and the same metabolites
in
different types of tissues may possess different relaxation times (e.g.,
brain tissue H 2 0 with T 2 = 70ms and CSF with T2 = 2s).
[1l]
has
showed
a large
scatter
41
Kreis et. al.
in the in vivo relaxation
time
measurements partially due to the contamination of CSF, arterial and
venous blood, as well as other small sources.
by multiple tissue structures,
which contains or is contaminated
spectral
For a spectral
signal will be just the sum of
signals
voxel
the
all of the
from
structures:
SIA = ISIAn
n
f An(l- exp(-
=
n
TR
TE
)
) )exp(T2An
T1An
(2-9)
where,
SIA = signal intensity of metabolite A,
SIAn = signal intensity of metabolite A from structure n,
fAn = fraction of total proton intensity contributed from structure n,
T1An, T2 An = T 1 and T 2 of metabolite A from structure n.
As one can expect, the signal intensity will
mono-exponential
decay
and
the
T,
no longer follow a simple
and
T2
relaxation
time
measurements will be unreliable.
Spectral overlap and J-coupling effects:
As mentioned
earlier in this chapter, the conditions to resolve
are determined
by their chemical
shifts
and
two
line
spectral
peaks
widths.
Some of the cerebral metabolites have very close chemical
shifts and it becomes difficult resolve them from each other.
situation is especially severe at lower field strengths like 1.5T.
This
For
example, the spectral peak of the methyl group of NAA (2.023 ppm)
overlaps with the peaks of GABA (1.91 ppm), NAAG (2.05 ppm), and
glutamate
(2.06
ppm).
Other
than
42
the
simple
singlet
spectral
some of the overlapping
overlaps,
spectral phases
are modulated
complexity makes
peaks
are J-coupled
and their
by the echo time TE.
This kind of
difficult.
Considering
the spectral quantification
both the spectral overlap and the J-coupling effects, equation
(2-9)
can be rewritten as:
SI =
=
SIn
n
Mon( 1 - exp(-
TR
Tln
) )exp(-
TE
T2n
(2-10)
)TEJn
where,
SIn= signal intensity from metabolite
M on = proton density of metabolite N
T1 n , T2 n = TI and T 2 of metabolite N,
J
= J-coupling effect
=1
for singlet peak
= 1/2 cos(2t J/2 TE)
for each doublet peak
= 1/4 cos(2x J TE)
= 1/2
= others
for the 2 side peaks of triplet
for the middle peak of triplet
for higher multiplicity
Table 2-2 is a list of some of the spectral
spectral
overlap.
43
groups
with possible
ppm
multiplicity
Mo (mM)
NAA (CH3)
NAAG
Glutamine (P-CH 2)
2.023
2.05
2.1
s
8
s
m
0.79
6.8
Glutamate (P -CH2)
2.14
m
6.8
GABA (3 -CH2)
1.91
qu
3.61
PCr/Cr(CH 3)
3.05
s
7.6
GABA (y-CH 2)
3.02
t
3.61
Choline (CH 3 )
3.25
s
1.6
3.2
dd
5
2.3
t
2.3
Metabolite
Group I
Group II
Group II myo-Inositol
Taurine
Table 2-2. List of major spectral overlap groups in 'H-MRS.
how the spectral overlap
Here in this chapter, we will demonstrate
and the J-multiplicity
reduce
the
Furthermore,
measurements
may degrade
accuracy
in
T2
the spectral
relaxation
we will describe
how the
lead to errors
in absolute
quality
time
errors
and thus
measurements.
in relaxation
metabolite
time
concentration
estimates.
Methods:
Computer
Simulation
All of the computer simulations were performed
Math Works, Inc.) programs.
44
with MATLAB (the
Spectral simulation :
Human
1H
cerebral
MR spectra
were
computer
rates (Ri
and relaxation
values of molar concentrations
preassigned
with
simulated
and R2) with most of the major cerebral metabolites detectable from
The absolute metabolite
'H MRS as indicated in table 2-1.
were
set
to
multiplicities,
physiological
[13].
and coupling constants
obtained from the literature
spin-echo
levels
magnetic field strength,
shifts
(ppm),
peaks were
of those spectral
[14]. The MATLAB program assumes
and allows one to specify
is performed
experiment
Chemical
amounts
a
the
spectral resolution (via spectral sweep and
The field inhomogeneity
number of data points), and echo time (TE).
is reflected as an extra factor in R2* (Hz) as expressed in equation (26). This program assumes perfect water and lipid suppression (zero
from water or lipid) and weak J-coupling only.
signal contamination
[15,16].
was not included in this simulation
contamination
Macromolecular
with higher order J coupling, this
For the spectral peaks
MATLAB program
simplified
the complex
spectral
formation
using double doublet peaks (i.e., split a single spectral
by
peak to be
doublet peaks with J1 spectral distance (ppm); then split each peak
to be doublet
again by J2 spectral
distance
Table
(ppm)).
2-3
illustrates the spectral elements used in this simulation.
Amplitude
spectra
of both
metabolite
spectral
lines)
multi-metabolite
and
individual
(sum
of all of the
metabolite
separately and coded with different colors.
45
spectra
were
plotted
Mo (mM)
metabolite
multiplicit
ppm
# of rotons
J
11
2.01
2.7
2.52
1
c
c
N/A
3.7, 15.9
9.8, 15.9
3
1
1
creatine
choline
taurine
7.6
1.6
2.3
4.4
3.05
3.25
3.44
3.27
3.55
c
1
1
3
3
c
3.9, 9.8
N/A
N/A
6.7
6.7
2.7, 9.9
1
2
9
2
2
2
myo-inositol
5
3.65
2
9.8
2
NAA
1
9.8
1
9.2
1
7.3
7.3, 7.3
1
6.8
1
7.3, 7.3
C
2.35 y-CH
1
7.3
t
3.78 a- CH2
7.3,
7.3
1
c
2.14 P- CH 2
6.8
1
3.6, 7.3
c
2.46 y-CH
1
7.3
4
4.12
0.7
7.3
3
2
1.33
Spectral elements used in Lorentzian line simulation.
2
3
3
c
3.77
3.28
3.80 a- CH,
2.10 P- CH 2
glutamate
glutamine
lactate
Table 2-3
T2 measurement errors:
For a simple singlet spectral peak without any overlapping peaks, the
signal intensities
from spin-echo
experiments
should follow mono-
decays as a function of echo time (TE) (equation (2-1)).
exponential
However, due to the spectral overlap and J-coupling effects, the total
signal intensity is not only likely to be a multi-exponential
decay but
also to oscillate in a sinusoidal-like form along TE axis [17]. We used
MATLAB to simulate
oscillation
this multi-exponential
phenomenon.
We paid
particular
decay
attention
prominent metabolite spectral groups in the typical
spectrum:
and sinusoidal
1H MR cerebral
NAA(methyl group, 2.023 ppm), creatine(3.05
choline(3.25
ppm).
Metabolites
to three
ppm), and
with chemical shifts less than 0.1
ppm away from any of the 3 major spectral locations were classified
46
to be the same spectral group as the major metabolite (Table 2-2).
The
MATLAB
programs
ignore
any
chemical
between the major and the minor metabolites
shift
differences
(e.g. major = NAA and
minors = NAAG, GABA, Glx) in the same spectral group and the total
spectral signal intensity of the spectral group was calculated
by summing up all of the metabolite
group.
simply
signals in the same spectral
The pure T 2 decay curves (as a function of TE) of the major
metabolite as well as the composite T2 decay curve from all of the
metabolites
are synthesized
The T 2 decay
curves
according to equation
separately
were
then
presented
(2-10)
in a semi-logarithm
fashion (log(SI) versus TE) and a least-squares
fitting program was
applied to the composite
the "measured
data set to estimate
value of the major metabolite.
T 2"
The T 2 error was then computed as a
percent deviation of the "measured
T 2" from the "pure
T," of the
major metabolite.
Absolute concentration measurement errors:
The
absolute
concentration
of the
estimated according to equation
cerebral
(2-5).
metabolite
can
be
However, this measurement
involves a lot of other measurements such as T2 measurements of the
internal
standard
Statistically,
the propagation
concentration
[A]
metabolite
2
and
the
of errors
one
of the
to
quantified.
be
measured
absolute
Ub can be expressed as equation (2-11):
2
G[A]
Mo
M
[A]
MO s
GMo
2
+
(2-11)
MO)A
where,
47
GMoOJ2QGSI)2Q
+TE
-o
SI
-2
MO
2
GT2
S-2T2ýT2)
,TI
2TR
1- exp(
1
)
j
U2
(2-12)
( Tj
We, again, utilized MATLAB to simulate the error properties
absolute concentration
the
standard
estimation.
metabolite
of this
In this case, water was used as
to measure
the NAA concentration.
percent error of the NAA concentration
was estimated
The
according to
equation (2-11) with the four variables : errors of T, of water (Gtlw),
T z of water ('t2w),
Tl of NAA (GOtlNAA), T 2 of NAA (G't2NAA).
The
simulation fixed 3 of those 4 variables and varied the 4th variable in
the range of error between
-100% and 100% with an increment step
of 10% error in the relaxation time.
study
the
property
of the
In this fashion, one is able to
concentration
error
caused
by
this
particular relaxation time error.
Phantom
and
in
vivo
spectra
In order to verify the computer simulation results, especially for the
T z measurement
errors, phantom
spectra as well as in vivo human
cerebral spectra were obtained with the same parameter
used in computer simulations.
settings as
All of the spectroscopy experiments
were conducted on a 1.5 T imager (Signa, GE). In order to get single
voxel spectra, a PRESS sequence was used to acquire data instead of
using spin-echo sequence as was used in computer simulation.
Two
second repetition times (TR) were used in both human and phantom
studies
and the echo tinie was stepped
from 51 to 304 ms with
approximately 10 to 17 ms increments for total of 18 points.
48
Shorter
but were not possible using
echo times ( TE <40 ms) were desirable
the GE PRESS sequence at the time.
For the phantom
of agar gel mixed with NAA (10mM ), glutamate
study, a bottle
(9.7mM), glutamine
(3.7mM), and GABA (3.29 mM) was used to simulate
the cerebral
For the in vivo study, spectra were taken from
metabolite contents.
occipital cortex of healthy human volunteers.
Results and Discussions:
Spectral
simulation:
The spectra were computer simulated
specifically for the
magnetic
field strengths of 1.5, 4.7, and 9.4T (the major magnets used at NMR
center,
Massachusetts
General
Hospital,
To simplify
MA).
the T 2 of NAA to
complicated metabolite compositions, we assumed
The spectra were
be 450 ms and the other metabolites to be 400 ms.
synthesized
the
either with short echo times (TE = 68 ms), intermediate
echo times (TE = 136ms) or long echo times (TE= 272ms).
echo times to be multiple of 68 ms is essential
magnetizations with J-modulation of 7.3 Hz.
spectra with reasonable
the
individual
with
spectra)
'presumable'major
mixing
field settings.
spectrum
different
color
those
Figure 2-1 shows typical
spectra with relatively bad shims (6R2 = 50 Hz).
the top box shows
to refocus
magnetic field homogeneity
under the three main magnetic
To set the
(the
codes
(8R2 = 10 Hz)
Figure 2-2
shows
In each diagram,
summation
to
of the
highlight
the
metabolite peaks ( NAA, Cr, and Cho) and the
bottom box shows the 'real'
spectra from the individual metabolites.
49
__Summed
~_~ Spectrum
NAA=450mS; R2(dB)-11 0Hz
Bo =1'5T, TE =68ms; T2_
-3
-3.5
-2.5
-2
-1
-1.5
0
-0.5
(Al)
ppm
Individual Specta
NAA
a-Gm,
a-Glu
Cho
b,rGim,
•
br Glu
Cr
Ino
lac
I
.u
4
-3
-3.5
-2
5
-1.5
-2
-1
-0.5
ppm
(A2)
Summed Spectrum
o -1.5T,TE =13•ms; T2_NAA-450ýms; R2(dB)=1OHz
4
-3.5
-3
-2.5
-2
-1.5
-1
I
-0,5
ppm
(B1)
Individual Specta
NAA
a-OGIm,
Cho
a- Glu
4
-3
-3.5
b,r-Gm,
b,r-Glu
Cr
-2.5
-1.5
-2
-1
-0.5
0
(B2)
ppm
Smunmed Spectrmn
Bo =1 .5T, TE -272ms; T2_NAA-450ms; R2(d_8)-=10HZ
4
--
-.
-j3
-
---
-r
-
-. 5.
nr
ppm
I
5 -
-4
a-Glm.
Cho
a-Glu
Ino
,
-3.5
U
-U.5
--I
(C1)
Individual Specta
L
A
b,r - GIm,
br-Glu
Cr
-
-3
N
tc
-2.5
-2
-1.5
-1
-0.5
PPm
0
(C2)
Figure 2-1(a) Spectra simulation: BO = 1.5T, TE = 68,136,and 272ms
(A,B,C respectively), AR2 = 10Hz (relatively good shim).
Summed Spect
Bo =4.7T, TE =68ms; T2_NAA=450ms; R2(d_B)=1 0Hz
10
5
0
-3.5
-3.5
-3
-2.5
-2
-5
-3
-2.5
-2
-1.5
1
05
I
-0.5
-1
ppm
(Al)
ppm
(A2)
Individual Specta
ISummed Specrum
Bo -4.7T, TE =136ms; T2_NAA=450ms; R2(d_B)=10Hz
10
,,.i
.a
0
1
-3.5
-3
-2.5
-2
-1.5
-1
-0.5
0
(B 1)
ppm
Individual Specta
I
105
5
-4
-rGrn.
a-Gbu
-3.5
byr-G*r
.
Cho
b,r-Glu
-3
-2.5
-2
-1.5
-1
-0.5
ppm
0
(B2)
Summed Spe
6
4
2
TE =2 2ms; T2 _AA=450ms; R2(dB)5=10Hz
=47T,
-o
-
8
lkitL
-
0
-2
7
1
_'
1
-3
'
_ ?_
-2.5
1
-2
-1.5
-~
-0.5
0
ppm
(C1)
Individual Specta
n
4 -
a-G
Co
Cr
C'
I
.
br-
NAA
G
2- nolac
-2
-4
-3.5
-3
-2.5
-2
-1.5
-1
-0.5
0
ppm
(C2)
Figure 2-1(b) Spectra simulation: BO = 4.7T, TE = 68,136,and 272ms
(A,B,C respectively), AR2 = 10Hz (relatively good shim).
Summed Spect
Bo =B.4T, TE -68ms; T2_gAA--450m; R2(d_B)=10Hz
-r
-3•
4
-2
-2.5
-3
-1
-1.5
ppm
NAA
I
I
a -Glun,
Cho
Ino I
*1
b,r-Glu
-I
I
S
I
-3
-3.5
I
b -
a-Glu .
-
(Al)
Individual Specta
I '
I.
0
-0'.5
I
-2.5
-2
-1.5
-1
-0.5
ppm
0
(A2)
ISumed Sperum
Bo =9 4T, TE =136ms; T2_ AA=450mls; R2(dB)=10Hz
1-
-
r
I-
KI
I
I
I
L.
ul
R
if
,nU
1
YY
rr
a
*In
-3.5
-3
-2.5
I
1
.
-2
-1.5
.
.
-1
-0.5
I
0
(B1)
ppm
Individual Specta
I
I
I-
'
lm
a-Glu
a-
Chon
I
i' N[
', A
AN
CV
nI
omD
o
-1
1m
b,r-br-Glol
G
U
0
.it
•
-3.5
-3
4LI•..
-2.5
-2
-1.5
-1
-0.5
(B2)
ppm
Is M s
Bo
I
=94T,
..
=2
2ms;
J
IL
JL A
-[
-!
T2_NAA=45
Hz
i
~I
IJ
TE
JIL ,
j,LJ,.[
4tI
Ar
P
-3.5
-4
-3
-2.5
-2
-1.5
-1
-0.5
ppm
Fil-T;Lm
a-ciU I Cho Cr
Ino
i 1
U[
...
_
··~
-3.5
~·
~
-3
I
i:;-G!m
br Clu
ii_
l
(Cl)
Individual Specta
Mh
..
11
I:.
II
uuy;r
-2.5
I
-2
-1.5
-1
-0.5
0
ppm
(C2)
Figure 2-1(c) Spectra simulation: BO = 9.4T, TE = 68,136,and 272ms
(A,B,C respectively), AR2 = 10Hz (relatively good shim).
I
I
_
I
I-
Summed Spectrum
I
. . . I
.I.
Bo
=1
ST,
TE
=68ms;
I..
Hz
"
/
-l /
-I
-?
____________________
krI/
>
-3
-3.5
I
I
-1.5
-1
I
I
-
-4
.. .
T2_NAA=45
-2.5
-2
I
-0.5
ppm
(Al)
Individual Specta
-
a-Glu I
Ino
0
Cho
rr-.
A
-I
b.r- Glu
cr
I
-4
-3.5
-3
-2.5
-2
-1.5
-1
-0.5
ppm
(A2)
Summed Spectrum
I
I
I
[
a
F-
i
-I
t
-.
~----~-
'--
-3.5
-4
I
K-
,-~(
I- V
i
Bo =15T, TE =136ms; T2_NAA=450ms; R2(d_B)=50Hz
-3
-2.5
-2
-1.5
-1
3
0
-0.5
(B 1)
ppm
Individual Specta
NA
2
UIn, oG
a-Glu
no
Gl
btr-ýGlu
/
I
(
0
,
-3.5
-1 /
-4
-3
-2.5
-2
-1.5
-1
-0.5
(B2)
ppm
Summed Spectrum
Bo =1I
5T, TE =2 2ms; T2_NIAA-450ms; R2(d_B)=50Hz
i/'•.
II
4
I
-3
-3.5
•
',,'
"
I
-2.5
I
I
-2
-1.5
I
-1
-0.5
(C1)
ppm
Individual Specta
II
INA- f
-
oCI
f IC
I
I
II
I
0
-
-1
-4
-
-~-C-
-3.5
-
-3
--
-2.5
-2
-1.5
-1
-0.5
ppm
(C2)
Figure 2-2(a) Spectra simulation: BO = 1.5T, TE = 68,136,and 272ms
(A,B,C respectively) , AR2 = 50Hz (relatively bad shim).
Summed Spectrum
Bo =4.7T, TE =6Bms; T2_N'AA=450ms; R2(d_B)'50Hz
'
2
0
-1
-3.5
-3
-2.5
-2
-1
-1.5
-0.5
ppm
(Al)
IndMdual Specta
2
-Glu
-
.
-J,
Cr
Cho
-3.5
-4
-
G
b,r-
Gl I
M,
Ino
-3
-2.5
-2
-1.5
-1
Spectr(A2)
Summed
Summed Spectrum
Bo =47T, TE =1 ms; T2
3
0
-0.5
AA=450mns; R2(dB)=50Hz
2
1
-j
0
-1
4
-3
-3.5
-2.5
-2
-1.5
-1
0
-0.5
ppm
(B1)
Individual Specta
a-Glu
'
Cho
b,r-Glu
-'noY
,
0
-1
-4
."
'
-3.5
lac
'.,
-3
-2.5
-2
-1.5
-1 -0.5
ppm
0
(B2)
Summed Spectrum
Bo =4 7T, TE =2•2ms; T2_NAA=450ms; RZ(d_B)=50Hz
32
1
'-3 III
0
-1
-4
-- ,0
"
-1
"'
--. 3
"
--
"
-1.0
- I
"'
-U..
U
(C1)
ppm
Individual Specta
INAA
3:
2 -a-Gu .l ChoC Cr
1-
no
-4
-3.5
bj1
b,r-GkL
-1
-3
-2.5
-2
-1.5
-1
-0.5
ppm
0
(C2)
Figure 2-2(b) Spectra simulation: BO = 4.7T, TE = 68,136,and 272ms
(A,B,C respectively) , AR2 = 50Hz (relatively bad shim).
Summed Spectrum
=9
-3
-3.5
4
4T,
TE
=68ms;
T2_NAA=450ms;
-2
-2.5
....
.....
..
Bo
R2(dB)
50Hz
-1
-1.5
-0.5
ppm
(Al)
Individual Specta
z.-GrQ,
a-Glu
Ino
-3
-3.5
4
bj -GIm,i
b,r-Glu
ChC, Cr
-2.5
lac
-2
-1 -0.5
-1.5
(A2)
ppm
Summed Spectrum
Bo =9.4T, TE =1•6ms; T2_NIAA=450rns; R2(d_B)=5Hz
1
-3
-3.5
-2.5
-2
-1
-1.5
-0.5
(B 1)
ppm
Individual Specta
N,r-Ai
-t
a-Glu
Ch
Ino
-3
-3.5
4
I
D
I
b,r -CGu
-2.5
Ilac
-2
-0.5
-1
-1.5
(B2)
ppm
Summed Spectrum
(B2)
=9'4T, TE =22ms; TZWAA=450ms; R2(d_B)--50Hz
ppm
'Bo
S
Summed Spectrum
-1 -4
I
I
-3.5
-3
I
I
I
-2.5
-2
-1.5
I
I
I
-0.5
-1
(Cl)
ppm
Individual Specta
ml, II
Gir-
2 -Glm
a-Glu
-1
-4
Cho
r
b,r-GIu
Sno
-3.5
lac
-3
-2.5
-2
-1.5
-0.5
-1
ppm
0
(C2)
Figure 2-2(c) Spectra simulation: BO = 9.4T, TE = 68,136,and 272ms
(A,B,C respectively) , AR2 = 50Hz (relatively bad shim).
(6R2 =10 Hz) --
For field
less than 8R2 of 10 Hz, the NAA methyl
group at
a.) Spectra with
inhomogeneities
T), this
is
peak
at
at the low field
However,
(Bo >= 4.7 T).
field strengths
metabolites
by other
contaminated
2.023 ppm is not severely
higher
shim
reasonable
contaminated
heavily
by
strengths
(Bo =1.5
glutamate,
glutamine, NAAG, and GABA. At an echo time of 68 ms,
those doublet and quadruplet
peaks with J equal to 7.3 Hz, such as
the quadruplet peaks of GABA at 1.91 ppm, will be out of phase and
In other words,
contribute no signal to the "observed NAA" peak.
the NAA peak at 2.023 ppm is less contaminated
by GABA at echo
On the other hand,
time of 68ms than echo times of 136 or 272ms.
the doublet peaks of lactate at 1.33 ppm will be out of phase, too,
and it would be a big loss to underestimate
this important
cerebral
metabolite, which is of particular interest to cerebral physiology and
studies of energy impairment.
Since the linewidth
8R2 =10 Hz and the spectral
amplitude
usually
much
higher
than
the
is so narrow with
NAA peak is
of the pure
amplitudes
of the
overlapped
metabolites, the pure NAA peak can thus be fit relatively accurately
when one has a good baseline.
However, for longer echo times such
as 136 ms and 272 ms, this NAA peak
is heavily
distorted
by
glutamate, glutamine, and GABA. Under this condition, it becomes
difficult
amounts
to fit the
pure
of glutamate,
NAA peak
glutamine
and
because
the
GABA will
non-negligible
modulate
the
The same chemical contamination
baseline level and spectral phase.
phenomena were observed for the creatine and choline peaks in low
field cases.
Comparing those spectra simulated at 1.5T with the 3
echo time settings, it is clear that the lower the echo time is, the
56
spectral
more
information
SNR is degraded
because
is presented
in the
spectra.
This is
at the longer echo times, especially
for
those metabolites with low proton densities and high relaxation rates
Nevertheless, an echo time of 68 ms is generally not desirable
(R2).
loss
the
because
of
possible
the
For other shorter echo times that
of macromolecules.
contamination
and
information
lactate
people routinely use such as TE = 20ms, our computer simulation did
not show great difference in the degree of spctral overlap in NAA, Cr,
However, this simulation with short
and Cho as in TE = 272ms cases.
echo time may not reflect the real spectra because our simulation did
which have relatively
not take the consideration
of macromolecules
short T 2 relaxation times.
Also, lipid which is at around
overlapped
generally
with lactate signal.
1.33ppm is
Since lipid has relatively
may only
short T 2 relaxation time (= 50ms), the lipid contamination
be problematic
in shorter
echo time instead
of longer echo time
(8R2 > 50Hz)--
The situation for the
experiments.
b.)
Spectra with bad shim
spectra with bad shim is much worse.
For a field inhomogeneity
of
8R2 = 50 Hz, it is almost impossible to separate the major metabolite
peaks (NAA, Cr, Cho) from the minor metabolites
Glutamine,
taurine,etc),
especially
at
low
(GABA, Glutamate,
magnetic
fields.
As
indicated in figure 2-2a, the fusion of the minor metabolites
disturbs
the baseline level and makes the spectral phase adjustment
difficult.
Moreover, it becomes hard to determine
Taking
the
methyl
NAA peak
as an
the real spectral linewidth.
example,
it is difficult
to
determine how wide the Lorentzian line should be to match the true
57
NAA peak in the top spectrum
of figure
Even for higher
2-2a.
magnetic field strengths, there is still certain degree of chemical shift
overlap.
For example,
it is tricky to isolate out the pure creatine
signal from the baseline due to the overlap of other chemicals.
This
is a particularly important issue because creatine is often used as the
internal
to measure
standard
metabolite
the absolute
or relative
concentrations of the other cerebral metabolites.
T,
errors:
measurement
Figure 2-3 shows the semi-log plots of the synthesized
intensity versus echo time (TE).
include
minor
NAA with
The contents
contributions
of this 'NAA' group
from
NAAG, glutamate,
The solid red lines represent
glutamine, and GABA.
'NAA' signal
the "pure" NAA
signal and the other color coded lines represent the "observed"NAA
signals which are contaminated
metabolites with
In this simulation, the T 2 value of one of the
different T 2 values.
minor metabolites
by the overlapping
is fixed (e.g., glutamate = 300ms in figure 2-3a)
while the T2s of the other 2 minor metabolites (e.g., GABA and NAAG)
were stepped
between
100 to 500 ms in 100ms
increments.
The
lines are coded to different saturation levels to represent the specific
T 2 values over the T2 span.
value is.
The darker the line is, the smaller the T 2
Because all of the minor metabolites in this 'NAA' group are
J-coupled, each single minor metabolite will have its signal decay in a
sinusoidal oscillation.
molar concentrations
Depending upon the ratio of the preassigned
among
these minor metabolites,
the summed
signal will cross the 'pure' NAA line at different TE points.
In other
words, not knowing the relative amount of those other metabolites, it
58
is impossible
'observed'
line.
to find the optimized
TE values
that the
NAA signal deviates the least from the 'pure' NAAdecay
Similar phenomena were found for
shown in figure 2-4.
get the
to ensure
"measured
'creatine' and 'choline' as
If we try to fit this oscillating decay curve to
T 2 " value,
can
T 2 errors
be
The
expected.
estimated T 2 error of NAA can be as big as 50% with reasonable
T2
settings of the minor metabolites ( = 360 ms).
Our experiments
phantoms
of both
in vivo
and
spectra showed similar oscillatory phenomena.
occipital
cortex
The phantom and in
vivo spectra turned out to match the computer synthesized
spectra
quite well even though the simulation assumed weak J-coupling only
and a single spin-echo rather than PRESS (which has two 180 degree
pulses).
Figure 2-5 shows
a stack plot of
figure 2-6 shows a stack plot of in vivo
spectra,
several
subsets
from
estimate the T2 value of NAA.
1200
ms depending
subsets.
upon
phantom
spectra.
spectra
and
In the human
the 18 TE data sets were
used
to
The results ranged between 400 ms to
which TE points were
included
in the
This confirms the observation from the computer simulation
and leaves the accuracy of cerebral metabolite
be suspect.
59
T z measurements
to
ýý pure NAASI
T2 (msec): NAA = 400, Glu = 350
T2 (ms) GABA NAAG
100
--
100
300
500
300
-
100
300
500
500
-
100
300
500
0
100
200 300
TE (msec)
400
500
(A)
pure NAASI
T2 (ms) GABA Glu
100 100
-
-300
----
(I)
0
100
200
300
TE (msec)
400
500
500
300 100
300
500
500 100
300
500
(B)
Figure 2-3 Signal intensity as a function of TE at around 2.023 ppm
which is dominated by NAA. The pure signal intensity of NAA is
presented as thick solid red line. The color curves represented the
"measured" signal intensity with contamination of other overlapping
compounds with different T2 value combination. (A) The "observed"
signal of NAA with fixed T2 of glutamate. (B) The "observed" signal
of NAA with fixed T2 of NAAG.
T2: Cr = 350 msec
4
-- pure Cr SI
T2 of GABA(ms)
-- 100
--- 200
-- 300
400
-~ 500
3
2
1
0
100
200 300
TE (msec)
400
500
(A)
T2: Cho = 350 ms
4
-
-I-
pure Cho SI
T2 (ms) Taurine myolno
3
-
100
-
300
*-
500
2
0
-1
-9
0
100
200
300
TE (msec)
400
100
300
500
100
300
500
100
300
500
500
(B)
Figure 2-4 Signal intensity as a function of TE at around 3.03 ppm (A)
and 3.23 ppm (B) which are dominated by creatine and choline,
respectively. The pure signals for Cr and Cho are presented as thick
solid red lines. The color curves represented the "measured" signal
intensity with contamination of other overlapping compounds.
NAA
10.0 mM
Glutamate
9.7 mM
Glutamine
3.5 mM
3.29 mM
GABA
TE = 68 ms
Figure 2-5:
TE = 272ms
TE = 136 ms
phantom spectra
Human Study: Occipital Cortex
N
1
ALiNi,
I
TE = 68 ms
Figure 2-6:
Ia--lm wII
".?m!I w
U111
w
,'
I
-
TE = 272ms
TE = 136 ms
occipital cortex spectra.
62
Measured
points
T2 (ms)
4 (longest)
1150
4 (better)
441
4 (shortest)
wI~C
18
:
$UFI'rT
I"1
I • 0[,"
"ill I'T
I-I1 " ]
I
1
# of TE
365
I
/0
476
measurement
concentration
Absolute
errors:
Correlations between errors of the NAA concentration and
internal
between
curves.
Figure
of relaxation
0 and
2-7
error variables
3 of them with 10% errors from the
(Gt2NAA/T2NAA] ), we assumed
stepped
molar
= [(tlw/Tlw, Ot2w/T2w, OtlNAA/T1NAA,
([Etlw, Et2w, EtlNAA, Et2NAA]
values
in
invariance
Out of the 4 relaxation
in the brain.
principle
relative
its
to
due
standard
concentration
Water was assumed to be the
were performed.
time measurements
relaxation
times
while
the 4th
variable
100% error to build the error
the
shows
simulation
results.
was
correlation
For a given
repetition time (TR) of 2 s, which is the TR used for the phantom and
in vivo studies, the water magnetizations will have enough time to be
close to fully recovered between excitations (TI water = 950 ms) and
thus the TI errors
concentration
of water
won't play an important
role in the
error of NAA (figure 2-7 a). On the other hand, since
the T, of NAA (= 1600 ms) is much longer, a two sec TR is not long
enough to allow the full recovery of the magnetization
of NAA and
thus the T, error of NAA will not be negligible in the concentration
estimate
(figure
concentration
2-7 b).
Figure 2-8
how TR effects
shows
estimate-the curves are steeper
NAA
as TR goes shorter.
For a TR value bigger than 2s, the concentration error induced by T,
NAA will become tolerable (the amount of concentration error caused
by every 1% increase in NAA T, error is smaller than 0.2%). Kreis et.
al. [10] has reported that the longitudinal relaxation times (TI) of the
major peaks in an in vivo proton spectrum
63
are close to each other
are less sensitive to the repetition
and the relative peak intensities
saturation
In other words, if an appropriate
times (TR).
is less dependent
used for corrections, the absolute concentration
repetition
factor is
on
The concentration
times greater than about 1.5 second.
error caused by T2 errors of water or NAA, in the other hand, are not
negligible no matter what TR and TE values have been chosen (figure
2-7
The error
c,d).
concentration estimates.
could
as
be
high
as
over
100%
in NAA
Therefore, the accuracy of T2 measurements
are much more important
than the accuracy of T, measurements.
This is bad because the T2 s are harder to measure than the Tis due to
J-modulation.
For all of the cases we represent
in figure 2-7, the
error curves from shorter echo times are in the lower part of the plot
than those with higher echo times.
However, this does not imply that
the smaller the echo time is, the more accurate
estimate
is.
The spectra
further contaminated
the
spectral
measurements,
with
shorter
echo times are frequently
by macromolecules[15],
composition
and
decrease
and will move the errors
the concentration
which will complicate
the
toward
accuracy
of
the lateral
upper
T2
corners along the concentration- T2 error curves( figure 2-7 c and d).
The concentration error is especially notable in the T2 water case due
to the steeper slope as shown in figure 2-7c.
Because the slope of
this error curve is so steep, a small increase in the water T 2 error
might triple the NAA concentration error.
64
Er ror in T1(NAA) = 10%, T2(NAA) = 10%
4AP
-
I &+'
C 120
o2
-
S100
._
C I
0
-h
S8O
~ID1~-us·sr
·-
IH~ _~-_..ra.--.-L
...1
~··n--as~-~n--·l-·-aI.._p
C
3 6C
z
1
0
z1
dc-g-g~_g_~k-ak~··9_e-·~c~·$~ig9-Bi~~
4C~---
~
----
--------
C
c 20
.
o
I
w
I
00
-i
I
I
I
I
I
I
I
-50
n
irT1
TE = 68 ms
---
I
I
I
I
orrE
of
I
I
0
I
I
I
50
water
--
I
)0
100
Error in T2 of water (%)
(%)
0
14
J
1
1
-
O
C
-
%10 0
TE = 272 ms
._ Error in T1 (water) = 10%, T2(water) = 10%
Error in T1 (water) = 10%, T2(water) = 10%
I iq, L
C
-*-
TE = 204 ms
----
-TE=136ms
-
C
-
C
e
8
3 60
0
( 4C
c
&M
L
IJ,
z
1ýA
z
P
-
C
C
.c_
C
100
3._
0
0
-50
Error in T1 of NAA (%)
100
3 m
u
SIf,, A A
o
orrE
T2
n
ir
kI
%OL\
Figure 2-7 Error in NAA concentration due to T1 and T2 values of NAA and water.
)
0
0/
Frrnr in Tl (wt.tprl = 100/, T9(wtp-rl = 1 n0
A
O
O
I
C
C
0
O
U1
)0
Error in T1 of NAA (%)
(A)
C
0
O
Ct
L
C
O
O
Z
C
L0
O
LU
)0
Error in T1 of NAA (%)
(B)
Figure 2-8 NAA concentration error due to different
TR settings. (A) TE = 68 ms, (B), TE = 272 ms.
Conclusions:
points
how
out
metabolites
c)
concentrations
macromolecular
the
of absolute
measurement
alternative
concentration
as an internal
(Mindex = SImetabolite /SIproton
will minimize
measurements
comparisons[18].
and
and
allow
Although
elusive.
will remain
density
density).
This metabolite
relatively
the absolute
from
the
An
weighted
to obtain a metabolite
error
and
shifts,
chemical
standard
the concentration
pulse
particular
to the overlap,
is to use signal from proton
solution
experiments
of the strongly
of the
used, b) all the chemicals contributing
sequence
"absolute"
accurate
it is to obtain
the influence
under
time
It also
estimates.
Unless one knows: a) the behavior
concentrations.
coupled
problematic
This
2-9).
of relaxation
accuracy
concentration
in the metabolite
measurements
1H spectra
synthesized
4.7T (figure
1.5T and
of the
importance
the
emphasizes
from
spectra
to real
similar
we have
simulations,
By using computer
index
index method
relaxation
time
accurate
inter-subject
metabolite
concentration
measurements has been a great desire and the 'holy grail' of 1 H MRS
over decades, relative concentration
more
useful
and
reasonable
67
measurements
for
may prove to be
quantitative
analysis.
(A) 1.5T
3.00
2.00
1.00
mm~
XI
0.00
-1.00
(B) 4.7T
PPM
(A) is from the occipital
In vivo spectra from 1.5T (A) and 4.7T(B).
Figure 2-9.
cortex of a HD patient and (B) is from the striatum of a rat with intrastriatal
injection of MPP+.
References:
1
K. L. Behar, J. A. den Hollander,
resonance
'H nuclear
High-resolution
Shulman, O. A. C. Petroff, J. W. Prichard,
magnetic
T. Ogino, R. G.
M. E. Stromski,
study of cerebral hypoxia
in vivo, Proc. Natl.
Acad. Sci. (USA), 80:4945 (1983)
2
T. Michaelis, K. D. Merboldt, W. Hanicke, M. L. Gyngell, H. Bruhn, J.
NMR spectra
brain
of human
1H
in localized
Frahm, On the identification of cerebral metabolites
in vivo, NMR Biomed., 4(2):90-98
(1991)
3
K. R. Thulborn, J. J. H. Ackerman, Absolute molar concentrations by
of in
for analysis
Bl, A Scheme
inhomogeneous
NMR in
vivo
metabolites, J. Magn. Reson. 55:357-371 (1983)
4
P. S. Tofts, S. Wray, A critical
metabolite
concentrations
of methods
assessment
of measuring
by NMR spectroscopy, NMR Biomed., 1:1-
10 (1988)
5
P. S. Tofts,
S. Wray,
metabolite
Direct
concentrations
in
using
vivo
31P nuclear
magnetic
spectroscopy, Biochem. Biophy. Acta, 886:399-405
6
of absolute
measurement
resonance
(1986)
0. A. C. Petroff, D. D. Spencer, J. R. Alger, J. W. Prichard, High-field
proton
magnetic
during
obtained
resonance
surgery
spectroscopy
of human
cerebrum
39:
1197-1202
Neurology,
for epilepsy,
(1989)
7
H. Bruhn, M. L. Gyngell, K. D. Merboldt,
J. Frahm,
Wauter,
Localized
stimulated
echoes;
high-resolution
initial
proton
application
Magn. Reson. Med., 9:79-93 (1989)
69
to
W. Hanicke,
NMR spectroscopy
human
brain
R.
using
in vivo,
8
J. Frahm,
in vivo. Relaxation
brain
the human
in different
NMR spectroscopy
Sauter, Localized proton
times
of
(1989)
P. A. Narayana, L. K. Fotedar, E. F. Jackson, T. P. Bohan, I. J. Butler,
vivo
in
Regional
K. S. Wolinsky,
(1989)
brain.
in the human
and
of water
T. Ernst, R. Kreis, B. D. Ross, Absolute quantitation
metabolites
resonance
magnetic
proton
spectroscopy of brain, J. Magn. Reson., 83:44-52
10
regions of
concentration
and
cerebral metabolites, Magn. Reson. Med., 11:47-63
9
R.
W. Hanicke,
K. D. Merboldt,
L. Gyngell,
H. Bruhn,
and
I. Compartments
water, J.
Magn. Reson., 102:1-8 (1993)
11
R. Kreis, T. Ernst, B. D. Ross, Development
vivo quantification
of metabolite
and
of the human
water content
K. L. Behar, T. Ogino, Assignment of resonance in the
13
with proton
1H spectrum
and J-resolved NMR
of rat brain by two-dimensional shfit correlated
spectroscopy, Magn. Reson. Med., 17:285-303
in
(1993)
magnetic resonance spectroscopy, MRM 30:424-437
12
brain:
(1991)
Neurobiology
P. L. McGeer, J. C. Eccles, E. G. McGeer, Molecular
of
the Mammalian Brain, Plenum Press, New York (1987)
14
15
T. Ernst, J. Henning,
spectroscopy
of major
21(1):82-96
(1991)
Coupling
macromolecule
resonances
in volume
metabolites,
brain
K.L. Behar D.L. Rothman,
effects
D. D. Spencer,
in
Magn.
1H
selevtive
Reson.
Med,
O.A. Petroff, Analysis
1H NMR spectra
of human
of
brain,
Magn. Reson. Med., 32(3): 294-302 (1994)
16
K.L. Behar
resonances
and
in the
Med., 30(1):
T. Ogino,
Characterization
1H NMR spectrum
38-44 (1993)
70
of macromolecule
of rat brain,
Magn. Reson.,
17
R. Kreis, T. Ernst, B. D. Ross, Absolute quantitation
in the human
metabolites
brain. II.
Metabolute
of water
and
concentrations,
J.
Schulz,
P.
Magn. Reson., 102:9 (1993)
18
E. Brouillet,
B.G. Jenkins,
M.F.
Kirschner,
analysis
illness
Beal,
B.R.
Y.I. Chen,
Rosen,
E. Storey,
Non-invasive
of focal excitotoxic lesions in models
using
16:450-461
spectroscopic
imaing,
(1996)
71
J.B.
neurochemical
of neurodegenerative
J Cereb. Blood Flow Metab,,
1H
Chapter 3
Studies
in
Spectroscopic
Resonance
Magnetic
Parkinson's Disease
Patients
Introduction and backg-round
Parkinson's
disease
(PD) is one of the most common
onset age of 58 years.
diseases in North America with an average
Statistically,
population
the
annual
and the prevalence
North America as in 1991
asymmetrically
[2]described
since then
etiology
[1].
rate is 300/100,000
The symptoms
PD symptoms
have
however,
been
received
72
population
of PD usually
in
starts
with PD in 1817
and
documented,
the
well
is still unclear.
of the etiology of PD was inspired
seven street drug abusers
20.5/100,000
Although James Parkinson
associated
the motor disorders
the
of PD is
rate
on one side of the body.
of this disease,
hypothesis
incidence
neurological
One popular
by human
an intravenous
evidence:
injection of I -
methyl-4-phenyl- 1,2,3,6-tetra-hydropyridine
idiopathic
(MPTP)
symptoms
resembling
Parkinson's
metabolite
of MPTP, 1-methyl-4-phenylpyridinium
and developed
disease[3].
The
ion (MPP+), is
taken up by dopaminergic reuptake system and is transported
to the
substantia
in the
nigra pars compacta (SNc) where
MPP + is a potent inhibitor of complex I in
matrix of mitochondria.
the electron
transport
mitochondrial
it accumulates
chain.
of MPP ÷ in the
The accumulation
matrix may lead to a reduction in ATP production
[4]
which may subsequently cause the death of the SNc neurons.
Several biochemical
observations
have shown a 30%-35% reduction
in complex I activity in post-mortem
patients but with
no abnormal
enzymatic
and IV in immunohistochemical
finding,
multiple
system
disorder
associated
staining
atrophy
activity
[3,8].
(MSA),
with dopaminergic
nigra[5,6,7] in PD
substantia
for complex III
In contrast to this
a similar
neurological
cell loss, appeared
to have
normal enzymatic activity in complex I [9]. The etiology of PD thus
has
been
dysfunction
hypothesized
to
have
of energy respiration.
phosphorylation
a
special
association
Since the cessation
may force pyruvate,
with
a
of oxidative
the final product of glycolysis
and the key element to enter TCA cycle, to be shunted to lactic acid
instead of acetyl-CoA [10], a dysfunction in the enzymatic
activity of
complex I may lead to extra accumulation of lactic acid.
Localized
'H MR spectroscopy
technique
to investigate
has
cerebral
recently
become
metabolites.
(NAA), which is a neuron-specific brain metabolite
a powerful
N-acetyl-aspartate
[11,12,13,14], has
been used as neuronal marker in 'H MRS and has been widely used
to verify the viability of the cerebral
73
neurons.
Detection of lactic
acid has been studied in 'H MRS for many years [15,16] and has been
[17,18].
function
for other cerebral
been
have
metabolites
Other 'H MRS detectable
as useful markers
proposed
to test the mitochondrial
marker
impairment
used as an energy
Creatine
conditions.
can serve as an internal standard to estimate the relative ratio of all
to
due
metabolites
cerebral
other
invariance
relative
its
The 'H-MRS
concentration in different disease states [see chapter 2].
the
to examine
applied
successfully
has been
technique
in
clinical
aspects and the possible etiologies of several neurological
diseases.
movement
disorder
Huntington's disease (HD) is a genetic inherited
as a neurological
which has been hypothesized
with
significant lactate elevation
observed
cortex.
localized
et. al. [17] applied
Jenkins
level
lactate
The elevated
ganglia.
and
'H-MRS to HD patients
and occipital
in the striatum
suggests
in the HD brain
in oxidative phosphorylation
abnormality
basal
II ~ IV) in the
(complex
impairment
energy
associated
disorder
an
and 'H MRS has shown a
promising ability to detect the possible energy impairment in human
brain.
metabolite
examine
in PD brains.
condition
in
metabolites
the energy
use of 'H MRS to investigate
We thus proposed
Although
like to
(SNc)
where
nigra
substantia
the
one would
histochemical studies in postmortem PD brains have found abnormal
dopaminergic
neuronal
spectra with reasonable
loss [19,20,21,22], it is difficult to acquire
quality from this area because
signal to noise ratio (due to the very small structure
possible
signal
perturbations
distortion
by
magnetic
size) and the
susceptibility
due to the high iron content in this organ.
SNc is the primary
system,
caused
of the low
degeneration
dopaminergic
of the
source
SNc cells
74
for the central
may
lead
Since the
nervous
to insufficient
cortex.
and frontal
such as striatum
pathway,
dopaminergic
telencephalic
of the meso-
structures
in the downstream
innervation
dopamine
The deficit in the dopamine innervation of these downstream
cerebral structures is the cause of the motor and/or mental disorder
Although there is no evidence in PD patients
in Parkinson's disease.
of massive neuronal
complex I proteins
mitochondrial
found
have
studies
staining
of
complex
III
mutations
in
with PD and the basal
DNA that may be associated
in oxidative phosphorylation
to defects
ganglia may be vulnerable
in
blot and decreased
in a Western
Genetic
[23,24].
terminals
decreased
show
does
tissue
striatal
the neostriatum,
activity
loss other than the dopaminergic
[25,26]. We hence propose to examine the energy respiration
hypothesis.
impairment
spectroscopy
particularly
the
energy
We propose to use localized in vivo
'H MR
to examine
the
the resonances
of
ppm and lactate at 1.33 ppm.
provide
information
further
SNc to verify
tiny
of the
instead
ganglia
of basal
status
in
metabolites
striatal
the methyl
PD brains,
group of NAA at 2.023
This 'H-MRS metabolite study should
the biochemical
understand
to help
etiology of Parkinson's disease.
Methods
disease
patients
Idiopathic
Parkinson's
volunteers
were included in this study.
and
age-matched
The range of the disease
severity of this patient group is listed in Table 3-1
normal volunteers
patients.
Electric Co.) with
and receiver.
Most of the
of the
PD
on a 1.5T whole body magnet
a quadrature
The subjects'
75
.
or close relatives
the spouses
MR studies were performed
(Signa, General
transmitter
were
normal
head
coil as
RF
heads were packed tightly
head motion during the scan as the
inside the head coil to prevent
Coronal
may change critically with only a slight shift in position.
matter contrast from a fast spin-echo
images with good grey/white
used
to locate the
A point resolved
spectroscopy
(TR/TE = 287 / 3.4 ms, 16 echo trains)
spectral voxel in the striatal areas.
(PRESS) with water suppression
sequence
were
was used to acquire the
ms, number
striatal spectra (TR/TE = 2 s/272
of averages
Spectra of the PD patients were either taken unilaterally
> 400).
(n = 23) or
Twelve out of 19 normal controls
from both of the striata(n=20).
also had the spectra obtained from both striata rather
than a single
can only excite a rectangular
Since the PRESS sequence
striatum.
voxel
ratio inside the spectral
position and the grey/white-matter
voxel, the spectral voxel was positioned to cover most of the caudate
nucleus, putaman,
and white matter (internal capsule)
to exclude part of the ventricle
from
this
avoid.
structures
from the heterogeneous
of signal
degree
a certain
thus
and
voxel
spectral
contamination
However, it is almost impossible
globus pallidus.
is impossible
to
The spectral compositions for white matter and grey matter
are different.
Therefore,
one has to pay particular attention
amount of spectral contamination
from white matter.
to the
A small voxel
size (= 4.0+0.6 cc) thus becomes critical and necessary to estimate the
striatal
metabolite
levels accurately
distortion by the white matter.
creates few problems
spectra.
Previous
to avoid the possible
In contrast, cerebrospinal
and can plausibly
studies
[27] have
be ignored
shown
that
spectral
fluid (CSF)
in the striatal
CSF does
not
contribute much of spectral signals in 'H MRS due to the pulsation of
the CSF. The striatal spectra acquired from the imager were then
transferred
to a SUN UNIX workstation
76
and were analyzed by using
commercial
Research
spectroscopy
analysis
software
Data were processed
Inc., Syracuse, NY).
Methods
NMR1 (New
using zero fill,
baseline correction, exponential filtering and Fourier transformation.
Each spectral peak was then fit to a Lorentzian line and the integral
value of the peak was used as the index of the striatal
metabolite
level.
Table 3-1 Clinical characteristics of the PDpatients participating the
'H MRS studies. Total number of PD patients is 22.
Results
normal
and 20 age-matched
Among the spectra of 23 PD patients
volunteers, we found that the metabolite ratio of creatine (3.02 ppm)
to NAA (2.023 ppm) was quite consistent (42±0.5% of NAA, 2 tailed T
test:
Since
P > 0.95).
the
spectral
peak
at around
ppm
2.02
(dominated by the methyl group of NAA) has higher signal intensity
distortion
and less
baseline
3.02ppm
(dominated
creatine
than
by creatine),
as the internal
the
signal
spectral
we chose to use NAA instead of
to yield higher
standard
relative metabolite measurements.
was
used
to
examine
the
precision
in the
The use of NAA as the internal
standard is legitimate since there is no observable
brain atrophy in the PD patients.
at around
striatal
or global
A two tailed student t statistic test
significance
of
differences
metabolites between the PD and the control groups.
77
in
the
Average
When
the
averaged
result
from
the
lactate
to NAA
together
(Table
spectra-
individual
the
3-2),
individual
from
ratios
average
spectra
lactate
level
were
was
significantly elevated in the PD group compared to the control group
(59% higher; p value < 0.0004). This measurement treated each single
spectrum as an individual entity regardless of which hemisphere
the
spectrum came from (the first affected striatum or not) and whether
or not more than
one spectrum
other 'H-MRS detectable
was taken
metabolites
from the subjects.
No
were found with a significant
difference in the PD group compared to the control group. Figure 3-1
shows pairs of spectra from PD patients and normal controls.
PD
Control
# of subjects
23
20
P value
# of spectra
age (yrs)
Lac/NAA (%)
Cr/NAA (%)
Cho/NAA (%)
Glx/NAA (%)
Asp/NAA(%)
43
62 ± 10
12.33 ± 0.59
42.86 + 0.46
51.95 ± 1.03
13.58 ±0.69
10.16 ± 0.50
30
58 ± 12
7.77 ± 0.34
42.92 ± 0.66
47.86 ± 0.99
15.45 ± 0.98
10.31 ± 0.78
> 0.3
< 0.004
> 0.95
> 0.15
> 0.4
> 0.9
Table 3-2
Cerebral metabolite levels measured from PD patients and
age-matched normal control volunteers. Lac: lactate, Cr: Creatine,Cho:
choline, Glx: glutamate and glutamine, Asp: aspartate. (metabolite
ratio: mean ± standard error; age: mean ± standard deviation)
78
Cho
NAA
Lac
3.002,00
(C)
(A)
1.00 0.00 -1.00
3.00 2.00
1.00 0.00 -1.00
PPM
3.00 2.00
3.00 2.00
1.00 0.00 -1.00
PPM
1.00 0.00 -1.00
PPM
PPM
PPM
3.00 2.00
1.00 0,00 -1.00
3.00
2.00
1.00 0.00 -1.00
PPM
(F).
(E)
(C)and age matched normal controls (D)
(B)
Figure 3-1. Striatal spectra from PD patients (A)
Asymmetric
of
in the two
lactate levels
the
striata of
the
subject-
lactate
to NAA
ratio
groups,
control
PD and
the
between
in the
differences
significant
data showed
the averaged
Although
the
fell into 2 categories -
distribution of the individual ratios, however,
If a Lactate/NAA ratio of
high versus low lactate groups(Figure 3-2).
10% (one standard deviation above the mean of the lactate level in
the normal control group) is taken as a cutoff between low and high
values, 21 out 41 PD spectra belong to the high lactate level group
while only 5 out of 29 normal control spectra are in this high lactate
group.
The lactate levels do not correlate with age, disease severity,
DOPA dose administrated,
closer examination
Upon
nor the duration of PD (Table 3-3).
of the double
taken
spectra
from
the
same
subjects (20 out of 23 PD patients and 9 out of 20 normal controls),
lactate levels in the two striata
we found significantly asymmetric
for both PD and control
asymmetric
statistical
Figure 3-3
groups.
spectra from the PD patients
results
of the double
spectra
shows
and Table 3-4 shows the
sorted
versus 'small' lac/NAA sides of the same subject.
the statistical results as a bar graph.
side seems to be mainly the left striatum
80
according
to 'big'
Figure 3-4 show
Only the 'Big' side showed
significant elevation in lactate ( p <0.045).
and 17 out of 18 in PD patients).
2 sets of the
Interestingly,
a
the "big"
(8 out 9 in control group
50
40
30
20
n
1
21
B
n=5
13
10
n = 24
n =20 -1
PD
Control
Figure 3-2 Distribution of the individual lactate to NAA ratios in the
42 PD and 33 control spectra from 24 PD patients
control subjects.
81
and 22 normal
NAA
Lac
(B) Patient I: Right
(A) Patinet I: Left
3.00
2.00
1.00
0.00 -1.00
3.00
2.00
Lac
3.00
2.00
1.00
PPM
1.00
0.00 -1.00
PPM
PPM
(D) Patient II: Right
(C) Patient II : Left
0.00 -1.00
3.00
2.00
1.00
0.00 -1.00
PPM
Asymmetric lactate level in the striatal spectra.
Figure 3-3.
from PD patient I and (C) and (D) are from PD
are
(B)
(A) and
Note the left striatum has higher lactate than the
patient II.
right side.
Lactate/NAA (%)
age (yrs)
Duration of PD (yrs)
DOPA dose (mg/day)
Hoehn-Yahr stage
Low (<10%)
58+12Ns
8.2±0.9 NS
500±89 NS
1.8±0.1 NS
High (>10%)
60+10
7.9±1.2
510+65
1.9±0.2
Table 3-3 Correlations of the PD lactate to NAA ratios with clinical
parameters.
NS: not significant (unpaired t test, 2-tailed)
Lactate/NAA (%)
big side
small side
PD
n=20
Control
n=9
P (PD vs Control)
P
(small v.s. big)
age
8.17±0.50
17.85±1.17
< 0.001
61±10
6.11±0.29
10.85±0.85
< 0.02
56+5
> 0.17
< 0.05
> 0.4
Table 3-4 Metabolite levels from both striata
When the metabolite
lactate
levels (relative
levels were grouped
to NAA), both
of the same subject.
as to small versus
the PD and control
showed asymmetric lactate amounts in the striata.
showed no significant
difference
between
big
groups
The 'small' group
PD and control groups.
However, the 'big' group showed significant elevation of lactate in the
PD group.
83
25
20
C1
0
w5
0
C
Big Side
Small Side
A
Figure 3-4 Lactate/NAA
when
ratio comparison
the spectra
both striata are broken into small versus big lactate side.
from
The lactate
level is significantly higher on the 'Big' side in the PD but not the
control group (*p < 0.045).
Elevation
Aspartate
cerebral metabolite
Although there was no other 'H-MRS detectable
beside lactate showing a significant change in the PD group (Table 3 2),
of the
some
modulation
elevated
those
at around
aspartate.
spectra
distinguished.
not narrow
PD spectra
with
showed
however
suspicious
may result
from
Figure 3-5 shows some of these spectra.
For
2.8 ppm. This modulation
narrow
linewidths,
a peak
could
be roughly
However, for most of the cases, the linewidths were
enough to resolve the 2.8 ppm peak from the creatine
peak (3.0 ppm) resulting in an asymmetric
peak.
some
shoulder on the creatine
This adds to the difficulty of accurate quantification
the creatine
peak and the aspartate
84
peak.
This aspartate
of both
peak,
however, could potentially be better resolved from the creatine peak
and hence quantified more accurately at higher magnetic fields such
as 3T or 4T.
The finding of this suspicious aspartate
that
involve
PD may
which may provide
precisely
establish
completely the
more
complicated
valuable
information
the
etiology
neurological
worth
of PD and
pathological changes in PD.
85
also
peak reveals
modulations
pursuing
to more
to verify
more
(A)
3.00
2.00
0.00
1.00
-1.00
PPM
(B)
3.00
2.00
1.00
0.00
-1.00
PPM
(C)
3.00
2.00
1.00
0.00
-1.00
PPM
2.8 ppm may
around
at
peak
A
Figure 3-5.
[indicated
aspartate
in
elevation
an
represent
are from
spectra
striatal
3
These
by arrows] .
3 PD patients(A,B,C).
Discussion
Morphology
considerations:
SNR
and
in
reported
been
have
phosphorylation
Defects in mitochondrial
various movement disorders, such as Huntington's disease (HD) and
In PD patients, deficiencies
Parkinson's disease (PD).
study
Our 'H-MRS
I
[28], skeletal muscle [29,30], and brain
have been found in platelets
[3 1,32].
in complex
of a
hypothesis
the
supported
has
possible mitochondrial respiration deficiency in striatum by showing
elevation
significant
level
lactate
striatal
of the
in PD patients.
However, Holshouser et. al. [33] did not find significant elevation
the
lactate
striatal
in their
multicenter
studies
'H-MRS
on
in
PD
However, we believe we have reasonable criticism of their
patients.
results. The Holshouser et. al. multicenter
voxel size of 8 ml to cover
the striatum
studies claimed to use a
with less than 10 to 20 %
of brain
study
Morphological
contamination.
non-gray
matter
structures
reveals that the total volume of a normal adult striatum
(whole brain)
hemisphere.
is about
17 ± 2.48 ml [34], or about 8 ml in one
However, the shape of the striatum is complicated and
extends over [3cm, 3cm, 3.5cm] in the [AP, ML, DV] directions [35].
In other
words,
the 7 to 10 ml of striatal
volume
distributed over a big volume of approximately
voxel of 8 ml Holshouser
et. al. reported
30 ml.
The cubic
to use in their
studies thus can not possibly cover less than
matter.
is nonevenly
10 to 20 % of white
Our 'H-MRS study used much smaller voxel in size (
ml) to cover most of the body of caudate nucleus, putaman,
of globus pallidus only.
contamination
of white
'H-MRS
4 ± 0.4
and part
This small voxel can still not avoid the
matter
such
87
as
internal
capsule,
by our
estimation
the
physiological
restricted
contamination
demand
and
is
about
consumption
%.
30
The
of energy
neuronal
is essentially
to gray matter only and any elevation in lactate level is
expected be located in gray matter.
contamination,
the
'H-MRS
detectable
lactate
reduced by the degree of the contamination
30% contamination
amount
is to
be
of white matter, e.g., a
of white matter in the spectral voxel may bring
the ratio Lactate to NAA of 20% (pure grey matter)
(whole voxel).
of voxel
With the consideration
down
to 14%
The other notable problem is the issue of signal to
noise ratio (SNR).
In theory, any measurable
quantity
has to have
SNR bigger than 1. In most of the spectral analysis, only the SNR of
has been estimated since NAA is in general the most prominent
NAA
For a given SNR of NAA, there is a
component in the 'H-MR spectra.
minimum detectable lactate to NAA ratio which is:
Lactate
NAA min
(%)=
100
(3-1)
SNRNAA
In other words, for spectrum
with given SNR of NAA (SNRNAA), the
lactate to NAA ratio is only reliable when the value is bigger than the
values obtained from equation 3-1.
Although Holshouser et. al. used
a lactate sensitive echo time (TE = 136ms) which should yield nice
negative doublet peaks of lactate at 1.33 ppm, the criteria they used
to screen the spectra were not strict enough to detect the minimum
lactate level smaller than 33% of NAA (one of the criteria for them to
screen the spectra is SNR >3). In our 'H-MRS study, we used much
stricter and flexible criteria to screen the spectral data for the lactate
level. Figure 3-6 shows a scatter plot of (Lactate/NAA) to SNRNAA with
the
solid
line
indicating
the
theoretically
88
minimum
detectable
(lactate/NAA)
separates
as a function
the spectral
data
of SNRNAA .
This SNR-(Lac/NAA)
into 2 distributions:
any
data
line
point
falling into the area to the right side of the solid line is reliable, while
any data point falling into the left side of this line is problematic.
We
further classified the area to the left side of the theoretical line to 2
subsets with the SNRNAA of 12.5 as the boundary
deviation
below
the mean
SNRNAA in normal
vertical dashed line in Figure 3-6:
line (= 1 standard
control
group)-the
area A for SNR > 12.5 and area B
for SNR < 12.5. A signal to noise ratio of NAA of 12.5 ( one standard
deviation
above
the mean
of SNRNAA) will yield the
minimum detectable Lac/NAA of 8.5%.
theoretically
Therefore, it would be safe to
float those data points in area A (indicated as V in Figure 3-6) to the
values right below the SNR-Lac/NAA line (indicated as 0 in Figure 3 6).
However, any data point in area B has too much uncertainty
in
the lactate level and has to be dropped off from this study (indicated
as x in Figure 3-6).
With the confidence of our spectral quality, we
can be sure now that our detection of the lactate elevation in the PD
group is reliable at least with respect to SNR.
89
30
25
S20
z 15
a,
5O10
5
0
0
Figure 3-6
5
10
15
20
25
30
35
SNR of NAA Peak at 2.023 ppm
40
Distribution of Lactate to NAA ratio versus signal to noise
ratio of NAA.
The solid line indicates
the theoretically
minimum
detectable Lactate to NAA ratio as a function of the SNR of NAA.
Any
data point to the right of the solid line (area C) is reliable. The dashed
line indicates I standard deviation below the mean SNRNA in normal
control group.
Any data point in area B will be discarded
(with
symbol x) and any point in area A will be floated to the value right
below the theoretical line (float from symbol V to symbol 0).
Therapeutic
Implication:
If the mitochondrial
respiration
deficiency
hypothesis
is true, the
finding of elevation in PD lactate level may provide some therapeutic
strategies
to
mitochondrial
slow
progression
of
PD,
such
function using metabolic cofactors.
disease, Koroshetz et al [36] has administrated
90
as
boosting
the
In Huntington's
several mitochondrial
respiration
with
boosters such as co-enzyme Q (QO1)
elevated
medication
basal
'H-MRS
striatal/occipital
examinations
in
to those HD patients
lactate
those
levels.
The
HD patients
showed
significant drop in the previously elevated lactate levels.
striatum
may
only be one of the
neurodegenerative
process
downstream
in PD, an energy
decrease the rate of degeneration
Although
structures
in the
boost may
in the striatum
post-
help
to
and thus stop the
worsen of the movement disorder which PD patients may suffer.
Asymmetric
Lactate
The asymmetric
levels
:
lactate data brings
up some interesting
about how the elderly human brain functions.
patients,
the high
lactate
side happened
Similar findings have been reported
studies
of
distribution
hypothesis
Huntington's
to be the
(HD)[36],
left striatum.
et. al. in 'H-MRS
although
the
can be proposed
that the left and right striata
different
degrees
vulnerability
A
in the
of vulnerability
the striata encounter certain energetic stress.
when
In other words, the
of the striatum may correlate with the working/firing
frequency that the striatum has performed
most people are left hemisphere
has long been hypothesized
The left striatum
over its life time.
Since
dominant and the left hemisphere
to govern the mental computation
thus has been hypothesized
heavier working load than the right striatum.
stress
or activation
and
this
may
asymmetric lactate levels in the two striata.
91
and
to have a
The higher neuronal
firing rate in the left striatum may lead to greater vulnerability
energy
age
in HD patients was much lower than the PD patients.
human brain may possess
language.
In most of the PD
by Jenkins
disease
questions
be the
cause
with
of the
Conclusions
The in vivo chemical/ metabolite study of the human brain using 'H MRS has provided
information
valuable
to evaluate
the neuronal
Here we used this technique
status in various neurological diseases.
to explore the possibility that prolonged energy stress may lead to
mitochondrial impairment and cause dysfunction in the striata of the
The finding from this 'H-MRS study may help provide
PD patients.
to
clues
the
researchers
etiology
of Parkinson's
and
disease
may
help
to design more advanced and sophisticated experiments
for a better understanding
the dysfunction
of the normal striatal function as well as
Such designs may include the
in the PD patients.
monitoring of the dynamics of the lactate levels or regional cerebral
blood flow (rCBF) and volume (rCBV) changes in the striatum during
of certain motor
such as the performance
physiological challenges
tasks. However, unlike in HD, PD may not represent
process
and
may
neurotransmission
be
in
involved
as well. The results
system
study provide information
multiple
a single disease
modulation
from
our
of
the
'H-MRS
about possible energy defects in PD, but
many other hypotheses and techniques need to be studied before the
etiology of PD is understood.
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96
An Animal Model of Parkinson's Disease
Chapter 4
using
6-hydroxydopamine
Rats
---
Anatomical and Metabolite
Imaging
Resonance
Magnetic
in
Lesions
Studies
Introduction and background
Dysfunction of the nigrostriatal
recognized
to
be
involved
dopaminergic
idiopathic
in
system has been long
Parkinson's
disease.
Postmortem histological studies in PD brains have found significant
loss of the pigmented dopaminergic
neurons
nigra
in the substantia
pars compacta (SNc) with little apparent atrophy in the striatum (str)
[1,2,3,4,5].
The
SNc is
the
primary
dopaminergic neurotransmission system.
the pigmented
striatum
dopaminergic
through
the
dopamine
source
Dopamine is synthesized
cells in SNc and transported
forebrain
medial
97
of the
bundle
in
to the
(MFB),
the
anterograde
Thus,
pathway [6,7].
dopaminergic
nigrostriatal
degeneration from the SNc to the neostriatum is a distinct possibility.
has been widely used in animal models to mimic the
neurotoxin,
depletion of Parkinson's disease' [8,9]. Injection of 6-
dopaminergic
OHDA into the medial forebrain
bundle can selectively
dopaminergic neurons without interruption
of
neurotransmitters
other
and
is
thus
OHDA lesion is performed
to
able
the
ablate
[8]. When the 6 -
while
stimulation
the
This
striatum remains intact and is essentially normal.
contralateral
the
only the ipsilateral striatum
unilaterally,
in dopaminergic
abnormality
the
destroy
of many of the functions
dopaminergic innervation from the SNc to striatum
suffers
dopaminergic
known
well
a
(6-OHDA),
6-hydroxydopamine
unilateral abnormality makes it possible to inspect the survivability
of the dopaminergic
behavioral
terminals
and to have
stimulation)
striatum
the animals
sacrificing
without
tests
in the ipsilateral
comparison
an internal
by using
(pharmacological
between
the 2
striata (intact versus ipsilaterally lesioned striata) in the same rat as
well as make external comparisons among individual rats.
behavioral
established
amphetamine
degree
of the
unilateral
(5 mg/kg,
test
a method
loss quantitatively
6-OHDA lesioning
stimulation
pharmacological
s.c.) provides
dopaminergic
to the
using
(see
The well
to estimate
in
appendix).
of
the
the
striatum
The
6-OHDA
animal model provides the opportunity to investigate the connections
iSix-hydroxydopamine is an analogue of dopamine with high affinity for the catecholamine
uptake systems. It can be administered either via the cerebrospinal fluid or directly into
brain parenchyma and the neurotoxin will be transported into catecholaminergic neurons.
Pretreatment of animals with desipramine (DMI) blocks the uptake of 6-OHDA into
norepinephrine containing terminals and constrains the neurotoxin to the dopaminecontaining terminals [1,6,17,18,19].
98
between :
1. the hypothesis that the neuronal loss in SNc is induced
by a possible energy
impairment
in the mitochondria
and 2. the fact that the striatal dysfunction
insufficient
dopaminergic
supply
propose using unilaterally
the global striatal physiology.
global
in PD is caused by the
SNc [1,14,19].
6-OHDA lesioned
the possible association between
possible
from
physiological
[10,11,12,13],
animals
the dopaminergic
We thus
to investigate
innervation
and
In this chapter, we will to examine the
and
neurological
abnormalities
striata, such as the changes in the macro cellular water
in
properties
using T 2 and diffusion weighted images, the major brain metabolite
levels using spectroscopic
imaging
(chemical
NAA and lactate, and the hemodynamic
shift images,
properties
CSI) of
(rCBF and rCBV)
by using bolus injection of Gd-DTPA [15,16]. In the next chapter, we
will propose to examine a possible abnormality
the
coupling
between
the
in neuronal activity-
dopamine-receptor
activity
and
the
regional hemodynamics in response to pharmacological stimulation.
Methods
Animals
Sprague-Dawley rats were used for all studies.
studied as well as 6-OHDA lesioned
MFB, rats
were
ketamine,
5mg/kg
first
ones.
anesthetized
with
Control animals were
To make lesions in the
ketamine
xylazine, 2.5mg/kg acepromazine).
(50
mg/kg
Desipramine
(20 mg/kg, i.p.) was injected intraperineally (i.p.) 30 minutes prior to
the
6-OHDA
dopaminergic
animals
were
infusion
to restrict
the
effect
of 6-OHDA
to
the
neurons[17,18,19, also see footnote i in page 98]. The
then
placed
in a sterotaxic
99
device
and
received
unilateral
of 6-OHDA into the
injection
(Coordinates:
animals were transferred
After the intracranial
testing
and
surgery,
back to the animal facility and allowed to
recover for at least 3 weeks" [20,21].
rotational
bundle
8 jtg/2gl, iced
[AP, ML, DV] = [-4.5,+1.2,-7.6],
stabilized with ascorbic acid [6,17]).
forebrain
medial
to screen
out
those
Behavior
neuronal
amphetamine
was given subcutaneously
placed in a computerized
rotameter
less
then
by
90%
For this, 5 mg/kg
[22].
loss in striatum
dopaminergic
with
rats
was assayed
(s.c.) and the animal
(San Diego Instruments,
was
CA) to
record the number of turns ipsilateral or contralateral to the lesioned
side for 90 minutes
in 10 minute
intervals
(see
appendix).
The
criterion for inclusion in the study was > 600 net ipsilateral turns per
90 min. intervals.
This has been shown to correlate with >90% loss
of nigrostriatal dopaminergic innervation [23].
MR
Studies:
on a 4.7 T GE Omega CSI imaging
The MR studies were performed
system.
All of the 6-OHDA lesioned animals were anesthetized
a halothane/N 2 0/0
using
a 35
mm
2
mixture
birdcage
(1% h'alothane
coil.
in volume) and imaged
All animals
regulated using a circulating water blanket
with
were
temperature
at 380 C over the whole
study period.
Although the 6-OHDA can produce acute insults in the dopaminergic neurons, the
supersensitivity (upregulation) in the postsynaptic striatal neurons does not begin to appear
until one to two weeks after the lesions. Therefore, the behavioral tests can only be used to
screen out the improperly lesioned animals at least 3 weeks after the 6-OHDA lesions.
100
Anatomical Images:
weighted sequences
were used to screen for possible abnormalities
The parameters used were:
such as edema or intracellular swelling.
1) T 2 -weighted
2.2mm
and diffusion-
sequences
T 2-weighted
spin-echo
Both conventional
images -- TR/TE 3s/80ms,
8 slices,
with
in plane
slice separation
and 2.3mm
slice thickness
2 average,
resolution of 0.195mm x 0.390mm.
slice thickness
slices with 2.2mm
plane resolution
[Gx,Gy,Gz]
and 2.3mm
x 0.390mm,
0.195mm
= [4,4,0]gauss/cm,
2 average, 4
2.2s/53ms,
-TR/TE
images
2) Diffusion-weighted
slice separation,
diffusion
gradients
were
500
s/cm 2
b
value
=
In
order
to
equivalent
in
(8=12.5ms, A = 20.5ms).
3)
Hemodynamic
hemodynamic
properties
rCBV and rCBF were
-
Measurement
of the lesioned striatum,
made
study
measurements
using bolus injections
the
of
of 0.2mmol/kg
Gd(DTPA) (Magnevist, Schering AG, Berlin) [15,16] and rapid gradient
echo imaging with 1.28 s/image temporal resolution (TR/TE 20/7ms;
Ernst Angle RF).
Chemical Shift of metabolite Images :
The metabolite images were acquired by using a spin-echo chemical
shift sequence
(TR/TE = 2s/272ms).
This CSI sequence
uses
binomial 1800 RFpulse with null point at 4.7 ppm to suppress
water signal [24,25] and with the maximum
ppm (the methyl group of NAA).
using
short-TI
inversion
inversion time (TI) [26].
recovery
the
RF power set at 2.023
Lipid suppression
(STIR)
a
technique
was done by
with
varied
The initial TI value was optimized according
101
to individual animal to minimize the lipid signal at the Ky = 0 line.
The inversion recovery waiting time (Ti) was then stepped through
the (Kx, Ky) domain (phase encodings) with 600gs increment.
dimensional
chemical shift images
[f,x,y] were obtained
Three
through
x
and y phase encodings with in plane resolution = 2.18mm x 2.18mm
(fov
35mm
resolution
with
=
x/y
phase
encodings)
7.18Hz (spectral
width
of 4000HZ with 512
digitization).
matrix
16x16
and
the
spectral
points
The raw data (t, K x , Ky) were zero filled to 32 by 3 2
in the
Kx and
Ky domain,
individual
spectral
line
was
modulated by a low pass sine filter and then 3D Fourier transform is
applied
to reconstruct
the 3D chemical shift images
[f,x,y].
metabolite images (NAA and lactate) were reconstructed
together
the spectral
lines containing
and viewed along the frequency
possible
spatial
inhomogeneities
axis
[f]
at
distortion
metabolite
axes (a [x,y] image).
of the
spectral
by adding
lines
peak
To avoid the
due
to
field
cross the imaging field (i.e., a shift in the spectral
different
inhomogeneities
the desired
The
spatial
coordinate
ABo), a homemade
program
[x,y]
due
to
field
is applied to trace the
spectral location of the metabolite peaks through the spatial domain
to correct for this distortion
frequency axis).
The NAA image was reconstructed
group signal centered
reconstructed
assuming
(i.e., correct the shift in the spectral
from
the planes
at 2.023
the
ppm
spectral
and
peak
corresponding
analyzed.
102
the
centered
from the methyl
lactate
image
at 1.33
to the linewidth
ppm
was
by
of the peak
Data
Analysis-
The anatomical
overlaid
images
(T 2 or diffusion
were
images)
(CSI) and hemodynamic
onto the metabolite
images to segment
weighted
(rCBV, rCBF)
and the
the outer contour of the hemispheres
The abnormality indices, labn , were then determined as:
striata.
(4-1)
labn = (Slips s tr - SIcont str) / SIcont str x 100
indices labn from each
The abnormality
animal were
pooled
then
together to investigate the statistical differences between the control
and ipsilateral striata (paired student t test).
Results:
images:
Water
abnormality
There was no obvious
on either
the T2 or diffusion
This
weighted images of the 6-OHDA lesioned animals (Figure 4-1).
indicates
that
the
loss of the
dopaminergic
striatum may just lead to functional impairment
morphological modification.
innervation
in the
but not to a global
This indicates that although there is a
in the dopaminergic
supply to the
striatal area, there are no gross tissue abnormalities.
In contrast to
certain degree
of abnormality
this normalcy, other neurotoxins, such as malonate or MPP + induced
striatal lesions, show very early striatal lesions that are detectable
using
diffusion
weighted
images
and the mature
likely to be missed on T 2 weighted
images [27].
lesions
are
not
The hemodynamic
maps (rCBF and rCBV, figure 4-2) from the bolus injection of GdDTPA showed no significant differences in the resting states between
the two hemispheres
(and striata), either.
103
The resting
rCBV was
slightly higher in the lesioned striatum but this was not significant (
= 5 ± 27%, p > 0.85) and the rCBF was about equal in both striata (
-
1 ± 11%, p >0.4). This suggests that there may not be much change
in the resting
state
of regional
change in vascularization
forebrain
bundle.
hemodynamics
the
significant
due to the 6-OHDA lesions in the medial
The stable resting
rCBV and rCBF status
important and essential in the investigation of
between
or any
pharmacological
stimulation
are
the neuronal coupling
and
the
hemodynamic
response, which we will discuss further in the next chapter (chapter
5).
104
T2 Weighted Images
ADC Images
Figure 4-1. T2 and ADC weighted image of rat with unilateral
lesioning in the medial forebrain bundle. There is no global
abnormality in the hemisphere ipsilateral to the lesioning.
L: Lesioned side, C: Control side.
CBV
Control
side
CBF
6-OHDA Control
lesion
side
6-OHDA
lesion
Control side
Lesioned side
Significant level
1.05 ± 0.27
0.99 ± 0.11
p > 0 .8 5
p> 0.45
Figure 4-2. Bolus injection of Gd-DTPA shows normal
baseline state in hemodynamic properties (CBV and
CBF, n = 9).
2)
The
Metabolite
images:
metabolite
images,
information.
other
the
on
increase of the lactate level in
There was no apparent
either striatum, ipsilateral or countralateral
neuronal
marker
healthy
neurons,
striatum
however,
showed
(figure 4-3; average
to the MFB lesion. The
(NAA), which
N-acetylaspartate
different
provide
hand,
= 9.61
is abundant
in the
a decrease
in
ipsilateral
± 0.01%, p < 0.0003;
range
between 5 to 21 % signal decrease compared to the control striatum).
Curiously, this signal depletion in the ipsilateral striata extends to the
medial bottom part of the brain.
Since the slice thickness
for the
metabolite images is 7 mm, and thus we lose the spatial resolution in
axis, the signal loss of this tail (marked
the antero-poster0
with a
white arrow in figure 4-3) is suspected to be the track of the medial
Similar NAA signal loss and lactate elevation along
forebrain bundle.
this tail have
injection
been
(unpublished
intrastriatally
found
data
in the MPP + lesions
via intrastriatal
from B. G. Jenkins).
MPP ÷ injected
will follow the reuptake
dopamine
and be shunted
forebrain
bundle.
pathway
to the substantia
106
of the endogenous
nigra via the medial
NAA Image
Control
side
6-OHDA
lesion
Figure 4-3. NAA image on a 6-OHDA lesioned rat. There is
significant NAA signal loss in the striatum ipsilateral to the
Note the signal loss extending to the
6-OHDA lesioning.
medial-ventral part of the brain may indicate the tract of
the medial forebrain bundle.
Discussion:
have demonstrated
diffusion weighted images, several investigators
terminals ipsilateral to the 6-OHDA lesion
the afferent dopaminergic
reduced
supersensitivity,
include:
alterations
Such
cells.
striatal
balance
in the biochemical
could lead to many other alterations
the
to the striatum
deficit
projection
striatum.
control
normal
the
to
compared
90%
a large dopaminergic
However,
showed that
staining with tyrosin-hydroxylase
Immunohistochemical
were
lesioning.
6-OHDA
following
changes
histological
global
in the T 2 and
global changes
no apparent
Although we detected
of
i.e.
upregulation of the postsynaptic dopaminergic receptors (DI and D2),
and higher turnover
rates in acetylcholine (ACh), serotonin
and the remaining DA. Zhou et. al. [28] demonstrated
diminished
in the striatal
sparsely
and
delineation between
innervation
induced
the two.
pallidus
sprouting
for the growth
with
a sharp
fibers
into
the
of 5-HT fibers between
vague.
that 6-OHDA lesions may trigger
an inhibition
(5-HT) fibers
of the dopaminergic
5-HT
of
the striatum and globus pallidus became
remove
densely
The interruption
dopaminergic territories, and the delineation
also indicates
are
Normally, the 5-HT fibers innervate the
the globus
the
neurons
area while the serotonin
showed increased sprouting.
striatum
that following
dopaminergic
of the SNc via 6-OHDA, the
lesioning
(5-HT),
Current evidence
trophic
of 5-HT neurons
signals
or
which may
initiate the sprouting of 5-HT in the nigra as well as in the striatum
[28,29]. Although the role and the mechanism
and rebalance
of various neurotransmitter
dopaminergic
dennervation
of the remodulation
systems after the striatal
is not clear, it may play an important
108
role
the
in maintaining
of the
integrity
function
striatal
after
lesioning of the nigrostriatal pathway.
Although
the
is highly
strata
ipsilateral
in the
NAA depletion
significant compared to the control striata in our study, literature has
significant
not reported
lesioned animals [30] or from postmortem
brains.
striata
loss in the
neuronal
of 6-OHDA
histology of idiopathic PD
This raises the question that the NAA loss in our metabolite
study is caused by the degeneration of those dopaminergic terminals,
or that there are some other factors which may lead to the NAA
That the NAA signal in the striatum ipsilateral to the 6 -
signal loss.
to the normal
surprising
appear
may
striatum
contralateral
compared
15% depleted
OHDA lesion is less than
immunohistological studies with tyrosine hydroxylase
normal brains show a relatively high population
since
staining in the
of the TH-positive
cell clusters covering the striatal slices (6-OHDA kills cells that stain
However, since more than 90% of
positive for tyrosine hydroxylase).
the striatal neurons are gabaergic instead of dopaminergic
and the
overlap of Nissl-stain and TH-stain show that the high TH-positive
cells
are
at the
clustered
[31,32,33].
heterogeneous
In
other
areas
words,
with
low
striatal
the
Nissel-stain
cell
density
organization
is
and the average volume density does not reflect real
local neuron density
(neuron
counts).
The dopaminergic
neurons,
which seems to be diffuse and widely covering the striatal section in
the TH-stain,
may not be the major fraction
of the total
striatal
neuron population.
In this sense, the 9.6% NAA in the ipsilateral
striatum depletion
from the chemical shift images may just reflect
the net neuronal
dopaminergic
loss after accounting
cells, the resprouting
109
for the degeneration
of the
of the 5-HT fibers which might
Or,
be expected to increase NAA, and the real neuronal population.
since NAA is higher in neurons with long axonal projections
such as
the loss of the SNc
the SNc neurons, the NAA loss may just represent
Dopaminergic terminals[34].
is an
The other possible factor leading to the NAA signal depletion
increase in susceptibility mismatch, which may be induced by extra
iron
in the
accumulation
discussed
in
1, one
death
the
of the
et. al. also showed
Oestreicher
SNc and
the
in the SNc may
dopaminergic
neurons
and may introduce an extra susceptibility
rate of NAA, it may
the R2* relaxation
we
etiology
of
lead to or
[5,3 5,3 6,37 ].
that 6-OHDA lesions in the medial
forebrain bundle lead to higher iron contents in SNc[38].
paramagnetic
As
striatum.
about
hypothesis
is that iron deposition
Parkinsonism
accelerate
chapter
ipsilateral
Since iron is
factor into
lead to the NAA signal
depletion if there is extra iron in the ipsilateral striatum.
This extra
AR2 * term may not play an important role in the water images (T 2
and diffusion weighted images) due to the much shorter T 2 of water.
We have performed
water R2 * measurements
in one of our 6-OHDA
lesioned animals and found no significant changes in R2 *.
For the energy impairment marker
lactate, there was no significant
difference between striata in our 6-OHDA lesioned animals (both are
in the baseline levels).
OHDA lesioned
forebrain
bundle
animals
However, as we could only examine the 6 at a minimum
of 3 days after the medial
lesioning, any lactate generated
during cell death
would likely have been washed out by the time we performed
chemical shift images.
the
Jenkins et. al. [27] has shown the general
lactate time courses involved in striatal
110
excitotoxicity.
The lactate
to build
level started
while
up steadily
impairment
the energy
occurred with intracranial injection of the neuronal toxins malonate
or MPP +.
Then the lactate
to
started
decline
approximately
to
the
1 ~ 24 hours.
baseline
course
the
over
level
and
value
a maximum
level reached
of
The 6-OHDA lesioning of the medial
forebrain bundle is likely to be similar to these models and hence b y
the time we were able to acquire
have
missed
the
the metabolite
and
of lactate
peak
impairment stage with no lactate left.
only
in PD is a slower process,
post-
the
detected
However, the 'H-MRS with PD
patients showed elevation of striatal lactate level.
progress
images, we may
Since the disease
the chance
we may have
to
monitor the lactate level over a much longer time period than in the
animal model with acute lesioning.
The lactate level shown in PD
patients may indicate the slow energy impairment process instead of
the near completion of cell death.
Conclusions:
The 6-OHDA lesions in the medial forebrain
dopaminergic
supply from the SNc to striatum
of the symptoms
of Parkinsonism
lesions induce an acute abnormality
in rats.
bundle
interrupt
and can mimic some
However,
the 6-OHDA
in the dopaminergic
supply
the striatal area in contrast to the chronic slow degenerative
in idiopathic Parkinsonism.
the
to
process
The 1H MRS studies in the idiopathic PD
patients (chapter 3) showed elevated striatal lactate levels while the
6-OHDA animal model showed no significant elevation.
The cause of
of a progressive
degenerative
this difference
process
may be the result
of PD versus
the
acute
insult
of the
6-OHDA model.
Nonetheless, neither the PD patients nor the 6-OHDA animals showed
111
brain
or anatomical
atrophy
controls.
to normal
compared
abnormalities
may
of Parkinsonism
This suggests that the abnormalities
not be induced by a massive striatal neuronal abnormality
but may
just caused by the malfunction of the minor neuronal population, the
more
the coupling
to investigate
we have
deeply,
To explore
in the striatum.
terminals
dopaminergic
receptor
hemodynamic
responses.
In the next chapter
use the 6-OHDA model again to investigate
regional hemodynamic
induced
the
by
cerebral
the neuronal
activity-
stimulation
d-amphetamine
agonist
the
5), we will
(chapter
coupling by the pharmacological
dopaminergic
issues
between
regional
the
and
system
dopaminergic
these
and
the
dopaminergic reuptake blocker CFT.
Appendix -- Behavioral Rotational Test
After unilateral 6-OHDA lesioning in the medial forebrain bundle, the
insufficient dopamine
an
supersensitivity,
dopamine receptors.
increase
in
number
the
of
postsynaptic
The degree of dopamine-receptor
each striatum will be proportional
of dopaminergic
to the number
will induce
supply to the ipsilateral striatum
coupling in
(though not necessarily linearly)
receptors
on the postsynaptic
side.
Since each hemispheric motor cortex circuit of the brain controls the
motion on the contralateral
ipsilaterally
amphetamine
side of the body, the animals will turn
to the 6-OHDA lesioning
(a dopamine releaser
to the loss of dopaminergic
with
the stimulation
and reuptake
innervation
of D-
blocker [39]) due
in this striatum.
More than
600 ipsilateral turns in 90 minutes indicates more than 90% loss of
the dopaminergic
OHDA lesioning.
innervation
in the striatum
ipsilateral
to the 6-
While more than 900 ipsilateral turns in 90 minutes
112
indicates
injected
lesioning,
[40,41,42], the ipsilateral
SNc of an animal with a
more
will synthesize
bundle
partial lesion of the medial forebrain
According
to the lesion.
the animal will turn contralateral
to the literature
to the 6-OHDA
ipsilateral
built up in the striatum
supersensitivity
if
to the
due
agonist,
dopamine
the
apomorphine,
with
hand,
On the other
loss.
more than 95% innervation
dopamine than the normal level to compensate for the insufficiency
dopamine
from
transport
in dopamine
level
SNc to
Therefore,
the
show
any
not
may
striatum
ipsilateral
in the
striatum.
abnormality until the loss of the dopaminergic neuronal terminals in
the striatum
This contention
is greater than 85%.
has been tested
experimentally [see fig 3 in ref. 40].
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6(1990)
Chapter 5
MRI (ph MRI)
Pharmacological
Introduction
Detection of specific
neurotransmitter
activity
longitudinally could be of great assistance
therapy
pathology and subsequent
instance, the destruction
the striatum
for understanding
fetal cell transplantation
brain
For
of many disease processes.
of the nigral dopaminergic
in Parkinson's
and
non-invasively
disease and subsequent
would be greatly
innervation
therapy
of
using
aided by a means
of
following progression of the graft. Studies of receptor binding can be
performed
in vivo
using
positron
imaging, or can be performed
emission
post mortem
tomography
(PET)
using autoradiography.
These techniques allow one to use receptor agonists or antagonists to
directly map out the receptor density
of these binding sites in the
brain and also allow for investigation
of the drug induced neuronal
119
blood flow
cerebral
in regional
metabolic changes such as changes
(rCBF) or regional glucose utilization (CMRglu) induced by stimulation
PET is
However
[1,2,3,4,5].
d-amphetamine
as
such
of drugs
due to
compromised in its ability to evaluate humans longitudinally
magnetic
functional
brain
in
to a revolution
mapping.
These
neuronal
activity,
has
The technique
[6,7,8].
sensitive to these parameters
in MR signal
to changes
leading
hemodynamics
and
metabolism
T,-based
BOLD or
either
using
resonance
are based upon the coupling between
techniques
intensity
led
has
techniques
The technique of
involved in the in vivo studies.
the radioactivity
been labeled as functional MRI or fMRI and has been widely applied
activity
The use of fMRI to perform
pharmacological ligands is also possible.
nature.
measurable
response
measured
MR
by
techniques,
to
or PET, it is necessary
autoradiography
to obtain
due to the large doses necessary
However,
response
is due
repeatable
due to its non-invasive
is tempting
such experiments
to activation
context which are relevant
pCO2) needs
problem
to understanding
fMRI
response
binding or indirect effects
the
the coupling between
be
delineation
the receptor
is
(such
One approach
to be investigated.
would
hemodynamics,
potential
the
neurotransmitter
that
of the neurotransmitter
neurotransmitter binding and the hemodynamic response.
whether
to
compared
ascertain
a
There are a number of issues which arise in this
system in question.
of
using
of neurons
stimulation
by task activation,
induced
to neuronal
In addition
studies.
brain functional
to task initiated
of the
binding,
120
due
The issue
to
direct
as a change
to
correlations
neurotransmitter
in
solving
this
between
the
release
and
exist for studying these events
Several useful techniques
behavior.
including PET imaging [9,10] and microdialysis [1 1].
We have chosen to study the dopamine system to examine the utility
of fMRI for investigation of neurotransmitter activation for a number
density
instance, shows great regional specificity with high receptor
in
the
frontal
exist.
models of dopaminergic
animal
reproducible
These
are
models
binding
interest
great
of
addition,
In
ganglia.
basal
and
cortex
for
glutamatergic,
unlike the
system,
The dopamine
of reasons.
and
in
very
ablation
studying
neurodegeneration (Parkinson's disease) as well as, for instance, drug
The ability to selectively and unilaterally
abuse (cocaine addiction).
denervate
system
the dopaminergic
using 6-hydroxydopamine
(6-
to measure
the
experiment
OHDA) also allows
for a controlled
efficacy of whether
or not the phMRI response
dopamine.
is due directly
There is a large body of autoradiographic
to
data on the
response of both rCBF and glucose utilization rates to dopaminergic
ligands such as d-amphetamine,
utilized two different
the dopamine concentration
from the transporter
which appears
more specific [15,16].
to pharmacological
We have
D-amphetamine
is a
to work by increasing
in the synapse by release of dopamine
receptor
tropane, or
antagonist
ligands.
dopaminergic
dopamine release compound
transporter
of behavioral
data on the 6-OHDA model [12,13].
and autoradiographic
fluoropheny)
as well as a wealth
[14].
1 1 C-WIN
whose
35,428 compound) is a dopamine
effects
We demonstrate
stimulation
CFT (23-carbomethoxy-33-(4-
are much
here that the fMRI response
(hereafter
tight coupling to the regional distribution
121
like cocaine, only
dubbed
phMRI) shows a
of dopamine transporters
using
PET and
to the
release
of dopamine
as
studied
using
6-OHDA
were
microdialysis.
Methods
Animal
preparation-
Unilateral
medial
forebrain
bundle
lesions
with
processed and tested as mentioned in the animal preparation
session
The successfully lesioned animals (n = 18) and normal
in chapter 4.
control rats (n = 5) were scheduled for both PET and MRI studies. Six
dopaminergic fetal cell transplanted rats were included in this study,
too, to validate the tight correlation
and the dopaminergic
can
be
assessed
by
between
The success of the graft
neuronal activities.
behavioral
the phMRI responses
testing
using
the
amphetamine
rotational test described in the last chapter (see appendix in chapter
4).
Unfortunately, due to the tremendously long preparation time for
the 6-OHDA lesioning plus the fetal cell transplantation,
no baseline
studies (MR or PET ) could be done in the graft recipients before the
neuronal
MR
transplantation.
Measurements-
All MR measurements
imager (Fremont, CA).
coil under
animals
were
performed
Animals were imaged using a 35mm birdcage
halothane/N20/02
were
on a 4.7T GE Omega CSI
temperature
anesthesia
regulated
(1.5%
using
halothane).
a circulating
blanket at 38 0 C and were monitored for arterial
All
water
oxygen saturation
using a Nonin pulse oximeter (Minneapolis, MN) during imaging.
The
immobility
the
homogeneous
of the
animal
during
the
drug
stimulation
and
signal intensity of the gradient echo images over the
122
whole brain tissue were two critical conditions for the success of the
To attain these goals, the animal heads were shaved
phMRI studies.
dental
with either
and wrapped
hydroplastic
gel or water-soluble
bathroom adhesive caulk to immobilize the head and to reduce the
rats were imaged using conventional
repeatedly
acquired
Ernst angle RF,
gradient recalled echo images
(TR/TE 400ms/15ms,
area
striatal
the
from
The
interface.
created by the tissue-air
differences
susceptibility
At least 1 5
3 or 5 slices with slice thickness 1.5mm).
stable base line image sets were acquired before the pharmacological
Three
stimulation.
tropane
carbomethoxy-3p3-(4-fluoropheny)
compound,
MA) were
RBI, Cambridge,
while the images were being
oxygenation saturation
artery
was connected
(CFT, or
then
WIN
23-
35,428
intravenously
given
acquired, the imaging was continued
post injection.
for 90 to 180 minutes
0.75mg/kg
or
D-amphetamine
mg/kg
to the blood
In addition
monitoring, an arterial line from the femoral
to a cardiac monitoring
device
(706 patient
monitor, Ivy Biomedical System Inc.) in the nonlesion control group.
and heart
The mean blood pressure
minutes
and
the arterial
rate were
every
recorded
blood gases (pO2, PCO 2, and
3
pH) were
sampled in 15 minute intervals. However not all animals, especially
for the rats with 6-OHDA lesions and the group with grafts, could
have blood sample or blood pressure monitored during MR scanning
due to the impossibility of performing the necessary
survival studies
with the surgery required to insert the femoral arterial line.
In addition to the MR study, another control population
of animals
was studied on bench tops under similar physical conditions as the
in-magnet
studies
(using
1.5%
123
halothane
anesthesia,
body
etc).
monitored,
regulated,
Measurements
of blood gases (pO2 , pCO 2, and pH), heart rate and
blood pressure
blood
oxygenation
temperature
were obtained
the same fashion as the in-magnet
studies over two-three hour time periods including baseline and post
pharmacological
i.v.
amphetamine
(3mg/kg
stimulations
or
0.75mg/kg CFT i.v.).
PET
Measurements-
The same
animals
were
by phMRI
tomography
emission
positron
studied
(PET).
also
scanned
Anesthetized
using
animals
were
placed on an imaging table. The head was secured in a customized
Plexiglas head holder equipped with ear and mouth bars designed to
reproducible
ensure
dopamine transporters were performed
as a tracer.
Imaging
head positioning in PET.
studies
of
labeled CFT
using carbon-11
For PET imaging 0.8-1.2 mCi of the labeled compound
(specific activity 800-1600 mCi/mmol) was administered into the tail
vein.
Serial dynamic imaging was acquired over the rat brain for 60-
90 min using 4.5 mm steps
between
and counts
were
for
15-60s intervals depending upon how far along the 20 min
half life "C decay curve the measurement
was
block averaged
performed
using
a home-built
high
occurred.
PET Imaging
resolution
single
ring
tomograph, PCR-I described earlier [17]. The resolution of PCR-I for
a point source at the center is 4.5 mm and the sensitivity
is 46,000
Hz for a source of 20 cm in diameter with a concentration of 1 mc/cc.
The overall efficiency is 64% of the theoretical maximum for a plane
thickness
corresponding
to the 2 cm high
crystals.
The
plane
thickness of 4.5 mm used in this study was obtained by the use of
cylindrical collimators which limit the effective height of the crystals.
124
attenuation,
Imaging data were corrected for uniformity,
decay and
Images were reconstructed using a ramp filter with
acquisition time.
a cut off value of 1.0 and convolution back projection.
--
Microdialysis
The extracellular
striatum
was accessed
literature
by using microdialysis.
lateral
dorsal
For amphetamine
of dopamine
data
we used the existing
stimulation,
from the
concentration
dopamine
from
release
For CFT stimulation, home made glass dialysate
[11,18,19].
probes were implanted into the dorsal lateral striatum in the normal
The dialysates were collected in 20 minute time blocks.
control rats.
The same CFT protocol as in the fMRI study was followed in the
microdialysis
injection.)
study (40 minutes
baseline and 90 minutes
post CFT
HPLC was used to analyze the dopamine concentration in
the dialysis.
Unfortunately,
because
procedure, we can not perform
of the invasiveness
of this
longitudinally
and we
microdialysis
used a different set of control animals than the fMRI study.
Data
Analysis-
The activation
maps of the phMRI data sets were
statistics
Komolgorov-Smirnov
images.
Two approaches
were
and overlaid
utilized
on the
to define
generated
gradient
using
echo
the activation.
Areas of activation as defined by the statistical maps were calculated
using automated segmentation of the maps at a predefined
of p < 0.01 (determined
be significant).
by requiring
threshold
at least four activated pixels to
The percent signal changes were also measured
these areas as well.
In order to determine
in
specificity, the anatomic
structures were segmented using a rat brain atlas [20]. The fraction
125
activated, as determined
of each structure
the ipsilateral
and
both
the
measured
then
were
hemispheres
contralateral
between
The differences
was then calculated.
from the statistical maps
using
percent signal change and the areas as
Contralateral - Ipsilateral
Contralateral
These numbers were then compared with the PET data by averaging
together
MRI slices
1.5 mm
the three
to obtain
same
the
slice
thickness as in the PET studies.
since there
of the PET data,
For analysis
to represent
are assumed
areas such as striatum.
and
in other brain
Regions of interest (ROI) including left and
were drawn and the percent activity
right striatum and cerebellum
of the injected dose was calculated.
transporter
binding
non-specific
hence can be used to normalize the signal responses
dopamine
specific
occurred in the cerebellum, the signal changes
dopaminergic binding
in cerebellum
little
is very
Binding ratios of "C-CFT into the
calculated
were
according
the
following
formula:
Percent Activity
(Percent Activity
Binding rati
# of Pixel
]str -
# of Pixel
BinPercent
ding ratio Activity
# of Pixel
Icerebellum
)cerebellum
The values of the binding ratios were averaged
between
55 and 6 5
min (so as not to include the initial activity due largely to flow) and
compared to the values of hemodynamic
well as to rotational
behavior
indices from MR studies as
using the amphetamine
described above.
126
stimulation
Results
The injection of either amphetamine
or CFT caused an increase
in
signal intensity using BOLD imaging that was regionally localized to
areas of the brain that are high in dopaminergic
shown in Fig. 5-1.
(but not parietal
receptor density as
These areas include cingulate and frontal cortex
cortex)
and striatum.
Initially,
we used higher
doses of amphetamine which led to non-specific increases in all areas
of the
brain
amphetamine
produces
as also reported
[21].
The
by
dose
an phMRI response
Silva et
of 3mg/kg
al. using
i.v.
20
mg/kg
of amphetamine
which is similar to that produced
0.75 mg/kg of CFT. The regional specificity of brain activation
by
was
measured by automated segmentation analysis of the statistical maps
generated by analysis of the time courses and measuring the fraction
of the anatomic structure activated.
5-2.
These results are shown in Fig.
It is clear that the frontal cortex and the striatum are the areas
which show the largest
dopaminergic innervation.
activation,
in keeping
with
why the parietal cortex activation
volume
averaging
Figure 5-3 illustrates
overlapped
large
Parietal cortex which is relatively low in
dopamine innervation has relatively low fMRI response.
partial
their
is not zero may be due to the
of our relatively
the parietal
The reason
thick
slice (1.5
cortex in the mid-striatal
with the striatal area in the more posterior
overlap is 10% in the parietal area.
127
mm).
slice is
slice.
The
(A)
D-Amphetamine: 3mg/kg i.v.
10-14
10-12
10-10
10-8
10-6
10
to
4
•8
wr
v
, 4
P7Q
a-ia
E
2
Ei
Q!
5-
44
Time (min)
Time (min)
B ImP.I
(
0-9
0-7
0 -5
3
0
2
AU
PMA
0
[]
ID
iC
.P"
VM
!
S-J50
0
50
Time (min)
100
150
M3-
Time (min)
Figure 5-1 Statistical maps of increased BOLD signal change in
normal control rats after injection with (A)3 mg/kg amphetamine or
(B)0.75 mg/kg CFT. Changes in blood pressure and pCO 2 as
measured in the magnet are shown with the percent signal changes.
Note the BOLD signal change is delineated from blood pressure and
pCO2 changes.
1'lM
''"'
IUU
.4 ,~,'
80
Tr
60
• ll11
• 40
20
•
•
CFT
Amphetamine
Figure 5-2 Bar graph showing the fraction of anatomic area activated
by either amphetamine ( n = 18) or CFT (n= 12). Segmentation was
performed as described in text. A schematic of the areas segmented
is shown. 1. frontal cortex, 2. striatum, 3. cingulate cortex, 4. parietal
crotex.
10% of the parietal cortex
isoverlapped with the striatum
from the more posterior slice
Fig. 5-3 Partial volume averaging in the parietal cortex. Yellow
area with black line indicates the contour of the slice from the
mid-striatum. Red line indicates the contour from the more
posterior slice. The slice thickness in this study is 1.5 mm with 0
mm gape between slices.
Analysis of the time courses show that the response of the phMRI is
transient,
peaking
at about
40 min
to the
to
We thus performed
a
Shown in figure 5-4 are the effects
of
of the effect.
reveal much about the nature
of experiments.
back
This time course has the potential
baseline in about 70-90 min.
number
and decreasing
amphetamine and CFT on blood gases and blood pressure.
It is clear
that the time courses for these changes do not parallel those of the
phMRI response as seen in figure 5-1.
The effect is clearly not due
spike seen in the blood pressure
to increased pCO2 or the transient
curves.
In order
to show
that
the time
course
of fMRI signal changes
parallels the time course for release of dopamine in the dorsal-lateral
striatum,
corresponding
5-5,
the fMRI time courses with time bins
we block averaged
to the microdialysis data points.
the fMRI time
course
is virtually
release of dopamine measured
well correlated
to the percent
superimposable
by microdialysis
change
concentration (Fig. 5-5 B).
131
As shown in figure
with
the
(Fig. 5-5 A) and is
in extracellular
dopamine
Amphetamine 3mg/kg i.v. n=12
Amphetamine 3mg/kg i.v. n = 4
~···1
'-'"''
· 7·.
1
.
1
. .
A)
110
.
.
.
.
(B)
105
I4-
100
95
90
85
8RO
-4 0
''''~
a
-20
60
40
20
1
I
I
I
I
0
80
0() 9N...
-4
,...~....
......
· · ·80
60
40
20
0
-20
Time Post Injection (min)
Time Post Injection (min)
CFT 0.75mg/kg i.v. n =4
CFT 0.75 mg/kg, n = 7
120
I
115
(D)
110
t1105
E100
,m ,
_
n .
.
.
.
. m
_
_
.
m
-
95
90
8R5
-40
-20
0
20
40
Time Post Injection (min)
60
90
*:)
-60
-40
-20
0
20
40
60
Time Post Injection (min)
Figure 5-4 Changes inblood pressure and pC02 for all the animals
pooled for amphetamines and CFT.
phMRI n = 1
(A) -4-- Microdialysis*
(B)
in
N5
3000
8
2500
2000
b$4
=
f 03
1500
2
1000 n
~
'I"
11111
. .
I
.
.
1
.
.
.
.
.
.
.
-
phMRI, n = 9
500
0
c1
.
-20 0 20 40 60 80 100 120
0
- phMRI, n = 1
-- Microdialysis, n = 4
h
(D)
1200
phMRI, n = 9
5
E~
,
ba
y = -26 + 4,4 In(x), p < 0.08
4
800
st
dr
EL
U
3
400
a
,,
1000IUUU 15UU 2UUU 25UU
Percent [DA] change (%)
Time Post Injection (min)
(C)
5UU
I
bJ,
o
a
0
t
a
· · I···i··
.
- -1..
- - .r1 r .-4U -ZU U
1)111)11.)111)111
. ..
..
..
..
.
.
.
.
AA
20 40 60 80 100
Time Post Injection (min)
°=
1
AA
400 500 600 700 800 900 1000
Percent [DA] change (%)
Figure 5-5 phMRI time points are block averaged with time bins
corresponding to the microdialysis data points. (A) amphetamine (C)
CFT stimulation. (B)and (D)show the correlation between the
percent phMRI signal changes versus percent increase in dopamine
release.
Destruction
input
of the dopaminergic
to the
frontal
cortex
striatum should lead to an ablation of the phMRI response.
shown
in Fig. 5-6 using both
amphetamine
typical of the greatest, average
and CFT.
and least differences
and
This is
Responses
between
the
ipsilateral and contralateral sides are shown in this figure in order to
get an indication
of the
Two time
measured.
animals shown
courses
range
of the signal changes
of responses
seen
that the response is much decreased
in the
on the
To prove this was not due to an artifact, such as loss
of regional blood flow or hemodynamics,
parameters
and
in Fig. 5-6 are presented in Fig. 5-7 for amphetamine
and CFT. It is apparent
ipsilateral side.
reproducibility
we measured
these latter
(rCBV and rCBF) using bolus injections of Gd(DTPA) [22,
23]. There was no statistically significant difference in the resting
rCBV or rCBF between
decreased
the two
phMRI response
hemispheres
is not due to a decrease
stimulus values of these parameters
in the previous chapter.
indicating
that
the
in the pre-
on the ipsilateral side as shown
These data are presented,
along with rCBV
and rCBF images in Fig. 5-8. In order to compare the phMRI response
to behavioral
correspond
changes,
we time binned
to the behavioral
changes in the striata
the fMRI time courses
data (figure 5-9 A).
ipsilateral
to
The fMRI signal
to the lesioning
parallel the time
courses of the rotational behavior in these animals.
Figure 5-9 B also
shows that the time course for the behavioral data of the unilaterally
lesioned rats also correlates
well with the extracellular
concentration.
134
dopamine
3 mg/kg D-amphetamine i.v. injection
0
-8
-7
-6
-5
-4
(A)
0
-3
0.75 mg/kg CFT i.v. injection
0
-8
-7
-6
-5
-4
(B)
o
Figure 5-6 phMRI responses of the unilaterally lesioned rats
with stimulation of (A) amphetamine (B)CFT. Shown here
are rats with good to bad phMRI reponses (top to bottom).
-3
.p Contralateral
4- Ipsilateral
(A)
•.
a
4
\r,,
3
2
ert
U
01
4)
3-'1
4)
1
0
-1
-2
_I
i0
--
Time Post Injection (min)
(B)
10
-4 Contralateral
4- Ipsilateral
8
6
4
2
0
-2
-A
-50
0
50
100
150
Time Post Injection (min)
Figure 5-7 Time courses in the ipsilateral and contralateral
striata in one of the 6-OHDA lesioned animals.(A) amphemine
3mg/kg (B) CFT 0.75 mg/kg
CBV
CBF
CBF
CBV
Control Lesion
Control Lesion
Lesioned striatum / Control Striatum (n= 9)
CBV = 1.05 _+0.27
p = 0.88
CBF = 0.99 + 0.11
p = 0.47
Figure 5-8 Maps of rCBV and rCBF generated using bolus injection
of Gd(DTPA) in the resting state (no amphetamine or CFT) in 6OHDA lesioned animals. Note there is no significant differences
between the lesioned and control sides indicating that 6-OHDA does
not cause destruction of the vascular bed. See chapter 4.
T"
-e-
Illlllllll
Net Ipsilateral Turns
-*- ph MRI, contralateral striatum
(A)
4
0M
Ln
riE
ph MRI, ipsilateral striatum
"4
r
120
T
n=12
80
3
..
I II,
III
. I
i
I I I
II.,.
I
I I
20 40 60 80
-20 0
Time Post Injection (min)
-* turns (n=
*
% [DA]
i:
-9
100
12)
4 A
0
t't~#
30VV
0
R
-- 0
-40
.0~
9Q
NNW
20
0
-1
('D
CI.
10A
"do
40
1
~0
c0
60
2
(B)
z
100
0
250(
20
200(
0
0
0.
oo,
150(
100(
0
50(
o Bc
a
Time Post Injection (min)
Figure 5-9 (A) phMRI response to amphetamine stimulation in a
unilaterally lesioned rat (with time bins corresponding to the
behavioral data points): the contralateral striatal time coures
parallels to the behavioral data. (B)Correlation between dopamine
release and behavioral data post amphetamine stimulation.
In
to investigate
order
transporter
and
receptor
experiments
on
the
the
same
we
phMRI
animals
was, in general,
dopamine
to
decided
studied
very
the
between
The comparison between
techniques above.
phMRI results
relationship
the
using
run
the
consistent.
In the
compared to the unlesioned, control animals (Fig. 5-10).
change
between
difference
(contralateral
in
lesioned
striatum,
The phMRI
this finding as is also seen in Fig. 5-8.
results faithfully reproduce
average
phMRI
the PET results and the
animals a loss of "C-CFT uptake is noted in the ipsilateral
The
PET
the
the
describing
parameter
ipsilateral
and
-ipsilateral)/contralateral)
the
contralateral
percent
striatum
was 39 ± 4% for PET
and
the decrease in phMRI response to amphetamine or CFT stimulus was
39± 16% (n=12; p < .001)
and 42± 18 % (n=18; p < 0.001) respectively
when measured as change in areas (figure 5-11). However, there is a
large decrease in this asymmetry
post transplantation
[PET = 20.90±
8.64 % (n=9; p >0.25), fMRI with CFT = 15.76± 3.66 % (n=9; p >0.35)].
Obviously
because
the
grafts
much
are
smaller
good correlation
transporter
the PET images
total
clearly
The
animals.
indicates
that
the
to the dopamine
is well reflected by the metabolic response as shown by
Although
correspond
the rats were
to those
nigro-striatal
innervation,
data had
screened
with greater
number of rotations between
behavioral
for all transplanted
binding of both CFT and amphetamine
decreased
phMRI.
with
recovery
the
even though there
striatum, we may not get return of full symmetry
was a full behavioral
than
there
than
were
animals.
any correlation
by rotational
data
to
95% loss of dopaminergic
clear
differences
in
the
Thus, we looked to see if the
with
the imaging
data.
We
compared loss of CFT binding in the ipsilateral striatum using PET or
139
metabolic
using
response
Ipsilateral)]/Contralateral)
phMRI
The correlation
as
[(Contralateral
between
the
PET, the
phMRI and the behavioral data (rotation) is shown in Fig. 5-12.
It is
clear that there is a weak, but significant, correlation
between
the
There was a strong correlation
between
the
rotation and the PET.
CFT and rotation, but none with
amphetamine.
140
PET
ph MRI
-8
-7
-6
-5
-4
-3
Figure 5-10 PET images and the corresponding phMRI images from a
6-OHDA lesioned and a neuronal transplanted animal. PET images
come from a slice centered on the striatum. There is good
correspondence between the PET images of CFT uptake and the
phMRI images of the metabolic response, with the exception of the
cortical regions.
Percent of striatal
I area activated
CFT
11C-CFT binding
phMR Amphetamine
I
PET [
60
50
UCI
0PN
*ImU
0Et
"
·c
40
30
20
10
Lesion only Rats
Transplanted Rats
Figure 5-11 There is big asymmetric response in the two
striata in the lesioned only rats. The asymmetry is greatly
reduced in the neuron transplanted rats.
500
I
80
60
40
Ir)
o
0)
cU
c
20
100
1000
1500
2000
I
y = 28 + 0.01 X; p < 0.15
11C-CFT
PET
I
I
80 y = -28 + 0.05 X; p < 0.03
60
40
20
0
80 Sy = 25 - 0.004 X; p > 0.75
60
40
20
I*
P
I
0
1500
500
1000
Turns in 90 Mins
CFT
ph MRI
Amphetamine
ph MRI
2000
Figure 5-12 Correlations between the PET and the phMRI and
the behavioral data.
Discussion:
We have demonstrated
for examination
effects
of the
We utilized
stimulation.
receptor
task
during
function
of brain
fMRI studies
appropriate
that the techniques commonly employed
a well
also
are
activation
for
of neurotransmitter
model
characterized
of
dopamine denervation to show that the metabolic response resulting
from stimulation of the dopamine system is well circumscribed to
and
those areas of the brain containing large dopamine innervation
that
both
for
of issues which
before studies of this nature
become routine
neurotransmitter systems in general.
a metabolic
response
in
system
dopamine
the
particular;
to neurotransmitter
the large doses necessary
This problem
other
and
The fact that we are observing
binding rather
binding itself, as in PET, creates some problems.
effects.
dopamine
nigro-striatal
There are a number
connections are denervated.
need to be addressed
the
when
is ablated
response
the
than
the
One problem is that
to see an effect may lead to non-specific
is especially
system
acute in the dopamine
because of the possibility of stimulating other monoamine systems in
the brain using a compound
problem is understanding
hemodynamics
like amphetamine
the coupling between
Another
[1,2,3,4].
the changes in the
with the changes in dopamine concentrations.
If the
is only coupled by very indirect means
to the
metabolic response
neurotransmitter
release then the phMRI technique
is not as useful
as if there is a direct correlation.
Our results shed light on these problems.
First, the spatial specificity
we observed in our phMRI results with high fMRI responses
brain
areas
with
high
dopaminergic
144
innervation
and
in those
the
nice
that the
data indicate
fMRI and microdialysis
between
correlation
fMRI effect is primarily due to dopamine and not other monoamine
systems which would lead to more non-specific cortical activity.
This
is indeed what we observed with higher doses of amphetamine
and
has
of CBF [1,2,3,4].
studies
in autoradiographic
seen
also been
Secondly, we showed that the time course for the signal changes are
of simple
not reflective
physiological
like
changes
hypercapnia,
further indicating that the response is due to a metabolic response to
using the well known model of 6-OHDA also showed
the striatum
using
in the ipsilateral
denervation
due
not
was also
striatum
in the
more credence to the origins of the phMRI signal differences
ipsilateral
percent
both the
identical.
and
striatum.
contralateral
differences
and
The mean
of the
values
- Ipsilateral)/Contralateral)
((Contralateral
PET binding
to
of 11C-labeled CFT, thus lending
using PET imaging
demonstrated
was
on the lesioned side
impairment of vascularization or hemodynamics
The dopaminergic
rCBV and rCBF
of resting
this
both
for
side
ipsilateral
of Gd(DTPA) showed
injection
bolus
Measurements
and CFT.
amphetamine
on the
ablated
nearly
was
as the change in signal
was due to dopamine
that the response
intensity
input to
Denervation of the dopaminergic
binding.
neurotransmitter
phMRI percent
signal
changes
for
were
While it is not clear at this time how to turn PET binding
into
measurements
blood
flow
changes,
the
nonetheless
tight
coupling between loss of CFT binding as measured by PET and loss of
BOLD signal
change
using
the
6-OHDA model
indicates
that
the
coupling between these two parameters may be quantitative.
In this regards
it would
be instructive
to evaluate
other
pulse
sequences to examine rCBF using T,-type techniques [7,21,24] as well
145
as
glucose
and
consumption
are
there
If
BOLD.
the
that
conclusion
to the
came
but
mode for
in an interleaved
in these experiments
stimulation,
amphetamine
as
We initially tried using IR
differences in these two signal responses.
and GE sequences
up
show
should
these
utilization
oxygen
flow,
between
dissociations
statistical power was not sufficient given the relatively low SNR of
the images.
Use of EPI techniques would alleviate this problem, but
our current
implementation
for temporal
resolution
of EPI involves
(1 mm vs. 0.25
resolution
of spatial
a tradeoff
in plane
mm
resolution) which makes it difficult to adequately segment structures
such as the cingulate and the accumbens in the small rat brain.
The time course noted for the phMRI signal changes are quite similar
particular,
time
the peak
using
measured
release
for dopamine
In
[11,18,19].
measurements
microdialysis
using
seen
to those
similar doses of amphetamine corresponds exactly to the peak of the
phMRI response at about 40 minutes.
This implies that the phMRI
response is due to the release of dopamine in the striatum.
The sum of these facts, and the loss of phMRI response after 6-OHDA
lesioning,
strongly
suggests
that
response
metabolic
the
primarily to the release of dopamine in the striatum.
it is instructive
phMRI.
to compare
the
behavioral
correlation between
In this regards
and PET data
and 5-12 there
As shown in Figs. 5-9, 5-11
is due
to the
was a good
the PET, rotation and CFT phMRI results.
This
was somewhat surprising as, according to the literature, rotations of
greater
than
should
600turns/90mins
dopamine cell loss in the striatum
screened
to manifest
[12,25].
this behavior,
146
reflect
the
greater
than
95%
Since all our rats were
amphetamine
results
we
obtained (i.e. no correlation with behavior) were what we expected.
transporter
phMRI using CFT implies that the some dopamine
still be present
literature,
as when
for this in the microdialysis
extracellular
measures
one
may
loss of the pre-synaptic
in spite of the supposed
There is some precedent
terminals.
with PET and
of behavior
correlations
we observe
The fact that
levels
dopamine
versus percent dopamine neuronal depletion, there is a good deal of
scatter - even for animals with nearly complete lesioning (see Fig. 3
variability
in ref. 19 ). This same inter-animal
behavioral/histological
observed
[25].
studies
has also
finding
This
in an
of CFT binding
in PET studies
has also been seen in
MPTP model
where CFT uptake is sometimes
Parkinsonism in primates
been
of
still seen,
even after lesioning (Brownell et al., unpublished ).
by the phMRI technique
manifested
The sensitivity
useful tool for pre-screening
stages.
of Parkinson's
disease
may make it a
in the earlier
Since symptoms of PD do not seem to occur until roughly 80%
of the dopaminergic innervation of the striatum is destroyed, there is
to changes that are on this order.
the possibility of being sensitive
This technique
may
also prove
useful
for evaluation
of other
dopamine ligands such as D1 and D2 agonists and antagonists.
ability of MRI to be used longitudinally
The
may enable
and repeatedly
one to study up and down-regulation of the dopamine receptors over
time.
abuse.
This could be of great interest
In
addition,
neurotransmitter
the
systems
possibility
in phenomena
such as drug
of examination
is quite feasible and will prove
fruitful area for future investigations.
147
of
other
to be a
Conclusions:
agonist CFT and
We show here that use of the dopamine transporter
release agonist, amphetamine,
the dopamine
response
features:
to
stimulation
dopaminergic
time
course
for release
3) The response
microdialysis;
with
correlated
in
transporter
dopaminergic
the
the
has
following
density; 2) The time course follows
in
is selectively
ablated
via
striatum
via unilateral
6-OHDA; 4) The response is
PET and
vivo
histologic
of
measures
binding as measured using "C-CFT; and 5)
is restored
response
the
of dopamine
lesioning of the nigral-striatal tract using
well
which
1) The response is regionally specific to those areas of the
brain with highest dopaminergic
the
cause a hemodynamic
in
those
same
animals
using
neural
transplantation of fetal mesencephalon cells into the striatum.
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151
rotational
behavior.
and Future Work
Summary
Thesis
Chapter 6
The primary objectives of this thesis were to investigate and verify
neurodegenerative
disease (PD) to evaluate the possible neurochemical
the neuronal
responses
on Parkinson's
We focused primarily
processes.
caused by disturbance
modulation
and
of the dopaminergic
system in the movement disorder of PD.
Although the Parkinsonian
and described
unclear.
and
have been
symptoms
since the 19th
century,
well documented
the etiology
of PD is still
Human evidence includes drug abusers who received MPTP
subsequently
developed
idiopathic PD[1]. Further studies
signs
symptoms
resembling
showed that MPP +, the metabolite
of MPTP, can block the enzymatic
electron transport chain.
and
activity
of complex
I in the
This human evidence suggests that PD may
152
of mitochondrial
with dysfunction
be associated
in the
respiration
Blockade of the electron transport chain may lead
basal ganglia [2,3].
to cessation of the TCA cycle, which in turn leads to accumulation of
This provides us an opportunity to monitor the lactate
lactic acid [4].
The lactate level in the
levels in the PD brains by using 'H-MRS.
using 'H-MRS
resting state of a normal brain is nearly undetectable
and any extra accumulation
lactate in the resting state
of cerebral
may indicate an abnormality in energy respiration. In this thesis we
tried to measure
in PD brains and normal
the cerebral metabolites
age-matched subjects by using 'H-MRS.
the
Although
energy
impairment
regarding
information
this neuronal
process
mechanism
involved
in basal
provide
valuable
the question
with
is associated
order
In
is yet to be answered.
disorders
may
degeneration,
neuronal
degenerative
theory
ganglia function
of how
movement
to understand
better,
one has
the
to
Postmortem studies
investigate the neurotransmitter system as well.
of PD brain show neuronal loss in the substantia nigra pars compacta
(SNc) [5,6]. Functionally, the neuronal loss in the SNc will lead to
alterations
in the striatal dopaminergic
from both PD patients
and animal
innervation.
models
However, data
of PD show
that
th e
Parkinsonian symptoms will not start to show up until there is about
80% to 90% loss of the dopaminergic
[5,6,7].
To mimic the abnormal
innervation
dopaminergic
in the striatum
innervation
in the
striatum, we used a well studied animal model with injection of 6hydroxydopamine
(6-OHDA) to selectively
the striatal dopaminergic innervation [8,9].
and unilaterally
deplete
This animal model allows
us to investigate 1) the possible striatal metabolite changes after the
153
regional
the
assess
metabolites
striatal
The
in brain.
activity
dopaminergic
to
2)
and
denervation,
dopaminergic
can be
In this thesis, we chose to use
assessed using 'H-MRS technique.
3D-
chemical shift imaging technique (3D-CSI) which allowed us not only
the
assess
distribution
information
metabolite
cerebral
but
the
also
over the whole brain. The regional
of those metabolites
MRI (fMRI)
activity can be assessed using functional
dopaminergic
We dubbed
ligands.
of specific dopaminergic
with the stimulation
spatial
this technique as pharmacological MRI or phMRI.
The main goals of this thesis
cerebral
levels
metabolite
are
using
evaluate
the
2) to validate
the
1) to accurately
'H-MRS, and
activity. In
possibility of using fMRI to assess the neurotransmitter
order
to evaluate
the
metabolite
cerebral
level
accurately,
we
investigated in this thesis the conditions that may alter the accuracy
of spectral analysis. We used computer simulations to synthesize 'HMR
under
spectra
relaxation
Only accurate
comparison
times
spectral
and
absolute
analysis
in the cerebral
field
strengths
the possible errors involved
We also evaluated
homogeneities.
both
magnetic
various
concentration
allows
us to have
and
in
measurements.
a reasonable
metabolite changes between
the normal
and disease states.
Technical Concerns in using 1H-MRS to explore the striatal
metabolites
The utilization of 'H-MRS to quantify the metabolic concentration
the striatum
in
involves several technical difficulties, such as spectral
154
low
overlap,
measurements.
ratios
noise
to
signal
We used computer simulations
metabolite
overlap,
spectral
relaxation
spin echo experiments
how
times and the
it would
be
times T, and T 2 because
of
and
J-
that
[10,11]
is usually achieved
signal intensity
and the
exponential decay as function of TE.
by multi-echo
will be a mono-
With spectral overlap, the signal
decay is no longer a mono-exponential.
coupling, the decay curve
to demonstrate
contamination
macromolecular
The T 2 measurement
coupling.
rate
We found
concentrations.
difficult to obtain accurate
relaxation
and
of the relaxation
each factor alters the estimation
absolute
(SNR)
When the overlap involves J-
oscillates in a sinusoidal
fashion
which
even more complicated.
makes the T 2 relaxation time measurement
Our computer simulations showed that the T 2 error could be as high
as over 100% and the error does not depend
on the number
of TE
and there are no magic TE values
points chose in the measurement
one can use to minimize this error.
When propagating
the relaxation time error into the estimation
the metabolite concentration,
of
we found that the T, error of NAA is
more critical than the T, errors of water.
The percent error in the
NAA concentration caused by the T, error of NAA is between 45% to
65% as a function of TEs.
The error essentially
comes from the
saturation factor and the longer the T, value is (T1NAA
greater
error
ErrorTINAA
will be in the concentration
> [NAA] ErrorTiwater ).
minimized by using a longer TR value.
the concentration
> Twater),
measurement
This T, error
factor
errors propagated
155
([NAA]
could
be
When TR is longer than 3 sec,
error due to T, error can be ignored.
none of the concentration
the
However,
from the T 2 errors
of
The T2 errors can make the error of
water or NAA can be neglected.
Therefore, the accuracy
the NAA concentration as high as over 100%.
of T 2 measurement
is much more important than the accuracy
of T,
measurement in estimating the absolute metabolite concentration.
Although our computer simulated spectra showed great similarity to
and the phantom spectra, our simulation was only based
the in vivo
upon the assumption of weak J-coupling and lack of macromolecular
components.
Future work would add in these two factors
to the
to investigate
the J-
simulations.
computer
It is also important
coupling effects under the influence of different pulse sequences.
In conclusion, absolute metabolite concentration
measurements
may
reliable.
We
have been a great objective but are not practically
the
relative
concentration
believe
that
provide
more useful and reasonable
measurements
comparisons
[12] may
for quantitative
analysis, especially for inter-laboratory comparisons.
1H-MRS
in PD patients and animal model of PD
The hypothesis that disruption of the electron transport chain in the
basal ganglia may be responsible for the movement
was
assessed
examine
by monitoring
the striatal
lactate
controls
the
lactic acid level.
level
using
in both
a
disorder in PD
We chose
to
PD patients
and age-
localized
'H-PRESS
spatially
matched
normal
sequence.
We also examined the lactate level in an animal model of
PD (6-OHDA lesioning) using a 3D-CSI technique.
In the human
study, we found that the striatal lactate level in PD
patients was elevated 59% compared to normal controls (p <0.0004).
156
'H-MRS
No other
major
significant
differences
striatal
the
PD and
between
for both PD patients
comparing
the lactate
and normal
the
control
normal
in the two
Just
controls (p < 0.02).
side, the PD patients
levels on the largest
showed a significant increase
showed
metabolite
We also found a significant lactate asymmetry
groups.
striata
detectable
to the
in the lactate level compared
most of
Interestingly,
normal controls (64.51% increase, p <0.05).
the subjects (PD and normal controls) have a higher lactate level in
are left hemisphere
Since most of the people
the left striatum.
has long been hypothesized
dominant, and the left hemisphere
to
The left
govern the mental computation, language, and motor tasks.
striatum thus has been hypothesized to have a heavier working load
than the right striatum.
The higher neuronal
firing incident rate in
the left striatum may lead to higher vulnerability with energy insults
and this may be the cause of the asymmetric lactate levels in the two
striata
for
However,
the
animals,
we
the
striatum
ipsilateral
elevation
in
significant
lactate
lesioning.
This may be due to the fact that
lesioning causes
cell death too rapidly
acute
the
find
not
did
6-OHDA lesioned
to
the
6-OHDA
(i.e. within 24 hours),
and
lactate may only be elevated during the first few days post-lesioning.
Since we only studied
these
animals
at least three
lesioning, we may not have seen lactate for this reason.
the neurodegeneration
process
in PD patients
which allows us to monitor
weeks
after
In contrast,
is a much slower progressive
the dynamic
lactate level in a much wider time window.
changes
in the
However, the neuronal
marker N-acetylaspartate (NAA) showed a significant decrease in the
157
striatum ipsilateral to the 6-OHDA lesioning (9.61%, p <0.0003; range
5-21%).
of the dopamine
of the degeneration
the rebalance
finding
histological
the animal NAA loss and the human
may represent
The mismatch
cell loss in the striatum.
not find any significant
between
PD brain did
of postmortem
Although histological studies
terminals and the resprouting of the serotonin (5-HT) fibers [13,14].
words,
In other
the NAA loss may just reflect the net neuronal
after the 6-OHDA assault.
(cell bodies) in the striatum
population
It has been found that PD
iron deposition in the ipsilateral striatum.
patients
have
animals
and 6-OHDA lesioned
due to the extra
mismatch
The other possibility is a susceptibility
high
relatively
iron
deposits in their brain [15,16]. Iron is paramagnetic
and can induce
AR2* changes and alter the signal intensity of NAA.
This extra AR2*
term may not play an important role in the water images (T 2 and
diffusion
weighted
T2* of water
due to the much shorter
images)
than NAA.
Overall, 'H-MRS provides
metabolites.
access
reliable
in vivo cerebral
to the
Although our studies showed elevated lactate levels in
PD patients, which did not correlate with age, disease severity, DOPA
dose administered of the duration of PD, there are still other possible
clinical variables which may influence the lactate levels.
PD patients
were
movement disorder.
brain
metabolism
taking
continuously
medication
Most of the
to reduce
the
Whether or not these medications can alter the
and
how
it would
long
metabolism
to return to the baseline
medication
is unclear.
Although
be
for the
neuronal
level after withdrawal
we would like to examine
158
of the
naive
difficult
practically
and disabling side effects patients may suffer during
uncomfortable
the medication off period.
applications
Finally we can ask if there are any clinical
Can we use 'H-MRS as a diagnostic
of this technique.
detection
tool for early
important
possible
the
to
due
reasons,
ethical
for
it is very
of the drug medication,
patients without any interference
clinical
an
therapeutic
possible
investigate
to
tool
Can it be
disease?
of Parkinson's
Since PD is a disease associated with aging, and many
interventions?
due to natural aging
problems
elderly persons may suffer neuronal
and Alzheimer's disease, it is possible that the detection of a small
It is known that PD symptoms don't show up unless
rather than PD.
degenerative
of the neuronal
but not be present as an early symptomatic sign.
of use to measure
lactate
for aid in the
is
may only
level in PD patients
Thus, the high lactate
indicate the near completion
in the striatum
innervation
more than 80% of the dopaminergic
lost [5,6,7].
conditions
may be associated with other neuronal
lactate elevation
process
However, it may be
of possible
evaluation
therapies by estimating the lactate levels pre- and post- medication;
electron
using perhaps
the
Furthermore,
understand
physiological
the
lactate
striatal
challenges,
transport
chain
may
level
function
cofactors
provide
a way
in PD patients.
PD brains may have
as CoQ10.
such
greater
to
Under
help
to
certain
difficulty
in
providing enough energy to perform tasks, and thus the lactate level
during neuronal stimulation may be different than normal subjects.
This hypothesis
remains to be investigated
for future studies.
For
instance, Scholz et. al. [17] has shown several motor tasks which will
activate basal ganglia in fMRI studies in the normal controls.
159
One can
lactate level before, during, and after the motor tasks
monitor
both PD patients
and
combine motor tasks
of the
response
normal
and functional
basal
In other
controls.
ganglia
spectroscopy
possible
under
in
one can
words,
the
to evaluate
stress
energetic
postulated to occur in PD.
Validation of usini pharmacological MRI (DhMRI) to assess the
donaminergic neuronal activity
PD is the neurodegenerative
disease associated with dysfunction
and dopaminergic
energy
respiration
[5,6,7].
To better understand
innervation
the dopaminergic
(PET)
or
possibility
temporal
this
to examine
same
resolution).
to probe the
Unlike positron emission tomography
autoradiography,
the
striatum
functions in PD, we
stimulation
used functional MRI with pharmacological
dopaminergic system (phMRI).
in the
in
technique
phMRI
animals
The phMRI technique
provides
longitudinally
a
(better
also provides
better
spatial resolution when compared to PET. In this thesis, we used Damphetamine
[18] and CFT [19,20] as our specific probes.
Both
ligands can increase the synaptic dopamine concentration transiently.
In order to validate that the phMRI response to the stimulation of Damphetamine
or CFT is truly linked to the dopaminergic
system, we
have to answer two questions:
1) Can we detect the metabolic response
to direct neurotransmitter
stimulation by using fMRI?
2) Is this fMRI response specific to a neurotransmitter system?
160
To answer these questions, we designed experiments
which showed
1) the BOLD signal changes differed from the blood pCO2 and global
blood pressure changes, 2) phMRI responses are regionally specific to
those
brain
areas
phMRI responses
obtained
with highest
correlated
3) the
innervation,
dopaminergic
maps
"C-CFT binding
well with the
correlated
via PET, 4) the phMRI time courses
with the
extracellular dopamine release assessed by microdialysis.
Further to verify that the phMRI responses were truly linked to the
dopaminergic
system,
we tested
this technique
animals-normal control rats, unilaterally
on 3 groups
dopaminergic
denervated
We demonstrated
rats.
rats, and dopamine fetal cell transplanted
of
that the phMRI responses were symmetric in the normal control rats,
ablated
unilaterally
and
later
restored
transplanted
at the
graft
striatum,
denervated
in the dopaminergically
site in the
fetal
dopamine
cell
rats.
Although the results from our phMRI experiments
strongly suggest
that the phMRI responses are linked to the dopaminergic system, we
Does
are still not clear what caused the BOLD or rCBV changes.
amphetamine
[21,22]?
or CFT modulate mainly the Dl or D2 receptor system
Do the positive BOLD signal changes represent excitatory or
inhibitory neuronal activity?
side
or
the
dopaminergic
systems
postsynaptic
system
side?
Can
induce alterations
such as serotonin
26] as well?
Is the effect mainly on the presynaptic
the
modulation
in other
of
the
neurotransmitter
[23,24], GABA [25], or glutamate
[25,
Is this phMRI response associated with any alteration in
the gene expression
such as c-fos [27,28]?
161
Are there
any clinical
To continue
of this techniques?
application
these
investigating
question, we may need to modulate the fMRI signals by combining
of DI/D2
various agonists and/or antagonists. Pre- or post-treatment
agonists or antagonists
may
phMRI experiments
receptor
dopamine
or CFT stimulation
with the amphetamine
help us to monitor
systems.
response
to pure Dl or D2 agonists/antagonists
whether
or not we can obtain
signal changes
activity.
the
phMRI
stimulation
to see
corresponding
One of the advantages
as in the
phMRI response
the phMRI
This can help us to understand
cases.
amphetamine/CFT
similar
two major
to these
also monitor
We can
to excitatory
neuronal
or inhibitory
of using this pure D1/D2 stimulation
of the
protocol is that we can again correlate the spatial distribution
phMRI responses
in
to the binding maps obtained via PET [29].
The
gene responses to stimulation in the dopaminergic system has been a
Scientists have been trying to investigate
popular
topic recently.
various
mRNA responses
such as cocaine
to drug stimulation
and
amphetamine. The c-fos gene has been drawing a lot of attention due
Histological
to its instant response to cocaine or amphetamine[30,31].
studies showed that c-fos expression
after the D-amphetamine
striatum
increases in the dorsal lateral
stimulation
the spatial distribution of the phMRI response
[32], which reflects
quite well.
Whether
or not the phMRI response is due to the increase in c-fos expression
or
simply
reflects
investigation.
the
dopaminergic
Antisense
innervation
oligonucleotides
have
needs
been
further
found
to
eliminate the c-fos expression [33] and one can possibly combine the
phMRI
antisense
amphetamine
study
oligonucleotides
with
the
intrastriatal
injection
of
to reveal the role of c-fos in the phMRI
162
signal changes.
Although all of our 6-OHDA lesioned animals passed
the behavioral test for at least 90% dopaminergic
striatum unilaterally,
the difference
denervation
in the
of the phMRI responses in the
two striata (when calculated as percent striatum activated), however,
is only about 40% instead of 90%.
Castaneda et. al. [34] showed great
variance in the release of dopamine under the condition of over 90%
loss of the striatal
dopamine
phMRI measurement
terminals
response
quantity
Our
of dopamine
terminals
in
between
the phMRI
function quantitatively,
the phMRI
If we can build
may help
3 in ref. 7).
of intact dopaminergic
and the dopaminergic
technique
the real
may reflect
released instead of the number
the striatum.
(figure
the
1) to detect
linkage
loss of dopaminergic
the early
innervation
in the elderly brain before any clinical indication of PD
symptoms,
2) to determine
transplanted
the
successful
sprouting
fetal cells over time when neuronal
rate
of the
transplantation
is
performed on PD patients.
Conclusions
The data presented in this thesis demonstrate the ability of magnetic
resonance
techniques
to
neurodegenerative process.
explore
various
aspects
of
the
We have demonstrated the ability of 'H-
MRS to investigate the striatal metabolite state in the PD brain.
We
support
the
have
found
that
the
lactate
levels
hypothesis of energy impairment.
in PD patients
We also demonstrated
the ability
of phMRI to asses neuronal activity.
This phMRI technique may not
only help
of PD but
to explore
the
etiology
functions of various neurotransmitter
163
systems
also to assess
the
in both normal
and
states.
disease
have
great
monitor
We believe the techniques
suffering from
possibly
could
that
the
improve
and
strategies
treatment
information
to furnish
potential
in this thesis
presented
care
of patients
diseases.
neurodegenerative
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