Magnetic resonance Magnetic Resonance Imaging to Assess Tissue Oxygenation and Redox Status

1. Magnetic Resonance Imaging to Assess Tissue
Oxygenation and Redox Status
.
Hyperpolarized (by EPR) MRI
Electron Paramagnetic Resonance (EPR) Imaging
Redox Sensitive Paramagnetic Contrast Agents in MRI
Presented by
NIVEDITHA G
1st sem Mpharm
NARGUND COLLEGE OF PHARMACY

2. EPR spectroscopy is similar to NMR
spectroscopy.
NMR spectroscopy detects nuclei with magnetic
moments.
ex: 1H, 13C, 19F, 31P etc.
EPR spectroscopy detects species with unpaired
electrons.
ex: free radicals, transition metal complexes
At a given magnetic field, EPR is more sensitive
than NMR
Electron Paramagnetic Resonance

3. NMR spectroscopy detects nuclei with magnetic moments.
ex: 1H, 13C, 19F, 31P etc.
EPR spectroscopy is similar to NMR spectroscopy.
EPR spectroscopy detects species with unpaired electrons.
ex: free radicals, transition metal complexes
At a given magnetic field, EPR is more sensitive than NMR
In MRI, proton NMR spectra are used for anatomic imaging.
Reason: simple NMR spectrum.
For EPR Imaging we need species with simple EPR spectrum.
candidates: free radicals

4. Can we image free radicals in biological systems?
Spatially-resolved (anatomical) information can be
obtained using EPR imaging, similar to MRI
MRI EPR imaging
Spin Probes Tissue protons Free radicals
(endogenous) (>50 M) (<< nM)
Probe Stability Ideal < nanoseconds
No suitable endogenous spin probes exist for EPR imaging.
Non-toxic exogenous paramagnetic probes needed for in vivo EPR imaging.

5. Molecular Oxygen Provides Contrast to Paramagnetic Probes in
EPRI
• Molecular oxygen is paramagnetic and provides contrast to paramagnetic probes.
This causes spectral broadening (increase in line width)
Line width changes from oxygen contrast > 200 % in EPR
In NMR such changes may be ~10%
• It is possible to image spatial distribution of paramagnetic spin
probe by EPR and obtain pO2 information
Oxygen (pO2)
0 25 50 75 100
EPR
Line-width
(mG)
0
200
400
600
EPR Line Width vs pO2

6. Desirable Features
Chemical and spectroscopic:
Water soluble
Kinetically and metabolically stable
Single and narrow line resonance
Linewidth  pO2
Toxicology:
Non-toxic at concentrations required for imaging
In vivo life time •
imaging time
Pharmacologic:
EPR Imaging with infusible probes may provide a more
global assessment of pO2

7. Overhauser, A; Phys. Rev. (1953)
Dynamic nuclear polarization
“…applicable to conduction electrons in alkali metals”
Hyperpolarized MRI using EPR and Paramagnetic Contrast Agents
Lurie DJ, Bussell DM, Bell LH, Mallard JR ,
Proton- Electron Double Magnetic-Resonance Imaging of Free-Radical Solutions
J. Magnetic Resonance 76, 366-370 (1988)
C
F F
F
.
Physical Basis for Hyperpolarization of Nuclei
Dynamic Nuclear Polarization with Paramagnetic Agents
Overhauser Effect

8. Trityl Radicals
Gomberg, M; JACS. (1900)
Triphenyl methyl: A case of trivalent carbon
…. “I reserve the field for myself.”
Golman, K et. al.
Overhauser-enhanced MR imaging (OMRI)
Acta Radiologica 39, 10-17(1998)
Contrast Agents for Hyperpolarization
of 1H, 13C

9. Contrast Agent - Trityl Radical
• Mouse MTD = 2.5 - 5 mmol/kg
• T1/2 in mouse blood and
kidney: 9-10 min
• Linewidth: 100 - 300 mG
Oxygen tension: 0 - 21 %
.
C F
F
F
OX063
F =
CH2OH
CH2OH
HOCH2
COO- Na+
S
S
S
S
HOCH2
Nycomed Imaging
Nycomed Amersham
Amersham
GE

10. Overhauser MRI
Combination of EPRI + MRI
MRI for anatomy and EPR for Spectral information
MR Imaging of Hyperpolarized Water Protons by EPR
Low magnetic field (~10 mT)
Uses Free Radical Paramagnetic contrast agents
Coil tuned to both NMR and EPR frequencies

11. Scanner
• Field strengths and Pulse Frequencie
EPR NMR
Magnet (mT): 8 15
Resonators: 226 MHz 640 kHz
• Magnet and resonator dimensions
Resonator Magnet
Diameter (cm): 2.5 80
Length (cm): 8 125
• Frequency Encoding
Gradient: 1.5 G/cm
• Phase Encoding
Gradient 64 steps

12. Int
0.6
1.2
1.8
2.4
3.0
MRI – Contrast Agent MRI + Contrast Agent
Kidney
Bladder
Tumor
Image Intensity enhanced by:
1) Contrast Agent concentration
2) Hypoxic/Ischemic regions

13. % Oxygen in
breathing air
Time
(min.)
0:45 6:20 12:00 17:30 23:20
21% 9.5% 21% 9.5% 21%
0
100
mm Hg
Oxygen Mapping with OMRI
Rat, respiratory model
1.5 mmol/kg
Oxygen Maps from OMRI Correspond with Changes in Tissue Oxygenation
Dynamic changes in pO2 can be monitored

14. 20
40
60
80
100
120
140
0
20
40
60
80
100
120
140
0
(Oxygen,
mm
Hg)
(Oxygen,
mm
Hg)
Air
Carbogen

15. Overhauser MRI/Summary
Currently implemented in mice, rats.
For human applications:
Contrast agents/Safety
SAR/Localized coils

16. Challenges in Imaging of 13C labeled molecules with MRI.
1) Lower concentration compared to protons
2) Lower magnetic moment than protons (25%)
3) Lower Polarization than protons (25%)
Hyperpolarized 13C MRI
Implications for Molecular Imaging

17. 1H 13C 13C Hyperpolarized
C, M 80 0.1 0.1
g (MHz/T) 42.5 10.7 10.7
Polariz., P 1.10-5 2.10-6 0.5
c g P 0.034 2.14.10-6 0.535
Sensitivity Considerations in MRI of 1H and 13C at 3 Tesla
Magnetic Field
Molecular Imaging With Endogenous Substances.
Golman et al: PNAS Vol 100, 10158-10163, 2003.
Golman et al: PNAS Vol 100, 10435-10439, 2003.

18. Strategy for Hyperpolarization of 13C labeled Molecules by EPR
for in vivo imaging
• Mix the 13C labeled compound with trityl radical
• Freeze to 1. 4 K and put it in 2.7 T magnet
• Irradiate with 95 GHz radiation (EPR)
• Thaw it to room temperature, inject and image in< 2min!
Golman et al PNAS 2003
Ardenkjaer et al PNAS 2003

19. The DNP 13C polariser
3.35 T and 1.2K
13C
13C
13C
13C
13C
13C
13C
13C
e-
e-
13C
13C
13C
13C
13C
13C
13C
13C
13C
13C
13C
13C
13C
13C
13C
13C
13C
e-
13C
13C
13C
13C
13C
13C
13C
13C
13C
13C
13C
13C
13C
13C
13C
13C
13C
13C
13C
13C
13C
13C
13C
13C
Pe= 94% and PC= 0.086%
Solid material doped with
unpaired electrons
Microwaves transfer
the polarisation
from electrons to nuclei
3. 35 T
95 GHz
1.2 K
Pellet of 13C
molecule doped
with
paramagnetic
substance

20. A)
B)
NMR Spectrum of 13C Urea (natural Abundance)
9.4 Tesla (400 MHz)
Single Shot NMR Spectrum
Acquisition time: 1s
Polarization of 13C Urea 20%
13C Urea at normal polarization
Acquisition time: 65 Hours
Polarization: 7.5 ppm

21. 0
20
40
60
80
100
120
0 10 20 30 40 50 60 70
Time (s)
Signal Loss of hyperpolarized 13C Urea in a mouse after iv bolus
There is ~ minute for image data
acquisition before polarization is lost.

22. Metabolic fates of
pyruvate
Oxaloacetate Acetyl-CoA
Pyruvate
Transamination
Carboxylation Oxidative
decarboxylation
Reduction
Pyruvate is converted into lactate, alanine,
oxaloacetate or Acetyl-CoA depending on the
needs of the tissues.
With suitable hyperpolarized molecules,
it is possible to distinguish breakdown
products based on their chemical shifts
by 13C MRI.

24. 3D surface rendering
In vivo metabolic mapping
using 13C-pyruvate

25. Imaging pO2 using EPR Imaging and
paramagnetic molecules
Direct detection of contrast agent by EPRI
Image collection using static magnetic field gradients
Images of spin probe distribution
pO2 maps obtained by T2* weighted imaging

26. frequency
FT
pulse width
10 - 100 ns
dead time
0.3 - 1.0 µs
1.0 - 4.0 µs
FID
TIME DOMAIN EPR EXPERIMENTS
The EPR Signals last ~ microseconds after RF pulse

27. Gradient ramping and pseudo-echo
r
FT
Frequency ( 1D spatial profile)
t
time
Gx
Gy
Gz
EPR/Single Point Imaging
After the dead time, the phase of one point after a fixed time delay is monitored at
different gradient magnitudes per direction.
The resultant envelope is equivalent of a Gradient Recalled Echo.
FT of this envelop gives the spatial projection
Resolution independent of line width.
Fourier reconstruction possible with static gradients
We implemented Fourier Imaging Capabilities in EPRI.

28. Time (ns) Frequency (MHz)
FT
EPR Experiment in Time-Domain.
Line width, n = 1/(p T2)
Dead-time
We have Developed EPR Instrumentation with
nanosecond time resolution

29. EPR can be used for pO2 imaging by post-processing for T2* effects.
t
time
cm
Int.
Using several times points in the echo for image reconstruction
it is possible to estimate oxygen dependent line width of the
contrast agent
Gx

30. 0 % 1 %
2.5 %
5 %
Oxygen Provides Excellent T2 Contrast to
Paramagnetic Contrast Agents
Phantom Schematic
mm
mm
Intensity Image
-20 -15 -10 -5 0 5 10 15 20
-20
-15
-10
-5
0
5
10
15
20
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
mm
mm
Line Width Mapping
-20 -15 -10 -5 0 5 10 15 20
-20
-15
-10
-5
0
5
10
15
20
150
160
170
180
190
200
210
220
230
240
250
pO2 dependent T2 Map
150
160
170
180
190
200
210
220
230
240
250
0 1 2 3 4 5
Oxygen concentration %
Lw
in
mG

31. Intensity Image
Sagital View ( 1mm Slices)

32. Line Width Image
Sagital View ( 1mm Slices)

33. N
O
R
N
O H
R
.
Reduction
Oxidation
n = 1, piperidine nitroxides eg. Tempol, Tempo, Tempone
n = 0, pyrrolidine nitroxides. Eg. Carbamoyl proxyl, carboxy proxyl
(n) (n)
Nitroxide radical Hydroxylamine
Paramagnetic Diamagnetic
Provides enhancement in T1 based MRI Does not provide T1 contrast in MRI
Nitroxides can provide redox status dependent contrast in MRI

34. %Difference in Intensity Green: +; Red: -
Evo = 1/60
Tempol-Induced T1 contrast in
Tumor Bearing Mouse
Time Course of Tempol-Induced
Image Intensity in Tumor Bearing
Mouse
Tempol-Induced T1 contrast in Tumor Bearing Mouse changes with time after administration.
Time-intensity profiles in muscle and tumor are different
Evo = 1/60
GEFI: TR75, TE3, FA45
Evo = 1/60 Evo = 8/60 Evo = 15/60
Evo = 22/60 Evo = 29/60 Evo = 60/60
Tumor Normal
B C D
E F G
A

35. y = -0.8592x + 5.2778
R2
= 0.9954
0
1
2
3
4
0 1 2 3 4 5 6 7 8
Time (min)
ln(%
change)
y = -0.2735x + 3.126
R2
= 0.7954
0
1
2
3
4
0 1 2 3 4 5 6 7 8
Time (min)
ln(%
change) Tumor
Normal
Change in Tempol-induced MR Intensity Enhancement as a Function of Time
Intensity change with time is faster in tumor than normal tissue.
Nitroxides are reduced faster in tumors than in normal tissue

36. MSME: TR4000, TE450
Evo = 1/60
GEFI: TR75, TE3, FA45
Evo = 1/60 Evo = 8/60 Evo = 15/60
Evo = 22/60 Evo = 29/60 Evo = 60/60
%Difference
Time Course of 3-CP-Induced
Image Intensity in Tumor Bearing
Mouse
3CP-Induced T1 contrast in
Tumor Bearing Mouse
3CP-Induced T1 contrast in Tumor Bearing Mouse changes with time after administration.
Time-intensity profiles in muscle and tumor are different
Evo = 1/60
Tumor Normal
GEFI: TR75, TE3, FA45

37. y = -0.1074x + 4.074
R2
= 0.9978
0
1
2
3
4
0 5 10 15 20
Time (min)
ln(%
change)
y = -0.0698x + 3.9395
R2
= 0.9933
0
1
2
3
4
0 5 10 15 20
Time (min)
ln(%
change) Tumor
Normal

38. John Cook, Ph. D
Deva Devasahayam, MS (EE)
Fuminori Hyodo, Ph. D
Janusz Koscielniak, Ph. D
Atsuko Matsumoto M.S
Ken-Ichiro Matsumoto, Ph. D
Sankaran Subramanian, Ph. D
James B. Mitchell, Ph. D
David Wink, Ph. D
Angelo Russo, Ph. D, M. D
Amram Samuni, Ph. D
Klaes Golman, Amersham, Sweden
Jan-Henrik Ardenkjaer, Amersham,
Sweden
ACKNOWLEDGEMENTS