# Presentation: 498623

## 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**
**Murali C Krishna**
**Radiation Biology Branch**
**Center for Cancer Research**
**National Cancer Institute**
**NIH, Bethesda, MD **

## Electron Paramagnetic Resonance

**NMR spectroscopy detects nuclei with magnetic moments.**
**ex: ****1****H, ****13****C, ****19****F, ****31****P 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**

## NMR spectroscopy detects nuclei with magnetic moments.

**ex: ****1****H, ****13****C, ****19****F, ****31****P 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***

## 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.
- 		* *

## 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
** **

**EPR Line Width vs pO****2**

## Linewidth  pO2

**Desirable Features**

***Chemical and spectroscopic:***

**Water soluble**
**Kinetically and metabolically stable**
**Single and narrow line resonance**
**Linewidth ******** pO****2**

***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 pO****2**

## Physical Basis for Hyperpolarization of Nuclei

**Physical Basis for Hyperpolarization of Nuclei**
**Dynamic Nuclear Polarization with Paramagnetic Agents**
**Overhauser Effect**

## Contrast Agents for Hyperpolarization of 1H, 13C   

**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 ****1****H, ****13****C   **

## .

**Contrast Agent - Trityl Radical **

**•  ****Mouse MTD = 2.5 - 5 mmol/kg**
** **
**  ****T****1/2**** in mouse blood and  **
**              ****kidney: 9-10 min**
**  ****Linewidth:            ****100  -  300 mG**
**   ****Oxygen tension:****      0  -  21 %**

**.**

**C**

- 

- 

- 

**OX063**

- 

**CH****2****OH**

**CH****2****OH**

**HOCH****2**

**COO****-  ****Na****+**

**S**

**S**

**S**

**S**

**HOCH****2**

***Nycomed Imaging***
***Nycomed Amersham***
***Amersham***
***GE***

## 	Overhauser MRI

- 	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

## Scanner

**Scanner**

**7**

** ****Field strengths and Pulse Frequencies**
**	****	****     ****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**

## Image Intensity enhanced by:

**Image Intensity enhanced by:**
**	****1) Contrast Agent concentration**
**	****2) Hypoxic/Ischemic regions**

## Oxygen Mapping with OMRI

**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 pO****2**** can be monitored **

## (Oxygen, mm Hg)

**20**

**40**

**60**

**80**

**100**

**120**

**140**

**0**

**20**

**40**

**60**

**80**

**100**

**120**

**140**

**0**

**(Oxygen, mm Hg)**

**(Oxygen, mm Hg)**

**Air**

**Carbogen**

## Overhauser MRI/Summary

**Currently implemented in mice, rats.**
**For human applications:**
**Contrast agents/Safety**
**SAR/Localized coils**

## 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 ****13****C MRI   **
**Implications for Molecular Imaging**

## 		1H		13C		13C Hyperpolarized

- C, M		80		0.1		0.1
-    (MHz/T)	42.5		10.7		10.7
- Polariz., P	1.10-5		2.10-6 		0.5
- c  P		0.034		2.14.10-6 	0.535

**Sensitivity Considerations in MRI of ****1****H and ****13****C 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.

## Strategy for Hyperpolarization of 13C labeled Molecules by EPR

**for in vivo imaging**
**    ****Mix the ****13****C 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**

## The DNP 13C polariser

- The DNP 13C polariser

**3. 35 T**
**95 GHz**
**1.2 K**

**Pellet of 13C molecule doped with paramagnetic substance**

## A)

- B)

**NMR Spectrum of ****13****C 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

## Signal Loss of hyperpolarized 13C Urea in a mouse after iv bolus

**Signal Loss of hyperpolarized ****13****C Urea in a mouse after iv bolus**

**There is  ~ minute for image data acquisition before polarization is lost.**

## Metabolic fates of pyruvate

**Alanine**

**Oxaloacetate**

**Lactate**

**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 ****13****C MRI.**

## Slide 23

## In vivo metabolic mapping using 13C-pyruvate

- In vivo metabolic mapping using 13C-pyruvate

## 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  **
**pO****2**** maps obtained by T****2***** weighted imaging**

## The EPR Signals last ~ microseconds after RF pulse

**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**

## r

**r**

**FT**

**   ****Frequency  ( 1D  spatial  profile)**

****

**RF pulse**

**Dead**
**time**

**time**

**G****x**

**G****y**

**G****z**

- 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.**

## FT

**Time (ns)**

**Frequency (MHz)**

***FT***

** **

**EPR Experiment in Time-Domain.**

**Line width, ******** = 1/(********T2)**

**Dead-time**

**We have Developed EPR Instrumentation with nanosecond time resolution**

## EPR can be used for pO2 imaging by post-processing for T2* effects.

****

**RF pulse**

**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

**G****x**

## Oxygen Provides Excellent T2 Contrast to Paramagnetic Contrast Agents  

**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**

**pO****2**** dependent T2 Map**

**0**

**1**

**2**

**3**

**4**

**5**

**Oxygen concentration %**

**Lw in mG**

## Intensity Image

- Sagital View ( 1mm Slices)

## Line Width Image

- Sagital View ( 1mm Slices)

## N

**N**

- O

- R

**N**

- O

- H

- R

**.**

- R

- e

- d

- u

- c

- t

- i

- o

- n

- O

- x

- i

- d

- a

- t

- i

- o

- n

- 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

## B

**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**

**Tumor**

**Normal**

**B**

**C**

**D**

**E**

**F**

**G**

**A**

## Tumor

**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 **

## MSME: TR4000, TE450

**Evo = 1/60**

**GEFI: TR75, TE3, FA45**

**%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**

## Tumor

**Tumor**

**Normal**

## ACKNOWLEDGEMENTS

**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**