**6. Functional MRI with hyperpolarized 129Xe**

As hyperpolarized xenon is a MRI signal source with properties very different from those generated from water-protons, hyperpolarized 129Xe MRI may yield structural and functional information not detectable by conventional proton-based MRI methods. This subsection shows that the differential distribution of hyperpolarized 129Xe in the cerebral cortex of the rat following a pain stimulus evoked in the animal's forepaw. Areas of higher hyperpolarized 129Xe signal corresponded to those areas previously demonstrated by conventional functional MRI (fMRI) methods as being activated by a forepaw pain stimulus. It demonstrated that the percent increase in hyperpolarized 129Xe signal over baseline was 13 - 28%, which is more sensitive than the conventional fMRI based on the blood oxygen level dependence (BOLD) (2-4%) (Zhou et al., 2011b).

Ex-vivo hyperpolarization of 129Xe is detectable by magnetic resonance spectroscopy (MRS) and MRI in animals and humans, although the resulting *in vivo* signal to noise ratio (SNR) of the hyperpolarized 129Xe signal is not as great as the signal produced by protons in conventional MRI, hyperpolarized 129Xe has several unique characteristics which may endow it with advantages in some imaging applications, including brain imaging (Zhou et.al, 2011a). The nuclear magnetic resonance frequency range (chemical shift) of hyperpolarized 129Xe *in vivo* is large compared to protons (200 ppm vs. 5 ppm respectively) and is also substantially affected by the local chemical environment, providing a means to detect localized physiological changes and biochemical binding events. In particular, the chemical shift experienced by 129Xe in the presence of oxygen (O2) is substantial and may offer a means to image changes in tissue O2 concentration that result from changes in neuronal activity. Xenon is also an ideal perfusion tracer (Betz, 1972) and inhaled nonradioactive xenon gas has been used to detect disease induced alterations in cerebral blood flow with high anatomical specificity (Gur, 1982). Because xenon is not intrinsic to biological tissue, hyperpolarized 129Xe produces virtually no background signal, which, in turn, results in high contrast hyperpolarized 129Xe MR images (Swanson, 1997).

#### **6.1 Rat brain functional hyperpolarized 129Xe imaging experiment**

In the rat brain function study, hyperpolarized 129Xe MRI was performed in rats to investigate the distribution of the hyperpolarized 129Xe signal following a well-established paradigm for producing anatomically localized neuronal activity. Six rats were intubated and connected to a ventilator that controlled the delivery of oxygen and hyperpolarized 129Xe gas. Male Spragur-Dawley rats weighing between 200-250g were placed on animal respirator, with tidal volume of 3ml O2 (3% isoflurane included) supplied for each breath. Immediately prior to the acquisition of CSI images, the animal was ventilated with alternate breaths of 100% hyperpolarized 129Xe and 98% O2: 2% isoflurane. The breath-hold period during the delivery of each hyperpolarized 129Xe breath was 2 seconds.

MRI for imaging stroke, and further demonstrates its capacity to serve as a complementary tool to proton MRI for the study of the pathophysiology during brain hypoperfusion. More importantly, these results indicate the possibility of the use of *in vivo* MRI to diagnose brain disease employing inhaled hyperpolarized gas, eliminating the potential adverse effects to

As hyperpolarized xenon is a MRI signal source with properties very different from those generated from water-protons, hyperpolarized 129Xe MRI may yield structural and functional information not detectable by conventional proton-based MRI methods. This subsection shows that the differential distribution of hyperpolarized 129Xe in the cerebral cortex of the rat following a pain stimulus evoked in the animal's forepaw. Areas of higher hyperpolarized 129Xe signal corresponded to those areas previously demonstrated by conventional functional MRI (fMRI) methods as being activated by a forepaw pain stimulus. It demonstrated that the percent increase in hyperpolarized 129Xe signal over baseline was 13 - 28%, which is more sensitive than the conventional fMRI based on the blood oxygen level

Ex-vivo hyperpolarization of 129Xe is detectable by magnetic resonance spectroscopy (MRS) and MRI in animals and humans, although the resulting *in vivo* signal to noise ratio (SNR) of the hyperpolarized 129Xe signal is not as great as the signal produced by protons in conventional MRI, hyperpolarized 129Xe has several unique characteristics which may endow it with advantages in some imaging applications, including brain imaging (Zhou et.al, 2011a). The nuclear magnetic resonance frequency range (chemical shift) of hyperpolarized 129Xe *in vivo* is large compared to protons (200 ppm vs. 5 ppm respectively) and is also substantially affected by the local chemical environment, providing a means to detect localized physiological changes and biochemical binding events. In particular, the chemical shift experienced by 129Xe in the presence of oxygen (O2) is substantial and may offer a means to image changes in tissue O2 concentration that result from changes in neuronal activity. Xenon is also an ideal perfusion tracer (Betz, 1972) and inhaled nonradioactive xenon gas has been used to detect disease induced alterations in cerebral blood flow with high anatomical specificity (Gur, 1982). Because xenon is not intrinsic to biological tissue, hyperpolarized 129Xe produces virtually no background signal, which, in turn, results

In the rat brain function study, hyperpolarized 129Xe MRI was performed in rats to investigate the distribution of the hyperpolarized 129Xe signal following a well-established paradigm for producing anatomically localized neuronal activity. Six rats were intubated and connected to a ventilator that controlled the delivery of oxygen and hyperpolarized 129Xe gas. Male Spragur-Dawley rats weighing between 200-250g were placed on animal respirator, with tidal volume of 3ml O2 (3% isoflurane included) supplied for each breath. Immediately prior to the acquisition of CSI images, the animal was ventilated with alternate breaths of 100% hyperpolarized 129Xe and 98% O2: 2% isoflurane. The breath-hold period

the patient resulting from the injection of gadolinium-based contrast agents.

**6. Functional MRI with hyperpolarized 129Xe** 

dependence (BOLD) (2-4%) (Zhou et al., 2011b).

in high contrast hyperpolarized 129Xe MR images (Swanson, 1997).

**6.1 Rat brain functional hyperpolarized 129Xe imaging experiment** 

during the delivery of each hyperpolarized 129Xe breath was 2 seconds.

High resolution proton images were taken of the rat head to provide an anatomical reference for hyperpolarized 129Xe images. In order to evaluate the distribution of hyperpolarized 129Xe in brain following an external sensory stimulus, we acquired MRS images before and after a pain producing stimulus that has a well-defined functional response that can be measured using traditional fMRI techniques. A baseline hyperpolarized 129Xe spectroscopic image was acquired from a coronal slice centered at the level of the anatomical reference slice.

The maximal, steady-state 129Xe brain signal occurred within 15 seconds after starting the ventilation with hyperpolarized 129Xe. Once verification of the xenon signal in the brain, a baseline 129Xe chemical shift image (CSI) was acquired that was centred in the plane corresponding to the proton reference image. Low flip angle used for CSI acquisition insured minimal loss of hyperpolarized 129Xe signal due to RF destruction, and the relatively long TR allowed continuous delivery of hyperpolarized 129Xe to the tissue, a steady–state concentration of hyperpolarized 129Xe was maintained in the brain thereby insuring constant signal intensity across the k-space. In a subset of animals (n=3) the animal's left forepaw was injected with a vehicle solution during baseline. Following acquisition of the baseline CSI image, the animal was ventilated for 10 minutes with O2 (isoflurane to allow for complete clearance of 129Xe magnetization from the brain). After that, the chemical irritant capsaicin (20 ul of 3 mg/ml) was injected into the animal's right forepaw (n= 6), and a second CSI was acquired.

#### **6.2 Experiment results and discussion**

A robust hyperpolarized 129Xe spectroscopic signal with one primary peak at 194.7 ppm developed within 15 seconds after starting the ventilation with hyperpolarized 129Xe. In order to determine the extent of hyperpolarized 129Xe distribution throughout the rat brain, a magnetic resonance spectroscopic image was acquired of the primary peak during the administration of hyperpolarized 129Xe (Figure 4). The four smaller resonances did not have sufficient SNR to produce spectroscopic images. Figure 4a shows an hyperpolarized 129Xe image taken in the axial plane. Addition of a color look-up table (Figure 4b) aided in visually delineating areas of low and high SNR. Figure 4c show a 1 mm proton slice in which the olfactory bulbs and cerebellum are visible. Overlay of the hyperpolarized 129Xe spectroscopic image onto the proton reference image (Figure 4d) revealed that the steadystate hyperpolarized 129Xe signal originated from within the brain tissue and further demonstrated a pattern of hyperpolarized 129Xe distribution throughout the brain with varying signal intensity in different brain regions.

Three of the six animals studied received a vehicle injection (saline) to the left forepaw immediately prior to the acquisition of the baseline image. 10 minutes after acquisition of the baseline hyperpolarized 129Xe MRS, the animal's right forepaw was injected with the chemical irritant capsaicin (20 ml of 3 mg/ml), and a second hyperpolarized 129Xe spectroscopic image was acquired. Responses from three individual animals are shown in Figure 5. Whereas baseline images showed some hyperpolarized 129Xe signal intensity in cortical and sub-cortical brain regions (Figure 5, left panel), images acquired following administration of capsaicin showed both higher hyperpolarized 129Xe signal intensity and an increased area of distribution within the brain (Figure 5, right panel). Superimposition of a rat brain atlas (Figure 5a) revealed that areas of hyperpolarized 129Xe signal increase occurred both bilaterally and

Hyperpolarized Xenon Brain MRI 139

In spite of the as yet unrefined nature of this imaging modality, our results indicate that hyperpolarized 129Xe MRI may have use as a probe for brain physiology and function. Because xenon is not inherent in the body, the substantial challenges resulting from high background signal in 1H fMRI may be somewhat reduced. Extracting meaningful data from 1H fMRI experiments is labour intensive, and requires a large number of subjects and image acquisitions. Extensive image post-processing is required and the influence that different post-processing steps play on the final data set achieved is actively debated. Conversely, hyperpolarized 129Xe MRI showed patterns of brain activation consistent with those obtained using 1H fMRI, using only a single set of images (one baseline and one post stimulus image) obtained from six animals. The magnitude of the signal difference between baseline and stimulus conditions for hyperpolarized 129Xe (13–28%) was comparable to differences typically obtained with conventional BOLD fMRI (2 to 29%) (Bock et al., 1998; Silva et al., 1999; Mandeville et al., 1999; Tuor et al., 2000) using a rat forepaw activation

This subsection will summarize the current progress as previously described, and comment the future research directions and applications in the brain imaging. Conventional MRI focuses mainly on the nuclear spin of the proton because it is ubiquitous in most parts of the human body. However, certain organs have a low proton spin density attributable to the large volume of air dispersed throughout the tissue. The low sensitivity of traditional magnetic resonance has motivated the development of techniques using hyperpolarized noble gases for NMR and MRI. Xenon has the unique characteristic of being soluble in many fluids and biological tissues, such as water, blood, lung tissue, and white and gray matter. Being a trace element in the atmosphere, xenon has no natural background signal in the human body. Therefore, dissolved-phase xenon MRI and molecular imaging could provide rich information related to biological and physiological changes beyond void space lung imaging. Efforts have demonstrated the value of dissolved xenon MRI in the study of lung gas exchange (Swanson et.al, 1999), and brain perfusion (Swanson et al., 1997; Kilian et al., 2004; Zhou et al., 2008, 2011a, 2011b) and function (Mazzanti et al., 2011). Recently, xenonbased molecular imaging has been demonstrated by using cryptophane-containing biosensors (Hilty et al., 2006). Sensitivity enhancement using a chemical amplification technique, hyperpolarized xenon chemical exchange saturation transfer (Hyper-CEST) (Schröder et al., 2006), allows imaging at low concentrations; however, for *in vivo* applications the small filling factor of a region of interest in the body relative to the NMR coil is a significant factor limiting sensitivity. In such cases remote detection methods (Hilty et.al, 2005) can provide dramatic improvements in sensitivity. In remote detection, the normal NMR coil that contains the full region of interest is used to encode spectroscopic and spatial information, then stores it as longitudinal magnetization. These encoded spins then

paradigm.

**7. Outlook of hyperpolarized 129Xe brain MRI** 

flow into a second coil with an optimized filling factor for detection.

Remote detection can overcome the filling factor issue of dissolved xenon MRI, although a low concentration of xenon in solution can be another significant impediment to highly sensitive detection. It has been shown that the solvated xenon signal can be amplified by xenon polarization transfer contrast, in which the dissolved-phase xenon from either lungs or brains is selectively saturated, and through exchange, the gas-phase signal is attenuated.

contralaterally in areas of the brain known to be involved in the processing of forepaw pain information, including the anterior cingulate and somatosensory cortices.

Fig. 4. Hyperpolarized 129Xe signal distribution in the rat brain. (3a) hyperpolarized 129Xe CSI image acquired with a 2D CSI pulse sequence from rat head under normal breathing conditions (slice thickness 10 mm). (3b) same image with false color applied. Warmer colors indicate increased hyperpolarized 129Xe signal intensity. (3c) Proton MRI of a rat head showing a 1 mm coronal slice through the brain acquired with a RARE pulse sequence. (3d) Proton image shown with overlay of hyperpolarized 129Xe MRI, in which only hyperpolarized 129Xe signal with an SNR above 2 are shown. FOV was 25 mm.

Fig. 5. Hyperpolarized 129Xe fMRI data from three animals. The hyperpolarized 129Xe signal is shown as a false color overlay on the corresponding 1 mm thick coronal proton reference image taken from the same animal. The left panel shows hyperpolarized 129Xe signal intensity during baseline and the right panel shows hyperpolarized 129Xe signal intensity after injection of capsaicin 20 ul (3 mg/ml) into the right forepaw. Color scale represents SNR and only signal with SNR above 2 are shown.

contralaterally in areas of the brain known to be involved in the processing of forepaw pain

Fig. 4. Hyperpolarized 129Xe signal distribution in the rat brain. (3a) hyperpolarized 129Xe CSI image acquired with a 2D CSI pulse sequence from rat head under normal breathing conditions (slice thickness 10 mm). (3b) same image with false color applied. Warmer colors indicate increased hyperpolarized 129Xe signal intensity. (3c) Proton MRI of a rat head showing a 1 mm coronal slice through the brain acquired with a RARE pulse sequence. (3d) Proton image shown with overlay of hyperpolarized 129Xe MRI, in which only hyperpolarized 129Xe signal with an SNR above 2 are shown. FOV was 25 mm.

Fig. 5. Hyperpolarized 129Xe fMRI data from three animals. The hyperpolarized 129Xe signal is shown as a false color overlay on the corresponding 1 mm thick coronal proton reference image taken from the same animal. The left panel shows hyperpolarized 129Xe signal intensity during baseline and the right panel shows hyperpolarized 129Xe signal intensity after injection of capsaicin 20 ul (3 mg/ml) into the right forepaw. Color scale represents

SNR and only signal with SNR above 2 are shown.

information, including the anterior cingulate and somatosensory cortices.

In spite of the as yet unrefined nature of this imaging modality, our results indicate that hyperpolarized 129Xe MRI may have use as a probe for brain physiology and function. Because xenon is not inherent in the body, the substantial challenges resulting from high background signal in 1H fMRI may be somewhat reduced. Extracting meaningful data from 1H fMRI experiments is labour intensive, and requires a large number of subjects and image acquisitions. Extensive image post-processing is required and the influence that different post-processing steps play on the final data set achieved is actively debated. Conversely, hyperpolarized 129Xe MRI showed patterns of brain activation consistent with those obtained using 1H fMRI, using only a single set of images (one baseline and one post stimulus image) obtained from six animals. The magnitude of the signal difference between baseline and stimulus conditions for hyperpolarized 129Xe (13–28%) was comparable to differences typically obtained with conventional BOLD fMRI (2 to 29%) (Bock et al., 1998; Silva et al., 1999; Mandeville et al., 1999; Tuor et al., 2000) using a rat forepaw activation paradigm.
