Targeted Imaging of Lung Cancer with Hyperpolarized 129Xe MRI Using Surface-Modified Iron Oxide Nanoparticles as Molecular Contrast Agents.

Hyperpolarized 129Xe (HP 129Xe) MRI enables functional imaging of various lung diseases but has been scarcely applied to lung cancer imaging. The aim of this study is to investigate the feasibility of targeted imaging of lung cancer with HP 129Xe MRI using surface-modified iron oxide nanoparticles (IONPs) as molecular targeting contrast agents. A mouse model of lung cancer (LC) was induced in nine mice by intra-peritoneal injection of urethane. Three months after the urethane administration, the mice underwent lung imaging with HP 129Xe MRI at baseline (0 h). Subsequently, the LC group was divided into two sub-groups: mice administered with polyethylene glycol-coated IONPs (PEG-IONPs, n = 4) and folate-conjugated dextran-coated IONPs (FA@Dex-IONPs, n = 5). The mice were imaged at 3, 6, and 24 h after the intravenous injection of IONPs. FA@Dex-IONPs mice showed a 25% reduction in average signal intensity at cancer sites at 3 h post injection, and a 24% reduction at 24 h post injection. On the other hand, in PEG-IONPs mice, while a signal reduction of approximately 28% was observed at cancer sites at 3 to 6 h post injection, the signal intensity was unchanged from that of the baseline at 24 h. Proton MRI of LC mice (n = 3) was able to detect cancer five months after urethane administration, i.e., later than HP 129Xe MRI (3 months). Furthermore, a significant decrease in averaged 1H T2 values at cancer sites was observed at only 6 h post injection of FA@Dex-IONPs (p < 0.05). As such, the targeted delivery of IONPs to cancer tissue was successfully imaged with HP 129Xe MRI, and their surface modification with folate likely has a high affinity with LC, which causes overexpression of folate receptors.

Quantitative assessment of regional lung ventilation in emphysematous mice using hyperpolarized 129Xe MRI with a continuous flow hyperpolarizing system.

BACKGROUND: Lung ventilation function in small animals can be assessed by using hyperpolarized gas MRI. For these experiments a free breathing protocol is generally preferred to mechanical ventilation as mechanical ventilation can often lead to ventilation lung injury, while the need to maintain a gas reservoir may lead to a partial reduction of the polarization. PURPOSE: To evaluate regional lung ventilation of mice by a simple but fast method under free breathing and give evidence for effectiveness with an elastase instilled emphysematous mice. ANIMAL MODEL: Emphysematous mice. MATERIALS AND METHODS: A Look-Locker based saturation recovery sequence was developed for continuous flow hyperpolarized (CF-HP) 129Xe gas experiments, and the apparent gas-exchange rate, k', was measured by the analysis of the saturation recovery curve. RESULTS: In mice with elastase-induced mild emphysema, reductions of 15-30% in k' values were observed as the results of lesion-induced changes in the lung. DATA CONCLUSION: The proposed method was applied to an emphysematous model mice and ventilation dysfunctions have been approved as a definite decrease in k' values, supporting the usefulness for a non-invasive assessment of the lung functions in preclinical study by the CF-HP 129Xe experiments.

Analysis of NMR Adsorption Isotherms of Zeolite ZSM-5: Adsorption Profiles Derived from the Pressure and Temperature Dependences of 129Xe NMR Chemical Shift and Signal Intensity.

129Xe NMR spectroscopy of nanomaterials, such as zeolites, can provide valuable information on the nanostructure and physicochemical properties of adsorption. In the present study the pressure and temperature dependences of the 129Xe NMR chemical shift and the signal intensity were investigated in detail with a zeolite ZSM-5. The pressure dependence of the signal intensity at constant temperature was analyzed based on the Langmuir and Dubinin-Radushkevich (D-R) models, from which the thermodynamic parameters and energetic profiles of adsorption were obtained together with information concerning the nanospace size. From this isotherm analysis the coverage, θ, was calculated and used for isotherm analysis of the chemical shift. The θ dependence of the chemical shift was successfully fitted by an exponential function, and the results were discussed in relation to the chemical shift at zero coverage, that at full coverage and the curvature of the exponential function. The chemical shift data reported with the zeolites NaA and KA, where separated signals were observed for the different number of encapsulated Xe atoms in the α cage, were analyzed and discussed collectively.

Inflammation during Lung Cancer Progression and Ethyl Pyruvate Treatment Observed by Pulmonary Functional Hyperpolarized 129Xe MRI in Mice.

This study aimed to assess the suitability of hyperpolarized 129Xe (HPXe) MRI for noninvasive longitudinal evaluation of pulmonary function in preclinical lung cancer models. A mouse model of lung cancer (LC) was induced in 5 mice by intraperitoneal injection of urethane, while a negative-control (NC) mice (N = 5) was prepared by injection of saline solution. Longitudinal HPXe MRI was performed over a 5-month period to monitor lung ventilation and gas exchange. The treatment efficacy of ethyl pyruvate (EP), an anti-inflammatory drug, to the mouse LC model was monitored using HPXe MRI by commencing administration of EP pre (early-phase) and 1-month post (late-phase) injection of urethane (N = 5 mice for each group). Gas-exchange function in LC mice was significantly reduced at 1-month after urethane injection compared with NC mice administered with saline (P < 0.01). Thereafter, it remained consistently lower than that of the NC group for the full 5-month measurement period. In contrast, the ventilation function of the LC model mice was not significantly different to that of the NC mice. Histological analysis revealed alveolar epithelial hyperplasia in LC mice alveoli at 1 month after urethane injection, and adenoma was confirmed 3 months after the injection. The early- and late-phase EP interventions were found to improve HPXe MRI metrics (reduced at 1 month postinjection of urethane) and significantly inhibit tumor growth. These results suggest that HPXe MRI gas-exchange metrics can be used to quantitatively assess changes in the precancerous lesion microenvironment and to evaluate therapeutic efficacy in cancer. Thus, HPXe MRI can be utilized to noninvasively monitor pulmonary pathology during LC progression and can visualize functional changes during therapy.

Multiplexed 129Xe HyperCEST MRI Detection of Genetically Reconstituted Bacterial Protein Nanoparticles in Human Cancer Cells.

Gas vesicle nanoparticles (GVs) are gas-containing protein assemblies expressed in bacteria and archaea. Recently, GVs have gained considerable attention for biotechnological applications as genetically encodable contrast agents for MRI and ultrasonography. However, at present, the practical use of GVs is hampered by a lack of robust methodology for their induction into mammalian cells. Here, we demonstrate the genetic reconstitution of protein nanoparticles with characteristic bicone structures similar to natural GVs in a human breast cancer cell line KPL-4 and genetic control of their size and shape through expression of reduced sets of humanized gas vesicle genes cloned into Tol2 transposon vectors, referencing the natural gas vesicle gene clusters of the cyanobacteria planktothrix rubescens/agardhii. We then report the utility of these nanoparticles as multiplexed, sensitive, and genetically encoded contrast agents for hyperpolarized xenon chemical exchange saturation transfer (HyperCEST) MRI.

Adipose Tissue-Derived Stem Cells Have the Ability to Differentiate into Alveolar Epithelial Cells and Ameliorate Lung Injury Caused by Elastase-Induced Emphysema in Mice.

Chronic obstructive pulmonary disease is a leading cause of mortality globally, with no effective therapy yet established. Adipose tissue-derived stem cells (ADSCs) are useful for ameliorating lung injury in animal models. However, whether ADSCs differentiate into functional cells remains uncertain, and no study has reported on the mechanism by which ADSCs improve lung functionality. Thus, in this study, we examined whether ADSCs differentiate into lung alveolar cells and are able to ameliorate lung injury caused by elastase-induced emphysema in model mice. Here, we induced ADSCs to differentiate into type 2 alveolar epithelial cells in vitro. We demonstrated that ADSCs can differentiate into type 2 alveolar epithelial cells in an elastase-induced emphysematous lung and that ADSCs improve pulmonary function of emphysema model mice, as determined with spirometry and 129Xe MRI. These data revealed a novel function for ADSCs in promoting repair of the damaged lung by direct differentiation into alveolar epithelial cells.

Stability Enhancement of 129Xe Hyperpolarizing System Using Alkali Metal Vapor in Spin-Exchange Optical Pumping Cell to Achieve High NMR Sensitivity.

Hyperpolarized (HP) 129Xe NMR and MRI have enabled 129Xe studies with extraordinarily enhanced sensitivity, stimulating new developments in magnetic resonance in chemistry, physics, biology and medicine. However, the standard method of HP 129Xe production inevitably demands Rb vapor for the excitation, which has made the method very sensitive to impurities such as water or oxygen. This is the case especially in the recirculating system. In the present study, stability of the hyperpolarizing system is discussed by proposing the "cell decay constant", which symbolizes the decay rate of the NMR signal obtained from the system. The cell decay constant is effectively decreased to 1/3 by introducing separated chambers and mechanical stirring of the alkali metals used in the system, making it effective for accumulating FIDs over 30 to 100 h. The newly developed hyperpolarizing system has been successfully applied for newly detecting a broad signal at 190 ppm with an industrial material Nanofiber.

Ethyl Pyruvate Improves Pulmonary Function in Mice with Bleomycin-induced Lung Injury as Monitored with Hyperpolarized 129Xe MR Imaging.

PURPOSE: High Mobility Group Box1 (HMGB1), which is one of the damage-associated molecular pattern molecules relating to various inflammatory diseases, has gained interest as a therapeutic target because of its involvement in wound healing processes. In the present study, we investigated HMGB1 as a potential therapeutic target in a model of lung fibrosis using a preclinical hyperpolarized 129Xe (HPXe) MRI system. METHODS: Lung injury was induced by intra-peritoneal injection of bleomycin (BLM) in 19 mice. Three weeks post-injection (when fibrosis was confirmed histologically), administration of ethyl pyruvate (EP) and alogliptin (ALG), which are down- and up-regulators of HMGB1, respectively, was commenced in six and seven of the 19 mice, respectively, and continued for a further 3 weeks. A separate sham-instilled group was formed of five mice, which were administered with saline for 6 weeks. Over the second 3-week period, the effects of disease progression and pharmacological therapy in the four groups of mice were monitored by HPXe MRI metrics of fractional ventilation and gas-exchange function. RESULTS: Gas-exchange function in BLM mice was significantly reduced after 3 weeks of BLM challenge compared to sham-instilled mice (P < 0.05). Ethyl pyruvate was found to improve HPXe MRI metrics of both ventilation and gas exchange, and repair tissue damage (assessed histologically), to a similar level as sham-instilled mice (P < 0.05), whilst ALG treatment caused no significant improvement of pulmonary function. CONCLUSION: This study demonstrates the down-regulator of HMGB1, EP, as a potential therapeutic agent for pulmonary fibrosis, as assessed by a non-invasive HPXe MRI protocol.

Continuous flow production of concentrated hyperpolarized xenon gas from a dilute xenon gas mixture by buffer gas condensation.

We present a new method for the continuous flow production of concentrated hyperpolarized xenon-129 (HP 129Xe) gas from a dilute xenon (Xe) gas mixture with high nuclear spin polarization. A low vapor pressure (i.e., high boiling-point) gas was introduced as an alternative to molecular nitrogen (N2), which is the conventional quenching gas for generating HP 129Xe via Rb-Xe spin-exchange optical-pumping (SEOP). In contrast to the generally used method of extraction by freezing Xe after the SEOP process, the quenching gas separated as a liquid at moderately low temperature so that Xe was maintained in its gaseous state, allowing the continuous delivery of highly polarized concentrated Xe gas. We selected isobutene as the candidate quenching gas and our method was demonstrated experimentally while comparing its performance with N2. Isobutene could be liquefied and removed from the Xe gas mixture using a cold trap, and the concentrated HP 129Xe gas exhibited a significantly enhanced nuclear magnetic resonance (NMR) signal. Although the system requires further optimization depending on the intended purpose, our approach presented here could provide a simple means for performing NMR or magnetic resonance imaging (MRI) measurements continuously using HP 129Xe with improved sensitivity.

Treatment response of ethyl pyruvate in a mouse model of chronic obstructive pulmonary disease studied by hyperpolarized 129 Xe MRI.

PURPOSE: The purpose of this work was to investigate disease progression and treatment response in a murine model of chronic obstructive pulmonary disease (COPD) using a preclinical hyperpolarized 129 Xe (HPXe) magnetic resonance imaging (MRI) strategy. METHODS: COPD phenotypes were induced in 32 mice by 10 weeks of exposure to cigarette smoke (CS) and lipopolysaccharide (LPS). Efficacy of ethyl pyruvate (EP), an anti-inflammatory drug, was investigated by administering EP to 16 of the 32 mice after 6 weeks of CS and LPS exposure. HPXe MRI was performed to monitor changes in pulmonary function during disease progression and pharmacological therapy. RESULTS: HPXe metrics of fractional ventilation and gas-exchange function were significantly reduced after 6 weeks of CS and LPS exposure compared to sham-instilled mice administered with saline (P < 0.05). After this observation, EP administration was started in 16 of the 32 mice and continued for 4 weeks. EP was found to improve HPXe MRI metrics to a similar level as in sham-instilled mice (P < 0.01). Histological analysis showed significant alveolar tissue destruction in the COPD group, but relatively normal alveolar structure in the EP and sham-instilled groups. CONCLUSION: This study demonstrates the potential efficacy of EP for COPD therapy, as assessed by a noninvasive, translatable 129 Xe MRI procedure. Magn Reson Med 78:721-729, 2017. © 2016 International Society for Magnetic Resonance in Medicine.

Hyperpolarized (129) Xe MRI using isobutene as a new quenching gas.

The use of a quenching gas, isobutene, with a low vapor pressure was investigated to enhance the utility of hyperpolarized (129) Xe (HP Xe) MRI. Xenon mixed with isobutene was hyperpolarized using a home-built apparatus for continuously producing HP Xe. The isobutene was then readily liquefied and separated almost totally by continuous condensation at about 173 K, because the vapor pressure of isobutene (0.247 kPa) is much lower than that of Xe (157 kPa). Finally, the neat Xe gas was continuously delivered to mice by spontaneous inhalation. The HP Xe MRI was enhanced twofold in polarization level and threefold in signal intensity when isobutene was adopted as the quenching gas instead of N2 . The usefulness of the HP Xe MRI was verified by application to pulmonary functional imaging of spontaneously breathing mice, where the parameters of fractional ventilation (ra ) and gas exchange (fD ) were evaluated, aiming at future extension to preclinical studies. This is the first application of isobutene as a quenching gas for HP Xe MRI.

Development and Characterization of Novel LipoCEST Agents Based on Thermosensitive Liposomes.

PURPOSE: To develop a novel probe for chemical exchange saturation transfer magnetic resonance imaging (CEST MRI) based on thermosensitive liposomes (lipoCEST) for theranostics, in which diagnostics and therapy are integrated into a single platform. METHODS: We developed two kinds of lipoCEST agents. The first kind encapsulated dysprosium (Dy)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-Na·3NaCl, terbium-DOTA-Na·3NaCl, or thulium-DOTA-Na·3NaCl into the inner cavity of thermosensitive liposomes, while the second kind encapsulated Dy-DOTA-Na and incorporated amphiphilic metal complex [thulium-diethylenetriamine pentaacetic acid-bis (stearylamide) (Tm-DTPA-BSA)] as a membrane constituent. The nuclear magnetic resonance (NMR)- and Z-spectra of these lipoCEST agents were acquired at various temperatures on a 9.4T MRI scanner. To investigate their applicability to the drug release induced by hyperthermia, we also encapsulated a fluorescent dye (calcein) into the inner cavity of liposomes and measured calcein release after warming them. RESULTS: The intra- and extraliposomal water signals could be differentiated in all agents from their NMR- and Z-spectra. The agent incorporating Tm-DTPA-BSA showed the largest chemical shift (approximately 15 ppm) derived from the intraliposomal water protons. The calcein retained in this agent was successfully released at 44°C. The agent incorporating 30 mol% of Tm-DTPA-BSA in its membrane released more calcein at 42-44ºC than that of the agent incorporating 10 mol%. CONCLUSION: We developed novel thermosensitive lipoCEST agents and characterized them. Our preliminary results suggest that they are useful and can be applied to theranostics.

Regional fractional ventilation mapping in spontaneously breathing mice using hyperpolarized ¹²9Xe MRI.

The feasibility of ventilation imaging with hyperpolarized (HP) (129) Xe MRI has been investigated for quantitative and regional assessment of ventilation in spontaneously breathing mice. The multiple breath ventilation imaging technique was modified to the protocol of spontaneous inhalation of HP (129) Xe delivered continuously from a (129) Xe polarizer. A series of (129) Xe ventilation images was obtained by varying the number of breaths before the (129) Xe lung imaging. The fractional ventilation, r, was successfully evaluated for spontaneously breathing mice. An attempt was made to detect ventilation dysfunction in the emphysematous mouse lung induced by intratracheal administration of porcine pancreatic elastase (PPE). As a result, the distribution of fractional ventilation could be visualized by the r map. Significant dysfunction of ventilation was quantitatively identified in the PPE-treated group. The whole-lung r value of 0.34 ± 0.01 for control mice (N = 4) was significantly reduced, to 0.25 ± 0.07, in PPE-treated mice (N = 4) (p = 0.038). This study is the first application of multiple breath ventilation imaging to spontaneously breathing mice, and shows that this methodology is sensitive to differences in the pulmonary ventilation. This methodology is expected to improve simplicity as well as noninvasiveness when assessing regional ventilation in small rodents.

Small animal imaging with hyperpolarized 129Xe magnetic resonance.

High-sensitivity nuclear magnetic resonance (NMR) of gaseous atoms realized by using a hyperpolarization technique is an attractive research tool used in a wide range of areas, such as physics, chemistry, material science and biomedical imaging. One of the most promising applications of this technology is the use as a noninvasive diagnostic tool for pulmonary diseases, where hyperpolarized (HP) noble gases, (3)He and (129)Xe, play a role as gaseous (i.e. inhalable) contrast agents of magnetic resonance imaging (MRI). During the last two decades, lung MRI with HP gases has become widely applicable from mouse to human. In this review we present a brief overview of recent progress made by our group in the development of HP (129)Xe MR measurements, while focusing on the methodology for probing pulmonary dysfunctions in mice.

Validation of noninvasive morphological and diffusion imaging in mouse emphysema by micro-computed tomography and hyperpolarized (129)Xe magnetic resonance imaging.

Animal disease models are pivotal in investigating the pathogenesis of emphysema and developing novel drugs, but the modalities to evaluate murine emphysema models have been of limited validity and sensitivity. In this study, we evaluated hyperpolarized (129)Xe magnetic resonance imaging (MRI) and micro-computed tomography (micro-CT) compared with traditional methods, such as plethysmography and histology. Elastase-treated mice and adiponectin knockout mice were used as murine emphysema models to evaluate these modalities. Three weeks after elastase administration, significant and heterogeneous emphysema was evaluated according to the mean linear intercept and plethysmography parameters. Notably, the distribution of low-density areas, as examined by micro-CT, correlated with the mean linear intercept and plethysmography parameters in whole lungs. These correlations were also observed in regional areas. Furthermore, we introduced hyperpolarized (129)Xe MRI, which can evaluate gas exchange between the alveoli and blood during spontaneous breathing. Parameters of gas exchange (fD) and alveolar size (Vs/Va) were significantly decreased in elastase-treated mice, and moderately correlated with the plethysmography parameters. Of importance, we could detect a decrease of the fD value in low-density areas with micro-CT, suggesting that gas exchange decreased in emphysematous lesions. Likewise, these parameters (fD and Vs/Va) were also decreased in adiponectin knockout mice, which exhibit emphysema with a homogeneous distribution. We demonstrated the feasibility of (129)Xe MRI and micro-CT in combination with traditional modalities. These noninvasive modalities provide complementary data that can be used for repeated estimations of regional gas exchange and lung morphology.

Direct imaging of hyperpolarized 129Xe alveolar gas uptake in a mouse model of emphysema.

MRI of hyperpolarized (129)Xe dissolved in pulmonary tissues, and blood has the potential to offer a new tool for regional evaluation of pulmonary gas exchange and perfusion; however, the extremely short T2* and low magnetization density make it difficult to acquire the image. In this study, an ultrashort echo-time sequence was introduced, and its feasibility to quantitatively assess emphysema-like pulmonary tissue destruction by a combination of dissolved- and gas-phase (129)Xe lung MRI was investigated. The ultrashort echo-time has made it possible to acquire dissolved (129)Xe images with reasonably high spatial resolution of 0.625 × 0.625 mm(2) and to obtain T2* of 0.67 ± 0.30 ms in a spontaneously breathing mouse at 9.4 T. The regional dynamic alveolar gas uptake as well as subsequent transport by pulmonary blood flow was also visualized. The ratio of (129)Xe magnetization that diffused into the septa relative to the gas-phase magnetization F was regionally evaluated. The mean F value of elastase-treated mice was 2.28 ± 0.46%, which was significantly reduced from that of control mice 3.41 ± 0.48% (P = 0.0052). This reflects the reduced uptake efficiency due to alveolar tissue destruction and is correlated with the histologically derived alveolar surface-to-volume ratio.

Development of a fast method for quantitative measurement of hyperpolarized 129Xe dynamics in mouse brain.

A fast method has been established for the precise measurement and quantification of the dynamics of hyperpolarized (HP) xenon-129 ((129)Xe) in the mouse brain. The key technique is based on repeatedly applying radio frequency (RF) pulses and measuring the decrease of HP (129)Xe magnetization after the brain Xe concentration has reached a steady state due to continuous HP (129)Xe ventilation. The signal decrease of the (129)Xe nuclear magnetic resonance (NMR) signal was well described by a simple theoretical model. The technique made it possible to rapidly evaluate the rate constant α, which is composed of cerebral blood flow (CBF), the partition coefficient of Xe between the tissue and blood (λ(i)), and the longitudinal relaxation time (T(1i)) of HP (129)Xe in the brain tissue, without any effect of depolarization by RF pulses and the dynamics in the lung. The technique enabled the precise determination of α as 0.103 ± 0.018 s(-1) (± SD, n = 5) on healthy mice. To investigate the potential of this method for detecting physiological changes in the brain of a kainic acid (KA) -induced mouse model of epilepsy, an attempt was made to follow the time course of α after KA injection. It was found that the α value changes characteristically with time, reflecting the change in the physiological state of the brain induced by KA injection. By measuring CBF using (1)H MRI and (129)Xe dynamics simultaneously and comparing these results, it was suggested that the reduction of T(1i), in addition to the increase of CBF due to KA-induced epilepsy, are possible causes of the change in (129)Xe dynamics. Thus, the present method would be useful to detect a pathophysiological state in the brain and provide a novel tool for future brain study.

Origin of dissolved-phase hyperpolarized 129Xe signal in the mouse chest based on experimental evidence from extensive magnetic resonance measurements.

Pulmonary study using hyperpolarized (HP) (129)Xe gas as an imaging medium must focus on dissolved-phase signals to make the most of the characteristic affinity of xenon for biological tissues, including blood. However, the spectral pattern of these signals differs between mice and other animals, including rats, canines, and humans. Dissolved-phase study has been reported only scarcely in mice, so spectral assignment has been an important subject for HP (129)Xe magnetic resonance (MR) spectroscopy (MRS) and MR imaging for its wider application. We performed MRS, including magnetization transfer experiments, and MR imaging studies to confirm the origin of dissolved-phase signals of mice ex vivo and in vivo and obtained evidence to assign dissolved-phase signals at 192 ppm for epicardial fat, 196 ppm for lung parenchyma, and 200 ppm for blood. These results were the first to show the possibility of fast exchange of xenon between plasma and red blood cells.

Hyperpolarized 129Xe lung MRI in spontaneously breathing mice with respiratory gated fast imaging and its application to pulmonary functional imaging.

In the present study, a balanced steady-state free precession pulse sequence combined with compressed sensing was applied to hyperpolarized (129) Xe lung imaging in spontaneously breathing mice. With the aid of fast imaging techniques, the temporal resolution was markedly improved in the resulting images. Using these protocols and respiratory gating, (129) Xe lung images in end-inspiratory and end-expiratory phases were obtained successfully. The application of these techniques for pulmonary functional imaging made it possible to simultaneously evaluate regional ventilation and gas exchange in the same animal. A comparative study between healthy and elastase-induced mouse models of emphysema showed abnormal ventilation as well as gas exchange in elastase-treated mice.

Hyperpolarized 129Xe MR imaging with balanced steady-state free precession in spontaneously breathing mouse lungs.

PURPOSE: We investigated the characteristics of hyperpolarized (HP) (129)Xe magnetic resonance (MR) imaging obtained from balanced steady-state free precession (SSFP) measurement of mouse lungs, especially under spontaneous breathing, and compared the results with those obtained using traditional spoiled gradient echo (SPGR) method, focusing on improved signal-to-noise ratio (SNR) and reduced total acquisition time. METHODS: We calculated magnetization response of the HP (129)Xe gas for the balanced SSFP sequence under spontaneous breathing to derive optimal conditions for the imaging experiment. We then placed an anesthetized mouse in the magnet (9.4T) supplied with oxygen gas and a mixture of HP (129)Xe gas supplied from a continuous-flow hyperpolarizing system. We obtained an axial plane image of the lung through balanced SSFP and SPGR sequences, changing the various magnetic resonance (MR) imaging parameters, and measured the SNR of these images. RESULTS: We demonstrated the clear dependence of image intensity on flip angle and number of shots. The SNR was higher in balanced SSFP than in SPGR and 2.3-fold higher compared at each maximum. In contrast, total acquisition time in balanced SSFP was shortened to about one-eighth that of SPGR using a one-shot acquisition mode. CONCLUSION: In HP (129)Xe MR imaging of the lung of a spontaneously breathing mouse, balanced SSFP sequence with multi-shot and centric order acquisition provides higher SNR in a shorter acquisition time than SPGR.