Hirohiko Imai

Biochemical and Functional Characterization of RNF213 (Mysterin) R4810K, a Susceptibility Mutation of Moyamoya Disease, in Angiogenesis In Vitro and In Vivo

Background P.R4810K of RNF213 (mysterin: rs112735431), which is an AAA+ ATPase, is the susceptibility polymorphism for moyamoya disease (MMD) in East Asians. However, the role of RNF213 R4810K in the etiology of MMD is unknown. Methods and Results To clarify the role of RNF213 in known angiogenic pathways, RNF213 expression was analyzed in endothelial cells (ECs) treated with several angiogenic and antiangiogenic factors, including interferons (IFNs). RNF213 was upregulated by IFN-β through signal transducer and activator of transcription x in the promoter and mediated antiangiogenic activity of IFN-β. RNF213 wild-type (WT) overexpression could not lower angiogenesis without IFN-β, but RNF213 R4810K overexpression could. To correlate biochemical function as ATPase and the role of RNF213 oligomer formation with antiangiogenic activity, we investigated the effects of mutations in the AAA+ module. A mutation of the Walker B motif (WEQ), which stabilizes oligomerization, inhibited angiogenesis, but AAA+ module deletion, which cannot initiate oligomerization, did not. Intriguingly, R4810K, similar to WEQ, decreased ATPase activity, suggesting its antiangiogenic activity through stabilizing oligomers. To confirm the antiangiogenic effect of RNF213 upregulation in vivo, vascular EC- or smooth muscle cell-specific Rnf213 R4757K (R4810K ortholog) or WT transgenic (Tg) mice were exposed to hypoxia. Cerebral angiogenesis by hypoxia was suppressed in EC-specific Rnf213 R4757K Tg mice, whereas it was not suppressed in other mice. Conclusions This study suggests the importance of inflammatory signals as environmental factors and R4810K carriers for susceptibility to cerebral hypoxia. A specific inhibitor of ATP binding to the first AAA+ could be a promising therapeutic candidate for MMD.

Noninvasive detection of pulmonary tissue destruction in a mouse model of emphysema using hyperpolarized 129Xe MRS under spontaneous respiration.

In the present study, a chemical shift saturation recovery method in hyperpolarized 129Xe MR spectroscopy measurements was applied to two groups of spontaneously breathing mice, an elastase‐induced emphysema model and a control group. Parameters detected were those related to lung structures and functions, such as alveolar septal thickness, h, the ratio of the alveolar septal volume relative to gas space volume, Vs/Va, and the transit time of blood through the gas exchange region, τ. To investigate the potential of these parameters as biomarkers, an attempt was made to detect physiologic changes in the lungs of elastase‐treated mice. Our results showed that Vs/Va was significantly reduced in elastase‐treated mice, reflecting emphysema‐like destruction of the alveolar wall. Compared with histologic results, this degree of reduction was shown to reflect the severity of wall destruction. On the other hand, significant changes in other parameters, h and τ, were not shown. This study is the first application of hyperpolarized 129Xe MR spectroscopy to a mouse model of emphysema and shows that the Vs/Va volume ratio is an effective biomarker for emphysema that could become useful in drug research and development through noninvasive detection of pathologic changes in small rodents. Magn Reson Med, 2010.

Hyperpolarized 129 Xe MRI of the mouse lung at a low xenon concentration using a continuous flow-type hyperpolarizing system.

To apply a continuous flow‐type hyperpolarizing (CF‐HP) system to lung imaging and investigate the feasibility of hyperpolarized 129Xe MRI at a low xenon concentration.

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 129Xe 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, 129Xe 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. Copyright

Magnetic Resonance Imaging of Tumor with a Self-Traceable Phosphorylcholine Polymer

Polymers are concentration-amplified with respect to the monomeric units. We show here that a phosphorylcholine polymer enriched with (13)C/(15)N at the methyl groups is self-traceable by multiple-resonance (heteronuclear-correlation) NMR in tumor-bearing mice inoculated with the mouse rectal cancer cell line (colon 26). Preliminary measurements indicated that the present polymeric nanoprobe was satisfactorily distinguished from lipids and detectable with far sub-micromolar spectroscopic and far sub-millimolar imaging sensitivities. Detailed ex vivo and in vivo studies for the tumor-bearing mice administered the probe with a mean molecular weight of 63,000 and a mean size of 13 nm, revealed the following: (1) this probe accumulates in the tumor highly selectively (besides renal excretion) and efficiently (up to 30% of the injected dose), (2) the tumor can thus be clearly in vivo imaged, the lowest clearly imageable dose of the probe being 100 mg/kg or 2.0 mg/20-g mouse, and (3) the competition between renal excretion and tumor accumulation is size-controlled; that is, the larger (higher molecular-weight) and smaller (lower molecular-weight) portions of the probe undergo tumor accumulation and renal excretion, respectively. The observed size dependence suggests that the efficient tumor-targeting of the present probe is stimulated primarily by the so-called enhanced permeability and retention (EPR) effect, that is, size-allowed invasion of the probe into the tumor tissue via defective vascular wall. Self-traceable polymers thus open an important area of magnetic resonance imaging (MRI) of tumors and may provide a highly potential tool to visualize various delivery/localization processes using synthetic polymers.

Morphogenesis of the Inner Ear at Different Stages of Normal Human Development

This study examined the external morphology and morphometry of the human embryonic inner ear membranous labyrinth and documented its three‐dimensional position in the developing embryo using phase‐contrast X‐ray computed tomography and magnetic resonance imaging. A total of 27 samples between Carnegie stage (CS) 17 and the postembryonic phase during trimester 1 (approximately 6–10 weeks after fertilization) were included. The otic vesicle elongated along the dorso‐ventral axis and differentiated into the end lymphatic appendage and cochlear duct (CD) at CS 17. The spiral course of the CD began at CS18, with anterior and posterior semicircular ducts (SDs) forming prominent circles with a common crus. The spiral course of the CD comprised more than two turns at the postembryonic phase, at which time the height of the CD was evident. A linear increase was observed in the length of anterior, posterior, and lateral SDs, in that order, and the length of the CD increased exponentially over the course of development. Bending in the medial direction was observed between the cochlear and vestibular parts from the latero‐caudal view, with the angle decreasing during development. The position of the inner ear was stable throughout the period of observation on the lateral to ventral side of the rhombencephalon, caudal to the pontine flexure, and adjacent to the auditory ganglia. The plane of the lateral semicircular canal was approximately 8.0°–14.6° with respect to the cranial caudal (z‐)axis, indicating that the orientation of the inner ear changes during growth to adulthood. Anat Rec, 298:2081–2090, 2015.

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

MRI of hyperpolarized 129Xe 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 T  2* 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 129Xe lung MRI was investigated. The ultrashort echo‐time has made it possible to acquire dissolved 129Xe images with reasonably high spatial resolution of 0.625 × 0.625 mm2 and to obtain T  2* 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 129Xe 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. Magn Reson Med, 2013.

Hyperpolarized 129Xe dynamic study in mouse lung under spontaneous respiration: Application to murine tumor B16BL6 melanoma

This is a study on the analysis of hyperpolarized (HP) (129)Xe dynamics applied in the lung of a pathological model mouse under spontaneous respiration. A novel parameter k(1)k(2) - a product of the rate constant for Xe transfer from gas to dissolved phase (k(1)) and from dissolved to gas phase (k(2)) - was shown to be derived successfully from the analysis of the HP (129)Xe dynamic MR experiment in the mouse lung under spontaneous respiration with the aid of a selective pre-saturation technique. A comparative study using healthy mice and model mice induced with lung cancer (by injection of murine tumor B16BL6 melanoma) was performed and a significant difference was found in the k(1)k(2) values of the two groups, that is, 0.020+/-0.007s(-2) (n=4) for healthy mice and 0.032+/-0.04s(-2) (n=3) for lung cancer model mice (p=0.04). Thus, the parameter obtained by our proposed method is considered useful for detection of lung tumors.

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

The feasibility of ventilation imaging with hyperpolarized (HP) 129Xe 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 129Xe delivered continuously from a 129Xe polarizer. A series of 129Xe ventilation images was obtained by varying the number of breaths before the 129Xe 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. Copyright

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 (129Xe) in the mouse brain. The key technique is based on repeatedly applying radio frequency (RF) pulses and measuring the decrease of HP 129Xe magnetization after the brain Xe concentration has reached a steady state due to continuous HP 129Xe ventilation. The signal decrease of the 129Xe 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 (λi), and the longitudinal relaxation time (T1i) of HP 129Xe 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 1H MRI and 129Xe dynamics simultaneously and comparing these results, it was suggested that the reduction of T1i, in addition to the increase of CBF due to KA‐induced epilepsy, are possible causes of the change in 129Xe 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. Copyright