Visualization of Reissner's membrane in the mouse inner ear using highly sensitive magnetic resonance imaging analysis.

Although research on hearing loss, including the identification of causative genes, has become increasingly active, the pathogenic mechanism of hearing loss remains unclear. One of the reasons for this is that the structure of the inner ear of mice, which is commonly used as a genetically modified animal model, is too small and complex, making it difficult to accurately capture abnormalities and dynamic changes in vivo. Especially, Reissner's membrane is a very important structure that separates the perilymph and endolymph of the inner ear. This malformation or damage induces abnormalities in hearing and balance. Until now, imaging analyses, such as magnetic resonance imaging (MRI) and computed tomography, are performed to investigate the inner ear structure in vivo; however, it has been difficult to analyze the small inner ear structure of mice owing to resolution. Therefore, there is an urgent need to develop an image analysis method that can accurately capture the structure of the inner ear of mice including Reissner's membrane, both dynamically and statically. This study aimed to investigate whether it is possible to accurately capture the structure (e.g., Reissner's membrane) and abnormalities of the inner ear of mice using an 11.7 T MRI. By combining two types of MRI methods, in vivo and ex vivo, we succeeded for the first time in capturing the fine structure of the normal mouse inner ear, such as the Reissner's membrane, and inflammatory lesions of otitis media mouse models in detail and accurately. In the future, we believe that understanding the state of Reissner's membrane during living conditions will greatly contribute to the development of research on inner ear issues, such as hearing loss.

Identification of an atypical monocyte and committed progenitor involved in fibrosis.

Monocytes and macrophages comprise a variety of subsets with diverse functions. It is thought that these cells play a crucial role in homeostasis of peripheral organs, key immunological processes and development of various diseases. Among these diseases, fibrosis is a life-threatening disease of unknown aetiology. Its pathogenesis is poorly understood, and there are few effective therapies. The development of fibrosis is associated with activation of monocytes and macrophages. However, the specific subtypes of monocytes and macrophages that are involved in fibrosis have not yet been identified. Here we show that Ceacam1+Msr1+Ly6C-F4/80-Mac1+ monocytes, which we term segregated-nucleus-containing atypical monocytes (SatM), share granulocyte characteristics, are regulated by CCAAT/enhancer binding protein ß (C/EBPß), and are critical for fibrosis. Cebpb deficiency results in a complete lack of SatM. Furthermore, the development of bleomycin-induced fibrosis, but not inflammation, was prevented in chimaeric mice with Cebpb-/- haematopoietic cells. Adoptive transfer of SatM into Cebpb-/- mice resulted in fibrosis. Notably, SatM are derived from Ly6C-FcεRI+ granulocyte/macrophage progenitors, and a newly identified SatM progenitor downstream of Ly6C-FcεRI+ granulocyte/macrophage progenitors, but not from macrophage/dendritic-cell progenitors. Our results show that SatM are critical for fibrosis and that C/EBPß licenses differentiation of SatM from their committed progenitor.

Accurate Estimation of the Duration of Testicular Ischemia Using Creatine Chemical Exchange Saturation Transfer (CrCEST) Imaging.

BACKGROUND: In the management of testicular torsion, estimating the duration of testicular ischemia is essential for deciding on an appropriate surgical treatment, but there are currently limited evaluation methods. PURPOSE: To perform testicular creatine chemical exchange saturation transfer (CrCEST) imaging and to evaluate its ability to accurately estimate the duration of testicular ischemia. STUDY TYPE: Prospective. ANIMAL MODEL: C57BL/6 control mice (n = 6) and testicular ischemia models induced by clamping the spermatic cord (n = 14). Eight of testicular ischemia models were serially imaged at two or three timepoints and a total of 26 images of ischemic testis were obtained. The ischemic duration ranged from 6-42 hours. FIELD STRENGTH/SEQUENCE: 11.7T vertical-bore MRI/segment fast low-angle shot acquisition for CEST. ASSESSMENT: CrCEST imaging was performed and the magnetization transfer ratio for the CrCEST effect (MTRCr** ) was calculated in control mice and testicular ischemia models. Correlation analysis between the duration of testicular ischemia and MTRCr** decline was performed. STATISTICAL TESTS: Paired t-test, and Pearson's correlation analysis. RESULTS: In control mice, the CrCEST effect in testes was significantly more than five times higher than that in skeletal muscle. MTRCr** did not differ significantly between the right and left testes (8.6 ± 0.8 vs. 8.3 ± 0.6, P = 0.96). In testicular ischemia models, MTRCr** of ischemic testes was significantly lower than that of controls (4 ± 2 vs. 8.9 ± 0.6, P < 0.001). Correlation analysis revealed a strong linear correlation between MTRCr** decline and the duration of ischemia (r = 0.96, P < 0.001). DATA CONCLUSION: A decreased CrCEST effect in ischemic testes correlated well with ischemic duration. Testicular CrCEST imaging was useful for accurately estimating the duration of testicular ischemia. LEVEL OF EVIDENCE: 2 TECHNICAL EFFICACY STAGE: 2.

Visualization of Spatial Distribution of Spermatogenesis in Mouse Testes Using Creatine Chemical Exchange Saturation Transfer Imaging.

BACKGROUND: When determining treatment strategies for male infertility, it is important to evaluate spermatogenesis and its spatial distribution in the testes. PURPOSE: To investigate the usefulness of creatine chemical exchange saturation transfer (CrCEST) imaging for evaluating spermatogenesis and its spatial distribution. STUDY TYPE: Prospective. ANIMAL MODEL: C57BL/6 control mice (n = 5) and model mice of male infertility induced by whole testis X-ray irradiation (n = 11) or localized X-ray irradiation to lower regions of testes (n = 3). FIELD STRENGTH/SEQUENCE: A 11.7-T vertical-bore magnetic resonance imaging (MRI)/segmented fast low-angle shot acquisition for CEST. ASSESSMENT: The magnetization transfer ratio for the CrCEST effect (MTRCr* ) was calculated in each testis of the control mice and X-ray irradiation model mice at 10, 15, 20, and 30 days after irradiation. Correlation analysis was performed between MTRCr* and Johnsen's score, a histological score for spermatogenesis. In the localized X-ray irradiation model, regional MTRCr* and Johnsen's score were calculated for correlation analysis. STATISTICAL TESTS: Unpaired t-test, one-way analysis of variance with Tukey's HSD test and Pearson's correlation analysis. A P value < 0.05 was considered statistically significant. RESULTS: In the irradiation model, CrCEST imaging revealed a significant linear decrease of MTRCr* after irradiation (control, 8.7 ± 0.6; 10 days, 7.9 ± 0.8; 15 days, 6.5 ± 0.6; 20 days, 5.4 ± 1.0; 30 days, 4.4 ± 0.8). A significant linear correlation was found between MTRCr* and Johnsen's score (Pearson's correlation coefficient (r) = 0.79). In the localized irradiation model, CrCEST imaging visualized a significant regional decrease of MTRCr* in the unshielded region (shielded, 6.9 ± 0.7; unshielded, 4.9 ± 1.0), and a significant linear correlation was found between regional MTRCr* and Johnsen's score (r = 0.78). DATA CONCLUSION: Testicular CrCEST effects correlated well with spermatogenesis. CrCEST imaging was useful for evaluating spermatogenesis and its spatial distribution. EVIDENCE LEVEL: 2 TECHNICAL EFFICACY: Stage 2.

Original experimental rat model of blast-induced mild traumatic brain injury: a pilot study.

OBJECTIVE: Diagnosing blast-induced mild traumatic brain injury (mTBI) is difficult due to minimal imaging findings. This study aimed to establish a rat model of behavioral abnormality caused by blast-induced mTBI and detect new findings for therapeutic intervention. METHODS: We used a bench-top blast wave generator with the blast wave exiting through a 20-mm I.D. nozzle aimed at the focused target. The blast wave was directed at the head of male Wistar rats under general anesthesia positioned prone 2.5 cm below the nozzle. Peak shock wave pressure was 646.2 ± 70.3 kPa. RESULTS: After blast injury, mTBI rats did not show the findings of brain hemorrhage or contusion macroscopically and on hematoxylin-eosin-stained frozen sections but did show anorexia and weight loss in the early post-injury phase. Behavioral experiments revealed short-term memory impairment at 2 weeks and depression-like behavior at 2 and 6 weeks. Diffusion-weighted ex vivo MRI showed high-intensity areas in layers of the bilateral hippocampus. Immunohistochemical analysis revealed accumulation of reactive microglia and GFAP-positive astrocytes in the same region and loss of NeuN-positive neurons in the hippocampal pyramidal cell layer. CONCLUSIONS: This model can reflect the pathophysiology of blast-induced mTBI and could potentially be used to develop therapeutic interventions in the future.

Mouse skeletal muscle creatine chemical exchange saturation transfer (CrCEST) imaging at 11.7T MRI.

BACKGROUND: Creatine chemical exchange saturation transfer (CrCEST) imaging is expected to be a novel evaluation method of muscular energy metabolism. PURPOSE: To develop CrCEST imaging of mouse skeletal muscle and to validate this technique by measuring changes in Cr concentration of ischemic hindlimbs. STUDY TYPE: Prospective. ANIMAL MODEL: C57BL/6 mice (n = 6), mild hindlimb ischemic mice (n = 6), and severe hindlimb ischemic mice (n = 6). FIELD STRENGTH/SEQUENCE: Magnetic resonance angiography (MRA), CrCEST imaging, and phosphorus magnetic resonance spectroscopy (31 P MRS) obtained at 11.7T. ASSESSMENT: MRA and 31 P MRS were performed to confirm the presence of ischemia following the compression by rubber tourniquet. CrCEST imaging was performed and magnetization transfer ratio asymmetry (MTRasym ), which reflects Cr concentration, and was calculated in severe ischemia models, mild ischemia models, and control mice. Follow-up CrCEST imaging was performed after the release of ischemia in the mild ischemia models. STATISTICAL TESTS: Mean ± SD, one-way analysis of variance (ANOVA) with Tukey's HSD test, unpaired or paired t-test. RESULTS: MRA revealed the loss of blood flow of the femoral artery in the ischemic hindlimb. 31 P MRS revealed different degrees of PCr decrease in severe and mild ischemic hindlimb (n = 3 per group, normal hindlimb: 1.0 ± 0, mild ischemic hindlimb: 0.77 ± 0.13, severe ischemic hindlimb: 0 ± 0). CrCEST imaging inversely revealed a significant stepwise increase in the MTRasym ratio of ischemic hindlimbs compared with controls (control, mild ischemia, and severe ischemia; 0.99 ± 0.04, 1.36 ± 0.08, and 1.59 ± 0.23, respectively, P < 0.0001). In addition, follow-up CrCEST imaging after the release of ischemia revealed normalization of the MTRasym ratios (recovered hindlimb: 1.01 ± 0.05). DATA CONCLUSION: We demonstrated an increase in the MTRasym of ischemic hindlimbs, along with a decrease of PCr. We demonstrated the normalization of MTRasym after the release of ischemia and developed CrCEST imaging of mouse skeletal muscle. LEVEL OF EVIDENCE: 2 Technical Efficacy: Stage 2 J. Magn. Reson. Imaging 2020;51:563-570.

Regulatory roles of epithelial-mesenchymal interaction (EMI) during early and androgen dependent external genitalia development.

Development of external genitalia (ExG) has been a topic of long mystery in the field of organogenesis research. Early stage male and female of mouse embryos develop a common genital tubercle (GT) in the perineum whose outgrowth extends distally from the posterior cloacal regions. Concomitant with GT outgrowth, the cloaca is divided into urogenital sinus and anorectum by urorectal septum (URS) internally. The outgrowth of the GT is associated with the formation of endodermal epithelial urethral plate (UP) attached to the ventral epidermis of the GT. Such a common developmental phase is observed until around embryonic day 15.5 (E15.5) morphologically in mouse embryogenesis. Various growth factor genes, such as Fibroblast growth factor (Fgf) and Wnt genes are expressed and function during GT formation. Since the discovery of key growth factor signals and several regulatory molecules, elucidation of their functions has been achieved utilizing mouse developmental models, conditional gene knockout mouse and in vitro culture. Analyses on the phenotypes of such mouse models have revealed that several growth factor families play fundamental roles in ExG organogenesis based on the epithelial-mesenchymal interaction (EMI). More recently, EMI between developing urethral epithelia and its bilateral mesenchyme of later stages is also reported during subsequent stage of androgen-dependent male-type urethral formation in the mouse embryo. Mafb, belonging to AP-1 family and a key androgen-responsive mesenchymal gene, is identified and starts to be expressed around E14.5 when masculinization of the urethra is initiated. Mesenchymal cell condensation and migration, which are regulated by nonmuscle myosin, are shown to be essential process for masculinization. Hence, studies on EMI at various embryonic stages are important not only for early but also for subsequent masculinization of the urethra. In this review, a dynamic mode of EMI for both early and late phases of ExG development is discussed.

Critical role of Trib1 in differentiation of tissue-resident M2-like macrophages.

Macrophages consist of at least two subgroups, M1 and M2 (refs 1-3). Whereas M1 macrophages are proinflammatory and have a central role in host defence against bacterial and viral infections, M2 macrophages are associated with responses to anti-inflammatory reactions, helminth infection, tissue remodelling, fibrosis and tumour progression. Trib1 is an adaptor protein involved in protein degradation by interacting with COP1 ubiquitin ligase. Genome-wide association studies in humans have implicated TRIB1 in lipid metabolism. Here we show that Trib1 is critical for the differentiation of F4/80(+)MR(+) tissue-resident macrophages--that share characteristics with M2 macrophages (which we term M2-like macrophages)--and eosinophils but not for the differentiation of M1 myeloid cells. Trib1 deficiency results in a severe reduction of M2-like macrophages in various organs, including bone marrow, spleen, lung and adipose tissues. Aberrant expression of C/EBPα in Trib1-deficient bone marrow cells is responsible for the defects in macrophage differentiation. Unexpectedly, mice lacking Trib1 in haematopoietic cells show diminished adipose tissue mass accompanied by evidence of increased lipolysis, even when fed a normal diet. Supplementation of M2-like macrophages rescues the pathophysiology, indicating that a lack of these macrophages is the cause of lipolysis. In response to a high-fat diet, mice lacking Trib1 in haematopoietic cells develop hypertriglyceridaemia and insulin resistance, together with increased proinflammatory cytokine gene induction. Collectively, these results demonstrate that Trib1 is critical for adipose tissue maintenance and suppression of metabolic disorders by controlling the differentiation of tissue-resident M2-like macrophages.

Cerebrospinal fluid mitochondrial DNA in neuromyelitis optica spectrum disorder.

BACKGROUND: Neuromyelitis optica spectrum disorder (NMOSD) is an inflammatory disease of the central nervous system. Although complement-dependent astrocyte damage mediated by anti-aquaporin 4 autoantibody (AQP4-Ab) is well acknowledged to be the core of NMOSD pathogenesis, additional inflammatory cascades may contribute to the establishment of lesion formation. Thus, in this study, we investigated the possible pathogenic role of immune-reactive mitochondrial DNA (mtDNA) in cerebrospinal fluid (CSF) of NMOSD patients. METHODS: Using quantitative polymerase chain reaction, we measured extracellular mtDNA levels in CSF of NMOSD patients positive for AQP4-Ab. Patients with multiple sclerosis or other neurological diseases were examined as controls. Pre- and post-treatment extracellular mtDNA levels were also compared in the NMOSD group. Extracellular mtDNA release from human astrocytes was analyzed in vitro utilizing NMOSD sera, and interleukin (IL)-1ß production was measured in supernatants of mixed glial cells stimulated with DNA fraction of CSF derived from NMOSD patients. Furthermore, specific innate immune pathways mediating the IL-1ß production by mtDNA were investigated in peripheral blood mononuclear cells with selective inhibitors of Toll-like receptor 9 (TLR9) and NOD-like receptor protein 3 (NLRP3) inflammasomes. RESULTS: Extracellular mtDNA level was specifically elevated in acute phase of NMOSD CSF. In vitro studies provided the evidence that mtDNA is released from human astrocytes by NMOSD sera. In addition, DNA fraction isolated from NMOSD CSF promoted secretion of IL-1ß from mixed glial cells. Selective inhibition of TLR9 and NLRP3 inflammasomes revealed that mtDNA-mediated IL-1ß production depends on specific innate immune pathways. CONCLUSION: Extracellular mtDNA is specifically elevated in the CSF of patients with acute phase NMOSD, and mtDNA released by AQP4-Ab-mediated cellular damage elicits the innate immune cascades via TLR9 and NLRP3 inflammasomes pathways. Our study highlights mtDNA-mediated innate immune pathways as a novel therapeutic target for future treatment of NMOSD patients.

Apparent brain temperature imaging with multi-voxel proton magnetic resonance spectroscopy compared with cerebral blood flow and metabolism imaging on positron emission tomography in patients with unilateral chronic major cerebral artery steno-occlusive disease.

PURPOSE: The purpose of the present study was to determine whether apparent brain temperature imaging using multi-voxel proton magnetic resonance (MR) spectroscopy correlates with cerebral blood flow (CBF) and metabolism imaging in the deep white matter of patients with unilateral chronic major cerebral artery steno-occlusive disease. METHODS: Apparent brain temperature and CBF and metabolism imaging were measured using proton MR spectroscopy and 15O-positron emission tomography (PET), respectively, in 35 patients. A set of regions of interest (ROIs) of 5 × 5 voxels was placed on an MR image so that the voxel row at each edge was located in the deep white matter of the centrum semiovale in each cerebral hemisphere. PET images were co-registered with MR images with these ROIs and were re-sliced automatically using image analysis software. RESULTS: In 175 voxel pairs located in the deep white matter, the brain temperature difference (affected hemisphere - contralateral hemisphere: ΔBT) was correlated with cerebral blood volume (CBV) (r = 0.570) and oxygen extraction fraction (OEF) ratios (affected hemisphere/contralateral hemisphere) (r = 0.641). We excluded voxels that contained ischemic lesions or cerebrospinal fluid and calculated the mean values of voxel pairs in each patient. The mean ΔBT was correlated with the mean CBF (r = - 0.376), mean CBV (r = 0.702), and mean OEF ratio (r = 0.774). CONCLUSIONS: Apparent brain temperature imaging using multi-voxel proton MR spectroscopy was correlated with CBF and metabolism imaging in the deep white matter of patients with unilateral major cerebral artery steno-occlusive disease.

A Novel Peroxisome Proliferator-activated Receptor (PPAR)α Agonist and PPARγ Antagonist, Z-551, Ameliorates High-fat Diet-induced Obesity and Metabolic Disorders in Mice.

A novel peroxisome proliferator-activated receptor (PPAR) modulator, Z-551, having both PPARα agonistic and PPARγ antagonistic activities, has been developed for the treatment of obesity and obesity-related metabolic disorders. We examined the effects of Z-551 on obesity and the metabolic disorders in wild-type mice on the high-fat diet (HFD). In mice on the HFD, Z-551 significantly suppressed body weight gain and ameliorated insulin resistance and abnormal glucose and lipid metabolisms. Z-551 inhibited visceral fat mass gain and adipocyte hypertrophy, and reduced molecules involved in fatty acid uptake and synthesis, macrophage infiltration, and inflammation in adipose tissue. Z-551 increased molecules involved in fatty acid combustion, while reduced molecules associated with gluconeogenesis in the liver. Furthermore, Z-551 significantly reduced fasting plasma levels of glucose, triglyceride, free fatty acid, insulin, and leptin. To elucidate the significance of the PPAR combination, we examined the effects of Z-551 in PPARα-deficient mice and those of a synthetic PPARγ antagonist in wild-type mice on the HFD. Both drugs showed similar, but weaker effects on body weight, insulin resistance and specific events provoked in adipose tissue compared with those of Z-551 as described above, except for lack of effects on fasting plasma triglyceride and free fatty acid levels. These findings suggest that Z-551 ameliorates HFD-induced obesity, insulin resistance, and impairment of glucose and lipid metabolisms by PPARα agonistic and PPARγ antagonistic activities, and therefore, might be clinically useful for preventing or treating obesity and obesity-related metabolic disorders such as insulin resistance, type 2 diabetes, and dyslipidemia.

Brain temperature measured by 1H-magnetic resonance spectroscopy in acute and subacute carbon monoxide poisoning.

INTRODUCTION: Brain temperature (BT) is associated with the balance between cerebral blood flow and metabolism according to the "heat-removal" theory. The present study investigated whether BT is abnormally altered in acute and subacute CO-poisoned patients by using (1)H-magnetic resonance spectroscopy (MRS). METHODS: Eight adult CO-poisoned patients underwent 3-T magnetic resonance imaging in the acute and subacute phases after CO exposure. MRS was performed on deep cerebral white matter in the centrum semiovale, and MRS-based BT was estimated by the chemical shift difference between water and the N-acetyl aspartate signal. We defined the mean BT + 1.96 standard deviations of the BT in 15 healthy controls as the cutoff value for abnormal BT increases (p < 0.05) in CO-poisoned patients. RESULTS: BT of CO-poisoned patients in both the acute and subacute phases was significantly higher than that of the healthy control group. However, BT in the subacute phase was significantly lower than in the acute phase. On the other hand, no significant difference in body temperature was observed between acute and subacute CO-poisoned patients. BT weakly correlated with body temperature, but this correlation was not statistically significant (rho = 0.304, p = 0.2909). CONCLUSIONS: The present results suggest that BT in CO-poisoned patients is abnormally high in the acute phase and remains abnormal in the subacute phase. BT alteration in these patients may be associated with brain perfusion and metabolism rather than other factors such as systemic inflammation and body temperature.

Early pathological alterations of lower lumbar cords detected by ultrahigh-field MRI in a mouse multiple sclerosis model.

Magnetic resonance imaging (MRI) is widely employed for the diagnosis of multiple sclerosis (MS). However, sometimes, the lesions found by MRI do not correlate with the neurological impairments observed in MS patients. We recently showed autoreactive T cells accumulate in the fifth lumbar cord (L5) to pass the blood-brain barrier and cause inflammation in the central nervous system of experimental autoimmune encephalomyelitis (EAE) mice, an MS model. We here investigated this early event using ultrahigh-field MRI. T2-weighted image signals, which conform to the water content, increased in L4 and L5 during the development of EAE. At the same time, the sizes of L4 and L5 changed. Moreover, angiographic images of MRI showed branch positions of the blood vessels in the lower lumbar cords were significantly altered. Interestingly, EAE mice showed occluded and thickened vessels, particularly during the peak phase, followed by reperfusion in the remission phase. Additionally, demyelination regions of some MS patients had increased lactic acid content, suggesting the presence of ischemic events. These results suggest that inflammation-mediated alterations in the lower lumbar cord change the homeostasis of the spinal cord and demonstrate that ultrahigh-field MRI enables the detection of previously invisible pathological alterations in EAE.

Pharmacological MRI response to a selective dopamine transporter inhibitor, GBR12909, in awake and anesthetized rats.

Pharmacological magnetic resonance imaging (phMRI) is a powerful tool for imaging the effects of drugs on brain activity. In preclinical phMRI studies, general anesthesia used for minimizing head movements is thought to influence the phMRI responses to drugs. In this study we investigated the phMRI responses to a selective dopamine transporter (DAT) inhibitor, GBR12909, and a dopamine (DA) releaser, d-amphetamine (AMPH), in the isoflurane anesthetized and awake rats using a relative cerebral blood volume (rCBV) method. AMPH (1 mg/kg i.p.) caused an increase in rCBV in the dopaminergic circuitry in the both anesthetized and awake rats. The striatal rCBV change was correlated with the change of the striatal DA concentration induced by AMPH in the both anesthetized and awake rats. GBR12909 (10 mg/kg i.p.) caused a positive rCBV response and showed a similar regional pattern of rCBV response to AMPH in the awake rats, and the correlation between the change of the striatal rCBV and the striatal DA concentration was observed. However, in the anesthetized rats, GBR12909 induced a widespread negative rCBV response, whereas an increase in striatal DA concentration was observed. These findings indicate that phMRI responses to activation of DA neurotransmission by GBR12909 or AMPH are overall identical in the awake state, while the phMRI response to a DAT inhibitor, GBR12909 but not to AMPH was changed by isoflurane anesthesia. For the evaluation of neuroactive drugs using phMRI, isoflurane anesthesia might be complicated the interpretation of pharmacodynamic effects of drugs in preclinical studies.

Activatable 19F MRI nanoparticle probes for the detection of reducing environments.

(19)F magnetic resonance imaging (MRI) probes that can detect biological phenomena such as cell dynamics, ion concentrations, and enzymatic activity have attracted significant attention. Although perfluorocarbon (PFC) encapsulated nanoparticles are of interest in molecular imaging owing to their high sensitivity, activatable PFC nanoparticles have not been developed. In this study, we showed for the first time that the paramagnetic relaxation enhancement (PRE) effect can efficiently decrease the (19)F NMR/MRI signals of PFCs in silica nanoparticles. On the basis of the PRE effect, we developed a reduction-responsive PFC-encapsulated nanoparticle probe, FLAME-SS-Gd(3+) (FSG). This is the first example of an activatable PFC-encapsulated nanoparticle that can be used for in vivo imaging. Calculations revealed that the ratio of fluorine atoms to Gd(3+) complexes per nanoparticle was more than approximately 5.0×10(2), resulting in the high signal augmentation.

Multifunctional core­shell silica nanoparticles for highly sensitive (19)F magnetic resonance imaging.

19F magnetic resonance imaging (19F MRI) is useful for monitoring particular signals from biological samples, cells, and target tissues, because background signals are missing in animal bodies. Therefore, highly sensitive 19F MRI contrast agents are in great demand for their practical applications. However, we have faced the following challenges: 1) increasing the number of fluorine atoms decreases the solubility of the molecular probes, and 2) the restriction of the molecular mobility attenuates the 19F MRI signals. Herein, we developed novel multifunctional core­shell nanoparticles to solve these issues. They are composed of a core micelle filled with liquid perfluorocarbon and a robust silica shell. These core­shell nanoparticles have superior properties such as high sensitivity, modifiability of the surface, biocompatibility, and sufficient in vivo stability. By the adequate surface modifications, gene expression in living cells and tumor tissue in living mice were successfully detected by 19F MRI.

[Application of Ultra-High-Field Magnetic Resonance Imaging to the Central Nervous System].

The development of high-performance magnetic resonance imaging (MRI) scanners is ongoing. The strength of the magnetic field is the most important factor in the use of this technology. Ultra-high magnetic fields provide many benefits, including high spatial and temporal resolution. In this chapter, we describe the characteristics and images obtained using ultra-high-field MRI.

Non-invasive differentiation of hepatic steatosis and steatohepatitis in a mouse model using nitroxyl radical as an MRI-contrast agent.

Drug-induced steatohepatitis is considered more serious than drug-induced hepatic steatosis, so that differentiating between the two is crucial in drug development. In addition, early detection of drug-induced steatohepatitis is considered important since recovery is possible with drug withdrawal. However, no method has been established to differentiate between the two. In the development of drug-induced steatohepatitis, reactive oxygen species (ROS) is excessively generated in the liver. It has been reported that ROS can be monitored with electron spin resonance (ESR) and dynamic nuclear polarization-magnetic resonance imaging (DNP-MRI) by using nitroxyl radicals, which are known to participate in various in vivo redox reactions. The decay/reduction rate, which is an index for monitoring nitroxyl radicals, has been reported to be increased in tissues with excessive ROS levels other than liver, but decreased in methionine choline deficient (MCD) diet-induced steatohepatitis with excess ROS. Therefore, looking to differentiate between drug-induced hepatic steatosis and steatohepatitis, we examined whether the reduction rate decreases in steatohepatitis other than the MCD-diet induced disease and whether the decrease could be detected by MRI. We used STAM™ mice in which hepatic steatosis and steatohepatitis developed sequentially under diabetic conditions. 3-carbamoyl-PROXYL (CmP), one of the nitroxyl radicals, was injected intravenously during the MRI procedure and the reduction rate was calculated. The reduction rate was significantly higher in early steatohepatitis than in hepatic steatosis and the control. Excess ROS in early steatohepatitis was detected by an immunohistochemical marker for ROS. Therefore, it was indicated that the increase or decrease in the reduction rate in steatohepatitis differs depending on the model, and early steatohepatitis could be noninvasively differentiated from hepatic steatosis using CmP in MRI. Since the change in direction of the reduction rate in steatohepatitis in clinical studies could be predicted by confirming the reduction rate in preclinical studies, the present method, which can be used consistently in clinical and preclinical studies, warrants consideration as a candidate monitoring method for differentiating between early drug-induced steatohepatitis and hepatic steatosis in drug development.

A mouse model of autoimmune inner ear disease without endolymphatic hydrops.

Autoimmune inner ear disease (AIED) is an organ-specific disease characterized by irreversible, prolonged, and progressive hearing and equilibrium dysfunctions. The primary symptoms of AIED include asymmetric sensorineural hearing loss accompanied by vertigo, aural fullness, and tinnitus. AIED is divided into primary and secondary types. Research has been conducted using animal models of rheumatoid arthritis (RA), a cause of secondary AIED. However, current models are insufficient to accurately analyze vestibular function, and the mechanism underlying the onset of AIED has not yet been fully elucidated. Elucidation of the mechanism of AIED onset is urgently needed to develop effective treatments. In the present study, we analyzed the pathogenesis of vertigo in autoimmune diseases using a mouse model of type II collagen-induced RA. Auditory brain stem response analysis demonstrated that the RA mouse models exhibited hearing loss, which is the primary symptom of AIED. In addition, our vestibulo-oculomotor reflex analysis, which is an excellent vestibular function test, accurately captured vertigo symptoms in the RA mouse models. Moreover, our results revealed that the cause of hearing loss and vestibular dysfunction was not endolymphatic hydrops, but rather structural destruction of the organ of Corti and the lateral semicircular canal ampulla due to an autoimmune reaction against type II collagen. Overall, we were able to establish a mouse model of AIED without endolymphatic hydrops. Our findings will help elucidate the mechanisms of hearing loss and vertigo associated with AIED and facilitate the development of new therapeutic methods.

Transplantation of Human Embryonic Stem Cell-Derived Pericyte-Like Cells Transduced with Basic Fibroblast Growth Factor Promotes Angiogenic Recovery in Mice with Severe Chronic Hindlimb Ischemia.

Critical limb ischemia (CLI) is a state of severe peripheral artery disease, with no effective treatment. Cell therapy has been investigated as a therapeutic tool for CLI, and pericytes are promising therapeutic candidates based on their angiogenic properties. We firstly generated highly proliferative and immunosuppressive pericyte-like cells from embryonic stem (ES) cells. In order to enhance the angiogenic potential, we transduced the basic fibroblast growth factor (bFGF) gene into the pericyte-like cells and found a significant enhancement of angiogenesis in a Matrigel plug assay. Furthermore, we evaluated the bFGF-expressing pericyte-like cells in the previously established chronic hindlimb ischemia model in which bone marrow-derived MSCs were not effective. As a result, bFGF-expressing pericyte-like cells significantly improved blood flow in both laser Doppler perfusion imaging (LDPI) and dynamic contrast-enhanced MRI (DCE-MRI). These findings suggest that bFGF-expressing pericyte-like cells differentiated from ES cells may be a therapeutic candidate for CLI.