Characterisation of N-glycans in the epithelial-like tissue of the rat cochlea

Nonomura Y., Sawamura S., Hanzawa K., et al. Characterisation of N-glycans in the epithelial-like tissue of the rat cochlea. Scientific Reports 9, 1551 (2019); https://doi.org/10.1038/s41598-018-38079-0.Membrane proteins (such as ion channels, transporters, and receptors) and secreted proteins are essential for cellular activities. N-linked glycosylation is involved in stability and function of these proteins and occurs at Asn residues. In several organs, profiles of N-glycans have been determined by comprehensive analyses. Nevertheless, the cochlea of the mammalian inner ear, a tiny organ mediating hearing, has yet to be examined. Here, we focused on the stria vascularis, an epithelial-like tissue in the cochlea, and characterised N-glycans by liquid chromatography with mass spectrometry. This hypervascular tissue not only expresses several ion transporters and channels to control the electrochemical balance in the cochlea but also harbours different transporters and receptors that maintain structure and activity of the organ. Seventy-nine N-linked glycans were identified in the rat stria vascularis. Among these, in 55 glycans, the complete structures were determined; in the other 24 species, partial glycosidic linkage patterns and full profiles of the monosaccharide composition were identified. In the process of characterisation, several sialylated glycans were subjected sequentially to two different alkylamidation reactions; this derivatisation helped to distinguish α2,3-linkage and α2,6-linkage sialyl isomers with mass spectrometry. These data should accelerate elucidation of the molecular architecture of the cochlea

Hearing Loss Controlled by Optogenetic Stimulation of Nonexcitable Nonglial Cells in the Cochlea of the Inner Ear

Light-gated ion channels and transporters have been applied to a broad array of excitable cells including neurons, cardiac myocytes, skeletal muscle cells and pancreatic β-cells in an organism to clarify their physiological and pathological roles. Nonetheless, among nonexcitable cells, only glial cells have been studied in vivo by this approach. Here, by optogenetic stimulation of a different nonexcitable cell type in the cochlea of the inner ear, we induce and control hearing loss. To our knowledge, deafness animal models using optogenetics have not yet been established. Analysis of transgenic mice expressing channelrhodopsin-2 (ChR2) induced by an oligodendrocyte-specific promoter identified this channel in nonglial cells—melanocytes—of an epithelial-like tissue in the cochlea. The membrane potential of these cells underlies a highly positive potential in a K+-rich extracellular solution, endolymph; this electrical property is essential for hearing. Illumination of the cochlea to activate ChR2 and depolarize the melanocytes significantly impaired hearing within a few minutes, accompanied by a reduction in the endolymphatic potential. After cessation of the illumination, the hearing thresholds and potential returned to baseline during several minutes. These responses were replicable multiple times. ChR2 was also expressed in cochlear glial cells surrounding the neuronal components, but slight neural activation caused by the optical stimulation was unlikely to be involved in the hearing impairment. The acute-onset, reversible and repeatable phenotype, which is inaccessible to conventional gene-targeting and pharmacological approaches, seems to at least partially resemble the symptom in a population of patients with sensorineural hearing loss. Taken together, this mouse line may not only broaden applications of optogenetics but also contribute to the progress of translational research on deafness

The asparagine 533 residue in the outer pore loop region of the mouse PKD2L1 channel is essential for its voltage-dependent inactivation

Voltage-dependent inactivation of ion channels contributes to the regulation of the membrane potential of excitable cells. Mouse polycystic kidney disease 2-like 1 (PKD2L1) forms voltage-dependent nonselective cation channels, which are activated but subsequently inactivated in response to membrane depolarization. Here, we found that the mutation of an asparagine 533 residue (N533Q) in the outer pore loop region of PKD2L1 caused a marked increase in outward currents induced by depolarization. In addition, the tail current analysis demonstrated that the N533Q mutants are activated during depolarization but the subsequent inactivation does not occur. Interestingly, the N533Q mutants lacked the channel activation triggered by the removal of stimuli such as extracellular alkalization and heating. Our findings suggest that the N533 residue in the outer pore loop region of PKD2L1 has a key role in the voltage-dependent channel inactivation.status: publishe

FDG healthy volunteer imaging with the world’s first helmet-type brain PET

[Purpose] We developed the world’s first helmet-type time-of-flight (TOF) PET, which had a hemispherical detector arrangement. The developed system is characterized as its high-performance and compactness as well as an unusual seat-type design which enables keeping the subject’s eye view wide. Here, we report the basic performance characteristics and the first FDG imaging results of healthy volunteers. [Methods] The helmet-type PET consisted of 54 detectors. The 45 detectors were arranged to form a hemisphere and the other 9 detectors were placed to form a half-ring behind the neck. The detector was composed of 12×12 lutetium fine silicate (LFS) scintillators (4×4×10 mm3) connected to a 12×12 silicon photomultiplier (SiPM) array with one-to-one coupling. The energy window was 450–590 keV and the coincidence time window was 3.6 ns. Image reconstruction was performed using 3D-OSEM with 4 iterations and 8 subsets. The performance was characterized based on the NEMA NU2 standards. The coincidence timing resolution was measured using a 22Na point source. 18F-FDG PET and MRI were performed for 11 normal control subjects. The PET data were measured for 10 minutes starting at 45 minutes after injection of 285±23 MBq. [Results] The coincidence timing resolution was 235 ps. With the sensitivity gain of 5.7 (= 20-cm-diameter object / 3.5 cm TOF localization), the effective sensitivity was 24 cps/kBq and the effective noise-equivalent count ratio was 120 kcps@9 kBq/mL. The spatial resolution of the FBP image was 2.9 mm at the central position (1 cm offset) of the field-of-view. For the human FDG PET images, the gray matter was clearly visualized with a high contrast to the white matter. Small brain nuclei such as the substantia nigra, the red nucleus, and the superior colliculus were able to be identified.[Conclusion] We have successfully demonstrated first human imaging on the helmet-type PET.第78回日本放射線技術学会総会学術大

Initial results of a mouse brain PET insert with a staggered 3-layer DOI detector

Small animal positron emission tomography (PET) requires a submillimeter resolution for better quantification of radiopharmaceuticals. On the other hand, depth-of-interaction (DOI) information is essential to preserve the spatial resolution while maintaining the sensitivity. Recently, we developed a staggered 3-layer DOI detector with 1 mm crystal pitch and 15 mm total crystal thickness, but we did not demonstrate the imaging performance of the DOI detector with full ring geometry. In this study we present initial imaging results obtained for a mouse brain PET prototype developed with the staggered 3-layer DOI detector. The prototype had 53 mm inner diameter and 11 mm axial field-of-view. The PET scanner consisted of 16 DOI detectors each of which had a staggered 3-layer LYSO crystal array (4/4/7 mm) coupled to a 4×4 SiPM array. The physical performance was evaluated in terms of the NEMA NU 4 2008 protocol. The measured spatial resolutions at the center and 15 mm radial offset were 0.67 mm and 1.56 mm for filtered-back-projection, respectively. The peak absolute sensitivity of 0.74% was obtained with an energy window of 400-600 keV. The resolution phantom imaging results show the clear identification of a submillimetric rod pattern with the ordered-subset expectation maximization algorithm. The inter-crystal scatter rejection using a narrow energy window could enhance the resolvability of a 0.75 mm rod significantly. In an animal imaging experiment, the detailed mouse brain structures such as cortex and thalamus were clearly identified with high contrast. In conclusion, we successfully developed the mouse brain PET insert prototype with a staggered 3-layer DOI detector

First prototyping of a dedicated PET system with the hemisphere detector arrangement

A strong demand is expected for high-sensitivity, high-resolution and low-cost brain positron emission tomography (PET) imaging for early diagnosis of dementia, as well as for general neuroscience studies. Therefore, we have proposed novel geometries of a hemisphere detector arrangement for high-sensitivity brain imaging, in which an add-on detector at the chin positionor neck position helps in sensitivity uniformity improvement. In this study, we developed the first prototype system for proof-of-concept using four-layer depth-of-interaction detectors, each of which consisted of 16 × 16 × 4 Zr-doped GSO crystals with dimensions of 2.8 × 2.8 × 7.5 mm3 and a high-sensitivity 64-channel flat-panel photomultiplier tube. We used 47 detectors to form a hemisphere detector with a hemisphere shape of 25 cm inner diameter and 50 cm outer diameter, and we used seven detectors for each of the add-on detectors. The total detector number of 54 was about one-fourth that of a typical whole-body PET scanner. The hemisphere detector for the prototype system was realized by multiple rings having different numbers of detectors and a cross-shaped top detector unit covering the top. Performance evaluation showed uniform spatial resolutions of 3–4 mm by the filtered back-projection method. Imaging tests of a hot-rod phantom done with an iterative method were able to resolve 2.2 mm rods. Peak sensitivity was measured as more than 10% at a region near the top of the head, which was achieved with the help of the top detector unit. In addition, using the prototype system, we performed the first FDG clinical test with a healthy volunteer. The results showed that the proposed geometries had high potential for realizing high-sensitivity, high-resolution, and low-cost brain PET imaging. As for the add-on detector position, it was shown that the neck position resulted in higher sensitivity and wider field of view (FOV) than the chin position because the add-on detector at the neck position can be placed continuously to the hemisphere detector and close to the FOV