Masatoshi Nagaya

In vivo imaging of axonal transport of mitochondria in the diseased and aged mammalian CNS

Significance The lack of intravital imaging of axonal transport of mitochondria in the living mammalian CNS precludes the characterization of transport dynamics in the diseased and aged mammalian CNS. Here we report minimally invasive intravital multiphoton imaging of mouse retinal ganglion cells that offers sequential time-lapse images of mitochondria transported in a single axon with submicrometer resolution. We show highly dynamic axonal transport of mitochondria in the mammalian CNS in vivo under physiological conditions and characterize disturbances of mitochondrial transport in a mouse glaucoma model and age-related changes in mitochondrial transport. Our method is useful for characterizing the dynamics of axonal transport of mitochondria and the dynamics of other submicrometer structures in the diseased and aged mammalian CNS in vivo. The lack of intravital imaging of axonal transport of mitochondria in the mammalian CNS precludes characterization of the dynamics of axonal transport of mitochondria in the diseased and aged mammalian CNS. Glaucoma, the most common neurodegenerative eye disease, is characterized by axon degeneration and the death of retinal ganglion cells (RGCs) and by an age-related increase in incidence. RGC death is hypothesized to result from disturbances in axonal transport and in mitochondrial function. Here we report minimally invasive intravital multiphoton imaging of anesthetized mouse RGCs through the sclera that provides sequential time-lapse images of mitochondria transported in a single axon with submicrometer resolution. Unlike findings from explants, we show that the axonal transport of mitochondria is highly dynamic in the mammalian CNS in vivo under physiological conditions. Furthermore, in the early stage of glaucoma modeled in adult (4-mo-old) mice, the number of transported mitochondria decreases before RGC death, although transport does not shorten. However, with increasing age up to 23–25 mo, mitochondrial transport (duration, distance, and duty cycle) shortens. In axons, mitochondria-free regions increase and lengths of transported mitochondria decrease with aging, although totally organized transport patterns are preserved in old (23- to 25-mo-old) mice. Moreover, axonal transport of mitochondria is more vulnerable to glaucomatous insults in old mice than in adult mice. These mitochondrial changes with aging may underlie the age-related increase in glaucoma incidence. Our method is useful for characterizing the dynamics of axonal transport of mitochondria and may be applied to other submicrometer structures in the diseased and aged mammalian CNS in vivo.

Plasma-activated medium suppresses choroidal neovascularization in mice: a new therapeutic concept for age-related macular degeneration

Choroidal neovascularization (CNV) is the main pathogenesis of age-related macular degeneration (AMD), which leads to severe vision loss in many aged patients in most advanced country. CNV compromises vision via hemorrhage and retinal detachment on account of pathological neovascularization penetrating the retina. Plasma medicine represents the medical application of ionized gas “plasma” that is typically studied in the field of physical science. Here we examined the therapeutic ability of plasma-activated medium (PAM) to suppress CNV. The effect of PAM on vascularization was assessed on the basis of human retinal endothelial cell (HREC) tube formation. In mice, laser photocoagulation was performed to induce CNV (laser-CNV), followed by intravitreal injection of PAM. N-Acetylcysteine was used to examine the role of reactive oxygen species in PAM-induced CNV suppression. Fundus imaging, retinal histology examination, and electroretinography (ERG) were also performed to evaluate PAM-induced retinal toxicity. Interestingly, HREC tube formation and laser-CNV were both reduced by treatment with PAM. N-acetylcysteine only partly neutralized the PAM-induced reduction in laser-CNV. In addition, PAM injection had no effect on regular retinal vessels, nor did it show retinal toxicity in vivo. Our findings indicate the potential of PAM as a novel therapeutic agent for suppressing CNV.

Histamine H4 receptor as a new therapeutic target for choroidal neovascularization in age-related macular degeneration

The present treatment for choroidal neovascularization (CNV) associated with age‐related macular degeneration (AMD) is not sufficient. Hence, we examined the therapeutic efficacy of reducing histamine H4 receptor expression on CNV in mice.

NEAR-INFRARED REFLECTANCE IMAGING IN EYES WITH ACUTE ZONAL OCCULT OUTER RETINOPATHY.

Purpose: To investigate the confocal scanning laser ophthalmoscopic images obtained with near-infrared (IR) light in eyes with acute zonal occult outer retinopathy (AZOOR). Methods: Observational case series. The medical records of 12 eyes of 10 patients with AZOOR were reviewed. Scanning laser ophthalmoscopic images obtained from the AZOOR eyes were compared with images obtained by spectral-domain optical coherence tomography, by fundus autofluorescence, and by an adaptive optics fundus camera. Results: In 8 of 12 eyes, abnormal hyporeflective areas were detected in the IR images, and the other 4 eyes did not have specific abnormalities in the IR images. The boundaries of the abnormal hyporeflective areas corresponded with the border of the irregularity of photoreceptor inner segment ellipsoid band in the spectral-domain optical coherence tomography images. The cone mosaics of the adaptive optics fundus image were disrupted in the abnormal hyporeflective area of the IR image. However, the areas of fundus autofluorescence abnormalities did not coincide with the hyporeflective areas in the IR images. Conclusion: The presence of hyporeflective areas in the IR images of patients with AZOOR suggests impairment of the photoreceptors area. The IR images would be useful to evaluate eyes with AZOOR.

Pikachurin Protein Required for Increase of Cone Electroretinogram B-Wave during Light Adaptation.

In normal eyes, the amplitude of the b-wave of the photopic ERGs increases during light adaptation, but the mechanism causing this increase has not been fully determined. The purpose of this study was to evaluate the contribution of receptoral and post-receptoral components of the retina to this phenomenon. To accomplish this, we examined the ERGs during light adaptation in Pikachurin null-mutant (Pika -/-) mice, which have a misalignment of the bipolar cell dendritic tips to the photoreceptor ribbon synapses. After dark-adaptation, photopic ERGs were recorded from Pika -/- and wild type (WT) mice during the first 9 minutes of light adaptation. In some of the mice, post-receptoral components were blocked pharmacologically. The photopic b-waves of WT mice increased by 50% during the 9 min of light adaptation as previously reported. On the other hand, the b-waves of the Pika -/- mice decreased by 20% during the same time period. After blocking post-receptoral components, the b-waves were abolished from the WT mice, and the ERGs resembled those of the Pika -/- mice. The extracted post-receptoral component increased during light adaptation in the WT mice, but decreased for the first 3 minutes to a plateau in Pika -/- mice. We conclude that the normal synaptic connection between photoreceptor and retinal ON bipolar cells, which is controlled by pikachurin, is required for the ERGs to increase during light-adaptation. The contributions of post-receptoral components are essential for the photopic b-wave increase during the light adaptation.