Ayami Nakanishi

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.

Pathologic Changes of Cone Photoreceptors in Eyes With Occult Macular Dystrophy

PURPOSE Occult macular dystrophy (OMD) is an inherited retinal disease characterized by a progressive decrease of vision and appearance of normal fundus. To determine the pathologic features of OMD, we investigated the alternation of the photoreceptors using quantitative image analysis. METHODS We studied 22 eyes of 11 OMD patients. Three of them had a mutation (R45W) in RP1L1. The relative intensities of the ellipsoid zone in the spectral-domain optical coherence tomography (SD-OCT) images and the density of the cone photoreceptors in the adaptive optics (AO) fundus images of the OMD patients were compared to those of normal controls. RESULTS The relative intensities of the ellipsoid zone in the SD-OCT images of patients with OMD were significantly lower (P < 0.001) by an average of 16% compared to that of the normal controls. Normal cone mosaics were not observed in the AO images of the macula in the eyes with OMD. The mean ± SD of cone density of the 9 OMD patients was 1970 ± 884 cells/mm2 at 2°, 1124 ± 483 cells/mm2 at 3°, and 1288 ± 715 cells/mm2 at 4° nasal to the fovea. The cone densities at 2°, 3°, and 4° nasal to the fovea of OMD were significantly lower than those of the normal controls (P < 0.001). CONCLUSIONS A sparse array of cone photoreceptors with significantly reduced density of the macula is one of the morphologic features of OMD.

Restoration of Cone Interdigitation Zone Associated With Improvement of Focal Macular ERG After Fovea-Off Rhegmatogenous Retinal Reattachment

PURPOSE To determine whether a correlation exists between the parameters of the focal macular ERGs (FMERGs) and the microstructural changes of the photoreceptors after successful surgery for fovea-off rhegmatogenous retinal detachment (RRD). METHODS Twenty eyes of 20 patients who had undergone successful surgery to reattach the retina in eyes with fovea-off RRD were studied. Focal macular ERGs and spectral-domain OCT (SD-OCT) were recorded at 1 and 6 months after the surgery. Changes of the components of the FMERGs, as well as changes of the SD-OCT parameters including the length of the external limiting membrane (ELM), ellipsoid zone (EZ), cone interdigitation zone (CIZ), and size of the outer photoreceptor area (between ELM and RPE), were determined. RESULTS During the postoperative period, the mean amplitudes of the a-waves increased by 1.4 times and the b-waves by 1.7 times. Spectral-domain OCT showed that the mean length of the EZ and CIZ and the size of the outer photoreceptor area had increased significantly at 6 months. The degree of the increase in the CIZ and outer photoreceptor area was significantly correlated with the increase in the amplitudes of the b-waves of the FMERGs (r = 0.56, P = 0.042, r = 0.57, P = 0.040, respectively; Spearman rank correlation test). However, the length of the EZ was not significantly correlated with the increase of the b-waves. CONCLUSIONS A restoration of the EZ alone might not be enough to improve the FMERGs, and a restoration of the EZ accompanied by that of the CIZ was essential for the recovery of the FMERGs after fovea-off RRD.

Novel RP1L1 Variants and Genotype-Photoreceptor Microstructural Phenotype Associations in Cohort of Japanese Patients With Occult Macular Dystrophy.

PURPOSE To determine the clinical and genetic characteristics of Japanese patients with occult macular dystrophy (OMD) in a nationwide multicenter study. METHODS Twenty-three patients from 21 families with clinically diagnosed OMD were studied at 10 institutions throughout Japan. Ophthalmologic examinations including spectral-domain optic coherence tomography were performed. Patients were classified into two phenotype groups: a classical group having both blurred ellipsoid zone and absence of interdigitation zone of the photoreceptors, and a nonclassical group lacking at least one of these two features. Whole-exome sequencing, direct sequencing, and in silico molecular analysis were performed to detect the pathogenic RP1L1 variants. Statistical associations between the phenotype and genotypes based on the presence of pathogenic RP1L1 variants were investigated. RESULTS There were 12 families with the classical findings and 9 families with the nonclassical findings. Nine pathogenic RP1L1 missense variants were identified in 12 families (57%) including three reported variants, namely, p.R45W, p.S1199C, and p.G1200A, and six novel variants, p.G221R, p.T1194M, p.T1196I, p.G1200D, p.G1200V, and p.V1201G. The pathogenic missense variants in seven families (33%) were located between amino acid numbers 1196 and 1201. A significant association was found between the photoreceptor microstructural phenotypes and molecular genotypes. CONCLUSIONS The spectrum of the morphologic phenotypes and pathogenic RP1L1 variants was documented in a well-characterized Japanese cohort with OMD. A unique motif including six amino acids (1196-1201) downstream of the doublecortin domain could be a hot spot for RP1L1 pathogenic variants. The significant association of the morphologic phenotypes and genotypes indicates that there are two types of pathophysiology underlying the occult macular dysfunction syndrome: a hereditary OMD with the classical phenotype (Miyakes disease), and a nonhereditary OMD-like syndrome with progressive occult maculopathy.

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.

Longitudinal study of visual field changes determined by Humphrey Field Analyzer 10-2 in patients with Retinitis Pigmentosa

The aim of this study is to determine the progress of the visual field defects obtained by the Humphrey Field Analyzer 10-2 program (HFA 10-2) in patients with retinitis pigmentosa (RP). The medical records of 45 eyes of 45 RP patients who had at least 3 visual field tests were reviewed. Linear mixed models were used to follow the changes of the mean deviation and the average sensitivity of 4, 12, and 20 points in three concentric squares, designated as S4, S12, and S20. The median follow-up time was 3.86 years [range: 1.93 to 9.86, IQR (Interquartile range): 3.01 to 4.93]. The median number of the visual field tests was 3 (range: 3 to 15, IQR: 3 to 4). The mean change of the MD was −0.46 dB/year (−5.80%/year). When the patients were grouped by the average initial MD, the less advanced group had slower progressions than the more advanced group in S4, S12, and S20. These results should be useful in understanding the pathological changes of RP in the central visual field.

Contributions of Second- and Third-Order Retinal Neurons to Cone Electroretinograms After Loss of Rod Function in Rhodopsin P347L Transgenic Rabbits

Purpose To determine the contribution of second- and third-order retinal neurons to the photopic electroretinograms (ERGs) after the degeneration of the rods in rhodopsin P347L transgenic rabbits (Tg). Methods Four wild-type (WT) rabbits and four Tg rabbits were studied at 18 months of age. The photopic ERGs elicited at stimulus onset and offset were analyzed. To block different retinal pathways, 2-amino-4-phosphonobutyric acid (APB), 6-cyano-7-nitroquinoxaline-2, 3 (1H,4H)-dione (CNQX), tetrodotoxin (TTX), and N-methyl-DL-aspartic acid (NMDA) were injected intravitreally. Digital subtraction of the postdrug ERGs from the predrug ERGs was used to determine the contributions of the ON-components blocked by APB, the OFF-components blocked by CNQX, and the third-order neurons blocked by TTX+NMDA. Results Contribution of the cone photoreceptors to the photopic ERGs in Tg rabbits was approximately 10% of that in WT rabbits. The amplitudes of the positive waves of the ON-components at stimulus onset in Tg rabbits were approximately one-half as large as those in WT. On the other hand, the amplitudes of the positive waves of the OFF-components at stimulus offset in Tg rabbits were approximately 1.4 to 2.3 times larger than those in WT. Transgenic rabbits had a positive wave at stimulus offset, which was reduced after the TTX+NMDA injection. Conclusions A reduced ON-component and an augmented OFF-component with abnormal responses of the third-order neurons contributed to the cone ERGs after the loss of rod function in Tg rabbits. Our results suggest a complex synaptic remodeling of the residual retinal cells in the advanced stage in Tg rabbits.

Associations Between Outer Retinal Structures and Focal Macular Electroretinograms in Patients With Retinitis Pigmentosa

Purpose Our earlier study showed that the width of the intact ellipsoid zone (EZ) of the photoreceptors was significantly but weakly correlated with the amplitudes of the focal macular ERGs (FMERGs). The aim of this study was to determine a microstructure of the photoreceptors in the spectral-domain optical coherence tomographic (SD-OCT) images that was more strongly correlated with the FMERG parameters in eyes with retinitis pigmentosa (RP). Methods This was a retrospective, observational study. The medical records of 65 patients with RP were reviewed. FMERGs were elicited by a 15-degree stimulus spot. The width of the EZ and the outer segment (OS) area surrounded by EZ and retinal pigment epithelium in the SD-OCT images within 15 degrees of the fovea were evaluated. Spearman correlation tests and multiple stepwise regression analyses were performed. Results There was a strong correlation between the amplitudes of FMERGs and the EZ width (r = 0.68 for a-wave amplitude; r = 0.64 for b-wave amplitude), and also between the amplitudes of the FMERGs and the OS area (r = 0.69 for a-wave amplitude; r = 0.67 for b-wave amplitude). However, some patients had long EZ widths but had severely reduced FMERGs. Multiple stepwise regression analyses showed that the OS area was the only significant independent predictor of the amplitudes of FMERGs (P < 0.001). Conclusions The OS area might be a better morphological structure to use to predict the physiological function of the macula.

Differences in ocular findings in two siblings: one with complete and other with incomplete achromatopsia

PurposePatients with complete achromatopsia (ACHM) lack cone function, and patients with incomplete ACHM have relatively good visual acuity with residual color vision. The pathological mechanism(s) underlying incomplete ACHM has not been determined. The purpose of this study was to determine the pathophysiology of ACHM in two siblings: one with complete ACHM and the other with incomplete ACHM.MethodsThe medical charts of the two siblings were reviewed.ResultsThe sibling with incomplete ACHM had decimal visual acuities that ranged from 0.4 to 0.6 and had moderate color blindness in both eyes. Her younger brother was diagnosed with complete ACHM and was not able to hold fixation, had severe pendular nystagmus, visual acuity that ranged from 0.08 to 0.1, and severe color vision abnormalities in both eyes. Optical coherence tomography (OCT) showed that the ellipsoid zone (EZ) was disruptive in the macular region in both patients. However, careful examination of the OCT images in the incomplete ACHM patient showed a high-density EZ in the central fovea. Adaptive optics (AO) fundus imaging of the sibling with incomplete ACHM revealed sparse cone mosaics remaining within 1° of the foveal center with no mosaics visible outside the central fovea. AO fundus imaging could not be performed in Case 2 because of the severe nystagmus.ConclusionOur results showed that cone mosaics were present in the central fovea in the sibling with incomplete ACHM patient. This may explain the better visual acuity and color vision in this sibling.