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 timelapse 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.mammalian CNS | in vivo imaging | mitochondrial axonal transport | neurodegeneration | aging

To evaluate functional impairment in the corneal endothelium of eyes in patients with diabetes mellitus, after small-incision cataract surgery.Methods: Evaluation was performed in 93 eyes in patients with type 2 diabetes mellitus (diabetic group) and 93 eyes in patients without diabetic mellitus (nondiabetic group) who underwent cataract surgery. Using a topography system, the corneal thickness in the central area was measured before surgery and 1 day, 1 week, and 1 month after surgery. Corneal endothelial cells were counted using a noncontact specular microscope.Results: No significant differences in any preoperative measures were observed between the diabetic and non-diabetic groups. The increase in corneal thickness 1 month after surgery was significantly higher in the diabetic group than in the nondiabetic group (P=.03). The corneal endothelial cell losses 1 day and 1 week after surgery were significantly higher in the diabetic group than in the nondiabetic group (after 1 day, P = .03; and after 1 week, P =.04). Conclusion:Compared with nondiabetic eyes, eyes of patients with diabetes mellitus showed more damage in corneal endothelial cells due to cataract surgery and a delay in the postoperative recovery of corneal edema.

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Oxidative stress is one of the causative factors in progression and etiology of agerelated cataract. Peroxiredoxin 6 (Prdx6), a savior for cells from internal or external environmental stresses, plays a role in cellular signaling by detoxifying reactive oxygen species (ROS) and thereby controlling gene regulation. Using targeted inactivation of the Prdx6 gene, we show that Prdx6-deficient lens epithelial cells (LECs) are more vulnerable to UV-triggered cell death, a major cause of skin disorders including cataractogenesis, and these cells display abnormal protein profiles. PRDX6-depleted LECs showed phenotypic changes and formed lentoid body, a characteristic of terminal cell differentiation and epithelial-mesenchymal transition. Prdx6 Ϫ/Ϫ LECs exposed to UV-B showed higher ROS expression and were prone to apoptosis compared with wild-type LECs, underscoring a protective role for Prdx6. Comparative proteomic analysis using fluorescence-based difference gel electrophoresis along with mass spectrometry and database searching revealed a total of 13 proteins that were differentially expressed in Prdx6 Ϫ/Ϫ cells. Six proteins were upregulated, whereas expression of seven proteins was decreased compared with Prdx6LECs. Among the cytoskeleton-associated proteins that were highly expressed in Prdx6-deficient LECs was tropomyosin (Tm)2␤. Protein blot and real-time PCR validated dramatic increase of Tm2␤ and Tm1␣ expression in these cells. Importantly, Prdx6 ϩ/ϩ LECs showed a similar pattern of Tm2␤ protein expression after transforming growth factor (TGF)-␤ or H2O2 treatment. An extrinsic supply of PRDX6 could restore Tm2␤ expression, demonstrating that PRDX6 may attenuate adverse signaling in cells and thereby maintain cellular homeostasis. Exploring redox-proteomics (Prdx6 Ϫ/Ϫ ) and characterization and identification of abnormally expressed proteins and their attenuation by PRDX6 delivery should provide a basis for development of novel therapeutic interventions to postpone ROS-mediated abnormal signaling deleterious to cells or tissues.peroxiredoxin; oxidative stress; proteomics; tropomyosin OXIDATIVE STRESS has been identified as one of the major causes of age-related diseases, including cataracts (1, 45, 62, 74 -76, 82). Oxidative stress-induced etiology and progression of diseases may result either from diminished natural antioxidants such as catalase, glutathione peroxidase, and peroxiredoxins (PRDXs) due to aging or from increased generation of reactive oxygen species (ROS) by exposure to environmental factors, X-rays, chemicals, toxins, and ultraviolet (UV) radiation. Environmental stressors such as these have been heavily implicated in the etiology and progression of several diseases by inducing ROS-mediated oxidative stress.UV radiation exposure induces several complex sets of acute or chronic responses that can lead to initiation of diseases through overproduction of ROS. ROS modify cellular signaling. Modulation of signal transduction pathways includes changes in gene expression (18). However, investigation...

The purpose of this study was to compare the surgical outcomes of vitrectomy with conventional internal limiting membrane (C-ILM) peeling to that with the inverted ILM (I-ILM) flap technique for large macular holes (MHs). This was a retrospective chart review of consecutive cases with a large MH at nine hospitals in Japan. Among the 1342 eyes, 165 eyes of 165 cases met the inclusion criteria. The results for medium-large MHs with a diameter 400-550 μm were compared to that of eyes with an extra-large MH with a diameter >550 μm. In addition, the results of C-ILM peeling were compared to that of the I-ILM technique. In medium-large MHs, the closure rate was 95.2% (59/62) by C-ILM peeling and 100% (19/19) by the I-ILM technique. In extra-large MHs, the closure rate was 88.4% (38/43) by C-ILM peeling and 100% (41/41) by I-ILM. Although the difference between the two methods was not significant, the I-ILM technique was successful in 100% of the cases. Multiple logistic regression analysis showed the best-corrected visual acuity (BCVA) at 6 months was significantly associated with the preoperative BCVA, MH size, age and sex of the patient. Analyses of the eyes with extra-large MHs show that the surgical success is high, and the I-ILM technique is more effective for closure in eyes with extra-large MHs.