Intermitochondrial signaling regulates the uniform distribution of stationary mitochondria in axons.

In the central nervous system (CNS), many neurons develop axonal arbors that are crucial for information processing. Previous studies have demonstrated that premature axons contain motile and stationary mitochondria, and their balance is important for axonal arborization. However, the mechanisms by which neurons determine the positions of stationary mitochondria as well as their turnover remain to be elucidated. We observed that the distribution of stationary mitochondrial spots along the unmyelinated and nonsynaptic axons is not random but rather relatively uniform both in primary cultured neurons and in tissues. Intriguingly, whereas the positions of each mitochondrial spot changed over time, the overall distribution remained uniform. In addition, local inactivation of mitochondria by KillerRed mediated chromophore-assisted light inactivation (CALI) inhibited the translocation of mitochondrial spots in adjacent axonal regions, suggesting that functional mitochondria enhance the motility of other mitochondria in the vicinity. Signals of ATP:ADP sensor, PercevalHR indicated that the ATP:ADP ratio was relatively high around mitochondria, and treating axons with phosphocreatine (PCr), which supplies ATP, reduced the immobile mitochondria induced by the local mitochondrial inactivation. In a mathematical model, we found that the ATP gradient generated by mitochondria, and ATP dependent regulation of mitochondrial motility could establish uniform mitochondrial distribution. These observations suggest that axons in the CNS possess the system that distributes mitochondria uniformly, and intermitochondrial signaling contribute to the regulation. In addition, our results suggest the possibility that ATP might be one of the molecules mediating the signaling.

Histone acetyltransferase and Polo-like kinase 3 inhibitors prevent rat galactose-induced cataract.

Diabetic cataracts can occur at an early age, causing visual impairment or blindness. The detailed molecular mechanisms of diabetic cataract formation remain incompletely understood, and there is no well-documented prophylactic agent. Galactose-fed rats and ex vivo treatment of lenses with galactose are used as models of diabetic cataract. To assess the role of histone acetyltransferases, we conducted cataract prevention screening with known histone acetyltransferase (HAT) inhibitors. Ex vivo treatment with a HAT inhibitor strongly inhibited the formation of lens turbidity in high-galactose conditions, while addition of a histone deacetylase (HDAC) inhibitor aggravated turbidity. We conducted a microarray to identify genes differentially regulated by HATs and HDACs, leading to discovery of a novel cataract causative factor, Plk3. Plk3 mRNA levels correlated with the degree of turbidity, and Plk3 inhibition alleviated galactose-induced cataract formation. These findings indicate that epigenetically controlled Plk3 influences cataract formation. Our results demonstrate a novel approach for prevention of diabetic cataract using HAT and Plk3 inhibitors.

Randomised Clinical Trial for Postoperative Complications after Ex-PRESS Implantation versus Trabeculectomy with 2-Year Follow-Up.

We compared complications between Ex-PRESS implantation and trabeculectomy for 2 years after surgery. Sixty-four open-angle glaucoma eyes were randomly assigned to treatment with trabeculectomy (n = 32) or Ex-PRESS implantation (n = 32). The primary outcomes were postoperative complications, including reduction of the endothelial cell density (ECD) of the cornea, cataract progression and the frequency of other late postoperative complications. The Ex-PRESS group had significantly greater reduction of postoperative corneal ECD than the trabeculectomy group did at 2 years after surgery (P = 0.026). Among the corneal areas measured using specular microscopy, the superior area, where the Ex-PRESS tube was inserted, had significantly more severe corneal ECD reduction than the inferior area after 2 years (-17.6% in superior area and -11.7% in inferior area, P = 0.04). More cataract progression occurred in the trabeculectomy group than in the Ex-PRESS group (P = 0.04). Twelve eyes (37.5%) in the trabeculectomy group and 4 eyes (12.5%) in the Ex-PRESS group underwent cataract surgery (P = 0.019). The total number of other postoperative complications between 3 months and 2 years was significantly higher in the trabeculectomy group than in the Ex-PRESS group (P = 0.02). Although Ex-PRESS implantation might be associated with an increased rate of corneal endothelial cell loss compared with trabeculectomy, it is beneficial for preventing cataract progression after filtering surgery.

Effect of 0.05% Difluprednate Ophthalmic Emulsion on Proinflammatory Cytokine Levels After Retinal Laser Photocoagulation in Rabbits.

PURPOSE: We aimed to evaluate the effect of the topical application of a strong corticosteroid, difluprednate, on the levels of inflammatory and angiogenic cytokine in the vitreous and aqueous humor after laser photocoagulation. METHODS: Pigmented rabbits were treated with retinal laser photocoagulation and divided into 4 groups, namely control (no additional treatment), topical application of difluprednate 0.05%, betamethasone sodium phosphate 0.1%, and sub-Tenon injection of triamcinolone acetonide (STTA). Samples of vitreous and aqueous humor were collected on posttreatment days 0, 1, 7, and 14. The levels of intraocular vascular endothelial growth factor (VEGF), interleukin-6 (IL-6), intercellular adhesion molecule-1 (ICAM-1), and monocyte chemotactic protein-1 (MCP-1) were measured using an immunoassay. Intraocular pressure (IOP) was monitored in each group. RESULTS: VEGF, IL-6, ICAM-1, and MCP-1 were significantly elevated on day 1 and were reduced in both the vitreous and aqueous humor following topical application of difluprednate and STTA. Topical betamethasone reduced their levels in the aqueous humor but not in the vitreous. A significant increase of IOP induced by difluprednate returned to control levels after withdrawal of administration. CONCLUSION: Although the elevation of IOP was an expected, manageable side effect, topical application of difluprednate was more effective than STTA and betamethasone for reducing inflammatory cytokine levels after laser treatment.

Effect of Microtubule Disruption on Dynamics of Acidic Organelles in the Axons of Primary Cultured Retinal Ganglion Cells.

PURPOSE: Axonal transport is fundamental to autophagy in neuronal cells. To understand its biological significance in various conditions, it is necessary to monitor the process of autophagy. However, monitoring methods are often limited to static analyses, such as protein expression and histological observations. Autophagy has multistep process and is highly dynamic; therefore, additional techniques are necessary to study autophagy. In this study, we quantified the dynamics of autophagy-related organelle transport under conditions of dynamic instability and catastrophic disruption of microtubules using in vitro live imaging. MATERIALS AND METHODS: Retinal ganglion cells (RGCs) were isolated from postnatal day 3 Sprague-Dawley rats by immunopanning. After 7 days of culture, acidic organelles were stained by LysoTracker. Dynamics of acidic organelles was quantified using kymographs. Colchicine was used to induce microtubule disruption. Movement of acidic organelles was observed at five time points: before, and at 6, 24, 72, and 120 h after colchicine stimulation. Ethidium homodimer-1 (EthD-1) was used to determine cell viability. RESULTS: The status of axonal transport of acidic organelles (n = 363) from 27 RGCs was classified into four categories: anterograde (1.4%), retrograde (90%), stationary (8.0%), and fluttering (0.28%). Six hours after the induction of microtubule disruption in 14 of 27 RGCs, almost all acidic organelles (n = 236) were stationary. All acidic components had completely stopped moving 24 h later. At 72 h after stimulation, axonal fragmentation, and shrinking and disappearance of soma were observed in 71% of RGCs. Finally, the remaining RGCs became positive for EthD-1. In the control (13 of 27 RGCs), axonal transport was maintained for 120 h and EthD-1-positive RGCs were not observed. CONCLUSION: Almost all acidic organelles were transported retrogradely along the axon, which was inhibited by colchicine. Understanding the dynamics of acidic organelles may provide useful parameters for characterizing autophagy of neuronal cells in pathophysiological conditions.

Photocoagulation of the Retinal Nonperfusion Area Prevents the Expression of the Vascular Endothelial Growth Factor in an Animal Model.

Purpose: The purpose of this study was to evaluate whether photocoagulation of the retinal nonperfusion area suppresses ocular vascular endothelial growth factor (VEGF) expression in a rabbit retinal vein occlusion (RVO) model. Methods: The retinas of pigmented rabbits were made ischemic by a laser on the main branch of retinal veins following intravenous injection of Rose Bengal. The eyes were enucleated before treatment and at 1, 7, and 14 days after laser occlusion. VEGF protein levels in the vitreous humor, sensory retina, and retinal pigment epithelium-choroid were measured with enzyme-linked immunosorbent assay. In situ hybridization of VEGF messenger RNA was performed to detect the location of VEGF expression in the sensory retina. Results: In the vitreous body, the VEGF protein level in the RVO group, but not that in the RVO + panretinal photocoagulation group, significantly increased on day 14. In the retina, the VEGF protein level in the RVO + panretinal photocoagulation group was significantly higher than that in the RVO group on day 1, but was significantly lower than that in the RVO group on days 7 and 14. In the in situ hybridization analysis, the RVO group showed a high expression of VEGF in the inner nuclear and ganglion cell layers on days 7 and 14. In contrast, VEGF expression in the RVO + panretinal photocoagulation group was strongly suppressed in both the inner nuclear and ganglion cell layers on days 7 and 14. Conclusions: This study is the first using an animal RVO model to demonstrate that laser photocoagulation of the retinal nonperfusion area suppresses VEGF-A expression in the retina.

Panretinal Photocoagulation Using Short-Pulse Laser Induces Less Inflammation and Macular Thickening in Patients with Diabetic Retinopathy.

We compared the effect of panretinal photocoagulation (PRP) using short-pulse laser (SPL) and conventional laser, regardless of the number of spots, in terms of their effect on the progression of diabetic macular edema (DME) and anterior flare intensity (AFI) in patients with high-risk nonproliferative diabetic retinopathy (non-PDR). Forty-two eyes of 42 patients were subjected to PRP using the conventional argon laser (Conv group) or SPL (SPL group). CRT and AFI levels in the SPL group were significantly lower than those in the Conv group (CRT at 4, 6, and 10 weeks; AFI at 6, 10, and 18 weeks). Eyes of rabbits were photocoagulated using conventional laser with 500 spots (Conv 500s), SPL with 500 spots (SPL 500s), or 1000 spots (SPL 1000s). Vascular endothelial growth factor (VEGF), interleukin-6 (IL-6), intercellular adhesion molecule-1 (ICAM-1), and monocyte chemotactic protein-1 (MCP-1) levels in vitreous humor were measured using an immunoassay. Compared to conventional laser, VEGF, IL-6, and MCP-1 levels were significantly lower in the SPL 1000s and SPL 500s groups. In patients with high-risk non-PDR, SPL has a greater preventive effect on the progression of DME and AFI and produces less inflammatory cytokines than conventional lasers.

Comparison of subconjunctival scarring after microincision vitrectomy surgery using 20-, 23-, 25- and 27-gauge systems in rabbits.

PURPOSE: To compare subconjunctival scarring after vitrectomy in rabbit eyes using different gauge systems by analysing anterior segment optical coherence tomography (AS-OCT) images and histological sections. METHODS: Vitrectomy using 20-, 23-, 25- and 27-gauge systems was performed for rabbits. Anterior segment optical coherence tomography (AS-OCT) images of the incision sites were obtained before and at day 1, 7 and 1 month after surgery. We measured the thickness of conjunctival epithelium, stroma, Tenon's capsule and total conjunctiva of these three layers, then determined the preservation rates of the borderlines between each layer. Surgical invasion was estimated by histological observation. RESULTS: The thickness of total conjunctiva, or the thickness of both conjunctival stroma and Tenon's capsule in the 20-gauge group was significantly thicker than that in the 27-gauge group at day 1 and day 7 after surgery. Preservation rates of the conjunctival stroma/Tenon's capsule borderline were significantly lower in the 20-gauge group than in the 25- and 27-gauge groups at day 1, day 7 and 1 month. Preservation rates of the Tenon's capsule/sclera borderline were significantly lower in the 20-gauge group than in the 25- and 27-gauge groups at 1 month. In the 27-gauge group, the number of α-smooth muscle actin-positive fibroblasts was significantly smaller than in the 20-gauge group at day 7. CONCLUSION: Based on the finding of AS-OCT and histology, micro incision vitreous surgery, especially using 27-gauge, contributed to less subconjunctival scarring postoperatively. Therefore, the 27-gauge pars plana vitrectomy (PPV) may be a more effective technique for preserving the structure of conjunctiva.

Neuroprotective effect of bilberry extract in a murine model of photo-stressed retina.

Excessive exposure to light promotes degenerative and blinding retinal diseases such as age-related macular degeneration and retinitis pigmentosa. However, the underlying mechanisms of photo-induced retinal degeneration are not fully understood, and a generalizable preventive intervention has not been proposed. Bilberry extract is an antioxidant-rich supplement that ameliorates ocular symptoms. However, its effects on photo-stressed retinas have not been clarified. In this study, we examined the neuroprotective effects of bilberry extract against photo-stress in murine retinas. Light-induced visual function impairment recorded by scotopic and phototopic electroretinograms showing respective rod and cone photoreceptor function was attenuated by oral administration of bilberry extract through a stomach tube in Balb/c mice (750 mg/kg body weight). Bilberry extract also suppressed photo-induced apoptosis in the photoreceptor cell layer and shortening of the outer segments of rod and cone photoreceptors. Levels of photo-induced reactive oxygen species (ROS), oxidative and endoplasmic reticulum (ER) stress markers, as measured by real-time reverse transcriptase polymerase chain reaction, were reduced by bilberry extract treatment. Reduction of ROS by N-acetyl-L-cysteine, a well-known antioxidant also suppressed ER stress. Immunohistochemical analysis of activating transcription factor 4 expression showed the presence of ER stress in the retina, and at least in part, in Müller glial cells. The photo-induced disruption of tight junctions in the retinal pigment epithelium was also attenuated by bilberry extract, repressing an oxidative stress marker, although ER stress markers were not repressed. Our results suggest that bilberry extract attenuates photo-induced apoptosis and visual dysfunction most likely, and at least in part, through ROS reduction, and subsequent ER stress attenuation in the retina. This study can help understand the mechanisms of photo-stress and contribute to developing a new, potentially useful therapeutic approach using bilberry extract for preventing retinal photo-damage.

The effect of triamcinolone acetonide or bevacizumab on the levels of proinflammatory cytokines after retinal laser photocoagulation in pigmented rabbits.

Although laser photocoagulation is a gold standard for the treatment of retinal ischemic diseases, thermal burn induces the inflammation and the progression of macular edema. To prevent this complication, combination therapy using anti-vascular endothelial growth factor (VEGF) drugs or steroids is clinically utilized, however the mechanisms are still unclear. In this study, we aimed to evaluate the changes in inflammatory and angiogenic cytokine levels in aqueous humor and vitreous body after intravitreal injection of bevacizumab (IVB) or triamcinolone (IVTA), as well as sub-Tenon injection of triamcinolone (STTA) after retinal laser photocoagulation in rabbits. Pigmented rabbits were treated with retinal laser photocoagulation and divided into 4 groups, namely Control (no additional treatment), IVB, IVTA or STTA accordingly. Samples of vitreous and aqueous humor were collected on post-treatment days 0, 1, 7 and 14. The levels of intraocular VEGF, interleukin-6 (IL-6), intercellular adhesion molecule-1 (ICAM-1) and monocyte chemotactic protein-1 (MCP-1) were measured using an immunoassay. The levels of VEGF, IL-6, ICAM-1 and MCP-1 were significantly elevated 1 day after laser treatment. IVTA and STTA significantly reduced the increase in the levels of VEGF, IL-6, ICAM-1 and MCP-1, while IVB reduced that of VEGF only in aqueous humor and vitreous body. The protein amount in the aqueous humor transiently increased 1 day after laser, and was significantly prevented by IVTA or STTA but not IVB. Data showed that bevacizumab only reduced intraocular VEGF after laser, while triamcinolone suppressed both the expression of VEGF and proinflammatory cytokines. We propose that these cytokine profiles may play an important role in the pathogenesis of inflammation after photocoagulation and the underlying mechanism of treatment with anti-VEGF drug and steroids.

Prospective observational study of conjunctival scarring after phacoemulsification.

PURPOSE: To examine whether anterior segment-optical coherence tomography (AS-OCT) can detect conjunctival scarring after transscleral phacoemulsification and whether temporal transscleral phacoemulsification causes scarring in the superior conjunctiva. METHODS: Transscleral phacoemulsification was performed in the superior conjunctiva (superior incision group) or the temporal conjunctiva (temporal incision group). Anterior segment-optical coherence tomography (AS-OCT) images of the superior conjunctiva were obtained before and after surgery. We quantified the thickness of the conjunctiva and preservation rates of the borderlines among the subconjunctival layers. The relationship between the AS-OCT images and histology was evaluated in rabbit eyes with phacoemulsification. RESULTS: Each group comprised 25 patients. At ≤1 month after surgery, the superior conjunctiva was significantly thicker in the superior incision group than the temporal incision group (1 day, 7 days, and 1 month after surgery; p < 0.0001, p < 0.0001, and p < 0.001 respectively); however, there were no significant differences between the two groups at ≥2 months. The borderline of the conjunctival stroma/Tenon's capsule and the borderline of the Tenon's capsule/sclera were preserved significantly better in the temporal incision group after surgery (p < 0.0001). The cell densities in the conjunctiva of the rabbit temporal incision group were unchanged after surgery, whereas the rabbit superior incision group had significantly more neutrophils (p = 0.0001) and myofibroblasts (p < 0.0001) in the superior conjunctiva than the temporal incision group. CONCLUSIONS: Anterior segment-optical coherence tomography (AS-OCT) images can detect conjunctival scarring after transscleral phacoemulsification. The layer structures in the superior conjunctiva are unaffected by temporal transscleral incision.

Randomized Clinical Trial for Early Postoperative Complications of Ex-PRESS Implantation versus Trabeculectomy: Complications Postoperatively of Ex-PRESS versus Trabeculectomy Study (CPETS).

We compared early postoperative complications between trabeculectomy and Ex-PRESS implantation. Enrolled patients with 39 primary open-angle or 25 exfoliative glaucoma were randomly assigned to receive trabeculectomy (trabeculectomy group) or Ex-PRESS implantation (Ex-PRESS group). Primary outcomes were early postoperative complications, including postoperative anterior chamber inflammation, frequencies of hyphema, flat anterior chamber, choroidal detachment, hypotonic maculopathy, and the change of visual acuity. The postoperative flare values in trabeculectomy group were higher than those in the Ex-PRESS group (overall, P = 0.004; and 10 days, P = 0.02). Hyphema occurred significantly more frequently in the trabeculectomy group (P = 0.0025). There were no significant differences of the other primary outcomes between the two groups. Additionally, duration of anterior chamber opening was significantly shorter in the Ex-PRESS group (P = 0.0002) and the eyes that had iris contact with Ex-PRESS tube had significantly shallower anterior chambers than did the eyes without the iris contact (P = 0.013). The Ex-PRESS implantation prevented early postoperative inflammation and hyphema in the anterior chamber and shortened the duration of anterior chamber opening. Iris contact with the Ex-PRESS tube occurred more frequently in eyes with open-angle glaucoma and shallow anterior chambers.

Angiopoietin-like Protein 2 Is a Multistep Regulator of Inflammatory Neovascularization in a Murine Model of Age-related Macular Degeneration.

Choroidal neovascularization (CNV) is a pathogenic process of age-related macular degeneration, a vision-threatening disease. The retinal pigment epithelium and macrophages both influence CNV development. However, the underlying mechanisms remain obscure. Here, we focus on Angptl2 (angiopoietin-like protein 2), a cytokine involved in age-related systemic diseases. Angptl2 was originally identified as an adipocytokine and is also expressed in the eye. Using a laser-induced CNV model, we found thatAngptl2KO mice exhibited suppressed CNV development with reduced macrophage recruitment and inflammatory mediator induction. The mediators monocyte chemotactic protein-1, interleukin-1ß (Il-1ß),Il-6, matrix metalloprotease-9 (Mmp-9), and transforming growth factor-ß1 (Tgf-ß1) that were up-regulated during CNV development were all suppressed in the retinal pigment epithelium-choroid of CNV models generated in theAngptl2KO mice. Bone marrow transplantation using wild-type and KO mice suggested that both bone marrow-derived and host-derived Angptl2 were responsible for macrophage recruitment and CNV development. Peritoneal macrophages derived fromAngptl2KO mice expressed lower levels of the inflammatory mediators. In the wild-type peritoneal macrophages and RAW264.7 cells, Angptl2 induced the mediators via integrins α4 and ß2, followed by the downstream activation of NF-κB and ERK. The activation of NF-κB and ERK by Angptl2 also promoted macrophage migration. Therefore, Angptl2 from focal tissue might trigger macrophage recruitment, and that from recruited macrophages might promote expression of inflammatory mediators including Angptl2 in an autocrine and/or paracrine fashion to facilitate CNV development. Angptl2 might therefore represent a multistep regulator of CNV pathogenesis and serve as a new therapeutic target for age-related macular degeneration.

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

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.

Blue light-induced inflammatory marker expression in the retinal pigment epithelium-choroid of mice and the protective effect of a yellow intraocular lens material in vivo.

Oxidative stress in the retinal pigment epithelium (RPE) is a well-accepted pathogenic change in vision-threatening diseases such as age-related macular degeneration. One source of oxidative stress is excessive light exposure, which causes excessive activation of the visual cycle. Because short wavelength light (blue light) has more energy, it is reported to be more harmful to photoreceptor cells than the other wavelengths of light. However, the biological effect of blue light in the RPE of living animals and the protective effect of a yellow intraocular lens (IOL) material that blocks blue light is still obscure. Therefore, we compared the pathogenic effect in the RPE-choroid complexes of mice exposed to light in a box made of a clear or a yellow IOL material. We measured the level of reactive oxygen species (ROS) using 2', 7'-dichlorodihydrofluorescein diacetate, the mRNA levels of inflammatory cytokines and a macrophage marker by real-time polymerase chain reaction, and the protein level of monocyte chemotactic protein-1 (MCP-1) by ELISA. The ROS level after light exposure was suppressed in the RPE-choroids of light-exposed mice in the yellow IOL material box. In parallel, all the inflammatory cytokines that we measured and a macrophage marker were also suppressed in the RPE-choroids of light-exposed mice in the yellow IOL material box. Therefore, a yellow IOL material suppressed, and thus blue light exacerbated, the increase in the ROS level and inflammatory cytokine expression as well as macrophage recruitment in the RPE-choroid in vivo after light exposure.

Altered Transport Velocity of Axonal Mitochondria in Retinal Ganglion Cells After Laser-Induced Axonal Injury In Vitro.

PURPOSE: Axonal damage precedes retinal ganglion cell (RGC) apoptosis in glaucomatous optic neuropathy. Because mitochondria in RGC axons are damaged before cell death, we examined axonal mitochondrial transport dynamics after axonal injury. METHODS: Mitochondria in rat-cultivated RGCs were stained with rhodamine 123. After axonal injury induced using a laser microdissection system, axonal transport was evaluated by time-lapse imaging. The RGC apoptosis was detected using ethidium homodimer-1 on day 3 after axonal injury. RESULTS: The rate of stationary mitochondria in RGC axons significantly increased from 37.6% before axonal injury to 70.6% at 5 minutes (P < 0.001) and 63.6% at 18 hours (P < 0.001) after axonal injury. The mean axonal mitochondrial transport velocity in RGCs transiently deteriorated from 0.48 ± 0.01 µm/s before axonal injury to 0.37 ± 0.02 µm/s at 5 minutes after axonal injury (P < 0.001). However, 23.5% of RGCs showed recovered axonal transport velocity at 18 hours after injury. On day 3 after axonal injury, RGCs with the recovery of axonal transport did not undergo apoptosis, whereas 69.2% of RGCs without the recovery of axonal transport underwent apoptosis (P = 0.029). CONCLUSIONS: Axonal injury disrupts mitochondrial transport in RGC axons. Irreversible decreased axonal mitochondrial transport velocity may be useful to predict RGC apoptosis after axonal injury.

Association of macular pigment optical density with serum concentration of oxidized low-density lipoprotein in healthy adults.

PURPOSE: To analyze the association between macular pigment optical density (MPOD), which reflects lutein (L), zeaxanthin (Z), and meso-zeaxanthin (MZ) in the macula, and background characteristics. METHODS: Fifty-five healthy adult volunteers were analyzed. Macular pigment optical density was measured using a heterochromatic flicker photometry technique, and serum concentrations of carotenoids and lipoproteins were by high-performance liquid chromatography and enzyme-linked immunosorbent assay, respectively. Dietary intake of nutrient was determined by a validated self-administered questionnaire on ingestion frequency. RESULTS: Macular pigment optical density was positively correlated with serum concentrations of L and Z and dietary L intake and inversely correlated with serum oxidized low-density lipoprotein (LDL). Although MPOD decreased with age (95% confidence interval, -0.011 to -0.002; correlation coefficient, -0.269; P = 0.007), serum L/Z and dietary L intake did not. In contrast, serum oxidized LDL was positively correlated with age (95% confidence interval, 0.69-2.34; correlation coefficient, 0.333; P = 0.0004). After adjusting for age, sex, and oxidized LDL, serum L was positively correlated with MPOD (95% confidence interval, 0.88-1.69; P = 0.000001). After adjusting for age, sex, and serum L, serum oxidized LDL was inversely correlated with MPOD (95% confidence interval, -0.002 to -0.0004; P = 0.006). CONCLUSION: Macular pigment optical density was inversely correlated with serum oxidized LDL. Further study to know the impact of oxidized LDL on MPOD may be warranted.

AMPK-NF-κB axis in the photoreceptor disorder during retinal inflammation.

Recent progress in molecular analysis has revealed the possible involvement of multiple inflammatory signaling pathways in pathogenesis of retinal degeneration. However, how aberrant signaling pathways cause tissue damage and dysfunction is still being elucidated. Here, we focus on 5'-adenosine monophosphate (AMP)-activated protein kinase (AMPK), originally recognized as a key regulator of energy homeostasis. AMPK is also modulated in response to inflammatory signals, although its functions in inflamed tissue are obscure. We investigated the role of activated AMPK in the retinal neural damage and visual function impairment caused by inflammation. For this purpose, we used a mouse model of lipopolysaccharide-induced inflammation in the retina, and examined the effects of an AMPK activator, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR). During inflammation, activated AMPK in the neural retina was decreased, but AICAR treatment prevented this change. Moreover, the electroretinogram (ERG) a-wave response, representing photoreceptor function, showed visual dysfunction in this model that was prevented by AICAR. Consistently, the model showed shortened photoreceptor outer segments (OSs) with reduced levels of rhodopsin, a visual pigment concentrated in the OSs, in a post-transcriptional manner, and these effects were also prevented by AICAR. In parallel, the level of activated NF-κB increased in the retina during inflammation, and this increase was suppressed by AICAR. Treatment with an NF-κB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ) preserved the rhodopsin level during inflammation, suppressing NF-κB. These findings indicated that AMPK activation by AICAR and subsequent NF-κB inhibition had a protective effect on visual function, and that AMPK activation played a neuroprotective role during retinal inflammation.

Phase II enzyme induction by a carotenoid, lutein, in a PC12D neuronal cell line.

The mechanism by which lutein, a carotenoid, acts as an antioxidant in retinal cells is still not fully understood. Here, lutein treatment of a neuronal cell line (PC12D) immediately resulted in reduced intracellular ROS levels, implying that it has a direct role in ROS scavenging. Significantly, lutein treatment also induced phase II antioxidative enzyme expression, probably via a nuclear factor-like 2 (Nrf2) independent pathway. This latter mechanism could explain why lutein acts diversely to protect against oxidative/cytotoxic stress, and why it is physiologically involved in the human neural tissue, such as the retina.

Angiotensin II type 1 receptor blockade suppresses light-induced neural damage in the mouse retina.

Exposure to light contributes to the development and progression of retinal degenerative diseases. However, the mechanisms underlying light-induced tissue damage are not fully understood. Here, we examined the role of angiotensin II type 1 receptor (AT1R) signaling, which is part of the renin-angiotensin system, in light-induced retinal damage. Light-exposed Balb/c mice that were treated with the AT1R blockers (angiotensin II receptor blockers; ARBs) valsartan, losartan, and candesartan before and after the light exposure exhibited attenuated visual function impairment, compared to vehicle-treated mice. This effect was dose-dependent and observed across the ARB class of inhibitors. Further evaluation of valsartan showed that it suppressed a number of light-induced retinal effects, including thinning of the photoreceptor cell layer caused by apoptosis, shortening of the photoreceptor cell outer segment, and increased levels of reactive oxygen species (ROS). The role of ROS in retinal pathogenesis was investigated further using the antioxidant N-acetyl-l-cysteine (NAC). Treatment of light-exposed mice with NAC before the light exposure suppressed the visual function impairment and photoreceptor cell histological changes due to apoptosis. Moreover, treatment with valsartan or NAC suppressed the induction of c-fos (a component of the AP-1 transcription factor) and the upregulation of fasl (a proapoptotic molecule whose transcript is regulated downstream of AP-1). Our results suggest that AT1R signaling mediates light-induced apoptosis, by increasing the levels of ROS and proapoptotic molecules in the retina. Thus, AT1R blockade may represent a new therapeutic approach for preventing light-induced retinal neural tissue damage.