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Leber's hereditary optic neuropathy, the most frequent mitochondrial disease due to mitochondrial DNA point mutations in complex I, is characterized by the selective degeneration of retinal ganglion cells, leading to optic atrophy and loss of central vision prevalently in young males. The current study investigated the reasons for the higher prevalence of Leber's hereditary optic neuropathy in males, exploring the potential compensatory effects of oestrogens on mutant cell metabolism. Control and Leber's hereditary optic neuropathy osteosarcoma-derived cybrids (11778/ND4, 3460/ND1 and 14484/ND6) were grown in glucose or glucose-free, galactose-supplemented medium. After having shown the nuclear and mitochondrial localization of oestrogen receptors in cybrids, experiments were carried out by adding 100 nM of 17β-oestradiol. In a set of experiments, cells were pre-incubated with the oestrogen receptor antagonist ICI 182780. Leber's hereditary optic neuropathy cybrids in galactose medium presented overproduction of reactive oxygen species, which led to decrease in mitochondrial membrane potential, increased apoptotic rate, loss of cell viability and hyper-fragmented mitochondrial morphology compared with control cybrids. Treatment with 17β-oestradiol significantly rescued these pathological features and led to the activation of the antioxidant enzyme superoxide dismutase 2. In addition, 17β-oestradiol induced a general activation of mitochondrial biogenesis and a small although significant improvement in energetic competence. All these effects were oestrogen receptor mediated. Finally, we showed that the oestrogen receptor β localizes to the mitochondrial network of human retinal ganglion cells. Our results strongly support a metabolic basis for the unexplained male prevalence in Leber's hereditary optic neuropathy and hold promises for a therapeutic use for oestrogen-like molecules. [ABSTRACT FROM AUTHOR]

Idebenone, the only approved treatment for Leber hereditary optic neuropathy (LHON), promotes recovery of visual function in up to 50% of patients, but we can neither predict nor understand the non-responders. Idebenone is reduced by the cytosolic NAD(P)H oxidoreductase I (NQO1) and directly shuttles electrons to respiratory complex III, bypassing complex I affected in LHON. We show here that two polymorphic variants drastically reduce NQO1 protein levels when homozygous or compound heterozygous. This hampers idebenone reduction. In its oxidized form, idebenone inhibits complex I, decreasing respiratory function in cells. By retrospectively analyzing a large cohort of idebenone-treated LHON patients, classified by their response to therapy, we show that patients with homozygous or compound heterozygous NQO1 variants have the poorest therapy response, particularly if carrying the m.3460G>A/MT-ND1 LHON mutation. These results suggest consideration of patient NQO1 genotype and mitochondrial DNA mutation in the context of idebenone therapy., Competing Interests: Declaration of interests M.R., P.B., M. Carbonelli, G.A., C.L.M., C.B.C., T.K., and V.C. are involved in clinical trials with idebenone (Santhera Pharmaceuticals and Chiesi Farmaceutici) in LHON patients. M.R., A.A.S., P.B., M. Carbonelli, G.A., C.L.M., C.B.C., T.K., and V.C. are involved in gene therapy trials with Lumevoq (GenSight Biologics) in LHON patients. A.A.S., C.L.M., T.K., and V.C. have received research support, speaker honoraria, consulting fees, and travel reimbursement from Santhera Pharmaceuticals, Chiesi GmbH, and GenSight Biologics. None of these activities are related to conduct of this study or writing of the manuscript., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Leber's hereditary optic neuropathy (LHON), a disease associated with a mitochondrial DNA mutation, is characterized by blindness due to degeneration of retinal ganglion cells (RGCs) and their axons, which form the optic nerve. We show that a sustained pathological autophagy and compartment-specific mitophagy activity affects LHON patient-derived cells and cybrids, as well as induced pluripotent-stem-cell-derived neurons. This is variably counterbalanced by compensatory mitobiogenesis. The aberrant quality control disrupts mitochondrial homeostasis as reflected by defective bioenergetics and excessive reactive oxygen species production, a stress phenotype that ultimately challenges cell viability by increasing the rate of apoptosis. We counteract this pathological mechanism by using autophagy regulators (clozapine and chloroquine) and redox modulators (idebenone), as well as genetically activating mitochondrial biogenesis (PGC1-α overexpression). This study substantially advances our understanding of LHON pathophysiology, providing an integrated paradigm for pathogenesis of mitochondrial diseases and druggable targets for therapy., Competing Interests: Declaration of interests V.C. and C.L.M. are both involved in clinical trials with idebenone (Santhera Pharmaceuticals) in LHON patients; V.C., C.L.M., and A.A.S. are involved in gene therapy trials with Lumevoq (GenSight Biologics) in LHON patients. V.C. and A.A.S. also serve as consultants in the advisory board of Chiesi Farmaceutici., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Age-related macular degeneration (AMD) is a progressive eye disease and the most common cause of blindness among the elderly. AMD is characterized by early atrophy of the choriocapillaris and retinal pigment epithelium (RPE). Although AMD is a multifactorial disease with many environmental and genetic risk factors, a hallmark of the disease is the origination of extracellular deposits, or drusen, between the RPE and Bruch membrane. Human retinal G-protein-coupled receptor (RGR) gene generates an exon-skipping splice variant of RGR-opsin (RGR-d; NP_001012740) that is a persistent component of small and large drusen. Herein, the findings show that abnormal RGR proteins, including RGR-d, are pathogenic in an animal retina with degeneration of the choriocapillaris, RPE, and photoreceptors. A frameshift truncating mutation resulted in severe retinal degeneration with a continuous band of basal deposits along the Bruch membrane. RGR-d produced less severe disease with choriocapillaris and RPE atrophy, including focal accumulation of abnormal RGR-d protein at the basal boundary of the RPE. Degeneration of the choriocapillaris was marked by a decrease in endothelial CD31 protein and choriocapillaris breakdown at the ultrastructural level. Fundus lesions with patchy depigmentation were characteristic of old RGR-d mice. RGR-d was mislocalized in cultured cells and caused a strong cell growth defect. These results uphold the notion of a potential hidden link between AMD and a high-frequency RGR allele., (Copyright © 2021 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background/objectives: Choroidal thinning has been suggested in Leber's hereditary optic neuropathy (LHON). No study has been conducted of the choroid in relation to the retinal ganglion cell-inner plexiform layer (RGC-IPL). We sought to measure choroidal thickness in chronic LHON and to correlate thickness changes with the RGC-IPL., Subjects/methods: Chronic LHON, 11778 mitochondrial DNA (mtDNA) mutation, patients (26 eyes; mean age: 35.1 ± 16.1 years) were prospectively recruited at Doheny Eye Center, University of California Los Angeles from March 2016 to July 2017. Age-matched healthy controls (27 eyes; mean age: 32.4 ± 11.1 years) were enroled for comparison. Swept-source optical coherence tomography (SS-OCT) imaging was performed in chronic LHON patients and compared with age-matched healthy controls., Results: The macular choroid was significantly thinner in chronic LHON (250.5 ± 62.2 μm) compared with controls (313.9 ± 60.2 μm; p < 0.0001). The peripapillary choroid was also significantly thinner in chronic LHON (135.7 ± 51.4 μm) compared with controls (183.0 ± 61.8 μm, p < 0.001). Choroidal thickness strongly correlated with retinal nerve fibre layer (RNFL) thickness in both the macular (R 2  = 0.72; 95% CI, 0.57-0.84) and peripapillary regions (R 2  = 0.53; 95% CI, 0.31-0.70). Choroidal thickness was also significantly correlated with macular RGC-IPL thickness (R 2  = 0.51; 95% CI, 0.26-0.73)., Conclusions: Choroidal thinning in chronic LHON correlated strongly with both RNFL and RGC-IPL thicknesses. These findings may suggest a pathophysiological mechanism involving vascular pathology of the choroid in relation to the retinal ganglion cell complex in LHON.

In autosomal dominant optic atrophy (ADOA), caused by mutations in the mitochondrial cristae biogenesis and fusion protein optic atrophy 1 (Opa1), retinal ganglion cell (RGC) dysfunction and visual loss occur by unknown mechanisms. Here, we show a role for autophagy in ADOA pathogenesis. In RGCs expressing mutated Opa1, active 5' AMP-activated protein kinase (AMPK) and its autophagy effector ULK1 accumulate at axonal hillocks. This AMPK activation triggers localized hillock autophagosome accumulation and mitophagy, ultimately resulting in reduced axonal mitochondrial content that is restored by genetic inhibition of AMPK and autophagy. In C. elegans, deletion of AMPK or of key autophagy and mitophagy genes normalizes the axonal mitochondrial content that is reduced upon mitochondrial dysfunction. In conditional, RGC specific Opa1-deficient mice, depletion of the essential autophagy gene Atg7 normalizes the excess autophagy and corrects the visual defects caused by Opa1 ablation. Thus, our data identify AMPK and autophagy as targetable components of ADOA pathogenesis.

[This corrects the article DOI: 10.1371/journal.pone.0232785.].

Background: Alzheimer's disease (AD) pathology precedes symptoms and its detection can identify at-risk individuals who may benefit from early treatment. Since the retinal nerve fiber layer (RNFL) is depleted in established AD, we tested whether its thickness can predict whether cognitively healthy (CH) individuals have a normal or pathological cerebrospinal fluid (CSF) Aß42 (A) and tau (T) ratio., Methods: As part of an ongoing longitudinal study, we enrolled CH individuals, excluding those with cognitive impairment and significant ocular pathology. We classified the CH group into two sub-groups, normal (CH-NAT, n = 16) or pathological (CH-PAT, n = 27), using a logistic regression model from the CSF AT ratio that identified >85% of patients with a clinically probable AD diagnosis. Spectral-domain optical coherence tomography (OCT) was acquired for RNFL, ganglion cell-inner plexiform layer (GC-IPL), and macular thickness. Group differences were tested using mixed model repeated measures and a classification model derived using multiple logistic regression., Results: Mean age (± standard deviation) in the CH-PAT group (n = 27; 75.2 ± 8.4 years) was similar (p = 0.50) to the CH-NAT group (n = 16; 74.1 ± 7.9 years). Mean RNFL (standard error) was thinner in the CH-PAT group by 9.8 (2.7) μm; p < 0.001. RNFL thickness classified CH-NAT vs. CH-PAT with 87% sensitivity and 56.3% specificity., Conclusions: Our retinal data predict which individuals have CSF biomarkers of AD pathology before cognitive deficits are detectable with 87% sensitivity. Such results from easy-to-acquire, objective and non-invasive measurements of the RNFL merit further study of OCT technology to monitor or screen for early AD pathology., Competing Interests: The authors have declared that no competing interests exist.

Introduction: Previous in vivo optical coherence tomography studies have proposed the retinal choroid as a potential oculovascular biomarker for Alzheimer's disease (AD). However, the clinical use of the choroid as a purported surrogate marker remains poorly understood. We pursued a histopathological approach to assess choroidal thickness and vascular morphology in severe disease., Methods: Human postmortem tissues from 8 patients with AD (mean age: 80.1 ± 12.7 years) and from 11 age-matched controls (mean age: 78.4 ± 16.57 years) were analyzed. Thickness, area, and vascularity of the retinal choroid and its sublayers were measured from the nasal and temporal quadrants of the superior retina., Results: Nasally, the choroid was thinner in the patients with AD than in the controls (22% thickness reduction; P  < .001), but to our surprise, the choroid was thicker in the patients with AD than in the controls (~60% increase; P  < .03) within the macula, temporally. The choroidal area was also significantly greater in the patients with AD than in the controls (~60% increase; P  < .03). Choroidal thickening in AD was strongly correlated with the stromal vessel number (R 2  = 0.96, P  < .001)., Discussion: We found significant differences in the retinal choroid by layer and by region, nasally and temporally with respect to the optic nerve. Intriguingly, the choroid was markedly thicker in the central macular region and was strongly associated with vessel number in the stromal vascular layer. These quantified histological findings in severe disease expand our understanding of vascular pathology in AD and suggest vascularity as a potential biomarker supplementary to thickness when evaluating the retinal choroid in AD., (© 2019 The Authors.)

Purpose: To provide a histopathologic, morphometric analysis of the retina in Alzheimer's disease (AD)., Methods: Human postmortem retinas from eight patients with AD (mean age: 80 ± 12.7 years) and from 11 age-matched controls (mean age: 78 ± 16.57 years) were analyzed. The retinas were sampled from the superior quadrant on both the temporal and nasal sides with respect to the optic nerve. Thickness of the inner and outer layers involving the retinal nerve fiber layer (RNFL), retinal ganglion cell layer (RGCL), inner plexiform layer (IPL), inner nuclear layer (INL), and outer nuclear layer (ONL) were measured and compared between controls and AD. A total of 16 measurements of retinal thickness were acquired for each layer., Results: RNFL thinning supero-temporally was significant closest to the optic nerve (∼35% thickness reduction; P < 0.001). Supero-nasally, RNFL was thinner throughout all points (∼40% reduction; P < 0.001). Supero-temporally, RGCL thinning was pronounced toward the macula (∼35% thickness reduction; P < 0.001). Supero-nasally, RGCL showed uniform thinning throughout (∼35% reduction; P < 0.001). IPL thinning supero-temporally was statistically significant in the macula (∼15% reduction; P < 0.01). Supero-nasal IPL featured uniform thinning throughout (∼25% reduction; P < 0.001). Supero-temporally, INL and ONL thinning were pronounced toward the macula (∼25% reduction; P < 0.01). Supero-nasally, INL and ONL were thinner throughout (∼25% reduction; P < 0.01)., Conclusions: Our study revealed marked thinning in both the inner and outer layers of the retina. These quantified histopathologic findings provide a more comprehensive understanding of the retina in AD than previously reported.

The optic nerve and the cells that give origin to its 1.2 million axons, the retinal ganglion cells (RGCs), are particularly vulnerable to neurodegeneration related to mitochondrial dysfunction. Optic neuropathies may range from non-syndromic genetic entities, to rare syndromic multisystem diseases with optic atrophy such as mitochondrial encephalomyopathies, to age-related neurodegenerative diseases such as Alzheimer's and Parkinson's disease where optic nerve involvement has, until recently, been a relatively overlooked feature. New tools are available to thoroughly investigate optic nerve function, allowing unparalleled access to this part of the central nervous system. Understanding the molecular pathophysiology of RGC neurodegeneration and optic atrophy, is key to broadly understanding the pathogenesis of neurodegenerative disorders, for monitoring their progression in describing the natural history, and ultimately as outcome measures to evaluate therapies. In this review, the different layers, from molecular to anatomical, that may contribute to RGC neurodegeneration and optic atrophy are tackled in an integrated way, considering all relevant players. These include RGC dendrites, cell bodies and axons, the unmyelinated retinal nerve fiber layer and the myelinated post-laminar axons, as well as olygodendrocytes and astrocytes, looked for unconventional functions. Dysfunctional mitochondrial dynamics, transport, homeostatic control of mitobiogenesis and mitophagic removal, as well as specific propensity to apoptosis may target differently cell types and anatomical settings. Ultimately, we can envisage new investigative approaches and therapeutic options that will speed the early diagnosis of neurodegenerative diseases and their cure., (© The Author 2017. Published by Oxford University Press.)

Melanopsin retinal ganglion cells (mRGCs) are intrinsically photosensitive RGCs deputed to non-image forming functions of the eye such as synchronization of circadian rhythms to light-dark cycle. These cells are characterized by unique electrophysiological, anatomical and biochemical properties and are usually more resistant than conventional RGCs to different insults, such as axotomy and different paradigms of stress. We also demonstrated that these cells are relatively spared compared to conventional RGCs in mitochondrial optic neuropathies (Leber's hereditary optic neuropathy and Dominant Optic Atrophy). However, these cells are affected in other neurodegenerative conditions, such as glaucoma and Alzheimer's disease. We here review the current evidences that may underlie this dichotomy. We also present our unpublished data on cell experiments demonstrating that melanopsin itself does not explain the robustness of these cells and some preliminary data on immunohistochemical assessment of mitochondria in mRGCs., (Copyright © 2017 Elsevier B.V. and Mitochondria Research Society. All rights reserved.)

There is increasing awareness on the role played by circadian rhythm abnormalities in neurodegenerative disorders such as Alzheimer's disease (AD) and Parkinson's disease (PD). The characterization of the circadian dysfunction parallels the mounting evidence that the hallmarks of neurodegeneration also affect the retina and frequently lead to loss of retinal ganglion cells (RGCs) and to different degrees of optic neuropathy. In the RGC population, there is the subgroup of cells intrinsically photosensitive and expressing the photopigment melanopsin [melanopsin-containing retinal ganglion cells (mRGCs)], which are now well known to drive the entrainment of circadian rhythms to the light-dark cycles. Thus, the correlation between the pathological changes affecting the retina and mRGCs with the circadian imbalance in these neurodegenerative diseases is now clearly emerging, pointing to the possibility that these patients might be amenable to and benefit from light therapy. Currently, this connection is better established for AD and PD, but the same scenario may apply to other neurodegenerative disorders, such as Huntington's disease. This review highlights similarities and differences in the retinal/circadian rhythm axis in these neurodegenerative diseases posing a working frame for future studies.

Purpose: Intravitreal vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) produced florid retinal neovascularization and hemorrhage in the rabbit. This study seeks to determine whether sustained subchoroidal release of both VEGF and bFGF can induce robust choroidal neovascularization (CNV) in the rabbit., Methods: Subchoroidal implantation through the sclera of polymeric pellets containing both 15 μg VEGF and 15 μg bFGF was performed on adult pigmented male Dutch belted rabbits (N = 6) and NZW albinos (N = 8). As negative controls, blank pellets with no growth factors were implanted in both Dutch belted rabbits (N = 6) and NZW albino rabbits (N = 4). Development of CNV was documented weekly over a 4-week period with indirect ophthalmoscopy, color fundus photography, and fluorescein angiography. Eyes were enucleated and prepared for histologic and immunohistochemical analyses at the end of the study. Amounts of VEGF and bFGF that were released in vitro from the pellets were measured by ELISA., Results: In all eyes with subchoroidal implants containing both VEGF and bFGF, strong fluorescein leakage was observed at 2, 3, and 4 weeks (P < 0.005); no leakage was seen initially in week 1. Negative control groups with blank implants showed no fluorescein leakage throughout the 4-week study period. Histologic analysis confirmed the presence of experimental CNV. New subretinal blood vessel growth occurred in all eyes with VEGF/bFGF implants. Negative control eyes with blank implants showed no vascular changes. In vitro sustained release of both VEGF and bFGF was confirmed by ELISA., Conclusion: Sustained subchoroidal release of both VEGF and bFGF produced experimental CNV rapidly in the rabbit. Understanding how these growth factors induce CNV may suggest novel therapeutic strategies in the large rabbit eye.

Objective: Melanopsin retinal ganglion cells (mRGCs) are photoreceptors driving circadian photoentrainment, and circadian dysfunction characterizes Alzheimer disease (AD). We investigated mRGCs in AD, hypothesizing that they contribute to circadian dysfunction., Methods: We assessed retinal nerve fiber layer (RNFL) thickness by optical coherence tomography (OCT) in 21 mild-moderate AD patients, and in a subgroup of 16 we evaluated rest-activity circadian rhythm by actigraphy. We studied postmortem mRGCs by immunohistochemistry in retinas, and axons in optic nerve cross-sections of 14 neuropathologically confirmed AD patients. We coimmunostained for retinal amyloid β (Aβ) deposition and melanopsin to locate mRGCs. All AD cohorts were compared with age-matched controls., Results: We demonstrated an age-related optic neuropathy in AD by OCT, with a significant reduction of RNFL thickness (p = 0.038), more evident in the superior quadrant (p = 0.006). Axonal loss was confirmed in postmortem AD optic nerves. Abnormal circadian function characterized only a subgroup of AD patients. Sleep efficiency was significantly reduced in AD patients (p = 0.001). We also found a significant loss of mRGCs in postmortem AD retinal specimens (p = 0.003) across all ages and abnormal mRGC dendritic morphology and size (p = 0.003). In flat-mounted AD retinas, Aβ accumulation was remarkably evident inside and around mRGCs., Interpretation: We show variable degrees of rest-activity circadian dysfunction in AD patients. We also demonstrate age-related loss of optic nerve axons and specifically mRGC loss and pathology in postmortem AD retinal specimens, associated with Aβ deposition. These results all support the concept that mRGC degeneration is a contributor to circadian rhythm dysfunction in AD., (© 2015 American Neurological Association.)

Purpose: The proteomic profile of vitreous from second-trimester human embryos and young adults was characterized using mass spectrometry and analyzed for changes in protein levels that may relate to structural changes occurring during this time. This vitreous proteome was compared to previous reports to confirm proteins already identified and reveal novel ones., Methods: Vitreous from 17 human embryos aged 14 to 20 weeks gestation (WG) and from a 12-, a 14-, a 15-, and a 28-year-old was individually analyzed using tandem mass spectrometry-based proteomics. Peptide spectral count associations with embryonic age were assessed using a general linear model of fold changes and Spearman's rank correlation. Differences between embryonic and young adult vitreous proteomes were also compared. Immunohistochemistry was used to evaluate three proteins in five additional fetal (10-18 WG) human eyes., Results: There were 1217 proteins identified in fetal and young adult human vitreous, 206 after quantile normalization and variance filtering. In embryos, the peptide counts of 37 proteins changed significantly from 14 to 20 WG: 75.7% increased, 24.3% decreased. Immunohistochemistry confirmed the absence of clusterin and cadherin in 10 and 14 WG eyes and their presence at 18 WG. Comparing embryonic to young adult vitreous, 47 proteins were significantly higher or lower. A total of 768 proteins not previously identified in the literature are presented., Conclusions: Proteins previously unreported in the human vitreous were identified. The human vitreous proteome undergoes significant changes during embryogenesis and young adulthood. A number of protein levels change considerably during the second trimester, with the majority decreasing.

The belly spot and tail (Bst(+/-)) mouse phenotype is caused by mutations of the ribosomal protein L24 (Rpl24). Among various phenotypes in Bst(+/-) mice, the most interesting are its retinal abnormalities, consisting of delayed closure of choroid fissures, decreased ganglion cells and subretinal vascularization. We further characterized the Bst(+/-) mouse and investigated the underlying molecular mechanisms to assess the feasibility of using this strain as a model for stem cell therapy of retinal degenerative diseases due to retinal ganglion cell (RGC) loss. We found that, although RGCs are significantly reduced in retinal ganglion cell layer in Bst(+/-) mouse, melanopsin(+) RGCs, also called ipRGCs, appear to be unchanged. Pupillary light reflex was completely absent in Bst(+/-) mice but they had a normal circadian rhythm. In order to examine the pathological abnormalities in Bst(+/-) mice, we performed electron microscopy in RGC and found that mitochondria morphology was deformed, having irregular borders and lacking cristae. The complex activities of the mitochondrial electron transport chain were significantly decreased. Finally, for subretinal vascularization, we also found that angiogenesis is delayed in Bst(+/-) associated with delayed hyaloid regression. Characterization of Bst(+/-) retina suggests that the Bst(+/-) mouse strain could be a useful murine model. It might be used to explore further the pathogenesis and strategy of treatment of retinal degenerative diseases by employing stem cell technology., (© 2015. Published by The Company of Biologists Ltd.)

Purpose: Macular pucker (MP) and macular hole (MH) are vitreomaculopathies treated by vitrectomy and membrane peel. The complication of postoperative central scotoma can be associated with significant reduction in visual acuity (VA). We seek to determine whether retinal nerve fiber layer (RNFL) disruption is the pathophysiologic basis of this defect. Mitigating clinical circumstances also were sought., Methods: Eleven eyes from 10 pseudophakic patients who had undergone vitrectomy with peeling for either MH or MP were studied with clinical measures, including optical coherence tomography (OCT). Membrane specimens were evaluated by immunohistochemistry for neurofilament, a marker for the inner retina. Ten eyes from 10 pseudophakic patients who underwent repeat surgery for persistent or recurrent pathology were evaluated to determine the relationship between the timing of reoperation and clinical outcome., Results: Cases with a postoperative central scotoma (N=4) had worse VA (~20/600) compared to those without (N=7, ~20/30, P=0.01). Eyes with a central scotoma had significantly reduced RNFL thickness in the temporal quadrant (53.67 vs. 72.33 μm, P=0.05) by OCT. A central scotoma was associated with more disruption of the inner retina on immunohistochemistry (P=0.03). In patients with persistent or recurrent pathology, waiting six months before reoperation resulted in better functional outcomes (P=0.03)., Conclusions: Central scotomata and poor VA were associated with disruption of the RNFL during membrane peeling. Affected patients have RNFL thinning and signs of optic neuropathy, for which we propose the term inner retinal optic neuropathy (IRON). In patients requiring reoperation, waiting six months between surgeries may reduce the risk of IRON., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

Leber's hereditary optic neuropathy is a maternally inherited blinding disease caused as a result of homoplasmic point mutations in complex I subunit genes of mitochondrial DNA. It is characterized by incomplete penetrance, as only some mutation carriers become affected. Thus, the mitochondrial DNA mutation is necessary but not sufficient to cause optic neuropathy. Environmental triggers and genetic modifying factors have been considered to explain its variable penetrance. We measured the mitochondrial DNA copy number and mitochondrial mass indicators in blood cells from affected and carrier individuals, screening three large pedigrees and 39 independently collected smaller families with Leber's hereditary optic neuropathy, as well as muscle biopsies and cells isolated by laser capturing from post-mortem specimens of retina and optic nerves, the latter being the disease targets. We show that unaffected mutation carriers have a significantly higher mitochondrial DNA copy number and mitochondrial mass compared with their affected relatives and control individuals. Comparative studies of fibroblasts from affected, carriers and controls, under different paradigms of metabolic demand, show that carriers display the highest capacity for activating mitochondrial biogenesis. Therefore we postulate that the increased mitochondrial biogenesis in carriers may overcome some of the pathogenic effect of mitochondrial DNA mutations. Screening of a few selected genetic variants in candidate genes involved in mitochondrial biogenesis failed to reveal any significant association. Our study provides a valuable mechanism to explain variability of penetrance in Leber's hereditary optic neuropathy and clues for high throughput genetic screening to identify the nuclear modifying gene(s), opening an avenue to develop predictive genetic tests on disease risk and therapeutic strategies.

Purpose: Alzheimer's disease (AD) is the most common neurodegenerative disorder characterized by progressive decline in learning, memory, and executive functions. In addition to cognitive and behavioral deficits, vision disturbances have been reported in early stage of AD, well before the diagnosis is clearly established. To further investigate ocular abnormalities, a novel AD transgenic rat model was analyzed., Methods: Transgenic (Tg) rats (TgF344-AD) heterozygous for human mutant APPswe/PS1ΔE9 and age-matched wild type (WT) rats, as well as 20 human postmortem retinal samples from both AD and healthy donors were used. Visual function in the rodent was analyzed using the optokinetic response and luminance threshold recording from the superior colliculus. Immunohistochemistry on retinal and brain sections was used to detect various markers including amyloid-β (Aβ) plaques., Results: As expected, Aβ plaques were detected in the hippocampus, cortex, and retina of Tg rats. Plaque-like structures were also found in two AD human whole-mount retinas. The choroidal thickness was significantly reduced in both Tg rat and in AD human eyes when compared with age-matched controls. Tg rat eyes also showed hypertrophic retinal pigment epithelial cells, inflammatory cells, and upregulation of complement factor C3. Although visual acuity was lower in Tg than in WT rats, there was no significant difference in the retinal ganglion cell number and retinal vasculature., Conclusions: In this study, we observed pathological changes in the choroid and in RPE cells in the TgF344-AD rat model; choroidal thinning was observed further in human AD retina. Along with Ab deposition, the inflammatory response was manifested by microglial recruitment and complement activation. Further studies are needed to elucidate the significance and mechanisms of these pathological changes [corrected].

Mitochondrial dysfunction in Wolfram Syndrome (WS) is controversial and optic neuropathy, a cardinal clinical manifestation, is poorly characterized. We here describe the histopathological features in postmortem retinas and optic nerves (ONs) from one patient with WS, testing the hypothesis that mitochondrial dysfunction underlies the pathology. Eyes and retrobulbar ONs were obtained at autopsy from a WS patient, and compared with those of a Leber hereditary optic neuropathy (LHON) patient and one healthy control. Retinas were stained with hematoxylin & eosin for general morphology and ONs were immunostained for myelin basic protein (MBP). Immunostained ONs were examined in four "quadrants": superior, inferior, nasal, and temporal. The WS retinas displayed a severe loss of retinal ganglion cells in the macular region similar to the LHON retina, but not in the control. The WS ONs, immunostained for MBP, revealed a zone of degeneration in the temporal and inferior quadrants. This pattern was similar to that seen in the LHON ONs but not in the control. Thus, the WS patient displayed a distinct pattern of optic atrophy observed bilaterally in the temporal and inferior quadrants of the ONs. This arrangement of axonal degeneration, involving primarily the papillomacular bundle, closely resembled LHON and other mitochondrial optic neuropathies, supporting that mitochondrial dysfunction underlies its pathogenesis., (Copyright © 2013 Elsevier B.V. All rights reserved.)

An animal model of Leber hereditary optic neuropathy (LHON) was produced by introducing the human optic atrophy mtDNA ND6 P25L mutation into the mouse. Mice with this mutation exhibited reduction in retinal function by elecroretinogram (ERG), age-related decline in central smaller caliber optic nerve fibers with sparing of larger peripheral fibers, neuronal accumulation of abnormal mitochondria, axonal swelling, and demyelination. Mitochondrial analysis revealed partial complex I and respiration defects and increased reactive oxygen species (ROS) production, whereas synaptosome analysis revealed decreased complex I activity and increased ROS but no diminution of ATP production. Thus, LHON pathophysiology may result from oxidative stress.

Purpose: Leber's hereditary optic neuropathy (LHON), a mitochondrial disease, has clinical manifestations that reflect the initial preferential involvement of the papillomacular bundle (PMB). The present study seeks to predict the order of axonal loss in LHON optic nerves using the Nerve Fiber Layer Stress Index (NFL-S(I)), which is a novel mathematical model., Methods: Optic nerves were obtained postmortem from four molecularly characterized LHON patients with varying degrees of neurodegenerative changes and three age-matched controls. Tissues were cut in cross-section and stained with p-phenylenediamine to visualize myelin. Light microscopic images were captured in 32 regions of each optic nerve. Control and LHON tissues were evaluated by measuring axonal dimensions to generate an axonal diameter distribution map. LHON tissues were further evaluated by determining regions of total axonal depletion., Results: A size gradient was evident in the control optic nerves, with average axonal diameter increasing progressively from the temporal to nasal borders. LHON optic nerves showed an orderly loss of axons, starting inferotemporally, progressing centrally, and sparing the superonasal region until the end. Values generated from the NFL-S(I) equation fit a linear regression curve (R(2) = 0.97; P < 0.001)., Conclusions: The quantitative histopathologic data from this study revealed that the PMB is most susceptible in LHON, supporting clinical findings seen early in the course of disease onset. The present study also showed that the subsequent progression of axonal loss within the optic nerve can be predicted precisely with the NFL-S(I) equation. The results presented provided further insight into the pathophysiology of LHON.

Purpose: Neuron-specific enolase (NSE) is a biomarker for neuronal stress. Leber's hereditary optic neuropathy (LHON) is a mitochondrial disease affecting retinal ganglion cells (RGC). These RGCs and their axons in the retinal nerve fiber layer (RNFL) and optic nerve head may show subclinical pathology in unaffected mutation carriers, or undergo cell death in affected patients. We hypothesize that increased levels of blood NSE may characterize LHON carriers as a biomarker of ongoing RGC stress., Methods: Serum was obtained from 74 members of a Brazilian pedigree with LHON carrying the homoplasmic 11778/ND4 mitochondrial DNA mutation. Classified by symptoms and psychophysical metrics, 46/74 patients were unaffected mutation "carriers," 14/74 were "affected," and 14/74 were "off-pedigree" controls. Serum NSE levels were determined by ELISA specific for the γ subunit of NSE., Results: Serum NSE concentrations in carriers (27.17 ± 39.82 μg/L) were significantly higher than affected (5.66 ± 4.19 μg/L; P = 0.050) and off-pedigree controls (6.20 ± 2.35 μg/L; P = 0.047). Of the 14/46 (30.4 %) carriers with significantly elevated NSE levels (mean = 75.8 ± 42.3 μg/L), 9/14 (64.3%) were male. Furthermore, NSE levels were nearly three times greater in asymptomatic male carriers (40.65 ± 51.21 μg/L) than in asymptomatic female carriers (15.85 ± 22.27 μg/L; P = 0.034)., Conclusions: Serum NSE levels are higher in LHON carriers compared with affected and off-pedigree individuals. A subgroup of mostly male carriers had significantly elevated serum NSE levels. Thus, male carriers are at higher risk for LHON-related neuronal stress.

Objective: To evaluate the safety and efficacy of a new therapeutic agent, EPI-743, in Leber hereditary optic neuropathy (LHON) using standard clinical, anatomic, and functional visual outcome measures., Design: Open-label clinical trial., Setting: University medical center. Patients  Five patients with genetically confirmed LHON with acute loss of vision were consecutively enrolled and treated with the experimental therapeutic agent EPI-743 within 90 days of conversion. Intervention  During the course of the study, 5 consecutive patients received EPI-743, by mouth, 3 times daily (100-400 mg per dose)., Main Outcome Measures: Treatment effect was assessed by serial measurements of anatomic and functional visual indices over 6 to 18 months, including Snellen visual acuity, retinal nerve fiber layer thickness measured by optical coherence tomography, Humphrey visual fields (mean decibels and area with 1-log unit depression), and color vision. Treatment effect in this clinical proof of principle study was assessed by comparison of the prospective open-label treatment group with historical controls., Results: Of 5 subjects treated with EPI-743, 4 demonstrated arrest of disease progression and reversal of visual loss. Two patients exhibited a total recovery of visual acuity. No drug-related adverse events were recorded., Conclusions: In a small open-label trial, EPI-743 arrested disease progression and reversed vision loss in all but 1 of the 5 consecutively treated patients with LHON. Given the known natural history of acute and rapid progression of LHON resulting in chronic and persistent bilateral blindness, these data suggest that the previously described irreversible priming to retinal ganglion cell loss may be reversed.

Leber's hereditary optic neuropathy (LHON) is characterized by retinal ganglion cell (RGC) degeneration with the preferential involvement of those forming the papillomacular bundle. The optic nerve is considered the main pathological target for LHON. Our aim was to investigate the possible involvement of the post-geniculate visual pathway in LHON patients. We used diffusion-weighted imaging for in vivo evaluation. Mean diffusivity maps from 22 LHON visually impaired, 11 unaffected LHON mutation carriers and 22 healthy subjects were generated and compared at level of optic radiation (OR). Prefrontal and cerebellar white matter were also analyzed as internal controls. Furthermore, we studied the optic nerve and the lateral geniculate nucleus (LGN) in post-mortem specimens obtained from a severe case of LHON compared to an age-matched control. Mean diffusivity values of affected patients were higher than unaffected mutation carriers (P<0.05) and healthy subjects (P<0.01) in OR and not in the other brain regions. Increased OR diffusivity was associated with both disease duration (B = 0.002; P<0.05) and lack of recovery of visual acuity (B = 0.060; P<0.01). Post-mortem investigation detected atrophy (41.9% decrease of neuron soma size in the magnocellular layers and 44.7% decrease in the parvocellular layers) and, to a lesser extent, degeneration (28.5% decrease of neuron density in the magnocellular layers and 28.7% decrease in the parvocellular layers) in the LHON LGN associated with extremely severe axonal loss (99%) in the optic nerve. The post-geniculate involvement in LHON patients is a downstream post-synaptic secondary phenomenon, reflecting de-afferentation rather than a primary neurodegeneration due to mitochondrial dysfunction of LGN neurons.

Purpose: To characterize optic nerve and retinal changes in a patient with end-stage retinitis pigmentosa (RP) with an implanted active epiretinal array., Methods: A 74-year-old man with end-stage X-linked RP underwent implantation of an epiretinal array over the macula in the right eye and subsequent stimulation until his death at 5 years and 3 months after implantation. The optic nerves from this study patient, as well as those from two age-matched normal patients and two age-matched RP patients, were morphometrically analyzed against two different sets of criteria and compared. The retina underlying the array in the study patient was also morphometrically analyzed and compared with corresponding regions of the retina in the age-matched RP patients., Results: Optic nerve total axon counts were significantly lower in the study patient and RP patients than in normal patients. However, there was no significant difference when comparing total axon counts from the optic nerve corresponding to the patient's implanted right eye versus the optic nerves from the RP patients (P = 0.59 and P = 0.61 using the two different criteria). Degenerated axon data quantified damage and did not show increased damage in the optic nerve quadrant that retinotopically corresponded to the site of epiretinal array implantation and stimulation. Except for the tack site, there was no significant difference when comparing the retina underlying the array and the corresponding perimacular regions of two RP patients., Conclusions: Long-term implantation and electrical stimulation with an epiretinal array did not result in damage that could be appreciated in a morphometric analysis of the optic nerve and retina.

Background: Alzheimer disease (AD) is associated with optic nerve degeneration, yet the underlying pathophysiology of this disease and the optic nerve disorder remain poorly understood. Low-density lipoprotein receptor-related protein (LRP) is implicated in the pathogenesis of AD by mediating the transport of amyloid-β (Aβ) out of the brain into the systemic circulation. As a key player in the reaction to central nervous system injury, astrocytes associate with LRP in AD. This study investigates the role of LRP and astrocytes in the pathogenesis of AD optic neuropathy., Methods: To investigate the role of LRP and astrocytes in the pathogenesis of AD optic neuropathy, we conducted immunohistochemical studies on postmortem optic nerves in AD patients (n = 11) and age-matched controls (n = 10) to examine the presence of LRP. Quantitative analyses using imaging software were used to document the extent of LRP in neural tissues. Axonal integrity was assessed by performing immunohistochemistry on the subjects' optic nerves with an antibody to neurofilament (NF) protein. Double-immunofluorescence labeling was performed to investigate whether LRP colocalized with astrocytes, expressing glial fibrillary acidic protein., Results: LRP expression was decreased in AD optic nerves compared to that in controls (P < 0.001). LRP immunoreactivity was observed in the microvasculature and perivascularly in close proximity to the astrocytic processes. Colocalization of LRP in the astrocytes of optic nerves was also demonstrated. The presence of optic neuropathy was confirmed in the AD optic nerves by demonstrating greatly reduced immunostaining for NF protein as compared to controls., Conclusions: The reduction of LRP in the AD degenerative optic nerves supports the hypothesis that LRP may play a role in the pathophysiology of AD optic neuropathy.

Background: Leber hereditary optic neuropathy (LHON) is a mitochondrial DNA (mtDNA) genetic disorder characterized by profound bilateral loss of central vision due to selective loss of retinal ganglion cells. Most patients with LHON do not have complaints related to the peripheral nervous system. We investigated possible qualitative and quantitative histological changes in the peripheral nerve of a patient with LHON as compared to normal controls., Methods: Brachial plexus specimens were obtained at necropsy from a patient with LHON carrying the 3460/ND1 mtDNA mutation and age-matched controls without known history of neurological disease. The nerves were evaluated by light microscope coupled to a digital camera-based morphometric analysis and electron microscopy., Results: Extensive axonal degeneration of the large heavily myelinated fibers was found in the brachial plexus from the patient with LHON. In LHON nerve fascicles, we counted over 10 times as many degenerated profiles as found in the control nerve fascicles., Conclusions: Microscopic examination of the brachial plexus in the patient with LHON clearly demonstrated a significant pattern of neurodegeneration. Our study suggests that peripheral neuropathy may be a subclinical feature associated with LHON.

In the last decade, there was the seminal discovery of melanopsin-expressing retinal ganglion cells (mRGCs) as a new class of photoreceptors that subserve the photoentrainment of circadian rhythms and other non-image forming functions of the eye. Since then, there has been a growing research interest on these cells, mainly focused on animal models. Only recently, a few studies have started to address the relevance of the mRGC system in humans and related diseases. We recently discovered that mRGCs resist neurodegeneration in two inherited mitochondrial disorders that cause blindness, i.e. Leber hereditary optic neuropathy and dominant optic atrophy. The mechanism leading to mRGCs sparing in these blinding disorders, characterized by extensive and selective loss of RGCs, is currently unknown and under investigation. Other studies reported on mRGCs in glaucoma, on genetic variation of the melanopsin gene (OPN4) in seasonal affective disorder and on the role of mRGCs in migraineous photophobia. Our own data and studies from others have shown a significant reduction of mRGCs with aging. We anticipate that these studies will lead to many other investigations addressing the role of mRGCs and circadian photoreception in the pathogenesis of circadian and sleep abnormalities in neurodegenerative disorders., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Mitochondrial optic neuropathies, that is, Leber hereditary optic neuropathy and dominant optic atrophy, selectively affect retinal ganglion cells, causing visual loss with relatively preserved pupillary light reflex. The mammalian eye contains a light detection system based on a subset of retinal ganglion cells containing the photopigment melanopsin. These cells give origin to the retinohypothalamic tract and support the non-image-forming visual functions of the eye, which include the photoentrainment of circadian rhythms, light-induced suppression of melatonin secretion and pupillary light reflex. We studied the integrity of the retinohypothalamic tract in five patients with Leber hereditary optic neuropathy, in four with dominant optic atrophy and in nine controls by testing the light-induced suppression of nocturnal melatonin secretion. This response was maintained in optic neuropathy subjects as in controls, indicating that the retinohypothalamic tract is sufficiently preserved to drive light information detected by melanopsin retinal ganglion cells. We then investigated the histology of post-mortem eyes from two patients with Leber hereditary optic neuropathy and one case with dominant optic atrophy, compared with three age-matched controls. On these retinas, melanopsin retinal ganglion cells were characterized by immunohistochemistry and their number and distribution evaluated by a new protocol. In control retinas, we show that melanopsin retinal ganglion cells are lost with age and are more represented in the parafoveal region. In patients, we demonstrate a relative sparing of these cells compared with the massive loss of total retinal ganglion cells, even in the most affected areas of the retina. Our results demonstrate that melanopsin retinal ganglion cells resist neurodegeneration due to mitochondrial dysfunction and maintain non-image-forming functions of the eye in these visually impaired patients. We also show that in normal human retinas, these cells are more concentrated around the fovea and are lost with ageing. The current results provide a plausible explanation for the preservation of pupillary light reaction despite profound visual loss in patients with mitochondrial optic neuropathy, revealing the robustness of melanopsin retinal ganglion cells to a metabolic insult and opening the question of mechanisms that might protect these cells.

Although Alzheimer's disease (AD) has been shown to be associated with a true primary optic neuropathy, the underlying pathophysiology of this disease and in particular the optic nerve disorder is still poorly understood. The receptor for advanced glycation end products (RAGE) has been implicated in the pathogenesis of AD by mediating the transport of plasma amyloid-beta into the brain. Once ligated, RAGE can play a role in signal transduction, leading to amplification and perpetuation of inflammatory processes. As a key player in the reaction to CNS injury, astrocytes have been shown to associate with RAGE in a number of diseases, including AD. To investigate the role of RAGE and astrocytes in the pathogenesis of AD optic neuropathy, we conducted immunohistochemical studies to examine the presence of RAGE in donor eyes from patients with AD (n = 10) and controls (n = 3). Both qualitative observation and quantitative analyses using imaging software were used to document the extent of RAGE in the neural tissues. The intensity and extent of RAGE expression was more prominent in AD nerves compared to controls (P < 0.05). The RAGE immunoreactivity was observed in the microvasculature and in close proximity to astrocytic processes. While RAGE immunoreactivity increased with age, the increase was more precipitous in the AD group compared to the controls. The up-regulation of RAGE in the AD optic nerves indicates that RAGE may play a role in the pathophysiology of AD optic neuropathy.

Since the early days of mitochondrial medicine, it has been clear that optic atrophy is a very common and sometimes the singular pathological feature in mitochondrial disorders. The first point mutation of mitochondrial DNA (mtDNA) associated with the maternally inherited blinding disorder, Leber's hereditary optic neuropathy (LHON), was recognized in 1988. In 2000, the other blinding disorder, dominant optic atrophy (DOA) Kjer type, was found associated with mutations in the nuclear gene OPA1 that encodes a mitochondrial protein. Besides these two non-syndromic optic neuropathies, optic atrophy is a prominent feature in many other neurodegenerative diseases that are now recognized as due to primary mitochondrial dysfunction. We will consider mtDNA based syndromes such as LHON/dystonia/Mitochondrial Encephalomyopahty Lactic Acidosis Stroke-like (MELAS)/Leigh overlapping syndrome, or nuclear based diseases such as Friedreich ataxia (mutations in FXN gene), deafness-dystonia-optic atrophy (Mohr-Tranebjerg) syndrome (mutations in TIMM8A), complicated hereditary spastic paraplegia (mutations in SPG7), DOA "plus" syndromes (mutations in OPA1), Charcot-Marie-Tooth type 2A (CMT2A) with optic atrophy or hereditary motor and sensory neuropathy type VI (HMSN VI) (mutations in MFN2), and Costeff syndrome and DOA with cataract (mutations in OPA3). Thus, genetic errors in both nuclear and mitochondrial genomes often lead to retinal ganglion cell death, a specific target for mitochondrial mediated neurodegeneration. Many mechanisms have been studied and proposed as the bases for the pathogenesis of mitochondrial optic neuropathies including bioenergetic failure, oxidative stress, glutamate toxicity, abnormal mitochondrial dynamics and axonal transport, and susceptibility to apoptosis.

Purpose: To determine the profile of neurodegeneration in Leber hereditary optic neuropathy (LHON)., Methods: We quantitated serum levels of phosphorylated neurofilament heavy chain (pNF-H) in a Brazilian pedigree of 16 affected patients and 59 carriers with LHON, both molecularly characterized as harboring the G to A mutation at nucleotide 11,778 of the mitochondrial genome. The association of subject characteristics to pNF-H levels was studied with multiple regression; pNF-H data were square-root transformed to effect normality of distribution of residuals. Relationships between the square-root of pNF-H and age and sex were investigated within groups with Pearson correlation and the two-sample t-test. Linear regression was used to assess the difference between groups and to determine if the relationship of age was different between affected individuals and carriers. Results of plotting pNF-H levels by age suggested a nonlinear, quadratic association so age squared was used in the statistical analysis. ANCOVA was used to assess the influence of age and group on pNF-H levels., Results: In the carrier group, there was a significant correlation of square-root pNF-H (mean=0.24 ng/ml(2)) with age (r=0.30, p=0.022) and a stronger correlation with quadratic age (r=0.37, p=0.003). With a higher mean pNF-H (0.33 ng/ml(2)) for the affected group, correlations were of similar magnitude, although they were not statistically significant: age (r=0.22, p=0.42), quadratic age (r=0.22, p=0.45). There was no correlation between age and pNF-H levels (mean=0.34 ng/ml(2)) in the off-pedigree group: age (r=0.03, p=0.87), quadratic age (r=0.04, p=0.84). There was no difference between sexes and pNF-H levels in any of the groups (affected, p=0.65; carriers, p=0.19; off-pedigree, p=0.93)., Conclusions: Elevated pNF-H released into the serum of some affected LHON patients may suggest that axonal degeneration occurs at some point after loss of visual function. Increases in pNF-H levels of carriers with increasing age, not seen in off-pedigree controls, may suggest subtle subclinical optic nerve degeneration.

Purpose: To characterize a model of optic nerve axonal degeneration induced by tumor necrosis factor (TNF)-alpha and to determine the role of nuclear factor (NF)-kappaB p65 in axonal degeneration., Methods: Groups of rats were euthanatized at 1 day, 1 or 2 weeks, or 1 or 2 months after intravitreal injection of TNF-alpha. Morphometric analyses of neurofilament- or Thy-1-positive cells, retinal ganglion cells (flat preparations stained with cresyl violet or retrograde labeling with a neurotracer), the number of axons, immunostaining for myelin basic protein, and TUNEL assays were performed. Levels of NF-kappaB p65 protein in retina and optic nerve were determined by Western blot analysis and immunohistochemistry. The effects of antisense oligodeoxynucleotide (AS ODN) against NF-kappaB p65 and helenalin, an inhibitor of NF-kappaB p65 activation, on TNF-alpha-induced optic nerve degeneration were determined by counting the number of axons., Results: Intravitreal injections of TNF-alpha induced obvious axonal loss and extensive degeneration of the axons from 2 weeks to 2 months after injection, whereas significant retinal ganglion cell loss was noted only at 2 months after injection. NF-kappaB p65 was increased in the optic nerve but not in the retina and was found to colocalize with ED-1 and Iba1, markers of microglia. Inhibition of NF-kappaB p65 with AS ODN or helenalin significantly ameliorated the effects of TNF-alpha-mediated axonal loss., Conclusions: TNF-alpha causes axonal degeneration with probable delayed loss of retinal ganglion cell bodies. NF-kappaB p65 may play a pivotal role in axonal degeneration, with the possible involvement of microglial cells.

Background: Relative sparing of the pupillary reflexes in patients with leber's hereditary optic neuropathy (LHON) has been observed clinically. This study sought to test histologically whether retino-pupillary fibers are spared in LHON., Methods: Di-I, a fluorescein dye that allows anterograde labeling of axons, was injected into the brachium of the superior colliculus in post-mortem brain from a patient diagnosed with LHON (3460 mutation) and a normal control brain. After 4 weeks, serial fragmatome sections were obtained in the pretectal area and further stained with propidium iodide (PI stains DNA) to delineate the pretectal nuclei in the dorsal midbrain. Examination was performed under a confocal microscope. Optic nerves obtained from the above subjects were cut, mounted and stained with p-phenylenediamine (PPD) and trichrome stain for digital morphometry., Results: Di-I-labeled fibers were visible on all sections from the superior colliculus to the pretectum in the LHON and the control specimens, as were the nuclei in the cell bodies stained with PI. There was mild attenuation of the afferent pretectal fibers in LHON, but this was not as dramatic as the attenuation of the total population of fibers in the LHON optic nerve., Conclusions: In our LHON patient, the preservation of retinofugal fibers to the pretectum lends support to the clinical observation of relatively preserved pupillary function in LHON.

Aims: To compare the ultrastructural aspects of human extraocular muscles in two types of mitochondrial disease: chronic progressive external ophthalmoplegia (CPEO) and Leber's hereditary optic neuropathy (LHON)., Methods: Muscle samples of the medial rectus obtained from surgery in a sporadic case of CPEO associated with deleted mitochondrial DNA, and post mortem in a case of 3460/ND1 LHON were processed for electron microscopy (EM). The medial rectus from an autoptic time to fixation matched control was used to exclude postmortem artefacts., Results: The CPEO specimen revealed focal areas of disruption and abnormalities of mitochondria in some muscle fibres, creating a "mosaic-like" pattern. In the LHON specimen a diffuse increase in both number and size of mitochondria (mean diameter 0.85 mum v 0.65 mum of control, p<0.0001) with swollen appearance and disorganised cristae filled all spaces of sarcoplasmic reticulum. In some areas the excessive number of mitochondria slightly distorted myofibrils., Conclusion: EM investigation of extraocular muscles in CPEO and LHON reveals marked differences. A "mosaic-like" pattern caused by a selective damage of muscle fibres was evident in CPEO, whereas a diffuse increase in mitochondria with preservation of myofibrils characterised the LHON case. These ultrastructural changes may relate to the different expression of the two diseases, resulting in ophthalmoplegia in CPEO and normal eye movements in LHON.

Aim: To investigate the correlation between retinal nerve fibre layer (RNFL) thickness and optic nerve head (ONH) size in normal white subjects by means of optical coherence tomography (OCT)., Methods: 54 eyes of 54 healthy subjects aged between 15 and 54 underwent peripapillary RNFL thickness measurement by a series of three circular scans with a 3.4 mm diameter (Stratus OCT, RNFL Thickness 3.4 acquisition protocol). ONH analysis was performed by means of six radial scans centred on the optic disc (Stratus OCT, Fast Optic Disc acquisition protocol). The mean RNFL values were correlated with the data obtained by ONH analysis., Results: The superior, nasal, and inferior quadrant RNFL thickness showed a significant correlation with the optic disc area (R = 0.3822, p = 0.0043), (R = 0.3024, p = 0.026), (R = 0.4048, p = 0.0024) and the horizontal disc diameter (R = 0.2971, p = 0.0291), (R = 0.2752, p = 0.044), (R = 0.3970, p = 0.003). The superior and inferior quadrant RNFL thickness was also positively correlated with the vertical disc diameter (R = 0.3774, p = 0.0049), (R = 0.2793, p = 0.0408). A significant correlation was observed between the 360 degrees average RNFL thickness and the optic disc area and the vertical and horizontal disc diameters of the ONH (R = 0.4985, p = 0.0001), (R = 0.4454, p = 0.0007), (R = 0.4301, p = 0.0012)., Conclusions: RNFL thickness measurements obtained by Stratus OCT increased significantly with an increase in optic disc size. It is not clear if eyes with large ONHs show a thicker RNFL as a result of an increased amount of nerve fibres or to the shorter distance between the circular scan and the optic disc edge.

Mitochondria are increasingly recognized as central players in the life and death of cells and especially of neurons. The energy-dependence of retinal ganglion cells (RGC) and their axons, which form the optic nerve, is singularly skewed. In fact, while mitochondria are very abundant in the initial, unmyelinated part of the axons anterior to the lamina cribrosa, their number suddenly decreases as the myelin sheath begins more posteriorly. The vascular system also presents different blood-brain barrier properties anterior and posterior to the lamina, possibly reflecting the different metabolic needs of the optic nerve head (unmyelinated) and of the retrobulbar optic nerve (myelinated). Mitochondrial biogenesis occurs within the cellular somata of RGC in the retina. It needs the coordinated interaction of nuclear and mitochondrial genomes. Mitochondria are then transported down the axons and distributed where they are needed. These locations are along the unmyelinated portion of the nerve, under the nodes of Ranvier in the retrobulbar nerve, and at the synaptic terminals. Efficient transportation of mitochondria depends on multiple factors, including their own energy production, the integrity of the cytoskeleton and its protein components (tubulin, etc.), and adequate myelination of the axons. Any dysfunction of these systems may be of pathological relevance for optic neuropathies with primary or secondary involvement of mitochondria. Leber's hereditary optic neuropathy (LHON) is the paradigm of mitochondrial optic neuropathies where a primary role for mitochondrial dysfunction is certified by maternal inheritance and association with specific mutations in the mitochondrial DNA (mtDNA). Clinical phenocopies of this pathology are represented by the wide array of optic neuropathies associated with vitamin depletion, toxic exposures, alcohol and tobacco abuse, and use of certain drugs. Moreover, the recent identification of mutations in the nuclear gene OPA1 as the causative factor in dominant optic atrophy (DOA, Kjer's type) brought the unexpected finding that this gene encodes for a mitochondrial protein, suggesting that DOA and LHON may be linked by similar pathogenesis. Polymorphisms in this very same gene may be associated with normal tension glaucoma (NTG), which might be considered a genetically determined optic neuropathy that again shows similarities with both LHON and DOA. Exciting new developments come from first examples of mitochondrial optic neuropathies in animal models that are genetically determined or are the result of ingenious engineering of mitochondrial gene expression, or from biochemical manipulations of the respiratory complexes. Even more exciting is the first successful attempt to correct the LHON-related complex I dysfunction by the allotopic nuclear expression of the recoded mitochondrial gene. There is hope that the genetic complexities, biochemical dysfunctions, and integrated anatomical-physiological cellular relationships will soon be precisely delineated and that promising therapeutic and prophylactic strategies will be proposed.

Purpose: To apply new methods in magnetic resonance imaging (MRI) in resolving the histoarchitecture of the human optic nerve obtained from normal individuals and a Leber's hereditary optic neuropathy (LHON) case., Design: Small case series--clinicopathologic correlation., Method: Three optic nerves were obtained from two normal subjects, aged 69 and 70, and a LHON/3460 patient, aged 75. The posterior pole of the eye with attached optic nerves was fixed in buffered paraformaldehyde and placed into a 10-mm quartz tube. Images were acquired in a Bruker AMX500 12 Tesla microimaging system. The three-dimensional data were acquired with 512 x 256 x 256 points, yielding a final isotopic resolution of 30 microm., Results: The sclera, choroids, and retina were easily distinguished. The nerve fiber layer was seen to enter the optic disc and traverse the lamina cribrosa (LC). The resolution of the image of the optic nerve head was such that the LC was visualized as multiple stacked plates. The fibers emerged from glial columns in the LC as distinct fascicles and could be made out to change appearance as they became myelinated and expanded in the more posterior nerve. The ophthalmic artery and vein were visualized, as were the optic nerve arachnoid and dural sheaths. In the Leber's case, the LC plates seemed collapsed or compressed. The axonal bundles were atrophic and the pial-collagen septae markedly thickened. The entire nerve had shrunk, creating space under the arachnoid, down and around the central ophthalmic artery and vein., Conclusions: These results demonstrate the feasibility of using extremely high-resolution magnetic resonance imaging (microMRI) to examine the three-dimensional (30 microm) images of the human optic nerve. Several atrophic lesions, normally visible only by histopathologic examination, were visualized in the Leber's optic nerve. microMRI may eventually permit the in vivo visualization of lesions in or about the optic nerve.

Selective degeneration of the smallest fibers (papillo-macular bundle) of the human optic nerve occurs in a large number of optic neuropathies characterized primarily by loss of central vision. The pathophysiology that underlies this peculiar pattern of cell involvement probably reflects different forms of genetic and acquired mitochondrial dysfunction. Maternally inherited Leber's hereditary optic neuropathy (LHON), dominant optic atrophy (Kjer disease), the optic atrophy of Leigh's syndrome, Friedreich ataxia and a variety of other conditions are examples of inherited mitochondrial disorders with different etiologies. Tobacco-alcohol amblyopia (TAA), the Cuban epidemic of optic neuropathy (CEON) and other dietary (Vitamins B, folate deficiencies) optic neuropathies, as well as toxic optic neuropathies such as due to chloramphenicol, ethambutol, or more rarely to carbon monoxide, methanol and cyanide are probably all related forms of acquired mitochondrial dysfunction. Biochemical and cellular studies in LHON point to a partial defect of respiratory chain function that may generate either an ATP synthesis defect and/or a chronic increase of oxidative stress. Histopathological studies in LHON cases and a rat model mimicking CEON revealed a selective loss of retinal ganglion cells (RGCs) and the corresponding axons, particularly in the temporal-central part of the optic nerve. Anatomical peculiarities of optic nerve axons, such as the asymmetric pattern of myelination, may have functional implications on energy dependence and distribution of mitochondrial populations in the different sections of the nerve. Histological evidence suggests impaired axonal transport of mitochondria in LHON and in the CEON-like rat model, indicating a possible common pathophysiology for this category of optic neuropathies. Histological evidence of myelin pathology in LHON also suggests a role for oxidative stress, possibly affecting the oligodendrocytes of the optic nerves.

Purpose: Leber's hereditary optic neuropathy (LHON), though known to be due to 1 of 3 pathogenic mtDNA point mutations (nucleotide positions 11,778, 3460, and 14,484), usually manifests itself acutely in young adulthood with a stereotypical presentation of dyschromatopsia, loss of central vision, and loss of the papillomacular bundle nerve fiber layer. Histopathologic investigations have demonstrated devastating losses of axons with relative sparing of the most peripherally placed fibers in the optic nerves. This study was designed to morphometrically investigate the nerve fiber spectrum from ultrastructural studies of optic nerves obtained from 2 patients with LHON., Methods: Two cases of LHON were molecularly characterized and the optic nerves from these cases studied by light microscopy and electron microscopy. Montages were made of electron micrographs cut orthogonal to fibers obtained from the periphery of each optic nerve, and these were then used for the measurement of each axon (short and long axis) and its myelin sheath. From this, a spectrum of nerve fiber layer was generated, yielding axon caliber profiles that could be compared between optic nerves., Results: The total depletion of optic nerve fiber population in the 2 cases of LHON varied from 95% to 99%. Those fibers that were spared were limited to the peripheral optic nerve. The nerve fiber layer spectra of these remaining fibers showed a marked diminution of the first peak of axons of less than 1 micron in diameter, with relative emphasis of a second peak of axons of about 2 microns in diameter. In comparison to normal controls, this reflected a preferential loss of the smallest axons corresponding to the P-cell population., Conclusions: The clinical features of dyschromatopsia and central scotoma (with preservation of pupils) in LHON suggests the selective loss of the P-cell population known to subserve these (and not pupil) functions. This also correlates well with the fundus findings of early losses of the papillomacular bundle. The present study extends these findings to demonstrate a relative preservation of the M-cells in the optic nerve as reflected by the nerve fiber spectral profile. This selective loss of smaller fibers and their corresponding smaller retinal ganglion cells may, in addition to explaining the clinical features in LHON, provide valuable insights as to the exact pathophysiologic mechanisms by which mitochondrial impairment may induce apoptosis in vulnerable neurons.

Purpose: To develop and characterize a new model of galactose-induced cataract formation in young, 3- to 4-week-old Hartley guinea pigs., Methods: Experimental animals were fed 50% galactose in powdered guinea pig chow containing 0.5 g ascorbate/kg diet. Control animals were fed normal powdered guinea pig chow (0.5 g ascorbate/kg diet). Lenses from all animals were subjected to photo-slit-lamp examination, light microscopic analysis, and high-pressure liquid chromatography (HPLC) analysis of polyol content., Results: Photo-slit-lamp examination indicated initial opacities in equatorial subcapsular region between 3 and 5 days in all galactose-fed animals (20/20); opacities progressed toward the anterior pole when diet was extended to 14 days. Histologic analysis of the equatorial changes confirmed progressive cataract formation consisting of small intra-fibrillar vacuoles in the pre-equatorial region (3 days), an increased number of enlarged and coalesced vacuoles (6 days), and progressive tissue swellings with cellular disruption and signs of epithelial multilayering (14 days). The anterior epithelium showed increased cell height and swelling after 3 days of the galactose diet. HPLC analysis of lens tissue indicated progressive accumulation of galactitol, 18 mM after 3 days, which plateaued to about 30 mM between 6 and 14 days. The level of myo-inositol dropped from a control value of 2.8 +/- 0.7 mM to 1.5 +/- 0.7 mM after 3 days, and was nearly undetectable after 14 days of the galactose diet., Conclusions: The current study suggests that the guinea pig model may serve as a valuable new tool to study sugar-induced cataract formation and to characterize the early morphologic and biochemical events in cataractogenesis.