Natural History of Conversion of Leber's Hereditary Optic Neuropathy: A Prospective Case Series.

PURPOSE: To illustrate the natural history of Leber's hereditary optic neuropathy (LHON). DESIGN: Prospective observational case series. PARTICIPANTS: The Soave-Brazil pedigree of m.11778G>A/ND4 mitochondrial DNA LHON mutation. METHODS: A prospectively acquired database of the Soave-Brazil pedigree was reviewed. Data from 285 individuals were included in the database over a 15-year period. The pedigree was reviewed for unaffected mutation carriers who converted to affected status, 6 patients with LHON were identified. The medical records were reviewed 1 year preconversion to 1 year postconversion for visual acuity (logarithm of the minimum angle of resolution [logMAR]), Humphrey Visual Field (HVF) mean deviation (MD), and retinal nerve fiber layer (RNFL) thickness, as measured by Cirrus (Carl Zeiss, Oberkochen, Germany) optic coherence tomography (OCT). The RNFL thickness values were normalized for age. Visual acuity, HVF, and processed RNFL data from each of the 12 eyes were then sorted into 2-month time periods relative to the date of conversion, within which they were averaged. MAIN OUTCOME MEASURES: The main outcome measures were visual acuity, HVF MD, and RNFL thickness. RESULTS: Decreased visual acuity preceded conversion by up to 2 months and then declined up to 8 months postconversion. Decrease in HVF MD occurred at least 4 months preceding conversion, after which values decreased until plateau at 6 to 8 months. Average RNFL thickness was above normal baseline thickness in all 4 quadrants as measured by OCT at the time of conversion. Increase in RNFL thickness preceded conversion as early as 4 to 6 months, peaked at conversion, and decreased until individual plateaus. The temporal quadrant was first to be involved, then the inferior and superior quadrants, and the nasal quadrant showed the latest and least changes. CONCLUSIONS: Subclinical changes preceded the date of conversion and may reflect the complicated nature of identifying the date of conversion in LHON. Early increases in RNFL preceding conversion suggest that structural changes precede clinically significant vision loss. Asynchronous quadrant involvement supports a previously published mathematical model. The natural history of LHON is not a subacute process, as previously believed, but progresses more slowly, taking up to 8 months to plateau.

Efficient mitochondrial biogenesis drives incomplete penetrance in Leber's hereditary optic neuropathy.

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.

Development and initial experience with a colored perfluorocarbon liquid for intraocular tamponade in vitreoretinal surgery.

PURPOSE: To present the development and initial experience of a novel colored perfluorocarbon liquid (PFCL) in vitreoretinal surgery. METHODS: This was an experimental laboratory study and prospective human interventional study. F6H8 (Fluoron GmbH) was colored by adding 0.3 g/L blue anthraquinone dye. Subsequently, 20% colored F6H8 was prepared by mixing with perfluorooctane or perfluorodecalin (Fluoron GmbH). The novel product is not yet FDA approved for human application. In the laboratory, the colored PFCL was covered with 1) uncolored PFCL, 2) BSS, and 3) silicone oil. Cell toxicity was evaluated in L929 mouse fibroblasts using a growth inhibition assay. Porcine ex vivo eyes were evaluated after vitrectomy followed by intravitreal and subretinal colored PFCL infusion. A pilot, prospective, noncomparative interventional study was conducted in patients with retinal detachment with proliferative vitreoretinopathy (PVR). RESULTS: The density of the colored PFLC mixture was 1.664 g/cm for perfluorooctane and 1.802 g/cm for perfluorodecalin. There was no relevant cell growth inhibition with any concentration of colored PFCL tested. Experiments in pigs revealed that infusion of the colored PFCL caused neither staining of the internal limiting membrane nor intravitreal residual droplets. In the prospective study, 9 eyes (75%) underwent surgery for rhegmatogenous retinal detachment with at least grade C PVR. The colored PFCL enabled retinal break examination and detection of residual intravitreal droplets in all surgeries. There was no case of separation or leakage of the dye from the PFCL solution that could have caused unwanted staining of the vitreous or epiretinal surface. CONCLUSION: The colored PFCL enabled intraoperative maneuvers such as endolaser use. In addition, removal of the colored PFCL was easily achieved at the end of surgery.

Investigation of the retinal biocompatibility of acid violet for chromovitrectomy.

PURPOSE: The primary objective was to investigate the retinal biocompatibility of acid violet (AV) as a vital dye for chromovitrectomy. The secondary objective was to evaluate the capacity of AV to stain the anterior capsule of the lens. METHODS: An amount of 0.05 ml of 0.25 g/l and 0.5 g/l AV was injected intravitreally in the OD, while balanced salt solution (BSS) was applied in the OS for control. Clinical examination and histology with light microscopy (LM) were performed after 7 days. Retinal cell layers were evaluated for morphologic alterations and number of cells. The electroretinographic (ERG) changes were assessed at baseline and 7 days. In another part of the study, 0.1 ml of 0.25 g/l AV was injected into the anterior chamber of ex-vivo porcine eyes, and its capacity to stain the anterior capsule was determined. Cadaveric eyes were used to test the capacity of AV to stain the internal limitant membrane (ILM) during vitrectomy. RESULTS: The gross histopathologic appearance of the retina, choroids, sclera, and optic nerve was within normal limits, without any signs of severe retinal necrosis or cystic degeneration. AV caused no substantial retinal alterations in retinal layers by LM at either the lower or higher dose when compared with the control eye. The injection of AV did not induce considerable ERG alterations. The violet dye stained the anterior capsule after anterior chamber injection and the ILM, allowing a safer capsulorrhexis and vitrectomy. CONCLUSION: Acid violet may be safe for the retina at concentrations of 0.25 and 0.50 g/l after intravitreous injection, and may be used as a vital dye for staining the anterior capsule and the ILM.

Posterior hyaloid detachment and internal limiting membrane peeling assisted by anthocyanins from acai fruit (Euterpe oleracea) and 10 other natural vital dyes: experimental study in cadaveric eyes.

PURPOSE: The purpose of this study was to determine whether natural dyes facilitate posterior hyaloid detachment (posterior vitreous detachment [PVD]) and retinal internal limiting membrane (ILM) peeling in human eyes. METHODS: Open-sky vitrectomy with posterior hyaloid and ILM removal was performed in 86 human cadaveric eyes. After core vitrectomy, 11 different dyes were injected into the vitreous cavity to aid hyaloid detachment and ILM removal. The dyes were allowed to settle on the macula for 5 minutes after PVD and were removed by mechanical aspiration. Intraocular forceps were used for ILM peeling, which was confirmed by light microscopy of the peeled tissue. Acai fruit (Euterpe oleracea) extract and 10 additional dyes from plants or animal sources were tested: pomegranate (Punica granatum), logwood (Haematoxylum campechianum), chlorophyll extract from alfalfa (Medicago sativa), cochineal (Dactylopius coccus), hibiscus (Hibiscus rosa-sinensis), indigo (Indigofera tinctoria), paprika (Capiscum annuum), turmeric (Curcuma longa), old fustic (Maclura tinctoria), and grape (Vitis vinifera). RESULTS: The dyes facilitated PVD and ILM peeling. Acai fruit (E. oleracea) extract, logwood (H. campechianum), cochineal (D. coccus), and old fustic (M. tinctoria) facilitated PVD in all cases; dye-assisted PVD was compared with triamcinolone-assisted PVD performed previously in a comparative model. Acai fruit (E. oleracea) extract, cochineal (D. coccus), and chlorophyll extract from alfalfa (M. sativa) showed the best capability for ILM staining; dye-assisted ILM removal was compared with the ILM peeling guided by indocyanine green staining performed previously in a comparative model. Light microscopy confirmed the ILM removal in all cases. CONCLUSION: Anthocyanin dye of the acai fruit (E. oleracea) and the dyes from cochineal (D. coccus) and chlorophyll extract from alfalfa (M. sativa) resulted in the best capability for posterior hyaloid and ILM staining in human cadaveric eyes and may be a useful tool for vitreoretinal surgery.

Toxicity and retinal penetration of infliximab in primates.

PURPOSE: To evaluate the retinal penetration and toxicity of two doses of intravitreal infliximab in primates. METHODS: Ten marmosets (Callithrix jacchus) were given intravitreal injection of 100 µg or 400 µg of infliximab, and balanced salt solution served as control. At baseline and after 24 hours (5 animals) and 7 days (the other 5), the eyes were examined by electroretinography. They were then killed (at 24 hours and 7 days) and assessed by light microscopy and transmission electron microscopy for toxicity and immunohistochemistry, using a biotinylated anti-human immunoglobulin G, to evaluate retinal penetration. RESULTS: There was no difference over 50% of the electroretinography b-wave between baseline and the time points studied in all animals. Light and electron microscopy, and electroretinography analysis, showed no signs of toxicity in any of the animals. Strong presence of infliximab was observed in all retinal layers 7 days after intravitreal injection at both doses (100 and 400 µg). CONCLUSION: Infliximab at doses of 100 and 400 µg seemed to cause no damage to the retina 24 hours and 7 days after its intravitreal injection, and deeply penetrated all its layers, in primates. These results encourage future perspectives for the treatment of chronic inflammatory diseases of the retina in humans.

Retinal vascular occlusion in patients with the Covid-19 virus.

BACKGROUND: The coronavirus disease (COVID-19) can cause acute respiratory distress syndrome with dyspnea, anosmia, fever, and cough. Few studies describing ocular findings have been reported. The current case series, reports the clinical findings and natural history of patients with retinal vascular occlusion after COVID-19 infection. CASE PRESENTATIONS: Patients from multiple Brazilian hospitals who had clinical and laboratory diagnoses of COVID-19 with retinal vein or arterial occlusion were analyzed retrospectively. The baseline demographics, clinical presentations of COVID-19, comorbidities, risk factors for thromboembolic events, and use of anticoagulant drugs were reviewed. The relevant clinical findings associated with the retinal vascular occlusive event, management, and outcomes were reported. Fourteen cases of retinal vascular occlusion within 3 months of the laboratory confirmed COVID-19 infection were identified. Three of which required hospitalization for COVID-19 management. Eight cases had central retinal vein occlusion, three branch retinal vein occlusion, one hemispheric retinal vein occlusion, and two central arterial occlusion. The mean patient age at presentation was 48 years; the visual acuity ranged from light perception to 20/20. Nine patients received intravitreal injections of anti-angiogenic drugs and one received ketorolac tromethamine drops for the management of secondary macular edema; four were untreated. CONCLUSIONS: COVID-19 patients may rarely have ocular manifestations of the disease. It was presented a case series of vascular occlusion events that may be related to COVID-19 infection, since these thrombotic events are actively involved in the disease pathophysiology. These cases emphasize the need for further investigation of ocular complications associated with this disease.

Pathological mitophagy disrupts mitochondrial homeostasis in Leber's hereditary optic neuropathy.

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.

Melanopsin retinal ganglion cells are resistant to neurodegeneration in mitochondrial optic neuropathies.

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.

Retinal and ocular toxicity in ocular application of drugs and chemicals--part I: animal models and toxicity assays.

AIMS: Experimental retinal research has gained great importance due to the ophthalmic pharmacotherapy era. An increasing number of drugs are constantly released into the market for the treatment of retinal diseases. In this review, animal species, animal models and toxicity assays in retinal research are discussed. METHODS: An extensive search of the literature was performed to review various aspects of the methods of investigation of drug toxicity. The different types of animal species, as well as single animal models available for the evaluation of safety and efficacy of retinal pharmacotherapy, were identified. In addition, a large variety of reported laboratory techniques were critically examined. RESULTS: In vitro studies are the first-line experiments for the development of a new drug for retinal diseases, using retinal pigment epithelial cells and other cell lines. The next step involves in vivo animal studies where nonhuman primates are considered the gold standard. However, cost and legal issues make their use difficult. Mice and rats provide genetically controlled models for investigations. Pigs, dogs and cats represent good large-size animal models, while rabbits are one of the most used species for retinal toxicity evaluations. Various laboratory methods were identified, including light microscopy, electron microscopy, electroretinography and new emerging methods, such as optical coherence tomography and scanning laser ophthalmoscopy for experimental purposes. CONCLUSIONS: A great number of animal species and models are available that simulate retinal diseases and provide experimental data for further human use. Work with animal models should include properly designed toxicity assays to obtain reliable results for safety and efficacy.

The Photopic Negative Response: An Objective Measure of Retinal Ganglion Cell Function in Patients With Leber's Hereditary Optic Neuropathy.

Purpose: The photopic negative response (PhNR) is a slow negative component of a flash photopic full-field ERG that has been shown to be specific for retinal ganglion cell (RGC) activity. Direct evaluation of RGC function is desirable in patients with Leber's hereditary optic neuropathy (LHON) in which the loss of central acuity can make it difficult to monitor patients with standard metrics. The purpose of this study was to evaluate the use of PhNR as an objective noninvasive clinical metric in LHON. Methods: Full-field photopic ERG recordings were collected in subjects with the mt.11778G>A/ND4 LHON mutation using a red on blue stimulus. The PhNR was identified using a computer-based automated detection system, and data were manually examined to remove movement artifacts. Results: The PhNR amplitude was compared between controls (n = 13), carriers (n = 17), and affected (n = 6). Mean PhNR amplitude decreased significantly across groups (P < 0.0001). Post hoc Tukey's test revealed a significant decrease in PhNR amplitude between carriers and controls (P < 0.05) and between carriers and affected (P < 0.01). Conclusions: We are able to demonstrate that the PhNR amplitude is significantly decreased in patients affected by LHON compared to carriers in a well-described pedigree. Surprisingly, there was also a decrease in PhNR in carriers, suggesting potential subclinical RGC dysfunction in some carriers. This is important in patients affected with LHON who typically have a dense central scotoma. The PhNR may be a useful objective outcome measure for future clinical trials.

Use of impression cytology for the detection of unsuspected ocular surface squamous neoplasia cells in pterygia.

Purpose: To evaluate the agreement between the methodologies of impression cytology (IC) and histopathology regarding epithelial lesions clinically diagnosed as pterygium and also regarding the detection of unsuspected and associated ocular surface squamous neoplasia (OSSN). Methods: Thirty-two Brazilian patients were included and IC was performed on all pterygia before excision. Histopathogical examination was considered the gold standard and was performed by two experienced ocular pathologists in which consensus existed regarding pterygia diagnosis. IC accuracy was assessed by sensitivity and specificity with a 95% confidence interval. Results: From the 32 primary lesions studied, histopathological examination confirmed the diagnosis of pterygium without atypical cells in 19 cases (60%) and showed unsuspected and associated OSSN cells in 13 cases (40%). IC demonstrated one false-negative and one false-positive result for atypia. Statistical analysis showed an estimated sensitivity of 92%, specificity of 94%, positive predictive value of 92%, and negative predictive value of 94%. Conclusion: IC demonstrated high agreement with histopathological analysis in the detection of atypical epithelial cells in unsuspected OSSN in Brazilian pterygia patients.

Lutein and zeaxanthin toxicity with and without brilliant blue in rabbits.

PURPOSE: To evaluate the safety profile of solutions containing lutein and zeaxanthin alone or associated with brilliant blue (BB). METHODS: Twenty-eight New Zealand rabbits were used to evaluate 4 concentrations of the various dye solutions: 0.5% lutein/zeaxanthin; 0.5% lutein/zeaxanthin associated with 0.0125% BB; 0.3% lutein/zeaxanthin associated with 0.025% BB; and 0.25% lutein/zeaxanthin associated with 0.05% BB. The pHs of the dye solutions ranged from 6.5 to 7.2 and the osmolarities from 280 to 320 mOsm/mL. Each rabbit had 0.1 mL of one of the dyeing solutions injected into the vitreous cavity of the right eye, while balanced salt solution (BSS) was injected into the left eye as the control. Scotopic electroretinography responses were recorded in all eyes at different time points. The animals were sacrificed at 1 and 7 days after injection; the eyes were analyzed by light and transmission electron microscopy. RESULTS: No significant (P>0.05) differences were seen in the a- and b-wave amplitudes among groups at any given point in time. Light and electron microscopy findings showed no significant abnormalities either, and were similar to the histological findings after intravitreal BSS injection. CONCLUSIONS: Lutein and zeaxanthin alone or in association with BB showed a good safety profile in this experimental model.

Retinal biocompatibility of brilliant blue g with deuterated water for chromovitrectomy.

PURPOSE: To investigate the retinal biocompatibility of Brilliant Blue G with deuterated water (BBG-D2O) as a vital dye for chromovitrectomy. METHODS: In this animal study, 0.05 mL of 0.25 g/L Brilliant Blue G (BBG) associated with 0.13 mL/mL of deuterium oxide (D2O) was injected intravitreally in the right eye and the same amount of balanced salt solution (BSS) was injected similarly in the left eye of rabbits. Clinical examination and histology with light microscopy were performed after seven days. Retinal cell layers were evaluated for morphologic alterations. Electroretinographic (ERG) changes were also assessed at baseline and 7 days after the injections. RESULTS: A total of 6 rabbits were included in the study. The gross histopathologic appearance of the retina, choroid, sclera and optic nerve was within normal limits without any sign of severe retinal necrosis or cystic degeneration. Light microscopy showed that BBG-D2O caused no substantial alterations in retinal layers as compared to control eyes. The injection of BBG-D2O did not induce considerable functional ERG alterations. CONCLUSION: Intravitreal injection of BBG-D2O 0.25 g/L seems to induce no retinal toxicity as documented by lack of functional and histological changes.

Effects of light exposure, pH, osmolarity, and solvent on the retinal pigment epithelial toxicity of vital dyes.

PURPOSE: To investigate the in vitro effect of pH, osmolarity, solvent, and light interaction on currently used and novel dyes to minimize dye-related retinal toxicity. DESIGN: Laboratory investigation. METHODS: Retinal pigment epithelium (RPE) human cells (ARPE-19) were exposed for 10 minutes to different pH solutions (4, 5, 6, 7, 7.5, 8, and 9) and glucose solutions (2.5%, 5.0%, 10%, 20%, 40%, and 50%) with osmolarity from 142 to 2530 mOsm, with and without 0.5 mg/mL trypan blue. R28 cells were also incubated with glucose (150, 310, and 1000 mOsm) and mannitol used as an osmotic control agent in both experiments. Dye-light interaction was assessed by incubating ARPE-19 for 10 minutes with trypan blue, brilliant blue, bromophenol blue, fast green, light green, or indigo carmine (0.05 mg/mL diluted in balanced saline solution) in the presence of high-brightness xenon and mercury vapor light sources. RESULTS: Solutions with nonphysiologic pH, below 7 and above 7.5, proved to be remarkably toxic to RPE cells with or without trypan blue. Also, all glucose solutions were deleterious to RPE (P < .001) even in iso-osmolar range. No harmful effect was found with mannitol solutions. Among the dyes tested, only light green and fast green were toxic to ARPE-19 (P < .001). Light exposure did not increase RPE toxicity either with xenon light or mercury vapor lamp. CONCLUSIONS: Solutions containing glucose as a dye solvent or nonphysiologic pH should be used with care in surgical situations where the RPE is exposed. Light exposure under present assay conditions did not increase the RPE toxicity.

The pupil light reflex in Leber's hereditary optic neuropathy: evidence for preservation of melanopsin-expressing retinal ganglion cells.

PURPOSE: To investigate the pupillary light reflex (PLR) of patients with severe loss of vision due to Leber's Hereditary Optic Neuropathy (LHON) in the context of a proposed preservation of melanopsin-expressing retinal ganglion cells (mRGCs). METHODS: Ten LHON patients (7 males; 51.6 ± 14.1 years), with visual acuities ranging from 20/400 to hand motion perception and severe visual field losses, were tested and compared with 16 healthy subjects (7 males; 42.15 ± 15.4 years) tested as controls. PLR was measured with an eye tracker and the stimuli were controlled with a Ganzfeld system. Pupil responses were measured monocularly, to 1 second of blue (470 nm) and red (640 nm) flashes with 1, 10, 100, and 250 cd/m² luminances. The normalized amplitude of peak of the transient PLR and the amplitude of the sustained PLR at 6 seconds after the flash offset were measured. In addition, optical coherence topography (OCT) scans of the peripapillary retinal nerve fiber layer were obtained. RESULTS: The patient's peak PLR responses were on average 15% smaller than controls (P < 0.05), but 5 out of 10 patients had amplitudes within the range of controls. The patients' sustained PLRs were comparable with controls at lower flash intensities, but on average, 27% smaller to the 250 cd/m² blue light, although there was considerable overlap with the PLR amplitudes of control. All patients had severe visual field losses and the retinal nerve fiber layer thickness was reduced to a minimum around the optic disc in 8 of the 10 patients. CONCLUSIONS: The PLR is maintained overall in LHON patients despite the severity of optic atrophy. These results are consistent with previous evidence of selective preservation of mRGCs.

Optic nerve histopathology in a case of Wolfram Syndrome: a mitochondrial pattern of axonal loss.

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.

Mathematically modeling the involvement of axons in Leber's hereditary optic neuropathy.

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.

Neuron-specific enolase is elevated in asymptomatic carriers of Leber's hereditary optic neuropathy.

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.