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Two major constituents of exfoliation material, fibrillin-1 and lysyl oxidase-like 1 (encoded by FBN1 and LOXL1), are implicated in exfoliation glaucoma, yet their individual contributions to ocular phenotype are minor. To test the hypothesis that a combination of FBN1 mutation and LOXL1 deficiency exacerbates ocular phenotypes, the pan-lysyl oxidase inhibitor β-aminopropionitrile (BAPN) was used to treat adult wild-type (WT) mice and mice heterozygous for a missense mutation in Fbn1 (Fbn1 C1041G /+ ) for 8 weeks and their eyes were examined. Although intraocular pressure did not change and exfoliation material was not detected in the eyes, BAPN treatment worsened optic nerve and axon expansion in Fbn1 C1041G/+ mice, an early sign of axonal damage in rodent models of glaucoma. Disruption of elastic fibers was detected only in Fbn1 C1041G/+ mice, which increased with BAPN treatment, as shown by histologic and immunohistochemical staining of the optic nerve pia mater. Transmission electron microscopy showed that Fbn1 C1041G/+ mice had fewer microfibrils, smaller elastin cores, and a lower density of elastic fibers compared with WT mice in control groups. BAPN treatment led to elastin core expansion in both WT and Fbn1 C1041G/+ mice, but an increase in the density of elastic fiber was confined to Fbn1 C1041G/+ mice. LOX inhibition had a stronger effect on optic nerve and elastic fiber parameters in the context of Fbn1 mutation, indicating the Marfan mouse model with LOX inhibition warrants further investigation for exfoliation glaucoma pathogenesis., Competing Interests: Disclosure Statement None declared., (Copyright © 2024 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

To enable rapid propagation of action potentials, axons are ensheathed by myelin, a multilayered insulating membrane formed by oligodendrocytes. Most of the myelin is generated early in development, resulting in the generation of long-lasting stable membrane structures. Here, we explored structural and dynamic changes in central nervous system myelin during development. To achieve this, we performed an ultrastructural analysis of mouse optic nerves by serial block face scanning electron microscopy (SBF-SEM) and confocal time-lapse imaging in the zebrafish spinal cord. We found that myelin undergoes extensive ultrastructural changes during early postnatal development. Myelin degeneration profiles were engulfed and phagocytosed by microglia using exposed phosphatidylserine as one "eat me" signal. In contrast, retractions of entire myelin sheaths occurred independently of microglia and involved uptake of myelin by the oligodendrocyte itself. Our findings show that the generation of myelin early in development is an inaccurate process associated with aberrant ultrastructural features that require substantial refinement., (© 2023 Djannatian et al.)

The morphometric assessment of the corneal subbasal nerve plexus (SNP) by confocal microscopy holds great potential as a sensitive biomarker for various ocular and systemic conditions and diseases. Automated wide-field montages (or large-area mosaic images) of the SNP provide an opportunity to overcome the limited field of view of the available imaging systems without the need for manual, subjective image selection for morphometric characterization. However, current wide-field montaging solutions usually calculate the mosaic image after the examination session, without a reliable means for the clinician to predict or estimate the resulting mosaic image quality during the examination. This contribution describes a novel approach for a real-time creation and visualization of a mosaic image of the SNP that facilitates an informed evaluation of the quality of the acquired image data immediately at the time of recording. In cases of insufficient data quality, the examination can be aborted and repeated immediately, while the patient is still at the microscope. Online mosaicking also offers the chance to identify an overlap of the imaged tissue region with previous SNP mosaic images, which can be particularly advantageous for follow-up examinations., (© 2022. The Author(s).)

Purpose: Netarsudil, a Rho kinase inhibitor with norepinephrine transport inhibitory effect, lowers intraocular pressure, however, its effect on axon damage remains to be elucidated. The aim of the current study was to investigate the effect of netarsudil on TNF-induced axon loss and to examine whether it affects phosphorylated-AMP-activated kinase (p-AMPK) and autophagy in the optic nerve., Methods: Intravitreal administration of TNF or TNF with netarsudil was carried out on rats and quantification of axon number was determined. Electron microscopy determined autophagosome numbers. Localization of p-AMPK expression was examined by immunohistochemistry. The changes in p62, LC3-II, and p-AMPK levels were estimated in the optic nerve by immunoblot analysis. The effect of an AMPK activator A769662 or an AMPK inhibitor dorsomorphin on axon number was evaluated., Results: Morphometric analysis revealed apparent protection by netarsudil against TNF-induced axon degeneration. Netarsudil increased autophagosome numbers inside axons. Netarsudil treatment significantly upregulated optic nerve LC3-II levels in both the TNF-treated eyes and the control eyes. Increased p62 protein level induced by TNF was significantly ameliorated by netarsudil. The netarsudil administration alone lessened p62 levels. Netarsudil significantly upregulated the optic nerve p-AMPK levels. A769662 exhibited obvious axonal protection against TNF-induced damage. A769662 treatment upregulated LC3-II levels and the increment of p62 level induced by TNF was significantly ameliorated by A769662. Immunohistochemical analysis revealed that p-AMPK is present in axons. Netarsudil-mediated axonal protection was significantly suppressed by dorsomorphin administration., Conclusions: Netarsudil upregulated p-AMPK and autophagy. Netarsudil-mediated axonal protection may be associated with upregulated p-AMPK.

Wolfram syndrome (WS), also known as a DIDMOAD (diabetes insipidus, early-onset diabetes mellitus, optic nerve atrophy and deafness) is a rare autosomal disorder caused by mutations in the Wolframin1 ( WFS1 ) gene. Previous studies have revealed that glucagon-like peptide-1 receptor agonist (GLP1 RA) are effective in delaying and restoring blood glucose control in WS animal models and patients. The GLP1 RA liraglutide has also been shown to have neuroprotective properties in aged WS rats. WS is an early-onset, chronic condition. Therefore, early diagnosis and lifelong pharmacological treatment is the best solution to control disease progression. Hence, the aim of this study was to evaluate the efficacy of the long-term liraglutide treatment on the progression of WS symptoms. For this purpose, 2-month-old WS rats were treated with liraglutide up to the age of 18 months and changes in diabetes markers, visual acuity, and hearing sensitivity were monitored over the course of the treatment period. We found that treatment with liraglutide delayed the onset of diabetes and protected against vision loss in a rat model of WS. Therefore, early diagnosis and prophylactic treatment with the liraglutide may also prove to be a promising treatment option for WS patients by increasing the quality of life.

Wolfram syndrome (WS) is a monogenic progressive neurodegenerative disease and is characterized by various neurological symptoms, such as optic nerve atrophy, loss of vision, cognitive decline, memory impairment, and learning difficulties. GLP1 receptor agonist liraglutide and BDNF mimetic 7,8-dihydroxyflavone (7,8-DHF) have had protective effect to visual pathway and to learning and memory in different rat models of neurodegenerative disorders. Although synergistic co-treatment effect has not been reported before and therefore the aim of the current study was to investigate liraglutide, 7,8-DHF and most importantly for the first time their co-treatment effect on degenerative processes in WS rat model. We took 9 months old WS rats and their wild-type (WT) control animals and treated them daily with liraglutide, 7,8-DHF or with the combination of liraglutide and 7,8-DHF up to the age of 12.5 months (n = 47, 5-8 per group). We found that liraglutide, 7,8-DHF and their co-treatment all prevented lateral ventricle enlargement, improved learning in Morris Water maze, reduced neuronal inflammation, delayed the progression of optic nerve atrophy, had remyelinating effect on optic nerve and thereby improved visual acuity in WS rats compared to WT controls. Thus, the use of the liraglutide, 7,8-DHF and their co-treatment could potentially be used as a therapeutic intervention to induce neuroprotection or even neuronal regeneration.

Glaucoma is a progressive chronic retinal degenerative disease and a leading cause of global irreversible blindness. This disease is characterized by optic nerve damage and retinal ganglion cell (RGC) death. The current treatments available target the lowering of intraocular pressure (IOP), the main risk factor for disease onset and development. However, in some patients, vision loss progresses despite successful IOP control, indicating that new and effective treatments are needed, such as those targeting the neuroprotection of RGCs. Adenosine A 3 receptor (A 3 R) activation confers protection to RGCs following an excitotoxic stimulus. In this work, we investigated whether the activation of A 3 R could also afford protection to RGCs in the laser-induced ocular hypertension (OHT) model, a well-characterized animal model of glaucoma. The intravitreal injection of 2-Cl-IB-MECA, a selective A 3 R agonist, abolished the alterations induced by OHT in the negative and positive components of scotopic threshold response (STR) without changing a- and b-wave amplitudes both in scotopic and photopic conditions. Moreover, the treatment of OHT eyes with the A 3 R agonist promoted the survival of RGCs, attenuated the impairment in retrograde axonal transport, and improved the structure of the optic nerve. Taking into consideration the beneficial effects afforded by 2-Cl-IB-MECA, we can envisage that A 3 R activation can be considered a good therapeutic strategy to protect RGCs from glaucomatous damage.

Mitochondrial dysfunction mediated loss of respiration, oxidative stress, and loss of cellular homeostasis contributes to the neuronal and axonal degenerations permanent loss of function in experimental autoimmune encephalomyelitis model (EAE) of multiple sclerosis (MS). To address the mitochondrial dysfunction mediated visual loss in EAE mice, self-complementary adeno-associated virus (scAAV) containing the NADH-dehydrogenase type-2 (NDI1) complex I gene was intravitreally injected into the mice after the onset of visual defects. Visual function assessed by pattern electroretinogram (PERGs) showed progressive loss of function in EAE mice were improved significantly in NDI1 gene therapy-treated mice. Serial optical coherence tomography (OCT) revealed that progressive thinning of inner retinal layers in EAE mice was prevented upon NDI1 expression. The 45% optic nerve axonal and 33% retinal ganglion cell (RGC) loss contributed to the permanent loss of visual function in EAE mice were ameliorated by NDI1-mediated prevention of mitochondrial cristae dissolution and improved mitochondrial homeostasis. In conclusion, targeting the dysfunctional complex I using NDI1 gene can be an approach to address axonal and neuronal loss responsible for permanent disability in MS that is unaltered by current disease modifying drugs.

The use of ex vivo compound action potential (CAP) recordings from intact optic nerves is an ideal model to study white matter function without the influence of gray matter. Here, we describe how freshly dissected optic nerves are placed in a humidified recording chamber and how evoked CAPs are recorded and monitored in real time for up to 10 h. Evoked CAP recordings allow for white matter to be studied under acute challenges such as anoxia, hypoxia, aglycemia, and ischemia.

Optic nerve sheath diameter (ONSD) is used clinically as a noninvasive measure for elevated intracranial pressure (ICP). This study had two purposes: to investigate the immediate effects optic nerve sheath (ONS) dilation post-ICP increase on trabecular fibers connecting the optic nerve to the ONS and to document any changes in these fibers 30 days post-increased ICP. In a swine model, ICP was increased by inflating a Foley catheter balloon in the epidural space. Three control pigs received the catheter insertion without inflation (no increase in ICP) and four experimental pigs received the catheter with inflation (increased ICP). The control and two randomly selected pigs with increased ICP were euthanized immediately after the procedure. The two other pigs were euthanized 30 days post-catheter inflation. For all pigs, the ONS was removed and imaged using a scanning electron microscope, calculating percent porosity values. Porosity values for the experimental groups (Immediately measured [IM] μ = 0.5749; Delayed measured [DM] μ = 0.5714) were larger than the control group (μ = 0.4336) and statistically significant (IM vs. Control, p  = 0.0018; DM vs. Control, p  = 0.0092). There was no significant difference ( p  = 0.9485) in porosity of the DM group when compared with the IM group. This study demonstrated that the trabecular fibers immediately post-increased ICP (ONS dilation) were more porous than the control and remained statistically different (more porous) after 30 days. These results suggest a structural change that occurs in the ONS with elevations in ICP.

Optic nerve atrophy represents the most common form of hereditary optic neuropathies leading to vision impairment. The recently described Bosch-Boonstra-Schaaf optic atrophy (BBSOA) syndrome denotes an autosomal dominant genetic form of neuropathy caused by mutations or deletions in the NR2F1 gene. Herein, we describe a mouse model recapitulating key features of BBSOA patients-optic nerve atrophy, optic disc anomalies, and visual deficits-thus representing the only available mouse model for this syndrome. Notably, Nr2f1-deficient optic nerves develop an imbalance between oligodendrocytes and astrocytes leading to postnatal hypomyelination and astrogliosis. Adult heterozygous mice display a slower optic axonal conduction velocity from the retina to high-order visual centers together with associative visual learning deficits. Importantly, some of these clinical features, such the optic nerve hypomyelination, could be rescued by chemical drug treatment in early postnatal life. Overall, our data shed new insights into the cellular mechanisms of optic nerve atrophy in BBSOA patients and open a promising avenue for future therapeutic approaches., (© 2019 The Authors. Published under the terms of the CC BY 4.0 license.)

Purpose: The aim of this study was to compare the ultrasonographic optic nerve sheath diameter (ONSD) in different grades of papilledema and in controls and to evaluate ONSD in atrophic papilledema/optic atrophy when raised ICP was suspected., Methods: Prospective cross-sectional case-control study. Following an ocular examination, papilledema was graded clinically using modified Frisén's grading. An ultrasonographic cross section of the retrobulbar optic nerve was obtained with a posterior transverse scan. Independent t-test and analysis of variance were the statistical tools used in the study., Results: The study included 55 cases and 55 age- and gender-matched controls; mean (± standard deviation) age was 37.17 (±11.25) years and male: female ratio was 49:61. There was a statistically significant difference in the mean ultrasonographic ONSD between cases [4.89 (±0.65) mm] and controls [3.12 (±0.22) mm] (P < 0.001). There was a significant difference in the mean ONSD across Frisén's grades of papilledema (P < 0.001). The mean ONSD in atrophic papilledema was 6.2 (±0.75) mm., Conclusion: In the presence of symptoms, ultrasonographic ONSD >4 mm is diagnostic of papilledema. Ultrasonographic ONSD correlates well with the severity of papilledema and can be used to follow-up patients with chronically elevated ICP. It is useful in detecting raised ICP in the presence of optic atrophy and to distinguish true papilledema from pseudopapilledema., Competing Interests: There are no conflicts of interest

Glaucomatous neurodegeneration represents one of the major causes of irreversible blindness worldwide. Yet, the detailed molecular mechanisms that initiate optic nerve damage and retinal ganglion cell (RGC) loss are not fully understood. Members of the protein tyrosine phosphatase (PTP) superfamily are key players in numerous neurodegenerative diseases. In order to investigate the potential functional relevance of the PTP megakaryocyte 2 (Meg2) in retinal neurodegeneration, we analyzed Meg2 knockout (KO) and heterozygous (HET)-synonym protein-tyrosine phosphatase non-receptor type 9 (Ptpn9)-mice. Interestingly, via global microarray and quantitative real-time PCR (RT-qPCR) analyses of Meg2 KO and HET retinae, we observed a dysregulation of several candidate genes that are highly associated with retinal degeneration and intraocular pressure (IOP) elevation, the main risk factor for glaucoma. Subsequent IOP measurements in Meg2 HET mice verified progressive age-dependent IOP elevation. Ultrastructural analyses and immunohistochemistry showed severe optic nerve degeneration accompanied by a dramatic loss of RGCs. Additionally, HET mice displayed reactive micro-/macrogliosis and early activation of the classical complement cascade with pronounced deposition of the membrane attack complex (MAC) in the retina and optic nerve. When treated with latanoprost, significant IOP lowering prevented RGC loss and microglial invasion in HET mice. Finally, electroretinogram (ERG) recordings revealed reduced a- and b-wave amplitudes, indicating impaired retinal functionality in Meg2 HET mice. Collectively, our findings indicate that the heterozygous loss of Meg2 in mice is sufficient to cause IOP elevation and glaucomatous neurodegeneration. Thus, Meg2 HET mice may serve as a novel animal model to study the pathomechanism involved in the onset and progression of glaucoma.

Hypoglycemia is a common iatrogenic consequence of type 1 diabetes therapy that can lead to central nervous system injury and even death if untreated. In the absence of clinically effective neuroprotective drugs we sought to quantify the putative neuroprotective effects of imposing hypothermia during the reperfusion phase following aglycemic exposure to central white matter. Mouse optic nerves (MONs), central white matter tracts, were superfused with oxygenated artificial cerebrospinal fluid (aCSF) containing 10 mmol/L glucose at 37°C. The supramaximal compound action potential (CAP) was evoked and axon conduction was assessed as the CAP area. Extracellular lactate was measured using an enzyme biosensor. Exposure to aglycemia, simulated by omitting glucose from the aCSF, resulted in axon injury, quantified by electrophysiological recordings, electron microscopic analysis confirming axon damage, the extent of which was determined by the duration of aglycemia exposure. Hypothermia attenuated injury. Exposing MONs to hypothermia during reperfusion resulted in improved CAP recovery compared with control recovery measured at 37°C, an effect attenuated in alkaline aCSF. Hypothermia decreases pH implying that the hypothermic neuroprotection derives from interstitial acidification. These results have important clinical implications demonstrating that hypothermic intervention during reperfusion can improve recovery in central white matter following aglycemia., (© 2019 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

Fireflies (Coleoptera: Lampyridae) have distinct visual systems at different stages of development. Larvae have stemmata and adults have compound eyes. Adults use compound eyes to mediate photic communication during courtship. Larvae do not manifest this behavior, yet they are bioluminescent. We investigated the structure of stemmata in Photuris firefly larvae to identify anatomical substrates (i.e., rhabdomeres) conferring visual function. Stemmata were located bilaterally on the antero-lateral surfaces of the head. Beneath the ~ 130 µm diameter lens, we identified a pigmented eye-cup. At its widest point, the eye-cup was ~ 150 µm in diameter. The optic nerve exited the eye-cup opposite the lens. Two distinct regions, asymmetric in size and devoid of pigmentation, were characterized in stemmata cross-sections. We refer to these regions as lobes. Each lobe contained a rhabdom of a radial network of rhabdomeres. Pairs of rhabdomeres formed interdigitating microvilli contributed from neighboring photoreceptor cell bodies. The optic nerve contained 88 axons separable into two populations based on size. The number of axons in the optic nerve together with distinct rhabdoms suggests these structures were formed from 'fusion stemmata.' This structural specialization provides an anatomical substrate for future studies of visually mediated behaviors in Photuris larvae.

The optic nerve conveys information about the outside world from the retina to multiple subcortical relay centers. Until recently, the optic nerve was widely believed to be incapable of re-growing if injured, with dire consequences for victims of traumatic, ischemic, or neurodegenerative diseases of this pathway. Over the past 10-20 years, research from our lab and others has made considerable progress in defining factors that normally suppress axon regeneration and the ability of retinal ganglion cells, the projection neurons of the retina, to survive after nerve injury. Here we describe research from our lab on the role of inflammation-derived growth factors, suppression of inter-cellular signals among diverse retinal cell types, and combinatorial therapies, along with related studies from other labs, that enable animals with optic nerve injury to regenerate damaged retinal axons back to the brain. These studies raise the possibility that vision might one day be restored to people with optic nerve damage.

The regenerative capacity of CNS tracts has ever been a great hurdle to regenerative medicine. Although recent studies have described strategies to stimulate retinal ganglion cells (RGCs) to regenerate axons through the optic nerve, it still remains to be elucidated how these therapies modulate the inhibitory environment of CNS. Thus, the present work investigated the environmental content of the repulsive axon guidance cues, such as Sema3D and its receptors, myelin debris, and astrogliosis, within the regenerating optic nerve of mice submitted to intraocular inflammation + cAMP combined to conditional deletion of PTEN in RGC after optic nerve crush. We show here that treatment was able to promote axonal regeneration through the optic nerve and reach visual targets at twelve weeks after injury. The Regenerating group presented reduced MBP levels, increased microglia/macrophage number, and reduced astrocyte reactivity and CSPG content following optic nerve injury. In addition, Sema3D content and its receptors are reduced in the Regenerating group. Together, our results provide, for the first time, evidence that several regenerative repulsive signals are reduced in regenerating optic nerve fibers following a combined therapy. Therefore, the treatment used made the CNS microenvironment more permissive to regeneration.

Eye irritation assessment is compulsory to anticipate health risks in military personnel exposed to riot control agents such as capsaicin, the principal constituent of oleoresin capsicum, or pepper sprays. The present work investigates certain fundamental yet unaddressed pharmacological manifestations on ocular exposure to capsaicin. Ocular pharmacology of capsaicin was studied using acute eye irritation (AEI), bovine corneal opacity and permeability (BCOP) assay, corneal fluorescein staining and indirect ophthalmoscopy studies, transcorneal permeation, Schirmer tear secretion test, nerve conduction velocity study and enzyme-linked immunosorbent assay (ELISA). Additionally, histopathology and scanning electron microscopy (SEM) of bovine corneas and rat optic nerves were done to further estimate capsaicin induced morphological variations. Our findings demonstrated that AEI, BCOP, corneal fluorescein staining and indirect ophthalmoscopy were useful in assessing capsaicin induced ocular irritation; AEI and BCOP also contributed towards indicating the eye irritation potential of capsaicin as per the United Nations Globally Harmonized System of Classification and Labelling of Chemicals categorization. Additional experimental observations include considerable transcorneal permeation of capsaicin, capsaicin induced reduction in tear secretions and nerve conduction velocity and increased expression of proinflammatory cytokines by ELISA. Histopathology and SEM were favourable techniques for the detection of capsaicin induced ocular physiological modifications.

Following injury to the central nervous system, axons and myelin distinct from the initial injury site undergo changes associated with compromised function. Quantifying such changes is important to understanding the pathophysiology of neurotrauma; however, most studies to date used 2 dimensional (D) electron microscopy to analyse single sections, thereby failing to capture changes along individual axons. We used serial block face scanning electron microscopy (SBF SEM) to undertake 3D reconstruction of axons and myelin, analysing optic nerves from normal uninjured female rats and following partial optic nerve transection. Measures of axon and myelin dimensions were generated by examining 2D images at 5 µm intervals along the 100 µm segments. In both normal and injured animals, changes in axonal diameter, myelin thickness, fiber diameter, G-ratio and percentage myelin decompaction were apparent along the lengths of axons to varying degrees. The range of values for axon diameter along individual reconstructed axons in 3D was similar to the range from 2D datasets, encompassing reported variation in axonal diameter attributed to retinal ganglion cell diversity. 3D electron microscopy analyses have provided the means to demonstrate substantial variability in ultrastructure along the length of individual axons and to improve understanding of the pathophysiology of neurotrauma.

Purpose: Gene therapy of retinal ganglion cells (RGCs) has promise as a powerful therapeutic for the rescue and regeneration of these cells after optic nerve damage. However, early after damage, RGCs undergo atrophic changes, including gene silencing. It is not known if these changes will deleteriously affect transduction and transgene expression, or if the therapeutic protein can influence reactivation of the endogenous genome., Methods: Double-transgenic mice carrying a Rosa26-(LoxP)-tdTomato reporter, and a mutant allele for the proapoptotic Bax gene were reared. The Bax mutant blocks apoptosis, but RGCs still exhibit nuclear atrophy and gene silencing. At times ranging from 1 hour to 4 weeks after optic nerve crush (ONC), eyes received an intravitreal injection of AAV2 virus carrying the Cre recombinase. Successful transduction was monitored by expression of the tdTomato reporter. Immunostaining was used to localize tdTomato expression in select cell types., Results: Successful transduction of RGCs was achieved at all time points after ONC using AAV2 expressing Cre from the phosphoglycerate kinase (Pgk) promoter, but not the CMV promoter. ONC promoted an increase in the transduction of cell types in the inner nuclear layer, including Müller cells and rod bipolar neurons. There was minimal evidence of transduction of amacrine cells and astrocytes in the inner retina or optic nerve., Conclusions: Damaged RGCs can be transduced and at least some endogenous genes can be subsequently activated. Optic nerve damage may change retinal architecture to allow greater penetration of an AAV2 virus to transduce several additional cell types in the inner nuclear layer.

There is increasing clinical evidence that the eye is not only affected by intraocular pressure (IOP), but also by intracranial pressure (ICP). Both pressures meet at the optic nerve head of the eye, specifically the lamina cribrosa (LC). The LC is a collagenous meshwork through which all retinal ganglion cell axons pass on their way to the brain. Distortion of the LC causes a biological cascade leading to neuropathy and impaired vision in situations such as glaucoma and idiopathic intracranial hypertension. While the effect of IOP on the LC has been studied extensively, the coupled effects of IOP and ICP on the LC remain poorly understood. We investigated in-vivo the effects of IOP and ICP, controlled via cannulation of the eye and lateral ventricle in the brain, on the LC microstructure of anesthetized rhesus monkeys eyes using the Bioptigen spectral-domain optical coherence tomography (OCT) device (Research Triangle, NC). The animals were imaged with their head upright and the rest of their body lying prone on a surgical table. The LC was imaged at a variety of IOP/ICP combinations, and microstructural parameters, such as the thickness of the LC collagenous beams and diameter of the pores were analyzed. LC microstructure was confirmed by histology. We determined that LC microstructure deformed in response to both IOP and ICP changes, with significant interaction between the two. These findings emphasize the importance of considering both IOP and ICP when assessing optic nerve health.

The purpose of the present study was to characterize the metabolic profile of the visual cortex in streptozotocin-induced Type 1 diabetic rats by means of in vivo proton MRS. Several metabolite concentration ratios in the visual cortex were calculated. In addition, postmortem histologic analyses for retinal ganglion cell (RGC) loss, optic nerve injury and visual cortex alterations were monitored. The results showed that diabetes induced several changes in visual cortex metabolites, such as reduced N-acetylaspartate, glutamate, γ-aminobutyric acid, taurine and choline-containing compound levels. Nevertheless, myo-inositol levels increased significantly as compared with controls. Remarkable RGC loss and optic nerve degeneration were observed by morphological analysis. Moreover, the results showed significant neuronal loss and glial activation in the visual cortex. These findings indicated that, besides vascular abnormalities, neuronal loss and degeneration in the visual pathway were induced due to disrupted glucose homeostasis in diabetes. Metabolic or functional abnormalities were induced in cerebral neurons of the visual cortex by diabetes., (Copyright © 2017 John Wiley & Sons, Ltd.)

ε-Toxin is a pore forming toxin produced by Clostridium perfringens types B and D. It is synthesized as a less active prototoxin form that becomes fully active upon proteolytic activation. The toxin produces highly lethal enterotoxaemia in ruminants, has the ability to cross the blood-brain barrier (BBB) and specifically binds to myelinated fibers. We discovered that the toxin induced a release of ATP from isolated mice optic nerves, which are composed of myelinated fibers that are extended from the central nervous system. We also investigated the effect of the toxin on compound action potentials (CAPs) in isolated mice optic nerves. When nerves were stimulated at 100 Hz during 200 ms, the decrease of the amplitude and the area of the CAPs was attenuated in the presence of ε-toxin. The computational modelling of myelinated fibers of mouse optic nerve revealed that the experimental results can be mimicked by an increase of the conductance of myelin and agrees with the pore forming activity of the toxin which binds to myelin and could drill it by making pores. The intimate ultrastructure of myelin was not modified during the periods of time investigated. In summary, the acute action of the toxin produces a subtle functional impact on the propagation of the nerve action potential in myelinated fibers of the central nervous system with an eventual desynchronization of the information. These results may agree with the hypothesis that the toxin could be an environmental trigger of multiple sclerosis (MS).

Purpose: To examine retinal changes after vitrectomy with internal limiting membrane (ILM) peeling, we used a cynomolgus monkey model and focused on surgical damages of ILM peeling for long observational period of 3 years., Methods: Vitrectomy was performed followed by ILM peeling similar to clinical settings in humans. Ultrastructural changes of the retina were investigated by light, transmission, and scanning electron microscopy at 3 months and 3 years after ILM peeling., Results: Ultrastructural study showed that the ILM peeled area was still clearly recognized after 3 years. The Müller cell processes covered most of the retina; however, the nerve fiber layer was partly uncovered and exposed to the vitreous space. The arcuate linear nerve fiber bundles were observed as comparable with dissociated optic nerve fiber layer appearance. Small round retinal surface defects were also observed around macula, resembling the dimple sign. Forceps-related retinal thinning was also found on the edge of ILM peeling, where we started peeling with fine forceps., Conclusion: The ultrastructural studies showed that most of ILM peeling area was covered with glial cells during wound healing processes. Retinal changes were found comparable with dissociated optic nerve fiber layer appearance or dimple sign, which were clinically observed with optical coherence tomography.

Folate, an essential micronutrient, is a critical cofactor in one-carbon metabolism for many cellular pathways including DNA synthesis, metabolism and maintenance. Folate deficiency has been associated with an increased risk of neurological disease, cancer and cognitive dysfunction. Dihydrofolate reductase (DHFR) is a key enzyme to regulate folate metabolism, however folate/DHFR activity in oligodendrocyte development has not been fully understood. Here we show that folate enhances oligodendrocyte maturation both in vitro and in vivo, which is accompanied with upregulation of oligodendrocyte-specific DHFR expression. On the other hand, pharmacological inhibition of DHFR by methotrexate (MTX) causes severe defects in oligodendrocyte survival and differentiation, which could be reversed by folate intake. We further demonstrate that folate activates a metabolic regulator AMPKα to promote oligodendrocyte survival and differentiation. Moreover, activation of AMPKα partially rescues oligodendrocyte defects caused by DHFR-inhibition both in vitro and in vivo. Taken together, these findings identify a previously uncharacterized role of folate/DHFR/AMPKα axis in regulating oligodendrocyte survival and myelination during CNS development.

Purpose: Preliminary assessment of a new prototype ultrasound-based hypersonic vitrector (HV) by qualitatively examining the histopathological changes in the retina and vitreous body after pars plana vitrectomy (PPV) and its ability to fragment vitreous collagen., Methods: Fourteen porcine cadaveric eyes, 20 eyes in live swine and six human cadaveric eyes underwent PPV using the HV or a pneumatic guillotine vitrector (GV). An additional 4 porcine crystalline lenses were touched with either the HV or GV for 1 minute. Following PPV, human vitreous was removed and processed for electron microscopy (EM). Eyes and lenses were fixed and sectioned for light microscopy (LM)., Results: There were no macroscopic retinal or optic nerve defects associated with either HV or GV PPVs. Cadaveric retinal specimens showed separation of the inner limiting membrane (ILM) and vacuolization and fragmentation at the nerve fiber layer (NFL) and the ganglion cell layer (GCL). ILM fragmentation and separation were found after PPV in live swine with both vitrectors. Small disruptions of the posterior capsule or structural lens defects were found after HV touch. The EM analysis revealed more fragmentation of human vitreous collagen fibrils after HV compared to GV PPV., Conclusions: LM and EM analysis of retina, vitreous, and crystalline lens after PPV showed similar morphological changes using the HV or the GV. Vitreous fragmentation appeared more effective with the HV. Overall this study suggests that the HV may be a promising new technology. More work is needed to quantitatively assess its safety and efficacy.

A lack of sufficient oligodendrocyte myelination contributes to remyelination failure in demyelinating disorders. miRNAs have been implicated in oligodendrogenesis; however, their functions in myelin regeneration remained elusive. Through developmentally regulated targeted mutagenesis, we demonstrate that miR-219 alleles are critical for CNS myelination and remyelination after injury. Further deletion of miR-338 exacerbates the miR-219 mutant hypomyelination phenotype. Conversely, miR-219 overexpression promotes precocious oligodendrocyte maturation and regeneration processes in transgenic mice. Integrated transcriptome profiling and biotin-affinity miRNA pull-down approaches reveal stage-specific miR-219 targets in oligodendrocytes and further uncover a novel network for miR-219 targeting of differentiation inhibitors including Lingo1 and Etv5. Inhibition of Lingo1 and Etv5 partially rescues differentiation defects of miR-219-deficient oligodendrocyte precursors. Furthermore, miR-219 mimics enhance myelin restoration following lysolecithin-induced demyelination as well as experimental autoimmune encephalomyelitis, principal animal models of multiple sclerosis. Together, our findings identify context-specific miRNA-regulated checkpoints that control myelinogenesis and a therapeutic role for miR-219 in CNS myelin repair., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Electron microscopy (EM) for biological samples, developed in the 1940-1950s, changed our conception about the architecture of eukaryotic cells. It was followed by a period where EM applied to cell biology had seemingly fallen asleep, even though new methods with important implications for modern EM were developed. Among these was the discovery that samples can be preserved by chemical fixation and most importantly by rapid freezing without the formation of crystalline ice, giving birth to the world of cryo-EM. The past 15-20 years are hallmarked by a tremendous interest in EM, driven by important technological advances. Cryo-EM, in particular, is now capable of revealing structures of proteins at a near-atomic resolution owing to improved sample preparation methods, microscopes and cameras. In this review, we focus on the challenges associated with the imaging of membranes by EM and give examples from the field of host-pathogen interactions, in particular of virus-infected cells. Despite the advantages of imaging membranes under native conditions in cryo-EM, conventional EM will remain an important complementary method, in particular if large volumes need to be imaged., (© 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.)

Purpose: A dissociated optic nerve fibre layer (DONFL) is a characteristic change noted in inner retinal morphology after internal limiting membrane (ILM) peeling. It is thought to be due to trauma to Muller cells as the ILM is peeled from their attached end plates. In this study, we aimed to determine the extent and size of Muller cell debris on the retinal side of excised ILM and assess whether this correlated with the extent of DONFL observed postoperatively., Method: Prospective single centre study of a consecutive series of patients undergoing macular hole surgery. Transmission electron microscopy of the ILM was used to assess Muller cell debris and postoperative spectral domain optical coherence tomography (SD-OCT) to assess the extent of DONFL. A variety of other pre- and postoperative features was also included., Results: Thirty-nine patients were analysed. There were retinal dimples characteristic of DONFL detected on SD-OCT in all 39 eyes. The portion of the retinal side of the ILM specimen covered by cellular debris ranged from 12% to 49%, with a median of 28%. Using linear regression, the percentage of retinal debris, the size of the debris and the postoperative visual acuity were significantly positively associated with the DONFL score. The total R squared for the model was 63.9%., Conclusion: The extent of DONFL observed postoperatively can be partly explained by the amount of retinal side cellular debris on the retinal side of the peeled ILM. Surgical strategies which minimize this material could reduce the extent of DONFL., (© 2016 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.)

Purpose: Dominant optic atrophy (MIM No. 165500) is a blinding condition related to mutations in OPA1, a gene encoding a large GTPase involved in mitochondrial inner membrane dynamics. Although several mouse models mimicking the disease have been developed, the pathophysiological mechanisms responsible for retinal ganglion cell degeneration remain poorly understood., Methods: Using a targeted metabolomic approach, we measured the concentrations of 188 metabolites in nine tissues, that is, brain, three types of skeletal muscle, heart, liver, retina, optic nerve, and plasma in symptomatic 11-month-old Opa1delTTAG/+ mice., Results: Significant metabolic signatures were found only in the optic nerve and plasma of female mice. The optic nerve signature was characterized by altered concentrations of phospholipids, amino acids, acylcarnitines, and carnosine, whereas the plasma signature showed decreased concentrations of amino acids and sarcosine associated with increased concentrations of several phospholipids. In contrast, the investigation of 3-month-old presymptomatic Opa1delTTAG/+ mice showed no specific plasma signature but revealed a significant optic nerve signature in both sexes, although with a sex effect. The Opa1delTTAG/+ versus wild-type optic nerve signature was characterized by the decreased concentrations of 10 sphingomyelins and 10 lysophosphatidylcholines, suggestive of myelin sheath alteration, and by alteration in the concentrations of metabolites involved in neuroprotection, such as dimethylarginine, carnitine, spermine, spermidine, carnosine, and glutamate, suggesting a concomitant axonal metabolic dysfunction., Conclusions: Our comprehensive metabolomic investigations revealed in symptomatic as well as in presymptomatic Opa1delTTAG/+ mice, a specific sensitiveness of the optic nerve to Opa1 insufficiency, opening new routes for protective therapeutic strategies.

Electron paramagnetic resonance (EPR) spin-label oximetry allows the oxygen permeability coefficient to be evaluated across homogeneous lipid bilayer membranes and, in some cases, across coexisting membrane domains without their physical separation. The most pronounced effect on oxygen permeability is observed for cholesterol, which additionally induces the formation of membrane domains. In intact biological membranes, integral proteins induce the formation of boundary and trapped lipid domains with a low oxygen permeability. The effective oxygen permeability coefficient across the intact biological membrane is affected not only by the oxygen permeability coefficients evaluated for each lipid domain but also by the surface area occupied by these domains in the membrane. All these factors observed in fiber cell plasma membranes of clear human eye lenses are reviewed here.

Mitochondrial transport is crucial for neuronal and axonal physiology. However, whether and how it impacts neuronal injury responses, such as neuronal survival and axon regeneration, remain largely unknown. In an established mouse model with robust axon regeneration, we show that Armcx1, a mammalian-specific gene encoding a mitochondria-localized protein, is upregulated after axotomy in this high regeneration condition. Armcx1 overexpression enhances mitochondrial transport in adult retinal ganglion cells (RGCs). Importantly, Armcx1 also promotes both neuronal survival and axon regeneration after injury, and these effects depend on its mitochondrial localization. Furthermore, Armcx1 knockdown undermines both neuronal survival and axon regeneration in the high regenerative capacity model, further supporting a key role of Armcx1 in regulating neuronal injury responses in the adult central nervous system (CNS). Our findings suggest that Armcx1 controls mitochondrial transport during neuronal repair., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Opalin, a central nervous system-specific myelin protein phylogenetically unique to mammals, has been suggested to play a role in mammalian-specific myelin. To elucidate the role of Opalin in mammalian myelin, we disrupted the Opalin gene in mice and analyzed the impacts on myelination and behavior. Opalin-knockout (Opalin-/-) mice were born at a Mendelian ratio and had a normal body shape and weight. Interestingly, Opalin-/- mice had no obvious abnormalities in major myelin protein compositions, expression of oligodendrocyte lineage markers, or domain organization of myelinated axons compared with WT mice (Opalin+/+) mice. Electron microscopic observation of the optic nerves did not reveal obvious differences between Opalin+/+ and Opalin-/- mice in terms of fine structures of paranodal loops, transverse bands, and multi-lamellae of myelinated axons. Moreover, sensory reflex, circadian rhythm, and locomotor activity in the home cage, as well as depression-like behavior, in the Opalin-/- mice were indistinguishable from the Opalin+/+ mice. Nevertheless, a subtle but significant impact on exploratory activity became apparent in Opalin-/- mice exposed to a novel environment. These results suggest that Opalin is not critical for central nervous system myelination or basic sensory and motor activities under conventional breeding conditions, although it might be required for fine-tuning of exploratory behavior., Competing Interests: The authors have declared that no competing interest exist.

Unlabelled: The impact of aging on CNS white matter (WM) is of general interest because the global effects of aging on myelinated nerve fibers are more complex and profound than those in cortical gray matter. It is important to distinguish between axonal changes created by normal aging and those caused by neurodegenerative diseases, including multiple sclerosis, stroke, glaucoma, Alzheimer's disease, and traumatic brain injury. Using three-dimensional electron microscopy, we show that in mouse optic nerve, which is a pure and fully myelinated WM tract, aging axons are larger, have thicker myelin, and are characterized by longer and thicker mitochondria, which are associated with altered levels of mitochondrial shaping proteins. These structural alterations in aging mitochondria correlate with lower ATP levels and increased generation of nitric oxide, protein nitration, and lipid peroxidation. Moreover, mitochondria-smooth endoplasmic reticulum interactions are compromised due to decreased associations and decreased levels of calnexin and calreticulin, suggesting a disruption in Ca(2+) homeostasis and defective unfolded protein responses in aging axons. Despite these age-related modifications, axon function is sustained in aging WM, which suggests that age-dependent changes do not lead to irreversible functional decline under normal conditions, as is observed in neurodegenerative diseases., Significance Statement: Aging is a common risk factor for a number of neurodegenerative diseases, including stroke. Mitochondrial dysfunction and oxidative damage with age are hypothesized to increase risk for stroke. We compared axon-myelin-node-mitochondrion-smooth endoplasmic reticulum (SER) interactions in white matter obtained at 1 and 12 months. We show that aging axons have enlarged volume, thicker myelin, and elongated and thicker mitochondria. Furthermore, there are reduced SER connections to mitochondria that correlate with lower calnexin and calreticulin levels. Despite a prominent decrease in number, elongated aging mitochondria produce excessive stress markers with reduced ATP production. Because axons maintain function under these conditions, our study suggests that it is important to understand the process of normal brain aging to identify neurodegenerative changes., (Copyright © 2016 the authors 0270-6474/16/369990-12$15.00/0.)

The aim of this paper is to study the morphology and the distribution of the monoamine oxidase enzymatic system in the optic nerve of 4 month-old Wistar (young) and 28 month-old Wistar (old) rats. The optic nerve was harvested from 20 young and old rats. The segment of optic nerve was divided longitudinally into two pieces, each 0.1 mm in length. The first piece was used for transmission electron microscopy. The second piece was stained with histochemical reaction for monoamine oxidase. The agerelated changes in the optic nerve of rats include micro-anatomical details, ultrastructure and monoamine oxidase histochemical staining. A strong decrease of the thin nerve fibers and a swelling of the thick ones can be observed in optic nerve fibers of old rats. Increased monoamine oxidase histochemical staining of the optic nerve of aged rats is well demonstrated. The increase of meningeal shealth and the decrease of thin nerve fibers of the optic nerve in old rats are well documented. Morphological, ultrastructural and histochemical changes observed in optic nerve fibers of the old rats show a close relation with aging.

Unlabelled: Myelin controls the time required for an action potential to travel from the neuronal soma to the axon terminal, defining the temporal manner in which information is processed within the CNS. The presence of myelin, the internodal length, and the thickness of the myelin sheath are powerful structural factors that control the velocity and fidelity of action potential transmission. Emerging evidence indicates that myelination is sensitive to environmental experience and neuronal activity. Activity-dependent modulation of myelination can dynamically alter action potential conduction properties but direct functional in vivo evidence and characterization of the underlying myelin changes is lacking. We demonstrate that in mice long-term monocular deprivation increases oligodendrogenesis in the retinogeniculate pathway but shortens myelin internode lengths without affecting other structural properties of myelinated fibers. We also demonstrate that genetically attenuating synaptic glutamate neurotransmission from retinal ganglion cells phenocopies the changes observed after monocular deprivation, suggesting that glutamate may constitute a signal for myelin length regulation. Importantly, we demonstrate that visual deprivation and shortened internodes are associated with a significant reduction in nerve conduction velocity in the optic nerve. Our results reveal the importance of sensory input in the building of myelinated fibers and suggest that this activity-dependent alteration of myelination is important for modifying the conductive properties of brain circuits in response to environmental experience., Significance Statement: Oligodendrocyte precursor cells differentiate into mature oligodendrocytes and are capable of ensheathing axons with myelin without molecular cues from neurons. However, this default myelination process can be modulated by changes in neuronal activity. Here, we show, for the first time, that experience-dependent activity modifies the length of myelin internodes along axons altering action potential conduction velocity. Such a mechanism would allow for variations in conduction velocities that provide a degree of plasticity in accordance to environmental needs. It will be important in future work to investigate how these changes in myelination and conduction velocity contribute to signal integration in postsynaptic neurons and circuit function., (Copyright © 2016 the authors 0270-6474/16/366937-12$15.00/0.)

The APP-PS1ΔE9 mouse model of Alzheimer's disease (AD) exhibits age dependent amyloid β (Aβ) plaque formation in their central nervous system due to high expression of mutated human APP and PSEN1 transgenes. Here we evaluated Aβ deposition and changes in soluble Aβ accumulation in the retinas of aged APP-PS1 mice using a combination of immunofluorescence, retinal flat mounts and western blotting techniques. Aβ accumulation in the retina has previously been shown to be associated with retinal ganglion cell apoptosis in animal models of glaucoma. This study investigated changes in the inner retinal function and structure in APP-PS1 mice using electrophysiology and histological approaches respectively. We report for the first time a significant decline in scotopic threshold response (STR) amplitudes which represents inner retinal function in transgenic animals compared to the wild type counterparts (p<0.0001). Thinning of the retina particularly involving inner retinal layers and reduction in axonal density in the optic nerve was also observed. TUNEL staining was performed to examine neuronal apoptosis in the inner retina. Intraocular pressure (IOP) measurements showed that APP-PS1ΔE9 mice had a slightly elevated IOP, but the significance of this finding is not yet known. Together, these results substantiate previous observations and highlight that APP-PS1ΔE9 mice show evidence of molecular, functional and morphological degenerative changes in the inner retina., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Background: Devic's neuromyelitis optica (NMO) is an autoimmune astrocytopathy, associated with central nervous system inflammation, demyelination, and neuronal injury. Several studies confirmed that autoantibodies directed against aquaporin-4 (AQP4-IgG) are relevant in the pathogenesis of NMO, mainly through complement-dependent toxicity leading to astrocyte death. However, the effect of the autoantibody per se and the exact role of intrathecal AQP4-IgG are still controversial., Methods: To explore the intrinsic effect of intrathecal AQP4-IgG, independent from additional inflammatory effector mechanisms, and to evaluate its clinical impact, we developed a new animal model, based on a prolonged infusion of purified immunoglobulins from NMO patient (IgG(AQP4+), NMO-rat) and healthy individual as control (Control-rat) in the cerebrospinal fluid (CSF) of live rats., Results: We showed that CSF infusion of purified immunoglobulins led to diffusion in the brain, spinal cord, and optic nerves, the targeted structures in NMO. This was associated with astrocyte alteration in NMO-rats characterized by loss of aquaporin-4 expression in the spinal cord and the optic nerves compared to the Control-rats (p = 0.001 and p = 0.02, respectively). In addition, glutamate uptake tested on vigil rats was dramatically reduced in NMO-rats (p = 0.001) suggesting that astrocytopathy occurred in response to AQP4-IgG diffusion. In parallel, myelin was altered, as shown by the decrease of myelin basic protein staining by up to 46 and 22 % in the gray and white matter of the NMO-rats spinal cord, respectively (p = 0.03). Loss of neurofilament positive axons in NMO-rats (p = 0.003) revealed alteration of axonal integrity. Then, we investigated the clinical consequences of such alterations on the motor behavior of the NMO-rats. In a rotarod test, NMO-rats performance was lower compared to the controls (p = 0.0182). AQP4 expression, and myelin and axonal integrity were preserved in AQP4-IgG-depleted condition. We did not find a major immune cell infiltration and microglial activation nor complement deposition in the central nervous system, in our model., Conclusions: We establish a link between motor-deficit, NMO-like lesions and astrocytopathy mediated by intrathecal AQP4-IgG. Our study validates the concept of the intrinsic effect of autoantibody against surface antigens and offers a model for testing antibody and astrocyte-targeted therapies in NMO.

Myelinated axons efficiently transmit information over long distances. The apposed myelin sheath confers favorable electrical properties, but restricts access of the axon to its extracellular milieu. Therefore, axonal metabolic support may require specific axo-myelinic communication. Here we explored activity-dependent glutamate-mediated signaling from axon to myelin. 2-Photon microscopy was used to image Ca(2+) changes in myelin in response to electrical stimulation of optic nerve axons ex vivo. We show that optic nerve myelin responds to axonal action potentials by a rise in Ca(2+) levels mediated by GluN2D and GluN3A-containing NMDA receptors. Glutamate is released from axons in a vesicular manner that is tetanus toxin-sensitive. The Ca(2+) source for vesicular fusion is provided by ryanodine receptors on axonal Ca(2+) stores, controlled by L-type Ca(2+) channels that sense depolarization of the internodal axolemma. Genetic ablation of GluN2D and GluN3A subunits results in greater lability of the compact myelin. Our results support the existence of a novel synapse between the axon and its myelin, suggesting a means by which traversing action potentials can signal the overlying myelin sheath. This may be an important physiological mechanism by which an axon can signal companion glia for metabolic support or adjust properties of its myelin in a dynamic manner. The axo-myelinic synapse may contribute to learning, while its disturbances may play a role in the pathophysiology of central nervous system disorders such as schizophrenia, where subtle abnormalities of myelinated white matter tracts have been shown in the human, or to frank demyelinating disorders such as multiple sclerosis., (Copyright © 2015 Elsevier Inc. All rights reserved.)

We have used X-ray phase contrast tomography to resolve the structure of uncut, entire myelinated optic, saphenous and sciatic mouse nerves. Intrinsic electron density contrast suffices to identify axonal structures. Specific myelin labeling by an osmium tetroxide stain enables distinction between axon and surrounding myelin sheath. Utilization of spherical wave illumination enables zooming capabilities which enable imaging of entire sciatic internodes as well as identification of sub-structures such as nodes of Ranvier and Schmidt-Lanterman incisures., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Purpose: Autophagy is a critical process, compromised in neurodegenerative disease, by which terminally differentiated cells like neurons manage cytoskeletal and organelle turnover. How autophagy relates to associated neurodegenerative pathologies remain unclear. We examined autophagy in optic neuropathy by investigating cytoskeletal degradation, mitochondria, and autophagic vesicles in the DBA2/J mouse model of glaucoma exhibiting differing levels of axon transport functionality., Methods: DBA/2J and DBA/2J(wt-gpnmb) control mice 11 to 14 months of age were injected with cholera toxin-B (CTB) to assay anterograde axonal transport. Axonal mitochondria and autophagic vesicles were analyzed with respect to transport integrity in proximal and distal optic nerve using serial block face scanning electron microscopy (3D EM)., Results: Several indices varied significantly between the DBA/2J and DBA/2J(wt-gpnmb) mice, including mitochondrial volume, average number of autophagic vesicles per axon, and mitochondrial cristae. However, there were no differences in mitochondrial cristae for axons with functional versus dysfunctional CTB transport, suggesting that mitochondrial dysfunction precedes overt transport blockade. Anterograde transport failure was accompanied by a dissociation of the relationship between mitochondrial and axon volumes. Autophagic vesicle profiles were significantly increased in optic nerve with transport deficit, consistent with greater autophagic activity. Mitochondria within autophagosomes, indicative of mitophagy, were observed in both proximal and distal axons., Conclusions: Loss of anterograde transport in DBA/2J optic nerve is concomitant with diminished mitochondrial volume, increased cytoskeletal breakdown and autophagic activity, and accumulation of autophagic profiles, including signs of mitophagy, in proximal optic nerve. Axons with transport deficit are metabolically underserved, though not necessarily from mitophagy.

Objective: To evaluate morphological parameters of optic disc and retinal nerve fiber layer (RNFL) examined with confocal laser tomography (HRT3) and laser polarimetry (GDx-VCC) in a normal population, and analyze correlations of these parameters with demographic variables., Patients and Methods: Cross-sectional study in the context of a glaucoma screening campaign in the primary care center of Barcelona. The individuals selected were non-hypertensive Mediterranean Caucasians with risk for glaucoma development (individuals≥60 years old or≥40 years old with family history of glaucoma or intraocular pressure or myopia>3diopter). All subjects underwent a complete ophthalmic examination, confocal laser tomography (HRT3) and scanning laser polarimetry (GDX-VCC), subjects with results within normal limits only being included. Structural parameters were analyzed along with age, refraction, and pachymetry based on the Spearman rank correlation test., Results: A total of 224 subjects included, with a mean age of 63.4±11.1 years. Disc areas, excavation and ring area were 2.14±0.52mm(2), 0.44±0.34mm (2) and 1.69±0.38mm(2), respectively. The mean RNFL (GDX) was 55.9±6.9μm. Age was correlated with lower ring volume, highest rate of cup shape measure, largest mean and maximum cup depth, lower nerve fiber index (NFI) and RNFL (all p-values below .05)., Conclusion: The mean values and distribution of several parameters of the papilla and the RNFL in normal Mediterranean Caucasians population are presented. A loss of thickness of the RNFL, ring thinning, and enlarged cup was observed with increased age., (Copyright © 2014 Sociedad Española de Oftalmología. Published by Elsevier España, S.L.U. All rights reserved.)

Objective: To evaluate the morphology of optic nerve and retina in the model of the rat non-arteritic anterior ischemic optic neuropathy (rNAION)., Methods: Experimental study. Thirty-six SD rats were randomly divided into four groups: 6 for naïve group, 3 for laser group, 3 for Rose Bengal (RB) group and 24 for rNAION group. After rNAION was induced by RB and laser, the retina and optic nerve were observed by Hematoxylin-Eosin (HE) staining and the optic nerve was investigated by transmission electron microscope and toludidine blue staining at different time points., Results: With these methods, significant changes were only found in the RB-laser induced eyes. The damage only occurred in the retinal ganglion cell (RGC) layer without any loss of the other layers of retina in the rNAION. Toludine blue-stained optic nerve showed that there was a loss of axons centrally and largely intact axons were spared in the peripheral nerve. More and more degenerative axons were observed with the transmission electron microscope as time went on., Conclusions: The morphological changes of HE staining, toludine blue staining and transmission electron microscopic finding were consistent with each other. The results made a contribution to further study.

Purpose: We sought to assess the effect of two different internal limiting membrane [ILM] peeling techniques carried out during surgery for idiopathic macular holes on the postoperative extent of a dissociated optic nerve fibre layer appearance [DONFL]., Methods: We collected prospective data of surgical records, videos, and pre- and postoperative imaging of a consecutive series of patients undergoing surgery for idiopathic macular hole with one of two surgeons. One surgeon used a forceps pinch-peel technique to peel the ILM, whereas the other surgeon used a diamond dusted membrane scraper. The extent of any DONFL was measured using spectral domain optical coherence tomography and blue reflectance imaging at three months postoperatively. A proportion of the ILMs removed were examined with transmission electron microscopy., Results: Fifty-seven patients were studied, with 41 in the forceps group and 16 in the scraper group. The groups were well matched, with no significant difference in any preoperative parameters. Some degree of DONFL was observed on the 3-month blue reflectance images in 88 % of the forceps group and 100 % of the scraper group [p = 0.14]. There was a significant difference in the total number of depressions in the nerve fibre layer typical of DONFL on OCT between the two groups [p = 0.001], and general regression analysis showed that the peeling technique used had the only significant association with the degree of DONFL observed. Electron microscopy showed large patches of cellular debris on the retinal side of the peeled ILM in 3 out of 4 cases in the scraper group and 1 out of 12 cases in the forceps group., Conclusion: ILM peeling technique and possibly other surgeon-specific factors appear to influence the extent of DONFL observed after ILM peeling macular hole surgery.

If "the connectome" represents a complete map of anatomical and functional connectivity in the brain, it should also include glia. Glia define and regulate both the brain's anatomical and functional connectivity over a broad range of length scales, spanning the whole brain to subcellular domains of synaptic interactions. This Perspective article examines glial interactions with the neuronal connectome (including long-range networks, local circuits, and individual synaptic connections) and highlights opportunities for future research. Our understanding of the structure and function of the neuronal connectome would be incomplete without an understanding of how all types of glia contribute to neuronal connectivity and function, from single synapses to circuits., (Copyright © 2015 Elsevier Inc. All rights reserved.)

We present the histopathological findings of a naturally mummified eye from the Peruvian Lambayeque culture (900-1,200 AD), in which rehydration, light microscopy, and scanning electron microscopy allowed a detailed analysis of several eye tissues including the eyelids, sclera, and optic nerve, the latter showing evidence of hemorrhage likely related to the documented strangulation as the cause of death. We conclude that histopathological analysis of rehydrated mummified tissues can provide valuable information from fragile eye structures including the optic nerve, and these findings can be useful from a forensic point of view.

Several psychiatric disorders are associated with aberrant white matter development, suggesting oligodendrocyte and myelin dysfunction in these diseases. There are indications that radial glial cells (RGCs) are involved in initiating myelination, and may contribute to the production of oligodendrocyte progenitor cells (OPCs) in the dorsal cortex. Liver X receptors (LXRs) are involved in maintaining normal myelin in the central nervous system (CNS), however, their function in oligodendrogenesis and myelination is not well understood. Here, we demonstrate that loss of LXRβ function leads to abnormality in locomotor activity and exploratory behavior, signs of anxiety and hypomyelination in the corpus callosum and optic nerve, providing in vivo evidence that LXRβ deletion delays both oligodendrocyte differentiation and maturation. Remarkably, along the germinal ventricular zone-subventricular zone and corpus callosum there is reduced OPC production from RGCs in LXRβ(-/-) mice. Conversely, in cultured RGC an LXR agonist led to increased differentiation into OPCs. Collectively, these results suggest that LXRβ, by driving RGCs to become OPCs in the dorsal cortex, is critical for white matter development and CNS myelination, and point to the involvement of LXRβ in psychiatric disorders.

Introduction: In the human demyelinating central nervous system (CNS) disease multiple sclerosis, remyelination promotes recovery and limits neurodegeneration, but this is inefficient and always ultimately fails. Furthermore, these regenerated myelin sheaths are thinner and shorter than the original, leaving the underlying axons potentially vulnerable. In rodent models, CNS remyelination is more efficient, so that in young animals (but not old) the number of myelinated axons is efficiently restored to normal, but in both young and old rodents, regenerated myelin sheaths are still short and thin. The reasons for these differences in remyelination efficiency, the thinner remyelinated myelin sheaths compared to developmental myelin and the subsequent effect on the underlying axon are unclear. We studied CNS remyelination in the highly regenerative adult zebrafish (Danio rerio), to better understand mechanisms of what we hypothesised would be highly efficient remyelination, and to identify differences to mammalian CNS remyelination, as larval zebrafish are increasingly used for high throughput screens to identify potential drug targets to improve myelination and remyelination., Results: We developed a novel method to induce a focal demyelinating lesion in adult zebrafish optic nerve with no discernible axonal damage, and describe the cellular changes over time. Remyelination is indeed efficient in both young and old adult zebrafish optic nerves, and at 4 weeks after demyelination, the number of myelinated axons is restored to normal, but internode lengths are short. However, unlike in rodents or in humans, in young zebrafish these regenerated myelin sheaths were of normal thickness, whereas in aged zebrafish, they were thin, and remained so even 3 months later. This inability to restore normal myelin thickness in remyelination with age was associated with a reduced macrophage/microglial response., Conclusion: Zebrafish are able to efficiently restore normal thickness myelin around optic nerve axons after demyelination, unlike in mammals. However, this fails with age, when only thin myelin is achieved. This gives us a novel model to try and dissect the mechanism for restoring myelin thickness in CNS remyelination.

The optic nerve is a unique part of the central nervous system. It lacks neuronal cell bodies and consists of axons of the retinal ganglion cells together with the supporting neuroglial cells. In the present study, aging of the optic nerve was studied in female Sprague-Dawley rats aged 3, 12, 24 and 30 months old, ultrastructurally, immunohistochemically and morphometrically trying to answer the question why aging is a common risk factor for many ocular diseases especially glaucoma. Additionally, studying the optic nerve aging offered a good opportunity to gain further insight into the effects of aging on white matter. Both nerve fibers and neuroglial cells demonstrated several age related changes which were more profound in 30 months old rats. Optic nerve axons displayed watery degeneration and dark degeneration. Myelin disturbances including widening, whorls, splitting and vacuolations of the myelin lamellae were also observed. Neuroglial cells appeared to be more frequent than in younger rats especially microglia cells and developed dense cytoplasmic inclusions. GFAP-positive astrocytes delineated age-related progressive increase in number, size as well as length and thickness of their processes. CD68 immunohistochemical staining revealed age-related changes in the morphology, location and number of CD68 positive microglia cells., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Introduction: The neuronal ceroid lipofuscinoses constitute a group of fatal inherited lysosomal storage diseases that manifest in profound neurodegeneration in the CNS. Visual impairment usually is an early symptom and selective degeneration of retinal neurons has been described in patients suffering from distinct disease subtypes. We have previously demonstrated that palmitoyl protein thioesterase 1 deficient (Ppt1-/-) mice, a model of the infantile disease subtype, exhibit progressive axonal degeneration in the optic nerve and loss of retinal ganglion cells, faithfully reflecting disease severity in the CNS. Here we performed spectral domain optical coherence tomography (OCT) in Ppt1-/- and ceroid lipofuscinosis neuronal 3 deficient (Cln3-/-) mice, which are models of infantile and juvenile neuronal ceroid lipofuscinosis, respectively, in order to establish a non-invasive method to assess retinal alterations and monitor disease severity in vivo., Results: Blue laser autofluorescence imaging revealed increased accumulation of autofluorescent storage material in the inner retinae of 7-month-old Ppt1-/- and of 16-month-old Cln3-/- mice in comparison with age-matched control littermates. Additionally, optical coherence tomography demonstrated reduced thickness of retinae in knockout mice in comparison with age-matched control littermates. High resolution scans and manual measurements allowed for separation of different retinal composite layers and revealed a thinning of layers in the inner retinae of both mouse models at distinct ages. OCT measurements correlated well with subsequent histological analysis of the same retinae., Conclusions: These results demonstrate the feasibility of OCT to assess neurodegenerative disease severity in mouse models of neuronal ceroid lipofuscinosis and might have important implications for diagnostic evaluation of disease progression and therapeutic efficacy in patients. Moreover, the non-invasive method allows for longitudinal studies in experimental models, reducing the number of animals used for research.