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The mitogen-activated protein kinase (MAPK) pathway has been shown to be involved in both neurodevelopment and neurodegeneration. c-Jun N-terminal kinase (JNK), a MAPK important in retinal development and after optic nerve crush injury, is regulated by two upstream kinases: MKK4 and MKK7. The specific requirements of MKK4 and MKK7 in retinal development and retinal ganglion cell (RGC) death after axonal injury, however, are currently undefined. Optic nerve injury is an important insult in many neurologic conditions including traumatic, ischemic, inflammatory, and glaucomatous optic neuropathies. Mice deficient in Mkk4, Mkk7, and both Mkk4 and Mkk7 were generated. Immunohistochemistry was used to study the distribution and structure of retinal cell types and to assess RGC survival after optic nerve injury (mechanical controlled optic nerve crush (CONC)). Adult Mkk4- and Mkk7-deficient retinas had all retinal cell types, and with the exception of small areas of disrupted photoreceptor lamination in Mkk4-deficient mice, the retinas of both mutants were grossly normal. Deficiency of Mkk4 or Mkk7 reduced JNK signaling in RGCs after axonal injury and resulted in a significantly greater percentage of surviving RGCs 35 days after CONC as compared to wild-type controls (Mkk4: 51.5%, Mkk7: 29.1%, WT: 15.2%; p < 0.001). Combined deficiency of Mkk4 and Mkk7 caused failure of optic nerve formation, irregular retinal axonal trajectories, disruption of retinal lamination, clumping of RGC bodies, and dendritic fasciculation of dopaminergic amacrine cells. These results suggest that MKK4 and MKK7 may serve redundant and unique roles in molecular signaling important for retinal development and injury response following axonal insult.

Retinal ganglion cell axons of the DBA/2J mouse model of glaucoma, a model characterized by extensive neuroinflammation, preserve synaptic contacts with their subcortical targets for a time after onset of anterograde axonal transport deficits, axon terminal hypertrophy, and cytoskeletal alterations. Though retrograde axonal transport is still evident in these axons, it is unknown if they retain their ability to transmit visual information to the brain. Using a combination of in vivo multiunit electrophysiology, neuronal tract tracing, multichannel immunofluorescence, and transmission electron microscopy, we report that eye-brain signaling deficits precede transport loss and axonal degeneration in the DBA/2J retinal projection. These deficits are accompanied by node of Ranvier pathology - consisting of increased node length and redistribution of the voltage-gated sodium channel Na v 1.6 that parallel changes seen early in multiple sclerosis (MS) axonopathy. Further, with age, axon caliber and neurofilament density increase without corresponding changes in myelin thickness. In contrast to these findings in DBA/2J mice, node pathologies were not observed in the induced microbead occlusion model of glaucoma - a model that lacks pre-existing inflammation. After one week of systemic treatment with fingolimod, an immunosuppressant therapy for relapsing-remitting MS, DBA/2J mice showed a substantial reduction in node pathology and mild effects on axon morphology. These data suggest that neurophysiological deficits in the DBA/2J may be due to defects in intact axons and targeting node pathology may be a promising intervention for some types of glaucoma., (Copyright © 2018. Published by Elsevier Ltd.)

Purpose: Amyloid beta (Aβ) is known to contribute to the pathophysiology of retinal neurodegenerative diseases such as glaucoma. Effects of intravitreal Aβ(1-42) on retinal and optic nerve morphology in animal models have widely been studied but not those of Aβ(1-40). Hence, we evaluated the time- and dose-related effects of intravitreal Aβ(1-40) on retinal and optic nerve morphology. Since oxidative stress and brain derived neurotrophic factor (BDNF) are associated with Aβ-induced neuronal damage, we also studied dose and time-related effects of Aβ(1-40) on retinal oxidative stress and BDNF levels., Materials and Methods: Five groups of rats were intravitreally administered with vehicle or Aβ(1-40) in doses of 1.0, 2.5, 5 and 10 nmol. Animals were sacrificed and eyes were enucleated at weeks 1, 2 and 4 post-injection. The retinae were subjected to morphometric analysis and TUNEL staining. Optic nerve sections were stained with toluidine blue and were graded for neurodegenerative effects. The estimation of BDNF and markers of oxidative stress in retina were done using ELISA technique., Results and Conclusions: It was observed that intravitreal Aβ(1-40) causes significant retinal and optic nerve damage up to day 14 post-injection and there was increasing damage with increase in dose. However, on day 30 post-injection both the retinal and optic nerve morphology showed a trend towards normalization. The observations made for retinal cell apoptosis, retinal glutathione, superoxide dismutase activity and BDNF were in accordance with those of morphological changes with deterioration till day 14 and recovery by day 30 post-injection. The findings of this study may provide a guide for selection of appropriate experimental conditions for future studies.

Ophthalmology and visual health are new frontiers for postgenomic research and technologies such as proteomics. In this context, the optic nerve and retina extend as the outgrowth of the brain, wherein the latter receives the optical input and the former relays the information for processing. While efforts to understand the optic nerve proteome have been made earlier, there exists a lacuna in its biochemical composition and molecular functions. We report, in this study, a high-resolution mass spectrometry-based approach using an Orbitrap Fusion Tribrid mass spectrometer to elucidate the human optic nerve proteomic profile. Raw spectra were searched against NCBI Human RefSeq 75 database using SEQUEST HT and MASCOT algorithms. We identified nearly 35,000 peptides in human optic nerve samples, corresponding to 5682 proteins, of which 3222 proteins are being reported for the first time. Label-free quantification using spectral abundance pointed out to neuronal structural proteins such as myelin basic protein, glial fibrillary acidic protein, and proteolipid protein 1 as the most abundant proteins. We also identified several neurotransmitter receptors and postsynaptic density synaptosomal scaffold proteins. Pathway analysis revealed that a majority of the proteins are structural proteins and have catalytic and binding activity. This study is one of the largest proteomic profiles of the human optic nerve and offers the research community an initial baseline optic nerve proteome for further studies. This will also help understand the protein dynamics of the human optic nerve under normal conditions.

Objective: This study aimed to investigate iron deposition and thickness and signal changes in optic radiation (OR) by enhanced T 2 * -weighted angiography imaging (ESWAN) in patients with relapsing-remitting multiple sclerosis (RRMS) with unilateral and bilateral lesions or no lesions., Methods: Fifty-one RRMS patients (42 patients with a disease duration [DD] ≥ 2 years [group Mor], nine patients with a DD < 2 years [group Les]) and 51 healthy controls (group Con) underwent conventional magnetic resonance imaging (MRI) and ESWAN at 3.0 T. The mean phase value (MPV) of the OR was measured on the phase image, and thickness and signal changes of the OR were observed on the magnitude image., Results: The average MPVs for the OR were 1,981.55 ± 7.75 in group Mor, 1,998.45 ± 2.01 in group Les, and 2,000.48 ± 5.53 in group Con. In group Mor, 28 patients with bilateral OR lesions showed bilateral OR thinning with a heterogeneous signal, and 14 patients with unilateral OR lesions showed ipsilateral OR thinning with a heterogeneous signal. In the remaining nine patients without OR lesions and in group Con, the bilateral OR had a normal appearance. In the patients, a negative correlation was found between DD and OR thickness and a positive correlation was found between MPV and OR thickness., Conclusions: We confirmed iron deposition in the OR in the RRMS patients, and the OR thickness was lower in the patients than in the controls., Key Points: • Enhanced T 2 * -weighted magnetic resonance angiography (ESWAN) provides new insights into multiple sclerosis (MS). • Focal destruction of the optic radiation (OR) is detectable by ESWAN. • Iron deposition in OR can be measured on ESWAN phase image in MS patients. • OR thickness was lower in the patients than in the controls. • Iron deposition and thickness changes of the OR are associated with disease duration.

Repeated spike firing can transmit information at synapses and modulate spike timing, shape, and conduction velocity. These latter effects have been found to result from voltage-induced changes in ion currents and could alter the signals carried by axons. Here, we test whether Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) regulates spike propagation in adult rat optic nerve. We find that small-, medium-, and large-diameter axons bind anti-Thr286-phosphorylated CaMKII (pT286) antibodies and that, in isolated optic nerves, electrical stimulation reduces pT286 levels, spike propagation is hastened by CaMKII autophosphorylation and slowed by CaMKII dephosphorylation, single and multiple spikes slow propagation of subsequently activated spikes, and more frequent stimulation produces greater slowing. Likewise, exposing freely moving animals to flickering illumination reduces pT286 levels in optic nerves and electrically eliciting spikes in vivo in either the optic nerve or optic chiasm slows subsequent spike propagation in the optic nerve. By increasing the time that elapses between successive spikes as they propagate, pT286 dephosphorylation and activity-induced spike slowing reduce the frequency of propagated spikes below the frequency at which they were elicited and would thus limit the frequency at which axons synaptically drive target neurons. Consistent with this, the ability of retinal ganglion cells to drive at least some lateral geniculate neurons has been found to increase when presented with light flashes at low and moderate temporal frequencies but less so at high frequencies. Activity-induced decreases in spike frequency may also reduce the energy required to maintain normal intracellular Na + and Ca 2+ levels. SIGNIFICANCE STATEMENT By propagating along axons at constant velocities, spikes could drive synapses as frequently as they are initiated. However, the onset of spiking has been found to alter the conduction velocity of subsequent ("follower") spikes in various preparations. Here, we find that spikes reduce spike frequency in rat optic nerve by slowing follower spike propagation and that electrically stimulated spiking ex vivo and spike-generating flickering illumination in vivo produce net decreases in axonal Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) autophosphorylation. Consistent with these effects, propagation speed increases and decreases, respectively, with CaMKII autophosphorylation and dephosphorylation. Lowering spike frequency by CaMKII dephosphorylation is a novel consequence of axonal spiking and light adaptation that could decrease synaptic gain as stimulus frequency increases and may also reduce energy use., (Copyright © 2018 the authors 0270-6474/18/388087-19$15.00/0.)

RNA-binding proteins Lin28a/b regulate cellular growth and tissue regeneration. Here, we investigated the role of Lin28 in the control of axon regeneration in postmitotic neurons. We find that Lin28a/b are both necessary and sufficient for supporting axon regeneration in mature sensory neurons through their regulatory partners, let-7 microRNAs (miRNAs). More importantly, overexpression of Lin28a in mature retinal ganglion cells (RGCs) produces robust and sustained optic nerve regeneration. Additionally, combined overexpression of Lin28a and downregulation of Pten in RGCs act additively to promote optic nerve regeneration, potentially by reducing the backward turning of regenerating RGC axons. Our findings not only reveal a vital role of Lin28 signaling in regulating mammalian axon regeneration but also identify a signaling pathway that can promote axon regeneration in the central nervous system (CNS)., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

In a rat ex vivo acute glaucoma model, high pressure (75 mmHg) causes swelling of ganglion cell axons and elevates levels of the endogenous steroids 24(S)-hydroxycholesterol (24SH) and allopregnanolone (AlloP). Furthermore, 24SH (0.1 µM) alone elevates AlloP levels via NMDA receptors. With this model, we now investigate possible interactions between 24SH and AlloP. We found that inhibition of AlloP synthesis with dutasteride under high pressure results in severe excitotoxicity in addition to axonal swelling. The excitotoxicity is prevented by exogenous AlloP but not 24SH, indicating that endogenous AlloP is crucial for protection. However, inhibition of 24SH synthesis with voriconazole induces severe excitotoxicity under normal pressure. Paradoxically, the excitotoxicity by voriconazole is better prevented by AlloP than 24SH. These findings suggest that inhibition of 24SH synthesis becomes excitotoxic in the absence of AlloP. We also observed that co-administration of sub-micromolar 24SH (0.1 µM) and AlloP (0.1 µM), concentrations that are only partially effective when administered alone, prevents axonal swelling under high pressure. This apparent enhanced protection indicates strong interaction between the two neurosteroids to preserve neuronal integrity, with 24SH contributing to AlloP synthesis via NMDA receptors and with AlloP playing an essential role in neuroprotection via GABA A receptors.

Daily oscillations of brain and body states are under complex temporal modulation by environmental light and the hypothalamic suprachiasmatic nucleus (SCN), the master circadian clock. To better understand mediators of differential temporal modulation, we characterize neuropeptide releasate profiles by nonselective capture of secreted neuropeptides in an optic nerve horizontal SCN brain slice model. Releasates are collected following electrophysiological stimulation of the optic nerve/retinohypothalamic tract under conditions that alter the phase of the SCN activity state. Secreted neuropeptides are identified by intact mass via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). We found time-of-day-specific suites of peptides released downstream of optic nerve stimulation. Peptide release was modified differentially with respect to time-of-day by stimulus parameters and by inhibitors of glutamatergic or PACAPergic neurotransmission. The results suggest that SCN physiology is modulated by differential peptide release of both known and unexpected peptides that communicate time-of-day-specific photic signals via previously unreported neuropeptide signatures.

Tissues of the central nervous system (CNS), including the optic nerve (ON), are considered a-lymphatic. However, lymphatic structures have been described in the dura mater of human ON sheaths. Since it is known that lymphatic markers are also expressed by single non-lymphatic cells, these results need confirmation according to the consensus statement for the use of lymphatic markers in ophthalmologic research. The aim of this study was to screen for the presence of lymphatic structures in the adult human ON using a combination of four lymphatic markers. Cross and longitudinal cryo-sections of human optic nerve tissue (n = 12, male and female, postmortem time = 15.8 ± 5.5 h, age = 66.5 ± 13.8 years), were obtained from cornea donors of the Salzburg eye bank, and analyzed using immunofluorescence with the following markers: FOXC2, CCL21, LYVE-1 and podoplanin (PDPN; lymphatic markers), Iba1 (microglia), CD68 (macrophages), CD31 (endothelial cell, EC), NF200 (neurofilament), as well as GFAP (astrocytes). Human skin sections served as positive controls and confocal microscopy in single optical section mode was used for documentation. In human skin, lymphatic structures were detected, showing a co-localization of LYVE-1/PDPN/FOXC2 and CCL21/LYVE-1. In the human ON however, single LYVE-1+ cells were detected, but were not co-localized with any other lymphatic marker tested. Instead, LYVE-1+ cells displayed immunopositivity for Iba1 and CD68, being more pronounced in the periphery of the ON than in the central region. However, Iba1+/LYVE-1- cells outnumbered Iba1+/LYVE-1+ cells. PDPN, revealed faint labeling in human ON tissue despite strong immunoreactivity in rat ON controls, showing co-localization with GFAP in the periphery. In addition, pronounced autofluorescent dots were detected in the ON, showing inter-individual differences in numbers. In the adult human ON no lymphatic structures were detected, although distinct lymphatic structures were identified in human skin tissue by co-localization of four lymphatic markers. However, single LYVE-1+ cells, also positive for Iba1 and CD68 were present, indicating LYVE-1+ macrophages. Inter-individual differences in the number of LYVE-1+ as well as Iba1+ cells were obvious within the ONs, most likely resulting from diverse medical histories of the donors., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Using mice expressing green fluorescent protein (GFP) from a transgenic CD11c promoter we found that a controlled optic nerve crush (ONC) injury attracted GFP hi retinal myeloid cells to the dying retinal ganglion cells and their axons. However, the origin of these retinal myeloid cells was uncertain. In this study we use transgenic mice in conjunction with ONC, partial and full optic nerve transection (ONT), and parabiosis to determine the origin of injury induced retinal myeloid cells. Analysis of parabiotic mice and fate mapping showed that responding retinal myeloid cells were not derived from circulating macrophages and that GFP hi myeloid cells could be derived from GFP lo microglia. Comparison of optic nerve to retina following an ONC showed a much greater concentration of GFP hi cells and GFP lo microglia in the optic nerve. Optic nerve injury also induced Ki67 + cells in the optic nerve but not in the retina. Comparison of the retinal myeloid cell response after full versus partial ONT revealed fewer GFP hi cells and GFP lo microglia in the retina following a full ONT despite it being a more severe injury, suggesting that full transection of the optic nerve can block the migration of responding myeloid cells to the retina. Our results suggest that the optic nerve can be a reservoir for activated microglia and other retinal myeloid cells in the retina following optic nerve injury.

Optic nerve injury is a leading cause of irreversible visual impairment worldwide and can even cause blindness. Excessive activation of astrocytes has negative effects on the repair and recovery of retinal ganglion cells following optic nerve injury. However, the molecular and cellular mechanisms underlying astrocyte activation after optic nerve injury remain largely unknown. In the present study, we explored the effects of microRNA-21 (miR-21) on axon regeneration and flash visual evoked potential (F-VEP) and the underlying mechanisms of these effects based on astrocyte activation in the rat model of optic nerve crush (ONC). To the best of our knowledge, this article is the first to report that inhibition of miR-21 enhances axonal regeneration and promotes functional recovery in F-VEP in the rat model of ONC. Furthermore, inhibition of miR-21 attenuates excessive astrocyte activation and glial scar formation, thereby promoting axonal regeneration by regulating the epidermal growth factor receptor (EGFR) pathway. In addition, we observed that the expression of tissue inhibitor of metalloproteinase-3, a target gene of miR-21, was inhibited during this process. Taken together, these findings demonstrate that inhibition of miR-21 regulates the EGFR pathway, ameliorating excessive astrocyte activation and glial scar progression and promoting axonal regeneration and alleviating impairment in F-VEP function in a model of ONC. This study's results suggest that miR-21 may represent a therapeutic target for optic nerve injury., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

In glaucoma, latest studies revealed an involvement of the complement system with and without an elevated intraocular pressure. In the experimental autoimmune glaucoma model, immunization with antigens, such as S100B, lead to retinal ganglion cell (RGC) loss and optic nerve degeneration after 28 days. Here, we investigated the timeline of progression of the complement system, toll-like-receptor 4 (TLR4), and the transcription factor nucleus factor-kappa B (NFκB). Therefore, rats were immunized with S100B protein (S100) and analyzed at 3, 7, and 14 days. RGC numbers were comparable at all points in time, whereas a destruction of S100 optic nerves was noted at 14 days. A significant increase of mannose binding lectin (MBL) was observed in S100 retinas at 3 days. Subsequently, significantly more MBL + cells were seen in S100 optic nerves at 7 and 14 days. Accordingly, C3 was upregulated in S100 retinas at 14 days. An increase of interleukin-1 beta was noted in S100 aqueous humor samples at 7 days. In this study, activation of complement system via the lectin pathway was obvious. However, no TLR4 alterations were noted in S100 retinas and optic nerves. Interestingly, a significant NFκB increase was observed in S100 retinas at 7 and 14 days. We assume that NFκB activation might be triggered via MBL leading to glaucomatous damage.

Background: The oral immunomodulatory agent laquinimod is currently evaluated for multiple sclerosis (MS) treatment. Phase II and III studies demonstrated a reduction of degenerative processes. In addition to anti-inflammatory effects, laquinimod might have neuroprotective properties, but its impact on the visual system, which is often affected by MS, is unknown. The aim of our study was to investigate potential protective effects of laquinimod on the optic nerve and retina in an experimental autoimmune encephalomyelitis (EAE) model., Methods: We induced EAE in C57/BL6 mice via MOG 35-55 immunization. Animals were divided into an untreated EAE group, three EAE groups receiving laquinimod (1, 5, or 25 mg/kg daily), starting the day post-immunization, and a non-immunized control group. Thirty days post-immunization, scotopic electroretinograms were carried out, and mice were sacrificed for histopathology (HE, LFB), immunohistochemistry (MBP, Iba1, Tmem119, F4/80, GFAP, vimentin, Brn-3a, cleaved caspase 3) of the optic nerve and retina, and retinal qRT-PCR analyses (Brn-3a, Iba1, Tmem119, AMWAP, CD68, GFAP). To evaluate the effect of a therapeutic approach, EAE animals were treated with 25 mg/kg laquinimod from day 16 when 60% of the animals had developed clinical signs of EAE., Results: Laquinimod reduced neurological EAE symptoms and improved the neuronal electrical output of the inner nuclear layer compared to untreated EAE mice. Furthermore, cellular infiltration, especially recruited phagocytes, and demyelination in the optic nerve were reduced. Microglia were diminished in optic nerve and retina. Retinal macroglial signal was reduced under treatment, whereas in the optic nerve macroglia were not affected. Additionally, laquinimod preserved retinal ganglion cells and reduced apoptosis. A later treatment with laquinimod in a therapeutic approach led to a reduction of clinical signs and to an improved b-wave amplitude. However, no changes in cellular infiltration and demyelination of the optic nerves were observed. Also, the number of retinal ganglion cells remained unaltered., Conclusion: From our study, we deduce neuroprotective and anti-inflammatory effects of laquinimod on the optic nerve and retina in EAE mice, when animals were treated before any clinical signs were noted. Given the fact that the visual system is frequently affected by MS, the agent might be an interesting subject of further neuro-ophthalmic investigations.

BackgroundPelizaeus Merzbacher disease (PMD) is a dysmyelinating disorder of the central nervous system caused by impaired differentiation of oligodendrocytes. This study was prompted by findings that antimuscarinic compounds enhance oligodendrocyte differentiation and remyelination in vitro. One of these compounds, clemastine fumarate, is licensed for treatment of allergic conditions. We tested whether clemastine fumarate can promote myelination in two rodent PMD models, the myelin-deficient and the PLP transgenic rat.MethodsPups were treated with daily injections of clemastine (10-30 mg/kg/day) on postnatal days 1-21. Neurologic phenotypes and myelination patterns in the brain, optic nerves, and spinal cords were assessed using histological techniques.ResultsNo changes in neurological phenotype or survival were observed even at the highest dose of clemastine. Postmortem staining with Luxol fast blue and myelin basic protein immunohistochemistry revealed no evidence for improved myelination in the CNS of treated rats compared to vehicle-treated littermates. Populations of mature oligodendrocytes were unaffected by the treatment.ConclusionThese results demonstrate lack of therapeutic effect of clemastine in two rat PMD models. Both models have rapid disease progression consistent with the connatal form of the disease. Further studies are necessary to determine whether clemastine bears a therapeutic potential in milder forms of PMD.

Neuromyelitis optica (NMO) and myasthenia gravis (MG) are autoimmune diseases mediated by autoantibodies against either aquaporin 4 (AQP4) or acetylcholine receptor (AChR), respectively. Recently, we and others have reported an increased prevalence of NMO in patients with MG. To verify whether coexisting autoimmune disease may exacerbate experimental autoimmune MG, we tested whether active immunization with AQP4 peptides or passive transfer of NMO-Ig can affect the severity of EAMG. Injection of either AQP4 peptide or NMO-Ig to EAMG or to naive mice caused increased fatigability and aggravation of EAMG symptoms as expressed by augmented muscle weakness (but not paralysis), decremental response to repetitive nerve stimulation, increased neuromuscular jitter, and aberration of immune responses. Thus, our study shows increased disease severity in EAMG mice following immunization with the NMO autoantigen AQP4 or by NMO-Ig, mediated by augmented inflammatory response. This can explain exacerbation or increased susceptibility of patients with one autoimmune disease to develop additional autoimmune syndrome.

A variety of conditions ranging from glaucoma to blunt force trauma lead to optic nerve atrophy. Identifying signaling pathways for stimulating axon growth in the optic nerve may lead to treatments for these pathologies. Inhibiting signaling by the nogo-66 receptor 1 (NgR1) promotes the re-extension of axons following a crush injury to the optic nerve, and while NgR1 mRNA and protein expression are observed in the retinal ganglion cell (RGC) layer and inner nuclear layer, which retinal cell types express NgR1 remains unknown. Here we determine the expression pattern of NgR1 in the mouse retina by co-labeling neurons with characterized markers of specific retinal neurons together with antibodies specific for NgR1 or Green Fluorescent Protein expressed under control of the ngr1 promoter. We demonstrate that more than 99% of RGCs express NgR1. Thus, inhibiting NgR1 function may ubiquitously promote the regeneration of axons by RGCs. These results provide additional support for the therapeutic potential of NgR1 signaling in reversing optic nerve atrophy.

The optic nerve is made of highly specialized neurons and the energetic supply to their axons is crucial due to their great demand. The energy comes basically through the oxidative phosphorylation in the mitochondria, supported by glial cells metabolism. Mitochondrial dysfunction is a shared feature encountered within the optic neuropathies, including Leber's Hereditary Optic Neuropathy, Leigh's Syndrome, or Kjer's syndrome. In an effort to investigate the metabolic alterations produced within the optic nerve in a mutant mouse model of Neurological Visual Disease (NVD), a rapid, robust, and efficient one-single phase extraction methodology has been developed and validated for the GC-MS platform. Once the method was successfully validated for lactic acid and pyruvic acid as markers of an adequate optic nerve function, the protocol was applied to unveil the metabolomic signature of the wild-type mouse optic nerve. Along the chromatographic profile of the optic nerve, 94 peaks were identified and, to our knowledge, for the first time. Afterwards, a targeted metabolomics analysis was performed to quantify lactic acid and pyruvic acid in the NDV mice group (n = 8) and its corresponding wild-type (n = 8). Finally, an untargeted metabolomic study was carried out and univariate and multivariate data analyses showed 34 compounds modified in the optic nerve of the mouse with NVD mutation. Then, the metabolic reaction network of the identified metabolites highlighted alterations in the catabolism of proteins, TCA cycle, and urea cycle, reflecting a mitochondrial energetic dysfunction. Taken together, this metabolomic study has proven to be suited for the study of optic neuropathies., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Our goal was to quantify and characterize how the collagen fiber crimp waviness of the lamina cribrosa (LC) and peripapillary sclera (PPS) changes with intraocular pressure (IOP). Thirteen sheep (ovine) eyes were immersion and perfusion fixed while maintaining IOP at 0, 10, 15, 20, or 50 mmHg. Coronal optic nerve head (ONH) sections (30 µm) were imaged with polarized light microscopy (PLM) and analyzed for collagen fiber orientation and waviness (SD of fiber orientation). In the LC, the waviness of every LC beam was measured. In the PPS, at least 900 collagen bundles were measured per eye. Using the waviness at 50 mmHg IOP, we defined tissue-specific thresholds to determine the fraction of loaded or recruited fibers. We found that fiber waviness decreased with IOP (P < 0.001). At every IOP, the waviness of the collagen fibers, and the fraction of fibers recruited in the LC were smaller or equal than those of the PPS (P < 0.001). At 15 mmHg IOP, both LC and PPS had ¾ recruited fibers and ¼ left in reserve. The decreased waviness with IOP and associated fiber recruitment is experimental evidence of fiber-based nonlinear biomechanical behavior of the ONH. At all IOPs the PPS had an equal or larger fraction of fibers recruited than the LC. That both LC and PPS had the same fraction of recruited and reserve fibers at normal IOP suggests that this may be an optimal fraction of recruitment for healthy eyes. Whether this extends to human eyes remains unknown., Statement of Significance: Collagen fibers exhibit a natural waviness or crimp that largely determine the nonlinear biomechanics of soft tissue. Experimental measurements of crimp morphology in the sheep eye, and how it changes with intraocular pressure (IOP), however, are exceedingly sparse. We quantified how posterior eye crimp changes with increasing IOP. We found that the lamina cribrosa and peripapillary sclera have fundamentally different crimp, and with increasing IOP, have different proportions of fibers that straighten, or get recruited, versus remaining wavy, or in reserve. Interestingly, at physiologic IOP of 15 mmHg, both tissues had about ¾ fibers recruited and ¼ fibers in reserve, indicating there may be an optimal fraction of fibers., (Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Collapsin Response Mediator Protein 2 (CRMP2) is an intracellular protein involved in axon and dendrite growth and specification. In this study, CRMP2 was identified in a conditioned media derived from degenerated sciatic nerves (CM). On cultured rat hippocampal neurons, acute extracellular application of CM or partially purified recombinant CRMP2 produced an increase in cytoplasmic calcium. The increase in cytoplasmic calcium was mostly mediated through NMDA receptors, with a minor contribution of N-type VDCC, and it was maintained as long as CM was present. By using live-labeling of CRMP2, Ca 2+ channel binding domain 3 (CBD3) peptide derived from CRMP2, and recombinant CRMP2, we demonstrated that that this effect was mediated by an action on the extracellular side of the NMDA receptor. This is the first report of an extracellular action of CRMP2. Prolonged exposure to extracellular CRMP2, may contribute to neuronal calcium dysregulation and neuronal damage., (Copyright © 2018 IBRO. Published by Elsevier Ltd. All rights reserved.)

Purpose: To investigate the effect and mechanism of proresolving lipid mediator resolvin D1 (RvD1) on the corneal epithelium and the restoration of mechanical sensation in diabetic mice., Methods: Type 1 diabetes was induced in mice with intraperitoneal streptozocin injections. The healthy and diabetic mice underwent removal of the central corneal epithelium, and then 100 ng/ml RvD1 or its formyl peptide receptor 2 (FPR2) antagonist WRW4 was used to treat the diabetic mice. Regeneration of the corneal epithelium and nerves was observed with sodium fluorescein staining and whole-mount anti-β3-tubulin fluorescence staining. The inflammatory response level was measured with hematoxylin and eosin staining (inflammatory cell infiltration), enzyme-linked immunosorbent assay (tumor necrosis factor alpha and interleukin-1 beta content), myeloperoxidase activity, and fluorescence staining (macrophage content). The reactive oxygen species (ROS) and glutathione (GSH) levels were examined with incubation with fluorescent probes, and oxidative stress-related protein expression levels were evaluated with fluorescence staining and western blotting., Results: Topical application of RvD1 promoted regeneration of the corneal epithelium in diabetic mice, accompanied by the reactivation of signaling and inflammation resolution related to regeneration of the epithelium. Furthermore, RvD1 directly attenuated the accumulation of ROS and nicotinamide adenine dinucleotide phosphate oxidase 2/4 expression, while RvD1 enhanced GSH synthesis and reactivated the Nrf2-ARE signaling pathway that was impaired in the corneal epithelium in the diabetic mice. More interestingly, topical application of RvD1 promoted regeneration of corneal nerves and completely restored impaired mechanical sensitivity of the cornea in diabetic mice. In addition, the promotion of corneal epithelial wound healing by RvD1 in diabetic mice was abolished by its FPR2 antagonist WRW4., Conclusions: Topical application of RvD1 promotes corneal epithelial wound healing and the restoration of mechanical sensation in diabetic mice, which may be related to the lipid mediator's regulation of inflammation resolution, the reactivation of regenerative signaling in the epithelium, and the attenuation of oxidative stress.

Background: Glaucoma is a leading cause of irreversible blindness manifesting as an age-related, progressive optic neuropathy with associated retinal ganglion cell (RGC) loss. Mitogen-activated protein kinases (MAPKs: p42/44 MAPK, SAPK/JNK, p38 MAPK) are activated in various retinal disease models and likely contribute to the mechanisms of RGC death. Although MAPKs play roles in the development of retinal pathology, their action in the optic nerve head (ONH), where the initial insult to RGC axons likely resides in glaucoma, remains unexplored., Methods: An experimental paradigm representing glaucoma was established by induction of chronic ocular hypertension (OHT) via laser-induced coagulation of the trabecular meshwork in Sprague-Dawley rats. MAPKs were subsequently investigated over the following days for expression and activity alterations, using RT-PCR, immunohistochemistry and Western immunoblot., Results: p42/44 MAPK expression was unaltered after intraocular pressure (IOP) elevation, but there was a significant activation of this enzyme in ONH astrocytes after 6-24 h. Activated SAPK/JNK isoforms were present throughout healthy RGC axons but after IOP elevation or optic nerve crush, they both accumulated at the ONH, likely due to RGC axon transport disruption, and were subject to additional activation. p38 MAPK was expressed by a population of microglia which were significantly more populous following IOP elevation. However it was only significantly activated in microglia after 3 days, and then only in the ONH and optic nerve; in the retina it was solely activated in RGC perikarya., Conclusions: In conclusion, each of the MAPKs showed a specific spatio-temporal expression and activation pattern in the retina, ONH and optic nerve as a result of IOP elevation. These findings likely reflect the roles of the individual enzymes, and the cells in which they reside, in the developing pathology following IOP elevation. These data have implications for understanding the mechanisms of ocular pathology in diseases such as glaucoma., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Axonal injury is a common feature of central nervous system insults. Upregulation of amyloid precursor protein (APP) is observed following central nervous system neurotrauma and is regarded as a marker of central nervous system axonal injury. However, the underlying mechanism by which APP mediates neuronal death remains to be elucidated. Here, we used mouse optic nerve axotomy (ONA) to model central nervous system axonal injury replicating aspects of retinal ganglion cell (RGC) death in optic neuropathies. APP and APP intracellular domain (AICD) were upregulated in retina after ONA and APP knockout reduced Tuj1 + RGC loss. Pathway analysis of microarray data combined with chromatin immunoprecipitation and a luciferase reporter assay demonstrated that AICD interacts with the JNK3 gene locus and regulates JNK3 expression. Moreover, JNK3 was found to be upregulated after ONA and to contribute to Tuj1 + RGC death. APP knockout reduced the ONA-induced enhanced expression of JNK3 and phosphorylated JNK (pJNK). Gamma-secretase inhibitors prevented production of AICD, reduced JNK3 and pJNK expression similarly, and protected Tuj1 + RGCs from ONA-induced cell death. Together these data indicate that ONA induces APP expression and that gamma-secretase cleavage of APP releases AICD, which upregulates JNK3 leading to RGC death. This pathway may be a novel target for neuronal protection in optic neuropathies and other forms of neurotrauma.

Optic nerve injury is a common disease. The present study aimed to examine the possible role of microRNA‑204 (miR‑204) in optic nerve injury through the regulation of growth‑associated protein-43 (GAP‑43). Initially, optic nerve injury models were established in Sprague‑Dawley (SD) rats, and the function of miR‑204 was either enhanced or inhibited through injection of miR‑204 mimic and inhibitor, respectively. Subsequently, the mRNA and protein levels of miR‑204, GAP‑43, toll‑like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88) and nuclear factor‑κB (NF‑κB) were examined in retinal tissues using reverse transcription‑quantitative polymerase chain reaction and western blot analyses. The apoptosis of retinal tissue cells was also detected using a terminal deoxynucleotidyl transferase mediated dUTP nick end labeling assay. There was a significant increase in the level of miR‑204 in retinal blood vessels of the model SD rats, compared with that in the normal SD rats (P<0.05), and the expression of GAP‑43 was significantly decreased (P<0.05). The results confirmed that the expression of GAP‑43 was significantly reduced, compared with that in the normal control group when the rats were treated with miR‑204 mimic (P<0.05), which was similar to the result in the model group. By contrast, its expression of GAP‑43 was significantly increased when treated with the miR‑204 inhibitor (P<0.05). Compared with the normal control group, the expression levels of TLR4, MyD88 and NF‑κB were significantly increased in the miR‑204 mimic group and model group (P<0.05), whereas the same three factors in the miR‑204 inhibitor group were effectively inhibited, compared with those in the model group, and showed similar results to the normal control group. The apoptotic rates of retinal cells in the miR‑204 mimic group and model group were significantly increased, compared with that in the normal control group (P<0.05), whereas miR‑204 inhibitor effectively reversed the effects on apoptotic rate observed in the model group, showing similar results to those in the normal control group. Taken together, miR‑204 promoted the apoptosis of retinal cells through inhibiting GAP‑43, providing theoretical guidance for the function of GAP‑43 in retinal injury.

Glaucoma is characterized by a progressive loss of retinal ganglion cells and their axons, but the underlying biological basis for the accompanying neurodegeneration is not known. Accumulating evidence indicates that structural and functional abnormalities of astrocytes within the optic nerve head (ONH) have a role. However, whether the activation of cyclic adenosine 3',5'-monophosphate (cAMP) signaling pathway is associated with astrocyte dysfunction in the ONH remains unknown. We report here that the cAMP/protein kinase A (PKA) pathway is critical to ONH astrocyte dysfunction, leading to caspase-3 activation and cell death via the AKT/Bim/Bax signaling pathway. Furthermore, elevated intracellular cAMP exacerbates vulnerability to oxidative stress in ONH astrocytes, and this may contribute to axonal damage in glaucomatous neurodegeneration. Inhibition of intracellular cAMP/PKA signaling activation protects ONH astrocytes by increasing AKT phosphorylation against oxidative stress. These results strongly indicate that activation of cAMP/PKA pathway has an important role in astrocyte dysfunction, and suggest that modulating cAMP/PKA pathway has therapeutic potential for glaucomatous ONH degeneration.

Purpose: The present study is designed to identify the influences of genetic background on optic nerve regeneration using the two parental strains (C57BL/6J and DBA/2J) and seven BXD recombinant inbred mouse strains., Methods: To study regeneration in the optic nerve, Pten was knocked down in the retinal ganglion cells using adenoassociated virus (AAV) delivery of shRNA, and a mild inflammatory response was induced with an intravitreal injection of zymosan with CPT-cAMP. The axons of the retinal ganglion cells were damaged by optic nerve crush (ONC). Following a 12-day survival period, regenerating axons were labeled by cholera toxin B, and 2 days later, the regenerating axons within the optic nerve were examined. The number of axons at 0.5 mm and 1 mm from the crush site were counted. In addition, we measured the distance that five axons had grown down the nerve and the longest distance a single axon reached., Results: The analysis revealed a considerable amount of differential axonal regeneration across the seven BXD strains and the parental strains. There was a statistically significant difference (p=0.014 Mann-Whitney U test) in the regenerative capacity in the number of axons reaching 0.5 mm from a low of 236.1±24.4 axons in the BXD102 mice to a high of 759.8±79.2 axons in the BXD29 mice. There were also statistically significant differences (p=0.014 Mann-Whitney U test) in the distance axons traveled. Looking at a minimum of five axons, the shortest distance was 787.2±46.5 µm in the BXD102 mice, and the maximum distance was 2025.5±223.3 µm in the BXD29 mice., Conclusions: Differences in genetic background can have a profound effect on axonal regeneration causing a threefold increase in the number of regenerating axons at 0.5 mm from the crush site and a 2.5-fold increase in the distance traveled by at least five axons in the damaged optic nerve.

Elevated intracranial pressure (ICP) can result in multiple neurologic sequelae including vision loss. Inducible models of ICP elevation are lacking in model organisms, which limits our understanding of the mechanism by which increased ICP impacts the visual system. We adapted a mouse model for the sustained elevation of ICP and tested the hypothesis that elevated ICP impacts the optic nerve and retinal ganglion cells (RGCs). ICP was elevated and maintained for 2 weeks, and resulted in multiple anatomic changes that are consistent with human disease including papilledema, loss of physiologic cupping, and engorgement of the optic nerve head. Elevated ICP caused a loss of RGC somas in the retina and RGC axons within the optic nerve, as well as a reduction in both RGC electrical function and contrast sensitivity. Elevated ICP also caused increased hypoxia-inducible factor (HIF)-1 alpha expression in the ganglion cell layer. These experiments confirm that sustained ICP elevation can be achieved in mice and causes phenotypes that preferentially impact RGCs and are similar to those seen in human disease. With this model, it is possible to model human diseases of elevated ICP such as Idiopathic Intracranial Hypertension and Spaceflight Associated Neuro-ocular Syndrome.

Dominant optic atrophy (DOA) is a rare progressive and irreversible blinding disease which is one of the most frequent forms of hereditary optic neuropathy. DOA is mainly caused by dominant mutation in the OPA1 gene encoding a large mitochondrial GTPase with crucial roles in membrane dynamics and cell survival. Hereditary optic neuropathies are commonly characterized by the degeneration of retinal ganglion cells, leading to the optic nerve atrophy and the progressive loss of visual acuity. Up to now, despite increasing advances in the understanding of the pathological mechanisms, DOA remains intractable. Here, we tested the efficiency of gene therapy on a genetically-modified mouse model reproducing DOA vision loss. We performed intravitreal injections of an Adeno-Associated Virus carrying the human OPA1 cDNA under the control of the cytomegalovirus promotor. Our results provide the first evidence that gene therapy is efficient on a mouse model of DOA as the wild-type OPA1 expression is able to alleviate the OPA1-induced retinal ganglion cell degeneration, the hallmark of the disease. These results displayed encouraging effects of gene therapy for Dominant Optic Atrophy, fostering future investigations aiming at clinical trials in patients.

Glaucoma is a leading cause of irreversible blindness and characterized by progressive damage of retinal ganglion cells (RGCs). Growing evidences have linked impaired mitophagy with neurodegenerative diseases, while the E3 ubiquitin ligase parkin may play a key role. However, the pathophysiological relationship between parkin and glaucoma remains largely unknown. Using chronic hypertensive glaucoma rats induced by translimbal laser photocoagulation, we show here that the protein level of parkin and its downstream optineurin proteins were increased in hypertensive retinas. The ratio of LC3-II to LC3-I, the number of mitophagosomes, and unhealthy mitochondria were increased in hypertensive optic nerves. Overexpression of parkin by viral vectors increased RGC survival in glaucomatous rats in vivo and under excitotoxicity in vitro. It also promoted optineurin expression and improved mitochondrial health. In parkin-overexpressed glaucomatous rats, the ratio of LC3-II to LC3-I, LAMP1 level, and the number of mitophagosomes in optic nerve were decreased at 3 days, yet increased at 2 weeks following intraocular pressure (IOP) elevation. These findings demonstrate that dysfunction of mitophagy exist in RGCs of glaucomatous rats. Overexpression of parkin exerted a significant protective effect on RGCs and partially restored dysfunction of mitophagy in response to cumulative IOP elevation.

Astrocytes make up approximately 30% of all the cells in the mammalian central nervous system. They are not passive, as once thought, but are integral to brain physiology and perform many functions that are important for normal neuronal development and metabolism, synapse formation, synaptic transmission, and in repair following injury/disease. Astrocytes also communicate with neurons, blood vessels, and other types of glial cells. Astrocytes within the optic nerve head region play a key role in glaucomatous axon degeneration. In this chapter, we describe ways in which astrocytes of the optic nerve head can be visualized, beginning with basic immunohistochemical staining methods, to single-cell dye injections and then to transgenic animals. We will also discuss the pros and cons of each method. Many of the methods were initially developed to visualize brain astrocytes; in some cases, the method has translated well to astrocytes of the optic nerve, and in others, it remains unclear.

The RNA-binding protein, HuR, modulates mRNA processing and gene expression of several stress response proteins i.e. Hsp70 and p53 that have been postulated to be involved in the pathogenesis of glaucoma, a chronic optic neuropathy leading to irreversible blindness. We evaluated HuR protein expression in retinas and optic nerves of glaucomatous rats and human primary open angle glaucoma patients and its possible impact on stress response mechanisms. We found that the cytoplasmic content of HuR was reduced more extensively in glaucomatous retinas than in optic nerves and this was linked with a declined cytoplasmic Hsp70 level and p53 nuclear translocation. In the optic nerve, the p53 content was decreased as a feature of reactive gliosis. Based on our findings, we conclude that the alteration in the HuR content, observed both in rat glaucoma model and human glaucoma samples, affects post-transcriptionally the expression of genes crucial for maintaining cell homeostasis; therefore, we postulate that HuR may be involved in the pathogenesis of glaucoma., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Aims: Optic neuritis (ON) is an inflammatory disease of the optic nerve, which often occurs in patients with multiple sclerosis (MS) and leads to retinal ganglion cell (RGC) death and even severe visual loss. Valproic acid (VPA) is a short-chain branched fatty acid with anti-epileptic, neuro-protective and anti-inflammatory effects. Here, we examined the effects of VPA in experimental autoimmune encephalomyelitis (EAE) rats and explored the underlying mechanisms., Main Methods: EAE was induced by subcutaneous injection with myelin basic protein, emulsified with complete Freund's adjuvant and Mycobacterium tuberculosis H37Ra into the Lewis rats. Subsequently, animals in the VPA groups were treated orally with VPA (250 or 500 mg/kg) once a day for 13 days., Key Findings: VPA treatment significantly attenuated inflammation and microgliosis in optic nerve in EAE-ON rats, as evidenced by the decrease in the mRNA levels of interferon (INF)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-17, and inducible nitric oxide synthase (iNOS), the suppression in nuclear factor (NF)-κB signal pathway as well as the down-regulation of CD11b expression in optic nerve. Additionally, the apoptotic RGCs were remarkably increased in the EAE retina, which was inhibited by VPA treatment. Consistent with the TUNEL staining, VPA administration also obviously suppressed the ratio of Bax: Bcl-2 and the expression of cleaved caspase-3 and PARP in optic nerve in EAE rats., Significance: Our findings demonstrated that VPA treatment could prevent inflammation responses and RGC apoptosis in optic nerve in EAE-ON rats, suggesting that VPA may be available for optic nerve protection during ON., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

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.

The role of bradykinin B 1 receptors on the oxidative stress as measured by the levels of Na+/K+ ATPase activity, malondialdehyde (MDA) and glutathione (GSH) in male Wistar rat optic nerve and visual cortex area 1 and 4weeks after STZ treatment was studied. Rats were divided into 4 groups (n=6-7): 1. Controls (non-diabetics); 2. Diabetics (65mg/kg streptozotocin, STZ); 3. Diabetics injected with B 1 antagonist R-954 (2mg/Kg) during the last 3days of a one week period; 4. Diabetics injected with B 1 antagonist R-954 (2mg/Kg) during the last 3days of a 4week period. The results showed that plasma glucose levels increased by up to 4 fold in diabetic rats 1 or 4weeks following the STZ treatment. R-954 treatment did significantly decrease blood glucose levels. Levels of MDA was increased in the plasma of the 1 and 4week diabetic animals whereas the GSH levels were decreased. Both markers returned to normal following R-954 treatment. Na+/K+ ATPase activity significantly decreased in the optic nerve and visual cortex of diabetic rats at 1 and 4weeks but returned to normal following R-954 treatment. MDA levels increased markedly at 1 and 4weeks compared with control levels in the optic nerve but slightly in the visual cortex and returned to control levels in both tissues following R-954 treatment. GSH levels decreased in both tissues at 1 and 4weeks compared with control levels. Following administration of the selective BKB 1 R antagonist R-954, the levels of GSH returned to normal in both tissues of the 1 and 4week diabetic animals. These results showed that the inducible BKB 1 receptors are associated with the oxidative stress in the optic nerve and cortical visual area of diabetic rats and suggested that BKB 1 -R antagonist R-954 could have a beneficial role in the treatment of diabetic retinopathy., (Copyright © 2017. Published by Elsevier Ltd.)

A significant proportion of the astronauts who spend extended periods in microgravity develop ophthalmic abnormalities. Understanding this syndrome, called visual impairment and intracranial pressure (VIIP), has become a high priority for National Aeronautics and Space Administration, especially in view of future long-duration missions (e.g., Mars missions). Moreover, to ensure selection of astronaut candidates who will be able to complete long-duration missions with low risk of the VIIP syndrome, it is imperative to identify biomarkers for VIIP risk prediction. Here, we hypothesize that the optic nerve sheath response to alterations in intracranial pressure may be a potential predictive biomarker for optic disc edema in astronauts. If confirmed, this biomarker could be used for preflight identification of astronauts at risk for developing VIIP-associated optic disc edema.

In the current study, it was aimed to evaluate the changes in myelinated and unmyelinated nerve fibers in retinal ischemia‑reperfusion injuries caused by acute ocular hypertension and to determine the sequence of these changes. Adult healthy New Zealand white rabbits were randomized to the hemodynamic group [n=12; used to determine the optimal intraocular pressure (IOP) for the subsequent experiments] and the hypertension group (n=6; 70‑mmHg hypertension induced in one eye). IOP was adjusted using a cannula and saline. Doppler ultrasound was used to measure the velocity of the optic artery under different intraocular pressures. Immunohistochemistry for myelin basic protein (MBP) was performed. Apoptosis of retinal cells was detected by terminal deoxynucleotidyl transferase biotin‑dUTP nick end labeling (TUNEL) assay. Electron microscopy was used to investigate the changes in myelinated and unmyelinated nerve fibers. IOP of the hypertension eyes was maintained at 70.2±1.0 mmHg, while IOP of control eyes was 7‑14 mmHg. Doppler ultrasound demonstrated an obvious decline of peak systolic velocity and an increase of resistance index of retinal bloodstream under a 70‑mmHg IOP. MBP immunohistochemistry and electron microscopy demonstrated obvious injuries to the myelin fibers. TUNEL indicated a significantly higher apoptosis rate in the hypertension eyes compared with control eyes. The apoptosis rate of retinal ganglion cells and bipolar cells in unmyelinated regions was higher than in myelinated regions. In conclusion, an IOP of 70 mmHg led to incomplete retinal ischemia but was the threshold for retinal ischemia, leading to obvious injuries to the myelin fibers.

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

Traumatic optic neuropathy (TON) is a devastating cause of permanent visual loss following blunt injury to the head. Animal models for TON exist, but most fail to recapitulate the clinical scenario of closed head indirect trauma to the nerve and subsequent neurodegeneration. Thus, we developed a clinically-relevant animal model for TON using a novel ultrasonic pulse injury modality (sonication-induced TON; SI-TON). To trigger TON, a microtip probe sonifier was placed on the supraorbital ridge directly above the entrance of the optic nerve into the bony canal. An ultrasonic pulse was then delivered to the optic nerve. After injury, the number of RGCs in the retina as well as visual function measured by PERG steadily decreased over a two-week period. In the optic nerve, pro-inflammatory markers were upregulated within 6 hours following injury. Immunohistochemistry showed activation of microglia and infiltration of CD45-positive leukocytes in the optic nerve and initiation of a gliotic response. The SI-TON model is capable of delivering a non-contact concussive injury to the optic nerve and induce TON in mice. Thus, our data indicate that the SI-TON model reliably recapitulates the pathophysiology and progressive neurodegeneration seen in the human manifestation.

The sigma 1 receptor (S1R) is a unique transmembrane protein that has been shown to regulate neuronal differentiation and cellular survival. It is expressed within several cell types throughout the nervous system and visceral organs, including neurons and glia within the eye. S1R ligands are therapeutic targets for diseases ranging from neurodegenerative conditions to neoplastic disorders. However, effects of S1R activation and inhibition within glia cells are not well characterized. Within the eye, the astrocytes at the optic nerve head are crucial to the health and survival of the neurons that send visual information to the brain. In this study, we used the S1R-specific agonist, (+)-pentazocine, to evaluate S1R activation within optic nerve head-derived astrocytes (ONHAs). Treatment of ONHAs with (+)-pentazocine attenuated the level and duration of stress-induced ERK phosphorylation following oxidative stress exposure and promoted survival of ONHAs. These effects were specific to S1R activation because they were not observed in ONHAs that were depleted of S1R using siRNA-mediated knockdown. Collectively, our results suggest that S1R activation suppresses ERK1/2 phosphorylation and protects ONHAs from oxidative stress-induced death.

Studies by Bruce Ransom and colleagues have made a major contribution to show that white matter is susceptible to ischemia/hypoxia. White matter contains axons and the glia that support them, notably myelinating oligodendrocytes, which are highly vulnerable to ischemic-hypoxic damage. Previous studies have shown that metabotropic GluRs (mGluRs) are cytoprotective for oligodendrocyte precursor cells and immature oligodendrocytes, but their potential role in adult white matter was unresolved. Here, we report that group 1 mGluR1/5 and group 2 mGluR3 subunits are expressed in optic nerves from mice aged postnatal day (P)8-12 and P30-35. We demonstrate that activation of group 1 mGluR protects oligodendrocytes against oxygen-glucose deprivation (OGD) in developing and young adult optic nerves. In contrast, group 2 mGluR are shown to be protective for oligodendrocytes against OGD in postnatal but not young adult optic nerves. The cytoprotective effect of group 1 mGluR requires activation of PKC, whilst group 2 mGluR are dependent on negatively regulating adenylyl cyclase and cAMP. Our results identify a role for mGluR in limiting injury of oligodendrocytes in developing and young adult white matter, which may be useful for protecting oligodendrocytes in neuropathologies involving excitoxicity and ischemia/hypoxia.

Failure of oligodendrocyte precursor cells (OPCs) to differentiate and remyelinate axons is thought to be a major cause of the limited ability of the central nervous system to repair plaques of immune-mediated demyelination in multiple sclerosis (MS). Current therapies for MS aim to lessen the immune response in order to reduce the frequency and severity of attacks, but these existing therapies do not target remyelination or stimulate repair of the damaged tissue. Thus, the promotion of OPC differentiation and remyelination is potentially an important therapeutic goal. Previous studies have shown that a recombinant human-derived monoclonal IgM antibody, designated rHIgM22, promotes remyelination, particularly of the spinal cord in rodent models of demyelination. Here, we examined the effects of rHIgM22 in remyelination in the brain using the mouse model of cuprizone-induced demyelination, which is characterized by spontaneous remyelination. The myelination state of the corpus callosum of cuprizone-fed mice treated with rHIgM22 was examined immediately after the end of the cuprizone diet as well as at different time points during the recovery period with regular food, and compared with that of cuprizone-fed animals treated with either vehicle or human IgM isotype control antibody. Mice fed only regular food were used as controls. We demonstrate that treatment with rHIgM22 accelerated remyelination of the demyelinated corpus callosum. The remyelination-enhancing effects of rHIgM22 were found across different, anatomically distinct regions of the corpus callosum, and followed a spatiotemporal pattern that was similar to that of the spontaneous remyelination process. These enhancing effects were also accompanied by increased differentiation of OPCs into mature oligodendrocytes. Our data indicate strong remyelination-promoting capabilities of rHIgM22 and further support its therapeutic potential in MS., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

The Aryl hydrocarbon Receptor(AhR) is among the most important receptors which bind pollutants; however it also regulates signaling pathways independently of such exposure. We previously demonstrated that AhR is expressed during development of the central nervous system(CNS) and that its deletion leads to the occurrence of a congenital nystagmus. Objectives of the present study are to decipher the origin of these deficits, and to identify the role of the AhR in the development of the CNS. We show that the AhR-knockout phenotype develops during early infancy together with deficits in visual-information-processing which are associated with an altered optic nerve myelin sheath, which exhibits modifications in its lipid composition and in the expression of myelin-associated-glycoprotein(MAG), a cell adhesion molecule involved in myelin-maintenance and glia-axon interaction. In addition, we show that the expression of pro-inflammatory cytokines is increased in the impaired optic nerve and confirm that inflammation is causally related with an AhR-dependent decreased expression of MAG. Overall, our findings demonstrate the role of the AhR as a physiological regulator of myelination and inflammatory processes in the developing CNS. It identifies a mechanism by which environmental pollutants might influence CNS myelination and suggest AhR as a relevant drug target for demyelinating diseases.

Objective: To study the protective effect of electroacupuncture (EA) along the visual conductive pathway for the optic nerve tissue of anterior ischemic optic neuropathy (AION) in terms of the structure and apoptosis., Methods: The AION model of right eye was established with laser in 48 New Zealand white ear rabbits. All rabbits were randomly divided into a model group, an acupuncture group and an acupuncture combined with EA group, 16 rabbits in each one. Other 16 normal left eyes were selected as a blank group. Acupuncture and EA of 30 min were used in the corresponding groups for 3 days at the right "Cuanzhu" (BL 2), "Yuyao" (EX-HN 4) and "Qiaomingxue" (Extra), once a day. There was no intervention in the model group and the blank group. The morphological structure and retinal thickness of lining of the optic nerve were detected with HE stain. The expressions of the Bcl-2 and Bax in the retina were detected with immunohistochemistry. And the concentration of tumor necrosis factor-α (TNF-α) in the retina was detected with enzyme linked immunosorbent assay (ELISA)., Results: In the model group, the ganglion cell layer revealed hyperplasia and disorder, and the retina ganglion cells revealed loose arrangements, thin inner nuclear layers, and reduction of cell amounts, some long parts missing cells. However, the above structural damages were much weaker in the acupuncture group and acupuncture combined with EA group. The inner layer of the retina in the model group was thinner than that in the blank group ( P <0.05). That in the acupuncture combined with EA group showed significant better results than those in the acupuncture and model groups (both P <0.05), which was not statistically different from that in the normal group ( P >0.05). The Bcl-2 count in the model group was lower than that in the blank group ( P <0.05), and that in the acupuncture combined with EA group was better than those in the acupuncture and model groups (both P <0.05), not significantly different from that in the blank group ( P >0.05). The number of Bax in the model group was higher than that in the blank group ( P <0.05), and that in the acupuncture combined with EA group was lower than those in the acupuncture and model groups (both P <0.05), and was similar to that in the blank group ( P >0.05). Bcl-2/Bax in the model group was lower than that in the blank group ( P <0.05). The value in the acupuncture combined with EA group presented better than those in the acupuncture and model groups (both P <0.05), which had no difference from that in the blank group ( P >0.05). TNF-α in the model group was higher than that in the blank group ( P <0.05), and no such differences were detected between other groups and the model group (both P >0.05)., Conclusions: EA along the visual conductive pathway is protective to some extent for optic nerve tissue, which can increase the expression of Bcl-2 and reduce the expression of Bax so as to restrain ganglion cell apoptosis.

It is widely considered that intraocular pressure (IOP)-induced deformation within the neural tissue pores of the lamina cribrosa (LC) contributes to neurodegeneration and glaucoma. Our goal was to study how the LC microstructure and mechanical properties determine the mechanical insult to the neural tissues within the pores of the LC. Polarized light microscopy was used to measure the collagen density and orientation in histology sections of three sheep optic nerve heads (ONH) at both mesoscale (4.4μm) and microscale (0.73μm) resolutions. Mesoscale fiber-aware FE models were first used to calculate ONH deformations at an IOP of 30mmHg. The results were then used as boundary conditions for microscale models of LC regions. Models predicted large insult to the LC neural tissues, with 95th percentile 1st principal strains ranging from 7 to 12%. Pores near the scleral boundary suffered significantly higher stretch compared to pores in more central regions (10.0±1.4% vs. 7.2±0.4%; p=0.014; mean±SD). Variations in material properties altered the minimum, median, and maximum levels of neural tissue insult but largely did not alter the patterns of pore-to-pore variation, suggesting these patterns are determined by the underlying structure and geometry of the LC beams and pores. To the best of our knowledge, this is the first computational model that reproduces the highly heterogeneous neural tissue strain fields observed experimentally., Statement of Significance: The loss of visual function associated with glaucoma has been attributed to sustained mechanical insult to the neural tissues of the lamina cribrosa due to elevated intraocular pressure. Our study is the first computational model built from specimen-specific tissue microstructure to consider the mechanics of the neural tissues of the lamina separately from the connective tissue. We found that the deformation of the neural tissue was much larger than that predicted by any recent microstructure-aware models of the lamina. These results are consistent with recent experimental data and the highest deformations were found in the region of the lamina where glaucomatous damage first occurs. This study provides new insight into the complex biomechanical environment within the lamina., (Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Pathogenesis in aquaporin-4 immunoglobulin G (AQP4-IgG) seropositive neuromyelitis optica spectrum disorders (herein called NMO) involves complement-dependent cytotoxicity initiated by AQP4-IgG binding to astrocyte AQP4. We recently reported that rats lacking complement inhibitor protein CD59 were highly susceptible to development of NMO pathology in brain and spinal cord following direct AQP4-IgG administration (Yao and Verkman, Acta Neuropath Commun 2017, 5:15). Here, we report evidence that CD59 is responsible for protection of peripheral, AQP4-expressing tissues in seropositive NMO. Rats made seropositive by intraperitoneal injection of AQP4-IgG developed marked weakness by 24 h and died soon thereafter. Serum creatine phosphokinase at 24 h was >900-fold greater in seropositive CD59 -/- rats than in seropositive CD59 +/+ (or control) rats. AQP4-expressing cells in skeletal muscle and kidney, but not in stomach, of seropositive CD59 -/- rats showed injury with deposition of AQP4-IgG and activated complement C5b-9, and inflammation. Organ injury in seropositive CD59 -/- rats was prevented by a complement inhibitor. Significant pathological changes in seropositive CD59 -/- rats were not seen in optic nerve, spinal cord or brain, including circumventricular tissue. These results implicate a major protective role of CD59 outside of the central nervous system in seropositive NMO, and hence offer an explanation as to why peripheral, AQP4-expressing cells are largely unaffected in NMO.

Low-grade gliomas are one of the most common brain tumors in children, where they frequently form within the optic pathway (optic pathway gliomas; OPGs). Since many OPGs occur in the context of the Neurofibromatosis Type 1 (NF1) cancer predisposition syndrome, we have previously employed Nf1 genetically-engineered mouse (GEM) strains to study the pathogenesis of these low-grade glial neoplasms. In the light of the finding that human and mouse low-grade gliomas are composed of Olig2+ cells and that Olig2+ oligodendrocyte precursor cells (OPCs) give rise to murine high-grade gliomas, we sought to determine whether Olig2+ OPCs could be tumor-initiating cells for Nf1 optic glioma. Similar to the GFAP-Cre transgenic strain previously employed to generate Nf1 optic gliomas, Olig2+ cells also give rise to astrocytes in the murine optic nerve in vivo. However, in contrast to the GFAP-Cre strain where somatic Nf1 inactivation in embryonic neural progenitor/stem cells (Nf1flox/mut; GFAP-Cre mice) results in optic gliomas by 3 months of age in vivo, mice with Nf1 gene inactivation in Olig2+ OPCs (Nf1flox/mut; Olig2-Cre mice) do not form optic gliomas until 6 months of age. These distinct patterns of glioma latency do not reflect differences in the timing or brain location of somatic Nf1 loss. Instead, they most likely reflect the cell of origin, as somatic Nf1 loss in CD133+ neural progenitor/stem cells during late embryogenesis results in optic gliomas at 3 months of age. Collectively, these data demonstrate that the cell of origin dictates the time to tumorigenesis in murine optic glioma.

Developing strategies that promote axonal regeneration within the injured CNS is a major therapeutic challenge, as axonal outgrowth is potently inhibited by myelin and the glial scar. Although regeneration can be achieved using the genetic deletion of PTEN, a negative regulator of the mTOR pathway, this requires inactivation prior to nerve injury, thus precluding therapeutic application. Here, we show that, remarkably, fibroblast-derived exosomes (FD exosomes) enable neurite growth on CNS inhibitory proteins. Moreover, we demonstrate that, upon treatment with FD exosomes, Wnt10b is recruited toward lipid rafts and activates mTOR via GSK3β and TSC2. Application of FD exosomes shortly after optic nerve injury promoted robust axonal regeneration, which was strongly reduced in Wnt10b-deleted animals. This work uncovers an intercellular signaling pathway whereby FD exosomes mobilize an autocrine Wnt10b-mTOR pathway, thereby awakening the intrinsic capacity of neurons for regeneration, an important step toward healing the injured CNS., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Glaucoma is a leading cause of blindness, characterized by retinal ganglion cell (RGC) loss and optic nerve (ON) damage. Cumulative evidence suggests glial cell involvement in the degeneration of the ON and RGCs. We analyzed the contribution of microglial reactivity to early axoglial alterations of the ON in an induced model of ocular hypertension. For this purpose, vehicle or chondroitin sulfate (CS) were weekly injected into the eye anterior chamber from Wistar rats for different intervals. The amount of Brn3a(+) RGC significantly decreased in CS-injected eyes for 10 and 15 (but not 6) weeks. A reduction in anterograde transport of β-subunit cholera toxin was observed in the superior colliculus and the lateral geniculate nucleus contralateral to CS-injected eyes for 6 and 15 weeks. A disruption of cholera toxin β-subunit transport was observed at the proximal myelinated ON. A significant decrease in phosphorylated neurofilament heavy chain immunoreactivity, an increase in ionized calcium-binding adaptor molecule 1(+), ED1(+) (microglial markers), and glial fibrillary acidic protein (astrocytes) (+) area, and decreased luxol fast blue staining were observed in the ON at 6 and 15 weeks of ocular hypertension. Microglial reactivity involvement was examined through a daily treatment with minocycline (30 mg/kg, i.p.) for 2 weeks, after 4 weeks of ocular hypertension. Minocycline prevented the increase in ionized calcium-binding adaptor molecule 1(+), ED-1(+), and glial fibrillary acidic protein(+) area, the decrease in phosphorylated neurofilament heavy-chain immunoreactivity and luxol fast blue staining, and the deficit in anterograde transport induced by 6 weeks of ocular hypertension. Thus, targeting microglial reactivity might prevent early axoglial alterations in the glaucomatous ON. Cover Image for this issue: doi: 10.1111/jnc.13807., (© 2017 International Society for Neurochemistry.)

The present study aims to examine the origin of oligodendrocyte progenitor cells (OPCs) in the mouse optic nerve (ON) by labeling OPCs in the fetal forebrain. The labeling of OPCs in the ON was performed by injection of a retrovirus vector carrying the lacZ gene into the lateral ventricle, or by inducible Cre/loxP of Olig2-positive cells. The retrovirus labeling revealed that ventricular zone-derived cells of the fetal forebrain relocated to the ON and differentiated into oligodendrocytes. In addition, lineage tracing of Olig2-positive cells and whole-mount staining of PDGFRα-positive cells demonstrated that OPCs appeared by E12.5 in the preoptic area, and spread caudally to enter the ON. Our results also suggest that OPCs generated during the early stage are depleted from the ON after maturation.