Perivascular neurons instruct 3D vascular lattice formation via neurovascular contact.

The vasculature of the central nervous system is a 3D lattice composed of laminar vascular beds interconnected by penetrating vessels. The mechanisms controlling 3D lattice network formation remain largely unknown. Combining viral labeling, genetic marking, and single-cell profiling in the mouse retina, we discovered a perivascular neuronal subset, annotated as Fam19a4/Nts-positive retinal ganglion cells (Fam19a4/Nts-RGCs), directly contacting the vasculature with perisomatic endfeet. Developmental ablation of Fam19a4/Nts-RGCs led to disoriented growth of penetrating vessels near the ganglion cell layer (GCL), leading to a disorganized 3D vascular lattice. We identified enriched PIEZO2 expression in Fam19a4/Nts-RGCs. Piezo2 loss from all retinal neurons or Fam19a4/Nts-RGCs abolished the direct neurovascular contacts and phenocopied the Fam19a4/Nts-RGC ablation deficits. The defective vascular structure led to reduced capillary perfusion and sensitized the retina to ischemic insults. Furthermore, we uncovered a Piezo2-dependent perivascular granule cell subset for cerebellar vascular patterning, indicating neuronal Piezo2-dependent 3D vascular patterning in the brain.

Compartmentalized ciliation changes of oligodendrocytes in aged mouse optic nerve.

Primary cilia are microtubule-based sensory organelles that project from the apical surface of most mammalian cells, including oligodendrocytes, which are myelinating cells of the central nervous system (CNS) that support critical axonal function. Dysfunction of CNS glia is associated with aging-related white matter diseases and neurodegeneration, and ciliopathies are known to affect CNS white matter. To investigate age-related changes in ciliary profile, we examined ciliary length and frequency in the retinogeniculate pathway, a white matter tract commonly affected by diseases of aging but in which expression of cilia has not been characterized. We found expression of Arl13b, a marker of primary cilia, in a small group of Olig2-positive oligodendrocytes in the optic nerve, optic chiasm, and optic tract in young and aged C57BL/6 wild-type mice. While the ciliary length and ciliated oligodendrocyte cells were constant in young mice in the retinogeniculate pathway, there was a significant increase in ciliary length in the anterior optic nerve as compared to the aged animals. Morphometric analysis confirmed a specific increase in the ciliation rate of CC1+ /Olig2+ oligodendrocytes in aged mice compared with young mice. Thus, the prevalence of primary cilia in oligodendrocytes in the visual pathway and the age-related changes in ciliation suggest that they may play important roles in white matter and age-associated optic neuropathies.

High Altitude as a Risk Factor for the Development of Nonarteritic Anterior Ischemic Optic Neuropathy.

BACKGROUND: Episodic high-altitude exposure leads to optic disc edema and retinopathy. It is uncertain whether high-altitude exposure is a risk factor for nonarteritic anterior ischemic optic neuropathy (NAION). METHODS: We performed a single-center, retrospective, cross-sectional case study of 5 patients with high-altitude-associated NAION (HA-NAION) from April 2014 to April 2019. Main study parameters included known vascular risk factors for NAION, evolution of visual acuity, visual field, optic disc, and macula measurements. RESULTS: We studied 5 eyes of 5 patients with HA-NAION that occurred at 7,000-9,000 ft above sea level, 28 patients with classic NAION that developed at sea level (normal altitude NAION or NA-NAION), and 40 controls. All 5 patients with HA-NAION had clinically confirmed NAION by a neuro-ophthalmologist within 3-21 days of onset and comprehensive follow-up evaluations (average follow-up of 23 months). Other than high-altitude exposure, 4 of 5 patients had undiagnosed obstructive sleep apnea (OSA, apnea-hypopnea index 5.4-22.2) and 1 had systemic vascular risk factors. All patients had disc-at-risk in the contralateral eye. The best-corrected distance visual acuity was 20/20 to 20/70 (median logMAR 0) at presentation and 20/70 to counting finger (median logMAR 0) at ≥6 months. Automated static perimetry revealed average mean deviation of -18.6 dB at presentation and -22.1 dB at ≥6 months. The average retinal nerve fiber layer was 244 µm (80-348 µm) at onset and 59 µm (55-80 µm) at ≥6 months. The average ganglion cell complex thickness was 50 µm (43-54 µm) at onset and 52 µm (50-55 µm) at ≥6 months. The patients with OSA were started on home continuous positive airway pressure treatment. Visual outcomes were similar in patients with HA-NAION and NA-NAION. - After addressing all NAION risk factors, no new events occurred in the HA-NAION group within 2-8 years with or without repeat high-altitude exposure. CONCLUSIONS: NAION can occur under high-altitude conditions. HA-NAION is associated with relatively younger age at onset, disc-at-risk, and OSA. These patients exhibit a relatively progressive course of vision loss after initial onset and severe thinning of optic nerves on optical coherence tomography. Treatment for OSA is recommended, especially with repeated high-altitude exposure.

Upregulation of retinal VEGF and connexin 43 in murine nonarteritic anterior ischemic optic neuropathy induced with 577 nm laser.

Nonarteritic anterior ischemic optic neuropathy (NAION) is a common acute optic neuropathy and cause of irreversible vision loss in those older than 50 years of age. There is currently no effective treatment for NAION and the biological mechanisms leading to neuronal loss are not fully understood. Promising novel targets include glial cells activation and intercellular communication mediated by molecules such as gap junction protein Connexin 43 (Cx43), which modulate neuronal fate in central nervous system disorders. In this study, we investigated retinal glial changes and neuronal loss following a novel NAION animal model using a 577 nm yellow laser. We induced unilateral photochemical thrombosis using rose bengal at the optic nerve head vasculature in adult C57BL/6 mice using a 577 nm laser and performed morphometric analysis of the retinal structure using serial in vivo optical coherence tomography (OCT) and histology for glial and neuronal markers. One day after experimental NAION, in acute phase, OCT imaging revealed peripapillary thickening of the retinal ganglion cell complex (GCC, baseline: 79.5 ±â€¯1.0 µm, n = 8; NAION: 93.0 ±â€¯2.5 µm, n = 8, P < 0.01) and total retina (baseline: 202.9 ±â€¯2.4 µm, n = 8; NAION: 228.1 ±â€¯6.8 µm, n = 8, P < 0.01). Twenty-one days after ischemia, at a chronic phase, there was significant GCC thinning (baseline 78.3 ±â€¯2.1 µm, n = 6; NAION: 72.2 ±â€¯1.9 µm, n = 5, P < 0.05), mimicking human disease. Examination of molecular changes in the retina one day after ischemia revealed that NAION induced a significant increase in retinal VEGF levels (control: 2319 ±â€¯195, n = 5; NAION: 4549 ±â€¯683 gray mean value, n = 5, P < 0.05), which highly correlated with retinal thickness (r = 0.89, P < 0.05). NAION also led to significant increase in mRNA level for Cx43 (Gj1a) at day 1 (control: 1.291 ±â€¯0.38; NAION: 3.360 ±â€¯0.58 puncta/mm2, n = 5, P < 0.05), but not of glial fibrillary acidic protein (Gfap) at the same time (control: 2,800 ±â€¯0.59; NAION: 4,690 ±â€¯0.90 puncta/mm2 n = 5, P = 0.19). Retinal ganglion cell loss at day 21 was confirmed by a 30% decrease in Brn3a+ cells (control: 2,844 ±â€¯235; NAION: 2,001 ±â€¯264 cells/mm2, n = 4, P < 0.05). We described a novel protocol of NAION induction by photochemical thrombosis using a 577 nm laser, leading to retinal edema and VEGF increase at day 1 and RGCs loss at day 21 after injury, consistent with the pathophysiology of human NAION. Early changes in glial cells intercommunication revealed by increased Cx43+ gap junctions are consistent with a retinal glial role in mediating cell-to-cell signaling after an ischemic insult. Our study demonstrates an early glial response in a novel NAION animal model and reveals glial intercommunication molecules such as Cx43 as a promising therapeutic target in acute NAION.

Multicolor Imaging of Optic Disc Drusen.

ABSTRACT: Optic disc drusen (ODD) are calcified deposits at the anterior optic nerve that are often detectable by ophthalmic imaging, including optical coherence tomography and fundus autofluorescence imaging. Multicolor (MC) imaging is a novel modality that captures reflectance of blue, green, and near-infrared laser lights with confocal scanning laser ophthalmoscopy to rapidly acquire high-resolution reflectance images of the optic disc and retina. Here, we show an eye with 3 MC imaging features of ODD, including prominent green hyperreflectance of the optic disc, green sheathing of the papillary and peripapillary vasculature (arterioles > venules), and presence of orange superficial ODD. MC imaging can provide rapid high-resolution assessment of eyes with optic nerve head elevation to help distinguish pseudopapilledema vs papilledema in children and adults without dilation, and future large studies incorporating MC imaging will help determine its contribution in the diagnosis and monitoring of ODD and assessment of other causes of optic nerve head elevation.

Updates on ophthalmic imaging features of optic disc drusen, papilledema, and optic disc edema.

PURPOSE OF REVIEW: Optic nerve head elevation can be associated with vision loss. This review provides an update regarding key features of optic disc drusen (ODD) compared with papilledema from increased intracranial pressure and optic disc edema from other causes. RECENT FINDINGS: Clinical history and funduscopic examination are not sufficient to correctly diagnose different causes of optic nerve head elevation. Multimodal ophthalmic imaging is noninvasive and should be used as first-line diagnostic testing to distinguish optic disc edema or papilledema from pseudoedema. Advanced ophthalmic imaging, including enhanced depth imaging optical coherence tomography (EDI-OCT) and autofluorescence imaging, can visualize ODD at high resolution and determine whether there is optic disc edema. OCT angiography does not require contrast and can rapidly visualize papillary, peripapillary, and macular microvasculature and identify important vascular biomarker of ischemia and, potentially, visual prognosis. SUMMARY: Multimodal ophthalmic imaging can help in the diagnosis of ODD and optic disc edema and identify patients at high risk of vision loss and neurological issues in order to ensure appropriate diagnosis and treatment.

Variation in Evolving Optic Neuritis.

BACKGROUND: The typical natural history of optic neuritis is subjected to important exceptions. Recognition of these exceptions has led to valuable insights regarding specific etiologies of optic neuritis. Exceptions to the natural history of recovering optic neuritis are well-defined (e.g., chronic relapsing inflammatory optic neuropathy), but exceptions to the natural history of evolving optic neuritis are less so. METHODS: Medical records of patients illustrating an atypical course of evolving optic neuritis were reviewed in a retrospective manner. Each patient was treated by at least one of the authors. RESULTS: Four patients were identified who illustrated an atypical natural history of incipient optic neuritis. Diagnoses included idiopathic optic neuritis, seropositive neuromyelitis optica spectrum disease, anti-myelin oligodendrocyte glycoprotein antibody disease, and multiple sclerosis in 1 patient each. Features of interest included an atypical temporal relationship between development of pain and onset of clinical optic neuropathy, an unusually protracted duration of pain, and an unusually long duration of worsening optic neuropathy before stabilization. CONCLUSIONS: This case series illustrates the substantial clinical heterogeneity which may be observed in the evolution of optic neuritis. The temporal relationship between development of pain and onset of clinical optic neuropathy, the duration of pain, and duration of worsening optic neuropathy before stabilization are all subjected to significant variability. Although most patients with optic neuritis present with painful vision loss which progresses over 1 week or less, careful attention to the exceptions described herein may facilitate earlier recognition of diagnostically challenging cases.

Systemic hypoxia led to little retinal neuronal loss and dramatic optic nerve glial response.

Vision loss is a devastating consequence of systemic hypoxia, but the cellular mechanisms are unclear. We investigated the impact of acute hypoxia in the retina and optic nerve. We induced systemic hypoxia (10% O2) in 6-8w mice for 48 h and performed in vivo imaging using optical coherence tomography (OCT) at baseline and after 48 h to analyze structural changes in the retina and optic nerve. We analyzed glial cellular and molecular changes by histology and immunofluorescence and the impact of pretreatment with 4-phenylbutyric acid (4-PBA) in oligodendroglia survival. After 48 h hypoxia, we found no change in ganglion cell complex thickness and no loss of retinal ganglion cells. Despite this, there was significantly increased expression of CCAAT-enhancer-binding protein homologous protein (CHOP), a marker of endoplasmic reticulum stress, in the retina and optic nerve. In addition, hypoxia induced obvious increase of GFAP expression in the anterior optic nerve, where it co-localized with CHOP, and significant loss of Olig2+ oligodendrocytes. Pretreatment with 4-PBA, which has been shown to reduce endoplasmic reticulum stress, rescued total Olig2+ oligodendrocytes and increased the pool of mature (CC-1+) but not of immature (PDGFRa+) oligodendrocytes. Consistent with a selective vulnerability of the retina and optic nerve in hypoxia, the most striking changes in the 48 h murine model of hypoxia were in glial cells in the optic nerve, including increased CHOP expression in the astrocytes and loss of oligodendrocytes. Our data support a model where glial dysfunction is among the earliest events in systemic hypoxia - suggesting that glia may be a novel target in treatment of hypoxia.

Immunoinhibitory checkpoint deficiency in medium and large vessel vasculitis.

Giant cell arteritis (GCA) causes autoimmune inflammation of the aorta and its large branches, resulting in aortic arch syndrome, blindness, and stroke. CD4+ T cells and macrophages form organized granulomatous lesions in the walls of affected arteries, destroy the tunica media, and induce ischemic organ damage through rapid intimal hyperplasia and luminal occlusion. Pathogenic mechanisms remain insufficiently understood; specifically, it is unknown whether the unopposed activation of the immune system is because of deficiency of immunoinhibitory checkpoints. Transcriptome analysis of GCA-affected temporal arteries revealed low expression of the coinhibitory ligand programmed death ligand-1 (PD-L1) concurrent with enrichment of the programmed death-1 (PD-1) receptor. Tissue-residing and ex vivo-generated dendritic cells (DC) from GCA patients were PD-L1lo, whereas the majority of vasculitic T cells expressed PD-1, suggesting inefficiency of the immunoprotective PD-1/PD-L1 immune checkpoint. DC-PD-L1 expression correlated inversely with clinical disease activity. In human artery-SCID chimeras, PD-1 blockade exacerbated vascular inflammation, enriched for PD-1+ effector T cells, and amplified tissue production of multiple T-cell effector cytokines, including IFN-γ, IL-17, and IL-21. Arteries infiltrated by PD-1+ effector T cells developed microvascular neoangiogenesis as well as hyperplasia of the intimal layer, implicating T cells in the maladaptive behavior of vessel wall endogenous cells. Thus, in GCA, a breakdown of the tissue-protective PD1/PD-L1 checkpoint unleashes vasculitic immunity and regulates the pathogenic remodeling of the inflamed arterial wall.

LncRNA Gm2044 promotes 17ß-estradiol synthesis in mpGCs by acting as miR-138-5p sponge.

Long noncoding RNAs (lncRNAs) have been demonstrated to play vital roles in mammalian reproduction. Our previous research revealed that lncRNA Gm2044 is highly expressed in mouse spermatocytes and regulates male germ cell function. The gene annotation database BioGPS shows that Gm2044 is not only highly expressed in testicular tissue but also in ovarian tissue, which suggests that Gm2044 may be involved in female reproductive development. In this study, we confirmed that lncRNA Gm2044 promotes 17ß-estradiol synthesis in mouse pre-antral follicular granulosa cells (mpGCs). Furthermore, bioinformatics methods, western blot, and the luciferase assay proved that Gm2044 functions as a miR-138-5p sponge to inhibit the direct target of miR-138-5p, Nr5a1, which enhances 17ß-estradiol synthesis through cyp19a1 activation. Taken together, our results provide an insight into the mechanistic roles of lncRNA Gm2044 for 17ß-estradiol synthesis by acting as competing-endogenous RNAs to modulate the function of mpGCs. Studying the potential lncRNAs, which regulate estradiol release, will be beneficial for the diagnosis and treatment of steroid hormone-related disease.

Structure-Function Analysis of Nonarteritic Anterior Ischemic Optic Neuropathy and Age-Related Differences in Outcome.

BACKGROUND: The optic nerve head is vulnerable to ischemia leading to anterior ischemic optic neuropathy (AION), the most common acute optic neuropathy in those older than 50 years of age. METHODS: We performed a cross-sectional study of 55 nonarteritic anterior ischemic optic neuropathy (NAION) eyes in 34 patients to assess clinical outcome and perform structure-function correlations. RESULTS: The peak age of NAION onset was between 50 and 55 years. Sixty-seven percent of patients presented with their first event between the ages of 40 and 60 years, and 32% presented at ≤50 years. Those with NAION onset at age ≤50 years did not have significantly better visual outcome per logMAR visual acuity, automated perimetric mean deviation (PMD) or optical coherence tomography (OCT) measurements. Kaplan-Meier survival curve and multivariate Cox proportional regression analysis showed that age >50 years at NAION onset was associated with greater risk of second eye involvement, with hazard ratio of 20. Older age at onset was significantly correlated with greater thinning of the ganglion cell complex (GCC) (P = 0.022) but not with logMAR visual acuity, PMD, or thinning of retinal nerve fiber layer (RNFL). Using area under receiver operating characteristic curve analyses, we found that thinning of RNFL and GCC was best able to predict visual outcome, and that mean RNFL thickness >65 µm or macular GCC thickness >55 µm significantly correlated with good visual field outcome. CONCLUSIONS: We showed that NAION onset at age >50 years had a greater risk of second eye involvement. Patients with OCT mean RNFL thickness >65 µm and mean macular ganglion cell complex thickness >55 µm had better visual outcomes.

Analysis of genetic characteristics and reproductive risks of balanced complex chromosome rearrangement carriers in China.

To examine the type, characteristics and meiotic behavior of balanced complex chromosome rearrangements (CCRs) and their relationship with reproductive abnormalities in Chinese people, karyotype analyses were performed in 1063 couples with reproductive abnormalities using G-banding technology. Additional data were retrieved from a Chinese database and analyzed statistically with the karyotype and clinic data of CCRs. Two CCR carriers were found among the 1063 couples, and in all a total of 124 CCR carriers with the complete information were identified in the karyotype analysis and the database search. Our results showed that simple 3-way or 4-way translocations were the most common types, present in 64/124 (51.6%) of CCRs. Double two-way translocations accounted for 26.6% and exceptional CCRs accounted for 21.6% of total cases. General risk of 77.6% for spontaneous abortions and 9.7% for an abnormal child were calculated based on 339 pregnancies of 124 carriers. Pregnancy consequences could be significantly associated with the type of CCRs. Abnormal pregnancy was frequently associated with CCRs on chromosome 8, while dyszoospermia was frequently associated with CCRs on chromosome 1 among the males. The most frequent mode of segregation was 3:3 adjacent-1 (8/12) in 12 abnormal karyotypes. Short chromosomes (groups D-G) were involved in 46.2% of CCRs showing 3:2, 4:2 and 5:3 segregation ratios. In conclusion, carriers of balanced CCRs have a high risk of an abortion and/or a chromosomally unbalanced child. The incidence of spermatogenic defect in male CCR carriers is high, and male infertility is associated with CCRs. Hence, identifying the types of CCRs, chromosomes involved, translocated segments of chromosomes, etc. will provide crucial information for prenatal diagnosis and genetic counseling for carriers of balanced CCRs.

Accessory lateral rectus in a patient with normal ocular motor control.

Although supernumerary extraocular muscles are common in monkeys and other species, they are relatively rare in humans and typically are noted in the context of childhood strabismus. We present a case of an incidentally found unilateral accessory lateral rectus muscle in a 51-year-old woman with normal ocular motor control. In this patient, the accessory lateral rectus was approximately 10% the size of a normally sized lateral rectus muscle. It originated from the orbital apex, traveled between the optic nerve and the lateral rectus and attached to the superolateral aspect of the globe. This unique case demonstrates that accessory lateral rectus in humans may have no impact on eye movement and ocular alignment.

Development of a novel SupraChoroidal-to-Optic-NervE (SCONE) drug delivery system.

PURPOSE: Targeted drug delivery to the optic nerve head may be useful in the preclinical study and later clinical management of optic neuropathies, however, there are no FDA-approved drug delivery systems to achieve this. The purpose of this work was to develop an optic nerve head drug delivery technique. METHODS: Different strategies to approach the optic nerve head were investigated, including standard intravitreal and retroorbital injections. A novel SupraChoroidal-to-Optic-NervE (SCONE) delivery was optimized by creating a sclerotomy and introducing a catheter into the suprachoroidal space. Under direct visualization, the catheter was guided to the optic nerve head. India ink was injected. The suprachoroidal approach was performed in New Zealand White rabbit eyes in vivo (25 animals total). Parameters, including microneedle size and design, catheter design, and catheter tip angle, were optimized ex vivo and in vivo. RESULTS: Out of the candidate optic nerve head approaches, intravitreal, retroorbital, and suprachoroidal approaches were able to localize India ink to within 2 mm of the optic nerve. The suprachoroidal approach was further investigated, and after optimization, was able to deposit India ink directly within the optic nerve head in up to 80% of attempts. In eyes with successful SCONE delivery, latency and amplitude of visual evoked potentials was not different than the naïve untreated eye. CONCLUSIONS: SCONE delivery can be used for targeted drug delivery to the optic nerve head of rabbits without measurable toxicity measured anatomically or functionally. Successful development of this system may yield novel opportunities to study optic nerve head-specific drug delivery in animal models, and paradigm-shifting management strategies for treating optic neuropathies. TRANSLATIONAL RELEVANCE: Here we demonstrate data on a new method for targeted delivery to the optic nerve head, addressing a significant unmet need in therapeutics for optic neuropathies.

Therapeutic benefit of idebenone in patients with Leber hereditary optic neuropathy: The LEROS nonrandomized controlled trial.

Leber hereditary optic neuropathy (LHON) is a mitochondrial disease leading to rapid and severe bilateral vision loss. Idebenone has been shown to be effective in stabilizing and restoring vision in patients treated within 1 year of onset of vision loss. The open-label, international, multicenter, natural history-controlled LEROS study (ClinicalTrials.gov NCT02774005) assesses the efficacy and safety of idebenone treatment (900 mg/day) in patients with LHON up to 5 years after symptom onset (N = 199) and over a treatment period of 24 months, compared to an external natural history control cohort (N = 372), matched by time since symptom onset. LEROS meets its primary endpoint and confirms the long-term efficacy of idebenone in the subacute/dynamic and chronic phases; the treatment effect varies depending on disease phase and the causative mtDNA mutation. The findings of the LEROS study will help guide the clinical management of patients with LHON.

Effect of Low Dietary Folate on Mouse Spermatogenesis and Spindle Assembly Checkpoint Dysfunction May Contribute to Folate Deficiency-Induced Chromosomal Instability in Cultured Mouse Spermatogonia.

Folate, as the initial substrate in one-carbon metabolism, is involved in the synthesis of important substances such as DNA, RNA, and protein. Folate deficiency (FD) is associated with male subfertility and impaired spermatogenesis, yet the underlying mechanisms are poorly understood. In the present study, we established an animal model of FD to investigate the effect of FD on spermatogenesis. GC-1 spermatogonia were used as a model to investigate the effect of FD on proliferation, viability, and chromosomal instability (CIN). Furthermore, we explored the expression of core genes and proteins of spindle assembly checkpoint (SAC), a signaling cascade ensuring accurate chromosome segregation and preventing CIN during mitosis. Cells were maintained in medium containing 0, 20, 200, or 2000 nM folate for 14 days. CIN was measured by using a cytokinesis-blocked micronucleus cytome assay. We found that sperm counts decreased significantly (p < 0.001) and the rate of sperm with defects in the head increased significantly (p < 0.05) in FD diet mice. We also found, relative to the folate-sufficient conditions (2000 nM), cells cultured with 0, 20, or 200 nM folate exhibited delayed growth and increased apoptosis in an inverse dose-dependent manner. FD (0, 20, or 200 nM) significantly induced CIN (p < 0.001, p < 0.001, and p < 0.05, respectively). Moreover, FD significantly and inverse dose dependently increased the mRNA and protein expression of several key SAC-related genes. The results indicate that FD impairs SAC activity, which contributes to mitotic aberrations and CIN. These findings establish a novel association between FD and SAC dysfunction. Thus, FD-impaired spermatogenesis may be partly due to genomic instability and proliferation inhibition of spermatogonia.