Retinal Axons Research Articles

Intraocular Delivery of a Collagen Mimetic Peptide Repairs Retinal Ganglion Cell Axons in Chronic and Acute Injury Models.

Vision loss through the degeneration of retinal ganglion cell (RGC) axons occurs in both chronic and acute conditions that target the optic nerve. These include glaucoma, in which sensitivity to intraocular pressure (IOP) causes early RGC axonal dysfunction, and optic nerve trauma, which causes rapid axon degeneration from the site of injury. In each case, degeneration is irreversible, necessitating new therapeutics that protect, repair, and regenerate RGC axons. Recently, we demonstrated the reparative capacity of using collagen mimetic peptides (CMPs) to heal fragmented collagen in the neuronal extracellular milieu. This was an important step in the development of neuronal-based therapies since neurodegeneration involves matrix metalloproteinase (MMP)-mediated remodeling of the collagen-rich environment in which neurons and their axons exist. We found that intraocular delivery of a CMP comprising single-strand fractions of triple helix human type I collagen prevented early RGC axon dysfunction in an inducible glaucoma model. Additionally, CMPs also promoted neurite outgrowth from dorsal root ganglia, challenged in vitro by partial digestion of collagen. Here, we compared the ability of a CMP sequence to protect RGC axons in both inducible glaucoma and optic nerve crush. A three-week +40% elevation in IOP caused a 67% degradation in anterograde transport to the superior colliculus, the primary retinal projection target in rodents. We found that a single intravitreal injection of CMP during the period of IOP elevation significantly reduced this degradation. The same CMP delivered shortly after optic nerve crush promoted significant axonal recovery during the two-week period following injury. Together, these findings support a novel protective and reparative role for the use of CMPs in both chronic and acute conditions affecting the survival of RGC axons in the optic projection to the brain.

Neuroinflammation, Microglia and Implications for Retinal Ganglion Cell Survival and Axon Regeneration in Traumatic Optic Neuropathy.

Traumatic optic neuropathy (TON) refers to a pathological condition caused by a direct or indirect insult to the optic nerves, which often leads to a partial or permanent vision deficit due to the massive loss of retinal ganglion cells (RGCs) and their axonal fibers. Retinal microglia are immune-competent cells residing in the retina. In rodent models of optic nerve crush (ONC) injury, resident retinal microglia gradually become activated, form end-to-end alignments in the vicinity of degenerating RGC axons, and actively internalized them. Some activated microglia adopt an amoeboid morphology that engulf dying RGCs after ONC. In the injured optic nerve, the activated microglia contribute to the myelin debris clearance at the lesion site. However, phagocytic capacity of resident retinal microglia is extremely poor and therefore the clearance of cellular and myelin debris is largely ineffective. The presence of growth-inhibitory myelin debris and glial scar formed by reactive astrocytes inhibit the regeneration of RGC axons, which accounts for the poor visual function recovery in patients with TON. In this Review, we summarize the current understanding of resident retinal microglia in RGC survival and axon regeneration after ONC. Resident retinal microglia play a key role in facilitating Wallerian degeneration and the subsequent axon regeneration after ONC. However, they are also responsible for producing pro-inflammatory cytokines, chemokines, and reactive oxygen species that possess neurotoxic effects on RGCs. Intraocular inflammation triggers a massive influx of blood-borne myeloid cells which produce oncomodulin to promote RGC survival and axon regeneration. However, intraocular inflammation induces chronic neuroinflammation which exacerbates secondary tissue damages and limits visual function recovery after ONC. Activated retinal microglia is required for the proliferation of oligodendrocyte precursor cells (OPCs); however, sustained activation of retinal microglia suppress the differentiation of OPCs into mature oligodendrocytes for remyelination after injury. Collectively, controlled activation of retinal microglia and infiltrating myeloid cells facilitate axon regeneration and nerve repair. Recent advance in single-cell RNA-sequencing and identification of microglia-specific markers could improve our understanding on microglial biology and to facilitate the development of novel therapeutic strategies aiming to switch resident retinal microglia’s phenotype to foster neuroprotection.

Long-term Effect of Permanent Demyelination on Axonal Survival in Multiple Sclerosis.

Background and ObjectivesTo investigate the long-term effect of permanent demyelination on axonal attrition by examining an association between intereye asymmetry of the multifocal visual evoked potential (mfVEP) latency delay and subsequent thinning of retinal ganglion cell axons in patients with a long-standing history of unilateral optic neuritis (ON).MethodsOnly patients with a significant degree of chronic demyelination (intereye latency asymmetry >5 ms) were included in this study. The level of optic nerve demyelination was estimated at baseline by the latency delay of mfVEP, while the degree of axonal loss was assessed by thinning of the retinal nerve fiber layer (RNFL) thickness between baseline and follow-up visits. Low-contrast visual acuity (LCVA) was also evaluated at baseline and follow-up. Patients were examined twice with an average interval of 6.1 ± 1.4 years.ResultsFrom 85 examined patients with multiple sclerosis, 28 satisfied inclusion criteria. Latency of the mfVEP was delayed, and RNFL thickness was reduced in ON eyes compared with fellow eyes at both visits. There was significant correlation between latency asymmetry and baseline or follow-up intereye RNFL thickness asymmetry. Intereye asymmetry of LCVA at baseline correlated with baseline latency asymmetry of mfVEP and baseline asymmetry of RNFL thickness. Latency of the mfVEP in ON eyes improved slightly during the follow-up period, whereas latency of the fellow eye remained stable. By contrast, RNFL thickness significantly declined in both ON and fellow eyes during the follow-up period. The rate of RNFL thinning in ON eyes, however, was more than 2 times faster compared with the fellow eyes (p < 0.001). Furthermore, baseline latency asymmetry significantly correlated with the rate of RNFL thinning in ON eyes during the follow-up (p < 0.001), explaining almost half of the variability of temporal RNFL progression. For each millisecond of latency delay (i.e., ∼0.5 mm of demyelination along the optic nerve), temporal RNFL thickness was annually reduced by 0.05%.DiscussionOur study provides clear in vivo evidence that chronic demyelination significantly accelerates axonal loss. However, because this process is slow and its effect is mild, long-term monitoring is required to establish and confidently measure the neurodegenerative consequences of demyelination.

Oligodendrocyte precursor cells sculpt the visual system by regulating axonal remodeling

Many oligodendrocyte precursor cells (OPCs) do not differentiate to form myelin, suggesting additional roles of this cell population. The zebrafish optic tectum contains OPCs in regions devoid of myelin. Elimination of these OPCs impaired precise control of retinal ganglion cell axon arbor size during formation and maturation of retinotectal connectivity and degraded functional processing of visual stimuli. Therefore, OPCs fine-tune neural circuits independently of their canonical role to make myelin.

Topographic map formation and the effects of NMDA receptor blockade in the developing visual system

The development of functional topography in the developing brain follows a progression from initially coarse to more precisely organized maps. To examine the emergence of topographically organized maps in the retinotectal system, we performed longitudinal visual receptive field mapping by calcium imaging in the optic tectum of GCaMP6-expressing transgenic Xenopus laevis tadpoles. At stage 42, just 1 d after retinal axons arrived in the optic tectum, a clear retinotopic azimuth map was evident. Animals were imaged over the following week at stages 45 and 48, over which time the tectal neuropil nearly doubled in length and exhibited more precise retinotopic organization. By microinjecting GCaMP6s messenger ribonucleic acid (mRNA) into one blastomere of two-cell stage embryos, we acquired bilateral mosaic tadpoles with GCaMP6s expression in postsynaptic tectal neurons on one side of the animal and in retinal ganglion cell axons crossing to the tectum on the opposite side. Longitudinal observation of retinotopic map emergence revealed the presence of orderly representations of azimuth and elevation as early as stage 42, although presynaptic inputs exhibited relatively less topographic organization than the postsynaptic component for the azimuth axis. Retinotopic gradients in the tectum became smoother between stages 42 and 45. Blocking N-methyl-D-aspartate (NMDA) receptor conductance by rearing tadpoles in MK-801 did not prevent the emergence of retinotopic maps, but it produced more discontinuous topographic gradients and altered receptive field characteristics. These results provide evidence that current through NMDA receptors is dispensable for coarse topographic ordering of retinotectal inputs but does contribute to the fine-scale organization of the retinotectal projection.

Eye Removal in Living Zebrafish Larvae to Examine Innervation-dependent Growth and Development of the Visual System

Zebrafish exhibit remarkable life-long growth and regenerative abilities. For example, specialized stem cell niches established during embryogenesis support continuous growth of the entire visual system, both in the eye and the brain. Coordinated growth between the retinae and the optic tectum ensures accurate retinotopic mapping as new neurons are added in the eyes and brain. To address whether retinal axons provide crucial information for regulating tectal stem and progenitor cell behaviors such as survival, proliferation, and/or differentiation, it is necessary to be able to compare innervated and denervated tectal lobes within the same animal and across animals. Surgical removal of one eye from living larval zebrafish followed by observation of the optic tectum achieves this goal. The accompanying video demonstrates how to anesthetize larvae, electrolytically sharpen tungsten needles, and use them to remove one eye. It next shows how to dissect brains from fixed zebrafish larvae. Finally, the video provides an overview of the protocol for immunohistochemistry and a demonstration of how to mount stained embryos in low-melting-point agarose for microscopy.

Characterization of Axon Guidance Phenotypes in Wnt/PCP Mutant Mice.

Our lab showed that the Wnt family proteins can function as axon guidance molecules and the planar cell polarity (PCP) pathway mediates the function of Wnts in axon guidance. One of the key evidences was by identifying the axon guidance defects in knockout or conditional knockout animals. We utilized a variety of axon tracing and labeling techniques, including immunohistochemistry (IHC), DiI, BDA, and fluorescent reporters (GFP or tdTomato). These studies have primarily been conducted in spinal cord commissural axons, but have been applied to retinal ganglion cell axons, corticospinal tract axons, dopaminergic and serotonergic projections.

Live Imaging of RNA Transport and Translation in Xenopus Retinal Axons.

In neurons, specific mRNAs are transported into axons, where their local translation supports essential cellular functions. Over the years, our knowledge of the molecular mechanisms underlying axonal mRNA translation has rapidly expanded. However, tools to study mRNA localization and translation in real time with high spatial precision were not available until recently. Here, we present a live imaging approach to examine axonal mRNA trafficking and translation simultaneously in Xenopus retinal ganglion cells (RGCs), using in vitro synthesized fluorescently labeled mRNAs coupled with a genetically encoded protein tagging system to visualize synthesizing peptides at single-molecule resolution. We further describe the process of image analysis in detail, thus providing a methodology that can be used to investigate new research questions in the field.

Trend of Improvement in Visual Field Deficits after Transsphenoidal Endoscopic Surgery in Pituitary Tumour Patients: A Case Series

Abstract&#x0D; Introduction: Pituitary tumours account for approximately 15% of all brain tumours, and the growing tumours press against the optic chiasm, resulting in impairment of visual function manifested as visual field defects, decreased visual acuity, and decreased color vision . Compression of the optic chiasm by pituitary tumours generally results in selective loss of the temporal VFs, or bitemporal hemianopia, implying that the nasal retinal fibers are preferentially damaged. The reason for this preferential damage is not fully understood. Transsphenoidal surgery has been reported to safely reduce the pressure on the anterior visual pathway in most patients. Improvement in visual function may occur after transsphenoidal decompression of the chiasm. Because improvement in visual function may occur from a variety of proposed biologic processes.&#x0D; Case Series : The number of patients according to gender was 71% male (10 people) while 29% female (4 people). The age distribution was found mostly at the age of 40-50 years 36% (5 people). The most common clinical symptoms were field disturbances 85% (12 people). Patients complained of visual field disturbances for 1-2 years as many as 58% (7 people). Vision before surgery is /6 as much as 45% (4 people). Improvements in vision were found for 1 month postoperatively as much as 22% (2 people).&#x0D; Discussion : Compression of the optic chiasm by pituitary tumours generally results in selective loss of the temporal VFs, or bitemporal hemianopia, implying that the nasal retinal fibers are preferentially damaged. The minimally invasive transsphenoidal approach can be used effectively for 95% of pituitary tumours. Exceptions are those large tumours with significant temporal or anterior cranial fossa extension. In such circumstances, transcranial approaches are often more appropriate. Occasionally, combined transsphenoidal and transcranial approaches are used. Nevertheless, some surgeons extend the basic transsphenoidal exposure in order to remove some of these tumours and avoid a craniotomy . Potential mechanisms of axonal injury from a compressive lesion include direct disruption of conduction along the axon, impaired axoplasmic flow, demyelination with impaired signal conduction, and ischemia from compression or stretching of the blood supply of the chiasm by the tumour. An early fast phase of improvement is consistent with restoration of signal conduction along retinal ganglion cell axons after removal of a compressive lesion.In some individuals, we observed the rapid restoration of normal vision, which would be consistent with this hypothesis. In these individuals, a physiologic conduction block is presumably the main mechanism of injury.&#x0D; Conclusion: The pattern of improvement of visual function after decompression of the anterior visual pathways suggests three phases of improvement. Improvement in visual function may occur after transsphenoidal decompression of the chiasm. Because improvement in visual function may occur from a variety of proposed biologic processes, we sought to better define this potential for improvement.

Characterization of Retinal Architecture in Spinocerebellar Ataxia Type 3 and Correlation with Disease Severity.

Neurodegeneration affects the brain and peripheral nervous system in spinocerebellar ataxia type 3 (SCA3). As the retina is also involved, studying the retinal architecture in a cohort of patients could reveal clinically relevant biomarkers. The aim is to investigate retinal architecture in SCA3 to identify potential biomarkers. We evaluated 38 patients with SCA3 and 25 healthy age-matched controls, who underwent visual acuity assessment, intraocular pressure measurement, and fundoscopy and macular and peripapillary spectral domain optical coherence tomography (SD-OCT). We measured the peripapillary retinal nerve fiber layer (pRNFL) thickness in each quadrant of the temporal-superior-nasal-inferior-temporal chart and the macular layer thicknesses in each sector of the inner circle of the Early Treatment Diabetic Retinopathy Study (IC-ETDRS) grid. Linear regression analysis was employed to test the associations between retinal parameters and age, disease duration, CAG repeats, and SARA (Scale of the Assessment and Rating of Ataxia) and ICARS (International Cooperative Ataxia Rating Scale) scores in SCA3. In all sectors, except for the temporal quadrant, pRNFL was significantly thinner in SCA3 patients than in controls. Average total macular, ganglion cell layer (GCL), and inner plexiform layer (IPL) thicknesses were significantly decreased in SCA3 patients in comparison to controls. The average total macular thickness and the average thicknesses of RNFL, GCL, and IPL negatively correlated with ICARS scores, whereas average GCL and IPL thicknesses negatively correlated with SARA scores. The retinal ganglion cells, their dendrites, and axons are selectively affected in SCA3 patients. The RNFL, GCL, and IPL thicknesses in SD-OCT correlate with the clinical phenotype and represent potential biomarkers for future clinical trials and natural history studies. © 2021 International Parkinson and Movement Disorder Society.

Correlations between cognitive assessment and RNFL changes in OCT in healthy aging subjects

AbstractBackgroundThe retinal nerve fibre layer (RNFL) comprises retinal ganglion cell axons and link the outer neuroretina to the higher visual cortex. The retina is the only part of the central nervous system that can be visualised directly and non‐invasively by optical coherence tomography (OCT). The study aimed to examine whether changes in the visual pathway are correlated with various domains of cognitive function assessment. We evaluated correlation between retinal structural (OCT) and neuropsychological (trail making test, TMT; Boston naming test, BNT) measures in healthy aging subjects.MethodIn the present cross‐sectional clinical study, 74 healthy aging subjects were recruited from the Optic Nerve Decline and Cognitive Changes (ONDCC) study. All participants underwent OCT peripapillary ring scan protocol for RNFL analysis, TMT testing for attention and executive function and BNT neuropsychological examination for the detection of word‐retrieval or naming difficulties. Averaged RNFL thickness measures were used to analyse the relationship with TMT and BNT. Correlations between TMT, BNT and OCT parameters were analysed using partial correlation and controlled for age and gender (P &lt; 0.05).ResultThe mean (SD) age of the current cohort is 67.5 (± 9.0 years). At baseline, significant correlation was observed between TMT‐test A and nasal RNFL thickness (r = ‐0.33, P = 0.009). The BNT also demonstrated mild but significant positive correlations with global (r = 0.32, P = 0.01), nasal (r = 0.27, P = 0.03) and nasal inferior (r = 0.33, P = 0.01) sectoral RNFL thicknesses respectively.ConclusionThe results indicated a significant correlation between RNFL thickness and executive function domain of cognitive assessment. Sectoral RNFL was also significantly correlated with naming ability (BNT) across the cohort of healthy elderly. These findings suggest that the RNFL measures may parallel cognitive decline in the aging population. Further studies are planned in confirmed cognitively impaired subjects.

Implantable patches assembled with mesenchymal stem cells and gelatin/silk fibroin composite microspheres for the treatment of traumatic optic neuropathy

The therapeutic benefits of transplanting mesenchymal stem cells (MSCs) to the injury site of traumatic optic neuropathy (TON) represents a crucial, yet unresolved question, the answer to which may facilitate development of innovative therapies for TON, a frustrating disease with no effective treatment. Herein, we address this issue by showing that MSC transplantation to the injury site is able to promote optic nerve regeneration in terms of retinal ganglion cell survival and axon regeneration using MSC@MS, implantable patches assembled with MSCs and gelatin/silk fibroin composite microspheres, which integrate 3D culture and trypsin-free harvesting, and can increase in vivo retention of transplanted MSCs. In contrast, conventional bolus injection of MSC suspension is not potent enough to produce significant benefits. We also show MSCs in MSC@MS demonstrate superior gene expression profiles over monolayer culture after IGF-1 stimulation. In future study, microspheres in MSC@MS can further be harnessed to deliver therapeutic cargoes that boost the potency of MSCs or treat TON synergistically with MSCs, making MSC@MS a versatile platform to develop tailored MSC-based therapies for TON.

Human Pluripotent Stem Cell-Derived Neural Progenitor Cells Promote Retinal Ganglion Cell Survival and Axon Recovery in an Optic Nerve Compression Animal Model.

Human pluripotent stem cell-derived neural progenitor cells (NPCs) have the potential to recover from nerve injury. We previously reported that human placenta-derived mesenchymal stem cells (PSCs) have neuroprotective effects. To evaluate the potential benefit of NPCs, we compared them to PSCs using R28 cells under hypoxic conditions and a rat model of optic nerve injury. NPCs and PSCs (2 × 106 cells) were injected into the subtenon space. After 1, 2, and 4 weeks, we examined changes in target proteins in the retina and optic nerve. NPCs significantly induced vascular endothelial growth factor (Vegf) compared to age-matched shams and PSC groups at 2 weeks; they also induced neurofilaments in the retina compared to the sham group at 4 weeks. In addition, the expression of brain-derived neurotrophic factor (Bdnf) was high in the retina in the NPC group at 2 weeks, while expression in the optic nerve was high in both the NPC and PSC groups. The low expression of ionized calcium-binding adapter molecule 1 (Iba1) in the retina had recovered at 2 weeks after NPC injection and at 4 weeks after PSC injection. The expression of the inflammatory protein NLR family, pyrin domain containing 3 (Nlrp3) was significantly reduced at 1 week, and that of tumor necrosis factor-α (Tnf-α) in the optic nerves of the NPC group was lower at 2 weeks. Regarding retinal ganglion cells, the expressions of Brn3a and Tuj1 in the retina were enhanced in the NPC group compared to sham controls at 4 weeks. NPC injections increased Gap43 expression from 2 weeks and reduced Iba1 expression in the optic nerves during the recovery period. In addition, R28 cells exposed to hypoxic conditions showed increased cell survival when cocultured with NPCs compared to PSCs. Both Wnt/β-catenin signaling and increased Nf-ĸb could contribute to the rescue of damaged retinal ganglion cells via upregulation of neuroprotective factors, microglial engagement, and anti-inflammatory regulation by NPCs. This study suggests that NPCs could be useful for the cellular treatment of various optic neuropathies, together with cell therapy using mesenchymal stem cells.

Cytokine and Complement Response in the Glaucomatous βB1-CTGF Mouse Model.

Glaucoma is a complex neurodegenerative disease leading to a loss of retinal ganglion cells (RGCs) and optic nerve axons. An activation of the complement system seems to contribute to cell loss in this disease. Hence, we investigated a possible initiation of the complement system and the cytokine response in the βB1-CTGF glaucoma model. In these mice, intraocular pressure is elevated, which is the main glaucoma risk factor in patients, and RGC loss occurs at 15 weeks of age. Therefore, quantitative real-time PCR and immunohistological experiments were performed in 5-, 10-, and 15-week-old βB1-CTGF animals and their corresponding wildtypes (WT) to analyze the expression of several complement system factors. We could show that mRNA levels of the terminal complement pathway components C3 and C5 (Hc) were upregulated at 10 weeks. In accordance, more C3+ and membrane attack complex+ cells were observed in transgenic retinae. Further, the C5a receptor anaphylatoxin receptor (C5ar) and the complement component C5a receptor 1 (C5ar1; CD88) mRNA levels were upregulated in 10- and 15-week-old βB1-CTGF mice. Interestingly, all three activation routes of the complement system were elevated in βB1-CTGF mice at some age. Especially C1q, as a marker of the classical pathway, was significantly increased at all investigated ages. Furthermore, mRNA expression levels of interferon-γ (Infg) were upregulated at 5 weeks, while Cxcl1 and Cxcl2 mRNA levels were upregulated at 10 and 15 weeks. The mRNA levels of the chemokines Cxcl10 were increased at all ages in βB1-CTGF mice. These results lead to the assumption that in these transgenic mice, a complement activation mainly through the classical pathway as well as a cytokine response plays a major role in cell death.

Live imaging of retinotectal mapping reveals topographic map dynamics and a previously undescribed role for Contactin 2 in map sharpening.

Organization of neuronal connections into topographic maps is essential for processing information. Yet, our understanding of topographic mapping has remained limited by our inability to observe maps forming and refining directly in vivo. Here, we used Cre-mediated recombination of a new colorswitch reporter in zebrafish to generate the first transgenic model allowing the dynamic analysis of retinotectal mapping in vivo. We found that the antero-posterior retinotopic map forms early but remains dynamic, with nasal and temporal retinal axons expanding their projection domains over time. Nasal projections initially arborize in the anterior tectum but progressively refine their projection domain to the posterior tectum, leading to the sharpening of the retinotopic map along the antero-posterior axis. Finally, using a CRISPR-mediated mutagenesis approach, we demonstrate that the refinement of nasal retinal projections requires the adhesion molecule Contactin 2. Altogether, our study provides the first analysis of a topographic map maturing in real time in a live animal and opens new strategies for dissecting the molecular mechanisms underlying precise topographic mapping in vertebrates.

Serotonin transporter-mediated molecular axis regulates regional retinal ganglion cell vulnerability and axon regeneration after nerve injury.

Molecular insights into the selective vulnerability of retinal ganglion cells (RGCs) in optic neuropathies and after ocular trauma can lead to the development of novel therapeutic strategies aimed at preserving RGCs. However, little is known about what molecular contexts determine RGC susceptibility. In this study, we show the molecular mechanisms underlying the regional differential vulnerability of RGCs after optic nerve injury. We identified RGCs in the mouse peripheral ventrotemporal (VT) retina as the earliest population of RGCs susceptible to optic nerve injury. Mechanistically, the serotonin transporter (SERT) is upregulated on VT axons after injury. Utilizing SERT-deficient mice, loss of SERT attenuated VT RGC death and led to robust retinal axon regeneration. Integrin β3, a factor mediating SERT-induced functions in other systems, is also upregulated in RGCs and axons after injury, and loss of integrin β3 led to VT RGC protection and axon regeneration. Finally, RNA sequencing analyses revealed that loss of SERT significantly altered molecular signatures in the VT retina after optic nerve injury, including expression of the transmembrane protein, Gpnmb. GPNMB is rapidly downregulated in wild-type, but not SERT- or integrin β3-deficient VT RGCs after injury, and maintaining expression of GPNMB in RGCs via AAV2 viruses even after injury promoted VT RGC survival and axon regeneration. Taken together, our findings demonstrate that the SERT-integrin β3-GPNMB molecular axis mediates selective RGC vulnerability and axon regeneration after optic nerve injury.

Global and Regional Damages in Retinal Ganglion Cell Axon Bundles Monitored Non-Invasively by Visible-Light Optical Coherence Tomography Fibergraphy.

Retinal ganglion cells (RGCs) exhibit compartmentalized organization, receiving synaptic inputs through their dendrites and transmitting visual information from the retina to the brain through the optic nerve. Little is known about the structure of RGC axon bundles extending from individual RGC somas to the optic nerve head (ONH) and how they respond to disease insults. We recently introduced visible-light optical coherence tomography fibergraphy (vis-OCTF), a technique for directly visualizing and analyzing mouse RGC axon bundles in vivo In this study, we validated vis-OCTF's ability to quantify RGC axon bundles with an increased number of RGCs using mice deficient in BCL2-associated X protein (BAX-/-). Next, we performed optic nerve crush (ONC) injury on wild-type (WT) mice and showed that the changes in RGC axon bundle width and thickness were location-dependent. Our work demonstrates the potential of vis-OCTF to longitudinally quantify and track RGC damage at single axon bundle level in optic neuropathies.SIGNIFICANCE STATEMENT Nearly all clinical and preclinical studies measure the retinal nerve fiber (RNFL) thickness as the sole indicator of retinal ganglion cell (RGC) damage without investigating RGC axon bundles directly. We demonstrated visible-light optical coherence tomography fibergraphy (vis-OCTF) to directly quantify global and regional RGC axon bundle organizations in vivo as a new biomarker for RGC health. We validated in vivo vis-OCTF measures using both confocal microscopy of the immunostained flat-mounted retina and numerical simulations. Vis-OCTF for monitoring RGC axon bundle organization has the potential to bring new insight into RGC damage in optic neuropathies.

Effect of papaverine on axonal outgrowth of primary retinal ganglion cells of Sprague Dawley rats

Increasing the level of cyclic adenosine 3, 5′-monophosphate is an important mechanism for axon outgrowth and recovery of central nervous system function. This study aimed to investigate the effects of papaverine, a non-specific phosphodiesterase inhibitor, on axon outgrowth of primary retinal ganglion cells from Sprague Dawley rats. Experiments were performed on primary retinal ganglion cells extracted from Sprague Dawley rat pups within 48–72 h of birth. At 24 h after seeding, immunofluorescence was used to identify and calculate the purity of retinal ganglion cells isolated by an improved two-step immunopanning method developed by author Sujia Ma. The effects of a range of papaverine concentrations on axon outgrowth of primary retinal ganglion cells cultures were observed by immunofluorescence and measured by ImageJ software at three different time points: 24, 48, and 72 h. The ability of papaverine to enable retinal ganglion cells to overcome the inhibitory effects of glial scar component chondroitin sulfate proteoglycans was examined using chondroitin sulfate proteoglycans-coated culture plates. Rp-adenosine 3′,5′-cyclic monophosphorothioate triethylammonium salt, a blocking agent of cyclic adenosine 3, 5′-monophosphate, and dibutyryl cyclic adenosine 3, 5′-monophosphate, an analogue of cyclic adenosine 3, 5′-monophosphate, were used to explore the mechanism of papaverine in promoting retinal ganglion cells axon outgrowth. Our study shows 2 μg/mL papaverine concentration significantly promoted axon outgrowth in primary retinal ganglion cells and restored axon outgrowth of these cells on chondroitin sulfate proteoglycans. Axon outgrowth was blocked by Rp-adenosine 3′,5′-cyclic monophosphorothioate triethylammonium salt and obviously promoted by dibutyryl cyclic adenosine 3, 5′-monophosphate. Our study is the first to describe the use of papaverine to promote axon outgrowth of retinal ganglion cells. These results may help to expand the application of papaverine, and they provide a cytological basis for papaverine in the treatment of optic nerve injury caused by glaucoma and other diseases.

Bruch membrane opening-minimum rim width and retinal nerve fiber layer thickness in myopic children

Background/objectivesIt is debatable whether the decrease of retinal nerve fiber layer (RNFL) thickness in myopic people under 18 years of age is due to insufficient measurement techniques or to real physical loss of retinal ganglion cells and axons. Hence, to better understand the relationship between the degree of myopia and the neuroretinal rim (NR), we aim to investigate the NR in the eyes of healthy myopic children using the novel measurement algorithms of spectral-domain optical coherence tomography (SD-OCT). Subjects/methodsThis prospective, cross-sectional study includes 378 left eyes of 378 (301 female) participants divided into three groups according to their spherical equivalent (SE) refractive error (RE) [Group-1(G1), -1.00 ≤ SE ≤ 1.00 diopters (D); Group-2 (G2), -4.00 ≤ SE < -1.00 D; Group-3 (G3), SE<-4.00 D]. All participants underwent a full ophthalmic examination, including biometric and pachymetric measurements. Standard peripapillary RNFL, as well as the novel algorithms, Bruch's membrane opening-minimum rim width (BMO-MRW), and RNFL acquired using the anatomic positioning system (APS-RNFL) were obtained by SD-OCT. Nasal, temporal, temporal-inferior, temporal-superior, nasal-inferior, nasal-superior sectors’ and their general (global) averages were recorded. Rim areas and disc sizes were measured via confocal scanning laser ophthalmoscopy. Global and the six sectors’ averages were recorded. ResultsG1 consisted of 141 subjects, G2 consisted of 89, and G3 consisted of 48. The sex distribution (p = 0.112) and mean age (p = 0.129) of the groups were similar. The mean global averages of the standard RNFLs were 96 ± 14.4 µ in G1, 93.8 ± 12.9 µ in G2, and 86 ± 11.8 µ in G3. The mean global averages of the APS-RNFLs were 103.9 ± 97 µ in G1, 103.3 ± 10.6 µ in G2, and 102 ± 10.6 µ in G3. The mean global averages of the BMO-MRW were 374.4 ± 57.7 µ in G1, 373.2 ± 62.2 µ in G2, and 351.9 ± 63.9 µ in G3. For the global averages, APS-RNFL and BMO-MRW did not detect any difference between the three groups (p = 0.563, p = 0.089, respectively), but the standard RNFL did (p < 0.001). Standard RNFL and APS-RNFL were found to be well correlated; however, the correlations between BMO-MRW and standard RNFL or APS-RNFL were either absent or very weak. All three methods showed weak but significant negative correlations with high myopic spherical RE, especially those in the standard RNFL. Moderately negative correlations were found between BMO-MRW and disc size in all sectors (highest in the nasal sector; r = -0.387, p < 0.001). However, there was almost no significant relationship between disc size and standard RNFL or APS-RNFL. Moderately significant negative correlations were observed between the groups categorically and standard RNFL in almost all sectors, while this was much less with APS-RNFL and was not observed in almost any sector with BMO-MRW. ConclusionsWhen evaluating the NR in healthy myopic children, it was found that, in particular, BMO-MRW and APS-RNFL are less effected by RE degree compared to standard RNFL. BMO-MRW and APS-RNFL should be used on these children to avoid the possible misdiagnosis of glaucoma.

Retinal Ganglion Cell Axon Regeneration Requires Complement and Myeloid Cell Activity within the Optic Nerve.

Axon regenerative failure in the mature CNS contributes to functional deficits following many traumatic injuries, ischemic injuries, and neurodegenerative diseases. The complement cascade of the innate immune system responds to pathogen threat through inflammatory cell activation, pathogen opsonization, and pathogen lysis, and complement is also involved in CNS development, neuroplasticity, injury, and disease. Here, we investigated the involvement of the classical complement cascade and microglia/monocytes in CNS repair using the mouse optic nerve injury (ONI) model, in which axons arising from retinal ganglion cells (RGCs) are disrupted. We report that central complement C3 protein and mRNA, classical complement C1q protein and mRNA, and microglia/monocyte phagocytic complement receptor CR3 all increase in response to ONI, especially within the optic nerve itself. Importantly, genetic deletion of C1q, C3, or CR3 attenuates RGC axon regeneration induced by several distinct methods, with minimal effects on RGC survival. Local injections of C1q function-blocking antibody revealed that complement acts primarily within the optic nerve, not retina, to support regeneration. Moreover, C1q opsonizes and CR3+ microglia/monocytes phagocytose growth-inhibitory myelin debris after ONI, a likely mechanism through which complement and myeloid cells support axon regeneration. Collectively, these results indicate that local optic nerve complement-myeloid phagocytic signaling is required for CNS axon regrowth, emphasizing the axonal compartment and highlighting a beneficial neuroimmune role for complement and microglia/monocytes in CNS repair.SIGNIFICANCE STATEMENT Despite the importance of achieving axon regeneration after CNS injury and the inevitability of inflammation after such injury, the contributions of complement and microglia to CNS axon regeneration are largely unknown. Whereas inflammation is commonly thought to exacerbate the effects of CNS injury, we find that complement proteins C1q and C3 and microglia/monocyte phagocytic complement receptor CR3 are each required for retinal ganglion cell axon regeneration through the injured mouse optic nerve. Also, whereas studies of optic nerve regeneration generally focus on the retina, we show that the regeneration-relevant role of complement and microglia/monocytes likely involves myelin phagocytosis within the optic nerve. Thus, our results point to the importance of the innate immune response for CNS repair.