in-vivo-methods-to-assess-retinal-ganglion-cell-optic-nerve-function

<em>In Vivo</em> Methods to Assess Retinal Ganglion Cell and Optic Nerve Function and Structure in Large Animals

The optic nerve collects axons signals from the retinal ganglion cells and transmits visual signal to the brain. Large animal models of optic nerve injury ...

Wenzhou Medical University

The degeneration of Müller cells has been recognized to involve in the pathogenesis of diabetic retinopathy. However, the mechanism is not yet clear. This study is to explore the potential role of Cyr61, a secreted signaling protein in extracellular matrix, in inducing human Müller cell degeneration in diabetic retinopathy (DR). Twenty patients with proliferative diabetic retinopathy (PDR) and twelve non-diabetic patients were recruited for this study. Vitreous fluid was collected during vitrectomy surgery for Cyr61 ELISA. Human Müller cell line MIO-M1 were cultured to be subconfluent, and then treated with glucose (0-20 mM) or Cyr61 (0-300 ng/ml). Cyr61 expression induced by increasing concentrations of glucose was evaluated by RT-qPCR and Western blot. Effects of Cyr61 on Müller cells viability, migration and apoptosis were observed by MTT assay, Transwell assay, and TUNEL assay. Vitreous Cyr61 levels were observed to be 8-fold higher in patients with PDR (3576.92 ± 1574.58 pg/mL), compared with non-diabetic controls (436.14 ± 130.69 pg/mL). Interestingly, the active PDR group was significantly higher than the quiescent PDR group (P<0.01). In retinal Müller cells culture, high glucose significantly and dose-dependently elevated Cyr61 expression at both mRNA and protein levels. Cyr61 at high concentrations dose-dependently inhibited the viability and migration of Müller cells. TUNEL assay further revealed that high concentration of Cyr61 significantly promoted the cell apoptosis. In conclusion, these findings demonstrated for the first time that the expression of Cyr61 was elevated by high glucose in Müller cells, and Cyr61 inhibited cell viability and migration while induced apoptosis, suggesting the potential role of Cyr61 in Müller cell degeneration. The elevated Cyr61 levels in vitreous fluid of PDR patients further support its role in diabetic retinopathy (DR).

Potential role of Cyr61 induced degeneration of human Müller cells in diabetic retinopathy.

To assess macular microstructure in eyes with tilted disk syndrome (TDS) and determine the relationship between TDS foveal morphology and visual function.

MACULAR MICROSTRUCTURAL FEATURES IN CHILDREN WITH TILTED DISK SYNDROME EVALUATED BY SPECTRAL DOMAIN OPTICAL COHERENCE TOMOGRAPHY.

Microglia have been discovered to undergo repopulation following ablation. However, the functionality of repopulated microglia and the mechanisms regulating microglia repopulation are unknown. We examined microglial homeostasis in the adult mouse retina, a specialized neural compartment containing regular arrays of microglia in discrete synaptic laminae that can be directly visualized. Using in vivo imaging and cell-fate mapping techniques, we discovered that repopulation originated from residual microglia proliferating in the central inner retina that subsequently spread by centrifugal migration to fully recapitulate pre-existing microglial distributions and morphologies. Repopulating cells fully restored microglial functions including constitutive "surveying" process movements, behavioral and physiological responses to retinal injury, and maintenance of synaptic structure and function. Microglial repopulation was regulated by CX3CL1-CX3CR1 signaling, slowing in CX3CR1 deficiency and accelerating with exogenous CX3CL1 administration. Microglial homeostasis following perturbation can fully recover microglial organization and function under the regulation of chemokine signaling between neurons and microglia.

Repopulating retinal microglia restore endogenous organization and function under CX3CL1-CX3CR1 regulation.

Large animal models of optic nerve injury are essential for translating novel findings into effective therapies due to their similarity to humans in many respects. However, most current tests evaluating the integrity of retinal ganglion cells (RGCs) and optic nerve (ON) are based on rodent animal models. We aimed to evaluate and optimize the in vivo methods to assess RGCs and ON's function and structure in large animals in terms of reproducibility, simplicity and sensitivity. Both goats and rhesus macaques were employed in this study. By using goats, we found anesthesia with isoflurane or xylazine resulted in different effects on reproducibility of flash visual evoked potential (FVEP) and pattern electroretinogram (PERG). FVEP with the large-Ganzfeld stimulator was significantly more stable than that with mini-Ganzfeld stimulator. PERG with simultaneous binocular stimulation, with superior simplicity over separate monocular stimulation, was appliable in goats due to undetectable interocular crosstalk of PERG signals. After ON crush in goats, some FVEP components, PERG, OCT and PLR demonstrated significant changes, in line with the histological study. By using rhesus macaque, we found the implicit time of PVEP, FVEP and PERG were significantly more reproducible than amplitudes, and OCT and PLR demonstrated small intersession variation. In summary, we established an optimized system to evaluate integrity of RGCs and ON in large animals in vivo, facilitating usage of large animal models of optic nerve diseases.

In vivo evaluation of retinal ganglion cells and optic nerve's integrity in large animals by multi-modality analysis.

Multiple lines of investigation have demonstrated that inflammation plays significant roles in etiology of age-related macular degeneration (AMD). Although interventional trials in AMD therapy targeting inflammatory pathways have been conducted, they have not yet been successful and a detailed understanding as to why some have failed is still elusive. One limitation is the relative dearth of information on how immune cells interact with retinal cells to generate AMD phenotypes at each disease stage. Here, we summarize current research evidence and hypotheses regarding potential pathogenic roles of innate immune cells in the eye, which include resident retinal microglia, macrophages derived from infiltrating systemic monocytes, and macrophages resident in the choroid. We relate recent findings regarding the physiology, function, and cellular interactions involving innate immune cells in the retina and choroid to AMD-related processes, including: (1) drusen formation and regression, (2) the onset and spread of degeneration in late atrophic AMD, and (3) the initiation, growth, and exudation of neovascular vessels in late "wet" AMD. Understanding how innate immune cells contribute to specific AMD phenotypes can assist in generating a comprehensive view on the inflammatory etiology of AMD and aid in identifying anti-inflammatory therapeutic strategies and selecting appropriate clinical outcomes for the planned interventions.

Innate Immunity in Age-Related Macular Degeneration.

Large animal model of optic nerve crush (ONC) plays an important role in translating novel therapeutic strategies developed in rodent model to clinical application. Due to the poor accessibility of the optic nerve (ON) in humans and large animals, lateral orbitotomy is needed to expose the retrobulbar ON. This study was to explore the effects of ONC and ON exposure with lateral orbitotomy (sham surgery) on the outer retinal function and structure in goats by using standard flash electroretinogram (FERG) and spectral-domain optical coherence tomography (SD-OCT). We found that ONC led to a transient reduction in FERG amplitudes at 1 week post injury (wpi), which recovered gradually over 2 months afterwards. Sham surgery alone also caused a similar pattern of amplitude reduction in FERG, although not as significantly as ONC did. Transient outer retinal thickening following ONC occurred at 4 wpi (when progressive thinning of the ganglion cell complex began), peaked at 8 wpi, then recovered gradually at 12 wpi. In contrast, outer retinal thickness remained unchanged statistically 3 months after sham surgery. Fundus fluorescein angiography showed that neither ONC nor ON exposure with lateral orbitotomy significantly caused any significant delay or absence of central retinal vascular filling. In summary, ONC with lateral orbitotomy affects outer retinal function and structure transiently.

Article	Year
Potential role of Cyr61 induced degeneration of human Müller cells in diabetic retinopathy. PloS one\| PubMed ID: 25329584	2014
MACULAR MICROSTRUCTURAL FEATURES IN CHILDREN WITH TILTED DISK SYNDROME EVALUATED BY SPECTRAL DOMAIN OPTICAL COHERENCE TOMOGRAPHY. Retina (Philadelphia, Pa.)\| PubMed ID: 27404961	2017
Repopulating retinal microglia restore endogenous organization and function under CX3CL1-CX3CR1 regulation. Science advances\| PubMed ID: 29750189	2018
In vivo evaluation of retinal ganglion cells and optic nerve's integrity in large animals by multi-modality analysis. Experimental eye research\| PubMed ID: 32598972	2020
Innate Immunity in Age-Related Macular Degeneration. Advances in experimental medicine and biology\| PubMed ID: 33848000	2021
In vivo evaluation of outer retinal function and structure after retrobulbar optic nerve crush by lateral orbitotomy in goats. Experimental eye research\| PubMed ID: 34097904	2021

Yikui Zhang

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