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Neuroscience

Organotypic Retinal Explant Cultures from Macaque Monkey

Published: August 24th, 2022

DOI:

10.3791/64178

1Yunnan Eye Institute & Key Laboratory of Yunnan Province, Yunnan Eye Disease Clinical Medical Center, Affiliated Hospital of Yunnan University, Yunnan University, 2Institute for Ophthalmic Research, Eberhard-Karls-Universität Tübingen

Retinal explants obtained from wild-type macaques were cultured in vitro. Retinal degeneration and the cGMP-PKG signaling pathway was induced using the PDE6 inhibitor zaprinast. cGMP accumulation in the explants at different zaprinast concentrations was verified using immunofluorescence.

Hereditary retinal degeneration (RD) is characterized by progressive photoreceptor cell death. Overactivation of the cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) pathway in photoreceptor cells causes photoreceptor cell death, especially in models harboring phosphodiesterase 6b (PDE6b) mutations. Previous studies on RD have used mainly murine models such as rd1 or rd10 mice. Given the genetic and physiological differences between mice and humans, it is important to understand to which extent the retinas of primates and rodents are comparable. Macaques share a high level of genetic similarity with humans. Therefore, wild-type macaques (aged 1-3 years) were selected for the in vitro culture of retinal explants that included the retina-retinal pigment epithelium (RPE)-choroid complex. These explants were treated with different concentrations of the PDE6 inhibitor zaprinast to induce the cGMP-PKG signaling pathway and simulate RD pathogenesis. cGMP accumulation and cell death in primate retinal explants were subsequently verified using immunofluorescence and the TUNEL assay. The primate retinal model established in this study may serve for relevant and effective studies into the mechanisms of cGMP-PKG-dependent RD, as well as for the development of future treatment approaches.

Hereditary retinal degeneration (RD) is characterized by progressive photoreceptor cell death and is caused by mutations in a wide variety of pathogenic genes1. The end result of RD is vision loss and in the vast majority of cases the disease remains untreatable to this day. Therefore, it is important to study the cellular mechanisms leading to photoreceptor death using models that faithfully represent the human disease condition. Here, primate-based models are of particular interest due to their closeness to humans. Notably, such models may advance the development of appropriate therapeutic interventions that can halt or ....

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The animal study was reviewed and approved by the Ethics Review Committee of Institute of Zoology, Chinese Academy of Sciences (IACUC-PE-2022-06-002), and animal ethics review and animal protocol of Yunnan University (YNU20220149).

1. Preparation of retinal explants

  1. Obtain primate eyeballs from wild-type macaques, aged 1 to 3 years old, store in tissue storage solution, and transport on ice within 3 h of enucleation after the monkeys were sacrificed or after natura.......

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In this study, Macaque monkey retinal explant culture was performed using explants containing the retina-RPE-choroid complex (Figure 1, Supplementary Figure S1). Compared with the in vitro culture of retinal cells using the retina without the attached RPE and choroid, our explant culture facilitates better cell survival and accordingly, prolongs the survival of photoreceptor cells.

We used different concentrations of the PDE6 inhibitor za.......

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Visual phototransduction refers to the biological process by which light signals are converted to electrical signals by photoreceptor cells within the retina of the eye. Photoreceptor cells are polarized neurons capable of phototransduction, and there are two different types of photoreceptors termed rods and cones after the shapes of their outer segments. Rods are responsible for the scotopic vision and cones are responsible for photopic and high acuity vision. Hereditary RD relates to neurodegenerative diseases characte.......

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This study was supported by grants from the National Natural Science Foundation of China (No. 81960180), the Zinke heritage foundation, and the Charlotte and Tistou Kerstan Foundation, Yunnan Eye Disease Clinical Medical Center (ZX2019-02-01). We thank Prof. Longbao Lv (Institute of Zoology, Chinese Academy of Sciences, Kunming, China) for sharing the monkey eyeballs used in this study.

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NameCompanyCatalog NumberComments
Bovine Serum Albumin (BSA)SigmaB2064Blocking solution
CorticosteroneSigmaC2505Supplements of Complete Medium
DL-tocopherolSigmaT1539Supplements of Complete Medium
Donkey anti sheep, Alxea Fluor 488Life technologies corporationA11015Secondary antibody of cGMP
Ethanol-acetic acid solutionShyuanyeR20492Fixing liquid
Fetal Bovine SerumGemini900-108Blocking solution
Fluorescence microscopeCarl ZeissAxio Imager.M2Immunofluorescence imaging
GlutamineSigmaG8540Supplements of Complete Medium
GlutathioneSigmaG6013Supplements of Complete Medium
In Situ Cell Death Detection Kit, TMR redRoche12156792910TUNEL assay
InsulinSigma16634Supplements of Complete Medium
L-cysteine HClSigmaC7477Supplements of Complete Medium
Linoleic acidSigmaL1012Supplements of Complete Medium
MACS Tissue Storage SolutionMiltenyi130-100-008Optimized storage of fresh organ and tissue samples
Normal Donkey SerumSolarbioSL050Blocking solution
Paraformaldehyde(PFA)BiosharpBL539AFixing agent
PEN. / STREP. 100×MilliporeTMS-AB2-CPenicillin / Streptomycin antibiotics
Phosphate buffer saline(PBS)SolarbioP1010Buffer solution
Povidone-iodineShanghailikang310411Disinfector agent
ProgesteroneSigmaP8783Supplements of Complete Medium
Proteinase KMillpore539480Break down protein
R16 mediumLife technologies corporation074-90743ABasic medium
RetinolSigmaR7632Supplements of Complete Medium
Retinyl acetateSigmaR7882Supplements of Complete Medium
Sheep anti-cGMPJan de Vente, Maastricht University, the NetherlandsPrimary antibody of cGMP
SucroseGHTECH57-50-1Dehydrating agent
T3SigmaT6397Supplements of Complete Medium
Tissue-Tek medium (O.C.T. Compound)SAKURA4583Embedding medium
Tocopheryl acetateSigmaT1157Supplements of Complete Medium
TransferrinSigmaT1283Supplements of Complete Medium
TranswellCorning Incorporated3412Cell / tissue culture
Tris-buffer (TBS)SolarbioT1080Blocking buffer
Triton X-100Solarbio9002-93-1Surface active agent
VECTASHIELD Medium with DAPIVectorH-1200Mounting medium
Vitamin B1SigmaT1270Supplements of Complete Medium
Vitamin B12SigmaV6629Supplements of Complete Medium
Vitamin CSigmaA4034Supplements of Complete Medium
ZaprinastSigmaZ0878PDE6 inhibitor
Zeiss Imager M2 Microscope Zeiss, Oberkochen,Germanyupright microscope
LSM 900 Airyscanhigh resolution laser scanning microscope
Zeiss Axiocam Zeiss, Oberkochen,Germanydigital camera
Zeiss Axiovision4.7
Adobe
Illustrator CC 2021 (Adobe Systems Incorporated, San Jose, CA)
Primate eyeballs from wildtype macaqueKUNMING INSTITUTE OF ZOOLOGYSYXK (Equation 1) K2017 -0008
Super Pap Pen Pen (Liquid Blocker, Diado, 0010, Japan
TUNEL kit solution (REF12156792910, Roche,Germany),

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