Name: organotypic retinal explant cultures from macaque monkey
Uploaded: 2022-08-24T13:00:00
Description: Watch this Scientific Journal Video about Organotypic Retinal Explant Cultures from Macaque Monkey at JoVE.com

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.

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
<ol>
	<li>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...

<ol>
	<li>O'Neal, T. B., Luther, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> StatPearls. , (2022).</li><li>Power, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+mechanisms+of+hereditary+photoreceptor+degeneration+-+Focus+on+cGMP.">Cellular mechanisms of hereditary photoreceptor degeneration - Focus on cGMP.</a> Progress in Retinal and Eye Research. 74, 100772 (2020).</li><li>Paquet-Durand, F., Hauck, S. M., van Veen, T., Ueffing, M., Ekstr&#246;m, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PKG+activity+causes+photoreceptor+cell+death+in+two+retinitis+pigmentosa+models.">PKG activity causes photoreceptor cell death in two retinitis pigmentosa models.</a> Journal of Neurochemistry. 108 (3), 796-810 (2009).</li><li>Tolone, A., Belhadj, S., Rentsch, A., Schwede, F., Paquet-Durand, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cGMP+pathway+and+inherited+photoreceptor+degeneration:+Targets,+compounds,+and+biomarkers.">The cGMP pathway and inherited photoreceptor degeneration: Targets, compounds, and biomarkers.</a> Genes (Basel). 10 (6), 453 (2019).</li><li>Arango-Gonzalez, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+common+non-apoptotic+cell+death+mechanism+in+hereditary+retinal+degeneration.">Identification of a common non-apoptotic cell death mechanism in hereditary retinal degeneration.</a> PLoS One. 9 (11), 112142 (2014).</li><li>Mencl, S., Trifunovi&#263;, D., Zrenner, E., Paquet-Durand, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PKG-dependent+cell+death+in+661W+cone+photoreceptor-like+cell+cultures+(experimental+study).">PKG-dependent cell death in 661W cone photoreceptor-like cell cultures (experimental study).</a> Advances in Experimental Medicine and Biology. 1074, 511-517 (2018).</li><li>Power, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+spatiotemporal+mapping+reveals+divergent+cell+death+pathways+in+three+mouse+models+of+hereditary+retinal+degeneration.">Systematic spatiotemporal mapping reveals divergent cell death pathways in three mouse models of hereditary retinal degeneration.</a> Journal of Comparative Neurology. 528 (7), 1113-1139 (2020).</li><li>Sancho-Pelluz, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excessive+HDAC+activation+is+critical+for+neurodegeneration+in+the+rd1+mouse.">Excessive HDAC activation is critical for neurodegeneration in the rd1 mouse.</a> Cell Death &amp; Disease. 1 (2), 24 (2010).</li><li>Kulkarni, M., Trifunovi&#263;, D., Schubert, T., Euler, T., Paquet-Durand, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+dynamics+change+in+degenerating+cone+photoreceptors.">Calcium dynamics change in degenerating cone photoreceptors.</a> Human Molecular Genetics. 25 (17), 3729-3740 (2016).</li><li>Trifunovi&#263;, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=cGMP-dependent+cone+photoreceptor+degeneration+in+the+cpfl1+mouse+retina.">cGMP-dependent cone photoreceptor degeneration in the cpfl1 mouse retina.</a> Journal of Comparative Neurology. 518 (17), 3604-3617 (2010).</li><li>Samardzija, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HDAC+inhibition+ameliorates+cone+survival+in+retinitis+pigmentosa+mice.">HDAC inhibition ameliorates cone survival in retinitis pigmentosa mice.</a> Cell Death &amp; Differentiation. 28 (4), 1317-1332 (2021).</li><li>Sch&#246;n, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+therapy+successfully+delays+degeneration+in+a+mouse+model+of+PDE6A-linked+Retinitis+Pigmentosa+(RP43).">Gene therapy successfully delays degeneration in a mouse model of PDE6A-linked Retinitis Pigmentosa (RP43).</a> Human Gene Therapy. 28 (12), 1180-1188 (2017).</li><li>McGregor, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+therapy+restores+retinal+activity+in+primate+for+at+least+a+year+following+photoreceptor+ablation.">Optogenetic therapy restores retinal activity in primate for at least a year following photoreceptor ablation.</a> Molecular Therapy. 30 (3), 1315-1328 (2022).</li><li>Lingam, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=cGMP-grade+human+iPSC-derived+retinal+photoreceptor+precursor+cells+rescue+cone+photoreceptor+damage+in+non-human+primates.">cGMP-grade human iPSC-derived retinal photoreceptor precursor cells rescue cone photoreceptor damage in non-human primates.</a> Stem Cell Research &amp; Therapy. 12 (1), 464 (2021).</li><li>Das, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+cGMP-signalling+and+calcium-signalling+in+photoreceptor+cell+death:+perspectives+for+therapy+development.">The role of cGMP-signalling and calcium-signalling in photoreceptor cell death: perspectives for therapy development.</a> Pflugers Archiv. 473 (9), 1411-1421 (2021).</li><li>Hoon, M., Okawa, H., Della Santina, L., Wong, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+architecture+of+the+retina:+Development+and+disease.">Functional architecture of the retina: Development and disease.</a> Progress in Retinal and Eye Research. 42, 44-84 (2014).</li><li>Schnichels, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retina+in+a+dish:+Cell+cultures,+retinal+explants+and+animal+models+for+common+diseases+of+the+retina.">Retina in a dish: Cell cultures, retinal explants and animal models for common diseases of the retina.</a> Progress in Retinal and Eye Research. 81, 100880 (2021).</li><li>Maryam, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+organization+of+human+cGMP+specific+Phosphodiesterase+6+(PDE6):+Structural+implications+of+somatic+mutations+in+cancer+and+retinitis+pigmentosa.">The molecular organization of human cGMP specific Phosphodiesterase 6 (PDE6): Structural implications of somatic mutations in cancer and retinitis pigmentosa.</a> Computational and Structural Biotechnology Journal. 17, 378-389 (2019).</li><li>Huang, L., Kutluer, M., Adani, E., Comitato, A., Marigo, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+in+vitro+cellular+model+for+molecular+studies+of+retinitis+pigmentosa.">New in vitro cellular model for molecular studies of retinitis pigmentosa.</a> International Journal of Molecular Sciences. 22 (12), 6440 (2021).</li><li>Zhou, J., Rasmussen, M., Ekstr&#246;m, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=cGMP-PKG+dependent+transcriptome+in+normal+and+degenerating+retinas:+Novel+insights+into+the+retinitis+pigmentosa+pathology.">cGMP-PKG dependent transcriptome in normal and degenerating retinas: Novel insights into the retinitis pigmentosa pathology.</a> Experimental Eye Research. 212, 108752 (2021).</li></ol>

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...

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 delay photoreceptor cell death.
Previous research on the mechanisms of cell death in RD has demonstrated that the decrease or loss of phosphodiesterase 6 (PDE6) activity caused by RD-triggering gene mutations leads to reduced hydrolysis of cyclic guanosine monophosphate (cGMP)2,3. cGMP is a specific agonist of the cyclic nucleotide-gated ion channels (CNGCs) in the rod outer segments (ROSs) and is also a key molecule responsible for the conversion of light signals into electrical signals in vertebrate photoreceptor cells4. Reduced cGMP hydrolysis causes the accumulation of cGMP in ROSs, leading to the opening of CNGCs 5. Consequently, the phototransduction pathways are activated, resulting in an increase in cation concentrations in photoreceptor cells. This process imposes a metabolic burden on photoreceptors, which when overactivated, for instance, by mutations in PDE6, may cause cell death.
Many studies have shown that a significant overaccumulation of cGMP in photoreceptors of mouse models with different RD gene mutations may cause the activation of cGMP-dependent protein kinase (PKG)3,6. This leads to a substantial increase in dying, TUNEL-positive cells and a gradual thinning of the photoreceptor cell layer. Previous studies suggest that PKG overactivation caused by elevated cGMP levels is a necessary and sufficient condition for the induction of photoreceptor cell death2,5. Studies on different mouse models of RD have also shown that PKG activation induced by elevated cGMP levels in photoreceptors, leads to overactivation of downstream effectors such as poly-ADP-ribose polymerase 1 (PARP1), histone deacetylase (HDAC), and calpain2,7,8,9. This implies causal associations between these different target proteins and photoreceptor cell death.
However, previous research on the pathology, toxicopharmacology, and therapy of RD was mainly based on mouse models for RD10,11,12. Nevertheless, immense difficulties remain in the clinical translation of these results. This is owing to the considerable genetic and physiological differences between mice and humans, especially with respect to the retinal structure. In contrast, non-human primates (NHPs) also share a high degree of similarity with humans with respect to genetic characteristics, physiological patterns, and environmental factor regulation. For example, optogenetic therapy was investigated as a means to restore retinal activity in an NHP model13. Lingam and colleagues demonstrated that good manufacturing practice-grade human-induced pluripotent stem cell-derived retinal photoreceptor precursor cells may rescue cone photoreceptor damage in NHP14. Therefore, NHP models are important for the exploration of RD pathogenesis and the development of effective treatment methods. In particular, NHP models of RD, exhibiting pathogenic mechanisms similar to those in humans, could play a critical role in studies on the development and in vivo toxicopharmacology analysis of new drugs.
In view of the long life-cycle, high level of technical difficulties, and high cost involved in establishing in vivo primate models, we established an in vitro non-human primate (NHP) model using cultures of explanted macaque retina. First, wild-type macaques aged 1-3 years were selected for in vitro culture of retinal explants, which included the retina-RPE-choroid complex. Explants were then treated with different concentrations of the PDE6 inhibitor zaprinast (100 &#181;M, 200 &#181;M, and 400 &#181;M) to induce the cGMP-PKG signaling pathway. Photoreceptor cell death was quantified and analyzed using the TUNEL assay, and cGMP accumulation in explants was verified via immunofluorescence. Given the high degree of similarity with respect to cell distribution and morphology, retinal layer thickness, and other physiological characteristics of the retina between monkeys and humans, the establishment of the cGMP-PKG signaling pathway in the in vitro retinal model may facilitate future research on the pathogenesis of RD as well as studies into the development and toxicopharmacology of new drugs for RD treatment.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma</td>
			<td>B2064</td>
			<td>Blocking solution</td>
		</tr>
		<tr>
			<td>Corticosterone</td>
			<td>Sigma</td>
			<td>C2505</td>
			<td>Supplements of Complete Medium</td>
		</tr>
		<tr>
			<td>DL-tocopherol</td>
			<td>Sigma</td>
			<td>T1539</td>
			<td>Supplements of Complete Medium</td>
		</tr>
		<tr>
			<td>Donkey anti sheep, Alxea Fluor 488</td>
			<td>Life technologies corporation</td>
			<td>A11015</td>
			<td>Secondary antibody of cGMP</td>
		</tr>
		<tr>
			<td>Ethanol-acetic acid solution</td>
			<td>Shyuanye</td>
			<td>R20492</td>
			<td>Fixing liquid</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gemini</td>
			<td>900-108</td>
			<td>Blocking solution</td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>Carl Zeiss</td>
			<td>Axio Imager.M2</td>
			<td>Immunofluorescence imaging</td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>Sigma</td>
			<td>G8540</td>
			<td>Supplements of Complete Medium</td>
		</tr>
		<tr>
			<td>Glutathione</td>
			<td>Sigma</td>
			<td>G6013</td>
			<td>Supplements of Complete Medium</td>
		</tr>
		<tr>
			<td>In Situ Cell Death Detection Kit, TMR red</td>
			<td>Roche</td>
			<td>12156792910</td>
			<td>TUNEL assay</td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma</td>
			<td>16634</td>
			<td>Supplements of Complete Medium</td>
		</tr>
		<tr>
			<td>L-cysteine HCl</td>
			<td>Sigma</td>
			<td>C7477</td>
			<td>Supplements of Complete Medium</td>
		</tr>
		<tr>
			<td>Linoleic acid</td>
			<td>Sigma</td>
			<td>L1012</td>
			<td>Supplements of Complete Medium</td>
		</tr>
		<tr>
			<td>MACS Tissue Storage Solution</td>
			<td>Miltenyi</td>
			<td>130-100-008</td>
			<td>Optimized storage of fresh organ and tissue samples</td>
		</tr>
		<tr>
			<td>Normal Donkey Serum</td>
			<td>Solarbio</td>
			<td>SL050</td>
			<td>Blocking solution</td>
		</tr>
		<tr>
			<td>Paraformaldehyde(PFA)</td>
			<td>Biosharp</td>
			<td>BL539A</td>
			<td>Fixing agent</td>
		</tr>
		<tr>
			<td>PEN. / STREP. 100&#215;</td>
			<td>Millipore</td>
			<td>TMS-AB2-C</td>
			<td>Penicillin / Streptomycin antibiotics</td>
		</tr>
		<tr>
			<td>Phosphate buffer saline(PBS)</td>
			<td>Solarbio</td>
			<td>P1010</td>
			<td>Buffer solution</td>
		</tr>
		<tr>
			<td>Povidone-iodine</td>
			<td>Shanghailikang</td>
			<td>310411</td>
			<td>Disinfector agent</td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>Sigma</td>
			<td>P8783</td>
			<td>Supplements of Complete Medium</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Millpore</td>
			<td>539480</td>
			<td>Break down protein</td>
		</tr>
		<tr>
			<td>R16 medium</td>
			<td>Life technologies corporation</td>
			<td>074-90743A</td>
			<td>Basic medium</td>
		</tr>
		<tr>
			<td>Retinol</td>
			<td>Sigma</td>
			<td>R7632</td>
			<td>Supplements of Complete Medium</td>
		</tr>
		<tr>
			<td>Retinyl acetate</td>
			<td>Sigma</td>
			<td>R7882</td>
			<td>Supplements of Complete Medium</td>
		</tr>
		<tr>
			<td>Sheep anti-cGMP</td>
			<td>Jan de Vente, Maastricht University, the Netherlands</td>
			<td></td>
			<td>Primary antibody of cGMP</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>GHTECH</td>
			<td>57-50-1</td>
			<td>Dehydrating agent</td>
		</tr>
		<tr>
			<td>T3</td>
			<td>Sigma</td>
			<td>T6397</td>
			<td>Supplements of Complete Medium</td>
		</tr>
		<tr>
			<td>Tissue-Tek medium (O.C.T. Compound)</td>
			<td>SAKURA</td>
			<td>4583</td>
			<td>Embedding medium</td>
		</tr>
		<tr>
			<td>Tocopheryl acetate</td>
			<td>Sigma</td>
			<td>T1157</td>
			<td>Supplements of Complete Medium</td>
		</tr>
		<tr>
			<td>Transferrin</td>
			<td>Sigma</td>
			<td>T1283</td>
			<td>Supplements of Complete Medium</td>
		</tr>
		<tr>
			<td>Transwell</td>
			<td>Corning Incorporated</td>
			<td>3412</td>
			<td>Cell / tissue culture</td>
		</tr>
		<tr>
			<td>Tris-buffer (TBS)</td>
			<td>Solarbio</td>
			<td>T1080</td>
			<td>Blocking buffer</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Solarbio</td>
			<td>9002-93-1</td>
			<td>Surface active agent</td>
		</tr>
		<tr>
			<td>VECTASHIELD Medium with DAPI</td>
			<td>Vector</td>
			<td>H-1200</td>
			<td>Mounting medium</td>
		</tr>
		<tr>
			<td>Vitamin B1</td>
			<td>Sigma</td>
			<td>T1270</td>
			<td>Supplements of Complete Medium</td>
		</tr>
		<tr>
			<td>Vitamin B12</td>
			<td>Sigma</td>
			<td>V6629</td>
			<td>Supplements of Complete Medium</td>
		</tr>
		<tr>
			<td>Vitamin C</td>
			<td>Sigma</td>
			<td>A4034</td>
			<td>Supplements of Complete Medium</td>
		</tr>
		<tr>
			<td>Zaprinast</td>
			<td>Sigma</td>
			<td>Z0878</td>
			<td>PDE6 inhibitor</td>
		</tr>
		<tr>
			<td>Zeiss Imager M2 Microscope</td>
			<td>&#160;Zeiss, Oberkochen,Germany</td>
			<td></td>
			<td>upright microscope</td>
		</tr>
		<tr>
			<td>LSM 900 Airyscan</td>
			<td></td>
			<td></td>
			<td>high resolution laser scanning microscope</td>
		</tr>
		<tr>
			<td>Zeiss Axiocam</td>
			<td>&#160;Zeiss, Oberkochen,Germany</td>
			<td></td>
			<td>digital camera</td>
		</tr>
		<tr>
			<td>Zeiss Axiovision4.7</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Adobe</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Illustrator CC 2021 (Adobe Systems Incorporated, San Jose, CA)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Primate eyeballs from wildtype macaque</td>
			<td>KUNMING INSTITUTE OF ZOOLOGY</td>
			<td>SYXK (<img alt="Equation 1" src="/files/ftp_upload/64178/64178eq01.jpg" style="margin: auto;" />) K2017 -0008</td>
			<td></td>
		</tr>
		<tr>
			<td>Super Pap Pen Pen (Liquid Blocker, Diado, 0010, Japan</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TUNEL kit solution (REF12156792910, Roche,Germany),</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

organotypic retinal explant cultures from macaque monkey

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.

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...

Watch this Scientific Journal Video about Organotypic Retinal Explant Cultures from Macaque Monkey at JoVE.com

Organotypic Retinal Explant Cultures from Macaque Monkey

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 ...

The primate retinal model established in this study we offering the investigation of the comprehensive mechanism and the therapeutic development of cGMP-PKG dependent retinal deterioration. This individual nonhuman primate model reserves retinal tissue confirmation and intercellular interactions which enables better simulation of in vivo conditions. It also reduces the animal use and it shortens the experimental duration.Providing a control experimental approach for investigating retinal development or degeneration. Begin the preparation of the retinal explants by washing the eyeballs isolated from wild type macaques with 10 milliliters of 5%povidone iodine for 30 seconds. To avoid bacterial contamination, dip the eyeballs into a 5%penicillin streptomycin PBS solution for one minute before incubation in 10 milliliters of R16 medium for five minutes.Then immerse the eyeballs in 10 milliliters of protein Ace-K solution preheated at 37 degrees Celsius and incubated for two minutes. Perform next incubation in 10 milliliters of one to one R16 plus fetal bovine serum solution for five minutes. Give a final wash in 10 milliliters of fresh R16.Once done, separate the sclera, cornea, iris, lens and vitreous body. Then cut the retina from four sides with tweezers and scissors. Next, use a four milliliter tree fine blade to cut the retinal explant at a 1.5 millimeter from the nasal side, four millimeter from the temporal side and a two millimeter from the superior and inferior sides of the optic nerve head.Then using a six millimeter tree fine blade cut the central side of the retinal explant containing the optic disc and macula. Using a wide pipet, pull the retina explant containing the retina and choroid and place it in the middle of the insert with the photoreceptor side up. Fully submerge the retina in one milliliter of the complete medium on the plate below the membrane.Culture the retinal explants and place them in a 37 degree Celsius incubator with humidified 5%carbon dioxide. Give the appropriate drug concentration treatment to explants for four days. Use at least three explants per treatment condition and use the explants without the drug as a positive control.For fixing and cryo-sectioning, treat the retinal explants with one milliliter of paraform aldehyde for 45 minutes. After a brief rinsing with one milliliter of PBS serially incubate the explants in 10%sucrose for 10 minutes, 20%sucrose for 20 minutes, and 30%sucrose for 30 minutes. Cut the retinal explants uniformly with four quadrants in the periphery and six millimeters at the center.Then transfer the retinal explants into a tin box of about 1.5 by 1.5 by 1.5 centimeters with an optimal cutting temperature compound embedding medium covering the explant. Immediately, freeze the tissue sections in liquid nitrogen and place them in a 20 degrees refrigerator for storage. Next, trim the agar into a small block containing the tissue sample.Section the blocks into 10 micrometer blocks and place the sections on adhesion microscope slides using a soft brush. Then dry the sections for 45 minutes at 40 degrees Celsius and store them at minus 80 degrees Celsius until use. For cyclic guanosine monophosphate or cGMP staining, draw a hydrophobic ring around the dried retinal sections with a liquid blocker pen and cover the samples.Immerse the slides in 200 microliters of 0.3%PBS and Triton-X100 or PBST for 10 minutes followed by a one hour treatment in 200 microliters of blocking solution at room temperature. Next, incubate the sample slide overnight with the 200 microliters of primary antibody prepared in the blocking solution at four degrees Celsius and rinse it thrice with PBS for 10 minutes. Incubate the slide in 200 microliters of secondary antibody for one hour at room temperature.After three washes of PBS for 10 minutes cover the samples with an antifade mounting medium containing DAPI and store at four degrees Celsius for at least 30 minutes before imaging. Dry the slide at room temperature for 15 minutes and draw a water repellent barrier ring around the retinal tissue specimen with a liquid blocker pen. After incubation with 40 milliliters of PBS for 15 minutes treat the sections with 42 milliliters of 0.05 molar tris-buffer or TBS at 37 degrees Celsius.Aspirate the TBS, incubate the samples with six microliters of proteinase K for five minutes and wash slides thrice with PBS for five minutes each. Expose the slides to 40 milliliters of ethanol acetic acid solution in the chaplin. Cover it with a transparent sheet and incubate at minus 20 degrees Celsius for five minutes.Wash the slides thrice with TBS for five minutes each time. Expose the slides to 200 to 300 microliters of blocking solution or 1%BSA and incubate them in a humidity chamber for one hour. At approximately 130 microliters of TMR red solution per slide and after one hour, give two washes of 200 microliters of PBS for five minutes each.Place cover slides containing one to two drops of antifade mounting medium with DAPI over the samples and incubate the slides at four degrees Celsius for at least 30 minutes before imaging. After sequential staining with tunnel and cGMP proceed for light and fluorescence microscopy by adjusting the camera parameters. Capture the images of four fields from each section using the software with a digital camera at 20x magnification.Obtain representative pictures from the central areas of the retina using DAPI, EGFP and TMR with z-stack scanning and an interval of one micrometer and optional at 1.251 micrometers. In the retinal explants treated with different concentrations of the PDE6 inhibitors Zaprinast the number of tunnel and cGMP positive cells were strongly increased in the outer nuclear layer as compared with the control group. The high proportion of tunnel positive cells suggested that the increase in cGMP activity may enhance the pre nest induced retinal cell degeneration and cGMP accumulation plays a role in photoreceptor degeneration.Explant preparation must be carried out as soon as possible. Op animal scatter fives and any accumulation because it improves cell survival and enhance the status of fiber of bulk stack series. To a greatest extent visible the vitreous should be cleaned with steroids.Avoid contacting the retinal nerve fiber layers when transferring retinal explants to prevent mechanical cell injury. Please take note, to issue a smooth transfer of retinal explants to culture inserts the pet can be pre-soaked in something to keep it moist before transferring retinal tissues. Additionally, the entire process must be carried out in the steroid atmosphere to avoid contamination during in the process.

Research

JoVE Journal

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