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

本研究得到了国家自然科学基金（第81960180号）、津科遗产基金会和云南省眼病临床医学中心夏洛特和蒂斯托克斯坦基金会的资助（ZX2019-02-01）。我们感谢吕龙宝教授（中国科学院动物研究所，中国昆明）分享本研究中使用的猴眼球。

该动物研究通过了中国科学院动物研究所伦理评审委员会（IACUC-PE-2022-06-002）和云南大学动物伦理评审与动物方案（YNU20220149）的审评和批准。
1.视网膜外植体的制备
<ol><li>从1至3岁的野生型猕猴身上获得灵长类动物的眼球，储存在组织储存溶液中，并在猴子被处死或自然死亡后3小时内在剜除后3小时内在冰上运输。</li>
	<li>为了制备蛋白酶 K 溶液，将 25 mg 蛋白酶...

<ol>
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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>

视觉光转导是指光信号被眼睛视网膜内的感光细胞转换为电信号的生物过程。感光细胞是能够进行光转导的极化神经元，并且有两种不同类型的感光器在其外段的形状之后称为杆状和视锥。视杆细胞负责暗视觉，视锥细胞负责明视和高敏视力。遗传性RD涉及以进行性视网膜感光细胞死亡为特征的神经退行性疾病15。
目前对RD的研究主要基于RD的小鼠模型，包括...

遗传性视网膜变性（RD）的特征是进行性感光细胞死亡，由多种致病基因突变引起1。RD的最终结果是视力丧失，在绝大多数情况下，这种疾病至今仍无法治愈。因此，使用忠实代表人类疾病状况的模型研究导致光感受器死亡的细胞机制非常重要。在这里，基于灵长类动物的模型因其与人类的接近而特别令人感兴趣。值得注意的是，这些模型可能会促进适当的治疗干预措施的开发，这些干预措施可以阻止或延缓感光细胞死亡。
先前对RD中细胞死亡机制的研究表明，由RD触发基因突变引起的磷酸二酯酶6（PDE6）活性的降低或丧失导致环鸟苷单磷酸（cGMP）的水解减少2，3。cGMP是杆外段（ROS）中环核苷酸门控离子通道（CNGCs）的特异性激动剂，也是负责脊椎动物感光细胞中光信号转化为电信号的关键分子4。cGMP水解减少导致cGMP在ROS中积累，导致CNGCs的开放5。因此，光转导途径被激活，导致感光细胞中阳离子浓度的增加。这个过程给光感受器带来了代谢负担，当光感受器过度激活时，例如，通过PDE6的突变，可能导致细胞死亡。
许多研究表明，具有不同RD基因突变的小鼠模型的光感受器中cGMP的显着过度积累可能导致cGMP依赖性蛋白激酶（PKG）的活化3，6。这导致垂死的TUNEL阳性细胞的显着增加和感光细胞层的逐渐变薄。先前的研究表明，由cGMP水平升高引起的PKG过度激活是诱导感光细胞死亡的必要和充分条件2，5。对不同小鼠RD模型的研究还表明，光感受器中cGMP水平升高诱导的PKG活化导致下游效应物的过度激活，例如聚ADP-核糖聚合酶1（PARP1），组蛋白脱乙酰酶（HDAC）和钙蛋白酶2，7，8，9。这意味着这些不同的靶蛋白与感光细胞死亡之间存在因果关系。
然而，先前关于RD的病理学，毒理学和治疗的研究主要基于RD10，11，12的小鼠模型。然而，这些结果的临床转化仍然存在巨大的困难。这是由于小鼠和人类之间存在相当大的遗传和生理差异，特别是在视网膜结构方面。相比之下，非人灵长类动物（NHP）在遗传特征，生理模式和环境因素调节方面也与人类高度相似。例如，在NHP模型中，光遗传学疗法被研究为恢复视网膜活动的手段13。Lingam及其同事证明，良好生产规范级人诱导的多能干细胞衍生的视网膜感光器前体细胞可以挽救NHP14中的视锥光感受器损伤。因此，非人灵长类模型对于探索RD发病机制和开发有效的治疗方法具有重要意义。特别是，RD的非人灵长类模型表现出与人类相似的致病机制，可以在新药开发和体内毒理学分析研究中发挥关键作用。
鉴于建立 体内 灵长类动物模型生命周期长、技术难度高、成本高等问题，我们利用体外猕猴视网膜培养物建立了 体外 非人灵长类动物（NHP）模型。首先，选择1-3岁的野生型猕猴进行视网膜外植体体 外培养， 其中包括视网膜-RPE-脉络膜复合体。然后用不同浓度的PDE6抑制剂zaprinast （100 μM、200 μM 和 400 μM）处理外植体以诱导cGMP-PKG信号通路。使用TUNEL测定法定量和分析感光细胞死亡，并通过免疫荧光 验证 外植体中的cGMP积累。鉴于猴与人类在细胞分布和形态、视网膜层厚度等视网膜生理特征方面高度相似，在 体外 视网膜模型中建立cGMP-PKG信号通路可能有助于未来RD发病机制的研究以及RD治疗新药的开发和毒理学研究。

<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

遗传性视网膜变性（RD）的特征是进行性感光细胞死亡。光感受器细胞中环鸟苷单磷酸（cGMP）依赖性蛋白激酶（PKG）途径的过度激活会导致感光细胞死亡，特别是在携带磷酸二酯酶6b（PDE6b）突变的模型中。以前对RD的研究主要使用小鼠模型，如 rd1 或 rd10 小鼠。鉴于小鼠和人类之间的遗传和生理差异，重要的是要了解灵长类动物和啮齿动物的视网膜在多大程度上具有可比性。猕猴与人类具有高度的遗传相似性。因此，选择野生型猕猴（年龄1-3岁）进行视网膜外植体的 体外 培养，其中包括视网膜-视网膜色素上皮（RPE）-脉络膜复合物。用不同浓度的PDE6抑制剂扎普利纳斯特处理这些外植体，诱导cGMP-PKG信号通路并模拟RD发病机制。随后使用免疫荧光和TUNEL测定法验证灵长类动物视网膜外植体中的cGMP积累和细胞死亡。本研究建立的灵长类动物视网膜模型可用于cGMP-PKG依赖性RD机制的相关有效研究，以及未来治疗方法的开发。

在这项研究中，猕猴视网膜外植体培养是使用含有视网膜-RPE-脉络膜复合物的外植体进行的（图1， 补充图S1）。与使用没有附着RPE和脉络膜的视网膜细胞体 外 培养相比，我们的外植体培养有助于更好的细胞存活，从而延长感光细胞的存活期。
我们使用不同浓度的PDE6抑制剂扎普瑞斯特（100μM、200μM和400μM; 补充图S2）诱导...

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

Organotypic Retinal Explant Cultures from Macaque Monkey

从野生型猕猴获得的视网膜外植体在 体外培养。视网膜变性和cGMP-PKG信号通路使用PDE6抑制剂扎匹司特诱导。使用免疫荧光法验证不同浓度的扎普瑞斯特外植体中的cGMP积累。

猕猴的器官型视网膜外植体培养物

Hereditary retinal degeneration (RD) is characterized by progressive photoreceptor cell death. Overactivation of the cyclic guanosine monophosphate ...

本研究建立的灵长类动物视网膜模型为cGMP-PKG依赖性视网膜恶化的综合机制和治疗开发提供了研究。这种个体非人灵长类动物模型保留了视网膜组织确认和细胞间相互作用，可以更好地模拟体内条件。它还减少了动物的使用，缩短了实验时间。为研究视网膜发育或变性提供对照实验方法。通过用10毫升5%聚维酮碘洗涤从野生型猕猴中分离的眼球30秒开始制备视网膜外植体。为避免细菌污染，将眼球浸入5%青霉素链霉素PBS溶液中一分钟，然后在10毫升R16培养基中孵育五分钟。然后将眼球浸入10毫升在37摄氏度下预热的蛋白质Ace-K溶液中，孵育两分钟。在10毫升一对一R16加胎牛血清溶液中进行下一次孵育5分钟。在 10 毫升新鲜 R16 中进行最后一次洗涤。完成后，将巩膜、角膜、虹膜、晶状体和玻璃体分开。然后用镊子和剪刀从四个侧面剪下视网膜。接下来，使用四毫升的树细刀片在距鼻侧 1.5 毫米、距颞侧 4 毫米和距视神经头上侧和下侧 2 毫米处切割视网膜外植体。然后使用六毫米的树细刀片切割包含视盘和黄斑的视网膜外植体的中心侧。使用宽移液管，拉出包含视网膜和脉络膜的视网膜外植体，并将其放在插入物的中间，感光器面朝上。将视网膜完全浸没在膜下方板上的一毫升完整培养基中。培养视网膜外植体，并将它们放入37摄氏度的培养箱中，加湿的5%二氧化碳。对外植体给予适当的药物浓度治疗四天。每个治疗条件至少使用三个外植体，并使用没有药物的外植体作为阳性对照。对于固定和冷冻切片，用一毫升副形醛处理视网膜外植体45分钟。用一毫升PBS短暂冲洗后，将外植体在10%蔗糖中连续孵育10分钟，20%蔗糖20分钟，30%蔗糖孵育30分钟。均匀切割视网膜外植体，周围有四个象限，中心有六个毫米。然后将视网膜外植体转移到约1.5×1.5×1.5厘米的锡盒中，用覆盖外植体的最佳切割温度化合物包埋介质。立即将组织切片冷冻在液氮中，并将它们放入20度的冰箱中储存。接下来，将琼脂切成包含组织样品的小块。将块切成10微米块，并使用软刷将切片放在粘附显微镜载玻片上。然后将切片在 40 摄氏度下干燥 45 分钟，并将它们存放在零下 80 摄氏度直至使用。对于环鸟苷单磷酸或cGMP染色，用液体阻滞笔在干燥的视网膜切片周围画一个疏水环并覆盖样品。将载玻片浸入200微升0.3%PBS和Triton-X100或PBST中10分钟，然后在室温下在200微升封闭溶液中处理一小时。接下来，将样品载玻片与封闭溶液中制备的 200 μL 一抗在 4 摄氏度下孵育过夜，并用 PBS 冲洗三次 10 分钟。将载玻片在200微升二抗中在室温下孵育一小时。在PBS洗涤三次10分钟后，用含有DAPI的抗淬灭封片剂覆盖样品，并在成像前在4摄氏度下储存至少30分钟。在室温下将载玻片干燥15分钟，并用液体阻滞笔在视网膜组织标本周围画一个防水屏障环。用40毫升PBS孵育15分钟后，用42毫升0.05摩尔三缓冲液或在37摄氏度下处理切片。吸出TBS，将样品与6微升蛋白酶K孵育5分钟，并用PBS洗涤载玻片三次，每次5分钟。将载玻片暴露在卓别林中的40毫升乙醇乙酸溶液中。用透明片覆盖，在零下20摄氏度下孵育五分钟。用TBS清洗载玻片三次，每次五分钟。将载玻片暴露在200至300微升封闭溶液或1%BSA中，并在湿度室中孵育一小时。每张载玻片约130微升TMR红溶液，一小时后，给予两次200微升PBS洗涤，每次五分钟。将含有一到两滴带有DAPI的抗淬灭封片的盖玻片放在样品上，并在成像前将载玻片在4摄氏度下孵育至少30分钟。用隧道和cGMP顺序染色后，通过调整相机参数进行光学和荧光显微镜检查。使用带有数码相机的软件以20倍放大率从每个部分捕获四个场的图像。使用DAPI，EGFP和TMR进行z-stack扫描，从视网膜的中心区域获得代表性图片，间隔为1微米，可选为1.251微米。在用不同浓度的PDE6抑制剂Zaprinast处理的视网膜外植体中，与对照组相比，外核层的隧道和cGMP阳性细胞数量显着增加。隧道阳性细胞比例高表明，cGMP活性的增加可能增强巢前诱导的视网膜细胞变性，cGMP积累在感光细胞变性中起一定作用。外植体制备必须尽快进行。Op动物散射五和任何积累，因为它提高了细胞存活率并增强了散装堆叠系列纤维的地位。在最大程度可见的范围内，玻璃体应用类固醇清洁。转移视网膜外植体时避免接触视网膜神经纤维层，以防止机械细胞损伤。请注意，为了将视网膜外植体顺利转移到培养插入物中，可以在转移视网膜组织之前预先浸泡宠物以保持湿润。此外，整个过程必须在类固醇气氛中进行，以避免在此过程中受到污染。

Research

JoVE Journal

Neuroscience

Organotypic Hippocampal Slice Cultures

器官型海马切片培养

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

科研

神经科学

Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection

器官型小脑文化：细胞凋亡的挑战和检测

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. ...

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

器官型培养的神经元雪崩多电极阵列记录

The cortex is spontaneously active, even in the absence of any particular input or motor output.  During development, this activity is important for the ...

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

胚胎腹侧中脑器官型片文化：一个系统来研究多巴胺能神经元发展在体外</em

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

浦肯野细胞树突形态的器官切片培养分析

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly ...

Neural Explant Cultures from Xenopus laevis

Neural Explant Cultures from Xenopus laevis

的神经植体培养<em&gt;非洲爪蟾</em

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

器官切片文化研究少突胶质细胞动力学和髓鞘

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

神经血管重塑的分析Entorhino，海马脑切片培养

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional ...

Investigating Functional Regeneration in Organotypic Spinal Cord Co-cultures Grown on Multi-electrode Arrays

调查器官脊髓联合培养物中生长多电极阵列功能再生

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a ...