In Vivo Imaging of Subretinal Bleb-Induced Outer Retinal Degeneration in the Rabbit

Bartuma, Hammurabi; Petrus‐Reurer, Sandra; Aronsson, Monica; Westman, Sofie; André, Helder; Kvanta, Anders

doi:10.1167/iovs.14-16208

Cited by 29 publications

(47 citation statements)

References 27 publications

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“…12 Extending the observation time for up to 6 months, the observed changes to the outer neuroretina/RPE complex persisted and even became more pronounced (Fig. 1A).…”

Section: Resultsmentioning

confidence: 91%

“…Microsurgeries were performed on both eyes as previously described using a 3-port 25 G transvitreal pars plana technique (Alcon Accurus; Alcon Nordic, Copenhagen, Denmark). 12 Phosphate buffered saline (Thermo Fisher Scientific) or NaIO 3 (Sigma-Aldrich Corp., St. Louis, MO, USA) in PBS was drawn into a 1 mL syringe connected to an extension tube (operated by the assistant) and a 38 G polytip cannula (operated by the surgeon; MedOne Surgical, Inc., Sarasota, FL, USA). Then, 25 G trocars were inserted 1 mm from the limbus and an infusion cannula was connected to the lower temporal trocar.…”

Section: Subretinal Injectionmentioning

confidence: 99%

“…12 For immunostaining, slides were deparaffinized in xylene, dehydrated in graded alcohols, and rinsed with dH 2 O and Tris-buffered saline (TBS, pH 7.6). Then, 10 mM citrate buffer (trisodium citrate dehydrate, pH 6.0; SigmaAldrich Corp.) with 0.05% Tween-20 (Sigma-Aldrich Corp.) at 968C for 30 minutes, followed by 30 minutes of cooling at room temperature was used as antigen retrieval method.…”

Section: Histology and Tissue Immunostainingmentioning

confidence: 99%

“…9,10 Since optimal integration of transplanted hESC-RPE is critical for a fully successful regenerative treatment of GA, it is vital that integration issues are carefully explored in a relevant largeeyed preclinical model. 11 We recently demonstrated that subretinal bleb injections of physiologic salt solutions in the large-eyed rabbit cause robust photoreceptor loss and RPE alterations; that is, hallmark features of clinical GA. 12 We have shown further that subretinal suspension transplants of hESC-RPE derived in a xeno-free and defined manner on laminin (LN)-521, a component of Bruch's membrane, integrate as pigmented and functional monolayers, including phagocytic and photoreceptor rescue capacity. 13 We also reported that integration success varied between eyes, and postulated that such variability could be dependent partly on the degree of bleb-associated outer neuroretinal and RPE damage.…”

mentioning

confidence: 98%

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Integration of Subretinal Suspension Transplants of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells in a Large-Eyed Model of Geographic Atrophy

Petrus‐Reurer

Bartuma

Aronsson

et al. 2017

Invest. Ophthalmol. Vis. Sci.

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PURPOSE. Subretinal suspension transplants of human embryonic stem cell-derived retinal pigment epithelial cells (hESC-RPE) have the capacity to form functional monolayers in naive eyes. We explore hESC-RPE integration when transplanted in suspension to a large-eyed model of geographic atrophy (GA).METHODS. Derivation of hESC-RPE was performed in a xeno-free and defined manner. Subretinal bleb injection of PBS or sodium iodate (NaIO 3 ) was used to induce a GA-like phenotype. Suspensions of hESC-RPE were transplanted to the subretinal space of naive or PBS-/NaIO 3 -treated rabbits using a transvitreal pars plana technique. Integration of hESC-RPE was monitored by multimodal real-time imaging and by immunohistochemistry.RESULTS. Subretinal blebs of PBS or NaIO 3 caused different degrees of outer neuroretinal degeneration, RPE hyperautofluorescence, focal RPE loss, and choroidal atrophy; that is, hallmark characteristics of GA. In nonpretreated naive eyes, hESC-RPE integrated as subretinal monolayers with preserved overlying photoreceptors, yet not in areas with outer neuroretinal degeneration and native RPE loss. When transplanted to eyes with PBS-/NaIO 3 -induced degeneration, hESC-RPE failed to integrate. CONCLUSIONS.In a large-eyed preclinical model, subretinal suspension transplants of hESC-RPE did not integrate in areas with GA-like degeneration.

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“…12 Extending the observation time for up to 6 months, the observed changes to the outer neuroretina/RPE complex persisted and even became more pronounced (Fig. 1A).…”

Section: Resultsmentioning

confidence: 91%

Section: Subretinal Injectionmentioning

confidence: 99%

Section: Histology and Tissue Immunostainingmentioning

confidence: 99%

mentioning

confidence: 98%

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Integration of Subretinal Suspension Transplants of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells in a Large-Eyed Model of Geographic Atrophy

Petrus‐Reurer

Bartuma

Aronsson

et al. 2017

Invest. Ophthalmol. Vis. Sci.

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“…Such therapeutic approaches could allow for sustainable anti-VEGF treatments, and bypass the risks of multiple injections and development of geographic atrophy. Furthermore, we have shown that transplantation of human embryonic stem-derived RPE cells in a large-eyed animal model of geographic atrophy rescued retinal structure and preserved functionality of photoreceptors 47,48 . Combining the results presented here with the results from the transplantation studies, genetic manipulation of human embryonic stem-derived RPE cells to express negative regulatory loops of HIF-1α could present a clinically feasible approach for future treatment of both dry and neovascular forms of AMD.…”

Section: Discussionmentioning

confidence: 95%

Gene transfer of prolyl hydroxylase domain 2 inhibits hypoxia‐inducible angiogenesis in a model of choroidal neovascularization

André

Mammadzada

Tunik

et al. 2016

Acta Ophthalmologica

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Cellular responses to hypoxia are mediated by the hypoxia-inducible factors (HIF).In normoxia, HIF-α proteins are regulated by a family of dioxygenases, through prolyl and asparagyl hydroxylation, culminating in proteasomal degradation and transcriptional inactivation. In hypoxia, the dioxygenases become inactive and allow formation of HIF transcription factor, responsible for upregulation of hypoxia genes. In ocular neoangiogenic diseases, such as neovascular age-related macular degeneration (nAMD), hypoxia seems pivotal. Here, we investigate the effects of HIF regulatory proteins on the hypoxia pathway in retinal pigment epithelium (RPE) cells, critically involved in nAMD pathogenesis. Hypoxia is a stress situation triggering a multitude of responses that ensure survival of organisms to oxygen deprivation. Adaptation to hypoxia occurs by transcriptional upregulation of multiple genes involved in responses such as angiogenesis (e.g. vascular endothelial growth factor; VEGF), formation of red blood cells (e.g. erythropoietin), anaerobic metabolism (e.g. glycolytic enzymes and glucose transporters), and multiple others 1,2 . Gene induction in hypoxia is mediated by hypoxia-inducible factors (HIF), a family of heterodimeric transcription factors composed of an α -and a β -subunit capable of recognizing hypoxia-response elements (HRE) in the regulatory regions of hypoxia-inducible genes [3][4][5] . In contrast to the constitutive HIF-β , oxygen levels regulate HIF-α activity and protein stability. At normoxia, an asparagine residue within the C-terminal transactivation domain of HIF-α is hydroxylated by the factor inhibiting HIF-1 (FIH-1), impairing the recruitment of the coactivator CBP (cAMP response element binging protein) 6,7 . An additional modification by hydroxylation regulates HIF-α protein stability, in this instance by a family of prolyl hydroxylase domain proteins (PHD), that hydroxylate two distinct proline residues within HIF-α [8][9][10][11][12] . Hydroxylated prolines are the recognition signature for the E3 ubiquitin-ligase von Hippel-Lindau protein (VHL), leading to proteasome-mediated degradation of HIF-α [13][14][15][16][17][18][19] . HIF dioxygenases (PHDs and FIH-1) require molecular oxygen to hydroxylate HIF-α , and are considered the cellular oxygen sensors. Upon oxygen deprivation, the dioxygenases are rendered inactive allowing formation of the transcriptional active HIF. In certain tissues, as the cornea in the eye, avascularity is maintained under hypoxic conditions, illustrating a supplementary regulatory mechanism of HIF-α proteins. In the hypoxic cornea, the tissue-specific inhibitory PAS protein (IPAS; inhibitory Period-Arnt-Sim domain) binds HIF-α subunits and creates a DNA-abortive complex incapable of activating transcription 20,21 .

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Optimizing Donor Cellular Dissociation and Subretinal Injection Parameters for Stem Cell-Based Treatments

Scruggs

Jiao

Cranston

et al. 2019

Stem Cells Translational Medicine

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Subretinal delivery of stem cell‐derived retinal cells as a strategy to treat retinal degenerative blindness holds great promise. Currently, two clinical trials are underway in which human fetal retinal progenitor cells (RPCs) are being delivered to patients by intravitreal or subretinal injection to preserve or restore vision, respectively. With the advent of the induced pluripotent stem cell (iPSC), and in turn three‐dimensional derivation of retinal tissue, it is now possible to generate autologous RPCs for cell replacement. The purpose of this study was to evaluate the effect of commonly used cell isolation and surgical manipulation strategies on donor cell viability. iPSC‐RPCs were subjected to various conditions, including different dissociation and isolation methods, injection cannula sizes, and preinjection storage temperatures and times. The effects of commonly used surgical techniques on both host and donor cell viability were evaluated in Yucatan mini‐pigs (n = 61 eyes). We found a significant increase in cell viability when papain was used for RPC isolation. In addition, a significant decrease in cell viability was detected when using the 41G cannula compared with 31G and at storage times of 4 hours compared with 30 minutes. Although 96.4% of all eyes demonstrated spontaneous retinal reattachment following injection, retinal pigment epithelium (RPE) abnormalities were seen more frequently in eyes receiving injections via a 31G cannula; interestingly, eyes that received cell suspensions were relatively protected against such RPE changes. These findings indicate that optimization of donor cell isolation and delivery parameters should be considered when developing a subretinal cell replacement strategy. Stem Cells Translational Medicine 2019;8:797&809

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In Vivo Imaging of Subretinal Bleb-Induced Outer Retinal Degeneration in the Rabbit

Abstract: Subretinal blebs induce pronounced photoreceptor degeneration and RPE changes in the rabbit as demonstrated by in vivo imaging using SD-OCT, IR-cSLO, and BAF.

Cited by 29 publications

References 27 publications

Integration of Subretinal Suspension Transplants of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells in a Large-Eyed Model of Geographic Atrophy

Integration of Subretinal Suspension Transplants of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells in a Large-Eyed Model of Geographic Atrophy

Gene transfer of prolyl hydroxylase domain 2 inhibits hypoxia‐inducible angiogenesis in a model of choroidal neovascularization

Optimizing Donor Cellular Dissociation and Subretinal Injection Parameters for Stem Cell-Based Treatments

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