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Age-related macular degeneration (AMD), a leading cause of vision loss, primarily arises from the degeneration of retinal pigment epithelium (RPE) and photoreceptors. Current therapeutic options for dry AMD are limited. Encouragingly, cultured RPE cells on parylene-based biomimetic Bruch's membrane demonstrate characteristics akin to the native RPE layer. In this study, we cultivated human embryonic stem cell-derived polarized RPE (hESC-PRPE) cells on parylene membranes at both small- and large-scale settings, collecting conditioned supernatant, denoted as PRPE-SF. We conducted a comprehensive analysis of the morphology of the cultured hESC-RPE cells and the secreted growth factors in PRPE-SF. To evaluate the in vivo efficacy of these products, the product was administered via intravitreal injections of PRPE-SF in immunodeficient Royal College of Surgeons (iRCS) rats, a model for retinal degeneration. Our study not only demonstrated the scalability of PRPE-SF production while maintaining RPE cell phenotype but also showed consistent protein concentrations between small- and large-scale batches. We consistently identified 10 key factors in PRPE-SF, including BMP-7, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-6, MANF, PEDF, PDGF-AA, TGFß1, and VEGF. Following intravitreal administration of PRPE-SF, we observed a significant increase in the thickness of the outer nuclear layer (ONL) and photoreceptor preservation in iRCS rats. Furthermore, correlation analysis revealed that IGFBP-3, IGFBP-4, MANF, PEDF, and TGFß1 displayed positive associations with in vivo bioactivity, while GDF-15 exhibited a negative correlation. Overall, this study highlights the feasibility of scaling up PRPE-SF production on parylene membranes without compromising its essential constituents. The outcomes of PRPE-SF administration in an animal model of retinal degeneration present substantial potential for photoreceptor preservation. Moreover, the identification of candidate surrogate potency markers, showing strong positive associations with in vivo bioactivity, lays a solid foundation for the development of a promising therapeutic intervention for retinal degenerative diseases.
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Polímeros , Degeneração Retiniana , Epitélio Pigmentado da Retina , Xilenos , Humanos , Animais , Ratos , Epitélio Pigmentado da Retina/metabolismo , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Proteína 4 de Ligação a Fator de Crescimento Semelhante à Insulina , Degeneração Retiniana/metabolismoRESUMO
Retinal degenerative diseases, including age-related macular degeneration and retinitis pigmentosa, significantly contribute to adult blindness. The Royal College of Surgeons (RCS) rat is a well-established disease model for studying these dystrophies; however, molecular investigations remain limited. We conducted a comprehensive analysis of retinal degeneration in RCS rats, including an immunodeficient RCS (iRCS) sub-strain, using ocular coherence tomography, electroretinography, histology, and molecular dissection using transcriptomics and immunofluorescence. No significant differences in retinal degeneration progression were observed between the iRCS and immunocompetent RCS rats, suggesting a minimal role of adaptive immune responses in disease. Transcriptomic alterations were primarily in inflammatory signaling pathways, characterized by the strong upregulation of Tnfa, an inflammatory signaling molecule, and Nox1, a contributor to reactive oxygen species (ROS) generation. Additionally, a notable decrease in Alox15 expression was observed, pointing to a possible reduction in anti-inflammatory and pro-resolving lipid mediators. These findings were corroborated by immunostaining, which demonstrated increased photoreceptor lipid peroxidation (4HNE) and photoreceptor citrullination (CitH3) during retinal degeneration. Our work enhances the understanding of molecular changes associated with retinal degeneration in RCS rats and offers potential therapeutic targets within inflammatory and oxidative stress pathways for confirmatory research and development.
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Degeneração Macular , Degeneração Retiniana , Retinose Pigmentar , Cirurgiões , Humanos , Adulto , Animais , Ratos , RetinaRESUMO
PURPOSE: To create new immunodeficient Royal College of Surgeons (RCS) rats by introducing the defective MerTK gene into athymic nude rats. METHODS: Female homozygous RCS (RCS-p+/RCS-p+) and male nude rats (Hsd:RH-Foxn1mu, mutation in the foxn1 gene; no T cells) were crossed to produce heterozygous F1 progeny. Double homozygous F2 progeny obtained by crossing the F1 heterozygotes was identified phenotypically (hair loss) and genotypically (RCS-p+ gene determined by PCR). Retinal degenerative status was confirmed by optical coherence tomography (OCT) imaging, electroretinography (ERG), optokinetic (OKN) testing, superior colliculus (SC) electrophysiology, and by histology. The effect of xenografts was assessed by transplantation of human embryonic stem cell-derived retinal pigment epithelium (hESC-RPE) and human-induced pluripotent stem cell-derived RPE (iPS-RPE) into the eye. Morphological analysis was conducted based on hematoxylin and eosin (H&E) and immunostaining. Age-matched pigmented athymic nude rats were used as control. RESULTS: Approximately 6% of the F2 pups (11/172) were homozygous for RCS-p+ gene and Foxn1mu gene. Homozygous males crossed with heterozygous females resulted in 50% homozygous progeny for experimentation. OCT imaging demonstrated significant loss of retinal thickness in homozygous rats. H&E staining showed photoreceptor thickness reduced to 1-3 layers at 12 weeks of age. Progressive loss of visual function was evidenced by OKN testing, ERG, and SC electrophysiology. Transplantation experiments demonstrated survival of human-derived cells and absence of apparent immune rejection. CONCLUSIONS: This new rat animal model developed by crossing RCS rats and athymic nude rats is suitable for conducting retinal transplantation experiments involving xenografts.
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Modelos Animais de Doenças , Células-Tronco Embrionárias Humanas/transplante , Síndromes de Imunodeficiência/terapia , Células-Tronco Pluripotentes Induzidas/transplante , Distrofias Retinianas/terapia , Epitélio Pigmentado da Retina/transplante , Animais , Sobrevivência Celular , Eletrorretinografia , Feminino , Técnicas de Genotipagem , Sobrevivência de Enxerto/fisiologia , Células-Tronco Embrionárias Humanas/fisiologia , Humanos , Síndromes de Imunodeficiência/diagnóstico , Síndromes de Imunodeficiência/fisiopatologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Masculino , Fenótipo , Ratos , Ratos Nus , Retina/fisiopatologia , Distrofias Retinianas/diagnóstico , Distrofias Retinianas/fisiopatologia , Epitélio Pigmentado da Retina/fisiologia , Tomografia de Coerência Óptica , c-Mer Tirosina Quinase/genéticaRESUMO
Class C ß-lactamases poorly hydrolyze cephamycins (e.g., cefoxitin, cefotetan, and moxalactam). In the past 2 decades, a new family of plasmid-based AmpC ß-lactamases conferring resistance to cefoxitin, the FOX family, has grown to include nine unique members descended from the Aeromonas caviae chromosomal AmpC. To understand the basis for the unique cephamycinase activity in the FOX family, we determined the first X-ray crystal structures of FOX-4, apo enzyme and the acyl-enzyme with its namesake compound, cefoxitin, using the Y150F deacylation-deficient variant. Notably, recombinant expression of N-terminally tagged FOX-4 also yielded an inactive adenylylated enzyme form not previously observed in ß-lactamases. The posttranslational modification (PTM), which occurs on the active site Ser64, would not seem to provide a selective advantage, yet might present an opportunity for the design of novel antibacterial drugs. Substantial ligand-induced changes in the enzyme are seen in the acyl-enzyme complex, particularly the R2 loop and helix H10 (P289 to N297), with movement of F293 by 10.3 Å. Taken together, this study provides the first picture of this highly proficient class C cephamycinase, uncovers a novel PTM, and suggests a possible cephamycin resistance mechanism involving repositioning of the substrate due to the presence of S153P, N289P, and N346I substitutions in the ligand binding pocket.
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Antibacterianos/farmacologia , Proteínas de Bactérias/ultraestrutura , Cefoxitina/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Proteínas de Escherichia coli/ultraestrutura , beta-Lactamases/ultraestrutura , Aeromonas caviae/efeitos dos fármacos , Sequência de Aminoácidos , Antibacterianos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cefoxitina/metabolismo , Cristalografia por Raios X , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Testes de Sensibilidade Microbiana , Modelos Moleculares , Dados de Sequência Molecular , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/ultraestrutura , Processamento de Proteína Pós-Traducional , Alinhamento de Sequência , Espectrometria de Massas em Tandem , beta-Lactamases/genética , beta-Lactamases/metabolismoRESUMO
PURPOSE: A subretinal implant termed CPCB-RPE1 is currently being developed to surgically replace dystrophic RPE in patients with dry age-related macular degeneration (AMD) and severe vision loss. CPCB-RPE1 is composed of a terminally differentiated, polarized human embryonic stem cell-derived RPE (hESC-RPE) monolayer pre-grown on a biocompatible, mesh-supported submicron parylene C membrane. The objective of the present delivery study was to assess the feasibility and 1-month safety of CPCB-RPE1 implantation in Yucatán minipigs, whose eyes are similar to human eyes in size and gross retinal anatomy. METHODS: This was a prospective, partially blinded, randomized study in 14 normal-sighted female Yucatán minipigs (aged 2 months, weighing 24-35 kg). Surgeons were blinded to the randomization codes and postoperative and post-mortem assessments were performed in a blinded manner. Eleven minipigs received CPCB-RPE1 while three control minipigs underwent sham surgery that generated subretinal blebs. All animals except two sham controls received combined local (Ozurdex™ dexamethasone intravitreal implant) and systemic (tacrolimus) immunosuppression or local immunosuppression alone. Correct placement of the CPCB-RPE1 implant was assessed by in vivo optical coherence tomography and post-mortem histology. hESC-RPE cells were identified using immunohistochemistry staining for TRA-1-85 (a human marker) and RPE65 (an RPE marker). As the study results of primary interest were nonnumerical no statistical analysis or tests were conducted. RESULTS: CPCB-RPE1 implants were reliably placed, without implant breakage, in the subretinal space of the minipig eye using surgical techniques similar to those that would be used in humans. Histologically, hESC-RPE cells were found to survive as an intact monolayer for 1 month based on immunohistochemistry staining for TRA-1-85 and RPE65. CONCLUSIONS: Although inconclusive regarding the necessity or benefit of systemic or local immunosuppression, our study demonstrates the feasibility and safety of CPCB-RPE1 subretinal implantation in a comparable animal model and provides an encouraging starting point for human studies.
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Células-Tronco Embrionárias Humanas/transplante , Degeneração Macular/cirurgia , Epitélio Pigmentado da Retina/transplante , Transplante de Células-Tronco/métodos , Animais , Células Cultivadas , Modelos Animais de Doenças , Estudos de Viabilidade , Feminino , Humanos , Degeneração Macular/diagnóstico , Estudos Prospectivos , Epitélio Pigmentado da Retina/citologia , Suínos , Porco Miniatura , Tomografia de Coerência Óptica , Resultado do TratamentoRESUMO
BACKGROUND: The retinotopic map property of the superior colliculus (SC) is a reliable indicator of visual functional changes in rodents. Electrophysiological mapping of the SC using a single electrode has been employed for measuring visual function in rat and mouse disease models. Single electrode mapping is highly laborious requiring long-term exposure to the SC surface and prolonged anesthetic conditions that can adversely affect the mapping data. NEW METHOD: To avoid the above-mentioned issues, we fabricated a fifty-six (56) electrode multi-electrode array (MEA) for rapid and reliable visual functional mapping of the SC. Since SC is a dome-shaped structure, the array was made of electrodes with dissimilar tip lengths to enable simultaneous and uniform penetration of the SC. RESULTS: SC mapping using the new MEA was conducted in retinal degenerate (RD) Royal College of Surgeons (RCS) rats and rats with focal retinal damage induced by green diode laser. For SC mapping, the MEA was advanced into the SC surface and the visual activities were recorded during full-filed light stimulation of the eye. Based on the morphological examination, the MEA electrodes covered most of the exposed SC area and penetrated the SC surface at a relatively uniform depth. MEA mapping in RCS rats (n=9) demonstrated progressive development of a scotoma in the SC that corresponded to the degree of photoreceptor loss. MEA mapping in the laser damaged rats demonstrated the presence of a scotoma in the SC area that corresponded to the location of retinal laser injury. COMPARISON WITH EXISTING METHODS AND CONCLUSIONS: The use of MEA for SC mapping is advantageous over single electrode recording by enabling faster recordings and reducing anesthesia time. This study establishes the feasibility of the MEA technique for rapid and efficient SC mapping, particularly advantageous for evaluating therapeutic effects in retinal degenerate rat disease models.
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Escotoma , Colículos Superiores , Humanos , Ratos , Animais , Camundongos , Colículos Superiores/fisiologia , Retina/fisiologia , Luz , EletrodosRESUMO
Retinal degeneration, a leading cause of irreversible low vision and blindness globally, can be partially addressed by retina prostheses which stimulate remaining neurons in the retina. However, existing electrode-based treatments are invasive, posing substantial risks to patients and healthcare providers. Here, we introduce a completely noninvasive ultrasonic retina prosthesis, featuring a customized ultrasound two-dimensional array which allows for simultaneous imaging and stimulation. With synchronous three-dimensional imaging guidance and auto-alignment technology, ultrasonic retina prosthesis can generate programmed ultrasound waves to dynamically and precisely form arbitrary wave patterns on the retina. Neuron responses in the brain's visual center mirrored these patterns, evidencing successful artificial vision creation, which was further corroborated in behavior experiments. Quantitative analysis of the spatial-temporal resolution and field of view demonstrated advanced performance of ultrasonic retina prosthesis and elucidated the biophysical mechanism of retinal stimulation. As a noninvasive blindness prosthesis, ultrasonic retina prosthesis could lead to a more effective, widely acceptable treatment for blind patients. Its real-time imaging-guided stimulation strategy with a single ultrasound array, could also benefit ultrasound neurostimulation in other diseases.
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Cegueira , Retina , Próteses Visuais , Retina/diagnóstico por imagem , Retina/fisiologia , Animais , Cegueira/terapia , Cegueira/fisiopatologia , Degeneração Retiniana/terapia , Degeneração Retiniana/diagnóstico por imagem , Ondas Ultrassônicas , Humanos , Neurônios/fisiologia , Ultrassonografia/métodos , Visão Ocular/fisiologiaRESUMO
Functional ultrasound (fUS) flow imaging provides a non-invasive method for the in vivo study of cerebral blood flow and neural activity. This study used functional flow imaging to investigate rat brain's response to ultrasound and colored-light stimuli. Male Long-Evan rats were exposed to direct full-field strobe flashes light and ultrasound stimulation to their retinas, while brain activity was measured using high-frequency ultrasound imaging. Our study found that light stimuli, particularly blue light, elicited strong responses in the visual cortex and lateral geniculate nucleus (LGN), as evidenced by changes in cerebral blood volume (CBV). In contrast, ultrasound stimulation elicited responses undetectable with fUS flow imaging, although these were observable when directly measuring the brain's electrical signals. These findings suggest that fUS flow imaging can effectively differentiate neural responses to visual stimuli, with potential applications in understanding visual processing and developing new diagnostic tools.
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An ultrasound concave 2-D ring array transducer was designed for applications in visual stimulation of the retina with a long-term goal to restore vision in individuals with intact neurons but suffering blindness due to retinopathies. The array was synthesized and has a frequency of 20 MHz (0.075-mm wavelengths in water), 18-mm focal length (the curvature of the concave array), 1004 elements (with a pitch of 4.0 wavelengths), and inner and outer diameters of 9 and 14 mm, respectively. Wave patterns produced with the array at the focal distance were simulated. Results show that the wave patterns obtained can achieve a full-width-at-half-maximum (FWHM) resolution of 0.147 mm that is very close to the FWHM diffraction limit (0.136 mm). In addition, a scaled experiment at a lower frequency of 2.5 MHz was performed. The result is very close to those obtained with the simulations.
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Retina , Transdutores , Humanos , Desenho de Equipamento , Ultrassonografia/métodos , Imagens de Fantasmas , Retina/diagnóstico por imagemRESUMO
Retinal degenerative diseases, including age-related macular degeneration (AMD) and retinitis pigmentosa, lack effective therapies. Conventional monotherapeutic approaches fail to target the multiple affected pathways in retinal degeneration. However, the retinal pigment epithelium (RPE) secretes several neurotrophic factors addressing diverse cellular pathways, potentially preserving photoreceptors. This study explored human embryonic stem cell-derived, polarized RPE soluble factors (PRPE-SF) as a combination treatment for retinal degeneration. PRPE-SF promoted retinal progenitor cell survival, reduced oxidative stress in ARPE-19 cells, and demonstrated critical antioxidant and anti-inflammatory effects for preventing retinal degeneration in the Royal College of Surgeons (RCS) rat model. Importantly, PRPE-SF treatment preserved retinal structure and scotopic b-wave amplitudes, suggesting therapeutic potential for delaying retinal degeneration. PRPE-SF is uniquely produced using biomimetic membranes for RPE polarization and maturation, promoting a protective RPE secretome phenotype. Additionally, PRPE-SF is produced without animal serum to avoid immunogenicity in future clinical development. Lastly, PRPE-SF is a combination of neurotrophic factors, potentially ameliorating multiple dysfunctions in retinal degenerations. In conclusion, PRPE-SF offers a promising therapeutic candidate for retinal degenerative diseases, advancing the development of effective therapeutic strategies for these debilitating conditions.
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Degeneração Retiniana , Epitélio Pigmentado da Retina , Ratos , Humanos , Animais , Epitélio Pigmentado da Retina/metabolismo , Degeneração Retiniana/metabolismo , Secretoma , Retina/metabolismo , Células Fotorreceptoras/metabolismoRESUMO
The optic nerve is the second cranial nerve (CN II) that connects and transmits visual information between the retina and the brain. Severe damage to the optic nerve often leads to distorted vision, vision loss, and even blindness. Such damage can be caused by various types of degenerative diseases, such as glaucoma and traumatic optic neuropathy, and result in an impaired visual pathway. To date, researchers have not found a viable therapeutic method to restore the impaired visual pathway; however, in this paper, a newly synthesized model is proposed to bypass the damaged portion of the visual pathway and set up a direct connection between a stimulated visual input and the visual cortex (VC) using Low-frequency Ring-transducer Ultrasound Stimulation (LRUS). In this study, by utilizing and integrating various advanced ultrasonic and neurological technologies, the following advantages are achieved by the proposed LRUS model: 1. This is a non-invasive procedure that uses enhanced sound field intensity to overcome the loss of ultrasound signal due to the blockage of the skull. 2. The simulated visual signal generated by LRUS in the visual-cortex-elicited neuronal response in the visual cortex is comparable to light stimulation of the retina. The result was confirmed by a combination of real-time electrophysiology and fiber photometry. 3. VC showed a faster response rate under LRUS than light stimulation through the retina. These results suggest a potential non-invasive therapeutic method for restoring vision in optic-nerve-impaired patients using ultrasound stimulation (US).
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OBJECTIVE: To evaluate the feasibility of a new technique for the implantation of ultrathin substrates containing stem cell-derived retinal pigment epithelium (RPE) cells into the subretinal space of retina-degenerate Royal College of Surgeon (RCS) rats. METHODS: A platform device was used for the implantation of 4-µm-thick parylene substrates containing a monolayer of human embryonic stem cell-derived RPE (hESC-RPE). Normal Copenhagen rats (n = 6) and RCS rats (n = 5) were used for the study. Spectral-domain optical coherence tomography (SD-OCT) scanning and histological examinations were performed to confirm placement location of the implant. hESC-RPE cells attached to the substrate before and after implantation were evaluated using standard cell counting techniques. RESULTS: SD-OCT scanning and histological examination revealed that the substrates were precisely placed in the rat's subretinal space. The hESC-RPE cell monolayer that covered the surface of the substrate was found to be intact after implantation. Cell counting data showed that less than 2% of cells were lost from the substrate due to the implantation procedure (preimplantation count 2,792 ± 74.09 cells versus postimplantation count 2,741 ± 62.08 cells). Detailed microscopic examination suggested that the cell loss occurred mostly along the edges of the implant. CONCLUSION: With the help of this platform device, it is possible to implant ultrathin substrates containing an RPE monolayer into the rat's subretinal space. This technique can be a useful approach for stem cell-based tissue bioengineering techniques in retinal transplantation research.
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Células-Tronco Embrionárias/citologia , Polímeros , Distrofias Retinianas/terapia , Epitélio Pigmentado da Retina/transplante , Transplante de Células-Tronco , Engenharia Tecidual , Alicerces Teciduais , Xilenos , Animais , Contagem de Células , Estudos de Viabilidade , Humanos , Ratos , Ratos Mutantes , Retina/patologia , Distrofias Retinianas/diagnóstico , Tomografia de Coerência ÓpticaRESUMO
BACKGROUND: The main cause of progressive vision impairment in retinal degenerative diseases is the dysfunction of photoreceptors and the underlying retinal pigment epithelial cells. The inadequate regenerative capacity of the neural retina and lack of established therapeutic options demand the development of clinical-grade protocols to halt the degenerative process in the eye or replace the damaged cells by using stem cell-derived products. Recently, stem cell-based regenerative therapies have been at the forefront of clinical investigations for retinal dystrophies. OBJECTIVE: This article will review different stem cell-based therapies currently employed for retinal degenerative diseases, recent clinical trials, and major challenges in the translation of these therapies from bench to bedside. METHODOLOGY: A systematic literature review was conducted to identify potentially relevant articles published in MEDLINE/PubMed, Embase, ClinicalTrials.gov, Drugs@FDA, European Medicines Agency, and World Health Organization International Clinical Trials Registry Platform. RESULTS: Transplantation of healthy cells to replace damaged cells in the outer retina is a clinically relevant concept because the inner retina that communicates with the visual areas of the brain remains functional even after the photoreceptors are completely lost. Various methods have been established for the differentiation of pluripotent stem cells into different retinal cell types that can be used for therapies. Factors released from transplanted somatic stem cells showed trophic support and photoreceptor rescue during the early stages of the disease. Several preclinical and phase I/II clinical studies using terminally differentiated photoreceptor/retinal pigment epithelial cells derived from pluripotent stem cells have shown proof of concept for visual restoration in Age-related Macular Degeneration (AMD), Stargardt disease, and Retinitis Pigmentosa (RP). CONCLUSION: Cell replacement therapy has great potential for vision restoration. The results obtained from the initial clinical trials are encouraging and indicate its therapeutic benefits. The current status of the therapies suggests that there is a long way to go before these results can be applied to routine clinical practice. Input from the ongoing multicentre clinical trials will give a more refined idea for the future design of clinical-grade protocols to transplant GMP level HLA matched cells.
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Células-Tronco Pluripotentes , Degeneração Retiniana , Humanos , Retina , Degeneração Retiniana/terapia , Epitélio Pigmentado da Retina/transplante , Pigmentos da Retina , Transplante de Células-Tronco/métodosRESUMO
Optokinetic nystagmus (OKN) is a reflexive eye movement initiated by the motion of visual stimuli in the field of vision. The head-tracking movement associated with OKN is commonly used as a measure of visual function in rodents. To record OKN responses in normal and experimental rats, a simple and inexpensive apparatus has been developed. This setup uses two tablet screens to display the OKN visual stimulus consisting of high contrast black and white stripes generated using the OKN Stripes Visualization Web Application, a freely available software. The rat is placed inside a clear Plexiglass holder that limits movement so that the rat's head continuously faces the OKN display screen. The position of the rat holder can be changed to adjust the distance between the rat and the display screen. A micro-camera positioned above the rat holder is used to record the rat's visual activities. These recordings can be used for quantitative assessments. Based on the presence or absence of clear head-tracking, the OKN responses at different spatial frequencies can be determined. The collected data demonstrates a novel technique for reliable measurement of visual acuity in normal and retinal degenerate rats.
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Nistagmo Optocinético , Visão Ocular , Animais , Movimento , Ratos , Retina , Acuidade VisualRESUMO
Ultrasound neuromodulation is an emerging technology. A significant amount of effort has been devoted to investigating the feasibility of noninvasive ultrasound retinal stimulation. Recent studies have shown that ultrasound can activate neurons in healthy and degenerated retinas. Specifically, high-frequency ultrasound can evoke localized neuron responses and generate patterns in visual circuits. In this review, we recapitulate pilot studies on ultrasound retinal stimulation, compare it with other neuromodulation technologies, and discuss its advantages and limitations. An overview of the opportunities and challenges to develop a noninvasive retinal prosthesis using high-frequency ultrasound is also provided.
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Retina , Próteses Visuais , Retina/diagnóstico por imagem , Retina/fisiologia , UltrassonografiaRESUMO
Objective. Retinal degeneration involving progressive deterioration and loss of function of photoreceptors is a major cause of permanent vision loss worldwide. Strategies to treat these incurable conditions incorporate retinal prostheses via electrically stimulating surviving retinal neurons with implanted devices in the eye, optogenetic therapy, and sonogenetic therapy. Existing challenges of these strategies include invasive manner, complex implantation surgeries, and risky gene therapy. Methods and Results. Here, we show that direct ultrasound stimulation on the retina can evoke neuron activities from the visual centers including the superior colliculus and the primary visual cortex (V1), in either normal-sighted or retinal degenerated blind rats in vivo. The neuron activities induced by the customized spherically focused 3.1 MHz ultrasound transducer have shown both good spatial resolution of 250 µm and temporal resolution of 5 Hz in the rat visual centers. An additional customized 4.4 MHz helical transducer was further implemented to generate a static stimulation pattern of letter forms. Conclusion. Our findings demonstrate that ultrasound stimulation of the retina in vivo is a safe and effective approach with high spatiotemporal resolution, indicating a promising future of ultrasound stimulation as a novel and noninvasive visual prosthesis for translational applications in blind patients.
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Retinal degeneration, such as age-related macular degeneration (AMD), is a leading cause of blindness worldwide. A myriad of approaches have been undertaken to develop regenerative medicine-based therapies for AMD, including stem cell-based therapies. Rodents as animal models for retinal degeneration are a foundation for translational research, due to the broad spectrum of strains that develop retinal degeneration diseases at different stages. However, mimicking human therapeutic delivery of subretinal implants in rodents is challenging, due to anatomical differences such as lens size and vitreous volume. This surgical protocol aims to provide a guided method for transplanting implants into the subretinal space in rats. A user-friendly comprehensive description of the critical steps has been included. This protocol has been developed as a cost-efficient surgical procedure for reproducibility across different preclinical studies in rats. Proper miniaturization of a human-sized implant is required prior to conducting the surgical experiment, which includes adjustments to the dimensions of the implant. An external approach is used instead of an intravitreal procedure to deliver the implant to the subretinal space. Using a small sharp needle, a scleral incision is performed in the temporal superior quadrant, followed by paracentesis to reduce intraocular pressure, thereby minimizing resistance during the surgical implantation. Next, a balanced salt solution (BSS) injection through the incision is carried out to achieve focal retinal detachment (RD). Lastly, insertion and visualization of the implant into the subretinal space are conducted. Post-operative assessment of the subretinal placement of the implant includes imaging by spectral domain optical coherence tomography (SD-OCT). Imaging follow-ups ascertain the subretinal stability of the implant, before the eyes are harvested and fixated for histological analysis.
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Degeneração Macular , Degeneração Retiniana , Humanos , Ratos , Animais , Degeneração Retiniana/cirurgia , Degeneração Retiniana/patologia , Reprodutibilidade dos Testes , Modelos Animais de Doenças , Degeneração Macular/terapia , Tomografia de Coerência Óptica/métodosRESUMO
The retina is a complex and fragile photosensitive part of the central nervous system which is prone to degenerative diseases leading to permanent vision loss. No proven treatment strategies exist to treat or reverse the degenerative conditions. Recent investigations demonstrate that cell transplantation therapies to replace the dysfunctional retinal pigment epithelial (RPE) cells and or the degenerating photoreceptors (PRs) are viable options to restore vision. Pluripotent stem cells, retinal progenitor cells, and somatic stem cells are the main cell sources used for cell transplantation therapies. The success of retinal transplantation based on cell suspension injection is hindered by limited cell survival and lack of cellular integration. Recent advances in material science helped to develop strategies to grow cells as intact monolayers or as sheets on biomaterial scaffolds for transplantation into the eyes. Such implants are found to be more promising than the bolus injection approach. Tissue engineering techniques are specifically designed to construct biodegradable or non-degradable polymer scaffolds to grow cells as a monolayer and construct implantable grafts. The engineered cell construct along with the extracellular matrix formed, can hold the cells in place to enable easy survival, better integration, and improved visual function. This article reviews the advances in the use of scaffolds for transplantation studies in animal models and their application in current clinical trials.
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Retinal pigment epithelium (RPE) replacement therapy is evolving as a feasible approach to treat age-related macular degeneration (AMD). In many preclinical studies, RPE cells are transplanted as a cell suspension into immunosuppressed animal eyes and transplant effects have been monitored only short-term. We investigated the long-term effects of human Induced pluripotent stem-cell-derived RPE (iPSC-RPE) transplants in an immunodeficient Royal College of Surgeons (RCS) rat model, in which RPE dysfunction led to photoreceptor degeneration. iPSC-RPE cultured as a polarized monolayer on a nanoengineered ultrathin parylene C scaffold was transplanted into the subretinal space of 28-day-old immunodeficient RCS rat pups and evaluated after 1, 4, and 11 months. Assessment at early time points showed good iPSC-RPE survival. The transplants remained as a monolayer, expressed RPE-specific markers, performed phagocytic function, and contributed to vision preservation. At 11-months post-implantation, RPE survival was observed in only 50% of the eyes that were concomitant with vision preservation. Loss of RPE monolayer characteristics at the 11-month time point was associated with peri-membrane fibrosis, immune reaction through the activation of macrophages (CD 68 expression), and the transition of cell fate (expression of mesenchymal markers). The overall study outcome supports the therapeutic potential of RPE grafts despite the loss of some transplant benefits during long-term observations.
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Células-Tronco Pluripotentes Induzidas/transplante , Epitélio Pigmentado da Retina/transplante , Animais , Biomarcadores/metabolismo , Humanos , Implantes Experimentais , Luz , Polímeros , Ratos , Colículos Superiores/efeitos da radiação , Análise de Sobrevida , Visão Ocular/efeitos da radiação , XilenosRESUMO
End-stage age-related macular degeneration (AMD) and retinitis pigmentosa (RP) are two major retinal degenerative (RD) conditions that result in irreversible vision loss. Permanent eye damage can also occur in battlefields or due to accidents. This suggests there is an unmet need for developing effective strategies for treating permanent retinal damages. In previous studies, co-grafted sheets of fetal retina with its retinal pigment epithelium (RPE) have demonstrated vision improvement in rat retinal disease models and in patients, but this has not yet been attempted with stem-cell derived tissue. Here we demonstrate a cellular therapy for irreversible retinal eye injuries using a "total retina patch" consisting of retinal photoreceptor progenitor sheets and healthy RPE cells on an artificial Bruch's membrane (BM). For this, retina organoids (ROs) (cultured in suspension) and polarized RPE sheets (cultured on an ultrathin parylene substrate) were made into a co-graft using bio-adhesives [gelatin, growth factor-reduced matrigel, and medium viscosity (MVG) alginate]. In vivo transplantation experiments were conducted in immunodeficient Royal College of Surgeons (RCS) rats at advanced stages of retinal degeneration. Structural reconstruction of the severely damaged retina was observed based on histological assessments and optical coherence tomography (OCT) imaging. Visual functional assessments were conducted by optokinetic behavioral testing and superior colliculus electrophysiology. Long-term survival of the co-graft in the rat subretinal space and improvement in visual function were observed. Immunohistochemistry showed that co-grafts grew, generated new photoreceptors and developed neuronal processes that were integrated into the host retina. This novel approach can be considered as a new therapy for complete replacement of a degenerated retina.