scholarly journals The learning curve of murine subretinal injection among clinically trained ophthalmic surgeons

2021 ◽  
Author(s):  
Peirong Huang ◽  
Siddharth Narendran ◽  
Felipe Pereira ◽  
Shinichi Fukuda ◽  
Yosuke Nagasaka ◽  
...  
Keyword(s):  
Learning Curve ◽  
Success Rate ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Training System ◽  
Training Data ◽  
Surgeon Experience ◽  
Systematic Training ◽  
Cusum Analysis ◽  
Subretinal Injection

PURPOSE: Subretinal injection (SRI) in mice is widely used in retinal research, yet the learning curve (LC) of this surgically challenging technique is unknown. METHODS: To evaluate the LC for SRI in a murine model, we analyzed training data from 3 clinically trained ophthalmic surgeons from 2018 to 2020. Successful SRI was defined as either the absence of retinal pigment epithelium (RPE) degeneration after phosphate buffer saline injection and the presence of RPE degeneration after Alu RNA injection. Multivariable survival-time regression models were used to evaluate the association between surgeon experience and success rate, with adjustment for injection agents, and to calculate an approximate case number to achieve a 95% success rate. A Cumulative Sum (CUSUM) analysis was performed and plotted individually to monitor each surgeon's simultaneous performance. RESULTS: Despite prior microsurgery experience, the combined average success rate of the first 50 cases in mice was only 27%. The predicted SRI success rate did not reach a plateau above 95% until approximately 364 prior cases. Using the 364-training case as a "cutoff" point, the predicted probability of success before and after the 364th case was 65.38% and 99.32%, respectively (P < 0.0001). CUSUM analysis showed an initial upward slope and then remained within the decision intervals with an acceptable success rate set at 95% in the late stage. CONCLUSIONS: This study demonstrates the complexity and substantial LC for successful SRI in mice with high confidence. A systematic training system could improve the reliability and reproducibility of SRI-related experiments and improve the interpretation of experimental results using this technique. Translational Relevance: Our prediction model and monitor system allow objective quantification of technical proficiency in the field of subretinal drug delivery and gene therapy for the first time.

Challenges in non-viral ocular gene transfer

2007 ◽  
Vol 35 (1) ◽  
pp. 47-49 ◽  
Author(s):  
L. Peeters ◽  
N.N. Sanders ◽  
J. Demeester ◽  
S.C. De Smedt
Keyword(s):  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Viral Gene ◽  
Target Tissue ◽  
Long Distance ◽  
Promising Alternative ◽  
Gene Complexes ◽  
Poly Ethylene ◽  
Review Reports ◽  
Subretinal Injection

Nowadays, there is no effective treatment for many retinal disorders. Knowledge of the genetic basis of many severe ocular diseases may allow for alternative treatments by gene therapy. Non-viral gene complexes, such as lipo- and poly-plexes, can be delivered to the posterior segment, most often the target tissue, by intravitreal or subretinal injection. Since subretinal injections are very invasive, intravitreal injection is a promising alternative route to deliver gene complexes into the eye. However, the drawback of this technique is the relative long distance the complexes have to travel through the vitreous gel before they reach the retina. This mini-review reports on how non-viral gene complexes behave in vitreous. It especially focuses on how the coating of lipoplexes with poly(ethylene glycol) influences their behaviour in vitreous and the transfection of retinal pigment epithelium.

Semiquantitative Methods for GFP Immunohistochemistry and In Situ Hybridization to Evaluate AAV Transduction of Mouse Retinal Cells Following Subretinal Injection

2020 ◽  
pp. 019262332096480
Author(s):  
Gennadiy Bondarenko ◽  
Steven D. Sorden ◽  
Brian J. Christian ◽  
Sharron Webster ◽  
Alok K. Sharma
Keyword(s):  
In Situ Hybridization ◽  
Fluorescent Protein ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Photoreceptor Layer ◽  
Rna In Situ Hybridization ◽  
Semiquantitative Evaluation ◽  
Green Fluorescent ◽  
Subretinal Injection

The goal of this study was to develop methods for the evaluation of green fluorescent protein (GFP) and GFP transcript biodistribution in paraformaldehyde-fixed paraffin-embedded (PFPE) eye sections to assess the effectiveness of Adeno-associated virus (AAV) gene delivery in an experimental ocular toxicity study. Female C57BL/6NTac mice were administered AAV2-enhancedGFP vector once via subretinal injection. One group also received anti-inflammatory therapy (meloxicam). Immunohistochemistry (IHC) and RNA in situ hybridization (ISH) for GFP were performed on PFPE serial eye sections and evaluated using semiquantitative methods. On day 43, GFP labeling in both IHC and ISH sections was greatest in the retinal pigment epithelium, compared with other retinal layers in which expression was negative to moderate. Despite the presence of IHC GFP labeling in the photoreceptor layer (PRL) in some animals, only low numbers of transduced cells were detected by ISH in the PRL. Simultaneous analysis of IHC and ISH may be needed for comprehensive assessment of gene transduction and protein biodistribution. This study demonstrates approaches for semiquantitative evaluation of IHC and ISH that allow interpretation and reporting of GFP expression in toxicity studies.

scholarly journals Advantages of robotic assistance over a manual approach in simulated subretinal injections and its relevance for gene therapy

Gene Therapy ◽  
2021 ◽  
Author(s):  
Reza Ladha ◽  
Thijs Meenink ◽  
Jorrit Smit ◽  
Marc D. de Smet
Keyword(s):  
Gene Therapy ◽  
Clinical Success ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Genetic Diseases ◽  
Cost Effective ◽  
Robotic Assistance ◽  
Injection Duration ◽  
Retinal Hole ◽  
Subretinal Injection

AbstractSubretinal injection is a method for gene delivery to treat genetic diseases of the photoreceptors and retinal pigment epithelium. A reflux-free subretinal injection is important to allow effective, safe, and cost-effective gene therapy to the retina. We report on a comparison between manual and robotic assistance in simulated subretinal injections using an artificial retina model. Nine surgeons carried out the procedure with and without the Preceyes Surgical System, using an OPMI Lumera 700 Zeiss surgical microscope equipped with intra-operative optical coherence tomography. Success in creating a bleb without reflux, injection duration, drift, tremor, and increase in the diameter of the puncture hole were analyzed. Robotic assistance improved drift (median 16 vs 212 µm), tremor (median 1 vs 18 µm), enlargement of the retinal hole, and allowed for prolonged injection times (median 52 vs 29 sec). Robotic assistance allowed higher rate of bleb formation (8/9 vs 4/9 attempts) with a moderate reduction in reflux (7/9 vs 8/9 attempts) in this artificial model. Robotic assistance can significantly contribute to subretinal injections and provide quantifiable parameters in assessing surgical and clinical success of novel retinal gene therapies.

scholarly journals Functional and morphological analysis of the subretinal injection of retinal pigment epithelium cells

2012 ◽  
Vol 29 (2) ◽  
pp. 83-93 ◽  
Author(s):  
MAREN ENGELHARDT ◽  
CHINATSU TOSHA ◽  
VANDA S. LOPES ◽  
BRYAN CHEN ◽  
LISA NGUYEN ◽  
...  
Keyword(s):  
Retinal Pigment Epithelium ◽  
Host Cell ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Primary Cultures ◽  
Host Cells ◽  
Apical Surface ◽  
Noninvasive Methods ◽  
Rpe Cells ◽  
Subretinal Injection

AbstractReplacement of retinal pigment epithelium (RPE) cells by transplantation is a potential treatment for some retinal degenerations. Here, we used a combination of invasive and noninvasive methods to characterize the structural and functional consequences of subretinal injection of RPE cells. Pigmented cells from primary cultures were injected into albino mice. Recovery was monitored over 8 weeks by fundus imaging, spectral domain optical coherence tomography (sdOCT), histology, and electroretinography (ERG). sdOCT showed that retinal reattachment was nearly complete by 1 week. ERG response amplitudes were reduced after injection, with cone-mediated function then recovering better than rod function. Photoreceptor cell loss was evident by sdOCT and histology, near the site of injection, and is likely to have been the main cause of incomplete recovery. With microscopy, injected cells were identified by the presence of apical melanosomes. They either established contact with Bruch’s membrane, and thus became part of the RPE monolayer, or were located on the apical surface of the host’s cells, resulting in apposition of the basal surface of the injected cell with the apical surface of the host cell and the formation of a series of desmosomal junctions. RPE cell density was not increased, indicating that the incorporation of an injected cell into the RPE monolayer was concomitant with the loss of a host cell. The transplanted and remaining host cells contained large vacuoles of ingested debris as well as lipofuscin-like granules, suggesting that they had scavenged the excess injected and host cells, and were stressed by the high digestive load. Therefore, although significant functional and structural recovery was observed, the consequences of this digestive stress may be a concern for longer-term health, especially where RPE cell transplantation is used to treat diseases that include lipofuscin accumulation as part of their pathology.

High-resolution scanning electron microscopy (HRSEM) of mitochondria from various rat tissues

1988 ◽  
Vol 46 ◽  
pp. 14-15
Author(s):  
P.J. Lea ◽  
M.J. Hollenberg
Keyword(s):  
Electron Microscopy ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Rat Hepatocytes ◽  
Three Dimensions ◽  
Objective Lens ◽  
Rat Tissues ◽  
Thin Sections ◽  
Reconstruction Methods ◽  
Scanning Electron

Our current understanding of mitochondrial ultrastructure has been derived primarily from thin sections using transmission electron microscopy (TEM). This information has been extrapolated into three dimensions by artist's impressions (1) or serial sectioning techniques in combination with computer processing (2). The resolution of serial reconstruction methods is limited by section thickness whereas artist's impressions have obvious disadvantages.In contrast, the new techniques of HRSEM used in this study (3) offer the opportunity to view simultaneously both the internal and external structure of mitochondria directly in three dimensions and in detail.The tridimensional ultrastructure of mitochondria from rat hepatocytes, retinal (retinal pigment epithelium), renal (proximal convoluted tubule) and adrenal cortex cells were studied by HRSEM. The specimens were prepared by aldehyde-osmium fixation in combination with freeze cleavage followed by partial extraction of cytosol with a weak solution of osmium tetroxide (4). The specimens were examined with a Hitachi S-570 scanning electron microscope, resolution better than 30 nm, where the secondary electron detector is located in the column directly above the specimen inserted within the objective lens.

Ultrastructural Changes of Smoooth Endoplasmic Reticulum in the Retinal Pigment Epithelium by Choline Chloride

1972 ◽  
Vol 30 ◽  
pp. 58-59
Author(s):  
Kazushige Hirosawa ◽  
Eichi Yamada
Keyword(s):  
Endoplasmic Reticulum ◽  
Epithelial Cell ◽  
Smooth Endoplasmic Reticulum ◽  
Pigment Epithelium ◽  
Choline Chloride ◽  
Retinal Pigment ◽  
Ultrastructural Changes ◽  
Lower Vertebrate ◽  
Visual Cells ◽  
Pigment Epithelial

The pigment epithelium is located between the choriocapillary and the visual cells. The pigment epithelial cell is characterized by a large amount of the smooth endoplasmic reticulum (SER) in its cytoplasm. In addition, the pigment epithelial cell of some lower vertebrate has myeloid body as a specialized form of the SER. Generally, SER is supposed to work in the lipid metabolism. However, the functions of abundant SER and myeloid body in the pigment epithelial cell are still in question. This paper reports an attempt, to depict the functions of these organelles in the frog retina by administering one of phospholipid precursors.

High-voltage electron microscopy of subretinal scar formation

1992 ◽  
Vol 50 (1) ◽  
pp. 486-487
Author(s):  
G.E. Korte ◽  
M. Marko ◽  
G. Hageman
Keyword(s):  
Electron Microscopy ◽  
Monoclonal Antibody ◽  
Retinal Pigment Epithelium ◽  
Glial Cells ◽  
High Voltage ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Sodium Iodate ◽  
High Voltage Electron Microscopy ◽  
Voltage Electron

Sodium iodate iv. damages the retinal pigment epithelium (RPE) in rabbits. Where RPE does not regenerate (e.g., 1,2) Muller glial cells (MC) forma subretinal scar that replaces RPE. The MC response was studied by HVEM in 3D computer reconstructions of serial thick sections, made using the STEREC0N program (3), and the HVEM at the NYS Dept. of Health in Albany, NY. Tissue was processed for HVEM or immunofluorescence localization of a monoclonal antibody recognizing MG microvilli (4).

The photoreceptor cytoskeleton visualized

1992 ◽  
Vol 50 (1) ◽  
pp. 480-481
Author(s):  
Beth Burnside
Keyword(s):  
Outer Segment ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Cell Polarization ◽  
Synaptic Proteins ◽  
Cell Functions ◽  
Vertebrate Photoreceptor ◽  
Numerous Cell ◽  
Turn Over

The vertebrate photoreceptor provides a drammatic example of cell polarization. Specialized to carry out phototransduction at its distal end and to synapse with retinal interneurons at its proximal end, this long slender cell has a uniquely polarized morphology which is reflected in a similarly polarized cytoskeleton. Membranes bearing photopigment are localized in the outer segment, a modified sensory cilium. Sodium pumps which maintain the dark current critical to photosensory transduction are anchored along the inner segment plasma membrane between the outer segment and the nucleus.Proximal to the nucleus is a slender axon terminating in specialized invaginating synapses with other neurons of the retina. Though photoreceptor diameter is only 3-8u, its length from the tip of the outer segment to the synapse may be as great as 200μ. This peculiar linear cell morphology poses special logistical problems and has evoked interesting solutions for numerous cell functions. For example, the outer segment membranes turn over by means of a unique mechanism in which new disks are continuously added at the proximal base of the outer segment, while effete disks are discarded at the tip and phagocytosed by the retinal pigment epithelium. Outer segment proteins are synthesized in the Golgi near the nucleus and must be transported north through the inner segment to their sites of assembly into the outer segment, while synaptic proteins must be transported south through the axon to the synapse.The role of the cytoskeleton in photoreceptor motile processes is being intensely investigated in several laboratories.

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