We would like to thank the National Institute of Health (NIH) Funding Grant to the National Eye Institute (R01 EY030054) to Dr. Mohamed Al-Shabrawey. We would like to thank Kathy Wolosiewicz for helping us with the video narration. We would like to thank Dr. Ken Mitton of the Eye Research Institute&#39;s Pediatric Retinal Research lab, Oakland University, for his help during the usage of the surgical microscope and recording. This video was edited and directed by Dr. Khaled Elmasry.

All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at Oakland University, Rochester, MI, USA and followed the guidelines established by the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research.
1. Animal preparation
<ol>
	<li>Keep the animals housed under a constant temperature in a light-controlled environment. The temperature settings for the animal housing room should follow the guidelines set forth by the &#34;Guide for the Care and Use of Laboratory Animals&#34;. The temperature range for mice is between 18 &#176;C and 26 &#176;C. Keep the humidity levels controlled and set at about 42%.</li>
	<li>For this experiment, use male C57BL/6J mice (check the Table of Materials for details) that are more than 12 weeks of age (an adult mouse was used for this protocol). 
	NOTE: A wild-type strain was used in this protocol for the demonstration, as it did not have any genetic abnormalities affecting the retina. However, one could use other strains, including genetically manipulated strains, for the retinal explant culture.</li>
	<li>Provide 12 h light/dark cycles via lighting control in the animal housing rooms.</li>
	<li>Inspect every animal for health and adequate food and water intake twice per day during the week and once per day on the weekends. Make sure to provide them with fresh food and clean water.</li>
	<li>If the food and water levels are low during the week, rinse the water bottle and refill it. Add fresh food whenever needed. Change soiled cages daily or as needed for the health of the animals. 
	NOTE: To study the photoreceptors and light-induced changes in the retina, dark-adapt the mice overnight in a special dark box located in a dark room in the animal facility.</li>
	<li>Euthanize the animals in their home cage by CO2 inhalation overdose using compressed CO2 gas in cylinders connected to a CO2 chamber. Conduct death confirmation using a secondary method of euthanasia such as cervical dislocation.</li>
	<li>After completing the procedure, confirm the animal death by an appropriate method, such as confirming cardiac and respiratory arrest or observing fixed and dilated pupils of the animal.</li>
</ol>
2. Tissue preparation
<ol>
	<li>Once the animal is euthanized, clean the area with 70% ethanol. Then, open the eyelids with one hand so that the eye is visible. Apply pressure to the superior and inferior parts of the orbit with the opposite hand using curved forceps until the globe protrudes.</li>
	<li>To enucleate the eye, gently close the forceps on the posterior portion of the eye, and elevate in a continuous motion. Using forceps, hold the eyeball from the optic nerve. Make sure to use sterilized dissection tools for all the steps and to work under complete aseptic conditions.</li>
	<li>Immerse each eyeball in a 1.5 mL tube containing 1 mL of ice-cold HBSS with penicillin-streptomycin (10,000 U/mL). Wash the eyeball in HBSS twice for 5 min each.</li>
	<li>Transfer the eyeball to a 1.5-2 mL tube containing complete media (10% FBS, 2% B27, 1% N2, 1% L-glutamine, and 1% penicillin and streptomycin).</li>
</ol>
3. Tissue dissection
<ol>
	<li>Dissect out the retina carefully under an operating microscope, as illustrated sequentially in Figure 1.
	<ol>
		<li>Make a circumferential incision around the limbus to open the eyeball. Remove the cornea by circularly dissecting along the border of the cornea and the (limbal) incision, followed by removing the anterior segment, lens, and vitreous body, leaving an empty eye cup.</li>
		<li>By holding the region of the optic nerve head with fine forceps, peel off the outer coat of the eye gently. Pay great attention during the removal of the outer coat in order to not injure the neural retina and to ensure the retina remains totally intact. 
		NOTE: The retina must be carefully peeled away from the retinal pigment epithelium (RPE), and a single cut at the optic nerve head must be performed to detach the retina from the optic nerve. Visually ensure this by identifying the hole left by the optic nerve (the optic nerve head).</li>
		<li>Dissect the retina radially into four equal-sized pieces.</li>
	</ol>
	</li>
	<li>Follow the steps below for handling the small pieces of the retina to ensure proper orientation of the retinal culture.
	<ol>
		<li>Using a dissecting scissor blade, open just beneath the retinal piece (Table of Materials).</li>
		<li>Slowly lift the retinal piece with the tip of an open scissor blade, and gently transfer it to the culture plate.</li>
	</ol>
	</li>
	<li>Transfer the explants derived from the isolated retina separately onto cell culture inserts in a 12-well plate, each containing 300 &#181;L of retinal explant media, with the retinal ganglion cell (neuroretina) side facing up. 
	NOTE: Ensure that the media is DMEM media with N2 and B27 supplements. Add penicillin-streptomycin (10,000 U/mL) to the media as well (Table of Materials). The retina is formed of multiple layers of neuronal cells interconnected via synapses and supported from the outside by the pigmented layer of retinal pigment epithelium, which is the black layer that was removed during the dissection.</li>
	<li>Remove any remnants of the pigmented epithelium; however, these remnants can help in identifying the proper orientation of the explant. Ensure that the pigmented part is facing down and that the non-pigmented part is facing upward.</li>
	<li>Incubate the retinal explant culture in humidified incubators at 37 &#176;C and 5% CO2.</li>
	<li>Make sure that the retinal surface is facing upward. If the retinal piece is flipped or folded, try to adjust it gently to the correct orientation. 
	&#8203;NOTE: Pay great attention to the correct orientation of the retinal explant in the culture plate. Avoid any flipping or folding of the explant in the culture plate as this will result in failure of the proper growth of the retinal cells in culture.</li>
</ol>
4. Retinal explant culture
<ol>
	<li>Maintain the retinal explant cultures (prepared in steps 3.2-3.3) in humidified incubators at 37 &#176;C and 5% CO2.</li>
	<li>Change half of the media from each well every other day for 2 weeks. Do this by pipetting 150 &#181;L from the well carefully. Then, add 150 &#181;L of fresh media back into the well. Avoid flipping the explant when changing the media.</li>
	<li>Check the explant carefully under the microscope before putting the culture plate back into the incubator. Examine the integrity of the cells and the retinal architecture under a brightfield microscope. Make sure the explant is still oriented correctly. Use both the 4x and 20x objective lenses to examine the explant. 
	NOTE: Avoid the complete immersion of the explant in the media.</li>
</ol>
5. Immunohistochemistry
<ol>
	<li>After 2 weeks, fix the retinal explant by removing the media. Do this by first washing once with 200 &#181;L of 1x PBS and then adding 300 &#181;L of 4% paraformaldehyde in 0.1 M PBS, pH 7.4, to the well containing the explant and leaving it overnight. 
	NOTE: This step can be done without removing the culture inserts from the plate. The explants can also be transferred to a 500 &#181;L conical tube, and the washing and fixation can be done inside the tube.</li>
	<li>Transfer the explant to a 500 &#181;L conical tube containing 300 &#181;L of 1x PBS-2.5% Triton X for washing.</li>
	<li>Wash the fixed explants in the tube three times for 5 min each using medium velocity on a shaker (25 rpm).</li>
	<li>Block the expected nonspecific reactions by incubating the explant with 300 &#181;L of 1x blocking buffer containing 0.02% thimerosal for 1 h at room temperature prior to the antibody incubation.</li>
	<li>Incubate the fixed explants with the primary antibodies overnight at 4 &#176;C using medium velocity on a shaker. 
	NOTE: Detect the retinal endothelial cells in the fixed explant by staining with GSL I, BSL I antibody (lectin) (7:1,000). Detect the glial cells in the retinal explants by staining with rabbit glial fibrillary acidic protein (GFAP) antibody (1:250). Detect the retinal neurons by staining the explant with rabbit NeuN antibody (1:250)29,30. Incubate s{Tual-Chalot, 2013 #117}{Tual-Chalot, 2013 #116}{Tual-Chalot, 2013 #116}ome explants with the lectin, NeuN combination, and incubate others with the lectin, GFAP combination.</li>
	<li>The next day, remove the primary antibody from the tubes by aspiration with a pipette. Wash the explants with 1x PBS-2.5% Triton X three times for 5 min each using medium velocity on a shaker.</li>
	<li>Add the secondary antibodies: Goat anti-Rabbit IgG (1:500) (secondary for GFAP and NeuN) and Texas Red (7:1,000) (for the lectin). Incubate for 1 h at room temperature using medium velocity on a shaker. 
	NOTE: The secondary antibodies are light sensitive, so cover the tube with aluminum foil.</li>
	<li>Remove the secondary antibodies by aspiration, and then wash three times for 5 min each with 1x PBS-2.5% Triton X.</li>
	<li>Transfer the explant from the tube to a glass slide using a transfer pipette. Remove any excess liquid on the slide, and then add one drop of mounting media with DAPI to the slide.</li>
	<li>Cover the tissue with a coverslip. Do not allow any air bubbles between the coverslip and the tissue.</li>
	<li>Take the stained slides to the fluorescence microscope, and start taking the images. It is important to cover the stained slides using a slide box as the fluorescence staining is light-sensitive.</li>
</ol>

The authors have nothing to disclose.

Our lab has been studying the pathophysiological changes that promote retinal microvascular dysfunction for years31,32,33,34,35,36. Retinal explants are one of the techniques that can be of great value to use as a model for studying retinal diseases such as diabetic retinopathy or degenerative retinal diseases. Having a contr...

Different models have been presented to study retinal diseases, including both in vivo and in vitro models. The usage of animals in research is still a matter of continuous ethical and translational debate1. Animal models involving rodents such as mice or rats are commonly used in retinal research2,3,4. However, clinical concerns have arisen because of the different physiological functions of the retina in rodents compared to humans, such as the absence of the macula or differences in color vision5. The usage of human postmortem eyes for retinal research also has many problems, including but not limited to differences in the genetic backgrounds of the original samples, the donors&#39; medical history, and the donors&#39; previous environments or lifestyles6. Furthermore, the usage of in vitro models in retinal research has some drawbacks as well. Cell culture models used to study retinal diseases include the utilization of cell lines of human origin, primary cells, or stem cells7. The cell culture models used have been shown to have problems in terms of being contaminated, misidentified, or dedifferentiated8,9,10,11. Recently, retinal organoid technology has shown significant progress. However, the construction of highly complex retinas in vitro has several limitations. For example, retinal organoids do not have the same physiological and biochemical characteristics as mature in vivo retinas. To overcome this limitation, retinal organoid technology must integrate more biological and cellular features, including smooth muscle cells, vasculature, and immune cells like microglia12,13,14,15.
Organotypic retinal explants have emerged as a reliable tool for studying retinal diseases such as diabetic retinopathy and degenerative retinal diseases16,17,18,19. Compared to other existing techniques, the use of retinal explants supports both in vitro retinal cell cultures and also current in vivo animal models by adding a unique feature to study the cross-talk between various retinal cells under the same biochemical parameters and independent of systemic variables. The explant cultures allow different retinal cells to be kept together in the same environment, allowing the preservation of retinal intercellular interactions20,21,22. Moreover, a previous study showed that retinal explants were able to preserve the morphological structure and functionality of the cultured retinal cells23. Thus, retinal explants can provide a decent platform for investigating possible therapeutic targets for a wide variety of retinal diseases24,25,26. Retinal explant cultures provide a controllable technique and are very flexible substitute for existing mothods that allow numerous pharmacological manipulations and can uncover several molecular mechanisms27.
The overall goal of this paper is to present the retinal explant technique as a reasonable intermediate model system between in vitro cell cultures and in vivo animal models. This technique can mimic retinal functions in a better way than dissociated cells. As various retinal layers remain intact, the retinal intercellular interactions can be assessed in the lab under well-controlled biochemical conditions and independent of vascular system functioning28.

<table><tbody><tr><td>Adult C57Bl/6J mice&amp;nbsp;</td><td>The Jackson Laboratory, Bar Harbor, ME, 04609, USA</td><td>664</td><td></td></tr><tr><td>All-in-One Fluorescence Microscope&amp;nbsp;</td><td>KEYENCE CORPORATION OF AMERICA, IL, 60143, U.S.A.</td><td>BZ-X800</td><td></td></tr><tr><td>B27 supplements</td><td>Thermo scientific. Waltham, MA, 02451, USA</td><td>Gibco #17504-04</td><td></td></tr><tr><td>Blockade blocking solution&amp;nbsp;</td><td>Thermo scientific. Waltham, MA, 02451, USA</td><td>B10710</td><td></td></tr><tr><td>DMEM F12</td><td>Thermo scientific. Waltham, MA, 02451, USA</td><td>Gibco #11320033</td><td></td></tr><tr><td>Goat anti-Rabbit IgG.</td><td>Thermo scientific. Waltham, MA, 02451, USA</td><td>F-2765</td><td></td></tr><tr><td>GSL I, BSL I (Isolectin)</td><td>Vector Laboratories. Burlingame, CA 94010,USA</td><td>B-1105-2</td><td></td></tr><tr><td>Hanks Ballanced Salt Solution (HBSS)</td><td>Thermo scientific. Waltham, MA, 02451, USA</td><td>Gibco #14175095</td><td></td></tr><tr><td>Micro Scissors, 12 cm, Diamond Coated Blades</td><td>World Precision Instruments,FL 34240, USA&amp;nbsp;</td><td>Straight (503365)</td><td></td></tr><tr><td>N2 supplements</td><td>Thermo scientific. Waltham, MA, 02451, USA</td><td>Gibco #17502-048</td><td></td></tr><tr><td>Nunc Polycarbonate Cell Culture Inserts in Multi-Well Plates</td><td>Thermo scientific. Waltham, MA, 02451, USA</td><td>140652</td><td></td></tr><tr><td>Paraformaldehyde 4% in PBS</td><td>BBP, Ashland, MA, 01721 USA</td><td>C25N107</td><td></td></tr><tr><td>Penicillin-Streptomycin (10,000 U/mL)</td><td>Thermo scientific. Waltham, MA, 02451, USA</td><td>15140148</td><td></td></tr><tr><td>PROLONG DIAMOND ANTIFADE 4&amp;prime;,6-diamidino-2-phenylindole (DAPI).</td><td>Thermo scientific. Waltham, MA, 02451, USA</td><td>P36962</td><td></td></tr><tr><td>Rabbit Anti-NeuN Antibody</td><td>Abcam.,Cambridge, UK</td><td>ab177487</td><td></td></tr><tr><td>Rabbit Glial Fibrillary Acidic Protein (GFAP) Antibody</td><td>Dako,Carpinteria, CA 93013, USA.</td><td>Z0334</td><td></td></tr><tr><td>Texas Red</td><td>Vector Laboratories. Burlingame, CA 94010,USA</td><td>SA-5006-1</td><td></td></tr><tr><td>TritonX</td><td>BioRad Hercules, CA,&amp;nbsp; 94547,USA</td><td>1610407</td><td></td></tr></tbody></table>

retinal explant of the adult mouse retina as an ex vivo model for studying retinal neurovascular diseases

One of the challenges in retina research is studying the cross-talk between different retinal cells such as retinal neurons, glial cells, and vascular cells. Isolating, culturing, and sustaining retinal neurons in vitro have technical and biological limitations. Culturing retinal explants may overcome these limitations and offer a unique ex vivo model to study the cross-talk between various retinal cells with well-controlled biochemical parameters and independent of the vascular system. Moreover, retinal explants are an effective screening tool for studying novel pharmacological interventions in various retinal vascular and neurodegenerative diseases such as diabetic retinopathy. Here, we describe a detailed protocol for retinal explants' isolation and culture for an extended period. The manuscript also presents some of the technical problems during this procedure that may affect the desired outcomes and reproducibility of the retinal explant culture. The immunostaining of the retinal vessels, glial cells, and neurons demonstrated intact retinal capillaries and neuroglial cells after 2 weeks from the beginning of the retinal explant culture. This establishes retinal explants as a reliable tool for studying changes in the retinal vasculature and neuroglial cells under conditions that mimic retinal diseases such as diabetic retinopathy.

Survival of the neuronal and vascular retinal cells of the retinal explant in culture media ex vivo for an extended time 
By culturing a retinal explant utilizing our protocol, we were successful in maintaining different retinal cells that were viable for up to 2 weeks. To verify the presence of different retinal cells, immunofluorescence staining of the retinal explant using a neuronal cell marker (NeuN), glial cell marker (GFAP), and vascular marker (isol...

Watch this Scientific Journal Video about Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases at JoVE.com

Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases

This protocol presents and describes steps for the isolation, dissection, culturing, and staining of retinal explants obtained from an adult mouse. This method is beneficial as an ex vivo model for studying different retinal neurovascular diseases such as diabetic retinopathy.

Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases

One of the challenges in retina research is studying the cross-talk between different retinal cells such as retinal neurons, glial cells, and vascular ...

retinal-explant-adult-mouse-retina-as-an-ex-vivo-model-for-studying

Research

JoVE Journal

Medicine

4.0K Views.  Oakland University William Beaumont School of Medicine. This protocol presents and describes steps for the isolation, dissection, culturing, and staining of retinal explants obtained from an adult mouse. This method is beneficial as an ex vivo model for studying different retinal neurovascular diseases such as diabetic retinopathy.

Video: Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases

Organotypic Culture of Full-thickness Adult Porcine Retina

There is a recognized demand for in vitro models that can replace or reduce animal experiments. Porcine retina has a similar neuronal structure to human retina and is therefore a valuable species for studying mechanisms of human retinal injury and degenerative disease. Here we describe a cost-effective technique for organotypic culture of adult porcine retina isolated from eyes obtained from an abattoir. After removing the anterior segment, a trephine blade was used to create multiple neural retina-Bruch's membrane-RPE-choroid-sclera explants from the posterior segment of adult porcine eyes. A piece of sterile filter paper was used to lift the neural retina off from each explant. The filter paper-retina complex was cultured (photoreceptor side up) atop an insert, which was held away from the bottom of the culture dish by a custom-made stand. The stand allows for good circulation of the culture medium to both sides of the retina. Overall, this procedure is simple, reproducible, and permits preservation of native retinal structure for at least seven days, making it a useful model for a variety of morphological, pharmacological, and biochemical studies on mammalian retina.

Here we describe a cost-effective technique for organotypic culture of adult porcine retina for seven days. Briefly, a sterile filter paper was used to lift the neural retina off from the RPE and place photoreceptor side up on an insert raised by a custom-made stand.

There is a recognized demand for in vitro models that can replace or reduce animal experiments. Porcine retina has a similar neuronal structure to human ...

Neuroscience

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The M&#252;ller glia re-enter the cell cycle and produce neuronal progenitor cells that undergo subsequent rounds of cell divisions and differentiate into the lost neuronal cell types. Both M&#252;ller glia and neuronal progenitor cell nuclei replicate their DNA and undergo mitosis in distinct locations of the retina, i.e. they migrate between the basal Inner Nuclear Layer (INL) and the Outer Nuclear Layer (ONL), respectively, in a process described as Interkinetic Nuclear Migration (INM). INM has predominantly been studied in the developing retina. To examine the dynamics of INM in the adult regenerating zebrafish retina in detail, live-cell imaging of fluorescently-labeled M&#252;ller glia/neuronal progenitor cells is required. Here, we provide the conditions to isolate and culture dorsal retinas from Tg[gfap:nGFP]mi2004 zebrafish that were exposed to constant intense light for 35 h. We also show that these retinal cultures are viable to perform live-cell imaging experiments, continuously acquiring z-stack images throughout the thickness of the retinal explant for up to 8 h using multiphoton microscopy to monitor the migratory behavior of gfap:nGFP-positive cells. In addition, we describe the details to perform post-imaging analysis to determine the velocity of apical and basal INM. To summarize, we established conditions to study the dynamics of INM in an adult model of neuronal regeneration. This will advance our understanding of this crucial cellular process and allow us to determine the mechanisms that control INM.

Zebrafish retinal regeneration has mostly been studied using fixed retinas. However, dynamic processes such as interkinetic nuclear migration occur during the regenerative response and require live-cell imaging to investigate the underlying mechanisms. Here, we describe culture and imaging conditions to monitor Interkinetic Nuclear Migration (INM) in real-time using multiphoton microscopy.

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The ...

Developmental Biology

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Angiogenesis is the complex process of new blood vessel formation defined by the sprouting of new blood vessels from a pre-existing vessel network. Angiogenesis plays a key role not only in normal development of organs and tissues, but also in many diseases in which blood vessel formation is dysregulated, such as cancer, blindness and ischemic diseases. In adult life, blood vessels are generally quiescent so angiogenesis is an important target for novel drug development to try and regulate new vessel formation specifically in disease. In order to better understand angiogenesis and to develop appropriate strategies to regulate it, models are required that accurately reflect the different biological steps that are involved. The mouse neonatal retina provides an excellent model of angiogenesis because arteries, veins and capillaries develop to form a vascular plexus during the first week after birth. This model also has the advantage of having a two-dimensional (2D) structure making analysis straightforward compared with the complex 3D anatomy of other vascular networks. By analyzing the retinal vascular plexus at different times after birth, it is possible to observe the various stages of angiogenesis under the microscope. This article demonstrates a straightforward procedure for analyzing the vasculature of a mouse retina using fluorescent staining with isolectin and vascular specific antibodies.

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

The neonatal murine retina provides a well characterized physiological model of angiogenesis, which permits investigations of the roles of different genes or drugs that modulate angiogenesis in an in vivo context. Immunofluorescent staining to accurately visualize the vascular plexus is pivotal to the success of these types of studies.

Angiogenesis  is the complex process of new blood vessel formation defined by the sprouting  of new blood vessels from a pre-existing vessel network. ...

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). It is increasingly being recognized that several neurodegenerative diseases such as Alzheimer&#8217;s, multiple sclerosis, and amyotrophic lateral sclerosis present elements of vascular compromise. In addition, the most prominent causes of blindness in pediatric and working age populations (retinopathy of prematurity and diabetic retinopathy, respectively) are characterized by vascular degeneration and failure of physiological vascular regrowth. The aim of this technical paper is to provide a detailed protocol to study CNS vascular regeneration in the retina. The method can be employed to elucidate molecular mechanisms that lead to failure of vascular growth after ischemic injury. In addition, potential therapeutic modalities to accelerate and restore healthy vascular plexuses can be explored. Findings obtained using the described approach may provide therapeutic avenues for ischemic retinopathies such as that of diabetes or prematurity and possibly benefit other vascular disorders of the CNS.

The rodent retina has long been recognized as an accessible window to the brain. In this technical paper we provide a protocol that employs the mouse model of oxygen-induced retinopathy to study the mechanisms that lead to failure of vascular regeneration within the central nervous system after ischemic injury. The described system can also be harnessed to explore strategies to promote regrowth of functional blood vessels within the retina and CNS.

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). ...

Long-Term, Serum-Free Cultivation of Organotypic Mouse Retina Explants with Intact Retinal Pigment Epithelium

In ophthalmic research, there is a strong need for in vitro models of the neuroretina. Here, we present a detailed protocol for organotypic culturing of the mouse neuroretina&#160;with intact retinal pigment epithelium (RPE). Depending on the research question, retinas can be isolated from wild-type animals or from disease models, to study, for instance, diabetic retinopathy or hereditary retinal degeneration. Eyes from early postnatal day 2&#8211;9 animals are enucleated under aseptic conditions. They are partially digested in proteinase K to allow for a detachment of the choroid from the RPE. Under the stereoscope, a small incision is made in the cornea creating two edges from where the choroid and sclera can be gently peeled off from the RPE and neuroretina. The lens is then removed, and the eyecup is cut in four points to give it a four-wedged shape resembling a clover leaf. The tissue is finally transferred in a hanging drop into a cell culture insert holding a polycarbonate culturing membrane. The cultures are then maintained in R16 medium, without serum or antibiotics, under entirely defined conditions, with a medium change every second day.
The procedure described enables the isolation of the retina and the preservation of its normal physiological and histotypic context for culturing periods of at least 2 weeks. These features make organotypic retinal explant cultures an excellent model with high predictive value, for studies into retinal development, disease mechanisms, and electrophysiology, while also enabling pharmacological screening.

The protocol describes organotypic explants of mouse neuroretina, cultivated together with its retinal pigment epithelium (RPE), in R16 defined medium, free of serum and antibiotics. This method is relatively simple to perform, less expensive, and time-consuming when compared to in vivo experiments, and can be adapted to numerous experimental applications.

In ophthalmic research, there is a strong need for in vitro models of the neuroretina. Here, we present a detailed protocol for organotypic culturing of ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility

Advancements in ophthalmic imaging tools offer an unprecedented level of access to researchers working with animal models of neurovascular injury. To properly leverage this greater translatability, there is a need to devise reproducible methods of drawing quantitative data from these images. Optical coherence tomography (OCT) imaging can resolve retinal histology at micrometer resolution and reveal functional differences in vascular blood flow. Here, we delineate noninvasive vascular readouts that we use to characterize pathological damage post vascular insult in an optimized mouse model of retinal vein occlusion (RVO). These readouts include live imaging analysis of retinal morphology, disorganization of retinal inner layers (DRIL) measure of capillary ischemia, and fluorescein angiography measures of retinal edema and vascular density. These techniques correspond directly to those used to examine patients with retinal disease in the clinic. Standardizing these methods enables direct and reproducible comparison of animal models with clinical phenotypes of ophthalmic disease, increasing the translational power of vascular injury models.

In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility

Here, we present three data analysis protocols for fluorescein angiography (FA) and optical coherence tomography (OCT) images in the study of Retinal Vein Occlusion (RVO).

Advancements in ophthalmic imaging tools offer an unprecedented level of access to researchers working with animal models of neurovascular injury. To ...

Characterization of a Novel Human Organotypic Retinal Culture Technique

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal pigment epithelium-choroid (RPE-choroid) and sclera, the integrity of the fragile retina can easily be compromised. The aim of this study was to develop a novel HORC model that contains the retina, RPE-choroid and sclera to maintain retinal integrity when culturing retinal explants.
After cutting circumferentially along the limbus to remove iris and lens, four deep incisions were made to flatten the eyecup. In contrast to previous HORC protocols, a trephine was used to cut through not only the retina but also the RPE-choroid and sclera. The resultant triple-layered explants were cultured for 72 h. Hematoxylin and Eosin staining (H&#38;E) was used to assess anatomical structures and retinal explants were further characterized by immunohistochemistry (IHC) for apoptosis, M&#252;ller cell integrity and retinal inflammation. To confirm the possibility of disease induction, explants were exposed to high glucose (HG) and pro-inflammatory cytokines (Cyt), to mimic diabetic retinopathy (DR). The Luminex magnetic bead assay was used to measure DR-related cytokines released into the culture medium.
H&#38;E staining revealed distinct retinal lamellae and compact nuclei in retinal explants with the underlying RPE-choroid and sclera, while retinas without the underlying structures exhibited reduced thickness and severe nuclei loss. IHC results indicated absence of apoptosis and retinal inflammation as well as preserved M&#252;ller cell integrity. The Luminex&#160;assays showed significantly increased secretion of DR-associated pro-inflammatory cytokines in retinal explants exposed to HG + Cyt relative to baseline levels at 24 h.
We successfully developed and characterized a novel HORC protocol in which retinal integrity was preserved without apoptosis or retinal inflammation. Moreover, the induced secretion of DR-associated pro-inflammatory biomarkers when exposing retinal explants to HG + Cyt suggests that this model could be used for clinically translatable retinal disease studies.

This study aims to develop a novel human organotypic retinal culture (HORC) model that prevents compromising retinal integrity during explant handling. This is achieved by culturing the retina with the overlying vitreous and the underlying retinal pigment&#160;epithelium-choroid (RPE-choroid) and sclera.

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal ...

Retinal Pathophysiological Evaluation in a Rat Model

A posterior segment eye disease like diabetic retinopathy alters the physiology of the retina. Diabetic retinopathy is characterized by a retinal detachment, breakdown of the blood-retinal barrier (BRB), and retinal angiogenesis. An in vivo rat model is a valuable experimental tool to examine the changes in the structure and function of the retina. We propose three different experimental techniques in the rat model to identify morphological changes of retinal cells, retinal vasculature, and compromised BRB. Retinal histology is used to study the morphology of various retinal cells. Also, quantitative measurement is performed by retinal cell count and thickness measurement of different retinal layers. A BRB breakdown assay is used to determine the leakage of extraocular proteins from the plasma to vitreous tissue due to the breakdown of BRB. Fluorescence angiography is used to study angiogenesis and leakage of blood vessels by visualizing retinal vasculature using FITC-dextran dye.

Diabetic retinopathy is one of the leading causes of blindness. Histology, blood-retinal barrier breakdown assay, and fluorescence angiography are valuable techniques to understand the pathophysiology of the retina, which could further enhance the efficient drug screening against diabetic retinopathy.

A posterior segment eye disease like diabetic retinopathy alters the physiology of the retina. Diabetic retinopathy is characterized by a retinal ...

Transpupillary-Guided Trans-Scleral Transplantation of Subretinal Grafts in a Retinal Degeneration Mouse Model

Transplantation of photoreceptor cells and retinal pigment&#160;epithelial (RPE) cells provide a potential therapy for retinal degeneration diseases. Subretinal transplantation of therapeutic donor cells into mouse recipients is challenging due to the limited surgical space allowed by the small volume of the mouse eye. We developed a trans-scleral surgical transplantation platform with direct transpupillary vision guidance to facilitate the subretinal delivery of exogenous cells in mouse recipients. The platform was tested using retinal cell suspensions and three-dimensional retinal sheets collected from rod-rich Rho::EGFP mice and cone-rich OPN1LW-EGFP;NRL-/- mice, respectively. Live/dead assay showed low cell mortality for both forms of donor cells. Retinal grafts were successfully delivered into the subretinal space of a mouse model of retinal degeneration, Rd1/NS, with minimum surgical complications as detected by multimodal confocal scanning laser ophthalmoscope (cSLO) imaging. Two months post-transplantation, histological staining demonstrated evidence of advanced maturation of the retinal grafts into &#39;adult&#39; rods and cones (by robust Rho::EGFP, S-opsin, and OPN1LW:EGFP expression, respectively) in the subretinal space. Here, we provide a surgical platform that can enable highly accurate subretinal delivery with a low rate of complications in mouse recipients. This technique offers precision and relative ease of skill acquisition. Furthermore, the technique could be used not only for studies of subretinal cell transplantation but also for other intraocular therapeutic studies including gene therapies.

This protocol presents a transpupillary vision-guided trans-scleral approach to safely and precisely deliver subretinal cellular grafts, with a low rate of surgical complications, in mouse recipients with or without retinal degeneration.

Transplantation of photoreceptor cells and retinal pigment&#160;epithelial (RPE) cells provide a potential therapy for retinal degeneration diseases. ...

Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases

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