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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Adult C57Bl/6J mice&#160;</td>
			<td>The Jackson Laboratory, Bar Harbor, ME, 04609, USA</td>
			<td>664</td>
			<td></td>
		</tr>
		<tr>
			<td>All-in-One Fluorescence Microscope&#160;</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&#160;</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&#160;</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&#8242;,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,&#160; 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

3.7K 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

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the occurrence of subretinal hemorrhage can affect photoreceptor cell death in the detached retina. Hence, it is advantageous to create reproducible RDs without subretinal hemorrhage for evaluating photoreceptor cell death. We modified a previously reported method to create bullous and persistent RDs in a reproducible location with rare occurrence of subretinal hemorrhage. The critical step of this modified method is the creation of a self-sealing scleral incision, which can prevent leakage of sodium hyaluronate after injection into the subretinal space. To make the self-sealing scleral incision, a scleral tunnel is created, followed by scleral penetration into the choroid with a 30 G needle. Although choroidal hemorrhage may occur during this step, astriction with a surgical spear reduces the rate of choroidal hemorrhage. This method allows a more reproducible and reliable model of photoreceptor death in diseases that involve RD such as rhegmatogenous RD, retinopathy of prematurity, diabetic retinopathy, central serous chorioretinopathy, and age-related macular degeneration (AMD).

Creating experimental retinal detachments with a reproducible and sustained height of detachment, and without subretinal hemorrhage, is important for studying the pathophysiology of photoreceptor cell loss in retinal disease and evaluating potential therapeutic interventions. Here, we report such a method in detail.

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the ...

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

Quantitative Fundus Autofluorescence for the Evaluation of Retinal Diseases

The retinal pigment epithelium (RPE) is juxtaposed to the overlying sensory retina, and supports the function of the visual system. Among the tasks performed by the RPE are phagocytosis and processing of outer photoreceptor segments through lysosome-derived organelles. These degradation products, stored and referred to as lipofuscin granules, are composed partially of bisretinoids, which have broad fluorescence absorption and emission spectra that can be detected clinically as fundus autofluorescence with confocal scanning laser ophthalmoscopy (cSLO). Lipofuscin accumulation is associated with increasing age, but is also found in various patterns in both acquired and inherited degenerative diseases of the retina. Thus, studying its pattern of accumulation and correlating such patterns with changes in the overlying sensory retina are essential to understanding the pathophysiology and progression of retinal disease. Here, we describe a technique employed by our lab and others that uses cSLO in order to quantify the level of RPE lipofuscin in both healthy and diseased eyes.

The retinal pigment epithelium (RPE) supports the sensory retina through recycling visual cycle byproducts, which accumulate as lipofuscin. These products are autofluorescent and can be qualitatively imaged in vivo. Here, we describe a method to quantitatively image RPE lipofuscin using confocal scanning laser ophthalmoscopy.

The retinal pigment epithelium (RPE) is juxtaposed to the overlying sensory retina, and supports the function of the visual system. Among the tasks ...

A Step by Step Protocol for Subretinal Surgery in Rabbits

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in adults in industrially developed countries. RPE transplantation appears to be a promising therapy, as it may replace dysfunctional RPE, restore its function, and thereby vision.
Here we describe a method for transplanting a cultured RPE monolayer on a scaffold into the subretinal space (SRS) of rabbits. After vitrectomy xenotransplants were delivered into the SRS using a custom made shooter consisting of a 20-gauge metallic nozzle with a polytetrafluoroethylene (PTFE) coated plunger. The current technique evolved in over 150 rabbit surgeries over 6 years. Post-operative follow-up can be obtained using non-invasive and repetitive in vivo imaging such as spectral domain optical coherence tomography (SD-OCT) followed by perfusion-fixed histology.
The method has well-defined steps for easy learning and high success rate. Rabbits are considered a large eye animal model useful in preclinical studies for clinical translation. In this context rabbits are a cost-efficient and perhaps convenient alternative to other large eye animal models.

Retinal pigment epithelium (RPE) replacement strategies and gene-based therapy are considered for several retinal degenerative conditions. For clinical translation, large eye animal models are required to study surgical techniques applicable in patients. Here we present a rabbit model for subretinal surgery geared towards RPE transplantation, which is versatile and cost-efficient.

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in ...

An Acute Retinal Model for Evaluating Blood Retinal Barrier Breach and Potential Drugs for Treatment

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An inflammatory factor, histamine, is demonstrated to compromise vessel integrity in the cultured retina through positive staining of IgG outside of the blood vessels. The effects of histamine itself and those of candidate drugs for potential treatments, such as lipoxin A4, are assessed using three parameters: blood vessel leakage via IgG immunostaining, activation of M&#252;ller cells via GFAP staining and change in neuronal dendrites through staining for MAP2. Furthermore, the layered organization of the retina allows a detailed analysis of the processes of M&#252;ller and ganglion cells, such as changes in width and continuity. While the data presented is with swine retinal culture, the system is applicable to multiple species. Thus, the model provides a reliable tool to investigate the early effects of compromised retinal vessel integrity on different cell types and also to evaluate potential drug candidates for treatment.

A low-cost, easy-to-use and powerful system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach induced by histamine. Blood vessel leakage, M&#252;ller cell activation and the continuity of neuronal processes are utilized to assess the damage response and its reversal with a potential drug, lipoxin A4.

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An ...

Fluorescent Dye Labeling of Erythrocytes and Leukocytes for Studying the Flow Dynamics in Mouse Retinal Circulation

The retinal and choroidal blood flow dynamics may provide insight into the pathophysiology and sequelae of various ocular diseases, such as glaucoma, diabetic retinopathy, age-related macular degeneration (AMD) and other ocular inflammatory conditions. It may also help to monitor the therapeutic responses in the eye. The proper labeling of the blood cells, coupled with live-cell imaging of the labeled cells, allows for the investigation of the flow dynamics in the retinal and choroidal circulation. Here, we describe the standardized protocols of 1.5% indocyanine green (ICG) and 1% sodium fluorescein labeling of mice erythrocytes and leukocytes, respectively. Scanning laser ophthalmoscopy (SLO) was applied to visualize the labeled cells in the retinal circulation of C57BL/6J mice (wild type). Both methods demonstrated distinct fluorescently labeled cells in the mouse retinal circulation. These labeling methods can have wider applications in various ocular disease models.

Live-cell imaging of the labeled blood cells in ocular circulation can provide information about inflammation and ischemia in diabetic retinopathy and age-related macular degeneration. A protocol to label blood cells and image the labeled cells in the retinal circulation is described.

The retinal and choroidal blood flow dynamics may provide insight into the pathophysiology and sequelae of various ocular diseases, such as glaucoma, ...

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular diseases, such as diabetic retinopathy, hypertensive retinopathy, and possibly glaucoma. Therefore, retinal microvascular preparations are pivotal tools for physiological and pharmacological studies to delineate the underlying pathophysiological mechanisms and to design therapies for the diseases. Despite the wide use of mouse models in ophthalmic research, studies on retinal vascular reactivity are scarce in this species. A major reason for this discrepancy is the challenging isolation procedures owing to the small size of these retinal blood vessels, which is ~ &#8804; 30 &#181;m in luminal diameter. To circumvent the problem of direct isolation of these retinal microvessels for functional studies, we established an isolation and preparation technique that enables ex vivo studies of mouse retinal vasoactivity under near-physiological conditions. Although the present experimental preparations will specifically refer to the mouse retinal arterioles, this methodology can readily be employed to microvessels from rats.

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Many vision-threatening ocular diseases are associated with dysfunctional retinal microvessels. Therefore, the measurement of retinal arteriole responses is important to investigate the underlying pathophysiological mechanisms. This article describes a detailed protocol for mouse retinal arteriole isolation and preparation to assess the effects of vasoactive substances on vascular diameter.

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular ...

Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tg(fli:EGFP) Zebrafish Retinal Vasculature

Diabetic retinopathy is the leading cause of blindness among middle-aged adults. The rising prevalence of diabetes worldwide will make the prevention of diabetic microvascular complications one of the key research fields of the next decades. Specialized, targeted therapy and novel therapeutic drugs are needed to manage the increasing number of patients at risk of vision-loss. The zebrafish is an established animal model for developmental research questions with increasing relevance for modeling metabolic multifactorial disease processes. The advantages of the species allow for optimal visualization and high throughput drug screening approaches, combined with the strong ability to knock out genes of interest. Here, we describe a protocol which will allow easy analysis of the adult tg(fli:EGFP) zebrafish retinal vasculature as a fast read-out in settings of long-term vascular pathologies linked to neoangiogenesis or vessel damage. This is achieved via dissection of the zebrafish retina and whole-mounting of the tissue. Visualization of the exposed vessels is then achieved via confocal microscopy of the green EGFP reporter expressed in the adult retinal vasculature. Correct handling of the tissue will lead to better outcomes and less internal vessel breakage to assure the visualization of the unaltered vascular structure. The method can be utilized in zebrafish models of retinal vasculopathy linked to changes in the vessel architecture as well as neoangiogenesis.

Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tgfli:EGFP Zebrafish Retinal Vasculature

Here, we discuss a method protocol which will allow an easy analysis of the adult tg(fli:EGFP) zebrafish retinal vasculature as a fast read-out in settings of long-term vascular pathologies linked to neoangiogenesis and structural changes.

Diabetic retinopathy is the leading cause of blindness among middle-aged adults. The rising prevalence of diabetes worldwide will make the prevention of ...

Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic fatty liver disease, metabolic liver disease, and liver cancer) requires experimental manipulation of animal models and in vitro cell cultures. Both techniques have limitations, such as the requirement of large numbers of animals for in vivo manipulation. However, in vitro cell cultures do not reproduce the structure and function of the multicellular hepatic environment. The use of precision-cut liver slices is a technique in which uniform slices of viable mouse liver are maintained in laboratory tissue culture for experimental manipulation. This technique occupies an experimental niche that exists between animal studies and in vitro cell culture methods. The presented protocol describes a straightforward and reliable method to isolate and culture precision-cut liver slices from mice. As an application of this technique, ex vivo liver slices are treated with bile acids to simulate cholestatic liver injury and ultimately assess the mechanisms of hepatic fibrogenesis.

This protocol provides a simple and reliable method for the production of viable precision-cut liver slices from mice. The ex vivo tissue samples can be maintained under laboratory tissue culture conditions for multiple days, providing a flexible model to examine liver pathobiology.

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic ...

Oxygen-Induced Retinopathy Model for Ischemic Retinal Diseases in Rodents

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR model induction and its readouts in both mice and rats. Retinal neovascularization is induced in OIR by exposing rodent pups either to hyperoxia (mice) or alternating levels of hyperoxia and hypoxia (rats). The primary readouts of these models are the size of neovascular (NV) and avascular (AVA) areas in the retina. This preclinical in vivo model can be used to evaluate the efficacy of potential anti-angiogenic drugs or to address the role of specific genes in the retinal angiogenesis by using genetically manipulated animals. The model has some strain and vendor specific variation in the OIR induction which should be taken into consideration when designing the experiments.

Oxygen-induced retinopathy (OIR) can be used to model ischemic retinal diseases such as retinopathy of prematurity and proliferative diabetic retinopathy and to serve as a model for proof-of-concept studies in evaluating antiangiogenic drugs for neovascular diseases. OIR induces robust and reproducible neovascularization in the retina that can be quantified.

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR ...