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 housin...

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 ...

One of the challenges in retinal research is to study the crosstalk between different cells, such as retinal neurons, glial cells, and vascular cells. In particular, isolating and the culturing of retinal neurons is challenging. We believe that cultured retinal explant can overcome this limitation and offer a unique ex vivo system that allow us to study the crosstalk between different retinal cells, under well-controlled biochemical parameters, and independent of vascular system.Also, a retina explant is a prominent screening tool to study novel pharmacological interventions in various retinovascular and neurodegenerative diseases, such as diabetic retinopathy, which is characterized by vascular and neuronal injury. Our lab has been studying pathophysiological changes, promoting microvascular retinal dysfunction for years. Utilizing a wide variety of models including both in vivo and in vitro models enabled us to optimize a lot of techniques inside our lab.Here, we describe a detailed protocol for retinal explants derived from a wild-type adult mouse that can be kept in culture for two weeks. Keep the animals housed under constant temperature and light-controlled environment. Inspect every animal for health, adequate food, and water.Make sure to provide the animals with fresh food and clean water. Change soiled cages on a daily basis or as needed for the health of the animals. Use male black 6 mice of more than 12 weeks of age.Euthanize the animals in their home cage by carbon dioxide overdose inhalation. 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. Follow death confirmation by a secondary method of euthanasia such as cervical dislocation.Apply pressure on the orbit using curved forceps until the globe protrudes. Gently close the forceps at the posterior portion of the eye and elevate in a continuous motion. Immerse the eyeball in a 1.5-milliliter tube containing a one-milliliter ice-cold HBSS.Transfer the eyeball to a 1.5-or two-milliliter tube containing complete media. Now it is ready for dissection. This diagram illustrates the consecutive steps that will be shown for dissecting the mouse eyeball to create the retinal explant.Circumferential incision is made along the limbus to open the eyeball. The cornea is removed via dissecting along the limbal incision. The lens and the vitreous are then removed, leaving an empty eye cup.Via holding the region of the optic nerve head with the fine forceps, peeling off the outer coat of the eye is performed gently. Great attention during the removal of the outer coat in order not to injure the neural retina and have the retina totally intact. Then, the retina will be cut into smaller pieces.This will be a great advantage, because it will allow you to have an experiment where your control group and experimental groups are derived from the same animal. However, cutting the retina into small pieces will make handling of it to be more challenging. Therefore, we developed a technique for handling these small retinal pieces to ensure proper orientation of the retina culture.Go with the dissecting scissor blade open just beneath the retinal piece. Slowly elevate the retinal piece with the tip of an open scissor blade and gently transfer it to the culture plate. The explants will be separately transferred onto cell culture inserts in multi-well plates, each of which contain 300 microliters of retinal explant media with the neural retina side facing up.Retinal explant cultures will be maintained in humidified incubators at 37 degrees Celsius and 5%carbon dioxide. Make sure that the retinal surface is facing upward. If the retinal piece is flipped or folded, try to adjust it gently to the right orientation.Failure to place the retinal explant in the right orientation with the neural retina facing upward will result in failure of the proper growth of the retinal cells in culture. Here, we can see immunofluorescence staining the retinal explants stained in culture for two weeks. The explants were fixed and stained with NeuN for retinal neurons, GFAP for glial cells, and lectin for blood vessels.The staining showed well-developed retinal cells and retinal vessels. Here, we can see immunofluorescence staining of retinal explants fixed and stained with GFAP for glial cells and lectin for blood vessels. The images show defective staining and underdevelopment of both retinal cells and retinal vessels.This is because the retinal explant was folded and was not oriented correctly on the culture plate. Great attention should be taken for the proper orientation of the explant with the retina facing upward. Having the control group being derived from the same single animal as the treated group makes it of great advantage to have more reliable comparison of different experimental conditions.Retinal implants can provide a decent platform for investigating possible therapeutic targets for a wide variety of retinal diseases, such as diabetic retinopathy and degenerative retinal diseases.

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

Abstract