Thank David Yee for his technical assistance. The work was supported in part by the Massachusetts Lions Eye Research Foundation and the Boston University Chobanian &#38; Avedisian School of Medicine Wing Tat Lee Award.

All procedures were performed according to an animal use protocol approved by the Boston University Institutional Animal Care and Use Committee according to the NIH Guide for the Care and Use of Laboratory Animals and complies with the Association for Research in Vision and Ophthalmology (ARVO) statement for the use of animals in ophthalmic and vision research.
1. Experimental animals
<ol>
	<li>House C57BL/6J mice under standard conditions.</li>
</ol>
2. Preparing the required solution and infusion line
<ol>
	<li>In a 50 mL&#160;syringe, fill with sterilized 0.9% NaCl saline solution at one per mouse. Place the syringe at a height of 120 cm above the level of the bench surface.</li>
	<li>Connect the filled syringe to an infusion line and a stopcock with a 30 G needle attached to the end of the line. Flush out all air bubbles from the line.</li>
</ol>
3. Preparing the workspace
<ol>
	<li>Set up a surgical/dissecting microscope under the saline bottle.</li>
	<li>Create a bed to stabilize the mouse during the procedure by cutting out a depression of approximately 6 cm x 3 cm in a sponge and placing it securely in a styrofoam well or other flat container.</li>
</ol>
4. Anesthetizing the mouse
<ol>
	<li>Anesthetize the mouse with a standard mixture of ketamine and xylazine at a level to achieve deep anesthetization. 
	NOTE: In this protocol, a mixture of ketamine 100 mg/kg and xylazine 10 mg/kg was injected intraperitoneally to achieve adequate anesthesia depth.</li>
	<li>Achieve adequate anesthesia when there is no paw withdrawal in response to a toe pinch and no blink reflex when the cornea is gently touched. Administer additional anesthesia if the mouse shows any signs of pain, such as hyperventilating or limb movement, while anesthetized.</li>
</ol>
5. Dilating the iris
<ol>
	<li>Dilate the iris of the eye to be perfused with 1 drop of 1% tropicamide. Allow 5 min for dilation. Apply an ophthalmic ointment such as bacitracin to the eye that will not be perfused.</li>
</ol>
6. Anterior chamber cannulation
<ol>
	<li>Securely place the anesthetized mouse in the sponge bed under the microscope. Apply saline solution to the eyes to rinse any debris or fur from the eye surface.</li>
	<li>Use a pair of non-toothed forceps and gently propose one of the eyes. 
	NOTE: An experiment will use either the left or right eyes of the mouse, but never both on the same mouse.</li>
	<li>With the infusion line closed, cannulate the anterior chamber with the needle approximately 2 mm from the limbus. Ensure the needle pierces the cornea perpendicularly to the peripheral, curved cornea surface, then slightly flattened parallel to the plane of the iris. The cornea may be manipulated to advance the needle and maintain eye control. Avoid striking the iris or lens and enlarging the corneal wound, which can result in leakage. Details are further described&#160;by Buchi et al.2.</li>
</ol>
7. Ischemic phase
<ol>
	<li>Turn the stopcock to run the infusion line. Verify that there is no leakage through the corneal wound. Ensure that there is a gradual distension of the cornea as the intraocular pressure increases. 
	NOTE: If significant leakage does not seal as the cornea distends, adequate pressure will not be maintained, and the procedure will have to be on a different mouse.</li>
	<li>Check that the intraocular pressure has elevated to 90-100 mmHg with a tonometer. Secure the infusion line with tape, carefully ensuring the needle does not change position and start leaking.</li>
	<li>Apply an ophthalmic ointment such as bacitracin to the perfused eye to prevent dryness. Place the mouse away from the microscope and underneath a heating lamp to maintain normal body temperature and monitor for the next 60 min.</li>
	<li>Remeasure the intraocular pressure at 30 min before the procedure ends to ensure the intraocular pressure is between 90-100 mmHg.</li>
</ol>
8. Reperfusion phase
<ol>
	<li>After 60 min, return the mouse to the microscope. Bring the saline solution bottle down to the level of the mouse, normalizing the intraocular pressure.</li>
	<li>Measure the intraocular pressure with the tonometry to check that it is near normal, 20 mmHg. Once the intraocular pressure is normalized, carefully remove the needle, avoiding damage to the lens or iris.</li>
</ol>
9. Post-operative care
<ol>
	<li>Coat mouse eyes with any ophthalmic antibacterial ointment such as a bacitracin veterinary ophthalmic ointment.</li>
	<li>Monitor the mouse for 1-2 h on a heated surface until they fully recover from anesthesia. Do not leave the mouse unattended until it has regained sufficient consciousness to maintain sternal recumbency. Return the mice to cages and housing once fully recovered.</li>
</ol>
10. Enucleating the eye3
<ol>
	<li>Anesthetize the mouse first with a mixture of ketamine 100 mg/kg and xylazine 10 mg/kg injected intraperitoneally and then euthanize via cervical dislocation.</li>
	<li>Position the euthanized mouse on a flat, dry, and smooth surface. Rinse the eye with a few drops of saline solution.</li>
	<li>Apply gentle pressure to the lateral canthus with the forceps until the eyeball is displaced from the socket and the optic nerve is accessible. Using Westcott scissors, cut following the orbital margins to sever the extraocular muscles.</li>
	<li>Position the Westcott scissors to the most posterior aspect of the eye socket and sever the optic nerve.</li>
</ol>
11. Fixing the sample4
<ol>
	<li>Place the enucleated eye into 4% paraformaldehyde in 0.1 M PBS in a sealed vial. After 3 days, remove the eye, place it into a vial of 70% ethanol solution, and store it overnight.</li>
	<li>Remove the eyes and place them in a vial of 95% ethanol solution. Then, place the vial in a sealed vacuum for 15 min, remove it from the vacuum, and leave it in room air for 45 min.</li>
	<li>Repeat the vacuum process, sequentially placing the eye into the following solutions: 100% ethanol, 100% ethanol, 100% xylene solution, and 100% xylene solution.</li>
	<li>Remove the eye and place it into a clean, empty vial. Fill the vial with 60 &#176;C liquidized paraffin. Place the vial into a sealed 60 &#176;C vacuum oven for 30 min and then 30 min in the 60 &#176;C oven without vacuum.</li>
	<li>Remove the eye, place it into a clean, empty vial, and fill it with 60 &#176;C liquidized paraffin. Store the vial at 60 &#176;C overnight.</li>
</ol>
12. Embedding the sample
<ol>
	<li>Transfer the eye to a metal embedding mold and fill the mold with liquidized paraffin on a 60 &#176;C hotplate, orienting the eye in the desired direction to make sections through the optic disk. Allow the mold to cool at room temperature (RT), then store at 4 &#176;C.</li>
</ol>
13. Sectioning the sample5
<ol>
	<li>Transfer the embedded specimens to the microtome. Slowly trim the block until the eye is reached and the sections include the optic disc.</li>
	<li>Cut sections as ribbons of 5 &#181;m thickness and float them on a water bath. Lift the paraffin ribbon carefully using forceps, lay ribbons of three sections onto a slide, and let it dry overnight.</li>
	<li>Repeat this process until there are at least 8 slides for each eye with sections centered on the optic disc.</li>
</ol>
14. Staining the sections5
<ol>
	<li>Heat the slides in a 60 &#176;C oven for 1 h to melt the paraffin. Submerge the slides in RT xylene three times, for 5 min each.</li>
	<li>Submerge the slides in 100% ethanol three times, for 3 min each. Rinse the sections in tap water for 1 min.</li>
	<li>Incubate the sections in hematoxylin stain for 45 s. Rinse the sections in tap water for 2 min.</li>
	<li>Incubate the sections in the Bluing reagent for 1 min. Rinse the sections in tap water for 1 min.</li>
	<li>Incubate the sections in eosin for 15 s.</li>
	<li>Rinse the sections in 95% and 100% ethanol twice for 1 min each. Rinse the sections in 100% xylene four times, for 1 min each. Then, mount the slides with mounting media.</li>
</ol>
15. Histological analysis6
<ol>
	<li>Analyze each retinal section under the microscope and record the number of retinal folds, the percentage of cell loss in the various retinal layers (ganglion cell layer, inner nuclear layer, outer nuclear layer), the presence of vitreous or subretinal hemorrhage, and the extent of retinal pigment epithelial damage. Refer to Table 1 for details on the scoring criteria.</li>
</ol>

Mouse Model for Retinal Ischemia-Reperfusion Injury to Study Retinal Pathologies

<ol>
	<li>Lee, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+degeneration+induced+in+a+mouse+model+of+ischemia-reperfusion+injury+and+its+management+by+pemafibrate+treatment.">Retinal degeneration induced in a mouse model of ischemia-reperfusion injury and its management by pemafibrate treatment.</a> FASEB J. 36 (9), e22497 (2022).</li><li>Buchi, E. R., Suivaizdis, I., Fu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pressure-induced+retinal+ischemia+in+rats:+An+experimental+model+for+quantitative+study.">Pressure-induced retinal ischemia in rats: An experimental model for quantitative study.</a> Ophthalmologica. 203 (3), 138-147 (1991).</li><li>Aerts, J., Nys, J., Arckens, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+highly+reproducible+and+straightforward+method+to+perform+in+vivo+ocular+enucleation+in+the+mouse+after+eye+opening.">A highly reproducible and straightforward method to perform in vivo ocular enucleation in the mouse after eye opening.</a> J Vis Exp. (92), e51936 (2014).</li><li>Nayagam, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+for+processing+eyes+implanted+with+a+retinal+prosthesis+for+localized+histopathological+analysis.">Techniques for processing eyes implanted with a retinal prosthesis for localized histopathological analysis.</a> J Vis Exp. (78), e50411 (2013).</li><li>Pang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Step-by-step+preparation+of+mouse+eye+sections+for+routine+histology,+immunofluorescence,+and+RNA+in+situ+hybridization+multiplexing.">Step-by-step preparation of mouse eye sections for routine histology, immunofluorescence, and RNA in situ hybridization multiplexing.</a> STAR Protoc. 2 (4), 100879 (2021).</li><li>Shome, A., Mugisho, O. O., Niederer, R. L., Rupenthal, I. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+grading+system+for+experimental+autoimmune+uveitis+in+mice.">Comprehensive grading system for experimental autoimmune uveitis in mice.</a> Biomedicines. 11 (7), 2022 (2023).</li><li>Ng, T. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha-melanocyte-stimulating+hormone+maintains+retinal+homeostasis+after+ischemia/reperfusion.">Alpha-melanocyte-stimulating hormone maintains retinal homeostasis after ischemia/reperfusion.</a> Biomolecules. 14 (5), 525 (2024).</li><li>Lee, D., Kang, H., Yoon, K. Y., Chang, Y. Y., Song, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+model+of+retinal+hypoperfusion+injury+induced+by+unilateral+common+carotid+artery+occlusion.">A mouse model of retinal hypoperfusion injury induced by unilateral common carotid artery occlusion.</a> Exp Eye Res. 201, 108275 (2020).</li><li>Goit, R. K., Taylor, A. W., Lo, A. C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-inflammatory+alpha-melanocyte-stimulating+hormone+protects+retina+after+ischemia/reperfusion+injury+in+type+I+diabetes.">Anti-inflammatory alpha-melanocyte-stimulating hormone protects retina after ischemia/reperfusion injury in type I diabetes.</a> Front Neurosci. 16, 799739 (2022).</li><li>Chen, J., Caspi, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+and+functional+evaluation+of+ocular+inflammatory+disease+using+the+model+of+experimental+autoimmune+uveitis.">Clinical and functional evaluation of ocular inflammatory disease using the model of experimental autoimmune uveitis.</a> Methods Mol Biol. 1899, 211-227 (2019).</li></ol>

The authors have no disclosures.

The ischemia/reperfusion model provides a reproducible method for studying the mechanisms and pathology of retinal ischemia/reperfusion injury. This model has usefulness in studying the pathology of retinal ischemia/reperfusion injury, and for identifying therapeutic targets. Several critical steps in the protocol may pose challenges and require technical skills to complete successfully. One is the actual cannulation, which can take multiple attempts to master and has a risk of damage to other structures in the eye. Addi...

Ischemia/reperfusion injuries manifest in various retinal pathologies, encompassing diabetic retinopathy, glaucoma, retinal vascular occlusions, and related vaso-occlusive conditions. Given the retina's high oxygen demand, it is particularly susceptible to ischemia/reperfusion injury, a phenomenon implicated in the pathogenesis of diseases like diabetic retinopathy. This form of injury results in the demise of retinal ganglion cells (RGCs), morphological degeneration of the retina, compromised retinal function, and eventual vision impairment1. Modeling the ischemia/reperfusion is appropriate for studies on mechanisms and treatment responses in various retinal pathologies related to ischemia/reperfusion injuries.
We focused on refining a model for ischemia/reperfusion injury in the mouse eye. The anterior chamber cannulation model for pressure-induced retinal ischemia injury was first published by B&#252;chi et al. in 19912. They successfully increased the intraocular pressure to 110 mmHg for a controlled time. They found that the resulting retinal injury was consistent with findings similar to retinal and choroidal vascular occlusion. Due to its relatively simple methodology and cost-effective execution, it became a functional model for the study of retinal ischemic injury. We added the additional step of lowering the infusion source to the mouse's level before withdrawing the needle. This prevented forming a possible elevated pressure differential within the eye when the needle was removed, causing intraocular damage unrelated to the ischemia/reperfusion.
The aim was to create a controlled and replicable model for researching the mechanisms and pathology of retinal ischemia/reperfusion injury in a mouse model while minimizing procedural damage to the eye. This model offers a way to identify potential treatments and enhance our comprehension of retinal pathologies associated with vascular occlusion.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5% Proparacaine</td>
			<td>Sandoz</td>
			<td>61314-016-01</td>
			<td></td>
		</tr>
		<tr>
			<td>1% tropicamide</td>
			<td>Sumerset Therapeutics</td>
			<td>700069-016-01</td>
			<td></td>
		</tr>
		<tr>
			<td>30 G needle&#160;</td>
			<td>Becton Dickinson</td>
			<td>305106</td>
			<td></td>
		</tr>
		<tr>
			<td>4% paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>15700</td>
			<td></td>
		</tr>
		<tr>
			<td>Bluing reagent</td>
			<td>Fisher Scientific</td>
			<td>&#160;22-050-114</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6J mice</td>
			<td>Jackson Laboratories</td>
			<td>664</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting Microscope</td>
			<td>Olympus</td>
			<td>SZ61</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin stain</td>
			<td>Electron Microscopy Sciences</td>
			<td>26051-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin stain</td>
			<td>Electron Microscopy Sciences (Gill&#39;s #2)</td>
			<td>26030-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Imager</td>
			<td>Olympus</td>
			<td>Q-Color 5</td>
			<td></td>
		</tr>
		<tr>
			<td>Infusion line (included in the in vivo perfusion system)</td>
			<td>Braintree Scientific</td>
			<td>IV4140</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine&#160;</td>
			<td>Covetrus</td>
			<td>10004027</td>
			<td>Zoetis NDC# 00856440301</td>
		</tr>
		<tr>
			<td>Microscope&#160;</td>
			<td>Olympus</td>
			<td>CX-33</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Microm</td>
			<td>HM335S</td>
			<td></td>
		</tr>
		<tr>
			<td>Ophthalmic antibacterial ointment&#160;</td>
			<td>Henry Schein&#160;</td>
			<td>1410468</td>
			<td>Baush &#38; Lomb NDC# 2420879535</td>
		</tr>
		<tr>
			<td>Permount Mounting Media</td>
			<td>Fisher Scientific</td>
			<td>SP15-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Prism</td>
			<td>GraphPad</td>
			<td>10.3.1 for macOS</td>
			<td>data collection, statistical anlaysis, graphs</td>
		</tr>
		<tr>
			<td>Saline Solution</td>
			<td>KD Medical Inc</td>
			<td>50-103-1363</td>
			<td></td>
		</tr>
		<tr>
			<td>Stopcock (included in the in vivo perfusion system)</td>
			<td>Braintree Scientific</td>
			<td>IV4140</td>
			<td></td>
		</tr>
		<tr>
			<td>Tonometer</td>
			<td>iCare</td>
			<td>TA01i</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Covetrus</td>
			<td>1XYL006</td>
			<td>Covetrus NDC# 11695402401</td>
		</tr>
	</tbody>
</table>

induction of retinal ischemia-reperfusion injury in a mouse eye model

Ischemia-reperfusion injuries are known to cause a range of retinal pathologies, including diabetic retinopathy, glaucoma, retinal vascular occlusions, and other vaso-occlusive conditions. This manuscript presents a method for inducing ischemia-reperfusion injury in a mouse model. The method utilized anterior chamber cannulation attached to a saline reservoir, generating hydrostatic pressure to raise the intraocular pressure to 90-100 mmHg. This method effectively caused constriction of retinal capillaries to induce retinal ischemia. At the end of the ischemic period (60 min), the intraocular pressure was normalized (&#8804;20 mmHg) before removing the cannula from the anterior chamber to initiate reperfusion. Days after the ischemia/reperfusion procedure, the eyes were collected and sectioned for histological staining. The histopathology of the retinal sections was scored by evaluating eight parameters of retinal injury: folds, hemorrhage, deformation, cell loss in the ganglion cell, inner nuclear, outer nuclear, and photoreceptor layers, and damage to retinal pigment epithelial cells. This method provided a reproducible model to study the mechanisms and pathology of retinal ischemia/reperfusion injury. In addition, this model can facilitate the discovery of potential therapeutic targets to treat retinal ischemia/reperfusion injury, advancing the study of retinal pathologies and improving patient outcomes.

To assess the pathology of the retinas after the ischemia/reperfusion, eyes were collected from one group of mice 2 days after the procedure and from another group of mice 7 days after the procedure. The enucleated eyes were fixed in 4% paraformaldehyde, embedded in paraffin, and sliced into 5 &#181;m sections. The sections were stained with hematoxylin and eosin (H&#38;E) and imaged for histological examination (Figure 1). The stained histological images showed damage and loss of cells in t...

Induction of Retinal Ischemia-Reperfusion Injury in a Mouse Eye Model

This protocol models retinal ischemia-reperfusion injury in a mouse eye by inducing retinal ischemia via anterior chamber cannulation and intraocular pressure elevation, followed by intraocular pressure normalization to initiate the reperfusion.

Ischemia-reperfusion injuries are known to cause a range of retinal pathologies, including diabetic retinopathy, glaucoma, retinal vascular occlusions, ...

induction-of-retinal-ischemia-reperfusion-injury-in-a-mouse-eye-model

Research

JoVE Journal

Biology

259 Views.  Boston University Chobanian & Avedisian School of Medicine. This protocol models retinal ischemia-reperfusion injury in a mouse eye by inducing retinal ischemia via anterior chamber cannulation and intraocular pressure elevation, followed by intraocular pressure normalization to initiate the reperfusion.

Video: Induction of Retinal Ischemia-Reperfusion Injury in a Mouse Eye Model

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Isolation of Retinal Stem Cells from the Mouse Eye

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of non-pigmented inner and pigmented outer cell layers, and the clonal RSC colonies that arise from a single pigmented cell from the CE are made up of both pigmented and non-pigmented cells which can be differentiated to form all the cell types of the neural retina and the RPE. There is some controversy about whether all the cells within the spheres all contain at least some pigment4; however the cells are still capable of forming the different cell types found within the neural retina1-3. In some species, such as amphibians and fish, their eyes are capable of regeneration after injury5, however; the mammalian eye shows no such regenerative properties. We seek to identify the stem cell in vivo and to understand the mechanisms that keep the mammalian retinal stem cells quiescent6-8, even after injury as well as using them as a potential source of cells to help repair physical or genetic models of eye injury through transplantation9-12. Here we describe how to isolate the ciliary epithelial cells from the mouse eye and grow them in culture in order to form the clonal retinal stem cell spheres. Since there are no known markers of the stem cell in vivo, these spheres are the only known way to prospectively identify the stem cell population within the ciliary epithelium of the eye.

In this video, we will demonstrate how to isolate retinal stem cells from the ciliary epithelium of the mouse eye and grow them in culture to form clonal retinal spheres. The spheres that are isolated possess the cardinal properties of stem cells: self-renewal and multipotentiality.

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of ...

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed under anoxia for 20 hr&#160;(simulated ischemia), and subsequently moved to a normal atmosphere with food (simulated reperfusion). This experimental paradigm results in increased death and neuronal damage, and techniques are presented to assess organism viability, alterations to the morphology of touch neuron processes, as well as touch sensitivity, which represents the behavioral output of neuronal function. Finally, a method for constructing hypoxic incubators using common kitchen storage containers is described. The addition of a mass flow control unit allows for alterations to be made to the gas mixture in the custom incubators, and a circulating water bath allows for both temperature control and makes it easy to identify leaks. This method provides a low cost alternative to commercially available units.

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

A protocol is described that uses anoxia/starvation in C. elegans to model ischemia/reperfusion. Functional outcomes include increased mortality, visible abnormalities in GFP-labeled neuronal processes, and impaired behavioral responses that require neuronal function.

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the eye. It is a single sheet of pigmented cells that are hexagonally packed and connected by tight junctions. The main functions of the RPE include absorption of light, phagocytosis of the shed photoreceptor outer segments, spatial buffering of ions, transport of nutrients, ions and water as well as active involvement in the visual cycle. With such important and diverse functions, it is critically important to study the biology of RPE cells. A number of RPE cell lines have been established; however, passaged and immortalized cells are known to quickly lose some of the morphological and physiological characteristics of natural RPE cells. Thus, primary cells are more suitable for studying different aspects of RPE cell biology and function. Mouse primary RPE cell culture is very useful to researchers since mouse models are widely used in biological studies, however collecting RPE cells from mouse is also very challenging due to their small size. Here, we present a protocol for establishing primary mouse RPE cell cultures which includes enucleation and dissection of the eyes and isolation of the RPE sheets to yield the cells for culturing. This method enables efficient cell recovery. The RPE cells obtained from two mice&#160;can reach confluency on one 12 mm polyester membrane insert pre-loaded in culture plate after one week of culture and display some of the original properties of bona fide RPE cells such as hexagonal shape and pigmentation after two weeks of culture.

The retinal pigment epithelium (RPE) is a multi-functional epithelium of the eye. Here we present a protocol to establish primary cell cultures derived from the murine RPE.

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the ...

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to understand the pathophysiology of ocular diseases because of their similarities with human anatomy and physiology. Alterations in the retinal pigment epithelium (RPE), including changes in morphology and function, are common features shared by many ocular disorders. However, successful isolation and culture of primary mouse RPE cells is very challenging. This paper is an updated audiovisual version of the protocol previously published by Fernandez-Godino et al. in 2016 to efficiently isolate and culture primary mouse RPE cells. This method is highly reproducible and results in robust cultures of highly polarized and pigmented RPE monolayers that can be maintained for several weeks on Transwells. This model opens new avenues for the study of the molecular and cellular mechanisms underlying eye diseases. Moreover, it provides a platform to test therapeutic approaches that can be used to treat important eye diseases with unmet medical needs, including inherited retinal disorders and macular degenerations.

This protocol, which was originally reported by Fernandez-Godino et al. in 20161, describes a method to efficiently isolate and culture mouse RPE cells, which form a functional and polarized RPE monolayer within one week on Transwell plates. The procedure takes approximately 3 hours.

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to ...

Development of a Cell Co-Culture Model to Mimic Cardiac Ischemia/Reperfusion In Vitro

Ischemic heart disease is the leading cause of death and disability worldwide. Reperfusion causes additional injury beyond ischemia. Endothelial cells (ECs) can protect cardiomyocytes (CMs) from reperfusion injury through cell-cell interactions. Co-cultures can help investigate the role of cell-cell interactions. A mixed co-culture is the simplest approach but is limited as isolated treatments and downstream analyses of single cell types are not feasible. To investigate whether ECs can dose-dependently attenuate CM cell damage and whether this protection can be further optimized by varying the contact distance between the two cell lines, we used Mouse Primary Coronary Artery Endothelial Cells and Adult Mouse Cardiomyocytes to test three types of cell culture inserts which varied in their inter-cell layer distance at 0.5, 1.0, and 2.0 mm, respectively. In CMs-only, cellular injury as assessed by lactate dehydrogenase (LDH) release increased significantly during hypoxia and further upon reoxygenation when the distance was 2.0 mm compared to 0.5 and 1.0 mm. When ECs and CMs were in nearly direct contact (0.5 mm), there was only a mild attenuation of the reoxygenation injury of CMs following hypoxia. This attenuation was significantly increased when the spatial distance was 1.0 mm. With 2.0 mm distance, ECs attenuated CM injury during both hypoxia and hypoxia/reoxygenation, indicating that sufficient culture distancing is necessary for ECs to crosstalk with CMs, so that secreted signal molecules can circulate and fully stimulate protective pathways. Our findings suggest, for the first time, that optimizing the EC/CM co-culture spatial environment is necessary to provide a favorable in vitro model for testing the role of ECs in CM-protection against simulated ischemia/reperfusion injury. The goal of this report is to provide a step-by-step approach for investigators to use this important model to their advantage.

Development of a Cell Co-Culture Model to Mimic Cardiac Ischemia/Reperfusion In Vitro

Spatial distance is a key parameter in assessing hypoxia/reoxygenation injury in a co-culture model of separate endothelial and cardiomyocyte cell layers, suggesting, for the first time, that optimizing the co-culture spatial environment is necessary to provide a favorable in vitro model for testing the role of endothelial cells in cardiomyocyte protection.

Ischemic heart disease is the leading cause of death and disability worldwide. Reperfusion causes additional injury beyond ischemia. Endothelial cells ...

Generation of Hook Ischemia-Reperfusion Model using a Three-Day Developing Chick Embryo

Ischemia and reperfusion (I/R) disorders, such as myocardial infarction, stroke, and peripheral vascular disease, are a few of the leading causes of illness and death. Many in vitro and in vivo models are currently available for studying the I/R mechanism in disease or damaged tissues. However, to date, no in ovo I/R model has been reported, which would allow for a better understanding of I/R mechanisms and faster drug screening. This paper describes I/R modeling using a spinal needle customized hook in a 3-day chick embryo to understand I/R development and treatment mechanisms. Our model can be used to investigate anomalies at the DNA, RNA, and protein levels. This method is simple, quick, and inexpensive. The current model can be used independently or in conjunction with existing in vitro and in vivo I/R models.

This paper describes ischemia-reperfusion (I/R) modeling in a 3-day chick embryo using a spinal needle customized hook to better understand I/R development and treatment. This model is simple, quick, and inexpensive.

Ischemia and reperfusion (I/R) disorders, such as myocardial infarction, stroke, and peripheral vascular disease, are a few of the leading causes of ...

Primary Culture of Porcine Retinal Pigment Epithelial Cells

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. Multiple functions, including phagocytosis, nutrient/metabolite transportation, vitamin A metabolism, etc., are conducted by the RPE on a daily basis. RPE cells are terminally differentiated epithelial cells with little or no regenerative capacity. Loss of RPE cells results in multiple eye diseases leading to visual impairment, such as age-related macular degeneration. Therefore, the establishment of an in vitro culture model of primary RPE cells, which more closely resembles the RPE in vivo than cell lines, is critical for the characteristic and mechanistic studies of RPE cells. Considering the fact that the source of human eyeballs is limited, we create a protocol to culture primary porcine RPE cells. By using this protocol, RPE cells can be easily dissociated from adult porcine eyeballs. Subsequently, these dissociated cells attach to culture dishes/inserts, proliferate to form a confluent monolayer, and quickly re-establish key features of epithelial tissue in vivo within 2 wks. By qRT-PCR, it is demonstrated that primary porcine RPE cells express multiple signature genes at comparable levels with native RPE tissue, while the expressions of most of these genes are lost/highly reduced in human RPE-like cells, ARPE-19. Moreover, the immunofluorescence staining shows the distribution of tight junction, tissue polarity, and cytoskeleton proteins, as well as the presence of RPE65, an isomerase critical for vitamin A metabolism, in cultured primary cells. Altogether, we have developed an easy-to-follow approach to culture primary porcine RPE cells with high purity and native RPE features, which could serve as a good model to understand RPE physiology, study cell toxicities, and facilitate drug screenings.

Here, an easy-to-follow method to culture primary porcine retinal pigment epithelial cells in vitro is presented.

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. ...

Isolation of Primary Mouse Retinal Pigmented Epithelium Cells

The retinal pigmented epithelium (RPE) layer lies immediately behind the photoreceptors and harbors a complex metabolic system that plays several critical roles in maintaining the photoreceptors' function. Thus, the RPE structure and function are essential to sustain normal vision. This manuscript presents an established protocol for primary mouse RPE cell isolation. RPE isolation is a great tool to investigate the molecular mechanisms underlying RPE pathology in the different mouse models of ocular disorders. Furthermore, RPE isolation can help in comparing primary mouse RPE cells isolated from wild-type and genetically modified mice, as well as testing drugs that can accelerate the development of therapy for visual disorders. The manuscript presents a step-by-step RPE isolation protocol; the entire procedure, from enucleation to seeding, takes approximately 4 hours. The media shouldn't be changed for 5-7 days after seeding, to allow the growth of the isolated cells without disturbance. This process is followed by the characterization of morphology, pigmentation, and specific markers in the cells via immunofluorescence. Cells can be passaged a maximum of three or four times.

This manuscript describes a simplified protocol for the isolation of retinal pigmented epithelium (RPE) cells from mouse eyes in a stepwise manner. The protocol includes the enucleation and dissection of mouse eyes, followed by the isolation, seeding, and culturing of RPE cells.

The retinal pigmented epithelium (RPE) layer lies immediately behind the photoreceptors and harbors a complex metabolic system that plays several critical ...

Induction of Retinal Ischemia-Reperfusion Injury in a Mouse Eye Model

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An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

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Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

Development of a Cell Co-Culture Model to Mimic Cardiac Ischemia/Reperfusion In Vitro

Generation of Hook Ischemia-Reperfusion Model using a Three-Day Developing Chick Embryo

Primary Culture of Porcine Retinal Pigment Epithelial Cells

Isolation of Primary Mouse Retinal Pigmented Epithelium Cells