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JoVE Journal Medicine

Medicine

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

Published: December 9, 2022 doi: 10.3791/63966
Automatic Translation
1,2,3, 1,2, 1,2
1Eye Research Center and Department of Foundational Medical Studies, Oakland University William Beaumont School of Medicine, 2Eye Research Institute, Oakland University, 3Human Anatomy and Embryology Department, Mansoura Faculty of Medicine, Mansoura University

Summary

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.

Abstract

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.

Introduction

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' medical history, and the donors' 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.

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Protocol

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

  1. 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 "Guide for the Care and Use of Laboratory Animals". The temperature range for mice is between 18 °C and 26 °C. Keep the humidity levels controlled and set at about 42%.
  2. 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.
  3. Provide 12 h light/dark cycles via lighting control in the animal housing rooms.
  4. 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.
  5. 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.
  6. 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.
  7. 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.

2. Tissue preparation

  1. 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.
  2. 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.
  3. 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.
  4. 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).

3. Tissue dissection

  1. Dissect out the retina carefully under an operating microscope, as illustrated sequentially in Figure 1.
    1. 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.
    2. 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).
    3. Dissect the retina radially into four equal-sized pieces.
  2. Follow the steps below for handling the small pieces of the retina to ensure proper orientation of the retinal culture.
    1. Using a dissecting scissor blade, open just beneath the retinal piece (Table of Materials).
    2. Slowly lift the retinal piece with the tip of an open scissor blade, and gently transfer it to the culture plate.
  3. Transfer the explants derived from the isolated retina separately onto cell culture inserts in a 12-well plate, each containing 300 µL 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.
  4. 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.
  5. Incubate the retinal explant culture in humidified incubators at 37 °C and 5% CO2.
  6. 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.
    ​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.

4. Retinal explant culture

  1. Maintain the retinal explant cultures (prepared in steps 3.2-3.3) in humidified incubators at 37 °C and 5% CO2.
  2. Change half of the media from each well every other day for 2 weeks. Do this by pipetting 150 µL from the well carefully. Then, add 150 µL of fresh media back into the well. Avoid flipping the explant when changing the media.
  3. 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.

5. Immunohistochemistry

  1. After 2 weeks, fix the retinal explant by removing the media. Do this by first washing once with 200 µL of 1x PBS and then adding 300 µL 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 µL conical tube, and the washing and fixation can be done inside the tube.
  2. Transfer the explant to a 500 µL conical tube containing 300 µL of 1x PBS-2.5% Triton X for washing.
  3. Wash the fixed explants in the tube three times for 5 min each using medium velocity on a shaker (25 rpm).
  4. Block the expected nonspecific reactions by incubating the explant with 300 µL of 1x blocking buffer containing 0.02% thimerosal for 1 h at room temperature prior to the antibody incubation.
  5. Incubate the fixed explants with the primary antibodies overnight at 4 °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.
  6. 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.
  7. 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.
  8. Remove the secondary antibodies by aspiration, and then wash three times for 5 min each with 1x PBS-2.5% Triton X.
  9. 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.
  10. Cover the tissue with a coverslip. Do not allow any air bubbles between the coverslip and the tissue.
  11. 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.

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Representative Results

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 (isolectin-B4) was performed (Figure 2). The staining showed well-organized and well-developed retinal neurons and glial cells. Additionally, the retinal blood vessels were structurally intact, as indicated by the isolectin-B4 staining. The morphological integrity of the retinal explant, as demonstrated by the immunostaining, indicated that the retinal explant culture was successful to maintaining viable retinal cells that could be experimentally used as a model. The different experimental conditions to be studied using the explant can be evaluated and analyzed by immunofluorescence staining for multiple cellular markers using ImageJ software. Immunostaining allows the measurement of the levels of immunoreactivity of each cellular marker and of the number of specific cells, such as the number of retinal ganglion cells or photoreceptors, among various experimental groups.

Importance of correct orientation of the retinal explant in the culture plate for the experimental outcomes
The correct orientation of the retinal explant in the culture plate is achieved by incubating the neuroretina facing upward. On the other hand, failure of the retinal explant may result from flipping or folding the retinal tissue, which causes neuroretina to face downward. The immunofluorescence staining of the cultured retinal explant after 2 weeks using different retinal markers as the previous experiment (NeuN, GFAP, and isolectin-B4) demonstrated that the failure of proper explant orientation results in the failure of the proper development of the neurovascular element (Figure 3).

Figure 1
Figure 1: Steps of dissecting the eyeball to create the retinal explant. (a) The enucleated eyeball is immersed in the media immediately after being removed from the animal. (b) A circumferential incision is made along the limbus to open the eyeball. (c) The cornea is removed by dissecting along the limbal incision. (d) By holding the optic nerve with fine forceps, the outer coat of the eye is peeled off gently. (e) The lens, the vitreous, and the ciliary body are removed. (F) Only the neural retina is left behind. (g) Four radial incisions are made in the retina to facilitate it being cut in the next step. (h) The retina can be cut into two or four pieces and then be transferred to the cell culture plate containing the insert and the media. Please click here to view a larger version of this figure.

Figure 2
Figure 2: Successful retinal explant culture with preserved retinal structure. Immunofluorescence staining of the retinal explants that had stayed in culture for 2 weeks. The explants were fixed and stained with (a) NeuN for retinal neurons (green) and isolectin-B4 for blood vessels (red), and (b) GFAP for glial cells (green) and isolectin-B4 for blood vessels (red). The staining showed well-developed retinal cells and retinal vessels. Scale bar = 50 µm. Please click here to view a larger version of this figure.

Figure 3
Figure 3: Unsuccessful retinal explant culture with defective, under-developed retinal cells. Immunofluorescence staining of retinal explants that had stayed in culture for two weeks. The explants were fixed and stained with GFAP for glial cells (green) and isolectin-B4 for blood vessels (red). The images show defective staining and under-developed retinal cells and retinal vessels. The retinal explant was folded and was not oriented correctly in the culture plate. Great attention should be given to ensure the proper orientation of the explant with the retina facing upward. Failure to put the retinal explant in the right orientation will result in failure of the proper development of the explant. Scale bar = 100 µm Please click here to view a larger version of this figure.

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Discussion

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 control group derived from the same single retina or animal as the treated group or groups gives this technique a great advantage in terms of providing more reliable comparisons of different experimental conditions.

One of the major challenges during retinal explant preparation is the folding of the explant or wrong orientation in the culture plate with the retinal surface facing downward. Other researchers suggested pipetting the retina up and down within the transferring pipette to have it in the correct orientation in the plate37. However, in their protocol, they used the whole retina for a single explant, but in this protocol, a single retina was cut into two to four pieces, meaning the pieces are smaller and it is difficult to do this maneuver for the proper orientation. Cutting a single retina into two to four pieces allows four to eight retinal pieces to be obtained from the same animal that could be used for an experiment. This allows experiments in which the control group and experimental groups are derived from the same animal.

However, cutting the retina into small pieces makes the handling of it more challenging. Therefore, we developed a technique for handling these small retinal pieces to ensure proper orientation of the retinal culture. To do this, the open dissecting scissor blade is placed just beneath the retinal piece, with the retinal surface facing upward. If the retinal piece is flipped or folded, it is adjusted gently to the correct orientation. Slowly, the retinal piece is elevated with the tip of an open scissor blade and gently put in the culture plate. This simple technique ensures each retinal piece is in the correct orientation with the retina facing upward and promotes better outcomes for studying retinal vessels and neuroglial cells (Figure 2). Failure to place the retinal explant in the correct orientation with the neuroretina facing upward will result in failure of the proper growth of the retinal cells in culture, as shown in Figure 3. Proper training is highly recommended to avoid this technical error.

This protocol describes an organotypic retinal explant of an adult mouse retina; however, the technique has also previously been described for both immature mouse retinas37 and human retinas38. In a previous study, retinal explant cultures were described for studying vascular development, in which endothelial function manipulation was made feasible ex vivo27. As an example of the morphological assessment of a retinal explant, the retinal explant was left in culture for 2 weeks, and then immunostaining was performed using NeuN (neuronal cell marker), GFAP (glial cell marker), and isolectin-B4 (vascular marker). The staining showed that the retinal cells and blood vessels were well-developed and well-preserved in the explant (Figure 2). However, more immunohistochemistry staining data can be obtained to check the viability and functionality of other retinal cells in the explant as well.

The explant culture itself could be stressful for the retinal cells. However, the cells could be incubated under relatively normal conditions so that the morphological changes occurring under various experimental conditions, such as hyperglycemia or hypoxia, could be compared to the baseline condition. The explant, thus, represents a good tool for evaluating various retinal cells and testing their responses to different pharmacological agents or therapeutic targets simultaneously in a wide variety of retinal diseases.

Retinal neovascularization is a cardinal sign of ischemic retinopathy, such as in retinopathy of prematurity and diabetic retinopathy. We (Figure 2) and others27,39,40,41,42 could observe intact retinal vasculature in cultured explants for up to 2 weeks. Thus, the retinal explant has been used to study the pathophysiology of both normal and pathological retinal neovascularization41,42,43,44,45,46,47,48,49. Interestingly, a research group recorded VEGF-induced vascular sprouting in mouse retinal explants while investigating the proangiogenic factors that are elevated in proliferative diabetic retinopathy43. Moreover, retinal explants have been embedded in a three-dimensional gel, allowing for the study of the sprouting of retinal endothelial cells in a more detailed spatiotemporal orientation45,46,47.

In addition to morphological studies, retinal explants are also used to some extent to evaluate retinal functions50,51,52,53. Ex vivo electroretinogram (ERG) imaging has been performed on isolated mouse retinal explants, allowing for the study of the different functions of a variety of retinal cells, including both rod and cone photoreceptors54,55,56,57. Interestingly, Calbiague et al. reported that, in response to high glucose treatment, retinal explant cultures derived from either mice or rats showed an increasingly reduced thickness of the retinal layers58. However, retinal bipolar cells, which are among the earliest sensitive cells to diabetic conditions, kept not only their morphology but also their electrophysiological properties in culture for up to 2 weeks58. In summary, we believe that this protocol could be a base or a starting point for additional future studies to optimize the benefits of explant usage in eye research.

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Disclosures

The authors have nothing to disclose.

Acknowledgments

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'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.

Materials

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

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Retinal Explant Adult Mouse Retina Ex Vivo Model Retinal Neurovascular Diseases Retinal Research Crosstalk Between Cells Retinal Neurons Glial Cells Vascular Cells Cultured Retinal Explant Biochemical Parameters Vascular System Screening Tool Pharmacological Interventions Retinovascular Diseases Neurodegenerative Diseases Diabetic Retinopathy Vascular And Neuronal Injury Pathophysiological Changes Microvascular Retinal Dysfunction In Vivo Models In Vitro Models
Retinal Explant of the Adult Mouse Retina as an <em>Ex Vivo</em> Model for Studying Retinal Neurovascular Diseases
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Elmasry, K., Moustafa, M.,… More

Elmasry, K., Moustafa, M., Al-Shabrawey, M. Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases. J. Vis. Exp. (190), e63966, doi:10.3791/63966 (2022).

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