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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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.

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.

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 (isol...

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 contr...

Disclosures

The authors have nothing to disclose.

Acknowledgements

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

NameCompanyCatalog NumberComments
Adult C57Bl/6J mice The Jackson Laboratory, Bar Harbor, ME, 04609, USA664
All-in-One Fluorescence Microscope KEYENCE CORPORATION OF AMERICA, IL, 60143, U.S.A.BZ-X800
B27 supplementsThermo scientific. Waltham, MA, 02451, USAGibco #17504-04
Blockade blocking solution Thermo scientific. Waltham, MA, 02451, USAB10710
DMEM F12Thermo scientific. Waltham, MA, 02451, USAGibco #11320033
Goat anti-Rabbit IgG.Thermo scientific. Waltham, MA, 02451, USAF-2765
GSL I, BSL I (Isolectin)Vector Laboratories. Burlingame, CA 94010,USAB-1105-2
Hanks Ballanced Salt Solution (HBSS)Thermo scientific. Waltham, MA, 02451, USAGibco #14175095
Micro Scissors, 12 cm, Diamond Coated BladesWorld Precision Instruments,FL 34240, USA Straight (503365)
N2 supplementsThermo scientific. Waltham, MA, 02451, USAGibco #17502-048
Nunc Polycarbonate Cell Culture Inserts in Multi-Well PlatesThermo scientific. Waltham, MA, 02451, USA140652
Paraformaldehyde 4% in PBSBBP, Ashland, MA, 01721 USAC25N107
Penicillin-Streptomycin (10,000 U/mL)Thermo scientific. Waltham, MA, 02451, USA15140148
PROLONG DIAMOND ANTIFADE 4′,6-diamidino-2-phenylindole (DAPI).Thermo scientific. Waltham, MA, 02451, USAP36962
Rabbit Anti-NeuN AntibodyAbcam.,Cambridge, UKab177487
Rabbit Glial Fibrillary Acidic Protein (GFAP) AntibodyDako,Carpinteria, CA 93013, USA.Z0334
Texas RedVector Laboratories. Burlingame, CA 94010,USASA-5006-1
TritonXBioRad Hercules, CA,  94547,USA1610407

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