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.
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.
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.
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
2. Tissue preparation
3. Tissue dissection
4. Retinal explant culture
5. Immunohistochemistry
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: 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: 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: 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.
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.
The authors have nothing to disclose.
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.
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 |