To investigate the blood-retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease, we used several adeno-associated virus (AAV) variants as tools to label retinal neurons and glia. Virus mediated reporter gene expression is then used as an indicator of retinal barrier permeability.
Müller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes (ILM), and in conjunction with astrocytes, pericytes and endothelial cells they establish the blood-retinal barrier (BRB). BRB limits material transport between the bloodstream and the retina while the ILM acts as a basement membrane that defines histologically the border between the retina and the vitreous cavity. Labeling Müller cells is particularly relevant to study the physical state of the retinal barriers, as these cells are an integral part of the BRB and ILM. Both BRB and ILM are frequently altered in retinal disease and are responsible for disease symptoms.
There are several well-established methods to study the integrity of the BRB, such as the Evans blue assay or fluorescein angiography. However these methods do not provide information on the extent of BRB permeability to larger molecules, in nanometer range. Furthermore, they do not provide information on the state of other retinal barriers such as the ILM. To study BRB permeability alongside retinal ILM, we used an AAV based method that provides information on permeability of BRB to larger molecules while indicating the state of the ILM and extracellular matrix proteins in disease states. Two AAV variants are useful for such study: AAV5 and ShH10. AAV5 has a natural tropism for photoreceptors but it cannot get across to the outer retina when administered into the vitreous when the ILM is intact (i.e., in wild-type retinas). ShH10 has a strong tropism towards glial cells and will selectively label Müller glia in both healthy and diseased retinas. ShH10 provides more efficient gene delivery in retinas where ILM is compromised. These viral tools coupled with immunohistochemistry and blood-DNA analysis shed light onto the state of retinal barriers in disease.
Müller cells are the major glial component of the retina. Morphologically, they span the retina radially and their endfeet, in contact with the vitreous, face the ILM and secret components of the latter. The ILM is a basement membrane composed of about ten different extracellular matrix proteins (laminin, agrin, perlecan, nidogen, collagen and several heparin sulfate proteoglycans). During development, its presence is indispensable for retinal histogenesis, navigation of optic axons, and survival of ganglion cells1-3. However, ILM is unessential in adult retina and can be surgically removed in certain pathologies without causing retinal damage4. In gene therapy, this membrane becomes a physical barrier for efficient transduction of the retina using AAVs by intravitreal injection5.
Through the extensive arborization of their processes, Müller cells provide nutritional and regulatory support to both retinal neurons and vascular cells. Müller cells are also involved in the regulation of the retinal homeostasis, in the formation and maintenance of the BRB6. Tight junctions between retinal capillary endothelial cells, Müller cells, astrocytes and pericytes form the BRB. BRB prevents certain substances from entering the retina.In many diseases like diabetic retinopathy, retinal vein occlusion and respiratory diseases, hypoxia of the retina causes leakage through the BRB7-9. This rupture is associated with an increase in vascular permeability leading to vasogenic edema, retinal detachment and retinal damage.
Müller cells are tightly associated with blood vessels and the basement membrane, playing an important role in both BRB and ILM integrity. Consequently, labeling Müller glial cells is particularly relevant to the study of the physical state of these retinal barriers.
Classically, BRB permeability is measured using the Evans blue assay consisting of systemic injection of Evans blue dye, which binds non-covalently to plasma albumin. This assay measures the albumin leakage (protein of intermediate size, ~66 kDa) from blood vessels into the retina (see Protocols Section 5)10. Alternatively, the vascular leakage can be visualized by fluorescence retinal angiography attesting for leakage of fluorescein (small molecule, ~359 Da; see Protocols Section 6)11. Nevertheless, both methods allow evaluation of the BRB permeability to small molecules and proteins but they do not provide information about the ILM integrity.
Hence, to study BRB permeability, we used an AAV based method that gives information on the BRB permeability to larger molecules (e.g., AAV particles, 25 nm diameter). Indeed, our method can detect presence of AAV transgene in the blood, which would suggest that ~25 nm diameter particles would be able to infiltrate into the bloodstream. This method also provides information on the structure of the ILM and extracellular matrix proteins in pathological conditions. Two AAV variants are useful for such study: AAV5 and ShH10. Subretinally injected, AAV5 has a natural tropism for photoreceptors and retinal pigment epithelium12 but it cannot get across to the outer retina when administered into the vitreous in wild-type retinas with intact ILM5,13. ShH10 is an AAV variant that has been engineered to specifically target glial cells over neurons14,15. ShH10 selectively labels Müller cells in both healthy and diseased retinas with increased efficiency in retinas with compromised barriers16. These viral tools coupled with immuhistochemistry and blood-DNA analysis provide information on the state of retinal barriers and their involvement in disease (Figure 1).
All animals used in this study were cared for and handled according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
1. Production of Recombinant AAV (rAAV) by Transient Transfection of HEK-293 Cells17,18
NOTE: See McClure C, JoVE (2011)19.
2. Intravitreal Injection of AAV
3. Immunohistochemistry
4. PCR Analysis of Mouse Blood Samples
5. Optional Evans Blue Method
NOTE: Quantify vascular permeability by measuring albumin leakage from blood vessels into the retina using the Evans blue method10.
6. Optional Fluorescein Angiography
NOTE: Leakage from retinal blood vessels in mice can be visualized by intraperitoneal injection of sodium fluorescein.
We expect increased retinal transduction of Müller glial cells using ShH10 if the animal model shows perturbations in the structure of the ILM (Figure 2A–B). For example, we have shown that in absence of Dp71, ShH10 targets specifically but more efficiently Müller glial cells by intravitreal injection, indicating increased permeability of the ILM in this mouse line compared to wild-type mice16 (Figure 2C–F).
AAV5 can also be used as an indicator of ILM permeability. AAV5, ineffective by intravitreal injection in wild-type mice, becomes strongly effective in gene delivery to photoreceptors in mice with compromised retinal barriers such as the Dp71-null mice16 or in other retinal degenerations22 (Figure 2G–H). This further confirms the ILM disorganization and potential permeability increase in the extra-cellular matrix surrounding the retinal neurons.
We found that, the BRB breakdown of Dp71-null mice remains selective to AAV particles since no trace of AAV were found in blood samples of intravitreally injected Dp71-null mice16 (Figure 3).
Figure 1: Schematic representation of the general protocol. After AAV production, particles are injected into the vitreous or into the penile vein to anesthetized adult mice. Fundus images with Micron III camera are performed to follow the GFP expression. The GFP expression pattern is analyzed on retinal cryosections and flatmounted retinas thanks to immunohistochemistry and confocal images. The AAV passage through the BRB is assessed by PCR analysis on blood samples coming from intravitreally or intrapenially injected mice, in order to detect the presence of GFP sequence, indicator of the AAV particles presence into the bloodstream.
Figure 2: ILM permeability to AAV transduction. Confocal images of flatmounted wild-type and Dp71-null retinas labeled with a pan-laminin antibody (A, B) (scale bar: 50 µm). Fundus images showing GFP expression (C, D). Flatmounted retinas one month after ShH10-GFP intravitreal injection (E, F). Three dimensional reconstitution (E*) of the area indicated by an asterisk in E showing transduced Müller glial cells in green and astrocytes in red (anti-GFAP antibody). Flatmounted retinas one month after intravitreal injection of AAV5-GFP (G, H) (scale bar: 500 µm). Confocal image of the area indicated by an asterisk in H showing transduced photoreceptors in green and cone outer segments in red (PNA lectin staining) (H*). The left column shows results from wild-type mouse retinas and the middle column shows results from Dp71-null mouse retinas. The schema (I) represents AAV transduction of retina with compromised barriers as Dp71-null retina, through the vitreous. Abbreviations : CV, choroidal vessels; RPE, retinal pigment epithelium; PhR, photoreceptor cells (rods and cones); MGC, Müller glial cells; HC, horizontal cells; BC, bipolar cells; AC, amacrine cells; M, microglia; EC, endothelial cells; P, pericytes; GC, ganglion cells; A, astrocytes; ILM, inner limiting membrane; V, vitreous; AAV, adeno-associated virus. (Re-print with permission from Vacca et al., Glia (2013)16, #3483740951391).
Figure 3: BRB permeability to AAV particles. PCR amplification of GFP transgene extracted from mouse blood samples. Mice either injected intravitreally (IVT) with ShH10-GFP or intrapenially (IP) with AAV-GFP at different times after injection. Lane 5-18, odd numbers for wild-type mice and even numbers for Dp71-null mice; lane 2, 1 ng of the AAV plasmid, pTR-SB-smCBA-hGFP; lane 3, water; lanes 5-6, blood DNA before injection; lanes 7-8, 3 hr post-IVT; lanes 9-10, 24 hr post-IVT; lanes 11-12, 24 hr post-IP; lanes 13-14, 48 hr post-IVT; lanes 15-16, 48 hr post-IP; lanes 17-18, 72 hr post-IVT; lanes 19-20, 72 hr post-IP. Lanes 1 and 4, Invitrogen scale ladder (100 bp): 100, 200, 300, 400, 500, 600, 700, 800, 1,000, 1,500, 2,000, 3,000 bp. (Re-print with permission from Vacca et al., Glia (2013)16, #3483740951391).
The BRB regulates the exchange of molecules between blood and retina. Its breakdown is associated with various diseases such as diabetic retinopathy or age-related macular degeneration (AMD). We recently showed that in a dystrophin knock-out mouse, which displays permeable BRB, the retina becomes more permissive to gene delivery mediated by adeno-associated viral vectors (AAV). However, despite BRB permeability AAV particles injected intraocularly stay confined to the ocular compartment in this model. Our results indicate that gene therapy for diseases that display permeable BRB does not represent additional risks of systemic side effects. Furthermore, they support the fact that other barriers such as the ILM become compromised during retinal disease allowing better access to viral particles. Our findings lead us to think that AAV encoding reporter genes is an excellent tool to study BRB permeability and ILM integrity in animal models of retinal disease. Particularly, the AAV variant ShH10, which specifically labels Müller glia can be used as a tool to label retinal glia and report on the state of the retina in response to disease. ShH10 will selectively label Müller glia in both healthy and diseased retinas. However, both ShH10 and other AAVs will provide more efficient gene delivery in retinas with compromised barriers.
Some critical steps in applying the above-described protocols are (1) developing manual dexterity to perform the safest intravitreal injection, and (2) properly fixing the tissue before and after dissection. In studying retinal barriers, the intravitreal injection should be well executed that is – without touching the lens or the retina. Damaging the lens or detaching the retina influences the BRB permeability23,24. Additionally lens damage impedes fundus imaging. Touching the retina can rupture the ILM and damage the retinal structure. To avoid touching the retina, the experimenter should observe the tip of the Hamilton syringe in the middle of the vitreous cavity, in front of the retina. A non-toxic dye can be included (such as Phenol red or fluorescein) in the viral solution to aid in the observation of the fluid injection into the vitreous. Damage to the retina can be readily observed during fundus imaging following the injection procedure. Another critical step is the fixation of the tissue after sufficient expression levels are reached. After enucleation, eyes must be immediately immersed in fixative buffer and before tissue permeabilization a second fixation is necessary in order to prevent GFP leakage. It can be helpful to slit the cornea before immersing the entire eye in the fixative – this will give the fixative a chance to permeate into the vitreous. This step can increase the overall tissue stability.
Lastly, it is also important to inject enough AAV particles to transduce efficiently the retina and to observe GFP expression on eye fundus images. The optimal amount of AAV particles is about 1010-1011 vg per eye, below 1010 particles the GFP expression is not always observed by fundus imaging.
This technique will not give the possibility to directly observe the ILM or the vasculature beneath the BRB. However it provides a way to examine retinal barriers and their modification in disease states, in a way that was previously not possible using the Evans blue assay and fluorescein angiography. Using AAVs with specific retinal transduction properties, it is possible to get better insight into the state of the retina with respect to the glial cells, their extracellular matrix and ILM. After mastering this technique, one can test the efficacy of therapeutic strategies and their influence on BRB and retinal permeability and go towards a better understanding of disease progression and the possibility to restore normal conditions after treatment in a mouse model of retinal disease.
The authors have nothing to disclose.
We thank the imaging platform of the Institut de la Vision. We acknowledge the French Muscular Dystrophy Association (AFM) for a PhD fellowship to O.V. and Allergan INC. This work performed in the frame of the LABEX LIFESENSES [reference ANR-10-LABX-65] was supported by French state funds managed by the ANR. We thank Peggy Barbe, and Mélissa Desrosiers for technical assistance with AAV preparations. We are grateful to Stéphane Fouquet for excellent technical assistance in confocal microscopy and his expert input with the interpretation of the results.
Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
C57BL6J mice strain | JANVIER LABS | mice | |
Ketamine 500 | Virbac France | anesthetic | |
Xylazine Rompun 2% | Bayer Healthcare | anesthetic | |
Neosynephrine 5% Faure | Europhta | dilatant | |
Mydriaticum 0,5% | Thea | dilatant | |
Sterdex | Novartis | anti-inflammatory | |
Cryomatrix embedding resin | Thermo Scientific | 6769006 | |
Superfrost Plus Adhesion Slides | Thermo Scientific | 10143352 | slides |
anti-laminin | Sigma | L9393 | antibody |
anti-rhodopsin clone 4D2 | Millipore | MABN15 | antibody |
anti-glutamine synthetase clone GS-6 | Millipore | MAB302 | antibody |
Anti-Glial Fibrillary Acidic Protein | Dako | 334 | antibody |
PNA Lectin | Invitrogen | L32459 | probe |
Alexa fluor conjugated secondary antibodies | Invitrogen | antibody | |
Fluorsave reagent | Calbiochem | 345789 | mounting medium |
QIAmp DNA Micro Kit | QIAGEN | 56304 | |
GoTaq DNA polymerase | Promega | M3001 | |
Evans Blue dye | Sigma | E2129 | dye |
5 µm filter | Millipore | ||
Sodium Citrate | Sigma | S1804 | |
Citric acid | Sigma | C1909-2.5KG | |
Formamide spectrophotometric | Sigma | 295876-2L | |
Fluorescein | Sigma | F2456 | dye |
Micron III | Phoenix Research Labs | Microscopy system based on 3-CCD color camera, frame grabber, and off-the-shelf software enables researchers to image mouse retinas. | |
Insulin Syringes | Terumo | SS30M3109 | |
Syringe 10 µl Hamilton | Dutscher | 74487 | Seringue 1701 |
Needle RN G33, 25 mm, PST 2 | Fisher Scientific | 11530332 | Intravitreal Injection |
UltraMicroPump UMP3 | World Precision Instruments | UMP3 | Versatile injector uses microsyringes to deliver picoliter volumes |
UltraMicroPump (UMP3) (one) with SYS-Micro4 Controller | UMP3-1 | Digital controller | |
Binocular magnifier SZ76 | ADVILAB | ADV-76B2 | Zoom 0.66 x 5 x LEDs with stand epi and dia / Retinas dissection |
Spring scissors straight – 8,5cm | Bionic France S.a.r.l | 15003-08 | Retinas dissection |
Micro-ciseaux de Vannas courbe | 15004-08 | ||
Pince Dumont 5 | 11254-20 | ||
Veriti 96-Well Thermal Cycler | Life technologies | 4375786 | Thermocycler |
Ultrasonic cleaner | Laboratory Supplies | G1125P1T | |
Nanosep 30k omega tubes | VWR | ||
Speedvac | Fisher Scientific | SC 110 A | |
Spectrofluorometer | TECAN | infinite M1000 |