This article describes a well-established and reproducible lectin stain assay for the whole mount retinal preparations and the protocols required for the quantitative measurement of vascular parameters frequently altered in proliferative and non-proliferative retinopathies.
Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, gliosis and a progressive change in vascular function and structure. Although the onset of the retinopathies is characterized by subtle disturbances in visual perception, the modifications in the vascular plexus are the first signs detected by clinicians. The absence or presence of neovascularization determines whether the retinopathy is classified as either non-proliferative (NPDR) or proliferative (PDR). In this sense, several animal models tried to mimic specific vascular features of each stage to determine the underlying mechanisms involved in endothelium alterations, neuronal death and other events taking place in the retina. In this article, we will provide a complete description of the procedures required for the measurement of retinal vascular parameters in adults and early birth mice at postnatal day (P)17. We will detail the protocols to carry out retinal vascular staining with Isolectin GSA-IB4 in whole mounts for later microscopic visualization. Key steps for image processing with Image J Fiji software are also provided, therefore, the readers will be able to measure vessel density, diameter and tortuosity, vascular branching, as well as avascular and neovascular areas. These tools are highly helpful to evaluate and quantify vascular alterations in both non-proliferative and proliferative retinopathies.
The eyes are nourished by two arterio-venous system: the choroidal vasculature, an external vascular network that irrigates retinal pigmented epithelium and photoreceptors; and the neuro-retinal vasculature that irrigate the ganglion cells layer and the inner nuclear layer of the retina1. The retinal vasculature is an organized network of vessels that deliver nutrients and oxygen to the retinal cells and harvest waste products to ensure proper visual signaling transduction. This vasculature has some distinct features, including: the lack of autonomous innervation, the regulation of vascular tone by intrinsic retinal mechanisms and the possession of a complex retinal-blood barrier2. Therefore, retinal vasculature has been the focus of many researchers who have extensively studied not only vasculogenesis during the development, but also the alterations and the pathological angiogenesis that these vessels undergo in diseases3. The most common vascular changes observed in retinopathies are vessel dilatation, neovascularization, loss of vascular arborization and deformation of the retinal main vessels, which makes them more ziggaggy4,5,6. One or more of the described alterations are the earliest signs to be detected by clinicians. Vascular visualization provides a rapid, non-invasive, and inexpensive screening method7. The extensive study of the alterations observed in the vascular tree will determine whether the retinopathy is non-proliferative or proliferative and the further treatment. The non-proliferative retinopathies can manifest themselves with aberrant vascular morphology, decreased vascular density, acellular capillaries, pericytes death, macular edema, among others. In addition, proliferative retinopathies also develop increased vascular permeability, extracellular remodeling, and the formation of vascular tufts toward the vitreous cavity that easily breakdown or induce retinal detachment8.
Once detected, the retinopathy can be monitored through its vascular changes9,10. The progression of the pathology can be followed through the structural changes of the vessels, which clearly define stages of the disease11. The quantification of vascular alterations in these models allowed to correlate vessel changes and neuronal death and to test pharmacological therapies for patients in different phases of the disease.
In light of the above statements, we consider that the recognition and quantification of vascular alterations are fundamental in retinopathies studies. In this work, we will show how to measure different vascular parameters. To do that, we will employ two animal models. One of them is the Oxygen-induced retinopathy mouse model12, which mimics Retinopathy of Prematurity and some aspects of proliferative Diabetic Retinopathy13,14. In this model, we will measure avascular areas, neovascular areas and the dilatation and tortuosity of main vessels. In our laboratory, a Metabolic Syndrome (MetS) mouse model has been developed, which induces a non-proliferative retinopathy15. Here, we will evaluate vascular density and branching.
C57BL/6J mice were handled according to guidelines of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Experimental procedures were designed and approved by the Institutional Animal Care and Use Committee (CICUAL) of the Faculty of Chemical Sciences, National University of Córdoba (Res. HCD 1216/18).
1. Preparation of buffer solutions and reagents
2. Fluorescent lectin staining
3. Confocal microscopy visualization and microphotograph acquisition
4. Image processing
5. Quantification of avascular areas
6. Quantification of neovascular areas
7. Quantification of vessel diameter
8. Quantification of vessel tortuosity
9. Quantification of vascular branching
10. Quantification of vascular density
As described in the protocol section, from a single fluorescent staining assay you can obtain the vascular morphology and evaluate several parameters of interest quantitatively. The search of a specific alteration will depend on the type of retinopathy studied. In this article, avascular and neovascular areas, tortuosity, and dilatation were evaluated in a mouse model of proliferative retinopathy, whereas the vascular branching and density were analyzed in a MetS mouse model, which induces a non-proliferative retinopathy.
In the first experimental example, the Oxygen-Induced Retinopathy (OIR) mouse model, was employed, which resembles the Retinopathy of Prematurity and some features of the proliferative stage of Diabetic Retinopathy. In this model, litters are maintained with their nursing mother in a hyperoxic chamber from postnatal day (P)7 to P1218. Intravitreal injections were performed at P12 to determine the effectiveness of a drug as an antiangiogenic (condition named as Treatment). Mice were sacrificed at P17, the time point of the maximum neovessels formation19. Mice injected with vehicle were employed as control. Both samples were fixed and stained with Isolectin GSA-IB4 together. After confocal acquisition, images were analyzed with ImageJ FIJI software as indicated above. With the Stitching module integrated to Confocal Microscope, the complete whole mount was observed as a unique image (Figure 1). At P17, it is possible to observe the hyaloid vasculature, a transient vascular system essential during the intrauterine life (Figure 1, white arrows)20.
As a consequence of excessive oxygen provision in the hyperoxic chamber, OIR mice arrest the physiological vascular development and generate avascular areas in the retina. Then, the avascular areas are defined as the zones that lack retinal blood vessels (Figure 2). From the images acquired, avascular areas can be quantified as the sum of the regions without vessels divided by the total area of the retina. Areas with mechanical damage also show absence of vessels. To identify damaged areas, analyze the integrity of neuronal layers with Hoechst stain.
The avascular areas of the retina avoid oxygen delivery and, therefore, a strong pro-angiogenic response is set, inducing neovascularization16. Neovessels are small caliber vessels that originate from a pre-existing vessel of the superior vascular plexus. Their structure is disorganized, therefore, neovessels grow as tufts toward the vitreous cavity21. We calculated the area occupied by neovessels' tufts by quantifying the neovascular area of the retina divided by the total area of the retina (Figure 3). As neovessels' shape and size is variable, occasionally they can look similar to debris and artifacts. To distinguish neovessels, verify that the tuft grows from a mature vessel.
Vascular dilatation and tortuosity are other two frequent alterations, which have negative effects on vascular biology, as they produce turbulent blood circulation. For the analysis of the dilatation, we measured the main vessels diameter at three heights before the first branch22,23 (Figure 4). Researchers must define a predetermined distance to measure vessel diameter in order to reduce variability among results. Ideally, the furthest point from the optic nerve should be around 300 µm. We suggest performing the analysis and later take an average of at least six mice per condition.
Regarding to tortuosity, we measure the distance covered by the main vessels following its shape with respect to the straight distance between the optic nerve and the first branching point24 (Figure 5). As we can see in the image, there is heterogeneity in the sinuosity of the main vessels. To obtain a representative result, not less than six mice per condition must be quantified.
The latest parameters, vascular branching and density, were analyzed in the MetS model exhibiting non-proliferative retinopathy. In MetS, both lipids and carbohydrates derangements are associated to retinal pericytes and endothelial cells dysfunction and death, leading to the formation of acellular capillaries or a decreased vascular branching25. In our model, ApoE-KO mice were fed with fructose added to the drinking water, at a concentration of 10% w/v. Control animals just received tap water. After 4 months of diet, mice were sacrificed, and the retinas processed as indicated before. For the measurement of vascular branching, we defined the quantification area by drawing a concentric circle, and then we counted, one by one, the primary branches arising from a main central vessel (Figure 6).
From the images acquired, the vascular density was quantified as the area occupied by vessels divided by the area of the ROI (a square of 0.015 mm2, see flatten in Figure 7), which was positioned in different places in each microphotograph, as it was explained in the protocol section. Avoid quantifying areas with mechanical damage (Figure 7, white arrows). If there are more than two areas with punctures, the retina must be discarded.
Figure 1: Vascular staining of retinas whole mount labeled with Isolectin GSA-IB4: Representative confocal images of P17 OIR mice retinas injected with vehicle or treatment at P12. Fluorescent staining was performed in whole mount retinas with Isolectin GSA-IB4 Alexa 488 according to the protocol. Avascular areas (AV) and neovascular areas (NV) are indicated. Scale bar: 500 µm. White arrows show the remnant hyaloid vasculature. Yellow arrows show incomplete scanning areas during confocal image acquisition. Please click here to view a larger version of this figure.
Figure 2: Quantification of avascular areas: (A) Representative images of whole mount retinas at P17 showing GSA-IB4 vascular staining in OIR-vehicle and OIR-treatment mice. Areas with vaso-obliteration are indicated in yellow. Scale bar: 500 µm. (B) The AV area (%) was quantified as the ratio of central avascular area to whole retinal area. Two-tailed unpaired t-test was used for statistical comparison. Data presented as mean ± SEM. *** p < 0.001. At least six animals were employed for each condition in the survival time examined. Please click here to view a larger version of this figure.
Figure 3: Quantification of neovascular areas: (A) Representative images of whole mount retinas at P17 showing GSA-IB4 vascular staining in OIR-vehicle and OIR-treatment mice. Areas with neovascularization are indicated in white. Scale bar: 500 µm. (B) The NV area (%) was quantified as the ratio of the area occupied by neovessels to whole retinal area. Two-tailed unpaired t-test was used for statistical comparison. Data presented as mean ± SEM. *** p < 0.001. At least six animals were employed for each condition in the survival time examined. Please click here to view a larger version of this figure.
Figure 4: Quantification of vascular dilatation: (A) Representative images of whole mount retinas at P17 showing GSA-IB4 vascular staining in OIR-vehicle and OIR-treatment mice. Left panels: ROIs selected for quantification. Right panels: zoom of the ROIs, showing straight lines performed in a main vessel to measure the vessel diameter. Three lines were traced transversally to the vessel and averaged. Scale bar: 100 µm. (B) The vessel diameter (%) was quantified as the average diameter measured in major vessels of the retina. Two-tailed unpaired t-test was used for statistical comparison. Data presented as mean ± SEM. *p < 0.05. At least six animals were employed for each condition in the survival time examined. Please click here to view a larger version of this figure.
Figure 5: Determination of tortuosity index: (A) Representative images of whole mount retinas at P17 showing GSA-IB4 vascular staining in OIR-vehicle and OIR-treatment mice. Left panels: ROIs selected for quantification. Center panels: zoom of the ROIs, showing segmented lines traced in a main vessel from the optic nerve to the first branching point. Right panels: zoom of the ROIs, showing straight lines traced in a main vessel from the optic nerve to the first branching point. Scale bar: 100 µm. (B) The tortuosity index was obtained by dividing the distance of the segmented line to the distance of the straight line. Two-tailed unpaired t-test was used for statistical comparison. Data presented as mean ± SEM. ns, non-significant. At least six animals were employed for each condition in the survival time examined. Please click here to view a larger version of this figure.
Figure 6: Quantification of vascular branching: (A) Representative images of whole mount retinas showing Isolectin GSA-IB4 vascular staining in ApoE-KO and ApoE-KO + fructose fed mice. Circular quantification areas were defined in yellow. Scale bar: 100 µm, 100x magnification. (B) The number of branches was quantified as the number of primary branches from a main vessel, since the optic nerve to the periphery. Two-tailed unpaired t-test was used for statistical comparison. Data presented as mean ± SEM. ns, non-significant. At least six animals were employed for each condition. Please click here to view a larger version of this figure.
Figure 7: Quantification of vascular density: (A) Representative images of whole mount retinas showing Isolectin GSA-IB4 vascular staining in ApoE-KO and ApoE-KO + fructose fed mice. Square of 0.015 mm2 quantification area was defined in yellow. Scale bar: 100 µm, 100x magnification. (B) Vascular density was quantified as the ratio of vascular area to total ROI area, which was positioned nine times in different places in each microphotograph. White arrows show areas with mechanical damage. Two-tailed unpaired t-test was used for statistical comparison. Data presented as mean ± SEM. ns, non-significant. At least six animals were employed for each condition. Please click here to view a larger version of this figure.
Animal models of retinopathies are powerful tools for studying vascular development, remodeling, or pathological angiogenesis. The success of these studies in the field relies on the easy access to the tissue that allows to perform a wide variety of techniques, providing data from in vivo and postmortem mice26,27. Moreover, great correlation has been found between in vivo studies and clinical analysis, providing solid traceability and reliability to these models28. In this article, we present a simple and robust description of the steps to characterize the vascular network in mouse models of retinal vascular diseases. In the literature, readers will find other possible parameters to measure and parallel approaches to quantify those selected here. The compiled protocols have many benefits over others because they are reproducible, and they just require a free software to perform the analysis (Image J Fiji).
Moreover, these protocols are easy to be performed by students that do not have extensive knowledge about the program and most of the measurements do not need additional plugins (except from vascular density).
Sample extraction and fluorescence staining technique are simple and brief29. It is very important to work with fresh retinas, although they can be stored at 4 °C in PBS with inhibitors of bacterial growth. As the tissue is thin and soft, then old samples frequently break down during the incubation or when unfolding the sample before mounting. Besides, excessive fixation with 4% PFA turns the retina excessively rigid and brittle, but this does not affect the lectin staining. Isolectin GSA-IB4 staining allows to visualize the complete vascular network at every layer, including neovessels and proliferating endothelial cells. Other markers, as CD 31, requires the incubation of the sample with concentrated antibodies and they have more background. On the other hand, in angiography, the fluorescent dye tends to diffuse out of the vessels, which increases the variability in the vessel caliber and tortuosity quantification. One disadvantage of Isolectin GSA-IB4 is that it can also label microglial cells30. Care should be taken when carrying out intravitreal injections or other experimental procedures that induce excessive infiltration because the microglial cells can form agglomerates.
Image acquisition could be performed with any confocal microscope. The use of a motorized plate couple to a software that includes the Stitching module will provide complete visualization of the whole mounted retina in a single image. When selecting the retinal area, make sure all extremes of the sample are included, otherwise the photo will be incomplete (Figure 1, yellow arrows). This is not very relevant in the mouse model of OIR, as an example, because vascular alterations are near the optic nerve. While in the rat model of OIR, this mistake can lead to inaccurate quantification, as the avascular and neovascular areas are next to the limbus. Another possibility is to take individual retinal microphotographs. The user can later organize them as a puzzle with a proper software. It is not recommendable to use epifluorescence microscope, especially when analyzing branching, because vessels sprout throughout the whole thickness of the retina and some vessels will not be detected in a solely z stack.
During image processing with ImageJ FIJI, it is recommended not to update the software until all the images are quantified, in order to keep identical conditions in the software. The calibration of the images (assignment of pixel size in microns) is a critical step as these protocols imply the quantification of distances and areas. When familiarized with the program, the user can explore other tools to select the neovascular and the avascular area in addition to the wand tool. The polygon selection tool is particularly useful to measure avascular areas that have been cut in two parts at the mounting step. Other researchers have created specific plugins to automate the selection of neovessels based on the fluorescence intensity of these structures. These methods are faster and less laborious, although it is recommended to check that small less-shiny neovessels have been selected prior to quantification31. The areas of neovessels will fluoresce more intensely than the surrounding normal vessels. The Magic Wand tool selects areas that are similarly colored and the tolerance determines how similar in color are the pixels selected. If a neovessel is of similar intensity to normal vessels, the tolerance may be adjusted downward to increase the sensitivity to subtle intensity differences. Although neovessel selection in every sample will be performed with an identical threshold, some neovessels are characterized by a minor fluorescence intensity. In this case, a lower tolerance will be required for proper selection.
Tortuosity and dilatation are measurements of distance. A comprehensive study of the vascular tree is helpful to select vessels of identical type (arteries or veins) and caliber. We measure the dilatation in vessels at three heights as shown in Figure 4. The angle of the straight selection respect to the vessel wall is another relevant point to take into account. This should be as close as possible to 90° to avoid possible errors. When drawing the straight line, users can use the zoom tool to approach the vessel of interest.
Vascular branching can also be measured in two or more quantification areas of different heights, if desired. In this case, researchers should conserve the region of the retina analyzed (central, medium, or peripheral side of the optic nerve). Although branching is altered in several animal models of retinopathy, early stages of the retinopathy associated to MetS presented here still do not show such changes15. For extensive studies in retinal capillaries of the intermediate and deep plexus and vessel sprouting, 3D computational methods provide valuable data of subtle variations in vascular morphology and network32. Finally, the vascular density is determined by the area occupied by vessels with respect to the total area of a ROI selected. The measurement of this parameter requires the use of a plugin and an actualized version of ImageJ FIJI, where Auto Threshold and Geometry to Distance Map tools must be available. Despite these additional steps, the plugin is easy to use, reproducible and reliable. For the measurement of vascular density, a ROI of 0.015 mm2 was chosen, in order to make ten measurements of the fluorescence intensity in different regions of the retina. This size allowed us to cover the entire photo, thus obtaining a more representative average value and its deviation in each retina.
Overall, despite the fact that these collections of methods are not automated and require manual selection of the parameters, they are able to provide quantitative and rigorous data of the retinas. The main limitation regarding the above presented protocols for vascular measurements relies on the inter-examiner variability when analyzing the same image. To minimize this bias, it is important to pre-establish the general setting as vessel wall boundaries, identification of avascular areas and neovessels, and also exclusion criteria for damaged tissues. Regarding vessel tortuosity, an agreed starting point next to the optic nerve is required. For beginner users, it is highly recommended to average the data collected by two or more examiners. In trained users, no significant differences have been found in the measurement of various parameters, as long as agreement in the quantification criteria remain constant. Similarly, additional parameters can be added to the listed protocols, depending on the vascular alterations detected in the retinopathy. In non-proliferative retinopathies, the measurement of acellular capillary number, migrating pericytes, ratio of pericytes / endothelial cells number and blood retinal barrier permeability are the earliest parameters altered in the retinal vasculature33,34,35. For chronic models and mild retinopathies, it is advisable to include these additional measurements. A major number of parameters measured will provide a more faithful landscape of the events taking place in the vasculature. In summary, in this article, we have shown classical, well-established and reproducible techniques to quantify some of the most relevant parameters taken into account in the clinical practice.
The authors have nothing to disclose.
We thank Carlos Mas, María Pilar Crespo, and Cecilia Sampedro of CEMINCO (Centro de Micro y Nanoscopía Córdoba, CONICET-UNC, Córdoba, Argentina) for assistance in confocal microscopy, to Soledad Miró and Victoria Blanco for dedicated animal care and Laura Gatica for histological assistance. We also thank to Victor Diaz (Pro-Secretary of Institutional Communication of FCQ) for the video production and edition and Paul Hobson for his critical reading and language revision of the manuscript.
This article was funded by grants from Secretaría de Ciencia y Tecnología, Universidad Nacional de Córdoba (SECyT-UNC) Consolidar 2018-2021, Fondo para la Investigación Científica y Tecnológica (FONCyT), Proyecto de Investigación en Ciencia y Tecnología (PICT) 2015 N° 1314 (all to M.C.S.).
Aluminuim foil | |||
Bovine Serum Albumin | Merck | A4503 | quality |
Calcium chloride dihydrate | Merck | C3306 | |
Hydrochloric acid | Biopack | 9632.08 | |
Confocal Microscope FV1200 | Olympus | FV1200 | with motorized plate |
Covers | Paul Marienfeld GmnH & Co. | 111520 | |
Dissecting Microscope | NIKON | SMZ645 | |
Disodium-hydrogen-phosphate dihydrate | Merck | 119753 | |
200 µL tube | Merck | Z316121 | |
Filter paper | Merck | WHA5201090 | |
Incubator shaker GyroMini | LabNet International | S0500 | |
Isolectin GS-IB4 From Griffonia simplicifolia, Alexa Fluor 488 Conjugate | Invitrogen | I21411 | |
Poly(vinyl alcohol) (Mowiol 4-88) | Merck | 475904 | |
Paraformaldehyde | Merck | 158127 | |
pHmeter | SANXIN | PHS-3D-03 | |
Potassium chloride | Merck | P9541 | |
Potassium-dihydrogen phosphate | Merck | 1,04,873 | |
Slides | Fisher Scientific | 12-550-15 | |
Sodium chloride | Merck | S3014 | |
Sodium hydroxide | Merck | S5881 | |
Tris | Merck | GE17-1321-01 | |
Triton X-100 | Merck | X100-1GA | |
Vessel Analysis Fiji software | Mai Elfarnawany | https://imagej.net/Vessel_Analysis |