Quantification of Vascular Parameters in Whole Mount Retinas of Mice with Non-Proliferative and Proliferative Retinopathies

Summary

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.

Abstract

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.

Introduction

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 ziggaggy^4,5,6. 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 changes^9,10. 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 Retinopathy^13,14. 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.

Protocol

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

1. Preparation of 1x phosphate buffer saline (PBS): Add 8 g of sodium chloride (NaCl), 0.2 g of potassium chloride (KCl), 14.4 g of disodium-hydrogen-phosphate dihydrate (Na₂HPO₄ 2H₂O) and 0.24 g of potassium-dihydrogen phosphate (KH₂PO₄) to 800 mL of distilled water. Stir the solution until complete dissolution of the salts. Adjust the pH to 7.4 with chloride acid (HCl) and adjust the volume to 1 L with additional distilled water. Filter the solution and store it at 4 °C.
2. Preparation of 4% paraformaldehyde (PFA): Add 40 g of solid PFA to 800 mL of freshly prepared PBS. Stir the solution in a heating plate at a constant temperature of 60 °C. Add 1 M sodium hydroxide (NaOH) until PFA is completely dissolved, checking that the pH is maintained at a range of 7.2-7.4. Top up the volume to 1,000 mL with PBS and mix. Turn off the heating plate and filter the solution when its temperature is under 35 °C. Store the solution at 4 °C for up to 3 days.
   CAUTION: PFA is harmful if swallowed or if inhaled and is probably a mutagenic compound. Use gloves and face mask during the whole process and work in a safety cabin.
3. Preparation of 1x Tris-buffered saline (TBS): Add 6.1 g of Tris and 9 g of NaCl to 800 mL of distilled water. Stir the solution until complete dissolution of the salts. Adjust pH to 7.4 with HCl and adjust the volume to 1 L with additional distilled water. Filter the solution and store it at 4 °C.
4. Preparation of TBS- 0.1% Triton X-100: Add 0.1 mL of TritonX-100 to 100 mL of TBS. Mix gently and store it at 4 °C for up to 1 week.
   NOTE: As Triton is a very dense detergent, slightly cut the terminal of the tip for better pipetting.
5. Preparation of TBS-5%-Bovine Serum Albumin (BSA) -0.1% Triton-X-100: Weigh 5 g of BSA and add TBS- 0.1% triton X-100 solution up to a final volume of 100 mL. Stir gently. Aliquot and store at -20 °C for up to 2 months.
6. Isolectin GSA-IB4 Alexa fluor-488 conjugate (Isolectin GSA-IB4): Weight 36.75 mg of calcium chloride dihydrate (CaCl₂·2H₂O) and add it to 500 mL of freshly prepared PBS. Stir the solution with a stir bar. Pipette 500 µL of the solution of CaCl₂/PBS and add it to the vial containing 500 µg of lyophilized Isolectin GSA-IB4 powder. Mix gently and aliquot the solution in 0.5 mL tubes. Store them at -20 °C protected from light.
7. Poly(vinyl alcohol) in glycerol/Tris: Weigh 2.4 g of Poly(vinyl alcohol), add 6 g of glycerol and stir gently. Then, add 6 mL of water and continue stirring for several hours at room temperature. Add 12 mL of pre-warmed 0.2 M Tris solution (pH: 8.5) and heat at a constant temperature of 50 °C for 10 min and mix occasionally. Finally, centrifuge the solution at 5,000 x g for 15 min for clarification.
   ​NOTE: Let the solution cool at room temperature, aliquot it and store it at -20 °C.

2. Fluorescent lectin staining

1. Upon mice sacrifice by carbon dioxide (CO₂) inhalation, enucleate the eyes with scissors¹⁶. Fix the enucleated eyes with freshly prepared 4% PFA for 1 h at room temperature (RT).
   NOTE: Fixation can also be performed by incubating the eyes in 4% PFA overnight (ON) at 4 °C. Mice were sacrificed according to the guidelines of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the Institutional Animal Care and Use Committee (CICUAL) of the Faculty of Chemical Sciences, National University of Córdoba (Res. HCD 1216/18).
2. Under the dissecting microscope, remove the corneas with scissors by cutting along the limbus and dissect the whole retinas. Discard the anterior segment of the eye, and then separate the retina from the RPE-Choroid.
   NOTE: Make sure to remove the remaining debris and hyaloid vessels out of the retina with forceps.
3. Place the retinas in 200 µL tubes. Then, block and permeabilize the retinas in 100 µL of Tris-buffered saline (TBS) solution containing 5% Bovine Serum Albumin (BSA) and 0.1% Triton-X-100 during 6 h at 4 °C with gentle agitation in the shaker.
   NOTE: This step can be performed for 2 h at RT.
4. Then, invert the tube to place the retina in the cap and remove the blocking solution with a pipette.
5. Add 100 µL of the solution containing 0.01 µg/µL of Isolectin GSA-IB4 (GSA-IB4). Wrap the tubes with aluminum foil to protect the samples from light. Incubate the retinas with the lectin solution ON at 4 °C or for 2 h at RT with gentle agitation in the shaker.
6. Wash the retina with 100 µL of TBS containing 0.1% Triton-X-100 for 20 min with gentle agitation in the shaker. For proper washing, place the retina in the cap of the tube by inverting it. Return the tube to its standing position and remove the medium remaining in the container. Repeat this step three times.
7. Place the retina in a slide and add a drop of PBS. Under the dissecting microscope, perform four equally distant cuts from the edges of the retina toward the optic nerve.
8. To unfold the petals of the retina, use forceps or small pieces of filter paper. Ensure that the photoreceptor side is facing down.
9. Remove the remaining PBS of the slide with filter paper. Then, add a mounting medium (Poly(vinyl alcohol) in glycerol/Tris) and coverslip. Let it dry for 1 h at RT.
10. Store slides at 4 °C protected from light.
    ​NOTE: Retinas can also be stored in PBS at 4 °C until confocal microscopy visualization.

3. Confocal microscopy visualization and microphotograph acquisition

1. Before visualization, incubate the slides for 10 min at RT covered from light.
2. At the confocal microscope, place the slide in the plate face down.
3. Focus the superior plexus of the retinal vasculature and select an area to start the image acquisition.
4. Set general laser parameters in the software: Wavelength: 488 nm; Laser intensity; HV; Offset; Gain.
   NOTE: Laser intensity, PMT, Offset, and gain may vary depending on the conditions and the usage of the lamp. The optimal settings provide a clear image of the vessels avoiding the saturation in the sensitivity of the photomultiplier tube to have a linear relationship with the fluorescence signal.
5. Set other acquisition parameters: Mode; Size; Objective, without zoom; No Kalman; Step Size.
   NOTE: Identical acquisition conditions must be used for imaging the control and experimental samples.
6. Set the coordinates in x and y axis: Scan the retina in focus 2x. If the area shows vessels, select it by clicking twice the area in order to mark it for later image acquisition.
   NOTE: It is highly recommendable to select retinal area by following the superior vascular plexus as the fluorescence of bigger vessels is higher.
7. Move in the grid to a neighbor square, focus the retina, and select the area if it corresponds. Repeat this step until the selected area comprises the whole retina.
8. Select one area and set the coordinates in z axis to define the thickness extent to scan. To do this, visualize the retina in focus 2x; with the micrometer, go to the top of the retina and click on Set at the Start position. Then, move to the bottom of the retina and click on Set at the End position.
9. Repeat step 3.8 at every area selected in the grid.
   NOTE: To verify the scanned area in depth, press Go at the Start position. If vessels are still visualized, readjust the position by moving the micrometer, and then click on Set. Repeat the process at the End position.
10. Initialize automated scanning.
11. Once the scanning process finishes, open the image.
12. Select the Series format to record the image as a Mosaic and save it with the preferred extension. Finally, the stitched image will be shown on the screen.

4. Image processing

1. In the ImageJ FIJI software, go to the Menu Bar and click on File > Open. Select the image to process. In the Bio-Formats Import Options window, select the Display OME-XML metadata and click on OK.
   NOTE: Images with similar name should be saved in separate folders.
2. In the OME Metadata window, search for the pixel size information, named as PhysicalSizeX, PhysicalSizeY, and PhysicalSizeZ. Copy this data.
3. For image calibration, go to the Menu Bar and select Image > Properties. Copy the information of the image in step 4.2 and click on OK. The image opens as a window with several photos, where each one represents the microphotographs acquired at a z axis.
4. Assign a preferred lut to the image in order to consign a color to the fluorescent label. To do that, go to the Menu Bar and click on the LUT icon.
5. To visualize all stacks in a single image, go to the Menu Bar and select Image > Stacks > Z Project.
6. In the Z Projection window displayed, select all the stacks where vessels are visualized. In Projection Type, choose Average Intensity and click on OK.
   NOTE: The result of the sum of the stacks will be shown in a new image named AVG (image name).
7. Adjust the brightness and contrast in the resulting image to reduce the background and highlight vessels. Go to the Menu Bar and click on Image> Adjust> Brightness/Contrast. In the B and C windows move the bars until a better intensity is observed. Click on Apply and save the changes.
8. Copy the parameters selected for Brightness/Contrast; these must be identical for all images.
9. Prior to image quantification, define the parameters that are going to be measured. In the Menu Bar, go to Analyze > Set Measurements. In the procedures described here, it will be necessary to obtain Area and Perimeter (this is ultimately also needed to measure distances). Click on OK.
10. Download the plugin required for vessel density quantification¹⁷ and follow the installations instructions provided by the plugin.
11. Continue with the quantification of a specific vascular parameter.

5. Quantification of avascular areas

1. In the Menu Bar, choose the Wand Tool. Select the retinal area that lacks vessels.
   NOTE: Bigger or smaller areas can be selected depending on the tolerance. To adjust the tolerance, click on the wand tool icon twice. Mode employed: Legacy.
2. Press the T letter on the keyboard to record the selected area in the ROI Manager. Repeat steps 5.1 and 5.2 with all the avascular areas of the retina.
3. Click on Measure in the ROI manager to obtain the avascular area information. The program will display a new window with the information required. Copy the data in another program or save the file.
   NOTE: In some images, one may prefer to select the avascular areas manually. In this case, choose the Polygon Selection tool, draw short distances around the avascular area, and click on the image when a new distance is about to start. Close the selected area by clicking on the first point.
4. Repeat steps 5.1, 5.2, and 5.3 to measure the total area of the retina.
5. Calculate the avascular area of the retina as the sum of all the avascular areas measured divided by the total area of the retina.

6. Quantification of neovascular areas

1. In the Menu Bar, choose the Wand tool.
2. Zoom in the image to better visualize neovessels. Select the neovessels one by one with the wand tool.
   NOTE: Adjust the tolerance no higher than 20 to ensure the selection of a single neovessel. This tolerance level must remain constant during all image quantifications.
3. Press the T letter on the keyboard to record the selected area in the ROI Manager. Repeat steps 6.1 and 6.2 with all neovascular areas of the retina.
4. Click on Measure in the ROI manager to obtain the area data. The program will display a new window with the information required. Copy the data in another program or save the file.
5. Repeat steps 6.1, 6.2, and 6.3 to quantify the total area of the retina.
6. Calculate the neovascular area of the retina as the sum of all the neovessels' areas measured divided by the total area of the retina.

7. Quantification of vessel diameter

1. In the Menu Bar, select the Straight-Line Tool.
2. Zoom in the image to better visualize the vessel wall. Perform three transversal lines to the main vessel before the first branch. This must be done by drawing a line from the boundary of one wall of the vessel to the opposite.
   NOTE: The line must be perfectly perpendicular to the wall vessel to avoid quantification errors.
3. Press the T letter on the keyboard to record the selected area in the ROI Manager. Repeat steps 7.1 and 7.2 in all the vessels that need to be quantified.
4. Click on Measure in the ROI manager to obtain the distance data. The program will display a new window with the information required. Copy the data in another program or save the file.

8. Quantification of vessel tortuosity

1. In the Menu Bar, click on the Straight-Line Tool icon with the right button of the mouse and select Segmented Line or Freehand Line. Draw a line inside the vessel from the optic nerve until the first vessel ramification following the vascular shape.
   NOTE: The line must follow the vascular shape to avoid quantification errors.
2. Press the T letter on the keyboard to record the selected area in the ROI Manager.
3. In the Menu Bar, select the Straight-Line Tool. Draw a line from the optic nerve until the first vessel ramification.
4. Press the T letter on the keyboard to record the selected area in the ROI Manager.
5. Click on Measure in the ROI Manager to obtain the distances data. The program will display a new window with the information required. Copy the data in another program or save the file.
6. Calculate the tortuosity index as follows: distance obtained with Segmented line divided by the distance obtained with Straight line.

9. Quantification of vascular branching

1. With the Oval Tool of the Menu Bar, define an area applied equally to all experimental conditions. The selected area must be a concentric circle drawn around the optic nerve.
2. Press the T letter on the keyboard to record the selected area in the ROI Manager.
   NOTE: In this case, it is recommendable to save the ROI as the quantification is slow and the identical ROI must be used in all images. To do this, in the ROI Manager click on MORE > Save.
3. Manually, count the number of primary branches arising from main vessels inside the selection area.
4. Repeat steps 9.2 and 9.3 in neighbor areas, maintaining the optic nerve-periphery criteria.

10. Quantification of vascular density

1. Transform the image to 8-bit mode.
2. Go to Plugins in the Menu Bar, select Vessel Analysis > Vascular Density.
3. Define an area with the Square tool of the Menu Bar, where the vessel density needs to be measured. Click on OK.
4. The program will display a new window with the information required. Copy the data in another program or save the file.
5. Repeat steps 10.2, 10.3, and 10.4 in the neighboring areas, maintaining the ROI but placing it nine times encompassing the periphery and the center of the whole retina.

Representative Results

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 branch^22,23 (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.

Discussion

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 mice^26,27. 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 vasculature^33,34,35. 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.

Materials

Name	Company	Catalog Number	Comments
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