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

In retinopathies, vascular alterations are the first sign to be detected by clinicians. They follow the progression of the disease, and they choose a treatment according to these changes. Several animal models have been developed in order to study different stages of the pathology.

In this work, we are going to show how to measure different vascular alterations. For that, we are going to employ two animal models. One of these is the Oxygen-Induced Retinopathy mouse model, which reproduces the retinopathy of prematurity and some aspects of the proliferative diabetic retinopathy.

Here, we are going to measure avascular areas, neovascular areas, and the dilatation and tortuosity of the arterioles. In our laboratory, a metabolic syndrome mouse model was developed, which induces a nonproliferative retinopathy. Vascular density and branching were evaluated in this work.

Upon mice sacrifice, enucleatize with scissors. Fix enucleated eyes with freshly-prepared paraformaldehyde for one hour at room temperature. 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. Place the retinas in 200 microliter tubes. Then block and permeabilize the retinas in 100 microliters of TBS containing 5%bovine serum albumin Triton during six hours at four degrees with gentle agitation in shaker.

Then invert the tube to place the retina in the cup and remove the blocking solution with pipette. Add 100 microliter solution containing isolectin. Wrap the tubes with aluminum foil to protect samples from light.

Incubate the retinas with lectin solution overnight at four degrees with gentle agitation in shaker. Wash the retinas with 100 microliters of TBS-Triton for 20 minutes with gentle agitation in shaker. Repeat this step three times.

Place the retina in a slide and add a drop of PBS. Under dissecting microscope, perform four equally-distant cuts from the edges of the retina towards the optic nerve. To unfold the petals of the retina, use forceps or small pieces of filter paper.

Ensure that photoreceptor side is facing down. Remove the remaining PBS off the slide with filter paper. Then add a mounting median and coverslip.

Let it dry for one hour at room temperature. Store slides at four degrees protected from light. At the confocal microscope, place the slide in the plate face down.

Focus the superior plexus of the retinal vasculature and select an area to start the image acquisition. Set general laser parameters in the software. Set the coordinates in X, Y, and Z axes.

Initialize the scanning. Once scanning process finishes, open the image and save it with a preferred extension. In the ImageJ FIJI software, go to the Menu Bar and click File, Open.

Select the image to process. In the Bio-Formats Import Option window, select Display OME-XML Metadata and click OK.In the OME Metadata window, search for pixel size information. Copy this data.

For image calibration, go to the Menu Bar and select Image Properties. Copy the information and click OK.Assign a preferred lut to the image. To visualize all stack in a single image, go to the Menu Bar and select Image, Stacks, Z Project.

In the projection window displayed, select all the stacks where vessels are visualized. In Projection Type, choose Average Intensity and click OK.Go to the Menu Bar, Image, Adjust, Brightness/Contrast. Click Apply and save changes.

Copy the parameters selected for brightness and contrast. These must be identical for all images. In the Menu Bar, choose the Wand tool.

Select the retinal area that lacks formed vessels. Press T on the keyboard to record the select areas in the ROI Manager. Click Measure in the ROI Manager to obtain the avascular area information.

Repeat these steps to measure the total area of the retina. In the Menu Bar, choose the Wand tool. Zoom in the image to better visualize neovessels.

Select the neovessels one by one with the Wand tool. Press the T letter on the keyboard to record the selected area in the ROI Manager. Click Measure in the ROI Manager to obtain the area data.

In the Menu Bar, select the Straight Line tool. Zoom in the image to better visualize the vessel wall. Perform three transversal lines to the main vessel before the first branch.

Press T on the keyboard to record the selected areas in the ROI Manager. Click Measure in the ROI Manager to obtain the distance data. In the Menu Bar, click the Straight Line tool icon with the right button of the mouse and select Segmented Line.

Draw a line inside the vessel from the optic nerve until the first vessel ramification, following the vascular shape. Press the T letter on the keyboard to record the selected area in the ROI Manager. In the Menu Bar, select the Straight Line tool.

Draw a line from the optic nerve until the first vessel ramification. Press the T letter on the keyboard to record the selected area in the ROI Manager. Click Measure on the ROI Manager to obtain the distances data.

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. Press the T letter on the keyboard to record the selected area in the ROI Manager.

Manually, count the number of primary branches arising from main vessels inside the selection area. Transform the image to 8-bit mode. Go to Plugins in the Menu Bar, select Vessel Analysis, Vascular Density.

Define an area with the Square tool of the Menu Bar where you want to measure vessel density. Click OK.The program will display a new window with the information required. In the first experimental example, the Oxygen-Induced Retinopathy mouse model was employed.

Intravitreal injections were performed at postnatal day 12 to determine the effectiveness of a drug as an antiangiogenic. Mice injected with vehicle were employed as control. The avascular areas are defined as the zones that lacks retinal blood vessels.

From the images acquired, avascular areas can be quantified as the sum of the regions without vessels divided 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.

Neovessels are small caliber vessels that originate from pre-existing vessels of the superior vascular plexus. We calculated the area occupied by neovessels'tufts by quantifying the neovascular area of the retina occupied by the total area of the retina. 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. For the analysis of the dilatation, we measured the main vessel diameter at three heights before the first branch. 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 micrometers. We suggest to perform the analysis, and later, average of at least six animals per condition. Regarding to tortuosity, we measure the distance traveled by the main vessel following its shape, respect to the straight-line distance between the optic nerve and the first branching point.

As we can see in the image, there is heterogeneity in the sinuosity of 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 a metabolic syndrome model exhibiting non-proliferative retinopathy.

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. From the images acquired, the vascular density was quantified as the area occupied by vessels divided by the area of the ROI, which was positioned in different places in each microphotograph. Avoid quantifying areas with mechanical damage.

If there are more than two areas with punctures, the retina must be discarded. In summary, in this article, we have shown classical, well-established and reproducible techniques to quantify some of the most relevant parameters taking account in the clinical practice.