Preparation and Analysis of Histological Slides of Rat and Mouse Eyeballs to Evaluate the Retina

Our research focused on developing a simple step-by-step methodology for preparing histological slides and assessing the rodent retina. The results of a histological examination of the retina can be used for research on many of ophthalmic diseases, including neurodegenerative and vascular disorders. In the published literature, the description of the methodology for a histological examination of rodent eyeballs are very limited.

Step-by-step guides are lacking, which makes it difficult for beginners to recreate. We believe that this protocol will be useful for other researchers. Even though there are other methods that can be used to assess the morphology of the retina, including optical coherence tomography, the histopathological examination allows for the assessment of single cells and provides fewer artifacts.

Thus, it can prove to be more useful for researching the pathomechanisms of ophthalmic diseases. To begin, remove the paraformaldehyde and sucrose-fixed rat eyeballs from the freezer. Cut the eyeball in half along the sagittal plane with a sharp scalpel.

Then remove and discard the lens. Thaw the eyeball at room temperature. Move the two halves into a cassette, and rinse under running water for 20 minutes to wash out the sucrose.

Dehydrate the eyeball in increasing ethanol concentration for 15 minutes each under a fume hood, with final dehydration in acetone for seven minutes. To clear the eyeball, place it in xylene for seven minutes under a fume hood. Add some solid paraffin into a beaker, and place it into an incubator set to a temperature higher than the melting temperature specified by the paraffin manufacturer.

After the paraffin is fully melted, move the eyeball into the beaker and leave the beaker in the incubator for one hour. Stir the beaker every 10 to 15 minutes during incubation for paraffin infiltration into the tissue. Next, open the cassette and remove the top cover.

Choose a metal mold, and pour a small amount of paraffin to cover the bottom of the mold. Then remove the eyeball from the cassette with forceps, and place one eyeball half with the bottom of the cup up and the other down onto the paraffin in the mold. When the tissue is anchored in place, carefully cover the eyeball with paraffin and the cassette bottom to form a block.

Transfer the block on a cooling plate to fully solidify. After solidification, remove the block from the metal mold and place the paraffin blocks back onto the cooling plate for 15 minutes. Secure a paraffin block in the microtome.

Set the microtome to trim the excess paraffin from the block, and trim until the center of the eyeball is reached. After changing the microtome settings, cut thinner sections of 3.5 micrometers. Place the cut section onto a slide.

Put the slide into an incubator for 15 minutes for the paraffin to melt. Then place the slide in xylene. Following this, transfer the slide in 96%ethanol.

After ethanol treatment, wash the slide in running water for five minutes. Place the slide for two minutes in Mayer's hematoxylin followed by two minutes in Mayer's hematoxylin with a clean solution. Wash the slide twice with running water for five minutes each.

Add 1%eosin for two minutes, followed by ethanol for one minute and 30 seconds each. Put the slide for two minutes in xylene, followed by one minute xylene with another beaker with a clean solution. Seal the slide using an automated slide sealer.

To begin, on a computer, open the high-resolution scanned rat eyeball image and find the cross pupil cross sections. Magnify the selected cross section by scrolling with the mouse. Then rotate the dot in the right-hand corner to adjust the image orientation so the retina is horizontal.

Click on the magnification box in the top right-hand corner and select 20 times to find the thickest part of the retina. Next, right-click on the mouse to choose Annotate, and Ruler. Position the mouse cursor at the external limiting membrane in the thickest part of the retina, and click the left button to start the measurement.

Then move the cursor perpendicularly to the internal limiting membrane, and click the left button again to complete the measurement. Title the measurement as Retinal thickness, and tick the boxes to show the title and length. Next, using a ruler, measure the thickness of each retinal layer, including the outer nuclear layer, outer plexiform layer, inner nuclear layer, and inner plexiform layer, and label them accordingly.

To count the number of cells within the ganglion cell layer, select one section of the retina, and using the ruler, measure the chosen distance along the ganglion cell layer. To mark the cell bodies, press the right-hand button on the mouse and select Annotate, Saved, and 1 x RBC. Click on each cell identified as a round, nucleated hematin-stained body along the ganglion cell layer over the predetermined length of the retina.

Count the number of encircled cells, The retinal thickness, the thickness of individual layers, and the number of cells in the ganglion cell layer were measured and compared across individuals to assess the features of neurodegeneration in the rodent retina.