Fluorescent Dye Labeling of Erythrocytes and Leukocytes for Studying the Flow Dynamics in Mouse Retinal Circulation

The overall goal of this procedure is to visualize retinal circulation of blood cells in the mouse using a scanning laser ophthalmoscope. This method can help answer key questions about retinal diseases such as what is the retinal floor dynamics of WBCs and RBCs and how can their flow dynamics contribute to disease pathogenesis. The main advantages of this technique is that it can refelct on different disease phenotypes and it is also very easy to perform.

This procedure will be demonstrated by our team members, Praveen, Bo Bo, and Neha. To isolate the mouse blood cells layer freshly collected whole blood on top of a 1:1 polysucrose and sodium diatrizoatem solution in a two milliliter tube. Then centrifuge the tube for 30 minutes.

Next aspirate and discard the plasma supernatant. Then carefully collect the buffy coat layer composed of leukocytes and the underlying layer of erythrocytes in separate tubes. Next wash the erythrocytes with 1XPBS to remove any contaminants and resuspend them in one volume of PBS.

Then transfer half milliliter aliquots of the suspension into microcentrifuge tubes. Spin down the aliquots, discard the supernatant and add about 200 microliters of 40%1XPBS to the pelleted erythrocytes. Mix them well.

And incubate them at room temperature for five minutes. After five minutes, add 100 microliters of ICG to each suspension. Mix the tubes using inversions and then incubate them at room temperature for five minutes.

Next add 300 microliters of 1XPBS and incubate for an hour with gentle agitation at 37 degree Celsius. Then centrifuge the suspensions, keep the pelleted cells and resuspend them in one milliliter of 1XPBS. Keep washing the cells with 1XPBS until the supernatant is clear.

After decanting the supernatant add back one volume of 1%BSA and PBS to achieve 50%hematocrit of ICG labeled erythrocytes. Next process the buffy coat collection. First wash the cells once with 10 milliliters of 1XPBS to remove any contaminants.

Centrifuge the samples again and then resuspend the pellet in 900 microliters of 1XPBS. Next add 100 microliters of 10%sodium fluorescein and incubate the cells at room temperature for two minutes. Finally wash the labeled lymphocytes three times with 10 milliliters of 1XPBS.

Centrifuge and resuspend them in 100 microliters of 1XPBS. To prepare the ICG labeled erythrocytes for injection first dilute an aliquot for injection down to a 5%or 1%hematocrit. Anesthetize the mouse and dilate its pupils.

Then apply ophthalmic ointment to both eyes. And place a contact lens over one of the eyes. Now image the mouse using a confocal SLO camera with a positive 25 diopter lens to compensate for the eye's refraction.

Fresh perfectly direct the cornea to the optical head of the SLO machine. Next switch on the imaging module and to maneuver it until the optic nerve is at the center of the imaging screen. Then at a 30 degree angle take the infrared fundus images.

Next take an infrared fundus video. Turn on the 790 nanometer ICG filter set the camera mode to high speed addition keep the view angle at 15 to 30 degrees. Then set the ICG intensity to 85 for all readings.

On the control panel, click on the acquire button to take a baseline video at 8.8 to 15 frames per second for one minute. Next load 100 microliters of ICG labeled erythrocytes into an insulin syringe with a 30 gauge needle and inject the cells into the tail vein or inject them retroorbitally. Immediately after the injection, take a new infrared fundus image and a new ICG filter video.

After acquiring the videos export them in TIFF format for image analysis. Process the images using the freely available imaging software package, MHJ. After opening the file, set the frame interval of the image video for velocity measurements.

Then open the MtrackJ plugin. From the menu, select tracking and toggle the option for moving to next timeframe index after adding a point. Now locate a cell of interest and track through each frame.

From the menu, click add to add a new track and then click on the chosen cell. Then continue clicking on the current position of the cell as images advance from frame to frame. Once tracked change the display option for the trajectory from the displaying choices and choose to display only the track points at the current time.

Continue clicking until the trajectory of the cell is displayed and then save the track. Now proceed with tracking the next cell starting with the add command and proceeding as with the first cell. After tracking all the cells of interest open the display of all the track measurements using the measure option.

Then save the output for further analysis. Erythrocytes labeled with 1.5%ICG were visualized in the retinal circulation of C57 black 6J mice using 1%or 5%hematocrit, cells were distinguishable but in the 1%hematocrit the labeled cells were more easily visualized. With either injected concentration some erythrostasis was observable in the parapapillary region and it was more visible with the 5%hematocrit.

Leukocytes were labeled with 1%sodium fluorescein and visualized as green labeled cells using fluorescence microscopy. Some clumping was observed. Once mastered, this technique can be done in six hours if performed properly.

While attempting this procedure, it is important to dilate the eyes to acquire quality retinal images and videos.