The overall goal of this procedure is to measure retinal blood flow in mice. This is accomplished by first infusing fluorescent microspheres through the cannulated femoral vein of an anesthetized mouse. The second step is to video record the microspheres flowing through the mouse's retinal vessels.
Next, infuse fluorescent dextran through the femoral vein and capture video as it flows through the retinal vessels. The final step is to measure microsphere velocities and vessel diameters from the recorded videos for a calculation of retinal blood flow. A primary advantage of this technique is the ability to accurately measure the retina blood flow using a standard fluorescence microscope.
This method can help answer key questions in the retina field, such as the extent to which changes in blood flow contribute to diabetic retinopathy. Although we will be demonstrating this technique in the retina of a mouse. The general method can be used in other tissues and also in rats.
Sonicate a 1%stock solution of 1.9 micron diameter microspheres withdraw 10 microliters of the stock solution into a 300 microliter syringe, then dilute the microspheres by drawing 240 microliters of sterile saline into the syringe. Next, prepare a 50 microliter solution of fluorescent dextran, dissolved in enough sterile saline so that the injected dose will be five milligrams per kilogram. Then cover the dextran with aluminum foil until time for its fusion.
After anesthetizing the animal and testing for adequate anesthesia by a tow pinch, place a heating pad underneath the animal. Keep the eyes moist with phosphate buffered saline. Next, break off the sharp tip of a syringe needle by bending it back and forth with the hemostat.
Then place a 20 centimeter length of polyethylene tubing on the blunted tip of the needle and fill the tubing with the heparinized saline. Now prepare a 25 units per milliliter heparinized saline solution. Fill the syringe with the heparinized saline after shaving one side of the lower abdomen, make a one centimeter incision at the transition between the abdomen and leg to expose the femoral vein.
Then tie off the vessel just proximal to the widening of the femoral vein with a five T suture. Next, make a partial incision of one half the vessel diameter and insert the tubing into the vessel along its axis at a 20 to 30 degree angle above horizontal. Secure the tubing with the suture tied around the cannulated section.
To prepare the animal for microscopy, dilate the pupil of the eye with one drop of 1%tropic aide ophthalmic solution, followed by a drop of 2.5%hyper melos ophthalmic solution. Then cover the eye with a five millimeter glass cover slip. After placing the animal on a plexiglass board, position the board underneath the microscope objective of an upright microscope.
Place surgical gauze underneath the head to allow a direct axis through the objective to the central retina and optic disc. Using the Forex objective and a fluorescein filter, focus on the optic disc, keeping it in the center of the field of view. Next, infuse the fluorescent microsphere solution into the femoral vein at a rate of 250 microliters per minute per kilogram.
Using a camera exposure time of eight milliseconds, video record the viewable retina around the optic disc. Stop the microsphere infusion. After three minutes, some microsphere solution may remain unused.
Infuse the prepared solution of fluorescent dextran into the femoral vein as a bolus over five seconds. To begin measuring the diameters of the retinal vessels, maintain focus on the optic disc with the Forex objective. Then switch to a 10 x objective and 40 milliseconds exposure.
Time to improve resolution video four to seven arterials and four to seven venues in the superficial retina. Focus on one quadrant of the viewable retina at a time keeping the center of the optic disc in one corner of the field of view. Record each quadrant for only 10 to 15 seconds to minimize the possibility of phototoxicity.
Now, analyze the video to determine flow rates and total retinal blood flow. Using a micrometer scale, calibrate the video system for a conversion of microns to pixels With image processing software. Play back the recorded video from the video of the bolus infusion of fluorescent dextran.
Identify arterials and vees the arterials perfuse before the vees. Measure the diameters of each of the arterials and vees filled with fluorescent dextran. Averaging five measures of diameter per vessel along the viewable length.
Then for each of the same arterials and vees, measure the streak length of the fluorescent microspheres using 10 successive microsphere streaks in each vessel to avoid selection bias. Now divide the streak lengths by the exposure time to calculate the microsphere velocity and average the 10 velocities per vessel. Next, calculate the volumetric flow rate for each vessel.
The flow equals the mean velocity times pi times the diameter squared divided by four. Finally sum the arteriolar flow rates and separately the ular flow rates to obtain the total retinal blood flow.Shown. Here are single frames of video showing 1.9 micron fluorescent microspheres appearing as streaks as they move through the retinal vessels.
The middle image shows the orientation of the retinal vessels in the other panels of the figure. Note that some frames have multiple streaks. In this experiment, most of the fluorescent streaks are in the range of 120 to 170 microns in length, which corresponds to velocities of 1.5 to 2.1 centimeters per second.
These next images are from the bolus infusion of fluorescent dextran. In the first frames, arterials are seen filling with dextran. The vees fill after the dye makes its transit through the capillary beds.
Shown here is one of the 10 x images used to measure vessel diameters. The arteriolar diameters ranged from 47 to 55 microns and the venal diameters ranged from 50 to 65 microns. This figure shows flow measurements from a previous experiment in which both fluorescent microspheres and fluorescent red blood cells were injected into the same mouse to determine if similar vessel velocities and flows would be found.
As this figure indicates, the measured velocities and flows were very similar Once mastered, this technique can be done in less than one hour if it is performed properly. While performing this procedure, it is important to remember to reduce the fluorescent excitation of the dyes by limiting the time the microscope shutter is open. After watching this video, you should have a good understanding of how fluorescent dyes can be used to measure retinal blood flow in mice.
