The protocol provides a model to investigate mechanisms of vascular injury and subsequent development of edema in a non-invasive manner. The main advantage of this technique is that you can follow the injury live over time without surgical intervention. This provides avenues to test treatments more efficiently.
It can be challenging to learn how to perform proper tail veining and to accommodate the animal in the platform correctly. For both techniques I recommend being patient and taking things slowly. To begin, gently handle the fiber optic cable and connect it to the laser control box and the laser adaptor of the retinal imaging microscope, then turn on the retinal imaging microscope lamp box.
Turn on the computer and open the imaging program. Place a piece of white paper in front of the microscope eyepiece and adjust the white balance by clicking on Adjust in the imaging program. Turn on the laser control box by turning the key and following the instructions on the screen of the laser control box.
To verify the baseline laser power, set the laser control box to 50 milliwatts and 2, 000 milliseconds. Then turn on the laser and place the power meter in front of the eyepiece. Press the foot switch pedal to activate the laser, aiming for the laser power readout to be 13 to 15 milliwatts.
Then, using the laser control box screen, adjust the experimental laser power to 100 milliwatts and 1, 000 milliseconds, and turn off the laser. Pour 300 milliliters of water into a 500-milliliter beaker and warm the beaker in a microwave oven for one minute. Then place a gauze in the warm water.
Next place a two-month-old male mouse in a restrainer. Press the warm gauze into the mouse tail gently and look for the dilated veins. Using a 26-gauge needle, inject the appropriate amount of rose bengal according to the animal's weight and apply pressure on the injection site to prevent hematoma or bleeding before wiping the site.
Then release the mouse from the restrainer and return it to the cage. Allow eight minutes for the rose bengal to circulate before the injection of anesthetics. Turn on the heated mouse platform.
Then add one drop of phenylephrine and tropicamide in each eye of the mouse. After confirming anesthesia, add one drop of proparacaine hydrochloride per eye, then add eye ointment to both eyes. Next, inject 150 microliters of carprofen subcutaneously between the ears.
Then accommodate the mouse on the platform and adjust the platform until the view of the retinal fundus is clear and focused. Count the retinal veins and take an image of the fundus. Next, turn on the laser and aim towards a retinal vein approximately 375 micrometers from the optic disc.
Irradiate the vessel by pressing the foot switch and slightly moving the laser beam up to 100 micrometers. Repeat this step three times and move the laser beam after each pulse so that the irradiation is not focused in one spot. Repeat the irradiation on other major vessels to achieve two to three occlusions.
After irradiating the vessels, turn off the lamp and wait for 10 minutes. Then turn the lamp back on, count the number of veins occluded, and take an image of the fundus. After image acquisition and saline injection, add lubricant eyedrops and gel ointment to both eyes.
Watch the mouse as it recovers from anesthesia and return it to the cage with other animals only when fully recovered. The number of occlusions achieved immediately after laser irradiation was not different between animals that were lasered 10 and 20 minutes after rose bengal administration. The number of occlusions sustained up to one day after retinal vein occlusion significantly decreased in animals lasered 20 minutes after rose bengal administration independently of genotype.
Without modifying the experimental power of 100 milliwatts, a low baseline laser power of 11.5 milliwatts resulted in no occlusions. In contrast, a baseline laser output of 13.5 milliwatts resulted in successful occlusions. Four main types of occlusions occur after laser photocoagulation:fully occluded vessels, partially occluded vessels, partially reperfused, and fully reperfused vessels.
The irradiated vasculature of Tamoxifen-inducible endothelial cell Casp9 knockout mice spent more time in partially reperfused and partially occluded states than that of C57 Black 6 mice, which spent more time in fully occluded states. The occlusion state of the vessels changes rapidly within the first 10 minutes after laser photocoagulation and flame-shaped hemorrhages are observed 24 hours post-injury. Different ophthalmic pathologies occurred after retinal vein occlusion.
The level of retinal edema after retinal vein occlusion can be quantified in the injured eyes using optical coherence tomography images. The state of neuronal layers can also be determined by assessing the disorganization of retinal inner layers. An example of an optical coherence tomography image with the corresponding labels for each layer is shown here.
Measuring the baseline laser power is important. This readout will greatly impact the success of the occlusions and can be optimized. Additional methods such as OCT, ERG, and Optiger can be performed.
This will help address the effects of neurovascular injury on retinal neuronal integrity and visual pathway function. This technique paves the way to interrogate the molecular pathways driving neurovascular disease and, by following individual animals over time, provides data which can be more readily translated to human disease.
