The overall goal of this procedure is to detect abnormalities in the choroidal vasculature in mouse models of age-related macular degeneration using time course endo in green and geography. This is accomplished by first preparing the animals and die for injection. The second step is to perform tail vein injections of incy in green.
Next images of the eye are acquired in the early middle and late phases of incy in green angiography, which are then exported and analyzed. Ultimately, time course incy in green angiography may be used as a standard procedure for characterizing various types of lesions in the choroid of age-related macular degeneration. Mouse models.
The main advantage of this technique, our existing methods, like intraperitoneal injection of endo signin green, is that it provides high quality time course images that are comparable to human studies To begin the procedure, dress in the proper attire for working with animals. This includes putting on surgical gowns, sterile foot covers, hair, bonnets, face masks, and gloves. Then prepare the heating pad to be used during imaging by turning on the system, setting the temperature to 37 degrees Celsius and covering it with a sterile blue pad.
Next, turn on a hot plate and heat a beaker of water to 40 degrees Celsius. Prepare the imaging system prior to the tail vein injection by pre warming the laser and mounting a 55 degree lens onto the machine. Then open the imaging software and input the identifying information of the mouse for imaging under a new patient sheet.
Under device type, choose infrared mode. With the imaging system set up, dilate the mouse's eyes with a 1%tropic IDE ophthalmic solution, and wait five minutes. During this time, weigh the mouse to determine the amount of anesthesia that is required.
Then inject a mixture of ketamine, xylazine, and acepromazine intraperitoneal as described in the accompanying text protocol. And wait about five minutes until the mouse is fully anesthetized. Instead of covering the eyes of the anesthetized mouse with eye ointment, apply sterile PBS to the eyes every two minutes or insert specialized contact lenses to prevent them from drying out and developing cataracts.
Once anesthetized, place the tail of the mouse into 40 degrees Celsius water for 20 to 30 seconds in order to dilate the tail vein. Then use a one milliliter syringe equipped with a 32 gauge needle to draw up two microliters per gram of body weight of a one milligram per milliliter indocyanine green solution. Be careful not to introduce any air bubbles into the syringe.
Next, hold the tail with one hand so that the lateral tail vein is upward and wipe the tail with an alcohol swab to sterilize the area to be injected. Then inject the needle about two millimeters into the vein at a minimal angle with the bevel of the needle facing upward. Draw back on the syringe slightly and look for traces of blood flow into the needle hub, indicating that the needle was successfully inserted into the vein.
Then slowly inject the indocyanine green solution. There should be minimal resistance when injecting following injection. Remove the needle and apply an alcohol swab directly to the injection site for five to 10 seconds to stop any bleeding.
The mouse is then ready for imaging following tail vein injection. Quickly transfer the mouse to the previously prepared imaging system to take images during the early phase of the choroidal filling. First, set the image, focus on the vasculature of interest.
Focus between 35 and 45 diopters for the retinal vasculature and between 10 and 15 diopters for the choroidal vasculature. Then control for brightness using the control module, adjust the values for focus and brightness digitally, and once set, keep them constant for that type of shot. Also, keep the distance from the mouse eye to the camera constant.
To ensure reproducible image quality. Perform the initial screening by imaging the entire posterior of the eye from different angles. Start by aligning the circle shaped luminescence emitted by the indocyanine green dye with the camera's field of view.
To accomplish this, make left to right, up and down and in and out. Adjustments of the camera position until the entire image is in focus and has no dark areas. Record the optimal device settings for repeat procedures.
Once the vasculature is in focus, capture the image frames by pressing the round black button on the acquisition module. This button can also be used to reduce or enhance the signal of indoc and green for best image quality. To examine a particular lesion area based on the previous screening results, set up the imaging system using the optimal angle and device settings previously determined.
Keep the position of the eye view angle, focusing depth and other device settings fixed for the entire time.Course. Save the images by pressing the acquire button on the touchscreen panel of the acquisition module. Continue to acquire images in the early phase until retinal and choroidal circulations are at maximum brightness.
This can take up to four minutes. Then continue to acquire images in the middle phase at six to 15 minutes post-injection. As can be seen here, the choroidal vessels become less distinct in this phase and appear as diffuse fluorescence.
Additionally, choroidal lesions exhibiting hyperfluorescence emerge next acquire images in the late phase at 17 to 25 minutes post injection. During this phase, the hyperfluorescence fades and vessels are no longer visible. Also, the optic nerve head becomes black and the choroidal lesions have maximal contrast with the fading background.
After finishing the acquisition of images, apply a clear lubricant IGEL to the mouse eyes and leave the mouse on a heating pad for recovery. Once they have regained some movement, return mice to their cages and holding area. Finally, export all the images as image files or video files for further analysis.
Shown here is a comparison of sodium fluorescein based fluorescein angiography and endocyt in green angiography in imaging, mouse retinal and choroidal vasculature. While retinal vessels can be seen using both FA and ICGA, choroidal vessels can only be seen with ICGA. Here are images taken of the choroidal vasculature following tail vein injection of endo in green in the wild type mouse.
No dilations or lesions were found at any of the imaging stages in the HT A one transgenic mouse. However, both dilations and cluster type polypoidal lesions were found in the early stage, and several more lesions appeared in the middle phase and became clearer in the late stage to compare between tail vein and intraperitoneal injection of the indocyanine green on imaging quality to additional HRA one transgenic mice were imaged following IP injection. The choroidal vasculature is mostly invisible five minutes after injection for both mice and is invisible more than 12 minutes for the mouse.
In the lower panels, in addition to the root of injection pigment color also influences the quality of images. Here a pigmented and albino mouse were compared using time course IV incy in green, an geography. The albino mouse had much sharper images of both big and small choroidal vessels at the early and middle phases and would provide a good breeding background for further studies in this area After its development.
This technique paved the way for researchers in the field of vision research to explore choroid abnormalities in mouse models.
