The overall goal of this procedure is to recreate the hallmarks of neovascular age-related macular degeneration or a MD in an easy to study animal model. This method can be used to study different facets of neovascular A MD research, including how genetic alterations or pharmaceutical treatments can affect the disease or how the disease can manifest under different imaging modalities. The main advantage of this technique is that once mastered, it is easy to reproduce, allowing experiments to be done quickly and efficiently.
Generally, individuals new to this method will struggle because they lack the fine dexterity and coordination to be successful, both of which improve with practice. To prepare the laser station position, the laser and slit lamp where it can be easily accessed, turn on the laser and set it to the predetermined parameters. Here, the setting 75 micrometer, spot size, 100 milliwatt power, and 100 millisecond duration are used.
Next place the anesthesia animal warmer tissue wipes and eyedrops with an easy reach of the laser operator. After anesthetizing the first mouse, place it onto the animal warmer for approximately three to five minutes. Perform a toe pinch to ensure complete anesthetization.
Then using a rolled tissue wipe to prevent nasal aspiration of liquid roll-off. Place a 30 microliter drop of tetracaine hydrochloride into each eye. Wait two minutes for the solution to take effect.
Next, apply one drop of topical tropic aide to each eye in order to dilate the pupils. And wait two minutes for the solution to take effect. Now quickly place the mouse onto the mouse stage, and then set the stage onto the chin rest of the slit lamp.
Turn on the slit lamp and adjust it to the lowest light brightness. Assess the mouse's pupil dilation. If the pupil has not achieved 2.5 to three millimeters of dilation.
Return the mouse to the animal warmer or administer an additional drop of tropic aide following pupil dilation. Orient the mouse on the platform so that it lies horizontally perpendicular to the slit beam. Slightly turn the mouse so that it isn't at an approximately 170 degree angle.
With the head oriented closer to the laser operator, position the mouse as close as possible to the slit lamp while maintaining platform stability and leaving enough room for the operator to perform fine motor manipulations. Once the mouse is properly positioned, apply one drop of artificial tear solution to a 25 millimeter by 25 millimeter glass cover slip. Also, place one drop of this solution onto the mouse's untargeted eye to prevent dehydration.
Grip one corner of the cover slip between the thumb and pointer finger, and wrap the remaining three fingers around the mouse to support and stabilize both the animal and the operator's hand. Then carefully press the cover. Slip onto the mouse's eye to flatten the cornea.
Position the cover slip perpendicular to the laser beam path to prevent scatter or reflection of the beam. Looking through the slit lamp, use the free hand to adjust the focus until the retina comes into view. The retina should appear light yellow to red With distinct red blood vessels present slowly and carefully move the mouse's head or the cover slip until the optic nerve is observed.
The optic nerve will be yellow with multiple blood vessels radiating from it. Then turn on the laser focusing beam and maneuver it to focus on the retinal pigment epithelium or RPE of the eye fundus. If the focusing beam appears to be ovular or out of focus, adjust the slit lamp focus or cover slip.
When the focusing beam is appropriately adjusted, use the foot trigger to initiate laser administration. A bubble should form immediately following laser administration Lesions with hazy borders or eyes with hemorrhaging lesions should be excluded from analysis. Be sure to observe bubble formation and a clear sharply defined circular area indicating a focused laser burn.
Lesions that do not produce a bubble may not appropriately induce CNV. Create additional lesions at all desired choroidal neovascularization or CNV positions. Switch the laser to standby mode when not in use record in a lab notebook the location and result of each laser administration.
Repeat the laser administration for the mouse's second eye using a fresh cover slip and the opposite hand to stabilize the mouse. After completing all administrations, turn off the laser and the slit lamp, discard the cover slip and place the mouse onto the animal. Warmer macroscopically.
Inspect the eye for any corneal surface or cataract injury. Following recovery, return the mouse to its cage. CNVs quantified via analysis of flat mounted choroids following immunofluorescent staining here.
Fit c dextran labeling was done via perfusion immediately prior to animal sacrifice. This was followed by area quantification via thresholding in Image J.Here postmortem immuno staining with the endothelial cell marker. ISO selectin GS IB four was performed, followed by imaging via confocal microscopy.
3D reconstructions of the CNV lesion on FOSS and side view were generated. Optical coherence tomography imaging was also used to visualize the CNV lesions in vivo. Here, a cross-sectional image of the retina is shown with a CNV lesion circled in yellow.
An en FOSS image of the lesion is also shown, circled in yellow. Once mastered, this technique can be done in 10 minutes if it is performed properly. While attempting this procedure, it's important to make sure that the animal is properly anesthetized and to position your hands so that it's supported and stable.
After watching this video, you should have a good understanding of how to deliver focus. Laser burns to induce CNV, which can subsequently be studied in a variety of ways.
