The overall goal of this procedure is to explore strategies to promote regrowth of functional blood vessels within the retina and the CNS after ischemic injury. This is accomplished by first using a mouse model of oxygen induced retinopathy. Next compounds are delivered to the eye by intravitreal injections.
Vessel perfusion and barrier function are then verified by fluorescein angiography. The final step is to extract the eye and isolate the retina. The retina is then stained and mounted before imaging.
Ultimately, immunofluorescence microscopy is used to assess rates of vascular regeneration following intravitreal treatments or in mice with genetic modifications. The technique we're presenting provides a highly reproducible, easy system in which to assess vascular regeneration within the central nervous system. It's based on the Smith model of oxygen induced retinopathy and can be quite helpful in designing therapeutic strategies to remedy ischemia within the central nervous system.
This method can help answer key questions about the molecular mechanisms that drive or prevent functional regeneration of blood vessels. In the CNS. It can be used to study the impact of genes, pharmacological compounds, or elucidate pathological mechanisms that start vascular growth.
In addition to ocular diseases, this technique can be extrapolated to other areas of the CNS, such as the brain and spinal cord Practice is required to successfully inject compounds into the vitreous humor of mouse pops and to carefully handle fresh readiness. To begin place mouse pups at P seven and a CD one fostering mother into an oxygen chamber set at 75%oxygen five days later, remove the animals from the chamber and return them to normal housing conditions. Next at P 12 or P 14, intravitreal injections are performed to deliver compounds to the inner retina.
After anesthetizing mice, verify the effectiveness of the anesthesia by sequentially pinching the tail rear foot and ear with forceps. A beveled pulled glass needle attached to a 10 microliter syringe with a drop of epoxy resin should be prepared ahead of time. Locate the injection site at the posterior limbus of the eye.
Insert the needle at a 45 degree angle and inject the compound of interest. After applying ointment to the eye, return the mouse back to the cage with a foster mother to begin centrifuge the fluorescein Dexter solution and collect the supernat to prevent vessel constriction. Warm the fluorescein dextrin solution before injecting.
Next, confirm the depth of sedation in the anesthetized mouse. When ready, make a midline incision over the abdomen with dissecting scissors. After opening the rib cage and removing peripheral tissue from the heart, clamp the descending aorta.
Insert a 25 gauge needle filled with warmed fluorescein dextrin into the left ventricle and perfuse the solution. Within two minutes of the injection, decapitate the animal and place the head on its side. Remove the skin and eyelids covering the eye with dissecting scissors.
Next place curved forceps below the eye and gently pull it up until the optic nerve is severed. Turn the mouse's head onto the other side and to repeat these steps. To ensure better penetration of fixative, puncture a hole in the anterior chamber of the eye.
Using a 30 gauge needle, transfer the eyes to a tube containing 4%paraform, aldehyde, and fix for one hour at room temperature. After this time, remove the PFA and wash the eyes four times with ice cold PBS. Place the eyes in a Petri dish containing cold PBS and position them under a stereo microscope.
Once in place, remove any extra tissue surrounding the eye with microdissection scissors and cut the cornea. Use two pairs of forceps to peel the sclera away from the periphery towards the optic nerve. Pinch the lens with forceps and extract it from the eye cup.
Use one pair of forceps as a support and the other to grip and carefully raise and remove the lens. Detach the aloid vessels from the inner side of the retina using a small brush and forceps. Next, remove bundles of hyaloid vessels connected to the optic disc using forceps.
Transfer the dissected retinas to a two milliliter micro centrifuge tube containing PBS and place on ice. Prior to starting the staining procedure, the dissected retinas are first incubated overnight with gentle shaking at four degrees Celsius. In a solution, a fluorescently coupled iso selectin B four containing calcium chloride During the entire staining procedure, the tubes should be covered with aluminum foil to protect them from light on the following day.
Remove the staining solution and wash the retinas three times in PBS for 10 minutes. At room temperature, transfer the retinas photoreceptor side down onto a microscope. Slide while bracing the retina with a brush.
Make four deep equidistant radial incisions. Using a surgical scalpel, carefully flatten the quadrants and immerse the retina in mounting medium to photo bleaching carefully place a cover slip on the surface of the mounted retina without applying pressure and avoiding air bubbles. Finally, the slides are imaged and quantified.
For vaso obliteration, a dose dependent increase in vascular regeneration was observed after Trin one was injected at P 14 and regrowth. Assessed at P 17 vessels with compromised barrier function leak fluorescence after profusion with a fluorescent dextrin after an intravitreal injection of lentivirus. At P three, the inhibition of IRE one alpha dramatically enhanced vascular regeneration in the vascular growth phase at P 17.
Once these techniques are mastered, intravitreal injection, cardiac perfusion and retinal extraction should each take about 10 minutes if they're performed properly. While attempting this procedure, it's important to preserve the 3D structure of the eye, avoid an excessive pressure that can damage the retina and compromise results. Don't forget that working with animals, viruses, or bioactive compounds can be extremely hazardous.
Safety precautions and sterilized tools should always be used while performing this procedure.
