The overall goal of the experiment is to observe the effect of anti-angiogenic therapy in a mouse model of corneal neovascularization. This is achieved by briefly applying a one molar solution of sodium hydroxide to the mouse eye to induce a controlled level of corneal neovascularization. As a second step, the anti-angiogenic compound is administered daily, which inhibits the growth of new vessels into the injured cornea.
Next, the mouse is euthanized and the injured eye, harvested, dissected, and immune stained. In order to quantify the level of neovascularization and inflammation, the results show a quantifiable reduction in neovascularization based on both clinical observation and immuno staining. The implications of this technique extend towards new therapies for corneal blindness because it provides a reproducible level of neovascularization against which you can test new compounds and therapies.
So this method can provide insight into the pathologic cornea neovascularization. You can also be a model to explore the mechanisms of angiogenesis and inflammation in general. Demonstrating the procedure will bebo a poster in my laboratory To prepare for the alkali burn injury.
Begin by soaking a round piece of filter paper, approximately two millimeters in diameter in a solution of one molar sodium hydroxide. Then anesthetize a mouse with an injection of 100 milligrams per kilogram, ketamine, and five milligrams per kilogram xylazine. After one to two minutes, test the depth of anesthesia by gently pinching the toe or tail of the animal to ensure that there is no response.
Using an eyedropper topically, apply a drop of 0.5%propane hydrochloride to the corneal surface of the eye for local analgesia, and place the mouse under a surgical microscope. Next, use sterile forceps to pick up the piece of sodium hydroxide soaked filter paper and looking through the scope. Place the filter paper on the central cornea.
Leave it for 30 seconds to generate an acute alkali burn of approximately two by two millimeters squared. The second eye of the mouse is not injured and so serves as an internal control. After removing the filter paper, use a syringe to gently flush the eye with 10 milliliters of one XPBS twice to wash away any residual one molar sodium hydroxide.
After flushing, immediately apply a drop of one X Saha working solution topically to the cornea. Repeat this application three times per day for 14 days in the control mice After the alkali burn and a double rinse with PBS apply PBS diluted DMSO containing no saha daily for 14 days. During this two week period, give a liquid 3%Gentamycin solution daily to both the saha treated and control mice.
The clinical assessment consists of a daily examination of the mice in a blinded fashion under a surgical microscope. These at least two observers and record a final score that is the average of the score from the two observers. Score corneal opacity on a scale of zero to four, where zero equals completely clear, one equals slightly hazy with the iris and pupil.
Easily visible, two equals slightly opaque with the iris and pupil still detectable. Three equals opaque with the pupil hardly detectable, and four equals completely opaque with no view of the pupil. A comparison of the corneal opacity scoring is shown here.
Also, score neovascularization on a scale of zero to three. Note that all eyes have a ring of normal non-pathogenic vessels around the cornea. So a neovascularization score of zero means no neo vessels.
A score of one means neo vessels at the corneal limbus. Two means neo vessels spanning the corneal limbus and approaching the corneal center. And three means neo vessels spanning the corneal center.
A comparison of the neovascularization scoring is shown here. Add an additional score for vessel size on a scale of zero to three, where zero equals no neo vessels. One equals neo vessels barely detectable under the surgical scope.
Two equals neo vessels easily seen under the surgical scope, and three equals neo vessels easily seen without the microscope. A comparison of the vessel size scoring is shown here at seven days and 14 days. Use a digital camera to take representative images of the eye.
After euthanizing a mouse, fix the enucleated eye in 4%paraform aldehyde for at least one hour at four degrees Celsius, and then transfer the eye to one XPBS and use forceps to carefully remove excess tissue. With the aid of a surgical microscope, use a micro knife to perforate the peri corneal region of the eye. This should release fluid from the eye using a pair of surgical scissors, start the perforation and cut the anterior portion of the eye.
That is the cornea from the posterior portion. Transfer the cornea back into 4%paraldehyde for fixation overnight at four degrees Celsius. The next day.
Discard the 4%para formaldehyde according to regulations, and rinse the cornea three times with one XPBS to remove any residual paraform aldehyde. Then incubate the cornea in one x blocking buffer for at least two hours at room temperature to permeate the tissue and prevent non-specific binding of the primary antibody. Apply the primary antibody in one x blocking buffer at a 100 to 500 fold dilution and incubate at four degrees Celsius overnight the following day.
Wash the cornea six times with one x washing buffer for one hour each time. To fully remove any unbound primary antibody, this step is carried out at room temperature. Apply a secondary antibody in one x blocking buffer at a 500 to 1000 fold dilution and incubate at four degrees Celsius overnight.
The next day, wash three times with one XPBS for one hour each at room temperature to fully remove any unbound secondary antibody. After transferring the cornea into fresh one XPBS, use a surgical microscope to carefully make four incisions from the periphery towards the center. This should divide the cornea into four quadrants of approximately equal size.
The resulting shape should look rather like a butterfly and allow it to lie flat on a slide. Transfer the cornea to a slide and mount it with a mounting medium appropriate for fluorescent imaging for best results. The samples should be photographed immediately, but they may be stored for several weeks protected from light at four degrees Celsius.
Seven days after the alkali burn injury, the progression of corneal neovascularization can be clearly seen. This image shows neovascularization in an eye that was not treated with saha. In contrast, this eye was treated three times per day with the histone deacetylase inhibitor.Saha.
Note the difference in corneal opacity and neovascularization between the two eyes shown here are corneal flat mounts one week after alkali burn injury. These cornea were treated with the control P-B-S-D-M-S-O vehicle during that week. These next images are from cornea treated with saha after the alkali burn injury.
The green staining shows vascular endothelial cell staining with PE camm one and the red staining shows lymphatic endothelial cell staining with LYVE one. The saha treatment resulted in a dramatic decrease in both he angiogenesis and lymph angiogenesis flat mounting. The cornea allows staining with PE camm one, which serves as a marker for the blood vessels and LYVE one, which binds specifically to lymphatic vessels.
This image shows the merged PE camm one LYVE one staining, allowing comparison of he angiogenesis versus lymph angiogenesis, as well as a comparison of the differing cell morphology. Sagittal sections using F four 80. Staining of macrophages and dpi.
Staining of cell nuclei are shown here. This merged F four 80 and DPI image shows macrophage infiltration into the cornea of an alkali burned eye After its development. This technique paved the way for researchers in the field of ophthalmology to explore corneal neovascularization in mouse models.
