The overall goal of this retina preparation method is to use video microscopy to measure mouse retinal arterial reactivity. This method can help answer key questions in the field of retina blood flow regulation and may help further our understanding of the pathophysiology of various ocular diseases. The main advantage of this new technique is that it allows to measure retinal blood vessel reactivity at the very high optical resolution.
And at the same time, it does not need a lot of sophisticated equipment. Generally, individuals new to this method may struggle because it can be difficult to find the ophthalmic artery and its branches. I will demonstrate this procedure together with Jenia Kouchek Zadeh and Wael Omran, post graduate students from my laboratory.
Prepare for the experimental procedures by laying out the required instruments. Then use a pair of steel scissors to cut the skull of the mouse sagittally into two halves and use eye scissors to remove the skin and brain. Use the eye scissors to remove the skull bone until only the orbital bone is left and transfer the orbit into a dissection dish containing ice cold Krebs-Henseleit buffer on ice under a surgical microscope.
Cut the orbital bone with eye scissors and use type four precision tweezers and student Vannas spring scissors to gently remove the eye globe and orbital tissue. Using type five precision tweezers and Vannas capsulotomy scissors, carefully remove the Harderian gland, connective tissue, and extraocular muscles taking care not to damage the main branch of the ophthalmic artery. Use 10-0 Nylon sutures to ligate the orbital branches in the proximal end of the main branch of the ophthalmic arteries and use the type four precision tweezers to transfer the eye globe into a Petri dish containing 70%ethanol.
It is very important that all of the orbital branches of the ophthalmic artery are ligated in order to avoid leakage during the experiment. After 10 seconds, return the eye globe to the dissection dish. The cornea will appear whitish from the ethanol exposure.
Replace the buffer with fresh Krebs solution and use the microscope and a three gauge needle to puncture the peripheral cornea. Use the Vannas capsulotomy scissors to dissect the cornea and gently cut away the iris tissue. Then place one sharp point of the Vannas capsulotomy scissors between the choroid and retina and cut off the choroid and sclera.
To mount the retina into a custom-made perfusion chamber, first use a needle holder to push the end of a glass micropipette with a 100 micrometer diameter tip into the silicon tube of the chamber. Then connect the syringe to the other end of the tube to flush the micropipette tip of any debris. Fill the perfusion chamber with cold Krebs buffer and place the chamber under a stereomicroscope.
Place a transparent plastic platform with a 1.8 millimeter indentation on top of two 1.6 millimeter diameter steel wires within the perfusion chamber and place a stainless steel ring onto the platform. Use a spoon to transfer the retina with the optic nerve with the ophthalmic artery still attached onto the platform and remove the sutured end of the ophthalmic artery. Place two performed loops of 10-0 Nylon suture onto the micropipette and secure the artery to the micropipette with the sutures.
Use one fine-point tweezers to place the retina onto the transparent platform and use a second pair of fine-point tweezers to gently tear open the anterior lens capsula. Pull out the lens and use micro scissors to remove the entire lens capsula and the vitreous. Then make four radial incisions into the retina half the distance from the pars plana to the optic nerve and place the stainless steel ring onto the retina to fix the retina to the platform.
To prepare the retinal arterioles for analysis, transfer the perfusion chamber under a light microscope and select the 100X water immersion objective lens. Connect tubes to the in and outflow ports of a paracyclic pump and circulate the chamber with 37 degrees Celsius Krebs buffer oxygenated with 95%oxygen and carbonated with 5%carbon dioxide. Connect the silicon tube equipped with the micropipette to a reservoir silicon hose via a three-way stopcock and fill the reservoir with Krebs buffer to a height corresponding to 50 millimeters of Mercury.
Using the objectives, manually focus on the retinal surface to search for blood vessels or red blood cells. When a blood vessel is found, focus on the vessel and open the three-way stopcock. The diameter of the vessel should increase immediately and red blood cells should appear.
After a 30-minute equilibration period, the arterioles will develop weak intrinsic tones with a less than 10%luminal diameter reduction. Add a mouse retinal restricting reagent to the bath solution and confirm the vessel viability. Then circulate the pump to wash the agonist out of the bath and add fresh Krebs buffer to the chamber.
In this representative experiment, U46619, a thromboxane mimetic, produced concentration-dependent vasoconstrictor responses in retinal arterioles from wild type C57Bl/6J mice to an approximately 50%reduction in the luminal diameter from resting at the one times 10 to the negative sixth molar concentration. In arterioles preconstricted with the agonist, cumulative administration of acetylcholine evoked concentration-dependent increases in the luminal diameter to approximately 25%of the preconstricted diameter at a one times 10 to the negative fifth molar acetylcholine concentration indicative of an intact vascular endothelium. Once mastered, this technique can be performed in 90 to 120 minutes if it's performed properly.
While attempting these procedures, remember to ligate all of the retrobulbar arterial branches to immerse the eye in ethanol before opening up the globe to avoid torsion of the ophthalmic artery and finally to check the glass micropipette tips for occlusion. After watching this video, you should have a good understanding of how to prepare the mouse retina for arterial measurement with video microscopy in vitro.
