Our lab studies the molecular mechanisms underlying posterior capsule opacification, which is the most prevalent negative sequela of cataract surgery. This method has really given us some new fundamental insight into the response of remnant lens epithelial cells to cataract surgery. We found for the very first time that lens epithelial cells produce pro-inflammatory cytokines a day or two before any TGF beta signaling and the expression of fibrotic genes.
And we've taken this even further to show that the expression of alpha v beta eight integrin on lens epithelial cells is required for the activation of TGF beta, leading to later capsular bag fibrosis. And we've taken this even further to show that drugs that block alpha v beta eight integrin function block this pathway and prevent the fibrosis, suggesting that this is a new therapeutic target to prevent fibrotic PCO. PCO pathogenesis studies often focus on lens epithelial cell conversion to myofibroblasts via exogenous active TGF beta.
However, this doesn't address how surgery triggers the fibrotic response. This in vivo model allows one to follow how lens epithelial cells respond to injury within the ocular environment, revealing both cell autonomous and non-autonomous mechanisms. We provide a unique in vivo technique for studying lens epithelial cell response to surgery while utilizing advanced genetic tools.
In the long term, investigations can elucidate likely therapeutic targets for inflammatory management while leveraging our finding that alpha v beta eight integrin is a likely molecular target to prevent myofibroblast formation post cataract surgery. To begin, obtain anesthetizing and analgesic agents, dilation drops, sterile disposable scalpel, desired types of forceps, needles, syringes and balanced salt solution, or BSS. Sterilize the surgical instruments.
Prior to anesthesia, administer one drop each of 1%atropine and 2.5%phenylephrine to dilate the pupil of the eye undergoing surgery. Using a sterile 26 gauge half inch needle, prepare a syringe with sterile BSS. Then attach a sterile one inch 27 gauge blunt dispensing tip bent at 45 degrees, to the syringe.
Following the mouse's anesthesia, perform a toe pinch to confirm anesthesia depth, ensuring pupil dilation reaches two millimeters. Move the anesthetized mouse to the stage of a compound dissecting light microscope with a heating pad. Using a sterile surgical scalpel, make a one to 1.5 millimeter incision in the center of the cornea, ensuring that the anterior lens capsule is also incised.
Gently expel one drop of BSS above the cornea to wet the surface for further manipulation. Now, insert the dispensing tip through the central corneal incision into the lens capsule incision and gently move the needle in a circular motion while slowly releasing BSS to expel the lens fiber mass. Once the fiber mass adheres to the dispensing tip, gently remove the dispensing tip from the inside eye.
Clean it with a sterile towel to remove fiber cells and repeat until all lens fiber cells and lens nucleus are removed. Rinse the lens capsule with sterile BSS to remove any remaining fibers. Observe the translucent or glassy appearance to confirm that the capsule is inside the eye.
To close the corneal incision, first, hold one corneal flap using forceps in the non-dominant hand and push the needle with nylon thread through it. Then push it through the next corneal flap. Place one square knot stitch in the central part of the incision and trim the excess thread.
Place the bent needle through the corneal gap, either above or below the recently placed suture and gently expel BSS to reinflate the eye For postoperative care, immediately apply one drop of 0.5%erythromycin ophthalmic ointment directly above the suture, covering the entire incision. Administer buprenorphine intraperitoneally. Allow the mouse to recover from anesthesia in a cage, placed on a heating pad to optimize thermal support and monitor until it recovers.
