mouse lens fiber cell (lec) removal to mimic the cataract surgery

Replicating Cataract Surgical Techniques in a Mouse Model Through LEC Extraction

The lens is a highly organized, transparent tissue that refracts light to produce a clearly focused image onto the retina1,2,3. This specialized organ is surrounded by an uninterrupted basement membrane (the capsule), which isolates the lens from other parts of the eye. The inner anterior surface of the capsule anchors a monolayer of lens epithelial cells (LECs), which then differentiate at the lens equator into lens fiber cells, which comprise the vast majority of the lens3. A cataract occurs when the lens loses its transparency due to factors like aging, genetic mutation, UV radiation, oxidative stress, and ocular trauma4. Cataract is a leading cause of blindness worldwide, particularly in countries with poor medical care5. However, this condition is now readily treated by surgical removal of opaque lens cells by phacoemulsification in which the central lens capsule and attached lens epithelium are removed, followed by the use of a vibrating probe to break the cellular mass of the lens into smaller fragments that can be suctioned out; leaving behind a capsular bag with some attached equatorial LECs1. Vision is then most commonly restored by post-surgical implantation of an artificial intraocular lens (IOL).
While cataract surgery (CS) is a highly effective and minimally invasive treatment for cataracts, a patient&#39;s post-surgical recovery can be acutely hampered by the development of ocular inflammation5,6,8. This inflammation can cause post-surgical pain, retinal edema leading to retinal detachment, as well as exacerbation of other inflammatory and fibrotic conditions like uveitis and glaucoma4,7,8,9. Ocular inflammation post-CS (PCS) is generally treated with anti-inflammatory eye drops which are plagued by poor patient compliance or dropless cataract surgery that can lead to an increased prevalence of floaters8,10,11. In the long term, the outcomes of CS can be compromised by the development of posterior capsular opacification (PCO)12. PCO occurs when residual LECs left behind after surgery undergo a wound healing response, proliferate, and migrate onto the posterior lens capsule at&#160;months to years PCS contributing to secondary visual obstruction13,14.
Understanding the molecular mechanisms by which CS results in such acute and chronic responses represents a major challenge in the field, as much of the literature investigating PCO relies on the induction of LEC conversion to myofibroblasts in culture via treatment with active transforming growth factor beta (TGF&#946;)12,15. Human capsular bag models created from cadaver lenses cultured in vitro better reflect the biology of the lens PCS as LECs are cultured on their native basement membrane but are difficult to manipulate mechanistically, do not recapitulate the intraocular environment, and are inherently variable due to lens to lens (or donor to donor) variability16,17. Rabbit1,18 and non-human primate19,20&#160;in vivo models of cataract surgery overcome some of these issues but still are not easy to manipulate for mechanistic studies. Notably, a prior study of mammalian lens regeneration found significant lens regeneration within four weeks after lens fiber cells were removed from mice, leaving the lens epithelial cells and capsule behind, while no lens regrowth occurred when the entire lens was removed21,22. We subsequently streamlined this procedure and optimized it for the study of the acute response of LECs to lens fiber cell removal and their subsequent conversion to a mixed population of cells with myofibroblast and fiber cell properties.
Using this mouse model of cataract surgery, we have shown that lens epithelial cells drastically remodel their transcriptome by 6 hours PCS to produce numerous proinflammatory cytokines23, while they begin expressing fibrotic markers as early as 24 h PCS12,23, prior to the onset of canonical TGF&#946; signaling. Mechanistic experiments in knockout mice using this model revealed that cellular fibronectin expression by LECs is required for a sustained fibrotic response PCS, likely due to both its role in the assembly of the fibrotic ECM and cell signaling12. Other studies showed that &#945;V&#946;8-integrin is required for the transition of LECs to myofibroblasts due to its function in TGF&#946; activation, and the potential of this approach to identify anti-PCO therapeutic leads was confirmed as a &#945;V&#946;8-integrin antagonist also blocked LEC EMT15.
Here, we provide a detailed protocol describing how to remove the lens fiber cells from living mice (Figure 1and&#160;Figure 4) while leaving behind the lens capsule and LECs to model the LEC response to cataract surgery.&#160;

Author Spotlight: Unraveling the Molecular Mechanisms in PCO and Fibrosis Following Cataract Surgery

Modeling Cataract Surgery in Mice

Our lab studies the molecular mechanisms underlying posterior capsular 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-8 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-8 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-8 integrin is a likely molecular target to prevent myofibroblast formation post-cataract surgery.

Mouse Lens Fiber Cell (LEC) Removal to Mimic the 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.

mouse-lens-fiber-cell-lec-removal-to-mimic-the-cataract-surgery

Research

JoVE Journal

Biology

86 visualizações. Para começar, obtenha agentes anestésicos e analgésicos, gotas de dilatação, bisturi descartável estéril, tipos desejados de fórceps, agulhas, seringas e solução salina balanceada ou BSS. Esterilize os instrumentos cirúrgicos. Antes da anestesia, administre uma gota de 1% de atropina e 2,5% de fenilefrina para dilatar a pupila do olho submetido à cirurgia.Usando uma agulha estéril de calibre 26 de meia polegada, prepare uma seringa com BSS...