Abstract
Purpose :
Corneal scars originate from keratocyte-derived fibroblasts and myofibroblasts that are ultimately cleared through apoptosis or revert back to keratocytes. However, the precise timeline and what occurs once wound closes is not clear. We interrogate a mouse model expressing a keratocyte lineage-specific reporter to elucidate cell phenotype dynamics during scar maturation.
Methods :
The KeraRT/tetO-Cre/mTmG (I-Kera mTmG) mouse model expresses tdTomato in all keratocan-negative cells. In keratocan-expressing cells, a doxycycline-inducible Cre recombinase knocks down tdTomato expression and activates expression of eGFP. Once eGFP is expressed, daughter cells will maintain eGFP expression allowing keratocye lineage tracing. Twelve adult I-KeramTmG mice were anesthetized and a 1mm full-thickness keratotomy created. Three and 6 months after injury, mice received 1 week of oral doxycycline to induce Cre recombinase activity. Three mice received another round of oral doxycycline 10 months post-injury before being sacrificed. The enucleated eyes were embedded for sectioning or imaged as flat mounts. Immunostaining against collagens, and SHG (second harmonic generation) microscopy were done to assess collagen deposition and organization.
Results :
At three months and six months post-injury, few eGFP-positive cells were visualized at the borders of scars in tissue sections and flat mounts, with central (core) areas of scar containing few to no visible eGFP-positive cells. Second harmonic generation (SHG) microscopy showed thickened and irregularly arrenged collagen fibers, characteristic of stromal scars. At ten months post-injury, eGFP-positive cells can be seen throughout the scar in both peripheral and central sections but the scar core has a majority of tdTomato cells. Second harmonic generation (SHG) signal is comparable to that seen at six months suggesting that nor extracellular matrix organization neither cell phenotype has changed significanlty 10 months post injury in a scar.
Conclusions :
We demonstrate using in vivo experiments that a mature scar contains a fibroblast/myofibroblast phenotype that predominantly originated from keratocyte lineage. Reversion of fibroblasts to keratocyte or migration of new keratocytes into scars occur. The key question to answer is if the scar matrix can be changed by cells or if the scar matrix will dictate phenotype of migrating cells..
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.