Previous studies have shown that
TGF beta 1 induces activation and myofibroblast transformation of cultured rabbit corneal keratocytes. To determine whether
TGF beta has a similar function in vivo, we evaluated the effect of
TGF beta-
blocking antibodies on corneal
fibrosis after lamellar
keratectomy (LK) in the rabbit. A total of 51 rabbits received standard LK
wounds, and eyes were treated with 50 microliters of Celluvisc/PBS, containing 10, 50, or 100 micrograms of 1D11, a mouse monoclonal anti-
TGF beta-blocking antibody. Control
wounds received either 100 micrograms of an irrelevant mouse
monoclonal antibody or vehicle alone. At days 14, 28, 42, and 56, eyes were evaluated by in vivo confocal microscopy (CM) and the mice were killed for light microscopy (LM) and immunostaining with
antibodies to human
fibronectin. In vivo CM of LK
wounds clearly identified a disorganized layer that contained irregularly arranged fibroblasts and reflective extracellular matrix overlying normal corneal stroma. In a subset of 11 eyes stained with 5-(4,6-dichlorotriazinyl) aminofluorescein (
DTAF) immediately after injury, the thickness of the disorganized layer identified by in vivo CM significantly correlated with both anterior corneal
fibrosis (r = 0.627; p < 0.025) and depth of keratocyte activation (r = 0.8980; p < 0.0005), indicating that in vivo CM can be used quantitatively to assess anterior stromal
fibrosis. In eyes treated with an irrelevant
monoclonal antibody, in vivo corneal
fibrosis averaged 100 +/- 26 microns thick at day 14, whereas treatment with 10, 50, and 100 micrograms anti-
TGF beta significantly reduced (p < 0.0005) the anterior disorganization in a dose-dependent fashion to 101 +/- 32, 45 +/- 11, and 56 +/- 18 microns, respectively. Semiquantitative measurement of anti-
fibronectin staining within the
wound revealed that anti-
TGF beta significantly reduced the intensity of anti-
fibronectin staining in the anterior 50 microns of the corneal stroma (p < 0.003). These findings indicate that
TGF beta plays an important in vivo role in keratocyte activation and myofibroblast transformation. Furthermore, the in vivo use of
TGF beta-blocking antibody effects may allow modulation of corneal
fibrosis after
refractive surgery.