Connective tissue growth factor is not necessary for haze formation in excimer laser wounded mouse corneas

Abstract

We sought to determine if connective tissue growth factor (CTGF) is necessary for the formation of corneal haze after corneal injury. Mice with post-natal, tamoxifen-induced, knockout of CTGF were subjected to excimer laser phototherapeutic keratectomy (PTK) and the corneas were allowed to heal. The extent of scaring was observed in non-induced mice, heterozygotes, and full homozygous knockout mice and quantified by macrophotography. The eyes from these mice were collected after euthanization for re-genotyping to control for possible Cre-mosaicism. Primary corneal fibroblasts from CTGF knockout corneas were established in a gel plug assay. The plug was removed, simulating an injury, and the rate of hole closure and the capacity for these cells to form light reflecting cells in response to CTGF and platelet-derived growth factor B (PDGF-B) were tested and compared to wild-type cells. We found that independent of genotype, each group of mice was still capable of forming light reflecting haze in the cornea after laser ablation (p = 0.40). Results from the gel plug closure rate in primary cell cultures of knockout cells were not statistically different from serum starved wild-type cells, independent of treatment. Compared to the serum starved wild-type cells, stimulation with PDGF-BB significantly increased the KO cell culture's light reflection (p = 0.03). Most interestingly, both reflective cultures were positive for α-SMA, but the cellular morphology and levels of α-SMA were distinct and not in proportion to the light reflection seen. This new work demonstrates that corneas without CTGF can still form sub-epithelial haze, and that the light reflecting phenotype can be reproduced in culture. These data support the possibilities of growth factor redundancy and that multiple pro-haze pathways exist.

Fig 1. CTGF KO mouse corneas still form haze.

A) Post hoc genotyping results in the injury corneas reveals good recombination in the homozygous KO corneas, but some degree of Cre-mosaicism in the heterozygous corneas. B) Quantitative analysis via macrophotography reveals increase haze in the absence of CTGF. An ANOVA did not detect any statistical significance among the groups.

Fig 2. CTGF KO versus wild-type primary corneal fibroblast cultures.

A) The rate at which primary fibroblasts can cover a circular defect in a monolayer culture varies by cell type. B) Representative images for each of the test conditions at the first (left) and last (right) time point. C) The results of post hoc comparisons among the test conditions.

Fig 3. Pathological light reflection can be modeled in vitro in response to growth factor stimulation.

The light reflection is not grossly affected by immersion media, though those submersed in methanol did have more bubbles (small white dots at the periphery of the well).

Fig 4. Changes in light reflection between the two cell types when stimulated by growth factors.

A) A globally enhanced image of the replicated treatment wells showing grossly more light reflection in the PDGF-BB treated wells. B) Light scatter quantities normalized to wild type serum starved cells, with the results of the ANOVA. C) The p-value results from the post hoc comparisons among the treatment wells and cell types.

Fig 5. Light reflecting cells qualitative differences.

These images were equivalently globally enhanced for better visualization. A) serum starved cells and b) PDGF-BB treated. C&E) the light reflective cells in the main body of the wells. D&F) the remaining gel-plug hole which has yet to be completely closed. G) A detailed image of the 3 distinct portions of the PDGF-BB treated wells which shows very haze-like cell phenotype which surrounds cells without such a phenotype (upper-left), in contrast to the still acellular hole.

Fig 6. Light reflection and α-SMA levels are not correlated.

The phenotypic nature is grossly similar in that both reflect light and have α-SMA, but the arrangement and level of reflection are drastically different.