Results of intrastromal corneal ring segment implanted alone or combined with same-day corneal crosslinking and their correlation with preoperative corneal biomechanical strain from finite element analysis.

PURPOSE: To compare the results of intrastromal corneal ring segment (ICRS) alone or combined with same-day corneal crosslinking (CXL) and investigate the relationship of preoperative corneal biomechanics data on the outcomes. SETTING: Department of Ophthalmology of Federal University of Parana. DESIGN: Prospective nonrandomized interventional comparative study. METHODS: Forty-nine eyes of 44 keratoconus patients underwent ICRS only (n = 27, Group 1) or same day ICRS+CXL (n = 22, Group 2) and were followed up for at least 24 months. Visual acuity and preoperative and postoperative tomographic variables were compared between groups. Tomographic data were obtained with a dual Scheimpflug analyzer, and eye-specific finite-element models were used to derive 3 variables related to preoperative biomechanical strain (maximum principal strain [MPS]): mean MPS (mMPS), highest local MPS (hMPS), and position of the hMPS (hMPSx and hMPSy). The relationship between preoperative strain data and the change (∆, difference between postoperative and preoperative data) in tomographic parameters was also investigated. RESULTS: Steepest (K2) and maximum keratometry (Kmax), inferior-superior (I-S) index, coma, and cone location magnitude index (CLMI) significantly improved in both groups. Corrected distance visual acuity was significantly better after ICRS alone (P = .03), whereas corneal asymmetry measured through the I-S index was better after CXL+ICRS (P = .04). In Group 1, hMPSy significantly correlated with K2, tomographical cylinder, mean keratometry, and ∆spherical aberration, whereas mMPS significantly correlated with ∆eccentricity. In Group 2, hMPS significantly correlated with K2, Kmax, I-S index, and ∆coma, and hMPSy significantly correlated with I-S index and ∆coma. The mMPS significantly correlated with ∆CLMI. CONCLUSIONS: ICRS alone seems to be the most suitable option to improve visual acuity, whereas combined ICRS+CXL provided better corneal regularizing results. Preoperative peak strain (hMPS) was predictive of the extent of regularization and flattening after ICRS+CXL.

Stromal fibroblast-bone marrow-derived cell interactions: implications for myofibroblast development in the cornea.

The purpose of this study was to test the hypothesis that mouse corneal stromal fibroblast and bone marrow-derived cell interactions augment corneal myofibroblast generation and, if so, to study whether such interactions are mediated by paracrine or juxtacrine mechanisms. Mouse bone marrow-derived cells and mouse corneal stromal fibroblasts were obtained from both mice with green fluorescent protein (GFP) expressed in all cells and normal GFP- BL6 control mice. To study the interactions of the different cell types, GFP+ cells of one type were co-cultured with GFP- cells of the other type in Primaria plates (to monitor juxtacrine signaling) or Transwell System plates (to monitor paracrine effects mediated by soluble mediators). Both cell types were cultured at a cell density of 1 × 10(5) cells per ml. The percentage of alpha smooth muscle actin+ myofibroblasts was significantly higher (ANOVA, p<0.001) when bone marrow-derived cells and mouse corneal stromal fibroblasts were co-cultured compared to when bone marrow-derived cells and mouse corneal stromal fibroblasts were cultured alone (control). The in vitro studies using GFP+ corneal fibroblasts or GFP+ bone marrow-derived cells demonstrated conclusively that both cells types could transform into myofibroblasts. However, the percentage of alpha smooth muscle actinassds+ myofibroblasts generated from either cell type precursor was higher when both cells were co-cultured together (juxtacrine) as compared to when bone marrow-derived cells and mouse corneal stromal fibroblasts were co-culture in different compartments of Transwell System (paracrine). Thus, more alpha smooth muscle actin+ GFP+ myofibroblasts were generated from GFP+ corneal stromal fibroblasts when GFP- bone marrow-derived cells were present and more alpha smooth muscle actin+ GFP+ myofibroblasts were generated from GFP+ bone marrow-derived cells when GFP- corneal stromal fibroblasts were present. Polyclonal anti-human latency associated peptide (LAP) (transforming growth factor-ß1) neutralizing antibody (a-LAP) and/or transforming growth factor-ß type I receptor kinase inhibitor (LY-364947) inhibited the generation of alpha smooth muscle actin+ myofibroblasts from either precursor cell in Transwell System co-culture experiments. These data suggest that TGFß is a paracrine modulator that regulates the generation of myofibroblasts from either corneal fibroblasts or bone marrow-derived cell precursors.

Interleukin-1 receptor role in the viability of corneal myofibroblasts.

The purpose of this study was to investigate the role of interleukin-1 (IL-1) in modulating myofibroblast viability in mouse corneas with stromal opacity. Twenty-four female B6; 129S1-Il1r1tm1Roml/J homozygous IL-1RI knockout mice and 24 control B6129SF2/J mice were included in this study. Each mouse had opacity-generating irregular phototherapeutic keratectomy (PTK) performed with an excimer laser in one eye. Groups of 8 mice from each group were euthanized at one month, three months and six months after surgery and the eyes cryo-preserved. The contralateral eye served as unwounded control. Immunohistochemistry was performed for α-smooth muscle actin (SMA) in central sections of all corneas. The TUNEL assay for apoptosis was performed on 8 sections of four eyes from each group. No SMA+ cells were detected in the stroma of unwounded control or knockout corneas. SMA+ myofibroblast density was significantly higher (p < 0.001) in the IL-1RI knockout group than in the control group at one month, three and six months after irregular PTK. Mean TUNEL+ stromal cells in the anterior 50 µm of stroma was significantly lower in the IL-1RI knockout group compared to the control group at six months after irregular PTK (p = 0.04). These results corroborate the findings of recent in vitro work that demonstrated an antagonistic effect of TGFß and IL-1 on myofibroblast viability, and found that IL-1-triggered myofibroblast apoptosis was suppressed by TGFß. Thus, IL-1 is an important modulator of myofibroblast viability during corneal wound healing.

Effect of TGFß and PDGF-B blockade on corneal myofibroblast development in mice.

The purpose of this study was to investigate the role of transforming growth factor beta (TGFß) and/or platelet-derived growth factor-B (PDGF-B) blockade on the differentiation of vimentin and alpha-smooth muscle actin (αSMA)-expressing myofibroblasts associated with haze in mice. Mouse corneas had haze-generating irregular PTK (phototherapeutic keratectomy) and topical treatment with the vectors. Six study groups of PTK treated corneas, with four corneas per group in each experiment, were Group 1) treated with TGFß-KDEL vector interfering with TGFß signaling through anomalous sorting of cytokine bound to the expressed altered receptor; Group 2) treated with PDGF-B-KDEL vector interfering with PDGF signaling through anomalous sorting of cytokine bound to the expressed altered receptor; Group 3) treated with both TGFß-KDEL vector and PDGF-B-KDEL vector to interfere with signaling of both cytokines; Group 4) empty pGFPC1 vector; Group 5) empty pCMV vector; and Group 6) no vector treatment control. At one month after surgery, the corneas were analyzed by immunocytochemistry (IHC) for central stromal cells expressing myofibroblast markers vimentin and αSMA. The stroma of corneas treated with the TGFß-KDEL vector alone (p < 0.05) or both the TGFß-KDEL and PDGF-B-KDEL vectors (P < 0.05) had significantly lower density of vimentin-positive cells compared to the corresponding control group. The central stroma of corneas treated with the TGFß-KDEL vector (p < 0.05) or the PDGF-B-KDEL vector (p < 0.05) had lower density of αSMA-positive cells compared to the corresponding control group. The density of αSMA-positive stromal cells was also significantly lower (p < 0.05) when both the TGFß-KDEL and PDGF-B-KDEL and vectors were applied together compared to the corresponding control groups. This study provides in situ evidence that TGFß and PDGF-B have important roles in modulating myofibroblast generation in the mouse cornea after haze-associated injury.

Monocyte development inhibitor PRM-151 decreases corneal myofibroblast generation in rabbits.

This study investigated whether PRM-151 (Promedior, Inc., Malvern, PA), a recombinant form of human pentraxin-2 (PTX-2, also referred to as serum amyloid P, hSAP), that inhibits differentiation of circulating monocytes into fibrocytes and profibrotic macrophages, could modulate generation of myofibroblasts after opacity-producing corneal injury in rabbits, and, therefore, have potential to reduce or prevent haze after PRK. Nine diopter PRK for myopia was performed with the VISX S4 IR laser. Four groups of 6 animals were treated in masked fashion: Group 1: 30 µl of topical PRM-151 (20 mg/ml) 6 times a day for 5 days; Group 2: 30 µl topical vehicle 6 times a day for 5 days; Group 3: 200 µl sub-conjunctival PRM-151 (total injection of 4 mg) immediately after surgery and every other day until day 8; Group 4: 200 µl sub-conjunctival injections of vehicle according to the same schedule as group 3. At one month after PRK, the animals were euthanized and immunohistochemistry was performed for the myofibroblast marker α-smooth muscle actin (SMA). The density of SMA+ cells/400× field in the central stroma was determined in each cornea. Myofibroblast density at one month after surgery was significantly lower (p = 0.006) after sub-conjunctival PRM-151 treatment (5.8 ± 2.8 cells/400× stromal field) compared to sub-conjunctival vehicle treatment (15.3 ± 2.9 cells/400× stromal field). There was no significant (p = 0.27) decrease in stromal myofibroblasts triggered by topical PRM-151 treatment (11.8 ± 6.6 cells/400× stromal field) compared to the topical vehicle treatment (14.2.8 ± 6.2 cells/400× stromal field). PRM-151 inhibits myofibroblast generation when administered by sub-conjunctival injection, but not when administered topically, after opacity-producing corneal injury. This study provides additional confirmation that bone marrow-derived cells contribute to corneal myofibroblast generation.