The purpose of this study was to determine whether bone marrow-derived cells can differentiate into myofibroblasts, as defined by alpha smooth muscle actin (SMA) expression, that arise in the corneal stroma after irregular phototherapeutic keratectomy and whose presence within the cornea is associated with corneal stromal haze. C57BL/6J-GFP chimeric mice were generated through bone marrow transplantation from donor mice that expressed enhanced green fluorescent protein (GFP) in a high proportion of their bone marrow-derived cells. Twenty-four GFP chimeric mice underwent haze-generating corneal epithelial scrape followed by irregular phototherapeutic keratectomy (PTK) with an excimer laser in one eye. Mice were euthanized at 2 weeks or 4 weeks after PTK and the treated and control contralateral eyes were removed and cryo-preserved for sectioning for immunocytochemistry. Double immunocytochemistry for GFP and myofibroblast marker alpha smooth muscle actin (SMA) were performed and the number of SMA+GFP+, SMA+GFP−, SMA −GFP+ and SMA−GFP− cells, as well as the number of DAPI+ cell nuclei, per 400X field of stroma was determined in the central, mid-peripheral and peri-limbal cornea. In this mouse model, there were no SMA+ cells and only a few GFP+ cells detected in unwounded control corneas. No SMA+ cells were detected in the stroma at two weeks after irregular PTK, even though there were numerous GFP+ cells present. At 4 weeks after irregular PTK, all corneas developed mild to moderately severe corneal haze. In each of the three regions of the corneas examined, there were on average more than 9X more SMA+GFP+ than SMA+GFP− myofibroblasts. This difference was significant (p <0.01). There were significantly more (p <0.01) SMA−GFP+ cells, which likely include inflammatory cells, than SMA+GFP+ or SMA+GFP− cells, although SMA−GFP− cells represent the largest population of cells in the corneas. In this mouse model, the majority of myofibroblasts developed from bone marrow-derived cells. It is possible that all myofibroblasts in these animals developed from bone marrow-derived cells since mouse chimeras produced using this method had only 60% to 95% of bone marrow-derived cells that were GFP+ and it is not possible to achieve 100% chimerization. This model, therefore, cannot exclude the possibility of myofibroblasts also developed from keratocytes and/or corneal fibroblasts.

PURPOSE: To examine early postoperative wound healing in rabbit corneas that had LASIK flaps formed with three different models (15 KHz, 30 KhZ, and 60 KHz) of a femtosecond laser compared with flaps formed with a microkeratome. METHODS: Thirty-nine rabbit eyes were randomized to receive either no surgery or corneal flaps formed with one of the lasers or the microkeratome. Sixteen eyes also had lamellar cuts with no side cuts with the 30 KHz laser. Animals were sacrificed and corneas processed as frozen sections or fixed for transmission electron microscopy. Frozen sections were evaluated with the TUNEL assay to detect apoptosis, immunocytochemistry for Ki67 to detect cell mitosis, and immunocytochemistry for CD11b to detect mononuclear cells. RESULTS: Rabbit corneas that had flaps formed with the 15 KHz laser had significantly more stromal cell death, greater stromal cell proliferation, and greater monocyte influx in the central and peripheral cornea at 24 hours after surgery than corneas that had flaps formed with the 30 KHz or 60 KHz laser or the microkeratome. Results of the 60 KHz laser and microkeratome were not significantly different for any of the parameters at 24 hours, except for mitotic stromal cells at the flap margin. Transmission electron microscopy revealed that the primary mode of stromal cell death at 24 hours after laser ablation was necrosis. CONCLUSIONS: Stromal cell necrosis associated with femtosecond laser flap formation likely contributes to greater inflammation after LASIK performed with the femtosecond laser, especially with higher energy levels that result in greater keratocyte cell death. [J Refract Surg. 2007;23:667-676.]

Stromal keratocyte apoptosis has been well-characterized as an early initiating event of the corneal wound healing response, triggering subsequent cellular processes that include bone marrow-derived cell infiltration, proliferation and migration of residual keratocyte cells, and, in some circumstances, generation of myofibroblast cells. Recent studies, however, have suggested a more general role for apoptosis in the overall stromal wound healing response that includes modulation and termination functions. This review article highlights, and ties together, recent studies that have demonstrated the important role apoptosis likely plays in the weeks to months following an initial insult to the cornea -depending on the type and extent of corneal injury.

Previous studies have suggested that abnormal corneal wound healing in patients after photorefractive keratectomy (PRK) is associated with the appearance of myofibroblasts in the stroma between two and four weeks after surgery. The purpose of this study was to examine potential myofibroblast progenitor cells that might express other filament markers prior to completion of the differentiation pathway that yields α-smooth muscle actin (SMA)-expressing myofibroblasts associated with haze localized beneath the epithelial basement membrane after PRK. Twenty-four female rabbits that had -9 diopter PRK were sacrificed at 1 week, 2 weeks, 3 weeks or 4 weeks after surgery. Corneal rims were collected, frozen at -80 °C, and analyzed by immunocytochemistry using anti-vimentin, anti-desmin, and anti-SMA antibodies. Double immunostaining was performed for the co-localization of SMA with vimentin or desmin with SMA. An increase in vimentin expression in stromal cells is noted as early as 1 week after PRK in the rabbit cornea. As the healing response continues at two or three weeks after surgery, many stromal cells expressing vimentin also begin to express desmin and SMA. By 4 weeks after the surgery most, if not all, myofibroblasts express vimentin, desmin and SMA. Generalized least squares regression analysis showed that there was strong evidence that each of the marker groups differed in expression over time compared to the other two (p < 0.01). Intermediate filaments - vimentin and desmin co-exist in myofibroblasts along with SMA and may play an important role in corneal remodeling after photorefractive keratectomy. The earliest precursors of myofibroblasts destined to express SMA and desmin are detectible by staining for vimentin at 1 week after surgery.

PURPOSE-To analyze the effects of variations in femtosecond laser energy level on corneal stromal cell death and inflammatory cell influx following flap creation in a rabbit model. METHODS-Eighteenrabbits were stratified in three different groups according to level of energy applied for flap creation (six animals per group). Three different energy levels were chosen for both the lamellar and side cut: 2.7 μJ (high energy), 1.6 μJ (intermediate energy), and 0.5 μJ (low energy) with a 60 KHz, model II, femtosecond laser (IntraLase). The opposite eye of each rabbit served as a control. At the 24-hour time point after surgery, all rabbits were euthanized and the corneoscleral rims were analyzed for the levels of cell death and inflammatory cell influx with the terminal uridine deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and immunocytochemistry for monocyte marker CD11b, respectively. CONCLUSIONS-Higher energy levels trigger greater cell death when the femtosecond laser is used to create corneal flaps. Greater corneal inflammatory cell infiltration is observed with higher femtosecond laser energy levels. RESULTS-TheEarly clinical experience with the femtosecond laser has suggested good efficacy in creating LASIK flaps with fewer complications and better outcomes compared with microkeratomes from different manufacturers. [1][2][3][4][5][6] The corneal wound healing response after LASIK performed with the femtosecond laser has been associated with greater postoperative inflammation, mainly in the periphery, especially with the 15 kHz IntraLase laser (Model I and II; IntraLase, Irvine, Calif). 7 After LASIK performed in humans with the 15 kHz laser, increased inflammation was often observed with the slit lamp at the flap interface and also with immunohistochemical detection of inflammatory cells at the flap edge and in the central interface in rabbit models. 8 Studies with rabbit models performed in our laboratory 8 suggested that decreased femtosecond laser energy resulted in decreased keratocyte necrosis and, therefore, decreased inflammation. However, three different IntraLase femtosecond laser models were used in our earlier studies. Design differences between the different lasers made it impossible to make firm conclusions regarding increases in stromal cell death and inflammation being related to higher energy levels.In the present study, we investigated the effect of variations in side-cut and lamellar-cut energies with the 60 kHz model II IntraLase femtosecond laser on the early wound healing response after flap formation. Specifically, stromal cell death and inflammatory cell infiltration were measured using histological assays to determine whether these parameters varied with different energy levels using a single model of femtosecond laser. MATERIALS AND METHODSEighteen 12-to 15-week-old female New Zealand white rabbits weighing 2.5 to 3.0 kg each were included in this study. One eye of each rabbit was selected at random for surgery or control. All rabbits had flaps cut with the...

Myofibroblast development and haze generation in the corneal stroma is mediated by cytokines, including transforming growth factor beta (TGF β), and possibly other cytokines. This study examined the effects of stromal PDGF-β blockade on the development of myofibroblasts in response to -9.0 diopter photorefractive keratectomy in the rabbit. Rabbits that had haze generating photorefractive keratectomy (PRK, for 9 diopters of myopia) in one eye were divided into three different groups: stromal application of plasmid pCMV.PDGFRB.23KDEL expressing a subunit of PDGF receptor b (domains 2-3, which bind PDGF-B), stromal application of empty plasmid pCMV, or stromal application of balanced salt solution (BSS). The plasmids (at a concentration 1000 ng/ μl) or BSS was applied to the exposed stroma immediately after surgery and every 24 hours for 4-5 days until the epithelium healed. The group treated with pCMV.PDGFRB.23KDEL showed lower αSMA+ myofibroblast density in the anterior stroma compared to either control group (P≤ 0.001). Although there was also lower corneal haze at the slit lamp at one month after surgery, the difference in haze after PDGF-B blockade was not statistically significant compared to either control group. Stromal PDGF-B blockade during the early postoperative period following PRK decreases stromal αSMA+ myofibroblast generation. PDGF is an important modulator of myofibroblast development in the cornea.

PURPOSE: To evaluate differences related to ocular aberrations after customized LASIK for myopia using three different microkeratomes. METHODS: Charts of 410 patients who underwent customized LASIK with the Alcon LADARVision4000 excimer laser were reviewed. Patients were stratified according to the device used to create the flap: Moria M 2 mierokeratome, Bausch & Lomb Hansatome microkeratome, or Intra Lase laser. The difference between the wavefront pre- and postoperative value received a positive or a negative sign if the change occurred toward or away from zero, respectively, and it was compared to preoperative minus postoperative manifest refraction spherical equivalent (MRSE). RESULTS: Patients showed increase in the aberration level after LASIK with the three devices used in this study. Intra Lase spherical aberration change tended to be better than mechanical microkeratomes for higher MRSE values (Intra Lase compared to Hansatome, P^. 023 for MRSE values ^4.00 diopters [D]; IntraLase compared to Moria, P^. 015 for MRSE values ^2.00 D). For total aberrations, the improvement values for IntraLase tended to be higher than those for Moria (Intra Lase com pa red to Mo ria, P^. 021 for M RS E va lues 2=3.00 D). For total higher order aberrations, IntraLase values tended to be better than Moria and Hansatome microkeratomes (IntraLase compared to Hansatome, P^.047 for MRSE values between 3.00 and 8.00 D; IntraLase compared with Moria, P^. 002 for MRSE values 2=2.00 D). Change in coma root-mean-square was similar for the three groups. CONCLUSIONS: The findings suggest the femtosecond laser provides a better platform for LASIK than the commonly used microkeratomes analyzed in this study. [J Refract Surg. 2007;23:880-887.]

PURPOSE:To evaluate the performance of support vector machine, multi‐layer perceptron and radial basis function neural network as auxiliary tools to identify keratoconus from Orbscan II maps.METHODS:A total of 318 maps were selected and classified into four categories: normal (n = 172), astigmatism (n = 89), keratoconus (n = 46) and photorefractive keratectomy (n = 11). For each map, 11 attributes were obtained or calculated from data provided by the Orbscan II. Ten‐fold cross‐validation was used to train and test the classifiers. Besides accuracy, sensitivity and specificity, receiver operating characteristic (ROC) curves for each classifier were generated, and the areas under the curves were calculated.RESULTS:The three selected classifiers provided a good performance, and there were no differences between their performances. The area under the ROC curve of the support vector machine, multi‐layer perceptron and radial basis function neural network were significantly larger than those for all individual Orbscan II attributes evaluated (p<0.05).CONCLUSION:Overall, the results suggest that using a support vector machine, multi‐layer perceptron classifiers and radial basis function neural network, these classifiers, trained on Orbscan II data, could represent useful techniques for keratoconus detection.