Effect of Femtosecond Laser Energy Level on Corneal Stromal Cell Death and Inflammation

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: Eighteen rabbits 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 mu J (high energy), 1.6 mu J (intermediate energy), and 0.5 mu J (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 comeoscleral 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. RESULTS: The high energy group (31.9 +/- 7.1 [standard error of mean (SEM) 2.9]) had significantly more TUNEL positive cells in the central flap compared to the intermediate (22.2 +/- 1.9 [SEM 0.8], P=.004), low (17.9 +/- 4.0 [SEM 1.6], P <= .001), and control eye (0.06 +/- 0.02 [SEM 0.009], P <= .001) groups. The intermediate and low energy groups also had significantly more TUNEL positive cells than the control groups (P <= .001). The difference between the intermediate and low energy levels was not significant (P=.56). The mean for CD11b-positive cells/400x field at the flap edge was 26.1 +/- 29.3 (SEM 11.9), 5.8 +/- 4.1 (SEM 1.6), 1.6 +/- 4.1 (SEM 1.6), and 0.005 +/- 0.01 (SEM 0.005) for high energy, intermediate energy, low energy, and control groups, respectively. Only the intermediate energy group showed statistically more inflammatory cells than control eyes (P = .015), most likely due to variability between eyes. 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. [J Refract Surg. 2009;25:869-874.] doi:10.3928/1081597X-20090917-08

Dynamics of the expression of intermediate filaments vimentin and desmin during myofibroblast differentiation after 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 a-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 I week, 2 weeks, 3 weeks or 4 weeks after surgery. Corneal rims were collected, frozen at -80 degrees 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 I week after surgery. (C) 2009 Elsevier Ltd. All rights reserved.

Corneal stroma PDGF blockade and myofibroblast development

Myofibroblast development and haze generation in the corneal stroma is mediated by cytokines, including transforming growth factor-beta (TGF-beta), and possibly other cytokines. This study examined the effects of stromal PDGF-beta 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/mu l) or BSS was applied to the exposed stroma immediately after surgery and every 24 h for 4-5 days until the epithelium healed. The group treated with pCMV.PDGFRB.23KDEL showed lower alpha SMA+ 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 alpha SMA+ myofibroblast generation. PDGF is an important modulator of myofibroblast development in the cornea. (C) 2008 Elsevier Ltd. All rights reserved.

Scanning laser polarimetry with enhanced corneal compensation for detection of axonal loss in band atrophy of the optic nerve

PURPOSE: To compare the abilities of scanning laser polarimetry (SLP) with enhanced corneal compensation (ECC) and variable corneal compensation (VCC) modes for detection of retinal nerve fiber layer (RNFL) loss in eyes with band atrophy (BA) of the optic nerve. DESIGN. Cross-sectional study. METHODS: Thirty-seven eyes from 37 patients with BA and temporal visual field defect from chiasmal compression and 40 eyes from 40 healthy subjects were studied. Subjects underwent standard automated perimetry and RNFL measurements using an SLP device equipped with VCC and ECC. Receiver operating characteristic (ROC) curves were calculated for each parameter. Pearson correlation coefficients were obtained to evaluate the relationship between RNFL thickness parameters and severity of visual field loss, as assessed by the temporal mean defect. RESULTS: All RNFL thickness parameters were significantly lower in eyes with BA compared with normal eyes with both compensation modes. However, no statistically significant differences were observed in the areas under the ROC curves for the different parameters between GDx VCC and ECC (Carl Zeiss Meditec, Inc, Dublin, California, USA). Structure-function relationships also were similar for both compensation modes. CONCLUSIONS: No significant differences were found between the diagnostic accuracy of GDx ECC and that of VCC for detection of BA of the optic nerve. The use of GDx ECC does not seem to provide a better evaluation of RNFL loss on the temporal and nasal sectors of the peripapillary retina in subjects with BA of the optic nerve.

Comparison of corneal changes after phacoemulsification using BSS Plus versus Lactated Ringer`s irrigating solution: a prospective randomised trial

Background/aims To compare two intraocular irrigating solutions, Balanced Salt Solution Plus (BSS Plus) versus Lactated Ringer`s (Ringer), for the preservation of corneal integrity after phacoemulsification. Methods 110 patients undergoing phacoemulsification were randomised to either BSS Plus (n = 55) or Ringer (n = 55) as the irrigating solution. Patients were examined at baseline and at 1, 8, 15, 30 and 60 days postoperatively. Evaluations included specular microscopy to evaluate endothelial cell density (ECD) and endothelial cell size variability (CV), and corneal pachymetry for central corneal thickness (CCT) measurement. Results Groups were well balanced regarding baseline ECD, CV and CCT (p>0.05). There was no statistically significant difference between ECD reduction in group BSS Plus 13.1 +/- 2.0% and Ringer 9.2 +/- 1.9% (p<0.05) at day 60 or in any study visit. There was no statistically significant difference between CV increase in group BSS Plus 23.0 +/- 3.0% and Ringer 20.2 +/- 4.0% (p<0.05) at day 60 or in any study visit. CCT was significantly increased (p<0.05) at 1, 8, 15 and 30 days postoperatively, returning to baseline at 60 days in both groups. There was no significant difference in CCT increase in both groups at any visit. Interestingly, there were statistically significant correlations between ECD loss and phacoemulsification time (p<0.0001) and ECD loss and irrigation solution volume (p<0.0001) in the Ringer group, but not in the BSS Plus group. Conclusions Ringers solution was similar to BSS Plus for corneal preservation in atraumatic cataract surgery. However, our study demonstrates that there is a trend towards lower postoperative endothelial cell density for surgeries with longer phacoemulsification time and higher irrigation volumes if Ringer is used.

Regeneration of the Corneal Epithelium after Debridement of its Central Region: An Autoradiographic Study on Rabbits

Purpose: To investigate the proliferative behavior of the corneal and limbal epithelia after debridement on the central region of the rabbit cornea. Methods: After scraping a circular epithelial area, 5 mm in diameter, in the center of the cornea, (3)H-thymidine ((3)H-TdR) was injected intravitreally, and the rabbits killed from 1 to 49 days afterward. The cornea, together with the adjacent conjunctiva, was processed for autoradiography. Results: The regenerating epithelium at the center of the cornea exhibited high frequencies of labeled nuclei when compared to controls. The mitotic indexes for the limbus were comparable in experimental and control eyes. The unique basal stratum of the limbal epithelium exhibited quick proliferation and vertical migration in all eyes. Cells that remained labeled for four weeks or more were observed throughout the corneal epithelium, including its basal stratum, and this did not depend on epithelial damage. Conclusion: Corneal epithelium wounds are healed by sliding and proliferation of cells surrounding the epithelial gap without any evidence for the participation of the limbal epithelium. Daughter cells labeled with (3)H-TdR were visualized in all layers of the corneal epithelium up to 7 weeks after the DNA precursor injection. However, at this long interval, the only labeled cells in the limbus were in the suprabasal layers.

Morphological and autoradiographic studies on the corneal and limbal epithelium of rabbits

The investigation was centered on the morphological features of the conjunctiva-cornea transition (limbus) of the rabbit eye and the proliferative behavior of its epithelium. The eyes were processed for examination with light and electron microscopy, as well as for autoradiography after intravitreal injection of [H-3]thymidine ([H-3]TdR). At the sites of extraocular muscle insertion, the vascularization of the stroma extended to the peripheral cornea, and the limbal epithelium was thin with its basal stratum made up by clear cuboidal cells. In between the muscle insertions, the cuboidal clear cells, as well as the stroma blood vessels; were scarce. At the light microscope level, the basement membrane was distinct in the cornea but not in the limbus or the conjunctiva. Autoradiographs demonstrated that, at the limbus, the basal cells migrated very quickly to the suprabasal region and remained there up to the 28-day interval. Labeled cells were identified in all epithelial layers of the cornea, including the basal one, at 21 and 28 days but not in the limbal basal clear cells. The rate of renewal of conjunctival epithelium was similar to that observed for the transition with scarce clear cells. The high-resolution autoradiographs demonstrated that the basal cuboidal clear limbal cells exhibit a quick renewal and that they are not label-retaining cells. These latter ones were detected all over the corneal epithelium and in the suprabasal layers of the limbus up to 28 days, in physiological conditions, without the need of stimulation by damage to the corneal epithelium.

Intravitreal triamcinolone versus bevacizumab for treatment of refractory diabetic macular oedema (IBEME study)

Background/aims: The aim of this study was to compare the morphological and visual acuity outcomes associated with a single intravitreal injection of triamcinolone acetonide versus bevacizumab for the treatment of refractory diffuse diabetic macular oedema. Methods: Twenty-eight patients were randomly assigned to receive a single intravitreal injection of either 4 mg/0.1 ml triamcinolone acetonide or 1.5 mg/0.06 ml bevacizumab. Comprehensive ophthalmic evaluation was performed at baseline and at weeks 1, 4, 8 (+/- 1), 12 (+/- 2) and 24 (+/- 2) after treatment. Main outcome measures included central macular thickness measured with optical coherence tomography (OCT) and best corrected Early Treatment Diabetic Retinopathy Study (ETDRS) visual acuity. Results: Twenty-six patients (26 eyes) completed all study visits (two patients missed two consecutive study visits). Central macular thickness was significantly reduced in the intravitreal triamcinolone group compared with the bevacizumab group at weeks 4, 8, 12 and 24 (p<0.05). Logarithm of the minimum angle of resolution (LogMAR) best-corrected visual acuity was significantly higher at weeks 8 (0.69; similar to 20/100(+1)) and 12 (0.74; 20/100(-2)) in the intravitreal triamcinolone group compared with the bevacizumab group (weeks 8 (0.83; similar to 20/125(-1)) and 12 (0.86; 20/ 160(+2))) (p<0.05). Significant change from baseline in mean intraocular pressure (mmHg) was seen at week 4 (+2.25) only in the intravitreal triamcinolone group (p<0.0001). No patient had observed cataract progression during the study. Conclusions: One single intravitreal injection of triamcinolone may offer certain advantages over bevacizumab in the short-term management of refractory diabetic macular oedema, specifically with regard to changes in central macular thickness. The actual clinical relevance of our preliminary findings, however, remains to be determined in future larger studies.

The lectin KM+ induces corneal epithelial wound healing in rabbits

Neutrophil influx is essential for corneal regeneration (Gan et al. 1999). KM+, a lectin from Artocarpus integrifolia, induces neutrophil migration (Santos-de-Oliveira et al. 1994). This study aims at investigating a possible effect of KM+ on corneal regeneration in rabbits. A 6,0-mm diameter area of debridement was created on the cornea of both eyes by mechanical scraping. The experimental eyes received drops of KM+ (2.5 mu g/ml) every 2 h, The control eyes received buffer, The epithelial wounded areas of the lectin-treated and untreated eyes were stained with fluorescein, photographed and measured, The animals were killed 12 h (group 1, n = 5), 24 h (group 2, n = 10) and 48 h (group 3, n = 5) after the scraping. The corneas were analysed histologically (haematoxylin and eosin and immunostaining for proliferation cell nuclear antigen, p&3, vascular endothelial growth factor, c-Met and laminin). No significant differences were found at the epithelial gap between treated and control eyes in the group 1. However, the number of neutrophils in the wounded area was significantly higher in treated eyes in this group. Three control and seven treated eyes were healed completely and only rare neutrophils persisted in the corneal stroma in group 2. No morphological distinction was observed between treated and control eyes in group 3. In treated corneas of group 2, there was an increase in immunostaining of factors involved in corneal healing compared to controls, Thus, topical application of KM+ may facilitate corneal epithelial wound healing in rabbits by means of a mechanism that involves increased influx of neutrophils into the wounded area induced by the lectin.

Corneal endothelial response to induced myopia in the chicken

Purpose: To investigate the role of corneal endothelial surface enlargement in the chicken myopia model in inducing corneal endothelial changes. Methods: Lid suture was performed on one eye of 1-day-old cockerels. Five chickens were killed at 1 week, and four chickens killed at each of 3 weeks, 6 weeks, and 10 weeks postnatal. The endothelial morphology was obtained by flat mounting the endothelial surface and the subsequent digitisation. Comparisons were undertaken between the control unsutured eye and the lid-sutured eye endothelium, and between the central endothelial areas compared to the peripheral endothelial areas in both the myopic and the normal corneas. Calculation of the contribution to the endothelial change by hypertrophy and mitosis were calculated using Bahn's formula. Results: Total endothelial surface area increased significantly over time in the myopic model compared to control eyes but the mean cell area of endothelial cells remained the same for both the enlarged myopic endothelial surface area and in the normal controls. Sampling from the central and the peripheral corneal endothelial surface also disclosed no difference. The mean cell area did increase steadily with age but was the same for both normal and myopic corneas. Conclusions: It would appear that there are equal contributions from hypertrophy and mitosis in the myopic group and the normal corneal group with a slightly increasing trend towards mitotic activity in the myopic corneal endothelial layer.

Identifying the origin of single corneal cells by DNA fingerprinting - Part I - Implications for corneal limbal allografting

Purpose. To demonstrate that the combination of impression cytology and single cell DNA fingerprinting represents a powerful tool that is suitable for detecting transplanted cells after corneal limbal allografting. Methods, Fifty single cells were obtained by corneal impression cytology from 12 patients undergoing cataract surgery. Individual cells were isolated from samples by micromanipulation. Polymerase chain reaction and short tandem repeat profiling was used to obtain forensic standard DNA fingerprints from single cells. Blood samples taken at the time of impression cytology provided control fingerprints. Results, informative DNA fingerprints were obtained from all corneal samples and 66% (33 of 50 cells) of isolated single cells, Of all fingerprints obtained, most (91%, 30 of 33 fingerprints) corneal fingerprints matched corresponding blond sample fingerprints. At least one corneal fingerprint matched the corresponding blood sample fingerprint in 83% (10 of 12 patients) of the patients in the study, Conclusions. This extremely specific single cell DNA fingerprinting system permits accurate identification of individual corneal epithelial cells, allowing very reliable determination of their origin, which will enable host and donor cells to be distinguished from each other after keratolimbal allografting procedures. even if the host and donor are the same sex or siblings. These DNA fingerprinting methods allow assessment of quality and quantity of donor cell survival, as well as survival time. The extreme sensitivity and accuracy of the technique means that should contamination occur, it would be identified, thus ensuring meaningful results.

Preserved xenogenic amniotic membrane as a patch on the repair of superficial corneal ulcers in rabbits

The aim of this study was to evaluate the effects of canine amniotic membrane, previously preserved in glycerin, used as a patch on the repair of experimentally-made superficial corneal ulcers and to compare corneal epithelization between the treated and non-treated groups. Xenogeneic amniotic membranes were collected aseptically and preserved in 99% glycerin at room temperature. Each animal was anesthetized and submitted to superficial corneal keratectomy of the left eye. The treated group received a fragment of canine amniotic membrane as a patch, while the control group had no treatment. The treated group showed blepharospasm, ocular discharge and conjunctival congestion. The membrane accelerated corneal repair in the beginning of the process, however, it delayed its conclusion (p<0.05). Treated eyes showed greater vessel formation and decreased corneal transparency (p<0.05). The stroma of the control group was thicker than that of the treated group (p<0.05). We suggest that amniotic membrane used in this manner can be applied as a therapy for superficial corneal ulcers in the beginning phases of the repair process.

Usefulness of corneal esthesiometry for screening diabetic retinopathy

OBJECTIVE: To assess the usefulness of corneal esthesiometry for screening diabetic retinopathy. METHODS: A cross-sectional study was carried out comprising 575 patients attending a diabetic retinopathy-screening program in the city of São Paulo. Corneal esthesiometry was assessed with the Cochet-Bonnet esthesiometer. The presence of diabetic retinopathy was detected with indirect fundoscopy. The validity of corneal esthesiometry in identifying diabetic retinopathy was evaluated by the Receiver Operating Characteristic (ROC) curve. RESULTS: Sensitivity and specificity analyses of the corneal esthesiometry for detecting the stages of diabetic retinopathy using different cut-offs showed values less than 80%. The best indices (72.2% sensitivity and 57.4% specificity) were obtained for the identification of patients with proliferative diabetic retinopathy. CONCLUSIONS: In the study series, corneal esthesiometry was not a good indicator of diabetic retinopathy.

A one-dimensional prescribed curvature equation modeling the corneal shape

We prove existence, uniqueness, and stability of solutions of the prescribed curvature problem (u'/root 1 + u'(2))' = au - b/root 1 + u'(2) in [0, 1], u'(0) = u(1) = 0, for any given a > 0 and b > 0. We also develop a linear monotone iterative scheme for approximating the solution. This equation has been proposed as a model of the corneal shape in the recent paper (Okrasinski and Plociniczak in Nonlinear Anal., Real World Appl. 13:1498-1505, 2012), where a simplified version obtained by partial linearization has been investigated.