Biomedical subjects

W Matthew Petroll

Publications and source records attributed to W Matthew Petroll.

At least 19 recordsLinked to original sources

PubMed2007

Updates in Acanthamoeba keratitis.

PURPOSE: Acanthamoeba keratitis is a potentially blinding microbial disease that has been increasing in incidence during the past two decades. Prognosis of this serious disease had been dismal, but improvement in diagnosis, a better understanding of the natural course of the disease, and recent introduction of multiple and effective therapeutic agents have resulted in improvement of visual outcomes. METHODS: A review of literature pertaining to Acanthamoeba keratitis. RESULTS: Contact lens wear and exposure to contaminated water sources remain the most important risk factors; however, in vivo confocal microscopy and improved biomicroscopic screening have proven instrumental in accurate early diagnosis. Complications of Acanthamoeba keratitis include dacryoadenitis, corneal melting and scarring, severe secondary glaucoma, cataract, and chronic anterior segment inflammation that can rarely lead to reactive blinding retinal ischemia. Combination chemotherapeutic agents have been shown to be more effective than monotherapy, whereas rehabilitative surgery such as penetrating keratoplasty is best performed on a quiet eye free of ocular inflammation and with no residual amoebae. CONCLUSIONS: Increased suspicion by clinicians for Acanthamoeba and confocal microscopy have allowed more rapid and accurate diagnosis; treatment with multiple antiamoeba drugs is essential to disease resolution. Provided there are no residual amoebae after treatment, penetrating keratoplasty has been successful in visual rehabilitation. Secondary glaucoma occurs frequently and may require drainage procedures for control of intraocular pressure. Posterior complications are rare but may lead to ischemic retinitis.

Acanthamoeba↗

PubMed2006

Severe reactive ischemic posterior segment inflammation in acanthamoeba keratitis: a new potentially blinding syndrome.

OBJECTIVE: To describe a newly recognized clinical syndrome in Acanthamoeba keratitis consisting of severe reactive ischemic posterior segment vascular inflammation. DESIGN: Noncomparative, retrospective, single-institution observational case series. PARTICIPANTS: Five eyes of 5 patients with Acanthamoeba keratitis. METHODS: A retrospective review of the records of patients diagnosed with Acanthamoeba keratitis between January 1, 1995, and December 1, 2005, was conducted to identify those who underwent eventual enucleation. Five enucleated eyes of 118 eyes with Acanthamoeba keratitis were identified. MAIN OUTCOME MEASURES: History, clinical examination results, available laboratory study results, and histopathologic examination results. RESULTS: Histopathologic examination showed Acanthamoeba cysts in the cornea in 4 eyes, whereas it failed to demonstrate amebic cysts or trophozoites in the posterior segment of all eyes studied and unexpectedly revealed chronic chorioretinal inflammation with perivascular lymphocytic infiltration and diffuse neuroretinal ischemia in 4 of 5 eyes. Retinal artery thrombosis was present in 3 of the 4 involved eyes, and central retinal artery and vein thrombosis was found in 1 eye. Hematologic studies in 3 patients showed abnormal anticardiolipin antibody levels in 1 patient and factor V Leiden deficiency in another. CONCLUSIONS: Prolonged Acanthamoeba keratitis can result in a severe sterile ischemic posterior segment inflammation that is potentially blinding, especially in patients with underlying hypercoagulation disorders.

Acanthamoeba Keratitis↗

PubMed2006

Current concepts: contact lens related Pseudomonas keratitis.

Despite the development of silicone hydrogel lenses, Pseudomonas aeruginosa (PA) continues to be the leading cause of contact lens related microbial keratitis. Understanding the pathogenesis of PA-mediated corneal infection is critical to the development of new prevention and treatment strategies. Recently intracellular invasion of surface corneal epithelial cells by PA has been revisited as an important element in the infection process. This review identifies the mechanisms involved, and examines the roles of the lens, hypoxia alone, PA stain, cystic fibrosis transmembrane receptor protein (CFTR), and membrane lipid rafts in mediating intracellular invasion in both in vitro and in vivo conditions. Non-toxic blockade of raft formation in vitro or in vivo effectively abrogates PA internalization and may represent a unique, new strategy to prevent or ameliorate lens-related PA microbial keratitis.

Animals↗

PubMed2006

Quantitative assessment of local collagen matrix remodeling in 3-D culture: the role of Rho kinase.

The purpose of this study was to quantitatively assess the role of Rho kinase in modulating the pattern and amount of local cell-induced collagen matrix remodeling. Human corneal fibroblasts were plated inside 100-microm thick fibrillar collagen matrices and cultured for 24 h in media with or without the Rho kinase inhibitor Y-27632. Cells were then fixed and stained with phalloidin. Fluorescent (for f-actin) and reflected light (for collagen fibrils) 3-D optical section images were acquired using laser confocal microscopy. Fourier transform analysis was used to assess collagen fibril alignment, and 3-D cell morphology and local collagen density were measured using MetaMorph. Culture in serum-containing media induced significant global matrix contraction, which was inhibited by blocking Rho kinase (p<0.001). Fibroblasts generally had a bipolar morphology and intracellular stress fibers. Collagen fibrils were compacted and aligned parallel to stress fibers and pseudopodia. When Rho kinase was inhibited, cells had a more cortical f-actin distribution and dendritic morphology. Both local collagen fibril density and alignment were significantly reduced (p<0.01). Overall, the data suggests that Rho kinase-dependent contractile force generation leads to co-alignment of cells and collagen fibrils along the plane of greatest resistance, and that this process contributes to global matrix contraction.

Actins↗

PubMed2006

Confocal assessment of the corneal response to intracorneal lens insertion and laser in situ keratomileusis with flap creation using IntraLase.

PURPOSE: To assess the response of the cornea to hydrogel intracorneal lens (ICL) insertion or laser in situ keratomileusis (LASIK) with IntraLase (IntraLase Corp.) at the cellular level. SETTING: Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas, Texas, USA. METHODS: Twenty patients (29 eyes) were evaluated by in vivo confocal microscopy 1 to 6 months postoperatively: 20 eyes had LASIK with flap creation by IntraLase, and 9 eyes had ICL insertion (8 following IntraLase). RESULTS: For LASIK with IntraLase, keratocyte activation and/or interface haze was detected in 8 of 20 eyes. The remaining eyes had interface particles but no cell activation. Keratocyte activation was generally limited to a few cell layers adjacent to the interface. However, 2 patients exhibited multiple layers of activation and increased extracellular matrix (ECM) reflectivity (haze) surrounding the interface by confocal microscopy. Both patients also had clinical haze and photophobia. For ICLs, following insertion, 5 of 9 eyes had activated keratocytes adjacent to the implant surfaces. The largest amount of cell activation and ECM haze detected by confocal microscopy was in 2 patients with significant clinical haze. Structures with an epithelioid morphology were detected on some implant surfaces. Epithelial thickness was 33.3 microm +/- 2.3 (SD) in the ICL eyes and 49.2 +/- 6.5 microm in the LASIK with IntraLase eyes. CONCLUSIONS: Both LASIK with IntraLase and ICL insertion following IntraLase induced keratocyte activation, which may underlie clinical observations of haze in some patients. Intracorneal lens implant also induced thinning of the overlying corneal epithelium.

Biocompatible Materials↗

PubMed2006

Noninvasive corneal stromal collagen imaging using two-photon-generated second-harmonic signals.

PURPOSE: To investigate the feasibility of using femtosecond-pulse lasers to produce second-harmonic generated (SHG) signals to noninvasively assess corneal stromal collagen organization. SETTING: The Eye Institute, University of California, Irvine, California, USA. METHODS: Mouse, rabbit, and human corneas were examined by two-photon confocal microscopy using a variable-wavelength femtosecond lasers to produce SHG signals. Two types were detected: forward scattered and backward scattered. Wavelength dependence of the SHG signal was confirmed by spectral separation using the 510 Meta (Zeiss). To verify the spatial relation between SHG signals and corneal cells, staining of cytoskeletons and nuclei was performed. RESULTS: Second-harmonic-generated signal intensity was strongest with an excitation wavelength of 800 nm for all 3 species. Second-harmonic-generated forward signals showed a distinct fibrillar pattern organized into bands suggesting lamellae, while backscattered SHG signals appeared more diffuse and indistinct. Reconstruction of SHG signals showed two patterns of lamellar organization: highly interwoven in the anterior stroma and orthogonally arranged in the posterior stroma. Unique to the human cornea was the presence of transverse, sutural lamellae that inserted into Bowman's layer, suggesting an anchoring function. CONCLUSIONS: Using two-photon confocal microscopy to generate SHG signals from the corneal collagen provides a powerful new approach to noninvasively study corneal structure. Human corneas had a unique organizational pattern with sutural lamellae to provide important biomechanical support that was not present in mouse or rabbit corneas.

Aged, 80 and over↗

PubMed2006

Tandem scanning confocal corneal microscopy in the diagnosis of suspected acanthamoeba keratitis.

OBJECTIVE: To evaluate the role of in vivo corneal tandem scanning confocal microscopy (TSCM) in the definitive diagnosis of suspected Acanthamoeba keratitis (AK). DESIGN: Noncomparative interventional single-institution case series. METHOD: A retrospective case review of patients consecutively referred with suspected AK and undergoing corneal TSCM was performed. RESULTS: A total of 63 cases that met the inclusion criteria for the study were referred for diagnostic evaluation. Tandem scanning confocal microscopy demonstrated Acanthamoeba cysts/trophozoites in 54 cases and fungal hyphae in 2, whereas 1 case was positive for both Acanthamoeba and fungus. Culture of the cornea or contact lenses was carried out in 35 cases, 9 of which were positive for Acanthamoeba. Six of the TSCM-positive cases also underwent corneal biopsy, being positive for Acanthamoeba in only 2. Six patients were negative for Acanthamoeba on TSCM, the etiology being fungal in 1 case, as shown by subsequent culture. One patient was positive on culture for Acanthamoeba but falsely negative by TSCM, which was limited by poor cooperation during the examination. Two cases initially masqueraded as Acanthamoeba keratitis but showed fungus on TSCM. Mean follow-up was 14 months. CONCLUSION: In vivo corneal TSCM can establish the diagnosis of Acanthamoeba keratitis rapidly and noninvasively, particularly when conventional microbiology is inconclusive.

Acanthamoeba Keratitis↗

PubMed2006

Bcl-2 and Bax regulation of corneal homeostasis in genetically altered mice.

PURPOSE: The antiapoptotic protein Bcl-2 and its proapoptotic counterpart Bax may play an important role in the regulation of corneal epithelial renewal. To understand the physiologic importance of Bcl-2 and Bax in the healthy cornea, epithelial and stromal thickness, proliferation, and surface cell exfoliation rates were examined in the central cornea of genetically altered mice overexpressing Bcl-2 and lacking Bax. METHODS: Adult Bcl-2 transgenic (n = 55) and Bax knockout (n = 82) mice and wild-type controls were clinically prescreened at no less than 2 months of age for any ocular developmental abnormalities. Polymerase chain reaction was used to confirm genotype. Corneal epithelial renewal was examined by in vivo tandem scanning confocal microscopy to measure total corneal and sublayer thickness and by immunohistochemistry on whole mount corneal tissues to determine basal epithelial cell proliferation using 5-bromo-2-deoxyuridine (BrdU) and surface cell exfoliation with a Calcein AM-Ethidium homodimer assay (live/dead). Stained corneas were scanned with a laser scanning confocal microscope, and images were digitized, and cell counts were obtained. Levels of Bcl-2 protein were assessed by Western blots. RESULTS: No significant changes in proliferation or cell death were found in either group compared to wild-type littermate controls; however, epithelial and stromal thicknesses were greater in the Bcl-2 transgenic group compared to wild-type and decreased in the Bax knockout. Western blotting confirmed that there was no change in the level of Bcl-2 expression in the corneal epithelium in the Bcl-2 transgenic or Bax knockout strains. CONCLUSIONS: The significant difference in epithelial and stromal thickness suggests a functional role for Bcl-2 and Bax in the maintenance of corneal homeostasis; however, the lack of significant alterations in proliferation, cell exfoliation, and levels of Bcl-2 protein in the adult corneal epithelium suggest that these phenotypic changes are a result of a new stable homeostatic equilibrium (proliferation + migration = shedding). Additional experiments to delineate the role of Bcl-2 and Bax during development and in the adult cornea are necessary.

Animals↗

PubMed2006

Prolonged hypoxia induces lipid raft formation and increases Pseudomonas internalization in vivo after contact lens wear and lid closure.

PURPOSE: To investigate the effects of hypoxia on lipid raft formation and Pseudomonas aeruginosa internalization by the corneal epithelium with and without the physical effects of contact lens wear. METHODS: One eye of each rabbit was randomly fitted with a low-Dk rigid gas-permeable contact lens (LDCTL) or closed with sutures, with the other as a control. After 1 day or 3 days, the rabbits were killed and bacterial invasion was assessed by gentamicin survival assay. Lipid rafts were identified by staining with FITC-conjugated beta subunit of cholera toxin. Corneal epithelial Bcl-2 expression was detected by Western blotting; surface epithelial cell size and thickness (epithelium and stroma) were measured by confocal microscopy. RESULTS: One-day hypoxia induced no significant changes in P. aeruginosa internalization, Bcl-2 expression, or lipid raft formation except in one of four eyelid-closed eyes. After 3 days, P. aeruginosa internalization was increased significantly (P < 0.05) in LDCTL-wearing eyes and not significantly (P = 0.10) increased in eyelid-closed eyes. Both 3-day test conditions also induced lipid raft-forming cells that bound P. aeruginosa, albeit in different regions of the cornea (peripherally in LDCTL-wearing eyes and centrally in closed eyes); did not alter epithelial thickness or surface cell size; and appeared to decrease epithelial Bcl-2 expression. CONCLUSIONS: This is the first direct comparison in vivo between two different methods inducing hypoxia on the corneal surface. Association of P. aeruginosa internalization with lipid raft formation in both conditions suggests a critical link among prolonged hypoxia, lipid raft formation, and susceptibility to P. aeruginosa infection. However, different distribution patterns of lipid raft-forming cells suggest physical effects of contact lens wear may direct localization of lipid raft-associated P. aeruginosa internalization on the corneal surface.

Animals↗

PubMed2006

Confocal assessment of the effects of fourth-generation fluoroquinolones on the cornea.

PURPOSE: To evaluate the toxicity of fourth-generation fluoroquinolone antibiotic solutions on the rabbit corneal epithelium. METHODS: In vivo confocal microscopy was used to assess epithelial structure in 18 rabbits, and tight junction integrity of superficial epithelial cells was evaluated with ZO-1 labeling in 10 rabbits. Eyes were bathed with commercial solutions of moxifloxacin (Vigamox) or gatifloxacin (Zymar) solution for 3 minutes, rinsed with balanced salt solution, and immediately examined. Balanced salt solution rinsing alone served as the control. RESULTS: A decrease in epithelial cell size was observed after treatment with Zymar (P < 0.05, two-way repeated-measures analysis of variance), but not with Vigamox or the control. Normal ZO-1 organization was observed in controls and eyes treated with Vigamox. ZO-1 staining in eyes treated with Zymar was disrupted, patchy, and generally weaker than that in control eyes. CONCLUSIONS: After short-term, intensive exposure to Vigamox, corneal epithelial integrity and tight junction organization are maintained. Zymar induces a loss of superficial epithelial cells and breakdown of tight junctions under similar conditions.

Animals↗

PubMed2006

Evaluation of intrastromal lipid deposits after intacs implantation using in vivo confocal microscopy.

PURPOSE: To assess the structure and location of intrastromal lipid deposits after implantation of Intacs by using in vivo confocal microscopy. METHODS: Seven eyes of six patients were examined by in vivo confocal microscopy 5 years (n = 6) or 2 months (n = 1) after uncomplicated implantation of Intacs for the correction of mild myopia. Selected images from all corneal layers were qualitatively evaluated for structural changes, with special attention paid to areas surrounding the Intacs implants. RESULTS: In the peripheral cornea of eyes examined 5 years after surgery, epithelial and endothelial cell layers appeared normal. Tandem scanning confocal microscopy showed stromal haze surrounding the implants in all eyes examined, but no keratocyte activation was seen. Reflective amorphous or crystalline structures consistent with lipid deposition were detected in all eyes with long-term implantation of Intacs. Deposits were localized to the inner and outer edges of Intacs segments and to the region anterior to the implant. Confocal microscopy did not show any deposits in the eye examined 2 months after surgery, although the region anterior to the implant appeared hazy and edematous. Areas central to the implant appeared normal in all eyes. CONCLUSIONS: The mechanical and physiologic stresses introduced by the implantation of Intacs lead to the accumulation of lipid deposits in the extracellular matrix. By using in vivo confocal microscopy, the location and structure of these deposits can be determined.

Adult↗

PubMed2005

Quantitative assessment of ophthalmic viscosurgical device retention using in vivo confocal microscopy.

PURPOSE: To develop and apply a new laboratory method for in vivo quantitative assessment of the retention of ophthalmic viscosurgical devices (OVDs) following phacoemulsification. SETTING: Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas, Texas, USA. METHODS: Studies of both eyes of New Zealand White rabbits were performed. Six OVDs were evaluated: Provisc and Healon (both sodium hyaluronate 1%), Healon5 (sodium hyaluronate 2.3%), Amvisc Plus (sodium hyaluronate 1.6%), Viscoat (sodium hyaluronate 3%-chondroitin sulfate 4%), and a new viscous-dispersive OVD, DisCoVisc (sodium hyaluronate 3%-chondroitin sulfate 4%). The OVD was injected to fill the anterior chamber and a phacoemulsification needle inserted with the tip positioned just anterior to the lens capsule. Simulated phacoemulsification was performed for 1 minute using flow rates of 20, 40, and 60 mL/min; a vacuum level of 300 mm Hg; and ultrasound power of 60% using a Legacy phacoemulsification unit. The needle was removed, and silicone oil (1000 centistokes) was injected into the anterior chamber. The distance between the corneal endothelium and the OVD-silicone oil interface was measured using in vivo confocal microscopy through-focusing (CMTF). RESULTS: Significant differences in residual thickness were found between the OVDs tested. Specifically, the residual thickness of both DisCoVisc (mean 324.5 microm +/- 163.7 [SD]) and Viscoat (251.4 +/- 100.9 microm) was significantly greater than that of Provisc (9.5 +/- 16.7 microm), Healon (3.8 +/- 11.3 microm), Healon5 (0.6 +/- 2.4 microm), and Amvisc Plus (65.6 +/- 134.0 microm) (P < .05, Dunn test). Ophthalmic viscosurgical device retention was greatest with DisCoVisc; however, there was no statistically significant difference between DisCoVisc and Viscoat in residual thickness. The flow rate did not have a significant effect on the residual thickness (Friedman 2-way analysis of variance by ranks). CONCLUSIONS: Residual OVD thickness following simulated phacoemulsification could be quantitatively measured using in vivo CMTF. The results are consistent with human surgical experience in that the dispersive OVD (Viscoat) was better retained than the cohesive compounds. DisCoVisc, a new viscous-dispersive OVD, also showed retention compared with Viscoat under the experimental conditions.

Animals↗

PubMed2005

Central corneal thickness in patients with congenital aniridia.

PURPOSE: To compare the mean central corneal thickness (CCT) in patients with congenital aniridia to that of a group of age-matched control subjects. The findings of specular and confocal microscopy in a patient with aniridia are discussed. METHODS: The mean values of five consecutive pachymetry measurements of patients with aniridia and control subjects were used for analysis. Statistical analysis was performed with a Mann-Whitney rank sum test. Specular microscopy was performed on one patient with aniridia using a Konan Specular Microscope Noncon ROBO CA (Hyogo, Japan). Confocal microscopy through focusing was performed with the Tandem Scanning Confocal Microscope (Reston, VA). RESULTS: Mean CCT measured 691.8 +/- 75.4 mum for patients with aniridia (16 eyes of 10 patients) and 548.2 +/- 21.2 mum for control subjects (P < 0.001). Specular microscopy in a patient with aniridia showed normal endothelial cell counts and structure. Confocal microscopy through focusing of this patient showed normal-appearing keratocytes and a thick corneal stroma. CONCLUSIONS: Patients with congenital aniridia have significantly thicker corneas than do age-matched control subjects. This difference can have important implications for the treatment of those patients who develop secondary glaucoma. The increased CCT in patients with aniridia is not a result of endothelial dysfunction but appears to be the result of the production of a thickened but otherwise healthy cornea by the mutated PAX6 gene.

Adolescent↗

PubMed2004

Four-dimensional multiphoton confocal microscopy: the new frontier in cellular imaging.

This paper reviews new developments in microscopy that combine gene transfer technology, multiphoton confocal fluorescence microscopy, live cell imaging and digital imaging techniques that provide unique insights into the complex physiological processes involved in tissue function at the cellular and subcellular level. The evolution of this novel, new technology is discussed with particular attention to earlier achievements in noninvasive ocular surface imaging. The practical basis of confocal microscopy, multiphoton confocal fluorescence microscopy, and the vital fluorescent labeling of cells in living tissues are also discussed. Additionally, one application using retroviral gene transfer to express enhanced green fluorescent protein in living wound healing fibroblasts is presented as an example of how living biology can be studied in situ in four dimensions (x, y, z, time).

Journal Article↗

PubMed2004

Evaluation of the corneal effects of topical ophthalmic fluoroquinolones using in vivo confocal microscopy.

PURPOSE: To compare the effects of several fluoroquinolone antibiotics on the corneal epithelium and stroma using in vivo confocal microscopy. METHODS: Five antibiotic solutions were evaluated: 1) 0.3% ofloxacin (Oflox) solution with 0.005% benzalkonium chloride (BAC); 2) 0.3% gatifloxacin (Gati) solution with 0.005% BAC; 3) 0.3% ciprofloxacin (Cipro) solution with 0.006% BAC; 4) 0.5% levofloxacin (Levo) with 0.005% BAC; and 5) 0.5% moxifloxacin (Moxi) solution with no BAC. Preservative-free artificial tears (Tears) were used as a control. New Zealand white rabbits were used for this study (six per solution group). Ten days prior to exposure to any solution, central corneal epithelial thickness and stromal thickness were measured using in vivo confocal microscopy through focusing. Images of the superficial epithelium were also acquired. Both eyes of each rabbit then received one drop of the assigned solution six times the first day and then four times per day for 6 days. On day 7, in vivo confocal microscopy was repeated. RESULTS: A significant decrease in epithelial thickness was induced by 7 days of exposure to Levo, Gati, Oflox, and Cipro (P < 0.05, two-way repeated-measures ANOVA, Tukey test). Tears and Moxi, which do not contain BAC, did not induce significant changes in epithelial thickness. No significant changes in stromal thickness were detected (P = 0.266), and no keratocyte activation was observed for any of the solutions evaluated. CONCLUSION: We have previously used confocal microscopy to establish a correlation between epithelial thinning (due to superficial cell loss) and slight ocular irritation. The results of this study suggest that Moxi induces less damage to the corneal epithelium than other antibiotic solutions, perhaps because it does not contain BAC.

Animals↗

PubMed2004

Pseudomonas aeruginosa corneal binding after 24-hour orthokeratology lens wear.

PURPOSE: To examine the effect of short-term 24-hr orthokeratology lens (OKL) wear on Pseudomonas aeruginosa binding, epithelial surface cell morphology, epithelial sheet thickness, and stromal thickness in a rabbit model. METHODS: Seventeen New Zealand white rabbits were treated according to the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. Partial membranectomy was performed on all rabbits 1 week before the experiments. Baseline values for epithelial and stromal thickness and epithelial surface cell size were determined by in vivo confocal microscopy in one randomly chosen eye (n = 6). One week later, rabbits were fitted in the same eye with a hyper oxygen-transmissible OKL. Twenty-four hours later, confocal microscopy was repeated. The second group of rabbits (n = 6) was fitted with an OKL in one randomly chosen eye for 24 hr. P. aeruginosa binding to the corneal epithelium was assessed for the control corneas and those exposed to the test lens. Scanning electron microscopy was performed on a third group of rabbits to assess epithelial surface damage (n = 5). RESULTS: There was a statistically significant difference (P<0.001) in P. aeruginosa binding between the control (1.11 +/- 0.74 x 10(5) colony-forming units per cornea) and the OKL-wearing eyes (2.74 +/- 0.69 x 10(5) colony-forming units per cornea). The central epithelium thinned by 6.5% after lens wear (48.2 +/- 1.9 microm to 45 +/- 1.7 microm, P=0.005); however, central stromal thickness increased by 7.3% (322 +/- 22 microm to 345 +/- 29 microm, P=0.006). Compared with the baseline value, central epithelial cell size increased significantly from 1,253 +/- 140 mm(2) to 1,627 +/- 393 mm(2) (29.4%, P=0.02). Scanning electron microscopy showed increased surface epithelial damage associated with OKL wear. CONCLUSIONS: This prospective, masked, pilot study showed that 24-hr hyper oxygen-transmissible OKL wear induced a statistically significant increase in P. aeruginosa binding to the epithelium of the rabbit cornea, accompanied by central epithelial thinning, stromal thickening, and surface cell damage assessed by scanning electron microscopy. Collectively, the data suggest that despite adequate lens oxygen transmissibility, the mechanical pressure inherent in the OKL design exerted on the corneal surface appears to be associated with increased adherence of P. aeruginosa to surface corneal epithelial cells, which may pose an increased risk for lens-related microbial keratitis, especially in overnight (i.e., closed-eye) wearing conditions. Future studies are needed to determine whether these results are similar in human wear and how P. aeruginosa binding during OKL wear compares with other lens-wearing modalities, such as daily or continuous soft lens wear.

Animals↗

PubMed2004

Corneal fibroblasts respond rapidly to changes in local mechanical stress.

PURPOSE: To investigate the response of corneal fibroblasts to local changes in extracellular matrix (ECM) tension. METHODS: Rabbit and human corneal fibroblasts were plated inside fibrillar collagen matrices. After 18 to 72 hours, a glass microneedle was inserted into the ECM and either pushed toward a cell to reduce local tension, or pulled away to increase tension. Time-lapse differential interference contrast (DIC) imaging was performed both before and after needle micromanipulation. ECM displacements were quantified, and strain maps were generated by finite element modeling. In some experiments, cells were treated with the Rho-kinase inhibitor Y-27632 either 30 minutes before, or 1 hour after they were pushed with the microneedle. Changes in focal adhesion organization were also evaluated in a subset of cells expressing green fluorescent protein (GFP)-zyxin, by simultaneous fluorescent and DIC imaging. RESULTS: Pulling on the ECM resulted in initial cell elongation, followed by disengagement and retraction of pseudopodia. In contrast, pushing the ECM toward a cell induced rapid shortening (contraction), presumably since existing cellular forces were no longer counterbalanced by ECM tension. Pseudopodial extension (spreading) was then observed at both ends of the cell. The ECM was pulled inward during this secondary spreading, and rapid turnover of focal adhesions was observed along extending pseudopodia. Preincubation with Y-27632 or cytochalasin D blocked both the initial contractile and secondary spreading responses. CONCLUSIONS: Overall, the data suggest that corneal fibroblasts actively respond to increases or decreases in local matrix stress in an attempt to maintain tensional homeostasis (constant tension), and that this response may be mediated by Rho and/or Rac.

Amides↗

PubMed2003

Direct correlation of collagen matrix deformation with focal adhesion dynamics in living corneal fibroblasts.

The purpose of this study was to develop and apply a new model for investigating how the organization and movement of cell-matrix adhesion sites correlate with force generation by corneal fibroblasts on a fibrillar collagen extracellular matrix. Primary cultures of rabbit corneal fibroblasts were transfected using a vector encoding GFP-zyxin to allow visualization of adhesion sites. Cells were plated at low density on top of 100 micro m thick fibrillar collagen lattices embedded with 2 micro m diameter red fluorescent beads. Time-lapse imaging was performed at one minute intervals for up to 3 hours. At each time interval, GFP-zyxin, bead and DIC images were acquired in rapid succession using filter wheels. Cells were treated with cytochalasin D and/or Triton X-100 at the end of each experiment. The movements of adhesions and nearby matrix landmarks were measured and correlated from the time-lapse digital images, and the size, intensity and orientation of the adhesions were quantified. GFP-zyxin was detected in adhesions of transfected corneal fibroblasts as confirmed using vinculin counterstaining. Time-lapse imaging revealed extensions and retractions of cell processes and displacements of the fiduciary beads that were similar to control cells. Extending processes exhibited the most complex behavior, with new adhesions continuously forming at the leading edge while existing adhesions moved backward in a retrograde fashion. This process generated tractional forces as indicated by pulling in of the extracellular matrix in front of the cell. Interestingly, during extension, adhesions along the ventral surface of the cell body generally moved toward those at the tip, resulting in contractile-like shortening and matrix compression at the base of lamellipodia. Overall, a high correlation was found between both the magnitude (R=0.87, P<0.001) and direction (R=0.98, P<0.001) of the adhesions and nearby matrix displacements. Cytochalasin D induced rapid and reversible disassembly of adhesions, cell elongation and matrix relaxation, including decompression at the base of the lamellipodia. This new experimental model allows direct, dynamic assessment of cell-matrix interactions on a fibrillar collagen matrix. Our results are consistent with the previously described 'frontal towing' model of cell motility and demonstrate for the first time that this mechanism is employed by cells interacting with a fibrillar extracellular matrix.

Actins↗