Imaging of normal and pathologic joint synovium using nonlinear optical microscopy as a potential diagnostic tool.

An estimated 1.3 million people in the United States suffer from rheumatoid arthritis (RA). RA causes profound changes in the synovial membrane of joints, and without early diagnosis and intervention, progresses to permanent alterations in joint structure and function. The purpose of this study is to determine if nonlinear optical microscopy (NLOM) can utilize the natural intrinsic fluorescence properties of tissue to generate images that would allow visualization of the structural and cellular composition of fresh, unfixed normal and pathologic synovial tissue. NLOM is performed on rabbit knee joint synovial samples using 730- and 800-nm excitation wavelengths. Less than 30 mW of excitation power delivered with a 40×, 0.8-NA water immersion objective is sufficient for the visualization of synovial structures to a maximum depth of 70 µm without tissue damage. NLOM imaging of normal and pathologic synovial tissue reveals the cellular structure, synoviocytes, adipocytes, collagen, vascular structures, and differential characteristics of inflammatory infiltrates without requiring tissue processing or staining. Further study to evaluate the ability of NLOM to assess the characteristics of pathologic synovial tissue and its potential role for the management of disease is warranted.

A comparison of mass removal, thermal injury, and crater morphology of cortical bone ablation using wavelengths 2.79, 2.9, 6.1, and 6.45 microm.

BACKGROUND AND OBJECTIVE: Previous investigations have reported evidence of wavelength dependence on cortical bone ablation. This study used mid-infrared laser wavelengths generated by a free electron laser (FEL) and mass removal measurements to further examine the ablation efficiency of a wavelength (2.79 microm) not previously reported and three wavelengths (2.9, 6.1, and 6.45 microm) previously demonstrated by crater morphology alone to be efficient for cortical bone removal. STUDY DESIGN/MATERIALS AND METHODS: The wavelengths examined were provided by an FEL emitting 4 microseconds macropulses consisting of 1-2 picoseconds duration micropulses delivered at 350 picoseconds intervals. The mass removal measurements were conducted by a microbalance, and the collateral thermal injury and crater morphology of cortical bone were examined by light microscopy following standard histologic processing. RESULTS: The study demonstrated that the highest mass removal was achieved at lambda = 6.1 microm followed by, in order, lambda = 2.9, 6.45, and 2.79 microm. The zones of thermal injury and crater morphology created in cortical bone at the selected wavelengths were examined at the radiant exposure of 28.3 J/cm2. Ablation using lambda = 6.1 microm provided the largest crater size and the least collateral thermal injury. The greatest amount of collateral thermal injury was produced by lambda = 2.79 microm at both the sides and base of the ablation crater. CONCLUSIONS: The mass removal of cortical bone produced by FEL ablation at selected mid-IR wavelengths was measured as a function of incident radiant exposure. The ablation efficiency was found to be dependent upon wavelength. The lambda = 2.79 microm did not offer any improvement over the other wavelengths evaluated, suggesting that a potential shift in the dynamic optical properties of water during tissue irradiance with the FEL does not present an advantage to the cutting of cortical bone. The lambda = 6.1 microm provided the highest ablation efficiency with deepest crater and the least amount of collateral thermal injury.

Mid-IR laser ablation of articular and fibro-cartilage: a wavelength dependence study of thermal injury and crater morphology.

BACKGROUND AND OBJECTIVE: The aim of this study was to evaluate areas of collateral thermal injury and crater morphology for evidence of wavelength-dependent effects on the ablation of articular cartilage and fibro-cartilage (meniscus) using selected mid-IR wavelengths produced by a free electron laser. STUDY DESIGN/MATERIALS AND METHODS: Two types of cartilage, articular cartilage and fibro-cartilage were used in the study. The wavelengths (lambda) evaluated were 2.79, 2.9, 6.1, and 6.45 microm generated by a free electron laser (FEL) using a 4 microseconds macropulse configuration. The zone of thermal injury and crater morphology produced by laser ablation were examined by light microscopy following standard histologic processing. RESULTS: The zone of thermal injury and crater morphology created in cartilage by the FEL at selected mid-IR wavelengths were examined as a function of incident radiant exposure. Ablation using lambda = 6.1 microm provided the largest crater size for both articular and fibro-cartilage at all radiant exposures. For the zones of collateral thermal injury in articular cartilage, lambda = 6.1 microm produced the least thermal injury at the radiant exposure of 7.6 J/cm2. When the radiant exposure is increased to 20.4 J/cm2, both lambda = 6.1 and 6.45 microm produced less thermal injury than the ablation using lambda = 2.79 and 2.9 microm. The greatest amount of collateral thermal injury was produced by lambda = 2.79 microm for both tissue types. CONCLUSIONS: The results demonstrate that crater depth and collateral thermal injury produced in articular cartilage and fibro-cartilage are wavelength-dependent with 6.1 microm providing the largest craters at all radiant exposures. The least amount of thermal injury was created in articular cartilage using lambda = 6.1 microm at the radiant exposure of 7.6 J/cm2. Both 6.1 and 6.45 microm wavelengths demonstrated similar amount of thermal injury at 20 J/cm2 that was less than lambda = 2.79 and 2.9 microm at similar fluences. These observations are explained based on the absorption by water and protein in the tissue types studied. It is further observed that the use of crater dimensions may not provide a reliable estimate for the amount of tissue removal provided by an ablation procedure.

Femtosecond laser-assisted intracorneal keratoprosthesis implantation: a laboratory model.

PURPOSE: To demonstrate femtosecond laser-assisted intracorneal keratoprosthesis implantation and determine the mechanical stability as a function of intraocular pressure. METHODS: Eight human corneoscleral rims were mounted on an artificial anterior chamber. The femtosecond laser microkeratome was used to create a 2.5-mm diameter posterior corneal cap. A 7.2-mm-diameter lamellar stromal pocket was then created at mid-corneal depth. Finally, a 6-mm arc opening to the corneal surface was created at the periphery of the lamellar cut. The posterior lenticule was removed using corneal forceps and a 7.0-mm biopolymer keratoprosthesis was inserted into the stromal pocket. The surface wound was sealed using two 10-0 nylon sutures. A 3.0-mm anterior corneal opening was trephined to expose the keratoprosthesis. Intrachamber pressure was raised until wound leak was observed. RESULTS: Seven of the 8 implants withstood pressures of at least 135 mm Hg without implant extrusion. CONCLUSION: Femtosecond laser corneal dissection provides an alternative to more challenging manual dissection methods for keratoprosthesis implantation. Use of the femtosecond laser microkeratome will further refine keratoprosthesis surgical technique and may allow rapid and easy execution of the surgery.

Fluoroquinolone therapy in Mycobacterium chelonae keratitis after lamellar keratectomy.

PURPOSE: To characterize a rabbit model of Mycobacterium chelonae keratitis after lamellar keratectomy and assess the effectiveness of fluoroquinolone therapy. SETTING: University Laboratory, University of California, Irvine, California, USA. METHODS: Twenty-eight New Zealand white rabbits had unilateral lamellar keratectomy with placement of 2.5 x 10(5) colony-forming units of log-phase M chelonae under each flap. Eyes (7 per group) were randomized and treated with sterile balanced salt solution, gatifloxacin 0.3%, ciprofloxacin 0.3%, or levofloxacin 0.5% 4 times daily. Two masked observers examined all eyes on days 2, 5, and 7 and weekly for 4 weeks. Severity of disease and bacterial culture results were the main outcomes measured. The means and standard deviations were calculated, and differences between the groups were statistically analyzed. RESULTS: All eyes developed clinical disease. At the time the rabbits were killed, eyes treated with balanced salt solution, ciprofloxacin, levofloxacin, and gatifloxacin were culture positive in 6 (85.7%), 7 (100%), 6 (85.7%), and 3 (42.9%) of 7 eyes per group, respectively. Frequency of positive culture and the severity of clinical disease in gatifloxacin-treated eyes were significantly less (P < .05) than in the other groups combined. CONCLUSIONS: The rabbit model of M chelonae keratitis was successfully developed in our study. A fourth-generation quinolone (gatifloxacin) showed the best performance among the fluoroquinolones tested in our experimental approach. The fourth-generation fluoroquinolone, gatifloxacin, could be effectively used for the treatment of mycobacterial keratitis.

Acute endophthalmitis following cataract surgery: a systematic review of the literature.

OBJECTIVES: To determine the reported incidence of acute endophthalmitis following cataract extraction over time and to explore possible contributing factors, such as type of cataract incision. METHODS: A systematic review of English-language articles was conducted by performing a broad search of PubMed from 1963 through March 2003 using such terms as cataract extraction, endophthalmitis, and postoperative complication. Additional studies were identified from bibliographies of relevant articles and published proceedings. Surgical approach was recorded, when available. Pooled incidence rates and relative risks of developing endophthalmitis using different incision techniques were assessed. RESULTS: From 4916 unique, potentially relevant citations, 215 studies that addressed endophthalmitis and met the selection criteria were analyzed. A total of 3 140 650 cataract extractions were pooled resulting in an overall rate of 0.128% of postcataract endophthalmitis. However, the incidence of acute endophthalmitis changed over time, with a significant increase since 2000 compared with previous decades (relative risk, 2.44 [95% confidence interval, 2.27-2.61]). The rate of endophthalmitis was 0.265% in the 2000-2003 period, 0.087% in the 1990s, 0.158% in the 1980s, and 0.327% during the 1970s. Furthermore, an upward trend in rates after 1992 was noted, compared with 1991 and prior. Incision type appeared to significantly influence risk, as endophthalmitis following clear corneal cataract extraction during the 1992-2003 period was 0.189% compared with 0.074% (relative risk, 2.55 [95% confidence interval, 1.75-3.71]) for scleral incision and 0.062% (relative risk, 3.06 [95% confidence interval, 2.48-3.76]) for limbal incision. CONCLUSIONS: This systematic review indicates that the incidence of endophthalmitis associated with cataract extraction has increased over the last decade. This upward trend in endophthalmitis frequency coincides temporally with the development of sutureless clear corneal incisions.

A technique to harvest Descemet's membrane with viable endothelial cells for selective transplantation.

PURPOSE: To describe a surgical technique using an artificial anterior chamber to facilitate harvest of Descemet's membrane (DM) and endothelium for corneal endothelial cell transplantation. DESIGN: Laboratory investigation. METHODS: Corneoscleral buttons of seven human donor eyes were mounted endothelial side up on an artificial anterior chamber. Keeping the endothelial side with its usual concavity, a manual trephination was made on the posterior surface with a 9.0-mm trephine, inside the Schwalbe line and just past the DM in depth. The chamber was filled with air, causing the endothelial side of the donor cornea to assume a convex configuration. The DM along with its endothelium was separated from the posterior stroma using a blunt cyclodialysis spatula. Drops of trypan blue 0.3% and alizarin red S 0.2% (n = 6) were applied. The stained DMs were examined under a light microscope and photographed to calculate the percentage of endothelial cell damage. Histology was done on the unstained cornea. RESULTS: The DM carrying endothelium was successfully removed from the posterior stroma in all seven eyes. Although the DM appears to be very friable, all samples were removed in toto without rupture. Vital staining showed a mean endothelial cell loss of 8.46% (standard deviation (SD) 6.9). Direct light microscopy demonstrated the preservation of endothelial cell morphology. CONCLUSIONS: This technique appears to be a safe and straightforward method to harvest DM for endothelial cell transplantation. Further studies are underway to determine the optimal method of insertion of the obtained healthy DM with endothelial cells through small corneal incisions.

In vitro measurement of rabbit corneal epithelial thickness using ultrahigh resolution optical coherence tomography.

The objective of this study was to reproducibly measure corneal epithelial thickness centrally and at the limbus in the rabbit cornea using ultrahigh resolution optical coherence tomography (OCT). Twelve freshly enucleated New Zealand white rabbit eyes were kept in a moist chamber at 4 degrees C. An ultrahigh resolution OCT system with a spatial resolution of 1.3 microm was used to image the cornea and its component layers. The central and peripheral (limbal) regions of all the samples were scanned within 6 h of harvest in order to minimize the post-mortem degradation of the corneal epithelium. The thickness of the corneal epithelium was determined by measuring the pixel equivalents of the obtained image. Unpaired Student's t-test was used to evaluate differences. The epithelial thickness centrally was found to be 45.8 +/- 2.2 microm, and 37.6 +/- 1.4 microm at the limbus (P < 0.001). Rabbit corneal epithelium is thicker centrally than at the limbus when measured by ultrahigh resolution OCT. This technique will aid in delineating the pathophysiology of diseases of the anterior cornea.

Femtosecond laser posterior lamellar keratoplasty: a laboratory model.

PURPOSE: To evaluate feasibility of femtosecond laser application in posterior lamellar keratoplasty. METHODS: To evaluate the laser's effectiveness through opaque corneas, anterior corneal caps were resected from opaque corneas induced with 80% acetone solution. To evaluate the femtosecond laser posterior lamellar keratoplasty surgical procedure, human corneoscleral rims were mounted on an artificial anterior chamber. After corneal pachymetry, the femtosecond laser was used to create a 6-mm-diameter, 200-microm-thick endostromal lenticule. Access to the lenticule was provided by a small perilimbal surface opening, also created by the laser. The lenticule was removed using a pair of corneal forceps. A donor lenticule of similar dimensions was created, its endothelial surface coated with viscoelastic, inserted, and positioned on the recipient bed. Two sutures were placed to seal the small surface opening. RESULTS: The femtosecond laser produced an effective and smooth dissection through opaque corneas even at deeper settings. Graft transplantation was fairly simple and effective. CONCLUSION: Femtosecond laser posterior lamellar keratoplasty is a procedure that may provide an alternative to penetrating keratoplasty or the technically challenging manual posterior lamellar keratoplasty.

Inhibition of corneal neovascularization by a peroxisome proliferator-activated receptor-gamma ligand.

PURPOSE: To determine the efficacy of the peroxisome proliferator-activated receptor gamma agonist, pioglitazone, in inhibiting corneal neovascularization. METHODS: Twenty-six adult male Sprague-Dawley rats were randomly divided into three groups. Each group received intrastromal polymer micropellets containing one of the following: Group 1, no active ingredient (n=10); Group 2, vascular endothelial growth factor (VEGF) (n=7); Group 3, VEGF and pioglitazone (n=9). Neovascularization was evaluated 7 days after pellet implantation. After systemic India ink injection, digital photographs of the eyes were taken. The area and density of neovascularization were measured using imaging software. RESULTS: Mean area of neovascularization was 0.43+/-0.18 mm2 for Group 1, 2.87+/-0.48 mm2 for Group 2 and 2.10+/-0.22 mm2 for Group 3. Statistical analysis showed significant differences between Groups 1 and 2 and Groups 1 and 3. There was no significant difference between Groups 2 and 3. Mean density of neovascularization was 2.16+/-0.66 for Group 1, 27.14+/-2.93 for Group 2 and 12.02+/-2.24 for Group 3. All comparisons between groups were statistically significant (P<0.01). CONCLUSIONS: Pioglitazone is effective in decreasing the density of angiogenesis in a VEGF-induced neovascular rat cornea model. There is possibility of even greater effect with higher doses of the drug. Pioglitazone is a promising drug for the treatment of ocular neovascularization.

Mechanical stability of microkeratome-assisted intracorneal keratoprosthesis implantation.

OBJECTIVE: To develop a laboratory model to study intracorneal keratoprosthesis implantation. METHODS: A combination microkeratome and artificial anterior chamber system was used to create a hinged lamellar keratectomy on 13 human corneas. After reflecting the flap, the posterior stroma was trephined at either 2.5 or 3.0 mm. A model keratoprosthesis was positioned in the bed. The flap was sutured closed. Intrachamber pressure was increased, and wound leak pressure was recorded. The anterior corneal lamella was trephined at either 3.0 or 3.5 mm to expose the keratoprosthesis. Leak pressure was again determined. RESULTS: After keratoprosthesis placement and prior to anterior trephination, all 13 corneas were watertight at maximum attainable intrachamber pressures. With posterior/anterior trephination combinations of 2.5/3.0 mm, 2.5/3.5 mm, or 3.0/3.5 mm, mean +/- SD wound leak pressure occurred at 95 +/- 12 mm Hg, 32 +/- 7 mm Hg, or 59 +/- 12 mm Hg, respectively (P<.01). CONCLUSIONS: With a posterior trephination of 2.5 mm, there is significant keratoprosthesis-cornea interface destabilization between a 3.0- and 3.5-mm anterior trephination. For an anterior trephination of 3.5 mm, interface destabilization improves by increasing the posterior trephination to 3.0 mm. CLINICAL RELEVANCE: An intracorneal keratoprosthesis may be implanted using microkeratome assistance. Our laboratory model provides a useful method for examining a range of posterior and anterior trephination diameters and their effects on the mechanical stability of intracorneal keratoprosthesis placement.

193-nm excimer laser-induced fluorescence detection of fluoroquinolones in rabbit corneas.

OBJECTIVE: To measure the 193-nm excimer laser-induced fluorescence of fluoroquinolone-treated cadaver rabbit corneas. METHODS: Prior to ablation with a commercially available ophthalmic excimer laser (Nidek EC-5000; Nidek Technologies, Pasadena, Calif), 35 cadaver rabbit corneas were treated with topical sterile balanced salt solution, 0.3% tobramycin sulfate, or the fluoroquinolones-0.3% ofloxacin, 0.5% levofloxacin, 0.3% ciprofloxacin hydrochloride, or 0.3% gatifloxacin. The fluorescence generated from each ablated corneal layer was measured and used to identify the presence of antibiotic. This was achieved by training a partial least-squares model to discriminate between the fluorescence spectra of antibiotic-treated and antibiotic-free (healthy) cornea. Antibiotic concentrations down to 0.06 microg/mL were detected with high accuracy. Assuming a constant ablation rate of 0.3 microm per laser pulse, the number of corneal layers ablated to reach antibiotic-free cornea is used to calculate the penetration depth of the antibiotic. RESULTS: The mean +/- SD penetration to a detectable depth was as follows: 0.3% ofloxacin, 7.1 +/- 3.0 microm; 0.5% levofloxacin, 6.7 +/- 1.4 microm; 0.3% ciprofloxacin, 1.2 +/- 0.6 microm; and 0.3% gatifloxacin, 7.0 +/- 1.9 microm. The penetration depth of 0.3% tobramycin could not be determined because its fluorescence spectrum overlapped with that of the native cornea. CONCLUSIONS: Topical administration of fluoroquinolone-containing solutions results in measurable differences in laser-induced corneal fluorescence. Under these experimental conditions, 0.3% ofloxacin, 0.5% levofloxacin, and 0.3% gatifloxacin all appear to penetrate the epithelium significantly more than 0.3% ciprofloxacin (P<.02). Clinical Relevance Monitoring of laser-induced fluorescence may be helpful in determining the penetration depths and concentrations of topically applied fluoroquinolones within the cornea.

A portable microkeratome for harvesting the human anterior corneal surface.

A portable, gas-driven turbine microkeratome device capable of harvesting the entire anterior corneal surface for lamellar transplantation on human donor globes was evaluated. The device consisted of a modified LASIK microkeratome with an enlarged suction ring, head, and blade. Vacuum was achieved by a simple hand pump. Lamellar keratectomy was performed on 5 fresh human donor globes. Lenticule dimensions were measured on days 0, 3, 6, and 9 after storage in preservation media at 4 degrees C. On day 0, the obtained lenticules were 13.9 +/- 0.9 mm and 13.5 +/- 0.4 mm, vertical and horizontal diameters, respectively. The average central lenticule thickness was 152.2 +/- 52 microm. Each lenticule was uniform in thickness over 5 measurement points (P = 0.74). Repeat measurements of corneal thickness over the 9 days showed no statistically significant difference (P = 0.51). On day 9 lenticules were 14.6 +/- 0.3 mm and 14.6 +/- 0.4 mm, vertical and horizontal diameters, respectively. When day 0 was compared to day 9, vertical diameter also showed no statistically significant difference (P = 0.16), whereas horizontal diameter was significantly different (P < 0.001). This device proves to be an economical alternative to electric-powered systems for the harvest of transplantable corneal sections.

Viability of limbal epithelium after anterior lamellar harvesting using a microkeratome.

OBJECTIVE: To determine the viability in cold eye bank storage of different layers of central and limbal corneal epithelium, including the limbal basal stem cell population, on days 0, 3, 6, and 9 after harvest using a large diameter microkeratome system. METHODS: Twenty-two human whole globes not suitable for transplantation were obtained from an eye bank (San Diego Eye Bank, San Diego, California) and used for study. Large-diameter anterior corneal discs were prepared using a large diameter microkeratome and stained with calcein AM and an ethidium homodimer to differentiate live from dead cells, respectively. A laser confocal microscope and digital imaging were used to distinguish live (green) from dead (red) cells. Central and limbal epithelial regions were isolated and the middle and basal epithelial sections were cell counted by 3 independent observers. These sections were stored up to 9 days at 4 degrees C in an eye bank corneal storage medium. Differences were tested using nonparametric methods. MAIN OUTCOME MEASURES: The percentage of live cells in each of these epithelial layers was determined for up to 9 days in cold eye bank corneal storage medium. RESULTS: At all time points studied, the better protected basal epithelial layers displayed greater mean viability than the overlying middle epithelial layers. However, the difference was not statistically significant on all days. When comparing the basal epithelial viability of the limbal and central regions, after day 0 in 4 degrees C cold organ culture, the observed viability of the limbal basal epithelium, the purported location of the limbal epithelial stem cell region, was significantly greater than that of the central epithelium. On day 0, median limbal basal versus central basal epithelial viability were 100% (range, 71.7-100%) versus 98.4% (range, 88.9-100%) (P>0.05); on day 3, 100% (range, 64.3-100%) versus 63.4% (range, 13.6-95.5%) (P<0.0005); on day 6, 95.0% (range, 35.0-100%) versus 28.0% (range, 0-92.0%) (P<0.0005); and on day 9, 95.0% (range, 3.7-100%) versus 68.6% (range, 0-100%) (P<0.0005). CONCLUSIONS: After microkeratome harvesting, the limbal basal epithelium is significantly longer lived in cold eye bank storage than central basal epithelium and the middle layers of limbal and central epithelium. This longevity not only bodes well for organ storage of limbal grafts, but also confirms the hardiness of the stem cell region.

Changes in gene expression in response to mechanical strain in human scleral fibroblasts.

Scleral fibroblasts are involved in scleral remodeling during axial elongation in myopia. Mechanical load is a potent stimulator of gene expression. This study seeks to identify changes in gene expression of scleral fibroblasts in response to mechanical load and speculate on possible mechanisms of scleral remodeling in the development of myopia. Human scleral fibroblasts (HSFs) were mechanically stretched for 30 min and 24 hr. A gene microarray analysis was used to measure changes in gene expression. A total of 237 genes revealed differential and significant changes in expression (P<0.01) after 30 min of stretching. Of these, 28 unexpressed genes began to be expressed (turned on), while 31 expressed genes were no longer expressed (turned off). After 24 hr, 308 genes showed reproducible changes in expression (P<0.01), while 29 genes were turned on and 17 genes were turned off. After 30 min, 25 genes showed at least a threefold change in expression. These included genes for cell receptors, protein kinases, cell growth/differentiation factors, extracellular matrix (ECM) proteins, lipid metabolism, protein metabolism, transcription factors, binding proteins and water channels. After 24 hr, 21 genes showed at least a threefold change in expression. These included genes for cell receptors, protein kinases, cell growth/differentiation factors, lipid metabolism, ECM proteins, transcription factors, and carbohydrate metabolism. RT-PCR and Southern blotting confirmed the changes in expression of selected genes. In this study we identified a large number of early and late mechanical response genes in HSFs. These changes in gene expression will provide potential candidate genes that might be involved in scleral remodeling during axial elongation in myopia.

Biomechanical characterization of human amniotic membrane preparations for ocular surface reconstruction.

PURPOSE: To investigate the tensile and elastic properties of both commercially available and experimental human amniotic membrane preparations. METHOD: Nine preparations of human amniotic membrane were studied. The four dry preparations were untreated (nonirradiated, n = 20), and gamma (n = 25), low-dose (AmbioDry, Okto Ophtho Inc., Costa Mesa, Calif., USA, n = 20) and high-dose (n = 20) electron beam sterilized. The same dry membranes were moistened with balanced salt solution (n = 20, 34, 20 and 20, respectively). The ninth group consisted of thawed medium-frozen amniotic membrane (AmnioGraft, Bio-Tissue Inc., Miami, Fla., USA, n = 20). The membranes were cut into thin strips, loaded on a gram range load sensor, and stretched incrementally to the point of rupture. The modulus of elasticity, displacement until rupture and maximum tolerated stress were recorded and compared. RESULTS: The dry preparations exhibited higher moduli of elasticity when compared with the moist samples, with the low-dose electron beam-irradiated samples having the greatest mean modulus of elasticity overall and maintaining a high modulus of elasticity as a moist sample (p < 0.05). Moist nonirradiated preparations and thawed medium-frozen preparations stretched the farthest before rupture and experienced the greatest mean stresses at the point of rupture. While 3 of 4 membranes had greater stretch when moistened as compared to their dry counterparts, there was no difference in the membrane stiffness between dry and moistened low-dose electron beam-irradiated samples (p > 0.8). CONCLUSIONS: Low-dose electron beam-irradiated amnion appeared to maintain desirable elastic characteristics in transition from a dry to rehydrated state and may thus provide an easy-to-manipulate transplant tissue for ocular surface reconstruction. Moist nonirradiated and thawed medium-frozen tissues, however, may provide surgical advantages as they required greater forces to rupture.

Fluoroquinolone therapy in multiple-drug resistant staphylococcal keratitis after lamellar keratectomy in a rabbit model.

PURPOSE: To assess the effectiveness of a fourth-generation fluoroquinolone for prophylaxis against multiple drug-resistant staphylococcal keratitis after lamellar keratectomy in a rabbit model. DESIGN: Experimental study. METHODS: Twenty-eight New Zealand white rabbits underwent unilateral lamellar keratectomy using a manual microkeratome followed by the placement of 1000 colony-forming units (CFUs) of log-phase Staphylococcus aureus bacteria under each flap. Eyes (seven in each group) were randomized and treated with one of the following agents: sterile balanced salt solution, gatifloxacin (0.3%), ciprofloxacin (0.3%) or levofloxacin (0.5%) immediately and 6, 12, and 18 hours after surgery. Inflammation was graded by two masked observers at 24 and 48 hours, and the presence or absence of infectious infiltrates was determined. The means and standard deviations were calculated, and differences among the groups were statistically analyzed. RESULTS: There were no flap complications encountered during surgery. Eyes treated with ciprofloxacin, levofloxacin, and balanced salt solution developed infectious infiltrates in five of seven eyes per group. Gatifloxacin-treated eyes did not develop clinical infection and exhibited lower mean inflammation scores (P <.01 compared with the other groups). CONCLUSION: The fourth-generation fluoroquinolone, gatifloxacin, is an effective prophylaxis against the development of keratitis after lamellar keratectomy in rabbits with an organism resistant to methicillin, levofloxacin, and ciprofloxacin.

Endothelial lamellar keratoplasty using an artificial anterior chamber and a microkeratome.

OBJECTIVE: To compare postoperative astigmatic change and graft stability using 2 different donor button diameters in endothelial lamellar keratoplasty to treat corneal endothelial failure. METHODS: A 200- micro m-thick corneal flap keratectomy was performed in human donor corneoscleral rims (n = 20; 10 donors and 10 recipients) using an artificial anterior chamber and a manual microkeratome (ALTK System; Moria USA, Doylestown, Pa). After flap reflection, stromal bed trephination was performed to obtain a disc consisting of posterior stroma, Descemet membrane, and endothelium. Host beds of 7.0 mm and 7.25-mm (n = 5) or 7.50-mm (n = 5) donor buttons were obtained using a freehand trephine. The graft was secured with 8 interrupted sutures (10-0 nylon) in the stromal bed. The flap was sutured with 3 interrupted sutures. Transplanted corneas were submitted to increasing intrachamber pressures to detect graft stability, and preoperative and postoperative videokeratographic data were recorded to assess astigmatic change. RESULTS: The mean (SD) postoperative astigmatic change was 1.14 (3.17) diopters (D) in the 7.25-mm donor button group and 2.27 (1.77) D in the 7.50-mm donor button group (P =.69). Mean (SD) resisted pressures of 75.4 (44.81) mm Hg and 100.4 (46.86) mm Hg were observed in the 7.25-mm and 7.50-mm groups, respectively (P =.54). CONCLUSION: Both donor button sizes exhibited similar graft stability and astigmatic postoperative change in this experimental model. CLINICAL RELEVANCE: As endothelial lamellar keratoplasty becomes further developed as a clinical alternative to penetrating keratoplasty, this laboratory model system should be useful in evaluating different mechanical factors that contribute to graft success.

Fluorescence-guided laser removal of chemically damaged cornea.

PURPOSE: To correlate the observed fluorescence spectrum with the depth of ablation during 193 nm argon-fluoride excimer laser ablation of chemically damaged corneas. SETTING: Laser facility, Cedars-Sinai Medical Center, Los Angeles, California, USA. METHODS: Three cadaver New Zealand white rabbit corneas were exposed to 1 N hydrogen chloride for 10 seconds. The resultant opaque corneas were ablated to perforation using the excimer laser. Laser-induced fluorescence was collected at 45 degrees from incidence and channeled into an ultraviolet-visible spectrometer coupled to an optical multichannel analyzer reading a diode array detector. The detector recorded single-shot fluorescence spectra. The data were examined by principal component analysis, and the evolution of eigenvectors and their weighting coefficients were used to compare data among corneas. The results were correlated with histopathological sections. RESULTS: The eigenvalues of 3 principal components corresponded to 88.9%, 10.0%, and 0.4% of the data in acid-burned corneas. Compared to that in undamaged corneas, more information was stored in the first principal component and the third eigenvector was distinctly altered. Acid-scarred tissue blue shifted the dominant fluorescence peak compared to that in normal corneal tissue. CONCLUSIONS: After severe hydrogen chloride burn to the rabbit corneal surface, monitoring the dominant peak wavelength shift of excimer-laser-induced fluorescence can detect the transition between severely acid-damaged and underlying tissue.

A portable microkeratome-based anterior corneal surface harvesting device.

PURPOSE: To determine the reproducibility of anterior sclerokeratectomy using a portable nonelectric microkeratome-based device capable of harvesting the entire anterior corneal surface for lamellar transplantation. METHODS: A modified gas turbine-driven microkeratome (LSK One, Moria/Microtech, Doylestown, PA) with a redesigned head large enough to incorporate the whole human anterior corneal surface in a pass and was coupled to a manual vacuum pump. This instrument was tested on 25 fresh porcine globes divided into 2 groups (170-microm and 200-microm head). To assess cut reproducibility the physical dimensions (diameter and thickness) of the obtained lenticules were measured. RESULTS: The obtained lenticules were fairly circular (horizontal versus vertical diameters, p >0.2), with average diameters of 12.85 +/- 0.52 mm and 13.25 +/- 1.15 mm for the 170 and 200-microm heads, respectively. The average central lenticule thickness was 176.92 +/- 34.68 microm and 166.00 +/- 53.74 microm for the 170 and 200-microm heads, respectively. CONCLUSION: This new system presents an economical and portable alternative to electric-powered systems. In addition to being used by surgeons in the operating room, eye bank technicians in the field could theoretically use this system; including in developing countries where cost, availability of electricity, and portability are issues.