Paula Sweet

Femtosecond laser posterior lamellar keratoplasty: a laboratory model.

Purpose To evaluate feasibility of femtosecond laser application in posterior lamellar keratoplasty. Methods To evaluate the lasers 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-μm-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.

Verapamil potentiation of VP-16-213 in acute lymphatic leukemia and reversal of pleiotropic drug resistance

SummaryVerapamil, the calcium-influx-blocking agent, has previously been shown to have favorable interactions with antineoplastic drugs. Our study of human T cell acute lymphatic leukemia (ALL) GM3639 indicates that verapamil enhances the in vitro cytotoxicity of VP-16-213 against drug-sensitive ALL by reducing the concentration of VP-16-213, resulting in 50% cell viability from 104.5±26.6 nM to 46.0±2.7 nM (P<0.05). The addition of verapamil to VP-16-213 treatment of BDF/1 mice bearing L1210 leukemia increases their mean survival from 21.2±3.6 to 50.4±4.3 days (P<0.01) and the survival of CD2F/1 mice bearing P388 leukemia from 27.8±3.7 to 49.1±5.0 days (P<0.01). The 30-day survival is significantly increased in L1210 and P388 leukemia mice, and 60-day survival is significantly increased in P388 leukemic mice by verapamil.We developed a vincristine (VCR)-resistant subline of GM3639 T cell ALL, L23, by continuous exposure of drug-sensitive cells to VCR. This subline demonstrates pleiotropic cross resistance to VP-16-213 and daunorubicin. The addition of verapamil to VCR, to VP-16-213, and to daunorubicin completely restores responsiveness to these drugs, as indicated by the normalization of the VCR and VP-16-213 concentrations required for cytotoxicity and the concentration of daunorubicin required for inhibition of thymidine incorporation.

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.

Synergistic interaction of cyclosporin A and verapamil on vincristine and daunorubicin resistance in multidrug-resistant human leukemia cells in vitro

SummaryWe studied the effects of cyclosporin A and verapamil on the modulation of vincristine and daunorubicin resistance in a multidrug-resistant subline of human T-cell acute lymphatic leukemia GM3639. Our results show that cyclosporin A is more effective than verapamil as a modulator of the high degree of primary vincristine resistance and the low degree of daunorubicin cross-resistance expressed by this cell line. Isobologram analysis revealed that the combined modulators act synergistically in correcting both vincristine and daunorubicin resistance.

Comparison of cyclosporin A, verapamil, PSC-833 and cremophor EL as enhancing agents of VP-16 in murine lymphoid leukemias

Although verapamil, cyclosporin A. cremophor EL and PSC-833 are active as multidrug resistance modulators, there has been limited study of these compounds as possible chemotherapy enhancing agents against drug-sensitive tumors. We compared these agents as modifiers of VP-16 cytotoxicity in vitro and modifiers of VP-16 efficacy in vivo against drug-sensitive P388 and L1210 leukemias. Our study indicates that cyclosporin A enhances VP-16 cytotoxicity to a significantly greater extent than equimolar concentrations of verapamil or PSC-833. Although cremophor EL shows significantly greater activity than verapamil in VP-16 cytotoxicity enhancement in vitro, it is ineffective when added to VP-16 therapy of mice bearing L1210 leukemia.

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.

Corneal lenticule harvest using a microkeratome and an artificial anterior chamber system at high intrachamber pressure.

Purpose To evaluate the safety and accuracy of a manual microkeratome and an artificial anterior chamber used at high intrachamber pressure to harvest corneal lenticules for lamellar keratoplasty. Setting Department of Ophthalmology, University of California, Irvine, California, USA. Methods forty‐seven human eye‐bank corneoscleral rims were mounted on an artificial anterior chamber. a manual microkeratome was used to perform lamellar keratectomy at a mean intrachamber pressure of 95.8 mm hg ± 4.8 (sd). two thicknesses (300 &mgr;m and 360 &mgr;m microkeratome heads) and diameters (8.0 mm and 9.0 mm) were attempted, and the resultant lenticules were analyzed by pachymetry and digital photography Results In the 9.0 mm/360 &mgr;m group, corneal perforation occurred in 12 of 17 lenticules (71.2%). Except for this group, 24 of 30 corneas (80.0%) showed a less than 0.5 mm deviation from the expected diameter. Neither the horizontal nor the vertical diameter differences were statistically significant (P > .05). The difference between the horizontal and vertical diameters was within ±0.2 mm in 23 corneas (76.7%). Three (10.0%) corneal beds contained a slightly uneven keratectomy margin. Conclusions This system provided accurate and smooth lenticules for lamellar keratoplasty. The precision and accuracy of the obtained corneal lenticules were better than those in previous reports. However, the 9.0 mm diameter/360 &mgr;m thickness head with high intrachamber pressure should not be applied in the clinical setting without further testing. In this laboratory study, a minimal corneal thickness of 588 &mgr;m was required to avoid perforation.

A laboratory model for microkeratome-assisted posterior lamellar keratoplasty utilizing a running graft suture and a sutureless hinged flap.

Purpose. To evaluate changes in anterior corneal curvature and graft stability with different sized donor buttons in a laboratory model of posterior lamellar keratoplasty. Methods. Thirty-six human eye bank corneas (18 donors and 18 recipients) were mounted on an artificial anterior chamber. A manual microkeratome was used to create a hinged anterior lamellar keratectomy. A 7.0-mm diameter posterior lamellar disk (posterior stroma, Descemets membrane, and endothelium) was then trephinated from the recipient cornea. Three different sizes (7.0, 7.25, and 7.5 mm) of donor buttons were compared. They were sutured into the recipient bed with a running 10-0 nylon suture and covered by the host corneal flap. The flap was replaced without sutures. The resulting endokeratoplasty was analyzed by computerized videokeratography and tonometry. Results. Regular postoperative astigmatism was present in all cases. There was an average increase in astigmatism of 1.47 ± 1.49 diopters (D) postoperatively. The mean change in the average keratometry readings was −5.12 ± 6.12 D. The grafts and flaps maintained watertight seals with average leak pressures of 66.9 ± 46.4 mm Hg. Although donor buttons oversized by 0.5 mm had the least change in average keratometry reading, those oversized by 0.25 mm had the best stability at high pressure. All groups had little change in astigmatism. Conclusion. The optimal sized button of those tested would be either oversized by 0.25 or 0.5 mm. This new surgical technique may result in lower risk of high and irregular astigmatism in the management of corneal endothelial disorders.

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.

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 × 105 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.