Lisa A. Buckley

PHOTOCHEMISTRY OF DNA USING 193 nm EXCIMER LASER RADIATION

Photoproducts in double‐stranded DNA induced by 193 nm radiation have been investigated. Double‐stranded, supercoiled pBR322 DNA in buffered aqueous solution was exposed to varying fluences of 193 nm radiation from an ArF excimer laser. The quantum yields for formation of cyclobutylpyrimidine dimers, frank strand breaks and alkali labile sites were calculated from the conversion of supercoiled (Form I) DNA to relaxed (Form II) DNA after treatment with Micrococcus luteus dimer‐specific endonuclease, no treatment, or treatment with alkali and heat, respectively. The quantum yields were 1.65 (± 0.03) × 10−3 for pyrimidine dimers, 9.4 (± 3.2) × 10−5 for frank strand breaks and 9.6 (± 3.6) × 10−5 for alkali labile sites. The quantum yields for pyrimidine dimers and frank strand breaks were independent of Tris buffer and EDTA concentrations. The yields of frank strand breaks and alkali labile sites were not affected by 10 mM mannitol. The relative quantum yields for these DNA photoproducts induced by 193 nm radiation differed markedly from those produced by 254 nm radiation.

Laser thrombolysis in an in-vitro model

Stroke is the leading cause of disability and is the third leading cause of morbidity in the United States. Approved therapies are limited. A novel therapy utilizing laser energy delivered through a fluid core catheter in the ablation of embolic thrombus in acute stroke is currently being tested in a clinical trial. In order to optimize the efficiency of clot removal by this system, an in vitro method to measure the effects of varying the laser parameters on the efficiency of clot removal was developed. Included in this model is a new reconstituted clot target that has reproducible mechanical and optical properties which allow reliable spectrophotometric measurement of the mass ablated. In this study the parameters of energy and repetition rate were adjusted while the average power remained constant at approximately 100 mW. The energy levels and repetition rates tested were 36 mJ at 3 Hz, 25 mJ at 4 Hz, 20 mJ at 5 Hz, 15 mJ at 7 Hz, and 12.4 mJ at 8 Hz. We demonstrate that the efficiency of clot removal achieved at higher pulse energies and low repetition rates can be maintained at a lower energy and higher repetition rate. These results are in agreement with previous studies using gel phantoms. This study demonstrates the reproducibility of the reconstituted clot and supports the use of this model as a clinically relevant method to investigate the efficiency of laser thrombolysis.

A reconstituted in vitro clot model for evaluating laser thrombolysis.

AbstractBackground/Objective: Laser thrombolysis is the selective removal of thrombus from occluded blood vessels using laser energy. A reconstituted clot model with reproducible optical absorption properties was developed to evaluate the effect of various laser parameters on thrombus removal rate. Study Design/Materials and Methods: Reconstituted clots were made with known fibrinogen concentrations and hematocrits. Ex vivo clots were collected from ten swine. Four red gelatin phantoms were prepared. Mass removal rates and ablation efficiencies were determined using a 577 nm, 1 μsec pulsed dye laser. The ablation efficiencies of the three clot models were compared at an energy of 25 mJ and a repetition rate of 4 Hz. In addition, the reconstituted clot model was ablated as pulse energy and repetition rate were varied with average power held constant at 100 mW. Results: The mean ablation efficiency for ex vivo clots ranged from 0.4 ± 0.1 to 3.4 ± 0.7 μg/mJ/pulse, with significant differences between groups (ANOVA p < 0.05). Reconstituted clots of varied fibrinogen content had ablation efficiencies of 1.5 ± 0.2 to 1.6 ± 0.3 μg/mJ/pulse at this energy and repetition rate. Gelatin ablation efficiency was inversely proportional to protein content and ranged from 0.5 ± 0.3 to 2.0 ± 0.7 μg/mJ/pulse. Reconstituted clot mass removal rates (in μg/s) were clinically similar for settings ranging from 13 mJ at 8 Hz to 33 mJ at 3 Hz. Conclusions: The reconstituted model clot is a reproducible and biologically relevant thrombolysis target. Ex vivo clot lacks reproducibility between individuals and gelatin phantoms lack clinical relevance. At a constant average power, varying laser parameters did not affect mass removal rates to a clinically significant degree.

Characterization of an in vitro laser thrombolysis model

Laser thrombolysis (LT) is under investigation as a safe and rapid therapy for arterial recanalization in acute embolic stroke. Clot formation is a complex process affected by many factors that lead to differences in strength and hemoglobin concentration between samples formed from whole blood. The strength of thrombus formed in vivo also varies with age. We have developed a reproducible and clinically relevant reconstituted clot model for the in vitro assessment of a laser thrombolysis system. Laser thrombolysis experiments were performed using a 577 nm 1 microsecond(s) ec pulsed dye laser at an energy of approximately 25 mJ and a repetition rate of 4 Hz. Laser ablation, tensile strength, scanning electron microscopic, and histological comparisons are made with other in vitro clot models. We have found that the tensile strength of this clot model is directly proportional to the fibrinogen concentration. Laser ablation and tensile pull test studies demonstrate that LT ablation efficiency (in (mu) g/mJ/pulse) is not significantly affected by differences in clot tensile strength. This agrees with previous studies using dye and gelatin. Our results provide support for the effective use of this laser thrombolysis system for the removal of clots of varied age and strength.

Photochemotherapy of intimal hyperplasia using Psoralen activated by uv light in porcine model

Psoralen activated by UVA light (PUVA) was investigated as a means of inhibiting smooth muscle cell proliferation resulting from balloon injury. Twenty kilogram domestic swine were anesthetized and underwent balloon angioplasty to create a 133% overstretch injury. Assignments of treatment and control were randomized between the left anterior descending (LAD) and circumflex (LCX) coronaries arteries. The animals were given with 5 mg/kg of 8- methoxypsoralen eternally. Treatment vessels received 600 mJ/cm2 of 364 nm light during balloon inflation to activate the psoralen. Control vessels received drug and balloon injury only. Serum was obtained during the light delivery to assess psoralen levels. At 30 days, animals were sacrificed and the coronary arteries perfusion fixed. Five sections per vessel were analyzed morphometrically to determine percent intimal area and extent of injury. The restenosis injury index was 0.21 plus or minus .02 in treatment vessels and 0.14 plus or minus .01 in the controls with a p-value less than .02. In this large animal model of balloon angioplasty injury, psoralen activated by ultraviolet light increased intimal hyperplasia.

MP41-06 MULTIPOTENT ADULT PROGENITOR CELL TREATMENT PROMOTES BLADDER FUNCTION RECOVERY AND DECREASES MORBIDITY AND DURATION OF URINARY TRACT INFECTION IN SPINAL CORD INJURED RATS

groups compared with PBS group (Figure 1). Hematoxylin/eosin staining demonstrated that loss of urothelial integrity in PBS group was restored in IV and BL group. Neutrophil infiltration was also decreased in IV and BL group compared with PBS group. Histological examination revealed a significant decrease in the total number of infiltrated mast cells in IV and BL rats compared with the PBS rats (p<0.05), (Figure 2). CONCLUSIONS: This is the first study to investigate the efficacy of CHA1 for chronic IC model. Submucosal injection of CHA1 in chronic IC showed improvement of voiding pattern and histological restoration and this suggest that CHA1 might have a therapeutic potential for IC. Further studies are required to evaluate the mechanism of CHA1 treatment.