BACKGROUND: Studies about the mechanism of laser occlusion on the varicose great saphenous vein are rare.OBJECTIVE: To explore the ultrastructural changes of the great varicose saphnous vein after it was occluded with laser.DESIGN: An observational study.SETTING: Laser Department of the General Hospital of Chinese PLA .PARTICI PANTS: There were 42 patients with varicose great saphenous veins that were occluded with laser in clinic of the General Hospital of Chinese PLA from January to April 2004. The inclusive criteria: There must be obvious vein tangles beneath the undystrophic skin at ankle without apparent thrombus inside the vein. The patients involved should be voluntary to take part in the study. Finally 9 patients were enrolled in this study.INTERVENTION: The great saphenous vein was intravenously occluded with laser of 810 nm and the working power was 12 W and the exposure time was 1 s. The occluded vein sample was taken out 3 hours after the occlusion.MAINOUTCOME MEASURES: The ultrastrncture of the occluded vein was observed histopathologically. Normal vein and prereatment varicose vein served as control.RESULTS: The normal vein wall can be divided into 3 layers: the internal layer was composed of the simple endothelial cells; the median layer was composed of the smooth muscle cells, elastic fibers and collagenous fibers; the external layer was composed of the loose connective tissues. However, the internal layer of the varicose vein was incomplete, and the endothelial cells were loosely connected. The smooth muscle cells became hyperplasic, hypertrophic or atrophic. The elastic fibers decreased in number in contrast to the increase of collagenous fibers. After laser occlusion, in the vein lumen there was a large number of blood cells. The platelets became flattened with pseudopods and adhered to collagenous fibers. The endothelial cells and smooth muscle cells near the lumen were damaged and the cytoplasma leaked and fused with extracellular matrix. Broken collagenous and elastic fibers could be seen near the lumen and some were observed in the lumen. There was no structure change in the external layer and adjacent elastic fibers and collagenous fibers.CONCLUSION: Laser occlusion damaged the internal layer and part of median layer of the varicose vein, caused aggregation of the blood cells in the lumen and promoted the adhesion of platelets to vein walls.

Objective To dynamically observe photodamage of subcellular sites by use of laser scanning confocal microscopy (LSCM). Methods The samples were divided into four groups. Murine lung endothelial cells were subcultured and incubated with HMME for 24 hours. Then the cells were stained with rhodamine-123 for demonstration of mitochondria. LSCM was applied and organelle-cell fluorescence intensity ratio analysis was adopted to study the intracellular distribution of HMME. Then dynamic fluorescence images sequence of rhodamine-123 was collected. Results Rhodamine-123′s fluorescence images of cell sample with HMME was changed gradually during irradiation: the typical characteristic of mitochondria disappeared gradually with decreasing fluorescence intensity. The fluorescence of rhodamine-123 was diffused and distributed in nuclear, while rhodamine-123′s fluorescence images of cell sample without HMME was not changed. Conclusion Mitochondria and nucleus are photodamage sites by HMME-PDT; LSCM can be applied in dynamic observation of photodamage of subcelluar sites.

BACKGROUND:The authors' experiments of the earlier stage proved that the hypocrellin B photodynamic therapy(HB-PDT) can cause selective injuries to choriocapillaries. It is not known whether changing the therapeutic parameters would gain a different result regarding the choriocapillaries after the hypocrellin B photodynamic treatment for a month.OBJECTIVE: To observe the features of Chinchilla rabbit choriocapillaries after HB-PDT treatment and to probe into the research prospect of using HB-PDT to treat choroidal neovascularization (CNV) and of using green light as the light source for PDT.DESIGN: A single sample study.SETTING: Laser Department of the Chinese PLA General Hospital.MATERIALS: The trial was conducted at the Laser Department and the Department of Pathology of the Chinese PLA General Hospital as well as the Department of Photoelectric Engineering of Beijing University of Science and Engineering. The materials included photosensitizing agent hypocrellin B (HB), a green laser transmitter, fundus fluorescence camera and transmission electronic microscope.METHODS: The 532 nm green laser transmitter and slit-lamp microscope were connected by light fiber. Chinchilla rabbits of 2.5 to 3.5 kg was narcotized generally and HB(1. 0 mg/kg) was injected into the marginal ear vein. HB was excited with the green laser of 532 nm. The power density of the light spot on fundus was 300 mW/cm2, and the energy density 30 J/cm2. Laser was applied immediately after HB injection and the diameter of the light spot was 2 000 μm. Direct observation of retina, fluorescein fundus angiography and observation with light microscope and electronic microscope were conducted on the 1st, 7th and 28th days respectively after PDT to find the biological features of retina and the choroid.MAIN OUTCOME MEASURES: Non-selective injury of retina through direct observation of the fundus; obliteration of the choriocapillaries detected through fluorescein fundus angiography; the position and extent of non-selective injury in retina and the structural changes of the choriocapillaries observed through the light microscope; the ultrastructural changes of the fundus observed through the electronic microscope.RESULTS: One day after PDT, photodynamic thrombosis was formed in choriocapillaries being illuminated and the external layer of retina was apparently injured. On the 7th day, injury of endothelial cells of the choriocapillaries was aggravated without obvious changes of the main vessels of choroid. On the 28th days, fibrous tissue appeared where the choriocapillaries had been and the glass membrane became thickened. Repair and proliferation of RPE cells appeared in the laser illuminated area.CONCLUSION: The biological effect in the target area and non-selective injury in the non-target area began to appear from the 1st to the 7th day after PDT and continued to aggravate. That would be repaired by fibrous tissues from the 28th day. It deserves further studies to treat age-related macular degeneration or other diseases in fundus characterized with choroidal neovascularization.

Objective To observe the effects of 510. 6nm laser irradiation on the apoptosis rate of vascularendothelial cell and smooth muscle cell in rabbit. Methods After the abdominal aorta of rabbits were irradiated bydifferent dosages of copper vapor laser (100mW/cm~2?500s, 100mW/cm~2?1 000s, 150mW/cm~2?500s, 200mW/cm~2?500s, 200mW/cm~2?1 000s, 100mW/cm~2?500s), the apoptosis rate of vascular endothelial cells andsmooth muscle cells was detected by use of TUNEL staining. Results The apoptosis rate of vascular smooth musclecell in each laser group was 21. 2%, 24. 5%, 30. 8%, 37. 3%, and 34. 5% respectively, while that was 3. 4% inthe control group and there was significant difference between control group and each laser group (P 0. 05, respectively). Conclusion The irradiation of copper vapor laser would induce ahigh rate of apoptosis of vascular smooth muscle cell, while had no significant effect on endothelial cell at the same sit-uation.

AIM: To investigate the characterization of absorption of hematoporphyrin monomerthyl ether (HMME), a domestic new generation photosensitizer product, by activated T cells from human peripheral blood. METHODS: Evaluation was performed by flow cytometry on the effects of incubating concentration and time of HMME on absorption by activated T cells. Lymphocytes were separated from human peripheral blood by density gradient centrifugation with Ficoll and T cells were activated with polyclonal stimulators PHA and PDB+Ion. To analyze the effects of HMME incubating doses on the absorption of activated T cells, the cultural lymphocytes were incubated with a serial doses of HMME for 1 h and HMME absorption were measured by FACS after immuno-staining with anti-CD3 antibody. To test the impact of HMME incubating time on the absorption of activated T cells, the cultural lymphocytes were incubated with HMME for various times and HMME absorption were measured by FACS after immuno-staining with anti-CD3 antibody. RESULTS: The HMME absorption-dose curve and absorption-time curve were shifted to right and up in the activated T cells as compared to resting T cells. HMME absorptions of activated T cells were statistic significantly larger than that of resting T cells in the doses between 5 mg/L to 20 mg/L. HMME absorptions of either activated T cells or resting T cells underwent a gradual increase with the incubation-time in HMME at concentration of 10 mg/L. HMME absorptions of activated T cells were statistic significantly larger than that of resting T cells in the incubation-time between 15 to 60 min. CONCLUSION: The differences of HMME absorption between activated T cells and resting T cells depend on the incubation times and doses of HMME. HMME absorption of activated T cells are significantly larger than that of resting T cells in certain incubation-times and doses. These results suggest that incubation time and dose associated with HMME-PDT therapeutic windows will be created for selective deletion of activated T cells.

Objective To compare and evaluate Organelle-cell fluorescence intensity ratio analysis established in this study with other techniques to determine the subcellular localization of photosensitizers. Methods CCD fluorescence microscopy imaging system was applied and a kind of special organelle probe BODIPY was selected to label Golgi body. Directly observing, pseudo-color fusing, wave-shape comparing, correlation coefficient calcula-ting and Organelle-cell fluorescence intensity ratio analysis were adopted, respectively, to study the intracellular distribution of domestic photosensitizer Hematoporphyrin monomethyl ether (HMME). Results Fluorescence distributing modes of HMME and BODIPY were similar with each other. There ware yellow space-overlap areas in the fusion image. Wave body of changing curve of gray scale value of all pixels in the straight line in two images corresponding to space coordinate was similar with each other. Correlation coefficient in each pixel between fluorescence intensity of HMME and that of BODIPY was 0.602 4. With increasing of parameter m, namely degree of organelles congregated reducing, the average fluorescence intensity ratio (J_1/J_2) of Golgi complex was decreasing, the two parameters po-ssessed obvious relativity. P