Angioresistance of thermally modified cartilage grafts in the chick chorioallantoic membrane model.

BACKGROUND: The chick chorioallantoic membrane (CAM) model allows direct observation of vascularization acutely in explanted or cultured tissues in an immunologically isolated environment. In vivo, angioinvasion of the tissue matrix does not occur in viable cartilage tissue, whereas denatured or nonviable grafts are readily vascularized and/or resorbed. OBJECTIVE: To determine, using the CAM model, whether angioinvasion of thermally altered cartilage explants occurs acutely. MATERIALS AND METHODS: Porcine septal cartilage specimens were removed from freshly killed animals and divided into 3 groups (n = 10): an untreated control group, a group in which cartilage was boiled in isotonic sodium chloride solution (normal saline) for 1 hour, and a laser-irradiated group (Nd:YAG, lambda = 1.32 microm, 30.8 W/cm2, irradiation time = 10 seconds). Tissue specimens were then washed in antibiotic solutions, cut into small cubes (approximately 1.5 mm3), placed on the surface of 30 CAMs (7 days after fertilization), and allowed to incubate for an additional 7 days. After incubation, the membranes and specimens were fixed in situ with formaldehyde and then photographed using a dissection microscope. RESULTS: Examination with a dissecting microscope showed no obvious vascular invasion of the cartilage or loss of gross tissue integrity in any of the 3 experimental groups, although all specimens were completely enveloped by the CAM vascular network. No vascular invasion of the tissue matrix was observed histologically. CONCLUSION: These experiments demonstrate that cartilage specimens remain acutely resistant to angioinvasion or metabolism by the immunologically immature CAM whether native unmodified tissue, completely denatured (boiled), or thermally modified following laser irradiation.

Systemic application of photosensitizers in the chick chorioallantoic membrane (CAM) model: photodynamic response of CAM vessels and 5-aminolevulinic acid uptake kinetics by transplantable tumors.

The aim of this study is to modify the chick chorioallantoic membrane (CAM) model into a whole-animal tumor model for photodynamic therapy (PDT). By using intraperitoneal (i.p.) photosensitizer injection of the chick embryo, use of the CAM for PDT has been extended to include systemic delivery as well as topical application of photosensitizers. The model has been tested for its capability to mimic an animal tumor model and to serve for PDT studies by measuring drug fluorescence and PDT-induced effects. Three second-generation photosensitizers have been tested for their ability to produce photodynamic response in the chick embryo/CAM system when delivered by i.p. injection: 5-aminolevulinic acid (ALA), benzoporphyrin derivative monoacid ring A (BPD-MA), and Lutetium-texaphyrin (Lu-Tex). Exposure of the CAM vasculature to the appropriate laser light results in light-dose-dependent vascular damage with all three compounds. Localization of ALA following i.p. injections in embryos, whose CAMs have been implanted with rat ovarian cancer cells to produce nodules, is determined in real time by fluorescence of the photoactive metabolite protoporphyrin IX (PpIX). Dose-dependent fluorescence in the normal CAM vasculature and the tumor implants confirms the uptake of ALA from the peritoneum, systemic circulation of the drug, and its conversion to PpIX.

Photodynamic activity of lutetium-texaphyrin in a mouse tumor system.

BACKGROUND AND OBJECTIVE: New photosensitizers proposed for photodynamic therapy (PDT) treatment of tumors need to be evaluated in animal models to determine the parameters needed for treatment. They also need to be compared with existing photosensitizers for efficacy. We examined the PDT response to lutetium-texaphyrin (PCI-0123) in a mouse mammary adenocarcinoma model and compared it with the PDT response seen when using Photofrin. STUDY DESIGN/MATERIALS AND METHODS: DBA/2 mice with SMT-F tumors were used to explore PCI-0123 toxicity, laser light dose, and drug dose effects on PDT response and to determine the most effective time for light application. The PDT response of PCI-0123-treated tumors was compared with that of Photofrin-treated tumors. RESULTS: Treatment of tumors with 150 J/cm2 of 740 nm laser light 5-6 hr after PCI-0123 administration (40 mg/kg) resulted in a 100% response rate and a 55% cure rate. Tumors treated with 150 J/cm2 of 630 nm laser light 24 hr after Photofrin administration (10 mg/kg) resulted in a 67% response rate and a 16% cure rate. CONCLUSION: PCI-0123 was found to be a more effective photosensitizer than Photofrin.

Photodynamic parameters in the chick chorioallantoic membrane (CAM) bioassay for topically applied photosensitizers.

The relative efficacy of Photofrin-based photodynamic therapy (PDT) has been compared with that of the second-generation photosensitizers 5-aminolevulinic acid (ALA), sulfonated chloro-aluminum phthalocyanine (AlPcSn), benzoporphyrin derivative monoacid ring A (BPD-MA), and lutetium texaphyrin (Lutex). PDT-induced vascular damage in the chick chorioallantoic membrane (CAM) is measured following topical application of the photosensitizers. In order to make meaningful comparisons, care is taken to keep treatment variables the same. These include light dose (5 and 10 J/cm2), power density (33 and 100 mW/cm2), and drug uptake time (30 and 90 min). The drug dose ranges from 0.1 microgram/cm2 for BPD to 5000 micrograms/cm2 for ALA. Results are also analyzed statistically according to CAM vessel type (arterioles versus venules), vessel diameter, and vessel development (embryonic age). For each photosensitizer, the order of importance for the various PDT parameters is found to be unique. The differences between the sensitizers are most likely due to variation in biophysical and biochemical characteristics, biodistribution, and uptake kinetics.

In vitro and in vivo comparison of argon-pumped and diode lasers for photodynamic therapy using second-generation photosensitizers.

BACKGROUND AND OBJECTIVE: Three prototype microchannel-cooled stacked diode array lasers were compared with the currently used conventional argon ion laser-pumped tunable dye lasers for suitability as light sources in photodynamic therapy (PDT) treatment. STUDY DESIGN/MATERIALS AND METHODS: The PDT response of Chinese hamster ovary (CHO-K1) cells in culture and SMT-F tumor bearing mice treated with chloro-aluminum sulfonated phthalocyanin (CASPc), benzoporphyrin derivative mono-acid (BPD-MA), and lutetium texaphyrin (Lutex) was determined using each laser light source. Survival of the CHO cells was measured using a cloning assay. Tumor regression/eradication was used to assess response in the mice. RESULTS: Both sources of laser light produced comparable PDT responses in the two systems tested. CONCLUSION: It would be possible to replace the currently used argon ion laser-pumped dye laser systems with the diode lasers tested.

Differential vascular response to laser photothermolysis.

Individual blood vessels in the chick choriallantoic membrane were selectively coagulated through photothermolysis, using pulsed laser irradiation at 585 nm. Pulse durations were chosen to be 0.45 ms and 10 ms, which correspond to the thermal relaxation times in blood vessels of 30 microns and 150 microns diameter, respectively. The short pulses, at a light fluence F = 3 Jcm-2, caused permanent occlusion of vessels of 40 microns diameter or less, whereas larger caliber vessels (60-120 microns) required F = 4-5 Jcm-2. The long-duration pulses, at F = 7 Jcm-2, caused coagulation of the larger diameter vessels; the small-caliber vessels and capillaries showed resistance to photothermolysis and required multiple exposures to achieve coagulation. The fluence versus diameter (F versus d) relationship for coagulation was calculated for the two pulse durations. The energy deposited in a cylindrical absorber of diameter d by an optical field, incident perpendicular to the vessel, was expressed analytically and compared with the energy required to coagulate a blood vessel of the same lumen dimeter. When thermal diffusion is incorporated into the model, our findings can be accounted for quantitatively. This information will be of use for improving the laser treatment of port wine stains and other vasculopathies. A surprising observation was that arterioles were damaged at lower incident energy densities than venules having the same lumen diameter, despite the fact that absorbance in oxygen-rich and oxygen-poor blood is the same at 585 nm.

Demonstration of synergistic effects of hyperthermia and photodynamic therapy using the chick chorioallantoic membrane model.

The chick chorioallantoic membrane (CAM) model was used to study vascular effects of photodynamic therapy (PDT) and hyperthermia (HPT) and the synergism of these modalities. The CAM is a convenient medium for monitoring the modifications of the vasculature. It is possible to view the CAM and to examine structural changes of individual blood vessels in real time. Moreover, the CAM is a closed system which lends itself to mathematical modeling of the temporal and spatial temperature profile and in which HPT can be performed quantitatively and to a selected depth, using different lasers. A porphyrin-type photosensitizer solution was applied to areas of the CAM, defined by teflon O-rings placed on the surface. Uptake dynamics of the sensitizer into the CAM was determined by analyzing its fluorescence in vivo. The CAM area was irradiated with a dual-wavelength laser system composed of a dye laser at 644 nm (to induce PDT) and a CO2 laser at 10.6 microns (to bring about HPT). Damage to the CAM vasculature, due to combined PDT+HPT, was compared to the outcome of the separate modalities, and a synergistic effect of about 40% was observed.

Excimer laser radiation for endarterectomy of experimental atheromas.

Open laser endarterectomy produces a smooth arterial surface with welded distal end points. This report evaluates 308-nm excimer laser radiation for the laser endarterectomy operation. Arteriosclerotic New Zealand white rabbits (N = 15) were studied. A thoraco-abdominal exploration was performed, the aorta was isolated, heparin was administered, and multiple endarterectomies were performed in each rabbit. A line of laser craters was created at the proximal and distal ends of an atheroma. Laser radiation was used to connect the craters to form the proximal and distal end points. The atheromas were dissected from the aorta with laser light and the end points were fused. The aortas were removed for light and electron microscopy and the animals were sacrificed. Excimer radiation was delivered by a 600-microns fiber at 50 mJ/pulse, 120-ns pulses and either 15- or 20-Hz frequency. At 15 Hz excimer laser endarterectomies showed no perforations along the surface or at the end points. The surfaces were smooth but the end points were not welded in place. At 20 Hz, perforations were seen along 7/11 surfaces and at 5/11 end points. Excimer laser endarterectomy is best performed at 15 Hz. The end points, however, cannot be welded with excimer laser radiation.

Comparison of contact and free beam laser endarterectomy.

Free beam laser endarterectomy (LE) and contact laser endarterectomy (CLE) were compared in 15 arteriosclerotic New Zealand white rabbits. The rabbits underwent balloon catheter trauma to the thoracoabdominal aorta and were fed a 2% cholesterol diet for 18 weeks. Thoracoabdominal exploration was performed under general anesthesia and multiple endarterectomies were performed in each rabbit. Atheromas were dissected from arteries with laser radiation and end points were welded in place with laser radiation. LEs (N = 8) were performed with argon ion radiation delivered through a 400 microns fiberoptic. Power was kept constant at 1 W and the average fluence was 97.5 +/- 6.6 J/cm2. CLEs were performed with conical sapphire probes powered by either argon ion radiation (N = 12) or Nd-YAG radiation (N = 10). Power used was 1 W to 4 W for each laser. Average argon ion fluence was 117.8 +/- 3.1 J/cm2 and average Nd-YAG fluence was 611.1 +/- 34.4 J/cm2. Following the operations, aortas were removed, fixed, serially sectioned, and stained. Microscopic study revealed welded end points with LE but not with CLE. There were no perforations with LE. There were 11/12 perforations with argon ion CLE and 8/10 perforations with Nd-YAG CLE. Free beam laser endarterectomy is superior to contact laser endarterectomy for experimental atheromas.

Mutation and sister chromatid exchange induction in Chinese hamster ovary (CHO) cells by pulsed excimer laser radiation at 193 nm and 308 nm and continuous UV radiation at 254 nm.

We compared mutagenesis and sister chromatid exchange (SCE) induction by 193 nm and 308 nm pulsed excimer laser radiation with 254 nm low intensity continuous wave UV light in Chinese hamster ovary (CHO) cells in culture. The 254 nm radiation was most mutagenic of the radiations, in accordance with expectation, and also was most effective in increasing the level of SCEs. The 193 nm radiation was mutagenic at the ouabain resistance locus, but not at the HGPRT locus. However, 193 nm radiation was also strongly cytotoxic at energies producing measurable mutations. This radiation also caused a dose-related increase in SCEs. Pulsed excimer radiation at 308 nm was mutagenic at both loci, and also increased the incidence of SCEs. Comparison of the ratio of mutants/surviving cells at the D37 after radiation showed similar values for 254 nm and 308 nm at the HGPRT locus, but at the ouabain resistance locus, the ratio for the 308 nm radiation was about 5 times that for 254 nm radiation. These results indicate that some risk for mutagenesis may accompany the use of excimer radiation in the UVA region in therapeutic applications.

Comparison of continuous-wave lasers for endarterectomy of experimental atheromas.

The standard surgical lasers, argon ion, neodymium-yttrium aluminum garnet, and carbon dioxide, are often operated as continuous wave lasers with specific uses. Clinical trials of laser therapy for arteriosclerotic cardiovascular disease are underway with all three lasers. Therefore, we compared these three lasers under controlled experimental conditions. A thoracoabdominal exploration was performed in 17 arteriosclerotic rabbits. The aorta was isolated, heparin administered, and multiple endarterectomies were performed in each rabbit with each of the lasers. A line of laser craters was created at the proximal and distal ends of an atheroma. Continuous-wave laser radiation was used to connect the craters and thereby form proximal and distal end points. The plaques were dissected free from the aorta with laser light and the end points were fused by laser. The aortas were removed for light microscopy and the animals were killed. The endarterectomy surfaces and end points were serially sectioned and graded according to light microscopic findings (1 = worst, 4 = best). Argon ion laser endarterectomy (N = 16) required 106 +/- 10 J/cm2. The surface score was 3.5 and end point score 3.4. Neodymium-yttrium aluminum garnet laser endarterectomy (N = 13) required 1,289 +/- 115 J/cm2 with a surface score of 2.4 (p less than 0.001 from argon ion) and an end point score of 1.3 (p less than 0.001 from argon ion). Carbon dioxide laser endarterectomy (N = 9) required 30 +/- 5J/cm2 with a surface score of 2.0 (p less than 0.001 from argon ion) and and end point score of 1.6 (p less than 0.001 from argon ion). Perforation occurred in one of 16 argon ion studies (technical error, not laser), in 11 of 13 neodymium-yttrium aluminum garnet studies, and in six of nine carbon dioxide studies. This study demonstrates that of the currently available clinical continuous-wave lasers, the argon ion laser is superior for endarterectomy of experimental atheromas.

Photosensitization of experimental atheromas by porphyrins.

Arteriosclerotic arteries have been shown to fluoresce when treated with hematoporphyrin derivative. This study investigates the incorporation and distribution of a partially purified form of hematoporphyrin derivative (Photofrin II) in normal and arteriosclerotic rabbit aortas. A thoracoabdominal exploration was performed in 15 rabbits. Group I comprised normal rabbits, Group II normal rabbits given 5 mg/kg Photofrin II 48 hours before surgery, Group III arteriosclerotic rabbits and Group IV arteriosclerotic rabbits given 5 mg/kg Photofrin II 48 hours before surgery. Multiple aortic biopsy specimens for frozen section were taken from all rabbits. In addition, open laser endarterectomy (with an argon ion laser) was performed on Group III and Group IV rabbits. Frozen sections were studied by digital video fluorescence microscopy to determine the distribution of Photofrin II within the layers of the aortic wall. The fluorescence of the intima of Group IV rabbits was found to be significantly greater than that of the intima, internal elastic lamina, media or adventitia of the other groups (p less than 0.01) and significantly greater than that of the internal elastic lamina, media or adventitia of Group IV rabbits (p less than 0.01). When open laser endarterectomy was performed, Group III rabbits required 103 +/- 14 J/cm2 and Group IV required 33 +/- 3 J/cm2 (p less than 0.01). It is concluded that porphyrins are selectively localized within the intima of arteriosclerotic arteries. This localization sensitizes atheromas to argon ion laser light and facilitates laser endarterectomy.

The thrombogenic potential of argon ion laser endarterectomy.

The surface thrombogenicity of atheromas, conventional endarterectomy (CE), laser endarterectomy (LE), and laser angioplasty (LA) were compared in the rabbit arteriosclerosis model. Normal (N = 6) and arteriosclerotic (N = 15) rabbits underwent thoracoabdominal exploration. Multiple CEs and LEs were performed in 12 arteriosclerotic rabbits leaving a segment of intact atheroma between each endarterectomy. Multiple LAs were performed in three arteriosclerotic rabbits. Argon ion laser radiation was used for all laser procedures. Blood (0.05 ml) from normal rabbits was placed on the CE surface, LE surface, LA surface, atheroma, and normal intima and clotting times were determined. Surface thrombogenicity was calculated as the ratio of the clotting time of the CE, LE, LA, or atheroma to normal intima. Surface thrombogenicity was 1.0 +/- 0.03 for normal intima (control), 0.58 +/- 0.06 for atheromas (P less than 0.001), 0.46 +/- 0.08 for CE (P less than 0.001 from atheromas), 0.46 +/- 0.08 for LE (P = NS from CE), and 0.27 +/- 0.09 for LA (P less than 0.001 from CE and LE). The thrombogenicity of LE is the same as the thrombogenicity of CE. Both forms of endarterectomy are less thrombogenic than LA in the rabbit model.

Experimental arteriosclerosis treated by argon ion and neodymium-YAG laser endarterectomy.

The argon ion laser (488 and 514.5 nm) and the Nd-YAG laser (1.06 micron) have been used in most studies of laser radiation for treatment of arteriosclerotic cardiovascular disease because their beams can be directed through a delivery fiber. We compared the effects of argon ion and Nd-YAG lasers in vivo on arteriosclerotic rabbit aortas with open laser endarterectomy. A thoracoabdominal exploration was performed in 16 rabbits to isolate the aorta. Laser beams were directed through delivery fibers to produce a line of laser craters at the proximal and distal ends of an atheroma. The lines of laser craters were connected by continuous-wave laser radiation to loosen the plaque and the cleavage plane was developed within the media by continuous-wave laser radiation. End points were fused by continuous-wave laser radiation and the aortas were harvested for light and scanning electron microscopy. Argon ion laser endarterectomy (eight rabbits) required an energy density of 98 +/- 19 J/cm2. Nd-YAG laser endarterectomy (eight rabbits) required an energy density of 1147 +/- 129 J/cm2. Perforation did not occur with the argon ion laser but occurred in six of eight Nd-YAG experiments. Even depths of plaque removal resulted from argon ion laser endarterectomy but not from Nd-YAG laser endarterectomy. Gross and microscopic grading of the aortas showed that argon ion endarterectomy surfaces were significantly better than Nd-YAG surfaces (p less than .05). Similarly, argon ion end points were significantly better than Nd-YAG end points (p less than .01).(ABSTRACT TRUNCATED AT 250 WORDS)

Experimental arteriosclerosis treated by conventional and laser endarterectomy.

Open laser endarterectomy was compared to standard surgical endarterectomy in the rabbit arteriosclerosis model. The aorta was exposed by a thoracoabdominal exploration in 16 rabbits. In Group I (8 rabbits), a conventional endarterectomy (CE) was performed with standard vascular instruments. In Group II (8 rabbits), laser endarterectomy (LE) was performed with an argon ion laser (488 nm and 514.5 nm) at a power of 1.0 W. Aortas were fixed, serially sectioned (6 micron) and stained (H +/- E) following each procedure. Gross and light microscopic examination revealed identical results for the endarterectomy surfaces of CE and LE. The proper cleavage plane within the media was developed with both techniques and the remaining arterial wall was not damaged with either procedure. The end points of LE were consistently superior to those of CE because of phototherapy fusion. The LE end points were tapered and the intima was fused. Intimal flaps were seen in 2/8 CE experiments and the remaining end points exhibited an uneven transition. LE required an average energy density of 124 +/- 9 J/cm2. We conclude that LE provides a satisfactory method for the in vivo evaluation of laser radiation upon arteriosclerotic arteries. LE may be the way to begin clinical laser trials.

Laser endarterectomy.

Laser radiation can be applied directly at a diseased artery to dissect the plaque away from the media and perform an open endarterectomy. The appropriate cleavage plane within the media can be achieved with the laser beam for precise reconstruction of the artery. Laser endarterectomy offers a technique for the study of laser radiation upon arteriosclerosis and may become a new modification of a standard surgical technique.

Laser applications to arteriosclerosis: angioplasty, angioscopy, and open endarterectomy.

We have studied laser radiation of arteriosclerotic cardiovascular disease to determine the best technique for evaluation. Rabbits with arteriosclerosis were treated by intraluminal laser angioplasty (N = 8), laser angioscopy (N = 2), and open laser endarterectomy (N = 5). All studies were performed with an argon ion laser delivering energy through a 400-microns quartz fiber. Power delivered was varied in order to determine the best value for this animal model. Following angioplasty, perforation was seen in three rabbits (in one case, it was due to mechanical trauma from the fiber optic), and early thrombosis occurred in two rabbits. Plaques were vaporized by angioscopy, but the depth of laser beam penetration or angle of incidence could not be controlled. Open laser endarterectomy gave consistently good results with removal of an arteriosclerotic plaque within the media. The optimum power was found to be 1.0-2.0 W. Endarterectomy required an energy density of 100-140 J/cm2. We conclude that open laser endarterectomy can be a standardized technique for the in vivo evaluation of laser radiation upon arteriosclerotic lesions.

In vitro cellular effects of hematoporphyrin derivative.

Several in vitro cell systems were exposed to hematoporphyrin derivative (HPD): established lines of rat kangaroo epithelial kidney; normal mouse embryonic fibroblasts; and differentiated neonatal rat myocardial cells. The uptake of HPD (25 to 100 micrograms/ml) by individual cells occurred rapidly over a 2-hr period and leveled off by 24 hr. HPD was excreted from cells by 48 hr after exposure. However, a low level of HPD (above background) was maintained in cells for up to 4 days following cessation of exposure. Intracellular binding of HPD was to mitochondria as demonstrated by fluorescence microscopy. HPD was also shown to have a growth-inhibiting effect on rat kangaroo cells without added light. The growth effects on mouse cells were less marked.