## 4 Discussion  
Chorioretiinal endothelial cells from rhesus monkeys were used in this study. The rhesus monkey eye was found to resemble the human eye in various aspects. Mainly, the retina possesses a macula common only in monkeys, humans, and apes. The macula in rhesus monkeys and humans contains macular pigment, a foveal pit consisting of the maximum concentration of cones, a capillary-free region, and the dislocation of inner retinal layers and vessels. Further, rhesus monkeys and humans were also found to share common genes susceptible to age-related macular degeneration [74]. The Griess method is specific for measuring nitrite [75]. NO is a gaseous free radical with a half-life of a few seconds or less. So, NO is indirectly measured in the cell culture supernatant through the levels of the more stable NO metabolites, nitrite (NO\({}_{2}\)) and nitrate (NO-) [76; 77; 78; 79]. The mean minimum detectable dose was 0.25 \(\mu\)mol/L for the kit used. In this study, the total NO was measured using the Griess method by converting nitrate into nitrite using nitrate reductase. The nitrite was then detected as an azo dye through calorimetric detection [80; 81]. The PGI\({}_{2}\) kit used was non-species specific. The polyclonal antibody in the kit binds competitively in equal affinities with 6-keto-Prostaglandin F1\(\alpha\) (6k-PGF1\(\alpha\)) and 2,3-dinor-6-keto-Prostaglandin F1\(\alpha\) (2,3d-6k-PGF1\(\alpha\)) [82]. In the current study, we used phenol red solution as a blood substitute to provide optical absorption for PUT. The phenol red solution was previously used in studying the role of ultrasound in the biochemical production of endothelial cells [47]. The use of phenol red can avoid the blood flowing inside the channels to cause the sticking of blood cells to the channel wall. The spatial-peak temporal-average ultrasound intensities used in this study were 0.033, 0.833, and 3.333 W/cm\({}^{2}\), which are at the same order of magnitude as previous studies, and the increase in NO was obvious even at the lowest intensity ultrasound [71]. A previous study demonstrated that the NO level from endothelial cells will increase under low ultrasound pressures [72], which is consistent with our result from 0.1 to 1 MPa. The reduction in the NO level for ultrasound pressure from 1 to 2 MPa is likely due to the effect of strong cavitation activities, which can cause the death of endothelial cells [3], as demonstrated by the increase in apoptotic cells. Some inconsistencies in the measurement results, particularly at the high energy levels, could be an inherent feature of the cavitation-related effect. The cavitation threshold was at 1.3 MPa for ultrasound-only. Figure 11: Apoptosis of endothelial cells after being treated with 0.5 MPa ultrasound peak negative pressure and 15 mJ/cm\({}^{2}\) laser fluence. Apoptotic cells show green fluorescence, and live cells show little or no fluorescence. Similarly, the cavitation was observed for the 15 mJ/cm\({}^{2}\) and 0.5 MPa treatment group. Near or above the cavitation threshold, excessive cell death may occur locally. Because of the erratic behavior of cavitation, the results can vary in a large range. Our measurement showed that: (1) at a low energy level, ultrasound-only and laser-only generally increased the release of NO and PGI\({}_{2}\); and (2) ultrasound-only at a high energy level, or the combined laser and ultrasound (PUT), suppressed the release of NO and PGI\({}_{2}\). This reduced release of NO and PGI\({}_{2}\) could be correlated with cavitation activities in the vessel. Based on previous studies, the effect of PUT is primarily through the mechanical effect of the ultrasound wave, particularly cavitation. During PUT, the ultrasound pressure provided directly by the ultrasound transducer is superimposed by photoacoustic pressure, produced through the photoacoustic effect when the laser illuminated an optical absorptive sample.