Hernández-Alvarado, Freddy

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    Using propidium iodide to determine the mechanism involved for a temporary increase in permeability of a bullfrog urinary bladder due to ultrasonically induced cavitation
    (2013) Hernández-Alvarado, Freddy; Cancelos, Silvina; College of Engineering; Leonardi, Stefano; Díaz, Rubén; Department of Mechanical Engineering; Rodríguez, Enid
    The recent advantages in medical ultrasound, in therapeutic application suggest that in the future ultrasound can be a tool for a safe drug delivery. It was found that acoustically induced cavitation permeabilizes cell membranes therefore enhancing the delivery of drugs [Fechheimer et al., 1987], [Lui et al., 1998], [Ng and Liu 2002]. However, lack of control over the pressure field, and consequently on the location and intensity of the cavitation, has hindered the capability of understanding the fundamentals involved in this phenomenon [Pitt et al., 2004], [Levy et al., 1989], [Mitragotri et al., 1996], [Benson et al., 1991], [Tang et al 2001], [Hynynen 1991], [Schlicher et al., 2006], [Terahara et al., 2002]. It has been previously shown that under certain conditions, ultrasonically induced cavitation bubbles can cause a transient increase in permeability [Cancelos et al., 2010]. The objective of this research is to determine the necessary amount of cavitation events to had a temporary increase in permeability without a permanent damage to the biological tissue. In order to gain more insight on the mechanisms involved we propose the use of a DNA stain impermeable to the cell membrane, such as propidium iodide. Therefore, if the cell’s plasma membrane is damaged during the application of ultrasound, the propidium iodide will stain the nucleus of damaged cells and they will remain stained after the ultrasound is removed even when the tissue membrane recovers its integrity. Using imaging techniques it is possible to assess the percentage of mechanically damaged cells that later recovered. The results show that the initial permeability value of a bullfrog urinary bladder for Propidium Iodide was P0=(3.07±2.87) x10-5 cm/sec. An increase in the permeability was consistently found in all experiments where cavitation was present. For experiments where cavitation was absent, there was no evidence for an increase in permeability. In experiments where the permeability recovered its initial value after a 10 times increased during ultrasound exposure, the dissipative power threshold was between 1.5 Watts and 2.5 Watts and 6-10 cavitation events were counted. It was noticed, that after a cavitation event occurred, the permeability increased instantly and retained the same value until the ultrasound was removed. Also, no signs of disruption of the epithelial cell layer were observed. The images look like the normal control with some of the nucleuses stained with Propidium Iodide and are consistent with the fact that the epithelial cell layer recuperates after the ultrasound is removed. All experiments where there was no recuperation in permeability, are correlated with a higher content of air in the liquid, possibly leading to the formation of air bubbles that erode the tissue continuously.