Reduced activity of adenylyl cyclase 1 attenuates morphine induced hyperalgesia and inflammatory pain in mice.

Opioid tolerance, opioid-induced hyperalgesia during repeated opioid administration, and chronic pain are associated with upregulation of adenylyl cyclase activity. The objective of this study was to test the hypothesis that a reduction in adenylyl cyclase 1 (AC1) activity or expression would attenuate morphine tolerance and hypersensitivity, and inflammatory pain using murine models. To investigate opioid tolerance and opioid-induced hyperalgesia, mice were subjected to twice daily treatments of saline or morphine using either a static (15 mg/kg, 5 days) or an escalating tolerance paradigm (10-40 mg/kg, 4 days). Systemic treatment with an AC1 inhibitor, ST03437 (2.5-10 mg/kg, IP), reduced morphine-induced hyperalgesia in mice. Lumbar intrathecal administration of a viral vector incorporating a short-hairpin RNA targeting Adcy1 reduced morphine-induced hypersensitivity compared to control mice. In contrast, acute morphine antinociception, along with thermal paw withdrawal latencies, motor performance, exploration in an open field test, and burrowing behaviors were not affected by intrathecal Adcy1 knockdown. Knockdown of Adcy1 by intrathecal injection also decreased inflammatory mechanical hyperalgesia and increased burrowing and nesting activity after intraplantar administration of Complete Freund's Adjuvant (CFA) one-week post-injection.

In vitro and in vivo transfection of primary phagocytes via microbubble-mediated intraphagosomal sonoporation.

The professional phagocytes, such as macrophages and dendritic cells, are the subject of numerous research efforts in immunology and cell biology. The use of primary phagocytes in these investigations however, are limited by their inherent resistance to transfection with DNA constructs. As a result, the use of phagocyte-like immortalized cell lines is widespread. While these cell lines are transfection permissive, they are generally regarded as poor biological substitutes for primary phagocytes. By exploiting the phagocytic machinery of primary phagocytes, we developed a non-viral method of DNA transfection of macrophages that employs intraphagosomal sonoporation mediated by internalized lipid-based microbubbles. This approach enables the transfection of primary phagocytes in vitro, with a modest, but reliable efficiency. Furthermore, this methodology was readily adapted to transfect murine peritoneal macrophages in vivo. This technology has immediate application to current research efforts and has potential for use in gene therapy and vaccination strategies.

Decafluorobutane as a phase-change contrast agent for low-energy extravascular ultrasonic imaging.

Currently available microbubbles used for ultrasound imaging and therapeutics are limited to intravascular space due to their size distribution in the micron range. Phase-change contrast agents (PCCAs) have been proposed as a means to overcome this limitation, since droplets formed in the hundred nanometer size range might be able to extravasate through leaky microvasculature, after which they could be activated to form larger highly echogenic microbubbles. Existing PCCAs in the sub-micron size range require substantial acoustic energy to be vaporized, increasing the likelihood of unwanted bioeffects. Thus, there exists a need for PCCAs with reduced acoustic activation energies for use in imaging studies. In this article, it is shown that decafluorobutane, which is normally a gas at room temperature, can be incorporated into metastable liquid sub-micron droplets with appropriate encapsulation methods. The resulting droplets are activatable with substantially less energy than other favored PCCA compounds. Decafluorobutane nanodroplets may present a new means to safely extend ultrasound imaging beyond the vascular space.