Structural impact of hydrodynamic injection on mouse liver

Abstract

The impact of hydrodynamic injection on liver structure was evaluated in mice using various microscopic techniques. Upon hydrodynamic injection of approximately 9% of body weight by volume, the liver rapidly expanded, reaching maximal size at the end of the injection and returned to its original size in 30 min. Histological analysis revealed a swollen appearance in the peri-central region of the liver where delivery of genes and fluorescence-labeled markers was observed. Scanning and transmission electron microscopy showed enlargement and rupture of endothelium that in about 24-48 h regains its morphology and normal function as a barrier against infection by adenovirus viral particles. At the cellular level in hydrodynamically treated animals, four types of hepatocytes were seen: cells with normal appearance; cells with enriched vesicles in the cytoplasm; cells with lightly stained cytosol; and cells with significant dilution of the cytoplasm. In addition, red blood cells and platelets were observed in the space of Disse and even inside hepatocytes. Vesicle formation is triggered by hydrodynamic injection and resembles the process of macropinocytosis. These results, whereas confirming the physical nature of hydrodynamic delivery, are important for a better understanding of this efficient method for intrahepatic gene and small interfering RNA delivery.

Hydrodynamic gene delivery to the pig liver via an isolated segment of the inferior vena cava

“…If one extrapolates a peak portal venous pressure of 125 mm Hg for pig 3, this gives a range of 101-126 mm Hg and a median value of 120 mm Hg in the four pigs without outflow obstruction. These pressures are about fourfold higher than values previously reported in the mouse 7,11 and by our group in rats (GJ Sawyer et al, manuscript submitted for publication). Thus volumes and flow rates of DNA solution comparable to rodent models generated far higher intrahepatic vascular pressures, but yielded much lower levels of gene delivery in the pig.…”

“…Pressure build-up is therefore dependent on a reasonable rate of flow through the liver into the portal vein. A rapid rise in portal venous pressure has previously been observed in mice given systemic hydrodynamic gene delivery 7,11 and in rats given IVC segment gene delivery (GJ Sawyer et al, manuscript submitted for publication).…”

“…10 In mice, under optimal conditions, the liver more than doubles in size. 11 A major attraction of this approach is that it requires neither viral nor nonviral vectors, which greatly simplifies potential clinical application, in particular avoiding potentially lethal inflammatory responses to viral proteins. 12 Translation of the hydrodynamic procedure into clinical practice poses a number of problems.…”

DNA delivery to ‘ex vivo’ human liver segments

“…The exact mechanism of gene delivery mediated by hydrodynamic injection remains unknown, but two routes of entry of DNA plasmids into hepatocytes have been suggested: plasma membrane rupture 19 and increased membrane permeation, 5,8,20,21 possibly through endocytic vesicles. In the present study we confirm that hydrodynamic injection mediates an increase in sinusoid diameter, with an endothelium that combines both continuous and discontinuous zones, although in no case was generalized disruption observed.…”

Direct comparison of hepatocyte-specific expression cassettes following adenoviral and nonviral hydrodynamic gene transfer

“…Following hydrodynamic gene transfer, intracellular transgene DNA is trapped within macropinocytotic vesicles and in the cytoplasm of hepatocytes. [40][41][42][43] Therefore, the sharp drop of transgene DNA copy numbers between 1 and 24 h after hydrodynamic gene transfer most likely corresponds to the degradation of minicircle DNA before it reaches the nucleus. Whereas human apo A-I plasma levels at day 35 were significantly higher following adenoviral transfer with 5 Â 10 10 particles than after hydrodynamic transfer with 20 mg of minicircles, copy numbers in PC at day 35 after transfer were similar.…”