Table 1.
Biliary hydrodynamic injection parameters used in the acute pig studies.
Fig 1.
Fluoroscopy monitors the success of biliary hydrodynamic injection.
(A) The anatomy of the biliary tract and the successful seal of the balloon to prevent retrograde flow of contrast was confirmed. With placement of catheter inside the CHD and balloon inflated, bifurcation of the CHD into the right and left branches is observed. (B) Hydrodynamic delivery to all lobes was confirmed by measuring real-time fluoroscopy of the injection. Example fluoroscopic images of one hydrodynamic injection are provided, showing a time course of images during injection (30 mL in 2 mL/sec). At the completion of injection (15 seconds), all liver lobes contain detectable contrast.
Fig 2.
Vital signs monitored during biliary hydrodynamic injection demonstrate no significant changes.
All pigs had vital signs monitored under anesthesia during ERCP procedure. Values pre- and post-procedure are provided, representing possible physiologic perturbations from hydrodynamic injection. Heart rate (A), respiratory rate (B), pulse oximetry (C), end tidal CO2 (D), and mean arterial pressure (E) all demonstrated no significant (n.s.) changes from pre- to post-procedure (unpaired two-tailed, parametric t-test used, P<0.05).
Fig 3.
Intrabiliary pressure monitored during hydrodynamic injection elucidates differences among the injection parameters.
Baseline pressure within the biliary system is minimal before injection in all conditions (A-D). Shortly before injection, the balloon is inflated creating a seal which appears to have minimal effect on the measured pressure. Upon initiation of injection (solid black arrow, parameters provided above graphs), pressure increases to a short peak, before equilibration during flow at a slightly lower pressure. Cessation of injection (dashed black arrows) yields a sharp decrease in pressure. The deflation of balloon (solid grey arrows) drops pressure further, suggesting a measure of baseline hydrostatic pressure remains in the system after the injection is completed. Higher pressure was achieved at the highest flow rate (D).
Table 2.
Serum chemistry before and after repeated biliary hydrodynamic injections in pig liver was evaluated.
Fig 4.
Imaging and biochemical analysis support the safety profile of biliary hydrodynamic injection.
(A) Abdominal CT with contrast was performed on Day 1 post-procedure. The axial, sagittal and coronal images did not demonstrate any evidence of intra- or extrahepatic biliary dilation, hepatic infarction/necrosis, abnormal gallbladder dilation, or gallbladder inflammation. The systemic and portal venous systems were patent. (B) Transient, acute elevation in AST and total bilirubin in the three injected was noted, which were not observed in five other pigs with resolution of any biochemical abnormalities by day 1 post-injection. Pig #1 was color-coded as green dots, Pig #2 was color-coded as blue dots, Pig #3, which received multiply injections with the largest volume and highest injection speed, was color-coded as red dots.
Fig 5.
Histology of pig liver post-repeated hydrodynamic biliary injection shows evidence of hydrodynamic effects.
(A) H&E stains from low flow rate pig injections (flow rates: <5 mL/sec; Pig #1 depicted) or high flow rate pig injections (flow rate: 10 mL/sec; Pig #3 depicted) are illustrated, along with H&E from a normal, non-injected pig liver for comparison. Histology taken from livers 15 minutes post-injection showed significant dilation of hepatic sinusoids (black arrows) and formation of intracellular vesicles, but otherwise no areas of focal necrosis. (B) Histology of pig liver on Day 1 and Day 14 post-procedure showed normal liver histology. H&E staining on pigs euthanized on Day 1 and Day 14 post-procedure are illustrated with no obvious sinusoid spaces dilation observed. Scattered fluid-filled vesicles were observed more rarely on Day 1 post-procedure. No fluid-filled vesicles were noted on Day 14. Scale bar: 200 μm in 4X images; 50 μm in 20X images.
Fig 6.
Hydrodynamic biliary injection induces acute tissue structural changes in pigs that is similar to mouse hydrodynamic tail vein injection.
(A) A mouse was injected with 10% body fluid volume over 4–7 seconds through the tail vein and sacrificed 15 minutes later for comparison to the biliary hydrodynamic injection in pigs. Small fluid-filled cytoplasm vesicles (black arrows), scattered hepatocytes with dilute cytoplasm, and occasional hepatocytes with engulfed red blood cells are observed in the mouse liver. Scale bar: left, 50 μm; right, 20 μm. (B) Pig #3, which is a high flow rate injection pig, has largely similar changes, with larger fluid-filled cytoplasmic vesicles and more frequent hepatocytes with dilute cytoplasm. Scale bar: left, 50 μm; right, 2.0 μm.
Table 3.
Comparison of intravascular pressures achieved in previous hydrodynamic liver gene therapy studies.