Embolization of Portal Vein Branches Induces Hepatocyte Replication in Swine: A Potential Step in Hepatic Gene Therapy

Embolization of Portal Vein Branches Induces Hepatocyte Replication in Swine: A Potential Step in Hepatic Gene Therapy

James R. Duncan, MD, PhD²^,1^,2, Marshall E. Hicks, MD¹, Shi-Rong Cai, MD³^,4, Elizabeth M. Brunt, MD⁵ and Katherine P. Ponder, MD⁴

¹ Mallinckrodt Institute of Radiology (J.R.D., M.E.H.)
² Depts of Cell Biology and Physiology (J.R.D.)
³ Internal Medicine (S.R.C.), Washington Univ School of Medicine, St Louis, Mo
⁴ Depts of Biochemistry and Molecular Biophysics (S.R.C., K.P.P.)
⁵ Pathology (E.M.B.), St Louis Univ Medical School, Mo.

View larger version (83K):

[in a new window]

Figure 1. Animal 4. Embolization of portal vein branches. The portal vein in this minipig was accessed by means of transhepatic puncture, and a catheter was advanced into the main portal vein. A, Digital subtraction portogram obtained before embolization was performed shows the portal vein branches (arrows) supplying the left lobe and the majority of the median lobe. These branches were then embolized with PVA particles and appropriately sized embolization coils. B, Portogram obtained after embolization shows occlusion of the portal vein branches.

View larger version (156K):

[in a new window]

Figure 2. Animal 8. A–D, Contrast-enhanced CT scans of the liver in this minipig were obtained approximately 6 hours after embolization with coils (arrows in D) of the portal vein branches (EMB) supplying the median and left lobes. Contrast material was injected through the portal vein catheter. The images were obtained in an oblique axial plane to best demonstrate the enhancement difference between the right and left lobes. GB = gallbladder, PER = hepatic segments still perfused by the portal vein, ST = stomach.

View larger version (112K):

[in a new window]

Figure 3. Photomicrographs show bromodeoxyuridine incorporation into hepatocytes after embolization. Bromodeoxyuridine was injected either 7 days after (A, B) or immediately after (C, D) embolization of the portal vein branches supplying the left and median lobes. Liver tissue from the right (nonembolized) lobe was collected 4 hours later, and bromodeoxyuridine incorporation into hepatocyte nuclei was assessed as described in Materials and Methods. A, C, No counterstain was applied, and the dark reaction product indicating DNA synthesis is seen only in the tissue obtained 7 days after the embolization procedure (A). B, D, The sections have been stained with hematoxylin to better visualize the nuclei of all the cells in the section. (Original magnification, x125.)

View larger version (19K):

[in a new window]

Figure 4. Graph shows the time course of hepatocyte replication. The time course of hepatocyte replication in a series of pigs was assessed as described in Materials and Methods. ${circ}$ = replication in right (nonembolized) lobe of minipigs, • = replication in right (nonembolized) lobe of a domestic pig (animal 3), ${square}$ = replication in left (embolized) lobe, error bars = SEM for different regions of each lobe from an individual animal.

View larger version (27K):

[in a new window]

Figure 5. Graphs show laboratory values obtained before and after embolization. A series of blood samples were analyzed for evidence of hepatic or renal damage after embolization in two domestic swine (animals 2 and 3) and in four minipigs (animals 4–7). The upper and lower limits of normal levels are indicated by the dotted lines. The different animals are indicated by different symbols. ALT = alanine aminotransferase, AST = aspartate aminotransferase, u/l = IU/L.

View larger version (18K):

[in a new window]

Figure 6. Graphs shows indocyanine green clearance before and after embolization. The serum clearance rate of indocyanine green was measured as described in Materials and Methods. The clearance rates immediately before (Pre-embo) and after (Post-embo) embolization are shown for animal 4 (A) and animal 7 (B). • = clearance before embolization, ${circ}$ = clearance less than 1 hour after occlusion of the portal vein branches supplying 60%–70% of the liver.

View larger version (127K):

[in a new window]

Figure 7. Photomicrographs of tissue collected at autopsy and stained with the Masson trichrome technique provide an overview of the histologic results of embolization. A, B, Tissue from the right lobe (A, nonembolized) and left lobe (B, embolized) obtained 4 days after embolization of the portal vein branches supplying the left and median lobes in animal 6. C, D, Tissue from the right lobe (C, nonembolized) and left lobe (D, embolized) obtained 12 days after embolization of the portal vein branches supplying the left and median lobes in animal 4. The hepatic lobules (L) are normal in the right lobe (A and C). After embolization, changes seen in the left lobe (B and D) include fibrosis (arrows) separating atrophic lobules. (Original magnification, x50.)

View larger version (17K):

[in a new window]

Figure 8. Graph shows relative lobule size after embolization. The relative hepatic lobule volume in lobules from the nonembolized right ( ${circ}$ ) and embolized left (•) lobes was calculated as described in Materials and Methods.

View larger version (127K):

[in a new window]

Figure 9. Animal 3. Photomicrographs show an embolized segment from the left lobe obtained 3 days after embolization. A, Low-magnification image shows a PVA particle (arrowhead) within a portal vein branch. Horizontal arrow = a portal triad, vertical arrow = a central vein. (Hematoxylin-eosin stain; original magnification, x50.) B, Higher magnification image shows the portal triad in more detail. The PVA particle, a bile duct (BD), and a hepatic arterial branch (HA) are shown. An inflammatory cell infiltrate surrounds the PVA particle. (Hematoxylin-eosin stain; original magnification, x125.)

View larger version (171K):

[in a new window]

Figure 10. Animal 6. Photomicrograph shows nodular regenerative hyperplasia (NRH) in the left (embolized) lobe 4 days after embolization. The interface between nodular regenerative hyperplasia and normal liver can be seen. (Hematoxylin-eosin stain; original magnification, x200.)

View larger version (141K):

[in a new window]

Figure 11. Animal 3. Electron microscopic images of hepatocyte apoptosis 3 days after embolization. A, Tissue from the left lobe (embolized) shows cells with condensed nuclear material (arrowheads). These cells likely represent apoptotic hepatocytes adjacent to normal hepatocytes. (Original magnification, x2,200.) B, A representative image of tissue from the right lobe (nonembolized) shows normal hepatic ultrastructure. (Original magnification, x1,650.)