Volume 118, Issue 3, Pages 591-598 (March 2000) Synergistic growth factors enhance rat liver proliferation and enable retroviral gene transfer via a peripheral vein  Stuart J. Forbes*, Mike Themis‡, Malcolm R. Alison§, Ildiko Sarosi∥, Charles Coutelle‡, Humphrey J.F. Hodgson*  Gastroenterology  Volume 118, Issue 3, Pages 591-598 (March 2000) DOI: 10.1016/S0016-5085(00)70266-6 Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 Time course of hepatocyte proliferation after a single administration of KGF. Three groups of 3 rats received subcutaneous injections of KGF (1 mg/kg). Animals were killed at 6-hour intervals; 1 hour before death, all rats were injected with BrdU. Results represent mean ± SD. **P < 0.05. Gastroenterology 2000 118, 591-598DOI: (10.1016/S0016-5085(00)70266-6) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 Hepatocyte proliferation 24 hours after single and combined growth factor stimulation. Three groups of 4 rats were injected with either a single growth factor or both growth factors. Another group of 3 rats received injections of both growth factor diluents only. T3 (4 mg/kg) and KGF (1 mg/kg) were administered at 0 hours. All animals received BrdU at 23 hours and were killed at 24 hours. Results represent mean ± SD. **P < 0.05. Gastroenterology 2000 118, 591-598DOI: (10.1016/S0016-5085(00)70266-6) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 Cumulative hepatocyte proliferation observed after growth factor stimulation. T3 (4 mg/kg) together with KGF (1 mg/kg) were injected subcutaneously into a group of 6 rats. Two rats were injected with diluent only. BrdU was injected at 5 hourly intervals between 12 and 32 hours to label all hepatocytes entering S-phase. Animals were killed at 33 hours, and analysis of BrdU incorporation was made as previously noted. Results represent mean ± SD. Gastroenterology 2000 118, 591-598DOI: (10.1016/S0016-5085(00)70266-6) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 4 The proportion of hepatocytes expressing β-galactosidase 9 days after intraportal injection of the retrovirus/DOGS complex. Three groups of 4 rats received injections of either a single growth factor or a combination of growth factors. A group of 3 control rats received injections of the growth factor diluents only. At 24 hours, all rats were injected intraportally with 1 mL of retrovirus during temporary hepatic artery and portal vein occlusion. All rats were killed 9 days after vector administration. Ten-micrometer-thick frozen liver sections were incubated with X-gal, and the proportion of hepatocytes expressing β-galactosidase was counted from 2000 hepatocytes in 3 different areas. Results represent mean ± SD. **P < 0.05. Gastroenterology 2000 118, 591-598DOI: (10.1016/S0016-5085(00)70266-6) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 5 Transduction of rat liver in vivo after growth factor–induced hyperplasia and peripheral venous administration of retroviral vector. Three groups of 4 rats received an injection of one or both growth factors. Two rats were injected with diluent only. All rats received 3 tail vein injections of 1 mL of the retrovirus/DOGS complex at 18, 24, and 30 hours and were killed at day 10. Hepatic nuclear localized β-galactosidase was quantified as previously noted. Results represent mean ± SD. **P < 0.05. Gastroenterology 2000 118, 591-598DOI: (10.1016/S0016-5085(00)70266-6) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 6 Photomicrographs of (A) BrdU-labeled liver 24 hours after T3 administration showing predominantly midzonal labeled hepatocytes; (B) BrdU-labeled liver 24 hours after subcutaneous injection of KGF (1 mg/kg) showing hepatocyte labeling throughout much of the parenchyma, but not in the centrilobular areas; (C) BrdU-labeled liver 24 hours after combined T3 and KGF administration; (D) continuous hepatic labeling with BrdU after combined administration of T3 and KGF; and (E) β-galactosidase staining of liver 10 days after combined T3 and KGF injection and peripheral venous administration of the retrovirus/DOGS complex. Transduced hepatocytes are scattered throughout the hepatic parenchyma except in the area around the hepatic vein. pt, portal tract; hv, hepatic vein. (Original magnifications: A, 100×; B–E, 40×.) Gastroenterology 2000 118, 591-598DOI: (10.1016/S0016-5085(00)70266-6) Copyright © 2000 American Gastroenterological Association Terms and Conditions