Volume 124, Issue 1, Pages 172-183 (January 2003) New method of delivering gene-altered Kupffer cells to rat liver: Studies in an ischemia- reperfusion model  Matthias Froh, Michael D. Wheeler, Olivia Smutney, Zhi Zhong, Blair U. Bradford, Ronald G. Thurman  Gastroenterology  Volume 124, Issue 1, Pages 172-183 (January 2003) DOI: 10.1053/gast.2003.50002 Copyright © 2003 American Gastroenterological Association Terms and Conditions

Fig. 1 Experimental design. (A) Delivery of carbon-labeled Kupffer cells. (B) Injection of Kupffer cells transduced by adenovirus. (C) Ischemia-reperfusion model following supplementation of transduced Kupffer cells. Gastroenterology 2003 124, 172-183DOI: (10.1053/gast.2003.50002) Copyright © 2003 American Gastroenterological Association Terms and Conditions

Fig. 2 Localization of carbon-labeled Kupffer cells in the host liver and other organs after reinjection. Representative micrographs of (A) an untreated liver and (B) a liver directly injected with carbon and (C) liver, (D) lung, and (E) spleen after reinjection of carbon-labeled Kupffer cells. The tissue was harvested 24 hours after injection, processed for light microscopy, and counterstained with methylene blue as described in Materials and Methods section. Carbon appears as black dots within Kupffer cells (arrows). (original magnifications, A–C 400× and D,E 100×.) Gastroenterology 2003 124, 172-183DOI: (10.1053/gast.2003.50002) Copyright © 2003 American Gastroenterological Association Terms and Conditions

Fig. 3 Seeding efficiency of carbon-labeled Kupffer cells in the host liver after reinjection. (A) Number of carbon-labeled Kupffer cells localized in the host liver 24 hours after injection. Host animals with or without cyclosporin A pretreatment were injected with different amounts of Kupffer cells (15 × 106 cells and 30 × 106 cells, respectively). (B) Time course of carbon-labeled Kupffer cell (30 × 106 cells) uptake in the host liver after cyclosporin A pretreatment and reinjection. Carbon-labeled Kupffer cells were detected in liver sections counterstained with methylene blue by light microscopy followed by image analysis as described in Materials and Methods section. Livers directly injected with carbon were used as positive controls (calculation is based on the formula: [number of carbon-labeled Kupffer cells in recipient liver]/[relative number of carbon-labeled Kupffer cells in donor liver]). Data are expressed as mean ± SEM (n = 5; P < 0.05; a Represents a statistically significant difference compared with 3 × 107 reinjected Kupffer cells without cyclosporin A pretreatment. One-way ANOVA followed by Tukey's post hoc analysis). Gastroenterology 2003 124, 172-183DOI: (10.1053/gast.2003.50002) Copyright © 2003 American Gastroenterological Association Terms and Conditions

Fig. 4 RT-PCR and Western blot analysis for β-galactosidase expression in Kupffer cells. (A) An ~821-base-pair fragment for β-galactosidase was amplified from rat liver tissue cDNA by RT-PCR. β-Galactosidase and GAPDH (~988-base-pair fragment) mRNA as an internal control are shown following no treatment (negative control), Ad.lacZ infection, or injection of Ad.lacZ-infected Kupffer cells. (B) Seven μg of cytosolic protein from either parenchymal cells (PC) or Kupffer cells (KC) were subjected to Western blotting using an anti-β-galactosidase monoclonal antibody. Parenchymal cells and Kupffer cells were isolated from untreated animals (negative control), animals directly infected with Ad.lacZ, or injected with Ad.lacZ-infected Kupffer cells. Gastroenterology 2003 124, 172-183DOI: (10.1053/gast.2003.50002) Copyright © 2003 American Gastroenterological Association Terms and Conditions

Fig. 5 Immunohistochemical staining of β-galactosidase in host livers. Representative micrographs of (A) an untreated liver, (B) a liver directly infected with Ad.lacZ, and (C) a liver after reinjection of Ad.lacZ-infected Kupffer cells. Livers were harvested 24 hours after Kupffer cell injection and processed for light microscopy. Immunohistochemical staining for β-galactosidase was performed using a monoclonal antibody. Immune complexes were visualized by applying diaminobenzidine (arrows), as described in Materials and Methods section. (Original magnifications, 200×.) Gastroenterology 2003 124, 172-183DOI: (10.1053/gast.2003.50002) Copyright © 2003 American Gastroenterological Association Terms and Conditions

Fig. 6 Effects of gene-altered, reinjected Kupffer cells on superoxide dismutase (SOD) activity. SOD activity was measured in homogenized liver tissue 24 hours after ischemia-reperfusion as described in Materials and Methods section. SOD activity was estimated based on a standard curve generated using purified bovine erythrocyte SOD. Values are mean ± SEM (n = 5; P < 0.05; a Represents a statistically significant difference compared with saline-sham group; b Represents a statistically significant difference compared with saline-, injected Kupffer cell-, and injected Kupffer cell (Ad.lacZ) ischemia-reperfusion group. One-way ANOVA followed by Tukey's post hoc analysis). Gastroenterology 2003 124, 172-183DOI: (10.1053/gast.2003.50002) Copyright © 2003 American Gastroenterological Association Terms and Conditions

Fig. 7 Effects of repopulated, transduced Kupffer cells on increases in serum ALT levels following hepatic ischemia-reperfusion. Rats were injected with Kupffer cells, gene-altered Kupffer cells (Ad.lacZ or Ad.SOD1), or saline 24 hours before ischemia was initiated in host livers for 1 hour. (A) Eight and (B) 24 hours after reperfusion, serum was collected, and ALT levels were measured as described in Materials and Methods section. Data are expressed as mean ± SEM (n = 5; P < 0.05; a Represents a statistically significant difference compared with saline-injected animals, animals injected with Kupffer cells, and animals injected with altered Kupffer cells (Ad.lacZ) following ischemia-reperfusion. One-way ANOVA followed by Tukey's post hoc analysis). Gastroenterology 2003 124, 172-183DOI: (10.1053/gast.2003.50002) Copyright © 2003 American Gastroenterological Association Terms and Conditions

Fig. 8 Histopathological analysis of liver 24 hours after ischemia-reperfusion. Livers were harvested 24 hours after sham operation from (A) saline-injected animals or (B) animals supplemented with transduced Kupffer cells (Ad.SOD1) and 24 hours after ischemia-reperfusion from animals injected with (C) saline, (D) Kupffer cells or (E) Ad.lacZ-, (F) Ad.SOD1-infected Kupffer cells. Liver tissue was processed for light microscopy and stained with H&E. Necrotic foci are designated by arrows. Representative photomicrographs (n = 5). (Original magnifications 100×.) Gastroenterology 2003 124, 172-183DOI: (10.1053/gast.2003.50002) Copyright © 2003 American Gastroenterological Association Terms and Conditions