1. Volume 39, Issue 3, Pages 365-373 (September 2003)
Liver regeneration in a retrorsine/CCl4 – induced acute liver failure model: do bone marrow-derived cells contribute? 
Marc H Dahlke, Felix C Popp, Ferdinand H Bahlmann, Heiko Aselmann, Mark D Jäger, Michael Neipp, Pompiliu Piso, Jürgen Klempnauer, Hans J Schlitt 
Journal of Hepatology 
Volume 39, Issue 3, Pages (September 2003)
DOI: /S (03)

2. Fig. 1
Schematic representation of rat models used. In experiments analysing chimerism of liver cells, rats were treated according to protocol 1. Retrorsine was applied twice 2 weeks apart, followed by bone marrow transplantation 2 weeks after the second injection of retrorsine. Two weeks later animals with stable peripheral chimerism were injected with another dose of retrorsine. Two weeks thereafter liver failure was induced by CCl4 injection. Three days later, a second BMC infusion was carried out to increase the amount of circulating stem cells. (The third injection of retrorsine and the additional BMC infusion were skipped in LEW/LEW.7B chimeras, as only the inflammatory infiltrate was examined early after liver failure). In experiments analysing survival benefit protocol 2 was used. Rats were pretreated with retrorsine as above (two injections). CCl4 was injected 2 weeks after the last dose of retrorsine. Bone marrow cell isografts (or MHC class I mismatched grafts to be able to trace donor cells) were transplanted 6 h later without immunosuppression.
Journal of Hepatology  , DOI: ( /S (03) )

3. Fig. 2
Liver weight in restoration experiments compared to hepatocyte counts in H&E sections. Line graph: Liver weights of control animals (A) were compared to animals 14 days after treatment with 0.6 ml/kg BW of CCl4 alone (B), PH alone (C), retrorsine pretreatment alone (F), or retrorsine pretreatment with application of CCl4 (D) or PH (E). Box graphs: Total hepatocytes (with exclusion of other cell populations in liver tissue) per high power field were counted from at least six distinct sections of three different animals. A–F like above. No significant difference in liver weight can be observed in all study groups. Cellular hyperplasia, outlined by low cell count and equal liver weight, was found in retrorsine pretreated groups. When CCl4 was applied cell count was higher in average, most likely due to post-inflammatory destruction of liver architecture. Retrorsine alone did not influence cell count or liver weight in the present model.
Journal of Hepatology  , DOI: ( /S (03) )

4. Fig. 3
Influx of hematopoietic cells in the LEW/LEW.7B bone marrow transplantation model. First row of pictures: H&E stains of liver cryosections of stable LEW/LEW.7B irradiation chimeras treated with the retrorsine/CCl4 protocol. Day 0 (before application of CCl4), 1 and 3. Maximum of eosinophilic necrosis on day 3. Second row: Immunohistochemical stains. Influx of donor and recipient type granulocytes (HIS 48mAb), maximum on day 1 after hepatocyte destruction. Third and fourth row: CD45 RC positive cells (OX22, subpopulation of T cells) and αβ-T cell receptor positive cells, respectively. Only minimal changes over time.
Journal of Hepatology  , DOI: ( /S (03) )

5. Fig. 4
Quantitative analysis of influxing cells in the LEW/LEW.7B model (Comparing retrorsine pretreated and non-pretreated groups). Immunohistochemical stains (compare Fig. 1) were quantitatively analyzed by two independent examiners. Counts of positive cells per high powerfield are outlined in a time course. (A) HIS 41mAb positive cells (whole donor cells); (B) HIS 48mAb positive cells (granulocytes); (C) OX22 mAb positive cells (CD45 RC, subpopulation of T cells); and (D) R73 mAb (T cells). Massive influx of donor-derived cells can be observed very early after liver destruction (note different scale in A). No significant difference in retrorsine pretreated animals. Most of influxing cells are granulocytes (B). Stable cell numbers of OX22 positive cells (C). No relevant changes in alpha/beta T Cell receptor positive cells (D).
Journal of Hepatology  , DOI: ( /S (03) )

6. Fig. 5
Analysis of donor-derived cells in the LEW.1A/LEW.1WR2 bone marrow transplantation model. Serial cryosections of liver tissue were analyzed on day 25 of the retrorsine/CCl4 protocol in stable MHC class I mismatched irradiation chimeras (LEW.1A/LEW.1WR2). (A) Immunohistochemistry double staining, redish: donor antigen (anti-RT1Au mAb), blue: THY1 mAb (CD90). Coexpression in proximity to portal triads. (B) Serial section. Only few OX1 mAb (CD45) positive cells in the same region. (C) Serial section. Donor-derived cells are confirmed to be THY1 mAb positive in the next section. (D) Serial section, expression of donor antigen only (RT1Au mAb).
Journal of Hepatology  , DOI: ( /S (03) )

7. Fig. 6
Flow cytometry analysis of liver single cell suspensions in the LEW.1A/LEW.1WR2 bone marrow transplantation model. Whole blood mononuclear cells (erythrocytes lysed by NH4Cl) and liver NPCs (obtained by a collagenase/pronase perfusion method, compare above.) were analyzed by FACS. Cells of a control animal (non-chimeric, A and C), were compared to a stable irradiation chimera, (B and D). First primary antibody was OX1 mAb (CD45) in blood cell stains and THY1 mAb (CD90) was used in NPC stains. First secondary mAb was phycoerythrin conjugated goat anti mouse immunoglobulin in all stains, second antibody in all stains was FITC conjugated RT1Au mAb (direct technique). Stains A and C represent the chimeric state of the animals. THY1 positive NPCs were of donor origin in the stable chimera, whereas no THY1 positive NPCs of donor origin were detected in the non-chimeric animal.
Journal of Hepatology  , DOI: ( /S (03) )