*3.6. A Four-Week Period is Not Su*ffi*cient to Provoke Regression of TAA-Induced Liver Fibrosis*

Lastly, we aimed to study the effects of a fibrocyte depletion on hepatic fibrolysis and regeneration. FC were depleted during a 4-week regeneration period after 18 weeks of fibrosis induction (Figure 6a). As the TAA-model induced a favorable, marked fibrosis, representing typical pathological features known from human liver fibrosis as expected (Figure 2), we anticipated a considerable regeneration after 4 weeks. Serum ALT levels, in fact, suggest an ameliorated disease state (Figure 4b). Surprisingly, fibrosis was not reduced by a 4-week regeneration period after cessation of the noxe: Total liver hydroxyproline levels remained constant in the control group after the regeneration period (409.8 ± 35.5 vs. 413.4 ± 38.2 μg/g liver; Figure 6c). FC depletion during the regeneration period did not significantly alter either hydroxyproline content (Figure 6c) or serum ALT levels (Figure 6b). We chose to not further investigate the role of FC in fibrolysis and regeneration until a protocol leading to marked liver regeneration could be established for the TAA-model.

**Figure 5.** Liver integrity was preserved by fibrocyte ablation. (**a**) Fibrocyte ablation caused a considerable reduction in serum levels of alanine amino transferase (ALT). Median is depicted, Mann-Whitney *U* test was applied. (**b**) Quantitative real-time PCR showed an unchanged expression of *Fasl.* (**c**) Bcl-2 western blot and optical densitometry thereof hinted at a reduced expression of Bcl-2 in the fibrocyte-ablated group. Arbitrary unit. *SC n* = 5; *Ctrl, FC-Abl. n* = 6. (**d**) qPCR displayed an unchanged *Bcl2* expression. (**e**,**f**) Bax is expressed comparably on a protein and transcriptional level in the controland fibrocyte-ablated group. Arbitrary unit; *SC n* = 4; *Ctrl n* = 10; *FC-Abl. n* = 9; All western blot experiments were performed ≥2 times, representative blots are shown. Columns and error bars depict Mean + SEM.

**Figure 6.** A four-week period was not sufficient to provoke regression of fibrosis. (**a**) Schematic representation of the animal experiments. Fibrocytes were depleted in mice of the respective group via the administration of VCV during a four-week regeneration period. (**b**) The direct comparison of serum ALT levels of mice sacrificed during fibrogenesis (see Section 3.5) and after regeneration shows an ameliorated disease state four weeks after the last TAA-administration. FC depletion during regeneration did not alter ALT levels. Median is depicted, Mann-Whitney *U* test was applied. (**c**) The direct comparison of hepatic hydroxyproline content indicates that a resolution of fibrosis cannot be observed after a regeneration period as short as four weeks. FC depletion during regeneration did not influence hydroxyproline levels. Mean is depicted, an unpaired *t*-test (two-tailed) was applied.
