Distinct collagen resolution dynamics in early versus advanced liver fibrosis: a quantitative SHG/TPEF study in mice.

BACKGROUND & AIMS: Liver fibrosis regression occurs after the source of injury is removed, but its dynamics and histologic features remain poorly characterized. Second-harmonic generation/two-photon excited fluorescence (SHG/TPEF) microscopy enables quantitative, high-resolution collagen assessment. We employed SHG/TPEF to characterize spatiotemporal collagen remodeling during spontaneous regression in carbon tetrachloride (CCl4)-induced mouse models.

METHODS

Early- and advanced-stage liver fibrosis were established by intraperitoneal CCl4 injection for 8 or 12 weeks. After peak injury, mice recovered for 4 or 12 weeks. Liver sections were analyzed by Sirius red staining and SHG/TPEF imaging, which quantifies 100 collagen measurements.

RESULTS

SHG/TPEF imaging showed strong positive correlations with collagen proportionate area (CPA) quantified by Sirius red staining in both early- (P8: r = 0.96, P < 0.001) and advanced-stage (P12: r = 0.87, P < 0.001) fibrosis models. In early-stage fibrosis, both CPA and SHG percentage decreased significantly after 4 weeks of recovery (CPA: 1.21 ± 0.30 vs. 0.51 ± 0.15, P < 0.001; SHG: 2.13 ± 0.68 vs. 1.18 ± 0.13, P = 0.008), with no further reduction by 12 weeks (P = 0.651 and P = 0.778, respectively). In contrast, advanced-stage fibrosis showed progressive declines, with significant reductions in SHG percentage between 4 and 12 weeks (1.91 ± 0.10 vs. 1.36 ± 0.35, P = 0.023). Quantitative collagen measurements analysis revealed that 35 measurements, particularly collagen fiber number-related metrics, regressed significantly slower in P12R4 than in P8R4 mice (P < 0.05, fold change > 1.5). Notably, early-stage regression predominantly occurred within the first 4 weeks, involving total, portal, and septal collagen, whereas advanced-stage regression was delayed, occurring mainly between 4-12 weeks and affecting total, septal, and fibrillar collagen measurements.

CONCLUSIONS

SHG/TPEF provides a quantitative platform for monitoring regression dynamics in preclinical settings. Advanced fibrosis resolves with a delay, underscoring stage as a critical determinant in anti-fibrotic evaluation.