Dual-wavelength photoacoustic microscopy for evaluation of microcirculatory dysfunction and spatial heterogeneity in mice with liver fibrosis

doi:10.12122/j.issn.1673-4254.2026.05.21

Abstract

Abstract:

Objective To investigate microcirculatory dysfunction and spatial heterogeneity in mice with liver fibrosis using dual-wavelength high-resolution photoacoustic microscopy (PAM). Methods In a male C57BL/6 mouse model of CCl₄-induced liver fibrosis, multiparametric imaging of intrahepatic microcirculation was performed using dual-wavelength PAM. The acquired images were used for reconstruction of blood oxygen saturation (sO₂) distribution maps based on PAM signals and using a spectral unmixing algorithm. The portal area and central vein region within the hepatic lobule were defined as the main regions of interest, and Evans Blue dynamic extravasation analysis was used to assess vascular permeability and spatial heterogeneity of fibrosis progression. Intrahepatic microvascular blood flow velocity was measured using the Doppler bandwidth broadening method, and the local metabolic rate of oxygen (MRO₂) was calculated based on sO₂ parameters. Results Compared with normal mice, the mice with liver fibrosis exhibited disorganized intrahepatic microvascular network architecture, focal rarefaction, and impaired branching structures with significantly decreased intrahepatic microvascular sO₂ [(93.62±2.44)% vs (85.53±3.37)%, P<0.05). Evans Blue dynamic tracing showed increased extravasation in the liver fibrosis group, and tracer diffusion kinetics differed significantly between the portal area and central vein region within the hepatic lobule, indicating marked spatial heterogeneity. Microvascular blood flow velocity was significantly lower in liver fibrosis group than in the normal control group (3.43±0.32 vs 4.16±0.25 mm/s; P<0.05). The local MRO₂ showed a decreasing trend in the liver fibrosis group compared with the normal control group (0.40±0.026 mL·100 g^-1·min^-1vs 0.35±0.048 mL·100 g^-1·min^-1). Conclusion Multiparametric imaging based on PAM enables systematic and multidimensional assessment of microcirculatory structural and functional alterations in mice during liver fibrosis progression, highlighting significant reductions of intrahepatic microvascular sO₂ and blood flow velocity. This approach combined with spatial heterogeneity analysis provides important imaging evidence for staging assessment, mechanistic study, and therapeutic efficacy evaluation for liver fibrosis.

Key words: photoacoustic microscopy, liver fibrosis, microvascular permeability, spatial heterogeneity

Qiansong XIA, Yang QIU, Honghui LI, Liming NIE. Dual-wavelength photoacoustic microscopy for evaluation of microcirculatory dysfunction and spatial heterogeneity in mice with liver fibrosis[J]. Journal of Southern Medical University, 2026, 46(5): 1167-1174.

Figures/Tables 5

Fig.1 Schematic diagram of the dual-wavelength photoacoustic microscopic imaging system. CL: Convex lens; NDF: Neutral density filter; DM: Dichroic mirror; BS: Beam splitter; PD: Photodiode; OL: Objective lens; RAP: Right-angle prism; RP: Rhomboid prism; UT: Ultrasonic transducer; AMP: Amplifier; DAQ: Data acquisition unit; PC: Personal computer.

Fig.2 Dual-wavelength imaging of livers from normal and liver fibrosis mice. A: Images of liver vessels and blood oxygen saturation at 532 nm and 559 nm in normal and liver fibrosis mice. B, C: Histogram of blood oxygen saturation distribution in hepatic vessels of normal and liver fibrosis mice, respectively. Red dashed line indicates the average blood oxygen saturation value measured. The average sO2 values within the displayed representative image are shown in the histograms. D: Comparison of intrahepatic microvascular oxygen saturation between normal and liver fibrosis groups. *P<0.05. Data are presented as Mean±SD, n=3.

Fig.3 Detection of delayed Evens Blue exudation in normal mice liver.

Fig.4 Delayed detection of Evans Blue extravasation in the liver of mice with liver fibrosis. A: Evans Blue extravasation within 30 min post-injection. B, C: Changes in Evans Blue exudation in the portal and central regions of the liver lobules over time in normal mice and the mice with liver fibrosis, respectively. *P<0.05.

Fig.5 Blood flow and oxygen metabolism imaging of the livers of normal mice and mice with liver fibrosis. A: Blood flow measurement phantom image. B: Profile of blood flow speed across the dashed line in (A). C: Graph showing the relationship between the set flow rate and measured flow rate. D: Images of vascular structures, blood flow, and oxygen metabolism in the livers of normal mice and mice with liver fibrosis. E: Comparison of intrahepatic microvascular blood flow speed between the normal and liver fibrosis groups. F: Comparison of intrahepatic microvascular metabolic rate of oxygen between the normal and liver fibrosis groups. Data are presented as Mean±SD, n=3. *P<0.05.

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