A new conversation between radiology and pathology-identifying microvascular architecture in stages of cirrhosis via diffraction enhanced imaging in vitro

Abstract

Background/aim: Diffraction enhanced imaging (DEI) is a synchrotron radiation X-ray phase-contrast imaging technique that can better reveal the microstructure of biological soft tissues than conventional X-rays. The aim of this study is to investigate the angio-architectural changes of the liver during fibrosis, cirrhosis and its subsequent regression by applying synchrotron radiation based DEI.

Methods: DEI experiments were performed at the 4W1A station of Beijing Synchrotron Radiation Facility. Twenty-four Sprague-Dawley rats were induced with liver fibrosis by carbon tetrachloride (CCl4) for up to 10 weeks, after which spontaneous regression started and continued until week 30. Quantitative analysis of the DEI images yielded the mean vascular density and intercapillary distance, which was then re-confirmed by immunohistochemical analysis of CD34.

Results: Based on the DEI results, the mean vascular density was 1.4-fold higher in fibrotic rats (at week 6) and 2-fold higher in cirrhotic rats (at week 10) compared with the control (p<0.05). Accordingly, the intercapillary distance decreased to 563.89 ± 243.35 µm in fibrotic rats and 392.90 ± 92.68 µm in cirrhotic rats compared with 673.85 ± 214.16 µm in the control (p<0.05). During fibrosis regression at week 30, vascular density was 0.7-fold lower and intercapillary distance increased to 548.60 ± 210.94 µm as compared with cirrhotic rats (p<0.05).In parallel to the DEI results, immunohistochemical analysis of CD34 showed similar changes.

Conclusion: Synchrotron-based DEI can conduct radiological as well as pathological analysis. Our results are consistent with previous reports indicating that angiogenesis is directly proportional to fibrosis progression. Furthermore, by clarifying the vascular characteristics of liver diseases, DEI reveals that cirrhosis cannot fully reverse during fibrosis regression.

Figure 1. Experimental setup of Diffraction Enhanced Imaging (DEI).

Schematic illustration of (DEI) experimental setup at the 4W1A Station in the Beijing Synchrotron Radiation Facility (BSRF).

Figure 2

The analyzer crystal selects specifically X-rays which transmitted in a certain angle range incidence on the detector to produce high contrast images (A-C) relative to the position on the RC in (D). A schematic of RC with 15 keV X-rays and silicon [333] diffraction plane (D).

Figure 3

For every pixel, a sample RC and a reference RC can be obtained. The change of the peak, width, area interrelated to the refraction, scattering, absorption of the sample.

Figure 4. Illustration and quantification of microvasculature and fibrosis in corresponding stages of cirrhosis.

Macroscopic observations of liver lobe (A_1-4) from representative control, fibrotic, cirrhotic and regressive rats, with corresponding DEI vasculature images (B_1-4) and fibrosis stained by Sirius-Red (C_1-4). The vasculature of control (B₁) became more dense at week 6 (B₂), further increasing in number at week 10 (B₃). After regression for 30 weeks, the number of micro-vessels decreased (B₄). The vascular density (D), intercapillary distance (E) and fibrosis area (F) were summarized and expressed as mean ± SD. *p<0.05 compared with control group. ^# p<0.05 compared with cirrhotic group.

Figure 5. Angiogenesis or neo-vascularization was evaluated by immune- histochemical stain of CD34.

CD34 positive microvasculature increases with time; control (A), fibrosis stage at week 6 (B), cirrhosis stage at week 10 (C) and regressive stage after cession of CCl₄ for 30 weeks (D). Results are expressed as mean ± SD. *p<0.05 compared with the control group. ^# p<0.05 compared with the cirrhotic group.