Research ArticleFibrillar collagen scoring by second harmonic microscopy: A new tool in the assessment of liver fibrosis
Introduction
Chronic liver diseases are generally associated with extra-cellular matrix (ECM) over-production. Hepatic fibrosis is the result of a dynamic process characterized by an imbalance between fibrogenesis and fibrolysis responsible for the accumulation of fibrillar components in the liver such as fibrillar collagen [1]. The progression of human fibrosis may lead to cirrhosis and cancer with clinical complications followed by high morbidity and mortality rates particularly in chronic hepatitis B or C virus (HBV, HCV) infections [2], [3]. In order to determine the degree of fibrosis, several methods are based on the pathological examination of liver biopsies by a trained pathologist using numerical systems with increasing severity stages like Scheuer [4], Ishak [5] or Metavir [6] scores after ECM staining [7], [8]. The Metavir grading system appears well adapted for assessing chronic liver damage related to viral hepatitis. An alternative to the numerical scoring systems relies on a direct measurement of the amount of fibrotic deposits by morphometric image analysis [9], [10]. To date, histomorphometric methodologies are not well standardized and appear to be time consuming procedures compared to classic numerical systems.
The aim of this study was to develop an innovative optical method for an accurate quantification of human liver fibrosis. This original process is based on an application of multiphoton microscopy enabling the observation of unstained samples using endogenous sources of nonlinear signals such as Two-Photon Excitation Fluorescence (TPEF) and Second Harmonic Generation (SHG). SHG makes the specific detection of supramolecules possible with high crystalline triple-helix structures deprived of centrosymmetry at microscopic and mesoscopic scales [11], [12], [13], [14]. In biological tissues, fibrillar collagen displays these properties. Like myosin fibers and microtubules, fibrillar collagen can generate a two-photon coherent process selectively collected and recorded at precisely half-wavelength of excitation which is characteristic of the SHG signature [15], [16], [17], [18]. SHG microscopy has been successfully employed in various models and has revealed its high potential for imaging and analyzing fibrillar collagen in unstained tissues [19], [20], [21], [22]. Recent data also suggested that SHG microscopy could be an interesting approach in the assessment of fibro-proliferative diseases [23], [24].
In this study, we show that multiphoton microscopy using SHG provides a powerful tool for evaluating human liver fibrosis. We established a reliable and standardized method for the quantification of fibrillar collagen and analyzed the correlation between the Fibrosis-SHG index and the Fibrosis-Metavir score currently considered to be the gold standard in the clinical assessment of liver fibrosis.
Section snippets
Biopsies and histological data
The study cohort included 119 patients who underwent percutaneous biopsies (n = 73) or large surgical biopsies (n = 46). Patients presented chronic liver disease with various degrees of severity related to HBV or HCV infection in 8.22% and 90.41% of cases, respectively; 1.37% presented co-infection with HBV and HCV. Exclusion criterion was inadequate specimen (length of biopsy <20 mm and/or portal tract <5). The mean biopsy length (percutaneous) was 39.6 ± 8.8 cm and mean number of fragments 2.4 ± 1.5.
Multiphoton microscopy in human liver fibrosis
The SHG phenomenon within molecules exhibiting non-centrosymmetric organization is characterized by a change of the excitation energy from two infrared incident photons into one emerging visible photon at exactly twice the energy (precisely half of the excitation wavelength) (Fig. 1A). In order to illustrate the high potential of multiphoton microscopy, we first imaged liver with moderate fibrosis (F2-Metavir). Liver cryosections (100 μm thick) were made and imaged without staining, at
Discussion
Second harmonic microscopy presented a major challenge in the assessment of liver fibrosis. Our results demonstrate that SHG provides an original and powerful tool for the quantitative evaluation of fibrillar collagen deposits. We first validated the selectivity of SHG signals for fibrotic deposits (fibrillar collagen type I and III) in human liver by establishing a strong correlation between SHG and immunochemistry assays. According to the literature, immunolabelings of non-fibrillar collagen
Acknowledgements
The authors thank the Histo-Pathology Platform (IFR 140), Pascale Bellaud for technical support in histologic preparation and staining, as well as Nina Cutro-Kelly for critical reading of the manuscript. We also thank Michel Samson, Claire Piquet-Pellorce and Mariette Lisbonne (EA SeRAIC), Anne Renault (UMR-CNRS 6251) and Pixel platform (UMR-CNRS 6026) for constructive comments on this study. We thank the CNRS project “Interface Physique, Chimie, Biologie: soutien à la prise de risque” for
References (29)
- et al.
Projecting future complications of chronic hepatitis C in the United States
Liver Transpl
(2003) Classification of chronic viral hepatitis: a need for reassessment
J Hepatol
(1991)- et al.
Histological grading and staging of chronic hepatitis
J Hepatol
(1995) - et al.
A comparison of fibrosis progression in chronic liver diseases
J Hepatol
(2003) - et al.
Natural history of liver fibrosis progression in patients with chronic hepatitis C
Lancet
(1997) - et al.
Histopathological evaluation of liver fibrosis: quantitative image analysis vs semi-quantitative scores. Comparison with serum markers
J Hepatol
(1998) - et al.
Optimization of second-harmonic generation microscopy
Micron
(2001) - et al.
Simultaneous multichannel nonlinear imaging: combined two-photon excited fluorescence and second-harmonic generation microscopy
Micron
(2001) - et al.
Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues
Biophys J
(2002) - et al.
Second and third optical harmonic generation in type I collagen, by nanosecond laser irradiation over a broad spectral region
Opt Commun
(2000)
3-Dimensional imaging of collagen using second harmonic generation
J Struct Biol
Reversibility of liver fibrosis and cirrhosis following treatment for hepatitis C
Gastroenterology
Liver fibrosis
J Clin Invest
Evaluation of liver fibrosis: a concise review
Am J Gastroenterol
Cited by (156)
Cellular and Molecular Techniques
2023, MacSween's Pathology of the Liver, Eighth EditionSphingosine kinase 1 mediates sexual dimorphism in fibrosis in a mouse model of NASH
2022, Molecular Metabolism
- †
Present address: Laboratoire de Spectrométrie et Optique Laser, EA 938, Université de Bretagne Occidentale, 29238 Brest, France.