Harmony in liver fibrosis…
Authors: Pierre Bedossa
Journal: Journal of Hepatology (2010)

Fibrillar collagen scoring by second harmonic microscopy: A new tool in the assessment of liver fibrosisJournal of HepatologyVol. 52Issue 3PreviewImaging of supramolecular structures by multiphoton microscopy offers significant advantages for studying specific fibrillar compounds in biological tissues. In this study, we aimed to demonstrate the relevance of Second Harmonic Generation (SHG) for assessing and quantifying, without staining, fibrillar collagen in liver fibrosis. Full-Text PDF The recent controversy on the respective place of liver biopsy and non-invasive tools in chronic liver diseases has focused attention on the quantitative evaluation of fibrosis. As a basis for further discussion that liver biopsy is the best available standard for assessment of liver fibrosis, the article of Gailhouste et al. [[1]Gailhouste L. Le Grand Y. Odin C. Guyader D. Turlin B. Ezan F. et al.Fibrillar collagen scoring by second harmonic microscopy: A new tool in the assessment of liver fibrosis.J Hepatol. 2010; 52: 398-406Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar] in the present issue of the Journal of Hepatology shows that biopsy can tell us much more than a score, providing additional useful information on the texture of fibrosis which might have clinical relevance. Liver fibrosis is the accumulation of newly synthesized extracellular matrix (ECM) molecules within the liver. Several ECM molecules have been characterized and fibrillar collagen (mainly isotype I and III) are among the most important [[2]Schuppan D. Structure of the extracellular matrix in normal and fibrotic liver: collagens and glycoproteins.Semin Liver Dis. 1990; 10: 1-10Crossref PubMed Scopus (318) Google Scholar]. These molecules auto-assemble into a characteristic triple helical pattern that organize into highly stable fibrillar units. These fibrillar structures serve as a scaffold for the deposition and organization of other ECM molecules and are therefore of major physiopathological importance in the process of liver fibrosis development [[3]Hulmes D.J.S. Building collagen molecules, fibrils, and suprafibrillar structures.J Struct Biol. 2002; 137: 2-10Crossref PubMed Scopus (430) Google Scholar].To assess liver fibrosis, pathologists normally utilize specific staining methods that highlight fibrous tissue. Masson’s trichrome, Picrosirius red or reticulin stain are among the most commonly used methods, each of which is based on a more or less well-characterized chemical reaction. Although these staining methods allow for the overall detection of fibrosis, their biochemical specificity is poor or even unknown. Immunohistochemical procedures using antibodies directed against various ECM components allow for the identification of single ECM proteins; however, this approach is limited by antibody specificity and the restrictive use of frozen tissue sections for some cases. An additional disadvantage is that immunohistochemistry is not a quantitative technique.In this issue Gailhouste et al. propose an original approach for the analysis of liver fibrosis using a non-linear optical microscopy modality, i.e. multiphoton microscopy. Non-linear optical microscopy has significantly improved three-dimensional imaging of biological tissues in recent years [[4]Konig K. Multiphoton microscopy in life sciences.J Microsc. 2000; 200: 83-104Crossref PubMed Scopus (1178) Google Scholar]. It enables the observation of endogenous signals such as Two-Photon Excitation Fluorescence (TPEF) and Second Harmonic Generation (SHG) in unstained samples and deep within the tissue sample [[5]Zipfel W.R. Williams R.M. Christie R. Nikitin A.Y. Hyman B.T. Webb W.W. Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation.Proc Natl Acad Sci USA. 2003; 100: 7075-7080Crossref PubMed Scopus (1516) Google Scholar]. In short, while TPEF allows for the visualization of the liver background and lobular organization, the SHG displays the three-dimensional architecture of fibrillar collagen. The specificity of SGH for fibrillar collagen is based on its highly ordered supra-molecular organization [[6]Brown E. McKee T. diTomaso E. Pluen A. Seed B. Brown E. et al.Dynamic imaging of collagen and its modulation in tumors in vivo using second-harmonic generation.Nat Med. 2003; 9: 796-800Crossref PubMed Scopus (750) Google Scholar]. The apparent complexity of this imaging technology combined with the high resolution and quality of images provided in this study should not reduce this article to a technical achievement only. It is in fact a new vision into liver fibrosis evaluation. With this approach the authors were able to visualize and quantify fibrillar collagen at a high resolution scale in the context of its microenvironment without utilizing the conventional staining method that is followed by formalin-fixed paraffin-embedding. To validate the specificity of the ECM components detected by the SHG, the authors compare SHG images with immunohistochemical labeling and observe a close relationship between Type I and III collagen isotypes and the SHG signal, while immunostaining for non-fibrillar collagen did not co-localize. High penetration and accurate spatial resolution of multiphoton microscopy provided impressive images of a 3-D reconstruction of the ECM network arrangement in liver parenchyma at a micrometer scale.Why is this important? If clinical evidence of fibrosis regression is present, the triple helix and organization of fibrillar collagen gives it high stability and, once deposited in the liver, it may impair liver architecture permanently [[7]Gieling R.G. Burt A.D. Mann D.A. Fibrosis and cirrhosis reversibility – molecular mechanisms.Clin Liver Dis. 2008; 12: 915-937Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar]. While obtaining a global assessment of liver fibrosis with the scoring system is important, the evaluation of presence, amount, and distribution of fibrillar collagen in a liver biopsy might be crucial for predicting prognosis of chronic liver diseases. However, it should be kept in mind that formation of fibrillar collagen is a late event in fibrogenesis. Evaluation of fibrosis with SHG may only give a rather restricted view of fibrosis stages, possibly missing the early stages of the process. Therefore, SHG should be considered as an additional tool that can provide an added value by allowing for the assessment of the relative amounts of fibrillar collagen within fibrous tissue, and should be used in conjunction with standard histopathology for fibrosis scoring.The physical properties of the ECM have recently been assessed through stiffness evaluation with ultrasound (Fibroscan) or magnetic resonance imaging. Although this approach appeared relevant to screen for advanced liver fibrosis, the correlation between liver fibrosis as assessed by the standard histopathology scoring system and liver stiffness is relatively loose [8Friedrich-Rust M. Ong M.F. Martens S. Sarrazin C. Bojunga J. Zeuzem S. et al.Performance of transient elastography for the staging of liver fibrosis: a meta-analysis.Gastroenterology. 2008; 134: 960-974Abstract Full Text Full Text PDF PubMed Scopus (1182) Google Scholar, 9Manning D.S. Afdhal N.H. Diagnosis and quantitation of fibrosis.Gastroenterology. 2008; 134: 1670-1681Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar]. Intuitively, the high degree of reticulation of fibrillar collagen might directly impact liver stiffness. Therefore, studying the correlation between the SHG index and liver stiffness might provide us with a plethora of new information.To validate this new method of evaluation of liver fibrosis, the authors compare the SHG index with the METAVIR score. Unsurprisingly, they found that the SHG index increased with the METAVIR stage but that this increase was exponential rather than linear. This exponential correlation is highly similar to the correlation between METAVIR scores and stiffness as evaluated with Fibroscan. The same is true for the wide range in SHG index observed in cirrhotic patients which has also been reported when stiffness evaluation is concerned in liver cirrhosis. These results also support the possible correlation between liver stiffness and relative composition of ECM in fibrillar collagen as assessed with the SGH index.Gailhouste et al. underline the benefit of a linear scale as provided by the SGH index by comparison to histological scoring systems since a continuous scale provides greater accuracy than a semi-quantitative score. Morphometry is another method that allows for a linear measurement of global liver fibrosis on tissue sections stained with PicroSirius red. Several studies have shown that quantitation with morphometry also correlates with histological scoring of fibrosis [[10]Pilette C. Rousselet M.C. Bedossa P. Chappard D. Oberti F. Rifflet H. et al.Histopathological evaluation of liver fibrosis: quantitative image analysis vs semi-quantitative scores. Comparison with serum markers.J Hepatol. 1998; 28: 439-446Abstract Full Text PDF PubMed Scopus (191) Google Scholar]. SHG and morphometry are both automated methods that avoid the risk of inter-observer variation in evaluation of liver fibrosis, a potential drawback of semi-quantitative scoring systems which remains partly subjective and dependent on pathologist expertise [[11]Rousselet M.C. Michalak S. Dupre F. Croue A. Bedossa P. Saint-Andre J.P. et al.Sources of variability in histological scoring of chronic viral hepatitis.Hepatology. 2005; 41: 257-264Crossref PubMed Scopus (450) Google Scholar]. Since both techniques can be performed in the same biopsy, a correlation between the evaluation of liver fibrosis with morphometry and SHG would also be useful in order to assess the extent of discrepancies in fibrosis quantification with both methods.Whether SHG or morphometry is concerned, these quantitative approaches, although very accurate and linear, deserve further commentaries. Firstly, sampling error which is related to heterogeneity in fibrosis distribution might become a relevant drawback when the accuracy of the evaluation technique for fibrosis quantification increases. Although this drawback is rather limited when simple semi-quantitative scoring systems are concerned (METAVIR or Ishak scores of fibrosis), several studies have shown that sampling errors become a relevant limit when accurate techniques that evaluate fibrosis along a linear scale are concerned [[12]Bedossa P. Dargère D. Paradis V. Sampling variability of liver fibrosis in chronic hepatitis C.Hepatology. 2003; 38: 1449-1457Crossref PubMed Scopus (1950) Google Scholar]. Second, the amount of fibrosis, whether evaluated through fibrillar collagen (SGH) or global fibrosis staining (Sirius red), is only a part of the deleterious process that leads to cirrhosis. Architectural changes, vascular shunts, and liver cell regeneration are among the other associated features which might have a significant influence on the natural progression of liver disease. They are also evaluated when pathologists score biopsies; however, these lesions are not considered when automated evaluation is used.Finally, SGH is clearly not a routine technique that is designed to replace staging of fibrosis with scoring systems but appears rather as a complement to semi-quantitative histopathology of fibrosis. At present, the major challenge is to demonstrate that the SGH index might provide an added value in the prediction of liver disease prognosis specifically with respect to fibrosis regression. The recent controversy on the respective place of liver biopsy and non-invasive tools in chronic liver diseases has focused attention on the quantitative evaluation of fibrosis. As a basis for further discussion that liver biopsy is the best available standard for assessment of liver fibrosis, the article of Gailhouste et al. [[1]Gailhouste L. Le Grand Y. Odin C. Guyader D. Turlin B. Ezan F. et al.Fibrillar collagen scoring by second harmonic microscopy: A new tool in the assessment of liver fibrosis.J Hepatol. 2010; 52: 398-406Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar] in the present issue of the Journal of Hepatology shows that biopsy can tell us much more than a score, providing additional useful information on the texture of fibrosis which might have clinical relevance. Liver fibrosis is the accumulation of newly synthesized extracellular matrix (ECM) molecules within the liver. Several ECM molecules have been characterized and fibrillar collagen (mainly isotype I and III) are among the most important [[2]Schuppan D. Structure of the extracellular matrix in normal and fibrotic liver: collagens and glycoproteins.Semin Liver Dis. 1990; 10: 1-10Crossref PubMed Scopus (318) Google Scholar]. These molecules auto-assemble into a characteristic triple helical pattern that organize into highly stable fibrillar units. These fibrillar structures serve as a scaffold for the deposition and organization of other ECM molecules and are therefore of major physiopathological importance in the process of liver fibrosis development [[3]Hulmes D.J.S. Building collagen molecules, fibrils, and suprafibrillar structures.J Struct Biol. 2002; 137: 2-10Crossref PubMed Scopus (430) Google Scholar]. To assess liver fibrosis, pathologists normally utilize specific staining methods that highlight fibrous tissue. Masson’s trichrome, Picrosirius red or reticulin stain are among the most commonly used methods, each of which is based on a more or less well-characterized chemical reaction. Although these staining methods allow for the overall detection of fibrosis, their biochemical specificity is poor or even unknown. Immunohistochemical procedures using antibodies directed against various ECM components allow for the identification of single ECM proteins; however, this approach is limited by antibody specificity and the restrictive use of frozen tissue sections for some cases. An additional disadvantage is that immunohistochemistry is not a quantitative technique. In this issue Gailhouste et al. propose an original approach for the analysis of liver fibrosis using a non-linear optical microscopy modality, i.e. multiphoton microscopy. Non-linear optical microscopy has significantly improved three-dimensional imaging of biological tissues in recent years [[4]Konig K. Multiphoton microscopy in life sciences.J Microsc. 2000; 200: 83-104Crossref PubMed Scopus (1178) Google Scholar]. It enables the observation of endogenous signals such as Two-Photon Excitation Fluorescence (TPEF) and Second Harmonic Generation (SHG) in unstained samples and deep within the tissue sample [[5]Zipfel W.R. Williams R.M. Christie R. Nikitin A.Y. Hyman B.T. Webb W.W. Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation.Proc Natl Acad Sci USA. 2003; 100: 7075-7080Crossref PubMed Scopus (1516) Google Scholar]. In short, while TPEF allows for the visualization of the liver background and lobular organization, the SHG displays the three-dimensional architecture of fibrillar collagen. The specificity of SGH for fibrillar collagen is based on its highly ordered supra-molecular organization [[6]Brown E. McKee T. diTomaso E. Pluen A. Seed B. Brown E. et al.Dynamic imaging of collagen and its modulation in tumors in vivo using second-harmonic generation.Nat Med. 2003; 9: 796-800Crossref PubMed Scopus (750) Google Scholar]. The apparent complexity of this imaging technology combined with the high resolution and quality of images provided in this study should not reduce this article to a technical achievement only. It is in fact a new vision into liver fibrosis evaluation. With this approach the authors were able to visualize and quantify fibrillar collagen at a high resolution scale in the context of its microenvironment without utilizing the conventional staining method that is followed by formalin-fixed paraffin-embedding. To validate the specificity of the ECM components detected by the SHG, the authors compare SHG images with immunohistochemical labeling and observe a close relationship between Type I and III collagen isotypes and the SHG signal, while immunostaining for non-fibrillar collagen did not co-localize. High penetration and accurate spatial resolution of multiphoton microscopy provided impressive images of a 3-D reconstruction of the ECM network arrangement in liver parenchyma at a micrometer scale. Why is this important? If clinical evidence of fibrosis regression is present, the triple helix and organization of fibrillar collagen gives it high stability and, once deposited in the liver, it may impair liver architecture permanently [[7]Gieling R.G. Burt A.D. Mann D.A. Fibrosis and cirrhosis reversibility – molecular mechanisms.Clin Liver Dis. 2008; 12: 915-937Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar]. While obtaining a global assessment of liver fibrosis with the scoring system is important, the evaluation of presence, amount, and distribution of fibrillar collagen in a liver biopsy might be crucial for predicting prognosis of chronic liver diseases. However, it should be kept in mind that formation of fibrillar collagen is a late event in fibrogenesis. Evaluation of fibrosis with SHG may only give a rather restricted view of fibrosis stages, possibly missing the early stages of the process. Therefore, SHG should be considered as an additional tool that can provide an added value by allowing for the assessment of the relative amounts of fibrillar collagen within fibrous tissue, and should be used in conjunction with standard histopathology for fibrosis scoring. The physical properties of the ECM have recently been assessed through stiffness evaluation with ultrasound (Fibroscan) or magnetic resonance imaging. Although this approach appeared relevant to screen for advanced liver fibrosis, the correlation between liver fibrosis as assessed by the standard histopathology scoring system and liver stiffness is relatively loose [8Friedrich-Rust M. Ong M.F. Martens S. Sarrazin C. Bojunga J. Zeuzem S. et al.Performance of transient elastography for the staging of liver fibrosis: a meta-analysis.Gastroenterology. 2008; 134: 960-974Abstract Full Text Full Text PDF PubMed Scopus (1182) Google Scholar, 9Manning D.S. Afdhal N.H. Diagnosis and quantitation of fibrosis.Gastroenterology. 2008; 134: 1670-1681Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar]. Intuitively, the high degree of reticulation of fibrillar collagen might directly impact liver stiffness. Therefore, studying the correlation between the SHG index and liver stiffness might provide us with a plethora of new information. To validate this new method of evaluation of liver fibrosis, the authors compare the SHG index with the METAVIR score. Unsurprisingly, they found that the SHG index increased with the METAVIR stage but that this increase was exponential rather than linear. This exponential correlation is highly similar to the correlation between METAVIR scores and stiffness as evaluated with Fibroscan. The same is true for the wide range in SHG index observed in cirrhotic patients which has also been reported when stiffness evaluation is concerned in liver cirrhosis. These results also support the possible correlation between liver stiffness and relative composition of ECM in fibrillar collagen as assessed with the SGH index. Gailhouste et al. underline the benefit of a linear scale as provided by the SGH index by comparison to histological scoring systems since a continuous scale provides greater accuracy than a semi-quantitative score. Morphometry is another method that allows for a linear measurement of global liver fibrosis on tissue sections stained with PicroSirius red. Several studies have shown that quantitation with morphometry also correlates with histological scoring of fibrosis [[10]Pilette C. Rousselet M.C. Bedossa P. Chappard D. Oberti F. Rifflet H. et al.Histopathological evaluation of liver fibrosis: quantitative image analysis vs semi-quantitative scores. Comparison with serum markers.J Hepatol. 1998; 28: 439-446Abstract Full Text PDF PubMed Scopus (191) Google Scholar]. SHG and morphometry are both automated methods that avoid the risk of inter-observer variation in evaluation of liver fibrosis, a potential drawback of semi-quantitative scoring systems which remains partly subjective and dependent on pathologist expertise [[11]Rousselet M.C. Michalak S. Dupre F. Croue A. Bedossa P. Saint-Andre J.P. et al.Sources of variability in histological scoring of chronic viral hepatitis.Hepatology. 2005; 41: 257-264Crossref PubMed Scopus (450) Google Scholar]. Since both techniques can be performed in the same biopsy, a correlation between the evaluation of liver fibrosis with morphometry and SHG would also be useful in order to assess the extent of discrepancies in fibrosis quantification with both methods. Whether SHG or morphometry is concerned, these quantitative approaches, although very accurate and linear, deserve further commentaries. Firstly, sampling error which is related to heterogeneity in fibrosis distribution might become a relevant drawback when the accuracy of the evaluation technique for fibrosis quantification increases. Although this drawback is rather limited when simple semi-quantitative scoring systems are concerned (METAVIR or Ishak scores of fibrosis), several studies have shown that sampling errors become a relevant limit when accurate techniques that evaluate fibrosis along a linear scale are concerned [[12]Bedossa P. Dargère D. Paradis V. Sampling variability of liver fibrosis in chronic hepatitis C.Hepatology. 2003; 38: 1449-1457Crossref PubMed Scopus (1950) Google Scholar]. Second, the amount of fibrosis, whether evaluated through fibrillar collagen (SGH) or global fibrosis staining (Sirius red), is only a part of the deleterious process that leads to cirrhosis. Architectural changes, vascular shunts, and liver cell regeneration are among the other associated features which might have a significant influence on the natural progression of liver disease. They are also evaluated when pathologists score biopsies; however, these lesions are not considered when automated evaluation is used. Finally, SGH is clearly not a routine technique that is designed to replace staging of fibrosis with scoring systems but appears rather as a complement to semi-quantitative histopathology of fibrosis. At present, the major challenge is to demonstrate that the SGH index might provide an added value in the prediction of liver disease prognosis specifically with respect to fibrosis regression.