|
1
|
Leaker BD, Wang Y, Tam J, Anderson RR. Analysis of culture and RNA isolation methods for precision-cut liver slices from cirrhotic rats. Sci Rep 2024; 14:15349. [PMID: 38961190 PMCID: PMC11222550 DOI: 10.1038/s41598-024-66235-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/11/2023] [Accepted: 06/28/2024] [Indexed: 07/05/2024] Open
Abstract
Precision-cut liver slices (PCLS) are increasingly used as a model to investigate anti-fibrotic therapies. However, many studies use PCLS from healthy animals treated with pro-fibrotic stimuli in culture, which reflects only the early stages of fibrosis. The effects of different culture conditions on PCLS from cirrhotic animals has not been well characterized and there is no consensus on optimal methods. In this study, we report a method for the collection and culture of cirrhotic PCLS and compare the effect of common culture conditions on viability, function, and gene expression. Additionally, we compared three methods of RNA isolation and identified a protocol with high yield and purity. We observed significantly increased albumin production when cultured with insulin-transferrin-selenium and dexamethasone, and when incubated on a rocking platform. Culturing with insulin-transferrin-selenium and dexamethasone maintained gene expression closer to the levels in fresh slices. However, despite stable viability and function up to 4 days, we found significant changes in expression of key genes by day 2. Interestingly, we also observed that cirrhotic PCLS maintain viability in culture longer than slices from healthy animals. Due to the influence of matrix stiffness on fibrosis and hepatocellular function, it is important to evaluate prospective anti-fibrotic therapies in a platform that preserves tissue biomechanics. PCLS from cirrhotic animals represent a promising tool for the development of treatments for chronic liver disease.
Collapse
Affiliation(s)
- Ben D Leaker
- Health Sciences and Technology, Harvard-Massachusetts Institute of Technology, Cambridge, MA, USA.
- Wellman Center for Photomedicine, Massachusetts General Hospital, Thier Research Building, MGH, 55 Blossom Street, Boston, MA, USA.
| | - Yongtao Wang
- Division of Gastrointestinal and Oncologic Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA
- Liver Center, Division of Gastroenterology, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA
| | - Joshua Tam
- Wellman Center for Photomedicine, Massachusetts General Hospital, Thier Research Building, MGH, 55 Blossom Street, Boston, MA, USA
- Department of Dermatology, Harvard Medical School, Boston, MA, USA
| | - R Rox Anderson
- Wellman Center for Photomedicine, Massachusetts General Hospital, Thier Research Building, MGH, 55 Blossom Street, Boston, MA, USA
- Department of Dermatology, Harvard Medical School, Boston, MA, USA
| |
Collapse
|
|
2
|
Leaker BD, Sojoodi M, Tanabe KK, Popov YV, Tam J, Anderson RR. Increased susceptibility to ischemia causes exacerbated response to microinjuries in the cirrhotic liver. FASEB J 2024; 38:e23585. [PMID: 38661043 DOI: 10.1096/fj.202301438rr] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/14/2023] [Revised: 03/07/2024] [Accepted: 03/18/2024] [Indexed: 04/26/2024]
Abstract
Fractional laser ablation is a technique developed in dermatology to induce remodeling of skin scars by creating a dense pattern of microinjuries. Despite remarkable clinical results, this technique has yet to be tested for scars in other tissues. As a first step toward determining the suitability of this technique, we aimed to (1) characterize the response to microinjuries in the healthy and cirrhotic liver, and (2) determine the underlying cause for any differences in response. Healthy and cirrhotic rats were treated with a fractional laser then euthanized from 0 h up to 14 days after treatment. Differential expression was assessed using RNAseq with a difference-in-differences model. Spatial maps of tissue oxygenation were acquired with hyperspectral imaging and disruptions in blood supply were assessed with tomato lectin perfusion. Healthy rats showed little damage beyond the initial microinjury and healed completely by 7 days without scarring. In cirrhotic rats, hepatocytes surrounding microinjury sites died 4-6 h after ablation, resulting in enlarged and heterogeneous zones of cell death. Hepatocytes near blood vessels were spared, particularly near the highly vascularized septa. Gene sets related to ischemia and angiogenesis were enriched at 4 h. Laser-treated regions had reduced oxygen saturation and broadly disrupted perfusion of nodule microvasculature, which matched the zones of cell death. Our results demonstrate that the cirrhotic liver has an exacerbated response to microinjuries and increased susceptibility to ischemia from microvascular damage, likely related to the vascular derangements that occur during cirrhosis development. Modifications to the fractional laser tool, such as using a femtosecond laser or reducing the spot size, may be able to prevent large disruptions of perfusion and enable further development of a laser-induced microinjury treatment for cirrhosis.
Collapse
Affiliation(s)
- Ben D Leaker
- Health Sciences and Technology, Harvard-Massachusetts Institute of Technology, Cambridge, Massachusetts, USA
- Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, Massachusetts, USA
| | - Mozhdeh Sojoodi
- Division of Gastrointestinal and Oncologic Surgery, Massachusetts General Hospital, Boston, Massachusetts, USA
- Department of Surgery, Harvard Medical School, Boston, Massachusetts, USA
| | - Kenneth K Tanabe
- Division of Gastrointestinal and Oncologic Surgery, Massachusetts General Hospital, Boston, Massachusetts, USA
- Department of Surgery, Harvard Medical School, Boston, Massachusetts, USA
| | - Yury V Popov
- Division of Gastroenterology, Hepatology and Nutrition, Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA
- Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA
| | - Joshua Tam
- Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, Massachusetts, USA
- Department of Dermatology, Harvard Medical School, Boston, Massachusetts, USA
| | - R Rox Anderson
- Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, Massachusetts, USA
- Department of Dermatology, Harvard Medical School, Boston, Massachusetts, USA
| |
Collapse
|
|
3
|
Crawford JM, Bioulac-Sage P, Hytiroglou P. Structure, Function and Responses to Injury. MACSWEEN'S PATHOLOGY OF THE LIVER 2024:1-95. [DOI: 10.1016/b978-0-7020-8228-3.00001-6] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/03/2025]
|
|
4
|
Kandurova KY, Sumin DS, Mamoshin AV, Potapova EV. Deconvolution of the fluorescence spectra measured through a needle probe to assess the functional state of the liver. Lasers Surg Med 2023; 55:690-701. [PMID: 37300892 DOI: 10.1002/lsm.23695] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2023] [Revised: 05/24/2023] [Accepted: 05/29/2023] [Indexed: 06/12/2023]
Abstract
OBJECTIVES Currently, one of the most pressing issues for surgeons in the treatment of obstructive jaundice is the ability to assess the functional state of the liver and to detect and determine the degree of liver failure in a timely manner with simple and objective techniques. In this regard, the use of fluorescence spectroscopy method can be considered as one of the ways to improve the informativity of existing diagnostic algorithms in clinical practice and to introduce new diagnostic tools. Thus, the aim of the work was to study in vivo the functional state of liver parenchyma by the method of fluorescence spectroscopy implemented through a needle probe and assess the contribution of the main tissue fluorophores to reveal new diagnostic criteria. MATERIALS AND METHODS We compared data from 20 patients diagnosed with obstructive jaundice and 11 patients without this syndrome. Measurements were performed using a fluorescence spectroscopy method at excitation wavelengths of 365 and 450 nm. Data were collected using a 1 mm fiber optic needle probe. The analysis was based on the comparison of the results of deconvolution with the combinations of Gaussian curves reflecting the contribution of the pure fluorophores in the liver tissues. RESULTS The results showed a statistically significant increase in the contribution of curves reflecting NAD(P)H fluorescence, bilirubin, and flavins in the group of patients with obstructive jaundice. This and the calculated redox ratio values indicated that the energy metabolism of the hepatocytes may have shifted to glycolysis due to hypoxia. An increase in vitamin A fluorescence was also observed. It may also serve as a marker of liver damage, indicating impaired vitamin A mobilization from the liver due to cholestasis. CONCLUSIONS The results obtained reflect changes associated with shifts in the content of the main fluorophores characterizing hepatocyte dysfunction caused by accumulation of bilirubin and bile acids and after disturbance of oxygen utilization. The contributions of NAD(P)H, flavins, and bilirubin as well as vitamin A can be used for further studies as promising diagnostic and prognostic markers for the course of liver failure. Further work will include collecting fluorescence spectroscopy data in patients with different clinical effects of obstructive jaundice on postoperative clinical outcome after biliary decompression.
Collapse
Affiliation(s)
- Ksenia Y Kandurova
- Research and Development Center of Biomedical Photonics, Orel State University, Orel, Russia
| | - Dmitry S Sumin
- Research and Development Center of Biomedical Photonics, Orel State University, Orel, Russia
- Department of Interventional Radiology, Orel Regional Clinical Hospital, Orel, Russia
| | - Andrian V Mamoshin
- Research and Development Center of Biomedical Photonics, Orel State University, Orel, Russia
- Department of Interventional Radiology, Orel Regional Clinical Hospital, Orel, Russia
| | - Elena V Potapova
- Research and Development Center of Biomedical Photonics, Orel State University, Orel, Russia
| |
Collapse
|
|
5
|
Rodewald M, Bae H, Huschke S, Meyer-Zedler T, Schmitt M, Press AT, Schubert S, Bauer M, Popp J. In vivo coherent anti-Stokes Raman scattering microscopy reveals vitamin A distribution in the liver. JOURNAL OF BIOPHOTONICS 2021; 14:e202100040. [PMID: 33720518 DOI: 10.1002/jbio.202100040] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/02/2021] [Revised: 03/10/2021] [Accepted: 03/10/2021] [Indexed: 06/12/2023]
Abstract
Here we present a microscope setup for coherent anti-Stokes Raman scattering (CARS) imaging, devised to specifically address the challenges of in vivo experiments. We exemplify its capabilities by demonstrating how CARS microscopy can be used to identify vitamin A (VA) accumulations in the liver of a living mouse, marking the positions of hepatic stellate cells (HSCs). HSCs are the main source of extracellular matrix protein after hepatic injury and are therefore the main target of novel nanomedical strategies in the development of a treatment for liver fibrosis. Their role in the VA metabolism makes them an ideal target for a CARS-based approach as they store most of the body's VA, a class of compounds sharing a retinyl group as a structural motive, a moiety that is well known for its exceptionally high Raman cross section of the C═C stretching vibration of the conjugated backbone.
Collapse
Affiliation(s)
- Marko Rodewald
- Institute of Physical Chemistry and Abbe Center of Photonics, Friedrich Schiller University, Jena, Germany
- Leibniz Institute of Photonic Technology, Member of Leibniz Health Technologies, Jena, Germany
| | - Hyeonsoo Bae
- Leibniz Institute of Photonic Technology, Member of Leibniz Health Technologies, Jena, Germany
| | - Sophie Huschke
- Department of Anesthesiology and Intensive Care Medicine, Jena University Hospital, Jena, Germany
- Center for Sepsis Control and Care, Jena University Hospital, Jena, Germany
| | - Tobias Meyer-Zedler
- Institute of Physical Chemistry and Abbe Center of Photonics, Friedrich Schiller University, Jena, Germany
- Leibniz Institute of Photonic Technology, Member of Leibniz Health Technologies, Jena, Germany
| | - Michael Schmitt
- Institute of Physical Chemistry and Abbe Center of Photonics, Friedrich Schiller University, Jena, Germany
| | - Adrian Tibor Press
- Department of Anesthesiology and Intensive Care Medicine, Jena University Hospital, Jena, Germany
- Center for Sepsis Control and Care, Jena University Hospital, Jena, Germany
- Faculty of Medicine, Friedrich Schiller University, Jena, Germany
| | - Stephanie Schubert
- Institute of Pharmacy, Department of Pharmaceutical Technology and Biopharmacy, Friedrich Schiller University, Jena, Germany
| | - Michael Bauer
- Department of Anesthesiology and Intensive Care Medicine, Jena University Hospital, Jena, Germany
- Center for Sepsis Control and Care, Jena University Hospital, Jena, Germany
| | - Juergen Popp
- Institute of Physical Chemistry and Abbe Center of Photonics, Friedrich Schiller University, Jena, Germany
- Leibniz Institute of Photonic Technology, Member of Leibniz Health Technologies, Jena, Germany
| |
Collapse
|
|
6
|
Boissier N, Drasdo D, Vignon-Clementel IE. Simulation of a detoxifying organ function: Focus on hemodynamics modeling and convection-reaction numerical simulation in microcirculatory networks. INTERNATIONAL JOURNAL FOR NUMERICAL METHODS IN BIOMEDICAL ENGINEERING 2021; 37:e3422. [PMID: 33249746 DOI: 10.1002/cnm.3422] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/05/2019] [Revised: 10/09/2020] [Accepted: 11/09/2020] [Indexed: 06/12/2023]
Abstract
When modeling a detoxifying organ function, an important component is the impact of flow on the metabolism of a compound of interest carried by the blood. We here study the effects of red blood cells (such as the Fahraeus-Lindqvist effect and plasma skimming) on blood flow in typical microcirculatory components such as tubes, bifurcations and entire networks, with particular emphasis on the liver as important representative of detoxifying organs. In one of the plasma skimming models, under certain conditions, oscillations between states are found and analyzed in a methodical study to identify their causes and influencing parameters. The flow solution obtained is then used to define the velocity at which a compound would be transported. A convection-reaction equation is studied to simulate the transport of a compound in blood and its uptake by the surrounding cells. Different types of signal sharpness have to be handled depending on the application to address different temporal compound concentration profiles. To permit executing the studied models numerically stable and accurate, we here extend existing transport schemes to handle converging bifurcations, and more generally multi-furcations. We study the accuracy of different numerical schemes as well as the effect of reactions and of the network itself on the bolus shape. Even though this study is guided by applications in liver micro-architecture, the proposed methodology is general and can readily be applied to other capillary network geometries, hence to other organs or to bioengineered network designs.
Collapse
Affiliation(s)
- Noemie Boissier
- Inria, Paris, France
- Laboratoire Jacques-Louis Lions, Sorbonne Université, CNRS, Université de Paris, Paris, France
- IfADo - Leibniz Research Centre for Working Environment and Human Factors, Dortmund, Germany
| | - Dirk Drasdo
- Inria, Paris, France
- Laboratoire Jacques-Louis Lions, Sorbonne Université, CNRS, Université de Paris, Paris, France
- IfADo - Leibniz Research Centre for Working Environment and Human Factors, Dortmund, Germany
| | | |
Collapse
|
|
7
|
Immunolocalization of androgen and vitamin D receptors in the epididymis of mature ram ( Ovis aries). Saudi J Biol Sci 2020; 28:217-223. [PMID: 33424300 PMCID: PMC7783664 DOI: 10.1016/j.sjbs.2020.09.051] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2020] [Revised: 09/25/2020] [Accepted: 09/27/2020] [Indexed: 11/23/2022] Open
Abstract
This study illustrated the immunohistochemical distribution of androgen and vitamin D receptors of epididymis in 20 sexually mature ram (Rahmani breed) with average age ranged from (2_4) years and average weight ranged from (50_65kg). Androgen receptor was localized in the cytoplasm of both ciliated and non ciliated cells of efferent ductules, besides the principal cells via the entire epididymal duct. The principal cells of both corpus and proximal cauda epididymis showed the highest immunoreactivity to androgen receptors. Furthermore, vitamin D receptor was localized in the cytoplasm of all epithelium of the efferent ductules besides principal cells of all epididymal regions, however the immunoreaction was significantly higher in the efferent ductules, distal caput and distal cauda epididymis. In conclusion, these results suggest that the function of ram epididymis is regulated by both androgen and Vitamin D.
Collapse
|
|
8
|
Peng X, Li J, Wang M, Qu K, Zhu H. A novel AMPK activator improves hepatic lipid metabolism and leukocyte trafficking in experimental hepatic steatosis. J Pharmacol Sci 2019; 140:153-161. [PMID: 31253430 DOI: 10.1016/j.jphs.2019.05.008] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/15/2019] [Revised: 05/22/2019] [Accepted: 05/24/2019] [Indexed: 02/06/2023] Open
Abstract
A novel AMP-activated protein kinase (AMPK) activator, IMM-H007 (H007), has been reported to reduce serum lipid levels and inhibit lipid accumulation in the liver in hyperlipidemic animal models. However, how H007 ameliorates hepatic steatosis and inflammation remains unknown. In the present study, H007, at 200 mg/kg, reduced hepatic lipid levels and the levels of collagenous fiber in the liver in high-fat diet (HFD)-fed hamsters compared to those in the HFD group. Meanwhile, compared to the controls, H007 significantly inhibited sterol-regulatory element binding protein (SREBP)-1c and acetyl CoA carboxylase (ACC) expression by upregulating the AMPK activity, suppressing the saturated fatty acid accumulation and increasing polyunsaturated fatty acid synthesis in the liver. Compared to the controls, H007 treatment inhibited the expression of monocyte chemotactic protein (MCP-1) in fatty acid-treated HepG2 cells; suppressed leukocyte adherence and rolling on the liver microvasculature; and suppressed hepatic macrophage infiltration. H007 also suppressed the expression of nuclear factor-κB (NF-κB) p65 in fatty acid- and lipopolysaccharide-treated HepG2 cells compared to that in the controls by activating AMPK. These data suggested that H007 had a beneficial effect by improving the lipid composition in the liver and inhibiting inflammatory cell trafficking in the development of nonalcoholic fatty liver disease.
Collapse
Affiliation(s)
- Xueying Peng
- State Key Laboratory for Bioactive Substances and Functions of Natural Medicines, Beijing Key Laboratory of New Drug Mechanisms and Pharmacological Evaluation Study, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China
| | - Jin Li
- State Key Laboratory for Bioactive Substances and Functions of Natural Medicines, Beijing Key Laboratory of New Drug Mechanisms and Pharmacological Evaluation Study, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China; School of Public Health, The First Affiliated Hospital, Institute of Translational Medicine, Zhejiang University School of Medicine, Hangzhou, China
| | - Minjie Wang
- State Key Laboratory for Bioactive Substances and Functions of Natural Medicines, Beijing Key Laboratory of New Drug Mechanisms and Pharmacological Evaluation Study, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China; School of Basic Medical Sciences, The Center for Drug Screening, Center for New Drug Safety Evaluation and Research, Inner Mongolia Medical University, Inner Mongolia, China
| | - Kai Qu
- State Key Laboratory for Bioactive Substances and Functions of Natural Medicines, Beijing Key Laboratory of New Drug Mechanisms and Pharmacological Evaluation Study, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China
| | - Haibo Zhu
- State Key Laboratory for Bioactive Substances and Functions of Natural Medicines, Beijing Key Laboratory of New Drug Mechanisms and Pharmacological Evaluation Study, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China.
| |
Collapse
|
|
9
|
Saitou T, Takanezawa S, Ninomiya H, Watanabe T, Yamamoto S, Hiasa Y, Imamura T. Tissue Intrinsic Fluorescence Spectra-Based Digital Pathology of Liver Fibrosis by Marker-Controlled Segmentation. Front Med (Lausanne) 2019; 5:350. [PMID: 30619861 PMCID: PMC6297145 DOI: 10.3389/fmed.2018.00350] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/18/2018] [Accepted: 11/28/2018] [Indexed: 01/16/2023] Open
Abstract
Tissue intrinsic emission fluorescence provides useful diagnostic information for various diseases. Because of its unique feature of spectral profiles depending on tissue types, spectroscopic imaging is a promising tool for accurate evaluation of endogenous fluorophores. However, due to difficulties in discriminating those sources, quantitative analysis remains challenging. In this study, we quantitatively investigated spectral-spatial features of multi-photon excitation fluorescence in normal and diseased livers. For morphometrics of multi-photon excitation spectra, we examined a marker-controlled segmentation approach and its application to liver fibrosis assessment by employing a mouse model of carbon tetrachloride (CCl4)-induced liver fibrosis. We formulated a procedure of internal marker selection where markers were chosen to reflect typical biochemical species in the liver, followed by image segmentation and local morphological feature extraction. Image segmentation enabled us to apply mathematical morphology analysis, and the local feature was applied to the automated classification test based on a machine learning framework, both demonstrating highly accurate classifications. Through the analyses, we showed that spectral imaging of native fluorescence from liver tissues have the capability of differentiating not only between normal and diseased, but also between progressive disease states. The proposed approach provides the basics of spectroscopy-based digital histopathology of chronic liver diseases, and can be applied to a range of diseases associated with autofluorescence alterations.
Collapse
Affiliation(s)
- Takashi Saitou
- Department of Molecular Medicine for Pathogenesis, Graduate School of Medicine, Ehime University, Toon, Japan.,Translational Research Center, Ehime University Hospital, Toon, Japan.,Division of Bio-Imaging, Proteo-Science Center (PROS), Ehime University, Toon, Japan
| | - Sota Takanezawa
- Department of Molecular Medicine for Pathogenesis, Graduate School of Medicine, Ehime University, Toon, Japan
| | - Hiroko Ninomiya
- Department of Molecular Medicine for Pathogenesis, Graduate School of Medicine, Ehime University, Toon, Japan
| | - Takao Watanabe
- Department of Gastroenterology and Metabiology, Graduate School of Medicine, Ehime University, Toon, Japan
| | - Shin Yamamoto
- Department of Gastroenterology and Metabiology, Graduate School of Medicine, Ehime University, Toon, Japan.,Department of Lifestyle-related Medicine and Endocrinology, Graduate School of Medicine, Ehime University, Toon, Japan
| | - Yoichi Hiasa
- Department of Gastroenterology and Metabiology, Graduate School of Medicine, Ehime University, Toon, Japan
| | - Takeshi Imamura
- Department of Molecular Medicine for Pathogenesis, Graduate School of Medicine, Ehime University, Toon, Japan.,Translational Research Center, Ehime University Hospital, Toon, Japan.,Division of Bio-Imaging, Proteo-Science Center (PROS), Ehime University, Toon, Japan
| |
Collapse
|
|
10
|
Croce AC, Ferrigno A, Bottiroli G, Vairetti M. Autofluorescence-based optical biopsy: An effective diagnostic tool in hepatology. Liver Int 2018; 38:1160-1174. [PMID: 29624848 DOI: 10.1111/liv.13753] [Citation(s) in RCA: 39] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/11/2017] [Accepted: 03/27/2018] [Indexed: 12/15/2022]
Abstract
Autofluorescence emission of liver tissue depends on the presence of endogenous biomolecules able to fluoresce under suitable light excitation. Overall autofluorescence emission contains much information of diagnostic value because it is the sum of individual autofluorescence contributions from fluorophores involved in metabolism, for example, NAD(P)H, flavins, lipofuscins, retinoids, porphyrins, bilirubin and lipids, or in structural architecture, for example, fibrous proteins, in close relationship with normal, altered or diseased conditions of the liver. Since the 1950s, hepatocytes and liver have been historical models to study NAD(P)H and flavins as in situ, real-time autofluorescence biomarkers of energy metabolism and redox state. Later investigations designed to monitor organ responses to ischaemia/reperfusion were able to predict the risk of dysfunction in surgery and transplantation or support the development of procedures to ameliorate the liver outcome. Subsequently, fluorescent fatty acids, lipofuscin-like lipopigments and collagen were characterized as optical biomarkers of liver steatosis, oxidative stress damage, fibrosis and disease progression. Currently, serum AF is being investigated to improve non-invasive optical diagnosis of liver disease. Validation of endogenous fluorophores and in situ discrimination of cancerous from non-cancerous tissue belong to the few studies on liver in human subjects. These reports along with other optical techniques and the huge work performed on animal models suggest many optically based applications in hepatology. Optical diagnosis is currently offering beneficial outcomes in clinical fields ranging from the respiratory and gastrointestinal tracts, to dermatology and ophthalmology. Accordingly, this review aims to promote an effective bench to bedside transfer in hepatology.
Collapse
Affiliation(s)
- Anna Cleta Croce
- Institute of Molecular Genetics, Italian National Research Council (CNR), Pavia, Italy.,Department of Biology & Biotechnology, University of Pavia, Pavia, Italy
| | - Andrea Ferrigno
- Internal Medicine and Therapy Department, University of Pavia, Pavia, Italy
| | - Giovanni Bottiroli
- Institute of Molecular Genetics, Italian National Research Council (CNR), Pavia, Italy.,Department of Biology & Biotechnology, University of Pavia, Pavia, Italy
| | - Mariapia Vairetti
- Internal Medicine and Therapy Department, University of Pavia, Pavia, Italy
| |
Collapse
|
|
11
|
Crawford JM, Bioulac-Sage P, Hytiroglou P. Structure, Function, and Responses to Injury. MACSWEEN'S PATHOLOGY OF THE LIVER 2018:1-87. [DOI: 10.1016/b978-0-7020-6697-9.00001-7] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/03/2025]
|
|
12
|
Liebig M, Hassanzada A, Kämmerling M, Genz B, Vollmar B, Abshagen K. Microcirculatory disturbances and cellular changes during progression of hepatic steatosis to liver tumors. Exp Biol Med (Maywood) 2017; 243:1-12. [PMID: 29065724 DOI: 10.1177/1535370217738730] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Non-alcoholic fatty liver disease is closely associated with metabolic syndrome and comprises a pathological spectrum of liver disease ranging from steatosis to steatohepatitis and can progress to fibrosis/cirrhosis and hepatocellular carcinoma. In 2013, a mouse model was described that mimics non-alcoholic fatty liver disease progression from steatohepatitis to tumors in a short time span and with high incidence. As microcirculatory disturbances play a crucial role in liver disease, the suitability of the steatosis-inflammation-tumor model for microcirculatory studies was assessed. Herein, we present a comprehensive view on morphological, microvascular, cellular, and functional aspects of non-alcoholic fatty liver disease progression in the steatosis-inflammation-tumor model using intravital microscopy, biochemical, and histological techniques. Mice develop steatohepatitis, mild fibrosis, and liver tumors at ages of 6, 12, and 20 weeks, respectively. Non-alcoholic fatty liver disease progression was accompanied by several general aspects of disease severity like increasing liver/body weight index, non-alcoholic fatty liver disease activity score, and hepatocellular apoptosis. Intravital microscopic analysis revealed significant changes in hepatic microcirculation with increasing structural alterations, elevated leukocyte adherence, and impaired nutritive perfusion. Non-alcoholic fatty liver disease was further characterized by a lower sinusoidal density with a striking rise at 20 weeks. The characteristic microcirculatory changes make the model a convenient tool for analysis of microcirculation during progression from steatosis to liver tumor. Impact statement Significant alterations of microcirculation contribute to progression of NAFLD, a chronic liver disease with increasing medical and socio-economic impact. Characterization of microcirculation in a NAFLD model reflecting all relevant stages of disease progression was still missing. Thus, we evaluated microcirculatory and cellular changes in a steatosis-inflammation-tumor model using in vivo microscopy. Analyses revealed increasing structural alterations, elevated leukocyte-endothelial interaction, and impaired nutritive perfusion. Thus, this model is suitable for further studies investigating therapeutic approaches targeting these progressive microcirculatory disturbances.
Collapse
Affiliation(s)
- Marie Liebig
- 1 Institute for Experimental Surgery, University Medicine Rostock, Rostock 18057, Germany
| | - Alireza Hassanzada
- 1 Institute for Experimental Surgery, University Medicine Rostock, Rostock 18057, Germany
| | - Malte Kämmerling
- 1 Institute for Experimental Surgery, University Medicine Rostock, Rostock 18057, Germany
| | - Berit Genz
- 1 Institute for Experimental Surgery, University Medicine Rostock, Rostock 18057, Germany.,2 QIMR Berghofer Medical Research Institute, Brisbane QLD 4006, Australia
| | - Brigitte Vollmar
- 1 Institute for Experimental Surgery, University Medicine Rostock, Rostock 18057, Germany
| | - Kerstin Abshagen
- 1 Institute for Experimental Surgery, University Medicine Rostock, Rostock 18057, Germany
| |
Collapse
|
|
13
|
Croce AC, Ferrigno A, Di Pasqua LG, Berardo C, Bottiroli G, Vairetti M. NAD(P)H and Flavin Autofluorescence Correlation with ATP in Rat Livers with Different Metabolic Steady-State Conditions. Photochem Photobiol 2017; 93:1519-1524. [PMID: 28696576 DOI: 10.1111/php.12804] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2017] [Accepted: 06/12/2017] [Indexed: 12/16/2022]
Abstract
The monitoring of NAD(P)H and flavin autofluorescence (AF) is at the basis of numerous investigations on energy metabolism. Nevertheless, the ability of these AF biomarkers to accurately represent the energy currency, ATP, is poorly explored. Here, we focused on the AF/ATP correlation in lean and fatty livers with different steady-state metabolic conditions, achieved after organ isolation, preservation and recovery, in a likely dependence on both liver intrinsic metabolic features and externally induced perturbations. Within these eventual, various conditions, a significant correlation was detected between liver NAD(P)H and flavin AF, measured via fiber-optic probe, and biochemical ATP data, strengthening AF as biomarker of energy metabolism in steady-state conditions for wide-ranging experimental and diagnostic applications.
Collapse
Affiliation(s)
- Anna C Croce
- Institute of Molecular Genetics, Italian National Research Council (CNR), Pavia, Italy.,Department of Biology and Biotechnology, University of Pavia, Pavia, Italy.,Internal Medicine and Therapy (IMT), University of Pavia, Pavia, Italy
| | - Andrea Ferrigno
- Internal Medicine and Therapy (IMT), University of Pavia, Pavia, Italy
| | - Laura G Di Pasqua
- Internal Medicine and Therapy (IMT), University of Pavia, Pavia, Italy
| | - Clarissa Berardo
- Internal Medicine and Therapy (IMT), University of Pavia, Pavia, Italy
| | - Giovanni Bottiroli
- Institute of Molecular Genetics, Italian National Research Council (CNR), Pavia, Italy.,Department of Biology and Biotechnology, University of Pavia, Pavia, Italy.,Internal Medicine and Therapy (IMT), University of Pavia, Pavia, Italy
| | - Mariapia Vairetti
- Internal Medicine and Therapy (IMT), University of Pavia, Pavia, Italy
| |
Collapse
|
|
14
|
Croce AC, Ferrigno A, Bertone V, Piccolini VM, Berardo C, Di Pasqua LG, Rizzo V, Bottiroli G, Vairetti M. Fatty liver oxidative events monitored by autofluorescence optical diagnosis: Comparison between subnormothermic machine perfusion and conventional cold storage preservation. Hepatol Res 2017; 47:668-682. [PMID: 27448628 DOI: 10.1111/hepr.12779] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/18/2016] [Revised: 06/13/2016] [Accepted: 07/18/2016] [Indexed: 12/16/2022]
Abstract
AIMS Livers with moderate steatosis are currently recruited as marginal organs to face donor shortage in transplantation, even though lipid excess and oxidative stress increase preservation injury risk. Sensitive, real-time detection of liver metabolism engagement could help donor selection and preservation procedures, ameliorating the graft outcome. Hence, we investigated endogenous biomolecules with autofluorescence (AF) properties as biomarkers supporting the detection of liver oxidative events and the assessment of metabolic responses to external stimuli. METHODS Livers from male Wistar rats fed a 12-day methionine/choline-deficient (MCD) diet were subjected to AF spectrofluorometric analysis (fiber-optic probe, 366-nm excitation) before and after organ isolation, and following preservation (cold storage or 20°C machine perfusion) and reperfusion. RESULTS Innovative dynamic AF results on lipid oxidation to lipofuscin-like lipopigments, correlating with biochemical oxidative damage (thiobarbituric acid reactive substances) and antioxidant defense (glutathione) parameters, suggested lipid engagement in MCD livers counteracting reactive oxidizing species. The maintained MCD liver functionality was supported by limited changes in bilirubin AF spectral profile, reflecting bile composition balance, despite their intrinsic mitochondrial weakness, confirmed by adenosine 5'-triphosphate levels, and regardless of different preservation effects on energy metabolism revealed by conventional reduced forms of nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate and flavin AF data. CONCLUSION Autofluorescence showed that, after a relatively short time on an MCD diet, livers are still able to face oxidizing events and maintain a functional balance. These results strengthen AF as a supportive diagnostic tool in experimental hepatology, to characterize marginal livers in real time, monitor their response to ischemia/reperfusion, and investigate protective therapeutic agents.
Collapse
Affiliation(s)
- Anna Cleta Croce
- Istituto di Genetica Molecolare, Consiglio Nazionale delle Ricerche (IGM-CNR), San Matteo, Pavia, Italy.,Biology and Biotechnology Department, University of Pavia, Pavia, Italy
| | - Andrea Ferrigno
- Internal Medicine and Therapy Department, University of Pavia, Pavia, Italy
| | - Vittorio Bertone
- Biology and Biotechnology Department, University of Pavia, Pavia, Italy
| | - Valeria Maria Piccolini
- Istituto di Genetica Molecolare, Consiglio Nazionale delle Ricerche (IGM-CNR), San Matteo, Pavia, Italy
| | - Clarissa Berardo
- Internal Medicine and Therapy Department, University of Pavia, Pavia, Italy
| | | | - Vittoria Rizzo
- Molecular Medicine Department, University of Pavia and Istituto Ricovero e Cura Carattere Scientifico (IRCCS), San Matteo, Pavia, Italy
| | - Giovanni Bottiroli
- Istituto di Genetica Molecolare, Consiglio Nazionale delle Ricerche (IGM-CNR), San Matteo, Pavia, Italy.,Biology and Biotechnology Department, University of Pavia, Pavia, Italy
| | - Mariapia Vairetti
- Internal Medicine and Therapy Department, University of Pavia, Pavia, Italy
| |
Collapse
|
|
15
|
Peeters G, Debbaut C, Laleman W, Monbaliu D, Vander Elst I, Detrez JR, Vandecasteele T, De Schryver T, Van Hoorebeke L, Favere K, Verbeke J, Segers P, Cornillie P, De Vos WH. A multilevel framework to reconstruct anatomical 3D models of the hepatic vasculature in rat livers. J Anat 2016; 230:471-483. [PMID: 27995631 DOI: 10.1111/joa.12567] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 10/19/2016] [Indexed: 12/21/2022] Open
Abstract
The intricate (micro)vascular architecture of the liver has not yet been fully unravelled. Although current models are often idealized simplifications of the complex anatomical reality, correct morphological information is instrumental for scientific and clinical purposes. Previously, both vascular corrosion casting (VCC) and immunohistochemistry (IHC) have been separately used to study the hepatic vasculature. Nevertheless, these techniques still face a number of challenges such as dual casting in VCC and limited imaging depths for IHC. We have optimized both techniques and combined their complementary strengths to develop a framework for multilevel reconstruction of the hepatic circulation in the rat. The VCC and micro-CT scanning protocol was improved by enabling dual casting, optimizing the contrast agent concentration, and adjusting the viscosity of the resin (PU4ii). IHC was improved with an optimized clearing technique (CUBIC) that extended the imaging depth for confocal microscopy more than five-fold. Using in-house developed software (DeLiver), the vascular network - in both VCC and IHC datasets - was automatically segmented and/or morphologically analysed. Our methodological framework allows 3D reconstruction and quantification of the hepatic circulation, ranging from the major blood vessels down to the intertwined and interconnected sinusoids. We believe that the presented framework will have value beyond studies of the liver, and will facilitate a better understanding of various parenchymal organs in general, in physiological and pathological circumstances.
Collapse
Affiliation(s)
- Geert Peeters
- IBiTech - bioMMeda, Department of Electronics and Information Systems, Ghent University, Ghent, Belgium
| | - Charlotte Debbaut
- IBiTech - bioMMeda, Department of Electronics and Information Systems, Ghent University, Ghent, Belgium
| | - Wim Laleman
- Gastroenterology & Hepatology, University Hospitals Leuven, Leuven, Belgium.,Department of Clinical and Experimental Medicine, KU Leuven, Leuven, Belgium
| | - Diethard Monbaliu
- Abdominal Transplant Surgery, University Hospitals Leuven, Leuven, Belgium.,Department of Microbiology and Immunology, KU Leuven, Leuven, Belgium
| | - Ingrid Vander Elst
- Gastroenterology & Hepatology, University Hospitals Leuven, Leuven, Belgium.,Department of Clinical and Experimental Medicine, KU Leuven, Leuven, Belgium
| | - Jan R Detrez
- Laboratory of Cell Biology and Histology, Department of Veterinary Sciences, University of Antwerp, Antwerp, Belgium
| | - Tim Vandecasteele
- Department of Morphology, Faculty of Veterinary Medicine, Ghent University, Ghent, Belgium
| | - Thomas De Schryver
- Center for X-Ray Tomography, Department of Physics and Astronomy, Ghent University, Ghent, Belgium
| | - Luc Van Hoorebeke
- Center for X-Ray Tomography, Department of Physics and Astronomy, Ghent University, Ghent, Belgium
| | - Kasper Favere
- IBiTech - bioMMeda, Department of Electronics and Information Systems, Ghent University, Ghent, Belgium
| | - Jonas Verbeke
- IBiTech - bioMMeda, Department of Electronics and Information Systems, Ghent University, Ghent, Belgium
| | - Patrick Segers
- IBiTech - bioMMeda, Department of Electronics and Information Systems, Ghent University, Ghent, Belgium
| | - Pieter Cornillie
- Department of Morphology, Faculty of Veterinary Medicine, Ghent University, Ghent, Belgium
| | - Winnok H De Vos
- Laboratory of Cell Biology and Histology, Department of Veterinary Sciences, University of Antwerp, Antwerp, Belgium.,Cell Systems and Imaging, Department of Molecular Biotechnology, University of Ghent, Ghent, Belgium
| |
Collapse
|
|
16
|
Legesse FB, Heuke S, Galler K, Hoffmann P, Schmitt M, Neugebauer U, Bauer M, Popp J. Hepatic Vitamin A Content Investigation Using CoherentAnti-Stokes Raman Scattering Microscopy. Chemphyschem 2016; 17:4043-4051. [DOI: 10.1002/cphc.201600929] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/23/2016] [Indexed: 12/11/2022]
Affiliation(s)
- Fisseha Bekele Legesse
- Institute of Physical Chemistry and Abbe Center of Photonics; Friedrich Schiller University Jena; Helmholtzweg 4 07743 Jena Germany
- Leibniz Institute of Photonic Technology (IPHT) Jena e.V.; Albert-Einstein-Str. 9 07745 Jena Germany
| | - Sandro Heuke
- Institute of Physical Chemistry and Abbe Center of Photonics; Friedrich Schiller University Jena; Helmholtzweg 4 07743 Jena Germany
- Leibniz Institute of Photonic Technology (IPHT) Jena e.V.; Albert-Einstein-Str. 9 07745 Jena Germany
| | - Kerstin Galler
- Leibniz Institute of Photonic Technology (IPHT) Jena e.V.; Albert-Einstein-Str. 9 07745 Jena Germany
- Center for Sepsis Control and Care; Jena University Hospital; Erlanger Allee 101 07747 Jena Germany
| | - Patrick Hoffmann
- Leibniz Institute of Photonic Technology (IPHT) Jena e.V.; Albert-Einstein-Str. 9 07745 Jena Germany
- Center for Sepsis Control and Care; Jena University Hospital; Erlanger Allee 101 07747 Jena Germany
| | - Michael Schmitt
- Institute of Physical Chemistry and Abbe Center of Photonics; Friedrich Schiller University Jena; Helmholtzweg 4 07743 Jena Germany
| | - Ute Neugebauer
- Institute of Physical Chemistry and Abbe Center of Photonics; Friedrich Schiller University Jena; Helmholtzweg 4 07743 Jena Germany
- Leibniz Institute of Photonic Technology (IPHT) Jena e.V.; Albert-Einstein-Str. 9 07745 Jena Germany
- Center for Sepsis Control and Care; Jena University Hospital; Erlanger Allee 101 07747 Jena Germany
| | - Michael Bauer
- Center for Sepsis Control and Care; Jena University Hospital; Erlanger Allee 101 07747 Jena Germany
- Department of Anesthesiology and Intensive Care Medicine; Jena University Hospital; Am Klinikum 1 07747 Jena Germany
| | - Jürgen Popp
- Institute of Physical Chemistry and Abbe Center of Photonics; Friedrich Schiller University Jena; Helmholtzweg 4 07743 Jena Germany
- Leibniz Institute of Photonic Technology (IPHT) Jena e.V.; Albert-Einstein-Str. 9 07745 Jena Germany
- Center for Sepsis Control and Care; Jena University Hospital; Erlanger Allee 101 07747 Jena Germany
| |
Collapse
|
|
17
|
Ito D, Akamatsu N, Togashi J, Kaneko J, Arita J, Hasegawa K, Sakamoto Y, Kokudo N. Behavior and clinical impact of ascites after living donor liver transplantation: risk factors associated with massive ascites. JOURNAL OF HEPATO-BILIARY-PANCREATIC SCIENCES 2016; 23:688-696. [PMID: 27474897 DOI: 10.1002/jhbp.390] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/24/2016] [Accepted: 07/29/2016] [Indexed: 08/29/2023]
Abstract
BACKGROUND Massive ascites after living donor liver transplantation (LDLT), defined as small-for-size graft syndrome, is a risk factor for a poor prognosis. Few studies have reported factors associated with ascites and the relevant outcome after LDLT. METHODS Data from 413 adult patients that underwent LDLT were retrospectively analyzed. RESULTS Recipient age, preoperative albumin level, Child-Pugh score, preoperative ascites, graft volume, intraoperative blood loss, and duration of warm ischemic time and the anhepatic phase were significantly associated with the total amount of ascites between postoperative day (POD) 1 and POD14. Multivariate analysis identified preoperative ascites, intraoperative blood loss, and duration of anhepatic phase as factors. Massive ascites (ascitic fluid discharge >1,000 ml/day on POD14 after LDLT) occurred in 200 (48.4%) patients, and mild ascites occurred in the remaining 213 patients. Daily changes in the ascites volume differed between the two groups. Nevertheless, massive ascites itself did not have a critical impact on the patient short- and long-term outcomes when properly managed with rigorous diuretics and albumin administration. CONCLUSION Massive ascites is frequent after LDLT; however, the impact of it could be minimized with an appropriate management.
Collapse
Affiliation(s)
- Daisuke Ito
- Artificial Organ and Transplantation Division, Department of Surgery, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan
| | - Nobuhisa Akamatsu
- Artificial Organ and Transplantation Division, Department of Surgery, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan
| | - Junichi Togashi
- Artificial Organ and Transplantation Division, Department of Surgery, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan
| | - Junichi Kaneko
- Artificial Organ and Transplantation Division, Department of Surgery, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan
| | - Junichi Arita
- Artificial Organ and Transplantation Division, Department of Surgery, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan
| | - Kiyoshi Hasegawa
- Artificial Organ and Transplantation Division, Department of Surgery, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan
| | - Yoshihiro Sakamoto
- Artificial Organ and Transplantation Division, Department of Surgery, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan
| | - Norihiro Kokudo
- Artificial Organ and Transplantation Division, Department of Surgery, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan
| |
Collapse
|
|
18
|
Fischer MA, Brehmer K, Svensson A, Aspelin P, Brismar TB. Renal versus splenic maximum slope based perfusion CT modelling in patients with portal-hypertension. Eur Radiol 2016; 26:4030-4036. [DOI: 10.1007/s00330-016-4277-7] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2015] [Revised: 02/04/2016] [Accepted: 02/08/2016] [Indexed: 12/15/2022]
|
|
19
|
Wang H, Liang X, Mohammed YH, Thomas JA, Bridle KR, Thorling CA, Grice JE, Xu ZP, Liu X, Crawford DHG, Roberts MS. Real-time histology in liver disease using multiphoton microscopy with fluorescence lifetime imaging. BIOMEDICAL OPTICS EXPRESS 2015; 6:780-92. [PMID: 25798303 PMCID: PMC4361433 DOI: 10.1364/boe.6.000780] [Citation(s) in RCA: 32] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/29/2014] [Revised: 01/30/2015] [Accepted: 01/31/2015] [Indexed: 05/07/2023]
Abstract
Conventional histology with light microscopy is essential in the diagnosis of most liver diseases. Recently, a concept of real-time histology with optical biopsy has been advocated. In this study, live mice livers (normal, with fibrosis, steatosis, hepatocellular carcinoma and ischemia-reperfusion injury) were imaged by MPM-FLIM for stain-free real-time histology. The acquired MPM-FLIM images were compared with conventional histological images. MPM-FLIM imaged subsurface cellular and subcellular histopathological hallmarks of live liver in mice models at high resolution. Additional information such as distribution of stellate cell associated autofluorescence and fluorescence lifetime changes was also gathered by MPM-FLIM simultaneously, which cannot be obtained from conventional histology. MPM-FLIM could simultaneously image and quantify the cellular morphology and microenvironment of live livers without conventional biopsy or fluorescent dyes. We anticipate that in the near future MPM-FLIM will be evaluated from bench to bedside, leading to real-time histology and dynamic monitoring of human liver diseases.
Collapse
Affiliation(s)
- Haolu Wang
- Therapeutics Research Centre, School of Medicine, The University of Queensland, Princess Alexandra Hospital, Woolloongabba, QLD 4102,
Australia
- Department of Biliary-Pancreatic Surgery, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, 1630 S. Dongfang Road, Shanghai, 200127,
China
- These authors contributed equally to this work
| | - Xiaowen Liang
- Therapeutics Research Centre, School of Medicine, The University of Queensland, Princess Alexandra Hospital, Woolloongabba, QLD 4102,
Australia
- These authors contributed equally to this work
| | - Yousuf H. Mohammed
- Therapeutics Research Centre, School of Medicine, The University of Queensland, Princess Alexandra Hospital, Woolloongabba, QLD 4102,
Australia
| | - James A. Thomas
- Department of Gastroenterology, Princess Alexandra Hospital, School of Medicine, The University of Queensland, Woolloongabba, QLD 4102,
Australia
| | - Kim R. Bridle
- School of Medicine, The University of Queensland, Gallipoli Medical Research Foundation, Greenslopes Private Hospital, Greenslopes, QLD 4120,
Australia
| | - Camilla A. Thorling
- Therapeutics Research Centre, School of Medicine, The University of Queensland, Princess Alexandra Hospital, Woolloongabba, QLD 4102,
Australia
| | - Jeffrey E. Grice
- Therapeutics Research Centre, School of Medicine, The University of Queensland, Princess Alexandra Hospital, Woolloongabba, QLD 4102,
Australia
| | - Zhi Ping Xu
- Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St Lucia, QLD 4072,
Australia
| | - Xin Liu
- Therapeutics Research Centre, School of Medicine, The University of Queensland, Princess Alexandra Hospital, Woolloongabba, QLD 4102,
Australia
| | - Darrell H. G. Crawford
- School of Medicine, The University of Queensland, Gallipoli Medical Research Foundation, Greenslopes Private Hospital, Greenslopes, QLD 4120,
Australia
| | - Michael S. Roberts
- Therapeutics Research Centre, School of Medicine, The University of Queensland, Princess Alexandra Hospital, Woolloongabba, QLD 4102,
Australia
- School of Pharmacy and Medical Science, University of South Australia, Adelaide, SA 5001,
Australia
| |
Collapse
|
|
20
|
Croce AC, Bottiroli G. Autofluorescence spectroscopy and imaging: a tool for biomedical research and diagnosis. Eur J Histochem 2014; 58:2461. [PMID: 25578980 PMCID: PMC4289852 DOI: 10.4081/ejh.2014.2461] [Citation(s) in RCA: 331] [Impact Index Per Article: 30.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/21/2014] [Accepted: 12/04/2014] [Indexed: 12/18/2022] Open
Abstract
Native fluorescence, or autofluorescence (AF), consists in the emission of light in the UV-visible, near-IR spectral range when biological substrates are excited with light at suitable wavelength. This is a well-known phenomenon, and the strict relationship of many endogenous fluorophores with morphofunctional properties of the living systems, influencing their AF emission features, offers an extremely powerful resource for directly monitoring the biological substrate condition. Starting from the last century, the technological progresses in microscopy and spectrofluorometry were convoying attention of the scientific community to this phenomenon. In the future, the interest in the autofluorescence will certainly continue. Current instrumentation and analytical procedures will likely be overcome by the unceasing progress in new devices for AF detection and data interpretation, while a progress is expected in the search and characterization of endogenous fluorophores and their roles as intrinsic biomarkers.
Collapse
Affiliation(s)
- A C Croce
- Institute of Molecular Genetics of the National Research Council, University of Pavia.
| | | |
Collapse
|
|
21
|
Feng AC, Fan HL, Chen TW, Hsieh CB. Hepatic hemodynamic changes during liver transplantation: A review. World J Gastroenterol 2014; 20:11131-11141. [PMID: 25170200 PMCID: PMC4145754 DOI: 10.3748/wjg.v20.i32.11131] [Citation(s) in RCA: 40] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/03/2014] [Revised: 03/21/2014] [Accepted: 05/05/2014] [Indexed: 02/07/2023] Open
Abstract
Liver transplantation is performed in the recent decades with great improvements not only technically but also conceptually. However, there is still lack of consensus about the optimal hemodynamic characteristics during liver transplantation. The representative hemodynamic parameters include portal vein pressure, portal vein flow, and hepatic venous pressure gradient; however, there are still others potential valuable parameters, such as total liver inflow and hepatic artery flow. All the parameters are correlated closely and some internal modulating mechanisms, like hepatic arterial buffer response, occur to maintain stable hepatic inflow. To distinguish the unique importance of each hepatic and systemic parameter in different states during liver transplantation, we reviewed the published data and also conducted two transplant cases with different surgical strategies applied to achieve ideal portal inflow and pressure.
Collapse
|
|
22
|
Microcirculation changes during liver resection — A clinical study. Microvasc Res 2014; 94:47-51. [DOI: 10.1016/j.mvr.2014.05.002] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/28/2014] [Revised: 04/28/2014] [Accepted: 05/05/2014] [Indexed: 12/16/2022]
|
|
23
|
Integrated autofluorescence characterization of a modified-diet liver model with accumulation of lipids and oxidative stress. BIOMED RESEARCH INTERNATIONAL 2014; 2014:803491. [PMID: 25006587 PMCID: PMC4070497 DOI: 10.1155/2014/803491] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 02/27/2014] [Revised: 05/09/2014] [Accepted: 05/09/2014] [Indexed: 01/16/2023]
Abstract
Oxidative stress in fatty livers is mainly generated by impaired mitochondrial β-oxidation, inducing tissue damages and disease progression. Under suitable excitation, light liver endogenous fluorophores can give rise to autofluorescence (AF) emission, the properties of which depend on the organ morphofunctional state. In this work, we characterized the AF properties of a rat liver model of lipid accumulation and oxidative stress, induced by a 1–9-week hypercaloric methionine-choline deficient (MCD) diet administration. The AF analysis (excitation at 366 nm) was performed in vivo, via fiber optic probe, or ex vivo. The contribution of endogenous fluorophores involved in redox reactions and in tissue organization was estimated through spectral curve fitting analysis, and AF results were validated by means of different histochemical and biochemical assays (lipids, collagen, vitamin A, ROS, peroxidised proteins, and lipid peroxidation -TBARS-, GSH, and ATP). In comparison with the control, AF spectra changes found already at 1 week of MCD diet reflect alterations both in tissue composition and organization (proteins, lipopigments, and vitamin A) and in oxidoreductive pathway engagement (NAD(P)H, flavins), with a subsequent attempt to recover redox homeostasis. These data confirm the AF analysis potential to provide a comprehensive diagnostic information on negative effects of oxidative metabolism alteration.
Collapse
|
|
24
|
Balmer ML, Slack E, de Gottardi A, Lawson MAE, Hapfelmeier S, Miele L, Grieco A, Van Vlierberghe H, Fahrner R, Patuto N, Bernsmeier C, Ronchi F, Wyss M, Stroka D, Dickgreber N, Heim MH, McCoy KD, Macpherson AJ. The liver may act as a firewall mediating mutualism between the host and its gut commensal microbiota. Sci Transl Med 2014; 6:237ra66. [PMID: 24848256 DOI: 10.1126/scitranslmed.3008618] [Citation(s) in RCA: 347] [Impact Index Per Article: 31.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
A prerequisite for establishment of mutualism between the host and the microbial community that inhabits the large intestine is the stringent mucosal compartmentalization of microorganisms. Microbe-loaded dendritic cells trafficking through lymphatics are arrested at the mesenteric lymph nodes, which constitute the firewall of the intestinal lymphatic circulation. We show in different mouse models that the liver, which receives the intestinal venous blood circulation, forms a vascular firewall that captures gut commensal bacteria entering the bloodstream during intestinal pathology. Phagocytic Kupffer cells in the liver of mice clear commensals from the systemic vasculature independently of the spleen through the liver's own arterial supply. Damage to the liver firewall in mice impairs functional clearance of commensals from blood, despite heightened innate immunity, resulting in spontaneous priming of nonmucosal immune responses through increased systemic exposure to gut commensals. Systemic immune responses consistent with increased extraintestinal commensal exposure were found in humans with liver disease (nonalcoholic steatohepatitis). The liver may act as a functional vascular firewall that clears commensals that have penetrated either intestinal or systemic vascular circuits.
Collapse
Affiliation(s)
- Maria L Balmer
- Maurice Müller Laboratories (DKF), Universitätsklinik für Viszerale Chirurgie und Medizin Inselspital, University of Bern, Murtenstrasse 35, CH-3010 Bern, Switzerland
| | - Emma Slack
- Maurice Müller Laboratories (DKF), Universitätsklinik für Viszerale Chirurgie und Medizin Inselspital, University of Bern, Murtenstrasse 35, CH-3010 Bern, Switzerland
| | - Andrea de Gottardi
- Maurice Müller Laboratories (DKF), Universitätsklinik für Viszerale Chirurgie und Medizin Inselspital, University of Bern, Murtenstrasse 35, CH-3010 Bern, Switzerland
| | - Melissa A E Lawson
- Maurice Müller Laboratories (DKF), Universitätsklinik für Viszerale Chirurgie und Medizin Inselspital, University of Bern, Murtenstrasse 35, CH-3010 Bern, Switzerland
| | - Siegfried Hapfelmeier
- Institute for Infectious Diseases, University of Bern, Friedbühlstrasse 51, CH-3010 Bern, Switzerland
| | - Luca Miele
- Institute of Internal Medicine, Catholic University of Rome, Largo Agostino Gemelli 8, 00168 Rome, Italy
| | - Antonio Grieco
- Institute of Internal Medicine, Catholic University of Rome, Largo Agostino Gemelli 8, 00168 Rome, Italy
| | - Hans Van Vlierberghe
- Department of Hepatology and Gastroenterology, Ghent University, De Pintelaan 185, 9000 Ghent, Belgium
| | - René Fahrner
- Maurice Müller Laboratories (DKF), Universitätsklinik für Viszerale Chirurgie und Medizin Inselspital, University of Bern, Murtenstrasse 35, CH-3010 Bern, Switzerland
| | - Nicola Patuto
- Maurice Müller Laboratories (DKF), Universitätsklinik für Viszerale Chirurgie und Medizin Inselspital, University of Bern, Murtenstrasse 35, CH-3010 Bern, Switzerland
| | - Christine Bernsmeier
- Division of Gastroenterology and Hepatology, University Hospital Basel, Petersgraben 4, CH-4031 Basel, Switzerland
| | - Francesca Ronchi
- Maurice Müller Laboratories (DKF), Universitätsklinik für Viszerale Chirurgie und Medizin Inselspital, University of Bern, Murtenstrasse 35, CH-3010 Bern, Switzerland
| | - Madeleine Wyss
- Maurice Müller Laboratories (DKF), Universitätsklinik für Viszerale Chirurgie und Medizin Inselspital, University of Bern, Murtenstrasse 35, CH-3010 Bern, Switzerland
| | - Deborah Stroka
- Maurice Müller Laboratories (DKF), Universitätsklinik für Viszerale Chirurgie und Medizin Inselspital, University of Bern, Murtenstrasse 35, CH-3010 Bern, Switzerland
| | - Nina Dickgreber
- Institute of Pathology, University Bern, Murtenstrasse 31, Postfach 62, CH-3010 Bern, Switzerland
| | - Markus H Heim
- Division of Gastroenterology and Hepatology, University Hospital Basel, Petersgraben 4, CH-4031 Basel, Switzerland
| | - Kathy D McCoy
- Maurice Müller Laboratories (DKF), Universitätsklinik für Viszerale Chirurgie und Medizin Inselspital, University of Bern, Murtenstrasse 35, CH-3010 Bern, Switzerland
| | - Andrew J Macpherson
- Maurice Müller Laboratories (DKF), Universitätsklinik für Viszerale Chirurgie und Medizin Inselspital, University of Bern, Murtenstrasse 35, CH-3010 Bern, Switzerland.
| |
Collapse
|
|
25
|
Yang YY, Liu RS, Lee PC, Yeh YC, Huang YT, Lee WP, Lee KC, Hsieh YC, Lee FY, Tan TW, Lin HC. Anti-VEGFR agents ameliorate hepatic venous dysregulation/microcirculatory dysfunction, splanchnic venous pooling and ascites of NASH-cirrhotic rat. Liver Int 2014; 34:521-34. [PMID: 23998651 DOI: 10.1111/liv.12299] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/11/2013] [Accepted: 07/28/2013] [Indexed: 12/20/2022]
Abstract
BACKGROUND & AIMS Antivascular endothelial growth factor receptor (VEGFR) agents improve hepatic fibrosis and portal hypertension in cirrhosis. Detail interactions among recruited/activated leucocytes, hepatic angiogenesis and fibrogenesis, splanchnic blood pooling, decreased hepatic veins to fluctuated splanchnic blood volume (hepatic venous dysregulation), portal hypertensive syndrome and ascites have never explored in cirrhosis. Our study used two anti-VEGFR agents - brivanib and sorafenib - to elucidate the relationship between above abnormalities of nonalcoholic steatohepatitis (NASH)-cirrhotic rats. MATERIALS AND METHODS NASH-cirrhotic rats received 2-week brivanib, sorafenib or vehicle (NASH-cirrhotic+briv, NASH-cirrhotic+soraf and NASH-cirrhotic rats) were included for various measurements. RESULTS In comparison with NASH-cirrhotic rats, significant decreased plasma VEGF, fibroblast growth factor, platelet-derived growth factor, hepatic tumour necrosis factor (TNFα), IL-1β, IL-6, IL-17 were accompanied by decreased leucocyte mass/activity ((99 m) Tc-phytate and (18) F-FDG SPECT/PET/CT scans), hepatic leucocytes recruitment/microvascular density (fluorescence-enhanced intravital microscopy) and hydroxyproline content, and increased hepatic blood flow in NASH-cirrhotic+briv and NASH-cirrhotic+soraf rats. In addition, increased hepatic microvasculatures compliance-related improved buffering effect of portal vein to acute mannitol infusion was associated with decreased circulating nitric oxide and aldosterone, plasma volume expansion (dye dilution method), splanchnic blood pooling ((99 m) Tc-RBC SPECT/PET/CT scans), peripheral hypotension, portal hypertension and ascites in brivanib and sorafenib-treated NASH-cirrhotic rats. CONCLUSION Besides antifibrotic, antiangiogenic and portal hypertensive effects, chronic antagonism of anti-VEGFR with brivanib and sorafenib improves hepatic blood flow, hepatic venous dysregulation, inhibits leucocytes recruitment/activation, splanchnic blood pooling and ascites formation in NASH-cirrhotic rats. Thus, brivanib and sorafenib might be ideal therapeutic agents in cirrhotic patients suffering from severe haemodynamic disarrangement and ascites.
Collapse
Affiliation(s)
- Ying-Ying Yang
- Division of General Medicine, Taipei Veterans General Hospital, Taipei, Taiwan; Department of Medicine, National Yang-Ming University School of Medicine, Taipei, Taiwan
| | | | | | | | | | | | | | | | | | | | | |
Collapse
|
|
26
|
Reetz J, Genz B, Meier C, Kowtharapu BS, Timm F, Vollmar B, Herchenröder O, Abshagen K, Pützer BM. Development of Adenoviral Delivery Systems to Target Hepatic Stellate Cells In Vivo. PLoS One 2013; 8:e67091. [PMID: 23825626 PMCID: PMC3688967 DOI: 10.1371/journal.pone.0067091] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/23/2012] [Accepted: 05/15/2013] [Indexed: 12/19/2022] Open
Abstract
Hepatic stellate cells (HSCs) are known as initiator cells that induce liver fibrosis upon intoxication or other noxes. Deactivation of this ongoing remodeling process of liver parenchyma into fibrotic tissue induced by HSCs is an interesting goal to be achieved by targeted genetic modification of HSCs. The most widely applied approach in gene therapy is the utilization of specifically targeted vectors based on Adenovirus (Ad) serotype 5. To narrow down the otherwise ubiquitous tropism of parental Ad, two modifications are required: a) ablating the native tropism and b) redirecting the vector particles towards a specific entity solely present on the cells of interest. Therefore, we designed a peptide of the nerve growth factor (NGFp) with specific affinity for the p75 neurotrophin receptor (p75NTR) present on HSCs. Coupling of this NGFp to vector particles was done either via chemical conjugation using bifunctional polyethylene glycol (PEG) or, alternatively, by molecular bridging with a fusion protein specific for viral fiber knob and p75NTR. Both Ad vectors transmit the gene for the green fluorescent protein (GFP). GFP expression was monitored in vitro on primary murine HSCs as well as after systemic administration in mice with healthy and fibrotic livers using intravital fluorescence microscopy. Coupling of NGFp to Ad via S11 and/or PEGylation resulted in markedly reduced liver tropism and an enhanced adenoviral-mediated gene transfer to HSCs. Transduction efficiency of both specific Ads was uniformly higher in fibrotic livers, whereas Ad.GFP-S11-NGFp transduce activated HSCs better than Ad.GFP-PEG-NGFp. These experiments contribute to the development of a targeted gene transfer system to specifically deliver antifibrotic compounds into activated HSCs by systemically applied adenoviral vector modified with NGFp.
Collapse
Affiliation(s)
- Julia Reetz
- Institute of Experimental Gene Therapy and Cancer Research, Rostock University Medical School, Rostock, Germany
| | - Berit Genz
- Institute for Experimental Surgery, Rostock University Medical School, Rostock, Germany
| | - Claudia Meier
- Institute of Experimental Gene Therapy and Cancer Research, Rostock University Medical School, Rostock, Germany
| | - Bhavani S. Kowtharapu
- Institute of Experimental Gene Therapy and Cancer Research, Rostock University Medical School, Rostock, Germany
| | - Franziska Timm
- Institute for Experimental Surgery, Rostock University Medical School, Rostock, Germany
| | - Brigitte Vollmar
- Institute for Experimental Surgery, Rostock University Medical School, Rostock, Germany
| | - Ottmar Herchenröder
- Institute of Experimental Gene Therapy and Cancer Research, Rostock University Medical School, Rostock, Germany
| | - Kerstin Abshagen
- Institute for Experimental Surgery, Rostock University Medical School, Rostock, Germany
- * E-mail:
| | - Brigitte M. Pützer
- Institute of Experimental Gene Therapy and Cancer Research, Rostock University Medical School, Rostock, Germany
| |
Collapse
|
|
27
|
Iguchi H, Oda M, Yamazaki H, Yokomori H. Participation of aquaporin-1 in vascular changes and remodeling in cirrhotic liver. Med Mol Morphol 2013; 46:123-32. [PMID: 23549977 DOI: 10.1007/s00795-013-0039-7] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2012] [Accepted: 02/13/2013] [Indexed: 12/22/2022]
Abstract
The pathophysiology of arterial capillary proliferation accompanying fibrosis in human cirrhosis remains unclear. However, evidence regarding the molecules participating in the pathophysiological process has been accumulating. Water channel proteins known as aquaporins (AQP)s, notably AQP-1, appear to be involved in the arterial capillary proliferation in the cirrhotic liver.
Collapse
Affiliation(s)
- Hiroyoshi Iguchi
- Department of Radiology, Kitasato University Medical Center, Saitama, 364-8501, Japan
| | | | | | | |
Collapse
|
|
28
|
Timm F, Vollmar B. Heterogeneity of the intrahepatic portal venous blood flow: impact on hepatocyte transplantation. Microvasc Res 2012; 86:34-41. [PMID: 23220352 DOI: 10.1016/j.mvr.2012.11.008] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/12/2012] [Revised: 11/19/2012] [Accepted: 11/26/2012] [Indexed: 11/28/2022]
Abstract
BACKGROUND The poor repopulation rate of the liver by transplanted hepatocytes markedly hampers liver cell therapy, which might be due to a limited sequestration of cells within the hepatic microvasculature. We therefore present intravital fluorescence microscopic data of transplanted hepatocytes immediately after portal venous injection demonstrating their intrahepatic distribution. METHODS Male Wistar rats were transplanted with freshly isolated, rhodamine 123 labelled, primary rat hepatocytes. Cells (10(6) in 0.5 ml) were slowly injected via a catheter in the V. lienalis over 2 min. Their distribution in the left lateral liver lobe was visualized simultaneously as well as over the following 30 min by intravital fluorescence microscopy. In a second set of animals green fluorescent microspheres exhibiting a size of 15 μm were injected and observed identically. For further analyses of portal venous blood flow distribution sodium fluorescein was injected via the V. lienalis as well as via the V. jugularis. RESULTS In vivo imaging allowed the clear detection and observation of hepatocytes flowing into the liver and forming microemboli, which are trapped particularly in small distal portal branches. To a minor extent they were trapped as solitary cells in the periportal zone of sinusoids. Most interestingly, the distribution of cells within the liver was highly heterogeneous, as wide areas of acini were found free of transplanted cells after portal venous injection, while neighbouring areas showed disproportionately high hepatocyte occurrence. To further investigate this phenomenon sodium fluorescein was injected via the V. lienalis instead and an identical heterogeneous distribution pattern with clear anatomical borders defining highly, semi, and non-portal venous perfused liver acini could be observed. In contrast, systemic injection of sodium fluorescein via the V. jugularis in the same animals resulted in a homogenous dispersion within the liver. CONCLUSION Using in vivo fluorescence microscopy and exclusive portal venous injection of a fluorescent dye, we provide evidence for the existence of liver areas, differentially supplied by portal venous blood. As a consequence, hepatocytes transplanted via the portal tract are very heterogeneously distributed within the liver. This observation forces us to reconsider our current knowledge on (i) monitoring engrafted cells, (ii) the optimal hepatocyte number to be transplanted, (iii) portal hypertension after cell injection, and last but not least (iv) the optimal transplantation route. Moreover, the established model for in vivo visualization of transplanted hepatocytes allows development of new therapeutic strategies facilitating an improved engraftment of cells.
Collapse
Affiliation(s)
- Franziska Timm
- Institute for Experimental Surgery, University of Rostock, 18057 Rostock, Germany.
| | | |
Collapse
|
|
29
|
Abstract
The necessity of the liver being the organ responsible for metabolism of alcohol exposes it to many untoward toxic side effects. In the first instance of hepatic steatosis, fibrosis may occur indolently over years, slowly converting a greasy, steatotic liver into a cirrhotic liver. In the case of alcoholic hepatitis, brisk sinusoidal fibrosis may lead to more rapid development of cirrhosis, with the liver extensively subdivided by sublobular fibrous septa developing in the midst of extensive ongoing inflammation and hepatocellular destruction. Continued destruction of the parenchyma after cirrhosis has developed may produce a densely fibrotic organ with little remaining parenchyma.
Collapse
|
|
30
|
Yang YY, Tsai TH, Huang YT, Lee TY, Chan CC, Lee KC, Lin HC. Hepatic endothelin-1 and endocannabinoids-dependent effects of hyperleptinemia in nonalcoholic steatohepatitis-cirrhotic rats. Hepatology 2012; 55:1540-50. [PMID: 22183953 DOI: 10.1002/hep.25534] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/06/2011] [Accepted: 11/22/2012] [Indexed: 12/15/2022]
Abstract
UNLABELLED Leptin, the ob gene product, is a protein released from adipocytes and has been detected in fibrotic and cirrhotic livers. Leptin in brain has an inhibitory effect on food intake. Nonalcoholic steatohepatitis (NASH) is characterized by hyperleptinemia. This study explores the possible mechanisms of hyperleptinemia in relation to increased intrahepatic resistance (IHR) and portal hypertension in NASH cirrhotic rats. NASH cirrhotic rats with hyperleptinemia were induced in Zucker (fa/fa) and lean rats by feeding the animals a high fat/methionine-choline-deficient (HF/MCD) diet with and without exogenous administration of recombinant leptin. Portal venous pressure (PVP), IHR, plasma and hepatic levels of various substances, histopathology of the liver, the hepatic hydroxyproline content, and the expression of various hepatic protein and messenger RNA (mRNA) were measured. Hepatic microcirculatory dysfunction and the vasoconstrictive response to endothelin-1 were also observed using a liver perfusion system and intravital microscopy. Finally, the effect of leptin on hepatic stellate cells (HSCs) was evaluated. Both in HF/MCD-Zucker and HF/MCD+leptin lean rats, significant hepatic fibrogenesis and cirrhosis, marked portal hypertension, microcirculatory dysfunction, an enhanced vasoconstrictive response to endothelin-1, and an increased IHR were found to be associated with higher levels of hepatic endothelin-1 and endocannabinoids, expression levels of the cannabinoid type 1 receptor, endothelin-1 type A receptor (ET(A) R), activator protein-1, transforming growth factor beta (TGF-β)(1), osteopontin, tumor necrosis factor alpha (TNF-α), leptin, and the leptin receptor (OBRb). Interestingly, acute incubation of leptin directly increases the expression of ET(A) R, OBRb and activator protein-1 in HSCs. CONCLUSION An HF/MCD diet and hyperleptinemia increase hepatic endocannabinoids production, promote hepatic fibrogenesis, enhance the hepatic vasoconstrictive response to endothelin-1, and aggravate hepatic microcirculatory dysfunction; these events subsequently increase IHR and portal hypertension in NASH cirrhotic rats.
Collapse
Affiliation(s)
- Ying-Ying Yang
- Division of General Medicine, Department of Medicine, Taipei Veterans General Hospital, Taipei, Taiwan.
| | | | | | | | | | | | | |
Collapse
|
|
31
|
Zhang J, Chen WN. Inhibition of HBV-induced angiogenesis by ibuprofen: Role of HBx. Interv Med Appl Sci 2012. [DOI: 10.1556/imas.4.2012.1.5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022] Open
Abstract
AbstractChronic hepatitis B virus (HBV) carriers may develop hepatocellular carcinoma (HCC) by a wide range of mechanisms including angiogenesis. We show that HBV replication induces the expression of angiogenic proteins interleukin 6 (IL6) and cyclooxygenase-2 (Cox2). Interestingly, ibuprofen (a Cox2 inhibitor) is found to attenuate the levels of IL6 and Cox 2 which are induced by HBV replication.The mechanism of attenuation of angiogenic proteins by ibuprofen was further investigated. Our results show that HBx is involved in the increase of the expression of Cox2 through the NFκB pathway. However, the expression of Cox2 is decreased when the HBx-expressing cells are incubated with ibuprofen. The contrasting effect of HBx on Cox2 is found to be determined by differential dimer formation among the members of the NFκB family of proteins, including NFκB, RelA, and C-rel. Specifically, HBx alone results in dimer formation between NFκB and RelA, while the combined presence of HBx and ibuprofen leads to the formation of NFκB and C-rel. Additional information on the interaction network involving HBx, ibuprofen, and NFκB pathways is revealed by two-dimensional liquid chromatography-tandem mass spectrometry proteomics analysis. Taken together, our findings provide new insights on the angiogenesis induced by HBV replication.
Collapse
Affiliation(s)
- Jianhua Zhang
- 1 School of Chemical and Biomedical Engineering, College of Engineering, Nanyang Technological University, Singapore, Singapore
| | - Wei Ning Chen
- 1 School of Chemical and Biomedical Engineering, College of Engineering, Nanyang Technological University, Singapore, Singapore
- 2 School of Chemical and Biomedical Engineering, Nanyang Technological University, 62 Nanyag Drive, Singapore, 637459, Singapore
| |
Collapse
|
|
32
|
Abshagen K, Eipel C, Vollmar B. A critical appraisal of the hemodynamic signal driving liver regeneration. Langenbecks Arch Surg 2012; 397:579-90. [PMID: 22311102 DOI: 10.1007/s00423-012-0913-0] [Citation(s) in RCA: 50] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/17/2012] [Accepted: 01/20/2012] [Indexed: 12/25/2022]
Abstract
BACKGROUND Many aspects of the signaling mechanisms involved in the initiation of hepatic regeneration are under current investigation. Nevertheless, the actual mechanisms switching liver regeneration on and off are still unknown. Hemodynamic changes in the liver following partial hepatectomy have been suggested to be a primary stimulus in triggering liver regeneration. Most of the new knowledge about the impact of hemodynamic changes on liver regeneration is both conceptually important and directly relevant to clinical problems. PURPOSE The purpose of this review is therefore to exclusively address the hemodynamic signal driving the liver regeneration process.
Collapse
Affiliation(s)
- Kerstin Abshagen
- Institute for Experimental Surgery, University of Rostock, 18055 Rostock, Germany.
| | | | | |
Collapse
|
|
33
|
Feng H, Zhang J, Tan JYL, Sadrolodabaee L, Chen WN. Proteomics-related biomarkers for HBV-associated hepatocellular carcinoma: current status and future prospects. Future Virol 2012. [DOI: 10.2217/fvl.11.148] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
HBV infection is the major cause of the development of hepatocellular carcinoma (HCC). HCC is one of the most common malignancies in the world. The morbidity rate associated with HCC is mainly linked to late diagnosis. Thus, it is very important to discover prognostic factors that can act as biomarkers for preventing HCC development, and those that can act as therapeutic targets. Proteomics analysis has been applied to identify biomarkers from clinical HCC samples. In addition, the cell-based HBV replication and viral protein overexpression system, which provides a model of the cell at an early stage of viral infection, was also used to identify biomarkers. The proteins identified at this stage may be relevant to HBV-associated HCC prognosis. In this review, we discuss the current status of proteomics analysis in the discovery of cellular proteins and prognostic HCC biomarkers, with a special focus on cell metastasis and angiogenesis.
Collapse
Affiliation(s)
- Huixing Feng
- School of Chemical & Biomedical Engineering, Nanyang Technological University, Singapore 637459, Singapore
| | - Jianhua Zhang
- School of Chemical & Biomedical Engineering, Nanyang Technological University, Singapore 637459, Singapore
| | - Jane YL Tan
- School of Chemical & Biomedical Engineering, Nanyang Technological University, Singapore 637459, Singapore
| | - Laleh Sadrolodabaee
- School of Chemical & Biomedical Engineering, Nanyang Technological University, Singapore 637459, Singapore
| | - Wei Ning Chen
- School of Chemical & Biomedical Engineering, Nanyang Technological University, Singapore 637459, Singapore
| |
Collapse
|
|
34
|
Crawford JM, Burt AD. Anatomy, pathophysiology and basic mechanisms of disease. MACSWEEN'S PATHOLOGY OF THE LIVER 2012:1-77. [DOI: 10.1016/b978-0-7020-3398-8.00001-5] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/06/2025]
|
|
35
|
Alteration in the Pharmacokinetics of Hemoglobin-Vesicles in a Rat Model of Chronic Liver Cirrhosis Is Associated with Kupffer Cell Phagocyte Activity. J Pharm Sci 2011; 100:775-83. [DOI: 10.1002/jps.22286] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2010] [Revised: 06/07/2010] [Accepted: 06/08/2010] [Indexed: 12/18/2022]
|
|
36
|
Eipel C, Abshagen K, Vollmar B. Regulation of hepatic blood flow: The hepatic arterial buffer response revisited. World J Gastroenterol 2010; 16:6046-57. [PMID: 21182219 PMCID: PMC3012579 DOI: 10.3748/wjg.v16.i48.6046] [Citation(s) in RCA: 355] [Impact Index Per Article: 23.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
The interest in the liver dates back to ancient times when it was considered to be the seat of life processes. The liver is indeed essential to life, not only due to its complex functions in biosynthesis, metabolism and clearance, but also its dramatic role as the blood volume reservoir. Among parenchymal organs, blood flow to the liver is unique due to the dual supply from the portal vein and the hepatic artery. Knowledge of the mutual communication of both the hepatic artery and the portal vein is essential to understand hepatic physiology and pathophysiology. To distinguish the individual importance of each of these inflows in normal and abnormal states is still a challenging task and the subject of ongoing research. A central mechanism that controls and allows constancy of hepatic blood flow is the hepatic arterial buffer response. The current paper reviews the relevance of this intimate hepatic blood flow regulatory system in health and disease. We exclusively focus on the endogenous interrelationship between the hepatic arterial and portal venous inflow circuits in liver resection and transplantation, as well as inflammatory and chronic liver diseases. We do not consider the hepatic microvascular anatomy, as this has been the subject of another recent review.
Collapse
|
|
37
|
Croce AC, De Simone U, Freitas I, Boncompagni E, Neri D, Cillo U, Bottiroli G. Human liver autofluorescence: an intrinsic tissue parameter discriminating normal and diseased conditions. Lasers Surg Med 2010; 42:371-8. [PMID: 20583250 DOI: 10.1002/lsm.20923] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023]
Abstract
BACKGROUND AND OBJECTIVE Autofluorescence (AF) emission is an intrinsic parameter that can provide real-time information on morpho-functional properties of biological tissue, being strictly related with their biochemical composition and structural organization. The diagnostic potentials of AF-based techniques have been investigated on normal, fibrotic, and steatotic liver tissues, in reference to histological features as evidenced by specific histochemical stainings. MATERIALS AND METHODS AF emission under excitation at 366 nm has been examined on cryostatic tissue sections obtained from biopsies collected during surgical operation, by means of fluorescence imaging and microspectrofluorometric techniques. RESULTS NAD(P)H, collagen, and vitamin A were found to be the endogenous fluorophores characterizing normal, fibrotic, and steatotic liver tissue AF, respectively. The differences of their photo-physical properties, in terms of emission amplitude, spectral shape, and response to irradiation, give rise to modifications of overall AF signal collected from tissues that allow the liver conditions to be distinguished. CONCLUSION The study provides a valid premise for a development of AF-based optical biopsy techniques for a real-time discrimination of liver anatomo-pathological patterns.
Collapse
Affiliation(s)
- Anna C Croce
- Histochemistry and Cytometry Section, IGM-CNR, Pavia 27100, Italy
| | | | | | | | | | | | | |
Collapse
|
|
38
|
Apparent Diffusion Coefficient of Fibrosis and Regenerative Nodules in the Cirrhotic Liver at MRI. AJR Am J Roentgenol 2010; 194:1515-22. [DOI: 10.2214/ajr.09.3484] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
|
|
39
|
Administration of a low dose of sildenafil for 1 week decreases intrahepatic resistance in rats with biliary cirrhosis: the role of NO bioavailability. Clin Sci (Lond) 2010; 119:45-55. [PMID: 20132096 DOI: 10.1042/cs20090601] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Increasing NO bioavailability improves hepatic endothelial dysfunction, which ameliorates intrahepatic resistance and portal hypertension. Acute administration of sildenafil increases hepatic production of NO with a reduction in hepatic sinusoid resistance in cirrhotic patients and enhances the vasorelaxation response to NO in cirrhotic rat livers. However, the mechanisms were still unclear. Therefore, our present study aims to evaluate the effects and mechanisms of administration of sildenafil for 1 week on the hepatic microcirculation of cirrhotic rats. Cirrhosis was induced by bile duct ligation with sham-operated rats serving as normal controls. Intrahepatic resistance was evaluated by in situ liver perfusion. Expression of phospho-eNOS (endothelial NO synthase), iNOS (inducible NO synthase), phospho-Akt, PDE-5 (phosphodiesterase-5) and sGC (soluble guanylate cyclase) were determined by Western blot analysis. Biosynthesis of BH4 (tetrahydrobiopterin) and GTPCH-I (GTP cyclohydrolase I) activity were examined by HPLC. Intravital microscopy was used to observe the direct change in hepatic microcirculation. In cirrhotic rat livers, sildenafil treatment increased hepatic sinusoid volumetric flow, NO bioavailability, BH4, GTPCH-I activity, and the protein expression of phospho-Akt, phospho-eNOS and sGC. These events were associated with reduced protein expression of PDE-5, portal perfusion pressure and portal vein pressure. In contrast, sham rats did not produce any significant change in these measurements. In conclusion, sildenafil treatment improves endothelial dysfunction by augmenting NO bioavailability in the hepatic microcirculation.
Collapse
|
|
40
|
Vollmar B, Menger MD. The hepatic microcirculation: mechanistic contributions and therapeutic targets in liver injury and repair. Physiol Rev 2009; 89:1269-339. [PMID: 19789382 DOI: 10.1152/physrev.00027.2008] [Citation(s) in RCA: 368] [Impact Index Per Article: 23.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023] Open
Abstract
The complex functions of the liver in biosynthesis, metabolism, clearance, and host defense are tightly dependent on an adequate microcirculation. To guarantee hepatic homeostasis, this requires not only a sufficient nutritive perfusion and oxygen supply, but also a balanced vasomotor control and an appropriate cell-cell communication. Deteriorations of the hepatic homeostasis, as observed in ischemia/reperfusion, cold preservation and transplantation, septic organ failure, and hepatic resection-induced hyperperfusion, are associated with a high morbidity and mortality. During the last two decades, experimental studies have demonstrated that microcirculatory disorders are determinants for organ failure in these disease states. Disorders include 1) a dysregulation of the vasomotor control with a deterioration of the endothelin-nitric oxide balance, an arterial and sinusoidal constriction, and a shutdown of the microcirculation as well as 2) an overwhelming inflammatory response with microvascular leukocyte accumulation, platelet adherence, and Kupffer cell activation. Within the sequelae of events, proinflammatory mediators, such as reactive oxygen species and tumor necrosis factor-alpha, are the key players, causing the microvascular dysfunction and perfusion failure. This review covers the morphological and functional characterization of the hepatic microcirculation, the mechanistic contributions in surgical disease states, and the therapeutic targets to attenuate tissue injury and organ dysfunction. It also indicates future directions to translate the knowledge achieved from experimental studies into clinical practice. By this, the use of the recently introduced techniques to monitor the hepatic microcirculation in humans, such as near-infrared spectroscopy or orthogonal polarized spectral imaging, may allow an early initiation of treatment, which should benefit the final outcome of these critically ill patients.
Collapse
Affiliation(s)
- Brigitte Vollmar
- Institute for Experimental Surgery, University of Rostock, Rostock, Germany.
| | | |
Collapse
|
|
41
|
Zhang J, Niu D, Sui J, Ching CB, Chen WN. Protein profile in hepatitis B virus replicating rat primary hepatocytes and HepG2 cells by iTRAQ-coupled 2-D LC-MS/MS analysis: Insights on liver angiogenesis. Proteomics 2009; 9:2836-45. [DOI: 10.1002/pmic.200800911] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/24/2023]
|
|
42
|
Vanheule E, Geerts AM, Van Huysse J, Schelfhout D, Praet M, Van Vlierberghe H, De Vos M, Colle I. An intravital microscopic study of the hepatic microcirculation in cirrhotic mice models: relationship between fibrosis and angiogenesis. Int J Exp Pathol 2009; 89:419-32. [PMID: 19134051 DOI: 10.1111/j.1365-2613.2008.00608.x] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
This intravital fluorescence microscopy (IVFM) study validates cirrhotic mice models and describes the different intrahepatic alterations and the role of angiogenesis in the liver during genesis of cirrhosis. Cirrhosis was induced by subcutaneous injection of carbon tetrachloride (CCl(4)) and by common bile duct ligation (CBDL) in mice. Diameters of sinusoids, portal venules (PV), central venules (CV) and shunts were measured at different time points by IVFM. Thereafter, liver samples were taken for sirius red, CD31, Ki67, vascular endothelial growth factor (VEGF) and alpha-smooth muscle actin (alpha-SMA) evaluation by immunohistochemistry (IHC). In parallel with fibrogenesis, hepatic microcirculation was markedly disturbed in CCl(4) and CBDL mice with a significant decrease in sinusoidal diameter compared to control mice. In CCl(4) mice, CV were enlarged, with marked sinusoidal-free spaces around CV. In contrast, PV were enlarged in CBDL mice and bile lakes were observed. In both mice models, intrahepatic shunts developed gradually after induction. During genesis of cirrhosis using CD31 IHC we observed a progressive increase in the number of blood vessels within the fibrotic septa area and a progressively increase in staining by Ki67, VEGF and alpha-SMA of endothelial cells, hepatocytes and hepatic stellate cells respectively. In vivo study of the hepatic microcirculation demonstrated a totally disturbed intrahepatic architecture, with narrowing of sinusoids in both cirrhotic mice models. The diameters of CV and PV increased and large shunts, bypassing the sinusoids, were seen after both CCl(4) and CBDL induction. Thus present study shows that there is angiogenesis in the liver during cirrhogenesis, and this is probably due partially to an increased production of VEGF.
Collapse
Affiliation(s)
- Eline Vanheule
- Department of Hepatology and Gastroenterology, Ghent University Hospital, Ghent, Belgium.
| | | | | | | | | | | | | | | |
Collapse
|
|
43
|
Yang YY, Huang YT, Lin HC, Lee FY, Lee KC, Chau GY, Loong CC, Lai CR, Lee SD. Thalidomide decreases intrahepatic resistance in cirrhotic rats. Biochem Biophys Res Commun 2009; 380:666-72. [PMID: 19285019 DOI: 10.1016/j.bbrc.2009.01.160] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/01/2009] [Accepted: 01/26/2009] [Indexed: 12/15/2022]
Abstract
Increased intrahepatic resistance (IHR) within cirrhotic liver is caused by increased endotoxemia, cytokines tumor necrosis factor-alpha (TNF-alpha), vasoconstrictor thromboxane A(2) (TXA(2)), and disrupted microvasculatures. We evaluated the effects of thalidomide-related inhibition of TNF-alpha upon the hepatic microcirculation of cirrhosis in rats. Portal venous pressure (PVP), hepatic TNF-alpha, expression of thromboxane synthase (TXS), and leukocyte common antigen (LCA) were measured in bile-duct-ligated (BDL) rats receiving 1 month of thalidomide (BDL-thalido rats). Portal perfusion pressure (PPP), IHR, and hepatic TXA(2) production were measured in the isolated liver perfusion system. Intravital microscopy was used to examine hepatic microvascular disruptions. In BDL-thalido rats, PVP, PPP, IHR, hepatic TXA(2) and TNF-alpha, hydroxyproline content, expression of TXS and LCA, and LPS-induced leukocyte recruitment were significantly decreased. Conversely, hepatic microvascular density and perfused sinusoids were significantly increased. Thalidomide decreased PVP and IHR by reducing hepatic TXA(2) and improving hepatic microvascular disruptions in rats with biliary cirrhosis.
Collapse
Affiliation(s)
- Ying-Ying Yang
- Division of General Medicine, Taipei Veterans General Hospital, Taipei, Taiwan
| | | | | | | | | | | | | | | | | |
Collapse
|
|
44
|
Koeppel TA, Mihaljevic N, Kraenzlin B, Loehr M, Jesenofsky R, Post S, Palma P. Enhanced iNOS Gene Expression in the Steatotic Rat Liver after Normothermic Ischemia. Eur Surg Res 2007; 39:303-11. [PMID: 17595544 DOI: 10.1159/000104401] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2006] [Accepted: 03/23/2007] [Indexed: 12/23/2022]
Abstract
BACKGROUND Impaired hepatic microcirculation in the steatotic liver has been identified as a considerable factor for increased vulnerability after ischemia/reperfusion (I/R). Changes in regulation and synthesis of vasoactive mediators, such as nitric oxide (NO) and endothelin (ET-1), may result in functional impairment of postischemic sinusoidal perfusion. The aim of the current study was to assess the impact of I/R injury on postischemic gene expression of NO and ET-1 in steatotic livers. MATERIALS AND METHODS Male Sprague-Dawley rats with or without hepatic steatosis (induced by carbon tetrachloride treatment) were subjected to normothermic I/R injury. Steady-state mRNA levels were assessed using RT-PCR to study the expression of genes encoding ET-1, NO synthase (endothelial cell NO synthase and inducible NO synthase, iNOS). Immunohistochemistry was performed for detection of iNOS. RESULTS I/R injury was followed by increased iNOS gene expression (RT-PCR/immunohistochemistry) in animals with hepatic steatosis, predominately in hepatocytes with fatty degeneration. A mild increase in mRNA levels for ET-1 was found in steatotic rat livers. I/R induced a further increase in ET-1 gene expression in some but not all reperfused steatotic livers. CONCLUSIONS We show an enhanced gene expression of iNOS in postischemic steatotic rat livers. Hepatocytes with fatty degeneration appear to be the major source for NO generation. Furthermore, I/R may also induce ET-1 gene expression. Dysregulation of sinusoidal perfusion by NO and ET-1 is therefore likely to contribute to I/R injury of the steatotic liver.
Collapse
Affiliation(s)
- Thomas A Koeppel
- Department of Surgery, Klinikum Mannheim gGmbH, University Hospital, Faculty of Medicine of the University of Heidelberg, Mannheim, Germany
| | | | | | | | | | | | | |
Collapse
|
|
45
|
Kollmar O, Corsten M, Scheuer C, Vollmar B, Schilling MK, Menger MD. Portal branch ligation induces a hepatic arterial buffer response, microvascular remodeling, normoxygenation, and cell proliferation in portal blood-deprived liver tissue. Am J Physiol Gastrointest Liver Physiol 2007; 292:G1534-42. [PMID: 17347450 DOI: 10.1152/ajpgi.00503.2006] [Citation(s) in RCA: 49] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
Portal branch ligation (PBL) may prevent liver failure after extended hepatic resection. However, clinical studies indicate that tumors within the ligated lobe develop accelerated growth. Although it is well known that tumor growth depends on the host's microvascularization, there is no information about how PBL affects the hepatic microcirculation. Our aims were to determine hepatic artery response, liver microcirculation, tissue oxygenation, and cell proliferation after PBL. Therefore, we used intravital multifluorescence microscopy, laser-Doppler flowmetry, immunohistochemistry, and biochemical techniques to examine microcirculatory responses, microvascular remodeling, and cellular consequences after left lateral PBL in BALB/c mice. During the first 7 days, PBL induced a reduction of left hilar blood flow by approximately 50%. This resulted in 80% sinusoidal perfusion failure, significant parenchymal hypoxia, and liver atrophy. After 14 days, however, left hilar blood flow was found to be restored. However, remodeling of the microvasculature included a rarefaction of the sinusoidal network, however, without substantial perfusion failure, compensated by a hepatic arterial buffer response and significant sinusoidal dilatation. This resulted in normalization of tissue oxygenation, indicating arterialization of the ligated lobe. Interestingly, late microvascular remodeling was associated with increased endothelial nitric oxide synthase expression, significant hepatocellular proliferation, and weight gain of the ligated lobe. Thus PBL induces only an initial microcirculatory failure with liver atrophy, followed by a hepatic arterial buffer response, microvascular remodeling, normoxygenation, and hepatocellular proliferation. This may explain the accelerated tumor progression occasionally observed in patients after PBL.
Collapse
Affiliation(s)
- Otto Kollmar
- Dept. of General, Visceral, Vascular, and Pediatric Surgery, Univ. of Saarland, D-66421 Homburg/Saar, Germany.
| | | | | | | | | | | |
Collapse
|
|
46
|
Eipel C, Eisold M, Schuett H, Vollmar B. Inhibition of Heme Oxygenase-1 Protects Against Tissue Injury in Carbon Tetrachloride Exposed Livers. J Surg Res 2007; 139:113-20. [PMID: 17275847 DOI: 10.1016/j.jss.2006.09.016] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2006] [Revised: 08/29/2006] [Accepted: 09/08/2006] [Indexed: 01/22/2023]
Abstract
BACKGROUND/AIMS During the metabolism of the hepatotoxin carbon tetrachloride (CCl(4)) by cytochrome P450, heme, and free radicals are released. Heme oxygenase (HO-1) is an enzyme that is induced by heme as well as oxidative stress and has been reported to be involved in mediating protection against toxic liver injury. The purpose of the present study was to specify the role of HO-1 in CCl(4)-hepatotoxicity. METHODS AND RESULTS We could demonstrate an up-regulation of HO-1 protein in CCl(4)-exposed liver tissue that reaches its maximum after 6 to 12 h, along with intrahepatic leukocyte accumulation and tissue injury. When animals were pretreated with hemin for augmentation of HO-1 expression, CCl(4)-exposure was associated with a reduction of intrahepatic leukocyte accumulation, while inhibition of CCl(4)-induced HO-1 expression by tin protoporphyrin-IX (SnPP-IX) enhanced leukocytic response. Of interest, however, liver morphology, transaminases, and bile flow as parameters of hepatocellular integrity and excretory function did not concur with reduced leukocyte numbers in the hepatic microcirculation, and revealed best organ function and tissue preservation in case of HO-1 inhibition by SnPP-IX. In contrast, hemin-treated CCl(4)-exposed livers demonstrated pathologic enzyme release and cholestasis. CONCLUSIONS Taken together, inhibition of HO-1 in CCl(4)-hepatotoxicity protected the liver, while higher HO-1 activity harmed liver tissue, most probably due to interference of the HO-1 pathway with CCl(4)-dependent metabolism via cytochrome P450 and heme overload-associated toxicity.
Collapse
Affiliation(s)
- Christian Eipel
- Institute of Experimental Surgery, University of Rostock, Rostock, Germany
| | | | | | | |
Collapse
|
|
47
|
Amon M, Laschke MW, Harder Y, Vollmar B, Menger MD. Impact of severity of local soft-tissue trauma on long-term manifestation of microcirculatory and microlymphatic dysfunctions. ACTA ACUST UNITED AC 2006; 61:924-32. [PMID: 17033564 DOI: 10.1097/01.ta.0000195979.25659.fe] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
BACKGROUND The present study aimed at quantitatively evaluating the impact of severity of local trauma on manifestation of soft-tissue injury-associated microcirculatory and microlymphatic dysfunctions in a chronic model that allowed repeated analyses by intravital fluorescence microscopy. METHODS C57BL/6 mice were chronically instrumented with dorsal skinfold chambers and subjected to mild (180 J/m2, n = 6), moderate (270 J/m2, n = 6), or severe trauma (450 J/m2, n = 6; 540 J/m2, n = 6). Nontraumatized animals served as controls (sham; n = 8). Intravital microscopy was performed before and at 5 minutes, 1 hour, 8 hours, 24 hours, 3 days, and 5 days after trauma, and included the analysis of (1) blood and lymph microvessel rupture, (2) hematoma formation and lymph leakage, (3) arteriolar and venular constriction, (4) capillary perfusion failure, (5) arteriolar and venular leukocyte adhesion, and (6) interstitial edema formation. RESULTS Mild trauma did not induce any changes of microcirculatory and microlymphatic functions. Moderate trauma did not affect lymphatics but provoked arteriolar constriction, capillary perfusion failure, leukocyte-endothelial cell interactions, and minor blood vessel ruptures with hematoma formation. These alterations, however, recovered within the first 24 hours after trauma. Severe trauma also did not affect the lymphatic microvasculature, but resulted in massive hematoma formation, arteriolar constriction, and capillary perfusion failure, which was associated with marked arteriolar and venular leukocyte recruitment and edema formation, and which did not recover to normal over a 5-day observation period. CONCLUSION Only severe trauma of > 450 J/m2 provokes irreversible microcirculatory dysfunction in soft tissue, however, without affecting the integrity of lymphatic microvessels. Of interest, trauma-induced microcirculatory alterations are neither dominated solely by microcirculatory dysfunction nor by leukocytic inflammation. Instead, both pathologies develop in parallel, generating a vicious circle, which may be responsible for the compromised healing of severely traumatized soft tissue frequently observed in clinical practice.
Collapse
Affiliation(s)
- Michaela Amon
- Institute for Clinical and Experimental Surgery, University of Saarland, Homburg/Saar, Germany.
| | | | | | | | | |
Collapse
|
|
48
|
Abshagen K, Eipel C, Menger MD, Vollmar B. Comprehensive analysis of the regenerating mouse liver: an in vivo fluorescence microscopic and immunohistological study. J Surg Res 2006; 134:354-62. [PMID: 16500681 DOI: 10.1016/j.jss.2006.01.002] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2005] [Revised: 12/19/2005] [Accepted: 01/03/2006] [Indexed: 11/19/2022]
Abstract
BACKGROUND Regeneration of tissue is a fundamental parameter of the liver's response to injury and is essential for post-operative recovery from hepatic resection. An adequate microvascular supply of both remnant tissue and newly formed cell clusters is necessary for the accurate restoration of the organ. However, a comprehensive analysis of hepatic architecture and microcirculation during liver regeneration is missing. METHODS Using intravital high resolution multifluorescence microscopy and histological techniques we analyzed the regenerating liver of mice at days 3, 5, and 8 after 68% hepatectomy. RESULTS Within the 8-day course of regeneration, liver weight restored to approximately 90% of its original mass. During the 8-day period of observation density of hepatocytes and Ito cells was constantly found reduced from 4,200 and 800 cells/mm(2) in non-hepatectomized control livers to approximately 2,500 and approximately 500 cells/mm(2). A transient decline of sinusoidal density was partly compensated by recruiting all sinusoids for perfusion with a final perfusion index of 79, 89, and 98% at days 3, 5, and 8 after resection. Increase of sinusoidal diameters preserved functional vessel area after resection, guaranteeing an adequate tissue oxygenation, as verified by low parenchymal NADH autofluorescence. The number of PCNA expressing hepatocytes rose 11-, 4-, and 2-fold at days 3, 5, and 8 after resection, followed by a maximum proliferation of sinusoidal lining cells at day 5. Cell apoptosis slightly increased, most probably in response to the restoration and to eliminate redundant cells. Liver regeneration was not associated with enhanced leukocyte-endothelial cell interaction, disproving inflammation as a relevant trigger for the regeneration. CONCLUSION The present data contribute to a better understanding of the complex vascular and cellular mechanisms during liver regeneration to develop strategies fighting against impeded liver growth.
Collapse
Affiliation(s)
- Kerstin Abshagen
- Department of Experimental Surgery, University of Rostock, Rostock, Germany
| | | | | | | |
Collapse
|
|
49
|
Kollmar O, Scheuer C, Menger MD, Schilling MK. Macrophage inflammatory protein-2 promotes angiogenesis, cell migration, and tumor growth in hepatic metastasis. Ann Surg Oncol 2006; 13:263-75. [PMID: 16424980 DOI: 10.1245/aso.2006.03.096] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2005] [Accepted: 08/22/2005] [Indexed: 11/18/2022]
Abstract
BACKGROUND In a mouse model of hepatic metastasis, we herein analyzed whether the CXC chemokine macrophage inflammatory protein (MIP)-2, a functional analogue of the human interleukin 8, stimulates tumor cell migration in vitro and angiogenesis and tumor growth in vivo. METHODS By using chemotaxis chambers, CT26.WT colorectal tumor cell adhesion and migration were studied under stimulation with different concentrations of MIP-2. To evaluate angiogenesis and tumor growth in vivo, 1 x 10(5) CT26.WT cells were implanted into the left liver lobe of syngeneic BALB/c mice, and 10, 100, and 1000 nM of MIP-2 or phosphate-buffered saline (controls) was injected into the peritumoral area. After 7 days, angiogenesis, proliferation, tumor growth, apoptosis, cleaved caspase 3, and CXCR-2 expression were analyzed by using intravital fluorescence microscopy, histology, immunohistochemistry, and fluorescence-activated cell sorting. RESULTS In vitro, 98.8% of unstimulated CT26.WT cells showed CXCR-2 receptor expression. In the chemotaxis assays, MIP-2 provoked a dose-dependent increase of cell migration and a most pronounced cell adhesion at a dose of 100 nM. In vivo, MIP-2, in particular in a dose of 100 or 1000 nM, induced a significant increase of tumor capillary density and a marked widening of the angiogenic front at the tumor margin. Capillaries of the angiogenic front, but not of the tumor center, showed significant dilation, thus indicating a pronounced action of vascular endothelial growth factor. Tumor volume was significantly increased, in particular after 100 nM of MIP-2 stimulation, when compared with phosphate-buffered saline-treated controls, whereas only 1000 nM of MIP-2-treated animals additionally showed a higher frequency of apoptotic cell death within the tumor margin. CONCLUSIONS Our study indicates for the first time that the CXC chemokine MIP-2 promotes angiogenesis and growth of colorectal CT26.WT hepatic metastasis.
Collapse
Affiliation(s)
- Otto Kollmar
- Department of General, Visceral, Vascular and Pediatric Surgery, University of Saarland, D-66421 Homburg-Saar, Germany.
| | | | | | | |
Collapse
|
|
50
|
Maksan SM, Ulger Z, Gebhard MM, Schmidt J. Impact of antithrombin III on hepatic and intestinal microcirculation in experimental liver cirrhosis and bowel inflammation: An in vivo analysis. World J Gastroenterol 2005; 11:4997-5001. [PMID: 16124052 PMCID: PMC4321916 DOI: 10.3748/wjg.v11.i32.4997] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To analyze the hepatic and intestinal microcirculation in an animal model of liver cirrhosis and inflammatory bowel disease (IBD) and to characterize the anti-inflammatory action of antithrombin III (ATIII) on leukocyte kinetics and liver damage.
METHODS: Hepatic and intestinal microcirculation was investigated by intravital videomicroscopy. Standardized models of experimental chronic liver cirrhosis and bowel inflammation were employed. Animals were divided into four groups (n = 6/group): controls, animals with cirrhosis, animals with cirrhosis and IBD, animals with cirrhosis and IBD treated with ATIII.
RESULTS: Cirrhosis facilitated leukocyte rolling and sticking in hepatic sinusoids (1.91±0.28 sticker/µm vs 0.5±0.5 sticker/µm in controls, P<0.05). The effect enhanced in animals with cirrhosis and IBD (5.4±1.65 sticker/µm), but reversed after ATIII application (3.97±1.04 sticker/µm, P<0.05). Mucosal blood flow showed no differences in cirrhotic animals and controls (5.3±0.31 nL/min vs 5.4±0.25 nL/min) and was attenuated in animals with cirrhosis and IBD significantly (3.49±0.6 nL/min). This effect was normalized in the treatment group (5.13±0.4 nL/min, P<0.05). Enzyme values rose during development of cirrhosis and bowel inflammation, and reduced after ATIII application (P<0.05).
CONCLUSION: Liver cirrhosis in the presence of IBD leads to a significant reduction in mucosal blood flow and an increase in hepatic leukocyte adherence with consecutive liver injury, which can be prevented by administration of ATIII.
Collapse
|