Advertisement

Understanding liver immunology using intravital microscopy

Open AccessPublished:June 05, 2015DOI:https://doi.org/10.1016/j.jhep.2015.05.027

      Summary

      The liver has come a long way since it was considered only a metabolic organ attached to the gastrointestinal tract. The simultaneous ascension of immunology and intravital microscopy evidenced the liver as a central axis in the immune system, controlling immune responses to local and systemic agents as well as disease tolerance. The multiple hepatic cell populations are organized in a vascular environment that promotes intimate cellular interactions, including initiation of innate and adaptive immune responses, rapid leukocyte recruitment, pathogen clearance and production of a variety of immune mediators. In this review, we focus on the advances in liver immunology supported by intravital microscopy in diseases such as isquemia/reperfusion, acute liver injury and infections.

      Abbreviations:

      CCD (Charge coupled devices), CMOS (Complementary metal-oxide semiconductors), DILI (Drug-induced liver injury), KC (Kupffer cell), LS (Laser scanning), LSEC (Liver sinusoidal endothelial cells), NET (Neutrophil extracellular traps), OPS (Orthogonal polarization spectral), SD (Spinning-disk), SDF (Stream dark field), SHG (Second harmonic generation), SIM (Structured illumination microscopy), SPECT (Sporozoite microneme protein essential for cell traversal), STED (Stimulated emission depletion), STORM (Stochastic optical reconstruction microscopy)

      Keywords

      Historical perspective of intravital microscopy

      Humans always wanted to see farther and better. The oldest recognized magnifying device is the “Nimrud lens”, which dates back more than 2700 years and it was supposedly used as a magnifying glass and to produce heat by concentrating the sunlight. In this context, these first lenses might have been also used to cauterize small wounds, linking them immediately to medical and biological sciences [

      Museum TB. The Nimrud Lens/The Layard Lens. January 26th 2015 [cited; Available from: <http://www.britishmuseum.org/research/collection_online/collection_object_details.aspx?objectId=369215&partId=1>.

      ]. These lenses were further adapted in many ways, culminating in the development of first telescopes and microscopes. The invention of the microscope (ca.1600) and its improvements in the last half-century were essential to breakthrough discoveries in many fields of scientific investigation. Such findings in microvascular morphology and physiology built the basis for elucidation of the leukocyte recruitment cascade [
      • Hwa C.
      • Aird W.C.
      The history of the capillary wall: doctors, discoveries, and debates.
      ]. Apparently, the conventional histopathology techniques using fixed tissues were insufficient to describe the dynamics of biological processes, which generated a demand for in vivo imaging strategies. Thus, the terminology “intravital microscopy” (IVM) was coined for the adaptation and use of a microscope to image cells and tissues in live animals. Due to inefficient light sources and limitations in imaging solid organs at that time, the first live tissues visualized were from thin and transparent organs such as frog’s interdigital webbing or tongue. The first report of intravital microscopy dates back from 1846 in Marshall Hall (1790–1857) and August Waller (1814–1870) observations in frog’s capillaries. Their discoveries were seminal for the initial understanding of endothelial physiology. Also visualizing the microcirculation, Julius Cohnheim (1839–1884) demonstrated that leukocytes immigrate across the vessel wall to the interstitial space following stimuli [
      • Hwa C.
      • Aird W.C.
      The history of the capillary wall: doctors, discoveries, and debates.
      ]. Researchers have explored the fact that different species may be used for intravital microscopy, leading to live imaging of worms, fish, insects, amphibians, reptiles and small mammalians.
      The application of IVM in hepatology is more recent probably due to the solid structure of the liver and its location inside the peritoneal cavity, which make liver imaging more difficult. Nevertheless, the vast hepatic microvasculature and unique diversity of cell types provide a rich field for intravital studies in both health and disease [
      • Crispe I.N.
      The liver as a lymphoid organ.
      ]. In the past two decades, the development of new imaging techniques, dyes and fluorescent proteins that favor IVM resulted in an explosion of possibilities to mechanistically investigate liver biology down to the molecular level (Fig. 1). Liver IVM has been employed in studies of transplantation, ischemia/reperfusion injury, cancer, acute/chronic toxic injuries, fibrosis, infections, hemorrhagic shock and sepsis. In this review, we will focus on how in vivo imaging using different microscopy modalities has enhanced our understanding of liver immunology.
      Figure thumbnail gr1
      Fig. 1Number of entries on Pubmed containing the words “liver” and “intravital” from 1950 to 2014. Note that the abrupt increase in published papers in 1990’s is coincident with the increased availability of fluorescence and laser scanning confocal microscopes. Source: Alexandru Dan Corlan. Medline trend: automated yearly statistics of PubMed results for any query, 2004. Web resource at URL:http://dan.corlan.net/medline-trend.html. Accessed: 2012-02-14. (Archived by WebCite at http://www.webcitation.org/65RkD48SV).

      Imaging modalities for liver intravital microscopy

      The liver can be imaged by intravital microscopy with most standard microscopes or even by confocal probes that fit in a surgical needle [
      • Mennone A.
      • Nathanson M.H.
      Needle-based confocal laser endomicroscopy to assess liver histology in vivo.
      ,
      • Shieh F.K.
      • Drumm H.
      • Nathanson M.H.
      • Jamidar P.A.
      High-definition confocal endomicroscopy of the common bile duct.
      ]. As the pioneers in IVM evaluated translucent organs, the liver edge can be imaged in a similar way, since it is considerably thin [
      • McClugage Jr., S.G.
      • McCuskey R.S.
      “In vivo” microscopic study of the response of the hepatic microvascular system to carbon tetrachloride poisoning.
      ]. In this way, a regular bright field microscope would suffice to evaluate general leukocyte trafficking and behavior (Fig. 2 and Supplemental Video 1). However, deeper mechanistic approaches often require evaluation of specific cell types, prompting the use of antibodies to particular surface antigens (i.e., F4/80 in mouse monocytes and macrophages, CD3 in lymphocytes, etc.) or fluorescent protein expressing cells (Supplemental Video 2). In this case, fluorescence microscopes enable the identification of different markers (or cells) in the same animal. More recently, laser scanning confocal microscopes and newer alternatives such as spinning-disk and multi-photon microscopes emerged as higher quality intravital imaging options [
      • Jenne C.N.
      • Wong C.H.
      • Zemp F.J.
      • McDonald B.
      • Rahman M.M.
      • Forsyth P.A.
      • et al.
      Neutrophils recruited to sites of infection protect from virus challenge by releasing neutrophil extracellular traps.
      ,
      • McDonald B.
      • McAvoy E.F.
      • Lam F.
      • Gill V.
      • de la Motte C.
      • Savani R.C.
      • et al.
      Interaction of CD44 and hyaluronan is the dominant mechanism for neutrophil sequestration in inflamed liver sinusoids.
      ,
      • McDonald B.
      • Urrutia R.
      • Yipp B.G.
      • Jenne C.N.
      • Kubes P.
      Intravascular neutrophil extracellular traps capture bacteria from the bloodstream during sepsis.
      ,
      • Honda M.
      • Takeichi T.
      • Asonuma K.
      • Tanaka K.
      • Kusunoki M.
      • Inomata Y.
      Intravital imaging of neutrophil recruitment in hepatic ischemia-reperfusion injury in mice.
      ,
      • Ritsma L.
      • Steller E.J.
      • Ellenbroek S.I.
      • Kranenburg O.
      • Rinkes I.H. Borel
      • van Rheenen J.
      Surgical implantation of an abdominal imaging window for intravital microscopy.
      ] (Fig. 3 and Supplemental Videos 3 and 4). Confocal microscopes provide high-resolution fluorescence imaging since they use a pinhole to exclude out of focus light [
      • White J.G.
      • Amos W.B.
      • Fordham M.
      An evaluation of confocal versus conventional imaging of biological structures by fluorescence light microscopy.
      ,
      • van Meer G.
      • Stelzer E.H.
      • Wijnaendts-van-Resandt R.W.
      • Simons K.
      Sorting of sphingolipids in epithelial (Madin-Darby canine kidney) cells.
      ] offering live tissue imaging even of thick and complex samples. However, as laser scanning confocal raster scan the sample point-by-point, faster events are challenging to capture. Field scanning confocals such as the spinning-disk microscope utilize multiple pinholes [
      • Wang E.
      • Babbey C.M.
      • Dunn K.W.
      Performance comparison between the high-speed Yokogawa spinning disc confocal system and single-point scanning confocal systems.
      ], enabling faster phenomena such as platelets flowing in the bloodstream or calcium waves in the liver parenchyma to be imaged with accuracy. Multi-photon microscopes are also beneficial for IVM because of their ability of imaging at great tissue depths (hundreds of micrometers) [
      • Denk W.
      • Strickler J.H.
      • Webb W.W.
      Two-photon laser scanning fluorescence microscopy.
      ]. In multi-photon microscopy, only fluorophores in the in-focus-plane are excited, therefore excluding the need for pinholes while reducing phototoxicity and bleaching. Multi-photon excitation has been used to visualize tumorigenesis [
      • Ellenbroek S.I.
      • van Rheenen J.
      Imaging hallmarks of cancer in living mice.
      ], angiogenesis [
      • Brown E.B.
      • Campbell R.B.
      • Tsuzuki Y.
      • Xu L.
      • Carmeliet P.
      • Fukumura D.
      • et al.
      In vivo measurement of gene expression, angiogenesis and physiological function in tumors using multiphoton laser scanning microscopy.
      ] and related deep tissue processes that were previously difficult to assess due to inefficient fluorescence excitation and detection using standard confocal techniques. There are alternative imaging techniques such as light sheet microscopy, which provide optical sectioning capability. However, traditional light sheet microscopes involve a unique optical geometry, which poses challenges to sample mounting and therefore sample choices. Nevertheless, as these techniques evolve, intravital imaging using light sheet microscopy may become feasible in the future. With the large variety of imaging techniques currently available, choosing the best imaging approach relies on what the main research question is and the available budget (Table 1). Multi-photon and spinning-disk microscopes generally have higher costs, although they are more effective and versatile for intravital imaging. Nonetheless, reliable data can be acquired by adapting regular fluorescence or laser scanning confocal microscopes for intravital imaging [
      • Marques P.E.
      • Antunes M.M.
      • David B.A.
      • Pereira R.V.
      • Teixeira M.M.
      • Menezes G.B.
      Imaging liver biology in vivo using conventional confocal microscopy.
      ].
      Figure thumbnail gr2
      Fig. 2Possibilities of imaging acquisition using different microscopes and fluorescent probes. Liver intravital was initially performed using very simple microscopes and analyzed by naked eye. With the advent of modern microscopes and acquisition devices, liver structures could be better imaged. Using filters, white light source can generate specific wavelengths to excite fluorophores that can be organic dyes (i.e. fluorescein, DAPI), fluorescently labeled antibodies (i.e. PE-conjugated IgG), quantum dots and fluorescent proteins (i.e. GFP, RFP). These same probes can be imaged using confocal microscopes, which use pinholes to acquire images with higher resolution. The two confocal modalities available are laser scanning (LS) and spinning-disk (SD) microscopes. Multi-photon lasers can excite the same fluorophores but it allows imaging of deeper structures. Another advantage of multi-photon excitation is the opportunity of second harmonic generation (SHG) when imaging non-linear materials such as collagens. More recently, super-resolution systems here represented by structured illumination microscopy (SIM), stochastic optical reconstruction microscopy (STORM) and stimulated emission depletion (STED) microscopy have become available and may be considered the future of in vivo imaging once the current limitations are surpassed. Images derived from these techniques can be captured by photomultiplier tubes (PMT), charge coupled devices (CCD) or complementary metal–oxide–semiconductors (CMOS). Further, off-line analyzes can be performed using one of the various image quantification softwares available.
      Figure thumbnail gr3
      Fig. 3Liver intravital microscopy performed using different imaging modalities. (A) Mouse liver was imaged using a regular inverted microscope equipped with white light. Images were acquired with a regular camera (bright field). Note that only blood flow can be visualized (see also ). Using the same mouse, 5 mg of FITC-conjugated albumin was injected i.v. and liver was imaged using a fluorescence microscope (white light filtered by a 488 nm filter, ). In sequence, the same mouse was imaged in a laser scanning confocal microscope and the improvement in resolution can be observed: the liver microvasculature is better visualized and red blood cells are seen in negative passing within the vessels (). Also, using a motorized Z-section scan, it is possible to render 3D images to better visualize and quantify liver structure. (B) Another advantage of confocal microscopy is the opportunity to merge multiple images acquired in different channels (endothelium – BV421-conjugated anti-PECAM-1, BD Biosciences, blue channel; Lysm-eGFP neutrophils, green channel; Kupffer cells – PE-conjugated anti-F4/80, eBiosciences, red channel). In this way, the interaction of different cells, structures or molecules can be simultaneously imaged in the same experimental setting. Mice: C57BL/6 and Lysm-eGFP were from Centro de Bioterismo in Universidade Federal de Minas Gerais (CEBIO – UFMG, Brazil). All animal studies were approved by the Animal Care and Use Committee at UFMG (CEBIO 051/2011).
      Table 1Advantages and limitations of recent intravital liver imaging technologies. Considering the particularities of the experimental design and available budget, the choice of the best imaging technique might be based on a balance between how fast and deep the target phenomenon occurs. Faster phenomenon might need a spinning-disk confocal setup to be imaged, and an event that occurs deeper in the tissues might only be visible using a multi-photon confocal. Alternatively, conventional laser scanning microscopes might be sufficient for slower and more superficial imaging protocols.
      Recent advances in the field of super-resolution microscopy pose interesting possibilities for IVM (Fig. 2). Techniques such as SIM (structured illumination microscopy), which utilizes structured illumination, provide two fold improvement over the conventional diffraction limit without compromising cell health or speed. SIM is currently limited to relatively thin samples, but as technology is constantly improved, it will be very interesting to adapt SIM for IVM. Indeed, multi-photon excitation has been applied to SIM to provide deeper penetration depths [
      • Chen B.C.
      • Legant W.R.
      • Wang K.
      • Shao L.
      • Milkie D.E.
      • Davidson M.W.
      • et al.
      Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution.
      ]. SIM in its standard form can be utilized to “complement” conventional IVM techniques to examine structures inside thin samples (e.g. cremaster muscle or mesentery) at higher resolution. Additional super-resolution techniques such as STORM (stochastic optical reconstruction microscopy) which utilize Gaussian fitting of emission points, provide even greater resolution improvements of ∼ten fold over conventional microscopy techniques, enabling resolution down to the single molecule level. However, achieving ∼ten fold improvement in resolution requires longer imaging times and more prolonged exposure of cells to light, limiting their utility in live imaging experiments. This limitation also applies to other super-resolution techniques like STED (stimulated emission depletion microscopy) that provide similar improvements in resolution.
      Fluorescent labeling of hepatic cells and structures is not usually an issue when performing IVM. The same antibodies and probes used for immunofluorescence or flow cytometry are normally effective for IVM as well. Therefore, the imaging possibilities are virtually limitless as it is feasible to label multiple cells simultaneously (Fig. 3). In this sense, one can visualize collagen and extracellular matrix proteins [
      • Guc E.
      • Fankhauser M.
      • Lund A.W.
      • Swartz M.A.
      • Kilarski W.W.
      Long-term intravital immunofluorescence imaging of tissue matrix components with epifluorescence and two-photon microscopy.
      ], blood flow [
      • Pires D.A.
      • Marques P.E.
      • Pereira R.V.
      • David B.A.
      • Gomides L.F.
      • Dias A.C.
      • et al.
      Interleukin-4 deficiency protects mice from acetaminophen-induced liver injury and inflammation by prevention of glutathione depletion.
      ], bile canaliculi [
      • Li F.C.
      • Liu Y.
      • Huang G.T.
      • Chiou L.L.
      • Liang J.H.
      • Sun T.L.
      • et al.
      In vivo dynamic metabolic imaging of obstructive cholestasis in mice.
      ], sinusoidal endothelial cells (LSECs) [
      • Marques P.E.
      • Amaral S.S.
      • Pires D.A.
      • Nogueira L.L.
      • Soriani F.M.
      • Lima B.H.
      • et al.
      Chemokines and mitochondrial products activate neutrophils to amplify organ injury during mouse acute liver failure.
      ], hepatocytes [
      • Hsu Y.C.
      • Huang H.P.
      • Yu I.S.
      • Su K.Y.
      • Lin S.R.
      • Lin W.C.
      • et al.
      Serine protease hepsin regulates hepatocyte size and hemodynamic retention of tumor cells by hepatocyte growth factor signaling in mice.
      ], stellate cells [
      • Zhang X.Y.
      • Sun C.K.
      • Wheatley A.M.
      A novel approach to the quantification of hepatic stellate cells in intravital fluorescence microscopy of the liver using a computerized image analysis system.
      ], Kupffer cells [
      • Marques P.E.
      • Oliveira A.G.
      • Pereira R.V.
      • David B.A.
      • Gomides L.F.
      • Saraiva A.M.
      • et al.
      Hepatic DNA deposition drives drug-induced liver injury and inflammation in mice.
      ], neutrophils [
      • McDonald B.
      • Pittman K.
      • Menezes G.B.
      • Hirota S.A.
      • Slaba I.
      • Waterhouse C.C.
      • et al.
      Intravascular danger signals guide neutrophils to sites of sterile inflammation.
      ], platelets [
      • Hoffmeister K.M.
      • Felbinger T.W.
      • Falet H.
      • Denis C.V.
      • Bergmeier W.
      • Mayadas T.N.
      • et al.
      The clearance mechanism of chilled blood platelets.
      ], lymphocytes [
      • Siegmund K.
      • Lee W.Y.
      • Tchang V.S.
      • Stiess M.
      • Terracciano L.
      • Kubes P.
      • et al.
      Coronin 1 is dispensable for leukocyte recruitment and liver injury in concanavalin A-induced hepatitis.
      ], NKT cells [
      • Lee W.Y.
      • Moriarty T.J.
      • Wong C.H.
      • Zhou H.
      • Strieter R.M.
      • van Rooijen N.
      • et al.
      An intravascular immune response to Borrelia burgdorferi involves Kupffer cells and iNKT cells.
      ], and even cellular components such as DNA [
      • Marques P.E.
      • Oliveira A.G.
      • Pereira R.V.
      • David B.A.
      • Gomides L.F.
      • Saraiva A.M.
      • et al.
      Hepatic DNA deposition drives drug-induced liver injury and inflammation in mice.
      ]. Also, liver function can be assessed using fluorescent reagents, such as hepatocyte viability using Rhodamine 6G [
      • Pires D.A.
      • Marques P.E.
      • Pereira R.V.
      • David B.A.
      • Gomides L.F.
      • Dias A.C.
      • et al.
      Interleukin-4 deficiency protects mice from acetaminophen-induced liver injury and inflammation by prevention of glutathione depletion.
      ,
      • Li F.C.
      • Huang G.T.
      • Lin C.J.
      • Wang S.S.
      • Sun T.L.
      • Lo S.Y.
      • et al.
      Apical membrane rupture and backward bile flooding in acetaminophen-induced hepatocyte necrosis.
      ], hepatocyte bile secretion using carboxyfluorescein diacetate (CFDA) [
      • Li F.C.
      • Huang G.T.
      • Lin C.J.
      • Wang S.S.
      • Sun T.L.
      • Lo S.Y.
      • et al.
      Apical membrane rupture and backward bile flooding in acetaminophen-induced hepatocyte necrosis.
      ], endothelial viability using formaldehyde-treated albumin [
      • Ito Y.
      • Bethea N.W.
      • Abril E.R.
      • McCuskey R.S.
      Early hepatic microvascular injury in response to acetaminophen toxicity.
      ], and Kupffer cell (KC) phagocytosis using fluorescent latex beads [
      • Lee W.Y.
      • Moriarty T.J.
      • Wong C.H.
      • Zhou H.
      • Strieter R.M.
      • van Rooijen N.
      • et al.
      An intravascular immune response to Borrelia burgdorferi involves Kupffer cells and iNKT cells.
      ].
      Imaging acquisition requires detection and recording devices. The most common acquisition devices for laser scanning confocals are photomultiplier tubes (PMTs), but charge coupled devices (CCD) and complementary metal-oxide semiconductors (CMOS) are also widely used for field scanning confocals and epi-fluorescence microscopes for their high signal-to-noise and high-speed acquisition properties [
      • Phan T.G.
      • Bullen A.
      Practical intravital two-photon microscopy for immunological research: faster, brighter, deeper.
      ] (Fig. 2). Acquisition is usually followed by image analysis in a computer equipped with specific softwares. At this point, images and videos can be translated into data, for example: cell counts, velocity, track length, area measurements, interactions, and many other parameters [
      • Marques P.E.
      • Antunes M.M.
      • David B.A.
      • Pereira R.V.
      • Teixeira M.M.
      • Menezes G.B.
      Imaging liver biology in vivo using conventional confocal microscopy.
      ,
      • Marques P.E.
      • Oliveira A.G.
      • Pereira R.V.
      • David B.A.
      • Gomides L.F.
      • Saraiva A.M.
      • et al.
      Hepatic DNA deposition drives drug-induced liver injury and inflammation in mice.
      ].
      A simple microscope setup for liver IVM may be composed of a conventional laser scanning confocal microscope loaded with three lasers (405, 488, and 543 nm). This equipment allows imaging in three different channels, therefore three different structures can be simultaneously investigated. This microscope is relatively low cost when compared to multi-photon microscopes, but is indicated for imaging of static or slow events (i.e., cell adhesion, necrosis, perfusion). Also, PMTs rather than CCD cameras and manually controlled devices rather than software-controlled devices can be chosen to reduce overall equipment cost.

      General surgical procedures for liver IVM

      There are different surgical procedures to image liver in vivo, differing mainly on imaging protocol duration. For longer experimental periods (days to months), static imaging windows might be surgically implanted in the liver region and the same animal might be imaged several times over the experiment. This was likely adapted from intravital studies in other tissues, such as in neuroscience experiments using cranial windows for longitudinal brain imaging [
      • Ritsma L.
      • Steller E.J.
      • Ellenbroek S.I.
      • Kranenburg O.
      • Rinkes I.H. Borel
      • van Rheenen J.
      Surgical implantation of an abdominal imaging window for intravital microscopy.
      ]. Despite the reduced animal number involved in the experiments and the advantages of the prolonged imaging period, this modality may involve a technically challenging step that requires implantation of a custom-made imaging window. On the other hand, for shorter procedures (minutes to hours) the general surgical procedure is based on a midline laparotomy, removal of the peritoneal skin and muscle, with further externalization of a small hepatic segment (usually the apex of right lobe). In this case, the surgical procedures are simplified and the animal is maintained anesthetized during the imaging procedure [
      • McDonald B.
      • McAvoy E.F.
      • Lam F.
      • Gill V.
      • de la Motte C.
      • Savani R.C.
      • et al.
      Interaction of CD44 and hyaluronan is the dominant mechanism for neutrophil sequestration in inflamed liver sinusoids.
      ,
      • Marques P.E.
      • Antunes M.M.
      • David B.A.
      • Pereira R.V.
      • Teixeira M.M.
      • Menezes G.B.
      Imaging liver biology in vivo using conventional confocal microscopy.
      ,
      • McDonald B.
      • Pittman K.
      • Menezes G.B.
      • Hirota S.A.
      • Slaba I.
      • Waterhouse C.C.
      • et al.
      Intravascular danger signals guide neutrophils to sites of sterile inflammation.
      ,
      • Marques P.E.
      • Oliveira A.G.
      • Pereira R.V.
      • David B.A.
      • Gomides L.F.
      • Saraiva A.M.
      • et al.
      Hepatic DNA deposition drives drug-induced liver injury and inflammation in mice.
      ,
      • Hickey M.J.
      • Westhorpe C.L.
      Imaging inflammatory leukocyte recruitment in kidney, lung and liver – Challenges to the multi-step paradigm.
      ]. Due to the invasive nature of the surgical approach, animals are usually killed at the end of the imaging process. In order to image small rodents (i.e. mice or small rats), it may be necessary to use a handcraft stage allowing positioning of the animal under the microscope appropriately. These procedures were recently described in details, and can be adapted to image different organs in the peritoneal cavity other than the liver [
      • Marques P.E.
      • Antunes M.M.
      • David B.A.
      • Pereira R.V.
      • Teixeira M.M.
      • Menezes G.B.
      Imaging liver biology in vivo using conventional confocal microscopy.
      ]. More recently, the implementation of intensive care monitoring expanded intravital imaging to up to six hours and opened the possibility of maintaining mice viable for longer time periods (up to 12 h) [
      • Heymann F.
      • Niemietz P.M.
      • Peusquens J.
      • Ergen C.
      • Kohlhepp M.
      • Mossanen J.C.
      • et al.
      Long term intravital multiphoton microscopy imaging of immune cells in healthy and diseased liver using CXCR6.Gfp reporter mice.
      ]. In any case, due to the invasive nature of the surgical approach, animals must be sacrificed at the end of the imaging process, which precludes the documentation of long-term biological phenomena.
      Regarding its application in humans, IVM is still under development and novel devices are becoming available for use. Most techniques are non-invasive, for example, patient hemodynamic status can be predicted by imaging sublingual microcirculation with orthogonal polarization spectral (OPS) imaging or side-stream dark field (SDF), which can be incorporated into a hand-held device for clinical use [
      • Bezemer R.
      • Bartels S.A.
      • Bakker J.
      • Ince C.
      Clinical review: clinical imaging of the sublingual microcirculation in the critically ill – Where do we stand?.
      ]. Also, there are reports of successful mapping of skin vasculature networks using Correlation Mapping Optical Coherence Tomography (cmOCT) [
      • Enfield J.
      • Jonathan E.
      • Leahy M.
      In vivo imaging of the microcirculation of the volar forearm using correlation mapping optical coherence tomography (cmOCT).
      ]. Considering this, intravital imaging of human microvasculature might become a powerful clinical tool in the future, especially when available for assessment of the vast hepatic vasculature and broad range of liver diseases.

      Applications of IVM in basic investigation on liver diseases

      Hepatic ischemia and reperfusion injury (I/R)

      A number of surgical procedures in the liver demand the interruption of hepatic blood flow (ischemia) for different times, especially during liver transplantation, resection of tumors or extensive liver trauma. During ischemia, the lower oxygen and nutrient concentrations cause cell damage, which will be proportional to the ischemic period. Once the circulation is restored (reperfusion), leukocytes are attracted to the liver where they encounter a pro-inflammatory milieu rich in cytokines, lipid mediators and damage-associated molecular patterns (DAMPs) [
      • Peralta C.
      • Jimenez-Castro M.B.
      • Gracia-Sancho J.
      Hepatic ischemia and reperfusion injury: effects on the liver sinusoidal milieu.
      ]. These molecules will sustain leukocyte infiltration and activation, which can amplify tissue injury [
      • Chen G.Y.
      • Nunez G.
      Sterile inflammation: sensing and reacting to damage.
      ,
      • Rock K.L.
      • Latz E.
      • Ontiveros F.
      • Kono H.
      The sterile inflammatory response.
      ]. Using multi-color imaging IVM, it was observed that I/R and the associated inflammatory response lead to a widespread destruction of liver parenchymal and microvascular structure [
      • Honda M.
      • Takeichi T.
      • Asonuma K.
      • Tanaka K.
      • Kusunoki M.
      • Inomata Y.
      Intravital imaging of neutrophil recruitment in hepatic ischemia-reperfusion injury in mice.
      ]. Neutrophil infiltration and platelet adhesion in presinusoidal arterioles and postsinusoidal venules is critical in the pathogenesis of liver damage during early reperfusion. Interruption of blood flow dramatically enhances platelet-endothelial cell interactions after reperfusion in early timepoints (∼eight–nine-fold in arterioles/venules and ∼five fold in sinusoids), which is mediated by tight interaction between platelet ICAM-1 with sinusoid-deposited fibrinogen [
      • Khandoga A.
      • Biberthaler P.
      • Enders G.
      • Axmann S.
      • Hutter J.
      • Messmer K.
      • et al.
      Platelet adhesion mediated by fibrinogen-intercellular adhesion molecule-1 binding induces tissue injury in the postischemic liver in vivo.
      ]. Interestingly, even without employing exogenous fluorescent agents, it is possible to assess hepatocyte metabolic status in vivo by visualizing mitochondria autofluorescence (λ excitation = 445 nm and λ emission = 458–630 nm), which strongly correlates with tissue perfusion status [
      • Lu H.H.
      • Wu Y.M.
      • Chang W.T.
      • Luo T.
      • Yang Y.C.
      • Cho H.D.
      • et al.
      Molecular imaging of ischemia and reperfusion in vivo with mitochondrial autofluorescence.
      ]. In fact, autofluorescence recovery strongly correlated with the restoration of microcirculatory blood flow during reperfusion. Also, increase in cellular NADH can be monitored directly in vivo by IVM (λ excitation = 330–390 nm and λ emission ⩾430 nm) as a measurement of impaired oxygen supply [
      • Vollmar B.
      • Burkhardt M.
      • Minor T.
      • Klauke H.
      • Menger M.D.
      High-resolution microscopic determination of hepatic NADH fluorescence for in vivo monitoring of tissue oxygenation during hemorrhagic shock and resuscitation.
      ].
      Liver intravital studies were especially important to elucidate the peculiarities of liver leukocyte recruitment and to define the role of adhesion molecules in post-ischemic areas (Fig. 4). It is well accepted that I/R causes up-regulation of adhesion molecules that support leukocyte emigration to the liver during reperfusion. In fact, antibodies against ICAM-1, a glycoprotein that binds to β2-integrins to mediate firm adhesion, have a profound beneficial effect on liver damage mediated by leukocytes during I/R [
      • Vollmar B.
      • Glasz J.
      • Menger M.D.
      • Messmer K.
      Leukocytes contribute to hepatic ischemia/reperfusion injury via intercellular adhesion molecule-1-mediated venular adherence.
      ]. On the other hand, the role for selectins was disputed for years. Selectins (CD62) are cell adhesion molecules that bind to sugar moieties and are responsible to the early interactions between leukocytes and endothelium. Due to the lower affinity interactions with its ligand PSGL-1 (P-selectin glycoprotein ligand-1), selectins mediate mainly leukocyte tethering and rolling on the vessel wall [
      • Ley K.
      • Kansas G.S.
      Selectins in T-cell recruitment to non-lymphoid tissues and sites of inflammation.
      ]. These observations were made in tissues such as muscle and skin, however, leukocyte-endothelium interactions are more frequent in narrower vessels (5–8 μm diameter), so it was not expected a major role for selectins in the initial recruitment steps [
      • Lee W.Y.
      • Kubes P.
      Leukocyte adhesion in the liver: distinct adhesion paradigm from other organs.
      ]. In fact, in a model of liver inflammation with bacterial peptide (fMLP), deletion or blockage of P, E or L-selectins have no effect on leukocyte rolling or adhesion in sinusoids [
      • Wong J.
      • Johnston B.
      • Lee S.S.
      • Bullard D.C.
      • Smith C.W.
      • Beaudet A.L.
      • et al.
      A minimal role for selectins in the recruitment of leukocytes into the inflamed liver microvasculature.
      ]. Controversially, deletion of P-selectin abolishes leukocyte rolling and adhesion in terminal hepatic venules in mice submitted to I/R, suggesting a primary role for selectins in leukocyte adhesion in post sinusoidal venules but not sinusoids itself [
      • Sawaya Jr., D.E.
      • Zibari G.B.
      • Minardi A.
      • Bilton B.
      • Burney D.
      • Granger D.N.
      • et al.
      P-selectin contributes to the initial recruitment of rolling and adherent leukocytes in hepatic venules after ischemia/reperfusion.
      ]. In line with this, the procedure for experimental liver ischemia cause disruption of intestinal blood flow when the superior mesenteric artery is occluded, causing both organs to be affected. In this case, toxic products derived from the injured gut can reach the liver, triggering an inflammatory response. Kubes et al. demonstrated that if the intestinal circulation is bypassed and re-routed directly to the liver – abolishing the intestinal effects of hepatic I/R – leukocyte rolling and adhesion is independent of selectins [
      • Wong J.
      • Johnston B.
      • Lee S.S.
      • Bullard D.C.
      • Smith C.W.
      • Beaudet A.L.
      • et al.
      A minimal role for selectins in the recruitment of leukocytes into the inflamed liver microvasculature.
      ]. Therefore, the beneficial effects of anti-selectin therapies might arrive from their protective role on other vasculatures, including the intestines. These data established that the leukocyte recruitment paradigm in the liver during inflammation might different from the other organs (Fig. 4).
      Figure thumbnail gr4
      Fig. 4The general leukocyte recruitment paradigm and its peculiarities in the liver. (A) In most tissues, leukocytes (i.e. neutrophils) initially tether the endothelium adjacent to the inflammatory focus, where they start to roll until firm adhesion. Once adhered to the vessel wall, leukocytes crawl towards “hot spots” within the lumen, and finally, following a chemotactic gradient, they reach the extravascular compartment to accumulate in areas rich in chemokines, bacterial products or necrosis-derived molecules. (B) Due to the narrow and vast capillary network in the liver, leukocytes are usually in intimate contact with the sinusoidal endothelial cells. Therefore, once these cells tether the vessel walls on inflamed regions, different mechanisms immediately arrest them. Cells can become mechanically trapped in the sinusoids in a mechanism independent of adhesion molecules. Also, different adhesion molecules might be involved if a systemic inflammatory response is occurring (i.e. endotoxemia) or if the stimulus is restricted to a specific are of the liver (i.e. focal injury).

      Acute liver injury

      Drugs and natural products can be directly or indirectly cytotoxic to liver cells. Drug-induced liver injury (DILI) may be caused by a variety of “over-the-counter” medications, including analgesics, antibiotics, phytotherapics, and weight control drugs. In this context, DILI is the leading cause of acute liver failure and drug withdraw from clinical trials, therefore representing a major health problem [
      • Bernal W.
      • Wendon J.
      Acute liver failure.
      ]. Methodologically, DILI can be acutely induced by administration of a high dose of different chemicals, including acetaminophen (APAP) [
      • Marques P.E.
      • Antunes M.M.
      • David B.A.
      • Pereira R.V.
      • Teixeira M.M.
      • Menezes G.B.
      Imaging liver biology in vivo using conventional confocal microscopy.
      ,
      • Pires D.A.
      • Marques P.E.
      • Pereira R.V.
      • David B.A.
      • Gomides L.F.
      • Dias A.C.
      • et al.
      Interleukin-4 deficiency protects mice from acetaminophen-induced liver injury and inflammation by prevention of glutathione depletion.
      ,
      • Marques P.E.
      • Amaral S.S.
      • Pires D.A.
      • Nogueira L.L.
      • Soriani F.M.
      • Lima B.H.
      • et al.
      Chemokines and mitochondrial products activate neutrophils to amplify organ injury during mouse acute liver failure.
      ], thioacetamide (TAA) [
      • Gomides L.F.
      • Marques P.E.
      • Faleiros B.E.
      • Pereira R.V.
      • Amaral S.S.
      • Lage T.R.
      • et al.
      Murine model to study brain, behavior and immunity during hepatic encephalopathy.
      ], carbon tetrachloride (CCl4) [
      • Jaeschke H.
      • Williams C.D.
      • McGill M.R.
      • Xie Y.
      • Ramachandran A.
      Models of drug-induced liver injury for evaluation of phytotherapeutics and other natural products.
      ], ethanol [
      • McCuskey R.S.
      • Bethea N.W.
      • Wong J.
      • McCuskey M.K.
      • Abril E.R.
      • Wang X.
      • et al.
      Ethanol binging exacerbates sinusoidal endothelial and parenchymal injury elicited by acetaminophen.
      ], halothane [
      • Proctor W.R.
      • Chakraborty M.
      • Chea L.S.
      • Morrison J.C.
      • Berkson J.D.
      • Semple K.
      • et al.
      Eosinophils mediate the pathogenesis of halothane-induced liver injury in mice.
      ], alpha-amanitin [
      • Jedicke N.
      • Struever N.
      • Aggrawal N.
      • Welte T.
      • Manns M.P.
      • Malek N.P.
      • et al.
      Alpha-1-antitrypsin inhibits acute liver failure in mice.
      ] and others. Due to the vast available literature about DILI, here we will focus on APAP and Concanavalin-A induced liver injury.
      The visualization of hepatic microvasculature, liver structure and the immune responses in living animals has provided substantial amount of mechanistic data on different DILI models. Intravenous administration of fluorescent high molecular weight proteins (albumin or dextran), as well as sinusoidal endothelium staining with fluorescence-conjugated antibodies (anti-PECAM1/CD31 or anti-VCAM-1/CD106) are used to evidence hepatic vasculature and perfusion status, since these probes will only stain viable vessels. Also, toxic hepatocyte injury leads to many cellular derangements, including membrane instability, DNA damage and impaired calcium homeostasis, which may be visualized using IVM by several ways [
      • Hinson J.A.
      • Roberts D.W.
      • James L.P.
      Mechanisms of acetaminophen-induced liver necrosis.
      ]. In line with this, Li and colleagues [
      • Li F.C.
      • Huang G.T.
      • Lin C.J.
      • Wang S.S.
      • Sun T.L.
      • Lo S.Y.
      • et al.
      Apical membrane rupture and backward bile flooding in acetaminophen-induced hepatocyte necrosis.
      ] demonstrated that APAP administration led to basal membrane disruption in hepatocytes, which developed into a severe canalicular membrane rupture and backward flooding of bile into the cell, a determinant event for hepatocyte death. These severe changes lead to hepatocyte blebbing and displacement into the sinusoidal blood flow, showing for the first time a mechanism for hepatocyte content release during toxic liver injury in vivo. Dying hepatocytes may release several intracellular contents, one of which is ATP, a known NLRP3 inflammasome activator. During APAP-induced liver injury, ATP is released by damaged cells and stimulates calcium waves in neighboring hepatocytes, increasing liver injury and inflammation [
      • Amaral S.S.
      • Oliveira A.G.
      • Marques P.E.
      • Quintao J.L.
      • Pires D.A.
      • Resende R.R.
      • et al.
      Altered responsiveness to extracellular ATP enhances acetaminophen hepatotoxicity.
      ]. Remarkably, treatment with an ATP-degrading enzyme (apyrase) significantly reduced cytokine production and neutrophil recruitment in vivo.
      APAP damages cells other than hepatocytes. Liver sinusoidal endothelial cells (LSECs) injury can be observed in earlier time points, even before hepatocytes, in a glutathione depletion-dependent manner [
      • DeLeve L.D.
      • Wang X.
      • Kaplowitz N.
      • Shulman H.M.
      • Bart J.A.
      • van der Hoek A.
      Sinusoidal endothelial cells as a target for acetaminophen toxicity. Direct action versus requirement for hepatocyte activation in different mouse strains.
      ]. This leads to a premature hepatic microvascular injury [
      • Ito Y.
      • Bethea N.W.
      • Abril E.R.
      • McCuskey R.S.
      Early hepatic microvascular injury in response to acetaminophen toxicity.
      ] that contributes to total damage induced by APAP. Interestingly, centrilobular sinusoids are already congested two hours after intoxication, which was related to LSEC swelling, erythrocyte retention in Disse’s space and overall reduction in sinusoidal diameter. Using the same IVM setup, McCuskey’s group found that inhibition of iNOS [
      • Ito Y.
      • Abril E.R.
      • Bethea N.W.
      • McCuskey R.S.
      Role of nitric oxide in hepatic microvascular injury elicited by acetaminophen in mice.
      ] or MMPs [
      • Ito Y.
      • Abril E.R.
      • Bethea N.W.
      • McCuskey R.S.
      Inhibition of matrix metalloproteinases minimizes hepatic microvascular injury in response to acetaminophen in mice.
      ] reduces toxic liver injury mostly by restoring sinusoidal perfusion and inhibiting erythrocyte extravasation into Disse’s space. Conversely, acute ethanol administration significantly increases APAP toxicity by worsening the parameters described above, while promoting intense KC activation [
      • Ito Y.
      • Abril E.R.
      • Bethea N.W.
      • McCuskey R.S.
      Ethanol binging enhances hepatic microvascular responses to acetaminophen in mice.
      ]. Following, Pires et al. [
      • Pires D.A.
      • Marques P.E.
      • Pereira R.V.
      • David B.A.
      • Gomides L.F.
      • Dias A.C.
      • et al.
      Interleukin-4 deficiency protects mice from acetaminophen-induced liver injury and inflammation by prevention of glutathione depletion.
      ] described the hepatic vascular destruction caused by APAP intoxication using R6G and FITC-conjugated albumin. APAP caused loss of hepatocyte viability and sinusoidal architecture, which were reduced in interleukin-4 (IL-4) deficient mice. Acute toxic overdose from APAP or TAA can cause severe centrilobular necrosis in the first 12 to 24 h. It is conventionally evaluated using histology; however, recent studies proposed strategies to visualize and quantify liver necrosis in vivo by intravenous administration of DNA-binding probes (Sytox green, propidium iodide or DAPI). These dyes bind to DNA deposited within necrotic areas, being very useful for intravital assessment of cell death [
      • Marques P.E.
      • Antunes M.M.
      • David B.A.
      • Pereira R.V.
      • Teixeira M.M.
      • Menezes G.B.
      Imaging liver biology in vivo using conventional confocal microscopy.
      ,
      • Gomides L.F.
      • Marques P.E.
      • Faleiros B.E.
      • Pereira R.V.
      • Amaral S.S.
      • Lage T.R.
      • et al.
      Murine model to study brain, behavior and immunity during hepatic encephalopathy.
      ]. We have proposed this method to quantify liver necrosis in vivo after toxic injury with APAP and TAA, but interestingly, we also observed an intravascular DNA deposition that covered almost half of total liver area [
      • Marques P.E.
      • Oliveira A.G.
      • Pereira R.V.
      • David B.A.
      • Gomides L.F.
      • Saraiva A.M.
      • et al.
      Hepatic DNA deposition drives drug-induced liver injury and inflammation in mice.
      ] and correlated with injury severity. These studies validate IVM as a useful tool to study vascular and cellular alterations during liver injury in vivo.
      Leukocytes have a distinct accumulation pattern within the liver during DILI (Fig. 4). Under homeostatic conditions, liver is broadly inhabited by KC. This strategic location favors removal of pathogens [
      • Balmer M.L.
      • Slack E.
      • de Gottardi A.
      • Lawson M.A.
      • Hapfelmeier S.
      • Miele L.
      • et al.
      The liver may act as a firewall mediating mutualism between the host and its gut commensal microbiota.
      ], macromolecules and senescent cells from the circulation [
      • Hoffmeister K.M.
      • Felbinger T.W.
      • Falet H.
      • Denis C.V.
      • Bergmeier W.
      • Mayadas T.N.
      • et al.
      The clearance mechanism of chilled blood platelets.
      ]. KC can be evidenced in vivo by fluorescent antibody staining (anti-F4/80 or anti-CD68) and by uptake of intravenously injected fluorescent beads, which are immediately phagocytized by them [
      • Ito Y.
      • Bethea N.W.
      • Abril E.R.
      • McCuskey R.S.
      Early hepatic microvascular injury in response to acetaminophen toxicity.
      ]. In sharp contrast, neutrophils are not present in the liver under normal conditions, being rapidly recruited after insults. As mentioned above, neutrophils skip selectin-mediated rolling, jumping to direct adhesion or even mechanical trapping when migrating to the liver (Fig. 4) [
      • Hickey M.J.
      • Westhorpe C.L.
      Imaging inflammatory leukocyte recruitment in kidney, lung and liver – Challenges to the multi-step paradigm.
      ]. Neutrophils have a particular behavior during injury: they accumulate in great numbers in the liver, subsequently migrating specifically to the interior of necrotic zones at the peak of APAP toxicity [
      • Marques P.E.
      • Oliveira A.G.
      • Pereira R.V.
      • David B.A.
      • Gomides L.F.
      • Saraiva A.M.
      • et al.
      Hepatic DNA deposition drives drug-induced liver injury and inflammation in mice.
      ]. Such directional migration is lost once neutrophils reach necrosis, and these cells shift to a random “patrolling” behavior. Conversely, KCs remain stationary in viable parenchyma areas, outside necrosis. As demonstrated previously by others [
      • Ju C.
      • Reilly T.P.
      • Bourdi M.
      • Radonovich M.F.
      • Brady J.N.
      • George J.W.
      • et al.
      Protective role of Kupffer cells in acetaminophen-induced hepatic injury in mice.
      ,
      • Ishida Y.
      • Kondo T.
      • Kimura A.
      • Tsuneyama K.
      • Takayasu T.
      • Mukaida N.
      Opposite roles of neutrophils and macrophages in the pathogenesis of acetaminophen-induced acute liver injury.
      ], while KC may have a regulatory and protective role during toxic challenge, emigrating neutrophils amplify liver injury by playing a major role in inflammation [
      • Marques P.E.
      • Amaral S.S.
      • Pires D.A.
      • Nogueira L.L.
      • Soriani F.M.
      • Lima B.H.
      • et al.
      Chemokines and mitochondrial products activate neutrophils to amplify organ injury during mouse acute liver failure.
      ,
      • Marques P.E.
      • Oliveira A.G.
      • Pereira R.V.
      • David B.A.
      • Gomides L.F.
      • Saraiva A.M.
      • et al.
      Hepatic DNA deposition drives drug-induced liver injury and inflammation in mice.
      ].
      In addition to DILI, there are models to directly induce immune-mediated liver injury, such as the administration of Concanavalin-A (Con-A) [
      • Knolle P.A.
      • Gerken G.
      • Loser E.
      • Dienes H.P.
      • Gantner F.
      • Tiegs G.
      • et al.
      Role of sinusoidal endothelial cells of the liver in concanavalin A-induced hepatic injury in mice.
      ], a lectin isolated from the jack-bean, or using a combination of D-galactosamine with lipopolysaccharide (Gal/LPS) [
      • Kuhla A.
      • Eipel C.
      • Abshagen K.
      • Siebert N.
      • Menger M.D.
      • Vollmar B.
      Role of the perforin/granzyme cell death pathway in D-Gal/LPS-induced inflammatory liver injury.
      ]. These models induce acute hepatitis mediated by T cells, NKT cells, neutrophils and KCs, associated with intense cytokine secretion in the liver. Similar to DILI, Con-A induced hepatic injury leads to hemodynamic alterations such as erythrocyte retention, platelet aggregation, reduced blood perfusion and sinusoidal injury [
      • Knolle P.A.
      • Gerken G.
      • Loser E.
      • Dienes H.P.
      • Gantner F.
      • Tiegs G.
      • et al.
      Role of sinusoidal endothelial cells of the liver in concanavalin A-induced hepatic injury in mice.
      ,
      • Miyazawa Y.
      • Tsutsui H.
      • Mizuhara H.
      • Fujiwara H.
      • Kaneda K.
      Involvement of intrasinusoidal hemostasis in the development of concanavalin A-induced hepatic injury in mice.
      ]. T CD4+ lymphocytes are thought to be central mediators of injury; however, Bonder et al. [
      • Bonder C.S.
      • Ajuebor M.N.
      • Zbytnuik L.D.
      • Kubes P.
      • Swain M.G.
      Essential role for neutrophil recruitment to the liver in concanavalin A-induced hepatitis.
      ] demonstrated that neutrophil recruitment precedes T cell arrival to Con-A challenged livers. In addition, neutrophils were directly activated by Con-A, leading to L-selectin shedding and increased ROS production. Interestingly, neutrophil accumulation into the liver governs subsequent CD4+ T cell recruitment, being a crucial step for Con-A induced hepatitis. In another report, P-selectin was shown to regulate leukocyte rolling in periportal and centrilobular vessels, but not in sinusoids [
      • March S.
      • Garcia-Pagan J.C.
      • Massaguer A.
      • Pizcueta P.
      • Panes J.
      • Engel P.
      • et al.
      P-selectin mediates leukocyte rolling in concanavalin-A-induced hepatitis.
      ]. In line with this, two adhesion molecules, α4β1 integrin and vascular adhesion adhesion protein 1 (VAP-1) are required for the recruitment of Th1 and Th2 lymphocytes to Con-A inflamed liver, respectively [
      • Bonder C.S.
      • Norman M.U.
      • Swain M.G.
      • Zbytnuik L.D.
      • Yamanouchi J.
      • Santamaria P.
      • et al.
      Rules of recruitment for Th1 and Th2 lymphocytes in inflamed liver: a role for alpha-4 integrin and vascular adhesion protein-1.
      ]. For the first time, evidence against unspecific physical trapping of leukocytes inside the liver during disease was presented. Also, it is important to highlight that different populations of the same leukocyte subset (Th1 and Th2 lymphocytes) use distinct molecules to adhere to liver sinusoids, linking leukocyte functionality to specific adhesion molecule expression.

      Focal liver injury and inflammation

      As mentioned before, few neutrophils are present in the liver under normal conditions, but rapidly accumulate following necrosis. Mice that express eGFP under control of the lysozyme M promoter (Lysm-eGFP mouse) have highly fluorescent neutrophils and have been extensively used to investigate neutrophil behavior within the liver. It has been shown that during sterile cell death neutrophils adhere and crawl inside liver sinusoids using β2-integrins, chasing an intravascular gradient of chemoattractants towards the necrotic area [
      • McDonald B.
      • Pittman K.
      • Menezes G.B.
      • Hirota S.A.
      • Slaba I.
      • Waterhouse C.C.
      • et al.
      Intravascular danger signals guide neutrophils to sites of sterile inflammation.
      ,
      • Marques P.E.
      • Oliveira A.G.
      • Pereira R.V.
      • David B.A.
      • Gomides L.F.
      • Saraiva A.M.
      • et al.
      Hepatic DNA deposition drives drug-induced liver injury and inflammation in mice.
      ]. Once reaching the chemotaxis zone, a CXCR2 chemokine-rich area surrounding necrosis, neutrophils shift to sense a gradient of mitochondria-derived formyl peptides, presumably coming from the necrotic hepatocytes. Such hierarchic migration mechanism is crucial for a precise infiltration within dead cell area, where chemokine concentrations are very low or absent [
      • McDonald B.
      • Pittman K.
      • Menezes G.B.
      • Hirota S.A.
      • Slaba I.
      • Waterhouse C.C.
      • et al.
      Intravascular danger signals guide neutrophils to sites of sterile inflammation.
      ].
      In contrast, the molecules that govern neutrophil emigration seem to be different in systemic inflammation (Fig. 4). CD44-hyaluronan interaction is the major adhesive mechanism for neutrophils in the liver during endotoxemic shock [
      • McDonald B.
      • McAvoy E.F.
      • Lam F.
      • Gill V.
      • de la Motte C.
      • Savani R.C.
      • et al.
      Interaction of CD44 and hyaluronan is the dominant mechanism for neutrophil sequestration in inflamed liver sinusoids.
      ,
      • Menezes G.B.
      • Lee W.Y.
      • Zhou H.
      • Waterhouse C.C.
      • Cara D.C.
      • Kubes P.
      Selective down-regulation of neutrophil Mac-1 in endotoxemic hepatic microcirculation via IL-10.
      ]. However, when a local gradient of fMLP was used, CD44-hyaluronan adhesiveness was not necessary for neutrophil trapping within sinusoids, being replaced by ICAM-1/CD11b as the major adhesive route. Corroborating this, previous LPS administration, which downregulates liver integrins in an IL-10-dependent mechanism [
      • Menezes G.B.
      • Lee W.Y.
      • Zhou H.
      • Waterhouse C.C.
      • Cara D.C.
      • Kubes P.
      Selective down-regulation of neutrophil Mac-1 in endotoxemic hepatic microcirculation via IL-10.
      ], precluded neutrophil crawling towards the fMLP gradient. Therefore, depending on the magnitude of the inflammatory response (systemic vs. local), neutrophils can rapidly shift their adhesive mechanisms. Recently a novel mouse lineage was generated using the neutrophil-specific locus Ly6G and the fluorescent protein tdTomato. This model was baptized “Catchup” [
      • Hasenberg A.
      • Hasenberg M.
      • Mann L.
      • Neumann F.
      • Borkenstein L.
      • Stecher M.
      • et al.
      Catchup: a mouse model for imaging-based tracking and modulation of neutrophil granulocytes.
      ], and a variant strain with stronger red fluorescence was described as CatchupIVM-red. Hopefully, this new model will provide exciting insights in neutrophil recruitment and function in multiple liver diseases.
      Using the same focal thermal injury model, the mechanisms of monocyte infiltration in a sterile hepatic injury in vivo were recently elucidated. Using a mouse with two fluorescence reports (CX3CR1-GFP and CCR2-RFP) [
      • Saederup N.
      • Cardona A.E.
      • Croft K.
      • Mizutani M.
      • Cotleur A.C.
      • Tsou C.L.
      • et al.
      Selective chemokine receptor usage by central nervous system myeloid cells in CCR2-red fluorescent protein knock-in mice.
      ], it was observed that CCR2hiCX3CR1low monocytes were recruited in initial time points and persisted for at least 48 h. Interestingly, these cells shifted their phenotypes from CCR2hiCX3CR3low to CX3CR3hiCCR2low and this was essential for liver injury repair [
      • Dal-Secco D.
      • Wang J.
      • Zeng Z.
      • Kolaczkowska E.
      • Wong C.H.
      • Petri B.
      • et al.
      A dynamic spectrum of monocytes arising from the in situ reprogramming of CCR2+ monocytes at a site of sterile injury.
      ]. These studies are strong examples of the applications of liver intravital microscopy as a tool of basic investigation.

      Liver response to bacterial infections and endotoxemia

      It has been long appreciated that the liver functions as a filter to prevent infection (or infection-derived products) from reaching systemic circulation. Despite the global importance of viral hepatitis, IVM studies on viral infections in the liver are still scarce in the literature. Therefore, due to space limitation, we will focus on bacterial and parasitical diseases.
      The high bacterial clearance capacity of the liver and liver-resident macrophages was documented decades ago [
      • Benacerraf B.
      • Sebestyen M.M.
      • Schlossman S.
      A quantitative study of the kinetics of blood clearance of P32-labelled Escherichia coli and Staphylococci by the reticuloendothelial system.
      ,
      • Gregory S.H.
      • Wing E.J.
      Neutrophil-Kupffer cell interaction: a critical component of host defenses to systemic bacterial infections.
      ]. However, only more recently the fundamental role of KC in controlling bacteria dissemination could be visualized. By using intravital microscopy of the liver, it was demonstrated that Mycobacterium bovis and Borrelia burgdorferi are rapidly removed from the circulation by KC (84, 21). Indeed, elimination of KC by clodronate liposomes resulted in bacteria flowing freely throughout the circulation for up to 72 h. Besides its phagocytic capacity, KC are also important for the elimination of bacteria. Mounting evidence suggests that these cells may need aid from other cell types to fully clear bacteria. In this sense, KC that caught bacteria will produce a set of cytokines and/or chemokines to recruit or immobilize other cells types that are similarly needed for bacteria eradication. For example, the elimination of Bacillus cereus and methicillin-resistant Staphylococcus aureus were shown to be dependent on the interaction of platelets with KC that shifted from a transient “touch-and-go” interaction to a more stable nucleation following the catching of bacteria [
      • Wong C.H.
      • Jenne C.N.
      • Petri B.
      • Chrobok N.L.
      • Kubes P.
      Nucleation of platelets with blood-borne pathogens on Kupffer cells precedes other innate immunity and contributes to bacterial clearance.
      ]. It is interesting to note that a similar cooperation mechanism was previously described for neutrophils and iNKT cells following the infection by different bacteria [
      • Gregory S.H.
      • Wing E.J.
      Neutrophil-Kupffer cell interaction: a critical component of host defenses to systemic bacterial infections.
      ,
      • Gregory S.H.
      • Sagnimeni A.J.
      • Wing E.J.
      Bacteria in the bloodstream are trapped in the liver and killed by immigrating neutrophils.
      ]. Kupffer cell-iNKT cell cooperation was fundamental for the limitation of B. burgdorferi dissemination [
      • Lee W.Y.
      • Moriarty T.J.
      • Wong C.H.
      • Zhou H.
      • Strieter R.M.
      • van Rooijen N.
      • et al.
      An intravascular immune response to Borrelia burgdorferi involves Kupffer cells and iNKT cells.
      ]. Upon Borrelia infection, KC secrete chemokines that acted through CXCR3 to induce iNKT cell recruitment and CD1d-dependent clustering. Activated iNKT cell secreted IFN-γ and contributed to limit bacteria dissemination to joints, bladder and heart. This cooperation may also be relevant to limit the dissemination of other bacterial types. For example, mice submitted to a stroke model died from infection that could be prevented by antibiotic pre-treatment. This was attributed to a modulation of the patrolling behavior of iNKT cells [
      • Geissmann F.
      • Cameron T.O.
      • Sidobre S.
      • Manlongat N.
      • Kronenberg M.
      • Briskin M.J.
      • et al.
      Intravascular immune surveillance by CXCR6+ NKT cells patrolling liver sinusoids.
      ] that became static and Th2-polarized after stroke [
      • Wong C.H.
      • Jenne C.N.
      • Lee W.Y.
      • Leger C.
      • Kubes P.
      Functional innervation of hepatic iNKT cells is immunosuppressive following stroke.
      ]. Additionally, KC may recruit circulating monocytes to form granulomas in response to Mycobacterium bovis-BCG [
      • Egen J.G.
      • Rothfuchs A.G.
      • Feng C.G.
      • Winter N.
      • Sher A.
      • Germain R.N.
      Macrophage and T cell dynamics during the development and disintegration of mycobacterial granulomas.
      ]. Formation of this granuloma was shown to be dependent on TNF-α and recruitment of both liver-resident macrophages and circulating monocytes. In this sense, a recent study using intravital microscopy demonstrated that the tolerogenic nature of Kupffer cell antigen presentation to T cells could be used to prevent unwanted antigen-specific glomerulonephritis, but this tolerogenic potential is lost upon liver injury, implicating that liver accumulation of inflammatory cells reprograms Kupffer cells [
      • Heymann F.
      • Peusquens J.
      • Ludwig-Portugall I.
      • Kohlhepp M.
      • Ergen C.
      • Niemietz P.
      • et al.
      Liver inflammation abrogates immunological tolerance induced by Kupffer cells.
      ].
      Furthermore, in vivo imaging was a powerful tool to describe the occurrence of neutrophil extracellular traps (NETs) in response to both endotoxin and bacteria [
      • McDonald B.
      • Urrutia R.
      • Yipp B.G.
      • Jenne C.N.
      • Kubes P.
      Intravascular neutrophil extracellular traps capture bacteria from the bloodstream during sepsis.
      ,
      • Kolaczkowska E.
      • Jenne C.N.
      • Surewaard B.G.
      • Thanabalasuriar A.
      • Lee W.Y.
      • Sanz M.J.
      • et al.
      Molecular mechanisms of NET formation and degradation revealed by intravital imaging in the liver vasculature.
      ]. NETs are webs of nuclear DNA ligated with various proteins with microbicidal activity, released by activated neutrophils, that can function as an additional mechanism of microbe killing [
      • Nauseef W.M.
      • Borregaard N.
      Neutrophils at work.
      ].

      Liver response in parasitic diseases

      The liver can also be a target for many parasitic diseases as many helminthes and protozoans reside in the liver or have an obligatory hepatic stage. Among these, malaria-causing Plasmodium protozoans are the most widely studied by use of intravital liver imaging. Static microscopy techniques associated with in vitro models of infection, lead to the suggestion of an active process of cell penetration used by these parasites to infect host cells, especially macrophages and hepatocytes [
      • Mota M.M.
      • Pradel G.
      • Vanderberg J.P.
      • Hafalla J.C.
      • Frevert U.
      • Nussenzweig R.S.
      • et al.
      Migration of Plasmodium sporozoites through cells before infection.
      ,
      • Pradel G.
      • Frevert U.
      Malaria sporozoites actively enter and pass through rat Kupffer cells prior to hepatocyte invasion.
      ,
      • Vanderberg J.P.
      • Chew S.
      • Stewart M.J.
      Plasmodium sporozoite interactions with macrophages in vitro: a videomicroscopic analysis.
      ]. At least for in vitro hepatocytes, this process was associated with the rapid opening and closing of cell membrane, with leakage of intracellular content and eventual cell death [
      • Mota M.M.
      • Pradel G.
      • Vanderberg J.P.
      • Hafalla J.C.
      • Frevert U.
      • Nussenzweig R.S.
      • et al.
      Migration of Plasmodium sporozoites through cells before infection.
      ]. Later, the parasite-encoded protein SPECT (sporozoite microneme protein essential for cell traversal) was shown to be essential for Plasmodium berghei active passage through cells and for parasite colonization of the liver in vivo [
      • Ishino T.
      • Yano K.
      • Chinzei Y.
      • Yuda M.
      Cell-passage activity is required for the malarial parasite to cross the liver sinusoidal cell layer.
      ].
      Despite evidence, a real glimpse into the dynamics of Plasmodium infection of liver cells was only achieved later, in a pioneer intravital microscopy study [
      • Frevert U.
      • Engelmann S.
      • Zougbede S.
      • Stange J.
      • Ng B.
      • Matuschewski K.
      • et al.
      Intravital observation of Plasmodium berghei sporozoite infection of the liver.
      ]. Time-lapse imaging of P. berguei infection showed that parasites rapidly stop at the sinusoids and actively move along the sinusoidal cell layer close to a KC. Parasites then penetrate and trespass KC to reach the inner hepatocytes. Analysis of parasite motion showed that KC penetration was preceded by a delay, which did not occur during hepatocyte penetration. Additionally, Plasmodium transit inside KC was significantly slower than inside hepatocytes. Together, these observations suggest that Plasmodium parasites use different mechanisms for invasion of different cell types (96, 98). In fact, SPECT was shown to be essential for hepatocyte infection in the presence of KC, while dispensable when KC were depleted [
      • Ishino T.
      • Yano K.
      • Chinzei Y.
      • Yuda M.
      Cell-passage activity is required for the malarial parasite to cross the liver sinusoidal cell layer.
      ]. Subsequently, it was demonstrated that merozoites are released into the circulation inside merosomes, large membrane-bound vesicles derived from the infected host cells (99), preventing merozoites from being trapped by KC lining the sinusoids. Following studies then described the dynamics of T cell-mediated immunity towards liver stage parasites [
      • Cockburn I.A.
      • Amino R.
      • Kelemen R.K.
      • Kuo S.C.
      • Tse S.W.
      • Radtke A.
      • et al.
      In vivo imaging of CD8+ T cell-mediated elimination of malaria liver stages.
      ,
      • Kimura K.
      • Kimura D.
      • Matsushima Y.
      • Miyakoda M.
      • Honma K.
      • Yuda M.
      • et al.
      CD8+ T cells specific for a malaria cytoplasmic antigen form clusters around infected hepatocytes and are protective at the liver stage of infection.
      ]. Intravital imaging showed that CD8+ T cells accumulate around infected hepatocytes and recruit more cells to the infected sites, resulting in the formation of large clusters that kill intracellular forms of Plasmodium.
      Imaging of parasite liver infections was also carried out to analyze dynamics of granuloma formation during Leishmania and Schistosoma infection [
      • Beattie L.
      • Peltan A.
      • Maroof A.
      • Kirby A.
      • Brown N.
      • Coles M.
      • et al.
      Dynamic imaging of experimental Leishmania donovani-induced hepatic granulomas detects Kupffer cell-restricted antigen presentation to antigen-specific CD8 T cells.
      ,
      • Girgis N.M.
      • Gundra U.M.
      • Ward L.N.
      • Cabrera M.
      • Frevert U.
      • Loke P.
      Ly6Chigh monocytes become alternatively activated macrophages in schistosome granulomas with help from CD4+ cells.
      ]. Both studies provided evidences that granulomas are very dynamic structures. In Schistosoma granulomas, for instance, tracking of CX3CR1-GFP+ cells provided evidences that sinusoid-patrolling monocytes stopped at sites of Schistosoma egg deposition, while the same cells presented increased speed in the vicinity of fully mature granulomas, suggesting that they are attracted to sites of granuloma formation [
      • Girgis N.M.
      • Gundra U.M.
      • Ward L.N.
      • Cabrera M.
      • Frevert U.
      • Loke P.
      Ly6Chigh monocytes become alternatively activated macrophages in schistosome granulomas with help from CD4+ cells.
      ]. Moreover, using fluorescently labeled antibodies, it was found that CX3CR1-GFP+ cells were comprised of both Ly6Chigh and Ly6Cint/low populations of which only the former presented patrolling behavior in uninfected animals. Acquisition of patrolling behavior by infiltrating CX3CR1-GFP+ Ly6Chigh cells in the set of Schistosoma infection shows that different cell populations can switch intravascular behavior depending on the inflammatory microenvironment.
      The dynamic nature of granulomas was further confirmed using a visceral leishmaniasis model [
      • Beattie L.
      • Peltan A.
      • Maroof A.
      • Kirby A.
      • Brown N.
      • Coles M.
      • et al.
      Dynamic imaging of experimental Leishmania donovani-induced hepatic granulomas detects Kupffer cell-restricted antigen presentation to antigen-specific CD8 T cells.
      ]. In this study, evidences supported a role of KC in presenting antigens inside the granuloma to CD8+ T cells. Analysis of the dynamics of T cell accumulation showed that both antigen-specific and non-specific T cells entered the granuloma, engaging contact with KC. However, only antigen-specific T cells contacting antigen-loaded KC had their speed and contact duration altered. Moreover, antigen-specific T cells were less likely to leave the granuloma. This selective behavior of antigen-specific T cells suggest a mechanism of preferential accumulation of relevant T cells within the infection site, contributing to more efficient antigen presentation and protective immunity.

      Concluding remarks

      The liver is a target organ of many infectious agents, either directly, as a consequence of liver stage in the pathogen life cycle, or finally as a consequence of pathogen dissemination. The liver is also target in many sterile diseases such as ischemia/reperfusion and toxic hepatitis. In any case, a better understanding of the processes that takes place in the liver in response to each of these stimuli is necessary for developing new therapeutic strategies. This fundamental knowledge can be obtained by using various techniques, among which the dynamic imaging tools provided by intravital microscopy recently emerged as a very powerful option to researchers. Despite this potential in providing new insights into pathogenesis and disease control mechanisms, liver intravital microscopy based studies in relevant models are still scarce. There are several unmeet needs, including the application of new super-resolution techniques in IVM, or devices that can image non-invasively with higher resolution, similar to what is achieved with experimental live imaging protocols (i.e. sub-cellular scale). The relatively unexplored venues that can be revealed by this technique thus make intravital imaging a very promising and exciting field in hepatology.

      Financial support

      CAPES , CNPq , FAPERJ and FAPEMIG / PRONEX for financial support.

      Conflict of interest

      The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

      Author’s contributions

      PEM, AGO, LC, HAPN and GBM prepared the figures and wrote the manuscript.

      Acknowledgements

      We would like to CAPES, CNPq and FAPEMIG/PRONEX for financial support.

      Supplementary data

      References

      1. Museum TB. The Nimrud Lens/The Layard Lens. January 26th 2015 [cited; Available from: <http://www.britishmuseum.org/research/collection_online/collection_object_details.aspx?objectId=369215&partId=1>.

        • Hwa C.
        • Aird W.C.
        The history of the capillary wall: doctors, discoveries, and debates.
        Am J Physiol Heart Circ Physiol. 2007; 293: H2667-H2679
        • Crispe I.N.
        The liver as a lymphoid organ.
        Annu Rev Immunol. 2009; 27: 147-163
        • Mennone A.
        • Nathanson M.H.
        Needle-based confocal laser endomicroscopy to assess liver histology in vivo.
        Gastrointest Endosc. 2011; 73: 338-344
        • Shieh F.K.
        • Drumm H.
        • Nathanson M.H.
        • Jamidar P.A.
        High-definition confocal endomicroscopy of the common bile duct.
        J Clin Gastroenterol. 2012; 46: 401-406
        • McClugage Jr., S.G.
        • McCuskey R.S.
        “In vivo” microscopic study of the response of the hepatic microvascular system to carbon tetrachloride poisoning.
        Microvasc Res. 1971; 3: 354-360
        • Jenne C.N.
        • Wong C.H.
        • Zemp F.J.
        • McDonald B.
        • Rahman M.M.
        • Forsyth P.A.
        • et al.
        Neutrophils recruited to sites of infection protect from virus challenge by releasing neutrophil extracellular traps.
        Cell Host Microbe. 2013; 13: 169-180
        • McDonald B.
        • McAvoy E.F.
        • Lam F.
        • Gill V.
        • de la Motte C.
        • Savani R.C.
        • et al.
        Interaction of CD44 and hyaluronan is the dominant mechanism for neutrophil sequestration in inflamed liver sinusoids.
        J Exp Med. 2008; 205: 915-927
        • McDonald B.
        • Urrutia R.
        • Yipp B.G.
        • Jenne C.N.
        • Kubes P.
        Intravascular neutrophil extracellular traps capture bacteria from the bloodstream during sepsis.
        Cell Host Microbe. 2012; 12: 324-333
        • Honda M.
        • Takeichi T.
        • Asonuma K.
        • Tanaka K.
        • Kusunoki M.
        • Inomata Y.
        Intravital imaging of neutrophil recruitment in hepatic ischemia-reperfusion injury in mice.
        Transplantation. 2013; 95: 551-558
        • Ritsma L.
        • Steller E.J.
        • Ellenbroek S.I.
        • Kranenburg O.
        • Rinkes I.H. Borel
        • van Rheenen J.
        Surgical implantation of an abdominal imaging window for intravital microscopy.
        Nat Protoc. 2013; 8: 583-594
        • White J.G.
        • Amos W.B.
        • Fordham M.
        An evaluation of confocal versus conventional imaging of biological structures by fluorescence light microscopy.
        J Cell Biol. 1987; 105: 41-48
        • van Meer G.
        • Stelzer E.H.
        • Wijnaendts-van-Resandt R.W.
        • Simons K.
        Sorting of sphingolipids in epithelial (Madin-Darby canine kidney) cells.
        J Cell Biol. 1987; 105: 1623-1635
        • Wang E.
        • Babbey C.M.
        • Dunn K.W.
        Performance comparison between the high-speed Yokogawa spinning disc confocal system and single-point scanning confocal systems.
        J Microsc. 2005; 218: 148-159
        • Denk W.
        • Strickler J.H.
        • Webb W.W.
        Two-photon laser scanning fluorescence microscopy.
        Science. 1990; 248: 73-76
        • Ellenbroek S.I.
        • van Rheenen J.
        Imaging hallmarks of cancer in living mice.
        Nat Rev Cancer. 2014; 14: 406-418
        • Brown E.B.
        • Campbell R.B.
        • Tsuzuki Y.
        • Xu L.
        • Carmeliet P.
        • Fukumura D.
        • et al.
        In vivo measurement of gene expression, angiogenesis and physiological function in tumors using multiphoton laser scanning microscopy.
        Nat Med. 2001; 7: 864-868
        • Marques P.E.
        • Antunes M.M.
        • David B.A.
        • Pereira R.V.
        • Teixeira M.M.
        • Menezes G.B.
        Imaging liver biology in vivo using conventional confocal microscopy.
        Nat Protoc. 2015; 10: 258-268
        • Chen B.C.
        • Legant W.R.
        • Wang K.
        • Shao L.
        • Milkie D.E.
        • Davidson M.W.
        • et al.
        Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution.
        Science. 2014; 346: 1257998
        • Guc E.
        • Fankhauser M.
        • Lund A.W.
        • Swartz M.A.
        • Kilarski W.W.
        Long-term intravital immunofluorescence imaging of tissue matrix components with epifluorescence and two-photon microscopy.
        J Vis Exp. 2014; https://doi.org/10.3791/51388
        • Pires D.A.
        • Marques P.E.
        • Pereira R.V.
        • David B.A.
        • Gomides L.F.
        • Dias A.C.
        • et al.
        Interleukin-4 deficiency protects mice from acetaminophen-induced liver injury and inflammation by prevention of glutathione depletion.
        Inflamm Res. 2014; 63: 61-69
        • Li F.C.
        • Liu Y.
        • Huang G.T.
        • Chiou L.L.
        • Liang J.H.
        • Sun T.L.
        • et al.
        In vivo dynamic metabolic imaging of obstructive cholestasis in mice.
        Am J Physiol Gastrointest Liver Physiol. 2009; 296: G1091-G1097
        • Marques P.E.
        • Amaral S.S.
        • Pires D.A.
        • Nogueira L.L.
        • Soriani F.M.
        • Lima B.H.
        • et al.
        Chemokines and mitochondrial products activate neutrophils to amplify organ injury during mouse acute liver failure.
        Hepatology. 2012; 56: 1971-1982
        • Hsu Y.C.
        • Huang H.P.
        • Yu I.S.
        • Su K.Y.
        • Lin S.R.
        • Lin W.C.
        • et al.
        Serine protease hepsin regulates hepatocyte size and hemodynamic retention of tumor cells by hepatocyte growth factor signaling in mice.
        Hepatology. 2012; 56: 1913-1923
        • Zhang X.Y.
        • Sun C.K.
        • Wheatley A.M.
        A novel approach to the quantification of hepatic stellate cells in intravital fluorescence microscopy of the liver using a computerized image analysis system.
        Microvasc Res. 2000; 60: 232-240
        • Marques P.E.
        • Oliveira A.G.
        • Pereira R.V.
        • David B.A.
        • Gomides L.F.
        • Saraiva A.M.
        • et al.
        Hepatic DNA deposition drives drug-induced liver injury and inflammation in mice.
        Hepatology. 2015; 61: 348-360https://doi.org/10.1002/hep.27216
        • McDonald B.
        • Pittman K.
        • Menezes G.B.
        • Hirota S.A.
        • Slaba I.
        • Waterhouse C.C.
        • et al.
        Intravascular danger signals guide neutrophils to sites of sterile inflammation.
        Science. 2010; 330: 362-366
        • Hoffmeister K.M.
        • Felbinger T.W.
        • Falet H.
        • Denis C.V.
        • Bergmeier W.
        • Mayadas T.N.
        • et al.
        The clearance mechanism of chilled blood platelets.
        Cell. 2003; 112: 87-97
        • Siegmund K.
        • Lee W.Y.
        • Tchang V.S.
        • Stiess M.
        • Terracciano L.
        • Kubes P.
        • et al.
        Coronin 1 is dispensable for leukocyte recruitment and liver injury in concanavalin A-induced hepatitis.
        Immunol Lett. 2013; 153: 62-70
        • Lee W.Y.
        • Moriarty T.J.
        • Wong C.H.
        • Zhou H.
        • Strieter R.M.
        • van Rooijen N.
        • et al.
        An intravascular immune response to Borrelia burgdorferi involves Kupffer cells and iNKT cells.
        Nat Immunol. 2010; 11: 295-302
        • Li F.C.
        • Huang G.T.
        • Lin C.J.
        • Wang S.S.
        • Sun T.L.
        • Lo S.Y.
        • et al.
        Apical membrane rupture and backward bile flooding in acetaminophen-induced hepatocyte necrosis.
        Cell Death Dis. 2011; 2: e183
        • Ito Y.
        • Bethea N.W.
        • Abril E.R.
        • McCuskey R.S.
        Early hepatic microvascular injury in response to acetaminophen toxicity.
        Microcirculation. 2003; 10: 391-400
        • Phan T.G.
        • Bullen A.
        Practical intravital two-photon microscopy for immunological research: faster, brighter, deeper.
        Immunol Cell Biol. 2010; 88: 438-444
        • Hickey M.J.
        • Westhorpe C.L.
        Imaging inflammatory leukocyte recruitment in kidney, lung and liver – Challenges to the multi-step paradigm.
        Immunol Cell Biol. 2013; 91: 281-289
        • Heymann F.
        • Niemietz P.M.
        • Peusquens J.
        • Ergen C.
        • Kohlhepp M.
        • Mossanen J.C.
        • et al.
        Long term intravital multiphoton microscopy imaging of immune cells in healthy and diseased liver using CXCR6.Gfp reporter mice.
        J Vis Exp. 2015; https://doi.org/10.3791/52607
        • Bezemer R.
        • Bartels S.A.
        • Bakker J.
        • Ince C.
        Clinical review: clinical imaging of the sublingual microcirculation in the critically ill – Where do we stand?.
        Crit Care. 2012; 16: 224
        • Enfield J.
        • Jonathan E.
        • Leahy M.
        In vivo imaging of the microcirculation of the volar forearm using correlation mapping optical coherence tomography (cmOCT).
        Biomed Opt Express. 2011; 2: 1184-1193
        • Peralta C.
        • Jimenez-Castro M.B.
        • Gracia-Sancho J.
        Hepatic ischemia and reperfusion injury: effects on the liver sinusoidal milieu.
        J Hepatol. 2013; 59: 1094-1106
        • Chen G.Y.
        • Nunez G.
        Sterile inflammation: sensing and reacting to damage.
        Nat Rev Immunol. 2010; 10: 826-837
        • Rock K.L.
        • Latz E.
        • Ontiveros F.
        • Kono H.
        The sterile inflammatory response.
        Annu Rev Immunol. 2010; 28: 321-342
        • Khandoga A.
        • Biberthaler P.
        • Enders G.
        • Axmann S.
        • Hutter J.
        • Messmer K.
        • et al.
        Platelet adhesion mediated by fibrinogen-intercellular adhesion molecule-1 binding induces tissue injury in the postischemic liver in vivo.
        Transplantation. 2002; 74: 681-688
        • Lu H.H.
        • Wu Y.M.
        • Chang W.T.
        • Luo T.
        • Yang Y.C.
        • Cho H.D.
        • et al.
        Molecular imaging of ischemia and reperfusion in vivo with mitochondrial autofluorescence.
        Anal Chem. 2014; 86: 5024-5031
        • Vollmar B.
        • Burkhardt M.
        • Minor T.
        • Klauke H.
        • Menger M.D.
        High-resolution microscopic determination of hepatic NADH fluorescence for in vivo monitoring of tissue oxygenation during hemorrhagic shock and resuscitation.
        Microvasc Res. 1997; 54: 164-173
        • Vollmar B.
        • Glasz J.
        • Menger M.D.
        • Messmer K.
        Leukocytes contribute to hepatic ischemia/reperfusion injury via intercellular adhesion molecule-1-mediated venular adherence.
        Surgery. 1995; 117: 195-200
        • Ley K.
        • Kansas G.S.
        Selectins in T-cell recruitment to non-lymphoid tissues and sites of inflammation.
        Nat Rev Immunol. 2004; 4: 325-335
        • Lee W.Y.
        • Kubes P.
        Leukocyte adhesion in the liver: distinct adhesion paradigm from other organs.
        J Hepatol. 2008; 48: 504-512
        • Wong J.
        • Johnston B.
        • Lee S.S.
        • Bullard D.C.
        • Smith C.W.
        • Beaudet A.L.
        • et al.
        A minimal role for selectins in the recruitment of leukocytes into the inflamed liver microvasculature.
        J Clin Invest. 1997; 99: 2782-2790
        • Sawaya Jr., D.E.
        • Zibari G.B.
        • Minardi A.
        • Bilton B.
        • Burney D.
        • Granger D.N.
        • et al.
        P-selectin contributes to the initial recruitment of rolling and adherent leukocytes in hepatic venules after ischemia/reperfusion.
        Shock. 1999; 12: 227-232
        • Bernal W.
        • Wendon J.
        Acute liver failure.
        N Engl J Med. 2013; 369: 2525-2534
        • Gomides L.F.
        • Marques P.E.
        • Faleiros B.E.
        • Pereira R.V.
        • Amaral S.S.
        • Lage T.R.
        • et al.
        Murine model to study brain, behavior and immunity during hepatic encephalopathy.
        World J Hepatol. 2014; 6: 243-250
        • Jaeschke H.
        • Williams C.D.
        • McGill M.R.
        • Xie Y.
        • Ramachandran A.
        Models of drug-induced liver injury for evaluation of phytotherapeutics and other natural products.
        Food Chem Toxicol. 2013; 55: 279-289
        • McCuskey R.S.
        • Bethea N.W.
        • Wong J.
        • McCuskey M.K.
        • Abril E.R.
        • Wang X.
        • et al.
        Ethanol binging exacerbates sinusoidal endothelial and parenchymal injury elicited by acetaminophen.
        J Hepatol. 2005; 42: 371-377
        • Proctor W.R.
        • Chakraborty M.
        • Chea L.S.
        • Morrison J.C.
        • Berkson J.D.
        • Semple K.
        • et al.
        Eosinophils mediate the pathogenesis of halothane-induced liver injury in mice.
        Hepatology. 2013; 57: 2026-2036
        • Jedicke N.
        • Struever N.
        • Aggrawal N.
        • Welte T.
        • Manns M.P.
        • Malek N.P.
        • et al.
        Alpha-1-antitrypsin inhibits acute liver failure in mice.
        Hepatology. 2014; 59: 2299-2308
        • Hinson J.A.
        • Roberts D.W.
        • James L.P.
        Mechanisms of acetaminophen-induced liver necrosis.
        Handb Exp Pharmacol. 2010; : 369-405
        • Amaral S.S.
        • Oliveira A.G.
        • Marques P.E.
        • Quintao J.L.
        • Pires D.A.
        • Resende R.R.
        • et al.
        Altered responsiveness to extracellular ATP enhances acetaminophen hepatotoxicity.
        Cell Commun Signal. 2013; 11: 10
        • DeLeve L.D.
        • Wang X.
        • Kaplowitz N.
        • Shulman H.M.
        • Bart J.A.
        • van der Hoek A.
        Sinusoidal endothelial cells as a target for acetaminophen toxicity. Direct action versus requirement for hepatocyte activation in different mouse strains.
        Biochem Pharmacol. 1997; 53: 1339-1345
        • Ito Y.
        • Abril E.R.
        • Bethea N.W.
        • McCuskey R.S.
        Role of nitric oxide in hepatic microvascular injury elicited by acetaminophen in mice.
        Am J Physiol Gastrointest Liver Physiol. 2004; 286: G60-G67
        • Ito Y.
        • Abril E.R.
        • Bethea N.W.
        • McCuskey R.S.
        Inhibition of matrix metalloproteinases minimizes hepatic microvascular injury in response to acetaminophen in mice.
        Toxicol Sci. 2005; 83: 190-196
        • Ito Y.
        • Abril E.R.
        • Bethea N.W.
        • McCuskey R.S.
        Ethanol binging enhances hepatic microvascular responses to acetaminophen in mice.
        Microcirculation. 2004; 11: 625-632
        • Balmer M.L.
        • Slack E.
        • de Gottardi A.
        • Lawson M.A.
        • Hapfelmeier S.
        • Miele L.
        • et al.
        The liver may act as a firewall mediating mutualism between the host and its gut commensal microbiota.
        Sci Transl Med. 2014; 6: 237ra266
        • Ju C.
        • Reilly T.P.
        • Bourdi M.
        • Radonovich M.F.
        • Brady J.N.
        • George J.W.
        • et al.
        Protective role of Kupffer cells in acetaminophen-induced hepatic injury in mice.
        Chem Res Toxicol. 2002; 15: 1504-1513
        • Ishida Y.
        • Kondo T.
        • Kimura A.
        • Tsuneyama K.
        • Takayasu T.
        • Mukaida N.
        Opposite roles of neutrophils and macrophages in the pathogenesis of acetaminophen-induced acute liver injury.
        Eur J Immunol. 2006; 36: 1028-1038
        • Knolle P.A.
        • Gerken G.
        • Loser E.
        • Dienes H.P.
        • Gantner F.
        • Tiegs G.
        • et al.
        Role of sinusoidal endothelial cells of the liver in concanavalin A-induced hepatic injury in mice.
        Hepatology. 1996; 24: 824-829
        • Kuhla A.
        • Eipel C.
        • Abshagen K.
        • Siebert N.
        • Menger M.D.
        • Vollmar B.
        Role of the perforin/granzyme cell death pathway in D-Gal/LPS-induced inflammatory liver injury.
        Am J Physiol Gastrointest Liver Physiol. 2009; 296: G1069-G1076
        • Miyazawa Y.
        • Tsutsui H.
        • Mizuhara H.
        • Fujiwara H.
        • Kaneda K.
        Involvement of intrasinusoidal hemostasis in the development of concanavalin A-induced hepatic injury in mice.
        Hepatology. 1998; 27: 497-506
        • Bonder C.S.
        • Ajuebor M.N.
        • Zbytnuik L.D.
        • Kubes P.
        • Swain M.G.
        Essential role for neutrophil recruitment to the liver in concanavalin A-induced hepatitis.
        J Immunol. 2004; 172: 45-53
        • March S.
        • Garcia-Pagan J.C.
        • Massaguer A.
        • Pizcueta P.
        • Panes J.
        • Engel P.
        • et al.
        P-selectin mediates leukocyte rolling in concanavalin-A-induced hepatitis.
        Liver Int. 2005; 25: 1053-1060
        • Bonder C.S.
        • Norman M.U.
        • Swain M.G.
        • Zbytnuik L.D.
        • Yamanouchi J.
        • Santamaria P.
        • et al.
        Rules of recruitment for Th1 and Th2 lymphocytes in inflamed liver: a role for alpha-4 integrin and vascular adhesion protein-1.
        Immunity. 2005; 23: 153-163
        • Menezes G.B.
        • Lee W.Y.
        • Zhou H.
        • Waterhouse C.C.
        • Cara D.C.
        • Kubes P.
        Selective down-regulation of neutrophil Mac-1 in endotoxemic hepatic microcirculation via IL-10.
        J Immunol. 2009; 183: 7557-7568
        • Hasenberg A.
        • Hasenberg M.
        • Mann L.
        • Neumann F.
        • Borkenstein L.
        • Stecher M.
        • et al.
        Catchup: a mouse model for imaging-based tracking and modulation of neutrophil granulocytes.
        Nat Methods. 2015; 12: 445-452https://doi.org/10.1038/nmeth.3322
        • Saederup N.
        • Cardona A.E.
        • Croft K.
        • Mizutani M.
        • Cotleur A.C.
        • Tsou C.L.
        • et al.
        Selective chemokine receptor usage by central nervous system myeloid cells in CCR2-red fluorescent protein knock-in mice.
        PLoS One. 2010; 5: e13693
        • Dal-Secco D.
        • Wang J.
        • Zeng Z.
        • Kolaczkowska E.
        • Wong C.H.
        • Petri B.
        • et al.
        A dynamic spectrum of monocytes arising from the in situ reprogramming of CCR2+ monocytes at a site of sterile injury.
        J Exp Med. 2015; 212: 447-456https://doi.org/10.1084/jem.20141539
        • Benacerraf B.
        • Sebestyen M.M.
        • Schlossman S.
        A quantitative study of the kinetics of blood clearance of P32-labelled Escherichia coli and Staphylococci by the reticuloendothelial system.
        J Exp Med. 1959; 110: 27-48
        • Gregory S.H.
        • Wing E.J.
        Neutrophil-Kupffer cell interaction: a critical component of host defenses to systemic bacterial infections.
        J Leukoc Biol. 2002; 72: 239-248
        • Wong C.H.
        • Jenne C.N.
        • Petri B.
        • Chrobok N.L.
        • Kubes P.
        Nucleation of platelets with blood-borne pathogens on Kupffer cells precedes other innate immunity and contributes to bacterial clearance.
        Nat Immunol. 2013; 14: 785-792
        • Gregory S.H.
        • Sagnimeni A.J.
        • Wing E.J.
        Bacteria in the bloodstream are trapped in the liver and killed by immigrating neutrophils.
        J Immunol. 1996; 157: 2514-2520
        • Geissmann F.
        • Cameron T.O.
        • Sidobre S.
        • Manlongat N.
        • Kronenberg M.
        • Briskin M.J.
        • et al.
        Intravascular immune surveillance by CXCR6+ NKT cells patrolling liver sinusoids.
        PLoS Biol. 2005; 3: e113
        • Wong C.H.
        • Jenne C.N.
        • Lee W.Y.
        • Leger C.
        • Kubes P.
        Functional innervation of hepatic iNKT cells is immunosuppressive following stroke.
        Science. 2011; 334: 101-105
        • Egen J.G.
        • Rothfuchs A.G.
        • Feng C.G.
        • Winter N.
        • Sher A.
        • Germain R.N.
        Macrophage and T cell dynamics during the development and disintegration of mycobacterial granulomas.
        Immunity. 2008; 28: 271-284
        • Heymann F.
        • Peusquens J.
        • Ludwig-Portugall I.
        • Kohlhepp M.
        • Ergen C.
        • Niemietz P.
        • et al.
        Liver inflammation abrogates immunological tolerance induced by Kupffer cells.
        Hepatology. 2015; ([Epub ahead of print])https://doi.org/10.1002/hep.27793
        • Kolaczkowska E.
        • Jenne C.N.
        • Surewaard B.G.
        • Thanabalasuriar A.
        • Lee W.Y.
        • Sanz M.J.
        • et al.
        Molecular mechanisms of NET formation and degradation revealed by intravital imaging in the liver vasculature.
        Nat Commun. 2015; 6: 6673
        • Nauseef W.M.
        • Borregaard N.
        Neutrophils at work.
        Nat Immunol. 2014; 15: 602-611
        • Mota M.M.
        • Pradel G.
        • Vanderberg J.P.
        • Hafalla J.C.
        • Frevert U.
        • Nussenzweig R.S.
        • et al.
        Migration of Plasmodium sporozoites through cells before infection.
        Science. 2001; 291: 141-144
        • Pradel G.
        • Frevert U.
        Malaria sporozoites actively enter and pass through rat Kupffer cells prior to hepatocyte invasion.
        Hepatology. 2001; 33: 1154-1165
        • Vanderberg J.P.
        • Chew S.
        • Stewart M.J.
        Plasmodium sporozoite interactions with macrophages in vitro: a videomicroscopic analysis.
        J Protozool. 1990; 37: 528-536
        • Ishino T.
        • Yano K.
        • Chinzei Y.
        • Yuda M.
        Cell-passage activity is required for the malarial parasite to cross the liver sinusoidal cell layer.
        PLoS Biol. 2004; 2: E4
        • Frevert U.
        • Engelmann S.
        • Zougbede S.
        • Stange J.
        • Ng B.
        • Matuschewski K.
        • et al.
        Intravital observation of Plasmodium berghei sporozoite infection of the liver.
        PLoS Biol. 2005; 3: e192
        • Cockburn I.A.
        • Amino R.
        • Kelemen R.K.
        • Kuo S.C.
        • Tse S.W.
        • Radtke A.
        • et al.
        In vivo imaging of CD8+ T cell-mediated elimination of malaria liver stages.
        Proc Natl Acad Sci U S A. 2013; 110: 9090-9095
        • Kimura K.
        • Kimura D.
        • Matsushima Y.
        • Miyakoda M.
        • Honma K.
        • Yuda M.
        • et al.
        CD8+ T cells specific for a malaria cytoplasmic antigen form clusters around infected hepatocytes and are protective at the liver stage of infection.
        Infect Immun. 2013; 81: 3825-3834
        • Beattie L.
        • Peltan A.
        • Maroof A.
        • Kirby A.
        • Brown N.
        • Coles M.
        • et al.
        Dynamic imaging of experimental Leishmania donovani-induced hepatic granulomas detects Kupffer cell-restricted antigen presentation to antigen-specific CD8 T cells.
        PLoS Pathog. 2010; 6: e1000805
        • Girgis N.M.
        • Gundra U.M.
        • Ward L.N.
        • Cabrera M.
        • Frevert U.
        • Loke P.
        Ly6Chigh monocytes become alternatively activated macrophages in schistosome granulomas with help from CD4+ cells.
        PLoS Pathog. 2014; 10: e1004080