In vivo imaging of systemic transport and elimination of xenobiotics and endogenous molecules in mice
We describe a two-photon microscopy-based method to evaluate the in vivo systemic transport of compounds. This method comprises imaging of the intact liver, kidney and intestine, the main organs responsible for uptake and elimination of xenobiotics and endogenous molecules. The image quality of the acquired movies was sufficient to distinguish subcellular structures like organelles and vesicles. Quantification of the movement of fluorescent dextran and fluorescent cholic acid derivatives in different organs and their sub-compartments over time revealed significant dynamic differences. Calculated half-lives were similar in the capillaries of all investigated organs but differed in the specific sub-compartments, such as parenchymal cells and bile canaliculi of the liver, glomeruli, proximal and distal tubules of the kidney and lymph vessels (lacteals) of the small intestine. Moreover, tools to image immune cells, which can influence transport processes in inflamed tissues, are described. This powerful approach provides new possibilities for the analysis of compound transport in multiple organs and can support physiologically based pharmacokinetic modeling, in order to obtain more precise predictions at the whole body scale.
KeywordsDrug transport Intravital imaging Bile canaliculi Hepatocyte Lacteal Glomerulus Renal tubules
Physiologically based pharmacokinetic
Liver sinusoidal endothelial cell
Selection of reporter mice, fluorescent marker dyes and antibodies for intravital imaging
Two-photon excitation range (nm)
Emission maximum (nm)
Tie2 × mT/mG reporter
Tissue morphology differentiating endothelial cells
LysM × mT/mG reporter
Tissue morphology differentiating macrophages and granulocytes
Fluorescent marker dyes
Mitochondrial membrane potential
Mitochondrial membrane potential
Bile acid transport
Fluorescein-labeled dextran (10 kDa)
Macrophages and endothelial cells
Alexa 546-labeled wheat germ agglutinin
Infiltrating myeloid cells
NK cells and innate lymphoid cells
Resident and infiltrating macrophages
All recordings presented were taken from living, anaesthetized mice with intact organs exhibiting all biological processes in vivo. The videos presented represent raw data without any image processing like image registration or de-shaking. The quality of the recordings was sufficient for elementary quantifications by defining regions of interests in order to achieve kinetic data of the transport processes. The technique we describe facilitates in vivo recording to study systemic transport and excretion processes in the most important organs.
Materials and methods
In vivo recordings were acquired using a custom modified inverted LSM MP7 (Zeiss, Jena, Germany) provided with a two-photon Chameleon Ultra II laser with a tuning range from 680 to 1080 nm and a maximum power of approximately 3.5 W at 800 nm. For detection in channel 1, a non-descanned detector with a short-pass filter at 485 was used. Channels 2 and 3 were equipped with a gallium arsenide phosphide (GaAsP) detector with a band-pass filter at 500–550 and 565–610, respectively. Images were acquired with an LD C-Apochromat 40×/1.1 water immersion objective. The temperature of the specimen was controlled with a matt coated incubation chamber (Solent Scientific, Segensworth, UK).
For in vivo imaging, 6- to 10-week-old male mice were initially anaesthetized with an intraperitoneal injection of ketamine (64 mg/kg), xylazine (7.2 mg/kg), acepromazine (1.7 mg/kg) and buprenorphine (0.08 mg/kg). When mice did not show reflexes after pinching feet, the abdomen was shaved and covered with olive oil to avoid disturbance by cut hair in the field of imaging. For liver preparation, the abdominal skin was removed and a small midline incision below the sternum was made with surgical scissors. Subsequently, the falciform ligament was cautiously cut in order to dissociate the liver. The left liver lobe was carefully exposed by gently pushing the abdomen, and subsequently, the mouse was placed in a lateral position onto a 49 mm × 74 mm cover slip 0.17–0.16 mm thickness (Ocon 159, Logitech, Glasgow, UK). The cover slip was put in a custom-made image platform from Plexiglas and fit into the stage of the microscope. The exposed liver was then covered with saline-soaked gauze. The temperature control of the incubation chamber was set at 36 °C. The quality of mouse preparation was verified by checking the breathing rate of the mouse and by the regular blood flow in the observed region. For the preparation of the kidney, the mouse was placed on its right side and a small incision made in the left flank region. The convex side of the left kidney was exposed via gentle pressure on the abdominal wall. In order to avoid dryness, the exposed kidney was covered with saline-moistened gauze during imaging. For intestine imaging, an incision was made on the right side of the abdominal wall. The intestine was gently pulled out using a moistened cotton-tipped applicator. To reduce peristalsis, the intestine was incubated in a 1% atropine sulfate bath for 10 min. Subsequently, the intestine was placed in a glass bottom microwell dishes (MatTek corporation, MA, USA) containing 0.5% atropine sulfate bath during the entire recording period. In case of recordings exceeding 1 h, the mice were kept on maintenance anesthesia via a respiratory mask using an evaporator (IAS3801, FMI GmbH, Seeheim-Jugenheim, Germany) with 1–2% isoflurane and a subcutaneous injection of 0.5 ml saline was given.
Reporters and markers
Wild-type mice used for this study were C57CL/B6N (Janvier Labs, Le Genest-Saint-Isle, France). mT/mG mice (Muzumdar et al. 2007) (#007676), Tie2Cre mice (Koni et al. 2001) (#004128) and LysMCre mice (Clausen et al. 1999) (#004781), all on the B6 background, were all obtained from the Jackson Laboratories, CA, USA. Tie2Cre and LysMCre mice were crossed with mT/mG mice to create cell-specific eGFP reporter mice.
Fluorescent markers or fluorescently labeled antibodies were injected via the tail vein before surgery or during recording via lateral tail vein catheters (SAI-infusion, IL, USA). Fluorescent marker dyes were TMRE and rhodamine 123, which had been solved in 50% methanol/PBS at a concentration of 1.2 mg/ml and 1 mg/ml, respectively (Thermo Scientific, MA, USA), cholyl-lysyl-fluorescein (CLF) in PBS at a concentration of 1 mg/ml (BD Biosciences, California, USA), fluorescein-labeled dextran 10,000 MW in PBS at a concentration of 10 mg/ml (Thermo Scientific), green fluorescent latex particles at a concentration of 50 mg/ml (Thermo scientific), Alexa 546-conjugated wheat germ agglutinin in PBS at a concentration of 0.5 mg/ml (Thermo Scientific) and Hoechst 33342 in PBS at a concentration of 10 mg/ml (Thermo Scientific). All injected antibodies including anti-mouse CD11b, anti-mouse F4/80, anti-mouse NK1.1 and anti-mouse Ly6G (eBioscience, CA, USA) were phycoerythrin conjugated and administered in 100 µl PBS.
For induction of acute liver damage, mice were injected intraperitoneally with 300 mg/kg APAP (Sigma-Aldrich, Taufkirchen, Germany) after overnight fasting. Three days after injection, livers of the mice were prepared for intravital imaging. In order to induce liver fibrosis, mice were injected intraperitoneally twice a week with 1 g/kg CCl4 (Carl Roth, Karlsruhe, Germany) for 2 months. Four days after, the last injection mice were prepared for intravital imaging.
Quantification of compound uptake
Recorded videos were quantified with the Zen software (Zeiss). Fluorescence intensity was quantified in defined regions of interest.
CLF and dextran half-lives in the quantified fluorescence intensities were determined by linear regression with MATLAB (version 126.96.36.199613; The MathWorks, Inc., Natick, MA). Pharmacokinetic analyses were performed with the MoBi® Toolbox for MATLAB (version 6.0.3; Bayer Technology Services GmbH, Leverkusen, Germany). PK-Sim® and MoBi® are freely available for non-commercial academic use. To this end, the quantified fluorescence intensities of CLF and dextran were treated as pharmacokinetic plasma concentration profiles and analyzed accordingly for their pharmacokinetic properties.
Liver: from sinusoids to bile canaliculi
The functional unit of the liver is the lobule, which displays a specific architecture consisting of the parenchymal hepatocytes, the endothelial cells lining the sinusoids, the stellate cells and the resident macrophages called Kupffer cells (Fig. 2a).
Sinusoidal endothelial cells
Specific visualization of sinusoidal endothelial cells (LSECs) in fluorescence microscopy can be achieved with Tie2 × mT/mG reporter mice. These mice express membrane-targeted eGFP in the LSEC, while membranes of the other cell types of the liver remain red (Video 3). The LSECs line the sinusoidal lumen and their nuclei show only a weak Hoechst signal compared to hepatocytes. Remarkably, in Tie2 × mT/mG reporter mice further cells in the sinusoidal lumen also express eGFP. Among them, platelets appear as small flakes rapidly flowing through the sinusoids, which is helpful for identification of the direction of the blood flow. These platelets can form transient aggregates (Video 3, 21–34 min). LSEC can also be stained by fluorescently labeled latex nanoparticles. Particles with a diameter larger than 500 nm are known to be exclusively taken up by macrophages. In contrast, smaller sized particles, below 100 nm, are taken up by macrophages as well as by LSEC (Fig. 3c). While fluorescent latex nanoparticles visualize both LSEC and Kupffer cells, lectins such as wheat germ agglutinin preferentially bind to glycoproteins of LSEC (Video 6). Injection of fluorescently labeled lectins is therefore an additional method that can be used to mark the sinusoidal network.
In order to investigate the resident macrophages in the liver by intravital imaging, LysM × mT/mG reporter mice can be used (Video 4). These mice also allow the visualization of other immune cell types that express LysM, including monocytes and granulocytes. While the green fluorescent Kupffer cells remain stationary, their protrusions show highly dynamic micromotility. Macrophages of the liver can also be visualized by intravenous injection of fluorescently labeled marker antibodies such as F4/80 (Fig. 3d).
Functional imaging of hepatic transport
Calculated half-lives for CLF and dextran in different compartments of the liver, kidney and intestine
Half-life for CLF (min)
Half-life for dextran (min)
Intravenous injection of dextran, clinically used as plasma volume expander, showed uptake into hepatocytes and vesicular transport into bile canaliculi. Although excretion of dextran via bile has been reported (Pupyshev and Maiborodina 2002), direct observation of its vesicular transport across hepatocytes has not yet been possible. An as yet unknown feature of bile salt transport is its transient enrichment in the LSEC/Space of Dissé before CLF signal appears in the hepatocytes (Fig. 4b, middle). This might be due to bile salt adsorption to extracellular matrix components in the Dissé space, prior to active transport through the hepatocyte membrane. Compound enrichment in close proximity to the carriers of the basolateral hepatocyte membranes may improve uptake into hepatocytes. Unfortunately, the resolution of classical two-photon imaging is not sufficient to differentiate the Dissé space from the very thin endothelial cells. Therefore, it remains on known whether transient CLF accumulation occurs in the endothelial cells or in the Dissé space.
Kidney: from glomeruli to distal tubules
Functional imaging of glomerular filtration and tubular transport
Quantification of the CLF signal showed rapid filtration from glomerular capillaries into Bowman’s space (Fig. 6d); whereby, the CLF in circulation is efficiently eliminated with a half-life of 2.3 min (Table 2). After filtration into Bowman’s space, CLF appeared in the proximal and later in the distal tubules. In the tubular lumen, a distinct retention by the brush border was evident, indicating a subsequent reabsorption mechanism for bile acids. Approximately 9 min after injection, CLF in the distal tubules was concentrated by a factor of two compared to the highest fluorescence intensity in the proximal tubules.
Due to its molecular properties, the renal elimination of fluorescently labeled dextran revealed significant differences compared to CLF. The filtration into Bowman’s space was less efficient due to its molecular size, with a threefold higher half-life (Fig. 6c; Video 8). After passing the Bowman’s space, dextran entered the proximal and the distal tubules showing significantly less retention in the brush border of the tubular cells (Fig. 6c). A striking feature was the long persistence of dextran in the distal tubules where no half-lives could be calculated for the time of imaging. The fluorescence intensity of dextran increased in the distal tubules approximately sixfold compared to the proximal tubules (Fig. 6e).
Recently, intestinal lacteals have attracted much attention (Bernier-Latmani et al. 2015; Choe et al. 2015; Nurmi et al. 2015). Lacteals represent specialized lymphatic vessels in the center of each villus. They are essential for absorption of fatty acids and lipid soluble vitamins. Moreover, they play a key role in gut immune responses. Recently, an intravital imaging technique for intestinal lacteals has been published, which allowed, for the first time, analysis of lipid drainage and contractility of intestinal lymphatic vessels (Choe et al. 2015). In contrast to the here established two-photon-based imaging of intact intestine, this study required surgical opening of the intestinal lumen. This is important, because cutting the intestine compromises blood flow in the capillaries. The technique used here allows imaging of compound transport between intestinal capillaries and lacteals. Interestingly, penetration into lacteals showed large differences between the tested compounds. Dextran crossed the blood-lacteal barrier within seconds and was significantly enriched in lacteals (Video 12; Fig. 9e). In contrast, the blood-lacteal barrier was virtually impermeable for the fluorescein-coupled bile acid CLF. This differential permeability of the blood-lacteal barrier may be important for design of drugs aimed at influencing the intestinal immune response.
Immune cells imaging
The LysM × mT/mG reporter mouse represents an appropriate tool to study neutrophil infiltration (Video 4). After induction of physical tissue damage, neutrophils swarm into the damaged region within minutes. While the burned tissue region is densely covered with neutrophils, they also show prolonged residence in the undamaged vicinity (Video 13). Alternatively, neutrophils can be studied by injection of fluorophore-coupled Ly6G antibodies (Fig. 10b).
In order to study infiltration of macrophages during acute liver damage, acetaminophen (APAP) intoxication was applied. These infiltrating myeloid cells (potentially including monocytes, macrophages and dendritic cells) differ from the resident Kupffer cells by the relatively high expression of CD11b (Ramachandran et al. 2012; Zigmond et al. 2014). Intravenous injection of fluorophore-coupled CD11b antibodies into mice 3 days after APAP administration reveals massive infiltration of myeloid cells into the dead cell area compared to control tissue (Fig. 10c, d).
Circulating natural killer (NK) cells can be visualized by intravenous injection of fluorophore-labeled NK1.1 antibodies. In healthy livers, only a relatively small number of NK cells are present in the sinusoids (Fig. 10e). An experimental condition where NK cells play an important role is fibrosis, which can be induced in the liver by chronic administration of carbon tetrachloride (CCl4). In the fibrotic liver, NK cells infiltrate into the tissue (Fig. 10f) in order to induce apoptosis of myofibroblasts during fibrosis recovery (Fasbender et al. 2016).
It should be considered that antibody-based visualization of immune cells might alter functionality of the labeled cells or trigger further immune cell responses by the bound antibody; therefore, this approach might best be limited to short-term imaging. In contrast, recording based on reporter mice should be the first choice when prolonged imaging is required.
The presented two-photon-based imaging setup describes intravital imaging of the most important organs involved in uptake and elimination of endogenous molecules as well as xenobiotics. Before using fluorescently labeled compounds in such studies, it must be ensured that the modified compound behaves similar to the original, as it was reported for CLF (de Waart et al. 2010).
Under optimal conditions image resolution sufficient for tracing individual vesicles, cell organelles such as mitochondria or even endocytic processes can be achieved. This opens possibilities to gain a deeper understanding of transport processes. For example, in the presented data CLF transport from the liver sinusoids to bile canaliculi includes a transient enrichment in a narrow space close to the basolateral hepatocyte membrane, which was not detected before. Also analysis of the transport from the intestinal capillaries to lacteals has not been possible in uncut intestine in the past, due to limitations of laser penetration and scattering of light. Two-photon imaging has been applied previously to study glomerular filtration (Nakano et al. 2012; Sandoval and Molitoris 2013) and transport in renal tubules (Wagner et al. 2016). The new technique described here allows simultaneous imaging of glomerular capillaries, Bowman’s space and proximal and distal tubules. This offers the opportunity to model excretion by the nephron as a function of secretion, reuptake and flow in each of these compartments simultaneously.
The presented studies of compound transport in tissues can, in particular, support physiologically based computational models. Currently, first-in-human trials represent a critical step in drug development, because of interspecies differences and difficulties of extrapolation from mouse to human (Thiel et al. 2015). Recently, PBPK modeling for cross-species extrapolation has been introduced, which improved the accuracy of prediction by incorporating target species-specific model parameter domains, such as species-specific physiology, plasma protein binding, transport kinetics and metabolism (Thiel et al. 2015). A limitation is that current PBPK models usually consider individual organs only as single compartments. However, the present study shows that transport occurs in various sub-compartments of tissues, which can be independently quantified. The measurements provide an important systems view on drug distribution within the organism: On the one hand, the blood concentrations in the different organs reflect drug concentration profiles in the overall blood pool of the body. On the other hand, the profiles in the specific sub-compartments enable a quantitative assessment of drug distribution within specific tissue compartments. Such measurements allow for the targeted quantification of specific physiological processes in detailed computational models of the liver (Ghallab et al. 2016; Hoehme et al. 2010; Schwen et al. 2014; Vartak et al. 2016), the kidney (Thomas 2009) or the intestine (Thelen et al. 2011). Another interesting application could be validation and refinement of distribution models describing organ-plasma partitioning in PBPK modeling (Jones et al. 2009). In this regard, it should be noted that the given profiles may be used for the derivation of biliary and renal clearance rates, as such enabling the quantitative assessment of drug excretion. These measurements therefore provide important information to key physiological processes underlying drug pharmacokinetics, i.e., distribution and excretion.
Many compounds such as bile acids, drugs and toxins undergo enterohepatic circulation and may be modified by the microbiota of the intestine or by the metabolism of the hepatocytes (Ridlon et al. 2015; Roberts et al. 2002). This kind of modification can alter pharmacological activities and transport efficiency. Measuring compound uptake in the intestine as well as in the hepatocytes in vivo as described in this study can give information on intestinal impermeability, hepatic uptake and body clearance of the parent substance and its metabolite, which may be dependent upon the properties of the liver-gut axis.
An attractive future application of the introduced systemic imaging method is to integrate spatial–temporal fluorescence image correlation methods in vivo such as fluorescence correlation spectroscopy (FCS) (Elson 2001) and raster image correlation spectroscopy (RICS) (Brown et al. 2008). The recording setup fulfills many important preconditions for FCS and RICS such as stable focus, fast imaging sequences and low phototoxicity. These techniques allow the determination of absolute concentrations of fluorescent test compounds in the analyzed compartments. Besides compound concentrations, correlation spectroscopy can yield further interesting data on protein binding, flux, diffusion coefficients and orientation of transport (Brown et al. 2008; Rossow et al. 2010).
In conclusion, the two-photon-based imaging toolbox presented here allows quantitative spatial–temporal analysis of physiological as well as pathophysiological processes in intact liver, kidney and intestine.
This study was supported by the BMBF, Germany, funded by projects LiSyM (FKZ 031L0052), Lebersimulator (031A355A) and the UK Medical Research Council (PMK; G1000230).
AG and RR contributed to conception and design; acquisition of data; analysis and interpretation of data; drafting and revising the article. LB helped in drafting and revising the article. GG helped with acquisition of data. LK contributed to analysis and interpretation of data; drafting and revising the article. PK and JH helped with conception and design; analysis and interpretation of data; drafting and revising the article.
Compliance with ethical standards
Conflict of interest
Lars Kuepfer is employed by Bayer AG, the company developing the PBPK modeling tools PK-Sim and MoBi.
Animal experiments were approved by the German animal ethics committee and the Animal Welfare and Ethics Review Body of the University of York. Studies in the UK were conducted under UK Home Office License (PPL 60/4377).
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