Non invasive in vivo investigation of hepatobiliary structure and function in STII medaka (Oryzias latipes): methodology and applications
© Hardman et al; licensee BioMed Central Ltd. 2008
Received: 20 March 2007
Accepted: 06 October 2008
Published: 06 October 2008
A novel transparent stock of medaka (Oryzias latipes; STII), recessive for all pigments found in chromatophores, permits transcutaneous imaging of internal organs and tissues in living individuals. Findings presented describe the development of methodologies for non invasive in vivo investigation in STII medaka, and the successful application of these methodologies to in vivo study of hepatobiliary structure, function, and xenobiotic response, in both 2 and 3 dimensions.
Using brightfield, and widefield and confocal fluorescence microscopy, coupled with the in vivo application of fluorescent probes, structural and functional features of the hepatobiliary system, and xenobiotic induced toxicity, were imaged at the cellular level, with high resolution (< 1 μm), in living individuals. The findings presented demonstrate; (1) phenotypic response to xenobiotic exposure can be investigated/imaged in vivo with high resolution (< 1 μm), (2) hepatobiliary transport of solutes from blood to bile can be qualitatively and quantitatively studied/imaged in vivo, (3) hepatobiliary architecture in this lower vertebrate liver can be studied in 3 dimensions, and (4) non invasive in vivo imaging/description of hepatobiliary development in this model can be investigated.
The non-invasive in vivo methodologies described are a unique means by which to investigate biological structure, function and xenobiotic response with high resolution in STII medaka. In vivo methodologies also provide the future opportunity to integrate molecular mechanisms (e.g., genomic, proteomic) of disease and toxicity with phenotypic changes at the cellular and system levels of biological organization. While our focus has been the hepatobiliary system, other organ systems are equally amenable to in vivo study, and we consider the potential for discovery, within the context of in vivo investigation in STII medaka, as significant.
The majority of our understanding of vertebrate hepatobiliary disease and toxicity has been derived from mammalian liver studies [1–5]. We know comparatively less about piscine biliary disease and toxicity, though we are beginning to gain greater insight into piscine hepatobiliary structure/function relationships [6–17]. Because our understanding of the piscine biliary system has lagged, particularly in a comparative sense, our ability to interpret and communicate biliary disease and toxicity in aquatic species has remained limited. By example, cholestasis (impaired and/or inhibited bile transport) has never been described in fish, a fact more representative of our lack of understanding/investigation, as opposed to the lack of occurrence of this physiological response.
Because the vertebrate liver is a common target organ of toxicity, largely due to the emergence and prevalence of modern xenobiotics (e.g., environmental contaminants, pharmaceuticals) in society over the last century [18–24], and the fact that various vertebrate systems (fish, rodent, avian, primate, human) are applied to understanding the mechanisms and modes of actions involved in xenobiotic induced injury, a betterment of our comparative understanding, and ability to investigate and interpret hepatotoxicity in the piscine hepatobiliary system, is essential. By example; the presence of personal care products and pharmaceuticals (PCPPs) [25–32], persistent environmental contaminants (POPs) [21, 22, 33–35], and the widespread application of antibacterial agents, pesticides, and hormones in aquaculture [36–39] present ecotoxicological and, because of the human consumption of fish, human health related concerns, that are likely to persist for decades. These and other environmental contaminants necessitate enhancement of our ability to understand hepatobiliary disease and toxicity in piscine species.
Advancement of our understanding of the piscine hepatobiliary system is not relegated to environmental concerns alone. Because of their many advantages (e.g., small size, relative ease of care and use, potentially higher statistical power due to large study cohorts), small fish animal models such as medaka, zebrafish, and fathead minnow have seen increasing application in biomedical research (e.g., carcinogenesis, mutagenesis, functional genomics, toxicogenomics) [40–45].
The see-through medaka (Oryzias latipes; STII), recessive for all pigment genes of chromatophores (iridophores, melanophores, xanthophores and leucophores) , is a unique small fish animal model that enables high resolution (< 1 μm) in vivo imaging of biological structure and function at virtually all levels of organization, from subcellular to gross anatomical [12, 47]. Exhibiting no expression of leucophores and melanophores, and minimal expression of xanthophores and iridophores, STII medaka are essentially transparent throughout their life cycle. In embryo, larval and juvenile STII medaka (from 3 to 60 days post fertilization (dpf)) it is possible to image internal cells, tissues and organs, and generate three-dimensional (3D) reconstructions from in vivo imaging. We present here an overview of our findings from in vivo investigations in STII medaka that demonstrate the utility of this experimental in vivo system. Specifically, we show that: phenotypic response to xenobiotic exposure can be investigated/imaged in vivo with high resolution (< 1 μm); hepatobiliary transport of solutes from blood (sinusoid) to bile (intrahepatic biliary passageways) can be qualitatively and quantitatively studied in vivo; hepatobiliary development in this model can be described/investigated via non invasive in vivo observations; and hepatobiliary structure/function in this lower vertebrate liver can be studied in 3D.
Our purpose here is to share in vivo methodologies and present examples of applications of these methodologies to in vivo investigation. First we describe the in vivo methodologies developed, and then give specific examples of the successful application of these methodologies to the evaluation of hepatobiliary structure, function, development, and xenobiotic response, in both 2D and 3D. Because much of our recent research has focused on the piscine hepatobiliary system, namely biliary system toxicity, this organ system will be emphasized in the examples provided.
For decades color mutant strains of medaka (Oryzias latipes), acquired from natural and commercially available populations, have been maintained in the Laboratory of Freshwater Fish Stocks at Nagoya University, Japan. Cross breeding from these stocks was used to produce a stable "transparent" strain of medaka , from which our STII medaka colony, maintained at Duke University since 2002, was derived. Fish care and maintenance was provided daily in accordance with Duke University IACUC approved animal protocols (A117-07-04; A141-06-04; A173-03-05). Brood stock were housed in a charcoal filtrated and UV treated recirculating system (City of Durham, NC water) maintained at 25 +/- 5°C. Water chemistries were maintained at; pH (7.0–7.4), dissolved oxygen (6–7 ppm), ammonia (0–0.5 ppm), nitrite (0–0.5 ppm) and nitrate (0–10 ppm). Brood stock were maintained on a diel cycle of 16:8 hr light:dark. Unconsumed diet, detritus and associated algal material were removed from brood stock tanks daily. Eggs and egg clusters, collected daily, were separated, cleaned in embryo rearing medium (ERM), and individual fertilized eggs maintained in ERM at 25°C for stock maintenance. Medaka larvae were fed a commercial ration of ground (100 μm) Otohime β daily (Ashby Aquatics, West Chester, PA). In addition, all brood stock fish diets were supplemented with Artemia nauplia (hatched brine shrimp) at least once per day, seven days per week.
Widefield microscopy was performed on a Zeiss Axioskopp equipped with DAPI/TRITC/FITC fluorescence filter cube set (DAPI/UV: Ex 360–380 nm/Em All Vis > 400 nm, FITC: Ex 450–490 nm/Em 515–565 nm, TRITC: Ex 528–552 nm/Em 578–632 nm), Zeiss Plan Neofluar 5×/0.15, 10×/0.3, 20×/0.5, 40×/0.85 pol, and 100×/1.3 oil objectives, Photometrics CoolSnap digital imaging system (2048 × 2048-element array) and IP Lab (V. 3.0) image acquisition software (Scanalytics). A xenon lamp was used for excitation. For confocal fluorescence microscopy a Zeiss 510 Meta system with Zeiss LSM 5 Axiovision image acquisition software, Argon and HeNe laser, and Carl Zeiss C-apochromatic 40×/1.2 and C-apochromatic 10×/0.45 objectives, was used. For brightfield microscopy a Nikon Eclipse E600 with a Nikon DXM 1200 digital capture system, halogen light source, Nikon plan neo-fluor 10×/0.3 wd16, plan neo-fluor 20×/0.5 wd2.1, plan neo-fluor 40×/0.75 wd0.72, and plan apo 60×/1.4 wd0.21 (oil) objectives, was used. A Nikon SZM 1500 with a Nikon DXM 1200 digital capture system, Nikon HR plan apo 1× WD54 and Nikon HR 0.5× WD136 objectives was also employed for brightfield dissecting microscopy. All transmission electron microscopy (TEM) was performed at the Laboratory for Advanced Electron and Light Optical Methods (LAELOM), College of Veterinary Medicine, North Carolina State University, on an FEI/Philips EM 208S Transmission Electron Microscope.
Image analysis and compilation was performed with EclipseNet (Nikon, USA), Adobe Photoshop (Adobe, Inc.), ImageJ (NIH, V1.32j), IP Lab software (Scanalytics, Inc., version 3.55), and Zeiss Image Browser (Carl Zeiss). All 3 dimensional reconstructions and analyses were performed with Amira 3D (Mercury Computer Systems, Berlin).
Chemicals and fluorescent probes
Fluorescent probe specifications
Initial/Peak Assimilation Time (min)
In vivo CYP3A activity. Uptake via gill, in vivo metabolism in gill and gut, good for investigating blood to bile transport, IHBPs, EHBPs and intestinal lumen.
In vivo imaging of CYP1A activity. Uptake via gill, in vivo metabolism in gill and liver. CYP 1A2, 2E Substrate
In vivo labeling of DNA, RNA. Good for apoptosis, interacts with DNA and RNA by intercalation or electrostatic attractions.
BODIPY 505/515: 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene
5 μM-100 nM
Uptake via gill, active transport through hepatobiliary system, concentrative blood to bile transport, and secretion from gall bladder into gut lumen. Good for elucidation of gill, IHBP, EHBP, intestinal lumen. Non-Polar, Lipophilic.
BODIPY FL C5-ceramide: N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)sphingosine
5 μM–100 nM
Putative passive transport in vivo. Uptake via gill, transport through cardiovascular and hepatobiliary systems and secretion from gall bladder into gut lumen.
In vivo Bodipy verapamil localized to hepatocytic cytosol in discrete vesicles. Transport to bile was not observed in the time frame assayed, which was 90 minutes.
BODIPY ® 493/503: 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY ® 493/503)
Uptake via gill, active transport through hepatobiliary system, concentrative blood to bile transport, and secretion from gall bladder into gut lumen. Good for elucidation of gill, IHBPs, EHBPs, intestinal lumen. Lipophilic, amphiphilic.
BODIPY ® 581/591 C5-HPC (Phosphocholine) PC: 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine
OIn vivo labeling of intrahepatic and extrahepatic biliary system. Uptake via gill. Hepatobiliary transport to gut lumen. Diffuse fluorescence in hepatocyte cytosol.
CellTrace™ Oregon Green ® 488 carboxylic acid diacetate, succinimidyl ester (carboxy-DFFDA, SE) *cell permeant* *mixed isomers
In vivo labeling of hepatic nuclei, potential for detection of apoptosis
DAPI: 4',6-diamidino-2-phenylindole, dihydrochloride
0.3 – 3.0 μM
In vivo nuclear labeling of virtually all cell types associated with gill, gut, liver, and cardiovascular system.
DAPI Diacetate: 4',6-diamidino-2-phenylindole, diacetate
0.3 – 3.0 μM
DAPI diacetate is water-soluble form. In vivo nuclear labeling.
Fluorescein-5-isothiocyanate (FITC 'Isomer I')
1 μM – 50 nM
Excellent in vivo probe for elucidating biliary system. In vivo labeling of intrahepatic and extrahepatic biliary system. Hepatobiliary transport to intestinal lumen. Uptake via gill.
MitoTracker ® Green FM
In vivo labeling of hepatocytes.
SYTO ® 16 green fluorescent nucleic acid stain
10 nM-1 μM
In vivo nuclear labeling of epithelia and endothelia, putative in vivo probe for apoptosis.
SYTO ® 27 green fluorescent nucleic acid stain
10 nM-1 μM
SYTOX ® Orange nucleic acid stain
YO-PRO ® -1 iodide (491/509)
YOYO-1 Iodide (491/509)
2 – 5 nM
In vivo accumulated in interstitial spaces, some labeling of vasculature. Typically used for assays for cell enumeration, cell proliferation and cell cycle.
Reference hepatotoxicants α-napthylisothiocyanate (ANIT) and diethylnitrosamine (DEN) were used for comparative study of responses of the hepatobiliary system. ANIT is a well described biliary toxicant in rodent models that induces hallmark responses in the mammalian liver, namely: cytotoxicity in biliary epithelium of bile ductules and ducts, cholestasis [48–51], and biliary tree arborization (biliary epithelial cell hyperplasia) [52–54]. DEN, a complete carcinogen, is a widely employed reference hepatotoxicant that has been used in fish .
ANIT Exposures: Cohorts (10 to 30 fish) of STII medaka were exposed to aqueous ANIT to target hepatocytes and biliary epithelia for toxic response. Exposures were in aqueous bath during various stages of development, under acute and chronic exposure conditions. Controls consisted of untreated medaka, and those that received DMSO (ANIT solvent). Studies were designed to evaluate hepatobiliary structure/function during the onset, progression, and recovery from ANIT induced changes. All exposures were done in 750 ml wide-bottom glass rearing beakers at 25°C, 16 hr light/8 hr dark cycle. Aqueous bath exposure medium was 1:3, ERM:de-ionized water. Acute exposures: medaka were subjected to single exposures of ANIT at concentrations from 0.25 μM to 10 μM ANIT and assessed at multiple time points (e.g., 5 minutes, 15 minutes...6, 12, 24, 48, 72 and 96 hrs, and at day 10, 30 and 60 post exposure). Chronic exposures: medaka were re-exposed every 3 days, or once weekly (static renewal baths), and examined at the same points given for acute exposures. DEN exposure was for 48 hrs at 200 ppm in aqueous bath, after which time medaka were reared for 10 months under normal conditions. At given time points during ANIT and DEN exposure regimes, subpopulations of medaka were removed from an exposure cohort for in vivo investigations, histological, immunohistochemical, and transmission electron microscopy (TEM) studies.
In vivo methodologies
As an overview, in vivo investigation in STII medaka can be considered to be comprised of three primary components, which describe a "system" of study; (1) sedation, (2) microscopy/imaging technologies, and (3) fluorescent probes.
To facilitate in vivo imaging of larval and older STII medaka (> 8 dpf) non lethal sedation was necessary, particularly for cellular level investigations. Optimal sedation was achieved with 10 μM tricaine methane sulfonate (MS-222). STII medaka were placed in a solution of 10 μM MS-222 (ERM as solvent), as soon as medaka were anaesthetized (immotile) they were removed from the sedative bath and placed in fresh ERM:deionized water (1:3). Sedated medaka were then placed on a glass depression well slide with enough ERM:deionized water to fill the depression well (~400 μl), oriented in a preferred anatomical position, and the depression well slide was sealed with a cover-slip. For optimal image clarity medaka were positioned so the dermis was in contact with the coverslip, which minimized optical distortion that may result from image diffraction through the fluid space between dermis and coverslip. Once mounted on a depression well glass slide medaka were imaged live with brightfield, and widefield and laser scanning confocal fluorescence microscopy (LSCM). Each was found to offer specific advantages, which will be illustrated through the in vivo findings presented. A brief overview of considerations for each imaging modality follows.
LSCM, due to higher planar resolution, was optimal for quantitative investigations into in vivo hepatobiliary transport (see Figures 2, 3, 4, 5, 7 and 8 for comparison). LSCM also offered the advantage of acquisition of confocal stacks of up 100 μm in depth, at planes of section from 0.5 μm to 2 μm. Further, individual confocal stacks could be joined, allowing for final in vivo confocal image stacks of up 300 μm in depth (the maximal depth, we found, at which high quality in vivo imaging could be performed with the system used). In summary, while widefield was sufficient for qualitative study/imaging of hepatobiliary structure, function and transport, accurate quantitative study/imaging required the finer spatial resolution provided by confocal microscopy. Examples illustrating the differences between widefield and confocal microscopy are given throughout the findings presented.
Fluorescent probes and endogenous fluorophores
The use of fluorescent probes in living medaka greatly enhanced our ability to investigate hepatobiliary structure/function and xenobiotic response in vivo. While fluorescent probes have been widely employed in vitro (e.g., cell culture based investigations), few commercially available fluorophores were, at the time these studies were undertaken, characterized for in vivo use in fish. Fluorophores were selected based on their biocompatibility and utility in analysis of hepatobiliary morphology and transport, and are given in Table 1. That a probe would exhibit uptake via aqueous bath was a priority for selection (we preferred to avoid use of intraperitoneal injection).
For brevity, a discussion of fluorescent probes most frequently employed for elucidation of cell/tissue/organ structure and function is given. Metabolic substrates 7-benzyloxyresorufin (7-BR, CYP3A substrate) and 7-ethoxyresorufin (7-ER, CYP1A substrate) were found valuable in vivo probes for investigating CYP3A and CYP1A expression in organs and tissues, and for elucidating the intra- and extrahepatic biliary system, as well as gut lumen. Both probes, non-fluorescent in their native state, are metabolically activated (de-alkylation of 7-BR, de-ethylation of 7-ER) by their respective CYP enzymes to the anionic fluorescent metabolite resorufin. Both probes have been widely employed in vitro [56, 57], and 7-ER has been employed in vivo in Fundulus heteroclitus and medaka [58–61]. An example of the in vivo application of 7-benzyloxyresorufin, elucidating in vivo CYP3A metabolism, transport, and hepatic morphology, is given in Figure 2.
Sphingolipids are a structurally diverse class of compounds composed of a polar head group and two nonpolar tails (akin to phospholipids), and are naturally occurring compounds found in all plants and animals. As such, we considered the potential for fluorescently-labeled sphingolipids to be incorporated into live cells, and found two fluorescent sphingosines, β-Bodipy C5-HPC and Bodipy FL C5 ceramide, to be useful for in vivo study in STII medaka. β-Bodipy C5-HPC, a fluorescently-labeled phosphocholine, proved biocompatible and was found optimal for elucidating epithelia, endothelia, and hepatobiliary transport. β-Bodipy C5-HPC labeled virtually all epithelia throughout the body of STII medaka. While in vivo kinetics for fluorescently-labeled sphingosines like β-Bodipy C5-HPC and Bodipy FL C5 ceramide are currently unknown, and little information exists on their in vivo metabolic properties, our observations suggest β-Bodipy C5-HPC is recognized as an endogenous sphingolipid, as β-Bodipy C5-HPC was readily incorporated into virtually all cell membranes under aqueous exposure conditions. For instance, β-Bodipy C5-HPC was not only taken up by virtually all epithelia, but also observed to cross the blood brain barrier (the only fluorophore observed to do so), labeling neural bundles in the medulla of medaka (Figure 3). β-Bodipy C5-HPC appeared to be taken up across gill epithelium, and transported through the cardiovascular system to the liver and gut. A small organic cation, the in vivo characteristics of this probe suggested concentrative transport of the fluorophore from blood to bile (hepatic), with fairly rapid (15 minutes post aqueous exposure) accumulation of the fluorophore in bile. While empirical and quantitative studies suggest concentrative vectorial transport of this fluorophore, in vivo kinetics will need further study and confirmation.
The in vivo kinetics of Bodipy FL C5 ceramide appeared more consistent with passive diffusion across cell membranes. Uptake and distribution of the fluorophore was observed to be substantially slower (30 – 45 minutes slower) than β-Bodipy C5-HPC. Where the fluorescent phosphocholine saw uptake and concentration in intrahepatic biliary passageways as early as 15 minutes post administration (aqueous bath), the fluorescent ceramide was not observed to accumulate (peak fluorescence) in the hepatobiliary system until ~45 minutes post exposure.
Cardiovascular transport of Bodipy FL C5 ceramide was much more distinct than that of β-Bodipy C5-HPC, with marked and prolonged residence time in blood plasma, and distinct labeling of endothelium and red blood cells. Bodipy FL C5 ceramide, because of its apparently slower (passive?) uptake, and accumulation in the cytosol of virtually all cell types, was found optimal for investigating epithelial cell morphology in vivo. This fluorophore also exhibited a more even distribution (concentration) across blood plasma, cytosol, and intrahepatic bile passageways. Hence, Bodipy FL C5 ceramide proved highly effective for elucidating cell morphology in vivo, allowing imaging of sinusoids, hepatocytes, and intrahepatic biliary passageways, simultaneously.
The highly lipophilic Bodipy 505/515 and 493, like β-Bodipy C5-HPC, were also found to be effective fluorophores for investigating hepatobiliary transport and bile secretion in vivo (Figure 3). Both probes allowed elucidation of the intrahepatic biliary passageways, extrahepatic biliary passageways, gut lumen, and intestinal lumen (e.g., mucosal folds of the gut).
Fluorescein isothiocyanate (FITC) was also utilized for elucidating the biliary architecture. This small organic cation showed rapid uptake, with marked differential distribution between blood and bile, and transport into biliary passageways. Because of the difference in concentration of FITC (as well as other fluorophores) in blood and bile on a temporal scale, this allowed for high resolution in vivo investigation into biliary morphology, bile transport, and xenobiotic response. (Note: the temporal variation in blood and bile concentrations is the result of fluorophore uptake and transport kinetics, determined by the physical and chemical properties of the fluorophore, and passive and active transmembrane transport kinetics in gill epithelia and hepatocytes.)
Several probes were found to be useful for in vivo nuclear labeling, these were: DAPI 4',6-diamidino-2-phenylindole, dihydrochloride; DAPI Diacetate 4',6-diamidino-2-phenylindole, diacetate; and the SYTO® and YO-PRO® series of fluorescent probes from Invitrogen (Figure 8). The ability to label and visualize nuclei of individual cells in organs and tissues proved valuable not only for structural elucidation (particularly in 3D), but for differentiation between nuclei and cellular organelles (described later in the xenobiotic response section).
Flavoproteins: flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN)
Pyridine Nucleotides: nicotinamide dinucleotide (NAD), nicotinamide dinucleotide phosphate (NADP), the reduced states fluoresce – NADPH, NADH
Hydroxykynurenine glucoside (3-HGK)
Our studies found UV/DAPI excitation to be highly useful in elucidation of morphological features, otherwise difficult to visualize. For instance, UV/DAPI excitation was highly effective for visualizing vasculature (blood plasma was relatively non fluorescent compared to individual epithelial, endothelial and red blood cells). UV/DAPI excitation was also useful for visualizing individual epithelial cells in vivo (e.g., hepatocyte morphology). Hence, the innate autofluorescence properties of cells/tissues can be utilized for investigation of a variety of structural and functional, as well as diagnostic purposes. Further, savings in time and materials may be realized, negating the need to administer exogenous fluorophores to elucidate cellular and tissue morphology. Note however that UV excitation wavelengths (< 400 nm) are known to be cytotoxic and special care must be taken when employing these methods.
At other excitation/emission wavelengths (TRITC/FITC) general tissue autofluorescence was less distinct than that observed with DAPI/UV. However, autofluorescence of bile fluid was marked under both TRITC and FITC illumination. Because of this TRITC/FITC excitation/emission could be employed to detect the onset of bile synthesis (endogenous bile fluorescence), and to observe bile transport in vivo. Of interest, we observed bile autofluorescence to vary on a temporal scale, a fact which merits further detailed study. While TRITC/FITC excitation resulted in distinct autofluorescence of intra- and extrahepatic bile passageways, as well as gall bladder, it was not consistently observed, suggesting variations in bile composition, perhaps due to varying concentrations of endogenous fluorophores (in bile) previously discussed (e.g., NADP, FADH, porphyrin compounds). For this reason, it was not possible to fully utilize bile autofluorescence as an indicator of hepatobiliary transport of specific bile constituents, though significant potential exists in applying fluorescence studies to the examination of bile transport.
Quantitation of hepatobiliary transport in vivo
For accurate quantitation of transport several key elements were necessary, and merit a brief discussion. One of the primary issues was standardization of microscopy techniques [65–67]. Exposure times while imaging (e.g., fluorophore quenching), laser intensity (excitation), fluorophore concentration in the exposure bath, and imaging depth in vivo (e.g., how deep into the tissues you are imaging), were each found to influence and define the digital image information captured in vivo, and this, is turn, affected quantitation of digital information. Perhaps one of the most important factors was the time of image acquisition. Most fluorophores used for investigation of blood to bile transport were observed to be assimilated by medaka fairly rapidly (10–20 minutes for initial uptake from aqueous bath). Peak/maximal fluorescence (a proxy for fluorophore saturation) occurred some 15 to 90 minutes later, depending on the fluorophore used. Hence, in developing in vivo methodology for quantifying hepatobiliary transport, several key elements were/are required. First, uptake, distribution and transport of individual fluorophores should be fully characterized for standardization of protocols. This would entail characterizing temporal changes, as well as the effect of fluorophore concentration (aqueous bath), on uptake and transport kinetics. In our investigations rigorous study and thorough determination of fluorophore kinetics in vivo was not possible due to the lack of a dedicated confocal instrument, and time. In lieu of this type of characterization, observed peak fluorescence times, and a time frame subsequent to peak fluorescence that allowed for in vivo imaging and quantitation of transport, was determined for each fluorophore employed.
We provide in Table 1 times at which fluorescence was first observed in the biliary passageways of the liver (initial fluorophore uptake), and the time when fluorescence peaked (maximal saturation). After peak fluorescence was achieved, fluorophore concentration in vivo (fluorescence) was assumed to be at a steady state as long as medaka remained under constant exposure to the fluorophore in aqueous medium. At this point, transport kinetics were assumed to have reached equilibrium (at the exposure concentrations given, under chronic exposure conditions). For our studies it was decided image acquisition would occur from the onset of peak fluorescence (saturation), or the time when fluorescence intensity peaks at a given exposure concentration (unique for each fluorophore), and for a predefined period of time following the onset of peak fluorescence; for our studies, 60 minutes. Hence, imaging was done at the onset peak fluorescence and for, on average, no more than 60 minutes post peak fluorescence. With the above factors accounted for, we found in vivo quantitation of hepatobiliary transport possible and a potentially valuable diagnostic tool.
For quantitation, digital images from in vivo investigations were converted to both RGB 32 bit color, and 8 bit grayscale. These were then analyzed in ImageJ using the following techniques. Regions of interest (ROIs) were defined for sinusoid lumen, hepatocyte cytosol, and canalicular/bile preductular lumens. ROIs were measured for fluorescence intensity (brightness value for RGB and grayscale value for grayscale) in both RGB 32 bit color, and 8 bit grayscale images. In the image under study multiple ROIs were randomly selected for; sinusoidal space, cytosol space and bile space. For instance, in a confocal stack, acquired from in vivo imaging, multiple ROIs were measured in each of the three compartments, resulting in repeated measures. Fluorescence intensity values, as well as the total area measured for sinusoid, cytosol and bile space, were imported to Excel and Statview for statistical analyses. Means were taken for each set of measures (sinusoid, cytosol, bile space) and used for plots/graphs. Descriptive statistics as well as bivariate analyses were used for quantitation.
Three-dimensional reconstructions from in vivo imaging
The fluorescent probes 7-benzyloxyresorufin (7-BR), β-Bodipy C5-HPC, Bodipy FL C5-ceramide, DAPI, and fluorescein isothiocyanate (FITC) were administered to living STII medaka in aqueous bath (see Table 1) to elucidate specific components of the hepatobiliary system (e.g., hepatocytes, endothelial cells, biliary epithelia, bile passageways). Individual medaka were exposed to fluorophores (aqueous bath) at the following concentrations and durations: 7-BR (10–50 μM, 10 to 30 minutes), β-Bodipy C5-HPC (30 nM–10 μM, 10 to 30 minutes), Bodipy FL C5-ceramide (500 nM–5 μM, 10 to 44 minutes), FITC (1 nM – 50 μM, 10 to 30 minutes). After 15 – 60 minutes of fluorophore exposure STII medaka were sedated, mounted on depression well glass slides with cover slip and imaged live using LSCM, at various stages of development (4–60 dpf). Confocal stacks from in vivo imaging of the hepatobiliary system were then imported into the 3D rendering and analytical software, Amira 3D. Confocal stacks were comprised of 0.5 μm to 2 μm slices (space between individual images/scans), though 0.7 μm was most commonly employed. Stacks were typically 90 to 120 μm thick (depth of scan), though in some instances these were combined to create stacks of up to 200 μm in thickness. The 3D reconstructions, manually created in Amira 3D, were then used for architectural, morphometric and volumetric analyses.
Results and discussion
In vivo description of hepatobiliary development
In vivo investigations permitted detailed study and description of normal development of the medaka hepatobiliary system, which included observation/description of hallmark events such as organogenesis, the onset of bile synthesis and transport, and metamorphosis of the liver from an embryonic to adult phenotype (Figure 4). In vivo investigations also permitted direct observation of hepatic architecture at the cellular level at various developmental stages, observations which, coupled with 3D reconstructions of the liver, led to important insights. For instance, characterization of variations in parenchymal architecture between embryonic and larval stages, and the comparative similarities and differences in vertebrate liver conceptual models . Figures 2, 4 [and Additional files 1, 2, 3, 4, 5, 6, 7, 8 and 9] provide examples of results from in vivo investigations into hepatobiliary development (unpublished studies), investigations that were fundamental to characterizing normalcy (in vivo) and to subsequent investigations into adaptive vs. toxic responses of the liver to xenobiotic exposure (see In vivo investigation of xenobiotic response).
Quantitation of hepatobiliary transport in vivo
To put these studies into context a brief overview of hepatobiliary transport is provided. Transport of solutes from blood to bile is a vital liver function. It is through bile synthesis and transport that xenobiotics of environmental origin and endogenous metabolic by-products are either safely removed from the system, or, with systemic accumulation, result in morbidity and mortality [4, 68–74]. Inhibition/impairment of bile transport (cholestasis) commonly results in morbidity and mortality in mammals. Little is known about bile transport in piscine species, and the relationship of impaired transport function to disease and toxicity in these organisms. While it is becoming increasingly apparent that many piscine species share bile synthetic and transport mechanisms with their mammalian counterparts [14, 75, 76], no studies on impaired/inhibited bile transport (cholestasis) in fish exist. Here we show methods that can be used to perform in vivo assessment of bile transport, and present initial findings employing these methodologies.
Quantitative analysis of blood to bile transport in vivo (STII medaka, 12 dpf): β-bodipy C5 phosphocholine (HPC), fluorescein Isothiocyanate (FTIC) and bodipy C5 ceramide.
F-test: Hypothesized Ratio = 1
C5 Ceramide: FITC
C5 Ceramide: HPC
Three-dimensional in vivo investigations: hepatobiliary architecture
Non invasive in vivo imaging in STII medaka allowed the generation of 3D models of the hepatobiliary system (Movies 1 – 9), under conditions of normalcy and toxicity. Using LSCM, in tandem with exogenous fluorophores, we were able to elucidate hepatocellular, biliary and vascular components of the liver in 3D. Three-dimensional investigations yielded important insights into medaka hepatobiliary structure/function, which may not have been possible using standard 2D methodologies alone (e.g., histological, ultrastructural). For instance, 3D analyses revealed that: the hepatic parenchyma in medaka is organized through a hexagonal structural motif (polyhedral tessellation), evident in the fine structure of the biliary system; the biliary system is an interconnected network of canaliculi and bile preductules; the canaliculo-preductular network perfuses the majority of the liver corpus (~95%) uniformly, with equidiameter intrahepatic biliary passageways (IHBPs) (1–2 μm) observed throughout the liver; larger bile ductules and ducts were observed only at the liver hilus, and consequently an arborizing biliary tree was absent, seen only in the rudimentary branching of intrahepatic ducts from the hepatic duct; parenchymal architecture is a predominantly 2 cell thick muralium, though tubule-like formations may also comprise the muralium; the livers of these small fish are replete with BPDECs, the putative mammalian correlary of bipotential progenitor/stem cells; BPDECs and hepatocytes form unique junctional complexes that create bile passageways (bile pre-ductules). BPDECs occupy the center of these junctional complexes, surrounded by bile pre-ductules. Collectively, these findings characterized the 3D architecture of the medaka hepatobiliary system and improve our comparative understanding of vertebrate liver structure and function. These findings also raised interesting questions regarding the "functional unit" of the vertebrate liver, suggesting the hepatobiliary system in medaka can be, as a conceptual model, considered a single functional unit of the vertebrate liver, akin to an individual unit of the mammalian lobule .
Hepatobiliary morphometrics from in vivo based 3D investigations.
IHBP Segment Length (μm)
IHBP Diameter (μm)
BPD Segment Length (μm)
8 – 40 dpf
Hepatocyte Diameter (μm)
8 – 40 dpf
(height variance of 20 μm to > 100 μm)
Sinusoid Diameter (height variance of 8 μm to 31 μm)
Hepatic Vein Diameter (μm)
Portal Vein Diameter (μm)
Hepatobiliary volumetrics from in vivo based 3D investigations.
Intrahepatic Biliary Passageways
Stack size: 115 × 115 × 50 μm, sections: 1 μm, scaling/voxel size: 0.2 × 0.2 × 1 μm, 3D volume analysis: ~50% total liver volume
Volume μm 3
SA μm 2
Intrahepatic Biliary Passageways
Stack size: 230 × 230 × 90 μm, sections: 1 μm, scaling/voxel size: 0.45 × 0.45 × 1 μm, 3D volume analysis: ~50% total liver volume
Volume μm 3
SA μm 2
Intrahepatic Biliary Passageways
Stack size: 325 × 325 × 178 μm, sections: 1 μm, scaling/voxel size: 0.64 × 0.64 × 2 μm, 3D volume analysis: ~15% total liver volume
Comparative hepatobiliary volumetric studies.
Volumetric assessment of α-napthylisothiocyanate (ANIT) exposed livers
30 dpf 2.5 μM ANIT, 48 hpd
Intrahepatic Biliary Passageways
Stack size: 230 × 230 × 89 μm, 1 μm sections, scaling/voxel size 0.45 × 045 × 1 μm, 3D volume analysis ~30% total liver volume
30 dpf 1 μm ANIT, 48 hpd
Volume μm 3
SA μm 2
Intrahepatic Biliary Passageways
Stack size: 325 × 325 × 89 μm, 1 μm section, scaling/voxel size 0.64 × 0.64 × 1 μm. ~30% total liver volume
Mean: 8 – 30 dpf
2.5 μM ANIT 30 dpf
1 μM ANIT 30 dpf
CTRL vs. Treated
Intrahepatic Biliary Passageways
In vivo investigation of xenobiotic response
With normalcy characterized (in vivo phenotypes of cells/tissues, hepatobiliary development, 3D parenchymal architecture, evaluations of blood to bile transport), it was then possible to apply this experimental system to the investigation of toxic response in vivo (see also ). To do so we used two reference hepatotoxicants; α-napthyl isothiocyanate (ANIT) and diethylnitrosamine (DEN). In vivo investigations (responses to xenobiotic exposure) were correlated with histopathology, ultrastructure and immunohistochemistry for validation/characterization of xenobiotic response/toxicity.
An example of non invasive in vivo serial analysis of the adult consequence of early life stage exposure to DEN is given in Figure 1. Shown is an in vivo assessment of neoplastic response 10 months post exposure to DEN. Histopathology revealed the tumor to be comprised of mixed neoplasms of hepatocellular and biliary origin, and with foci of biliary hyperplasia.
In vivo evaluation of responses of the liver to ANIT exposure revealed distinct dose dependent phenotypic changes, these included: (1) canalicular attenuation and dilation in response to 1 – 3 μM acute aqueous ANIT exposure; (2) bile preductular lesions in response to 2 – 5 μM chronic ANIT exposure; (3) hydropic vacuolation, at ANIT concentrations of 2 – 8 μM ANIT, which resulted in a distinct "pebbling" of the liver when evaluated in vivo; and (4) chronic passive hepatic congestion, an end stage response of the liver associated with high mortality, at 6 – 8 μM ANIT (see Figures 7, 8 and 9). In vivo observations were correlated with ex vivo histological and electron microscopic studies to aid in interpretation of in vivo findings and to verify affected cell types. Lastly, volumetric analyses of 3D reconstructions from ANIT treated medaka suggested a possible reduction in bile flow, as well as choleresis, with no changes to other volumetric liver indices (Tables 4, 5 and 7). In vivo 3D morphometric and volumetric indices were consistent with both in vivo (2D) and ex vivo findings (ultrastructural studies), revealing accuracy and standardization of quantitative assessments across in vivo and ex vivo techniques. These findings, while largely descriptive, suggest ANIT induced changes in the medaka hepatobiliary system are: (1) similar to ANIT induced changes described in rat liver, and (2) consistent with responses commonly observed in cholestasis in mammalian livers.
We have described the development and application of non invasive in vivo methodologies to the study of biological structure, function and xenobiotic response in STII medaka. The development of this in vivo investigatory "system" encompassed the validation and application of exogenous fluorescent probes, and endogenous fluorescence, for the in vivo study of biological structure, function, and xenobiotic response, in STII medaka, in both 2D and 3D contexts. With confocal microscopy, high resolution (< 1 μm) in vivo imaging was achieved (e.g., subcellular).
Of particular interest is the ability to investigate biological structure/function relationships, and xenobiotic response, in 3D, and to potentially integrate molecular mechanisms of toxicity with system level phenotypic changes, in a 3D context. Significant advances have been achieved over the last decade in 3D elucidation of structure/function relationships at the molecular/protein levels of biological organization. However, similar information on organ system structure and function at the cellular level has lagged. Elucidation of biological structure/function relationships in a real world 3D context is vital to advance our interpretive and diagnostic capabilities (e.g., normalcy and disease/toxicity), further our comparative understanding of organ system ontogeny, and integrate genetic and molecular information with system levels of biological organization. The latter is of particular importance. One of the main challenges facing life sciences is the integration and interpretation of genomic, proteomic, and metabolomic information in relation to the complex physiological system in which "omic" mechanisms operate. As these findings suggest, STII medaka provide a unique means by which to potentially integrate mechanisms of toxicity (e.g., genomic, proteomic function) with system level responses and phenotypic changes, in vivo (the overarching goal of our laboratory).
The findings presented also demonstrate in vivo quantitation of hepatobiliary transport is possible, suggesting STII medaka provide a novel means by which to investigate piscine hepatobiliary transport, and the effects of toxins/toxicants, as well as genetic disorders, on biliary transport mechanisms. Because hepatobiliary transport may be impaired by a variety of pharmaceuticals and environmental contaminants, the ability to investigate altered organ system function in vivo is a valuable tool that should prove valuable to the future study of piscine biliary transport.
Collectively these findings demonstrate the ability to study, with high resolution, normalcy and disease/toxicity in vivo in the hepatobiliary system of living medaka, a capability that provides a valuable diagnostic and investigatory tool. The reviewed findings presented here, in conjunction with earlier studies [11, 16, 77–85], have, we feel, significantly advanced our comparative understanding of the piscine liver, and we consider the potential for discovery, within the context of in vivo investigation in STII medaka, as significant.
Thanks to Dr. David Miller, Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, Research Triangle Park, for providing access to their laser scanning confocal microscopy facility, and special thanks to the Duke University Integrated Toxicology Program. This publication was made possible by Grant Number 1 RO1 RR018583-02 from the National Center for Research Resources (NCRR), a component of the National Institutes of Health (NIH). Its contents are solely the responsibility of the authors and do not necessarily represent the official views of NCRR or NIH.
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