In vivo imaging of malaria parasites in the murine liver

The form of the malaria parasite inoculated by the mosquito, called the sporozoite, transforms inside the host liver into thousands of a new form of the parasite, called the merozoite, which infects erythrocytes. We present here a protocol to visualize in vivo the behavior of Plasmodium berghei parasites in the hepatic tissue of the murine host. The use of GFP-expressing parasites and a high-speed spinning disk confocal microscope allows for the acquisition of four-dimensional images, which provide a time lapse view of parasite displacement and development in tissue volumes. These data can be analyzed to give information on the early events of sporozoite penetration of the hepatic tissue, that is, sporozoite gliding in the liver sinusoids, crossing the sinusoidal barrier, gliding in the parenchyma and traversal of hepatocytes, and invasion of a final hepatocyte, as well as the terminal events of merosome and merozoite release from infected hepatocytes. Combined with the use of mice expressing fluorescent cell types or cell markers, the system will provide useful information not only on the primary infection process, but also on parasite interactions with the host immune cells in the liver.     

Microcirculation in the murine liver: a computational fluid dynamic model based on 3D reconstruction from in vivo microscopy

The liver is organized in hexagonal functional units – termed lobules – characterized by a rather peculiar blood microcirculation, due to the presence of a tangled network of capillaries – termed sinusoids. A better understanding of the hemodynamics that governs liver microcirculation is relevant to clinical and biological studies aimed at improving our management of liver diseases and transplantation.Herein, we built a CFD model of a 3D sinusoidal network, based on in vivo images of a physiological mouse liver obtained with a 2-photon microscope. The CFD model was developed with Fluent 16.0 (ANSYS Inc., Canonsburg, PA), particular care was taken in imposing the correct boundary conditions representing a physiological state. To account for the remaining branches of the sinusoids, a lumped parameter model was used to prescribe the correct pressure at each outlet. The effect of an adhered cell on local hemodynamics is also investigated for different occlusion degrees.The model here proposed accurately reproduces the fluid dynamics in a portion of the sinusoidal network in mouse liver. Mean velocities and mass flow rates are in agreement with literature values from in vivo measurements. Our approach provides details on local phenomena, hardly described by other computational studies, either focused on the macroscopic hepatic vasculature or based on homogeneous porous medium model.     

Three-dimensional magnetic resonance imaging for the study of ovarian function in a bovine in vitro model

Three-dimensional magnetic resonance imaging coupled with maximum intensity projection display, a technique usually reserved for magnetic resonance imaging angiography, is useful for the study of ovarian follicular growth. The ovaries of 19 cows were examined each day by transrectal ultrasonography. From these data, the precise phase of the ovarian cycle was determined and cows were ovariectomized on day 3 of wave one (n = 5), on day 6 of wave one (n = 4), on day 1 of wave two (n = 4), >/= 17 days after ovulation (n = 5), and on the day of ovulation (n = 1). The excised ovaries were examined by magnetic resonance imaging using a fast imaging with steady state precession imaging sequence with maximum intensity projection reconstruction, displayed as a cine-loop of the ovaries rotating in space. This provided the clearest view among the three principal three-dimensional steady state data acquisition approaches tried; the follicles and other ovarian structures could be distinguished unambiguously. Results from the bovine model indicate that the acuity of the three-dimensional fast imaging with steady state precession technique has potential application in in vivo intravaginal imaging in women for studying normal and pathological ovarian function.     

Ex vivo imaging of motor axon dynamics in murine triangularis sterni explants

We provide a protocol that describes an explant system that allows the dynamics of motor axons to be imaged. This method is based on nerve-muscle explants prepared from the triangularis sterni muscle of mice, a thin muscle that covers the inside of the thorax. These explants, which can be maintained alive for several hours, contain long stretches of peripheral motor axons including their terminal arborizations and neuromuscular junctions. Explants can be prepared from transgenic mouse lines that express fluorescent proteins in neurons or glial cells, which enables direct visualization of their cellular and subcellular morphology by fluorescence microscopy. Time-lapse imaging then provides a convenient and reliable approach to follow the dynamic behavior of motor axons, their surrounding glial cells and their intracellular organelles with high temporal and spatial resolution. Triangularis sterni explants can be prepared in 15 min, imaged ex vivo for several hours and processed for immunohistochemistry in about 2 h.     

Functional in vivo imaging of cysteine cathepsin activity in murine model of inflammation

Near-infrared fluorophore (NIRF)-labeled imaging probes are becoming increasingly important in bio-molecular imaging applications, that is, in animal models for tumor imaging or inflammation studies. In this study we showed that the previously introduced chemical concept of ‘Reverse Design’ represents an efficient strategy for the generation of selective probes for cysteine proteases from chemically optimized protease inhibitors for investigations in proteomic lysates as well as for in vivo molecular imaging studies. The newly developed activity-based probe AW-091 was demonstrated to be highly selective for cathepsin S in vitro and proved useful in monitoring cysteine cathepsin activity in vivo, that is, in zymosan-induced mouse model of inflammation. AW-091 showed higher signal-to-background ratios at earlier time points than the commercially available polymer-based ProSense680 (VisEn Medical) and thus represents an efficient new tool for studying early proteolytic processes leading to various diseases, including inflammation, cancer, and rheumatoid arthritis. In addition, the fluorescent signal originating from the cleaved AW-091 was shown to be reduced by the administration of an anti-inflammatory drug, dexamethasone and by the cathepsin inhibitor E-64, providing a valuable system for the evaluation of small-molecule inhibitors of cathepsins.     

Antitumor effects of IL-6 on murine liver tumor cells in vivo

IL-6 is a pleiotropic cytokine that is capable of modulating the diverse functions of hepatocytes such as acute phase responses and inflammation in the liver. To learn its antitumor effects in vivo, the cDNA of IL-6 was transfected into murine liver cells, TIB cells. IL-6-transfected TIB cells (TIB73-IL-6 or TIB75-IL-6) produced much higher levels of IL-6 compared with vector-transfected TIB cells (TIB73-vec or TIB75-vec). To investigate the effects of IL-6 on TIB tumor growth in vivo, IL-6-transfected TIB cells or vector-transfected TIB cells were injected subcutaneously into syngeneic mice. Vector-transfected TIB cells grew rapidly 3 weeks after injection, but IL-6-transfected TIB cells did not grow at all for up to 6 weeks. Pathologically, IL-6-transfected TIB cells demonstrated a severe necrosis and apoptotic pattern. Taken together, these results indicate that IL-6 functions as a growth inhibiting factor in vivo, and another biological role of IL-6 in the liver is suggested.     

In vivo imaging of immunotoxin treatment using Katushka-transfected A-431 cells in a murine xenograft tumour model

Purpose

Preclinical in vivo analyses of treatment responses are an important prerequisite to evaluate new therapeutics. Molecular in vivo imaging in the far red (FR)/near infra red (NIR) is a promising method, as it enables measurements at different time points in individual animals, thereby reducing the number of animals required, while increasing statistical significance. Here, we show the establishment of a method to monitor response to treatment using fluorescent cells, expressing the epidermal growth factor receptor (EGFR), a target already used in therapy.

Methods

We transfected A-431 tumour cells with the far red–emitting protein Katushka (Kat2), resulting in strong fluorescence allowing for the monitoring of tumour growth when implanted in BALB/c nu/nu mice with a CRi Maestro in vivo imager. We targeted A-431 cells with a previously reported immunotoxin (IT), consisting of the anti-EGFR antibody single-chain variable fragment (scFv) 425, fused to Pseudomonas aeruginosa Exotoxin A’ (ETA’). In addition, EGFR expression was verified using the 425(scFv) conjugated to a NIR dye BG-747 through a SNAP-tag linker.

Results

The results show the feasibility to evaluate response to treatment in vivo by FR imaging, while at the same location detecting EGFR expression. Treatment with 425(scFv)-ETA’ resulted in decelerated tumour growth, while not affecting the overall health of the animals. This is in contrast to treatment with Doxorubicin, which, although decreasing the tumour size, resulted in poor health.

Conclusions

We developed a novel method to non-invasively determine treatment responses by in vivo imaging of multiple parameters which showed the efficacy of 425(scFv)-ETA’.     

Quantitative isolation and in vivo imaging of malaria parasite liver stages

The liver stages of Plasmodium, the causative agent of malaria, are the least explored forms in the parasite's life cycle despite their recognition as key vaccine and drug targets. In vivo experimental access to liver stages of human malaria parasites is practically prohibited and therefore rodent model malaria parasites have been used for in vivo studies. However, even in rodent models progress in the analysis of liver stages has been limited, mainly due to their low abundance and associated difficulties in visualisation and isolation. Here, we present green fluorescent protein (GFP)-tagged Plasmodium yoelii rodent malaria parasite liver infections in BALB/c mice as an excellent quantitative model for the live visualisation and isolation of the so far elusive liver stages. We believe P. yoelii GFP-tagged liver stages allow, for the first time, the efficient quantitative isolation of intact early and late liver stage-infected hepatocyte units by fluorescence activated cell sorting. GFP-tagged liver stages are also well suited for intravital imaging, allowing us for the first time to visualise them in real time. We identify previously unrecognised features of liver stages including vigorous parasite movement and expulsion of 'extrusomes'. Intravital imaging thus reveals new, important information on the malaria parasite's transition from tissue to blood stage.     

Intracranial implantation with subsequent 3D in vivo bioluminescent imaging of murine gliomas

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The cell line was originally induced by intracranial injection of 3-methyl-cholantrene into a C57BL/6 syngeneic mouse strain (1); therefore, immunologically competent C57BL/6 mice can be used. While we use GL261, the following protocol can be used for the implantation and monitoring of any intracranial mouse tumor model. GL261 cells were engineered to stably express firefly luciferase (GL261-luc). We also created the brighter GL261-luc2 cell line by stable transfection of the luc2 gene expressed from the CMV promoter. C57BL/6-cBrd/cBrd/Cr mice (albino variant of C57BL/6) from the National Cancer Institute, Frederick, MD were used to eliminate the light attenuation caused by black skin and fur. With the use of albino C57BL/6 mice; in vivo imaging using the IVIS Spectrum in vivo imaging system is possible from the day of implantation (Caliper Life Sciences, Hopkinton, MA). The GL261-luc and GL261-luc2 cell lines showed the same in vivo behavior as the parental GL261 cells. Some of the shared histological features present in human GBMs and this mouse model include: tumor necrosis, pseudopalisades, neovascularization, invasion, hypercellularity, and inflammation (1). Prior to implantation animals were anesthetized by an intraperitoneal injection of ketamine (50 mg/kg), xylazine (5 mg/kg) and buprenorphine (0.05 mg/kg), placed in a stereotactic apparatus and an incision was made with a scalpel over the cranial midline. A burrhole was made 0.1 mm posterior to the bregma and 2.3mm to the right of the midline. A needle was inserted to a depth of 3mm and withdrawn 0.4 mm to a depth of 2.6 mm. Two μl of GL261-luc or GL261-luc2 cells (10(7) cells/ml) were infused over the course of 3 minutes. The burrhole was closed with bonewax and the incision was sutured. Following stereotactic implantation the bioluminescent cells are detectable from the day of implantation and the tumor can be analyzed using the 3D image reconstruction feature of the IVIS Spectrum instrument. Animals receive a subcutaneous injection of 150 μg luciferin /kg body weight 20 min prior to imaging. Tumor burden is quantified using mean tumor bioluminescence over time. Tumor-bearing mice were observed daily to assess morbidity and were euthanized when one or more of the following symptoms are present: lethargy, failure to ambulate, hunched posture, failure to groom, anorexia resulting in >10% loss of weight. Tumors were evident in all of the animals on necropsy.     

In vivo micro-CT imaging of liver lesions in small animal models

Three-dimensional micro computed tomography (microCT) offers the opportunity to capture images liver structures and lesions in mice with a high spatial resolution. Non-invasive microCT allows for accurate calculation of vessel tortuosity and density, as well as liver lesion volume and distribution. Longitudinal monitoring of liver lesions is also possible. However, distinguishing liver lesions from variations within a normal liver is impossible by microCT without the use of liver- or tumor-specific contrast-enhancing agents. The combination of microCT for morphologic imaging with functional imaging, such as positron emission tomography (PET) or single photon emission tomography (SPECT), offers the opportunity for better abdominal imaging and assessment of structure discrepancies visible by functional imaging.This paper describes methods of current microCT imaging options for imaging of liver lesions compared to other imaging techniques in small animals.     

In vivo liver and lung targeting of adriamycin encapsulated in glutaraldehyde-treated murine erythrocytes

Treatment of adriamycin-loaded erythrocytes from B6D2F1 mice with 0.1% glutaraldehyde produced the following effects: a considerable decrease in the in vitro leakage of the unmodified drug and a selective liver (and, to a lesser extent, lung) uptake of the encapsulated drug (70% of the injected dose) compared to drug leakage from, and tissue distribution of, carrier erythrocytes not treated with glutaraldehyde. The liver vascular bed was not saturated by five daily intravenous injections of 20 microliters of glutaraldehyde-treated erythrocytes, which allows a total dosage of 200 micrograms of the drug (half the LD50 value) to be administered. No appreciable liver damage results from extensive and prolonged uptake of glutaraldehyde-treated carrier erythrocytes. Entrapment of adriamycin within erythrocytes along with glutaraldehyde treatment of the carrier cells seems to be a promising therapeutic strategy against liver (and lung) tumors.     

Micro-CT based experimental liver imaging using a nanoparticulate contrast agent: a longitudinal study in mice

Background

Micro-CT imaging of liver disease in mice relies on high soft tissue contrast to detect small lesions like liver metastases. Purpose of this study was to characterize the localization and time course of contrast enhancement of a nanoparticular alkaline earth metal-based contrast agent (VISCOVER ExiTron nano) developed for small animal liver CT imaging.

Methodology

ExiTron nano 6000 and ExiTron nano 12000, formulated for liver/spleen imaging and angiography, respectively, were intravenously injected in C57BL/6J-mice. The distribution and time course of contrast enhancement were analysed by repeated micro-CT up to 6 months. Finally, mice developing liver metastases after intrasplenic injection of colon carcinoma cells underwent longitudinal micro-CT imaging after a single injection of ExiTron nano.

Principal Findings

After a single injection of ExiTron nano the contrast of liver and spleen peaked after 4–8 hours, lasted up to several months and was tolerated well by all mice. In addition, strong contrast enhancement of abdominal and mediastinal lymph nodes and the adrenal glands was observed. Within the first two hours after injection, particularly ExiTron nano 12000 provided pronounced contrast for imaging of vascular structures. ExiTron nano facilitated detection of liver metastases and provided sufficient contrast for longitudinal observation of tumor development over weeks.

Conclusions

The nanoparticulate contrast agents ExiTron nano 6000 and 12000 provide strong contrast of the liver, spleen, lymph nodes and adrenal glands up to weeks, hereby allowing longitudinal monitoring of pathological processes of these organs in small animals, with ExiTron nano 12000 being particularly optimized for angiography due to its very high initial vessel contrast.     

Three-dimensional surface imaging from CT scans for the study of craniofacial dysmorphology

Computer-assisted medical imaging technologies provide new tools for the study of congenital craniofacial deformities. Three-dimensional surface reconstructions have been developed to simplify the interpretation and improve the utility of CT scans of the head. While 3-D reconstructions initially were applied to assist clinical management of craniofacial deformities, these images now are finding utility in the study of unique anomalies, the definition of group characteristics for dysmorphic heads, the differentiation of similar phenotypes, and the documentation of the effects of cranial surgery on craniofacial growth. These findings should assist the formulation and evaluation of hypotheses regarding mechanisms of congenital malformation and deformation.     

A micro-computed tomography study of the trabecular bone structure in the femoral head

The goal of this study was to characterize the trabecular microarchitecture of the femoral head using micro-computed tomography (ICT). Femoral head specimens were obtained from subjects following total hip replacement. Cylindrical cores from the specimens were scanned to obtain 3-D images with an isotropic resolution of 26 Im. Bone structural parameters were evaluated on a per millimeter basis: relative bone volume (BV/TV), trabecular number (Tb.N), thickness (Tb.Th) and separation (Tb.Sp), structure model index (SMI), and connectivity (Conn.D). The ICT data show that the first two millimeters, starting at the joint surface, are characterized by more plate-like trabeculae, and are significantly denser than the underlying trabecular bone. Regional differences in the trabecular architecture reveal that the superior pole has significantly higher BV/TV, Tb.N and Tb.Th values, with lower Tb.Sp compared to the inferior and side poles. Because subchondral bone is essential in the load attenuation of joints, the difference in bone structure between the subchondral and trabecular bone might arise from the different functions each have within joint-forming bones. The denser trabecular structure of the superior pole as compared to the inferior pole can be interpreted as a functional adaptation to higher loading in this area.