SWIP—a stabilized window for intravital imaging of the murine pancreas

Pancreatitis and pancreatic ductal adenocarcinoma (PDAC) are grave illnesses with high levels of morbidity and mortality. Intravital imaging (IVI) is a powerful technique for visualizing physiological processes in both health and disease. However, the application of IVI to the murine pancreas presents significant challenges, as it is a deep, compliant, visceral organ that is difficult to access, easily damaged and susceptible to motion artefacts. Existing imaging windows for stabilizing the pancreas during IVI have unfortunately shown poor stability for time-lapsed imaging on the minutes to hours scale, or are unable to accommodate both the healthy and tumour-bearing pancreata. To address these issues, we developed an improved stabilized window for intravital imaging of the pancreas (SWIP), which can be applied to not only the healthy pancreas but also to solid tumours like PDAC. Here, we validate the SWIP and use it to visualize a variety of processes for the first time, including (1) single-cell dynamics within the healthy pancreas, (2) transformation from healthy pancreas to acute pancreatitis induced by cerulein, and (3) the physiology of PDAC in both autochthonous and orthotopically injected models. SWIP can not only improve the imaging stability but also expand the application of IVI in both benign and malignant pancreas diseases.     

3D reconstruction of dental specimens from 2D histological images and microCT-scans

Direct comparison of experimental and theoretical results in biomechanical studies requires a careful reconstruction of specimen surfaces to achieve a satisfactory congruence for validation. In this paper a semi-automatic approach is described to reconstruct triangular boundary representations from images originating from, either histological sections or microCT-, CT- or MRI-data, respectively. In a user-guided first step, planar 2D contours were extracted for every material of interest, using image segmentation techniques. In a second step, standard 2D triangulation algorithms were used to derive high quality mesh representations of the underlying surfaces. This was accomplished by converting the 2D meshes into 3D meshes by a novel lifting procedure. The meshes can be imported as is into finite element programme packages such as Marc/Mentat or COSMOS/M. Accuracy and feasibility of the algorithm is demonstrated by reconstructing several specimens as examples and comparing simulated results with available measurements performed on the original objects.     

Some characteristics of the histological structure of the fetal bovine pancreas

Some properties of histological structure of fetal bovine pancreas were demonstrated using light microscopic methods. The different forms of acino-insular complexes were described: 1) acino-insular complexes with single B-cells including epithelial layer of acini; 2) acino-insular complexes with segmental (sector) localization of insular cell groups; 3) acino-insular complexes with small and more large groups of endocrine cells timely contacted with acini; 4) acino-insular complexes at the stage of separation of endocrine cell groups (microislets) from acini. The consideration of acino-insular complexes in morphogenesis of bovine endocrine pancreas in discussed.     

Accuracy of computer-aided geometric 3D reconstruction based on histological serial microgrinding preparation

Purpose: For our research on computer-optimised and automated cochlear implant surgery, we pursue a model-based approach to overcome the limitations of currently available clinical imaging modalities. A serial cross section preparation procedure has been developed and evaluated concerning accuracy to serve for modelling of a digital anatomic atlas to make delicate soft tissue structures available for pre-operative planning.

Methods: A special grinding tool was developed allowing the setting of a specific amount of abrasion as equidistant slice thickness was considered a crucial step. Additionally, each actual abrasion was accurately measured and used during three-dimensional reconstruction of the serial cross-sectional images obtained via digital photo documentation after each microgrinding step. A well-known reference object was prepared using this procedure and evaluated in terms of accuracy.

Results: Reconstruction of the whole sample was achieved with an error less than 0.4%, and the edge lengths in the direction of abrasion could be reconstructed with an average error of 0.6 ± 0.3 mm; both prove the realisation of equidistant abrasion. Using artificial registration fiducials and a custom-made algorithm for image alignment, parallelism and rectangularity could be preserved with average errors less than 0.4° ± 0.3°.

Conclusion: We present a systematic, practicable and reliable method for the geometrically accurate reconstruction of anatomical structures, which is especially suitable for the middle and inner ear anatomy including soft tissue structures. For the first time, the quality of such a reconstruction process has been quantified and successfully proven for its usability.     

A leukemia virus-related protein in the murine pancreas

A protein that has been detected in the granules of islet cells in the murine pancreas is similar but not identical to the endogenous murine leukemia virus envelope protein gp70. The pancreatic protein was detected by several immunological methods using both polyclonal and monoclonal anti-murine gp70. On purification by affinity chromatography, it was shown to be different from murine gp70 in its subcellular location and its molecular size and the size of its precursor and by the effect of various reagents on its immunological activity as determined by the ELISA assay.     

Enucleation and reconstruction of murine L-cell fibroblasts

Two types of teflon chambers are suggested for the procedures of enucleation and reconstruction of cells cultivated in the monolayer. The first chamber simplifies the enucleation and permits performing it on the standard cover glasses with a small volume of medium with cytochalasin B. The second chamber permits purifying the karyoplast fraction from intact cells by their adhesion on the cover glasses. Then the karyoplasts are sedimented by centrifugation on cytoplasts located on the cover glasses. This procedure promotes sticking of fragments and their subsequent fusion in the presence of polyethylene glycol. In total the number of reconstructed cells increases many times.     

On the mechanism of ribonuclease secretion in the murine exocrine pancreas

Summary In the rat pancreas, normal diurnal and pilocarpine-induced variations in the protein, ribonuclease (RNase), and water content were studied. The results show a considerable variation in the dirunal content of RNase, whereas the protein and water concentrations remained constant. Furthermore, the experiments provide evidence that RNase is secreted by means of a mechanism partly independent of other proteins and that every zymogen granule does not contain the same amount of the enzyme. The earlier presumption that there are at least two intracellular storage forms of the enzyme is confirmed and the intracellular variability in the capacity to attract anti-RNase antibodies is explained.     

3-Dimensional organization of the N-terminal vinculin head fragment

The cytoskeleton-associated protein vinculin is composed of a globular head and an elongated tail domain. The protein can be cleaved by V8 protease treatment into two fragments with apparent molecular masses of 90 and 29/27 kDa, respectively. So far, no high-resolution data on the tertiary structure of the N-terminal 90-kDa fragment are available. We analyzed the 90-kDa fragment in detail, using electron spectroscopic imaging in conjunction with modelling experiments. The front view projection of this fragment appears roughly rhomboidal, with 4 intensity maxima arranged at the vertices and a stain-filled region in the center. Based on a detailed examination of different particle projections, a 3-dimensional model was constructed which appears as a flattened tetrahedron. A comparison of the 90-kDa fragment with the intact protein allows for a correlation between the subdomain organization of the vinculin head and the biochemically defined V8 protease cleavage sites (aa 851 and 857).     

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis ¹. In some cancers (e.g. glioma ²), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality ³. It has been estimated that invasion and metastasis are responsible for 90% of cancer deaths ⁴. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment ⁵.A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling ⁶. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes ⁷. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer ⁸.We present a refined method ⁹ for examining the contributions of candidate proteins to collective invasion ¹⁰. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.     

Quantitative 3-dimensional imaging of murine neointimal and atherosclerotic lesions by optical projection tomography

Objective

Traditional methods for the analysis of vascular lesion formation are labour intensive to perform - restricting study to ‘snapshots’ within each vessel. This study was undertaken to determine the suitability of optical projection tomographic (OPT) imaging for the 3-dimensional representation and quantification of intimal lesions in mouse arteries.

Methods and Results

Vascular injury was induced by wire-insertion or ligation of the mouse femoral artery or administration of an atherogenic diet to apoE-deficient mice. Lesion formation was examined by OPT imaging of autofluorescent emission. Lesions could be clearly identified and distinguished from the underlying vascular wall. Planimetric measurements of lesion area correlated well with those made from histological sections subsequently produced from the same vessels (wire-injury: R² = 0.92; ligation-injury: R² = 0.89; atherosclerosis: R² = 0.85), confirming both the accuracy of this methodology and its non-destructive nature. It was also possible to record volumetric measurements of lesion and lumen and these were highly reproducible between scans (coefficient of variation = 5.36%, 11.39% and 4.79% for wire- and ligation-injury and atherosclerosis, respectively).

Conclusions

These data demonstrate the eminent suitability of OPT for imaging of atherosclerotic and neointimal lesion formation, providing a much needed means for the routine 3-dimensional analysis of vascular morphology in studies of this type.     

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.     

Techniques for histological reconstruction of brain stem recording sites.

The masseter muscle of Sprague-Dawley rats was injected with 10 mg of horseradish peroxidase. The electrical activity of the trigeminal motor nucleus was investigated 24-36 hours later using micropipettes filled with a 2.0 M NaCl-7% fast green FCF solution. A parallel series of penetrations at 200 micron intervals was made through the motor complex at known depths. The grid formed from these penetrations was reconstructed using the following techniques. Marks were made at known depths in one or more of the tracts by iontophoresing fast green from the microelectrode tip with a hyperpolarizing current of 10 muA for 10 minutes. To assure proper alignment of the tissue containing the green marks, two agar-India ink plugs were placed caudal and parallel to the recording sites. The frozen tissue was sectioned on a microtome, first through the agar plugs and then through the tissue containing the green marks. The tissue could be incubated for the peroxidase reaction product or stained for Nissl substance. These combined procedures offer a means to correlate the structure and function of brain stem nuclear groups.     

Dynamics of embryonic pancreas development using real-time imaging

Current knowledge about developmental processes in complex organisms has relied almost exclusively on analyses of fixed specimens. However, organ growth is highly dynamic, and visualization of such dynamic processes, e.g., real-time tracking of cell movement and tissue morphogenesis, is becoming increasingly important. Here, we use live imaging to investigate expansion of the embryonic pancreatic epithelium in mouse. Using time-lapse imaging of tissue explants in culture, fluorescently labeled pancreatic epithelium was found to undergo significant expansion accompanied by branching. Quantification of the real-time imaging data revealed lateral branching as the predominant mode of morphogenesis during epithelial expansion. Live imaging also allowed documentation of dynamic beta-cell formation and migration. During in vitro growth, appearance of newly formed beta-cells was visualized using pancreatic explants from MIP-GFP transgenic animals. Migration and clustering of beta-cells were recorded for the first time using live imaging. Total beta-cell mass and concordant aggregation increased during the time of imaging, demonstrating that cells were clustering to form "pre-islets". Finally, inhibition of Hedgehog signaling in explant cultures led to a dramatic increase in total beta-cell mass, demonstrating application of the system in investigating roles of critical embryonic signaling pathways in pancreas development including beta-cell expansion. Thus, pancreas growth in vitro can be documented by live imaging, allowing visualization of the developing pancreas in real-time.     

Chronobiological serial sections for core temperature monitoring after transplantation of the murine pancreas

In order to study any early sign of rejection of pancreas transplantation, rhythmometry was carried out on female adult inbred Lewis rats. Animals previously kept in continuous light, more or less synchronized in frequency by cyclic human activities, were transferred to a regimen of light (L) and darkness (D) alternating at 12-h intervals in single cages at the room temperature of 24 +/- 1 degrees C, with food and water ad libitum. At this time under ether anesthesia, temperature transensors were implanted in healthy rats and rats rendered diabetic by the administration of streptozotocin. Some of the diabetic rats were left untreated; casual blood sampling showed gross hyperglycemia. Other rats were treated by pancreatic grafts from ethionine-prepared donors, either by isografts (in rats of the Lewis strain) or by allografts (of the pancreas from inbred Fischer rats transplanted to Lewis rats). Intraperitoneal temperature was telemetered at 10-min intervals for 3 weeks following transplantation. Urine volumes were determined from rats housed in metabolic cages. Data were analyzed rhythmometrically. Chronobiological serial sections and single cosinors served this purpose. Following sensor implantation and transfer to an LD 12:12 regimen, the adjustment of the thermal acrophase consistently near the middle of the daily dark span occurred within approximately 7 days in healthy rats and in streptozotocin-diabetic rats cured by isograft. Thermal acrophase adjustment was slower for animals rendered diabetic by streptozotocin and left untreated or for animals thus rendered diabetic which had rejected the pancreatic allograft (as documented by hyperglycemia in casually sampled blood). The eventual synchronization of the circadian temperature rhythm of allografted rats differed from one rat to the other and, for some allografted animals, from the consistent synchronization of the circadian rhythm in telemetered intraperitoneal temperature of diabetic and non-diabetic Lewis rats. The acrophase of the circadian rhythm in urine volume of healthy rats or of a rat with a pancreatic isograft (which cured a prior streptozotocin-induced diabetes) differed with statistical significance from those of rats with untreated diabetes, some in this state after the rejection of a pancreatic allograft. Both urine volume and core temperature are ready marker rhythms, not only for rats but also for human beings. Both variables can be self-monitored by the cooperation of instructed but not necessarily extensively educated patients. Temperature, in particular, can also be monitored with automatic devices and alterations of certain of its rhythm characteristics may signal changes preceding fever. The use of such admittedly unspecific yet eminently practical and possibly informative marker rhythmometry awaits clinical testing.     

Embryogenesis of the murine endocrine pancreas; early expression of pancreatic polypeptide gene.

By immunofluorescence on cytospin preparations and on semithin sections of mouse pancreatic buds, we have found glucagon and pancreatic polypeptide (PP)-containing cells at embryonal day 10.5 (E 10.5) in dorsal buds and at E 11.5 in ventral buds. Insulin-containing cells appear in dorsal buds at E 11.5, and one to two days later in ventral buds. Somatostatin-containing cells are detectable from E 13.5 in both dorsal and ventral buds. A quantitative analysis shows that up to E 15.5, PP-containing cells are relatively abundant in both buds. By PCR amplification of oligo(dT)-primed cDNAs prepared from total pancreatic RNA, we also detect PP mRNA from E 10.5 onwards, thus confirming the early expression of the PP gene in the developing mouse pancreas. Analysis of endocrine cells in situ suggests three major patterns of cell distribution in embryonic pancreas. First, individual hormone-containing cells are located within the epithelium of pancreatic ducts. In both dorsal and ventral buds, the majority of these endocrine cells contain PP, but many also contain glucagon, insulin or somatostatin. Secondly, clusters of endocrine cells are found in the pancreatic interstitium. Many of these cells contain both glucagon and PP which, by immunogold labelling of consecutive thin sections, can be shown to co-exist within individual secretory granules. Finally, starting on E 18.5, typical islets are formed with centrally located B cells and with the adult 'one cell-one hormone' phenotype. These results suggest an intriguing ontogenic relationship between A- and PP-cells, and also indicate that PP-containing cells may occupy a hitherto unexpected place in the lineage of endocrine islet cells.