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The liver has a unique regenerative capability upon injury or partial resection. The regeneration process comprises a complex interplay between parenchymal and non-parenchymal cells and is tightly regulated at different scales. Thus, we investigated liver regeneration using multi-scale methods by combining non-invasive imaging with immunohistochemical analyses. In this context, non-invasive imaging can provide quantitative data of processes involved in liver regeneration at organ and body scale. We quantitatively measured liver volume recovery after 70% partial hepatectomy (PHx) by micro computed tomography (µCT) and investigated changes in the density of CD68+ macrophages by fluorescence-mediated tomography (FMT) combined with µCT using a newly developed near-infrared fluorescent probe. In addition, angiogenesis and tissue-resident macrophages were analyzed by immunohistochemistry. Based on the results, a model describing liver regeneration and the interactions between different cell types was established. In vivo analysis of liver volume regeneration over 21 days after PHx by µCT imaging demonstrated that the liver volume rapidly increased after PHx reaching a maximum at day 14 and normalizing until day 21. An increase in CD68+ macrophage density in the liver was detected from day 4 to day 8 by combined FMT-µCT imaging, followed by a decline towards control levels between day 14 and day 21. Immunohistochemistry revealed the highest angiogenic activity at day 4 after PHx that continuously declined thereafter, whereas the density of tissue-resident CD169+ macrophages was not altered. The simulated time courses for volume recovery, angiogenesis and macrophage density reflect the experimental data describing liver regeneration after PHx at organ and tissue scale. In this context, our study highlights the importance of non-invasive imaging for acquiring quantitative organ scale data that enable modeling of liver regeneration.

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Vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR)-targeted therapies predominantly affect nascent, immature tumor vessels. Since platelet-derived growth factor receptor (PDGFR) blockade inhibits vessel maturation and thus increases the amount of immature tumor vessels, we evaluated whether the combined PDGFR inhibition by nilotinib and VEGFR2 blockade by DC101 has synergistic therapy effects in a desmoplastic breast cancer xenograft model. In this context, besides immunohistological evaluation, molecular ultrasound imaging with BR55, the clinically used VEGFR2-targeted microbubbles, was applied to monitor VEGFR2-positive vessels noninvasively and to assess the therapy effects on tumor angiogenesis. DC101 treatment alone inhibited tumor angiogenesis, resulting in lower tumor growth and in significantly lower vessel density than in the control group after 14 days of therapy. In contrast, nilotinib inhibited vessel maturation but enhanced VEGFR2 expression, leading to markedly increased tumor volumes and a significantly higher vessel density. The combination of both drugs led to an almost similar tumor growth as in the DC101 treatment group, but VEGFR2 expression and microvessel density were higher and comparable to the controls. Further analyses revealed significantly higher levels of tumor cell-derived VEGF in nilotinib-treated tumors. In line with this, nilotinib, especially in low doses, induced an upregulation of VEGF and IL-6 mRNA in the tumor cells in vitro, thus providing an explanation for the enhanced angiogenesis observed in nilotinib-treated tumors in vivo. These findings suggest that nilotinib inhibits vessel maturation but counteracts the effects of antiangiogenic co-therapy by enhancing VEGF expression by the tumor cells and stimulating tumor angiogenesis.

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A modified silicone injection procedure was used for visualization of the hepatic vascular tree. This procedure consisted of in-vivo injection of the silicone compound, via a 26 G catheter, into the portal or hepatic vein. After silicone injection, organs were explanted and prepared for ex-vivo micro-CT (µCT) scanning. The silicone injection procedure is technically challenging. Achieving a successful outcome requires extensive microsurgical experience from the surgeon. One of the challenges of this procedure involves determining the adequate perfusion rate for the silicone compound. The perfusion rate for the silicone compound needs to be defined based on the hemodynamic of the vascular system of interest. Inappropriate perfusion rate can lead to an incomplete perfusion, artificial dilation and rupturing of vascular trees. The 3D reconstruction of the vascular system was based on CT scans and was achieved using preclinical software such as HepaVision. The quality of the reconstructed vascular tree was directly related to the quality of silicone perfusion. Subsequently computed vascular parameters indicative of vascular growth, such as total vascular volume, were calculated based on the vascular reconstructions. Contrasting the vascular tree with silicone allowed for subsequent histological work-up of the specimen after µCT scanning. The specimen can be subjected to serial sectioning, histological analysis and whole slide scanning, and thereafter to 3D reconstruction of the vascular trees based on histological images. This is the prerequisite for the detection of molecular events and their distribution with respect to the vascular tree. This modified silicone injection procedure can also be used to visualize and reconstruct the vascular systems of other organs. This technique has the potential to be extensively applied to studies concerning vascular anatomy and growth in various animal and disease models.

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BACKGROUND: Liver regeneration consists of cellular proliferation leading to parenchymal and vascular growth. This study complements previous studies on cellular proliferation and weight recovery by (1) quantitatively describing parenchymal and vascular regeneration, and (2) determining their relationship. Both together are needed to (3) characterize the underlying growth pattern. METHODS: Specimens were created by injecting a polymerizing contrast agent in either portal or hepatic vein in normal or regenerating livers after 70% partial hepatectomy. 3D image data were obtained through micro-CT scanning. Parenchymal growth was assessed by determining weight and volume of the regenerating liver. Vascular growth was described by manually determined circumscribed parameters (maximal vessel length and radius of right inferior portal/hepatic vein), automatically determined cumulative parameters (total edge length and total vascular volume), and parameters describing vascular density (total edge length/volume, vascular volume fraction). The growth pattern was explored by comparing the relative increase of these parameters to the increase expected in case of isotropic expansion. RESULTS: Liver volume recovery paralleled weight recovery and reached 90% of the original liver volume within 7 days. Comparing radius-related vascular parameters immediately after surgical resection and after virtual resection in-silico revealed a slight increase, possibly reflecting the effect of resection-induced portal hyperperfusion. Comparing length-related parameters between post-operative day 7 and after virtual resection showed similar vascular growth in both vascular systems investigated. In contrast, radius-related parameters increased slightly more in the portal vein. Despite the seemingly homogeneous 3D growth, the observed vascular parameters were not compatible with the hypothesis of isotropic expansion of liver parenchyma and vascular structures. CONCLUSION: We present an approach for the quantitative analysis of the vascular systems of regenerating mouse livers. We applied this technique for assessing the hepatic growth pattern. Prospectively, this approach can be used to investigate hepatic vascular regeneration under different conditions.

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BACKGROUND/AIM: Liver size regulation is based on the balance between hepatic regeneration and atrophy. To achieve a better understanding of intrahepatic size regulation, we explored the size regulation of a portally deprived liver lobe on a liver subjected to concurrent portal vein ligation (PVL) and partial hepatectomy (PHx). MATERIALS AND METHODS: Using a surgical rat model consisting of right PVL (rPVL) plus 70% PHx, we evaluated the size regulation of liver lobes 1, 2, 3, and 7 days after the operation in terms of liver weight and hepatocyte proliferation. Portal hyperperfusion was confirmed by measuring portal flow. The portal vascular tree was visualized by injection of a contrast agent followed by CT imaging of explanted livers. Control groups consisted of 70% PHx, rPVL, and sham operation. RESULTS: The size of the ligated right lobe increased to 1.4-fold on postoperative day 7 when subjected to rPVL + 70% PHx. The right lobe increased to 3-fold when subjected to 70% PHx alone and decreased to 0.3-fold when subjected to rPVL only. The small but significant increase in liver weight after the combined procedure was accompanied by a low proliferative response. In contrast, hepatocyte proliferation was undetectable in the right lobe undergoing atrophy after PVL only. The caudate lobe in the rPVL + 70% PHx group increased to 4.6-fold, which is significantly more than in the other groups. This increase in liver weight was paralleled by persisting portal hyperperfusion and a prolonged proliferative phase of 3 days. CONCLUSIONS: A discontinued portal blood supply does not always result in atrophy of the ligated lobe. The concurrent regenerative stimulus induced by 70% PHx seemed to counteract the local atrophy after a simultaneously performed rPVL, leading to a low but prolonged regenerative response of the portally deprived liver lobe. This observation supports the conclusion that portal flow is not necessary for liver regeneration. The persisting portal hyperperfusion may be crucial for the specific kinetics of prolonged liver regeneration after rPVL + 70% PHx in the portally supplied caudate lobe. Both observations deserve more attention regarding the underlying mechanism in further studies.

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BACKGROUND: We adapted the anatomically oriented parenchyma-preserving resection technique for associating liver partition with portal vein ligation (PVL) for staged hepatectomy (ALPPS) in rats and examined the role of revascularization in intrahepatic size regulation. METHODS: We performed the procedures based on anatomic study. The ALPPS procedure consisted of a 70% PVL (occluding the left median, left lateral, and right lobes), parenchymal transection (median lobe) and partial (10%) hepatectomy (PHx; caudate lobe). The transection effect was evaluated by measuring the extent of hepatic atrophy or regeneration of individual liver lobes in the ALPPS and control groups (70% PVL and 10% PHx without transection). The survival rates after stage II resection and collateral formation within the portal vein system was examined. RESULTS: Anatomic study revealed a close spatial relationship between the demarcation line and the middle median hepatic vein. This enabled placing the transection plane without injuring the hepatic vein. Transection was achieved via stepwise clamping, followed by 2-3 parenchyma-preserving piercing sutures on both sides of the clamp. Ligated liver lobes atrophy was significantly enhanced after ALPPS compared with the control group. In contrast, both a significantly greater relative weight of the regenerated lobe and proliferation index on the first postoperative day were observed. All animals tolerated stage II-resection without complications. Portoportal collaterals were only observed in the control group. CONCLUSION: We developed an anatomically precise technique for parenchymal transection. The lack of a dense vascular network between the portalized and deportalized lobes may play an important role in accelerating regeneration and atrophy augmentation.

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INTRODUCTION: The intra-hepatic vascular anatomy in rodents, its variations and corresponding supplying and draining territories in respect to the lobar structure of the liver have not been described. We performed a detailed anatomical imaging study in rats and mice to allow for further refinement of experimental surgical approaches. METHODS: LEWIS-Rats and C57Bl/6N-Mice were subjected to ex-vivo imaging using µCT. The image data were used for semi-automated segmentation to extract the hepatic vascular tree as prerequisite for 3D visualization. The underlying vascular anatomy was reconstructed, analysed and used for determining hepatic vascular territories. RESULTS: The four major liver lobes have their own lobar portal supply and hepatic drainage territories. In contrast, the paracaval liver is supplied by various small branches from right and caudate portal veins and drains directly into the vena cava. Variations in hepatic vascular anatomy were observed in terms of branching pattern and distance of branches to each other. The portal vein anatomy is more variable than the hepatic vein anatomy. Surgically relevant variations were primarily observed in portal venous supply. CONCLUSIONS: For the first time the key variations of intrahepatic vascular anatomy in mice and rats and their surgical implications were described. We showed that lobar borders of the liver do not always match vascular territorial borders. These findings are of importance for the design of new surgical procedures and for understanding eventual complications following hepatic surgery.

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Fluorescence-mediated tomography (FMT) enables longitudinal and quantitative determination of the fluorescence distribution in vivo and can be used to assess the biodistribution of novel probes and to assess disease progression using established molecular probes or reporter genes. The combination with an anatomical modality, e.g., micro computed tomography (µCT), is beneficial for image analysis and for fluorescence reconstruction. We describe a protocol for multimodal µCT-FMT imaging including the image processing steps necessary to extract quantitative measurements. After preparing the mice and performing the imaging, the multimodal data sets are registered. Subsequently, an improved fluorescence reconstruction is performed, which takes into account the shape of the mouse. For quantitative analysis, organ segmentations are generated based on the anatomical data using our interactive segmentation tool. Finally, the biodistribution curves are generated using a batch-processing feature. We show the applicability of the method by assessing the biodistribution of a well-known probe that binds to bones and joints.

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A sacrificial templating process using lithographically printed minimal surface structures allows complex de novo geo-metries of delicate hydrogel materials. The hydrogel scaffolds based on cellulose and chitin nanofibrils show differences in terms of attachment of human mesenchymal stem cells, and allow their differentiation into osteogenic outcomes. The approach here serves as a first example toward designer hydrogel scaffolds viable for biomimetic tissue engineering.

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The liver has the unique capability of regeneration from various injuries. Different animal models and in vitro methods are used for studying the processes and mechanisms of liver regeneration. Animal models were established either by administration of hepatotoxic chemicals or by surgical approach. The administration of hepatotoxic chemicals results in the death of liver cells and in subsequent hepatic regeneration and tissue repair. Surgery includes partial hepatectomy and portal vein occlusion or diversion: hepatectomy leads to compensatory regeneration of the remnant liver lobe, whereas portal vein occlusion leads to atrophy of the ipsilateral lobe and to compensatory regeneration of the contralateral lobe. Adaptation of modern radiological imaging technologies to the small size of rodents made the visualization of rodent intrahepatic vascular anatomy possible. Advanced knowledge of the detailed intrahepatic 3D anatomy enabled the establishment of refined surgical techniques. The same technology allows the visualization of hepatic vascular regeneration. The development of modern histological image analysis tools improved the quantitative assessment of hepatic regeneration. Novel image analysis tools enable us to quantify reliably and reproducibly the proliferative rate of hepatocytes using whole-slide scans, thus reducing the sampling error. In this review, the refined rodent models and the newly developed imaging technology to study liver regeneration are summarized. This summary helps to integrate the current knowledge of liver regeneration and promises an enormous increase in hepatological knowledge in the near future.

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