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Electro Groom Horse Vacuum Portable Vacuum CleanerIHC was improved with an optimized clearing technique ( CUBIC) that extended the imaging depth for confocal microscopy more than five‐fold. The VCC and micro‐ CT scanning protocol was improved by enabling dual casting, optimizing the contrast agent concentration, and adjusting the viscosity of the resin ( PU4ii). We have optimized both techniques and combined their complementary strengths to develop a framework for multilevel reconstruction of the hepatic circulation in the rat. Nevertheless, these techniques still face a number of challenges such as dual casting in VCC and limited imaging depths for IHC. Previously, both vascular corrosion casting ( VCC) and immunohistochemistry ( IHC) have been separately used to study the hepatic vasculature. Although current models are often idealized simplifications of the complex anatomical reality, correct morphological information is instrumental for scientific and clinical purposes. In contrast to other organs, it comprises two afferent vessels, namely the hepatic artery (HA) and the valveless portal vein (PV). It has a unique macro‐ and microvascular system, profoundly determining blood flow distribution and regulation. We believe that the presented framework will have value beyond studies of the liver, and will facilitate a better understanding of various parenchymal organs in general, in physiological and pathological circumstances.The liver is the largest internal organ of the human body (Mitra & Metcalf, 2012). Our methodological framework allows 3D reconstruction and quantification of the hepatic circulation, ranging from the major blood vessels down to the intertwined and interconnected sinusoids. Segmentation algorithms were typically optimized for the venous systems, as the hepatic artery required higher imaging resolutions (Soler et al. 2002) to modeling the impact of partial hepatectomy (Debbaut et al. 2015), the algorithmic generation of realistic hepatic vascular systems (Schwen & Preusser, 2012), preoperative liver surgery planning (Soler et al. This assumption is rather imprecise in the case of rats, whose portal vein has been shown to trifurcate (Martins & Neuhaus, 2007).At the microanatomical level, the prevailing theory concerning the hepatic substructure is the classic hexagonal lobule, established by Kiernan ( 1833). 2006 Schwen & Preusser, 2012). Furthermore, ramification patterns were usually assumed to consist merely of bifurcations (Op Den Buijs et al. Cirrhosis), it is also vital to segment the hepatic arterial network to gain more insight in the pathogenesis. However, in the case of impaired blood flow conditions (e.g. Liver cirrhosis).Previously, vascular corrosion casting (VCC) in combination with high‐resolution micro‐CT (μCT) scanning provided unique insight into the hepatic vasculature, covering both macro‐ and microcirculation. ( 2012a).More detailed 3D anatomical knowledge of the entire liver vasculature across different length scales is therefore essential to understand its function, its hemodynamics, and its role in various liver pathologies (e.g. Characteristic for all these models is that they represent a simplification of the true complexity of the three‐dimensional (3D) structure of the liver microcirculation as demonstrated by Debbaut et al. 2012c).In addition to the hexagonal lobule model, several other functional liver units have been published in the literature. However, this blood flow cascade remains a simplification of the complex reality (McCuskey, 2000 Kan & Madoff, 2008 Debbaut et al. Postbox for mac serial magnet torrentAllowing flow only from the HA to the PV and not vice versa. It was hypothesized that these arteriolo‐portal venular shunts (occurring between branches of approximately 50 μm diameter) function as a one‐way valve‐like mechanism, i.e. ( 2014) noted functional evidence of the existence of shunts between the HA and PV in rats in normal and various pathological conditions. ( 2012b) experienced technical difficulties while dual casting rat livers, and geometrical data on all vascular trees (HA, PV, and HV) could only be gathered by splitting the protocol over two rat livers (one to obtain the PV and HV systems, and one to retrieve the HA system). Current limitations of the casting technique include perfusion difficulties, especially for microcirculatory perfusion, and the reactivity of casting resins with other chemical compounds and surrounding tissue (Krucker et al. 2011, 2012b, 2014 Peeters et al. With the advent of chemical clearing technologies and advanced 3D immunohistochemistry, various methods emerged to overcome both limits (Richardson & Lichtman, 2015).As both VCC (with μCT scanning) and IHC (with confocal imaging) still face a number of challenges and more detailed morphological data on the liver vasculature is needed, the aim of this study was to optimize and combine the VCC and IHC protocols in a rat liver model. A bottleneck of conventional immunohistochemistry and confocal microscopy, however, is the limited penetration depth of antibodies and photons. 3D reconstructions of microvascular hepatic networks using IHC in mice have shown that anatomical geometries with a height up to 100 μm can be achieved (Hoehme et al. This method can also provide information about structures other than the microvascular network, such as connective tissue and the biliary network (Hammad et al. Confocal microscopy of immunostained liver tissue has proven to be applicable for imaging 3D geometries at the microlevel down to a resolution of 0.2 μm (White et al. Simultaneous casting of HA and PV) may be problematic when contrast‐based differentiation between the supplying blood vessels is desired. Combining the complementarity of both techniques enabled us to set up multilevel models, which allow the macro‐ as well as the microvasculature of the rat liver to be examined accurately in 3D. IHC methods, on the other hand, were tailored specifically to the rat vascular system and supplemented with a chemical clearing technology to increase the visualization depth. 2012b) included usage of another casting resin (PU4ii) and contrast agent (Lipiodol), adapting the casting sequence (sequentially), and increasing the reproducibility of the VCC protocol. Differences with previous work (Debbaut et al. After cannulation, the thoracic aorta (TA) and renal arteries (RA) were clamped to force the arterially injected resin to flow into the HA. Additional measures to support precise catheterization included ligatures, tissue glue and clamping of the PV proximal to the inserted catheter. The PV and abdominal aorta (AA) were cannulated with a 14‐G and a 22‐G catheter (Terumo, Leuven, Belgium), respectively. Heparin (0.3 mL 5000 U mL −1) (Heparine Leo, Leo Pharma, Lier, Belgium) was injected intrasplenically with a 26‐gauge (G) needle (Terumo, Leuven, Belgium) to prevent clotting. Next, the upper thorax and abdomen were shaved, and a combined midline thoracotomy and laparotomy was performed to expose the liver. ( 1994), to present and quantify the branching topology in a logical manner.Animals ( n = 2) were anesthetized by intraperitoneal injection of 130 μL 100 g −1 pentobarbital (Nembutal, Ceva Sante Animale, Brussels, Belgium).
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