Sheep head frame validation for CT and MRI studies

Abstract IntroductionsAim of EDEN 2020 project’s Milestone 5 is the development of a steerable catheter for CED system in glioblastoma therapy. The VET group is involved in realization and validation of the proper animal model.Materials and methodsIn this part of the study two fresh sheep’s head from the local slaughter were used.The heads were located into an ad hoc Frame system based on anatomical measures and CT images, producted by Renishaw plc partner in this project. The frame was adapted and every components were checked for the ex vivo validation tests.CT imaging was taken in Lodi at Università degli studi di Milano, Facoltà di Medicina Veterinaria, with CT scanner and MRI imaging was taken in La Cittadina, CremonaResultsSystem validation was approved by the ex vivo trial.The frame system doesn’t compromise the imaging acquisition in MRI and CT systems.Every system components are functional to their aims.DiscussionThe Frame system is adapted to the sheep head. It is composed by elements able to lock the head during the imaging acquisition. Frame system is characterized by a support base helpings the animals to keep the head straight forward during imaging time, under general anesthesia. The design of these device support the airways anatomy, avoiding damaging or obstruction of airflows during anesthesia period.The role of elements like mouth bar and ovine head pins is to lock the head in a stable position during imaging acquisition; fixing is guaranteed by V shape head pins, that are arranged against the zygomatic arches. Lateral compression forces to the cranium, and the V shape pins avoid the vertical shifting of the head and any kind of rotations. (fig. 1

Sheep brain atlas creation. Diffusion tensor imaging and Scanning electron microscope in sheep brain analysis

IntroductionsAim of EDEN 2020 project is the development of a steerable catheter for CED system in glioblastoma therapy. The VET group is involved in realization and validation of the proper animal model.For surgical planning purpose a Diffusion Tensor Imaging (DTI) of white matter tracts in the sheep is necessary to identify the target points useful for the catheter introduction.The analysis of the sheep brain under a Scanning Electron Microscope (SEM) is required to understand any alterations due to the catheter introduction and to fluids injection during CED administration. Materials and methodsAnimals were treated in accordance with the European Communities Council directive (86/609/EEC), to the laws and regulations on animal welfare enclosed in D.L.G.S. 26/2014A total of five 70 kg female, one year old, sheep were used for the study.All animals, under general anesthesia, underwent to Magnetic Resonance Imaging (MRI) acquisition. MRI scanner used was Philips Ingenia 1.5 Tesla system.Once the DTI imaging were acquired the animals were euthanased, sheep brain was collected and samples of white matter tracts obtained with disposable biopsy punches of 1.5-2 mm of diameter.The samples were fixed, stained in Osmium tetroxide (OsO4) and then embedded with two different protocols (cold curing vs thermal curing) in resin for the Focused Ion Beam (FIB) SEM analyses. Results and discussionAll the DTI images were uploaded to TrackVis software and major white matter fiber tracts analysed. Corticospinal tract, visual radiation, fornix and fronto-occipital fasciculus were identified.Corticospinal tract was identified as major white matter tract in sheep brain and useful as target area for the research aims.For the SEM analysis the thermal protocol was recognised as better curing methods for the research purpose than cold curing one. ConclusionThe data acquired in this study are still submitted to analysis. AcknowledgmentThe project has received funding from the European Union’s EU Research and Innovation programme Horizon 2020 (no 688279)

Isolated slaughterhouse liver as model for normothermic perfusion after warm and cold ischemia: single case report

AbstractLiver transplantation is an ultimate procedure in patients suffering end-stage liver diseases. In these last years the donation after cardiac death (DCD) has increased the pool of potential liver donors. Different studies and procedures are involved in the prevention of the main ischemic problems during the reconditioning and resuscitation of the marginal livers. Normothermic extracorporeal liver perfusion (NELP) avoids prolonged cold storage damage that is the main cause of steatosis and biliary tract ischemia in transplanted patiens. Different porcine models have been studied and developed to understand the ischemia mechanism and to select the better technique for NELP.We conducted our study using a DCD pig liver model collected from slaughterhouse. Using extracorporeal membrane oxygenation, 2000 ml of total fluid containing autologous blood, lidocaine, heparin, antibiotics, glucose 10 % solution and flunixin, the NELP was achieved. The liver was perfused over 7 hours after 48 hours of cold storage (4C°), using Eurocollins solution. During the liver withdrawal in the slaughterhouse 20 minutes were waited to simulate the warm ischemia (WI) time. Histological samples, swab for bacterial grow, blood sample, temperature and pulse oximetry saturation were collected to assess the liver viability and function. These analyses revealed stable metabolism throughout perfusion identifying a cycles 2 hours length, coinciding with recovery of oxygen uptake rates to fresh liver, as described in literature.In summary the preliminary established model of isolated hemoperfused slatherhouse liver reveals the important role of the relation between cold storage and normothermic perfusion. Moreover this preliminary study justifies further investigation of the optimization of the treatment protocols and perfusion media

Derivation of canine hepatocyte in vitro models to study Branched-Chain Amino Acid effects on liver functions.

Branched chain amino acids (BCAA), have been shown to affect human gene expression, proteinmetabolism, apoptosis, and regeneration of hepatocytes. Furthermore, they have been demonstratedto inhibit proliferation of liver cancer cells in vitro, and to be essential for lymphocyte proliferation.In veterinary medicine, the use of BCAAs as integration of a normal dietary plan, is likely to be a validchoice for the same benefit found in human clinical nutrition, although this aspect is still debated.Indeed, long-term oral supplementation with BCAAs in the prevention of liver fibrosis and injury in thedog's liver is still unclear. Aim of the present study will be to determine how BCAAs preserve liverfunctions in vitro. To this purpose we have selected and set up three different in vitro models: hepaticdog cells and canine hepatocellular carcinoma cells plated in 2D monolayer and hepatic dog cellscultured onto 3D scaffolds, obtained from decellularized rabbit liver. All cells adhered and proliferatedonce plated. Cells grown in monolayer quickly entered G0 end arrested growth, ELISA test confirmedtheir ability to produce albumin. Cells grown on scaffold vigorously replicated and showed theircapability to recellularize ECM rabbit liver. These results, although preliminary, demonstrate that theculture conditions used well preserved the original phenotype and function and further support thepossibility to use in vitro models to successfully study BCAA efficacy in dog

Unveiling IL-33/ST2 Pathway Unbalance in Cardiac Remodeling Due to Obesity in Zucker Fatty Rats

Obesity is an epidemic condition linked to cardiovascular disease severity and mortality. Fat localization and type represent cardiovascular risk estimators. Importantly, visceral fat secretes adipokines known to promote low-grade inflammation that, in turn, modulate its secretome and cardiac metabolism. In this regard, IL-33 regulates the functions of various immune cells through ST2 binding and—following its role as an immune sensor to infection and stress—is involved in the pro-fibrotic remodeling of the myocardium. Here we further investigated the IL-33/ST2 effects on cardiac remodeling in obesity, focusing on molecular pathways linking adipose-derived IL-33 to the development of fibrosis or hypertrophy. We analyzed the Zucker Fatty rat model, and we developed in vitro models to mimic the adipose and myocardial relationship. We demonstrated a dysregulation of IL-33/ST2 signaling in both adipose and cardiac tissue, where they affected Epac proteins and myocardial gene expression, linked to pro-fibrotic signatures. In Zucker rats, pro-fibrotic effects were counteracted by ghrelin-induced IL-33 secretion, whose release influenced transcription factor expression and ST2 isoforms balance regulation. Finally, the effect of IL-33 signaling is dependent on several factors, such as cell types’ origin and the balancing of ST2 isoforms. Noteworthy, it is reasonable to state that considering IL-33 to have a unique protective role should be considered over-simplistic

Unveiling IL-33/ST2 Pathway Unbalance in Cardiac Remodeling Due to Obesity in Zucker Fatty Rats

Obesity is an epidemic condition linked to cardiovascular disease severity and mortality. Fat localization and type represent cardiovascular risk estimators. Importantly, visceral fat secretes adipokines known to promote low-grade inflammation that, in turn, modulate its secretome and cardiac metabolism. In this regard, IL-33 regulates the functions of various immune cells through ST2 binding and—following its role as an immune sensor to infection and stress—is involved in the pro-fibrotic remodeling of the myocardium. Here we further investigated the IL-33/ST2 effects on cardiac remodeling in obesity, focusing on molecular pathways linking adipose-derived IL-33 to the development of fibrosis or hypertrophy. We analyzed the Zucker Fatty rat model, and we developed in vitro models to mimic the adipose and myocardial relationship. We demonstrated a dysregulation of IL-33/ST2 signaling in both adipose and cardiac tissue, where they affected Epac proteins and myocardial gene expression, linked to pro-fibrotic signatures. In Zucker rats, pro-fibrotic effects were counteracted by ghrelin-induced IL-33 secretion, whose release influenced transcription factor expression and ST2 isoforms balance regulation. Finally, the effect of IL-33 signaling is dependent on several factors, such as cell types’ origin and the balancing of ST2 isoforms. Noteworthy, it is reasonable to state that considering IL-33 to have a unique protective role should be considered over-simplistic

In vivo MRI measurement of microstructural constraints for direct drug delivery within the brain

Brain tissue microstructure may influence the efficient delivery of therapeutics within the brain. Diffusion Tensor Imaging (DTI) enables the depiction of tissue properties in vivo, and thus is potentially relevant for planning convection-enhanced delivery (CED) within the brain. We report on the quantitative assessment of the distribution of a Gadolinium solution infused by CED within the brain of a live ovine model. Infusate distributions were measured at multiple timepoints and compared to microstructural properties as depicted by DTI, thus demonstrating the impact of tissue features and catheter positioning on drug distribution in vivo