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.

Morphometric study of the ventricular indexes in healthy ovine BRAIN using MRI.

BACKGROUND: Sheep (Ovis aries) have been largely used as animal models in a multitude of specialties in biomedical research. The similarity to human brain anatomy in terms of brain size, skull features, and gyrification index, gives to ovine as a large animal model a better translational value than small animal models in neuroscience. Despite this evidence and the availability of advanced imaging techniques, morphometric brain studies are lacking. We herein present the morphometric ovine brain indexes and anatomical measures developed by two observers in a double-blinded study and validated via an intra- and inter-observer analysis. RESULTS: For this retrospective study, T1-weighted Magnetic Resonance Imaging (MRI) scans were performed at 1.5 T on 15 sheep, under general anaesthesia. The animals were female Ovis aries, in the age of 18-24 months. Two observers assessed the scans, twice time each. The statistical analysis of intra-observer and inter-observer agreement was obtained via the Bland-Altman plot and Spearman rank correlation test. The results are as follows (mean ± Standard deviation): Indexes: Bifrontal 0,338 ± 0,032 cm; Bicaudate 0,080 ± 0,012 cm; Evans' 0,218 ± 0,035 cm; Ventricular 0,241 ± 0,039 cm; Huckman 1693 ± 0,174 cm; Cella Media 0,096 ± 0,037 cm; Third ventricle ratio 0,040 ± 0,007 cm. Anatomical measures: Fourth ventricle length 0,295 ± 0,073 cm; Fourth ventricle width 0,344 ± 0,074 cm; Left lateral ventricle 4175 ± 0,275 cm; Right lateral ventricle 4182 ± 0,269 cm; Frontal horn length 1795 ± 0,303 cm; Interventricular foramen left 1794 ± 0,301 cm; Interventricular foramen right 1,78 ± 0,317 cm. CONCLUSIONS: The present study provides baseline values of linear indexes of the ventricles in the ovine models. The acquisition of these data contributes to filling the knowledge void on important anatomical and morphological features of the sheep brain.

Soluble Receptor for Advanced Glycation End Products: A Protective Molecule against Intramyocardial Lipid Accumulation in Obese Zucker Rats?

Most of the obesity-related complications are due to ectopic fat accumulation. Recently, the activation of the cell-surface receptor for advanced glycation end products (RAGE) has been associated with lipid accumulation in different organs. Nevertheless, the role of RAGE and sRAGE, the soluble form that prevents ligands to activate RAGE, in intramyocardial lipid accumulation is presently unknown. To this aim, we analyzed whether, in obesity, intramyocardial lipid accumulation and lipid metabolism-related transcriptome are related to RAGE and sRAGE. Heart and serum samples were collected from 10 lean (L) and 10 obese (OB) Zucker rats. Oil red staining was used to detect lipids on frozen heart sections. The lipid metabolism-related transcriptome (84 genes) was analyzed by a specific PCR array. Heart RAGE expression was explored by real-time RT-PCR and Western blot analyses. Serum levels of sRAGE (total and endogenous secretory form (esRAGE)) were quantified by ELISA. Genes promoting fatty acid transport, activation, and oxidation in mitochondria/peroxisomes were upregulated in OB hearts. Intramyocardial lipid content did not differ between OB and L rats, as well as RAGE expression. A slight increase in epicardial adipose tissue was observed in OB hearts. Total sRAGE and esRAGE concentrations were significantly higher in OB rats. sRAGE may protect against obesity-induced intramyocardial lipid accumulation by preventing RAGE hyperexpression, therefore allowing lipids to be metabolized. EAT also played a protective role by working as a buffering system that protects the myocardium against exposure to excessively high levels of fatty acids. These observations reinforce the potential role of RAGE pathway as an interesting therapeutic target for obesity-related complications, at least at the cardiovascular level.

High Penetration of Paclitaxel in Abdominal Wall of Rabbits after Hyperthermic Intraperitoneal Administration of Nab-Paclitaxel Compared to Standard Paclitaxel Formulation.

PURPOSE: Paclitaxel (PTX) is currently used in combination with cisplatin for Hyperthermic Intraperitoneal Chemotherapy (HIPEC) for the treatment of peritoneal carcinomatosis. Albumin-bound PTX is a promising new drug for HIPEC because of its easy solubility in aqueous perfusion medium and possibly because of the tendency of albumin to cross physiological barriers and accumulate in tumor tissue. METHODS: We tested the feasibility of using nab-paclitaxel in rabbits treated by HIPEC for 60 min compared with the classical formulation at an equivalent PTX dose. Samples of perfusate and blood were collected at different time points and peritoneal tissues were collected at the end of perfusion. PTX concentrations were determined by HPLC. The depth of paclitaxel penetration through the peritoneal barrier was assessed by mass spectrometry imaging. RESULTS: PTX after nab-paclitaxel treatment penetrated up to 0.63 mm in the peritoneal wall, but after CRE-paclitaxel, it was not detectable in the peritoneum. Moreover, the peritoneal concentration after nab-paclitaxel was five times that after paclitaxel classical formulation. Despite the high levels reached in the peritoneum, systemic exposure of PTX was low. CONCLUSIONS: Our results show that nab-paclitaxel penetrates into the abdominal wall better than CRE-paclitaxel, in terms of effective penetration and peritoneal tissue concentration.

On the Use of the Platelet Activity State Assay for the In Vitro Quantification of Platelet Activation in Blood Recirculating Devices for Extracorporeal Circulation.

We designed an experimental setup to characterize the thrombogenic potential associated with blood recirculating devices (BRDs) used in extracorporeal circulation (ECC). Our methodology relies on in vitro flow loop platelet recirculation experiments combined with the modified-prothrombinase platelet activity state (PAS) assay to quantify the bulk thrombin production rate of circulated platelets, which correlates to the platelet activation (PA) level. The method was applied to a commercial neonatal hollow fiber membrane oxygenator. In analogous hemodynamic environment, we compared the PA level resulting from multiple passes of platelets within devices provided with phosphorylcholine (PC)-coated and noncoated (NC) fibers to account for flow-related mechanical factors (i.e., fluid-induced shear stress) together with surface contact activation phenomena. We report for the first time that PAS assay is not significantly sensitive to the effect of material coating under clinically pertinent flow conditions (500 mL/min), while providing straightforward information on shear-mediated PA dynamics in ECC devices. Being that the latter is intimately dependent on local flow dynamics, according to our results, the rate of thrombin production as measured by the PAS assay is a valuable biochemical marker of the selective contribution of PA in BRDs induced by device design features. Thus, we recommend the use of PAS assay as a means of evaluating the effect of modification of specific device geometrical features and/or different design solutions for developing ECC devices providing flow conditions with reduced thrombogenic impact.

Reconstruction of ovine axonal cytoarchitecture enables more accurate models of brain biomechanics.

There is an increased need and focus to understand how local brain microstructure affects the transport of drug molecules directly administered to the brain tissue, for example in convection-enhanced delivery procedures. This study reports a systematic attempt to characterize the cytoarchitecture of commissural, long association and projection fibres, namely the corpus callosum, the fornix and the corona radiata, with the specific aim to map different regions of the tissue and provide essential information for the development of accurate models of brain biomechanics. Ovine samples are imaged using scanning electron microscopy combined with focused ion beam milling to generate 3D volume reconstructions of the tissue at subcellular spatial resolution. Focus is placed on the characteristic cytological feature of the white matter: the axons and their alignment in the tissue. For each tract, a 3D reconstruction of relatively large volumes, including a significant number of axons, is performed and outer axonal ellipticity, outer axonal cross-sectional area and their relative perimeter are measured. The study of well-resolved microstructural features provides useful insight into the fibrous organization of the tissue, whose micromechanical behaviour is that of a composite material presenting elliptical tortuous tubular axonal structures embedded in the extra-cellular matrix. Drug flow can be captured through microstructurally-based models using 3D volumes, either reconstructed directly from images or generated in silico using parameters extracted from the database of images, leading to a workflow to enable physically-accurate simulations of drug delivery to the targeted tissue.

Prostatic leiomyosarcoma in a dog treated using a multimodal approach.

Prostatic leiomyosarcoma is an uncommon tumor encountered in male dogs, with only 2 cases reported in the veterinary literature with no follow-up described. A 12-year-old male intact German Wirehaired Pointer presented for evaluation of straining to defecate and urinate. Whole body computed tomography (CT) examination identified a spherical multicavitary expansile mass arising from the prostate gland and severely obliterating the pelvic canal. Partial subcapsular prostatectomy was performed, and histological and immunohistochemical results were consistent with prostatic leiomyosarcoma. Metronomic cyclophosphamide and nonsteroidal anti-inflammatory drugs were administered as adjuvant chemotherapy. Follow-up CT 10 months later indicated no signs of recurrence or metastasis. To the best of our knowledge, this patient represents the first report of successful multidisciplinary treatment consisting of partial subcapsular prostatectomy and adjuvant chemotherapy for prostatic leiomyosarcoma in a dog. After 15 months of follow-up, the patient remained recurrence-free without metastasis.

Circulating miRNome of Trachemys scripta after elective gonadectomy under general anesthesia.

Post-surgical management is an important issue in veterinary medicine, requiring biomarkers with high sensitivity and specificity for timely and effective treatment. Emerging evidence suggests that miRNAs are promising stress- and pain-related markers. The aims were to profile the circulating miRNA signature in plasma of turtles (Trachemys scripta) and point out potential candidate biomarkers to assess the status of the animal. The plasma of female turtles underwent surgical gonadectomy were collected 24 h pre-surgery, and 2.5 h and 36 h post-surgery. The expression of miRNAs was profiled by Next Generation Sequencing and the dysregulated miRNAs were validated using RT-qPCR. The diagnostic value of miRNAs was calculated by ROC curves. The results showed that 14 miRNAs were differentially expressed over time. RT-qPCR validation highlighted that 2-miR-499-3p and miR-203-5p-out of 8 miRNAs tested were effectively modulated. The Area Under the Curve (AUC) of miR-203-5p was fair (AUC 0.7934) in discriminating pre- and 36 h post-surgery samples and poor for other time points; the AUC of miR-499-3p was excellent (AUC 0.944) in discriminating pre-surgery and 2.5 h post-surgery samples, and fair in discriminating pre-surgery and 36 h post-surgery (AUC 0.7292) and 2.5 h and 36 h post-surgery (AUC 0.7569) samples. In conclusion, we demonstrated for the first time that miRNAs profile changes in plasma of turtles underwent surgical oophorectomy and identified miR-203-5p and miR-499-3p as potential candidate biomarkers to assess animals' status. Further studies are necessary to confirm their diagnostic value and to investigate functional and mechanistic networks to improve our understanding of the biological processes.

Development and in vivo assessment of a novel MRI-compatible headframe system for the ovine animal model.

BACKGROUND: The brain of sheep has primarily been used in neuroscience as an animal model because of its similarity to the human brain, in particular if compared to other models such as the lissencephalic rodent brain. Their brain size also makes sheep an ideal model for the development of neurosurgical techniques using conventional clinical CT/MRI scanners and stereotactic systems for neurosurgery. METHODS: In this study, we present the design and validation of a new CT/MRI compatible head frame for the ovine model and software, with its assessment under two real clinical scenarios. RESULTS: Ex-vivo and in vivo trial results report an average linear displacement of the ovine head frame during conventional surgical procedures of 0.81 mm for ex-vivo trials and 0.68 mm for in vivo tests, respectively. CONCLUSIONS: These trial results demonstrate the robustness of the head frame system and its suitability to be employed within a real clinical setting.

7-T MRI tracking of mesenchymal stromal cells after lung injection in a rat model.

BACKGROUND: Mesenchymal stromal cells (MSCs) are able to migrate and engraft at sites of inflammation, injuries, and tumours, but little is known about their fate after local injection. The purpose of this study is to perform MSC tracking, combining in vivo 7-T magnetic resonance imaging (MRI) and histological assessment, following lung injection in a rat model. METHODS: Five lungs were injected with ferumoxide-labelled MSCs and five with perfluorocarbon-labelled MSCs and underwent 7-T MRI. MRI acquisitions were recorded immediately (T0), at 24 h (T24) and/or 48 h (T48) after injection. For each rat, labelled cells were assessed in the main organs by MRI. Target organs were harvested under sterile conditions from rats sacrificed 0, 24, or 48 h after injection and fixed for histological analysis via confocal and structured illumination microscopy. RESULTS: Ferumoxide-labelled MSCs were not detectable in the lungs, whereas they were not visible in the distant sites. Perfluorocarbon-labelled MSCs were seen in 5/5 injected lungs at T0, in 1/2 at T24, and in 1/3 at T48. The fluorine signal in the liver was seen in 3/5 at T0, in 1/2 at T24, and in 2/3 at T48. Post-mortem histology confirmed the presence of MSCs in the injected lung. CONCLUSIONS: Ferumoxide-labelled cells were not seen at distant sites; a linear decay of injected perfluorocarbon-labelled MSCs was observed at T0, T24, and T48 in the lung. In more than half of the experiments, perfluorocarbon-labelled MSCs scattering to the liver was observed, with a similar decay over time as observed in the lung.

In vivo Diffusion Tensor Magnetic Resonance Tractography of the Sheep Brain: An Atlas of the Ovine White Matter Fiber Bundles.

Diffusion Tensor Magnetic Resonance Imaging (DTI) allows to decode the mobility of water molecules in cerebral tissue, which is highly directional along myelinated fibers. By integrating the direction of highest water diffusion through the tissue, DTI Tractography enables a non-invasive dissection of brain fiber bundles. As such, this technique is a unique probe for in vivo characterization of white matter architecture. Unraveling the principal brain texture features of preclinical models that are advantageously exploited in experimental neuroscience is crucial to correctly evaluate investigational findings and to correlate them with real clinical scenarios. Although structurally similar to the human brain, the gyrencephalic ovine model has not yet been characterized by a systematic DTI study. Here we present the first in vivo sheep (ovis aries) tractography atlas, where the course of the main white matter fiber bundles of the ovine brain has been reconstructed. In the context of the EU's Horizon EDEN2020 project, in vivo brain MRI protocol for ovine animal models was optimized on a 1.5T scanner. High resolution conventional MRI scans and DTI sequences (b-value = 1,000 s/mm2, 15 directions) were acquired on ten anesthetized sheep o. aries, in order to define the diffusion features of normal adult ovine brain tissue. Topography of the ovine cortex was studied and DTI maps were derived, to perform DTI tractography reconstruction of the corticospinal tract, corpus callosum, fornix, visual pathway, and occipitofrontal fascicle, bilaterally for all the animals. Binary masks of the tracts were then coregistered and reported in the space of a standard stereotaxic ovine reference system, to demonstrate the consistency of the fiber bundles and the minimal inter-subject variability in a unique tractography atlas. Our results determine the feasibility of a protocol to perform in vivo DTI tractography of the sheep, providing a reliable reconstruction and 3D rendering of major ovine fiber tracts underlying different neurological functions. Estimation of fiber directions and interactions would lead to a more comprehensive understanding of the sheep's brain anatomy, potentially exploitable in preclinical experiments, thus representing a precious tool for veterinaries and researchers.

Simple and Quick Method to Obtain a Decellularized, Functional Liver Bioscaffold.

The development of new approaches for organ transplantation has become crucial in the last years. In particular, organ engineering, involving the preparation of acellular matrices that provide a natural habitat for reseeding with an appropriate population of cells, is an attractive although technically demanding approach. We here describe a method that allows for the derivation of functional in vitro hepatic organoids and that does not require a previous selection of all the parenchymal hepatocytes and non-parenchymal cells, namely, Kupffer cells, liver endothelial cells, and hepatic stellate cells. The procedure also replaces the costly standard collagenase perfusion step with a trypsin-based enzymatic digestion that results in high-yield decellularization. A combination of physical and chemical treatments through deep immersion and intraluminal infusion of two different consecutive solutions is used: (1) deionized water (DI) and (2) DI + Triton X 1% + ammonium hydroxide (NH4OH) 0.1%. This ensures the isolation of the hepatic constructs that reliably maintain original architecture and ECM components while completely removing cellular DNA and RNA. The procedure is fast, simple, and cheap and warrants an optimal organoid functionality that may find applications in both toxicological and transplantation studies.

Expression of c-kit proto-oncogene in canine mastocytoma: a kinetic study using real-time polymerase chain reaction.

KIT receptor, the c-kit gene product, is thought to play a major role in canine mastocytoma, one of the most common neoplastic diseases in dogs. In the present study, the expression of c-kit proto-oncogene in blood and in tumor biopsies from 41 dogs with histologically confirmed mastocytoma at different grades of cellular differentiation and 5 negative control dogs was investigated using real-time (quantitative) reverse transcription-polymerase chain reaction (RRT-PCR). The animals were followed up for over 1 year after surgery in order to characterize the kinetics of c-kit expression in blood. Transcript mRNAs extracted from blood at different time points after surgery and from tumor tissue surgically removed from each dog were used in a quantitative RRT-PCR assay targeting the extracellular coding region of the c-kit gene. Tissues constitutively expressing c-kit (brain and spleen) were used as positive controls. Levels of expression of c-kit were higher in tumor biopsies than in blood; the blood level decreased in the patients between 1 and 3 months after surgery. No KIT expression was detected in blood from the 5 dogs not affected by mastocytoma (negative controls). The RRT-PCR appears to be a suitable method for sensitive and quantitative detection of c-kit gene expression in canine blood and neoplastic tissues. Although c-kit expression levels measured by RRT-PCR do not correlate with prognosis, they confirm that surgery remains the main treatment to reduce circulating mastocytes and that circulating mast cells can be detected even in benign highly differentiated forms of mastocytoma such as grade I.

A study of mutations in the c-kit gene of 32 dogs with mastocytoma.

Mutations in the intracellular juxtamembrane domain of the c-kit gene in 32 dogs with different grades of histologically confirmed mastocytoma were studied. Transcript RNAs extracted from neoplastic tissue surgically collected from dogs of different breeds and from a negative control were reverse transcribed into complementary DNA and amplified by polymerase chain reaction. The region corresponding to the c-kit juxtamembrane domain was sequenced and compared with GenBank sequences. Two different types of mutations were identified within exon 11: a previously underscribed single-nucleotide substitution and a 6-bp deletion. The c-kit juxtamembrane domain sequences of all dogs were grouped in 3 clusters. No mutations were detected in tissues constitutively expressing c-kit (cerebellum and spleen), obtained from dogs not affected by mastocytoma (controls). All the substitutions were found in dogs bearing grade I or II mast cell tumors; the deletion was detected in 1 dog with grade II mastocytoma.

Stem cell transplantation effectively occludes bronchopleural fistula in an animal model.

BACKGROUND: Bronchopleural fistula after lung resection still represents a challenging life-threatening complication for thoracic surgeons. Considering its extremely high mortality rate, an effective treatment is urgently required. Our project investigated the hypothesis of experimental bronchopleural fistula closure by bronchoscopic injection of autologous bone marrow-derived mesenchymal stem cells into the cavity of the fistula, evaluating its feasibility and safety in a large animal model. METHODS: An experimental bronchopleural fistula was created in 9 goats after right upper tracheal lobectomy. The animals were randomly assigned to two groups: one received autologous bone marrow-derived mesenchymal stem cell bronchoscopic transplantation; the other received standard bronchoscopic fibrin glue injection. RESULTS: All animals receiving bronchoscopic stem cell transplantation presented fistula closure by extraluminal fibroblast proliferation and collagenous matrix development; none (0%) died during the study period. All animals receiving standard treatment still presented bronchopleural fistula; 2 of them (40%) died. Findings were confirmed by pathology examination, computed tomography, and magnetic resonance imaging. CONCLUSIONS: Bronchoscopic transplantation of bone marrow-derived mesenchymal stem cells effectively closes experimental bronchopleural fistula by extraluminal fibroblast proliferation and collagenous matrix development. Stem cells may play a crucial role in the treatment of postresectional bronchopleural fistula after standard lung resection. Although these results provide a basis for the development of clinical therapeutic strategies, the exact mechanism by which they are obtained is not yet completely clear; further studies are required to understand exactly how stem cells work in this field.

Morphologic features of biocompatibility and neoangiogenesis onto a biodegradable tracheal prosthesis in an animal model.

We evaluated a newly designed bioresorbable polymer (Degrapol) tracheal prosthesis in an in-vivo angiogenesis-inducing animal model focusing on the specific tissue reaction, the neo-angiogenesis and also the eventual cathepsin B role during the polymer degradation. Fifteen rabbits were divided into three groups (2, 6 and 8 weeks) and our tube-shaped porous prosthesis was implanted using the common carotid artery and the internal jugular vein as vascular pedicle. Optical and electron microscopy, immunohistochemistry and immunocytochemistry were performed at the end of each period, showing cells and fibrils, in direct contact with the Degrapol scaffold, strongly increased with time. Blood vessel neoformation was visible with CD31 expression localized at the endothelial cells forming the neovascular walls. Over time many of them differentiate in muscle fibers as validated by the expression of alpha-smooth muscle actin (SMA). Few inflammatory cells, expressing CD14, were visible while most cells adopting a pronounced spreading phenotype showed a strong positivity for cathepsin B. We concluded that this bioresorbable polymer provided a good substrate for fibrous tissue deposition with an excellent degree of neo-angiogenesis. Also, cathepsin B seems to contribute to the polymer degradation and particularly to neovascularization by stimulating capillary-like tubular structures and cell proliferation.

Prefabricated tracheal prosthesis with partial biodegradable materials: a surgical and tissue engineering evaluation in vivo.

Large and circumferential tracheal defects remain at this time an unsolved problem for reconstructive surgery. Many types of prosthetic and tissue grafts have been used but with limited comfortable results. Major complications are anastomotic dehiscence, graft ischemia and stenosis due to the poor vascularization of the prosthetic complex. We studied the in vivo behaviour of a prefabricated flap composed of a partially bioresorbable tracheal prosthesis and an arterio-venous vascular carrier. The prosthesis was made of a tubular skeleton of knitted Dacron (20 microm porosity) embedded within a bioresorbable poly-lactic-co-glycolic acid polymer (PLA(75)GA(25)) covering both sides. Fifteen New Zealand White rabbits were divided in three groups, depending on the time of examination (30, 90 and 180 days post-implantation). The prosthesis was implanted in the visceral space of the neck using the common carotid trunk and the internal jugular vein as vascular pedicle. The histological, immunohistochemical, and ESEM analyses of collected samples, showed a time-dependent process of tissue neoformation and neovascularization on the prosthetic material with a significant increase from 30 to 90 days post-implantation. In contrast, there was no statistically significant difference in the fibrovascular connective deposition from 90 to 180 days. This finding indicated the three months time as the best period for the tissue deposition and consequent hypothetical orthotopic transplantation of the prosthesis. Further in vivo studies are intended to confirm the results.