An in vivo pig model for testing novel PET radioligands targeting cerebral protein aggregates

Positron emission tomography (PET) has become an essential clinical tool for diagnosing neurodegenerative diseases with abnormal accumulation of proteins like amyloid-β or tau. Despite many attempts, it has not been possible to develop an appropriate radioligand for imaging aggregated α-synuclein, which is seen in, e.g., Parkinson's Disease. Access to a large animal model with α-synuclein pathology would critically enable a more translationally appropriate evaluation of novel radioligands. We here established a pig model with cerebral injections of α-synuclein preformed fibrils or brain homogenate from postmortem human brain tissue from individuals with Alzheimer's disease (AD) or dementia with Lewy body (DLB) into the pig's brain using minimally invasive surgery and validated against saline injections. In the absence of a suitable α-synuclein radioligand, we validated the model with an unselective amyloid-β tracer [11C]PIB, which has a high affinity for β-sheet structures in aggregates. Gadolinium-enhanced MRI confirmed that the blood-brain barrier function was intact. A few hours post-injection, pigs were PET scanned with [11C]PIB. Quantification was done with Logan invasive graphical analysis and simplified reference tissue model 2 using the occipital cortex as a reference region. After the scan, we retrieved the brains to confirm successful injection using autoradiography and immunohistochemistry. We found four times higher [11C]PIB uptake in AD-homogenate-injected regions and two times higher uptake in α-synuclein-preformed-fibrils-injected regions compared to the saline-injected regions. The [11C]PIB uptake was the same in the occipital cortex, cerebellum, DLB-homogenate, and saline-injected regions. With its large brains and ability to undergo repeated PET scans as well as neurosurgical procedures, the pig provides a robust, cost-effective, and good translational model for assessment of novel radioligands including, but not limited to, proteinopathies.

Collagenase-Induced Patellar Tendinopathy with Neovascularization: First Results towards a Piglet Model of Musculoskeletal Embolization

Background: Therapeutic strategies targeting neovessels responsible for musculoskeletal chronic pain have emerged, including neovessels embolization. Our study aimed to develop a large animal model of patellar tendinopathy with neovascularization. Methods: Nine 3-month-old male piglets (18 patellar tendons) received percutaneous injections of increasing doses of collagenase (0 to 50 mg) at day 0 (D0). Tendinopathy was evaluated by ultrasound (D7 and D14). Neovascularization was evaluated visually and on angiographies. Bonar score was used for histological analysis (D14). Correlations were evaluated using Spearman’s rank (Rs) test. Results: Research protocol was well tolerated. All tendons were enlarged with a median increase of 31.58% [25–40.28] at D7 (p = 0.244) at D7 and 57.52% [48.41–91.45] at D14 (p = 0.065). Tendons with collagenase injection had more hypoechoic changes, with one tendon rupture (p = 0.012). Neovascularization was reported above 5 mg collagenase (p < 0.01) at D7 and D14 with dose-related neovessels induction (Rs = 0.8, p < 0.001). The Bonar score increased above 5 mg collagenase, correlated with the dose (Rs = 0.666, p = 0.003). Conclusions: The study shows the feasibility, safety and reproducibility of this new large animal model of patellar tendinopathy with neovascularization after collagenase injection. It will allow studying new treatments on direct embolization of neovessels by endovascular approach.

Regeneration of Subcutaneous Cartilage in a Swine Model Using Autologous Auricular Chondrocytes and Electrospun Nanofiber Membranes Under Conditions of Varying Gelatin/PCL Ratios

The scarcity of ideal biocompatible scaffolds makes the regeneration of cartilage in the subcutaneous environment of large animals difficult. We have previously reported the successful regeneration of good-quality cartilage in a nude mouse model using the electrospun gelatin/polycaprolactone (GT/PCL) nanofiber membranes. The GT/PCL ratios were varied to generate different sets of membranes to conduct the experiments. However, it is unknown whether these GT/PCL membranes can support the process of cartilage regeneration in an immunocompetent large animal model. We seeded swine auricular chondrocytes onto different GT/PCL nanofiber membranes (GT:PCL = 30:70, 50:50, and 70:30) under the sandwich cell-seeding mode. Prior to subcutaneously implanting the samples into an autologous host, they were cultured in vitro over a period of 2 weeks. The results revealed that the nanofiber membranes with different GT/PCL ratios could support the process of subcutaneous cartilage regeneration in an autologous swine model. The maximum extent of homogeneity in the cartilage tissues was achieved when the G5P5 (GT: PC = 50: 50) group was used for the regeneration of cartilage. The formed homogeneous cartilage tissues were characterized by the maximum cartilage formation ratio. The extents of the ingrowth of the fibrous tissues realized and the extents of infiltration of inflammatory cells achieved were found to be the minimum in this case. Quantitative analyses were conducted to determine the wet weight, cartilage-specific extracellular matrix content, and Young’s modulus. The results indicated that the optimal extent of cartilage formation was observed in the G5P5 group. These results indicated that the GT/PCL nanofiber membranes could serve as a potential scaffold for supporting subcutaneous cartilage regeneration under clinical settings. An optimum GT/PCL ratio can promote cartilage formation.

Automatic Air Volume Control System for Ventilation of Two Patients Using a Single Ventilator: A Large Animal Model Study

The COVID-19 pandemic outbreak led to a global ventilator shortage. Hence, different strategies to use a single ventilator to support multiple patients are considered. A mechatronic system Ventil divides and automatically controls gas volume pumped through two channels and was successfully validated in independent lung ventilation. We used Ventil in a series of experiments on a large animal model to verify its usability for ventilation in two patients using a single ventilator. The results of investigations on 12 pigs showed that the physiological level of respiratory parameters was maintained for 24 hours. Application of Ventil did not lead to injuries in the lungs, as indicated by CT scan analysis. We conclude that ventilation using Ventil can be considered safe in patients subjected to deep sedation without spontaneous breathing efforts.

In-vitro and In-vivo Feasibility Study for a Mobile VV-ECMO and ECCO2R System

Extracorporeal membrane oxygenation (ECMO) is an established rescue therapy for patients with chronic respiratory failure waiting for lung transplantation (LTx). The therapy inherent immobilization may result in fatigue, consecutive deteriorated prognosis and even lost eligibility for transplantation. We conducted a feasibility study on a novel system designed for the deployment of a mobile ECMO device, enabling physical exercise of awake patients prior to LTx. The system comprises a novel mobile oxygenator with a directly connected blood pump, a double lumen cannula, gas blender and supply, as well as control, and energy management. In-vitro experiments included tests regarding performance, efficiency, and blood damage. A reduced system was tested in vivo for feasibility using a novel large animal model. Six anesthetized pigs were first positioned in supine position, followed by a 45&deg; angle, simulating an upright position of the patients. We monitored performance and vital parameters. All in-vitro experiments showed good performance for the respective subsystems and the integrated system. The acute invivo trials of 8h duration confirmed the results. The novel mobile ECMO-system enables adequate oxygenation and decarboxylation sufficient for, e.g., physical exercise of designated LTx-recipients. These results are promising and suggest further preclinical studies on safety and efficacy to facilitate translation into clinical application.

The efficacy and safety of pinocembrin in a sheep model of bleomycin-induced pulmonary fibrosis

The primary flavonoid, pinocembrin, is thought to have a variety of medical uses which relate to its reported anti-oxidant, anti-inflammatory, anti-microbial and anti-cancer properties. Some studies have reported that this flavonoid has anti-fibrotic activities. In this study, we investigated whether pinocembrin would impede fibrosis, dampen inflammation and improve lung function in a large animal model of pulmonary fibrosis. Fibrosis was induced in two localized lung segments in each of the 10 sheep participating in the study. This was achieved via two infusions of bleomycin delivered bronchoscopically at a two-week interval. Another lung segment in the same sheep was left untreated, and was used as a healthy control. The animals were kept for a little over 5 weeks after the final infusion of bleomycin. Pinocembrin, isolated from Eucalyptus leaves, was administered to one of the two bleomycin damaged lung segments at a dose of 7 mg. This dose was given once-weekly over 4-weeks, starting one week after the final bleomycin infusion. Lung compliance (as a measure of stiffness) was significantly improved after four weekly administrations of pinocembrin to bleomycin-damaged lung segments. There were significantly lower numbers of neutrophils and inflammatory cells in the bronchoalveolar lavage of bleomycin-infused lung segments that were treated with pinocembrin. Compared to bleomycin damaged lung segments without drug treatment, pinocembrin administration was associated with significantly lower numbers of immuno-positive CD8+ and CD4+ T cells in the lung parenchyma. Histopathology scoring data showed that pinocembrin treatment was associated with significant improvement in inflammation and overall pathology scores. Hydroxy proline analysis showed that the administration of pinocembrin did not reduce the increased collagen content that was induced by bleomycin in this model. Analyses of Masson’s Trichrome stained sections showed that pinocembrin treatment significantly reduced the connective tissue content in lung segments exposed to bleomycin when compared to bleomycin-infused lungs that did not receive pinocembrin. The striking anti-inflammatory and modest anti-fibrotic remodelling effects of pinocembrin administration were likely linked to the compound’s ability to improve lung pathology and functional compliance in this animal model of pulmonary fibrosis.