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.

A Dystrophin Exon-52 Deleted Miniature Pig Model of Duchenne Muscular Dystrophy and Evaluation of Exon Skipping

Duchenne muscular dystrophy (DMD) is a lethal X-linked recessive disorder caused by mutations in the DMD gene and the subsequent lack of dystrophin protein. Recently, phosphorodiamidate morpholino oligomer (PMO)-antisense oligonucleotides (ASOs) targeting exon 51 or 53 to reestablish the DMD reading frame have received regulatory approval as commercially available drugs. However, their applicability and efficacy remain limited to particular patients. Large animal models and exon skipping evaluation are essential to facilitate ASO development together with a deeper understanding of dystrophinopathies. Using recombinant adeno-associated virus-mediated gene targeting and somatic cell nuclear transfer, we generated a Yucatan miniature pig model of DMD with an exon 52 deletion mutation equivalent to one of the most common mutations seen in patients. Exon 52-deleted mRNA expression and dystrophin deficiency were confirmed in the skeletal and cardiac muscles of DMD pigs. Accordingly, dystrophin-associated proteins failed to be recruited to the sarcolemma. The DMD pigs manifested early disease onset with severe bodywide skeletal muscle degeneration and with poor growth accompanied by a physical abnormality, but with no obvious cardiac phenotype. We also demonstrated that in primary DMD pig skeletal muscle cells, the genetically engineered exon-52 deleted pig DMD gene enables the evaluation of exon 51 or 53 skipping with PMO and its advanced technology, peptide-conjugated PMO. The results show that the DMD pigs developed here can be an appropriate large animal model for evaluating in vivo exon skipping efficacy.

Differential Effects of Insulin-Deficient Diabetes Mellitus on Visceral vs. Subcutaneous Adipose Tissue—Multi-omics Insights From the Munich MIDY Pig Model

Adipose tissue (AT) is no longer considered to be responsible for energy storage only but is now recognized as a major endocrine organ that is distributed across different parts of the body and is actively involved in regulatory processes controlling energy homeostasis. Moreover, AT plays a crucial role in the development of metabolic disease such as diabetes. Recent evidence has shown that adipokines have the ability to regulate blood glucose levels and improve metabolic homeostasis. While AT has been studied extensively in the context of type 2 diabetes, less is known about how different AT types are affected by absolute insulin deficiency in type 1 or permanent neonatal diabetes mellitus. Here, we analyzed visceral and subcutaneous AT in a diabetic, insulin-deficient pig model (MIDY) and wild-type (WT) littermate controls by RNA sequencing and quantitative proteomics. Multi-omics analysis indicates a depot-specific dysregulation of crucial metabolic pathways in MIDY AT samples. We identified key proteins involved in glucose uptake and downstream signaling, lipogenesis, lipolysis and β-oxidation to be differentially regulated between visceral and subcutaneous AT in response to insulin deficiency. Proteins related to glycogenolysis, pyruvate metabolism, TCA cycle and lipogenesis were increased in subcutaneous AT, whereas β-oxidation-related proteins were increased in visceral AT from MIDY pigs, pointing at a regionally different metabolic adaptation to master energy stress arising from diminished glucose utilization in MIDY AT. Chronic, absolute insulin deficiency and hyperglycemia revealed fat depot-specific signatures using multi-omics analysis. The generated datasets are a valuable resource for further comparative and translational studies in clinical diabetes research.

Abstract 10679: Tissue-to-Arterial Carbon Dioxide Partial Pressure Gradient as a Useful Measure for Monitoring Tissue Perfusion in a Pig Model of Hemorrhagic Shock and Resuscitation

Introduction: Transcutaneous CO 2 (tcPCO 2 ) and arterial CO 2 (artPCO 2 ) become decoupled during shock. Aim: To test the hypotheses the gradient between tcPCO 2 and artPCO 2 (tc-artPCO 2 ) can be an early, sensitive measure to detect inadequate tissue perfusion in a pig model of hemorrhage shock. Methods: Six female pigs were used. A transcutaneous monitor was attached to the ear for measuring transcutaneous O 2 (tcPO 2 ) and tcPCO 2 . Pulmonary artery catheter and the pulse index continuous cardiac output (PiCCO) were instrumented for monitoring a variety of hemodynamic parameters. To induce massive hemorrhagic shock, blood was withdrawn stepwisely. Then, animals were resuscitated in stages with transfusions of the stored blood. The parameters were measured at the timings of 10, 20, and 30 ml/kg of blood withdrawals and the completions of 10, 20, and 30 ml/kg of blood transfusion . Levels of systemic oxygen delivery (DO 2 ) were also calculated at all measurement points. Results: Hemorrhage and blood transfusion impacted hemodynamic and laboratory data, such as cardiac output (CO), stroke volume, MAP, heart rate, pulmonary artery wedge pressure, global end-diastolic volume, hemoglobin, and arterial lactate. The tc-artPCO 2 markedly increased as CO decreased ( Figure A ). The critical level of DO 2 (DO 2crit ) was defined as 11.72 ml/kg/min according to tcPO 2 (a threshold as 30 mmHg). There was significant correlation between tc-artPCO 2 and DO 2 (r = -0.83, P<.0001). ROC analyses revealed that the AUCs to predict DO 2crit for tc-artPCO 2 , shock index (SI), and lactate were 0.94 (95% CI, 0.87-1.00), 0.78 (0.63-0.93), and 0.65 (0.47-0.82), respectively. The AUC for tc-artPCO 2 was greater with respect to the prediction of DO 2crit than for SI (P<.05) ( Figure B ). Conclusions: The tc-artPCO 2 strongly correlated with CO and DO 2 during hemorrhage shock and resuscitation. The less-invasive tc-artPCO 2 monitoring can sensitively detect systemic inadequate O2 supply in hemorrhagic shock.