The conversion of endogenous alpha-synuclein (asyn) to pathological asyn-enriched aggregates is a hallmark of Parkinson’s disease (PD). These inclusions can be detected in the central and enteric nervous system (ENS). Moreover, gastrointestinal symptoms can appear up to 20 years before the diagnosis of PD. The dual-hit hypothesis posits that pathological asyn aggregation starts in the ENS, and retrogradely spreads to the brain. In this study, we tested this hypothesis by directly injecting preformed asyn fibrils into the duodenum wall of wild-type rats and transgenic rats with excess levels of human asyn. We provide a meticulous characterization of the bacterial artificial chromosome (BAC) transgenic rat model with respect to initial propagation of pathological asyn along the parasympathetic and sympathetic pathways to the brainstem, by performing immunohistochemistry at early time points post-injection. Induced pathology was observed in all key structures along the sympathetic and parasympathetic pathways (ENS, autonomic ganglia, intermediolateral nucleus of the spinal cord (IML), heart, dorsal motor nucleus of the vagus, and locus coeruleus (LC)) and persisted for at least 4 months post-injection. In contrast, asyn propagation was not detected in wild-type rats, nor in vehicle-injected BAC rats. The presence of pathology in the IML, LC, and heart indicate trans-synaptic spread of the pathology. Additionally, the observed asyn inclusions in the stomach and heart may indicate secondary anterograde propagation after initial retrograde spreading. In summary, trans-synaptic propagation of asyn in the BAC rat model is fully compatible with the “body-first hypothesis” of PD etiopathogenesis. To our knowledge, this is the first animal model evidence of asyn propagation to the heart, and the first indication of bidirectional asyn propagation via the vagus nerve, i.e., duodenum-to-brainstem-to-stomach. The BAC rat model could be very valuable for detailed mechanistic studies of the dual-hit hypothesis, and for studies of disease modifying therapies targeting early pathology in the gastrointestinal tract.

Positron emission tomography (PET) provides a means of studying physiological and pharmacological processes as they occur in the living brain. Mice, rats, dogs, cats, pigs and non-human primates are often used in studies using PET. They are commonly anaesthetized with ketamine, propofol or isoflurane in order to prevent them from moving during the imaging procedure. The use of anaesthesia in PET studies suffers, however, from the drawback of possibly altering central neuromolecular mechanisms. As a result, PET findings obtained in anaesthetized animals may fail to correctly represent normal properties of the awake brain. Here, we review findings of PET studies carried out either in both awake and anaesthetized animals or in animals given at least two different anaesthetics. Such studies provide a means of estimating the extent to which anaesthesia affects the outcome of PET neuroimaging in animals. While no final conclusion can be drawn concerning the ‘best’ general anaesthetic for PET neuroimaging in laboratory animals, such studies provide findings that can enhance an understanding of neurobiological mechanisms in the living brain.

In modern nature conservation and rewilding there is a need for controlling the movements of large grazers in extensively managed areas. The inflexibility of physical fencing can be a limitation in nature management, and the physical boundaries created by physical fencing can have detrimental effects on wildlife. Virtual fencing systems provide boundaries without physical structures. These systems utilise collars with GPS technology to track animals and deliver auditory or electric cues to encourage the animals to stay within the predefined boundaries. This study aims to assess the use of virtual fencing (Nofence©) to keep twelve Angus cows (Bos taurus) within a virtual enclosure without compromising their welfare. As such, the study examines inter-individual differences between the cows as well as their herd behaviour, when reacting and learning to respond appropriately to virtual fencing. Moreover, the activity of the cows was used as an indicator of welfare. The virtual fencing was successful in keeping the herd within the designated area. Moreover, the cattle learned to avoid the virtual border and respond to auditory cues, where the cows received significantly more auditory warning and electric impulses per week throughout the first 14 days than the remaining 125 days (p < 0.001). The cows were found to express both inter-individual differences (p < 0.001) and herd behaviour. The cattle did not express any significant changes in their activity upon receiving an electrical impulse from the collar. Thus, indicating that there were little to no acute welfare implications associated with the use of virtual fencing in this study. This study clearly supports the potential for virtual fencing as a viable alternative to physical electric fencing. However, it also shows that both individual differences in personality and herd structure should be considered when selecting individuals for virtual fencing.

Brain cholinergic function has been previously studied with PET but little effort has been devoted to imaging peripheral organs. Many disorders, including diabetes and Parkinson disease, are associated with autonomic dysfunction including parasympathetic denervation. Nonneuronal cholinergic signaling is also involved in immune responses to infections and in cancer pathogenesis. 5-(11)C-methoxy-donepezil, a noncompetitive acetylcholinesterase ligand, was previously validated for imaging cerebral levels of acetylcholinesterase. In the present study, we explored the utility of (11)C-donepezil for imaging acetylcholinesterase densities in peripheral organs, including the salivary glands, heart, stomach, intestine, pancreas, liver, and spleen. With autoradiography, we determined binding affinities and levels of nonspecific (11)C-donepezil binding to porcine tissues. Radiation dosimetry was estimated by whole-body PET of a single human volunteer. Biodistribution and kinetic analyses of (11)C-donepezil time-activity curves were assessed with dynamic PET scans of 6 healthy human volunteers. A single pig with bacterial abscesses was PET-scanned to explore (11)C-donepezil uptake in infections. Autoradiography showed high (11)C-donepezil binding (dissociation constant, 6-39 nM) in pig peripheral organs with low nonspecific signal. Radiation dosimetry was favorable (effective dose, 5.2 μSv/MBq). Peripheral metabolization of (11)C-donepezil was low (>90% unchanged ligand at 60 min). Slow washout kinetics were seen in the salivary glands, heart, intestines, pancreas, and prostate. A linear correlation was seen between (11)C-donepezil volumes of distribution and standardized uptake values, suggesting that arterial blood sampling may not be necessary for modeling uptake kinetics in future (11)C-donepezil PET studies. High standardized uptake values and slow washout kinetics were seen in bacterial abscesses. (11)C-donepezil PET is suitable for imaging acetylcholinesterase densities in peripheral organs. Its uptake may potentially be quantitated with static whole-body PET scans not requiring arterial blood sampling. We also demonstrated high (11)C-donepezil binding in bacterial abscesses. We propose that (11)C-donepezil PET imaging may be able to quantify the parasympathetic innervation of organs but also detect nonneuronal cholinergic activity in infections.

Aging is the biggest risk factor for developing Parkinson's disease (PD), the second most common neurodegenerative disorder. Several animal models have been developed to explore the pathophysiology underlying neurodegeneration and the initiation and spread of alpha-synuclein-related PD pathology, and to investigate biomarkers and therapeutic strategies. However, bench-to-bedside translation of preclinical findings remains suboptimal and successful disease-modifying treatments remain to be discovered. Despite aging being the main risk factor for developing idiopathic PD, most studies employ young animals in their experimental set-up, hereby ignoring age-related cellular and molecular mechanisms at play. Consequently, studies in young animals may not be an accurate reflection of human PD, limiting translational outcomes. Recently, it has been shown that aged animals in PD research demonstrate a higher susceptibility to developing pathology and neurodegeneration, and present with a more disseminated and accelerated disease course, compared to young animals. Here we review recent advances in the investigation of the role of aging in preclinical PD research, including challenges related to aged animal models that are limiting widespread use. Overall, current findings indicate that the use of aged animals may be required to account for age-related interactions in PD pathophysiology. Thus, although the use of older animals has disadvantages, a model that better represents clinical disease within the elderly would be more beneficial in the long run, as it will increase translational value and minimize the risk of therapies failing during clinical studies. Furthermore, we provide recommendations to manage the challenges related to aged animal models.