Ageing promotes pathological alpha-synuclein propagation and autonomic dysfunction in wild-type rats.

Neuronal aggregates of misfolded alpha-synuclein protein are found in the brain and periphery of patients with Parkinson's disease. Braak and colleagues have hypothesized that the initial formation of misfolded alpha-synuclein may start in the gut, and then spread to the brain via peripheral autonomic nerves hereby affecting several organs, including the heart and intestine. Age is considered the greatest risk factor for Parkinson's disease, but the effect of age on the formation of pathology and its propagation has not been studied in detail. We aimed to investigate whether propagation of alpha-synuclein pathology from the gut to the brain is more efficient in old versus young wild-type rats, upon gastrointestinal injection of aggregated alpha-synuclein. Our results demonstrate a robust age-dependent gut-to-brain and brain-to-gut spread of alpha-synuclein pathology along the sympathetic and parasympathetic nerves, resulting in age-dependent dysfunction of the heart and stomach, as observed in patients with Parkinson's disease. Moreover, alpha-synuclein pathology is more densely packed and resistant to enzymatic digestion in old rats, indicating an age-dependent maturation of alpha-synuclein aggregates. Our study is the first to provide a detailed investigation of alpha-synuclein pathology in several organs within one animal model, including the brain, skin, heart, intestine, spinal cord and autonomic ganglia. Taken together, our findings suggest that age is a crucial factor for alpha-synuclein aggregation and complete propagation to heart, stomach and skin, similar to patients. Given that age is the greatest risk factor for human Parkinson's disease, it seems likely that older experimental animals will yield the most relevant and reliable findings. These results have important implications for future research to optimize diagnostics and therapeutics in Parkinson's disease and other age-associated synucleinopathies. Increased emphasis should be placed on using aged animals in preclinical studies and to elucidate the nature of age-dependent interactions.

Intrinsic Heart Regeneration in Adult Vertebrates May be Strictly Limited to Low-Metabolic Ectotherms.

The heart has a high-metabolic rate, and its "around-the-clock" vital role to sustain life sets it apart in a regenerative setting from other organs and appendages. The landscape of vertebrate species known to perform intrinsic heart regeneration is strongly biased toward ectotherms-for example, fish, salamanders, and embryonic/neonatal ectothermic mammals. It is hypothesized that intrinsic heart regeneration is exclusively limited to the low-metabolic hearts of ectotherms. The biomedical field of regenerative medicine seeks to devise biologically inspired regenerative therapies to diseased human hearts. Falsification of the ectothermy dependency for heart regeneration hypothesis may be a crucial prerequisite to meaningfully seek inspiration in established ectothermic regenerative animal models. Otherwise, engineering approaches to construct artificial heart components may constitute a more viable path toward regenerative therapies. A more strict definition of regenerative phenomena is generated and several testable sub-hypotheses and experimental avenues are put forward to elucidate the link between heart regeneration and metabolism. Also see the video abstract here https://youtu.be/fZcanaOT5z8.

Lymph Nodes Draining Infections Investigated by PET and Immunohistochemistry in a Juvenile Porcine Model.

BACKGROUND: [18F]FDG and [11C]methionine accumulate in lymph nodes draining S. aureus -infected foci. The lymph nodes were characterized by weight, [11C]methionine- and [18F]FDG-positron emissions tomography (PET)/computed tomography (CT), and immunohistochemical (IHC)-staining. METHODS: 20 pigs inoculated with S. aureus into the right femoral artery were PET/CT-scanned with [18F]FDG, and nine of the pigs were additionally scanned with [11C]methionine. Mammary, medial iliac, and popliteal lymph nodes from the left and right hind limbs were weighed. IHC-staining for calculations of area fractions of Ki-67, L1, and IL-8 positive cells was done in mammary and popliteal lymph nodes from the nine pigs. RESULTS: The pigs developed one to six osteomyelitis foci. Some pigs developed contiguous infections of peri-osseous tissue and inoculation-site abscesses. Weights of mammary and medial iliac lymph nodes and their [18F]FDG maximum Standardized Uptake Values (SUVFDGmax) showed a significant increase in the inoculated limb compared to the left limb. Popliteal lymph node weight and their FDG uptake did not differ significantly between hind limbs. Area fractions of Ki-67 and IL-8 in the right mammary lymph nodes and SUVMetmax in the right popliteal lymph nodes were significantly increased compared with the left side. CONCLUSION: The PET-tracers [18F]FDG and [11C]methionine, and the IHC- markers Ki-67 and IL-8, but not L1, showed increased values in lymph nodes draining soft tissues infected with S. aureus. The increase in [11C]methionine may indicate a more acute lymph node response, whereas an increase in [18F]FDG may indicate a more chronic response.

Radiotracers for Bone Marrow Infection Imaging.

INTRODUCTION: Radiotracers are widely used in medical imaging, using techniques of gamma-camera imaging (scintigraphy and SPECT) or positron emission tomography (PET). In bone marrow infection, there is no single routine test available that can detect infection with sufficiently high diagnostic accuracy. Here, we review radiotracers used for imaging of bone marrow infection, also known as osteomyelitis, with a focus on why these molecules are relevant for the task, based on their physiological uptake mechanisms. The review comprises [67Ga]Ga-citrate, radiolabelled leukocytes, radiolabelled nanocolloids (bone marrow) and radiolabelled phosphonates (bone structure), and [18F]FDG as established radiotracers for bone marrow infection imaging. Tracers that are under development or testing for this purpose include [68Ga]Ga-citrate, [18F]FDG, [18F]FDS and other non-glucose sugar analogues, [15O]water, [11C]methionine, [11C]donepezil, [99mTc]Tc-IL-8, [68Ga]Ga-Siglec-9, phage-display selected peptides, and the antimicrobial peptide [99mTc]Tc-UBI29-41 or [68Ga]Ga-NOTA-UBI29-41. CONCLUSION: Molecular radiotracers allow studies of physiological processes such as infection. None of the reviewed molecules are ideal for the imaging of infections, whether bone marrow or otherwise, but each can give information about a separate aspect such as physiology or biochemistry. Knowledge of uptake mechanisms, pitfalls, and challenges is useful in both the use and development of medically relevant radioactive tracers.

Preclinical Testing of Radiopharmaceuticals for the Detection and Characterization of Osteomyelitis: Experiences from a Porcine Model.

The development of new and better radioactive tracers capable of detecting and characterizing osteomyelitis is an ongoing process, mainly because available tracers lack selectivity towards osteomyelitis. An integrated part of developing new tracers is the performance of in vivo tests using appropriate animal models. The available animal models for osteomyelitis are also far from ideal. Therefore, developing improved animal osteomyelitis models is as important as developing new radioactive tracers. We recently published a review on radioactive tracers. In this review, we only present and discuss osteomyelitis models. Three ethical aspects (3R) are essential when exposing experimental animals to infections. Thus, we should perform experiments in vitro rather than in vivo (Replacement), use as few animals as possible (Reduction), and impose as little pain on the animal as possible (Refinement). The gain for humans should by far exceed the disadvantages for the individual experimental animal. To this end, the translational value of animal experiments is crucial. We therefore need a robust and well-characterized animal model to evaluate new osteomyelitis tracers to be sure that unpredicted variation in the animal model does not lead to a misinterpretation of the tracer behavior. In this review, we focus on how the development of radioactive tracers relies heavily on the selection of a reliable animal model, and we base the discussions on our own experience with a porcine model.

Attempts to Target Staphylococcus aureus Induced Osteomyelitis Bone Lesions in a Juvenile Pig Model by Using Radiotracers.

Background [18F]FDG Positron Emission Tomography cannot differentiate between sterile inflammation and infection. Therefore, we, aimed to develop more specific radiotracers fitted for differentiation between sterile and septic infection to improve the diagnostic accuracy. Consequently, the clinicians can refine the treatment of, for example, prosthesis-related infection. METHODS: We examined different target points; Staphylococcus aureus biofilm (68Ga-labeled DOTA-K-A9 and DOTA-GSGK-A11), bone remodeling ([18F]NaF), bacterial cell membranes ([68Ga]Ga-Ubiquicidin), and leukocyte trafficking ([68Ga]Ga-DOTA-Siglec-9). We compared them to the well-known glucose metabolism marker [18F]FDG, in a well-established juvenile S. aureus induced osteomyelitis (OM) pig model. RESULTS: [18F]FDG accumulated in the OM lesions seven days after bacterial inoculation, but disappointingly we were not able to identify any tracer accumulation in OM with any of the supposedly more specific tracers. CONCLUSION: These negative results are, however, relevant to report as they may save other research groups from conducting the same animal experiments and provide a platform for developing and evaluating other new potential tracers or protocol instead.

Evidence for bidirectional and trans-synaptic parasympathetic and sympathetic propagation of alpha-synuclein in rats.

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.

3Rs as part of preclinical neuropsychiatric translational crisis, and ARRIVE guidelines as part of solution.

OBJECTIVE: The ongoing translational and reproducibility crisis dominates preclinical research today as results from animal studies often disappoint when transferred to human clinical studies. This problem is especially relevant in the field of brain diseases and translational neuropsychiatry. METHODS: Here, we discuss if the 3R concept could be part of the translational crisis. RESULTS: The focus has been on the second R, which is to reduce the variation between the experimental animals, so that the number of animals per study can be reduced. However, the risk of obtaining false results has also increased. We, therefore, recommend that researchers use a broader perspective as also suggest by Russell and Burch who founded the 3Rs when considering the 3R concept, which involves the translational aspects described in detail in their 3R book from 1959. CONCLUSION: This may together with systematic reviews and well-designed and well-performed animal studies and accurate reporting of the results indeed contribute to solving the translational crisis in preclinical research.

Segmental innervation of the Göttingen minipig hind body. An electrophysiological study.

The Göttingen minipig is being used increasingly in biomedical research. The anatomical structure of the porcine peripheral nervous system has been extensively characterized, but no equivalent to the dermatome map, which is so valuable in human neurophysiological research, has been created. We characterized the medullar segmental skin and muscle innervations of the minipig hind body, using neurophysiological methodology. Six adult minipigs underwent unilateral laminectomy from L2 to S3, exposing the nerve roots. The skin of the hind part of the body was divided into 36 predefined fields, based on anatomical landmarks for consistent reproducibility. We recorded the evoked potential in each exposed nerve root L2-S3 for cutaneous stimulation of each skin field, mapping the sensory innervation of the entire hind body. We subsequently recorded the motor response in seven predefined muscles during sequential stimulation of the L2-S3 nerve roots. We obtained a clear sensory evoked potential in the nerve roots during stimulation of the skin fields, allowing us to map the sensory innervation of the minipig hind body. Neurophysiological data from skin stimulation and muscle recordings enabled us to map the sensory innervation of the Göttingen minipig hind body and provide information about muscular innervation. The skin fields were sensory innervated by more than one root. The muscles each had one dominant root with minor contribution from neighboring roots. This is consistent with experimental data from human studies.

In vivo quantification of glial activation in minipigs overexpressing human α-synuclein.

Parkinson's disease is characterized by a progressive loss of substantia nigra (SN) dopaminergic neurons and the formation of Lewy bodies containing accumulated alpha-synuclein (α-syn). The pathology of Parkinson's disease is associated with neuroinflammatory microglial activation, which may contribute to the ongoing neurodegeneration. This study investigates the in vivo microglial and dopaminergic response to overexpression of α-syn. We used positron emission tomography (PET) and the 18 kDa translocator protein radioligand, [11 C](R)PK11195, to image brain microglial activation and (+)-α-[11 C]dihydrotetrabenazine ([11 C]DTBZ), to measure vesicular monoamine transporter 2 (VMAT2) availability in Göttingen minipigs following injection with recombinant adeno-associated virus (rAAV) vectors expressing either mutant A53T α-syn or green fluorescent protein (GFP) into the SN (4 rAAV-α-syn, 4 rAAV-GFP, 5 non-injected control minipigs). We performed motor symptom assessment and immunohistochemical examination of tyrosine hydroxylase (TH) and transgene expression. Expression of GFP and α-syn was observed at the SN injection site and in the striatum. We observed no motor symptoms or changes in striatal [11 C]DTBZ binding potential in vivo or striatal or SN TH staining in vitro between the groups. The mean [11 C](R)PK11195 total volume of distribution was significantly higher in the basal ganglia and cortical areas of the α-syn group than the control animals. We conclude that mutant α-syn expression in the SN resulted in microglial activation in multiple sub- and cortical regions, while it did not affect TH stains or VMAT2 availability. Our data suggest that microglial activation constitutes an early response to accumulation of α-syn in the absence of dopamine neuron degeneration.

Cerebral blood flow measured by positron emission tomography during normothermic cardiopulmonary bypass: an experimental porcine study.

BACKGROUND: Mean arterial blood pressure (MAP) and/or pump flow during normothermic cardiopulmonary bypass (CPB) are the most important factors of cerebral perfusion. The aim of this study was to explore the influence of CPB blood flow on cerebral blood flow (CBF) measured by dynamic positron emission tomography (PET) using 15O-labelled water with no pharmacological interventions to maintain the MAP. METHODS: Eight pigs (69-71 kg) were connected to normothermic CPB. After 60 minutes (min) with a CPB pump flow of 60 mL/kg/min, the pigs were changed to either 35 mL/kg/min or 47.5 mL/kg/min for 60 min and, thereafter, all the pigs returned to 60 mL/kg/min for another 60 min. The MAP was measured continuously and the CBF was measured by positron emission tomography (PET) during spontaneous circulation and at each CPB pump flow after 30 min of steady state. RESULTS: Two pigs were excluded due to complications. CBF increased from spontaneous circulation to a CPB pump flow of 60 mL/kg/min. A reduction in CPB pump flow to 47.5 mL/kg/min (n=3) resulted in only minor changes in CBF while a reduction to 35 mL/kg/min (n=3) caused a pronounced change (correlation coefficient (R2) 0.56). A return of CPB pump flow to 60 mL/kg/min was followed by an increase in CBF, except in the one pig with the lowest CBF during low flow (R2=0.44). CBF and MAP were not correlated (R2=0.20). CONCLUSION: In this experimental porcine study, a relationship was observed between pump flow and CBF under normothermic low-flow CPB. The effect of low pump flow on MAP showed substantial variations, with no correlation between CBF and MAP.

Genetically modified pig models for neurodegenerative disorders.

Increasing incidence of neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease has become one of the most challenging health issues in ageing humans. One approach to combat this is to generate genetically modified animal models of neurodegenerative disorders for studying pathogenesis, prognosis, diagnosis, treatment, and prevention. Owing to the genetic, anatomic, physiologic, pathologic, and neurologic similarities between pigs and humans, genetically modified pig models of neurodegenerative disorders have been attractive large animal models to bridge the gap of preclinical investigations between rodents and humans. In this review, we provide a neuroanatomical overview in pigs and summarize and discuss the generation of genetically modified pig models of neurodegenerative disorders including Alzheimer's diseases, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis, spinal muscular atrophy, and ataxia-telangiectasia. We also highlight how non-invasive bioimaging technologies such as positron emission tomography (PET), computer tomography (CT), and magnetic resonance imaging (MRI), and behavioural testing have been applied to characterize neurodegenerative pig models. We further propose a multiplex genome editing and preterm recloning (MAP) approach by using the rapid growth of the ground-breaking precision genome editing technology CRISPR/Cas9 and somatic cell nuclear transfer (SCNT). With this approach, we hope to shorten the temporal requirement in generating multiple transgenic pigs, increase the survival rate of founder pigs, and generate genetically modified pigs that will more closely resemble the disease-causing mutations and recapitulate pathological features of human conditions.

Using Activity Measures and GNSS Data from a Virtual Fencing System to Assess Habitat Preference and Habitat Utilisation Patterns in Cattle.

There has been an increased focus on new technologies to monitor habitat use and behaviour of cattle to develop a more sustainable livestock grazing system without compromising animal welfare. One of the currently used methods for monitoring cattle behaviour is tri-axial accelerometer data from systems such as virtual fencing technology or bespoke monitoring technology. Collection and transmission of high-frequency accelerometer and GNSS data is a major energy cost, and quickly drains the battery in contemporary virtual fencing systems, making it unsuitable for long-term monitoring. In this paper, we explore the possibility of determining habitat preference and habitat utilisation patterns in cattle using low-frequency activity and location data. We achieve this by (1) calculating habitat selection ratios, (2) determining daily activity patterns, and (3) based on those, inferring grazing and resting sites in a group of cattle wearing virtual fencing collars in a coastal setting with grey, wooded, and decalcified dunes, humid dune slacks, and salt meadows. We found that GNSS data, and a measure of activity, combined with accurate mapping of habitats can be an effective tool in assessing habitat preference. The animals preferred salt meadows over the other habitats, with wooded dunes and humid dune slacks being the least preferred. We were able to identify daily patterns in activity. By comparing general trends in activity levels to the existing literature, and using a Gaussian mixture model, it was possible to infer resting and grazing behaviour in the different habitats. According to our inference of behaviour the herd predominantly used the salt meadows for resting and ruminating. The approach used in this study allowed us to use GNSS location data and activity data and combine it with accurate habitat mapping to assess habitat preference and habitat utilisation patterns, which can be an important tool for guiding management decisions.

Element Concentrations and Histopathology of Liver and Kidney in West Greenland Ringed Seals (Pusa hispida).

Ringed seals are consumed in Greenland and are therefore included as a key biomonitoring species with the focus on pollution exposure and health effects. Ringed seals in Central West Greenland (Qeqertarsuaq) and in North West Greenland (Qaanaaq) were analyzed for metal concentrations in the liver and histological changes in the liver and kidney. The mean liver concentration of mercury in Qaanaaq was 3.73 ± 5.01 µg/g ww (range: 0.28-23.29 µg/g ww), and the mean cadmium concentration was 7.80 ± 8.95 µg/g ww (range: 0.013-38.79 µg/g ww). For Qeqertarsuaq, the liver concentration of mercury was 1.78 ± 1.70 µg/g ww (range: 0.45-8.00 µg/g ww) and the mean cadmium concentration was 11.58 ± 6.32 µg/g ww (range: 0.11-25.45 µg/g ww). Age had a positive effect on the liver concentrations of metals, while no effect was found for sex or histological changes. The prevalence of histological changes in liver tissue decreased in the following order: random pattern mononuclear cell infiltration (92.1%), portal cell infiltration (68.4%), hepatic intracellular fat (18.4%), portal fibrosis (7.9%), focal hepatic fibrosis (7.9%), bile duct hyperplasia/fibrosis (7.9%) and lipid granuloma (2.6%). For kidney tissue, the prevalence of histological changes decreased in the following order: glomerular mesangial deposits (54.1%) > glomerular basement membrane thickening (45.9%) > THD (40%) > tubular hyaline casts (14.0%) > glomerular atrophy (13.5%) > dilated tubules (13.5%) > glomerular hyper-cellularity (10.8%) > mononuclear cell infiltrations (8.1%).

Spatial Distribution and Hierarchical Behaviour of Cattle Using a Virtual Fence System.

Interest in virtual fencing has increased due to its flexibility for agriculture and rewilding. However, systems like Nofence© require large financial investments, and the need for individual collars complicates large-scale use. If cattle herds maintain cohesive groups around leading individuals, fewer collars could be used, thereby enhancing cost efficiency. This study investigates the pattern in spatial distribution in a herd of 17 Angus cows on Fanø in Denmark with GPS locations, using a Nofence© system. The aim of this paper is to determine how individuals position themselves in a herd, spatially, and identify a pattern in ranks. The method used in this study examines the distances between an individual to the rest of the herdmates using nearest neighbour and descriptive statistics. Contrary to expectations, this study did not reveal a distinct pattern in herd distribution. While some tendencies in spatial distribution patterns were observed, only a low concordance could be found (W=0.15,p<0.001), indicating great variability in the cattle's ranks. A cumulative curve of the ranks estimated over the entire periods, however, allowed a rough estimation of the hierarchy and allowed identification of the highest-ranked cows, making the use of a cumulative curve a possible solution to finding the high-ranked cows. This research underscores the complexity of cattle social structures and highlights the need for extended observation periods and alternative methodologies to enhance the cost-effectiveness and scalability of virtual fencing in agricultural and rewilding contexts.

Effects of Social Facilitation and Introduction Methods for Cattle on Virtual Fence Adaptation.

Agricultural industries rely on physical fences to manage livestock. However, these present practical, financial, and ecological challenges, which may be solved using virtual fencing. This study aimed to identify how experienced cattle through social facilitation and the introduction method influence inexperienced cattle. Based on three stocks held in Fanø, Denmark, containing 12, 17 and 13 Angus (Bos taurus), we examined the virtual fence learning in three case studies using one gradual introduction with zero experienced cattle (A) and two different instant introductions with one (B) and ten (C) experienced cattle. Gradual introduction had the virtual fence moved 20 m every other day for eleven days, and in the two instant introductions, the physical fence was removed in one day. Warnings and impulses were recorded during an 11-day learning period and a 26-day post-learning period, using the impulses per warning to quantify if the cattle adapted. Case studies A and B showed a significant reduction in the warnings and impulses, but only A showed a significant reduction in the impulses per warning when comparing the learning period to the post-learning period. Due to the non-standardised experiments, it was not possible to conclude if the number of experienced cattle or the introduction method had an effect on the results.

Special Challenges in PET Imaging of Ectothermic Vertebrates.

The bulk of biomedical positron emission tomography (PET)-scanning experiments are performed on mammals (ie, rodents, pigs, and dogs), and the technique is only infrequently applied to answer research questions in ectothermic vertebrates such as fish, amphibians, and reptiles. Nevertheless, many unique and interesting physiological characteristics in these ectothermic vertebrates could be addressed in detail through PET. The low metabolic rate of ectothermic animals, however, may compromise the validity of physiological and biochemical parameters derived from the images created by PET and other scanning modalities. Here, we review some of the considerations that should be taken into account when PET scanning fish, amphibians, and reptiles. We present specific results from our own experiments, many of which remain previously unpublished, and we draw on examples from the literature. We conclude that knowledge on the natural history and physiology of the species studied and an understanding of the limitations of the PET scanning techniques are necessary to avoid the design of faulty experiments and erroneous conclusions.

Facing Danger: Exploring Personality and Reactions of European Hedgehogs (Erinaceus europaeus) towards Robotic Lawn Mowers.

The populations of European hedgehog (Erinaceus europaeus) are in decline, and it is essential that research identifies and mitigates the factors causing this. Hedgehogs are increasingly sharing habitats with humans, being exposed to a range of dangers in our backyards. Previous research has documented that some models of robotic lawn mowers can cause harm to hedgehogs. This study explored the personality and behaviour of 50 live hedgehogs when facing an approaching, disarmed robotic lawn mower. By combining a novel arena and novel object test, we found that 27 hedgehogs could be categorised as "shy" and 23 as "bold", independently of sex and age. The encounter tests with a robotic lawn mower showed that the hedgehogs positioned themselves in seven different ways. Personality did not affect their reactions. Adult hedgehogs tended to react in a shyer manner, and the hedgehogs, generally, acted less boldly during their second encounter with the robotic lawn mower. Additionally, our results show that bold individuals reacted in a more unpredictable way, being more behaviourally unstable compared to the shy individuals. This knowledge will be applied in the design of a standardised hedgehog safety test, eventually serving to produce and approve hedgehog-friendly robotic lawn mowers.

Testing the Impact of Robotic Lawn Mowers on European Hedgehogs (Erinaceus europaeus) and Designing a Safety Test.

Previous research has established that some models of robotic lawn mowers are potentially harmful to hedgehogs. As the market for robotic lawn mowers is expanding rapidly and the populations of European hedgehogs (Erinaceus europaeus) are in decline, it is important to investigate this risk further to understand the potential threat which some robotic lawn mowers may pose to hedgehogs. We tested 19 models of robotic lawn mowers in collision with hedgehog cadavers to measure their effect on hedgehogs. Our results showed that some models of robotic lawn mowers may injure hedgehogs, whereas others are not harmful to them. Apart from one single incidence, all robotic lawn mowers had to physically touch the hedgehog carcasses to detect them. Larger hedgehog cadavers were less likely to be "injured", with height being the most influential measure of size. The firmness of the tested hedgehog cadavers (frozen or thawed) did not influence the outcome of the collision tests. Neither the position of the hedgehog cadavers nor the selected technical features of the lawn mowers affected the probability of injury. Based on the results, we designed a standardised safety test to measure the effect of a specific model of robotic lawn mower on hedgehogs.

A self-unfolding proximity enabling device for oral delivery of macromolecules.

Oral delivery of macromolecules remains highly challenging due to their rapid degradation in the gastrointestinal tract and poor absorption across the tight junctions of the epithelium. In the last decade, researchers have investigated several medical devices to overcome these challenges using various approaches, some of which involve piercing through the intestine using micro and macro needles. We have developed a new generation of medical devices called self-unfolding proximity enabling devices, which makes it possible to orally deliver macromolecules without perforating the intestine. These devices protect macromolecules from the harsh conditions in the stomach and release their active pharmaceutical ingredients in the vicinity of the intestinal epithelium. One device version is a self-unfolding foil that we have used to deliver insulin and nisin to rats and pigs respectively. In our study, this device has shown a great potential for delivering peptides, with a significant increase in the absorption of solid dosage of insulin by ∼12 times and nisin by ∼4 times in rats and pigs, respectively. With the ability to load solid dosage forms, our devices can facilitate enhanced absorption of minimally invasive oral macromolecule formulations.