Effect of atropine and propofol on the minimum anaesthetic concentration of isoflurane in the freshwater turtle Trachemys scripta (yellow-bellied slider).

OBJECTIVE: To determine if the administration of atropine would reduce the measured minimum anaesthetic concentration of isoflurane (MACisoflurane) in freshwater turtles - the yellow-bellied slider (Trachemys scripta scripta). STUDY DESIGN: Paired, blinded, randomized, prospective studies of 1) the effect of atropine in isoflurane anaesthetized freshwater turtles (T. scripta scripta) and 2) the effect of atropine in yellow-bellied sliders in which anaesthesia was induced with propofol and maintained with isoflurane. ANIMALS: T. scripta scripta (n = 8), female, adult. METHODS: Atropine (2 mg kg-1) or an isovolumetric control injection of saline was administered intraperitoneally 15 minutes prior to induction of anaesthesia with isoflurane. Individual MACisoflurane was then determined by end-tidal gas analysis in a bracketing design by an experimenter blinded to the administered drug, with a 2 week washout period. The experiment was repeated, with atropine (2 mg kg-1) or saline administered intravascularly in combination with propofol for anaesthetic induction. Linear mixed modelling was used to determine the effects of atropine and propofol on the individual MACisoflurane. Data are presented as mean ± standard deviation. RESULTS: Premedication with atropine significantly reduced MACisoflurane (p = 0.0039). In isoflurane-induced T. scripta scripta, MACisoflurane decreased from 4.2 ± 0.4% to 3.3 ± 0.8% when atropine had been administered. Propofol as an induction agent had a MAC-sparing effect (p < 0.001) such that MACisoflurane following propofol and a control injection of saline was 2.3 ± 1.0%, which decreased further to 1.5 ± 0.8% when propofol was combined with atropine. CONCLUSIONS AND CLINICAL RELEVANCE: Atropine, presumably by inhibiting parasympathetically mediated pulmonary artery constriction, decreases right-to-left cardiac shunting and the MACisoflurane in yellow-bellied sliders, and thereby may facilitate control of inhalant anaesthesia. Propofol can be used for induction of anaesthesia and reduces the required concentration of inhaled anaesthesia assessed 1.5 hours following induction.

The ongoing cut-down of the Amazon rainforest threatens the climate and requires global tree planting projects: A short review.

The Amazon rainforest has sustained human existence for more than 10,000 years. Part of this has been the way that the forest controls regional climate including precipitation important for the ecosystem as well as agroforestry and farming. In addition, the Amazon also affects the global weather systems, so cutting down the rainforest significantly increases the effects of climate change, threatening the world's biodiversity and causing local desertification and soil erosion. The current fire activities and deforestation in the Amazon rainforest therefore have consequences for global sustainability. In the light of this, the current decisions made in Brazil regarding an increase in Amazon deforestation require policy changes if the global ecosystems and biodiversity are not to be set to collapse. There is only one way to move forward and that is to increase efforts in sustainable development of the region including limitation in deforestation and to continuously measure and monitor the development. The G7 countries have offered Brazil financial support for at least 20 million euros for fighting the forest fires but the president denies receiving such financial support and says that it is more relevant to raise new forests in Europe. In fact, this is exactly what is happening in Denmark and China in order to reduce climate change. Such activities should be global and include South America, Europe, Africa and Asia where deforestation is important issue. Forest restoration reduces climate change, desertification, and preserves both the regional tropical and global environment if the wood is not burned at a later stage but instead used in e.g. roads as filling material. Changes are therefore needed through improved international understanding and agreements to better avoid the global climate changes, from cutting down the precious rainforest before it is too late as rainforest cannot be re-planted.

Environmental management of two of the world's most endangered marine and terrestrial predators: Vaquita and cheetah.

Two of the world most endangered marine and terrestrial species are at the brink of extinction. The vaquita (Phocoena sinus) is the smallest existing cetacean and the population has declined to barely 22 individuals now remaining in Mexico's Gulf of California. With the ongoing decline, it is likely to go extinct within few years. The primary threat to this species has been mortality as a result of by-catch from gillnet fishing as well as environmental toxic chemicals and disturbance. This has called for the need to establish a National Park within the Gulf of California to expand essential habitat and provide the critical ecosystem protection for vaquita to thrive and multiply, given that proper conservation enforcement and management of the park are accomplished. In the terrestrial environment, the cheetah (Acinonyx jubatus) is reduced to a low number worldwide with the Iran subpopulation currently listed as Critically Endangered and the Indian subpopulation already extinct. There is a need for conservation efforts due to habitat loss, but also an indication of the conspicuous threat of illegal trade and trafficking from Africa and Arab countries in the Middle East. Funds have also been set up to provide refuges for the cheetah by working directly with farmers and landowners, which is a critical movement in adaptive management. These are the potential options for the preservation and possibly the expansion of the overall vaquita and cheetah populations.

Discussion: Early life and lessons learned from mass extinctions.

The origin of life is surrounded by great mystery and difficult to answer since we only know the existence of life from our own globe Earth. However, it is important to understand how life arose and developed if we should understand biodiversity and mass extinctions to day. The present Discussion try shed light on this and discuss the newest finding and theories.

Pig slurry needs modifications to be a sustainable fertilizer in crop production.

Pig slurry from modern high-technological farms contain copper and zinc, which is under suspicion of being co-factors in the development of antibiotic resistance. In addition, these two elements contaminate the surrounding environment when spread in large quantities. Therefore, biogas plants and biomembranes should be used to remove hazardous substances such as copper, zink, antibiotics and antibiotic resistant bacteria before pig slurry is used as a fertilizer in crop production. That would allow recycling of phosphorous in a way that only to a limited extent affect the health of the environment.

Kinetic Modelling of [68Ga]Ga-DOTA-Siglec-9 in Porcine Osteomyelitis and Soft Tissue Infections.

BACKGROUND: [68Ga]Ga-DOTA-Siglec-9 is a positron emission tomography (PET) radioligand for vascular adhesion protein 1 (VAP-1), a protein involved in leukocyte trafficking. The tracer facilitates the imaging of inflammation and infection. Here, we studied the pharmacokinetic modelling of [68Ga]Ga-DOTA-Siglec-9 in osteomyelitis and soft tissue infections in pigs. METHODS: Eight pigs with osteomyelitis and soft tissue infections in the right hind limb were dynamically PET scanned for 60 min along with arterial blood sampling. The fraction of radioactivity in the blood accounted for by the parent tracer was evaluated with radio-high-performance liquid chromatography. One- and two-tissue compartment models were used for pharmacokinetic evaluation. Post-mortem soft tissue samples from one pig were analysed with anti-VAP-1 immunofluorescence. In each analysis, the animal's non-infected left hind limb was used as a control. RESULTS: Tracer uptake was elevated in soft tissue infections but remained low in osteomyelitis. The kinetics of [68Ga]Ga-DOTA-Siglec-9 followed a reversible 2-tissue compartment model. The tracer metabolized quickly; however, taking this into account, produced more ambiguous results. Infected soft tissue samples showed endothelial cell surface expression of the Siglec-9 receptor VAP-1. CONCLUSION: The kinetics of [68Ga]Ga-DOTA-Siglec-9 uptake in porcine soft tissue infections are best described by the 2-tissue compartment model.

Preclinical evaluation of potential infection-imaging probe [68 Ga]Ga-DOTA-K-A9 in sterile and infectious inflammation.

The development of bacteria-specific infection radiotracers is of considerable interest to improve diagnostic accuracy and enabling therapy monitoring. The aim of this study was to determine if the previously reported radiolabelled 1,4,7,10-tetraazacyclododecane-N,N',N″,N‴-tetraacetic acid (DOTA) conjugated peptide [68 Ga]Ga-DOTA-K-A9 could detect a staphylococcal infection in vivo and distinguish it from aseptic inflammation. An optimized [68 Ga]Ga-DOTA-K-A9 synthesis omitting the use of acetone was developed, yielding 93 ± 0.9% radiochemical purity. The in vivo infection binding specificity of [68 Ga]Ga-DOTA-K-A9 was evaluated by micro positron emission tomography/magnetic resonance imaging of 15 mice with either subcutaneous Staphylococcus aureus infection or turpentine-induced inflammation and compared with 2-deoxy-2-[18 F]fluoro-D-glucose ([18 F]FDG). The scans showed that [68 Ga]Ga-DOTA-K-A9 accumulated in all the infected mice at injected doses ≥3.6 MBq. However, the tracer was not found to be selective towards infection, since the [68 Ga]Ga-DOTA-K-A9 also accumulated in mice with inflammation. In a concurrent in vitro binding evaluation performed with a 5-carboxytetramethylrhodamine (TAMRA) fluorescence analogue of the peptide, TAMRA-K-A9, the microscopy results suggested that TAMRA-K-A9 bound to an intracellular epitope and therefore preferentially targeted dead bacteria. Thus, the [68 Ga]Ga-DOTA-K-A9 uptake observed in vivo is presumably a combination of local hyperemia, vascular leakiness and/or binding to an epitope present in dead bacteria.

CARDIOVASCULAR EFFECTS OF ALFAXALONE AND PROPOFOL IN THE BULLFROG, LITHOBATES CATESBEIANUS.

Alfaxalone is becoming a popular anesthetic for nonmammalian vertebrates, but the physiological effects of its administration remain largely unknown in these taxa. Therefore, the cardiovascular responses to a clinically relevant dose of alfaxalone (10 mg/kg) are reported in the bullfrog ( Lithobates catesbeianus), following intramuscular (IM) and intravascular (IV) administration (via a femoral artery catheter) and compared with an IV dose of propofol, another parenteral GABA (γ-aminobutyric acid) agonist in common veterinary use as an induction agent. Heart rate (HR) and mean arterial blood pressure (MAP) (assessed by direct measurement from the catheter) are reported from under undisturbed conditions to assess both the direct effects of the drugs and the interaction with the stress of handling associated with IM injection of alfaxalone where IM administration is possible. Alfaxalone caused HR to increase significantly for over 45 min in both groups from a baseline of approximately 30 beats/min. This was significantly different from the lack of significant HR response on the IV administration of propofol. MAP increased in the peri-injection period with both routes of administration for alfaxalone but after IV use decreased significantly from 10 min following administration. Propofol did not affect blood pressure after 5 min from injection. Assessment of immobilization following intramuscular injection of alfaxalone in a pilot study was in accordance with the literature, as it provided no antinociception as a sole agent but did produce sedation and loss of righting reflex.

Whole-Body Biodistribution, Dosimetry, and Metabolite Correction of [11C]Palmitate: A PET Tracer for Imaging of Fatty Acid Metabolism.

INTRODUCTION: Despite the decades long use of [11C]palmitate positron emission tomography (PET)/computed tomography in basic metabolism studies, only personal communications regarding dosimetry and biodistribution data have been published. METHODS: Dosimetry and biodistribution studies were performed in 2 pigs and 2 healthy volunteers by whole-body [11C]palmitate PET scans. Metabolite studies were performed in 40 participants (healthy and with type 2 diabetes) under basal and hyperinsulinemic conditions. Metabolites were estimated using 2 approaches and subsequently compared: Indirect [11C]CO2 release and parent [11C]palmitate measured by a solid-phase extraction (SPE) method. Finally, myocardial fatty acid uptake was calculated in a patient cohort using input functions derived from individual metabolite correction compared with population-based metabolite correction. RESULTS: In humans, mean effective dose was 3.23 (0.02) µSv/MBq, with the liver and myocardium receiving the highest absorbed doses. Metabolite correction using only [11C]CO2 estimates underestimated the fraction of metabolites in studies lasting more than 20 minutes. Population-based metabolite correction showed excellent correlation with individual metabolite correction in the cardiac PET validation cohort. CONCLUSION: First, mean effective dose of [11C]palmitate is 3.23 (0.02) µSv/MBq in humans allowing multiple scans using ∼300 MBq [11C]palmitate, and secondly, population-based metabolite correction compares well with individual correction.

Cholinergic PET imaging in infections and inflammation using 11C-donepezil and 18F-FEOBV.

INTRODUCTION: Immune cells utilize acetylcholine as a paracrine-signaling molecule. Many white blood cells express components of the cholinergic signaling pathway, and these are up-regulated when immune cells are activated. However, in vivo molecular imaging of cholinergic signaling in the context of inflammation has not previously been investigated. METHODS: We performed positron emission tomography (PET) using the glucose analogue 18F-FDG, and 11C-donepezil and 18F-FEOBV, markers of acetylcholinesterase and the vesicular acetylcholine transporter, respectively. Mice were inoculated subcutaneously with Staphylococcus aureus, and PET scanned at 24, 72, 120, and 144 h post-inoculation. Four pigs with post-operative abscesses were also imaged. Finally, we present initial data from human patients with infections, inflammation, and renal and lung cancer. RESULTS: In mice, the FDG uptake in abscesses peaked at 24 h and remained stable. The 11C-donepezil and 18F-FEOBV uptake displayed progressive increase, and at 120-144 h was nearly at the FDG level. Moderate 11C-donepezil and slightly lower 18F-FEOBV uptake were seen in pig abscesses. PCR analyses suggested that the 11C-donepezil signal in inflammatory cells is derived from both acetylcholinesterase and sigma-1 receptors. In humans, very high 11C-donepezil uptake was seen in a lobar pneumonia and in peri-tumoral inflammation surrounding a non-small cell lung carcinoma, markedly superseding the 18F-FDG uptake in the inflammation. In a renal clear cell carcinoma no 11C-donepezil uptake was seen. DISCUSSION: The time course of cholinergic tracer accumulation in murine abscesses was considerably different from 18F-FDG, demonstrating in the 11C-donepezil and 18F-FEOBV image distinct aspects of immune modulation. Preliminary data in humans strongly suggest that 11C-donepezil can exhibit more intense accumulation than 18F-FDG at sites of chronic inflammation. Cholinergic PET imaging may therefore have potential applications for basic research into cholinergic mechanisms of immune modulation, but also clinical applications for diagnosing infections, inflammatory disorders, and cancer inflammation.

When local anesthesia becomes universal: Pronounced systemic effects of subcutaneous lidocaine in bullfrogs (Lithobates catesbeianus).

Sodium channel blockers are commonly injected local anesthetics but are also routinely used for general immersion anesthesia in fish and amphibians. Here we report the effects of subcutaneous injection of lidocaine (5 or 50mgkg-1) in the hind limb of bullfrogs (Lithobates catesbeianus) on reflexes, gular respiration and heart rate (handled group, n=10) or blood pressure and heart rate via an arterial catheter (catheterized group n=6). 5mgkg-1 lidocaine did not cause loss of reflexes or change in heart rate in the handled group, but was associated with a reduction in gular respiratory rate (from 99±7 to 81±17breathsmin-1). 50mgkg-1 lidocaine caused a further reduction in respiratory rate to 59±15breathsmin-1, and led to a progressive loss of righting reflex (10/10 loss by 40min), palpebral reflex (9/10 loss at 70min), and contralateral toe pinch withdrawal (9/10 loss at 70min). Reflexes were regained over 4h. Systemic sedative effects were not coupled to local anti-nociception, as a forceps pinch test at the site of injection provoked movement at the height of the systemic effect (tested at 81±4min). Amphibians are routinely subject to general anesthesia via exposure to sodium channel blockers such as MS222 or benzocaine, however caution should be exercised when using local injectable lidocaine in amphibians, as it appears to dose-dependently cause sedation, without necessarily preventing local nociception for the duration of systemic effects.

Exploring the radiosynthesis and in vitro characteristics of [68 Ga]Ga-DOTA-Siglec-9.

Vascular adhesion protein 1 is a leukocyte homing-associated glycoprotein, which upon inflammation rapidly translocates from intracellular sources to the endothelial cell surface. It has been discovered that the cyclic peptide residues 283-297 of sialic acid-binding IgG-like lectin 9 (Siglec-9) "CARLSLSWRGLTLCPSK" bind to vascular adhesion protein 1 and hence makes the radioactive analogues of this compound ([68 Ga]Ga-DOTA-Siglec-9) interesting as a noninvasive visualizing marker of inflammation. Three different approaches to the radiosynthesis of [68 Ga]Ga-DOTA-Siglec-9 are presented and compared with previously published methods. A simple, robust radiosynthesis of [68 Ga]Ga-DOTA-Siglec-9 with a yield of 62% (non decay-corrected) was identified, and it had a radiochemical purity >98% and a specific radioactivity of 35 MBq/nmol. Furthermore, the protein binding and stability of [68 Ga]Ga-DOTA-Siglec-9 were analyzed in vitro in mouse, rat, rabbit, pig, and human plasma and compared with in vivo pig results. The plasma in vitro protein binding of [68 Ga]Ga-DOTA-Siglec-9 was the lowest in the pig followed by rabbit, human, rat, and mouse. It was considerably higher in the in vivo pig experiments. The in vivo stability in pigs was lower than the in vitro stability. Despite considerable species differences, the observed characteristics of [68 Ga]Ga-DOTA-Siglec-9 are suitable as a positron emission tomography tracer.

Mice and minipigs with compromised expression of the Alzheimer's disease gene SORL1 show cerebral metabolic disturbances on hyperpolarized [1-13C]pyruvate and sodium MRI.

The sortilin-related receptor 1 (SORL1) gene, encoding the cellular endosomal sorting-related receptor with A-type repeats (SORLA), is now established as a causal gene for Alzheimer's disease. As the latest addition to the list of causal genes, the pathophysiological effects and biomarker potential of SORL1 variants remain relatively undiscovered. Metabolic dysfunction is, however, well described in patients with Alzheimer's disease and is used as an imaging biomarker in clinical diagnosis settings. To understand the metabolic consequences of loss-of-function SORL1 mutations, we applied two metabolic MRI technologies, sodium (23Na) MRI and MRI with hyperpolarized [1-13C]pyruvate, in minipigs and mice with compromised expression of SORL1. At the age analysed here, both animal models display no conventional imaging evidence of neurodegeneration but show biochemical signs of elevated amyloid production, thus representing the early preclinical disease. With hyperpolarized MRI, the exchange from [1-13C]pyruvate to [1-13C]lactate and 13C-bicarbonate was decreased by 32 and 23%, respectively, in the cerebrum of SORL1-haploinsufficient minipigs. A robust 11% decrease in the sodium content was observed with 23Na-MRI in the same minipigs. Comparably, the brain sodium concentration gradually decreased from control to SORL1 haploinsufficient (-11%) to SORL1 knockout mice (-23%), suggesting a gene dose dependence in the metabolic dysfunction. The present study highlights that metabolic MRI technologies are sensitive to the functional, metabolic consequences of Alzheimer's disease and Alzheimer's disease-linked genotypes. Further, the study suggests a potential avenue of research into the mechanisms of metabolic alterations by SORL1 mutations and their potential role in neurodegeneration.

Gross pathology and liver mercury concentrations in harbour porpoises, harbour seals and grey seals in Denmark, Northern Europe.

Here we report the first investigation of gross pathology and mercury (Hg) in liver tissue from harbour porpoises, harbour seals and grey seals from Denmark, Northern Europe. Mercury concentrations ranged between 0.2 and 248 µg/g wet weight (ww) with highest concentrations found in grey seals and subadult harbour seals from the Baltic Sea, with no relationship to body condition. Necropsy findings across all three species decreasing in the following order: pneumonia (n = 60) > respiratory parasitism (n = 56) > wounds (n = 18) > GI-parasites (stomach nematodes and/or parasitic colitis) (n = 16) > ectoparasites (skin lice) (n = 12) > hepatic parasites/lesions (n = 8) > focal alopecia (n = 5) > nephropathy (n = 4) > middle ear complex parasites (n = 3) > nasal parasites (n = 2). Heart and/or lung worms were significantly highest in subadult harbour porpoises, GI parasites and nephropathy significantly lowest in subadult harbour seals and focal alopecia significantly highest in subadult harbour seals. Most cases of pneumonia were associated with respiratory parasites (68 %), while nine cases of wounds led to signs of septicaemia. Significant positive relationships were observed between Hg and the presence of respiratory parasites in subadult harbour porpoises and between Hg and the presence of focal alopecia, nephropathy, and gastrointestinal parasites in subadult harbour seals. Levels of Hg were in the categories for low risk (16-64 µg/g ww) in 18 %, moderate risk (64-83 µg/g ww) in 3 %, high risk (83-123 µg/g ww) in 2 % and severe risk (>123 µg/g ww) in 3 % of all individuals for health effects in marine mammals. In conclusion, using marine mammals as integrative sentinel species for Danish North Sea, Inner Danish Waters and the Baltic Sea ecosystems provides monitoring of ocean health in terms of multiple stressors such as anthropogenic contaminants and infectious diseases all being important in the context of global change.

Preclinical Imaging Studies: Protocols, Preparation, Anesthesia, and Animal Care.

Today preclinical PET imaging connects laboratory research with clinical applications. Here PET clearly bridges the gap, as nearly identical imaging protocols can be applied to both animal and humans. However, some hurdles exist and researchers must be careful, partly because the animals are usually anesthetized during the scans, while human volunteers are awake. This review is based on our own experiences of some of the most important pitfalls and how to overcome them. This includes how studies should be designed, how to select the right anesthesia and monitoring. The choice of anesthesia is quite crucial, as it may have a greater influence on the results than the effect of the tested procedures. Monitoring is necessary, as the animals cannot fully maintain homeostasis during anesthesia, and reliable results are dependent on a stable physiology. Additionally, it is important to note that rodents, in particular, are prone to rapidly becoming hypothermic. Thus, the selection of an appropriate anesthetic and monitoring protocol is crucial for both obtaining accurate results and ensuring animal welfare. Prior to imaging, catheters for tracer administration and, if necessary, blood sampling should be implanted. The administration of tracers should be done in a manner that minimizes interference with the scans, and the same applies to any serial blood sampling. The limited blood volume and organ size of rodents should also be taken into consideration when planning experiments. Finally, if the animal needs to be awakened after the scan, proper care must be taken to ensure their welfare.