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Prostaglandin E(2) receptors (EP-Rs) belong to the family of heterotrimeric G protein-coupled ectoreceptors with seven transmembrane domains. They can be subdivided into four subtypes according to their ligand-binding and G protein-coupling specificity: EP1 couple to G(q), EP2 and EP4 to G(s), and EP3 to G(i). The EP4-R, in contrast to the EP3beta-R, shows rapid agonist-induced desensitization. The agonist-induced desensitization depends on the presence of the EP4-R carboxyl-terminal domain, which also confers desensitization in a G(i)-coupled rEP3hEP4 carboxyl-terminal domain receptor hybrid (rEP3hEP4-Ct-R). To elucidate the possible mechanism of this desensitization, in vivo phosphorylation stimulated by activators of second messenger kinases, by prostaglandin E(2), or by the EP3-R agonist M&B28767 was investigated in COS-7 cells expressing FLAG-epitope-tagged rat EP3beta-R (rEP3beta-R), hEP4-R, or rEP3hEP4- Ct-R. Stimulation of protein kinase C with phorbol-12-myristate-13-acetate led to a slight phosphorylation of the FLAG- rEP3beta-R but to a strong phosphorylation of the FLAG-hEP4-R and the FLAG-rEP3hEP4-Ct-R, which was suppressed by the protein kinase A and protein kinase C inhibitor staurosporine. Prostaglandin E(2) stimulated phosphorylation of the FLAG- hEP4-R in its carboxyl-terminal receptor domain. The EP3-R agonist M&B28767 induced a time- and dose-dependent phosphorylation of the FLAG-rEP3hEP4-Ct-R but not of the FLAG-rEP3beta-R. Agonist-induced phosphorylation of the FLAG- hEP4-R and the FLAG-rEP3hEP4-Ct-R were not inhibited by staurosporine, which implies a role of G protein-coupled receptor kinases (GRKs) in agonist-induced receptor phosphorylation. Overexpression of GRKs in FLAG-rEP3hEP4-Ct-R- expressing COS-7 cells augmented the M&B28767-induced receptor phosphorylation and receptor sequestration. These findings indicate that phosphorylation of the carboxyl-terminal hEP4-R domain possibly by GRKs but not by second messenger kinases may be involved in rapid agonist-induced desensitization of the hEP4-R and the rEP3hEP4-Ct-R.
Background and Purpose Recent studies suggested a role for PGE2 in the expression of the chemokine IL-8. PGE2 signals via four different GPCRs, EP1-EP4. The role of EP1 and EP4 receptors for IL-8 induction was studied in HEK293 cells, overexpressing EP1 (HEK-EP1), EP4 (HEK-EP4) or both receptors (HEK-EP1 + EP4). Experimental Approach IL-8 mRNA and protein induction and IL-8 promoter and NF-?B activation were assessed in EP expressing HEK cells. Key Results In HEK-EP1 and HEK-EP1 + EP4 but not HEK or HEK-EP4 cells, PGE2 activated the IL-8 promoter and induced IL-8 mRNA and protein synthesis. Stimulation of HEK-EP1 + EP4 cells with an EP1-specific agonist activated IL-8 promoter and induced IL-8 mRNA and protein, whereas a specific EP4 agonist neither activated the IL-8 promoter nor induced IL-8 mRNA and protein synthesis. Simultaneous stimulation of HEK- EP1 + EP4 cells with both agonists activated IL-8 promoter and induced IL-8 mRNA to the same extent as PGE2. In HEK-EP1 + EP4 cells, PGE2-mediated IL-8 promoter activation and IL-8 mRNA induction were blunted by inhibition of I?B kinase. PGE2 activated NF-?B in HEK-EP1, HEK-EP4 and HEK-EP1 + EP4 cells. In HEK-EP1 + EP4 cells, simultaneous activation of both receptors was needed for maximal PGE2-induced NF-?B activation. PGE2-stimulated NF-?B activation by EP1 was blocked by inhibitors of PLC, calcium-signalling and Src-kinase, whereas that induced by EP4 was only blunted by Src-kinase inhibition. Conclusions and Implications These findings suggest that PGE2-mediated NF-?B activation by simultaneous stimulation of EP1 and EP4 receptors induces maximal IL-8 promoter activation and IL-8 mRNA and protein induction.
Background & purpose: Recent studies suggested a role of prostaglandin E-2 (PGE(2)) in the expression of the chemokine IL-8 by monocytes. The function of EP4 receptor for TNF alpha-induced IL-8 expression was studied in monocytic cell lines. Experimental approach: IL-8 mRNA and protein induction as well as IL-8 promoter activity and transcription factor activation were assessed in monocytic cell lines, primary blood mononuclear cells (PBMC) and transgenic HEK293 cells expressing the EP4 receptor. Key results: In monocytic cell lines THP-1, MonoMac and U937 PGE(2) had only a marginal impact on IL-8 induction but strongly enhanced TNFa-induced IL-8 mRNA and protein synthesis. Similarly, in PBMC IL-8 mRNA induction was larger by simultaneous stimulation with TNF alpha and PGE(2) than by either stimulus alone. The EP4 receptor subtype was the most abundant EP receptor in all three cell lines and in PBMC. Stimulation of THP-1 cells with an EP4 specific agonist enhanced TNF alpha-induced IL-8 mRNA and protein formation to the same extent as PGE(2). In HEK293 cells expressing EP4, but not in wild type HEK293 cells lacking EP4, PGE(2) enhanced TNFainduced IL-8 protein and mRNA synthesis. In THP-1 cells, the enhancement of TNF alpha-mediated IL-8 mRNA induction by PGE(2) was mimicked by a PICA-activator. Furthermore in these cells PGE(2) induced expression of transcription factor C/EBPS, enhanced NF-KB activation by TNFa and inhibited TNF alpha-mediated AP-1 activation. PGE(2) and TNF alpha synergistically activated transcription factor CREB, induced C/EBPS expression and enhanced the activity of an IL-8 promoter fragment containing-223 bp upstream of the transcription start site. Conclusions and implications: These findings suggest that a combined stimulation of TNF alpha and PGE(2)/EP4 signal chains in monocytic cells leads to maximal IL-8 promoter activity, as well as IL-8 mRNA and protein induction, by activating the PICA/CREB/C/EB1313 as well as NF-kappa B signal chains.
Botulinum neurotoxin (BoNT) is used for the treatment of a number of ailments. The activity of the toxin that is isolated from bacterial cultures is frequently tested in the mouse lethality assay. Apart from the ethical concerns inherent to this assay, species-specific differences in the affinity for different BoNT serotypes give rise to activity results that differ from the activity in humans. Thus, BoNT/B is more active in mice than in humans. The current study shows that the stimulus-dependent release of a luciferase from a differentiated human neuroblastoma–based reporter cell line (SIMA-hPOMC1-26-Gluc) was inhibited by clostridial and recombinant BoNT/A to the same extent, whereas both clostridial and recombinant BoNT/B inhibited the release to a lesser extent and only at much higher concentrations, reflecting the low activity of BoNT/B in humans. By contrast, the genetically modified BoNT/B-MY, which has increased affinity for human synaptotagmin, and the BoNT/B protein receptor inhibited luciferase release effectively and with an EC50 comparable to recombinant BoNT/A. This was due to an enhanced uptake into the reporter cells of BoNT/B-MY in comparison to the recombinant wild-type toxin. Thus, the SIMA-hPOMC1-26-Gluc cell assay is a versatile tool to determine the activity of different BoNT serotypes providing human-relevant dose-response data.
Botulinum neurotoxin (BoNT) is used for the treatment of a number of ailments. The activity of the toxin that is isolated from bacterial cultures is frequently tested in the mouse lethality assay. Apart from the ethical concerns inherent to this assay, species-specific differences in the affinity for different BoNT serotypes give rise to activity results that differ from the activity in humans. Thus, BoNT/B is more active in mice than in humans. The current study shows that the stimulus-dependent release of a luciferase from a differentiated human neuroblastoma–based reporter cell line (SIMA-hPOMC1-26-Gluc) was inhibited by clostridial and recombinant BoNT/A to the same extent, whereas both clostridial and recombinant BoNT/B inhibited the release to a lesser extent and only at much higher concentrations, reflecting the low activity of BoNT/B in humans. By contrast, the genetically modified BoNT/B-MY, which has increased affinity for human synaptotagmin, and the BoNT/B protein receptor inhibited luciferase release effectively and with an EC50 comparable to recombinant BoNT/A. This was due to an enhanced uptake into the reporter cells of BoNT/B-MY in comparison to the recombinant wild-type toxin. Thus, the SIMA-hPOMC1-26-Gluc cell assay is a versatile tool to determine the activity of different BoNT serotypes providing human-relevant dose-response data.
Prostaglandin (PG)F₂α has previously been shown to increase glucose output from perfused livers and isolated hepatocytes, where it stimulated glycogen phosphorylase via an inositol-trisphosphatedependent signal pathway. In this study, PGF₂α binding sites on hepatocyte plasma membranes, that might represent the putative receptor, were characterized. Binding studies could not be performed with intact hepatocytes, because PGF₂α accumulated within the cells even at 4°C. The intracellular accumulation was an order of magnitude higher than binding to plasma membranes. Purified hepatocyte plasma membranes had a high-affinity/low-capacity and a low-affinity/highcapacity binding'site for PGF₂α. The respective binding constants for the high-affinity site were Kd = 3 nM and Bmax = 6 fmol/mg membrane protein, and for the low-affinity site Kd = 426 nM and Bmax = 245 fmol/mg membrane protein. Specific PGF₂α binding to the low-affinity site, but not to the high-affinity site, could be enhanced most potently by GTP[γS] followed by GDP[ϐS] and GTP, but not by ATP[γS] or GMP. PGF₂α competed most potently with [³H]PGF₂α for specific binding to hepatocyte plasma membranes, followed by PGD₂ and PGE₂. Since the low-affinity PGF₂α-binding site had a Kd in the concentration range in which PG had previously been shown to be half-maximally active, and since this binding site showed a sensitivity to GTP, it is concluded that it might represent the receptor involved in the PGF₂α signal chain in hepatocytes. A biological function of the high-affinity site is currently not known.
Die Expansion des renalen Tubulointerstitiums aufgrund einer Akkumulation zellulärer Bestandteile und extrazellulärer Matrix ist eine charakteristische Eigenschaft der chronischen Nierenerkrankung (CKD) und führt zu einer Progression der Erkrankung in Richtung eines terminalen Nierenversagens. Die Fibroblasten Proliferation und ihre Transformation hin zum sekretorischen Myofibroblasten-Phänotyp stellen hierbei Schlüsselereignisse dar. Signalprozesse, die zur Induktion der Myofibroblasten führen, werden aktiv beforscht um anti-fibrotische Therapieansätze zu identifizieren. Das anti-inflammatorische Protein Annexin A1 und sein Rezeptor Formyl-Peptid Rezeptor 2 (FPR2) wurden in verschiedenen Organsystemen mit der Regulation von Fibroblastenaktivität in Verbindung gebracht, jedoch wurden ihre Expression und Funktion bei renalen fibrotischen Erkrankungen bisher nicht untersucht. Ziel der aktuellen Studie war daher die Untersuchung der renalen Annexin A1- und FPR2-Expression in einem Tiermodell des chronischen Nierenversagens, sowie die Charakterisierung der funktionellen Rolle von Annexin A1 in der Regulation des Fibroblasten Phänotyps und ihrer Syntheseleistung. Dazu wurden neugeborene Sprague-Dawley Ratten in den ersten zwei Wochen ihres Lebens entweder mit Vehikel oder mit einem Angiotensin II Typ I Rezeptor Antagonisten behandelt und ohne weitere Intervention bis zu einem Alter von 11 Monaten (CKD Ratten) gehalten. Die Regulation und Lokalisation von Annexin A1 und FPR2 wurden mit Hilfe von Real-Time PCR und Immunhistochemie erfasst. Annexin A1- und FPR2-exprimierende Zellen wurden weiter durch Doppelimmunfluoreszenzfärbungen charakterisiert. Gefärbt wurde mit Antikörpern gegen endotheliale Zellen (rat endothelial cell antigen), Makrophagen (CD 68), Fibroblasten (CD73) und Myofibroblasten (alpha-smooth muscle actin (α-sma)). Zellkulturstudien wurden an immortalisierten renalen kortikalen Fibroblasten aus Wildtyp- und Annexin A1-defizienten Mäusen, sowie an etablierten humanen und murinen renalen Fibrolasten durchgeführt. Eine Überexpression von Annexin A1 wurde durch eine stabile Transfektion erreicht. Die Expression von Annexin A1, α-sma und Kollagen 1α1 wurde durch Real-Time PCR, Western Blot und Immuhistochemie erfasst. Die Sekretion des Annexin A1 Proteins wurde nach TCA-Fällung des Zellkulturüberstandes im Western Blot untersucht. Wie zu erwarten zeigten die CKD Ratten eine geringere Anzahl an Nephronen mit deutlicher glomerulären Hypertrophie. Der tubulointerstitielle Raum war durch fibrilläres Kollagen, aktivierte Fibroblasten und inflammatorische Zellen expandiert. Parallel dazu war die mRNA Expression von Annexin A1 und Transforming growth factor beta (TGF-β) signifikant erhöht. Die Annexin A1-Lokalisation mittels Doppelimmunfluorsezenz identifizierte eine große Anzahl von CD73-positiven kortikalen Fibroblasten und eine Subpopulation von Makrophagen als Annexin A1-positiv. Die Annexin A1-Menge in Myofibroblasten und renalen Endothelien war gering. FPR2 konnte in der Mehrzahl der renalen Fibroblasten, in Myofibroblasten, in einer Subpopulation von Makrophagen und in renalen Epithelzellen nachgewiesen werden. Eine Behandlung der murinen Fibroblasten mit dem pro-fibrotischen Zytokin TGF-β führte zu einem parallelen Anstieg der α-sma-, Kollagen 1α1- und Annexin A1-Biosynthese und zu einer gesteigerten Sekretion von Annexin A1. Eine Überexpression von Annexin A1 in murinen Fibroblasten reduzierte das Ausmaß der TGF-β induzierten α-sma- und Kollagen 1α1-Biosynthese. Fibroblasten aus Annexin A1-defizienten Mäusen zeigten einen starken Myofibroblasten-Phänotyp mit einer gesteigerten Expression an α-sma und Kollagen 1α1. Der Einsatz eines Peptidantagonisten des FPR2 (WRW4) resultierte in einer Stimulation der α-sma-Biosynthese, was die Vermutung nahe legte, dass Annexin A1 FPR2-vermittelt anti-fibrotische Effekte hat. Zusammenfassend zeigen diese Ergebnisse, dass renale kortikale Fibroblasten eine Hauptquelle des Annexin A1 im renalen Interstitium und einen Ansatzpunkt für Annexin A1-Signalwege in der Niere darstellen. Das Annexin A1/FPR2-System könnte daher eine wichtige Rolle in der Kontrolle des Fibroblasten Phänotyp und der Fibroblasten Aktivität spielen und daher einen neuen Ansatz für die anti-fibrotischen pharmakologischen Strategien in der Behandlung des CKD darstellen.
Intercropping is widespread in small-holder farming systems in tropical regions and is also practiced in the cultivation of indigenous vegetables, to alleviate the multiple burdens of malnutrition. Due to interspecific competition and/or complementation between intercrops, intercropping may lead to changes in plants accumulation of minerals and secondary metabolites and hence, alter nutritional quality for consumers. Intercropping aims to intensify land productivity, while ensuring that nutritional quality is not compromised. This study aimed to investigate changes in minerals and secondary plant metabolites in intercropped Brassica carinata and Solanum scabrum, two important African indigenous vegetables, and evaluated the suitability of this combination for dryer areas. B. carinata and S. scabrum were grown for 6 weeks under controlled conditions in a greenhouse trial. Large rootboxes (8000 cm(3) volume) were specifically designed for this experiment. Each rootbox was planted with two plants, either of the same plant species (mono) or one of each plant species (mixed). A quartz sand/soil substrate was used and fertilized adequately for optimal plant growth. During the last 4 weeks of the experiment, the plants were either supplied with optimal (65% WHC) or low (30% WHC) irrigation, to test the effect of a late-season drought. Intercropping increased total glucosinolate content in B. carinata, while maintaining biomass production and the contents of other health related minerals in both B. carinata and S. scabrum. Moreover, low irrigation led to an increase in carotene accumulation in both mono and intercropped S. scabrum, but not in B. carinata, while the majority of kaempferol glycosides and hydroxycinnamic acid derivatives of both species were decreased by intercropping and drought treatment. This study indicates that some health-related phytochemicals can be modified by intercropping or late-season drought, but field validation of these results is necessary before definite recommendation can be made to stakeholders.
Mechanistic studies on the adverse effects of manganese overexposure in differentiated LUHMES cells
(2022)
Manganese (Mn) is an essential trace element, but overexposure is associated with toxicity and neurological dysfunction. Accumulation of Mn can be observed in dopamine-rich regions of the brain in vivo and Mn-induced oxidative stress has been discussed extensively. Nevertheless, Mn-induced DNA damage, adverse effects of DNA repair, and possible resulting consequences for the neurite network are not yet characterized. For this, LUHMES cells were used, as they differentiate into dopaminergic-like neurons and form extensive neurite networks. Experiments were conducted to analyze Mn bioavailability and cytotoxicity of MnCl2, indicating a dose-dependent uptake and substantial cytotoxic effects. DNA damage, analyzed by means of 8-oxo-7,8-dihydro-2'-guanine (8oxodG) and single DNA strand break formation, showed significant dose- and time-dependent increase of DNA damage upon 48 h Mn exposure. Furthermore, the DNA damage response was increased which was assessed by analytical quantification of poly(ADP-ribosyl)ation (PARylation). Gene expression of the respective DNA repair genes was not significantly affected. Degradation of the neuronal network is significantly altered by 48 h Mn exposure. Altogether, this study contributes to the characterization of Mn-induced neurotoxicity, by analyzing the adverse effects of Mn on genome integrity in dopaminergic-like neurons and respective outcomes.
Quantitative Bioimaging to Investigate the Uptake of Mercury Species in Drosophila melanogaster
(2015)
The uptake of mercury species in the model organism Drosophila melanogaster was investigated by elemental bioimaging using laser ablation-inductively coupled plasma mass spectrometry (LA-ICPMS). The mercury distribution in Drosophila melanogaster was analyzed for the three species mercury(II) chloride, methylmercury chloride, and thimerosal after intoxication. A respective analytical method was developed and applied to the analysis of the entire Drosophila melanogaster first, before a particular focus was directed to the cerebral areas of larvae and adult flies. For quantification of mercury, matrix-matched standards based on gelatin were prepared. Challenges of spatially dissolved mercury determination, namely, strong evaporation issues of the analytes and an inhomogeneous distribution of mercury in the standards due to interactions with cysteine containing proteins of the gelatin were successfully addressed by complexation with meso-2,3-dimercaptosuccinic acid (DMSA). No mercury was detected in the cerebral region for mercury(II) chloride, whereas both organic species showed the ability to cross the blood brain barrier. Quantitatively, the mercury level in the brain exceeded the fed concentration indicating mercury enrichment, which was approximately 3 times higher for methylmercury chloride than for thimerosal.
Arsenic-containing lipids (arsenolipids) are natural products of marine organisms such as fish, invertebrates, and algae, many of which are important seafoods. A major group of arsenolipids, namely, the arsenic-containing hydrocarbons (AsHC), have recently been shown to be cytotoxic to human liver and bladder cells, a result that has stimulated interest in the chemistry and toxicology of these compounds. In this study, elemental laser ablation-inductively coupled plasma mass spectrometry (LA-ICPMS) and molecular matrix-assisted laser desorption/ionization (MALDI-)MS were used to image and quantify the uptake of an AsHC in the model organism Drosophila melanogaster. Using these two complementary methods, both an enrichment of arsenic and the presence of the AsHC in the brain were revealed, indicating that the intact arsenolipid had crossed the blood-brain barrier. Simultaneous acquisition of quantitative elemental concentrations and molecular distributions could allow new insight into organ-specific enrichment and possible transportation processes of arsenic-containing bioactive compounds in living organisms.
Im Sinne des Refinements von Tierversuchen sollen alle Bedingungen während der Zucht, der Haltung und des Transports von zu Versuchszwecken gehaltenen Tieren und alle Methoden während des Versuchs so verbessert werden, dass die verwendeten Tiere ein minimales Maß an potentiellem Distress, Schmerzen oder Leiden erfahren. Zudem soll ihr Wohlbefinden durch die Möglichkeit des Auslebens speziesspezifischer Verhaltensweisen und die Anwendung tierschonender Verfahren maximal gefördert werden. Zur Etablierung von Grundsätzen des Refinements sind grundlegende Kenntnisse über die physiologischen Bedürfnisse und Verhaltensansprüche der jeweiligen Spezies unabdingbar. Die Experimentatoren sollten das Normalverhalten der Tiere kennen, um potentielle Verhaltensabweichungen, wie Stereotypien, zu verstehen und interpretieren zu können. Standardisierte Haltungsbedingungen von zu Versuchszwecken gehaltenen Mäusen weichen in diversen Aspekten von der natürlichen Umgebung ab und erfordern eine gewisse Adaptation. Ist ein Tier über einen längeren Zeitraum unfähig, sich an die gegebenen Umstände anzupassen, können abnormale Verhaltensweisen, wie Stereotypien auftreten. Stereotypien werden definiert als Abweichungen vom Normalverhalten, die repetitiv und ohne Abweichungen im Ablauf ausgeführt werden, scheinbar keiner Funktion dienen und der konkreten Umweltsituation nicht immer entsprechen.
Bisher war unklar, in welchem Ausmaß stereotypes Verhalten den metabolischen Phänotyp eines Individuums beeinflusst. Ziel dieser Arbeit war es daher, das stereotype Verhalten der FVB/NJ-Maus erstmals detailliert zu charakterisieren, systematisch zusammenzutragen, welche metabolischen Konsequenzen dieses Verhalten bedingt und wie sich diese auf das Wohlbefinden der Tiere und die Verwendung stereotyper Tiere in Studien mit tierexperimentellem Schwerpunkt auswirken.
Der Versuch begann mit der Charakterisierung der mütterlichen Fürsorge in der Parentalgeneration. Insgesamt wurden 35 Jungtiere der F1-Generation vom Absatz an, über einen Zeitraum von 11 Wochen einzeln gehalten, kontinuierlich beobachtet, bis zum Versuchsende wöchentlich Kotproben gesammelt und das Körpergewicht bestimmt. Zusätzlich erfolgten begleitende Untersuchungen wie Verhaltenstests und die Erfassung der physischen Aktivität und metabolischer Parameter. Anschließend wurden u.a. die zerebralen Serotonin- und Dopamingehalte, fäkale Glucocorticoidlevels, hepatisches Glykogen und muskuläre Glykogen- und Triglyceridlevels bestimmt.
Nahezu unabhängig von der mütterlichen Herkunft entwickelte sich bei mehr als der Hälfte der 35 Jungtiere in der F1-Generation stereotypes Verhalten. Diese Daten deuten darauf hin, dass es keine Anzeichen für das Erlernen oder eine direkte genetische Transmission stereotypen Verhaltens bei der FVB/NJ-Maus gibt. Über den gesamten Beobachtungszeitraum zeichneten sich die stereotypen FVB/NJ-Mäuse durch ein eingeschränktes Verhaltensrepertoire aus. Zu Gunsten der erhöhten Aktivität und des Ausübens stereotypen Verhaltens lebten sie insgesamt weniger andere Verhaltensweisen (Klettern, Graben, Nagen) aus. Darüber hinaus waren Stereotypien sowohl im 24-Stunden Open Field Test als auch in der Messeinrichtung der indirekten Tierkalorimetrie mit einer erhöhten Aktivität und Motilität assoziiert, während die circadiane Rhythmik nicht divergierte. Diese erhöhte körperliche Betätigung spiegelte sich in den niedrigeren Körpergewichtsentwicklungen der stereotypen Tiere wieder. Außerdem unterschieden sich die Körperfett- und Körpermuskelanteile.
Zusammenfassend lässt sich sagen, dass das Ausüben stereotypen Verhaltens zu Differenzen im metabolischen Phänotyp nicht-stereotyper und stereotyper FVB/NJ-Mäuse führt. Im Sinne der „Guten Wissenschaftlichen Praxis“ sollte das zentrale Ziel jedes Wissenschaftlers sein, aussagekräftige und reproduzierbare Daten hervorzubringen. Jedoch können keine validen Resultate von Tieren erzeugt werden, die in Aspekten variieren, die für den vorgesehenen Zweck der Studie nicht berücksichtigt wurden. Deshalb sollten nicht-stereotype und stereotype Individuen nicht innerhalb einer Versuchsgruppe randomisiert werden. Stereotype Tiere demzufolge von geplanten Studien auszuschließen, würde allerdings dem Gebot des zweiten R’s – der Reduction – widersprechen. Um Refinement zu garantieren, sollte der Fokus auf der maximal erreichbaren Prävention stereotypen Verhaltens liegen. Diverse Studien haben bereits gezeigt, dass die Anreicherung der Haltungsumwelt (environmental enrichment) zu einer Senkung der Prävalenz von Stereotypien bei Mäusen führt, dennoch kommen sie weiterhin vor. Daher sollte environmental enrichment zukünftig weniger ein „Kann“, sondern ein „Muss“ sein – oder vielmehr: der Goldstandard. Zudem würde eine profunde phänotypische Charakterisierung dazu beitragen, Mausstämme zu erkennen, die zu Stereotypien neigen und den für den spezifischen Zweck am besten geeigneten Mausstamm zu identifizieren, bevor ein Experiment geplant wird.
This case report addresses the problem of underreporting negative results and adverse side effects in animal testing. We present our findings regarding a hyperphagic mouse model associated with unforeseen high mortality. The results outline the necessity of reporting detailed information in the literature to avoid duplication. Obese mouse models are essential in the study of obesity, metabolic syndrome and diabetes mellitus. An experimental model of obesity can be induced by the administration of gold thioglucose (GTG). After transcending the blood-brain barrier, the GTG molecule interacts with regions of the ventromedial hypothalamus, thereby primarily targeting glucose-sensitive neurons. When these neurons are impaired, mice become insensitive to the satiety effects of glucose and develop hyperphagia. In a pilot study for optimising dosage and body weight development, C57BL/6 mice were treated with GTG (0.5 mg/g body weight) or saline, respectively. Animals were provided a physiological amount of standard diet (5 g per animal) for the first 24 hours after treatment to prevent gastric dilatation. Within 24 hours after GTG injection, all GTG-treated animals died of gastric overload and subsequent circulatory shock. Animals developed severe attacks of hyperphagia, and as the amount of provided chow was restricted, mice exhibited unforeseen pica and ingested bedding material. These observations strongly suggest that restricted feeding is contraindicated concerning GTG application. Presumably, the impulse of excessive food intake was a strong driving force. Therefore, the actual degree of suffering in the GTG-induced model of hyperphagia should be revised from moderate to severe.
Color matters
(2018)
Concerning standardization of laboratory animal husbandry, only exiguous changes of habitat can potentially influence animal physiology or results of behavioral tests. Routinely, mice chow is dyed when different types of diets are dispensed. Given the fact that the dye itself has no effects on food odor or flavor, we wanted to test the hypothesis that the color of chow has an impact on food uptake in mice. Twelve-week-old male mice of different strains (C57BL/6J, DBA/2J, C3H/HeJ, BALB/cJ; n = 12/strain) were single-housed in PhenoMaster (R) cages. After acclimatization standard mice chow in different colors was administered. Food intake was monitored as a two-alternative choice test of different color combinations. All animals had an average food intake of 3 g/d and no preferences were observed when a combination of identically colored food was offered. Preference tests yielded significant aversion to blue food and significant attraction to yellow and green food in C57BL/6 and DBA/2J mice. In C3H/HeJ and BALB/cJ mice no color-related pattern occurred. Selected mice strains have known differences concerning functionality of their visual sense. C57BL/6 and DBA/2 mice are considered to be normal sighted at testing age, BALB/c is representative for albino strains and C3H mice carry mutations resulting in retinal alterations. Results suggesting that normal-sighted mice would be selective concerning food color when given the choice. Nevertheless, this does not influence overall quantity of food intake when animals were provided solely with food colored with a single dye. Moreover, visually impaired mice showed no color-related food preferences.
Background & Aims: Exosomes are small membrane vesicles involved in intercellular communication. Hepatocytes are known to release exosomes, but little is known about their biological function. We sought to determine if exosomes derived from hepatocytes contribute to liver repair and regeneration after injury. Methods: Exosomes derived from primary murine hepatocytes were isolated and characterized biochemically and biophysically. Using cultures of primary hepatocytes, we tested whether hepatocyte exosomes induced proliferation of hepatocytes in vitro. Using models of ischemia/reperfusion injury and partial hepatectomy, we evaluated whether hepatocyte exosomes promote hepatocyte proliferation and liver regeneration in vivo. Results: Hepatocyte exosomes, but not exosomes from other liver cell types, induce dose-dependent hepatocyte proliferation in vitro and in vivo. Mechanistically, hepatocyte exosomes directly fuse with target hepatocytes and transfer neutral ceramidase and sphingosine kinase 2 (SK2) causing increased synthesis of sphingosine-1-phosphate (S1P) within target hepatocytes. Ablation of exosomal SK prevents the proliferative effect of exosomes. After ischemia/reperfusion injury, the number of circulating exosomes with proliferative effects increases. Conclusions: Our data shows that hepatocyte-derived exosomes deliver the synthetic machinery to form S1P in target hepatocytes resulting in cell proliferation and liver regeneration after ischemia/reperfusion injury or partial hepatectomy. These findings represent a potentially novel new contributing mechanism of liver regeneration and have important implications for new therapeutic approaches to acute and chronic liver disease. (C) 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.
Exosomes are small membrane vesicles released by different cell types, including hepatocytes, that play important roles in intercellular communication. We have previously demonstrated that hepatocyte-derived exosomes contain the synthetic machinery to form sphingosine-1-phosphate (S1P) in target hepatocytes resulting in proliferation and liver regeneration after ischemia/reperfusion (I/R) injury. We also demonstrated that the chemokine receptors, CXCR1 and CXCR2, regulate liver recovery and regeneration after I/R injury. In the current study, we sought to determine if the regulatory effects of CXCR1 and CXCR2 on liver recovery and regeneration might occur via altered release of hepatocyte exosomes. We found that hepatocyte release of exosomes was dependent upon CXCR1 and CXCR2. CXCR1-deficient hepatocytes produced fewer exosomes, whereas CXCR2-deficient hepatocytes produced more exosomes compared to their wild-type controls. In CXCR2-deficient hepatocytes, there was increased activity of neutral sphingomyelinase (Nsm) and intracellular ceramide. CXCR1-deficient hepatocytes had no alterations in Nsm activity or ceramide production. Interestingly, exosomes from CXCR1-deficient hepatocytes had no effect on hepatocyte proliferation, due to a lack of neutral ceramidase and sphingosine kinase. The data demonstrate that CXCR1 and CXCR2 regulate hepatocyte exosome release. The mechanism utilized by CXCR1 remains elusive, but CXCR2 appears to modulate Nsm activity and resultant production of ceramide to control exosome release. CXCR1 is required for packaging of enzymes into exosomes that mediate their hepatocyte proliferative effect.