Chemo- and optogenetic activation of hypothalamic Foxb1-expressing neurons and their terminal endings in the rostral-dorsolateral PAG leads to tachypnea, bradycardia, and immobility.

Foxb1 -expressing neurons occur in the dorsal premammillary nucleus (PMd) and further rostrally in the parvafox nucleus, a longitudinal cluster of neurons in the lateral hypothalamus of rodents. The descending projection of these Foxb1+ neurons end in the dorsolateral part of the periaqueductal gray (dlPAG). The functional role of the Foxb1+ neuronal subpopulation in the PMd and the parvafox nucleus remains elusive. In this study, the activity of the Foxb1+ neurons and of their terminal endings in the dlPAG in mice was selectively altered by employing chemo- and optogenetic tools. Our results show that in whole-body barometric plethysmography, hM3Dq-mediated, global Foxb1+ neuron excitation activates respiration. Time-resolved optogenetic gain-of-function manipulation of the terminal endings of Foxb1+ neurons in the rostral third of the dlPAG leads to abrupt immobility and bradycardia. Chemogenetic activation of Foxb1+ cell bodies and ChR2-mediated excitation of their axonal endings in the dlPAG led to a phenotypical presentation congruent with a 'freezing-like' situation during innate defensive behavior.

Assessment of liver function by gadoxetic acid avidity in MRI in a model of rapid liver regeneration in rats.

BACKGROUND: This animal study investigates the hypothesis of an immature liver growth following ALPPS (associating liver partition and portal vein ligation for staged hepatectomy) by measuring liver volume and function using gadoxetic acid avidity in magnetic resonance imaging (MRI) in models of ALPPS, major liver resection (LR) and portal vein ligation (PVL). METHODS: Wistar rats were randomly allocated to ALPPS, LR or PVL. In contrast-enhanced MRI scans with gadoxetic acid (Primovist®), liver volume and function of the right median lobe (=future liver remnant, FLR) and the deportalized lobes (DPL) were assessed until post-operative day (POD) 5. Liver functionFLR/DPL was defined as the inverse value of time from injection of gadoxetic acid to the blood pool-corrected maximum signal intensityFLR/DPL multiplied by the volumeFLR/DPL. RESULTS: In ALPPS (n = 6), LR (n = 6) and PVL (n = 6), volumeFLR and functionFLR increased proportionally, except on POD 1. Thereafter, functionFLR exceeded volumeFLR increase in LR and ALPPS, but not in PVL. Total liver function was significantly reduced after LR until POD 3, but never undercuts 60% of its pre-operative value following ALPPS and PVL. DISCUSSION: This study shows for the first time that functional increase is proportional to volume increase in ALPPS using gadoxetic acid avidity in MRI.

What to consider for ECG in mice-with special emphasis on telemetry.

Genetically or surgically altered mice are commonly used as models of human cardiovascular diseases. Electrocardiography (ECG) is the gold standard to assess cardiac electrophysiology as well as to identify cardiac phenotypes and responses to pharmacological and surgical interventions. A variety of methods are used for mouse ECG acquisition under diverse conditions, making it difficult to compare different results. Non-invasive techniques allow only short-term data acquisition and are prone to stress or anesthesia related changes in cardiac activity. Telemetry offers continuous long-term acquisition of ECG data in conscious freely moving mice in their home cage environment. Additionally, it allows acquiring data 24/7 during different activities, can be combined with different challenges and most telemetry systems collect additional physiological parameters simultaneously. However, telemetry transmitters require surgical implantation, the equipment for data acquisition is relatively expensive and analysis of the vast number of ECG data is challenging and time-consuming. This review highlights the limits of non-invasive methods with respect to telemetry. In particular, primary screening using non-invasive methods can give a first hint; however, subtle cardiac phenotypes might be masked or compensated due to anesthesia and stress during these procedures. In addition, we detail the key differences between the mouse and human ECG. It is crucial to consider these differences when analyzing ECG data in order to properly translate the insights gained from murine models to human conditions.

The AP-1 transcription factor Fosl-2 drives cardiac fibrosis and arrhythmias under immunofibrotic conditions.

Fibrotic changes in the myocardium and cardiac arrhythmias represent fatal complications in systemic sclerosis (SSc), however the underlying mechanisms remain elusive. Mice overexpressing transcription factor Fosl-2 (Fosl-2tg) represent animal model of SSc. Fosl-2tg mice showed interstitial cardiac fibrosis, disorganized connexin-43/40 in intercalated discs and deregulated expression of genes controlling conduction system, and developed higher heart rate (HR), prolonged QT intervals, arrhythmias with prevalence of premature ventricular contractions, ventricular tachycardias, II-degree atrio-ventricular blocks and reduced HR variability. Following stimulation with isoproterenol Fosl-2tg mice showed impaired HR response. In contrast to Fosl-2tg, immunodeficient Rag2-/-Fosl-2tg mice were protected from enhanced myocardial fibrosis and ECG abnormalities. Transcriptomics analysis demonstrated that Fosl-2-overexpression was responsible for profibrotic signature of cardiac fibroblasts, whereas inflammatory component in Fosl-2tg mice activated their fibrotic and arrhythmogenic phenotype. In human cardiac fibroblasts FOSL-2-overexpression enhanced myofibroblast signature under proinflammatory or profibrotic stimuli. These results demonstrate that under immunofibrotic conditions transcription factor Fosl-2 exaggerates myocardial fibrosis, arrhythmias and aberrant response to stress.

Protocol for a systematic review of good surgical practice guidelines for experimental rodent surgery.

Objective: Surgery is an integral part of many experimental studies. Aseptic and minimal invasive surgical technique and optimal perioperative and post-operative care are prerequisites to achieve surgical success and best possible animal welfare outcomes. Good surgical practice cannot only improve the animal's postoperative recovery, but also study outcome and validity. There seems to be a lack of implementation of good surgical practice during rodent surgery. The aim of this systematic review is to identify, critically evaluate and compare the currently recommended standards and underlying guidelines for rodent surgery-and finally to compile a comprehensive guideline of good surgical practice for rodent surgery. Search strategy: PubMed, Embase and Web of Science were searched to identify guidelines published in peer-reviewed journals. To identify grey literature and unpublished guidelines, we will perform a Google search for published guidelines and search laboratory animal sciences books for relevant book chapters. Additionally, we will conduct a survey among animal researchers enquiring about the guidelines they use. Screening and study selection: For publications retrieved by the systematic search, unique references are screened by two reviewers, first for eligibility based on title and abstract and subsequently for final inclusion based on full text. Eligibility of books is based on title and content, final inclusion based on chapter full text. Guidelines are either retrieved by Google searches or a survey. Google searches will be conducted by at least four of the authors. Thereafter, guidelines will be screened by two of the authors. Data extraction and synthesis: We will extract data from publications, book chapters and guidelines. Based on the extracted data, we will perform a descriptive synthesis of the bibliographical details, guideline development and endorsement, and the prevalence of individual recommendations, including subgroup analysis of the guidance per continent or country and differences between peer-reviewed versus non-peer-reviewed guidance.

Error-prone protein synthesis recapitulates early symptoms of Alzheimer disease in aging mice.

Age-related neurodegenerative diseases (NDDs) are associated with the aggregation and propagation of specific pathogenic protein species (e.g., Aß, α-synuclein). However, whether disruption of synaptic homeostasis results from protein misfolding per se rather than accumulation of a specific rogue protein is an unexplored question. Here, we show that error-prone translation, with its frequent outcome of random protein misfolding, is sufficient to recapitulate many early features of NDDs, including perturbed Ca2+ signaling, neuronal hyperexcitability, and mitochondrial dysfunction. Mice expressing the ribosomal ambiguity mutation Rps9 D95N exhibited disrupted synaptic homeostasis resulting in behavioral changes reminiscent of early Alzheimer disease (AD), such as learning and memory deficits, maladaptive emotional responses, epileptiform discharges, suppressed circadian rhythmicity, and sleep fragmentation, accompanied by hippocampal NPY expression and cerebral glucose hypometabolism. Collectively, our findings suggest that random protein misfolding may contribute to the pathogenesis of age-related NDDs, providing an alternative framework for understanding the initiation of AD.

Premature aging in mice with error-prone protein synthesis.

The main source of error in gene expression is messenger RNA decoding by the ribosome. Translational accuracy has been suggested on a purely correlative basis to positively coincide with maximum possible life span among different rodent species, but causal evidence that translation errors accelerate aging in vivo and limit life span is lacking. We have now addressed this question experimentally by creating heterozygous knock-in mice that express the ribosomal ambiguity mutation RPS9 D95N, resulting in genome-wide error-prone translation. Here, we show that Rps9 D95N knock-in mice exhibit reduced life span and a premature onset of numerous aging-related phenotypes, such as reduced weight, chest deformation, hunchback posture, poor fur condition, and urinary syndrome, together with lymphopenia, increased levels of reactive oxygen species-inflicted damage, accelerated age-related changes in DNA methylation, and telomere attrition. Our results provide an experimental link between translational accuracy, life span, and aging-related phenotypes in mammals.

Random errors in protein synthesis activate an age-dependent program of muscle atrophy in mice.

Random errors in protein synthesis are prevalent and ubiquitous, yet their effect on organismal health has remained enigmatic for over five decades. Here, we studied whether mice carrying the ribosomal ambiguity (ram) mutation Rps2-A226Y, recently shown to increase the inborn error rate of mammalian translation, if at all viable, present any specific, possibly aging-related, phenotype. We introduced Rps2-A226Y using a Cre/loxP strategy. Resulting transgenic mice were mosaic and showed a muscle-related phenotype with reduced grip strength. Analysis of gene expression in skeletal muscle using RNA-Seq revealed transcriptomic changes occurring in an age-dependent manner, involving an interplay of PGC1α, FOXO3, mTOR, and glucocorticoids as key signaling pathways, and finally resulting in activation of a muscle atrophy program. Our results highlight the relevance of translation accuracy, and show how disturbances thereof may contribute to age-related pathologies.

The NO-donor MPC-1011 stimulates angiogenesis and arteriogenesis and improves hindlimb ischemia via a cGMP-dependent pathway involving VEGF and SDF-1α.

BACKGROUND AND AIMS: Peripheral arterial disease (PAD) is an important cause of morbidity and mortality with little effective medical treatment currently available. Nitric oxide (NO) is crucially involved in organ perfusion, tissue protection and angiogenesis. METHODS: We hypothesized that a novel NO-donor, MPC-1011, might elicit vasodilation, angiogenesis and arteriogenesis and in turn improve limb perfusion, in a hindlimb ischemia model. Hindlimb ischemia was induced by femoral artery ligation in Sprague-Dawley rats, which were randomized to receive either placebo, MPC-1011, cilostazol or both, up to 28 days. Limb blood flow was assessed by laser Doppler imaging. RESULTS: After femoral artery occlusion, limb perfusion in rats receiving MPC-1011 alone or in combination with cilostazol was increased throughout the treatment regimen. Capillary density and the number of arterioles was increased only with MPC-1011. MPC-1011 improved vascular remodeling by increasing luminal diameter in the ischemic limb. Moreover, MPC-1011 stimulated the release of proangiogenic cytokines, including VEGF, SDF1α and increased tissue cGMP levels, reduced platelet activation and aggregation, potentiated proliferation and migration of endothelial cells which was blunted in the presence of soluble guanylyl cyclase inhibitor LY83583. In MPC-1011-treated rats, Lin-/CD31+/CXCR4+ cells were increased by 92.0% and Lin-/VEGFR2+/CXCR4+ cells by 76.8% as compared to placebo. CONCLUSIONS: Here we show that the NO donor, MPC-1011, is a specific promoter of angiogenesis and arteriogenesis in a hindlimb ischemia model in an NO-cGMP-VEGF- dependent manner. This sets the basis to evaluate and confirm the efficacy of such therapy in a clinical setting in patients with PAD and impaired limb perfusion.

Elevated FGF23 and disordered renal mineral handling with reduced bone mineralization in chronically erythropoietin over-expressing transgenic mice.

Fibroblast Growth Factor 23 (FGF23) is a phosphaturic factor causing increased renal phosphate excretion as well as suppression of 1,25 (OH)2-vitamin D3. Highly elevated FGF23 can promote development of rickets and osteomalacia. We and others previously reported that acute application of erythropoietin (EPO) stimulates FGF23 production. Considering that EPO is clinically used as chronic treatment against anemia, we used here the Tg6 mouse model that constitutively overexpresses human EPO in an oxygen-independent manner, to examine the consequences of long-term EPO therapy on mineral and bone metabolism. Six to eight weeks old female Tg6 mice showed elevated intact and C-terminal fragment of FGF23 but normal plasma levels of PTH, calcitriol, calcium and phosphate. Renal function showed moderate alterations with higher urea and creatinine clearance and mild albuminuria. Renal phosphate excretion was normal whereas mild hypercalciuria was found. Renal expression of the key proteins TRPV5 and calbindin D28k involved in active calcium reabsorption was reduced in Tg6 mice. Plasma levels of the bone turnover marker osteocalcin were comparable between groups. However, urinary excretion of deoxypyridinoline (DPD) was lower in Tg6 mice. MicroCT analysis showed reduced total, cortical, and trabecular bone mineral density in femora from Tg6 mice. Our data reveal that chronic elevation of EPO is associated with high FGF23 levels and disturbed mineral homeostasis resulting in reduced bone mineral density. These observations imply the need to study the impact of therapeutically applied EPO on bone mineralization in patients, especially those suffering from chronic kidney disease.

Differential Impact of Dietary Branched Chain and Aromatic Amino Acids on Chronic Kidney Disease Progression in Rats.

The metabolism of dietary proteins generates waste products that are excreted by the kidney, in particular nitrogen-containing urea, uric acid, ammonia, creatinine, and other metabolites such as phosphates, sulfates, and protons. Kidney adaptation includes an increase in renal plasma flow (RPF) and glomerular filtration rate (GFR) and represents a burden for diseased kidneys increasing the progression rate of CKD. The present study aimed at identifying potential differences between amino acid (AA) groups constituting dietary proteins regarding their impact on RPF, GFR, and CKD progression. We utilized the well-established 5/6 nephrectomy (5/6 Nx) CKD model in rats and submitted the animals for 5 weeks to either the control diet (18% casein protein) or to diets containing 8% casein supplemented with 10% of a mix of free amino acids, representing all or only a subset of the amino acids contained in casein. Whereas the RPF and GFR measured in free moving animals remained stable during the course of the diet in rats receiving the control mix, these parameters decreased in animals receiving the branched chain amino acid (BCAA) supplementation and increased in the ones receiving the aromatic amino acids (AAAs). In animals receiving essential amino acids (EAAs) containing both BCAAs and AAAs, there was only a small increase in RPF. The kidneys of the 5/6 Nx rats receiving the BCAA diet showed the strongest increase in smooth muscle actin and collagen mRNA expression as a result of higher level of inflammation and fibrosis. These animals receiving BCAAs also showed an increase in plasma free fatty acids pointing to a problem at the level of energy metabolism. In contrast, the animals under AAA diet showed an activation of AMPK and STAT3. Taken together, our results demonstrate that subsets of EAAs contained in dietary proteins, specifically BCAAs and AAAs, exert contrasting effects on kidney functional parameters and CKD progression.

Kidney Mass Reduction Leads to l-Arginine Metabolism-Dependent Blood Pressure Increase in Mice.

BACKGROUND: Uninephrectomy (UNX) is performed for various reasons, including kidney cancer or donation. Kidneys being the main site of l-arginine production in the body, we tested whether UNX mediated kidney mass reduction impacts l-arginine metabolism and thereby nitric oxide production and blood pressure regulation in mice. METHODS AND RESULTS: In a first series of experiments, we observed a significant increase in arterial blood pressure 8 days post-UNX in female and not in male mice. Further experimental series were performed in female mice, and the blood pressure increase was confirmed by telemetry. l-citrulline, that is used in the kidney to produce l-arginine, was elevated post-UNX as was also asymmetric dimethylarginine, an inhibitor of nitric oxide synthase that competes with l-arginine and is a marker for renal failure. Interestingly, the UNX-induced blood pressure increase was prevented by supplementation of the diet with 5% of the l-arginine precursor, l-citrulline. Because l-arginine is metabolized in the kidney and other peripheral tissues by arginase-2, we tested whether the lack of this metabolic pathway also compensates for decreased l-arginine production in the kidney and/or for local nitric oxide synthase inhibition and consecutive blood pressure increase. Indeed, upon uninephrectomy, arginase-2 knockout mice (Arg-2-/-) neither displayed an increase in asymmetric dimethylarginine and l-citrulline plasma levels nor a significant increase in blood pressure. CONCLUSIONS: UNX leads to a small increase in blood pressure that is prevented by l-citrulline supplementation or arginase deficiency, 2 measures that appear to compensate for the impact of kidney mass reduction on l-arginine metabolism.

Effect of AP102, a subtype 2 and 5 specific somatostatin analog, on glucose metabolism in rats.

PURPOSE: Somatostatin analogs are widely used to treat conditions associated with hormonal hypersecretion such as acromegaly and metastatic neuroendocrine tumors. First generation somatostatin analogs, such as octreotide and lanreotide, have high affinity for somatostatin receptor subtype 2 (SSTR2), but have incomplete efficacy in many patients. Pasireotide targets multiple SSTRs, having the highest affinity for SSTR5, but causes hyperglycemia and diabetes mellitus in preclinical and clinical studies. AP102 is a new somatostatin analogs with high affinity at both SSTR2 and SSTR5. We aimed to characterize the effects of AP102 vs. pasireotide on random and dynamic glucose levels, glucoregulatory hormone concentrations and growth axis measures in healthy Sprague-Dawley rats. METHODS: Three doses of each compound were evaluated under acute conditions (1, 10, and 30 µg/kg s.c.), and two doses during a chronic (4-week) infusion (3 and 10 µg/kg/h s.c.). RESULTS: Neither acute nor chronic AP102 administration altered blood glucose concentrations or dynamic responses following an intraperitoneal glucose tolerance test. In contrast, acute and chronic pasireotide dosing increased random and post-intraperitoneal glucose tolerance test blood glucose measures, compared to vehicle-treated controls. Both AP102 and pasireotide acutely suppressed growth hormone levels, although insulin-like growth factor-1 and somatic growth was suppressed to a greater extent with pasireotide. CONCLUSIONS: AP102 is a new dual SSTR2/SSTR5-specific somatostatin analog that acutely reduces growth hormone but does not cause hyperglycemia during acute or chronic administration in a healthy rat model. Further studies in diabetic animals and in humans are necessary to determine the potential utility of AP102 in the clinical setting.

Mutations in NONO lead to syndromic intellectual disability and inhibitory synaptic defects.

The NONO protein has been characterized as an important transcriptional regulator in diverse cellular contexts. Here we show that loss of NONO function is a likely cause of human intellectual disability and that NONO-deficient mice have cognitive and affective deficits. Correspondingly, we find specific defects at inhibitory synapses, where NONO regulates synaptic transcription and gephyrin scaffold structure. Our data identify NONO as a possible neurodevelopmental disease gene and highlight the key role of the DBHS protein family in functional organization of GABAergic synapses.

Deletion of Rictor in brain and fat alters peripheral clock gene expression and increases blood pressure.

The mammalian target of rapamycin complex 2 (mTORC2) contains the essential protein RICTOR and is activated by growth factors. mTORC2 in adipose tissue contributes to the regulation of glucose and lipid metabolism. In the perivascular adipose tissue, mTORC2 ensures normal vascular reactivity by controlling expression of inflammatory molecules. To assess whether RICTOR/mTORC2 contributes to blood pressure regulation, we applied a radiotelemetry approach in control and Rictor knockout (Rictor(aP2KO)) mice generated using adipocyte protein-2 gene promoter-driven CRE recombinase expression to delete Rictor. The 24-hour mean arterial pressure was increased in Rictor(aP2KO) mice, and the physiological decline in mean arterial pressure during the dark period was impaired. In parallel, heart rate and locomotor activity were elevated during the dark period with a pattern similar to blood pressure changes. This phenotype was associated with mild cardiomyocyte hypertrophy, decreased cardiac natriuretic peptides, and their receptor expression in adipocytes. Moreover, clock gene expression was reduced or phase-shifted in perivascular adipose tissue. No differences in clock gene expression were observed in the master clock suprachiasmatic nucleus, although Rictor gene expression was also lower in brain of Rictor(aP2KO) mice. Thus, this study highlights the importance of RICTOR/mTORC2 for interactions between vasculature, adipocytes, and brain to tune physiological outcomes, such as blood pressure and locomotor activity.

Tibio-femoral joint contact forces in sheep.

Although the sheep has become a standard model for understanding the mechanical conditions that occur after injury and investigating surgical treatments such as osteochondral defect healing and ligament reconstruction, no study has yet evaluated the contact forces that occur in the sheep tibio-femoral joint in vivo. In this study, bone pins, together with reflective markers, were used to measure the 3D kinematics of three sheep hind limbs, simultaneously with the ground reaction forces during repetitions of gait trials. Joint contact forces were then calculated using inverse dynamics and optimisation techniques. Whilst average peak axial tibio-femoral contact forces of 2.1 body weight (BW) were calculated across the 3 sheep, only small medio-lateral and antero-posterior shear forces, averaging 0.7 BW, were determined. Average knee flexion angles ranging from 49 degrees to 70 degrees were observed. From the forces determined in this study, we have provided a better understanding of the mechanical loading environment that occurs in sheep. This has important implications for the interpretation of knee studies in quadrupeds and their relevance to the clinical situation.

Osteoclastic activity begins early and increases over the course of bone healing.

Osteoclasts are specialised bone-resorbing cells. This particular ability makes osteoclasts irreplaceable for the continual physiological process of bone remodelling as well as for the repair process during bone healing. Whereas the effects of systemic diseases on osteoclasts have been described by many authors, the spatial and temporal distribution of osteoclasts during bone healing seems to be unclear so far. In the present study, healing of a tibial osteotomy under standardised external fixation was examined after 2, 3, 6 and 9 weeks (n = 8) in sheep. The osteoclastic number was counted, the area of mineralised bone tissue was measured histomorphometrically and density of osteoclasts per square millimetre mineralised tissue was calculated. The osteoclastic density in the endosteal region increased, whereas the density in the periosteal region remained relatively constant. The density of osteoclasts within the cortical bone increased slightly over the first 6 weeks, however, there was a more rapid increase between the sixth and ninth weeks. The findings of this study imply that remodelling and resorption take place already in the very early phase of bone healing. The most frequent remodelling process can be found in the periosteal callus, emphasising its role as the main stabiliser. The endosteal space undergoes resorption in order to recanalise the medullary cavity, a process also started in the very early phase of healing at a low level and increasing significantly during healing. The cortical bone adapts in its outward appearance to the surrounding callus structure. This paradoxic loosening is caused by the continually increasing number and density of osteoclasts in the cortical bone ends. This study clearly emphasises the osteoclastic role especially during early bone healing. These cells do not simply resorb bone but participate in a fine adjusted system with the bone-producing osteoblasts in order to maintain and improve the structural strength of bone tissue.