Leveraging interindividual variability in threat conditioning of inbred mice to model trait anxiety.

Trait anxiety is a major risk factor for stress-induced and anxiety disorders in humans. However, animal models accounting for the interindividual variability in stress vulnerability are largely lacking. Moreover, the pervasive bias of using mostly male animals in preclinical studies poorly reflects the increased prevalence of psychiatric disorders in women. Using the threat imminence continuum theory, we designed and validated an auditory aversive conditioning-based pipeline in both female and male mice. We operationalised trait anxiety by harnessing the naturally occurring variability of defensive freezing responses combined with a model-based clustering strategy. While sustained freezing during prolonged retrieval sessions was identified as an anxiety-endophenotype behavioral marker in both sexes, females were consistently associated with an increased freezing response. RNA-sequencing of CeA, BLA, ACC, and BNST revealed massive differences in phasic and sustained responders' transcriptomes, correlating with transcriptomic signatures of psychiatric disorders, particularly post-traumatic stress disorder (PTSD). Moreover, we detected significant alterations in the excitation/inhibition balance of principal neurons in the lateral amygdala. These findings provide compelling evidence that trait anxiety in inbred mice can be leveraged to develop translationally relevant preclinical models to investigate mechanisms of stress susceptibility in a sex-specific manner.

Aerosol exposure of staff during dental treatments: a model study.

BACKGROUND: Due to exposure to potentially infectious aerosols during treatments, the dental personnel is considered being at high risk for aerosol transmitted diseases like COVID-19. The aim of this study was to evaluate aerosol exposure during different dental treatments as well as the efficacy of dental suction to reduce aerosol spreading. METHODS: Dental powder-jet (PJ; Air-Flow®), a water-cooled dental handpiece with a diamond bur (HP) and water-cooled ultrasonic scaling (US) were used in a simulation head, mounted on a dental unit in various treatment settings. The influence of the use of a small saliva ejector (SE) and high-volume suction (HVS) was evaluated. As a proxy of aerosols, air-born particles (PM10) were detected using a Laser Spectrometer in 30 cm distance from the mouth. As control, background particle counts (BC) were measured before and after experiments. RESULTS: With only SE, integrated aerosol levels [median (Q25/Q75) µg/m3 s] for PJ [91,246 (58,213/118,386) µg/m3 s, p < 0.001, ANOVA] were significantly increased compared to BC [7243 (6501/8407) µg/m3 s], whilst HP [11,119 (7190/17,234) µg/m3 s, p > 0.05] and US [6558 (6002/7066) µg/m3 s; p > 0.05] did not increase aerosol levels significantly. The use of HVS significantly decreased aerosol exposure for PJ [37,170 (29,634/51,719) µg/m3 s; p < 0.01] and HP [5476 (5066/5638) µg/m3 s; p < 0.001] compared to SE only, even reaching lower particle counts than BC levels for HP usage (p < 0.001). CONCLUSIONS: To reduce the exposure to potentially infectious aerosols, HVS should be used during aerosol-forming dental treatments.

Reliability of genomic variants across different next-generation sequencing platforms and bioinformatic processing pipelines.

BACKGROUND: Next Generation Sequencing (NGS) is the fundament of various studies, providing insights into questions from biology and medicine. Nevertheless, integrating data from different experimental backgrounds can introduce strong biases. In order to methodically investigate the magnitude of systematic errors in single nucleotide variant calls, we performed a cross-sectional observational study on a genomic cohort of 99 subjects each sequenced via (i) Illumina HiSeq X, (ii) Illumina HiSeq, and (iii) Complete Genomics and processed with the respective bioinformatic pipeline. We also repeated variant calling for the Illumina cohorts with GATK, which allowed us to investigate the effect of the bioinformatics analysis strategy separately from the sequencing platform's impact. RESULTS: The number of detected variants/variant classes per individual was highly dependent on the experimental setup. We observed a statistically significant overrepresentation of variants uniquely called by a single setup, indicating potential systematic biases. Insertion/deletion polymorphisms (indels) were associated with decreased concordance compared to single nucleotide polymorphisms (SNPs). The discrepancies in indel absolute numbers were particularly prominent in introns, Alu elements, simple repeats, and regions with medium GC content. Notably, reprocessing sequencing data following the best practice recommendations of GATK considerably improved concordance between the respective setups. CONCLUSION: We provide empirical evidence of systematic heterogeneity in variant calls between alternative experimental and data analysis setups. Furthermore, our results demonstrate the benefit of reprocessing genomic data with harmonized pipelines when integrating data from different studies.

Clinical significance and risk factors for new onset and recurring atrial fibrillation following cardiac surgery - a retrospective data analysis.

BACKGROUND: Although mortality after cardiac surgery has significantly decreased in the last decade, patients still experience clinically relevant postoperative complications. Among others, atrial fibrillation (AF) is a common consequence of cardiac surgery, which is associated with prolonged hospitalization and increased mortality. METHODS: We retrospectively analyzed data from patients who underwent coronary artery bypass grafting, valve surgery or a combination of both at the University Hospital Muenster between April 2014 and July 2015. We evaluated the incidence of new onset and intermittent/permanent AF (patients with pre- and postoperative AF). Furthermore, we investigated the impact of postoperative AF on clinical outcomes and evaluated potential risk factors. RESULTS: In total, 999 patients were included in the analysis. New onset AF occurred in 24.9% of the patients and the incidence of intermittent/permanent AF was 59.5%. Both types of postoperative AF were associated with prolonged ICU length of stay (median increase approx. 2 days) and duration of mechanical ventilation (median increase 1 h). Additionally, new onset AF patients had a higher rate of dialysis and hospital mortality and more positive fluid balance on the day of surgery and postoperative days 1 and 2. In a multiple logistic regression model, advanced age (odds ratio (OR) = 1.448 per decade increase, p < 0.0001), a combination of CABG and valve surgery (OR = 1.711, p = 0.047), higher C-reactive protein (OR = 1.06 per unit increase, p < 0.0001) and creatinine plasma concentration (OR = 1.287 per unit increase, p = 0.032) significantly predicted new onset AF. Higher Horowitz index values were associated with a reduced risk (OR = 0.996 per unit increase, p = 0.012). In a separate model, higher plasma creatinine concentration (OR = 2.125 per unit increase, p = 0.022) was a significant risk factor for intermittent/permanent AF whereas higher plasma phosphate concentration (OR = 0.522 per unit increase, p = 0.003) indicated reduced occurrence of this arrhythmia. CONCLUSIONS: New onset and intermittent/permanent AF are associated with adverse clinical outcomes of elective cardiac surgery patients. Different risk factors implicated in postoperative AF suggest different mechanisms might be involved in its pathogenesis. Customized clinical management protocols seem to be warranted for a higher success rate of prevention and treatment of postoperative AF.

NAPE-PLD deletion in stress-TRAPed neurons results in an anxiogenic phenotype.

Anandamide (AEA) is an endogenous ligand of the cannabinoid CB1 and CB2 receptors, being a component of the endocannabinoid signaling system, which supports the maintenance or regaining of neural homeostasis upon internal and external challenges. AEA is thought to play a protective role against the development of pathological states after prolonged stress exposure, including depression and generalized anxiety disorder. Here, we used the chronic social defeat (CSD) stress as an ethologically valid model of chronic stress in male mice. We characterized a genetically modified mouse line where AEA signaling was reduced by deletion of the gene encoding the AEA synthesizing enzyme N-acyl-phosphatidylethanolamine-hydrolyzing phospholipase D (NAPE-PLD) specifically in neurons activated at the time of CSD stress. One week after the stress, the phenotype was assessed in behavioral tests and by molecular analyses. We found that NAPE-PLD deficiency in neurons activated during the last three days of CSD stress led to an increased anxiety-like behavior. Investigating the molecular mechanisms underlying this phenotype may suggest three main altered pathways to be affected: (i) desensitization of the negative feedback loop of the hypothalamic-pituitary-adrenal axis, (ii) disinhibition of the amygdala by the prefrontal cortex, and (iii) altered neuroplasticity in the hippocampus and prefrontal cortex.

beeRapp: an R shiny app for automated high-throughput explorative analysis of multivariate behavioral data.

Summary: Animal behavioral studies typically generate high-dimensional datasets consisting of multiple correlated outcome measures across distinct or related behavioral domains. Here, we introduce the BEhavioral Explorative analysis R shiny APP (beeRapp) that facilitates explorative and inferential analysis of behavioral data in a high-throughput fashion. By employing an intuitive and user-friendly graphical user interface, beeRapp empowers behavioral scientists without programming and data science expertise to perform clustering, dimensionality reduction, correlational and inferential statistics and produce up to thousands of high-quality output plots visualizing results in a standardized and automated way. Availability and implementation: The code and data underlying this article are available at https://github.com/anmabu/beeRapp.

Long-term molecular differences between resilient and susceptible mice after a single traumatic exposure.

BACKGROUND AND PURPOSE: Post-traumatic stress disorder (PTSD) is a heterogeneous disorder induced by trauma, resulting in severe long-term impairments of an individual's mental health. PTSD does not develop in every individual and, thus, some individuals are more resilient. However, the underlying molecular mechanisms are poorly understood. Here, we aimed to elucidate these processes. EXPERIMENTAL APPROACH: We used a single-trauma PTSD model in mice to induce long-term maladaptive behaviours and profiled the mice 4 weeks after trauma into resilient or susceptible individuals. The classification of phenotype was based on individual responses in different behavioural experiments. We analysed microbiome, circulating endocannabinoids, and long-term changes in brain phospholipid and transcript levels. KEY RESULTS: We found many molecular differences between resilient and susceptible individuals across multiple molecular domains, including lipidome, transcriptome and gut microbiome. Some differences were stable even several weeks after the trauma, indicating the long-term impact of traumatic stimuli on the organism's physiology. Furthermore, the integration of these multilayered molecular data revealed that resilient and susceptible individuals have very distinct molecular signatures across various physiological systems. CONCLUSION AND IMPLICATIONS: Trauma induced individual-specific behavioural responses that, in combination with a longitudinal characterisation of mice, could be used to identify distinct sub-phenotypes within the trauma-exposed group. These groups differed significantly not only in their behaviour but also in specific molecular aspects across a variety of tissues and brain regions. This approach may reveal new targets and predictive biomarkers for the pharmacological treatment and prognosis of stress-related disorders. LINKED ARTICLES: This article is part of a themed issue on New discoveries and perspectives in mental and pain disorders. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.17/issuetoc.

A Resilience Related Glial-Neurovascular Network Is Transcriptionally Activated after Chronic Social Defeat in Male Mice.

Upon chronic stress, a fraction of individuals shows stress resilience, which can prevent long-term mental dysfunction. The underlying molecular mechanisms are complex and have not yet been fully understood. In this study, we performed a data-driven behavioural stratification together with single-cell transcriptomics of the hippocampus in a mouse model of chronic social defeat stress. Our work revealed that in a sub-group exhibiting molecular responses upon chronic stress, the dorsal hippocampus is particularly involved in neuroimmune responses, angiogenesis, myelination, and neurogenesis, thereby enabling brain restoration and homeostasis after chronic stress. Based on these molecular insights, we applied rapamycin after the stress as a proof-of-concept pharmacological intervention and were able to substantially increase stress resilience. Our findings serve as a data resource and can open new avenues for further understanding of molecular processes underlying stress response and for targeted interventions supporting resilience.

Voluntary Wheel Running Did Not Alter Gene Expression in 5xfad Mice, but in Wild-Type Animals Exclusively after One-Day of Physical Activity.

Physical activity is considered a promising preventive intervention to reduce the risk of developing Alzheimer's disease (AD). However, the positive effect of therapeutic administration of physical activity has not been proven conclusively yet, likely due to confounding factors such as varying activity regimens and life or disease stages. To examine the impact of different routines of physical activity in the early disease stages, we subjected young 5xFAD and wild-type mice to 1-day (acute) and 30-day (chronic) voluntary wheel running and compared them with age-matched sedentary controls. We observed a significant increase in brain lactate levels in acutely trained 5xFAD mice relative to all other experimental groups. Subsequent brain RNA-seq analysis did not reveal major differences in transcriptomic regulation between training durations in 5xFAD mice. In contrast, acute training yielded substantial gene expression changes in wild-type animals relative to their chronically trained and sedentary counterparts. The comparison of 5xFAD and wild-type mice showed the highest transcriptional differences in the chronic and sedentary groups, whereas acute training was associated with much fewer differentially expressed genes. In conclusion, our results suggest that different training durations did not affect the global transcriptome of 3-month-old 5xFAD mice, whereas acute running seemed to induce a similar transcriptional stress state in wild-type animals as already known for 5xFAD mice.

N6 -Methyladenosine Modification in Chronic Stress Response Due to Social Hierarchy Positioning of Mice.

Appropriately responding to stressful events is essential for maintaining health and well-being of any organism. Concerning social stress, the response is not always as straightforward as reacting to physical stressors, e.g., extreme heat, and thus has to be balanced subtly. Particularly, regulatory mechanisms contributing to gaining resilience in the face of mild social stress are not fully deciphered yet. We employed an intrinsic social hierarchy stress paradigm in mice of both sexes to identify critical factors for potential coping strategies. While global transcriptomic changes could not be observed in male mice, several genes previously reported to be involved in synaptic plasticity, learning, and anxiety-like behavior were differentially regulated in female mice. Moreover, changes in N6-methyladenosine (m6A)-modification of mRNA occurred associated with corticosterone level in both sexes with, e.g., increased global amount in submissive female mice. In accordance with this, METTL14 and WTAP, subunits of the methyltransferase complex, showed elevated levels in submissive female mice. N6-adenosyl-methylation is the most prominent type of mRNA methylation and plays a crucial role in processes such as metabolism, but also response to physical stress. Our findings underpin its essential role by also providing a link to social stress evoked by hierarchy building within same-sex groups. As recently, search for small molecule modifiers for the respective class of RNA modifying enzymes has started, this might even lead to new therapeutic approaches against stress disorders.

The gut microbiota instructs the hepatic endothelial cell transcriptome.

The gut microbiota affects remote organ functions but its impact on organotypic endothelial cell (EC) transcriptomes remains unexplored. The liver endothelium encounters microbiota-derived signals and metabolites via the portal circulation. To pinpoint how gut commensals affect the hepatic sinusoidal endothelium, a magnetic cell sorting protocol, combined with fluorescence-activated cell sorting, was used to isolate hepatic sinusoidal ECs from germ-free (GF) and conventionally raised (CONV-R) mice for transcriptome analysis by RNA sequencing. This resulted in a comprehensive map of microbiota-regulated hepatic EC-specific transcriptome profiles. Gene Ontology analysis revealed that several functional processes in the hepatic endothelium were affected. The absence of microbiota influenced the expression of genes involved in cholesterol flux and angiogenesis. Specifically, genes functioning in hepatic endothelial sphingosine metabolism and the sphingosine-1-phosphate pathway showed drastically increased expression in the GF state. Our analyses reveal a prominent role for the microbiota in shaping the transcriptional landscape of the hepatic endothelium.

Applying univariate vs. multivariate statistics to investigate therapeutic efficacy in (pre)clinical trials: A Monte Carlo simulation study on the example of a controlled preclinical neurotrauma trial.

BACKGROUND: Small sample sizes combined with multiple correlated endpoints pose a major challenge in the statistical analysis of preclinical neurotrauma studies. The standard approach of applying univariate tests on individual response variables has the advantage of simplicity of interpretation, but it fails to account for the covariance/correlation in the data. In contrast, multivariate statistical techniques might more adequately capture the multi-dimensional pathophysiological pattern of neurotrauma and therefore provide increased sensitivity to detect treatment effects. RESULTS: We systematically evaluated the performance of univariate ANOVA, Welch's ANOVA and linear mixed effects models versus the multivariate techniques, ANOVA on principal component scores and MANOVA tests by manipulating factors such as sample and effect size, normality and homogeneity of variance in computer simulations. Linear mixed effects models demonstrated the highest power when variance between groups was equal or variance ratio was 1:2. In contrast, Welch's ANOVA outperformed the remaining methods with extreme variance heterogeneity. However, power only reached acceptable levels of 80% in the case of large simulated effect sizes and at least 20 measurements per group or moderate effects with at least 40 replicates per group. In addition, we evaluated the capacity of the ordination techniques, principal component analysis (PCA), redundancy analysis (RDA), linear discriminant analysis (LDA), and partial least squares discriminant analysis (PLS-DA) to capture patterns of treatment effects without formal hypothesis testing. While LDA suffered from a high false positive rate due to multicollinearity, PCA, RDA, and PLS-DA were robust and PLS-DA outperformed PCA and RDA in capturing a true treatment effect pattern. CONCLUSIONS: Multivariate tests do not provide an appreciable increase in power compared to univariate techniques to detect group differences in preclinical studies. However, PLS-DA seems to be a useful ordination technique to explore treatment effect patterns without formal hypothesis testing.

Impact of Acute and Chronic Amyloid-ß Peptide Exposure on Gut Microbial Commensals in the Mouse.

Alzheimer's disease (AD) is the most common form of dementia. Besides its cognitive phenotype, AD leads to crucial changes in gut microbiome composition in model mice and in patients, but the reported data are still highly inconsistent. Therefore, we investigated chronic effects of AD-characteristic neurotoxic amyloid-ß (Aß) peptides as provided by transgenic overexpression (5xFAD mouse model) and acute effects due to oral application of Aß on gut microbes. Astonishingly, one-time feeding of wild type mice with Aß42 provoked immediate changes in gut microbiome composition (ß diversity) as compared to controls. Such obvious changes were not observed when comparing 5xFAD mice with wild type littermates. However, acute as well as chronic exposure to Aß significantly affected the abundance of numerous individual operational taxonomic units. This provides first evidence that acute in vivo exposure to Aß results in a shift in the enteric microbiome. Furthermore, we suggest that chronic exposure to Aß might trigger an adaptive response of gut microbiota which could thereby result in dysbiosis in model mice but also in human patients.

α-Linolenic Acid-Rich Diet Influences Microbiota Composition and Villus Morphology of the Mouse Small Intestine.

α-Linolenic acid (ALA) is well-known for its anti-inflammatory activity. In contrast, the influence of an ALA-rich diet on intestinal microbiota composition and its impact on small intestine morphology are not fully understood. In the current study, we kept adult C57BL/6J mice for 4 weeks on an ALA-rich or control diet. Characterization of the microbial composition of the small intestine revealed that the ALA diet was associated with an enrichment in Prevotella and Parabacteroides. In contrast, taxa belonging to the Firmicutes phylum, including Lactobacillus, Clostridium cluster XIVa, Lachnospiraceae and Streptococcus, had significantly lower abundance compared to control diet. Metagenome prediction indicated an enrichment in functional pathways such as bacterial secretion system in the ALA group, whereas the two-component system and ALA metabolism pathways were downregulated. We also observed increased levels of ALA and its metabolites eicosapentanoic and docosahexanoic acid, but reduced levels of arachidonic acid in the intestinal tissue of ALA-fed mice. Furthermore, intestinal morphology in the ALA group was characterized by elongated villus structures with increased counts of epithelial cells and reduced epithelial proliferation rate. Interestingly, the ALA diet reduced relative goblet and Paneth cell counts. Of note, high-fat Western-type diet feeding resulted in a comparable adaptation of the small intestine. Collectively, our study demonstrates the impact of ALA on the gut microbiome and reveals the nutritional regulation of gut morphology.

Germ-free housing conditions do not affect aortic root and aortic arch lesion size of late atherosclerotic low-density lipoprotein receptor-deficient mice.

The microbiota has been linked to the development of atherosclerosis, but the functional impact of these resident bacteria on the lesion size and cellular composition of atherosclerotic plaques in the aorta has never been experimentally addressed with the germ-free low-density lipoprotein receptor-deficient (Ldlr-/- ) mouse atherosclerosis model. Here, we report that 16 weeks of high-fat diet (HFD) feeding of hypercholesterolemic Ldlr-/- mice at germ-free (GF) housing conditions did not impact relative aortic root plaque size, macrophage content, and necrotic core area. Likewise, we did not find changes in the relative aortic arch lesion size. However, late atherosclerotic GF Ldlr-/- mice had altered inflammatory plasma protein markers and reduced smooth muscle cell content in their atherosclerotic root plaques relative to CONV-R Ldlr-/- mice. Neither absolute nor relative aortic root or aortic arch plaque size correlated with age. Our analyses on GF Ldlr-/- mice did not reveal a significant contribution of the microbiota in late aortic atherosclerosis.

The Microbiota Promotes Arterial Thrombosis in Low-Density Lipoprotein Receptor-Deficient Mice.

Atherosclerotic plaque development depends on chronic inflammation of the arterial wall. A dysbiotic gut microbiota can cause low-grade inflammation, and microbiota composition was linked to cardiovascular disease risk. However, the role of this environmental factor in atherothrombosis remains undefined. To analyze the impact of gut microbiota on atherothrombosis, we rederived low-density lipoprotein receptor-deficient (Ldlr-/- ) mice as germfree (GF) and kept these mice for 16 weeks on an atherogenic high-fat Western diet (HFD) under GF isolator conditions and under conventionally raised specific-pathogen-free conditions (CONV-R). In spite of reduced diversity of the cecal gut microbiome, caused by atherogenic HFD, GF Ldlr-/- mice and CONV-R Ldlr-/- mice exhibited atherosclerotic lesions of comparable sizes in the common carotid artery. In contrast to HFD-fed mice, showing no difference in total cholesterol levels, CONV-R Ldlr-/- mice fed control diet (CD) had significantly reduced total plasma cholesterol, very-low-density lipoprotein (VLDL), and LDL levels compared with GF Ldlr-/- mice. Myeloid cell counts in blood as well as leukocyte adhesion to the vessel wall at the common carotid artery of GF Ldlr-/- mice on HFD were diminished compared to CONV-R Ldlr-/- controls. Plasma cytokine profiling revealed reduced levels of the proinflammatory chemokines CCL7 and CXCL1 in GF Ldlr-/- mice, whereas the T-cell-related interleukin 9 (IL-9) and IL-27 were elevated. In the atherothrombosis model of ultrasound-induced rupture of the common carotid artery plaque, thrombus area was significantly reduced in GF Ldlr-/- mice relative to CONV-R Ldlr-/- mice. Ex vivo, this atherothrombotic phenotype was explained by decreased adhesion-dependent platelet activation and thrombus growth of HFD-fed GF Ldlr-/- mice on type III collagen.IMPORTANCE Our results demonstrate a functional role for the commensal microbiota in atherothrombosis. In a ferric chloride injury model of the carotid artery, GF C57BL/6J mice had increased occlusion times compared to colonized controls. Interestingly, in late atherosclerosis, HFD-fed GF Ldlr-/- mice had reduced plaque rupture-induced thrombus growth in the carotid artery and diminished ex vivo thrombus formation under arterial flow conditions.