Transcriptomic and proteomic profiling of young and old mice in the bleomycin model reveals high similarity.

The most common preclinical, in vivo model to study lung fibrosis is the bleomycin-induced lung fibrosis model in 2- to 3-mo-old mice. Although this model resembles key aspects of idiopathic pulmonary fibrosis (IPF), there are limitations in its predictability for the human disease. One of the main differences is the juvenile age of animals that are commonly used in experiments, resembling humans of around 20 yr. Because IPF patients are usually older than 60 yr, aging appears to play an important role in the pathogenesis of lung fibrosis. Therefore, we compared young (3 months) and old mice (21 months) 21 days after intratracheal bleomycin instillation. Analyzing lung transcriptomics (mRNAs and miRNAs) and proteomics, we found most pathways to be similarly regulated in young and old mice. However, old mice show imbalanced protein homeostasis as well as an increased inflammatory state in the fibrotic phase compared to young mice. Comparisons with published human transcriptomic data sets (GSE47460, GSE32537, and GSE24206) revealed that the gene signature of old animals correlates significantly better with IPF patients, and it also turned human healthy individuals better into "IPF patients" using an approach based on predictive disease modeling. Both young and old animals show similar molecular hallmarks of IPF in the bleomycin-induced lung fibrosis model, although old mice more closely resemble several features associated with IPF in comparison to young animals.

Deep Learning-Based Detection of Endothelial Tip Cells in the Oxygen-Induced Retinopathy Model.

Proliferative retinopathies, such as diabetic retinopathy and retinopathy of prematurity, are leading causes of vision impairment. A common feature is a loss of retinal capillary vessels resulting in hypoxia and neuronal damage. The oxygen-induced retinopathy model is widely used to study revascularization of an ischemic area in the mouse retina. The presence of endothelial tip cells indicates vascular recovery; however, their quantification relies on manual counting in microscopy images of retinal flat mount preparations. Recent advances in deep neural networks (DNNs) allow the automation of such tasks. We demonstrate a workflow for detection of tip cells in retinal images using the DNN-based Single Shot Detector (SSD). The SSD was designed for detection of objects in natural images. We adapt the SSD architecture and training procedure to the tip cell detection task and retrain the DNN using labeled tip cells in images of fluorescently stained retina flat mounts. Transferring knowledge from the pretrained DNN and extensive data augmentation reduced the amount of required labeled data. Our system shows a performance comparable to the human level, while providing highly consistent results. Therefore, such a system can automate counting of tip cells, a readout frequently used in retinopathy research, thereby reducing routine work for biomedical experts.

Automatization and improvement of µCT analysis for murine lung disease models using a deep learning approach.

BACKGROUND: One of the main diagnostic tools for lung diseases in humans is computed tomography (CT). A miniaturized version, micro-CT (µCT) is utilized to examine small rodents including mice. However, fully automated threshold-based segmentation and subsequent quantification of severely damaged lungs requires visual inspection and manual correction. METHODS: Here we demonstrate the use of densitometry on regions of interest (ROI) in automatically detected portions of the lung, thus avoiding the need for lung segmentation. Utilizing deep learning approaches, the middle part of the lung is found in a µCT-stack and a ROI is placed in the left and the right lobe. RESULTS: The intensity values within the ROIs of the µCT images were collected and subsequently used for the calculation of different lung-related parameters, such as mean lung attenuation (MLA), mode, full width at half maximum (FWHM), and skewness. For validation, the densitometric approach was correlated with histological readouts (Ashcroft Score, Mean Linear Intercept). CONCLUSION: We here show an automated tool that allows rapid and in-depth analysis of µCT scans of different murine models of lung disease.

Three-dimensional accurate detection of lung emphysema in rats using ultra-short and zero echo time MRI.

Emphysema is a life-threatening pathology that causes irreversible destruction of alveolar walls. In vivo imaging techniques play a fundamental role in the early non-invasive pre-clinical and clinical detection and longitudinal follow-up of this pathology. In the present study, we aimed to evaluate the feasibility of using high resolution radial three-dimensional (3D) zero echo time (ZTE) and 3D ultra-short echo time (UTE) MRI to accurately detect lung pathomorphological changes in a rodent model of emphysema.Porcine pancreas elastase (PPE) was intratracheally administered to the rats to produce the emphysematous changes. 3D ZTE MRI, low and high definition 3D UTE MRI and micro-computed tomography images were acquired 4 weeks after the PPE challenge. Signal-to-noise ratios (SNRs) were measured in PPE-treated and control rats. T2* values were computed from low definition 3D UTE MRI. Histomorphometric measurements were made after euthanizing the animals. Both ZTE and UTE MR images showed a significant decrease in the SNR measured in PPE-treated lungs compared with controls, due to the pathomorphological changes taking place in the challenged lungs. A significant decrease in T2* values in PPE-challenged animals compared with controls was measured using UTE MRI. Histomorphometric measurements showed a significant increase in the mean linear intercept in PPE-treated lungs. UTE yielded significantly higher SNR compared with ZTE (14% and 30% higher in PPE-treated and non-PPE-treated lungs, respectively).This study showed that optimized 3D radial UTE and ZTE MRI can provide lung images of excellent quality, with high isotropic spatial resolution (400 µm) and SNR in parenchymal tissue (>25) and negligible motion artifacts in freely breathing animals. These techniques were shown to be useful non-invasive instruments to accurately and reliably detect the pathomorphological alterations taking place in emphysematous lungs, without incurring the risks of cumulative radiation exposure typical of micro-computed tomography.

Modulation of vascular contraction via soluble guanylate cyclase signaling in a novel ex vivo method using rat precision-cut liver slices.

Fibrotic processes in the liver of non-alcoholic steatohepatitis (NASH) patients cause microcirculatory dysfunction in the organ which increases blood vessel resistance and causes portal hypertension. Assessing blood vessel function in the liver is challenging, necessitating the development of novel methods in normal and fibrotic tissue that allow for drug screening and translation toward pre-clinical settings. Cultures of precision cut liver slices (PCLS) from normal and fibrotic rat livers were used for blood vessel function analysis. Live recording of vessel diameter was used to assess the response to endothelin-1, serotonin and soluble guanylate cyclase (sGC) activation. A cascade of contraction and relaxation events in response to serotonin, endothelin-1, Ketanserin and sGC activity could be established using vessel diameter analysis of rat PCLS. Both the sGC activator BI 703704 and the sGC stimulator Riociguat prevented serotonin-induced contraction in PCLS from naive rats. By contrast, PCLS cultures from the rat CCl4 NASH model were only responsive to the sGC activator, thus establishing that the sGC enzyme is rendered non-responsive to nitric oxide under oxidative stress found in fibrotic livers. The role of the sGC pathway for vessel relaxation of fibrotic liver tissue was identified in our model. The obtained data shows that the inhibitory capacities on vessel contraction of sGC compounds can be translated to published preclinical data. Altogether, this novel ex vivo PCLS method allows for the differentiation of drug candidates and the translation of therapeutic approaches towards the clinical use.

Intermittent exposure to whole cigarette smoke alters the differentiation of primary small airway epithelial cells in the air-liquid interface culture.

Cigarette smoke (CS) is the leading risk factor to develop COPD. Therefore, the pathologic effects of whole CS on the differentiation of primary small airway epithelial cells (SAEC) were investigated, using cells from three healthy donors and three COPD patients, cultured under ALI (air-liquid interface) conditions. The analysis of the epithelial physiology demonstrated that CS impaired barrier formation and reduced cilia beat activity. Although, COPD-derived ALI cultures preserved some features known from COPD patients, CS-induced effects were similarly pronounced in ALI cultures from patients compared to healthy controls. RNA sequencing analyses revealed the deregulation of marker genes for basal and secretory cells upon CS exposure. The comparison between gene signatures obtained from the in vitro model (CS vs. air) with a published data set from human epithelial brushes (smoker vs. non-smoker) revealed a high degree of similarity between deregulated genes and pathways induced by CS. Taken together, whole cigarette smoke alters the differentiation of small airway basal cells in vitro. The established model showed a good translatability to the situation in vivo. Thus, the model can help to identify and test novel therapeutic approaches to restore the impaired epithelial repair mechanisms in COPD, which is still a high medical need.

Seeding induced by alpha-synuclein oligomers provides evidence for spreading of alpha-synuclein pathology.

Lewy bodies, alpha-synuclein (alpha-syn) immunopositive intracellular deposits, are the pathological hallmark of Parkinson's disease (PD). Interestingly, Lewybody-like structures have been identified in fetal tissue grafts about one decade after transplantation into the striatum of PD patients. One possible explanation for the accelerated deposition of alpha-syn in the graft is that the aggregation of alpha-syn from the host tissue to the graft is spread by a prion disease-like mechanism. We discuss here an in vitro model which might recapitulate some aspects of disease propagation in PD. We found here that in vitro-generated alpha-syn oligomers induce transmembrane seeding of alpha-syn aggregation in a dose- and time-dependent manner. This effect was observed in primary neuronal cultures as well as in neuronal cell lines. The seeding oligomers were characterized by a distinctive lithium dodecyl sulfate-stable oligomer pattern and could be generated in a dynamic process out of pore-forming oligomers. We propose that alpha-syn oligomers form as a dynamic mixture of oligomer types with different properties and that alpha-syn oligomers can be converted into different types depending on the brain milieu conditions. Our data indicate that extracellular alpha-syn oligomers can induce intracellular alpha-syn aggregation, therefore we hypothesize that a similar mechanism might lead to alpha-syn pathology propagation.

Deep learning enables pathologist-like scoring of NASH models.

Non-alcoholic fatty liver disease (NAFLD) and the progressive form of non-alcoholic steatohepatitis (NASH) are diseases of major importance with a high unmet medical need. Efficacy studies on novel compounds to treat NAFLD/NASH using disease models are frequently evaluated using established histological feature scores on ballooning, inflammation, steatosis and fibrosis. These features are assessed by a trained pathologist using microscopy and assigned discrete scores. We demonstrate how to automate these scores with convolutional neural networks (CNNs). Whole slide images of stained liver sections are analyzed using two different scales with four CNNs, each specialized for one of four histopathological features. A continuous value is obtained to quantify the extent of each feature, which can be used directly to provide a high resolution readout. In addition, the continuous values can be mapped to obtain the established discrete pathologist-like scores. The automated deep learning-based scores show good agreement with the trainer - a human pathologist.

Automated classification of the behavior of rats in the forced swimming test with support vector machines.

The forced swimming test of rats or mice is a frequently used behavioral test to evaluate compounds for antidepressant activity in vivo. The aim of this study was to replace the human observer, needed to score and analyze the behavior of animals, by a fully automated method. For this purpose, in a first step from a video recording of each rat, an activity profile was calculated, from which subsequently a set of meaningful properties was extracted. This set was finally used to train a Support Vector Machine (SVM). Furthermore, specialized kernel functions, namely the so-called time resolved p-spectrum and modified optimal assignment kernels, were developed to calculate the similarity of activity profiles. Our method allows for a very reliable discrimination of animals treated with antidepressants of different classes (tricyclics imipramine and desipramine as well as selective serotonin reuptake inhibitor, SSRI, fluoxetine) versus a vehicle-treated group. Moreover, our technique is able to classify between tricyclic antidepressants and SSRIs. The results of this work enabled the development of an automated and highly accurate behavior classification system.

Deep neural network based histological scoring of lung fibrosis and inflammation in the mouse model system.

Preclinical studies of novel compounds rely on quantitative readouts from animal models. Frequently employed readouts from histopathological tissue scoring are time consuming, require highly specialized staff and are subject to inherent variability. Recent advances in deep convolutional neural networks (CNN) now allow automating such scoring tasks. Here, we demonstrate this for the case of the Ashcroft fibrosis score and a newly developed inflammation score to characterize fibrotic and inflammatory lung diseases. Sections of lung tissue from mice exhibiting a wide range of fibrotic and inflammatory states were stained with Masson trichrome. Whole slide scans using a 20x objective were acquired and cut into smaller tiles of 512x512 pixels. The tiles were subsequently classified by specialized CNNs, either an "Ashcroft fibrosis CNN" or an "inflammation CNN". For the Ashcroft fibrosis score the CNN was fine-tuned by using 14000 labelled tiles. For the inflammation score the CNN was trained with 3500 labelled tiles. After training, the Ashcroft fibrosis CNN achieved an accuracy of 79.5% and the inflammation CNN an accuracy of 80.0%. An error analysis revealed that misclassifications are almost exclusively with neighboring scores, which reflects the inherent ambiguity of parts of the data. The variability between two experts was found to be larger than the variability between the CNN classifications and the ground truth. The CNN generated Ashcroft score was in very good agreement with the score of a pathologist (r2 = 0.92). Our results demonstrate that costly and time consuming scoring tasks can be automated and standardized with deep learning. New scores such as the inflammation score can be easily developed with the approach presented here.

Age-related pathology after adenoviral overexpression of the leucine-rich repeat kinase 2 in the mouse striatum.

Mutations in leucine-rich repeat kinase 2 (LRRK2) age-dependently cause Parkinson's disease and are associated with several inflammatory diseases. So far, the potential role of LRRK2 expression in glial cells as mediators of neuroinflammation and the influence of aging have not been investigated in viral vector-based LRRK2 animal models. In this study, we compared the effect of striatal injection of high-capacity adenoviral vectors expressing either a kinase-overactive LRRK2 with the familial G2019S mutation or a kinase-inactive LRRK2 variant in young and old C57BL/6J mice. The intrinsic adenovirus tropism guided preferentially glial transduction, and the vector design led to stable expression for at least 6 months. In histopathological analysis, young mice expressing either LRRK2 variant presented with transient vacuolization of striatal white fiber tracts accompanied by accumulation of microglial cells and astrogliosis, but inflammation resolved without permanent damage. Old mice had a stronger and prolonged inflammatory reaction and experienced permanent damage in form of partial neuron loss after 3 months exclusively in case of LRRK2_G2019S expression. The autophagic receptor p62 accumulated in cells with high levels of either LRRK2 variant, even more so in old mice. We conclude that the aging mouse brain is more susceptible to LRRK2-associated pathology, and in this model, glial LRRK2 expression significantly contributed to neuroinflammation, ultimately causing neurodegeneration.

The selective mGlu5 receptor antagonist MTEP, similar to NMDA receptor antagonists, induces social isolation in rats.

It has repeatedly been shown that uncompetitive N-methyl-D-aspartate (NMDA) receptor antagonists can mimic certain aspects of positive and negative symptoms of schizophrenia in human volunteers and laboratory animals. The purpose of the present study was to expand these findings and to determine whether the selective metabotropic glutamate receptor subtype 5 (mGluR5) antagonist, MTEP (3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine), could induce similar effects in Wistar rats. First, MTEP (1.0-10.0 mg/kg; intraperitoneally) after acute and subchronic (daily for 5 days) administration as well as the uncompetitive antagonists of the NMDA receptor of either high affinity, phencyclidine (0.5-4.0 mg/kg; subcutaneously (s.c.)) and (+)-MK-801 (0.03-0.25 mg/kg; s.c.), or low-moderate affinity, ketamine (2.0-16.0 mg/kg; s.c.) and memantine (0.15-20.0 mg/kg; s.c.), following daily administration for 3 days were tested in the social interaction test to determine their ability to reproduce the negative and positive symptoms measured by social isolation and stereotyped behavior, respectively. Second, the compounds were tested in the motility test following acute administration to determine their ability to induce locomotor hyperactivity reflecting the positive symptoms. In line with previous findings, all examined NMDA receptor antagonists produced social interaction deficits, locomotor hyperactivity, and stereotypy except memantine. Notably, this study found that MTEP following both acute and subchronic administration dose-dependently induced social isolation, but did not cause either locomotor hyperactivity or stereotypy. These data demonstrate that social behavior deficits in rats can be caused by both the blockade of the NMDA receptor and the inhibition of mGluR5, whereas mGluR5 antagonists may not independently be able to mimic the positive symptoms.

Functional Proton MRI in Emphysematous Rats.

OBJECTIVE: To demonstrate the feasibility of proton magnetic resonance imaging (MRI) ventilation-related maps in rodents for the evaluation of lung function in the presence of pancreatic porcine elastase (PPE)-induced emphysema. MATERIALS AND METHODS: Twelve rats were equally divided into 3 groups: group 1 (no administration of PPE); group 2 (PPE selectively only in the left lung); and group 3 (PPE administered in both lungs). Magnetic resonance imaging (MRI) and computed tomographic (CT) data were acquired at baseline, at 2 weeks and 4 weeks after administration, after which the animals were euthanized. The MRI protocol comprised a golden angle 2-dimensional ultrashort echo time MRI sequence [echo time, 0.343 millisecond (ms); repetition time, 120 ms; 12 slides with thickness, 1 mm; acquisition time, 30 minutes], from which inspiration and expiration images were reconstructed after the extraction of a self-gating signal. Inspiration images were registered to images at expiration, and expansion maps were created by calculating the specific difference in signal intensity. The lungs were segmented, and the mean specific expansion (MSE) calculated as an established surrogate for fractional ventilation. Computed tomographic data provided lung density (peak of the Hounsfield unit histogram, HU_P), whereas histology provided the mean linear intercept for each lung. RESULTS: Two weeks after administration, the control group had a mean MSE in both lungs corresponding to 96% of the baseline. Group 2 had 85% of the baseline, and group 3 had 57%. Considering the PPE-treated lungs alone, a significant reduction in MSE of 27% at 2 weeks and 40% at 4 weeks was found with respect to nontreated lungs. Significant correlations between HU_P and MSE were found at all time points (baseline: r = 0.606, P = 0.0017; 2 weeks: r = 0.837, P ≤ 0.0001; 4 weeks: r = 0.765, P < 0.0001; all time points: r = 0.739, P < 0.0001). Mean linear intercept values significantly correlated both with MRI MSE (r = -0.770, P < 0.0001) and with CT HU_P (r = -0.882, P < 0.0001). DISCUSSION: The calculated ventilation-related maps showed a reduction of function in the PPE-treated lungs, both compared to the nontreated lungs and to the baseline values. Moreover, a good agreement between MRI-measured MSE, CT, and histology data quantitatively supports the presence of ventilation deficit in emphysematous lungs.In this work, we have demonstrated the feasibility of ventilation-related maps from non-contrast-enhanced H lung MRI, which were capable of tracking changes in lung function over time in emphysematous rats.

Gene delivery to adipose tissue using transcriptionally targeted rAAV8 vectors.

In recent years, the increasing prevalence of obesity and obesity-related co-morbidities fostered intensive research in the field of adipose tissue biology. To further unravel molecular mechanisms of adipose tissue function, genetic tools enabling functional studies in vitro and in vivo are essential. While the use of transgenic animals is well established, attempts using viral and non-viral vectors to genetically modify adipocytes in vivo are rare. Therefore, we here characterized recombinant Adeno-associated virus (rAAV) vectors regarding their potency as gene transfer vehicles for adipose tissue. Our results demonstrate that a single dose of systemically applied rAAV8-CMV-eGFP can give rise to remarkable transgene expression in murine adipose tissues. Upon transcriptional targeting of the rAAV8 vector to adipocytes using a 2.2 kb fragment of the murine adiponectin (mAP2.2) promoter, eGFP expression was significantly decreased in off-target tissues while efficient transduction was maintained in subcutaneous and visceral fat depots. Moreover, rAAV8-mAP2.2-mediated expression of perilipin A - a lipid-droplet-associated protein - resulted in significant changes in metabolic parameters only three weeks post vector administration. Taken together, our findings indicate that rAAV vector technology is applicable as a flexible tool to genetically modify adipocytes for functional proof-of-concept studies and the assessment of putative therapeutic targets in vivo.

Human primary co-culture angiogenesis assay reveals additive stimulation and different angiogenic properties of VEGF and HGF.

Therapeutic angiogenesis aims to induce blood vessel growth in acute or chronic ischemic tissues and has gained tremendous interest over the last years. To study factors and combinations thereof that potentially induce or modify angiogenesis and to evaluate their therapeutic potential, various in vitro assays have been developed. Although endothelial cells have attracted most attention in these assays, they alone cannot complete vessel maturation since extracellular matrix (ECM) components and mesenchymal cells also play an important role in vascular development. To address this complexity we focussed on a human co-culture angiogenesis assay comprising primary endothelial cells as well as primary ECM-producing fibroblasts. In this assay HGF and VEGF as single factors and combined were tested for the potential to induce an angiogenic response, which was detected by image analysis assessing the area, length and branches of the formed vascular structures. The results show that the cytokines HGF and VEGF both promote angiogenesis in this co-culture assay by inducing distinguishable patterns of vascular structures. VEGF increases the length, area and branch point number of induced vessels whereas HGF mediates exclusively vascular area growth resulting in vascular structures of enlarged diameter. Moreover, the combination of both cytokines results in an additive increase of vascular diameter.