The PEDtracker: An Automatic Staging Approach for Drosophila melanogaster Larvae

The post-embryonal development of arthropod species, including crustaceans and insects, is characterized by ecdysis or molting. This process defines growth stages and is controlled by a conserved neuroendocrine system. Each molting event is divided in several critical time points, such as pre-molt, molt, and post-molt, and leaves the animals in a temporarily highly vulnerable state while their cuticle is re-hardening. The molting events occur in an immediate ecdysis sequence within a specific time window during the development. Each sub-stage takes only a short amount of time, which is generally in the order of minutes. To find these relatively short behavioral events, one needs to follow the entire post-embryonal development over several days. As the manual detection of the ecdysis sequence is time consuming and error prone, we designed a monitoring system to facilitate the continuous observation of the post-embryonal development of the fruit fly Drosophila melanogaster. Under constant environmental conditions we are able to observe the life cycle from the embryonic state to the adult, which takes about 10 days in this species. Specific processing algorithms developed and implemented in Fiji and R allow us to determine unique behavioral events on an individual level—including egg hatching, ecdysis and pupation. In addition, we measured growth rates and activity patterns for individual larvae. Our newly created RPackage PEDtracker can predict critical developmental events and thus offers the possibility to perform automated screens that identify changes in various aspects of larval development. In conclusion, the PEDtracker system presented in this study represents the basis for automated real-time staging and analysis not only for the arthropod development

The Panopticon—Assessing the Effect of Starvation on Prolonged Fly Activity and Place Preference

Animal behaviours are demonstrably governed by sensory stimulation, previous experience and internal states like hunger. With increasing hunger, priorities shift towards foraging and feeding. During foraging, flies are known to employ efficient path integration strategies. However, general long-term activity patterns for both hungry and satiated flies in conditions of foraging remain to be better understood. Similarly, little is known about how permanent contact chemosensory stimulation affects locomotion. To address these questions, we have developed a novel, simplistic fly activity tracking setup— the Panopticon. Using a 3D-printed Petri dish inset, our assay allows recording of walking behaviour, of several flies in parallel, with all arena surfaces covered by a uniform substrate layer. We tested two constellations of providing food: (i) in single patches and (ii) omnipresent within the substrate layer. Fly tracking is done with FIJI, further assessment, analysis and presentation is done with a custom-built MATLAB analysis framework. We find that starvation history leads to a long-lasting reduction in locomotion, as well as a delayed place preference for food patches which seems to be not driven by immediate hunger motivation

Morpho-Functional 1H-MRI of the Lung in COPD: Short-Term Test-Retest Reliability

Purpose Non-invasive end-points for interventional trials and tailored treatment regimes in chronic obstructive pulmonary disease (COPD) for monitoring regionally different manifestations of lung disease instead of global assessment of lung function with spirometry would be valuable. Proton nuclear magnetic resonance imaging (1H-MRI) allows for a radiation-free assessment of regional structure and function. The aim of this study was to evaluate the short-term reproducibility of a comprehensive morpho-functional lungMRI protocol in COPD. Materials and Methods 20 prospectively enrolled COPD patients (GOLD I-IV) underwent 1H-MRI of the lung at 1.5T on two consecutive days, including sequences for morphology, 4D contrast-enhanced perfusion, and respiratory mechanics. Image quality and COPD-related morphological and functional changes were evaluated in consensus by three chest radiologists using a dedicated MRI-based visual scoring system. Test-retest reliability was calculated per each individual lung lobe for the extent of large airway (bronchiectasis, wall thickening, mucus plugging) and small airway abnormalities (tree in bud, peripheral bronchiectasis, mucus plugging),consolidations, nodules, parenchymal defects and perfusion defects. The presence of tracheal narrowing, dystelectasis, pleural effusion, pulmonary trunk ectasia, right ventricular enlargement and, finally, motion patterns of diaphragma and chest wall were addressed. Results Median global scores [10(Q1:8.00;Q3:16.00) vs. 11(Q1:6.00;Q3:15.00)] as well as category subscores were similar between both timepoints, and kappa statistics indicated "almost perfect" global agreement (kappa = 0.86, 95% CI = 0.81-0.91). Most subscores showed at least "substantial" agreement of MRI1 and MRI2 (kappa = 0.64-1.00),whereas the agreement for the diagnosis of dystelectasis/effusion (kappa = 0.42, 95% CI = 0.00-0.93) was "moderate" and of tracheal abnormalities (kappa = 0.21, 95% CI = 0.00-0.75) "fair". Most MRI acquisitions showed at least diagnostic quality at MRI1 (276 of 278) and MRI2 (259 of 264). Conclusion Morpho-functional 1H-MRI can be obtained with reproducible image quality and high short-term test-retest reliability for COPD-related morphological and functional changes of the lung. This underlines its potential value for the monitoring of regional lung characteristics in COPD trials

Functional Lung MRI in Chronic Obstructive Pulmonary Disease: Comparison of T1 Mapping, Oxygen-Enhanced T1 Mapping and Dynamic Contrast Enhanced Perfusion

Purpose Monitoring of regional lung function in interventional COPD trials requires alternative end-points beyond global parameters such as FEV1. T1 relaxation times of the lung might allow to draw conclusions on tissue composition, blood volume and oxygen fraction. The aim of this study was to evaluate the potential value of lung Magnetic resonance imaging (MRI) with native and oxygen-enhanced T1 mapping for the assessment of COPD patients in comparison with contrast enhanced perfusion MRI. Materials and Methods 20 COPD patients (GOLD I-IV) underwent a coronal 2-dimensional inversion recovery snapshot flash sequence (8 slices/lung) at room air and during inhalation of pure oxygen, as well as dynamic contrast-enhanced first-pass perfusion imaging. Regional distribution of T1 at room air (T1), oxygen-induced T1 shortening (Delta T1) and peak enhancement were rated by 2 chest radiologists in consensus using a semi-quantitative 3-point scale in a zone-based approach. Results Abnormal T1 and Delta T1 were highly prevalent in the patient cohort. T1 and Delta T1 correlated positively with perfusion abnormalities (r = 0.81 and r = 0.80;p&0.001), and with each other (r = 0.80;p< 0.001). In GOLD stages I and II Delta T1 was normal in 16/29 lung zones with mildly abnormal perfusion (15/16 with abnormal T1). The extent of T1 (r = 0.45;p< 0.05), T1 (r = 0.52;p< 0.05) and perfusion abnormalities (r = 0.52;p< 0.05) showed a moderate correlation with GOLD stage. Conclusion Native and oxygen-enhanced T1 mapping correlated with lung perfusion deficits and severity of COPD. Under the assumption that T1 at room air correlates with the regional pulmonary blood pool and that oxygen-enhanced T1 reflects lung ventilation, both techniques in combination are principally suitable to characterize ventilation-perfusion imbalance. This appears valuable for the assessment of regional lung characteristics in COPD trials without administration of i. v. contrast

Reproducibility of pulmonary magnetic resonance angiography in adults with muco-obstructive pulmonary disease

Background Recent studies support magnetic resonance angiography (MRA) as a diagnostic tool for pulmonary arterial disease. Purpose To determine MRA image quality and reproducibility, and the dependence of MRA image quality and reproducibility on disease severity in patients with chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF). Material and Methods Twenty patients with COPD (mean age 66.5 ± 8.9 years; FEV1% = 42.0 ± 13.3%) and 15 with CF (mean age 29.3 ± 9.3 years; FEV1% = 66.6 ± 15.8%) underwent morpho-functional chest magnetic resonance imaging (MRI) including time-resolved MRA twice one month apart (MRI1, MRI2), and COPD patients underwent non-contrast computed tomography (CT). Image quality was assessed visually using standardized subjective 5-point scales. Contrast-to-noise ratio (CNR) and signal-to-noise ratio (SNR) were measured by regions of interest. Disease severity was determined by spirometry, a well-evaluated chest MRI score, and by computational CT emphysema index (EI) for COPD. Results Subjective image quality was diagnostic for all MRA at MRI1 and MRI2 (mean score = 4.7 ± 0.6). CNR and SNR were 4 43.8 ± 8.7 and 50.5 ± 8.7, respectively. Neither image quality score nor CNR or SNR correlated with FEV1% or chest MRI score for COPD and CF (r = 0.239–0.248). CNR and SNR did not change from MRI1 to MRI2 (P = 0.434–0.995). Further, insignificant differences in CNR and SNR between MRA at MRI1 and MRI2 did not correlate with FEV1% nor chest MRI score in COPD and CF (r = −0.238–0.183), nor with EI in COPD (r = 0.100–0.111). Conclusion MRA achieved diagnostic quality in COPD and CF patients and was highly reproducible irrespective of disease severity. This supports MRA as a robust alternative to CT in patients with underlying muco-obstructive lung disease

Echo Time-Dependent Observed Lung T-1 in Patients With Chronic Obstructive Pulmonary Disease in Correlation With Quantitative Imaging and Clinical Indices

Background There is a clinical need for imaging-derived biomarkers for the management of chronic obstructive pulmonary disease (COPD). Observed pulmonary T-1 (T-1(TE)) depends on the echo-time (TE) and reflects regional pulmonary function. Purpose To investigate the potential diagnostic value of T-1(TE) for the assessment of lung disease in COPD patients by determining correlations with clinical parameters and quantitative CT. Study Type Prospective non-randomized diagnostic study. Population Thirty COPD patients (67.7 +/- 6.6 years). Data from a previous study (15 healthy volunteers [26.2 +/- 3.9 years) were used as reference. Field Strength/Sequence Study participants were examined at 1.5 T using dynamic contrast-enhanced three-dimensional gradient echo keyhole perfusion sequence and a multi-echo inversion recovery two-dimensional UTE (ultra-short TE) sequence for T-1(TE) mapping at TE1-5 = 70 mu sec, 500 mu sec, 1200 mu sec, 1650 mu sec, and 2300 mu sec. Assessment Perfusion images were scored by three radiologists. T-1(TE) was automatically quantified. Computed tomography (CT) images were quantified in software (qCT). Clinical parameters including pulmonary function testing were also acquired. Statistical Tests Spearman rank correlation coefficients (rho) were calculated between T-1(TE) and perfusion scores, clinical parameters and qCT. A P-value -0.69) were found. Overall, correlations were strongest at TE2, weaker at TE1 and rarely significant at TE4-TE5. Data Conclusion In COPD patients, the increase of T-1(TE) with TE occurred at shorter TEs than previously found in healthy subjects. Together with the lack of correlation between T-1 and clinical parameters of disease at longer TEs, this suggests that T-1(TE) quantification in COPD patients requires shorter TEs. The TE-dependence of correlations implies that T-1(TE) mapping might be developed further to provide diagnostic information beyond T-1 at a single TE. Level of Evidence 2 Technical Efficacy Stage

Quantification of pulmonary perfusion abnormalities using DCE-MRI in COPD: comparison with quantitative CT and pulmonary function

Objectives Pulmonary perfusion abnormalities are prevalent in patients with chronic obstructive pulmonary disease (COPD), are potentially reversible, and may be associated with emphysema development. Therefore, we aimed to evaluate the clinical meaningfulness of perfusion defects in percent (QDP) using DCE-MRI. Methods We investigated a subset of baseline DCE-MRIs, paired inspiratory/expiratory CTs, and pulmonary function testing (PFT) of 83 subjects (age = 65.7 +/- 9.0 years, patients-at-risk, and all GOLD groups) from one center of the COSYCONET COPD cohort. QDP was computed from DCE-MRI using an in-house developed quantification pipeline, including four different approaches: Otsu's method, k-means clustering, texture analysis, and 80(th) percentile threshold. QDP was compared with visual MRI perfusion scoring, CT parametric response mapping (PRM) indices of emphysema (PRMEmph) and functional small airway disease (PRMfSAD), and FEV1/FVC from PFT. Results All QDP approaches showed high correlations with the MRI perfusion score (r = 0.67 to 0.72, p < 0.001), with the highest association based on Otsu's method (r = 0.72, p < 0.001). QDP correlated significantly with all PRM indices (p < 0.001), with the strongest correlations with PRMEmph (r = 0.70 to 0.75, p < 0.001). QDP was distinctly higher than PRMEmph (mean difference = 35.85 to 40.40) and PRMfSAD (mean difference = 15.12 to 19.68), but in close agreement when combining both PRM indices (mean difference = 1.47 to 6.03) for all QDP approaches. QDP correlated moderately with FEV1/FVC (r = - 0.54 to - 0.41, p < 0.001). Conclusion QDP is associated with established markers of disease severity and the extent corresponds to the CT-derived combined extent of PRMEmph and PRMfSAD. We propose to use QDP based on Otsu's method for future clinical studies in COPD

Design and application of an MR reference phantom for multicentre lung imaging trials

Introduction As there is an increasing number of multicentre lung imaging studies with MRI in patients, dedicated reference phantoms are required to allow for the assessment and comparison of image quality in multi-vendor and multi-centre environments. However, appropriate phantoms for this purpose are so far not available commercially. It was therefore the purpose of this project to design and apply a cost-effective and simple to use reference phantom which addresses the specific requirements for imaging the lungs with MRI. Methods The phantom was designed to simulate 4 compartments (lung, blood, muscle and fat) which reflect the specific conditions in proton-MRI of the chest. Multiple phantom instances were produced and measured at 15 sites using a contemporary proton-MRI protocol designed for an in vivo COPD study at intervals over the course of the study. Measures of signal- and contrast-to-noise ratio, as well as structure and edge depiction were extracted from conventionally acquired images using software written for this purpose. Results For the signal to noise ratio, low intra-scanner variability was found with 4.5% in the lung compartment, 4.0% for blood, 3.3% for muscle and 3.7% for fat. The inter-scanner variability was substantially higher, with 41%, 32%, 27% and 32% for the same order of compartments. In addition, measures of structure and edge depiction were found to both vary significantly among several scanner types and among scanners of the same model which were equipped with different gradient systems. Conclusion The described reference phantom reproducibly quantified image quality aspects and detected substantial inter-scanner variability in a typical pulmonary multicentre proton MRI study, while variability was greater in lung tissue compared to other tissue types. Accordingly, appropriate reference phantoms can help to detect bias in multicentre in vivo study results and could also be used to harmonize equipment or data

Towards quantitative perfusion MRI of the lung in COPD: The problem of short-term repeatability

Purpose 4D perfusion magnetic resonance imaging (MRI) with intravenous injection of contrast agent allows for a radiation-free assessment of regional lung function. It is therefore a valuable method to monitor response to treatment in patients with chronic obstructive pulmonary disease (COPD). This study was designed to evaluate its potential for monitoring short-term response to hyperoxia in COPD patients. Materials and methods 19 prospectively enrolled COPD patients (median age 66y) underwent paired dynamic contrast-enhanced 4D perfusion MRI within 35min, first breathing 100% oxygen (injection 1, O-2) and then room air (injection 2, RA), which was repeated on two consecutive days (day 1 and 2). Post-processing software was employed to calculate mean transit time (MTT), pulmonary blood volume (PBV) and pulmonary blood flow (PBF), based on the indicator dilution theory, for the automatically segmented whole lung and 12 regions of equal volume. Results Comparing O-2 with RA conditions, PBF and PBV were found to be significantly lower at O-2, consistently on both days (p<10-8). Comparing day 2 to day 1, MTT was shorter by 0.59 +/- 0.63 s (p<10-8), PBF was higher by 22 +/- 80 ml/min/100ml (p<3.10-4), and PBV tended to be lower by 0.2 +/- 7.2 ml/100ml (p = 0.159) at both, RA and O-2, conditions. Conclusion The second injection (RA) yielded higher PBF and PBV, which apparently contradicts the established hypothesis that hyperoxia increases lung perfusion. Quantification of 4D perfusion MRI by current software approaches may thus be limited by residual circulating contrast agent in the short-term and even the next day