Inter-experimental Variability Research Articles

Outcomes of CEM43 in Predicting Thermal Damage Induced by Focal Laser Ablation in Controlled Ex Vivo Experiments: A Comparison to Histology and MRI.

Focal laser ablation (FLA) serves as a targeted therapy for prostate cancer (PCa). Clinical studies have demonstrated significant variations in ablation volumes with consistent fiber configurations. Consequently, a prediction model is needed for the safe application of FLA in treating PCa. This study aimed to evaluate the reproducibility of FLA-induced temperature profiles in controlled ex vivo experiments using clinical laser treatment protocols. Additionally, it sought to examine the effectiveness of the CEM43 model in predicting the zone of irreversible damage (ZID) and to compare these findings with outcomes derived from the Arrhenius model. Freshly excised postmortem human prostate and porcine liver specimens were used for controlled ex vivo ablation. Tissues were secured in a Perspex sample holder for precise placement of the laser fiber and thermocouples. FLA was conducted with a 1064-nm Nd:YAG laser at 3 W in continuous-wave mode for 10 min. Pre- and post-FLA 3D T1-weighted 7 T MRI scans were obtained to assess the treatment area. Whole-mount hematoxylin and eosin histological slides were prepared and digitized. On histology, the ZID was defined as the total of vaporized, carbonized, and coagulated tissue. A 2D thermal development map was created from temperature data, using bi-cubic interpolation. The cumulative equivalent thermal isoeffect dose at 43°C in minutes (CEM43) model was applied to predict the ZID, with 240 equivalent minutes (240-CEM43) used as the damage threshold. Additionally, the Arrhenius thermal model was used for comparison of CEM43 results. Predicted ZIDs were compared to MRI and histology. FLA treatment was performed on ex vivo human prostate samples (n = 2) and porcine liver specimens (n = 5). For human prostate tissue, FLA did not result in an identifiable ZID upon histological macroscopic examination or a lesion on MRI. Ex vivo porcine liver samples showed a clearly demarcated oval-shaped hyperintense lesion surrounding the laser fiber tip on post-FLA MRI. The MRI lesion (range 1.6-2.1 cm2) corresponded with the shape and location of the ZID on histology, but was smaller (median 1.7 vs. 3.2, p = 0.02). Histological examination of porcine liver samples revealed ZIDs ranging from 2.1 to 4.1 cm2, whereas 240-CEM43-predicted ZIDs ranged from 3.3 to 3.8 cm2. Although the median 240-CEM43-predicted ZID was not significantly larger than the histology ZID (3.8 vs. 3.2 cm2, p = 0.22), it tended to overpredict the histological results in most experiments. The median Arrhenius-predicted ZID was similar to the histologicalZID (3.2 vs. 3.2 cm2, p = 0.56), but varied in size when comparing individual experiments (range 2.5-3.2 cm2). FLA on ex vivo human prostate showed no thermal damage on histopathology or MRI. Ex vivo porcine liver FLA resulted in identifiable ZID on histology and lesions on MRI. 240-CEM43 generally overestimated the ZID and had less variability compared to histology. Results from the Arrhenius model were in better agreement with the histology findings, but still did not predict the individual FLA-induced histological thermal damage. Inter-experiment ZID variability underlines the need for developing a more comprehensive predictive dosimetry model for FLA in PCa treatment.

Establishment of a Magnetically Controlled Scalable Nerve Injury Model.

Animal models of peripheral nerve injury (PNI) serve as the fundamental basis for the investigations of nerve injury, regeneration, and neuropathic pain. The injury properties of such models, including the intensity and duration, significantly influence the subsequent pathological changes, pain development, and therapeutic efficacy. However, precise control over the intensity and duration of nerve injury remains challenging within existing animal models, thereby impeding accurate and comparative assessments of relevant cases. Here, a new model that provides quantitative and off-body controllable injury properties via a magnetically controlled clamp, is presented. The clamp can be implanted onto the rat sciatic nerve and exert varying degrees of compression under the control of an external magnetic field. It is demonstrated that this model can accurately simulate various degrees of pathology of human patients by adjusting the magnetic control and reveal specific pathological changes resulting from intensity heterogeneity that are challenging to detect previously. The controllability and quantifiability of this model may significantly reduce the uncertainty of central response and inter-experimenter variability, facilitating precise investigations into nerve injury, regeneration, and pain mechanisms.

Wave Excitation Tests on a Fixed Sphere

Wave excitation tests on a fixed sphere with the center at the still water level were carried out with three different physical wave basin setups. The tests were completed as a continued effort of the working group OES Wave Energy Converters Modelling Verification and Validation to increase confidence in numerical models of wave energy converters by generation of accurate benchmarks datasets for numerical model validation. An idealized testcase with wave excitation of a fixed sphere to be used with the benchmarks was formulated. The three investigated physical wave basin setups included: 1) a six degree-of-freedom load cell mounted to the top of the sphere, 2) a bending beam force transducer mounted to the top of the sphere, and 3) a system of six pretensioned wires mounted to the top and bottom of the sphere with force transducers attached to each wire. The aim of the present paper is to identify the best representation of the idealized testcase. To this end, the three experimental setups are inter-compared in terms of dynamic properties, sensitivity, and disturbances of the water phase from the presence of measurement equipment. Low inter-experiment variability was disclosed, i.e., 5-8% depending on wave-nonlinearity, indicating accurate representations of the idealized testcase across all setups. Setup 3 was found to be the more accurate representation and further work with this setup to release a public benchmark dataset was planned.

An electric field modeling study with meta-analysis to understand the antidepressant effects of transcranial direct current stimulation (tDCS).

Transcranial direct current stimulation (tDCS) has mixed effects for major depressive disorder (MDD) symptoms, partially owing to large inter-experimental variability in tDCS protocols and their correlated induced electric fields (E-fields). We investigated whether the E-field strength of distinct tDCS parameters was associated with antidepressant effect. A meta-analysis was performed with placebo-controlled clinical trials of tDCS enrolling MDD patients. PubMed, EMBASE, and Web of Science were searched from inception to March 10, 2023. Effect sizes of tDCS protocols were correlated with E-field simulations (SimNIBS) of brain regions of interest (bilateral dorsolateral prefrontal cortex [DLPFC] and bilateral subgenual anterior cingulate cortex [sgACC]). Moderators of tDCS responses were also investigated. A total of 20 studies were included (21 datasets, 1,008 patients), using 11 distinct tDCS protocols. Results revealed a moderate effect for MDD (g = 0.41, 95%CI 0.18-0.64), while cathode position and treatment strategy were found to be moderators of response. A negative association between effect size and tDCS-induced E-field magnitude was seen, with stronger E-fields in the right frontal and medial parts of the DLPFC (targeted by the cathode) leading to smaller effects. No association was found for the left DLPFC and the bilateral sgACC. An optimized tDCS protocol is proposed. Our results highlight the need for a standardized tDCS protocol in MDD clinical trials.

Do more with Less: Improving High Parameter Cytometry Through Overnight Staining.

Recent advances in flow cytometry have allowed high-dimensional characterization of biological phenomena, enabling breakthroughs in a multitude of fields. Despite the appreciation of the unique properties of antigens and fluorophores in high-parameter panel design, staining conditions are often standardized for short surface stains, regardless of antibody affinity or antigen accessibility. Here, we demonstrate how increasing antibody incubation times can lead to substantial improvements in sensitivity, maintaining specificity, and reducing background, while also significantly reducing the costs of high-parameter cytometry panels. Furthermore, overnight staining reduces the influence of interexperimental variability, assisting accurate pooling over experiments over extended time courses. We provide guidance on how to optimize staining conditions for diverse antigens, including how different fixation strategies can affect epitope accessibility. Overnight staining can thus substantially improve the resolution, repeatability, and cost-effectiveness of high-parameter cytometry. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC.

Acute toxicity of pesticide mixtures to honey bees is generally additive, and well predicted by Concentration Addition

Understanding the frequency of non-additive effects of pesticides (synergism and antagonism) is important in the context of risk assessment. The goal of this study was to investigate the prevalence of non-additive effects of pesticides to honey bees (Apis mellifera). We investigated a large set of mixtures including insecticides and fungicides of different chemical modes of action and classes. The mixtures included represent a relevant sample of pesticides that are currently used globally. We investigated whether the experimental toxicity of the mixtures could be predicted based on the Concentration Addition (CA) model for acute contact and oral adult bee toxicity tests. We measured the degree of deviation from the additivity predictions of the experimental toxicity based on the well-known Mixture Deviation Ratio (MDR). Further, we investigated the appropriate MDR thresholds that should be used for the identification of non-additive effects based on acceptable rates for false positive (alpha) and true positive (beta) findings. We found that a deviation factor of MDR = 5 is a sound reference for labeling potential non-additive effects in acute adult bee experimental designs when assuming a typical Coefficient of Variation (CV%) = 100 in the determination of the LD50 of a pesticide (a factor of 2× deviation in the LD 50 resulting from inter-experimental variability). We found that only 2.4 % and 9 % of the mixtures evaluated had an MDR > 5 and MDR < 0.2, respectively. The frequency and magnitude of deviation from additivity found for bees in this study are consistent with those of other terrestrial and aquatic taxa. Our findings suggest that additivity is a good baseline for predicting the toxicity of pesticide mixtures to bees, and that the rare cases of synergy of pesticide mixtures to bees are not random but have a mechanistic basis.

Aggregation of cryopreserved mid-hindgut endoderm for more reliable and reproducible hPSC-derived small intestinal organoid generation.

SummaryA major technical limitation hindering the widespread adoption of human pluripotent stem cell (hPSC)-derived gastrointestinal (GI) organoid technologies is the need for de novo hPSC differentiation and dependence on spontaneous morphogenesis to produce detached spheroids. Here, we report a method for simple, reproducible, and scalable production of small intestinal organoids (HIOs) based on the aggregation of cryopreservable hPSC-derived mid-hindgut endoderm (MHE) monolayers. MHE aggregation eliminates variability in spontaneous spheroid production and generates HIOs that are comparable to those arising spontaneously. With a minor modification to the protocol, MHE can be cryopreserved, thawed, and aggregated, facilitating HIO production without de novo hPSC differentiation. Finally, aggregation can also be used to generate antral stomach organoids and colonic organoids. This improved method removes significant barriers to the implementation and successful use of hPSC-derived GI organoid technologies and provides a framework for improved dissemination and increased scalability of GI organoid production.

Gene Expression Transcriptome Profile Validation: A Critical Appraisal of Methods and Signature Analyses

<h3>Purpose</h3> The aim of our study is to assess the reliability of transcriptome profile platform outcomes between different tissue preservation conditions, RNA extraction methods and heart-failure-related genes, minimizing intra- and inter-experimental variability. <h3>Methods</h3> Our cohort of study is composed by 6 FFPE endomyocardial biopsies (EMBs) and 6 fresh frozen (FF) heart tissues samples belonging to the same adult end-stage heart failure patient, limiting inter-patient variability bias. Two major RNA extraction procedure were tested for FF and FFPE tissues. Then, we performed a high-throughput microarray expression profiling without a bias of transcript selection. We analyzed mRNAs expression through R programming and transcriptome analysis software, exploring >20000 transcripts. We refined the transcriptome analysis selecting, from a literature systematic review appraisal 30 heart-failure related genes, coupling the overlapping genes in the differential expression and network analysis. <h3>Results</h3> We compared the analysis of up- and down-regulated genes between the groups, addressing both the different RNA extraction procedure and tissue preservation, while sharing the same transcriptome platform. The differential expressed genes (DEG) analyzed had fold change set at 2 and -2, and a cut-off threshold of FDR adjusted p-value < 0,05. Heatmap showed a great samples' clusterization according to preservation and extraction methods. Only 284 genes passed filter criteria in FFPE inter-groups comparison of extraction methods, while 52 differed in FFPE vs. FF, showing a great overlap of transcriptome signature, corroborated by the heart-failure related set of genes. The network analysis highlighted that most of the DEG are strongly associated (p-value adjusted <0.004) to the dysregulation of reticulum and collagen related cell component, in accordance with heart failure stage. <h3>Conclusion</h3> To the best of our knowledge, this is the first study in heart transplantation fields comparing genetic extraction and preservation methods for microarray expression profile evaluation correlated with pathophysiological mechanism. A proof of concept that RNAs from FFPE-EMBs, and not only FF, are feasible for microarray expression profile evaluation, and derived transcriptome profiles have a strong potential for research and diagnostic clinical application.

Evaluation of the Speed, Accuracy and Precision of the QuickMIC Rapid Antibiotic Susceptibility Testing Assay With Gram-Negative Bacteria in a Clinical Setting.

The rapidly changing landscape of antimicrobial resistance poses a challenge for empirical antibiotic therapy in severely ill patients and highlights the need for fast antibiotic susceptibility diagnostics to guide treatment. Traditional methods for antibiotic susceptibility testing (AST) of bacteria such as broth microdilution (BMD) or the disc diffusion method (DDM) are comparatively slow and show high variability. Rapid AST methods under development often trade speed for resolution, sometimes only measuring responses at a single antibiotic concentration. QuickMIC is a recently developed lab-on-a-chip system for rapid AST. Here we evaluate the performance of the QuickMIC method with regard to speed, precision and accuracy in comparison to traditional diagnostic methods. 151 blood cultures of clinical Gram-negative isolates with a high frequency of drug resistance were tested using the QuickMIC system and compared with BMD for 12 antibiotics. To investigate sample turnaround time and method functionality in a clinical setting, another 41 clinical blood culture samples were acquired from the Uppsala University Hospital and analyzed on site in the clinical laboratory with the QuickMIC system, and compared with DDM for 8 antibiotics routinely used in the clinical laboratory. The overall essential agreement between MIC values obtained by QuickMIC and BMD was 83.4%, with an average time to result of 3 h 2 min (SD: 24.8 min) for the QuickMIC method. For the clinical dataset, the categorical agreement between QuickMIC and DDM was 96.8%, whereas essential and categorical agreement against BMD was 91.0% and 96.7%, respectively, and the total turnaround time as compared to routine diagnostics was shown to be reduced by 40% (33 h vs. 55 h). Interexperiment variability was low (average SD: 44.6% from target MIC) compared to the acceptable standard of ±1 log2 unit (i.e. -50% to +100% deviation from target MIC) in BMD. We conclude that the QuickMIC method can provide rapid and accurate AST, and may be especially valuable in settings with high resistance rates, and for antibiotics where wildtype and antibiotic-resistant bacteria have MIC distributions that are close or overlapping.

Mesenchymal Stem Cell and Monocyte Co-cultures.

Transwell co-cultures are critical to study cell-to-cell communication through the release of factors between different cells allowing for the simultaneous assessment of treatment effects on one cell type (e.g., Cell A) and their impact on another cell type (e.g., Cell B). This allows for the simultaneous assessment of two different cell types and the factors they secrete under the same treatment conditions, which minimizes interexperimental variability, demonstrates causation rather than association, and enhances the translatability of the findings to the in vivo condition. Here we describe transwell co-cultures of human mesenchymal stem cells (MSCs) and peripheral blood monocytes or pre-osteoclasts to assess osteoblast-mediated actions on osteoclastogenesis.

Evaluation of Tissue Stem Cell-Derived Human Intestinal Organoids, a Physiologically Relevant Model to Evaluate Cytochrome P450 Induction in Gut.

Induction of cytochrome P450 can cause drug-drug interactions and efficacy failure. Induction risk in liver and gut is typically inferred from experiments with plated hepatocytes. Organoids are physiologically relevant, multicellular structures originating from stem cells. Intestinal stem cell-derived organoids retain traits of normal gut physiology, such as an epithelial barrier and cellular diversity. Matched human enteroid and colonoid lines, generated from ileal and colon biopsies from two donors, were cultured in extracellular matrix for 3 days, followed by a single 48-hour treatment with rifampin, omeprazole, CITCO, and phenytoin at concentrations that induce target genes in hepatocytes. After treatment, mRNA was analyzed for induction of target genes. Rifampin induced CYP3A4; estimated EC50 and maximal fold induction were 3.75 µM and 8.96-fold, respectively, for ileal organoids and 1.40 µM and 11.3-fold, respectively, for colon organoids. Ileal, but not colon, organoids exhibited nifedipine oxidase activity, which was induced by rifampin up to 14-fold. The test compounds did not increase mRNA expression of CYP1A2, CYP2B6, multidrug resistance transporter 1 (P-glycoprotein), breast cancer resistance protein, and UDP-glucuronosyltransferase 1A1 in ileal organoids. Whereas omeprazole induced CYP3A4 (up to 5.3-fold, geometric mean, n = 4 experiments), constitutive androstane receptor activators phenytoin and CITCO did not. Omeprazole failed to induce CYP1A2 mRNA but did induce CYP1A1 mRNA (up to 7.7-fold and 15-fold in ileal and colon organoids, respectively, n = 4 experiments). Despite relatively high intra- and interexperimental variability, data suggest that the model yields induction responses that are distinct from hepatocytes and holds promise to enable evaluation of CYP1A1 and CYP3A4 induction in gut. SIGNIFICANCE STATEMENT: An adult intestinal stem cell-derived organoid model to test P450 induction in gut was evaluated. Testing several prototypical inducers for mRNA induction of P450 isoforms, UDP-glucuronosyltransferase 1A1, P-glycoprotein, and breast cancer resistance protein with both human colon and ileal organoids resulted in a range of responses, often distinct from those found in hepatocytes, indicating the potential for further development of this model as a physiologically relevant gut induction test system.

Breaking the circularity in circular analyses: Simulations and formal treatment of the flattened average approach.

There has been considerable debate and concern as to whether there is a replication crisis in the scientific literature. A likely cause of poor replication is the multiple comparisons problem. An important way in which this problem can manifest in the M/EEG context is through post hoc tailoring of analysis windows (a.k.a. regions-of-interest, ROIs) to landmarks in the collected data. Post hoc tailoring of ROIs is used because it allows researchers to adapt to inter-experiment variability and discover novel differences that fall outside of windows defined by prior precedent, thereby reducing Type II errors. However, this approach can dramatically inflate Type I error rates. One way to avoid this problem is to tailor windows according to a contrast that is orthogonal (strictly parametrically orthogonal) to the contrast being tested. A key approach of this kind is to identify windows on a fully flattened average. On the basis of simulations, this approach has been argued to be safe for post hoc tailoring of analysis windows under many conditions. Here, we present further simulations and mathematical proofs to show exactly why the Fully Flattened Average approach is unbiased, providing a formal grounding to the approach, clarifying the limits of its applicability and resolving published misconceptions about the method. We also provide a statistical power analysis, which shows that, in specific contexts, the fully flattened average approach provides higher statistical power than Fieldtrip cluster inference. This suggests that the Fully Flattened Average approach will enable researchers to identify more effects from their data without incurring an inflation of the false positive rate.

Fluorescent Multiplex Cell Rolling Assay: Simultaneous Capturing up to Seven Samples in Real-Time Using Spectral Confocal Microscopy.

The parallel plate flow chamber assay is widely utilized to study physiological cell-cell adhesive interactions under dynamic flow that mimics the bloodstream. In this technique, the cells are perfused under defined shear stresses over a monolayer of endothelial cells (expressing homing molecules, e.g., selectins) or a surface (expressing recombinant homing molecules). However, with the need to study multiple samples and multiple parameters per sample, using a traditional bright-field microscope-based flow assay allows only one sample at a time to be analyzed, resulting in high interexperiment variability, the need for normalization, waste of materials, and significant consumption of time. We developed a multiplexing approach using a three-color fluorescence staining method, which allowed for up to seven different combination signatures to be run at one time. Using this fluorescent multiplex cell rolling (FMCR) assay, each sample is labeled with a different signature of emission wavelengths and mixed with other samples just minutes before the flow run. Subsequently, real-time images are acquired in a single pass using a line-scanning spectral confocal microscope. To illustrate the glycan-dependent binding of E-selectin, a central molecule in cell migration, to its glycosylated ligands expressed on myeloid-leukemic cells in flow, the FMCR assay was used to analyze E-selectin-ligand interactions following the addition (fucosyltransferase-treatment) or removal (deglycosylation) of key glycans on the flowing cells. The FMCR assay allowed us to analyze the cell-adhesion events from these different treatment conditions simultaneously in a competitive manner and to calculate differences in rolling frequency, velocity, and tethering capability of cells under study.

Validation of an in Vitro Mismatch Repair Assay Used in the Functional Characterization of Mismatch Repair Variants

A significant proportion of DNA-mismatch repair (MMR) variants are classified as of unknown significance, precluding diagnosis. The invitro MMR assay is used to assess their MMR capability, likely the most important function of an MMR protein. However, the robustness of the assay, crucial for its use in the clinical setting, has been rarely evaluated. The aim of the present work was to validate an invitro MMR assay approach to the functional characterization of MMR variants, as a first step to meeting quality standards of diagnostic laboratories. The MMR assay was optimized by testing a variety of reagents and experimental conditions. Reference materials and standard operating procedures were established. To determine the intra- and interexperimental variability of the assay and its reproducibility among centers, independent transfections of six previously characterized MLH1 variants were performed in two independent laboratories. Reagents and conditions optimal for performing the invitro MMR assay were determined. The validated assay demonstrated no significant intra- or interexperimental variability and good reproducibility between centers. We set up a robust invitro MMR assay that can provide relevant invitro functional evidence for MMR variant pathogenicity assessment, eventually improving the molecular diagnosis of hereditary cancer syndromes associated with MMR deficiency.

Corrigendum to: Standardisation of the in vitro comet assay: influence of lysis time and lysis solution composition on the detection of DNA damage induced by X-rays

The alkaline comet assay, in vivo and in vitro, is currently used in several areas of research and in regulatory genotoxicity testing. Several efforts have been made in order to decrease the inter-experimental and inter-laboratory variability and increase the reliability of the assay. In this regard, lysis conditions are considered as one of the critical variables and need to be further studied. Here, we tested different times of lysis (from no lysis to 1 week) and two different lysis solutions in human lymphoblast (TK6) cells unexposed or exposed to X-rays. Similar % tail DNA values were obtained independently of the time of lysis employed for every X-ray dose tested and both lysis solutions. These results, taken together with our previous ones with methyl methanesulfonate and H2O2, which showed clear lysis-time dependence, support that the influence of the lysis time in the comet assay results depends on the type of lesion being detected; some DNA lesions may spontaneously give rise to apurinic or apyrimidinic (AP) sites during the lysis period, which can be converted into strand breaks detectable with the comet assay. Testing different times of lysis would be useful to increase the sensitivity of the comet assay and to ensure the detection of DNA lesions of an unknown compound, thereby providing some insight into the chemical nature of the lesions induced. However, the same lysis conditions (i.e. lysis time and lysis solution) should be used when comparing results between different experiments or laboratories.

Implementation and experimental validation of surface electromyogram and force model of Tibialis Anterior muscle for examining muscular factors.

This study reports a surface electromyogram and force of contraction model. The objective was to investigate the effect of changes in the size, type and number of motor units in the Tibialis Anterior muscle to surface electromyogram and force of dorsiflexion. A computational model to simulate surface electromyogram and associated force of contraction by the Tibialis Anterior muscle was developed. This model was simulated for isometric dorsiflexion, and comparative experiments were conducted for validation. Repeated simulations were performed to investigate the different parameters and evaluate inter-experimental variability. An equivalence statistical test and the Bland-Altman method were used to observe the significance between the simulated and experimental data. Simulated and experimentally recorded data had high similarity for the three measures: maximal power of power spectral density (p < 0.0001), root mean square of surface electromyogram (p < 0.0001) and force recorded at the footplate (p < 0.03). Inter-subject variability in the experimental results was in-line with the variability in the repeated simulation results. This experimentally validated computational model for the surface electromyogram and force of the Tibialis Anterior muscle is significant as it allows the examination of three important muscular factors associated with ageing and disease: size, fibre type and number of motor units.

Organotypic Culture of Neonatal Murine Inner Ear Explants.

The inner ear is a complex organ containing highly specialised cell types and structures that are critical for sensing sound and movement. In vivo, the inner ear is difficult to study due to the osseous nature of the otic capsule and its encapsulation within an intricate bony labyrinth. As such, mammalian inner ear explants are an invaluable tool for the study and manipulation of the complex intercellular connections, structures, and cell types within this specialised organ. The greatest strength of this technique is that the complete organ of Corti, or peripheral vestibular organs including hair cells, supporting cells and accompanying neurons, is maintained in its in situ form. The greatest weakness of in vitro hair cell preparations is the short time frame in which the explanted tissue remains viable. Yet, cochlear explants have proven to be an excellent experimental model for understanding the fundamental aspects of auditory biology, substantiated by their use for over 40 years. In this protocol, we present a modernised inner ear explant technique that employs organotypic cell culture inserts and serum free media. This approach decreases the likelihood of explant damage by eliminating the need for adhesive substances. Serum free media also restricts excessive cellular outgrowth and inter-experimental variability, both of which are side effects of exogenous serum addition to cell cultures. The protocol described can be applied to culture both cochlear and vestibular explants from various mammals. Example outcomes are demonstrated by immunohistochemistry, hair cell quantification, and electrophysiological recordings to validate the versatility and viability of the protocol.

Open-CSAM, a new tool for semi-automated analysis of myofiber cross-sectional area in regenerating adult skeletal muscle

Adult skeletal muscle is capable of complete regeneration after an acute injury. The main parameter studied to assess muscle regeneration efficacy is the cross-sectional area (CSA) of the myofibers as myofiber size correlates with muscle force. CSA analysis can be time-consuming and may trigger variability in the results when performed manually. This is why programs were developed to completely automate the analysis of the CSA, such as SMASH, MyoVision, or MuscleJ softwares. Although these softwares are efficient to measure CSA on normal or hypertrophic/atrophic muscle, they fail to efficiently measure CSA on regenerating muscles. We developed Open-CSAM, an ImageJ macro, to perform a high throughput semi-automated analysis of CSA on skeletal muscle from various experimental conditions. The macro allows the experimenter to adjust the analysis and correct the mistakes done by the automation, which is not possible with fully automated programs. We showed that Open-CSAM was more accurate to measure CSA in regenerating and dystrophic muscles as compared with SMASH, MyoVision, and MuscleJ softwares and that the inter-experimenter variability was negligible. We also showed that, to obtain a representative CSA measurement, it was necessary to analyze the whole muscle section and not randomly selected pictures, a process that was easily and accurately be performed using Open-CSAM. To conclude, we show here an easy and experimenter-controlled tool to measure CSA in muscles from any experimental condition, including regenerating muscle.

Three Optimized Methods for In Situ Quantification of Progenitor Cell Proliferation in Embryonic Kidneys Using BrdU, EdU, and PCNA.

Background:Nephron progenitor cells derived from the metanephric mesenchyme undergo a complex balance of self-renewal and differentiation throughout kidney development to give rise to the mature nephron. Cell proliferation is an important index of progenitor population dynamics. However, accurate and reproducible in situ quantification of cell proliferation within progenitor populations can be technically difficult to achieve due to the complexity and harsh tissue treatment required of certain protocols.Objective:To optimize and compare the performance of the 3 most accurate S phase–specific labeling methods used for in situ detection and quantification of nephron progenitor and ureteric bud cell proliferation in the developing kidney, namely, 5-bromo-2’-deoxyuridine (BrdU), 5-ethynyl-2’-deoxyuridine (EdU), and proliferating cell nuclear antigen (PCNA).Methods:Protocols for BrdU, EdU, and PCNA were optimized for fluorescence labeling on paraformaldehyde-fixed, paraffin-embedded mouse kidney tissue sections, with co-labeling of nephron progenitor cells and ureteric bud with Six2 and E-cadherin antibodies, respectively. Image processing and analysis, including quantification of proliferating cells, were carried out using free ImageJ software.Results:All 3 methods detect similar ratios of nephron progenitor and ureteric bud proliferating cells. The BrdU staining protocol is the lengthiest and most complex protocol to perform, requires tissue denaturation, and is most subject to interexperimental signal variability. In contrast, bound PCNA and EdU protocols are relatively more straightforward, consistently yield clear results, and far more easily lend themselves to co-staining; however, the bound PCNA protocol requires substantive additional postexperimental analysis to distinguish the punctate nuclear PCNA staining pattern characteristic of proliferating cells.Conclusions:All 3 markers exhibit distinct advantages and disadvantages in quantifying cell proliferation in kidney progenitor populations, with EdU and PCNA protocols being favored due to greater technical ease and reproducibility of results associated with these methods.

A deep learning based method for large-scale classification, registration, and clustering of in-situ hybridization experiments in the mouse olfactory bulb

BackgroundThe Allen Mouse Brain Atlas allows study of the brain’s molecular anatomy at cellular scale, for thousands genes. To fully leverage this resource, one must register histological images of brain tissue – a task made challenging by the brain’s structural complexity and heterogeneity, as well as inter-experiment variability. New methodWe have developed a deep-learning based methodology for classification and registration of thousands of sections of brain tissue, using the mouse olfactory bulb (OB) as a case study. ResultsWe trained a convolutional neural network (CNN) to derive an image similarity measure for in-situ hybridization experiments, and embedded these in a low-dimensional feature space to guide the design of registration templates. We then compiled a high quality, registered atlas of gene expression for the OB (the first such atlas for the OB, to our knowledge). As proof-of-principle, the atlas was clustered using non-negative matrix factorization to reveal canonical expression motifs, and to identify novel, lamina-specific marker genes. Comparison with existing methodsOur method leverages virtues of CNNs for a set of important problems in molecular neuroanatomy, with performance comparable to existing methods. ConclusionThe atlas we have complied allows for intra- and inter-laminar comparisons of gene expression patterns in the OB across thousands of genes, as well identification of canonical expression profiles through clustering. We anticipate that this will be a useful resource for investigators studying the bulb’s development and functional topography. Our methods are publicly available for those interested in extending them to other brain areas.