Primatologist: A modular segmentation pipeline for macaque brain morphometry.

Because they bridge the genetic gap between rodents and humans, non-human primates (NHPs) play a major role in therapy development and evaluation for neurological disorders. However, translational research success from NHPs to patients requires an accurate phenotyping of the models. In patients, magnetic resonance imaging (MRI) combined with automated segmentation methods has offered the unique opportunity to assess in vivo brain morphological changes. Meanwhile, specific challenges caused by brain size and high field contrasts make existing algorithms hard to use routinely in NHPs. To tackle this issue, we propose a complete pipeline, Primatologist, for multi-region segmentation. Tissue segmentation is based on a modular statistical model that includes random field regularization, bias correction and denoising and is optimized by expectation-maximization. To deal with the broad variety of structures with different relaxing times at 7 T, images are segmented into 17 anatomical classes, including subcortical regions. Pre-processing steps insure a good initialization of the parameters and thus the robustness of the pipeline. It is validated on 10 T2-weighted MRIs of healthy macaque brains. Classification scores are compared with those of a non-linear atlas registration, and the impact of each module on classification scores is thoroughly evaluated.

giRAff: an automated atlas segmentation tool adapted to single histological slices.

Conventional histology of the brain remains the gold standard in the analysis of animal models. In most biological studies, standard protocols usually involve producing a limited number of histological slices to be analyzed. These slices are often selected into a specific anatomical region of interest or around a specific pathological lesion. Due to the lack of automated solutions to analyze such single slices, neurobiologists perform the segmentation of anatomical regions manually most of the time. Because the task is long, tedious, and operator-dependent, we propose an automated atlas segmentation method called giRAff, which combines rigid and affine registrations and is suitable for conventional histological protocols involving any number of single slices from a given mouse brain. In particular, the method has been tested on several routine experimental protocols involving different anatomical regions of different sizes and for several brains. For a given set of single slices, the method can automatically identify the corresponding slices in the mouse Allen atlas template with good accuracy and segmentations comparable to those of an expert. This versatile and generic method allows the segmentation of any single slice without additional anatomical context in about 1 min. Basically, our proposed giRAff method is an easy-to-use, rapid, and automated atlas segmentation tool compliant with a wide variety of standard histological protocols.

A general deep learning framework for neuron instance segmentation based on Efficient UNet and morphological post-processing.

Recent studies have demonstrated the superiority of deep learning in medical image analysis, especially in cell instance segmentation, a fundamental step for many biological studies. However, the excellent performance of the neural networks requires training on large, unbiased dataset and annotations, which is labor-intensive and expertise-demanding. This paper presents an end-to-end framework to automatically detect and segment NeuN stained neuronal cells on histological images using only point annotations. Unlike traditional nuclei segmentation with point annotation, we propose using point annotation and binary segmentation to synthesize pixel-level annotations. The synthetic masks are used as the ground truth to train the neural network, a U-Net-like architecture with a state-of-the-art network, EfficientNet, as the encoder. Validation results show the superiority of our model compared to other recent methods. In addition, we investigated multiple post-processing schemes and proposed an original strategy to convert the probability map into segmented instances using ultimate erosion and dynamic reconstruction. This approach is easy to configure and outperforms other classical post-processing techniques. This work aims to develop a robust and efficient framework for analyzing neurons using optical microscopic data, which can be used in preclinical biological studies and, more specifically, in the context of neurodegenerative diseases. Code is available at: https://github.com/MIRCen/NeuronInstanceSeg.

Multiscale segmentation- and error-guided iterative convolutional neural network for cerebral neuron segmentation in microscopic images.

This article uses microscopy images obtained from diverse anatomical regions of macaque brain for neuron semantic segmentation. The complex structure of brain, the large intra-class staining intensity difference within neuron class, the small inter-class staining intensity difference between neuron and tissue class, and the unbalanced dataset increase the difficulty of neuron semantic segmentation. To address this problem, we propose a multiscale segmentation- and error-guided iterative convolutional neural network (MSEG-iCNN) to improve the semantic segmentation performance in major anatomical regions of the macaque brain. After evaluating microscopic images from 17 anatomical regions, the semantic segmentation performance of neurons is improved by 10.6%, 4.0%, 1.5%, and 1.2% compared with Random Forest, FCN-8s, U-Net, and UNet++, respectively. Especially for neurons with brighter staining intensity in the anatomical regions such as lateral geniculate, globus pallidus and hypothalamus, the performance is improved by 66.1%, 23.9%, 11.2%, and 6.7%, respectively. Experiments show that our proposed method can efficiently segment neurons with a wide range of staining intensities. The semantic segmentation results are of great significance and can be further used for neuron instance segmentation, morphological analysis and disease diagnosis. Cell segmentation plays a critical role in extracting cerebral information, such as cell counting, cell morphometry and distribution analysis. Accurate automated neuron segmentation is challenging due to the complex structure of brain, the large intra-class staining intensity difference within neuron class, the small inter-class staining intensity difference between neuron and tissue class, and the unbalanced dataset. The proposed multiscale segmentation- and error-guided iterative convolutional neural network (MSEG-iCNN) improve the segmentation performance in 17 major anatomical regions of the macaque brain. HIGHLIGHTS: Cell segmentation plays a critical role in extracting cerebral information, such as cell counting, cell morphometry and distribution analysis. Accurate automated neuron segmentation is challenging due to the complex structure of brain, the large intra-class staining intensity difference within neuron class, the small inter-class staining intensity difference between neuron and tissue class, and the unbalanced dataset. The proposed multiscale segmentation- and error-guided iterative convolutional neural network (MSEG-iCNN) improve the segmentation performance in 17 major anatomical regions of the macaque brain.

Automated Atlas-based Segmentation of Single Coronal Mouse Brain Slices using Linear 2D-2D Registration.

A significant challenge for brain histological data analysis is to precisely identify anatomical regions in order to perform accurate local quantifications and evaluate therapeutic solutions. Usually, this task is performed manually, becoming therefore tedious and subjective. Another option is to use automatic or semi-automatic methods, among which segmentation using digital atlases co-registration. However, most available atlases are 3D, whereas digitized histological data are 2D. Methods to perform such 2D-3D segmentation from an atlas are required. This paper proposes a strategy to automatically and accurately segment single 2D coronal slices within a 3D volume of atlas, using linear registration. We validated its robustness and performance using an exploratory approach at whole-brain scale.

Evaluation of Deep Learning Topcoders Method for Neuron Individualization in Histological Macaque Brain Section.

Cell individualization has a vital role in digital pathology image analysis. Deep Learning is considered as an efficient tool for instance segmentation tasks, including cell individualization. However, the precision of the Deep Learning model relies on massive unbiased dataset and manual pixel-level annotations, which is labor intensive. Moreover, most applications of Deep Learning have been developed for processing oncological data. To overcome these challenges, i) we established a pipeline to synthesize pixel-level labels with only point annotations provided; ii) we tested an ensemble Deep Learning algorithm to perform cell individualization on neurological data. Results suggest that the proposed method successfully segments neuronal cells in both object-level and pixel-level, with an average detection accuracy of 0.93.

Evaluation of automated segmentation algorithms for neurons in macaque cerebral microscopic images.

Accurate cerebral neuron segmentation is required before neuron counting and neuron morphological analysis. Numerous algorithms for neuron segmentation have been published, but they are mainly evaluated using limited subsets from a specific anatomical region, targeting neurons of clear contrast and/or neurons with similar staining intensity. It is thus unclear how these algorithms perform on cerebral neurons in diverse anatomical regions. In this article, we introduce and reliably evaluate existing machine learning algorithms using a data set of microscopy images of macaque brain. This data set highlights various anatomical regions (e.g., cortex, caudate, thalamus, claustrum, putamen, hippocampus, subiculum, lateral geniculate, globus pallidus, etc.), poor contrast, and staining intensity differences of neurons. The evaluation was performed using 10 architectures of six classic machine learning algorithms in terms of typical Recall, Precision, F-score, aggregated Jaccard index (AJI), as well as a performance ranking of algorithms. F-score of most of the algorithms is superior to 0.7. Deep learning algorithms facilitate generally higher F-scores. U-net with suitable layer depth has been evaluated to be excellent classifiers with F-score of 0.846 and 0.837 when performing cross validation. The evaluation and analysis indicate the performance gap among algorithms in various anatomical regions and the strengths and limitations of each algorithm. The comparative result highlights at the same time the importance and difficulty of neuron segmentation and provides clues for future improvement. To the best of our knowledge, this work is the first comprehensive study for neuron segmentation in such large-scale anatomical regions. Neuron segmentation plays a critical role in extracting cerebral information, such as neuron counting and neuron morphological analysis. Accurate automated cerebral neuron segmentation is a challenging task due to different kinds, poor contrast, staining intensity differences, and fuzzy boundaries of neurons. The comprehensive evaluation and analysis of performance among existing machine learning algorithms in diverse anatomical regions allows to make clear of the strengths and limitations of state-of-the-art algorithm. The comprehensive study provides clues for future improvement and creation of automated methods.

Automated Individualization of Size-Varying and Touching Neurons in Macaque Cerebral Microscopic Images.

In biomedical research, cell analysis is important to assess physiological and pathophysiological information. Virtual microscopy offers the unique possibility to study the compositions of tissues at a cellular scale. However, images acquired at such high spatial resolution are massive, contain complex information, and are therefore difficult to analyze automatically. In this article, we address the problem of individualization of size-varying and touching neurons in optical microscopy two-dimensional (2-D) images. Our approach is based on a series of processing steps that incorporate increasingly more information. (1) After a step of segmentation of neuron class using a Random Forest classifier, a novel min-max filter is used to enhance neurons' centroids and boundaries, enabling the use of region growing process based on a contour-based model to drive it to neuron boundary and achieve individualization of touching neurons. (2) Taking into account size-varying neurons, an adaptive multiscale procedure aiming at individualizing touching neurons is proposed. This protocol was evaluated in 17 major anatomical regions from three NeuN-stained macaque brain sections presenting diverse and comprehensive neuron densities. Qualitative and quantitative analyses demonstrate that the proposed method provides satisfactory results in most regions (e.g., caudate, cortex, subiculum, and putamen) and outperforms a baseline Watershed algorithm. Neuron counts obtained with our method show high correlation with an adapted stereology technique performed by two experts (respectively, 0.983 and 0.975 for the two experts). Neuron diameters obtained with our method ranged between 2 and 28.6 µm, matching values reported in the literature. Further works will aim to evaluate the impact of staining and interindividual variability on our protocol.

A validation dataset for Macaque brain MRI segmentation.

Validation data for segmentation algorithms dedicated to preclinical images is fiercely lacking, especially when compared to the large number of databases of Human brain images and segmentations available to the academic community. Not only is such data essential for validating methods, it is also needed for objectively comparing concurrent algorithms and detect promising paths, as segmentation challenges have shown for clinical images. The dataset we present here is a first step in this direction. It comprises 10 T2-weighted MRIs of healthy adult macaque brains, acquired on a 7 T magnet, along with corresponding manual segmentations into 17 brain anatomic labelled regions spread over 5 hierarchical levels based on a previously published macaque atlas (Calabrese et al., 2015) [1]. By giving access to this unique dataset, we hope to provide a reference needed by the non-human primate imaging community. This dataset was used in an article presenting a new primate brain morphology analysis pipeline, Primatologist (Balbastre et al., 2017) [2]. Data is available through a NITRC repository (https://www.nitrc.org/projects/mircen_macset).

Voxel-Based Statistical Analysis of 3D Immunostained Tissue Imaging.

Recently developed techniques to visualize immunostained tissues in 3D and in large samples have expanded the scope of microscopic investigations at the level of the whole brain. Here, we propose to adapt voxel-based statistical analysis to 3D high-resolution images of the immunostained rodent brain. The proposed approach was first validated with a simulation dataset with known cluster locations. Then, it was applied to characterize the effect of ADAM30, a gene involved in the metabolism of the amyloid precursor protein, in a mouse model of Alzheimer's disease. This work introduces voxel-based analysis of 3D immunostained microscopic brain images and, therefore, opens the door to localized whole-brain exploratory investigation of pathological markers and cellular alterations.

High-throughput 3D whole-brain quantitative histopathology in rodents.

Histology is the gold standard to unveil microscopic brain structures and pathological alterations in humans and animal models of disease. However, due to tedious manual interventions, quantification of histopathological markers is classically performed on a few tissue sections, thus restricting measurements to limited portions of the brain. Recently developed 3D microscopic imaging techniques have allowed in-depth study of neuroanatomy. However, quantitative methods are still lacking for whole-brain analysis of cellular and pathological markers. Here, we propose a ready-to-use, automated, and scalable method to thoroughly quantify histopathological markers in 3D in rodent whole brains. It relies on block-face photography, serial histology and 3D-HAPi (Three Dimensional Histology Analysis Pipeline), an open source image analysis software. We illustrate our method in studies involving mouse models of Alzheimer's disease and show that it can be broadly applied to characterize animal models of brain diseases, to evaluate therapeutic interventions, to anatomically correlate cellular and pathological markers throughout the entire brain and to validate in vivo imaging techniques.

ADAM30 Downregulates APP-Linked Defects Through Cathepsin D Activation in Alzheimer's Disease.

Although several ADAMs (A disintegrin-like and metalloproteases) have been shown to contribute to the amyloid precursor protein (APP) metabolism, the full spectrum of metalloproteases involved in this metabolism remains to be established. Transcriptomic analyses centred on metalloprotease genes unraveled a 50% decrease in ADAM30 expression that inversely correlates with amyloid load in Alzheimer's disease brains. Accordingly, in vitro down- or up-regulation of ADAM30 expression triggered an increase/decrease in Aß peptides levels whereas expression of a biologically inactive ADAM30 (ADAM30(mut)) did not affect Aß secretion. Proteomics/cell-based experiments showed that ADAM30-dependent regulation of APP metabolism required both cathepsin D (CTSD) activation and APP sorting to lysosomes. Accordingly, in Alzheimer-like transgenic mice, neuronal ADAM30 over-expression lowered Aß42 secretion in neuron primary cultures, soluble Aß42 and amyloid plaque load levels in the brain and concomitantly enhanced CTSD activity and finally rescued long term potentiation alterations. Our data thus indicate that lowering ADAM30 expression may favor Aß production, thereby contributing to Alzheimer's disease development.

Robust supervised segmentation of neuropathology whole-slide microscopy images.

Alzheimer's disease is characterized by brain pathological aggregates such as Aß plaques and neurofibrillary tangles which trigger neuroinflammation and participate to neuronal loss. Quantification of these pathological markers on histological sections is widely performed to study the disease and to evaluate new therapies. However, segmentation of neuropathology images presents difficulties inherent to histology (presence of debris, tissue folding, non-specific staining) as well as specific challenges (sparse staining, irregular shape of the lesions). Here, we present a supervised classification approach for the robust pixel-level classification of large neuropathology whole slide images. We propose a weighted form of Random Forest in order to fit nonlinear decision boundaries that take into account class imbalance. Both color and texture descriptors were used as predictors and model selection was performed via a leave-one-image-out cross-validation scheme. Our method showed superior results compared to the current state of the art method when applied to the segmentation of Aß plaques and neurofibrillary tangles in a human brain sample. Furthermore, using parallel computing, our approach easily scales-up to large gigabyte-sized images. To show this, we segmented a whole brain histology dataset of a mouse model of Alzheimer's disease. This demonstrates our method relevance as a routine tool for whole slide microscopy images analysis in clinical and preclinical research settings.

Segmentation of small animal PET/CT mouse brain scans using an MRI-based 3D digital atlas.

The work reported in this paper aimed at developing and testing an automated method to calculate the biodistribution of a specific PET tracer in mouse brain PET/CT images using an MRI-based 3D digital atlas. Surface-based registration strategy and affine transformation estimation were considered. Such an approach allowed overcoming the lack of anatomical information in the inner regions of PET/CT brain scans. Promising results were obtained in one mouse (on two scans) and will be extended to a neuroinflammation mouse model to characterize the pathology and its evolution. Major improvements are expected regarding automation, time computation, robustness and reproducibility of mouse brain segmentation. Due to its generic implementation, this method could be successfully applied to PET/CT brain scans of other species (rat, primate) for which 3D digital atlases are available.