Acute ischemic STROKE - from laboratory to the Patient's BED (STROKELABED): A translational approach to reperfusion injury. Study Protocol.

Cerebral edema (CE) and hemorrhagic transformation (HT) are frequent and unpredictable events in patients with acute ischemic stroke (AIS), even when an effective vessel recanalization has been achieved. These complications, related to blood-brain barrier (BBB) disruption, remain difficult to prevent or treat and may offset the beneficial effect of recanalization, and lead to poor outcomes. The aim of this translational study is to evaluate the association of circulating and imaging biomarkers with subsequent CE and HT in stroke patients with the dual purpose of investigating possible predictors as well as molecular dynamics underpinning those events and functional outcomes. Concurrently, the preclinical study will develop a new mouse model of middle cerebral artery (MCA) occlusion and recanalization to explore BBB alterations and their potentially harmful effects on tissue. The clinical section of the study is based on a single-center observational design enrolling consecutive patients with AIS in the anterior circulation territory, treated with recanalization therapies from October 1, 2015 to May 31, 2020. The study will employ an innovative evaluation of routine CT scans: in fact, we will assess and quantify the presence of CE and HT after stroke in CT scans at 24 h, through the quantification of anatomical distortion (AD), a measure of CE and HT. We will investigate the relationship of AD and several blood biomarkers of inflammation and extracellular matrix, with functional outcomes at 3 months. In parallel, we will employ a newly developed mouse model of stroke and recanalization, to investigate the emergence of BBB changes 24 h after the stroke onset. The close interaction between clinical and preclinical research can enhance our understanding of findings from each branch of research, enabling a deeper interpretation of the underlying mechanisms of reperfusion injury following recanalization treatment for AIS.

Deep learning-based localization algorithms on fluorescence human brain 3D reconstruction: a comparative study using stereology as a reference.

3D reconstruction of human brain volumes at high resolution is now possible thanks to advancements in tissue clearing methods and fluorescence microscopy techniques. Analyzing the massive data produced with these approaches requires automatic methods able to perform fast and accurate cell counting and localization. Recent advances in deep learning have enabled the development of various tools for cell segmentation. However, accurate quantification of neurons in the human brain presents specific challenges, such as high pixel intensity variability, autofluorescence, non-specific fluorescence and very large size of data. In this paper, we provide a thorough empirical evaluation of three techniques based on deep learning (StarDist, CellPose and BCFind-v2, an updated version of BCFind) using a recently introduced three-dimensional stereological design as a reference for large-scale insights. As a representative problem in human brain analysis, we focus on a 4 -cm 3 portion of the Broca's area. We aim at helping users in selecting appropriate techniques depending on their research objectives. To this end, we compare methods along various dimensions of analysis, including correctness of the predicted density and localization, computational efficiency, and human annotation effort. Our results suggest that deep learning approaches are very effective, have a high throughput providing each cell 3D location, and obtain results comparable to the estimates of the adopted stereological design.

Gold-Hydrogel Nanocomposites for High-Resolution Laser-Based 3D Printing of Scaffolds with SERS-Sensing Properties.

Although visible light-based stereolithography (SLA) represents an affordable technology for the rapid prototyping of 3D scaffolds for in vitro support of cells, its potential could be limited by the lack of functional photocurable biomaterials that can be SLA-structured at micrometric resolution. Even if innovative photocomposites showing biomimetic, bioactive, or biosensing properties have been engineered by loading inorganic particles into photopolymer matrices, main examples rely on UV-assisted extrusion-based low-resolution processes. Here, SLA-printable composites were obtained by mixing a polyethylene glycol diacrylate (PEGDA) hydrogel with multibranched gold nanoparticles (NPs). NPs were engineered to copolymerize with the PEGDA matrix by implementing a functionalization protocol involving covalent grafting of allylamine molecules that have C═C pendant moieties. The formulations of gold nanocomposites were tailored to achieve high-resolution fast prototyping of composite scaffolds via visible light-based SLA. Furthermore, it was demonstrated that, after mixing with a polymer and after laser structuring, gold NPs still retained their unique plasmonic properties and could be exploited for optical detection of analytes through surface-enhanced Raman spectroscopy (SERS). As a proof of concept, SERS-sensing performances of 3D printed plasmonic scaffolds were successfully demonstrated with a Raman probe molecule (e.g., 4-mercaptobenzoic acid) from the perspective of future extensions to real-time sensing of cell-specific markers released within cultures. Finally, biocompatibility tests preliminarily demonstrated that embedded NPs also played a key role by inducing physiological cell-cytoskeleton rearrangements, further confirming the potentialities of such hybrid nanocomposites as groundbreaking materials in laser-based bioprinting.

Group ICA of wide-field calcium imaging data reveals the retrosplenial cortex as a major contributor to cortical activity during anesthesia.

Large-scale cortical dynamics play a crucial role in many cognitive functions such as goal-directed behaviors, motor learning and sensory processing. It is well established that brain states including wakefulness, sleep, and anesthesia modulate neuronal firing and synchronization both within and across different brain regions. However, how the brain state affects cortical activity at the mesoscale level is less understood. This work aimed to identify the cortical regions engaged in different brain states. To this end, we employed group ICA (Independent Component Analysis) to wide-field imaging recordings of cortical activity in mice during different anesthesia levels and the awake state. Thanks to this approach we identified independent components (ICs) representing elements of the cortical networks that are common across subjects under decreasing levels of anesthesia toward the awake state. We found that ICs related to the retrosplenial cortices exhibited a pronounced dependence on brain state, being most prevalent in deeper anesthesia levels and diminishing during the transition to the awake state. Analyzing the occurrence of the ICs we found that activity in deeper anesthesia states was characterized by a strong correlation between the retrosplenial components and this correlation decreases when transitioning toward wakefulness. Overall these results indicate that during deeper anesthesia states coactivation of the posterior-medial cortices is predominant over other connectivity patterns, whereas a richer repertoire of dynamics is expressed in lighter anesthesia levels and the awake state.

Mapping brain state-dependent sensory responses across the mouse cortex.

Sensory information must be integrated across a distributed brain network for stimulus processing and perception. Recent studies have revealed specific spatiotemporal patterns of cortical activation for the early and late components of sensory-evoked responses, which are associated with stimulus features and perception, respectively. Here, we investigated how the brain state influences the sensory-evoked activation across the mouse cortex. We utilized isoflurane to modulate the brain state and conducted wide-field calcium imaging of Thy1-GCaMP6f mice to monitor distributed activation evoked by multi-whisker stimulation. Our findings reveal that the level of anesthesia strongly shapes the spatiotemporal features and the functional connectivity of the sensory-activated network. As anesthesia levels decrease, we observe increasingly complex responses, accompanied by the emergence of the late component within the sensory-evoked response. The persistence of the late component under anesthesia raises new questions regarding the potential existence of perception during unconscious states.

Optogenetic confirmation of transverse-tubular membrane excitability in intact cardiac myocytes.

T-tubules (TT) form a complex network of sarcolemmal membrane invaginations, essential for well-co-ordinated excitation-contraction coupling (ECC) and thus homogeneous mechanical activation of cardiomyocytes. ECC is initiated by rapid depolarization of the sarcolemmal membrane. Whether TT membrane depolarization is active (local generation of action potentials; AP) or passive (following depolarization of the outer cell surface sarcolemma; SS) has not been experimentally validated in cardiomyocytes. Based on the assessment of ion flux pathways needed for AP generation, we hypothesize that TT are excitable. We therefore explored TT excitability experimentally, using an all-optical approach to stimulate and record trans-membrane potential changes in TT that were structurally disconnected, and hence electrically insulated, from the SS membrane by transient osmotic shock. Our results establish that cardiomyocyte TT can generate AP. These AP show electrical features that differ substantially from those observed in SS, consistent with differences in the density of ion channels and transporters in the two different membrane domains. We propose that TT-generated AP represent a safety mechanism for TT AP propagation and ECC, which may be particularly relevant in pathophysiological settings where morpho-functional changes reduce the electrical connectivity between SS and TT membranes. KEY POINTS: Cardiomyocytes are characterized by a complex network of membrane invaginations (the T-tubular system) that propagate action potentials to the core of the cell, causing uniform excitation-contraction coupling across the cell. In the present study, we investigated whether the T-tubular system is able to generate action potentials autonomously, rather than following depolarization of the outer cell surface sarcolemma. For this purpose, we developed a fully optical platform to probe and manipulate the electrical dynamics of subcellular membrane domains. Our findings demonstrate that T-tubules are intrinsically excitable, revealing distinct characteristics of self-generated T-tubular action potentials. This active electrical capability would protect cells from voltage drops potentially occurring within the T-tubular network.

Optical Clearing and Labeling for Light-sheet Fluorescence Microscopy in Large-scale Human Brain Imaging.

Despite the numerous clearing techniques that emerged in the last decade, processing postmortem human brains remains a challenging task due to its dimensions and complexity, which make imaging with micrometer resolution particularly difficult. This paper presents a protocol to perform the reconstruction of volumetric portions of the human brain by simultaneously processing tens of sections with the SHORT (SWITCH - H2O2 - Antigen Retrieval - 2,2'-thiodiethanol [TDE]) tissue transformation protocol, which enables clearing, labeling, and sequential imaging of the samples with light-sheet fluorescence microscopy (LSFM). SHORT provides rapid tissue clearing and homogeneous multi-labeling of thick slices with several neuronal markers, enabling the identification of different neuronal subpopulations in both white and grey matter. After clearing, the slices are imaged via LSFM with micrometer resolution and in multiple channels simultaneously for a rapid 3D reconstruction. By combining SHORT with LSFM analysis within a routinely high-throughput protocol, it is possible to obtain the 3D cytoarchitecture reconstruction of large volumetric areas at high resolution in a short time, thus enabling comprehensive structural characterization of the human brain.

A modular and adaptable analysis pipeline to compare slow cerebral rhythms across heterogeneous datasets.

Neuroscience is moving toward a more integrative discipline where understanding brain function requires consolidating the accumulated evidence seen across experiments, species, and measurement techniques. A remaining challenge on that path is integrating such heterogeneous data into analysis workflows such that consistent and comparable conclusions can be distilled as an experimental basis for models and theories. Here, we propose a solution in the context of slow-wave activity (<1 Hz), which occurs during unconscious brain states like sleep and general anesthesia and is observed across diverse experimental approaches. We address the issue of integrating and comparing heterogeneous data by conceptualizing a general pipeline design that is adaptable to a variety of inputs and applications. Furthermore, we present the Collaborative Brain Wave Analysis Pipeline (Cobrawap) as a concrete, reusable software implementation to perform broad, detailed, and rigorous comparisons of slow-wave characteristics across multiple, openly available electrocorticography (ECoG) and calcium imaging datasets.

Microplastics reach the brain and interfere with honey bee cognition.

Scientific research on the impact of microplastics (MPs) in terrestrial systems is still emerging, but it has confirmed adverse health effects in organisms exposed to plastics. Although recent studies have shown the toxicological effects of individual MPs polymers on honey bees, the effects of different polymer combinations on cognitive and behavioural performance remain unknown. To fill this knowledge gap, we investigated the effects of oral exposure to spherical MPs on cognitive performance and brain accumulation in the honey bee Apis mellifera. We evaluated the acute toxicity, after a two-day exposure, of polystyrene (PS - 4.8-5.8 µm) and plexiglass (Poly(methyl methacrylate), or PMMA - 1-40 µm) MPs, and a combination of the two (MIX), at two environmentally relevant and one higher concentration (0.5, 5 and 50 mg L-1) and analysed their effects on sucrose responsiveness and appetitive olfactory learning and memory. We also used fluorescent thermoset amino formaldehyde MPs (1-5 µm) to explore whether microspheres of this diameter could penetrate the insect blood-brain barrier (BBB), using Two-Photon Fluorescence Microscopy (TPFM) in combination with an optimized version of the DISCO clearing technique. The results showed that PS reduced sucrose responsiveness, while PMMA had no significant effect; however, the combination had a marked negative effect on sucrose responsiveness. PMMA, PS, and MIX impaired bee learning and memory in bees, with PS showing the most severe effects. 3D brain imaging analysis using TFPM showed that 1-5 µm MPs penetrated and accumulated in the brain after only three days of oral exposure. These results raise concerns about the potential mechanical, cellular, and biochemical damage that MPs may cause to the central nervous system.

Segmentation of supragranular and infragranular layers in ultra-high resolution 7T ex vivo MRI of the human cerebral cortex.

Accurate labeling of specific layers in the human cerebral cortex is crucial for advancing our understanding of neurodevelopmental and neurodegenerative disorders. Leveraging recent advancements in ultra-high resolution ex vivo MRI, we present a novel semi-supervised segmentation model capable of identifying supragranular and infragranular layers in ex vivo MRI with unprecedented precision. On a dataset consisting of 17 whole-hemisphere ex vivo scans at 120 µm, we propose a multi-resolution U-Nets framework (MUS) that integrates global and local structural information, achieving reliable segmentation maps of the entire hemisphere, with Dice scores over 0.8 for supra- and infragranular layers. This enables surface modeling, atlas construction, anomaly detection in disease states, and cross-modality validation, while also paving the way for finer layer segmentation. Our approach offers a powerful tool for comprehensive neuroanatomical investigations and holds promise for advancing our mechanistic understanding of progression of neurodegenerative diseases.

A cellular resolution atlas of Broca's area.

Brain cells are arranged in laminar, nuclear, or columnar structures, spanning a range of scales. Here, we construct a reliable cell census in the frontal lobe of human cerebral cortex at micrometer resolution in a magnetic resonance imaging (MRI)-referenced system using innovative imaging and analysis methodologies. MRI establishes a macroscopic reference coordinate system of laminar and cytoarchitectural boundaries. Cell counting is obtained with a digital stereological approach on the 3D reconstruction at cellular resolution from a custom-made inverted confocal light-sheet fluorescence microscope (LSFM). Mesoscale optical coherence tomography enables the registration of the distorted histological cell typing obtained with LSFM to the MRI-based atlas coordinate system. The outcome is an integrated high-resolution cellular census of Broca's area in a human postmortem specimen, within a whole-brain reference space atlas.

Optimization of highly inclined illumination for diffraction-limited and super-resolution microscopy.

In HILO microscopy, a highly inclined and laminated light sheet is used to illuminate the sample, thus drastically reducing background fluorescence in wide-field microscopy, but maintaining the simplicity of the use of a single objective for both illumination and detection. Although the technique has become widely popular, particularly in single molecule and super-resolution microscopy, a limited understanding of how to finely shape the illumination beam and of how this impacts on the image quality complicates the setting of HILO to fit the experimental needs. In this work, we build up a simple and comprehensive guide to optimize the beam shape and alignment in HILO and to predict its performance in conventional fluorescence and super-resolution microscopy. We model the beam propagation through Gaussian optics and validate the model through far- and near-field experiments, thus characterizing the main geometrical features of the beam. Further, we fully quantify the effects of a progressive reduction of the inclined beam thickness on the image quality of both diffraction-limited and super-resolution images and we show that the most relevant impact is obtained by reducing the beam thickness to sub-cellular dimensions (< 3 µm). Based on this, we present a simple optical solution that exploits a rectangular slit to reduce the inclined beam thickness down to 2.6 µm while keeping a field-of-view dimension suited for cell imaging and allowing an increase in the number of localizations in super-resolution imaging of up to 2.6 folds.

Multilayered Bioorthogonal SERS Nanoprobes Selectively Aggregating in Human Fluids: A Smart Optical Assay for ß-Amyloid Peptide Quantification.

Alzheimer's disease (AD) is a debilitating neurological condition characterized by cognitive decline, memory loss, and behavioral skill impairment, features that worsen with time. Early diagnosis will likely be the most effective therapy for Alzheimer's disease since it can ensure timely pharmacological treatments that can reduce the irreversible progression and delay the symptoms. Amyloid ß-peptide 1-42 (Aß (1-42)) is considered one of the key pathological AD biomarkers that is present in different biological fluids. However, Aß (1-42) detection still relies on colorimetric and enzyme-linked immunoassays as the gold standard characterized by low accuracy or high costs, respectively. In this context, optical detection techniques based on surface-enhanced Raman spectroscopy (SERS) through advanced nanoconstructs are promising alternatives for the development of novel rapid and low-cost methods for the targeting of Aß pathological biomarkers in fluids. Here, a multilayered nanoprobe constituted by bioorthogonal Raman reporters (RRs) embedded within two layers of gold nanoparticles (Au@RRs@AuNPs) has been developed and successfully validated for specific detection of Aß (1-42) in the human cerebrospinal fluid (CSF) with sensitivity down to pg/mL. The smart double-layer configuration enables us to exploit the outer gold NP surfaces for selective absorption of targeted peptide whose concentration controls the aggregation behavior of Au@RRs@AuNPs, proportionally reflected in Raman intensity changes, providing high specificity and sensitivity and representing a significant step ahead of the state of the art on SERS for clinical analyses.

Nogo-A antibody delivery through the olfactory mucosa mitigates experimental autoimmune encephalomyelitis in the mouse CNS.

Systemic administration of Nogo-A-neutralizing antibody ameliorates experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. However, the blood-brain barrier (BBB) is a major obstacle limiting the passage of systemically applied antibody to the CNS. To bypass the BBB, in the present study we tested the intranasal route of administration by targeting the olfactory mucosa with the Nogo-A-blocking antibody 11C7 mAb in myelin oligodendrocyte glycoprotein-induced EAE. Antibodies were specifically administered onto the olfactory mucosa using a microcatheter. Antibody distribution was examined in the CNS by ELISA and light-sheet microscopy. The effects of 11C7 mAb on Nogo-A signaling were assessed by Western blotting. EAE-induced deficits were monitored daily. Demyelination was observed on spinal cord histological sections. Gene expression changes were followed by trancriptomic analyses. A sensitive capture ELISA revealed a rapid and widespread distribution of 11C7 mAb in the CNS, including the olfactory bulb, the cerebellum and the lumbar spinal cord, but not in the CSF. Light-sheet microscopy allowed to observe antibody accumulation in the parenchyma, thus demonstrating nose-to-brain transfer of IgG. At the functional level, the widespread penetration of 11C7 mAb in the CNS, including the thoracolumbar spinal cord, resulted in the improvement of motor symptoms and in the preservation of myelin in the spinal cord of EAE mice. This was accompanied by Nogo-A signaling downregulation, as reflected by the decreased level of phosphorylated cofilin observed by Western blotting in the cerebellum. In the brain of EAE score-matched animals, 11C7 modified the expression of genes that can influence neurotransmission and cognitive functions, independently of the demyelination phenotype in the spinal cord. In conclusion, our data show the feasibility of olfactory mucosa-directed administration for the delivery of therapeutic antibodies targeting CNS antigens in EAE mice.

Brain-wide neuron quantification toolkit reveals strong sexual dimorphism in the evolution of fear memory.

Fear responses are functionally adaptive behaviors that are strengthened as memories. Indeed, detailed knowledge of the neural circuitry modulating fear memory could be the turning point for the comprehension of this emotion and its pathological states. A comprehensive understanding of the circuits mediating memory encoding, consolidation, and retrieval presents the fundamental technological challenge of analyzing activity in the entire brain with single-neuron resolution. In this context, we develop the brain-wide neuron quantification toolkit (BRANT) for mapping whole-brain neuronal activation at micron-scale resolution, combining tissue clearing, high-resolution light-sheet microscopy, and automated image analysis. The robustness and scalability of this method allow us to quantify the evolution of activity patterns across multiple phases of memory in mice. This approach highlights a strong sexual dimorphism in recruited circuits, which has no counterpart in the behavior. The methodology presented here paves the way for a comprehensive characterization of the evolution of fear memory.

Fiber-Based SERS-Fluidic Polymeric Platforms for Improved Optical Analysis of Liquids.

Downsizing surface-enhanced Raman spectroscopy (SERS) within microfluidic devices has opened interesting perspectives for the development of low-cost and portable (bio)sensors for the optical analysis of liquid samples. Despite the research efforts, SERS-fluidic devices still rely either on the use of expensive bulky set-ups or on polymeric devices giving spurious background signals fabricated via expensive manufacturing processes. Here, polymeric platforms integrating fluidics and optics were fabricated with versatile designs allowing easy coupling with fiber-based Raman systems. For the first time, anti-fouling photocurable perfluoropolyether (PFPE) was explored for high-throughput SERS-integrating chip fabrication via replica molding of negative stamps obtained through standard and advanced fabrication processes. The PFPE devices comprised networks of channels for fluid handling and for optical fiber housing with multiple orientations. Embedded microfeatures were used to control the relative positioning of the fibers, thus guaranteeing the highest signal delivering and collection. The feasibility of PFPE devices as fiber-based SERS fluidic platforms was demonstrated through the straightforward acquisition of Raman-SERS spectra of a mixture of gold nanoparticles as SERS substrates with rhodamine 6G (Rh6G) at decreasing concentrations. In the presence of high-performing gold nanostars, the Rh6G signal was detectable at dilutions down to the nanomolar level even without tight focusing and working at low laser power-a key aspect for analyte detection in real-world biomedical and environmental applications.

Imaging Approaches to Investigate Pathophysiological Mechanisms of Brain Disease in Zebrafish.

Zebrafish has become an essential model organism in modern biomedical research. Owing to its distinctive features and high grade of genomic homology with humans, it is increasingly employed to model diverse neurological disorders, both through genetic and pharmacological intervention. The use of this vertebrate model has recently enhanced research efforts, both in the optical technology and in the bioengineering fields, aiming at developing novel tools for high spatiotemporal resolution imaging. Indeed, the ever-increasing use of imaging methods, often combined with fluorescent reporters or tags, enable a unique chance for translational neuroscience research at different levels, ranging from behavior (whole-organism) to functional aspects (whole-brain) and down to structural features (cellular and subcellular). In this work, we present a review of the imaging approaches employed to investigate pathophysiological mechanisms underlying functional, structural, and behavioral alterations of human neurological diseases modeled in zebrafish.

APP and Bace1: Differential effect of cholesterol enrichment on processing and plasma membrane mobility.

High cholesterol levels are a risk factor for the development of Alzheimer's disease. Experiments investigating the influence of cholesterol on the proteolytic processing of the amyloid precursor protein (APP) by the ß-secretase Bace1 and on their proximity in cells have led to conflicting results. By using a fluorescence bioassay coupled with flow cytometry we found a direct correlation between the increase in membrane cholesterol amount and the degree of APP shedding in living human neuroblastoma cells. Analogue results were obtained for cells overexpressing an APP mutant that cannot be processed by α-secretase, highlighting the major influence of cholesterol enrichment on the cleavage of APP carried out by Bace1. By contrast, the cholesterol content was not correlated with changes in membrane dynamics of APP and Bace1 analyzed with single molecule tracking, indicating that the effect of cholesterol enrichment on APP processing by Bace1 is uncoupled from changes in their lateral diffusion.

A Guide to Perform 3D Histology of Biological Tissues with Fluorescence Microscopy.

The analysis of histological alterations in all types of tissue is of primary importance in pathology for highly accurate and robust diagnosis. Recent advances in tissue clearing and fluorescence microscopy made the study of the anatomy of biological tissue possible in three dimensions. The combination of these techniques with classical hematoxylin and eosin (H&E) staining has led to the birth of three-dimensional (3D) histology. Here, we present an overview of the state-of-the-art methods, highlighting the optimal combinations of different clearing methods and advanced fluorescence microscopy techniques for the investigation of all types of biological tissues. We employed fluorescence nuclear and eosin Y staining that enabled us to obtain hematoxylin and eosin pseudo-coloring comparable with the gold standard H&E analysis. The computational reconstructions obtained with 3D optical imaging can be analyzed by a pathologist without any specific training in volumetric microscopy, paving the way for new biomedical applications in clinical pathology.

Dysplasia and tumor discrimination in brain tissues by combined fluorescence, Raman, and diffuse reflectance spectroscopies.

Identification of neoplastic and dysplastic brain tissues is of paramount importance for improving the outcomes of neurosurgical procedures. This study explores the combined application of fluorescence, Raman and diffuse reflectance spectroscopies for the detection and classification of brain tumor and cortical dysplasia with a label-free modality. Multivariate analysis was performed to evaluate classification accuracies of these techniques-employed both in individual and multimodal configuration-obtaining high sensitivity and specificity. In particular, the proposed multimodal approach allowed discriminating tumor/dysplastic tissues against control tissue with 91%/86% sensitivity and 100%/100% specificity, respectively, whereas tumor from dysplastic tissues were discriminated with 89% sensitivity and 86% specificity. Hence, multimodal optical spectroscopy allows reliably differentiating these pathologies using a non-invasive, label-free approach that is faster than the gold standard technique and does not require any tissue processing, offering the potential for the clinical translation of the technology.