Development of an optical microscopy platform for high-throughput screening of human brain tissue

One of the greatest scientific challenges of our day is to gain a complete understanding of the human brain. Despite the substantial advantages that came with the introduction of automatic histology instrumentation, traditional protocols still suffer from significant drawbacks. Such limitations are inherent to the two-dimensional nature of the classic slide-based protocol including low sensitivity for sparse features, difficult assessment of dimensions, alteration of morphology, visual artifacts (different orientation or distribution), and destruction of the tissue specimen. All these problems hinder a thorough examination of the sample. To overcome the limitations of traditional protocols, the present project aims to develop a novel methodology to perform a 3D analysis of human brain tissue at the micrometer scale using high-resolution light sheet fluorescence microscopy (LSFM). Several advantages of LSFM are non-destructive optical sectioning, fast acquisition times, micrometric resolution, high penetration depths, and 3D tomography. The new LSFM apparatus will allow the analysis of brain samples from healthy subjects and subjects affected by focal cortical dysplasias obtaining detailed information about the cerebral cytoarchitecture.

Development of optical clearing and staining methods for multi-target investigation of human brain tissue cytoarchitecture

3D reconstruction with LSFM requires the sample to be transparent. The excitation light and the emitted fluorescence need to penetrate and exit the sample. However, biological tissues are opaque because of light scattering, therefore a clearing step is necessary. The principle of tissue clearing relies on the homogenization of the refractive index inside and outside the sample. In this project, we developed a new tissue-clearing protocol called SHORT (SWITCH – H2O2 – antigen Retrieval – TDE), based on the TDE clearing method and the SWITCH immunohistochemistry technique to stain the cellular structures of clarified samples while highlighting the contrast between nuclei and cytoplasm.

Now that the sample preparation is completed to scale up the analysis for routine use, the sample treatment will be parallelized. Moreover, to enlarge the panel of biological markers analyzable with the LSFM, a large plethora of staining will be tested and applied. Various antibodies for specific staining will be used to characterize both healthy and affected subjects. Finally, multi-round and multi-staining approaches will be investigated to obtain different staining on the same sample.

Artificial intelligence to extract information from large-scale microscopy images

Once brain imaging has been performed, it is of paramount importance to automate the process of understanding the contents of the obtained 3D images. A first step is counting and localizing the nuclei of the set of marked neurons. Despite its apparent simplicity, the task is non trivial because of the image resolution that can be afforded for large portions of the human brain, and because of the large contrast variability that characterizes the acquired images. During the last few years, deep learning has shown remarkable progress in the automated analysis of various types of medical and biological images. However, this is often achieved thanks to a particular form of learning, called supervised learning, where the available data has been labeled by hand with the variables that need to be predicted. In our case, this would amount to asking human experts to annotate large enough portions of brain images by putting markers in correspondence of every nucleous. The result is what is technically known as supervision. This step, unfortunately, is very laborious and error prone. From a broader perspective of artificial intelligent systems, this approach to learning also appears to share little similarities to human learning. Babies for example do not learn to read handwritten text using thousands of examples where a teacher explicitly tells them what are the characters in and handwritten document. Machines trained by supervised learning however do. Recent advances in the area of self-supervised learning allow AI researchers to develop machines that acquire experience without requiring a teaching signal. The resulting machines perform well even with a very limited amount of supervision. These techniques need specialization in their application to the brain images that are produced within the project and the work carried out by DINFO goes precisely in this direction.

Large-scale mapping of human brain from biobanks

The combination of optical tissue clearing with advanced microscopy techniques such as LSFM can address the issues of more traditional histology protocols, enabling 3D histology, and providing a higher level of anatomical insight. The approach presented here will be used to analyze tissues affected by various pathologies to provide insight into the disease, eliminating human biases and obtaining a more precise diagnostic evaluation. But will be used also to investigate the anatomical architecture of healthy samples arrived from biobanks to provide a cell census of the various brain areas, ensuring a complete description of the organ.

Large-scale genomic analysis of human developmental encephalopathies (DE) for causative genes identification and functional characterization of pathogenic mechanisms.

We aim to expand the knowledge of genetic causes of developmental encephalopathies (DE). To this purpose, we will analyze the DNA (the molecule carrying the instructions for the development, functioning, growth, and reproduction of all organisms) extracted from biological samples (e.g. blood or saliva) of patients and their parents through whole exome (i.e. the protein-coding regions of the genome) or whole genome (i.e. the entire DNA sequence of the cell) sequencing. In patients with focal epilepsy who have been subjected to epilepsy surgery, we will also analyze a small sample of the epileptic brain tissue removed during surgery to search for somatic pathogenic variants (i.e. pathogenic variants that are present only in the brain), which represent the main cause of this type of epilepsy.

Comprehensive classification of pathological human brain tissue

We will characterize epileptogenic tissue surgically removed from patients with drug resistant epilepsy due to focal brain malformations at the morphological and structural level through anatomopathological studies. For a more comprehensive characterization of the epileptic tissue, we will integrate anatomopathological data with genetic data.

Transcriptomics analysis of dysplastic human brain tissue

Transcriptomics is the analysis of the RNA transcripts produced by the genes and provides a link between the genome (all genetic information of an organism), the proteome (the set of expressed proteins in a given type of cell or organism, at a given time, under defined conditions), and the phenotype (the set of observable characteristics or traits of a cell or an organism). We will compare the transcriptomics profile of aberrant cells isolated from epileptogenic tissue surgically removed from patients with drug resistant focal epilepsy carrying mutations in known causative genes versus mutation negative samples. Such studies will allow identifying novel focal epilepsy causative genes and provide indications on possible targets for novel personalized therapeutic strategies.

Characterizing, in neuronal and electroporated models, morphologic abnormalities and electrophysiological dysfunction caused by somatic and constitutional mutations

In order to explore the effects of the mutations we will identify in patients using large-scale genomic analysis, we will express them in cellular and animal models and carry out morphological and electrophysiological analyses. For selected mutations associated with severe epileptic encephalopathies, we will perform such analyses also in human neurons obtained reprogramming cells isolated from patients’ skin. The use of reprogrammed human neurons has the potential to further enhance our knowledge of pathways underlying the developmental process of the human brain, the current knowledge of which has predominantly come from animal studies postmortem tissue.

Development of high-throughput optical systems for functional and structural mapping of the nervous system in zebrafish larvae

The possibility to link the behaviour and the neuronal activity in a causative manner is of great importance for the study of vertebrate brain. However, it is necessary to perform this analysis in a brain-wide fashion, since the neuronal activity generating the behaviour is largely distributed. This would not be possible for the human subjects, at least not without giving up the fine spatio-temporal resolution. Instead, thanks to the optical transparency provided by the zebrafish larva model, the detailed characterization of the vertebrate brain becomes possible. By carefully selecting the most evolutionarily conserved behavioural patterns and by thoughtful inference, it will be possible to extend a selection of the obtained results to the more general case of the vertebrate brain, human included.

In order to reach this goal, we will use an integrated optical system that we have developed in our laboratory. This system is able to “read” the activity of each neuron in the larval brain five times each second. Moreover, this system is also able to simultaneously “write” the activity of a large group of arbitrarily-chosen neurons displaced across the brain. In addition this optical system allows for non-invasive imaging without detrimental effects on the larva. To evoke behaviour-related nervous activity or to observe the effect of stimulated nervous activity on behaviorally-relevant settings, the larva can also be immersed in a virtual reality environment during the imaging and stimulation session.

Expansion of zebrafish animal facility

Zebrafish is a small fresh water tropical fish popular with home aquarium owners for its elegant look and active social behaviors. In the last decades, this fish has gained enormous popularity in several areas of scientific research, both in basic biology and in biomedicine. Reproduction takes place with deposition of hundreds of eggs by each couple. The egg, embryos and larvae during the first weeks of development are fully transparent, allowing researcher to study in great detail the processes of development of all organs and tissues, as well as their pathological alterations. With the development of efficient tools for molecular biology, the sequencing of the zebrafish genome and the availability of powerful microscopy techniques, therefore, this fish has become a great model in developmental biology, neuroscience and many other fields. For example it is the only vertebrate model in which all neurons can be simultaneously imaged to measure their activity and study the workings of neural circuits in vivo. Furthermore, its use in drug screening promises to revolutionize the drug discovery process and to facilitate the advent of personalized medicine. In order to use this great vertebrate model in research it is necessary to establish a facility for rearing adults and larvae. The Department of Biology has developed a facility capable of hosting up to 48 different lines of fish (for example carrying mutations interesting for the study of pathology or the imaging of neuronal activity in vivo) for a total of about 1500 fish. With the project Human Brain Optical Mapping (humanbrainmap) the Biology departartment has increased the capacity of the facility with an added 20 tanks, for as many new transgenic lines and a total of 2000 fish. The facility is also equipped with microinjection setup for generation of new transgenic lines, fluorescence stereomicroscope for mutant screening and a Daniovision setup for real-time measurement of zebrafish larvae behavior. The improvement allows a strong synergy with all other partners of the project in the development of several lines of research.

Generation of zebrafish transgenic lines for novel functional and pharmacological studies

The study of specific cell type involved in the development and maintenance of pain is of particular interest in the development of targeted therapy, thus reducing off-target effects. Schwann cells and oligodendrocytes are the glia cells that wrap peripheral and central nerves respectively. In addition to their established role as a protective barrier, Schwann cells/oligodendrocyte have recently emerged as key cellular element to sustain neuronal damage and chronic pain. Recent observations report how intracellular mechanisms in Schwann cells play a fundamental role in the modulation of pain mechanisms. The zebrafish represents an unprecedented tool, hitherto poorly explored, to identify the role of central and peripheral glial cells in chronic pain.

To investigate the role of Schwann cells in chronic pain in zebrafish, we will use different fluorescent molecules able to reach with and monitor the variation of the intracellular second messengers involved in pain pathways over time at the cellular level in sensory neurons and Schwann cells. Then, the genetically modified zebrafish will be stimulated with proalgesic substances, and will be monitored by microscopy, by evaluating the fluorescence variations determined by fluorescent molecules and indicative for a specific intracellular pathway activation.

Pilot study on the use zebrafish for drug screening in autism related disorders

Creatine transporter deficiency (CTD) is an X-linked inherited metabolic disease presenting with cerebral creatine deficiency, early intellectual disability, autistic behavior and epilepsy. Although rare, CTD is a severe health care problem since it is a chronic condition with a strong impact on patients’ quality of life and no cure is available. We aim to provide a new model for in vivo screening of novel candidate molecules to ameliorate symptoms.

Pilot study on zebrafish to understand the role of the Schwann cell/oligodendrocyte lineage in the peripheral and central processing of tissue damage and chronic pain

After the development of the genetic models and the study of the intracellular pathways stimulated by proalgesic mediators, behavioral activity of transgenic zebrafish larvae will be studied in vivo following the administration of proalgesic substances. We will also develop a model of neuropathic pain in zebrafish to study chronic pain mechanisms. The TRPA1 channel, which is abundantly expressed in primary sensory neurons and has been recently identified in Schwann cells/oligodendrocytes, will be also explored in these models, by using selective channel blockers or genetic deletion. Behavioral test will be carried out by the assessment of spontaneous locomotor activity, light-dark preference (phototaxis), cognitive responses, social behaviors using a specific instrument (DanioVision system) able to recorder all these activities in zebrafish.

Pilot study on the use zebrafish and human organoids for getting insight into the brain-heart axis: developmental and (patho)physiological aspects

The study of organs formation and function requires huge amounts of efforts and multidisciplinary expertizes because in them many biological processes are involved and controlled by an evolutionarily conserved network of genes. In addition, the final morphological/physiological result of an organ and its proper function is also due to epigenetic processes, external forces and environmental factors.

The new frontiers of scientific investigation in development and physiology have shifted versus different and integrated aspects of organ physiopathology, analysing complex signals among organs communication overtime. One of the final aims of this task is exploring the brain-heart axis, getting deeper insight into the neuronal mechanisms controlling heartbeat and dysrhythmia, elucidating new ways to analyse and understand the molecular and functional basis of human arrhythmias.

Dissemination activity, Biomedical Devices, Functional data analysis

Research and scientific activities are, often, perceived as distant by non-experts, which restricts all potential interactions between these areas. Develop methods for public outreach represents a great opportunity to enrich both ends. This website, social media and other instruments will be used to spread results and inform potential participants about incoming experiments. The collaboration with a public accessible environment, like the Museum of Science in Florence, will be crucial to reach our goal.

We would like to collect biomedical signals through non-invasive, wearable sensors available on the market. The signals recorded will allow us to detect the cognitive/emotional state during different stimuli and situations. Once the said state will be defined, it will be possible to identify critical aspects in various activity that will be optimized. Artistic shows, education, teamwork and rehabilitation are just a few of the dozens of areas that could benefit from this approach. Imagine an artistic show optimized to obtain the highest emotive impact possible.

The evaluation of the potential of low-cost wearable sensors could also open new roads for screening possibilities available in low-income countries.

We will also focus on the development of new sensors based on hyperspectral imaging (HSI) to expand the available pool of information. HSI is an optical technique that uses light at different wavelengths to assess the pathophysiological state of biological tissues. Lot of human tissues have light-absorbing components that are strictly correlated to their metabolism, oxygenation degree and health status in general. Given its non-invasiveness and real-time measurement capabilities, this will be an excellent complement to our setup.