Human CASPR2 Antibodies Reversibly Alter Memory and the CASPR2 Protein Complex.

OBJECTIVE: The encephalitis associated with antibodies against contactin-associated proteinlike 2 (CASPR2) is presumably antibody-mediated, but the antibody effects and whether they cause behavioral alterations are not well known. Here, we used a mouse model of patients' immunoglobulin G (IgG) transfer and super-resolution microscopy to demonstrate the antibody pathogenicity. METHODS: IgG from patients with anti-CASPR2 encephalitis or healthy controls was infused into the cerebroventricular system of mice. The levels and colocalization of CASPR2 with transient axonal glycoprotein 1 (TAG1) were determined with stimulated emission depletion microscopy (40-70µm lateral resolution). Hippocampal clusters of Kv1.1 voltage-gated potassium channels (VGKCs) and GluA1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) were quantified with confocal microscopy. Behavioral alterations were assessed with standard behavioral paradigms. Cultured neurons were used to determine the levels of intracellular CASPR2 and TAG1 after exposure to patients' IgG. RESULTS: Infusion of patients' IgG, but not controls' IgG, caused memory impairment along with hippocampal reduction of surface CASPR2 clusters and decreased CASPR2/TAG1 colocalization. In cultured neurons, patients' IgG led to an increase of intracellular CASPR2 without affecting TAG1, suggesting selective CASPR2 internalization. Additionally, mice infused with patients' IgG showed decreased levels of Kv1.1 and GluA1 (two CASPR2-regulated proteins). All these alterations and the memory deficit reverted to normal after removing patients' IgG. INTERPRETATION: IgG from patients with anti-CASPR2 encephalitis causes reversible memory impairment, inhibits the interaction of CASPR2/TAG1, and decreases the levels of CASPR2 and related proteins (VGKC, AMPAR). These findings fulfill the postulates of antibody-mediated disease and provide a biological basis for antibody-removing treatment approaches. ANN NEUROL 2022;91:801-813.

Deficiency of the ywhaz gene, involved in neurodevelopmental disorders, alters brain activity and behaviour in zebrafish.

Genetic variants in YWHAZ contribute to psychiatric disorders such as autism spectrum disorder and schizophrenia, and have been related to an impaired neurodevelopment in humans and mice. Here, we have used zebrafish to investigate the mechanisms by which YWHAZ contributes to neurodevelopmental disorders. We observed that ywhaz expression was pan-neuronal during developmental stages and restricted to Purkinje cells in the adult cerebellum, cells that are described to be reduced in number and size in autistic patients. We then performed whole-brain imaging in wild-type and ywhaz CRISPR/Cas9 knockout (KO) larvae and found altered neuronal activity and connectivity in the hindbrain. Adult ywhaz KO fish display decreased levels of monoamines in the hindbrain and freeze when exposed to novel stimuli, a phenotype that can be reversed with drugs that target monoamine neurotransmission. These findings suggest an important role for ywhaz in establishing neuronal connectivity during development and modulating both neurotransmission and behaviour in adults.

Three-Photon Infrared Stimulation of Endogenous Neuroreceptors in Vivo.

To interrogate neural circuits and crack their codes, in vivo brain activity imaging must be combined with spatiotemporally precise stimulation in three dimensions using genetic or pharmacological specificity. This challenge requires deep penetration and focusing as provided by infrared light and multiphoton excitation, and has promoted two-photon photopharmacology and optogenetics. However, three-photon brain stimulation in vivo remains to be demonstrated. We report the regulation of neuronal activity in zebrafish larvae by three-photon excitation of a photoswitchable muscarinic agonist at 50 pM, a billion-fold lower concentration than used for uncaging, and with mid-infrared light of 1560 nm, the longest reported photoswitch wavelength. Robust, physiologically relevant photoresponses allow modulating brain activity in wild-type animals with spatiotemporal and pharmacological precision. Computational calculations predict that azobenzene-based ligands have high three-photon absorption cross-section and can be used directly with pulsed infrared light. The expansion of three-photon pharmacology will deeply impact basic neurobiology and neuromodulation phototherapies.

Few-cycle all-fiber supercontinuum laser for ultrabroadband multimodal nonlinear microscopy.

Temporally coherent supercontinuum sources constitute an attractive alternative to bulk crystal-based sources of few-cycle light pulses. We present a monolithic fiber-optic configuration for generating transform-limited temporally coherent supercontinuum pulses with central wavelength at 1.06 µm and duration as short as 13.0 fs (3.7 optical cycles). The supercontinuum is generated by the action of self-phase modulation and optical wave breaking when pumping an all-normal dispersion photonic crystal fiber with pulses of hundreds of fs duration produced by all-fiber chirped pulsed amplification. Avoidance of free-space propagation between stages confers unequalled robustness, efficiency and cost-effectiveness to this novel configuration. Collectively, the features of all-fiber few-cycle pulsed sources make them powerful tools for applications benefitting from the ultrabroadband spectra and ultrashort pulse durations. Here we exploit these features and the deep penetration of light in biological tissues at the spectral region of 1 µm, to demonstrate their successful performance in ultrabroadband multispectral and multimodal nonlinear microscopy.

The Capillary Morphogenesis Gene 2 Triggers the Intracellular Hallmarks of Collagen VI-Related Muscular Dystrophy.

Collagen VI-related disorders (COL6-RD) represent a severe form of congenital disease for which there is no treatment. Dominant-negative pathogenic variants in the genes encoding α chains of collagen VI are the main cause of COL6-RD. Here we report that patient-derived fibroblasts carrying a common single nucleotide variant mutation are unable to build the extracellular collagen VI network. This correlates with the intracellular accumulation of endosomes and lysosomes triggered by the increased phosphorylation of the collagen VI receptor CMG2. Notably, using a CRISPR-Cas9 gene-editing tool to silence the dominant-negative mutation in patients' cells, we rescued the normal extracellular collagen VI network, CMG2 phosphorylation levels, and the accumulation of endosomes and lysosomes. Our findings reveal an unanticipated role of CMG2 in regulating endosomal and lysosomal homeostasis and suggest that mutated collagen VI dysregulates the intracellular environment in fibroblasts in collagen VI-related muscular dystrophy.

A computational diffusion model to study antibody transport within reconstructed tumor microenvironments.

BACKGROUND: Antibodies revolutionized cancer treatment over the past decades. Despite their successfully application, there are still challenges to overcome to improve efficacy, such as the heterogeneous distribution of antibodies within tumors. Tumor microenvironment features, such as the distribution of tumor and other cell types and the composition of the extracellular matrix may work together to hinder antibodies from reaching the target tumor cells. To understand these interactions, we propose a framework combining in vitro and in silico models. We took advantage of in vitro cancer models previously developed by our group, consisting of tumor cells and fibroblasts co-cultured in 3D within alginate capsules, for reconstruction of tumor microenvironment features. RESULTS: In this work, an experimental-computational framework of antibody transport within alginate capsules was established, assuming a purely diffusive transport, combined with an exponential saturation effect that mimics the saturation of binding sites on the cell surface. Our tumor microenvironment in vitro models were challenged with a fluorescent antibody and its transport recorded using light sheet fluorescence microscopy. Diffusion and saturation parameters of the computational model were adjusted to reproduce the experimental antibody distribution, with root mean square error under 5%. This computational framework is flexible and can simulate different random distributions of tumor microenvironment elements (fibroblasts, cancer cells and collagen fibers) within the capsule. The random distribution algorithm can be tuned to follow the general patterns observed in the experimental models. CONCLUSIONS: We present a computational and microscopy framework to track and simulate antibody transport within the tumor microenvironment that complements the previously established in vitro models platform. This framework paves the way to the development of a valuable tool to study the influence of different components of the tumor microenvironment on antibody transport.

3D and 4D Image Fusion: Coping with Differences in Spectroscopic Modes among Hyperspectral Images.

Image fusion is often oriented to solve differences in spatial scale and orientation among different spectroscopic platforms. However, an additional problem arises when the nature of the spectroscopic information differs in dimensionality as well. Indeed, most imaging systems, e.g., Raman, IR, MS, etc., allow acquisition of 3D images, with a linear spectrum per pixel, but new platforms have emerged, such as the recent excitation-emission fluorescence imaging platforms that provide 4D images, with a 2D spectral landscape per pixel. A proper 3D/4D image fusion needs to take into account the difference in the dimension of the spectral information and in the underlying models of both measurements (bilinear for 3D images and trilinear for 4D images). This work solves this image fusion problem through a new dedicated variant of the multivariate curve resolution-alternating least squares (MCR-ALS) algorithm for multiset analysis based on the incorporation of a hybrid bilinear/trilinear model that can handle the image fused structure preserving the natural behavior of the 3D and 4D imaging techniques coupled. The example is illustrated on the fusion of real 3D Raman and 4D fluorescence images recorded on cross sections of rice leaf samples.

Engineering Polar Oxynitrides: Hexagonal Perovskite BaWON2.

Non-centrosymmetric polar compounds have important technological properties. Reported perovskite oxynitrides show centrosymmetric structures, and for some of them high permittivities have been observed and ascribed to local dipoles induced by partial order of nitride and oxide. Reported here is the first hexagonal perovskite oxynitride BaWON2 , which shows a polar 6H polytype. Synchrotron X-ray and neutron powder diffraction, and annular bright-field in scanning transmission electron microscopy indicate that it crystalizes in the non-centrosymmetric space group P63 mc, with a total order of nitride and oxide at two distinct coordination environments in cubic and hexagonal packed BaX3 layers. A synergetic second-order Jahn-Teller effect, supported by first principle calculations, anion order, and electrostatic repulsions between W6+ cations, induce large distortions at two inequivalent face-sharing octahedra that lead to long-range ordered dipoles and spontaneous polarization along the c axis. The new oxynitride is a semiconductor with a band gap of 1.1 eV and a large permittivity.

Nanopatterns of Surface-Bound EphrinB1 Produce Multivalent Ligand-Receptor Interactions That Tune EphB2 Receptor Clustering.

Here we present a nanostructured surface able to produce multivalent interactions between surface-bound ephrinB1 ligands and membrane EphB2 receptors. We created ephrinB1 nanopatterns of regular size (<30 nm in diameter) by using self-assembled diblock copolymers. Next, we used a statistically enhanced version of the Number and Brightness technique, which can discriminate-with molecular sensitivity-the oligomeric states of diffusive species to quantitatively track the EphB2 receptor oligomerization process in real time. The results indicate that a stimulation using randomly distributed surface-bound ligands was not sufficient to fully induce receptor aggregation. Conversely, when nanopatterned onto our substrates, the ligands effectively induced a strong receptor oligomerization. This presentation of ligands improved the clustering efficiency of conventional ligand delivery systems, as it required a 9-fold lower ligand surface coverage and included faster receptor clustering kinetics compared to traditional cross-linked ligands. In conclusion, nanostructured diblock copolymers constitute a novel strategy to induce multivalent ligand-receptor interactions leading to a stronger, faster, and more efficient receptor activation, thus providing a useful strategy to precisely tune and potentiate receptor responses. The efficiency of these materials at inducing cell responses can benefit applications such as the design of new bioactive materials and drug-delivery systems.

Interference with Clp protease impairs carotenoid accumulation during tomato fruit ripening.

Profound metabolic and structural changes are required for fleshy green fruits to ripen and become colorful and tasty. In tomato (Solanum lycopersicum), fruit ripening involves the differentiation of chromoplasts, specialized plastids that accumulate carotenoid pigments such as ß-carotene (pro-vitamin A) and lycopene. Here, we explored the role of the plastidial Clp protease in chromoplast development and carotenoid accumulation. Ripening-specific silencing of one of the subunits of the Clp proteolytic complex resulted in ß-carotene-enriched fruits that appeared orange instead of red when ripe. Clp-defective fruit displayed aberrant chromoplasts and up-regulated expression of nuclear genes encoding the tomato homologs of Orange (OR) and ClpB3 chaperones, most probably to deal with misfolded and aggregated proteins that could not be degraded by the Clp protease. ClpB3 and OR chaperones protect the carotenoid biosynthetic enzymes deoxyxylulose 5-phosphate synthase and phytoene synthase, respectively, from degradation, whereas OR chaperones additionally promote chromoplast differentiation by preventing the degradation of carotenoids such as ß-carotene. We conclude that the Clp protease contributes to the differentiation of chloroplasts into chromoplasts during tomato fruit ripening, acting in co-ordination with specific chaperones that alleviate protein folding stress, promote enzyme stability and accumulation, and prevent carotenoid degradation.

Synaptic phosphorylated α-synuclein in dementia with Lewy bodies.

Dementia with Lewy bodies is characterized by the accumulation of Lewy bodies and Lewy neurites in the CNS, both of which are composed mainly of aggregated α-synuclein phosphorylated at Ser129. Although phosphorylated α-synuclein is believed to exert toxic effects at the synapse in dementia with Lewy bodies and other α-synucleinopathies, direct evidence for the precise synaptic localization has been difficult to achieve due to the lack of adequate optical microscopic resolution to study human synapses. In the present study we applied array tomography, a microscopy technique that combines ultrathin sectioning of tissue with immunofluorescence allowing precise identification of small structures, to quantitatively investigate the synaptic phosphorylated α-synuclein pathology in dementia with Lewy bodies. We performed array tomography on human brain samples from five patients with dementia with Lewy bodies, five patients with Alzheimer's disease and five healthy control subjects to analyse the presence of phosphorylated α-synuclein immunoreactivity at the synapse and their relationship with synapse size. Main analyses were performed in blocks from cingulate cortex and confirmed in blocks from the striatum of cases with dementia with Lewy bodies. A total of 1 318 700 single pre- or postsynaptic terminals were analysed. We found that phosphorylated α-synuclein is present exclusively in dementia with Lewy bodies cases, where it can be identified in the form of Lewy bodies, Lewy neurites and small aggregates (<0.16 µm3). Between 19% and 25% of phosphorylated α-synuclein deposits were found in presynaptic terminals mainly in the form of small aggregates. Synaptic terminals that co-localized with small aggregates of phosphorylated α-synuclein were significantly larger than those that did not. Finally, a gradient of phosphorylated α-synuclein aggregation in synapses (pre > pre + post > postsynaptic) was observed. These results indicate that phosphorylated α-synuclein is found at the presynaptic terminals of dementia with Lewy bodies cases mainly in the form of small phosphorylated α-synuclein aggregates that are associated with changes in synaptic morphology. Overall, our data support the notion that pathological phosphorylated α-synuclein may disrupt the structure and function of the synapse in dementia with Lewy bodies.

Tomato fruit carotenoid biosynthesis is adjusted to actual ripening progression by a light-dependent mechanism.

Carotenoids are isoprenoid compounds that are essential for plants to protect the photosynthetic apparatus against excess light. They also function as health-promoting natural pigments that provide colors to ripe fruit, promoting seed dispersal by animals. Work in Arabidopsis thaliana unveiled that transcription factors of the phytochrome-interacting factor (PIF) family regulate carotenoid gene expression in response to environmental signals (i.e. light and temperature), including those created when sunlight reflects from or passes though nearby vegetation or canopy (referred to as shade). Here we show that PIFs use a virtually identical mechanism to modulate carotenoid biosynthesis during fruit ripening in tomato (Solanum lycopersicum). However, instead of integrating environmental information, PIF-mediated signaling pathways appear to fulfill a completely new function in the fruit. As tomatoes ripen, they turn from green to red due to chlorophyll breakdown and carotenoid accumulation. When sunlight passes through the flesh of green fruit, a self-shading effect within the tissue maintains high levels of PIFs that directly repress the master gene of the fruit carotenoid pathway, preventing undue production of carotenoids. This effect is attenuated as chlorophyll degrades, causing degradation of PIF proteins and boosting carotenoid biosynthesis as ripening progresses. Thus, shade signaling components may have been co-opted in tomato fruit to provide information on the actual stage of ripening (based on the pigment profile of the fruit at each moment) and thus finely coordinate fruit color change. We show how this mechanism may be manipulated to obtain carotenoid-enriched fruits.

Multiphoton imaging with blue-diode-pumped SESAM-modelocked Ti:sapphire oscillator generating 5 nJ 82 fs pulses.

We report a directly blue diode pumped Ti:Sapphire oscillator that generates 5 nJ pulses. This is five times higher pulse energy than previously reported for a directly diode pumped Ti:sapphire laser. With 460 mW of average power at 92 MHz and 82 fs pulses, its peak power reaches 61 kW, also several times higher the value than previously published. Direct diode pumping significantly reduces the complexity and therefore the footprint and the cost of the laser, while SESAM modelocking ensures reliable selfstarting and robust operation. Such a laser is ideally suited for biomedical imaging and nanostructuring applications. As a demonstration of sufficient peak power for microscopy applications, we perform different modalities of nonlinear microscopy of biological samples.

Identification of Individual Exosome-Like Vesicles by Surface Enhanced Raman Spectroscopy.

Exosome-like vesicles (ELVs) are a novel class of biomarkers that are receiving a lot of attention for the detection of cancer at an early stage. In this study the feasibility of using a surface enhanced Raman spectroscopy (SERS) based method to distinguish between ELVs derived from different cellular origins is evaluated. A gold nanoparticle based shell is deposited on the surface of ELVs derived from cancerous and healthy cells, which enhances the Raman signal while maintaining a colloidal suspension of individual vesicles. This nanocoating allows the recording of SERS spectra from single vesicles. By using partial least squares discriminant analysis on the obtained spectra, vesicles from different origin can be distinguished, even when present in the same mixture. This proof-of-concept study paves the way for noninvasive (cancer) diagnostic tools based on exosomal SERS fingerprinting in combination with multivariate statistical analysis.

A transcriptome-proteome integrated network identifies endoplasmic reticulum thiol oxidoreductase (ERp57) as a hub that mediates bone metastasis.

Bone metastasis is the most common distant relapse in breast cancer. The identification of key proteins involved in the osteotropic phenotype would represent a major step toward the development of new prognostic markers and therapeutic improvements. The aim of this study was to characterize functional phenotypes that favor bone metastasis in human breast cancer. We used the human breast cancer cell line MDA-MB-231 and its osteotropic BO2 subclone to identify crucial proteins in bone metastatic growth. We identified 31 proteins, 15 underexpressed and 16 overexpressed, in BO2 cells compared with parental cells. We employed a network-modeling approach in which these 31 candidate proteins were prioritized with respect to their potential in metastasis formation, based on the topology of the protein-protein interaction network and differential expression. The protein-protein interaction network provided a framework to study the functional relationships between biological molecules by attributing functions to genes whose functions had not been characterized. The combination of expression profiles and protein interactions revealed an endoplasmic reticulum-thiol oxidoreductase, ERp57, functioning as a hub that retained four down-regulated nodes involved in antigen presentation associated with the human major histocompatibility complex class I molecules, including HLA-A, HLA-B, HLA-E, and HLA-F. Further analysis of the interaction network revealed an inverse correlation between ERp57 and vimentin, which influences cytoskeleton reorganization. Moreover, knockdown of ERp57 in BO2 cells confirmed its bone organ-specific prometastatic role. Altogether, ERp57 appears as a multifunctional chaperone that can regulate diverse biological processes to maintain the homeostasis of breast cancer cells and promote the development of bone metastasis.

Two-photon fluorescence imaging with 30 fs laser system tunable around 1 micron.

We developed a low-cost, low-noise, tunable, high-peak-power, ultrafast laser system based on a SESAM-modelocked, solid-state Yb tungstate laser plus spectral broadening via a microstructured fiber followed by pulse compression. The spectral selection, tuning, and pulse compression are performed with a simple prism compressor. The output pulses are tunable from 800 to 1250 nm, with the pulse duration down to 25 fs, and average output power up to 150 mW, at 80 MHz pulse repetition rate. We introduce the figure of merit (FOM) for the two-photon and multi-photon imaging (or other nonlinear processes), which is a useful guideline in discussions and for designing the lasers for an improved microscopy signal. Using a 40 MHz pulse repetition rate laser system, with twice lower FOM, we obtained high signal-to-noise ratio two-photon fluorescence images with or without averaging, of mouse intestine section and zebra fish embryo. The obtained images demonstrate that the developed system is capable of nonlinear (TPE, SHG) imaging in a multimodal operation. The system could be potentially used in a variety of other techniques including, THG, CARS and applications such as nanosurgery.

Dynamics of macrophage polarization support Salmonella persistence in a whole living organism.

Numerous intracellular bacterial pathogens interfere with macrophage function, including macrophage polarization, to establish a niche and persist. However, the spatiotemporal dynamics of macrophage polarization during infection within host remain to be investigated. Here, we implement a model of persistent Salmonella Typhimurium infection in zebrafish, which allows visualization of polarized macrophages and bacteria in real time at high resolution. While macrophages polarize toward M1-like phenotype to control early infection, during later stages, Salmonella persists inside non-inflammatory clustered macrophages. Transcriptomic profiling of macrophages showed a highly dynamic signature during infection characterized by a switch from pro-inflammatory to anti-inflammatory/pro-regenerative status and revealed a shift in adhesion program. In agreement with this specific adhesion signature, macrophage trajectory tracking identifies motionless macrophages as a permissive niche for persistent Salmonella. Our results demonstrate that zebrafish model provides a unique platform to explore, in a whole organism, the versatile nature of macrophage functional programs during bacterial acute and persistent infections.

Detection of capillary abnormalities in early diabetic retinopathy using scanning laser ophthalmoscopy and optical coherence tomography combined with adaptive optics.

This study tested if a high-resolution, multi-modal, multi-scale retinal imaging instrument can provide novel information about structural abnormalities in vivo. The study examined 11 patients with very mild to moderate non-proliferative diabetic retinopathy (NPDR) and 10 healthy subjects using fundus photography, optical coherence tomography (OCT), OCT angiography (OCTA), adaptive optics scanning laser ophthalmoscopy (AO-SLO), adaptive optics OCT and OCTA (AO-OCT(A)). Of 21 eyes of 11 patients, 11 had very mild NPDR, 8 had mild NPDR, 2 had moderate NPDR, and 1 had no retinopathy. Using AO-SLO, capillary looping, inflections and dilations were detected in 8 patients with very mild or mild NPDR, and microaneurysms containing hyperreflective granular elements were visible in 9 patients with mild or moderate NPDR. Most of the abnormalities were seen to be perfused in the corresponding OCTA scans while a few capillary loops appeared to be occluded or perfused at a non-detectable flow rate, possibly because of hypoperfusion. In one patient with moderate NPDR, non-perfused capillaries, also called ghost vessels, were identified by alignment of corresponding en face AO-OCT and AO-OCTA images. The combination of multiple non-invasive imaging methods could identify prominent microscopic abnormalities in diabetic retinopathy earlier and more detailed than conventional fundus imaging devices.

TANGO1 inhibitors reduce collagen secretion and limit tissue scarring.

Uncontrolled secretion of ECM proteins, such as collagen, can lead to excessive scarring and fibrosis and compromise tissue function. Despite the widespread occurrence of fibrotic diseases and scarring, effective therapies are lacking. A promising approach would be to limit the amount of collagen released from hyperactive fibroblasts. We have designed membrane permeant peptide inhibitors that specifically target the primary interface between TANGO1 and cTAGE5, an interaction that is required for collagen export from endoplasmic reticulum exit sites (ERES). Application of the peptide inhibitors leads to reduced TANGO1 and cTAGE5 protein levels and a corresponding inhibition in the secretion of several ECM components, including collagens. Peptide inhibitor treatment in zebrafish results in altered tissue architecture and reduced granulation tissue formation during cutaneous wound healing. The inhibitors reduce secretion of several ECM proteins, including collagens, fibrillin and fibronectin in human dermal fibroblasts and in cells obtained from patients with a generalized fibrotic disease (scleroderma). Taken together, targeted interference of the TANGO1-cTAGE5 binding interface could enable therapeutic modulation of ERES function in ECM hypersecretion, during wound healing and fibrotic processes.

Quantitative imaging of microtubule alteration as an early marker of axonal degeneration after ischemia in neurons.

Neuronal death can be preceded by progressive dysfunction of axons. Several pathological conditions such as ischemia can disrupt the neuronal cytoskeleton. Microtubules are basic structural components of the neuronal cytoskeleton that regulate axonal transport and neuronal function. Up-to-date, high-resolution observation of microtubules in living neuronal cells is usually accomplished using fluorescent-based microscopy techniques. However, this needs exogenous fluorescence markers to produce the required contrast. This is an invasive procedure that may interfere with the microtubule dynamics. In this work, we show, for the first time to our knowledge, that by using the endogenous (label-free) contrast provided by second harmonic generation (SHG) microscopy, it is possible to identify early molecular changes occurring in the microtubules of living neurons under ischemic conditions. This is done by measuring the intensity modulation of the SHG signal as a function of the angular rotation of the incident linearly polarized excitation light (technique referred to as PSHG). Our experiments were performed in microtubules from healthy control cultured cortical neurons and were compared to those upon application of several periods of oxygen and glucose deprivation (up to 120 min) causing ischemia. After 120-min oxygen and glucose deprivation, a change in the SHG response to the polarization was measured. Then, by using a three-dimensional PSHG biophysical model, we correlated this finding with the structural changes occurring in the microtubules under oxygen and glucose deprivation. To our knowledge, this is the first study performed in living neuronal cells that is based on direct imaging of axons and that provides the means of identifying the early symptoms of ischemia. Live observation of this process might bring new insights into understanding the dynamics and the mechanisms underlying neuronal degeneration or mechanisms of protection or regeneration.