Neurite orientation dispersion and density imaging reveals abnormal white matter and glymphatic function in active young boxers.

The neurological effects and underlying pathophysiological mechanisms of sports-related concussion (SRC) in active young boxers remain poorly understood. This study aims to investigate the impairment of white matter microstructure and assess changes in glymphatic function following SRC by utilizing neurite orientation dispersion and density imaging (NODDI) on young boxers who have sustained SRC. A total of 60 young participants were recruited, including 30 boxers diagnosed with SRC and 30 healthy individuals engaging in regular exercise. The assessment of whole-brain white matter damage was conducted using diffusion metrics, while the evaluation of glymphatic function was performed through diffusion tensor imaging (DTI) analysis along the perivascular space (DTI-ALPS) index. A two-sample t-test was utilized to examine group differences in DTI and NODDI metrics. Spearman correlation and generalized linear mixed models were employed to investigate the relationship between clinical assessments of SRC and NODDI measurements. Significant alterations were observed in DTI and NODDI metrics among young boxers with SRC. Additionally, the DTI-ALPS index in the SRC group exhibited a significantly higher value than that of the control group (left side: 1.58 vs. 1.48, PFDR = 0.009; right side: 1.61 vs. 1.51, PFDR = 0.02). Moreover, it was observed that the DTI-ALPS index correlated with poorer cognitive test results among boxers in this study population. Repetitive SRC in active young boxers is associated with diffuse white matter injury and glymphatic dysfunction, highlighting the detrimental impact on brain health. These findings highlight the importance of long-term monitoring of the neurological health of boxers.

Neph1 is required for neurite branching and is negatively regulated by the PRRXL1 homeodomain factor in the developing spinal cord dorsal horn.

The cell-adhesion molecule NEPH1 is required for maintaining the structural integrity and function of the glomerulus in the kidneys. In the nervous system of Drosophila and C. elegans, it is involved in synaptogenesis and axon branching, which are essential for establishing functional circuits. In the mammalian nervous system, the expression regulation and function of Neph1 has barely been explored. In this study, we provide a spatiotemporal characterization of Neph1 expression in mouse dorsal root ganglia (DRGs) and spinal cord. After the neurogenic phase, Neph1 is broadly expressed in the DRGs and in their putative targets at the dorsal horn of the spinal cord, comprising both GABAergic and glutamatergic neurons. Interestingly, we found that PRRXL1, a homeodomain transcription factor that is required for proper establishment of the DRG-spinal cord circuit, prevents a premature expression of Neph1 in the superficial laminae of the dorsal spinal cord at E14.5, but has no regulatory effect on the DRGs or on either structure at E16.5. By chromatin immunoprecipitation analysis of the dorsal spinal cord, we identified four PRRXL1-bound regions within the Neph1 introns, suggesting that PRRXL1 directly regulates Neph1 transcription. We also showed that Neph1 is required for branching, especially at distal neurites. Together, our work showed that Prrxl1 prevents the early expression of Neph1 in the superficial dorsal horn, suggesting that Neph1 might function as a downstream effector gene for proper assembly of the DRG-spinal nociceptive circuit.

Shootin1 Regulates Retinal Ganglion Cell Neurite Development: Insights From an RGC Direct Somatic Cell Reprogramming Model.

Purpose: Retinal ganglion cells (RGCs) connect the retina to the brain. Proper development of the axons and dendrites of RGCs is the basis for these cells to function as projection neurons to deliver visual information to the brain. The purpose of this study was to investigate the function of Shtn1 (which encodes shootin1) in RGC neurite development. Methods: Immunofluorescence (IF) was used to characterize the expression pattern of marker genes. An in vitro direct somatic cell reprogramming system was used to generate RGC-like neurons (iRGCs), which was subsequently used to study the function of Shtn1. Short-hairpin RNAs (shRNAs) were used to knock down Shtn1, and the coding sequence (CDS) of Shtn1 was used to overexpress the gene. Lentiviruses were used to deliver shRNAs or CDSs into iRGCs. The patch clamp technique was used to measure the electrophysiological properties of the iRGCs. RNA sequencing (RNA-seq) was used to examine transcriptome expression. Results: Using IF, we demonstrated that shootin1 is distinctively expressed in RGCs during the period in which RGCs actively develop and adjust the connections of their neurites with upstream and downstream neurons. Using the iRGC system, we demonstrated that Shtn1 promotes the growth and complexity of neurites and thus the electrophysiological maturation, of iRGCs. RNA-seq analyses showed that Shtn1 may also regulate gene expression and neurogenesis in iRGCs. Conclusions: Shtn1 promotes RGC neurite development. These findings improve our understanding of the molecular machinery governing RGC neurite development and may help to optimize future RGC regeneration methods.

Microstructural associations between locus coeruleus, cortical, and subcortical regions are modulated by astrocyte reactivity: a 7T MRI adult lifespan study.

The locus coeruleus-norepinephrine system plays a key role in supporting brain health along the lifespan, notably through its modulatory effects on neuroinflammation. Using ultra-high field diffusion magnetic resonance imaging, we examined whether microstructural properties (neurite density index and orientation dispersion index) in the locus coeruleus were related to those in cortical and subcortical regions, and whether this was modulated by plasma glial fibrillary acidic protein levels, as a proxy of astrocyte reactivity. In our cohort of 60 healthy individuals (30 to 85 yr, 50% female), higher glial fibrillary acidic protein correlated with lower neurite density index in frontal cortical regions, the hippocampus, and the amygdala. Furthermore, under higher levels of glial fibrillary acidic protein (above ~ 150 pg/mL for cortical and ~ 145 pg/mL for subcortical regions), lower locus coeruleus orientation dispersion index was associated with lower orientation dispersion index in frontotemporal cortical regions and in subcortical regions. Interestingly, individuals with higher locus coeruleus orientation dispersion index exhibited higher orientation dispersion index in these (sub)cortical regions, despite having higher glial fibrillary acidic protein levels. Together, these results suggest that the interaction between locus coeruleus-norepinephrine cells and astrocytes can signal a detrimental or neuroprotective pathway for brain integrity and support the importance of maintaining locus coeruleus neuronal health in aging and in the prevention of age-related neurodegenerative diseases.

Automated Analysis of Neuronal Morphology in 2D Fluorescence Micrographs through an Unsupervised Semantic Segmentation of Neurons.

Brain function emerges from a highly complex network of specialized cells that are interlinked by billions of synapses. The synaptic connectivity between neurons is established between the elongated processes of their axons and dendrites or, together, neurites. To establish these connections, cellular neurites have to grow in highly specialized, cell-type dependent patterns covering extensive distances and connecting with thousands of other neurons. The outgrowth and branching of neurites are tightly controlled during development and are a commonly used functional readout of imaging in the neurosciences. Manual analysis of neuronal morphology from microscopy images, however, is very time intensive and prone to bias. Most automated analyses of neurons rely on reconstruction of the neuron as a whole without a semantic analysis of each neurite. A fully-automated classification of all neurites still remains unavailable in open-source software. Here we present a standalone, GUI-based software for batch-quantification of neuronal morphology in two-dimensional fluorescence micrographs of cultured neurons with minimal requirements for user interaction. Single neurons are first reconstructed into binarized images using a Hessian-based segmentation algorithm to detect thin neurite structures combined with intensity- and shape-based reconstruction of the cell body. Neurites are then classified into axon, dendrites and their branches of increasing order using a geodesic distance transform of the cell skeleton. The software was benchmarked against a published dataset and reproduced the phenotype observed after manual annotation. Our tool promises accelerated and improved morphometric studies of neuronal morphology by allowing for consistent and automated analysis of large datasets.

Neuritogenic glycosaminoglycan hydrogels promote functional recovery after severe traumatic brain injury.

Objective.Severe traumatic brain injury (sTBI) induced neuronal loss and brain atrophy contribute significantly to long-term disabilities. Brain extracellular matrix (ECM) associated chondroitin sulfate (CS) glycosaminoglycans promote neural stem cell (NSC) maintenance, and CS hydrogel implants have demonstrated the ability to enhance neuroprotection, in preclinical sTBI studies. However, the ability of neuritogenic chimeric peptide (CP) functionalized CS hydrogels in promoting functional recovery, after controlled cortical impact (CCI) and suction ablation (SA) induced sTBI, has not been previously demonstrated. We hypothesized that neuritogenic (CS)CP hydrogels will promote neuritogenesis of human NSCs, and accelerate brain tissue repair and functional recovery in sTBI rats.Approach.We synthesized chondroitin 4-Osulfate (CS-A)CP, and 4,6-O-sulfate (CS-E)CP hydrogels, using strain promoted azide-alkyne cycloaddition (SPAAC), to promote cell adhesion and neuritogenesis of human NSCs,in vitro; and assessed the ability of (CS-A)CP hydrogels in promoting tissue and functional repair, in a novel CCI-SA sTBI model,in vivo. Main results.Results indicated that (CS-E)CP hydrogels significantly enhanced human NSC aggregation and migration via focal adhesion kinase complexes, when compared to NSCs in (CS-A)CP hydrogels,in vitro. In contrast, NSCs encapsulated in (CS-A)CP hydrogels differentiated into neurons bearing longer neurites and showed greater spontaneous activity, when compared to those in (CS-E)CP hydrogels. The intracavitary implantation of (CS-A)CP hydrogels, acutely after CCI-SA-sTBI, prevented neuronal and axonal loss, as determined by immunohistochemical analyses. (CS-A)CP hydrogel implanted animals also demonstrated the significantly accelerated recovery of 'reach-to-grasp' function when compared to sTBI controls, over a period of 5-weeks.Significance.These findings demonstrate the neuritogenic and neuroprotective attributes of (CS)CP 'click' hydrogels, and open new avenues for the development of multifunctional glycomaterials that are functionalized with biorthogonal handles for sTBI repair.

Implication of thermal signaling in neuronal differentiation revealed by manipulation and measurement of intracellular temperature.

Neuronal differentiation-the development of neurons from neural stem cells-involves neurite outgrowth and is a key process during the development and regeneration of neural functions. In addition to various chemical signaling mechanisms, it has been suggested that thermal stimuli induce neuronal differentiation. However, the function of physiological subcellular thermogenesis during neuronal differentiation remains unknown. Here we create methods to manipulate and observe local intracellular temperature, and investigate the effects of noninvasive temperature changes on neuronal differentiation using neuron-like PC12 cells. Using quantitative heating with an infrared laser, we find an increase in local temperature (especially in the nucleus) facilitates neurite outgrowth. Intracellular thermometry reveals that neuronal differentiation is accompanied by intracellular thermogenesis associated with transcription and translation. Suppression of intracellular temperature increase during neuronal differentiation inhibits neurite outgrowth. Furthermore, spontaneous intracellular temperature elevation is involved in neurite outgrowth of primary mouse cortical neurons. These results offer a model for understanding neuronal differentiation induced by intracellular thermal signaling.

Recent advances in enhances peripheral nerve orientation: the synergy of micro or nano patterns with therapeutic tactics.

Several studies suggest that topographical patterns influence nerve cell fate. Efforts have been made to improve nerve cell functionality through this approach, focusing on therapeutic strategies that enhance nerve cell function and support structures. However, inadequate nerve cell orientation can impede long-term efficiency, affecting nerve tissue repair. Therefore, enhancing neurites/axons directional growth and cell orientation is crucial for better therapeutic outcomes, reducing nerve coiling, and ensuring accurate nerve fiber connections. Conflicting results exist regarding the effects of micro- or nano-patterns on nerve cell migration, directional growth, immunogenic response, and angiogenesis, complicating their clinical use. Nevertheless, advances in lithography, electrospinning, casting, and molding techniques to intentionally control the fate and neuronal cells orientation are being explored to rapidly and sustainably improve nerve tissue efficiency. It appears that this can be accomplished by combining micro- and nano-patterns with nanomaterials, biological gradients, and electrical stimulation. Despite promising outcomes, the unclear mechanism of action, the presence of growth cones in various directions, and the restriction of outcomes to morphological and functional nerve cell markers have presented challenges in utilizing this method. This review seeks to clarify how micro- or nano-patterns affect nerve cell morphology and function, highlighting the potential benefits of cell orientation, especially in combined approaches.

Advanced Hollow Cathode Discharge Plasma Treatment of Unique Bilayered Fibrous Nerve Guidance Conduits for Enhanced/Oriented Neurite Outgrowth.

Despite all recent progresses in nerve tissue engineering, critical-sized nerve defects are still extremely challenging to repair. Therefore, this study targets the bridging of critical nerve defects and promoting an oriented neuronal outgrowth by engineering innovative nerve guidance conduits (NGCs) synergistically possessing exclusive topographical, chemical, and mechanical cues. To do so, a mechanically adequate mixture of polycaprolactone (PCL) and polylactic-co-glycolic acid (PLGA) was first carefully selected as base material to electrospin nanofibrous NGCs simulating the extracellular matrix. The electrospinning process was performed using a newly designed 2-pole air gap collector that leads to a one-step deposition of seamless NGCs having a bilayered architecture with an inner wall composed of highly aligned fibers and an outer wall consisting of randomly oriented fibers. This architecture is envisaged to afford guidance cues for the extension of long neurites on the underlying inner fiber alignment and to concurrently provide a sufficient nutrient supply through the pores of the outer random fibers. The surface chemistry of the NGCs was then modified making use of a hollow cathode discharge (HCD) plasma reactor purposely designed to allow an effective penetration of the reactive species into the NGCs to eventually treat their inner wall. X-ray photoelectron spectroscopy (XPS) results have indeed revealed a successful O2 plasma modification of the inner wall that exhibited a significantly increased oxygen content (24 → 28%), which led to an enhanced surface wettability. The treatment increased the surface nanoroughness of the fibers forming the NGCs as a result of an etching effect. This effect reduced the ultimate tensile strength of the NGCs while preserving their high flexibility. Finally, pheochromocytoma (PC12) cells were cultured on the NGCs to monitor their ability to extend neurites which is the base of a good nerve regeneration. In addition to remarkably improved cell adhesion and proliferation on the plasma-treated NGCs, an outstanding neural differentiation occurred. In fact, PC12 cells seeded on the treated samples extended numerous long neurites eventually establishing a neural network-like morphology with an overall neurite direction following the alignment of the underlying fibers. Overall, PCL/PLGA NGCs electrospun using the 2-pole air gap collector and O2 plasma-treated using an HCD reactor are promising candidates toward a full repair of critical nerve damage.

Type I collagen concentration affects neurite outgrowth of adult rat DRG explants by altering mechanical properties of hydrogels.

Type I collagen is a predominant fibrous protein that makes up the extracellular matrix. Collagen enhances cell attachment and is commonly used in three-dimensional culture systems, to mimic the native extracellular environment, for primary sensory neurons such as dorsal root ganglia (DRG). However, the effects of collagen concentration on adult rat DRG neurite growth have not been assessed in a physiologically relevant, three-dimensional culture. This study focuses on the effects of type I collagen used in a methacrylated hyaluronic acid (MAHA)-laminin-collagen gel (triple gel) on primary adult rat DRG explants in vitro. DRGs were cultured in triple gels, and the neurite lengths and number of support cells were quantified. Increased collagen concentration significantly reduced neurite length but did not affect support cell counts. Mechanical properties, fiber diameter, diffusivity, and mesh size of the triple gels with varying collagen concentration were characterized to further understand the effects of type I collagen on hydrogel property that may affect adult rat DRG explants. Gel stiffness significantly increased as collagen concentration increased and is correlated to DRG neurite length. Collagen concentration also significantly impacted fiber diameter but there was no correlation with DRG neurite length. Increasing collagen concentration had no significant effect on mesh size and diffusivity of the hydrogel. These data suggest that increasing type I collagen minimizes adult rat DRG explant growth in vitro while raising gel stiffness. This knowledge can help develop more robust 3D culture platforms to study sensory neuron growth and design biomaterials for nerve regeneration applications.

A deep learning approach for automation in neurite tracing and cell size estimation from differential contrast images under healthy and hypoxic condition.

Chronic hypoxia is known to be a major cause of neurite length retraction followed be degeneration. Specifically, laser scanning confocal microscopy (LSCM) based-contrast imaging is used for monitoring neuronal morphology under hypoxic condition. Although imaging of neurons using LSCM via differential contrast imaging (DIC) is a powerful tool to identify the neuronal states under degenerative condition, fully automated quantification of neurite length and cell shape remains challenging. In this context, we propose an integrated framework that combines panorama imaging of neuronal morphology using LSCM, and deep learning model that allows automated tracing of neurites and cell shape. First, we establish an in vitro hypoxic model using cobalt chloride treatment of N2A cells and perform the large-scale imaging using DIC optics. Next, we tested the performance of U-Net, U-Net++ and FCN architecture using DIC images, where U-Net and U-Net++ demonstrates robustness and accuracy in tracing neurite length and segmentation of cell shape. The result shows that the U-Net++ is able to depict the difference in cell size and neurite length for control and chronic hypoxic condition. The proposed method was also validated and compared with other CNN models including FCN and U-Net. Moreover, the analysis indicates a significant alteration of cell shape and neurite length under hypoxic condition via deep-learning based automated cell segmentation.Clinical Relevance-The proposed framework assumes importance where quantification of neurite length and cell shape from a large dataset remains challenging due to time-consuming manual segmentation by experts. Specially, the framework based on labeling of a small dataset (15-20 images) can be used to identify the neuronal state under neurodegeneration and image-based assessment of neuroprotective drugs.

ANDA: an open-source tool for automated image analysis of in vitro neuronal cells.

BACKGROUND: Imaging of in vitro neuronal differentiation and measurements of cell morphologies have led to novel insights into neuronal development. Live-cell imaging techniques and large datasets of images have increased the demand for automated pipelines for quantitative analysis of neuronal morphological metrics. RESULTS: ANDA is an analysis workflow that quantifies various aspects of neuronal morphology from high-throughput live-cell imaging screens of in vitro neuronal cell types. This tool automates the analysis of neuronal cell numbers, neurite lengths and neurite attachment points. We used chicken, rat, mouse, and human in vitro models for neuronal differentiation and have demonstrated the accuracy, versatility, and efficiency of the tool. CONCLUSIONS: ANDA is an open-source tool that is easy to use and capable of automated processing from time-course measurements of neuronal cells. The strength of this pipeline is the capability to analyse high-throughput imaging screens.

Changes in Cortical Microstructure of the Human Brain Resulting from Long-Term Motor Learning.

The mechanisms subserving motor skill acquisition and learning in the intact human brain are not fully understood. Previous studies in animals have demonstrated a causal relationship between motor learning and structural rearrangements of synaptic connections, raising the question of whether neurite-specific changes are also observable in humans. Here, we use advanced diffusion magnetic resonance imaging (MRI), sensitive to dendritic and axonal processes, to investigate neuroplasticity in response to long-term motor learning. We recruited healthy male and female human participants (age range 19-29) who learned a challenging dynamic balancing task (DBT) over four consecutive weeks. Diffusion MRI signals were fitted using Neurite Orientation Dispersion and Density Imaging (NODDI), a theory-driven biophysical model of diffusion, yielding measures of tissue volume, neurite density and the organizational complexity of neurites. While NODDI indices were unchanged and reliable during the control period, neurite orientation dispersion increased significantly during the learning period mainly in primary sensorimotor, prefrontal, premotor, supplementary, and cingulate motor areas. Importantly, reorganization of cortical microstructure during the learning phase predicted concurrent behavioral changes, whereas there was no relationship between microstructural changes during the control phase and learning. Changes in neurite complexity were independent of alterations in tissue density, cortical thickness, and intracortical myelin. Our results are in line with the notion that structural modulation of neurites is a key mechanism supporting complex motor learning in humans.SIGNIFICANCE STATEMENT The structural correlates of motor learning in the human brain are not fully understood. Results from animal studies suggest that synaptic remodeling (e.g., reorganization of dendritic spines) in sensorimotor-related brain areas is a crucial mechanism for the formation of motor memory. Using state-of-the-art diffusion magnetic resonance imaging (MRI), we found a behaviorally relevant increase in the organizational complexity of neocortical microstructure, mainly in primary sensorimotor, prefrontal, premotor, supplementary, and cingulate motor regions, following training of a challenging dynamic balancing task (DBT). Follow-up analyses suggested structural modulation of synapses as a plausible mechanism driving this increase, while colocalized changes in cortical thickness, tissue density, and intracortical myelin could not be detected. These results advance our knowledge about the neurobiological basis of motor learning in humans.

Defective neurite elongation and branching in Nibp/Trappc9 deficient zebrafish and mice.

Loss of function in transport protein particles (TRAPP) links a new set of emerging genetic disorders called "TRAPPopathies". One such disorder is NIBP syndrome, characterized by microcephaly and intellectual disability, and caused by mutations of NIBP/TRAPPC9, a crucial and unique member of TRAPPII. To investigate the neural cellular/molecular mechanisms underlying microcephaly, we developed Nibp/Trappc9-deficient animal models using different techniques, including morpholino knockdown and CRISPR/Cas mutation in zebrafish and Cre/LoxP-mediated gene targeting in mice. Nibp/Trappc9 deficiency impaired the stability of the TRAPPII complex at actin filaments and microtubules of neurites and growth cones. This deficiency also impaired elongation and branching of neuronal dendrites and axons, without significant effects on neurite initiation or neural cell number/types in embryonic and adult brains. The positive correlation of TRAPPII stability and neurite elongation/branching suggests a potential role for TRAPPII in regulating neurite morphology. These results provide novel genetic/molecular evidence to define patients with a type of non-syndromic autosomal recessive intellectual disability and highlight the importance of developing therapeutic approaches targeting the TRAPPII complex to cure TRAPPopathies.

Co-culture system of human skin equivalents with mouse neural spheroids.

This study describes a co-culture system of human skin equivalents (HSEs) and dorsal root ganglion (DRG) neurons. We prepared spheroids of mouse DRG neurons with or without Schwann cells (SCs). Spheroids comprising DRG neurons and SCs showed longer neurite extensions than those comprising DRG neurons alone. Neurite extension of more than 1 mm was observed from spheroids cultured inside HSEs, whereas neurite extension was primarily observed on the surface of HSEs from spheroids cultured on HSEs. We propose that our model may be a useful tool for studying neurite extension in the human skin.

A mathematical study of the efficacy of possible negative feedback pathways involved in neuronal polarization.

Neuronal polarization, a process wherein nascent neurons develop a single long axon and multiple short dendrites, can occur within in vitro cell cultures without environmental cues. This is an apparently random process in which one of several short processes, called neurites, grows to become long, while the others remain short. In this study, we propose a minimum model for neurite growth, which involves bistability and random excitations reflecting actin waves. Positive feedback is needed to produce the bistability, while negative feedback is required to ensure that no more than one neurite wins the winner-takes-all contest. By applying the negative feedback to different aspects of the neurite growth process, we demonstrate that targeting the negative feedback to the excitation amplitude results in the most persistent polarization. Also, we demonstrate that there are optimal ranges of values for the neurite count, and for the excitation rate and amplitude that best maintain the polarization. Finally, we show that a previously published model for neuronal polarization based on competition for limited resources shares key features with our best-performing minimal model: bistability and negative feedback targeted to the size of random excitations.

Neuronal contact predicts connectivity in the C. elegans brain.

Axons must project to particular brain regions, contact adjacent neurons, and choose appropriate synaptic targets to form a nervous system. Multiple mechanisms have been proposed to explain synaptic partnership choice. In a "lock-and-key" mechanism, first proposed by Sperry's chemoaffinity model,1 a neuron selectively chooses a synaptic partner among several different, adjacent target cells, based on a specific molecular recognition code.2 Alternatively, Peters' rule posits that neurons indiscriminately form connections with other neuron types in their proximity; hence, neighborhood choice, determined by initial neuronal process outgrowth and position, is the main predictor of connectivity.3,4 However, whether Peters' rule plays an important role in synaptic wiring remains unresolved.5 To assess the nanoscale relationship between neuronal adjacency and connectivity, we evaluate the expansive set of C. elegans connectomes. We find that synaptic specificity can be accurately modeled as a process mediated by a neurite adjacency threshold and brain strata, offering strong support for Peters' rule as an organizational principle of C. elegans brain wiring.

Upside-Down Preference in the Forskolin-Induced In Vitro Differentiation of 50B11 Sensory Neurons: A Morphological Investigation by Label-Free Non-Linear Microscopy.

In this study, we revealed a peculiar morphological feature of 50B11 nociceptive sensory neurons in in vitro culture related to the forskolin-induced differentiation of these cells growing upside-down on cover glass supports. Multi-photon non-linear microscopy was applied to monitor increased neurite arborization and elongation. Under live and unstained conditions, second harmonic generation (SHG) microscopy could monitor microtubule organization inside the cells while also correlating with the detection of cellular multi-photon autofluorescence, probably derived from mitochondria metabolites. Although the differentiated cells of each compartment did not differ significantly in tubulin or multi-photon autofluorescence contents, the upturned neurons were more elongated, presenting a higher length/width cellular ratio and longer neurites, indicative of differentiated cells. SHG originating from the axons' microtubules represented a proper tool to study neurons' inverted culture in live conditions without exogenous staining. This work represents the first instance of examining neuronal cell lines growing and differentiated in an upside-down orientation, allowing a possible improvement of 50B11 as a model in physiology studies of sensory neurons in peripheric nervous system disease (e.g., Fabry disease, Friedreich ataxia, Charcot-Marie-Tooth, porphyria, type 1 diabetes, Guillain-Barré syndrome in children) and analgesic drug screening.

Neurite growth induced by red light-caused intracellular reactive oxygen species production through cytochrome c oxidase activation.

The applications of red-light photobiomodulation (PBM) to enhance neurite growth have been proposed for many years. However, the detailed mechanisms require further studies. In the present work we used a focused red-light spot to illuminate the junction of the longest neurite and the soma of a neuroblastoma cell (N2a), and demonstrated enhanced neurite growth at 620 nm and 760 nm with adequate illumination energy fluences. In contrast, 680 nm light showed no effect on neurite growth. The neurite growth was accompanied with the increase of intracellular reactive oxygen species (ROS). Using Trolox to reduce the ROS level, this red light-induced neurite growth was hindered. Suppressing the activities of cytochrome c oxidase (CCO) by using either a small-molecule inhibitor or siRNA abrogated the red light-induced neurite growth. These results suggest that red light-induced ROS production through the activation of CCO could be beneficial for neurite growth.