Neuropathology

To detect brain atrophy, begin with slides carrying the deparaffinized brain sections from neurodegenerative mouse models at various ages.

To visualize these NPs, take a deparaffinized, fixed human brain section.

Add double-distilled water to wash the sections.

Treat the tissue with osmium tetroxide to stabilize the cell structures, including lipofuscin.

Homogenize the tissue to release proteins into the buffer. The protease inhibitors block protease activity, preventing protein degradation.

Add a loading buffer containing a detergent, a reducing agent, and a tracking dye.

Incubate with a proteolytic enzyme to digest the normal prions, leaving the resistant aggregates intact.

Incubate with hydrogen peroxide solution to inhibit endogenous peroxidases and prevent non-specific staining.

These sections contain neurofibrillary tangles—abnormal aggregates of tau protein found within hippocampal neurons.

To visualize heparan sulfate, begin with a diseased human hippocampal tissue section containing amyloid plaques.

Centrifuge the sample to pellet cellular debris and collect the supernatant.

This protocol outlines the extraction of proteins from neuronal cancer cells. The cells are washed with a buffer and lysed via enzymatic and mechanical disruption. The extracted protein-rich supernatant is collected after centrifugation.

This video demonstrates the procedure for fluorescently staining rat cortical neurons for microscopy. It targets F-actin and dendritic proteins to analyze structural integrity and calculate F-actin density, which is crucial for studying neuronal function and health.

This video demonstrates the protocol for isolating amyloid fibrils from mouse brain tissue extract using density gradient centrifugation and digestion of non-amyloid proteins.

This video outlines the generation of a neuronal injury model using reactive oxygen species (ROS) in mouse primary cerebellar granule neurons. It details the process of inducing oxidative stress by adding high concentrations of hydrogen peroxide. ROS exposure causes DNA strand breaks and genomic instability. Additionally, the oxidation of cellular proteins impairs their functions, while lipid peroxidation compromises membrane integrity. This cascade leads to cytochrome c release and caspase...

This video demonstrates a method to assess the neurotoxic effects of nanoparticles using microglia. It outlines the steps for activating microglia with nanoparticles, filtering conditioned media to isolate microglia-derived pro-inflammatory cytokines, and testing hypothalamic neuron toxicity with the conditioned media.

The video demonstrates inducing neurodegeneration in Drosophila larva segmental nerves via mechanical injury. The ventral cuticle of anesthetized larvae is pinched using forceps to cause injury to the segmental nerves. The injured larvae are maintained with food, allowing the injury to cause degeneration of the motor neurons in the nerves, causing disruption of their neuromuscular junctions (NMJs).

This video demonstrates a procedure to create a rat model of Parkinson's disease by injecting the neurotoxin 6-hydroxydopamine (6-OHDA) into the brain. The neurotoxin infusion into the medial forebrain bundle (MFB) specifically targets dopaminergic neurons responsible for motor control, leading to their destruction and replicating Parkinson's disease symptoms.

In this video, a stepwise protocol for Human embryonic stem cells into neural organoids is demonstrated. The method involves inducing neuronal rosette formation, followed by controlled maturation and final culturing on a hydrophobic membrane to achieve fully developed neural organoids.

This video demonstrates the differentiation of human embryonic stem cells (hESCs) into dopaminergic neurons, to provide a model for studying Parkinson's disease. The hESCs are cultured in a neural induction medium in a microwell plate to form 3D neurospheres. These are then cultured in a neural induction medium to promote the differentiation into dopaminergic progenitor neurons. The final maturation of the neurons occurs in a culture insert under air-liquid interface conditions, resulting in 3D...

This video demonstrates an in vitro assessment of neuronal growth cone collapse by amyloid-β (Aβ) aggregates. Treating the neurons with Aβ aggregates causes cytoskeletal destabilization, leading to the collapse of growth cones at the tip of the axons. The treated cells are compared with control cells to assess the extent of growth cone collapse.

The video demonstrates a procedure for preparing human cortical brain slices from Alzheimer's disease patients for analyzing the deposition of amyloid-beta (Aβ) peptides using Matrix-assisted Laser Desorption/Ionization Imaging Mass Spectrometry (MALDI-IMS). The steps include preparing the slides, treating the tissue to enhance ionization, and analyzing the spatial distribution of amyloid-beta peptides.

This video demonstrates the immunostaining of neurons treated with alpha-synuclein (αS) aggregates. The neurons are first treated with αS aggregates. They are then fixed, permeabilized, and treated with a blocking solution. Primary antibodies are added, followed by a fluorescently labeled secondary antibodies and a nuclear stain. The sample is then mounted and observed under a microscope.

This video demonstrates a procedure to induce retinal degeneration in adult zebrafish using laser injury. The laser targets photoreceptor cells in the retina, triggering cell death and a degenerative process. This damage activates Müller glia (MG) cells, leading to their dedifferentiation into stem cell progenitors that aid in retinal regeneration.

This video demonstrates the procedure of injecting monomeric amyloid peptides into the forebrain of adult zebrafish to generate amyloid toxicity, mimicking the neuronal damage seen in Alzheimer's...

This video demonstrates the protocol for measuring odorant receptor activation in transfected cells using a cyclic adenosine monophosphate (cAMP) biosensor and luminescence detection. The method is applied to study receptor-ligand interactions and signaling pathways in olfactory neurons.

This video demonstrates the procedure for injecting amyloid-beta peptides, aluminum chloride, and a neuroinflammatory growth factor, which collectively aids in toxic amyloid plaque formation in the hippocampus region. This causes memory impairment in the rat, characteristic of Alzheimer's disease.

This video demonstrates the precise stereotaxic infusion of amyloid beta into the dentate gyrus to establish a mouse model of Alzheimer's disease. Accurate coordinate determination and controlled delivery of amyloid beta proteins result in neuronal degradation, disrupting memory processing and contributing to Alzheimer's disease development.

This video demonstrates the injection of alpha-synuclein fibrils into a mouse to induce neuroinflammation. The fibrils are injected intraperitoneally into a transgenic mouse expressing mutated alpha-synuclein proteins. Upon reaching the central nervous system, the fibrils enter neurons and induce aggregation of the mutated proteins. The aggregates spread to neighboring neurons, microglia, and astrocytes. Recognition of the aggregates triggers microglia to induce neuroinflammation, damaging the...

This video demonstrates the procedure for in vivo imaging of C. elegans expressing prion-like proteins tagged with red fluorescent protein. The protocol involves immobilizing nematodes on an agarose pad for time-lapse confocal microscopy, revealing protein aggregation and transport within and between cells. This technique is crucial for studying protein aggregation related to neurodegenerative diseases.

This video demonstrates the process of injecting a viral vector into the rat substantia nigra to model Parkinson's disease. It outlines the steps for preparing the rat, microinjecting the viral vector into the substantia nigra, and the recovery procedure.

This video explains the process of isolating and enriching detergent-insoluble protein aggregates from frozen postmortem human brain tissue. It demonstrates key steps, including homogenization, detergent treatment, sonication, and ultracentrifugation to separate and purify the insoluble aggregates. The final step involves solubilizing the aggregates in urea buffer for further analysis.

This video demonstrates imaging and quantification of protein aggregates in genetically modified Drosophila brains. Brains are fixed, washed, and treated with an antifading agent before mounting. Images are captured using a confocal microscope and quantified to observe the transmission of mutant protein aggregates.

This video demonstrates a protocol for investigating the co-localization of orexin and cannabinoid receptors in normal and diet-induced obesity (DIO) adult zebrafish brain tissue using immunofluorescence.

This video demonstrates the process of quantifying alpha-synuclein accumulation in embryonic mouse dopamine neurons pre-treated with alpha-synuclein pre-formed fibrils. It outlines the steps for fixing, staining, and quantifying the dopamine neurons containing alpha-synuclein aggregates using immunofluorescence.

This video demonstrates how to measure proteasome activity in different subcellular compartments of the lateral amygdala from fear-conditioned and control rat brains using a fluorogenic peptide assay. The proteasome cleaves the fluorogenic peptide substrate, releasing fluorescent molecules, which are quantified using a plate reader to assess proteasome activity in the nuclear and synaptic protein fractions. Increased nuclear activity suggests changes in gene expression, while reduced synaptic...

This video demonstrates the process of inducing levodopa-induced dyskinesias in a Parkinson's rat model by administering levodopa alongside a dopamine conversion inhibitor. Repeated injections overstimulate the brain neurons, resulting in motor dysfunction.

This video demonstrates the induction of orofacial neuropathic pain in a rat model by ligating the infraorbital nerve. This results in nerve damage and inflammation, causing abnormal pain signaling and mimicking chronic pain conditions, which can be used to study the orofacial pain assay.

This video demonstrates the method of detecting aging neurons in cortical organoid sections by staining for beta-galactosidase activity, which is elevated in senescent cells. The enzyme cleaves a substrate to produce a blue product, followed by microscopic observation of the stained neurons.

This video demonstrates the generation of a focal demyelination mouse model using a two-point Lysophosphatidylcholine (LPC) injection. The skull of an anesthetized mouse is exposed. The injection coordinates are determined, and a hole is drilled to enable LPC injections at targeted sites. LPC induces focal demyelination of neurons via inflammation, while a two-point injection ensures its sustainability.

This video demonstrates the process of visualizing a biocytin-filled interneuron in a rat hippocampal section, highlighting its neurochemical identity through the use of specific antibodies and fluorescence microscopy.

This video demonstrates the immunohistochemical staining for S-100 protein in rectal suction biopsy sections. Antigen-retrieved sections are blocked and treated with primary, followed by secondary antibodies against S-100, a marker of Hirschsprung's disease. Chromogenic substrate is added to stain the targeted cells. Samples are dehydrated and mounted before visualizing under the microscope.

This video demonstrates the method for inducing demyelination in mouse spinal cord neurons using lysolecithin injections, followed by remyelination through the migration and differentiation of oligodendrocyte precursor cells. The process impairs and then gradually restores neuronal signal transmission.

This video demonstrates the process of transducing human neuronal cells with a lentivirus encoding a mutant human tau protein tagged with yellow fluorescent protein (YFP) to study protein aggregation. It outlines the steps from viral entry to mutant tau expression, culminating in the formation of visible cytoplasmic tau aggregates in the neurons.

This video demonstrates the labeling of neuronal cells with antibodies for mitochondrial respiratory chain complexes, mitochondrial DNA-associated protein, and mitochondrial outer membrane protein, followed by flow cytometry to quantify their levels.

This video demonstrates the procedure of measuring γ-secretase activity in genetically modified human cells using a tetracycline-inducible system. The process involves inducing APP gene expression with tetracycline, generating amyloid-β peptides, and activating luciferase production. The bioluminescence resulting from the luciferase-substrate reaction is then measured, reflecting γ-secretase activity.

This protocol outlines the use of immunohistochemistry to identify locus coeruleus regions in mouse brain sections. By targeting dopamine β-hydroxylase with green fluorophore-labeled antibodies, norepinephrine-producing neurons are visualized as green fluorescence under a microscope.

The video demonstrates the process of using a microdialysis probe to collect extracellular proteins from the interstitial fluid (ISF) of a mouse brain. A microdialysis probe, equipped with a high-molecular-weight cut-off semi-permeable membrane, is inserted into the brain of an anesthetized mouse via an implanted guide cannula. The probe, connected to pumps, allows a physiological buffer to perfuse the brain tissue and mix with the ISF. Proteins in the ISF diffuse across the semi-permeable...

This video demonstrates the method of inducing chronic stress in a mouse through repeated immobilization, leading to corticosterone release and reduced glutamate receptor activity in neurons, which results in depression-like behavior.

This video demonstrates the procedure to prepare and treat rat brain hemislices with a neurotoxic peptide to mimic neurodegeneration. The protocol allows for analyzing neurodegenerative events by directly comparing control and treated slices in the same anatomical plane.

This video demonstrates the use of transfected dorsal root ganglion cells to study neurotransmitter release by stimulating them with a neuropeptide receptor agonist.

This video demonstrates the immunohistochemical staining of cholinergic fibers in the nucleus basalis of the Meynert region of mouse brain sections. Sections are blocked and treated with primary against choline acetyltransferase, followed by biotinylated secondary antibodies. Avidin-biotin-peroxidase complexes are added, which bind to secondary antibodies, followed by the chromogenic substrate. Sections are then dehydrated and mounted before visualizing under the microscope.

This video demonstrates the assessment of dopamine uptake in synaptosomes harvested from mouse brains. It outlines steps for preparing synaptosomes for dopamine uptake via dopamine transporters, treating them with varying concentrations of radiolabeled dopamine, isolating the synaptosomes, and measuring radioactivity to quantify dopamine uptake.

This video demonstrates the procedure for staining fixed rat brain slices containing biocytin-filled interneurons. The protocol includes incubation with avidin-biotin complex (ABC), staining with HRP and chromogenic substrate, preserving structures with osmium tetroxide, and embedding the tissue in resin for imaging.

This video demonstrates the detection of serotonin receptor RNA synthesis using oligonucleotide probes in rat brain tissue affected by serotonin syndrome. The protocol includes tissue pretreatment, probe hybridization, and enzymatic detection to visualize and quantify serotonin receptor RNA expression under a microscope.

In this video, a mouse cerebellar brain slice was used to investigate synaptic interactions between parallel fibers, climbing fibers, and Purkinje cells, which are essential for motor control. Stimulation of climbing fibers induced long-term depression (LTD) in Purkinje cells, weakening synaptic strength and resulting in reduced response to parallel fiber stimulation.

This video demonstrates the immunofluorescence staining of a transgenic Drosophila brain for single-cell imaging of glial cells. The brain expresses membrane-targeted proteins fused with different combinations of antigenic epitopes under the control of a glial-specific recombinase system. The isolated and fixed brains are stained with primary antibodies specific to the antigenic epitopes, followed by fluorophore-conjugated secondary antibodies. This labeling enables the simultaneous imaging of...

This video demonstrates a stereotactically-guided surgical procedure for ablating the auditory cortex in a rat. The steps include identifying anatomical landmarks in an anesthetized rat secured in a stereotactic frame, creating a window on the skull above the auditory cortex, aspirating the cortical layers, and providing post-operative care to assess the impact of the ablation on sound processing.

In this video, we describe the procedure to perform fluorescence staining using fluorescence microscopy to demonstrate phalloidin staining of neural crest cells cultured on hydrogels of varying stiffness to visualize and analyze cytoskeletal organization.

This video demonstrates the crosslinking of neuronal surface proteins using Bis-sulfosuccinimidyl suberate or BS3. Isolated and chilled mouse brain is sectioned coronally. The desired region is extracted from the sections, minced, and treated with BS3. BS3 crosslinks surface proteins like GABA receptors, leaving internal proteins unaltered. The reaction is quenched with glycine before processing the samples for further analysis.

The video demonstrates a technique for measuring glucose uptake in Drosophila larval motor neurons using a fluorescence resonance energy transfer (FRET)-based intracellular glucose sensor. After dissecting the larvae to expose the ventral nerve cord, the motor neurons are visualized through the fluorescence emitted by the sensor's donor fluorophore. When stimulated with glucose, FRET between the sensor's donor and acceptor fluorophores triggers the emission of fluorescence from the acceptor,...

This video demonstrates the procedure for immunostaining a fixed mouse brain tissue section with fluorescently labeled astrocytes using primary and secondary antibodies and capture the images of the stained astrocytes through confocal microscopy.

This video demonstrates how to isolate Purkinje cells from a human post-mortem cerebellar tissue using laser capture microdissection.

This video demonstrates a detailed step-by-step protocol for preparing frozen mouse brain tissue sections for downstream analyses.

This video demonstrates the development of a pro-inflammatory and degenerative organ culture model for early-stage intervertebral disc (IVD) disease using TNF-α. It outlines the procedure for injecting TNF-α into the intervertebral disc and explains the mechanisms through which TNF-α induces inflammation and degradation of the IVD.

This video demonstrates the dissociation of frozen rat brain tissue into a single-cell suspension using enzymatic digestion and mechanical trituration. The prepared cells are fixed and permeabilized with ethanol for downstream applications like flow cytometry or gene expression analysis.

The video demonstrates the analysis of the palmitoylation state of cell membrane proteins isolated from mouse brain tissue. The process involves exposing the non-palmitoylated cysteines and blocking them, then using a polymer that binds to the palmitoylated cysteines, aiding in assessing the palmitoylation state of the isolated proteins.

This video demonstrates a method for fabricating polymer microbeads that encapsulate neurotoxic amyloid-beta-secreting cells. The cell-polymer suspension is processed through a microbead fabrication device, with polymer gelation achieved via calcium cross-linking, forming spherical microbeads around the cells. The polymer allows cell proliferation while allowing controlled, long-term amyloid-beta release, simulating its accumulation in Alzheimer's disease.

The video demonstrates a step-by-step procedure for symmetrically dissecting a fixed postmortem human brain, preparing smaller tissue blocks for further analysis.

This video demonstrates the procedure to assess mitochondrial calcium retention capacity by monitoring real-time changes in fluorescence as mitochondria uptake calcium. This assay is useful for studying mitochondrial function and calcium-related processes, including apoptosis and neurodegenerative diseases.

This video demonstrates the effect of continuous drug infusion into the mouse brain on blood glucose levels. Glucose metabolism is assessed by collecting blood from a fasted mouse's tail and measuring glucose levels before and after oral glucose administration. Insulin tolerance is evaluated by injecting insulin in the mouse, with glucose levels recorded at specific intervals.

In this video, an anesthetized rat undergoes an ocular procedure where magnetic beads are injected into the anterior chamber under a magnetic field to block the iridocorneal angle. This blockage obstructs the trabecular meshwork, preventing aqueous humor drainage and leading to increased intraocular pressure, damaging the retinal ganglion cells and optic nerve. This develops a glaucoma-like condition.

Source: Kummari, E., et. al., Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area., J. Vis. Exp. (2015)In this video, we demonstrate the immunohistochemistry protocol for dopamine neurons in brain tissue sections for subsequent laser capture microdissection.

Source: Bennett, S. A., et al., Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models. J. Vis. Exp. (2019).This video demonstrates the procedure of assessing histone modifications in yeast cells overexpressing neurodegenerative proteins. The steps include cell lysis, centrifugation, electrophoresis, membrane transfer, and antibody-based detection of modified histones.

Source: Yan, Z., et al. Evaluating the Anti-depression Effect of Xiaoyaosan on Chronically-stressed Mice. J. Vis. Exp. (2019).The video demonstrates the process of establishing a mouse model of chronic stress-induced depression and assessing depressive behaviors. The chronic application of various stressors triggers the release of corticosterone, leading to neuronal over-excitation and eventual neuronal death, which results in depression-like symptoms in the mice.

Source: Bétemps, D., et al., Detection of Disease-associated α-synuclein by Enhanced ELISA in the Brain of Transgenic Mice Overexpressing Human A53T Mutated α-synuclein. J. Vis. Exp. (2015).This video demonstrates the procedure to detect neurodegenerative disease-associated protein α-synuclein in brain tissue from transgenic mice using ELISA. The steps include tissue homogenization, centrifugation, and ELISA detection of α-synuclein protein to quantify its disease-associated forms.

Source: Wulf, M., et.al. Laser Microdissection-Based Protocol for the LC-MS/MS Analysis of the Proteomic Profile of Neuromelanin Granules. J. Vis. Exp. (2021).This video demonstrates the analysis of peptide samples from neuromelanin granules (NMGs) isolated from human postmortem brain tissue. The peptides are first separated using high-performance liquid chromatography (HPLC) and then analyzed by mass spectrometry to determine their mass-to-charge ratios (m/z). Targeted peptides are fragmented...

Source: Zhi, L. et al., Measurement of Oxygen Consumption Rate in Acute Striatal Slices from Adult Mice. J. Vis. Exp. (2022).This video demonstrates how to measure mitochondrial oxygen consumption rate (OCR) in mouse striatal brain slices. It details the steps for preparing the tissue plate and sensor cartridge, loading them into the flux analyzer, and explains the cellular processes underlying OCR patterns.

Source: Josiah, S. S., et al. Study of the Functions and Activities of Neuronal K-Cl Co-Transporter KCC2 Using Western Blotting. J. Vis. Exp. (2022).The video demonstrates the steps for detecting and quantifying phosphorylated potassium-chloride cotransporter 2 (KCC2) protein in a cell lysate. This process involves immunoprecipitation to isolate phosphorylated KCC2 and chemiluminescence to quantify the isolated protein.

Source: Lani-Louzada, R., et al. Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents. J. Vis. Exp. (2022).This video demonstrates the quantification of retinal ganglion cells from chemically fixed rat retinas subjected to full-circle cauterization of the limbal vascular plexus. The process includes permeabilization, antibody staining, and nuclear counterstaining, followed by visualization under an epifluorescence microscope to assess the impact of...

Source: Jensen, V. N., et al. Repeated Measurement of Respiratory Muscle Activity and Ventilation in Mouse Models of Neuromuscular Disease. J. Vis. Exp. (2017).This video demonstrates the method for recording simultaneous plethysmography and electromyography signals in a freely moving ALS mouse model. The procedure involves using implanted electrodes and a transmitter to capture muscle activity, alongside a plethysmography chamber to monitor breathing patterns, to investigate the role of...

Source: Jiang, X., et al. Inducement and evaluation of a murine model of experimental myopia. J. Vis. Exp. (2019)This video demonstrates a procedure for inducing myopia in a mouse through the installation of a custom lens frame. The frame is secured to the exposed skull with dental adhesive, and concave lenses are inserted to shift the focal point behind the retina. This setup stimulates eyeball elongation leading to myopia.

Source: Jolly, S., et.al. Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections. J. Vis. Exp. (2016).This video demonstrates the use of double fluorescence in situ hybridization to visualize the expression of two RNA targets in mouse brain sections. The protocol involves sequential detection of RNA using probes labeled with fluorescein isothiocyanate and digoxigenin, followed by enzymatic signal...

Source: Baleriola, J., et. al., Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization. J. Vis. Exp. (2015)This video demonstrates the detection of mRNA within cholinergic axons in fixed mouse hippocampal slices using fluorescent in situ hybridization and antibody staining. Fluorescence microscopy reveals the spatial localization of probe-tagged mRNAs and choline acetyltransferase in cholinergic neurons.

Source: Zhang, J., et al. High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses. J. Vis. Exp. (2016)This video demonstrates an electron microscopy technique for visualizing neurotransmitter receptor localization in retinal synapses. The retinal sections contain retinal ganglion cells that exhibit membrane-bound neurotransmitter receptors and are labeled with cholera toxin subunit B (CTB). Upon labeling the markers with gold nanoparticle-conjugated...

Source: de March, C. A. et. al., Real-time In Vitro Monitoring of Odorant Receptor Activation by an Odorant in the Vapor Phase. J. Vis. Exp. (2019)This video demonstrates the method to monitor the real-time activation of odorant receptors in recombinant mammalian cells using luminescence measurement. Odorant binding triggers G-protein activation and cAMP production, activating glosensor proteins that emit luminescence upon substrate binding, providing a quantitative measure of receptor response.

Source: Alluri, H., et al. Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction. J. Vis. Exp. (2015).The video demonstrates the fluorescent localization of tight junction proteins and stress fibers in rat brain microvascular endothelial cells (RBMECs). In the stressed monolayer, fluorescently labeled antibodies are used to tag the tight junction proteins, while fluorophore-tagged phalloidin labels the stress...

Source: Eid, L. & Parent, M. Preparation of Non-human Primate Brain Tissue for Pre-embedding Immunohistochemistry and Electron Microscopy. J. Vis. Exp. (2017).This video demonstrates the preparation of aldehyde-fixed non-human primate brain tissue using pre-embedding immunohistochemistry for electron microscopy. It outlines the steps for immunolabeling a specific neuronal protein, followed by a peroxidase reaction that produces a colored precipitate, enabling visualization of the target...

Source: Evilsizor, M. N., et. al., Primer for Immunohistochemistry on Cryosectioned Rat Brain Tissue: Example Staining for Microglia and Neurons. J. Vis. Exp. (2015)This video demonstrates a step-by-step immunohistochemistry staining procedure on cryosectioned rat brain tissue to visualize the spatial arrangement of microglia and neurons.

Source: Kirchner, K.N., et al., A Hydrophobic Tissue Clearing Method for Rat Brain Tissue. J. Vis. Exp. (2020).This video demonstrates the method of hydrophobic tissue clearing of a rat brain tissue sample. The sample is sequentially dehydrated in methanol, treated with a dichloromethane and methanol mixture to dissolve lipids and pigments, and transferred to dibenzyl ether for complete tissue clearing, enabling detailed imaging without thin sectioning.

To detect these bacteria, take a deparaffinized, rehydrated, bacteria-infected human brain section with exposed protein antigens and inactivated endogenous phosphatase.

The virus infects pyramidal neurons in the cerebral cortex, and expressed viral proteins localize within intracytoplasmic granular structures known as inclusion bodies.

Wash to remove the paraffin, then rehydrate the tissue using decreasing alcohol concentrations.

This video demonstrates the isolation of cell-surface and intracellular proteins from an astrocyte culture. The astrocyte culture is placed on ice to inhibit endocytosis, and then the cell-surface proteins are labeled with a biotinylation reagent. The cells are lysed to release the biotinylated cell-surface proteins and non-biotinylated intracellular proteins. Finally, streptavidin-coated beads are used to separate the biotinylated cell-surface proteins from the intracellular proteins.

This video demonstrates the intracerebroventricular injection process for studying murine cytomegalovirus (MCMV) distribution in the neonatal mouse brain. MCMV, engineered to express an enhanced green fluorescent protein (EGFP) for visibility, is injected into the lateral ventricle of a mouse pup's brain. The injected virus circulates through the cerebrospinal fluid, infecting cells in the periventricular region, including the marginal zone, choroid plexus, and subventricular zone.

This video demonstrates the establishment of an in vitro model of Zika virus infection in a developing brain. Human cerebral organoids containing rosettes of stem cells, neural progenitor cells (NPCs), and neurons are infected with the Zika virus. The virus infects NPCs and triggers apoptosis, disrupting organoid development.

The video demonstrates the microinjection of lipopolysaccharides (LPS) into the brains of zebrafish larvae to induce neuroinflammation. The larvae are positioned on an agarose-lined dish, the needle is inserted into the brain ventricles, and the LPS is injected. The injected LPS triggers an immune response that leads to neuroinflammation.

This video demonstrates the evaluation of mitochondrial health in genetically modified astrocytes, which express a fluorescent protein within their mitochondria. The protein helps to track changes in mitochondrial health as its fluorescence changes from green to red, indicating stress or damage. Images are captured and analyzed using various parameters.

This video demonstrates the procedure of injecting chimeric HIV viruses into the cortex of a rat's brain, where the virus RNA integrates into the host genome, replicates, and spreads to establish an active HIV infection in the experimental model.

This video demonstrates the generation of a mouse model with a CNS infection. A suckling pup is injected with the virus both intracranially and intraperitoneally. The virus spreads, infecting the CNS causing neuronal cell death and inflammation. The pup is assessed for symptoms of infection as the disease progresses.

This video demonstrates the generation of neuroinflammation in a mouse model. An anesthetized mouse with micro-wounds is taken, and an inoculum of pseudorabies virus (PRV) is applied to the wounds. The virus infects the peripheral nerves, spreads to the dorsal root ganglia (DRG), and further infects the spinal cord and brain. Infection of neurons in the nervous system leads to an immune response that induces neuronal damage, leading to neuroinflammation.

The video demonstrates intracerebral injection of Zika virus suspension in an adult mouse to induce neurodegeneration. An anesthetized mouse is secured in a stereotaxic frame, and a hole is drilled into the exposed skull for injection. A suspension of the Zika virus is injected into the brain tissue. The viral infection causes inflammation, leading to neurodegeneration.

This video demonstrates the quantification of the infective bacterial load from the perfused mouse brain infected with Listeria monocytogenes.

This video demonstrates a method to study permeation through an in vitro blood-brain barrier (BBB) model using astrocytes and endothelial cells on a membrane insert, with free FITC and FITC-loaded ferritin. FITC-ferritin bound to receptors on endothelial cells and crossed the membrane barrier to the lower chamber while free FITC showed restricted permeation and remained in the upper chamber.

This video demonstrates a procedure to infect human fetal neural stem cells with Zika virus. The infected cells can be used for further studies.

This video demonstrates an in vitro model to study pathogens crossing the blood-brain barrier (BBB). Microvascular endothelial cells, which express tight junctions, form an endothelial layer in the upper chamber, while neurons, astrocytes, and microglia represent the brain parenchyma in the lower chamber, separated by a porous membrane. Virus infection in the BBB model mimics the pathogen crossing from blood to the brain. Upon introducing a neurotropic virus, it crosses the BBB via...

Source: Funes, S., et. al., Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes. J. Vis. Exp. (2022)This video demonstrates an assay to study the phagocytic capacity of microglia-like cells using pH-sensitive fluorescent dye-labeled synaptosomes.

Immerse the tissue in xylene to dissolve the paraffin.

Intravenously inject fluorophore-tagged albumin, which crosses the damaged BBB, reaching the brain.

The tissue shows amyloid fibrils and abnormal protein aggregates in the cerebral vessel walls, which are key indicators of neurological disorders.

MCCH is characterized by reduced blood flow to the brain. This limits oxygen and nutrient supply to the endothelial cell layer of cerebral blood vessels, damaging the cells.

Suspend the cell mixture in an extracellular matrix or ECM solution.

Make a neck incision to expose and loosely ligate the bilateral common carotid arteries.

This video demonstrates the method of inducing experimental autoimmune encephalomyelitis (EAE) by injecting myelin-derived peptides with an adjuvant to activate autoreactive T cells, which migrate to the brain. Pertussis toxin is administered to increase blood-brain barrier permeability, allowing these T cells and other immune cells to enter the brain. These immune cells cause demyelination, leading to impaired nerve transmission and development of EAE.

This video demonstrates a procedure for assessing blood-brain barrier disruption in an EAE mouse model. Fluorescent dyes of different sizes are injected retro-orbitally and circulate through the brain's blood vessels. The smaller dye leaks through gaps into the brain tissue, while the larger dye enters only if the disruption is severe, correlating with the extent of barrier damage.

This video demonstrates a procedure to induce tissue damage or infarction in the pons of an anesthetized rat using electrical stimulation.

In this video, fertilized zebrafish embryos are dechorionated under a stereo microscope and then exposed to a cholesterol synthesis inhibitor, leading to the development of blood vessel ruptures and brain hemorrhages in the larvae. Hemorrhagic larvae, identifiable by distinct red spots, are subsequently separated under a microscope for further analysis.

This video demonstrates the process of inducing blood-brain barrier (BBB) disruption and cerebral microhemorrhages in a rat model using lipopolysaccharide (LPS). It highlights the sequence of immune responses that lead to increased BBB permeability and subsequent brain tissue damage.

This video demonstrates an assay to detect Cerebral microhemorrhages (CMHs) in a rat model. CMHs are brain bleeds due to blood-brain barrier disruption, identified by leaked red blood cells (RBCs). In a rat model, fixed brain tissue is cryoprotected, sectioned, and stained with hematoxylin-eosin for RBCs, allowing microscopic visualization of CMHs. Leaked RBCs appear as red-orange under the microscope.

The video demonstrates the procedure for creating a mouse model to study neonatal hypoxic-ischemic brain injury. It outlines the steps for surgically inducing ischemia and subjecting the animal to hypoxia to exacerbate brain damage. Finally, it shows how to assess the resulting brain lesions, ensuring the mouse model is prepared for further scientific investigation.

This video demonstrates how to characterize the extracellular space in a brain slice by using ion-selective and iontophoresis microelectrodes to measure and compare electrical signals. This process helps determine the space's volume fraction and degree of twisting.

In this video, a Multielectrode Array (MEA) coated with glutamate Oxidase is implanted into the brain tissue of an anesthetized mouse. Glutamate solution is injected into the extracellular space where the MEA detects the glutamate levels, enabling real-time monitoring of glutamate uptake by the astrocytes.

In this video, a genetically modified mouse pup carrying the Ccm2 gene flanked by loxP sites and Cre recombinase fused to an estrogen receptor is used to induce cerebral cavernous malformations (CCMs). The administration of 4-hydroxytamoxifen activates Cre in endothelial cells, leading to the deletion of Ccm2 and the formation of CCMs in the brain.

This video demonstrates the procedure for isolating brain cells and infiltrated immune cells from ischemic mouse brain tissues by enzymatic digestion, followed by the removal of cell debris and myelin by density gradient centrifugation, to study inflammation responses in stroke research.

The video demonstrates the induction of subarachnoid hemorrhage via endovascular perforation in a mouse model. An incision is made in the neck region of the anesthetized mouse to access the common carotid artery and its bifurcation. A filament is inserted through the arteries to perforate an intracranial artery branch, inducing subarachnoid hemorrhage.

This video demonstrates a method to generate a rat model of neonatal intraventricular hemorrhage. In this procedure, an anesthetized rat pup is injected with hemoglobin into the lateral ventricle of the brain. The hemoglobin causes oxidative stress and releases heme, simulating intraventricular hemorrhage. The resulting damage to brain tissues, driven by reactive oxygen species and inflammatory cytokines, leads to ventricular enlargement, a common consequence of intraventricular hemorrhage.

This video demonstrates the procedure of performing permanent distal middle cerebral artery occlusion, or MCAO, with the common carotid artery, or CCA ligation, in aged rats to model ischemic stroke. This blocks blood flow to the brain, leading to neuronal death and generating a model to study stroke mechanisms and potential treatments.

This video demonstrates the procedure for establishing a subarachnoid hemorrhage rat model. An anesthetized rat is implanted with probes to measure intracranial pressure and cerebral blood flow. The target site is identified, and the needle is inserted at a pre-chiasmatic position. Autologous blood is then injected into the subarachnoid space, increasing intracranial pressure and decreasing cerebral blood flow, establishing the model.

This video demonstrates an experimental investigation of lymphocyte transmigration across a blood-brain barrier (BBB) endothelium model. A monolayer of human brain microvascular endothelial cells (HBMECs) is generated in vitro, and peripheral blood mononuclear cells (PBMCs) containing lymphocytes are added to the monolayer. The cells are then incubated to allow lymphocyte transmigration, and the migrated cells are quantified using flow cytometry.

This video demonstrates the assessment of the infarct zone and brain edema in brain slices from rats subjected to middle cerebral artery occlusion. Infarcted areas are identified using redox staining, while a blue filter and threshold function in image analysis software quantify the infarct zone and edema in the affected hemisphere.

This video demonstrates a method for assessing transendothelial electrical resistance (TEER) in mouse brain microvascular endothelial cells in vitro. A high TEER value indicates intact endothelial cell junctions, while the addition of activated T cells lowers the TEER value, reflecting disruption of the junctional barrier.

This video demonstrates a procedure for renal artery sympathetic denervation in an angiotensin II-induced hypertensive mouse model, using phenol to disrupt sympathetic nerve function and reduce blood pressure.

This video demonstrates the process of inducing ischemic stroke in a rat model by occluding the middle cerebral artery (MCA) using a thread bolt. The procedure involves ligating the carotid arteries, inserting a thread bolt into the external carotid artery, and blocking blood flow from the MCA to the brain, leading to neuronal death and mimicking an ischemic stroke.

This video demonstrates the procedure of replicating a thrombotic stroke in a mouse model by blocking the right common carotid artery and exposing the mouse to a low-oxygen environment. This process reduces blood and oxygen flow to the brain, causing blood clot formation and brain damage.

This video demonstrates the precise technique for inducing a white matter stroke in an anesthetized mouse by injecting a nitric oxide inhibitor at targeted brain coordinates within the white matter. This leads to blood vessel constriction and inhibition of blood supply, resulting in neuronal damage and a brain stroke.

Source: Wanderer, S., et al. Arterial Pouch Microsurgical Bifurcation Aneurysm Model in the Rabbit. J. Vis. Exp. (2020)This video demonstrates the creation of an experimental aneurysm model in a rabbit using an elastase-treated vessel pouch. After excising a segment of the right CCA, the pouch is treated with elastase, then sutured to the left CCA to restore blood flow. The resulting stress on the weakened pouch walls induces aneurysm formation.

Source: Sharma, T. P., et al., Translaminar Autonomous System Model for the Modulation of Intraocular and Intracranial Pressure in Human Donor Posterior Segments. J. Vis. Exp. (2020).This video demonstrates the procedure of preparing a human eye to study the effects of translaminar pressure on retinal ganglion cell signaling by simulating intraocular and intracranial pressures in a translaminar autonomous system.

Source: Lani-Louzada, R., et al., Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents. J. Vis. Exp. (2022)This video demonstrates the induction and monitoring of elevated intraocular pressure (IOP) in a rat model. The process involves cauterizing the limbal blood vessels, resulting in vessel occlusion and elevated IOP. The rat is then allowed to recover, and IOP is regularly monitored in both the experimental and control eyes for comparison.

Source: Jungen, C., et al. Impact of Intracardiac Neurons on Cardiac Electrophysiology and Arrhythmogenesis in an Ex Vivo Langendorff System. J. Vis. Exp. (2018).The video demonstrates an ex vivo technique for stimulating the intracardiac nervous system to assess the heart’s susceptibility to arrhythmia. Using a perfusion apparatus, an isolated mouse heart is perfused with an oxygenated buffer. The intracardiac nervous system is then stimulated at a high frequency to induce arrhythmias.

Source: Hakim, M. A. et al., Isolation and Functional Analysis of Arteriolar Endothelium of Mouse Brain Parenchyma. J. Vis. Exp. (2022).This video demonstrates the functional analysis of an arteriolar endothelial tube isolated from a mouse brain. It outlines the steps for measuring intracellular calcium levels and membrane potential, both at baseline and in response to pharmacological stimulation.

Source: Tang, X. et. al., Massive Pontine Hemorrhage by Dual Injection of Autologous Blood. J. Vis. Exp. (2021)This protocol describes the controlled infusion of autologous blood into the pontine region of a rat's brain to model massive pontine hemorrhage. The purpose is to simulate a localized brain bleed by increasing localized pressure.

Source: Alluri, H., et.al. Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction. J. Vis. Exp. (2015).The video demonstrates the impact of oxygen-glucose deprivation and subsequent reoxygenation on rat brain microvascular endothelial cells (RBMECs) by simulating hypoxic and nutrient-deprived conditions. These conditions result in ATP depletion, increased intracellular calcium, and the generation of reactive...

Source: Gucciardo, E., et. al., An Ex Vivo Tissue Culture Model for Fibrovascular Complications in Proliferative Diabetic Retinopathy. J. Vis. Exp. (2019)This video details an immunostaining protocol for ex vivo cultures of fibrovascular tissue embedded in fibrin gel. This enables detailed visualization of vascular changes associated with proliferative diabetic...

Source: Talley Watts, L., et al. Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke. J. Vis. Exp. (2015)This video demonstrates a technique to induce blood clot formation in a mouse brain. An anesthetized mouse with a cranial window and a thinned skull undergoes photothrombosis through the activation of a photosensitive dye using high-intensity light. This process generates reactive oxygen species, which damage the vessel walls and trigger clot...

Source: Kuts, R. et al. Laser-Induced Brain Injury in the Motor Cortex of Rats. J. Vis. Exp. (2020)This video demonstrates assessing blood-brain barrier (BBB) integrity following laser-induced brain injury in rats. A tracking dye is injected into the tail vein, allowing it to circulate to the brain and leak into the tissue through the damaged BBB. The brain is then extracted and processed to release the dye. The fluorescence intensity of the dye is measured to quantify the extent of...

Astrocytes secrete signaling molecules that enhance the maturation and differentiation of stem cells into neurons and astrocytes.

This video shows a method to test interneuron migration in response to chemoattractive cues from periventricular endothelial cells (PVECs). Interneurons are placed in a central compartment, with PVECs and control cells on either side. Increased migration towards PVECs indicates a positive response to their chemoattractive signals.

This video demonstrates the immunofluorescence staining of cerebellar organoid slices to identify specific target antigens expressed in various cerebellar neurons, which are indicative of organoid maturation.

This video demonstrates the method of identifying neurons from a mixed culture of cerebellar granule neurons and glial cells using dual staining with fluorescein diacetate (FDA) and propidium iodide (PI), where live neurons appear green and dead neurons appear red.

This video demonstrates a detailed immunostaining protocol to visualize phenotypic defects in the mushroom bodies of mutant Drosophila brains. The protocol involves using antibodies against Fas2, a protein involved in axonal guidance that facilitates the proper morphological and functional development of the mushroom body.

This video demonstrates the procedure for staining newly proliferated neural stem cells in brain tissue sections using EdU incorporation, followed by immunofluorescence to visualize specific proteins, enhancing the understanding of cell dynamics and proliferation.

This video demonstrates the immunofluorescence staining of cryosections from mouse embryonic craniofacial tissues, using fluorescence microscopy to assess nuclear marker expression and determine cell proliferation and signaling activity.

This video demonstrates how neuronal vesicles are labeled with specific fluorescent proteins to visualize their movement. First, a plasmid coding for a fluorescent protein is incubated with a transfection agent and then introduced into a culture of neurons. Subsequently, images of the neurons are acquired and analyzed to track the vesicles' movement.

This video demonstrates the measurement of retinoic acid (RA) secretion from neurosphere cells using RA reporter cells. It outlines the steps involved in co-culturing neurosphere cells with RA reporter cells and performing a colorimetric RA assay to measure RA levels.

This video demonstrates the immunostaining of the embryonic murine gastrointestinal tract to visualize and analyze the neuronal population distribution using immunofluorescence and confocal microscopy.

This video demonstrates the viral labeling and transplantation of medial ganglionic eminence (MGE) cells for in vivo studies. It outlines the steps involved in transducing MGE cells with a viral reporter, transplanting these transduced cells into the cortex of a mouse pup, and enabling reporter expression in specific interneuron subtypes.

This video demonstrates the isolation of oxytocin receptor-rich nuclei from the neonatal rat brain. Coronal slicing, followed by identification and precise punching of oxytocin receptor-rich brain nuclei, enables the effective extraction of oxytocin receptor-rich nuclei for further analysis.

Source: Pericoli, G. et. al., Live-3D-Cell Immunocytochemistry Assays of Pediatric Diffuse Midline Glioma. J. Vis. Exp. (2021)This video demonstrates live cell imaging to observe pediatric diffuse midline glioma cell migration using immunohistochemical labeling with anti-CD44 antibodies.

Source: Jantzie, L. L., et. al. Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats. J. Vis. Exp. (2015)This video demonstrates a protocol to induce hypoxic-ischemic and inflammatory conditions in fetal rats by restricting uterine blood flow and injecting LPS, modeling neuroinflammation and disrupted brain development relevant to preterm birth studies.

Source: Larpthaveesarp, A., et. al. Transient Middle Cerebral Artery Occlusion Model of Neonatal Stroke in P10 Rats. J. Vis. Exp. (2017)This video demonstrates the establishment of the Middle Cerebral Artery Occlusion model in a rat pup. The internal carotid artery (ICA) is exposed, and a ligature is secured to restrict blood flood. An occlude is inserted through the ICA to reach the Middle Cerebral Artery. The pup is then allowed to recover before further analysis.

The disease triggers autoreactive immune cells to attack myelin proteins, degrading the myelin sheath surrounding neuronal axons.

The control section exhibits axons with tightly packed myelin sheaths containing concentric plasma membrane layers. The injured section exhibits decompacted myelin with large gaps.

Stress impairs oligodendrocyte function, disrupting the lipid-rich myelin sheath surrounding neuronal axons in the medial prefrontal cortex, a brain region regulating emotion.

This video demonstrates the immunoprecipitation of DNA fragments crosslinked with target protein from stem cell-derived motor neuron lysates using target protein-specific antibody-coated magnetic beads.

This video demonstrates a protocol for assessing the structural integrity of neuromuscular junctions in the dorsal longitudinal muscles of Drosophila. The procedure involves staining neurons and muscles with two distinct antibodies and comparing fluorescence patterns between mutant and wild-type flies. A reduction in neuronal staining in mutant flies indicates neurodegeneration, while the muscle structure remains intact.

This video demonstrates evaluating the neurodegenerative effects of a test chemical on Caenorhabditis elegans with fluorescently tagged neurons. Normal neurons show bright fluorescence of intact soma and processes, while diminished fluorescence, swollen soma, and punctuated processes indicate neurodegeneration.

This video demonstrates a method for establishing a small fiber neuropathy model in mice using the TRPV1 agonist resiniferatoxin (RTX). It outlines the steps involved in administering RTX to mice and explains how RTX induces neuronal death in small-diameter sensory neurons, thereby establishing the neuropathy model.

In this video, a mouse model of multiple sclerosis is established using experimental autoimmune encephalomyelitis (EAE). The mouse is injected with an emulsion containing a neuronal peptide and an adjuvant, triggering an immune response that activates autoreactive T cells. This is followed by an injection of pertussis toxin (PTX) to increase blood-brain barrier permeability, allowing the autoreactive T-cells to infiltrate the brain. This infiltration initiates a signaling cascade that...

This video demonstrates the procedure of inducing paralysis by transferring activated Th17 cells targeting aquaporin-4 into a mouse model. In the central nervous system, the activated Th17 cells interact with aquaporin-4-expressing astrocytes, triggering an immune response that leads to tail and limb paralysis in the mouse.

This video demonstrates the process of assessing motor dysfunction in a mouse model through the administration of Pertussis toxin and an inoculum containing myelin-mimicking peptides and adjuvants, followed by treadmill tests to assess motor function and evaluate the resulting nerve damage due to immune activation and demyelination.

This video demonstrates the staircase test to assess motor function in a hemiparkinsonian rat with a right brain lesion. The lesion disrupts dopamine signaling, impairing left paw control, which results in more pellets remaining on the left stairs, indicating motor dysfunction.

This video demonstrates the use of a radial locomotion assay to evaluate motor dysfunction in C. elegans expressing either wild-type or ALS-mutant human TDP-43 protein. Worms with varying levels of wild-type or mutant TDP-43 expression are allowed to crawl, and their movement is measured by the radial distance traveled from the central point on the assay plate. The assay distinguishes between mild, moderate, and severe motor impairments based on the distance traveled by the different C. elegans...

This video demonstrates assessing the muscle using cultured motor neurons, Schwann cells, and myotubes, followed by treatment with a neuromuscular blocker to inhibit contraction. Color-coded time-motion graphics show a color-coded time-motion graphic. Red indicates the fastest movement, and blue indicates the slowest.

This video demonstrates the visualization of protein-protein interactions at the Drosophila neuromuscular junction (NMJ) using Proximity Ligation Assay (PLA). After isolating the body walls, interacting proteins are probed with primary antibodies and PLA-conjugated secondary antibodies. The proximity of the interacting proteins facilitates the ligation of probes with connector oligonucleotides, forming circular DNA, which is then amplified by rolling circle amplification. Fluorescent probes...

Source: Wagner, K. et al., Studying Membrane Protein Trafficking in Drosophila Photoreceptor Cells Using eGFP-Tagged Proteins. J. Vis. Exp. (2022).This video demonstrates the visualization of membrane protein trafficking in Drosophila photoreceptor cells using eGFP-tagged TRPL proteins. It details the preparation of transgenic flies for imaging, the capture of fluorescence images of photoreceptor cells, and the quantification of TRPL trafficking by measuring relative eGFP fluorescence in the...

Source: Deseure, K., et al. Chronic Constriction Injury of the Rat's Infraorbital Nerve (IoN-CCI) to Study Trigeminal Neuropathic Pain. J. Vis. Exp. (2015).This video demonstrates a method to induce neuropathic pain in a rat model by constricting the infraorbital nerve. After exposing the infraorbital nerve and tying ligatures to constrict it, the immune response at the injury site increases the excitability of neighboring neurons, which transmit pain signals to the brain.

Source: Brakkee, E. M., et al. Surgery and Behavioral Testing in the Tibial Neuroma Transposition Model in Rats. J. Vis. Exp. (2023).This video demonstrates a procedure to generate a rat tibial neuroma transposition (TNT) model. After exposing the tibial nerve in the hind leg of an anesthetized rat, transect the nerve. Next, create a subcutaneous tunnel, thread the severed nerve through the tunnel, and suture it in place. In the absence of a target for reinnervation, disorganized axonal...

Source: Lee, D. et. al., Modified Experimental Conditions for Noise-Induced Hearing Loss in Mice and Assessment of Hearing Function and Outer Hair Cell Damage. J. Vis. Exp. (2023)This video demonstrates the procedure of noise-induced hearing loss in mice. High-volume sound damages hair cells in the inner ear and disrupts auditory pathways, resulting in hearing impairment.

Source: Lee, D., et. al. Modified Experimental Conditions for Noise-Induced Hearing Loss in Mice and Assessment of Hearing Function and Outer Hair Cell Damage. J. Vis. Exp. (2023)This video demonstrates the measurement of auditory brainstem response (ABR) in a mouse. Electrodes are placed on the head, behind the ear, and near the tail of an anesthetized mouse. Acoustic stimuli at various intensities are delivered, and electric signals generated are recorded as waveforms.

Source: Olmstead, D. N., et al., Facial Nerve Axotomy in Mice: A Model to Study Motoneuron Response to Injury. J. Vis. Exp. (2015).This video demonstrates the facial nerve crush procedure in a mouse, where blunt dissection through key landmarks exposes the facial nerve trunk at the stylomastoid foramen. Forceps are then used to compress the nerve trunk, inducing axonal damage and disrupting neuronal signal transmission.

Source: Cheah, M., et al. Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration. J. Vis. Exp. (2017).This video demonstrates a step-by-step procedure for injecting a viral suspension into the dorsal root ganglia (DRGs) and inducing a controlled axonal injury via a dorsal root crush in an anesthetized rat. The protocol involves exposing the DRGs, delivering a growth-promoting viral vector to facilitate axonal regeneration, and severing axonal...

To detect apoptotic cell death using the TUNEL assay, treat the brain slice with the enzyme proteinase-K.

Using fluorescence microscopy, visualize the injured nerve, which exhibits a progressive increase in axonal swellings and disconnections.

The drug encapsulation enhances tumor cell specificity and reduces neurotoxicity.

This video demonstrates the delivery of Traumatic Brain Injury (TBI) in mice. After exposing the skull, the TBI site is marked. The mouse is then positioned under the TBI device. The impact is delivered by lifting the metal rod and allowing it to drop freely onto the impactor, which is in contact with the skull.

This video demonstrates immunofluorescence staining of rat hippocampal neurons cultured within a microfluidic chip to analyze neuronal maturation and connectivity.

This video demonstrates the generation of a neuronal injury model in mouse primary cerebellar granule neurons. It outlines the steps involved in inducing neuronal injury using NMDA, an excitatory neurotransmitter receptor agonist, and glycine, a co-agonist. NMDA and glycine bind to specific neuronal receptors, causing their overstimulation and resulting in a massive intracellular calcium influx, which leads to neuronal cell damage and death.

This video demonstrates the immunofluorescence staining of a mouse brain slice to quantify the distribution of a specific presynaptic protein. The process involves blocking nonspecific sites, applying primary antibodies for the target protein and a reference marker, followed by secondary antibody labeling and nuclear counterstaining. The reference marker ensures accurate quantification by providing a consistent baseline, and the distribution of the presynaptic protein is analyzed across brain...

This video demonstrates a method to investigate zebrafish larval brain injury using large-scale scanning transmission electron microscopy (STEM), a technique that combines elements of both scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to produce high-resolution images. It outlines the steps involved in preparing samples and the acquisition of high-resolution STEM images to identify brain injury.

This video demonstrates a surgical protocol on a rat model, detailing steps from anesthesia preparation to facial nerve injury induction. This protocol is useful for studying nerve regeneration and surgical techniques in medical research.

This video demonstrates the procedure for inducing an open-skull traumatic brain injury in a mouse by making a midline skull incision, drilling through the skull and removing the bone flap to expose the brain cortex, and applying high pressure to induce trauma. The wound is then sutured to prepare the mouse model for subsequent experiments.

This video demonstrates the procedure for inducing brain death in a mouse model by increasing controlled intracranial pressure through a balloon catheter, mimicking natural brain death.

This video demonstrates a procedure to generate precise, controlled brain injury in the rat motor cortex using a laser.

This video demonstrates the biotinylation and fluorescent labeling of modified sialoglycoproteins in primary neural stem and progenitor cells, as well as neurons, and their visualization using fluorescence microscopy.

In this video, a shock tube device is used to generate a shockwave by rupturing a polymer diaphragm through controlled pressurization. The shockwave impacts brain slices contained in a secured bag, inducing traumatic injury to the brain slice.

This video demonstrates the procedure for preparing cultured neurons for optical analysis of recycled synaptic vesicles. The process involves fluorescence tagging of synaptic vesicle proteins and immunostaining to label and visualize the recycled vesicles within the neurons.

This video demonstrates the process of inducing stretch injury on human induced pluripotent stem cell-derived neurons (hiPSCNs). The cells are taken in a bottomless multi-well plate with a silicone membrane attached. Controlled mechanical stress is applied on the membrane to induce cytoskeletal disruption and cell membrane damage, inducing a stretch injury phenotype in the cells.

This video demonstrates a procedure to inflict a closed-head traumatic injury in anesthetized Drosophila flies using a strike device with a gas-propelled impactor. The injury affects neuronal function and impairs walking ability.

This video demonstrates the cortical stab injury procedure in mice, involving skull exposure and brain lesion creation. It induces reactive astrogliosis, a neuroprotective response of astrocytes, to study CNS trauma, inflammation, and repair mechanisms.

The video demonstrates a method to generate a mouse model of traumatic brain injury (TBI) using a lateral fluid percussion device (LFPI). Upon anesthetizing the mouse, the brain is exposed by creating a window on the skull. A saline-filled cannula is attached to the window and is then connected to the LFPI. The pendulum of the device is released to create a pressure pulse that inflicts a traumatic brain injury.

This video demonstrates the procedure for inducing traumatic brain injury (TBI) in a mouse model. An anesthetized mouse with an exposed skull is taken to create an opening in the skull, and then a controlled cortical injury is induced using an impact device. The injury damages the brain tissue, causing impaired cognitive function and motor coordination.

This video demonstrates the surgical procedure for performing a thoracic spinal cord hemisection in rats. It outlines the steps to expose the T8 spinal cord segment, perform the hemisection to disrupt neural tracts on one side, and provide post-operative care.

This video demonstrates a method for inducing targeted neuronal injury in Drosophila larvae using a high-power laser. After positioning the larva under a microscope, a low-power laser scans for fluorescent neurons, and then the laser power is increased to induce localized damage. The injury is confirmed by increased fluorescence and a crater at the injury site.

The video demonstrates the use of a High-Impact Trauma (HIT) device to induce closed-head traumatic brain injury (TBI) in Drosophila. TBI causes mechanical brain trauma affecting neuronal function, which over time leads to neurodegeneration and mortality.

This video demonstrates the induction of penetrating traumatic brain injury in the drosophila brain. Newly emerged flies are selected, anesthetized, and positioned under a fluorescence-equipped stereo microscope. The GFP-expressing mushroom body is identified, and a Minutien pin is inserted to cause injury. The flies are allowed to recover for further studies.

Source: Shandra, O. et al., Inducing Post-Traumatic Epilepsy in a Mouse Model of Repetitive Diffuse Traumatic Brain Injury. J. Vis. Exp.

This video demonstrates the induction of traumatic brain injury (TBI) in a rat using a lateral fluid percussion injury (FPI) device. The procedure involves creating a hole in the skull of an anesthetized rat to expose the dura mater, onto which a cannula is attached. The rat is then connected to the device, which generates a pressure wave that reaches the brain, mimicking TBI by causing neural cell death and vascular...

Source: Yang, Z., et.al. A Repetitive Concussive Head Injury Model in Mice. J. Vis. Exp. (2016).This video demonstrates the induction of repetitive concussive head injuries in a mouse using an electromagnetic impact device. The procedure involves positioning the impact tip between the interfrontal and lambdoid sutures on the skull of an anesthetized mouse and using a probe tip to precisely set the impact depth. The impact is delivered at a controlled velocity and depth to simulate mild...

Source: Logsdon, A. F., et al. Low-intensity Blast Wave Model for Preclinical Assessment of Closed-head Mild Traumatic Brain Injury in Rodents. J. Vis. Exp. (2020)This video demonstrates the induction of closed-head mild traumatic brain injury in a mouse model using a pressurized gas shock tube setup. The generated pressure wave ruptures the polyester membrane, delivering a shockwave to the mouse’s head that results in rapid brain movement within the skull, causing a mild traumatic brain injury.

Source: Macheda, T., et. al. Electromagnetic Controlled Closed-Head Model of Mild Traumatic Brain Injury in Mice. J. Vis. Exp. (2022)This video demonstrates the procedure to induce a closed-head injury in a mouse model using a stereotaxic frame. The controlled impact replicates mild traumatic brain injury, allowing researchers to study neurological effects and recovery by measuring post-injury reflexes and behavior.

Source: Elzat, E. Y. Y., et al., Establishing a Mouse Contusion Spinal Cord Injury Model Based on a Minimally Invasive Technique. J. Vis. Exp. (2022).This video demonstrates the method for inducing a contusion injury at the T9 level spinal cord in a mouse model, using a spinal cord injury coaxial platform. A weight is dropped on the T9 level spinal cord that creates a contusion that immediately causes tissue discoloration and swelling.

Source: Lakshman, N., et al. A Neurosphere Assay to Evaluate Endogenous Neural Stem Cell Activation in a Mouse Model of Minimal Spinal Cord Injury. J. Vis. Exp. (2018).This video demonstrates the activation and migration of neural stem cells (NSCs) in a mouse spinal cord injury model. To expose the spinal cord, a vertebra from the thoracic region of the spine is removed from an anesthetized mouse. A superficial injury is then created in the white matter of the spinal cord using a bent needle,...

Source: Kumar, S., et. al. Mouse Model of Pressure Ulcers After Spinal Cord Injury. J. Vis. Exp. (2019)This video demonstrates a detailed induction of spinal cord injury in mice. The anesthetized mouse is dissected to expose T9-T10 vertebrae, and their spinous processes are removed. The exposed spinal cord is then transected to inflict the injury. The surgical area is then closed, and the mouse is allowed to recover.

This video demonstrates a method to assess target protein internalization in the mouse cortical astrocytes using biotinylation, followed by cell lysis, streptavidin-based protein extraction, denaturation, and Western blot analysis to confirm successful internalization.

This video demonstrates a method to assess protein activity using hippocampal neurons over patterned protein zones. Neurons exhibit repulsion in test protein zones and attachment in control protein zones, highlighting the effects of attractive or repulsive protein activity.

This video demonstrates glaucoma induction in an anesthetized rat by injecting hypertonic saline into the episcleral vein. The saline enters the trabecular meshwork via Schlemm's canal, causing tissue scarring, which blocks aqueous humor outflow and raises intraocular pressure. The elevated pressure damages retinal ganglion cells, leading to glaucoma.

Homogenize the tissue with a homogenizer to release proteins, including glutamic acid decarboxylase, or GAD enzyme isoforms, essential for brain function.

This video demonstrates the procedure of surgically accessing the optic nerve in an anesthetized animal and precisely delivering lysolecithin to induce focal demyelination, creating neuropathological conditions for further study.

Incubate the slice in aCSF containing fluorescent false neurotransmitters or FFNs.

This video demonstrates the induction of handling-induced seizures in a mouse infected with Theiler's Murine Encephalomyelitis Virus (TMEV). The infected mouse, which has hyperexcitable neurons, is exposed to external physical stimuli. This exposure causes stress, leading to abnormal electrical activity in the neurons, which results in seizures.

This video demonstrates the procedure to induce heat-induced seizures in a transgenic mouse with epilepsy-related gene mutations by gradually increasing body temperature in a controlled chamber. This protocol is used to study seizure mechanisms and potential therapies for epilepsy.

This video demonstrates the induction of ictal events in coronal cortical slices from a transgenic mouse expressing light-activated cation channels in cortical excitatory pyramidal neurons. The slice is placed in a recording chamber, and a recording electrode is inserted into the superficial cortical layer. Upon perfusion with a seizure-inducing drug that blocks potassium channels, the neurons become hyperexcitable and generate spontaneous ictal events. To generate on-demand ictal events, a...

This video demonstrates the process of inducing epilepsy in mice using pentylenetetrazole (PTZ) injections, a GABA-A receptor antagonist. Repeated intraperitoneal administration of PTZ progressively lowers the seizure threshold by inhibiting chloride ion influx, disrupting inhibitory neurotransmission, and leading to hyperexcitability in neurons that trigger seizures. This process results in kindling and the development of spontaneous seizures, mimicking chronic epilepsy.

Source: Soukupová, M., et al. Microdialysis of Excitatory Amino Acids During EEG Recordings in Freely Moving Rats. J. Vis. Exp. (2018)This video demonstrates the assessment of extracellular excitatory neurotransmitter levels in rats with chronic epilepsy induction. After implanting a microdialysis probe into the brain and confirming the absence of seizures using electroencephalography (EEG), extracellular fluid is collected to evaluate elevated neurotransmitter levels in epileptic rats. A...

Source: Schönherr, S., et. al. Combined Optogenetic and Freeze-fracture Replica Immunolabeling to Examine Input-specific Arrangement of Glutamate Receptors in the Mouse Amygdala. J. Vis. Exp. (2016)This video demonstrates the immunolabelling of freeze-fractured replicas of mouse brain tissue for electron microscopy. The fractured and coated replicas are digested to expose receptors. Primary and gold-tagged secondary antibodies are added to label specific neuronal membrane receptors. The replica...

In this video, glioblastoma and microglia cells are mixed with a polymer matrix solution and transferred to a porous chamber insert where a gel forms, embedding the cells. Microglia stimulate the migration of glioblastoma cells through the porous insert, mimicking tumor cell invasion into surrounding tissue, Cells are then fixed and prepared for further analysis.

In this video, a method is demonstrated to enable the transfer of fluorescently labeled mitochondria into glioblastoma stem cells (GSCs) through centrifugation. The successful uptake and functional integration of the donor mitochondria are confirmed by detecting the fluoresce using confocal microscopy.

Source: Guyon, J., et al. A 3D Spheroid Model for Glioblastoma. J. Vis. Exp. (2020)The video demonstrates the generation of a three-dimensional spheroid model of tumor cell invasion. Spheroids are formed from human brain tumor cells and embedded in a collagen matrix. The tumor cells at the spheroid periphery adopt an invasive phenotype, secreting proteases to degrade the collagen and advancing to invade the surrounding matrix.

Source: Lim, M., et al. Modeling Brain Metastasis by Internal Carotid Artery Injection of Cancer Cells. J. Vis. Exp. (2022)In this video, an anesthetized mouse undergoes a surgical procedure in which cancer cells are injected into the common carotid artery (CCA) to facilitate targeted delivery to the brain. The procedure involves precise suturing and manipulation of the carotid arteries to direct the injected cells toward the internal carotid artery (ICA), promoting tumor formation in the brain...

Source: Ear, P. H., et. al. Establishment and Characterization of Small Bowel Neuroendocrine Tumor Spheroids. J. Vis. Exp. (2019)This video demonstrates the immunofluorescence staining of small bowel neuroendocrine tumor spheroids. The spheroids are fixed, permeabilized, and blocked and are then incubated with primary and secondary antibodies. A dye is added to stain nuclei before mounting and visualizing them under a fluorescent microscope.

Source: Mudduluru, G., et. al. Impedance-based Real-time Measurement of Cancer Cell Migration and Invasion. J. Vis. Exp. (2020)This video demonstrates an impedance-based assay to measure glioblastoma cell invasion and migration. Cells degrade the extracellular matrix, move through microporous membranes, and adhere to electrodes, generating impedance. This real-time analysis reflects their invasive potential for cancer research applications.

Source: Bolcaen, J., et. al., PET and MRI Guided Irradiation of a Glioblastoma Rat Model Using a Micro-irradiator. J. Vis. Exp. (2017)This video demonstrates the procedure for establishing a glioblastoma rat model by stereotactically injecting brain tumor-causing glioblastoma cells into the prefrontal cortex.

Source: Mendez, F. M. et al., Native Chromatin Immunoprecipitation Using Murine Brain Tumor Neurospheres. J. Vis. Exp. (2018).This video demonstrates the isolation of protein-DNA complexes from neurosphere cells derived from a mouse brain tumor using native chromatin immunoprecipitation. It details the steps for chromatin preparation, immunoprecipitation, washing, and protein digestion to release DNA for downstream analysis.

Source: Núñez, F. J., et al. Evaluation of Biomarkers in Glioma by Immunohistochemistry on Paraffin-Embedded 3D Glioma Neurosphere Cultures. J. Vis. Exp. (2019).This video demonstrates an immunohistochemical technique for labeling tumor biomarkers in paraffin-embedded brain tumor neurospheres. The neurosphere sections undergo paraffin removal and rehydration, followed by treatment to expose biomarker sites. Primary and secondary antibodies are then applied to label these biomarkers. The...