Protocols in Current Issue
Protocols in Past Issues
0 Q&A 403 Views Jul 5, 2024

Vascular cognitive impairment (VCI) is a syndrome defined as cognitive decline caused by vascular disease and is associated with various types of dementia. Chronic cerebral hypoperfusion (CCH) is one of the major contributors to VCI. Among the various rodent models used to study CCH-induced VCI, we have found the mouse bilateral common carotid artery stenosis (BCAS) model to be highly suitable. Here, we introduce the BCAS model of C57BL/6J mice generated using microcoils with an internal diameter of 0.18 mm. To produce the mouse BCAS model, the bilateral common carotid arteries are isolated from the adhering tissues and vagus nerves and twined around the microcoils. This model shows cognitive impairment and white matter lesions preceding neuronal dysfunction around postoperative day 28, which is similar to the human clinical picture. Overall, the mouse BCAS model will continue to be useful in studying CCH-induced VCI.

0 Q&A 1517 Views Feb 20, 2024

Astrocytes are increasingly recognized for their important role in neurodegenerative diseases like amyotrophic lateral sclerosis (ALS). In ALS, astrocytes shift from their primary function of providing neuronal homeostatic support towards a reactive and toxic role, which overall contributes to neuronal toxicity and cell death. Currently, our knowledge on these processes is incomplete, and time-efficient and reproducible model systems in a human context are therefore required to understand and therapeutically modulate the toxic astrocytic response for future treatment options. Here, we present an efficient and straightforward protocol to generate human induced pluripotent stem cell (hiPSC)-derived astrocytes implementing a differentiation scheme based on small molecules. Through an initial 25 days, hiPSCs are differentiated into astrocytes, which are matured for 4+ weeks. The hiPSC-derived astrocytes can be cryopreserved at every passage during differentiation and maturation. This provides convenient pauses in the protocol as well as cell banking opportunities, thereby limiting the need to continuously start from hiPSCs. The protocol has already proven valuable in ALS research but can be adapted to any desired research field where astrocytes are of interest.

Key features

• This protocol requires preexisting experience in hiPSC culturing for a successful outcome.

• The protocol relies on a small molecule differentiation scheme and an easy-to-follow methodology, which can be paused at several time points.

• The protocol generates >50 × 106 astrocytes per differentiation, which can be cryopreserved at every passage, ensuring a large-scale experimental output.

Graphical overview

0 Q&A 604 Views Feb 20, 2024

Dopaminergic (DAergic) neurodegeneration in the substantia nigra pars compacta of the human brain is the pathological feature associated with Parkinson’s disease (PD). Drosophila also exhibits mobility defects and diminished levels of brain dopamine on exposure to neurotoxicants mimicking PD. Our laboratory demonstrated in a Drosophila model of sporadic PD that there is no decrease in DAergic neuronal number; instead, there is a significant reduction in tyrosine hydroxylase (TH) fluorescence intensity (FI). Here, we present a sensitive assay based on the quantification of FI of the secondary antibody (ab). As the FI is directly proportional to the amount of TH synthesis, its reduction under PD conditions denotes the decrease in the TH synthesis, suggesting DAergic neuronal dysfunction. Therefore, FI quantification is a refined and sensitive method to understand the early stages of DAergic neurodegeneration. FI quantification is performed using the ZEN 2012 SP2 single-user software; a license must be acquired to utilize the imaging system to interactively control image acquisition, image processing, and analysis. This method will be of good use to biologists, as it can also be used with little modification to characterize the extent of degeneration and changes in the level of degeneration in response to drugs in different cell types. Unlike the expensive and cumbersome confocal microscopy, the present method will be an affordable option for fund-constrained neurobiology laboratories.

Key features

• Allows characterizing the incipient DAergic and other catecholaminergic neurodegeneration, even in the absence of loss of neuronal cell body.

• Great alternative for the fund-constrained neurobiology laboratories in developing countries to utilize this method in different cell types and their response to drugs/nutraceuticals.

Graphical overview

0 Q&A 452 Views Oct 5, 2023

Many single nucleotide polymorphisms (SNPs) identified by genome-wide association studies exert their effects on disease risk as expression quantitative trait loci (eQTL) via allele-specific expression (ASE). While databases for probing eQTLs in tissues from normal individuals exist, one may wish to ascertain eQTLs or ASE in specific tissues or disease-states not characterized in these databases. Here, we present a protocol to assess ASE of two possible target genes (GPNMB and KLHL7) of a known genome-wide association study (GWAS) Parkinson’s disease (PD) risk locus in postmortem human brain tissue from PD and neurologically normal individuals. This was done using a sequence of RNA isolation, cDNA library generation, enrichment for transcripts of interest using customizable cDNA capture probes, paired-end RNA sequencing, and subsequent analysis. This method provides increased sensitivity relative to traditional bulk RNAseq-based and a blueprint that can be extended to the study of other genes, tissues, and disease states.

Key features

• Analysis of GPNMB allele-specific expression (ASE) in brain lysates from cognitively normal controls (NC) and Parkinson’s disease (PD) individuals.

• Builds on the ASE protocol of Mayba et al. (2014) and extends application from cells to human tissue.

• Increased sensitivity by enrichment for desired transcript via RNA CaptureSeq (Mercer et al., 2014).

• Optimized for human brain lysates from cingulate gyrus, caudate nucleus, and cerebellum.

Graphical overview

0 Q&A 393 Views Jun 20, 2023

There are more than 40 types of spinocerebellar ataxia (SCA), most of which are caused by abnormal expansion of short tandem repeats at various gene loci. These phenotypically similar disorders require molecular testing at multiple loci by fluorescent PCR and capillary electrophoresis to identify the causative repeat expansion. We describe a simple strategy to screen for the more common SCA1, SCA2, and SCA3 by rapidly detecting the abnormal CAG repeat expansion at the ATXN1, ATXN2, and ATXN3 loci using melting curve analysis of triplet-primed PCR products. Each of the three separate assays employs a plasmid DNA carrying a known repeat size to generate a threshold melt peak temperature, which effectively distinguishes expansion-positive samples from those without a repeat expansion. Samples that are screened positive based on their melt peak profiles are subjected to capillary electrophoresis for repeat sizing and genotype confirmation. These screening assays are robust and provide accurate detection of the repeat expansion while eliminating the need for fluorescent PCR and capillary electrophoresis for every sample.

0 Q&A 637 Views May 5, 2023

Management of neuropathic pain is notoriously difficult; current analgesics, including anti-inflammatory- and opioid-based medications, are generally ineffective and can pose serious side effects. There is a need to uncover non-addictive and safe analgesics to combat neuropathic pain. Here, we describe the setup of a phenotypic screen whereby the expression of an algesic gene, Gch1, is targeted. GCH1 is the rate-limiting enzyme in the de novo synthesis of tetrahydrobiopterin (BH4), a metabolite linked to neuropathic pain in both animal models and in human chronic pain sufferers. Gch1 is induced in sensory neurons after nerve injury and its upregulation is responsible for increased BH4 levels. GCH1 protein has proven to be a difficult enzyme to pharmacologically target with small molecule inhibition. Thus, by establishing a platform to monitor and target induced Gch1 expression in individual injured dorsal root ganglion (DRG) neurons in vitro, we can screen for compounds that regulate its expression levels. This approach also allows us to gain valuable biological insights into the pathways and signals regulating GCH1 and BH4 levels upon nerve injury. This protocol is compatible with any transgenic reporter system in which the expression of an algesic gene (or multiple genes) can be monitored fluorescently. Such an approach can be scaled up for high-throughput compound screening and is amenable to transgenic mice as well as human stem cell–derived sensory neurons.

Graphical overview

0 Q&A 402 Views May 5, 2023

A basic function of the nervous system is to confer the ability to detect external stimuli and generate appropriate behavioral and physiological responses. These can be modulated when parallel streams of information are provided to the nervous system and neural activity is appropriately altered. The nematode Caenorhabditis elegans utilizes a simple and well characterized neural circuit to mediate avoidance or attraction responses to stimuli, such as the volatile odorant octanol or diacetyl (DA), respectively. Aging and neurodegeneration constitute two important factors altering the ability to detect external signals and, therefore, changing behavior. Here, we present a modified protocol to assess avoidance or attraction responses to diverse stimuli in healthy individuals and Caenorhabditis elegans models associated with neurodegenerative diseases.

0 Q&A 875 Views Apr 5, 2023

Microinflammation enhances the permeability of specific blood vessel sites through an elevation of local inflammatory mediators, such as interleukin (IL)-6 and tumor necrosis factor (TNF)-α. By a two-dimensional immunohistochemistry analysis of tissue sections from mice with experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS), we previously showed that pathogenic immune cells, including CD4+ T cells, specifically accumulate and cause microinflammation at the dorsal vessels of the fifth lumbar cord (L5), resulting in the onset of disease. However, usual pathological analyses by using immunohistochemistry on sections are not effective at identifying the microinflammation sites in organs. Here, we developed a new three-dimensional visualization method of microinflammation using luminescent gold nanoclusters (AuNCs) and the clear, unobstructed brain/body imaging cocktails and computational analysis (CUBIC) tissue-clearing method. Our protocol is based on the detection of leaked AuNCs from the blood vessels due to an enhanced vascular permeability caused by the microinflammation. When we injected ultrasmall coordinated Au13 nanoclusters intravenously (i.v.) to EAE mice, and then subjected the spinal cords to tissue clearing, we detected Au signals leaked from the blood vessels at L5 by light sheet microscopy, which enabled the visualization of complex tissue structures at the whole organ level, consistent with our previous report that microinflammation occurs specifically at this site. Our method will be useful to specify and track the stepwise development of microinflammation in whole organs that is triggered by the recruitment of pathogenic immune cells at specific blood vessels in various inflammatory diseases.

0 Q&A 727 Views Mar 20, 2023

The electroencephalogram (EEG) is a powerful tool for analyzing neural activity in various neurological disorders, both in animals and in humans. This technology has enabled researchers to record the brain’s abrupt changes in electrical activity with high resolution, thus facilitating efforts to understand the brain’s response to internal and external stimuli. The EEG signal acquired from implanted electrodes can be used to precisely study the spiking patterns that occur during abnormal neural discharges. These patterns can be analyzed in conjunction with behavioral observations and serve as an important means for accurate assessment and quantification of behavioral and electrographic seizures. Numerous algorithms have been developed for the automated quantification of EEG data; however, many of these algorithms were developed with outdated programming languages and require robust computational hardware to run effectively. Additionally, some of these programs require substantial computation time, reducing the relative benefits of automation. Thus, we sought to develop an automated EEG algorithm that was programmed using a familiar programming language (MATLAB), and that could run efficiently without extensive computational demands. This algorithm was developed to quantify interictal spikes and seizures in mice that were subjected to traumatic brain injury. Although the algorithm was designed to be fully automated, it can be operated manually, and all the parameters for EEG activity detection can be easily modified for broad data analysis. Additionally, the algorithm is capable of processing months of lengthy EEG datasets in the order of minutes to hours, reducing both analysis time and errors introduced through manual-based processing.

0 Q&A 618 Views Jan 20, 2023

Lysosomes play a central role in signaling, nutrient sensing, response to stress, and the degradation and recycling of cellular content. Defects in lysosomal digestive enzymes or structural components can impair lysosomal function with dire consequences to the cell, such as neurodegeneration. A number of methods exist to assess lysosomal stress in the model Drosophila, such as specific driver and reporter strains, transmission electron microscopy, and the investigation of gene expression. These methods, however, can be time consuming and, in some cases, costly. The procedure described here provides a quick, reliable, and low-cost approach to measure lysosomal stress in the Drosophila brain. Using fluorescence confocal microscopy and the LysoTracker staining, this protocol allows for the direct measurement of lysosome size and number. This method can be used to assess lysosomal stress under a number of different genetic and environmental scenarios in the Drosophila brain.

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