Name: sample preparation for endopeptidomic analysis in human cerebrospinal fluid
Uploaded: 2017-12-04T13:00:00
Description: Watch this Scientific Journal Video about Sample Preparation for Endopeptidomic Analysis in Human Cerebrospinal Fluid at JoVE.com

Many thanks to Tanveer Batth and colleagues for advice in setting up the pre-fractionation method.
This work was supported by funding from the Swedish Research Council, the Wallstr&#246;m and Sj&#246;blom Foundation, the Gun and Bertil Stohne Foundation Stiftelse, the Magnus Bergwall Foundation, the &#197;hl&#233;n Foundation, Alzheimerfonden, Demensf&#246;rbundet, Stiftelsen f&#246;r Gamla Tj&#228;narinnor, the Knut and Alice Wallenberg Foundation, Frimurarestiftelsen, and FoU-V&#228;stra G&#246;talandsregionen.
The main recipients of funding for this project were Kaj Blennow, Henrik Zetterberg and Johan Gobom.

&#160;The protocol described below is a refined version of the one used in a previous study where a large amount of endogenous peptides were identified in human CSF15. Updates to the original protocol involve minor alterations to the chemical pre-treatment of CSF as well as optimisation of the gradient used for offline high-pH RP HPLC pre-fractionation.
Ethical considerations
All studies of Swedish patient and control materia...

<ol>
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T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanding+the+cerebrospinal+fluid+endopeptidome.">Expanding the cerebrospinal fluid endopeptidome.</a> Proteomics. 17 (5), (2017).</li><li>Guldbrandsen, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-depth+characterization+of+the+cerebrospinal+fluid+(CSF)+proteome+displayed+through+the+CSF+proteome+resource+(CSF-PR).">In-depth characterization of the cerebrospinal fluid (CSF) proteome displayed through the CSF proteome resource (CSF-PR).</a> Mol Cell Proteomics. 13 (11), 3152-3163 (2014).</li><li>Kroksveen, A. C., Opsahl, J. A., Aye, T. T., Ulvik, R. J., Berven, F. 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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthogonality+of+separation+in+two-dimensional+liquid+chromatography.">Orthogonality of separation in two-dimensional liquid chromatography.</a> Anal Chem. 77 (19), 6426-6434 (2005).</li><li>Batth, T. S., Francavilla, C., Olsen, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Off-line+high-pH+reversed-phase+fractionation+for+in-depth+phosphoproteomics.">Off-line high-pH reversed-phase fractionation for in-depth phosphoproteomics.</a> J Proteome Res. 13 (12), 6176-6186 (2014).</li><li>Yang, F., Shen, Y., Camp, D. G., Smith, R. 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The introduction of an high-pH RP HPLC pre-fractionation step to a previously developed protocol for recovery of endogenous peptides by molecular weight ultrafiltration11 reduced relative sample complexity and thereby allowed for a 5-fold larger sample volume to be studied. This, in turn, increased the concentration of the subset of peptides present in each fraction and thereby improved the chances of detecting low abundant peptides.
By performing an identification stra...

Biomarkers in cerebrospinal fluid (CSF) are currently transforming research into neurodegenerative disorders. In Alzheimer&#39;s disease, the most common neurodegenerative disorder, affecting over 60 million people worldwide1,2, a biomarker triplet consisting of the peptide amyloid beta, microtubule-stabilizing protein tau, and a phosphorylated tau form, can detect the disease with high sensitivity and specificity, and has been included in the diagnostic research criteria3. In other neurodegenerative diseases, such as Parkinson&#39;s disease and Multiple Sclerosis, proteomic studies have identified numerous biomarker candidates, some of which are currently under evaluation in clinical studies4,5,6.
Alongside proteins, CSF also contains an abundance of endogenous peptides7,8,9,10,11,12. Constituting cleavage products of many brain-derived proteins, these peptides also represent a potentially important source of disease biomarkers. To increase the inventory of identified endogenous peptides in human CSF and enable CSF endopeptidomic analyses in clinical studies, a method was developed for sample preparation and LC-MS analysis (a brief protocol scheme has been included in Figure 1).The application of this method in a recent study resulted in the identification of nearly 16,400 endogenous CSF peptides in pooled CSF samples from several individuals of non-specific diagnosis, expanding the known CSF endopeptidome ten-fold13. The method can optionally be used in conjunction with isobaric labelling approach for quantification.
Sample Preparation
The main source of protein mass in CSF is plasma constituents (e.g. albumin and immunoglobulins) passing over the blood brain barrier14,15. Their high abundance hampers the detection of low-abundant, brain-derived sample components. Endogenous peptides can be readily separated from the high-abundant proteins, thereby allowing a significantly larger volume of CSF peptide extract to be used for LC-MS analysis, thereby enabling detection of lower-abundant peptides.
In the protocol presented here, molecular weight cut-off (MWCO) filtration was used to separate the CSF peptides from the protein fraction; a method that has been used in several previous studies8,9,10,11,12,16. The filtration step was followed by an offline RP HPLC pre-fractionation step performed over a high-pH mobile phase gradient. By performing two RP HPLC steps in tandem, with pH being the main distinction, the difference in selectivity between the two steps results mainly from altered peptide retention as a consequence of different peptide charge states. The application of high-pH peptide pre-fractionation prior to LC-MS under acidic conditions has proven efficient in increasing peptide identification17,18, and even to be superior for this purpose in complex biological samples compared to more orthogonal separation modes19, such as strong cat-ion exchange (SCX) and RP20. To shorten the analysis time, a concatenation scheme was used, pooling every 12th fraction (e.g., fractions 1, 13, 25, 37, and 49), which due to the high resolving power of RP HPLC still largely avoided co-elution of peptides from different fractions in the LC-MS step20,21.
Peptide identification
Peptide identification in peptidomic studies differs from that of proteomic studies in that no enzyme cleavage can be specified in the database search, and as a consequence, identification rates are usually lower11. A recent study13&#160;showed that the identification rates for endogenous peptides obtained with Sequest and Mascot were substantially improved when the default scoring algorithm of the respective software program was modified using the adaptive scoring algorithm Percolator, indicating that optimal scoring algorithms for endogenous peptides differ from that of tryptic peptides13. In that study, identification based on automatic peptide de novo sequencing using the software PEAKS (BSI) was found to be complementary to the two fragment ion fingerprinting-based search engines, resulting in a significantly larger set of identified peptides.

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	<tbody>
		<tr height="38">
			<td height="38" style="height:38px;width:547px;">&#160;1 M Triethylammonium bicarbonate</td>
			<td style="width:339px;">Fluka, Sigma-Aldrich</td>
			<td style="width:339px;">17902-100ML</td>
			<td style="width:339px;">TEAB</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">&#160;8 M Guanidinium hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>G7294-100ML</td>
			<td>GdnHCl</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:547px;">Tris(2-carboxyethyl)phosphine hydrochloride</td>
			<td>Pierce</td>
			<td>20490</td>
			<td>TCEP</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Iodoacetamide</td>
			<td>SIGMA</td>
			<td>I1149-5G</td>
			<td>IAA</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Hydroxylamine 50% (w/w)</td>
			<td>Sigma-Aldrich</td>
			<td>457804-50ML</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Acetonitrile, Far UV, HPLC gradient grade</td>
			<td>Sigma-Aldrich</td>
			<td>271004-2L</td>
			<td>AcN</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Formic acid</td>
			<td>Fluka, Sigma-Aldrich&#160;</td>
			<td>56302-1mL-F</td>
			<td>FA</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Triflouroacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>T6508-10AMP</td>
			<td>TFA</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Ammonium hydroxide solution</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>30501-1L-1M</td>
			<td>NH4OH</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-30 membrane</td>
			<td>Merck Millipore</td>
			<td>UFC903024</td>
			<td>MWCO-filter</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Sep-Pak C18, 100 mg</td>
			<td>Waters</td>
			<td>WAT023590</td>
			<td>SPE-column</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Resprep 12-port SPE Manifold&#160;</td>
			<td>Restek</td>
			<td>26077</td>
			<td>Vacuum manifold</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">TMT10plex Isobaric Label Reagent Set</td>
			<td>Thermo Fisher Scientific</td>
			<td>90110</td>
			<td>TMT10plex</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">UltiMate 3000 RSLCnano LC System</td>
			<td>Dionex</td>
			<td>5200.0356</td>
			<td>Online sample separation</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Ultimate 3000 RPLC Rapid Separation Binary System</td>
			<td>Dionex</td>
			<td>IQLAAAGABHFAPBMBEZ</td>
			<td>Offline high-pH fractionation</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Orbitrap Fusion Tribrid mass spectrometer</td>
			<td>Thermo Scientific</td>
			<td style="width:339px;">IQLAAEGAAPFADBMBCX</td>
			<td>Mass spectrometer for sample analysis</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Proteome Discoverer 2.0&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>IQLAAEGABSFAKJMAUH</td>
			<td>Proteomics search platform</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Mascot v2.4</td>
			<td>Matrix Science</td>
			<td style="width:339px;">&#160;-&#160;</td>
			<td>Proteomics search engine</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Sequest HT</td>
			<td>Thermo</td>
			<td style="width:339px;">&#160;-&#160;</td>
			<td>Proteomics search engine</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">PEAKS v7.5&#160;</td>
			<td>Bioinformatic Solutions Inc.)</td>
			<td style="width:339px;">&#160;-&#160;</td>
			<td>Proteomics search engine</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Acclaim PepMap 100, 75 &#181;m x 2 cm, C18, 100 &#197; pore size, 3 &#181;m particle size</td>
			<td>Thermo Fisher Scientific</td>
			<td>164535</td>
			<td>Trap column (nano HPLC)</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Acclaim PepMap C18, 75 &#181;m x 500 mm, 100&#197; pore size,&#160;2 &#181;m particle size</td>
			<td>Thermo Fisher Scientific</td>
			<td>164942</td>
			<td>Separation Column (nano HPLC)</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Savant SpeedVac High Capacity Concentrators</td>
			<td>Thermo Fisher Scientific</td>
			<td>SC210A-230</td>
			<td>SpeedVac/Vacuum concentrator</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">XBridge Peptide BEH C18 Column, 130&#197;, 3.5 &#181;m, 2.1 mm X 250 mm</td>
			<td>Waters</td>
			<td>186003566</td>
			<td>Separation Column (micro HPLC)</td>
		</tr>
	</tbody>
</table>

sample preparation for endopeptidomic analysis in human cerebrospinal fluid

This protocol describes a method developed to identify endogenous peptides in human cerebrospinal fluid (CSF). For this purpose, a previously developed method based on molecular weight cut-off (MWCO) filtration and mass spectrometric analysis was combined with an offline high-pH reverse phase HPLC pre-fractionation step.
Secretion into CSF is the main pathway for removal of molecules shed by cells of the central nervous system. Thus, many processes in the central nervous system are reflected in the CSF, rendering it a valuable diagnostic fluid. CSF has a complex composition, containing proteins that span a concentration range of 8 - 9 orders of magnitude. Besides proteins, previous studies have also demonstrated the presence of a large number of endogenous peptides. While less extensively studied than proteins, these may also hold potential interest as biomarkers.
Endogenous peptides were separated from the CSF protein content through MWCO filtration. By removing a majority of the protein content from the sample, it is possible to increase the sample volume studied and thereby also the total amount of the endogenous peptides. The complexity of the filtrated peptide mixture was addressed by including a reverse phase (RP) HPLC pre-fractionation step at alkaline pH prior to LC-MS analysis. The fractionation was combined with a simple concatenation scheme where 60 fractions were pooled into 12, analysis time consumption could thereby be reduced while still largely avoiding co-elution.
Automated peptide identification was performed by using three different peptide/protein identification software programs and subsequently combining the results. The different programs were complementary rather than comparable with less than 15% of the identifications overlapped between the three.

The method described here has been applied and evaluated in three studies prior to the introduction of sample pre-fractionation (Table 1). The first study used offline LC for spotting CSF fractions on a MALDI target plate and resulted in 730 identified endogenous peptides11. In the two following studies, isobaric labelling was employed. Primarily in a case/control study for identification and characterisation of potential biomarkers in the CSF endopeptidome and proteome simultaneously<sup class="x...

Watch this Scientific Journal Video about Sample Preparation for Endopeptidomic Analysis in Human Cerebrospinal Fluid at JoVE.com

Sample Preparation for Endopeptidomic Analysis in Human Cerebrospinal Fluid

A method for mass spectrometric analysis of endogenous peptides in human cerebrospinal fluid (CSF) is presented. By employing molecular weight cut-off filtration, chromatographic pre-fractionation, mass spectrometric analysis and a subsequent combination of peptide identification strategies, it was possible to expand the known CSF peptidome nearly ten-fold compared to previous studies.

This protocol describes a method developed to identify endogenous peptides in human cerebrospinal fluid (CSF). For this purpose, a previously developed ...

The goal of this protocol is to prepare and analyze human cerebrospinal fluid samples for LC-MS analysis of endogenous peptides.A peptide pre-fractionation step prior to LC-MS increases the number of identified peptides and enables detection of relatively low abundance CSF peptides.Endogenous peptides in CSF are calcium molecules that have not been extensively explored and may potentially include important new biomarkers of neurodegenerative disorders.This method enables extensive characterization of the CSF peptidome by mass spectrometry.The main advantages of this method are that it enables injection of low abundance CSF peptides and is compatible with multiplex isobaric labeling techniques, making it applicable for clinical studies.So far we have mainly applied this method to the analysis of cerebrospinal fluid.But we have preliminary data indicating that with minor modifications, it can also be used for analysis of plasma and tissue samples.Begin by thawing 1.5 milliliter aliquots of CSF at room temperature on roller mixer.Once thawed, transfer the contents to 10 milliliter polypropylene tubes.And add 80 microliters of one molar TAB as a buffering agent.Then add 0.65 milliliters of a eight molar guanidinium hydrochloride to dissolve protein aggregates and vortex gently at room temperature for 10 minutes.Next, at 60 microliters of 200 millimolar aqueous TCEP, and incubate at 55

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Neuroscience

Isolation of Cerebrospinal Fluid from Rodent Embryos for use with Dissected Cerebral Cortical Explants

The CSF is a complex fluid with a dynamically varying proteome throughout development and in adulthood. During embryonic development, the nascent CSF differentiates from the amniotic fluid upon closure of the anterior neural tube. CSF volume then increases over subsequent days as the neuroepithelial progenitor cells lining the ventricles and the choroid plexus generate CSF. The embryonic CSF contacts the apical, ventricular surface of the neural stem cells of the developing brain and spinal cord. CSF provides crucial fluid pressure for the expansion of the developing brain and distributes important growth promoting factors to neural progenitor cells in a temporally-specific manner. To investigate the function of the CSF, it is important to isolate pure samples of embryonic CSF without contamination from blood or the developing telencephalic tissue. Here, we describe a technique to isolate relatively pure samples of ventricular embryonic CSF that can be used for a wide range of experimental assays including mass spectrometry, protein electrophoresis, and cell and primary explant culture. We demonstrate how to dissect and culture cortical explants on porous polycarbonate membranes in order to grow developing cortical tissue with reduced volumes of media or CSF. With this method, experiments can be performed using CSF from varying ages or conditions to investigate the biological activity of the CSF proteome on target cells.

The ventricular cerebrospinal fluid (CSF) bathes the neuroepithelial and cerebral cortical progenitor cells during early brain development in the embryo. Here we describe the method developed to isolate ventricular CSF from rodent embryos of different ages in order to investigate its biological function. In addition, we demonstrate our cerebral cortical explant dissection and culture technique that allows for explant growth with minimal volumes of culture medium or CSF.

The CSF is a complex fluid with a dynamically varying proteome throughout development and in adulthood. During embryonic development, the nascent CSF ...

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other neurological diseases at an increasing speed. However, little is known about the molecular mechanisms that underlie these disorders. The genetic, molecular, and behavioral toolbox of Drosophila melanogaster makes this model organism particularly useful to characterize new disease genes and mechanisms in a high-throughput manner. Nevertheless, high-throughput screens require efficient and reliable assays that, ideally, are cost-effective and allow for the automatized quantification of traits relevant to these disorders. The island assay is a cost-effective and easily set-up method to evaluate Drosophila locomotor behavior. In this assay, flies are thrown onto a platform from a fixed height. This induces an innate motor response that enables the flies to escape from the platform within seconds. At present, quantitative analyses of filmed island assays are done manually, which is a laborious undertaking, particularly when performing large screens.
This manuscript describes the &#34;Drosophila Island Assay&#34; and &#34;Island Assay Analysis&#34; algorithms for&#160;high-throughput, automated data processing and quantification of island assay data. In the setup, a simple webcam connected to a laptop collects an image series of the platform while the assay is performed. The &#34;Drosophila Island Assay&#34; algorithm developed for the open-source software Fiji processes these image series and quantifies, for each experimental condition, the number of flies on the platform over time. The &#34;Island Assay Analysis&#34; script, compatible with the free software R, was developed to automatically process the obtained data and to calculate whether treatments/genotypes are statistically different. This greatly improves the efficiency of the island assay and makes it a powerful readout for basic locomotion and flight behavior. It can thus be applied to large screens investigating fly locomotor ability, Drosophila models of movement disorders, and drug efficacy.

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

The island assay is a relatively new, cost-effective assay that can be used to evaluate the basic locomotor behavior of Drosophila melanogaster. This manuscript describes algorithms for automatic data processing and objective quantification of island assay data, making this assay a sensitive, high-throughput readout for large genetic or pharmacological screens.

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other ...

An Improved Method for Collection of Cerebrospinal Fluid from Anesthetized Mice

The cerebrospinal fluid (CSF) is a valuable body fluid for analysis in neuroscience research. It is one of the fluids in closest contact with the central nervous system and thus, can be used to analyze the diseased state of the brain or spinal cord without directly accessing these tissues. However, in mice it is difficult to obtain from the cisterna magna due to its closeness to blood vessels, which often contaminate samples. The area for CSF collection in mice is also difficult to dissect to and often only small samples are obtained (maximum of 5-7 &#181;L or less). This protocol describes in detail a technique that improves on current methods of collection to minimize contamination from blood and allow for the abundant collection of CSF (on average 10-15 &#181;L can be collected). This technique can be used with other dissection methods for tissue collection from mice, as it does not impact any tissues during CSF extraction. Thus, the brain and spinal cord are not affected with this technique and remain intact. With greater CSF sample collection and purity, more analyses can be used with this fluid to further aid neuroscience research and better understand diseases affecting the brain and spinal cord.

This protocol describes an improved technique for the abundant collection of cerebrospinal fluid (CSF) with no contamination from blood. With greater sample collection and purity, more analyses can be performed using CSF to further our understanding of diseases that affect the brain and spinal cord.

The cerebrospinal fluid (CSF) is a valuable body fluid for analysis in neuroscience research. It is one of the fluids in closest contact with the central ...

In Vivo Imaging of Cerebrospinal Fluid Transport through the Intact Mouse Skull using Fluorescence Macroscopy

Cerebrospinal fluid (CSF) flow in rodents has largely been studied using ex vivo quantification of tracers. Techniques such as two-photon microscopy and magnetic resonance imaging (MRI) have enabled in vivo quantification of CSF flow but they are limited by reduced imaging volumes and low spatial resolution, respectively. Recent work has found that CSF enters the brain parenchyma through a network of perivascular spaces surrounding the pial and penetrating arteries of the rodent cortex. This perivascular entry of CSF is a primary driver of the glymphatic system, a pathway implicated in the clearance of toxic metabolic solutes (e.g., amyloid-&#946;). Here, we illustrate a new macroscopic imaging technique that allows real-time, mesoscopic imaging of fluorescent CSF tracers through the intact skull of live mice. This minimally-invasive method facilitates a multitude of experimental designs and enables single or repeated testing of CSF dynamics. Macroscopes have high spatial and temporal resolution and their large gantry and working distance allow for imaging while performing tasks on behavioral devices. This imaging approach has been validated using two-photon imaging and fluorescence measurements obtained from this technique strongly correlate with ex vivo fluorescence and quantification of radio-labeled tracers. In this protocol, we describe how transcranial macroscopic imaging can be used to evaluate glymphatic transport in live mice, offering an accessible alternative to more costly imaging modalities.

Transcranial optical imaging allows wide-field imaging of cerebrospinal fluid transport in the cortex of live mice through an intact skull.

Cerebrospinal fluid (CSF) flow in rodents has largely been studied using ex vivo quantification of tracers. Techniques such as two-photon microscopy and ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...

Cochlear Surface Preparation in the Adult Mouse

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous etiologies such as exposure to excessive noise, the use of ototoxic medications, bacterial or viral ear infections, head injuries, and the aging process. Loss of sensory hair cells is a common pathological feature of the varieties of acquired hearing loss. Additionally, the inner hair cell synapse can be damaged by mild insults. Therefore, surface preparations of cochlear epithelia, in combination with immunolabeling techniques and confocal imagery, are a very useful tool for the investigation of cochlear pathologies, including losses of ribbon synapses and sensory hair cells, changes in protein levels in hair cells and supporting cells, hair cell regeneration, and determination of report gene expression (i.e., GFP) for verification of successful transduction and identification of transduced cell types. The cochlea, a bony spiral-shaped structure in the inner ear, holds the auditory sensory end organ, the organ of Corti (OC). Sensory hair cells and surrounding supporting cells in the OC are contained in the cochlear duct and rest on the basilar membrane, organized in a tonotopic fashion with high-frequency detection occurring in the base and low-frequency in the apex. With the availability of molecular and genetic information and the ability to manipulate genes by knockout and knock-in techniques, mice have been widely used in biological research, including in hearing science. However, the adult mouse cochlea is miniscule, and the cochlear epithelium is encapsulated in a bony labyrinth, making microdissection difficult. Although dissection techniques have been developed and used in many laboratories, this modified microdissection method using cell and tissue adhesive is easier and more convenient. It can be used in all types of adult mouse cochleae following decalcification.

This article presents a modified cochlear surface preparation method that requires decalcification and use of a cell and tissue adhesive to adhere the pieces of cochlear epithelia to 10 mm round cover slips for immunohistochemistry in adult mouse cochleae.

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous ...

A Pilot Study on the Repetitive Transcranial Magnetic Stimulation of A&#946; and Tau Levels in Rhesus Monkey Cerebrospinal Fluid

Previous studies have demonstrated that a non-invasive light-flickering regime and auditory tone stimulation could affect A&#946; and tau metabolism in the brain. As a non-invasive technique, repetitive transcranial magnetic stimulation (rTMS) has been applied for the treatment of neurodegenerative disorders. This study explored the effects of rTMS on A&#946; and tau levels in rhesus monkey cerebrospinal fluid (CSF). This is a single-blind, self-controlled study. Three different frequencies (low frequency, 1 Hz; high frequencies, 20 Hz and 40 Hz) of rTMS were used to stimulate the bilateral-dorsolateral prefrontal cortex (DLPFC) of the rhesus monkey. A catheterization method was used to collect CSF. All samples were subjected to liquid chip detection to analyze CSF biomarkers (A&#946;42, A&#946;42/A&#946;40, tTau, pTau). CSF biomarker levels changed with time after stimulation by rTMS. After stimulation, the A&#946;42 level in CSF showed an upward trend at all frequencies (1 Hz, 20 Hz, and 40 Hz), with more significant differences for the high-frequencies (p &lt; 0.05) than for the low frequency. 
After high-frequency rTMS, the total Tau (tTau) level of CSF immediately increased at the post-rTMS timepoint (p &lt; 0.05) and gradually decreased by 24 h. Moreover, the results showed that the level of phosphorylated Tau (pTau) increased immediately after 40 Hz rTMS (p &lt; 0.05). The ratio of A&#946;42/A&#946;40 showed an upward trend at 1 Hz and 20 Hz (p &lt; 0.05). There was no significant difference in the tau levels with low-frequency (1 Hz) stimulation. Thus, high-frequencies (20 Hz and 40 Hz) of rTMS may have positive effects on A&#946; and tau levels in rhesus monkey CSF, while low-frequency (1 Hz) rTMS can only affect A&#946; levels.

A Pilot Study on the Repetitive Transcranial Magnetic Stimulation of A&amp;#946; and Tau Levels in Rhesus Monkey Cerebrospinal Fluid

Here, we describe the procedure for a pilot study to explore the effect of repetitive transcranial magnetic stimulation with different frequencies (1 Hz/20 Hz/40 Hz) on A&#946; and tau metabolism in rhesus monkey cerebrospinal fluid.

Previous studies have demonstrated that a non-invasive light-flickering regime and auditory tone stimulation could affect A&#946; and tau metabolism in ...

Comprehensive Understanding of Inactivity-Induced Gait Alteration in Rodents

It is well known that disuse affects neural systems and that joint motions become altered; however, which outcomes properly exhibit these characteristics is still unclear. The present study describes a motion analysis approach that utilizes three-dimensional (3D) reconstruction from video captures. Using this technology, disuse-evoked alterations of walking performances were observed in rodents exposed to a simulated microgravity environment by unloading their hindlimb by their tail. After 2 weeks of unloading, the rats walked on a treadmill, and their gait motions were captured with four charge-coupled device (CCD) cameras. 3D motion profiles were reconstructed and compared to those of control subjects using the image processing software. The reconstructed outcome measures successfully portrayed distinct aspects of distorted gait motion: hyperextension of the knee and ankle joints and higher position of the hip joints during the stance phase. Motion analysis is useful for several reasons. First, it enables quantitative behavioral evaluations instead of subjective observations (e.g., pass/fail in certain tasks). Second, multiple parameters can be extracted to fit specific needs once the fundamental datasets are obtained. Despite hurdles for broader application, the disadvantages of this method, including labor intensity and cost, may be alleviated by determining comprehensive measurements and experimental procedures.

The present protocol describes three-dimensional motion tracking/evaluation to depict gait motion alteration of rats after exposure to a simulated disuse environment.

It is well known that disuse affects neural systems and that joint motions become altered; however, which outcomes properly exhibit these characteristics ...

Cerebrospinal Fluid and Blood Withdrawal from Rats with Concurrent EEG Recording

Sampling Cerebrospinal Fluid and Blood from Lateral Tail Vein in Rats During EEG Recordings

Because the composition of body fluids reflects many physiological and pathological dynamics, biological liquid samples are commonly obtained in many experimental contexts to measure molecules of interest, such as hormones, growth factors, proteins, or small non-coding RNAs. A specific example is the sampling of biological liquids in the research of biomarkers for epilepsy. In these studies, it is desirable to compare the levels of molecules in cerebrospinal fluid (CSF) and in plasma, by withdrawing CSF and plasma in parallel and considering the time distance of the sampling from and to seizures. The combined CSF and plasma sampling, coupled with video-EEG monitoring in epileptic animals, is a promising approach for the validation of putative diagnostic and prognostic biomarkers. Here, a procedure of combined CSF withdrawal from cisterna magna and blood sampling from the lateral tail vein in epileptic rats that are continuously video-EEG monitored is described. This procedure offers significant advantages over other commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, and reduced time of anesthesia. Additionally, it can be used to obtain CSF and plasma samples in both tethered and telemetry EEG recorded rats, and it may be used repeatedly across multiple days of experiment. By minimizing the stress due to sampling by shortening isoflurane anesthesia, measures are expected to reflect more accurately the true levels of investigated molecules in biofluids. Depending on the availability of an appropriate analytical assay, this technique may be used to measure the levels of multiple, different molecules while performing EEG recording at the same time.

Author Spotlight: Obtaining High-Quality CSF and Blood Samples for Epilepsy Biomarker Discovery 

The protocol shows repeated cerebrospinal fluid and blood collections from epileptic rats performed in parallel with continuous video-electroencephalogram (EEG) monitoring. These are instrumental for exploring possible links between changes in various body fluid molecules and seizure activity.

Because the composition of body fluids reflects many physiological and pathological dynamics, biological liquid samples are commonly obtained in many ...

Optimizing Sample Preparation Process for Transmission Electron Microscopy of Neuromuscular Junctions in Drosophila Larvae

The Drosophila neuromuscular junction (NMJ) has emerged as a valuable model system in the field of neuroscience. The application of confocal microscopy at the Drosophila NMJ enables researchers to acquire synaptic information, encompassing both quantitative data on synapse abundance and detailed insights into their morphology. However, the diffuse distribution and limited visual range of the TEM present challenges for the ultrastructural analysis. This study introduces an innovative and efficient sample preparation method that surpasses the conventional approach. The procedure begins by placing a metal mesh at the base of a flat-bottomed bottle or test tube, followed by positioning fixed larvae samples onto the mesh. An additional mesh is placed over the samples, ensuring that they are positioned between the two meshes. The fixed samples are thoroughly dehydrated and infiltrated before proceeding with the embedding procedure. Then embedding of the samples in epoxy resin is performed in a flat sheet manner, which allows for the preparation of muscles for positioning and sectioning. Benefiting from these steps, all the muscles of Drosophila larvae can be visualized under light microscopy, therey facilitating subsequent positioning and sectioning. Excess resin is removed after locating the 6th and 7th muscles of body segments A2 and A3. Serial ultra-thin sectioning of the 6th or 7th muscle is performed.

Optimizing Sample Preparation Process for Transmission Electron Microscopy of Neuromuscular Junctions in Drosophila Larvae

This report provides a new sample preparation procedure for visualizing neuromuscular junctions in Drosophila Larvae. This method is more effective in preventing the curling of the samples compared to the traditional method and is particularly useful for Drosophila neuromuscular junction ultrastructural analysis.

The Drosophila neuromuscular junction (NMJ) has emerged as a valuable model system in the field of neuroscience. The application of confocal microscopy at ...