Name: in vivo microdialysis method to collect large extracellular proteins from brain interstitial fluid with high-molecular weight cut-off probes
Uploaded: 2018-09-26T12:30:00
Description: Watch this Scientific Journal Video about In Vivo Microdialysis Method to Collect Large Extracellular Proteins from Brain Interstitial Fluid with High-molecular Weight Cut-off Probes at JoVE.com

This work was supported by &#39;&#39;Grant-in-Aid for Scientific Research on Innovative Areas (Brain Protein Aging and Dementia Control)(15H01552) from MEXT and Grant-in-Aid for Young Scientists (B) (16K20969). The author thanks Dr. David M. Holtzman and Dr. John R. Cirrito for the technical advices during the development of this method.

All animal studies were reviewed and approved by the Institutional Animal Care and Use Committee of the Graduate School of Medicine at the University of Tokyo.
1. Pre-surgical Procedure
<ol>
	<li>Before starting surgery, wipe everything with 70% ethanol to maintain sterile conditions.&#160;Thermal support using a heating pad is recommended.</li>
	<li>Anesthetize the mice by intraperitoneal injection of chloral hydrate (400 mg/kg). Confirm anesthetization by performing a toe pinch.&#160;Use o...

<ol>
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D., 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+vivo+measurement+of+apolipoprotein+E+from+the+brain+interstitial+fluid+using+microdialysis.">In vivo measurement of apolipoprotein E from the brain interstitial fluid using microdialysis.</a> Molecular Neurodegeneration. 8 (1), 13 (2013).</li><li>Emmanouilidou, E., 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=GABA+transmission+via+ATP-dependent+K++channels+regulates+&#945;-synuclein+secretion+in+mouse+striatum.">GABA transmission via ATP-dependent K+ channels regulates &#945;-synuclein secretion in mouse striatum.</a> Brain. 139 (3), 871-890 (2016).</li><li>Takeda, S., 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=Seed-competent+high-molecular-weight+tau+species+accumulates+in+the+cerebrospinal+fluid+of+Alzheimer's+disease+mouse+model+and+human+patients.">Seed-competent high-molecular-weight tau species accumulates in the cerebrospinal fluid of Alzheimer's disease mouse model and human patients.</a> Annals of Neurology. 80 (3), 355-367 (2016).</li><li>Yamada, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+Microdialysis+of+brain+interstitial+fluid+for+the+determination+of+extracellular+tau+levels.">In vivo Microdialysis of brain interstitial fluid for the determination of extracellular tau levels.</a> Methods in Molecular Biology. 1523, 285-296 (2017).</li><li>Kang, J. -. 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W., 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=Neuronal+activity+regulates+the+regional+vulnerability+to+amyloid-&#946;+deposition.">Neuronal activity regulates the regional vulnerability to amyloid-&#946; deposition.</a> Nature Neuroscience. 14 (6), 750-756 (2011).</li><li>Yamada, K., 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=Neuronal+activity+regulates+extracellular+tau+in+vivo.">Neuronal activity regulates extracellular tau in vivo.</a> Journal of Experimental Medicine. 211 (3), 387-393 (2014).</li><li>Pooler, A. M., Phillips, E. C., Lau, D. H. W., Noble, W., Hanger, D. 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Microdialysis with high molecular weight cut off membranes has to be operated by a push-pull mode, thus it is critical that the flow rate is accurate and constant. The inaccuracy in the flow rates can be the cause of air bubble generation and inconsistency in the sample concentration. If the flow is inconsistent, check all connections for leakage. If the problem still persists, it may be necessary to re-start with new probes and tubings.
Microdilaysis probes are continuously perfused by the pe...

ISF comprises 15-20% of total brain volume and offers a microenvironment critical for signal transduction, substrate transport and waste clearance1. Therefore, the ability of collecting ISF from living animals will provide greater implications for various biological processes as well as disease mechanism. In vivo microdialysis is one of the few methods that sample and quantify extracellular molecules from ISF from awake, freely moving animals and thereby serves as a useful tool in neuroscience research field2,3. In this method, microdialysis probes with semipermeable membranes are inserted in the brain and perfused with perfusion buffer at the relatively slow flow rate (0.1-5 &#181;L/min). During this perfusion, extracellular molecules in ISF passively diffuse into the probe according to the concentration gradient and collect as a dialysate. Although this article focuses on the method to sample ISF in the brain, both the principle and the method can be applied to other organs by appropriate modification if necessary.
Microdialysis was first employed in the early 1960s, and since then it has been extensively used to collect small molecules including amino acids or neurotransmitters in brain. However, recent commercial availability of microdialysis probes with high-molecular weight cut off membranes (100 kDa-3 MDa) has extended its application to relatively larger proteins in ISF as well4,5,6,7. The studies using these probes has led to the finding that proteins such as tau or &#945;-synuclein that were long thought to be exclusive cytoplasmic are also physiologically present in ISF4,5,8.
One of the difficulties using microdialysis probes with large cut off membranes (typically over 1,000 kDa) is that they are more susceptible to ultrafiltration fluid loss due to the inner pressure accumulated in the probes. Microdialysis probes used here have a unique structure to avoid this issue. The pressure will not be built up due to this structure, thus microdialysis with these probes should be operated in a &#34;push-pull&#34; mode using a syringe pump to perfuse the probes (=push) and a roller/peristaltic pump to collect the dialysate coming from the probe outlet (=pull)9 (Although it needs both push and pull pumps, due to pressure cancelling vent holes present in the probes, the system is technically only driven by the pull pump). This article starts with the stereotaxic surgery of a guide cannula implantation and describes how to set up microdialysis lines in order to collect ISF through microdialysis probes with 1,000 kDa cut-off membranes.

<table>
	<tbody>
		<tr>
			<td>The Univentor 820 Microsampler</td>
			<td>Univentor</td>
			<td>8303002</td>
			<td>Refrigerated fraction collector</td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>KD scientific</td>
			<td>KDS-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Roller pump</td>
			<td>Eicom microdialysis</td>
			<td>ERP-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Raturn Stand-Alone System</td>
			<td>BASi</td>
			<td>MD-1409</td>
			<td>Free-moving system</td>
		</tr>
		<tr>
			<td>Dual species cage kit</td>
			<td>BASi</td>
			<td>CX-1600</td>
			<td></td>
		</tr>
		<tr>
			<td>AtmosLM Microdialysis probe (shaft length 8 mm, membrane length 2 mm)</td>
			<td>Eicom microdialysis</td>
			<td>PEP-8-02</td>
			<td>Shaft length for a probe, a guide, a dumy probe and a stereotaxic adaptor should be identical.</td>
		</tr>
		<tr>
			<td>Microdialysis guide (shaft length 8 mm)</td>
			<td>Eicom microdialysis</td>
			<td>PEG-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Microdialysis dummy probe (shaft length 8 mm)</td>
			<td>Eicom microdialysis</td>
			<td>PED-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Bone screw</td>
			<td>BASi</td>
			<td>MD-1310</td>
			<td></td>
		</tr>
		<tr>
			<td>Super bond C&#38;B set</td>
			<td>Sunmedical</td>
			<td></td>
			<td>Dental cement</td>
		</tr>
		<tr>
			<td>Small animal Stereotaxic Instrument with digital display console</td>
			<td>Kopf</td>
			<td>Model 940</td>
			<td>Stereotaxic apparatus</td>
		</tr>
		<tr>
			<td>Mouse and neonatal rat adaptor</td>
			<td>Stoelting</td>
			<td>51625</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard Ear Bars and Rubber Tips for Mouse Stereotaxic</td>
			<td>Stoelting</td>
			<td>51648</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin solution from bovine serum</td>
			<td>Sigma</td>
			<td>A7284-50ML</td>
			<td>30% BSA solution</td>
		</tr>
		<tr>
			<td>FEP tubing (70 cm)</td>
			<td>Eicom microdialysis</td>
			<td>JF-10-70</td>
			<td>Internal volume = 0.5 &#181;L/cm</td>
		</tr>
		<tr>
			<td>Teflon tubing (50 cm)</td>
			<td>Eicom microdialysis</td>
			<td>JT-10-50</td>
			<td>Internal volume = 0.08 &#181;L/cm</td>
		</tr>
		<tr>
			<td>Byton tube</td>
			<td>Eicom microdialysis</td>
			<td>JB-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Intramedic luer stab adaptor 23G</td>
			<td>BD</td>
			<td>427565</td>
			<td>Blunt end needle</td>
		</tr>
		<tr>
			<td>Roller tube</td>
			<td>Eicom microdialysis</td>
			<td>RT-5S</td>
			<td>Internal volume = 4 &#181;L</td>
		</tr>
		<tr>
			<td>Cap nut</td>
			<td>Eicom microdialysis</td>
			<td>AC-5</td>
			<td></td>
		</tr>
		<tr>
			<td>0.25 mL microcentrifuge tube with cap</td>
			<td>QSP</td>
			<td>503-Q</td>
			<td>Tubes for fraction collector</td>
		</tr>
		<tr>
			<td>Sterotaxic adaptor (shaft length 8 mm)</td>
			<td>Eicom microdialysis</td>
			<td>PESG-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Connection needle</td>
			<td>Eicom microdialysis</td>
			<td>RTJ</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse animal collar</td>
			<td>BASi</td>
			<td>MD-1365</td>
			<td></td>
		</tr>
		<tr>
			<td>High Speed Rotary Micromotor kit</td>
			<td>FOREDOM</td>
			<td>K.1070</td>
			<td>Drill</td>
		</tr>
		<tr>
			<td>Picrotoxin</td>
			<td>Sigma</td>
			<td>P1675</td>
			<td></td>
		</tr>
		<tr>
			<td>Screw driver for bone screws</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton swab</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical clipper</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo microdialysis method to collect large extracellular proteins from brain interstitial fluid with high-molecular weight cut-off probes

In vivo microdialysis is a powerful technique to collect ISF from awake, freely-behaving animals based on a dialysis principle. While microdialysis is an established method that measures relatively small molecules including amino acids or neurotransmitters, it has been recently used to also assess dynamics of larger molecules in ISF using probes with high molecular weight cut off membranes. Upon using such probes, microdialysis has to be run in a push-pull mode to avoid pressure accumulated inside of the probes. This article provides step-by-step protocols including stereotaxic surgery and how to set up microdialysis lines to collect proteins from ISF. During microdialysis, drugs can be administered either systemically or by direct infusion into ISF. Reverse microdialysis is a technique to directly infuse compounds into ISF. Inclusion of drugs in the microdialysis perfusion buffer allows them to diffuse into ISF through the probes while simultaneously collecting ISF. By measuring tau protein as an example, the author shows how its levels are altered upon stimulating neuronal activity by reverse microdialysis of picrotoxin. Advantages and limitations of microdialysis are described along with the extended application by combining other in vivo methods.

To stimulate or inhibit neuronal activity in reverse microdialysis11,12,13, picrotoxin, GABAA receptor antagonist or tetrodotoxin, Na+ channel blocker have been used. It has been shown that tau release is stimulated by increase of neuronal activity13,14. Consistent with these previous observations, when 50 &#181;M pi...

Watch this Scientific Journal Video about In Vivo Microdialysis Method to Collect Large Extracellular Proteins from Brain Interstitial Fluid with High-molecular Weight Cut-off Probes at JoVE.com

In Vivo Microdialysis Method to Collect Large Extracellular Proteins from Brain Interstitial Fluid with High-molecular Weight Cut-off Probes

In vivo microdialysis has enabled collection of molecules present in brain interstitial fluid (ISF) from awake, freely-behaving animals. In order to analyze relatively large molecules in ISF, the current article specifically focuses on the microdialysis protocol using probes with high molecular weight cut off membranes.

In Vivo Microdialysis Method to Collect Large Extracellular Proteins from Brain Interstitial Fluid with High-molecular Weight Cut-off Probes

In vivo microdialysis is a powerful technique to collect ISF from awake, freely-behaving animals based on a dialysis principle. While microdialysis is an ...

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