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 of meloxicam SR at induction and buprenorphine upon recovery BID for at least 24 h&#160;is recommended.</li>
	<li>Shave the hair with a surgical clipper. Fix a mouse in the mouse and neonatal rat adaptor using ear bars and a nose clamp. 
	NOTE: It is critical to make sure that mouse head is secured at this stage and does not move side-by-side.</li>
	<li>Apply vet ointment on eyes to prevent dryness while under anesthesia.</li>
</ol>
2. Stereotaxic Surgery for Guide Cannula Implantation
<ol>
	<li>Set the mouse and neonatal rat adaptor on the stereotaxic apparatus. Make an incision sagittally on the skin over the skull using a scalpel.&#12288;Wipe off blood and the connective tissue on the skull using a damp cotton swab.</li>
	<li>Determine the coordinate for brain region of interest using brain atlas. Before starting measurements, make sure that midline is straight so that the drill bit can be moved A-P and remain on the midline suture the entire time. 
	NOTE: This article uses the coordinate (A/P: -3.1 mm, M/L: -2.5 mm, D/V: -1.2 mm, 12 degrees) to target posterior hippocampus.</li>
	<li>Level skull A-P
	<ol>
		<li>Attach a drill on a manipulator of stereotaxic frame.Lower the drill until it gently touches lambda and record its ventral coordinate. Repeat this procedure for bregma. 
		NOTE: When the skull is leveled, the vertical measurement of bregma is equal to that of lambda. If not, adjust the height of nose clamp accordingly. After the skull gets leveled, record the anterior/posterior, lateral coordinate of bregma.</li>
	</ol>
	</li>
	<li>Level skull left-right
	<ol>
		<li>Move the drill from bregma to the coordinate (A/P: -3.1 mm, M/L: +2.0 mm), lower the drill to the skull and record the vertical coordinate. Then repeat this procedure for the coordinate (A/P: -3.1 mm, M/L: -2.0 mm). 
		NOTE: If the skull is leveled, these vertical measurements of two equidistant points from the midline are equal. If not, adjust the height of the ear bars.</li>
	</ol>
	</li>
	<li>Drill a burr hole carefully at the target coordinate (A/P: -3.1 mm, M/L: -2.5 mm) to implant a guide cannula. If the diameter of a burr hole is not large enough for a guide cannula implantation, drill another hole that overlaps with the first one. Drill another hole on the right (contralateral side) parietal bone and insert a bone screw, which helps to secure dental cement in 1.10 (See Figure 1B).</li>
	<li>Cut a circular locking piece from the back side of lid of a 1.5 mL centrifuge tube by a razor blade and make a &#39;&#39;crown&#39;&#39;. This crown is used to prevent dental cement from spreading to the skin. Place it on the skull so that the burr holes made in step 2.5 stay within the circle (See Figure 1).</li>
	<li>Set up a stereotaxic assembly by putting a guide cannula on the shorter arm of a stereotaxic adaptor and fasten it using a cap nut. Set the longer arm of the stereotaxic adaptor on the electrode clamp. Attach it on the manipulator of the stereotaxic apparatus (See Figure 1A).</li>
	<li>Rotate the D-V stereotax assembly on the manipulator arm by 12 degrees (See Figure 1B). Move the guide cannula to the burr hole made in 1.6. Insert the guide cannula slowly into the brain by lowering it by 1.2 mm. 
	NOTE: The angle of the probe is specific to the hippocampus; other regions may require other angles or no angle at all. Consult a brain atlas for precise coordinates. Dry the surface of the skull, because if not the cement will not stick and cement cap can become dislodged</li>
	<li>Add the dental cement within the crown to fully cover both the metal part of the guide cannula and the bone screw enough to secure them. Apply additional dental cement if there is a part of skull exposed.</li>
	<li>Wait until dental cement is completely dried (~12-20 min). Remove the stereotaxic adaptor from the electrode clamp. Remove the cap nut and replace the stereotaxic adaptor with a dummy probe and fasten the cap nut.</li>
	<li>Release the mouse from the stereotaxic apparatus and house the mouse alone in an individual cage. 
	NOTE: The mouse should not be left unattended until it has regained sufficient consciousness to maintain sternal recumbency. Check the mouse daily until the day of microdialysis. The mouse receives NSAIDs such as carprofen if it appears to be in pain. Waiting 2 weeks is necessary for sleep-wake studies so the mouse habituates to the new environment10, but other types of studies may require shorter recovery periods (e.g., 1-2 days).</li>
</ol>
3. Microdialysis Setup
<ol>
	<li>Quality-check of probes: Fill a disposable 1ml syringe with distilled waterand connect it to the outlet (shorter port) of a probe using a byton tube. Cover the vent holes with the fingers and depress the syringe plunger gently to infuse water to the probe. Check that water appears from the probe inlet and there is no leakage on the surface of a microdialysis membrane.
	<ol>
		<li>Activation of probes: Submerge the membranes of a probe in ethanol (70-100%) for two seconds. Then, infuse distilled water into the probe again with a syringe again.</li>
	</ol>
	</li>
	<li>Preparation of perfusion buffer: In order to avoid adhesion of target molecules to the tubings,add BSA by diluting 30% BSA solution to 4% with artificial CSF (1.3 mM CaCl2, 1.2 mM MgSO4, 3 mM KCl, 0.4 mM KH2PO4, 25 mM NaHCO3, 122 mM NaCl, pH=7.35), which closely matches electrolyte concentration in CSF, immediately prior to use. Filter the perfusion buffer through a syringe filter unit with 0.1 &#181;m pore size . 
	NOTE: 4% BSA improves recovery for sticking proteins but can severely limit delivery of compounds, especially for compounds that have high BSA-binding. This is one advantage of using lower concentration of BSA (such as 0.15%) in certain instances3. Note that BSA can aggregate very easily when agitated by vortex or stir plate. These aggregates can clog probes or membrane pores. Use caution when preparing a BSA solution to limit this aggregation</li>
	<li>Prepare two separate lines for inlet and outlet (See Figure 1C) and connect both lines with a connection needle. Fill a disposable 3 mL syringe filled with perfusion buffer and connect it with the inlet end of the tubing using a blunt-end needle. Fill the entire tubing with perfusion buffer by running the syringe pump.</li>
	<li>Stop the syringe pump and replace the connection needle between inlet line and outlet line with an activated microdialysis probe from step 3.3 (See Figure 1C). Before this replacement, put the cap nut on the probe.</li>
	<li>Mount the roller tube in the outlet tubing on the roller pump. Start the syringe pump at 10 &#181;L/min and then the roller pump at the slightly slower flow rate (9.5-9.8 &#181;L/min). Make sure to remove all air bubbles in the entire tubing, which may influence the recovery when they enter the probe.</li>
	<li>Anesthetize the mouse from step 1.12 in the same way as in step 1.2. Put the mouse collar around its neck. Remove the cap nut and dummy probe and slowly insert a microdialysis probe from 3.4 through the guide cannula and fasten the cap nut.</li>
	<li>Place the mouse in the cage connected to a free-moving system and tether the mouse with the collar. Keep running both syringe pump and roller pump at the indicated flow rate in step 3.5 for at least 1 h. 
	NOTE: To achieve ISF collection from awake animals, this article uses a free-moving system, where the cage itself responds to an animal&#39;s movement to keep tubings from twisting. Alternatively, liquid swivels available from various companies can be also used.</li>
	<li>Stop the roller pump first and then the syringe pump. Set the desired flow rate. Run the syringe pump 20% faster than the roller pump. 
	NOTE: For instance, if you run at 1 &#181;L/min, operate the syringe pump at 1.2 &#181;L/min. Optimal flow rate should be determined empirically for each molecule.</li>
	<li>Collecting ISF samples: place the free end of outlet tubing on the refrigerated fraction collector. 
	NOTE: Appropriate sample volume varies depending on assays used for analysis.</li>
	<li>After the completion of the experiment, remove the probe.&#160;(Anesthetize the mouse as needed.)&#160; Handle mouse recovery the same way as in step 2.11. Analyze the collected ISF by methods such as HPLC or ELISA.</li>
	<li>To wash the entire tubing after microdialysis, connect inlet and outlet tubings again by replacing the probe with the connection needle and run the diluted bleach in the entire tubing and then flush it with water. Dry and store it for repeated use. 
	NOTE: Other types of tubings are acceptable, however, BSA in perfusion buffer can clog the tubings with small diameter, thus these tubings are considered as single-use. The tubings can be worn down or clogged after multiple uses, so make sure that the flow rate is consistent every time before use.</li>
</ol>

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	<li>Lei, Y., Han, H., Yuan, F., Javeed, A., Zhao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+brain+interstitial+system:+Anatomy,+modeling,+in+vivo+measurement,+and+applications.">The brain interstitial system: Anatomy, modeling, in vivo measurement, and applications.</a> Progress in Neurobiology. 157, 230-246 (2017).</li><li>Kushikata, T., Hirota, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuropeptide+microdialysis+in+free-moving+animals.">Neuropeptide microdialysis in free-moving animals.</a> Methods in Molecular Biology. 789, 261-269 (2011).</li><li>Cirrito, J. R., 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+assessment+of+brain+interstitial+fluid+with+microdialysis+reveals+plaque-associated+changes+in+amyloid-beta+metabolism+and+half-life.">In vivo assessment of brain interstitial fluid with microdialysis reveals plaque-associated changes in amyloid-beta metabolism and half-life.</a> The Journal of Neuroscience. 23 (26), 8844-8853 (2003).</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=Assessment+of+&#945;-synuclein+secretion+in+mouse+and+human+brain+parenchyma.">Assessment of &#945;-synuclein secretion in mouse and human brain parenchyma.</a> PLoS One. 6 (7), 1-9 (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=In+vivo+microdialysis+reveals+age-dependent+decrease+of+brain+interstitial+fluid+tau+levels+in+P301S+human+tau+transgenic+mice.">In vivo microdialysis reveals age-dependent decrease of brain interstitial fluid tau levels in P301S human tau transgenic mice.</a> The Journal of Neuroscience. 31 (37), 13110-13117 (2011).</li><li>Ulrich, J. 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. -. 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=Amyloid-&#946;+dynamics+are+regulated+by+orexin+and+the+sleep-wake+cycle.">Amyloid-&#946; dynamics are regulated by orexin and the sleep-wake cycle.</a> Science. 326 (November), 1005-1008 (2009).</li><li>Cirrito, J. R., 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=Synaptic+activity+regulates+interstitial+fluid+amyloid-beta+levels+in+vivo.">Synaptic activity regulates interstitial fluid amyloid-beta levels in vivo.</a> Neuron. 48 (6), 913-922 (2005).</li><li>Bero, A. 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. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+release+of+endogenous+tau+is+stimulated+by+neuronal+activity.">Physiological release of endogenous tau is stimulated by neuronal activity.</a> EMBO Reports. 14 (4), 389-394 (2013).</li><li>Castellano, J. M., 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=Human+apoE+isoforms+differentially+regulate+brain+amyloid-&#946;+peptide+clearance.">Human apoE isoforms differentially regulate brain amyloid-&#946; peptide clearance.</a> Science Translational Medicine. 3 (89), 89ra57 (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=Analysis+of+in+vivo+turnover+of+tau+in+a+mouse+model+of+tauopathy.">Analysis of in vivo turnover of tau in a mouse model of tauopathy.</a> Molecular Neurodegeneration. 10 (1), 55 (2015).</li><li>Taylor, H., 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=Investigating+local+and+long-range+neuronal+network+dynamics+by+simultaneous+optogenetics,+reverse+microdialysis+and+silicon+probe+recordings+in+vivo.">Investigating local and long-range neuronal network dynamics by simultaneous optogenetics, reverse microdialysis and silicon probe recordings in vivo.</a> Journal of Neuroscience Methods. 235, 83-91 (2014).</li></ol>

The author has nothing to disclose.

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 perfusion buffer. Therefore, there is not sufficient time for extracellular molecules to reach a full equilibrium in the perfusion buffer. As a consequence, the concentration in the dialysate is much lower than its actual concentration in ISF. In order to estimate the true concentration of molecules in ISF, a zero flow method is often used3,5,15. This method measures the concentration by altering the flow rate. There is an inverse relationship between the concentration and the flow rate. Therefore, the concentration of target molecules can be determined by extrapolating the exponential curve back to the theoretical zero flow, which represents the perfect recovery of the molecules. However, the recovery can be influenced by various factors such as interaction with membranes or hydrophobicity of molecules or interaction with other proteins, and one should keep in mind that the concentration at the theoretical zero may not be necessarily equivalent to its true concentration in ISF.
A probe insertion causes acute local injury in brain. From this reason, the first several fractions are typically excluded from further analysis. In fact, the levels of ISF tau protein in hippocampus are not stable in the first 9-15 hour likely because the acute injury causes non-specific release of tau into ISF (the author&#39;s unpublished observation). Recovery after probe insertion injury varies from target to target. Pilot studies should be performed to determine when each target reaches a steady-state level to determine when samples should be analyzed. This determines the &#34;start point&#34; for microdialysis sampling. An endpoint for sampling is when the target is no longer at the steady state (typically 3-5 days after microdialysis probe implantation; not guide implantation). Start and end for each target should be determined empirically by each lab for each target.
Microdialysis is especially useful when combined with drug treatment. Drugs can be administered systemically or directly infused into ISF. Reverse microdialysis is one method that directly infuses small compounds via probes. Dialysis occurs bi-directionally. Therefore, inclusion of drugs in the perfusion buffer allows its diffusion though microdialysis probes to the surrounding tissue. This method enabled the local administration of small compound and simultaneous collection of ISF. Reverse microdialysis is less invasive compared to a pressure injection through cannula and can maintain more constant concentration of drugs in target area.
Before starting reverse microdialysis, the internal volume of entire tubing should be taken into account to calculate the timing of treatment and sample collection. For example, the internal volume of entire tubing used in this article is 91.5 &#181;L (Refer to the Table of Materials for internal volume of each tubing). Therefore, when the flow rate is 1.0 &#181;L/min, it takes 91.5 min for the perfusion buffer containing drugs to reach the fraction collector. On the other hand, when drugs are delivered systemically, the inner volume of the outlet tubing (56.5 &#181;L in this case) should be considered.
In vivo microdialysis method offers several advantages over other techniques. For example, while brain homogenates likely consist of both extracellular and intracellular proteins, microdialysis specifically collects extracellular proteins from ISF. Second, a single microdialysis probe can collect ISF at the different time points from the same animal. Target concentration in each sample can be normalized to a % baseline. This normalizes absolute concentration between animals so relative differences can be compared. This increases power of a study and typically reduces animal number needed for an experiment when comparing relative differences in protein levels following drug administration/manipulation. On the other hand, the major limitation of microdialysis is the restriction of the size of the target molecule.
Microdialysis can be combined with other in vivo techniques. For example, performing microdialysis in regulatable transgenic mice can estimate in vivo turnover of target protein16. The combination with EEG, measurement was used to measure electric activity during reverse microdialysis11. And optogenetics enabled manipulation of neuronal activity in brain while simultaneously collecting ISF17.

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&amp;amp;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 &amp;micro;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 &amp;micro;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 &amp;micro;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 picrotoxin (PTX) was administered via reverse microdialysis (see Discussion for more details) in awake C57B6/J mice increased ISF endogenous tau levels measured by ELISA compared to vehicle control (DMSO) (Figure 2).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57869/57869fig1.jpg" /> 
Figure 1: Surgery setup and the construction of microdialysis circuits. (A) Set up a stereotaxic assembly for a guide implantation using a cap nut, a stereotaxic adaptor and a guide cannula. (B) Stereotaxic surgery to implant a guide cannula. The guide cannula is inserted in the brain at 12 degrees with respect to vertical. (C) Construction of an inlet tubing and an outlet tubing. Inlet line consists of 70 cm FEP tubing (JF-70) and the outlet line consists of JF-70 and the roller tube and 1/2 of FEP tubing. The connection needles are used for each connection. (D) Once the entire tubing is filled with perfusion buffer, stop the pump and replace the connection needle between inlet line and outlet line with an activated microdialysis probe. Make sure to connect the end of inlet tubing to the inlet port of a probe (longer port) and connect the end of outlet tubing with outlet port of a probe (shorter port). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57869/57869fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57869/57869fig2.jpg" /> 
Figure 2: Representative data showing ISF tau changes upon picrotoxin reverse microdialysis. Picrotoxin (PTX, 50 &#181;M) or DMSO were delivered into the hippocampus of C57B6/J mice via reverse microdialysis. Picrotoxin rapidly increased ISF endogenous tau levels from its basal levels (i.e., the average tau concentrations during 3 h before PTX administration) compared to DMSO treatment. Data is shown as mean&#177;SEM, n = 3 for DMSO, n = 4 for PTX. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57869/57869fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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Neuroscience

14.5K Views.  University of Tokyo. 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.

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

Survivable Stereotaxic Surgery in Rodents 

The ability to measure extracellular basal levels of neurotransmitters in the brain of awake animals allows for the determination of effects of different systemic challenges (pharmacological or physiological) to the CNS. For example, one can directly measure how the animal's midbrain dopamine projections respond to dopamine-releasing drugs like d-amphetamine or natural stimuli like food. In this video, we show you how to implant guide cannulas targeting specific sites in the rat brain, how to insert and implant a microdialysis probe and how to use high performance liquid chromatography coupled with electrochemical detection (HPLC-EC) to measure extracellular levels of oxidizable neurotransmitters and metabolites. Local precise introduction of drugs through the microdialysis probe allows for refined work on site specificity in a compound s mechanism of action. This technique has excellent anatomical and chemical resolution but only modest time resolution as microdialysis samples are usually processed every 20-30 minutes to ensure detectable neurotransmitter levels. Complementary ex vivo tools (i.e., slice and cell culture electrophysiology) can assist with monitoring real-time neurotransmission.

Survivable Stereotaxic Surgery in Rodents

The monitoring of extracellular neurotransmitter levels in distinct brain regions of freely moving animals offers insights on the link between neurotransmitter release and behavior. In vivo microdialysis coupled with electrochemical detection provides excellent anatomical and chemical resolution; and information on how basal neurotransmission is altered by pharmacological or physiological manipulations.

The ability to measure extracellular basal levels of neurotransmitters in the brain of awake animals allows for the determination of effects of different ...

Biology

Consensus Brain-derived Protein, Extraction Protocol for the Study of Human and Murine Brain Proteome Using Both 2D-DIGE and Mini 2DE Immunoblotting

Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or pathological conditions. Basically, this technique is based on the separation of proteins according to their isoelectric point in a first step, and secondly according to their molecular weights by SDS polyacrylamide gel electrophoresis (SDS-PAGE). In this report an optimized sample preparation protocol for little amount of human post-mortem and mouse brain tissue is described. This method enables to perform both two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and mini 2DE immunoblotting. The combination of these approaches allows one to not only find new proteins and/or protein modifications in their expression thanks to its compatibility with mass spectrometry detection, but also a new insight into markers validation. Thus, mini-2DE coupled to western blotting permits to identify and validate post-translational modifications, proteins catabolism and provides a qualitative comparison among different conditions and/or treatments. Herein, we provide a method to study components of protein aggregates found in AD and Lewy body dementia such as the amyloid-beta peptide and the alpha-synuclein. Our method can thus be adapted for the analysis of the proteome and insoluble proteins extract from human brain tissue and mice models too. In parallel, it may provide useful information for the study of molecular and cellular pathways involved in neurodegenerative diseases as well as potential novel biomarkers and therapeutic targets.

A common protein extraction protocol using urea/thiourea/SDS buffer for human and mice brain tissue allows indentification of proteins by 2D-DIGE and their subsequent characterization by mini 2DE immunoblotting. This method enables one to obtain more reproducible and reliable results from human biopsies and experimental models.

Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or ...

Custom-made Microdialysis Probe Design

Microdialysis is a commonly used technique in neuroscience research. Therefore commercial probes are in great demand to monitor physiological, pharmacological and pathological changes in cerebrospinal fluid. Unfortunately, commercial probes are expensive for research groups in public institutions. In this work, a probe assembly is explained in detail to build a reliable, concentric, custom-made microdialysis probe for less than $10. The microdialysis probe consists of a polysulfone membrane with a molecular cut-off of 30 kDa. Probe in vitro recoveries of substances with different molecular weight (in the range of 100-1,600 Da) and different physicochemical properties are compared. The probe yields an in vitro recovery of approximately 20% for the small compounds glucose, lactate, acetylcholine and ATP. In vitro recoveries for neuropeptides with a molecular weight between 1,000-1,600 Da amount to 2-6%. Thus, while the higher molecular weight of the neuropeptides lowered in vitro recovery values, dialysis of compounds in the lower range (up to 500 Da) of molecular weights has no great impact on the in vitro recovery rate. The present method allows utilization of a dialysis membrane with other cut-off value and membrane material. Therefore, this custom-made probe assembly has the advantage of sufficient flexibility to dialyze substances in a broad molecular weight range. Here, we introduce a microdialysis probe with an exchange length of 2 mm, which is applicable for microdialysis in mouse and rat brain regions. However, dimensions of the probe can easily be adapted for larger exchange lengths to be used in larger animals.

Microdialysis is a commonly used technique in neuroscience research. Therefore commercial probes are in great demand.In this work a probe assembly is explained in detail to build a reliable, concentric, custom-made microdialysis probe for less than $10.

Microdialysis is a commonly used technique in neuroscience research. Therefore commercial probes are in great demand to monitor physiological, ...

A Simple and Reproducible Method to Prepare Membrane Samples from Freshly Isolated Rat Brain Microvessels

The blood-brain barrier (BBB) is a dynamic barrier tissue that responds to various pathophysiological and pharmacological stimuli. Such changes resulting from these stimuli can greatly modulate drug delivery to the brain and, by extension, cause considerable challenges in the treatment of central nervous system (CNS) diseases. Many BBB changes that affect pharmacotherapy, involve proteins that are localized and expressed at the level of endothelial cells. Indeed, such knowledge on BBB physiology in health and disease has sparked considerable interest in the study of these membrane proteins. From a basic science research standpoint, this implies a requirement for a simple but robust and reproducible method for isolation of microvessels from brain tissue harvested from experimental animals. In order to prepare membrane samples from freshly isolated microvessels, it is essential that sample preparations be enriched in endothelial cells but limited in the presence of other cell types of the neurovascular unit (i.e., astrocytes, microglia, neurons, pericytes). An added benefit is the ability to prepare samples from individual animals in order to capture the true variability of protein expression in an experimental population. In this manuscript, details regarding a method that is utilized for isolation of rat brain microvessels and preparation of membrane samples are provided. Microvessel enrichment, from samples derived, is achieved by using four centrifugation steps where dextran is included in the sample buffer. This protocol can easily be adapted by other laboratories for their own specific applications. Samples generated from this protocol have been shown to yield robust experimental data from protein analysis experiments that can greatly aid the understanding of BBB responses to physiological, pathophysiological, and pharmacological stimuli.

Here, a method for isolation of rat brain microvessels and for the preparation of membrane samples is described. This protocol has the clear advantage of producing enriched microvessel samples with acceptable protein yield from individual animals. Samples can then be used for robust protein analyses at the brain microvascular endothelium.

The blood-brain barrier (BBB) is a dynamic barrier tissue that responds to various pathophysiological and pharmacological stimuli. Such changes resulting ...

Microdialysis of Excitatory Amino Acids During EEG Recordings in Freely Moving Rats

Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain interstitial space with the behavior and/or with the specific outcome of a pathology (e.g., seizures for epilepsy). When studying epilepsy, the microdialysis technique is often combined with short-term or even long-term video-electroencephalography (EEG) monitoring to assess spontaneous seizure frequency, severity, progression and clustering. The combined microdialysis-EEG is based on the use of several methods and instruments. Here, we performed in vivo microdialysis and continuous video-EEG recording to monitor glutamate and aspartate outflow over time, in different phases of the natural history of epilepsy in a rat model. This combined approach allows the pairing of changes in the neurotransmitter release with specific stages of the disease development and progression. The amino acid concentration in the dialysate was determined by liquid chromatography. Here, we describe the methods and outline the principal precautionary measures one should take during in vivo microdialysis-EEG, with particular attention to the stereotaxic surgery, basal and high potassium stimulation during microdialysis, depth electrode EEG recording and high-performance liquid chromatography analysis of aspartate and glutamate in the dialysate. This approach may be adapted to test a variety of drug or disease induced changes of the physiological concentrations of aspartate and glutamate in the brain. Depending on the availability of an appropriate analytical assay, it may be further used to test different soluble molecules when employing EEG recording at the same time.

Here, we describe a method for in vivo microdialysis to analyze aspartate and glutamate release in the ventral hippocampus of epileptic and non-epileptic rats, in combination with EEG recordings. Extracellular concentrations of aspartate and glutamate may be correlated with the different phases of the disease.

Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain ...

Quantitative Measurement of Intrathecally Synthesized Proteins in Mice

Cerebrospinal fluid (CSF), a fluid found in the brain and the spinal cord, is of great importance to both basic and clinical science. The analysis of the CSF protein composition delivers crucial information in basic neuroscience research as well as neurological diseases. One caveat is that proteins measured in CSF may derive from both intrathecal synthesis and transudation from serum, and protein analysis of CSF can only determine the sum of these two components. To discriminate between protein transudation from the blood and intrathecally produced proteins in animal models as well as in humans, CSF protein profiling measurements using conventional protein analysis tools must include the calculation of the albumin CSF/serum quotient (Qalbumin), a marker of the integrity of the blood-brain interface (BBI), and the protein index (Qprotein/Qalbumin), an estimate of intrathecal protein synthesis. This protocol illustrates the entire procedure, from CSF and blood collection to quotients and indices calculations, for the quantitative measurement of intrathecal protein synthesis and BBI impairment in mouse models of neurological disorders.

Elevated spinal fluid protein levels can either be the result of diffusion of plasma protein across an altered blood-brain barrier or intrathecal synthesis. An optimized testing protocol is presented in this article that helps to discriminate both cases and provides quantitative measurements of intrathecally synthesized proteins.

Cerebrospinal fluid (CSF), a fluid found in the brain and the spinal cord, is of great importance to both basic and clinical science. The analysis of the ...

Real-Time Dynamic Collection of Hippocampal Extracellular Fluid from Conscious Rats Using a Microdialysis System

A variety of central nervous system (CNS) diseases are associated with changes in the composition of hippocampal extracellular fluid (HECF). However, difficulty in obtaining HECF in real time from conscious rats has long restricted the evaluation of CNS disease progression and the effectiveness of ethnomedicine therapy. Encouragingly, a brain microdialysis technique can be used for continuous sampling with the advantages of dynamic observation, quantitative analysis, and a small sampling size. This allows the monitoring of changes in the extracellular fluid content for compounds from traditional herbs and their metabolites in the brain of living animals. The aim of this study was thus to accurately implant a cerebrospinal fluid microdialysis probe into the hippocampal region of Sprague Dawley (SD) rats with a three-dimensional brain stereotaxic apparatus, cutting off molecular weights greater than 20 kDa. The high-quality HECF was then obtained from conscious rats using a microdialysis sampling control system with an adjustable sampling rate from 2.87 nL/min - 2.98 mL/min. In conclusion, our protocol provides an efficient, rapid, and dynamic method to obtain HECF in awake rats with the help of microdialysis technology, which provides us with unlimited possibilities to further explore the pathogenesis of CNS-related diseases and evaluate drug efficacy.

The protocol here provides a detailed real-time dynamic sampling of extracellular fluid from the hippocampus of awake rats using a microdialysis system.

A variety of central nervous system (CNS) diseases are associated with changes in the composition of hippocampal extracellular fluid (HECF). However, ...

Efficient Isolation of Brain sEVs from Minimal Tissue for Biomarker Discovery

Harnessing the Power of MicroRNA Cargoes in Small Extracellular Vesicles Released from Fresh-Frozen Human Brain Sections

Small extracellular vesicles (sEVs) are crucial mediators of cell-cell communication, transporting diverse cargoes like proteins, lipids, and nucleic acids (microRNA, mRNA, DNA). The microRNA sEV cargo has potential utility as a powerful non-invasive disease biomarker due to sEV&#39;s ability to traverse biological barriers (e.g., blood-brain barrier) and become accessible through various body fluids. Despite numerous studies on sEV biomarkers in body fluids, identifying tissue or cell-specific sEV subpopulations remains challenging, particularly from the brain. Our study addresses this challenge by adapting existing methods to isolate sEVs from minimal amounts of frozen human brain sections using size exclusion chromatography (SEC).
After ethical approval, approximately 250 &#181;g of fresh-frozen human brain tissue (obtained from Manchester Brain Bank [UK]) was sliced from the 3 donor tissues and incubated in collagenase type 3/Hibernate-E solution, with intermediate agitation, followed by serial centrifugation and filtration steps. Then, sEVs were isolated using the SEC method and characterized by following MISEV guidelines. Before isolating RNA from within these sEVs, the solution was treated with Proteinase-K and RNase-A to remove any non-sEV extracellular RNA. The RNA quantity and quality were checked and processed further for qPCR and small RNA sequencing experiments.
The presence of sEVs was confirmed through fluorescence nanoparticle tracking analysis (fNTA) and western blot for surface markers (CD9, CD63, CD81). Size distribution (50-200 nm) was confirmed by NTA and electron microscopy. The total RNA concentration within lysed sEVs ranged from 3-9 ng/&#181;L and was used for successful quantification by qPCR for selected candidate microRNAs. Small RNA sequencing on MiSeq provided high-quality data (Q &#62;32) with 1.4-5 million reads per sample.
This method enables efficient isolation and characterization of sEVs from minimal brain tissue volumes, facilitating non-invasive biomarker research and holds promise for equitable disease biomarker studies, offering insights into neurodegenerative diseases and potentially other disorders.

Here, we establish a protocol using minimal amounts of fresh-frozen brain tissue sections and an accessible high-speed centrifugation method coupled with size exclusion chromatography to obtain small extracellular vesicles as sources for microRNA (miRNA) biomarkers for neurological disorders.

Small extracellular vesicles (sEVs) are crucial mediators of cell-cell communication, transporting diverse cargoes like proteins, lipids, and nucleic ...

Isolation of the Brain Secretome from Ex Vivo Brain Slice Cultures

The brain secretome consists of proteins either actively secreted or shed from the cell surface by proteolytic cleavage in the extracellular matrix of the nervous system. These proteins include growth factor receptors and transmembrane proteins, among others, covering a broad spectrum of roles in the development and normal functioning of the central nervous system. The current procedure to extract the secretome from cerebrospinal fluid is complicated and time-consuming, and it is difficult to isolate these proteins from experimental animal brains. In this study, we present a novel protocol for isolating the brain secretome from mouse brain slice cultures. First, the brains were isolated, sliced, and cultured ex vivo. The culture medium was then filtered and concentrated for isolating proteins by centrifugation after a few days. Finally, the isolated proteins were resolved using sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently probed for purity characterization by western blot. This isolation procedure of the brain secretome from ex vivo brain slice cultures can be used to investigate the effects of the secretome on a variety of neurodevelopmental diseases, such as autism spectrum disorders.

Isolation of the Brain Secretome from Ex Vivo Brain Slice Cultures

The brain secretome plays a pivotal role in the development and normal functioning of the central nervous system. In this article, we provide a detailed protocol for isolation of the brain secretome from ex vivo brain slice cultures.

The brain secretome consists of proteins either actively secreted or shed from the cell surface by proteolytic cleavage in the extracellular matrix of the ...

Microdialysis of Interstitial Fluid from a Mouse Brain to Isolate Extracellular Proteins

Source: Yamada, K. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/57869">In Vivo Microdialysis Method to Collect Large Extracellular Proteins from Brain Interstitial Fluid with High-molecular Weight Cut-off Probes.</a> J. Vis. Exp. (2018)
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 membrane and are collected for analysis.

Source: Yamada, K. In Vivo Microdialysis Method to Collect Large Extracellular Proteins from Brain Interstitial Fluid with High-molecular Weight Cut-off ...

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

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Survivable Stereotaxic Surgery in Rodents

Consensus Brain-derived Protein, Extraction Protocol for the Study of Human and Murine Brain Proteome Using Both 2D-DIGE and Mini 2DE Immunoblotting

Custom-made Microdialysis Probe Design

A Simple and Reproducible Method to Prepare Membrane Samples from Freshly Isolated Rat Brain Microvessels

Microdialysis of Excitatory Amino Acids During EEG Recordings in Freely Moving Rats

Quantitative Measurement of Intrathecally Synthesized Proteins in Mice

Real-Time Dynamic Collection of Hippocampal Extracellular Fluid from Conscious Rats Using a Microdialysis System

Harnessing the Power of MicroRNA Cargoes in Small Extracellular Vesicles Released from Fresh-Frozen Human Brain Sections

Isolation of the Brain Secretome from Ex Vivo Brain Slice Cultures

Microdialysis of Interstitial Fluid from a Mouse Brain to Isolate Extracellular Proteins