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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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 (bis in die, twice a day) 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. 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 a 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 the 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 (Anteroposterior [A/P]: -3.1 mm, mediolateral [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&#160;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&#160;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&#160;Figure 1A).</li>
	<li>Rotate the dorsal-ventral (D-V) stereotax assembly on the manipulator arm by 12 degrees (See&#160;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 nonsteroidal anti-inflammatory drugs (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 environment, but other types of studies may require shorter recovery periods (e.g.,&#160;1-2 days).</li>
</ol>
3. Microdialysis Setup
<ol>
	<li>Quality-check of probes: Fill a disposable 1ml syringe with distilled water and 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 cerebrospinal fluid (aCSF: 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% bovine serum albumin (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 instances. 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&#160;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 a 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&#160;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 high-performance liquid chromatography (HPLC) or enzyme-linked immunosorbent assay (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>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>

microdialysis of interstitial fluid from a mouse brain to isolate extracellular proteins

Source: Yamada, K. <a href="https://app.jove.com/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.

<img alt="Figure 1" src="/files/ftp_upload/23125/23125_Figure1.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...

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

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

Take an anesthetized mouse implanted with a guide cannula in its brain.
A dummy probe inside the cannula prevents the entry of tissue debris.
Place a collar around the mouse&#39;s neck.
Replace the dummy probe with a microdialysis probe containing a specialized semi-permeable membrane, which allows the flow of molecules with a high molecular weight.
The probe is connected to pumps through inlet and outlet ports to regulate the flow of a physiological buffer.
Tether the mouse inside a cage using the collar, allow it to recover, and initiate the flow.
The buffer enters the brain tissue through the inlet and mixes with the interstitial fluid, or ISF, in the extracellular space, which contains proteins involved in cellular communication.
The components diffuse across the membrane and are collected through the outlet in fractions.
The collected ISF, containing extracellular proteins, is ready for analysis.

microdialysis-interstitial-fluid-from-mouse-brain-to-isolate

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Neurodegenerative Diseases

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

Vidéo: Microdialysis of Interstitial Fluid from a Mouse Brain to Isolate Extracellular Proteins - Concept

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.

Microdialyse d’acides aminés excitateurs durant les enregistrements EEG en se déplaçant librement des Rats

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

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Neurosciences

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.

Collecte dynamique en temps réel de liquide extracellulaire hippocampique de rats conscients à l’aide d’un système de microdialyse

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

Dynamic Continuous Blood Extraction from Rat Heart via Noninvasive Microdialysis Technique

Dynamic analysis of blood components is of great importance in understanding cardiovascular diseases and their related diseases, such as myocardial infarction, arrhythmia, atherosclerosis, cardiogenic pulmonary edema, pulmonary embolism, and cerebral embolism. At the same time, it is urgent to break through the continuous heart blood sampling technique in live rats to evaluate the effectiveness of distinctive ethnic medicine therapy. In this study, a blood microdialysis probe was implanted in the right jugular vein of rats in a precise and noninvasive surgical procedure. Cardiac blood samples were then collected at a rate of 2.87 nL/min to 2.98 mL/min by connecting to an online microdialysis sample collection system. Even more momentously, the acquired blood samples can temporarily be stored in microdialysis containers at 4 &#176;C. The microdialysis-based online continuous blood collection program from rat heart has greatly guaranteed the quality of blood samples, advancing and invigorating the scientific rationality of the research on systemic cardiovascular diseases and evaluating ethnomedicine therapy from the perspective of hematology.

Dynamic Continuous Blood Extraction from Rat Heart via Noninvasive Microdialysis Technique

The present protocol describes a simple and efficient method for the real-time and dynamic collection of rat heart blood using the microdialysis technique.

Extraction dynamique continue de sang du cœur de rat par une technique de microdialyse non invasive

Dynamic analysis of blood components is of great importance in understanding cardiovascular diseases and their related diseases, such as myocardial ...

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

Transcription

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

Microdialyse d’acides aminés excitateurs durant les enregistrements EEG en se déplaçant librement des Rats

Collecte dynamique en temps réel de liquide extracellulaire hippocampique de rats conscients à l’aide d’un système de microdialyse

Extraction dynamique continue de sang du cœur de rat par une technique de microdialyse non invasive