Name: an improved method for collection of cerebrospinal fluid from anesthetized mice
Uploaded: 2018-03-19T13:00:00
Description: Watch this Scientific Journal Video about An Improved Method for Collection of Cerebrospinal Fluid from Anesthetized Mice at JoVE.com

This work was supported by the National Natural Science Foundation of China (81650110527, 81371400) and National Key Basic Research Program of China (2013CB530900).

All animal experiments were performed in accordance with the policies of the Society for Neuroscience (USA) and Fudan University (Shanghai, China) ethical committees. This procedure is for a non-survival surgery.
1. Setup of CSF Collection Apparatus
<ol>
	<li>Pull the glass capillary (inner diameter 0.75 mm, outer diameter 1.0 mm) using a micropipette puller (as shown in Liu et al.10; sharpened capillary is shown in Figure 1).</l...

<ol>
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F., 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=Changes+in+amyloid-&#946;+and+Tau+in+the+cerebrospinal+fluid+of+transgenic+mice+overexpressing+amyloid+precursor+protein.">Changes in amyloid-&#946; and Tau in the cerebrospinal fluid of transgenic mice overexpressing amyloid precursor protein.</a> Sci. Transl. Med. 5 (194), 194re2 (2013).</li><li>Oshio, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+cerebrospinal+fluid+production+and+intracranial+pressure+in+mice+lacking+choroid+plexus+water+channel+Aquaporin-1.">Reduced cerebrospinal fluid production and intracranial pressure in mice lacking choroid plexus water channel Aquaporin-1.</a> FASEB J. , (2004).</li></ol>

This protocol describes in detail a technique which improves on current methods10 of CSF collection to minimize contamination from blood and allow for the abundant collection of CSF (on average ~ 10-15 &#181;L can be obtained) from mice. When breaking the capillary tip, the tip&#160;of the capillary should not be too small (as then the CSF will be extracted very slowly) or too big (will not be fine enough to collect the CSF and tissue can become lodged in the capillary). Care should be taken when ...

The CSF is a valuable body fluid for analysis in neuroscience research. The CSF is primarily made from blood plasma, containing few cells (no red blood cells and only a few white blood cells) and is almost protein-free. It is one of the fluids in close contact with the central nervous system (CNS) and it can pass many electrolytes out from the brain and spinal cord to the peripheral system. In humans, CSF samples can be collected to aid in diagnosing disease or for research purposes in clinical trials, as a spinal tap (or lumbar puncture) is a minor, invasive procedure: the CSF fluid can reflect changes in the CNS without having to directly access these tissues. Thus, in recent years, for research purposes in the clinic, CSF samples have been obtained from patients of neurodegenerative diseases such as Alzheimer&#39;s disease and other dementias1,2,3. There are many biomarker assays which have been developed using CSF samples to potentially aid in diagnosing diseases in the clinic2,3. However, there is much debate on the reliability of these assays to produce consistent, sensitive results to specifically diagnose disease4,5. So, there is a great need for the development of better assays and targets, which can be found in the CSF, to aid in producing a standard technique to diagnose neurodegenerative diseases with greater sensitivity and specificity. Due to the potential importance of human CSF samples in disease, the collection of CSF from rodents in neuroscience research is also of interest.
Mice are important animals in biological and medical research and allow for the testing of potential therapeutic compounds and proof-of-concept studies before human clinical trials. However, in mice it is difficult to obtain CSF samples due to its closeness to the brain in a small animal, as the usual method of CSF collection in mice is to obtain it via the cisterna magna, an opening between the cerebellum and dorsal surface of the medulla oblongata. This causes difficulty in collecting CSF samples as this area is difficult to dissect to and in close proximity to blood vessels, increasing the risk of contamination from blood cells. Due to these difficulties, most researchers can only obtain a small amount of CSF for analysis (usually stated as 5-7 &#181;L) and the contamination of CSF samples by blood cells is a primary concern for analyses6,7,8,9. Blood contamination can obscure results and not truly reflect the state of the CNS. Furthermore, limited sample collected can impact research as the usual amount collected from mice is enough for only one measurement (in duplicate or triplicate) using enzyme-linked immunosorbent assay (ELISA). Thus, CSF samples are usually pooled from multiple mice in order to have enough sample to run multiple assays. Developing a protocol for the abundant, uncontaminated collection of CSF from mice is greatly desired and will be beneficial in improving neuroscience research using rodents.
In this protocol, a technique for the abundant (an average of 10-15 &#181;L) collection of CSF from anesthetized mice is described in detail and improves on a currently known method of CSF collection to minimize contamination from blood10. A robust protocol for CSF collection will aid in the development of CSF-based biomarker assays, which could be used to aid in diagnosing disease, as well as improve research into the mechanisms that underlie diseases affecting the CNS.

<table>
	<tbody>
		<tr>
			<td>Chloral hydrate (used as anesthetic)</td>
			<td>Sinopharm Chemicals Reagen Co. Ltd.</td>
			<td>30037517</td>
			<td>CAS number 302-17-0.</td>
		</tr>
		<tr>
			<td>Dissecting scissors</td>
			<td>66 vision technology</td>
			<td>54002</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting curved forceps</td>
			<td>66 vision technology</td>
			<td>53072</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting straight forceps</td>
			<td>66 vision technology</td>
			<td>53070</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse adapter (with ear bars)</td>
			<td>Made in-house.</td>
			<td>N/A</td>
			<td>Similar equipment available from World Precision Instruments.</td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Meiji Labax</td>
			<td>Model 15381</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>World Precision Instruments</td>
			<td>M3301</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic base for micromanipulator</td>
			<td>Kanetec</td>
			<td>MB-K</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass capillaries</td>
			<td>World Precision Instruments</td>
			<td>1B100-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Sutter Instruments</td>
			<td>Model P-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringes (1ml)</td>
			<td>Tansoole</td>
			<td>02024692</td>
			<td>For 1ml.</td>
		</tr>
		<tr>
			<td>Microtubes (1.5ml)</td>
			<td>Axygen</td>
			<td>MCT-150-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitor Cocktail Set III EDTA-free</td>
			<td>Calbiochem</td>
			<td>539134</td>
			<td></td>
		</tr>
		<tr>
			<td>Human A&#946;42 ELISA kit</td>
			<td>Invitrogen</td>
			<td>KHB3441</td>
			<td></td>
		</tr>
		<tr>
			<td>Piping (teflon tubing)</td>
			<td>World Precision Instruments</td>
			<td>MMP-KIT</td>
			<td>Obtained from a microinjection kit and attached to the capillary holder and syringe.</td>
		</tr>
		<tr>
			<td>Mini centrifuge</td>
			<td>Tiangen Biotech</td>
			<td>OSE-MC8</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton buds</td>
			<td>Obtained from any household store/pharmacy.</td>
			<td>N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Using the procedure outlined here (Figure 1 and Figure 2), the CSF immediately collected in the capillary should be clear (Figure 2E), not pink or red. If there is a pink to red tinge to the fluid collected in the capillary, then there was contamination with blood.
As an example of the application of the CSF sample collected with this metho...

Watch this Scientific Journal Video about An Improved Method for Collection of Cerebrospinal Fluid from Anesthetized Mice at JoVE.com

An Improved Method for Collection of Cerebrospinal Fluid from Anesthetized Mice

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 ...

The overall goal of this procedure is to improve collection of cerebrospinal fluid from mice without contamination from blood. This method provides an easy and reliable way to collect CSF for monitoring the biomarkers of neural diseases. The main advantage of this technique is that the addition with the micromanipulator to aid in CSF collection from mice decreases the contamination of blood during collection.Begin by placing a pulled glass capillary into a capillary holder which is firmly mounted on a micromanipulator. Next, while viewing through a dissecting microscope, use straight forceps to break the tip of the sharpened capillary so that the inner diameter of the broken tip is about 10 to 20 microns. Then attach a thin tube and a syringe to the other end of the capillary holder, connect the thin tube and the syringe with a three-way valve and shift the three-way valve to open.Then plunge the syringe to expel any contaminants and create positive pressure in the capillary tube. Repeat this a few times by disconnecting and reconnecting the syringe to the three-way valve. Then draw up air to 100 to 200 microliters before the final reconnection of the syringe with the three-way valve.Move the micromanipulator with the glass capillary to the side to prevent damage during the surgical procedure. Before beginning the surgery, ensure that the mouse has been fully anesthetized by pinching all four limbs and observing the absence of a reaction. Then use dissecting scissors to cut and trim the hair at the back of the head of the mouse from above the eyes between the ears to the bottom of the neck.Secure the head of the mouse using a stereotaxic frame mouse adapter. Make sure the head cannot move and is secured tightly in the adapter by gently pushing down onto the head of the mouse. Use scissors and curved forceps to cut through the mouse skin across the back of the neck and then down the middle of the skull between the ears and eyes of the mouse.Pull the skin and tissue apart and out of the area over the cisterna magna. While viewing through the dissecting microscope, carefully dissect away the last thin layers of muscle over the base of the skull using forceps and move these layers of muscle to the side and out of the area of interest. Once the dura over the cisterna magna is exposed, use a wet cotton bud to wipe the membrane clean and then use a dry cotton bud to dry the area.The cisterna magna appears triangular with one to two large blood vessels running through the area. Either side of or between the blood vessels is optimal for capillary insertion and CSF collection. Once the cisterna magna is exposed, use the micromanipulator to align the glass capillary to the back of the mouse head so that the sharpened point is just behind the membrane at a 30 to 45 degree angle.Next, plunge the syringe once or a few times and then move the plunger back 100 to 200 microliters to make sure there are no contaminants and there is negative pressure in the glass capillary. Then move the capillary tip closer to the membrane until resistance can be seen, but without puncturing the membrane. Knock on the micromanipulator controls to gently tap the capillary tube through the membrane.CSF should automatically be drawn into the capillary tube once the opening has been punctured. Leave the capillary in place to slowly collect CSF. If the CSF has stopped flowing, slowly draw the syringe up approximately 50 microliters to create more negative pressure in the capillary.Ten to 15 microliters of CSF is collected in 10 to 30 minutes. Once the desired volume of CSF has been collected, close the tubing by shifting the three-way valve to close and remove the syringe connected to the tubing. Open and then close the tubing to create equalized pressure in the capillary, the tubing, and the area outside the capillary.Then reconnect the syringe before gently removing the glass capillary from the mouse using micromanipulator. Next, place the collection tube containing one microliter of 20X protease inhibitor under the sharpened point of the glass capillary. Open the tubing and gently plunge the syringe.The CSF should flow out of the capillary and into the collection tube. After collection, pulse centrifuge the CSF to mix the sample with the protease inhibitor at the bottom of the collection tube. CSF samples can be aliquoted and then stored for further analysis at minus 80 degrees Celsius.CSF collected from APP/PS1 mice demonstrated a significant increase in levels of human A beta 42 at 12 months of age. In wild type mice, no human A beta 42 was detected. Once mastered, this procedure can be done in one hour if performed properly.While attempting this procedure, it is important to remember to avoid blood vessels when inserting the capillary and to only make minor adjustments with the micromanipulator during CSF collection to avoid contamination with blood.

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