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).</li>
	<li>Place a sharpened glass capillary into the capillary holder firmly mounted on a micromanipulator (Figure 1A).</li>
	<li>Break the tip of the sharpened capillary with straight forceps under a dissecting microscope, so that the inner diameter of the broken tip is about 10-20 &#181;m (Figure 1C).</li>
	<li>Attach a thin tube and a syringe to the other end of the capillary holder, and connect the thin tube and the syringe with a three-way valve.</li>
	<li>Shift the three-way valve to open and plunge the syringe to blow air from the syringe through the tubing to the glass capillary to expel any contaminants and create positive pressure in the capillary tube.</li>
	<li>Repeat this a few times by dis-connecting and re-connecting the syringe to the three-way valve, and drawing the syringe up ~ 100-200 &#181;L before the last re-connection of the syringe with the three-way valve (Figure 1B).</li>
	<li>Move the micromanipulator with the glass capillary to the side to prevent damage during mouse dissection.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56774/56774fig1.jpg" /> 
Figure 1. Microscope, micromanipulator, and capillary setup. (A) Microscope and micromanipulator with capillary attached setup, as well as a closer image of (B) the connected syringe and three-way valve to open (shown here) or close the piping, and (C) an unbroken (right) and broken (left) capillary tip ready for CSF collection. Once broken, the tip of the capillary should have an inner diameter of 0.75 mm, outer diameter of 1.0 mm. <a href="//cloudfront.jove.com/files/ftp_upload/56774/56774fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Mouse Dissection
<ol>
	<li>Anesthetize the mouse. 
	NOTE: For this protocol, C57BL/6 and amyloid precursor protein/presenilin 1 (APP/PS1) transgenic (Alzheimer&#39;s disease mouse model) mice were used and anesthetized with 4% chloral hydrate (in dH20) at 10 &#181;L/g of mouse weight via intraperitoneal injection.</li>
	<li>When fully anesthetized, ensure that the mouse is adequately anesthetized.&#160;The withdrawal response to toe pinch, by slightly extending the hind limbs and pinching the toes and watching for a withdrawal response, is performed to assure adequate anesthesia.&#160;Check by pinching all four limbs; if there is no reaction the mouse is properly anesthetized.&#160;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 using dissecting scissors (Figure 2A). Alternatively, clippers or depilatory cream can be used to aid in hair removal.</li>
	<li>Secure the head of the mouse using a mouse adapter (back of the head facing up with the nose pointed down at ~ 45 &#176;, Figure 2A). Fix ear bars between the ears and eyes of the mouse and tighten. Make sure the head cannot move and is secured tightly in the adapter by gently pushing down onto the head of the mouse.</li>
	<li>Recheck withdrawal response to toe pinch prior to making surgical incision and regularly thereafter to assure surgical plane of anesthesia. Visually note any change in respiratory rate during the procedure to indicate changes in planes of anesthesia. A clean, non-aseptic, technique can be used for this short non-survival surgery. Using scissors and curved forceps, cut through the skin and the first layer of muscle until the base of the skull is exposed with only a thin layer of muscle. 
	NOTE: Cut through the mouse skin across the back of the neck and then cut the skin down the middle of the skull between the ears and eyes of the mouse. Skin and tissue can then be pulled apart and out of the area over the cisterna magna.</li>
	<li>While viewing through the dissecting microscope,&#160;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. If there is bleeding, use cotton buds to remove and help stop the bleeding.</li>
	<li>Once the dura over the cisterna magna is exposed (triangular in shape with usually 1-2 large blood vessels running through the area; either side of or between the blood vessels is optimal for capillary insertion and CSF collection, Figure 2B), use a wet cotton bud to wipe the membrane clean and then use a dry cotton bud to dry the area.</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56774/56774fig2.jpg" /> 
Figure 2. Mouse and dissection setup with representative images of capillary insertion for CSF collection. Setup of mouse ready for CSF extraction with (A) shaved head clamped in place for dissection and (B) detailed image (10x view) of a dissected mouse dura covering the cisterna magna (dashed arrow shows a blood vessel running through the area and solid arrow shows area optimal for capillary insertion). Detailed images (10x view) of (C) sharpened tip of glass capillary aligned against dura of cisterna magna, (D) the point at which the capillary almost punctures the dura, with some resistance from dura, and (E) the capillary tip tapped through the dura to collect CSF. CSF should be a clear liquid collected in the glass capillary (dotted arrow). All scale bars at the bottom right of each microscope image indicate 100 &#181;m. <a href="//cloudfront.jove.com/files/ftp_upload/56774/56774fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. CSF Collection from Anesthetized Mouse
<ol>
	<li>Align the glass capillary to the back of the mouse head so that the sharpened point is just behind the membrane at a ~ 30-45 &#176; angle (Figure 2C).</li>
	<li>Plunge the syringe once or a few times and draw the syringe up ~ 100-200 &#181;L (to make sure there are no contaminants and there is negative&#160;pressure in the glass capillary).</li>
	<li>Using the micromanipulator, move the capillary tip closer to the membrane until resistance can be seen, but not puncturing it (Figure 2D).</li>
	<li>Once resistance is seen between the tip of the capillary and the membrane, gently tap the capillary tube through the membrane by knocking on the micromanipulator controls. Use the microscope to see the sharpened point of the capillary puncture into the cisterna magna. CSF should automatically be drawn into the capillary tube once the opening has been punctured (Figure 2E).</li>
	<li>Leave the capillary to slowly collect CSF. If the CSF has stopped drawing out, draw the syringe up a small amount slowly (~ 50 &#181;L) to create more negative pressure in the capillary to extract out the CSF. 
	NOTE: Alternatively, the capillary can be gently and slowly pulled out of the brain using the fine micromanipulator controls to allow CSF to flow into the capillary again. A total amount of ~ 10-15 &#181;L of CSF (~ 3-4 cm in the capillary) takes ~ 10-30 min, and occasionally 20 &#181;L or more can be collected. Although not absolutely necessary, it is recommended to use a heat pad&#160;during CSF collection. CSF collection was performed at room temperature (~ 23 &#176;C).</li>
	<li>Once the amount of desired CSF is collected, close the tubing by shifting the three-way valve to close (to stop drawing out the CSF).</li>
	<li>Take out the syringe connected to the tubing, and open and then close the tubing (to create equalized pressure in the capillary, tubing, and the area outside the capillary).&#160;Reconnect the syringe with the three-way valve, but do not plunge the syringe.</li>
	<li>Gently remove the glass capillary from the mouse using the micromanipulator.</li>
	<li>Place the collection tube (1.5 mL microtubes with 1 &#181;L of 20x protease inhibitor) under the sharpened point of the glass capillary.</li>
	<li>Open the tubing and gently plunge the syringe: the CSF should flow out of the capillary and into the collection tube.</li>
	<li>After collection, quickly centrifuge (pulse spin for 5 s at maximal speed using a mini centrifuge) the CSF to mix the sample with the protease inhibitor at the bottom of the collection tube. 
	NOTE: CSF samples can be aliquoted and then stored for further analysis at -80 &#176;C. The rest of the mouse can be dissected for other tissues, such as brain and spinal cord. The mouse is still alive at this stage and thus blood can also be collected, if needed.</li>
</ol>

<ol>
	<li>Anoop, A., Singh, P. K., Jacob, R. S., Maji, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CSF+Biomarkers+for+Alzheimer's+Disease+Diagnosis.">CSF Biomarkers for Alzheimer's Disease Diagnosis.</a> Int. J. Alzheimers. Dis. 2010, 1-12 (2010).</li><li>Blennow, K., Hampel, H., Weiner, M., Zetterberg, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebrospinal+fluid+and+plasma+biomarkers+in+Alzheimer+disease.">Cerebrospinal fluid and plasma biomarkers in Alzheimer disease.</a> Nat. Rev. Neurol. 6 (3), 131-144 (2010).</li><li>Schoonenboom, N. S. 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=Cerebrospinal+fluid+markers+for+differential+dementia+diagnosis+in+a+large+memory+clinic+cohort.">Cerebrospinal fluid markers for differential dementia diagnosis in a large memory clinic cohort.</a> Neurology. 78 (1), 47-54 (2012).</li><li>Molinuevo, J. L., 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=The+clinical+use+of+cerebrospinal+fluid+biomarker+testing+for+Alzheimer's+disease+diagnosis:+A+consensus+paper+from+the+Alzheimer's+Biomarkers+Standardization+Initiative.">The clinical use of cerebrospinal fluid biomarker testing for Alzheimer's disease diagnosis: A consensus paper from the Alzheimer's Biomarkers Standardization Initiative.</a> Alzheimer's Dement. 10 (6), 808-817 (2014).</li><li>Fagan, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CSF+biomarkers+of+Alzheimer's+disease:+impact+on+disease+concept,+diagnosis,+and+clinical+trial+design.">CSF biomarkers of Alzheimer's disease: impact on disease concept, diagnosis, and clinical trial design.</a> Adv. Geriatr. 2014, 1-14 (2014).</li><li>Ramautar, 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=Metabolic+profiling+of+mouse+cerebrospinal+fluid+by+sheathless+CE-MS.">Metabolic profiling of mouse cerebrospinal fluid by sheathless CE-MS.</a> Anal. Bioanal. Chem. 404 (10), 2895-2900 (2012).</li><li>Liu, L., Herukka, S., Minkeviciene, R., Vangreon, T., Tanila, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longitudinal+observation+on+CSF+A&#946;42+levels+in+young+to+middle-aged+amyloid+precursor+protein/presenilin-1+doubly+transgenic+mice.">Longitudinal observation on CSF A&#946;42 levels in young to middle-aged amyloid precursor protein/presenilin-1 doubly transgenic mice.</a> Neurobiol. Dis. 17 (3), 516-523 (2004).</li><li>Schelle, J., 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=Prevention+of+tau+increase+in+cerebrospinal+fluid+of+APP+transgenic+mice+suggests+downstream+effect+of+BACE1+inhibition.">Prevention of tau increase in cerebrospinal fluid of APP transgenic mice suggests downstream effect of BACE1 inhibition.</a> Alzheimer's Dement. , (2016).</li><li>You, J. -. S., Gelfanova, V., Knierman, M. D., Witzmann, F. A., Wang, M., Hale, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+blood+contamination+on+the+proteome+of+cerebrospinal+fluid.">The impact of blood contamination on the proteome of cerebrospinal fluid.</a> Proteomics. 5 (1), 290-296 (2005).</li><li>Liu, L., Duff, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Technique+for+Serial+Collection+of+Cerebrospinal+Fluid+from+the+Cisterna+Magna+in+Mouse.">A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse.</a> J. Vis. Exp. (21), (2008).</li><li>Maia, L. 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>

The authors have nothing to disclose.

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&amp;beta;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 ...

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Research

JoVE Journal

Neuroscience

61.7K Views.  University of Chinese Academy of Sciences, Chinese Academy of Science. Dit protocol beschrijft een verbeterde techniek voor de overvloedige hoeveelheid cerebrospinale vloeistof (CSF) met geen besmetting uit bloed. Met grotere sample collectie en zuiverheid, kunnen meer analyses worden uitgevoerd met behulp van CSF om ons begrip van ziekten die de hersenen en het ruggenmerg beïnvloeden.

Video: An Improved Method for Collection of Cerebrospinal Fluid from Anesthetized Mice

A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse 

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is pathologically characterized by extracellular deposition of &#946;-amyloid peptide (A&#946;) and intraneuronal accumulation of hyperphosphorylated tau protein. Because cerebrospinal fluid (CSF) is in direct contact with the extracellular space of the brain, it provides a reflection of the biochemical changes in the brain in response to pathological processes. CSF from AD patients shows a decrease in the 42 amino-acid form of A&#946; (A&#946;42), and increases in total tau and hyperphosphorylated tau, though the mechanisms responsible for these changes are still not fully understood. Transgenic (Tg) mouse models of AD provide an excellent opportunity to investigate how and why A&#946; or tau levels in CSF change as the disease progresses. Here, we demonstrate a refined cisterna magna puncture technique for CSF sampling from the mouse. This extremely gentle sampling technique allows serial CSF samples to be obtained from the same mouse at 2-3 month intervals which greatly minimizes the confounding effect of between-mouse variability in A&#946; or tau levels, making it possible to detect subtle alterations over time. In combination with A&#946; and tau ELISA, this technique will be useful for studies designed to investigate the relationship between the levels of CSF A&#946;42 and tau, and their metabolism in the brain in AD mouse models. Studies in Tg mice could provide important validation as to the potential of CSF A&#946; or tau levels to be used as biological markers for monitoring disease progression, and to monitor the effect of therapeutic interventions. As the mice can be sacrificed and the brains can be examined for biochemical or histological changes, the mechanisms underlying the CSF changes can be better assessed. These data are likely to be informative for interpretation of human AD CSF changes.

A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse

Transgenic (Tg) mouse models of AD provide an excellent opportunity to investigate how and why A&#946; or tau levels in CSF change as the disease progresses in human patients. Here, we demonstrate a refined cisterna magna puncture technique for serial CSF sampling from the mouse.

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is pathologically characterized by extracellular deposition of &#946;-amyloid ...

Biology

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

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

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

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

Cannula Implantation into the Cisterna Magna of Rodents

Cisterna magna cannulation (CMc) is a straightforward procedure that enables direct access to the cerebrospinal fluid (CSF) without operative damage to the skull or the brain parenchyma. In anesthetized rodents, the exposure of the dura mater by blunt dissection of the neck muscles allows the insertion of a cannula into the cisterna magna (CM). The cannula, composed either by a fine beveled needle or borosilicate capillary, is attached via a polyethylene (PE) tube to a syringe. Using a syringe pump, molecules can then be injected at controlled rates directly into the CM, which is continuous with the subarachnoid space. From the subarachnoid space, we can trace CSF fluxes by convective flow into the perivascular space around penetrating arterioles, where solute exchange with the interstitial fluid (ISF) occurs. CMc can be performed for acute injections immediately following the surgery, or for chronic implantation, with later injection in anesthetized or awake, freely moving rodents. Quantitation of tracer distribution in the brain parenchyma can be performed by epifluorescence, 2-photon microscopy, and magnetic resonance imaging (MRI), depending on the physico-chemical properties of the injected molecules. Thus, CMc in conjunction with various imaging techniques offers a powerful tool for assessment of the glymphatic system and CSF dynamics and function. Furthermore, CMc can be utilized as a conduit for fast, brain-wide delivery of signaling molecules and metabolic substrates that could not otherwise cross the blood brain barrier (BBB).

Here we describe a protocol to perform cisterna magna cannulation (CMc), a minimally invasive way to deliver tracers, substrates and signaling molecules into the cerebrospinal fluid (CSF). Combined with different imaging modalities, CMc enables glymphatic system and CSF dynamics assessment, as well as brain-wide delivery of various compounds.

Cisterna magna cannulation (CMc) is a straightforward procedure that enables direct access to the cerebrospinal fluid (CSF) without operative damage to ...

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

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

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

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

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 ...

Intraventricular Drug Delivery and Sampling for Pharmacokinetics and Pharmacodynamics Study

Although the blood-brain barrier (BBB) protects the brain from foreign entities, it also prevents some therapeutics from crossing into the central nervous system (CNS) to ameliorate diseases or infections. Drugs are administered directly into the CNS in animals and humans to circumvent the BBB. The present protocol describes a unique way of treating brain infections through intraventricular delivery of antibiotics, i.e., polymyxins, the last-line antibiotics to treat multi-drug resistant Gram-negative bacteria. A straightforward stereotaxic surgery protocol was developed to implant a guide cannula reaching into the lateral ventricle in rats. After a recovery period of 24 h, rats can be injected consciously and repeatedly through a cannula that is fitted to the guide. Injections can be delivered manually as a bolus or infusion using a microinjection pump to obtain a slow and controlled flow rate. The intraventricular injection was successfully confirmed with Evans Blue dye. Cerebrospinal fluid (CSF) can be drained, and the brain and other organs can be collected. This approach is highly amenable for studies involving drug delivery to the CNS and subsequent assessment of pharmacokinetic and pharmacodynamic activity.

Delivery of therapeutics directly into the central nervous system is one way of circumventing the blood-brain barrier. The present protocol demonstrates intracerebroventricular injection for subsequent collection of cerebrospinal fluid and bodily organs. This facilitates the investigation of drug pharmacokinetics and pharmacodynamics in animal models for developing new treatments.

Although the blood-brain barrier (BBB) protects the brain from foreign entities, it also prevents some therapeutics from crossing into the central nervous ...

Mapping Cerebral Blood Flow and Electrical Fields: Electrode Placement in Mice

Placement of Extracranial Stimulating Electrodes and Measurement of Cerebral Blood Flow and Intracranial Electrical Fields in Anesthetized Mice

The detection of cerebral blood flow (CBF) responses to various forms of neuronal activation is critical for understanding dynamic brain function and variations in the substrate supply to the brain. This paper describes a protocol for measuring CBF responses to transcranial alternating current stimulation (tACS). Dose-response curves are estimated both from the CBF change occurring with tACS (mA) and from the intracranial electric field (mV/mm). We estimate the intracranial electrical field based on the different amplitudes measured by glass microelectrodes within each side of the brain. In this paper, we describe the experimental setup, which involves using either bilateral laser Doppler (LD) probes or laser speckle imaging (LSI) to measure the CBF; as a result, this setup requires anesthesia for the electrode placement and stability. We present a correlation between the CBF response and the current as a function of age, showing a significantly larger response at higher currents (1.5 mA and 2.0 mA) in young control animals (12-14 weeks) compared to older animals (28-32 weeks) (p &lt; 0.005 difference). We also demonstrate a significant CBF response at electrical field strengths &lt;5 mV/mm, which is an important consideration for eventual human studies. These CBF responses are also strongly influenced by the use of anesthesia compared to awake animals, the respiration control (i.e., intubated vs. spontaneous breathing), systemic factors (i.e., CO2), and local conduction within the blood vessels, which is mediated by pericytes and endothelial cells. Likewise, more detailed imaging/recording techniques may limit the field size from the entire brain to only a small region. We describe the use of extracranial electrodes for applying tACS stimulation, including both homemade and commercial electrode designs for rodents, the concurrent measurement of the CBF and intracranial electrical field using bilateral glass DC recording electrodes, and the imaging approaches. We are currently applying these techniques to implement a closed-loop format for augmenting the CBF in animal models of Alzheimer's disease and stroke.

Author Spotlight: Stimulation-Based Approach to Improve Cerebral Blood Flow in Alzheimer's Model 

We describe a protocol for assessing dose-response curves for extracranial stimulation in terms of brain electrical field measurements and a relevant biomarker-cerebral blood flow. Since this protocol involves invasive electrode placement into the brain, general anesthesia is needed, with spontaneous breathing preferred rather than controlled respirations.

The detection of cerebral blood flow (CBF) responses to various forms of neuronal activation is critical for understanding dynamic brain function and ...

Cerebrospinal Fluid and Blood Withdrawal from Rats with Concurrent EEG Recording

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

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

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

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

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

Osmotic Minipump Implantation for Increasing Glucose Concentration in Mouse Cerebrospinal Fluid

Diabetes increases the risk of cognitive decline and impairs brain function. Whether or not this relationship between high glucose and cognitive deficits is causal remains elusive. Moreover, whether these deficits are mediated by an increase in glucose levels in cerebrospinal fluid (CSF) and/or blood is also unclear. There are very few studies investigating the direct effects of high CSF glucose levels on central nervous system (CNS) function, especially on learning and memory, since current diabetes models are not sufficiently developed to address such research questions. This article describes a method to chronically increase CSF glucose levels for 4 weeks by continuously infusing glucose into the lateral ventricle using osmotic minipumps in mice. The protocol was validated by measuring glucose levels in CSF. This protocol increased CSF glucose levels to ~328 mg/dL after infusion of a 50% glucose solution at a 0.25 &#181;L/h flow rate, compared to a CSF glucose concentration of ~56 mg/dL in mice that received artificial cerebrospinal fluid (aCSF). Furthermore, this protocol did not affect blood glucose levels. Therefore, this method can be used to determine the direct effects of high CSF glucose on brain function or a specific neural pathway independently of changes in blood glucose levels. Overall, the approach described here will facilitate the development of animal models for testing the role of high CSF glucose in mediating features of Alzheimer&#39;s disease and/or other neurodegenerative disorders associated with diabetes.

This article describes a detailed protocol to increase glucose concentration in the cerebrospinal fluid (CSF) of mice. This approach can be useful for studying the effects of high CSF glucose on neurodegeneration, cognition, and peripheral glucose metabolism in mice.

Diabetes increases the risk of cognitive decline and impairs brain function. Whether or not this relationship between high glucose and cognitive deficits ...

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

Place the collar around the neck of the anesthetized guide cannula implanted animal, and remove the cap nut and dummy probe. Slowly insert the microdialysis probe through the guide cannula, and fasten the cap nut.
Then, place the mouse in a cage connected to a free moving system, and tether the mouse with the collar. After at least one hour, sequentially stop the roller pump and the syringe pump, restarting the pumps with the syringe pump set to 20% faster than the roller pump, and place the free end of the outlet tubing on a refrigerated fraction collector to collect the brain ISF. When the appropriate experimental volume has been obtained, remove the probe, and handle the mouse recovery as just demonstrated.

An Improved Method for Collection of Cerebrospinal Fluid from Anesthetized Mice

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