This work was supported by the Novo Nordisk Foundation and National Institute of Neurological Disorders and Stroke, NINDS/NIH (M.N.). A.L.R.X. and S.H-R are recipients of a postdoctoral fellowship and a PhD scholarship from the Lundbeck Foundation, respectively.

All procedures were performed in accordance with the European Directive 2010/63/EU for animal research and were approved by the Animal Experiments Council under the Danish Ministry of Environment and Food (2015-15-0201-00535).
1. Procedure for Cannulation
<ol>
	<li>Cannula preparation 
	NOTE: Avoid touching the cannula with non-sterile gloves.
	<ol>
		<li>Break off the beveled metal tip of a 30G dental needle using a needle holder.</li>
		<li>Using a needle holder, prepare the cannula b...

<ol>
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The authors have nothing to disclose.

We have presented a protocol that describes a detailed procedure for cisterna magna cannulation (CMc), which offers a straightforward method to deliver labeled molecules to the CSF compartment. CMc allows the subsequent visualization of CSF dynamics, both in vivo and ex vivo, using different imaging modalities or histology.
One of the main advantages of the CMc technique lies in its direct access to the subarachnoid space without the need to expose the brain by craniotomy. By...

Cerebrospinal fluid (CSF) bathes the central nervous system (CNS) throughout the ventricular system and along the subarachnoid spaces, an anatomically defined space in continuum with the ventricles, which surrounds the brain and the spinal cord. One of the main functions of the CSF is to provide a route for clearance of metabolites and solutes from the brain parenchyma. Clearance is facilitated via the recently discovered glymphatic system1, the brain analog to the peripheral lymphatic system. Herein, we describe and discuss the cisterna magna cannulation (CMc), a minimally invasive method for the direct delivery of molecules into the CSF. CMc is the key method for studying the glymphatic function. Furthermore, CMc can also be applied for the study of CSF dynamics and for a fast, brain-wide delivery of non-blood brain barrier (BBB) permeable molecules into the brain parenchyma, along the perivascular space.
The CMc exploits physiological principles of CSF movement dynamics through the CNS to deliver labeled tracer molecules or drugs into the CSF-filled space of the cisterna magna (CM). Molecules are injected through a cannula implanted into the atlanto-occipital dural membrane covering the CM. Molecules are then carried by CSF bulk flow into the brain parenchyma via the paravascular space1. Tracer or contrast agent injected via the CMc follows the movement of CSF, which allows the assessment of CSF movement and glymphatic influx by quantifying intensity levels of labeled molecules that enter the brain parenchyma. CMc is compatible with different imaging techniques including epifluorescence, 2-photon microscopy, and magnetic resonance imaging (MRI). Also, this assessment can be performed both in vivo or ex vivo. Importantly, CMc allows for the visualization of the glymphatic system under anesthesia or during natural sleep, as well as in awake, freely moving animals.
The CMc technique can be utilized to study different aspects of fluid dynamics in the CSF, but has proven to be particularly useful for studying the glymphatic system. Glymphatic activity drives the convective flow of CSF from the periarterial space via aquaporin-4 (AQP-4) water channels, which are tethered in the membrane of astrocytic vascular-wrapping endfeet. The convective flow enables the interchange of CSF and interstitial fluid (ISF) within the brain parenchyma. CSF/ISF containing metabolic waste and solutes is then removed from the brain parenchyma via the perivenous space2,3. Ultimately, CSF/ISF reaches the periphery via the recently described dural lymphatic vessels4,5. The glymphatic system has been shown crucial for the clearance of harmful waste metabolites such as amyloid-&#946;2. Further, glymphatic clearance is impaired in aging6, after traumatic brain injury7, and in animal models of diabetes8 and Alzheimer&#39;s disease9. Notably, glymphatic activity is state dependent, showing significantly higher activity during sleep or anesthesia in comparison to wakefulness1. Indeed, young anesthetized animals exhibit the highest glymphatic activity. Thus, experimental quantification of glymphatic activity is critical when studying its role in health and disease.
Several studies have addressed CSF dynamics and its interchange with interstitial fluid (ISF) in the brain parenchyma. However, the methods by which labeled molecules are delivered are rather invasive, triggering brain parenchyma damage and changes in the intracranial pressure (ICP) (see review10). Some examples are intraventricular or intraparenchymal injections which involve craniotomy or drilling of a burr hole in the skull. These procedures have been shown to alter ICP, thus disrupting glymphatic function2. Also, such invasive methods induce astrogliosis and increase AQP-4 immunoreactivity in the brain parenchyma damaged area and its surroundings11,12. As astrocytes and AQP-4 are key elements of the glymphatic system, the CMc is the method of choice for its studies. The major advantages of CMc in comparison to more invasive procedures are the maintenance of an intact skull and brain parenchyma, avoiding ICP alterations and astrogliosis, respectively. Thus, CMc in conjunction with different imaging tools opens for a wide range of possibilities to study not only the glymphatic system, but also the dynamics and mechanisms of fluid flow in homeostasis, as well as in animal models of neurological diseases.
The cisterna magna cannulation (CMc) procedure allows easy and direct access to the cerebrospinal fluid (CSF). By injecting different molecules (e.g. fluorescent tracers, MRI contrast agents) the experimenter can track their movement within the CSF compartment and assess the activity of the glymphatic system. The following protocol describes both the acute CMc, for injections immediately following the surgery, and chronic implantation of the cannula, in which the animal recovers from the surgical procedure for a later injection. The most important difference between the acute and chronic implantation is that the chronic implantation allows for the study of glymphatic activity in awake mice.

<table border="0" cellpadding="0" cellspacing="0" width="1153">
	<tbody>
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			<td height="21" style="height:21px;width:485px;">SOPIRA Carpule 30G 0.3 x 12mm</td>
			<td style="width:245px;">Kulzer</td>
			<td style="width:257px;">AA001</td>
			<td style="width:167px;"></td>
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			<td height="21" style="height:21px;width:485px;">Polyethylene Tubing 0.024&#8221; OD x 0.011&#8221; ID</td>
			<td style="width:245px;">Scandidact</td>
			<td>PE10-CL-500</td>
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			<td style="width:245px;">Chirana T. Injecta</td>
			<td>CHINS01</td>
			<td></td>
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			<td style="width:245px;">Meda AS</td>
			<td style="width:257px;">no catalogue number, see link in comments</td>
			<td>http://www.meda.dk/behandlingsomrader/desinfektion/desinfektion-af-hud/klorhexidin-sprit-medic-05/</td>
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			<td>Vitrex Medical A/S</td>
			<td>520213</td>
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			<td>Ophtha</td>
			<td>145250</td>
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			<td height="42" style="height:42px;width:485px;">Wooden applicator, Double cotton bud (&#216; appr. 4 - 5 mm, length appr. 12 mm)</td>
			<td style="width:245px;">Heinz Herenz</td>
			<td style="width:257px;">1032018</td>
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			<td>Medicom</td>
			<td>A18005</td>
			<td></td>
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			<td height="21" style="height:21px;">Ferric chloride 10% solution</td>
			<td style="width:245px;">Algeos</td>
			<td style="width:257px;">NV0382</td>
			<td></td>
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			<td height="21" style="height:21px;">Kimtech Science Precision Wipes Tissue Wipers</td>
			<td style="width:245px;">Kimberly Clark Professional</td>
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			<td height="24" style="height:24px;">Loctite Super Glue Precision 5g</td>
			<td style="width:245px;">Loctite</td>
			<td style="width:257px;">no catalogue number, see link in comments</td>
			<td>http://www.loctite-consumer.dk/da/produkter/superglue-liquid.html&#160;</td>
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			<td height="21" style="height:21px;">Insta-Set CA Accelerator</td>
			<td style="width:245px;">Bob Smith Industries</td>
			<td style="width:257px;">BSI-152</td>
			<td></td>
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			<td height="21" style="height:21px;">Dental Cement Powder</td>
			<td style="width:245px;">A-M Systems</td>
			<td style="width:257px;">525000</td>
			<td></td>
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			<td height="21" style="height:21px;">Surgical weld&#160;</td>
			<td style="width:245px;">Kent Scientific Corporation</td>
			<td style="width:257px;">INS750391</td>
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			<td height="24" style="height:24px;width:485px;">Hamilton syringe GASTIGHT&#174; , 1700 series, 1710TLL, volume 100 &#956;L, PTFE Luer lock</td>
			<td>Hamilton syringes</td>
			<td style="width:257px;">1710TLL</td>
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			<td style="width:245px;">KD Scientific</td>
			<td style="width:257px;">788130</td>
			<td></td>
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			<td height="21" style="height:21px;width:485px;">Paraformaldehyde powder, 95%</td>
			<td style="width:245px;">Sigma Aldrich</td>
			<td style="width:257px;">158127</td>
			<td></td>
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			<td height="21" style="height:21px;width:485px;">Phosphate buffered saline (PBS; 0.01M; pH 7.4)</td>
			<td style="width:245px;">Sigma Aldrich</td>
			<td style="width:257px;">P3813</td>
			<td></td>
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			<td style="width:245px;">Thermo Fisher Scientific</td>
			<td style="width:257px;">O34784</td>
			<td></td>
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			<td height="24" style="height:24px;width:485px;"> 
			DAPI (diamidino-2-phenylindole) Solution (1 mg/mL)</td>
			<td style="width:245px;">Thermo Fisher Scientific</td>
			<td style="width:257px;">62248</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:485px;">Dextran, Fluorescein, 3000 MW, Anionic</td>
			<td style="width:245px;">Thermo Fisher Scientific</td>
			<td style="width:257px;">D3305</td>
			<td></td>
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			<td height="21" style="height:21px;">E-Z Anesthesia EZ-7000 Classic System</td>
			<td style="width:245px;">E-Z Systems</td>
			<td>EZ-7000</td>
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			<td style="width:245px;">ScanVet</td>
			<td>55226</td>
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			<td style="width:245px;">ScanVet</td>
			<td style="width:257px;">545349</td>
			<td></td>
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			<td height="21" style="height:21px;width:485px;">Ketaminol Vet 100 mg/mL (ketamine)</td>
			<td>Intervet International BV</td>
			<td style="width:257px;">511519</td>
			<td></td>
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			<td height="42" style="height:42px;width:485px;">Rompin Vet 20 mg/mL (xylazin)</td>
			<td style="width:245px;">KVP Pharma + Veterin&#228;r Produkte GmbH</td>
			<td style="width:257px;">148999</td>
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			<td height="21" style="height:21px;width:485px;">Xylocain 20 mg/mL (lidocain)</td>
			<td style="width:245px;">AstraZeneca</td>
			<td style="width:257px;">158543</td>
			<td></td>
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			<td height="21" style="height:21px;width:485px;">Marcain 2.5 mg/mL (bupivacain)</td>
			<td style="width:245px;">AstraZeneca</td>
			<td style="width:257px;">123918</td>
			<td></td>
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			<td height="21" style="height:21px;width:485px;">Bupaq Vet 0.3 mg/mL (buprenorphine)</td>
			<td style="width:245px;">Richter Pharma AG</td>
			<td style="width:257px;">185159&#160;</td>
			<td></td>
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	</tbody>
</table>

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).

Upon fixation of mice or rats in a stereotaxic frame, the neck muscles around the occipital crest region are bluntly dissected to expose the cisterna magna (CM). The triangular structure of the CM is readily recognized between the caudal portion of the cerebellum and the medulla (Figure 1A-1C). The cannula is inserted 1 - 2 mm into the CM by gently piercing the atlanto-occipital membrane (Figure 1D). The dura mem...

Watch this Scientific Journal Video about Cannula Implantation into the Cisterna Magna of Rodents at JoVE.com

Cannula Implantation into the Cisterna Magna of Rodents

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 ...

The overall goal of this surgical procedure is to enable direct delivery of tracers, substrates and signaling molecules into the cerebrospinal fluid. This method can help to answer key questions in the neuroscience filed regarding CSF and ISF dynamics in the brain. This method allows the delivery of different molecules, tracers, active metabolic substrates into the CSF, cerebrospinal fluid, compartment without damaging the skull or the brain parenchyma.The implications of this technique extend towards neurological diseases because it allows the evaluation of CSF-ISF fluxes in the brain and also the clearance of metabolites and toxic proteins from the brain parenchyma. So this method provides insight into the dynamics of the CSF-ISF fluxes and additionally we can use it as a fast, brain-wide delivery route for signaling molecules and molecular substrates that could not otherwise pass the blood-brain barrier. To prepare the cannula, break of the beveled metal tip of a 30 gauge dental needle using a needle holder.Then, insert the beveled metal tip into a 30 centimeter length of PE-10 tubing filled with aCSF. Next, use a one milliliter syringe fitted with a 30 gauge needle to flush the cannula with artificial cerebrospinal fluid. The cannula is then ready for use in the surgery.After anesthetizing the mouse, position the animal over a heating pad on a stereotaxic apparatus. Check for a lack of pedal reflex with a toe pinch, and wait for the animal's respiration to become slow and steady. Next, shave the head and neck.Then, wipe away the fur and sterilize the exposed skin. First, scrub the skin with alcohol. Then make two applications of 0.5 per cent chlorohexidine or two per cent iodine solution.For chronic cannulations, also administer analgesics at the cannulation site. Now, fix the mouse in the stereotaxic frame, making use of either the intraneural arch or the zygomatic arch. Then, tilt the head slightly so that it forms an angle of 120 degrees to the body.Next, apply ophthalmic ointment and reapply it as needed during the surgery. Find the occipital crest, which is the part of the skull protruding immediately above the neck muscles. Lift the overlying skin using a pair of tweezers and cut an almond-shaped piece of skin of approximately one centimeter along the midline.Then, using the occipital crest as a reference point, pull apart the superficial connective tissue to expose the neck muscles below. Use cotton swabs or eye spears to control any resultant bleeding. To proceed, separate the muscles at the midline by carefully running the tweezers down the middle of the incision site in the anterior to posterior axis.Then, with a pair of curved forceps in each hand, join the tips in the middle near the bottom of the skull and pull the muscles aside to expose the cisterna magna. The cisterna magna looks like a tiny inverted triangle, outlined by the cerebellum above and medulla below. Using a surgical eye spear or cotton swab, wipe the dural membrane covering the CM.Next, with curved tweezers in the dominant hand, grasp the cannula near the tube-covered needle.Then, place the middle finger of the other hand on the ear bar to create a stable rest for the cannula. Now, at the center of the CM, puncture the dura at 45 degrees relative to the mouse head. Only insert the needle to where the bevel is under the dura to avoid penetrating into the cerebellum or medulla.If necessary, clean off any leaked CSF with a spear or swab. Then, apply a few drops of cyanoacrylate onto the dural membrane surrounding the cannula and add a drop of glue accelerator to cure the glue immediately. Next, combine dental cement and cyanoacrylate glue.Then, cover the entire incision site and quickly apply a drop of glue accelerator to cure. For injection in mice with acute cisterna magna cannulation, proceed directly to injection of CSF tracers with the aid of an injection pump. For chronic cannulation, cut the tubing down to two or three centimeters and seal it with a surgical weld to retain the inter-cranial pressure levels.Then, administer carprofen. Now, house the mouse alone and keep the cage over a heating pad until the mouse has regained sternal recumbency. To prepare the cannula, break off the beveled metal tip of a 30 gauge dental needle using a needle holder.Then, insert the beveled metal tip into a 30 centimeter length of PE-10 tubing filled with aCSF. Using a 30 gauge needle, flush the tubing. Attach the 30 gauge needle to a 100 microliter syringe filled with distilled water and then attach this to a syring pump.Using the pump, withdraw enough air to create a one centimeter bubble in the cannula. Then, dip the needle into the CSF tracer solution and withdraw 12 microliters into the cannula. Cut the tubing attached to the cannula up to three to four centimeters length and quickly connect the CSF tracer-filled tubing with the implanted cannula with the aid of fine forceps.To inject CSF tracers into the CM of anesthetized animals, use a syringe pump to deliver tracers at one microliter per minute for five to ten minutes. Then allow the tracers to circulate for 30 minutes without moving the cannula. Gently restrain the animal and cut off approximately one centimeter of the tubing from the implanted cannula.Then, quickly connect the two cannulas, using the aid of a fine forceps and a pair of curved tweezers if necessary. Now, inject one microliter of tracer per minute over seven to twelve minutes, using a syring pump. After the injection, let the tracer circulate for 30 minutes while taking the necessary measures to ensure that the cannula remains undisturbed and the tubing remains attached.Then proceed with harvesting the brain. Mouse brains were injected with CSF tracers into the CM as described. A macroscopic dorsal view shows the distribution of the tracers in the subarachnoid cisterns of the cerebellopontine region in the olfactory bulb and in the paravascular space along the middle cerebral arteries.In the ventral portion of the brain, a macroscopic view shows the CSF tracer distribution along the Circle of Willis. Histological sections of CM-injected brains further reveal the paravascular distribution of tracers within the brain parenchyma. Mice injected under anesthesia or while asleep had much more tracer distribution than mice injected while awake and freely moving in their home cage.After watching this video, you should gain a good knowledge of how to perform cisterna magna cannulation that will allow you to deliver chemicals or molecules of interest into the cerebrospinal fluid compartment without any operative damage to the skull or to the brain parenchyma. Once mastered, this technique can be performed in between 20 to 30 minutes if performed properly. While doing this procedure, it is important to apply the correct amount of pressure when puncturing the dura mater to avoid major CSF leakage that could interfere with the intercranial pressure.After its development, this cisterna magna cannulation has paved the way for studies in the CSF-ISF clearance in the brain via the so-called lymphatic system that nowadays is widely used in different animal models of neurological diseases.

cannula-implantation-into-the-cisterna-magna-of-rodents

Research

JoVE Journal

Neuroscience

42.5K vistas.  University of Copenhagen. The overall goal of this surgical procedure is to enable direct delivery of tracers, substrates and signaling molecules into the cerebrospinal fluid. This method can help to answer key questions in the neuroscience filed regarding CSF and ISF dynamics in the brain. This method allows the delivery of different molecules, tracers, active metabolic substrates into the CSF, cerebrospinal fluid, compartment without damaging the skull or the brain parenchyma.The implications of this techniqu...