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 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).
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-β2. Further, glymphatic clearance is impaired in aging6, after traumatic brain injury7, and in animal models of diabetes8 and Alzheimer'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.
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
2. Injection of CSF Tracers via Acute Implanted CM Cannula in Anesthetized Animals
NOTE: For injection of CSF tracers via acute implanted CM cannula in anesthetized animals, immediately after step 8 from previous section, proceed to CSF tracer injection as described below.
3. Injection of CSF Tracers via Chronically Implanted CM cannula in Awake Animals
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 membrane is a tough structure and the insertion of the cannula is improved by tilting the animal head by a 120° in relation to the body. With the aid of an injection pump, differently labeled molecules are then injected into the cisterna magna at controlled rates (Figure 1E). After an interval to allow CSF tracer circulation, animals are euthanized. The brain is carefully dissected and fixed by immersion in 4% PFA o/n at 4 °C. Macroscopic dorsal views of brains harvested from CM-injected rodents show the distribution of CSF tracers in the subarachnoid cisterns of the cerebellum, in the olfactory bulb and in the paravascular space along the middle cerebral arteries (MCAs) (Figure 1F). In the ventral portion of the brain, macroscopic views show CSF tracer distribution along the Circle of Willis (Figure 1G). Histological sections of CM-injected brains further reveal the paravascular distribution of tracers within the brain parenchyma. Mice injected under anesthesia (Figure 1H) (or during natural sleep, see1) show a remarkable increase in tracer distribution into the brain parenchyma in comparison to mice injected while awake and freely moving in their home cage (Figure 1I).
Figure 1: Injection of tracers into the cisterna magna. (A) Schematic overview of the mouse head and brain showing the location of the cisterna magna (CM) in relation to the brain and cranial structures. (B) Photomicrograph of exposed CM after the surrounding neck muscles have been bluntly dissected and pushed to the sides. (C) Higher magnification of the area depicted in B (black rectangle), showing the inverted triangular structure of the CM (dashed line) and its location in relation to the surrounding structures, i.e. occipital crest, cerebellum, and medulla. (D) Photomicrography of the cannula inserted into the CM. (E) Scheme of the lateral view of the mouse fixed head, slightly tilted at an angle of 120° in relation to the body. Inset of dashed rectangle delimited area shows the scheme of a parasagittal view scheme of a parasagittal view of the mouse brain with the cannula inserted into the CM, as outlined in E. A syringe, which is coupled to an injection pump, is used to deliver CSF tracers or contrast agents into the CM through a tube connected to a fine 30G needle. Representative images of a whole mouse brain at 30 min after the end of CM injection with a fluorescent tracer seen from the dorsal (F) and ventral (G) aspects. (H, I,) Representative coronal brain sections counterstained with DAPI (4',6-diamidino-2-phenylindole; 1 µg/mL in PBS) of mice injected with CSF tracers into the CM under anesthetized (H) and wakefulness (I), 30 min after the end of CM injection at a rate of 1 µL/min of a 5 µL volume of a mixture of ovalbumin-AF647 conjugate (OA, 45kDa; 2% in aCSF) and dextran-FITC conjugate (DEX, 3kDa; 2% in aCSF). Scale bars, 5 mm for B, C, F, G; 2 mm for D, and 500 µm for H, I. Cb, cerebellum; CM, cisterna magna; Ctx, cortex; and OB, olfactory bulb. Please click here to view a larger version of this figure.
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 not requiring a cranial window or penetration of the brain parenchyma with a needle tip, CMc allows the delivery of molecules into the CSF compartment and the assessment of the glymphatic system by a minimally invasive procedure, with only brief disturbance of intracranial pressure (ICP).
Notably, the injection into the CM is downstream of the main sources of CSF, the choroid plexi located in the ventricular system (lateral, third and fourth ventricles). From the lateral ventricles, CSF flows to the third ventricle via the intraventricular foramina (the foramen of Monro) and from the third to fourth ventricles via the cerebral aqueduct (the aqueduct of Sylvius) to the brain stem and spinal cord (reviewed in3). CSF reaches the subarachnoid space via the CM by flowing through the median aperture (or the foramen of Magendie), and thus CMc injections bypass the entire ventricular system. However, while this may be problematic in some models of CSF/ISF dynamics through the ventricles, direct injection of tracers into the ventricles requires invasive surgical procedures such as drilling of burr holes in the skull windows, and the application of ventricular injections substantially disrupt the ICP13. Likewise, pressure injection of tracers into the subarachnoid space13,14 in our hands abolishes the flux of CSF tracers along the paravascular space. In contrast, even though CMc entails puncture of the dural membrane, ICP is only transiently perturbed and is quickly restored2.
Using the CMc, glymphatic activity can be measured in anesthetized animals after acute CMc, as well as in awake animals, observing a 24-hour recovery period upon cannula implantation. Acute CMc is suited for combination with 2-photon imaging, which provides detailed information about glymphatic activity within cortex to a depth of approximately 200 µm1,2. Importantly, acute CMc also affords the advantage of supporting unbiased MRI studies, where tracer distribution is followed dynamically, relative to an individual baseline image acquired before the initiation of CSF tracer injection15,16,17. For MRI, the dental needle used for CMc should be replaced by a borosilicate capillary (approximately 1 cm length, tip diameter of approximately 20 µm) attached to the PE tubing.
In contrast to acute cannulation, chronic CMc allows the experimenter to perform CSF tracer injection in animals during natural sleep or under anesthesia, as well as in awake, freely moving animals. This is a crucial factor since glymphatic activity is highly state-dependent; tracer influx to the parenchyma is much greater in animals that were injected under anesthesia or asleep than in animals that were injected in the awake state1. In addition, animals with a chronically implanted cannula can receive CSF tracer in their home cage, thus minimizing confounding factors due to effects of stress and arousal on glymphatic activity. For chronic injections under anesthesia, a mixture of ketamine/xylazine (100 mg/kg; 10 mg/kg, respectively) is recommended. Isoflurane at concentrations above 1.5% induces brain swelling and does not enhance glymphatic activity compared to the awake state1. Note that after CMc implantation, animals should be single housed, in order to assure that CMc implanted animals will not damage the cannula of each other. Also, since the CMc chronic implantation is a recovery surgical procedure, it should be performed under sterile conditions and animals should receive post-operational analgesics.
Importantly, CMc can be used as a method to deliver CSF tracers in mice as well as in rats, with minimal modifications to the protocol. Appropriate anesthetics dose should be administered and the maximum volume of CSF tracer that is injected in rats is 30 µL, due to the differences in the size of the ventricular and subarachnoid spaces between the two species.
Despite its procedural simplicity, some training and practice is required for the experimenter to successfully perform CMc. Since the CM varies in size in between species and individual animals, it is advisable to practice the recognition of its structure. Practicing the procedure using Evans Blue (2% in aCSF) allows the experimenter to confirm correct needle insertion. Occasionally, a vessel will be located directly at the midline of the CM, whereupon the needle should be inserted adjacent to the vessel, but as close as possible to the midline. These cases should be noted, for later confirmation that tracers are evenly distributed, despite the off-center placement of the needle tip. Importantly, the atlanto-occipital membrane covering the CM is mechanically tough, and sufficient pressure should be applied to insert the beveled needle tip. However, it is critical that the pressure applied does not result in plunging the needle tip into the medulla or the cerebellum. To facilitate needle insertion into the CM, the head of animals should be tilted downwards at an angle of 120° relative to the body, which stretches the membrane. Importantly, caution should be taken not to obstruct respiration by this head flexion. If the needle tip should enter the cerebellum, tracers will be retained in the tissue and fail to distribute throughout the subarachnoid space. Damage to the medulla is frequently fatal, whereas cerebellum damage in chronic cannulations can result in prostration and general abnormalities in the behavior of the animals. To minimize the risk of this eventuality, needles with a smaller bevel length can be used.
When moving the muscles in the neck region that covers the dura membrane to insert the cannula into the CM, bleeding can occur. Cotton swabs can be used to absorb the bleeding, but alternatively, ferric chloride solution can be applied. Ferric chloride has a hemostatic effect18, and also triggers the stiffening of neck muscles around the incision site, thus helping to obtain correct insertion of the needle into the CM. Ferric chloride also dries out the skull and dural membrane, presenting better surfaces for adhesion of the cannula. For CMc, apply 1 – 2 drops of ferric chloride solution (10%) (approximately 1 mL) into a cotton swab and dab the neck muscles and the occipital crest. However, topical ferric chloride may possibly seep through the membrane into the CSF, with unknown effects on brain homeostasis. If the use of ferric chloride is a matter of concern, one can instead use wound retractors to keep open the incision site. Careful removal of wound retractors after applying the cyanoacrylate glue avoids inadvertent attachment to the incision site.
CMc is a straightforward and reproducible procedure to deliver molecules directly into the CSF compartment. Since CMc is minimally invasive, it is the preferred method for the visualization of the glymphatic system and can be combined with different imaging modalities such as epifluorescence and 2-photon microscopy or MRI. Thus, CMc represents a great tool for studies of fluid dynamics, namely CSF and ISF, and also of brain fluid clearance. Due to the macroscopic coverage of the glymphatic system, CMc has the potential to be used to deliver molecules brain-wide.
The authors have nothing to disclose.
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.
SOPIRA Carpule 30G 0.3 x 12mm | Kulzer | AA001 | |
Polyethylene Tubing 0.024” OD x 0.011” ID | Scandidact | PE10-CL-500 | |
30G x ½” 0.3 x 12 mm Luer-Lock | Chirana T. Injecta | CHINS01 | |
Chlorhexidine 0.5% (chlorhexidine digluconate) | Meda AS | no catalogue number, see link in comments | http://www.meda.dk/behandlingsomrader/desinfektion/desinfektion-af-hud/klorhexidin-sprit-medic-05/ |
Alcohol Swab 70% Isopropyl Alcohol 30 x 60mm | Vitrex Medical A/S | 520213 | |
Viskoese Oejendraeber Ophtha | Ophtha | 145250 | |
Wooden applicator, Double cotton bud (Ø appr. 4 – 5 mm, length appr. 12 mm) | Heinz Herenz | 1032018 | |
Eye spears | Medicom | A18005 | |
Ferric chloride 10% solution | Algeos | NV0382 | |
Kimtech Science Precision Wipes Tissue Wipers | Kimberly Clark Professional | 05511 | |
Loctite Super Glue Precision 5g | Loctite | no catalogue number, see link in comments | http://www.loctite-consumer.dk/da/produkter/superglue-liquid.html |
Insta-Set CA Accelerator | Bob Smith Industries | BSI-152 | |
Dental Cement Powder | A-M Systems | 525000 | |
Surgical weld | Kent Scientific Corporation | INS750391 | |
Hamilton syringe GASTIGHT® , 1700 series, 1710TLL, volume 100 μL, PTFE Luer lock | Hamilton syringes | 1710TLL | |
LEGATO 130 Syringe pump | KD Scientific | 788130 | |
Paraformaldehyde powder, 95% | Sigma Aldrich | 158127 | |
Phosphate buffered saline (PBS; 0.01M; pH 7.4) | Sigma Aldrich | P3813 | |
Ovalbumin, Alexa Fluor 647 Conjugate | Thermo Fisher Scientific | O34784 | |
DAPI (diamidino-2-phenylindole) Solution (1 mg/mL) |
Thermo Fisher Scientific | 62248 | |
Dextran, Fluorescein, 3000 MW, Anionic | Thermo Fisher Scientific | D3305 | |
E-Z Anesthesia EZ-7000 Classic System | E-Z Systems | EZ-7000 | |
Attane Isofluran 1000 mg/g | ScanVet | 55226 | |
Euthanimal 200mg/mL (sodium pentobarbital) | ScanVet | 545349 | |
Ketaminol Vet 100 mg/mL (ketamine) | Intervet International BV | 511519 | |
Rompin Vet 20 mg/mL (xylazin) | KVP Pharma + Veterinär Produkte GmbH | 148999 | |
Xylocain 20 mg/mL (lidocain) | AstraZeneca | 158543 | |
Marcain 2.5 mg/mL (bupivacain) | AstraZeneca | 123918 | |
Bupaq Vet 0.3 mg/mL (buprenorphine) | Richter Pharma AG | 185159 |