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 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 ....

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

1. Cannula preparation
   NOTE: Avoid touching the cannula with non-sterile gloves.
   1. Break off the beveled metal tip of a 30G dental needle using a needle holder.
   2. Using a needle holder, prepare the cannula by inserting the beveled metal tip (approximately 0.3 cm) into a 30-cm length of PE10 tubing (Polyethylene Tubing 0.024" OD x 0.011" ID) filled....

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.......

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.......

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.