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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Animal Preparation
<ol>
	<li>Sedate a pig by intramuscular (i.m.) injection of tiletamine (3.75 mg/kg) and zolazepam (3.75 mg/kg) and dexmedetomidine (37.5 &#956;g/kg). Wait for it to become unconscious.</li>
	<li>Prepare an intravenous line by inserting a 20 G cannula into the ear vein. 
	NOTE: Make sure the cannula is in t...

locating the cisterna magna in a pig model: a surgical method to access the cisterna magna in a pig model for direct cannulation

Source: B&#232;chet, N. B., et al. <a href="https://www.jove.com/v/62641">Direct Cannula Implantation in the Cisterna Magna of Pigs.</a> J. Vis. Exp. (2021).
In this video, we describe a method to access the cisterna magna in a pig model. The cisterna magna is a preferred site for studies concerning cerebrospinal fluid.

<img alt="Figure 1" src="/files/ftp_upload/20821/20821_Figure1.jpg" /> 
Figure 1. Cisterna magna cannulation in pigs. (A)&#160;Pig prepped prior to the start of the surgery and marked where dermal incisions will be performed starting from the occipital crest (OC) then posterior to thoracic vertebrae (TV) and lateral to each ear base (EB). (B)&#160;Head in the relaxed position with the trapezius, semispinalis capitus biventer and semi...

Locating the Cisterna Magna in a Pig Model: A Surgical Method to Access the Cisterna Magna in a Pig Model for Direct Cannulation

Source: B&#232;chet, N. B., et al. Direct Cannula Implantation in the Cisterna Magna of Pigs. J. Vis. Exp. (2021).
In this video, we describe a method to ...

The cisterna magna is an opening in the subarachnoid space, wherein cerebrospinal fluid, or CSF, produced in the ventricular system, drains. This makes the cisterna magna a preferred target for studying CSF circulation.
To access the cisterna magna in a pig model, prep the anesthetized animal in a prone position. Mark the surface anatomy of the animal, starting from the occipital crest towards the thoracic vertebra and each ear base to outline a path for the dermal incisions. Start by making an incision from the occipital crest to the first thoracic vertebra.
Next, incise along the shoulders to further slit the skin. Finally, make incisions from the occipital crest towards the base of each ear. Now, retract the incised skin to expose the underlying neck muscles. Next, dissect the neck muscles and pull them away to reveal the atlas or the first cervical vertebra and the skull base. Next, maneuver the animal&#39;s head in an appropriate direction to open the space between the back of the skull and the atlas to access the cisterna magna.&#160;
The cisterna magna can now be directly cannulated for further studies.&#160;

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Research

JoVE Encyclopedia of Experiments

Biology

Large Animal Models

Porcine

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.

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 ...

JoVE Journal

Neuroscience

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 ...

A Pilot Study on the Repetitive Transcranial Magnetic Stimulation of A&#946; and Tau Levels in Rhesus Monkey Cerebrospinal Fluid

Previous studies have demonstrated that a non-invasive light-flickering regime and auditory tone stimulation could affect A&#946; and tau metabolism in the brain. As a non-invasive technique, repetitive transcranial magnetic stimulation (rTMS) has been applied for the treatment of neurodegenerative disorders. This study explored the effects of rTMS on A&#946; and tau levels in rhesus monkey cerebrospinal fluid (CSF). This is a single-blind, self-controlled study. Three different frequencies (low frequency, 1 Hz; high frequencies, 20 Hz and 40 Hz) of rTMS were used to stimulate the bilateral-dorsolateral prefrontal cortex (DLPFC) of the rhesus monkey. A catheterization method was used to collect CSF. All samples were subjected to liquid chip detection to analyze CSF biomarkers (A&#946;42, A&#946;42/A&#946;40, tTau, pTau). CSF biomarker levels changed with time after stimulation by rTMS. After stimulation, the A&#946;42 level in CSF showed an upward trend at all frequencies (1 Hz, 20 Hz, and 40 Hz), with more significant differences for the high-frequencies (p &lt; 0.05) than for the low frequency. 
After high-frequency rTMS, the total Tau (tTau) level of CSF immediately increased at the post-rTMS timepoint (p &lt; 0.05) and gradually decreased by 24 h. Moreover, the results showed that the level of phosphorylated Tau (pTau) increased immediately after 40 Hz rTMS (p &lt; 0.05). The ratio of A&#946;42/A&#946;40 showed an upward trend at 1 Hz and 20 Hz (p &lt; 0.05). There was no significant difference in the tau levels with low-frequency (1 Hz) stimulation. Thus, high-frequencies (20 Hz and 40 Hz) of rTMS may have positive effects on A&#946; and tau levels in rhesus monkey CSF, while low-frequency (1 Hz) rTMS can only affect A&#946; levels.

Here, we describe the procedure for a pilot study to explore the effect of repetitive transcranial magnetic stimulation with different frequencies (1 Hz/20 Hz/40 Hz) on A&#946; and tau metabolism in rhesus monkey cerebrospinal fluid.

Previous studies have demonstrated that a non-invasive light-flickering regime and auditory tone stimulation could affect A&#946; and tau metabolism in ...

Locating the Cisterna Magna in a Pig Model: A Surgical Method to Access the Cisterna Magna in a Pig Model for Direct Cannulation

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