This work was supported by the Knut and Alice Wallenberg Foundation, Hj&#228;rnfonden, Wenner Gren Foundations, and the Crafoord foundation.

All procedures were carried out in accordance with the European directive 2010/63/EU and were approved by Malm&#246;-Lund Ethical committee on Animal Research (Dnr 5.8.18-05527/2019) and conducted according to the CODEX guidelines of the Swedish Research Council.
1. Preparation
<ol>
	<li>Tracer
	<ol>
		<li>Prepare artificial CSF (126 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 2 mM MgCl2, 2 mM CaCl2, 10 mM glucose, 26 mM NaHCO3; pH 7.4)</li>
	...

<ol>
	<li>Redzic, Z. B., Segal, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+the+choroid+plexus+and+the+physiology+of+the+choroid+plexus+epithelium.">The structure of the choroid plexus and the physiology of the choroid plexus epithelium.</a> Advanced Drug Delivery Reviews. 56 (12), 1695-1716 (2004).</li><li>Sakka, L., Coll, G., Chazal, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+and+physiology+of+cerebrospinal+fluid.">Anatomy and physiology of cerebrospinal fluid.</a> European Annals of Otorhinolaryngology, Head and Neck Diseases. 128 (6), 309-316 (2011).</li><li>Nedergaard, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Garbage+truck+of+the+brain.">Garbage truck of the brain.</a> Science. 340 (6140), 1529-1530 (2013).</li><li>Iliff, J. 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=A+Paravascular+pathway+facilitates+csf+flow+through+the+brain+parenchyma+and+the+clearance+of+interstitial+solutes,+including+amyloid+B.">A Paravascular pathway facilitates csf flow through the brain parenchyma and the clearance of interstitial solutes, including amyloid B.</a> Science Translational Medicine. 4 (147), (2012).</li><li>Xie, 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=Sleep+drives+metabolite+clearance+from+the+Adult+Brain.">Sleep drives metabolite clearance from the Adult Brain.</a> Science. 342 (6156), 373-378 (2013).</li><li>Mestre, H., 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=Aquaporin-4-dependent+glymphatic+solute+transport+in+the+rodent+brain.">Aquaporin-4-dependent glymphatic solute transport in the rodent brain.</a> eLife. 7, 40070 (2018).</li><li>Ringstad, G., 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=Brain-wide+glymphatic+enhancement+and+clearance+in+humans+assessed+with+MRI.">Brain-wide glymphatic enhancement and clearance in humans assessed with MRI.</a> JCI Insight. 3 (13), 121537 (2018).</li><li>Lundgaard, I., Wang, W., Eberhardt, A., Vinitsky, H. S., Cameron, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beneficial+effects+of+low+alcohol+exposure,+but+adverse+effects+of+high+alcohol+intake+on+glymphatic+function.">Beneficial effects of low alcohol exposure, but adverse effects of high alcohol intake on glymphatic function.</a> Scientific Reports. , 1-16 (2018).</li><li>Munk, A. S., 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=PDGF-B+is+required+for+development+of+the+glymphatic+system.">PDGF-B is required for development of the glymphatic system.</a> Cell Reports. 26 (11), 2955-2969 (2019).</li><li>Plog, B. A., 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=Transcranial+optical+imaging+reveals+a+pathway+for+optimizing+the+delivery+of+immunotherapeutics+to+the+brain.">Transcranial optical imaging reveals a pathway for optimizing the delivery of immunotherapeutics to the brain.</a> JCI Insight. 3 (20), 1-15 (2018).</li><li>Bechet, N. B., 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=Light+sheet+fluorescence+micrscopy+of+optically+cleared+brains+for+studying+the+glymphatic+system.">Light sheet fluorescence micrscopy of optically cleared brains for studying the glymphatic system.</a> Journal of Cerebral Blood Flow &amp; Metabolism. 40 (10), 1975-1986 (2020).</li><li>Xavier, A. L. 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=Cannula+implantation+into+the+cisterna+magna+of+rodents.">Cannula implantation into the cisterna magna of rodents.</a> Journal of Visualized Experiments. (135), e57378 (2018).</li><li>Ramos, 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=Cisterna+magna+injection+in+rats+to+study+glymphatic+function.">Cisterna magna injection in rats to study glymphatic function.</a> Methods in Molecular Biology. 1938, (2019).</li><li>Sweeney, A. 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=in+vivo+imaging+of+cerebrospinal+fluid+transport+through+the+intact+mouse+skull+using+fluorescence+macroscopy.">in vivo imaging of cerebrospinal fluid transport through the intact mouse skull using fluorescence macroscopy.</a> Journal of visualized experiments. (149), e59774 (2019).</li><li>Eide, P. K., Ringstad, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MRI+with+intrathecal+MRI+gadolinium+contrast+medium+administration:+A+possible+method+to+assess+glymphatic+function+in+human+brain.">MRI with intrathecal MRI gadolinium contrast medium administration: A possible method to assess glymphatic function in human brain.</a> Acta Radiologica Open. 4 (11), 205846011560963 (2015).</li><li>Ringstad, G., Vatnehol, S. A. S., Eide, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glymphatic+MRI+in+idiopathic+normal+pressure+hydrocephalus.">Glymphatic MRI in idiopathic normal pressure hydrocephalus.</a> Brain. 140 (10), 2691-2705 (2017).</li><li>Kornum, B. R., Knudsen, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cognitive+testing+of+pigs+(Sus+scrofa)+in+translational+biobehavioral+research.">Cognitive testing of pigs (Sus scrofa) in translational biobehavioral research.</a> Neuroscience and Biobehavioral Reviews. 35 (3), 437-451 (2011).</li><li>B&#232;chet, N. B., Shanbhag, N. C., Lundgaard, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glymphatic+function+in+the+gyrencephalic+brain.">Glymphatic function in the gyrencephalic brain.</a> Journal of Cerebral Blood Flow &amp; Metabolism. , (2021).</li><li>Raghunandan, A., 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=Bulk+flow+of+cerebrospinal+fluid+observed+in+periarterial+spaces+is+not+an+artifact+of+injection.">Bulk flow of cerebrospinal fluid observed in periarterial spaces is not an artifact of injection.</a> bioRxiv. , (2020).</li><li>D'Angelo, A., 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=Spinal+fluid+collection+technique+from+the+atlanto-occipital+space+in+pigs.">Spinal fluid collection technique from the atlanto-occipital space in pigs.</a> Acta Veterinaria Brno. 78 (2), 303-305 (2009).</li><li>Ma, Q., 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=Rapid+lymphatic+efflux+limits+cerebrospinal+fluid+flow+to+the+brain.">Rapid lymphatic efflux limits cerebrospinal fluid flow to the brain.</a> Acta Neuropathologica. 137 (1), 151-165 (2019).</li><li>Hablitz, L. 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=Increased+glymphatic+influx+is+correlated+with+high+EEG+delta+power+and+low+heart+rate+in+mice+under+anesthesia.">Increased glymphatic influx is correlated with high EEG delta power and low heart rate in mice under anesthesia.</a> Science Advances. 5 (2), 5447 (2019).</li><li>Mestre, H., 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=Flow+of+cerebrospinal+fluid+is+driven+by+arterial+pulsations+and+is+reduced+in+hypertension.">Flow of cerebrospinal fluid is driven by arterial pulsations and is reduced in hypertension.</a> Nature Communications. 9 (1), 4878 (2018).</li><li>Pleticha, 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=Pig+lumbar+spine+anatomy+and+imaging-guided+lateral+lumbar+puncture:+A+new+large+animal+model+for+intrathecal+drug+delivery.">Pig lumbar spine anatomy and imaging-guided lateral lumbar puncture: A new large animal model for intrathecal drug delivery.</a> Journal of Neuroscience Methods. 216 (1), 10-15 (2013).</li></ol>

Herein, is described, a detailed protocol to perform the direct cannulation of the cisterna magna in pigs, including the necessary preparation, surgical procedure, tracer infusion and extraction of the brain. This requires someone with experience and certification for working with large animals. If carried out correctly, this allows for the delivery of desired molecules with surety directly into the CSF, after which a series of different advanced light imaging modalities can be used to explore CSF distribution and glymph...

Cerebrospinal fluid (CSF) is an ultrafiltrate of blood which is found within and around the central nervous system (CNS)1,2. Apart from giving buoyancy to the brain or absorbing damaging mechanical forces, CSF also plays a pivotal role in clearing metabolic waste from the CNS3. Waste clearance is facilitated by the recently characterized glymphatic system which permits the convective flow of CSF through the brain parenchyma via perivascular spaces (PVS), which encircle penetrating arteries3,4,5. This process has been shown to be dependent on aquaporin-4 (AQP4), a water channel expressed primarily on the astrocytic endfeet, bound to the PVS4,6. The study of the glymphatic system is achieved by both in vivo and ex vivo imaging, using either advanced light microscopy or magnetic resonance imaging (MRI), following the introduction of a fluorescent/radioactive tracer or contrast agent into the CSF7,8,9,10,11.
An effective way to introduce a tracer into the CSF without incurring damage to the brain parenchyma is through cisterna magna cannulation (CMc)12,13. A large majority of all glymphatic studies, thus far have been carried out in rodents and avoided in higher mammals because of the invasiveness of CMc coupled to the practical simplicity of working with a small mammal. Additionally, the thin skulls of mice permit in vivo imaging without the need for a cranial window and subsequently allow for an uncomplicated brain extraction11,14. Experiments carried out in humans have yielded a valuable macroscopic in vivo data on the glymphatic function, but relied on intrathecal tracer injections in the distal lumbar spine and, furthermore, utilize MRI which does not yield sufficient resolution to capture the microanatomy the glymphatic system7,15,16. Understanding the architecture and extent of the glymphatic system in higher mammals is essential for its translation to humans. In order to facilitate glymphatic translation to humans, it is important to apply techniques that are carried out in rodents to higher mammals so as to allow for direct comparisons of the glymphatic system across species of increasing cognition and brain complexity17. Pig and human brains are gyrencephalic, possessing a folded neuroarchitecture, while rodent brains are lissencephalic, thereby having substantial difference among each other. In terms of the overall size, pig brains are, also, more comparable to humans, being 10-15 times smaller than the human brain, while mouse brains are 3,000 times smaller18. By better understanding the glymphatic system in large mammals, it may be possible to utilize the human glymphatic system for future therapeutic intervention in conditions such as stroke, traumatic brain injury and neurodegeneration. Direct CMc in pigs in vivo is a method that allows for the high-resolution light microscopy of the glymphatic system in a higher mammal. Furthermore, due to the size of the pigs used, it is possible to apply monitoring systems similar to those used in human surgeries making it feasible to tightly document and regulate vital functions in order to assess how these contribute to the glymphatic function.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.01% azide in PBS</td>
			<td>Sigmaaldrich</td>
			<td>S2002</td>
			<td></td>
		</tr>
		<tr>
			<td>18G needle</td>
			<td>Mediq</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1ml Syringe</td>
			<td>FischerSci</td>
			<td>15849152</td>
			<td></td>
		</tr>
		<tr>
			<td>20G cannula</td>
			<td>Mediq</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>22G cannula</td>
			<td>Mediq</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>4% paraformaldehyde</td>
			<td>Sigmaaldrich</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>Anatomical forceps</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin Alexa-Fluor 647 Conjugate</td>
			<td>ThermoFischer</td>
			<td>A34785</td>
			<td>2 vials (10mg)</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigmaaldrich</td>
			<td>C1016</td>
			<td></td>
		</tr>
		<tr>
			<td>Chisel</td>
			<td>ClasOhlson</td>
			<td>40-8870</td>
			<td></td>
		</tr>
		<tr>
			<td>Dental cement</td>
			<td>Agnthos</td>
			<td>7508</td>
			<td></td>
		</tr>
		<tr>
			<td>compact saw</td>
			<td>ClasOhlson</td>
			<td>40-9517</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigmaaldrich</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr>
			<td>Hammer</td>
			<td>ClasOhlson</td>
			<td>40-7694</td>
			<td></td>
		</tr>
		<tr>
			<td>Insta-Set CA Accelerator</td>
			<td>BSI-Inc</td>
			<td>BSI-151</td>
			<td></td>
		</tr>
		<tr>
			<td>IV line TAP, 3-WAYS with 10cm extension</td>
			<td>Bbraun</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigmaaldrich</td>
			<td>P9333</td>
			<td></td>
		</tr>
		<tr>
			<td>Marker pen</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigmaaldrich</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>MilliQ water</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCL</td>
			<td>Sigmaaldrich</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Sigmaaldrich</td>
			<td>S8282</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigmaaldrich</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>No. 20 scalpel blade</td>
			<td>Agnthos</td>
			<td>BB520</td>
			<td></td>
		</tr>
		<tr>
			<td>No. 21 Scalpel blade</td>
			<td>Agnthos</td>
			<td>BB521</td>
			<td></td>
		</tr>
		<tr>
			<td>No. 4 Scalpel handle</td>
			<td>Agnthos</td>
			<td>10004-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Saline</td>
			<td>Mediq</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Salmon knife</td>
			<td>Fiskers</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Self-retaining retractors</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Superglue</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical curved scissors</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical forceps</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical towel clamps</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
	</tbody>
</table>

direct cannula implantation in the cisterna magna of pigs

The glymphatic system is a waste clearance system in the brain that relies on the flow of cerebrospinal fluid (CSF) in astrocyte-bound perivascular spaces and has been implicated in the clearance of neurotoxic peptides such as amyloid-beta. Impaired glymphatic function exacerbates disease pathology in animal models of neurodegenerative diseases, such as Alzheimer's, which highlights the importance of understanding this clearance system. The glymphatic system is often studied by cisterna magna cannulations (CMc), where tracers are delivered directly into the cerebrospinal fluid (CSF). Most studies, however, have been carried out in rodents. Here, we demonstrate an adaptation of the CMc technique in pigs. Using CMc in pigs, the glymphatic system can be studied at a high optical resolution in gyrencephalic brains and in doing so bridges the knowledge gap between rodent and human glymphatics.

Once the pig is unconscious, it is palpated, and its surface anatomy is marked, starting at the occipital crest (OC) and working towards the thoracic vertebrae (TV) and each ear base (EB). It is along these lines that the dermal incisions are made (Figure 1A). The three muscle layers including trapezius, semispinalis capitus biventer and semispinalis capitus complexus are resected and held open by two sets of self-retaining retractors to expose the cisterna magna (CM) (F...

Watch this Scientific Journal Video about Direct Cannula Implantation in the Cisterna Magna of Pigs at JoVE.com

Direct Cannula Implantation in the Cisterna Magna of Pigs

This article presents a step-by-step protocol for the direct cannula implantation in the cisterna magna of pigs.

The glymphatic system is a waste clearance system in the brain that relies on the flow of cerebrospinal fluid (CSF) in astrocyte-bound perivascular spaces ...

This protocol is important as it allows for investigation of the glymphatic system in a large mammal, thereby bringing us closer to understanding the glymphatic system in humans. There are two advantages to this technique. Introduction of traces into the cisterna magna avoids any direct damage to the brain, which is in contrast to intraventricular injections.Secondly, this is a direct approach which allows immediate visualization of the cannulation and knowing whether or not it has been successful. Begin by placing the pig in a prone position. Palpate the back of its head and neck to locate and mark the occipital crest, the spine of the first thoracic vertebrae, and the base of each ear.Draw a straight line between the crest and the vertebrae along the longitudinal axis. Then following the base of the skull, draw two lines from the crest to the base of each ear. Carefully clamp the animal's tail and watch for a reflex to see if it is in a deep sleep.Begin by making a dermal incision along the longitudinal line down to the muscle using a scalpel with a number 21 blade, then extend two perpendicular incisions further along the shoulders, each 10 to 15 centimeters in length. Starting from the occipital crest, make dermal incisions along the line down to the base of each ear. Use anatomical forceps to grip the skin corners formed at the occipital crest, then run the scalpel blade lightly over the fascia to separate the skin from the underlying muscle.Resect the skin along each of the five incisions to visualize parts of the trapezius muscle. Use the scalpel to make a longitudinal incision approximately one centimeter deep where the trapezius comes together at the midline, then perform a blunt dissection along the longitudinal cut in the muscles using a combination of straight and curved surgical forceps, which will separate the bellies of the trapezius and underlying semispinalis capitis biventer muscle. Sever any persisting muscle fibers with a scalpel and continue to perform blunt dissection until the semispinalis capitis complexus becomes visible.Move along the posterior aspect of the skull and sever the origins of the trapezius and semispinalis capitis biventer muscles. To separate the two muscles longitudinally, use the surgical forceps to perform blunt dissection until the semispinalis capitis complexus is fully visible. Then use the self-retaining retractors to retract the trapezius and semispinalis capitis biventer muscles.Use the scalpel to make a one centimeter deep longitudinal incision where the bellies of the semispinalis capitis complexus come together at the midline. Perform a blunt dissection using surgical forceps working along the longitudinal cut between the muscle bellies until both the atlas and axis are palpable. Move along the posterior aspect of the skull, severing the origins of the semispinalis capitis complexus muscles.Use the scalpel and blunt dissection to separate the muscle longitudinally from the underlying vertebrae, then use another set of self-retaining retractors to retract the semispinalis capitis complexus muscles. Use a scalpel to carefully remove any remaining tissue overlying the region where the atlas meets the skull base. Place one arm onto the animal's neck and one finger at the juncture of the atlas and the skull, then simultaneously elevate the head and flex the neck while palpating with the finger to reveal the cisterna magna.Ensure that one person elevates and flexes the head and neck of the animal while the other palpates for the cisterna magna, making a note of its anatomical location. Introduce a 22 gauge cannula slowly and carefully into the cisterna magna through the dura at an angle oblique to the longitudinal axis, then retract the needle from the cannula and place a cap on the lock. Start with applying super glue and an accelerator where the cannula enters the tissue, then apply the dental cement.Wait five minutes for the cement to harden. Remove the cap carefully from the cannula. Attach the cannula to the male end of the IV line tap with the tracer using a 10 centimeter extension.Inject the tracer slowly at a rate of 100 microliters per minute either by hand or using a micro infusion pump. Remove the IV line tap and replace it with the cap. Check if the tracer is visible pulsating at the base of the cannula.Then place sandbags under the neck of the animal to maintain some flexion. Release the head and leave the animal in a resting prone position. Release the self-retaining retractors and replace the muscles.Use the surgical towel clamps to bring the skin together over the muscles. First use gauze and then a blanket to cover the towel clamps and the incision to limit heat loss. Allow the tracer to circulate for the desired amount of time.Stitched macroscopic images of the brain's dorsal surface can provide detailed insights into the distribution patterns of the tracer across the sulci and fissures. Similar images from the brain's ventral and lateral surfaces can provide information about the tracer distribution in the temporal lobe and the lateral fissure. Images of the brain surface of higher magnification produced using a stereoscope can help visualize the tracer in the PVS along the arteries.Macroscopic coronal brain sections provide insight into the depth of tracer penetration in the interhemispheric fissure and subcortical tracer distribution and structure, such as the hippocampus and striatum. Immunohistochemical staining for AQP4, glial fibrillary acidic protein, and smooth muscle actin showed that the tracer localized within the PVS, as well as moved into the brain parenchyma. Astrocyte foot processes that form the outer surface of the PVS are identified using AQP4 and glial fibrillary acidic protein staining.The endothelial cells that form the inner surface of the PVS are stained using lectin and GLUT-1 stain. SMA staining identifies arteries and arterioles and can be used to show that PVS influx occurs along the arteries instead of veins, which constitutes the basic physiology of normal glymphatic function. Going forward, we hope to explore the glymphatic system at a high resolution in large mammal in the context of neuropathology such as stroke and traumatic brain injury.

direct-cannula-implantation-in-the-cisterna-magna-of-pigs

Research

JoVE Journal

Neuroscience

3.4K visualizações.  Lund University. Este protocolo é importante, pois permite a investigação do sistema glifático em um grande mamífero, aproximando-nos da compreensão do sistema glicêntico em humanos. Há duas vantagens nessa técnica. A introdução de traços na cisterna magna evita qualquer dano direto ao cérebro, o que contrasta com as injeções intraventriculares.Em segundo lugar, trata-se de uma abordagem direta que permite a visualização imediata da cannulação e saber se ela foi ou não bem sucedida....