This work was funded by the National Institute of Neurological Disorders and Stroke and the National Institute on Aging (US National Institutes of Health; R01NS100366 and RF1AG057575 to MN), the Fondation Leducq Transatlantic Networks of Excellence Program, and the EU Horizon 2020 research and innovation programme (grant no. 666881; SVDs@target). We would also like to thank Dan Xue for expert assistance with graphic illustrations.

All experiments were approved by the University Committee on Animal Resources (UCAR, Protocol No. 2011-023) at the University of Rochester and performed according to the NIH Guide for the Care and Use of Laboratory Animals.
1. Preparing the cisterna magna cannula, head plate, and head holder
<ol>
	<li>Sterilize all surgical instruments and head plates before surgery. 
	NOTE: Fluorescent tracers are delivered directly into CSF via a cisterna magna cannulation. For detailed instructions o...

<ol>
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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=Impairment+of+glymphatic+pathway+function+promotes+tau+pathology+after+traumatic+brain+injury.">Impairment of glymphatic pathway function promotes tau pathology after traumatic brain injury.</a> The Journal of Neuroscience. 34 (49), 16180-16193 (2014).</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+beta.">A paravascular pathway facilitates CSF flow through the brain parenchyma and the clearance of interstitial solutes, including amyloid beta.</a> Science Translational Medicine. 4 (147), (2012).</li><li>Louveau, 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=Structural+and+functional+features+of+central+nervous+system+lymphatic+vessels.">Structural and functional features of central nervous system lymphatic vessels.</a> Nature. 523 (7560), 337-341 (2015).</li><li>Peng, W., 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=Suppression+of+glymphatic+fluid+transport+in+a+mouse+model+of+Alzheimer's+disease.">Suppression of glymphatic fluid transport in a mouse model of Alzheimer's disease.</a> Neurobiology of Disease. 93, 215-225 (2016).</li><li>Da Mesquita, ., 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=Functional+aspects+of+meningeal+lymphatics+in+ageing+and+Alzheimer's+disease.">Functional aspects of meningeal lymphatics in ageing and Alzheimer's disease.</a> Nature. 560 (7717), 185-191 (2018).</li><li>Gaberel, T., 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=Impaired+glymphatic+perfusion+after+strokes+revealed+by+contrast-enhanced+MRI:+a+new+target+for+fibrinolysis.">Impaired glymphatic perfusion after strokes revealed by contrast-enhanced MRI: a new target for fibrinolysis.</a> Stroke. 45 (10), 3092-3096 (2014).</li><li>Xavier, A. 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T., 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=Impairment+of+paravascular+clearance+pathways+in+the+aging+brain.">Impairment of paravascular clearance pathways in the aging brain.</a> Annals of Neurology. 76 (6), 845-861 (2014).</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>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), 373-377 (2013).</li><li>Plog, B. 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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=Brain-wide+pathway+for+waste+clearance+captured+by+contrast-enhanced+MRI.">Brain-wide pathway for waste clearance captured by contrast-enhanced MRI.</a> Journal of Clinical Investigation. 123 (3), 1299-1309 (2013).</li><li>Davoodi-Bojd, E., 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=Modeling+glymphatic+system+of+the+brain+using+MRI.">Modeling glymphatic system of the brain using MRI.</a> Neuroimage. 188, 616-627 (2019).</li><li>Lee, 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=The+Effect+of+Body+Posture+on+Brain+Glymphatic+Transport.">The Effect of Body Posture on Brain Glymphatic Transport.</a> The Journal of Neuroscience. 35 (31), 11034-11044 (2015).</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), (2019).</li><li>Rasmussen, M. K., Mestre, H., 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=The+glymphatic+pathway+in+neurological+disorders.">The glymphatic pathway in neurological disorders.</a> The Lancet Neurology. 17 (11), 1016-1024 (2018).</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, (2018).</li><li>Monai, 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=Calcium+imaging+reveals+glial+involvement+in+transcranial+direct+current+stimulation-induced+plasticity+in+mouse+brain.">Calcium imaging reveals glial involvement in transcranial direct current stimulation-induced plasticity in mouse brain.</a> Nature Communications. 7, 11100 (2016).</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>Schindelin, 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=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Ren, Z., 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=Hit+&amp;+Run'+model+of+closed-skull+traumatic+brain+injury+(TBI)+reveals+complex+patterns+of+post-traumatic+AQP4+dysregulation.">Hit &amp; Run' model of closed-skull traumatic brain injury (TBI) reveals complex patterns of post-traumatic AQP4 dysregulation.</a> Journal of Cerebral Blood Flow &amp; Metabolism. 33 (6), 834-845 (2013).</li><li>Plog, B. 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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=Transcranial+amelioration+of+inflammation+and+cell+death+after+brain+injury.">Transcranial amelioration of inflammation and cell death after brain injury.</a> Nature. 505 (7482), 223-228 (2014).</li><li>Xu, H. T., Pan, F., Yang, G., Gan, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choice+of+cranial+window+type+for+in+vivo+imaging+affects+dendritic+spine+turnover+in+the+cortex.">Choice of cranial window type for in vivo imaging affects dendritic spine turnover in the cortex.</a> Nature Neuroscience. 10 (5), 549-551 (2007).</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>Silasi, G., Xiao, D., Vanni, M. P., Chen, A. C., Murphy, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intact+skull+chronic+windows+for+mesoscopic+wide-field+imaging+in+awake+mice.">Intact skull chronic windows for mesoscopic wide-field imaging in awake mice.</a> Journal of Neuroscience Methods. 267, 141-149 (2016).</li></ol>

The authors have nothing to disclose.

We have described a detailed protocol for performing transcranial CSF imaging in live mice using commercially-available fluorescent macroscopes and tracers. This technique is simple and minimally-invasive, yet quantitative. In vivo imaging correlates well with sensitive methods such as liquid scintillation counting of radio-labeled tracers including 3H-dextran and 14C-inulin after CM delivery, and with ex vivo coronal section quantification10,18</...

Cerebrospinal fluid (CSF) bathes the brain and spinal cord and is involved in maintaining homeostasis, supplying nutrients, and regulating intracranial pressure1. CSF in the subarachnoid space enters the brain through a network of perivascular spaces (PVS) surrounding cortical pial arteries and then flows down along penetrating arterioles2. Once in the parenchyma, CSF exchanges with interstitial fluid (ISF), carrying harmful metabolites such as amyloid-&#946; (A&#946;) and tau protein aggregates out of the brain through low resistance white matter tracts and perivenous spaces2,3. This pathway is dependent on astroglial aquaporin-4 (AQP4) channels and has therefore been termed the glial-lymphatic (glymphatic) system4. Waste products of the neuropil are ultimately cleared from the CSF-ISF through lymphatic vessels near cranial nerves and in the meninges out towards the cervical lymph nodes5. The failure of this system has been implicated in several neurologic diseases such as Alzheimer&#8217;s disease6,7, traumatic brain injury3, and ischemic and hemorrhagic stroke8.&#160;
CSF transport can be visualized by infusing tracers into the cisterna magna (CM)9,10 and glymphatic studies in the past have mainly utilized two-photon microscopy4,11,12,13, magnetic resonance imaging (MRI)14,15,16,17, and ex vivo imaging3,6,11,18 to evaluate tracer kinetics. Two-photon microscopy is a suitable method for detailed imaging of CSF tracers in PVSs and the parenchyma due to its high spatial resolution, however, it has a narrow field of view and requires an invasive cranial window or thinning of the skull. Ex vivo imaging, in combination with immunohistochemistry, enables multilevel analyses ranging from single cells up to the whole brain19. However, the process of perfusion-fixation that is required to observe the post-mortem tissue produces profound changes in CSF flow direction and collapses the PVS, significantly altering the distribution and the location of the tracers12. Finally, while MRI can track CSF flow throughout the entire murine and human brain, it lacks spatial and temporal resolution of perivascular flow.
A new technique, transcranial macroscopic imaging, solves some of these limitations by enabling wide-field imaging of perivascular CSF transport in the entire dorsal cortex of living mice. This type of imaging is done with an epifluorescent macroscope using a multiband filter cube, tunable LED light source, and high-efficiency CMOS camera10. These set-ups are able to resolve PVSs up to 1-2 mm below the skull surface and can detect fluorophores up to 5-6 mm below the cortical surface while leaving the skull entirely intact10. Multiband filters and LEDs that can quickly tune the excitation wavelength enable the use of multiple fluorophores allowing CSF to be labeled with tracers of different molecular weights and chemical properties in the same experiment.
This procedure requires a simple, minimally invasive surgery to expose the skull and place a light-weight head plate to stabilize the head during the imaging session. Tracers can be delivered into the CM without drilling into the skull or penetrating the cortical tissue with pipettes or cannulas9,20. Both CM cannulas and head plates remain stable for several days to weeks and facilitate more complex experimental designs compared to the classical end-point visualization. This protocol describes how transcranial macroscopic imaging is used to study glymphatic system function following acute or chronic injection of fluorescent CSF tracer into the CM of anesthetized/sleeping or awake mice.

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			<td>University of Rochester Vivarium</td>
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			<td>100 &#181;L Gastight Syringe Model 1710 TLL, PTFE Luer Lock</td>
			<td>Hamilton Company</td>
			<td>81020</td>
			<td></td>
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			<td>A-M Systems Dental Cement Powder</td>
			<td>Fisher Scientific</td>
			<td>NC9991371</td>
			<td></td>
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			<td></td>
			<td></td>
			<td>University of Rochester Vivarium</td>
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			<td>Chlorhexidine</td>
			<td>Prevantics</td>
			<td>B10800</td>
			<td></td>
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			<td>CMOS Camera</td>
			<td>Hammamatsu</td>
			<td>ORCA Flash 4.0</td>
			<td></td>
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			<td>Head Plate</td>
			<td>University of Rochester</td>
			<td>No catalog #</td>
			<td>Custom made at the machine shop at the University of Rochester</td>
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			<td>High-Temperature Cautery</td>
			<td>Bovie Medical Corporation</td>
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			<td>Bob Smith Industries</td>
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			<td>Vet One</td>
			<td>502017</td>
			<td>University of Rochester Vivarium</td>
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			<td>Ketamine</td>
			<td></td>
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			<td>Strong Memorial Hospital Pharmacy</td>
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			<td>Elmer&#39;s Products, Inc</td>
			<td>No catalog #, see link in comments</td>
			<td>https://www.amazon.com/Krazy-Glue-KG48348MR-Advance-Multicolor/dp/B000BKO6DG</td>
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			<td>Micropore Surgical tape</td>
			<td>Fisher Scientific</td>
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			<td>Paraformaldehyde</td>
			<td>Sigma-aldrich</td>
			<td>P6148</td>
			<td></td>
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			<td>PE10 - Polyethylene .011&#34; x .024&#34; per ft., 100 ft. continuous</td>
			<td>Braintree Scientific</td>
			<td>PE10 100 FT</td>
			<td></td>
		</tr>
		<tr>
			<td>Pump 11 Elite Infusion Only Dual Syringe</td>
			<td>Harvard Apparatus</td>
			<td>70-4501</td>
			<td></td>
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			<td>PURALUBE VET OINTMENT</td>
			<td>Dechra</td>
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			<td>Fisher Scientific</td>
			<td>22-029-553</td>
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			<td>Research Macro Zoom Microscope</td>
			<td>Olympus</td>
			<td>MVX10</td>
			<td></td>
		</tr>
		<tr>
			<td>Simple Head Holder Plate (for mice)</td>
			<td>Narishige International USA Inc</td>
			<td>MAG-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Single-use Needles, BD Medical</td>
			<td>VWR</td>
			<td>BD305106</td>
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			<td>Sterile Alcohol Prep Pads</td>
			<td>Fisher Scientific</td>
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			<td>PRIOR Lumen 1600-LED</td>
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			<td></td>
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			<td>Xylazine</td>
			<td></td>
			<td></td>
			<td>University of Rochester Vivarium</td>
		</tr>
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in vivo imaging of cerebrospinal fluid transport through the intact mouse skull using fluorescence macroscopy

Cerebrospinal fluid (CSF) flow in rodents has largely been studied using ex vivo quantification of tracers. Techniques such as two-photon microscopy and magnetic resonance imaging (MRI) have enabled in vivo quantification of CSF flow but they are limited by reduced imaging volumes and low spatial resolution, respectively. Recent work has found that CSF enters the brain parenchyma through a network of perivascular spaces surrounding the pial and penetrating arteries of the rodent cortex. This perivascular entry of CSF is a primary driver of the glymphatic system, a pathway implicated in the clearance of toxic metabolic solutes (e.g., amyloid-&#946;). Here, we illustrate a new macroscopic imaging technique that allows real-time, mesoscopic imaging of fluorescent CSF tracers through the intact skull of live mice. This minimally-invasive method facilitates a multitude of experimental designs and enables single or repeated testing of CSF dynamics. Macroscopes have high spatial and temporal resolution and their large gantry and working distance allow for imaging while performing tasks on behavioral devices. This imaging approach has been validated using two-photon imaging and fluorescence measurements obtained from this technique strongly correlate with ex vivo fluorescence and quantification of radio-labeled tracers. In this protocol, we describe how transcranial macroscopic imaging can be used to evaluate glymphatic transport in live mice, offering an accessible alternative to more costly imaging modalities.

CSF influx is imaged on an epifluorescent macroscope (Figure 1A), which allows for mesoscopic imaging of CSF tracer transport in the murine cortex. The whole-skull head plate permits the visualization of the rostral nasal bones, both frontal and parietal bones in the center, and the rostral portion of the interparietal bone caudally (Figure 1B). During imaging, the nasofrontal, sagittal, coronal, and lambdoid sutures can be readily identified (<strong class="xfi...

Watch this Scientific Journal Video about In Vivo Imaging of Cerebrospinal Fluid Transport through the Intact Mouse Skull using Fluorescence Macroscopy at JoVE.com

In Vivo Imaging of Cerebrospinal Fluid Transport through the Intact Mouse Skull using Fluorescence Macroscopy

Transcranial optical imaging allows wide-field imaging of cerebrospinal fluid transport in the cortex of live mice through an intact skull.

Cerebrospinal fluid (CSF) flow in rodents has largely been studied using ex vivo quantification of tracers. Techniques such as two-photon microscopy and ...

This new technique for real-time mesoscopic imaging of florescent CSF traces through the intact skull of live mice can be used to evaluate glymphatic transport. The main advantage is that this technique enables dynamic measurements of in-vivo intracisternal tracers at a fraction of the cost of other imaging modalities. The procedures required to place the head plate on cisterna magna cannula are simple and minimally invasive but require practice to be performed correctly.Visual demonstration of the critical steps of this method is required to maximize the image quality and to enable reproducible comparisons between different mice and experimental groups. After confirming a lack of response to toe pinch wet the neck and head with sterile water and shave the moistened fur. Wipe the exposed skin with an alcohol swab to remove any residual hair and place the mouse in a stereotactic frame on top of a temperature controlled pad.Apply ointment to the animals eyes and clean the exposed skin with a Chlorhexidine swab. After 2 minutes remove the Chlorhexidine with an alcohol wipe and apply an iodone solution. When the iodone has dried inject subcutaneous analgesia into the top of the skull and neck.Starting at the part of the neck that covers the occipital crust, make a midline cut in the overlying skin continuing rostrally toward the intraorbital line. Extend the incision laterally to the border where the temporal muscle inserts into the skull and remove all of the skin of the fusiform incision to expose both the frontal and parietal bones. Irrigate the skull with sterile saline, and use cotton swabs to clean the surface until it is free of debris and hair.After inserting the cisterna magna cannula apply a mixture of dental cement and cyanoacrylate glue to the ventral side of the head plate around the border. Place the head plate onto the skull so that the anterior border of the plate aligns with the posterior tip of the nasal bone and the posterior border aligns with the anterior aspect of the interparietal bone making sure that the sagittal suture is centered and straight relative to the window. It is important to make sure that the head plate is secured and that it does not obstruct the field of view of the cisterna magna cannulation.Use a couple of drops of glue accelerator to fix the position of the head plate and fill any remaining gaps with the cement mixture. Glue the cisterna magna cannula to the head plate and use the head plate to place the mouse into the head holder in a fixed position. Carefully place the mouse and the infusion pump attached to the cannula on a cart for transport to the macroscope and place the head holder on the stage of the macroscope.Make sure that the line from the syringe pump to the cisterna magna cannula is not taut and that it has no kinks. Observe the respiratory rate and pink coloration of the mucus membranes to confirm a good oxygenation. Turn on the macroscope camera and the light emitting diode and start the live mode.Adjust the magnification of the imaging field so that the nasofrontal suture at the top of the field and lambdoid suture at the bottom can clearly be visualized. Once in place tape the head holder to the macroscope stage and focus the macroscope on the exposed skull until the focal plane is located on the lateral sides of the parietal bones posterior to the coronal sutures. For CSF tracer infusion set the infusion pump to the appropriate rate and volume and set the appropriate excitation wavelength and exposure time for the tracer for each channel.Then check that the triggering function on the macroscope is correct before simultaneously initiating the tracer infusion and the imaging. During imaging the naso frontal, sagittal, coronal, and lambdoid sutures can be readily identified. Once the CSF tracer has been infused into the cisterna magna the tracer fluorescence is first observed in large pools of subarachnoid CSF at the olfactofrontal cistern and the quadrigeminal cistern near the pineal recess and eventually surrounding the middle cerebral arteries.CSF tracers then enter their brain along peri vascular spaces of the cortical peal branches of the middle cerebral artery. CSF transport imaging after traumatic brain injury reveals tracer initially at the olfactofrontal cistern but the glymphatic influx along the cortical perivascular spaces is completely abolished on the side of the injury. Quantitative analysis at the in-vivo images demonstrates that the ipsilateral influx area is decreased nearly a third compared to the contralateral hemisphere.To avoid motion artifacts and the changes in the focal plane remember to correctly secure the head plate and the head plate holder and check the anesthesia level of the animal. After the experiment in-vivo imaging results can be further validated by quantifying the tracer penetration using the actual This technique allows the study of the glymphatic system in a physiological and minimally invasive manner and can help address future questions about CSF hydrodynamics in health and disease.

in-vivo-imaging-cerebrospinal-fluid-transport-through-intact-mouse

Research

JoVE Journal

Neuroscience

13.4K visualizações.  University of Rochester Medical Center. Esta nova técnica para imagens mesoscópicas em tempo real de traços florescente de CSF através do crânio intacto de ratos vivos pode ser usada para avaliar o transporte glifático. A principal vantagem é que essa técnica permite medições dinâmicas de rastreadores intracisternais in-vivo a uma fração do custo de outras modalidades de imagem. Os procedimentos necessários para colocar a placa da cabeça na cisterna magna cânula são simples e minimamente invasivos, mas exigem que a...