Name: a bedside, single burr hole approach to multimodality monitoring in severe brain injury
Uploaded: 2019-03-26T12:30:00
Description: Watch this Scientific Journal Video about A Bedside, Single Burr Hole Approach to Multimodality Monitoring in Severe Brain Injury at JoVE.com

The authors wish to acknowledge the leadership of Dr. Norberto Andaluz (University of Louisville) for his role in spearheading this technique. We also wish to acknowledge the hard work of the neurosurgical residents who refined the technique and the neurocritical care nursing staff who have embraced this new technique for the benefit of their patients.

This protocol was developed as a standard of care. The retrospective use of data gathered during the course of care was approved through a waiver of informed consent by the University of Cincinnati&#8217;s Institutional Review Board.
1. Patient Selection
<ol>
	<li>Identify patient with acute brain injury (traumatic brain injury, stroke). 
	NOTE: Collaborative discussion between surgical and intensive care teams is critical to ensure that there is consensus on which acut...

<ol>
	<li>Jones, P. 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=Measuring+the+burden+of+secondary+insults+in+head-injured+patients+during+intensive+care.">Measuring the burden of secondary insults in head-injured patients during intensive care.</a> Journal of Neurosurgical Anesthesiology. 6 (1), 4-14 (1994).</li><li>Juul, N., Morris, G. F., Marshall, S. B., Marshall, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracranial+hypertension+and+cerebral+perfusion+pressure:+influence+on+neurological+deterioration+and+outcome+in+severe+head+injury.+The+Executive+Committee+of+the+International+Selfotel+Trial.">Intracranial hypertension and cerebral perfusion pressure: influence on neurological deterioration and outcome in severe head injury. The Executive Committee of the International Selfotel Trial.</a> Journal of Neurosurgery. 92 (1), 1-6 (2000).</li><li>Carney, N., 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=Guidelines+for+the+Management+of+Severe+Traumatic+Brain+Injury,+Fourth+Edition.">Guidelines for the Management of Severe Traumatic Brain Injury, Fourth Edition.</a> Neurosurgery. 80 (1), 6-15 (2017).</li><li>Hawthorne, C., Piper, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+of+intracranial+pressure+in+patients+with+traumatic+brain+injury.">Monitoring of intracranial pressure in patients with traumatic brain injury.</a> Frontiers in Neurology. 5, 121 (2014).</li><li>Binz, D. D., Toussaint, L. G., Friedman, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemorrhagic+complications+of+ventriculostomy+placement:+a+meta-analysis.">Hemorrhagic complications of ventriculostomy placement: a meta-analysis.</a> Neurocritical Care. 10 (2), 253-256 (2009).</li><li>Bauer, D. F., Razdan, S. N., Bartolucci, A. A., Markert, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meta-analysis+of+hemorrhagic+complications+from+ventriculostomy+placement+by+neurosurgeons.">Meta-analysis of hemorrhagic complications from ventriculostomy placement by neurosurgeons.</a> Neurosurgery. 69 (2), 255-260 (2011).</li><li>Poca, M. -. A., Sahuquillo, J., Arribas, M., B&#225;guena, M., Amor&#243;s, S., Rubio, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiberoptic+intraparenchymal+brain+pressure+monitoring+with+the+Camino+V420+monitor:+reflections+on+our+experience+in+163+severely+head-injured+patients.">Fiberoptic intraparenchymal brain pressure monitoring with the Camino V420 monitor: reflections on our experience in 163 severely head-injured patients.</a> Journal of Neurotrauma. 19 (4), 439-448 (2002).</li><li>Koskinen, L. -. O. D., Grayson, D., Olivecrona, 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+complications+and+the+position+of+the+Codman+MicroSensorTM+ICP+device:+an+analysis+of+549+patients+and+650+Sensors.">The complications and the position of the Codman MicroSensorTM ICP device: an analysis of 549 patients and 650 Sensors.</a> Acta Neurochirurgica. 155 (11), 2141-2148 (2013).</li><li>Badri, 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=Mortality+and+long-term+functional+outcome+associated+with+intracranial+pressure+after+traumatic+brain+injury.">Mortality and long-term functional outcome associated with intracranial pressure after traumatic brain injury.</a> Intensive Care Medicine. 38 (11), 1800-1809 (2012).</li><li>Yuan, 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=Impact+of+intracranial+pressure+monitoring+on+mortality+in+patients+with+traumatic+brain+injury:+a+systematic+review+and+meta-analysis.">Impact of intracranial pressure monitoring on mortality in patients with traumatic brain injury: a systematic review and meta-analysis.</a> Journal of Neurosurgery. 122 (3), 574-587 (2015).</li><li>Shen, 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=Effects+of+Intracranial+Pressure+Monitoring+on+Mortality+in+Patients+with+Severe+Traumatic+Brain+Injury:+A+Meta-Analysis.">Effects of Intracranial Pressure Monitoring on Mortality in Patients with Severe Traumatic Brain Injury: A Meta-Analysis.</a> PloS One. 11 (12), e0168901 (2016).</li><li>Chesnut, R. 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=A+trial+of+intracranial-pressure+monitoring+in+traumatic+brain+injury.">A trial of intracranial-pressure monitoring in traumatic brain injury.</a> The New England Journal of Medicine. 367 (26), 2471-2481 (2012).</li><li>Aries, M. J. 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=Continuous+determination+of+optimal+cerebral+perfusion+pressure+in+traumatic+brain+injury.">Continuous determination of optimal cerebral perfusion pressure in traumatic brain injury.</a> Critical Care Medicine. 40 (8), 2456-2463 (2012).</li><li>Vespa, P., 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=Metabolic+crisis+occurs+with+seizures+and+periodic+discharges+after+brain+trauma.">Metabolic crisis occurs with seizures and periodic discharges after brain trauma.</a> Annals of Neurology. 79 (4), 579-590 (2016).</li><li>Hartings, J. 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=Spreading+depolarisations+and+outcome+after+traumatic+brain+injury:+a+prospective+observational+study.">Spreading depolarisations and outcome after traumatic brain injury: a prospective observational study.</a> The Lancet. Neurology. 10 (12), 1058-1064 (2011).</li><li>Okonkwo, D. O., 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+Oxygen+Optimization+in+Severe+Traumatic+Brain+Injury+Phase-II:+A+Phase+II+Randomized+Trial.">Brain Oxygen Optimization in Severe Traumatic Brain Injury Phase-II: A Phase II Randomized Trial.</a> Critical Care Medicine. 45 (11), 1907-1914 (2017).</li><li>Foreman, B., Ngwenya, L. B., Stoddard, E., Hinzman, J. M., Andaluz, N., Hartings, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+and+Reliability+of+Bedside,+Single+Burr+Hole+Technique+for+Intracranial+Multimodality+Monitoring+in+Severe+Traumatic+Brain+Injury.">Safety and Reliability of Bedside, Single Burr Hole Technique for Intracranial Multimodality Monitoring in Severe Traumatic Brain Injury.</a> Neurocritical Care. , (2018).</li><li>Stuart, R. 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=Intracranial+multimodal+monitoring+for+acute+brain+injury:+a+single+institution+review+of+current+practices.">Intracranial multimodal monitoring for acute brain injury: a single institution review of current practices.</a> Neurocritical Care. 12 (2), 188-198 (2010).</li><li>Talving, P., 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=Intracranial+pressure+monitoring+in+severe+head+injury:+compliance+with+Brain+Trauma+Foundation+guidelines+and+effect+on+outcomes:+a+prospective+study.">Intracranial pressure monitoring in severe head injury: compliance with Brain Trauma Foundation guidelines and effect on outcomes: a prospective study.</a> Journal of Neurosurgery. 119 (5), 1248-1254 (2013).</li><li>Aiolfi, A., Benjamin, E., Khor, D., Inaba, K., Lam, L., Demetriades, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+Trauma+Foundation+Guidelines+for+Intracranial+Pressure+Monitoring:+Compliance+and+Effect+on+Outcome.">Brain Trauma Foundation Guidelines for Intracranial Pressure Monitoring: Compliance and Effect on Outcome.</a> World Journal of Surgery. 41 (6), 1543-1549 (2017).</li><li>Pinggera, D., Petr, O., Putzer, G., Thom&#233;, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+I+do+it/Technical+note:+Adjustable+and+Rigid+Fixation+of+Brain+Tissue+Oxygenation+Probe+(LICOX)+in+Neurosurgery+-+from+bench+to+bedside.">How I do it/Technical note: Adjustable and Rigid Fixation of Brain Tissue Oxygenation Probe (LICOX) in Neurosurgery - from bench to bedside.</a> World Neurosurgery. 117, 62-64 (2018).</li><li>Gardner, P. A., Engh, J., Atteberry, D., Moossy, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemorrhage+rates+after+external+ventricular+drain+placement.">Hemorrhage rates after external ventricular drain placement.</a> Journal of Neurosurgery. 110 (5), 1021-1025 (2009).</li><li>Maniker, A. H., Vaynman, A. Y., Karimi, R. J., Sabit, A. O., Holland, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemorrhagic+complications+of+external+ventricular+drainage.">Hemorrhagic complications of external ventricular drainage.</a> Neurosurgery. 59 (4 Suppl 2), (2006).</li><li>Dreier, J. P., 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=Recording,+analysis,+and+interpretation+of+spreading+depolarizations+in+neurointensive+care:+Review+and+recommendations+of+the+COSBID+research+group.">Recording, analysis, and interpretation of spreading depolarizations in neurointensive care: Review and recommendations of the COSBID research group.</a> Journal of Cerebral Blood Flow and Metabolism: Official Journal of the International Society of Cerebral Blood Flow and Metabolism. 37 (5), 1595-1625 (2017).</li></ol>

This article provides the practical elements of a method for introducing multiple probes into the brain follow acute brain injury in order to facilitate a multimodal approach to understanding the physiology underlying secondary brain injury. The existing Brain Trauma Foundation guidelines suggest the use of intracranial pressure monitoring in specific patients after trauma (Level IIb)3, although there is evidence to suggest that this is variably practiced even at high-volume level I trauma centers...

Severe brain injuries such as traumatic brain injury (TBI) or subarachnoid hemorrhage may result in coma, a clinical state in which patients do not respond to their environment. Neurosurgeons and neurointensivists rely heavily on the clinical neurological exam, but severe brain injuries may make it impossible to detect changes related to the brain&#39;s physiologic environment: elevations in intracranial pressure (ICP), decreases in cerebral blood flow, or nonconvulsive seizures and spreading depolarizations. These physiologic disturbances can lead to further injury, termed secondary brain injury.
After severe traumatic brain injury, elevations in ICP are common and may result in decreased blood flow and therefore secondary brain injury and neurodeterioration. Elevations in ICP have been documented in up to 89% of patients1 and neurodeterioration occurs in one-quarter, increasing mortality from 9.6% to 56.4%2. Therefore, the measurement of ICP is the most commonly used biomarker for the development of secondary brain injury and has a Level IIb recommendation from the Brain Trauma Foundation3.
The measurement of ICP was pioneered over 50 years ago4 using catheters that were introduced through a twist drill craniostomy (often referred to interchangeably as a burr hole) typically created in the frontal bone at the mid-pupillary line just anterior to the coronal suture and passed into the ventricles. However, these external ventricular drainage catheters (EVDs) require midline anatomy, which is not always present after severe brain injuries, and misplacement can potentially damage deep structures such as the thalamus. Although EVDs allow drainage of CSF as a potential treatment option, the hemorrhage rates from EVDs are 6&#8211;7% on average5,6.
Intraparenchymal pressure monitors are introduced via burr hole and are common alternatives and adjuncts to EVDs with hemorrhage rates of 3&#8211;5%7,8. These are smaller probes that sit 2&#8211;3 cm under the inner table of the skull, and allow for continuous measurement of pressure but without an option to drain cerebrospinal fluid, as do EVDs. Existing cohort studies9 and meta-analyses10,11 suggest that targeting ICP as a marker of secondary brain injury may improve survival; however, a randomized controlled trial comparing treatment of ICP based on neurological exam alone vs. measured ICP failed to demonstrate benefit12.
Advances in neurosurgery and neurointensive care have led to an understanding that brain physiology is more complicated than ICP alone. It has been demonstrated that autoregulatory function within the brain is impaired after brain injury13, leading to changes in the regulation of regional cerebral blood flow (rCBF). Further, the burden of nonconvulsive seizures14 and spreading depolarizations15 are being recognized using recordings from intracranial electroencephalography (iEEG) electrodes. Strategies to improve brain tissue oxygen (PbtO2) were shown to be a target for therapy and proved feasible in a large, multicenter Phase II clinical trial16.
This article describes a technique that allows for the simultaneous measurement of multiple modalities &#8212; including ICP, PbtO2, rCBF, and iEEG &#8212; using a simple, single burr hole placed at the bedside in patients with severe acute brain injuries requiring intensive care. Patient selection and surgical approach to this technique are included. This technique specifically allows for the placement of multiple probes to provide targeted monitoring of multiple physiologic parameters that may provide a more sensitive and specific early warning system for secondary brain injuries.

<table>
	<tbody>
		<tr>
			<td>Cranial Access Kit</td>
			<td>Integra LifeSciences</td>
			<td>NA</td>
			<td>Cranial Access kit</td>
		</tr>
		<tr>
			<td>Neurovent PTO</td>
			<td>Qflow 500</td>
			<td>NA</td>
			<td>ICP/PBtO2 catheter</td>
		</tr>
		<tr>
			<td>Qflow 500 Perfusion Probe</td>
			<td>Hemedex, Inc</td>
			<td>#H0000-1600</td>
			<td>rCBF catheter</td>
		</tr>
		<tr>
			<td>Qflow 500 Titanium Bolt</td>
			<td>Hemedex, Inc</td>
			<td>#H0000-3644</td>
			<td>Cranial access bolt</td>
		</tr>
		<tr>
			<td>Spencer Depth Electrode</td>
			<td>Ad-Tech Medical Instrument Corporation</td>
			<td>NA</td>
			<td>iEEG</td>
		</tr>
	</tbody>
</table>

a bedside, single burr hole approach to multimodality monitoring in severe brain injury

Intracranial pressure (ICP) monitoring is a cornerstone of the intensive care management of patients with severe acute brain injuries, including traumatic brain injury. While elevations in ICP are common, data regarding the measurement and treatment of these ICP elevations are conflicting. There is increasing recognition that changes in the balance between supply and demand of brain tissue are critically important and therefore the measurement of multiple modalities is required. Approaches are not standard, and therefore this article provides a description of a bedside, single burr hole approach to multimodality monitoring that allows the passage of probes designed to measure not only ICP but brain tissue oxygen, blood flow, and intracranial electroencephalography. Patient selection criteria, operative procedures, and practical considerations for securing probes during critical care are described. This method is readily performed, safe, secure, and flexible for the adoption of a variety of multimodality monitoring approaches aimed at detecting or preventing secondary brain injuries.

Experience in using this approach in 43 patients with severe TBI was recently published17. Patient selection limits the number of those eligible, but focusing on only those with TBI at a level I trauma center led to approximately 2 patients per month. This number is predicated on hospital volume and may increase if additional acute brain injuries are considered for monitoring, such as those with hemorrhagic stroke.
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Watch this Scientific Journal Video about A Bedside, Single Burr Hole Approach to Multimodality Monitoring in Severe Brain Injury at JoVE.com

A Bedside, Single Burr Hole Approach to Multimodality Monitoring in Severe Brain Injury

A method of recording multimodality monitoring signals in patients with severe brain injuries using a bedside, single burr hole technique is described.

Intracranial pressure (ICP) monitoring is a cornerstone of the intensive care management of patients with severe acute brain injuries, including traumatic ...

The placement of a quad lumen monitor can allow for the collection of different types of data including intracranial pressure, brain tissue oxygenation, cerebral blood flow, and electrical activity. Our protocol allows different types of information to be collected in severe traumatic brain injury patients at the bedside using a simple procedure involving a single burr hole. Immobilize the head securely to ensure that it does not move during burr hole placement.Rolled towels and tape can be used to help secure the patient's head. Begin by identifying the correct location for the bolt placement. 11 centimeters from the nasion or one centimeter anterior to the coronal suture, and two to three centimeters laterally at about the mid-pupillary line.Sterilize the area with Betadine solution and use 1%lidocaine with epinephrine for local analgesia. While the Betadine is drying, thread each probe through a locking nut, and subsequently insert each probe through one of the lumens of the bolt. Place the intracranial pressure brain tissue oxygen probe preferentially in the tallest lumen.The other probes can be fit through any of the remaining lumens. Confirm that the distance from the end of the bolt to the tip of each probe is two and a half to three centimeters, and advance the depth electrode until the most proximal electrode is just outside the end of the bolt. Once the probe has been placed the appropriate distance from the end of the bolt, tighten the locking nut on the lumen of the bolt, and then on the probe itself to lock the probe in place.Then loosen the nut from the lumen and remove each probe with its locking nut in place, placing each probe on the sterile table next to the bolt. When all of the probes are ready, use a scalpel to make a one to two centimeter incision in the marked region, and use a blunt tipped instrument to separate the subgaleal tissues to expose the periosteum. Use a hex bit to tighten a 5.3 millimeter drill bit to the cranial drill, and place the drill perpendicular to the skull.Using continuous pressure while rotating the instrument, drill until there is a tactile change in pressure, and continue drilling with counter upward support to avoid plunging the drill into the cortex. Remove the drill and clear the burr hole of any bone chips or debris. Use a scalpel to incise the dura in a cruciate fashion and confirm that the dura is completely open.To insert the cranial bolt, hold the bolt by the plastic wings and thread the bolt through the burr hole with a firm clockwise twisting motion. Insert the thinnest pre-measured probe until the locking nut meets the lumen, followed by the rest of the probes. Insert the depth electrode with the stylet in place, and tighten the electrode on the lumen.Then gently loosen the locking nut from the probe, just enough to remove the stylet, before re-tightening the nut. When all of the probes have been inserted, have available personnel connect the intracranial pressure brain tissue oxygen probe to the bedside monitor, to assess the intracranial pressure and brain tissue oxygen. Then use durable tape to gently loop each probe to secure it to its lumen to create strain resistance.If desired, wrap the probes and bolt with sterile gauze at the completion of the procedure. All probes project through the bolt into the brain parenchyma within millimeters of each other. Here, scout computed tomography, or CT coronal and sagittal images, demonstrating the trajectory of probes at approximately 1.5 and two to three centimeters respectively below the inner table of the skull are shown.In this axial CT image, a probe placed after non-surgical severe traumatic brain injury with excellent placement can be observed. Note that with standard windowing, relatively dense probes may obscure subtle peri-probe hematomas. This axial CT image of a probe placed after surgical severe traumatic brain injury illustrates the location of a bolt and probes contralateral to the hemicraniectomy site.These incorrectly placed probes after non-surgical severe traumatic brain injury are approaching the frontal horn of the lateral ventricle, indicating that they are greater than three centimeters below the inner table of the skull, which may affect measurements obtained by the probes. Place the probes at the proper depth from the bottom of the bolt and tighten the locking nuts tightly enough that the probes do not get dislodged without becoming damaged. If intracranial pressures are high and refractory to treatment, an external ventricular drain can also be placed to drain cerebrospinal fluid and relieve intracranial pressure.A continuous scalp electroencephalography electrode can be placed to correlate potential seizure activity with the depth electrode findings.

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Neuroscience

A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are characteristic to neurons in both normal and diseased brain. Towards this, a great deal of effort has been placed on building high-resolution neuroanatomical maps1-3. With the expansion of molecular genetics and advances in light microscopy has come the ability to query not only neuronal morphologies, but also the molecular and cellular makeup of individual neurons and their associated networks4. Major advances in the ability to mark and manipulate neurons through transgenic and gene targeting technologies in the rodent now allow investigators to 'program' neuronal subsets at will5-6. Arguably, one of the most influential contributions to contemporary neuroscience has been the discovery and cloning of genes encoding fluorescent proteins (FPs) in marine invertebrates7-8, alongside their subsequent engineering to yield an ever-expanding toolbox of vital reporters9. Exploiting cell type-specific promoter activity to drive targeted FP expression in discrete neuronal populations now affords neuroanatomical investigation with genetic precision.
Engineering FP expression in neurons has vastly improved our understanding of brain structure and function. However, imaging individual neurons and their associated networks in deep brain tissues, or in three dimensions, has remained a challenge. Due to high lipid content, nervous tissue is rather opaque and exhibits auto fluorescence. These inherent biophysical properties make it difficult to visualize and image fluorescently labelled neurons at high resolution using standard epifluorescent or confocal microscopy beyond depths of tens of microns. To circumvent this challenge investigators often employ serial thin-section imaging and reconstruction methods10, or 2-photon laser scanning microscopy11. Current drawbacks to these approaches are the associated labor-intensive tissue preparation, or cost-prohibitive instrumentation respectively.
Here, we present a relatively rapid and simple method to visualize fluorescently labelled cells in fixed semi-thick mouse brain slices by optical clearing and imaging. In the attached protocol we describe the methods of: 1) fixing brain tissue in situ via intracardial perfusion, 2) dissection and removal of whole brain, 3) stationary brain embedding in agarose, 4) precision semi-thick slice preparation using new vibratome instrumentation, 5) clearing brain tissue through a glycerol gradient, and 6) mounting on glass slides for light microscopy and z-stack reconstruction (Figure 1).
For preparing brain slices we implemented a relatively new piece of instrumentation called the 'Compresstome' VF-200 (http://www.precisionary.com/products_vf200.html). This instrument is a semi-automated microtome equipped with a motorized advance and blade vibration system with features similar in function to other vibratomes. Unlike other vibratomes, the tissue to be sliced is mounted in an agarose plug within a stainless steel cylinder. The tissue is extruded at desired thicknesses from the cylinder, and cut by the forward advancing vibrating blade. The agarose plug/cylinder system allows for reproducible tissue mounting, alignment, and precision cutting. In our hands, the 'Compresstome' yields high quality tissue slices for electrophysiology, immunohistochemistry, and direct fixed-tissue mounting and imaging. Combined with optical clearing, here we demonstrate the preparation of semi-thick fixed brain slices for high-resolution fluorescent imaging.

Here we describe a rapid and simple method to image fluorescently labeled cells in semi-thick brain slices. By fixing, slicing, and optically clearing brain tissue we describe how standard epifluorescent or confocal imaging can be used to visualize individual cells and neuronal networks within intact nervous tissue.

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are ...

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion channel, and transporter cell surface presentation on a minutes time scale.&#160;There is a broad diversity of mechanisms that control endocytic trafficking of individual proteins. Studies investigating the molecular underpinnings of trafficking have primarily relied upon surface biotinylation to quantitatively measure changes in membrane protein surface expression in response to exogenous stimuli and gene manipulation.&#160;However, this approach has been mainly limited to cultured cells, which may not faithfully reflect the physiologically relevant mechanisms at play in adult neurons.&#160;Moreover, cultured cell approaches may underestimate region-specific differences in trafficking mechanisms.&#160;Here, we describe an approach that extends cell surface biotinylation to the acute brain slice preparation.&#160;We demonstrate that this method provides a high-fidelity approach to measure rapid changes in membrane protein surface levels in adult neurons.&#160;This approach is likely to have broad utility in the field of neuronal endocytic trafficking.

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Neuronal membrane trafficking dynamically controls plasma membrane protein availability and significantly impacts neurotransmission.&#160;To date, it has been challenging to measure neuronal endocytic trafficking in adult neurons.&#160;Here, we describe a highly effective, quantitative method to measure rapid changes in surface protein expression ex vivo in acute brain slices.

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies using Drosophila have contributed important breakthroughs in understanding neurological processes. Thus, with the goal of understanding the cellular and molecular basis of TBI pathologies in humans, we developed the High Impact Trauma (HIT) device to inflict closed head TBI in flies. Flies subjected to the HIT device display phenotypes consistent with human TBI such as temporary incapacitation and progressive neurodegeneration. The HIT device uses a spring-based mechanism to propel flies against the wall of a vial, causing mechanical damage to the fly brain. The device is inexpensive and easy to construct, its operation is simple and rapid, and it produces reproducible results. Consequently, the HIT device can be combined with existing experimental tools and techniques for flies to address fundamental questions about TBI that can lead to the development of diagnostics and treatments for TBI. In particular, the HIT device can be used to perform large-scale genetic screens to understand the genetic basis of TBI pathologies.

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Here we describe a method to inflict closed head traumatic brain injury (TBI) in Drosophila. This method provides a gateway to investigate the cellular and molecular mechanisms that underlie TBI pathologies using the vast array of experimental tools and techniques available for flies.

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies ...

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. Single-molecule imaging is gaining momentum as it provides exceptional access to the visualization of individual molecules in living cells. Here, we describe a technique that we developed to perform single-particle tracking photo-activated localization microscopy (sptPALM) in Drosophila larvae. Synaptic communication relies on key presynaptic proteins that act by docking, priming, and promoting the fusion of neurotransmitter-containing vesicles with the plasma membrane. A range of protein-protein and protein-lipid interactions tightly regulates these processes and the presynaptic proteins therefore exhibit changes in mobility associated with each of these key events. Investigating how mobility of these proteins correlates with their physiological function in an intact live animal is essential to understanding their precise mechanism of action. Extracting protein mobility with high resolution in vivo requires overcoming limitations such as optical transparency, accessibility, and penetration depth. We describe how photoconvertible fluorescent proteins tagged to the presynaptic protein Syntaxin-1A can be visualized via slight oblique illumination and tracked at the motor nerve terminal or along the motor neuron axon of the third instar Drosophila larva.

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

Here we illustrate how single molecule photo-activated localization microscopy can be carried out on the motor nerve terminal of a live Drosophila melanogaster third instar larva.

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. ...

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, very few have been widely accepted. Here, we propose&#160;a new procedure&#160;adapted from the well-known ex vivo brain slice technique, which offers a closer in vivo-like scenario than in vitro preparations, for investigating the early events triggering cell degeneration, as observed in Alzheimer&#39;s disease (AD). This variation consists of simple and easily reproducible steps, which enable preservation of the anatomical cytoarchitecture of the selected brain region and its local functionality in a physiological milieu. Different anatomical areas can be obtained from the same brain, providing the opportunity to perform multiple experiments with the treatments in question in a site-, dose-, and time-dependent manner. Potential limitations&#160;which could affect the outcomes related to this methodology are related to the conservation of the tissue, i.e., the maintenance of its anatomical integrity during the slicing and incubation steps and the section thickness, which can influence the biochemical and immunohistochemical analysis. This approach can be employed for different purposes, such as exploring molecular mechanisms involved in physiological or pathological conditions, drug screening, or dose-response assays. Finally, this protocol could also reduce the number of animals employed in behavioral studies. The application reported here has been recently described and tested for the first time on ex vivo rat brain slices containing the basal forebrain (BF), which is one of the cerebral regions primarily affected in AD. Specifically, it has been demonstrated that the administration of a toxic peptide derived from the C-terminus of acetylcholinesterase (AChE) could prompt an AD-like profile, triggering, along the antero-posterior axis of the BF, a differential expression of proteins altered in AD, such as the alpha7 nicotinic receptor (&#945;7-nAChR), phosphorylated Tau (p-Tau), and amyloid beta (A&#946;).

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

We present a method which can&#160;provide further insights into the early events underlying neurodegeneration and based on the established ex-vivo brain technique, combining the advantages of in vivo and in vitro experiments. Moreover, it represents a unique opportunity for direct comparison of treated and untreated group in the same anatomical plane.

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...

A Novel In Vitro Model of Blast Traumatic Brain Injury

Traumatic brain injury is a leading cause of death and disability in military and civilian populations. Blast traumatic brain injury results from the detonation of explosive devices, however, the mechanisms that underlie the brain damage resulting from blast overpressure exposure are not entirely understood and are believed to be unique to this type of brain injury. Preclinical models are crucial tools that contribute to better understand blast-induced brain injury. A novel in vitro blast TBI model was developed using an open-ended shock tube to simulate real-life open-field blast waves modelled by the Friedlander waveform. C57BL/6N mouse organotypic hippocampal slice cultures were exposed to single shock waves and the development of injury was characterized up to 72 h using propidium iodide, a well-established fluorescent marker of cell damage that only penetrates cells with compromised cellular membranes. Propidium iodide fluorescence was significantly higher in the slices exposed to a blast wave when compared with sham slices throughout the duration of the protocol. The brain tissue injury is very reproducible and proportional to the peak overpressure of the shock wave applied.

A Novel In Vitro Model of Blast Traumatic Brain Injury

This paper describes a novel model of primary blast traumatic brain injury. A compressed-air driven shock tube is used to expose in vitro mouse hippocampal slice cultures to a single shock wave. This is a simple and rapid protocol generating a reproducible brain tissue injury with a high throughput.

Traumatic brain injury is a leading cause of death and disability in military and civilian populations. Blast traumatic brain injury results from the ...

A Revised Surgical Approach to Induce Endolymphatic Hydrops in the Guinea Pig

Endolymphatic hydrops is an enlargement of scala media that is most often associated with Meniere&#39;s disease, though the pathophysiologic mechanism(s) remain unclear. In order to adequately study the attributes of endolymphatic hydrops, such as the origins of low-frequency hearing loss, a reliable model is needed. The guinea pig is a&#160;good model because it hears in the low-frequency regions that are putatively affected by endolymphatic hydrops. Previous research has demonstrated that endolymphatic hydrops can be induced surgically via intradural or extradural approaches that involve drilling on the endolymphatic duct and sac. However, whether it was possible to create an endolymphatic hydrops model using an extradural approach that avoided dangerous drilling on the endolymphatic duct and sac was unknown. The objective of this study was to demonstrate a revised extradural approach to induce experimental endolymphatic hydrops at 30 days post-operatively by obliterating the endolymphatic sac and injuring the endolymphatic duct with a fine pick. The sample size consisted of seven guinea pigs. Functional measurements of hearing were made and temporal bones were subsequently harvested for histologic analysis. The approach had a success rate of 86% in achieving endolymphatic hydrops. The risk of cerebrospinal fluid leak was minimal. No perioperative deaths or injuries to the posterior semicircular canal occurred in the sample. The presented method demonstrates a safe and reliable way to induce endolymphatic hydrops at a relatively quick time point of 30 days. The clinical implications are that the presented method provides a reliable model to further explore the origins of low-frequency hearing loss that can be associated endolymphatic hydrops.

This article demonstrates an extradural approach to obliterate the guinea pig endolymphatic sac and injure the endolymphatic duct with a fine pick in order to induce experimental endolymphatic hydrops.

Endolymphatic hydrops is an enlargement of scala media that is most often associated with Meniere&#39;s disease, though the pathophysiologic mechanism(s) ...

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to intracranial delivery, intra-arterial administration of NSCs is less invasive and produces a more diffuse distribution of NSCs within the brain parenchyma. Further, intra-arterial delivery allows the first-pass effect in the brain circulation, lessening the potential for trapping of cells in peripheral organs, such as liver and spleen, a complication associated with peripheral injections. Here, we detail the methodology, in both mice and rats, for delivery of NSCs through the common carotid artery (mouse) or external carotid artery (rat) to the ipsilateral hemisphere after an ischemic stroke. Using GFP-labeled NSCs, we illustrate the widespread distribution achieved throughout the rodent ipsilateral hemisphere at 1 d, 1 week and 4 weeks after postischemic delivery, with a higher density in or near the ischemic injury site. In addition to long-term survival, we show evidence of differentiation of GFP-labeled cells at 4 weeks. The intra-arterial delivery approach described here for NSCs can also be used for administration of therapeutic compounds, and thus has broad applicability to varied CNS injury and disease models across multiple species.

A method for delivering neural stem cells, adaptable for injecting solutions or suspensions, through the common carotid artery (mouse) or external carotid artery (rat) after ischemic stroke is reported. Injected cells are distributed broadly throughout the brain parenchyma and can be detected up to 30 d after delivery.

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...

Electromagnetic Controlled Closed-Head Model of Mild Traumatic Brain Injury in Mice

Highly reproducible animal models of traumatic brain injury (TBI), with well-defined pathologies, are needed for testing therapeutic interventions and understanding the mechanisms of how a TBI alters brain function. The availability of multiple animal models of TBI is necessary to model the different aspects and severities of TBI seen in people. This manuscript describes the use of a midline closed head injury (CHI) to develop a mouse model of mild TBI. The model is considered mild because it does not produce structural brain lesions based on neuroimaging or gross neuronal loss. However, a single impact creates enough pathology that cognitive impairment is measurable at least 1 month after injury. A step-by-step protocol to induce a CHI in mice using a stereotaxically guided electromagnetic impactor is defined in the paper. The benefits of the mild midline CHI model include the reproducibility of the injury-induced changes with low mortality. The model has been temporally characterized up to 1 year after the injury for neuroimaging, neurochemical, neuropathological, and behavioral changes. The model is complementary to open skull models of controlled cortical impact using the same impactor device. Thus, labs can model both mild diffuse TBI and focal moderate-to-severe TBI with the same impactor.

The protocol describes mild traumatic brain injury in a mouse model. In particular, a step-by-step protocol to induce a mild midline closed head injury and the characterization of the animal model is fully explained.

Highly reproducible animal models of traumatic brain injury (TBI), with well-defined pathologies, are needed for testing therapeutic interventions and ...