We thank the dedicated work from our nurses and surgical technicians in making the advance of this technique a possibility.

NOTE: The protocol follows the guidelines of and was approved by the Brigham human research ethics committee.
1. Equipment and positioning
<ol>
	<li>Use an open Jackson table for the procedure. Ensure the availability of both frameless stereotactic navigation and intraoperative neuromonitoring with lower extremity electromyography (EMGs), which are critical to the success of the case. 
	&#8203;NOTE: An open Jackson table allows the abdominal viscera to fall away from the spine during the lateral approach.</li>
	<li>Place the patient in the prone position with legs extended. Pay special attention to the hip and/or thigh pads on the side that the interbody spacer will be introduced. If required, shift these pads caudally before the procedure begins if they crowd into the anticipated lateral entry point below the patient&#39;s lowest ribs.</li>
</ol>
2. Initial posterior approach and posterolateral instrumentation
<ol>
	<li>Expose the posterior elements first via a midline incision over the target levels. Open the fascia in standard fashion, and dissect the paraspinal musculature off the bony elements, including the eventual pedicle screw entry points. Next, place a spinous clamp and have the radiology technician bring in the O-arm to obtain an intraoperative computed tomography scan to allow for stereotactic navigation.</li>
	<li>Next, place pedicle screws at the appropriate levels in a standard fashion with navigation assistance. 
	&#8203;NOTE: Doing so at this step ensures that the pedicle screws are placed before the navigation is disturbed, either inadvertently during the case or intentionally by the placement of the interbody cages. Moreover, placement of the screws can aid in the lateral discectomy portion of the case.</li>
</ol>
3. Lateral approach and interbody cage placement
<ol>
	<li>Next, start the lateral approach. Using the navigation, mark a skin incision on the flank, positioning it such that it will bring the surgeon perpendicularly across the mid-point of the target disc space (or allow for multiple such trajectories if inserting interbody cages at multiple levels).
	<ol>
		<li>At this point, rotate the patient&#39;s bed away for a more comfortable working position for the surgeon (i.e., &#34;airplaning&#34;). Similarly, consider using a sitting stool to drop the surgeon&#39;s working angle to allow for a more comfortable approach (Figure 1).</li>
	</ol>
	</li>
	<li>Make a 2&#39;&#39; to 3&#39;&#39; incision in the patient&#39;s flank, parallel to the patient&#39;s ribs. Dissect through the subcutaneous fat and external oblique fascia using electrocautery. Next, dissect with a pair of Metzenbaum scissors to spread open the external oblique, internal oblique, and transverse abdominus muscles and gain access to the retroperitoneal space.</li>
	<li>Once the potential retroperitoneal space is encountered, use fingers for blunt dissection of the space to feel the peritoneal cavity pulling away through the force of gravity and then rapidly encounter the bulk of the psoas muscle overlying the spine. Feel the transverse process as a landmark posteriorly. Use fingers for further blunt dissection to separate the retroperitoneal cavity more thoroughly from the lateral spine surface, especially in the cranial-caudal direction to minimize the chance of inadvertently entering the peritoneal cavity in the subsequent steps.</li>
	<li>Next, place a table-mounted, lighted, lateral-access retractor system just superficial&#160;to the psoas muscle (Figure 2).
	<ol>
		<li>To enter the psoas, first, use a navigation-guided fenestrated probe to select an optimal entry point and approach angle into the target disc space. Then, place A K-wire through the fenestrated probe into the disc space to secure the access.</li>
		<li>Place sequential dilators over the probe superficial to the psoas muscle until finally, the table-mounted retractor system is brought in and secured.</li>
		<li>Connect the light source to the retractor blades. Then, open the retractor blades in the cranial-caudal and anterior-posterior directions to visualize the surgical area directly.</li>
	</ol>
	</li>
	<li>Next, dissect the psoas muscle under direct vision using long Panfield 4 and long Kittner dissectors to expose enough disc space to accommodate the width of the cage (generally 18 mm). If required, use EMG monitoring to monitor the lumbosacral space. 
	NOTE: Under direct vision, the lumbosacral plexus nerves traveling on the surface of the psoas muscle are easily identified and avoided to minimize injury to those nerves. Traversing the psoas in a separate step, under direct vision with the lighted retractor system, allows for greater ability to avoid damage to the lumbosacral plexus.
	<ol>
		<li>Once the disc space is fully exposed, place two pairs of pins in the cranial and caudal vertebral bodies to keep the surgical corridor through the psoas muscle open. 
		NOTE: The pins keep the psoas muscle (and the associated plexus nerves) clear of the surgical corridor, thus providing a comfortable and safe surgical environment. It eliminates the muscle creeping problem commonly encountered with the expandable tubular retractor system (Figure 3).</li>
	</ol>
	</li>
	<li>Ensure that the disc space is adequately exposed in both the cranial-caudal and the anterior-posterior dimensions. Perform an annulotomy with a #15 blade, and perform an initial discectomy using pituitary rongeurs and curettes.
	<ol>
		<li>During this step, break the annulus on the contralateral side using a navigated cobb elevator to release the space and facilitate a larger interbody cage placement and scoliosis correction when needed.
		<ol>
			<li>Insert the navigated cobb elevator into the disc space. Under the navigation guidance, advance the tip of the cobb elevator beyond the contralateral disc border and &#34;pop&#34; it through the contralateral annulus for annulus release. 
			NOTE: Due to the cobb elevator being navigated, the location of the tip of the cobb elevator can be tracked at all times. Therefore, take care not to violate the annulus on either the anterior or posterior side to protect the great vessels and the thecal sac, respectively.</li>
		</ol>
		</li>
		<li>Of note, with simultaneous access to the posterior spine while this is going on, place the pedicle screws in distraction at this time if needed. 
		NOTE: This not only facilitates entrance into particularly narrow and collapsed disc spaces but also allows for the placement of a larger interbody spacer than would be possible otherwise.</li>
	</ol>
	</li>
	<li>Afterwards, use sequentially larger navigated shavers and navigated cage trials to prepare the disc space further. Take care to avoid violating the bony endplates. Once an appropriately sized cage trial is determined, insert the corresponding interbody cage (Conduit lateral Interbody cage) with navigation guidance (Figure 4). Before inserting the cage, fill the cage with allograft bone chips or any grafting materials of the surgeon&#39;s choice.</li>
	<li>Remove the pins holding back the psoas muscle and achieve hemostasis. At this point, shift the lighted retractor system to another target level if multiple interbody cages are intended to be placed. Otherwise, remove the system and close the muscle, fascia, and skin in a layered fashion.</li>
</ol>
4. Completion of the posterior portion
<ol>
	<li>Should further posterior decompression be needed (e.g., laminectomy), perform it at this point. 
	&#8203;NOTE: This can also be performed simultaneously during some portions of the lateral procedure if a second qualified operator is available (Figure 1).</li>
	<li>Finally, place rods to connect the pedicle screws, decorticate the spine, and place a morselized bone graft in a standard fashion. Routinely place vancomycin powder in the cavity, place wound drains, and use liposomal bupivacaine in the back musculature. Perform the closure in the standard layered fashion, including muscle, fascia, subcutaneous tissues, and skin. 
	NOTE: Closure can be performed simultaneously with the closure of the lateral incision should it fit with the workflow of the individual case.</li>
</ol>

<ol>
	<li>Ozgur, B. M., Aryan, H. E., Pimenta, L., Taylor, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extreme+lateral+interbody+fusion+(XLIF):+a+novel+surgical+technique+for+anterior+lumbar+interbody+fusion.">Extreme lateral interbody fusion (XLIF): a novel surgical technique for anterior lumbar interbody fusion.</a> The Spine Journal. 6 (4), 435-443 (2006).</li><li>Kwon, B., Kim, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lateral+lumbar+interbody+fusion:+indications,+outcomes,+and+complications.">Lateral lumbar interbody fusion: indications, outcomes, and complications.</a> Journal of the American Academy of Orthopaedic Surgeons. 24 (2), 96-105 (2016).</li><li>Rodgers, W. B., Gerber, E. J., Patterson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraoperative+and+early+postoperative+complications+in+extreme+lateral+interbody+fusion:+an+analysis+of+600+cases.">Intraoperative and early postoperative complications in extreme lateral interbody fusion: an analysis of 600 cases.</a> Spine. 36 (1), 26-32 (2011).</li><li>Pimenta, L., Turner, A. W. L., Dooley, Z. A., Parikh, R. D., Peterson, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanics+of+lateral+interbody+spacers:+going+wider+for+going+stiffer.">Biomechanics of lateral interbody spacers: going wider for going stiffer.</a> The Scientific World Journal. 2012, 381814 (2012).</li><li>Ploumis, 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=Biomechanical+comparison+of+anterior+lumbar+interbody+fusion+and+transforaminal+lumbar+interbody+fusion.">Biomechanical comparison of anterior lumbar interbody fusion and transforaminal lumbar interbody fusion.</a> Journal of Spinal Disorders &amp; Techniques. 21 (2), 120-125 (2008).</li><li>Blizzard, D. J., Thomas, 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=MIS+single-position+lateral+and+oblique+lateral+lumbar+interbody+fusion+and+bilateral+pedicle+screw+fixation:+feasibility+and+perioperative+results.">MIS single-position lateral and oblique lateral lumbar interbody fusion and bilateral pedicle screw fixation: feasibility and perioperative results.</a> Spine. 43 (6), 440-446 (2018).</li><li>Ouchida, 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=Simultaneous+single-position+lateral+interbody+fusion+and+percutaneous+pedicle+screw+fixation+using+O-arm-based+navigation+reduces+the+occupancy+time+of+the+operating+room.">Simultaneous single-position lateral interbody fusion and percutaneous pedicle screw fixation using O-arm-based navigation reduces the occupancy time of the operating room.</a> European Spine Journal. 29 (6), 1277-1286 (2020).</li><li>Lamartina, C., Berjano, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prone+single-position+extreme+lateral+interbody+fusion+(Pro-XLIF):+preliminary+results.">Prone single-position extreme lateral interbody fusion (Pro-XLIF): preliminary results.</a> European Spine Journal. 29, 6-13 (2020).</li><li>Quiceno, 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=Single+position+spinal+surgery+for+the+treatment+of+grade+II+spondylolisthesis:+A+technical+note.">Single position spinal surgery for the treatment of grade II spondylolisthesis: A technical note.</a> Journal of Clinical Neuroscience. 65, 145-147 (2019).</li><li>Buckland, A. 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=Single+position+circumferential+fusion+improves+operative+efficiency,+reduces+complications+and+length+of+stay+compared+with+traditional+circumferential+fusion.">Single position circumferential fusion improves operative efficiency, reduces complications and length of stay compared with traditional circumferential fusion.</a> The Spine Journal. , (2020).</li></ol>

Y.L. is a consultant for Depuy Synthes. S.E.H, S.G., K.H., N.K declare no competing financial interests.

This study provides a detailed protocol for a prone, single-position, 3D-navigation-guided lateral lumbar interbody fusion (Pro-LLIF). Pro-LLIF permits concurrent access to the anterior and posterior spine and does not require patient repositioning, unlike the two-stage OLIF or XLIF approach9. This single-position approach has been associated with decreased operative time, anesthesia time, and surgical staffing requirements, presenting physical and financial benefits8,...

First described as extreme lateral interbody fusion (XLIF) in 2006, the lateral lumbar interbody fusion approach (LLIF) utilizes a transpsoas approach to the vertebral body1. The LLIF presents several operative advantages over other traditional approaches. First, the LLIF is one of the least invasive interbody fusion approaches, minimizing perioperative tissue damage and blood loss, as well as postoperative pain and length of hospital stay2,3. The LLIF allows for the placement of larger interbody spacers, which confers a greater likelihood of fusion and greater disc height distraction4,5. 
Several LLIF protocols are currently employed, each of which presents limitations. The two-stage approach requires two patient positions for cage placement and posterior screw fixation, respectively. This protocol may increase intraoperative time and anesthetic exposure as the surgeon must wait for patient repositioning between the first and second stages of the procedure. Single-position LLIF variants have also been developed to improve the two-position process. Using a stand-alone LLIF technique forgoes the posterior component of the LLIF surgery and thus negates the need for patient repositioning. However, this technique precludes direct posterior decompression and the added stability of pedicle screw placement. Performing the entire surgery in the lateral position has also been described, but this introduces additional ergonomic challenges for the surgeon6,7. 
A prone single-position approach effectively decreases operative time, thus speeding patients' recovery. Below, the protocol for performing a prone single-position approach for simultaneous access to the anterior and posterior lumbar spine is outlined. Unlike a previously described variation of this approach, 3D navigation is employed to guide both the lateral approach and the interbody cage placement8. Finally, this article includes a case series of the first 10 patients who underwent this prone, lateral lumbar interbody fusion (Pro-LLIF) procedure at the authors' institution.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>CONDUIT Lateral Lumbar Implants</td>
			<td>DePuy Synthes</td>
			<td></td>
			<td>EIT Cellular Titanium Interbody</td>
		</tr>
		<tr>
			<td>COUGAR LS Lateral Spreaders</td>
			<td>DePuy Synthes</td>
			<td></td>
			<td>Lateral Spreaders: 6, 8, 10, 12, 16 mm</td>
		</tr>
		<tr>
			<td>COUGAR LS Lateral Trials</td>
			<td>DePuy Synthes</td>
			<td></td>
			<td>Parallel Trial, 18 x 6 mm</td>
		</tr>
		<tr>
			<td>COUGAR LS Lateral Trials</td>
			<td>DePuy Synthes</td>
			<td></td>
			<td>Lordotic Trials, 18 x 8 mm 18 x 10 mm 18 x 12 mm 18 x 14 mm</td>
		</tr>
		<tr>
			<td>DePuy Synthes ATP/Lateral Discetomy Instruments</td>
			<td>Avalign Technologies LLC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dual Lead Awl Tip Taps 4.35 mm &#8211; 10 mm</td>
			<td>DePuy Synthes</td>
			<td></td>
			<td>Navigation Enabled Instruments used with Medtronic StealthStation Navigation System</td>
		</tr>
		<tr>
			<td>EXPEDIUM 5.5 System</td>
			<td>DePuy Synthes</td>
			<td></td>
			<td>with VIPER Cortical Fix Screws</td>
		</tr>
		<tr>
			<td>EXPEDIUM Driver Shaft T20 5.5</td>
			<td>DePuy Synthes</td>
			<td></td>
			<td>Navigation Enabled Instruments used with Medtronic StealthStation Navigation System</td>
		</tr>
		<tr>
			<td>EXPEDIUM Drive Sleeve 5.5</td>
			<td>DePuy Synthes</td>
			<td></td>
			<td>Navigation Enabled Instruments used with Medtronic StealthStation Navigation System</td>
		</tr>
		<tr>
			<td>Phantom XL3 Lateral Access System</td>
			<td colspan="2">TeDan Surgical Innovations, LLC</td>
			<td>Lateral Access retractor (includes dilators and LED Lightsource)</td>
		</tr>
		<tr>
			<td>PIPELINE LS LATERAL Fixation Pins</td>
			<td>DePuy Synthes</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>The R Project, R package version 4.0, MatchIt package</td>
			<td></td>
			<td></td>
			<td>propensity-score matching</td>
		</tr>
		<tr>
			<td>SENTIO MMG Lateral Probe</td>
			<td>DePuy Synthes</td>
			<td></td>
			<td>Lateral Access Probe</td>
		</tr>
		<tr>
			<td>SENTIO MMG Stim Clip</td>
			<td>DePuy Synthes</td>
			<td></td>
			<td>attaches to insilated dilators, conducting triggered EMG while rotating 360 degrees</td>
		</tr>
		<tr>
			<td>VIPER 2 1.45 mm Guidewire, Sharp</td>
			<td>DePuy Synthes</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

three-dimensional navigation-guided, prone, single-position, lateral lumbar interbody fusion technique

Lateral interbody fusion provides a significant biomechanical advantage over the traditional transforaminal lumbar interbody fusion due to the large implant size and optimal implant position. However, current methods for lateral interbody cage placement require either a two-staged procedure or a single lateral decubitus position that precludes surgeons from having either full access to the posterior spine for direct decompression or comfortable pedicle screw placement.
Herein is one institution's experience with 10 cases of a prone single-position approach for simultaneous access to the anterior and posterior lumbar spine. This allows both lateral lumbar interbody cage placement, direct posterior decompression, and pedicle screw placement, all in one position. Three-dimensional (3D) navigation is utilized for increased precision in both approaching the lateral spine and interbody cage placement. The traditional blind psoas muscle tubular dilation was also modified. Tubular retractors and lateral vertebral body retractor pins were used to minimize the risks to the lumbar plexus.

Cohort demographics 
Ten consecutive patients underwent the Pro-LLIF procedure from August 2020 to February 2021. The eligibility criteria for this procedure were ages 18 and older and symptomatic degenerative spondylosis with spinal instability (spondylolisthesis or degenerative scoliosis) from L2 to L5, requiring interbody fusion. Per the institution&#39;s standard of care, all patients had trialed and failed a course of conservative management. The exclusion criteria were patients excluded from o...

Watch this Scientific Journal Video about Three-dimensional Navigation-guided, Prone, Single-position, Lateral Lumbar Interbody Fusion Technique at JoVE.com

Three-dimensional Navigation-guided, Prone, Single-position, Lateral Lumbar Interbody Fusion Technique

The single-position, prone, lateral approach allows for both lateral lumbar interbody placement and direct posterior decompression with pedicle screw placement in one position.

Lateral interbody fusion provides a significant biomechanical advantage over the traditional transforaminal lumbar interbody fusion due to the large ...

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3.1K Views.  Brigham and Women’s Hospital, Harvard Medical School. The single-position, prone, lateral approach allows for both lateral lumbar interbody placement and direct posterior decompression with pedicle screw placement in one position.

Video: Three-dimensional Navigation-guided, Prone, Single-position, Lateral Lumbar Interbody Fusion Technique

Dissection and Culture of Mouse Dopaminergic and Striatal Explants in Three-Dimensional Collagen Matrix Assays

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum1. Reciprocally, medium spiny neurons in the striatum that give rise to the striatonigral (direct) pathway innervate the substantia nigra2. The development of these axon tracts is dependent upon the combinatorial actions of a plethora of axon growth and guidance cues including molecules that are released by neurites or by (intermediate) target regions3,4. These soluble factors can be studied in vitro by culturing mdDA and/or striatal explants in a collagen matrix which provides a three-dimensional substrate for the axons mimicking the extracellular environment. In addition, the collagen matrix allows for the formation of relatively stable gradients of proteins released by other explants or cells placed in the vicinity (e.g. see references 5 and 6). Here we describe methods for the purification of rat tail collagen, microdissection of dopaminergic and striatal explants, their culture in collagen gels and subsequent immunohistochemical and quantitative analysis. First, the brains of E14.5 mouse embryos are isolated and dopaminergic and striatal explants are microdissected. These explants are then (co)cultured in collagen gels on coverslips for 48 to 72 hours in vitro. Subsequently, axonal projections are visualized using neuronal markers (e.g. tyrosine hydroxylase, DARPP32, or &beta;III tubulin) and axon growth and attractive or repulsive axon responses are quantified. This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of mesostriatal and striatonigral axon growth and guidance during development. Using this assay, it is also possible to assess other (intermediate) targets for dopaminergic and striatal axons or to test specific molecular cues.

Explants from the midbrain dopamine system and striatum are used in a collagen matrix assay for the in vitro analysis of mesostriatal and striatonigral pathway development. In this assay axonal outgrowth and guidance can be manipulated and quantified. It can also be modified for assessing other regions or molecular cues.

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum1. ...

Three Dimensional Vestibular Ocular Reflex Testing Using a Six Degrees of Freedom Motion Platform

The vestibular organ is a sensor that measures angular and linear accelerations with six degrees of freedom (6DF). Complete or partial defects in the vestibular organ results in mild to severe equilibrium problems, such as vertigo, dizziness, oscillopsia, gait unsteadiness nausea and/or vomiting. A good and frequently used measure to quantify gaze stabilization is the gain, which is defined as the magnitude of compensatory eye movements with respect to imposed head movements. To test vestibular function more fully one has to realize that 3D VOR ideally generates compensatory ocular rotations not only with a magnitude (gain) equal and opposite to the head rotation but also about an axis that is co-linear with the head rotation axis (alignment). Abnormal vestibular function thus results in changes in gain and changes in alignment of the 3D VOR response.
Here we describe a method to measure 3D VOR using whole body rotation on a 6DF motion platform. Although the method also allows testing translation VOR responses 1, we limit ourselves to a discussion of the method to measure 3D angular VOR. In addition, we restrict ourselves here to description of data collected in healthy subjects in response to angular sinusoidal and impulse stimulation.
Subjects are sitting upright and receive whole-body small amplitude sinusoidal and constant acceleration impulses. Sinusoidal stimuli (f = 1 Hz, A = 4&deg;) were delivered about the vertical axis and about axes in the horizontal plane varying between roll and pitch at increments of 22.5&deg; in azimuth. Impulses were delivered in yaw, roll and pitch and in the vertical canal planes. Eye movements were measured using the scleral search coil technique 2. Search coil signals were sampled at a frequency of 1 kHz.
The input-output ratio (gain) and misalignment (co-linearity) of the 3D VOR were calculated from the eye coil signals 3.
Gain and co-linearity of 3D VOR depended on the orientation of the stimulus axis. Systematic deviations were found in particular during horizontal axis stimulation. In the light the eye rotation axis was properly aligned with the stimulus axis at orientations 0&deg; and 90&deg; azimuth, but gradually deviated more and more towards 45&deg; azimuth.
The systematic deviations in misalignment for intermediate axes can be explained by a low gain for torsion (X-axis or roll-axis rotation) and a high gain for vertical eye movements (Y-axis or pitch-axis rotation (see Figure 2). Because intermediate axis stimulation leads a compensatory response based on vector summation of the individual eye rotation components, the net response axis will deviate because the gain for X- and Y-axis are different.
In darkness the gain of all eye rotation components had lower values. The result was that the misalignment in darkness and for impulses had different peaks and troughs than in the light: its minimum value was reached for pitch axis stimulation and its maximum for roll axis stimulation.
Case Presentation
Nine subjects participated in the experiment. All subjects gave their informed consent. The experimental procedure was approved by the Medical Ethics Committee of Erasmus University Medical Center and adhered to the Declaration of Helsinki for research involving human subjects.
Six subjects served as controls. Three subjects had a unilateral vestibular impairment due to a vestibular schwannoma. The age of control subjects (six males and three females) ranged from 22 to 55 years. None of the controls had visual or vestibular complaints due to neurological, cardio vascular and ophthalmic disorders.
The age of the patients with schwannoma varied between 44 and 64 years (two males and one female). All schwannoma subjects were under medical surveillance and/or had received treatment by a multidisciplinary team consisting of an othorhinolaryngologist and a neurosurgeon of the Erasmus University Medical Center. Tested patients all had a right side vestibular schwannoma and underwent a wait and watch policy (Table 1; subjects N1-N3) after being diagnosed with vestibular schwannoma. Their tumors had been stabile for over 8-10 years on magnetic resonance imaging.

A method is described to measure three-dimensional vestibulo ocular reflexes (3D VOR) in humans using a six degrees of freedom (6DF) motion simulator. The gain and misalignment of the 3D angular VOR provide a direct measure of the quality of vestibular function. Representative data on healthy subjects are provided

The vestibular organ is a sensor that measures angular and linear accelerations with six degrees of freedom (6DF). Complete or partial defects in the ...

Three-dimensional Confocal Analysis of Microglia/macrophage Markers of Polarization in Experimental Brain Injury

After brain stroke microglia/macrophages (M/M) undergo rapid activation with dramatic morphological and phenotypic changes that include expression of novel surface antigens and production of mediators that build up and maintain the inflammatory response. Emerging evidence indicates that M/M are highly plastic cells that can assume classic pro-inflammatory (M1) or alternative anti-inflammatory (M2) activation after acute brain injury. However a complete characterization of M/M phenotype marker expression, their colocalization and temporal evolution in the injured brain is still missing.	
	Immunofluorescence protocols specifically staining relevant markers of M/M activation can be performed in the ischemic brain. Here we present immunofluorescence-based protocols followed by three-dimensional confocal analysis as a powerful approach to investigate the pattern of localization and co-expression of M/M phenotype markers such as CD11b, CD68, Ym1, in mouse model of focal ischemia induced by permanent occlusion of the middle cerebral artery (pMCAO). Two-dimensional analysis of the stained area reveals that each marker is associated to a defined M/M morphology and has a given localization in the ischemic lesion. Patterns of M/M phenotype marker co-expression can be assessed by three-dimensional confocal imaging in the ischemic area. Images can be acquired over a defined volume (10 &mu;m z-axis and a 0.23 &mu;m step size, corresponding to a 180 x 135 x 10 &mu;m volume) with a sequential scanning mode to minimize bleed-through effects and avoid wavelength overlapping. Images are then processed to obtain three-dimensional renderings by means of Imaris software. Solid view of three dimensional renderings allows the definition of marker expression in clusters of cells. We show that M/M have the ability to differentiate towards a multitude of phenotypes, depending on the location in the lesion site and time after injury.

A way to gain new insights into the complexity of the brain inflammatory response is presented. We describe immunofluorescence-based protocols followed by three-dimensional confocal analysis to investigate the pattern of co-expression of microglia/macrophage phenotype markers in a mouse model of focal ischemia.

After brain stroke microglia/macrophages (M/M) undergo  rapid activation with dramatic morphological and phenotypic changes that  include expression of ...

Protocol for Three-dimensional Confocal Morphometric Analysis of Astrocytes

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify their functional and structural properties, a condition called reactive astrogliosis. Here, a protocol for assessment of the morphological properties of astrocytes is presented. This protocol includes quantification of 12 different parameters i.e. the surface area and volume of the tissue covered by an astrocyte (astrocyte territory), the entire astrocyte including branches, cell body, and nucleus, as well as total length and number of branches, the intensity of fluorescence immunoreactivity of antibodies used for astrocyte detection, and astrocyte density (number/1,000 &#181;m2). For this purpose three-dimensional (3D) confocal microscopic images were created, and 3D image analysis software such as Volocity 6.3 was used for measurements. Rat brain tissue exposed to amyloid beta1-40 (A&#946;1-40) with or without a therapeutic&#160;intervention was used to present the method. This protocol can also be used for 3D morphometric analysis of other cells from either in vivo or in vitro conditions.

Astrocytes in the CNS change their functional and structural properties in response to harmful stimuli. This report presents a protocol for assessment of three-dimensional astrocyte morphology in diseased conditions or after therapeutic interventions.

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify ...

Three-dimensional Imaging and Analysis of Mitochondria within Human Intraepidermal Nerve Fibers

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to isolate nerve-specific mitochondria and evaluate disease-induced alterations of mitochondria in the distal tip of sensory nerves. The protocol combines fluorescence immunohistochemistry, confocal microscopy and 3D image analysis techniques to visualize and quantify nerve-specific mitochondria. Detailed parameters are defined throughout the procedures in order to provide a concrete example of how to use these techniques to isolate nerve-specific mitochondria. Antibodies were used to label nerve and mitochondrial signals within tissue sections of skin punch biopsies, which was followed by indirect immunofluorescence to visualize nerves and mitochondria with a green and red fluorescent signal respectively. Z-series images were acquired with confocal microscopy and 3D analysis software was used to process and analyze the signals. It is not necessary to follow the exact parameters described within, but it is important to be consistent with the ones chosen throughout the staining, acquisition and analysis steps. The strength of this protocol is that it is applicable to a wide variety of circumstances where one fluorescent signal is used to isolate other signals that would otherwise be impossible to study alone.

This protocol uses three-dimensional (3D) imaging and analysis techniques to visualize and quantify nerve-specific mitochondria. The techniques are applicable to other situations where one fluorescent signal is used to isolate a subset of data from another fluorescent signal.

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to ...

Large-scale Three-dimensional Imaging of Cellular Organization in the Mouse Neocortex

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical properties, synaptic connections, and in vivo functions, but their basic functional and anatomical organization from cellular to network scale is poorly understood. Here we describe a method for the three-dimensional imaging of fluorescently-labeled neurons across large areas of the brain for the investigation of the cortical cellular organization. Specific types of neurons are labeled by the injection of fluorescent retrograde neuronal tracers or expression of fluorescent proteins in transgenic mice. Block brain samples, e.g., a hemisphere, are prepared after fixation, made transparent with tissue clearing methods, and subjected to fluorescent immunolabeling of the specific cell types. Large areas are scanned using confocal or two-photon microscopes equipped with large working distance objectives and motorized stages. This method can resolve the periodic organization of the cell type-specific microcolumn functional modules in the mouse neocortex. The procedure can be useful for the study of three-dimensional cellular architecture in the diverse brain areas and other complex tissues.

Here we describe a procedure for tissue clearing, fluorescent labeling, and large-scale imaging of mouse brain tissue which, thereby, enables visualization of the three-dimensional organization of cell types in the neocortex.

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical ...

Three-Dimensional Shape Modeling and Analysis of Brain Structures

Statistical shape analysis of brain structures has been used to investigate the association between their structural changes and pathological processes. We have developed a software package for accurate and robust shape modeling and group-wise analysis. Here, we introduce an pipeline for the shape analysis, from individual 3D shape modeling to quantitative group shape analysis. We also describe the pre-processing and segmentation steps using open software packages. This practical guide would help researchers save time and effort in 3D shape analysis on brain structures.

We introduce a semi-automatic protocol for shape analysis on brain structures, including image segmentation using open software, and further group-wise shape analysis using an automated modeling package. Here, we demonstrate each step of the 3D shape analysis protocol with hippocampal segmentation from brain MR images.

Statistical shape analysis of brain structures has been used to investigate the association between their structural changes and pathological processes. ...

Three-Dimensional Motor Nerve Organoid Generation

A fascicle of axons is one of the major structural motifs observed in the nervous system. Disruption of axon fascicles could cause developmental and neurodegenerative diseases. Although numerous studies of axons have been conducted, our understanding of formation and dysfunction of axon fascicles is still limited due to the lack of robust three-dimensional in vitro models. Here, we describe a step-by-step protocol for the rapid generation of a motor nerve organoid (MNO) from human induced pluripotent stem (iPS) cells in a microfluidic-based tissue culture chip. First, fabrication of chips used for the method is described. From human iPS cells, a motor neuron spheroid (MNS) is formed. Next, the differentiated MNS is transferred into the chip. Thereafter, axons spontaneously grow out of the spheroid and assemble into a fascicle within a microchannel equipped in the chip, which generates an MNO tissue carrying a bundle of axons extended from the spheroid. For the downstream analysis, MNOs can be taken out of the chip to be fixed for morphological analyses or dissected for biochemical analyses, as well as calcium imaging and multi-electrode array recordings. MNOs generated with this protocol can facilitate drug testing and screening and can contribute to understanding of mechanisms underlying development and diseases of axon fascicles.

This protocol provides a comprehensive procedure to fabricate human iPS cell-derived motor nerve organoid through spontaneous assembly of a robust bundle of axons extended from a spheroid in a tissue culture chip.

A fascicle of axons is one of the major structural motifs observed in the nervous system. Disruption of axon fascicles could cause developmental and ...

Transtubular Endoscopic Posterolateral Decompression for L5-S1 Lumbar Lateral Disc Herniation

We report a novel technique for C-arm free transtubular L5 nerve decompression under CT-based navigation to reduce the radiation hazard. This procedure is performed under general anesthesia and neuromonitoring. The patient is placed in a prone position on an operating carbon table. A navigation reference frame is placed percutaneously into the contralateral sacroiliac joint or spinous process. Then, CT scan images are obtained. After instrument registration, the L5-S1 foraminal level is confirmed with a navigated probe, and the entry point is marked. Using an approximately 2 cm skin incision, the subcutaneous tissue and muscles are dissected. The navigated first dilator is aimed at the L5-S1 Kambin&#39;s triangle, and sequential dilation is performed. The 18 mm tube is used and fixed to the frame. The bone around the Kambin&#39;s triangle is removed with a navigated burr. For lateral disc herniation, the L5 nerve root is identified and retracted, and the disc fragment is removed. The navigation-guided tubular endoscopic decompression is an effective procedure. There is no radiation hazard to the surgeon or the operating room staff.

Presented here is a novel technique of C-arm free transtubular posterolateral decompression for lumbar foraminal stenosis and lateral disc herniation under O-arm navigation.

We report a novel technique for C-arm free transtubular L5 nerve decompression under CT-based navigation to reduce the radiation hazard. This procedure is ...

Production of Human Neurogenin 2-Inducible Neurons in a Three-Dimensional Suspension Bioreactor

The derivation of neuronal lineage cells from human induced pluripotent stem cells (hiPSCs) marked a milestone in brain research. Since their first advent, protocols have been continuously optimized and are now widely used in research and drug development. However, the very long duration of these conventional differentiation and maturation protocols and the increasing demand for high-quality hiPSCs and their neural derivatives raise the need for the adoption, optimization, and standardization of these protocols to large-scale production. This work presents a fast and efficient protocol for the differentiation of genetically modified, doxycycline-inducible neurogenin 2 (iNGN2)-expressing hiPSCs into neurons using a benchtop three-dimensional (3D) suspension bioreactor.
In brief, single-cell suspensions of iNGN2-hiPSCs were allowed to form aggregates within 24 h, and neuronal lineage commitment was induced by the addition of doxycycline. Aggregates were dissociated after 2 days of induction and cells were either cryopreserved or replated for terminal maturation. The generated iNGN2 neurons expressed classical neuronal markers early on and formed complex neuritic networks within 1 week after replating, indicating an increasing maturity of neuronal cultures. In summary, a detailed step-by-step protocol for the fast generation of hiPSC-derived neurons in a 3D environment is provided that holds great potential as a starting point for disease modeling, phenotypic high-throughput drug screenings, and large-scale toxicity testing.

This article describes a protocol for the generation of human induced pluripotent stem cell-derived neurons in a benchtop 3D suspension bioreactor.

The derivation of neuronal lineage cells from human induced pluripotent stem cells (hiPSCs) marked a milestone in brain research. Since their first ...