Beyond Conventional Stereotaxic Targeting: Using Multiplanar Computed Tomography, Optical Coherence Tomography and Intracranial/Extravascular Ultrasound of the Subarachnoid Space to Locate the Rhesus Vestibular Nerve for Single Unit Recording

By Shiyao Dong

A thesis submitted to Johns Hopkins University in conformity with the requirements for the degree of Master of Science in Engineering

Baltimore, Maryland August, 2016

© 2016 Shiyao Dong All Rights Reserv Abstract

Single-neuron electrophysiologic recording of activity in the vestibular nerve and vestibular nuclei of alert rhesus monkeys is a uniquely informative technique in the arsenal of neurophysiologists who seek to elucidate adaptive neuronal signal processing that mediate learning within vestibulo-ocular and vestibulo-spinal reflex pathways. The traditional approach to targeting these structures with a recording microelectrode - stereotactic guidance based on atlas coordinates - has changed little over the past half century and is made difficult by the vestibular nerve’s mobility, mechanical compliance, small caliber and relatively long distance from where a microelectrode enters the cranium

(typically at the base of a recording chamber surgically affixed to the parietal aspect of the skull). The goal of the present study was to determine whether imaging techniques that have recently gained prominence in clinical care might augment or replace the traditional neurophysiologic method. Toward that end, we evaluated 3D multiplanar computed tomography (3DCT), optical coherence tomography (OCT) and an intracranial/cisternal adaptation of intravascular ultrasound (IVUS) as adjuncts to facilitate targeting the rhesus vestibular nerve. Image guidance using CT scans acquired after recording chamber implantation proved to be a simple and useful complement to traditional atlas-based stereotaxis; however, atlas-based stereotaxis, 3DCT, OCT and IVUS offer complementary advantages and disadvantages for targeting cranial nerves. Although OCT and IVUS ultimately proved needlessly complex for our application, adaptation of those techniques for intracranial/cisternal or intracranial imaging of cranial nerves, spinal nerves and other structures adjacent to cerebrospinal fluid spaces may hold promise for intraoperative guidance during minimal-access rhizotomy, biopsy and other neurosurgical procedures.

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Keywords: CT-guidance, vestibular nerve, stereotactic surgery, OCT, ultrasound

Thesis Review Committee:

Advisor: Charles C. Della Santina, Ph.D. M.D.

Thesis Reviewer: Chenkai Dai, Ph.D. M.D. Kathleen Cullen, Ph.D.

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Acknowledgement

I really appreciate those people who help me a lot throughout my Master research, especially members of Vestibular NeuroEngineering Laboratory. Without their help, this thesis project wouldn’t be possible. I would like to thank Dr. Paul Bottomley, Dr. Jon Resar,

Dr. Caroline Garret, Dr. Yu Chen, Cardiac Cath lab at Johns Hopkins Hospital, Shashank

Sathyanarayana Hegde, Kelly Lane, and Nicole McIntosh for the help with equipment and animals. I would like to thank Pengyu Ren for the help with animal surgery. I would like to thank Dr. Chenkai Dai for the valuable advice and helpful guidance on my research project. I would like thank Dr. Kathleen Cullen for reviewing my thesis.

Most of all, I’m really thankful to Dr. Charley Della Santina, my friendly, helpful and responsible advisor, mentor and friend. He shared his experience and vision with me, and provided me with essential skills and tools for research. His guidance helped me get through lots of difficulties during my master research.

I would like to thank my family who taught me, loved me and supported me throughout my life endeavors.

The project described was supported by NIH/NIDCD (R01-DC2390, R01DC9255) and donors to the Johns Hopkins Vestibular NeuroEngineering Laboratory.

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Table of contents

Abstract ...... ii Acknowledgement ...... iv Table of contents ...... v List of tables...... vii List of Figures ...... viii Chapter 1 Introduction ...... 1 1.1 Challenge in Single-Unit Recording in Rhesus Vestibular Nerve ...... 3 1.2 Current approaches to targeting a cranial nerve ...... 7 1.2.1 Conventional Stereotaxic Procedure ...... 8 1.2.2 Image-guided Stereotaxis ...... 9 1.3 Potential imaging techniques for nerve targeting ...... 12 1.3.1 Intravascular Ultrasound ...... 13 1.3.2 Optical Coherence Tomography ...... 15 1.4 Project Aims ...... 20 Chapter 2 Post-operative CT Guidance for Single-unit Recording in Rhesus Vestibular Nerve ...... 22 2.1 Experimental Subjects ...... 22 2.2 Materials and Methods ...... 22 2.2.1 Recording Chamber Implantation Surgical Procedures ...... 23 2.2.2 CT Imaging and Analysis ...... 26 2.3 Results...... 31 2.3.1 Measurement of Vestibular Nerve Location ...... 31 2.3.2 Experimental Validation and Errors Calibration ...... 32 2.4 Discussion ...... 37 Chapter 3 Intracranial/Extravascular Application of OCT and IVUS for Imaging the Subarachnoid Space ...... 41 3.1 Experimental Subjects ...... 41 3.1.1 Phantom ...... 41 3.1.2 Chinchilla Subject ...... 41 3.1.3 Rhesus Monkey Subjects ...... 42 3.2 Surgical Procedures ...... 42 3.2.1 Chinchilla Surgery ...... 42

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3.3 Intracranial/Extravascular Optical Coherence Tomography ...... 43 3.3.1 Method ...... 43 3.3.2 Results ...... 46 3.4 Intracranial/Extravascular Ultrasound Imaging of the Subarachnoid Space . 52 3.4.1 Method ...... 52 3.4.2 Results ...... 52 3.5 Discussion ...... 55 Chapter 4 Conclusion ...... 58 4.1 Post-Operative CT Guidance ...... 58 4.2 Intracranial Imaging of the Subarachnoid Space Using OCT and IVUS ...... 58 4.3 Future Directions ...... 59 Reference List ...... 61 Curriculum Vitae ...... 68

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List of tables

Table 1-1 Comparison of OCT and IVUS ...... 17

Table 2-1. General information of experimental subjects ...... 22

Table 2-2. Location of porus acusticus in CT stereotaxic coordinate...... 36

Table 2-3. Location of porus acusticus in CT chamber coordinate ...... 36

Table 3-1.Comparison of OCT and IC/EVUS ...... 55

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List of Figures

Figure 1-1. Rhesus Monkey Brain Atlas (Interaural -6.00 mm) ...... 5

Figure 1-2. Rhesus Monkey Brain Atlas (Interaural -5.55 mm) ...... 6

Figure 1-3.MRI-guided stereotaxic framework overview ...... 10

Figure 1-4. IVUS images of aortic dissection ...... 14

Figure 1-5 Basic schematic of OCT working principle ...... 16

Figure 1-6. A camera image of the OCT probe on top of the human basal ganglia ...... 18

Figure 1-7. OCT images of a tympanic membrane (TM) ...... 19

Figure 2-1. Stereotaxic frame parameters ...... 25

Figure 2-2. CT 3D reconstruction in stereotaxic head coordinates ...... 28

Figure 2-3. CT 3D reconstruction in recording chamber coordinate (left ear) ...... 29

Figure 2-4. XY Micromanipulator for single-unit recording ...... 30

Figure 2-5. Region of interest in CT 3D reconstruction ...... 33

Figure 2-6. Measurement of inner acoustic opening location in CT stereotaxic coordinate

(Animal ID: RhF234D) ...... 34

Figure 2-7. Measurement of inner acoustic opening location in CT chamber coordinate

(Animal ID: RhF234D) ...... 35

Figure 2-8.Location of porus acusticus in transverse plane of chamber coordinate ...... 38

Figure 3-1. Placement of OCT catheter inside penne embedded in gelatin ...... 44

Figure 3-2.Simulation of placing OCT catheter through the floor of bulla in chinchilla right ear under camera view ...... 45

Figure 3-3. OCT image of a penne in the gelatin phantom ...... 47

Figure 3-4.OCT image of inner acoustic canal in chinchilla ...... 48

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Figure 3-5.Location of OCT catheter placement into the internal auditory canal and cerebellopontine angle of a chinchilla placed just after euthanasia and then dissected after

OCT image acquisition...... 49

Figure 3-6.Rhesus monkey inner acoustic opening on left side ...... 50

Figure 3-7.A slice of OCT side-view scan when catheter is placed in CSF space in the post-mortem rhesus monkey on left side ...... 51

Figure 3-8.IC/EVUS side-view image of penne embedded in gelatin phantom ...... 53

Figure 3-9. Images of subarcuate parafloccular recess obtained using intracranial/extravascular ultrasound in the cerebellopontine angle of a rhesus monkey specimen...... 54

Figure 3-10.Comparison of OCT and IC/EVUS in gelatin phantom ...... 57

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Chapter 1 Introduction

In normal individuals, the two vestibular labyrinths provide the central nervous system (CNS) with sensation of head rotation and linear accelerations due to both gravity and translational motion. These sensory inputs drive compensatory reflexes that maintain posture and stabilize gaze so as to maximize visual acuity during head movement. Bilateral loss of vestibular sensation due to ototoxic injury or other insults to both labyrinths is disabling. Patients suffer from chronic disequilibrium, increased frequency of falls and unstable vision during head movements typical of common daily activities like walking and driving.[1-3] Most individuals with mild or moderate loss eventually compensate through rehabilitative exercises that augment residual function, but those with profound loss who fail to compensate have no good treatments options. There are approximately

64000 U.S adults who experience chronic, symptomatic profound loss of bilateral sensation with no solution for relief.[4, 5] No clinical-approved treatment beyond rehabilitation exists for these patients. Vestibular implants, which are neuroelectronic vestibular prostheses designed to restore motion-modulated vestibular nerve activity, should provide a solution to people with bilateral vestibular deficiency who experience chronic dizziness even after rehabilitation, by restoring sensation of head motion and gravitational orientation that normally drive vestibular reflexes.[6-10]

Normally, the eyes rotate opposite the direction of head rotation in order to stabilize images on retinae. The angular vestibulo-ocular reflex (aVOR) drives the compensatory eye rotation. Sensory input to the aVOR is provided by three mutually orthogonal semicircular canals (SCC) in each inner ear’s vestibular labyrinth. In each SCC’s ampullary

1 nerve, the firing rates of vestibular afferents are modulated by the component of head angular velocity about that SCC’s axis.[11-14] By measuring the resulting eye movement in 3D, relative excitation of the ampullary nerves driving the reflex can be estimated.[15-

19] Suzuki, Cohen et al. demonstrated that delivering pulses of electrical stimulation to the ampullary nerve via wire electrodes can elicit an aVOR about axes of rotation similar to those elicited by the hydrodynamic excitation of the individual canals.[13, 20-26] The vestibular prosthesis modulates electrical stimulation of surviving vestibular afferents based on motion sensor input. A vestibular prosthesis directly stimulates vestibular afferent fibers, which synapse on regions of the CNS that generate vestibular-mediated reflexes.

Thus, the vestibular prosthesis can partly replace function of the lost labyrinth with an electronic solution.

The Vestibular NeuroEngineering Laboratory (VNEL) at Johns Hopkins has done significant work with the Johns Hopkins Multichannel Vestibular Prosthesis (MVP) from its initial development,[6, 27, 28] including reduction in size and power consumption,[29] reduction in channel cross-talk,[30] developing an understanding of the effect of stimulus parameters,[31] and conducting chronic stimulation experiments.[32, 33] After initial development in small mammals, VNEL translated MVP technology to rhesus monkeys, demonstrating similarly favorable outcomes. VNEL recently initiated a first-in-human clinical trial of a vestibular implant based on a modified cochlear implant.[34]

One of the key remaining challenges to vestibular prosthesis development is vestibulo-ocular reflex (VOR) misalignment due to current spread to vestibular afferent fibers beyond those in the targeted nerve branch.[30] When current spread incurs spurious excitation of adjacent vestibular nerve branches, the prosthesis cannot selectively stimulate

2 each ampullary nerve. Higher current stimuli can increase the aVOR magnitude, but meanwhile the eye rotation axis increasingly deviates from the ideal because of spurious stimulation of other vestibular nerve branches.[30]

The 3D VOR axis is a good estimate of relative activity in the 3 ampullary nerves of the implanted ear, but that 3-value vector is a greatly simplified estimate of neuron activity.

Single-unit recording, a more direct assay of measuring single-unit neuronal activity, is needed to guide further optimization of designs that reduce VOR misalignment. By combining single-unit recording in rhesus vestibular nerve during MVP stimulation with

3D VOR data, we can better understand current spread and refine a rigorous biophysical model of neuronal activation by current spread.

1.1 Challenge in Single-Unit Recording in Rhesus Vestibular Nerve

Accurate targeting is required for single-unit recording in the rhesus vestibular nerve; however, targeting the vestibular nerve for electrophysiology is a challenging task. Also known as the eighth cranial nerve, the vestibulocochlear nerve splits into two large divisions: the cochlear nerve and the vestibular nerve. The peripheral parts of the eighth nerve travel a short distance medially from their respective neurosensory epithelia to ganglia (i.e., clusters of nerve cell bodies near the vestibular labyrinth [Scarpa’s ganglion] and within the central spiraling modiolus of the cochlea [the spiral ganglion]). From there, the central part of the nerve travels through the internal auditory meatus (also called the internal auditory canal, or IAC) alongside the facial nerve. Departing the IAC at the porus acusticus (also called the internal acoustic meatus), the eighth nerve traverses a cerebrospinal fluid filled space in the cerebellopontine angle before entering the brainstem

3 at the junction of the pons and medulla lateral to the facial nerve. The diameter of vestibulocochlear nerve is about 0.9 mm in rhesus and 1 mm in human. [35-38] The length of the vestibulocochlear nerve, from the glial-Schwann junction, where cochlear nerve and vestibular nerve join together within the IAC, to the brainstem, is 10-13 mm in human.

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Figure 1-1. Rhesus Monkey Brain Atlas (Interaural -6.00 mm)

Before entering brainstem, the rhesus vestibulocochlear nerve is about 45 mm inferior to bregma in stereotactic coordinates. Adapted from: Paxinos, G., X.-F. Huang, and A.W. Toga, The rhesus monkey brain in stereotaxic coordinates. 2000.

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Figure 1-2. Rhesus Monkey Brain Atlas (Interaural -5.55 mm)

After entering brainstem, the rhesus vestibular nerve is about 44 mm inferior to bregma in stereotactic coordinates. Adapted from: Paxinos, G., X.-F. Huang, and A.W. Toga, The rhesus monkey brain in stereotaxic coordinates. 2000.

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and 7-10 mm in females. Figure 1-1 shows a section of rhesus monkey brain from a stereotaxic atlas by Paxinos, Huang and Toga,[39] illustrating the position of the vestibulocochlear nerve before entering the brainstem. Figure 1-2 shows another section, illustrating the position of vestibulocochlear nerve after entering brainstem. Rhesus monkeys have similar size of vestibulocochlear nerve, the average diameter of which is about 0.9 mm.[38] Only the portion of vestibulocochlear nerve that exits the IAC before entering brainstem is available for placing recording electrode in, which is shorter than 10 mm. The superior-inferior distance from vestibulocochlear nerve to bregma (confluence of sutures of frontal and parietal bones) according to the Paxinos et al. stereotaxic atlas is about 44-45 mm.[39] Thus, the small size of the vestibular nerve, long distance to the nerve from parietal cranium surface (where a recording electrode must enter the cranium in our preparation), and inter-subject difference among rhesus monkeys combine to make targeting the vestibular nerve difficult.

1.2 Current approaches to targeting a cranial nerve

Conventional stereotaxic guidance based on atlas coordinates has long been successfully employed by investigators experienced in that technique to locate small targets in brain and to perform action such as injection, stimulation and implantation, etc. The development of computed tomography (CT) and more recently magnetic resonance imaging (MRI) has greatly facilitated the conventional stereotactic procedure.

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1.2.1 Conventional Stereotaxic Procedure

Conventional stereotaxic surgery includes three main components: (1) a stereotaxic planning system including atlas and coordinate calculator, (2) a stereotaxic apparatus and

(3) a method for stereotaxic localization. Each available rhesus monkey brain atlas is based one single animal,[39-41] which precludes correction for inter-subject variability, apart from simply scaling all dimensions according to the relative length of the distance between the left and right bony ear canal entrances (as determined by ear bar settings on a stereotactic frame). In a typical stereotactic atlas, the three dimensions are: lateral-medial, anterior-posterior and superior-inferior. Rhesus monkey brain atlases often use the bony external auditory meatus, the inferior orbital ridges, and the bregma as landmarks. The simple orthogonal stereotaxic apparatus uses a set of three Cartesian coordinates (x, y and z) in an orthogonal frame of reference. Reid’s horizontal (Z) plane passes through the interaural axis and the lowest point of the cephalic edge of each infraorbital ridge. Reid’s coronal (X) plane is perpendicular to the Z plane and contains the interaural axis. Reid’s midsagittal (Y) plane is perpendicular to the other two planes and lies along the head’s plane of symmetry. Positive X, Y and Z are anterior, left and superior. Guide-bars in x, y and z directions, fitted with high precision Vernier scales allow a neurophysiologist or neurosurgeon to position the point of probe or electrode inside the brain, at the calculated coordinates for the target structure, through a small craniotomy.

Single-unit recording of electrophysiological activity from individual afferent neurons within the rhesus vestibular nerve has long been traditionally guided by atlas-based stereotaxic planning.[42-44] To determine how deep the electrode should be inserted to get the vestibular nerve, experienced researchers in field of vestibular study listen to the

8 characteristic sounds of neural activity when fed into an audio monitor.[45-47] They approach the vestibular nerve through the floccular complex, which is identified by its eye movement-related neural activity. As the recording microelectrode is advanced inferiorly via a trajectory starting at posterosuperolateral parietal bone and passing through the flocculus, entry into the vestibular nerve is typically preceded by a silence, indicating that the electrode has left the cerebellum and is passing through cerebrospinal fluid in the subarachnoid space of the cerebellopontine angle. After pushing all the way through and then exiting the nerve, the microelectrode may travel a short distance (typically less than a few mm) before hitting mater over the petrous part of the temporal bone, and event that can blunt or otherwise damage the electrode tip and which is sometimes signified by abrupt onset of 60 Hz noise in the output of the microelectrode preamplifier.

1.2.2 Image-guided Stereotaxis

In 1978, Russel Brown, an American physician, invented a device known as the N- localizer.[48-55] The N-localizer enables guidance of stereotaxic surgery or radiosurgery using tomographic images that are obtained via computed tomography (CT),[56-59] magnetic resonance imaging (MRI),[60-62] or positron emission tomography (PET).[63,

64] Surgical precision is significantly improved because CT, MRI and PET permit accurate visualization of intracranial anatomic detail. These imaging techniques can display intracranial anatomy in 3-D images, allowing surgeons to register a patient’s cranial image space to stereotaxic physical space with the help of external markers before surgery.

Surgeons now commonly use intracranial images preoperatively for stereotaxic planning or during surgical interventions as guidance, and several commercial systems for image- guided navigation during sinus and intracranial surgery are available.

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Functional magnetic resonance imaging (fMRI) allows localization of brain regions specialized for different perceptual and higher cognitive functions; however, the spatial resolution with which fMRI can identify different levels of physiologic activity is orders of magnitude more coarse than is possible with single-unit (i.e., individual neuron) electro- physiological recording of neural activity using tungsten needle microelectrodes or glass micropipettes. Ohayon et al. designed a novel framework for MRI-stereotaxic registration and chamber placement for precise electrode guidance to recording site defined in MRI space.[65] Figure 1-3 shows overview of the frame. The framework allows positioning a recording chamber, according to pre-surgery planning, and is not limited to

Figure 1-3.MRI-guided stereotaxic framework overview

A region of interest is selected for targeting and a virtual chamber is placed. MRI scan is performed after markers are attached. The software calculates the parameters to align the manipulators. Adapted from[65].

10 vertical penetrations. It also permits implantation of chambers while the animal is simply head fixed in the primate chair, detached from the stereotaxic frame.

CT-guided stereotaxic targeting has been used in humans for implantation of deep brain stimulation electrodes and for craniofacial surgery.[66, 67] Eggers et al. proposed intraoperative CT-guidance for craniofacial surgery with a fully automated registration, which allows surgeons to navigate the operation without delay of patient-to-image registration.[67] Neurosurgeons and neurotologists have increasingly embraced preoperative virtual planning and intraoperative navigation to reduce the risk of complications and to increase the efficiency and confidence with which they can work near critical anatomic structures.[68] Similarly, neuroscience researchers should benefit from

CT-aided stereotaxis to refine their approaches to recording neurophysiologic activity of small, hard-to-reach intracranial structures like the vestibular nerve.

Although experienced surgeons can complete procedures without image guidance, the ability to precisely target and/or identify anatomic points of interest is cost-effective in scenarios that require the surgeon to target a hard-to-find structure while avoiding large blood vessels and other vital or fragile structures.[69] Similarly, although experienced neurophysiologists can find their electrophysiologic targets through a combination of frame-based stereotaxis, systematic sampling of a 3D grid volume encompassing the expected location of the nerve of interest, directed search relying on the location of neurons with recognizable firing patterns, patience and luck, image-guidance should provide a useful adjunct to traditional stereotactic techniques.

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Realizing this, Watanabe et al. developed a new system which introduces the idea of CT-guided stereotaxis into conventional open neurosurgery[70]. Using metal markers, they coregistered a preoperative CT imaging data set (for high resolution bone anatomy) with patient’s head. They reported that this system makes it possible for a surgeon to identify the operating site by obtaining real-time positional information in surgery translated back onto CT images. However, for targeting the rhesus vestibular nerve, that approach might suffer from errors arising during recording chamber implantation surgery.

CT scans obtained before chamber implantation may not be sufficient to accurately target the nerve in each recording experiment after surgery. A more efficient solution involves first surgically attaching a rigid recording chamber to the skull (using traditional stereotactic targeting), then performing a high-resolution CT and 3D reconstructions along mutually orthogonal planes that either contain or are perpendicular to the recording chamber’s axis. We evaluated that approach in the present study.

1.3 Potential imaging techniques for nerve targeting

In addition to CT and MRI guidance, biomedical imaging techniques such as intravascular ultrasound (IVUS) and optical coherence tomography (OCT) can image soft tissue with better resolution, which could possibly help visualize vestibular nerve intraoperatively or even directly observe the microelectrode as it is advanced into the nerve.

Stereotaxic procedures that require insertion of needle-type instrument into the brain serve crucial roles in neurosurgery. The procedures are employed as aids to diagnosis in management of various medical conditions including biopsy of tumors, assessment of vessel wall plaques, and placement of electrodes that are used for signal recording and deep brain stimulation (DBS).[71-73] Catheter-based IVUS and OCT have both established

12 roles in interventional cardiology as visualization aids during stent implantation. The small size of IVUS and OCT catheters and the high resolution of these imaging modalities gives these two intravascular imaging technologies potential to be applied in the subarachnoid space to provide guidance for targeting cranial and spinal nerves.

1.3.1 Intravascular Ultrasound

The concept of intravascular ultrasound was first introduced by Born et al. in

1972.[74] IVUS is a catheter based system that allows physicians to acquire images of diseased vessels from inside the artery. In the late 1980s, commercially available IVUS catheter-based probes were used in both animals and humans.[75] Intravascular ultrasound imaging, working in the range of 20-45 MHz, is the most commonly used intravascular imaging modality (after dye-contrasted cine or flat panel CT fluoroscopic angiography) for diagnosing coronary artery diseases or coronary heart diseases.[76] Imaging through blood with large penetration (>5 mm) makes IVUS able to facilitate vessel modeling and plaque morphology.[77, 78] The use of IVUS is currently being incorporated into several modalities that will offer more real-time information in both the aorta and the treatment of peripheral vascular disease.[76, 79-83] Figure 1-4 illustrates IVUS images along the imaging catheter inside the subclavian artery.[81]

IVUS can provide a cross-sectional image of the vessel or other tissue within the range, with resolution of 100-200 um and the imaging depth around 10 mm [81, 84]. These features endow IVUS with potential to be applied outside the vasculature, deployed via a recording microelectrode’s guide tube trocar through brain tissue and into cerebrospinal fluid spaces, where it can be used for image guidance as an aid for nerve targeting in single-

13 unit recording or intracranial neurosurgery. An analogous approach might be used in the cranium, in which IVUS might be renamed intracranial/extravascular ultrasound

(IC/EVUS).

Figure 1-4. IVUS images of aortic dissection

In aortic dissection, entry tear is the initial intimal tear that causes the blood to flow between the layers of the wall of the aorta. These images, adapted from [81], show that IVUS can images a vessel wall with sufficient resolution to identify an entry tear.

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1.3.2 Optical Coherence Tomography

Optical coherence tomography (OCT) is a recently developed biomedical imaging that has accelerated the research in the fields of medicine and biology. The capability of

OCT to perform high resolution, cross-sectional tomographic imaging of microstructure in biological tissues has led this technology to be applied to surgical guidance and medical diagnostics. Compared with IVUS, OCT provides higher resolution of around 10-20 um.[84-86] Like IVUS, needles and probes have been developed to integrate OCT into intravascular catheters.

In essence, OCT probes detect and report the occurrence of reflection backward along a beam path extending like a laser beam from and back to the OCT sensor.

Interferometry allows detection of these reflections and determination of how far each reflection-generating boundary is from the sensor (Figure 1-5). The light source has a bandwidth in the near-infrared spectrum with central wavelengths ranging from 1,250 to

1,350 nm. Tissue penetration, and therefore the depth over which OCT images tissue, is limited to 1-3 mm.[87] Rapid, repetitive and systematic reorientation of the OCT sensor allows a system to scan either a slice of tissue perpendicular to the catheter (side-viewing

OCT) or a conic section of tissue concentric with the catheter (forward-viewing OCT).

Side-view imaging OCT probes generate a 2D image of a disk of tissue perpendicular to the axis of the OCT probe’s introducing catheter. A side-viewing system consists of an optical fiber, a focusing lens and a prism or mirror for lateral deflection of light. The entire apparatus spins within a surrounding catheter, allowing the OCT sensor’s single line of sight to be scanned like a submarine periscope or an air traffic control radar

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Figure 1-5 Basic schematic of OCT working principle system. A constant-velocity “pull-back” of the rotating sensor along the axis of the catheter allows one to sample many disc slices and then reconstruct them to image a 3D cylindrical volume.[88, 89] In addition to intravascular imaging of vessel walls, side-view imaging probes are used to image gland ducts and other hollow organs.[87]

Compared with side imaging probes, forward imaging probes are more challenging to construct, because 3D image construction cannot easily be done by back-forth “pull- back” translation. To acquire the 3D image of object, for example, the scanning is realized by a piezoelectric transducer, galvanometer scanners or a microelectromechanical system.

[90-92] The scanning device in a forward imaging endoscope is usually placed at the distal end of the system. That makes it very difficult to maintain the diameter small and have a large working distance (WD) and broad field of view (FOV). If these technical challenges can be overcome, Forward-viewing OCT seems especially well-suited to providing useful image guidance during intracranial stereotaxic procedures, because the guide tube through

16 which the OCT imaging system is introduced is nominally pointing at the target one wants to see.

Forward-scanning OCT (also called endoscopic OCT, or EOCT) has been applied to detect diseases of the tympanic membrane and the middle ear, such as otitis media or cholesteatoma.[94-96] Burkhardt et al. designed a 3D forward imaging EOCT at a large

WD with a large FOV and acquired images of tympanic membrane (Figure 1-7).[97]

Although a forward-scanning needle probe can be used to provide real-time feedback along the line of sight as a microelectrode guide tube is inserted, the forward-viewing OCT systems described so far do not provide as much field of view as side-view imaging systems.

Chen et al. at University of Maryland, College Park designed a forward-imaging needle- type OCT probe for stereotaxic procedures intended to overcome this problem by using a gradient-index (GRIN) rod lens, which has a gradual variation of refractive index and provides variable length focus without the weight, size and fragility of a traditional lens,

(Figure 1-6).[93].

Table 1-1 summarizes performance parameters for IVUS and endoscopic OCT, which are promising techniques for real-time guidance for intracranial surgery.[87]

Modality Axial Resolution Lateral Resolution Penetration depth Smallest Catheter size OCT 12-18 um 20-90 um 1.5-3 mm ~ 1 mm IVUS 150-200 um 150-300 um 4-8 mm ~ 1 mm

Table 1-1 Comparison of OCT and IVUS

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Figure 1-6. A camera image of the OCT probe on top of the human basal ganglia

The top yellow bar shows the full track of OCT reconstruction along the probe insertion direction. Labels: extreme capsule (ex), claustrum (Cl), external capsule (ec), putamen (PUT), lateral medullary lamina (lml), globus pallidus externa (GPe) and globus pallidus interna (GPi).Adapted from [93].

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Figure 1-7. OCT images of a tympanic membrane (TM)

(A) The red arrow points at the umbo, the deepest point of the TM. (B) Manubrium of malleus (MM). (C) Top view OCT image of the TM. (D) Top view camera picture, the umbo (*) and the MM (**).(E) 3-D OCT image of the TM. Scale bars:1 mm. Adapted from [97].

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1.4 Project Aims

Although experienced neuroscience researchers can successfully target the rhesus vestibular nerve for single-unit recording using traditional atlas-based stereotaxis, finding the nerve can be a challenging and laborious task that requires weeks of months of hunting with a microelectrode, systematically making passes through a grid of tracks surrounding the trajectory estimated using skull landmarks and a stereotactic atlas from a different animal. Differences between atlas and experimental subjects, small size of the nerve and long distance to the nerve from craniotomy combine to make it take long time targeting the nerve before recording electrophysiological activity. Thus, there is a need for a guidance method to help locate the nerve more efficiently.

The aim of this project was to evaluate a method of image guidance based on post- operative CT scans (obtained after surgical placement of a recording chamber on the parietal calvarium) in searching for vestibular afferents that:

1. individualizes targeting for the specific subject under study, rather than relying

on an atlas created from a different specimen;

2. provides direct guidance to help calibrate the placement of recording

microelectrode; and is

3. accessible to researchers with less experienced in stereotaxic procedures.

Although our focus was on image guidance in the form of CT 3D reconstructions, which relies on technology now widely available in most academic medical centers, we also explored catheter-based imaging techniques including intravascular ultrasound (IVUS)

20 and optical coherence tomography (OCT) to facilitate targeting the vestibular nerve more precisely following the CT guidance.

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Chapter 2 Post-operative CT Guidance for Single-unit

Recording in Rhesus Vestibular Nerve

We performed multiplanar CT scans on rhesus monkeys after recording chamber implantation. Locations of the internal acoustic opening for both sides, where the vestibular nerve bundle leaves the internal auditory canal to traverse the cerebellopontine angle on its way from the inner ear to the brainstem, were measured with respect to recording chamber coordinates for recording navigation and in stereotaxic coordinate for comparison. Details of the experiment procedure are described in respective sections.

2.1 Experimental Subjects

Five rhesus monkeys were used for experiments, which were performed in accordance with a protocol approved by the Johns Hopkins Animal Care and Use

Committee. Details about the five rhesus monkeys are shown in Table 2-1.

No. Animal ID Weight (kg) Sex Implanted Ears IVUS test OCT test 1 RhF234D 5.2 F Both No No 2 RhF247E 5.3 F Both No No 3 RhM46xH 4.5 M Both No No 4 RhF4617J 6.0 F Both Yes(post- Yes(post- mortem) mortem) 5 RhM54AM 6.0 M Both No No

Table 2-1. General information of experimental subjects 2.2 Materials and Methods

All experiments were performed in accordance with protocols approved by the

Johns Hopkins Animal Care and Use Committee, which is accredited by the Association

22 for the Assessment and Accreditation of Laboratory Animal Care (AAALAC) International and consistent with European Community Directive 86/609/EEC.

2.2.1 Recording Chamber Implantation Surgical Procedures

Five rhesus monkeys weighting 4-7kg were used for evaluating CT guidance to facilitating single unit recording from vestibular nerve afferent neurons. Surgical procedures were carried out under strict sterile conditions. Animals were sedated with ketamine (0.2 mg/kg), treated with prophylactic IV cefazolin, endotracheally intubated and maintained under deep anesthesia via inhalation of 1.5-5 % isoflurane in oxygen. Surgical preparation was done in stages so as to limit the operative time and shorten the recovery period for any single anesthetic exposure. In the first surgery, a head-restraining cap was permanently affixed on the calvarium using dental acrylic affixed to multiple titanium bone screws.

Before chamber implantation, we positioned a skull from a monkey of body size similar to the animal on which we planned to operate in the stereotaxic frame, securing it in place using ear bars, an incisor bar and a nose clamp. We then positioned a chamber holder so that its axis passed from ipsilateral parietal cortex through the cerebellum to the porus acusticus (i.e., the medial opening of the internal auditory canal) while avoiding major blood vessels (Figure 2-1). Stereotaxic manipulator parameters were recorded for later use during chamber implantation surgery.

We then positioned the animal to be studied on the stereotactic frame. Using the previously determined settings, we positioned a 1.9 cm diameter titanium recording chamber over the parietal calvarium and marked its location on the scalp and, after skin

23 incision and elevation, on the skull. A ~1 cm diameter craniotomy was drilled within the region of calvarium encircled by the mark, and the chamber was cemented in place using dental cement (3M ESPE ProTemp®II or OrthoJet®). A hairless, split-thickness skin graft was harvested, meshed and applied to the exposed dura, then held in place using antibiotic- impregnated GelFoam® and sterile packing.[98] The packing was removed and replaced every other day starting 7 days post-op and continuing until the dura was completely covered by skin. This technique can yield a clean, dry, easy-to-maintain recording chamber floor that is almost entirely free of granulation tissue, significantly reducing the risk of infection, blood loss, CSF leakage and animal distress that might otherwise occur with daily debridement. The skin grafts act as a biologic dressing, sealing itself closed after guide tube removal.

A second recording chamber was placed using the same techniques, typically on the same day and in a position approximately at the mirror image across the midsagittal plane from the first chamber.

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Figure 2-1. Stereotaxic frame parameters

Stereotaxic setting parameters: (a) XY-slide for right and left, Medial-Lateral slide for right and left. (b) Vertical azimuth for right, horizontal azimuth for right. (c) Vertical azimuth for left, horizontal azimuth for left and upper slide.

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2.2.2 CT Imaging and Analysis

Monkeys maintained under propofol IV infusion anesthesia and endotracheally intubated were imaged in a high-resolution CT scanner (Toshiba Aquilion One). A guide tube containing a tungsten recording microelectrode was inserted along the axis of each recording chamber, advanced through skin graft, dura, parietal/occipital cortex, tentorium, and cerebellum toward the presumed location of the porus acusticus, then left in place temporarily while a CT scan was performed using a bone algorithm, step scan mode, a 120 kV source collimated to 0.5-mm slice thickness at full-width half maximum, and a (120 mm)2 region of interest imaged on a 512 x 512 voxel/slice matrix, creating voxels of 0.4 x

0.4 x 0.3 mm.

To define the location of the porus acusticus, we used two different coordinate systems (Figure 2-2 and Figure 2-3). We used stereotactic coordinates using Reid’s planes.

Reid’s horizontal (Z) plane passes through the interaural axis and the lowest point of the cephalic edge of each infraorbital ridge. Reid’s coronal (X) plane is perpendicular to the Z plane and contains the interaural axis. Reid’s midsagittal (Y) plane is perpendicular to the other two planes and lies along the head’s plane of symmetry. Positive X, Y and Z are anterior, left and superior. To define locations with respect to each of the two cylindrical recording chambers, we defined a chamber coordinate system resulting in which the Zc axis for a given chamber is the axis of that chamber, the Yc axis is perpendicular to Zc of the same chamber and intersects the Zc axis of the other chamber, and Xc is perpendicular

26 to both Zc and Yc. Positive Xc, Yc and Zc are approximately anterior, left and superior with respect to the skull and the stereotactic coordinate system.

In single-unit recording experiments, we placed an X-Y micromanipulator (Figure

2-4) on the chamber with its X, Y-axis in alignment with the chamber coordinate. Based on the location of target measured in CT chamber coordinate, we changed micromanipulator settings and passed tungsten electrodes through a guide tube to target the ipsilateral porus acusticus.

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Figure 2-2. CT 3D reconstruction in stereotaxic head coordinates

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Figure 2-3. CT 3D reconstruction in recording chamber coordinate (left ear)

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Figure 2-4. XY Micromanipulator for single-unit recording

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2.3 Results

2.3.1 Measurement of Vestibular Nerve Location

As described previously, the vestibular nerve the petrous temporal bone’s posteromedial face via the porus acusticus (inner acoustic opening). Therefore, we used

CT reconstructions to measure the location of porus acusticus as the landmark for targeting the nerve.

In the rhesus monkey skull anatomy, another constant feature is a subarcuate parafloccular recess, which is close to the porus acusticus on the petrous part of the temporal bone and similar in size (Figure 2-5). The recess, within which a portion of the cerebellar paraflocculus sits encircled by the three semicircular canals of the ipsilateral labyrinth, is posterior and superior to the porus acusticus. For different individuals, the distance between subarcuate parafloccular recess and porus acusticus may be different, but the relative position is almost the same. This relation served as our criterion to distinguish these two similar structures and find the location of porus acusticus. Once the porus acusticus is identified in CT transverse view, we choose one point as our target location for measurement, which would be about 0.5 mm medial of the porus acusticus and far enough from nearby bone surfaces to reduce the risk that the recording electrode may hit the bone in later experiment. Figure 2-6 and Figure 2-7 show the example of measurement in CT scan. Same measurement method has been applied to all five experimental subjects. Table

2-2 and table 2-3 shows the measurement results. The internal auditory canals are roughly coaxial with the external bony auditory canals (where the stereotaxic ear bars go) and the porus acusticus in rhesus is about 1-1.1 cm off midline. Since internal acoustic opening is

31 a hole instead of a single point, one cannot uniquely define or measure its precise location in CT scan. We measured the location estimated as the center of the nerve exiting the porus acusticus twice per ear and took the average as the final result. The distances from porus acusticus to the chamber axis in X-Y plane on both sides for five monkeys were all smaller than the chamber radius, indicating that the traditional method of stereotactic targeting we used for chamber placement was adequate to at least ensure that the nerve would be accessible via each chamber, if only one knows where to look.

2.3.2 Experimental Validation and Errors Calibration

As described previously, based on measurements of vestibular nerve location in chamber coordinates, we inserted recording electrode through guide tube navigated by the

XY micromanipulator to target ipsilateral vestibular nerve. If we didn’t successfully target the nerve at the first time, we would pull out and insert the electrode several times or change

XY micromanipulator setting by 0.1 mm per time, then reinsert electrode through guide tube. We found the vestibular nerve and successfully recorded afferent units in a single afternoon of electrophysiologic recording during the first experiment after chamber implantation for monkey 54A and for monkey 4617. Previously, without using CT guidance, it usually took us 3-4 months to find the vestibular nerve through a systematic electrophysiologic survey of activity in a volume about the location expected from traditional stereotactic targeting.

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Figure 2-5. Region of interest in CT 3D reconstruction

In CT 3D reconstruction :(a) skull base with guide tube in left side chamber. (b) view of left lateral skull with guide tube from medial side. (c) view through craniotomy from left chamber floor.

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Figure 2-6. Measurement of inner acoustic opening location in CT stereotaxic coordinate (Animal ID: RhF234D)

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Figure 2-7. Measurement of inner acoustic opening location in CT chamber coordinate (Animal ID: RhF234D)

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Table 2-2. Location of porus acusticus in CT stereotaxic coordinate

Location of internal acoustic opening on left and right side in five monkeys measured from 3D reconstruction in stereotaxic coordinate (cm). Lat+ = lateral, Med- = medial, Ant+ = anterior, Pos- = Posterior, Sup+ = superior, Inf- = inferior

Table 2-3. Location of porus acusticus in CT chamber coordinate

Location of internal acoustic opening on left and right side in five monkeys measured from 3D reconstruction in chamber coordinate (cm). X+/X- = X-axis, Y+/Y- = Y-axis, Z+/Z- = Z-axis.

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2.4 Discussion

Guidance using 3D reconstructions of a CT scan obtained after initial placement of a recording chamber using traditional stereotactic targeting is an efficient and effective method that helps target the rhesus vestibular nerve for single-unit recording. We accomplish this by registering the location of the porus acusticus relative to a chamber’s axis identified in CT scans and determining parameters for a micromanipulator XY stage mounted on the chamber that navigates and holds a guide tube and recording electrode.

Although a low sample number precludes a statistically rigorous comparison between the traditional stereotaxic method alone and traditional stereotaxis augmented by post- operative CT guidance, the latter was more efficient in our experience, With the help of the post-operative CT guidance, vestibular afferent neurons were precisely located in the first experiment after chamber implantation in the two rhesus monkeys for which we used this technique,.

CT guidance using 3D reconstructions obtained after surgical attachment of a recording chamber to the calvarium provides a safety net in case of inaccuracies of stereotactic targeting (e.g., due to differences between the study subject and atlas), and it gives the surgeon flexibility. For example, if a large meningeal vessel is in the path that had been planned for a guide tube, the surgeon can reorient the chamber on the fly during surgery without fear of later failing to find the nerve.

In the rhesus brain stereotaxic atlas we use,[39] the position the portion of vestibular nerve that comes out of porus acusticus is about: posterior 0.06 cm, inferior 0.10

37 cm and lateral 0.85 cm. Compared with the atlas, in our results, the position is more anterior and superior, which shows quite obvious inter-subject difference.

Although we still used a traditional stereotactic approach to initially place the recording chambers, our method does not require that a chamber be very accurately oriented, as long as the target lies along a path accessible by a guide tube passed parallel to the chamber’s axis via a craniotomy. As Figure 2-8 shows, in all five monkeys, the distances from porus acusticus to the chamber axis are all smaller than the chamber radius, which means the nerve can be reached within the range of chamber. Navigation of electrode is all based on the CT scan measurements.

Figure 2-8.Location of porus acusticus in transverse plane of chamber coordinate

Part (a) shows the location of porus acusticus in chamber coordinate on left side for five rhesus monkeys, part (b) shows the right side. Red dots represent targets. The black dot represents chamber axis, and the black ring shows the location of the ~3.5 cm diameter chamber.

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The CT method also provides accurate and precise information regarding the distance from parietal dura to the vestibular nerve, which is another parameter needed to identify the location of the nerve efficiently. This saves time and reduces the risk of damage to the animal’s brainstem and to the recording microelectrodes, because it permits one to quickly and reliably pass the guide tube until it stops just before the nerve while not injuring the nerve or adjacent vessels. Inserting electrodes with image guidance can therefore reduce complications compared to standard stereotaxic surgery and therefore reduce the risk to an animal subject. Moreover, by helping to maximize data yields per animal, CT guidance can reduce the number of animals needed to obtain data for a given experiment.

Despite the advantages of CT guidance, several modes of targeting failure remain.

First, the guide tube may bend or tilt subtly when penetrating scar tissue near the craniotomy skull surface, which causes the end of guide tube to move away from planned position. The tendency for this to occur can be reduced by ensuring the guide tube is rigid, held to the XY stage in a way that ensures it remains perpendicular to the stage, and filed so that its tip is concentrically beveled. (Typical slant-beveled spinal needles tend to bend as they are advanced through tissue.) Second, the outer diameter of tungsten microelectrodes (120 um) is typically much smaller than the inner diameter of guide tube

(1mm), so a microelectrode advanced beyond the tip of a guide tube may bend when penetrating hard scar tissue or being squeezed by the core of tissue that may enter the guide tube lumen as the guide tube is inserted. Third, one can accurately target the vestibulocochlear nerve with a guide tube and yet not selectively impale individual axons in a way that yields high signal-to-noise recordings. Because the cell bodies of vestibular nerve afferents are in the fundus of the internal auditory canal, they are shielded by bone

39 and inaccessible to microelectrodes inserted via a parietal craniotomy. Tungsten microelectrodes with tips of ~1-3 um may be too large to isolate vestibular primary afferent axons with diameter less than ~10 um. Fourth, one may accurately target the 7th/8th cranial nerve bundle but miss the vestibular divisions by being ~200 um too anterior, instead hitting only facial nerve motor neurons and cochlear afferents. Finally, changes in head orientation can cause the brainstem to shift position within the skull. The effect of such movement should be minimized by targeting the nerve where it leaves the bone at the porus acusticus. However, clinical magnetic resonance imaging scans acquired with patients lying supine commonly show the 7th/8th nerve complex resting against the posterior lip of the porus (C Della Santina, personal communication), suggesting that a point coaxial with the internal auditory canal may not necessarily lying within the center of the nerve.

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Chapter 3 Intracranial/Extravascular Application of OCT and

IVUS for Imaging the Subarachnoid Space

We explored two real-time imaging techniques that are currently applied to cardiology intervention surgery: intravascular optical coherence and intravascular ultrasound. We sought to determine whether these highly refined intravascular technologies, which are already approved by the United States Food & Drug

Administration for intravascular use in interventional cardiology and interventional neuroradiology, might be adapted for intracranial/extravascular imaging of structures in the subarachnoid space.

3.1 Experimental Subjects

3.1.1 Phantom

We made a phantom for image testing by embedding spaghetti and penne pasta in a bowl full of red gelatin to simulate the CSF space. The inner diameter of penne pasta is about 5mm and the thickness of penne wall is about 1.5 mm.

3.1.2 Chinchilla Subject

An immediately post-mortem adult wild-type 450g female chinchilla (Chinchilla lanigera) was used for experiments, which were performed just after euthanasia following a separate, terminal single unit recording experiment performed under deep general anesthesia in accordance with a protocol approved by the Johns Hopkins Animal Care and

Use Committee.

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3.1.3 Rhesus Monkey Subjects

A post-mortem rhesus monkey (RhF4617J) was used for IVUS and OCT experiments, which was performed in accordance with a protocol (Protocol#: PR12M318) approved by the Johns Hopkin Animal Care and Use Committee.

3.2 Surgical Procedures

3.2.1 Chinchilla Surgery

A chinchilla was used to study OCT immediately after euthanasia following a terminal single-unit recording experiment performed as part of a different study. The chinchilla had been anesthetized with 3-5% isoflurane anesthesia and sterilely prepped.

Skin was locally anesthetized (0.5% bupivacaine with 1:100K epinephrine). Enrofloxacin antibiotic prophylaxis was given. A patch of skin was excised between the mastoid bullae, and holes were drilled in the roof of each bulla using otologic drill. GelFilm® and/or

GelFoam® were used to shield the middle ear and ossicles from the mastoid bulla. Dental cement (3M ESPE ProTemp®II or OrthoJet®) was used to conformally fill the superomedial recess of each bulla, with the cement joining across the midline adjacent to the skull to form a rigid cast around the central portion of the skull. A phenolic post was embedded in the cement and oriented 10o pitched back a plane tangent to the central portion of the visible skull (so that when animals was restrained with the post in the 45o pitch-nose- down slot of a Plexiglass restraint, the chinchilla’s mean horizontal semicircular canal axis was Earth vertical).

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Immediately following the single unit experiment, the animal was euthanized under deep inhalational anesthesia. A hole was then opened on the floor of bulla 1mm anterior and medial to the anterior semicircular canal ampulla using an otologic drill, to create a port into the internal auditory canal for inserting an OCT imaging catheter.

3.3 Intracranial/Extravascular Optical Coherence Tomography

3.3.1 Method

For the intracranial OCT imaging experiment, we used a St. Jude Medical® C7XR unit with a C7 Dragonfly IV imaging catheter with OCT flush, 100 frames/sec, pullback rate 2cm/s. the outer diameter of catheter is 0.9mm. The imaging resolution is about 20um in greatest dimension. The system is available to perform pullback side-viewing image scan with length of 55mm.

First, we performed an OCT imaging test in the penne-gelatin phantom to simulate the situation of cerebrospinal fluid (CSF) space around porus acusticus. Figure 3-1 shows the OCT images obtained by inserting a 30 mm-length of the catheter roughly along the axis of penne inside the gelatin. We triggered the pullback scanning and the transducer moved backward while scanning.

For the chinchilla experiment, we performed an OCT imaging scan to examine the inner acoustic canal in an animal immediately after euthanasia by passing the catheter through a hole opened on the floor of bulla via which the 7th/8th nerve complex had been accessed for single unit recording as part of a separate research protocol (Figure 3-2).

(Whereas we approach the rhesus vestibular nerve for single unit recording via the parietal cranium, we typically approach the chinchilla internal auditory canal via the bulla, so the 43 never is directly visible in the latter case.) The chinchilla head was then dissected to determine where the OCT probe had been.

Next, we placed the catheter near the left porus acusticus in a rhesus monkey skull and performed the image scanning to visualize its bone structure in OCT.

Finally, we passed an OCT catheter inside CSF through the guide tube navigated by the CT measurement of target in a post-mortem monkey head (RhF4617J).

Figure 3-1. Placement of OCT catheter inside penne embedded in gelatin

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Figure 3-2.Simulation of placing OCT catheter through the floor of bulla in chinchilla right ear under camera view

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3.3.2 Results

Stacks of OCT side-view images were acquired with imaging diameter of 5mm, which nicely constructed a 3D image of the penne pasta (Figure 3-3). The tiny circle in the center of image is the catheter around the OCT probe. The larger, oval orange curve with fading shade outside it is the imaging of interface between gelatin and wall of penne. The infrared light hardly penetrates the penne’s inner wall and mostly reflects back or is absorbed by the pasta, so the outer wall/gelatin boundary is not detected by OCT.

An OCT 3D reconstruction was acquired via pullback scanning after the catheter was passed through the floor of the bulla and into the internal auditory canal and cerebellopontine angle of a chinchilla immediately post-mortem (Figure 3-4). We can clearly identify two black holes near each other on the surface of some dense matter which is likely to be the bone. The landmark in 3D reconstructed image is identical to the real bone structure, which was exposed after dissecting the chinchilla’s head (Figure 3-5).

OCT 3D reconstruction (Figure 3-6 b) was also done after pullback scanning near porus acusticus in a rhesus monkey skull (Figure 3-6 a), which clearly shows the porus acusticus and subarcuate parafloccular recess compared with CT 3D reconstruction (Figure

3-6 c).

Unfortunately, in the OCT image acquired from scanning in the post-mortem rhesus monkey assisted by post-operative CT guidance (Figure 3-7), we can hardly identify any structures. In one of the image scans, a dark blurry area close to the shields of catheters could be CSF since no reflection occurred there. The areas that have bright boundary and fade in radial gradient could be either soft tissue or bone. We could not confidently relate

46 them to any anatomic landmarks that would help find the porus acusticus or the vestibular nerve.

Figure 3-3. OCT image of a penne in the gelatin phantom

Top left section shows the 3D reconstruction of part of the penne’s inner wall within the pullback scanning range. Top right section shows the slice of side-view image, location of which is indicated by the white circle in the left section. Lower section shows a cross-sectional image along catheter axis, orientation of which is indicated by the yellow in the top left section.

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Figure 3-4.OCT image of inner acoustic canal in chinchilla

Two black holes can be identified as porus on the surface of dense matter, facing toward a small space free of reflection.

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Figure 3-5.Location of OCT catheter placement into the internal auditory canal and cerebellopontine angle of a chinchilla placed just after euthanasia and then dissected after OCT image acquisition.

Red circle is the area of internal auditory canal, where vestibular afferent nerves of different semicircular canals join together. The view is from the contralateral side, after removal of the brain and brainstem.

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Figure 3-6.Rhesus monkey inner acoustic opening on left side

Rhesus monkey inner acoustic canal: (a) endoscopic photograph of an OCT catheter placed near internal acoustic opening on bony skull specimen, (b) Optical coherence tomography shows bone/CSF interfaces, (c) CT 3D reconstruction.

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Figure 3-7.A slice of OCT side-view scan when catheter is placed in CSF space in the post-mortem rhesus monkey on left side

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3.4 Intracranial/Extravascular Ultrasound Imaging of the

Subarachnoid Space

3.4.1 Method

For the intracranial/extravascular ultrasound (IC/EVUS) experiment, we used a

Volcano CORE® mobile unit, operating at 10 frames/sec with the Volcano Eagle-Eye®

Platinum IVUS imaging catheter. The outer diameter of catheter is 1.1mm. The system is available to perform side-viewing scans and construct a cross-sectional image centered on the transducer. The imaging resolution is about 200 um in greatest dimension. The ultrasound unit can provide a real-time side-view image with imaging diameter of 10mm but it doesn’t have the automatic pullback function we used with our OCT imaging unit.

First, we performed IC/EVUS imaging test in the pasta-gelatin phantom to simulate the situation of cerebrospinal fluid (CSF) space around porus acusticus by inserting the catheter roughly along the axis of penne inside the gelatin, which is similar to the approach in the OCT experiment. We then used post-operative CT guidance on a rhesus monkey head specimen with implanted chambers and passed the IC/EVUS catheter through brain tissue through the guide tube navigated by the CT measurement of target. We manually pulled back the catheter. Due to constraints on availability of OCT and IVUS equipment for use with animal specimens, we could not perform IC/EVUS in a live animal.

3.4.2 Results

As Figure 3-8 shows, the ultrasound penetrates the penne and a cross-sectional image of penne is readily detected. The small bright white circle is identified as the shield

52 of catheter and the larger bright circle-like shape is identified as the transverse view of penne. In one of the ultrasound images scanned in CSF space on the right side in the rhesus monkey head specimen (Figure 3-9 a), we found an angular structure formed by two short bright edges.

Figure 3-8.IC/EVUS side-view image of penne embedded in gelatin phantom

The boundary between the outer wall of the penna and the gelatin can be clearly identified, which cannot be seen in OCT due to light absorption.

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Figure 3-9. Images of subarcuate parafloccular recess obtained using intracranial/extravascular ultrasound in the cerebellopontine angle of a rhesus monkey specimen.

Subarcuate parafloccular recess in (a) IC/EVUS side-view scan (b) CT 3D reconstruction (c) CT cross- sectional image (d) CT cross-sectional image with measurement of the angular structure

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The angle is approximately 62o, and lengths of two edges are about 4mm and 5mm after scaling to real space. We retrieved 3D CT reconstruction of the specimen (Figure 3-9 b), and found that the subarcuate parafloccular recess had a sharp-angle opening. A cross- sectional CT image (Figure 3-9 c) shows a similar structure as seen in the IC/EVUS image.

As measured in CT scan, the angle is approximately 60° and the lengths of bone edges are about 4.9mm and 5.3mm (Figure 3-9 d). Measurements from the IC/EVUS image and the

CT image are basically the same, which suggests that IC/EVUS can identify bone/CSF boundaries like the porus acusticus and subarcuate parafloccular recess, if they have already been seen on a CT 3D reconstruction. However, even with advance knowledge of the anatomy from the CT dataset, we could not identify any structure that appeared to be the vestibular nerve on IC/EVUS.

3.5 Discussion

Comparing the OCT and IC/EVUS images of the penne-gelatin phantom (Figure

3-10), we can see that OCT has better spatial resolution than ultrasound, but IC/EVUS is able to penetrate the penne and provides both a larger imaging depth and definition of the outer pasta/gelatin boundary. Summary of comparison is shown in Table 3-1.

Techniques Catheter Model Imaging Depth Resolution Catheter Automatic Images Diameter Pullback through Function optical opaque material OCT C7 Dragonfly 5 mm in ~20 um 2.7F Yes No (St. Jude diameter (0.9mm) Medical) IC/EVUS Eagle-Eye 10 mm in ~200 um 3.5F No Yes Platinum diameter (1.2 mm)

Table 3-1.Comparison of OCT and IC/EVUS

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In the immediately postmortem chinchilla specimen, OCT 3D reconstruction apparently showed the porus acusticus and subarcuate parafloccular fossa, which although unnecessary in that experimental preparation (we can already see the chinchilla 7th/8th nerve directly), served as a useful proof of concept. Unfortunately, our attempts at performing

OCT imaging inside CSF space in the post-mortem rhesus monkey did not result in images from which we could identify the vestibular nerve, porus acusticus, or other anatomic landmarks. There are several possible causes for the failure. First, soft tissue and possibly blood in CSF make the OCT light reflect back and generate blurring artifacts, which limits the field of view and penetration depth. Regardless of any other source of failure, the major limitation of OCT for our application was the shallow penetration depth. Second, the guide tube might not have been pointed at the right target, so the porus acusticus or subarcuate parafloccular recess might have been completely out of the imaging range. Third, inability to image along the guide tube axis with our side-viewing OCT system precluded seeing where the guide tube was headed. Finally, the lack of OCT catheters designed and intended for our intended use required that we instead use a clinical model of OCT catheter intended for coronary artery imaging that would not allow us to advance the OCT imaging unit to the end of the unit’s protective catheter sheath. To overcome this, we had to cut the end of the sheath, allowing bodily fluids to back fill into the catheter and perhaps cloud the image.

As for IC/EVUS, although its resolution is not as high as OCT, it has larger penetration through soft tissue. The IC/EVUS images we obtained (Figure 3-9) clearly show the bone boundaries of the subarcuate parafloccular recess, as confirmed by CT scan.

IC/EVUS provided clear contrast between bone and other tissue, which can help identify bone landmarks that can help one target the vestibular nerve if the catheter is within 10 mm

56 of the porus acusticus. However, the IVUS probe is slightly larger than the 1.0 mm inner diameter guide tube we normally use.

Figure 3-10.Comparison of OCT and IC/EVUS in gelatin phantom

Images of the penne in gelatin: (a) camera view of the penne in gelatin (b) OCT side-view image of the penne (c) IC/EVUS side-view image of the penne

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Chapter 4 Conclusion

4.1 Post-Operative CT Guidance

Compared with the conventional stereotaxic method, augmenting traditional stereotaxis with post-operative CT guidance as presented in this thesis was more efficient and more effective in helping researchers find the rhesus vestibular nerve. This method doesn’t completely depend on an atlas or require the monkey to be physically attached to the stereotaxic frame in recording stage, which allows for more flexibility in surgical technique and allows a neurophysiologist to find the vestibular nerve despite inter-subject anatomic differences. Moreover, this method provides intuitive image guidance on where and how deep the guide tube and electrode should be inserted to reach the target. Having accurate measurements from a CT scan greatly reduces the risk of overinsertion, so one can avoid inadvertently cutting the nerve during guide tube insertion or damaging the microelectrode by driving it into a bone wall. CT imaging units with adequate resolution are widely available in academic medical centers, and 3D reconstruction software is readily available to research labs.

4.2 Intracranial Imaging of the Subarachnoid Space Using OCT and

IVUS

OCT and ultrasound within CSF spaces like the cerebellopontine angle offer complementary advantages for targeting cranial nerves. When passed through the guide tube of a single-unit recording set-up, optical coherence tomography probes can provide direct view of intracranial tissue and bone structures through a small craniotomy.

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Unfortunately, in our experience, optical blurring and a shallow penetration depth limited the ability to visualize the porus acusticus or the vestibular nerve in post-mortem and live rhesus monkeys using OCT.

Our intracranial adaptation of IVUS offered a larger imaging depth and ability to see through optically opaque or translucent material (probably including nerve, dura and brain), but its resolution is not as high as optical coherence tomography. The large penetration depth enables it to detect bone structures beyond soft tissues within the range.

Visualization of the bone landmarks in US images has the potential to serve as real-time guidance for targeting cranial nerves in electrophysiologic experiments.

Adaptation of intravascular OCT and IVUS systems for intracranial/extravascular imaging of cranial and spinal nerves may hold promise for intraoperative guidance in minimal-access rhizotomy and other clinical procedures that require precise spatial targeting of neural structures through narrow channels. Further development of OCT is needed in imaging penetration. It would be great if OCT and IVUS are combined in one modality for targeting, which will have large imaging penetration depth for detecting more structures and high resolution for detecting soft tissues.

4.3 Future Directions

Although the post-operative CT guidance can already give us direct and precise guidance to reach the porus acusticus, imprecision in our estimate of the exact direction and size of vestibular nerve coming out of the porus acusticus still make targeting a challenge. To visualize the vestibular nerve, coregistering 3D reconstructions of both CT and MRI imaging could be considered. To perform MRI after chamber implantation, the

59 chambers and guide tube would need to be replaced by non-ferrous, nonmagnetic material.

Recent progress in intravascular MRI [99-101] also holds promise as another means by which a high resolution imaging unit can be introduced via a guide tube into the subarachnoid space, whether for targeting the vestibular nerve in rhesus monkeys or for analogous neurosurgical procedures in humans.

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Curriculum Vitae

Shiyao Dong was born in Xi’an, Shaanxi, China on February 1st, 1992. After graduating from the Gaoxin No.1 High School, he attended the Zhejiang University. In

2010, he graduated from the Zhejiang University with a B.S in Biomedical Engineering.

His research interests include: biomedical engineering and data mining.

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