Optical coherence tomography guided neurosurgical procedures in small rodents

Journal of Neuroscience Methods 176 (2009) 85–95

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Optical coherence tomography guided neurosurgical procedures in small rodents M. Samir Jafri a,b,∗ , Rebecca Tang b , Cha-Min Tang a,b a b

Research Service, Department of Veterans Affairs Medical Center, Baltimore, MD, United States Department of Neurology, University of Maryland School of Medicine, Baltimore, MD, United States

a r t i c l e

i n f o

Article history: Received 12 May 2008 Received in revised form 19 August 2008 Accepted 20 August 2008 Keywords: Stereotaxis Epilepsy Hippocampus Lesion Stem cell Gene therapy Imaging Surgical techniques Optical coherence tomography Rodent Rat Neurosurgery Substantia nigra Parkinson’s disease

a b s t r a c t The delivery of therapeutic agents directly to targets deep within the brain is becoming an important tool in the treatment of a variety of neurological disorders. Currently, the standard method to accomplish this is by using stereotactic procedures. While this existing method is adequate for many experimental situations, it is essentially a blind procedure that cannot provide real-time feedback on whether the actual location deviated from the intended location or whether the therapeutic agent was actually delivered. Here we describe an optical guidance technique that is designed to work in conjunction with existing stereotactic procedures to provide the needed real-time feedback for therapeutic delivery in live animals. This real-time feedback is enabled by a technology called catheter-based optical coherence tomography (OCT). In this study we show that OCT can provide real-time position feedback based on microanatomic landmarks from the live rodent brain. We show that OCT can provide the necessary guidance to perform microsurgery such as the selective transection of the Schaffer collateral inputs to the CA1 region of the hippocampus with minimal perturbation of overlying structures. We also show that OCT allows visual monitoring of the successful delivery of viral vectors to specific subregions of the hippocampus. Published by Elsevier B.V.

1. Introduction Efforts towards finding treatments for neurological conditions ranging from epilepsy to degenerative disorders such as Parkinson’s, Alzheimer’s and Lou Gehrig’s disease have led to exciting possibilities in gene and cell-based therapies. One under appreciated technical hurdle for these therapies is the means to precisely and reliably deliver the therapeutic agent into the brain region of interest. The precision and reliability by which the therapeutic agents are delivered can affect clinical trial outcomes. For example, when nerve growth factor (NGF) was delivered intraventricularly rather than into the cortical structures in patients with Alzheimer’s disease (AD), the trial had to be discontinued because of severe debilitating side effects at sites other than the intended brain structures, particularly NGF-induced back pain (Eriksdotter-Jonhagen et al., 1998). In the failed glial-cell derived neurotrophic factor (GDNF) trials for Parkinson’s disease (PD),

∗ Corresponding author at: Department of Neurology, University of Maryland School of Medicine, 12th floor Bressler Building, 655 West Baltimore Street, Baltimore, MD 21201-1595, United States. Tel.: +1 410 706 2384; fax: +1 410 706 0186. E-mail address: [email protected] (M.S. Jafri). 0165-0270/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.jneumeth.2008.08.038

many in the field felt that the reason for the failures was that GDNF may have backtracked along the sides of the large catheter or that the catheters had been placed in the wrong location. Three catheters in two patients were mispositioned and required additional surgery for repositioning (Lang et al., 2006). Also, it was discovered at the completion of the study during explantation that three of the infusion catheters in two test patients had migrated out of the putamen (Lang et al., 2006). Adverse events in GDNF treated subjects compared to controls included paraesthesia (65% GDNF vs. 18% control) and headache (29% vs. 6%) (Lang et al., 2006). The cause of these side effects is unknown but may have resulted from the spreading of GDNF away from the target area. Unless one can obtain direct evidence of proper location and confirmation of proper delivery, there will always be lingering doubts on the interpretation of negative results. The purpose of this project is to provide the proof of principle for an image-guided delivery method that can provide the precision, reliability, and confidence level for an increasingly important procedure in translational neuroscience. The current method for delivering therapeutic agents to the brain is through standard stereotactic procedures that targets a predetermined coordinate within the skull. In humans the coordinate is determined by a pre-operative MRI or CT. For rodents, the coordinate is directly obtained from a stereotactic atlas. Standard

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stereotactic procedures are essentially blind procedures because the surgeon does not visualize the target directly. The analogy can be made of a pilot making an instrument landing in the dark without runway lights or radar while using only GPS and the altimeter. There is no question that a smart pilot can accomplish such a task. But even a smart and experienced pilot will not know when there is an unexpected obstacle on the runway under such a situation. For the neurosurgeon the obstacle can be an at risk blood vessel directly in front of the advancing stereotactic probe. A more problematic challenge for the neurosurgeon is that the brain can frequently shift during surgery due to CSF leakage. The only pilots who have to deal with a similar problem are those landing on an aircraft carrier. A real-time image guided procedure that can identify local landmarks would be able to respond to the unexpected and compensate for shifting targets. In the case of delivery of stem cell and gene therapy, the ability to monitor the movement of minute volumes of injected solutions is an additional challenge. When delivering volumes of a few microliters or less, there is no good way to verify whether the therapeutic agents actually exited the needle tip. This may be the result of unexpected air or other compliance in the system or blockage of the needle. Increasing pressure or volume of settings often causes excess delivery or even tissue damage or dissection from the release of the built up pressure. Furthermore, it is not currently possible to determine whether the therapeutic agent tracked up along the outside of the needle or dissected into a tissue plane or into the ventricle. We have previously introduced the use of catheter-based optical coherence tomography (OCT) in ex vivo brain tissue (Jafri et al., 2005). OCT is an emerging clinical imaging technology that has mainly been used in ophthalmology. Optical coherence tomography is an optical ranging technology that uses infrared light (1300 nm) in a way similar to the use of sound waves in ultrasound medical devices. The unique features of catheter-based OCT for guidance of stereotactic neurosurgical procedures are that it provides (1) spatial resolution that is ∼100-fold higher than clinical MRIs, (2) temporal resolution of full frame images at up to 100 frames per second, (3) geometry that allows the probe to be introduced through a small catheter and (4) the ability to couple with an injection needle providing visual confirmation of delivery of minute volumes to specific targets in the CNS. Here we describe an in vivo study using a rodent model to demonstrate the use of OCT to image structures in the brain and to target and visualize delivery of marker beads and viral vectors in vivo in real time. We have shown targeted delivery of marker beads to the rat substantia nigra, delivery of viral vectors to the dentate gyrus, subiculum and CA1 region of the hippocampus and lesioning of the Schaffer collateral pathway in the hippocampus without destroying overlying structures. We have also shown monitoring of delivery of volumes as small as 0.2 ␮l. 2. Materials and methods 2.1. Optical coherence tomography The prototype OCT system used in this study was built by LightLab Imaging (Westford, MA) for neuroimaging research. The system consists of an imaging engine, probe interface unit (PIU), rotary OCT probe, computer and display (Fig. 1A). Connected to the computer via serial interface, the imaging engine houses the light source and other key electro-optical components. Light from the broadband light source is split into reference and sample beams by an efficient polarization-diversity interferometer. The broadband light source consists of a pair of superluminescent diodes (SLEDs) with different central wavelengths whose outputs were combined to produce 18 mW of polarized single-mode light at a peak wavelength of

1300 nm, with a FWHM bandwidth of 65 nm. As determined by the FWHM width of the coherence function measured in air, the axial resolution of the OCT system in tissue is approximately 10 ␮m. Light from a red laser diode is co-launched with the near-infrared beam through a wavelength-division multiplexer to enable visualization of the beam at the distal end of the OCT probe. To form a circumferential image, a motor-driven rotary coupler within the probe interface unit rotates the optical fiber inside the imaging core (Fig. 1B). Probes can be made with differing lengths from the PIU to imaging tip depending on the distance the probe must be inserted. Longer probes have less image stability and are easier to damage. The probes used in these studies are 100 cm long, but good images have been obtained with probes of over 200 cm. The PIU includes a mechanism for pulling the rotating fiber back within the imaging catheter at a constant speed over a maximum distance of 5 cm. The sample beam transmits through the rotating fiber and focuses on the tissue. After passing back into the interferometer, the light backscattered from the tissue mixes with separate reference beams in orthogonal polarization states. The optical delay line in the reference arm, which consists of multi-faceted spiral-shaped mirror rotated by an air-bearing motor, scans a distance of 4.5 mm in air (about 3.3 mm in tissue) at a user-selected repetition rate between 1000 and 4000 lines/s. Interference signals from a pair of photodetectors are processed by on-board digital-signal processors before being sent to the main computer for scan formatting and display. In the present study, images were acquired at approximately 3–10 frames/s, with each frame consisting of 256 lines. Frame rates of up to 20 Hz are currently available. The OCT probe employed in this study was based on a fiber-optic imaging core with a unique design (Swanson et al., 2002). A microoptical lens/beam deflector assembly at the tip of the catheter consists of a segment of multimode fiber interposed between segments of coreless single-mode fiber, composed of silica glass with a tailored refractive-index profile, the multimode fiber segment acts as a converging lens. To deflect the focused beam perpendicular to the long axis of the fiber, one end of the distal coreless segment is polished at a 45◦ angle. The lens assembly is about 1 mm long and has the same diameter (125 ␮m) as the single-mode fiber to which it is attached. Because the elements of the lens assembly are fusion-spliced via arc welds, its mechanical strength is similar to that of an untreated fiber. For this study, the micro-lens assembly was configured to provide a focal spot diameter of 15 ␮m (FWHM) at a working distance of approximately 1 mm from the center of the probe. The outer diameter of the probe inserted in the brain was 0.36 mm, small enough to insert through 24-gauge thin-wall stainless tubing (Jafri et al., 2005). In these experiments the OCT probe was inserted in one barrel of a dual guide tube with either an injection needle or a cutting wire in a second thinner barrel (Fig. 1C, E and F). Details of these devices are given in their appropriate sections below. The guide tube was held in a micromanipulator mounted on a rodent stereotactic frame (David Kopf Instruments, Tujunga, CA). The probe holder was constructed in-house and designed to hold the OCT probe firmly in place once positioned in the guide tube. The needle or wire in the other barrel was held in place, but was adjustable allowing it to be advanced when needed and in the case of the cutting wire, rotated as well. Fig. 1D shows the relative size of the OCT probe (d) and the fused silica injection needle (c) compared to a guide cannula (a), a recording electrode (b) and a stimulus electrode (e) used in human deep brain stimulation surgeries. 2.2. General surgical procedure All procedures performed were approved by the University of Maryland, Baltimore (UMB), Institutional Animal Care and Use

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Fig. 1. OCT apparatus. (A) Schematic representation of the recording set up. (B) The probe interface unit is a free-standing component. The probe is shown exiting on the left and looping back on top of unit (arrows). (C) Manipulator designed to hold the double-barreled tube containing the OCT probe and either the fused silica injection needle or the nitinol cutting wire. White dashed box is expanded in (E) and (F). (D) The OCT probe (d) and fused silica needle (c) are small compared to the cannula (a), recording electrode (b) and stimulating electrode (e) used in human deep brain stimulation procedures. (E) Dual tubes showing OCT probe and curved, extended nitinol wire. The manipulator allows the nitinol wire to be rotated independently within its tube to perform cutting function. (F) Dual tubes showing OCT probe and fused silica injection needle. Scale bar (E) and (F) 1 mm.

Committee (IACUC). The animals were fed (food and water ad libitum) and housed (12 h light/dark cycle) in a facility maintained by UMB Veterinary Services. All procedures were performed on adult Sprague–Dawley rats (250–350 g, Charles River). Adult Sprague–Dawley rats were anesthetized using ketamine (40–80 mg/kg, i.p.)/xylazine (5–10 mg/kg i.p.) and given buprenorphine (0.02–0.05 mg/kg s.q.) prior to surgery for preemptive pain management. They were maintained at 37–39 ◦ C on a waterproof, adjustable temperature, electric warming pad (Kaz Inc., Southborough, MA) during the procedure. During the surgery a petroleum-based ophthalmic ointment, Puralube (Pharmaderm, Florham Park, NJ), was applied on the eyes to prevent them from drying out or getting scratched by debris or hair. Anesthetized animals were placed in a small animal stereotactic frame. A small (1 cm) incision was made in the scalp extending from the brow ridge to the base of the skull, scraping any muscle back and drilling a small hole in the skull with a hand-held electric drill (Dremel, 2 mm miniature burr). A thin double-barreled guide cannula (0.4 and 0.5 mm in O.D.) was inserted through the burr hole in the skull and advanced partly towards the target brain structure. An OCT imaging probe, 0.35 mm in diameter, was placed in one of the two guide cannulae (Fig. 1C, Dd, E and F). The tip of the OCT imaging probe was advanced about 3–5 mm past the end of the cannula. The second guide cannula was used for either the injection needle or the nitinol cutting wire. In all experiments, voice recordings were made as the OCT video was recorded continuously while advancing the probe, injecting solutions, and retracting the probe.

Voice recordings are captured along with the video and stored in the same file so images and events can be easily correlated. 2.3. General injection procedure for microspheres or viral vectors A fused silica needle (35G, 100 ␮m i.d., 50 cm in length, WPI Inc., Sarasota FL, Fig. 1C, D and F) was placed within the second thinner guide tube (Fig. 1Dc and F) and attached to a 50 ␮l Hamilton syringe installed in a digitally controlled syringe pump. The needle was not advanced beyond the opening of the guide tube. The guide tube was advanced towards the target structure in the brain with the OCT imaging probe providing high resolution, realtime feedback of the precise position of the OCT tip in the rat brain. When the desired position in the brain was reached, after a 2-min waiting period, the pump was activated to deliver 1 ␮l of solution per location at a rate 0.5 ␮l/min by a calibrated stepper motor controlled syringe pump for a total injection time of 2 min. Visualization of the microbubbles on OCT was used to confirm the success of the injection. If microbubbles were not seen, the injection was continued until the microbubbles were seen and 1 ␮l volume was delivered. After an additional 2-min waiting period the assembly was retracted by 0.25 mm followed by an additional 1-min waiting period. The rest periods used before and after injection were to avoid track back of injected solution. The assembly continued to be retracted 0.25 mm at a time separated by 1 min intervals until the target structure was cleared as seen on the OCT image. The assembly was then withdrawn slowly until clear of the animal.

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2.4. Delivery of microspheres to substantia nigra Fluorescent microspheres (0.39 ␮m dia, Duke Scientific, Fremont, CA) were mixed with microbubbles (1:1, Vevo Micromarker contrast agent, VisualSonics, Toronto, ON) to allow visualization of the injected material on OCT and loaded into the Hamilton syringe. A hole was drilled through the skull at a point 3 mm posterior and 2 mm lateral to bregma. The arm of the frame, used to hold the OCT/needle assembly, was set up vertically and lined up with the hole in the skull. The landmarks visualized by OCT in order were: cortex, corpus callosum, hippocampus, thalamus and substantia nigra (SN). The SN was distinguished by its characteristic striated appearance. The target region was a point 1 mm beyond the entry into the SN from the capsule, an easily discernable boundary on OCT. The injections were performed as described above. 2.5. Delivery of viral vector The procedure for delivering viral vectors is similar to that used for microspheres. The viral vectors were mixed with microbubbles to track their delivery. Adeno-associated virus (AAV2.CMV.EGFP, Penn Vector Core, Gene Therapy, University of Pennsylvania) expressing enhanced green fluorescent protein was used. The injections consisted of 1010 viral particles in 1 ␮l or less at a rate of 0.5 ␮l/min as described above. To target hippocampal regions, a hole was drilled through the skull at a point 7 mm posterior and 7 mm lateral to bregma. The arm of the stereotactic frame was used to hold the OCT/needle assembly. It was set up angled 45◦ posterior to anterior from perpendicular with respect to the midline at an angle 30◦ above horizontal and lined up with the hole in the skull. The landmarks visualized by OCT in order were: cortex, corpus callosum, ventricle, alveus and finally the hippocampus. The hippocampus was distinguished by the characteristic appearance of the cell layers that were clearly visible on OCT. The target regions of the subiculum, dentate gyrus and CA1 region of the hippocampus were visually identified. The location was noted and indicated on the OCT image for comparison with the histology. Eight weeks were allowed after injection for expression of EGFP reporter. 2.6. Hippocampal lesioning The goal was to lesion the Schaffer collaterals without cutting the cortex or the axonal output of the CA1 neurons. OCT provides the capability to identify critical anatomic landmarks in the hippocampus and allows monitoring of the precise location of the lesion being created in real-time. Under OCT guidance precise lesions in the hippocampus were made using a retractable, thin curved cutting probe. The hippocampus was targeted as described under “delivery of viral vector section.” The target region was a point between the CA1 and CA3 regions of the hippocampus. The location was noted and indicated on the OCT image for comparison with the histology. The cutting probe was stereotactically inserted within 2 mm of the OCT imaging probe. This cutting probe consisted of a thin curved nitinol wire (0.006–0.011 in.-in-diameter; radius of curvature, 2–3 mm) placed inside a hollow straight stainless guide tube (0.016–0.020 in.-in-diameter, Fig. 1C). Nitinol wire is a curved, flexible, “shape-memory” wire made of a nickel titanium alloy. The wire was withdrawn into the straight cannula until the target was reached. Upon reaching the target, the wire was extended out of the guide tube. The shape memory characteristic of the nitinol wire allowed it to return to its curvature when extended out of the straight guide tube, projecting lateral to the axis of the probe. The wire was visible on OCT. Cuts in the brain were made by extend-

ing the curved nitinol wire beyond the tip of the straight hollow tubing and either rotating the wire or moving the entire assembly forward and back along its axis. After the wire was withdrawn back into the cannula the lesion was visible (Fig. 3A). The assembly was then retracted. The advantage of this procedure is that little damage is created at sites other than the intended site. After making the desired cuts, the curved wire was retracted into the straight tubing and the entire assembly removed. 2.7. Tissue fixation At the end of each experiment, rats were treated with a lethal dose of Pentobarbital (100 mg/kg i.p.) and perfused through the heart with 100 ml saline followed by 200 ml 4% paraformaldehyde (PFA). After removal the brain was immersed in 4% PFA at 4 ◦ C for an additional 24–48 h. Cryostat sections were prepared and mounted on slides for visualization. 3. Results 3.1. OCT provides location feedback in vivo Standard stereotactic methodology is fundamentally a blind procedure based on dead reckoning to reach a pre-determined coordinate typically based on “historic” information. That information may be an atlas when working with rodents or a pre-operative MRI when working with primates. OCT can be used in conjunction with standard stereotactic procedures. It provides real-time feedback of its actual location relative to well defined anatomic landmarks. Optical guidance for stereotactic procedures relies on the ability to detect optically the junctions between gray and white matter and to use them as landmarks for the target. OCT-assisted stereotaxis was used to guide injection of colored beads into rat substantia nigra (Fig. 2). Fig. 2A shows a coronal section of unfixed rat brain through which the needle was inserted indicating the track used to target the substantia nigra (sn). The cortex (ctx), corpus callosum (cc), hippocampus (hip) and thalamus (th) are identified in the photograph (Fig. 2A) as well as in the accompanying anatomical schematic (Fig. 2B). The blue line in both these images illustrates the orientation of the track used to obtain the B-mode and L-mode images shown in Fig. 2C (described below). OCT images through a comparable brain were recorded in realtime video at 3.1 Hz as the probe was advanced through the tissue (Fig. 2C). Information acquired by OCT can be displayed in two basic formats. The scans are displayed and recorded as a sequence of two-dimensional tomograms (“B-mode”), each showing a single 360◦ sweep of the light beam (Fig. 2C, left and right columns). In this format the characteristic distribution of gray and white matter, the blood vessel pattern and the degree of light backscattering of each structure together provide an optical signature of the structure. Alternately, the intensity of the averaged backscattered light can be quantified and color coded to create a modified version of a longitudinal-mode (“L-mode”) projection, which is commonly employed in intravascular ultrasound imaging. Color-coded light intensity is displayed as a function of radial distance and axial position as the probe is advanced in one direction (Fig. 2C, center). To obtain the L-mode images in these figures, the radially integrated intensity above a selectable threshold (80% of maximum) was displayed on a color scale. This mode is particularly helpful for precisely mapping the position of gray–white matter borders and the position of the probe relative to these borders. Myelinated fiber tracts, such as the corpus callosum, typically appear bright in OCT images (Fig. 2C, corpus callosum) and as a highly intense signal indicated in red in the L-mode. Their

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Fig. 2. Structure can be clearly identified by OCT. (A) Photograph of a coronal section of rat brain showing the cortex (ctx), corpus callosum (cc), hippocampus (hip), thalamus (th) and substantia nigra (sn). Blue line indicates approximate OCT track shown in (C). (B) Schematic diagram of OCT track shown in (C). (C) The left and right columns show individual B-mode OCT images obtained during a single 360◦ rotation of the light beam. These structures were imaged while the probe was slowly advanced approximately along the track shown in (A) and (B) in a comparable rat brain. The gray matter of the cortex gives an image with low intensity, but deep penetration while the white matter of the cc gives a bright signal, characterized by strong backscattering and poor penetration. The image of the substantia nigra reflects the gray and white matter striations of this structure. These images are described in the text. Scale bar, 0.5 mm. The L-mode projection of OCT (center) displays the absolute averaged intensity of backscattered light and depth of penetration as the probe is linearly advanced. For interpretation compare the L-mode data to actual anatomic structures shown in (A) and (B) and see text. (D) OCT-targeted injection of labeled beads into the substantia nigra (red dot). Scale bar, 1 mm.

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Fig. 3. Focal lesioning in vivo using OCT. (A) OCT image of a rat hippocampus showing lesion of Schaffer collaterals without lesioning overlying structures. (B) Postmortem section of rat brain verifying accuracy of lesion. Hippocampal CA1, CA3, dentate (d) and subiculum (s) are indicated. Scale bar, 0.5 mm.

bright appearance on OCT demonstrates that myelinated fibers are also strong backscatterers of light in the near-infrared spectrum (∼1300 nm). Penetration of light into the myelinated fibers is shallow (less than a few hundred micrometers) since healthy white matter behaves predominately as a specular reflector. In contrast, gray matter disperses the light and is a weak reflector, resulting in an image with low intensity, but deep penetration. An example of this type of tissue is shown in the B-mode image of the cortex and the corresponding region of the L-mode projection (Fig. 2C). The boundary between the cortex and the corpus callosum is shown on the L-mode projection by the distinct high intensity red signal. The B-mode image of the hippocampus has a characteristic appearance that reflects its structure (Figs. 2C, 3, 4 and 5). Hippocampal imaging is described in detail in the sections on lesioning and stem cell delivery. The thalamus is a mixture of gray and white matter. Both the B-mode images and the L-mode projection represent these structural characteristics with a signal that has intermediate brightness and penetration as compared with pure gray or white matter regions. Although there is not a distinct fiber tract immediately ventral to the substantia nigra (SN), entrance into the substantia nigra is apparent on OCT. There is a marked increase in the depth of light penetration upon entering the SN. The SN is associated with thick ribbons of white matter tracts seen in the B-mode image of the SN (Fig. 3C). The actual location of OCT-guided delivery of colored microbeads to the SN is shown in a sagittal section (Fig. 2D, different animal from Fig. 2A). The tissue was harvested 48–72 h after surgery with the animals behaving normally at the time. 3.2. OCT can guide focal lesioning In many instances it may be preferable to create a lesion in a deep brain structure without destroying structures superficial to it. For example, current rodent models of epilepsy consist of either percussion injury to the brain or surgical lesioning using a scalpel to cut from the surface of the brain to the hippocampus to create a Schaffer collateral lesion. In the percussion model, the force necessary is strong enough to kill roughly half the rats immediately with only about 50% of the remaining animals developing epilepsy. Both techniques result in widespread trauma (hemorrhage, cell death, ischemia, deafferentation, etc.) making it difficult to dissect and identify which parameter is most critical for the generation of epilepsy. If the goal is to cut the Schaffer collaterals, it is preferable to avoid cutting the cortex and axonal output of the CA1 neurons. OCT offers a means of generating this type of focal lesion allowing examination of the role of specific pathways in epilepsy in vivo.

This OCT image of a lesioned rat hippocampus (Fig. 3A) clearly shows structural detail similar to that seen in anatomical sections (Fig. 3B). The probe is passing through the dentate gyrus (Fig. 3A circle). As described above, the brightest areas are the densest bands of white matter while the darker areas consist mostly of gray matter. The CA1, CA3, dentate (d) and subiculum (s) are easily discernable (labeled in Fig. 3B). The very dark band to the upper left of the probe indicates the lesion in vivo in real-time immediately after lesioning (Fig. 3A). The anatomical section (Fig. 3B) shows the location of the lesion through the Schaffer collaterals in post-mortem tissue. Operated animals have survived over 2 months. Sham operated animals underwent the identical procedure except that the cutting wire was never extended and have shown no effects of the surgery. The lesioned animals show no unexpected effects of the surgery beyond what was expected from the lesion. 3.3. OCT can guide precise delivery of viral vectors Another area where precise targeting is necessary is the delivery of therapeutic agents, in particular gene therapy. The ability to deliver gene therapy reliably and reproducibly to precise targets and, more importantly, to avoid mis-delivery, will open up a plethora of new therapeutic options. As shown above, the substantia nigra, subthalamic nucleus and specific regions of the hippocampus as well as other areas can be confirmed as targets in real time. As an example, adeno-associated virus carrying enhanced green fluorescent protein (AAV2-EGFP) was delivered to either the dentate, subiculum or CA1 region of a rat hippocampus (Fig. 4). In each pair of images, the OCT image at the injection site is shown on the right (Fig. 4B, D and F) and a photomicrograph showing the fluorescent cells superimposed on the corresponding brightfield image is on the left (Fig. 4A, C and E). Each injection was 0.4 ␮l of AAV2-EGFP (10−10 particles/␮l) at a rate of 0.03 ␮l/s by a calibrated stepper motor controlled syringe pump. In the OCT image of the dentate gyrus, the characteristic triangular structure is seen below the probe (Fig. 4B). The circle to the left of the probe is the injection needle. The location of the transfected cells exactly matches the OCT image of the needle tip with respect to the structure of the dentate (Fig. 4A). Similarly, the tissue structure and needle position shown in the OCT images of the subiculum (Fig. 4C) and CA1 region (Fig. 4E) correlate with the location of the transfected cells (Fig. 4D and F, respectively). These animals were maintained for 8 weeks post-surgery with no ill effects on their health.

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Fig. 4. Precise targeting of viral vector delivery using OCT. Photomicrographs of AAV2-EGFP expression and brightfield images in rat hippocampal dentate gyrus (A), subiculum (C) and CA1 region (E). Corresponding OCT images show real-time structural images of dentate gyrus (B), subiculum (D) and CA1 region (F). The dark circle to the left of the probe is the injection needle. Note the correlation between the position of the injection needle and the gene expression. Scale bars, 0.5 mm.

3.4. OCT can confirm delivery success and monitor delivery volume When delivering therapeutic agents to the brain, it is desirable to deliver small volumes to minimize damage to the tissue around the injection site and to limit the spread to small target regions. One of the problems with delivery of very small volumes is knowing whether the intended volume is delivered or if there is any

delivery at all. This is particularly a problem with stem cell delivery where the cells can clump and block the very fine gauge needles. The resolution of the OCT allows visualization of sub-microliter volumes of injected fluid providing direct real-time confirmation of the success and location of delivery. In addition, the delivery can be monitored to determine whether the injected fluid leaks out of the intended target region or tracks back along the needle track, both well-known problems with injections in the brain.

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Fig. 5. OCT can monitor delivery in vivo. OCT image of rat hippocampus before (A) and after (B) delivery of 0.2 ␮l injection of microbubble solution (B arrow). OCT injection of 0.2 ␮l microbubble solution at a tissue plane in rat hippocampus (C) shows spreads of microbubbles along the plane (D arrows). Scale bars, 0.5 mm.

To achieve this, a contrast agent must be added to the injection mixture. Microbubbles, such as those used for ultrasound, are nontoxic, inert, and give a strong signal on OCT (Fig. 5B and D arrows). Confirmation of delivery of volumes as small as 0.2 ␮l appears as a bright signal (Fig. 5B and D arrows). The tip of the injection needle is just shy of being even with the image plane so the needle is not in view in this image. Using small volumes, it is possible to tightly control the spread of the delivered agent to a small area as seen in the AAV2-EGFP expression in the hippocampus (Fig. 4). A video of this injection can be seen in Supplementary video A. Another issue that can complicate delivery in brain tissue is movement of the injected solution through tissue planes or leakage out of structures into spaces such as the ventricles. The tip of the injection needle can be seen at about 10 o’clock to the OCT probe in this live anesthetized rat hippocampus (Fig. 5C). Injection of 0.2 ␮l of microbubble solution at 0.03 ␮l/s results in lateral spreading along a tissue plane in rat hippocampus (Fig. 5D arrows). The tip of the needle can be seen to the left of the probe casting a shadow. For this experiment, the tip of the needle was deliberately placed at a tissue plane to demonstrate the ability of OCT to identify and precisely target fine structures and to show that realtime monitoring can help identify these types of events even when delivering small volumes.

4. Discussion Catheter-based OCT can be a unique and useful tool for the delivery of agents into the brains in live animal studies. This is the first study to use catheter-based optical coherence tomography to guide stereotactic neurosurgical procedures in vivo. Real-time OCT images show structural detail that allows identification of anatomical structures deep within the brain. Under OCT guidance in live rats we have (1) targeted delivery of minute volumes to the substantia nigra, (2) created focal lesions in the hippocampus without destroying the overlying brain structures and (3) monitored delivery and seen expression of a viral vector to the dentate gyrus, subiculum and CA1 region of the hippocampus. OCT has not been used extensively to examine brain tissue. Our own previous study in human brain (Jafri et al., 2005) demonstrated the ability of this catheter-based system to differentiate between grey and white matter in autopsy human brain. Jeon et al. (2006) use a form of OCT microscopy to scan structures in ex vivo rat brain slices and noted the contrast between the OCT signal from grey and white matter. In this study we have extended these results to imaging in vivo survival studies and to targeted procedures, either lesioning or delivery of agents to the brain. There is an increasing interest in the development of gene and stem cell therapeutic techniques for the treatment of disorders of

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the brain. At this developmental level it is important to be able to use rodent models for discovery and refinement of the potential therapeutic. This study shows that catheter-based OCT can be a unique and useful tool both for developing models with targeted lesions and for delivery of agents into the brains in live animal studies. In many cases, the success of targeting and delivery of an agent in rodent brains is not known for days or weeks after the procedure or sometimes not until autopsy. It is often difficult to determine in vivo whether an animal is not responding to the treatment or simply had a poorly targeted injection. With OCT, accuracy of targeting and confirmation of delivery can be established at the time of surgery removing uncertainty and improving the efficiency of the experiments. 4.1. Clinical applications For most neurodegenerative disorders, such as PD and AD, as well as for posttraumatic epilepsy and cognitive deficits, current treatment consists primarily of palliative measures. However, on the horizon are novel therapeutic avenues, for example, gene and stem cell therapy, that may lead to neuroprotection and regeneration. Delivery of neurotrophic factors (e.g., GDNF, NGF or others) has been shown to be effective in animal models of PD, AD, epilepsy and traumatic brain injury. One of the main hurdles that remains is to be able to reliably and reproducibly deliver these agents to precise targets deep within the brain (Wirth and Ylä-Herttuala, 2006). These targets include the substantia nigra and subthalamic nucleus (During et al., 2001) as well as the hippocampus. Moreover, errors in delivery can lead to adverse consequences if the wrong part of the brain is given some of these agents. A number of studies targeting GDNF gene therapy to the substantia nigra in both rats and monkeys have shown aberrant sprouting outside this region (Kirik et al., 2000; Georgievska et al., 2002; Kordower, 2003; Hurelbrink and Barker, 2004). Furthermore, it is not currently possible to determine whether the therapeutic agent tracked up along the outside of the needle or dissected into a tissue plane or into the ventricle. The results presented here show that catheter-based OCT can address these concerns. In addition, because the OCT images in real time, adjustments can be made at the time of surgery decreasing the need for repeated surgical procedures. Another neurosurgical procedure that can benefit from OCT guidance is the placement of deep brain stimulating electrodes. Deep brain stimulation is in use for the treatment of Parkinson’s disease, dystonia and chronic pain (reviewed in Kern and Kumar, 2007) and is being studied for use in depression (Schlaepfer and Lieb, 2005; Mayberg et al., 2005; Schlaepfer et al., 2007), obsessive compulsive disorder (Nuttin et al., 1999), epilepsy (Velasco et al., 1995; Velisek et al., 2002; Veliskova and Moshe, 2006) and phantom limb pain (Kringelbach et al., 2007). The targets for these procedures include deep brain structures such as specific nuclei of the thalamus, globus pallidus pars interna, subthalamic nucleus, substantia nigra pars reticulate, and zona incerta (Kern and Kumar, 2007). Currently these procedures are guided by time consuming and complicated electrophysiological recording techniques. OCT passes require only a few minutes per pass and the images can be viewed and interpreted in real time. Decreasing the time necessary for the mapping procedure will make these procedures less strenuous, safer and much less expensive for the patient. To effectively utilize OCT in the clinic, its limited depth of imaging must be overcome by combining it with other tools. With an imaging depth of only 1–2 mm catheter-based OCT is an excellent choice for small animal brains, but would be lost within the human brain. Nevertheless, OCT L-mode displays are particularly good for marking position of gray–white matter borders and can serve to

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identify deviations from predicted axial positions. The radial OCT images are better suited for identifying deviations in lateral position (Jafri et al., 2005). These gray–white matter boundaries provide excellent feedback on position within structures. On the other hand, MRI can readily image the entire brain but cannot provide realtime information or fine structural details. Currently, therapeutic delivery is achieved by positioning the needle tip to a stereotactic coordinate determined by a pre-operative MRI or from a stereotactic atlas. Thus, MRI provides the macroscopic historic location information, while OCT provides microscopic real-time anatomic information. Ideally, if these two modalities can be successfully merged into a seamless system, investigators will have a powerful tool to confidently and safely pursue a variety of stereotactic neurosurgical procedures. We are currently working to integrate OCT with frameless MRI technology to achieve this. To further improve the precision of targeting, we are developing a high-resolution, interactive three-dimensional atlas of the human brain. We have developed a combination of histological and optical techniques and begun to map important nuclei and their surrounding white matter tracts of the basal ganglia. The deformable atlas can be mapped onto the patient’s preoperative MRI, and surgical progress can be precisely localized in real time by comparing OCT images with atlas predictions. Finally, many of the advancements in OCT described in the next section may also improve image penetration, clarity or detail. 4.2. OCT is an evolving technology OCT is rapidly evolving and improving. Led by improvements and innovations in ophthalmology, cardiology, and oncology where OCT is clinically used, changes in both hardware and software have resulted in better image quality, faster scans and thinner probes. As OCT moves into the clinic for neurological, ovarian and prostate imaging, advancements are likely to follow (reviewed in Zysk et al., 2007b). The development of new light sources of different wavelengths can lead to better tissue penetration and improved contrast. Advances in light source and detector technologies have made Fourier domain detection techniques such as spectral-domain OCT (SD-OCT) and swept-source OCT (SS-OCT) allow video rates of over 100 frames/s and improved image quality with significantly reduced movement-induced artifacts during scans (Yun et al., 2006; Schmitt et al., 2007). These modalities provide an improved ability to generate 3D volumetric images (Hitzenberger et al., 2003). These improvements have also opened up the feasibility of ultra high resolution OCT (UHR OCT) that allows sub-2-␮m axial resolution (Bizheva et al., 2003; Unterhuber et al., 2004). With UHR OCT single cells, their projections and subcellular organelles have been imaged (Povazay et al., 2002; Bizheva et al., 2004, 2005). Probe technology can also be improved. Probes can be made thinner by using smaller optical fibers with more durable outer sleeves that still maintain flexibility. Alternatively rigid probes may be developed to obviate the need for a guide cannula in brain procedures (Zysk et al., 2007a). Our laboratory is currently working on development of a forward-scanning needle-type probe. Functional OCT, or optophysiology, allows detection of changes in living cells upon stimulation. In ophthalmology changes in the tissue reflectivity of the photoreceptor layer and plexiform layer of dark-adapted retinas to light stimulation have been measured (Bizheva et al., 2006). Other physiological functions such as depolarization (Stepnoski et al., 1991) and membrane swelling (Srinivas et al., 2003) also cause detectable changes in optical properties opening the door to future functional imaging in the brain. Other areas of advancement for OCT include development of contrast agents, magnetomotive OCT, and new computed imag-

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ing methods. In this study, we have shown that microbubbles are a potential non-toxic contrast enhancing agent for OCT. Fluorescent and bioluminescent probes cannot be visualized on OCT. Contrast agents for OCT must either scatter or absorb the OCT signal resulting in positive or negative contrast enhancement, respectively. Nanoparticles, microspheres and dyes of a variety of sizes are being evaluated (Lee et al., 2003; Boppart et al., 2005). In magnetomotive OCT, magnetic iron oxide nanoparticles loaded in tissue are perturbed by a magnetic field resulting in light-scattering changes detectable on OCT (Oldenburg et al., 2005, 2008). One promising example of a new computational imaging method is called Interferometric Synthetic Aperture Microscopy (ISAM), in which out-of-focus features can be resolved from phase information (Ralston et al., 2007, 2008). 4.3. Summary The advantages of OCT for guidance of stereotactic neurosurgical procedures are (1) real-time high resolution spatial imaging capability, (2) a geometry that allows the probe to be introduced through a small catheter and coupled with an injection needle and (3) the ability to visually confirm delivery of minute volumes to specific targets in the CNS. The limitations of OCT are the relatively small field of view and the low optical contrast in many other biological tissues. Since many targets within the brain are small and have distinctive structural landmarks within or around them, the use of OCT guidance for neurosurgical targeting takes advantage of many of the strengths of catheter-based OCT. In addition to guidance and confirmation of targeted delivery of stem cell or gene therapy, OCT can be used to guide and confirm placement of deep-brain stimulating electrodes or in-dwelling catheters for drug delivery as well as provide a means to identify blood vessels along the surgical track in real time. In summary, we have shown catheter-based OCT to be an effective means of precise targeting in the brain of a living rat. In addition to providing secondary confirmation of location at the time of the intervention, we illustrate that OCT guidance provides the ability to perform tasks that cannot otherwise be carried out (microsurgical lesioning of hippocampal tracts, and monitoring of injection). In the long term, these results suggest that catheter-based OCT may potentially be an effective tool for use in future therapeutic interventions in the human brain. Acknowledgements This material is based upon work supported by Type II Merit Review to MSJ and a Merit Review and REAP award to C-MT from the Office of Research and Development, Biomedical Laboratory R&D Service, Department of Veterans Affairs. This study was also supported by a RO1 from NIBIB (C-MT). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jneumeth.2008.08.038. References Bizheva K, Pflug R, Hermann B, Povazay B, Sattmann H, Qiu P, et al. Optophysiology: depth-resolved probing of retinal physiology with functional ultrahigh-resolution optical coherence tomography. Proc Natl Acad Sci USA 2006;103:5066–71. Bizheva K, Povazay B, Hermann B, Sattmann H, Drexler W, Mei M, et al. Compact, broad-bandwidth fiber laser for sub-2-microm axial resolution optical coherence tomography in the 1300-nm wavelength region. Opt Lett 2003;28: 707–9.

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