Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging

Summary

We present a method of creating a thinned-skull cortical window (TSCW) in a mouse model for in vivo OCT imaging of the cerebral cortex.

Abstract

Optical coherence tomography (OCT) is a biomedical imaging technique with high spatial-temporal resolution. With its minimally invasive approach OCT has been used extensively in ophthalmology, dermatology, and gastroenterology^1-3. Using a thinned-skull cortical window (TSCW), we employ spectral-domain OCT (SD-OCT) modality as a tool to image the cortex in vivo. Commonly, an opened-skull has been used for neuro-imaging as it provides more versatility, however, a TSCW approach is less invasive and is an effective mean for long term imaging in neuropathology studies. Here, we present a method of creating a TSCW in a mouse model for in vivo OCT imaging of the cerebral cortex.

Introduction

Since its introduction in the early 1990's, OCT has been used extensively for biological imaging of tissue structure and function². OCT generates cross-sectional images by measuring echo time delay of backscattered light⁴ by implementing low coherence light source with a fiber-optic Michelson interferometer^2,4. SD-OCT, also known as Fourier domain OCT (FD-OCT), was first introduced in 1995⁵ and offers a superior imaging modality compared with traditional time domain OCT (TD-OCT). In SD-OCT, the reference arm is kept stationary resulting in a high speed and ultra high resolution image acquisition^6-9.

Presently, TSCW models have been used largely for in vivo brain imaging applications of two-photon microscopy in place of a traditional craniotomy. These TSCW have been used concurrently with a custom skull plate or a glass cover slip^10-13 to provide additional imaging stability. In our studies, we have observed that accessories such as these are not necessary for OCT imaging when a TSCW is used. Therefore, the lack of a skull plate or glass cover slips allows for a wider range of imaging window size as they may interfere with the optical beam and alter OCT images.

A thinned-skull preparation has proven to be advantageous in imaging studies of the brain using two-photon microscopy^10-13. In our experiments, we utilize a SD-OCT system to image the cortex in vivo through a TSCW. Our custom SD-OCT imaging setup contains a broadband, low-coherence light source consisting of two superluminescent diodes (SLD) centered at 1295 nm with a bandwidth of 97 nm resulting in an axial and lateral resolution of 8 μm and 20 μm, respectively¹⁴. With our optical imaging device, we envision that imaging through a TSCW has great potential in identifying and visualizing structures and functions in the optically dense brain tissue.

Protocol

1. Surgical Preparation

1. Female CD 1 mice between the ages of 6-8 weeks were used in our experiments.
2. Anesthetize the mouse with an intraperitoneal injection of a ketamine and xylazine combination (80 mg/kg ketamine/10 mg/kg xylazine). Place the mouse on a homeothermic pad to ensure optimal body temperature at ~37 °C. Continuously monitor level of anesthesia by testing animal's reflexes (e.g., pinching foot with blunt forceps) and inject more anesthesia when necessary.
3. Lubricate both eyes with an artificial tear ointment. Remove hairs on the scalp using a razor and remove residual hair using 70% alcohol prep pads. Apply a thin layer of Nair hair removal cream over the scalp and wait 2 min for it to take effect. Gently wipe away Nair and remaining hair using saline moistened cotton swabs and alcohol prep pads. Scalp should now be completely hairless.
4. Disinfect scalp using a betadine swab stick and clean with 70% ethanol prep pads.
5. Carefully wrap the animal in surgical drapes to ensure optimal body temperature of ~37 °C and mount the animal onto a stereotaxic frame to immobilize the skull. Lightly tap the skull to ensure its stability. A list of materials used is provided in Table 1.

2. Thinned-skull Cortical Window Preparation

1. Start the incision at the midline point between the eyes. Continue caudally to the midline point between the ears. Part the skin with forceps.
2. Locate the area to be thinned under a dissecting microscope and gently remove the fascia using tweezers. Dry the skull with sterile cotton swabs before creating the thinned cortical window. In our experiments, we created a 4 × 4 mm thinned cranial window ~1 mm posterior and lateral to bregma.
3. Begin thinning the skull using a round carbide bur with drill bit size 0.75 mm in a surgical hand drill using light sweeping motion only. Do not apply direct pressure onto the skull. Stop drilling every 20-30 sec to remove bone dust using sterile saline and cotton swabs and to avoid overheating the skull. The saline will also aid in dissipating the heat throughout the skull.
4. Once the outer layer of the compact bone is completely removed the middle spongy bone layer should now be visible. There may be some slight bleeding as blood vessels are more apparent in the spongy bone layer. Switch to a green stone bur and continue drilling using extra caution as the spongy layer is more delicate. The green stone bur will remove less bone material while creating evenness throughout the cranial window. Stop drilling occasionally to remove bone dust and to cool the skull.
5. Finally, when the skull has become more transparent and vasculature on the brain is now visible, begin polishing the skull using a polishing bur. This will allow a more precise thinning while smoothing down the skull. Check thinness of the skull by gently tapping on it with forceps. Stop polishing when the skull becomes slightly flexible.
6. The thinned cranial window should now be completely smooth and reflective and ready for imaging (Figure 1). Due to the nature of highly scattering tissues of the brain, the skull should be thinned to at least 55 μm for optimal depth penetration. A list of materials used is provided in Table 1.

3. Optical Coherence Tomography Imaging

1. After surgery is complete, check animal's breathing rate and reflexes to ensure proper level of anesthesia and administer additional anesthesia if necessary. Remove animal from the stereotaxic frame, keep animal wrapped in surgical drapes, and transport animal to the imaging station.
2. Before imaging check signs for reflexes and apply additional artificial tear if needed. Mount animal on to the stereotaxic frame to secure the skull.
3. Place animal under OCT camera and position the TSCW under the optical beam (Figure 2). A cross-sectional view of the skull and brain can now be visualized (Figure 3).
4. Data acquisition can begin once area of interest is located. For imaging purposes, we use galvo mirrors to achieve an imaging window with a width of 4.0 mm. An imaging depth of 2 mm was obtained with 6 mW of incident power and a focal point 1 mm below the thinned skull. Each cross-sectional area consisted of 2,048 axial scans with an acquisition rate of 0.14 sec per image.
5. Volumetric scans of the brain can also be obtained by collecting a series of 2D cross-sectional images by using two sets of galvo mirrors for x-y scanning with the first galvo mirror scanning the beam in the sagittal direction and the second galvo mirror scanning in the coronal direction.

Results

After creating a thinned window over the cerebral cortex the vasculature should now be more visually prominent (Figure 1) and will allow for a deeper imaging depth (up to 1 mm). The right cortex is thinned to approximately 55 μm as compared to a normal skull measured at 140 μm (Figure 1) and provides greater optical clarity. Further thinning to 10-15 μm is possible¹¹ however not necessary as the use of glass cover slips and skull plates are not implemented in our expe...

Discussion

Imaging with OCT and a thinned-skull is a novel neuro-imaging technique that has only been recently investigated^{15, 16}. In our experiments, we demonstrated the feasibility of SD-OCT imaging through a TSCW in a mouse model in vivo. From our results, the skull is thinned to approximately 55 μm and the penetration depth is obtained at approximately 1 mm with image resolution of 8 μm and 20 μm in the axial and lateral direction, respectively. In the signal intensity profile, OCT imaging through a...

Materials

Name	Company	Catalog Number	Comments
Ketamine	Phoenix Pharmaceuticals	57319-542-02
Xylazine	Akorn, Inc.	139-236
Artificial Tears Ointment	Rugby	0536-6550-91
Nair	Church Dwight Co., Inc.	4010130
Sterile Alcohol Prep Pad	Kendall Healthcare	6818
Cotton Tipped Applicators	Fisherbrand	23-400-115
Betadine Solution Swabstick	Purdue Products	67618-153-01
Saline Solution, .9%	Phoenix Pharmaceuticals	57319-555-08
Stereotactic Frame	Stoelting
High Speed Surgical Hand Drill	Foredom		38,000 rpm
Carbide Round Bur	Stoelting		0.75 mm
Dura-Green Stones	Shofu		Shank: HP Shape: BA1
CompoMaster Coarse & CompoMaster Polisher	Shofu		Shape: Mini-Pt.
SpaceDrapes	Braintree Scientific, Inc.