Name: Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging (Video) | JoVE Journal
Uploaded: 2012-11-19T13:00:00.000+00:00
Duration: 7 min 28 s
Description: Learn the thinned-skull cortical window technique for in vivo optical coherence tomography imaging in mice. This less invasive method preserves brain viability while enabling high-resolution structural and functional brain studies.

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5 minutes

This work was supported by the UC Discovery Proof of Concept grant and by the NIH (R00 EB007241). The authors would also like to thank Jacqueline Hubbard for her assistance in this experiment.

1. Surgical Preparation
<ol>
	<li>Female CD 1 mice between the ages of 6-8 weeks were used in our experiments.</li>
	<li>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 &deg;C. Continuously monitor level of anesthesia by testing animal's reflexes (e.g., pinching foot with blunt forceps) and inject more anesthesia when necessary.</li>
	<li>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.</li>
	<li>Disinfect scalp using a betadine swab stick and clean with 70% ethanol prep pads.</li>
	<li>Carefully wrap the animal in surgical drapes to ensure optimal body temperature of ~37 &deg;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.</li>
</ol>
2. Thinned-skull Cortical Window Preparation
<ol>
	<li>Start the incision at the midline point between the eyes. Continue caudally to the midline point between the ears. Part the skin with forceps.</li>
	<li>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 &times; 4 mm thinned cranial window ~1 mm posterior and lateral to bregma.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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 &mu;m for optimal depth penetration. A list of materials used is provided in Table 1.</li>
</ol>
3. Optical Coherence Tomography Imaging
<ol>
	<li>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.</li>
	<li>Before imaging check signs for reflexes and apply additional artificial tear if needed. Mount animal on to the stereotaxic frame to secure the skull.</li>
	<li>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).</li>
	<li>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.</li>
	<li>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.</li>
</ol>

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<li>Yang, G., Pan, F., Parkhurst, C. N., Grutzendler, J., Gan, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Thinned-skull+cranial+window+technique+for+long-term+imaging+of+the+cortex+in+live+mice%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">Thinned-skull cranial window technique for long-term imaging of the cortex in live mice.</a> Nature Protocols. 5, (2010).</li>
<li>Lu, M., Majewska, S., K, A., Gelbard, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Thin-skull+Window+Technique+for+Chronic+Two-photon+In+vivo+Imaging+of+Murine+Microglia+in+Models+of+Neuroinflammation%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation.</a> J. Vis. Exp. (43), e2059(2010).</li>
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<li>Liang, C., Wierwille, J., Moreira, T., Schwartzbauer, G., Jafri, M. S., Tang, C., Chen, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+forward-imaging+needle-type+OCT+probe+for+image+guided+stereotactic+procedures%5BTitle%5D)+AND+%22Opt+Express%22%5BJournal%5D)">A forward-imaging needle-type OCT probe for image guided stereotactic procedures.</a> Opt Express. 19, 26283-26294 (2011).</li>
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No conflicts of interest declared.

Imaging with OCT and a thinned-skull is a novel neuro-imaging technique that has only been recently investigated15, 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 &mu;m and the penetration depth is obtained at approximately 1 mm with image resolution of 8 &mu;m and 20 &mu;m in the axial and lateral direction, respectively. In the signal intensity profile, OCT imaging through a...

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 interferometer2,4. 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 acquisition6-9.
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 slip10-13 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 microscopy10-13. 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 &mu;m and 20 &mu;m, 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.

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

thinned-skull cortical window technique for in vivo optical coherence tomography imaging

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

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 gastroenterology1-3. 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.

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 experiments (...

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.

Optical coherence tomography (OCT) is a biomedical imaging technique  with high spatial-temporal resolution. With its minimally invasive approach OCT  has ...

The overall goal of this procedure is to create a thin skull cranial window in a mouse model for in vivo optical coherence tomography imaging of the brain. This is accomplished by first anesthetizing the animal and preparing it for surgery. Next, thin the skull using various dental burrs.The final step is to obtain OCT images of the brain in vivo through the thin skull cranial window. Ultimately, brain structural and functional information can be obtained from the resulting OCT images. The main advantage of this technique over existing methods such as craniectomy or cover slip windows, is that finical window is less invasive and more likely to maintain the biological integrity and Viability of the brain.The implications of this technique extend towards the diagnosis of neurological disorders because OCT provides high spatial and temporal resolution and provides superior depth penetration when compared to confocal and two photon microscopy. Generally, individuals new to this method will struggle to create a uniformly thin skull surface without introducing skull fractures or subdural bleeding. To begin this procedure, anesthetize a CD one mouse between the ages of six to eight weeks with an intraperitoneal injection of ketamine and xylazine combination.Then place the mouse on a homeo themic pad or blanket to ensure the optimal body temperature at approximately 37 degrees Celsius during the surgical preparation, continuously monitor the level of anesthesia by testing the animal's reflexes and inject more anesthesia when necessary. Next, lubricate both eyes with the artificial tear ointment. Remove the hair on the scalp with the clippers.Then remove the residual clipped hair with a wedded pad or swab. After that, apply a thin layer of depilatory cream over the scalp. After two minutes, gently wipe away the cream and the remaining hair using saline, moistened cotton swabs and alcohol prep pads.Now disinfect the scalp using a Betadine swab stick and clean with 70%ethanol prep pads. Then mount the animal onto a stereotaxic frame to immobilize the skull. Lightly tap the skull to ensure its stability.In this procedure, start the scalp incision at the midline point between the eyes. Continue coddly to the midline point between the ears part, the skin flaps with forceps. Next, locate the area to be thinned under a dissecting microscope.Use forceps to scrape the skull to remove any residual fascia. Then dry the skull with sterile cotton swabs before creating a four by four millimeter thinned cortical window about one millimeter posterior and lateral torema. Use a surgical hand drill with a round carbide burr in a light sweeping motion to thin the skull.Do not apply direct pressure to the skull. Stop drilling every 20 to 30 seconds 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.Once the outer layer of the compact bone is completely removed, the middle spongy bone layer should be visible. There may be slight bleeding as blood vessels are more apparent in the spongy bone layer. Switch to a green stone burr and continue drilling using extra caution.As the spongy layer is more delicate, the green stone burr will remove less bone material while insuring evenness throughout the cranial window. Stop drilling occasionally to remove bone dust and to cool the skull. Finally, when the skull has become more transparent and the vasculature on the brain is visible, begin to polish the skull using a polishing bur check the thickness of the skull by gently tapping on it with forceps stop polishing.When the skull becomes slightly flexible, the thinned cranial window should now be completely smooth and reflective and ready for imaging. Due to the nature of highly scattering tissues of the brain, the skull should be thinned to at least 55 microns for optimal depth penetration. After the surgery is complete, check the animal's breathing rate via visual inspection and reflexes to ensure proper level of anesthesia and administer additional anesthesia If necessary, place the animal under the OCT camera, then position the thin skull cranial window under the optical beam.A cross sectional view of the skull and brain can now be visualized. Data acquisition can begin. Once the area of interest is located for imaging purposes, we scanned the OCT beam over an imaging cortical window with a width of 4.0 millimeters and obtain an imaging depth of two millimeters with six milliwatts of incident power and a focal 0.1 millimeter below the thin skull.Each cross-sectional area consisted of 2048 axial scans with an acquisition rate of 0.14 seconds per image. This particular method has allowed us to identify specific structures in our OCT cross-sectional images. This figure shows a para sagittal OCT image of the cortex under a normal skull, and here shows a para sagittal OCT image under a thin skull.The structures of the brain are more visually apparent under a thin skull cranial window as opposed to a normal skull. Additionally, a coronal cross-sectional OCT image is also obtained to facilitate in identifying midline structures. Volumetric scans of the brain can also be obtained by collecting a series of 2D cross-sectional images by using two sets of gal O mirrors for XY scanning with the first gal O mirror scanning the beam in the sagittal direction, and the second gal mirror scanning the coronal direction.Once mastered, this technique can be completed in 10 minutes If it is performed properly. While attempting this procedure is important to uniformly thin the skull to ensure adequate signal penetration during OCT imaging, After watching this video, you should have a good understanding of how to prepare a thin skull cortical window for OCT imaging purposes.

thinned-skull-cortical-window-technique-for-vivo-optical-coherence

Research

JoVE Journal

Neuroscience

We describe a method for repeatedly visualizing murine microglia and circulating monocytes in vivo over hours, days or weeks using transcranial two-photon microscopy. We demonstrate how to prepare a thinned-skull window that allows intermittent observation of quiescent microglia that can be activated by adjacent stereotactic injection of the HIV-1 regulatory protein Tat.

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

In this video and supplemental material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation.

Chronic Imaging of Mouse Visual Cortex Using a Thinned-skull Preparation

We present a method to form an imaging window in the mouse skull that spans millimeters and is stable for months without inflammation of the brain. This method is well suited for longitudinal studies of blood flow, cellular dynamics, and cell/vascular structure using two-photon microscopy.

A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain

By combining a polished and reinforced thin-skull (PoRTS) cranial window and glioblastoma (GBM) cell injection, we can observe glioma initiation and growth from injected GBM cells in the brain of a live mouse longitudinally.

Imaging Glioma Initiation In Vivo Through a Polished and Reinforced Thin-skull Cranial Window

Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Here, we describe a semi-invasive optical microscopy approach for the induction of a Rose Bengal photothrombotic clot in the somatosensory cortex of a mouse in vivo.  The technical aspects of the imaging procedure are described from induction of a photothrombotic event to application and data collection.

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke

This manuscript describes a procedure to track the remodeling of the cerebrovasculature during amyloid plaque accumulation in vivo using longitudinal two-photon microscopy. A thinned-skull preparation enables the visualization of fluorescent dyes to assess the progression of cerebrovascular damage in a mouse model of Alzheimer's disease.

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

Presented here is a protocol for the implantation of a chronic cranial window for the longitudinal imaging of brain cells in awake, head-restrained mice.

Implantation of a Cranial Window for Repeated In Vivo Imaging in Awake Mice

We introduced a novel imaging technique called Optical Multilayer Interference Tomography (OMLIT), which enables unbiased imaging of all cells in brain specimens at the mesoscale and can be seamlessly integrated into the imaging workflow of tape-based serial scanning electron microscopy on the same sample.

A Scanning Electron Microscopy-Compatible Optical Imaging Method for Mesoscopic All-Cell Brain Mapping

Learn the thinned-skull cortical window technique for in vivo optical coherence tomography imaging in mice. This less invasive method preserves brain viability while enabling high-resolution structural and functional brain studies.

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

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