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>Lubricat...

<ol>
	<li>Bizheva, K., Unterhuber, A., Hermann, B., Povazay, B., Sattmann, H., Drexler, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+ex+vivo+and+in+vitro+brain+morphology+in+animal+models+with+ultrahigh+resolution+optical+coherence+tomography.">Imaging ex vivo and in vitro brain morphology in animal models with ultrahigh resolution optical coherence tomography.</a> Journal of Biomedical Optics. 9, 719-724 (2004).</li><li>Fujimoto, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+coherence+tomography+for+ultrahigh+resolution+in+vivo+imaging.">Optical coherence tomography for ultrahigh resolution in vivo imaging.</a> Nature Biotechnology. 21, 1361-1367 (2003).</li><li>Wantanabe, H., Rajagopalan, U. M., Nakamichi, Y., Igarashi, K. M., Kadono, H., Tanifuji, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Swept+source+optical+coherence+tomography+as+a+tool+for+real+time+visualization+and+localization+of+electrodes+used+in+electrophysiological+studies+of+brain+in+vivo.">Swept source optical coherence tomography as a tool for real time visualization and localization of electrodes used in electrophysiological studies of brain in vivo.</a> Biomedical Optics Express. 2, 3129-3134 (2011).</li><li>Huang, D., Swanson, E. A., Lin, C. P., Schuman, J. S., Stinson, W. G., Chang, W., Hee, M. R., Flottee, T., Gregory, K., Puliafito, C. A., Fujimoto, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+coherence+tomography.">Optical coherence tomography.</a> Science. 254, 1178-1181 (1991).</li><li>Mitsui, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+range+of+optical+reflectometry+with+spectral+interferometry.">Dynamic range of optical reflectometry with spectral interferometry.</a> Japanese Journal of Applied Physics. 38, 6133-6137 (1999).</li><li>de Boer, J. F., Cense, B., Park, B. H., Pierce, M. C., Tearney, G. J., Bouma, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+signal-to-noise+ratio+in+spectral-domain+compared+with+time-domain+optical+coherence+tomograhy.">Improved signal-to-noise ratio in spectral-domain compared with time-domain optical coherence tomograhy.</a> Optics Letters. 28, 2067-2069 (2003).</li><li>de Boer, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+5.">Ch. 5.</a> Optical Coherence Tomography: Technology and Applications. , (2008).</li><li>Choma, M. A., Sarunic, M. V., Yang, C., Izatt, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitivity+advantage+of+swept+source+and+fourier+domain+optical+coherence+tomography.">Sensitivity advantage of swept source and fourier domain optical coherence tomography.</a> Optics Express. 11, 2183-2189 (2003).</li><li>Leitgeb, R. A., Drexler, W., Unterhuber, A., Hermann, B., Bajraszewski, T., Le, T., Stingl, A., Fercher, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrahigh+resolution+fourier+domain+optical+coherence+tomography.">Ultrahigh resolution fourier domain optical coherence tomography.</a> Optics Express. 12, 2156-2165 (2004).</li><li>Drew, P. J., Shih, A. Y., Driscoll, J. D., Knutsen, P. M., Blinder, P., Davalos, D., Akassoglou, K., Tsai, P. S., Kleinfeld, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+optical+access+through+a+polished+and+reinforced+thinned+skull.">Chronic optical access through a polished and reinforced thinned skull.</a> Nature Methods. 7, 981-984 (2010).</li><li>Shih, A. Y., Mateo, C., Drew, P. J., Tsai, P. S., Kleinfeld, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Polished+and+Reinforced+Thinned-skull+Window+for+Long-term+Imaging+of+the+Mouse.">A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse.</a> J. Vis. Exp. 61, e3742 (2012).</li><li>Yang, G., Pan, F., Parkhurst, C. N., Grutzendler, J., Gan, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thinned-skull+cranial+window+technique+for+long-term+imaging+of+the+cortex+in+live+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Thin-skull+Window+Technique+for+Chronic+Two-photon+In+vivo+Imaging+of+Murine+Microglia+in+Models+of+Neuroinflammation.">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><li>Wang, Y., Oh, C. M., Oliveira, M. C., Islam, M. S., Ortega, A., Park, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GPU+accelerated+real-time+multi-functional+spectral-domain+optical+coherence+tomography+system+at+1300nm.">GPU accelerated real-time multi-functional spectral-domain optical coherence tomography system at 1300nm.</a> Optics Express. 20, 14797-14813 (2012).</li><li>Aguirre, A. D., Chen, Y., Fujimoto, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depth-resolved+imaging+of+functional+activation+in+the+rat+cerebral+cortex+using+optical+coherence+tomography.">Depth-resolved imaging of functional activation in the rat cerebral cortex using optical coherence tomography.</a> Opt. Lett. 31, 3459-3461 (2006).</li><li>Chen, Y., Aguirre, A. D., Ruvinskaya, L., Devor, A., Boas, D. A., Fujimoto, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+coherence+tomography+(OCT)+reveals+depth-resolved+dynamics+during+functional+brain+activation.">Optical coherence tomography (OCT) reveals depth-resolved dynamics during functional brain activation.</a> Journal of Neuroscience Methods. 178, 162-173 (2009).</li><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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+forward-imaging+needle-type+OCT+probe+for+image+guided+stereotactic+procedures.">A forward-imaging needle-type OCT probe for image guided stereotactic procedures.</a> Opt Express. 19, 26283-26294 (2011).</li><li>Srinivasan, V. J., Sakadzic, S., Gorczynska, I., Ruvinskaya, S., Wu, W., Fugimoto, J. G., Boas, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+cerebral+blood+flow+with+optical+coherence+tomography.">Quantitative cerebral blood flow with optical coherence tomography.</a> Optics Express. 18, 2477-2494 (2010).</li><li>Galetta, K. M., Calabresi, P. A., Frohman, E. M., Balcer, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+Coherence+Tomography+(OCT):+imaging+the+visual+pathway+as+a+model+for+neurodegeneration.">Optical Coherence Tomography (OCT): imaging the visual pathway as a model for neurodegeneration.</a> The Journal of the American Society for Experimental NeuroTherapeutics. 8, 117-132 (2011).</li><li>Seigo, M. A., Sotirchos, E. S., Newsome, S., Babiarz, A., Eckstein, C., Ford, E., Oakley, J. D., Syc, S. B., Frohman, T. C., Ratchford, J. N., Balcer, L. J., Frohman, E. M., Calabresi, P. A., Saidha, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+assessment+of+retinal+neuronal+layers+in+multiple+sclerosis+with+maual+and+automated+optical+coherence+tomography+segementation+techniques.">In vivo assessment of retinal neuronal layers in multiple sclerosis with maual and automated optical coherence tomography segementation techniques.</a> J. Neurol. , (2012).</li><li>Frohman, E. M., Fujimoto, J. G., Frohman, T. C., Calabresi, P. A., Cutter, G., Balcer, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+coherence+tomography:+a+window+into+the+mechanisms+of+multiple+sclerosis.">Optical coherence tomography: a window into the mechanisms of multiple sclerosis.</a> Nature Clinical Practice. 4, 664-675 (2008).</li><li>Gill, A. S., Rajneesh, K. F., Owen, C. M., Yeh, J., Hsu, M., Binder, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+optical+detection+of+cerebral+edema+in+vivo.">Early optical detection of cerebral edema in vivo.</a> J. Neurosurg. 114, 470-477 (2011).</li></ol>

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 border="1">
	<tbody>
		<tr>
			<td>Materials</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Phoenix Pharmaceuticals</td>
			<td>57319-542-02</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Akorn, Inc.</td>
			<td>139-236</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Artificial Tears Ointment</td>
			<td>Rugby</td>
			<td>0536-6550-91</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Nair</td>
			<td>Church &amp; Dwight Co., Inc.</td>
			<td>4010130</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sterile Alcohol Prep Pad</td>
			<td>Kendall Healthcare</td>
			<td>6818</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cotton Tipped Applicators</td>
			<td>Fisherbrand</td>
			<td>23-400-115</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Betadine Solution Swabstick</td>
			<td>Purdue Products</td>
			<td>67618-153-01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Saline Solution, .9%</td>
			<td>Phoenix Pharmaceuticals</td>
			<td>57319-555-08</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Stereotactic Frame</td>
			<td>Stoelting</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>High Speed Surgical Hand Drill</td>
			<td>Foredom</td>
			<td>&nbsp;</td>
			<td>38,000 rpm</td>
		</tr>
		<tr>
			<td>Carbide Round Bur</td>
			<td>Stoelting</td>
			<td>&nbsp;</td>
			<td>0.75 mm</td>
		</tr>
		<tr>
			<td>Dura-Green Stones</td>
			<td>Shofu</td>
			<td>&nbsp;</td>
			<td>Shank: HP 
			Shape: BA1</td>
		</tr>
		<tr>
			<td>CompoMaster Coarse &amp; CompoMaster Polisher</td>
			<td>Shofu</td>
			<td>&nbsp;</td>
			<td>Shape: Mini-Pt.</td>
		</tr>
		<tr>
			<td>SpaceDrapes</td>
			<td>Braintree Scientific, Inc.</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

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 &mu;m as compared to a normal skull measured at 140 &mu;m (Figure 1) and provides greater optical clarity. Further thinning to 10-15 &mu;m is possible11 however not necessary as the use of glass cover slips and skull plates are not implemented in our expe...

Watch this Scientific Journal Video about Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging at JoVE.com

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

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.

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 ...

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

Research

JoVE Journal

Neuroscience

15.0K visualizações.  University of California, Riverside. Apresenta-se um método de criação de uma janela diluído crânio-cortical (TSCW) em um modelo de rato para In vivo Outubro de imagem do córtex cerebral.