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>

<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>

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 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

15.1K 次观看.  University of California, Riverside. 我们提出一个方法创建一个稀疏的头骨皮质的窗口（TSCW）在小鼠模​​型

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

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

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or thinned-skull 3 preparations. While the open-skull technique is very versatile, it is not optimal for studying microglia because it is invasive and can cause microglial activation. Even though the thinned-skull approach is minimally invasive, the repeated re-thinning of skull required for chronic imaging increases the risks of tissue injury and microglial activation and allows for a limited number of imaging sessions. Here we present a chronic thin-skull window method for monitoring murine microglia in vivo over an extended period of time using two-photon microscopy. We demonstrate how to prepare a stable, accessible, thinned-skull cortical window (TSCW) with an apposed glass coverslip that remains translucent over the course of three weeks of intermittent observation. This TSCW preparation is far more immunologically inert with respect to microglial activation than open craniotomy or repeated skull thinning and allows an arbitrary number of imaging sessions during a time period of weeks. We prepare TSCW in CX3CR1 GFP/+ mice 4 to visualize microglia with enhanced green fluorescent protein to &le;150 &mu;m beneath the pial surface. We also show that this preparation can be used in conjunction with stereotactic brain injections of the HIV-1 neurotoxic protein Tat, adjacent to the TSCW, which is capable of inducing durable microgliosis. Therefore, this method is extremely useful for examining changes in microglial morphology and motility over time in the living brain in models of HIV Associated Neurocognitive Disorder (HAND) and other neurodegenerative diseases with a neuroinflammatory component.

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

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.

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or ...

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

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The technique presented here allows the imaging of the same area of the brain at several time points (chronic imaging) with microscopic resolution allowing the tracking of dendritic spines which are the small structures that represent the majority of postsynaptic excitatory sites in the CNS. The ability to clearly resolve fine cortical structures over several time points has many advantages, specifically in the study of brain plasticity in which morphological changes at synapses and circuit remodeling may help explain underlying mechanisms. In this video and supplementary material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation. The thinned-skull preparation is a minimally invasive approach, which avoids potential damage to the dura and/or cortex, thus reducing the onset of an inflammatory response. When this protocol is performed correctly, it is possible to clearly monitor changes in dendritic spine characteristics in the intact brain over a prolonged period of time.

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

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The ...

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

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form a polished and reinforced thinned skull (PoRTS) window in the mouse skull that spans several millimeters in diameter and is stable for months. The skull is thinned to 10 to 15 &mu;m in thickness with a hand held drill to achieve optical clarity, and is then overlaid with cyanoacrylate glue and a cover glass to: 1) provide rigidity, 2) inhibit bone regrowth and 3) reduce light scattering from irregularities on the bone surface. Since the skull is not breached, any inflammation that could affect the process being studied is greatly reduced. Imaging depths of up to 250 &mu;m below the cortical surface can be achieved using two-photon laser scanning microscopy. This window is well suited to study cerebral blood flow and cellular function in both anesthetized and awake preparations. It further offers the opportunity to manipulate cell activity using optogenetics or to disrupt blood flow in targeted vessels by irradiation of circulating photosensitizers.

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.

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

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

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity. As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses. Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain. Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo. This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex. Therefore, images can be acquired immediately after surgery is performed. The experimental procedure can be performed repetitively over various time intervals ranging from hours to years. The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions.

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

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.

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in vivo&#8212;rapidly, repetitively, and at a high resolution. This protocol describes OCT imaging in the mouse retina as a powerful tool to study optic neuropathies (OPN). The OCT system is an interferometry-based, non-invasive alternative to common post mortem histological assays. It provides a fast and accurate assessment of retinal thickness, allowing the possibility to track changes, such as retinal thinning or thickening. We present the imaging process and analysis with the example of the Opa1delTTAG mouse line. Three types of scans are proposed, with two quantification methods: standard and homemade calipers. The latter is best for use on the peripapillary retina during radial scans; being more precise, is preferable for analyzing thinner structures. All approaches described here are designed for retinal ganglion cells (RGC) but are easily adaptable to other cell populations. In conclusion, OCT is efficient in mouse model phenotyping and has the potential to be used for the reliable evaluation of therapeutic interventions.

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

This manuscript describes a protocol for the in vivo imaging of the mouse retina with high-resolution spectral domain optical coherence tomography (SD-OCT). It focuses on retinal ganglion cells (RGC) in the peripapillary region, with several scanning and quantifying approaches described.

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...

Using Optical Coherence Tomography and Optokinetic Response As Structural and Functional Visual System Readouts in Mice and Rats

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for the investigation of processes of neurodegeneration, neuroprotection and neuro-repair involving the visual system, as these often correlate well with retinal changes. As a functional readout, visually evoked compensatory eye and head movements are commonly used in experimental models involving the visual function. Combining both techniques allows a quantitative in vivo investigation of structure and function, which can be used to investigate the pathological conditions or to evaluate the potential of novel therapeutics. A great benefit of the presented techniques is the possibility to perform longitudinal analyses allowing the investigation of dynamic processes, reducing variability and cuts down the number of animals needed for the experiments. The protocol described aims to provide a manual for acquisition and analysis of high quality retinal scans of mice and rats using a low cost customized holder with an option to deliver inhalational anesthesia. Additionally, the proposed guide is intended as an instructional manual for researchers using optokinetic response (OKR) analysis in rodents, which can be adapted to their specific needs and interests.

A detailed protocol for the assessment of structural and visual readouts in rodents by optical coherence tomography and optokinetic response is presented. The results provide valuable insights for ophthalmologic as well as neurologic research.

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for ...

Retinal Vascular Reactivity as Assessed by Optical Coherence Tomography Angiography

The vascular supply to the retina has been shown to dynamically adapt through vasoconstriction and vasodilation to accommodate the metabolic demands of the retina. This process, referred to as retinal vascular reactivity (RVR), is mediated by neurovascular coupling, which is impaired very early in retinal vascular diseases such as diabetic retinopathy. Therefore, a clinically feasible method of assessing vascular function may be of significant interest in both research and clinical settings. Recently, in vivo imaging of the retinal vasculature at the capillary level has been made possible by the FDA approval of optical coherence tomography angiography (OCTA), a noninvasive, minimal risk and dyeless angiography method with capillary level resolution. Concurrently, physiological and pathological changes in RVR have been shown by several investigators. The method shown in this manuscript is designed to investigate RVR using OCTA with no need for alterations to the clinical imaging procedures or device. It demonstrates real time imaging of the retina and retinal vasculature during exposure to hypercapnic or hyperoxic conditions. The exam is easily performed with two personnel in under 30 min with minimal subject discomfort or risk. This method is adaptable to other ophthalmic imaging devices and the applications may vary based on the composition of the gas mixture and patient population. A strength of this method is that it allows for an investigation of retinal vascular function at the capillary level in human subjects in vivo. Limitations of this method are largely those of OCTA and other retinal imaging methods including imaging artifacts and a restricted dynamic range. The results obtained from the method are OCT and OCTA images of the retina. These images are amenable to any analysis that is possible on commercially available OCT or OCTA devices. The general method, however, can be adapted to any form of ophthalmic imaging.

This article describes a method for measuring retinal vasculature reactivity in vivo with human subjects using a gas breathing provocation technique to deliver vasoactive stimuli while acquiring retinal images.

The vascular supply to the retina has been shown to dynamically adapt through vasoconstriction and vasodilation to accommodate the metabolic demands of ...

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

To fully understand the cellular physiology of neurons and glia in behaving animals, it is necessary to visualize their morphology and record their activity in vivo in behaving mice. This paper describes a method for the implantation of a chronic cranial window to allow for the longitudinal imaging of brain cells in awake, head-restrained mice. In combination with genetic strategies and viral injections, it is possible to label specific cells and regions of interest with structural or physiological markers. This protocol demonstrates how to combine viral injections to label neurons in the vicinity of GCaMP6-expressing astrocytes in the cortex for simultaneous imaging of both cells through a cranial window. Multiphoton imaging of the same cells can be performed for days, weeks, or months in awake, behaving animals. This approach provides researchers with a method for viewing cellular dynamics in real time and can be applied to answer a number of questions in neuroscience.

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

To fully understand the cellular physiology of neurons and glia in behaving animals, it is necessary to visualize their morphology and record their ...