We thank Emily A. Kelly for the head plate design.
We are grateful for the support of NIH awards to H.A.G.: PO1 MH64570, RO1 MH56838, RO1 MH078989, R21 MH03851 and the generous support of the Geoffrey Waasdorp Pediatric Neurology Fund without which this work would not have been possible. A.K.M is funded by the NIH (EY019277), Whitehall Foundation, the Alfred P. Sloan Foundation and a Career Award in the Biomedical Sciences from the Burroughs Wellcome Fund. M.E.T. is funded by a Fonds de la recherche en sante du Quebec (FRSQ) postdoctoral training award. D.F.M. is a trainee in the Medical Scientist Training Program, funded by NIH T32 GM07356 and T32 AI049815.

1. Preparing the Animal for Imaging
<ol>
<li>In our experiments, we use adult CX3CR1GFP/+ mice that express GFP in mononuclear cell lineages, including microglia.4 Different strains of mice can be used to meet the needs of a specific project.</li>
<li>Anesthetize the mouse with an intraperitoneal injection of a three drug cocktail consisting of 0.05 mg/kg Fentanyl, 5.0 mg/kg Midazolam, and 0.5 mg/kg Medetomidine.5 Surgical preparation for TSCW starts after the animal no longer responds to painful stimuli, such as a tail pinch. Throughout the surgery and imaging, we keep the animal on a heating pad to prevent post-anesthesia hypothermia. If necessary, a booster dose of one-third the original dose of the anesthetic cocktail can be given to restore the original anesthetic plane.</li>
<li>Cover the animal's eyes with a protective ophthalmic ointment to keep the eyes moist during anesthesia, which suppresses the animal's blink reflex. Remove the hair from the scalp of the animal using a razor, shears, scissors, or chemical methods. Disinfect the scalp using a 10% povidone-iodine solution and 70% ethanol. All surgical instruments need to be autoclaved, sterilized with a glass-bead sterilizer, or disinfected with 70% ethanol.</li>
<li>Make a midline incision of the scalp using small scissors, starting 4-5 mm caudal to the skull and advancing forward to the front of the eyes. It is important that this incision be long enough that the skin will not interfere with the gluing of the head plate that is used to stabilize the mouse head during two-photon imaging (Figure 1). Identify the area to be thinned under a dissecting scope. Avoid areas of interest directly located over cranial sutures, as the skull is less stable in these areas and underlying large vessels and meninges will interfere with imaging.</li>
<li>Apply 100% ethanol followed by 10% solution of ferric chloride with a sterile cotton swab to dry the membranes on top of the skull and scrape them away using fine tweezers or a razor blade. Failure to remove the membranes results in unstable head plate attachment.</li>
<li>Place a thin layer of the Permabond 910 glue around the edges of the viewing window underneath the head plate. Place the head plate coated with glue over the area of interest on the animal's skull with light pressure (Figure 1). It is important that the head plate is only glued to the bone of the skull. Any skin or membranes that are caught between the head plate and the skull will decrease the stability of the bond. Apply a small amount of acrylate around the viewing window using a syringe to instantly bond the glue. Finally, apply a small amount of Loctite 454 to the edges of the viewing window to prevent leaks.</li>
<li>Screw the head plate to the animal holder (Figure 1) and check the stability of the head plate under a dissection scope by lightly probing with a pair of forceps. There should be no movement of the skull relative to the head plate. Place a drop of saline on the viewing window to ensure there are no leaks.</li>
</ol>
2. Preparing the Thinned Skull Cortical Window
<ol>
<li>In preparation for thinning the skull, dry the skull using a combination of sterile cotton swabs and compressed air. We initially thin the skull with a sterile IRF 007 drill bit in a Microtorque II drill set at 4000 rpm. Very gently, begin thinning a 2-2.5 mm diameter circular area of the skull. Use only light sweeping motions nearly parallel to the skull; use no direct downward pressure. Stop drilling every 20-30 seconds to remove bone dust using the compressed air. These breaks in drilling allow the skull to cool so there is no heat-induced damage to the underlying brain tissue.
<ol class="lalpha">
<li>	As the drilling progresses, notice the transition to the moister spongy bone layer (i.e. the "diploe"). Once this layer is reached, exercise extra caution with the drill.</li>
</ol></li>
<li>Occasionally check the thinness of the skull by placing saline over the thinned area and viewing under the dissecting scope. Saline will further enable heat dissipation. As the skull is thinned, smaller vasculature becomes visible.</li>
<li>Use a sterile dental microblade to achieve the final thinning under saline, as the microblade provides much more tactile feedback about the stability of the skull. This allows for much thinner preparations than with the drill alone. From our experience, the optimal skull thickness is 10-30 &mu;m.
<ol class="lalpha">
<li>	It is important to check the thinness of the skull under epifluorescence multiple times during the first few attempts at making a thin skull window. The sharpness and depth of visible microglia and vasculature give a good indication as to when the window is ready for imaging.</li>
</ol></li>
<li>	Once the window is ready, glue a custom-made rectangular piece of #0 coverglass with a dimension of less than 2 mm on each side over the window. Using a 1.0 mm diameter glass pipette, place a small drop of cyanoacrylate glue on the thinned skull area and carefully lower down the small piece of coverslip, then press gently against the skull to squeeze out excess amount of the glue. It is important to avoid bubble formation between the glass and the glue since trapped air will cause the area underneath to become optically opaque. The cyanoacrylate glue is used because it remains transparent when dry and also prevents bone and membrane regrowth keeping the skull under the window thin and translucent. If there is any glue on top of the cover glass, use the microblade to carefully remove it once the cover glass is in place and the glue is dry.</li>
<li>An analgesic (for example, Buprenorphine 2 mg/kg sub-cutaneously) is administered immediately after the surgery and re-administered at any signs of pain, including reluctance to move, eat or drink, weight loss, salivation, piloerection, respiratory sounds, etc.</li>
</ol>
3. Two-photon Imaging
<ol>
<li>In preparation for two-photon imaging, cover the TSCW with saline and locate the area of interest under epifluorescence (Figure 2). To enable subsequent imaging of the area, take a picture using a photographic camera.</li>
<li>For two-photon imaging, we use a custom-made two-photon microscope6 with a Ti:Sapphire laser tuned to 920 nm. Fluorescence is detected by using a photomultiplier tube in whole-field detection mode and a 580/180 emission filter. A 20X water-immersion lens (0.95 N.A.) is used throughout the imaging session. The maximum output of the laser power at the objective is set between 50 and 65 mW.</li>
<li>Select an area of interest in cortical layers I or II (up to 150 &mu;m below the pial surface). To facilitate re-imaging, take pictures of the same field of view at 1X, 2X, and 3X digital zoom. Blood vessels can serve as landmarks to easily find the same field of view during subsequent imaging sessions. Under a zoom of 3X, multiple z-stacks with 80-90 Z-steps in each stack and a 0.69 &mu;m step can be acquired every 5 minutes for up to 30 minutes, which enables a good sampling of microglial morphology and behavior over time (Figure 3). Between acquisitions of z-stacks, one should verify the level of saline over the TSCW, as well as the animal's depth of anesthesia.</li>
</ol>
4. Injection of the HIV-1 Neurotoxin, Tat
<ol>
<li>First, silanize the inner lining of a 35 gauge needle and 10 &mu;L Microvolume syringe to prevent Tat deposition. Withdraw the silanizing solution (Sigmacote) until it fills both the needle and syringe. Allow the solution to sit for 5 minutes at room temperature. Empty the solution from the syringe, remove the plunger, and allow the syringe and needle to dry completely. Finally, wash the syringe and needle thoroughly with deionized water.</li>
<li>Perform a small craniotomy 0.5 mm in diameter either 3 mm rostral or lateral to the TSCW where two-photon images have been obtained, using a smaller drill bit and carefully thinning the skull until a pair of forceps with sharp tips can remove the thinned layer of bone away easily. We use a 10 &mu;L Microvolume syringe fitted with 35 gauge needle controlled by an Ultramicropump III syringe pump mounted on a 3-axis micromanipulator and after lining up the needle tip to the craniotomy, it is carefully advanced to a depth of 700 to 900 &mu;m below the pial surface where 3 &mu;L Tat1-72 (or other pro-inflammatory agents) or saline (or control vehicle) is delivered at 80 nL/min.</li>
</ol>
5. Animal Housing
<ol>
<li>If the animal is to be imaged multiple times in a single day, cover the open area of the skull with a mixture of opthalmic ointment and Vaseline to prevent discomfort or damage to the skull in between imaging sessions. Disconnect the support bridge from the small head-plate (see Figure 1B) and place the animal in a warmed cage with easily accessible food and water. All animals are to be housed singly after surgery at all times. Once the imaging sessions for an animal are complete for a given day, carefully remove the entire head-plate and suture the skin back over the skull with #6 or smaller suture. Again, house the animals singly with easily accessible food and water between experimental days. Check the animals daily for any signs of stress, pain, or infection.</li>
</ol>
6. Representative Results
 <img src="/files/ftp_upload/2059/2059fig1.jpg" alt="Figure 1" /> 
Figure 1. Stabilization of animal for surgery and two-photon imaging (A) and head plate design (B).
 <img src="/files/ftp_upload/2059/2059fig2.jpg" alt="Figure 2" /> 
Figure 2. GFP-labeled microglia visualized with epifluorescence.
 <img src="/files/ftp_upload/2059/2059fig3.jpg" alt="Figure 3" /> 
Figure 3. GFP-labeled microglia visualized with two-photon imaging after implantation of the TSCW, approximately 50 &mu;m below the pial surface. The single two-photon sections taken 15-30 minutes after implantation of the TSCW (day 0), as well as 7 and 17 days later, demonstrate that the windows stays clear while microglia remain inactivated in absence of inflammatory insults. The z-projections taken 6 and 24 hours post-injection of the HIV neurotoxin tat exhibit impressive morphologic changes of activated microglia. Scale bar: 10 &mu;m.

<ol>
	<li>Mostany, R., &amp;amp, P. o. r. t. e. r. a. -. C. a. i. l. l. i. a. u., C, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+craniotomy+surgery+procedure+for+chronic+brain+imaging.">A craniotomy surgery procedure for chronic brain imaging.</a> J Vis Exp. 12, (2008).</li><li>Holtmaat, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=high-resolution+imaging+in+the+mouse+neocortex+through+a+chronic+cranial+window.">high-resolution imaging in the mouse neocortex through a chronic cranial window.</a> Nat Protoc. 4, (2009).</li><li>Yang, G. <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 Protoc. 5, 213-21 (2010).</li><li>Jung, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+fractalkine+receptor+CX(3)CR1+function+by+targeted+deletion+and+green+fluorescent+protein+reporter+gene+insertion.">Analysis of fractalkine receptor CX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion.</a> Mol Cell Biol. 20, 4106-41 (2000).</li><li>Mrsic-Flogel, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homeostatic+regulation+of+eye-specific+responses+in+visual+cortex+during+ocular+dominance+plasticity.">Homeostatic regulation of eye-specific responses in visual cortex during ocular dominance plasticity.</a> Neuron. 54, 961-96 (2007).</li><li>Majewska, A., Yiu, G., &amp;amp, Y. u. s. t. e., R, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+custom-made+two-photon+microscope+and+deconvolution+system.">A custom-made two-photon microscope and deconvolution system.</a> Pflugers Arch. 441, 2-3 (2000).</li><li>Sharer, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathology+of+HIV-1+infection+of+the+central+nervous+system.">Pathology of HIV-1 infection of the central nervous system.</a> A review. J Neuropathol Exp Neurol. 51, (1992).</li><li>Everall, I. P., Aliberti, J., &amp;amp, B. a. l. c. e. r. z. y. k., M, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+density+in+the+superior+frontal+and+temporal+gyri+does+not+correlate+with+the+degree+of+human+immunodeficiency+virus-associated+dementia.">Neuronal density in the superior frontal and temporal gyri does not correlate with the degree of human immunodeficiency virus-associated dementia.</a> Acta Neuropathol (Berl. 88, 538-53 (1994).</li><li>Masliah, E., Banati, R. B., &amp;amp, Q. u. i. n. l. a. n., M, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+injury+is+a+pathological+substrate+for+human+immunodeficiency+virus-related+cognitive+disorders.">Dendritic injury is a pathological substrate for human immunodeficiency virus-related cognitive disorders.</a> Ann Neurol. 42, 963-96 (1997).</li><li>Everall, I. P., Banati, R. B., &amp;amp, U. p. e. n. d. e. r., B, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+synaptic+density+is+reduced+in+mild+to+moderate+human+immunodeficiency+virus+neurocognitive+disorder.">Cortical synaptic density is reduced in mild to moderate human immunodeficiency virus neurocognitive disorder.</a> Brain Pathol. 9, (1999).</li><li>Glass, J. D., Fedor, H., Wesselingh, S. L., &amp;amp, M. c. A. r. t. h. u. r., C, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunocytochemical+quantitation+of+human+immunodeficiency+virus+in+the+brain:+correlations+with+dementia.">Immunocytochemical quantitation of human immunodeficiency virus in the brain: correlations with dementia.</a> Ann Neurol. 38, (1995).</li><li>Feng, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+neuronal+subsets+in+transgenic+mice+expressing+multiple+spectral+variants+of+GFP.">Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP.</a> Neuron. 28, (2000).</li></ol>

No conflicts of interest declared.

There is growing consensus that the actual substrate for HIV-1 associated neurocognitive deficits (HAND) is destruction of synaptic architecture, presumably caused by pro-inflammatory mediators released from HIV-1 infected mononuclear phagocytes. Microglial activation, multinucleated giant cells, and gliosis due to astrocytic hypertrophy are considered nonspecific hallmarks of HIV-1 infection and inflammation in the CNS 7. In contrast, the severity of premortem neurologic disease has been correlated with loss of synaptic complexity, as well as macrophage burden in the CNS 8,9,10,11. However, dynamic interactions between microglia, peripheral infiltrating macrophages, and neurons in patients with HAND simply cannot be modeled using conventional static processing obtained from conventional immunocytochemical studies. Recent studies from our laboratories have suggested that at least some of these interactions between peripheral and central mononuclear cells and neurons can be modeled in part by stereotactic injection of the HIV-1 protein Tat1-72 into brain parenchyma (Lu, Marker, Tremblay, Qi, and Gelbard, unpublished data). We therefore decided to apply in vivo two photon imaging to examine the interplay between monocyte-derived macrophages, brain resident microglia and neurons, in a tractable small animal model for neuroAIDS. While open-skull or thinned-skull preparations afford a single examination of cortical architecture for a brief period of time (hours), investigators interested in the time course of subacute events cannot use these preparations without artifactually activating microglia/macrophage due to surgical manipulation of the calvarial window. Here we demonstrate a simple way of circumventing these limitation by gluing a tiny piece of glass coverslip over the thinned skull area preventing the calvarium from regenerating in the TSCW as well as maintaining translucency for &ge;3 weeks. We further demonstrate that this technique allows us to monitor the behavior of peripheral monocytes entering cerebral microvasculature as well as brain-resident microglia in response to stereotactic injection of HIV Tat1-72 in the cortex of mice heterozygous for CX3CR1/GFP (to identify cells of mononuclear lineage). This technique can easily be adapted to use on mice with different genetic backgrounds; for example, we routinely use heterozygous CX3CR1/GFP X Thy-1 YFP 12 mice to investigate interactions between monocyte-derived macrophages, brain resident microglia and neurons in in vivo models of neuroinflammation relevant to HAND. Our TSCW preparation allows investigators to study cell-cell interactions between the periphery and CNS that may occur over periods of weeks, in which the animal can be repeatedly imaged before and after experimental treatment. Thus, each animal can serve as its own control. One caveat for this TSCW model is that the target cells under investigation need to be either genetically engineered to express enhanced fluorescent proteins (eXFP) or be labeled with a fluorescent dye without mechanical breach of the calvarium; and that eXFP expression must be robust enough to allow cellular architecture to be visualized after repeated imaging sessions over a period of weeks.

<table><tbody><tr><td>Fentanyl Citrate</td><td>Hospira Inc.</td><td></td><td></td></tr><tr><td>Midazolam hydrochloride</td><td>Baxter Internationl Inc.</td><td></td><td></td></tr><tr><td>Medetomidine hydrocholoride (Domitor)</td><td>Pfizer Pharma GmbH</td><td></td><td></td></tr><tr><td>Heating pads</td><td>Beyond Bodi Heat</td><td></td><td></td></tr><tr><td>Tobramycin and dexamethasone ophthalmic ointment (TobraDex)</td><td>Alcon</td><td></td><td></td></tr><tr><td>Povidone-iodine 10% solution</td><td>Betadine Puredue Pharma</td><td></td><td></td></tr><tr><td>Ferric chloride 10% solution</td><td>Ricca Chemical Company</td><td>3120-32</td><td></td></tr><tr><td>Methyl cyanoacrylate 910</td><td>Permabond</td><td></td><td></td></tr><tr><td>Cyanoacrylate 454</td><td>Loctite</td><td></td><td></td></tr><tr><td>TEETS denture material crosslinking methyl methacrylate</td><td>Co-Oral-lte Dental Mfg CO</td><td></td><td></td></tr><tr><td>Microtorque II handpiece kit</td><td>Pearson Assessments</td><td>R14-0002</td><td></td></tr><tr><td>IRF 007 drill bits</td><td>Fine Science Tools</td><td>19008-07</td><td></td></tr><tr><td>Norland bendable microblade full radius</td><td>Salvin Dental</td><td>BLAD-NORLAND-69</td><td></td></tr><tr><td>Cyanoacrylate</td><td>The Original Superglue</td><td></td><td></td></tr><tr><td>#0 glass coverslip</td><td>Electron Microscopy Sciences</td><td>63750-01</td><td></td></tr><tr><td>10 &amp;mu;l Microvolume syringe</td><td>World Precision Instruments, Inc.</td><td>ILS010LT</td><td></td></tr><tr><td>UltraMicroPump III</td><td>World Precision Instruments, Inc.</td><td>UMP3-1</td><td></td></tr><tr><td>35 gauge NanoFil needle</td><td>World Precision Instruments, Inc.</td><td>NL35BL-2</td><td></td></tr><tr><td>Ball Mill, Carbide bits #1/4</td><td>World Precision Instruments, Inc.</td><td>501860</td><td></td></tr><tr><td>Sigmacote</td><td>Sigma-Aldrich</td><td>SL2-100</td><td></td></tr><tr><td>Photomultiplier tube </td><td>Hamamatsu Corp.</td><td>HC125-02</td><td></td></tr><tr><td>Ti:Sapphire laser Mai-Tai</td><td>Newport Corp.</td><td></td><td></td></tr></tbody></table>

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.

Watch this Scientific Journal Video about A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation at JoVE.com

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.

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 ...

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Universidad de Cartagena UDEC

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23.6K Views.  University of Rochester. 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.

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

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 ...

Acute Brain Trauma in Mice Followed By Longitudinal Two-photon Imaging

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no effective treatment for it yet. Limitations of currently used animal models impede understanding of the pathology mechanism. Multiphoton microscopy allows studying cells and tissues within intact animal brains longitudinally under physiological and pathological conditions. Here, we describe two models of acute brain injury studied by means of two-photon imaging of brain cell behavior under posttraumatic conditions. A selected brain region is injured with a sharp needle to produce a trauma of a controlled width and depth in the brain parenchyma. Our method uses stereotaxic prick with a syringe needle, which can be combined with simultaneous drug application. We propose that this method can be used as an advanced tool to study cellular mechanisms of pathophysiological consequences of acute trauma in mammalian brain in vivo. In this video, we combine acute brain injury with two preparations: cranial window and skull thinning. We also discuss advantages and limitations of both preparations for multisession imaging of brain regeneration after trauma.

Acute brain trauma is a severe injury that has no adequate treatment to date. Multiphoton microscopy allows studying longitudinally the process of acute brain trauma development and probing therapeutical strategies in rodents. Two models of acute brain trauma studied with in vivo two-photon imaging of brain are demonstrated in this protocol.

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no ...

Medicine

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 ...

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon microscopy in Thy1-GFP transgenic mouse line. This approach creates a possibility to investigate the correlation of behavioural manipulations with changes in neuronal morphology in vivo.
The cranial window implantation procedure was considered to be limited only to the easily accessible cortex regions such as the barrel field. Our approach allows visualization of neurons in the highly vascularized RSC. RSC is an important element of the brain circuit responsible for spatial memory, previously deemed to be problematic for in vivo two-photon imaging.
The cranial window implantation over the RSC is combined with an injection of mCherry-expressing recombinant adeno-associated virus (rAAVmCherry) into the dorsal hippocampus. The expressed mCherry spreads out to axonal projections from the hippocampus to RSC, enabling the visualization of changes in both presynaptic axonal boutons and postsynaptic dendritic spines in the cortex. 
This technique allows long-term monitoring of experience-dependent structural plasticity in RSC.

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in mouse retrosplenial cortex using in vivo two photon microscopy in Thy1-GFP transgenic line. This approach is combined with injection of mCherry-expressing adeno-associated virus into dorsal hippocampus. These techniques allow long-term monitoring of experience-dependent structural plasticity in RSC.

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon ...

Precise Brain Mapping to Perform Repetitive In Vivo Imaging of Neuro-Immune Dynamics in Mice

The central nervous system (CNS) is regulated by a complex interplay of neuronal, glial, stromal, and vascular cells that facilitate its proper function. Although studying these cells in isolation in vitro or together ex vivo provides useful physiological information; salient features of neural cell physiology will be missed in such contexts. Therefore, there is a need for studying neural cells in their native in vivo environment. The protocol detailed here describes repetitive in vivo two-photon imaging of neural cells in the rodent cortex as a tool to visualize and study specific cells over extended periods of time from hours to months. We describe in detail the use of the grossly stable brain vasculature as a coarse map or fluorescently labeled dendrites as a fine map of select brain regions of interest. Using these maps as a visual key, we show how neural cells can be precisely relocated for subsequent repetitive in vivo imaging. Using examples of in vivo imaging of fluorescently-labeled microglia, neurons, and NG2+ cells, this protocol demonstrates the ability of this technique to allow repetitive visualization of cellular dynamics in the same brain location over extended time periods, that can further aid in understanding the structural and functional responses of these cells in normal physiology or following pathological insults. Where necessary, this approach can be coupled to functional imaging of neural cells, e.g., with calcium imaging. This approach is especially a powerful technique to visualize the physical interaction between different cell types of the CNS in vivo when genetic mouse models or specific dyes with distinct fluorescent tags to label the cells of interest are available.

This protocol describes a chronic cranial window implantation technique that can be used for longitudinal imaging of neuro-glio-vascular structures, interactions, and function in both healthy and diseased conditions. It serves as a complementary alternative to the transcranial imaging approach that, while often preferred, possesses some critical limitations.

The central nervous system (CNS) is regulated by a complex interplay of neuronal, glial, stromal, and vascular cells that facilitate its proper function. ...

Immunology and Infection

In Vivo Chronic Two-Photon Imaging of Microglia in the Mouse Hippocampus

Microglia, the only immune cells resident in the brain, actively participate in neural circuit maintenance by modifying synapses and neuronal excitability. Recent studies have revealed the differential gene expression and functional heterogeneity of microglia in different brain regions. The unique functions of the hippocampal neural network in learning and memory may be associated with the active roles of microglia in synapse remodeling. However, inflammatory responses induced by surgical procedures have been problematic in the two-photon microscopic analysis of hippocampal microglia. Here, a method is presented that enables the chronic observation of microglia in all layers of the hippocampal CA1 through an imaging window. This method allows the analysis of morphological changes in microglial processes for more than 1 month. Long-term and high-resolution imaging of the resting microglia requires minimally invasive surgical procedures, appropriate objective lens selection, and optimized imaging techniques. The transient inflammatory response of hippocampal microglia may prevent imaging immediately after surgery, but the microglia restore their quiescent morphology within a few weeks. Furthermore, imaging neurons simultaneously with microglia allows us to analyze the interactions of multiple cell types in the hippocampus. This technique may provide essential information about microglial function in the hippocampus.

In Vivo Chronic Two-Photon Imaging of Microglia in the Mouse Hippocampus

This paper describes a method for chronic in vivo observation of the resting microglia in the mouse hippocampal CA1 using precisely controlled surgery and two-photon microscopy.

Microglia, the only immune cells resident in the brain, actively participate in neural circuit maintenance by modifying synapses and neuronal ...

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in the brain sense various biological conditions in the periphery and transmit the signals to neurons. Microglia, immune cells in the brain, are involved in synaptic development and plasticity. Therefore, the contribution of microglia to neural circuit construction in response to the internal state of the body should be tested critically by intravital imaging of the relationship between microglial dynamics and neuronal activity.
Here, we describe a technique for the simultaneous imaging of microglial dynamics and neuronal activity in awake mice. Adeno-associated virus encoding R-CaMP, a gene-encoded calcium indicator of red fluorescence protein, was injected into layer 2/3 of the primary visual cortex in CX3CR1-EGFP transgenic mice expressing EGFP in microglia. After viral injection, a cranial window was installed onto the brain surface of the injected region. In vivo two-photon imaging in awake mice 4 weeks after the surgery demonstrated that neural activity and microglial dynamics could be recorded simultaneously at the sub-second temporal resolution. This technique can uncover the coordination between microglial dynamics and neuronal activity, with the former responding to peripheral immunological states and the latter encoding the internal brain states.

Here, we describe a protocol combining adeno-associated virus injection with cranial window implantation for simultaneous imaging of microglial dynamics and neuronal activity in awake mice.

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in ...

Intravital Imaging of Fluorescent Protein Expression in Mice with a Closed-Skull Traumatic Brain Injury and Cranial Window Using a Two-Photon Microscope

The goal of this protocol is to demonstrate how to longitudinally visualize the expression and localization of a protein of interest within specific cell types of an animal's brain, upon exposure to exogenous stimuli. Here, the administration of a closed-skull traumatic brain injury (TBI) and simultaneous implantation of a cranial window for subsequent longitudinal intravital imaging in mice is shown. Mice are intracranially injected with an adeno-associated virus (AAV) expressing enhanced green fluorescent protein (EGFP) under a neuronal specific promoter. After 2 to 4 weeks, the mice are subjected to a repetitive TBI using a weight drop device over the AAV injection location. Within the same surgical session, the mice are implanted with a metal headpost and then a glass cranial window over the TBI impacting site. The expression and cellular localization of EGFP is examined using a two-photon microscope in the same brain region exposed to trauma over the course of months.

This study demonstrates delivery of a repetitive traumatic brain injury to mice and simultaneous implantation of a cranial window for subsequent intravital imaging of a neuron-expressed EGFP using two-photon microscopy.

The goal of this protocol is to demonstrate how to longitudinally visualize the expression and localization of a protein of interest within specific cell ...

Two-Photon Microscopy for Studying Microglial Process Attraction Toward a Compound in a Mouse Brain Slice

<a href='https://appjove.unicartagenaproxy.elogim.com/v/58788/two-photon-imaging-microglial-processes-attraction-toward-atp-or'>Source: Etienne, F. et al. Two-photon Imaging of Microglial Processes' Attraction Toward ATP or Serotonin in Acute Brain Slices.&#160;J. Vis. Exp. (2019)</a>This video demonstrates the use of two-photon microscopy to study the attraction of microglial processes toward an injected compound in brain slices. It provides an overview of preparing mouse brain slices, injecting a chemoattractant test compound, and imaging fluorescently labeled microglia to visualize their responses.

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

Summary

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Chapters in this video

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

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

Acute Brain Trauma in Mice Followed By Longitudinal Two-photon Imaging

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

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

Precise Brain Mapping to Perform Repetitive In Vivo Imaging of Neuro-Immune Dynamics in Mice

In Vivo Chronic Two-Photon Imaging of Microglia in the Mouse Hippocampus

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice

Intravital Imaging of Fluorescent Protein Expression in Mice with a Closed-Skull Traumatic Brain Injury and Cranial Window Using a Two-Photon Microscope

Two-Photon Microscopy for Studying Microglial Process Attraction Toward a Compound in a Mouse Brain Slice