We thank members of the Eyo lab for discussing the ideas presented in this manuscript. We thank Dr. Justin Rustenhoven from the Kipnis Lab at the University of Virginia for the gift of NG2DsRed mice33. This work is supported by funding from the National Institute of Neurological Disorders and Stroke of the National Institute of Health to U.B.E (K22 NS104392).

All steps are in accordance with the guidelines set and approved by the Institutional Animal Care and Use Committee of the University of Virginia.
1. Mouse preparation for cranial window implantation
NOTE: Various transgenic mouse lines with florescent tags are suitable for imaging.
<ol>
	<li>Use CX3CR1GFP/+ mice26 to visualize microglia in vivo. Typically, juvenile to young adult 4 to 10-week-old mice that weigh 17-25 g are used. 
	NOTE: Although, this approach is even apt for pre-weaned mice, the need to return the mice to their cage with their mothers for feeding, may complicate recovery if the mother does not take adequate care of pups post-surgery. Therefore, the use of mice post-weaning is recommended.</li>
	<li>Anesthetize the mouse using isoflurane (5% flow in oxygen for induction for 1 min) in an anesthetic chamber. Check that the mouse doesn&#8217;t show any movement or twitching responses to toe and/or tail pinches. Take the mouse out of the chamber and in open air thoroughly shave the hair on the head between the ears from about the eye level to the top of the neck region using a hair trimmer. 
	NOTE: The concentration of isoflurane used would depend on the size of the induction chamber. Therefore, for smaller chambers, 3-4% isoflurane can be used to effectively induce anesthesia while larger chambers will require up to 5%.</li>
	<li>Move the mouse to the stereotactic surgery station nose cone for anesthesia (1.5-2% for maintenance for the surgery), stabilize its head using ear bars, and maintain the mouse on a heating pad to keep the body temperature warm.</li>
	<li>Lubricate both eyes with eye ointment. Inject 100 &#181;L of 0.25% bupivicaine (to provide local analgesia to the mouse that will last 8-12 h) and 100 &#181;L of 4 mg/mL dexamethasone (to reduce the inflammation that may result from the surgery procedure) subcutaneously at the incision site. Allow the mouse to sit for at least 5 min before moving to the next step.</li>
	<li>Clean the shaved head with three alternating swabs of betadine and 70% alcohol. Make a midline scalp incision using surgical blade or scissors extending from the back of the skull region between the ears to the frontal area between the eyes. The remaining skin is cut to expose the skull.&#160;</li>
	<li>Clean the connective tissue located between the scalp and the underlying skull with 3% hydrogen peroxide (H2O2) and localize the brain area to be imaged with stereotactic coordinates. 
	NOTE: There is often some bleeding (step 1.5) from the incision on the skull surface. This bleeding usually resolves by itself within 3-5 min. Cleaning with the peroxide helps. Prior bupivacaine treatment (step 1.4) is also noted to limit the amount of bleeding during this time.</li>
</ol>
2. Mouse cranial window implantation surgery
<ol>
	<li>Drill a circular opening ~4 mm into the skull using a dental drill bit (0.7 mm tip diameter) and carefully remove this portion of the skull using pointed forceps. For imaging the somatosensory cortex of 6-8-week-old mice, locate the center of the craniotomy at -2.5 posterior and &#177; 2.0 lateral to bregma. During drilling, regularly moisten the skull with sterile saline and cotton swabs to cool the brain, clean off bone debris and soften the skull bone for eventual removal. 
	NOTE: The coordinates for the craniotomy would vary depending on the region of interest and the age of the mice.</li>
	<li>After the skull is removed, carefully place a small coverglass (size #0 at 0.1 &#177; 0.02 mm thickness) moistened with saline in the craniotomy. Dry off excess saline using a sterile wipe.</li>
	<li>Using a pointed applicator (such as a pipette tip or the pointed end of a broken wooden cotton swab stick), apply cyanoacrylate glue around the window and allow it to attach to the brain and skull. Apply the primer glue to the rest of the skull and cure it with a curing light for 20-40 s. Prepare a well around the window with the final glue and cure with a curing light for 20-40 s.</li>
	<li>Glue a small head plate on to the skull on the contralateral hemisphere of the craniotomy first with the primer glue as a primer and then with the final glue. Cure both with the curing light for 20-40 s each. 
	NOTE: Sutures are not needed if the skull is totally covered with the glue during this procedure.</li>
</ol>
3. Post-surgery care
<ol>
	<li>Allow the mouse to wake up in the absence of anesthesia (recovery done on a heating pad shortens the recovery time) and return it to its home cage once fully awake. Inject one subcutaneous dose of buprenorphine SR (0.5 mg/kg) as post-operative analgesia that is sufficient for 72 h.</li>
	<li>To facilitate a healthy recovery from the surgery, provide the mouse an extra soft food, which can be in the form of regular solid chow in water to soften the chow or food in the form of a gel. 
	NOTE: A one-time provision of the soft food immediately after the surgery is sufficient.</li>
	<li>Monitor the mouse daily for health and proper recovery for the first 72 h of the surgery procedure. Afterwards, perform imaging from as early as 2 weeks from the window implantation surgery. 
	NOTE: If done well, mice recover well showing normal ambulatory behaviors, sufficient cage exploration, good hydration, stable weight gain and extensive interactions with other mice in the cage and other items in the cage. Mice showing lethargy, dehydration and greater than 10% weight loss following the surgery are euthanized and removed from the study.</li>
</ol>
4. Two-photon brain mapping for initial imaging
<ol>
	<li>Anesthetize the mouse (Isoflurane, 5 % induction and 1.5 % maintenance). Stabilize the head using screws to mount the headplate on the two-photon microscope stage, being maintained on a heating plate at 35 &#730;C. Inject intraperitoneally 100 &#181;L of blood vessel dye such as Rhodamine B (2 mg/mL). 
	NOTE: Imaging could also be done in awake mice without anesthesia. However, recent studies indicate that anesthesia affects microglial surveillance dynamics27,28,29 and that head fixation for two photon imaging in awake mice increases stress even during chronic imaging for at least 25 days (see Juczewski et al., 2020)30.</li>
	<li>Clean the surface of the cranial window gently using a cotton swab dabbed in 70% ethanol. Put a few drops of water or saline on the cranial window and lower the objective lens into the solution since the objective is an immersion lens.</li>
	<li>Hand-draw a coarse map to denote the major blood vessel landmarks in a lab notebook while looking through the eyepiece by epiflorescence. Use this drawing to identify the specific regions during two photon imaging. Alternatively, take pictures of the blood vessels either through a camera fitted to the microscope or through a hand-held camera or phone. 
	NOTE: These hand-drawn images and pictures are to facilitate revisiting the same broad regions under the microscope before two photon imaging. These are not precise image mapping.</li>
	<li>Under two photon imaging, collect images of florescent cells and vessels as needed. Take careful notes with appropriate coordinates to ensure that that the precise regions can be revisited for subsequent imaging. Collect several fields of view in this initial imaging session e.g. acquire z-stack images every 1-2 &#181;m through a volume of tissue. 
	NOTE: While collecting images by two photon, the blood vessel landmarks are used for coarse mapping. If fine mapping is needed, YFP-labeled dendrites from Thy1-YFP31 mice are used.
	<ol>
		<li>Use these recommended parameters for imaging: a wavelength of 880-900 nm is optimal; for GFP and/or dsRed / Rhodamine excitation, a 565 nm dichroic mirror with 525/50 nm (green channel) and 620/60 nm (red channel) emission filters are used; for GFP and YFP separation, a 509 nm dichroic mirror with 500/15 and 537/26 nm emission filters are used; the power at the brain is maintained at 25 mW or below; image resolution is 1024 x 1024 pixels, the field of view taken with a 25X 0.9 NA objective at a 1.5X zoom factor is 295.24 x 295.24 &#181;m.</li>
	</ol>
	</li>
	<li>At the end of the imaging, take the mouse off the stage, allow it to wake up from anesthesia and return to its home cage until a future imaging session.</li>
</ol>
5. Two-photon imaging and re-imaging
<ol>
	<li>For future subsequent imaging sessions, which could be anywhere from a few hours to months after the initial imaging session, anesthetize the mouse (Isoflurane, 5% induction and 1.5% maintenance), mount on the two-photon microscope, maintain on a heating plate and re-inject 100 &#181;l a blood vessel dye such as Rhodamine B (2 mg/mL).</li>
	<li>Open the previously obtained images in ImageJ and, using these images as well as the notes from the previous session, identify the previously&#160;imaged&#160;areas and carefully re-image them.&#160;</li>
	<li>Repeat this for as long as the imaging window is clear or as essential for the extent of the study.</li>
</ol>

The authors have nothing to disclose.

The advent of in vivo two-photon imaging has opened opportunities to explore the plethora of cellular interactions and dynamics that occur in the healthy brain. Initial studies focused on using the open skull craniotomy approach to image neuronal dendrites by both acute and chronic imaging37,38. This can also be used to elucidate neuroimmune interactions in the brain. This protocol describes a method for the reliable imaging of fluorescently tagged cells (especia...

The central nervous system (CNS) is governed by a complex interplay of interactions between various resident cell types including neurons, glia and vessel-associated cells. Traditionally, neural cells were studied in isolated, co-cultured1,2,3,4,5 (in vitro) or excised brain tissue (ex vivo)6,7,8,9,10 contexts. However, there is need to further understand neural cell behavior and interactions in the native environment of the intact brain in vivo. In this protocol, we describe a method to map in vivo regions of interest and precisely re-image those regions in future imaging sessions to track the complex interactions between the various CNS cell types over extended periods of time.
The development of in vivo imaging approaches has provided significant gains for the proper understanding of neural function11,12,13,14,15. Specifically, these approaches provide several advantages over traditional in vitro and ex vivo approaches. First, in vivo imaging systems have physiologically relevant cell and tissue components such as the vasculature with the full repertoire of cellular interactions to provide a complete understanding of neural network physiology. Second, recent findings suggest that when removed from their native environment, certain neural cells (such as microglia) lose important features of their identity and thus physiology16,17 which can be preserved in the in vivo setting. Third, in vivo imaging systems provide the opportunity for stable longitudinal investigations of weeks to months to study CNS cellular interactions. Finally, given the growing evidence for contributions from the peripheral immune system18,19 and the microbiome20,21 in CNS physiology, in vivo systems provide a platform to interrogate such contributions and effects on CNS cells. Thus, approaches that employ longitudinal in vivo imaging to study neuro-immune physiology and interactions in healthy, injured, and diseased states are a great complementary addition to traditional approaches.
In this protocol, we describe a reliable approach to image different cell types in the brain including microglia, neurons and NG2+ cells as examples. Two approaches to visualize neural cells in vivo have been developed: the thinned skull approach and the open skull with a cranial window approach. Although thinned skull approaches are in use and are preferred because they overcome some of the disadvantages of the open skull approach such as glial cell activation, higher-than-physiological spine dynamics and the use of anti-inflammatory agents22,23,24,25, thinned skull approaches also show a few critical drawbacks. First, the thinning procedure is a very delicate procedure that many researchers find difficult to perfect especially when re-thinning is necessary. This is the case because it is often difficult for experimenters to ascertain that they have thinned the skull to a ~20 &#181;m depth. Second, for adequate comparisons between mice, thinning would need to be identical and a variety of thinning success between imaging sessions or mice could complicate visualization of neural structures. Third, when employed for longitudinal imaging, animals with thinned skulls can only be used for a limited number of sessions when re-thinning of the skull is employed. Forth, since some of the bone tissue still remains, clarity in depth of imaging could be compromised from the thinned skull approach allowing for great visualization of more superficial but not as much with deeper regions. In the light of this, deeper brain structures such as the hippocampus, cannot be successfully imaged with the thinned skull approach. These considerations raise the need for alternative and complementary approaches that could overcome these concerns.
Alternative to the thinned skull approach, the open skull window implantation approach uses a procedure in which the skull is replaced with an optically clear glass coverslip. This allows for a near-unlimited number of imaging sessions. Moreover, given the replacement of the skull with the glass coverslip, this method allows for a clear viewing window of fluorescently tagged brain cells for extensive periods of times from hours to months and, therefore, can be employed to study cell activity and interactions that are relevant for physiology, aging and pathology.
Overall, we detail steps that can be followed to do implant chronic cranial windows through a stereotaxic craniotomy that enables in vivo imaging of brain regions of interest. We also describe how the grossly stable brain vasculature or the fluorescently labeled dendrites could be used to generate a coarse or a fine map, respectively of the brain regions of interest. This approach can then be used for repeated imaging over several sessions. The importance of this technique, therefore, lies in its ability to image the long-term changes or stasis in brain elements including the arrangement, morphology, and interactions of the different cellular types.

<table><tbody><tr><td>Coverglass (3mm)</td><td>Warner Instruments</td><td>64-0726</td><td></td></tr><tr><td>Cyanoacrylate glue (Krazy Glue)</td><td>Amazon</td><td>https://www.amazon.com/Krazy-Glue-Original-Purpose-Instant/dp/B07GSF31WZ/ref=sr_1_2?keywords=krazy+glue&amp;amp;qid=1583856837&amp;amp;s=pet-supplies&amp;amp;sr=8-2</td><td></td></tr><tr><td>Demi Ultra LED Curing Light System</td><td>Dental Health Products, Inc</td><td>910860-1</td><td></td></tr><tr><td>Dental Drill</td><td>Osada: www.osadausa.edu</td><td>EXL-M40</td><td></td></tr><tr><td>Drill Bit</td><td>Fine Science Tools</td><td>#19008-07</td><td></td></tr><tr><td>Eye Ointment</td><td>Henry Schien</td><td>1338333</td><td></td></tr><tr><td>iBond Total Etch (Primer glue)</td><td>Chase Dental Supply (Heraeus Kulzer)</td><td>66040094</td><td></td></tr><tr><td>Rhodamine B</td><td>Millipore Sigma</td><td>81-88-9 (R6626)</td><td></td></tr><tr><td>Tetris Evoflow glue (Final glue)</td><td>Top Dent (Ivoclar Vivadent)</td><td>#595956</td><td></td></tr><tr><td>Wahl Brav Mini+</td><td>Amazon</td><td>https://www.amazon.com/Wahl-Professional-Animal-BravMini-41590-0438/dp/B00IN24ILE/ref=asc_df_B00IN24ILE/?tag=hyprod-20&amp;amp;linkCode=df0&amp;amp;hvadid=167141013968&amp;amp;hvpos=&amp;amp;hvnetw=g&amp;amp;hvrand=12368793083893626704&amp;amp;hvpone=&amp;amp;hvptwo=&amp;amp;hvqmt=&amp;amp;hvdev=c&amp;amp;hvdvcmdl=&amp;amp;hvlocint=&amp;amp;hvlocphy=9008337&amp;amp;hvtargid=pla-332197544154&amp;amp;psc=1</td><td></td></tr></tbody></table>

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.

To visualize microglial dynamics in vivo, double transgenic CX3CR1GFP/+:Thy1YFP mice were used. The Thy1-YFP H line is used as opposed to the Thy1-GFP M line to avoid florescence overlap of microglia (GFP) and neurons (YFP). Alternative approaches could use a reporter line in which microglia are labeled with e.g., tdTomato and then the Thy1-GFP M line can be used. A drawback of the H line is that YFP labels a lot of neurons and the label increases with increasing age (personal observation). The M li...

Watch this Scientific Journal Video about Precise Brain Mapping to Perform Repetitive In Vivo Imaging of Neuro-Immune Dynamics in Mice at JoVE.com

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

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. ...

precise-brain-mapping-to-perform-repetitive-vivo-imaging-neuro-immune

Research

JoVE Journal

Immunology and Infection

7.6K Views.  University of Virginia. 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.

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

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 ...

Neuroscience

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo 4-7. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions 8-17. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task.
We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

A minimally invasive protocol to stabilize the mouse spinal column and perform repetitive in vivo spinal cord imaging using two-photon microscopy is described. This method combines a spinal stabilization device and an anesthetic regimen to minimize respiratory-induced movements and produce raw imaging data that require no alignment or other post-processing.

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has ...

Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, neutrophils, T cell subsets, B cells, natural killer (NK) and NKT cells. Intravital microscopy of the liver for monitoring immune cell migration is particularly challenging due to the high requirements regarding sample preparation and fixation, optical resolution and long-term animal survival. Yet, the dynamics of inflammatory processes as well as cellular interaction studies could provide critical information to better understand the initiation, progression and regression of inflammatory liver disease. Therefore, a highly sensitive and reliable method was established to study migration and cell-cell-interactions of different immune cells in mouse liver over long periods (about 6 hr) by intravital two-photon laser scanning microscopy (TPLSM) in combination with intensive care monitoring.
The method provided includes a gentle preparation and stable fixation of the liver with minimal perturbation of the organ; long term intravital imaging using multicolor multiphoton microscopy with virtually no photobleaching or phototoxic effects over a time period of up to 6 hr, allowing tracking of specific leukocyte subsets; and stable imaging conditions due to extensive monitoring of mouse vital parameters and stabilization of circulation, temperature and gas exchange.
To investigate lymphocyte migration upon liver inflammation CXCR6.gfp knock-in mice were subjected to intravital liver imaging under baseline conditions and after acute and chronic liver damage induced by intraperitoneal injection(s) of carbon tetrachloride (CCl4).
CXCR6 is a chemokine receptor expressed on lymphocytes, mainly on Natural Killer T (NKT)-, Natural Killer (NK)- and subsets of T lymphocytes such as CD4 T cells but also mucosal associated invariant (MAIT) T cells1. Following the migratory pattern and positioning of CXCR6.gfp+ immune cells allowed a detailed insight into their altered behavior upon liver injury and therefore their potential involvement in disease progression.

Stable intravital high-resolution imaging of immune cells in the liver is challenging. Here we provide a highly sensitive and reliable method to study migration and cell-cell-interactions of immune cells in mouse liver over long periods (about 6 hours) by intravital multiphoton laser scanning microscopy in combination with intensive care monitoring.

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, ...

Intravital Imaging of Neutrophil Priming Using IL-1&#946; Promoter-driven DsRed Reporter Mice

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that enhances the phagocytic functionality of neutrophils. Although extensive studies have unveiled the existence and importance of neutrophil priming during infection and injury, means of visualizing this process in vivo have been unavailable. The protocol provided enables monitoring of the dynamic process of neutrophil priming in living animals by combining three methodologies: 1) DsRed reporter signal &#8212; used as a measure of priming 2) in vivo neutrophil labeling &#8212; achieved by injection of fluorescence-conjugated anti-lymphocyte antigen 6G (Ly6G) monoclonal antibody (mAb) and 3) intravital confocal imaging. Several critical steps are involved in this protocol: oxazolone-induced mouse ear skin inflammation, appropriate sedation of animals, repeated injections of anti-Ly6G mAb, and prevention of focus drift during imaging. Although a few limitations have been observed, such as the limit of continuous imaging time (~ 8 hr) in one mouse and the leakage of fluorescein isothiocyanate-dextran from blood vessels in the inflammatory state, this protocol provides a fundamental framework for intravital imaging of primed neutrophil behavior and function, which can easily be expanded to examination of other immune cells in mouse inflammation models.

This current protocol employs fluorescent reporters, in vivo labeling, and intravital imaging techniques to enable monitoring of the dynamic process of neutrophil priming in living animals.

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that ...

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 ...

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 ...

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 ...

Imaging CD19+ B Cells in an Experimental Autoimmune Encephalomyelitis Mouse Model using Positron Emission Tomography

Multiple sclerosis (MS) is the most common demyelinating central nervous system (CNS) disease affecting young adults, often resulting in neurological deficits and disability as the disease progresses. B lymphocytes play a complex and critical role in MS pathology and are the target of several therapeutics in clinical trials. Currently, there is no way to accurately select patients for specific anti-B cell therapies or to non-invasively quantify the effects of these treatments on B cell load in the CNS and peripheral organs. Positron emission tomography (PET) imaging has enormous potential to provide highly specific, quantitative information regarding the in vivo spatiotemporal distribution and burden of B cells in living subjects.
This paper reports methods to synthesize and employ a PET tracer specific for human CD19+ B cells in a well-established B cell-driven mouse model of MS, experimental autoimmune encephalomyelitis (EAE), which is induced with human recombinant myelin oligodendrocyte glycoprotein 1-125. Described here are optimized techniques to detect and quantify CD19+ B cells in the brain and spinal cord using in vivo PET imaging. Additionally, this paper reports streamlined methods for ex vivo gamma counting of disease-relevant organs, including bone marrow, spinal cord, and spleen, together with high-resolution autoradiography of CD19 tracer binding in CNS tissues.

Imaging CD19+ B Cells in an Experimental Autoimmune Encephalomyelitis Mouse Model using Positron Emission Tomography

This paper details methodology for radiolabeling a human-specific anti-CD19 monoclonal antibody and how to use it to quantify B cells in the central nervous system and peripheral tissues of a mouse model of multiple sclerosis using in vivo PET imaging, ex vivo gamma counting, and autoradiography approaches.

Multiple sclerosis (MS) is the most common demyelinating central nervous system (CNS) disease affecting young adults, often resulting in neurological ...

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

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Chapters in this video

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

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

Intravital Imaging of Neutrophil Priming Using IL-1β Promoter-driven DsRed Reporter Mice

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

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

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

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice

Imaging CD19⁺ B Cells in an Experimental Autoimmune Encephalomyelitis Mouse Model using Positron Emission Tomography