The authors would like to acknowledge the Ligue Francaise contre l&#39;&#233;pilepsie (to M.A-L), Institut National de la Sant&#233; et de la Recherche M&#233;dicale Grant AVENIR R12087FS (to F.J), grant from the university of Montpellier (to F.J) and grant from Federation pour la Recherche sur le Cerveau (to N.M). We acknowledge the technical assistance of Chrystel Lafont at the IPAM in vivo imaging core platform facility of Montpellier. We also thank Mary Vernov (Weill Cornell Medical College) for proofreading the manuscript.

Mice are allowed ad libitum access to food, water, and maintained on a 12-hr light-dark cycle. All procedures involving laboratory animals conformed to National and European laws and were approved by the French Ministry for Education and Scientific Research (CEEA-LR-00651-01). A total of 6 transgenic 5xFAD and 4 littermate wildtype (WT) control mice were used for this procedure.
1. Pre-operative Preparation
<ol>
	<li>Intraperitoneally (IP) inject Methoxy-X04 (10 mg/kg) 48 hr before surgery to label A&#946; deposits11.</li>
	<li>Prepare sterile artificial cerebrospinal fluid (aCSF) (120 mM NaCl, 26.2 mM NaHCO3, 2.5 mM KCl, 1 mM NaH2PO4, 1.3 mM MgCl2, 10 mM glucose; bubble with 95% O2/ 5% CO2 before adding 2.5 mM CaCl2).</li>
	<li>Sterilize surgical tools (scissors, forceps, razor blade and drill bit), using an autoclave or a hot bead sterilizer before aseptic surgery.</li>
	<li>Clean the surgical bench and microscope plate with 70% ethanol and cover with a clean absorbent cloth.</li>
	<li>Inject ketamine/xylazine (75&#160;mg/kg and 10&#160;mg/kg, respectively) and the analgesic ketoprofen (2.5 mg/kg)&#160;IP.</li>
	<li>Check the appropriate level of anesthesia with paw or tail pinches.</li>
	<li>Apply sterile ophthalmic ointment to the eyes and shave the fur (nose to ears) while avoiding the whiskers. A depilatory cream can be used as long as it is followed by carefully rinsing with water to prevent skin irritation. Shave the remaining fur with a razor blade. Sterilize the skin with povidone-iodine antiseptic solution.</li>
	<li>Place the mouse under the binocular microscope and make a skin incision from the ears to the nose. Pull the skin sideways to expose the skull. Incise the periosteum and repeatedly rinse the skull with sterile aCSF to stop possible bleeding.</li>
</ol>
2. Vasculature Labeling and Thinned Skull Window Preparation (40 min)
<ol>
	<li>Remove the eye ointment with a cotton tip. Apply a drop of topical ophthalmic anesthetic (tetracaine 1%)&#160;and gently pressure the skin around the eye socket to achieve protrusion.</li>
	<li>Inject Fluorescein isothiocyanate (FITC) conjugated with Dextran (70 kDa) (100 mg/kg or 50 &#181;l of a 50 mg/ml solution; hypodermic insulin needle) in the retro-orbital sinus following the procedure described by Yardeni and colleagues 12. Apply sterile ophthalmic ointment to eyes.</li>
	<li>Ensure that the skin is retracted and the skull is clean and dry around the selected imaging area. Apply a small amount of cyanoacrylic glue around the window of the head mount device (consisting of stacked razor-blades, see Figure 1), and glue around the target region applying gentle pressure for several seconds 10.</li>
	<li>Pull the ends of the skin to the edge of the window of the head mount device, making sure the area is dry. Secure the skin and the edge of the window with a small amount of veterinary grade cyanoacrylic glue and wait until dry (Figure 1).</li>
	<li>Secure the animal at the stage using the head-mount device. Rinse with sterile aCSF and ensure that the well between the skin and head-mount device is perfectly sealed. Wrap the mouse in a survival blanket to maintain normothermia.</li>
	<li>Perform thinning of the skull in aCSF solution as previously described10. Note that the scalp is not completely removed. Regularly change the aCSF to clear bone debris and to avoid tissue overheating. Any remaining bone debris can be removed with brief air puffs.&#160;The aCSF solution also attenuates vibrations created by drilling. 
	NOTE: The mouse skull thickness varies between 80 to 150 &#181;m depending on the age of the animal and the region of interest.</li>
	<li>Using a dental drill (at medium speed and a 0.7 mm burr) start thinning the skull surface in an area 1.0 mm in diameter using a regular vertical motion parallel to the skull and remove most of the spongy bone (first 40 - 70 &#181;m). remember to replace aCSF every 20-30 sec&#160;and limit uninterrupted drilling to&#160; 3-4 sec.&#160;Note that bone near sutures is highly vascularized. Apply aCSF presoaked gelfoam to stop eventual bleeding.</li>
	<li>Use a sharp disposable ophthalmic microsurgical blade to continue with the thinning of the skull, being careful not to apply excessive pressure. The final region is of about 0.5 mm in diameter and with a thickness of 20 - 35 &#181;m. 
	NOTE: Due to bone regrowth and scarring, it is necessary to further thin the window at each imaging session.</li>
</ol>
3. Two-photon Microscopy (45 min)
<ol>
	<li>If necessary, inject with 19 mg/kg ketamine&#160;to maintain adequate anesthesia.&#160;</li>
	<li>Transfer the mouse (fixed to the headmount stage) under the two-photon laser microscope. Using a 20X water immersion objective with a numerical aperture of 1.0, locate the thinned cranial window at the center of the optical field using the epi-fluorescent lamp. Ensure that the objective is always immersed in aCSF.</li>
	<li>Start laser scanning with a mode locked pulsed laser (Ti-Sapphire 680-1040nm). Set excitation wavelength at 750 nm to detect the emitted fluorescence of the methoxy-XO4 and FITC-dextran in the blue and green channels respectively. 
	NOTE: The microscope has two beam splitters at 506 nm and 555 nm. Blue light is captured by an NDD detector equipped with a 470 &#177; 12 nm filter. Green light is captured by a high sensitivity GaAsP detector equipped with a 525 &#177; 25 nm filter. The maximal laser power emitted from the objective should not exceed 10 mW to avoid laser-induced tissue damage.</li>
	<li>Acquire a low magnification stack (500 &#181;m x 500 &#181;m, 512 x 512 pixels; 2 &#181;m step) at a 0.7X numerical zoom to create a 3D map for the precise relocalization of the ROI at later time points.</li>
	<li>Acquire a mosaic of 4 high digital magnification images with a 2X numerical zoom (200 &#181;m x 200 &#181;m, 512 x 512 pixels; 1 &#181;m step). Using the imaging software move the window in 200 &#181;m steps to capture all regions. Depth of stacks is typically 250 &#181;m, starting from the pial surface in each image of the mosaic.</li>
	<li>Before removing the mouse from the microscope, take a picture or make a hand-drawn 2D map of the pial vasculature for future relocalization of the imaging field.</li>
</ol>
4. Recovery and Re-imaging
<ol>
	<li>Remove the head-mount device by applying traction while holding the skull underneath. If any glue remains attached to the skull or skin, scrape it off carefully with fine forceps. Suture the scalp and place the mouse in a heated cage to monitor recovery from anesthesia before returning it to the home cage. Apply topical antibiotic cream to the suture and inject ketoprofen (2.5 mg/kg) IP the following 2 days.</li>
	<li>Repeat steps 1.1 to 2.5 for repeated imaging sessions. If necessary, shave the bone with a microsurgical blade to ensure optimal quality of the image. 
	NOTE: Additional thinning with the dental drill may be necessary when the interval between imaging sessions is longer than one month.</li>
	<li>Position the mouse underneath the microscope using epifluorescent light according to the 2D map of the pial vasculature taken from the previous session (step 3.6).</li>
	<li>Start two-photon laser scanning and adjust microscope stage to achieve alignment at the micrometer scale using the 3D map obtained in step 3.4. Proceed to detailed imaging as described in step 3.5.</li>
</ol>
5. Post-acquisition Three-dimensional Reconstruction and Image Analysis
<ol>
	<li>Obtain three-dimensional reconstructions of the vasculature and the amyloid depositions using the 3D image analysis software such as IMARIS (version used 8.0 and 7.7.2).</li>
	<li>Use the Normalize Layer plugin on the image processing menu and contrast change submenu to obtain ideal contrast of the two channels in the entire depth of the image stack.</li>
	<li>Open the images from the same region at all time points simultaneously and always process them in parallel in order to compare the different time points.</li>
	<li>Trace vessels using the filament tracer module.
	<ol>
		<li>Click on the filament icon to create a new filament, skip the automatic tracer and choose the auto-path method.</li>
		<li>Be sure the selected channel corresponds to the vascular image and using the select function of the pointer, click at a vessel bifurcation that is conserved across the imaging sessions while holding the shift + control keys to determine a starting point. Use the select function of the pointer and click while holding the shift key to select the end points of the filaments. 
		NOTE: Comparison of the obtained skeletons will show structural changes of the vasculature.</li>
	</ol>
	</li>
	<li>Perform plane by plane analysis to track new plaques. Apply surface module to the blue channel for tracking the growth of A&#946; plaques. Click on the surface icon to create a new surface and proceed with the automated surface creation. Use the thresholding method to set and subtract the background signal. The statistics menu provides data relative to the surface of individual amyloid deposits.</li>
</ol>

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Authors have no competing financial interests.

The open-skull technique for in vivo two-photon microscopy offers the advantage of unlimited imaging sessions of large imaging fields13,14. However, this technique also produces inflammation in the region of interest14, often incompatible or impacting neuro-vascular read-outs15. On the contrary, the thinned skull transcranial technique does not result in neuro-inflammation, enabling reliable imaging of the cerebrovascular structures and plaque accumulation10,14. A seco...

The brain vasculature is a multi-cellular structure, which is anatomically and functionally coupled to neurons. A dynamic remodeling of vessels occurs throughout brain development and during the progression of pathologies of the central nervous system (CNS) 1,2. It is widely accepted that cerebrovascular damage is a hallmark of several CNS diseases, including epilepsy, Alzheimer&#39;s disease (AD), traumatic brain injury and encephalitis 3,4. Therefore, tracking cerebrovascular changes in vivo becomes significant when modeling CNS diseases, from onset and into chronic phases. As cerebrovascular modifications often occur concomitantly with neuronal damage or plasticity, imaging of the neuro-vasculature represents a key entry point to decipher CNS disease pathophysiology.
This protocol describes a longitudinal two-photon based procedure to track the remodeling of the cerebrovasculature in a mouse model of AD, a progressive pathology marked by cerebrovascular defects on large and small caliber vessels due to amyloidogenic plaque deposition 5-7. This procedure allows for the visualization of amyloid deposits and tracking of their position and growth with respect to neurovascular remodeling throughout the course of the disease. Vital fluorescent dyes are injected before each imaging session for the visualization of the cerebrovasculature and amyloid plaques in transgenic AD mice8. Repeated imaging sessions of a ROI through a thinned skull transcranial window is non-invasive and the method of choice to assess neurovascular remodeling in the living mouse brain 2,5,9,10.
The procedure below outlines the surgical protocol, image acquisition and processing. The early progression of cerebral amyloid angiopathy (CAA) mostly at large leptomeningeal and penetrating arterioles is characterized.

<table><tbody><tr><td>methoxy-X04</td><td>tocris</td><td>4920</td><td>use 10 mg/kg</td></tr><tr><td>FITC-Dextran 70 Kda</td><td>sigma</td><td>46945</td><td>use 100 mg/kg</td></tr><tr><td>gelfoam/Bloxang</td><td>Bausch and Lomb</td><td></td><td></td></tr><tr><td>micorsurgical blade</td><td>surgistar</td><td>6900</td><td>must be sharp and not dented</td></tr><tr><td>povidone-iodine</td><td>betadine</td><td></td><td>antisceptic solution</td></tr><tr><td>binocular stereomicroscope</td><td>olympus</td><td>SX10</td><td>optimal image contrast is crucial for this procedure</td></tr><tr><td>2-photon microscope</td><td>zeiss</td><td></td><td>Zeiss LSM 710mp</td></tr><tr><td>fine scissors-toughcut</td><td>Fine science tools</td><td>14058-09</td><td>this scissors are optimized for cutting skin and soft tissue</td></tr></tbody></table>

longitudinal in vivo imaging of the cerebrovasculature: relevance to cns diseases

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular plasticity or damage occurring overtime and in relation to neuronal activity or blood flow. In vivo two-photon microscopy allows the study of the structural and functional plasticity of large cellular units in the living brain. In particular, the thinned-skull window preparation allows the visualization of cortical regions of interest (ROI) without inducing significant brain inflammation. Repetitive imaging sessions of cortical ROI are feasible, providing the characterization of disease hallmarks over time during the progression of numerous CNS diseases. This technique accessing the pial structures within 250 &#956;m of the brain relies on the detection of fluorescent probes encoded by genetic cellular markers and/or vital dyes. The latter (e.g., fluorescent dextrans) are used to map the luminal compartment of cerebrovascular structures. Germane to the protocol described herein is the use of an in vivo marker of amyloid deposits, Methoxy-O4, to assess Alzheimer&#39;s disease (AD) progression. We also describe the post-acquisition image processing used to track vascular changes and amyloid depositions. While focusing presently on a model of AD, the described protocol is relevant to other CNS disorders where pathological cerebrovascular changes occur.

This protocol describes a method for visualizing the cerebrovasculature and amyloid deposits overtime. Fluorescent dyes were injected to label amyloid depositions (methoxy-XO4) 11 and to fill the cerebrovascular lumen (FITC-Dextran)1. 3D image analysis software modules were used to create 3D images of a constant field of view captured at consecutive time points. Representative images obtained in the somatosensory cortex of 5XFAD mice5 (genetic model of AD)...

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

This manuscript describes a procedure to track the remodeling of the cerebrovasculature during amyloid plaque accumulation in vivo using longitudinal two-photon microscopy. A thinned-skull preparation enables the visualization of fluorescent dyes to assess the progression of cerebrovascular damage in a mouse model of Alzheimer&#39;s disease.

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

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular ...

longitudinal-vivo-imaging-cerebrovasculature-relevance-to-cns

Research

JoVE Journal

Medicine

7.1K Views.  Institute of Functional Genomics.  Dit manuscript beschrijft een procedure om de herinrichting van de cerebrovasculature volgen tijdens amyloïde plaque accumulatie in vivo met behulp van longitudinale twee-foton microscopie. Een uitgedunde schedel bereiden voor de visualisatie van fluorescente kleurstoffen om de progressie van cerebrovasculaire schade te beoordelen in een muizenmodel van de ziekte van Alzheimer. 

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

Non-invasive Parenchymal, Vascular and Metabolic High-frequency Ultrasound and Photoacoustic Rat Deep Brain Imaging

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central nervous system of small animals. However, transdermal and transcranial neuroimaging is frequently affected by low sensitivity, image aberrations and loss of space resolution, requiring scalp or even skull removal before imaging. To overcome this challenge, a new protocol is presented to gain significant insights in brain hemodynamics by photoacoustic and high-frequency ultrasounds imaging with the animal skin and skull intact. The procedure relies on the passage of ultrasound (US) waves and laser directly through the fissures that are naturally present on the animal cranium. By juxtaposing the imaging transducer device exactly in correspondence to these selected areas where the skull has a reduced thickness or is totally absent, one can acquire high quality deep images and explore internal brain regions that are usually difficult to anatomically or functionally describe without an invasive approach. By applying this experimental procedure, significant data can be collected in both sonic and optoacoustic modalities, enabling to image the parenchymal and the vascular anatomy far below the head surface. Deep brain features such as parenchymal convolutions and fissures separating the lobes were clearly visible. Moreover, the configuration of large and small blood vessels was imaged at several millimeters of depth, and precise information were collected about blood fluxes, vascular stream velocities and the hemoglobin chemical state. This repertoire of data could be crucial in several research contests, ranging from brain vascular disease studies to experimental techniques involving the systemic administration of exogenous chemicals or other objects endowed with imaging contrast enhancement properties. In conclusion, thanks to the presented protocol, the US and PA techniques become an attractive noninvasive performance-competitive means for cortical and internal brain imaging, retaining a significant potential in many neurologic fields.

The present work describes a new protocol to perform non-invasive high-frequency ultrasound and photoacoustic based imaging on rat brain, to efficiently visualize deep subcortical regions and their vascular patterns by directing signals on skull foramina naturally present on animal cranium.

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central ...

Neuroscience

Quantification of Cerebral Vascular Architecture using Two-photon Microscopy in a Mouse Model of HIV-induced Neuroinflammation

Human Immunodeficiency Virus 1 (HIV-1) infection frequently results in HIV-1 Associated Neurocognitive Disorders (HAND), and is characterized by a chronic neuroinflammatory state within the central nervous system (CNS), thought to be driven principally by virally-mediated activation of microglia and brain resident macrophages. HIV-1 infection is also accompanied by changes in cerebrovascular blood flow (CBF), raising the possibility that HIV-associated chronic neuroinflammation may lead to changes in CBF and/or in cerebral vascular architecture. To address this question, we have used a mouse model for HIV-induced neuroinflammation, and we have tested whether long-term exposure to this inflammatory environment may damage brain vasculature and result in rarefaction of capillary networks. In this paper we describe a method to quantify changes in cortical capillary density in a mouse model of neuroinflammatory disease (HIV-1 Tat transgenic mice). This generalizable approach employs in vivo two-photon imaging of cortical capillaries through a thin-skull cortical window, as well as ex vivo two-photon imaging of cortical capillaries in mouse brain sections. These procedures produce images and z-stack files of capillary networks, respectively, which can be then subjected to quantitative analysis in order to assess changes in cerebral vascular architecture.

This paper describes a method by which the vascular architecture in the brain can be quantified using in vivo and ex vivo two-photon microscopy.

Human Immunodeficiency Virus 1 (HIV-1) infection frequently results in HIV-1 Associated Neurocognitive Disorders (HAND), and is characterized by a chronic ...

Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

Focal cerebral ischemia (i.e., ischemic stroke) may cause major brain injury, leading to a severe loss of neuronal function and consequently to a host of motor and cognitive disabilities. Its high prevalence poses a serious health burden, as stroke is among the principal causes of long-term disability and death worldwide1. Recovery of neuronal function is, in most cases, only partial. So far, treatment options are very limited, in particular due to the narrow time window for thrombolysis2,3. Determining methods to accelerate recovery from stroke remains a prime medical goal; however, this has been hampered by insufficient mechanistic insights into the recovery process. Experimental stroke researchers frequently employ rodent models of focal cerebral ischemia. Beyond the acute phase, stroke research is increasingly focused on the sub-acute and chronic phase following cerebral ischemia. Most stroke researchers apply permanent or transient occlusion of the MCA in mice or rats. In patients, occlusions of the MCA are among the most frequent causes of ischemic stroke4. Besides proximal occlusion of the MCA using the filament model, surgical occlusion of the distal MCA is probably the most frequently used model in experimental stroke research5. Occlusion of a distal (to the branching of the lenticulo-striate arteries) MCA branch typically spares the striatum and primarily affects the neocortex. Vessel occlusion can be permanent or transient. High reproducibility of lesion volume and very low mortality rates with respect to the long-term outcome are the main advantages of this model. Here, we demonstrate how to perform a chronic cranial window (CW) preparation lateral to the sagittal sinus, and afterwards how to surgically induce a distal stroke underneath the window using a craniotomy approach. This approach can be applied for sequential imaging of acute and chronic changes following ischemia via epi-illuminating, confocal laser scanning, and two-photon intravital microscopy.

Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

Surgical occlusion of a distal middle cerebral artery branch (MCAo) is a frequently used model in experimental stroke research. This manuscript describes the basic technique of permanent MCAo, combined with the insertion of a lateral cranial window, which offers the opportunity for longitudinal intravital microscopy in mice.

Focal cerebral ischemia (i.e., ischemic stroke) may cause major brain injury, leading to a severe loss of neuronal function and consequently to a host of ...

Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional multispectral diffuse reflectance imaging system. This system is constructed from a broadband white light source, a motorized filter wheel with a set of narrowband interference filters, a light guide, a collecting lens, a video zoom lens, and a monochromatic charged-coupled device (CCD) camera. An ellipsoidal cranial window is made in the skull bone of a rat under isoflurane anesthesia to capture in vivo multispectral diffuse reflectance images of the cortical surface. Regulation of the fraction of inspired oxygen using a gas mixture device enables the induction of different respiratory states such as normoxia, hyperoxia, and anoxia. A Monte Carlo simulation-based multiple regression analysis for the measured multispectral diffuse reflectance images at nine wavelengths (500, 520, 540, 560, 570, 580, 600, 730, and 760 nm) is then performed to visualize the two-dimensional maps of hemodynamics and the light scattering properties of the in vivo rat brain.

Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional multispectral diffuse reflectance imaging system.

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional ...

An In Vivo Duo-color Method for Imaging Vascular Dynamics Following Contusive Spinal Cord Injury

Spinal cord injury (SCI) causes significant vascular disruption at the site of injury. Vascular pathology occurs immediately after SCI and continues throughout the acute injury phase. In fact, endothelial cells appear to be the first to die after a contusive SCI. The early vascular events, including increased permeability of the blood-spinal cord barrier (BSCB), induce vasogenic edema and contribute to detrimental secondary injury events caused by complex injury mechanisms. Targeting the vascular disruption, therefore, could be a key strategy to reduce secondary injury cascades that contribute to histological and functional impairments after SCI. Previous studies were mostly performed on postmortem samples and were unable to capture the dynamic changes of the vascular network. In this study, we have developed an in vivo duo-color two-photon imaging method to monitor acute vascular dynamic changes following contusive SCI. This approach allows detecting blood flow, vessel diameter, and other vascular pathologies at various sites of the same rat pre- and post-injury. Overall, this method provides an excellent venue for investigating vascular dynamics.

An In Vivo Duo-color Method for Imaging Vascular Dynamics Following Contusive Spinal Cord Injury

We introduce an in vivo imaging method using two different fluorescent dyes to track dynamic spinal vascular changes following a contusive spinal cord injury in adult Sprague-Dawley rats.

Spinal cord injury (SCI) causes significant vascular disruption at the site of injury. Vascular pathology occurs immediately after SCI and continues ...

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 Imaging of Cerebrospinal Fluid Transport through the Intact Mouse Skull using Fluorescence Macroscopy

Cerebrospinal fluid (CSF) flow in rodents has largely been studied using ex vivo quantification of tracers. Techniques such as two-photon microscopy and magnetic resonance imaging (MRI) have enabled in vivo quantification of CSF flow but they are limited by reduced imaging volumes and low spatial resolution, respectively. Recent work has found that CSF enters the brain parenchyma through a network of perivascular spaces surrounding the pial and penetrating arteries of the rodent cortex. This perivascular entry of CSF is a primary driver of the glymphatic system, a pathway implicated in the clearance of toxic metabolic solutes (e.g., amyloid-&#946;). Here, we illustrate a new macroscopic imaging technique that allows real-time, mesoscopic imaging of fluorescent CSF tracers through the intact skull of live mice. This minimally-invasive method facilitates a multitude of experimental designs and enables single or repeated testing of CSF dynamics. Macroscopes have high spatial and temporal resolution and their large gantry and working distance allow for imaging while performing tasks on behavioral devices. This imaging approach has been validated using two-photon imaging and fluorescence measurements obtained from this technique strongly correlate with ex vivo fluorescence and quantification of radio-labeled tracers. In this protocol, we describe how transcranial macroscopic imaging can be used to evaluate glymphatic transport in live mice, offering an accessible alternative to more costly imaging modalities.

Transcranial optical imaging allows wide-field imaging of cerebrospinal fluid transport in the cortex of live mice through an intact skull.

Cerebrospinal fluid (CSF) flow in rodents has largely been studied using ex vivo quantification of tracers. Techniques such as two-photon microscopy and ...

Ex Vivo Pressurized Hippocampal Capillary-Parenchymal Arteriole Preparation for Functional Study

From subtle behavioral alterations to late-stage dementia, vascular cognitive impairment typically develops following cerebral ischemia. Stroke and cardiac arrest are remarkably sexually dimorphic diseases, and both induce cerebral ischemia. However, progress in understanding the vascular cognitive impairment, and then developing sex-specific treatments, has been partly limited by challenges in investigating the brain microcirculation from mouse models in functional studies. Here, we present an approach to examine the capillary-to-arteriole signaling in an ex vivo hippocampal capillary-parenchymal arteriole (HiCaPA) preparation from mouse brain. We describe how to isolate, cannulate, and pressurize the microcirculation to measure arteriolar diameter in response to capillary stimulation. We show which appropriate functional controls can be used to validate the HiCaPA preparation integrity and display typical results, including testing potassium as a neurovascular coupling agent and the effect of the recently characterized inhibitor of the Kir2 inward rectifying potassium channel family, ML133. Further, we compare the responses in preparations obtained from male and female mice. While these data reflect functional investigations, our approach can also be used in molecular biology, immunochemistry, and electrophysiology studies.

The present manuscript details how to isolate hippocampal arterioles and capillaries from the mouse brain and how to pressurize them for pressure myography, immunofluorescence, biochemistry, and molecular studies.

From subtle behavioral alterations to late-stage dementia, vascular cognitive impairment typically develops following cerebral ischemia. Stroke and ...

In Vivo Fiber-Coupled Pre-Clinical Confocal Laser-scanning Endomicroscopy (pCLE) of Hippocampal Capillaries in Awake Mice

The goal of this protocol is to describe fiber-optic-bundle-coupled pre-clinical confocal laser-scanning endomicroscopy (pCLE) in its specific application to elucidate capillary blood flow effects during seizures, driven by mural cells. In vitro and in vivo cortical imaging have shown that capillary constrictions driven by pericytes can result from functional local neural activity, as well as from drug application, in healthy animals. Here, a protocol is presented on how to use pCLE to determine the role of microvascular dynamics in neural degeneration in epilepsy, at any tissue depth (specifically in the hippocampus). We describe a head restraint technique that has been adapted to record pCLE in awake animals, to address potential side-effects of anesthetics on neural activity. Using these methods, electrophysiological and imaging recordings can be conducted over several hours in deep neural structures of the brain.

In Vivo Fiber-Coupled Pre-Clinical Confocal Laser-scanning Endomicroscopy pCLE of Hippocampal Capillaries in Awake Mice

Whereas multiphoton imaging is only effective at limited depths from the tissue&#8217;s surface, it is possible to achieve 3 &#181;m resolution imaging at any depth via pCLE. Here, we present a protocol to conduct pCLE imaging to measure microvascular dynamics in the hippocampus of ictal and wild-type mice.

The goal of this protocol is to describe fiber-optic-bundle-coupled pre-clinical confocal laser-scanning endomicroscopy (pCLE) in its specific application ...

A Needle Model for Vascular Cognitive Impairment Studies in Mice

A Mouse Model for Vascular Cognitive Impairment and Dementia Based on Needle-guided Asymmetric Bilateral Common Carotid Artery Stenosis

Vascular cognitive impairment and dementia (VCID) results from vascular brain injury. Given VCID&#39;s high incidence, which is expected to continue rising as the population ages, it is critical to establish a robust animal model for the disease. This paper presents a novel method of creating a mouse model of VCID that is based on asymmetric bilateral common carotid artery stenosis, which mimics human chronic cerebral hypoperfusion caused by carotid atherosclerosis.
Briefly, common carotid arteries (CCAs) are ligated to different gauge needles (32 G for the right CCA and 34 G for the left CCA) using 7-0 silk sutures followed by immediate needle removal. The remaining suture rings cause persistent blood flow reduction and long-term cognitive impairment associated with white matter injury, microinfarcts, and reactive gliosis, thus closely mimicking the pathogenesis of VCID. Importantly, in this needle model, the clinical representations do not revert with time, providing reliable long-term cognitive impairment. Moreover, the survival rate 24 weeks post surgery was 81.6%, which is higher compared to the other established models of VCID with a similar level of blood flow reduction.
Additional advantages include low material cost and compatibility with MRI to monitor brain injury in live animals since no metal is implanted. The main challenge in employing the needle model of VCID is the requirement for developing advanced surgical skills since mouse CCAs are less than 0.6 mm in diameter and are very fragile. High-quality visual representation of the surgery will thus help researchers to master this technique and advance our understanding of VCID, potentially leading to the development of novel therapeutic modalities to decrease the devastating cognitive decline associated with VCID.

The needle method of asymmetric bilateral common carotid artery stenosis is proposed to create a mouse model for vascular cognitive impairment and dementia. It results in longer-term outcomes compared to previously established models and is compatible with live MRI. Visual representation demonstrating the procedure provides guidance for mastering the surgery.

Vascular cognitive impairment and dementia (VCID) results from vascular brain injury. Given VCID&#39;s high incidence, which is expected to continue ...

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

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