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 border="0" cellpadding="0" cellspacing="0" width="627">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">methoxy-X04</td>
			<td style="width:125px;">tocris</td>
			<td align="right" style="width:119px;">4920</td>
			<td style="width:167px;">use 10 mg/Kg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FITC-Dextran 70Kda</td>
			<td style="width:125px;">sigma</td>
			<td align="right" style="width:119px;">46945</td>
			<td>use 100 mg/Kg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">gelfoam/Bloxang</td>
			<td style="width:125px;">Bausch and Lomb</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">micorsurgical blade</td>
			<td style="width:125px;">surgistar</td>
			<td align="right" style="width:119px;">6900</td>
			<td>must be sharp and not dented</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">povidone-iodine</td>
			<td style="width:125px;">betadine</td>
			<td style="width:119px;"></td>
			<td>antisceptic solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">binocular stereomicroscope</td>
			<td style="width:125px;">olympus</td>
			<td style="width:119px;">SX10</td>
			<td>optimal image contrast is crucial for this procedure</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2-photon microscope</td>
			<td style="width:125px;">zeiss</td>
			<td style="width:119px;"></td>
			<td>Zeiss LSM 710mp</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">fine scissors-toughcut</td>
			<td style="width:125px;">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 ...

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7.0K Views.  Institute of Functional Genomics. 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's disease.

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

The Measurement and Treatment of Suppression in Amblyopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current estimates put the prevalence of amblyopia at approximately 1-3%1-3, the majority of cases being monocular2. Amblyopia is most frequently caused by ocular misalignment (strabismus), blur induced by unequal refractive error (anisometropia), and in some cases by form deprivation.
Although amblyopia is initially caused by abnormal visual input in infancy, once established, the visual deficit often remains when normal visual input has been restored using surgery and/or refractive correction. This is because amblyopia is the result of abnormal visual cortex development rather than a problem with the amblyopic eye itself4,5 . Amblyopia is characterized by both monocular and binocular deficits6,7 which include impaired visual acuity and poor or absent stereopsis respectively. The visual dysfunction in amblyopia is often associated with a strong suppression of the inputs from the amblyopic eye under binocular viewing conditions8. Recent work has indicated that suppression may play a central role in both the monocular and binocular deficits associated with amblyopia9,10 .
Current clinical tests for suppression tend to verify the presence or absence of suppression rather than giving a quantitative measurement of the degree of suppression. Here we describe a technique for measuring amblyopic suppression with a compact, portable device11,12 . The device consists of a laptop computer connected to a pair of virtual reality goggles. The novelty of the technique lies in the way we present visual stimuli to measure suppression. Stimuli are shown to the amblyopic eye at high contrast while the contrast of the stimuli shown to the non-amblyopic eye are varied. Patients perform a simple signal/noise task that allows for a precise measurement of the strength of excitatory binocular interactions. The contrast offset at which neither eye has a performance advantage is a measure of the "balance point" and is a direct measure of suppression. This technique has been validated psychophysically both in control13,14 and patient6,9,11 populations.
In addition to measuring suppression this technique also forms the basis of a novel form of treatment to decrease suppression over time and improve binocular and often monocular function in adult patients with amblyopia12,15,16 . This new treatment approach can be deployed either on the goggle system described above or on a specially modified iPod touch device15.

Amblyopia is a developmental disorder of the visual cortex that is often accompanied by strong suppression of one eye. We present a new technique for measuring and treating interocular suppression in patients with amblyopia that can be deployed using virtual reality goggles or a portable iPod Touch device.

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current ...

The Use of Pharmacological-challenge fMRI in Pre-clinical Research: Application to the 5-HT System

Pharmacological MRI (phMRI) is a new and promising method to study the effects of substances on brain function that can ultimately be used to unravel underlying neurobiological mechanisms behind drug action and neurotransmitter-related disorders, such as depression and ADHD. Like most of the imaging methods (PET, SPECT, CT) it represents a progress in the investigation of brain disorders and the related function of neurotransmitter pathways in a non-invasive way with respect of the overall neuronal connectivity. Moreover it also provides the ideal tool for translation to clinical investigations. MRI, while still behind in molecular imaging strategies compared to PET and SPECT, has the great advantage to have a high spatial resolution and no need for the injection of a contrast-agent or radio-labeled molecules, thereby avoiding the repetitive exposure to ionizing radiations. Functional MRI (fMRI) is extensively used in research and clinical setting, where it is generally combined with a psycho-motor task. phMRI is an adaptation of fMRI enabling the investigation of a specific neurotransmitter system, such as serotonin (5-HT), under physiological or pathological conditions following activation via administration of a specific challenging drug.
The aim of the method described here is to assess brain 5-HT function in free-breathing animals. By challenging the 5-HT system while simultaneously acquiring functional MR images over time, the response of the brain to this challenge can be visualized. Several studies in animals have already demonstrated that drug-induced increases in extracellular levels of e.g. 5-HT (releasing agents, selective re-uptake blockers, etc) evoke region-specific changes in blood oxygenation level dependent (BOLD) MRI signals (signal due to a change of the oxygenated/deoxygenated hemoglobin levels occurring during brain activation through an increase of the blood supply to supply the oxygen and glucose to the demanding neurons) providing an index of neurotransmitter function. It has also been shown that these effects can be reversed by treatments that decrease 5-HT availability16,13,18,7. In adult rats, BOLD signal changes following acute SSRI administration have been described in several 5-HT related brain regions, i.e. cortical areas, hippocampus, hypothalamus and thalamus9,16,15. Stimulation of the 5-HT system and its response to this challenge can be thus used as a measure of its function in both animals and humans2,11.

The goal of this technique is to assess serotonin (5-HT) neurotransmitter function in the live and free-breathing animal with pharmacological magnetic resonance imaging (phMRI) and an intravenous challenge with a selective serotonin reuptake inhibitor (SSRI), fluoxetine.

Pharmacological MRI (phMRI) is a new and promising method to study the effects of substances on brain function that can ultimately be used to unravel ...

Intranasal Administration of CNS Therapeutics to Awake Mice

Intranasal administration is a method of delivering therapeutic agents to the central nervous system (CNS). It is non-invasive and allows large molecules that do not cross the blood-brain barrier access to the CNS. Drugs are directly targeted to the CNS with intranasal delivery, reducing systemic exposure and thus unwanted systemic side effects1. Delivery from the nose to the CNS occurs within minutes along both the olfactory and trigeminal neural pathways via an extracellular route and does not require drug to bind to any receptor or axonal transport2. Intranasal delivery is a widely publicized method and is currently being used in human clinical trials3.
Intranasal delivery of drugs in animal models allows for initial evaluation of pharmacokinetic distribution and efficacy. With mice, it is possible to administer drugs to awake (non-anesthetized) animals on a regular basis using a specialized intranasal grip. Awake delivery is beneficial because it allows for long-term chronic dosing without anesthesia, it takes less time than with anesthesia, and can be learned and done by many people so that teams of technicians can dose large numbers of mice in short periods. Efficacy of therapeutics administered intranasally in this way to mice has been demonstrated in a number of studies including insulin in diabetic mouse models 4-6 and deferoxamine in Alzheimer's mouse models. 7,8
The intranasal grip for mice can be learned, but is not easy and requires practice, skill, and a precise grip to effectively deliver drug to the brain and avoid drainage to the lung and stomach. Mice are restrained by hand using a modified scruff in the non-dominant hand with the neck held parallel to the floor, while drug is delivered with a pipettor using the dominant hand. It usually takes 3-4 weeks of acclimating to handling before mice can be held with this grip without a stress response. We have prepared this JoVE video to make this intranasal delivery technique more accessible.

A method to intranasally administer drugs to awake mice for the purpose of targeting the brain is described. This method allows for repeat dosing over long periods using intranasal administration of drug without anesthesia, and nose-to-brain delivery with minimal systemic exposure.

Intranasal administration is a method of delivering  therapeutic agents to the central nervous system (CNS). It is  non-invasive and allows large ...

In vivo Macrophage Imaging Using MR Targeted Contrast Agent for Longitudinal Evaluation of Septic Arthritis

Macrophages are key-cells in the initiation, the development and the regulation of the inflammatory response to bacterial infection. Macrophages are intensively and increasingly recruited in septic joints from the early phases of infection and the infiltration is supposed to regress once efficient removal of the pathogens is obtained. The ability to identify in vivo macrophage activity in an infected joint can therefore provide two main applications: early detection of acute synovitis and monitoring of therapy.
In vivo noninvasive detection of macrophages can be performed with magnetic resonance imaging using iron nanoparticles such as ultrasmall superparamagnetic iron oxide (USPIO). After intravascular or intraarticular administration, USPIO are specifically phagocytized by activated macrophages, and, due to their magnetic properties, induce signal changes in tissues presenting macrophage infiltration. A quantitative evaluation of the infiltrate is feasible, as the area with signal loss (number of dark pixels) observed on gradient echo MR images after particles injection is correlated with the amount of iron within the tissue and therefore reflects the number of USPIO-loaded cells.
We present here a protocol to perform macrophage imaging using USPIO-enhanced MR imaging in an animal model of septic arthritis, allowing an initial and longitudinal in vivo noninvasive evaluation of macrophages infiltration and an assessment of therapy action.

In vivo Macrophage Imaging Using MR Targeted Contrast Agent for Longitudinal Evaluation of Septic Arthritis

We demonstrate how to perform macrophage MR imaging using ultrasmall superparamagnetic contrast agent (USPIO) in septic arthritis, allowing an initial and longitudinal in vivo non-invasive evaluation of macrophages infiltration and an assessment of therapy efficacy.

Macrophages are key-cells in the initiation, the development and the regulation of the inflammatory response to bacterial infection. Macrophages are ...

Diffusion Tensor Magnetic Resonance Imaging in the Analysis of Neurodegenerative Diseases

Diffusion tensor imaging (DTI) techniques provide information on the microstructural processes of the cerebral white matter (WM) in vivo. The present applications are designed to investigate differences of WM involvement patterns in different brain diseases, especially neurodegenerative disorders, by use of different DTI analyses in comparison with matched controls. 	
DTI data analysis is performed in a variate fashion, i.e. voxelwise comparison of regional diffusion direction-based metrics such as fractional anisotropy (FA), together with fiber tracking (FT) accompanied by tractwise fractional anisotropy statistics (TFAS) at the group level in order to identify differences in FA along WM structures, aiming at the definition of regional patterns of WM alterations at the group level. Transformation into a stereotaxic standard space is a prerequisite for group studies and requires thorough data processing to preserve directional inter-dependencies. The present applications show optimized technical approaches for this preservation of quantitative and directional information during spatial normalization in data analyses at the group level. On this basis, FT techniques can be applied to group averaged data in order to quantify metrics information as defined by FT. Additionally, application of DTI methods, i.e. differences in FA-maps after stereotaxic alignment, in a longitudinal analysis at an individual subject basis reveal information about the progression of neurological disorders. Further quality improvement of DTI based results can be obtained during preprocessing by application of a controlled elimination of gradient directions with high noise levels.	
In summary, DTI is used to define a distinct WM pathoanatomy of different brain diseases by the combination of whole brain-based and tract-based DTI analysis.

Diffusion tensor imaging (DTI) basically serves as an MRI-based tool to identify in vivo the microstructure of the brain and pathological processes due to neurological disorders within the cerebral white matter. DTI-based analyses allow for application to brain diseases both at the group level and in single subject data.

Diffusion tensor imaging (DTI) techniques provide information on the microstructural processes of the cerebral white matter (WM) in vivo. The present ...

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

Intravital imaging has emerged as an indispensable tool in biological research. In the process, many imaging techniques have been developed to study different biological processes in animals non-invasively. However, a major technical limitation in existing intravital imaging modalities is the inability to combine non-invasive, longitudinal imaging with single-cell resolution capabilities. We show here how transplantation into the anterior chamber of the eye circumvents such significant limitation offering a versatile experimental platform that enables non-invasive, longitudinal imaging with cellular resolution in vivo. We demonstrate the transplantation procedure in the mouse and provide representative results using a model with clinical relevance, namely pancreatic islet transplantation. In addition to enabling direct visualization in a variety of tissues transplanted into the anterior chamber of the eye, this approach provides a platform to screen drugs by performing long-term follow up and monitoring in target tissues. Because of its versatility, tissue/cell transplantation into the anterior chamber of the eye not only benefits transplantation therapies, it extends to other in vivo applications to study physiological and pathophysiological processes such as signal transduction and cancer or autoimmune disease development.

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

A new approach combining intraocular transplantation and confocal microscopy enables longitudinal, non-invasive real-time imaging with single-cell resolution within grafted tissues in vivo. We demonstrate how to transplant pancreatic islets into the anterior chamber of the mouse eye.

Intravital imaging has emerged as an indispensable tool in biological research.  In the process, many imaging techniques have been developed to study ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

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 ...

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest improved outcome measures in both experimental and clinical studies. Although the BMMSC are implanted into the tendon lesion in large numbers (usually 10 - 20 million cells), only a relatively small number survive (&#60;10%) although these can persist for up to 5 months after implantation. This appears to be a common observation in other species where BMMSC have been implanted into other tissues and it is important to understand when this loss occurs, how many survive the initial implantation process and whether the cells are cleared into other organs. Tracking the fate of the cells can be achieved by radiolabeling the BMMSC prior to implantation which allows non-invasive in vivo imaging of cell location and quantification of cell numbers.
This protocol describes a cell labeling procedure that uses Technetium-99m (Tc-99m), and tracking of these cells following implantation into injured flexor tendons in horses. Tc-99m is a short-lived (t1/2 of 6.01 hr) isotope that emits gamma rays and can be internalized by cells in the presence of the lipophilic compound hexamethylpropyleneamine oxime (HMPAO). These properties make it ideal for use in nuclear medicine clinics for the diagnosis of many different diseases. The fate of the labeled cells can be followed in the short term (up to 36 hr) by gamma scintigraphy to quantify both the number of cells retained in the lesion and distribution of the cells into lungs, thyroid and other organs. This technique is adapted from the labeling of blood leukocytes and could be utilized to image implanted BMMSC in other organs.

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

This protocol describes the radiolabeling of equine mesenchymal stem cells and their implantation into tendon injuries in the horse in order to determine cell survival and tissue distribution using gamma scintigraphy.

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest ...

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to image cellular events in an intact animal. Additionally, the ability to induce physiological disease states such as stroke in vivo increases its utility. The technique described herein allows for physiological assessment of cellular responses within the CNS following a stroke and can be adapted for other pathological conditions being studied. The technique presented uses laser excitation of the photosensitive dye Rose Bengal in vivo to induce a focal ischemic event in a single blood vessel. 
The video protocol demonstrates the preparation of a thin-skulled cranial window over the somatosensory cortex in a mouse for the induction of a Rose Bengal photothrombotic event keeping injury to the underlying dura matter and brain at a minimum. Surgical preparation is initially performed under a dissecting microscope with a custom-made surgical/imaging platform, which is then transferred to a confocal microscope equipped with an inverted objective adaptor. Representative images acquired utilizing this protocol are presented as well as time-lapse sequences of stroke induction. This technique is powerful in that the same area can be imaged repeatedly on subsequent days facilitating longitudinal in vivo studies of pathological processes following stroke.

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke

Here, we describe a semi-invasive optical microscopy approach for the induction of a Rose Bengal photothrombotic clot in the somatosensory cortex of a mouse in vivo. The technical aspects of the imaging procedure are described from induction of a photothrombotic event to application and data collection.

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to ...