Name: an in vivo blood-brain barrier permeability assay in mice using fluorescently labeled tracers
Uploaded: 2018-02-26T15:00:00
Description: Watch this Scientific Journal Video about An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers at JoVE.com

The authors would like to acknowledge Sphingonet consortium funded by the Leduq foundation for supporting this work. This work was also supported by the Collaborative Research Center &#34;Vascular differentiation and remodeling&#34; (CRC/ Transregio23, Project C1) and by the 7. FP, COFUND, Goethe International Postdoc Programme GO-IN, No. 291776 funding. We further acknowledge Kathleen Sommer for her technical assistance with mice handling and genotyping.

All animals were handled with utmost care minimizing pain or discomfort during the procedure. This procedure follows the animal care guidelines of our institution and has been approved by the local committee (Regierungspraesidium Darmstadt, approval number FK/1044).
A schematic of the work steps for in vivo permeability assay in mice is shown in Figure 1. The details of each step are described below.
1. Animal Handling
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Blood-brain barrier dysfunction is associated with a number of neurological disorders, including primary and secondary brain tumors or stroke. BBB breakdown is often associated with life-threatening CNS edema. The elucidation of the molecular mechanisms that trigger the opening or closure of the BBB is therefore of therapeutic significance in neurological disorders and commonly investigated by researchers. However, methods to investigate BBB permeability in vivo reported in the literature, are often associated w...

The blood-brain barrier (BBB) consists of the microvascular endothelial cells (ECs) supported by closely associated pericytes (PCs), which are ensheathed in the basal lamina, and astrocytes (ACs) that envelop the basement membrane with their end-feet1,2. ECs interact with several cell types that support and regulate the barrier function, primarily ACs and PCs, and also neurons and microglia, all of which together form the neurovascular unit (NVU). The NVU is critical for the function of the BBB, which limits the transport of blood-borne toxins and pathogens from entering the brain. This function is a result of tight-junction molecules such as claudin-5, occludin, zonula occludens-1, which are present between ECs and also due to the action of transporters such as p-glycoprotein (P-gp) that efflux molecules that enter the endothelium back into the vessel lumen1,2,3. The BBB however allows for the transport of essential molecules such as nutrients (glucose, iron, amino acids) by specific transporters expressed on the EC plasma membranes1,2,3. The EC layer is highly polarized with respect to the distribution of the various transporters between the luminal (blood-facing) and abluminal (brain-facing membranes) to allow for the specific and vectorial transport function4,5. While the BBB is protective with respect to tightly regulating the CNS milieu, it is a major challenge for CNS drug delivery in diseases such as Parkinson&#39;s with a functional BBB. Even in neurological diseases with BBB dysfunction, it cannot be assumed that the brain drug delivery is increased particularly as the barrier dysfunction could include damage to the specific transporter targets for example as in Alzheimer&#39;s disease (AD). In AD, several amyloid beta transporters such as LRP1, RAGE, P-gp are known to be dysregulated and hence targeting these transporters might be futile6,7,8. The BBB is impaired in several neurological diseases such as stroke, AD, meningitis, multiple-sclerosis, and in brain tumors9,10,11. Restoring the barrier function is a crucial part of the therapeutic strategy and thus its assessment is critical.
In this work, we have described an objective and quantitative protocol for permeability assay in rodents that we successfully applied to several mouse lines both transgenic and experimental disease models10,12,13,14. The method is based on a simple intraperitoneal injection of fluorescent tracers followed by perfusion of the mice to remove the tracers from the vascular compartment. Brain and other organs are collected post perfusion and permeability assessed by an objective and absolute permeability index based on fluorescence measurements of tissue homogenates in a plate reader. All raw fluorescence values are corrected for the background using tissue homogenates or serum from sham animals that do not receive any tracer. Ample normalizations are included for serum volume, serum fluorescence, and the weight of the tissues, thus yielding permeability index that is absolute and comparable between experiments and tissue types. For ease of comparison between groups, the absolute permeability index values can be readily transformed to ratios as we had performed previously12. Concurrently, stored hemi-brains and kidney could be utilized for tracer visualization by fluorescence microscopy10. The classic fluorescence microscopy could be valuable in obtaining regional difference in permeability albeit cumbersome due to subjective selection of tissue sections and images for a semi-quantitative analysis. The detailed steps are presented in the protocol and notes are added where appropriate. This provides the necessary information for successfully performing the in vivo permeability assay in mice that can be scaled to other small animals. The assay can be applied to many kinds of tracers allowing for the charge and the size based permeability assessment by a combination of tracers with distinct fluorescence spectra.

<table>
	<tbody>
		<tr>
			<td>Tetramethyl Rhodamine (TMR) dextran 3kD</td>
			<td>Thermosfisher</td>
			<td>D3308</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein isothiocyanate (FITC) dextran 3kD</td>
			<td>Thermosfisher</td>
			<td>D3306</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine (Ketavet)</td>
			<td>Zoetis</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine (Rompun)</td>
			<td>Bayer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.9% Saline</td>
			<td>Fresenius Kabi Deutschland GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1X PBS</td>
			<td>Gibco</td>
			<td>10010-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-tek O.C.T compound</td>
			<td>Sakura Finetek</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>37% Formaldhehyde solution</td>
			<td>Sigma</td>
			<td>252549-1L</td>
			<td>prepare a 4% solution</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin, fraction V</td>
			<td>Roth</td>
			<td>8076.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>rat anti CD31 antibody, clone MEC 13.3</td>
			<td>BD Pharmingen</td>
			<td>553370</td>
			<td></td>
		</tr>
		<tr>
			<td>goat anti rat alexa 568</td>
			<td>Molecular Probes</td>
			<td>A-11077</td>
			<td></td>
		</tr>
		<tr>
			<td>goat anti rat alexa 488</td>
			<td>Molecular Probes</td>
			<td>A-11006</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Molecular Probes</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>Aqua polymount</td>
			<td>Polyscience Inc</td>
			<td>18606</td>
			<td></td>
		</tr>
		<tr>
			<td>21-gauge butterfly needle</td>
			<td>BD</td>
			<td>387455</td>
			<td></td>
		</tr>
		<tr>
			<td>serum collection tube</td>
			<td>Sarstedt</td>
			<td>41.1500.005</td>
			<td></td>
		</tr>
		<tr>
			<td>2mL eppendorf tubes</td>
			<td>Sarstedt</td>
			<td>72.695.500</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimtech precision wipes tissue wipers</td>
			<td>Kimberley-Clark Professional</td>
			<td>05511</td>
			<td></td>
		</tr>
		<tr>
			<td>384-well black plate</td>
			<td>Greiner</td>
			<td>781086</td>
			<td></td>
		</tr>
		<tr>
			<td>slides superfrost plus</td>
			<td>Thermoscientific</td>
			<td>J1800AMNZ</td>
			<td></td>
		</tr>
		<tr>
			<td>PTFE pestle</td>
			<td>Wheaton</td>
			<td>358029</td>
			<td></td>
		</tr>
		<tr>
			<td>electric overhead stirrer</td>
			<td>VWR</td>
			<td>VWR VOS 14</td>
			<td></td>
		</tr>
		<tr>
			<td>plate reader</td>
			<td>Tecan</td>
			<td>Infinite M200</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Microm GmbH</td>
			<td>HM 550</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon C1 Spectral Imaging confocal Laser Scanning Microscope System</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>peristaltic perfusion system</td>
			<td>BVK Ismatec</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>microcentrifuge</td>
			<td>eppendorf</td>
			<td>5415R</td>
			<td></td>
		</tr>
	</tbody>
</table>

an in vivo blood-brain barrier permeability assay in mice using fluorescently labeled tracers

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains brain homeostasis. The principal sites of the barrier are endothelial cells of the brain capillaries whose barrier function results from tight intercellular junctions and efflux transporters expressed on the plasma membrane. This function is regulated by pericytes and astrocytes that together form the neurovascular unit (NVU). Several neurological diseases such as stroke, Alzheimer&#39;s disease (AD), brain tumors are associated with an impaired BBB function. Assessment of the BBB permeability is therefore crucial in evaluating the severity of the neurological disease and the success of the treatment strategies employed.
We present here a simple yet robust permeability assay that have been successfully applied to several mouse models both, genetic and experimental. The method is highly quantitative and objective in comparison to the tracer fluorescence analysis by microscopy that is commonly applied. In this method, mice are injected intraperitoneally with a mix of aqueous inert fluorescent tracers followed by anesthetizing the mice. Cardiac perfusion of the animals is performed prior to harvesting brain, kidneys or other organs. Organs are homogenized and centrifuged followed by fluorescence measurement from the supernatant. Blood drawn from the cardiac puncture just before perfusion serves for normalization purpose to the vascular compartment. The tissue fluorescence is normalized to the wet weight and serum fluorescence to obtain a quantitative tracer permeability index. For additional confirmation, the contralateral hemi-brain preserved for immunohistochemistry can be utilized for tracer fluorescence visualization purposes.

We have recently shown that angiopoietin-2 (Ang-2) gain-of-function (GOF) mice have higher brain vascular permeability than control mice in healthy conditions10. In stroke-induced mice, it was also shows that the GOF mice had bigger infarct sizes and greater permeability than the control littermates. These results show a critical role of Ang-2 in permeability at the BBB. The protocol therefore utilized the GOF mice and compared them to control littermates to descri...

Watch this Scientific Journal Video about An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers at JoVE.com

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Here we present a mouse brain vascular permeability assay using intraperitoneal injection of fluorescent tracers followed by perfusion that is applicable to animal models of blood-brain barrier dysfunction. One hemi-brain is used for assessing permeability quantitatively and the other for tracer visualization/immunostaining. The procedure takes 5 - 6 h for 10 mice.

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains ...

The overall goal of this procedure is to assess the permeability of brain vasculature in mice using fluorescently-labeled tracers to assess blood-brain barrier dysfunction in animal models. This method can help answer key questions in the blood-brain barrier field pertaining to neuro-vascular permeability in diseases and its recovery upon therapy. The main advantage of this method is that it is simple and quantitative.It gives the possibility for concurrent assessment of permeability by fluorometry, which is quantitative, and also fluorescence microscopy for regional defenses. The implications of this technique extend towards diagnosis and therapy of several neurological disorders, including brain tumors, stroke, and Alzheimer's disease because these diseases are associated with life-threatening brain edema and improvement of BBD function as a key therapeutic target. Demonstrating part of the procedure will be Miss Jadranka Macas, a scientific technology officer at the institute.Intraperitoneally inject mice with 100 microliters of two-millimolar tracer solution. Choose lysine-fixable tracers, such as dextrans labeled with FITC and TMR with distinct excitation emission spectra for minimal interference between the dyes. If combining tracers, inject 200 microliters of combined tracer solution intraperitoneally.Include vehicle-only injections to serve as sham controls for autofluorescence background subtraction. After each injection, allow five minutes before deeply anesthetizing the mice according to the approved institutional protocols. Prepare the animals for cardiac perfusion 10 minutes after anesthetic administration.Check the absence of paw twitch response to ensure that the mouse has reached a surgical plane of anesthesia. Lay the mouse to be perfused on its back and apply 80%ethanol on the skin of the abdominal area. After using small scissors to create a two-centimeter incision in the abdominal wall, separate the liver from the diaphragm, and then slowly cut through the diaphragm, exposing the plural cavity.Cut the ribcage bilaterally and fix the cut sternum to expose the left ventricle. Insert a 21-gauge butterfly needle connected to a peristaltic perfusion system in the posterior of the left ventricle. Puncture the right atrium and then use a one-milliliter pipette tip with the end cut off to quickly collect the 200 to 300 microliters of released blood and transfer to a serum collection tube.Store the collected blood on ice. As soon as the blood is collected, switch on the perfusion system and perfuse the animal for three minutes with room temperature calcium and magnesium ion-free PBS. Assess perfusion quality by noting the color of the liver and kidneys, which appear pale after perfusion.After harvesting the brain, verify that there is no blood visible in vessels of the meninges. The sample should be excluded from the permeability analysis if the perfusion quality is poor. Use a scalpel to separate the brain into two hemi-brains.Then dissect the cerebellum and olfactory lobe from one hemi-brain, and transfer the hemi-cerebrum to a two-milliliter tube. Place one harvested kidney to another two-milliliter tube. Immediately place the samples on dry ice.Natively embed the remaining kidney and hemi-brain with cerebellum and olfactory lobes intact in TissueTech optimal cutting temperature compound, and put it on dry ice. Lastly, after centrifuging the blood samples at 10, 000 G for 10 minutes at four degrees Celsius, transfer the serum supernatants to 1.5-milliliter tubes and place them in the dry ice container. Transfer the samples to the minus 80 degrees Celsius freezer as homogenization efficiency increases after freeze-thawing.Place the hemi-cerebrum and kidney samples on ice to thaw, and then weigh the tubes containing the organs. From these weights, subtract the mean value of approximately 20 empty tubes to get the tissue weight. Add 300 microliters of cold PBS to the tube containing kidney and 200 microliters to the tubes containing hemi-cerebrum.Homogenize each sample in the original microfuge tube with a PTFE pestle attached to an electric overhead stirrer, using about 15 strokes. Store the homogenized samples on ice, protected from light. Rinse the pestle with PBS in between samples and wipe dry before proceeding to the next sample.When all samples have been homogenized, centrifuge for 20 minutes at 15, 000 G and four degrees Celsius. Following centrifugation, transfer the supernatants to new 1.5-milliliter tubes on ice before proceeding to fluorescence measurement and quantification. Transfer the supernatant to a 384-well plate and insert the plate into fluorescence reader.Make sure the right excitation and emission wavelengths are included for the tracer and set the gain to Optimal. Start the measurement, and export the data as a spreadsheet to perform the calculations as described in the protocol. On the day of staining, thaw 10-micron cryostat sections mounted on slides at 37 degrees Celsius for 10 minutes.Then, fix the sections with 4%paraformaldehyde for 10 minutes at room temperature, followed by quick-washing in PBS. Permeabilize and block non-specific binding in the sections by incubating in sterile PBS containing 1%PSA and 0.5%Triton X-100 for one hour at room temperature. After blocking, incubate the slides with a one-to-100 dilution of CD31 primary antibody for 1.5 hours at room temperature.After washing with PBS three times for five minutes each, perform secondary antibody incubation for one hour at room temperature with species-specific fluorescently labeled antibodies. Mount the stained sections with Aqua-Poly/Mount and leave overnight for polymerization at room temperature in the dark. Finally, acquire images using a spectral imaging confocal laser scanning microscope system.In order to establish the tracer circulation time for the permeability assay, a long circulation time of two hours is compared to a short circulation time of 15 minutes post tracer injection. The longer circulation time of two hours leads to low amounts of tracers accumulation in kidney compared to a short 15-minute circulation time potentially due to a high clearance FITC dextran shown in green from the vascular compartment. This effect is even more dramatic in the brain due to the tight blood-brain barrier.CD31 staining seen in the red channel confirmed the presence of vessels in the kidney at both time points examined and in the brain. Once mastered, this technique can be performed within five to six hours for 10 mice with two scientists working in tandem. While attempting this technique, it is important to remember to use tracers of right molecular weight, chart and fluorescence characteristics in order to answer the questions regarding the blood-brain barrier permeability.Following this procedure, other methods like immuno-histochemistry for BBD and disease fill on factors can be performed to answer additional questions, like severity and localization of disease site.

Research

JoVE Journal

Neuroscience

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

An Assay for Permeability of the Zebrafish Embryonic Neuroepithelium

The brain ventricular system is conserved among vertebrates and is composed of a series of interconnected cavities called brain ventricles, which form ...

Assessment of Blood-brain Barrier Permeability by Intravenous Infusion of FITC-labeled Albumin in a Mouse Model of Neurodegenerative Disease

Disruption of blood-brain barrier (BBB) integrity is a common feature for different neurological and neurodegenerative diseases. Although the interplay ...

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier ...

A Benchtop Approach to the Location Specific Blood Brain Barrier Opening using Focused Ultrasound in a Rat Model

Stereotaxic surgery is the gold standard for localized drug and gene delivery to the rodent brain. This technique has many advantages over systemic ...

Electrocorticographic Recording of Cerebral Cortex Areas Manipulated Using an Adeno-Associated Virus Targeting Cofilin in Mice

The use of electrocorticographic (ECoG) recordings in rodents is relevant to sleep research and to the study of a wide range of neurological conditions. ...

Lineage Tracing of Inducible Fluorescently-Labeled Stem Cells in the Adult Mouse Brain

A telomerase reverse transcriptase (Tert) lineage-tracing mouse line was developed to investigate the behavior and fate of adult tissue stem cells, by ...

Transendothelial Resistance Measurement to Assess Cell Barrier Integrity

Barrier Functional Integrity Recording on bEnd.3 Vascular Endothelial Cells via Transendothelial Electrical Resistance Detection

Author Spotlight: Applications of TEER Detection to Assess Cell Barrier Integrity 

The blood-brain barrier (BBB) is a dynamic physiological structure composed of microvascular endothelial cells, astrocytes, and pericytes. By coordinating ...

A 3D Blood-Brain Barrier Model Derived from Human Pluripotent Stem-Cells

Reconstruction of the Blood-Brain Barrier In Vitro to Model and Therapeutically Target Neurological Disease

Author Spotlight: A Personalized Approach Towards Investigating Alzheimer's Disease Using an In Vitro Blood-Brain Barrier Model 

The blood-brain barrier (BBB) is a key physiological component of the central nervous system (CNS), maintaining nutrients, clearing waste, and protecting ...