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
<ol>
	<li>Preparation and administration of tracers and anesthetics
	<ol>
		<li>Prepare labeled tubes and molds for tissue embedding at least a day before the permeability assay. Maintain sterile conditions throughout the protocol by working under clean fume hood and using tools cleaned with 80 % ethanol. Use male or female wild-type (WT) or angiopoietin-2 (Ang-2) gain-of-function (GOF) CD1 mice in the mature adult age group of 3-6 months for the current protocol. Perform genotyping of the mice as described previously 10.&#160; 
		Note: 80% alcohol will not result in sterile instruments but will minimize contamination of the samples prepared.</li>
		<li>Dilute all the tracers in sterile PBS into 2 mM stocks and store the aliquots protected from light at -20 &#176;C.</li>
		<li>Choose tracers such as (Tetramethyl Rhodamine) TMR and (fluorescein isothiocyanate) FITC with distinct excitation/emission spectra and that are lysine fixable resulting in minimal interference between the dyes both by fluorescence microscopy (using the TMR or FITC filter respectively) and by fluorometry in a plate reader for the permeability assay. 
		NOTE: TMR dextran 3 kD and FITC dextran 3 kD used in the study are both lysine fixable and hence could be also used for immunohistochemical analysis post-fixation with aldehydes in order to investigate regional differences.</li>
		<li>Handle all animals with utmost care following the institutional care guidelines. Inject intraperitoneally each mouse with 100 &#181;L tracer solution that can be increased up to 200 &#181;L when an additional tracer is combined using 100 &#181;L of each tracer in a 1:1 mix10,13. Inject at least one animal with PBS alone instead of the tracers to serve as sham control for autofluorescence background subtraction.</li>
		<li>5 min after tracer injection, anesthetize the animal with an i.p injection of Ketamine and Xylazine (100 mg and 5-10 mg in 0.9 % Saline per kg body weight respectively, 150 &#181;L of the cocktail per 25 g mouse). 
		NOTE: Vet ointment was not applied on eyes during the procedure as the period between animal anesthesia and perfusion/sacrifice was brief (10 minutes) where any drying of the eyes was not observed.</li>
	</ol>
	</li>
	<li>Blood collection and cardiac perfusion
	<ol>
		<li>Prepare the animals for cardiac perfusion 10 min after anesthetic administration. Check the absence of paw twitch response in order to ensure that the animal reached a surgical plane of anesthesia.</li>
		<li>Lay the animals on their back and apply 80 % ethanol on the skin of abdominal area. Using small scissors, open up the abdominal wall with a small incision (2 cm) just beneath the rib cage. Separate liver from the diaphragm and then slowly cut through the diaphragm exposing the pleural cavity 15.</li>
		<li>Cut the rib cage bilaterally and fix the cut sternum in order to expose the left ventricle. Insert a 21-gauge butterfly needle connected to a peristaltic perfusion system in the posterior of the left ventricle.</li>
		<li>Puncture the right atrium and quickly (within 10 s) collect 200-300 &#181;L of blood released into the chest cavity using 1 mL pipette tips (with end cut-off) in serum collection tubes and store it on ice.&#160; 
		Note: This perfusion procedure is non-survival.</li>
		<li>As soon as the blood is collected, switch on the perfusion system (10-12 rpm, 5 mL/min) and perfuse the animal for 3 min with warm (RT) 1x PBS (free of Ca2+/Mg2+ ions). 
		NOTE: PBS containing Ca2+/Mg2+ ions can be utilized for better heart activity during the perfusion. The total amount of PBS used for perfusion is in the range of 15-20 mL.</li>
		<li>Assess perfusion quality by noting the color of liver, kidneys, which appear white/pale after perfusion. 
		NOTE: Kidney or liver perfusion do not necessarily indicate brain perfusion as the arteries (carotid/vertebral) reaching the brain from aorta might get ruptured during the preparation in step 3 particularly during the atrial puncture.</li>
	</ol>
	</li>
</ol>
2. Tissue Processing
<ol>
	<li>Organ collection and storage
	<ol>
		<li>At the end of the perfusion, confirm death of&#160;the animal by cervical dislocation and harvest brain and kidneys. Verify brain perfusion by the color of the brains (no visible blood in vessels of the meninges), so as to exclude the animals from permeability analysis when perfusion quality is not good. Separate the brain into 2 hemibrains using a scalpel (Figure 1).</li>
		<li>Dissect with a scalpel one hemibrain free of olfactory lobes and cerebellum and transfer the hemicerebrum to a 2-mL tube. Store hemi-cerebellum in an additional tube if required for cerebellar permeability assessment.</li>
		<li>Transfer a single kidney to another 2-mL tube. Store the samples immediately on dry ice.</li>
		<li>Embed natively the remaining kidney and hemi-brain (comprising the cerebellum and olfactory lobes) in tissue-tek optimal cutting temperature (O.C.T) compound on dry ice.</li>
		<li>In the end, centrifuge the blood samples stored on ice in serum collection tubes at 10, 000 g, 10 min at 4 &#176;C. Transfer serum supernatants to 1.5 mL tubes and place them in the dry ice container.</li>
		<li>Transfer all the samples collected on dry-ice to -80 &#176;C freezer until further processing. 
		NOTE: It is important to freeze the samples before proceeding to the homogenization steps as homogenization efficiency increases after as freeze thawing.</li>
	</ol>
	</li>
	<li>Homogenization and centrifugation
	<ol>
		<li>Thaw on ice the hemi-cerebrum and kidney samples frozen down at -80 &#176;C and weigh the tubes containing the organs. From these weights, subtract the mean value of several empty tubes (about 20) to get the tissue weight. Add 300 &#181;L and 200 &#181;L of cold 1X PBS to the tubes containing kidney and hemi-cerebrum, respectively. 
		NOTE: For lower amounts of tissue (like hemi-cerebellum, below 100 mg) weighing the individual tubes is recommended.</li>
		<li>Homogenize each sample in the original eppendorf tube with a polytetrafluoroethylene (PTFE) pestle attached to an electric overhead stirrer (1, 000 rpm) by performing about 15 strokes (1 stroke = 1 up and 1 down). Rinse the pestle with PBS in between samples and wipe dry before proceeding to the next sample. 
		NOTE: The use of detergents during homogenization was avoided due to potential interference with fluorometry. However, intracellular tracer is also detected in this protocol as the tissue is thoroughly homogenized, which is more efficient after one round of freeze thawing.</li>
		<li>Store the homogenized samples on ice protected from light and centrifuge all together in the end at 15,000 g, 20 min, 4 &#176;C in a tabletop centrifuge. Transfer supernatants to a new 1.5 mL tubes on ice for immediate fluorometry or at -80 &#176;C to be analyzed later.</li>
	</ol>
	</li>
	<li>Fluorescence measurement and quantification
	<ol>
		<li>If frozen at the end of the previous step, thaw on ice the tissue supernatant and serum samples stored at -80 &#730;C protecting them from light with the foil.</li>
		<li>Pipette 50 &#181;L of diluted serum (30 &#181;L 1x PBS + 20 &#181;L serum) or tissue supernatants (as is) into a 384-well black plate. Include at least one sample from sham animal for serum and tissue supernatants to ensure proper background subtraction, as this tissue homogenate background is normally higher than that of PBS used as the diluent. 
		NOTE: An average of 3 sham animals can be used for autofluorescence although a very little variability in sham autofluorescence values (data not shown) was observed. The amount of serum used can be reduced by diluting it with PBS, which can also increase the signal to noise ratio for samples with low fluorescence such as the brain samples. Upto 10 &#181;L serum was tested diluting it with 40 &#181;L PBS. Make sure that no bubbles are present in the wells.</li>
		<li>Insert the 384-well black plate into the plate reader and open a new script selecting fluorescence measurement.</li>
		<li>Set the gain to optimal and use excitation/emission (nm) values of 550/580 or 490/520 for TMR or FITC dyee respectively and start the measurement to obtain the raw fluorescence units (RFUs).</li>
		<li>Use the raw fluorescence units (RFUs) from the plate reader to calculate the permeability index (PI) after subtracting the corresponding sham values.
		<ol>
			<li>*Permeability Index (mL/g) =(Tissue RFUs/g tissue weight)/(Serum RFUs/mL serum)</li>
		</ol>
		</li>
		<li>Example calculation for brain permeability index (PI) of the animal GOF1 (Table 1):
		<ol>
			<li>GOF1 brain RFUs - SHAM brain RFUs = 154 - 22.5 = 131.5</li>
			<li>GOF1 serum RFUs - SHAM serum RFUs = 38305 - 27 = 38278</li>
			<li>Brain weight (g) = 0.195</li>
			<li>Serum volume (ml) = 0.02</li>
			<li>Brain Permeability Index (10-3 mL/g) = (131.5/0.195)/(38278/0.02) = 0.352 
			NOTE: The permeability index calculations yield values that are absolute and comparable between experiments and tissue types. These however can be presented as ratios for ease of comparison between 2 groups 12. This can be achieved by dividing the PI of each animal in both groups with the mean PI of control group, driving the control group mean through 1 yet keeping the inter animal variation in the control group. This transformation provides values for experimental group (or groups) relative to control group set to 1.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Immunofluorescence visualization of tracer
	<ol>
		<li>Cut the kidney/hemi-brain blocks embedded natively in tissue-tek compound (O.C.T) (step 2.1) into 10 &#181;m sections on a cryostat set to -20 &#176;C and transfer the sections to slides placed at room temperature. Once the sections are dried (about 30 min), transfer slides to -80 &#176;C freezer until use.</li>
		<li>On the day of staining, thaw the slides at 37 &#176;C for 10 min, and then fix the sections with 4% paraformaladehyde (PFA) for 10 min at room temperature followed by quick washing in PBS.</li>
		<li>Incubate the sections in permeabilization/blocking buffer made of sterile PBS containing 1 % BSA and 0.5 % Triton X-100 pH 7.5 for 1 h at room temperature.</li>
		<li>Incubate slides for 1.5 h at room temperature with CD31 primary antibody (0.5 mg/ml, clone MEC 13.3), diluted in 1:100 in buffer containing sterile PBS, 0.5 % BSA, 0.25 % Triton X-100 (pH 7.2) followed by three 5 min washes in PBS.</li>
		<li>Perform secondary antibody incubation in the above buffer for 1 h at room temperature with species-specific fluorescently labeled antibodies diluted 1:500 (2 mg/mL). For CD31, goat anti-rat Alexa 568 or Alexa 488 can be used at a dilution of 1:500. Include DAPI (300 &#181;M) in the secondary antibody mix (1:1,000 dilution from the stock) to stain for the nuclei.</li>
		<li>Mount the stained sections with aqua polymount and leave it overnight for polymerization at room temperature in dark.</li>
		<li>Acquire images using a spectral imaging confocal laser scanning microscope system. Analyze images by NIS elements software (version 4.3). Software such as Photoshop can be used for generation of montage figures.</li>
	</ol>
	</li>
</ol>

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The authors declare that they have no competing financial interests.

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 ...

an-vivo-blood-brain-barrier-permeability-assay-mice-using

Research

JoVE Journal

Neuroscience

24.3K Views.  Goethe University Hospital. 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.

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

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 blood-brain barrier (BBB) consisting of EC, astrocyte end-feet, pericytes and the basal membrane is responsible for the protection and homeostasis of the brain parenchyma. In vitro BBB models are common tools to study the structure and function of the BBB at the cellular level. A considerable number of different in vitro BBB models have been established for research in different laboratories to date. Usually, the cells are obtained from bovine, porcine, rat or mouse brain tissue (discussed in detail in the review by Wilhelm et al. 1). Human tissue samples are available only in a restricted number of laboratories or companies 2,3. While primary cell preparations are time consuming and the EC cultures can differ from batch to batch, the establishment of immortalized EC lines is the focus of scientific interest.
Here, we present a method for establishing an immortalized brain microvascular EC line from neonatal mouse brain. We describe the procedure step-by-step listing the reagents and solutions used. The method established by our lab allows the isolation of a homogenous immortalized endothelial cell line within four to five weeks. The brain microvascular endothelial cell lines termed cEND 4 (from cerebral cortex) and cerebEND 5 (from cerebellar cortex), were isolated according to this procedure in the F&ouml;rster laboratory and have been effectively used for explanation of different physiological and pathological processes at the BBB. Using cEND and cerebEND we have demonstrated that these cells respond to glucocorticoid- 4,6-9 and estrogen-treatment 10 as well as to pro-infammatory mediators, such as TNFalpha 5,8. Moreover, we have studied the pathology of multiple sclerosis 11 and hypoxia 12,13 on the EC-level. The cEND and cerebEND lines can be considered as a good tool for studying the structure and function of the BBB, cellular responses of ECs to different stimuli or interaction of the EC with lymphocytes or cancer cells.

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

This method describes how to isolate and immortalize microvascular endothelial cells from mouse brain. We describe a step-by-step protocol starting from the homogenization of brain tissue, digestion steps, seeding and immortalization of the cells. Usually, it takes about five weeks to obtain a homogenous, immortalized microvascular endothelial cell line.

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 technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

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 during the earliest stages of brain development and are maintained throughout the animal's life. The brain ventricular system is found in vertebrates, and the ventricles develop after neural tube formation, when the central lumen fills with cerebrospinal fluid (CSF) 1,2. CSF is a protein rich fluid that is essential for normal brain development and function3-6.
In zebrafish, brain ventricle inflation begins at approximately 18 hr post fertilization (hpf), after the neural tube is closed. Multiple processes are associated with brain ventricle formation, including formation of a neuroepithelium, tight junction formation that regulates permeability and CSF production. We showed that the Na,K-ATPase is required for brain ventricle inflation, impacting all these processes 7,8, while claudin 5a is necessary for tight junction formation 9. Additionally, we showed that "relaxation" of the embryonic neuroepithelium, via inhibition of myosin, is associated with brain ventricle inflation.
To investigate the regulation of permeability during zebrafish brain ventricle inflation, we developed a ventricular dye retention assay. This method uses brain ventricle injection in a living zebrafish embryo, a technique previously developed in our lab10, to fluorescently label the cerebrospinal fluid. Embryos are then imaged over time as the fluorescent dye moves through the brain ventricles and neuroepithelium. The distance the dye front moves away from the basal (non-luminal) side of the neuroepithelium over time is quantified and is a measure of neuroepithelial permeability (Figure 1). We observe that dyes 70 kDa and smaller will move through the neuroepithelium and can be detected outside the embryonic zebrafish brain at 24 hpf (Figure 2).
This dye retention assay can be used to analyze neuroepithelial permeability in a variety of different genetic backgrounds, at different times during development, and after environmental perturbations. It may also be useful in examining pathological accumulation of CSF. Overall, this technique allows investigators to analyze the role and regulation of permeability during development and disease.

We describe a live whole animal quantitative measurement for permeability of the embryonic zebrafish brain. The technique analyzes the ability to retain cerebrospinal fluid and molecules of different molecular weights within the neural tube lumen and quantifies their movement out of the ventricles. This method is useful for determining differences in epithelial permeability and maturation during development and disease.

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

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

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 between perturbed BBB homeostasis and the pathogenesis of brain disorders needs further investigation, the development and validation of a reliable procedure to accurately detect BBB alterations may be crucial and represent a useful tool for potentially predicting disease progression and developing targeted therapeutic strategies.
Here, we present an easy and efficient procedure for evaluating BBB leakage in a neurodegenerative condition like that occurring in a preclinical mouse model of Huntington disease, in which defects in the permeability of BBB are clearly detectable precociously in the disease. Specifically, the high molecular weight fluorescein isothiocyanate labelled (FITC)-albumin, which is able to cross the BBB only when the latter is impaired, is acutely infused into a mouse jugular vein and its distribution in the vascular or parenchymal districts is then determined by fluorescence microscopy.
Accumulation of green fluorescent-albumin in the brain parenchyma functions as an index of aberrant BBB permeability and, when quantitated by using Image J processing software, is reported as Green Fluorescence Intensity.

In this study, we present an easy and efficient procedure for evaluating the disruption of the blood-brain barrier under neurodegenerative conditions. To achieve our objective, we infused high molecular weight fluorescein isothiocyanate labelled (FITC)-albumin into mouse jugular vein and evaluated its leakage into the brain parenchyma by fluorescence microscopy.

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

Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier (BBB). Rats with induced ischemic stroke were established and used as in vivo models to investigate the functional integrity of the BBB. Spectrophotometric detection of Evans blue (EB) in the brain samples with ischemic injury could provide reliable justification for the research and development of novel therapeutic modalities. This method generates reproducible results, and is applicable in any laboratory without a need for special equipment. Here, we present a visualized and technical guideline on the detection of the extravasation of EB following induction of ischemic stroke in rats.

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

The overall goal of this procedure is to provide a highly reproducible technique for in vivo assessment of the blood-brain barrier disruption in rat models 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 ...

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal dysfunction and loss. Fluorescence-based imaging tools and technologies enable unprecedented analysis of subcellular neurobiological processes, yet there is still a need for unbiased, reproducible, and accessible approaches for extracting quantifiable data from imaging studies. We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-based imaging studies using Drosophila models of neurodegeneration. Specifically, we describe an easy-to-follow, semi-automated approach using Fiji/ImageJ to analyze two cellular processes: first, we quantify protein aggregate content and profile in the Drosophila optic lobe using fluorescent-tagged mutant huntingtin proteins; and second, we assess autophagy-lysosome flux in the Drosophila visual system with ratiometric-based quantification of a tandem fluorescent reporter of autophagy. Importantly, the protocol outlined here includes a semi-automated segmentation step to ensure all fluorescent structures are analyzed to minimize selection bias and to increase resolution of subtle comparisons. This approach can be extended for the analysis of other cell biological structures and processes implicated in neurodegeneration, such as proteinaceous puncta (stress granules and synaptic complexes), as well as membrane-bound compartments (mitochondria and membrane trafficking vesicles). This method provides a standardized, yet adaptable reference point for image analysis and quantification, and could facilitate reliability and reproducibility across the field, and ultimately enhance mechanistic understanding of neurodegeneration.

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-imaging-based cell biological studies of protein aggregation and autophagic flux in the central nervous system of Drosophila models of neurodegeneration.

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal ...

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 delivery including precise localization to a target brain region and reduction of off target side effects. However, stereotaxic surgery is highly invasive which limits its translational efficacy, requires long recovery times, and provides challenges when targeting multiple brain regions. Focused ultrasound (FUS) can be used in combination with circulating microbubbles to transiently open the blood brain barrier (BBB) in millimeter sized regions. This allows intracranial localization of systemically delivered agents that cannot normally cross the BBB. This technique provides a noninvasive alternative to stereotaxic surgery. However, to date this technique has yet to be widely adopted in neuroscience laboratories due to the limited access to equipment and standardized methods. The overall goal of this protocol is to provide a benchtop approach to FUS BBB opening (BBBO) that is affordable and reproducible and can therefore be easily adopted by any laboratory.

Focused ultrasound with microbubble agents can open the blood brain barrier focally and transiently. This technique has been used to deliver a wide range of agents across the blood brain barrier. This article provides a detailed protocol for the localized delivery to the rodent brain with or without MRI guidance.

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

A High-Throughput Image-Guided Stereotactic Neuronavigation and Focused Ultrasound System for Blood-Brain Barrier Opening in Rodents

The blood-brain barrier (BBB) has been a major hurdle for the treatment of various brain diseases. Endothelial cells, connected by tight junctions, form a physiological barrier preventing large molecules (&#62;500 Da) from entering the brain tissue. Microbubble-mediated focused ultrasound (FUS) can be used to induce a transient local BBB opening, allowing larger drugs to enter the brain parenchyma.
In addition to large-scale clinical devices for clinical translation, preclinical research for therapy response assessment of drug candidates requires dedicated small animal ultrasound setups for targeted BBB opening. Preferably, these systems allow high-throughput workflows with both high-spatial precision as well as integrated cavitation monitoring, while still being cost effective in both initial investment and running costs.
Here, we present a bioluminescence and X-ray guided stereotactic small animal FUS system that is based on commercially available components and fulfills the aforementioned requirements. A particular emphasis has been placed on a high degree of automation facilitating the challenges typically encountered in high-volume preclinical drug evaluation studies. Examples of these challenges are the need for standardization in order to ensure data reproducibility, reduce intra-group variability, reduce sample size and thus comply with ethical requirements and decrease unnecessary workload. The proposed BBB system has been validated in the scope of BBB opening facilitated drug delivery trials on patient-derived xenograft models of glioblastoma multiforme and diffuse midline glioma.

The blood-brain barrier (BBB) can be temporarily disrupted with microbubble-mediated focused ultrasound (FUS). Here, we describe a step-by-step protocol for high-throughput BBB opening in vivo using a modular FUS system accessible for non-ultrasound experts.

The blood-brain barrier (BBB) has been a major hurdle for the treatment of various brain diseases. Endothelial cells, connected by tight junctions, form a ...

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

Summary

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Chapters in this video

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

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

An Assay for Permeability of the Zebrafish Embryonic Neuroepithelium

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

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

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

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

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

A High-Throughput Image-Guided Stereotactic Neuronavigation and Focused Ultrasound System for Blood-Brain Barrier Opening in Rodents