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

<ol>
	<li>Abbott, N. J., R&#246;nnb&#228;ck, L., Hansson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte-endothelial+interactions+at+the+blood-brain+barrier.">Astrocyte-endothelial interactions at the blood-brain barrier.</a> Nature reviews. Neuroscience. 7 (1), 41-53 (2006).</li><li>Zhao, Z., Nelson, A. R., Betsholtz, C., Zlokovic, B. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+Dysfunction+of+the+Blood-Brain+Barrier.">Establishment and Dysfunction of the Blood-Brain Barrier.</a> Cell. 163 (5), 1064-1078 (2015).</li><li>Obermeier, B., Daneman, R., Ransohoff, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development,+maintenance+and+disruption+of+the+blood-brain+barrier.">Development, maintenance and disruption of the blood-brain barrier.</a> Nature Medicine. 19 (12), 1584-1596 (2013).</li><li>Devraj, K., Klinger, M. E., Myers, R. L., Mokashi, A., Hawkins, R. A., Simpson, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GLUT-1+glucose+transporters+in+the+blood-brain+barrier:+differential+phosphorylation.">GLUT-1 glucose transporters in the blood-brain barrier: differential phosphorylation.</a> Journal of neuroscience research. 89 (12), 1913-1925 (2011).</li><li>Banks, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+blood-brain+barrier+to+blood-brain+interface:+new+opportunities+for+CNS+drug+delivery.">From blood-brain barrier to blood-brain interface: new opportunities for CNS drug delivery.</a> Nature reviews. Drug discovery. 15 (4), 275-292 (2016).</li><li>Zlokovic, B. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurovascular+pathways+to+neurodegeneration+in+Alzheimer's+disease+and+other+disorders.">Neurovascular pathways to neurodegeneration in Alzheimer's disease and other disorders.</a> Nature reviews. Neuroscience. 12 (12), 723-738 (2011).</li><li>Paganetti, P., Antoniello, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+efflux+of+amyloid-&#946;+peptides+through+the+blood-brain+barrier+by+muscarinic+acetylcholine+receptor+inhibition+reduces+pathological+phenotypes+in+mouse+models+of+brain+amyloidosis.">Increased efflux of amyloid-&#946; peptides through the blood-brain barrier by muscarinic acetylcholine receptor inhibition reduces pathological phenotypes in mouse models of brain amyloidosis.</a> Journal of Alzheimer's disease: JAD. 38 (4), 767-786 (2014).</li><li>Devraj, K., Poznanovic, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BACE-1+is+expressed+in+the+blood-brain+barrier+endothelium+and+is+upregulated+in+a+murine+model+of+Alzheimer's+disease.">BACE-1 is expressed in the blood-brain barrier endothelium and is upregulated in a murine model of Alzheimer's disease.</a> Journal of cerebral blood flow and metabolism: official journal of the International Society of Cerebral Blood Flow and Metabolism. 36 (7), 1281-1294 (2016).</li><li>Daneman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+blood-brain+barrier+in+health+and+disease.">The blood-brain barrier in health and disease.</a> Annals of neurology. 72 (5), 648-672 (2012).</li><li>Gurnik, S., Devraj, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiopoietin-2-induced+blood-brain+barrier+compromise+and+increased+stroke+size+are+rescued+by+VE-PTP-dependent+restoration+of+Tie2+signaling.">Angiopoietin-2-induced blood-brain barrier compromise and increased stroke size are rescued by VE-PTP-dependent restoration of Tie2 signaling.</a> Acta neuropathologica. 131 (5), 753-773 (2016).</li><li>Scholz, A., Harter, P. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cell-derived+angiopoietin-2+is+a+therapeutic+target+in+treatment-naive+and+bevacizumab-resistant+glioblastoma.">Endothelial cell-derived angiopoietin-2 is a therapeutic target in treatment-naive and bevacizumab-resistant glioblastoma.</a> EMBO Molecular Medicine. 8 (1), 39-57 (2016).</li><li>Gross, S., Devraj, K., Feng, Y., Macas, J., Liebner, S., Wieland, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleoside+diphosphate+kinase+B+regulates+angiogenic+responses+in+the+endothelium+via+caveolae+formation+and+c-Src-mediated+caveolin-1+phosphorylation.">Nucleoside diphosphate kinase B regulates angiogenic responses in the endothelium via caveolae formation and c-Src-mediated caveolin-1 phosphorylation.</a> Journal of cerebral blood flow and metabolism: official journal of the International Society of Cerebral Blood Flow and Metabolism. 37 (7), 2471-2484 (2017).</li><li>Ziegler, N., Awwad, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#946;-Catenin+Is+Required+for+Endothelial+Cyp1b1+Regulation+Influencing+Metabolic+Barrier+Function.">&#946;-Catenin Is Required for Endothelial Cyp1b1 Regulation Influencing Metabolic Barrier Function.</a> The Journal of neuroscience: the official journal of the Society for Neuroscience. 36 (34), 8921-8935 (2016).</li><li>Vutukuri, R., Brunkhorst, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alteration+of+sphingolipid+metabolism+as+a+putative+mechanism+underlying+LPS-induced+BBB+disruption.">Alteration of sphingolipid metabolism as a putative mechanism underlying LPS-induced BBB disruption.</a> Journal of Neurochemistry. , (2017).</li><li>Gage, G. J., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+Animal+Perfusion+Fixation+for+Rodents.">Whole Animal Perfusion Fixation for Rodents.</a> J Vis Exp. (65), e3564 (2012).</li><li>Hoffmann, A., Bredno, J., Wendland, M., Derugin, N., Ohara, P., Wintermark, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+and+Low+Molecular+Weight+Fluorescein+Isothiocyanate+(FITC)-Dextrans+to+Assess+Blood-Brain+Barrier+Disruption:+Technical+Considerations.">High and Low Molecular Weight Fluorescein Isothiocyanate (FITC)-Dextrans to Assess Blood-Brain Barrier Disruption: Technical Considerations.</a> Translational stroke research. 2 (1), 106-111 (2011).</li><li>Armulik, A., Genov&#233;, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pericytes+regulate+the+blood-brain+barrier.">Pericytes regulate the blood-brain barrier.</a> Nature. 468 (7323), 557-561 (2010).</li><li>Daneman, R., Zhou, L., Kebede, A. A., Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pericytes+are+required+for+blood-brain+barrier+integrity+during+embryogenesis.">Pericytes are required for blood-brain barrier integrity during embryogenesis.</a> Nature. 468 (7323), 562-566 (2010).</li><li>Bell, R. D., Winkler, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pericytes+control+key+neurovascular+functions+and+neuronal+phenotype+in+the+adult+brain+and+during+brain+aging.">Pericytes control key neurovascular functions and neuronal phenotype in the adult brain and during brain aging.</a> Neuron. 68 (3), 409-427 (2010).</li><li>Banks, W. A., Gray, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipopolysaccharide-induced+blood-brain+barrier+disruption:+roles+of+cyclooxygenase,+oxidative+stress,+neuroinflammation,+and+elements+of+the+neurovascular+unit.">Lipopolysaccharide-induced blood-brain barrier disruption: roles of cyclooxygenase, oxidative stress, neuroinflammation, and elements of the neurovascular unit.</a> Journal of Neuroinflammation. 12, 223 (2015).</li><li>Krause, G., Winkler, L., Mueller, S. L., Haseloff, R. F., Piontek, J., Blasig, I. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+function+of+claudins.">Structure and function of claudins.</a> Biochimica et biophysica acta. 1778 (3), 631-645 (2008).</li><li>Johansson, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood-Brain+Barrier:+Role+of+Brain+Endothelial+Surface+Charge+and+Glycocalyx.">Blood-Brain Barrier: Role of Brain Endothelial Surface Charge and Glycocalyx.</a> Ischemic Blood Flow in the Brain. , 33-38 (2001).</li><li>Fu, B. M., Li, G., Yuan, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Charge+effects+of+the+blood-brain+barrier+on+the+transport+of+charged+molecules.">Charge effects of the blood-brain barrier on the transport of charged molecules.</a> The FASEB Journal. 22 (1 Supplement), (2008).</li><li>Goebl, N. A., Babbey, C. M., Datta-Mannan, A., Witcher, D. R., Wroblewski, V. J., Dunn, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+Fc+receptor+mediates+internalization+of+Fc+in+transfected+human+endothelial+cells.">Neonatal Fc receptor mediates internalization of Fc in transfected human endothelial cells.</a> Molecular biology of the cell. 19 (12), 5490-5505 (2008).</li><li>Lopez-Quintero, S. V., Ji, X. -. Y., Antonetti, D. A., Tarbell, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+three-pore+model+describes+transport+properties+of+bovine+retinal+endothelial+cells+in+normal+and+elevated+glucose.">A three-pore model describes transport properties of bovine retinal endothelial cells in normal and elevated glucose.</a> Investigative ophthalmology &amp; visual science. 52 (2), 1171-1180 (2011).</li><li>Hallmann, R., Mayer, D. N., Berg, E. L., Broermann, R., Butcher, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+mouse+endothelial+cell+surface+marker+is+suppressed+during+differentiation+of+the+blood+brain+barrier.">Novel mouse endothelial cell surface marker is suppressed during differentiation of the blood brain barrier.</a> Developmental dynamics: an official publication of the American Association of Anatomists. 202 (4), 325-332 (1995).</li></ol>

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...

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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 ...

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Research

JoVE Journal

Neuroscience

24.4K 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

Predicting In Vivo Payloads Delivery using a Blood-brain Tumor-barrier in a Dish

Highly selective by nature, the blood-brain barrier (BBB) is essential for the brain homeostasis in physiological conditions. However, in the context of brain tumors, the molecular selectivity of BBB also shields the neoplastic cells by blocking the delivery of peripherally administered chemotherapies. The development of novel drugs (including nanoparticles) targeting malignant brain tumors ideally requires the use of preclinical animal models to study the drug&#8217;s transcytosis and antitumor efficacy. In order to comply with the 3R principle (refine, reduce, and replace) to reduce the number of laboratory animals in experimental setup and perform the high-throughput screening of a large library of antitumor agents, we developed a reproducible in vitro human and murine mimic of the blood-brain tumor-barrier (BBTB) using three-layered cultures of endothelial cells, astrocytes, and patient-derived glioblastoma spheres. For higher scalability and reproducibility, commercial cell lines or immortalized cells have been used in tailored conditions to allow the formation of a barrier resembling the actual BBB. Here we describe a protocol to obtain a BBTB mimic by culturing endothelial cells in contact with astrocytes at specific cell densities on inserts. This BBTB mimic can be used, for instance, for the quantification and confocal imaging of the nanoparticle passage through the endothelial and astrocytic barriers, in addition to the evaluation of the tumor cell targeting within the same assay. Moreover, we show that the obtained data can be used to predict the behavior of nanoparticles in preclinical animal models. In a broader perspective, this in vitro model could be adapted to other neurodegenerative diseases for the determination of the passage of new therapeutic molecules through the BBB and/or be supplemented with brain organoids to directly evaluate the efficacy of drugs.

Drug targeting to central nervous system tumors is a major challenge. Here we describe a protocol to produce an in vitro mimic of the blood-brain tumor-barrier using murine and/or human cells and discuss their relevance for the predictability of central nervous system tumor targeting in vivo.

Highly selective by nature, the blood-brain barrier (BBB) is essential for the brain homeostasis in physiological conditions. However, in the context of ...

Cancer Research

An in vivo Assay to Test Blood Vessel Permeability

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under physiologic conditions the endothelium is impermeable to albumin, so Evans blue bound albumin remains restricted within blood vessels. In pathologic conditions that promote increased vascular permeability endothelial cells partially lose their close contacts and the endothelium becomes permeable to small proteins such as albumin. This condition allows for extravasation of Evans Blue in tissues. A healthy endothelium prevents extravasation of the dye in the neighboring vascularized tissues. Organs with increased permeability will show significantly increased blue coloration compared to organs with intact endothelium. The level of vascular permeability can be assessed by simple visualization or by quantitative measurement of the dye incorporated per milligram of tissue of control versus experimental animal/tissue. Two powerful aspects of this assay are its simplicity and quantitative characteristics. Evans Blue dye can be extracted from tissues by incubating a specific amount of tissue in formamide. Evans Blue absorbance maximum is at 620 nm and absorbance minimum is at 740 nm. By using a standard curve for Evans Blue, optical density measurements can be converted into milligram dye captured per milligram of tissue. Statistical analysis should be used to assess significant differences in vascular permeability.

An in vivo Assay to Test Blood Vessel Permeability

We are presenting an in vivo assay to test blood vessel permeability. This assay is based on intravenous injection of a dye and subsequent visualization of its diffusion into interstitial spaces.

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under ...

Medicine

Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm&#183;cm2 on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 &#177;&#160;0.11 x 10-3 cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.

Setting-up an In Vitro Model of Rat Blood-brain Barrier BBB: A Focus on BBB Impermeability and Receptor-mediated Transport

The aim of the present study was to validate the reproducibility of an in vitro BBB model involving a rat syngeneic co-culture of endothelial cells and astrocytes. The endothelial cell monolayer presented high TEER and low LY permeability. Expression of specific TJ proteins, functional responses to inflammation and functionality of transporters and receptors were assessed.

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and ...

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 ...

Visualizing Impairment of the Endothelial and Glial Barriers of the Neurovascular Unit during Experimental Autoimmune Encephalomyelitis In Vivo

The neurovascular unit (NVU) is composed of microvascular endothelial cells forming the blood-brain barrier (BBB), an endothelial basement membrane with embedded pericytes, and the glia limitans composed by the parenchymal basement membrane and astrocytic end-feed embracing the abluminal aspect of central nervous system (CNS) microvessels. In addition to maintaining CNS homeostasis the NVU controls immune cell trafficking into the CNS. During immunosurveillance of the CNS low numbers of activated lymphocytes can cross the endothelial barrier without causing BBB dysfunction or clinical disease. In contrast, during neuroinflammation such as in multiple sclerosis or its animal model experimental autoimmune encephalomyelitis (EAE) a large number of immune cells can cross the BBB and subsequently the glia limitans eventually reaching the CNS parenchyma leading to clinical disease. 
Immune cell migration into the CNS parenchyma is thus a two-step process that involves a sequential migration across the endothelial and glial barrier of the NVU employing distinct molecular mechanisms. If following their passage across the endothelial barrier, T cells encounter their cognate antigen on perivascular antigen-presenting cells their local reactivation will initiate subsequent mechanisms leading to the focal activation of gelatinases, which will enable the T cells to cross the glial barrier and enter the CNS parenchyma. Thus, assessing both, BBB permeability and MMP activity in spatial correlation to immune cell accumulation in the CNS during EAE allows to specify loss of integrity of the endothelial and glial barriers of the NVU. 
We here show how to induce EAE in C57BL/6 mice by active immunization and how to subsequently analyze BBB permeability in vivo using a combination of exogenous fluorescent tracers. We further show, how to visualize and localize gelatinase activity in EAE brains by in situ zymogaphy coupled to immunofluorescent stainings of BBB basement membranes and CD45+ invading immune cells.

Here, we present protocols to investigate impairment of the neurovascular unit during experimental autoimmune encephalomyelitis in vivo. We specifically address how to determine blood-brain barrier permeability and gelatinase activity involved in leukocyte migration across the glia limitans.&#160;

The neurovascular unit (NVU) is composed of microvascular endothelial cells forming the blood-brain barrier (BBB), an endothelial basement membrane with ...

Immunology and Infection

Fluorescent Dye-Based Visualization of Blood-Brain Barrier Breakdown in Mice

Evaluation of Blood-Brain Barrier Breakdown in a Mouse Model of Mild Traumatic Brain Injury

Fluorescent dyes are used to determine the extent of dye extravasation that occurs due to blood-brain barrier (BBB) breakdown. Labeling with these dyes is a complex process influenced by several factors, such as the concentration of dyes in the blood, permeability of brain vessels, duration of dye extravasation, and reduction in dye concentration in the tissue due to degradation and diffusion. In a mild traumatic brain injury model, exposure to blast-induced shock waves (BSWs) triggers BBB breakdown within a limited time window. To determine the precise sequence of BBB breakdown, Evans blue, and fluorescein isothiocyanate-dextran were injected intravascularly and intracardially into mice at various time points relative to BSW exposure. The distribution of dye fluorescence in brain slices was then recorded. Differences in the distribution and intensity between the two dyes revealed the spatiotemporal sequence of BBB breakdown. Immunostaining of the brain slices showed that astrocytic and microglial responses correlated with the sites of BBB breakdown. This protocol has broad potential for application in studies involving different BBB breakdown models.

A method was developed to visualize dye extravasation due to blood-brain barrier (BBB) breakdown by administering two fluorescent dyes to mice at different time points. The use of glycerol as a cryoprotectant facilitated immunohistochemistry on the same sample.

Fluorescent dyes are used to determine the extent of dye extravasation that occurs due to blood-brain barrier (BBB) breakdown. Labeling with these dyes is ...

In Vivo Vascular Permeability Detection in Mouse Submandibular Gland

Saliva plays an important role in oral and overall health. The intact endothelial barrier function of blood vessels enables saliva secretion, whereas the endothelial barrier dysfunction is related to many salivary gland secretory disorders. The present protocol describes an in vivo paracellular permeability detection method to evaluate the function of endothelial tight junctions (TJs) in mouse submandibular glands (SMG). First, fluorescence-labeled dextrans with different molecular weights (4 kDa, 40 kDa, or 70 kDa) were injected into the angular veins of mice. Afterward, the unilateral SMG was dissected and fixed in the customized holder under a two-photon laser-scanning microscope, and then images were captured for blood vessels, acini, and ducts. Utilizing this method, the real-time dynamic leakage of the different-sized tracers from blood vessels into the basal sides of the acini and even across the acinar epithelia into the ducts was monitored to evaluate the alteration of the endothelial barrier function under physiological or pathophysiological conditions.

In Vivo Vascular Permeability Detection in Mouse Submandibular Gland

In the present protocol, the endothelial barrier function of the submandibular gland (SMG) was evaluated by injecting different molecular weighted fluorescent tracers into the angular veins of test animal models in vivo under a two-photon laser-scanning microscope.

Saliva plays an important role in oral and overall health. The intact endothelial barrier function of blood vessels enables saliva secretion, whereas the ...

Biology

Assessment of Gut Barrier Integrity in Mice Using Fluorescein-Isothiocyanate-Labeled Dextran

Gut barrier integrity is a hallmark of intestinal health. While gut barrier integrity can be assessed using indirect markers such as the measurement of plasma inflammatory markers and bacterial translocation to the spleen and lymph nodes, the gold standard directly quantifies the ability of selected molecules to traverse the gut mucosal layer toward systemic circulation. This article uses a non-invasive, cost-effective, and low-burden technique to quantify and follow in real time the intestinal permeability in mice using fluorescein-isothiocyanate-labeled dextran (FITC-dextran). Prior to oral supplementation with FITC-dextran, the mice are fasted. They are then gavaged with FITC-dextran diluted in phosphate-buffered saline (PBS). One hour after the gavage, the mice are subjected to general anesthesia using isoflurane, and the in vivo fluorescence is visualized in an imaging chamber. This technique aims to assess residual fluorescence in the abdominal cavity and the hepatic uptake, which is suggestive of portal migration of the fluorescent probe. Blood and stool samples are collected 4 h after oral gavage, and the mice are sacrificed. Plasma and fecal samples diluted in PBS are then plated, and the fluorescence is recorded. The concentration of FITC-dextran is then calculated using a standard curve. In previous research, in vivo imaging has shown that fluorescence rapidly spreads to the liver in mice with a weaker gut barrier induced by a low-fiber diet, while in mice supplemented with fiber to strengthen the gut barrier, the fluorescent signal is retained mostly in the gastrointestinal tract. In addition, in this study, control mice had elevated plasma fluorescence and reduced fluorescence in the stool, while inversely, inulin-supplemented mice had higher levels of fluorescence signals in the gut and low levels in the plasma. In summary, this protocol provides qualitative and quantitative measurements of intestinal permeability as a marker for gut health.

In the present study, fluorescein-isothiocyanate-labeled (FITC) dextran is administered to mice via oral gavage to evaluate intestinal permeability both in vivo and in plasma and fecal samples. As gut barrier function is affected in many disease processes, this direct and quantitative assay can be used in diverse research areas.

Gut barrier integrity is a hallmark of intestinal health. While gut barrier integrity can be assessed using indirect markers such as the measurement of ...

Determining Blood-Brain Barrier Disruption Using Fluorescent Dyes in an EAE Mouse Model

Source: Tietz, S. M., et al., <a href="https://app.jove.com/v/59249">Visualizing Impairment of the Endothelial and Glial Barriers of the Neurovascular Unit during Experimental Autoimmune Encephalomyelitis In Vivo.</a> J. Vis. Exp. (2019).
This video demonstrates a procedure for assessing blood-brain barrier disruption in an EAE mouse model. Fluorescent dyes of different sizes are injected retro-orbitally and circulate through the brain&#39;s blood vessels. The smaller dye leaks through gaps into the brain tissue, while the larger dye enters only if the disruption is severe, correlating with the extent of barrier damage.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. In ...

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Neuropathology

Cerebrovascular Diseases

Blood-Brain Barrier Mimic Permeability Assay: An In Vitro Assay to Assess the Permeability of BBB Mimic to Tracer Molecules

Source: Le Joncour, V. et al. <a href="https://www.jove.com/v/59384/predicting-vivo-payloads-delivery-using-blood-brain-tumor-barrier">Predicting In Vivo Payloads Delivery using a Blood-Brain Tumor-Barrier in a Dish.</a> J. Vis. Exp. (2019)
This video describes a permeability assay to evaluate the permeability of in vitro blood-brain barrier using sodium fluorescein molecules.

Source: Le Joncour, V. et al. Predicting In Vivo Payloads Delivery using a Blood-Brain Tumor-Barrier in a Dish. J. Vis. Exp. (2019)
This video describes a ...

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

Summary

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Chapters in this video

Predicting In Vivo Payloads Delivery using a Blood-brain Tumor-barrier in a Dish

An in vivo Assay to Test Blood Vessel Permeability

Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport

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

Visualizing Impairment of the Endothelial and Glial Barriers of the Neurovascular Unit during Experimental Autoimmune Encephalomyelitis In Vivo

Evaluation of Blood-Brain Barrier Breakdown in a Mouse Model of Mild Traumatic Brain Injury

In Vivo Vascular Permeability Detection in Mouse Submandibular Gland

Assessment of Gut Barrier Integrity in Mice Using Fluorescein-Isothiocyanate-Labeled Dextran

Determining Blood-Brain Barrier Disruption Using Fluorescent Dyes in an EAE Mouse Model

Blood-Brain Barrier Mimic Permeability Assay: An In Vitro Assay to Assess the Permeability of BBB Mimic to Tracer Molecules