This work was supported by &#34;Fondazione Neuromed&#34; and funded by the Italian Ministry of Health &#34;Ricerca Corrente&#34; to V.M.

All procedures on animals were approved by the IRCCS Neuromed Animal Care Review Board and by &#34;Istituto Superiore di Sanit&#224;&#34; (permit number: 1163/2015- PR) and were in accordance with the guidelines of the EU Directive 2010/63/EU for animal experiments.
1. Preparation of FITC-albumin Solution to be Injected into Jugular Vein
NOTE: All experiments were carried out in manifest (11-week old) HD R6/2 mice and in age and gender-matched wild-type (WT) control littermates.
<ol>
	<li>Dissolve FITC-albumin powder (see Table of Materials) in phosphate buffered saline (PBS) at 10 mg/ml. 
	NOTE: For optimal results, this should be prepared fresh for each labelling reaction.</li>
</ol>
2. Preparation of Equipment to Be Used During Surgery
<ol>
	<li>Lay out the previously autoclaved surgical instruments (see Table of Materials).</li>
	<li>Clean the surgical bench with alcohol (70% ethanol solution). Prepare the surgical area (45 cm x 45 cm) with a surgical sterile disposable towel drape and set up the operating stereomicroscope.</li>
</ol>
3. Surgical Procedure for FITC-albumin Infusion into the Jugular Vein
<ol>
	<li>Record mouse body weight for anesthesia.</li>
	<li>Anesthetize mouse with an intraperitoneal injection (i.p) of an appropriate dose of Ketamine (100 mg/kg) - xylazine (10 mg/kg) solution.</li>
	<li>Monitor animal sedation by gentle toe pinch withdraw reflex as demonstrated in Walantus et al.11,12.</li>
	<li>Place the anesthetized mouse in a face-up supine position and fix the four legs and the tail on the surgical workbench with adhesive tape.</li>
	<li>After the mouse is secured, carefully shave the appropriate surgical margin on the neck with an electric razor (see Table of Materials) and disinfect it with povidone-iodine solution and 70% ethanol.</li>
	<li>Dry the exposed shaved surface with cotton swabs.</li>
	<li>Gently pull up the shaved skin and make a 1.5 cm long longitudinal incision, by scalpel, in the midclavicular line starting midway on the thorax and extending to just below the neck.</li>
	<li>Carefully stretch apart connective tissue with forceps under microscopic observation (5X magnification) to expose the external jugular vein.</li>
	<li>Ensure a sufficient space on the right and on the left of the jugular vein to facilitate the insertion of the 30&#189; G needle for the infusion of the FITC-albumin solution.</li>
	<li>Inject the animal intravenously (right jugular vein) slowly (over 3 min) with 100 &#181;L of 10 mL/kg FITC-albumin by using the 30&#189; G needle.</li>
	<li>After 15 minutes, euthanize the mouse by decapitation, rapidly remove the brain from the skull as previously described13, and immerse it for 15 min in at least 10x the volume of the brain itself of pre-chilled isopentane.</li>
	<li>Move the isopentane frozen brain into a pre-chilled tube and store it into a -80 &#176;C freezer until histological sectioning.</li>
</ol>
4. Histological Sectioning of Brain Infused with FITC-albumin
<ol>
	<li>Embed frozen brain in optimal cutting temperature (OCT) compound (see Table of Materials) prior to cryostat sectioning.</li>
	<li>Serially cut the brain into 20 &#181;m thick sections, with a cryostat (see Table of Materials), and mount them onto microscope glass slides.</li>
	<li>Perform the FITC-Albumin/laminin co-staining by using a specific anti-laminin antibody (see Table of Materials) as previously described6.
	<ol>
		<li>Briefly, incubate sections with the primary antibody anti-laminin (pAb 1:1000) over night and proceed with appropriate secondary antibody conjugated to Cy3.</li>
		<li>Coverslip slides with mounting medium containing 4&#39;,6-diamidino-2-phenylindole (DAPI) (absorption peak = 360 nm; emission peak = 460 nm (see Table of Materials).</li>
		<li>Leave the slide to dry overnight at 4 &#176;C in the dark.</li>
		<li>Observe the fluorescence staining under a fluorescence microscope (see Table of Materials) at 20X magnification equipped with both FITC (absorption peak = 495 nm; emission peak = 525 nm) and Cy3 filters (absorption peak = 550 nm; emission peak = 570 nm).
		<ol>
			<li>To view the samples immediately after the staining, secure the coverslip at the corners using nail polish and examine them under the microscope as above (4.3.3).</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
5. Setting Up the Fluorescence Microscope and Acquisition of Colour Images
<ol>
	<li>Set up the fluorescence microscope as follows.
	<ol>
		<li>Turn on the fluorescence lamp about 10 minutes before use. Turn on the microscope, the connected computer, and the digital camera. Run the image analysis software (see Table of Materials).</li>
		<li>Use the appropriate objective for optimal signal collection and spatial resolution, and select appropriate filters.</li>
	</ol>
	</li>
	<li>Acquisition of Colour Images
	<ol>
		<li>Select the [live] command on the image analysis software (see Table of Materials) and set up the optimal illumination parameters for each filter.</li>
		<li>Select the [freeze] command to take the single channel colour image and add the scale bar (&#39;Edit&#62;Burn Scale&#39;)</li>
		<li>Save each image in the &#39;.tiff&#39; format.</li>
		<li>Merge channels into a single image by selecting &#39;File&#62;Merge channel&#39; and save it in the</li>
		<li>tiff&#39; format.</li>
		<li>Acquire a minimum of four twenty-four bit color pictures (2200 &#181;m x 3400 &#181;m) per brain slice (6 sections per slide).</li>
	</ol>
	</li>
</ol>
6. Assessment of FITC-albumin Extravasation
<ol>
	<li>Analyze the fluorescence intensity for each single channel by using the freely available ImageJ14,15.</li>
	<li>Normalize the total fluorescent signal intensity of the FITC-albumin by the total CY3-Laminin fluorescent intensity per each image and report the extravasated albumin outside the vessels as Green Fluorescence Intensity.</li>
</ol>

<ol>
	<li>Obermeier, B., Verma, A., 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=The+blood-brain+barrier.">The blood-brain barrier.</a> Handb Clin Neurol. 133, 39-59 (2016).</li><li>Serlin, Y., Shelef, I., Knyazer, B., Friedman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+and+physiology+of+the+blood-brain+barrier.">Anatomy and physiology of the blood-brain barrier.</a> Semin Cell Dev Biol. 38, 2-6 (2015).</li><li>Moretti, 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=Blood-brain+barrier+dysfunction+in+disorders+of+the+developing+brain.">Blood-brain barrier dysfunction in disorders of the developing brain.</a> Front Neurosci. 9, 40 (2015).</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>Drouin-Ouellet, J., 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=Cerebrovascular+and+blood-brain+barrier+impairments+in+Huntington's+disease:+Potential+implications+for+its+pathophysiology.">Cerebrovascular and blood-brain barrier impairments in Huntington's disease: Potential implications for its pathophysiology.</a> Ann Neurol. 78 (2), 160-177 (2015).</li><li>Di Pardo, 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=Impairment+of+blood-brain+barrier+is+an+early+event+in+R6/2+mouse+model+of+Huntington+Disease.">Impairment of blood-brain barrier is an early event in R6/2 mouse model of Huntington Disease.</a> Sci Rep. 7, 41316 (2017).</li><li>Lecler, A., Fournier, L., Diard-Detoeuf, C., Balvay, D. <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+Leakage+in+Early+Alzheimer+Disease.">Blood-Brain Barrier Leakage in Early Alzheimer Disease.</a> Radiology. 282 (3), 923-925 (2017).</li><li>van de Haar, H. J., 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=Blood-Brain+Barrier+Leakage+in+Patients+with+Early+Alzheimer+Disease.">Blood-Brain Barrier Leakage in Patients with Early Alzheimer Disease.</a> Radiology. 282 (2), 615 (2017).</li><li>Fernandez-Lopez, D., 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=Blood-brain+barrier+permeability+is+increased+after+acute+adult+stroke+but+not+neonatal+stroke+in+the+rat.">Blood-brain barrier permeability is increased after acute adult stroke but not neonatal stroke in the rat.</a> J Neurosci. 32 (28), 9588-9600 (2012).</li><li>Yang, Y., Rosenberg, G. A. <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+breakdown+in+acute+and+chronic+cerebrovascular+disease.">Blood-brain barrier breakdown in acute and chronic cerebrovascular disease.</a> Stroke. 42 (11), 3323-3328 (2011).</li><li>Walantus, W., Castaneda, D., Elias, L., Kriegstein, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+utero+intraventricular+injection+and+electroporation+of+E15+mouse+embryos.">In utero intraventricular injection and electroporation of E15 mouse embryos.</a> J Vis Exp. (6), e239 (2007).</li><li>Szot, G. L., Koudria, P., Bluestone, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+pancreatic+islets+into+the+kidney+capsule+of+diabetic+mice.">Transplantation of pancreatic islets into the kidney capsule of diabetic mice.</a> J Vis Exp. (9), e404 (2007).</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), (2012).</li><li>McCloy, R. 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=Partial+inhibition+of+Cdk1+in+G+2+phase+overrides+the+SAC+and+decouples+mitotic+events.">Partial inhibition of Cdk1 in G 2 phase overrides the SAC and decouples mitotic events.</a> Cell Cycle. 13 (9), 1400-1412 (2014).</li><li>Burgess, 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=Loss+of+human+Greatwall+results+in+G2+arrest+and+multiple+mitotic+defects+due+to+deregulation+of+the+cyclin+B-Cdc2/PP2A+balance.">Loss of human Greatwall results in G2 arrest and multiple mitotic defects due to deregulation of the cyclin B-Cdc2/PP2A balance.</a> Proc Natl Acad Sci U S A. 107 (28), 12564-12569 (2010).</li><li>Krueger, M., Hartig, W., Reichenbach, A., Bechmann, I., Michalski, D. <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+breakdown+after+embolic+stroke+in+rats+occurs+without+ultrastructural+evidence+for+disrupting+tight+junctions.">Blood-brain barrier breakdown after embolic stroke in rats occurs without ultrastructural evidence for disrupting tight junctions.</a> PLoS One. 8 (2), e56419 (2013).</li><li>Hirano, A., Kawanami, T., Llena, J. 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The authors have no conflicts of interest to disclose

The technique we describe here is primarily useful for detecting BBB leakage under brain disease conditions. BBB dysfunction is gaining attention as a common feature of diverse neurologic disorders4. We previously used this approach to describe the early derangement of BBB integrity in a mouse model of a rare neurodegenerative disease like HD6.
This method takes advantage of the relative simplicity and effectiveness of the procedure, which provid...

An erratum was issued for: <a href="https://www.jove.com/video/56389/assessment-blood-brain-barrier-permeability-intravenous-infusion-fitc">Assessment of Blood-brain Barrier Permeability by Intravenous Infusion of FITC-labeled Albumin in a Mouse Model of Neurodegenerative Disease</a>. One of the author&#39;s names was corrected from:
Maglione Vittorio
to:
Vittorio Maglione

An erratum was issued for: <a href="https://www.jove.com/video/56389/assessment-blood-brain-barrier-permeability-intravenous-infusion-fitc">Assessment of Blood-brain Barrier Permeability by Intravenous Infusion of FITC-labeled Albumin in a Mouse Model of Neurodegenerative Disease</a>. One of the author's names was corrected.

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

Homeostasis within the central nervous system (CNS) is a prerequisite for the proper communication and function of the neuronal cells. The CNS parenchyma is tightly sealed off from the periphery by the endothelial blood-brain barrier (BBB), which represents the interface between the peripheral bloodstream and the brain and plays a pivotal role in the cross-talk between these two districts1,2. The BBB is a complex and dynamic three-dimensional structure mainly composed of specialized micro-vessel endothelial cells (ECs) linked to each other through intercellular junctional complexes - tight junctions (TJs)- and surrounded by pericytes, neuron endings and astrocyte foot processes1,2.
Under physiological conditions, the extremely low permeability of the intact BBB ensures the strict regulation of the transport of nutrients and other molecules into and out of the brain, and provides the CNS with a unique protection from changes occurring in the composition of the blood that might influence neural activity and against potential peripheral insults1,2,3.
Disruption of BBB integrity and its enhanced permeability has long been known to constitute a key feature for many neurological and neurodegenerative disorders4 including Huntington&#39;s Disease (HD)5,6, however, whether such a dysfunction is a causative phenomenon or a propagative event in the course of the disease is still unclear. The timing of BBB breakdown also remains elusive, however, emerging evidence by our group and others indicates that disrupted BBB integrity does not represent a late event in the disease progression, but rather an early step6,7,8, which may have long-term consequences.
With this in mind, it is important to precociously reveal BBB breakdown in neurodegeneration in order to develop strategies useful to predict disease progression and brain damage and to develop alternative and more targeted interventions capable of successfully mitigating the clinical consequences of such a disruption. Reliable imaging of BBB impairment is, therefore, of major importance in both experimental research and clinical management of brain diseases.
In this paper, we describe a successful and simple procedure for the evaluation of BBB permeability in a HD mouse model by using the high molecular weight fluorescein isothiocyanate labelled-albumin (FITC-albumin). Extravasation of FITC-albumin, which normally cannot cross the barrier, into the brain parenchyma was measured as an index of BBB leakage. This technique is readily adaptable to rats and to other pathological conditions characterized by cerebrovasculature impairment9,10.

<table><tbody><tr><td>Albumin-fluorescin isothiocyanate conjiugate</td><td>SIGMA</td><td>A9771-100MG</td><td></td></tr><tr><td>pAb anti-Laminin</td><td>Novus Biologicals</td><td>NB300-144</td><td></td></tr><tr><td>CY3 anti-rabbit made in goat</td><td>MILLIPORE</td><td>AP132</td><td></td></tr><tr><td>SUPERFROST PLUS</td><td>Thermo Scientific</td><td>J1800AMNZ</td><td></td></tr><tr><td>Cover Slips 24 X 50 mm</td><td>Thermo Scientific (DIAPATH)</td><td>61050</td><td></td></tr><tr><td>Kilik Optimal Cutting Temperature (OCT) compound</td><td>Bio Optica</td><td>05-9801</td><td></td></tr><tr><td>VECTASHIELD Mounting Media</td><td>VECTOR</td><td>H-1500</td><td>Mounting media with DAPI</td></tr><tr><td>iNSu/Light Insulin Disposible Syringe</td><td>RAYS Health &amp;amp; Safety</td><td>INS1ML26G13</td><td></td></tr><tr><td>30G 1/2&quot;</td><td>BD Microlance</td><td>304000</td><td>Needle for Insulin disposible Syringe</td></tr><tr><td>Scalpel Handle</td><td>F.S.T.</td><td>91003-12</td><td></td></tr><tr><td>#22 Disposable Scalpel blads</td><td>F.S.T.</td><td>10022-00</td><td></td></tr><tr><td>Fine Iris scissors 10.5 cm</td><td>F.S.T.</td><td>14094-11</td><td></td></tr><tr><td>Dumont Forceps #5745 45&amp;deg; 0.10 x 0.06 mm</td><td>F.S.T.</td><td>11251-35</td><td></td></tr><tr><td>Graefe Forceps 10 cm</td><td>F.S.T.</td><td>11051-10</td><td></td></tr><tr><td>Dumont Forceps #5 0.1 X 0.06 mm</td><td>F.S.T.</td><td>11251-20</td><td></td></tr><tr><td>Medical patch</td><td>Medicalis</td><td>34788</td><td></td></tr><tr><td>Sterile disposable towel drape</td><td>Dispotech</td><td>TVO50VE</td><td></td></tr><tr><td>Stereoscopic Microscope</td><td>NIKON</td><td>SMZ 745 T</td><td></td></tr><tr><td>Optic Illuminator LED light (C-FLED2)</td><td>NIKON</td><td>1003167</td><td>Optic Illuminator for Stereoscopic Micrscope</td></tr><tr><td>Eclipse Ni-U Microscope</td><td>Nikon</td><td>932162</td><td>Epifluorescence Microscope</td></tr><tr><td>Microscope digital Camera</td><td>Nikon</td><td>DS-Ri2</td><td>Microscope camera</td></tr><tr><td>Intenslight</td><td>Nikon</td><td>C-HGFI</td><td>Microscope lamp</td></tr><tr><td>NIS-Elements 64 bit</td><td>Nikon</td><td>AR 4.40.00</td><td>Analysis Software</td></tr><tr><td>Electric Razor</td><td>Gemei</td><td>GM-3007</td><td></td></tr></tbody></table>

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.

Proper infusion of FITC-albumin into the jugular vein results in the extravasation of the green fluorescence tracer from the bloodstream into the brain parenchymawhen the BBB is compromised6. Under physiological conditions, distribution of infused fluorescent albumin is restricted to the inside of the brain blood vessels and no signal in the surrounding perivascular area and/or brain parenchyma is detectable (Figure 1A, micrographs on ...

Watch this Scientific Journal Video about Assessment of Blood-brain Barrier Permeability by Intravenous Infusion of FITC-labeled Albumin in a Mouse Model of Neurodegenerative Disease at JoVE.com

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

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 ...

assessment-blood-brain-barrier-permeability-intravenous-infusion-fitc

Research

JoVE Journal

Neuroscience

9.9K Views.  IRCCS Neuromed. In deze studie presenteren wij een gemakkelijke en efficiënte procedure voor de beoordeling van de verstoring van de bloed - hersenbarrière neurodegeneratieve omstandigheden. Onze om doel te bereiken, we geïnfundeerd ultrahoog moleculair gewicht fluoresceïne isothiocyanaat geëtiketteerd (FITC)-albumine in muis jugular ader en de lekkage in de hersenen parenchym door fluorescentie microscopie geëvalueerd.

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

In Vitro Permeation of FITC-loaded Ferritins Across a Rat Blood-brain Barrier: a Model to Study the Delivery of Nanoformulated Molecules

Brain microvascular endothelial cells, supported by pericytes and astrocytes endfeet, are responsible for the low permeation of large hydrosoluble drugs through the blood-brain barrier (BBB), causing difficulties for effective pharmacological therapies. In recent years, different strategies for promoting brain targeting have aimed to improve drug delivery and activity at this site, including innovative nanosystems for drug delivery across the BBB. In this context, an in vitro approach based on a simplified cellular model of the BBB provides a useful tool to investigate the effect of nanoformulations on the trans-BBB permeation of molecules. This study describes the development of a double-layer BBB, consisting of co-cultured commercially available primary rat brain microvascular endothelial cells and astrocytes. A multiparametric approach for the validation of the model, based on the measurement of the transendothelial electrical resistance and the apparent permeability of a high molecular weight dextran, is also described. As proof of concept for the employment of this BBB model to study the effect of different nanoformulations on the translocation of fluorescent molecules across the barrier, we describe the use of fluorescein isothiocyanate (FITC), loaded into ferritin nanoparticles. The ability of ferritins to improve the trans-BBB permeation of FITC was demonstrated by flux measurements and confocal microscopy analyses. The results suggest this is a useful system for validating nanosystems for delivery of drugs across the BBB.

In Vitro Permeation of FITC-loaded Ferritins Across a Rat Blood-brain Barrier: a Model to Study the Delivery of Nanoformulated Molecules

A method to establish an in vitro model of blood-brain barrier based on a co-culture of rat brain microvascular endothelial cells and astrocytes is described and validated. This system proved to be a valid tool to study the effect of nanoformulation on the trans-barrier permeation of fluorescent molecules.

Brain microvascular endothelial cells, supported by pericytes and astrocytes endfeet, are responsible for the low permeation of large hydrosoluble drugs ...

Bioengineering

Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week protocol for repetitive hypoxic preconditioning (RHP) induces long-term protection against central nervous system (CNS) injury in a mouse model of focal ischemic stroke. RHP consists of 9 stochastic exposures to hypoxia that vary in both duration (2 or 4 hr) and intensity (8% and 11% O2). RHP reduces infarct volumes, blood-brain barrier (BBB) disruption, and the post-stroke inflammatory response for weeks following the last exposure to hypoxia, suggesting a long-term induction of an endogenous CNS-protective phenotype. The methodology for the dual quantification of infarct volume and BBB disruption is effective in assessing neurovascular protection in mice with RHP or other putative neuroprotectants. Adult male Swiss Webster mice were preconditioned by RHP or duration-equivalent exposures to 21% O2 (i.e. room air). A 60 min transient middle cerebral artery occlusion (tMCAo) was induced 2 weeks following the last hypoxic exposure. Both the occlusion and reperfusion were confirmed by transcranial laser Doppler flowmetry. Twenty-two hr after reperfusion, Evans Blue (EB) was intravenously administered through a tail vein injection. 2 hr later, animals were sacrificed by isoflurane overdose and brain sections were stained with 2,3,5- triphenyltetrazolium chloride (TTC). Infarcts volumes were then quantified. Next, EB was extracted from the tissue over 48 hr to determine BBB disruption after tMCAo. In summary, RHP is a simple protocol that can be replicated, with minimal cost, to induce long-term endogenous neurovascular protection from stroke injury in mice, with the translational potential for other CNS-based and systemic pro-inflammatory disease states.

This protocol describes repetitive hypoxic preconditioning, or brief exposures to systemic hypoxia that reduce infarct volumes and blood-brain barrier disruption following transient middle cerebral artery occlusion in mice. It also details dual quantification of infarct volume and blood-brain barrier disruption after stroke to assess the efficacy of neurovascular protection.

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week ...

Medicine

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.

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.

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

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

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 ...

Highly Sensitive Measurement of Glomerular Permeability in Mice with Fluorescein Isothiocyanate-polysucrose 70

The loss of albumin in urine (albuminuria) predicts cardiovascular outcome. Under physiological conditions, small amounts of albumin are filtered by the glomerulus and reabsorbed in the tubular system up until the absorption limit is reached. Early increases in pathological albumin filtration may, thus, be missed by analyzing albuminuria. Therefore, the use of tracers to test glomerular permselectivity appears advantageous. Fluorescently labeled tracer fluorescein isothiocyanate (FITC)-polysucrose (i.e., FITC-Ficoll), can be used to study glomerular permselectivity. FITC-polysucrose molecules are freely filtered by the glomerulus but not reabsorbed in the tubular system. In mice and rats, FITC-polysucrose has been investigated in models of glomerular permeability by using technically complex procedures (i.e., radioactive measurements, high-performance liquid chromatography [HPLC], gel filtration). We have modified and facilitated a FITC-polysucrose tracer-based protocol to test early and small increases in glomerular permeability to FITC-polysucrose 70 (size of albumin) in mice. This method allows repetitive urine analyses with small urine volumes (5 &#181;L). This protocol contains information on how the tracer FITC-polysucrose 70 is applied intravenously and urine is collected via a simple urinary catheter. Urine is analyzed via a fluorescence plate reader and normalized to a urine concentration marker (creatinine), thereby avoiding technically complex procedures.

Here, we present a protocol to test glomerular permeability in mice using a highly sensitive, nonradioactive tracer. This method allows repetitive urine analyses with small urine volumes.

The loss of albumin in urine (albuminuria) predicts cardiovascular outcome. Under physiological conditions, small amounts of albumin are filtered by the ...

Analysis of Blood-Brain Barrier Models via Immunofluorescence Staining

Use of the MicroSiM (&#181;SiM) Barrier Tissue Platform for Modeling the Blood-Brain Barrier

The microSiM (&#181;SiM) is a membrane-based culture platform for modeling the blood-brain barrier (BBB). Unlike conventional membrane-based platforms, the&#160;&#181;SiM provides experimentalists with new capabilities, including live cell imaging, unhindered paracrine signaling between &#39;blood&#39; and &#39;brain&#39; chambers, and the ability to directly image immunofluorescence without the need for the extraction/remounting of membranes. Here we demonstrate the basic use of the platform to establish monoculture (endothelial cells) and co-culture (endothelial cells and pericytes) models of the BBB using ultrathin nanoporous silicon-nitride membranes. We demonstrate compatibility with both primary cell cultures and human induced pluripotent stem cell (hiPSC) cultures. We provide methods for qualitative analysis of BBB models via immunofluorescence staining and demonstrate the use of the &#181;SiM for the quantitative assessment of barrier function in a small molecule permeability assay. The methods provided should enable users to establish their barrier models on the platform, advancing the use of tissue chip technology for studying human tissues.

Author Spotlight: Advancing the Use of Tissue Chip Technology for Studying Human Tissues 

This report provides protocols for assembly, cell culture, and assays on the &#181;SiM platform for the construction of blood-brain barrier models.

The microSiM (&#181;SiM) is a membrane-based culture platform for modeling the blood-brain barrier (BBB). Unlike conventional membrane-based platforms, ...

Measuring Post-Stroke Cerebral Edema, Infarct Zone and Blood-Brain Barrier Breakdown in a Single Set of Rodent Brain Samples

One of the most common causes of morbidity and mortality worldwide is ischemic stroke. Historically, an animal model used to stimulate ischemic stroke involves middle cerebral artery occlusion (MCAO). Infarct zone, brain edema and blood-brain barrier (BBB) breakdown are measured as parameters that reflect the extent of brain injury after MCAO. A significant limitation to this method is that these measurements are normally obtained in different rat brain samples, leading to ethical and financial burdens due to the large number of rats that need to be euthanized for an appropriate sample size. Here we present a method to accurately assess brain injury following MCAO by measuring infarct zone, brain edema and BBB permeability in the same set of rat brains. This novel technique provides a more efficient way to evaluate the pathophysiology of stroke.

This protocol describes a novel technique of measuring the three most important parameters of ischemic brain injury on the same set of rodent brain samples. Using only one brain sample is highly advantageous in terms of ethical and economic costs.

One of the most common causes of morbidity and mortality worldwide is ischemic stroke. Historically, an animal model used to stimulate ischemic stroke ...

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 ...

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 ...

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

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