Name: assessment of blood-brain barrier permeability by intravenous infusion of fitc-labeled albumin in a mouse model of neurodegenerative disease
Uploaded: 2017-11-08T14:00:00
Description: 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

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 l...

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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...

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>
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			<td>pAb anti-Laminin</td>
			<td>Novus Biologicals</td>
			<td>NB300-144</td>
			<td></td>
		</tr>
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			<td>CY3 anti-rabbit made in goat</td>
			<td>MILLIPORE</td>
			<td>AP132</td>
			<td></td>
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			<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>
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		<tr>
			<td>iNSu/Light Insulin Disposible Syringe</td>
			<td>RAYS Health &#38;Safety</td>
			<td>INS1ML26G13</td>
			<td></td>
		</tr>
		<tr>
			<td>30G 1/2&#34;</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>
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			<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>
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		<tr>
			<td>Dumont Forceps #5745 45&#176; 0.10 x 0.06 mm</td>
			<td>F.S.T.</td>
			<td>11251-35</td>
			<td></td>
		</tr>
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			<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>
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</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 ...

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