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...

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 &#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>
		<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&#176; 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 ...

The overall goal of this procedure is to assess the brain-blood barrier permeability in a mouse model of degenerative disorder by intrajugular injection of FITC-labeled albumin. This method can help answer the key questions in the study of brain homeostasis. It can be particularly useful to investigate any possible link between blood-brain barrier damage and neurodegeneration.The main advantage of this technique is that it is fast and easy to perform and can be applied, also, to larger animal models and to other neuro-pathological settings. Demonstrating the surgical procedure of intrajugular injection of FITC-albumen will be Luca Capocci. Salvatore Castaldo will demonstrate the histological sectioning of a brains.And Enrico Amico will demonstrate the procedure for micrograph acquisition. Begin by preparing FITC-albumen solution by dissolving FITC-albumen powder in phosphate buffered saline at 10 milligrams per milliliter. Lay out the autoclaved surgical instruments.Clean the surgical bench with 70%ethanol and place a surgical sterile disposable drape. Then set up the operating stereo microscope. After anesthetizing the mouse with an intraperitoneal injection of an appropriate does of ketamine xylazine solution, monitor animal sedation by giving a gentle toe pinch and ensuring the absence of the withdrawal reflex.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. After the mouse is secured, carefully shave the appropriate surgical margin on the neck and disinfect it with povidone-iodine solution in 70%ethanol. Dry the exposed shaved surface with cotton swabs.Next, after making a 1.5 centimeter long longitudinal incision in the mid-clavicular line, starting midway on the thorax and extending to just below the neck, carefully stretch apart connective tissue with forceps under microscopic observation to expose the external jugular vein. Ensure sufficient space on the right and on the left of the jugular vein to facilitate the insertion of the 30 and a half gauge needle for the infusion of the FITC-albumen solution. Inject 100 microliters of 10 milligrams per milliliter FITC-albumen in the right jugular vein over three minutes.This is the most critical step during the procedure. Incorrect insertion of the needle can cause irreversible damage of the vein wall and compromise the entire experiment. Fifteen minutes after injection, after euthanizing the mouse by decapitation, rapidly remove the brain from the skull and immerse it in a volume of pre-chilled isopentane, that is ten times that of the brain, for 15 minutes.Move the isopentane frozen brain into a pre-chilled tube and store it in a 80 degrees Celsius freezer until histological sectioning. Embed frozen brain in optimal cutting temperature compound prior to cryostat sectioning. Cut the brain into 20 micron thick sections with a cryostat and mount them onto microscope glass slides.Perform the FITC-albumin laminin co-staining by using a specific anti-laminin antibody, as previously described. Cover slip slides with mounting medium containing DAPI and leave the slide to dry overnight at four degrees Celsius in the dark. The next day, observe the fluorescence staining under a fluorescence microscope at 20 times magnification, equipped with both FITC and Cy3 filters.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.Use the appropriate objective for optimal signal collection and spatial resolution and select appropriate filters. Acquire a minimum of four, 24-bit color exposures per brain slice. Analyze the fluorescence intensity for each single channel, using the freely available ImageJ software.The following representative fluorescence micrographs of brain cryosections show differential distribution of green fluorescent albumin under both physiological and pathological conditions. The red staining, shown here, represents the vascular marker, laminin, under physiological conditions and under conditions of blood-brain barrier compromise. These merged images show co-localization of green fluorescent albumin and laminin, along with DAPI staining of nuclei in the intact and compromised blood-brain barriers.This graph displays the quantification of the green fluorescence emitted by FITC-albumin, which is reported as the green fluorescence intensity in both healthy and compromised conditions. While attempting this procedure, it's important to remember to freshly prepare each single reagent and predispose the surgical bench. After watching this video, you may have a good understanding of how to assess the permeability of the blood-brain barrier in an animal model of neurodegenerative disease, by measuring the affixation of the green fluorescent albumin into brain parenchyma.

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

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Neuroscience

9.7K 조회수.  IRCCS Neuromed. The overall goal of this procedure is to assess the brain-blood barrier permeability in a mouse model of degenerative disorder by intrajugular injection of FITC-labeled albumin. This method can help answer the key questions in the study of brain homeostasis. It can be particularly useful to investigate any possible link between blood-brain barrier damage and neurodegeneration.The main advantage of this technique is that it is fast and easy to perform and can be applied, also, to larger...