We thank the imaging platform of the Institut de la Vision. We acknowledge the French Muscular Dystrophy Association (AFM) for a PhD fellowship to O.V. and Allergan INC. This work performed in the frame of the LABEX LIFESENSES [reference ANR-10-LABX-65] was supported by French state funds managed by the ANR. We thank Peggy Barbe, and M&#233;lissa Desrosiers for technical assistance with AAV preparations. We are grateful to St&#233;phane Fouquet for excellent technical assistance in confocal microscopy and his expert input with the interpretation of the results.

All animals used in this study were cared for and handled according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
1. Production of Recombinant AAV (rAAV) by Transient Transfection of HEK-293 Cells17,18
NOTE: See McClure C, JoVE (2011)19.
<ol>
	<li>Purify a large-scale plasmid preparation (at least 1 mg/ml) of the AAV vector plasmids. Use 3 plasmids. The AAV helper plasmid carrying the replication and capsid genes without the AAV inverted terminal repeats ITRs, the transgene expression cassette carrying the AAV ITR&#160;referred to as the transfer plasmid, and the plasmid supplying the adenovirus helper genes E2, E4, and VA RNA genes referred to as pHELPER.</li>
	<li>Transfect 293 cells with these 3 plasmids using polyethyleneimine (or other transfection reagent). Above 80% of transfection efficiency is necessary for good viral yields.</li>
	<li>Purify recombinant AAV from 293 cell lysates on an iodixanol gradient. 15%, 25%, 40%, and 60% iodixanol is used to obtain the gradient. Collect 40% iodixanol fraction containing AAV particles, concentrate the fraction after ultracentrifugation at 500,000 x g for 1 hr and buffer exchange against PBS (phosphate buffered saline)&#160;with 0.001% Pluronic.</li>
	<li>To determine viral genomic titer20, perform a DNAse ProteinaseK digest followed by QPCR. The forward and reverse primers amplify the 62 bp region located in the AAV2 ITR. For the plasmid standard, the probe is labeled with FAM and has a black hole quencher (BHQ).</li>
	<li>Carry out qPCR (quantitative polymerase chain reaction)&#160;&#160;in a final volume of 25 &#956;l using 340 nM reverse ITR primer, 100 nM forward ITR primer, 100 nM AAV2 ITR probe, 0.4 mM dNTPs, 2 mM MgCl2, 1x Platinum Taq Buffer, 1U Platinum Taq and 5 &#956;l template (standard, sample and no template control).</li>
	<li>For the standard, use the plasmid for transfection in 7 x 10-fold serial dilutions (5 &#181;l each of 2 x 109 to 2 x 103 genomes/ml resulting in a final concentration from 1010 to 104 genomes/ml).</li>
	<li>Begin the program by an initial denaturation step at 95 &#176;C for 15 min followed by 40 cycles of denaturation at at 95 &#176;C for 1 min and annealing/extension at 60 &#176;C for 1 min. Store at 4 &#176;C for a short-term storage or at -20 &#176;C for long storage periods.</li>
</ol>
2. Intravitreal Injection of AAV
<ol>
	<li>Anesthetize C57BL6J mice with ketamine (50 mg/kg) and xylazine (10 mg/kg) and verify the loss of righting reflex, the withdrawal reflex and tail pinch response indicating a proper anesthetization. Dilate pupils by corneal application of neosynephrine 5% and mydriaticum 0.5% eye drops. Use vet ointment on eyes to prevent dryness while under anesthesia.</li>
	<li>Pass an ultrafine 30 G disposable needle through the equator and next to the limbus, into the vitreous cavity (see Chiu K, JoVE (2007)21). Inject 1 &#181;l stock containing 1 to 4 x 1011 vp of ShH10-GFP or AAV5-GFP with direct observation of the needle in the center of the vitreous cavity (Figure 1). Use one eye as a control for ILM experiments. Inject both eyes for BRB experiments. Apply an anti-inflammatory and antibacterial topical treatment on injected eyes.</li>
	<li>Dilate pupils with neosynephrine and mydriaticum eye drops for imaging. Perform fundus examinations with an eye fundus camera to follow GFP (Green Fluorescent Protein)&#160;expression 1 week, 2 weeks, 1 month, and 2 months after intravitreal injection (Figure 1). Sacrifice mice by CO2 inhalation 1 to 2 months post-injection.</li>
</ol>
3. Immunohistochemistry
<ol>
	<li>For retinal cryosections (Figure 1), dissect enucleated eyes to remove lens and cornea, and immersion fix in 4% paraformaldehyde for 1 hr.
	<ol>
		<li>Cryoprotect the previously fixed eyes in 10% sucrose for 1 hr at room temperature, 20% sucrose for another hr at room temperature. Cryoprotect in 30% sucrose solution, overnight at 4 &#176;C. Freeze and embed eyecups in embedding resin such as Cryo-matrix. Make 10 &#181;m cryosections and mount on slides specially treated for frozen tissue sections and that improve tissue adherence during staining.</li>
		<li>Permeabilize sections with 0.1% Triton X100 in PBS pH 7.4&#160;for 5 min. Wash 2x in PBS and block for 1 hr at room temperature in PBS with 1% BSA, 0.1% Tween 20.</li>
	</ol>
	</li>
	<li>For retinal flatmounts (Figure 1), fix enucleated eyes in 4% paraformaldehyde for 15 min to facilitate the dissection.
	<ol>
		<li>In 15-30 min, dissect the eyes to remove the cornea and lens. Separate the retina from the retinal pigment epithelium (RPE) and sclera by cutting around the ora serrata and the optic nerve. Immerse in 4% paraformaldehyde for another 30 min to fix the retina and the fluorescent protein in transduced retinal cells (fixation must not exceed 24 hr). Wash 5 min in sterile PBS, pH 7.4.</li>
		<li>Change the PBS to blocking buffer (PBS with 1% BSA, 2% normal goat or donkey serum, 0.5% Triton X-100), and incubate in blocking buffer for either 4 hr at room temperature or 4 &#176;C overnight.</li>
	</ol>
	</li>
	<li>Incubate tissues with primary antibodies in blocking buffer for either 4 hr at room temperature or at 4 &#176;C overnight. Wash 3x with PBS. 
	NOTE: The antibodies used are as follows: anti-laminin (1/1,000) labeling the ILM, anti-rhodopsin clone 4D2 (1/500) labeling rods, anti-glutamine synthetase clone GS-6 (1/1,500) labeling M&#252;ller cells, PNA Lectin (1/40) labeling cones.</li>
	<li>Incubate tissues with 1:500 dilution of alexa fluor conjugated secondary antibodies in blocking buffer for 1 hr (cryosections) or 2 hr (flatmounts) at room temperature and protected from light. Wash 3x with PBS.</li>
	<li>Make relieving cuts to the retina and flatmount it onto a glass slide with either the photoreceptor or retinal ganglion cell (RGC) side facing upwards. Add aqueous mountingmedium, apply coverslip and store at 4 &#176;C.</li>
	<li>Perform confocal microscopy on laser-scanning confocal microscope. Acquire images sequentially, line by line, to reduce excitation and emission crosstalk. Define step size according to the Nyquist&#8211;Shannon sampling theorem. Use exposure settings that minimize oversaturated pixels in the final images. Process 12-bit images with FIJI, project Z-sections on a single plane using maximum intensity under Z-project function and finally convert to 8-bit RGB color mode.</li>
</ol>
4. PCR Analysis of Mouse Blood Samples
<ol>
	<li>Inject 100 &#181;l stock containing 1 to 4 x 1011 particles of an AAV-GFP into the penile vein of anesthetized C57BL6J mice (5% of isoflurane for induction in a close chamber and 2.5% of isoflurane through a nose cone during surgery) using an insulin syringe with an ultra-fine 6 mm needle. This animal will serve as a positive control of circulating AAV particles.</li>
	<li>Sample blood from mouse tail before injection and 3 hr, 24 hr, 2 days, and 3 days after ShH10 intravitreal injection or after intrapenile injection (Figure 1). About 10-20 &#181;l is collected in heparin tubes. Sacrifice mice by CO2 inhalation after completion of the procedure.</li>
	<li>Extract genomic DNA from blood samples using the extraction kit of your choice.</li>
	<li>Perform PCR amplification of genomic DNA. For this protocol use the following primer pair: GFP, sense 5&#8217;-CGACACAATCTGCCCTTTCG-3&#8217;, antisense 5&#8217;-CATGGACGAGCTGTACAAGGGA-3&#8217;. The following PCR program was performed: 95 &#176;C 2 min (1 cycle); 95 &#176;C 45 sec, 55 &#176;C 1 min, 72 &#176;C 45 sec (30 cycles); 72&#176;C 5 min (1 cycle); 4 &#176;C hold.</li>
</ol>
5. Optional Evans Blue Method
NOTE: Quantify vascular permeability by measuring albumin leakage from blood vessels into the retina using the Evans blue method10.
<ol>
	<li>Prepare Evans Blue dye by dissolution in a normal saline solution (6 mg/ml), sonication for 5 min in an ultrasonic cleaner, and filtration through a 5 &#181;m filter.</li>
	<li>Anesthetize C57BL6J mice (isoflurane inhalation, see step 4.1), check the loss of righting reflex, the withdrawal reflex and tail pinch response indicating a proper anesthetization, and inject Evans blue (45 mg/kg) through the penile vein (Figure 1). Check that paws, muzzle, and ears of mice become clearly blue following Evans blue injection, confirming the uptake and distribution of the dye.</li>
	<li>Anesthetize C57BL6J mice 3 hr after injection of the dye with ketamine (50 mg/kg) and xylazine (10 mg/kg) and verify the loss of righting reflex indicating a proper anesthetization. Sample intracardially about 500 &#181;l of blood in heparin tubes and perfuse mice for 2 min via the left ventricle with a citrate buffer (0.05 M, pH 3.5; dissolve 7.8 g of citric acid and 3.8 g of sodium citrate in 1 L of distilled water) pre-warmed to 37 &#176;C to prevent vasoconstriction. Mice are sacrificed via the perfusion.</li>
	<li>After perfusion, enucleate and carefully dissect both eyes under an operating microscope to collect the retinas (see Section 3.2). Dry retinas in a centrifugal evaporator overnight, weigh, and extract the Evans blue dye by incubating retinas with 100 &#956;l of formamide for 18 hr at 65 &#176;C with shaking (100 x g). Centrifuge retina samples in 30K omega filter tubes at 20,798.5 x g for 2 hr at 4 &#176;C and centrifuge blood samples at 20,798.5 x g for 15 min at 4 &#176;C.</li>
	<li>Use both supernatants to measure absorbance. Determine absorbance of each sample by subtracting the absorbance maximum for Evans blue at 620 nm to the absorbance minimum at 740 nm. Calculate Evans blue concentration in plasma and retina from a standard curve of Evans blue in formamide. Express BRB permeability in microliter of Evans blue per gram of dry retina per hour (&#181;l plasma/g retinal dry wt/hr).</li>
</ol>
6. Optional Fluorescein Angiography
NOTE: Leakage from retinal blood vessels in mice can be visualized by intraperitoneal injection of sodium fluorescein.
<ol>
	<li>Anesthetize C57BL6J mice (isoflurane inhalation, see step 4.1) and dilate pupils with eye drops (neosynephrine and mydriaticum). Use vet ointment on eyes to prevent dryness while under anesthesia.</li>
	<li>Inject intraperitoneally 0.01-0.02 ml of 10% sodium fluorescein in 0.1 ml sterile saline for fluorescein angiography.</li>
	<li>Collect images of both eyes after injection using an eye fundus camera. Sacrifice mice by CO2 inhalation after the procedure. 
	NOTE: 20 sec are necessary for the retinal vessels to become visible on angiography. Images have to be captured between 3-10 min maximum after fluorescein injection. The leakage spots (if present) are observable after 2.5 min. At later time points the image quality is lost due to sodium fluorescein diffusing into the vitreous, thus only images shortly obtained after are used for analysis.</li>
</ol>

<ol>
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The authors have nothing to disclose.

The BRB regulates the exchange of molecules between blood and retina. Its breakdown is associated with various diseases such as diabetic retinopathy or age-related macular degeneration (AMD). We recently showed that in a dystrophin knock-out mouse, which displays permeable BRB, the retina becomes more permissive to gene delivery mediated by adeno-associated viral vectors (AAV). However, despite BRB permeability AAV particles injected intraocularly stay confined to the ocular compartment in this model. Our results indicat...

M&#252;ller cells are the major glial component of the retina. Morphologically, they span the retina radially and their endfeet, in contact with the vitreous, face the ILM and secret components of the latter. The ILM is a basement membrane composed of about ten different extracellular matrix proteins (laminin, agrin, perlecan, nidogen, collagen and several heparin sulfate proteoglycans). During development, its presence is indispensable for retinal histogenesis, navigation of optic axons, and&#160;survival of ganglion cells1-3. However, ILM is unessential in adult retina and can be surgically removed in certain pathologies without causing retinal damage4. In gene therapy, this membrane becomes a physical barrier for efficient transduction of the retina using AAVs by intravitreal injection5.
Through the extensive arborization of their processes, M&#252;ller cells provide nutritional and regulatory support to both retinal neurons and vascular cells. M&#252;ller cells are also involved in the regulation of the retinal homeostasis, in the formation and maintenance of the BRB6. Tight junctions between retinal capillary endothelial cells, M&#252;ller cells, astrocytes and pericytes form the BRB. BRB prevents certain substances from entering the retina.In many diseases like diabetic retinopathy, retinal vein occlusion and respiratory diseases, hypoxia of the retina causes leakage through the BRB7-9. This rupture is associated with an increase in vascular permeability leading to vasogenic edema, retinal detachment and retinal damage.
M&#252;ller cells are tightly associated with blood vessels and the basement membrane, playing an important role in both BRB and ILM integrity. Consequently, labeling M&#252;ller glial cells is particularly relevant to the study of the physical state of these retinal barriers.
Classically, BRB permeability is measured using the Evans blue assay consisting of systemic injection of Evans blue dye, which binds non-covalently to plasma albumin. This assay measures the albumin leakage (protein of intermediate size, ~66 kDa) from blood vessels into the retina (see Protocols Section 5)10. Alternatively, the vascular leakage can be visualized by fluorescence retinal angiography attesting for leakage of fluorescein (small molecule, ~359 Da; see Protocols Section 6)11. Nevertheless, both methods allow evaluation of the BRB permeability to small molecules and proteins but they do not provide information about the ILM integrity.
Hence, to study BRB permeability, we used an AAV based method that gives information on the BRB permeability to larger molecules (e.g., AAV particles, 25 nm diameter). Indeed, our method can detect presence of AAV transgene in the blood, which would suggest that ~25 nm diameter particles would be able to infiltrate into the bloodstream. This method also provides information on the structure of the ILM and extracellular matrix proteins in pathological conditions. Two AAV variants are useful for such study: AAV5 and ShH10. Subretinally injected, AAV5 has a natural tropism for photoreceptors and retinal pigment epithelium12 but it cannot get across to the outer retina when administered into the vitreous in wild-type retinas with intact ILM5,13. ShH10 is an AAV variant that has been engineered to specifically target glial cells over neurons14,15. ShH10 selectively labels M&#252;ller cells in both healthy and diseased retinas with increased efficiency in retinas with compromised barriers16. These viral tools coupled with immuhistochemistry and blood-DNA analysis provide information on the state of retinal barriers and their involvement in disease (Figure 1).

<table border="0" cellpadding="0" cellspacing="0" width="643">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Name of Material/ Equipment</td>
			<td style="width:107px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:193px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">C57BL6J mice strain</td>
			<td style="width:107px;">JANVIER LABS</td>
			<td style="width:119px;"></td>
			<td>mice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Ketamine 500</td>
			<td style="width:107px;">Virbac France</td>
			<td style="width:119px;"></td>
			<td>anesthetic</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:225px;">Xylazine Rompun 2%</td>
			<td style="width:107px;">Bayer Healthcare</td>
			<td style="width:119px;"></td>
			<td>anesthetic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Neosynephrine 5% Faure</td>
			<td style="width:107px;">Europhta</td>
			<td style="width:119px;"></td>
			<td>dilatant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Mydriaticum 0,5%</td>
			<td style="width:107px;">Thea</td>
			<td style="width:119px;"></td>
			<td>dilatant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Sterdex</td>
			<td style="width:107px;">Novartis</td>
			<td style="width:119px;"></td>
			<td>anti-inflammatory</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:225px;">Cryomatrix embedding resin</td>
			<td style="width:107px;">Thermo Scientific</td>
			<td style="width:119px;">6769006</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:225px;">Superfrost Plus Adhesion Slides</td>
			<td style="width:107px;">Thermo Scientific</td>
			<td style="width:119px;">10143352</td>
			<td>slides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">anti-laminin&#160;</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:119px;">L9393</td>
			<td>antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">anti-rhodopsin clone 4D2&#160;</td>
			<td style="width:107px;">Millipore</td>
			<td style="width:119px;">MABN15</td>
			<td>antibody</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:225px;">anti-glutamine synthetase clone GS-6&#160;</td>
			<td style="width:107px;">Millipore</td>
			<td style="width:119px;">MAB302</td>
			<td>antibody</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:225px;">Anti-Glial Fibrillary Acidic Protein</td>
			<td style="width:107px;">Dako</td>
			<td style="width:119px;">334</td>
			<td>antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">PNA Lectin&#160;</td>
			<td style="width:107px;">Invitrogen</td>
			<td style="width:119px;">L32459</td>
			<td>probe</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:225px;">Alexa fluor conjugated secondary antibodies&#160;</td>
			<td style="width:107px;">Invitrogen</td>
			<td style="width:119px;"></td>
			<td>antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Fluorsave reagent</td>
			<td style="width:107px;">Calbiochem</td>
			<td style="width:119px;">345789</td>
			<td>mounting medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">QIAmp DNA Micro Kit&#160;</td>
			<td style="width:107px;">QIAGEN</td>
			<td style="width:119px;">56304</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">GoTaq DNA polymerase</td>
			<td style="width:107px;">Promega</td>
			<td style="width:119px;">M3001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Evans Blue dye&#160;</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:119px;">E2129&#160;</td>
			<td>dye</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">5 &#181;m filter&#160;</td>
			<td style="width:107px;">Millipore</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Sodium Citrate&#160;</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:119px;">S1804</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Citric acid&#160;</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:119px;">C1909-2.5KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Formamide spectrophotometric&#160;</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:119px;">295876-2L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Fluorescein</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:119px;">F2456&#160;</td>
			<td>dye</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:225px;">Micron III</td>
			<td style="width:107px;">Phoenix Research Labs</td>
			<td style="width:119px;"></td>
			<td style="width:193px;">Microscopy system based on 3-CCD color camera, frame grabber, and off-the-shelf software enables researchers to image mouse retinas.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Insulin Syringes</td>
			<td style="width:107px;">Terumo</td>
			<td style="width:119px;">SS30M3109&#160;</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Syringe 10 &#181;l Hamilton</td>
			<td style="width:107px;">Dutscher</td>
			<td style="width:119px;">74487</td>
			<td>Seringue 1701</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:225px;">Needle RN G33, 25 mm, PST 2&#160;</td>
			<td style="width:107px;">Fisher Scientific</td>
			<td style="width:119px;">11530332</td>
			<td>Intravitreal Injection</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:225px;">UltraMicroPump UMP3</td>
			<td rowspan="2" style="width:107px;">World Precision Instruments</td>
			<td style="width:119px;">UMP3</td>
			<td style="width:193px;">Versatile injector uses microsyringes to deliver picoliter volumes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:225px;">UltraMicroPump (UMP3) (one) with SYS-Micro4 Controller</td>
			<td style="width:119px;">UMP3-1</td>
			<td>Digital controller</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:225px;">Binocular magnifier SZ76</td>
			<td style="width:107px;">ADVILAB</td>
			<td style="width:119px;">ADV-76B2</td>
			<td style="width:193px;">Zoom 0.66 x 5 x LEDs with stand epi and dia / Retinas dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Spring scissors straight - 8,5cm</td>
			<td rowspan="3" style="width:107px;">Bionic France S.a.r.l</td>
			<td style="width:119px;">15003-08</td>
			<td rowspan="3" style="width:193px;">Retinas dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Micro-ciseaux de Vannas courbe</td>
			<td style="width:119px;">15004-08</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Pince Dumont 5</td>
			<td style="width:119px;">11254-20</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:225px;">Veriti 96-Well Thermal Cycler</td>
			<td style="width:107px;">Life technologies</td>
			<td style="width:119px;">4375786</td>
			<td>Thermocycler</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:225px;">Ultrasonic cleaner&#160;</td>
			<td style="width:107px;">Laboratory Supplies</td>
			<td style="width:119px;">G1125P1T</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Nanosep 30k omega tubes&#160;</td>
			<td style="width:107px;">VWR</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:225px;">Speedvac</td>
			<td style="width:107px;">Fisher Scientific</td>
			<td style="width:119px;">SC 110 A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Spectrofluorometer&#160;</td>
			<td style="width:107px;">TECAN&#160;</td>
			<td style="width:119px;">infinite M1000</td>
			<td></td>
		</tr>
	</tbody>
</table>

using adeno-associated virus as a tool to study retinal barriers in disease

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes (ILM), and in conjunction with astrocytes, pericytes and endothelial cells they establish the blood-retinal barrier (BRB). BRB limits material transport between the bloodstream and the retina while the ILM acts as a basement membrane that defines histologically the border between the retina and the vitreous cavity. Labeling M&#252;ller cells is particularly relevant to study the physical state of the retinal barriers, as these cells are an integral part of the BRB and ILM. Both BRB and ILM are frequently altered in retinal disease and are responsible for disease symptoms.
There are several well-established methods to study the integrity of the BRB, such as the Evans blue assay or fluorescein angiography. However these methods do not provide information on the extent of BRB permeability to larger molecules, in nanometer range. Furthermore, they do not provide information on the state of other retinal barriers such as the ILM. To study BRB permeability alongside retinal ILM, we used an AAV based method that provides information on permeability of BRB to larger molecules while indicating the state of the ILM and extracellular matrix proteins in disease states. Two AAV variants are useful for such study: AAV5 and ShH10. AAV5&#160;has a natural tropism for photoreceptors but it cannot get across to the outer retina when administered into the vitreous when the ILM is intact (i.e., in wild-type retinas). ShH10 has a strong tropism towards glial cells and will selectively label M&#252;ller glia in both healthy and diseased retinas. ShH10 provides more efficient gene delivery in retinas where ILM is compromised. These viral tools coupled with immunohistochemistry and blood-DNA analysis shed light onto the state of retinal barriers in disease.

We expect increased retinal transduction of M&#252;ller glial cells using ShH10 if the animal model shows perturbations in the structure of the ILM (Figure 2A-B). For example, we have shown that in absence of Dp71,&#160;ShH10 targets specifically but more efficiently M&#252;ller glial cells by intravitreal injection, indicating increased permeability of the ILM in this mouse line compared to wild-type mice16 (Figure 2C-F).
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Watch this Scientific Journal Video about Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease at JoVE.com

Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

To investigate the blood-retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease, we used several adeno-associated virus (AAV) variants as tools to label retinal neurons and glia. Virus mediated reporter gene expression is then used as an indicator of retinal barrier permeability.

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes ...

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15.0K Views.  Sorbonne Universtés, UPMC Univ Paris 06, UMR_S 968. To investigate the blood-retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease, we used several adeno-associated virus (AAV) variants as tools to label retinal neurons and glia. Virus mediated reporter gene expression is then used as an indicator of retinal barrier permeability.

Video: Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a number of gene therapy clinical trials. More recently, a number of additional AAV serotypes have also been isolated, which have been shown to exhibit selective tissue-tropism in various small and large animal models1. Of the 10 most commonly used AAV serotypes, AAV3 is by far the least efficient in transducing cells and tissues in vitro as well as in vivo.
However, in our recently published studies, we have documented that AAV3 vectors transduce human liver cancer - hepatoblastoma (HB) and hepatocellular carcinoma (HCC) - cell lines extremely efficiently because AAV3 utilizes human hepatocyte growth factor receptor as a cellular co-receptor for binding and entry in these cells2,3.
In this article, we describe the steps required to achieve high-efficiency transduction of human liver cancer cells by recombinant AAV3 vectors carrying a reporter gene. The use of recombinant AAV3 vectors carrying a therapeutic gene may eventually lead to the potential gene therapy of liver cancers in humans.

In this article, we describe the identification of the adeno-associated virus serotype 3 (AAV3) as the most efficient vector for targeting human liver cancer cells.

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a ...

Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being analyzed 1, 2. This classical banding technique, however, is not reliable in identifying complex chromosomal aberrations such as those associated with cancer. To overcome the limitations of the banding technique, Spectral Karyotyping (SKY) is introduced to provide much reliable information on chromosome abnormalities.
SKY is a multicolor fluorescence in-situ hybridization (FISH) technique to detect metaphase chromosomes with spectral microscope 3, 4. SKY has been proven to be a valuable tool for the cytogenetic analysis of a broad range of chromosome abnormalities associated with a large number of genetic diseases and malignancies 5, 6. SKY involves the use of multicolor fluorescently-labelled DNA probes prepared from the degenerate oligonucleotide primers by PCR. Thus, every chromosome has a unique spectral color after in-situ hybridization with probes, which are differentially labelled with a mixture of fluorescent dyes (Rhodamine, Texas Red, Cy5, FITC and Cy5.5). The probes used for SKY consist of up to 55 chromosome specific probes 7-10.
The procedure for SKY involves several steps (Figure 1). SKY requires the availability of cells with high mitotic index from normal or diseased tissue or blood. The chromosomes of a single cell from either a freshly isolated primary cell or a cell line are spread on a glass slide. This chromosome spread is labeled with a different combination of fluorescent dyes specific for each chromosome. For probe detection and image acquisition,the spectral imaging system consists of sagnac interferometer and a CCD camera. This allows measurement of the visible light spectrum emitted from the sample and to acquire a spectral image from individual chromosomes. HiSKY, the software used to analyze the results of the captured images, provides an easy identification of chromosome anomalies. The end result is a metaphase and a karyotype classification image, in which each pair of chromosomes has a distinct color (Figure 2). This allows easy identification of chromosome identities and translocations. For more details, please visit Applied Spectral Imaging website (<a href="http://www.spectral-imaging.com/" target="_blank">http://www.spectral-imaging.com/</a>).
SKY was recently used for an identification of chromosome segregation defects and chromosome abnormalities in humans and mice with Autosomal Dominant Polycystic Kidney Disease (ADPKD), a genetic disease characterized by dysfunction in primary cilia 11-13. Using this technique, we demonstrated the presence of abnormal chromosome segregation and chromosomal defects in ADPKD patients and mouse models 14. Further analyses using SKY not only allowed us to identify chromosomal number and identity, but also to accurately detect very complex chromosomal aberrations such as chromosome deletions and translocations (Figure 2).

Spectral Karyotyping (SKY) is an advanced cytogenetics technique to identify genomic and chromosomal aberrations. This technique takes advantage of chromosome painting probes, which allow classification of all chromosomes. SKY can also identify complex chromosome aberrations and segregation defects in mice and humans with various diseases, including polycystic kidney disease.

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being ...

Osteopathic Manipulative Treatment as a Useful Adjunctive Tool for Pneumonia

Pneumonia, the inflammatory state of lung tissue primarily due to microbial infection, claimed 52,306 lives in the United States in 20071 and resulted in the hospitalization of 1.1 million patients2. With an average length of in-patient hospital stay of five days2, pneumonia and influenza comprise significant financial burden costing the United States $40.2 billion in 20053. Under the current Infectious Disease Society of America/American Thoracic Society guidelines, standard-of-care recommendations include the rapid administration of an appropriate antibiotic regiment, fluid replacement, and ventilation (if necessary). Non-standard therapies include the use of corticosteroids and statins; however, these therapies lack conclusive supporting evidence4. (Figure 1)

Osteopathic Manipulative Treatment (OMT) is a cost-effective adjunctive treatment of pneumonia that has been shown to reduce patients&#8217; length of hospital stay, duration of intravenous antibiotics, and incidence of respiratory failure or death when compared to subjects who received conventional care alone5. The use of manual manipulation techniques for pneumonia was first recorded as early as the Spanish influenza pandemic of 1918, when patients treated with standard medical care had an estimated mortality rate of 33%, compared to a 10% mortality rate in patients treated by osteopathic physicians6. When applied to the management of pneumonia, manual manipulation techniques bolster lymphatic flow, respiratory function, and immunological defense by targeting anatomical structures involved in the these systems7,8, 9, 10.

The objective of this review video-article is three-fold: a) summarize the findings of randomized controlled studies on the efficacy of OMT in adult patients with diagnosed pneumonia, b) demonstrate established protocols utilized by osteopathic physicians treating pneumonia, c) elucidate the physiological mechanisms behind manual manipulation of the respiratory and lymphatic systems. Specifically, we will discuss and demonstrate four routine techniques that address autonomics, lymph drainage, and rib cage mobility: 1) Rib Raising, 2) Thoracic Pump, 3) Doming of the Thoracic Diaphragm, and 4) Muscle Energy for Rib 1.5,11

Pneumonia is one of the top ten leading causes of death in the U.S. Osteopathic manipulative techniques (OMT) may be utilized as an adjunctive therapy in the treatment of pneumonia to enhance biomechanical and immune function; and ultimately, to reduce hospital stay, duration of antibiotics, incidence of respiratory failure, and mortality. In this video-article, we will review randomized controlled studies on the use of OMT in pneumonia patients, as well as demonstrate specific hands-on techniques that are routinely practiced by osteopathic physicians when treating pulmonary infections.

Pneumonia, the inflammatory state of lung tissue primarily due to microbial infection, claimed 52,306 lives in the United States in 20071 and resulted in ...

Fundus Photography as a Convenient Tool to Study Microvascular Responses to Cardiovascular Disease Risk Factors in Epidemiological Studies

The microcirculation consists of blood vessels with diameters less than 150 &#181;m. It makes up a large part of the circulatory system and plays an important role in maintaining cardiovascular health. The retina is a tissue that lines the interior of the eye and it is the only tissue that allows for a non-invasive analysis of the microvasculature. Nowadays, high-quality fundus images can be acquired using digital cameras. Retinal images can be collected in 5 min or less, even without dilatation of the pupils. This unobtrusive and fast procedure for visualizing the microcirculation is attractive to apply in epidemiological studies and to monitor cardiovascular health from early age up to old age.
Systemic diseases that affect the circulation can result in progressive morphological changes in the retinal vasculature. For example, changes in the vessel calibers of retinal arteries and veins have been associated with hypertension, atherosclerosis, and increased risk of stroke and myocardial infarction. The vessel widths are derived using image analysis software and the width of the six largest arteries and veins are summarized in the Central Retinal Arteriolar Equivalent (CRAE) and the Central Retinal Venular Equivalent (CRVE). The latter features have been shown useful to study the impact of modifiable lifestyle and environmental cardiovascular disease risk factors.
The procedures to acquire fundus images and the analysis steps to obtain CRAE and CRVE are described. Coefficients of variation of repeated measures of CRAE and CRVE are less than 2% and within-rater reliability is very high. Using a panel study, the rapid response of the retinal vessel calibers to short-term changes in particulate air pollution, a known risk factor for cardiovascular mortality and morbidity, is reported. In conclusion, retinal imaging is proposed as a convenient and instrumental tool for epidemiological studies to study microvascular responses to cardiovascular disease risk factors.

Retinal image analysis is an unobtrusive procedure for visualizing the microcirculation. The impact of cardiovascular disease risk factors can result in changes of retinal vessel calibers. The procedures to acquire fundus images and the steps for calculating the vessel calibers are described.

The microcirculation consists of blood vessels with diameters less than 150 &#181;m. It makes up a large part of the circulatory system and plays an ...

Adapting Human Videofluoroscopic Swallow Study Methods to Detect and Characterize Dysphagia in Murine Disease Models

This study adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating translational dysphagia research. Successful outcomes are dependent upon three critical components: test chambers that permit self-feeding while standing unrestrained in a confined space, recipes that mask the aversive taste/odor of commercially-available oral contrast agents, and a step-by-step test protocol that permits quantification of swallow physiology. Elimination of one or more of these components will have a detrimental impact on the study results. Moreover, the energy level capability of the fluoroscopy system will determine which swallow parameters can be investigated. Most research centers have high energy fluoroscopes designed for use with people and larger animals, which results in exceptionally poor image quality when testing mice and other small rodents. Despite this limitation, we have identified seven VFSS parameters that are consistently quantifiable in mice when using a high energy fluoroscope in combination with the new murine VFSS protocol. We recently obtained a low energy fluoroscopy system with exceptionally high imaging resolution and magnification capabilities that was designed for use with mice and other small rodents. Preliminary work using this new system, in combination with the new murine VFSS protocol, has identified 13 swallow parameters that are consistently quantifiable in mice, which is nearly double the number obtained using conventional (i.e., high energy) fluoroscopes. Identification of additional swallow parameters is expected as we optimize the capabilities of this new system. Results thus far demonstrate the utility of using a low energy fluoroscopy system to detect and quantify subtle changes in swallow physiology that may otherwise be overlooked when using high energy fluoroscopes to investigate murine disease models.

This study successfully adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating translational dysphagia research.

This study adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating ...

Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on animal models. The identification of PD-associated genes has led to the development of genetic PD models. Most transgenic &#945;-SYN mouse models develop gradual &#945;-SYN pathology but fail to display clear dopaminergic cell loss and dopamine-dependent behavioral deficits. This hurdle was overcome by direct targeting of the substantia nigra with viral vectors overexpressing PD-associated genes. Local gene delivery using viral vectors provides an attractive way to express transgenes in the central nervous system. Specific brain regions can be targeted (e.g. the substantia nigra), expression can be induced in the adult setting and high expression levels can be achieved. Further, different vector systems based on various viruses can be used. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based alpha-synuclein animal model for Parkinson&#39;s disease.

Development of an Alpha-synuclein Based Rat Model for Parkinson&#039;s Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

This manuscript describes how viral vector-mediated local gene delivery provides an attractive way to express transgenes in the central nervous system. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based animal model for Parkinson's disease.

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on ...

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a condition that typically involves damage to or loss of the delicate mechanosensory structures of the inner ear. Sophisticated in vitro and ex vivo assays have emerged in recent years, enabling the screening of an increasing number of potentially therapeutic compounds while minimizing resources and accelerating efforts to develop cures for SNHL. Though homogenous cultures of certain cell types continue to play an important role in current research, many scientists now rely on more complex organotypic cultures of murine inner ears, also known as cochlear explants. The preservation of organized cellular structures within the inner ear facilitates the in situ evaluation of various components of the cochlear infrastructure, including inner and outer hair cells, spiral ganglion neurons, neurites, and supporting cells. Here we present the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The careful preparation of these explants facilitates the identification of mechanisms that contribute to SNHL and constitutes a valuable tool for the hearing research community.

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

The goal of this protocol is to demonstrate the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The technique can be utilized as an in vitro screening tool in hearing research.

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a ...

Using Retinal Imaging to Study Dementia

The retina offers a unique &#8220;window&#8221; to study pathophysiological processes of dementia in the brain, as it is an extension of the central nervous system (CNS) and shares prominent similarities with the brain in terms of embryological origin, anatomical features and physiological properties.&#160; The vascular and neuronal structure in the retina can now be visualized easily and non-invasively using retinal imaging techniques, including fundus photography and optical coherence tomography (OCT), and quantified semi-automatically using computer-assisted analysis programs. Studying the associations between vascular and neuronal changes in the retina and dementia could improve our understanding of dementia and, potentially, aid in diagnosis and risk assessment.&#160; This protocol aims to describe a method of quantifying and analyzing retinal vasculature and neuronal structure, which are potentially associated with dementia. This protocol also provides examples of retinal changes in subjects with dementia, and discusses technical issues and current limitations of retinal imaging.

The retina shares prominent similarities with the brain and thus represents a unique window to study vasculature and neuronal structure in the brain non-invasively. This protocol describes a method to study dementia using retinal imaging techniques. This method can potentially aid in diagnosis and risk assessment of dementia.

The retina offers a unique &#8220;window&#8221; to study pathophysiological processes of dementia in the brain, as it is an extension of the central ...

Adeno-Associated Virus-Mediated Delivery of CRISPR for Cardiac Gene Editing in Mice

The clustered, regularly interspaced, short, palindromic repeat (CRISPR) system has greatly facilitated genome engineering in both cultured cells and living organisms from a wide variety of species. The CRISPR technology has also been explored as novel therapeutics for a number of human diseases. Proof-of-concept data are highly encouraging as exemplified by recent studies that demonstrate the feasibility and efficacy of gene editing-based therapeutic approach for Duchenne muscular dystrophy (DMD) using a murine model. In particular, intravenous and intraperitoneal injection of the recombinant adeno-associated virus (rAAV) serotype rh.74 (rAAVrh.74) has enabled efficient cardiac delivery of the Staphylococcus aureus CRISPR-associated protein 9 (SaCas9) and two guide RNAs (gRNA) to delete a genomic region with a mutant codon in exon 23 of mouse Dmd gene. This same approach can also be used to knock out the gene-of-interest and study their cardiac function in postnatal mice when the gRNA is designed to target the coding region of the gene. In this protocol, we show in detail how to engineer rAAVrh.74-CRISPR vector and how to achieve highly efficient cardiac delivery in neonatal mice.

Here we provide a detailed protocol to carry out in vivo cardiac gene editing in mice using recombinant Adeno-Associated Virus(rAAV)-mediated delivery of CRISPR. This protocol offers a promising therapeutic strategy to treat dystrophic cardiomyopathy in Duchenne muscular dystrophy and can be used to generate cardiac-specific knockout in postnatal mice.

The clustered, regularly interspaced, short, palindromic repeat (CRISPR) system has greatly facilitated genome engineering in both cultured cells and ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...