Leon Tai is funded by University of Illinois Chicago start-up funds.

All experiments follow the University of Illinois, Chicago Institutional Animal Care and Use Committee protocols.
1. General Preparation
NOTE: The brain microvascular endothelial cell line (hCMEC/D3) is an extensively characterized immortalized human BEC line14,15,16,18,19. Culture the hCMEC/D3 cells on tissue culture flasks coated with collagen Type I (calf skin, 1:20 dilution of 0.1% solution in Hank&#39;s Balanced Salt Solution (HBSS) containing Ca2+ and Mg2+) in basal Endothelial Growth Basal Medium (EBM-2) containing 2-5% Fetal Bovine Serum (FBS), 10% ascorbic acid, 10% gentamicin sulphate, 25% hydrocortisone and 1/4 of the total volume of the supplied growth factor supplements per 500 mL of media [vascular endothelium growth factor (VEGF), epidermal growth factor (EGF), insulin-like growth factor 1 (IGF-1) and human basic fibroblastic growth factor (bFGF), see Table of Materials]. 
NOTE: EBM-2 medium with FBS and growth factors is referred to as &#34;EBM-2 complete&#34;. EBM-2 without FBS and supplements is referred to as &#34;EBM-2 basal&#34;. At full confluence, the hCMEC/D3 cells are ~1 x 105 cells/cm2.
<ol>
	<li>Passage the hCMEC/D3 cells at a ratio of 1:5 by first incubating with HBSS for 3 min without Ca2+ and Mg2+ followed by detachment with 5 mL trypsin/EDTA (0.25%) for 5 min. Neutralize the trypsin using EBM-2 complete (1:1 neutralization), and centrifuge at 290 x g for 5 min at 4 &#176;C; discard the supernatant. Resuspend the pellet in EBM-2 complete and re-plate at 1:5 ratio.</li>
	<li>Culture the primary human pericytes and astrocytes according to the supplier&#39;s protocol [Table of Materials]. Culture the pericytes in pericyte basal medium with FBS and pericyte growth supplement.</li>
	<li>Culture the human astrocytes in astrocyte medium containing astrocyte growth factors. Culture both the pericytes and the astrocytes in tissue culture flasks coated with 3 mL of poly-L-lysine (PLL) for cell adherence and utilize the cells between passages 2 and 5.</li>
	<li>Euthanize 7 mice and remove the brain stems and cerebella with forceps; detach the meninges by carefully rolling the brains on gauze. Mince the remaining brain tissue in a Petri dish in Minimal Essential Medium (MEM) with HEPES (5 mL per brain) with a sterile razor blade. Isolate the primary mouse BECs from 2-month-old C57BL/6J mice according to the referenced protocol20.</li>
	<li>Transfer the minced brain tissue to a 15 mL conical tube. Centrifuge at 290 x g for 5 min at 4 &#176;C. Remove the supernatant and incubate the tissue in a papain (833.33 &#181;L per brain) and DNase (41.7 &#181;L per brain) solution, in a 37 &#176;C water bath for 1 h, to digest the tissue.</li>
	<li>Triturate the homogenate sequentially through 19 and 21 gauge needles, mix at a 1:1 ratio with a 22% Bovine Serum Albumin (BSA) solution, and centrifuge at 1360 x g for 10 min at 4 &#176;C.</li>
	<li>Resuspend the resulting pellet in 1 mL EBM-2 complete and centrifuge at 290 x g for 5 min at 4 &#176;C. Resuspend again in 1 mL EBM-2 complete and plate the cells (1 mL/well) on 6-well plates coated with collagen (~1 brain per well).</li>
	<li>Replace the media 24 h later with EBM-2 complete containing 4 &#181;g/mL puromycin hydrochloride; replace with EBM-2 complete after 2 days. The primary BECs are cultured as for the hCMEC/D3 cells and are at full confluence at 1 x 105 cells/cm2.</li>
	<li>Prepare a 100 &#181;M of oA&#946;, 24 h before the assay. 
	NOTE: oA&#946; is considered the disease relevant form of A . For oA&#946;, use the well-characterized preparations described by Dahlgren et al.21.</li>
	<li>Thaw the basement membrane stock solution overnight at 4 &#176;C and aliquot into sterile PCR tube strips (8 tubes) at 140 &#181;L basement membrane per tube. Refreeze each strip and store at -20 &#176;C. Thaw one strip per 96-well plate at 4 &#176;C, 24 h prior to the assay. Pre-cool the pipette tips to avoid solidification. 
	NOTE: All handling of the basement membrane matrix must be carried out on ice to avoid rapid solidification. As 10 &#181;L per well of basement membrane is used on the day of the assay, each strip is sufficient for one 96-well plate.</li>
	<li>Incubate fully confluent BECs in EBM-2 basal, 24 h prior to the assay. 
	NOTE: The rationale is: EBM-2 complete contains supplemented factors and FBS with the purpose of promoting optimal cell growth; however, the same molecular pathways that promote cell growth are also important for brain endothelial cell coverage and dynamics, both in the presence and absence of a stressor. We therefore serum and supplement starve the BECs to reduce the confounding effect of residual activation of cellular signaling pathways. Of note, the cells are briefly (5 min) exposed to FBS during the neutralization of trypsinized cells prior to the assay. In contrast to BECs, the pericytes and astrocytes are not serum starved, due to the requirement of different factors for growth and the relative instability of these cell types in response to serum starvation (Tai laboratory, unpublished observations). 
	NOTE: EBM-2 basal is utilized during the assay for the single and triple culture meshwork formation and disruption assays. This is critical to prevent confounding stressor or treatment dependent signaling.</li>
</ol>
2. Meshwork-like Formation and Disruption Assays
<ol>
	<li>Single BEC culture assays: 
	NOTE: Three different paradigms for the single cultures of BECs are shown in Figure 1A. Each paradigm is designed to assess the response of BECs in different models of vessel coverage. Paradigm 1 is meshwork formation. This paradigm is designed to examine the effects of stressors and/or treatments on angiogenesis and meshwork formation. BECs, oA&#946;, and treatments are all added to the plate at the 0 h time point. Paradigm 2 is prevention of meshwork disruption. Cells are plated in the presence of a growth factor or drug and incubated for 4 h to form meshwork-like structures. A stressor, oA&#946; in this case, is added after 4 h. This paradigm assesses the ability of a treatment to prevent stressor-induced damage to preformed meshwork-like structures. Paradigm 3 is simultaneous treatment of meshwork disruption. Cells are plated and allowed to form meshwork-like structures for 4 h. Treatments and oA&#946; are then added simultaneously at the 4 h time point, assessing the ability of the preformed cell network to respond to various treatments. All the paradigms follow similar common steps, except with differences in the timing of treatment addition.
	<ol>
		<li>On the day of the assay, pipette the basement membrane matrix at 10 &#181;L/well into the bottom of 96-well plates and allow to set for 1 h at 37 &#176;C.</li>
		<li>Suspend the lyophilized green cell tracking dye (see Table of Materials) in 10 &#181;L DMSO to generate a 10-mM stock solution, and dilute to 10 &#181;M (1:1000) in EBM-2 basal. Remove the media from the fully confluent flask and replace with EBM-2 basal containing the green cell tracking dye (5 mL for a 75 cm2 flask). Preload BECs with the green cell tracking dye, 20 min prior to the start of the assay.</li>
		<li>Incubate the cells for 20-30 min at 37 &#176;C, remove the medium with cell tracking dye, and detach the cells as described in Step 1.1. Neutralize the media with EBM-2 containing 10% FBS and centrifuge at 240 x g for 5 min at 4 &#176;C. Resuspend the pellet in 1 mL of EBM-2 basal.</li>
		<li>Plate the BECs in the 96-well plate at 10,000 cells/well in EBM-2 basal (0 h time point in all paradigms). 
		NOTE: Depending on the paradigm being utilized, treatments, stressors, or vehicle controls are added at the specified time points. In all paradigms, the medium is harvested for subsequent analysis (e.g. ELISA analysis) and cells are fixed at 24 h with freshly prepared 4% paraformaldehyde in Phosphate Buffered Serum (PBS). As an alternative approach to determine the term effects of oA&#946; on mesh formation, the protocol could be modified to pre-incubate confluent cell culture flasks with oA&#946;, and then perform the meshwork formation paradigms (+/-oA&#946;). 
		NOTE: The final volume in each well for all paradigms is 70 &#181;L. All treatments (oA&#946;, EGF, etc.) are added to their respective wells at a 1:20 dilution i.e. 3.5 &#181;L per treatment. For Paradigm 1, the cells are added in a cell suspension solution at 63 &#181;L/well. Immediately oA&#946;, desired treatments, and controls are added at 3.5 &#181;L/well each, giving a total of 70 &#181;L. For Paradigm 2, 63 &#181;L of cell suspension and 3.5 &#181;L of treatment (e.g. 100 ng/mL EGF) are added at the 0 h time point. After 4 h incubation, 3.5 &#181;L of an oA&#946; stock is added (e.g. for 100 nM final oA&#946; concentration, 3.5 &#181;L of 2 &#181;M oA&#946; stock). For Paradigm 3, 63 &#181;L of cell suspension is added at the 0 h time point and at 4 h, oA&#946; and desired treatments are added at 3.5 &#181;L/well each, giving a total of 70 &#181;L. These volumes can be adjusted according to treatments. For example, if only one treatment is required, the cell suspension volume can be adjusted to 66.5 &#181;L (maintaining 10,000 cells/well) and the treatment volume can remain at 3.5 &#181;L.</li>
	</ol>
	</li>
	<li>Triple culture assay: 
	NOTE: In addition to the single culture assays of BECs, we have developed a triple culture assay paradigm (Figure 1B) to determine the response of BECs, pericytes, and astrocytes within preformed networks to relevant stressors (e.g.oA&#946;). BECs are plated in the presence of desired treatments at 0 h. At 4 h, pericytes are gently added to the plate. At 7 h, astrocytes are added to the plate, followed by the addition of a stressor at 11 h. Cells are then incubated until the 24 h time point. 
	NOTE: For the triple culture assays, it is important to consider timing to ensure all cells are confluent at the same time. Human astrocytes and pericytes should be cultured 1 week before the assay, whereas the hCMEC/D3 cells need to be plated or passaged 4-5 days before the assay date. Further, it is important to use low passage (2-5) primary cells to avoid phenotypic drift.
	<ol>
		<li>Add the green cell tracking dye working solution to the hCMEC/D3 cells 20 min prior to starting the assay. Plate the hCMEC/D3 cells at 10,000 cells/well in EBM-2 basal (0 h time point). The volumes used are 45 &#181;L of cell suspension and 3.5 &#181;L of treatment (i.e. final EGF concentrations of 50 ng/mL, 100 ng/mL, or 1,000 ng/mL from stocks that are 20 times more concentrated).</li>
		<li>Following the 3.5 h incubation at 37 &#176;C, add the blue cell tracking dye working solution (10 &#181;M cell tracker blue in EBM-2 basal) to the human primary pericytes. At the 4 h time point (~30 min incubation with cell tracking dye), gently add 2,000 pericytes/well to the plate containing the hCMEC/D3 in a volume of 6 &#181;L/well.</li>
		<li>Following an additional 2.5 h incubation (total 6.5 h from 0 h time point), add the orange cell tracking dye working solution (10 &#181;M cell tracker orange in EBM-2 basal) to the human primary astrocytes. At the 7 h time point, gently add 10,000 astrocytes/well to the plate in 12 &#181;L volume. Therefore, the overall cellular ratio for endothelial cells:pericytes:astrocytes is 5:1:5. Further, the volume achieved to this point is 66.5 &#181;L.</li>
		<li>Add oA&#946; (3.5 &#181;L of 100 &#181;M stock) 4 h later (total 11 h from 0 h time point).</li>
		<li>Fix the cells 13 h later in freshly prepared 4% paraformaldehyde in PBS. 
		Caution: Wear protective clothing, gloves and eyeglasses while handling paraformaldehyde.</li>
	</ol>
	</li>
</ol>
3. Quantification
<ol>
	<li>Capture fluorescence images of the whole well of the 96-well plate at 1.6X magnification with a 2 s exposure time at 50% maximum power using a dissecting microscope. 
	NOTE: Equivalent microscopes can be utilized; however, it is critical to capture the entire well for comprehensive analysis.</li>
	<li>For quantitative analysis, process the images using the ImageJ angiogenesis analyzer plugin22 to quantify the number of branches, number of meshes, and total cell length (these readouts are automatically tabulated by the software) as described below. A detailed visual protocol of this step is provided in Figure 2.
	<ol>
		<li>Open fiji ImageJ by clicking on the software icon.</li>
		<li>Click on the double red forward arrows located at the end of the toolbar, then click &#34;Angiogenesis Analyzer&#34;.</li>
		<li>Select the folder with image files, click &#34;Open&#34;.</li>
		<li>When the &#34;settings for analysis&#34; box appears, click &#34;OK&#34;.</li>
		<li>When the &#34;processing images&#34; box appears, which indicates the number of images to be processed, click &#34;OK&#34;.</li>
		<li>When the &#34;batch progress window&#34; box appears, which indicates the estimated processing time. During this time the program quantifies the outputs including the number of branches, number of meshes, and total cell length. 
		NOTE: Once all the images are quantified, the results file will appear in the original image folder as a spreadsheet document.</li>
		<li>Select the spreadsheet file containing results and compare the number of branches, meshes and total cell length between groups. 
		NOTE: In the triple culture assay ImageJ is further utilized to analyze pericyte/astrocyte coverage and number. Images are thresholded by a blinded experimenter and the &#34;analyze particles&#34; function utilized to generate readouts. Fixed cells and tube-like structures can also be immunostained for A&#946;17 or other markers.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

The methods described can be utilized to address several fundamental biological questions surrounding cerebrovascular coverage24. Specifically, they can identify which receptors and signaling pathways play a role in angiogenesis, vessel coverage in cancer tissue, and peripheral endothelial cells relevant to the brain. Examples include angiogenic growth factor receptors, nitric oxide, mitogen activated protein kinase signaling and calcium signaling25,<sup class="x...

The blood-brain barrier (BBB) of cerebral capillaries is the largest interface of blood-to-brain contact and plays a central role in the homeostasis of the central nervous system (CNS)1,2. Dynamic processes at the BBB prevent the uptake of unwanted molecules from the blood, remove waste products from the CNS, supply essential nutrients and signaling molecules to the CNS, and modulate neuroinflammation1,2,3,4,5. BBB damage is prevalent during aging and several neurodegenerative disorders including Alzheimer&#39;s disease (AD), multiple sclerosis and stroke1,2,3,4,5,6. Therefore, BBB dysfunction may play a key role in neurodegenerative disorders, including as a therapeutic target.
Maintaining vessel coverage is important for the homeostatic functions of the BBB. However, in vivo and in vitro data conflict on whether the processes involved in neurodegenerative disorders cause higher or lower BBB coverage6,7,8,9,10,11,12,13, particularly in AD. Therefore, there is a strong rationale for the development of in vitro models using relevant cell types to assess and more comprehensively understand the dynamics of BBB coverage. Cerebral capillaries are composed of astrocytes, pericytes and brain endothelial cells (BECs)3. All cell types contribute to the function of the BBB through structural support and via the secretion of effector molecules such as angiogenic growth factors, cytokines and chemokines that act in paracrine- and autocrine-like fashion. However, the major effector cells of the BBB are BECs3. In general, the cell culture techniques for assessing BBB function are permeability assays performed on cells grown on filter inserts, or assessing levels of key BEC proteins, both after the addition of stressors14,15,16. Although important, these assays do not focus on the cerebrovascular coverage.
Here, our previous methods17 are detailed to assess BEC coverage and meshwork-like structures using single cultures of immortalized human BECs, single cultures of primary mouse BECs, and a humanized triple culture model (BECs, astrocytes and pericytes) of the BBB. The goal was to demonstrate the detrimental effect of oA&#946;, which is considered an important contributor to AD progression, on BEC coverage. The protective effect of the epidermal growth factor (EGF) highlights the potential of the technique as a therapeutic screening tool. The technique has several broad applications for basic and applied research including: 1) delineating the role of specific pathways on angiogenesis and vessel coverage, 2) evaluating the effects of disease and aging-relevant factors on angiogenesis and vessel coverage, and 3) identifying pharmacological targets.

<table border="0" cellpadding="0" cellspacing="0" width="847">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">hCMEC/D3 cells</td>
			<td style="width:183px;">Milipore</td>
			<td style="width:119px;">SCC066</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">EBM-2 basal media&#160;</td>
			<td style="width:183px;">Lonza</td>
			<td>CC-3156</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Collagen Type 1</td>
			<td style="width:183px;">ThermoFisher</td>
			<td style="width:119px;">A1064401&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">HBSS, calcium, magnesium, no phenol red</td>
			<td style="width:183px;">ThermoFisher</td>
			<td style="width:119px;">14025092</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">HBSS, no calcium, no magnesium, no phenol red</td>
			<td style="width:183px;">ThermoFisher</td>
			<td style="width:119px;">14175095</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Trypsin-EDTA (0.25%)</td>
			<td style="width:183px;">ThermoFisher</td>
			<td style="width:119px;">25200056</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:379px;">Final concentrations of the SingleQuot growth factor supplements for EBM2 media</td>
			<td style="width:183px;">Lonza</td>
			<td>CC-4147</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5% FBS</td>
			<td style="width:183px;">Lonza</td>
			<td>CC-4147</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10% Ascorbic acid</td>
			<td style="width:183px;">Lonza</td>
			<td>CC-4147</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10% Gentamycin sulphate</td>
			<td style="width:183px;">Lonza</td>
			<td>CC-4147</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">25% Hydrocortisone</td>
			<td style="width:183px;">Lonza</td>
			<td>CC-4147</td>
			<td></td>
		</tr>
		<tr height="78">
			<td height="78" style="height:78px;width:379px;">1/4 volume of the supplied growth factors: fibroblast growth factor, epidermal growth factor, insulin-like growth factor, vascular endothelial growth factor</td>
			<td style="width:183px;">Lonza</td>
			<td>CC-4147</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Puromycin hydrochloride</td>
			<td style="width:183px;">VWR</td>
			<td style="width:119px;">80503-312</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">MEM-HEPES&#160;</td>
			<td style="width:183px;">Thermo Scientific&#160;</td>
			<td style="width:119px;">12360-038</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Papain cell dissociation system (papain and DNase1)</td>
			<td style="width:183px;">Worthington Biochemical</td>
			<td style="width:119px;">LK003150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Human pericytes</td>
			<td style="width:183px;">Sciencell</td>
			<td>1200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Pericyte basal media</td>
			<td style="width:183px;">Sciencell</td>
			<td>1201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Pericyte growth supplement</td>
			<td style="width:183px;">Sciencell</td>
			<td>1252</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Human Astrocytes</td>
			<td style="width:183px;">Sciencell</td>
			<td>1800</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Astrocyte media</td>
			<td style="width:183px;">Sciencell</td>
			<td>1801</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Astrocyte growth supplement</td>
			<td style="width:183px;">Sciencell</td>
			<td>1852</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Basement membrane (Matrigel Growth Factor Reduced)&#160;</td>
			<td style="width:183px;">Corning</td>
			<td style="width:119px;">356231</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Angiogenesis m-plates (96-well)</td>
			<td style="width:183px;">ibidi</td>
			<td style="width:119px;">89646</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Human Epidermal growth factor&#160;</td>
			<td style="width:183px;">Shenendoah Biotechnology</td>
			<td style="width:119px;">100-26</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CellTracker green&#160;</td>
			<td style="width:183px;">ThermoFisher</td>
			<td>C7025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CellTracker orange</td>
			<td style="width:183px;">ThermoFisher</td>
			<td>C34551</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CellTracker blue&#160;</td>
			<td style="width:183px;">ThermoFisher</td>
			<td>C2110</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Poly-l-lysine</td>
			<td style="width:183px;">Sciencell</td>
			<td>0403</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">10% Neutral Buffered Formalin</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td>HT5012-60ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">C57BL mice</td>
			<td style="width:183px;">Jackson Laboratory</td>
			<td style="width:119px;">na</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">PCR tube strips</td>
			<td style="width:183px;">GeneMate</td>
			<td style="width:119px;">T-3014-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Zeiss stereo discover v.8 dissecting microscope</td>
			<td style="width:183px;">Zeiss</td>
			<td style="width:119px;">na</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro assays to assess blood-brain barrier mesh-like vessel formation and disruption

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by astrocytes, pericytes and brain endothelial cells (BECs). Here, we detail methods to assess BBB coverage using single cultures of immortalized human BECs, single cultures of primary mouse BECs, and a humanized triple culture model (BECs, astrocytes and pericytes) of the BBB. To highlight the applicability of the assays to disease states, we describe the effect of oligomeric amyloid-&#946; (oA&#946;), which is an important contributor to Alzheimer&#39;s disease (AD) progression, on BBB coverage. Further, we utilize the epidermal growth factor (EGF) to illuminate the drug screening potential of the techniques. Our results show that single and triple cultured BECs form meshwork-like structures under basal conditions, and that oA&#946; disrupts this cell meshwork formation and degenerates the preformed mesh structures, but EGF blocks this disruption. Thus, the techniques described are important for dissecting fundamental and disease-relevant processes that modulate BBB coverage.

In single cultures, both the hCMEC/D3 cells (Figure 3A) and the primary mouse BECs (Figure 3B) form meshwork-like structures throughout the well. The structures are characterized by a meshwork of interlinked nodes (Figure 3). In all the paradigms described (Figure 1), the meshwork-like structures are similar after 24 h in the control groups, forming ~20 meshwork-like str...

Watch this Scientific Journal Video about In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption at JoVE.com

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Maintaining blood-brain barrier coverage is key for the homeostasis of the central nervous system. This protocol describes in vitro techniques to delineate the fundamental and pathological processes that modulate blood-brain barrier coverage.

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by ...

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JoVE Journal

Medicine

8.4K Views.  University of Illinois at Chicago. Maintaining blood-brain barrier coverage is key for the homeostasis of the central nervous system. This protocol describes in vitro techniques to delineate the fundamental and pathological processes that modulate blood-brain barrier coverage.

Video: In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a significant obstacle to pharmaceutical treatments of brain disorders as it limits the passage of molecules from the vasculature into the brain tissue to molecules less than approximately 500 Da in size6. FUS induced BBB disruption (BBBD) is temporary and reversible4 and has an advantage over chemical means of inducing BBBD by being highly localized. FUS induced BBBD provides a means for investigating the effects of a wide range of therapeutic agents on the brain, which would not otherwise be deliverable to the tissue in sufficient concentration. While a wide range of ultrasound parameters have proven successful at disrupting the BBB2,5,7, there are several critical steps in the experimental procedure to ensure successful disruption with accurate targeting. This protocol outlines how to achieve MRI-guided FUS induced BBBD in a rat model, with a focus on the critical animal preparation and microbubble handling steps of the experiment.

Microbubble-mediated focused ultrasound disruption of the blood-brain barrier (BBB) is a promising technique for non-invasive targeted drug delivery in the brain1-3. This protocol outlines the experimental procedure for MRI-guided transcranial BBB disruption in a rat model.

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a ...

The Measurement and Treatment of Suppression in Amblyopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current estimates put the prevalence of amblyopia at approximately 1-3%1-3, the majority of cases being monocular2. Amblyopia is most frequently caused by ocular misalignment (strabismus), blur induced by unequal refractive error (anisometropia), and in some cases by form deprivation.
Although amblyopia is initially caused by abnormal visual input in infancy, once established, the visual deficit often remains when normal visual input has been restored using surgery and/or refractive correction. This is because amblyopia is the result of abnormal visual cortex development rather than a problem with the amblyopic eye itself4,5 . Amblyopia is characterized by both monocular and binocular deficits6,7 which include impaired visual acuity and poor or absent stereopsis respectively. The visual dysfunction in amblyopia is often associated with a strong suppression of the inputs from the amblyopic eye under binocular viewing conditions8. Recent work has indicated that suppression may play a central role in both the monocular and binocular deficits associated with amblyopia9,10 .
Current clinical tests for suppression tend to verify the presence or absence of suppression rather than giving a quantitative measurement of the degree of suppression. Here we describe a technique for measuring amblyopic suppression with a compact, portable device11,12 . The device consists of a laptop computer connected to a pair of virtual reality goggles. The novelty of the technique lies in the way we present visual stimuli to measure suppression. Stimuli are shown to the amblyopic eye at high contrast while the contrast of the stimuli shown to the non-amblyopic eye are varied. Patients perform a simple signal/noise task that allows for a precise measurement of the strength of excitatory binocular interactions. The contrast offset at which neither eye has a performance advantage is a measure of the "balance point" and is a direct measure of suppression. This technique has been validated psychophysically both in control13,14 and patient6,9,11 populations.
In addition to measuring suppression this technique also forms the basis of a novel form of treatment to decrease suppression over time and improve binocular and often monocular function in adult patients with amblyopia12,15,16 . This new treatment approach can be deployed either on the goggle system described above or on a specially modified iPod touch device15.

Amblyopia is a developmental disorder of the visual cortex that is often accompanied by strong suppression of one eye. We present a new technique for measuring and treating interocular suppression in patients with amblyopia that can be deployed using virtual reality goggles or a portable iPod Touch device.

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current ...

Rat Model of Blood-brain Barrier Disruption to Allow Targeted Neurovascular Therapeutics

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain barrier1. The barrier selectively excludes molecules from crossing between the blood and the brain based upon their size and charge. This function can impede the delivery of therapeutics for neurological disorders. A number of chemotherapeutic drugs, for example, will not effectively cross the blood-brain barrier to reach tumor cells2. Thus, improving the delivery of drugs across the blood-brain barrier is an area of interest.
The most prevalent methods for enhancing the delivery of drugs to the brain are direct cerebral infusion and blood-brain barrier disruption3. Direct intracerebral infusion guarantees that therapies reach the brain; however, this method has a limited ability to disperse the drug4. Blood-brain barrier disruption (BBBD) allows drugs to flow directly from the circulatory system into the brain and thus more effectively reach dispersed tumor cells. Three methods of barrier disruption include osmotic barrier disruption, pharmacological barrier disruption, and focused ultrasound with microbubbles. Osmotic disruption, pioneered by Neuwelt, uses a hypertonic solution of 25% mannitol that dehydrates the cells of the blood-brain barrier causing them to shrink and disrupt their tight junctions. Barrier disruption can also be accomplished pharmacologically with vasoactive compounds such as histamine5 and bradykinin6. This method, however, is selective primarily for the brain-tumor barrier7. Additionally, RMP-7, an analog of the peptide bradykinin, was found to be inferior when compared head-to-head with osmotic BBBD with 25% mannitol8. Another method, focused ultrasound (FUS) in conjunction with microbubble ultrasound contrast agents, has also been shown to reversibly open the blood-brain barrier9. In comparison to FUS, though, 25% mannitol has a longer history of safety in human patients that makes it a proven tool for translational research10-12.
In order to accomplish BBBD, mannitol must be delivered at a high rate directly into the brain's arterial circulation. In humans, an endovascular catheter is guided to the brain where rapid, direct flow can be accomplished. This protocol models human BBBD as closely as possible. Following a cut-down to the bifurcation of the common carotid artery, a catheter is inserted retrograde into the ECA and used to deliver mannitol directly into the internal carotid artery (ICA) circulation. Propofol and N2O anesthesia are used for their ability to maximize the effectiveness of barrier disruption13. If executed properly, this procedure has the ability to safely, effectively, and reversibly open the blood-brain barrier and improve the delivery of drugs that do not ordinarily reach the brain 8,13,14.

Blood-brain barrier disruption aids the delivery of certain drugs to the brain. Mannitol delivered intra-arterially shrinks cells surrounding blood vessels in order to physically disrupt the barrier.

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain ...

An in vivo Assay to Test Blood Vessel Permeability

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

An in vivo Assay to Test Blood Vessel Permeability

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

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

Permanent Cerebral Vessel Occlusion via Double Ligature and Transection

Stroke is a leading cause of death, disability, and socioeconomic loss worldwide. The majority of all strokes result from an interruption in blood flow (ischemia) 1. Middle cerebral artery (MCA) delivers a great majority of blood to the lateral surface of the cortex 2, is the most common site of human stroke 3, and ischemia within its territory can result in extensive dysfunction or death 1,4,5. Survivors of ischemic stroke often suffer loss or disruption of motor capabilities, sensory deficits, and infarct. In an effort to capture these key characteristics of stroke, and thereby develop effective treatment, a great deal of emphasis is placed upon animal models of ischemia in MCA.
Here we present a method of permanently occluding a cortical surface blood vessel. We will present this method using an example of a relevant vessel occlusion that models the most common type, location, and outcome of human stroke, permanent middle cerebral artery occlusion (pMCAO). In this model, we surgically expose MCA in the adult rat and subsequently occlude via double ligature and transection of the vessel. This pMCAO blocks the proximal cortical branch of MCA, causing ischemia in all of MCA cortical territory, a large portion of the cortex. This method of occlusion can also be used to occlude more distal portions of cortical vessels in order to achieve more focal ischemia targeting a smaller region of cortex. The primary disadvantages of pMCAO are that the surgical procedure is somewhat invasive as a small craniotomy is required to access MCA, though this results in minimal tissue damage. The primary advantages of this model, however, are: the site of occlusion is well defined, the degree of blood flow reduction is consistent, functional and neurological impairment occurs rapidly, infarct size is consistent, and the high rate of survival allows for long-term chronic assessment.

Permanent Cerebral Vessel Occlusion via Double Ligature and Transection

We describe a highly reproducible method for the permanent occlusion of a rodent major cerebral blood vessel. This technique can be accomplished with very little peripheral damage, minimal blood loss, a high rate of long-term survival, and consistent infarct volume commensurate with the human clinical population.

Stroke is a leading cause of death, disability, and socioeconomic loss worldwide. The majority of all strokes result from an interruption in blood flow ...

Stretch in Brain Microvascular Endothelial Cells (cEND) as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

Due to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying mechanisms involved in it are useful for treatment. There are both in vivo and in vitro methods available for this purpose. In vivo models can mimic actual head injury as it occurs during TBI. However, in vivo techniques may not be exploited for studies at the cell physiology level. Hence, in vitro methods are more advantageous for this purpose since they provide easier access to the cells and the extracellular environment for manipulation.
Our protocol presents an in vitro model of TBI using stretch injury in brain microvascular endothelial cells. It utilizes pressure applied to the cells cultured in flexible-bottomed wells. The pressure applied may easily be controlled and can produce injury that ranges from low to severe. The murine brain microvascular endothelial cells (cEND) generated in our laboratory is a well-suited model for the blood brain barrier (BBB) thus providing an advantage to other systems that employ a similar technique. In addition, due to the simplicity of the method, experimental set-ups are easily duplicated. Thus, this model can be used in studying the cellular and molecular mechanisms involved in TBI at the BBB.

Stretch in Brain Microvascular Endothelial Cells cEND as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

In vitro traumatic brain injury models are being developed to reproduce in vivo brain deformation. Stretch-induced injury has been employed for astrocytes, neurons, glial cells, aortic, and brain endothelial cells. However, our system uses a blood brain barrier (BBB) model that possesses properties constituting a legitimate model of the BBB to establish an in vitro&#160;TBI model.

Due to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying ...

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

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

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

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

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

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic cerebrovascular accidents account for 80% of all strokes. A common cause of IR injury is the rapid inflow of fluids following an acute/chronic occlusion of blood, nutrients, oxygen to the tissue triggering the formation of free radicals.
Ischemic stroke is followed by blood-brain barrier (BBB) dysfunction and vasogenic brain edema. Structurally, tight junctions (TJs) between the endothelial cells play an important role in maintaining the integrity of the blood-brain barrier (BBB). IR injury is an early secondary injury leading to a non-specific, inflammatory response. Oxidative and metabolic stress following inflammation triggers secondary brain damage including BBB permeability and disruption of tight junction (TJ) integrity.
Our protocol presents an in vitro example of oxygen-glucose deprivation and reoxygenation (OGD-R) on rat brain endothelial cell TJ integrity and stress fiber formation. Currently, several experimental in vivo models are used to study the effects of IR injury; however they have several limitations, such as the technical challenges in performing surgeries, gene dependent molecular influences and difficulty in studying mechanistic relationships. However, in vitro models may aid in overcoming many of those limitations. The presented protocol can be used to study the various molecular mechanisms and mechanistic relationships to provide potential therapeutic strategies. However, the results of in vitro studies may differ from standard in vivo studies and should be interpreted with caution.

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is associated with a high rate of morbidity and mortality. The goal of the in vitro model of oxygen-glucose deprivation and reoxygenation (OGD-R) described here is to assess the effects of ischemia reperfusion injury on a variety of cells, particularly in blood-brain barrier (BBB) endothelial cells.

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic ...

The 4-vessel Sampling Approach to Integrative Studies of Human Placental Physiology In Vivo

The human placenta is highly inaccessible for research while still in utero. The current understanding of human placental physiology in vivo is therefore largely based on animal studies, despite the high diversity among species in placental anatomy, hemodynamics and duration of the pregnancy. The vast majority of human placenta studies are ex vivo perfusion studies or in vitro trophoblast studies. Although in vitro studies and animal models are essential, extrapolation of the results from such studies to the human placenta in vivo is uncertain. We aimed to study human placenta physiology in vivo at term, and present a detailed protocol of the method. Exploiting the intraabdominal access to the uterine vein just before the uterine incision during planned cesarean section, we collect blood samples from the incoming and outgoing vessels on the maternal and fetal sides of the placenta. When combining concentration measurements from blood samples with volume blood flow measurements, we are able to quantify placental and fetal uptake and release of any compound. Furthermore, placental tissue samples from the same mother-fetus pairs can provide measurements of transporter density and activity and other aspects of placental functions in vivo. Through this integrative use of the 4-vessel sampling method we are able to test some of the current concepts of placental nutrient transfer and metabolism in vivo, both in normal and pathological pregnancies. Furthermore, this method enables the identification of substances secreted by the placenta to the maternal circulation, which could be an important contribution to the search for biomarkers of placenta dysfunction.

The 4-vessel Sampling Approach to Integrative Studies of Human Placental Physiology In Vivo

We present a detailed method to study human placental physiology in vivo at term. The method combines blood sampling from the incoming and outgoing vessels on the maternal and fetal sides of the placenta with ultrasound measurements of volume blood flow and placental tissue sampling.

The human placenta is highly inaccessible for research while still in utero. The current understanding of human placental physiology in vivo is therefore ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

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The 4-vessel Sampling Approach to Integrative Studies of Human Placental Physiology In Vivo

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells