This work was supported by the following grants: AHA 16SDG31170008 to X.R. and NIH P20 GM109098, P01 AG027956, and U54 GM104942 to J.W.S.

NOTE: The scheme of the protocol is shown in Figure 1, including a timeline of the procedures.
1. Culture and Passage of CVE Cells
<ol>
	<li>Culture CVE cells (bEnd.3 cells) in complete growth medium (high glucose Dulbecco&#39;s Modified Eagle&#39;s Medium, DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin in a T75 cm2 tissue culture flask. Grow at 37 &#176;C in a 5% CO2 incubator.</li>
	<li>Remove and discard the cell culture medium. Rinse the cell layer with 0.25% Trypsin-0.03% EDTA solution, and then add 1 - 2 mL of Trypsin-EDTA solution to the flask and keep the flask at 37 &#176;C for 3 - 5 min. Observe the cells under an inverted microscope until the cell layer is detached. Add 10 mL of complete growth medium and aspirate the cells by gently pipetting.</li>
	<li>Decant the fluid and cells into a 15-mL centrifuge tube and spin at 700 x g for 5 min to remove cellular debris.</li>
	<li>Discard the supernatant from the centrifuge tube. Add 10 mL of complete growth medium and re-suspend the cell pellet. Spin the cells at 700 x g for 5 min.</li>
	<li>Discard the supernatant from the centrifuge tube. Re-suspend the cell pellet in complete growth medium at a concentration of 2.0 &#215; 105 cells/mL (determined by counting using a hemocytometer; exclude dead cells by Trypan-blue staining). 
	NOTE: Freshly-thawed cells are sub-cultured for at least 3 passages for experiments.</li>
</ol>
2. Seed and Treat Cells on Cell Culture Plates
<ol>
	<li>Seed cells on a 96-well extracellular flux cell culture plate: 80 &#181;L/well. Place the cell culture plate at 37 &#176;C in a 5% CO2 incubator. Note: 16 &#215; 103 cells per well will reach 60 - 80% confluency within 24 h.</li>
	<li>If the cells are being exposed to a chemical, add the treatments once the cells are attached to the plate bottom (8 - 24 h after seeding). 
	NOTE: For transfection experiments, do not add antibiotics in complete growth medium.</li>
</ol>
3. Assessment of Mitochondrial Function Using the Bioanalyzer
<ol>
	<li>Prior to the start of the assay, hydrate an extracellular flux sensor cartridge with calibrant by adding 150 &#181;L of extracellular flux calibrant solution to the plate and incubating O/N at 37 &#176;C without CO2.</li>
	<li>Using the plate washer station, change the cell culture medium into pH 7.0 extracellular flux assay medium. Incubate the cells at 37 &#176;C without CO2 for 30 - 60 min.</li>
	<li>Prepare working solutions of 8 &#181;M oligomycin, 4.5 &#181;M FCCP, 10 &#181;M rotenone, and 10 &#181;M antimycin A in DMEM. Load 25 &#181;L of the stock solutions into cartridge injection ports: Port A, oligomycin; Port B, FCCP; Port C, rotenone and antimycin A with extracellular flux assay medium. Implement the assay protocol as described in Table 1.</li>
	<li>Load the hydrated cartridge into the bioanalyzer and perform the calibration by clicking the &#34;Start&#34; button. After the calibration is complete, remove the cartridge bottom plate and load the cell plate by clicking the &#34;Unload Cartridge&#34; prompt. Continue the assay until the end of all measurements.</li>
	<li>Open the data file and obtain the rate values from the bioanalyzer. Export the data to a spreadsheet file.</li>
</ol>
4. Data Analyses
NOTE: The calculations for data analyses are formulated below. The values are obtained from the bioanalyzer instrument as the OCR presented in Figure 2.
<ol>
	<li>To calculate Basal respiration, subtract the non-mitochondrial respiration (measurements 10 - 12) from the basal value (measurement 3). 
	NOTE: Basal respiration = Basal - non-mitochondrial respiration = Rate &#9314; - Rate &#9321;~&#9323;.</li>
	<li>To calculate ATP production, subtract the ATP-linked respiration (measurements 4 - 6) from the basal value (measurement 3). 
	NOTE: ATP production = Basal - ATP-linked respiration = Rate &#9314; - Rate &#9315;~&#9317;.</li>
	<li>To calculate Maximal respiration, subtract the non-mitochondrial respiration (measurements 10 - 12) from the Maximum respiration (measurements 7 - 9). 
	NOTE: Maximal respiration = Maximum respiration - non-mitochondrial respiration = Rate &#9318;~&#9320; - Rate &#9321;~&#9323;.</li>
	<li>To calculate Spare capacity, subtract the basal value (measurement 3) from the Maximum respiration (measurements 7 - 9). 
	NOTE: Spare capacity = Maximum respiration - Basal = Rate &#9318;~&#9320; - Rate &#9314;.</li>
	<li>To calculate Proton leak, subtract the non-mitochondrial respiration (measurement 10 - 12) from the ATP-linked respiration (measurement 4). 
	NOTE: Proton leak = ATP-linked respiration - non-mitochondrial respiration = Rate &#9315; - Rate &#9321;~&#9323;.</li>
</ol>

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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tert-butylhydroquinone+compromises+survival+in+murine+experimental+stroke.">Tert-butylhydroquinone compromises survival in murine experimental stroke.</a> Neurotoxicol Teratol. 54, 15-21 (2016).</li><li>Modis, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+bioenergetics+is+regulated+by+PARP1+under+resting+conditions+and+during+oxidative+stress.">Cellular bioenergetics is regulated by PARP1 under resting conditions and during oxidative stress.</a> Biochem Pharmacol. 83, 633-643 (2012).</li><li>Brown, R. C., Morris, A. P., O'Neil, R. 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The authors declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Open Access fees for this article were provided by Agilent Technologies.

This protocol represents a method for evaluating of the bioenergetic phenotype in cerebral vascular endothelial cells. It serves as a basic assay for the endothelial cell mitochondrial evaluation, and it is optimal for experiments designed to investigate the mechanisms of stimuli that may affect the mitochondrial signaling pathway in CVE cells. It also provides a method for testing potential therapeutics for BBB-disruption-associated diseases.
Critical Steps within the Protocol
Extracellular flux bioanalyzers have the ability to measure OCR in real time. In this assay, identifying the appropriate cell density is critical. If the cell density is below 8 &#215; 103 cells per well, the basal OCR is too low to be analyzed (Figure 2 and Figure 3); if the cell density is above 32 &#215; 103 cells per well, the cells do not respond to oligomycin or FCCP treatments (Figure 2 and Figure 3). The optimal cell density for this cell line in our experimental model is 16 &#215; 103 cells per well, which demonstrates replicable results and sensitivity to stimuli7 and treatments10,14. If a 24-well bioanalyzer is used, new titrations of the cell density would be required for the assay.
It is documented that CVEs have a large volume of mitochondria compared with other endothelial cells or tissue types15,16, suggesting the need for greater energy production and fewer cells for measuring bioenergetics metabolism. We have tested several types of brain endothelial cells, such as primary and immortalized murine cerebrovascular endothelial cells7 and immortalized human cerebrovascular endothelial cells (unpublished data). These cells required similar cell densities to reach the acceptable OCR (basal respiration OCR within 40 - 160 pmol/min for 96-well plates and 50 - 400 pmol/min for 24-well plates) when the bioenergetics assay was performed. Kaczara et al. reported measuring bioenergetic metabolism in human umbilical vein endothelial cells (HUVEC), which used a higher cell density17.
Our group did not evaluate vessel cells from other tissue or organs. Theoretically, the bioanalyzer could potentially be used on any cell type, but it requires optimizing the assay for the cell density, cell culture medium, and culture conditions (some specific cells may require coated plates to grow well). It is required that the experiments are completed to ensure the OCR rate is within the acceptable range. If the OCR in endothelial cells from other organs is lower than CVEs from brains, increasing the cell density can help to get within an acceptable OCR range. Alternatively, if cells are not using oxidative phosphorylation as their main source of energy, the bioanalyzer would not be an optimal assay.
The bioanalyzer allows for the sequential disruption of the electron transport chain, based on the order in which the reagents are applied. First, oligomycin is applied, which inhibits mitochondrial Complex V (ATP synthase). Second, FCCP, an electron uncoupler, is applied, which leads to the disruption of the proton gradient. Finally, rotenone and antimycin A, which inhibit mitochondrial Complexes I and III, respectively, are applied to lead to a total inhibition of electron flow. The order of drug exposure is essential because the drugs block the specific electron transport chain reaction, and the sequential changes can be measured to reflect the mitochondrial function.
Modifications and Troubleshooting
To evaluate mitochondrial response to stimuli in CVE cells, it is recommended that the stimuli are applied to cells after the cells are adhered to the cell culture plate bottom (see the timeline shown in Figure 1; it usually takes at least 6 h). To obtain replicable results by treatments, it is extremely important to keep the cell density consistent. If pretreatments are designed prior to cell seeding, cell density for each pre-treatment should be carefully measured, excluding the dead cells for seeding. If transfection is included in the experimental design, refer to the manufacturer&#39;s transfection protocol. Demonstrated in data presented in Figure 4, miR-34a was transfected into the CVE cells using a lipofectamine transfection kit, which requires antibiotics-free cell culture medium and a mito-stress test to assess the changes in metabolic function with overexpression of miR-34a. The doses of plasmid may also be optimized, as previously published13. Another change that can be made to the protocol is changing the concentrations of the reagents. A titration curve of oligomycin, FCCP, and/or rotenone and Antimycin A can be completed.
In addition, if this assay is used for high throughput analyses of various drugs, it is suggested to include a parallel assay to measure cell viability and cell proliferation, which was described in our previous publications7,13. The OCR values are significantly affected by cell number (Figures 2 and 3), and the cell proliferation and viability assays benefit the normalization of the data. However, the completion of these assays are at the discretion of each individual laboratory.
Limitations of the Technique
The major limitation of this protocol is that we only used an in vitro cell culture model in the study. Currently, there are no available ex vivo models or animal models that may address endothelial cell mitochondrial function. New models are expected to be developed for the evaluation of bioenergetic function in vivo and ex vivo in future studies.
Another limitation is the extension of the barrier assessments following the bioenergetics measures. The experiments cannot be performed because, first, after the completion of the bioenergetic measurements, the cell viability is diminished following complete disruption of the electron transport chain; second, specific cell culture plates and inserts are required to detect bioenergetics values and do not fit inserts for barrier assessments. Therefore, there are not many functional assays that can be completed after the bioenergetics assay. However, it is expected that special devices can be developed to perform functional assays prior to the bioenergetics assay.
Significance of the Technique with Respect to Existing/Alternative Methods
Previous techniques to evaluate mitochondrial function necessitated the isolation of the mitochondria from the cells18. This new technique using the bioanalyzer allows for measurement of mitochondrial activity in intact cells, which preserves more of the cellular environment than evaluating isolated mitochondria.
Future Applications or Directions after Mastering This Technique
This protocol is designed and developed for the bEnd.3 cell line, but it is also compatible with primary cerebral vascular endothelial (pCVE) cells7 or other endothelium, and we have demonstrated this in our previous publication using pCVE cells7. When other types of endothelium are used, the coating of the culture plate and the use of growth factors may be required. However, the titration assay is recommended for other cell types as well. This protocol offers a general method to be adopted in the evaluation of the bioenergetics of CVE cells, and it may be further applied to mechanism studies or therapeutic responses in this manner.

It is widely recognized that the blood-brain-barrier (BBB) formed by cerebral vascular endothelial (CVE) cells has very distinct and unique functions paramount to vascular biology. These endothelial cells use mitochondria to generate most of the cellular supply of adenosine triphosphate (ATP) as a source of chemical energy. In addition to providing ATP for the CVE cells, mitochondria regulate various cellular processes in vascular endothelial cells, such as cellular signaling1-4, apoptosis5, and control of the cell cycle and cell growth6. Dysregulation of mitochondrial bioenergetic function in CVE cells may affect mitochondrial biogenesis, leading to impaired vascular endothelium activity, and exacerbate cerebrovascular diseases and neurodegenerative disorders, e.g., stroke7,8 and Alzheimer&#39;s disease (AD)9. We have demonstrated that insults to CVE cells following exposure to lipopolysaccharide (LPS) impair mitochondrial function in CVE cells and worsen stroke outcomes7. Tert-butylhydroquinone (tBHQ) increases stroke mortality by interfering with oxidative phosphorylation in CVE cells10. Therefore, the quantitative model of bioenergetic function in CVE cells not only pilots a series of mechanism-related studies for central nervous system (CNS) diseases, but also provides a breakthrough in drug screening of therapeutics targeting mitochondrial function in vascular biology.
Isolated mitochondria have been used to measure bioenergetic function. We7 and others11 have reported assessments of cellular bioenergetics in intact CVE cells by using an extracellular flux bioanalyzer. The analyzer provides the advantage of endogenous cellular bioenergetic assessment. This sensitive and consistent assay measures the mitochondrial metabolism of CVE cells and can be used to evaluate bioenergetic impairment by stimuli, investigate the mechanisms of mitochondrial signaling pathways, and screen drugs or treatments that affect mitochondrial function in CVE cells. The Oxygen Consumption Rate (OCR) is recorded by the bioanalyzer using a pharmacological profiling approach by combining the use of four mitochondrial disruptors: oligomycin, carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP), rotenone, and antimycin A. In this protocol, we detail an optimized approach that allows for the measurement and calculation of mitochondrial functional parameters in CVE cells, including basal mitochondrial respiration, ATP production, proton leak, maximal respiration, and spare respiratory capacity, in a single assay.

<table><tbody><tr><td>bEnd.3 cell line</td><td>ATCC</td><td>CRL-2299</td><td>25 - 30 passages</td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium (DMEM)</td><td>ATCC</td><td>30-2002</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Atlanta Biologicals</td><td>S12450</td><td>10% final concentration</td></tr><tr><td>Penicillin/Steptomycin</td><td>Hyclone</td><td>SV30010</td><td>1&amp;times;100 stocking</td></tr><tr><td>0.25% trypsin, 0.03% EDTA solution</td><td>Corning</td><td>25-053-CI</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Corning</td><td>25-000-CI</td><td>1.0 &amp;micro;M final concentration</td></tr><tr><td>Glucose</td><td>Sigma</td><td>CAS 50-99-7</td><td>25 mM final concentration</td></tr><tr><td>Oligomycin</td><td>Sigma</td><td>O4876</td><td>1.0 &amp;micro;M final concentration</td></tr><tr><td>Carbonilcyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP)</td><td>Sigma</td><td>C2920</td><td>0.5 &amp;micro;M final concentration</td></tr><tr><td>Rotenone</td><td>Sigma</td><td>R8875</td><td>1.0 &amp;micro;M final concentration</td></tr><tr><td>Antimycin A</td><td>Sigma</td><td>CAS 1397-94-0</td><td>1.0 &amp;micro;M final concentration</td></tr><tr><td>Plate wash station</td><td>Seahorse Bioscience</td><td></td><td></td></tr><tr><td>Extracellular flux bioanayzer&amp;nbsp;&amp;nbsp;</td><td>Seahorse Bioscience</td><td>XFe96</td><td></td></tr><tr><td>Extracellular flux cell culture plate</td><td>Seahorse Bioscience</td><td>102416-100</td><td></td></tr><tr><td>Extracellular flux sensor cartridge</td><td>Seahorse Bioscience</td><td>102416-100</td><td></td></tr><tr><td>Extracellular flux calibrant solution</td><td>Seahorse Bioscience</td><td>100840-000</td><td></td></tr><tr><td>Extracellular flux assay medium</td><td>Seahorse Bioscience</td><td>102365-100</td><td>PH buffered prior to assay</td></tr></tbody></table>

evaluation of bioenergetic function in cerebral vascular endothelial cells

The integrity of the blood-brain-barrier (BBB) is critical to prevent brain injury. Cerebral vascular endothelial (CVE) cells are one of the cell types that comprise the BBB; these cells have a very high-energy demand, which requires optimal mitochondrial function. In the case of disease or injury, the mitochondrial function in these cells can be altered, resulting in disease or&#160;the opening of the BBB. In this manuscript, we introduce a method to measure mitochondrial function in CVE cells by using whole, intact cells and a bioanalyzer. A mito-stress assay is used to challenge the cells that have been perturbed, either physically or chemically, and evaluate their bioenergetic function. Additionally, this method also provides a useful way to screen new therapeutics that have direct effects on mitochondrial function. We have optimized the cell density necessary to yield oxygen consumption rates that allow for the calculation of a variety of mitochondrial parameters, including ATP production, maximal respiration, and spare capacity. We also show the sensitivity of the assay by demonstrating that the introduction of the&#160;microRNA, miR-34a, leads to a pronounced and detectable decrease in mitochondrial activity. While the data shown in this paper is optimized for the bEnd.3 cell line, we have also optimized the protocol for primary CVE cells, further suggesting the utility in preclinical and clinical models.

To assess the bioenergetic function of CVE cells in response to oxidative stress, we chose the murine brain microvascular endothelia cell line bEnd.3, which displays the same comparative barrier characteristics as primary brain microvascular endothelial cells12. Given that the kinetics and relative intensity of response are varied among the various cell types, the first series of experiments were designed to obtain measurable OCR levels by identifying the optimal number of bEnd.3 cells to use in the assay for metabolic profiling, shown in Figure 2. Following the evaluation, the data are quantified and presented in Figure 3. Basal respiration, maximal respiration, and spare respiratory capacity showed a proportional response with cell density. However, ATP production decreased when 64 &#215; 103 cells per well were selected, suggesting that the over-confluent cell culture is not suitable for this experiment. The optimal cell densities occur between 8 - 32 &#215; 103 cells per well, based on the response to mitochondrial disruptors.
For subsequent experiments, a seeding density of 16 &#215; 103 cells per well was used to allow for optimal detection of changes in OCR. Using 16 &#215; 103 cells per well, we observed the expected responses in OCR and demonstrated that the microRNA miR-34a reduces mitochondrial function in CVE cells (Figure 4). We have previously reported that miR-34a reduced oxidative phosphorylation in these cells13. The repeated experiments also showed that maximal respiration and spare capacity were significantly decreased by the overexpression of miR-34a in CVEs at 24 h post-transfection, even though the basal respiration, ATP production, and proton leak had no significant changes (Figure 4). These data demonstrate&#160;the sensitivity of the bioanalyzer to detect changes in mitochondrial function in CVEs.
<img alt="Figure 1" src="/files/ftp_upload/54847/54847fig1.jpg" /> 
Figure 1. Strategic Planning for the Experiment. The timeline for the cell, plate, and cartridge preparation is indicated. <a href="http://cloudflare.jove.com/files/ftp_upload/54847/54847fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54847/54847fig2.jpg" /> 
Figure 2. Representative Raw Data of Bioenergetic Function in CVE Cells with Various Cell Densities. Oxygen consumption rates were measured in CVE cells with different cell densities. Data represent mean &#177; SD (n = 5); OCR: Oxygen Consumption Rate; FCCP: carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone; Rot/Anti-A: rotenone and antimycin A. &#9312;, &#9313;, and &#9314; indicate basal respiration; &#9315;, &#9316;, and &#9317; indicate ATP linked respiration; &#9318;, &#9319;, and &#9320; indicate maximum respiration; &#9321;, &#9322;, and &#9323; indicate non-mitochondrial respiration. <a href="http://cloudflare.jove.com/files/ftp_upload/54847/54847fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54847/54847fig3.jpg" /> 
Figure 3. Representative Results of Bioenergetic Function in CVE Cells with Various Cell Densities. Basal respiration, ATP production, maximal respiration, and spare capacity are calculated from the raw data generated by the bioenergetics functional assay in Figure 2 using the formulas indicated in Section 4. Data represent mean &#177; SD (n = 5); OCR: Oxygen Consumption Rate. <a href="http://cloudflare.jove.com/files/ftp_upload/54847/54847fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54847/54847fig4.jpg" /> 
Figure 4: Representative Results of Decreased Bioenergetic Function by miR-34a. (A) Raw data of Mitochondrial Function following overexpression of miR-34a at 24 h post-transfection. (B) Basal respiration, ATP production, maximal respiration, and spare capacity are calculated from the raw data generated by the bioenergetics functional assay in Figure 4A; the parameters are calculated by the formulas indicated in Section 4. Overexpression of miR-34a reduces mitochondrial function in CVE cells at 24 h post-transfection. Data represent mean &#177; SD (n = 5); OCR: Oxygen Consumption Rate. ****, P &#60; 0.0001. <a href="http://cloudflare.jove.com/files/ftp_upload/54847/54847fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Table 1" src="/files/ftp_upload/54847/54847table1.jpg" /> 
Table 1. The Instrument Run Protocol.

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Evaluation of Bioenergetic Function in Cerebral Vascular Endothelial Cells

Endothelial cell mitochondria are critical to maintain blood-brain-barrier integrity. We introduce a protocol to measure bioenergetic function in cerebral vascular endothelial cells.

The integrity of the blood-brain-barrier (BBB) is critical to prevent brain injury. Cerebral vascular endothelial (CVE) cells are one of the cell types ...

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Research

JoVE Journal

Neuroscience

9.2K Views.  West Virginia University. Endothelial cell mitochondria are critical to maintain blood-brain-barrier integrity. We introduce a protocol to measure bioenergetic function in cerebral vascular endothelial cells.

Video: Evaluation of Bioenergetic Function in Cerebral Vascular Endothelial Cells

Non-invasive Assessment of Microvascular and Endothelial Function

The authors have utilized capillaroscopy and forearm blood flow techniques to investigate the role of microvascular dysfunction in pathogenesis of cardiovascular disease. Capillaroscopy is a non-invasive, relatively inexpensive methodology for directly visualizing the microcirculation. Percent capillary recruitment is assessed by dividing the increase in capillary density induced by postocclusive reactive hyperemia (postocclusive reactive hyperemia capillary density minus baseline capillary density), by the maximal capillary density (observed during passive venous occlusion). Percent perfused capillaries represents the proportion of all capillaries present that are perfused (functionally active), and is calculated by dividing postocclusive reactive hyperemia capillary density by the maximal capillary density. Both percent capillary recruitment and percent perfused capillaries reflect the number of functional capillaries. The forearm blood flow (FBF) technique provides accepted non-invasive measures of endothelial function: The ratio FBFmax/FBFbase is computed as an estimate of vasodilation, by dividing the mean of the four FBFmax values by the mean of the four FBFbase values. Forearm vascular resistance at maximal vasodilation (FVRmax) is calculated as the mean arterial pressure (MAP) divided by FBFmax. Both the capillaroscopy and forearm techniques are readily acceptable to patients and can be learned quickly.
The microvascular and endothelial function measures obtained using the methodologies described in this paper may have future utility in clinical patient cardiovascular risk-reduction strategies. As we have published reports demonstrating that microvascular and endothelial dysfunction are found in initial stages of hypertension including prehypertension, microvascular and endothelial function measures may eventually aid in early identification, risk-stratification and prevention of end-stage vascular pathology, with its potentially fatal consequences.

Capillaroscopy is a non-invasive, relatively inexpensive methodology for directly visualizing the microcirculation. The forearm blood flow technique provides accepted non-invasive measures of endothelial function.

The authors have utilized capillaroscopy and forearm blood flow techniques to investigate the role of microvascular dysfunction in pathogenesis of ...

Medicine

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The blood-brain barrier (BBB) consisting of EC, astrocyte end-feet, pericytes and the basal membrane is responsible for the protection and homeostasis of the brain parenchyma. In vitro BBB models are common tools to study the structure and function of the BBB at the cellular level. A considerable number of different in vitro BBB models have been established for research in different laboratories to date. Usually, the cells are obtained from bovine, porcine, rat or mouse brain tissue (discussed in detail in the review by Wilhelm et al. 1). Human tissue samples are available only in a restricted number of laboratories or companies 2,3. While primary cell preparations are time consuming and the EC cultures can differ from batch to batch, the establishment of immortalized EC lines is the focus of scientific interest.
Here, we present a method for establishing an immortalized brain microvascular EC line from neonatal mouse brain. We describe the procedure step-by-step listing the reagents and solutions used. The method established by our lab allows the isolation of a homogenous immortalized endothelial cell line within four to five weeks. The brain microvascular endothelial cell lines termed cEND 4 (from cerebral cortex) and cerebEND 5 (from cerebellar cortex), were isolated according to this procedure in the F&ouml;rster laboratory and have been effectively used for explanation of different physiological and pathological processes at the BBB. Using cEND and cerebEND we have demonstrated that these cells respond to glucocorticoid- 4,6-9 and estrogen-treatment 10 as well as to pro-infammatory mediators, such as TNFalpha 5,8. Moreover, we have studied the pathology of multiple sclerosis 11 and hypoxia 12,13 on the EC-level. The cEND and cerebEND lines can be considered as a good tool for studying the structure and function of the BBB, cellular responses of ECs to different stimuli or interaction of the EC with lymphocytes or cancer cells.

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

This method describes how to isolate and immortalize microvascular endothelial cells from mouse brain. We describe a step-by-step protocol starting from the homogenization of brain tissue, digestion steps, seeding and immortalization of the cells. Usually, it takes about five weeks to obtain a homogenous, immortalized microvascular endothelial cell line.

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The ...

Ultrasound Assessment of Endothelial-Dependent Flow-Mediated Vasodilation of the Brachial Artery in Clinical Research

The vascular endothelium is a monolayer of cells that cover the interior of blood vessels and provide both structural and functional roles. The endothelium acts as a barrier, preventing leukocyte adhesion and aggregation, as well as controlling permeability to plasma components. Functionally, the endothelium affects vessel tone.
Endothelial dysfunction is an imbalance between the chemical species which regulate vessel tone, thombroresistance, cellular proliferation and mitosis. It is the first step in atherosclerosis and is associated with coronary artery disease, peripheral artery disease, heart failure, hypertension, and hyperlipidemia.
The first demonstration of endothelial dysfunction involved direct infusion of acetylcholine and quantitative coronary angiography. Acetylcholine binds to muscarinic receptors on the endothelial cell surface, leading to an increase of intracellular calcium and increased nitric oxide (NO) production. In subjects with an intact endothelium, vasodilation was observed while subjects with endothelial damage experienced paradoxical vasoconstriction.
There exists a non-invasive, in vivo method for measuring endothelial function in peripheral arteries using high-resolution B-mode ultrasound. The endothelial function of peripheral arteries is closely related to coronary artery function. This technique measures the percent diameter change in the brachial artery during a period of reactive hyperemia following limb ischemia.
This technique, known as endothelium-dependent, flow-mediated vasodilation (FMD) has value in clinical research settings. However, a number of physiological and technical issues can affect the accuracy of the results and appropriate guidelines for the technique have been published. Despite the guidelines, FMD remains heavily operator dependent and presents a steep learning curve. This article presents a standardized method for measuring FMD in the brachial artery on the upper arm and offers suggestions to reduce intra-operator variability.

Endothelial dysfunction is associated with numerous disease states and is predictive of adverse cardiovascular events in humans. Flow-mediated vasodilation (FMD) is a non-invasive ultrasound method of evaluating endothelial function. Methodological choices and operator experience may affect results. A systematic approach to FMD in human studies is discussed here.

The vascular endothelium is a monolayer of cells that cover the interior of blood vessels and provide both structural and functional roles. The ...

A Methodological Approach to Non-invasive Assessments of Vascular Function and Morphology

The endothelium is the innermost lining of the vasculature and is involved in the maintenance of vascular homeostasis. Damage to the endothelium may predispose the vessel to atherosclerosis and increase the risk for cardiovascular disease. Assessments of peripheral endothelial function are good indicators of early abnormalities in the vascular wall and correlate well with assessments of coronary endothelial function. The present manuscript details the important methodological steps necessary for the assessment of microvascular endothelial function using laser Doppler imaging with iontophoresis, large vessel endothelial function using flow-mediated dilatation, and carotid atherosclerosis using carotid artery ultrasound. A discussion on the methodological considerations for each of the techniques is also presented, and recommendations are made for future research.

The present article describes the methodological considerations for several non-invasive assessments of vascular function and morphology that are commonly used in medical research to assess different stages of atherosclerosis.

The endothelium is the innermost lining of the vasculature and is involved in the maintenance of vascular homeostasis.  Damage to the endothelium may ...

Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have identified a special population of vascular cells, the periventricular endothelial cells, that play a critical role in the migration and distribution of forebrain GABAergic interneurons during embryonic development. This, coupled with their cell-autonomous functions, alludes to novel roles of periventricular endothelial cells in the pathology of neuropsychiatric disorders like schizophrenia, epilepsy, and autism. Here, we have described three different in vitro assays that collectively evaluate the functions of periventricular endothelial cells and their interaction with GABAergic interneurons. Use of these assays, particularly in a human context, will allow us to identify the link between periventricular endothelial cells and brain disorders. These assays are simple, low cost, and reproducible, and can be easily adapted to any adherent cell type.

We present three simple in vitro assays-the long-distance migration assay, the co-culture migration assay, and chemo-attraction assay-that collectively evaluate the functions of human stem cell derived periventricular endothelial cells and their interaction with GABAergic interneurons.

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have ...

A Simple Bioassay for the Evaluation of Vascular Endothelial Growth Factors

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive expression of families of related receptors, a broad range of related ligands and the difficulty of dealing with primary cultures of specialized endothelial cells. Here we describe a bioassay for the detection of ligands to the vascular endothelial growth factor receptor-2 (VEGFR-2), a key transducer of signals that promote angiogenesis and lymphangiogenesis. A cDNA encoding a fusion of the extracellular (ligand-binding) region of VEGFR-2 with the transmembrane and cytoplasmic regions of the erythropoietin receptor (EpoR) is expressed in the factor-dependent cell line Ba/F3. This cell line grows in the presence of interleukin-3 (IL-3) and withdrawal of this factor results in death of the cells within 24 hr. Expression of the VEGFR-2/EpoR receptor fusion provides an alternative mechanism to promote survival and potentially proliferation of stably transfected Ba/F3 cells in the presence of a ligand capable of binding and cross-linking the extracellular portion of the fusion protein (i.e., one that can cross-link the VEGFR-2 extracellular region). The assay can be performed in two ways: a semi-quantitative approach in which small volumes of ligand and cells permit a rapid result in 24 hr, and a quantitative approach involving surrogate markers of a viable cell number. The assay is relatively easy to perform, is highly responsive to known VEGFR-2 ligands and can accommodate extracellular inhibitors of VEGFR-2 signaling such as monoclonal antibodies to the receptor or ligands, and soluble ligand traps.

We describe a simple cell-based bioassay for detecting, quantifying and monitoring the activity of members of the vascular endothelial growth factor family of ligands. The assay uses chimeric receptors expressed in a factor-dependent cell line to provide a semi-quantitative or quantitative assessment of receptor binding and cross-linking by the ligand.

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive ...

Biology

Transendothelial Resistance Measurement to Assess Cell Barrier Integrity

Barrier Functional Integrity Recording on bEnd.3 Vascular Endothelial Cells via Transendothelial Electrical Resistance Detection

The blood-brain barrier (BBB) is a dynamic physiological structure composed of microvascular endothelial cells, astrocytes, and pericytes. By coordinating the interaction between restricted transit of harmful substances, nutrient absorption, and metabolite clearance in the brain, the BBB is essential in preserving central nervous system homeostasis. Building in vitro models of the BBB is a valuable tool for exploring the pathophysiology of neurological disorders and creating pharmacological treatments. This study describes a procedure for creating an in vitro monolayer BBB cell model by seeding bEnd.3 cells into the upper chamber of a 24-well plate. To assess the integrity of cell barrier function, the conventional epithelial cell voltmeter was used to record the transmembrane electrical resistance of normal cells and CoCl2-induced hypoxic cells in real-time. We anticipate that the above experiments will provide effective ideas for the creation of in vitro models of BBB and drugs to treat disorders of central nervous system diseases.

Author Spotlight: Applications of TEER Detection to Assess Cell Barrier Integrity 

This protocol describes a dependable and efficient in vitro model of the brain blood barrier. The method uses mouse cerebral vascular endothelial cells bEnd.3 and measures transmembrane electrical resistance.

The blood-brain barrier (BBB) is a dynamic physiological structure composed of microvascular endothelial cells, astrocytes, and pericytes. By coordinating ...

Simultaneous Measurements of Intracellular Calcium and Membrane Potential in Freshly Isolated and Intact Mouse Cerebral Endothelium

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the lumen of blood vessels and command changes in vascular diameter as needed to meet the metabolic demand of neurons. Primary endothelial-dependent signaling pathways of hyperpolarization of membrane potential (Vm) and nitric oxide typically operate in parallel to mediate vasodilation and thereby increase blood flow. Although integral to coordinating vasodilation over several millimeters of vascular length, components of endothelium-derived hyperpolarization (EDH) have been historically difficult to measure. These components of EDH entail intracellular Ca2+ [Ca2+]i increases and subsequent activation of small- and intermediate conductance Ca2+-activated K+ (SKCa/IKCa) channels.
Here, we present a simplified illustration of the isolation of fresh endothelium from mouse cerebral arteries; simultaneous measurements of endothelial [Ca2+]i and Vm using Fura-2 photometry and intracellular sharp electrodes, respectively; and a continuous superfusion of salt solutions and pharmacological agents under physiological conditions (pH 7.4, 37 &#176;C). Posterior cerebral arteries from the Circle of Willis are removed free of the posterior communicating and the basilar arteries. Enzymatic digestion of cleaned posterior cerebral arterial segments and subsequent trituration facilitates removal of adventitia, perivascular nerves, and smooth muscle cells. Resulting posterior cerebral arterial endothelial &#34;tubes&#34; are then secured under a microscope and examined using a camera, photomultiplier tube, and one to two electrometers while under continuous superfusion. Collectively, this method can simultaneously measure changes in endothelial [Ca2+]i and Vm in discrete cellular locations, in addition to the spreading of EDH through gap junctions up to millimeter distances along the intact endothelium. This method is expected to yield a high-throughput analysis of the cerebral endothelial functions underlying mechanisms of blood flow regulation in the normal and diseased brain.

Demonstrated here are protocols for (1) freshly isolating intact cerebral endothelial "tubes" and (2) simultaneous measurements of endothelial calcium and membrane potential during endothelium-derived hyperpolarization. Further, these methods allow for pharmacological tuning of endothelial cell calcium and electrical signaling as individual or interactive experimental variables.

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the ...

Isolation and Functional Analysis of Arteriolar Endothelium of Mouse Brain Parenchyma

Cerebral blood flow is conveyed by vascular resistance arteries and downstream parenchymal arterioles. Steady-state vascular resistance to blood flow increases with decreasing diameter from arteries to arterioles that ultimately feed into capillaries. Due to their smaller size and location in the parenchyma, arterioles have been relatively understudied and with less reproducibility in findings than surface pial arteries. Regardless, arteriolar endothelial cell structure and function&#8212;integral to the physiology and etiology of chronic degenerative diseases&#8212;requires extensive investigation. In particular, emerging evidence demonstrates that compromised endothelial function precedes and exacerbates cognitive impairment and dementia.
In the parenchymal microcirculation, endothelial K+ channel function is the most robust stimulus to finely control the spread of vasodilation to promote increases in blood flow to areas of neuronal activity. This paper illustrates a refined method for freshly isolating intact and electrically coupled endothelial &#34;tubes&#34; (diameter, ~25 &#181;m) from mouse brain parenchymal arterioles. Arteriolar endothelial tubes are secured during physiological conditions (37 &#176;C, pH 7.4) to resolve experimental variables that encompass K+ channel function and their regulation, including intracellular Ca2+ dynamics, changes in membrane potential, and membrane lipid regulation. A distinct technical advantage versus arterial endothelium is the enhanced morphological resolution of cell and organelle (e.g., mitochondria) dimensions, which expands the usefulness of this technique. Healthy cerebral perfusion throughout life entails robust endothelial function in parenchymal arterioles, directly linking blood flow to the fueling of neuronal and glial activity throughout precise anatomical regions of the brain. Thus, it is expected that this method will significantly advance the general knowledge of vascular physiology and neuroscience concerning the healthy and diseased brain.

Intensive preparation of intact mouse cerebral endothelial &#34;tubes&#34; from cerebral parenchymal arterioles is illustrated for studying cerebral blood flow regulation. Further, we demonstrate the experimental strengths of this endothelial study model for fluorescence imaging and electrophysiology measurement of key cellular signaling pathways, including changes in intracellular [Ca2+] and membrane potential.

Cerebral blood flow is conveyed by vascular resistance arteries and downstream parenchymal arterioles. Steady-state vascular resistance to blood flow ...

Impedance-Based Biosensors in Investigating Brain Endothelial Barrier Strength

Measuring Changes in Brain Endothelial Barrier Integrity with Two Impedance-based Biosensors in Response to Cancer Cells and Cytokines

The blood-brain barrier (BBB) protects the brain parenchyma against harmful pathogens in the blood. The BBB consists of the neurovascular unit, comprising pericytes, astrocytic foot processes, and tightly adhered endothelial cells. Here, the brain endothelial cells form the first line of barrier against blood-borne pathogens. In conditions like cancer and neuroinflammation, circulating factors in the blood can disrupt this barrier. Disease progression significantly worsens post barrier disruption, which permits access to or impairment of regions of the brain. This significantly worsens the prognoses, particularly due to limited treatment options available at the level of the brain. Hence, emerging studies aim to investigate potential therapeutics that can prevent these detrimental factors in the blood from interacting with the brain endothelial cells.
The commercially available Electric Cell-Substrate Impedance Sensing (ECIS) and cellZscope instruments measure the impedance across cellular monolayers, such as the BBB endothelium, to determine their barrier strength. Here we detail the use of both biosensors in assessing brain endothelial barrier integrity upon the addition of various stimuli. Crucially, we highlight the importance of their high-throughput capability for concurrent investigation of multiple variables and biological treatments.

Author Spotlight: Studying Brain Endothelial Barrier in Metastatic Cancer Using Impedance-Based Biosensors

Here we demonstrate the technique of using impedance-based biosensors: ECIS and cellZscope, for measuring brain endothelial barrier strength. We detail the preparation and technique of adding various stimuli to an in vitro model of the brain endothelium. We measure, record, and give a representative analysis of the findings.

The blood-brain barrier (BBB) protects the brain parenchyma against harmful pathogens in the blood. The BBB consists of the neurovascular unit, comprising ...

Evaluation of Bioenergetic Function in Cerebral Vascular Endothelial Cells

Summary

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Chapters in this video

Non-invasive Assessment of Microvascular and Endothelial Function

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

Ultrasound Assessment of Endothelial-Dependent Flow-Mediated Vasodilation of the Brachial Artery in Clinical Research

A Methodological Approach to Non-invasive Assessments of Vascular Function and Morphology

Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons

A Simple Bioassay for the Evaluation of Vascular Endothelial Growth Factors

Barrier Functional Integrity Recording on bEnd.3 Vascular Endothelial Cells via Transendothelial Electrical Resistance Detection

Simultaneous Measurements of Intracellular Calcium and Membrane Potential in Freshly Isolated and Intact Mouse Cerebral Endothelium

Isolation and Functional Analysis of Arteriolar Endothelium of Mouse Brain Parenchyma

Measuring Changes in Brain Endothelial Barrier Integrity with Two Impedance-based Biosensors in Response to Cancer Cells and Cytokines