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>

<ol>
	<li>Quintero, M., Colombo, S. L., Godfrey, A., Moncada, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria+as+signaling+organelles+in+the+vascular+endothelium.">Mitochondria as signaling organelles in the vascular endothelium.</a> Proc Natl Acad Sci U S A. 103, 5379-5384 (2006).</li><li>Liu, Y., Li, H., Bubolz, A. H., Zhang, D. X., Gutterman, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cytoskeletal+elements+are+critical+for+flow-mediated+dilation+in+human+coronary+arterioles.">Endothelial cytoskeletal elements are critical for flow-mediated dilation in human coronary arterioles.</a> Med Biol Eng Comput. 46, 469-478 (2008).</li><li>Al-Mehdi, A. B., 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=Perinuclear+mitochondrial+clustering+creates+an+oxidant-rich+nuclear+domain+required+for+hypoxia-induced+transcription.">Perinuclear mitochondrial clustering creates an oxidant-rich nuclear domain required for hypoxia-induced transcription.</a> Sci Signal. 5, ra47 (2012).</li><li>Mironov, S. L., Ivannikov, M. V., Johansson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=[Ca2+]i+signaling+between+mitochondria+and+endoplasmic+reticulum+in+neurons+is+regulated+by+microtubules.+From+mitochondrial+permeability+transition+pore+to+Ca2+-induced+Ca2++release.">[Ca2+]i signaling between mitochondria and endoplasmic reticulum in neurons is regulated by microtubules. From mitochondrial permeability transition pore to Ca2+-induced Ca2+ release.</a> J Biol Chem. 280, 715-721 (2005).</li><li>Sutendra, G., 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=The+role+of+Nogo+and+the+mitochondria-endoplasmic+reticulum+unit+in+pulmonary+hypertension.">The role of Nogo and the mitochondria-endoplasmic reticulum unit in pulmonary hypertension.</a> Sci Transl Med. 3, 88ra55 (2011).</li><li>Mitra, K., Wunder, C., Roysam, B., Lin, G., Lippincott-Schwartz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+hyperfused+mitochondrial+state+achieved+at+G1-S+regulates+cyclin+E+buildup+and+entry+into+S+phase.">A hyperfused mitochondrial state achieved at G1-S regulates cyclin E buildup and entry into S phase.</a> Proc Natl Acad Sci U S A. 106, 11960-11965 (2009).</li><li>Doll, D. 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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 border="0" cellpadding="0" cellspacing="0" width="840">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="26">
			<td height="26" style="height:27px;width:377px;">bEnd.3 cell line</td>
			<td style="width:147px;">ATCC</td>
			<td style="width:119px;">CRL-2299</td>
			<td style="width:197px;">25-30 passages</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:377px;">Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM)</td>
			<td style="width:147px;">ATCC</td>
			<td style="width:119px;">30-2002</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:377px;">Fetal bovine serum</td>
			<td style="width:147px;">Atlanta Biologicals</td>
			<td style="width:119px;">S12450</td>
			<td>10% in final</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:377px;">Penicillin/Steptomycin</td>
			<td style="width:147px;">Hyclone</td>
			<td style="width:119px;">SV30010</td>
			<td>1&#215;100 stocking</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:377px;">0.25% trypsin 0.03% EDTA solution</td>
			<td style="width:147px;">Corning</td>
			<td style="width:119px;">25-053-CI</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:377px;">Sodium pyruvate</td>
			<td style="width:147px;">Corning</td>
			<td style="width:119px;">25-000-CI</td>
			<td>1.0 &#181;M in final</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:377px;">Glucose</td>
			<td style="width:147px;">Sigma</td>
			<td style="width:119px;">CAS 50-99-7</td>
			<td>25 mM in final</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:377px;">Oligomycin</td>
			<td style="width:147px;">Sigma</td>
			<td style="width:119px;">O4876</td>
			<td>1.0 &#181;M in final</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:377px;">Carbonilcyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP)</td>
			<td style="width:147px;">Sigma</td>
			<td style="width:119px;">C2920</td>
			<td>0.5 &#181;M in final</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:377px;">Rotenone</td>
			<td style="width:147px;">Sigma</td>
			<td style="width:119px;">R8875</td>
			<td>1.0 &#181;M in final</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:377px;">Antimycin A</td>
			<td style="width:147px;">Sigma</td>
			<td style="width:119px;">CAS 1397-94-0</td>
			<td>1.0 &#181;M in final</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:377px;">Plate wash station</td>
			<td style="width:147px;">Seahorse Bioscience</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:377px;">Extracellular flux bioanayzer&#160;&#160;</td>
			<td style="width:147px;">Seahorse Bioscience</td>
			<td style="width:119px;">XFe96</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:377px;">Extracellular flux cell culture plate</td>
			<td style="width:147px;">Seahorse Bioscience</td>
			<td style="width:119px;">102416-100</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:377px;">Extracellular flux sensor cartridge</td>
			<td style="width:147px;">Seahorse Bioscience</td>
			<td style="width:119px;">102416-100</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:377px;">Extracellular flux calibrant solution</td>
			<td style="width:147px;">Seahorse Bioscience</td>
			<td style="width:119px;">100840-000</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:377px;">Extracellular flux assay medium</td>
			<td style="width:147px;">Seahorse Bioscience</td>
			<td style="width:119px;">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 ...

evaluation-bioenergetic-function-cerebral-vascular-endothelial

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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

Studying Synaptic Vesicle Pools using Photoconversion of Styryl Dyes

The fusion of synaptic vesicles with the plasma membrane (exocytosis) is a required step in neurotransmitter release and neuronal communication. The vesicles are then retrieved from the plasma membrane (endocytosis) and grouped together with the general pool of vesicles within the nerve terminal, until they undergo a new exo- and endocytosis cycle (vesicle recycling). These processes have been studied using a variety of techniques such as electron microscopy, electrophysiology recordings, amperometry and capacitance measurements. Importantly, during the last two decades a number of fluorescently labeled markers emerged, allowing optical techniques to track vesicles in their recycling dynamics. One of the most commonly used markers is the styryl or FM dye 1; structurally, all FM dyes contain a hydrophilic head and a lipophilic tail connected through an aromatic ring and one or more double bonds (Fig. 1B). A classical FM dye experiment to label a pool of vesicles consists in bathing the preparation (Fig. 1Ai) with the dye during the stimulation of the nerve (electrically or with high K+). This induces vesicle recycling and the subsequent loading of the dye into recently endocytosed vesicles (Fig. 1Ai-iii). After loading the vesicles with dye, a second round of stimulation in a dye-free bath would trigger the FM release through exocytosis (Fig. 1Aiv-v), process that can be followed by monitoring the fluorescence intensity decrease (destaining).

Although FM dyes have contributed greatly to the field of vesicle recycling, it is not possible to determine the exact localization or morphology of individual vesicles by using conventional fluorescence microscopy. For that reason, we explain here how FM dyes can also be used as endocytic markers using electron microscopy, through photoconversion. The photoconversion technique exploits the property of fluorescent dyes to generate reactive oxygen species under intense illumination. Fluorescently labeled preparations are submerged in a solution containing diaminobenzidine (DAB) and illuminated. Reactive species generated by the dye molecules oxidize the DAB, which forms a stable, insoluble precipitate that has a dark appearance and can be easily distinguished in electron microscopy 2,3. As DAB is only oxidized in the immediate vicinity of fluorescent molecules (as the reactive oxygen species are short-lived), the technique ensures that only fluorescently labeled structures are going to contain the electron-dense precipitate. The technique thus allows the study of the exact location and morphology of actively recycling organelles.

FM dyes have been of invaluable help in the understanding of synaptic dynamics. FMs are normally followed under the fluorescent microscope during different stimulation conditions. However, photoconversion of FM dyes combined with electron microscopy allows the visualization of distinct synaptic vesicle pools, among other ultrastructure components, in synaptic boutons.

The fusion of synaptic vesicles with the plasma membrane (exocytosis) is a required step in neurotransmitter release and neuronal communication. The ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Isolation of Primary Murine Brain Microvascular Endothelial Cells

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional complex of tight and adherens junctions. Together with other cell-types such as astrocytes or pericytes, they form the neurovascular unit (NVU), which specifically regulates the interchange of fluids, molecules and cells between the peripheral blood and the CNS. Through this complex and dynamic system BMECs are involved in various processes maintaining the homeostasis of the CNS. A dysfunction of the BBB is observed as an essential step in the pathogenesis of many severe CNS diseases. However, specific and targeted therapies are very limited, as the underlying mechanisms are still far from being understood. 
Animal and in vitro models have been extensively used to gain in-depth understanding of complex physiological and pathophysiological processes. By reduction and simplification it is possible to focus the investigation on the subject of interest and to exclude a variety of confounding factors. However, comparability and transferability are also reduced in model systems, which have to be taken into account for evaluation. The most common animal models are based on mice, among other reasons, mainly due to the constantly increasing possibilities of methodology. In vitro studies of isolated murine BMECs might enable an in-depth analysis of their properties and of the blood-brain-barrier under physiological and pathophysiological conditions. Further insights into the complex mechanisms at the BBB potentially provide the basis for new therapeutic strategies.
This protocol describes a method to isolate primary murine microvascular endothelial cells by a sequence of physical and chemical purification steps. Special considerations for purity and cultivation of MBMECs as well as quality control, potential applications and limitations are discussed.

Brain microvascular endothelial cells (BMEC) are interconnected by specific junctional proteins forming a highly regulated barrier separating blood and the central nervous system (CNS), the so-called blood-brain-barrier (BBB). The isolation of primary murine brain microvascular endothelial cells, as discussed in this protocol, enables detailed in vitro studies of the BBB.

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional ...

Quantification of Cerebral Vascular Architecture using Two-photon Microscopy in a Mouse Model of HIV-induced Neuroinflammation

Human Immunodeficiency Virus 1 (HIV-1) infection frequently results in HIV-1 Associated Neurocognitive Disorders (HAND), and is characterized by a chronic neuroinflammatory state within the central nervous system (CNS), thought to be driven principally by virally-mediated activation of microglia and brain resident macrophages. HIV-1 infection is also accompanied by changes in cerebrovascular blood flow (CBF), raising the possibility that HIV-associated chronic neuroinflammation may lead to changes in CBF and/or in cerebral vascular architecture. To address this question, we have used a mouse model for HIV-induced neuroinflammation, and we have tested whether long-term exposure to this inflammatory environment may damage brain vasculature and result in rarefaction of capillary networks. In this paper we describe a method to quantify changes in cortical capillary density in a mouse model of neuroinflammatory disease (HIV-1 Tat transgenic mice). This generalizable approach employs in vivo two-photon imaging of cortical capillaries through a thin-skull cortical window, as well as ex vivo two-photon imaging of cortical capillaries in mouse brain sections. These procedures produce images and z-stack files of capillary networks, respectively, which can be then subjected to quantitative analysis in order to assess changes in cerebral vascular architecture.

This paper describes a method by which the vascular architecture in the brain can be quantified using in vivo and ex vivo two-photon microscopy.

Human Immunodeficiency Virus 1 (HIV-1) infection frequently results in HIV-1 Associated Neurocognitive Disorders (HAND), and is characterized by a chronic ...

Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal cord of the majority of these fiber systems have not been thoroughly investigated in any species. Serotonergic fibers, which project to the spinal cord, have been studied in rats and opossums on histological sections and their functional significance has been deduced based on their fiber termination pattern in the spinal cord. With the development of CLARITY and CUBIC techniques, it is possible to investigate this fiber system and its distribution in the spinal cord, which is likely to reveal previously unknown features of serotonergic supraspinal pathways. Here, we provide a detailed protocol for imaging the serotonergic fibers in the mouse spinal cord using the combined CLARITY and CUBIC techniques. The method involves perfusion of a mouse with a hydrogel solution and clarification of the tissue with a combination of clearing reagents. Spinal cord tissue was cleared in just under two weeks, and the subsequent immunofluorescent staining against serotonin was completed in less than ten days. With a multi-photon fluorescent microscope, the tissue was scanned and a 3D image was reconstructed using Osirix software.

Supraspinal projections are important for pain perception and other behaviors, and serotonergic fibers are one of these fiber systems. The present study focused on the application of the combined CLARITY/CUBIC protocol to the mouse spinal cord in order to investigate the termination of these serotonergic fibers.

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal ...

Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional multispectral diffuse reflectance imaging system. This system is constructed from a broadband white light source, a motorized filter wheel with a set of narrowband interference filters, a light guide, a collecting lens, a video zoom lens, and a monochromatic charged-coupled device (CCD) camera. An ellipsoidal cranial window is made in the skull bone of a rat under isoflurane anesthesia to capture in vivo multispectral diffuse reflectance images of the cortical surface. Regulation of the fraction of inspired oxygen using a gas mixture device enables the induction of different respiratory states such as normoxia, hyperoxia, and anoxia. A Monte Carlo simulation-based multiple regression analysis for the measured multispectral diffuse reflectance images at nine wavelengths (500, 520, 540, 560, 570, 580, 600, 730, and 760 nm) is then performed to visualize the two-dimensional maps of hemodynamics and the light scattering properties of the in vivo rat brain.

Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional multispectral diffuse reflectance imaging system.

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional ...

Rat Model of Widespread Cerebral Cortical Demyelination Induced by an Intracerebral Injection of Pro-Inflammatory Cytokines

Multiple sclerosis (MS) is the most common immune-mediated disease of the central nervous system (CNS) and progressively leads to physical disability and death, caused by white matter lesions in the spinal cord and cerebellum, as well as by demyelination in grey matter. Whilst conventional models of experimental allergic encephalomyelitis are suitable for the investigation of the cell-mediated inflammation in the spinal and cerebellar white matter, they fail to address grey matter pathologies. Here, we present the experimental protocol for a novel rat model of cortical demyelination allowing the investigation of the pathological and molecular mechanisms leading to cortical lesions. The demyelination is induced by an immunization with low-dose myelin oligodendrocyte glycoprotein (MOG) in an incomplete Freund&#39;s adjuvant followed by a catheter-mediated intracerebral delivery of pro-inflammatory cytokines. The catheter, moreover, enables multiple rounds of demyelination without causing injection-induced trauma, as well as the intracerebral delivery of potential therapeutic drugs undergoing a preclinical investigation. The method is also ethically favorable as animal pain and distress or disability are controlled and relatively minimal. The expected timeframe for the implementation of the entire protocol is around 8 - 10 weeks.

The protocol presented here allows the reproduction of a widespread grey matter demyelination of both cortical hemispheres in adult male Dark Agouti rats. The method comprises of intracerebral implantation of a catheter, subclinical immunization against myelin oligodendrocyte glycoprotein, and intracerebral injection of a pro-inflammatory cytokine mixture through the implanted catheter.

Multiple sclerosis (MS) is the most common immune-mediated disease of the central nervous system (CNS) and progressively leads to physical disability and ...

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.

The zebrafish is a model system that has many valuable features including optical clarity, rapid external development, and, of particular importance to the field of hearing and balance, externally located sensory hair cells. This article outlines how transgenic zebrafish can be used to assay both hair-cell mechanosensation and presynaptic function in toto.

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory ...

Isolation of Primary Murine Skeletal Muscle Microvascular Endothelial Cells

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal muscles regulating the exchange of fluids and nutrients as well as the immune response against infectious agents by controlling immune cell migration. For these functions, MMEC form a functional &#34;myovascular unit&#34; (MVU), with further cell types, such as fibroblasts, pericytes and skeletal muscle cells. Consequently, a dysfunction of MMEC and therefore the MVU contributes to a vast variety of myopathies. However, regulatory mechanisms of MMEC in health and disease remain insufficiently understood and their elucidation precedes more specific treatments for myopathies. The isolation and in-depth investigation of primary MMEC functions in the context of the MVU might facilitate a better understanding of these processes.
This article provides a protocol to isolate primary murine MMEC of the skeletal muscle by mechanical and enzymatic dissociation including purification and culture maintenance steps.

Microvascular endothelial cells of skeletal muscles (MMEC) shape the inner wall of muscle capillaries and regulate both, exchange of fluids/molecules and migration of (immune) cells between muscle tissue and blood. Isolation of primary murine MMEC, as described here, enables comprehensive in vitro investigations of the &#34;myovascular unit&#34;.

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal ...