We thank Charles Hewitt for excellent technical assistance while establishing equipment and supplies needed for the current protocols. We thank Drs. Sean M. Wilson and Christopher G. Wilson, from the LLU Center for Perinatal Biology, for providing us with an additional inverted microscope and electrometer, respectively. This research has been supported by National Institutes of Health grant R00-AG047198 (EJB) and Loma Linda University School of Medicine new faculty start-up funds. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Before conducting the following experiments, ensure that all animal care use and protocols are approved by the Institutional Animal Care and Use Committee (IACUC) and performed in accord with the National Research Council&#39;s &#34;Guide for the Care and Use of Laboratory Animals&#34; (8th Edition, 2011) and the ARRIVE guidelines. The IACUC of Loma Linda University has approved all protocols used for this manuscript for male and female C57BL/6 mice (age range: 3 to 30 mo).
<p class="jove...

<ol>
	<li>Longden, T. A., Hill-Eubanks, D. C., Nelson, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion+channel+networks+in+the+control+of+cerebral+blood+flow.">Ion channel networks in the control of cerebral blood flow.</a> Journal of Cerebral Blood Flow &amp; Metabolism. 36 (3), 492-512 (2016).</li><li>Chen, B. R., Kozberg, M. G., Bouchard, M. B., Shaik, M. A., Hillman, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+role+for+the+vascular+endothelium+in+functional+neurovascular+coupling+in+the+brain.">A critical role for the vascular endothelium in functional neurovascular coupling in the brain.</a> Journal of the American Heart Association. 3 (3), e000787 (2014).</li><li>Iadecola, C., Yang, G., Ebner, T. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+endothelial+intermediate+conductance+KCa+channels+in+cerebral+EDHF-mediated+dilations.">Role of endothelial intermediate conductance KCa channels in cerebral EDHF-mediated dilations.</a> American Journal of Physiology-Heart and Circulatory Physiology. 285 (4), H1590-H1599 (2003).</li><li>Behringer, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+and+electrical+signaling+in+arterial+endothelial+tubes:+New+insights+into+cellular+physiology+and+cardiovascular+function.">Calcium and electrical signaling in arterial endothelial tubes: New insights into cellular physiology and cardiovascular function.</a> Microcirculation. 24 (3), (2017).</li><li>Endemann, D. H., Schiffrin, E. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+Aging+on+Calcium+Signaling+and+Membrane+Potential+in+Endothelium+of+Resistance+Arteries:+A+Role+for+Mitochondria.">Impact of Aging on Calcium Signaling and Membrane Potential in Endothelium of Resistance Arteries: A Role for Mitochondria.</a> The Journal of Gerontology, Series A: Biological Sciences and Medical Sciences. 72 (12), 1627-1637 (2017).</li><li>Behringer, E. J., 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=Calcium+and+electrical+dynamics+in+lymphatic+endothelium.">Calcium and electrical dynamics in lymphatic endothelium.</a> The Journal of Physiology. 595 (24), 7347-7368 (2017).</li><li>Simmers, M. B., Pryor, A. W., Blackman, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arterial+shear+stress+regulates+endothelial+cell-directed+migration,+polarity,+and+morphology+in+confluent+monolayers.">Arterial shear stress regulates endothelial cell-directed migration, polarity, and morphology in confluent monolayers.</a> American Journal of Physiology-Heart and Circulatory Physiolog. 293 (3), H1937-H1946 (2007).</li><li>Sandow, S. L., Grayson, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limits+of+isolation+and+culture:+intact+vascular+endothelium+and+BKCa.">Limits of isolation and culture: intact vascular endothelium and BKCa.</a> American Journal of Physiology-Heart and Circulatory Physiology. 297 (1), H1-H7 (2009).</li><li>Diaz-Otero, J. M., Garver, H., Fink, G. D., Jackson, W. F., Dorrance, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging+is+associated+with+changes+to+the+biomechanical+properties+of+the+posterior+cerebral+artery+and+parenchymal+arterioles.">Aging is associated with changes to the biomechanical properties of the posterior cerebral artery and parenchymal arterioles.</a> American Journal of Physiology-Heart and Circulatory Physiology. 310 (3), H365-H375 (2016).</li><li>Kochukov, M. Y., Balasubramanian, A., Abramowitz, J., Birnbaumer, L., Marrelli, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+endothelial+transient+receptor+potential+C3+channel+is+required+for+small+conductance+calcium-activated+potassium+channel+activation+and+sustained+endothelial+hyperpolarization+and+vasodilation+of+cerebral+artery.">Activation of endothelial transient receptor potential C3 channel is required for small conductance calcium-activated potassium channel activation and sustained endothelial hyperpolarization and vasodilation of cerebral artery.</a> Journal of the American Heart Association. 3 (4), (2014).</li><li>Zhang, L., Papadopoulos, P., Hamel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+TRPV4+channels+mediate+dilation+of+cerebral+arteries:+impairment+and+recovery+in+cerebrovascular+pathologies+related+to+Alzheimer's+disease.">Endothelial TRPV4 channels mediate dilation of cerebral arteries: impairment and recovery in cerebrovascular pathologies related to Alzheimer's disease.</a> British Journal of Pharmacology. 170 (3), 661-670 (2013).</li><li>Socha, M. 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In light of recent developments6,15,16,17, we now demonstrate the method to isolate mouse cerebral arterial endothelium in preparation for simultaneous measurement of [Ca2+]i and Vm underlying EDH consistently for ~2 h at 37 &#176;C. Although technically difficult, we can measure cell-to-cell coupling as well (see reference6, Figure 1). I...

Blood flow throughout the brain is regulated by the coordination of vasodilation among cerebral arteries and arterioles in vascular networks1. Endothelial cells lining cerebral resistance arteries command changes in vascular diameter as needed to meet the metabolic demand of neurons1,2,3. In particular, during endothelium-derived hyperpolarization (commonly known as EDH), intracellular Ca2+ ([Ca2+]i) and electrical signaling in endothelial cells coordinate vasodilation among endothelial cells and their surrounding smooth muscle cells through gap junctions for arterial relaxation4. Physiological initiation of EDH sequentially entails stimulation of Gq-coupled receptors (GPCRs), an increase in [Ca2+]i, and activation of endothelial small- and intermediate-Ca2+-activated K+ (SKCa/IKCa) channels to hyperpolarize cerebral endothelial membrane potential (Vm)5,6,7. Thus, the intimate relationship of endothelial [Ca2+]i and Vm is integral to blood flow regulation and indispensable to cardio- and cerebrovascular function6,8. Throughout the broader literature, numerous studies have reported the association of vascular endothelial dysfunction with the development of chronic diseases (e.g., hypertension, diabetes, heart failure, coronary artery disease, chronic renal failure, peripheral artery disease)9,10, indicating the significance of studying endothelial function in physiological as well as pathological conditions.
Vascular endothelium is integral to the production of hyperpolarization, vasodilation, and tissue perfusion and thus, examination of its native cellular properties is crucial. As a general study model, preparation of the mouse arterial endothelial tube model has been published before for skeletal muscle11,12, gut13, lung14, and recently for the brain6. Studies of simultaneous [Ca2+]i and Vm measurements in particular have been published for skeletal muscle arterial endothelium15,16 as well as lymphatic vessel endothelium17. In addition to primary studies utilizing the endothelial tube approach, a comprehensive review of its advantages and disadvantages8 can be consulted to determine if this experimental tool is appropriate for a specific study. In brief, an advantage is that the key physiological components of endothelial cell function are retained (e.g., Ca2+ influx and intracellular release, hyperpolarization of Vm up to the Nernst potential for K+&#160;via SKCa/IKCa activation, and endothelial intercellular coupling via gap junctions) without confounding factors such as perivascular nerve input, smooth muscle voltage-gated channel function and contractility, circulation of blood, and hormonal influences8. In contrast, commonly used cell culture approaches introduce significant alterations in morphology18 and ion channel expression19 in a manner that can greatly obfuscate comparisons to physiological observations determined ex vivo or in vivo. Limitations include a lack of integration with other essential components for regulating blood flow, such as smooth muscle and restricted flexibility in an experimental schedule, as this model is optimally tested within 4 h of intact vascular segment isolation from the animal.
Building from a previous video protocol authored by Socha and Segal12 and recent experimental developments in the interim6,15,16, we hereby demonstrate the isolation of fresh endothelium from posterior cerebral arteries and simultaneous measurements of endothelial [Ca2+]i and Vm using Fura-2 photometry and intracellular sharp electrodes, respectively. Additionally, this experiment entails continuous superfusion of salt solutions and pharmacological agents during physiological conditions (pH 7.4, 37 &#176;C). We chose the posterior cerebral artery, as it yields isolated endothelium with the structural integrity (cells coupled through gap junctions) and sufficient dimensions (width &#8805;50 &#181;m, length &#8805;300 &#181;m) amenable for intra- and intercellular signaling along and among endothelial cells. In addition, studies of the rodent posterior cerebral artery are substantially represented in the literature and encompass examination of fundamental endothelial signaling mechanisms, vascular development/aging, and pathology20,21,22. This experimental application is expected to yield a high-throughput analysis of cerebral endothelial function (and dysfunction) and will thereby allow for significant advancements in the understanding of blood flow regulation throughout aging and the development of neurodegenerative disease.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich (St. Louis, MO, USA)</td>
			<td>G7021</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M2670</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>223506</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma</td>
			<td>S8045</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Sigma</td>
			<td>A2383</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>ThermoFisher Scientific (Pittsburgh, PA, USA)</td>
			<td>A466250</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase (Type H Blend)</td>
			<td>Sigma</td>
			<td>C8051</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithioerythritol</td>
			<td>Sigma</td>
			<td>D8255</td>
			<td></td>
		</tr>
		<tr>
			<td>Papain</td>
			<td>Sigma</td>
			<td>P4762</td>
			<td></td>
		</tr>
		<tr>
			<td>Elastase</td>
			<td>Sigma</td>
			<td>E7885</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium iodide</td>
			<td>Sigma</td>
			<td>P4170</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr>
			<td>Fura-2 AM dye</td>
			<td>Invitrogen, Carlsbad, CA, USA</td>
			<td>F14185</td>
			<td></td>
		</tr>
		<tr>
			<td>Recirculating chiller (Isotemp 500LCU)</td>
			<td>ThermoFisher Scientific</td>
			<td>13874647</td>
			<td></td>
		</tr>
		<tr>
			<td>Plexiglas superfusion chamber&#160;</td>
			<td>Warner Instruments, Camden, CT, USA</td>
			<td>RC-27</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslip bottom (2.4&#160;&#215;&#160;5.0&#160;cm)</td>
			<td>ThermoFisher Scientific</td>
			<td>12-548-5M</td>
			<td></td>
		</tr>
		<tr>
			<td>Anodized aluminum platform (diameter: 7.8&#160;cm)&#160;</td>
			<td>Warner Instruments</td>
			<td>PM6 or PH6</td>
			<td></td>
		</tr>
		<tr>
			<td>Compact aluminum stage&#160;</td>
			<td>Siskiyou, Grants Pass, OR, USA</td>
			<td>8090P</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Siskiyou</td>
			<td>&#160;MX10</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscopes&#160;</td>
			<td>Zeiss, NY, USA</td>
			<td>Stemi 2000 &#38; 2000-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiber optic light sources</td>
			<td>&#160;Schott, Mainz, Germany &#38; KL200, Zeiss</td>
			<td>Fostec 8375</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon inverted microscope</td>
			<td>Nikon Instruments Inc, Melville, NY, USA</td>
			<td>Ts2</td>
			<td></td>
		</tr>
		<tr>
			<td>Phase contrast objectives&#160;</td>
			<td>Nikon Instruments Inc</td>
			<td>&#160;(Ph1 DL; 10X &#38; 20X)</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent objectives&#160;</td>
			<td>Nikon Instruments Inc</td>
			<td>20X (S-Fluor), and 40X (Plan Fluor)</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon inverted microscope</td>
			<td>Nikon Instruments Inc</td>
			<td>Eclipse TS100</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsyringe pump controller (Micro4 )&#160;</td>
			<td>World Precision Instruments (WPI), Sarasota, FL, USA</td>
			<td>SYS-MICRO4</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibration isolation table</td>
			<td>Technical Manufacturing, Peabody, MA, USA</td>
			<td>&#160;Micro-g</td>
			<td></td>
		</tr>
		<tr>
			<td>Amplifiers</td>
			<td>Molecular Devices, Sunnyvale, CA, USA</td>
			<td>Axoclamp 2B &#38; Axoclamp 900A</td>
			<td></td>
		</tr>
		<tr>
			<td>Headstages&#160;</td>
			<td>Molecular Devices</td>
			<td>HS-2A &#38; HS-9A</td>
			<td></td>
		</tr>
		<tr>
			<td>Function generator&#160;</td>
			<td>EZ Digital, Seoul, South Korea</td>
			<td>FG-8002</td>
			<td></td>
		</tr>
		<tr>
			<td>Data Acquision System</td>
			<td>Molecular Devices, Sunnyvale, CA, USA</td>
			<td>Digidata 1550A</td>
			<td></td>
		</tr>
		<tr>
			<td>Audible Baseline Monitors</td>
			<td>Ampol US LLC, Sarasota, FL, USA</td>
			<td>&#160;BM-A-TM</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital Storage Oscilloscope</td>
			<td>Tektronix, Beaverton, Oregon, USA</td>
			<td>&#160;TDS 2024B</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence System Interface, ARC Lamp + Power Supply, Hyperswitch, PMT</td>
			<td>Molecular Devices, Sunnyvale, CA, USA</td>
			<td>IonOptix Systems</td>
			<td></td>
		</tr>
		<tr>
			<td>Temperature Controller&#160;&#160;</td>
			<td>Warner Instruments</td>
			<td>TC-344B or C</td>
			<td></td>
		</tr>
		<tr>
			<td>Inline Heater&#160;</td>
			<td>Warner Instruments</td>
			<td>SH- 27B</td>
			<td></td>
		</tr>
		<tr>
			<td>Valve Controller&#160;</td>
			<td>Warner Instruments</td>
			<td>VC-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Inline Flow Control Valve</td>
			<td>Warner Instruments</td>
			<td>&#160;FR-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Electronic Puller&#160;</td>
			<td>Sutter Instruments, Novato, CA, USA</td>
			<td>P-97 or P-1000&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Microforge</td>
			<td>Narishige, East Meadow, NY, USA</td>
			<td>&#160;MF-900</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate Glass Tubes (Trituration)</td>
			<td>World Precision Instruments (WPI), Sarasota, FL, USA</td>
			<td>1B100-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate Glass Tubes (Pinning)</td>
			<td>Warner Instruments</td>
			<td>G150T-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate Glass Tubes (Sharp Electrodes)</td>
			<td>Warner Instruments</td>
			<td>GC100F-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe Filter (0.22 &#181;m)&#160;&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>722-2520</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Petri Dish + Charcoal Sylgard</td>
			<td>Living Systems Instrumentation, St. Albans City, VT, USA</td>
			<td>DD-90-S-BLK</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas Style Scissors (3 mm &#38; 9.5&#160;mm)</td>
			<td>World Precision Instruments</td>
			<td>555640S, 14364</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors 3 &#38; 7 mm blades</td>
			<td>Fine Science Tools (or FST), Foster City, CA, USA</td>
			<td>Moria MC52 &#38; 15000-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Sharpened fine-tipped forceps&#160;</td>
			<td>FST</td>
			<td>Dumont #5 &#38; Dumont #55</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The schematic demonstration of the protocol described above is shown in the attached figures. A brain isolated from a young adult male C57BL/6N mouse (5 months) is shown in Figure 1A. Posterior cerebral arteries are carefully isolated from the Circle of Willis, removed without connective tissue, and cut into segments (Figure 1B-D). From partially digested arterial segments, the intact endothelial tube is produced...

Watch this Scientific Journal Video about Simultaneous Measurements of Intracellular Calcium and Membrane Potential in Freshly Isolated and Intact Mouse Cerebral Endothelium at JoVE.com

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

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 ...

This method can help answer key questions in the field of cerebral vascular physiology as it relates to cerebral blood flow regulation during aging and development of chronic diseases. The primary advantage of this technique is that it allows simultaneous measurement of intracellular calcium and membrane potential of cerebral arterial endothelium in it's native form under physiological conditions. To isolate the mouse brain, under the microscope remove the skin and hair over the skull.And remove excessive blood with cold calcium-free PSS. Next, make an incision using only the tips of standard dissection scissors. Starting from the occipital bone and extending up through the nasal bone of the skull.Then open she skull carefully along the incision, using coarse-tipped forceps and separate the connective tissue to expose the brain. Gently wash the isolated brain with cold calcium-free PSS in a beaker to remove the blood. Place the brain ventral side up in a chamber containing cold dissection solution for the isolation of cerebral arteries.To isolate the cerebral arteries, secure the isolated brain in cold dissection solution by inserting two stainless steel pins through it, into a charcoal-infused silicon polymer coating at the bottom of a glass Petri dish. Subsequently, surgically isolate the posterior cerebral arteries about 0.3 to 0.5 centimeter segments from the posterior communicating and basilar arteries. Use stainless steel pins to secure both isolated posterior cerebral arteries in the dissection solution in the Petri dish.Then clean the isolated posterior cerebral arteries carefully by removing connective tissue using sharpened fine-tipped forceps. Cut the intact arteries into one to two millimeter segments for enzymatic digestion. In this procedure, prepare the trituration apparatus using a microscope, a camera, and an aluminum stage holding a chamber and micromanipulators.Secure a microsyringe with a pump controller adjacent to the stage and the specimen. Next completely backfill a titration pipette with mineral oil and secure it over the microsyringe piston. Then using the microsyringe with the pump controller withdraw about 130 nanoliters of dissociation solution into the pipette while ensuring the absence of air bubbles.Subsequently, place intact arterial segments into one milliliter of dissociation solution with required concentrations of enzymes in a 10 milliliter glass tube. Incubate at 34 degrees Celsius for 10 to 12 minutes for partial digestion. Following digestion, replace the enzyme solution with five milliliters of fresh dissociation solution.Using a one milliliter pipette, transfer one segment into the chamber containing dissociation solution at room temperature. Next place the pipette into the dissociation solution in the chamber and position it close to one end of the digested vessel. Set a rate within the range of two to five nanoliters per second on the pump controller for gentle trituration.While viewing through 100 times to 200 times magnification withdraw and eject the arterial segment to dissociate smooth muscle cells while producing an endothelial tube. If necessary, carefully use fine-tipped forceps to separate dissociated adventitia and internal elastic lamina from the endothelial tube. Confirm that all smooth muscle cells are dissociated and that only endothelial cells remain as intact tube.Using micromanipulators, secure each end of the endothelial tube on the glass cover slip of the superfusion chamber using borosilicate glass pinning pipettes. Wash out the dissociated adventitia and smooth muscle cells from the chamber. And replace the dissociation solution with two molar more calcium chloride PSS.Transfer the mobile platform with secured endothelial tube onto the microscope of superfusion and experimental rig. Then use six clean 50 milliliter reservoirs for continuous delivery of PSS and respective drug solutions during the experiment. Use the in-line flow control valve to manually set the flow rate as consistent as laminer flow while matching flow feed to vacuum suction.Deliver PSS to the chamber for the superfusion of the endothelial tube for at least five minutes before recording the background data and dye loading. To measure the membrane potential simultaneously with intracellular calcium concentration, pull a sharp electrode and backfill it with two molar potassium chloride to record from cells loaded with fura-2 dye. To study the intracellular coupling via dye transfer, backfill the microelectrode with 0.1%propidium iodide dissolved in two molar potassium chloride.Next place the electrode over a silver wire coated with chloride in the pipette holder attached to an electrometer head stage secured with a micromanipulator. Use the micromanipulator to briefly position the tip of the electrode into the flowing PSS in the chamber, while viewing through the four times objective. Subsequently, set the resting membrane potential to zero as consistent with grounded bath potential.If desired, use audible baseline monitors linked to electrometers to associate sound pitch with potential recordings. Afterward, increase magnification to 400 times using a 40 times objective and position the tip of electrode just over the cell of the endothelial tube. Adjust the photometric window using the photometry software to focus on about 50 to 80 endothelial cells.Then gently place the electrode into one of the cells of the endothelial tube using the micromanipulator and wait at least two minutes for the resting membrane potential to stabilize. Once resting membrane potential is stable at its expected value, turn on the photomultiplier tube on the fluorescence interface in the absence of light and begin acquisition of the intracellular calcium concentration by exciting Fura-2 alternately at 340 and 380 nanometers while collecting florescence emission at 510 nanometers. Once simultaneous measurements of membrane potential and intracellular calcium concentration are established, allow about five minutes for the superfusion of endothelial tube with PSS at a constant laminar flow rate before the application of drugs.Apply the drug prepared in PSS to the superfusion chamber at a constant flow rate. During drug treatment measure the membrane potential in the F340/F380 ratios simultaneously. Once the experiment is done, withdraw the electrode from the cell.Then stop respective recordings of membrane potential and intracellular calcium concentration and save the files for data analysis. This is a differential interference contrast image of a mouse cerebral arterial endothelial tube, adjusted for experiment in photometric window using a 40 times objective. A sharp electrode is placed into a cell, as shown at the top of the image.Here are the examples of raw traces for simultaneous measurement of F340/F380 ratio and membrane potential in response to 100 micromolar ATP. Following this procedure, other methods like confocal fluorescent microscopy, can be performed in order to answer additional questions regarding microdomain signaling of endothelial calcium, and reactive oxygen species, as examples. In general this technique will continue to pave the way for researches in cellular physiology to explore vascular function and aging in the blood and lymphatic circulation.

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Research

JoVE Journal

Biology

8.0K visualizações.  Loma Linda University. Este método pode ajudar a responder a perguntas-chave no campo da fisiologia vascular cerebral no que se refere à regulação do fluxo sanguíneo cerebral durante o envelhecimento e desenvolvimento de doenças crônicas. A principal vantagem desta técnica é que permite a medição simultânea do cálcio intracelular e potencial de membrana do endotélio arterial cerebral em sua forma nativa sob condições fisiológicas. Para isolar o cérebro do rato, sob o microscópio remova a pele e o...