The authors would like to thank Jules Morin for insightful comments on the manuscript. This research was funded by awards from the CADASIL Together We Have Hope non-profit organization, the Center for Women&#39;s Health and Research, and the NHLBI R01HL136636 (FD).

All experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Colorado, Anschutz Medical Campus and were performed according to the guidelines from the National Institutes of Health.
1. Solutions
<ol>
	<li>Use MOPS-buffered saline for the dissection and to keep samples at 4 &#176;C before their utilization. Do not gas the solution. Prepare MOPS buffered saline with following composition: 135 mM NaCl, 5 mM KCl, 1 mM KH2PO4, 1 mM MgSO4, 2.5 mM CaCl2, 5 mM glucose, 3 mM MOPS, 0.02 mM EDTA, 2 mM pyruvate, 10 mg/mL bovine serum albumin, pH 7.3 at 4 &#176;C.</li>
	<li>Use artificial cerebrospinal fluid (aCSF) as the bath solution and pipette solution. Gas both aCSF and Ca2+-free aCSF with 5% CO2, 20% O2, and N2 balance. Prepare the solution with the following composition: 125 mM NaCl, 3 mM KCl, 26 mM NaHCO3, 1.25 mM NaH2PO4, 1 mM MgCl2, 4 mM glucose, 2 mM CaCl2, pH 7.3 (with aeration with 5% CO2, 20% O2, and N2 balance).</li>
	<li>Obtain the maximal dilation in nominally Ca2+-free aCSF (0 mM [Ca2+]o, 5 mM EGTA).</li>
</ol>
2. Organ chamber preparation
<ol>
	<li>Insert borosilicate glass capillaries (outside diameter = 1.2 mm; inside diameter = 0.69 mm; length = 10 cm) into a glass puller. Pull the capillary to make a long, thin tip at one end.</li>
	<li>To one side of the chamber, add a cannula that can connect to a miniature peristaltic pump to luminally pressurize the vessel. Under a dissection microscope, break the tip of the cannula so that it is small enough to fit the vessel of interest, but large enough to allow solutions to flow through the tip. Ensure that the tip is roughly 10-15 &#181;m in diameter.</li>
	<li>Fill the cannula using a syringe with an attached 0.22 &#181;m filter with oxygenated aCSF. Make sure there are no air bubbles or debris in the cannula.</li>
	<li>Add two more cannulas to the opposite side of the chamber. Do not break their tips.</li>
</ol>
3. Hippocampus dissection and isolation
<ol>
	<li>Euthanize and decapitate a mouse. For this experiment, use an 8-week-old C57BL6/J mouse to compare differences between males and females. Inject the mouse with pentobarbital and decapitate with surgical scissors.</li>
	<li>Using small dissection scissors, cut the skin along the midline at the top of its head. Move the skin off to the sides.</li>
	<li>Starting at the caudal side of the skull, cut the skull along the midline until the olfactory bulbs are reached. Remove portions of the skull until the cerebrum is exposed.</li>
	<li>Slowly remove the brain, starting near the nose of the mouse. Separate the brain from the olfactory bulbs, cranial nerves, and spinal cord by cutting through the structures with the small dissection scissors.</li>
	<li>Place the brain in a dissection plate with enough MOPS solution to completely submerge it. Using a dissection microscope, place the brain in the center of the dissection plate with the ventral side facing down.</li>
	<li>Using a razor blade, cut the brain in half along the longitudinal fissure. Hold the blade so that the sharp edge is parallel to the bottom of the dissection plate. Press the blade through the brain in one stroke. Move one hemisphere to the side of the plate.</li>
	<li>Perform the following steps for each hemisphere separately or in parallel.
	<ol>
		<li>Place one hemisphere in the center of the plate so the midline is facing down. Then use the razor blade to cut along the transverse fissure to remove the cerebellum and brain stem. Push the blade straight through the tissue.</li>
		<li>Rotate the hemisphere so that the medial side is facing up (Figure 1A). Use one spatula to hold the brain in place. Using a second spatula, insert the tip below the corpus callosum and scoop underneath to remove the thalamus, septum, and hypothalamus, covering the hippocampus (Figure 1B).</li>
		<li>Ensure that the hippocampus is now visible as a curved structure near the posterior side of the cerebrum. Using one spatula to hold the cerebrum in place, use the second spatula to scoop the hippocampus out of the cerebrum (Figure 1C).</li>
	</ol>
	</li>
	<li>Transfer the hippocampi to a new dissection plate filled about halfway with MOPS solution. Discard the rest of the brain.</li>
</ol>
4. Hippocampal arteriole isolation
<ol>
	<li>Complete the following steps for each hippocampus.
	<ol>
		<li>Pin down one of the hippocampi using small pins at each end of the section. The hippocampal artery is facing up.</li>
		<li>Using very sharp forceps, gently stretch small sections of the hippocampus. This will loosen the tissue surrounding the arterioles, making it easier to dissect them.</li>
		<li>Search through the dorsal hippocampal tissue to identify the external transverse artery (Figure 1C)16,27.</li>
		<li>Gently grab the external transverse artery and slowly pull it away from the tissue to collect the arterioles and capillaries perfusing the CA3 region of the hippocampus.</li>
		<li>Once there are no more vessels to be removed from the tissue, discard the hippocampi. Keep the vessels on ice in plates while not in use.</li>
	</ol>
	</li>
</ol>
5. Hippocampal capillary-parenchymal arteriole cannulation
<ol>
	<li>Find an arteriole with a branch that ends with capillaries. Transfer it to the organ chamber. Ensure that the arteriole is about 15-30 &#181;m when fully dilated (Figure 1D).</li>
	<li>Carefully mount the blood vessel by pushing the cannula tip through the arteriole wall below the target area. Carefully slide the vessel onto the cannula until there is enough tissue to place the tie on.</li>
	<li>Make a loose knot with 12-0 nylon sutures so that it fits over the blood vessel and cannula. Use a half-hitch knot to secure ties. Then pull the ends to tighten the knot and secure the arteriole to the cannula. Remove any extraneous vessel branches below the tie by gently pulling them with forceps.</li>
	<li>Make another tie and secure it on the other end of the arteriole to seal it.</li>
	<li>Lower the cannula with the attached vessel until it is flat against the coverslip on the bottom of the chamber. Be careful not to lower the cannula too much or it will break.</li>
	<li>Using one cannula on the opposite side of the chamber, lower it so that the point of it pins down the tie on the end of the arteriole.</li>
	<li>Use the third cannula to pin down the capillary branch to the coverslip. Place the tip close to the end of the branch while leaving the ends of the capillaries exposed (not underneath the cannula).</li>
</ol>
6. Pressure myography
<ol>
	<li>Move the chamber from the dissection scope to the microscope with the recording software.</li>
	<li>Connect the inflow and outflow tubing to the chamber for perfusion. Start the perfusion with heated aCSF (37 &#176;C) at a flow rate of 4 mL/min.</li>
	<li>Attach the pressurizing cannula to a peristaltic pump paired with a pressure transducer and bring the internal pressure to 20 mm Hg.</li>
	<li>Start the recording software. Adjust the microscope and imaging settings to achieve the clearest image possible. Begin the recording once the settings are optimized for the detection software.</li>
	<li>Increase the pressure of the vessel up to 40 mm Hg while recording the arterial diameter with an edge detection software.</li>
	<li>Allow ~15-20 min to wash MOPS solution out of the chamber with aCSF, and to let the HiCaPA preparation to equilibrate and develop myogenic tone.</li>
	<li>To test the viability of a vessel, apply 1 &#181;M NS309 solution to the bath perfusion (see Figure 2A,B and the Representative Results section). The arteriolar segment must dilate, demonstrating about 30-40% myogenic tone as previously described3,14,20.</li>
</ol>
7. Focal stimulation of capillary ends
<ol>
	<li>Once the baseline tone for the arteriole has been established and the endothelial function assessed, test the response of capillary stimulation.</li>
	<li>Using a glass puller, make cannulas so that there is a fine point at one end. Break the tip off a cannula so that the drug tested can flow through the tip smoothly at 5 psi.</li>
	<li>Fill the cannula with the drug solution of interest and add it to a 3-axis micromanipulator attached to the microscope. Connect the tubing from the pressure ejection system to the cannula.</li>
	<li>Slowly lower the cannula into the bath near the capillaries, being careful not to hit any part of the vessel or hardware in the chamber. Maneuver the tip of the cannula next to the ends of the capillaries without touching them. Keep the tip of the cannula just off the coverslip to prevent the vessel from being stimulated if the cannula leaks.</li>
	<li>When ready to stimulate the capillaries, lower the cannula to the coverslip and just next to the capillaries. Activate the pressure ejection system with the desired ejection time (here 20 s). Once the stimulation is finished, raise the cannula slightly to avoid further stimulation.</li>
	<li>Repeat stimulation as necessary. Change the pressure ejection system cannula to test different drug compounds.</li>
	<li>To confirm that only the capillaries are being stimulated, fill the cannula with 1 &#181;M NS309 solution and repeat the above steps. 
	NOTE: Capillary endothelial cells do not express K+ channels activated by NS309, so the arteriole must not respond to the stimulation. If the arteriole dilates, then the cannula will need to be repositioned or the diameter of the hole will need to be smaller (see Figure 2A,B and the Representative Results section).</li>
</ol>

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The authors have nothing to disclose.

The pressurized HiCaPA (hippocampal capillary-parenchymal arteriole) preparation described in the present manuscript is an extension of our well-established procedure to isolate, pressurize, and study parenchymal arterioles29. We recently reported that Kir2.1 channels in brain capillary endothelial cells sense increases in [K+]o associated with neural activation, and generate an ascending hyperpolarizing signal that dilates upstream arterioles20. Revealing this previously unanticipated role for the capillaries has been possible in part by developing the CaPA preparation from cortical microcirculation20,21. This manuscript presents a similar experimental approach but from a deeper and more restricted structure of the mouse brain to describe a simple and reproducible approach to investigate capillary-to-arteriole signaling during neurovascular coupling.
The brain microcirculation is exquisitely fragile and certain practices, especially minimizing stretching and handling of the vessels, must be used to ensure the survival of the arterioles and capillaries. The spontaneous development of the myogenic tone is the first indicator of a preparation&#39;s viability30. The endothelial function can then be assessed by adding the SK and IK channels&#39; agonist NS309 to the bath solution, which should cause near maximum dilation. In case of a failure to develop tone or response to the bath application of NS309, the preparation should be replaced with another one. NS309 is also used to test the spread of the focal capillary stimulation. Because capillary endothelial cells lack SK and IK channels20, local delivery of NS309 onto capillaries by pressure ejection should have no effect on upstream arteriolar diameter as displayed in Figure 2, illustrating that compounds do not accidentally stimulate the arteriole. Once these steps are validated, capillary-to-arteriole signaling can be tested.
Here we examined electrical signaling by stimulating capillaries with aCSF containing 10 mM K+. However, different signaling modalities can be explored using the present approach by stimulating capillaries with different known vasoactive agents or neurotransmitters. Another benefit of this preparation is the possibility to investigate and eventually compare NVC between different animals and between different brain regions. This is particularly interesting because the brain is not uniformly targeted by cerebrovascular pathologies31,32. A general limitation of the approach presented here is that by isolating the microcirculation, crucial components of the neurovascular unit, such as neurons and astrocytes, are lost. Other preparations, such as the cranial window for in vivo CBF imaging, maintain the structure of the intact neurovascular unit and are more appropriate to study NVC in an intact system. However, in the cranial window preparation, parenchymal arterioles are difficult to image without specific equipment, like a multiphoton microscope, and deeper regions, such as the hippocampus, remain difficult to image. In this regard, the approach developed in the Filosa laboratory using luminal flow to induce myogenic tone in brain slices represents an elegant link between brain slice and in vivo approaches33. However, the surrounding nervous tissue can limit the penetration of a drug applied topically, increasing its off-target potential and making interpretations difficult, because several cell types are exposed to the drugs. We primarily developed our ex vivo approach to address these potential issues. In conclusion, multiple approaches should be used in conjunction to fully study NVC.
In summary, the present report describes an ex vivo intact preparation of pressurized hippocampal arterioles and capillaries that allows the effects of pharmacological and biological agents to be tested on functional parameters at discrete positions along the capillary-arteriole continuum.

The pial circulation on the surface of the brain has been the object of much study, partly because of its experimental accessibility. However, the topology of the cerebral vasculature creates distinct regions. In contrast to the robust pial network rich in anastomoses with substantial capacity for redirecting the blood flow, the intracerebral parenchymal arterioles (PAs) present limited collateral supply, each of them perfusing a discrete volume of nervous tissue1,2. This creates a bottleneck effect on the blood flow which, combined with unique physiological features3,4,5,6,7,8, makes intracerebral arterioles a crucial site for cerebral blood flow (CBF) regulation9,10. Despite the technical challenges inherent to the isolation and cannulation of PAs, the last decade has seen an increased interest in ex vivo functional studies using pressurized vessels11,12,13,14,15,16,17. One of the reasons for this increased interest is the considerable research effort conducted on neurovascular coupling (NVC), the mechanism sustaining the brain functional hyperemia18.
Regionally, CBF can rapidly increase following local neural activation19. The cellular mechanisms and signaling properties controlling NVC are incompletely understood. However, we identified a previously unanticipated role for the brain capillaries during NVC in sensing neural activity and translating it into a hyperpolarizing electrical signal to dilate upstream arterioles20,21,22. Action potentials23,24 and opening of large-conductance Ca2+-activated K+ (BK) channels on the astrocytic endfeet25,26 increase the interstitial potassium ion concentration [K+]o, which results in activation of strong inward rectifier K+ (Kir) channels in the vascular endothelium of capillaries. This channel is activated by external K+ but also by hyperpolarization itself. Spreading through gap junctions, the hyperpolarizing current then regenerates in adjacent capillary endothelial cells up to the arteriole, where it causes myocyte relaxation and CBF increase20,21. The study of this mechanism led us to develop a pressurized capillary-parenchymal arteriole (CaPA) preparation to measure the arteriolar diameter during capillary stimulation with vasoactive agents. The CaPA preparation is composed of a cannulated intracerebral arteriole segment with an intact, downstream capillary ramification. The capillary ends are compressed against the chamber glass bottom by a micropipette, which occludes and stabilizes the entire vascular formation20,21.
We previously made instrumental innovations by imaging CaPA preparations from the mouse cortex20,21 and arterioles from the rat amygdala13 and hippocampus16,17. As the hippocampal vasculature receives more attention due to its susceptibility to pathological conditions, here we provide a step-by-step method for CaPA preparation from the mouse hippocampus (HiCaPA) that can not only be used in functional NVC studies but also in molecular biology, immunochemistry, and electrophysiology.

<table><tbody><tr><td>0.22&amp;micro;m Syringe Filters</td><td>CELLTREAT Scientific Products</td><td>229751</td><td></td></tr><tr><td>12-0 Nylon (12cm) Black</td><td>Microsurgery Instruments, Inc</td><td>S12-0 NYLON</td><td></td></tr><tr><td>Automatic Temperature Controller</td><td>Warner Instruments</td><td>TC-324B</td><td></td></tr><tr><td>Borosilicate Glass O.D.: 1.2 mm, I.D.: 0.68 mm</td><td>Sutter Instruments</td><td>B120-69-10</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma-Aldrich</td><td>A7030</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt; dihydrate</td><td>Sigma-Aldrich</td><td>C3881</td><td></td></tr><tr><td>D-(+)-Glucose</td><td>Sigma-Aldrich</td><td>G5767</td><td></td></tr><tr><td>Dissection Scope</td><td>Olympus</td><td>SZ11</td><td></td></tr><tr><td>ECOLINE VC-MS/CA 4-12 &amp;mdash; complete Pump with Drive and MS/CA 4-12 pump-head</td><td>Ismatec</td><td>ISM 1090</td><td></td></tr><tr><td>EGTA</td><td>Sigma-Aldrich</td><td>E4378</td><td></td></tr><tr><td>Fine Scissors - Sharp</td><td>Fine Science Tools</td><td>14063-09</td><td></td></tr><tr><td>Inline Water Heater</td><td>Warner Instruments</td><td>SH-27B</td><td></td></tr><tr><td>Integra&amp;trade; Miltex&amp;trade;Tissue Forceps</td><td>Fisher Scientific</td><td>12-460-117</td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P9333</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>P5379</td><td></td></tr><tr><td>Magnesium sulfate heptahydrate</td><td>Sigma-Aldrich</td><td>M1880</td><td></td></tr><tr><td>MgCl Anhydrous</td><td>Sigma-Aldrich</td><td>M8266</td><td></td></tr><tr><td>Micromanipulator</td><td>Narishige</td><td>MN-153</td><td></td></tr><tr><td>ML 133 hydrochloride</td><td>Tocris</td><td>4549</td><td></td></tr><tr><td>MOPS</td><td>Sigma-Aldrich</td><td>M1254</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S9625</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S9638</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S8875</td><td></td></tr><tr><td>NS309</td><td>Tocris</td><td>3895</td><td></td></tr><tr><td>Picospritzer III - Intracellular Microinjection Dispense Systems, 2-channel</td><td>Parker Hannifin</td><td>052-0500-900</td><td></td></tr><tr><td>Pressure Servo Controller with Peristaltic Pump</td><td>Living Systems Instrumentation</td><td>PS-200</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Sigma-Aldrich</td><td>P3662</td><td></td></tr><tr><td>Super Fine Forceps</td><td>Fine Science Tools</td><td>11252-20</td><td></td></tr><tr><td>Surgical Scissors - Sharp-Blunt</td><td>Fine Science Tools</td><td>14001-13</td><td></td></tr><tr><td>Vertical Micropipette Puller</td><td>Narishige</td><td>PP-83</td><td></td></tr></tbody></table>

ex vivo pressurized hippocampal capillary-parenchymal arteriole preparation for functional study

From subtle behavioral alterations to late-stage dementia, vascular cognitive impairment typically develops following cerebral ischemia. Stroke and cardiac arrest are remarkably sexually dimorphic diseases, and both induce cerebral ischemia. However, progress in understanding the vascular cognitive impairment, and then developing sex-specific treatments, has been partly limited by challenges in investigating the brain microcirculation from mouse models in functional studies. Here, we present an approach to examine the capillary-to-arteriole signaling in an ex vivo hippocampal capillary-parenchymal arteriole (HiCaPA) preparation from mouse brain. We describe how to isolate, cannulate, and pressurize the microcirculation to measure arteriolar diameter in response to capillary stimulation. We show which appropriate functional controls can be used to validate the HiCaPA preparation integrity and display typical results, including testing potassium as a neurovascular coupling agent and the effect of the recently characterized inhibitor of the Kir2 inward rectifying potassium channel family, ML133. Further, we compare the responses in preparations obtained from male and female mice. While these data reflect functional investigations, our approach can also be used in molecular biology, immunochemistry, and electrophysiology studies.

Endothelial small-conductance (SK) and intermediate-conductance (IK) Ca2+-sensitive K+ channels exert a dilatory influence on the diameter of PAs. Bath application of 1 &#181;M NS309, a synthetic IK and SK channel agonist, caused near maximal dilation (Figure 2A,B). However, capillary endothelial cells lack IK and SK channels and did not hyperpolarize in response to NS30920. As a result, stimulating capillary ends with 1 &#181;M NS309 by focal pressure ejection (20 s, 5 psi) did not cause upstream arteriolar dilation (Figure 2A,B). This result indicates that NS309 did not reach the arteriole in the HiCaPA preparations and could be used as a control to assess the spatial restriction of the compound applied onto capillaries by pressure ejection.
This preparation was fundamentally designed for the measurement of inside-out electrical signaling from capillaries to PAs. Using the HiCaPA preparation, we applied aCSF containing 10 mM K+ onto the capillary ends and measured an upstream arteriolar dilation (Figure 2A,C) as we previously did in CaPA preparations from the cortical vasculature20. We then investigated, for the first time to our knowledge, capillary-to-arteriole electrical signaling in female mice using HiCaPA preparations. Arteriolar dilation evoked by capillary stimulation with 10 mM K+ did not differ between preparations from male and female mice (Figure 2A,C).
Finally, another fundamental benefit of this approach is the possibility to apply pharmacological tools in the bath before capillary stimulation. Here we tested the effect of ML133, a recently developed Kir2 inhibitor28. Addition of 10 &#181;M ML133 to the bath perfusion virtually abolished capillary-induced arteriolar dilation in response to 10 mM K+ in HiCaPA preparations from both male and female mice (Figure 2A,C). This last result suggests that the Kir2.1 channel mediates electrical signaling in female cerebral vasculature as we previously described in the cortical microcirculation of the male brain.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60676/60676fig1.jpg" /> 
Figure 1: Methodology for isolation and pressurization of hippocampal capillary-parenchymal arterioles (HiCaPA) preparation from mouse. (A) Freshly isolated brain is cut in half in the sagittal plane following the interhemispheric fissure and placed with the medial side facing up. (B) The thalamus, septum, and hypothalamus are gently removed to reveal the hippocampus. (C) The hippocampus is carefully removed. (D) Arterioles with capillary trees are isolated from the hippocampus and one end of the arteriolar segment is cannulated with a micropipette connected to a pressurizing system, and the other end is occluded. Capillary ends are sealed and maintained against the coverslip with the tip of a glass pipette. Internal diameter is monitored with an edge detection system in one or several regions of the arteriole. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60676/60676fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60676/60676fig2.jpg" /> 
Figure 2: Focal stimulation of capillaries with 1 &#181;M NS309 has no effect on upstream arteriolar diameter, unlike stimulation with aCSF containing 10 mM K+. (A) Representative recording of the upstream arteriolar diameter showing the effect of bath application of 1 &#181;M NS309 followed by successive capillary ends stimulation (20 s, 5 psi) with 1 &#181;M NS309 and with aCSF containing 10 mM K+ in the absence or presence of the Kir2 channel inhibitor ML133. Application of 10 mM K+ onto capillaries produced a rapid upstream arteriolar dilation that was blocked by 10 &#181;M ML133. NS309 did not cause dilation. The absence of upstream arteriolar dilation in response to capillary stimulation with NS309 illustrates that pressure-ejected compounds do not reach the arteriole. (B) Summary data showing diameter changes induced by 1 &#181;M NS309 applied in the bath or on the capillary ends (n = 14; ****p &#60; 0.0001, paired t-test). (C) Summary data showing arteriolar diameter changes induced by 10 mM K+ applied directly onto the capillaries in HiCaPA preparations from male (n = 6) or female (n = 8) mice before and after 10 &#181;M ML133 was applied in the bath (***p &#60; 0.0005, n.s. = nonsignificant, unpaired t-test). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60676/60676fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Ex Vivo Pressurized Hippocampal Capillary-Parenchymal Arteriole Preparation for Functional Study at JoVE.com

Ex Vivo Pressurized Hippocampal Capillary-Parenchymal Arteriole Preparation for Functional Study

The present manuscript details how to isolate hippocampal arterioles and capillaries from the mouse brain and how to pressurize them for pressure myography, immunofluorescence, biochemistry, and molecular studies.

From subtle behavioral alterations to late-stage dementia, vascular cognitive impairment typically develops following cerebral ischemia. Stroke and ...

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6.8K Views.  University of Colorado Anschutz Medical Campus. The present manuscript details how to isolate hippocampal arterioles and capillaries from the mouse brain and how to pressurize them for pressure myography, immunofluorescence, biochemistry, and molecular studies.

Video: Ex Vivo Pressurized Hippocampal Capillary-Parenchymal Arteriole Preparation for Functional Study

The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional deficits in the affected patients. Despite intensive research efforts, there is still no effective treatment option available that reduces neuronal injury and protects neurons in the ischemic areas from delayed secondary death. Research in this area typically involves the use of elaborate and problematic animal models. Entorhino-hippocampal organotypic slice cultures challenged with oxygen and glucose deprivation (OGD) are established in&#160;vitro models which mimic cerebral ischemia. The novel aspect of this study is that changes of the brain blood vessels are studied in addition to neuronal changes and the reaction of both the neuronal compartment and the vascular compartment can be compared and correlated. The methods presented in this protocol substantially broaden the potential applications of the organotypic slice culture approach. The induction of OGD or hypoxia alone can be applied by rather simple means in organotypic slice cultures and leads to reliable and reproducible damage in the neural tissue. This is in stark contrast to the complicated and problematic animal experiments inducing stroke and ischemia in vivo. By broadening the analysis to include the study of the reaction of the vasculature could provide new ways on how to preserve and restore brain functions. The slice culture approach presented here might develop into an attractive and important tool for the study of ischemic brain injury and might be useful for testing potential therapeutic measures aimed at neuroprotection.

A protocol for entorhino-hippocampal organotypic slice cultures, which allows reproducing many aspects of ischemic brain injury, is presented. By studying changes of the neurovasculature in addition to changes in the neurons, this protocol is a versatile tool to study plastic changes in neural tissue after injury.

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional ...

Isolation and Cannulation of Cerebral Parenchymal Arterioles

Intracerebral parenchymal arterioles (PAs), which include parenchymal arterioles, penetrating arterioles and pre-capillary arterioles, are high resistance blood vessels branching out from pial arteries and arterioles and diving into the brain parenchyma. Individual PA perfuse a discrete cylindrical territory of the parenchyma and the neurons contained within. These arterioles are a central player in the regulation of cerebral blood flow both globally (cerebrovascular autoregulation) and locally (functional hyperemia). PAs are part of the neurovascular unit, a structure that matches regional blood flow to metabolic activity within the brain and also includes neurons, interneurons, and astrocytes. Perfusion through PAs is directly linked to the activity of neurons in that particular territory and increases in neuronal metabolism lead to an augmentation in local perfusion caused by dilation of the feed PA. Regulation of PAs differs from that of better-characterized pial arteries. Pressure-induced vasoconstriction is greater in PAs and vasodilatory mechanisms vary. In addition, PAs do not receive extrinsic innervation from perivascular nerves &#8212; innervation is intrinsic and indirect in nature through contact with astrocytic endfeet. Thus, data regarding contractile regulation accumulated by studies using pial arteries does not directly translate to understanding PA function. Further, it remains undetermined how pathological states, such as hypertension and diabetes, affect PA structure and reactivity. This knowledge gap is in part a consequence of the technical difficulties pertaining to PA isolation and cannulation. In this manuscript we present a protocol for isolation and cannulation of rodent PAs. Further, we show examples of experiments that can be performed with these arterioles, including agonist-induced constriction and myogenic reactivity. Although the focus of this manuscript is on PA cannulation and pressure myography, isolated PAs can also be used for biochemical, biophysical, molecular, and imaging studies.

This manuscript describes a simple and reproducible protocol for isolation of intracerebral arterioles (a group of blood vessels encompassing parenchymal arterioles, penetrating arterioles and pre-capillary arterioles) from mice, to be used in pressure myography, immunofluorescence, biochemistry, and molecular studies.

Intracerebral parenchymal arterioles (PAs), which include parenchymal arterioles, penetrating arterioles and pre-capillary arterioles, are high resistance ...

Tuning in the Hippocampal Theta Band In Vitro: Methodologies for Recording from the Isolated Rodent Septohippocampal Circuit

This protocol outlines the procedures for preparing and recording from the isolated whole hippocampus, of WT and transgenic mice, along with recent improvements in methodologies and applications for the study of theta oscillations. A simple characterization of the isolated hippocampal preparation is presented whereby the relationship between internal hippocampal theta oscillators is examined together with the activity of pyramidal cells, and GABAergic interneurons, of the cornu ammonis-1 (CA1) and subiculum (SUB) areas. Overall, we show that the isolated hippocampus is capable of generating intrinsic theta oscillations in vitro and that rhythmicity generated within the hippocampus can be precisely manipulated by optogenetic stimulation of parvalbumin-positive (PV) interneurons. The in vitro isolated hippocampal preparation offers a unique opportunity to use simultaneous field and intracellular patch-clamp recordings from visually-identified neurons to better understand the mechanisms underlying theta rhythm generation.

Tuning in the Hippocampal Theta Band In Vitro: Methodologies for Recording from the Isolated Rodent Septohippocampal Circuit

Here, we present a protocol for recording rhythmic neuronal network theta and gamma oscillations from an isolated whole hippocampal preparation. We describe the experimental steps from extraction of the hippocampus to details of field, unitary and whole-cell patch clamp recordings as well as optogenetic pacing of the theta rhythm.

This protocol outlines the procedures for preparing and recording from the isolated whole hippocampus, of WT and transgenic mice, along with recent ...

Isolation of Cerebral Capillaries from Fresh Human Brain Tissue

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic strategies that hold the promise to enhance brain drug delivery, improve brain protection, and treat brain disorders. However, studying the human blood-brain barrier function is challenging. Thus, there is a critical need for appropriate models. In this regard, brain capillaries isolated from human brain tissue represent a unique tool to study barrier function as close to the human in vivo situation as possible. Here, we describe an optimized protocol to isolate capillaries from human brain tissue at a high yield and with consistent quality and purity. Capillaries are isolated from fresh human brain tissue using mechanical homogenization, density-gradient centrifugation, and filtration. After the isolation, the human brain capillaries can be used for various applications including leakage assays, live cell imaging, and immune-based assays to study protein expression and function, enzyme activity, or intracellular signaling. Isolated human brain capillaries are a unique model to elucidate the regulation of the human blood-brain barrier function. This model can provide insights into central nervous system (CNS) pathogenesis, which will help the development of therapeutic strategies for treating CNS disorders.

Isolated brain capillaries from human brain tissue can be used as a preclinical model to study barrier function under physiological and pathophysiological conditions. Here, we present an optimized protocol to isolate brain capillaries from fresh human brain tissue.

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic ...

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, very few have been widely accepted. Here, we propose&#160;a new procedure&#160;adapted from the well-known ex vivo brain slice technique, which offers a closer in vivo-like scenario than in vitro preparations, for investigating the early events triggering cell degeneration, as observed in Alzheimer&#39;s disease (AD). This variation consists of simple and easily reproducible steps, which enable preservation of the anatomical cytoarchitecture of the selected brain region and its local functionality in a physiological milieu. Different anatomical areas can be obtained from the same brain, providing the opportunity to perform multiple experiments with the treatments in question in a site-, dose-, and time-dependent manner. Potential limitations&#160;which could affect the outcomes related to this methodology are related to the conservation of the tissue, i.e., the maintenance of its anatomical integrity during the slicing and incubation steps and the section thickness, which can influence the biochemical and immunohistochemical analysis. This approach can be employed for different purposes, such as exploring molecular mechanisms involved in physiological or pathological conditions, drug screening, or dose-response assays. Finally, this protocol could also reduce the number of animals employed in behavioral studies. The application reported here has been recently described and tested for the first time on ex vivo rat brain slices containing the basal forebrain (BF), which is one of the cerebral regions primarily affected in AD. Specifically, it has been demonstrated that the administration of a toxic peptide derived from the C-terminus of acetylcholinesterase (AChE) could prompt an AD-like profile, triggering, along the antero-posterior axis of the BF, a differential expression of proteins altered in AD, such as the alpha7 nicotinic receptor (&#945;7-nAChR), phosphorylated Tau (p-Tau), and amyloid beta (A&#946;).

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

We present a method which can&#160;provide further insights into the early events underlying neurodegeneration and based on the established ex-vivo brain technique, combining the advantages of in vivo and in vitro experiments. Moreover, it represents a unique opportunity for direct comparison of treated and untreated group in the same anatomical plane.

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...

Preparing Acute Brain Slices from the Dorsal Pole of the Hippocampus from Adult Rodents

Whole-cell patch-clamp recordings from acute rodent brain slices are a mainstay of modern neurophysiological research, allowing precise measurement of cellular and synaptic properties. Nevertheless, there is an ever increasing need to perform correlated analyses between different experimental modes in addition to slice electrophysiology, for example: immunohistochemistry, molecular biology, in vivo imaging or electrophysiological recording; to answer evermore complex questions of brain function. However, making meaningful conclusions from these various experimental approaches is not straightforward, as even within relatively well described brain structures, a high degree of sub-regional variation of cellular function exists. Nowhere is this better exemplified than in the CA1 of the hippocampus, which has well-defined dorso-ventral properties, based on cellular and molecular properties. Nevertheless, many published studies examine protein expression patterns or behaviorally correlated in vivo activity in the dorsal extent of the hippocampus; and explain findings mechanistically with cellular electrophysiology from the ventro-medial region. This is further confounded by the fact that many acute slice electrophysiological experiments are performed in juvenile animals, when other experimental modes are performed in more mature animals. To address these issues, this method incorporates transcardial perfusion of mature (&#62;60 day old rodents) with artificial cerebrospinal fluid followed by preparation of modified coronal slices including the septal pole of the dorsal hippocampus to record from CA1 pyramidal cells. This process leads to the generation of healthy acute slices of dorsal hippocampus allowing for slice-based cellular electrophysiological interrogation matched to other measures.

The purpose of this protocol is to describe a method to produce slices of the dorsal hippocampus for electrophysiological examination. This procedure employs perfusion with chilled ACSF prior to slice preparation with a near-coronal slicing angle which allows for preservation of healthy principal neurons.

Whole-cell patch-clamp recordings from acute rodent brain slices are a mainstay of modern neurophysiological research, allowing precise measurement of ...

In Vivo Fiber-Coupled Pre-Clinical Confocal Laser-scanning Endomicroscopy (pCLE) of Hippocampal Capillaries in Awake Mice

The goal of this protocol is to describe fiber-optic-bundle-coupled pre-clinical confocal laser-scanning endomicroscopy (pCLE) in its specific application to elucidate capillary blood flow effects during seizures, driven by mural cells. In vitro and in vivo cortical imaging have shown that capillary constrictions driven by pericytes can result from functional local neural activity, as well as from drug application, in healthy animals. Here, a protocol is presented on how to use pCLE to determine the role of microvascular dynamics in neural degeneration in epilepsy, at any tissue depth (specifically in the hippocampus). We describe a head restraint technique that has been adapted to record pCLE in awake animals, to address potential side-effects of anesthetics on neural activity. Using these methods, electrophysiological and imaging recordings can be conducted over several hours in deep neural structures of the brain.

In Vivo Fiber-Coupled Pre-Clinical Confocal Laser-scanning Endomicroscopy pCLE of Hippocampal Capillaries in Awake Mice

Whereas multiphoton imaging is only effective at limited depths from the tissue&#8217;s surface, it is possible to achieve 3 &#181;m resolution imaging at any depth via pCLE. Here, we present a protocol to conduct pCLE imaging to measure microvascular dynamics in the hippocampus of ictal and wild-type mice.

The goal of this protocol is to describe fiber-optic-bundle-coupled pre-clinical confocal laser-scanning endomicroscopy (pCLE) in its specific application ...

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

The retina is a highly metabolically active tissue that requires a substantial blood supply. The retinal circulation supports the inner retina, while the choroidal vessels supply the photoreceptors. Alterations in retinal perfusion contribute to numerous sight-threatening disorders, including diabetic retinopathy, glaucoma and retinal branch vein occlusions. Understanding the molecular mechanisms involved in the control of blood flow through the retina and how these are altered during ocular disease could lead to the identification of new targets for the treatment of these conditions. Retinal arterioles are the main resistance vessels of the retina, and consequently, play a key role in regulating retinal hemodynamics through changes in luminal diameter. In recent years, we have developed methods for isolating arterioles from the rat retina which are suitable for a wide range of applications including cell physiology studies. This preparation has already begun to yield new insights into how blood flow is controlled in the retina and has allowed us to identify some of the key changes that occur during ocular disease. In this article, we describe methods for the isolation of rat retinal arterioles and include protocols for their use in patch-clamp electrophysiology, calcium imaging and pressure myography studies. These vessels are also amenable for use in PCR-, western blotting- and immunohistochemistry-based studies.

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

This manuscript describes a straightforward protocol for the isolation of arterioles from the rat retina that can be used in electrophysiological, calcium imaging and pressure myography studies.

The retina is a highly metabolically active tissue that requires a substantial blood supply. The retinal circulation supports the inner retina, while the ...

Biology

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 ...

Preparing Cerebral Endothelial Tubes from Mouse Parenchymal Arterioles

Source: Hakim, M. A., et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/63463">Isolation and Functional Analysis of Arteriolar Endothelium of Mouse Brain Parenchyma.</a> J. Vis. Exp. (2022)
This video demonstrates a method for isolating the endothelial tubes from mouse cerebral parenchyma. Through a combination of enzymatic digestion and mechanical dissociation, the process yields an intact tube with endothelial cells.

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee. 
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Ex Vivo Pressurized Hippocampal Capillary-Parenchymal Arteriole Preparation for Functional Study

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