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Method Article

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

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DOI:

10.3791/60676

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December 18th, 2019

 ,  , 
1Department of Anesthesiology, University of Colorado Anschutz Medical Campus, 2Department of Neurological Sciences, University of Vermont Larner College of Medicine, 3Department of Pharmacology, University of Colorado Anschutz Medical Campus

In This Article

Summary

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.

Abstract

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.

Introduction

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.

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Protocol

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

  1. Use MOPS-buffered saline for the dissection and to keep samples at 4 °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 °C.
  2. 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).
  3. Obtain the maximal dilation in nominally Ca2+-free aCSF (0 mM [Ca2+]o, 5 mM EGTA).

2. Organ chamber preparation

  1. 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.
  2. 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 µm in diameter.
  3. Fill the cannula using a syringe with an attached 0.22 µm filter with oxygenated aCSF. Make sure there are no air bubbles or debris in the cannula.
  4. Add two more cannulas to the opposite side of the chamber. Do not break their tips.

3. Hippocampus dissection and isolation

  1. 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.
  2. Using small dissection scissors, cut the skin along the midline at the top of its head. Move the skin off to the sides.
  3. 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.
  4. 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.
  5. 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.
  6. 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.
  7. Perform the following steps for each hemisphere separately or in parallel.
    1. 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.
    2. 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).
    3. 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).
  8. Transfer the hippocampi to a new dissection plate filled about halfway with MOPS solution. Discard the rest of the brain.

4. Hippocampal arteriole isolation

  1. Complete the following steps for each hippocampus.
    1. Pin down one of the hippocampi using small pins at each end of the section. The hippocampal artery is facing up.
    2. 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.
    3. Search through the dorsal hippocampal tissue to identify the external transverse artery (Figure 1C)16,27.
    4. 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.
    5. 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.

5. Hippocampal capillary-parenchymal arteriole cannulation

  1. Find an arteriole with a branch that ends with capillaries. Transfer it to the organ chamber. Ensure that the arteriole is about 15-30 µm when fully dilated (Figure 1D).
  2. 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.
  3. 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.
  4. Make another tie and secure it on the other end of the arteriole to seal it.
  5. 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.
  6. 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.
  7. 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).

6. Pressure myography

  1. Move the chamber from the dissection scope to the microscope with the recording software.
  2. Connect the inflow and outflow tubing to the chamber for perfusion. Start the perfusion with heated aCSF (37 °C) at a flow rate of 4 mL/min.
  3. Attach the pressurizing cannula to a peristaltic pump paired with a pressure transducer and bring the internal pressure to 20 mm Hg.
  4. 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.
  5. Increase the pressure of the vessel up to 40 mm Hg while recording the arterial diameter with an edge detection software.
  6. 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.
  7. To test the viability of a vessel, apply 1 µM 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.

7. Focal stimulation of capillary ends

  1. Once the baseline tone for the arteriole has been established and the endothelial function assessed, test the response of capillary stimulation.
  2. 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.
  3. 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.
  4. 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.
  5. 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.
  6. Repeat stimulation as necessary. Change the pressure ejection system cannula to test different drug compounds.
  7. To confirm that only the capillaries are being stimulated, fill the cannula with 1 µM 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).

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Results

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 µM 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 ca...

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Discussion

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. Revea...

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Disclosures

The authors have nothing to disclose.

Acknowledgements

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's Health and Research, and the NHLBI R01HL136636 (FD).

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
0.22µm Syringe FiltersCELLTREAT Scientific Products229751
12-0 Nylon (12cm) BlackMicrosurgery Instruments, IncS12-0 NYLON
Automatic Temperature ControllerWarner InstrumentsTC-324B
Borosilicate Glass O.D.: 1.2 mm, I.D.: 0.68 mmSutter InstrumentsB120-69-10
Bovine serum albuminSigma-AldrichA7030
CaCl2 dihydrateSigma-AldrichC3881
D-(+)-GlucoseSigma-AldrichG5767
Dissection ScopeOlympusSZ11
ECOLINE VC-MS/CA 4-12 — complete Pump with Drive and MS/CA 4-12 pump-headIsmatecISM 1090
EGTASigma-AldrichE4378
Fine Scissors - SharpFine Science Tools14063-09
Inline Water HeaterWarner InstrumentsSH-27B
Integra™ Miltex™Tissue ForcepsFisher Scientific12-460-117
KClSigma-AldrichP9333
KH2PO4Sigma-AldrichP5379
Magnesium sulfate heptahydrateSigma-AldrichM1880
MgCl AnhydrousSigma-AldrichM8266
MicromanipulatorNarishigeMN-153
ML 133 hydrochlorideTocris4549
MOPSSigma-AldrichM1254
NaClSigma-AldrichS9625
NaH2PO4Sigma-AldrichS9638
NaHCO3Sigma-AldrichS8875
NS309Tocris3895
Picospritzer III - Intracellular Microinjection Dispense Systems, 2-channelParker Hannifin052-0500-900
Pressure Servo Controller with Peristaltic PumpLiving Systems InstrumentationPS-200
Sodium pyruvateSigma-AldrichP3662
Super Fine ForcepsFine Science Tools11252-20
Surgical Scissors - Sharp-BluntFine Science Tools14001-13
Vertical Micropipette PullerNarishigePP-83

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Tags

Hippocampal Capillary ArterioleEx Vivo PreparationPressure MyographyArteriolar Diameter MeasurementCapillary StimulationKir2 Inhibitor ML133Potassium Neurovascular CouplingSex Specific ResponsesVessel Cannulation TechniqueMicrocirculation Isolation
Ex Vivo Pressurized Hippocampal Capillary-Parenchymal Arteriole Preparation for Functional Study
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