Isolation and Functional Analysis of Arteriolar Endothelium of Mouse Brain Parenchyma

Podsumowanie

Intensive preparation of intact mouse cerebral endothelial "tubes" 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.

Streszczenie

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—integral to the physiology and etiology of chronic degenerative diseases—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 "tubes" (diameter, ~25 µm) from mouse brain parenchymal arterioles. Arteriolar endothelial tubes are secured during physiological conditions (37 °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.

Wprowadzenie

Parenchymal arterioles directly deliver essential oxygen and nutrients throughout the brain¹. While interfacing with capillaries, highly vasoactive arterioles respond to retrograde signaling initiated by capillary ion channels that sense metabolic signals from specific neuronal regions². With brain parenchyma having historically received the bulk of investigation, a role for endothelial dysfunction has now emerged for clarifying pathological mechanisms associated with various cerebrovascular disorders that underlie dementia (e.g., ischemic stroke, Alzheimer's disease)^3,

Protokół

Experimenters should ensure that designated use of animals and associated protocols are approved by their Institutional Animal Care and Use Committee (IACUC) and performed in accordance with the National Research Council's "Guide for the Care and Use of Laboratory Animals" (8^th Edition, 2011) and the ARRIVE guidelines. The IACUC of Loma Linda University and the University of Arizona has approved all protocols used for this manuscript for C57BL/6N and 3xTg-AD mice (males and females; age range: 2-30 months). See Figure 1 as an overview of the isolation and examination of arteriolar endothelial tubes freshly ....

Wyniki

A demonstration of the protocol is shown in Figure 1 with arteriolar dissection and endothelial tube isolation steps as Figure 2 and Figure 3, respectively. Here, endothelial function was assessed by measuring [Ca²⁺]_i and V_m using Fura-2 photometry and sharp electrode electrophysiology (Figure 4A) in response to a pharmacological agent [2-methylthioadenosine diphosphate .......

Dyskusje

Growing evidence suggests that cerebrovascular disease (CVD), aging, and Alzheimer's disease are strongly correlated and are a current topic of dementia research^4,8,14,21. Thus, it is obvious that studies of the cerebrovascular network would have a broad impact on health while requiring continued extensive investigation during conditions of disease. As a significant point of vascular r.......

Materiały

Name	Company	Catalog Number	Comments
Amplifiers	Molecular Devices, Sunnyvale, CA, USA	Axoclamp 2B & Axoclamp 900A
Audible baseline monitors	Ampol US LLC, Sarasota, FL, USA	BM-A-TM
Bath Chiller (Isotemp 500LCU)	ThermoFisher Scientific	13874647
Borosilicate glass capillaries (Pinning)	Warner Instruments	G150T-6
Borosilicate glass capillaries (Sharp Electrodes)	Warner Instruments	GC100F-10
Borosilicate glass capillaries (Trituration)	World Precision Instruments (WPI), Sarasota, FL, USA	1B100-4
BSA: Bovine Serum Albumin	Sigma	A7906
CaCl2: Calcium Chloride	Sigma	223506
Collagenase (Type H Blend)	Sigma	C8051
Cover Glass (2.4 × 5.0 cm)	ThermoFisher Scientific	12-548-5M
Data Acquision Digitizer	Molecular Devices, Sunnyvale, CA, USA	Digidata 1550A
Dissection Dish (Glass Petri with Charcoal Sylgard bottom)	Living Systems Instrumentation, St. Albans City, VT, USA	DD-90-S-BLK
Dithioerythritol	Sigma	D8255
DMSO: Dimethyl Sulfoxide	Sigma	D8418
Elastase (porcine pancreas)	Sigma	E7885
Endoplasmic Reticulum Tracker (ER-Tracker Red, BODIPY TR Glibenclamide)	ThermoFisher Scientific	E34250
Fiber optic light sources	Schott, Mainz, Germany & KL200, Zeiss	Fostec 8375
Flow Control Valve	Warner Instruments	FR-50
Fluorescence system interface, ARC lamp & power supply, hyperswitch and PMT	Molecular Devices, Sunnyvale, CA, USA	IonOptix Systems
Forceps (Fine-tipped, sharpened)	FST	Dumont #5 & Dumont #55
Function Generator	EZ Digital, Seoul, South Korea	FG-8002
Fura-2 AM dye	Invitrogen, Carlsbad, CA, USA	F14185
Glucose	Sigma-Aldrich (St. Louis, MO, USA)	G7021
HCl: Hydrochloric Acid	ThermoFisher Scientific (Pittsburgh, PA, USA)	A466250
Headstages	Molecular Devices	HS-2A & HS-9A
HEPES: (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)	Sigma	H4034
Inline Solution Heater	Warner Instruments	SH-27B
KCl: Potassium Chloride	Sigma	P9541
MgCl2: Magnesium Chloride	Sigma	M2670
Microforge	Narishige, East Meadow, NY, USA	MF-900
Micromanipulator	Siskiyou	MX10
Micropipette puller (digital)	Sutter Instruments, Novato, CA, USA	P-97 or P-1000
Microscope (Nikon-inverted)	Nikon Instruments Inc, Melville, NY, USA	Ts2
Microscope (Nikon-inverted)	Nikon Instruments Inc	Eclipse TS100
Microscope objectives	Nikon Instruments Inc	20X (S-Fluor) and 40X (Plan Fluor)
Microscope platform (anodized aluminum; diameter, 7.8 cm)	Warner Instruments	PM6 or PH6
Microscope Stage (Aluminum)	Siskiyou, Grants Pass, OR, USA	8090P
Microsyringe Pump Controller	World Precision Instruments (WPI), Sarasota, FL, USA	SYS-MICRO4
MTA: 2-Methylthioadenosine diphosphate trisodium salt	Tocris	1624
NaCl: Sodium Chloride	Sigma	S7653
NaOH: Sodium Hydroxide	Sigma	S8045
Nuclear Stain (NucBlue Live ReadyProbes Reagent; Hoechst 33342)	ThermoFisher Scientific	R37605
Oscilloscope	Tektronix, Beaverton, Oregon, USA	TDS 2024B
Papain	Sigma	P4762
Phase contrast objectives	Nikon Instruments Inc	(Ph1 DL; 10X & 20X)
Plasma Membrane Stain (CellMask Deep Red)	ThermoFisher Scientific	C10046
Plexiglas superfusion chamber	Warner Instruments, Camden, CT, USA	RC-27
Scissors (3 mm & 7 mm blades)	Fine Science Tools (or FST), Foster City, CA, USA	Moria MC52 & 15000-00
Scissors (Vannas style; 9.5 mm & 3 mm blades)	World Precision Instruments	555640S, 14364
Stereomicroscopes	Zeiss, NY, USA	Stemi 2000 & 2000-C
Syringe filter (0.22 µm)	ThermoFisher Scientific	722-2520
Temperature Controller (Dual Channel)	Warner Instruments	TC-344B or C
Valve Control System	Warner Instruments	VC-6
Vibration Isolation Table	Technical Manufacturing, Peabody, MA, USA	Micro-g