In Vivo Three-Dimensional Two-Photon Microscopy to Study Conducted Vascular Responses by Local ATP Ejection Using a Glass Micro-Pipette

Summary

We present an optimized local ejection procedure using a glass micro-pipette and a fast two-photon hyperstack imaging method, which allows precise measurement of capillary diameter changes and investigation of its regulation in three dimensions.

Abstract

Maintenance of normal brain function requires a sufficient and efficient supply of oxygen and nutrition by a complex network of vessels. However, the regulation of cerebral blood flow (CBF) is incompletely understood, especially at the capillary level. Two-photon microscopy is a powerful tool widely used to study CBF and its regulation. Currently, this field is limited by the lack of in vivo two-photon microscopy studies examining (1) CBF responses in three-dimensions, (2) conducted vascular responses, and (3) localized interventions within the vascular network. Here, we describe a 3D in vivo method using two-photon microscopy to study conducted vascular responses elicited by local ejection of ATP with a glass micro-pipette. Our method uses fast and repetitive hyperstack two-photon imaging providing precise diameter measurements by maximal intensity projection of the obtained images. Furthermore, we show that this method can also be used to study 3D astrocytic calcium responses. We also discuss the advantages and limitations of glass micro-pipette insertion and two-photon hyperstack imaging.

Introduction

The brain has a high energy consumption rate. About 20% of the oxygen and 25% of the glucose consumed by the human body are dedicated to brain function, while the brain only occupies 2% of the total body mass. Maintenance of normal brain function requires a sufficient and efficient supply of oxygen and nutrition by blood flow in a complex network of vessels. Local brain activity and cerebral blood flow (CBF) are robustly coupled, depending on the functional properties of neurons, astrocytes, pericytes, smooth muscle cells (SMCs) and endothelial cells (ECs)¹. Recently, the first few orders of capillaries branching from penetrating arterioles hav....

Protocol

All procedures involving animals were approved by the Danish National Ethics Committee according to the guidelines set forth in the European Council’s Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes and were in compliance with the ARRIVE guidelines. This is a terminal procedure with the mice being euthanized prior to anesthetic recovery.

1. Pre-surgical preparation

1. Clean the surgical table and all the surrounding area with 70% ethanol. Thoroughly clean and dry the surgical tools before the surgery.
2. Anesthetize an NG2DsRed mo....

Discussion

One challenge for vascular studies is the precise measurement of vessel diameters. The method we describe here used a motorized piezo objective to make fast and repetitive hyperstack imaging by two-photon microscopy. Firstly, this method allows repeated examinations of the penetrating arteriole, 1^st order and 2^nd order capillaries without loss of focus and led to the discovery of slowly conducted vascular responses in capillaries in vivo. Secondly, in combination with a viral vector injection techni.......

Materials

Name	Company	Catalog Number	Comments
Agarose	Sigma–Aldrich	A6138	Apply upon exposed cortex for protection
Alexa 594	Life Technologies	A-10438	Stain puffing compound to red fluorescent color
ATP	Sigma-Aldrich	A9187	Vasodilator and vasoconstrictor, puffing compound
Cyanoacrylate glue and activator	Loctite	Adhesives and SF7452	Glue for metal piece and coverglass
Eye lubricant	Neutral Ophtha, Ophtha A/S, Denmark		Keep the mouse eyes moisterized
FITC-dextran	Sigma-Aldrich	FD500S	Blood serum dye, green fluorescent color
NG2DsRed mice	Jackson Laboratory	8241	These transgenic mice express an red fluorescent protein variant (DsRed) under the control of the mouse NG2 (Cspg4) promoter
pZac2.1 gfaABC1D-lck-GCaMP6f	Addgene	52924-AAV5	Astrocyte specific viral vectors carrying genetically encoded calcium indicators
TRITC-dextran	Sigma-Aldrich	52194	Blood serum dye, red fluorescent color
List of Equipments
Air pump	WPI	PV830	Give air pressure to pipette puffing
Blood gas analyzer	Radiometer	ABL 700	Measure levels of blood gases
Blood pressure monitor	World Precision Instruments	BP-1	Monitor aterial blood pressure
Body temperature controller	CWE	Model TC-1000	Keep the mouse body temperature in physiological range
Capnograph	Harvard Apparatus	Type 340	Monitor the end-expiratory CO2 from the mouse
Electrical stimulator	A.M.P.I.	ISO-flex	Apply whisker pad stimulation
Mechanical ventilator	Harvard Apparatus	D-79232	Mechanically ventilate the mouse in physiological range
Micropipette puller	Sutter Instrument	P-97
Two-photon microscope	Femtonics Ltd	Femto3D-RC
List of Surgical Instruments
Anatomical tweezer	Lawton	09-0007
Angled and balanced tweezer	S&T AG	00595 FRAS-18 RM-8
Iris scissor	Lawton	05-1450
Micro vascular clamp	S&T AG	462
Mouse vascular catheters	Verutech	100828