Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

Summary

A new ex vivo preparation for imaging the mouse spinal cord. This protocol allows for two-photon imaging of live cellular interactions throughout the spinal cord.

Abstract

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for live-cell imaging in the murine spinal cord. This technique is uniquely suited to analyze neural precursor cell (NPC) dynamics following transplantation into spinal cords undergoing neuroinflammatory demyelinating disorders. NPCs migrate to sites of axonal damage, proliferate, differentiate into oligodendrocytes, and participate in direct remyelination. NPCs are thereby a promising therapeutic treatment to ameliorate chronic demyelinating diseases. Because transplanted NPCs migrate to the damaged areas on the ventral side of the spinal cord, traditional intravital 2P imaging is impossible, and only information on static interactions was previously available using histochemical staining approaches. Although this method was generated to image transplanted NPCs in the ventral spinal cord, it can be applied to numerous studies of transplanted and endogenous cells throughout the entire spinal cord. In this article, we demonstrate the preparation and imaging of a spinal cord with enhanced yellow fluorescent protein-expressing axons and enhanced green fluorescent protein-expressing transplanted NPCs.

Introduction

Mouse models of demyelination, including experimental autoimmune encephalomyelitis (EAE) and intracranial infection with neuroadapted mouse hepatitis virus (MHV), are excellent tools to study molecular pathways and cellular interactions associated with disease. They have led to and supported the effectiveness of FDA approved pharmaceutical therapies, mainly targeting cessation of autoimmunity and inflammation¹. However, once endogenous remyelination has failed, the currently approved therapies do not effectively repair demyelinated lesions in the central nervous system. Therefore, repair-focused therapies at this stage of disease are critical for the allevi....

Protocol

NOTE: Ethics Statement: The protocol for animal handling was approved by the Institutional Animal Care and Use Committee (IACUC) of the University of California, Irvine, protocol #2010-2943.

1. Removal of Spinal Cord

1. Place paper towels wetted with ~100% liquid isoflurane, USP in euthanizing chamber and place dry paper towels on top. Place mouse in chamber on top of dry paper towels so mouse is not touching the isoflurane and make sure the chamber is covered. Wait at least one minute after cessation of breathing to ensure that the mouse is euthanized.
2. Perform a spinal transection of the neck to ensure the mouse is eutha....

Results

While the explanted spinal cord imaging protocol can be used to visualize any fluorescence within the spinal cord, our representative results demonstrate eGFP-NPC interactions with eYFP-axons. First, we show the embedded ventral spinal cord preparation in Figure 1A. Next, we show the 2P microscope setup and key components in Figure 1B. Figure 2 demonstrates eGFP and eYFP fluorescence in a single z-stack within the ventral spinal cord. Acquisition of consecutive z-stacks .......

Discussion

Real-time 2P imaging of intact tissue is required to investigate NPC kinetics and interactions following transplantation into the demyelinated mouse spinal cord. Intravital 2P imaging is commonly used to determine cellular dynamics on the dorsal side of the spinal cord in living mice, and has been used to study dorsal demyelination in demyelinating disease^17-19. However, because transplanted NPCs migrate to the ventral white matter, which lies too deep to image in situ using 2P microscopy, an ex vivo .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Isoflurane, USP	Piramal Critical Care, Inc	N/A
Fine scissors	Fine Science Tools	14060-09	sharp
scalpel blade #10	Fine Science Tools	10010-00
scalpel handle	Fine Science Tools	10003-12
Luer rongeurs	Fine Science Tools	16001-15
Graefe forceps	Fine Science Tools	11052-10
Vannas scissors	Fine Science Tools	15615-08
scalpel blade #11	Fine Science Tools	10011-00
RPMI-1640	Gibco	12-115F
agarose, low gelling temperature	Sigma	A9414-25G
Parafilm	Fisher Scientific	13-374-12
Vetbond (tissue adhesive)	3M	1469SB
22 mm square cover slip	Fisher Scientific	12-547
25X dipping objective, 1.1 NA	Nikon	CFI Apo LWD 25XW
Single inline solution heater	Warner Instruments	64-0102
520 nm single-edge dichroic beam splitter	Semrock	FF520-Di02-25x36	Brightline
560 nm single-edge dichroic beam splitter	Semrock	FF560-FDi01-25x36	Brightline
photomultiplier tubes	Hamamatsu	R928
C/L variable-speed tubing pump	Masterflex	77122-22
digital thermometer	Comar Instruments	3501
Chameleon Ultra Ti:Sapphire laser	Coherent	N/A
Slidebook 6 software	3i	N/A
Imaris 7.7 software	Bitplane	N/A