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.
Highlights
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
The kinetics of migration differ in response to environmental cues; neural precursor cell (NPC) transplanted into a non-damaged spinal cord have greater velocities than NPCs transplanted into a damaged spinal cord[6]
This study follows up a previous methods publication, which showed the procedure for transplanting enhanced green fluorescent proteinexpressing NPCs into the spinal cord of mice following JHMV strain of MHV-induced demyelination[14]
Summary
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 inflammation[1]. This study follows up a previous methods publication, which showed the procedure for transplanting enhanced green fluorescent protein (eGFP)expressing NPCs into the spinal cord of mice following JHMV strain of MHV-induced demyelination[14]. 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
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