This work was supported in part by National Institutes of Health (NIH) Grants R01 GM-41514 (to M.D.C.), R39 GM-048071 (to I.P.), and R01 NS-074987 (to T.E.L.) and the National Multiple Sclerosis Society (NMSS) Collaborative Center Research Award CA1058-A-8 (to C.M.W., T.E.L. and M.D.C.), NMSS Grant RG4925, NIH Training Grant T32-AI-060573 (to M.L.G.), NMSS Postdoctoral Fellowship FG 1960-A-1 (to J.G.W.), and funding from the George E. Hewitt Foundation for Medical Research (M.P.M.).

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
<ol>
	<li>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 minut...

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The authors have no competing interests.

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 disease17-19. However, because transplanted NPCs migrate to the ventral white matter, which lies too deep to image in situ using 2P microscopy, an ex vivo ...

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 alleviation of chronic symptoms and improvement of quality of life. Recently, neural precursor cells (NPCs) have come to the forefront as a potential regenerative therapeutic modality to target areas of inflammation and demyelination. Several studies have highlighted the ability of NPCs to induce endogenous remyelination and participate directly in remyelination2-8. Because NPCs are involved in direct remyelination, it is imperative to understand their kinetics and interactions with endogenous cells following transplantation. After transplantation, NPCs migrate ventrally to areas of white matter damage, then rostrally and caudally relative to the transplant site5,9. The kinetics of migration differ in response to environmental cues; NPCs transplanted into a non-damaged spinal cord have greater velocities than NPCs transplanted into a damaged spinal cord6. After a migratory period, transferred NPCs proliferate extensively, at a higher rate in a damaged spinal cord relative to an intact spinal cord6. Finally, the majority of NPCs differentiate into oligodendrocytes and initiate direct remyelination4,6,9.
The demyelinated lesion is complex and can include a diverse population of cells at various stages of activation. For example, an active multiple sclerosis (MS) lesion may include a significant population of activated T cells, M1 microglia and M1 macrophages, but a chronic silent MS lesion may be comprised primarily of reactive astrocytes with few inflammatory cells10-13. Because of the diversity of effector cells, two-photon (2P) imaging in mouse models of demyelination is an extremely useful tool to help understand local cellular interactions within the lesion. In MS and many extensively used MS research models, the majority of lesions are located on the ventral side of the spinal cord, a region inaccessible to intravital 2P imaging due to lesion depth and the high lipid content of the spinal cord. To circumvent these issues and study cell-cell interactions within lesions along the ventral spinal cord we have developed a simple ex vivo 2P imaging preparation6.
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 demyelination14. Five week old mice are infected with JHMV and transplanted with eGFP-NPCs at thoracic level 9 on day 14 post-infection. The protocol presented here provides detailed steps on how to extract the spinal cord, make an ex vivo agarose preparation, and image transplanted eGFP-NPC interactions with enhanced yellow fluorescent protein (eYFP)-expressing axons. Mice expressing eYFP under the neuronal-specific Thy1 promoter were used in this procedure15. Only some of the axons express eYFP, making it useful for imaging individual axons. Here we show spinal cords removed at 7 days post-transplant; however, spinal cords can be extracted at any time point following transplantation. While we show interactions of NPCs with damaged axons, our protocol can be used in combination with genetic fluorescent markers of other cell types to investigate a multitude of cellular interactions occurring throughout the mouse spinal cord.

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			<td height="21" style="height:21px;width:381px;">Name of Reagent/ Equipment</td>
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			<td style="width:167px;">Comments/Description</td>
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			<td height="25" style="height:25px;width:381px;">Isoflurane, USP</td>
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			<td height="21" style="height:21px;width:381px;">agarose, low gelling temperature</td>
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			<td height="21" style="height:21px;width:381px;">Parafilm</td>
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			<td height="21" style="height:21px;width:381px;">22 mm square cover slip</td>
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			<td height="21" style="height:21px;width:381px;">25x dipping objective, 1.1 NA</td>
			<td style="width:175px;">Nikon</td>
			<td style="width:136px;">CFI Apo LWD 25XW</td>
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			<td height="21" style="height:21px;width:381px;">520 nm single-edge dichroic beam splitter</td>
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			<td height="21" style="height:21px;width:381px;">560 nm single-edge dichroic beam splitter</td>
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			<td style="width:175px;">Coherent</td>
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			<td height="21" style="height:21px;width:381px;">Imaris 7.7 software</td>
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</table>

two-photon imaging of cellular dynamics in the mouse spinal cord

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.

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 ...

Watch this Scientific Journal Video about Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord at JoVE.com

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

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.

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for ...

two-photon-imaging-of-cellular-dynamics-in-the-mouse-spinal-cord

Research

JoVE Journal

Neuroscience

10.1K Görüntüleme.  University of California, Irvine.  Fare omuriliği görüntülenmesi için yeni bir ex vivo hazırlık. Bu protokol, omurilik boyunca canlı hücresel etkileşimlerin iki foton görüntüleme için izin verir. 