This work was supported by the National Multiple Sclerosis Society grant RG4595A1/T to DD and the NIH/NINDS grants NS051470, NS052189 and NS066361 to K.A. Figures and movies adapted and/or reprinted from Davalos et al., J Neurosci Methods. 2008 Mar 30;169(1):1-7 Copyright 2008, with permission from Elsevier.

1. Building the spinal stabilization device
<ol>
	<li>Order the Narishige STS-A Compact Spinal Cord Clamps and the Narishige MA-6N head holding adaptor.</li>
	<li>Custom design and make a stainless steel base plate to hold the two Narishige parts in alignment so that the animal's head is supported while its spinal column and tail are clamped. Keep in mind that the entire device should fit under the microscope lens usually on a lowered microscope stage.</li>
</ol>
2. Animal surgery
<ol>
	<li>Pr...

<ol>
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We have nothing to disclose.

The method described here allows for stable and repetitive in vivo imaging of densely populated fluorescent cellular structures in the spinal cord of anesthetized mice using two-photon microscopy. The achieved stability is a result of a custom-made spinal stabilization device and an anesthetic regimen that reduces respiratory-induced movement artifact. The spinal stabilization device allows breathing space underneath the mouse body and can be built using commercially available spinal clamps and head mounting pie...

<table border="1">
	<tbody>
		<tr>
			<td>	 Name of the reagent </td>
			<td>	 Company </td>
			<td>	 Catalogue number </td>
			<td>	 Comments </td>
		</tr>
		<tr>
			<td>Rhodamine B dextran</td>
			<td>Invitrogen</td>
			<td>D1841</td>
			<td>70 kDa, diluted in 
			ACSF (3% w/v)</td>
		</tr>
		<tr>
			<td>Ketamine HCl</td>
			<td>Bionichepharma</td>
			<td>NDC No: 67457-001-10</td>
			<td>Injectable, 50mg/ml</td>
		</tr>
		<tr>
			<td>Anased</td>
			<td>Lloyd Labs</td>
			<td>NADA No: 139-236</td>
			<td>Xylazine injectable, 
			20mg/ml</td>
		</tr>
		<tr>
			<td>Acepromazine</td>
			<td>Vedco</td>
			<td>NADA No: 117-531</td>
			<td>Injectable,10mg/ml</td>
		</tr>
		<tr>
			<td>Artificial tears 
			ointment</td>
			<td>Phoenix 
			pharmaceutical</td>
			<td>NDC No: 57319-760- 
			25</td>
			<td>Lubricant</td>
		</tr>
		<tr>
			<td>Betadine</td>
			<td>Fisher</td>
			<td>19-061617</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>McPherson-Westcott 
			Scissors</td>
			<td>World Precision 
			Instruments</td>
			<td>555500S</td>
			<td>Curved, blunt-tip 
			scissors</td>
		</tr>
		<tr>
			<td>Straight Forceps</td>
			<td>World Precision 
			Instruments</td>
			<td>555047FT</td>
			<td>Toothed tip forceps</td>
		</tr>
		<tr>
			<td>Small vessel cauterize</td>
			<td>Fine Science Tools</td>
			<td>18000-00</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Gelfoam</td>
			<td>Pharmacia,Pfizer Inc.</td>
			<td>Mixer Mill MM400</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Compact spinal cord 
			clamps</td>
			<td>Narishige</td>
			<td>STS-A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Head holding adaptor</td>
			<td>Narishige</td>
			<td>MA-6N</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Gelseal</td>
			<td>Amersham 
			Biosciences Corp.</td>
			<td>80-6421-43</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lactated Ringers</td>
			<td>Baxter Healthcare</td>
			<td>2B8609</td>
			<td>&nbsp;</td>
		</tr>

		<tr>
			<td>Buprenex</td>
			<td>Reckit Benckiser 
			Pharmaceuticals Inc.</td>
			<td>NDC No: 12496- 
			6757-1</td>
			<td>Buprenorphine, 
			injectable</td>
		</tr>
		<tr>
			<td>Baytril</td>
			<td>Bayer</td>
			<td>NADA 140-913</td>
			<td>Enrofloxacin, 
			antibacterial injectable 
			2.27% (20ml)</td>
		</tr>
		<tr>
			<td>Heating pad - Large</td>
			<td>Fine Science Tools</td>
			<td>21060-10</td>
			<td>&nbsp;</td>
		</tr>

	</tbody>
</table>

in vivo imaging of the mouse spinal cord using two-photon microscopy

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo 4-7. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions 8-17. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task.
We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.

Watch this Scientific Journal Video about In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy at JoVE.com

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

A minimally invasive protocol to stabilize the mouse spinal column and perform repetitive in vivo spinal cord imaging using two-photon microscopy is described. This method combines a spinal stabilization device and an anesthetic regimen to minimize respiratory-induced movements and produce raw imaging data that require no alignment or other post-processing.

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has ...

in-vivo-imaging-of-the-mouse-spinal-cord-using-two-photon-microscopy

Research

JoVE Journal

Neuroscience

23.6K visualizações.  University of California, San Francisco. Um protocolo minimamente invasiva para estabilizar a coluna vertebral do mouse e executar repetitivos In vivo Imagem da medula espinhal usando microscopia de dois fótons é descrito. Este método combina um dispositivo de estabilização da coluna vertebral e um regime anestésico para minimizar respiratória induzida por movimentos e produzir dados de imagem-primas que não necessitam de alinhamento ou de outras pós-processamento.