We thank C. Ricard, M-C. Amoureux, A. Jaouen, and S. Meijia-Gervacio for helpful discussions; M. Hocine and C. Meunier, and the staff of the animal and PicSIL imaging facilities of the IBDML for technical support. This work was supported by an institutional grant from Centre National de la Recherche Scientifique, and by grants from the Association de Recherche sur la Scl&#233;rose en Plaque (ARSEP) (to G.R. and K.K.F), Agence Nationale de la Recherche (ANR JCJC), F&#233;d&#233;ration de Recherche sur le Cerveau (FRC) Institut de Recherche sur la Moelle Epini&#232;re (IRME) (to FD). K.K.F. was supported by an ARSEP fellowship. Imaging was performed on the PIcSIL imaging facility of the IBDML.

1. Window and Support Structure Preparation
<ol>
	<li>Window preparation - prepare several windows of various sizes (width = 1.8-2.2 mm; length = 4.5-5.2 mm) for implantation.
	<ol>
		<li>Making the modified drill burr to cut the cover glass - using a generous amount of dental cement attach a drill burr to a shaft (in our case old Bulldog clamps). Be sure that the burr is ~45&#176; relative to the shaft for easier handling. 
		Note: diamond scribes also work well for cutting cover glass.</li>
		<li>Making slide-A ...

<ol>
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Neurosci. 10 (5), 549-551 (2007).</li><li>Holtmaat, A., Bonhoeffer, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term,+high-resolution+imaging+in+the+mouse+neocortex+through+a+chronic+cranial+window.">Long-term, high-resolution imaging in the mouse neocortex through a chronic cranial window.</a> Nat. Protoc. 4 (8), 1128-1144 (2009).</li><li>Dray, C., Rougon, G., Debarbieux, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+by+in+vivo+imaging+of+the+dynamics+of+vascular+and+axonal+networks+in+injured+mouse+spinal+cord.">Quantitative analysis by in vivo imaging of the dynamics of vascular and axonal networks in injured mouse spinal cord.</a> Proc. Natl. Acad. Sci. U.S.A. 106 (23), 9459-9464 (2009).</li><li>Kerschensteiner, M., Schwab, M. E., Lichtman, J. W., Misgeld, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> In vivo imaging of axonal degeneration and regeneration in the injured spinal. 11 (5), 572-577 (2005).</li><li>Ylera, B., Ert&#252;rk, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronically+CNS-injured+adult+sensory+neurons+gain+regenerative+competence+upon+a+lesion+of+their+peripheral+axon.">Chronically CNS-injured adult sensory neurons gain regenerative competence upon a lesion of their peripheral axon.</a> Curr. 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C., Helmchen, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+Ca2++imaging+of+dorsal+horn+neuronal+populations+in+mouse+spinal+cord.">In vivo Ca2+ imaging of dorsal horn neuronal populations in mouse spinal cord.</a> J. Physiol. 588 (18), 3397-3402 (2010).</li><li>Niki&#263;, I., Merkler, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+reversible+form+of+axon+damage+in+experimental+autoimmune+encephalomyelitis+and+multiple+sclerosis).">A reversible form of axon damage in experimental autoimmune encephalomyelitis and multiple sclerosis).</a> Nat. Med. 17 (4), 495-499 (2011).</li><li>Di Maio, A., Skuba, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+dorsal+root+regeneration:+rapid+immobilization+and+presynaptic+differentiation+at+the+CNS/PNS+border.">In vivo imaging of dorsal root regeneration: rapid immobilization and presynaptic differentiation at the CNS/PNS border.</a> J. Neurosci. 31 (12), 4569-4582 (2011).</li><li>Laffray, S., Pag&#232;s, S., Dufour, H., De Koninck, P., De Koninck, Y., C&#244;t&#233;, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptive+movement+compensation+for+in+vivo+imaging+of+fast+cellular+dynamics+within+a+moving+tissue.">Adaptive movement compensation for in vivo imaging of fast cellular dynamics within a moving tissue.</a> PloS One. 6 (5), (2011).</li><li>Fenrich, K. K., Weber, P., Hocine, M., Zalc, M., Rougon, G., Debarbieux, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+in+vivo+imaging+of+normal+and+pathological+mouse+spinal+cord+with+subcellular+resolution+using+implanted+glass+windows.">Long-term in vivo imaging of normal and pathological mouse spinal cord with subcellular resolution using implanted glass windows.</a> J. Physiol. 590 (16), 3665-3675 (2012).</li><li>Kuerten, S., Angelov, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparing+the+CNS+morphology+and+immunobiology+of+different+EAE+models+in+C57BL/6+mice+-+a+step+towards+understanding+the+complexity+of+multiple+sclerosis).">Comparing the CNS morphology and immunobiology of different EAE models in C57BL/6 mice - a step towards understanding the complexity of multiple sclerosis).</a> Ann. Anat. 190 (1), 1-15 (2008).</li><li>Mendel, I., Kerlero de Rosbo, N., Ben-Nun, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+myelin+oligodendrocyte+glycoprotein+peptide+induces+typical+chronic+experimental+autoimmune+encephalomyelitis+in+H-2b+mice:+fine+specificity+and+T+cell+receptor+V+beta+expression+of+encephalitogenic+T+cells.">A myelin oligodendrocyte glycoprotein peptide induces typical chronic experimental autoimmune encephalomyelitis in H-2b mice: fine specificity and T cell receptor V beta expression of encephalitogenic T cells.</a> Eur. J. Immunol. 25 (7), 1951-1959 (1995).</li><li>Farrar, M. J., Bernstein, I. M., Schlafer, D. H., Cleland, T. A., Fetcho, J. R., Schaffer, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+in+vivo+imaging+in+the+mouse+spinal+cord+using+an+implanted.">Chronic in vivo imaging in the mouse spinal cord using an implanted.</a> Nat. Methods. , (2012).</li><li>Moreno, B., Jukes, J. -. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+inflammation+induces+axon+injury+during+brain+inflammation.">Systemic inflammation induces axon injury during brain inflammation.</a> Ann. Neurol. 70 (6), 932-942 (2011).</li><li>Lassmann, H., van Horssen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+basis+of+neurodegeneration+in+multiple+sclerosis.">The molecular basis of neurodegeneration in multiple sclerosis.</a> FEBS Lett. 585 (23), 3715-3723 (2011).</li><li>Yenari, M. A., Han, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroprotective+mechanisms+of+hypothermia+in+brain+ischaemia.">Neuroprotective mechanisms of hypothermia in brain ischaemia.</a> Nat. Rev. Neurosci. 13 (4), 267-278 (2012).</li><li>Kalmbach, A. S., Waters, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+surface+temperature+under+a+craniotomy.">Brain surface temperature under a craniotomy.</a> J. Neurophysiol. 108 (11), 3138-3146 (2012).</li><li>P&#233;rez-Nievas, B. G., Garc&#237;a-Bueno, B., Madrigal, J. L. M., Leza, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+immobilisation+stress+ameliorates+clinical+score+and+neuroinflammation+in+a+MOG-induced+EAE+in+Dark+Agouti+rats:+mechanisms+implicated.">Chronic immobilisation stress ameliorates clinical score and neuroinflammation in a MOG-induced EAE in Dark Agouti rats: mechanisms implicated.</a> J. Neuroinflammation. 7, 60 (2010).</li><li>Bjartmar, C., Kidd, G., M&#246;rk, S., Rudick, R., Trapp, B. 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The authors declare that they have no competing financial interests.

We described a method for implanting and maintaining glass windows over the exposed spinal cords of adult mice to study the dynamic progression of EAE over time. A similar glass window implantation approach has been used to study spinal cord injury models13&#160;and may also be useful for studying other spinal cord pathologies such as glioblastomas,&#160;meningitis, or the cellular physiology of the normal spinal cord. For our method, vertebral stabilization was achieved by building an exoskeleton around four ...

Multiple sclerosis is a CNS auto-immune disease that causes progressive myelin and axon damage. The standard research model for CNS autoimmune demyelinating diseases is EAE in adult rodents. However, the dynamic cellular events underlying EAE progression remain largely unexplored. One approach is to use intravital two-photon microscopy to visualize cellular populations with fluorescent markers for extended periods of time in the spinal cords of adult mice with EAE.
Intravital two-photon microscopy is a powerful way to study cellular physiology, cellular interactions, and the dynamic progression of disease in living animals with subcellular resolution1. However, a major hurdle to the approach is that it requires optical access to the region of interest. For example, cranial glass windows are a well-established methodology for repeatedly imaging the same microstructures in the brain over several weeks2,3. Adaptation of this methodology to spinal cord imaging is, however, not straightforward and it requires several technical modifications to immobilize the dorsal spinal tissue within the articulated vertebrae.
Previously, optical access to the spinal cord required surgically reexposing the spinal cord at the beginning of each imaging session and suturing the skin closed at the end of each session4-12. These repeated surgeries are traumatic for the animal, limit the number and length of imaging sessions, and increases the probability of surgery related artifacts such as inflammation and damage to the spinal cord through mechanical perturbation. To overcome these shortcomings, we recently developed a protocol to implant glass windows over the exposed spinal cords of adult fluorescent transgenic mice with traumatic spinal cord injuries for long-term in vivo two-photon microscopy experiments without repeated surgeries13. Here we present a detailed adaptation of the implantation protocol in the context of its application to study the dynamic cellular events underlying the progression of EAE in adult mice.

<table border="0" cellpadding="0" cellspacing="0" width="875">
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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="38">
			<td height="38" style="height:39px;width:220px;">Bone removal drill</td>
			<td style="width:124px;">World Precision Instruments</td>
			<td style="width:156px;">IDEAL MICRO-DRILL</td>
			<td style="width:375px;">Also sold by Harvard Apparatus. 
			Any high quality surgical bone drill will suffice.</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;width:220px;">Drill burr (#1/4 or 1/2 Carbide Round Burr)</td>
			<td style="width:124px;">World Precision Instruments</td>
			<td style="width:156px;">501860 (#1/4) 
			501860 (#1/2)</td>
			<td>Also sold by Harvard Apparatus</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">Staples</td>
			<td>Lyreco</td>
			<td>5.002.567</td>
			<td style="width:375px;">Crown = 8 mm 
			Leg = 4 mm</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">Paperclip</td>
			<td></td>
			<td></td>
			<td>Any standard 25 mm paperclip</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">Needle drivers</td>
			<td>Fine Science Tools</td>
			<td>12003-15</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">Tissue scissors</td>
			<td>Fine Science Tools</td>
			<td>14028-10</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">Adson forceps</td>
			<td>Fine Science Tools</td>
			<td>11027-12</td>
			<td>1 x 2 teeth</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">Blunt forceps</td>
			<td>Fine Science Tools</td>
			<td>11293-00</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">Spinal forks</td>
			<td>Harvard Apparatus</td>
			<td>724813</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">Dental cement</td>
			<td>GACD</td>
			<td>12-565 &#38; 12-568</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">Silicone elastomer</td>
			<td style="width:124px;">World Precision Instruments</td>
			<td>KWIK-SIL</td>
			<td>Also avaiable through Coherent Scientific and Fisher Scientific</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">Cyanoacrylate</td>
			<td>Eleco-EFD</td>
			<td>Cyanolit 201</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">Microscope</td>
			<td>Zeiss</td>
			<td>7MP</td>
			<td style="width:375px;">Our microscope is in an upright configuration and is equipped with 5 NDD detectors.</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">Objective lens</td>
			<td>Zeiss</td>
			<td style="width:156px;">W Plan-Apochromat 20x/1.0 DIC M27 75mm</td>
			<td>Working distance = 1.8 mm</td>
		</tr>
	</tbody>
</table>

implanting glass spinal cord windows in adult mice with experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) in adult rodents is the standard experimental model for studying autonomic demyelinating diseases such as multiple sclerosis. Here we present a low-cost and reproducible glass window implantation protocol that is suitable for intravital microscopy and studying the dynamics of spinal cord cytoarchitecture with subcellular resolution in live adult mice with EAE. Briefly, we surgically expose the vertebrae T12-L2 and construct a chamber around the exposed vertebrae using a combination of cyanoacrylate and dental cement. A laminectomy is performed from T13 to L1, and a thin layer of transparent silicone elastomer is applied to the dorsal surface of the exposed spinal cord. A modified glass cover slip is implanted over the exposed spinal cord taking care that the glass does not directly contact the spinal cord. To reduce the infiltration of inflammatory cells between the window and spinal cord, anti-inflammatory treatment is administered every 2 days (as recommended by ethics committee) for the first 10 days after implantation. EAE is induced only 2-3 weeks after the cessation of anti-inflammatory treatment.
Using this approach we successfully induced EAE in 87% of animals with implanted windows and, using Thy1-CFP-23 mice (blue axons in dorsal spinal cord), quantified axonal loss throughout EAE progression. Taken together, this protocol may be useful for studying the recruitment of various cell populations as well as their interaction dynamics, with subcellular resolution and for extended periods of time. This intravital imaging modality represents a valuable tool for developing therapeutic strategies to treat autoimmune demyelinating diseases such as EAE.

Using chronically implanted glass windows in combination with intravital two-photon microscopy and EAE models may be a useful tool to understand the cellular mechanisms underlying autoimmune demyelinating disease pathology. Using our implantation method we found that most windows were clear at the time of EAE induction, and remained clear throughout EAE progression (Figure&#160;1c). Using this approach, we found that windows remained clear for the entire postimplantation period (average ~68 d&#177;7 s.e....

Watch this Scientific Journal Video about Implanting Glass Spinal Cord Windows in Adult Mice with Experimental Autoimmune Encephalomyelitis at JoVE.com

Implanting Glass Spinal Cord Windows in Adult Mice with Experimental Autoimmune Encephalomyelitis

We describe a method for implanting and maintaining glass windows over the exposed spinal cords of adult mice for studying experimental autoimmune encephalomyelitis. These windows provide chronic optical access to the spinal cord for monitoring cell dynamics at the subcellular level in live animals using two-photon microscopy.

Experimental autoimmune encephalomyelitis (EAE) in adult rodents is the standard experimental model for studying autonomic demyelinating diseases such as ...

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7.3K 次观看.  Aix Marseille University. We describe a method for implanting and maintaining glass windows over the exposed spinal cords of adult mice for studying experimental autoimmune encephalomyelitis. These windows provide chronic optical access to the spinal cord for monitoring cell dynamics at the subcellular level in live animals using two-photon microscopy.