Name: implanting glass spinal cord windows in adult mice with experimental autoimmune encephalomyelitis
Uploaded: 2013-12-21T14:00:00
Description: Watch this Scientific Journal Video about Implanting Glass Spinal Cord Windows in Adult Mice with Experimental Autoimmune Encephalomyelitis at JoVE.com

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. 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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. 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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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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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	<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.

玻璃植入脊髓的Windows在成年小鼠实验性自身免疫性脑脊髓炎

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

Research

JoVE Journal

Medicine

Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

建模在小鼠体内的行程 - 中东与灯丝模型大脑中动脉闭塞

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are ...

科研

医学

Acute and Chronic Tactile Sensory Testing after Spinal Cord Injury in Rats

在大鼠脊髓损伤后的急性和慢性触觉测试

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory ...

Controlled Cervical Laceration Injury in Mice

控制宫颈裂伤损伤小鼠

Use of genetically modified mice enhances our  understanding of molecular mechanisms underlying several neurological disorders  such as a spinal cord ...

A Contusion Model of Severe Spinal Cord Injury in Rats

挫伤模型脊髓严重损伤的保护作用

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is ...

Evaluation of Respiratory Muscle Activation Using Respiratory Motor Control Assessment (RMCA) in Individuals with Chronic Spinal Cord Injury

Evaluation of Respiratory Muscle Activation Using Respiratory Motor Control Assessment RMCA in Individuals with Chronic Spinal Cord Injury

评价呼吸肌慢性脊髓损伤个人使用呼吸电机控制评估（RMCA）

During breathing, activation of respiratory muscles is coordinated by integrated input from the brain, brainstem, and spinal cord. When this coordination ...

Controlled Cortical Impact Model for Traumatic Brain Injury

控制皮层影响模型创伤性脑损伤

Every year over a million Americans suffer a traumatic brain injury (TBI).  Combined with the incidence of TBIs worldwide, the physical, emotional, ...

A Radio-telemetric System to Monitor Cardiovascular Function in Rats with Spinal Cord Transection and Embryonic Neural Stem Cell Grafts

无线电遥测系统，以监测大鼠脊髓横断和胚胎神经干细胞移植在心血管功能

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter ...

Synergetic Use of Neural Precursor Cells and Self-assembling Peptides in Experimental Cervical Spinal Cord Injury

在实验颈脊髓损伤协同使用神经前体细胞，并自组装肽的

Spinal cord injuries (SCI) cause serious neurological impairment and psychological, economic, and social consequences for patients and their families. ...

Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury

在脊髓损伤的实验挫伤型号神经干细胞移植

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can ...

Contrast Enhanced Ultrasound Imaging for Assessment of Spinal Cord Blood Flow in Experimental Spinal Cord Injury

超声造影成像脊髓血流量的评估实验脊髓损伤

Reduced spinal cord blood flow (SCBF) (i.e., ischemia) plays a key role in traumatic spinal cord injury (SCI) pathophysiology and is accordingly an ...