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>
	<li>Kim, J. V., Dustin, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+response+to+focal+necrotic+injury+inside+the+blood-brain+barrier.">Innate response to focal necrotic injury inside the blood-brain barrier.</a> J. Immunol. 177 (8), 5269-5277 (2006).</li><li>Xu, H. -. T., Pan, F., Yang, G., Gan, W. -. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choice+of+cranial+window+type+for+in+vivo+imaging+affects+dendritic+spine+turnover+in+the+cortex.">Choice of cranial window type for in vivo imaging affects dendritic spine turnover in the cortex.</a> Nat. 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. Biol. 19 (11), 930-936 (2009).</li><li>Dibaj, P., Nadrigny, F., 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=NO+mediates+microglial+response+to+acute+spinal+cord+injury+under+ATP+control+in+vivo.">NO mediates microglial response to acute spinal cord injury under ATP control in vivo.</a> Glia. 58 (9), 1133-1144 (2010).</li><li>Dibaj, P., Steffens, H., 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+reveals+distinct+inflammatory+activity+of+CNS+microglia+versus+PNS+macrophages+in+a+mouse+model+for+ALS.">In Vivo imaging reveals distinct inflammatory activity of CNS microglia versus PNS macrophages in a mouse model for ALS.</a> PloS One. 6 (3), (2011).</li><li>Johannssen, H. 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. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurological+disability+correlates+with+spinal+cord+axonal+loss+and+reduced+N-acetyl+aspartate+in+chronic+multiple+sclerosis+patients.">Neurological disability correlates with spinal cord axonal loss and reduced N-acetyl aspartate in chronic multiple sclerosis patients.</a> Ann. Neurol. 48 (6), 893-901 (2000).</li></ol>

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.

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

implanting-glass-spinal-cord-windows-adult-mice-with-experimental

Research

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Medicine

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

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are very limited. To meet the need for novel therapeutic approaches, experimental stroke research frequently employs rodent models of focal cerebral ischaemia. Most researchers use permanent or transient occlusion of the middle cerebral artery (MCA) in mice or rats.
Proximal occlusion of the middle cerebral artery (MCA) via the intraluminal suture technique (so called filament or suture model) is probably the most frequently used model in experimental stroke research. The intraluminal MCAO model offers the advantage of inducing reproducible transient or permanent ischaemia of the MCA territory in a relatively non-invasive manner. Intraluminal approaches interrupt the blood flow of the entire territory of this artery. Filament occlusion thus arrests flow proximal to the lenticulo-striate arteries, which supply the basal ganglia. Filament occlusion of the MCA results in reproducible lesions in the cortex and striatum and can be either permanent or transient. In contrast, models inducing distal (to the branching of the lenticulo-striate arteries) MCA occlusion typically spare the striatum and primarily involve the neocortex. In addition these models do require craniectomy. In the model demonstrated in this article, a silicon coated filament is introduced into the common carotid artery and advanced along the internal carotid artery into the Circle of Willis, where it blocks the origin of the middle cerebral artery. In patients, occlusions of the middle cerebral artery are among the most common causes of ischaemic stroke. Since varying ischemic intervals can be chosen freely in this model depending on the time point of reperfusion, ischaemic lesions with varying degrees of severity can be produced. Reperfusion by removal of the occluding filament at least partially models the restoration of blood flow after spontaneous or therapeutic (tPA) lysis of a thromboembolic clot in humans.
In this video we will present the basic technique as well as the major pitfalls and confounders which may limit the predictive value of this model.

Filamentous occlusion of the Middle cerebral artery is a common model for studying ischemic stroke in mice.

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

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

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory testing in rat SCI models is needed. However, until recently, no single testing approach had been validated for SCI so that standardized methods have not been implemented across labs. Additionally, available testing methods could not be implemented acutely or when severe motor impairments existed, preventing studies of the development of SCI-induced allodynia3. Here we present two validated sensory testing methods using von Frey Hair (VFH) monofilaments which quantify changes in tactile sensory thresholds after SCI4-5. One test is the well-established Up-Down test which demonstrates high sensitivity and specificity across different SCI severities when tested chronically5. The other test is a newly-developed dorsal VFH test that can be applied acutely after SCI when allodynia develops, prior to motor recovery4-5. Each VFH monofilament applies a calibrated force when touched to the skin of the hind paw until it bends. In the up-down method, alternating VFHs of higher or lower forces are used on the plantar L5 dermatome to delineate flexor withdrawal thresholds. Successively higher forces are applied until withdrawal occurs then lower force VFHs are used until withdrawal ceases. The tactile threshold reflects the force required to elicit withdrawal in 50% of the stimuli. For the new test, each VFH is applied to the dorsal L5 dermatome of the paw while the rat is supported by the examiner. The VFH stimulation occurs in ascending order of force until at least 2 of 3 applications at a given force produces paw withdrawal. Tactile sensory threshold is the lowest force to elicit withdrawal 66% of the time. Acclimation, testing and scoring procedures are described. Aberrant trials that require a retest and typical trials are defined. Animal use was approved by Ohio State University Animal Care and Use Committee.

We describe two tactile sensory testing methods for acute or chronic periods of spinal cord injury in rats. These validated procedures can detect the development and maintenance of allodynia-like sensations.

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

Use of genetically modified mice enhances our understanding of molecular mechanisms underlying several neurological disorders such as a spinal cord injury (SCI). Freehand manual control used to produce a laceration model of SCI creates inconsistent injuries often associated with a crush or contusion component and, therefore, a novel technique was developed. Our model of cervical laceration SCI has resolved inherent difficulties with the freehand method by incorporating 1) cervical vertebral stabilization by vertebral facet fixation, 2) enhanced spinal cord exposure, and 3) creation of a reproducible laceration of the spinal cord using an oscillating blade with an accuracy of &plusmn;0.01 mm in depth without associated contusion. Compared to the standard methods of creating a SCI laceration such as freehand use of a scalpel or scissors, our method has produced a consistent lesion. This method is useful for studies on axonal regeneration of corticospinal, rubrospinal, and dorsal ascending tracts.

A novel technique to create a reproducible in vivo model of cervical spinal cord laceration injury in the mouse is described. This technique is based on spine stabilization by fixation of the cervical facets and laceration of the spinal cord using an oscillating blade with an accuracy of &plusmn;0.01 mm.

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

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is described to obtain a consistent model of severe SCI. Use of a stereotactic frame and computer controlled impactor allows for creation of reproducible injury. Hypothermia and urinary tract infection pose significant challenges in the post-operative period. Careful monitoring of animals with daily weight recording and bladder expression allows for early detection of post-operative complications. The functional results of this contusion model are equivalent to transection models. The contusion model can be utilized to evaluate the efficacy of both neuroprotective and neuroregenerative approaches.

A contusion model of severe spinal cord injury is described. Detailed pre-operative, operative and post-operative steps are described to obtain a consistent model.

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

During breathing, activation of respiratory muscles is coordinated by integrated input from the brain, brainstem, and spinal cord. When this coordination is disrupted by spinal cord injury (SCI), control of respiratory muscles innervated below the injury level is compromised1,2 leading to respiratory muscle dysfunction and pulmonary complications. These conditions are among the leading causes of death in patients with SCI3. Standard pulmonary function tests that assess respiratory motor function include spirometrical and maximum airway pressure outcomes: Forced Vital Capacity (FVC), Forced Expiratory Volume in one second (FEV1), Maximal Inspiratory Pressure (PImax) and Maximal Expiratory Pressure (PEmax)4,5. These values provide indirect measurements of respiratory muscle performance6. In clinical practice and research, a surface electromyography (sEMG) recorded from respiratory muscles can be used to assess respiratory motor function and help to diagnose neuromuscular pathology. However, variability in the sEMG amplitude inhibits efforts to develop objective and direct measures of respiratory motor function6. Based on a multi-muscle sEMG approach to characterize motor control of limb muscles7, known as the voluntary response index (VRI)8, we developed an analytical tool to characterize respiratory motor control directly from sEMG data recorded from multiple respiratory muscles during the voluntary respiratory tasks. We have termed this the Respiratory Motor Control Assessment (RMCA)9. This vector analysis method quantifies the amount and distribution of activity across muscles and presents it in the form of an index that relates the degree to which sEMG output within a test-subject resembles that from a group of healthy (non-injured) controls. The resulting index value has been shown to have high face validity, sensitivity and specificity9-11. We showed previously9 that the RMCA outcomes significantly correlate with levels of SCI and pulmonary function measures. We are presenting here the method to quantitatively compare post-spinal cord injury respiratory multi-muscle activation patterns to those of healthy individuals.

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

The purpose of this publication is to present our original work on a multi-muscle surface electromyographic approach to quantitatively characterize respiratory muscle activation patterns in individuals with chronic spinal cord injury using vector-based analysis.

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

Every year over a million Americans suffer a traumatic brain injury (TBI). Combined with the incidence of TBIs worldwide, the physical, emotional, social, and economical effects are staggering. Therefore, further research into the effects of TBI and effective treatments is necessary. The controlled cortical impact (CCI) model induces traumatic brain injuries ranging from mild to severe. This method uses a rigid impactor to deliver mechanical energy to an intact dura exposed following a craniectomy. Impact is made under precise parameters at a set velocity to achieve a pre-determined deformation depth. Although other TBI models, such as weight drop and fluid percussion, exist, CCI is more accurate, easier to control, and most importantly, produces traumatic brain injuries similar to those seen in humans. However, no TBI model is currently able to reproduce pathological changes identical to those seen in human patients. The CCI model allows investigation into the short-term and long-term effects of TBI, such as neuronal death, memory deficits, and cerebral edema, as well as potential therapeutic treatments for TBI.

Traumatic brain injuries (TBIs) remain a serious health problem. Using the controlled cortical impact surgery model, research on the effects of TBI and possible treatment methods may be performed.

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

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter connected to a radio transmitter into the femoral artery of rats that underwent a T4 spinal cord transection with or without grafting of embryonic brainstem-derived neural stem cells expressing green fluorescent protein. Compared to other methods such as cannula insertion or tail-cuff, telemetry is advantageous to continuously monitor blood pressure and heart rate in freely moving animals. It is also capable of long term multiple data acquisitions. In spinal cord injured rats, basal cardiovascular data under unrestrained condition and autonomic dysreflexia in response to colorectal distension were successfully recorded. In addition, cardiovascular parameters before and after SCI can be compared in the same rat if a transmitter is implanted before a spinal cord transection. One limitation of the described telemetry procedure is that implantation in the femoral artery may influence the blood supply to the ipsilateral hindlimb.

We present a protocol for using a radio-telemetric system to record cardiovascular parameters in T4 spinal cord transected rats eight weeks after embryonic brainstem neural stem cell grafting into the lesion site. Telemetry is an advanced technique to accurately evaluate cardiovascular function in conscious freely moving spinal cord injured rats.

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

Spinal cord injuries (SCI) cause serious neurological impairment and psychological, economic, and social consequences for patients and their families. Clinically, more than 50% of SCI affect the cervical spine1. As a consequence of the primary injury, a cascade of secondary mechanisms including inflammation, apoptosis, and demyelination occur finally leading to tissue scarring and development of intramedullary cavities2,3. Both represent physical and chemical barriers to cell transplantation, integration, and regeneration. Therefore, shaping the inhibitory environment and bridging cavities to create a supportive milieu for cell transplantation and regeneration is a promising therapeutic target4. Here, a contusion/compression model of cervical SCI using an aneurysm clip is described. This model is more clinically relevant than other experimental models, since complete transection or ruptures of the cord are rare. Also in comparison to the weight drop model, which in particular damage the dorsum columns, circumferential compression of the spinal cord appears advantageous. Clip closing force and duration can be adjusted to achieve different injury severity. A ring spring facilitates precise calibration and constancy of clip force. Under physiological conditions, synthetic self-assembling peptides (SAP) self-assemble into nanofibers and thus, are appealing for application in SCI5. They can be injected directly into the lesion minimizing damage to the cord. SAPs are biocompatible structures erecting scaffolds to bridge intramedullary cavities and thus, equip the damaged cord for regenerative treatments. K2(QL)6K2 (QL6) is a novel SAP introduced by Dong et al.6 In comparison to other peptides, QL6 self-assembles into &#946;-sheets at neutral pH6.14 days after SCI, after the acute stage, SAPs are injected into the center of the lesion and neural precursor cells (NPC) are injected into adjacent dorsal columns. In order to support cell survival, transplantation is combined with continuous subdural administration of growth factors by osmotic micro pumps for 7 days.

Treating cervical spinal cord injury with both self-assembling peptides (SAP) and neural precursor cells (NPC), together with growth factors, is a promising approach to promote regeneration and recovery. A contusion/compression aneurysm clip rat model of cervical SCI and combined treatment involving SAP injection and NPC transplantation is established.

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

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can be used both to investigate the biological responses to injury and to test potential therapies. Contusion or compression injury delivered to the surgically exposed spinal cord are the most widely used models of the pathology. In this report the experimental contusion is performed by using the Infinite Horizon (IH) Impactor device, which allows the creation of a reproducible injury animal model through definition of specific injury parameters. Stem cell transplantation is commonly considered a potentially useful strategy for curing this debilitating condition. Numerous studies have evaluated the effects of transplanting a variety of stem cells. Here we demonstrate an adapted method for spinal cord injury followed by tail vein injection of cells in CD1 mice. In short, we provide procedures for: i) cell labeling with a vital tracer, ii) pre-operative care of mice, iii) execution of a contusive spinal cord injury, and iv) intravenous administration of post mortem neural precursors. This contusion model can be utilized to evaluate the efficacy and safety of stem cell transplantation in a regenerative medicine approach.

Spinal cord injury is a traumatic condition that causes severe morbidity and high mortality. In this work we describe in detail a contusion model of spinal cord injury in mice followed by a transplantation of neural stem cells.

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

Reduced spinal cord blood flow (SCBF) (i.e., ischemia) plays a key role in traumatic spinal cord injury (SCI) pathophysiology and is accordingly an important target for neuroprotective therapies. Although several techniques have been described to assess SCBF, they all have significant limitations. To overcome the latter, we propose the use of real-time contrast enhanced ultrasound imaging (CEU). Here we describe the application of this technique in a rat contusion model of SCI. A jugular catheter is first implanted for the repeated injection of contrast agent, a sodium chloride solution of sulphur hexafluoride encapsulated microbubbles. The spine is then stabilized with a custom-made 3D-frame and the spinal cord dura mater is exposed by a laminectomy at ThIX-ThXII. The ultrasound probe is then positioned at the posterior aspect of the dura mater (coated with ultrasound gel). To assess baseline SCBF, a single intravenous injection (400 &#181;l) of contrast agent is applied to record its passage through the intact spinal cord microvasculature. A weight-drop device is subsequently used to generate a reproducible experimental contusion model of SCI. Contrast agent is re-injected 15 min following the injury to assess post-SCI SCBF changes. CEU allows for real time and in-vivo assessment of SCBF changes following SCI. In the uninjured animal, ultrasound imaging showed uneven blood flow along the intact spinal cord. Furthermore, 15 min post-SCI, there was critical ischemia at the level of the epicenter while SCBF remained preserved in the more remote intact areas. In the regions adjacent to the epicenter (both rostral and caudal), SCBF was significantly reduced. This corresponds to the previously described &#8220;ischemic penumbra zone&#8221;. This tool is of major interest for assessing the effects of therapies aimed at limiting ischemia and the resulting tissue necrosis subsequent to SCI.

Contrast Enhanced Ultrasound imaging is a reliable in-vivo tool for quantifying spinal cord blood flow in an experimental rat spinal cord injury model. This paper contains a comprehensive protocol for application of this technique in association with a contusion model of thoracic spinal cord injury.

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