Name: laminectomy and spinal cord window implantation in the mouse
Uploaded: 2019-10-23T12:30:00
Description: Watch this Scientific Journal Video about Laminectomy and Spinal Cord Window Implantation in the Mouse at JoVE.com

S.E. Lutz is supported by the National Center for Advancing Translational Sciences, National Institutes of Health, under Grant KL2TR002002 and University of Illinois Chicago College of Medicine start-up funds. Simon Alford is supported by RO1 MH084874. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. The authors thank Dritan Agalliu in the Department of Neurology at Columbia University Medical Center for the Tg eGFP:Claudin-5 mice, scientific discussions, and insights into the development of the surgical protocol and imaging applications. The authors thank Sunil P. Gandhi in the Department of Neurobiology and Behavior at University of California, Irvine for designing the first prototype of the stereotactic apparatus and animal temperature controller, discussion of the surgical protocol, and training in two-photon microscopy. The authors also thank Steve Pickens (W. Nuhsbaum, Inc.) for assistance in customizing the surgical stereomicroscope, and Ron Lipinski (Whale Manufacturing) for machining stereotactic parts.

All experiments follow the University of Illinois, Chicago Institutional Animal Care and Use Committee protocols. This is a terminal procedure.&#160;
1. Reagent Preparation
<ol>
	<li>Prepare artificial cerebral spinal fluid (aCSF) to contain 125 mM NaCl, 5 mM KCl, 10 mM Glucose, 10 mM HEPES, 2 mM MgCl2&#183;6H2O, 2 mM CaCl2&#183;2H2O in ddH2O. Sterile filter and freeze in individual-use aliquots. Warm aCSF in a water bath to 39 &#176;C b...

<ol>
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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=A+spinal+cord+window+chamber+model+for+in+vivo+longitudinal+multimodal+optical+and+acoustic+imaging+in+a+murine+model.">A spinal cord window chamber model for in vivo longitudinal multimodal optical and acoustic imaging in a murine model.</a> PLOS ONE. 8 (3), e58081 (2013).</li><li>Helmchen, F., Denk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+tissue+two-photon+microscopy.">Deep tissue two-photon microscopy.</a> Nature Methods. 2 (12), 932-940 (2005).</li><li>Liebner, S., 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=Functional+morphology+of+the+blood-brain+barrier+in+health+and+disease.">Functional morphology of the blood-brain barrier in health and disease.</a> Acta Neuropathologica. 135 (3), 311-336 (2018).</li><li>Shen, L., Weber, C. R., Turner, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tight+junction+protein+complex+undergoes+rapid+and+continuous+molecular+remodeling+at+steady+state.">The tight junction protein complex undergoes rapid and continuous molecular remodeling at steady state.</a> Journal of Cell Biology. 181 (4), 683-695 (2008).</li><li>Knowland, 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=Stepwise+recruitment+of+transcellular+and+paracellular+pathways+underlies+blood-brain+barrier+breakdown+in+stroke.">Stepwise recruitment of transcellular and paracellular pathways underlies blood-brain barrier breakdown in stroke.</a> Neuron. 82, 1-15 (2014).</li><li>Lutz, S. E., 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=Caveolin1+Is+Required+for+Th1+Cell+Infiltration,+but+Not+Tight+Junction+Remodeling,+at+the+Blood-Brain+Barrier+in+Autoimmune+Neuroinflammation.">Caveolin1 Is Required for Th1 Cell Infiltration, but Not Tight Junction Remodeling, at the Blood-Brain Barrier in Autoimmune Neuroinflammation.</a> Cell Reports. 21 (8), 2104-2117 (2017).</li><li>Harrison, M., 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=Vertebral+landmarks+for+the+identification+of+spinal+cord+segments+in+the+mouse.">Vertebral landmarks for the identification of spinal cord segments in the mouse.</a> Neuroimage. 68, 22-29 (2013).</li><li>Cupido, A., Catalin, B., Steffens, H., Kirchhoff, F., Bakota, L., Brandt, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Laser Scanning Microscopy and Quantitative Image Analysis of Neuronal Tissue. , 37-50 (2014).</li><li>Sekiguchi, K. J., 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=Imaging+large-scale+cellular+activity+in+spinal+cord+of+freely+behaving+mice.">Imaging large-scale cellular activity in spinal cord of freely behaving mice.</a> Nature Communications. 7, 11450 (2016).</li><li>Nadrigny, F., Le Meur, K., Schomburg, E. D., Safavi-Abbasi, S., Dibaj, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+laser-scanning+microscopy+for+single+and+repetitive+imaging+of+dorsal+and+lateral+spinal+white+matter+in+vivo.">Two-photon laser-scanning microscopy for single and repetitive imaging of dorsal and lateral spinal white matter in vivo.</a> Physiological Research. 66 (3), 531-537 (2017).</li><li>Adelsperger, A. R., Bigiarelli-Nogas, K. J., Toore, I., Goergen, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+Low-flow+Digital+Anesthesia+System+for+Mice+and+Rats.">Use of a Low-flow Digital Anesthesia System for Mice and Rats.</a> Journal of Visualized Experiments. (115), (2016).</li><li>Damen, F. W., Adelsperger, A. R., Wilson, K. E., Goergen, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+Traditional+and+Integrated+Digital+Anesthetic+Vaporizers.">Comparison of Traditional and Integrated Digital Anesthetic Vaporizers.</a> Journal of the American Association for Laboratory Animal Science. 54 (6), 756-762 (2015).</li><li>Miyamoto, K., 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=Selective+COX-2+inhibitor+celecoxib+prevents+experimental+autoimmune+encephalomyelitis+through+COX-2-independent+pathway.">Selective COX-2 inhibitor celecoxib prevents experimental autoimmune encephalomyelitis through COX-2-independent pathway.</a> Brain. 129 (Pt 8), 1984-1992 (2006).</li><li>Muthian, G., 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=COX-2+inhibitors+modulate+IL-12+signaling+through+JAK-STAT+pathway+leading+to+Th1+response+in+experimental+allergic+encephalomyelitis.">COX-2 inhibitors modulate IL-12 signaling through JAK-STAT pathway leading to Th1 response in experimental allergic encephalomyelitis.</a> Journal of Clinical Immunology. 26 (1), 73-85 (2006).</li></ol>

The method described here allows for stable imaging of the spinal cord in mice through a glass window. This method has been applied to assess BBB remodeling in transgenic eGFP:Claudin5+/- mice that express a fluorescent BBB tight junction protein, but it could be applied equally well for studies of any fluorescent proteins or cells in the spinal cord.
Multiple methods for laminectomy and spinal cord stabilization have been developed. All protocols address stabilizing the spinal cord during ima...

Transgenic animal models expressing fluorescent proteins, when combined with intravital microscopy, provide a powerful platform for addressing biology and pathophysiology. To apply these techniques to the spinal cord, specialized protocols are required to prepare the spinal cord for imaging. One such strategy is to conduct a laminectomy and spinal cord window implantation. The key features of an ideal laminectomy protocol for microscopy include preservation of native tissue structure and function, stability of the imaging field, quick processing time, and reproducibility of results. A particular challenge is to stabilize the imaging field against the motion induced by respiration and heartbeat. Multiple ex vivo and in vivo strategies have been reported to achieve these goals1,2,3,4,5. Most in vivo methods involve clamping the sides of the spinal column2,4 and is often followed by implanting a rigid metal apparatus3,4 for stability during surgery and downstream imaging applications. Clamping the spinal column can potentially compromise blood flow and induce blood-brain barrier (BBB) protein remodeling.
The purpose of this method is to make the intact spinal cord available for optical imaging in the living mouse while minimizing the invasiveness of the protocol and improving outcomes. We describe a single laminectomy and cover-glass implantation procedure paired with a minimally invasive oval plastic 3D-printed backplate that still achieves robust mechanical stability. The backplate is directly adhered to the anterior and posterior vertebral spines with dental cement. The backplate is equipped with lateral extension arms with screw holes that rigidly attach to the microscope stage via a metal arm. This effectively anchors the intact anterior and posterior vertebra to the microscope stage, providing mechanical resistance to the motion artifact that would otherwise be introduced by respiration and heartbeat. The method has been optimized for laminectomy of a single vertebra at thoracic level 12, omitting the clamps utilized in alternative strategies for stability during in vivo imaging. The procedure is rapid, taking approximately 30 min per mouse.
This protocol can be used to study disease mechanisms of the BBB. The BBB is a dynamic microvascular system comprised of endothelial cells, vascular smooth muscle, pericytes, and astrocyte foot processes that provide a highly selective environment for the central nervous system (CNS). Representative data depict the application of this protocol in transgenic mice engineered to express enhanced green fluorescent protein (eGFP):Claudin-5, a BBB tight junction protein. The provided backplate printing files can also be customized for alternative applications.

<table>
	<tbody>
		<tr>
			<td>3D printer</td>
			<td>Raise3D</td>
			<td>Pro2</td>
			<td>For printing backplates</td>
		</tr>
		<tr>
			<td>PLA 3D printing filament</td>
			<td>Inland</td>
			<td>PLA+-175-B</td>
			<td>Black plastic 3D printing material</td>
		</tr>
		<tr>
			<td>3D CAD software</td>
			<td>Dassault Systemes</td>
			<td>Solidworks software</td>
			<td>used to design 3D shapes</td>
		</tr>
		<tr>
			<td>3D printer software</td>
			<td>Raise3D</td>
			<td>Ideamaker software</td>
			<td>software used to interface with the 3D printer</td>
		</tr>
		<tr>
			<td>3D printed oval backplate</td>
			<td>custom</td>
			<td></td>
			<td>Stabilizing imaging field</td>
		</tr>
		<tr>
			<td>Surgical dissecting microscope</td>
			<td>Leica</td>
			<td>M205 C</td>
			<td>Equipped with Leica FusionOptics, Planapo 0.63x M-series objective, and gliding stage</td>
		</tr>
		<tr>
			<td>Microscope camera</td>
			<td>Leica</td>
			<td>MC170</td>
			<td>HD color camera for visualizing surgical field</td>
		</tr>
		<tr>
			<td>Gliding stage</td>
			<td>Leica</td>
			<td>10446301</td>
			<td>The gliding stage is constructed of two metal plates. The base plate is fixed. The upper plate slides on greased interface to allow rotational and linear movement.</td>
		</tr>
		<tr>
			<td>Surgical station and stabilization fork</td>
			<td>Whale Manufactoring</td>
			<td>custom</td>
			<td>Laminectomy</td>
		</tr>
		<tr>
			<td>SomnoSuite low-flow isoflurane delivery unit</td>
			<td>Kent Scientific</td>
			<td>SS-01</td>
			<td>Surgical anesthesia administration with integrated digitial vaporizer</td>
		</tr>
		<tr>
			<td>Stainless steel 1.5 inch mounting post</td>
			<td>ThorLabs</td>
			<td>P50/M</td>
			<td>For mounting surgical station onto optical table for two-photon imaging</td>
		</tr>
		<tr>
			<td>Counterbored Clamping Fork for 1.5&#34; mounting Post</td>
			<td>ThorLabs</td>
			<td>PF175</td>
			<td>For stabilizing surgical station mount onto optical table for two-photon imaging</td>
		</tr>
		<tr>
			<td>Ideal bone microdrill</td>
			<td>Harvard apparatus</td>
			<td>72-6065</td>
			<td>Thinning bone for laminectomy</td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>Fisher Scientific</td>
			<td>15-462-10</td>
			<td>Warming saline</td>
		</tr>
		<tr>
			<td>Cautery gun</td>
			<td>FST</td>
			<td>18010-00</td>
			<td>Cauterizing minor bleeds</td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Benchmark</td>
			<td>BF11222</td>
			<td>1.9&#8221; x 4.5&#8221; silicone heater with 20&#8221; Teflon leads, 10W, 5V</td>
		</tr>
		<tr>
			<td>K type thermocoupled rectal probe</td>
			<td>Physitemp</td>
			<td>RET3</td>
			<td>Measuring mouse body temperature</td>
		</tr>
		<tr>
			<td>petroleum jelly</td>
			<td>Sigma</td>
			<td>8009-03-8</td>
			<td>Lubricating rectal probe</td>
		</tr>
		<tr>
			<td>Feedback-regulated thermal controller</td>
			<td>custom</td>
			<td>NA</td>
			<td>Commercially available alternatives include the Physitemp TCAT series</td>
		</tr>
		<tr>
			<td>PVA Surgical eye spears</td>
			<td>Beaver-visitec international</td>
			<td>40400-8</td>
			<td>Absorbing blood</td>
		</tr>
		<tr>
			<td>Electric trimmer</td>
			<td>Wahl</td>
			<td>41590-0438</td>
			<td>Trimming mouse fur</td>
		</tr>
		<tr>
			<td>Blade, #11</td>
			<td>FST</td>
			<td>14002-14</td>
			<td>Surgical tool</td>
		</tr>
		<tr>
			<td>Forceps, #5</td>
			<td>FST</td>
			<td>11254-20</td>
			<td>Surgical tool</td>
		</tr>
		<tr>
			<td>Forceps, #4</td>
			<td>FST</td>
			<td>14002-14</td>
			<td>Surgical tool</td>
		</tr>
		<tr>
			<td>Titatnium toothed forceps</td>
			<td>WPI</td>
			<td>555047FT</td>
			<td>Surgical tool</td>
		</tr>
		<tr>
			<td>Titanium Iris scissors</td>
			<td>WPI</td>
			<td>555562S</td>
			<td>Surgical tool</td>
		</tr>
		<tr>
			<td>Vetbond tissue adhesive</td>
			<td>3M</td>
			<td>084-1469SB</td>
			<td>Preparing tissue surface for dental acrylic</td>
		</tr>
		<tr>
			<td>Ceramic mixing tray</td>
			<td>Jack Richeson</td>
			<td>420716</td>
			<td>Mixing dental acrylic agent with accelerant</td>
		</tr>
		<tr>
			<td>Orthojet dental acrylic</td>
			<td>Lang Dental</td>
			<td>1520BLK, 1503BLK</td>
			<td>Permanently bonding backplate to tissue</td>
		</tr>
		<tr>
			<td>Small round cover glass, #1 thickness, 3 mm</td>
			<td>Harvard apparatus</td>
			<td>64-0720</td>
			<td>optical window</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher Scientific</td>
			<td>7647-14-5</td>
			<td>For aCSF</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Fisher Scientific</td>
			<td>7447-40-7</td>
			<td>For aCSF</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Fisher Scientific</td>
			<td>50-99-7</td>
			<td>For aCSF</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>7365-45-9</td>
			<td>For aCSF</td>
		</tr>
		<tr>
			<td>MgCl2&#183;6H2O</td>
			<td>Fisher Scientific</td>
			<td>7791-18-6</td>
			<td>For aCSF</td>
		</tr>
		<tr>
			<td>CaCl2&#183;2H2O</td>
			<td>Fisher Scientific</td>
			<td>10035-04-8</td>
			<td>For aCSF</td>
		</tr>
		<tr>
			<td>Carprofen</td>
			<td>Rimadyl</td>
			<td>QM01AE91</td>
			<td>Analgesia</td>
		</tr>
		<tr>
			<td>Bacteriostatic water</td>
			<td>Henry Schein</td>
			<td>2587428</td>
			<td>Diluent for carprofen</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Henry Schein</td>
			<td>11695-6776-2</td>
			<td>Anesthesia</td>
		</tr>
		<tr>
			<td>Lactated ringer solution</td>
			<td>Baxter</td>
			<td>0338-0117-04</td>
			<td>Hydration for mouse</td>
		</tr>
		<tr>
			<td>Agarose High EEO</td>
			<td>Sigma</td>
			<td>A9793</td>
			<td>gel point 34-37 degrees C</td>
		</tr>
		<tr>
			<td>Opthalmic lubricating ointment</td>
			<td>Akwa Tears</td>
			<td>68788-0697</td>
			<td>Prevent corneal drying</td>
		</tr>
		<tr>
			<td>MOM Two-Photon Microscope</td>
			<td>Sutter</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

laminectomy and spinal cord window implantation in the mouse

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated digital vaporizer is utilized to achieve a stable plane of anesthesia at a low-flow rate of isoflurane. A single vertebral spine is removed, and a commercially available cover-glass is overlaid on a thin agarose bed. A 3D-printed plastic backplate is then affixed to the adjacent vertebral spines using tissue adhesive and dental cement. A stabilization platform is used to reduce motion artifact from respiration and heartbeat. This rapid and clamp-free method is well-suited for acute multi-photon fluorescence microscopy. Representative data are included for an application of this technique to two-photon microscopy of the spinal cord vasculature in transgenic mice expressing eGFP:Claudin-5 &#8212; a tight junction protein.

Implanted glass windows and intravital two-photon microscopy provides a useful tool for assessing dynamic changes in CNS proteins. The functional integrity of the BBB is influenced by the expression, subcellular localization, and turnover rates of tight junction proteins7. Previous studies have demonstrated that tight junction proteins undergo rapid and dynamic remodeling at steady state8. The currently described laminectomy and glass window...

Watch this Scientific Journal Video about Laminectomy and Spinal Cord Window Implantation in the Mouse at JoVE.com

Laminectomy and Spinal Cord Window Implantation in the Mouse

This protocol describes implantation of a glass window onto the spinal cord of a mouse to facilitate visualization by intravital microscopy.

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated ...

This method enables the examination of protein dynamics in vivo within the intact central nervous system to address questions in neuroscience and immunology. The main advantages of this procedure are an excellent motion stability for image acquisition, brief surgical time, reduced operator exposure to gaseous anesthesia, and inexpensive customizable backplates. Visual demonstration of this method is critical as it is difficult to remove the bone without damaging the closely underlying nervous system tissue during the laminectomy steps of the procedure.Before beginning the procedure, use 3D computer aided design software to create a model to the indicated dimensions. So we've got an object, which is the three dimensional object, that that's what we can start with. What I can then do is we can put settings on that for the printing version.So if I open Edit this, go into Advanced, open a window that enables me to look at all of these settings. Set the nozzle temperature to 205 degree Celsius, the bed temperature to 45 degrees Celsius, and the printing speed to 45 millimeters per second on a 3D printer. Use a 0.4 millimeter hot ent nozzle and a 0.2 millimeter layer height to print the back plates.Then assess the printed back plates visually for their structural integrity. Gross structural failures indicate printing defects. When the back plates are ready, place the head of an eight to 12 week old anesthetized mouse on a heat pad between the ear bars of a surgical restrainer and apply ointment to the animal's eyes.After confirming a lack of response to toe pinch, use a number 11 blade to make a 1.5 centimeter rostral to caudal midline incision over the lower thoracic upper lumbar region and separate the skin from the peritoneum. Use forceps to peel back any remaining transparent connective tissue under the skin to expose the superficial musculature. Displace the muscles with a foam surgical spear along with the remaining deeper muscular of the target vertebra.To create a seat for the back plate, clear the muscle from the posterior aspect of thoracic 11 and the anterior aspect of thoracic 13. Using forceps to remove the remaining muscle from the tendons as necessary. When all of the muscle has been removed, carefully cut the tendons with forceps until sufficient space for visualizing and manipulating the spinal cord is achieved.Confirm that the dura mater of the intervertebral space, the semitransparent laminar bone, the central superficial blood vessel under the bone, and the anterior radiating artery are clearly visible. Wet the region with warm artificial cerebral spinal fluid and use a micro-drill to thin the laminar bone using straight strokes parallel to the long axis of the spinal cord. Gently grasping the superficial spinous process with forceps, lift the vertebra.The bone should lift away easily. Use number four forceps to clear away any bone shards, controlling any bleeding with a surgical spear and gentle steady pressure as necessary. Then rinse the tissue with warm artificial cerebral spinal fluid.For cover glass implantation, gently apply a three millimeter borosilicate cover glass to the exposed cord. Use a small spatula to apply 39 degree Celsius warmed 2%agarose to the edge of the glass and allow capillary action to draw the agarose under the cover glass surface. Apply tissue adhesive to the exposed bony articular processes of the intact adjacent vertebra at the thoracic level 11 and 13 vertebral spines.Apply additional tissue adhesive in a ring around the laminectomy site over the adjacent tendon and the transverse process. Next, use a new spatula to apply dental cement mixed with accelerant onto the tissue adhesive layer and place a back plate onto the surgical field centered over the cover glass window. Applying multiple thin layers of dental cement results in a stronger back plate adhesion, but be sure to place the back plate quickly before the dental cement starts to dry.After allowing the dental cement to cure for 10 minutes, fill in the interior base and underside of the back place with additional adhesive. Applying the dental cement in thin layers can help reduce the risk of cement spreading to the cover glass and agarose, which can compromise the entire protocol. When the dental cement has dried, apply a forked back plate holder to the appropriate position over the window and screw the back plate into the back plate holder.Then apply saline to the back plate to test for leakage. The animal and the surgical platform can then be transferred to the optical table of a two photon microscope for imaging through the cover glass window. In this representative experiment, EGFP positive claudin-5 was visualized within the fluorescently labeled tight junctions throughout a vascular plexus.The clear delineation of the tight junction structures in the Z projection indicates that minimal XY image displacement is produced after a successful laminectomy, window placement, and back plate implantation. While attempting this procedure it's important to remember that this microsurgery is difficult and may take time to master. However once mastered, this surgery can be performed in approximately 30 minutes.Following this procedure, other methods, like two photon microscopy and in vivo imaging, can be performed to answer additional questions about neurovascular remodeling and other disease processes involving the spinal cord.

laminectomy-and-spinal-cord-window-implantation-in-the-mouse

Research

JoVE Journal

Neuroscience

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

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.

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

Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central nervous system. We demonstrate a stereotaxic injection technique that allows targeted gene expression or silencing in the dorsal horn of the mouse spinal cord. The surgical procedure is brief. It requires laminectomy of a single vertebra, providing for quick recovery of the animal and unimpaired motility of the spine. Controlled injection of a small vector suspension volume at low speed and use of a microsyringe with beveled glass cannula minimize the tissue lesion. The local immune response to the vector depends on the intrinsic properties of the virus employed; in our experience, it is minor and short-lived when a recombinant adeno-associated virus is used. A reporter gene such as enhanced green fluorescent protein facilitates monitoring spatial distribution of the vector, and the efficacy and cellular specificity of the transfection.

Viral vectors allow for targeted gene manipulation. We demonstrate a method for conditional gene expression or ablation in the mouse spinal cord, using stereotaxic injection of a viral vector into the dorsal horn, a prominent site of synaptic contact between primary somatosensory afferents and neurons of the central nervous system.

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central ...

Spinal Cord Transection in the Larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6&#160;weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2&#160;days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.

After spinal transection, adult zebrafish have functional recovery by six weeks post-injury. To take advantage of larval transparency and faster recovery, we present a method for transecting the larval spinal cord. After transection, we observe sensory recovery beginning at 2&#160;days post-injury, and C-bend movement by 3 days post-injury.

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability ...

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by repeated, time-lapse in vivo imaging in live rodents. These studies were made possible by straightforward surgical procedures, which enabled optical access for a prolonged period of time without repeat surgical procedures. In contrast, analogous studies of the spinal cord have been previously limited to only a few imaging sessions, each of which required an invasive surgery. As previously described, we have developed a spinal chamber that enables continuous optical access for upwards of 8 weeks, preserves mechanical stability of the spinal column, is easily stabilized externally during imaging, and requires only a single surgery. Here, the design of the spinal chamber with its associated surgical implements is reviewed and the surgical procedure is demonstrated in detail. Briefly, this video will demonstrate the preparation of the surgical area and mouse for surgery, exposure of the spinal vertebra and appropriate tissue debridement, the delivery of the implant and vertebral clamping, the completion of the chamber, the removal of the delivery system, sealing of the skin, and finally, post-operative care. The procedure for chronic in vivo imaging using nonlinear microscopy will also be demonstrated. Finally, outcomes, limitations, typical variability, and a guide for troubleshooting are discussed.

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

In this video, we describe a procedure for implanting a chronic optical imaging chamber over the dorsal spinal cord of a live mouse. The chamber, surgical procedure, and chronic imaging are reviewed and demonstrated.

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by ...

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.

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 ...

Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal cord of the majority of these fiber systems have not been thoroughly investigated in any species. Serotonergic fibers, which project to the spinal cord, have been studied in rats and opossums on histological sections and their functional significance has been deduced based on their fiber termination pattern in the spinal cord. With the development of CLARITY and CUBIC techniques, it is possible to investigate this fiber system and its distribution in the spinal cord, which is likely to reveal previously unknown features of serotonergic supraspinal pathways. Here, we provide a detailed protocol for imaging the serotonergic fibers in the mouse spinal cord using the combined CLARITY and CUBIC techniques. The method involves perfusion of a mouse with a hydrogel solution and clarification of the tissue with a combination of clearing reagents. Spinal cord tissue was cleared in just under two weeks, and the subsequent immunofluorescent staining against serotonin was completed in less than ten days. With a multi-photon fluorescent microscope, the tissue was scanned and a 3D image was reconstructed using Osirix software.

Supraspinal projections are important for pain perception and other behaviors, and serotonergic fibers are one of these fiber systems. The present study focused on the application of the combined CLARITY/CUBIC protocol to the mouse spinal cord in order to investigate the termination of these serotonergic fibers.

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to intracranial delivery, intra-arterial administration of NSCs is less invasive and produces a more diffuse distribution of NSCs within the brain parenchyma. Further, intra-arterial delivery allows the first-pass effect in the brain circulation, lessening the potential for trapping of cells in peripheral organs, such as liver and spleen, a complication associated with peripheral injections. Here, we detail the methodology, in both mice and rats, for delivery of NSCs through the common carotid artery (mouse) or external carotid artery (rat) to the ipsilateral hemisphere after an ischemic stroke. Using GFP-labeled NSCs, we illustrate the widespread distribution achieved throughout the rodent ipsilateral hemisphere at 1 d, 1 week and 4 weeks after postischemic delivery, with a higher density in or near the ischemic injury site. In addition to long-term survival, we show evidence of differentiation of GFP-labeled cells at 4 weeks. The intra-arterial delivery approach described here for NSCs can also be used for administration of therapeutic compounds, and thus has broad applicability to varied CNS injury and disease models across multiple species.

A method for delivering neural stem cells, adaptable for injecting solutions or suspensions, through the common carotid artery (mouse) or external carotid artery (rat) after ischemic stroke is reported. Injected cells are distributed broadly throughout the brain parenchyma and can be detected up to 30 d after delivery.

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...

Extraction and Enzymatic Purification of Microglia from the Mouse Spinal Cord

Rapid and Efficient Enrichment of Mouse Spinal Cord Microglia

The vertebral column defines a vertebrate and shapes the spinal canal, a cavity that encloses and safeguards the spinal cord. Proper development and function of the mammalian central nervous system rely significantly on the activity of resident macrophages known as microglia. Microglia display heterogeneity and multifunctionality, enabling distinct gene expression and behavior within the spinal cord and brain. Numerous studies have explored cerebral microglia function, detailing purification methods extensively. However, the purification of microglia from the spinal cord in mice lacks a comprehensive description. In contrast, the utilization of a highly purified collagenase, as opposed to an unrefined extract, lacks reporting within central nervous system tissues. In this study, the vertebral column and spinal cord were excised from 8-10 week-old C57BL/6 mice. Subsequent digestion employed a highly purified collagenase, and microglia purification utilized a density gradient. Cells underwent staining for flow cytometry, assessing viability and purity through CD11b and CD45 staining. Results yielded an average viability of 80% and a mean purity of 95%. In conclusion, manipulation of mouse microglia involved digestion with a highly purified collagenase, followed by a density gradient. This approach effectively produced substantial spinal cord microglia populations.

Author Spotlight: Microglia Research on Spinal Cord Heterogeneity and Purification 

Microglia are regarded as some of the most versatile cells in the body, capable of morphological and functional adaptation. Their heterogeneity and multifunctionality enable the maintenance of brain homeostasis, while also being linked to various neurological pathologies. Here, a technique for purifying spinal cord microglia is described.

The vertebral column defines a vertebrate and shapes the spinal canal, a cavity that encloses and safeguards the spinal cord. Proper development and ...