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 - using a standard microscope slide, tape a short piece of ruler paper (~2 cm) to the bottom of the slide so that you can see the ruler through the glass. Next, place a piece of tape along the edge of the 0 mm mark of the ruler to act as a fixture for the cover glass in the next step.</li>
		<li>Using the modified drill burr, slide-A, and an unmodified microscope slide (slide-B), position a piece of cover glass (no. 1.5 thickness) to the 0 mm mark on slide-A. Use slide-B to hold the cover glass in place and as a jig for the intended cut line. Cut the cover glass into a long rectangle of approximately 5 x 22 mm. Rotate the cut glass 90&#176; and cut the rectangle into smaller rectangle of ~2 x 5 mm.</li>
	</ol>
	</li>
	<li>Staple modifications - two modified staples will be implanted to support and fix the window chamber to the vertebrae (see section 2.7-8).
	<ol>
		<li>Using a standard small staple (crown = 8 mm; leg = 4 mm), bend the middle of the crown ~90&#176; so that the tips of the legs are nearly touching.</li>
		<li>Bend the tips of the legs outwards ~45&#176; at the half-way point, so that the distal half of the legs are parallel.</li>
		<li>Bend the distal half of the legs ~30&#176; upward relative to the plane of the staple. Place the modified staples into ethanol prior to surgery for sterilization. 
		Note: Staples can also be sterilized using ethylene oxide or autoclaved.</li>
	</ol>
	</li>
	<li>Paperclip modifications - prepare one modified paperclip to be implanted as part of the chamber (see sections 2.9-10) and to act as a holding clip for surgery and imaging.
	<ol>
		<li>Using a standard 25 mm paperclip, remove the small bend leaving an elongated U-shape.</li>
		<li>Using pliers or needle-drivers, reduce the diameter of the bend to ~2 mm while keeping the tips parallel.</li>
		<li>Bend the tips outward ~90&#176; at about 8-10 mm from the bend. Make any adjustments necessary so that it lies flat.</li>
	</ol>
	</li>
</ol>
2. Window Implantation
Note: All surgical and animal care procedures were conducted in accordance with the European Communities Council Directive and were approved by the National Animal Studies Committee of France (authorization no. 13,300).
<ol>
	<li>Anesthetize the mouse (Ket/Xyl; induction dose: 120 mg/kg, 12 mg/kg; supplemental dose, 40 mg/kg, 4 mg/kg), apply eye ointment to the eyes.</li>
	<li>Shave its back approximately ~2 cm wide by ~5 cm long, centered on the thoracic hump, and clean the exposed skin using iodine soap followed by iodine solution. Clean gloves by spraying with 90% ethanol.&#160;</li>
	<li>Using tissue scissors cut the dorsal skin longitudinally over vertebrae T11 to L1 (~2 cm). Using a scalpel, make bilateral longitudinal incisions between the spinous and transverse processes from T11 to L1.&#160;Note: If gloves or instruments touch the fur or skin of the animal, reclean the gloves or instrument using a cold sterilizer such as 90% ethanol or Germ-X.</li>
	<li>Suspend the mouse in the spinal fork stereotaxic - Begin by clamping the top right spinal fork into position. Grasp T11 using Adson forceps and position the T11 transverse process over the fixed spinal fork. While holding the vertebra in place, fasten the contralateral spinal fork under the contralateral transverse process.</li>
	<li>Using the same procedure as step 2.4, position L1 within the spinal fork.</li>
	<li>Using a scalpel (no. 23), gently scrape away the muscles between the spinous and transverse processes from T11 to L1 so that the laminae are clearly visible. For a better view of the pedicles of T11, cut the tendon projecting rostrally from the tip of the transverse process.</li>
	<li>Rinse the modified staples with PBS, and insert the tips of the staples lateral to the vertebral pedicles of T11 and L1 adjusting the distance between the tips so that they are lightly touching the pedicles. 
	Note: Use only PBS containing dexamethasone (0.1%), penicillin (1,000 U/ml), and streptomycin (1 mg/ml), to reduce postsurgical inflammation and to improve window clarity.</li>
	<li>Glue the staples in place with a small drop of cyanoacrylate at each insertion point and remove the mouse from the spinal forks.</li>
	<li>Apply a thin layer of cyanoacrylate to the cut edges of the skin and exposed muscles around the cleared vertebrae being careful to not glue the vertebrae. Bend the tips of the modified paperclip so that they enter under the protruding sections of the implanted staples and rest parallel to the long axis of the isolated vertebral column and leave in this position.</li>
	<li>Starting at the skin-muscle interface, apply several layers of dental cement to cover the entire opening except the exposed vertebrae, being sure to implant the paper clip securely within the dental cement.</li>
	<li>Once the dental cement is cured (5-15 min), attach the mouse to the &#39;clip holder&#39; by the exposed portion of the implanted paper clip. Using forceps, remove the muscle and other soft tissue between the spinous processes and laminae of the exposed vertebrae. 
	Note: Any device that can securely grasp the exposed portion of the paperclip without causing stress to the clip/chamber, and can be maneuvered to reorient the mouse throughout surgery can be used as a &#39;clip holder&#39;.</li>
	<li>Using the bone removal drill, thin the laminae of T12 and T13 bilaterally. To remove the dorsal bone, grasp the spinous process with blunt forceps and lift taking care to apply no downward force towards the spinal cord. If the bone does not easily come away, continue to thin the laminae until the spinous process can be easily removed.</li>
	<li>Using the bone removal drill, continue to shape the lateral edges of the vertebrae to form a support structure for the window. Gently remove all of the small bone fragments touching the dura mater using blunt forceps. Be sure to maintain the humidity of the dura mater using PBS.</li>
	<li>Choose one of the prepared windows that is slightly wider than the laminectomy opening and test how well it rests on the exposed bone. Ideally, the window will rest bilaterally on both vertebrae so that the middle of the window touches the dura mater (Figure&#160;1a). The window should not, however, compress the spinal cord even when pushing the window towards the spinal cord. If the window is unbalanced or rests above the dura, repeat step 2.13.</li>
	<li>Once satisfied with the laminectomy, place a small piece of autoclaved tissue-paper soaked in PBS onto the spinal cord to prevent drying of the dura and set the mouse aside.</li>
	<li>Cleaning the window - place the window in a solution of window cleaner for several seconds. Using a piece of microscope lens tissue, wipe the window dry. With the surgical microscope verify that the window is clean, and repeat the cleaning process if not. Place the window in a clean area with easy access for rapid retrieval and placement of the window in step 2.19.</li>
	<li>Retrieve the mouse and remove the tissue paper. Gently remove any debris from the dura using blunt forceps and allow the dura to dry until slightly tacky.</li>
	<li>Quickly fill the silicone (Kwik-sil) mixer tube and extrude silicone onto a clean slide until there are no bubbles emerging from the tip. Immediately administer a small bead of silicone along the midline of the exposed spinal cord.</li>
	<li>Retrieve the cleaned windows using blunt forceps and immediately place over the silicone. Using the blunt forceps, apply a small amount of pressure to the window to spread the silicone to the edges of the window and hold in place for ~1 min. 
	Note: Prior to mixing, silicone must be stored below 27 &#176;C otherwise the cure-time can increase significantly and it will not adhere to the window or dura mater leading to reduced optical clarity.</li>
	<li>Slowly release pressure from the window. If the window does not move, proceed immediately to step 2.21. If, however, the window rises when the pressure is released (i.e.&#160;the vertebrae move), proceed to step 2.21 while maintaining pressure on the window to hold it in place.</li>
	<li>Apply cyanoacrylate rostral and caudal of the window. If the cyanoacrylate does not cover the rostral and caudal edges of the window, use a toothpick to guide some of the liquid cyanoacrylate over these edges. Next, guide the glue along the lateral edges of the window (Figure&#160;1a) to cover all of the surrounding muscles. Note: The cyanoacrylate should cover the surface of the outer edges of the window. Too much cyanoacrylate can compromise optical clarity, not enough cyanoacrylate will result in air bubbles between the silicone and window.</li>
	<li>To protect the cyanoacrylate and increase structural integrity of the chamber, apply dental cement to all regions covered with cyanoacrylate (Figure 1a). As with the cyanoacrylate, start at the rostral and caudal edges and use a small wood stick to guide the dental cement along the edges of the window.</li>
</ol>
3. Postoperative Care, EAE Induction, and Window Maintenance
<ol>
	<li>Inject Dexamethasone (0.2 mg/kg) and Rimadyl (5 mg/kg) s.q. over the pelvic regions. Transfer the mouse to a warm cage (~26 &#176;C) with nesting material. Mice should be fully mobile, exploring their cage, and grooming after the effects of the anesthetic have fully subsided (usually 2-3 hr). House animals individually and make sure they have easy access to food and water for the first 1-2 days.&#160;Once the mouse begins to recover from anesthetic, inject buprenorphine (0.05 mg/kg) s.q. Following full recovery from anesthetic, the mouse should show no signs of pain.&#160;Note: Once fully recovered from anesthetic, any signs of pain or abnormal motor activity indicate that the window was not implanted correctly.</li>
	<li>Inject Dexamethasone(0.2 mg/kg) and Rimadyl (5 mg/kg) every 2 d for the first 10 d after surgery (Figure&#160;1a). To inject mice with windows, lightly sedate the animal with isoflurane in an induction chamber (~1.75% isoflurane in air for 1-2 min). Remove the mouse from the chamber and immediately inject the drugs. The mouse should be fully awake in &#60;1 min. 
	Note: Do not scruff mice with implanted windows since this can cause separation of the skin from the implant.&#160;It is possible to inject mice i.p. without sedation by allowing them to grab a cage lid with their forepaws and gently raising their hindlimbs. However, in this position the mouse will sometimes pull away causing stress to the implanted window. This can lead to bleeding between the window and spinal cord, thus compromising optical clarity.</li>
	<li>EAE can be induced at 2-3 weeks after the cessation of anti-inflammatory treatment (~3-4 weeks after surgery). Lightly sedate the mouse as described in step 3.2. Remove the mouse from the induction chamber and maintain the sedation with an isoflurane mask placed over the nose.</li>
	<li>Inject MOG35-55 peptide (75 &#181;g in Freund&#39;s adjuvant containing 800 &#181;g of mycobacterium tuberculosis) into 3 different s.q. locations (bilaterally over the femur, and at the base of the tail), and pertussis toxin (400 ng) i.p.14,15 
	Note: Mice with EAE require supplemental care compared to mice with windows only. Weigh EAE mice every day and be sure that they have easy access to food and water at all times. Supplement with a 3% agarose jelly containing to maintain animal hydration and weight. Glucose (1-3%) can also be added to the jelly for additional calories.</li>
	<li>Two days after EAE induction, repeat the sedation protocol outlined in step 3.2 and inject a second dose of pertussis toxin (400 ng) i.p.</li>
	<li>To image the spinal cord, first sedate the animal as described in step 3.2, and lightly anesthetize with ketamine/xylazine (100 mg/kg; 10 mg/kg) i.p. This will provide ~ 1 hr of imaging.</li>
	<li>For imaging sessions lasting &#60;2 hr, light anesthesia can be maintained with supplementary injections of ketamine/xylazine (50 mg/kg; 5 mg/kg) every 45-60 min. Alternatively, for longer imaging sessions, mice can be continuously sedated with isoflurane (0.75-1.25% isoflurane in humidified air). 
	Note: windows can be cleaned under a surgical microscope by gently wiping away debris with the corner of a tissue or a cotton-tipped swab. Do not use alcohol to clean the window because it can degrade some types of dental cement and it can cool the spinal cord.</li>
	<li>Once lightly anesthetized, the mouse is attached firmly to a clip holder by the exposed portion of the implanted paperclip (Figure&#160;1b).</li>
	<li>The spinal cord is aligned perpendicular to the objective lens by adjusting the positioning of the clip within the holder, adjusting the position of the holder, and adjusting the position of the stage. The dorsal surface of the spinal cord is rapidly imaged, and the dorsal vasculature is used to determine the orientation of the spinal cord.</li>
</ol>

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The authors declare that they have no competing financial interests.

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

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

<table><tbody><tr><td>Bone removal drill</td><td>World Precision Instruments</td><td>IDEAL MICRO-DRILL</td><td>Also sold by Harvard Apparatus.&lt;br/&gt; Any high quality surgical bone drill will suffice.</td></tr><tr><td>Drill burr (#1/4 or 1/2 Carbide Round Burr)</td><td>World Precision Instruments</td><td>501860 (#1/4)&lt;br/&gt; 501860 (#1/2)</td><td>Also sold by Harvard Apparatus</td></tr><tr><td>Staples</td><td>Lyreco</td><td>5.002.567</td><td>Crown = 8 mm&lt;br/&gt; Leg = 4 mm</td></tr><tr><td>Paperclip</td><td></td><td></td><td>Any standard 25 mm paperclip</td></tr><tr><td>Needle drivers</td><td>Fine Science Tools</td><td>12003-15</td><td></td></tr><tr><td>Tissue scissors</td><td>Fine Science Tools</td><td>14028-10</td><td></td></tr><tr><td>Adson forceps</td><td>Fine Science Tools</td><td>11027-12</td><td>1 x 2 teeth</td></tr><tr><td>Blunt forceps</td><td>Fine Science Tools</td><td>11293-00</td><td></td></tr><tr><td>Spinal forks</td><td>Harvard Apparatus</td><td>724813</td><td></td></tr><tr><td>Dental cement</td><td>GACD</td><td>12-565 &amp;amp; 12-568</td><td></td></tr><tr><td>Silicone elastomer</td><td>World Precision Instruments</td><td>KWIK-SIL</td><td>Also avaiable through Coherent Scientific and Fisher Scientific</td></tr><tr><td>Cyanoacrylate</td><td>Eleco-EFD</td><td>Cyanolit 201</td><td></td></tr><tr><td>Microscope</td><td>Zeiss</td><td>7MP</td><td>Our microscope is in an upright configuration and is equipped with 5 NDD detectors.</td></tr><tr><td>Objective lens</td><td>Zeiss</td><td>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.

Implantação de vidro Medular do Windows em camundongos adultos com Experimental Auto-imune Encephalomyelitis

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

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Research

JoVE Journal

Medicine

7.4K visualizações.  Aix Marseille University. We describe a method for implanting and maintaining glass windows over the exposed spinal cords of adult mice for studying experimental autoimmune encephalomyelitis. These windows provide chronic optical access to the spinal cord for monitoring cell dynamics at the subcellular level in live animals using two-photon microscopy.

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

Surgical Transplantation of Mouse Neural Stem Cells into the Spinal Cords of Mice Infected with Neurotropic Mouse Hepatitis Virus

Mice infected with the neurotropic JHM strain of mouse hepatitis virus (MHV) develop pathological and clinical outcomes similar to patients with the demyelinating disease Multiple Sclerosis (MS). We have shown that transplantation of NSCs into the spinal cords of sick mice results in a significant improvement in both remyelination and in clinical outcome. Cell replacement therapies for the treatment of chronic neurologic diseases are now a reality and in vivo models are vital in understanding the interactions between the engrafted cells and host tissue microenvironment. This presentation provides an adapted method for transplanting cells into the spinal cord of JHMV-infected mice. In brief, we provide a procedure for i) preparation of NSCs prior to transplant, ii) pre-operative care of mice, iii) exposure of the spinal cord via laminectomy, iv) stereotactic injection of NSCs, and iv) post-operative care.

The transplantation of mouse neural stem cells (NSCs) into the spinal cords of mice with established demyelination is detailed. The preparation of NSCs, the laminectomy of thoracic vertebra 9 (T9), and transplantation of NSCs is outlined along with the pre- and post-operative care of the mice.

Transplante de Células-Tronco cirúrgica do rato Neural na medula espinhal de camundongos infectados com o vírus da Hepatite neurotrófico rato

Mice infected with the neurotropic JHM strain of mouse hepatitis virus (MHV) develop pathological and clinical outcomes similar to patients with the ...

Neurociência

Systemic Injection of Neural Stem/Progenitor Cells in Mice with Chronic EAE

Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative neurology. This directive has arisen from the extensive evidence that brain repair is achieved after focal or systemic NPC transplantation in several preclinical models of neurological diseases.
These experimental data have identified the cell delivery route as one of the main hurdles of restorative stem cell therapies for brain diseases that requires urgent assessment. Intraparenchymal stem cell grafting represents a logical approach to those pathologies characterized by isolated and accessible brain lesions such as spinal cord injuries and Parkinson's disease. Unfortunately, this principle is poorly applicable to conditions characterized by a multifocal, inflammatory and disseminated (both in time and space) nature, including multiple sclerosis (MS). As such, brain targeting by systemic NPC delivery has become a low invasive and therapeutically efficacious protocol to deliver cells to the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system (CNS).
This alternative method of cell delivery relies on the NPC pathotropism, specifically their innate capacity to (i) sense the environment via functional cell adhesion molecules and inflammatory cytokine and chemokine receptors; (ii) cross the leaking anatomical barriers after intravenous (i.v.) or intracerebroventricular (i.c.v.) injection; (iii) accumulate at the level of multiple perivascular site(s) of inflammatory brain and spinal cord damage; and (i.v.) exert remarkable tissue trophic and immune regulatory effects onto different host target cells in vivo.
Here we describe the methods that we have developed for the i.v. and i.c.v. delivery of syngeneic NPCs in mice with experimental autoimmune encephalomyelitis (EAE), as model of chronic CNS inflammatory demyelination, and envisage the systemic stem cell delivery as a valuable technique for the selective targeting of the inflamed brain in regenerative neurology.

The transplantation of neural stem/progenitor cells (NPCs) holds great promises in regenerative neurology. The systemic delivery of NPCs has turned into effective, low invasive, and therapeutically very efficacious protocol to deliver stem cells in the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system.

Injeção sistêmica de células-tronco neurais / progenitoras em ratos com EAE crónica

Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative ...

Imunologia e Infecção

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.

Um Processo de Implantação de uma Câmara Spinal para Longitudinal<em&gt; In Vivo</em&gt; Criação de Imagens do Cordão do rato Spinal

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

<meta charset="utf8"></head><body>Desenvolvendo um modelo de camundongo eficiente e preciso para glioma da medula espinhal

Surgical Transplantation of Tumor Cells into the Spinal Cord of Mice

Spinal cord gliomas are commonly malignant tumors of the spinal cord, leading to a high rate of disability. However, uniform treatment guidelines and comprehensive data on spinal cord gliomas remain limited due to the lack of suitable preclinical animal models. Developing a simple and reproducible animal model has become essential for advancing basic and translational research. A murine model is ideal, as the murine spinal cord shares structural similarities with the human spinal cord. This protocol describes the generation of a reproducible murine model of spinal cord glioma by directly injecting tumor cells into the intervertebral space using the spinous process of the seventh cervical vertebra as a guide. Compared to other methods, this approach is more effective and convenient, involving a smaller incision, reduced invasiveness and blood loss, faster recovery, and more stable tumor formation. This model is expected to advance the understanding of disease mechanisms, optimize surgical strategies, and support the development of therapeutic drugs for spinal cord gliomas.

<meta charset="utf8"></head><body>Transplante cirúrgico de células tumorais na medula espinhal de camundongos

The present protocol describes the development of a reproducible murine model of spinal cord glioma by injecting tumor cells into the intervertebral space, offering a more effective and less invasive approach for research and therapeutic development.

Transplante cirúrgico de células tumorais na medula espinhal de camundongos

Spinal cord gliomas are commonly malignant tumors of the spinal cord, leading to a high rate of disability. However, uniform treatment guidelines and ...

Pesquisa do câncer

Visualizing Impairment of the Endothelial and Glial Barriers of the Neurovascular Unit during Experimental Autoimmune Encephalomyelitis In Vivo

The neurovascular unit (NVU) is composed of microvascular endothelial cells forming the blood-brain barrier (BBB), an endothelial basement membrane with embedded pericytes, and the glia limitans composed by the parenchymal basement membrane and astrocytic end-feed embracing the abluminal aspect of central nervous system (CNS) microvessels. In addition to maintaining CNS homeostasis the NVU controls immune cell trafficking into the CNS. During immunosurveillance of the CNS low numbers of activated lymphocytes can cross the endothelial barrier without causing BBB dysfunction or clinical disease. In contrast, during neuroinflammation such as in multiple sclerosis or its animal model experimental autoimmune encephalomyelitis (EAE) a large number of immune cells can cross the BBB and subsequently the glia limitans eventually reaching the CNS parenchyma leading to clinical disease. 
Immune cell migration into the CNS parenchyma is thus a two-step process that involves a sequential migration across the endothelial and glial barrier of the NVU employing distinct molecular mechanisms. If following their passage across the endothelial barrier, T cells encounter their cognate antigen on perivascular antigen-presenting cells their local reactivation will initiate subsequent mechanisms leading to the focal activation of gelatinases, which will enable the T cells to cross the glial barrier and enter the CNS parenchyma. Thus, assessing both, BBB permeability and MMP activity in spatial correlation to immune cell accumulation in the CNS during EAE allows to specify loss of integrity of the endothelial and glial barriers of the NVU. 
We here show how to induce EAE in C57BL/6 mice by active immunization and how to subsequently analyze BBB permeability in vivo using a combination of exogenous fluorescent tracers. We further show, how to visualize and localize gelatinase activity in EAE brains by in situ zymogaphy coupled to immunofluorescent stainings of BBB basement membranes and CD45+ invading immune cells.

Here, we present protocols to investigate impairment of the neurovascular unit during experimental autoimmune encephalomyelitis in vivo. We specifically address how to determine blood-brain barrier permeability and gelatinase activity involved in leukocyte migration across the glia limitans.&#160;

Visualizando o comprometimento das barreiras endoteliais e Glial da unidade Neurovascular durante encefalomielite auto-imune Experimental In Vivo

The neurovascular unit (NVU) is composed of microvascular endothelial cells forming the blood-brain barrier (BBB), an endothelial basement membrane with ...

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.

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

Implante de janela de laminectomia e medula espinhal no 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 ...

Implantation of a Cranial Window for Repeated In Vivo Imaging in Awake Mice

To fully understand the cellular physiology of neurons and glia in behaving animals, it is necessary to visualize their morphology and record their activity in vivo in behaving mice. This paper describes a method for the implantation of a chronic cranial window to allow for the longitudinal imaging of brain cells in awake, head-restrained mice. In combination with genetic strategies and viral injections, it is possible to label specific cells and regions of interest with structural or physiological markers. This protocol demonstrates how to combine viral injections to label neurons in the vicinity of GCaMP6-expressing astrocytes in the cortex for simultaneous imaging of both cells through a cranial window. Multiphoton imaging of the same cells can be performed for days, weeks, or months in awake, behaving animals. This approach provides researchers with a method for viewing cellular dynamics in real time and can be applied to answer a number of questions in neuroscience.

Implantation of a Cranial Window for Repeated In Vivo Imaging in Awake Mice

Presented here is a protocol for the implantation of a chronic cranial window for the longitudinal imaging of brain cells in awake, head-restrained mice.

Implantação de uma janela craniana para imagem repetida em vivo em ratos acordados

To fully understand the cellular physiology of neurons and glia in behaving animals, it is necessary to visualize their morphology and record their ...

Isolando todos os tipos de células residentes no SNC simultaneamente de camundongos autoimunes

Simultaneous Isolation of Principal Central Nervous System-Resident Cell Types from Adult Autoimmune Encephalomyelitis Mice

Experimental autoimmune encephalomyelitis (EAE) is the most common murine model for multiple sclerosis (MS) and is frequently used to further elucidate the still unknown etiology of MS in order to develop new treatment strategies. The myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55) EAE model reproduces a self-limiting monophasic disease course with ascending paralysis within 10 days after immunization. The mice are examined daily using a clinical scoring system. MS is driven by different pathomechanisms with a specific temporal pattern, thus the investigation of the role of central nervous system (CNS)-resident cell types during disease progression is of great interest. The unique feature of this protocol is the simultaneous isolation of all principal CNS-resident cell types (microglia, oligodendrocytes, astrocytes, and neurons) applicable in adult EAE and healthy mice. The dissociation of the brain and the spinal cord from adult mice is followed by magnetic-activated cell sorting (MACS) to isolate microglia, oligodendrocytes, astrocytes, and neurons. Flow cytometry was used to perform quality analyses of the purified single-cell suspensions confirming viability after cell isolation and indicating the purity of each cell type of approximately 90%. In conclusion, this protocol offers a precise and comprehensive way to analyze complex cellular networks in healthy and EAE mice. Moreover, required mice numbers can be substantially reduced as all four cell types are isolated from the same mice.

Autor em destaque: Estudando vias neuroinflamatórias por meio do isolamento simultâneo de todos os principais tipos de células residentes no SNC 

To date, protocols for the simultaneous isolation of all principal central nervous system-resident cell types from the same mouse are an unmet demand. The protocol shows a procedure applicable in na&#239;ve and experimental autoimmune encephalomyelitis mice to investigate complex cellular networks during neuroinflammation and simultaneously reduce the required mice numbers.

Isolamento Simultâneo dos Principais Tipos Celulares Residentes no Sistema Nervoso Central de Camundongos Adultos com Encefalomielite Autoimune

Experimental autoimmune encephalomyelitis (EAE) is the most common murine model for multiple sclerosis (MS) and is frequently used to further elucidate ...

Implantação de vidro Medular do Windows em camundongos adultos com Experimental Auto-imune Encephalomyelitis

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