The following agencies provided critical funding support for this study: MSTP T32 GM007250 (T.A.E.), 5T32EB7509 (D.S.B.), NCI R01 CA154656 (A.Y.H.), Dana Foundation (A.Y.H.), St. Baldrick&#8217;s Foundation (A.Y.H.), Alex&#8217;s Lemonade Stand Foundation (A.Y.H.), Gabrielle&#8217;s Angel Foundation (A.Y.H.) and Hyundai Hope-on-Wheels Program (A.Y.H.). The authors are thankful for the indispensable help of Jerry Silver and Sarah Busch in learning the Dorsal Column Crush (DCC) injury. The authors are also grateful to Jingquang You, Elisabeth Hare and Hongmei Hu for technical help with genotyping and Ross Anderson for mounting the spinal stabilizing unit.

NOTE: All animals should be housed and utilized in accordance with the animal care and use committee (IACUC) at the institution. All procedures described here are approved by the Case Western Reserve University IACUC.
1. Transgenic Animals
<ol>
	<li>Use commercially available fluorescent reporter mice to label axons (Thy-1 YFP H)50 and microglia / monocytes (CX3CR1GFP/GFP 51; see list of materials). These mice should be intercrossed and maintained as individual breeding colonies according to institutional animal care policies.</li>
</ol>
2. Bone Marrow Chimeras
<ol>
	<li>Identify animals that are at least 8 weeks of age for use as recipient animals.</li>
	<li>Place host animals in a circular irradiation holding cage designed to hold the mouse in a position to ensure even, whole body irradiation.</li>
	<li>Set timer on a cesium (Cs-137) irradiator to administer a whole-body radiation dosage of 800-1,000 Rads to the animals according to manufacturer&#8217;s instructions (examples shown here are 980 Rads).</li>
	<li>Return animals to their cages to rest for a minimum of 4 hr.</li>
	<li>Choose donor animals of the appropriate genotype (see Figure 2A) as the source of marrow progenitor cells. 
	NOTE: The appropriate donors are animals expressing different lineage-specific fluorescent reporters from the host to identify different cell populations, or one of the same genotype as controls.</li>
	<li>Fill one Petri dish with 70% alcohol, and a second dish with 10 ml Roswell Park Memorial Institute (RPMI) media and a 40 &#181;m cell strainer soaked in the media. Fill a 15 ml conical tube with RPMI media. Keep the dishes and tube on ice.</li>
	<li>Sacrifice the donor animals by CO2 asphyxiation according to institutional guidelines. Clean the skin and fur by soaking the entire animal with 70% ethanol until all fur is wet.</li>
	<li>Remove the skin from lower half of animal by cutting around the mid-section and pulling skin down over the hind legs to fully expose the leg muscles.</li>
	<li>Remove the tibia by anterior overextension at the knee joint to dislocate the bone, then using sterile forceps and scissors, peel and cut muscles away from both ends of the tibia. Place the bone in the Petri dish with alcohol for up to 30 sec then transfer to the cell strainer inside the Petri dish containing the media.</li>
	<li>Dislocate the hip joint and peel away the muscles attached to the femur, placed the femur briefly in alcohol and then place it in the same cell strainer inside the dish.</li>
	<li>In a tissue culture hood, use sterile scissors to cut off both ends of the bones and insert a 21 gauge needle on a 1 ml syringe into the end of the bone. Flush the marrow into the 15 ml conical with media. Repeat the procedures for the remaining bones.</li>
	<li>Using the back of the plunger from a 3 ml syringe, crush bone ends in the cell strainer on the petri dish to extract more cells.</li>
	<li>Place the filter on top of a 50 ml conical tube and transfer media with aspirated cells from the 15 ml conical tube. Use the back of the plunger to crush the marrow in order to obtain a single cell suspension. Wash the 15 ml conical tube, bone fragments and the cell strainer with 30 ml of media into a 50 ml conical tube.</li>
	<li>Centrifuge the cells for 5 min at 500 x g to pellet cells.</li>
	<li>Lyse red blood cells with 2 ml of Ammonium-Chloride-Potassium (ACK) lysis buffer for 2 min. Quench the lysis buffer with 10 ml of media containing 10% fetal bovine serum. Centrifuge at 500 x g for 5-10 min to pellet.</li>
	<li>Wash cells once in 10 ml of RPMI media and twice in saline, centrifuging at 500 x g for 5-10 min to pellet for each wash.</li>
	<li>Resuspend cells to a final concentration of 3 x 106 cells in 200 &#181;l of saline.</li>
	<li>Transfer 3 x 106 marrow cells via tail vein injection into irradiated recipient mice.</li>
	<li>Place acidified water (pH 3.0) in recipient cage and allow mice to recover. 
	NOTE: Mice that fail the transplantation will die within 10-14 days after the procedure.</li>
	<li>After 8 weeks, verify marrow reconstitution by examining peripheral blood immune cells using flow cytometry (Figure 2B-C).</li>
</ol>
3. Dorsal Column Crush Injury
NOTE: Choose appropriate chimera animals or double transgenic animals for surgery based on the experiment desired. Animals with either labeled resident or bone marrow derived cells were created in previous steps.
<ol>
	<li>Prepare and autoclave surgical tools, including rongeurs, Dumont #4 forceps, iris scissors, forceps for holding tissues, needle drivers and wound clips.</li>
	<li>Prepare medications to administer to the mice for pain control. Bupernex and Marcaine are commonly used for pain control. Use appropriate medications as required and approved by the Institutional Animal Care and Use Committee (IACUC).</li>
	<li>Induce anesthesia with 4% isoflurane and maintain with 1-2% isoflurane to achieve a breathing rate of 60-100 breaths per min. Apply eye lubricant to the animal&#8217;s eyes to prevent dryness under anesthesia and confirm lack of response by foot pinch. Maintain the temperature using a heating pad and monitor the temperature using a rectal probe.</li>
	<li>Shave the back of the mouse with an electric shaver over the targeted spinal cord level, and then swab the area three times with povidone-iodine followed by twice with 70% alcohol. Place the animal on a sterile drape, and cover the animal with another sterile drape with a hole cut in an appropriate location to visualize the surgical site. Maintain all tools on sterile drapes throughout the surgery.</li>
	<li>Cut the skin along the midline at the desired spinal level with iris scissors to expose the muscle on the back of the animal.</li>
	<li>Under a dissecting microscope, cut the back muscles, including the trapezius and the ligaments of the latissimus dorsi along the midline where they attach to the dorsal spinal processes, and expose the vertebral column.</li>
	<li>Remove the rotator muscles between the dorsal and transverse processes of the vertebra to reveal the lamina of the specific vertebra to be removed (T11 in this case). Identify T11 by the insertion of the last non-floating rib onto this process.</li>
	<li>Insert tips of rongeurs into the space above and below the process to be removed and lift slightly to make sure they are securely in place around the vertebral lamina but not too deep to injure the spine. Close rongeurs in one movement to remove the lamina.</li>
	<li>Enlarge the laminectomy as needed by using the rongeurs to remove small parts of the remaining vertebra.</li>
	<li>Measure 1 mm from the tips of a #4 forceps by holding them against a ruler and marking with a permanent marker. Measure a 1 mm in separation between the two tines by holding the forceps against the ruler and fix the maximum width of the tips to 1 mm by sliding a rubber forceps ring into place over the shaft of the forceps.</li>
	<li>Create a small hole by inserting a 30 gauge needle through the dura at the two insertion points for the tines of the forceps to access the spinal cord.</li>
	<li>Insert the forceps tines at a 90&#176; angle to the dura through the holes to the depth of 1 mm marked on the sides of the forceps, making sure the mark is above the dura. Pinch close the forceps and hold for 10 sec. Release, and repeat forceps squeeze two more times for a total of three holds. Remove forceps from the spinal cord.</li>
	<li>Soak a piece of absorbable gelatin compressed sponge in saline and place over the injury site.</li>
	<li>Using a running stitch, suture the muscle in place over the laminectomy and soaked gelatin sponge with #4 synthetic non-absorbable nylon sutures.</li>
	<li>Clip the skin in place using 5 mm wound clips.</li>
	<li>Inject 10 &#181;l Marcaine (1.0 mg/kg) in 490 &#181;l of saline subcutaneously around the incision site and 5 &#181;l Bupernex (0.1 mg/kg) intramuscularly into the gluteus muscles.</li>
	<li>Allow animal to recover on a warm pad and monitor for any difficulties in movement or signs of paralysis in the hind limbs. Do not leave animal unattended or in the presence of other animals until fully recovered from anesthesia. Do not use any animals that display observable motor deficits. 
	NOTE: Although this crush lesion can be performed at any level along the spinal cord, practically, T10&#8211;L4 pose fewer technical challenges for imaging with respect to breathing and stabilizing body movement using clamps. Clamp placement must be modified around the rib cage to image at thoracic levels.</li>
</ol>
4. Intravital Imaging
<ol>
	<li>Prepare and autoclave surgical tools, including Dumont #4 forceps, iris scissors, forceps for holding tissue, needle drivers and wound clips.</li>
	<li>Anesthetize the mouse as before with isoflurane. Induce anesthesia at 4-5% and maintain at 1-2% as needed to maintain a breath rate of 60-100 breaths per minute and a lack of response to foot pinch. Keep the mouse heated, monitor breath rate when not imaging and continuously monitor animal temperature with a rectal probe.</li>
	<li>Clean the skin around the wound clips with Betadine and then alcohol and remove the wound clips according to manufacturer instructions. Once wound clips are removed, remove hair with Nair if needed and clean the site again with povidone-iodine and 70% alcohol.</li>
	<li>Re-open the skin with scissors. Remove any remaining sutures from muscle and pull muscle apart with forceps, cutting with scissors if needed.</li>
	<li>Under a dissecting microscope, create pockets for the spinal cord clamps by cutting the muscle with scissors at the side of the transverse processes on the vertebra one vertebral level above and below the laminectomy.</li>
	<li>Place clamps around each of these vertebra and tighten. Adjust as needed to allow room for microscope objective to reach the spinal cord. Make sure that the spinal cord is parallel to the imaging platform, and maintain tissue stability for imaging. If breathing artifact is seen, readjust clamps accordingly to minimize motion in the imaging field.</li>
	<li>Cover the clamps with parafilm.</li>
	<li>Remove the absorbable gelatin compressed sponge from the spinal cord with forceps, delicately picking off as many pieces as possible, also removing as much scar tissue from over the meninges as possible. Cover exposed spinal cord with saline and new sponge if needed. 
	NOTE: If any bleeding occurs from the surrounding muscle, either staunch with sponge or cauterize as needed. However, take care not to touch the spinal cord with the cautery. Cover the spinal cord with either a piece of dampened tissue or gelatin compressed sponge when cauterizing.</li>
	<li>Create a well to hold the immersion fluid by first sealing the skin and tissue with Vetbond, and then using dental acrylic to build up a well between the two clamps of the spinal cord holder and the sides of the animal. Wait until the acrylic cures.</li>
	<li>Fill the well with artificial cerebral spinal fluid (aCSF) and check again for any bleeding around the surgery site.</li>
	<li>Optionally at this point, inject fluorescent vessel dyes intravenously by tail vein injection to highlight the blood vessels during imaging. 
	NOTE: Fluorescent dextran above 70 kDa is often used, although quantum dots and fluorescently labeled lectins also serve as useful vessel dyes. 50 &#181;l of 150,000 WM TRITC-dextran is typically injected intravenously.</li>
	<li>To obtain physiologically relevant immune cell motility, the temperature of the mouse must be maintained at 37 &#176;C. Move the mouse to a temperature controlled environment under the microscope for imaging and monitor the animal for correct anesthesia level by breath rate and foot pinch. Monitor the animal frequently during imaging to determine depth of anesthesia, aiming to achieve a respiratory rate of 60 - 100 breaths per minute.</li>
	<li>Locate the imaging site through the microscope eyepiece.</li>
	<li>Adjust the 2-photon laser scanning microscope to take a z-stack image every 30-60 sec to obtain dynamic imaging data.</li>
	<li>Once imaging session is completed remove the animal from the heated box.</li>
	<li>Loosen the spinal clamps and remove the mouse from the clamps. Pull the acrylic well away from the skin while removing the clamps.</li>
	<li>If the mouse is to be imaged again, place saline soaked gelatin compressed sponge over the spinal cord. Close the muscle and skin over the surgery site. Do not leave the animal unattended or in the presence of other animals while recovering from anesthesia. If the animal shows any signs of neurologic deficit after recovery, the animal should be euthanized. Otherwise, euthanize the mouse in a CO2 chamber before it emerges from anesthesia according to IACUC-approved euthanasia protocol.</li>
	<li>Analyze fluorescent images using image analysis software by following the manufacturer&#8217;s instructions.</li>
</ol>

The authors have nothing to disclose.

Imaging interactions of different cell types in their native tissue compartments during ensuing pathology in real-time has generated great interest. Within the dense network of the CNS, cell-cell contact and signaling with adjacent cells are essential for normal function and for understanding CNS pathology. Here, we described the use of 2-photon laser scanning microscopy for the observation of cellular movement within a mechanical lesion in the spinal cord. In addition to the quality of the surgery and tissue preparation, mechanical stability of the tissue is paramount for successful time-lapse microscopy, particularly the isolation of the spinal cord from breathing motion artifact. Stability can be assessed by looking for movement of the spinal cord under a dissecting microscope that corresponds to the heart rate or breathing of the animal. Stability should be assessed both while performing the surgery and also immediately before beginning imaging. If the animal is not stable, adjustments should be made to the placement and tightness of the spinal cord clamps. Cell-type specific fluorescent reporter mouse models coupled with bone marrow chimera approaches have allowed the identification of monocytic cells versus microglia and axons. Previous studies have revealed that blood derived monocytes and not microglia are responsible for secondary axonal damage after trauma using the methodology described here43. Dextran conjugated vessel dye allows for vessel identification both to provide landmarks and to identify breaches in vessel integrity. The selection of the appropriate fluorescent reporter mouse model is critical to allow the proper identification of the desired cell types to be imaged.
Development of fluorescent mouse models that are appropriate for the studies being undertaken is also critical to the success of experiments. Distinguishing between the two phagocytic populations of CX3CR1+/GFP cells in the CNS has traditionally been difficult. As shown here, a common immunological technique, irradiation chimeras, can be utilized to distinguish radio-resistant, CNS-resident microglia from bone marrow-derived macrophages. The irradiation procedure has the potential to damage the blood brain barrier and alter cell phenotypes, so use of this model should be considered carefully. The extent of these changes differs with irradiation dose as well as duration of recovery period, and their impact on different inflammatory models has not been fully studied. The effects of irradiation on CNS blood brain barrier can be minimized by shielding the head during irradiation, and this has been shown to decrease cell infiltration into the spinal cord after irradiation, even if the spinal cord is not directly shielded52. Here we have observed that the number of cells infiltrating into the CNS at steady state as a result of chimera generation is insignificant in contrast to the number of cells entering the lesion. Other alternative models to consider include drug-induced chimeras52 or parabiotic mouse models53.
Here, we have presented a specific, simple and reproducible small injury model that results in a lesion to the dorsal white matter of the spinal cord that is easy to image using the 2-photon microscopy. This method also provides a quantifiable lesion to assay the degree of axonal dieback in the dorsal columns that in the literature has been coupled with complementary fixed tissue analysis16,18,43,48,54. In this model, animals display only minimal deficits and require no special care after injury. Future studies utilizing the dorsal column crush injury and imaging techniques described here may be powerful screening tools to assess the efficacy of treatment for spinal cord injury. These treatments may include small molecule inhibitors, drugs, cell products, tissue grafts and combinatorial treatments. The interplay between macrophages, microglia and neurons are also likely to play a role in other disease models in the spinal cord, including multiple sclerosis, tumors, meningitis and amyotrophic lateral sclerosis, and this technique may be useful in the study of these diseases as well.

The inflammatory reaction to disease or injury in the central nervous system (CNS) is poorly understood, especially with regards to the interactions among immune and resident cells within the tissue. Investigations of these cellular interactions in the spinal cord are of particular interest in the living animal. The only easily accessible CNS white matter tract is in the dorsal columns of the spinal cord, making this an important area on which to focus efforts on improving feasible experimental approach. These investigations have been limited due to the technical difficulty in accessing and stabilizing the CNS for imaging. Live imaging in the spinal cord has been described previously1-13, however, few studies have addressed cellular movement in a spinal cord injury beyond a few hours after the initial insult. The traumatic spinal cord lesion is a complex environment, with neurons, astrocytes, fibroblasts, NG2 progenitor cells, and immune cells including microglia, neutrophils, macrophages, T cells, B cells and dendritic cells14,15. Macrophages are the subset of immune cells responsible for phagocytosis in the lesion, infiltrating from the circulation. The role of these phagocytic cells has been debated, with reports indicating that these cells can take on both damaging and protective roles in the injured tissue. These roles range from increasing secondary axonal dieback after an injury and acting in a phagocytic manner16-22, to taking on a wound healing phenotype and decreasing functional deficits in the injured animal21,23,24.
Previously, attempts to distinguish macrophages from microglia have relied primarily on morphology in healthy tissue. However, activated microglia and macrophages express many of the same markers and display indistinguishable morphology after injury25-29, making the separation of different activities of these cells difficult to study based on these factors alone30. These cells can be separated by differential expression of CD45 using flow cytometry33,34, although this approach is less useful in discriminating these cell types in vivo. Utilizing differential expression of CCR2 and CX3CR1 in microglia and macrophages has also been explored, although the dynamic changes in the expression of these markers as monocytes differentiate into macrophages can complicate accurate analysis35,36. Microglia are the resident immune cells in the CNS and arise from yolk sac progenitors during fetal development, while macrophages are derived from bone marrow progenitors and enter the injured CNS after an insult31,32. An alternative approach to using morphology to distinguish these two cell types is to replace the bone marrow with progenitors from a donor animal expressing a traceable marker in the macrophages derived from the donor progenitors, while preserving the CNS resident, recipient-derived microglia. These bone marrow chimeric models are commonly utilized in many other applications37-40. This method has unique caveats associated with damage to the integrity of the blood-brain barrier caused by irradiation or cytotoxic drugs used to eradicate marrow progenitors in the host, thereby limiting its use in certain applications. Recently, functional distinctions between microglia and macrophages in the CNS are beginning to be discerned by using flow cytometry, gene chip array analysis and chimerism methods24,26,41-44, which will prove very useful in the future. Although separating these two cell types remains difficult, understanding their unique functions is important in leading to more targeted treatments of disorders that involve both microglia and macrophages within the CNS.
Description of cellular interactions within the spinal cord lesion has primarily come from studies involving cell culture models. This is due to the challenges involved with imaging both the spinal cord lesion and immune cells together in a living animal. Current rodent models of spinal cord injury of varying type and severity include contusion models45,46, pin-prick injuries2, laceration47, and the dorsal column crush injury described here16,18,48. Inflammation in the meninges increases with the severity of injury and poses unique challenges for the implantation of windows or surgeries for serial imaging. Some of these challenges include the infiltration of phagocytic cells as well as the generation of fibrous tissue over the surgical site. Some of these issues can be overcome by creating smaller lesions and covering the exposed spinal cord with non-immunogenic surgical dressing between the dura and the paraspinous muscles to allow for subsequent re-opening surgery, as is done here to study the dorsal column crush injury. The ability to image only the dorsal portion of the spinal cord also limits the choice of injury, since contusion models primarily cause damage to the central cord, often sparing the most dorsal part of the dorsal columns, especially at early time points after injury45,49. Therefore, we describe a simple injury model that yields a clean, repeatable, rectangular shaped lesion that is useful for both the observation of cell movement within the lesion and for easy quantification of axonal position relative to the lesion.

<table><tbody><tr><td>CX3CR1 GFP mice</td><td>Jackson laboratories</td><td>5582</td><td></td></tr><tr><td>Thy-1 YFP H mice</td><td>Jackson laboratories</td><td>3782</td><td></td></tr><tr><td>Mouse pie cage</td><td>Braintree Scientific</td><td>MPC 2 set</td><td></td></tr><tr><td>60 mm&lt;sup&gt;2&lt;/sup&gt; Petri dish</td><td>Corning</td><td>430166</td><td>For bone marrow isolation</td></tr><tr><td>Falcon 40 &amp;mu;m cell filter</td><td>Fisher Scientific</td><td>08-771-1</td><td>For bone marrow isolation</td></tr><tr><td>1 x 15 ml and 1 x 50 ml conical tube</td><td>Fisher Scientific</td><td></td><td>For bone marrow isolation</td></tr><tr><td>21 gauge needle</td><td>BD</td><td>305177</td><td>For bone marrow isolation</td></tr><tr><td>1 ml syringe</td><td>BD</td><td>309628</td><td>For bone marrow isolation</td></tr><tr><td>ACK lysis buffer</td><td>Gibco</td><td>A10492-01</td><td>For bone marrow isolation</td></tr><tr><td>HBSS</td><td>Corning Cellgro</td><td>21-022-CV</td><td>For bone marrow isolation</td></tr><tr><td>RPMI media</td><td>Sigma</td><td>R8758</td><td>For bone marrow isolation</td></tr><tr><td>F4/80 antibody</td><td>Ebioscience</td><td>12-4801 PE</td><td>For FACS verification of chimeras</td></tr><tr><td>30 gauge insulin syringe</td><td>BD</td><td>328280</td><td>For tail vein injection</td></tr><tr><td>Spinal Cord Clamps</td><td>Narshinge</td><td>STS-A</td><td>For imaging</td></tr><tr><td>Ortho-Jet Dental Acrylic powder</td><td>Lang Dental</td><td>REF1320</td><td>For imaging</td></tr><tr><td>Ortho-Jet Dental Acrylic liquid</td><td>Lang Dental</td><td>REF1404</td><td>For imaging</td></tr><tr><td>Gelfoam gelatin sponges</td><td>Pfizer</td><td>9034201</td><td>For imaging</td></tr><tr><td>4-0 Ethilon sutures</td><td>Ethilon</td><td>1667G</td><td>For surgery</td></tr><tr><td>Reflex Clips, 7 mm</td><td>Kent Scientific</td><td>INS750344</td><td>For surgery. Sterilize before use</td></tr><tr><td>Iris Scissors</td><td>Fine Science Tools</td><td>14061-09</td><td>For surgery</td></tr><tr><td>45/5 Forceps, Dumoxel</td><td>Fine Science Tools</td><td>11251-35</td><td>For surgery</td></tr><tr><td>Rongeurs</td><td>Fine Science Tools</td><td>16021-14</td><td>For surgery</td></tr><tr><td>30 gauge needle</td><td>BD</td><td>305106</td><td>For making access holes in dura</td></tr><tr><td>Forceps Dumont #4 Biology tips</td><td>Dumont</td><td>11242-40</td><td>For surgery</td></tr><tr><td>Needle Drivers</td><td>Fine Science Tools</td><td>12002-12</td><td>For surgery</td></tr><tr><td>Michel Wound Clip Forceps</td><td>Kent Scientific</td><td>INS700753</td><td>For surgery</td></tr><tr><td>Wound clip remover</td><td>Fine Science Tools</td><td>12033-00</td><td>For surgery</td></tr><tr><td>O-rings for Forceps</td><td>Fine Science Tools</td><td>11200-00</td><td>For surgery, Often provided with #4 forceps, can also be ordered separately</td></tr><tr><td>Cordless Rechargable Animal trimmer</td><td>Wahl</td><td>Series 8900</td><td>for surgery</td></tr><tr><td>Vetbond</td><td>3M</td><td>1469SB</td><td>For surgery</td></tr><tr><td>Betadine Solution (10% Providine-Iodine Topical Solution)</td><td>Purdue Products L.P.</td><td>NDC 67618-150-08</td><td>For surgery</td></tr><tr><td>Nair Hair remover lotion</td><td>Church &amp;amp; Dwight Co., Inc.</td><td>NRSL-22329-05</td><td>For surgery</td></tr><tr><td>Isoflurane</td><td>Butler Schein</td><td>NDC 11695-6776-2</td><td>Drugs &amp;ndash; for surgery</td></tr><tr><td>Marcaine</td><td>Henry Schein</td><td>NDC 0409-1587-50</td><td>Drugs &amp;ndash; for surgery 1.0 mg/kg given subcutaneously</td></tr><tr><td>Bupernex</td><td>Henry Schein</td><td>121-7793</td><td>Drugs &amp;ndash; for surgery buprenorphine 0.1 mg/kg</td></tr><tr><td>aCSF</td><td>Chemicals from Sigma</td><td>119 mM NaCl&lt;br /&gt;26.2 mM NaHCO&lt;sub&gt;3&lt;/sub&gt;&lt;br /&gt;2.5 mM KCl&lt;br /&gt;1 mM NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;&lt;br /&gt;1.3 mM MgCl&lt;sub&gt;2&lt;/sub&gt;&lt;br /&gt;10 mM glucose</td><td>For surgery&lt;br /&gt;From Cold Spring Harbor Protocols</td></tr><tr><td>TRITC-dextran 150,000 MW</td><td>Sigma</td><td>T1287</td><td>For intravenous administration to label vasculature</td></tr></tbody></table>

intravital imaging of axonal interactions with microglia and macrophages in a mouse dorsal column crush injury

Traumatic spinal cord injury causes an inflammatory reaction involving blood-derived macrophages and central nervous system (CNS)-resident microglia. Intra-vital two-photon microscopy enables the study of macrophages and microglia in the spinal cord lesion in the living animal. This can be performed in adult animals with a traumatic injury to the dorsal column. Here, we describe methods for distinguishing macrophages from microglia in the CNS using an irradiation bone marrow chimera to obtain animals in which only macrophages or microglia are labeled with a genetically encoded green fluorescent protein. We also describe a injury model that crushes the dorsal column of the spinal cord, thereby producing a simple, easily accessible, rectangular lesion that is easily visualized in an animal through a laminectomy. Furthermore, we will outline procedures to sequentially image the animals at the anatomical site of injury for the study of cellular interactions during the first few days to weeks after injury.

A schematic diagram depicting the dorsal column crush lesion is shown in Figure 1A. After the lesion, the animal is prepared and stabilized for intravital microscopy using spinal clamps (Figure 1B). An image taken immediately after the dorsal column crush injury shows a clearly visible rectangular lesion with forceps tine insertion points that are clearly identifiable. Axons at the injury site are severed across the span of the entire dorsal column (Figure 1C). The integrity of blood vessels remains intact, and no evidence of vessel dye leakage was detected by fluorescence. To perform serial imaging of the same lesion over weeks, the muscles and skin can be sutured over the laminectomy for subsequent re-exposure. Similar lesion morphology is seen at later time points (Figures 1D, E). 5 days after injury, the forceps insertion sites are still visible but the lesion has begun to increase in size due to secondary injury caused by inflammation. The axons at the caudal end of the lesion have retracted from the initial site of the injury via a process called &#8220;axonal dieback&#8221;. The axons at the rostral end of the lesion exhibited Wallerian-like degeneration (Figure 1D, E). Between days 5 and 22 after injury, the size of the lesion has stabilized. Concurrently, a large influx of CX3CR1+ cells can be seen infiltrating in and around the crush lesion during these time points, although the identity of these cells (microglia versus macrophages) can not be distinguished in the preparation as shown in Figure 1.
In order to distinguish the behavior of microglia and macrophages within the crush lesion microenvironment, a radiation chimera model was used (outlined in Figure 2A). First, the bone marrow progenitor cells of a CX3CR1+/GFP mouse are replaced with non-fluorescent donor cells, thus only GFP+ CNS-resident microglia are visible by fluorescent imaging. The efficiency of marrow reconstitution can be confirmed by flow cytometry examination of the peripheral blood 8 weeks after marrow transplant (Figure 2B, C). In Figure 2B, F4/80 and GFP positive macrophages, highlighted in blue, are detected in a CX3CR1+/GFP &#8594; C57BL/6 chimeric mouse, in which some GFP negative but F4/80 positive monocytes are also present. In Figure 2C, no double positive F4/80 and GFP positive cells are left after replacement of CX3CR1+/GFP recipient bone marrow with wild type bone marrow. Before injury, microglia are evenly distributed within the spinal cord, displaying a resting, ramified morphology (Figure 2D). 8 days after injury, only a few microglia can be detected in and around the lesion. These microglia display an amoeboid morphology (Figure 2E). Second, bone marrow progenitor cells in a non-fluorescent mouse are replaced by marrow cells from a CX3CR1+/GFP mouse, therefore rendering bone marrow derived CX3CR1+ monocytes / macrophages visible by GFP fluorescence (Figure 2A). Before injury, the only GFP+ cells detected are largely perivascular, which have been reported to be bone marrow derived and continually turn over on a regular basis28 (Figure 2F). After a crush injury, CX3CR1+/GFP cells can be seen along the inside of blood vessels surrounding the lesion (Figure 2H-J), and the lesion center is filled with infiltrating CX3CR1+/GFP macrophages (Figure 2G).
Time lapse imaging of the dorsal columns can be performed for up to 6 hr. In Figure 3, we show a non-chimeric CX3CR1+/GFP mouse immediately after injury in which cells from the circulation can be seen moving out of the blood vessel towards the lesion core and moving within the injury site (Figure 3A, B; Supplemental Movie 1). Image analysis utilizing fluorescence intensity to identify cells can track the paths taken by macrophages (Figure 3A, B; Supplemental Movie 1). Statistics derived from the imaging analysis show the average motility of macrophages in the lesion is 3.6 &#181;m/min (Figure 3D). Finally, at 22 days after injury, axons, microglia and macrophages can still be detected within the lesion demonstrating the area of imaging is stable over long periods of time (Supplementary Movie 2).
<img alt="Figure 1" fo:content-width="7in" src="/files/ftp_upload/52228/52228fig1highres.jpg" width="600" /> 
Figure 1: Creation and sequential imaging of the dorsal column crush injury. (A) The dorsal column crush injury is performed by removing the dorsal process of the vertebra at the level of interest. Forceps are inserted into the dorsal column of the spinal cord 1 mm apart and are then closed 3 times to produce the lesion shown in purple. (B) The animal is then positioned with spinal cord clamps to obtain stability for intravital imaging. (C) Immediately after injury, the forceps insertion sites (red oval) and the rectangular crush injury volume (grey box) can be clearly identified. Axons in the dorsal roots can be seen (white arrows), as well as the injured ends of the axons on the caudal side of the lesion (filled arrow heads). (D) 5 days after injury, the forceps insertion sites (red oval), and the extent of the lesion (grey box) can still be easily visualized. The lesion has increased in size since the initial insult. Axons undergoing Wallerian-like degeneration can be seen rostral to the lesion (open arrow heads) in addition to axons in the dorsal roots (white arrows) and the ends of injured axons (white arrowheads). (E) 22 days after injury, the lesion site is still identifiable with similar dimensions to the injury after 5 days. Note the large number of CX3CR1+ cells in and around the injured site. Features labeled are the same as in (D). All scale bars = 200 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52228/52228fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" fo:content-width="7in" src="/files/ftp_upload/52228/52228fig2highres.jpg" width="600" /> 
Figure 2: Construction of chimeric mice to track CX3CR1+/GFP expressing CNS-resident microglia or bone marrow-derived macrophages in the dorsal column crush lesion. (A) A schematic diagram of chimeric mice generation with either CX3CR1+/GFP expressing microglia or macrophages. First, Thy-1 YFP H mice are irradiated and the bone marrow is reconstituted with marrow cells isolated from a CX3CR1+/GFP mouse, resulting in a chimeric mouse whose macrophages are GFP+. Second, Thy-1 YFP H / CX3CR1+/GFP mice are irradiated and the bone marrow is reconstituted with marrow cells isolated from a non-fluorescent mouse, producing a chimeric mouse whose microglia are GFP+. (B) Example of flow cytometry data with F4/80+ and CX3CR1 GFP+ cells in the blood from a chimeric animal with a CX3CR1+/GFP marrow donor transplanted into an irradiated C57BL/6 recipient. Cells derived from the CX3CR1 positive donors that are positive for both GFP and F4/80 are shown in the blue highlighted area. (C) Example of flow cytometry data with F4/80+ and CX3CR1 GFP+ cells in a chimeric animal with a C57BL6 donor marrow transplanted into an irradiated CX3CR1+/GFP recipient. Note the lack of double positive CX3CR1+/GFP and F4/80 cells within the blue highlighted area. (D) An intravital two-photon microscopic snapshot of a mouse from the first chimeric scheme, showing GFP+ microglia with ramified morphology within the dorsal column (arrow head). These cells are interspersed among axons (yellow) in the dorsal root and the dorsal column. Scale bar = 100 &#181;m. (E) Intravital imaging of the same mouse in (B) 8 days after dorsal column injury reveals a few CX3CR1+ microglia with large and amoeboid shaped cell bodies that are lacking processes (arrow head). Scale bar = 100 &#181;m. (F) Snapshot of a mouse from the second chimeric scheme in (A), showing scant GFP+ macrophages, within the CNS parenchyma. Scale bar = 100 &#181;m. (G) Intravital imaging of the same mouse in (F) 8 days after dorsal column injury reveals a large influx of macrophages in and around the lesion. Scale bar = 100 &#181;m. (H) A higher magnification picture of CX3CR1+ blood derived cells in contact with the vessels in the uninjured animal. Scale bar = 30 &#181;m. (I) A reconstruction of CX3CR1 cells in contact with the vessel, viewed from inside the vessel. Scale bar = 30 &#181;m. (J) A reconstruction of CX3CR1 cells in contact with the vessel, viewed from outside of the vessel. Scale bar = 20 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52228/52228fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" fo:content-width="7in" src="/files/ftp_upload/52228/52228fig2highres.jpg" width="600" /> 
Figure 3:. Representative cell tracking data in a non-chimeric CX3CR1+/GFP mouse immediately after dorsal column crush injury. (A) A snapshot of CX3CR1+ microglia and macrophages around damaged axons (yellow) from Supplementary Movie 1 immediately after a dorsal column crush injury. (B) The paths (grey) taken by the CX3CR1+ cells in (A) during a 110 min intravital imaging session. (C) A time coded representation of the tracks in (B). Tracks are colored based on their time within the whole 110 minute movie, as shown in the time bar. (D) Histogram of overall motility of the CX3CR1+ cells. All scale bars are 30 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52228/52228fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Movie 1: Representative intravital movie immediately after dorsal column crush injury in the double heterozygote Thy-1YFP/+ / CX3CR1GFP/+ mouse. Intravital spinal imaging was performed immediately after a dorsal column crush injury at T11 level. Right panel shows the movement of CX3CR1+ microglia and macrophages. The paths of the cell movement are shown as white tracks on the left panel. The injury is located to the upper left corner. CX3CR1+ cells can be seen moving into the site of the injury along these tracks. Axons are shown in yellow. Scale bar = 40 &#181;m. Total time: 110 min. Playback speed: 300x.
Supplementary Movie 2: Representative intravital movie at 22 days after dorsal column crush lesion in the double heterozygote Thy-1YFP/+ / CX3CR1GFP/+ mouse. Shown here is a representative movie taken in a mouse 22 days after the initial dorsal column crush injury at spinal level T11. The injury is located at the upper right corner of the frame. Axons (yellow) ascending rostrally are shown at the bottom right. The dorsal vein and blood vessels are labeled with TRITC-dextran (red). CX3CR1+ cells within the lesion move at a slower speed compared to those immediately after injury. Scale bar = 50 &#181;m. Total time: 90 min. Playback speed: 600x.

Watch this Scientific Journal Video about Intravital Imaging of Axonal Interactions with Microglia and Macrophages in a Mouse Dorsal Column Crush Injury at JoVE.com

Intravital Imaging of Axonal Interactions with Microglia and Macrophages in a Mouse Dorsal Column Crush Injury

Two-photon intravital imaging can be used to investigate interactions among different cell types in the spinal cord in their native tissue environment in a bone marrow chimeric animal with a dorsal column traumatic spinal cord crush injury.

Traumatic spinal cord injury causes an inflammatory reaction involving blood-derived macrophages and central nervous system (CNS)-resident microglia. ...

intravital-imaging-axonal-interactions-with-microglia-macrophages

Nanyang Technological University

Research

JoVE Journal

Neuroscience

11.4K Views.  Case Western Reserve University. Two-photon intravital imaging can be used to investigate interactions among different cell types in the spinal cord in their native tissue environment in a bone marrow chimeric animal with a dorsal column traumatic spinal cord crush injury. 

Video: Intravital Imaging of Axonal Interactions with Microglia and Macrophages in a Mouse Dorsal Column Crush Injury

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 ...

Live Imaging of Dorsal Root Axons after Rhizotomy

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. Regeneration of dorsal root (DR) axons into spinal cord is prevented at the dorsal root entry zone (DREZ), the interface between the CNS and PNS. Our understanding of the molecular and cellular events that prevent regeneration at DREZ is incomplete, in part because complex changes associated with nerve injury have been deduced from postmortem analyses. Dynamic cellular processes, such as axon regeneration, are best studied with techniques that capture real-time events with multiple observations of each living animal. Our ability to monitor neurons serially in vivo has increased dramatically owing to revolutionary innovations in optics and mouse transgenics. Several lines of thy1-GFP transgenic mice, in which subsets of neurons are genetically labeled in distinct fluorescent colors, permit individual neurons to be imaged in vivo1. These mice have been used extensively for in vivo imaging of muscle2-4 and brain5-7, and have provided novel insights into physiological mechanisms that static analyses could not have resolved. Imaging studies of neurons in living spinal cord have only recently begun. Lichtman and his colleagues first demonstrated their feasibility by tracking injured dorsal column (DC) axons with wide-field microscopy8,9. Multi-photon in vivo imaging of deeply positioned DC axons, microglia and blood vessels has also been accomplished10. Over the last few years, we have pioneered in applying in vivo imaging to monitor regeneration of DR axons using wide-field microscopy and H line of thy1-YFP mice. These studies have led us to a novel hypothesis about why DR axons are prevented from regenerating within the spinal cord11.
In H line of thy1-YFP mice, distinct YFP+ axons are superficially positioned, which allows several axons to be monitored simultaneously. We have learned that DR axons arriving at DREZ are better imaged in lumbar than in cervical spinal cord. In the present report we describe several strategies that we have found useful to assure successful long-term and repeated imaging of regenerating DR axons. These include methods that eliminate repeated intubation and respiratory interruption, minimize surgery-associated stress and scar formation, and acquire stable images at high resolution without phototoxicity.

An in vivo imaging protocol to monitor primary sensory axons following dorsal root crush is described. The procedures utilize wide-field fluorescence microscopy and thy1-YFP transgenic mice, and permit repeated imaging of axon regeneration over 4 cm in the PNS and axon interactions with the interface of the CNS.

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. ...

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.

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 ...

Medicine

Acute Brain Trauma in Mice Followed By Longitudinal Two-photon Imaging

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no effective treatment for it yet. Limitations of currently used animal models impede understanding of the pathology mechanism. Multiphoton microscopy allows studying cells and tissues within intact animal brains longitudinally under physiological and pathological conditions. Here, we describe two models of acute brain injury studied by means of two-photon imaging of brain cell behavior under posttraumatic conditions. A selected brain region is injured with a sharp needle to produce a trauma of a controlled width and depth in the brain parenchyma. Our method uses stereotaxic prick with a syringe needle, which can be combined with simultaneous drug application. We propose that this method can be used as an advanced tool to study cellular mechanisms of pathophysiological consequences of acute trauma in mammalian brain in vivo. In this video, we combine acute brain injury with two preparations: cranial window and skull thinning. We also discuss advantages and limitations of both preparations for multisession imaging of brain regeneration after trauma.

Acute brain trauma is a severe injury that has no adequate treatment to date. Multiphoton microscopy allows studying longitudinally the process of acute brain trauma development and probing therapeutical strategies in rodents. Two models of acute brain trauma studied with in vivo two-photon imaging of brain are demonstrated in this protocol.

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

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 ...

An In Vivo Duo-color Method for Imaging Vascular Dynamics Following Contusive Spinal Cord Injury

Spinal cord injury (SCI) causes significant vascular disruption at the site of injury. Vascular pathology occurs immediately after SCI and continues throughout the acute injury phase. In fact, endothelial cells appear to be the first to die after a contusive SCI. The early vascular events, including increased permeability of the blood-spinal cord barrier (BSCB), induce vasogenic edema and contribute to detrimental secondary injury events caused by complex injury mechanisms. Targeting the vascular disruption, therefore, could be a key strategy to reduce secondary injury cascades that contribute to histological and functional impairments after SCI. Previous studies were mostly performed on postmortem samples and were unable to capture the dynamic changes of the vascular network. In this study, we have developed an in vivo duo-color two-photon imaging method to monitor acute vascular dynamic changes following contusive SCI. This approach allows detecting blood flow, vessel diameter, and other vascular pathologies at various sites of the same rat pre- and post-injury. Overall, this method provides an excellent venue for investigating vascular dynamics.

An In Vivo Duo-color Method for Imaging Vascular Dynamics Following Contusive Spinal Cord Injury

We introduce an in vivo imaging method using two different fluorescent dyes to track dynamic spinal vascular changes following a contusive spinal cord injury in adult Sprague-Dawley rats.

Spinal cord injury (SCI) causes significant vascular disruption at the site of injury. Vascular pathology occurs immediately after SCI and continues ...

Precise Brain Mapping to Perform Repetitive In Vivo Imaging of Neuro-Immune Dynamics in Mice

The central nervous system (CNS) is regulated by a complex interplay of neuronal, glial, stromal, and vascular cells that facilitate its proper function. Although studying these cells in isolation in vitro or together ex vivo provides useful physiological information; salient features of neural cell physiology will be missed in such contexts. Therefore, there is a need for studying neural cells in their native in vivo environment. The protocol detailed here describes repetitive in vivo two-photon imaging of neural cells in the rodent cortex as a tool to visualize and study specific cells over extended periods of time from hours to months. We describe in detail the use of the grossly stable brain vasculature as a coarse map or fluorescently labeled dendrites as a fine map of select brain regions of interest. Using these maps as a visual key, we show how neural cells can be precisely relocated for subsequent repetitive in vivo imaging. Using examples of in vivo imaging of fluorescently-labeled microglia, neurons, and NG2+ cells, this protocol demonstrates the ability of this technique to allow repetitive visualization of cellular dynamics in the same brain location over extended time periods, that can further aid in understanding the structural and functional responses of these cells in normal physiology or following pathological insults. Where necessary, this approach can be coupled to functional imaging of neural cells, e.g., with calcium imaging. This approach is especially a powerful technique to visualize the physical interaction between different cell types of the CNS in vivo when genetic mouse models or specific dyes with distinct fluorescent tags to label the cells of interest are available.

This protocol describes a chronic cranial window implantation technique that can be used for longitudinal imaging of neuro-glio-vascular structures, interactions, and function in both healthy and diseased conditions. It serves as a complementary alternative to the transcranial imaging approach that, while often preferred, possesses some critical limitations.

The central nervous system (CNS) is regulated by a complex interplay of neuronal, glial, stromal, and vascular cells that facilitate its proper function. ...

Immunology and Infection

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in the brain sense various biological conditions in the periphery and transmit the signals to neurons. Microglia, immune cells in the brain, are involved in synaptic development and plasticity. Therefore, the contribution of microglia to neural circuit construction in response to the internal state of the body should be tested critically by intravital imaging of the relationship between microglial dynamics and neuronal activity.
Here, we describe a technique for the simultaneous imaging of microglial dynamics and neuronal activity in awake mice. Adeno-associated virus encoding R-CaMP, a gene-encoded calcium indicator of red fluorescence protein, was injected into layer 2/3 of the primary visual cortex in CX3CR1-EGFP transgenic mice expressing EGFP in microglia. After viral injection, a cranial window was installed onto the brain surface of the injected region. In vivo two-photon imaging in awake mice 4 weeks after the surgery demonstrated that neural activity and microglial dynamics could be recorded simultaneously at the sub-second temporal resolution. This technique can uncover the coordination between microglial dynamics and neuronal activity, with the former responding to peripheral immunological states and the latter encoding the internal brain states.

Here, we describe a protocol combining adeno-associated virus injection with cranial window implantation for simultaneous imaging of microglial dynamics and neuronal activity in awake mice.

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in ...

Intravital Imaging of Fluorescent Protein Expression in Mice with a Closed-Skull Traumatic Brain Injury and Cranial Window Using a Two-Photon Microscope

The goal of this protocol is to demonstrate how to longitudinally visualize the expression and localization of a protein of interest within specific cell types of an animal's brain, upon exposure to exogenous stimuli. Here, the administration of a closed-skull traumatic brain injury (TBI) and simultaneous implantation of a cranial window for subsequent longitudinal intravital imaging in mice is shown. Mice are intracranially injected with an adeno-associated virus (AAV) expressing enhanced green fluorescent protein (EGFP) under a neuronal specific promoter. After 2 to 4 weeks, the mice are subjected to a repetitive TBI using a weight drop device over the AAV injection location. Within the same surgical session, the mice are implanted with a metal headpost and then a glass cranial window over the TBI impacting site. The expression and cellular localization of EGFP is examined using a two-photon microscope in the same brain region exposed to trauma over the course of months.

This study demonstrates delivery of a repetitive traumatic brain injury to mice and simultaneous implantation of a cranial window for subsequent intravital imaging of a neuron-expressed EGFP using two-photon microscopy.

The goal of this protocol is to demonstrate how to longitudinally visualize the expression and localization of a protein of interest within specific cell ...

Intravital Imaging of Axonal Interactions with Microglia and Macrophages in a Mouse Dorsal Column Crush Injury

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