This work was supported by Institut National de la Sante et de la Recherche Medicale, Agence Nationale Recherche (ANR-17-CE14-0005-03), Federation pour la Recherche sur le Cerveau (FRC 2017), Carnot Maturation (to L.J.), Universidade Federal de Rio de Janiero (UFRJ for J.B.), NIH (R01EB016629-01) and the Yale School of Medicine. We acknowledge the ICM platforms: ICM-QUANT for cellular imaging and ICM-histomics for immunohistochemistry. All animal work was conducted at the PHENO-ICMice facility. The Core is supported by 2 &#8220;Investissements d&#8217;avenir&#8221; (ANR-10- IAIHU-06 and ANR-11-INBS-0011-NeurATRIS) and the &#8220;Fondation pour la Recherche M&#233;dicale&#8221;. We acknowledge Nicolas Renier for methodological advice and manuscript reading.

All in vivo procedures used in this study complied with all relevant ethical regulations for animal testing and research, in accordance to the European Community for experimental animal use guidelines (L358-86/609EEC). The study received ethical approval by the Ethical Committee of INSERM (n&#176;2016110111126651) and the Institutional Animal Care and Use Committee of ICM (Institut du Cerveau et de la Moelle &#233;pini&#232;re).
1. Preparation
<ol>
	<li>Prepare the following dissection tools for surgery: scalpel (1), microforceps (2), forceps (1), dissection scissors, and Michel suture clips. Prepare 26 G needles (0.45 mm x 13 mm), 1 mL syringe, and 10 &#181;L microsyringe.</li>
	<li>Pull microcapillaries with a single-step protocol at 67.5 &#176;C with a glass micropipette puller. Prepare two microcapillaries per injection.</li>
	<li>Prepare reagents for imaging lymphatic drainage (Table 1): Ovalbumin Alexa Fluor 555 conjugate (OVA-A555, 2 mg/mL in 1x phosphate buffered saline [PBS]) and anti-LYVE1 antibody (1 mg/mL in 1x PBS).</li>
	<li>Prepare antibodies (Table 1) for iDISCO+. For primary antibodies, use anti-LYVE1 rabbit polyclonal antibody (1:1,600) and anti-PROX1 goat polyclonal IgG antibody (1:2,000). For secondary antibodies, use Alexa Fluor donkey anti-rabbit-568, donkey anti-rabbit-647, and donkey anti-goat-647 (1:2,000).</li>
</ol>
2. Surgery procedures for intra-cisterna magna (ICM) and thoracolumbar (ThLb) injections
<ol>
	<li>Preparation of the animal for surgery
	<ol>
		<li>Use adult male and female C57BL6/J mice, 8&#8211;12-weeks-old.</li>
		<li>Inject the mouse intraperitoneally (IP) with 0.015 mg/mL buprenorphine solution diluted in 0.9% sodium chloride at 0.1 mg/kg, 15 min before surgery.</li>
		<li>Anesthetize the mouse in an induction box with 2&#8211;3% isoflurane gas.</li>
	</ol>
	</li>
	<li>Preparation of anesthetized animal for tracer injection
	<ol>
		<li>Place the anesthetized mouse and its heating pad on the stereotaxic apparatus. Use ear bars to hold the mouse&#39;s head, and lay down the body at a ~135&#176; angle to the head or immobilize the spinal cord at the ThLb vertebral level (Th12-L1) with a spinal adaptor. Pinch the tail or the paw with the forcep to check the efficiency of anesthesia.</li>
		<li>Inject IP 200 &#181;L of 0.9% sodium chloride for mouse hydration.</li>
		<li>Using a scalpel blade, make a skin incision, either in the occipital region toward the cervical region for ICM injection, or at the ThLb vertebral level (Th10-L3) for injection into the ThLb spinal parenchyma.</li>
	</ol>
	</li>
	<li>Tracer injection
	<ol>
		<li>Discard paracervical and paraspinal muscles covering the neck and the column to visualize the surface of the dura mater, the outermost layer of the meninges.</li>
		<li>Carefully punctate the central area of the dura mater and underlaying arachnoid with a 26 G needle.</li>
		<li>Microcapillary implantation
		<ol>
			<li>Cut 2 mm of the glass capillary tip (see step 1.2), then use the microcapillary connected to a cannula linked to a 10 &#181;L syringe to aspirate 2&#8722;8 &#181;L of the OVA-A555 or LYVE1 antibody.</li>
			<li>Introduce the microcapillary in the medial region of the dura mater at a 30&#176; angle for ICM injections or 10&#176; angle for ThLb spinal injections, and push it in to 1.5 mm below the dura mater. 
			NOTE: The ligament is punctured, but no laminectomy is performed.</li>
			<li>Add 10 &#181;L of surgical glue to close the incision around the glass capillary and wait for it to dry.</li>
		</ol>
		</li>
		<li>Slowly inject the fluorescent tracer at 1 &#181;L/min. Once the injection volume has been delivered, maintain the capillary in place for 1 min. Retract the microcapillary and add surgical glue to close the injection hole.</li>
	</ol>
	</li>
	<li>Postinjection, close the skin incisions with suture clips. Remove the mouse from the stereotaxic apparatus and place it in a postsurgery warming chamber at 37 &#176;C until it recovers.</li>
</ol>
3. Perfusion and tissue dissection
<ol>
	<li>At either 15 min or 45 min after CSF tracer injection, inject IP a lethal dose (100 &#181;L) of sodium pentobarbital. Pinch the tail or the paw to verify that there is no reflex.</li>
	<li>With dissection scissors, cut the skin and open the peritoneal layer from the lower abdomen region toward the thoracic cage. Open the thoracic cage with scissors to have access to the heart.</li>
	<li>Insert the 26 G needle in the left ventricule of the heart and start to perfuse with 20 mL of ice-cold 4% paraformaldehyde (PFA) in 1x PBS at 2 mL/min. Use scissors to rapidly cut the right atrium and release the perfusion fluid stream.</li>
	<li>Completely remove the skin with forceps and cut the four legs with scissors. Remove all the internal organs but be careful to preserve the LNs intact. 
	NOTE: Mandibular LNs are superficial, so take care to not remove them when cutting off the skin. Deep-cervical LNs (dcLNs) are located on each side of the trachea, in contact with the lateral surface of the internal jugular veins and close to the stereomastoid muscles.</li>
	<li>Cut the ribs to remove the vertebral column with the spinal cord inside from the cervical to the lumbar segments.</li>
	<li>Immerse the dissected tissues in ice-cold 4% PFA in 1x PBS in a 50 mL tube overnight (~18 h) at 4&#8201;&#176;C. Wash the fixed tissues 3x in 50 mL of 1x PBS for 5 min.</li>
</ol>
4. Fluorescence macroscopy of a vertebral segment
<ol>
	<li>Position the sample under the fluorescence stereozoom microscope with a camera (Table of Materials). Take an overview of the sample or zoom on a specific region.</li>
</ol>
5. Sample preparation for whole mount immunostaining
<ol>
	<li>Using a microtome blade, transversally cut the head-and-vertebral column at the occipital and cervical levels. 
	NOTE: This dissection allows isolation of the head and the cervical vertebral region from the rest of the vertebral column.</li>
	<li>With a microtome blade, cut the cervical, thoracolumbar, and sacral regions of the vertebral column transversally into segments of 2&#8722;4 vertebrae, about 0.5 cm size each.</li>
	<li>Isolate each segment in the order it was separated from the vertebral axis, along the entire length of the cervical and thoracic regions of the vertebral column. Preserve each sample in a tube of 2 mL 1x PBS.</li>
</ol>
6. iDISCO+ whole mount immunostaining of a vertebral segment
NOTE: The detailed description of the iDISCO+ protocol is accessible at http://www.idisco.info.
<ol>
	<li>Day 1: Tissue dehydration
	<ol>
		<li>Dehydrate vertebral tissue samples (i.e., a vertebral segment) by successive immersion in 20%, 40%, 60%, 80%, and 100% methanol in 1x PBS for 1 h with agitation.</li>
		<li>Incubate the samples overnight in a solution of 33% methanol/66% dichloromethane (DCM) at room temperature (RT) with agitation.</li>
	</ol>
	</li>
	<li>Day 2: Tissue bleaching
	<ol>
		<li>Wash samples 2x with 100% methanol for 1 h at RT. Incubate the samples in 5% H2O2 in methanol (30% H2O2 and methanol 1:5 v/v) at 4 &#176;C overnight.</li>
	</ol>
	</li>
	<li>Day 3: Decalcification and permeabilization step
	<ol>
		<li>Rehydrate the samples gradually in 80%, 60%, 40%, 20% methanol, then in 1x PBS (1 h in each solution) at RT with agitation.</li>
		<li>Decalcify the vertebrae by incubating samples in Morse&#8217;s solution (10% trisodium citrate and 45% formic acid 1:1 v/v) for 30 min at RT to preserve the bone structure.</li>
		<li>Rinse the samples 2x with 1x PBS and incubate 2x for 1 h in PTx2 solution (0.2% Triton X-100 in 1x PBS, renew for the second incubation) at RT with agitation. Then incubate the pretreated samples in permeabilization solution (PTx2 with 20% dimethyl sulfoxide [DMSO] and 2.3% w/v glycine) at 37&#8201;&#176;C for 24 h.</li>
	</ol>
	</li>
	<li>Day 4: Blocking step
	<ol>
		<li>Incubate samples in blocking solution (PTx2 with 6% donkey serum and 10% DMSO) at 37&#8201;&#176;C for 24 h.</li>
	</ol>
	</li>
	<li>Days 5&#8722;16: Whole mount immunolabeling
	<ol>
		<li>Incubate samples in primary antibody diluted in PTwH (1x PBS containing 0.2% Tween-20 and 0.1% heparin at 10 mg/mL in 1x PBS) with 5% DMSO/3% donkey serum at 37&#8201;&#176;C for 6 days. Wash samples 4&#8722;5x in PTwH at RT with agitation overnight.</li>
		<li>Incubate samples in secondary antibody dilutions in PTwH with 3% donkey serum at 37&#8201;&#176;C for 4 days. Wash samples in PTwH 4&#8722;5x at RT under agitation overnight before clearing.</li>
	</ol>
	</li>
	<li>Days 17 and 18: iDISCO+ tissue clearing
	<ol>
		<li>Dehydrate samples gradually by successive immersion in 1x PBS, then 20%, 40%, 60%, 80%, and 2x in 100% methanol (1 h in each solution). Incubate each sample overnight in a solution of 33% methanol/66% DCM.</li>
		<li>Wash 2x&#8201;in 100% DCM for 15&#8201;min to remove the methanol. Incubate in dibenzyl ether (DBE) without shaking until cleared (4 h) and then store in DBE at RT before imaging.</li>
	</ol>
	</li>
</ol>
7. LSFM imaging
<ol>
	<li>Image cleared samples in a transversal plane with the LSFM equipped with a 4x/0.3 objective.
	<ol>
		<li>Use a single sided three sheet illumination configuration, fixed x position (no dynamic focusing). Use LED lasers tuned to 561&#8201;nm, 100&#8201;mW; and 639&#8201;nm, 70&#8201;mW. Set the light sheet numerical aperture to 30%.</li>
		<li>Use different emission filters: 595/40 for Alexa Fluor-568 or -555, and -680/30 for Alexa Fluor-647.</li>
	</ol>
	</li>
	<li>Fill the microscope chamber with DBE.</li>
	<li>Aquire stacks with 2.5 &#956;m z steps and a 30 ms exposure time per step with the camera (Table of Materials). Use the x2 optical zoom for an effective magnification of (x8), 0.8 &#181;m/pixel, and perform the mosaic acquisitions with a 10% overlap on the full frame.</li>
	<li>Acquire images in .tif format with acquisition software and convert them into 3D format with a file conversion software.</li>
	<li>Reconstruct mosaics aquisition with stitcher software (Table of Materials). Open the images and move manually to reconstitute the whole mosaic picture, using the 10% overlap between images as a guideline.</li>
	<li>Use the 3D software (Table of Materials) to generate orthogonal projections of data, as shown in Figure 1, Figure 2, Figure 3, and Figure 4, and add a color code attribute to the lymphatic vessels and the other anatomical structures on display. Set a gamma correction of 1.47 to the raw data obtained from the LSFM according to the manufacturer&#8217;s instructions.</li>
</ol>

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The authors have nothing to disclose.

The iDISCO+/LSFM protocol provides unprecedented 3D views of the CNS-associated lymphatic network within its surrounding tissues at a cellular resolution level. This protocol is well adapted to medium size samples, not exeeding 1.5 cm3, due to the limitations of the LSFM optical system, the reduced working distance, and the large size of commercial objective lenses for high-resolution microscopy23. This limitation prevents capturing the whole brain-associated lymphatic system. It is important to note that the area of investigation has to be cautiously delimited and the tissues surrounding the CNS have to be carefully dissected in order to include the extracranial lymphatic vessels and LNs that contribute to the whole lymphatic circuitry (Table 2).
In addition to size and anatomical considerations, the complexity of surrounding mesenchymal tissues varies along the skull and vertebral column, which requires adaptation of the decalcification and preclearing treatment in order to obtain a homogeneous sample clarification and allow light beam propagation within a soft isotropic biological tissue. In the absence of bones, LFSM imaging of the brain or spinal cord tissues does not necessitate the decalcification step, and the final resolution of the captured images is optimal19. The above described protocol, which includes a mild decalcification step with Morse&#8217;s solution, is well adapted for LSFM imaging of the vertebral column as illustrated in Figure 1 and Figure 4. In contrast, the neck region displays a particularly complex bone anatomy in addition to multiple layers of muscles, fat, and glandular tissues, which reduce the quality of captured LSFM images, as reflected in Figure 3B. LSFM imaging of the neck and cervical area may thus be improved by a more stringent treatment of tissues; for example, with EDTA, as previously reported24. The decalcification step is therefore critical and decalcification conditions have to be previously tested for each antibody used before starting the full iDISCO+ protocol (Table 2).
While the iDISCO+/LSFM protocol allows generation of a 3D view of connecting circuits between the meningeal and epidural spaces and associated LNs, the direct quantitative analysis of lymphatic vasculature from LSFM-captured images is not feasible for the following reasons: 1) delineation of lymphatic vessel circuits is unreliable due to the discontinuous pattern of lymphatic marker expression, because membranar LYVE1 is hereterogenously distributed21 and PROX1 has a nuclear expression pattern22; 2) the heterogenous penetration of antibodies, as well as the anisotropy that may persist in the biological tissue due to incomplete and heterogenous decalcification and preclearing. LSFM imaging thus needs to be extended by virtual reality tools that enable interactive visualization and thus facilitate quantification of the lymphatic vasculature (www.syglass.io). It is also noteworthy that the precise description of the CNS-associated circuitry requires backing up LSFM information with high-resolution confocal data obtained by conventional immunolabeling on thin (5&#8211;10 &#181;m) cryostat or paraffin-embedded tissue sections, especially to precisely localize the position of lymphatic vessels with regards to the dura mater and the CSF, as reported previously11,14,18.
The iDISCO+/LSFM protocol allows three-dimensional visualization of the macromolecular drainage in the CNS-associated lymphatic system, as illustrated in Figure 3 and Figure 4. However, the functional assessment of lymphatic drainage requires, in addition to the recommendations on the iDISCO+/LSFM protocol detailed above, following a rigorous procedure, as the final outcome depends on the quality of the injection surgery, the choice of the delivery site, the type and injected volume of macromolecule marker used, and the time of sacrifice after tracer administration (Table 2). Due to variations of the tracer pattern between injected animals, the characterization of lymphatic drainage circuits requires large experimental groups (&#62;10 by injection condition). In the protocol presented, 1) the dura mater must be punctured before injection to prevent unwanted lesions and penetration into the CNS tissues; 2) the injected volume has to be less than 2 &#181;L to limit unwanted diffusion through the injection hole, along the injection capillary, into the epidural space or extravertebral tissues; 3) the deepness of injection capillary insertion has to be limited to 2 mm below the dura mater to avoid CNS injury or mistargeting in ICM and intraspinal injections, respectively. Note also that a complementary high-resolution confocal analysis of neighboring vertebral segments needs to be performed, as indicated above, to assess the presence of injected tracer inside of the lymphatic vessels. This analysis requires establishing the intensity profile plots for the tracer and the lymphatic marker on cross sections of marker-labeled lymphatic vessels. This approach has been previously used to demonstrate OVA555 uptake by ThLb lymphatics at 15 min after injection (Supplementary Figure 5F in Jacob et al.14). However, it has not been illustrated for the anti-LYVE1 tracer in the present study (Figure 4).
Among possible CSF tracers, OVA-A555 is an excellent option as it is resistant to the iDISCO+ protocol treatments and maintains high fluorescence for LSFM imaging. However, note that the type of tracer must be chosen in accordance with the analysis time point (Table 1 and Table 2). As reported above, OVA-A555 labeling of local vertebral lymphatic vessels is observed at 15 min after injection14. However, OVA-A555 is no longer detected in these local lymphatic circuits at 45 min after injection (Figure 3) in contrast to the anti-LYVE1 antibody (Figure 4).
To conclude, the iDISCO+/LSFM protocol is well adapted for investigating the 3D structure and drainage of the CNS-associated lymphatic system in physiological and pathological conditions such as CNS and vertebral column cancers, or vertebral bone and joint diseases. Although the full procedure is long and requires methodological rigor, it provides valuable, unique information when used with complementary analysis using virtual reality tools and high-resolution confocal imaging.

The CNS is surrounded by the CSF and overlaying layers of meninges, epidural tissue, and bones. Altogether, the CSF provides physical protection to the soft brain and spinal cord. It is mainly secreted by the choroid plexus and the meningeal membranes (i.e., the pia mater, the arachnoid, and the dura mater). The CSF-meningeal complex also establishes a functional interface between the CNS tissues and the rest of the body, thereby contributing to CNS homeostasis. First, the CSF penetrates the CNS parenchyma through the CNS para-arterial spaces and interacts dynamically with the interstitial fluid (ISF)1 via the glymphatic (glia-lymphatic) system, which consists of the paravascular spaces and the astrocyte end-feet membranes around the CNS vessels2,3,4. Metabolic waste and excess fluid are then ultimately cleared by intramural perivascular drainage directly from the brain parenchyma toward the systemic circulation3, as well as the paravenous spaces toward the CSF and via brain-draining lymphatic vessels, according to the glymphatic model2,4. CSF outflow is mainly via the lymphatic system, through the cribriform plate and associated extracranial lymphatic vessels5,6,7, as well as by the meningeal lymphatic vessels, which converge at the brain-draining LNs8,9,10,11,12 (Figure 1). An important, although secondary, role in CSF outflow is taken by the cranial arachnoid villi, which penetrate the lacuna of meningeal venous sinuses13.
The CFS drainage circuits have been extensively investigated through experimental approaches based on the injection of colored/fluorescent tracers into the CNS or CSF, followed by the imaging of the tracers&#8217; pattern inside the CNS and throughout the body organs and tissues at different timepoints after injection13. For a long time, the outflow of CSF was considered to be exclusively and directly taken in charge by the blood circulation, through arachnoid villi projecting into dural venous sinuses13. However, the CSF outflow is predominantly performed by the lymphatic vasculature, as recently shown by near-infrared (NIR) dynamic imaging of CSF-injected tracer transport in mice9,10. The CSF-draining lymphatic vessels then return lymph to the bloodstream via the right subclavian vein. Complementary approches have detected both extracranial6,7,13 and intracranial9,10,11,12 lymphatic exits of CSF-injected tracers and suggest that the CSF is absorbed by two lymphatic pathways, one external and the other one internal to the skull and vertebral column. The main part of CSF drainage rapidly occurs through lymphatic vessels located rostrally, outside of the skull in the nasal mucosa, through channels of the cribriform plate of the ethmoid bone3,6,13 and, caudally, outside of lumbosacral vertebral bones through dorsolateral routes that are not yet fully characterized7,14. In addition, in the meninges of the skull, lymphatic capillaries of the dura mater directy absorb CSF and meningeal immune cells toward dural lymphatic collectors that cross the skull bones and connect to CNS-draining LNs12,14. These meningeal lymphatic vessels play important roles in CNS pathophysiology, because brain meningeal lymphatics are altered upon aging and also impact the outcome of neurological brain diseases, including neurodegeneration, neuroinflammation, and brain cancer15,16,17. Therefore, the CNS-associated lymphatic vasculature (i.e., the dural and peripheral lymphatic vessels draining the CSF) may be a promising novel target to combat CNS diseases in humans.
Convergent studies performed with immunohistology and high-resolution magnetic resonance imaging demonstrated that the meningeal lymphatic vasculature also exists in primates, including common marmoset monkeys and humans7,11,13. Moreover, meningeal lymphatic vessels are not restricted to the skull, but extend within the vertebral column to the surface of spinal ganglia and rami13,18. Three-dimensional (3D) imaging of the vertebral column lymphatics preserving the overall anatomy of labeled vertebral and spinal samples, including overlying bones, muscles, ligaments, as well as neighboring visceral tissues, was recently performed14. The iDISCO+ protocol19,20 was used to immunolabel decalcified and cleared preparations of the whole vertebral column with lymphatic-specific antibodies against either the membrane receptor LYVE121 or the transcription factor PROX122. Image acquisition and analysis were then carried out with light sheet fluorescence microscopy (LSFM) and the Imaris software. LSFM allows for rapid and minimally invasive 3D imaging of large specimens by axial confinement of illumination, which results in reduced photobleaching and phototoxicity23.
The iDISCO+/LSFM approach allows characterization of the distinct layers of dural and epidural lymphatic vessels, and the connection of this vasculature to the extravertebral lymphatic circuits and the LNs neighboring the vertebral column. The protocol was applied to tissues previously injected with fluorescent tracers to demonstrate vertebral canal drainage. The present paper provides details on the iDISCO+/LSFM methodology to image the vertebral lymphatic vasculature and illustrates its relevance to CSF and epidural fluid drainage investigation.

<table>
	<tbody>
		<tr>
			<td>Consumables</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tubes: 0.2ml</td>
			<td>Eppendorf</td>
			<td>30124359</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tubes: 2ml</td>
			<td>Eppendorf</td>
			<td>30120094</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical centrifuge tubes: 15ml</td>
			<td>Falcon</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical centrifuge tubes: 50ml</td>
			<td>Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome blade 80mm</td>
			<td>Microm Microtech France</td>
			<td>F/MM35P</td>
			<td></td>
		</tr>
		<tr>
			<td>Needles 26G (0.45x13 mm)</td>
			<td>Terumo</td>
			<td>AN*2613R1</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe 1ml</td>
			<td>Terumo</td>
			<td>SS+01H1</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscopes and imaging softwares</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AxioZoom.V16 fluorescence stereo zoom microscope, equipped with an ORCA-Flash 4.0 digital sCMOS camera (Hamamatsu Photonics) or an OptiMOS sCMOS camera</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Imspector Microscope controller software, Version v144 (acquisiton software)</td>
			<td>Abberior instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Imaris File Converter x64 9.2.0(file convertion software) , Imaris stitcher software 9.2.0 (stitcher software), Imaris x64 9.2.0 (3D software)</td>
			<td>OXFORD instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LED lasers (OBIS) LVBT Laser module 2nd generayion</td>
			<td>COHERENT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultramicroscope II equipped with a sCMOS camera (Andor Neo) and a 4&#8201;&#215;&#8201;/0.3 objective lens (LVMI-Fluor WD6)</td>
			<td>LaVision Biotec</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 568 Donkey anti Rabbit</td>
			<td>Thermo Fisher</td>
			<td>A10042</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 Donkey anti goat</td>
			<td>Jackson ImmunoResearch</td>
			<td>705-605-147</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 Donkey anti Rabbit</td>
			<td>Jackson ImmunoResearch</td>
			<td>711-605-152</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-LYVE1 polyclonal antibody</td>
			<td>Angiobio</td>
			<td>#11-034</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-PROX1 goat polyclonal IgG antibody</td>
			<td>R&#38;D systems</td>
			<td>#AF2727</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine Injection Ampoules (Buprecare solution, 0.3mg/ml)</td>
			<td>Animalcare</td>
			<td>Ampule 1ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Dibenzyl Ether 100% (DBE)</td>
			<td>Sigma Aldrich</td>
			<td>108014</td>
			<td></td>
		</tr>
		<tr>
			<td>Dichloromethane 100% (DCM)</td>
			<td>Sigma Aldrich</td>
			<td>270997</td>
			<td></td>
		</tr>
		<tr>
			<td>Formic acid 99%</td>
			<td>CARLO ERBA</td>
			<td>405793</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma Aldrich</td>
			<td>G.7126</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparine sodium salt from porcine</td>
			<td>Sigma Aldrich</td>
			<td>H4784</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen peroxide solution (H2O2 30%)</td>
			<td>Sigma Aldrich</td>
			<td>H1009</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane (Iso-Vet 100%)</td>
			<td>Piramal</td>
			<td>NDC 66794-013-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol 100%</td>
			<td>Sigma Aldrich</td>
			<td>322415</td>
			<td></td>
		</tr>
		<tr>
			<td>Ovalbumin Alexa Fluor 555 Conjugate</td>
			<td>Invitrogen</td>
			<td>11549176</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffer Solution PBS (stock solution 10X)</td>
			<td>Euromedex</td>
			<td>ET330-A</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pentobarbital (Euthasol 400mg/mL)</td>
			<td>Dechra</td>
			<td>08718469445110</td>
			<td></td>
		</tr>
		<tr>
			<td>Tri-sodium citrate</td>
			<td>VWR</td>
			<td>6132-04-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical tools and equipments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anaesthesia system</td>
			<td>Univentor</td>
			<td>Univentor 410 Anaesthesia Unit</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass micropipette puller</td>
			<td>Narishige</td>
			<td>PC-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>CMA Microdialysis AB</td>
			<td>CMA 450 Temperature controller</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcapillaries (Glass Capillaries)</td>
			<td>Harvard Apparatus</td>
			<td>GC120-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Microforceps, forceps,dissection scissors and Michel Suture Clips (7.5&#8201;&#215;&#8201;1.75mm)</td>
			<td>Fine Science Tool</td>
			<td>12040-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel (sterile disposable scalpel 23)</td>
			<td>Swann-Norton</td>
			<td>0510</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotaxic apparatus</td>
			<td>KOPF</td>
			<td>Model 940</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe Hamilton 10&#181;l 701N</td>
			<td>Hamilton</td>
			<td>28618-U</td>
			<td></td>
		</tr>
		<tr>
			<td>Warm air System</td>
			<td>Vet-Tech LTD</td>
			<td>HE011</td>
			<td></td>
		</tr>
	</tbody>
</table>

three-dimensional imaging of the vertebral lymphatic vasculature and drainage using idisco+ and light sheet fluorescence microscopy

The lymphatic system associated with the central nervous system (CNS) includes the lymphatic vasculature that spins around the brain, the spinal cord, and its associated LNs. The CNS-associated lymphatic system is involved in the drainage of CSF macromolecules and meningeal immune cells toward CNS-draining LNs, thereby regulating waste clearance and immune surveillance within CNS tissues. Presented is a novel approach to obtain three-dimensional (3D) and cellular resolution images of CNS-associated lymphatics while preserving the integrity of their circuits within surrounding tissues. The iDISCO+ protocol is used to immunolabel lymphatic vessels in decalcified and cleared whole mount preparations of the vertebral column that are subsequently imaged with light sheet fluorescence microscopy (LSFM). The technique reveals the 3D structure of the lymphatic network connecting the meningeal and epidural spaces around the spinal cord to extravertebral lymphatic vessels. Provided are 3D images of the drainage circuits of molecular tracers previously injected into either the CSF via the cisterna magna or the thoracolumbar spinal parenchyma. The iDISCO+/LSFM approach brings unprecedented opportunities to explore the structure and function of the CNS-associated lymphatic system in neurovascular biology, neuroimmunology, brain and vertebral cancer, or vertebral bone and joint biology.

3D imaging of the vertebral lymphatic vasculature 
Figure 1 presents the steps of the iDISCO+/LSFM procedure and a LSFM image of lymphatic circuits inside the vertebral canal of iDISCO+-treated ThLb vertebrae. The combination of iDISCO+ with LSFM preserved the vertebral anatomy and captured a view of the lymphatic vascular network (i.e., the intravertebral vessels connected with the extravertebral vessels exiting dorsally and laterally from the vertebral body) within the surrounding bones, ligaments, muscles, and nerve ganglia.
Fluorescence macroscopy imaging of dcLN drainage 
In order to image macromolecule drainage in the CNS-associated lymphatic system, macromolecular tracers were administrated in vivo by injection into either the CSF or the spinal parenchyma. A macromolecular tracer can be easily delivered into the CSF at the cisterna magna. The cisterna magna is located between the cerebellum and the dorsal surface of the medulla oblongata, above the foramen magnum. Macromolecular tracer can also be injected into the spinal parenchyma by stereotactic surgery at different levels along the vertebral column.
The macromolecular tracers used were either directly labeled with a fluorophore or detected postmortem by immunohistochemistry with specific antibodies. Figure 2A illustrates the experimental plan for tracking OVA-A555, a red fluorescent and small molecular weight tracer (around 45 kDa), that was injected into either the CSF (Figure 2B) or the ThLb region of the spinal cord (Figure 2C).
At 45 min after the macromolecular tracer injection, the mice were sacrificed, perfused with 4% PFA, and processed to isolate dissected segments of the brain stem region of the head and the vertebral column that were decalcified and clarified. Macromolecule drainage was then readily assessed by fluorescence macroscopy imaging of the LNs that collect the CSF and epidural fluids. As shown in Figure 2, OVA-A555 injection into either the CSF (Figure 2B) or the ThLb (Figure 2C) region of the spinal cord resulted in OVA-A555 accumulation into the dcLNs at 45 min after injection. This observation indicates the uptake and drainage of fluorescent tracer by the lymphatic system; it is a prerequisite before pursuing the iDISCO+/LSFM procedure to image the vertebral lymphatic drainage.
3D imaging of macromolecule drainage in the vertebral lymphatic system 
Based on the detection of OVA-A555 labeling in dcLNs, the iDISCO+/LSFM procedure could be applied to decalcified and precleared vertebral samples isolated from OVA-A555-injected mice. This approach allowed the creation of a 3D map of the lymphatic drainage of CSF and spinal epidural fluid at a specific time point after tracer injection. This 3D mapping could be performed by imaging successive CNS segments, at each level of vertebral column, from the injection point.
Figure 3A shows the experimental design for OVA-A555 injection into the ThLb spinal parenchyma and the resulting 3D pattern of OVA-A555 distribution in a cervical and a thoracic vertebral segment, in conjunction with the lymphatic vasculature. At 45 min after OVA-A555 injection, OVA-A555 accumulation was detected in spinal cord tissues and dcLNs (white arrows in Figure 3B), in agreement with microscope observations illustrated in Figure 2. It was not detected, however, in the cervical and thoracic lymphatic vasculature labeled with anti-LYVE1 antibodies. The absence of CSF-injected tracer in the vertebral lymphatic vessels may be due to either a short persistence time of tracer inside lymphatic vessels or a lack of uptake of the tracer by the lymphatic vessels (Figure 3C).
To test the first hypothesis, the rabbit anti-LYVE1 antibody was used as a lymphatic endothelial cell tag to bind CNS-associated lymphatic vessels. The injected rabbit anti-LYVE1 antibody was thereafter detected by immunohistochemistry with an anti-rabbit secondary antibody, while lymphatic endothelial cells were immunolabeled with anti-PROX1 antibodies. Figure 4 represents the experimental design of anti-LYVE1 injection into the ThLb spinal parenchyma (Figure 4A), and the resulting 3D distribution pattern of LYVE1 antibodies in a ThLb segment close to the injection site, with respect to PROX1+ lymphatics (Figure 4B). Both vertebral lymphatics and their extravertebral lymphatic connections were labeled by injected anti-LYVE1 antibodies, which substantiated the tracer uptake by vertebral lymphatic vessels and the lymphatic drainage toward the extravertebral lymphatic system. LNs were lacking in the ThLb region and, thus, could not be visualized in the imaged segment. Furthermore, the discontinuous pattern of LYVE1 marker observed along lymphatic vessels likely reflected the discontinuous expression of LYVE1 in the lymphatic vasculature, as reported in previous studies14,21. Altogether, the results of the present study demonstrated that, at 45 min after tracer injection, LYVE1 antibody, but not OVA-A555, allowed detection of local vertebral lymphatic uptake and was preferrable to OVA-A555 as a persistent marker of local vertebral lymphatic drainage.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61099/61099fig01v2.jpg" /> 
Figure 1: Three-dimensional view of the vertebral lymphatic vasculature.&#160;(A) Schematic representation of the protocol. (B) Planar projection of a 3D view of the ThLb vertebral lymphatic vasculature from a dorsofrontal perspective. Lymphatic vessels were immunolabeled with the anti-PROX1 antibody (green) using the iDISCO+ protocol and then imaged by LSFM. Note the metameric-like pattern of the vasculature within the vertebral canal of three successive vertebrae (white arrowheads). In addition to semicircular dorsal vessels (white arrowheads), each vertebral network included ventral branches (yellow arrows), bilateral lateral exit pathways along the spinal rami and ganglia (white arrows), as well as a dorsal exit route at the midline (white double arrow). Vertebral networks were interconnected with a longitudinal vessel (purple arrows). For a complete description, see Jacob L. et al.18 SC = spinal cord; Asterisk = ventral vertebral body; D = dorsal; L = lateral; V = ventral; Scale bars = 300 &#181;m. <a href="https://www.jove.com/files/ftp_upload/61099/61099fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61099/61099fig02v2.jpg" /> 
Figure 2: dcLNs collected OVA-A555-labeled CSF Fluids.&#160;(A) Scheme of the experimental design. OVA-A555 was injected into either the ICM or the ThLb spinal parenchyma, then mice were sacrificed 45 min after injection. The samples were cleared and observed by fluorescence macroscope imaging (B,C). Fluorescence macroscope images of cervival vertebrae from ICM- (B) and ThLb- (C) injected mice. Note that OVA-A555 accumulated in the dcLNs (B,C, white arrowheads), the pial and paravascular spaces of the cervical spinal cord (B,C) and after ICM injection, in ventrolateral exit routes, likely along cervical nerves (B, white arrows). SC = spinal cord. Asterisk = ventral vertebral body; D = dorsal; L = lateral; V = ventral; Scale bars in B and C = 2 mm. <a href="https://www.jove.com/files/ftp_upload/61099/61099fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61099/61099fig03v2.jpg" /> 
Figure 3: Detection of OVA-A555-labeled CSF fluids in the vertebral lymphatic system.&#160;(A) Scheme of the experimental design. OVA-A555 was injected into the ThLb spinal parenchyma, then mice were sacrificed at 45 min after injection. The samples were treated with the iDISCO+ protocol and imaged with a LSFM. (B,C) Planar projections of LSFM-captured frontal 3D views of cervical (B) and thoracic (C) spine segments. OVA-A555 accumulation (red) was detected in spinal cord tissues and dcLNs (B, white arrow), as illustrated in Figure 2, but not in the cervical and thoracic lymphatic vasculature immunolabeled here with anti-LYVE1 antibodies (green). SC = spinal cord; Asterisk = ventral vertebral body; D = dorsal; L = lateral; V = ventral; Scale bars = 1 mm (B), 300 &#181;m (C). <a href="https://www.jove.com/files/ftp_upload/61099/61099fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61099/61099fig04v2.jpg" /> 
Figure 4: Detection of vertebral lymphatic drainage after intraspinal injection of anti-LYVE1 antibodies.&#160;(A) Scheme of the experimental design. Anti-LYVE1 antibodies were injected into the ThLb spinal parenchyma, then mice were sacrificed 45 min after injection. The samples were treated with the iDISCO+ protocol and imaged with a LSFM. (B). Planar projections of frontal 3D views of a ThLb (B) spine segment, captured with a LSFM. Anti-LYVE1 antibodies were detected with anti-rabbit antibodies (purple) and the lymphatic vasculature with anti-PROX1 antibodies (green). White vessels are PROX1+ lymphatics colabeled with anti-LYVE1 antibodies (B); these include vertebral (yellow arrows) and extravertebral (double yellow arrows) lymphatics. SC = spinal cord; Asterisk = ventral vertebral body; D = dorsal; L = lateral; V = ventral; Scale bars = 300 &#181;m (B). <a href="https://www.jove.com/files/ftp_upload/61099/61099fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagents</td>
			<td>Target</td>
			<td>Figure</td>
			<td>Protocol step</td>
			<td>Comment</td>
		</tr>
		<tr>
			<td>OVA-A555</td>
			<td>CSF tracer</td>
			<td>Figure 2 and Figure 3</td>
			<td>2. ICM or ThLb injection 
			7. LSFM imaging.</td>
			<td>Water soluble, easy to inject and high intense fluorescence</td>
		</tr>
		<tr>
			<td rowspan="2">Anti-Lyve1 antibody</td>
			<td>Membrane marker of LVs cells</td>
			<td>Figure 3</td>
			<td>6. iDISCO+ whole mount immnunostaining. 
			7. LSFM imaging.</td>
			<td rowspan="2">Efficient antibody to whole mount immnunostaining</td>
		</tr>
		<tr>
			<td>Tracer drainage of the dural and epidural LVs</td>
			<td>Figure 4</td>
			<td>2. ICM or ThLb injection 
			6. iDISCO+ whole mount immnunostaining 
			7. LSFM imaging</td>
		</tr>
		<tr>
			<td>Anti-Prox1 antibody</td>
			<td>Nuclear marker of LVs cells</td>
			<td>Figure 1 and Figure 4</td>
			<td>6. iDISCO+ whole mount immnunostaining 
			7. LSFM imaging</td>
			<td>Efficient antibody to whole mount immnunostaining</td>
		</tr>
	</tbody>
</table>
Table 1: Antibodies and tracers used in the study.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Problem</td>
			<td>Possible reason</td>
			<td>Solution</td>
		</tr>
		<tr>
			<td rowspan="3">Surgery for tracer injection</td>
			<td>Unwanted CNS tissue lesion</td>
			<td>1. Lack of control of glass capillary insertion 
			2. Incorrect depth of glass capillary insertion</td>
			<td>1. Punctate with care, but fully, the dura mater with a 26 G needle before glass capillary insertion. 
			2. Reduce the deepness of glass capillary insertion (&#60;1.5 mm from the dura mater). 
			3. Reduce the glass capillary diameter.</td>
		</tr>
		<tr>
			<td rowspan="2">Unwanted defilement of injected tracer into the epidural or extra-vertebral spaces</td>
			<td>Incorrect injection of tracer</td>
			<td>1. Check if the glass micro capillary has been well inserted into the punctate the dura mater. 
			2. Add surgical glue between glass microcapillary and the surrounded tissue before injecting tracer.</td>
		</tr>
		<tr>
			<td>Excessive volume of injected tracer</td>
			<td>Reduce the volume of injected tracer (&#60;2 &#181;L).</td>
		</tr>
		<tr>
			<td rowspan="2">iDISCO+ immunostaining</td>
			<td rowspan="2">Absence, heterogeneity or excessive background of labeling in the tissue</td>
			<td>1. Issues with the concentration of the primary antibody 
			2. Insufficient permeabilization 
			3. Insufficient washing 
			4. Insufficient clearing</td>
			<td>Increase the number and/or the time of incubation steps: permebilization, whashing, primary antibody and clearing. See http://www.idisco.info (FAQ AND TROUBLESHOOTING).</td>
		</tr>
		<tr>
			<td>Insufficient decalcification</td>
			<td>Use a more stringent decalcification treatment of sample with EDTA21 or Morse solution for head tissues especially.</td>
		</tr>
		<tr>
			<td rowspan="3">iDISCO+ clearing</td>
			<td rowspan="3">Samples are opaque or brown-colored</td>
			<td>Insufficient bleaching</td>
			<td>Use fresh H2O2 solution, increase volume and/or incubation time.</td>
		</tr>
		<tr>
			<td>Presence of oxidation</td>
			<td>Fill the tube completely to avoid the presence of air.</td>
		</tr>
		<tr>
			<td>Insufficient clearing</td>
			<td>Increase volume and/or incubation time. See http://www.idisco.info (FAQ AND TROUBLESHOOTING).</td>
		</tr>
		<tr>
			<td rowspan="2">Tracer detection</td>
			<td rowspan="2">Undetectable tracer</td>
			<td>Incorrect combination of the selected tracer</td>
			<td>Alexa 555, 594 and 647 fluorochromes are resistant to iDISCO+ protocol5,6,7,8,9,10. However, this is not the case for FITC, GFP, RFP fluorochromes.</td>
		</tr>
		<tr>
			<td>Sacrifice timepoint after injection</td>
			<td>Prefer OVA-A555 for short-term (15 min) drainage analysis in local vertebral lymphatics. For lymph nodes drainage analysis, OVA-A555 and Lyve1 antibody can be used for longer term (&#62;45 min) analysis.</td>
		</tr>
		<tr>
			<td rowspan="2">Imaging: capture and analysis</td>
			<td rowspan="2">Captured images of the lymphatic circuit are not satisfactory</td>
			<td>Dissection issue (lymph nodes are missing)</td>
			<td>Include carefully the vertebral/skull neighboring tissues in your dissected sample, according to the lymphatic circuit that you want to image.</td>
		</tr>
		<tr>
			<td>Imaging issue</td>
			<td>1. Modify the acquisition parameters of the LSFM: laser intensity, light-sheet numerical aperture, thickness of light-sheet, exposition time. 
			2. Place the sample in the support to reduce the path travelled by light through the tissue until the objective. 
			3. Be sure that there are no bubbles inside the tissue sample during acquisition.</td>
		</tr>
	</tbody>
</table>
Table 2: Troubleshooting advice for each step of the protocol, including possible issues and solutions.

Watch this Scientific Journal Video about Three-Dimensional Imaging of the Vertebral Lymphatic Vasculature and Drainage using iDISCO+ and Light Sheet Fluorescence Microscopy at JoVE.com

Three-Dimensional Imaging of the Vertebral Lymphatic Vasculature and Drainage using iDISCO+ and Light Sheet Fluorescence Microscopy

A protocol is presented combining tissue clearing with light sheet fluorescence microscopy (LSFM) to obtain three-dimensional and cellular resolution images of the lymphatic vessels and lymph nodes (LNs) collecting the cerebrospinal fluid (CSF) and spinal epidural fluid.

Three-Dimensional Imaging of the Vertebral Lymphatic Vasculature and Drainage using iDISCO+ and Light Sheet Fluorescence Microscopy

The lymphatic system associated with the central nervous system (CNS) includes the lymphatic vasculature that spins around the brain, the spinal cord, and ...

three-dimensional-imaging-vertebral-lymphatic-vasculature-drainage

Research

JoVE Journal

Immunology and Infection

9.9K Views.  Institut du Cerveau et de la Moelle Epinière. A protocol is presented combining tissue clearing with light sheet fluorescence microscopy (LSFM) to obtain three-dimensional and cellular resolution images of the lymphatic vessels and lymph nodes (LNs) collecting the cerebrospinal fluid (CSF) and spinal epidural fluid.

Video: Three-Dimensional Imaging of the Vertebral Lymphatic Vasculature and Drainage using iDISCO+ and Light Sheet Fluorescence Microscopy

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external environment. The cell can either recover from this insult, which requires active membrane repair processes, or else die depending on the amount of toxin exposure and cell type1. In addition, these toxins induce strong inflammatory responses in infected hosts through activation of immune cells, including macrophages, which produce an array of pro-inflammatory cytokines2. Many Gram positive bacteria produce cholesterol binding toxins which have been shown to contribute to their virulence through largely uncharacterized mechanisms.
Morphologic changes in the plasma membrane of cells exposed to these toxins include their sequestration into cholesterol-enriched surface protrusions, which can be shed into the extracellular space, suggesting an intrinsic cellular defense mechanism3,4. This process occurs on all cells in the absence of metabolic activity, and can be visualized using EM after chemical fixation4. In immune cells such as macrophages that mediate inflammation in response to toxin exposure, induced membrane vesicles are suggested to contain cytokines of the IL-1 family and may be responsible both for shedding toxin and disseminating these pro-inflammatory cytokines5,6,7. A link between IL-1&beta; release and a specific type of cell death, termed pyroptosis has been suggested, as both are caspase-1 dependent processes8. To sort out the complexities of this macrophage response, which includes toxin binding, shedding of membrane vesicles, cytokine release, and potentially cell death, we have developed labeling techniques and fluorescence microscopy methods that allow for real time visualization of toxin-cell interactions, including measurements of dysfunction and death (Figure 1). Use of live cell imaging is necessary due to limitations in other techniques. Biochemical approaches cannot resolve effects occurring in individual cells, while flow cytometry does not offer high resolution, real-time visualization of individual cells. The methods described here can be applied to kinetic analysis of responses induced by other stimuli involving complex phenotypic changes in cells.

Methods for purifying the cholesterol binding toxin streptolysin O from recombinant E. coli and visualization of toxin binding to live eukaryotic cells are described. Localized delivery of toxin induces rapid and complex changes in targeted cells revealing novel aspects of toxin biology.

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external ...

Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse

The lymphatic vascular system is an important component of the circulatory system that maintains fluid homeostasis, provides immune surveillance, and mediates fat absorption in the gut. Yet despite its critical function, there is comparatively little understanding of how the lymphatic system adapts to serve these functions in health and disease1. Recently, we have demonstrated the ability to dynamically image lymphatic architecture and lymph "pumping" action in normal human subjects as well as in persons suffering lymphatic dysfunction using trace administration of a near-infrared fluorescent (NIRF) dye and a custom, Gen III-intensified imaging system2-4. NIRF imaging showed dramatic changes in lymphatic architecture and function with human disease. It remains unclear how these changes occur and new animal models are being developed to elucidate their genetic and molecular basis. In this protocol, we present NIRF lymphatic, small animal imaging5,6 using indocyanine green (ICG), a dye that has been used for 50 years in humans7, and a NIRF dye-labeled cyclic albumin binding domain (cABD-IRDye800) peptide that preferentially binds mouse and human albumin8. Approximately 5.5 times brighter than ICG, cABD-IRDye800 has a similar lymphatic clearance profile and can be injected in smaller doses than ICG to achieve sufficient NIRF signals for imaging8. Because both cABD-IRDye800 and ICG bind to albumin in the interstitial space8, they both may depict active protein transport into and within the lymphatics. Intradermal (ID) injections (5-50 &mu;l) of ICG (645 &mu;M) or cABD-IRDye800 (200 &mu;M) in saline are administered to the dorsal aspect of each hind paw and/or the left and right side of the base of the tail of an isoflurane-anesthetized mouse. The resulting dye concentration in the animal is 83-1,250 &mu;g/kg for ICG or 113-1,700 &mu;g/kg for cABD-IRDye800. Immediately following injections, functional lymphatic imaging is conducted for up to 1 hr using a customized, small animal NIRF imaging system. Whole animal spatial resolution can depict fluorescent lymphatic vessels of 100 microns or less, and images of structures up to 3 cm in depth can be acquired9. Images are acquired using V++ software and analyzed using ImageJ or MATLAB software. During analysis, consecutive regions of interest (ROIs) encompassing the entire vessel diameter are drawn along a given lymph vessel. The dimensions for each ROI are kept constant for a given vessel and NIRF intensity is measured for each ROI to quantitatively assess "packets" of lymph moving through vessels.

Recently developed imaging techniques using near-infrared fluorescence (NIRF) may help elucidate the role the lymphatic system plays in cancer metastasis, immune response, wound repair, and other lymphatic-associated diseases.

The lymphatic  vascular system is an important component of the circulatory system that  maintains fluid homeostasis, provides immune surveillance, and ...

Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy

Many aspects of the infection of macrophages by Cryptococcus neoformans have been extensively studied and well defined. However, one particular interaction that is not clearly understood is non-lytic exocytosis. In this process, yeast cells are released into the extracellular space by a poorly understood mechanism that leaves both the macrophage and Cn viable. Here, we describe how to follow a large number of individually infected macrophages for a 24 hr infection period by time-lapsed microscopy. Infected macrophages are housed in a heating chamber with a CO2 atmosphere attached to a microscope that provides the same conditions as a cell-culture incubator. Live digital microscopy can provide information about the dynamic interactions between a host and pathogen that is not available from static images. Being able to visualize each infected cell can provide clues as to how macrophages handle fungal infections, and vice versa. This technique is a powerful tool in studying the dynamics that are behind a complex phenomenon.

Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy

We describe how to visualize macrophage-C. neoformans (Cn) interactions in real time, with specific emphasis on the process of non-lytic exocytosis using digital light microscopy. Using this technique individually infected macrophages can be studied to ascertain various aspects of this phenomenon.

Many aspects of the infection of macrophages by Cryptococcus neoformans have been extensively studied and well defined. However, one particular ...

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target cells. Inside the host cell the effector proteins manipulate cellular functions to the benefit of the bacteria. To better understand the control of type III secretion during host cell interaction, sensitive and accurate assays to measure translocation are required. We here describe the application of an assay based on the fusion of a Yersinia enterocolitica effector protein fragment (Yersinia outer protein; YopE) with TEM-1 beta-lactamase for quantitative analysis of translocation. The assay relies on cleavage of a cell permeant FRET dye (CCF4/AM) by translocated beta-lactamase fusion. After cleavage of the cephalosporin core of CCF4 by the beta-lactamase, FRET from coumarin to fluorescein is disrupted and excitation of the coumarin moiety leads to blue fluorescence emission. Different applications of this method have been described in the literature highlighting its versatility. The method allows for analysis of translocation in vitro and also in in vivo, e.g., in a mouse model. Detection of the fluorescence signals can be performed using plate readers, FACS analysis or fluorescence microscopy. In the setup described here, in vitro translocation of effector fusions into HeLa cells by different Yersinia mutants is monitored by laser scanning microscopy. Recording intracellular conversion of the FRET reporter by the beta-lactamase effector fusion in real-time provides robust quantitative results. We here show exemplary data, demonstrating increased translocation by a Y. enterocolitica YopE mutant compared to the wild type strain.

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Effector translocation into host cells via a type III secretion system is a common virulence strategy among gram-negative bacteria. A beta-lactamase effector fusion based assay for quantitative analysis of translocation was applied. In Yersinia infected cells, conversion of a FRET reporter by the beta-lactamase is monitored using laser scanning microscopy.

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target ...

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila express gustatory receptors1,2, ion channels3-6, and ionotropic receptors7 that are involved in the detection of volatile and non-volatile sensory cues. These neurons directly contact tastants such as food, noxious substances and pheromones and therefore influence many complex behaviors such as feeding, egg-laying and mating. Electrode recordings and calcium imaging have been widely used in insects to quantify the neuronal responses evoked by these tastants. However, electrophysiology requires specialized equipment and obtaining measurements from a single taste sensillum can be technically challenging depending on the cell-type, size, and position. In addition, single neuron resolution in Drosophila can be difficult to achieve since taste sensilla house more than one type of chemosensory neuron. The live calcium imaging method described here allows responses of single gustatory neurons in live flies to be measured. This method is especially suitable for imaging neuronal responses to lipid pheromones and other ligand types that have low solubility in water-based solvents.

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

The forelegs and proboscis of Drosophila contain a rich repertoire of gustatory sensory neurons. Here, we present a method using calcium imaging to measure physiological responses from sensory neurons in the foreleg and proboscis of live flies upon exogenous application of a gustatory pheromone.

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila ...

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo fluorescence optical imaging (OI), in two different mouse models of inflammation: a rheumatoid arthritis (RA) and a contact hypersensitivity reaction (CHR) model. Light with a wavelength in the near infrared (NIR) window (650 - 950 nm) allows a deeper tissue penetration and minimal signal absorption compared to wavelengths below 650 nm. The major advantages using fluorescence OI is that it is cheap, fast and easy to implement in different animal models.
Activatable fluorescent probes are optically silent in their inactivated states, but become highly fluorescent when activated by a protease. Activated MMPs lead to tissue destruction and play an important role for disease progression in delayed-type hypersensitivity reactions (DTHRs) such as RA and CHR. Furthermore, MMPs are the key proteases for cartilage and bone degradation and are induced by macrophages, fibroblasts and chondrocytes in response to pro-inflammatory cytokines. Here we use a probe that is activated by the key MMPs like MMP-2, -3, -9 and -13 and describe an imaging protocol for near infrared fluorescence OI of MMP activity in RA and control mice 6 days after disease induction as well as in mice with acute (1x challenge) and chronic (5x challenge) CHR on the right ear compared to healthy ears.

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

This paper explains the application of fluorescent imaging using an activatable optical imaging probe to visualize the in vivo activity of key matrix metalloproteinases in two different experimental models of inflammation.

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo ...

Visualizing Intracellular SNARE Trafficking by Fluorescence Lifetime Imaging Microscopy

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze membrane fusion within eukaryotic cells. The SNARE protein family consists of about 36 different members. Specific intracellular transport routes are catalyzed by specific sets of 3 or 4 SNARE proteins that thereby contribute to the specificity and fidelity of membrane trafficking. However, studying the precise function of SNARE proteins is technically challenging, because SNAREs are highly abundant and functionally redundant, with most SNAREs having multiple and overlapping functions. In this protocol, a new method for the visualization of SNARE complex formation in live cells is described. This method is based on expressing SNARE proteins C-terminally fused to fluorescent proteins and measuring their interaction by F&#246;rster resonance energy transfer (FRET) employing fluorescence lifetime imaging microscopy (FLIM). By fitting the fluorescence lifetime histograms with a multicomponent decay model, FRET-FLIM allows (semi-)quantitative estimation of the fraction of the SNARE complex formation at different vesicles. This protocol has been successfully applied to visualize SNARE complex formation at the plasma membrane and at endosomal compartments in mammalian cell lines and primary immune cells, and can be readily extended to study SNARE functions at other organelles in animal, plant, and fungal cells.

This protocol describes a new method allowing for the quantitative visualization of complex formation of SNARE proteins, based on F&#246;rster resonance energy transfer, and fluorescence lifetime imaging microscopy.

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze ...

Light-sheet Microscopy for Three-dimensional Visualization of Human Immune Cells

In vivo, activation, proliferation, and function of immune cells all occur in a three-dimensional (3D) environment, for instance in lymph nodes or tissues. Up to date, most in vitro systems rely on two-dimensional (2D) surfaces, such as cell-culture plates or coverslips. To optimally mimic physiological conditions in vitro, we utilize a simple 3D collagen matrix. Collagen is one of the major components of extracellular matrix (ECM) and has been widely used to constitute 3D matrices. For 3D imaging, the recently developed light-sheet microscopy technology (also referred to as single plane illumination microscopy) is featured with high acquisition speed, large penetration depth, low bleaching, and photocytotoxicity. Furthermore, light-sheet microscopy is particularly advantageous for long-term measurement. Here we describe an optimized protocol how to set up and handle human immune cells, e.g. primary human cytotoxic T lymphocytes (CTL) and natural killer (NK) cells in the 3D collagen matrix for usage with the light-sheet microscopy for live cell imaging and fixed samples. The procedure for image acquisition and analysis of cell migration are presented. A particular focus is given to highlight critical steps and factors for sample preparation and data analysis. This protocol can be employed for other types of suspension cells in a 3D collagen matrix and is not limited to immune cells.

Here, we present a protocol to visualize immune cells embedded in a three-dimensional (3D) collagen matrix using light-sheet microscopy. This protocol also elaborates how to track cell migration in 3D. This protocol can be employed for other types of suspension cells in the 3D matrix.

In vivo, activation, proliferation, and function of immune cells all occur in a three-dimensional (3D) environment, for instance in lymph nodes or ...

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling and cellular processes that control efferocytosis. This quantification can be difficult to perform as apoptotic cells are often efferocytosed piecemeal, thus necessitating methods which can accurately delineate between the efferocytosed portion of an apoptotic target versus residual unengulfed cellular fragments. The approach outlined herein utilizes dual-labeling approaches to accurately quantify the dynamics of efferocytosis and efferocytic capacity of efferocytes such as macrophages. The cytosol of the apoptotic cell is labeled with a cell-tracking dye to enable monitoring of all apoptotic cell-derived materials, while surface biotinylation of the apoptotic cell allows for differentiation between internalized and non-internalized apoptotic cell fractions. The efferocytic capacity of efferocytes is determined by taking fluorescent images of live or fixed cells and quantifying the amount of bound versus internalized targets, as differentiated by streptavidin staining. This approach offers several advantages over methods such as flow cytometry, namely the accurate delineation of non-efferocytosed versus efferocytosed apoptotic cell fractions, the ability to measure efferocytic dynamics by live-cell microscopy, and the capacity to perform studies of cellular signaling in cells expressing fluorescently-labeled transgenes. Combined, the methods outlined in this protocol serve as the basis for a flexible experimental approach that can be used to accurately quantify efferocytic activity and interrogate cellular signaling pathways active during efferocytosis.

Efferocytosis, the phagocytic removal of apoptotic cells, is required to maintain homeostasis and is facilitated by receptors and signaling pathways that allow for the recognition, engulfment, and internalization of apoptotic cells. Herein, we present a fluorescence microscopy protocol for the quantification of efferocytosis and the activity of efferocytic signaling pathways.

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...