Name: in vivo two-color 2-photon imaging of genetically-tagged reporter cells in the skin
Uploaded: 2019-07-11T13:30:00
Description: Watch this Scientific Journal Video about In Vivo Two-Color 2-Photon Imaging of Genetically-Tagged Reporter Cells in the Skin at JoVE.com

This work is supported by the grant NS096030 (MDB). We would also like to thank the imaging core manager Ved Prakash. We would also like to thank Olympus Discovery Center/Imaging Core facility at UT Dallas for providing equipment and support

Animal procedures were approved by The University of Texas at Dallas Institutional Animal Care and Use Committee and were in accordance with National Institutes of Health guidelines.&#160; All experiments were performed using 8-12-week-old male and female mice bred in-house on a C57BL/6 background. Transgenic mice with cre-recombinase driven by the fibroblast-specific protein-1 promoter were purchased commercially (Jackson, 012641) (FSP1cre)+/- and crossed with tdTomatolox-stop-lox&#160;mic...

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Arguably the most important steps of in vivo 2-photon imaging are: 1) Choosing the right genetic reporter mouse and fluorescently-tagged protein for the multi-photon setup and experimental needs21,22; 2) imaging the correct focal plane to have an accurate representation of the population of cells in the tissue23; 3) minimizing movement due to an improperly immobilized animal24; and 4) choosing when to analyze data q...

Lipopolysaccharide (LPS) is an endotoxin found in the outer membrane of gram-negative bacteria1. LPS has a high binding affinity for the toll-like receptor 4 (TLR4)/CD14/MD2 receptor complex2. TLR4 is a pattern recognition receptor commonly found on the outer membrane of a wide range of immune cells, mesenchymal cells, and a subset of sensory neurons3,4,5. Activation of TLR4 expressed on immune cells leads to MyD88-dependent and independent second messenger systems, ending with nuclear-factor kappa beta (NF&#954;B) translocation to the nucleus of the cell. This causes the prototypic immune cell to produce and release pro-inflammatory cytokines such as Interleukin (IL)-1&#946;, IL-6, and TNF-&#945;6. However, how other cell-types respond to TLR4 stimulation is not as clear. Fibroblasts have been implicated in a wide range of pathologies such as cancers and cystic fibrosis and have recently been shown to play a role monocyte chemo-attraction and promoting inflammation7,8,9. &#160;Our lab is interested in the role of fibroblasts in the development of acute and chronic pain, as early evidence suggests that factors released by fibroblasts (matrix metalloproteinases (MMPs), tissue inhibitor of metalloproteinases (TIMPs), and fibroblast growth factors (FGFs)) are involved in neuropathic pain10.
Activation states of cells can be determined by a variety of factors that include: induction of immediate-early genes, altered protein expression, cell proliferation, and morphological changes11,12,13. There are many techniques that exist to answer questions we may have about how activation of cells contributes to pathologies, but they all have their limitations. Prototypical immunohistochemistry uses fluorescently tagged antibodies to label proteins of interest in fixed tissue, which may be unspecific and often require significant troubleshooting before yielding fruitful results14. Western blotting is a useful technique when comparing levels of protein expression in post-mortem tissue; however, the histological component is lacking in this technique and researchers are unable to identify any changes in morphology15. RNA-Seq allows us to quantify the presence of messenger RNA in a sample which in many instances yields insightful data; however, the gap between transcription and translation makes it difficult to have temporal resolution after a stimulus16. Confocal imaging is useful in determining the expression and co-localization of proteins that exist in a cross-section of tissues17. Often, this is not representative of the entirety of the tissue sample. In contrast, multiphoton microscopes allow users to image roughly 1 mm deep into a sample, creating a comprehensive three-dimensional representation18. Therefore, we choose to focus on in vivo, 2-photon imaging preps, as data collected from these experiments are more directly relatable to the highly plastic and interconnected microenvironment of living tissue.
An advantage of studying protein-receptor interactions in vivo is that we can clearly capture how cells respond to a stimulus, real-time, in their native environment without the harmful and unpredictable influence of post-mortem tissue extraction19. In addition, longitudinal studies may be performed to assess cellular plasticity and priming that may occur because of activation.&#160; Using 2-photon imaging, we preserve the integrity of the microenvironment present when an external stimulus is applied. This protocol provides a way to identify uptake of molecules in fibroblasts following peripheral injection of fluorescently tagged LPS over the course of several hours in vivo and the role of TLR4 in fibroblast activation.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:1131px;">10x PBS, 4 L</td>
			<td style="width:185px;">Fisherbrand</td>
			<td style="width:116px;">BP3994</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">700 Series MICROLITER Syringes, Hamilton, Model 705 LT Syringe</td>
			<td>Hamilton</td>
			<td>80501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD Precision Glide Needle 30G</td>
			<td>VWR</td>
			<td>305106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blue Pad</td>
			<td>Fisherbrand</td>
			<td>1420665</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Filter Cube: Green/Red (BP 495-540 DM570 BP 575-645)</td>
			<td>Olympus</td>
			<td>FV30-FGR</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoflurane, USP 250 mL</td>
			<td>Vedco</td>
			<td>50989-150-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lipopolysaccharides from Escherichia Coli O111:B4 - FITC conjugate</td>
			<td>Sigma-Aldrich</td>
			<td>F3665-1MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Main scanner laser: Spectra Physics INSIGHT DS+ -OL pulsed IR LASER, tunable from 680 nm to 1300 nm, 120 fs pulse width at specimen plane</td>
			<td>Spectra Physics</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro Centrifuge Tubes, 1.5 mL</td>
			<td>VWR</td>
			<td>20170-333</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Multiphoton Microscope: Olympus MPE-RS TWIN</td>
			<td>Olympus</td>
			<td>MPE-RS TWIN</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Objective: Ultra 25x MPE water-immersion objective 1.05 NA, 2 mm WD</td>
			<td>Olympus</td>
			<td>XLPLN25XWMP2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Personal Lubricant Jelly (Gel)</td>
			<td>equate</td>
			<td>ZH727 2E F1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SGM-4 Stereotaxic Apparatus</td>
			<td>Narishige</td>
			<td>16030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SomnoSuite Low-Flow Anesthesia System</td>
			<td>Kent Scientific Corporation</td>
			<td>SS-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stimulation laser: Olympus-specific SPECTRA PHYSICS MAI TAI HP DEEP SEE-OL pulsed IR LASER, tunable from 690 nm to 1040 nm, 100 fs pulse width at specimen plane</td>
			<td>Spectra Physics</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo two-color 2-photon imaging of genetically-tagged reporter cells in the skin

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located in. Although traditional imaging techniques are useful in identifying protein interactions and morphology in fixed tissue, they are not able to give insight as to how quickly cells are able to bind and uptake proteins, and once activated how their morphology changes in vivo. In the present study, we ask 2 major questions: 1) what is the time-course of fibroblast activation via toll-like receptor-4 (TLR4) and lipopolysaccharide (LPS) interaction and 2) how do these cells behave once activated? Using 2-photon imaging, we have developed a novel technique to assess the ability of LPS-FITC to bind to its cognate receptor, TLR4, expressed on peripheral fibroblasts in the genetic reporter mouse line; FSP1cre; tdTomatolox-stop-lox&#160;in vivo. This unique approach allows researchers to create in-depth, time-lapse videos and/or pictures of proteins interacting with live cells that allows one to have an increased level of granularity in understanding how proteins can alter cellular behavior.

Initially, we injected LPS-FITC into the hind-paw of wild-type mice in order to visualize the uptake of LPS-FITC in all cell types present in the dermal layer of the paw. Having observed a myriad of cells in the dermal layer of the hind paw uptake fluorescently-tagged LPS in a wild type mouse (Video Figure 1, 2), we tried to specifically target fibroblasts as they are a primary focus in our research. Before imaging the paws of animals injected with LPS-FITC we wanted to be clear that there is no inherent...

Watch this Scientific Journal Video about In Vivo Two-Color 2-Photon Imaging of Genetically-Tagged Reporter Cells in the Skin at JoVE.com

In Vivo Two-Color 2-Photon Imaging of Genetically-Tagged Reporter Cells in the Skin

Morphological changes occur in immune responsive fibroblast cells following activation and promote alterations in cellular recruitment. Utilizing 2-photon imaging in conjunction with a genetically engineered Fibroblast-specific protein 1 (FSP1)-cre; tdTomato floxed-stop-floxed (TB/TB) mouse line and green fluorescently tagged lipopolysaccharide-FITC, we can illustrate highly specific uptake of lipopolysaccharide in dermal fibroblasts and morphological changes in vivo.

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located ...

Our protocol allows us to visualize how fluorescently tagged cells respond to an inflammatory peripheral stimulus in a live animal in real time. 2-Photon imaging allows visualization deep into the tissue of a live specimen, preserving the integrity of their cells and their microenvironment and providing an accurate representation of the biological system. Breathing moves the paw over time, causing blurriness and a loss of the focal plane.Be sure to affix the paw firmly with tape to a stable surface before imaging. In this you must be able to find appropriate plane of interest, make sure that objective is close to the paw without touching and is directly above injection site. Before beginning the procedure, turn on the multi-photon system and select the 25X subjective.Place a stereotaxic apparatus onto the stage of the multi-photon microscope and connect the apparatus to an anesthesia delivery machine. Place a piece of matte black paper on the surface of the apparatus as a connection point for the mouse paw. Set the resonance scanner with a fixed scan area of 512 by 512 micrometers.Tune the excitation lasers to the 930 and 1, 100 nanometer excitation wavelengths for green and red fluorescent protein signals respectively. Use a dichroic mirror of 690 to 1, 050 nanometers to direct the light path of both excitation lasers to the single objective allowing the 930 nanometer tuned excitation laser to be reflected to the main scanner and the 1, 100 nanometer tuned laser to pass directly into the main scanner. Then set the laser power of FITC to 5%and green fluorescent protein to 20%and turn off the overhead lights.For in vivo imaging, anesthetize the mouse and outlined in the text protocol. Confirm a lack of response to toe pinch in the anesthetized mouse and place the mouse in the stereotaxic apparatus with access to a nose cone. Use black tape to firmly affix the hind paw to the piece of black paper on areas both proximal and distal to the area of interest, making sure the plantar surface of the paw is unobstructed and facing up toward the objective.Place a generous amount of water based lubricant onto the plantar surface of the paw. Touch the objective to the lubricant to create a column of liquid between the paw and the objective. Use the FITC excitation light to focus into the dermal layer of the paw, confirming that the tandem dimer Tomato tagged fibroblasts can be visualized.Image the area of cells located just below the plantar surface of the hind paw with both lasers. Acquire a 15 minute time lapse of about five to 10 Z-slices at approximately one micrometer per slice to establish a baseline representation of the environment. When the baseline imaging has been obtained, load a 25 microliter glass Hamilton syringe with five micrograms of FITC conjugated lipopolysaccharide, or LPS, per 20 microliters of PBS.Administer the solution by intraplantar injection into the experimental hind paw without disturbing the paw position. Then image an area of cells located just below the plantar surface of the hind paw with both lasers. Acquire a 60 to 120 minute time lapse of about five to 10 Z-slices at approximately one micrometer per slice to identify the cell mediated intraplantar uptake of LPS FITC.As there is no inherent fluorescence by the cells within the dermal layer, a myriad of cells in the dermal layer of the hind paw can be observed taking fluorescently tagged LPS after intraplantar injection in a wild type mouse. After LPS FITC injection, only fibroblast specific protein one positive fibroblasts expressing toll like receptor four bind and uptake the injected protein with a high level of co-localization with a tandem dimer Tomato tag expressed by these cells. In contrast, mice that have toll like receptor four knocked out of the entire body do not bind and uptake LPS after injection.Indeed, cell silhouettes are visible after LPS FITC injection indicating that the drug is dispersing in the interstitial fluid around cells, but is not actually being bound by a receptor. The most important thing to remember in this protocol is to ensure that the paw's immobilized so that there are no distortions in the video due to movement or respiration. Using this procedure, the recruitment of fluorescently tagged cells to an area after injury and the morphological changes in a cell's response to a stimulus over time can be tracked.So 2-Photon imaging allows researchers to combine genetic reporter mice and fluorescently tagged compounds to assess what happens in a live animal after an injection.

Research

JoVE Journal

Bioengineering

Three-dimensional Optical-resolution Photoacoustic Microscopy

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Viral Nanoparticles for In vivo Tumor Imaging

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A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

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Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

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Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

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An In Vitro Organ Culture Model of the Murine Intervertebral Disc

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in ...