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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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 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.

Introduction

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 (NFκB) translocation to the nucleus of the cell. This causes the prototypic immune cell to produce and release pro-inflammatory cytokines such as Interleukin (IL)-1β, IL-6, and TNF-α6. 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.  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.  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.

Protocol

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.  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 mice, also purchased commercially (Jackson, 007914) and (FSP1cre)+/- ; tdTomatolox-stop-lox and FSP1cre-/-; tdTomatolox-stop-lox mice were bred in-house on a C57BL/6 background (Figure 1A,B,C). Fibroblast-specific protein 1 is an endogenous protein expressed on roughly 72% of fibroblast and represents an effective cre driver in dermal tissue20. Mice were group housed and given ad libitum access to food and water. Room temperature was maintained at 21 ± 2 °C. While we used C57BL/6 crossed male and female mice at 8-12 weeks, we do not believe the age, sex, or genetic background are necessary requirements to run multiphoton experiments. Mice were deeply anesthetized immediately after the experiment and euthanized.

1. Preparation of drugs

  1. Prepare a 5 µg/20 µL solution of lipopolysaccharide-FITC (See Table of Materials) in sterile 1x PBS (pH 7.4). Vortex the stock solution at medium intensity for minimally 30 seconds to ensure homogenous mixing for an equal concentration throughout the solution before pipetting (LPS is a glutinous molecule).  
    NOTE: Do not place LPS into a glass container, if possible, use siliconized microcentrifuge tubes. Keep on ice until use.

2. Imaging set-up

  1. Set up the multiphoton system (see Table of Materials) for two-color imaging. This requires the use of two separate excitation lasers (see Table of Materials), a GFP/RFP filter cube set (see materials table), and a 25x (1.05 NA) water-immersion objective (see Table of Materials).
    NOTE: Users can alter these settings to better suit alternative setups and multiphoton microscope capabilities. These are the parameters used in the experimental protocol. See Table of Materials for specific details.
  2. Place a stereotaxic apparatus (see Table of Materials) on the stage of the multiphoton microscope. Connect this to an anesthesia delivery machine (see Table of Materials) to ensure the animals are anesthetized for the duration of the experiment. Place a piece of matte black paper on the surface of the apparatus as a connection point for the mouse paw.
  3. Select the resonant scanner with a fixed scan area of 512 µm x 512 µm.
    NOTE: Do not perform the experiments in this protocol using a galvanometer scanner. Due to the slower sample rate, there will be distortion in z-stack time-lapse videos due to the respiration of the animal that is unavoidable.
  4. Tune the two excitation lasers to the excitation wavelengths of GFP and RFP, 930 nm and 1100 nm respectively, and direct the light path of both excitation lasers to the single objective using a dichroic mirror of 690-1,050 nm allowing the 930 nm-tuned excitation laser to be reflected to the main scanner and the 1,100 nm-tuned laser to pass directly into the main scanner (Figure 2).
    NOTE: It is possible to alter these excitation wavelengths depending on the user's setup.
  5. Set the laser power of FITC to 5% and GFP to 20%.
    NOTE: This setup provides an optimal signal-to-noise ratio (SNR) in these experiments. Detect signal via multi-alkali photo-multiplier tubes (PMTs); however, GaAsP detectors may be used instead in very high-sensitivity experiments.
  6. Prepare the room for imaging under dark conditions without stray light.

3. In vivo imaging

  1. Place the mouse into the induction chamber of the anesthesia delivery system and use 5% isoflurane (see Table of Materials) at a 2 L/min flow rate of oxygen to place the mouse under deep anesthesia.
     NOTE: It is highly recommended to wear all appropriate PPE during the experiment.
    1. Transfer the mouse to the stereotaxic apparatus with access to a nose cone to maintain anesthesia throughout the experiment. Reduce the isoflurane to 1.5%-2% and keep the flow rate constant.
    2. Firmly affix the hind paw to a piece of black paper with black tape on areas both proximal and distal to the area of interest (this reduces the effects of mouse respiration on image quality), making sure the plantar surface of the paw is unobstructed and facing up towards the objective. Ensure that the head of the mouse is stable within the nose cone attached to the apparatus.
      NOTE: Monitor the mouse for any signs of distress or dehydration throughout the experiment and adjust isoflurane accordingly.
  2. Place a generous amount of sterile water-based lubricant (gel, see Table of Materials) on the plantar surface of the paw and touch the objective to it in order to create a column of liquid between the paw and the objective.
  3. Use the FITC excitation light to focus into the dermal layer of the paw. Ensure that tdTomato-tagged fibroblasts are visualized before continuing on (this step is important in determining the correct focal plane to image).
    NOTE: The dermal layer of the paw is about 100-150 µm into the paw.
  4. Image the area of cells located just below the plantar surface of the hind paw with both the 930 nm and 1100 nm-tuned lasers and acquire a 15-minute time-lapse of about 5-10 z-slices at approximately 1 µm per slice to establish a representation of the environment prior to injection with LPS-FITC.
  5. Perform an intraplantar injection of 5 µg/20 µL LPS-FITC on the mouse’s hind paw using a 25 µL glass Hamilton syringe (see Table of Materials) and 30G needle (see Table of Materials), taking care to not disturb the position of the paw.
  6. Image an area of cells located just below the plantar surface of the hind paw with both the 930 nm and 1100 nm-tuned lasers and acquire a 60-120 minute time-lapse of about 5-10 z-slices at approximately 1 µm per slice to identify uptake of LPS-FITC by cells.

Results

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...

Discussion

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...

Disclosures

The authors declare that they have no competing financial interests.

Acknowledgements

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

Materials

NameCompanyCatalog NumberComments
10x PBS, 4 LFisherbrandBP3994
700 Series MICROLITER Syringes, Hamilton, Model 705 LT SyringeHamilton80501
BD Precision Glide Needle 30GVWR305106
Blue PadFisherbrand1420665
Filter Cube: Green/Red (BP 495-540 DM570 BP 575-645)OlympusFV30-FGR
Isoflurane, USP 250 mLVedco50989-150-15
Lipopolysaccharides from Escherichia Coli O111:B4 - FITC conjugateSigma-AldrichF3665-1MG
Main scanner laser: Spectra Physics INSIGHT DS+ -OL pulsed IR LASER, tunable from 680 nm to 1300 nm, 120 fs pulse width at specimen planeSpectra Physics
Micro Centrifuge Tubes, 1.5 mLVWR20170-333
Multiphoton Microscope: Olympus MPE-RS TWINOlympusMPE-RS TWIN
Objective: Ultra 25x MPE water-immersion objective 1.05 NA, 2 mm WDOlympusXLPLN25XWMP2
Personal Lubricant Jelly (Gel)equateZH727 2E F1
SGM-4 Stereotaxic ApparatusNarishige16030
SomnoSuite Low-Flow Anesthesia SystemKent Scientific CorporationSS-01
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 planeSpectra Physics

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