Visualization of Amino Acid Metabolites in Intestinal Tissues of Infected Mice

Samenvatting

Source: Specker, J. T., et al. Investigation of Microbial Cooperation via Imaging Mass Spectrometry Analysis of Bacterial Colonies Grown on Agar and in Tissue During Infection. J. Vis. Exp. (2022)

This video demonstrates the use of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry to visualize amino acid distribution in intestinal tissues from mice infected with Clostridioides difficile, alone or in combination with Enterococcus faecalis. The procedure involves tissue preparation, matrix application, laser ionization, and detection of amino acid ions to generate spatial ion images. The results reveal microbial cooperation, as E. faecalis alters the gut environment by releasing ornithine and depleting arginine, thereby enhancing C. difficile pathogenesis.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Preparation and shipment of noninfected control and C. difficile-infected mouse cecal tissues
   1. Inoculate 4-8-week-old C57BL/6 male mice with antibiotics (0.5 mg/mL cefoperazone or 0.5 mg/mL cefoperazone + 1 mg/mL vancomycin) in drinking water ad libitum for 5 days, followed by a 2-day recovery period and subsequent infection.
   2. Euthanize the animals by CO₂ asphyxiation and harvest the mouse cecum organ immediately. Embed in a 20% mixture of optimal cutting temperature (OCT) compound in distilled water.
   3. Place the samples on dry ice and then package and ship for analysis; store at -80 °C until analysis.

 

2. Cryosectioning of noninfected control versus C. difficile-infected mouse cecal tissues

1. Clean all cryosectioning equipment by rinsing with 100% ethanol and allow to dry before placing samples into the cryostat chamber. Prepare ITO-coated microscope slides by using an ohmmeter to identify the side of the slide with the conductive coating. Use a diamond-tipped scribe to etch and label the ITO-coated side of the microscope slide.
   NOTE: Proper personal protective equipment, including gloves, laboratory glasses, a lab coat, and cryostat sleeves, should be worn.
2. Perform cryosectioning on a research cryomicrotome. Transfer the OCT-embedded mouse ceca tissues from a -80 °C freezer to the cryomicrotome chamber (30 °C chamber temperature, -28 °C object temperature) on dry ice. Mount the tissue samples to be compared using imaging mass spectrometry (e.g., noninfected control vs. C. difficile-infected) on the same microscope slide to ensure identical sample preparation of both tissue types and enable accurate metabolite comparisons.
3. Inside the cryomicrotome chamber, mount the OCT-embedded tissue on a sample chuck using an additional OCT solution. After the OCT solution has solidified, fix the chuck to the specimen head. Begin cryosectioning at 10-50 µm increments to reach the desired tissue depth/plane of the organ.
4. Once an optimal cross-section of the tissue is reached, begin collecting sections at 12 µm thickness. Gently manipulate the slice using artist paintbrushes and place it onto a Teflon-coated microscope slide.
5. Roll the ITO-coated microscope slide on top of the tissue section to pick up the tissue from the Teflon-coated slide. Thaw mount the tissue section to the microscope slide by pressing the palm to the back of the ITO-coated slide. Continue to thaw the mount until the tissue section transitions from translucent to an opaque/matte texture.
6. Transfer the tissue-mounted microscope slides on dry ice to a dry box with desiccant for storage for same-day analysis, or store at -80 °C for longer-term storage.

3. Preparation of noninfected control versus C. difficile-infected mouse cecal tissues for matrix application and MALDI imaging mass spectrometry

1. Perform MALDI matrix application (30 °C nozzle temperature, eight passes, 0.1 mL/min flow rate, CC pattern, 0 s drying time, 10 psi).
2. Perform MALDI imaging mass spectrometry
3. Analyze the ion images from multiple biological replicates and compare the results for significance via intensity box plot comparisons using the SCiLS statistical software referenced above.
   NOTE: The appropriate statistical tests and comparisons will depend on the application and can include spatial segmentation, classification models, and comparative analysis for determining discriminative and correlated spectral features. Comparative analyses can include assigning p-values to significant features, generating principal component analyses for tissue comparisons, and visualizing box plots to identify variations. It is also useful to visualize the tissue using brightfield microscopy of the tissue section stained via hematoxylin and eosin (H&E) following imaging mass spectrometry. This enables clear identification of morphological features in the tissue and can be analyzed in consultation with a trained pathologist.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
0.2 μm Titan3 nylon syringe filters	Thermo Scientific	42225-NN
1,5-diaminonaphthalene MALDI matrix	Sigma Aldrich	2243-62-1
20 mL Henke Ject luer lock syringes	Henke Sass Wolf	4200.000V0
275i series convection vacuum gauge	Kurt J. Lesker company	KJL275807LL
7T solariX FTICR mass spectrometer equipped with a Smartbeam II Nd:YAG MALDI laser system (2 kHz, 355 nm)	Bruker Daltonics
Acetic acid solution, suitable for HPLC	Sigma Aldrich	64-19-7
Acetonitrile, suitable for HPLC, gradient grade, ≥99.9%	Sigma Aldrich	75-05-8
Ammonium hydroxide solution, 28% NH3 in H2O, ≥99.99% trace metals basis	Sigma Aldrich	1336-21-6
Autoclavable biohazard bags: 55 gal	Grainger	45TV10
Biohazard specimen transport bags (8 x 8 in.)	Fisher Scientific	01-800-07
Brain heart infusion broth	BD Biosciences	90003-040
C57BL/6 male mice	Jackson Laboratories
CanoScan 9000F Mark II photo and document scanner	Canon
CM 3050S research cryomicrotome	Leica Biosystems
Desiccator cabinet	Sigma Aldrich	Z268135
Diamond tip scriber, Electron Microscopy Sciences	Fisher Scientific	50-254-51
Drierite desiccant pellets	Drierite	21005
Ethanol, 200 Proof	Decon Labs	2701
flexImaging software	Bruker Daltonics
ftmsControl software	Bruker Daltonics
Glass vacuum trap	Sigma Aldrich	Z549460
HTX M5 TM robotic sprayer	HTX Technologies
Indium Tin Oxide (ITO)-coated microscope slides	Delta Technologies	CG-81IN-S115
In-line HEPA filter to vacuum pump	LABCONCO	7386500
Methanol, HPLC Grade	Fisher Chemical	67-56-1
MTP slide-adapter II	Bruker Daltonics	235380
Optimal cutting temperature (OCT) compound	Fischer Scientific	23-730-571
Peridox RTU Sporicide, Disinfectant and Cleaner	CONTEC	CR85335
PTFE (Teflon) printed slides, Electron Microscopy Sciences	VWR	100488-874
Rotary vane vacuum pump RV8	Edwards	A65401903
Tissue-Tek Accu-Edge Disposable High Profile Microtome Blades	Electron Microscopy Sciences	63068-HP
Transparent vacuum tubing	Cole Palmer	EW-06414-30
Ultragrade 19 vacuum pump oil	Edwards	H11025011
Variable voltage transformer	Powerstat
Water, suitable for HPLC	Sigma Aldrich	7732-18-5
Wide-mouth dewar flask	Sigma Aldrich	Z120790