Iraqi1 project protocol

Susceptibility to periodontitis in 4 inbred strains and 19 emerging lines (pre-CC) of the Collaborative Cross   (2013)

Iraqi FA
With: Shusterman A, Salyma Y, Nashef A, Soller M, Wilensky A, Mott R, Weiss EI, Houri-Haddad Y




Iraqi1 Protocol

Project protocol - Contents

Workflow and sampling

Step
Procedure
 Equipment
Data collected
1
 Prepare periodontal pathogenic bacteria Anerobic chamber, spectrophotometer
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2
 Mice treated to suppress normal oral flora, followed by a 3-day wash out (antibiotic free)  
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3

Cohort 1: Control mice treated with PBS; Cohort 2: Treated mice infected with P. gingivalis and F. nucleatum in PBS

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4 Mice euthanized 42 days post-infection; maxillae harvested for analysis Dissection kit, computerized tomography (microCT) Maxillary jaw alveolar bone volume

Equipment and supplies

  • Anaerobic bacterial chamber (85% N2, 5% H2, and 10% CO2)
  • Compact fan-beam-type computerized tomography (microCT 40, Scanc Medical, Bassersdorf SWITZERLAND)

Reagents and solutions

  • Porphyromonas gingivalis ATCC 381
  • Fusobacterium nucleatum PK 1594
  • Peptone yeast extract with hemin and Vitamin K (Wilkins Chalgren broth, Oxoid Ltd, UK)
  • Phosphate buffered saline (PBS)
  • Sulfamethoxazole
  • Trimethoprim
  • Carboxymethylose

Procedure: Bacterial preparation

    1. P. gingivalis and F. nucleatum are grown in peptone yeast extract containing hemin and vitamin K in an anaerobic chamber followed by three washes in PBS.
    2. Bacterial concentration is spectrophotometrically standardized to OD650nm = 0.1 for P. gingivalis (1010 bacteria/mL), and OD660nm = 0.26 for F. nucleatum (109 bacteria/mL).

Procedure: Bacterial infection and harvesting maxillae

    1. Mice are pre-treated with sulfamethoxazole/trimethoprim (0.08% and 0.016%, respectively) in drinking water for 10 days, followed by a three day wash out (no antibiotics).
    2. Treated mice are infected with 400 µL containing 109 bacteria/mL of each pathogen at days 0, 2, and 4, using 2% carboxymethycellulose in PBS.
    3. Control mice receive PBS only.
    4. Mice are euthanized at 42 days post-infection and maxillae harvested.

Procedure: microCT analysis

    1. Samples are placed in a cylindrical sample holder and approximately 200 microtomographic slices are obtained (12 µm increments) covering the entire bucco-palatal width of each hemi-jaw.
    2. Maxillary hemi-jaws are analyzed by compact fan-beam-type computerized tomography (microCT).
    3. For calculation of bone volume in control and treated mice, a reference line is set throughout the microtomographic slices at a distance from the cemento-enamel junction, chosen to be below the horizontal zone of destruction in treated mice.
    4. Bone volumes are recorded for control and treated mice.

Data collected by investigator

  • Maxillary jaw alveolar bone volume: uninfected, control volume
  • Maxillary jaw alveolar bone volume: infected, residual volume