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Haston1 project protocol

Susceptibility to radiation-induced fibrotic lung disease in 26 inbred strains of mice   (2012)

Haston CK
With: Paun A




Haston1 Protocol

Project protocol - Contents

Workflow and sampling

Step
Procedure
Equipment
Data collected
1
 Mice irradiated at 8-10 wks of age
Gamma irradiator
-
2
 Mice monitored over next 26 wks; euthanized when moribund
-
Number of days without respiratory distress
3 Bronchoalveolar lavage Cannulus Inflammatory cell counts
4 Histopathology Microscope and camera
Extent of fibrosis, mast cell counts, alveolitis score

Equipment and supplies

  • Gamma Cell Cesium-137
  • Dissection kit
  • Cannulus
  • Cytocentrifuge
  • Microtome
  • Microscope with digital camera
  • Image Pro Plus Software

Reagents and solutions

  • Phosphate buffered saline (PBS)
  • 10% neutral buffered formalin
  • Masson's Trichrome
  • Hematoxylin and eosin (H&E) kit (Hema-3 Stain Set)
  • Hematoxylin and eosin (H&E)
  • Toluidine blue

Procedure: Irradiation and monitoring

    1. Lung damage is elicited by whole thorax radiation exposure (18 Gray (Gy)); dose rate 0.7 Gy/min) using a Gamma irradiator.
    2. Mice are weighed weekly from 8 wks post-treatment; mice losing >20% of their body weight, and exhibiting distress through ruffled fur, accelerated breathing and hunched posture are euthanized. Mice not exhibiting distress during this time period are euthanized at 26 wks.

Procedure: Bronchoalveolar lavage and analysis

    1. At necropsy bronchoalveolar lavage (BAL) is performed by cannulating the trachea and retrieving cells from a single 1-mL injection of PBS.
    2. BAL fluid is centrifuged at 302 g for 10 min.
    3. Cells are resuspended in PBS and stained with an H&E kit.
    4. Cells are cytocentrifuged at 214.2 g for 3 min.
    5. Inflammatory cell counts are performed at 400x magnification.

Procedure: Histopathology

    1. At necropsy (after bronchoalveolar lavage), lungs are removed and the left lobe is perfused with 10% neutral buffered formalin and processed for histology using three methods described in steps 2, 3, 4.
    2. Lung sections are stained with Masson's Trichrome to determine the area of fibrosis using Image Pro Plus Software (fibrosis reported as percentage of entire lobe).
    3. Other sections are stained with Toluidine blue and positive cells (Mast cells) are counted and averaged across 10 fields of lung area (400x magnification).
    4. Other sections are stained with H&E to assess alveolitis using semi-quantitative histology.
    5. All scoring is completed by a researcher blinded to mouse strain and treatment.

Data collected by investigator

  • survival (days post irradiation without respiratory distress)
  • degree of fibrosis [average for untreated control mice = 0.0%]
  • BAL macrophages
  • BAL lymphocytes
  • BAL neutrophils
  • mast cells per mm2 of lung tissue [average for untreated control mice = 0.07]
  • alveolitis (inflammation index) [average for untreated control mice = 1.6%]