Acute Exposure to Particulate Air Pollution in Childhood Asthma

EPA Grant Number: R825702C013
Subproject: this is subproject number 013 , established and managed by the Center Director under grant R825702
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: Environmental Lung Disease Center (National Jewish Medical and Research Center)
Center Director: Mason, Robert J.
Title: Acute Exposure to Particulate Air Pollution in Childhood Asthma
Investigators: Chang, Ling-Yi
Current Investigators: Rabinovitch, Nathan , Gelfand, Erwin
Institution: National Jewish Medical and Research Center
EPA Project Officer: Chung, Serena
Project Period: February 16, 1998 through February 28, 2003 (Extended to February 28, 2004)
RFA: Environmental Lung Disease Center (National Jewish Medical and Research Center) (1998) RFA Text |  Recipients Lists
Research Category: Targeted Research

Objective:

The Core will be responsible for providing consultation and physical resources to carry out light and electron microscopic evaluations of cells and/or tissues being studied under the component projects. In addition to providing partial support in microscopy and pathology for all research projects, the Core provide electron microscopic service to projects 1: Particle induced lung inflammation and EC-SOD.

Functions

The specific function of the Morphology Core is to provide:

Appropriate Histopathology Facilities. Many of the component project involve exposure of animals. histopathology examinations of lung tissue are required for proper documentation and quality control. Other morphological experiments that can be conducted include examinations of cell structure (for instance, cytoskeleton), protein expression and protein localization.

Advice and Consultation. The morphometry/morphology Core will ensure availability of professional personnel with sufficient experience in both light and electron microscopy and in histopathological techniques. Dr. Ling-Yi Chang, the Core Director, has extensive experiences in experimental lung pathology, immunocytochemistry and morphometry using both light and EM microscopy.

Teakhing/Education. This Core will provide training for laboratory personnel in microscopy and pathological techniques. Technicians, students and fellows working on projects under this program who need access to light or electron microscopy will be trained by the Laboratory Researcher IV working in this Core.

Electron Microscopy Support for Project 1. Ultrastructural changes occurring in the terminal bronchioles and proximal ventilatory units after particle exposure described in Dr Crapo's investrigation of particle induced lung injury will require:

  • Plastic embedding
  • Ultrathin sectioning
  • Qualitative EM analysis of lung tissue

Histology Support for Projects 1, 2, 4 and 5 and the Pilot Projects 2 and 3. This unit will provide general histology and guidance on quantitation within the limits of its budget. If the demand signficantly exceeds the budget, the costs will have to revert to the individual projects.

Immunocytochemistry Support for Projects 4 and 1. Particle induced lung inflammation and No, induced alterations in the expressions of hexokinase and thioredoxin in the lung will be examined by light microscopic immunocytochemistry. These studies will require:

  • Immunolabeling of paraffin sections
  • Qualitative analysis of the localization of intensity of the immunostain

Quality Assurance. Quality assurance programs have been and will continue to be developed to ensure that procedures are constant between individuals and between groups. Individuals are not permitted to begin working on instruments prior to training and/or evaluation by qualified personnel. All equipment is calibrated against standards at regular intervals. Light and electron microscopes are aligned on a weekly basis.

Methods of Procedures

Each lung submitted for histologic studies will be inflation fixed unless otherwise noted. The choice of fixative depends on the histological procedure to be performed. When morphometric studies are involved, shrinkage of tissue is minimized by the choice of fixative having the correct osmolarity ( 1,2), usually 2% glutaraldehyde. The fixation will be carefully monitored to fix the lung at a uniform degree of inflation. The approaches used for either small or large animals and described the complications commonly encountered have been described in detail previously (3,4). After fixation, lung or lobe volume will be determined by water displacement. Lung for immunocytochemical investigations will be processed by a pamformaldehyde based fixative. Biopsies lung samples will be fixed by either immersion or injection of fixative with a fine needle into the specimen. Cell suspensions and cultured cells will be fixed by addition of a double strength of the fixative of choice.

Histology Four micron thick paraffin sections of the lung will be stained with hematoxylin and eosin, Sirius red (collagen) or Verhoeff-Van Gieson (elastic tissue) stains. All of the sections will be reviewed by Dr. Chang for both the extent and severity of tissue injury, with special emphasis placed on:

  • Inflammatory cell present
  • airway morphology, evidence of injury or remodeling
  • Interstitial cell reactions and connective tissue changes
  • Microvascular injury
  • Evidence of other intercurrent processes

Immunocytochemistry Paraffin sections will be processed for light microscopic staining of EC- SOD/NOS/nitrotyrosine (Crapo) or hexokinase/thioredoxin (White). Paraffin sections will be deparaffinize and hydrated and undergo anitigen retrieval procedure with trypsin or heating in citrate buffer (optional). Intrinsic peroxidase ativity and non-specific binding will be blocked by incubations with 1% H202 and 10% normal serum (from host species of the 2nd antibody) respectively. Specific protein localization will be achieved by incubation sequentially with the primary antibody, biotinylated secondary antibody, strepavidin conjugated to horseradish peroxidase, and DAB substrate. The slides will be counterstained with methyl green or H&E and examined for location and intensity of the aantibody reactions.

The procedures described above can be applied extensively in our laboratory to a variety of proteins/enzymes and can be adapted for proteins of interest in other projects (5-7). Fluorescent tagged secondary antibodies can be used instead of HRP.

Electron microscopic studies Ultrastructural studies of the lung will be performed on mouse lung tissue after particle exposures (Crapo). Randomly selected terminal airways and proximal ventilator-y units will be embedded in expoxy resin. Ultrathin sections will be cut and examined with a Philip CM10 electron microscope for the following changes:

  • Injury to ciliated and Clara cells in the terminal bronchioles
  • Thickening of bronchiolar and alveolar basement membranes
  • Injury to alveolar type I epithelium
  • Cellular or interstitial edema
  • Interstitial fibrosis

Other procedures We have established procedures for in situ hybridization for the identification of cells/cell types that express a specific mRNA (7,8), immuno-cytochemical labeling on 4 pm thick cryostat sections for proteins sensitive to fixative and high resolution immunocytochemistry with semithin (OS- 1 pm) and ultrathin sections of liquid nitrogen flash frozen tissue (6,9,10) as well as methods for light and EM morphometry (1 l- 13). These techniques can be adapted for use with any studies that found a need for morphological evaluations.

Quality Assurance

The methods used for histology, immunocytochenistry and electron microscopic procedures in our laboratory have been well documented. Precautionary procedures are installed throughout the investigation to ensure overall quality of the study. These procedures are outlined below:

  • Duplicate slides (for each staining procedure) are produced, one for the principal investigator, the other kept on file for the morphology core.
  • Postive and negative controls will be included for every immunocytochemical staining run.
  • Each specimen will be assigned a identification code. Record for each specimen with respect to experimental treatments, specimen collection and preparation conditions and interpretation of the pathology results will be kept on file.
  • Light microscopes are aligned on a monthly basis. The electron microscope is aligned on a weekly basis.
  • A calibration grid is taken with each separate set of micrographs to ensure that the correct magnification can be calculated.
  • Detailed records of all micrographs and negatives are kept on file.
  • Dr. Chang will review slides regularily with pathologist at the University of Colorado Health Science Center to ensure proper interpretation of lung pathology.

Resources

The facilities for this Core, which will be utilized by the majority of the proposed projects, occupy approximately ? square feet of space located in the Goodman building and in the Littman building. This include laboratories designed for processing tissue for light and electron microscopy. Available equipment includes:

1. Philip CM 10 electron microscope
2. Philip M400 electron microscope
3. Three Reichert ultramicrotomes, one with cryo attachments
4. Leica ultramicrotome with cryo attachments
5. Nikon Optiphot fluorescence light photomicrscope
6. Nikon Labphot photomicroscope
7. Wild double head dissecting microscope
8. Light microscopy morphometry work station attached to an Olympus LM microscope
9. LKB automated processor for plastic embedding
10. Automated processor for paraffin embedding
11. Plate/film processing Darkroom
12. Print processing dark room equipped with Durst enlarger and Kodak Royal print processor

In addition, two fluorescent confocal microscope equipped with the NIH image analysis program are located in the flow cytometry laboratory on the 10fh floor of the Goodman building. They are also available for use by investigators in this application.

Publications and Presentations:

Publications have been submitted on this subproject: View all 20 publications for this subprojectView all 132 publications for this center

Journal Articles:

Journal Articles have been submitted on this subproject: View all 5 journal articles for this subprojectView all 110 journal articles for this center

Supplemental Keywords:

Health, Scientific Discipline, PHYSICAL ASPECTS, Risk Assessments, Microbiology, Disease & Cumulative Effects, Physical Processes, Molecular Biology/Genetics, Biology, Histology, Electron Microscopy, Immunology, health effects, electron microscope, immune system effects, pathology, lung disease, airway disease, exposure, human exposure, immunocytochemistry, cell injury, histopathology, animal studies, exposure assessment, human health risk, immune response, protein expression

Progress and Final Reports:

  • 1998
  • 1999
  • 2000
  • 2001
  • 2002
  • 2003
  • Final Report

  • Main Center Abstract and Reports:

    R825702    Environmental Lung Disease Center (National Jewish Medical and Research Center)

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R825702C001 SP-A and SP-D in Environmental Lung Disease
    R825702C003 Adaptation to Nitrogen Dioxide: Role of Altered Glycolytic Pathway Enzyme Expression and NF-κB-Dependent Cellular Defenses Against Apoptosis
    R825702C005 Inhalation of Particulate Matter Alters the Allergic Airway Response to Inhaled Allergen
    R825702C006 Particle-Induced Lung Inflammation and Extracellular EC-SOD
    R825702C007 Indoor-Outdoor Relationships of Airborne Particle Count and Endotoxin Concentrations
    R825702C008 The Role of Mitochondrial DNA Mutations in Oxidant-Mediated Lung Injury
    R825702C009 Immunopathogenesis of Hypersensitivity Pneumonitis in the Mouse
    R825702C010 Activation of Natural T Lymphocytes by Diesel Exhaust Particulates Leads to Their Production of Interleukin-4 and TH2 Lymphocyte Differentiation to Allergen
    R825702C011 Latex Antigen Levels During Powdered and Powderless Glove Use
    R825702C012 Adjuvant Effects of Ozone in a Model of Allergen-Induced Airway Inflammation and Hyperresponsiveness
    R825702C013 Acute Exposure to Particulate Air Pollution in Childhood Asthma
    R825702C014 Mechanisms of Ozone Toxicity to the Lung