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Application of laser capture microdissection and protein microarray technologies in the molecular analysis of airway
Elizabeth Roberts1, Lu Charboneau, Virginia Espina
1Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina, USA.
Abstract:
Understanding the mechanisms by which various types of air pollution particles (particulate matter, PM) mediate adverse health effects would provide biological plausibility to epidemiological associations of increased rates of morbidity and mortality. The majority of information regarding the means by which PM generates lung injury has been derived from in vitro studies. However, it is unclear as to what extent these mechanisms can be extrapolated to the in vivo situation. Current methods to assess mechanisms of PM-induced lung injury make it difficult to obtain site-specific, sensitive, and comprehensive determinations of cellular and molecular pathology associated with PM-induced injury. In the present study, the ability of laser capture microdissection (LCM) and protein microarray technologies were assessed to examine the effect of residual oil fly ash (ROFA) exposure on airway intracellular signaling pathways and transcription factor activation. Sprague-Dawley rats were intratracheally instilled with 0.5 mg/rat of ROFA. LCM was used to recover airway cells and protein extracts derived from the microdissected airways were analyzed by protein microarray. ROFA exposure increased p-ERK:ERK and p-I kappa B:I kappa B, suggesting changes in cell growth, transformation, and inflammation within the airway. These results are consistent with previously reported in vitro findings, demonstrating for the first time the credibility of applying LCM and protein microarray technologies to assess acute lung injury induced by environmental air pollutants.
Insights
New methods using laser capture microdissection and protein microarrays reveal how air pollution particles cause lung injury in vivo. This study shows changes in cell growth and inflammation pathways after exposure to residual oil fly ash.
Area of Science:
- Environmental Health
- Toxicology
- Molecular Biology
Background:
- Epidemiological studies link air pollution particles (PM) to increased morbidity and mortality.
- In vitro studies suggest mechanisms of PM-induced lung injury, but in vivo relevance is unclear.
- Current methods for assessing PM-induced lung injury lack site-specificity and comprehensiveness.
Purpose of the Study:
- To evaluate laser capture microdissection (LCM) and protein microarray technologies for assessing in vivo lung injury.
- To investigate the effects of residual oil fly ash (ROFA) exposure on airway intracellular signaling and transcription factor activation.
- To establish the utility of LCM and protein microarrays for studying environmental air pollutant-induced lung injury.
Main Methods:
- Sprague-Dawley rats were exposed to residual oil fly ash (ROFA) via intratracheal instillation.
- Laser capture microdissection (LCM) was employed to isolate airway cells.
- Protein extracts from microdissected airways were analyzed using protein microarray technology.
Main Results:
- ROFA exposure led to increased levels of phosphorylated ERK and I kappa B relative to their total amounts (p-ERK:ERK and p-I kappa B:I kappa B).
- These molecular changes suggest alterations in cell growth, transformation, and inflammation within the airways.
- Findings align with previous in vitro observations, validating the in vivo approach.
Conclusions:
- Laser capture microdissection (LCM) combined with protein microarrays is a credible approach for assessing acute lung injury from environmental air pollutants.
- This methodology allows for site-specific and sensitive determination of molecular pathology in vivo.
- The study provides biological plausibility for epidemiological findings on air pollution and health.
