Immunocytochemical Study for Detection of Lactoglobulin in Alveolar Macrophage of Cow Milk Aspirated Mouse

Hong, Hye Suk; Byun, Sang Hyun; Chung, Young Hun; Lee, Chung Sik

Abstract

PURPOSE
It is difficult to detect small amount of aspiration into the lungs due to the lack of safe, sensitive and specific diagnostic tool. Recently, in animal studies, it has been reported that immunocytochemistry for lactoglobulin can be used to detect the minimal aspiration of cow milk. So, we tried to determine the difference between immunocytochemistry for lactoglobulin and Oil Red O stain of alveolar macrophages in cow milk aspirated mice.
METHODS
Fifty seven mice with 6-8 weeks old and 30-40 g weighing were used. Mice received either single or multiple intranasal instillation of 0.05 ml cow milk for study and saline for control under the anesthesia with ketamine and xylazine. The trachea of mouse was cannulated with 20G Jelco needle and then, mouse lungs were lavaged 3 times with 0.5 ml of phosphate buffer solution at 4 hours, 12 hours, 24 hours after the last milk or saline instillation. Cells in bronchoalveolar lavage fluid(BALF) were stained with Oil Red O and immunocytochemistry for beta-lactoglobulin.
RESULTS
After single aspiration of milk, no cellular difference was found in bronchoalveolar lavage fluid(BALF) when compared with saline aspirated group at 4 hours. But after repeated aspiration of milk, significant change was observed in the number of alveolar macrophage, neutrophil, lymphocyte and eosinophil. Immunocytochemical reactivity was not observed in alveolar macrophages of saline aspirated group. Lipid-laden alveolar macrophages were recovered rarely in Oil Red O staining. Immunocytochemical staining displayed stain-positive alveolar macrophages for beta-lactoglobulin at 4 hours after milk aspiration, it had a peak at 12 hours and decreased markedly at 24 hours. Immunocytochemical stain positive alveolar macrophages appeared similarly in number between single and repeated aspiration group.
CONCLUSION
These observations suggested that alveolar macrophages could be detected more easily on immunocytochemistry for lactoglobulin than Oil Red O stain and immunocytochemistry could be used as a sensitive & specific diagnostic method for the detection of milk aspiration.