Evaluation of cell proliferation in ear and lymph node using BrdU immunohistochemistry for mouse ear swelling test

The mouse ear swelling test (MEST) was developed as an alternative to guinea pig models for measuring the contact sensitization potential. However, the MEST relies on the quantitative measurement of ear swelling by micrometer as the means of determining the endpoint. The purpose of this study was to investigate the possibility of using cell proliferation in the ear and lymph node by bromodeoxyuridine (BrdU) immunohistochemistry as a reliable marker for MEST. Female Balb/c mice were treated by the topical application of various sensitizers, 2,4-dinitrochlorobenzene (DNCB), toluene diisocyanate (TDI) and α-hexylcinnamaldehyde (HCA) and an irritant, sodium lauryl sulfate (SLS) following the protocol of MEST. The proliferation of cells in the ear and auricular lymph node was analyzed by BrdU incorporations into cells. There were significant increases in the cell proliferations of the ear and auricular lymph node in mice treated with DNCB and TDI compared to the vehicle control. All allergens and the irritant were correctly identified by the MEST using BrdU immunohistochemistry of lymph node responses. The standard MEST assay showed positive results in the case of the strong sensitizers, DNCB and TDI. However, HCA and SLS were not correctly identified in the ear swelling assay. These results suggest that the measurement of cell proliferation in the auricular lymph node using BrdU immunohistochemistry could provide a reliable marker for MEST.ope