Student Research: Julie T. Wroble

Characterization of Ethylnitrosourea Treated CNS Micromass Cultures Using Immunocytochemical and Flow Cytometric Methods
Faculty Advisor: 


Micromass cultures of embryonic rat midbrain cells are composed of at least two morphologically distinct cell types: fibroblast0like cells and neurons. Observations in Dr. Faustman's lab have suggested that these two cell populations are differentially susceptible to toxicants. To investigate potential differencces between these populations, the immuno-localization of neuron-specific and cytoskeletal markers was determined in day 5 CNS cultures. These cultures were stained with anti sera to: A2B5 (GQ ganglioside), acetylated tubulin (AT), beta-tubulin (BT), c-neu, gamma-amino buturic acid (GABA), glial fibrillary acidic protein (GFAP), Microtubule Associated Protein 1 (MAP 1), MAP 2, MAP 5, meurofilament (NF-H), neuron-specific enolase (NSE), neural cell adhesion molecule (N-CAM), tau, and vimentim (VM). Our immunocytochemical studies show that antibodies to the following cellular components were specific for cells exhibiting neuronal morphology: A2B5, AT, GABA, MAP 2, MAP 5, NSE, N-CAM, and tau. c-neu stained neuronal cells with moderate specificity. BT, MAP 1, and NF-H stained both cell populations. GFAP and VM were not detectable in either cell population.

Based on these results, two neuron-specific antibodies, A2B5 and N-CAM, were used to examine the effects of toxicant exposed micromass cultures. The cultures were treated with two concentrations (214 uM and 427 uM) of ethylnitrosourea (ENU) and cultured for five days. Treated cultures were compared with untreated controls uwing morphological and immunocytochemical methods and flow cytometry. ENU-treated cultures exhibited dose dependent changes in morphology, including decreases in cell number and density; decreased extent of differentiation; and decreased staining by A2B5 and N-CAM. Using glow cytometry, two cell populations were characterized based on their size and granularity characteristics. Population 1 (P1) cells were smaller than Population 2 (P@) cells. The fraction of P1 cells was found to decrease with increasing ENU concentration. However, concurrent flow cytometric analysis using antibody markers revealed that equivalent percentages of total cells were positively labeled with A2B5 or N-CAM regardless of ENU concentration.

Therefore, immunocytochemical staining patterns support the hypothesis that neuronal cells are more susceptible to the adverse effects of ENU. Although ENU caused dose related decreases in the number of relatively small cells, the percentage of cells positively staining for neuron-specific markers was comparable in the two populations. Thus, flow cytometry observations using A2B5 and N-CAM did not support a selective effect of ENU on these cell populations. Differencecs in these two observations have been explored and may reflect loss of cells during harvesting for flow cytometric evaluation. Another explanation is that immunocytochemical results have been misleading; the unit density of fluorescence may appear brighter in neurons due to the morphological characteristics of the cells.