Effect of ACTH1-39 on the Oligodendroglial Differentiation Pathway with Regards to Myelin Membrane Sheet Formation
Objectives: This project investigates whether ACTH also induces changes in galactolipid expression and myelin membrane sheet extension, critical steps in OPC differentiation during myelin formation or remyelination.
Methods: OPCs were prepared from neonatal rat brains, and cultures were incubated for 24 hours with 200 nM ACTH1-39 or medium alone (control). For analysis of membrane sheet surface area, OPCs were fixed and immunostained with an antibody to galactolipids (A007). Imaging was performed on a Zeiss LSM 510 META in the WSU Bioimaging Core. Membrane sheet surface area and average fluorescence intensity were determined using PerkinElmer Volocity 3D Image Analysis Software. The surface area and fluorescence intensities of 23 untreated and 23 ACTH treated cells were then compared to determine whether ACTH1-39 had an effect on myelin membrane sheet formation.
Results: The average surface area for untreated control cells was 1951.7 ± 1039.2 µm2 and for ACTH treated cells 2449.3 ± 1152.7 µm2(p = 0.12). The average fluorescence intensity for untreated controls cells was 91.5 ± 38.4 and for ACTH treated cells 81.6 ± 32.4 (pixel intensities ranging from 0-255; p = 0.18). Total fluorescence per cell (surface area X fluorescence intensity) for control cells was 162,300 + 12,500 fluorescence units and for ACTH treated cells 188,700 + 17,780 fluorescence units (p=0.23)
Conclusions: Although the p-values were above the statistical significance threshold, the trends suggest that ACTH1-39 has an effect on accelerating OPC myelin sheet formation as seen by the larger average surface area and total galactolipid expression. Most OPCs in this analysis displayed membrane sheets, so the OPCs were relatively late in their differentiation pathway. The trend for this stage of OPCs might be significant with a larger sample size or longer exposure to ACTH, or more readily demonstrable with earlier stages of OPC development.
Support: Medical Student Research Scholarship Funds, 2015/2016 FCMSC Workforce of the Future Initiative (Kuo); Investigator Initiated Award. Mallinckrodt Pharmaceuticals Autoimmune and Rare Disease Division (formerly Questcor) and Parker Webber Endowed Chair, DMC Foundation/WSU (RPL).