An In Vitro Model for the Study of Cellular Pathophysiology in Globoid Cell Leukodystrophy

The overall goal of this procedure is to demonstrate the unique transformation of microglia into GLO cells in the presence of the toxin cycline. This is accomplished by first coating cover slips with extracellular matrix proteins. In the second step, glial cells are plated onto the cover slips, and then cycline and latex beads are administered to the cultures.

In the final step, the cells are stained by immunochemistry. Ultimately, the effects of the cycline on the nucleation and phagocytic ability of the microglial cells can be evaluated by immunofluorescent microscopy. The main advantage of this technique over existing methods like the use of microglial cell lines, is that the use of primary glial cultures mimics the hypothesized interactions between I astrocytes and microglia in brains of individuals with GLO void cell leukodystrophy or GLD demonstrating the procedure will be KO Claycomb, a graduate student from my laboratory.

To prepare the extracellular matrix or ECM coated cover slips, first soak circular cover slips with sterile hydrochloric acid in a sterile Petri dish. After 30 minutes, wash the cover slips three times with sterile water and then soak them in 95%ethanol for at least 20 minutes. After letting the cover slips air dry, add 150 to 250 microliter droplets of diluted ECM protein coating materials onto a sheet of paraform distributing the droplets so that cover slips will not overlap after placement.

Next, use forceps to gently place a cover slip onto each droplet and then place the paraform inside a cell culture hood with the sash closed after four to six hours, place the cover slips ECM coated side up into individual wells of a 24 well plate. Then wash each well with sterile PB S3 times and store the cover slips at four degrees Celsius. To prepare the glial cells for plating onto the cover slips aspirate the media from glial cell cultures and then gently wash the monolayers three times with 10 milliliters of sterile PBS.

Detach the cells with eight to 10 milliliters of trypsin EDTA after five to 10 minutes. At 37 degrees Celsius, add 20 milliliters of complete media to the flask to stop the reaction. Then transfer the cell suspension to a 50 milliliter conical tube and spin down the cells for 10 minutes.

At 300 Gs and room temperature, use a P 1000 to gently resuspend the palate in two milliliters of fresh, complete media. After counting, dilute the cells to approximately one times 10 to the fifth cells per milliliter. Now plate 500 microliters of the cells onto each of the ECM coated cover slips.

Add additional fresh complete media to each well and then incubate the plate at 37 degrees Celsius for three to five days. Once the cells have reached the desired confluence, add freshly prepared cycline to each well and gently swirl the culture plate to mix. After a week of cycline treatment, collect the media and fix the cells in 500 microliters of cold, 4%para formaldehyde per well at room temperature.

Then wash each well three times with PBS and store the plates at four degrees Celsius. To visualize the GLO cells by immuno cyto chemistry, first replace the PBS with blocking buffer and incubate the cells at room temperature. After an hour, incubate the cells in primary antisera against the microglia marker IO one for at least one hour at room temperature, and then wash each well three times in PBS for five minutes.

After incubating the cells with the appropriate fluorescence conjugated secondary antibody counterstain the cells with DAPI to identify the nuclei. Then after washing the cells as just demonstrated, use mounting medium to mount each cover slip onto individual microscope slides and view the slides on a fluorescent microscope for visual and quantitative analysis. To assess the phagocytic activity of the cycline treated microglia after cycline treatment, but before fixation, add FITC labeled latex beads to each G OID cell culture in accordance with the manufacturer's and instructions.

After 48 hours, fix the cells in PFA as just demonstrated, and then process the cells for immunochemistry as shown. Finally, assess the number of IBO one positive microglia that co localize with the fluorescently labeled beads by fluorescence microscopy immunochemical. Staining of microglia using IO one in conjunction with a nuclear counter stain enables the identification of large rounded multinucleated cells.

Note that in many instances, the gross enlargement of the size of the nucleus reflects the multinucleated status of these cells as observed in this image, cycline treatment also evokes a generalized activation of the microglia that is measurable by an increase in the PHA acidic activity of the mono nucleated microglia, as well as the multinucleated GLO cells. As observed in this representative image, the PHA Acidly active profiles of IBO one positive microglia can be readily identified during co-culture with oligodendrocyte progenitor cells. The implications of this technique extend toward the therapy of GLD as the formation of these multinucleated cells in response to cycline treatment is a defining characteristic of the neuropathology of GLD and the contribution of these cells to this pathology has not been explored in this way previously.