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Combined Effects of Macrophage Enrichment and Addition of Menadione on Myelinating Rat Spinal Cord Aggregate Cultures

Contact Person: Richard M. Devon (devonr@duke.usask.ca)

Materials and Methods

Aggregate cultures were prepared from spinal cords of embryonic day 15 Wistar rats as detailed in Devon (1987). Growth medium was DMEM/F12 (Sigma) supplemented with 10% fetal bovine serum (Hy-Clone) and 1% D-glucose (Sigma). Spinal cords were dissociated by enzymatic digestion and mechanical trituration. The cells were pelleted and then resuspended in 20 mL of growth medium containing 0.01% DNase. The cell suspension was divided into four equal aliquots, added to 25 mL Ehrlenmeyer flasks, gassed with 95% O2/5% CO2 and sealed with a rubber stopper. The flasks were placed on a gyratory platform (100 RPM) in a 37°C walk-in incubator. Cells were fed after 3 DIV by removing 2 mL of spent medium and adding 2 mL fresh growth medium. Cultures were fed every second day thereafter by complete medium replacement. The cultures were maintained for a minimum of 28 days to ensure extensive myelination throughout the aggregates.

Rat peritoneal macrophages were isolated from male Wistar rats by intraperitoneal injection (2.5 mL per 100 g body weight) of 1% thioglycollate according to the methods of Conrad (1981) and Meltzer (1981). Five days later, macrophages were collected by peritoneal lavage with cold Puck’s BSS and pelleted. Cells were resuspended in 20 mL of DMEM/F12 containing 10% heat inactivated FBS, 1% glucose and 0.025% penicillin-streptomycin (Sigma). Macrophages were purified by differential attachment to tissue culture plates and then labelled by overnight incubation in growth medium containing 0.25% colloidal gold labelled wheatgerm (WGA-Au, Sigma; Seeley and Field, 1988). Macrophages were incubated in a 0.08% trypsin-versene solution for 15 minutes, collected, washed twice with cold Puck’s BSS and then pelleted. They were resuspended in growth medium containing penicillin-streptomycin (0.025%) and added to the spinal cord cell suspension at a density of 1.5 x 106 macrophages for each 5 mL aliquot. Ehrlenmeyer flasks were maintained as outlined above. Four flasks were used each time; 3 flasks contained the different concentrations of menadione while one flask was designated as the control. Menadione was dissolved in growth medium and sterilized through a 0.22 µM filter. Concentrations of 5, 10 and 20 µM menadione were used in all experiments since it had been previously shown that 20µM of menadione caused cell damage in aggregate cultures (Devon and Juurlink, 1995). The aggregate cultures were sampled at 24, 48 and 96 hours and individual experiments performed in triplicate.

Aggregate cultures were fixed and processed for light and electron microscopy according to Devon (1997). Light microscope (LM) analysis was performed on 1% toluidine blue stained semi-thin sections using a Zeiss microscope at 600X magnification and photographs were taken using Kodak Tec Pan film (ISO 50). Ultra-thin sections (50 - 60 nm) were cut on a Reichert OmU3 ultramicrotome using an IVIC diamond knife (Caracas, Venezuela) and mounted on either 200 mesh copper grids or 1% Formvar coated 2 mm slot copper grids (Marivac). The sections were double stained with 4% uranyl acetate and Reynold’s lead citrate (1963) and viewed on a Phillips 410 electron microscope. Photographs taken at magnifications of 4300X and 9300X.

Stereological analysis of numerical density (Nv = the number of cells per unit volume) was performed at the LM level to quantify the effect of menadione exposure. Nv was calculated as N(4/3)pr2T where N is the number of cell nuclei counted, T is the thickness of the section (1µm) and r is the radius of the aggregate (Weibel, 1969). Changes to Nv reflected whether exposure to the various concentrations of menadione resulted in a preferential loss of one cell type compared to another. For each experiment, 3 sections of 3 different aggregates from each treatment group were examined at the LM level and all the cell nuclei counted and classified according to cell type. The results were statistically analysed for significance using a three way ANOVA comparing the numerical density of individual cell types against the different menadione concentrations and exposure times. Morphometric analysis of the nature and extent of myelin damage was also performed at the EM level. The average number of damaged profiles in each category was calculated for standard aggregate and macrophage enriched aggregate cultures to determine the effect of menadione exposure.

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Devon, R.M.; Bartnik, B.; Juurlink, B.H.J.; (1998). Combined Effects of Macrophage Enrichment and Addition of Menadione on Myelinating Rat Spinal Cord Aggregate Cultures. Presented at INABIS '98 - 5th Internet World Congress on Biomedical Sciences at McMaster University, Canada, Dec 7-16th. Available at URL http://www.mcmaster.ca/inabis98/oxidative/devon0759/index.html
© 1998 Author(s) Hold Copyright