STANDARDIZED METHODS TO BIOASSAY NEUROTROPHIC FACTORS FOR DOPAMINERGIC-NEURONS

The search for specific neurotrophic factors that will eventually be u sed to reduce or arrest the rare of degeneration of dopaminergic neuro ns in Parkinson's disease is being pursued by first testing the abilit y of putative compounds to increase the survival of dopaminergic neuro ns in primary cultures of the fetal, ventral mesencephalon. This resea rch has intensified in recent years, The experimental procedures used by different laboratories in these studies differ widely, and meaningf ul comparisons of the results obtained are accordingly difficult to ma ke. Some important experimental variables include the age of the fetal tissue used; the dissection technique used to isolate the ventral mes encephalon; the percentage of dopaminergic neurons present in the cult ure initially; handling of the tissue during dissection; the technique used to disperse the cells; the use of serum; the technique of platin g the cells; the attachment factors used; detachment and loss of cells during the staining procedure; the age of the cultures at the time of analysis; the uneven distribution of cells at the time of analysis an d the use of imaging techniques in the analysis, We show that when the E14 rat embryo is used, it is possible to consistently obtain a cultu re with 20% of tyrosine hydroxylase-positive neurons. Neither the plat ing density in the range of 7.8 x 10(3) to 1.25 x 10(5) cells/cm(2), n or the percentage of serum in the growth medium affected the percentag e of cells that expressed TH initially, at 4 or 12 h after plating, Wh en the cells were plated as 25 mu l droplets, called microislands (are a approximate to 12.5 mm(2)), and allowed to attach before additional growth medium was added, cell density remained uniform at the center o f the microisland for the duration of the culture, Restriction of the analysis of cell survival to the Center of the microisland therefore h elped to decrease the variability in counting that could occur when ce lls are dispersed over a larger area. In contrast, in an 8-well chambe r slide or 35 mm petri dish, in which the whole area is plated, cell d ensity was consistently higher at the edge (edge effect), versus the c entre, by a factor of about three, The use of microisland cultures als o has the additional benefit of increasing by a factor of about five t he number of individual cultures that can be set up per litter, and a proportionate reduction in the number of animals used per experiment, When the percentage of serum in the growth medium was 0% always, or 10 % for the first 12 h, and 0% thereafter, or 10% always, the number of TH-pos neurons per field (using a x 20 objective, column factor 1.25; area 320 mu m(2)) after 5 days in culture (DIV5) was < 1, 3-8 and 14-2 2, respectively. Under the same experimental conditions, the number of neurons (MAP2-positive) per field was 5-8, 18-30 and 45-65 (N = 10 in all cases), respectively, Serum deprivation therefore has a highly de leterious effect on neuronal survival in culture, We suggest that cult ures that were exposed to serum at any stage of the experiment, should not be referred to as 'serum-free', since even a brief exposure to se rum exerts a protective effect on neurons, and especially on dopaminer gic neurons. Instead, the percentage and kind of serum used, the exact usage, and the duration of exposure of the cells to serum should be s tated. Finally, it is suggested that where possible, an imaging system with manual count and journaling capabilities be used in the analysis . The methods described are illustrated by dose-response curves of the neurotrophic effects of BDNF, NGF-beta and IL-6 versus percentage sur vival on dopaminergic neurons, when grown in serum-free medium through out.