NONINVASIVE DOPAMINE DETERMINATION BY REVERSED-PHASE HPLC IN THE MEDIUM OF FREE-FLOATING ROLLER TUBE CULTURES OF RAT FETAL VENTRAL MESENCEPHALON - A TOOL TO ASSESS DOPAMINERGIC TISSUE PRIOR TO GRAFTING

The low availability of dopamine containing neurons for grafting in Pa rkinson's disease is a general problem. Free-floating roller tube (FFR T) cultures allow storage of fetal mesencephalic tissue prior to trans plantation. Preoperative functional testing permits to select an optim ized set of individual cultures for transplantation. Rat fetal ventral mesencephali (E13) were dissected out and divided into four equally s ized pieces each and individually prepared as FFRT cultures. After 4, 8, 12, and 16 days in vitro (DIV) the medium of each culture was colle cted during routine medium change and immediately stabilized. Dopamine was extracted and probes were determined with reversed phase HPLC usi ng electrochemical detection. After 16 DIV cultures were fixed and cel l counts performed in tyrosine hydroxylase (TH)-immunostained serial s ections. The mean dopamine content +/- SEM in culture conditioned medi a was at 4 DIV: 21 +/- 2 pg, n = 38; at 8 DIV: 37 +/- 4 pg, n = 40; at 12 DIV: 52 +/- 7 pg, n = 38; and at 16 DIV: 39 +/- 5 pg, n = 38. In a ll cultures devoid of dopamine after 4 and 8 DIV (12.5%) levels remain ed below detectability at 12 and 16 DIV. Cultures derived from the ros tral mesencephalon showed significantly higher dopamine values than th ose from the caudal mesencephalon at 12 DIV. The mean number of TH-imm unoreactive (-ir) cells/culture +/- SEM after 16 DIV was 556 +/- 51, n = 40. The correlation between TH-ir cell number (CN) and dopamine con tent of rostrally derived cultures at 16 DIV was: CN = 7.4 (dopamine [ pg]) + 248; R = 0.75; n = 19; p < 0.001. No dopamine was present in cu ltures without TH-ir cells. These results demonstrate that sequential noninvasive screening of dopamine in single cultures is feasible and t hat the dopamine content is correlated to the number of surviving TH-i r cells. This permits to select cultures rich in dopaminergic neurons for transplantation.