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A double-blinded study was conducted to evaluate the dose response of hemiparkinsonian rhesus monkeys to intracerebroventricular (ICV) injections of recombinant methionine human glial cell line-derived neurotrophic factor (GDNF). Thirty rhesus monkeys with stable hemiparkinsonian features were divided into six treatment groups (vehicle, 10, 30, 100, 300 and 1000 microg GDNF; n = 5/group). Each animal received 4 ICV administrations spaced at four week intervals. In addition, the animals were followed for 4 mo after the last injection. Standardized video taped behavioral tests were used to rate parkinsonian features using a nonhuman primate rating scale and assess side effects from treatment. Significant behavioral improvements were measured in animals receiving 100 to 1000 microg GDNF. One month after the last GDNF administration, parkinsonian features in animals receiving 100 and 1000 microg GDNF began to return to baseline levels. However, 300 microg GDNF recipients continued to display behavioral improvements. Parkinsonian features significantly improved were: bradykinesia, rigidity, posture and balance. The most common side effect was a transient weight loss after GDNF administration. Only one other side effect was observed, one animal receiving 1000 microg GDNF displayed dyskinetic movements. The results provide additional information for evaluating the possible clinical application of GDNF for treating Parkinson's disease.

We describe the methods and rationale for using rats with a partial unilateral lesion of the nigrostriatal system as an animal model for studying neural plasticity in both young and aged brains. The rats are lesioned with 6-hydroxydopamine injected into the substantia nigra or the medial forebrain bundle. Amphetamine- and apomorphine-induced rotational behaviors are tested 3 and 4 weeks following the lesion. Based on the rotational responses to amphetamine and apomorphine administration, animals can be classified into one of three groups: unaffected, partially lesioned, or severely lesioned. Animals classified as displaying unaffected rotational behavior are those that do not respond to either amphetamine or apomorphine stimulation. Partially lesioned animals rotate ipsilateral to the lesioned side upon amphetamine injection, but do not display a significant number of rotations in response to apomorphine administration. In contrast, severely lesioned rats rotate after both amphetamine and apomorphine injections. Cell counts reveal that the mean number of dopamine neurons in the ventral mesencephalon of partially lesioned animals is reduced to 40% of that of the intact side. Also in partially lesioned animals, dopamine concentrations on the lesion side are even more severely depleted, averaging about 20% of levels in the contralateral intact striatum. Striatal dopamine concentrations correlate well with the number of surviving dopamine neurons in the ventral mesencephalon (r2 = 0.66, P < 0.05). Amphetamine-induced rotation rates also show a moderate correlation with both striatal dopamine concentrations and mesencephalic dopamine neuron cell counts. Therefore, rotational behavior induced by amphetamine and apomorphine stimulation can be used to identify partially lesioned rats following unilateral 6-hydroxydopamine lesions. It is also possible to estimate the extent of nigrostriatal system damage from the rate of amphetamine-induced rotation.Copyright 1994, 1999 Academic Press

In order to maintain a chronic supply of growth factor for medulla cells in vitro, chromaffin cells from rat, African green monkeys and man were co-cultured with C6 glioma cells, which secrete growth factors that sustain sympathetic neurons in vitro. The response of chromaffin cells to coculture was compared to treatment of medullary cells with nerve growth factor (NGF) alone. Dispersed chromaffin cell preparations were obtained by a trypsin-collagenase procedure, and subjected to differential plating on collagen-coated surfaces. With both human and monkey tissue, non-chromaffin cells did attach to the culture plates and an enriched chromaffin cell population could be replated. Rat adrenal medulla cells survived very poorly in vitro and were not enriched in this procedure. Cultured human and monkey chromaffin cells survived as epithelial cells (50%) and showed neuritic outgrowth on 55 to 66% of the cells after eight days when treated with nerve growth factor (NGF). These cells showed strong catecholamine histofluorescence, tyrosine hydroxylase (TH) and dopamine beta hydroxylase (DBH) immunoreactivity. In contrast, only ten percent of adult rat chromaffin cells survived in culture, although NGF treatment rescued an additional 20% of the cells and induced neuritic outgrowth after one week in vitro. C6 glioma cells were treated with mitomycin C bromodeoxyuridine to inhibit mitosis and were plated with the various medulla cells in a one to one ratio. Both human and monkey chromaffin cells expressed extensive and enhanced neuritic arborization within eight days of co-culture, (64–82% respectively) and exhibited intimate contact with the glioma cells as seen at the ultrastructural level. Importantly, survival of adult rat adrenal medulla cells was enhanced to 50% or more with 40% of the cells extending neurites when co-cultured with glioma cells for seven days. Chromaffin cells from all three species reacted for TH, DBH and PNMT in co-culture and were histofluorescent. The majority of these cells were also immunoreactive for serotonin and enkephalin, while only 37% of chromaffin cells indicated the presence of NPY. These data indicate that adrenal medulla can be maintained in vitro as the neuronal phenotype when co-cultured with growth factor producing cells and that this strategy may be useful for in vivo transplantation studies.

Tunicamycin, an inhibitor of N-acetylglucosaminylpyrophosphopolyisoprenol-dependent glycosylation, was used to study the effect of glycosylation on the synthesis, post-translational modification, secretion and function of the complement proteins that are associated with the major histocompatibility complex in humans, mice and guinea pigs. Tunicamycin blocked glycosylation of pro-C4, C2 and factor B and inhibited secretion of the corresponding native complement proteins synthesized by guinea-pig peritoneal macrophages in tissue culture. In addition, underglycosylated pro-C4 was more rapidly catabolized intracellularly than the corresponding fully glycosylated pro-complement protein. C4 protein secreted by cells incubated with tunicamycin had approximately the same specific biological activity as the protein obtained from control culture media, suggesting that carbohydrate is not required for its activity in immune haemolysis. Direct studies of carbohydrate incorporation and the tunicamycin effect suggested an unequal distribution of sugar among the C4 subunits, with maximal incorporation of carbohydrate into alpha-, and less into the beta-chain of the native protein.