6-Hydroxydopamine-induced nerve terminal lesion of the nigrostriatal system may provide a partial lesion model of Parkinson's disease useful for the assessment of neuroprotective treatments and behavioral recovery after therapeutic intervention. The aim of the present study was to assess the retrograde degenerative changes in the dopaminergic neurons of the substantia nigra and the associated behavioral. and neurochemical consequences of intrastriatal injections of B-hydroxydopamine in young adult rats. Four groups of rats were stereotaxically injected in the right striatum with graded doses of 6-hydroxydopamine ranging from 0 to 20 mu g. Structural and functional deficits were quantified by tyrosine hydroxylase-immunoreactive nigral cell numbers, striatal dopamine content, skilled paw use, and drug-induced rotation. The results show that striatal 6-hydroxydopamine lesions produce dose-dependent decreases in striatal dopamine levels and tyrosine hydroxylase-immunoreactive cell numbers in the ipsilateral substantia nigra, accompanied by a significant long-lasting atrophy of the remaining dopaminergic neurons. Paw reaching test scores on the side contralateral to the lesion were non-linearly correlated with dopaminergic neuronal cell loss and exhibited a clear symptomatic threshold such that impaired paw use appeared only after >50% loss of nigral dopamine neurons or a reduction of 60-80% of striatal dopamine levels. The behavioral, cellular, and neurochemical effects of the nerve terminal lesion thus bear some resemblance to the early stages of Parkinson's disease, where the severity of motor impairment is correlated with the loss of dopamine in the striatum and dopaminergic neuronal loss in the substantia nigra.Rats with intrastriatal 6-hydroxydopamine lesions thus provide a model of progressive dopamine neuron degeneration useful not only for the exploration of neuroprotective therapeutic intervention but also for the study of mechanisms of functional and structural recovery after subtotal damage of the nigrostriatal dopamine system.

Glial cell line-derived neurotrophic factor (GDNF) has been shown to exert neuroprotective effects on dopamine (DA) neurons in vivo. Here we report long-term rescue of nigral DA neurons after delayed short-term GDNF administration in a rat lesion model that reproduces the slowly progressing degenerative process seen in Parkinson's disease. GDNF injected close to the substantia nigra provided near-complete protection and persistent survival of the lesioned nigral neurons for at least 4 months after discontinuation of GDNF treatment. Long-term rescue of the nigral cells, however, was not accompanied by any significant reinnervation of the lesioned striatal target or any signs of functional recovery in either drug-induced or spontaneous motor behaviors. We conclude that not only preservation of the nigral DA neurons but also restoration of striatal DA function is necessary for functional recovery in the rat Parkinson model.

Tissue storage prior to intracerebral transplantation would represent a major advantage when conducting clinical transplantation trials in that the procurement of the embryonic donor tissue and the timing of neurosurgery could be planned more efficiently. In the present study, the effects of storing rat embryonic striatal tissue at either +4°C or below freezing temperature prior to grafting to the adult striatum, were assessed with regard to transplant survival, morphology and integration. Eleven days following a unilateral injection of ibotenic acid into the head of the caudate-putamen, a control group of rats received grafts of striatal primordium prepared immediately after dissection from rat embryos (embryonic day 16). A second group of rat embryonic striatal tissue was stored at 4°C (hibernation) for 5 days and then transplanted. A third group of the striatal donor tissue was cryopreserved in liquid nitrogen for 5 days before implantation surgery. Six to seven weeks following transplantation surgery, the grafts were analysed in brain sections processed for acetylcholinesterase histochemistry, DARPP-32 (dopamine and cyclic AMP regulated phosphoprotein with a molecular weight of 32 kDa) and tyrosine hydroxylase (TH) immunocytochemistry. The mean total graft volume and the relative size of the AChE-positive regions were not significantly different between the three groups. Striatal-specific graft regions, positively stained for AChE and DARPP-32, generally exhibited TH immunoreactivity, suggesting that they had received dopaminergic afferents from the host brain. We conclude that embryonic rat striatal tissue can be cryopreserved or hibernated over 5 days without significant impairment in the yield of striatal neurons following intrastriatal implantation and without markedly affecting transplant morphology.

Disruption of dopaminergic neurotransmission in the striatum by neurotoxic lesions of the substantia nigra leads to increases in glutamic acid decarboxylase and proenkephalin messenger RNA expression, and to decreases in preprotackykinin (the precursor molecule for substance P) messenger RNA expression in the two populations of striatal medium-sized spiny projection neurons. These cells also express TrkB, the neurotrophin receptor for brain-derived neurotrophic factor and neurotrophin 4/5, and TrkC, the receptor for neurotrophin-3.(2,21) Since there is some indication that exogenous brain-derived neurotrophic factor can exert neuromodulatory effects in the basal ganglia,(1,24) we studied the effects of repeated intrastriatal injections of the four members of the neurotrophin family of neural growth factors, nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4/5 on the expression of striatal neurotransmitter-related genes in the unilaterally 6-hydroxydopamine-lesioned rat using in situ hybridization histochemistry. We found that 4 mu g/day of brain-derived neurotrophic factor or neurotrophin-4/5 when injected intrastriatally for eight consecutive days led to a normalization of the denervation-induced decrease of preprotachykinin messenger RNA when compared to animals injected with equivalent doses of nerve growth factor, neurotrophin-3, or vehicle. Neurotrophin-4/5 alone also normalized expression of messenger RNA encoding the 67 x 10(3) mol. wt isoform of glutamate decarboxylase, while none of the neurotrophins had a significant effect on preproenkephalin messenger RNA expression.These findings suggest that brain-derived neurotrophic factor and neurotrophin-4/5 can specifically counteract some of the effects of striatal dopamine denervation in a rat model of Parkinson's disease and may prove to be interesting candidates for the future study of neurotrophin/neurotransmitter-interactions in the basal ganglia.

Glial cell line-derived neurotrophic factor (GDNF) and transforming growth factor beta 3 (TGF-beta 3) are members of the TGF-beta superfamily with high neurotrophic activity on cultured nigral dopamine neurons. We investigated the effects of intracerebral administration of GDNF and TGF-beta 3 on the delayed cell death of the dopamine neurons in the rat substantia nigra following 6-hydroxydopamine lesions of dopaminergic terminals in the striatum. Fluorescent retrograde tracer injections and tyrosine hydroxylase immunocytochemistry demonstrated nigral degeneration with an onset 1 week after lesion, leading to extensive death of nigral neurons 4 weeks postlesion. Administration of recombinant human GDNF for 4 weeks over the substantia nigra at a cumulative dose of 140 micrograms, starting on the day of lesion, completely prevented nigral cell death and atrophy, while a single injection of 10 micrograms 1 week postlesion had a partially protective effect. Continuous administration of TGF-beta 3, starting on the day of lesion surgery, did not affect nigral cell death or atrophy. These findings support the notion that GDNF, but not TGF-beta 3, is a potent neurotrophic factor for nigral dopamine neurons in vivo.

When grafting human mesencephalic tissue to patients suffering from Parkinson's disease, the number of surviving dopamine (DA) neurons in the graft is probably crucial. It may be possible to increase the number of DA neurons available for grafting to a patient by pooling tissue from many human embryos collected over several days or by obtaining more DA neurons from each embryo. We have addressed these issues by cryopreserving human mesencephalic DA neurons prior to transplantation and also by grafting human embryonic diencephalic DA neurons. The effects of cryopreservation were assessed 4-15 weeks after xenografting ventral mesencephalic tissue into the DA-depleted striatum of immunosuppressed rats with unilateral 6-hydroxydopamine lesions of the mesostriatal pathway. Control rats grafted with fresh mesencephalic tissue displayed robust reductions in amphetamine-induced turning following transplantation. Functional effects of the cryopreserved mesencephalic grafts were only observed in the one rat out of nine which contained the largest graft in this group. The number of tyrosine hydroxylase immunoreactive neurons in animals transplanted with cryopreserved tissue was significantly reduced to 9% of fresh tissue control grafts. Morphological analysis showed that cryopreserved DA neurons were approximately 22% and 28% smaller regarding the length of the long and short axis, respectively, when compared to the neurons found in fresh grafts. In the second part of the study, the survival and function of human embryonic diencephalic DA neurons were examined following xenografting into the DA-depleted rat striatum. A reduction of motor asymmetry was observed in two out of seven diencephalon-grafted rats. This finding was consistent with a good graft survival in these particular rats, which both contained large grafts rich in tyrosine hydroxylase immunoreactive neurons. Moreover, there was immunopositive staining for graft-derived fibers in the rat striatum containing tyrosine hydroxylase and human neurofilament, both in rats grafted with mesencephalic and diencephalic DA neurons. These findings suggest that cryopreservation, using the current technique, is not a suitable storage method for use in clinical trials of DA neuron grafting in Parkinson's disease. On the other hand, the application of alternative sources of DA neurons may in the future develop into a strategy which can increase the number of neurons obtainable from each human embryo.

In order to elucidate the importance of the striatal GABAergic neurones in mediating functional effects of exogenously applied brain-derived neurotrophic factor (BDNF) in the basal ganglia, we performed daily injections of BDNF or vehicle into the dopamine-depleted striatum of unilaterally 6-hydroxydopamine-lesioned rats for 1 week. In situ hybridization revealed that BDNF exacerbated the lesion-induced up-regulation of preproenkephalin (PPE) mRNA, and completely reversed the lesion-induced decrease of preprotachykinin (PPT) mRNA. In contrast, striatal levels of trkB mRNA were not significantly affected by BDNF administration. Up-regulation of PPE and PPT mRNA was also observed in unlesioned BDNF-injected animals. We conclude that exogenously applied BDNF increases neuropeptide mRNA expression in striatal neurones independently of the presence of a dopaminergic innervation.

During the past few years ,,consensus statements'' on the adjuvant treatment of patients with breast cancer have been issued periodically (the last one in the spring of 1992). Many physicians have interpreted such a tabellary synopsis as a ''push-button'' treatment, which is, of course, beside the point, since the text of the original publications for several subgroups of patients defines this treatment as ''optional''. In fact, physicians cannot decide by themselves, which treatment a patient should have. All patients must be shown both sides of the medal, i. e. a small chance to live longer and the risk of side effects, and finally each individual patient has to decide for herself on the basis of ''informed consent''. In case of widespread acceptance, as mentioned by some authors, that nearly all patients should be treated in some adjuvant manner, a large group which does not need any adjuvant treatment at all, will remain undefined. This group comprises at least 50 % of all women ever treated for primary breast cancer. All physicians should consider it to be an urgent responsibility to define this group. This can be achieved only by means of studies, which include untreated or placebo-treated controls.

Brain-derived neurotrophic factor (BDNF) has been shown to promote the survival of dopaminergic neurons from the substantia nigra in cell culture. In order to assess whether a similar survival-promoting effect is present also in vivo, we grafted fetal nigral tissue to the dopamine-depleted striatum of 6-hydroxydopamine-lesioned rats receiving two-week intraventricular infusions or daily intrastriatal injections of BDNF, NGF, or vehicle. Wheninfused chronically at a high dose (12 μg/day) into the lateral ventricle, BDNF caused a behavioral syndrome of reduced food and water intake, body weight loss, and locomotor hyperactivity in comparison to NGF- and vehicle-infused graft recipients. NGF-infused graft recipients displayed a transient weight loss during the first week of infusion. At 15 days, amphetamine-induced turning was significantly attenuated to 3% of pregraft values in BDNF-infused recipients, whereas functional graft effects were not present in NGF- or vehicle-infused animals. Survival of tyrosine hydroxylase-immunoreactive graft cells, however, was similar in all treatment groups. Notably, NGF- and BDNF-infusions led to a significant size increase of cholinergic host neurons in the medial septal nucleus and the vertical limb of the diagonal band ipsilateral to the infusion, wheras there was no cholinergic neuron hypertophy in vehicle-infused animals. Daily intrastriatalinjections of BDNF (2 μg) produced no weight loss or locomotor hyperactivity, but also enhanced functional graft effects in BDNF-injected, as compared to vehicle-injected animals. Survival rates of grafted tyrosine hydroxylase-immunoreactive cells were, however, similar in both groups. We conclude that, using the administration methods chosen for this study, BDNF may enhance the functional capacity but not the survival of grafted dopamine neurons.

Dissociated ventral mesencephalon of 6 to 8-week-old human embryos were implanted by stereotaxic injection at different sites along the nigrostriatal pathway in adult rats, previously subjected to a 6-hydroxydopamine lesion of the intrinsic mesotelencephalic dopamine pathways. The recipients were immunosuppressed by daily injections of cyclosporin A to prevent rejection. At 13-20 weeks after transplantation, the implanted human neurons and their associated fiber outgrowths were visualized with a species-specific antibody recognizing human, but not rat, intermediary neurofilaments (HNF). From implants placed in the host rostral mesencephalic region, HNF-positive axonal projections were seen to extend in large numbers rostrally along the medial forebrain bundle and the internal capsule, and ramify within the caudate putamen, the ventral striatum and the amygdaloid nuclei (a distance of about 5-6 mm), and more sparsely in the frontal cortex and the olfactory bulb (a distance of about 10 mm). From implants placed in the internal capsule, abundant HNF-positive axons extended in the rostral, but not caudal, direction along the myelinated fiber bundles into the caudate putamen and the ventral striatum. Tyrosine hydroxylase (TH) immunohistochemistry revealed that the vast majority of the rostrally projecting HNF-positive axons were also TH-positive, and that the graft-derived axons gave rise to dense TH-positive terminal networks, above all in large areas of the previously denervated caudate putamen. From control implants of cortical neuroblasts, axonal projections were seen along the medial forebrain bundle and the internal capsule, but the axons were TH-negative and showed only sparse projections to the striatal areas. Instead, dense projections were seen, e.g., in the frontal cortex. The results demonstrate a remarkable ability of human mesencephalic neuroblasts to extend axons along the trajectories of the nigrostriatal and mesolimbocortical pathways to reach and innervate the principal striatal and limbic target areas in the forebrain. This shows that the basic requirements for the formation of long axonal pathways may be present in the adult mammalian central nervous system (CNS) at least for certain types of projection neurons. Furthermore, it implies that the developing human neuroblasts can escape the inhibitory features known to be present along myelinated growth trajectories in the adult mammalian brain. In addition, the present approach may offer new possibilities for functional neural grafting in the rat Parkinson model, since transplanted nigral neurons placed in their natural position within the rostral mesencephalon could provide an anatomically and functionally more integrated system than the standard model with ectopically placed intrastriatal nigral grafts.

In the present study we quantitatively assessed to what extent freeze-storage at liquid nitrogen temperature influences the survival and function of fetal mesencephalic grafts in the dopamine-depleted rat striatum. Ventral mesencephalic (VM) tissue was dissected from rat fetuses and stored overnight in a preservative medium at 4 °C (hibernation). It was grafted intrastriatally either as a fresh cell suspension or was frozen as tissue fragments or as a cell suspension after stepwise incubation in ascending concentrations of dimethyl-sulphoxide. Following a cryopreservation interval of 80 days in liquid nitrogen, the frozen samples were rapidly thawed, rinsed, and grafted. Cellular viabilities of graft cell suspensions, as assessed by ethidium bromide/acridine orange staining, were decreased from 90% in fresh tissue to 38-35% in frozen and thawed tissue. Amphetamine-induced turning behavior at 6 weeks post-grafting was significantly attenuated in hosts that had received fresh grafts or grafts that were frozen as tissue fragments. Tyrosine hydroxylase-(TH-) immunocytochemistry of recipient brains revealed significant decreases in TH-positive graft cell numbers in rats grafted with cryopreserved tissue (38–42% of fresh tissue). Moreover, the dye exclusion viability of thawed VM tissue was found to accurately predict the subsequent graft survival. There was no difference with respect to graft cell numbers between the two freezing methods employed, though block storage seems to be more simple from a practical point of view. The present study indicates that freezing in liquid nitrogen may be a feasible method for long-term storage of fetal neural tissue for grafting, although a marked decrease in graft survival and function of cryopreserved tissue must be taken into account.