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Previous studies have indicated that administration of glial cell line-derived neurotrophic factor (GDNF) counteracts neuronal death after stroke. However, in these studies damage was evaluated at most a few days after the insult. Here, we have explored the long-term consequences of two routes of GDNF delivery to the rat striatum prior to stroke induced by 30 min of middle cerebral artery occlusion (MCAO): striatal transduction with a recombinant lentiviral vector or transduction of the substantia nigra with a recombinant adeno-associated viral vector and subsequent anterograde transport of GDNF to striatum. Despite high GDNF levels, stereological quantification of striatal neuron numbers revealed no protection at 5 or 8 weeks after MCAO. In fact, anterograde GDNF delivery exacerbated neuronal loss. Moreover, supply of GDNF did not alleviate the striatum-related behavioral deficits. Thus, we demonstrate that the actions of GDNF after stroke are more complex than previously believed and that high levels of this factor, which are neuroprotective in models of Parkinson's disease, can increase ischemic damage. Our findings also underscore the need for quantitative assessment of long-term neuronal survival and behavioral changes to evaluate the therapeutic potential of factors such as GDNF.

Glial cell line-derived neurotrophic factor (GDNF) has shown potential as a treatment for Parkinson's disease. Recombinant adeno-associated viral vectors expressing the GDNF protein (rAAV-GDNF) have been used in rodent models of Parkinson's disease to promote functional regeneration after 6-OHDA lesions of the nigrostriatal system. The goal of the present study was to assess the anatomical and functional efficacy of rAAV-GDNF in the common marmoset monkey (Callithrix jacchus). rAAV-GDNF was injected into the striatum and substantia nigra 4 weeks prior to a unilateral 6-OHDA lesion of the nigrostriatal bundle. Forty percent of the dopamine cells in the lesioned substantia nigra of the rAAV-GDNF-treated monkeys survived, compared with 21% in the untreated monkeys. Fine dopaminergic fibres were observed microscopically in the injected striatum of some rAAV-GDNF-treated monkeys, suggesting that rAAV-GDNF treatment may have prevented, at least in part, the loss of dopaminergic innervation of the striatum. Protection of dopamine cells and striatal fibre innervation was associated with amelioration of the lesion-induced behavioural deficits. rAAV-GDNF-treated monkeys showed partial or complete protection not only in the amphetamine and apomorphine rotation but also in head position and the parkinsonian disability rating scale. Therefore, our study provides evidence for the behavioural and anatomical efficacy of GDNF delivered via an rAAV vector as a possible treatment for Parkinson's disease.

Defective handling of proteins is a central feature of major neurodegenerative diseases. The discovery that neuronal dysfunction or degeneration can be caused by mutations in single cellular proteins has given new opportunities to model the underlying disease processes by genetic modification of cells in vitro or by generation of transgenic animals carrying the disease-causing gene. Recent developments in recombinant viral-vector technology have opened up an interesting alternative possibility, based on direct gene transfer to selected subregions or subsets of neurons in the brain. Using the highly efficient adeno-associated virus or lentivirus vectors, recent reports have shown that overexpression of mutated human huntingtin or alpha-synuclein in neurons in the striatum or substantia nigra induces progressive neuropathology and neurodegeneration, similar to that seen in Huntington's and Parkinson's diseases. Targeted overexpression of disease-causing genes by recombinant viral vectors provides a new and highly flexible approach for in vivo modeling of neurodegenerative diseases, not only in mice and rats but also in primates.

To explore the role of brain-derived neurotrophic factor for survival and generation of striatal neurons after stroke, recombinant adeno-associated viral vectors carrying brain-derived neurotrophic factor or green fluorescent protein genes were injected into right rat substantia nigra 4-5 weeks prior to 30 min ipsilateral of middle cerebral artery occlusion. The brain-derived neurotrophic factor-recombinant adeno-associated viral transduction markedly increased the production of brain-derived neurotrophic factor protein by nigral cells. Brain-derived neurotrophic factor was transported anterogradely to the striatum and released in biologically active form, as revealed by the hypertrophic response of striatal neuropeptide Y-positive interneurons. Animals transduced with brain-derived neurotrophic factor-recombinant adeno-associated virus also exhibited abnormalities in body posture and movements, including tilted body to the right, choreiform movements of left forelimb and head, and spontaneous, so-called 'barrel' rotation along their long axis. The continuous delivery of brain-derived neurotrophic factor had no effect on the survival of striatal projection neurons after stroke, but exaggerated the loss of cholinergic, and parvalbumin- and neuropeptide Y-positive, gamma-aminobutyric acid-ergic interneurons. The high brain-derived neurotrophic factor levels in the animals subjected to stroke also gave rise to an increased number of striatal cells expressing doublecortin, a marker for migrating neuroblasts, and cells double-labelled with the mitotic marker, 5-bromo-2'-deoxyuridine-5'monophosphate, and early neuronal (Hu) or striatal neuronal (Meis2) markers. Our findings indicate that long-term anterograde delivery of high levels of brain-derived neurotrophic factor increases the vulnerability of striatal interneurons to stroke-induced damage. Concomitantly, brain-derived neurotrophic factor potentiates the stroke-induced neurogenic response, at least at early stages.

We used a high-titer recombinant adeno-associated virus (rAAV) vector to express WT or mutant human alpha-synuclein in the substantia nigra of adult marmosets. The alpha-synuclein protein was expressed in 90-95% of all nigral dopamine neurons and distributed by anterograde transport throughout their axonal and dendritic projections. The transduced neurons developed severe neuronal pathology, including alpha-synuclein-positive cytoplasmic inclusions and granular deposits; swollen, dystrophic, and fragmented neuritis; and shrunken and pyknotic, densely alpha-synuclein-positive perikarya. By 16 wk posttransduction, 30-60% of the tyrosine hydroxylase-positive neurons were lost, and the tyrosine hydroxylase-positive innervation of the caudate nucleus and putamen was reduced to a similar extent. The rAAV-alpha-synuclein-treated monkeys developed a type of motor impairment, i.e., head position bias, compatible with this magnitude of nigrostriatal damage. rAAV vector-mediated alpha-synuclein gene transfer provides a transgenic primate model of nigrostriatal alpha-synucleinopathy that is of particular interest because it develops slowly over time, like human Parkinson's disease (PD), and expresses neuropathological features (alpha-synuclein-positive inclusions and dystrophic neurites, in particular) that are similar to those seen in idiopathic PD. This model offers new opportunities for the study of pathogenetic mechanisms and exploration of new therapeutic targets of particular relevance to human PD.

Sustained neurotrophic factor treatment in neurodegenerative disorders such as Parkinson's disease is likely to affect both degenerating and intact neurons. To investigate the effect of long-term glial cell line-derived neurotrophic factor (GDNF) overexpression on intact nigrostriatal dopamine neurons, we injected a recombinant lentiviral vector encoding GDNF, or green fluorescent protein, in the right striatum of young adult rats. Thirteen months after viral injection GDNF levels were 4.5 ng/mg tissue in the striatum and 0.9 ng/mg in the substantia nigra as measured by ELISA, representing a 25-100-fold increase above control vector- or nontransduced tissue. GDNF overexpression significantly reduced tyrosine hydroxylase mRNA levels (by 39-72%) in the substantia nigra and ventral tegmental area neurons, and the optical density of tyrosine hydroxylase-immunoreactive innervation in the striatum was reduced by 25-52% with the most prominent reductions appearing caudally. No significant reduction was seen in striatal vesicular monoamine transporter 2-immunoreactivity or [3H]mazindole binding autoradiography to dopamine uptake sites, two other presynaptic markers in dopamine axon terminals. The striatal D1 and D2 receptor binding as determined by [3H]SCH23390 and [3H]spiperone binding, respectively, was unaltered relative to the intact side in both treatment groups. Preproenkephalin mRNA levels in postsynaptic striatal neurons, which increase upon removal of striatal dopamine, were also unaffected by the GDNF treatment. Taken together our findings indicate that sustained GDNF administration to intact nigrostriatal dopamine neurons selectively reduces tyrosine hydroxylase expression, without altering striatal dopamine transmission to the extent that compensatory changes in several other components related to dopamine storage and signalling occur.

The effects of sustained (up to 9 months) striatal overexpression of glial cell line derived neurotrophic factor (GDNF) on lesioned nigrostriatal dopamine (DA) neurons was studied using a recombinant lentiviral (rLV) vector to deliver GDNF into the striatum 4 weeks prior to the creation of an intrastriatal 6-hydroxydopamine lesion. The results of the amphetamine-induced rotation suggested an initial partial protection followed by a complete recovery, whereas the spontaneous motor behaviors remained impaired. There was a clear protection of the nigral tyrosine hydroxylase (TH)-positive neurons in the rLV-GDNF group compared to rats injected with the control vector encoding green fluorescent protein (GFP) (70 and 20% of the intact side, respectively). However, the striatal TH+ fiber density was equally reduced (to 20% of the intact side) in both groups. Further morphological analyses indicated that the nigrostriatal projections of the DA neurons were indeed preserved in the GDNF group. The axonal projections were visualized using two independent methods: First, retrograde labeling of the nigral cell bodies by intrastriatal Fluoro-Gold injections showed that the majority of rescued cells in the GDNF group had preserved axonal projections to striatum. Second, injections of a recombinant adeno-associated viral vector expressing GFP into the nigra was used to anterogradely fill the DA neurons and their projections with GFP protein. GFP immunostaining clearly demonstrated that the fibers of the nigral DA cells were preserved along the nigrostriatal pathway and innervated large parts of the striatum, but did not express TH at detectable levels. In addition, fiber sprouting was observed in the globus pallidus, entopeduncular nucleus, and substantia nigra, corresponding to areas where GDNF protein was released. The lack of functional recovery in the spontaneous motor behaviors may, at least in part, be explained by this extensive aberrant fiber sprouting in the downstream striatal target nuclei and/or decreased synthesis of dopamine in the striatum.

In the adult brain, new neurons are continuously generated in the subventricular zone and dentate gyrus, but it is unknown whether these neurons can replace those lost following damage or disease. Here we show that stroke, caused by transient middle cerebral artery occlusion in adult rats, leads to a marked increase of cell proliferation in the subventricular zone. Stroke-generated new neurons, as well as neuroblasts probably already formed before the insult, migrate into the severely damaged area of the striatum, where they express markers of developing and mature, striatal medium-sized spiny neurons. Thus, stroke induces differentiation of new neurons into the phenotype of most of the neurons destroyed by the ischemic lesion. Here we show that the adult brain has the capacity for self-repair after insults causing extensive neuronal death. If the new neurons are functional and their formation can be stimulated, a novel therapeutic strategy might be developed for stroke in humans.

In order to assess the role of striatal dopamine (DA) afferents in L-DOPA-induced dyskinesia, we have studied a large series of rats sustaining 2, 3, or 4 unilateral injections of 6-hydroxydopamine (6-OHDA) in the lateral striatum. This type of lesion produced a dose-dependent depletion of DA fibers in the caudate-putamen, which was most pronounced in the lateral aspects of this structure. An additional group of rats was injected with 6-OHDA in the medial forebrain bundle to obtain complete DA denervation on one side of the brain. During a course of chronic L-DOPA treatment, rats with intrastriatal 6-OHDA lesions developed abnormal involuntary movements (AIMs), which mapped onto striatal domains exhibiting at least approximately 90% denervation, as judged by DA transporter autoradiography. The denervated areas showed local upregulation of preproenkephalin and prodynorphin mRNA, and FosB-like immunoreactivity, which were highly correlated with the rats' AIM scores. When compared to completely DA-denervated animals, the rats with intrastriatal 6-OHDA lesions showed an overall lower incidence, lower severity and different topographic distribution of AIMs. The involvement of proximal limb and axial muscles in the abnormal movements was proportional to the spreading of the lesion from lateral towards medial aspects of the caudate-putamen. Locomotive AIMs were only seen in rats with complete lesions, but not in any of the animals with intrastriatal 6-OHDA (which showed > 5% DA fiber sparing in the medial striatum). Intrastriatally 6-OHDA-lesioned rats had a larger therapeutic window for L-DOPA than did rats with complete bundle lesions, since they exhibited an overall lower predisposition to dyskinesia but a similar degree of drug-induced motor improvement in a test of forelimb stepping. Our results are the first to demonstrate that selective and partial DA denervation in the sensorimotor part of the striatum can confer cellular and behavioral supersensitivity to L-DOPA, and that the phenomenology of L-DOPA-induced rat AIMs can be accounted for by the topography of DA denervation within the caudate-putamen.

Intrastriatal delivery of the tyrosine hydroxylase gene by viral vectors is being explored as a tool for local delivery of L-dopa in animals with lesions of the nigrostriatal pathway. The functional effects reported using this approach have been disappointing, probably because the striatal L-dopa levels attained have been too low. In the present study, we have defined a critical threshold level of L-dopa, 1.5 pmol/mg of tissue, that has to be reached to induce any significant functional effects. Using new generation high-titer recombinant adeno-associated virus vectors, we show that levels of striatal L-dopa production exceeding this threshold can be obtained provided that tyrosine hydroxylase is coexpressed with the cofactor synthetic enzyme, GTP-cyclohydrolase-1. After striatal transduction with this combination of vectors, substantial functional improvement in both drug-induced and spontaneous behavior was observed in rats with either complete or partial 6-hydroxydopamine lesions of the nigrostriatal pathway. However, complete reversal of motor deficits occurred only in animals in which part of the striatal dopamine innervation was left intact. Spared nigrostriatal fibers thus may convert L-dopa to dopamine and store and release dopamine in a more physiologically relevant manner in the denervated striatum to mediate better striatal output-dependent motor function. We conclude that intrastriatal L-dopa delivery may be a viable strategy for treatment and control of adverse side effects associated with oral L-dopa therapy such as on-off fluctuations and drug-induced dyskinesias in patients with Parkinson's disease.

Recombinant adeno-associated viral vectors display efficient tropism for transduction of the dopamine neurons of the substantia nigra. Taking advantage of this unique property of recombinant adeno-associated viral vectors, we expressed wild-type and A53T mutated human alpha-synuclein in the nigrostriatal dopamine neurons of adult rats for up to 6 months. Cellular and axonal pathology, including alpha-synuclein-positive cytoplasmic inclusions and swollen, dystrophic neurites similar to those seen in brains from patients with Parkinson's disease, developed progressively over time. These pathological alterations occurred preferentially in the nigral dopamine neurons and were not observed in other nondopaminergic neurons transduced by the same vectors. The degenerative changes were accompanied by a loss of 30-80% of the nigral dopamine neurons, a 40-50% reduction of striatal dopamine, and tyrosine hydroxylase levels that was fully developed by 8 weeks. Significant motor impairment developed in those animals in which dopamine neuron cell loss exceeded a critical threshold of 50-60%. At 6 months, signs of cell body and axonal pathology had subsided, suggesting that the surviving neurons had recovered from the initial insult, despite the fact that alpha-synuclein expression was maintained at a high level. These results show that nigral dopamine neurons are selectively vulnerable to high levels of either wild-type or mutant alpha-synuclein, pointing to a key role for alpha-synuclein in the pathogenesis of Parkinson's disease. Targeted overexpression of alpha-synuclein in the nigrostriatal system may provide a new animal model of Parkinson's disease that reproduces some of the cardinal pathological, neurochemical, and behavioral features of the human disease.

We used a recombinant lentiviral vector (rLV) for gene delivery of GDNF to the striatum, and assessed its neuroprotective effects in the intrastriatal 6-hydroxydopamine (6-OHDA) lesion model. The level of GDNF expression obtained with the rLV-GDNF vector was dose-related and ranged between 0.9-9.3 ng/mg tissue in the transduced striatum, as determined by ELISA, and 0.2-3.0 ng/mg tissue were detected in the ipsilateral substantia nigra (SN), due to anterograde transport of the GDNF protein. GDNF expression was apparent at 4 days and maintained for > 8 months after injection. Striatal delivery of rLV-GDNF efficiently protected the nigral dopamine (DA) neurons and their projection, against the 6-OHDA lesion (65-77% of intact side). Sprouting of the lesioned axons was observed along the nigrostriatal pathway, precisely corresponding to the areas containing anterogradely transported GDNF.

Previous studies have shown that the functional efficacy of intrastriatal transplants of fetal dopamine (DA) neurons in the rat Parkinson model depends on their ability to establish a new functional innervation of the denervated striatum. Here we report that the survival, growth, and function of the grafted DA neurons greatly depend on the severity of the lesion of the host nigrostriatal system. Fiber outgrowth, and to a lesser extent also cell survival, were significantly reduced in animals in which part of the intrinsic DA system was left intact. Moreover, graft-induced functional recovery, as assessed in the stepping, paw-use, and apomorphine rotation tests, was obtained only in severely lesioned animals, i.e., in rats with >70% DA denervation of the host striatum. Functional recovery seen in these animals in which the 6-hydroxydopamine (6-OHDA) lesion was confined to the striatum was more pronounced than that previously obtained in rats with complete lesions of the mesencephalic DA system, indicating that spared portions of the host DA system, particularly those innervating nonstriatal forebrain areas, may be necessary for the grafts to exert their optimal functional effect. These data have implications for the optimal use of fetal nigral transplants in Parkinson patients in different stages of the disease.

Here we studied the effects of glial cell line-derived neurotrophic factor (GDNF) in a rat model that represents the symptomatic stages of Parkinson's disease. GDNF was infused starting 2 weeks after an intrastriatal 6-hydroxydopamine (6-OHDA) lesion in order to halt the ongoing degeneration of the nigrostriatal dopaminergic neurons. GDNF or vehicle was infused in the striatum or the lateral ventricle via an osmotic minipump over a total 4-week period (2-6 weeks postlesion). Motor function was evaluated by the stepping, paw reaching and drug-induced motor asymmetry tests before the pump infusion was initiated, and was repeated once during (5 weeks postlesion) and twice after the withdrawal of the minipumps (7 and 11 weeks postlesion). We found that within two weeks following the lesion approximately 40% of the nigral TH-positive neurons were lost. In the vehicle infusion groups there was an additional 20% cell loss between 2 and 12 weeks after the lesion. This latter cell loss occurred mainly in the caudal part of the SN whereas the cell loss in the rostral SN was almost complete within the first two weeks. Ventricular GDNF infusion completely blocked the late degenerating neurons in the caudal SN and had long lasting behavioural effects on the stepping test and amphetamine rotation, extending to 6 weeks after withdrawal of the factor. Striatal infusion affected the motor behaviour transiently during the infusion period but the motor performance of these animals returned to baseline upon cessation of the GDNF delivery, and the delayed nigral cell loss was marginally affected. We conclude that intraventricular GDNF can successfully block the already initiated degenerative process in the substantia nigra, and that the effects achieved via the striatal route, when GDNF is given acutely after the lesion, diminish as the fibre terminal degeneration proceeds.

Here we studied whether glial cell line-derived neurotrophic factor (GDNF), given as a single bolus injection before an intrastriatal 6-hydroxydopamine (6-OHDA) lesion, can protect the nigrostriatal dopamine neurons against the toxin-induced damage and preserve normal motor functions in the lesioned animals. GDNF or vehicle was injected in the striatum (25 microg), substantia nigra (25 microg) or lateral ventricle (50 microg) 6 h before the 6-OHDA lesion (20 microg/3 microL). Motor function was evaluated by the stepping and drug-induced motor asymmetry tests. Lesioned animals given vehicle alone showed a clear ipsilateral-side bias in response to amphetamine (13 turns/min), a moderate contralateral-side bias to apomorphine (4.5 turns/min) and a moderate to severe stepping deficit on the contralateral forepaw (three to four steps, as compared with 11-13 steps on the unimpaired side). Injection of GDNF into the striatum had a significant protective effect both on nigrostriatal function (1-2 turns/min in the rotation tests and seven to eight steps in the stepping test), and the integrity of the nigrostriatal pathway, seen as a protection of both the cell bodies in the substantia nigra and the dopamine innervation in the striatum. Injection of GDNF in the nigra had a protective effect on the nigral cell bodies, but not the striatal innervation, and failed to provide any functional benefit. In contrast, intranigral GDNF had deleterious effects on both the striatal TH-positive fibre density and on drug-induced rotation tests. Intraventricular injection had no effect. We conclude that preservation of normal motor functions in the intrastriatal 6-OHDA lesion model requires protection of striatal terminal innervation, and that this can be achieved by intrastriatal, but not nigral or intraventricular, administration of GDNF.

Immature progenitor cells (generated by in vitro propagation) may provide a useful alternative to primary cells (from dissected embryonic tissue) for transplantation if their migratory and proliferative and differentiation properties can be controlled and directed in vivo. In this study E15 murine EGF-responsive progenitor cells were transplanted to the striatum of adult rats. Simultaneously, these animals received continuous infusion of either epidermal growth factor (EGF) or vehicle, to the lateral ventricle, for 8 days. In animals that received EGF, the transplanted progenitors migrated toward the lateral ventricle and proliferated, as evidenced by bromodeoxyuridine incorporation. Progenitor cells transplanted to rats that received vehicle infusions showed neither of these responses. In all animals, transplanted progenitors expressed an immature astrocyte or oligodendrocyte phenotype, the majority of cells being astrocytes. We conclude that EGF stimulates the migration and proliferation of murine progenitor cells in vivo, either directly or indirectly, but does not influence their phenotypic differentiation., (Copyright 2000 Academic Press.)

Previous studies have used recombinant adeno-associated viral (rAAV) vectors to deliver glial cell line-derived neurotrophic factor (GDNF) in the substantia nigra to protect the nigral dopamine (DA) neurons from 6-hydroxydopamine-induced damage. However, no regeneration or functional recovery was observed in these experiments. Here, we have used an rAAV-GDNF vector to express GDNF long-term (6 months) in either the nigral DA neurons themselves, in the striatal target cells, or in both of these structures. The results demonstrate that both nigral and striatal transduction provide significant protection of nigral DA neurons against the toxin-induced degeneration. However, only the rats receiving rAAV-GDNF in the striatum displayed behavioral recovery, accompanied by significant reinnervation of the lesioned striatum, which developed gradually over the first 4-5 months after the lesion. GDNF transgene expression was maintained at high levels throughout this period. These results provide evidence that rAAV is a highly efficient vector system for long-term expression of therapeutic proteins in the nigrostriatal system.

Glial cell line-derived neurotrophic factor (GDNF) has prominent survival-promoting effects on lesioned nigrostriatal dopamine neurons, but understanding of the conditions under which functional recovery can be obtained remains to be acquired. We report here the time course of nigrostriatal axon degeneration in the partial lesion model of Parkinson's disease and the morphological and functional effects of sequential administration of GDNF in the substantia nigra (SN) and striatum during the first 5 weeks postlesion. By 1 day postlesion, the nigrostriatal axons had retracted back to the level of the caudal globus pallidus. Over the next 6 days axonal retraction progressed down to the SN, and during the following 7 weeks 74% of tyrosine hydroxylase-positive (TH(+)) and 84% of retrogradely labeled nigral neurons were lost, with a more pronounced loss in the rostral part of the SN. GDNF administration protected 70 and 72% of the nigral TH(+) and retrogradely labeled cell bodies, respectively, but did not prevent the die-back of the lesioned nigrostriatal axons. Although clear signs of sprouting were observed close to the injection site in the striatum as well as in the globus pallidus, the overall DA innervation of the striatum [as measured by [(3)H]-N-[1-(2-benzo(b)thiopenyl)cyclohexyl]piperidine-binding autoradiography] was not improved by the GDNF treatment. Moreover, the lesion-induced deficits in forelimb akinesia and drug-induced rotation were not attenuated. We conclude that functional recovery in the partial lesion model depends not only on preservation of the nigral cell bodies, but more critically on the ability of GDNF to promote significant reinnervation of the denervated striatum., (Copyright 2000 Academic Press.)

The glial cell line-derived neurotrophic factor (GDNF)-family of neurotrophic factors consisted until recently of three members, GDNF, neurturin, and persephin. We describe here the cloning of a new GDNF-family member, neublastin (NBN), identical to artemin (ART), recently published (Baloh et al., 1998). Addition of NBN/ART to cultures of fetal mesencephalic dopamine (DA) neurons increased the number of surviving tyrosine hydroxylase (TH)-immunoreactive neurons by approximately 70%, similar to the maximal effect obtained with GDNF. To investigate the neuroprotective effects in vivo, lentiviral vectors carrying the cDNA for NBN/ART or GDNF were injected into the striatum and ventral midbrain. Three weeks after an intrastriatal 6-hydroxydopamine lesion only about 20% of the nigral DA neurons were left in the control group, while 80-90% of the DA neurons remained in the NBN/ART and GDNF treatment groups, and the striatal TH-immunoreactive innervation was partly spared. We conclude that NBN/ART, similarly to GDNF, is a potent neuroprotective factor for the nigrostriatal DA neurons in vivo., (Copyright 2000 Academic Press.)

We investigated here the effect of the novel glial cell line-derived neurotrophic factor (GDNF)-family member neurturin (NTN) on transplanted fetal dopamine (DA) neurons. Three groups of rats with complete unilateral 6-hydroxydopamine (6-OHDA) lesions of the nigrostriatal DA system received intrastriatal grafts of embryonic ventral mesencephalic tissue. Following transplantation animals received repeated injections of vehicle or NTN (0.3 microg or 3.0 microg) over three weeks posttransplantation. NTN-treated animals had significantly (1.8-fold) more tyrosine hydroxylase-immunoreactive (TH-IR) neurons. Graft volume, TH-IR cell volume and overall dopaminergic host reinnervation remained unchanged. Amphetamine-induced rotation was rapidly compensated in all grafted rats. We conclude that administration of NTN may be a powerful way to increase survival of transplanted fetal DA neurons.

Both glial cell line-derived neurotrophic factor (GDNF) and its recently discovered congener, neurturin (NTN), have been shown to exert neuroprotective effects on lesioned nigral dopamine (DA) neurons when administered at the level of the substantia nigra. In the present study, we have explored the relative in vivo potency of these two neurotrophic factors using two alternative routes of administration, into the striatum or the lateral ventricle, which may be more relevant in a clinical setting. In rats subjected to an intrastriatal (IS) 6-hydroxydopamine (6-OHDA) lesion, GDNF and NTN were injected every third day for 3 weeks starting on the day after the 6-OHDA injection. GDNF provided almost complete (90-92%) protection of the lesioned nigral DA neurons after both IS and intracerebroventricular (ICV) administration. NTN, by contrast, was only partially effective after IS injection (72% sparing) and totally ineffective after ICV injection. Although the trophic factor injections protected the nigral neurons from lesion-induced cell death, the level of expression of the phenotypic marker, tyrosine hydroxylase (TH), was markedly reduced in the rescued cell bodies. The extent of 6-OHDA-induced DA denervation in the striatum was unaffected by both types of treatment; consistent with this observation, the high rate of amphetamine-induced turning seen in the lesioned control animals was unaltered by either GDNF or NTN treatment. In the GDNF-treated animals, and to a lesser extent also after IS NTN treatment, prominent axonal sprouting was observed within the globus pallidus, at the level where the lesioned nigrostriatal axons are known to end at the time of onset of the neurotrophic factor treatment. The results show that GDNF is highly effective as a neuroprotective and axon growth-stimulating agent in the IS 6-OHDA lesion model after both IS and ICV administration. The lower efficacy of NTN after IS, and particularly ICV, administration may be explained by the poor solubility and diffusion properties at neutral pH.

Partial lesions of the nigrostriatal dopamine system have been investigated with respect to their ability to induce consistent long-lasting deficits in movement initiation and skilled forelimb use. In eight different lesion groups 6-hydroxydopamine (6-OHDA) was injected at one, two, three, or four sites into the lateral sector of the right striatum, in a total dose of 20-30 microgram. Impairments in movement initiation in a forelimb stepping test, and in skilled paw use in a paw-reaching test, was seen only in animals where the severity of the lesion exceeded a critical threshold, which was different for the different tests used: single (1 x 20 microgram) or two-site (2 x 10 microgram) injections into the striatum had only small affects on forelimb stepping, no effect on skilled paw use. More pronounced deficits were obtained in animals where the same total dose of 6-OHDA was distributed over three or four sites along the rostro-caudal extent of the lateral striatum or where the injections were made close to the junction of the globus pallidus. The results show that a 60-70% reduction in tyrosine hydroxylase (TH)-positive fiber density in the lateral striatum, accompanied by a 50-60% reduction in TH-positive cells in substantia nigra (SN), is sufficient for the induction of significant impairment in initiation of stepping. Impaired skilled paw-use, on the other hand, was obtained only with a four-site (4 x 7 microgram) lesion, which induced 80-95% reduction in TH fiber density throughout the rostrocaudal extent of the lateral striatum and a 75% loss of TH-positive neurons in SN. Drug-induced rotation, by contrast, was observed also in animals with more restricted presymptomatic lesions. The results indicate that the four-site intrastriatal 6-OHDA lesion may be a relevant model of the neuropathology seen in parkinsonian patients in a manifest symptomatic stage of the disease and may be particularly useful experimentally since it leaves a significant portion of the nigrostriatal projection intact which can serve as a substrate for regeneration and functional recovery in response to growth promoting and neuroprotective agents., (Copyright 1998 Academic Press.)

Glial cell line-derived neurotrophic factor (GDNF) exhibits potent effects on survival and function of midbrain dopaminergic (DA) neurons in a variety of models. Although other growth factors expressed in the vicinity of developing DA neurons have been reported to support survival of DA neurons in vitro, to date none of these factors duplicate the potent and selective actions of GDNF in vivo. We report here that neurturin (NTN), a homolog of GDNF, is expressed in the nigrostriatal system, and that NTN exerts potent effects on survival and function of midbrain DA neurons. Our findings indicate that NTN mRNA is sequentially expressed in the ventral midbrain and striatum during development and that NTN exhibits survival-promoting actions on both developing and mature DA neurons. In vitro, NTN supports survival of embryonic DA neurons, and in vivo, direct injection of NTN into the substantia nigra protects mature DA neurons from cell death induced by 6-OHDA. Furthermore, administration of NTN into the striatum of intact adult animals induces behavioral and biochemical changes associated with functional upregulation of nigral DA neurons. The similarity in potency and efficacy of NTN and GDNF on DA neurons in several paradigms stands in contrast to the differential distribution of the receptor components GDNF Family Receptor alpha1 (GFRalpha1) and GFRalpha2 within the ventral mesencephalon. These results suggest that NTN is an endogenous trophic factor for midbrain DA neurons and point to the possibility that GDNF and NTN may exert redundant trophic influences on nigral DA neurons acting via a receptor complex that includes GFRalpha1.

Chronic treatment with epidermal growth factor (EGF) stimulates growth of all wall layers of the urinary tract in pigs and rats. Herein, we investigated the acute effects of EGF on detrusor smooth muscle activity. For in vivo examination, awake rats received EGF (75 micrograms/kg) intravenously and detrusor smooth muscle activity was monitored cystometrically. The EGF bolus caused no alteration in diuresis but a doubling of the micturition frequency, a 25% increase in micturition pressures, and increased irregular baseline contractile activity. For in vitro examination detrusor smooth muscle strips were exposed to EGF (1 microgram/ml). EGF caused contraction and increase in the spontaneous activity. In conclusion, EGF increases rat detrusor smooth muscle contractile activity in vivo and in vitro. The finding suggests that a direct effect of EGF on bladder smooth muscles is part of the genesis to the growth of the detrusor smooth muscle observed after chronic EGF treatment.

The epidemiological survey of the foci of campylobacteriosis confirmed the activity of the "milk" factor in the transmission of this enteric infection. The proliferation of Campylobacter in milk corresponded to the general regularities of the growth of bacterial population in the closed system. Most of the Campylobacter strains under study retained their viability in cooled boiled milk for a longer time.

Intrastriatal 6-hydroxydopamine injections in rats induce partial lesions of the nigrostriatal dopamine (DA) system which are accompanied by a delayed and protracted degeneration of DA neurons within the substantia nigra. By careful selection of the dose and placement of the toxin it is possible to obtain reproducible and regionally defined partial lesions which are well correlated with stable functional deficits, not only in drug-induced behaviors but also in spontaneous motoric and sensorimotoric function, which are analogous to the symptoms seen in patients during early stages of Parkinson's disease. The intrastriatal partial lesion model has proved to be particularly useful for studies on the mechanisms of action of neurotrophic factors since it offers opportunities to investigate both protection of degenerating DA neurons during the acute phases after the lesion and stimulation of regeneration and functional recovery during the chronic phase of the postlesion period when a subset of the spared nigral DA neurons persist in an atrophic and dysfunctional state. In the in vivo experiments performed in this model glial cell line-derived neurotrophic factor (GDNF) has been shown to exert neurotrophic effects both at the level of the cell bodies in the substantia nigra and at the level of the axon terminals in the striatum. Intrastriatal administration of GDNF appears to be a particularly effective site for induction of axonal sprouting and regeneration accompanied by recovery of spontaneous sensorimotor behaviors in the chronically lesioned nigrostriatal dopamine system.

Data on some parameters of the epidemic process of campylobacteriosis in the Ukraine are presented. Campylobacteriosis patients were found to constitute 1.9 +/- 0.095% of all examined patients with acute enteric infections (AEI). No statistically significant difference in the proportion of campylobacteriosis among child and adult AEI patients was established. The proportion of sick persons at the period of the spring-summer rise in morbidity was 71.5%. A definite relationship between the epizootic and epidemic processes in campylobacteriosis was noted. In the Ukraine the predominant infective agent was Campylobacter jejuni belonging mainly to serotype Lio and biotype 1. Quite frequently campylobacteriosis was found to be accompanied by mixed infections.

The data on the cytopathogenic activity of campylobacteria isolated from different sources are presented. 84.7 % of the isolated campylobacteria have been shown to possess cytopathogenicity with respect to Hep-2 cell cultures. The greatest number of highly cytopathogenic strains (52.6 %) has been registered among clinical isolates of campylobacteria. At the same time 44.1 % of highly cytopathogenic strains have been isolated from chickens, which is indicative of the potential danger of poultry as the source of Campylobacter infection. In the course of the epidemiological surveillance of campylobacteriosis the determination of cytopathogenically active Campylobacter strains is necessary.

The results of the clinico-epidemiological study of campylobacteriosis in a concrete area (Kiev and the Kiev region) are presented. The proportion of campylobacteriosis cases was found to be 6.4% among patients hospitalized in connection with acute enteric infections. Hens were most frequently the source of human infection. Thus, at the local poultry farm the proportion of hens contaminated with bacteria of the genus Campylobacter was 44.8%. The possible routes of the spread of Campylobacter infection and the factors of its transmission were established. The most important element of the epidemiological marking of Campylobacter bacteria is the determination of their species and serotype.

Antibioticograms of different campylobacteria strains have been analyzed. It is shown possible to develop a system of epidemiological marking on this basis. With this purpose sensitivity of campylobacteria to gentamycin, canamycin, carbenicyllin, tetracylin and erythromycin has been studied. No statistical difference in the average markers of resistance in the studied groups of strains was observed. This permitted supposing that R-plasmids in "human" strains may be isolated not only from the human intestine microflora, but also from other sources (animals, birds, environmental objects) as well. There are found common R-spectra in different groups of strains (Gm Kb Tc Er; Kb and Kb Tc), which confirms the same infection source. The study of antibioticograms of campylobacteria which circulate among people, animals, birds and environmental objects permits revealing regularities of epidemic process in case of campylobacteriosis.

The influence of multiple antibiotic resistance of Campylobacter strains determined by R plasmids on their virulence was studied. It was shown that the strains with multiple resistance were mostly isolated from children with campylobacteriosis (26.5 per cent). The number of such strains isolated from the infected adults, hens and environmental objects amounted to 25.8, 23.3 and 21.4 per cent respectively. The difference of the resistance determinants in the tested strains was statistically insignificant. It was suggested that the R plasmids of the human strains could be as well detected in the strains from the infected hens and environmental objects. A chromosome-plasmid pattern of the Campylobacter resistance to kanamycin, tetracycline and erythromycin was determined. The analysis of the cytopathogenic activity of the plasmid-containing strains and their aplasmid clones revealed that this criterion of the virulence statistically significantly increased after the plasmid loss by the strains. It was concluded that the antibiotic resistant strains had a selective superiority while circulating in various ecological niches.

An analytical survey of literature on the problem of immunological aspects of campylobacteriosis is presented. Antigenic nature of various components of a bacterial cell of these agents is described. Schemes of serotyping of campylobacteria on the basis of thermolabile and thermostable antigens are analyzed. It is shown expedient to use serotyping for determination of an infection source in the epidemiological practice. Proceeding from the published data presented, a conclusion is made that it is necessary to develop the home schemes of serotyping. At the same time a problem on development of new proximate methods promoting immunological indication of campylobacteriosis antigens in various ecological niches is not less urgent and is to be taken into account when planning antiepidemiological measures against the campylobacteriosis infection.

Composition of fatty acids of total lipids in home strains of campylobacteria has been studied. Lipids of all the strains of C. jejuni and C. coli mainly consist of saturated fatty acids (from 75.7 to 78.7%) with predominance of tetradecanoic and hexadecanoic fatty acids. The level of unsaturated fatty acids is considerably lower (from 21.0 to 22.5%); These acids are mainly presented by hexadecene acid. Qualitative composition of fatty acids of total lipids in C. jejuni and C. coli does not permit using it for differentiation within these species.

An analytical review of literature on the problem of biological properties of campylobacteria is presented. Taxonomic characteristics of campylobacteriosis agents are given. Morphological and cultural peculiarities of campylobacteria are considered, their ability to form coccal forms is emphasized. Peculiar attention is paid to serotype and biotype characteristics of bacteria of the Campylobacter genus. It is shown expedient to develop the home schemes of sero- and biotypization. Data on biological properties of "new" agents of human campylobacteriosis (Campylobacter cinaedi, C. hypointestinalis, C. upsaliensis) are generalized for the first time in home literature. A conclusion is made that the study of biological properties of campylobacteria strains circulating in the Ukrainian territory as well as the development of efficient prophylactic and antiepidemic measures on this basis are urgent now.

Clinical course and epidemiological features of campylobacteriosis in Ukraine are described. The disease accounts for 11.2% of the total number of acute enteric infectious cases. Ratio of bacterial contamination of hens and their role in spread of campylobacteriosis have been established. Possible ways of transfer of the infection are discussed.

An analytical review of recent publications of home and foreign authors on the problem of laboratory diagnosis of campylobacteriosis is presented. The commercial nutrient media, methods of creation of the microaerophilic conditions for cultivation of campylobacter are presented. The filtration method is preferable for isolation of these agents from the studied material highly contaminated by accompanying microflora. A special attention is paid to immunodiagnosis of campylobacteriosis: agglutination reaction, coagglutination reaction, passive hemagglutination reaction, immunoenzyme and radioimmune analyses. Seroepidemiological examination of the staff at one of meat-packing factories in the Republic carried out by the method of indirect immunoenzyme analysis has revealed high levels of anticampylobacteriosis antibodies in 17.9% of examinees. The promising trends in perfection of the methods for laboratory diagnosis of campylobacteriosis are outlined.