David M. Yurek

BDNF enhances the functional reinnervation of the striatum by grafted fetal dopamine neurons.

Transplantation of fetal dopaminergic neurons to the striatum can ameliorate neurological deficits exhibited by experimental animals and human graft recipients with Parkinsons disease. Recovery, however, is incomplete due to suboptimal survival of grafted cells and limited synaptic integration with the host brain. A number of neurotrophic factors have recently been shown to promote the survival and differentiation of dopamine neurons in vitro. In the present study we examined the effects of one such factor, brain-derived neurotrophic factor (BDNF), on the development of fetal substantia nigra following transplantation to the dopamine-depleted striatum of adult rats. Infusion of BDNF greatly enhanced the reinnervation of the host striatum by the engrafted dopamine neurons, as determined by tyrosine hydroxylase immunostaining, and also increased the effect of the graft on locomotor behavior induced by amphetamine administration. These effects became apparent during the 4-week period of BDNF infusion and persisted for an additional 6 weeks following the termination of BDNF delivery. These findings demonstrate that BDNF exerts a significant effect on the functional reinnervation of the striatum by transplanted fetal dopamine neurons in the rat, and suggest that application of this factor might similarly improve the clinical efficacy of neural transplantation employed in the treatment for Parkinsons disease.

Differential expression of GDNF, BDNF, and NT-3 in the aging nigrostriatal system following a neurotoxic lesion

Protein levels for brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and glial cell line-derived neurotrophic factor (GDNF) were measured in the striatum and ventral midbrain of young and aged Brown Norway/F344 F1 (F344BNF(1)) hybrid rats following a unilateral 6-hydroxydopamine (6-OHDA) lesion of the nigrostriatal pathway. At 2 weeks post-lesion, protein levels of BDNF and GDNF were higher in the denervated striatum when compared to the intact striatum for young (4-5 months old) but not old (31-33 months old) rats. Interestingly, in old rats BDNF protein in the denervated striatum was significantly lower than that measured in the intact striatum. At the same time point BDNF protein levels in the ventral midbrain were higher on the lesioned versus intact side for both young and old rats while no significant side differences were detected for GDNF protein in the ventral midbrain of young or old rats. No significant differences in NT-3 protein levels were detected between the lesioned and intact sides for striatal or ventral midbrain regions in either young or old brain. While no significant age effects were detected for BDNF or NT-3 protein, young rats showed higher GDNF protein levels in both the striatum (lesioned or intact) and ventral midbrain (lesioned or intact) than old rats. These data show that two endogenous neurotrophic factors, BDNF and GDNF, are differentially affected by a 6-OHDA lesion in the aging nigrostriatal system with young brain showing a significant compensatory increase of these two factors in the denervated striatum while no compensatory increase is observed in aged brain.

Glial cell line-derived neurotrophic factor improves survival of dopaminergic neurons in transplants of fetal ventral mesencephalic tissue.

This study was designed to determine whether or not an exogenous source of glial cell line-derived neurotrophic factor (GDNF) could be delivered continuously into the denervated/transplanted striatum and stimulate the survival, growth, and function of fetal ventral mesencephalic tissue transplants. Adult male rats with unilateral 6-hydroxydopamine lesions received transplants of fetal ventral mesencephalic tissue into the denervated striatum. Immediately thereafter, osmotic pumps [Alzet 2002, 0.5 microliter/h] were attached to intracerebral cannula and either a citrate buffer alone [control] or r-methuGDNF [dissolved in sodium citrate buffer to a concentration of 0.45 microgram/microliter] was infused into a site approximately 1.0 mm lateral to the transplant for a 2-week period; one group of lesioned animals did not receive transplants but was infused with GDNF. The effect of GDNF on tyrosine hydroxylase-positive (TH+) fiber outgrowth from transplants was variable, and image analysis revealed no significant difference between the GDNF and citrate groups. In contrast, the mean number of TH+ cells bodies in transplants infused with GDNF [2,037 +/- 149, n = 8] vs citrate [663 +/- 160, n = 8] was statistically significant (P < 0.001); cell counts were made in every third brain section [35 micrometer]. Similarly, transplants infused with GDNF showed an over-compensatory effect to amphetamine-induced rotational behavior that was significantly lower than that observed in transplanted animals receiving citrate buffer infusions. Infusions of GDNF into the denervated striatum alone had no significant effect on amphetamine-induced rotational behavior or on TH fiber morphology in the lesioned striatum. Thus, a continuous infusion of GDNF can improve the survivability of dopaminergic neurons in transplants of fetal ventral mesencephalic tissue.

Age-related decline in striatal dopamine release and motoric function in Brown Norway/Fischer 344 hybrid rats

The Brown Norway/Fischer 344 F1 hybrid rats (F344BNF1) is a newer rat model and is emerging as an important rodent model of aging. In the present study we used motoric performance tests, intracerebral microdialysis, and neurochemical measures of postmortem brain tissue to investigate the effects of aging in young (4-5 months), middle-aged (18-19), and old (24-25 months) F344BNF1 hybrid rats. We observed that old F344BNF1 rats exhibited decreased motoric performance, and lower levels of spontaneous and d-amphetamine-induced locomotor activity than those observed in young F344BNF1 rats. Microdialysis measures of extracellular basal levels of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), and 4-hydroxy-3-methoxyphenylacetic acid (HVA) were significantly diminished in the striata of the middle-aged and old rats as compared to levels in young animals. In addition, d-amphetamine-evoked overflow of DA was significantly decreased in the middle-aged and aged rat striatum as compared to DA overflow in young F344BNF1 rats. Studies of postmortem brain tissue showed that the changes in overflow of DA correlated with significantly lower DA tissue content in ventral striatum and midbrain. Moreover, both dopamine turnover ratios (DOPAC/DA, HVA/DA) and the serotonin turnover ratio (5-HIAA/5-HT) were significantly elevated in the ventral striatum and nucleus accumbens. The results of this study demonstrate a correlation between reductions in striatal DA neurochemistry and diminished motor function in aged F344BNF1 rats.

Chronic levodopa impairs the recovery of dopamine agonist-induced rotational behavior following neural grafting

SummaryThe effect of chronic levodopa treatment on the function of embryonic mesencephalic tissue grafts was assessed in rats by monitoring rotational behavior elicited by dopamine (DA) agonists before and after neural grafting. Rats were given unilateral 6-hydroxydopamine (6-OHDA) lesions of the nigrostriatal pathway and baseline measures of rotational behavior induced by D1 receptor stimulation, D2 receptor stimulation, or amphetamine were determined. Subsequently, DA grafts were implanted into the lesioned striatum and chronic regimens of either saline or levodopa began one day after neural grafting and were continued for 7 weeks. Rotational behavior elicited by the D1 agonist, SKF 38393, was completely attenuated throughout the six week-period following the commencement of levodopa treatment, regardless of the absence or presence of a DA graft. Conversely, rotational behavior elicited by the D2 agonist, quinpirole, was significantly elevated in ungrafted animals receiving chronic levodopa. Grafted animals receiving chronic levodopa did not show a significant reduction in rotational behavior, whereas grafted animals receiving chronic saline showed a significant 67% reduction in quinpirole-induced rotational behavior. Amphetamine-induced rotational behavior was reduced in both levodopa and saline treated grafted animals, however grafted animals receiving chronic levodopa treatment showed a reduction of rotational behavior that was uncharacteristic and less compensatory than that observed in grafted animals receiving chronic saline treatment. Morphology of grafts indicate that there were areas of impaired neurite outgrowth of TH-positive fibers in animals treated with levodopa. The results of the present study suggest that the impaired recovery in quinpirole and amphetamine-induced rotational behavior in grafted animals receiving chronic levodopa treatment may be related to (1) impaired graft function, (2) an alteration in pre- and postsynaptic mechanisms in the host DAergic system, or (3) a combined effect of (1) and (2).

Embryonic mesencephalic grafts increase levodopa-induced forelimb hyperkinesia in parkinsonian rats.

Recent observations from clinical trials of neural grafting for Parkinsons disease (PD) have demonstrated that grafted dopamine neurons can worsen dyskinesias in some graft recipients. This deleterious side effect reveals a new challenge for neural transplantation, that of elucidating mechanisms underlying these postgraft dyskinesias. One problem facing this challenge is the availability of a cost‐effective and reliable animal model in which to pursue initial investigations. In the current study, we investigated the interaction of an embryonic ventral mesencephalic (VM) dopamine (DA) neuron graft on levodopa (LD)‐induced dyskinetic movements in unilaterally 6‐hydroxydopamine‐lesioned rats. Rats were administered LD (levodopa‐carbidopa, 50:5 mg/kg) twice daily for 6 weeks after either a sham graft or VM DA graft. Although a single solid graft of embryonic DA neurons can prevent progression of some lesioned‐induced behavioral abnormalities such as LD‐induced rotation and dystonia, it significantly increases hyperkinetic movements of the contralateral forelimb. This differential effect of grafted neurons on abnormal behavioral profiles is reminiscent of that reported in grafted patients with PD. Data from this study illustrate important similarities between this model of parkinsonism and PD in human patients that make it suitable for initial preclinical investigations into possible mechanisms underlying postgraft aggravation of dyskinetic movements.

Regional Levels of Lactate and Norepinephrine After Experimental Brain Injury

Abstract: The recently developed controlled cortical impact model of brain injury in rats may be an excellent tool by which to attempt to understand the neurochemical mechanisms mediating the pathophysiology of traumatic brain injury. In this study, rats were subjected to lateral controlled cortical impact brain injury of low grade severity; their brains were frozen in situ at various times after injury to measure regional levels of lactate, high energy phosphates, and norepinephrine. Tissue lactate concentration in the injury site left cortex was increased in injured animals by sixfold at 30 min and twofold at 2.5 h and 24 h after injury (p < 0.05). At all postinjury times, lactate concentration was also increased in injured animals by about twofold in the cortex and hippocampus adjacent to the injury site (p < 0.05). No significant changes occurred in the levels of ATP and phosphocreatine in most of the brain regions of injured animals. However, in the primary site of injury (left cortex), phosphocreatine concentration was decreased by 40% in injured animals at 30 min after injury (p < 0.05). The norepinephrine concentration was decreased in the injury site left cortex of injured animals by 38% at 30 min, 29% at 2.5 h, and 30% at 24 h after injury (p < 0.05). The level of norepinephrine was also reduced by ∼20% in the cortex adjacent to the injury site in injured animals. The present results suggest that controlled cortical impact brain injury produces disorder in the neuronal oxidative and norepinephrine metabolism.

Long-term Transgene Expression in the Central Nervous System Using DNA Nanoparticles

This study demonstrates proof of concept for delivery and expression of compacted plasmid DNA in the central nervous system. Plasmid DNA was compacted with polyethylene glycol substituted lysine 30-mer peptides, forming rod-like nanoparticles with diameters between 8 and 11 nm. Here we show that an intracerebral injection of compacted DNA can transfect both neurons and glia, and can produce transgene expression in the striatum for up to 8 weeks, which was at least 100-fold greater than intracerebral injections of naked DNA plasmids. Bioluminescent imaging (BLI) of injected animals at the 11th postinjection week revealed significantly higher transgene activity in animals receiving compacted DNA plasmids when compared to animals receiving naked DNA. There was minimal evidence of brain inflammation. Intrastriatal injections of a compacted plasmid encoding for glial cell line-derived neurotrophic factor (pGDNF) resulted in a significant overexpression of GDNF protein in the striatum 1-3 weeks after injection.

Striatal trophic factor activity in aging monkeys with unilateral MPTP-induced parkinsonism

Striatal trophic activity was assessed in female rhesus monkeys of advancing age rendered hemiparkinsonian by unilateral intracarotid administration of MPTP. Three age groups were analyzed: young adults (8-9.5 years) n=4, middle-aged adults (15-17 years) n=4, and aged adults (21-31 years) n=7. Fresh frozen tissue punches of caudate nucleus and putamen were collected 3 months after MPTP treatment and assayed for combined soluble striatal trophic activity, brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF). This time point was chosen in an effort to assess a relatively stable phase of the dopamine (DA)-depleted state that may model the condition of Parkinsons disease (PD) patients at the time of therapeutic intervention. Analyses were conducted on striatal tissue both contralateral (aging effects) and ipsilateral to the DA-depleting lesion (lesion x aging effects). We found that combined striatal trophic activity in the contralateral hemisphere increased significantly with aging. Activity from both middle-aged and aged animals was significantly elevated as compared to young adults. Following DA depletion, young animals significantly increased combined striatal trophic activity, but middle-aged and aged animals did not exhibit further increases in activity over their elevated baselines. BDNF levels in the contralateral hemisphere were significantly reduced in aged animals as compared to young and middle-aged subjects. With DA depletion, BDNF levels declined in young and middle-aged animals but did not change from the decreased baseline level in old animals. GDNF levels were unchanged with aging and at 3 months after DA depletion. The results are consistent with several conclusions. First, by middle age combined striatal trophic activity is elevated, potentially reflecting a compensatory reaction to ongoing degenerative changes in substantia nigra DA neurons. Second, in response to DA depletion, young animals were capable of generating a significant increase in trophic activity that was sustained for at least 3 months. This capacity was either saturated or was not sustained in middle-aged and aged animals. Third, the aging-related chronic increase in combined striatal trophic activity was not attributable to BDNF or GDNF as these molecules either decreased or did not change with aging.

Lesion-induced increase of BDNF is greater in the striatum of young versus old rat brain.

Young (4-5 month old) and old (32-34 month old) Brown Norway/F344 hybrid rats were given unilateral 6-OHDA lesions of the nigrostriatal pathway. Four weeks later tissue from the lesioned or intact striatum or ventral midbrain was dissected and analyzed for brain-derived neurotrophic factor (BDNF) protein levels using an enzyme-linked immunosorbent assay. BDNF protein content was greater in the lesioned striatum than in the intact striatum for all young rats, and the increased BDNF content in the lesioned striatum of young rats was directly correlated with severity of lesion as determined by rotational scores. BDNF content in the lesioned striatum increased in less than half of the old rats and was not significantly different than BDNF content in the intact striatum. BDNF content in the lesioned substantia nigra/ventral tegmental area (SN/VTA) was greater than BDNF content in the intact SN/VTA for both young and old rats. These data suggest that an age-related difference in activity of at least one neurotrophic factor, BDNF, occur within the denervated striatum following a neurotoxic lesion of the nigrostriatal pathway.