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Methamphetamine (meth) is a potent psychostimulant known to cause neurotoxicity. Clinical reports suggest meth abuse is a risk factor for Parkinson’s disease. We investigated changes in the blood–brain barrier and cerebral vasculature as a mechanism underlying this risk in rats treated acutely and trained to self-administer meth. We observed blood–brain barrier leakage in rats treated acutely with meth. Hypoperfusion in the striatum was detected with acute and chronic meth treatment and was associated with hypoxia. This was correlated with reductions in striatal tyrosine hydroxylase in rats trained to self-administer meth. These findings suggest a new mechanism of meth-induced neurotoxicity involving striatal vasoconstriction resulting in hypoxia and dopamine reductions leading to an increased risk for Parkinson’s disease for meth abusers.

Despite decades of research and the development of a large group of animal models, our understanding of the mechanisms responsible for the progressive loss of dopamine neurons in Parkinson's disease (PD) is unknown. So-called neuroprotective studies demonstrate that a vast group of molecules readily attenuate the dopamine (DA) neuron loss produced by DA neurotoxin insult. Despite these successes, these neuroprotective strategies have been surprisingly ineffective in patients. This may reflect the fact that the initial pathogenic event and the subsequent disease progression is a consequence of different mechanisms. As we began to think about this disconnect, we discovered that animals exposed to DA neurotoxins exhibited blood–brain barrier (BBB) dysfunction. If the BBB in PD patients is disrupted, then the barrier that normally segregates peripheral vascular factors from brain parenchyma is no longer present. Immune cells could then enter brain and produce a self-perpetuating (progressive) degenerative process. In this review, we propose that peripheral immunity contributes to the degenerative process of PD and may be responsible for the progressive nature of the disease. This hypothesis is supported by a broad and diverse literature that is just beginning to come together to suggest that PD is, in part, an autoimmune disease. In order to understand this hypothesis, the reader must question the conventional wisdom that the BBB is intact in PD, the brain is an immune privileged area, and that pathogenic insult and disease progression may reflect different mechanisms.

The blood–brain barrier (BBB) is a tightly regulated barrier in the central nervous system. Though the BBB is thought to be intact during neurodegenerative diseases such as Alzheimer's (AD) and Parkinson's disease (PD), recent evidence argues otherwise. Dysfunction of the BBB may be involved in disease progression, eliciting of peripheral immune response, and, most importantly, altered drug efficacy. In this review, we will give a brief overview of the BBB, its components, and their functions. We will critically evaluate the current literature in AD and PD BBB pathology resulting from insult, neuroinflammation, and neurodegeneration. Specifically, we will discuss alterations in tight junction, transport and endothelial cell surface proteins, and vascular density changes, all of which result in altered permeability. Finally, we will discuss the implications of BBB dysfunction in current and future therapeutics. Developing a better appreciation of BBB dysfunction in AD and PD may not only provide novel strategies in treatment, but will prove an interesting milestone in understanding neurodegenerative disease etiology and progression.

Animal models have been an essential tool for researchers and clinicians in their efforts to study and treat Parkinson's disease (PD). Thus, the various ways 6-hydroxydopamine is employed, the use of MPTP in rodents and nonhuman primates, the prenatal exposure to bacterial endotoxin, the postnatal exposure to environmental toxins such as paraquat and rotenone, the assessment of dopamine (DA) neurons in genetic knockout mouse, and even the behavioral analysis of fruit flies and worms have added significantly to our knowledge base of PD—or have they? Are these animal models manifesting a true model of PD? Have the 7786 published studies (to date) on PD with animal models led to a clearer understanding of its etiology, treatment, or progression? In this review we critically assess this question. We begin with a succinct history of the major contributions, which have led to the current animal models of PD. We then evaluate the primary issue of the progressive loss of DA neurons, which, except for a few studies, has not been addressed in animal models of PD, even though this is the major pathological characteristic of the disease. Lastly, we discuss the possibility that more than one risk factor for PD may be necessary to develop an animal model that shows synergy—the progressive loss of DA neurons. Thus, the multiple hit hypothesis of PD—that is, the effect of more then one risk factor—may be the start of new era in animal models of PD that is one step closer to mimicking the pathology of PD in humans.

We investigated whether in utero exposure to the Gram(−) bacteriotoxin lipopolysaccharide (LPS) induces dopamine (DA) neuron loss in rats. The proinflammatory cytokine tumor necrosis factor α (TNF‐α) kills DA neurons and is elevated in the brains of patients with Parkinson's disease (PD). LPS is a potent inducer of TNF‐α, and both are increased in the chorioamniotic environment of women who have bacterial vaginosis (BV) during pregnancy, suggesting that BV might interfere with the normal development of fetal DA neurons. Gravid female rats were injected intraperitoneally with either LPS or normal saline at embryonic day 10.5 and their pups were killed at postnatal day 21. The brains of the pups were assessed for DA and TNF‐α levels and DA cell counts in the mesencephalon using tyrosine hydroxylase immunoreactive (THir) cells as a DA neuron marker. Prenatal LPS exposure significantly reduced striatal DA (29%) and increased DA activity (72%) as well as TNF‐α (101%). Stereological cell counts in the mesencephalon were also significantly reduced (27%) by prenatal LPS exposure. Prenatal exposure to LPS, as might occur in humans with BV, produces a significant loss of THir cells in rats that is still present 33 days following a single injection of LPS. Since this cell loss is well past the normal phase of DA neuron apoptosis that occurs in early postnatal life, rats so exposed may have a permanent loss of DA neurons, suggesting that prenatal infections may represent risk factors for PD. © 2001 Movement Disorder Society.

We report on generation of dopamine neurons from long-term cultures of human fetal mesencephalic precursor cells. These CNS precursor cells were successfully expanded in vitro using the mitogens epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2). Incubation of these cultures in 3% atmospheric oxygen resulted in higher cellular yields than room air. Following incubation in differentiation media containing interleukin (IL)-1b (IL-1b), IL-11, leukemia inhibitory factor (LIF), and glial cell line-derived neurotrophic factor (GDNF), up to 1% of the precursor cells converted into cells immunoreactive for tyrosine hydroxylase (TH), a marker for dopamine neurons. The TH immunoreactive cells exhibited morphological and functional properties characteristic of dopamine neurons in culture. These precursor cells might serve as a useful source of human dopamine neurons for studying the development and degeneration of human dopamine neurons and may further serve as a continuous, on-demand source of cells for therapeutic transplantation in patients with Parkinson's disease.

Levels of the proinflammatory cytokine tumor necrosis factor α (TNFα) are increased in postmortem brain and cerebral spinal fluid from patients with Parkinson's disease (PD). This observation provides a basis for associating TNFα with neurodegeneration, but a specific toxicity in dopamine (DA) neurons has not been firmly established. Therefore, we investigated TNFα-induced toxicity in DA neurons by utilizing primary cultures of embryonic rat mesencephalon. Exposure to TNFα resulted in a dose-dependent decrease in DA neurons as evidenced by decreased numbers of tyrosine hydroxylase-immunoreactive (THir) cells. TNFα toxicity was selective for DA neurons in that neither glial cell counts nor the total number of neurons was decreased and no general cytotoxicity was evidenced by lactate dehydrogenase assay. Many of the cells which remained immunoreactive for TH had shrunken and rounded cell bodies with broken, blunted, or absent processes. However, TNFα-treated cultures also contained some THir cells which appeared to be undamaged and possibly resistant to TNFα-induced toxicity. Additionally, immunocytochemistry revealed basal expression of TNFα receptor 1 (p55, R1) and TNFα receptor 2 (p75, R2) on all cells within the mesencephalic cultures to some degree, even though only DA neurons were affected by TNFα treatment. These data strongly suggest that TNFα mediates cell death in a sensitive population of DA neurons and support the potential involvement of proinflammatory cytokines in the degeneration of DA neurons in PD.

This paper describes the development and validation of a method for the quantitative analysis of Topiramate (2,3:4,5‐bis‐O‐(1‐methylethylidene)‐β‐D‐fructopyranose sulfamate), a new antiepileptic drug, in human plasma using liquid‐liquid extraction followed by flow‐injection negative ion electrospray mass spectrometry. Using Prednisone (1,4‐pregnadiene‐17‐α,21‐diol‐3,11,20‐trione [10 µg/mL]) as an internal standard, calibration curves for Topiramate were linear over a range of 1 to 30 µg/mL in human plasma and were highly reliable (r2= 0.9991). The lower limit of quantitation of the assay was 2 µg/mL in human plasma. Precision (%CV <15%) and accuracy (<20%) for both intra‐ and inter‐day validations were satisfactory. The method has been used in clinical pharmacology research. Copyright © 2001 John Wiley & Sons, Ltd.

The structural analysis of sixteen molecular species of diacyl glycerophosphoethanolamine from fresh water snail Lymneae stagnaliskidney using chromatography and mass spectrometry is described in this paper. 1‐eicosadienoyl‐2‐eicosatetraenoyl‐sn‐glycero‐3‐phosphoethanolamine (PE 20:2‐20:4), 1‐eicosapentaenoyl‐2‐eicosadienoyl‐sn‐glycero‐3‐phosphoethanolamine (PE 20:5‐20:2) and 1‐eicosatrienoyl‐2‐eicosatetraenoyl‐sn‐glycero‐3‐phosphoethanolamine (PE 20:3‐20:4) as well as 1‐octadecanoyl‐2‐eicosatetraenoyl‐sn‐glycero‐3‐phosphoethanolamine (PE 18:0‐20:4), 1‐ocetadecenoyl‐2‐eicosatetraenoyl‐sn‐glycero‐3‐phosphoethanolamine (PE 18:1‐20:4) and 1‐octadecanoyl‐2‐eicosapentaneoyl‐sn‐glycero‐3‐phosphoethanolamine (PE 18:0‐20:5) were found as the major molecular species, and the first three were tentatively identified as the novel species present in this biological material. The presence of a relatively high content of 1,2‐dieicosenoyl‐sn‐glycero‐3‐phosphoethanolamine species (approximately 27% of total species) as well as the absence of 22‐carbon fatty acid containing and plasmalogen PE molecular species are remarkable in healthy Lymneae stagnaliskidney. Copyright © 1999 John Wiley & Sons, Ltd.

Topiramate, a sulfamate‐substituted monosaccharide (2,3:4,5‐bis‐O‐(1‐methylethylidene)‐β‐D‐fructopyranose sulfamate), is a new antiepileptic drug, which has been approved for adjunctive therapy in adult patients with partial‐onset seizures. Liquid‐liquid extraction followed by flow‐injection negative‐ion electrospray mass spectrometry was evaluated as a means for the quantitative analysis of Topiramate in human plasma. Prednisone (1,4‐pregnadiene‐17‐α,21‐diol‐3,11,20‐trione [15 µg/mL]) was used as the internal standard because its solubility and molecular weight are similar to those of Topiramate. Calibration curves for Topiramate were linear over a range of 1 to 30 µg/mL plasma (signal‐to‐noise ratio >4) and were highly reliable (r2= 0.994). This approach offers several advantages: (i) the extraction of Topiramate from human plasma using chloroform is simple and reproducible; (ii) the quantitative determination of Topiramate, in the presence of an internal standard, by flow‐injection negative‐ion electrospray mass spectrometry with selected‐ion recording, is rapid and accurate and does not require chromatographic separation; (iii) the assay possesses adequate sensitivity (2–25 µg/mL) for the quantitative analysis of Topiramate in plasma from patients. Copyright © 1999 John Wiley & Sons, Ltd.

9‐Fluorenylmethyloxycarbonyl (FMOC) derivatives of catecholamines, including dopamine (DA; 3,4‐dihydroxyphenethylamine), norepinephrine (NE; 2‐amino‐1‐(3,4‐dihydroxyphenyl)ethanol) and epinephrine (EPI; 1‐phenyl‐1‐hydroxy‐2‐methylaminopropane) as well as 3,4‐dihydroxybenzylamine (DHBA) have been analyzed using high performance liquid chromatography coupled to fluorometric detection and atmospheric pressure chemical ionization mass spectrometry at low femtomole levels. Structures of the derivatives have also been characterized by liquid chromatography/atmospheric pressure chemical ionization mass spectrometry and electrospray/tandem mass spectrometry. Protonated molecules of FMOC‐DA and FMOC‐DHBA as well as [M + H − H2O]+ions of FMOC‐NE and FMOC‐EPI appear in their conventional mass spectra, and abundant fragments characteristic of catecholamines dominated the spectra. Structurally diagnostic ions using several hundred femtomoles of the FMOC derivatives of catecholamine were observed. Collision‐induced dissociation (CID) of electrospray‐generated protonated molecules of FMOC‐DA and FMOC‐DHBA as well as [M + H − H2O]+ions of FMOC‐NE and FMOC‐EPI produced diagnostic product ion spectra. Liquid chromatography/atmospheric pressure chemical ionization mass spectrometric analysis of the FMOC derivatives of catecholamines and DHBA should prove useful in the separation and characterization of these compounds from biological materials. Copyright © 1999 John Wiley & Sons, Ltd.

The structural analysis of sixteen molecular species of diacyl glycerophosphoethanolamine from fresh water snail Lymneae stagnalis kidney using chromatography and mass spectrometry is described in this paper. 1-eicosadienoyl-2-eicosatetraenoyl-sn-glycero-3-phosphoethanolamine (PE 20:2-20:4), 1-eicosapentaenoyl-2-eicosadienoyl-sn-glycero-3-phosphoethanolamine (PE 20:5-20:2) and 1-eicosatrienoyl-2-eicosatetraenoyl-sn-glycero-3-phosphoethanolamine (PE 20:3-20:4) as well as 1-octadecanoyl-2-eicosatetraenoyl-sn-glycero-3-phosphoethanolamine (PE 18:0-20:4), 1-ocetadecenoyl-2-eicosatetraenoyl-sn-glycero-3-phosphoethanolamine (PE 18:1-20:4) and 1-octadecanoyl-2-eicosapentaneoyl-sn-glycero-3-phosphoethanolamine (PE 18:0-20:5) were found as the major molecular species, and the first three were tentatively identified as the novel species present in this biological material. The presence of a relatively high content of 1,2-dieicosenoyl-sn-glycero-3-phosphoethanolamine species (approximately 27% of total species) as well as the absence of 22-carbon fatty acid containing and plasmalogen PE molecular species are remarkable in healthy Lymneae stagnalis kidney. Copyright © 1999 John Wiley & Sons, Ltd.

Topiramate, a sulfamate-substituted monosaccharide (2,3:4,5-bis-O-(1-methylethylidene)-β-D-fructopyranose sulfamate), is a new antiepileptic drug, which has been approved for adjunctive therapy in adult patients with partial-onset seizures. Liquid-liquid extraction followed by flow-injection negative-ion electrospray mass spectrometry was evaluated as a means for the quantitative analysis of Topiramate in human plasma. Prednisone (1,4-pregnadiene-17-α,21-diol-3,11,20-trione [15 µg/mL]) was used as the internal standard because its solubility and molecular weight are similar to those of Topiramate. Calibration curves for Topiramate were linear over a range of 1 to 30 µg/mL plasma (signal-to-noise ratio >4) and were highly reliable (r² = 0.994). This approach offers several advantages: (i) the extraction of Topiramate from human plasma using chloroform is simple and reproducible; (ii) the quantitative determination of Topiramate, in the presence of an internal standard, by flow-injection negative-ion electrospray mass spectrometry with selected-ion recording, is rapid and accurate and does not require chromatographic separation; (iii) the assay possesses adequate sensitivity (2–25 µg/mL) for the quantitative analysis of Topiramate in plasma from patients. Copyright © 1999 John Wiley & Sons, Ltd.

The caudate, putamen, and cerebellum from five patients with Parkinson's disease (PD) and five normal, aged controls were studied to determine if cell-free extracts from these tissues influenced dopamine neuron growth in culture. Cultures incubated with extracts of the caudate and putamen, but not the cerebellum, from PD patients contained more tyrosine hydroxylase immunoreactive neurons than aged controls. These data suggest that the parkinsonian striatum compensates for dopamine loss by increasing neurotrophic factor production. Copyright 1993 Academic Press

Rat progenitor cells from the germinal region of the fetal mesencephalon were isolated and expanded in media containing the mitogen epidermal growth factor. These cells remained mitotically active (up to 8 months), were immunoreactive for the progenitor cell marker nestin, and were readily infected with the BAGα retrovirus. When incubated in complete media containing serum in poly-l-lysine-coated plates, these cells spontaneously converted to neurons and glia but rarely expressed the dopamine (DA) neuron phenotype. Nineteen different cytokines were screened for their ability to induce the DA phenotype and only interleukin (IL)-1 was found to induce the expression of the DA neuron marker tyrosine hydroxylase (TH). The addition of IL-1, IL-11, leukemia inhibitory factor (LIF), and glial cell line-derived neurotrophic factor (GDNF) were found to further increase the number of TH immunoreactive (TH-ir) cells. The addition of mesencephalic membrane fragments and striatal culture-conditioned media along with the cytokine mixture induced the expression of morphologically mature TH-ir cells that were also immunoreactive for dopa-decarboxylase, the DA transporter, and DA itself. The DA neuron cell counts were approximately 20–25% of the overall cell population and 50% of the neurofilament population. Astrocytes and oligodendrocytes were also present. These data suggest that hematopoietic cytokines participate in the development of the DA neuron phenotype. Parallels between the function of hematopoietic cytokines in bone marrow and the central nervous system may exist and be useful in understanding the factors which regulate the differentiation of neurons in the brain.

A technique for isolating mitotic progenitor cells from the embryonic rostral mesencephalon is described. Culture of the progenitor cells in complete media with subsequent staining for neuron specific enolase (NSE) revealed that only 0.6% of the cells were NSE immunoreactive. Coculturing the progenitor cells with established striatal cultures did not result in conversion of any of the cells to the dopamine neuron phenotype (tyrosine hydroxylase immunoreactive (THir) neurons). In contrast, co-culture of progenitor cells with established mesencephalic cultures produced a statistically significant, and in some cases (three of twelve), dramatic increase in the number of THir cells. The THir cells that were present had more pronounced process extension than those observed in mesencephalic mono-cultures. Culturing progenitor cells in transwell baskets that were continuously exposed to media but physically separated from established mesencephalic cultures growing underneath the baskets led to the conversion of only a few progenitor cells to THir neurons in four of twelve transwell studies suggesting that cell-cell contact between progenitor cells and mesencephalic cells is required for the conversion. This co-culture technique also increased the number of THir neurons in the mesencephalic cultures although the increase was not profound enough to explain the increase observed in traditional co-culture. These data suggest that mitotic progenitor cells can be isolated from fetal rat tissue and successfully converted to the dopamine neuron phenotype. Progenitor-mesencephalic co-culture appears to increase the number of THir cells in both tissue sources mediated in part by soluble factor(s) although cell-cell contact and presumably extracellular matrix proteins play a more substantial role. These progenitor cells may prove useful as a tissue source for transplantation procedures in Parkinson's disease.

ABSTRACT: Prenatal exposure to the dopamine (DA) agonist cocaine, even if limited to early gestation, is associated with impaired developmental outcome in the human infant. We investigated the possible role of neurotrophic factors in this process by evaluating 4- to 6-d-old New Zealand White rabbit pups (n = 14) born to cocaineexposed does (30 mg/kg/d s.c. from days 7 to 15 of a 32-d gestation) and control does (sterile H2O). Cocaine exposure reduced striatal dopamine by 46% (t = 2.31; p < 0.05) and striatal 3,4-dihydroxyphenyl acetic acid by 49% (t = 2.44; p < 0.05). The number of neuron-specific enolase immunoreactivc neurons in mcsenccphalic cultures incubated with striatal extracts from pups exposed to cocainewas reduced by 61% relative to the effect of striatal extracts from control pups (t = 4.84; p < 0.01). The present results suggest that the reduction in striatal dopamine observed may result from a cocaine-induced decrease in striatal trophic activity.

ABSTRACT: Most studying the consequences of prenatal cocaine (COC) exposure employ rodents or other multiparous organisms in their models. We have previously shown that when pregnant Sprague‐Dawley albino rats are administered a 30 mg/kg subcutaneous (s.c.) injection on embryonic day 15 (E15), fetal brain COC levels show a proximal‐to‐distal (in relation to the cervix) gradient that can vary by as much as 350%. The present study sought to determine whether this gradient translated into a similar gradient in brain dopamine (DA) levels. Pregnant rats were administered COC or saline (SAL) (30 mg/kg COC or 1 ml/kg SAL, b.i.d., E7‐E19). On E20, dams were anesthetized with halothane, the fetuses immediately removed, their brains excised, frozen and subsequently processed for DA, dihydroxyphenylacetic acid (DOPAC) or homovanillic acid (HVA). High‐performance liquid chromatography (HPLC) analysis revealed a proximal‐to‐distal gradient for DA in both COC‐ and SAL‐exposed fetuses. Average fetal DA levels per litter were significantly lower in COC‐exposed litters (57.39 ± 3.67 ng/hemibrain SAL; 48.29 ± 3.87 ng/hemibrain COC F7,1= 11.66, p<0.05). The gradients for DA were in opposite directions such that COC litters showed the lowest levels of DA in the most distal uterine positions, whereas SAL‐exposed litters showed the highest DA levels in the same location. These data suggest that a gradient in brain dopamine normally exists for fetuses based upon uterine position, and that cocaine can have selectively greater effects on this level as a function of fetal location.

We studied the effect of dopamine agonists (levodopa, apomorphine, lergotrile, and M-7 [2(dimethylamino)5,6-dihydroxytetralin] on myoclonic jumping behavior in young male guinea pigs. All these agents had a significant antagonistic effect on the frequency of this serotonin-mediated behavior. The duration of the antagonism corresponded in all cases to the duration of stereotyped chewing behavior induced by these agents alone. The dopamine antagonist haloperidol potentiated jumping behavior. Therefore myoclonic jumping behavior is influenced by dopaminergic mechanisms, and this behavior may be the result of interaction between dopaminergic and serotonergic activity. The role of dopaminergic mechanisms in human myoclonic disorders needs further clarification.