Discontinuation of methylphenidate after long-term exposure in nonhuman primates.

Attention-deficit hyperactivity disorder (ADHD) is a common human neurobehavioral disorder that usually begins in early childhood. Methylphenidate (MPH) has been used extensively as a first-line medicine for the treatment of ADHD. Since ADHD is often diagnosed in early childhood and can persist for the entire lifespan, individuals may take MPH for many years. Given that in the course of one's lifetime a person may stop taking MPH for periods of time, or may implement lifestyle changes that may reduce the need for MPH entirely, it is important to understand how cessation of MPH affects the adult brain following long-term use of MPH. The blockage of the dopamine transporter (DAT) and the norepinephrine transporter (NET) by MPH may help with ADHD symptoms by boosting monoamine levels in the synapse. In the present study, microPET/CT was used to investigate possible neurochemical alterations in the cerebral dopamine system after cessation of long-term MPH administration in nonhuman primates. MicroPET/CT images were collected from adult male rhesus monkeys 6 months after they stopped receiving vehicle or MPH following 12 years of chronic treatment. The neurochemical status of brain dopaminergic systems was evaluated using the vesicular monoamine transporter 2 (VMAT2) ligand [18F]-AV-133 and a tracer for imaging dopamine subtype 2 (D2) and serotonin subfamily 2 (5HT2) receptors, [18F]-FESP. Each tracer was injected intravenously and ten minutes later microPET/CT images were obtained over 120 min. The binding potential (BP) of each tracer in the striatum was obtained using the Logan reference tissue model with the cerebellar cortex time activity curve (TAC) as an input function. Brain metabolism was also evaluated using microPET/CT images of [18F]-FDG. [18F]-FDG was injected intravenously, and ten minutes later, microPET/CT images were obtained over 120 min. Radiolabeled tracer accumulation in regions of interest (ROIs) in the prefrontal cortex, temporal cortex, striatum, and cerebellum were converted into standard uptake values (SUVs). Compared to the vehicle control group, the BPs of [18F] AV-133 and [18F]-FESP in the striatum were not significantly altered in MPH treated groups. Additionally, no significant differences were detected in the SUVs of [18F]-FDG in the MPH treated group compared with control. This study demonstrates that 6 months after cessation of long-term, chronic MPH treatment, there are no significant neurochemical or neural metabolic changes in the central nervous system (CNS) of non-human primates (NHPs) and suggests that microPET imaging is helpful in assessing the status of biomarkers of neurochemical processes linked to chronic CNS drug exposure. (Supported by NCTR).

MicroPET/CT assessment of neurochemical effects in the brain after long-term methylphenidate treatment in nonhuman primates.

Methylphenidate (MPH) is a psychostimulant approved by the FDA to treatment Attention-Deficit Hyperactivity Disorder (ADHD). MPH is believed to exert its pharmacological effects via preferential blockade of the dopamine transporter (DAT) and the norepinephrine transporter (NET), resulting in increased monoamine levels in the synapse. We used a quantitative non-invasive PET imaging technique to study the effects of long-term methylphenidate use on the central nervous system (CNS). We conducted microPET/CT scans on young adult male rhesus monkeys to monitor changes in the dopaminergic system. We used [18F] AV-133, a ligand for the vesicular monoamine transporter 2 (VMAT2), and [18F]FESP a ligand for the D2 and 5HT2 receptors. In this study we evaluated the effects if chronic MPH treatment in the nonhuman primates (NHP). Two-year-old, male rhesus monkeys were orally administered MPH diluted in the electrolyte replenisher, Prang, twice a day, five days per week (M-F) over an 8-year period. The dose of MPH was gradually escalated from 0.15 mg/kg initially to 2.5 mg/kg/dose for the low dose group, and 1.5 mg/kg to 12.5 mg/kg/dose for the high dose group (Rodriguez et al., 2010). Scans were performed on Mondays, about 60 h after their last treatment, to avoid the acute effects of MPH. Tracers were injected intravenously ten minutes before microPET/CT scanning. Sessions lasted about 120 min. The Logan reference tissue model was used to determine the Binding Potential (BP) of each tracer in the striatum with the cerebellar cortex time activity curve as an input function. Both MP treatment groups had a lower [18F] AV-133 BP, although this failed to reach statistical significance. MPH treatment did not have a significant effect on The BP of [18F] FESP in the striatum. Long-term administration of MPH did not significant change any of the marker of monoamine function used here. These data suggest that, despite lingering concerns, long-term use of methylphenidate does not negatively impact monoamine function. This study also demonstrates that microPET imaging can distinguish differences in binding potentials of a variety of radiotracers in the CNS of NHPs. This approach may provide minimally-invasive biomarkers of neurochemical processes associated with chronic exposure to CNS medications. (Supported by NCTR).

Programming microphysiological systems for children's health protection.

Microphysiological systems (MPS) and computer simulation models that recapitulate the underlying biology and toxicology of critical developmental transitions are emerging tools for developmental effects assessment of drugs/chemicals. Opportunities and challenges exist for their application to alternative, more public health relevant and efficient chemical toxicity testing methods. This is especially pertinent to children's health research and the evaluation of complex embryological and reproductive impacts of drug/chemical exposure. Scaling these technologies to higher throughput is a key challenge and drives the need for in silico models for quantitative prediction of developmental toxicity to inform safety assessments. One example is cellular agent-based models, constructed from extant embryology, that produce data useful to simulate critical developmental transitions and thereby predict phenotypic consequences of disruption in silico. Biologically inspired MPS models built from human induced pluripotent stem (iPS)-derived cells and synthetic matrices that recapitulate organ-specific physiologies and native tissue architectures are providing exciting new research opportunities to advance the assessment of developmental toxicity and offer the possibility of deriving a full 'human on a chip' system, or a 'Homunculus.' Impact statement This 'commentary' summarizes research needs and opportunities for engineered MPS models for developmental and reproductive toxicity testing. Emerging concepts can be taken forward to a virtual tissue modeling framework for assessing chemical (and non-chemical) stressors on human development. These models will advance children's health research, both basic and translational and new ways to evaluate complex embryological and reproductive impacts of drug and chemical exposures to inform safety assessments.

Minimally invasive biomarkers of general anesthetic-induced developmental neurotoxicity.

The association of general anesthesia with developmental neurotoxicity, while nearly impossible to study in pediatric populations, is clearly demonstrable in a variety of animal models from rodents to nonhuman primates. Nearly all general anesthetics tested have been shown to cause abnormal brain cell death in animals when administered during periods of rapid brain growth. The ability to repeatedly assess in the same subjects adverse effects induced by general anesthetics provides significant power to address the time course of important events associated with exposures. Minimally-invasive procedures provide the opportunity to bridge the preclinical/clinical gap by providing the means to more easily translate findings from the animal laboratory to the human clinic. Positron Emission Tomography or PET is a tool with great promise for realizing this goal. PET for small animals (microPET) is providing valuable data on the life cycle of general anesthetic induced neurotoxicity. PET radioligands (annexin V and DFNSH) targeting apoptotic processes have demonstrated that a single bout of general anesthesia effected during a vulnerable period of CNS development can result in prolonged apoptotic signals lasting for several weeks in the rat. A marker of cellular proliferation (FLT) has demonstrated in rodents that general anesthesia-induced inhibition of neural progenitor cell proliferation is evident when assessed a full 2weeks after exposure. Activated glia express Translocator Protein (TSPO) which can be used as a marker of presumed neuroinflammatory processes and a PET ligand for the TSPO (FEPPA) has been used to track this process in both rat and nonhuman primate models. It has been shown that single bouts of general anesthesia can result in elevated TSPO expression lasting for over a week. These examples demonstrate the utility of specific PET tracers to inform, in a minimally-invasive fashion, processes associated with general anesthesia-induced developmental neurotoxicity. The fact that PET procedures are also used clinically suggests an opportunity to confirm in humans what has been repeatedly observed in animals.

MicroPET/CT assessment of FDG uptake in brain after long-term methylphenidate treatment in nonhuman primates.

Methylphenidate (MPH) is a psychostimulant commonly used for the treatment of Attention-Deficit Hyperactivity Disorder (ADHD). Since the long-term effects of this drug on the central nervous system (CNS) are not well understood, we conducted microPET/CT scans on young adult male rhesus monkeys (n=4/group) to gather information on brain metabolism using the uptake of [(18)F]Fluoro-2-deoxy-2-d-glucose (FDG) as a marker. Approximately two-year old, male rhesus monkeys were treated orally with MPH twice per day, five days per week (M-F) over a 6-year period. Subjects received MPH at either 2.5 or 12.5mg/kg/dose or vehicle (Prang). To minimize the acute effects of MPH on FDG uptake, microPET/CT scans were scheduled on Mondays before their first daily dosing of the week (approximately 68h since their last treatment). FDG (370±8.88MBq) was injected intravenously and 30min later microPET/CT images were obtained over 60min. Radiolabeled tracer accumulation in regions of interest (ROIs) in the prefrontal cortex, temporal cortex, striatum and cerebellum were converted into Standard Uptake Values (SUVs). Compared to the control group, the uptake of FDG in the cerebellum was significantly decreased in both the low and high dose groups. These preliminary data demonstrate that microPET imaging is capable of distinguishing differences in retention of FDG in the brains of NHPs treated chronically with MPH and suggests that this approach may provide a minimally invasive biomarker for exploring the effects of chronic MPH treatment on aspects of brain function.

Clinical outcomes of proximal row carpectomy versus four-corner arthrodesis for post-traumatic wrist arthropathy: a systematic review.

We conducted a systematic review of studies reporting clinical outcomes after proximal row carpectomy or to four-corner arthrodesis for scaphoid non-union advanced collapse or scapholunate advanced collapse arthritis. Seven studies (Levels I-III; 240 patients, 242 wrists) were evaluated. Significantly different post-operative values were as follows for four-corner arthrodesis versus proximal row carpectomy groups: wrist extension, 39 (SD 11º) versus 43 (SD 11º); wrist flexion, 32 (SD 10º) versus 36 (SD 11º); flexion-extension arc, 62 (SD 14º) versus 75 (SD 10º); radial deviation, 14 (SD 5º) versus 10 (SD 5º); hand grip strength as a percentage of contralateral side, 74% (SD 13) versus 67% (SD 16); overall complication rate, 29% versus 14%. The most common post-operative complications were non-union (grouped incidence, 7%) after four-corner arthrodesis and synovitis and clinically significant oedema (3.1%) after proximal row carpectomy. Radial deviation and post-operative hand grip strength (as a percentage of the contralateral side) were significantly better after four-corner arthrodesis. Four-corner arthrodesis gave significantly greater post-operative radial deviation and grip strength as a percentage of the opposite side. Wrist flexion, extension, and the flexion-extension arc were better after proximal row carpectomy, which also had a lower overall complication rate.

Anaesthetic neurotoxicity and neuroplasticity: an expert group report and statement based on the BJA Salzburg Seminar.

Although previously considered entirely reversible, general anaesthesia is now being viewed as a potentially significant risk to cognitive performance at both extremes of age. A large body of preclinical as well as some retrospective clinical evidence suggest that exposure to general anaesthesia could be detrimental to cognitive development in young subjects, and might also contribute to accelerated cognitive decline in the elderly. A group of experts in anaesthetic neuropharmacology and neurotoxicity convened in Salzburg, Austria for the BJA Salzburg Seminar on Anaesthetic Neurotoxicity and Neuroplasticity. This focused workshop was sponsored by the British Journal of Anaesthesia to review and critically assess currently available evidence from animal and human studies, and to consider the direction of future research. It was concluded that mounting evidence from preclinical studies reveals general anaesthetics to be powerful modulators of neuronal development and function, which could contribute to detrimental behavioural outcomes. However, definitive clinical data remain elusive. Since general anaesthesia often cannot be avoided regardless of patient age, it is important to understand the complex mechanisms and effects involved in anaesthesia-induced neurotoxicity, and to develop strategies for avoiding or limiting potential brain injury through evidence-based approaches.

Ketamine anesthesia during the first week of life can cause long-lasting cognitive deficits in rhesus monkeys.

Previously our laboratory has shown that ketamine exposure (24h of clinically relevant anesthesia) causes significant increases in neuronal cell death in perinatal rhesus monkeys. Sensitivity to this ketamine-induced neurotoxicity was observed on gestational days 120-123 (in utero exposure via maternal anesthesia) and on postnatal days (PNDs) 5-6, but not on PNDs 35-37. In the present study, six monkeys were exposed on PND 5 or 6 to intravenous ketamine anesthesia to maintain a light surgical plane for 24h and six control animals were unexposed. At 7 months of age all animals were weaned and began training to perform a series of cognitive function tasks as part of the National Center for Toxicological Research (NCTR) Operant Test Battery (OTB). The OTB tasks used here included those for assessing aspects of learning, motivation, color discrimination, and short-term memory. Subjects responded for banana-flavored food pellets by pressing response levers and press-plates during daily (M-F) test sessions (50 min) and were assigned training scores based upon their individual performance. As reported earlier (Paule et al., 2009) beginning around 10 months of age, control animals significantly outperformed (had higher training scores than) ketamine-exposed animals for approximately the next 10 months. For animals now over 3 and one-half years of age, the cognitive impairments continue to manifest in the ketamine-exposed group as poorer performance in the OTB learning and color and position discrimination tasks, as deficits in accuracy of task performance, but also in response speed. There are also apparent differences in the motivation of these animals which may be impacting OTB performance. These observations demonstrate that a single 24-h episode of ketamine anesthesia, occurring during a sensitive period of brain development, results in very long-lasting deficits in brain function in primates and provide proof-of-concept that general anesthesia during critical periods of brain development can result in subsequent functional deficits. Supported by NICHD, CDER/FDA and NCTR/FDA.

MicroPET imaging of ketamine-induced neuronal apoptosis with radiolabeled DFNSH.

Recent reports indicate that 6-12 h of ketamine anesthesia can trigger neuronal apoptosis in postnatal day (PND) 7 rats. In vitro, ex vivo, and confocal fluorescent imaging studies suggest that dansyl compounds can accumulate within the cytoplasm of the apoptotic cell. High-resolution positron emission tomography (microPET) imaging has been proposed as a minimally invasive method for detecting apoptosis in the rat brain. Compared with [(18)F]-labeled annexin V, which binds to externalized phosphatidylserine (PS) on the outer membrane of apoptotic cells, intracellular uptake of the dansylhydrazone of p-fluorobenzaldehyde (DFNSH) may lead to improved target-to-background contrast ratios. In this study, the effect of ketamine on the uptake and retention of [(18)F]-DFNSH in the rat brain was investigated using microPET imaging. On PND 7, rat pups in the experimental group were exposed, at 2-h intervals, to six subcutaneous injections of ketamine (20 mg/kg) and control rat pups received six injections of saline. On PND 35, [(18)F]-DFNSH (37 MBq) was injected into the tail vein of rats and microPET images were obtained over 2 h following the injection. Radiolabeled tracer accumulation in the region of interest (ROI) in the frontal cortex was converted into standard uptake values (SUVs). The radiotracer was quickly distributed into the brains of both ketamine- and saline-treated rats. Compared with the control group, the uptake of [(18)F]-DFNSH was significantly increased in the ROI, frontal cortex area of ketamine-treated rats. In addition, the wash-out duration of the tracer was prolonged in the ketamine-treated animals. This study demonstrates that microPET imaging is capable of distinguishing differences in retention of [(18)F]-DFNSH in ROI and suggests that this compound may serve as a minimally invasive biomarker of neuronal apoptosis in rodents.

Changes in gene expression after phencyclidine administration in developing rats: a potential animal model for schizophrenia.

Repeated administration of phencyclidine (PCP), an N-methyl-d-aspartate (NMDA) receptor antagonist, during development, may result in neuronal damage that leads to behavioral deficits in adulthood. The present study examined the potential neurotoxic effects of PCP exposure (10mg/kg) in rats on postnatal days (PNDs) 7, 9 and 11 and the possible underlying mechanism(s) for neurotoxicity. Brain tissue was harvested for RNA extraction and morphological assessments. RNA was collected from the frontal cortex for DNA microarray analysis and quantitative RT-PCR. Gene expression profiling was determined using Illumina Rat Ref-12 Expression BeadChips containing 22,226 probes. Based on criteria of a fold-change greater than 1.4 and a P-value less than 0.05, 19 genes including NMDAR1 (N-methyl-d-aspartate receptor) and four pro-apoptotic genes were up-regulated, and 25 genes including four anti-apoptotic genes were down-regulated, in the PCP-treated group. In addition, the schizophrenia-relevant genes, Bdnf (Brain-derived neurotrophic factor) and Bhlhb2 (basic helix-loop-helix domain containing, class B, 2), were significantly different between the PCP and the control groups. Quantitative RT-PCR confirmed the microarray results. Elevated neuronal cell death was further confirmed using Fluoro-Jade C staining. These findings support the hypothesis that neurodegeneration caused by PCP occurs, at least in part, through the up-regulation of NMDA receptors, which makes neurons possessing these receptors more vulnerable to endogenous glutamate. The changes in schizophrenia-relevant genes after repeated PCP exposure during development may provide important information concerning the validation of an animal model for this disorder.

Gene expression profiling in the developing rat brain exposed to ketamine.

Ketamine, a non-competitive N-methyl-d-aspartate (NMDA) receptor antagonist, is associated with accelerated neuronal apoptosis in the developing rodent brain. In this study, postnatal day (PND) 7 rats were treated with 20 mg/kg ketamine or saline in six successive doses (s.c.) at 2-h intervals. Brain frontal cortical areas were collected 6 h after the last dose and RNA isolated and hybridized to Illumina Rat Ref-12 Expression BeadChips containing 22,226 probes. Many of the differentially expressed genes were associated with cell death or differentiation and receptor activity. Ingenuity Pathway Analysis software identified perturbations in NMDA-type glutamate, GABA and dopamine receptor signaling. Quantitative polymerase chain reaction (Q-PCR) confirmed that NMDA receptor subunits were significantly up-regulated. Up-regulation of NMDA receptor mRNA signaling was further confirmed by in situ hybridization. These observations support our working hypothesis that prolonged ketamine exposure produces up-regulation of NMDA receptors and subsequent over-stimulation of the glutamatergic system by endogenous glutamate, triggering enhanced apoptosis in developing neurons.

The effects of L-carnitine on the combination of, inhalation anesthetic-induced developmental, neuronal apoptosis in the rat frontal cortex.

The anesthetic gas nitrous oxide (N2O) and the volatile anesthetic isoflurane (ISO) are commonly used in surgical procedures for human infants and in veterinary and laboratory animal practice to produce loss of consciousness and analgesia. Recent reports indicate that exposure of the developing brain to general anesthetics that block N-methyl-D-aspartate (NMDA) glutamate receptors or potentiate GABA(A) receptors can trigger widespread apoptotic neurodegeneration. In the present study, the question arises whether a relatively low dose of ISO alone or its combination with N2O entails significant risk of inducing enhanced apoptosis. In addition, the role of L-carnitine to attenuate these effects was also examined. Postnatal day 7 (PND-7) rat pups were exposed to N2O (75%) or a low dose of ISO (0.55%) alone, or N2O plus ISO for 2, 4, 6 or 8 h with or without L-carnitine. The neurotoxic effects were evaluated 6 h after completion of anesthetic administration. No significant neurotoxic effects were observed for the animals exposed to N2O or ISO alone. However, enhanced apoptotic cell death was apparent when N2O was combined with ISO at exposure durations of 6 h or more. Co-administration of L-carnitine (300 or 500 mg/kg, i.p.) effectively protected neurons from the anesthetic-induced damage. These data indicate that 6 h or more of inhaled anesthetic exposure consisting of a combination of N2O and ISO results in enhanced neuronal apoptosis, and L-carnitine effectively blocks the neuronal apoptosis caused by inhalation anesthetics in the developing rat brain.

L-carnitine protects neurons from 1-methyl-4-phenylpyridinium-induced neuronal apoptosis in rat forebrain culture.

1-Methyl-4-phenylpyridinium ion (MPP+), an inhibitor of mitochondrial complex I, has been widely used as a neurotoxin because it elicits a severe Parkinson's disease-like syndrome with an elevation of intracellular reactive oxygen species (ROS) and apoptosis. L-carnitine plays an integral role in attenuating the brain injury associated with mitochondrial neurodegenerative disorders. The present study investigates the effects of L-carnitine against the toxicity of MPP+ in rat forebrain primary cultures. Cells in culture were treated for 24 h with 100, 250, 500 and 1000 microM MPP+ alone or co-incubated with L-carnitine. MPP+ produced a dose-related increase in DNA fragmentation as measured by cell death ELISA (enzyme-linked immunosorbent assay), an increase in the number of TUNEL (terminal dUTP nick-end labeling)-positive cells and a reduction in the mitochondrial metabolism of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). No significant effect was observed with the release of lactate dehydrogenase (LDH), indicating that cell death presumably occurred via apoptotic mechanisms. Co-incubation of MPP+ with L-carnitine significantly reduced MPP+-induced apoptosis. Western blot analyses showed that neurotoxic concentrations of MPP+ decreased the ratio of BCL-X(L) to Bax and decreased the protein levels of polysialic acid neural cell adhesion molecules (PSA-NCAM), a neuron specific marker. L-carnitine blocked these effects of MPP+ suggesting its potential therapeutic utility in degenerative disorders such as Parkinson's disease, Alzheimer's disease, ornithine transcarbamylase deficiency and other mitochondrial diseases.

Selective alterations of transcription factors in MPP+-induced neurotoxicity in PC12 cells.

MPP(+) (1-methyl-4-phenylpyridinium; the active metabolite of the neurotoxin MPTP (1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine)) depletes dopamine (DA) content and elicits cell death in PC12 cells. However, the mechanism of MPP(+)-induced neurotoxicity is still unclear. In this study, the dose response and time-course of MPP(+)-induced DA depletion and decreased cell viability were determined in nerve growth factor (NGF)-differentiated PC12 cells. The alteration of transcription factors (TFs) induced by MPP(+) from a selected dose level and time point was then evaluated using protein/DNA-binding arrays. K-means clustering analysis identified four patterns of protein/DNA-binding changes. Three of the 28 TFs identified in PC12 cells increased by 100% (p53, PRE, Smad SBE) and 2 decreased by 50% (HSE, RXR(DR1)) of control with MPP(+) treatment. In addition, three TFs decreased within the range of 33-50% (TFIID, E2F1, CREB) and two TFs increased within the range of 50-100% (PAX-5, Stat4). An electrophoretic mobility shift assay (EMSA) was used to confirm the changes of p53 and HSE. The observed changes in TFs correlated with the alterations of DA and cell viability. The data indicates that selective transcription factors are involved in MPP(+)-induced neurotoxicity and it provides mechanistic information that may be applicable to animal studies with MPTP and clinical studies of Parkinson's disease.

The role of the N-methyl-D-aspartate receptor in ketamine-induced apoptosis in rat forebrain culture.

Recent data suggest that anesthetic drugs may cause widespread and dose-dependent apoptotic neurodegeneration during development. The window of vulnerability to this neurotoxic effect, particularly with N-methyl-D-aspartate (NMDA) antagonists such as ketamine, is restricted to the period of synaptogenesis. The purposes of this study are to determine whether treatment of forebrain cultures with ketamine results in a dose-related increase in neurotoxicity and whether upregulation of NMDA receptor subunit NR1 promotes ketamine-induced apoptosis. Forebrain cultures were treated for 12 h with 0.1, 1, 10 and 20 microM ketamine or co-incubated with NR1 antisense oligonucleotide (2 microM). After washout of the ketamine, cultures were kept in serum-containing medium (in presence of glutamate) for 24 h. Application of ketamine (10 and 20 microM) resulted in a substantial increase in DNA fragmentation as measured by cell death enzyme-linked immunosorbent assay, increased number of terminal dUTP nick-end labeling positive cells, and a reduction in mitochondrial metabolism of the dye 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide. No significant effect was seen in the release of lactate dehydrogenase, indicating that cell death presumably occurred via an apoptotic mechanism. Co-incubation of ketamine with NR1 antisense significantly reduced ketamine-induced apoptosis. Western analysis showed that neurotoxic concentrations of ketamine increased Bax and NR1 protein levels. NR1 antisense prevented this increase caused by ketamine, suggesting that ketamine-induced cell death is associated with a compensatory upregulation of the NMDA receptor. These data suggest that NR1 antisense offers neuroprotection from apoptosis in vitro, and that upregulation of the NR1 following ketamine administration is, at least, partially responsible for the observed apoptosis.

Selective alterations of gene expression in mice induced by MPTP.

1-methyl-4-phenyl-1,2,4,6,-tetrahydropyridine (MPTP) is a selective neurotoxin that produces striatal dopamine depletion resulting in parkinsonism like symptoms in humans and is, therefore, used to generate animal models for Parkinson's disease (PD). In this study, C57BL/6N mice were treated with MPTP acutely (3x20 mg/kg, 2-hour interval, one day injection). Mice were then sacrificed 24 hours after the last injection and brain tissue was collected for analysis. Significant decrease of striatal dopamine (DA) and the metabolites (DOPAC, HVA) was observed after MPTP treatment. MPTP also reduced protein expression of tyrosine hydroxylase (TH) in the striatum. Real time RT-PCR was used to examine selective genes of the dopaminergic system in the substantia nigra. Our data demonstrated that MPTP significantly decreased gene expression of TH, dopamine transporter (DAT), and vesicle monoamine transporter (VMAT), coinciding with the pattern of dopamine concentration changes and protein expression after MPTP treatment. Although a significant decrease of DA metabolites was observed in striatum, there was no change in the expression of monoamine oxidases (MAO-A, MAO-B) or catechol O-methyltransferase (COMT), indicating that these changes might be simply a consequence of reduced monoamine levels. In addition, gene expression of alpha-synuclein was also decreased with MPTP treatment, but there was no change in beta-synuclein and parkin. This is the first study using real-time PCR to indicate that MPTP selectively alters gene expression and provides information for clinical studies in PD. Future studies will focus on gene expression of other pathways that may be affected by MPTP treatment and investigation of gene expression in specific cell types in vivo using LCM technology.

Developmental neurotoxicity of ketamine: morphometric confirmation, exposure parameters, and multiple fluorescent labeling of apoptotic neurons.

Ketamine is a widely used pediatric anesthetic recently reported (C. Ikonomidou et al., 1999, Science 283, 70-74) to enhance neuronal death in neonatal rats. To confirm and extend these results, we treated four groups of PND 7 rats with seven sc doses, one every 90 min, of either saline, 10 mg/kg ketamine, 20 mg/kg ketamine, or a single dose of 20 mg/kg ketamine. The repeated doses of 20 mg/kg ketamine increased the number of silver-positive (degenerating) neurons in the dorsolateral thalamus to a degree comparable to previous results (Ikonomidou et al., 1999, Science 283, 70-74), i.e., 28-fold vs. 31-fold respectively. However, blood levels of ketamine immediately after the repeated 20 mg/kg doses were about 14 micrograms/ml, about seven-fold greater than anesthetic blood levels in humans (J. M. Malinovsky et al., 1996, Br. J. Anaesth. 77, 203-207; R. A. Mueller and R. Hunt, 1998, Pharmacol. Biochem. Behav. 60, 15-22). Levels of ketamine in blood following exposure to the multiple 10 mg/kg doses of ketamine or to a single 20 mg/kg dose ranged around 2-5 micrograms/ml; although these blood levels are close to an anesthetic level in humans, they failed to produce neurodegeneration. To investigate the mode of ketamine-induced neuronal death, coronal sections were stained with both Fluoro-Jade B (a green fluorescent stain selective for neurodegeneration) and DAPI (a blue DNA stain), as well as for caspase-3 (using an antisera labeled red with rhodamine). These histochemical results confirmed the developmental neurotoxicity of ketamine, demonstrated that Fluoro-Jade B (FJ-B), like silver methods, successfully stained degenerating neurons in neonatal rats, and indicated that ketamine acts by increasing the rate of neuronal apoptosis.