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The o-phthaldialdehyde precolumn derivatives of psychosine, sphinganine and sphingosine extracted from brain and spinal cord tissues were determined by high-performance liquid chromatography-fluorescence detection. This method was developed with the purpose of detecting an endogenous amount of psychosine, sphingosine and sphinganine using small aliquots of brain tissues and spinal cord in rats. These sphingolipid bases were extracted in various ratios of chloroform-methanol and several pH values. Recovery of the method is about 81% in 12 ng/tube (final volume, 320 microl), 90-95% in 45 ng/tube of sphingosine and sphinganine within 2-12% relative standard deviation. Detection limits of these sphingoid bases were about 0.05 pmol/mg brain tissue. In the forebrain, brainstem and spinal cord of rats at three different ages of postnatal days (PND) 1, PND 13 and 6 months old, the endogenous concentrations of psychosine, sphingosine and sphinganine were determined. From these results, this method is suitable for the determination of sphingoid bases in small aliquot of brain and spinal cord tissues.

Gestational cocaine (COC) exposure has been reported to alter behavior and possibly dopamine (DA) receptors. In this paper, we further examined the effects of prenatal COC (40 mg/Kg, s.c.) on DA receptor binding and the behavioral response to quinpirole, a DA D2 receptor agonist. In an attempt to elucidate possible mechanisms of such effects, we exposed pregnant dams to specific reuptake blockers; fluoxetine 12.5 mg/kg, a serotonin reuptake blocker; desipramine 10 mg/kg, a norepinephrine reuptake blocker; GBR-12909 10 mg/kg, a DA reuptake blocker; or to a local anesthetic, lidocaine 40 mg/kg. Drugs were administered once daily over gestational days 8–20. Control dams were injected with saline (SAL) or pair-fed to the COC group. Quinpirole challenge was performed in the offspring on post natal day 19. Two pups per litter were injected (s.c.) with 0.03 or 0.09 mg/kg quinpirole-HCl on post-natal day 19. The remaining pups in each litter were sacrificed for analysis of striatal DA receptors. Results showed that only COC exposure altered the behavioral response to the quinpirole challenge by increasing quinpirole-induced stereotypy and motor activity relative to SAL controls. DA receptor analysis showed no alteration in kD or BMAX for striatal D1 or D2 sites in any group. These results suggest that prenatal COC exposure produces alterations in function of the D2 receptor complex which are not reflected in kD or BMAX and that these effects are not fully mimicked by exposure to specific monoamine reuptake blockers or a local anesthetic.

Ibogaine (IBO) is a psychoactive indole alkaloid that has antiaddictive properties. However, treatment with IBO may lead to neurotoxicity, since IBO and its metabolites interact persistently with many neurotransmitter systems. Here, we recorded cortical electroencephalogram (EEG) signals from rats anesthetized with isoflurane. The heart rate (HR) was monitored via electrocardiogram (EKG) electrodes. After the baseline EEG was recorded, rats received one intraperitoneal (i.p.) dose of 50 mg/kg IBO. EEG signals were recorded for 2 hr. Rats were then sacrificed and brains dissected into frontal cortex (FC), caudate nucleus (CN), hippocampus (HIP), and brain stem (BS). The level of dopamine (DA), serotonin (5-HT), and their metabolites were determined by high-performance liquid chromatography with electrochemical detection (HPLC-ECD). Compared with baseline, a decrease in HR immediately after IBO injection and a decrease in delta, theta, alpha and beta power spectra frequency bands (1-4, 4-8, 8-13, 13-32 Hz) during the first 30 min after IBO administration was observed. EEG recovered within the next 15 min. In CN, the level of DA decreased and DA turnover rate increased significantly. The levels of 5-HT increased in FC. The pattern of EKG AND EEG response to IBO may be due to multiple receptor interactions of IBO.

The widespread usage of methylphenidate (MPH) in the pediatric population has received considerable attention due to its potential effect on child development. For the first time a physiologically based pharmacokinetic (PBPK) model has been developed in juvenile and adult humans and nonhuman primates to quantitatively evaluate species- and age-dependent enantiomer specific pharmacokinetics of MPH and its primary metabolite ritalinic acid. The PBPK model was first calibrated in adult humans using in vitro enzyme kinetic data of MPH enantiomers, together with plasma and urine pharmacokinetic data with MPH in adult humans. Metabolism of MPH in the small intestine was assumed to account for the low oral bioavailability of MPH. Due to lack of information, model development for children and juvenile and adult nonhuman primates primarily relied on intra- and interspecies extrapolation using allometric scaling. The juvenile monkeys appear to metabolize MPH more rapidly than adult monkeys and humans, both adults and children. Model prediction performance is comparable between juvenile monkeys and children, with average root mean squared error values of 4.1 and 2.1, providing scientific basis for interspecies extrapolation of toxicity findings. Model estimated human equivalent doses in children that achieve similar internal dose metrics to those associated with pubertal delays in juvenile monkeys were found to be close to the therapeutic doses of MPH used in pediatric patients. This computational analysis suggests that continued pharmacovigilance assessment is prudent for the safe use of MPH.

As a part of an Operant Test Battery (OTB), a set of five behavioral tasks was given to a group of children at the Arkansas Children's Hospital (ACH) and a group of rhesus monkeys at the National Center for Toxicological Research (NCTR) [4]. Through a separate process, at ACH, the children had been diagnosed as Attention Deficit Disorder (ADD), boarder line ADD, or healthy. Using the Self-Organizing Map neural network [1], the profiles of both healthy monkeys and children of varied ages with ADD were then generated and compared. The goal is to determine whether the animal model shares certain traits characteristics of ADD.

The present study was designed to investigate if chronic exposure to manganese (Mn) produces an effect on antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPx) activities and reduced glutathione (GSH) content in different regions of rat brain. Adult male Sprague-Dawley (CD) rats were dosed with 0, 2.5 or 5.0 mg MnCl2/kg, for 3 months (5 days/week). The activity of total superoxide dismutase did not vary significantly in any region of the brain with either 2.5 or 5.0 mg MnCl2/kg. A significant increase of Mn-superoxide dismutase (Mn-SOD) activity was attained in hippocampus, cerebellum and brain stem. The Cu, Zn-superoxide dismutase activity was reduced in all regions of the brain, however, reduction was not statistically significant. No significant effect of Mn on glutathione peroxidase activity was observed in any region of the brain. Glutathione content was significantly reduced in cerebellum, whereas, no change was observed in other brain regions. The results show that chronic exposure to manganese significantly increased the Mn-superoxide dismutase activity in selected brain regions. Therefore, increased Mn-SOD may enhance the antioxidant ability of the brain to reduce oxidative stress. © 1997 John Wiley & Sons, Ltd.

Fluoro-Jade is an anionic fluorochrome capable of selectively staining degenerating neurons in brain slices. The histochemical application of Fluoro-Jade results in a simple, sensitive and reliable method for staining degenerating neurons and their processes. The technique will detect neuronal degeneration resulting from exposure to a variety of neurotoxic insults. Fluoro-Jade can be combined with other fluorescent methodologies including immunofluorescence, fluorescent axonal tract tracing, and fluorescent Nissl counterstaining. Compared to conventional methodologies, Fluoro-Jade is a more sensitive and definitive marker of neuronal degeneration than hematoxylin and eosin (H&E) or Nissl type stains, while being comparably sensitive yet considerably simpler and more reliable than suppressed silver techniques. © 1997 Elsevier Science B.V. All rights reserved.

Experimental evidence suggests that oxidative and nitrative mechanisms account for much of the dopaminergic neuronal injury in Parkinson’s disease (PD). The ubiquitously expressed non-receptor tyrosine kinase c-Abl is activated by oxidative stress and thus, may play a role in redox-mediated neurodegeneration. Recently, we reported that c-Abl is activated in PD and that a c-Abl inhibitor mitigated neuronal damage in a PD animal model, suggesting a novel neuroprotective therapeutic approach. In the studies presented here, we evaluated the efficacy of a potent and clinically relevant second-generation irreversible Abl kinase inhibitor, INNO-406, as a therapeutic agent for PD. Our studies reveal that INNO-406 is capable of preventing the progression of dopaminergic neuronal damage in a toxin-induced C57 mouse model of PD. Using bovine brain microvessel endothelium as an in vitro blood-brain barrier (BBB) model, we detected rapid and significant transfer of INNO-406. Additionally, pharmacokinetic analyses demonstrated significant nanomolar concentrations of INNO-406 in brain in the presence or absence of MPTP administration, however, INNO-406 did not alter the brain levels of MPP+ in MPTP-treated mice. Finally, we showed that 10 mg/kg of INNO-406 given to C57 mice for one week before MPTP treatment (4×20 mg/kg i.p., every 2 h) and then for one week after MPTP treatment decreased the loss of dopamine in the striatum by 45% and the loss of TH+ neurons in substantia nigra pars compacts by 40%. This treatment regimen also abrogated activation of c-Abl, tyrosine phosphorylation of the Abl substrate and E3-ubiquitin ligase parkin, and accumulation of the toxic parkin substrate AIMP2. We propose that compounds of the INNO-406 class of Abl inhibitors will be useful new neuroprotective drugs for the treatment of PD-like pathology in preclinical systems that should be easily translated to the clinic.

Ibogaine (IBO) is an indole alkaloid that is reported to facilitate drug abstinence in substance abusers. Despite considerable investigation, the mechanism of IBO action in vivo and its suitability as a treatment for drug addiction remains unclear. The present study was designed to evaluate the time-course effects of acute IBO on neuroendocrine and neurochemical indices. Adult male rats were treated with i.p. saline or 50 mg/kg IBO and sacrificed 15, 30, 60, 120 min and 24 h later. Trunk blood was collected for hormone measures and brains were dissected for neurochemical analyses. IBO produced a rapid elevation in plasma prolactin that declined to control levels by 60 min. Corticosterone levels increased 15 min after drug administration, continued to increase for 120 min, but returned to control levels 24 h after dosing. IBO decreased dopamine (DA) concentrations in the striatum and frontal cortex at 30, 60 and 120 min after injection while DA metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), were elevated over the same time period. 24 h after IBO, DOPAC concentrations in striatum and HVA levels in the frontal cortex were below control values. Serotonin (5-HT) and its metabolite 5-hydroxyindole acetic acid (5-HIAA) were decreased at 60 min after IBO administration only in the striatum. These data indicate that a single injection of IBO produces a spectrum of effects that includes: (1) elevation of plasma prolactin and corticosterone, (2) short- and long-term effects on DA neurotransmission, and (3) modest, transient effects of 5-HT neurotransmission. The effects of IBO reported herein may have relevance to the anti-addictive properties of this drug, and this proposal warrants further investigation.

It has been postulated that differences in pharmacokinetics do not contribute to the well-known individual variability in response to amphetamine (AMPH), but this is yet to be investigated thoroughly. Therefore, rotational behavior of outbred rats (Sprague-Dawley, 4 months old) was recorded during microdialysis sessions and striatal microdialysate was analyzed concomitantly for AMPH and dopamine concentrations after a single injection of 2.5 mg/kg AMPH SC. Three hours later these rats received three doses of 5 mg/kg AMPH SC (spaced 2 h apart) and their brain temperature was recorded every 20 min. The most important findings were: 1) the increase in extracellular dopamine was highly correlated with the corresponding peak AMPH levels in the microdialysate; 2) the peak dopamine level in response to 2.5 mg/kg AMPH was predictive of the hyperthermic response observed during 3 × 5 mg/kg AMPH and 3) high versus low rotators differed neither in their AMPH nor in their dopamine extracellular striatal concentrations.

The effects of acute perinatal ischemia-hypoxia on fetal liver and brain energy metabolism, fetal brain total free fatty acid concentration and subsequent offspring behavior were investigated in rats. Ischemia-hypoxia was induced at term either by ligation of the uterine blood vessels or submersion of the entire uterine horn in warmed saline. Fetuses of the adjacent horn served as within-dam controls for all assessments and fetuses of dams which had not undergone the surgical stress served as independent controls for enzyme assays. Ischemia-hypoxia was associated with reduced activity of fatty acid synthase in the liver and brain. Total free fatty acid concentration significantly increased in the fetal hypoxic brain. Pups not used for enzyme analyses were cross-fostered for behavioral assessments. Relative to the enzymatic alterations, there were few behavioral alterations associated with ischemia-hypoxia. At postnatal day 30, rats made hypoxic by ligation of the uterine blood vessels had decreased caudate nucleus and brain stem weights relative to within-dam controls. At postnatal day 85, rats made hypoxic by submersion of the uterine horn had decreased olfactory bulb weight. The results of this study indicate an initial acute response to a brief period of ischemia-hypoxia at term pregnancy in the fetal rat brain and liver.

The combined effects of amphetamine (AMPH) and 3-nitropropionic acid (3-NPA) were investigated to determine how the energy depletion proposed to be produced by AMPH interacts with an inhibitor of mitochondrial respiration to produce striatal neurotoxicity. Neither two doses (2 h apart) of 3.75 mg/kg AMPH alone nor a single dose of 30 mg/kg 3-NPA i.p. produced neurotoxicity in the striatum or lowered striatal dopamine content in rat. Administration of 40 mg/kg of 3-NPA alone almost invariably produced either lethality or did not produce neurotoxicity in the striatum of surviving animals. However, 30 mg/kg of 3-NPA administered along with 2 doses of 3.75 mg/kg AMPH to 47 animals produced striatal damage in the 31 survivors with 15 of the surviving rats showing muscle rigidity/catatonia for several days after dosing, along with decreased food consumption. Thirteen of these 15 rats showed degeneration of axons and cell bodies in the medial caudate-putamen with minimal damage to the globus pallidus. However, two rats exhibited hindlimb paralysis and signs of axonal and neuronal soma degeneration in the thalamus and cerebellar nuclei as well as striatum. Sixteen of the rats given both AMPH and 3-NPA exhibited only torpidity and loss of muscle tone 1-3 h after dosing. Such rats showed no signs of neuronal cell degeneration in the striatum, but did show significant dopamine depletions (60% of control) and reductions in tyrosine hydroxylase immunoreactivity at 14 days postexposure. The mitochondrial dysfunction produced by 3-NPA combined with activation of neuronal pathways by AMPH may have predisposed terminals, axons and cell bodies in striatum to degeneration.

The need for biologically based, quantitative risk assessment procedures for noncancer endpoints such as neurotoxicity has been discussed in reports by the United States Congress (Office of Technology Assessment, OTA), National Research Council (NRC), and a federal coordinating council. According to OTA, current attention and resources allocated to health risk assessment research are inadequate and not commensurate with its impact on public health and the economy. Methods to include continuous rather than dichotomous data for neurotoxicity endpoints, biomarkers of exposure and effects, and pharmacokinetic and mechanistic data have been proposed for neurotoxicity risk assessment but require further review and validation before acceptance. The purpose of this symposium was to examine procedures to enhance the risk assessment process for neurotoxicants and to discuss techniques to make the process more quantitative. Accordingly, a review of the currently used safety factor risk assessment approach for neurotoxicants is provided along with specific examples of how this process may be enhanced with the use of the benchmark dose approach. The importance of including physiologically based pharmacokinetic data in the risk assessment process and specific examples of this approach is presented for neurotoxicants. The role of biomarkers of exposure and effect and mechanistic information in the risk assessment process are also addressed. Finally, quantitative approaches with the use of continuous neurotoxicity data are demonstrated and the outcomes compared to those generated by currently used risk assessment procedures.

The purpose of this study is to identify the incidence of capsular defects in patients undergoing revision hip arthroscopy. A radiographic and anatomical analysis of MR arthrograms of patients undergoing revision arthroscopy was performed to assess for the presence of capsular defect. Intra-operative images and findings were reviewed. Patients with persistent cam and pincer lesions were excluded. From October 2011 to October 2012, 25 patients underwent revision hip arthroscopy surgery, and 9 patients met our inclusion criteria. Within this series, all patients had post-surgical capsular irregularities and seven patients (78 %) had radiographic evidence of capsule and iliofemoral defects on MR arthrogram. Gross capsular defects were confirmed at revision surgery in two patients. The findings of this study demonstrate post-surgical radiographic and anatomical evidence of capsular defects in a select group of patients following hip arthroscopy. IV.

Nitrous Oxide (N2O), an N-methyl-D-aspartate (NMDA) receptor antagonist, and isoflurane (ISO), which acts on multiple receptors including postsynaptic gamma-aminobutyric acid (GABA) receptors, are frequently used inhalation anesthetics, alone or as a part of a balanced anesthetic regimen administered to pregnant women and to human neonates and infants requiring surgery. The current study investigated histological features and gene expression profiles in response to prolonged exposure to N2O or ISO alone, and their combination in developing rat brains. Postnatal day 7 rats were exposed to clinically-relevant concentrations of N2O (70%), ISO (1.0%) or N2O plus ISO (N2O + ISO) for 6 hours. The neurotoxic effects were evaluated and the brain tissues were harvested for RNA extraction 6 hours after anesthetic administration. The prolonged exposure to N2O + ISO produced elevated neuronal cell death as indicated by an increased number of TUNEL-positive cells in frontal cortical levels compared with control. No significant neurotoxic effects were observed in animals exposed to N2O or ISO alone. DNA microarray analysis revealed gene expression changes after N2O, ISO or N2O + ISO exposure. Differentially expressed genes (DEGs) from the N2O + ISO group were significantly associated with 45 pathways directly related to brain functions. Although the gene expression profiles from animals exposed to N2O or ISO alone were remarkably different from those of the control group, the pathways of these genes involved were not closely associated with neurons. These findings provide novel insights into the mechanisms by which N2O + ISO cause neurotoxicity in the developing brain, suggesting multiple factors are involved in the neuronal cell death-inducing effects (cascades) of N2O + ISO.

A possible role for NO modulation of dopamine (DA) release in the caudate/putamen (CPU) during methamphetamine (METH) exposure was investigated using in vivo microdialysis in rats. Inclusion of the nitric oxide synthase (NOS) inhibitors N G -nitro- l -arginine (NOARG), N G -nitro- l -arginine methyl ester (L-NAME) or D-NAME (less potent inhibitor) in the microdialysis buffer prior to METH minimally affected basal levels of DA, DOPAC or HVA in CPU microdialysate. However, L-NAME and NOARG produced concentration-dependent decreases of up to 64% (100 μM) in CPU DA levels in microdialysate during exposure to four doses of METH (5 mg/kg i.p./2h), with lesser effects on DOPAC or HVA. Reversal of the NOARG inhibition was produced by inclusion of 500 μM of either l -arginine or l -citrulline in the microdialysate. D-NAME (100 μM) minimally affected levels of DA or metabolites. Paradoxically, inclusion of from 20 to 2 μM of the NO x generators isosorbide dinitrate (ISON) or sodium nitroprusside (SNP) in the microdialysis buffer decreased DA and DOPAC levels in microdialysate during METH exposure. This paradox might result from the concentrations of NO x produced by SNP or ISON being great and not regionally specific resulting in inhibition of DA release and/or synthesis while the NO generated endogenously during METH exposure may have localized and site-specific actions. Alternatively, NO x may inhibit NOS or other enzymes in the NO synthesis pathway, thereby reducing levels of an intermediate (other than NO) which potentiates DA release. In their entirety, our results indicate that NO generation in the CPU may augment the release of DA during METH exposure.

Domoic acid is a potent excitotoxin produced by diatoms which is subsequently passed along the marine food chain. Its chemical structure and toxicological properties are similar to kainic acid. Like kainic acid, exposure results in extensive hippocampal degeneration. The effect of domoic acid on other primate brain structures, however, is less resolved. In an attempt to clarify this issue, the present study applied a degeneration specific histochemical technique (de Olmos' cupric-silver method) to reveal degeneration within the brains of domoic acid-dosed cynomolgus monkeys. Degenerating neuronal cell bodies and terminals were found not only within the hippocampus, but also within a number of other 'limbic' structures including the entorhinal cortex, the subiculum, the piriform cortex, the lateral septum, and the dorsal lateral nucleus of the thalamus. Although the hippocampus is a component of the original limbic circuit of Papez, other components such as the mammillary bodies, the anterior nucleus of the thalamus and the cingulate cortex contained no degeneration, while a number of more recently documented efferent targets of the hippocampal formation revealed extensive degeneration. The pattern of degeneration generally correlated with those regions containing high densities of kainate receptors.

As the incidence of human immunodeficiency virus (HIV) infection has increased in women over the past decade, the need for safe, effective therapy during pregnancy has increased concomitantly. Although dideoxynucleosides such as 3'-deoxy-3'-azidothymidine (AZT), 2',3'-dideoxyinosine (ddI), 2',3'-dideoxycytidine (ddC), and 2',3'-didehydro-3'-deoxythymidine have been approved for use in the general population, the administration, efficacy, and toxicity of these compounds during pregnancy and development are now being investigated. Initial human studies suggest that maternal use of AZT during pregnancy is well tolerated by both mother and child and provides a promising degree of protection from vertical HIV transmission to the infant. In vitro and animal models have greatly increased our understanding of the distribution and toxicity resulting from fetal dideoxynucleoside exposure. AZT, ddI, and ddC rapidly cross the placenta by simple diffusion but with different rates of transfer. In vivo data confirm the ...

A basic PBPK model for the dosimetry of organic acids in discrete areas of the brain was constructed using 2,4-D as a model compound. The PBPK model describes distribution of 2,4-D throughout the body and within discrete areas of the brain. The brain compartments in the model were the hypothalamus, caudate nucleus, hippocampus, forebrain, brainstem, and cerebellum. The remainder of the model consisted of two-body compartments and venous and arterial blood compartments. In the model, chemical uptake by the brain was membrane-limited by the blood-brain barrier (BBB) with saturable clearance from the cerebrospinal fluid (CSF) into the venous blood by the choroid plexus. The body has both a central and a deep compartment with saturable clearance from the central compartment. The model was used to examine the brain and CSF concentrations of 2,4-D as a function of plasma 2,4-D, as well as plasma time-course behavior using experimental data from rabbits given 2,4-D 40 or 100 mg/kg, IP or Na-2,4-D 40 mg/kg, i.v. administration. Model parameters were adjusted to fit the observed 2,4-D concentrations in blood and brain regions over a 2-h time frame. PBPK models could be a useful tool for evaluating the safety of this class of organic acids.

Advances in pediatric and obstetric surgery have resulted in an increase in the complexity, duration and number of anesthetic procedures. Currently, the general anesthetics that are used most often have either NMDA receptor blocking or GABA receptor activating properties. It has been reported that prolonged exposure of the developing brain to a clinically relevant concentration of anesthetics that have NMDA antagonist or GABA-mimetic properties, and/or their combinations, resulted in an extensive abnormal pattern of neuroapoptosis, and subsequent cognitive deficits in animals. Molecular imaging using positron emission tomography (PET) is a leading modality for obtaining non- or minimally invasive in vivo measurements of multiple biological processes in various organs. The development of microPET imaging applications has provided the ability to collect sensitive and quantitative three-dimensional molecular information from the living brains of a variety of animals. The main aim of this review was to describe molecular imaging approaches that have been used in the study of pediatric anesthetic-induced neuronal toxicity.

Ketamine is used as a general anesthetic, and recent data suggest that anesthetics can cause neurodegeneration and/or neuroprotection. The precise mechanisms are not completely understood. This review is to examine the work on ketamine and to address how developmental biology may be utilized when combined with biochemical, pathological, and pharmacokinetic assessments to produce a bridging model that may decrease the uncertainty in extrapolating preclinical data to human conditions. Advantages of using preclinical models to study critical issues related to ketamine anesthesia have been described. These include the relationships between ketamine-induced neurotoxicity/protection and the preclinical models/approaches in elucidating mechanisms associated with ketamine exposure. The discussions focus on the following: (1) the doses and time-course over which ketamine is associated with damage to, or protection of, neural cells, (2) how ketamine directs or signals neural cells to undergo apoptosis or necrosis, (3) how such exposures can trigger mitochondrial dysfunction, (4) how antioxidants and knockdowns of specific transcription modulators or receptors affect neurotoxicity induced by ketamine, and (5) whether the potential neural damage can be monitored after ketamine exposure in living animals using positron emission tomography.

Ketamine, a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist, is frequently used in pediatric general anesthesia. Accumulating evidence from animal experiments has demonstrated that ketamine causes neuronal cell death during the brain growth spurt. To elucidate the underlying mechanisms associated with ketamine-induced neuronal toxicity and search for approaches or agents to prevent ketamine's adverse effects on the developing brain, a primary nerve cell culture system was utilized. Neurons harvested from the forebrain of newborn rats were maintained under normal control conditions or exposed to either ketamine (10 µM) or ketamine plus L-carnitine (an antioxidant; 1-100 µM) for 24h, followed by a 24-h withdrawal period. Ketamine exposure resulted in elevated NMDA receptor (NR1) expression, increased generation of reactive oxygen species (ROS) as indicated by higher levels of 8-oxoguanine production, and enhanced neuronal damage. Coadministration of L-carnitine significantly diminished ROS generation and provided near complete protection of neurons from ketamine-induced cell death. NMDA receptors regulate channels that are highly permeable to calcium, and calcium imaging data demonstrated that neurons exposed to ketamine had a significantly elevated amplitude of calcium influx and higher intracellular free calcium concentrations ([Ca(2+)]i) evoked by NMDA (50 µM), compared with control neurons. These findings suggest that prolonged ketamine exposure produces an increase in NMDA receptor expression (compensatory upregulation), which allows for a higher/toxic influx of calcium into neurons once ketamine is removed from the system, leading to elevated ROS generation and neuronal cell death. L-Carnitine appears to be a promising agent in preventing or reversing ketamine's toxic effects on neurons at an early developmental stage.

The utility of hip arthroscopy has recently progressed beyond diagnostic to therapeutic purposes addressing central and peripheral compartment pathologies. Capsulotomy provides freedom of visualization and instrumentation. The contribution to hip stability of both dynamic and static hip structures is not fully understood. However, both basic science biomechanical and clinical outcome studies have exhibited a relevant role of the capsule in hip stability. Though rare, iatrogenic post-arthroscopy subluxation and dislocation have been reported. Therefore many surgeons have cautioned against aggressive capsulotomy or capsulectomy without repair, because of the potential for precipitation of iatrogenic hip instability. We typically perform a “T” capsulotomy and recommend complete capsular closure in conjunction with labral repair and osseous femoral and acetabular treatment. A safe, efficient, and effective method to accomplish complete capsular closure is presented to reduce iatrogenic postoperative hip instability.

Background: Osteochondral lesions of the talus (OLTs) are a common cause of ankle pain and disability. Current clinical guidelines favor autogenous or allogenic osteochondral grafting procedures for lesions larger than 10 mm in diameter because of increased failure rates in these larger lesions with arthroscopic debridement, curettage, and microfracture. There are currently no biomechanical data nor level I clinical data supporting this size threshold. Purpose: The purpose of this study was to determine the effect of OLT defect size on stress concentration, rim stress, and location of peak stress and whether a threshold defect size exists. Study Design: Descriptive laboratory study. Methods: Progressively larger medial OLTs were created (6, 8, 10, and 12 mm) in 8 fresh-frozen cadaveric ankle joints. With a calibrated Tekscan pressure sensor in the tibiotalar joint, an axial load of 686 N was applied, and pressure was recorded in neutral and 15° of plantar flexion with each defect size. Peak stress, contact area, peak and average rim stresses, and location of peak stress were determined. Results: The distance between peak stress and defect rim was significantly decreased with increasing defect size for lesions of 10 mm and larger. Total tibiotalar contact area was significantly decreased with increasing defect size and with ankle plantar flexion. While peak joint stress and peak rim stress were not affected by defect size or plantar flexion, average rim stress was significantly increased by plantar flexion. Conclusion: Reduction in contact area and shift in the location of peak stress with increasing defect size may contribute to articular cartilage degeneration, pain, and defect enlargement in patients with OLTs. There appears to be a threshold of 10 mm after which the distance between the rim of the defect and the peak stress decreases; however, there is no change in peak stress magnitude with increasing defect size. Clinical Relevance: The location of peak stress in the ankle joint becomes closer to the rim of the defect in OLTs at a threshold of 10 mm and greater in diameter. These data may have implications toward OLT size thresholds for surgical decision making in symptomatic lesions (ie, primary osteochondral transplantation procedure vs curettage and debridement). The ultimate goal is to determine whether there is a threshold defect size for primary osteoarticular graft techniques.

Aim: The purpose of the current study was to determine whether copper nanoparticles (Cu-NPs) can induce the release of proinflammatory mediators that influence the restrictive characteristics of the blood–brain barrier. Material & methods: Confluent rat brain microvessel endothelial cells (rBMECs) were treated with well-characterized Cu-NPs (40 or 60 nm). Cytotoxicity of the Cu-NPs was evaluated by cell proliferation assay (1.5–50 µg/ml). The extracellular concentrations of proinflammatory mediators (IL-1β, IL-2, TNF-α and prostaglandin E2) were evaluated by ELISA. Results: The exposure of Cu-NPs at low concentrations increases cellular proliferation of rBMECs, by contrast, high concentrations induce toxicity. Prostaglandin E2 release was significantly increased (threefold; 8 h) for Cu-NPs (40 and 60 nm). The extracellular levels of both TNF-α and IL-1β were significantly elevated following exposure to Cu-NPs. The P-apparent ratio, as an indicator of increased permeability of rBMEC was approximately twofold for Cu-NPs (40 and 60 nm). Conclusion: These data suggest that Cu-NPs can induce rBMEC, proliferation at low concentrations and/or induce blood–brain barrier toxicity and potential neurotoxicity at high concentrations. Original submitted 9 June 2011; Revised submitted 1 August 2011; Published online 16 February 2012

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes marked depletion of dopamine (DA) levels and reduction in the activity of tyrosine hydroxylase (TH) in the nigrostriatal DA pathway. In the brain, the enzyme monoamine oxidase B converts MPTP to 1-methyl-4-phenylpyridinium (MPP+) which enters DA terminals via DA uptake sites. Within the DA terminals, MPP+ blocks the mitochondrial complex I and causes ATP depletion. This is thought to be the main cause of MPTP-induced terminal degeneration. In addition, reactive oxygen species (ROS) generated after blockade of the complex I as well as those generated due to DA oxidation may participate in MPTP-induced dopaminotoxicity. The present study sought to determine if a single injection of a large dose of MPTP generates ROS. We also sought to determine if these changes as well as changes in DA levels were correlated and age-dependent. Toward that end, we have used C57/B6N male mice that were 22 days or 12 months old. These animals were injected with a single dose of MPTP (40 mg/kg, ip). Animals were sacrificed at various times after drug administration. MPTP produced no significant increase in ROS nor decreases in DA or HVA concentrations in the striatum of the younger mice. However, DOPAC concentrations were significantly decreased from 15-120 min after drug administration. In the older mice, MPTP caused significant increases in ROS from the beginning to the end of the study period. DA concentrations were decreased from 60 min onward. DOPAC concentrations were decreased significantly after 15-120 min while HVA concentrations were significantly increased after 60 and 120 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Elimination and metabolic profiles of the O -glucuronide conjugated products of pyrilamine and their nonconjugated O -dealkylated and N -desmethyl pyrilamine products were determined after the oral administration of ( 14 C)-pyrilamine maleate to Fischer 344 rats, B 6 C 3 F1 mice and female rhesus monkeys by stomach tube or i.v. The total cumulative urinary and fecal pyrilamine products were determined. The conjugated pyrilamine metabolites, isolated and identified were the glucuronide products of O -dealkylated pyrilamine and ring-hydroxylated pyrilamine, and the nonconjugated metabolites were predominately the N -desmethylpyrilamine and O -dealkylated pyrilamine and their ring-hydroxylated products. Statistically significant differences were observed in the percentages of the conjugated and nonconjugated metabolites of pyrilamine excreted by the three species studied.

Summary Knowledge is limited regarding the relative importance of the multiple factors involved in the vertical transmission of HIV infection; it will be many years before the intricacies of transmission are unraveled. A multifaceted approach to the scientific investigation of the factors involved as well as to therapeutic interventions to interrupt the transmission of maternal-fetal transmission of HIV are needed. Multiple agents will need to be investigated in parallel or in combination because of urgency of the problem is so great that developing potentially efficacious interventions cannot be postponed until all scientific questions have been researched. If any of the clinical trials are proven successful, the specimens accumulated from these trials, including samples on the mother, placental samples, and samples on the infant, should be analyzed intensively. These investigations may provide a key to unlocking the answers to the factors subserving protection from maternal-fetal transfer of the HIV infection.

The Effect of Dideoxycytidine on Lymphocyte Subpopulations in Nonhuman Primates. Taylor, L. D., Binienda, Z., Schmued, L., and Slikker, W., Jr. (1994). Fundam. Appl. Toxicol. 23, 434-438. In the present study, 2′,3′dideoxycytidine (ddC), which has antiretroviral activity, was given chronically to uninfected nonhuman primates to determine whether it produces adverse immunological or hematological effects. Nine healthy adult male rhesus monkeys were divided into three groups and given the following doses of ddC in a gelatin vehicle; group A, 0.06, 6.0, 3.0, and 1.5 mg/kg; group B, 0.6 mg/kg; group C, 0 mg/kg. Blood samples were collected for hematologic analysis and flow cytometric analyses of lymphocyte subpopulations. Chronic ddC exposure did not cause significant changes in the number of red blood cells, monocytes, or reticulocytes. The number of white blood cells and neutrophils increased and these changes were observed only in group A animals at the 1.5 mg/kg dose. The most significant alterations observed were decreases in the number of T helper cells (CD4) and B cells (CD20). CD4 + and CD20 + lymphocytes exhibited dose-related shifts that were reversible over time and after drug withdrawal. The results indicate that ddC has few hematologic effects but it does have profound but transient effects on the number of cells in lymphocyte subpopulations in normal primates.

Juvenile male rhesus monkeys treated with methylphenidate hydrochloride (MPH) to evaluate genetic and behavioral toxicity were observed after 14 mo of treatment to have delayed pubertal progression with impaired testicular descent and reduced testicular volume. Further evaluation of animals dosed orally twice a day with ( i ) 0.5 mL/kg of vehicle ( n = 10), ( ii ) 0.15 mg/kg of MPH increased to 2.5 mg/kg (low dose, n = 10), or ( iii ) 1.5 mg/kg of MPH increased to 12.5 mg/kg (high dose, n = 10) for a total of 40 mo revealed that testicular volume was significantly reduced ( P < 0.05) at months 15 to 19 and month 27. Testicular descent was significantly delayed ( P < 0.05) in the high-dose group. Significantly lower serum testosterone levels were detected in both the low- ( P = 0.0017) and high-dose ( P = 0.0011) animals through month 33 of treatment. Although serum inhibin B levels were increased overall in low-dose animals ( P = 0.0328), differences between groups disappeared by the end of the study. Our findings indicate that MPH administration, beginning before puberty, and which produced clinically relevant blood levels of the drug, impaired pubertal testicular development until ∼5 y of age. It was not possible to resolve whether MPH delayed the initiation of the onset of puberty or reduced the early tempo of the developmental process. Regardless, deficits in testicular volume and hormone secretion disappeared over the 40-mo observation period, suggesting that the impact of MPH on puberty is not permanent.

Systems biology has been defined as the iterative and integrative study of biological systems as they respond to perturbations. This chapter highlights the application of the systems biology approach to enhance the understanding of complex biological processes such as neurodegeneration in the developing brain. Although not yet fully delineated, the working model for anesthetic (e.g., ketamine)-induced neurodegeneration during development involves the modulation of normally occurring brain-sculpting mechanisms that control CNS development. Exposure of the developing mammal to anesthetics such as ketamine perturbs the endogenous N-methyl-d-aspartate (NMDA) receptor system and results in enhanced neuronal cell death. The working model is that prolonged ketamine exposure produces up-regulation of NMDA receptors and subsequent over-stimulation of the glutamatergic system by endogenous glutamate, triggering enhanced apoptosis of developing neurons. When the nervous system was perturbed with ketamine-induced anesthesia and gene expression changes were monitored, NMDA receptor genes were significantly up-regulated and this finding was confirmed by in situ hybridization studies. Systems biology, as applied to toxicology, provides a framework in which information can be arranged in the form of a biological model and various global datasets can be collected and integrated to determine whether they support the model. Discrepancies can be identified and hypotheses-driven studies conducted in order to address them. Thus, data generated via iteration of this process can be used to reformulate the model in light of the new data. Keywords: systems biology; toxicology; development; genomics; proteomics; metabolomics

We investigated and compared the concentration-dependent cytotoxicity of single-walled carbon nanotubes (SWCNTs) and SWCNTs functionalized with polyethylene glycol (SWCNT-PEGs) in neuronal PC12 cells at the biochemical, cellular, and gene expressional levels. SWCNTs elicited cytotoxicity in a concentration-dependent manner, and SWCNT-PEGs exhibited less cytotoxic potency than uncoated SWCNTs. Reactive oxygen species (ROS) were generated in both a concentration- and surface coating-dependent manner after exposure to these nanomaterials, indicating different oxidative stress mechanisms. More specifically, gene expression analysis showed that the genes involved in oxidoreductases and antioxidant activity, nucleic acid or lipid metabolism, and mitochondria dysfunction were highly represented. Interestingly, alteration of the genes is also surface coating-dependent with a good correlation with the biochemical data. These findings suggest that surface functionalization of SWCNTs decreases ROS-mediated toxicological response in vitro.

Stem cell transplantation promises new hope for the treatment of stroke although significant questions remain about how the grafted cells elicit their effects. One hypothesis is that transplanted stem cells enhance endogenous repair mechanisms activated after cerebral ischaemia. Recognizing that bilateral reorganization of surviving circuits is associated with recovery after stroke, we investigated the ability of transplanted human neural progenitor cells to enhance this structural plasticity. Our results show the first evidence that human neural progenitor cell treatment can significantly increase dendritic plasticity in both the ipsi- and contralesional cortex and this coincides with stem cell-induced functional recovery. Moreover, stem cell-grafted rats demonstrated increased corticocortical, corticostriatal, corticothalamic and corticospinal axonal rewiring from the contralesional side; with the transcallosal and corticospinal axonal sprouting correlating with functional recovery. Furthermore, we demonstrate that axonal transport, which is critical for both proper axonal function and axonal sprouting, is inhibited by stroke and that this is rescued by the stem cell treatment, thus identifying another novel potential mechanism of action of transplanted cells. Finally, we established in vitro co-culture assays in which these stem cells mimicked the effects observed in vivo. Through immunodepletion studies, we identified vascular endothelial growth factor, thrombospondins 1 and 2, and slit as mediators partially responsible for stem cell-induced effects on dendritic sprouting, axonal plasticity and axonal transport in vitro. Thus, we postulate that human neural progenitor cells aid recovery after stroke through secretion of factors that enhance brain repair and plasticity.

Phencyclidine (PCP) inhibits the uptake of the neurotransmitter dopamine (DA), and blocks N-methyl-D-aspartate (NMDA) receptor-regulated ion channels. PCP also binds to sigma receptors in vivo and in vitro in rat brain. Prolonged exposure to PCP in adults has been observed to reduce the number of PCP binding sites in brain. We designed these experiments to evaluate whether prolonged prenatal exposure to PCP produces alterations in the development of DA and NMDA systems in brain. To do so, we characterized the normal course of development of basal and stimulated DA release in striatal slices, the ontogeny of striatal DA concentrations, and the development of NMDA receptor channels and associated glutamate binding sites in frontal cortex. We compared these developmental profiles to those in rats exposed to prenatal PCP, in an attempt to characterize the effect of prenatal PCP exposure on the pattern of brain development. Pregnant CD rats were injected s.c. with either 0, 10 or 20 mg/kg PCP daily on gestational days 8 through 20. On postnatal days (PND) 8, 21, 45, or 100, rats were sacrificed and brain tissues isolated for in vitro assessment. In vitro [3H]DA release from striatal slices evoked by either 40 microM glutamate or 15 mM K+ increased over 250% from PND 8 to PND 45, and glutamate-stimulated release was still significantly below adult levels at PND 45. In contrast, D-methamphetamine (D-METH)-evoked [3H]DA release, frontal cortical glutamate binding sites and NMDA channels developed early, reaching adult levels on or before PND 21.(ABSTRACT TRUNCATED AT 250 WORDS)

Extracellular levels of dopamine (DA) and metabolites as well as serotonin [5-hydroxytryptamine (5-HT)] and 5-hydroxyindoleacetic acid (5-HIAA) were determined in the caudate putamen (CPU) of either 6- or 12-month-old female rats using microdialysis and high-performance liquid chromatography with electrochemical detection (HPLC-ED) before, during, and after four consecutive injections (given at 2-h intervals) of methamphetamine (METH). In 6-month-old rats administered 4 x 5 mg/kg METH at an environmental temperature (ET) of 23 degrees C, peak extracellular DA levels (between 50 and 150 rho g/10 microliters) were attained 30-45 min after each dose of METH while dihydroxyphenylacetic acid (DOPAC) decreased steadily after the first doses of METH until it reached a plateau at 50% of control (550-700 pg/10 microliters) levels. Increases in 5-HT levels during METH administrations paralleled DA increases while 5-HIAA decreases paralleled DOPAC decreases. The total CPU DA and 5-HT content of these rats was about 65% of control at 3 days post-METH. Reducing the ET to 4 degrees C during dosing decreased the peak and average DA levels attained during the 4 x 5 mg/kg METH administration to about 50% of that observed at a 23 degrees C ET. Increasing the dose to 4 x 10 mg/kg METH (4 degrees C ET) increased peak and average CPU DA levels to 200% that observed during 4 x 5 mg/kg METH at a 23 degrees C ET. However, no significant decreases in total CPU DA content of any rats dosed with METH at a 4 degrees C ET were observed 3 days post-METH. In 12-month-old rats dosed with 4 x 5 mg/kg METH (23 degrees C ET), the peak and average extracellular DA levels were only 30-60% that of 6-month-old rats. However, the CPU DA content of older rats was significantly decreased both 3 (30% control) and 14 (60% control) days post-METH. In summary, METH toxicity may not be predicted solely by the extracellular levels of DA attained during METH administration; age and ET also greatly influence METH neurotoxicity.

This report examined blood-brain barrier (BBB) related proinflammatory mediators and permeability changes in response to various sized gold nanoparticles (Au-NPs) (3, 5, 7, 10, 30 and 60 nm) in vitro using primary rat brain microvessel endothelial cells (rBMEC). The Au-NPs were characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS) and laser Doppler velocimetry (LDV). The accumulation of Au-NPs was determined spectrophotometrically. The rBMEC cytotoxicity of Au-NPs was evaluated by cell proliferation assay (XTT) (concentration range 0.24-15.63 μg/cm², for 24 h). The time-dependent changes (0, 2, 4 and 8 h) of several proinflammatory mediators (IL-1β, IL-2, TNFα and PGE₂) were evaluated by ELISA. The smaller Au-NPs (3-7 nm) showed higher rBMEC accumulation compared to larger Au-NPs (10-60 nm), while only moderate decreased cell viability was observed with small Au-NPs (3 nm) at high concentrations (≥ 7.8 μg/cm²). Even though slight changes in cell viability were observed with small Au-NPs, the basal levels of the various proinflammatory mediators remained unchanged with all treatments except LPS (positive control). rBMEC morphology appeared unaffected 24 h after exposure to Au-NPs with only mild changes in fluorescein permeability indicating BBB integrity was unaltered. Together, these data suggest the responses of the cerebral microvasculature to Au-NPs have a significant relationship with the Au-NPs unique size-dependent physiochemical properties.

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.

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.