The effects of long-term methylphenidate administration and withdrawal on progressive ratio responding and T2 MRI in the male rhesus monkey.

Methylphenidate is a frequently prescribed drug treatment for Attention-Deficit/Hyperactivity Disorder. However, methylphenidate has a mode of action similar to amphetamine and cocaine, both powerful drugs of abuse. There is lingering concern over the long-term safety of methylphenidate, especially in a pediatric population, where the drug may be used for years. We performed a long-term evaluation of the effects of chronic methylphenidate use on a behavioral measure of motivation in male rhesus monkeys. Animals were orally administered a sweetened methylphenidate solution (2.5 or 12.5 mg/kg, twice a day, Mon-Fri) or vehicle during adolescence and into adulthood. These animals were assessed on a test of motivation (progressive ratio responding), during methylphenidate treatment, and after cessation of use. Moreover, animals were evaluated with quantitative T2 MRI about one year after cessation of use. During the administration phase of the study animals treated with a clinically relevant dose of methylphenidate generally had a higher rate of responding than the control group, while the high dose group generally had a lower rate of responding. These differences were not statistically significant. In the month after cessation of methylphenidate, responding in both experimental groups dropped compared to their previous level of performance (p = 0.19 2.5 mg/kg, p = 0.06 12.5 mg/kg), and responding in the control animals was unchanged (p = 0.81). While cessation of methylphenidate was associated with an acute reduction in responding, group differences were not observed in the following months. These data suggest that methylphenidate did not have a significant impact on responding, but withdrawal from methylphenidate did cause a temporary change in motivation. No changes in T2 MRI values were detected when measured about one year after cessation of treatment. These data suggest that long-term methylphenidate use does not have a negative effect on a measure of motivation or brain function / microstructure as measured by quantitative T2 MRI. However, cessation of use might be associated with temporary cognitive changes, specifically alteration in motivation. Importantly, this study modeled use in healthy individuals, and results may differ if the same work was repeated in a model of ADHD.

Optimization of Detection of Gadodiamide Brain Retention in Rats Using Quantitative T2 Mapping and Intraperitoneal Administration.

BACKGROUND: Currently, the gadolinium retention in the brain after the use of contrast agents is studied by T1 -weighted magnetic resonance imaging (MRI) (T1 w) and T1 mapping. The former does not provide easily quantifiable data and the latter requires prolonged scanning and is sensitive to motion. T2 mapping may provide an alternative approach. Animal studies of gadolinium retention are complicated by repeated intravenous (IV) dosing, whereas intraperitoneal (IP) injections might be sufficient. HYPOTHESIS: T2 mapping will detect the changes in the rat brain due to gadolinium retention, and IP administration is equivalent to IV for long-term studies. STUDY TYPE: Prospective longitudinal. ANIMAL MODEL: A total of 31 Sprague-Dawley rats administered gadodiamide IV (N = 8) or IP (N = 8), or saline IV (N = 6) or IP (N = 9) 4 days per week for 5 weeks. FIELD STRENGTH/SEQUENCES: A 7 T, T1 w, and T2 mapping. ASSESSMENT: T2 relaxation and image intensities in the deep cerebellar nuclei were measured pre-treatment and weekly for 5 weeks. Then brains were assessed for neuropathology (N = 4) or gadolinium content using inductively coupled plasma mass spectrometry (ICP-MS, N = 12). STATISTICAL TESTS: Repeated measures analysis of variance with post hoc Student-Newman-Keuls tests and Hedges' effect size. RESULTS: Gadolinium was detected by both approaches; however, T2 mapping was more sensitive (effect size 2.32 for T2 vs. 0.95 for T1 w), and earlier detection (week 3 for T2 vs. week 4 for T1 w). ICP-MS confirmed the presence of gadolinium (3.076 ± 0.909 nmol/g in the IV group and 3.948 ± 0.806 nmol/g in the IP group). There was no significant difference between IP and IV groups (ICP-MS, P = 0.109; MRI, P = 0.696). No histopathological abnormalities were detected in any studied animal. CONCLUSION: T2 relaxometry detects gadolinium retention in the rat brain after multiple doses of gadodiamide irrespective of the route of administration. EVIDENCE LEVEL: 1 TECHNICAL EFFICACY: Stage 1.

Global Neurotoxicity: Quantitative Analysis of Rat Brain Toxicity Following Exposure to Trimethyltin.

The organotin, trimethyltin (TMT), is a highly toxic compound. In this study, silver-stained rat brain sections were qualitatively and quantitatively evaluated for degeneration after systemic treatment with TMT. Degenerated neurons were counted using image analysis methods available in the HALO image analysis software. Specific brain areas including the cortex, inferior and superior colliculus, and thalamus were quantitatively analyzed. Our results indicate extensive and widespread damage to the rat brain after systemic administration of TMT. Qualitative results suggest severe TMT-induced toxicity 3 and 7 days after the administration of TMT. Trimethyltin toxicity was greatest in the hippocampus, olfactory area, cerebellum, pons, mammillary nucleus, inferior and superior colliculus, hypoglossal nucleus, thalamus, and cerebellar Purkinje cells. Quantification showed that the optic layer of the superior colliculus exhibited significantly more degeneration compared to layers above and below. The inferior colliculus showed greater degeneration in the dorsal area relative to the central area. Similarly, in cortical layers, there was greater neurodegeneration in deeper layers compared to superficial layers. Quantification of damage in various thalamic nuclei showed that the greatest degeneration occurred in midline and intralaminar nuclei. These results suggest selective neuronal network vulnerability to TMT-related toxicity in the rat brain.

Ketamine-Induced Toxicity in Neurons Differentiated from Neural Stem Cells.

Ketamine is used as a general anesthetic, and recent data suggest that anesthetics can cause neuronal damage when exposure occurs during development. The precise mechanisms are not completely understood. To evaluate the degree of ketamine-induced neuronal toxicity, neural stem cells were isolated from gestational day 16 rat fetuses. On the eighth day in culture, proliferating neural stem cells were exposed for 24 h to ketamine at 1, 10, 100, and 500 µM. To determine the effect of ketamine on differentiated stem cells, separate cultures of neural stem cells were maintained in transition medium (DIV 6) for 1 day and kept in differentiation medium for another 3 days. Differentiated neural cells were exposed for 24 h to 10 µM ketamine. Markers of cellular proliferation and differentiation, mitochondrial health, cell death/damage, and oxidative damage were monitored to determine: (1) the effects of ketamine on neural stem cell proliferation and neural stem cell differentiation; (2) the nature and degree of ketamine-induced toxicity in proliferating neural stem cells and differentiated neural cells; and (3) to provide information regarding receptor expression and possible mechanisms underlying ketamine toxicity. After ketamine exposure at a clinically relevant concentration (10 µM), neural stem cell proliferation was not significantly affected and oxidative DNA damage was not induced. No significant effect on mitochondrial viability (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay) in neural stem cell cultures (growth medium) was observed at ketamine concentrations up to 500 µM. However, quantitative analysis shows that the number of differentiated neurons was substantially reduced in 10 µM ketamine-exposed cultures in differentiation medium, compared with the controls. No significant changes in the number of GFAP-positive astrocytes and O4-positive oligodendrocytes (in differentiation medium) were detected from ketamine-exposed cultures. The discussion focuses on: (1) the doses and time-course over which ketamine is associated with damage of neural cells; (2) how ketamine directs or signals neural stem cells/neural cells to undergo apoptosis or necrosis; (3) how functional neuronal transmitter receptors affect neurotoxicity induced by ketamine; and (4) advantages of using neural stem cell models to study critical issues related to ketamine anesthesia.

Protective effect of acetyl-L-carnitine on propofol-induced toxicity in embryonic neural stem cells.

Propofol is a widely used general anesthetic. A growing body of data suggests that perinatal exposure to general anesthetics can result in long-term deleterious effects on brain function. In the developing brain there is evidence that general anesthetics can cause cell death, synaptic remodeling, and altered brain cell morphology. Acetyl-L-carnitine (L-Ca), an anti-oxidant dietary supplement, has been reported to prevent neuronal damage from a variety of causes. To evaluate the ability of L-Ca to protect against propofol-induced neuronal toxicity, neural stem cells were isolated from gestational day 14 rat fetuses and on the eighth day in culture were exposed for 24h to propofol at 10, 50, 100, 300 and 600 µM, with or without L-Ca (10 µM). Markers of cellular proliferation, mitochondrial health, cell death/damage and oxidative damage were monitored to determine: (1) the effects of propofol on neural stem cell proliferation; (2) the nature of propofol-induced neurotoxicity; (3) the degree of protection afforded by L-Ca; and (4) to provide information regarding possible mechanisms underlying protection. After propofol exposure at a clinically relevant concentration (50 µM), the number of dividing cells was significantly decreased, oxidative DNA damage was increased and a significant dose-dependent reduction in mitochondrial function/health was observed. No significant effect on lactase dehydrogenase (LDH) release was observed at propofol concentrations up to 100 µM. The oxidative damage at 50 µM propofol was blocked by L-Ca. Thus, clinically relevant concentrations of propofol induce dose-dependent adverse effects on rat embryonic neural stem cells by slowing or stopping cell division/proliferation and causing cellular damage. Elevated levels of 8-oxoguanine suggest enhanced oxidative damage [reactive oxygen species (ROS) generation] and L-Ca effectively blocks at least some of the toxicity of propofol, presumably by scavenging oxidative species and/or reducing their production.

Inhalation Anesthesia-Induced Neuronal Damage and Gene Expression Changes in Developing Rat Brain.

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.

Ketamine-induced neuronal damage and altered N-methyl-D-aspartate receptor function in rat primary forebrain culture.

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.

A minimally invasive, translational biomarker of ketamine-induced neuronal death in rats: microPET Imaging using 18F-annexin V.

It has been reported that suppression of N-methyl-D-aspartate (NMDA) receptor function by ketamine may trigger apoptosis of neurons when given repeatedly during the brain growth spurt period. Because microPET scans can provide in vivo molecular imaging at sufficient resolution, it has been proposed as a minimally invasive method for detecting apoptosis using the tracer (18)F-labeled annexin V. In this study, the effect of ketamine on the metabolism and integrity of the rat brain were evaluated by investigating the uptake and retention of (18)F-fluorodeoxyglucose (FDG) and (18)F-annexin V using microPET imaging. On postnatal day (PND) 7, rat pups in the experimental group were exposed to six injections of ketamine (20 mg/kg at 2-h intervals) and control rat pups received six injections of saline. On PND 35, 37 MBq (1 mCi) of (18)F-FDG or (18)F-annexin V was injected into the tail vein of treated and control rats, and static microPET images were obtained over 1 (FDG) and 2 h (annexin V) following the injection. No significant difference was found in (18)F-FDG uptake in the regions of interest (ROIs) in the brains of ketamine-treated rats compared with saline-treated controls. The uptake of (18)F-annexin V, however, was significantly increased in the ROI of ketamine-treated rats. Additionally, the duration of annexin V tracer washout was prolonged in the ketamine-treated animals. These results demonstrate that microPET imaging is capable of distinguishing differences in retention of (18)F-annexin V in different brain regions and suggests that this approach may provide a minimally invasive biomarker of neuronal apoptosis in rats.

Prolonged exposure to ketamine increases neurodegeneration in the developing monkey brain.

Ketamine, a widely used pediatric anesthetic, has been associated with enhanced neuronal toxicity in the developing brain, but mechanisms and neuronal susceptibility to neurotoxic insult leading to neuronal cell death remain poorly defined. One of the main goals of this study was to determine whether there is a duration of ketamine-induced anesthesia below which no significant ketamine-induced neurodegeneration can be detected. Newborn rhesus monkeys (postnatal day 5 or 6) were administered ketamine intravenously for 3, 9 or 24h to maintain a steady anesthetic plane, followed by a 6-h withdrawal period. The 9- and 24-h durations were selected as relatively long and extremely long exposures, respectively, while the 3-h treatment more closely approximates a typical duration of pediatric general anesthesia. Animals were subsequently perfused under anesthesia and brain tissue was processed for analyses using silver and Fluoro-Jade C stains and caspase-3 immunostain. The results indicated that no significant neurotoxic effects occurred if the anesthesia duration was 3h. However, ketamine infusions for either 9 or 24h significantly increased neuronal cell death in layers II and III of the frontal cortex. Although a few caspase-3- and Fluoro-Jade C-positive neuronal profiles were observed in some additional brain areas including the hippocampus, thalamus, striatum and amygdala, no significant differences were detected between ketamine-treated and control monkeys in these areas after 3, 9 or 24h of exposure. These data show that treatment with ketamine up to 3h is without adverse effects as determined by nerve cell death. However, anesthetic durations of 9h or greater are associated with significant brain cell death in the frontal cortex. Thus, the threshold duration below which no neurotoxicity would be expected is somewhere between 3 and 9h.

Overlapping but distinct effects of genistein and ethinyl estradiol (EE(2)) in female Sprague-Dawley rats in multigenerational reproductive and chronic toxicity studies.

Genistein and ethinyl estradiol (EE(2)) were examined in multigenerational reproductive and chronic toxicity studies that had different treatment intervals among generations. Sprague-Dawley rats received genistein (0, 5, 100, or 500 ppm) or EE(2) (0, 2, 10, or 50 ppb) in a low phytoestrogen diet. Nonneoplastic effects in females are summarized here. Genistein at 500 ppm and EE(2) at 50 ppb produced similar effects in continuously exposed rats, including decreased body weights, accelerated vaginal opening, and altered estrous cycles in young animals. At the high dose, anogenital distance was subtly affected by both compounds, and a reduction in litter size was evident in genistein-treated animals. Genistein at 500 ppm induced an early onset of aberrant cycles relative to controls in the chronic studies. EE(2) significantly increased the incidence of uterine lesions (atypical focal hyperplasia and squamous metaplasia). These compound-specific effects appeared to be enhanced in the offspring of prior exposed generations.

Potential neurotoxicity of ketamine in the developing rat brain.

Ketamine, an N-methyl-D-aspartate (NMDA) receptor ion channel blocker, is a widely used anesthetic recently reported to enhance neuronal death in developing rodents and nonhuman primates. This study evaluated dose-response and time-course effects of ketamine, levels of ketamine in plasma and brain, and the relationship between altered NMDA receptor expression and ketamine-induced neuronal cell death during development. Postnatal day 7 rats were administered 5, 10, or 20 mg/kg ketamine using single or multiple injections (subcutaneously) at 2-h intervals, and the potential neurotoxic effects were examined 6 h after the last injection. No significant neurotoxic effects were detected in layers II or III of the frontal cortex of rats administered one, three, or six injections of 5 or 10 mg/kg ketamine. However, in rats administered six injections of 20 mg/kg ketamine, a significant increase in the number of caspase-3- and Fluoro-Jade C-positive neuronal cells was observed in the frontal cortex. Electron microscopic observations showed typical nuclear condensation and fragmentation indicating enhanced apoptotic characteristics. Increased cell death was also apparent in other brain regions. In addition, apoptosis occurred after plasma and brain levels of ketamine had returned to baseline levels. In situ hybridization also showed a remarkable increase in mRNA signals for the NMDA NR1 subunit in the frontal cortex. These data demonstrate that ketamine administration results in a dose-related and exposure-time dependent increase in neuronal cell death during development. Ketamine-induced cell death appears to be apoptotic in nature and closely associated with enhanced NMDA receptor subunit mRNA expression.

Protective effects of 7-nitroindazole on ketamine-induced neurotoxicity in rat forebrain culture.

Ketamine, a non-competitive N-methyl-d-aspartate (NMDA) receptor antagonist, is used as a pediatric anesthetic for surgical procedures. Recent data suggest that anesthetic drugs may cause neurodegeneration during development. The purpose of this study was to determine the dose and temporal response of ketamine using newborn rat forebrain cultures and also to determine if co-administration of 7-nitroindazole, a nitric oxide synthase (NOS) inhibitor, could protect or reverse ketamine-induced cell death. Neural cells collected from the rat forebrain were incubated for 24h with 1, 10 or 20 microM ketamine alone or with ketamine plus 1, 5, 10 or 20 microM 7-nitroindazole. Ketamine (10 microM) caused an increase in DNA fragmentation and elevated immunoreactivity to nitrotyrosine, a marked reduction in the expression of the neuronal marker polysialic acid neural cell adhesion molecule (PSA-NCAM) and in mitochondrial metabolism, as well as an increased Bax/BCL-XL ratio. No significant effect was observed in the release of lactate dehydrogenase (LDH). Ketamine-induced neurotoxic effects were effectively blocked by 7-nitroindazole (10 microM). These data indicate a role for nitric oxide in the enhanced degeneration induced by ketamine in vitro and also suggest that blocking neuronal nitric oxide synthase (nNOS) may help reduce the risk of ketamine in pediatrics.

Ketamine-induced neuronal cell death in the perinatal rhesus monkey.

Ketamine is widely used as a pediatric anesthetic. Studies in developing rodents have indicated that ketamine-induced anesthesia results in brain cell death. Additional studies are needed to determine if ketamine anesthesia results in brain cell death in the nonhuman primate and if so, to begin to define the stage of development and the duration of ketamine anesthesia necessary to produce brain cell death. Rhesus monkeys (N = 3 for each treatment and control group) at three stages of development (122 days of gestation and 5 and 35 postnatal days [PNDs]) were administered ketamine intravenously for 24 h to maintain a surgical anesthetic plane, followed by a 6-h withdrawal period. Similar studies were performed in PND 5 animals with 3 h of ketamine anesthesia. Animals were subsequently perfused and brain tissue processed for analyses. Ketamine (24-h infusion) produced a significant increase in the number of caspase 3-, Fluoro-Jade C- and silver stain-positive cells in the cortex of gestational and PND 5 animals but not in PND 35 animals. Electron microscopy indicated typical nuclear condensation and fragmentation in some neuronal cells, and cell body swelling was observed in others indicating that ketamine-induced neuronal cell death is most likely both apoptotic and necrotic in nature. Ketamine increased N-methyl-D-aspartate (NMDA) receptor NR1 subunit messenger RNA in the frontal cortex where enhanced cell death was apparent. Earlier developmental stages (122 days of gestation and 5 PNDs) appear more sensitive to ketamine-induced neuronal cell death than later in development (35 PNDs). However, a shorter duration of ketamine anesthesia (3 h) did not result in neuronal cell death in the 5-day-old monkey.

Blockade of N-methyl-D-aspartate receptors by ketamine produces loss of postnatal day 3 monkey frontal cortical neurons in culture.

Ketamine, an N-methyl-D-aspartate (NMDA) receptor antagonist, is used as a general pediatric anesthetic. Recent data suggest that anesthetic drugs may cause neurodegeneration during development. The purpose of this study was to determine the robustness of ketamine-induced developmental neurotoxicity using rhesus monkey frontal cortical cultures and also to determine if dysregulation of NMDA receptor subunits promotes ketamine-induced cell death. Frontal cortical cells collected from the neonatal monkey were incubated for 24 h with 1, 10, or 20 microM ketamine alone or with ketamine plus either NR1 antisense oligonucleotides or the nuclear factor kB translocation inhibitor, SN-50. Ketamine caused a marked reduction in the neuronal marker polysialic acid neural cell adhesion molecule and mitochondrial metabolism, as well as an increase in DNA fragmentation and release of lactate dehydrogenase. Ketamine-induced effects were blocked by NR1 antisenses and SN-50. These data suggest that NR1 antisenses and SN-50 offer neuroprotection from the enhanced degeneration induced by ketamine in vitro.