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.

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.

The use of in situ perfusion of the rat mesentery as a model to investigate vascular injury directly induced by drugs.

Exposure of the vasculature to vasodilators, pharmaceuticals and industrial chemicals may lead to injury of the blood vessel wall in animals. Vascular injury may begin with changes in the permeability of vascular endothelial cell and vessels, resulting in possible hemorrhage and edema leading subsequently to immune cell infiltration. The present study was undertaken to determine if the direct exposure of the Sprague Dawley rat mesenteric vasculature through the perfusion of aminophylline, fenoldopam, compound 48/80, histamine or serotonin has any such effects on the blood vessels, and if the two vital dyes Monastral blue B and Evans blue can be used to enhance the visualization of the vascular damage. Microscopic visualization was enhanced by the use of dyes and a variety of alterations of the perfused mesenteric vessels were detected, including varying degrees of mast cell degranulation, microvascular vasodilatation and increased vascular permeability. Macroscopic evidence of vascular damage was minimal. This study demonstrates that in situ perfusion of the rat mesentery is a simple and useful method to eliminate the influence of a variety of physiologic influences or homeostatic responses and can be used to further investigate drug-induced vascular damage.

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.

Cytochrome c: a non-invasive biomarker of drug-induced liver injury.

Limitations of existing biomarkers to detect liver injury in experimental animals highlight the need for additional tools to predict human toxicity. The utility of cytochrome c (cyt c) as a biomarker in serum and urine was evaluated in two rodent liver injury models. Adult Sprague-Dawley rats treated with acetaminophen or D-galactosamine (GalN) showed dose- and time-dependent histomorphological changes and TUNEL staining in liver consistent with hepatocellular necrosis, apoptosis and inflammation up to 72 h. Matching changes in serum alanine transaminase (ALT), aspartate transaminase (AST) and cyt c peaked at 24 h for either drug at the highest dose, cyt c falling rapidly at 48 hours with ALT and AST remained high. Intracellular transit of cyt c from mitochondria to the cytoplasm in damaged hepatocytes, and then to peripheral circulation, was observed by immunohistochemistry. Correlation coefficients between cyt c and serum diagnostic tests indicate the liver to be the primary source of cyt c. Urinary analysis for cyt c revealed time-dependent increase at 6 h, peaking at 24 h in GalN-treated rats in contrast with irregular patterns of urinary ALT and AST activity. Histological changes detected at 6 h preceded altered ALT, AST and cyt c at 12 and 18 h, respectively, in GalN-treated rats. These studies demonstrate cyt c to be a useful indicator of hepatic injury in rodents and support its utility as a non-invasive predictor of drug-induced hepatotoxicity, when utilized as a potential urinary biomarker.

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.

The utility of the K6/ODC transgenic mouse as an alternative short term dermal model for carcinogenicity testing of pharmaceuticals.

The use of transgenic rodents may overcome many limitations of traditional cancer studies. Regulatory perspectives continue to evolve as new models are developed and validated. The transgenic mouse, K6/ODC, develops epidermal tumors when exposed to genotoxic carcinogens. In this study, K6/ODC mice were evaluated for model fitness and health robustness in a 36-week study to determine oncogenic risk of residual DNA in vaccines from neoplastic cell substrates. K6/ODC and C57BL/6 mice were treated with T24-H-ras expression plasmid, carrier vector DNA, or saline topically or by subcutaneous injection. One group of K6/ODC mice received 7,12-dimethylbenz-[a]anthracene [DMBA] dermally. Only DMBA-treated mice developed papillomas by six weeks, increasing in incidence to 25 weeks. By week 11, many K6/ODC mice showed severe dehydration and dermal eczema. By week 32, (6/8) surviving K6/ODC mice showed loss of mobility and balance. Microscopic evaluation of tissues revealed dermal/sebaceous gland hyperplasia, follicular dystrophy, splenic atrophy, and amyloid deposition/neutrophilic infiltration within liver, heart, and spleen, in all K6/ODC mice. Pathology was not detected in C57BL/6 mice. Progressive adverse health, decreased survival, and failure to develop papillomas to the H-ras plasmid suggest that K6/ODC mice may be an inappropriate alternative model for detection of oncogenic DNA and pharmaceutical carcinogenicity testing.

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.

Increased neurobehavioral toxicity of styrene in protein-malnourished rats.

Influence of protein deficiency on the neurobehavioral toxicity of styrene during gestation and early infancy was studied in rats. Eye opening and fur growth were delayed in rat pups born to dams receiving a low protein diet. These pups also showed a delay in the development of surface and air righting reflexes and cliff avoidance response and a marginal increase in the levels of dopamine and serotonin receptors in comparison to those born to dams receiving a normal protein diet. Alterations in these parameters were more marked in pups born to dams exposed to styrene and receiving a low protein diet. In addition, these pups also showed a significant decrease in the activity of monoamine oxidase, Na+, K(+)-ATPase and succinic dehydrogenase as well as significant increases in motor activity and receptor sensitivity when compared to rat pups born to dams receiving a low protein diet. No significant alterations in behavioral and biochemical parameters were observed in the pups born to dams exposed to styrene and receiving a normal protein diet at this dose level. These results suggest that protein deficiency during early life renders the animals more susceptible to styrene.

Quinidine enhancement of digoxin toxicity in rats and minipigs.

The combined use of digoxin and quinidine has been associated with potentially fatal toxic reactions. The mechanisms involved in this drug interaction were investigated in Wistar rats and Hormel miniature pigs (minipigs). Infusions of digoxin alone (0.013 mg/min) and in combination with quinidine (0.051 mg/min) demonstrated that quinidine pretreatment significantly decreases the time of onset of arrhythmias and death in these animals as compared with digoxin alone. Similarly, when administered as a slow, bolus intravenous (i.v.) injection to either anesthetized or unanesthetized minipigs, quinidine pretreatment (10 mg/kg, i.v.) led to the rapid development of arrhythmias and death. Quinidine administration in both species also causes a dramatic reduction in blood pressure, and it appears that this hypotension may trigger the irregular rhythms of the heart once digoxin is administered. Electrocardiographic (ECG) evidence showed that the toxicity associated with digoxin administration alone was characterized by bradyarrhythmias and death was attributed to cardiac arrest. In contrast, quinidine pretreatment led to severe tachyarrhythmias and earlier death by ventricular fibrillation. These studies demonstrate the usefulness of these two species, particularly the minipigs, in assessing the magnitude of this drug interaction and also confirm the risks involved when these two drugs are combined.

Opiate mixed agonist-antagonist interactions with histamine antagonists vs. morphine.

Anecdotal reports of polydrug abuse in humans using tripelennamine and pentazocine prompted our investigation of drug interactions between tripelennamine, morphine and various synthetic mixed agonist-antagonists in mice. Pentazocine, nalbuphene and butorphanol, at doses of 4.0-8.0 mg/kg, all showed frank or borderline intrinsic antinociceptive activity and potentiated the tripelennamine response, whereas cyclazocine, an experimental compound with very strong mixed agonist-antagonist qualities at 5.0 mg/kg, showed intrinsic antinociceptive activity but was not potentiated by tripelennamine and actually blocked the tripelennamine response. In a comparative study, pentazocine, butorphanol and nalbuphene had no effect on morphine antinociception whereas cyclazocine completely abolished the antinociceptive effects of morphine. Cimetidine blocked the response of cyclazocine, but not nalbuphene, pentazocine or butorphanol. Our findings demonstrate that the mechanism of action of cyclazocine is significantly different from that of the other mixed agonist-antagonists studied. They also suggest possible histaminergic involvement in antinociception, as well as a locus for antinociception separate from the opiate receptor.

Effect of H1 blockers alone and in combination with morphine to produce antinociception in mice.

Antinociception was assessed in male CD-1 mice by a modification of Haffner's tail-clamp procedure. The H1 blockers, including an ethylenediamine (pyrilamine), an ethanolamine (diphenhydramine), a phenothiazine (methdilazine), a piperazine (cyclizine) and an alkylamine (chlorpheniramine), all produced antinociception when given alone to mice and also caused potentiation when combined with morphine.

The effect of week-long infusion of propranolol, via an osmotic minipump, on blood pressure, heart rate and vascular responses in spontaneously hypertensive rats.

Propranolol was infused in SHR subcutaneously for 7 days at two concentrations (either 3.75 or 7.5 mg kg-1 day) via a minipump. Mean blood pressure and heart rate measured under pentobarbitone anaesthesia on day 7 after implantation showed a significant dose-dependent decrease in both propranolol-treated groups. In the low-dose propranolol-treated rats, there was no change in contractile responses to phenylephrine over controls. In rats receiving the higher dose of propranolol there was a significant increase in the response to phenylephrine. There was no change in the relaxation response of any of the groups to isoprenaline. The results indicate that propranolol, while lowering blood pressure and heart rate, is also modifying the alpha-receptor response of the vascular wall in the spontaneously hypertensive rat.

Interaction of hexachlorophene and other compounds with spin-labeled brain membranes.

Experiments reported here demonstrate that hexachlorophene influences oxidation-reduction events inside the brain membrane, possibly via a free radical mechanism. This was shown by nitroxide spin label quenching inside the rat cerebellum membrane bilayer due to the interaction between hexachlorophene and peroxidase-hydrogen peroxide system. Prior addition of antioxidants, e.g., vitamin E or butylated hydroxytoluene, prevented such membrane-bound fatty acid spin label reduction, presumably due to their free radical scavenging abilities. The 5-doxyl stearic acid spin probe attached to the brain membranes did not exhibit any detectable changes in their ESR spectra nor, consequently, in the microviscosity of the membranes when exposed to up to 40 mM hexachlorophene.

Protection with butylated hydroxytoluene and other compounds against intoxication and mortality caused by hexachlorophene.

The antioxidants butylated hydroxytoluene (BHT) and ethoxyquin protected rats against intoxication and mortality normally produced by hexachlorophene (HCP, 100 mg/kg). BHT also prevented the elevation of cerebrospinal fluid pressure, a central nervous system effect of HCP poisoning. In addition, both phenobarbital and SKF-525A protected against HCP poisoning, with the barbiturate also offering significant protection against triethyltin. L-Ascorbic acid, vitamin E, N,N-diphenyl-p-phenylenediamine and reduced and oxidized glutathione over a range of doses were ineffective in preventing HCP lethality. The protective effect of phenobarbital against HCP and triethyltin intoxication further supports existing evidence of a common or similar mechanism of toxic action for these two structurally dissimilar compounds.

The effect of tripelennamine alone and in combination with opiates to produce antinociception in mice.

Antinociception (ANTI) was assessed in male CD-1 mice by a modification of Haffner's tail clamp procedure. Studies revealed that tripelennamine (Tp) alone produced antinociception (ANTI) in mice and also caused potentiation when combined with morphine (M) or nalbuphene (NB). Naloxone (Nx) only partially blocked the effect of Tp, but fully blocked M. Although atropine (At) had no intrinsic ANTI activity, it enhanced that of Tp but not M. Histamine (Hm) had no intrinsic ANTI activity, nor did it interact with either Tp or M. The partial abolition of Tp ANTI, in contrast to complete blockade of M effects with Nx, appears to indicate that Tp can stimulate the opiate receptor as well as another receptor for ANTI at a different locus. The combination of Tp with various opiates may have considerable abuse potential.

Alteration of sensitivity of adrenergic vascular responses after prolonged exposure to agonists via osmotic minipump.

A study was performed to determine whether a constant 1-week exposure to either alpha or beta agonists in vivo would allow alteration or manipulation of the responses of rat aortic alpha- and beta-adrenergic receptors. Osmotic minipumps delivering either phenylephrine, isoproterenol, or propranolol for 7 days at a dose of 3.2, 4.2, or 5.2 mg/kg/day, respectively, were implanted in male Holtzman rats under halothane anesthesia. Seven days later, rats were killed and aortic ring preparations were used to measure alpha- and beta-adrenergic responses. In phenylephrine-pretreated rats, alpha-adrenergic responses, as measured by contractions induced by phenylephrine, were markedly reduced (P less than 0.05) across a dose range of 10(-9) to 10(-6) M. In contrast, in these same phenylephrine-pretreated preparations, the beta-adrenergic responses involving isoproterenol-induced relaxation were significantly increased (P less than 0.05) across a dose range of 10(-7) to 10(-5) M. Isoproterenol pretreatment for 7 days resulted in a statistically significant reduction of beta-adrenergic aortic relaxation, whereas the alpha-adrenergic responses to phenylephrine remained unchanged compared with controls. Propranolol pretreatment had no effect on either alpha- or beta-adrenergic responses. These findings indicate that the alpha agonist-induced response after in vivo pretreatment induces reciprocal changes in the functionally related beta-adrenergic apparatus, and also suggest linkage between these two receptors. In contrast, the beta response appears to desensitize or downregulate in response to beta agonist exposure in a manner that seems to be independent of or to operate in the absence of an alteration of the alpha response.

Vascular reactivity to adrenergic agents and neuronal and vascular catecholamine levels in spontaneously hypertensive rats.

Spontaneously hypertensive rats (SHR) were compared to their controls, Wistar-Kyoto (WKY) rats, with respect to contractile responses of aortic preparations and catecholamine concentrations in aorta and superior cervical ganglion (SCG). Contractile responses of aortic preparations to norepinephrine were significantly lower in SHR than in WKY rats, but relaxation responses to isoproterenol were not different. Norepinephrine levels were elevated in SCG and aortas of SHR vs. WKY rats. The results suggest that there are important differences in vascular responses of SHR compared with WKY rats, and raise the question of whether these differences are reflective of transmitter levels and their effects on alpha-adrenergic receptor sensitivity. The results also suggest that lack of beta receptor involvement in the observed alpha receptor desensitization may be important for the development of hypertension in SHR.