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.

Sex differences in cytochrome P450 1B1, an estrogen-metabolizing enzyme, in the rhesus monkey telencephalon.

The metabolic enzyme CYP1B1 is a recently cloned member of the cytochrome P450 superfamily, expressed widely throughout primate tissue, including the CNS. Although CYP1B1 protein is known to metabolize estradiol to catecholestrogens in the uterus, its localization and function in brain have not yet been described. To better understand CYP1B1 distribution, we have combined in situ hybridization (ISH) for its mRNA with immunohistochemistry (IHC) for the CYP1B1 protein in selected brain regions of male and female adult rhesus monkeys (Macaca mulatta). Blocks of formalin-fixed tissue obtained from the frontal cortex, hippocampus, thalamus, and amygdala were processed and embedded in paraffin. They were then sectioned and stained as described for human tissue [Muskhelishvili, L., Thompson, P.A., Kusewitt, D.F., Wang, C., Kadlubar, F.F., 2001. In situ hybridization and immunohistochemical analysis of cytochrome P450 1B1 expression in human normal tissues. J. Histochem. Cytochem. 49, 229-236]. Results indicated widespread distribution of CYP1B1 mRNA in both male and female monkey frontal cortex, hippocampus, thalamus, and amygdala. In contrast, although CYP1B1 protein was co-localized with its mRNA in the female brains, it was primarily restricted to hippocampal pyramidal neurons in the male brains. These results suggest that CYP1B1 may subserve widespread metabolic functions in the female primate brain but have more restricted actions within the hippocampal pyramidal neurons of the male.

Fluoro-Jade C results in ultra high resolution and contrast labeling of degenerating neurons.

The causes and effects of neuronal degeneration are of major interest to a wide variety of neuroscientists. Paralleling this growing interest is an increasing number of methods applicable to the detection of neuronal degeneration. The earliest methods employing aniline dyes were methodologically simple, but difficult to interpret due to a lack of staining specificity. In an attempt to circumvent this problem, numerous suppressed silver methods have been introduced. However, these methods are labor intensive, incompatible with most other histochemical procedures and notoriously capricious. In an attempt to develop a tracer with the methodological simplicity and reliability of conventional stains but with the specificity of an ideal suppressed silver preparation, the Fluoro-Jade dyes were developed. Fluoro-Jade C, like its predecessors, Fluoro-Jade and Fluoro-Jade B, was found to stain all degenerating neurons, regardless of specific insult or mechanism of cell death. Therefore, the patterns of neuronal degeneration seen following exposure to either the glutamate agonist, kainic acid, or the inhibitor of mitochondrial respiration, 3-NPA, were the same for all of the Fluoro-Jade dyes. However, there was a qualitative difference in the staining characteristics of the three fluorochromes. Specifically, Fluoro-Jade C exhibited the greatest signal to background ratio, as well as the highest resolution. This translates to a stain of maximal contrast and affinity for degenerating neurons. This makes it ideal for localizing not only degenerating nerve cell bodies, but also distal dendrites, axons and terminals. The dye is highly resistant to fading and is compatible with virtually all histological processing and staining protocols. Triple labeling was accomplished by staining degenerating neurons with Fluoro-Jade C, cell nuclei with DAPI and activated astrocytes with GFAP immunofluoresence.

Neurohistochemical biomarkers of the marine neurotoxicant, domoic acid.

Domoic acid and its potent excitotoxic analogues glutamic acid and kainic acid, are synthesized by marine algae such as seaweed and phytoplankton. During an algal bloom, domoic acid may enter the food web through its consumption by a variety of marine organisms held in high regard as seafoods by both animals and humans. These seafoods include clams, mussels, oysters, anchovies, sardines, crabs, and scallops, among others. Animals, such as pelicans, cormorants, loons, grebes, sea otters, dolphins, and sea lions, which consume seafood contaminated with domoic acid, suffer disorientation and often death. Humans consuming contaminated seafood may suffer seizures, amnesia and also sometimes death. In addition to analytical measurement of domoic acid exposure levels in algae and/or seafood, it is useful to be able to identify the mode of toxicity through post-mortem evaluation of the intoxicated animal. In the present study, using the rat as an animal model of domoic acid intoxication, we compared histochemical staining of the limbic system and especially the hippocampus with degeneration-selective techniques (Fluoro-Jade and silver), a conventional Nissl stain for cytoplasm (Cresyl violet), a myelin-selective stain (Black-Gold), an astrocyte-specific stain (glial fibrillary acidic protein), early/immediate gene responses (c-Fos and c-Jun), as well as for heat shock protein (HSP-72) and blood-brain barrier integrity (rat IgG). The results demonstrate that the degeneration-selective stains are the biomarkers of domoic acid neurotoxicity that are the most useful and easy to discern when screening brain sections at low magnification. We also observed that an impairment of blood-brain barrier integrity within the piriform cortex accompanied the onset of domoic acid neurotoxicity.

3-nitropropionic acid inhibition of succinate dehydrogenase (complex II) activity in cultured Chinese hamster ovary cells: antagonism by L-carnitine.

3-Nitropropionic acid (3-NPA) is an inhibitor of the mitochondrial enzyme succinate dehydrogenase (SDH, a part of complex II) that links the tricarboxylic acid (TCA) cycle to the respiratory electron transport chain. 3-NPA inactivates SDH by covalently and irreversibly binding to its active site. We previously examined the effects of 3-NPA on the histochemical activity of SDH in vivo, by using the reduction of a yellow tetrazolium dye (nitro blue tetrazolium) to a blue formazan as an indicator. In studies of cultured cells, the related dye methylthiazoletetrazolium (MTT) has commonly been used as an indicator of the presence and number of viable cells; that is cells that are capable of producing energy via the TCA cycle. Here we observed that doses of 3-NPA as low as 10(-8) M inhibited formazan production in an in vitro model system using CHO cells. This effect was antagonized by l-carnitine, which greatly increased the production of formazan, indicating a considerable improvement in energy production by the cultured cells. CHO cells appear to be a convenient model for the evaluation of therapeutic compounds that may modulate cellular bioenergetics.

Dietary exposure to genistein increases vasopressin but does not alter beta-endorphin in the rat hypothalamus.

Genistein is a plant-derived estrogenic isoflavone commonly found in soy-based products such as soymilk and soy-based dietary supplements for treating menopausal symptoms, for example. Vasopressin is a neurosecretory nonapeptide synthesized primarily in neurons of the hypothalamus and secreted into the bloodstream from the posterior lobe of the pituitary. The endogenous opiate peptide beta-endorphin is synthesized both in neurons of the hypothalamus and in pituitary cells, primarily of the neurointermediate lobe. It has been reported that exposure to 17beta-estradiol or diethylstilbesterol increased the vasopressin content of the hypothalamus, and that estradiol valerate selectively damages hypothalamic beta-endorphin-containing neurons. Since little was known of the potential effects of estrogenic endocrine-disruptor compounds on hypothalamic neuropeptides, we fed Sprague-Dawley fetuses from day 7 in utero until sacrifice at postnatal day 77, with either a control diet (<1 ppm) or an experimental diet containing 25, 250, or 1250 ppm of genistein. We then conducted ELISA assays for hypothalamic content of both beta-endorphin and vasopressin immunoreactivity. Whereas there were no statistically reliable effects of dietary genistein on hypothalamic beta-endorphin content, vasopressin levels were significantly elevated in the 1250-ppm genistein group (p < 0.05). Elevated vasopressin levels may be associated with fluid balance, altered blood pressure, and cardiovascular effects. These data are consistent with the known actions of estradiol and may serve to explain our finding in a previous study that estrogenic endocrine-disruptors such as genistein increased sodium preference in rats exposed through their diet.

Ontogeny of the N-methyl-D-aspartate (NMDA) receptor system and susceptibility to neurotoxicity.

The NMDA receptor has been widely investigated in recent years as a target for the pharmacological management of seizures, pain and a variety of neurological disorders. Its role in normal central nervous system (CNS) activity and development, as well as in the development of CNS abnormalities and neurodegeneration has also been of interest. The NMDA receptor is one of three pharmacologically distinct subtypes of ionotropic receptor channels that are sensitive to the endogenous excitatory amino acid, L-glutamate. The ontogeny of the NMDA receptor, a multiple tetrameric and heteromeric channel complex with at least six known subunits, is controlled by three gene families and varies in developmental profile with species and regional brain area. NMDA receptors play a role in excitatory synaptic transmission, in the activity-dependent synaptic plasticity underlying learning and memory, and in pre- and postnatal CNS development, including brain cell differentiation, axonal growth and degeneration of unused neurons. The results of recent studies suggest that sustained alteration of NMDA receptor activation during critical periods of development may have deleterious effects on normal CNS development and function. Neonatal rats administered the NMDA receptor antagonists 2-amino-5-phosphonovalerate (AP5) and MK-801 during the first two weeks of life develop abnormal axonal arborization in the retinal connections to the superior colliculus, interfering with normal visual responses. Results from monkey studies suggest that chronic developmental exposure to high doses of a NMDA antagonist, remacemide, has pronounced and long-lasting effects on learning. Recent findings indicate that if NMDA receptors are blocked during a specific period in neonatal life (first two weeks postnatally in the rat), massive apoptotic neurodegeneration results, due not to excitotoxic overstimulation of neurons but to deprivation of stimulation. These observations require further laboratory evidence and support in order to establish their relevance to drug-induced human neurodevelopmental concerns. It is necessary to investigate the relevance of these findings in other animal species in addition to the rat, most notably, nonhuman primates, where neuronal cytoarchitecture and development are significantly different than the rodent but more like the human.

Quantitative three-dimensional reconstruction: feasibility for studies of sexually dimorphic hypothalamic development in rats.

The adult rat brain develops through an interplay of neuronal proliferation with programmed cell death. Sensory stimulation, as well as growth factors and steroids, may alter the balance between these competing processes. "Endocrine disrupters" (EDs) may do the same by mimicry or modulation of endogenous hormones. The sexually dimorphic nucleus (SDN) of the medial preoptic hypothalamus contains a high concentration of estrogen receptors (ERs). The SDN develops to a final adult volume, which may be used as an indication of the hormonal conditions during perinatal development. Although male rats have been repeatedly observed to have a greater adult SDN volume than female rats, variability between the actual measurements reported (both within and between laboratories) have been rather large. Exposure of female rats to testosterone (or excessive estradiol, beyond the binding capacity of alpha-fetoprotein) has been shown to masculinize them through a P450 aromatase that converts testosterone to estrogen in the SDN. Exposure of males to estradiol may feminize them at low doses through interference with the synthesis of their endogenous testosterone, which normally acts on SDN ERs following aromatization. We have employed computer-assisted reconstruction methods in order to render the SDN within the surrounding hypothalamus in 3-D for computation of its volume. Ongoing studies are investigating whether exposure through the diet to estrogenic endocrine disruptors such as genistein, nonylphenol, and ethinyl estradiol might produce effects similar to those of estradiol itself on the adult SDN.

Electroencephalographic, behavioral, and c-fos responses to acute domoic acid exposure.

Domoic acid, a potent excitotoxic analogue of glutamate and kainate, may cause seizures, amnesia, and sometimes death in humans consuming contaminated shellfish. Continuous behavioral observations and recordings of the electrocorticogram (ECoG, via bipolar, epidural electrodes) were obtained from nonanesthetized rats for 2 h after intraperitoneal injection with either saline, 2.2, or 4.4 mg/kg of domoic acid. Rats were then sacrificed for c-fos immunohistochemistry. Fast Fourier transformation (FFT) of the ECoG data to obtain the voltage as a function of frequency indicated that the lower frequency bands (theta, 4.75-6.75 Hz and delta, 1.25-4.50 Hz) were the first to respond, with a significant elevation by 30 min after the high dose of domoic acid. The lower dose of domoic acid also caused a significant elevation of ECoG voltage, but not until later in the session. Sixty minutes after dosing, the behavioral biomarkers of "ear scratching" and "rearing, praying" (RP) seizures became significantly elevated in the high-dose rats. The low-dose rats showed no significant alterations in behavior at any time during the session. In postmortem brains obtained immediately after the sessions, c-fos was activated in the anterior olfactory nucleus by both the low and high doses of domoic acid. However, only the high dose increased c-fos immunoreactivity in the hippocampus, affecting both the granule and pyramidal neurons. These data indicate that electroencephalographic and c-fos responses can be obtained at a dose of domoic acid that fails to activate the behavioral response most commonly used as a bioassay for this marine toxin: ear scratching with the ipsilateral foot.

Meal-induced increase in plasma gastrin immunoreactivity in the rhesus monkey (Macaca mulatta).

The hormonal response to food intake in rodents, dogs, and humans involves gastrin and cholecystokinin, structurally related peptides present in plasma, gut, and brain. In order to determine the time course of plasma gastrin response in a nonhuman primate, six overnight-fasted adult male rhesus monkeys were offered a meal of bananas (11g) and peanut butter (1 Tbsp) or a fresh water bottle in a crossover design. All animals completely consumed the meal within 10 min. Compared to non-fed control levels, plasma gastrin was significantly elevated (52 ± 11 pM vs. 32 ± 9 pM, means ± S. E. M.) from 10 to 45 min post-ingestion, but returned to near basal fasted level by 120 min. Levels of gastrin in tissue samples (n = 2) were highest in the antrum of the stomach, with decreasing amounts measured in the upper and lower duodenum, respectively. The results demonstrate that the plasma concentration and response to a meal of rhesus monkey gastrin are similar to those of other mammalian species. However, the high concentrations of gastrin found in duodenum are thus far unique to Macaca mulatta and humans.