Catherine E. Creeley

Isoflurane-induced Neuroapoptosis in the Neonatal Rhesus Macaque Brain

Background:Brief isoflurane anesthesia induces neuroapoptosis in the developing rodent brain, but susceptibility of non-human primates to the apoptogenic action of isoflurane has not been studied. Therefore, we exposed postnatal day 6 (P6) rhesus macaques to a surgical plane of isoflurane anesthesia for 5 h, and studied the brains 3 h later for histopathologic changes. Method:With the same intensity of physiologic monitoring typical for human neonatal anesthesia, five P6 rhesus macaques were exposed for 5 h to isoflurane maintained between 0.7 and 1.5 end-tidal Vol% (endotracheally intubated and mechanically ventilated) and five controls were exposed for 5 h to room air without further intervention. Three hours later, the brains were harvested and serially sectioned across the entire forebrain and midbrain, and stained immunohistochemically with antibodies to activated caspase-3 for detection and quantification of apoptotic neurons. Results:Quantitative evaluation of brain sections revealed a median of 32.5 (range, 18.0-48.2) apoptotic cells/mm3 of brain tissue in the isoflurane group and only 2.5 (range, 1.1-5.2) in the control group (difference significant at P = 0.008). Apoptotic neuronal profiles were largely confined to the cerebral cortex. In the control brains, they were sparse and randomly distributed, whereas in the isoflurane brains they were abundant and preferentially concentrated in specific cortical layers and regions. Conclusion:The developing non-human primate brain is sensitive to the apoptogenic action of isoflurane and displays a 13-fold increase in neuroapoptosis after 5 h exposure to a surgical plane of isoflurane anesthesia.

Propofol-induced apoptosis of neurones and oligodendrocytes in fetal and neonatal rhesus macaque brain

BACKGROUND Exposure of the fetal or neonatal non-human primate (NHP) brain to isoflurane or ketamine for 5 h causes widespread apoptotic degeneration of neurones, and exposure to isoflurane also causes apoptotic degeneration of oligodendrocytes (OLs). The present study explored the apoptogenic potential of propofol in the fetal and neonatal NHP brain. METHOD Fetal rhesus macaques at gestational age 120 days were exposed in utero, or postnatal day 6 rhesus neonates were exposed directly for 5 h to propofol anaesthesia (n=4 fetuses; and n=4 neonates) or to no anaesthesia (n=4 fetuses; n=5 neonates), and the brains were systematically evaluated 3 h later for evidence of apoptotic degeneration of neurones or glia. RESULTS Exposure of fetal or neonatal NHP brain to propofol caused a significant increase in apoptosis of neurones, and of OLs at a stage when OLs were just beginning to myelinate axons. Apoptotic degeneration affected similar brain regions but to a lesser extent than we previously described after isoflurane. The number of OLs affected by propofol was approximately equal to the number of neurones affected at both developmental ages. In the fetus, neuroapoptosis affected particularly subcortical and caudal regions, while in the neonate injury involved neocortical regions in a distinct laminar pattern and caudal brain regions were less affected. CONCLUSIONS Propofol anaesthesia for 5 h caused death of neurones and OLs in both the fetal and neonatal NHP brain. OLs become vulnerable to the apoptogenic action of propofol when they are beginning to achieve myelination competence.

Isoflurane-induced apoptosis of oligodendrocytes in the neonatal primate brain

Previously we reported that exposure of 6‐day‐old (P6) rhesus macaques to isoflurane for 5 hours triggers a robust neuroapoptosis response in developing brain. We have also observed (unpublished data) that isoflurane causes apoptosis of cellular profiles in the white matter that resemble glia. We analyzed the cellular identity of the apoptotic white matter profiles and determined the magnitude of this cell death response to isoflurane.

Lithium protects against anesthesia-induced developmental neuroapoptosis.

Background:Ethanol and anesthetic drugs trigger neuroapoptosis in the developing mouse brain. Recently, it was found that ethanol-induced neuroapoptosis is preceded by suppressed phosphorylation of extracellular signal-regulated protein kinase (ERK), and lithium counteracts both the phosphorylated ERK suppressant action and ethanol-induced neuroapoptosis. The current study was undertaken to address the following questions. (1) Do ketamine and propofol mimic ethanol in suppressing ERK phosphorylation? (2) If they do, does lithium prevent this suppressant action and also prevent these anesthetic drugs from triggering neuroapoptosis? Method:Postnatal day 5 mice were treated with propofol, ketamine, lithium, a combination of propofol or ketamine with lithium or saline, and their brains were prepared for Western blot analysis or histology. For Western blot, cytosolic lysates of caudate putamen were analyzed for expression of phosphorylated ERK and phosphorylated serine/threonine-specific protein kinase. For histology, brains were stained immunohistochemically with antibodies to activated caspase-3, and the density of activated caspase-3 positive cells was determined. Results:Ketamine and propofol suppressed phosphorylated ERK, and lithium counteracted both the phosphorylated ERK suppressant action and neuroapoptotic action of these anesthetic drugs. Conclusion:If further testing finds lithium to be safe for use in pediatric/obstetric medicine, administration of a single dose of lithium before anesthesia induction may be a suitable means of mitigating the risk of anesthesia-induced developmental neuroapoptosis.

Low Doses of Memantine Disrupt Memory in Adult Rats

Memantine, a drug recently approved for treatment of Alzheimer’s disease, has been characterized as a unique NMDA antagonist that confers protection against excitotoxic neurodegeneration without the serious side effects that other NMDA antagonists are known to cause. In the present study, we determined what dose of memantine is required to protect the adult rat brain against an NMDA receptor-mediated excitotoxic process and then tested that dose and a range of lower doses to determine whether the drug in this dose range is associated with significant side effects. Consistent with previous research, we found that memantine confers a neuroprotective effect beginning at an intraperitoneal dose of 20 mg/kg, a dose that we found, contrary to previous reports, produces locomotor disturbances severe enough to preclude testing for learning and memory effects. We then determined that, at intraperitoneal doses of 10 and 5 mg/kg, memantine disrupts both memory and locomotor behaviors. Rats treated with these doses performed at control-like levels in learning a hole-board task but were significantly impaired in demonstrating what they had learned when tested 24 h later. This impairment of memory retention was not state dependent in that it was demonstrable regardless of whether the rats were or were not exposed to memantine on the day of retention testing. We conclude that, in the adult rat, memantine behaves like other NMDA antagonists in that it is neuroprotective only at doses that produce intolerable side effects, including memory impairment.

The young: neuroapoptosis induced by anesthetics and what to do about it.

Millions of human fetuses, infants, and children are exposed to anesthetic drugs every year in the United States and throughout the world. Anesthesia administered during critical stages of neurodevelopment has been considered safe and without adverse long-term consequences. However, recent reports provide mounting evidence that exposure of the immature animal brain to anesthetics during the period of rapid synaptogenesis, also known as the brain growth spurt period, triggers widespread apoptotic neurodegeneration, inhibits neurogenesis, and causes significant long-term neurocognitive impairment. Herein, we summarize currently available evidence for anesthesia-induced pathological changes in the brain and associated long-term neurocognitive deficits and discuss promising strategies for protecting the developing brain from the potentially injurious effects of anesthetic drugs while allowing the beneficial actions of these drugs to be realized.

Isoflurane-induced Apoptosis of Neurons and Oligodendrocytes in the Fetal Rhesus Macaque Brain

Background:The authors have previously shown that exposure of the neonatal nonhuman primate (NHP) brain to isoflurane for 5 h causes widespread acute apoptotic degeneration of neurons and oligodendrocyte. The current study explored the potential apoptogenic action of isoflurane in the fetal NHP brain. Methods:Fetal rhesus macaques at gestational age of 120 days (G120) were exposed in utero for 5 h to isoflurane anesthesia (n = 5) or to no anesthesia (control condition; n = 4), and all regions of the brain were systematically evaluated 3 h later for evidence of apoptotic degeneration of neurons or glia. Results:Exposure of the G120 fetal NHP brain to isoflurane caused a significant increase in apoptosis of neurons and of oligodendrocytes at a stage when oligodendrocytes were just beginning to myelinate axons. The neuroapoptosis response was most prominent in the cerebellum, caudate, putamen, amygdala, and several cerebrocortical regions. Oligodendrocyte apoptosis was diffusely distributed over many white matter regions. The total number of apoptotic profiles (neurons + oligodendrocytes) in the isoflurane-exposed brains was increased 4.1-fold, compared with the brains from drug-naive controls. The total number of oligodendrocytes deleted by isoflurane was higher than the number of neurons deleted. Conclusions:Isoflurane anesthesia for 5 h causes death of neurons and oligodendrocytes in the G120 fetal NHP brain. In the fetal brain, as the authors previously found in the neonatal NHP brain, oligodendrocytes become vulnerable when they are just achieving myelination competence. The neurotoxic potential of isoflurane increases between the third trimester (G120) and the neonatal period in the NHP brain.

ETHANOL CAUSES AND LITHIUM PREVENTS NEUROAPOPTOSIS AND SUPPRESSION OF pERK IN THE INFANT MOUSE BRAIN

Transient exposure of immature animals during the brain growth spurt period to ethanol triggers neuroapoptosis in the developing brain. Here we report that lithium, when administered in a single, well-tolerated dose to infant mice, suppresses spontaneous neuroapoptosis that occurs naturally in the developing brain, and prevents ethanol from triggering neuroapoptosis. To explore lithiums mechanism of action, we focused on kinase signaling systems (ERK, Akt, JNK) that are believed to play a regulatory role in cell survival, and found that very rapidly after ethanol administration there is a suppression of ERK phosphorylation, and that lithium stimulates ERK phosphorylation and prevents ethanol from suppressing this phosphorylation process. Ethanol also suppressed pAKT, but lithium did not counteract this effect. We also found that ethanol activates the JNK system, but this cannot explain the neurotoxic action of ethanol, because JNK activation did not occur in the same neuronal populations that are killed by ethanol.

Alcohol-induced neuroapoptosis in the fetal macaque brain.

The ability of brief exposure to alcohol to cause widespread neuroapoptosis in the developing rodent brain and subsequent long-term neurocognitive deficits has been proposed as a mechanism underlying the neurobehavioral deficits seen in fetal alcohol spectrum disorder (FASD). It is unknown whether brief exposure to alcohol causes apoptosis in the fetal primate brain. Pregnant fascicularis macaques at various stages of gestation (G105 to G155) were exposed to alcohol for 8h, then the fetuses were delivered by caesarean section and their brains perfused with fixative and evaluated for apoptosis. Compared to saline control brains, the ethanol-exposed brains displayed a pattern of neuroapoptosis that was widespread and similar to that caused by alcohol in infant rodent brain. The observed increase in apoptosis was on the order of 60-fold. We propose that the apoptogenic action of alcohol could explain many of the neuropathological changes and long-term neuropsychiatric disturbances associated with human FASD.

Donepezil markedly potentiates memantine neurotoxicity in the adult rat brain

The NMDA antagonist, memantine (Namenda), and the cholinesterase inhibitor, donepezil (Aricept), are currently being used widely, either individually or in combination, for treatment of Alzheimers disease (AD). NMDA antagonists have both neuroprotective and neurotoxic properties; the latter is augmented by drugs, such as pilocarpine, that increase cholinergic activity. Whether donepezil, by increasing cholinergic activity, might augment memantines neurotoxic potential has not been investigated. In the present study, we determined that a dose of memantine (20mg/kg, i.p.), considered to be in the therapeutic (neuroprotective) range for rats, causes a mild neurotoxic reaction in the adult rat brain. Co-administration of memantine (20 or 30 mg/kg) with donepezil (2.5-10mg/kg) markedly potentiated this neurotoxic reaction, causing neuronal injury at lower doses of memantine, and causing the toxic reaction to become disseminated and lethal to neurons throughout many brain regions. These findings raise questions about using this drug combination in AD, especially in the absence of evidence that the combination is beneficial, or that either drug arrests or reverses the disease process.