Early-in-life isoflurane exposure alters resting-state functional connectivity in juvenile non-human primates.

BACKGROUND: Clinical studies suggest that anaesthesia exposure early in life affects neurobehavioural development. We designed a non-human primate (NHP) study to evaluate cognitive, behavioural, and brain functional and structural alterations after isoflurane exposure during infancy. These NHPs displayed decreased close social behaviour and increased astrogliosis in specific brain regions, most notably in the amygdala. Here we hypothesise that resting-state functional connectivity MRI can detect alterations in connectivity of brain areas that relate to these social behaviours and astrogliosis. METHODS: Imaging was performed in 2-yr-old NHPs under light anaesthesia, after early-in-life (postnatal days 6-12) exposure to 5 h of isoflurane either one or three times, or to room air. Brain images were segmented into 82 regions of interest; the amygdala and the posterior cingulate cortex were chosen for a seed-based resting-state functional connectivity MRI analysis. RESULTS: We found differences between groups in resting-state functional connectivity of the amygdala and the auditory cortices, medial premotor cortex, and posterior cingulate cortex. There were also alterations in resting-state functional connectivity between the posterior cingulate cortex and secondary auditory, polar prefrontal, and temporal cortices, and the anterior insula. Relationships were identified between resting-state functional connectivity alterations and the decrease in close social behaviour and increased astrogliosis. CONCLUSIONS: Early-in-life anaesthesia exposure in NHPs is associated with resting-state functional connectivity alterations of the amygdala and the posterior cingulate cortex with other brain regions, evident at the juvenile age of 2 yr. These changes in resting-state functional connectivity correlate with the decrease in close social behaviour and increased astrogliosis. Using resting-state functional connectivity MRI to study the neuronal underpinnings of early-in-life anaesthesia-induced behavioural alterations could facilitate development of a biomarker for anaesthesia-induced developmental neurotoxicity.

Frontal Electroencephalography Findings in Critically Ill COVID-19 Patients.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) negatively impacts the central nervous system, and studies using a full montage of electroencephalogram (EEG) electrodes have reported nonspecific EEG patterns associated with coronavirus disease 2019 (COVID-19) infection. The use of this technology is resource-intensive and limited in its implementation. In this descriptive pilot study, we report neurophysiological patterns and the potential prognostic capability of an abbreviated frontal EEG electrode montage in critically ill COVID-19 patients. MATERIALS AND METHODS: Patients receiving mechanical ventilation for SARS-CoV-2 respiratory failure were monitored with Sedline Root Devices using EEG electrodes were placed over the forehead. Qualitative EEG assessments were conducted daily. The primary outcome was mortality, and secondary outcomes were duration of endotracheal intubation and lengths of intensive care and hospitalization stay. RESULTS: Twenty-six patients were included in the study, and EEG discontinuity was identified in 22 (84.6%) patients. The limited sample size and patient heterogeneity precluded statistical analysis, but certain patterns were suggested by trends in the data. Survival was 100% (4/4) for those patients in which a discontinuous EEG pattern was not observed. The majority of patients (87.5%, 7/8) demonstrating activity in the low-moderate frequency range (7 to 17 Hz) survived compared with 61.1% (11/18) of those without this observation. CONCLUSIONS: The majority of COVID-19 patients showed signs of EEG discontinuity during monitoring with an abbreviated electrode montage. The trends towards worse survival among those with EEG discontinuity support the need for additional studies to investigate these associations in COVID-19 patients.

Does inflammation mediate behavioural alterations in anaesthesia-induced developmental neurotoxicity?

Anaesthesia exposure early in life potentially impairs neurobehavioural development. A recent study in the Journal investigated the possibility that progesterone mitigates anaesthesia-induced developmental neurotoxicity in neonatal rats exposed to sevoflurane. The novel findings show that the steroid hormone progesterone protects against development of behavioural alterations caused by sevoflurane. The protective mechanism is proposed to relate to anti-inflammatory properties of progesterone, which brings up important questions regarding the role of inflammation in mediating the neurobehavioural alterations in anaesthesia-induced developmental neurotoxicity. We discuss this mechanism and encourage new research that may clarify the underlying mechanisms of progesterone-induced protection and extend these findings into a translational model.

Astrogliosis in juvenile non-human primates 2 years after infant anaesthesia exposure.

BACKGROUND: Infant anaesthesia causes acute brain cell apoptosis, and later in life cognitive deficits and behavioural alterations, in non-human primates (NHPs). Various brain injuries and neurodegenerative conditions are characterised by chronic astrocyte activation (astrogliosis). Glial fibrillary acidic protein (GFAP), an astrocyte-specific protein, increases during astrogliosis and remains elevated after an injury. Whether infant anaesthesia is associated with a sustained increase in GFAP is unknown. We hypothesised that GFAP is increased in specific brain areas of NHPs 2 yr after infant anaesthesia, consistent with prior injury. METHODS: Eight 6-day-old NHPs per group were exposed to 5 h isoflurane once (1×) or three times (3×), or to room air as a control (Ctr). Two years after exposure, their brains were assessed for GFAP density changes in the primary visual cortex (V1), perirhinal cortex (PRC), hippocampal subiculum, amygdala, and orbitofrontal cortex (OFC). We also assessed concomitant microglia activation and hippocampal neurogenesis. RESULTS: Compared with controls, GFAP densities in V1 were increased in exposed groups (Ctr: 0.208 [0.085-0.427], 1×: 0.313 [0.108-0.533], 3×: 0.389 [0.262-0.652]), whereas the density of activated microglia was unchanged. In addition, GFAP densities were increased in the 3× group in the PRC and the subiculum, and in both exposure groups in the amygdala, but there was no increase in the OFC. There were no differences in hippocampal neurogenesis among groups. CONCLUSIONS: Two years after infant anaesthesia, NHPs show increased GFAP without concomitant microglia activation in specific brain areas. These long-lasting structural changes in the brain caused by infant anaesthesia exposure may be associated with functional alterations at this age.

Recent advances in understanding cognitive and behavioural alterations after early-in-life anaesthesia exposure and new mitigation/alternative strategies in preclinical studies.

PURPOSE OF REVIEW: Long-term behavioural and cognitive impairments after exposure to general anaesthetics during infancy is an intensely investigated and controversial topic. Recent clinical studies with prospective assessments associate exposure with long-term behavioural alterations rather than cognitive impairments. This review aims to provide an understanding of the long-term cognitive impairments and behavioural alterations found in recent animal studies and to summarize latest advances in strategies to protect against anaesthesia-induced developmental neurotoxicity (AIDN). RECENT FINDINGS: Preclinical studies, particularly those in nonhuman primates (NHPs), provide accumulating evidence that anaesthesia exposure during infancy is associated with long-term alterations in behaviour, but cognitive impairments are more controversial. Results from recent studies aiming to find mitigating strategies to reduce AIDN or to identify alternative anaesthetic agents include the co-administration of dexmedetomidine with the anaesthetic drugs or the alternative use of hypnotic neurosteroids without being harmful to the developing brain. SUMMARY: Recent findings in animal studies with translational relevance support the proposed association between early-in-life anaesthesia exposure and long-term alterations in behaviour. Studies aiming to prevent AIDN are promising and need evaluation in the NHP model. The careful design of subsequent translational studies will be critical to advance the field forward towards safer anaesthesia exposure in children.

Roadmap for Conducting Neuroscience Research in the COVID-19 Era and Beyond: Recommendations From the SNACC Research Committee.

The coronavirus disease 2019 (COVID-19) pandemic has impacted many aspects of neuroscience research. At the 2020 Society of Neuroscience in Anesthesiology and Critical Care (SNACC) Annual Meeting, the SNACC Research Committee met virtually to discuss research challenges encountered during the COVID-19 pandemic along with possible strategies for facilitating research activities. These challenges and recommendations are included in this Consensus Statement. The objectives are to: (1) provide an overview of the disruptions and challenges to neuroscience research caused by the COVID-19 pandemic, and; (2) put forth a set of consensus recommendations for strengthening research sustainability during and beyond the current pandemic. Specific recommendations are highlighted for adapting laboratory and human subject study activities to optimize safety. Complementary research activities are also outlined for both laboratory and clinical researchers if specific investigations are impossible because of regulatory or societal changes. The role of virtual platforms is discussed with respect to fostering new collaborations, scheduling research meetings, and holding conferences such that scientific collaboration and exchange of ideas can continue. Our hope is for these recommendations to serve as a valuable resource for investigators in the neurosciences and other research disciplines for current and future research disruptions.

Behavioural impairments after exposure of neonatal mice to propofol are accompanied by reductions in neuronal activity in cortical circuitry.

BACKGROUND: Both animal and retrospective human studies have linked extended and repeated general anaesthesia during early development with cognitive and behavioural deficits later in life. However, the neuronal circuit mechanisms underlying this anaesthesia-induced behavioural impairment are poorly understood. METHODS: Neonatal mice were administered one or three doses of propofol, a commonly used i.v. general anaesthetic, over Postnatal days 7-11. Control mice received Intralipid® vehicle injections. At 4 months of age, the mice were subjected to a series of behavioural tests, including motor learning. During the process of motor learning, calcium activity of pyramidal neurones and three classes of inhibitory interneurones in the primary motor cortex were examined in vivo using two-photon microscopy. RESULTS: Repeated, but not a single, exposure of neonatal mice to propofol i.p. caused motor learning impairment in adulthood, which was accompanied by a reduction of pyramidal neurone number and activity in the motor cortex. The activity of local inhibitory interneurone networks was also altered: somatostatin-expressing and parvalbumin-expressing interneurones were hypoactive, whereas vasoactive intestinal peptide-expressing interneurones were hyperactive when the mice were performing a motor learning task. Administration of low-dose pentylenetetrazol to attenuate γ-aminobutyric acid A receptor-mediated inhibition or CX546 to potentiate α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-subtype glutamate receptor function during emergence from anaesthesia ameliorated neuronal dysfunction in the cortex and prevented long-term behavioural deficits. CONCLUSIONS: Repeated exposure of neonatal mice to propofol anaesthesia during early development causes cortical circuit dysfunction and behavioural impairments in later life. Potentiation of neuronal activity during recovery from anaesthesia reduces these adverse effects of early-life anaesthesia.

Brain pathology caused in the neonatal macaque by short and prolonged exposures to anticonvulsant drugs.

Barbiturates and benzodiazepines are potent GABAA receptor agonists and strong anticonvulsants. In the developing brain they can cause neuronal and oligodendroglia apoptosis, impair synaptogenesis, inhibit neurogenesis and trigger long-term neurocognitive sequelae. In humans, the vulnerable period is projected to extend from the third trimester of pregnancy to the third year of life. Infants with seizures and epilepsies may receive barbiturates, benzodiazepines and their combinations for days, months or years. How exposure duration affects neuropathological sequelae is unknown. Here we investigated toxicity of phenobarbital/midazolam (Pb/M) combination in the developing nonhuman primate brain. Neonatal rhesus monkeys received phenobarbital intravenously, followed by infusion of midazolam over 5 (n = 4) or 24 h (n = 4). Animals were euthanized at 8 or 36 h and brains examined immunohistochemically and stereologically. Treatment was well tolerated, physiological parameters remained at optimal levels. Compared to naïve controls, Pb/M exposed brains displayed widespread apoptosis affecting neurons and oligodendrocytes. Pattern and severity of cell death differed depending on treatment-duration, with more extensive neurodegeneration following longer exposure. At 36 h, areas of the brain not affected at 8 h displayed neuronal apoptosis, while oligodendroglia death was most prominent at 8 h. A notable feature at 36 h was degeneration of neuronal tracts and trans-neuronal death of neurons, presumably following their disconnection from degenerated presynaptic partners. These findings demonstrate that brain toxicity of Pb/M in the neonatal primate brain becomes more severe with longer exposures and expands trans-synaptically. Impact of these sequelae on neurocognitive outcomes and the brain connectome will need to be explored.

Infant isoflurane exposure affects social behaviours, but does not impair specific cognitive domains in juvenile non-human primates.

BACKGROUND: Clinical studies show that children exposed to anaesthetics for short times at young age perform normally on intelligence tests, but display altered social behaviours. In non-human primates (NHPs), infant anaesthesia exposure for several hours causes neurobehavioural impairments, including delayed motor reflex development and increased anxiety-related behaviours assessed by provoked response testing. However, the effects of anaesthesia on spontaneous social behaviours in juvenile NHPs have not been investigated. We hypothesised that multiple, but not single, 5 h isoflurane exposures in infant NHPs are associated with impairments in specific cognitive domains and altered social behaviours at juvenile age. METHODS: Eight Rhesus macaques per group were anaesthetised for 5 h using isoflurane one (1×) or three (3×) times between postnatal days 6 and 12 or were exposed to room air (control). Cognitive testing, behavioural assessments in the home environment, and provoked response testing were performed during the first 2 yr of life. RESULTS: The cognitive functions tested did not differ amongst groups. However, compared to controls, NHPs in the 3× group showed less close social behaviour (P=0.016), and NHPs in the 1× group displayed increased anxiety-related behaviours (P=0.038) and were more inhibited towards novel objects (P<0.001). CONCLUSIONS: 5 h exposures of NHPs to isoflurane during infancy are associated with decreased close social behaviour after multiple exposures and more anxiety-related behaviours and increased behavioural inhibition after single exposure, but they do not affect the cognitive domains tested. Our findings are consistent with behavioural alterations in social settings reported in clinical studies, which may guide future research.

Neurotoxicity of sub-anesthetic doses of sevoflurane and dexmedetomidine co-administration in neonatal rats.

BACKGROUND: Preclinical studies suggest that exposures of infant animals to general anesthetics cause acute neurotoxicity and affect their neurobehavioral development representing a potential risk to human infants undergoing anesthesia. Alternative or mitigating strategies to counteract such adverse effects are desirable. Dexmedetomidine (DEX) is a clinically established sedative with potential neuroprotective properties. DEX ameliorates experimental brain injury as well as neurotoxicity caused by anesthetic doses of sevoflurane (SEVO) or other general anesthetics in infant animals. However, it is unknown whether DEX also is beneficial when given together with lower doses of these drugs. Here we tested the hypothesis that DEX co-administration with a sub-anesthetic dose of SEVO reduces responsiveness to external stimuli while also protecting against SEVO-induced brain cell apoptosis. METHOD: Rats were exposed on postnatal day 7 for 6 h to SEVO 1.1, 1.8, or 2.5% and were given intraperitoneal injections of saline or DEX at different doses (1-25 µg/kg) three times during the exposure. Responsiveness to external stimuli, respiratory rates, and blood gases were assessed. Apoptosis was determined in cortical and subcortical brain areas by activated caspase-3 immunohistochemistry. RESULTS: Rats exposed to SEVO 1.1% alone were sedated but still responsive to external stimuli whereas those exposed to SEVO 1.8% reached complete unresponsiveness. SEVO-induced brain cell apoptosis increased dose-dependently, with SEVO 1.1% causing a small increase in apoptosis above that in controls. Co-administration of DEX at 1 µg/kg did not alter the responsiveness to stimuli nor the apoptosis induced by SEVO 1.1%. In contrast, co-administration of DEX at 5 µg/kg or higher with SEVO 1.1% reduced responsiveness but potentiated apoptosis. CONCLUSIONS: In the neonatal rat model, co-administration of a clinically relevant dose of DEX (1 µg/kg) with a sub-anesthetic dose of SEVO (1.1%) does not affect the neurotoxicity of the anesthetic while co-administration of higher doses of DEX with SEVO 1.1% potentiates it.

Standards for preclinical research and publications in developmental anaesthetic neurotoxicity: expert opinion statement from the SmartTots preclinical working group.

In March 2019, SmartTots, a public-private partnership between the US Food and Drug Administration and the International Anesthesia Research Society, hosted a meeting attended by research experts, anaesthesia journal editors, and government agency representatives to discuss the continued need for rigorous preclinical research and the importance of establishing reporting standards for the field of anaesthetic perinatal neurotoxicity. This group affirmed the importance of preclinical research in the field, and welcomed novel and mechanistic approaches to answer some of the field's largest questions. The attendees concluded that summarising the benefits and disadvantages of specific model systems, and providing guidance for reporting results, would be helpful for designing new experiments and interpreting results across laboratories. This expert opinion report is a summary of these discussions, and includes a focused review of current animal models and reporting standards for the field of perinatal anaesthetic neurotoxicity. This will serve as a practical guide and road map for novel and rigorous experimental work.

Mild hypothermia ameliorates anesthesia toxicity in the neonatal macaque brain.

Sedatives and anesthetics can injure the developing brain. They cause apoptosis of neurons and oligodendrocytes, impair synaptic plasticity, inhibit neurogenesis and trigger long-term neurocognitive deficits. The projected vulnerable period in humans extends from the third trimester of pregnancy to the third year of life. Despite all concerns, there is no ethically and medically acceptable alternative to the use of sedatives and anesthetics for surgeries and painful interventions. Development of measures that prevent injury while allowing the medications to exert their desired actions has enormous translational value. Here we investigated protective potential of hypothermia against histological toxicity of the anesthetic sevoflurane in the developing nonhuman primate brain. Neonatal rhesus monkeys underwent sevoflurane anesthesia over 5 h. Body temperature was regulated in the normothermic (>36.5 °C), mild hypothermic (35-36.5 °C) and moderately hypothermic (<35 °C) range. Animals were euthanized at 8 h and brains examined immunohistochemically (activated caspase 3) and stereologically to quantify apoptotic neuronal and oligodendroglial death. Sevoflurane anesthesia was well tolerated at all temperatures, with oxygen saturations, end tidal CO2 and blood gases remaining at optimal levels. Compared to controls, sevoflurane exposed brains displayed significant apoptosis in gray and white matter affecting neurons and oligodendrocytes. Mild hypothermia (35-36.5 °C) conferred significant protection from apoptotic brain injury, whereas moderate hypothermia (<35 °C) did not. Hypothermia ameliorates anesthesia-induced apoptosis in the neonatal primate brain within a narrow temperature window (35-36.5 °C). Protection is lost at temperatures below 35 °C. Given the mild degree of cooling needed to achieve significant brain protection, application of our findings to humans should be explored further.

Quantitative ultrasound and apoptotic death in the neonatal primate brain.

Apoptosis is triggered in the developing mammalian brain by sedative, anesthetic or antiepileptic drugs during late gestation and early life. Whether human children are vulnerable to this toxicity mechanism remains unknown, as there are no imaging techniques to capture it. Apoptosis is characterized by distinct structural features, which affect the way damaged tissue scatters ultrasound compared to healthy tissue. We evaluated whether apoptosis, triggered by the anesthetic sevoflurane in the brains of neonatal rhesus macaques, can be detected using quantitative ultrasound (QUS). Neonatal (n = 15) rhesus macaques underwent 5 h of sevoflurane anesthesia. QUS images were obtained through the sagittal suture at 0.5 and 6 h. Brains were collected at 8 h and examined immunohistochemically to analyze apoptotic neuronal and oligodendroglial death. Significant apoptosis was detected in white and gray matter throughout the brain, including the thalamus. We measured a change in the effective scatterer size (ESS), a QUS biomarker derived from ultrasound echo signals obtained with clinical scanners, after sevoflurane-anesthesia in the thalamus. Although initial inclusion of all measurements did not reveal a significant correlation, when outliers were excluded, the change in the ESS between the pre- and post-anesthesia measurements correlated strongly and proportionally with the severity of apoptotic death. We report for the first time in vivo changes in QUS parameters, which may reflect severity of apoptosis in the brains of infant nonhuman primates. These findings suggest that QUS may enable in vivo studies of apoptosis in the brains of human infants following exposure to anesthetics, antiepileptics and other brain injury mechanisms.

Developmental Neurotoxicity: An Update.

In the section of "Developmental Neurotoxicity: An Update" of the Pediatric Anesthesia Neurodevelopmental Assessment (PANDA) symposium 2018 the speakers presented the current literature in translational and clinical research. Dr. Brambrink spoke about translational research in anesthetic neurotoxicity, beginning with discovery in the rodent model, then focusing on evidence from nonhuman primates. Dr. Waspe applied the methodology of Adverse Outcome Pathways from the field of toxicology to developmental neurotoxicity of anesthetics. Dr. O'Leary presented relevant clinical studies that were published in 2017 divided by a focus on academic performance, clinical outcomes or diagnoses, or neuropsychological testing.

An open label pilot study of a dexmedetomidine-remifentanil-caudal anesthetic for infant lower abdominal/lower extremity surgery: The T REX pilot study.

BACKGROUND: Concern over potential neurotoxicity of anesthetics has led to growing interest in prospective clinical trials using potentially less toxic anesthetic regimens, especially for prolonged anesthesia in infants. Preclinical studies suggest that dexmedetomidine may have a reduced neurotoxic profile compared to other conventional anesthetic regimens; however, coadministration with either anesthetic drugs (eg, remifentanil) and/or regional blockade is required to achieve adequate anesthesia for surgery. The feasibility of this pharmacological approach is unknown. The aim of this study was to determine the feasibility of a remifentanil/dexmedetomidine/neuraxial block technique in infants scheduled for surgery lasting longer than 2 hours. METHODS: Sixty infants (age 1-12 months) were enrolled at seven centers over 18 months. A caudal local anesthetic block was placed after induction of anesthesia with sevoflurane. Next, an infusion of dexmedetomidine and remifentanil commenced, and the sevoflurane was discontinued. Three different protocols with escalating doses of dexmedetomidine and remifentanil were used. RESULTS: One infant was excluded due to a protocol violation and consent was withdrawn prior to anesthesia in another. The caudal block was unsuccessful in two infants. Of the 56 infants who completed the protocol, 45 (80%) had at least one episode of hypertension (mean arterial pressure >80 mm Hg) and/or movement that required adjusting the anesthesia regimen. In the majority of these cases, the remifentanil and/or dexmedetomidine doses were increased although six infants required rescue 0.3% sevoflurane and one required a propofol bolus. Ten infants had at least one episode of mild hypotension (mean arterial pressure 40-50 mm Hg) and four had at least one episode of moderate hypotension (mean arterial pressure <40 mm Hg). CONCLUSION: A dexmedetomidine/remifentanil neuraxial anesthetic regimen was effective in 87.5% of infants. These findings can be used as a foundation for designing larger trials that assess alternative anesthetic regimens for anesthetic neurotoxicity in infants.

Anesthesia and the developing brain: A way forward for laboratory and clinical research.

All commonly used general anesthetics have been shown to cause neurotoxicity in animal models, including nonhuman primates. Opinion, however, remains divided over how cumulative evidence from preclinical and human studies in this field should be interpreted and its translation to current practices in pediatric anesthesia and surgery. A group of international experts in laboratory and clinical sciences recently convened in Genoa, Italy, to evaluate the current state of both laboratory and clinical research and discuss future directions for basic, translational, and clinical studies in this field. This paper describes those discussions and conclusions. A central goal identified was the importance of continuing to pursue laboratory research efforts to better understand the biological pathways underlying anesthesia neurotoxicity. The distinction between basic and translational experimental designs in this field was highlighted, and it was acknowledged that it will be important for future animal research to try to causally link structural changes with long-term cognitive abnormalities. While inherent limitations will continue to affect the ability of even large observational cohorts to determine if anesthesia impacts neurodevelopment or behavioral outcomes, the importance of conducting further large well-designed cohort studies was also emphasized. Adequately powered cohorts could clarify which populations are at increased risk, provide information on environmental and healthcare-related risk modifiers, and guide future interventional trials. If anesthetics cause structural or functional adverse neurological effects in young children, alternative or mitigating strategies need to be considered. While protective or mitigating strategies have been repeatedly studied in animals, there are currently no human data to support alternative anesthetic strategies in clinical practice. Lastly, it was noted that there is still considerable debate over the clinical relevance of anesthesia neurotoxicity, and the need to evaluate the impact of other aspects of perioperative care on neurodevelopment must also be considered.

Caffeine Augments Anesthesia Neurotoxicity in the Fetal Macaque Brain.

Caffeine is the most frequently used medication in premature infants. It is the respiratory stimulant of choice for apnea associated with prematurity and has been called the silver bullet in neonatology because of many proven benefits and few known risks. Research has revealed that sedative/anesthetic drugs trigger apoptotic death of neurons and oligodendrocytes in developing mammalian brains. Here we evaluated the influence of caffeine on the neurotoxicity of anesthesia in developing nonhuman primate brains. Fetal macaques (n = 7-8/group), at a neurodevelopmental age comparable to premature human infants, were exposed in utero for 5 hours to no drug (control), isoflurane, or isoflurane + caffeine and examined for evidence of apoptosis. Isoflurane exposure increased apoptosis 3.3 fold for neurons and 3.4 fold for oligodendrocytes compared to control brains. Isoflurane + caffeine caused neuronal apoptosis to increase 8.0 fold compared to control levels but did not augment oligoapoptosis. Neuronal death was particularly pronounced in the basal ganglia and cerebellum. Higher blood levels of caffeine within the range considered therapeutic and safe for human infants correlated with increased neuroapoptosis. Caffeine markedly augments neurotoxicity of isoflurane in the fetal macaque brain and challenges the assumption that caffeine is safe for premature infants.