Surgery, Anesthesia, and the Immature Brain

MILLIONS OF CHILDREN UNDERGO SURGERY with anesthesia every year. During the perioperative period they are exposed to a multitude of stressors capable of interfering with normal brain development. Pain, stress, inflammation, hypoxia, and ischemia have all been shown to adversely affect the immature central nervous system (CNS). However, recent findings from animal studies have indicated that sedatives and anesthetics—the very drugs used to reduce pain and stress—may themselves undesirably influence brain development by triggering structural and functional abnormalities. In fact, this phenomenon is currently one of the most intensely investigated laboratory research fields in anesthesiology and a passionately debated topic ( Fig. 25.1 ). To date, more than 400 animal studies have addressed the effects of anesthetics on the developing brain. However, translating these laboratory findings to humans in clinical settings has been complicated.

To begin with, human epidemiologic studies have found mixed evidence for an association between surgery with general anesthesia in childhood and subsequent neurodevelopmental abnormalities. Some studies identified more frequently occurring learning disabilities in children after surgery with general anesthesia early in life, whereas others have not. Although surgery or comorbidities may affect the developing brain, the mounting laboratory data demonstrating deleterious effects of anesthetic exposure without surgery have forced clinicians to consider the possibility that anesthetics may also play a role in this phenomenon. However, similar to the uncertainties surrounding animal studies, the interpretation of the human data is fraught with substantial limitations.

Any discussion of the long-term effects of anesthetics on the developing brain is further complicated by the fact that anesthetic drugs may be organ protective under certain conditions, and may indeed mitigate brain damage that is due to inflammatory responses, hypoxia-ischemia, or other insults that might occur during the perioperative period.

It is currently impossible to provide any definitive statements about the effects of anesthesia on human neurodevelopment. This chapter provides an overview of the current laboratory and clinical data concerning the affect of sedatives, anesthetics, and analgesics on the immature brain.

Background

About 170 years ago, William T.G. Morton conducted the first successful public demonstration in the Western hemisphere of a drug-induced, reversible coma during a surgical procedure at the Massachusetts General Hospital. After witnessing Morton's demonstration, Oliver Wendell Holmes called the phenomenon “anesthesia,” from the Greek words an- (without) and aisthēsis (sensibility), and it instantaneously revolutionized the field of surgery. The inscription on Morton's tombstone, “Inventor and Revealer of Inhalation Anesthesia: Before Whom, in All Time, Surgery was Agony; By Whom, Pain in Surgery was Averted and Annulled; Since Whom, Science has Control of Pain,” represents a powerful testament to the tremendously positive impact that anesthesiology has made on the field of medicine. The use of general anesthetics to facilitate surgery quickly spread around the world and according to recent estimates, anesthesia currently allows more than 230 million patients of all ages worldwide to undergo major surgical procedures every year. This positive impact is now tempered by the possibility that anesthetics may adversely affect normal brain development. During the first century of their use, general anesthetics were regarded with serious concern because they were combustible and carried substantial hemodynamic and respiratory adverse effects. Accordingly, up until 25 to 30 years ago, general anesthesia was rarely used in critically ill neonates because of the fear of myocardial depression and hemodynamic instability. Perioperative drug regimens were often limited to neuromuscular blocking agents and nitrous oxide. However, with the realization that unopposed pain exerts deleterious effects on the developing brain, that dramatic stress responses to painful stimulation are detectable even in preterm infants, and that modern anesthetics and analgesics can abolish these responses without substantial hemodynamic compromise, pediatric anesthesia, for the past three decades, has afforded critically ill neonates the benefits of amnesia, analgesia, and immobility during increasingly invasive surgeries. These surgical interventions have helped to save countless lives and preserve quality of life in this vulnerable population. All the while, the powerful coma induced by general anesthetics has been thought to be temporary and devoid of serious long-term adverse effects after emergence. This notion of reversibility is now being seriously questioned because of structural abnormalities detected in neonatal animals during and immediately after anesthetic exposure and long-term cognitive abnormalities reported in animals and some children exposed to anesthesia at a young age.

Normal Brain Development

To put the effects of anesthetic exposures into context of brain development, the natural developmental processes must be taken into consideration. The human brain undergoes a complex and extended process of enormous growth in cell number, synapses, and connections during the perinatal period and beyond. Combined with this expansion of cells and connections early in life, subsequently massive regressive processes also occur during normal brain development. These expanding and regressive processes allow the brain to fully develop and eventually execute tasks, such as talking, walking, reading, writing, calculating, acquiring social skills, perfecting fine motor dexterity, and accomplishing complex functions, including learning, abstract thinking, as well as planning and executing long-term objectives.

To accommodate these functions, the human brain initially undergoes a rapid growth in size and cell number both in utero and during the early postnatal period, and is then pared back to achieve an efficient network of about 100 billion neurons in the adult brain. At birth, the size of the immature brain is one-third that of the adult brain, doubling in weight within the first year of life, and reaching 90% of its adult size by 6 years of age. This dramatic growth spurt coincides with a remarkable overabundance of neurons and neuronal connections. In fact, less than half the neurons generated during development survive into adulthood. Superfluous neurons that lose in the competition for a limited amount of trophic factors are removed by programmed cell death.

After their rapid growth in number during early brain development, immature neurons subsequently form an excess of connections via synapses. Depending on the brain region, synaptic densities are maximum in infants and young toddlers between 3 and 15 months of age and will undergo a progressive reduction by about half during adolescence and into adulthood. Connections that are active with continued electrical and chemical signals are sustained, whereas those with little or no activity are lost. Axons are myelinated by oligodendrocytes, leading to maturation of the CNS.

In summary, brain architecture changes rapidly and dramatically throughout early life. Neuronal density is greatest during fetal life, and excess neurons are eliminated via apoptosis or programmed cell death, predominantly in utero, during the neonatal period and throughout infancy. Rapid growth of dendrites and synaptic connections occur during infancy and early childhood, and unneeded dendrites and synapses are trimmed back, predominantly during later childhood and adolescence.

While these processes occur at slightly different stages of development for different regions of the brain, the first several years after birth represent a critical period for the entire developing CNS. Recent findings in animals suggest that exposure to anesthetics or sedatives during this period may interfere with proper neuronal development, brain architecture, and subsequent function. Although the exact molecular mechanisms by which anesthetics afford their therapeutic properties of amnesia, analgesia, and immobility are incompletely understood, their interaction with a wide variety of ion channels, such as sodium, calcium, and potassium channels, as well as several cell membrane proteins, including γ-aminobutyric acid (GABA), glycine, glutamate ( N -methyl- d -aspartate [NMDA]), acetylcholine, and serotonin receptor systems, make it conceivable that anesthetics could create permanent abnormalities by interfering with important processes during critical windows of brain development. In fact, both GABA and NMDA receptors play critical roles in trophic signaling and in the regulation of neuronal maturation and programmed cell death. During early brain development, for example, GABA directs cell proliferation, neuroblast migration, and dendritic maturation. In turn, developmental NMDA receptor stimulation directly fosters survival and maturation of some neurons. It is therefore plausible that, by acting as modulators of these receptors, anesthetics might interfere with these critical developmental processes.

Effects of Anesthetic Exposure on the Developing Brain

Concerns regarding neurologic abnormalities after general anesthesia in young children were first raised more than half a century ago, when postoperative behavioral changes were observed after administration of vinyl ether, cyclopropane, or ethyl chloride for otolaryngologic surgery. However, these abnormalities were regarded as psychological in nature because they were alleviated by the timely administration of preoperative sedative drugs. Approximately two decades later the focus of research into the long-term effects of anesthetics shifted to animal models representing occupational exposure in pregnant health care workers. Delayed synaptogenesis and behavioral abnormalities were observed in neonatal rats born to rodent dams that were chronically exposed to subanesthetic doses of halothane during their entire pregnancy. However, interest in the effects of anesthetics on brain development in children was not elicited until a seminal study observed widespread neuronal degeneration after a prolonged exposure to ketamine in neonatal rat pups. This initial discovery led to numerous editorials and reviews, and more than 400 original articles (see Fig. 25.1 ) into the brain structural and/or functional effects of almost every sedative and anesthetic in current clinical use in a wide variety of immature animal species. However, no discussion about the effects of drug exposure of the developing brain would be complete without examining the effects of opioid analgesics in the developing brain. Accordingly, this chapter examines the specific cellular effects that sedatives, anesthetics, and analgesics trigger in the immature brain.

Apoptotic Cell Death

The most widely studied deleterious structural consequence of exposure to sedatives or anesthetics in immature animals is widespread apoptosis. Although neuronal apoptosis eliminates approximately 50% to 70% of neurons throughout the brain during the entire developmental period, this natural process affects only a small fraction of cells at any particular time point. Exposure to anesthetics or sedatives briefly, but dramatically, increases the number of apoptotic neurons ( Fig. 25.2 ). Some studies demonstrated up to a 68-fold increase in the density of degenerating neurons after a combination of anesthetics in newborn rats compared with control animals, although it remained unclear what fraction of the entire neuronal population these degenerating neurons represented. In newborn mice, a 6-hour exposure to a clinically relevant dose of isoflurane triggers apoptotic cell death in 2% of neurons in the superficial cortex, a substantially affected brain region at this age. Under normal conditions, less than 0.1% of neurons undergo physiologic apoptosis in this region in unanesthetized littermates. The exact mechanism and selectivity of the cell death process remains unknown as dying neurons are located immediately adjacent to seemingly unaffected neighboring cells ( Fig. 25.3 ). Increased neuroapoptosis has now been observed after in vitro and in vivo exposures to a wide variety of sedatives and anesthetics, including chloral hydrate, clonazepam, diazepam, midazolam, nitrous oxide, desflurane, enflurane, halothane, isoflurane, sevoflurane, ketamine, pentobarbital, phenobarbital, propofol, and xenon, as well as opioid receptor agonists in a wide variety of species, including fruit flies, nematodes, chicks, mice, rats, guinea pigs, piglets, and rhesus monkeys ( E-Table 25.1 ). Selective stains such as cupric silver and Fluoro-Jade (EMD Millipore, Billerica, MA) have confirmed the cellular demise in neurons that positively stained for activated caspase 3, the central executioner enzyme of the apoptotic cascade.

FIGURE 25.3, Neurons affected by apoptotic cell death following anesthetic exposure are surrounded by seemingly unaffected cells. Representative photomicrograph from a 7-day-old mouse following a 6-hour exposure to 1.5% isoflurane, showing neocortical cells stained for the apoptotic cell death marker activated caspase 3 ( blue , top ), the neuronal marker NeuN ( red , middle ), and a merged image of the two stains ( bottom ). The majority of dying cells are identified as postmitotic neurons, as indicated by the purple cells coexpressing activated caspase 3 and NeuN in the bottom ( arrows ), whereas some cells expressing caspase 3 are NeuN-negative ( arrowheads ). Importantly, cells affected by anesthetic-induced neuroapoptosis are surrounded by numerous, seemingly unaffected neurons. Scale bar = 10 µm.

E-TABLE 25.1

Representative Preclinical Studies Examining Structural and Neurocognitive Effects of Anesthetic Exposure Early in Life

Anesthetic Agent	Dose and Duration	Species, Age	Pathology	Study
Buprenorphine	1.5 mg/kg per day to dam	Rat, E7–E21 or P10	Decrease in striatal nerve growth factor	Wu et al.
Chloral hydrate	50–300 mg/kg × 1	Mouse, P5	Increased neuroapoptosis, ameliorated by lithium	Cattano et al.
Clonazepam	0.5–4 mg/kg × 1	Rat, P7	Increased neurodegeneration	Bittigau et al.
Clonazepam	0.5–4 mg/kg × 1	Rat, P7	Increased neurodegeneration	Ikonomidou et al.
Desflurane	7% for 30–120 minutes	Rat, P16	No increase in neurodegeneration or gross changes in dendritic arborization, increase in number of dendritic spines	Briner et al.
Desflurane	12% for 6 hours	Mouse, cortical neuronal culture	No increase in expression of proapoptotic factors, no accumulation of reactive oxygen species, no activation of apoptosis pathways	Zhang et al.
Desflurane	7.4% for 6 hours	Mouse, P7–P8	Increased neuroapoptosis compared with no anesthesia, but similar to equianesthetic doses of isoflurane or sevoflurane	Istaphanous et al.
Desflurane	8% for 6 hours	Mouse, P6	Increased neuroapoptosis, compared with sevoflurane and isoflurane, and long-term impairment in memory function	Kodama et al.
Desflurane	9% for 2 hours/day × 3 days	Mouse, P6–P8	No increase in inflammatory markers or learning impairment in adulthood	Shen et al.
Dexmedetomidine	1–75 µg/kg × 3	Rat, P7	No increase in neuroapoptosis	Sanders et al.
Dexmedetomidine	25 µg/g every 2 hours × 3	Rat, P7	No increase in cortical neuroapoptosis	Sanders et al.
Dexmedetomidine	75 µg/kg IP × 1 or 25 µg/kg every 2 hours × 3	Rat, P7	No increase in neuroapoptosis in hippocampus, dexmedetomidine ameliorated isoflurane-induced neuroapoptosis	Li et al.
Dexmedetomidine	5 or 10 µg/kg ×1	Rat, P7	Unaltered hippocampal synaptic function in young adulthood	Tachibana et al.
Dexmedetomidine	3 or 30 µg/kg per hour for 12 hours to dam	Cynomolgus monkey, E120	Minimal neuroapoptosis in frontal cortex at both doses	Koo et al.
Dexmedetomidine	25 µg/kg daily × 3	Rat, P7–9	No increase in apoptotic neurons in hippocampus, learning and memory in adulthood unaltered	Duan et al.
Dexmedetomidine	75 µg/kg × 1 or 25 µg/kg × 3	Rat, P7	No increase in apoptosis observed in hippocampus	Li et al.
Dexmedetomidine	75 µg/kg × 1	Rat, P7	No significant increase in neuroapoptosis in hippocampus	Liao et al.
Dexmedetomidine	30 or 45 µg/kg every 90 minutes × 6	Rat, P7	Cellular degeneration and apoptosis in somatosensory cortex and thalamus, but not limbic thalamus	Pancaro et al.
Diazepam	10–30 mg/kg × 1	Rat, P7	Increased neurodegeneration	Ikonomidou et al.
Diazepam	5–30 mg/kg × 1	Rat, P7	Increased neurodegeneration above 10 mg/kg, prevented by flumazenil	Bittigau et al.
Diazepam	5 mg/kg × 1	Mouse, P10	No increased neurodegeneration in cortex, but increased in thalamus, no subsequent behavioral or learning deficits	Fredriksson et al.
Diazepam	20 mg/kg on P6 and on P8	Rat, P6 and P8	Inhibition of neurogenesis	Stefovska et al.
Enflurane	2%–4% for 0.5 hour	Mouse, prenatal E6–E17	Learning impairment in adulthood	Chalon et al.
Fentanyl	50 µg/kg per hour for 72 hours	Rat, P14	Diminished morphine antinociceptive sensitivity in juvenile and adult animals	Thornton et al.
Fentanyl	0.1, 1, or 10 µg/kg × 3	Mouse, P4 and P5	Significant exacerbation of ibotenate-induced white-matter brain lesions at highest dose	Laudenbach et al.
Fentanyl	30 µg/kg + 15 µg/kg per hour for 4 hours	Swine, P5	Slightly increased neuronal apoptosis, not statistically significantly different from control	Rizzi et al.
Fentanyl	90 µg/kg × 1	Male rat, P14	Long-term anxiolytic-like behavior when tested in young adulthood	Medeiros et al.
Halothane	10 ppm for 40 hours/week	Rat, conception to P60	Learning deficits and decrease in synaptic density	Quimby et al.
Halothane	10 ppm for 40 hours/week	Rat, conception to P60	Defects in visual and spatial learning tasks	Quimby et al.
Halothane	2.5% for 2 hours	Rat, prenatal E3, E10, or E17	More errors in maze task in adult animals exposed on E3 and E10, but not at E17	Smith et al.
Halothane	50–200 ppm continuously	Rat, conception to P28	Dose-dependent decrease in cerebral synapse density	Uemura et al.
Halothane	25–100 ppm continuously	Rat, conception to P60	Decrease in apical and basal dendritic length and numbers	Uemura et al.
Halothane	25–100 ppm continuously	Rat, conception to P60	Decrease in synaptic density, no learning impairment	Uemura et al.
Halothane	1%–2% for 0.5 hour	Mouse, prenatal E6–E17	Learning impairment in adulthood	Chalon et al.
Heroin	10 mg/kg per day to dam	Mouse, E8–E18	Hyperactivity and spatial learning deficits in adulthood	Yanai et al.
Heroin	10 mg/kg per day to dam	Mouse, E9–E18	Increased neuronal apoptosis and impaired spatial learning and memory in juvenile animals	Wang et al.
Heroin	500 µg/mL	Rat, E16–E17 cortical neuronal cultures	Decrease in neuronal viability, increase in apoptosis and DNA fragmentation	Cunha-Oliveira et al.
Isoflurane	0.2–0.3 mmol/L	Mouse, P14 and P60 hippocampal slices	Block of synaptic transmission more pronounced in P14 slices compared with older animals	Simon et al.
Isoflurane + nitrous oxide + midazolam	0.75% + 75% + 9 mg/kg for 6 hours	Rat, P7	Increased neuroapoptosis and neurodegeneration, adult learning impairment	Jevtovic-Todorovic et al.
Isoflurane	0.75%–1.5% for 6 hours	Rat, P7	Increased neurodegeneration	Jevtovic-Todorovic et al.
Isoflurane + midazolam + thiopental	1.5% for 4 hours + 1 mg/kg + 7 mg/kg to ewe	Sheep, prenatal E120	No increase in neuroapoptosis, as assessed 6 days postanesthesia	McClaine et al.
Isoflurane + nitrous oxide + midazolam	0.75% + 75% + 9 mg/kg for 2–6 hours	Rat, P1–P14	Increased neuroapoptosis following exposure on P7, not P1–P3 or P10–P14	Yon et al.
Isoflurane + nitrous oxide + midazolam	0.55% + 75% + 1 mg/kg for 4 hours	Swine, P5	Increased neurodegeneration in neocortex	Rizzi et al.
Isoflurane	1.5% for 1–5 hours	Rat, organotypic slice culture	Increased neurodegeneration only after 5 hours of exposure	Wise-Faberowski et al.
Isoflurane	2.4% for 24 hours	Rat, primary cortical neuron culture	Increased apoptosis	Wei et al.
Isoflurane + nitrous oxide + midazolam	0.75% + 75% + 9 mg/kg for 2–6 hours	Rat, P7	Increased neuroapoptosis, ameliorated by estradiol	Lu et al.
Isoflurane + nitrous oxide + midazolam	0.75% + 75% + 9 mg/kg for 6 hours	Rat, P7	Increased neuroapoptosis, ameliorated by melatonin	Yon et al.
Isoflurane	0.75% for 4 hours	Mouse, P5	Increased neuroapoptosis, ameliorated by pilocarpine	Olney et al.
Isoflurane	0.75% for 6 hours	Rat, P7, and mouse organotypic hippocampal slices	Increased neuroapoptosis, exacerbated by nitrous oxide and ameliorated by xenon	Ma et al.
Isoflurane	2.4% for 24 hours	Rat, primary cortical neuron culture	Increased cellular degeneration, blocked by isoflurane or halothane preconditioning	Wei et al.
Isoflurane	1.3% for 6 hours	Rat, prenatal E21	Decrease in neuroapoptosis, no impaired learning, improved memory retention in juvenile animals	Li et al.
Isoflurane + nitrous oxide + midazolam	0.75% + 75% + 9 mg/kg for 6 hours	Rat, P7	Decreased neuronal density in multiple brain areas in adulthood	Nikizad et al.
Isoflurane + nitrous oxide + midazolam	0.55% + 75% + 1 mg/kg for 4 hours	Guinea pig, prenatal E20–E50	Increased neuroapoptosis between E35 and E40 and decreased adult neuronal density, not after E50 or in fentanyl control group	Rizzi et al.
Isoflurane	0.75% for 4 hours, 1.5% for 2 hours, or 2% for 1 hour	Mouse, P5–P7	Increased neuroapoptosis	Johnson et al.
Isoflurane + nitrous oxide	0.75% + 75% for 6 hours	Rat, P7	Increased neuroapoptosis in spinal cord	Sanders et al.
Isoflurane	Dose not specified	Mouse, P4	Increased neuroapoptosis, ameliorated by hypothermia	Olney et al.
Isoflurane + nitrous oxide	0.55% + 75% or individually for 2–8 hours	Rat, P7	Increased neuroapoptosis and neurodegeneration after exposure of 6 hours or longer, ameliorated by l -carnitine, no increase by either agent administered individually	Zou et al.
Isoflurane	1%–2% for 10 minutes	Mouse, P0	Neonatal and adolescent impairment in tests of body coordination and adult learning; decreased hippocampal cellularity and volume, more pronounced in males	Rothstein et al.
Isoflurane	1.2% for 12 hours	Rat, primary cortical neuron culture	Increased cytotoxicity, ameliorated by xestospongin C	Wang et al.
Isoflurane	0.75% for 6 hours	Rat, P7	Increased neuroapoptosis, no effect on short-term, but impairment of long-term memory, ameliorated by dexmedetomidine	Sanders et al.
Isoflurane	0.75% for 6 hours	Mouse, P8 organotypic hippocampal slices	Increased neuroapoptosis, ameliorated by dexmedetomidine, not reversed by gabazine	Sanders et al.
Isoflurane	0.75% for 6 hours	Rat, P7	Increased cortical neuroapoptosis, reduced by dexmedetomidine coadministration	Sanders et al.
Isoflurane	1.5% for 6 hours	Mouse, P7	Increased neurodegeneration early after exposure, no adult learning impairment, no decreased adult neuronal density	Loepke et al.
Isoflurane	1 MAC for 1–4 hours	Rat, P7	Widespread brain cell death following exposure for >2 hours, not 1 hour; adult neurocognitive deficits only after 4-hour exposure	Stratmann et al.
Isoflurane	1 MAC for 4 hours	Rat, P7	Decreased progenitor proliferation, deficits in fear conditioning and memory impairment in adulthood	Stratmann et al.
Isoflurane	3.4% for 4 hours	Rat, neuronal progenitor culture, harvest P2, 2–12 weeks in culture	No neurodegeneration or activation of caspases 3 or 7, decrease in caspase 9, inhibition of proliferation, preferential differentiation to neurons	Stratmann et al.
Isoflurane	1.4%	Rat, cortical and hippocampal neuron culture, harvest P1, DIC 5	Increased neuroapoptosis, reduction in dendritic spines and number of synapses, ameliorated by administration of tPA or inhibition of p75 ^NTR	Head et al.
Isoflurane + nitrous oxide + midazolam	0.75% + 75% + 9 mg/kg for 6 hours	Rat, P7	Decreased synaptic density in subiculum 2 weeks after treatment	Lunardi et al.
Isoflurane	1.6% for 5 hours	Rhesus monkey, P5	Increase in apoptotic cell death of neurons in neocortex and immature oligodendrocytes in white matter	Olney et al.
Isoflurane	1.3% or 3% for 1 hour to dam	Rat, E21	No increase in neuroapoptosis or S100β blood levels following lower dose, but both increased following higher dose	Wang et al.
Isoflurane	1.7% for 35 minutes × 4 days	Rat, P14 and mouse, P14	No increased immediate hippocampal neuronal cell death, no difference in adult learning of easy task, impairment in adult learning of difficult learning and recognition tasks	Zhu et al.
Isoflurane	1.6% for 5 hours	Rhesus monkey, P5	Increase in neocortical apoptotic cell death	Brambrink et al.
Isoflurane	1.5% for 30–120 minutes	Rat, P16	No increase in neurodegeneration or gross changes in dendritic arborization, increase in number of dendritic spines	Briner et al.
Isoflurane	0.75% for 6 hours	Mouse, P7	Increased neuroapoptosis and serum levels of S100β immediately following exposure, no long-term impairment in learning and memory tests	Liang et al.
Isoflurane	2% for 2 hours and/or caffeine 80 mg/kg IP	Mouse, P4	Increased neuroapoptosis following caffeine, worse than following isoflurane exposure, demonstrating potentiation following combination exposure	Yuede et al.
Isoflurane	1.5% for 6 hours	Mouse, P7–P8	Increased neuroapoptosis compared with no anesthesia, but similar to equianesthetic doses of desflurane or sevoflurane	Istaphanous et al.
Isoflurane	2% for 90 minutes for each time point	Mouse, P10, P20, P30, P60, and P90	No behavioral or cognitive abnormalities in adulthood	Kulak et al.
Isoflurane + nitrous oxide	0.75% isoflurane + 70% nitrous oxide for 6 hours	Rat, P7	Increased neuroapoptosis and subsequent altered cognitive function, which were ameliorated by xenon pretreatment	Shu et al.
Isoflurane + nitrous oxide	0.75% isoflurane + 70% nitrous oxide for 6 hours	Rat, organotypic hippocampal slice culture	Increased neuroapoptosis, which was ameliorated by xenon pretreatment	Shu et al.
Isoflurane + nitrous oxide + midazolam	0.55% + 75% + 1 mg/kg for 4 hours	Swine, P5	Increased neuroapoptosis in cerebral cortex compared with control or a fentanyl-treated comparison group	Rizzi et al.
Isoflurane	3% for 24 hours	Rat, astroglial culture	Impairment in growth of immature glia, delay in glial maturation, without effects on total actin or GFAP levels	Lunardi et al.
Isoflurane	1.5% for induction + 1% for 4 hours	Mouse, 1 month	Transient decrease in dendritic filopodia elimination in somatosensory cortex, without effects on dendritic spine density	Yang et al.
Isoflurane	0.7%, 1.4%, or 2.8% for 6 hours	Rat, embryonic neuronal stem cells	No effect on neural stem cell viability. Two higher concentrations inhibited cell proliferation	Culley et al.
Isoflurane	1.5% or 5% for 6–12 hours	Mouse, cortical neuronal culture	No cytotoxicity observed, even when adding clinical doses of nitrous oxide or ketamine	Campbell et al.
Isoflurane	1.4% from 15 minutes to 4 hours	Mouse, primary neuronal culture and hippocampal slice culture	Increased apoptosis and cytoskeletal destabilization	Lemkuil et al.
Isoflurane + intrathecal bupivacaine	1% for 1 or 6 hours	Rat, P7, P14, P21	Neuroapoptosis in brain and spinal cord after 6 hours of exposure, but not 1 hour; intact spontaneous motor performance in adulthood	Yahalom et al.
Isoflurane and/or nitrous oxide	1% and/or 70% for 8 hours	Rhesus monkey, P5–P6	No neuroapoptosis observed following either agent alone, neuroapoptosis increased following anesthetic combination	Zou et al.
Isoflurane + nitrous oxide + midazolam	0.75% + 75% + 9 mg/kg for 6 hours	Rat, P7	Enlargement and impairment of structural integrity of mitochondria and decreased density in subiculum, as well as increased autophagic activity	Sanchez et al.
Isoflurane + nitrous oxide	0.75% isoflurane + 70% nitrous oxide for 6 hours	Rat, P7	Increased neuroapoptosis and subsequent altered cognitive function, which were ameliorated by xenon pretreatment	Shu et al.
Isoflurane	Titrated between 0.7% and 1.5% isoflurane for 5 hours	Rhesus monkey, P6	Widespread apoptotic cell death of neurons and oligodendrocytes (6.3% of all myelinating oligodendrocytes in forebrain)	Brambrink et al.
Isoflurane	Isoflurane 1.5% for 6 hours	Mouse, P7	Quantification of apoptotic neuronal cell death in superficial layers of visual cortex finds almost 3% of neurons affected following exposure, compared with less than 0.1% of neurons undergoing natural apoptosis in control animals	Istaphanous et al.
Isoflurane	2% for 6 hours	Mouse, P6	Increased neuroapoptosis, but no long-term impairment in memory function	Kodama et al.
Isoflurane	1.5% for 6 hours	Mouse, P7, P21, or P49	Increased apoptosis in olfactory bulb at all ages and dentate gyrus at the two older time points; neurons younger than 15 days of age particularly vulnerable	Hofacer et al.
Isoflurane	Titrated between 1% and 1.5% for 5 hours to dam	Rhesus monkey, E120	Increased neuroapoptosis, predominantly in cerebellum, caudate, putamen, and amygdala, as well as diffuse apoptotic oligodendrocyte death	Creeley et al.
Isoflurane	0.75% for 6 hours	Rat, P7	Neuronal apoptosis in hippocampus, dose-dependently inhibited by dexmedetomidine	Li et al.
Isoflurane	0.75% for 6 hours	Rat, P7	Increased neuroapoptosis in hippocampus, inhibited by dexmedetomidine coadministration	Liao et al.
Isoflurane	1.5% for 6 hours	Mouse, P7, P21, or P49	Differential degrees of neuroapoptosis among brain regions dependent on age during exposure; sustained vulnerability into adulthood in regions with continued neurogenesis	Deng et al.
Isoflurane	1.5% for 6 hours	Rats, P7	Apoptotic marker caspase 3 increase in cortex and hippocampus was ameliorated by preconditioning with a 30-minute exposure to 1.5% isoflurane on P6	Peng et al.
Isoflurane	2% for 1 hour	Mouse, P7	Apoptotic cell death increased in inner nuclear layer of retina	Cheng et al.
Isoflurane	Titrated between 1.5% and 3% for 5 hours	Rhesus monkey, P6	Increased neuroapoptosis and oligodendrocytic apoptosis was reduced by lithium coadministration	Noguchi et al.
Ketamine	20 mg/kg × 7	Rat, P7	Increased neurodegeneration	Ikonomidou et al.
Ketamine	25–75 mg/kg × 1–7	Rat, P7	Increased neurodegeneration only after 7 repeated doses of 25 mg/kg	Hayashi et al.
Ketamine	50 mg/kg × 1	Mouse, P10	Cortical neurodegeneration; hypo- and hyperactivity, abnormal habituation in adulthood	Fredriksson et al.
Ketamine	50 mg/kg × 1	Mouse, P10	Neurodegeneration, worsened by coadministration of diazepam	Fredriksson et al.
Ketamine	50 mg/kg × 1	Mouse, P10	Neurodegeneration and impairment in memory tasks in adulthood	Fredriksson et al.
Ketamine	10–25 mg/kg × 1–7	Rat, P7	Neurodegeneration observed after highest dose, plasma levels 7 times higher than during human anesthesia	Scallet et al.
Ketamine	1.25–40 mg/kg × 1	Mouse, P7	Neurodegeneration after doses of 5 mg/kg or higher, no gross neurobehavioral abnormalities 7 days after treatment	Rudin et al.
Ketamine	0.1–20 µmol/L for 6–48 hours	Rat, forebrain neuron culture	Increased DNA fragmentation after higher doses for longer periods of time	Wang et al.
Ketamine	10–40 mg/kg × 1	Mouse, P7	Increased neurodegeneration following doses of 20 mg/kg or higher, worsened by midazolam coadministration	Young et al.
Ketamine	1–20 µmol/L for 2–24 hours	Rhesus monkey, forebrain neuron culture	Increased DNA fragmentation and decreased mitochondrial function after higher doses for longer periods of time	Wang et al.
Ketamine	0.01–40 µg/mL for 1–48 hours	Rat, GABAergic neuron culture	Neuronal cell loss and decrease in dendritic length and branching with higher concentrations for longer exposure times	Vutskits et al.
Ketamine	100 µmol/L for 48 hours	Rat, cortical neuron culture	Increased apoptosis, ameliorated by NMDA, IGF-1, alsterpaullone, or iodoindirubin	Takadera et al.
Ketamine	0.1–30 µmol/L for 24 hours	Rat, cortical neuron culture	Decreased cell viability and increased DNA fragmentation, ameliorated by erythropoietin	Shang et al.
Ketamine	20–50 mg/kg per hour × 24 hours	Rhesus monkey, E122, P5, or P35	Increased neurodegeneration in two younger age groups, ketamine plasma levels higher than in humans	Slikker et al.
Ketamine	10–10 µg/mL for 1–24 hours	Rat, GABAergic neuron culture	Neuronal cell loss with higher concentrations or longer exposure times	Vutskits et al.
Ketamine	2.5 mg/kg × 2 for 4 days	Rat, P1–P4	Minimal neurodegeneration, but ketamine ameliorated pain-induced neurodegeneration and subsequent learning abnormalities	Anand et al.
Ketamine	25 mg/kg × 1	Mouse, P10	No increase in neurodegeneration, but subsequent learning impairment, worsened by coadministration of thiopental or propofol	Fredriksson et al.
Ketamine	1–20 µmol/L for 24 hours	Rat, forebrain neuron culture	Increased DNA fragmentation after higher doses, blocked by nitroindazole	Wang et al.
Ketamine	5–25 mg/kg × 1	Mouse, P10	Dose-dependent alterations in levels of developmentally expressed proteins and adult behavioral abnormalities	Viberg et al.
Ketamine	2.5 mg/kg × 2 for 4 days	Rat, P1–P4	No increase in neuronal cell death, no change in adult learning and exploratory behavior and ameliorated pain-induced neurodegeneration and behavioral abnormalities	Rovnaghi et al.
Ketamine	20 mg/kg × 6	Rat, P7	Decreased body weight 1–3 days after exposure, no abnormal spontaneous behavior observed during first 4 days	Boctor et al.
Ketamine	30 mg/kg + 15 mg/kg every 90 minutes × 2	Mouse, P15–P90	No increase in neurodegeneration, impairment in dendritic spine development following exposure on P20 or younger	Vutskits et al.
Ketamine	40 mg/kg	Mouse, P5	Increased neuroapoptosis, ameliorated by lithium coadministration	Straiko et al.
Ketamine	5–20 mg/kg × 1–6	Rat, P7	Increased apoptosis and neurodegeneration only observed following 6 repeated doses of 20 mg/kg	Zou et al.
Ketamine	20 mg/kg × 7	Rat, P7	Increased apoptosis and neurodegeneration, decreased weight gain, increase in BDNF and TrkB cDNA	Ibla et al.
Ketamine	20–50 mg/kg per hour for 3–25 hours	Rhesus monkey, P5–P6	Neuronal degeneration only in neocortex following 9-hour exposure or longer, no degeneration in deeper brain areas	Zou et al.
Ketamine	30 mg/kg + 15 mg/kg every 90 minutes × 2	Mouse, P15	Increase in dendritic spine density and decrease in spine head diameter in the somatosensory cortex and hippocampus	De Roo et al.
Ketamine	100 mg/kg per day × 5 days	Mouse, P8–P12	Impaired dendritic spine maturation immediately following exposure, which subsided within 2 weeks	Tan et al.
Ketamine or ( S )-ketamine	1–8 mmol/L or 0.6–4 mmol/L for 24 hours	Human, neuroblastoma cells	Increased apoptosis, but up to 80% less cell death following ( S )-ketamine	Braun et al.
Ketamine	5–20 mg/kg × 5	Rat, P7	Increased neuroapoptosis in neocortex and thalamus, increase in cell cycle proteins	Soriano et al.
Ketamine	0.1–1000 µmol/L × 6–48 hours	Rat, cortical neuron culture	Increase in expression of caspase 3 and cell cycle proteins	Soriano et al.
Ketamine	20 mg/kg every 2 hours × 6	Rat, P7	Increased neurodegeneration in frontal cortex	Shi et al.
Ketamine	3–15 mg/kg intrathecal L4-6	Rat, P3, P7, or P21	Increased neuronal cell death and subsequent altered spinal cord function after injection at P3, but not at P7 or P21	Walker et al.
Ketamine	20 mg/kg bolus + 20–50 mg/kg per hour for 24 hours	Rhesus monkey, P5–P6	Decreased performance of the National Center for Toxicological Research Operant Testing Battery and motivation	Paule et al.
Ketamine + fentanyl	20 mg/kg + 90 µg/kg	Rat, P14	Long-term hyperactivity and behavioral changes indicating lower levels of anxiety	Madeiros et al.
Ketamine	20 mg/kg every 2 hours × 6	Rat, P7	Greater accumulation of a radioactive tracer and more cell death in frontal cortex in adulthood	Zhang et al.
Ketamine + xylazine	42.5 + 4.3 mg/kg every 90 minutes × 3	Mouse, 1 month	Transient increase in dendritic filopodia formation in somatosensory cortex, without effects on dendritic spine density	Yang et al.
Ketamine	100 µmol/L, 1 mmol/L, or 3 mmol/L	Mouse, cortical neuronal culture	Cytotoxicity only observed in doses of 1 mmol/L or higher	Campbell et al.
Ketamine	18.4–86.5 mg/kg per hour for 5 hours to dam	Rhesus monkey, E120	Increased neuroapoptosis, especially in cerebellum, caudate-putamen, and nucleus accumbens	Brambrink et al.
Ketamine	18.4–56.0 mg/kg per hour for 5 hours	Rhesus monkey, P6	Increased neuroapoptosis, especially in basal ganglia and thalamus	Brambrink et al.
Ketamine	75 mg/kg daily × 3	Rat, P7–9	Increase in apoptotic neurons in hippocampus, impaired learning and memory in adulthood, both effects ameliorated by coadministration of dexmedetomidine	Duan et al.
Ketamine	20–50 mg/kg per hour for 12 hours to dam	Cynomolgus monkey, E120	Neuroapoptosis in frontal cortex	Koo et al.
Ketamine	1–500 µM for 24 hours	Rat, neural stem cell culture	No oxidative DNA damage, mitochondrial toxicity, or impaired proliferation observed following exposure to clinically relevant concentration. However, number of differentiated neurons reduced	Slikker et al.
Ketamine	20 mg/kg every 90 minutes × 6	Rat, P7	Significant cellular degeneration and apoptosis in limbic thalamus, but not somatosensory cortex or thalamus	Pancaro et al.
Methadone	10 mg/kg per day to dam	Rat, E5–E21	Decreased brain monoamines in juveniles and hyperresponsiveness in adulthood	Rech et al.
Methadone	10–15 mg/kg per day to dam	Rat, E8–E21	Increased excitability tested in juvenile animals using the acoustic startle reflex	Hutchings et al.
Methadone	9 mg/kg per day to dam	Rat, E7–E21	Increased noradrenergic activity in hippocampus of male juveniles	Robinson et al.
Methadone	9 mg/kg per day	Rat, P1–P10	Reduced activity of dopaminergic neurons in frontal cortex of juvenile males	Robinson et al.
Methadone	9 mg/kg per day to dam	Rat, E7–E21	Reduction in nerve growth factor, disruption of cholinergic neuronal activity	Robinson et al.
Methadone	9 mg/kg per day to dam	Rat, E7–E21 or P10	Reduction in nerve growth factor in striatum	Wu et al.
Midazolam	9 mg/kg × 1	Rat, P7	No increase in neurodegeneration	Jevtovic-Todorovic et al.
Midazolam	9 mg/kg × 1	Mouse, P7	Increased neuroapoptosis	Young et al.
Midazolam	9 mg/kg × 1	Mouse, P5	Increased neuroapoptosis	Olney et al.
Midazolam	3–9 mg/kg × 1	Rat, P1–P14	No increase in neuroapoptosis	Yon et al.
Midazolam	0.25–25 µg/mL	Rat, GABAergic neuron culture	No effect on neuronal survival	Vutskits et al.
Midazolam	25 mg/kg + 15 mg/kg every 90 minutes × 2	Mouse, P15–P90	No increase in neurodegeneration, impairment in dendritic spine development following exposure on P20 or younger	Vutskits et al.
Midazolam	25 mg/kg + 15 mg/kg every 90 minutes × 2	Mouse, P15	Increase in dendritic spine density and decrease in spine head diameter in the somatosensory cortex and hippocampus	De Roo et al.
Midazolam	50 mg/kg	Mouse, P10	No impairment of learning and memory in adulthood	Xu et al.
Midazolam	50 mg/kg per day × 5 days	Mouse, P8–P12	Impaired dendritic spine maturation immediately following exposure, which subsided within 2 weeks	Tan et al.
Midazolam	25 mg/kg IP injections for 6 hours	Rats, P5 or P15	Increased neuroapoptosis in medial prefrontal cortex at both age points without long-term measurable diminution in neuronal density	Osterop et al.
Morphine	10 mg/kg per day	Rat, E12–P5	Gender- and region-specific changes in µ-opioid receptor density, decrease in neuronal density, and reduction in dendritic growth, partially reversed by naltrexone	Hammer et al.
Morphine	5 mg/kg per day	Rat, P1–P4	Decreased µ-opioid receptor density in striatum following exposure	Tempel el al.
Morphine	20 mg/kg per day to dam, except for E11: 10 mg/kg per day	Rat, E11–E18	Decreased µ-opioid receptor density in adulthood	Rimanoczy et al.
Morphine	1 µmol/L for 1–2 days	Mouse, P5–P6 cerebellar neuronal precursor culture	Marked decrease in DNA synthesis, without effect on cell survival	Hauser et al.
Morphine	5 mg/kg × 3 doses, then 10 mg/kg twice a day to dam	Rat, E11–E18	Impairment in spatial learning in adulthood	Slamberova et al.
Morphine	20 mg/kg	Mouse, P4–P5	Suppression of DNA synthesis in astrocytes	Stiene-Martin et al.
Morphine	1–100 µmol/L for 5 days	Human, GW16–22 fetal microglial, astrocyte, and neuronal cultures	Progressive increase in apoptosis in neurons after 2-day exposure, or microglia after 3 days, but not in astrocytes. Apoptosis blocked by naloxone	Hu et al.
Morphine	2 mg/kg per day to dam, progressively increased by 2 mg/kg per day	Rat, E0–P21	Impairment in spatial learning in young adulthood	Yang et al.
Morphine	20 mg/kg per day	Chick, E12–E16	Impairment in long-term memory formation	Che et al.
Morphine	2 mg/kg per day	Rat, P3–P7	Impairment in adult cognitive function	McPherson et al.
Morphine	0.5, 1, or 3 mg/kg	Rat, P7	Increased pain behavior to a pain challenge several days after morphine injection	Zissen et al.
Morphine	15 µmol/L for 7 days	Mouse, E16 hippocampal neuronal culture	Increased apoptosis, decreased neuronal density	Svensson et al.
Morphine	4 mg/kg per day	Mouse, P5–P9	Impairment in reward-mediated learning in adulthood	Boasen et al.
Morphine	5 mg/kg × 3 doses, then 10 mg/kg twice a day to dam	Rat, E11–E18	Decreased synaptic plasticity and impaired learning in juveniles	Niu et al.
Morphine	2 mg/kg per day to dam, progressively increased by 2 mg/kg per day, maximum 14 mg/kg per day	Rat, E0–P21	Decreased performance in learning tasks in adulthood	Lin et al.
Morphine	5 µg/kg per day × 8 days	Rat, P8–P14	Increased nociceptive response throughout adulthood, which could be blocked by ketamine	Rozisky et al.
Morphine	10 mg/kg × 1	Rat, P7 or P15	No increase in neuronal apoptosis in deeper layers of medial prefrontal cerebral cortex	Massa et al.
Morphine	10 mg/kg twice daily × 14 days	Rat, P1–14	Long-term decrease in threshold for thermal stimulation and temporary learning impairment	Craig et al.
Nitrous oxide (hyperbaric)	50%–150% for 2–6 hours	Rat, P7	No increase in neurodegeneration	Jevtovic-Todorovic et al.
Nitrous oxide (hyperbaric)	50%–150% for 2–6 hours	Rat, P1–P14	No increase in neuroapoptosis	Yon et al.
Nitrous oxide	75% for 6 hours	Rat, P7, and mouse organotypic hippocampal slices	No increase in neuroapoptosis in rats, increased neuroapoptosis in mice	Ma et al.
Pentobarbital	5–10 mg/kg × 1	Rat, P7	Increased neurodegeneration	Bittigau et al.
Pentobarbital	0.5, 5, or 50 µg/mL	Rat, PC12 pheochromocytoma cell culture	Dose-dependent inhibition of apoptotic cell death triggered by oxygen-glucose deprivation	Morimoto et al.
Pentobarbital	10–20 mg/kg	Rat, P7	Impairment in spatial learning; however, hypercarbia and hypoxia observed during exposure	Tachibana et al.
Phenobarbital	40–100 mg/kg	Rat, P7	Increased neuroapoptosis, ameliorated by β-estradiol	Bittigau et al.
Phenobarbital	50 mg/kg	Rat, P7	Increased neuroapoptosis, ameliorated by β-estradiol	Asimiadou et al.
Phenobarbital	30 mg/kg × 2	Mouse, P6	Long-term alterations in brain protein expression	Kim et al.
Phenobarbital	75 mg/kg	Rat, P7–P8	Increased neuroapoptosis, enhanced by lamotrigine administration	Katz et al.
Phenobarbital	25 mg/kg	Mouse, P0	Neonatal and adolescent impairment in tests of body coordination and adult learning; decreased hippocampal cellularity and volume, more pronounced in males	Rothstein et al.
Phenobarbital	50 mg/kg (P6), 40 mg/kg (P8)	Rat, P6 and P8	Inhibition of neurogenesis	Stefovska et al.
Propofol	0.5–10 µg/mL for 8 hours	Rat, GABAergic neuron culture	Dose-dependent decrease in GABAergic enzyme GAD after higher doses	Honegger et al.
Propofol	10–100 µg/mL for 2 hours to 10 days	Rat, dissociated neuronal culture P1 or P7	Increased cell death in P1 neurons following higher doses, no evidence for neurotoxic effects in organotypic hippocampal culture on P7	Spahr-Schöpfer et al.
Propofol	1–50 µg/mL	Rat GABAergic neuron culture	Increased neuronal cell death following highest dose, altered dendritic development following lowest dose	Vutskits et al.
Propofol	5–500 µmol/L for 2–24 hours	Chick, neuron explant culture	Dose-dependent neurite growth cone collapse	Al-Jahdari et al.
Propofol	10–60 mg/kg × 1	Mouse, P10	Increased neurodegeneration following highest dose	Fredriksson et al.
Propofol	25–300 mg/kg × 1	Mouse, P5–P7	Increased neuroapoptosis following doses >50 mg/kg	Cattano et al.
Propofol + fentanyl	6 mg/kg per hour + 10 µg/kg per hour for 24 hours	Swine, P0	No increased neurodegeneration	Gressens et al.
Propofol	5 µmol/L for 5 hours	Rat, hippocampal neuron culture	Increased neuroapoptosis	Kahraman et al.
Propofol	50 mg/kg + 25 mg/kg every 90 minutes × 2	Mouse, P15–P90	No increase in neurodegeneration, impairment in dendritic spine development following exposure on P20 or younger	Vutskits et al.
Propofol	25 mg/kg	Rat, P7	Increased neuroapoptosis, decrease in nerve growth factor	Pesic et al.
Propofol	50–100 mg/kg	Mouse, P5	Increased neuroapoptosis, ameliorated by lithium coadministration	Straiko et al.
Propofol	0.01–1 mg/mL for 3–48 hours	Rat, primary cortical neuron culture, harvested E18	Highest concentration improved cell viability after up to 6 hours of exposure, but reduced cell viability after more than 12 hours	Berns et al.
Propofol	30 mg/kg every 90 minutes × 3	Rat, P6	Increase in neurodegeneration in some brain areas, especially in parts of thalamus and subiculum, subtle neurologic impairment in adult animals	Bercker et al.
Propofol	50 mg/kg + 25 mg/kg every 90 minutes × 2	Mouse, P15	Increase in dendritic spine density and decrease in spine head diameter in the somatosensory cortex and hippocampus	De Roo et al.
Propofol	40 mg/kg + 20 mg/kg per hour for 6 hours	Rat, P5, P10, P15, P20, or P30	Decrease in dendritic spine density following exposure between P5 and P10, but increased density at P15–P30	Briner et al.
Propofol	10 or 60 mg/kg	Mouse, P10	Reduction in BDNF, no changes in adult spontaneous behavior, but reduced sedative effect of diazepam in adulthood	Pontén et al.
Propofol	50 mg/kg × 7 days	Rat, P7–P14	No increase in neuroapoptosis, only in hypoxic condition	Tu et al.
Propofol	100 mg/kg × 1	Mouse, P5	Increased neuroapoptosis in hippocampus	Pearn et al.
Propofol	3 µmol/L for 6 hours	Mouse, neuron culture, harvested P1–P3, DIC 5–7	Increased neuroapoptosis, mediated by p75 neurotrophin receptor activation	Pearn et al.
Propofol	300–450 µg/kg per minute for 5 hours	Rhesus monkey, E120 or P6	Apoptotic cell death of oligodendrocytes and neurons; neuroapoptosis predominantly in subcortical and caudal regions during fetal exposure and in neocortex during neonatal exposure	Creeley et al.
Propofol	0.9–50 µg/mL for 1–48 hours	Rat, hippocampal astrocyte culture	Dose-dependent increase in apoptosis and reduction in astrocytic viability	Sun et al.
Propofol		Rat, E20	Increased neuroapoptosis and subsequent learning impairment, attenuated by dexmedetomidine coadministration	Li et al.
Sevoflurane	4% for 24 hours	Rat, primary cortical neuron culture	No increase in apoptosis	Wei et al.
Sevoflurane	2% for 12 hours	Rat, primary cortical neuron culture	No increase in cytotoxicity	Wang et al.
Sevoflurane	1.7% for 2 hours	Mouse, P7	Increased neuroapoptosis	Zhang et al.
Sevoflurane	3% for 6 hours	Mouse, P6	Increased neuroapoptosis, impairments in fear conditioning and social interaction in adulthood	Satomoto et al.
Sevoflurane	4% or 8% for 12 or 6 hours, respectively	Rat, primary cortical neuron culture, E18	No difference in cell viability compared with untreated controls	Berns et al.
Sevoflurane	3%–5% for 6 hours	Rat, P6	No increase in neurodegeneration immediately after exposure, no long-term neurologic impairment	Bercker et al.
Sevoflurane	2.1% for 0.5–6 hours	Rat, P4–P8	Seizure activity during exposure and neuroapoptosis partially blocked by administration of bumetanide	Edwards et al.
Sevoflurane	2.5% for 30–120 minutes	Rat, P16	No increase in neurodegeneration or gross changes in dendritic arborization, increase in number of dendritic spines	Briner et al.
Sevoflurane	1.1% for 6 hours	Mouse, P7	Increased neuroapoptosis immediately following exposure, no long-term impairment in learning and memory tests	Liang et al.
Sevoflurane	2.1% or 3% for 2 hours or 6 hours	Mouse, P6	Increased neuroapoptosis following the longer, but not the shorter, exposure time. Transgenic Alzheimer disease mice more susceptible	Lu et al.
Sevoflurane	2.9% for 6 hours	Mouse, P7–P8	Increased neuroapoptosis compared with no anesthesia, but similar to equianesthetic doses of desflurane or isoflurane	Istaphanous et al.
Sevoflurane	3.8% for 6 hours	Mouse, P6	Increased neuroapoptosis, less than with desflurane and isoflurane, and long-term impairment in memory function	Kodama et al.
Sevoflurane	1 MAC × 4 hours	Rat, P7	Memory impairment, alleviated by environmental enrichment	Shih et al.
Sevoflurane	3% for 2 hours daily × 3	Mouse, P6–8	Immediate increase in neuroinflammation and subsequent cognitive impairment, ameliorated by environmental enrichment or antiinflammatory treatment (ketorolac)	Shen et al.
Sevoflurane	1.2% for 6 hours	Rat, P7	No increased neuronal apoptosis detected in hippocampus	Li et al.
Sevoflurane	2.5%–3.5% for 6 hours	Mouse, P6–7	Neuroapoptosis immediately following exposure and long-term memory impairment; normal subsequent social behavior, anxiety level, and vocalization	Chung et al.
Sevoflurane	2%–2.5% for 4 hours × 3	Rhesus monkey, between P6 and P28	Exaggerated anxiety behavior in juveniles following repeated anesthetic exposures in infancy	Raper et al.
Sevoflurane	2.5% for 9 hours	Rhesus monkey, P5–6	Increased neuronal degeneration	Liu et al.
Sevoflurane	2%–2.5% for 4 hours × 3	Rhesus monkey, 3 exposures during first 6 postnatal weeks	No differences in maternal behavior towards infants observed	Raper et al.
Sufentanil	2 µg/kg × 3	Mouse, P4 and P5	No exacerbation of ibotenate-induced white-matter brain lesions	Laudenbach et al.
Thiopental	5–25 mg/kg × 1	Mouse, P10	No increase in neurodegeneration by itself, but exacerbation and learning abnormalities in conjunction with ketamine	Fredriksson et al.
Xenon	75% for 6 hours	Rat, P7 and mouse organotypic hippocampal slices	No increase in neuroapoptosis	Ma et al.
Xenon	70% for 4 hours	Mouse, P7	Increased neuroapoptosis, but decreased isoflurane-induced neuroapoptosis	Cattano et al.
Xenon	50% for 24 hours + fentanyl infusions	Swine, P0	No neuroapoptosis detected	Sabir et al.
Xenon	0.75, 1, or 2 MAC for 6 hours	Rat, hippocampal slice cultures	Increased cell death following two higher doses in CA1, CA3, and dentate	Brosnan et al.

BDNF, brain-derived neurotrophic factor; DIC, days in culture ; E, embryological day; GAD, glutamic acid decarboxylase; GFAP , glial fibrillary acidic protein; GW, gestational week; IGF-1, insulin-like growth factor 1; IP , intraperitoneal; MAC, minimum alveolar concentration; P , postnatal day; tPA, tissue plasminogen activator; p75 ^NTR , p75 neurotrophin receptor TrkB , tyrosine-related kinase B.

Apoptosis represents an inherent, energy-consuming process using a cascade of enzymes called caspases. Apoptosis is highly conserved among species and culminates in self-destruction and elimination of cells, even under physiologic conditions, when these cells are functionally redundant or potentially detrimental to the organism. It involves an orderly breakdown of the cell that includes chromatin aggregation, nuclear and cytoplasmic condensation, and partitioning of cytoplasmic and nuclear material into apoptotic bodies for subsequent phagocytosis, without an extensive inflammatory response. That contrasts with features observed during necrosis, for example during ischemia, which includes energy failure, cellular swelling, membrane rupture, and release of cytoplasmic content into the extracellular compartment, followed by an inflammatory response. However, there seems to exist substantial overlap and common pathways between cell death processes, such as apoptosis, necrosis, and autophagy, which have previously been thought to be entirely separate. Apoptosis, which has also been termed cellular suicide or programmed cell death, is extensively used during tissue homeostasis, endocrine-dependent tissue atrophy, and normal embryogenesis (e.g., cardiac sculpting, ablation of tail tissue as part of tadpole metamorphosis in amphibians, or elimination of interdigital mesenchymal tissue of fingers and toes). Similarly, brain cells are produced in excess during normal brain development and are eliminated in large number during normal brain maturation in rodents, nonhuman primates, and humans. This physiologic apoptotic cell death is critical to establish proper brain structure and function, and any disruption of this process can lead to massive brain malformations and intrauterine demise. In the developing brain, apoptotic cell death can also be triggered by pathologic, extrinsic factors, such as hypoxia and ischemia. It currently remains unclear whether anesthesia-induced neuroapoptosis accelerates physiologic programmed cell death or whether it eliminates cells not destined to die, as in pathologic apoptosis.

Animal studies have initially identified a narrow window of susceptibility to neuronal cell death induced by several anesthetic drugs, such as the NMDA antagonist ketamine, the GABA agonist isoflurane, or ethanol (a combined NMDA antagonist and GABA agonist). Ketamine-induced neuronal demise is most pronounced during exposure between 5 and 7 days of age in neonatal rodents or before 6 days of age in monkeys. Similarly, a dramatic increase in neuronal apoptosis was detected in the cortex, thalamus, and amygdala of 3- to 10 day-old rodents after prolonged exposure to isoflurane, but minimal cell death was detected in 1-day-old animals or those older than 10 days of age. However, data from the laboratory of one of the authors challenge the notion that anesthetic-induced neuroapoptosis is limited to a narrow age range. In mice, neuronal apoptosis was similarly observed in the cortex and hippocampus of neonatal animals after a prolonged exposure to isoflurane; however, vulnerability extended into adulthood in brain regions with ongoing neurogenesis, such as the dentate and olfactory bulb. This finding could be explained by the fact that neurons were found particularly vulnerable to isoflurane-induced apoptosis before 14 days of cellular age and therefore would be expected to occur throughout the life of the animal in neurogenic niches containing neurons of this particular age. Surprisingly, however, other studies suggest that intrauterine exposure to clinical doses of isoflurane in prenatal rats may actually decrease physiologic apoptosis and improve subsequent memory retention, whereas only supraclinical doses of isoflurane induced neuroapoptosis in this setting. Whereas chronic opioid exposure in immature animal models can lead to long-term neurodegeneration, a recent study found no neuronal cell death immediately after a brief exposure to morphine in newborn rats. Additional research is needed to elucidate the timing of exposure on cellular demise and the differential effects of anesthetics and analgesics on brain structural integrity.

Long-Term Brain Cellular Viability, Neurologic Function, and Behavior

To answer the important question of whether anesthetics simply hasten natural apoptosis or whether they induce pathologic apoptosis, long-term neuronal density and neurologic function have to be assessed in adult animals exposed to anesthesia as neonates. If exposure to anesthetics or analgesics only temporarily accelerated physiologic apoptosis, one would expect normal cellular density and function in adulthood. Conversely, permanent neuronal cell loss and long-term neurocognitive impairment after anesthetic exposure early in life would suggest that anesthesia-induced neuronal apoptosis may be pathologic in nature and that the organism was unable to compensate for the neonatal cell loss by postexposure neuronal plasticity and repair. To address these questions, several studies measured neurologic function, assessed behavior, and/or determined neuronal density in adult animals after anesthetic exposure in the neonatal period. Results from these studies, however, are conflicting. A number of studies reported long-term neurocognitive or behavioral abnormalities after neonatal exposure to enflurane, halothane, isoflurane, sevoflurane, propofol, or ketamine, or to a combination of isoflurane, nitrous oxide, and midazolam. ^a

a References .

Importantly, however, many of these studies only observed abnormalities in very specific tests or subsets of neurocognitive batteries, whereas many other neurobehavioral domains remained intact. For example, a 6-hour exposure to midazolam, isoflurane, and nitrous oxide in neonatal rats transiently impaired learning in a water maze task in young adulthood and in older animals, whereas in the same animals, several other tests of behavior and learning, including acoustic startle response, sensorimotor tests, spontaneous behavior in an open field, and learning and memory in the radial arms maze, remained unimpaired. Similarly, after a 4-hour exposure to one minimum alveolar concentration (1 MAC) of isoflurane in 7-day-old rats, long-term memory retention was abnormal at two time points, whereas performance at several other time points, as well as in other tests of learning and memory, remained intact. Accordingly, the relevance of these temporary and limited learning deficits remains unclear. Similar to humans, the performance of rodents and primates in learning tasks depends to a great extent on maternal behavior and rearing conditions, making them strong confounders during neurocognitive testing. Moreover, another obvious and important factor in neurocognitive testing is the verification of similar degrees of motivation when comparing separate groups of animals. For example, a 24-hour exposure to ketamine sedation early in life impaired subsequent performance of rhesus monkeys in learning and memory tests, in addition to decreasing their motivation to perform these tasks.

Studies of prolonged opioid administration in immature animals have also found evidence for long-term impairment in learning tasks, as well as altered pain responses in adult animals after exposure to morphine, fentanyl, heroin, or methadone early in life. ^a

a References .

Conversely, several other investigations have observed no neurologic abnormalities after administration of midazolam, isoflurane, sevoflurane, or ketamine, even when using complex neurologic tests in neonatal animals. ^b

b References .

It remains to be determined whether these differential findings are attributable to the anesthetic doses, exposure times, species, or are related to the specific type of neurologic test used or the timing of the exposure or the assessment. Interestingly, escalating exposure times of isoflurane in neonatal rats caused neuronal apoptosis beginning at 2 hours of anesthesia, but no evidence of long-term neurologic abnormalities until 4 hours of anesthesia. In another study, a 6-hour exposure to isoflurane caused significant apoptosis immediately after exposure in neonatal mice but resulted in no measurable long-term deficits in performance of complex neurologic tests as adult animals. Moreover, in this study, brain neuronal density in adulthood was not diminished in regions significantly affected by anesthesia-induced neuroapoptosis compared with unanesthetized littermates. Studies in mice exposed to 6 hours of isoflurane at 21 days of age did not observe subsequent diminution in dentate granule cells, despite considerable apoptotic cell death immediately after exposure. These findings could either suggest that isoflurane may only accelerate physiologic apoptosis or that the developing brain's plasticity and capacity for repair can compensate for a pathologic insult early in life. Conversely, a study in similarly aged rats observed a permanent elimination of neurons, as well as neurologic abnormalities in adult animals, after exposure to isoflurane, nitrous oxide, and midazolam as neonates, suggesting that either the specific combination of anesthetic drugs (isoflurane alone vs. the combination exposure) or species differences (rats vs. mice) could affect relationships between neonatal neuroapoptosis and long-term function and neuronal density. Alternatively, these conflicting results may be explained by the dissimilar testing environment because neurocognitive tests are not easily transferable among laboratories. Yet another explanation holds that neonatal apoptosis may not be causally linked to adult neurocognitive performance at all, as evidenced by substantial apoptotic cell death observed immediately after carbon dioxide–induced hypercarbia in unanesthetized neonatal rats, which lacked long-term neurologic sequelae.

Effects on Neurogenesis and Gliogenesis

The generation of new neurons, or neurogenesis, as well as new astrocytes—gliogenesis—is most active in the immature brain in utero or soon after birth. Accordingly, several studies have investigated the effects of anesthetic exposure, primarily isoflurane, on progenitor viability and rates of neurogenesis. Although isoflurane did not kill neural progenitor cells in vitro, 3.4% isoflurane for 4 hours decreased the rate of neuronal proliferation and increased neuronal fate selection. These findings have been confirmed as 2.8% isoflurane for 6 hours had no effect on neural stem cell viability, whereas larger doses inhibited cell proliferation. In vivo studies in newborn mice failed to find significant cell death in radial glial-type progenitor cells in the dentate gyrus immediately after a 6-hour isoflurane exposure, although more mature neuroblasts made up a substantial number of cells affected by anesthesia-induced apoptotic cell death. Morphine, on the other hand, did not affect cell survival in a murine cerebellar neuronal precursor culture, although, exposure decreased DNA synthesis.

Isoflurane also impairs the growth of cultured immature astrocytes and delays their maturation after a 24-hour administration of 3% isoflurane, although even this extreme exposure showed no effect on cell viability. Morphine, although increasing apoptosis in neurons and microglia, did not affect astrocytes in a study involving human fetal cell cultures.

Alterations in Dendritic Architecture

The immature brain accumulates an overabundance of neuronal connections in infancy, and the number of dendrites and synapses dramatically decreases after the first year of life. Several studies have examined the effects of a wide variety of anesthetics, such as propofol, isoflurane, sevoflurane, desflurane, midazolam, and ketamine on dendritic arborization and synaptic architecture. ^c

c References .

A common theme in these studies is that anesthetics can affect dendritic arborization and synaptic density, and that the direction of any change, either an increase or a decrease in the number of dendritic spines, depends on the age at which the animals were exposed to anesthetics and therefore the developmental state of the brain. During the first 2 weeks of life, anesthetic exposure can lead to a decrease in synaptic and dendritic spine density in small rodents, whereas exposure beyond this age can lead to an increase in the number of dendrites. Other studies suggest that this differential effect may depend on the maturational switch of GABA from excitation to inhibition, which is related to the switch from the immature form of the potassium-chloride-cotransporter NKCC1 to the mature KCC2. However, the permanence of these dendritic changes remains controversial because some studies have observed only a transient effect after ketamine, midazolam, or isoflurane anesthesia exposure early in life.

Decrease in Trophic Factors

Isoflurane- or propofol-based anesthesia in neonatal animals has been associated with a decrease in brain-derived neurotrophic factor (BDNF), a protein integral to neuronal survival, growth, and differentiation. The cellular mechanism involves a reduction in tissue plasminogen activator and plasmin, which converts proBDNF to BDNF. Accordingly, isoflurane has been found to trigger proBDNF/p75 ^NTR (p75 neurotrophin receptor) complex–mediated apoptosis in neonatal mice. Similarly, prolonged exposure to opioid receptor agonists early in life alters nerve growth factors in the immature brain.

Degeneration of Mitochondria

Ultrastructural morphologic abnormalities have been reported in mitochondria of pyramidal neurons in the subiculum of 7 day-old rats following 6 hours of isoflurane, nitrous oxide, and midazolam. A morphometric analysis demonstrated mitochondrial enlargement, impaired structural integrity, and decreased mitochondrial density, indicating protracted mitochondrial injury after drug exposure. Moreover, an ultrastructural examination with electron microscopy revealed increased autophagy, a form of cell death.

Abnormal Reentry Into Cell Cycle

Ketamine induces reentry of postmitotic neurons into the cell cycle in immature rats. This could be one trigger for apoptotic cell death, since postmitotic neurons lose the ability of neuronal progenitor cells to enter the cell cycle during proliferation and are committing programmed cell death when forced to reenter the cell cycle.

Destabilization of the Cytoskeleton

Structural integrity of the cellular cytoskeleton is critical for proper neuronal morphology and function. Actin is one of the major components of the cytoskeleton of all eukaryotic cells and participates in important cellular processes, including cell signaling, motility, and division. It is also essential for the formation of dendritic spines. Isoflurane has been found to depolymerize actin in neurons and astrocytes, initiating cytoskeletal destabilization, impairment of astrocyte morphologic differentiation and maturation, as well as neuronal apoptosis.

Effects on the Developing Spinal Cord

Most animal studies have focused on the effects of general anesthetics and sedatives on the developing brain. However, it is important to also consider the developmental impact of anesthetics on the spinal cord. One study observed increased neuroapoptosis in the lumbar spinal cord of 7-day-old rats after 6 hours of 0.75% isoflurane with 75% nitrous oxide. Increased neurodegeneration was also documented in brain and spinal cord after 6 hours of 1% isoflurane in a similar model, but not after 1 hour of anesthesia or following spinal administration of bupivacaine. Intrathecal ketamine can also cause neuroapoptosis in the developing spinal cord of 3-day-old rats, but not of 7-day-old rats. Preservative-free ketamine caused long-term alterations in spinal cord function and gait disturbances, whereas in a separate study, even high-dose intrathecal morphine produced no signs of spinal cord toxicity.

Putative Mechanisms for Neurotoxicity

The exact mechanisms that trigger the previously listed effects of anesthetics and sedatives on the immature brain and spinal cord remain unresolved. Elucidating these mechanisms will be critical in establishing the relevance of these findings for pediatric anesthesia and neonatal critical care medicine, as well as for developing mitigating interventions, if necessary. The current, overarching hypothesis is that anesthetics and sedatives interfere with normal GABA _A and NMDA receptor–mediated activity, which are the putative targets for unconsciousness, amnesia, and immobility, and at the same time are essential for mammalian CNS development. Administering GABA _A -receptor agonists and/or NMDA-receptor antagonists could potentially cause abnormal neuronal inhibition during a vulnerable period in neuronal development, triggering apoptosis in susceptible neurons, which in turn leads to neurocognitive impairment and decreased neuronal density into adulthood. Other lines of evidence suggest that the NMDA receptor–blocking properties of ketamine may upregulate NMDA receptors, rendering neurons more susceptible to excitotoxic injury caused by endogenous glutamate action on the increased number of receptors immediately after withdrawal of the anesthetic such as proposed for ketamine. However, several observations partly contradict both hypotheses; neuronal cell death has been reported during exposure to anesthetics and not only after their discontinuation. Moreover, several anesthetics with minimal NMDA-receptor interaction, such as propofol and barbiturates, have demonstrated robust neurotoxic properties, whereas the neurotoxic potency of the NMDA-antagonist xenon has been found to be limited, therefore casting doubt on receptor upregulation as the sole mechanism for anesthetic neurotoxicity. In terms of abnormal neuronal inhibition being the main trigger for apoptosis in developing neurons, GABA _A -receptor stimulation indeed decreases neuronal activity in the mature brain; however, it also causes excitation in developing neurons, thereby contradicting the inhibition hypothesis. In immature neurons, intracellular chloride (Cl ⁻ ) concentration is high; thus GABA-induced opening of Cl ⁻ channels allows this anion to exit the cell, leading to membrane depolarization. On the other hand, the intracellular Cl ⁻ concentration is low in mature neurons. When anesthetics open Cl ⁻ channels in mature neurons, ions enter the cell, thereby hyperpolarizing the membrane. This reversal of the cellular Cl ⁻ gradient occurs as a result of a switch from the immature Na ⁺ -K ⁺ -2Cl ⁻ cotransporter 1 (NKCC1) to the mature brain form, K ⁺ -Cl ⁻ cotransporter 2 (KCC2). Along these lines, studies in neonatal rats demonstrated excitatory properties in the brain and episodes of epileptic seizures during sevoflurane anesthesia. Isoflurane has also been shown to cause an excessive release of Ca ²⁺ from the endoplasmic reticulum via overactivation of inositol 1,4,5-trisphosphate receptors (InsP3Rs) in neonatal rats in vivo and in vitro. A similar mechanism may be linked to the production of Alzheimer-associated increases in β-amyloid protein levels after anesthesia. Although xenon and hypothermia cause neuronal inhibition, they do not exacerbate isoflurane-induced neuronal cell death as expected by the cumulative inhibition of neurons, but rather significantly reduce it.

Importantly, evidence indicates that equianesthetic concentrations of the three contemporary inhalational anesthetics cause similar degrees of neuroapoptosis, suggesting that it is the anesthetic depth and not the specific doses or end-tidal concentrations of the anesthetics that determines cytotoxic potency. However, other studies have failed to link the anesthetic and the apoptotic mechanisms. Specifically, although racemic ketamine and ( S )-ketamine both elicit their anesthetic effects via NMDA-receptor blockade, ( S )-ketamine induced up to 80% less cell death in vitro compared with equipotent doses of racemic ketamine. Moreover, concomitant administration of the GABA _A -receptor antagonist gabazine failed to attenuate neuroapoptosis induced by the GABA agonist isoflurane, whereas administration of the α ₂ -agonist dexmedetomidine did. Decreases in anesthetic-induced neuronal activity may therefore be less important than the disruption of the neuronal balance of excitation and inhibition, as demonstrated by studies that examined anesthesia-induced dendritic morphologic changes in mice. In a mechanistic study of brain development, simultaneous blockade of excitatory and inhibitory activity with tetrodotoxin did not lead to structural changes during synaptogenesis that would have been expected from a causative relationship between neuronal inhibition and structural abnormalities; the administration of either GABA _A -agonistic or NMDA-antagonistic compounds alone did alter synaptogenesis.

It is not entirely clear at this time whether cytotoxicity is a direct effect of the anesthetic itself, of any anesthetic by-products, or if it is related to physiologic derangements observed during anesthesia in small rodents. Hypercarbia can trigger widespread neuroapoptosis, even in unanesthetized neonatal rats exposed to increased partial pressures of carbon dioxide. Whereas apoptotic cell death was quantitatively indistinguishable from neurodegeneration in isoflurane-treated litter mates, which were also hypercarbic; neurocognitive impairment in adults was observed only in the isoflurane-treated animals. However, widespread apoptotic neurodegeneration observed in anesthetized nonhuman primates, where carbon dioxide tensions were controlled by tracheal intubation and ventilation, suggests that metabolic derangements may not be sufficient to explain the structural abnormalities observed in immature animal species. Lastly, experimental models of neurodegeneration have implicated reentry of postmitotic neurons into the cell cycle, leading to cell death. Ketamine exposure has been found to induce aberrant cell cycle reentry, leading to apoptotic cell death in the developing rat brain. However, a causative link between neuronal degeneration immediately after exposure and subsequent cognitive abnormalities observed into adulthood have yet to be firmly established.

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