1. Anesthesia and the Developing Brain
Yaacov Gozal, M.D.
Associate Professor of Anesthesiology
Hebrew University-Hadassah Medical School
Chair, Dept. of Anesthesiology, Perioperative Medicine and Pain Treatment
Director, Operating Rooms
Shaare Zedek Medical Center
Jerusalem, Israel

2. This is part of the hospital
This is part of the hospital. The three lower levels were built underground. Even in times of military attack, the hospital is able to function since vital areas such as the operating rooms, the emergency room, the pharmacy and the medical and the sterile supply are below ground.

3. I live in Jerusalem, a city which combines the old and the new
I live in Jerusalem, a city which combines the old and the new. King David established it as his capital more than 3000 years ago.
We can see the Old City with the Dome of the Rock, the Western Wall, and the Jerusalem’s bridge of the Chords. The bridge curves into Jerusalem’s western entrance. It was designed by the famous spanish architect and engineer Santiago Calatrava. It is really a new addition to a city of ancient symbols.

4. Introduction Anesthesia: safe intervention
For more than 150 yrs: brain returns to the same state as before the anesthetic
Recent yrs: neurotoxic effects of anesthetics
Long lasting
Cognitive defects
In general, anesthesia is considered a safe intervention, allowing complex surgical procedures in seriously ill patients of all groups.
Providing general anesthesia to young children has become so safe that families and caregivers have little to worry about in terms of the immediate risks of the anesthetic and the immediate postoperative period.
For more than 150 years of anesthetic practice, it was believed that as a general anesthetic wears off, the brain would return to the same state as before the anesthetic.
In recent years, animal experiments have provided evidence of the neurotoxic effects of anesthetics, resulting in long lasting cognitive deficits. These toxic effects are mainly detectable at the extremes of ages, both in preterm babies and newborns, but also in aged animals.
In this lecture, we will try to review the current literature relating to the animal experiments, the possible transfer of the experimental data and the current clinical data.

5. Jevtovic-Todorovic V, et al. J Neurosci 2003; 23:876-882
Animal Studies
Combined anesthetic (Midazolam, N2O, Isoflurane)
7-day-old rats for 6 hours
Impairment of long-term potentiation in the hippocampus
Progressive deficit in spatial recognition tasks: 4 weeks and 4.5 months after anesthesia!!!
Jevtovic-Todorovic V, et al. J Neurosci 2003; 23:
In 2003, Vesna Jevtovic-Todorovic and colleagues from Charlottesville, Virginia published their seminal work in the Journal of Neurosciences.
They found that a combined anesthetic (midazolam, nitrous oxide, and Isoflurane) administered to 7-day-old rats for 6 hours killed neurons in the developing brain and caused long-term impairment of brain function.
They showed that long-term potentiation in the hippocampus was impaired in anesthetized rats.
We have to remember that the hippocampus is a brain structure important for learning and memory.
More importantly, these authors demonstrated a progressive deficit in spatial recognition tasks administered both 4 weeks and 4.5 months after anesthesia.

6. Factors Affecting the Toxicity of Anesthetics
Timing of Exposure:
Neurons: especially vulnerable during the “brain growth spurt”
Varies by species:
Rats: 7th to 10th postnatal day
Rhesus monkeys: 5th to 16th postnatal day
Humans: last trimester until the 3rd year of life
In rhesus monkeys:
Ketamine for 24 hrs at the end of pregnancy: apoptosis in the fetus
Same in 5-day-old
On day 35: no apoptosis
Slikker W Jr, et al. Toxilogical Sciences 2007; 98:
Three main factors affect the toxicity of anesthetics:
The first is the timing of exposure. The neurotoxicity of anesthetics is highly dependent on the stage of development.
Neurons seem to be especially vulnerable during the “brain growth spurt” (the period of intensive development and differentiation).
The timing of this varies by species: in rats, it lasts from the 7th to the 10th (up to 17th) postnatal day. The rhesus monkeys from the 5th to the 16th postnatal day. In the human brain, the period is thought to last from the last trimester until the 3rd year of life.
For example, in rhesus monkeys, a connection between the age of the animals at the time of anesthesia and neuronal damage has been demonstrated.
In their work published in Toxological Sciences, William Slikker from The National Center for Toxicological Research in Silverspring, Maryland showed that anesthesia with ketamine for 24 hrs at the end of pregnancy resulted in neuronal apoptosis (programmed cell death) in the fetus as detected by immunocytochemistery. Apoptosis was also detectable when 5-day-old rhesus monkeys received ketamine for 24 hrs.
In contrast, ketamine anesthesia after the termination of brain development on day 35 after birth did not induce apoptosis.

7. Factors Affecting the Toxicity of Anesthetics
Frequency and Duration of Anesthetic Exposure:
Apoptosis increases as a function of duration or repetition of the anesthetic
Single dose of ketamine in 7-day-old rats: no apoptosis
Repeated administration or for more than 6 hrs: increase in apoptosis
Pediatric Anesthesia 2002; 12:
1 hr Isoflurane: no neurotoxicity
2 hr application: increase in neuronal apoptosis
Anesthesiology 2009; 110:
Ketamine for 3 hrs in in 5-6-day-old monkeys: no apoptosis
5, 9, 24 hr application: significant apoptosis
Anesthesiology 2012; 116:
The second factor is the frequency and duration of anesthetic exposure. Apoptosis increases as a function of duration or repetition of the anesthetic.
For instance, a single dose of ketamine given to 7-day-old rats did not induce apoptosis, In contrast, repeated administration of the drug or anesthesia given for more than 6 hrs resulted in a significant increase in neuronal apoptosis.
Likewise, a 1 hr anesthetic with Isoflurane was not neurotoxic, whereas a 2 hr application was associated with an increase in neuronal apoptosis.
This is similar to findings in5-6 day-old rhesus monkeys where anesthesia with ketamine for 3 hrs did not induce apoptosis, but significant apoptotic neurons were detectable after administration for 5, 9 or 24 hrs.

8. Factors Affecting the Toxicity of Anesthetics
Dose Dependency:
Increasing the dose of anesthetic increases:
The number of apoptotic neurons
The degree of developmental impairment
The degree of cellular differentiation and synaptogenesis
Anesth Analg 2011;113:
Anesthesiology 2005; 102:
Thirdly, numerous animal experiments reveal a clear dose-dependent element to toxicity.
Increasing the dose of anesthetic increases the number of apoptotic neurons, the degree of developmental impairment, cellular differentiation and synaptogenesis

9. Possible Mechanisms of Anesthetic-Induced Neurodegeneration
Apoptosis:
Intrinsic Pathway:
Initiated in response to signals from within the cell
Results in the release of pro-apoptotic proteins from the mitochondrium
Extrinsic Pathway: activated via “death” receptors (TNF receptor family)
Nature 1996; 384:
Neuroscience 2005;135:
Anesth Analg 2008; 106:
Neuroscience Letters 2008; 447:
Anesthesiology 2010; 112:
Now, we will rapidly discuss the possible mechanisms of anesthetic-induced neurodegeneration
Animal experiments have revealed that anesthetics induce neurotoxic damage by a number of mechanisms
Anesthetic might induce apoptosis but they also influence neuronal development, differentiation and neuronal plasticity
In vitro and In Vivo studies have revealed that anesthetics induce apoptosis via 2 possible pathways termed the intrinsic and extrinsic pathways
The intrinsic pathway is iniatiated in response to signals from within the cell and results in the release of pro-apoptotic proteins from the mitochondria
The extrinsic pathway is activated via “death” receptors (tumor necrosis factor (TNF) receptor family)

10. Possible Mechanisms of Anesthetic-Induced Neurodegeneration
Influence on Neuronal Differentiation, Synaptogenesis and Network Formation:
Isoflurane for 35 min on 4 consecutive days
Young mice and rats (14th day), adult rats (60th day)
Results:
Impaired memory performance in the young animals
More pronounced as the animals grew older
Reduction in hippocampal stem cells
Persistently reduced neurogenesis
Unaffected adult animals
Zhu C, et al. J Cereb Blood Flow Met 2010; 30:
Anesthetics also influence neuronal differentiation, synaptogenesis and network formation on various levels.
For example, Changlian Zhu from the University of Gothenburg in Sweden showed that the administration of isoflurane for 35 minutes on 4 consecutive days in young mice and rats (14th day) and in adult rats (60th day) impaired memory performance in the young animals. These effects became more pronounced as the animals grew older.
The learning disabilities were accompanied by a reduction in hippocampal stem cells and persistently reduced neurogenesis. Adult animals were unaffected.

11. Possible Mechanisms of Anesthetic-Induced Neurodegeneration
Activation of Reactive Oxygen Species:
By propofol, sevoflurane or isoflurane
Mitochondrial dysfunction
Energy breakdown of the neuron
Anesthesiology 2011; 115:
Neurobiology of Disease 2012; 45:
Another possible mechanism is the activation of reactive oxygen species by anesthetics like propofol, sevoflurane or isoflurane resulting in mitochondrial dysfunction and in an energy breakdown of the neuron

12. Possible Mechanisms of Anesthetic-Induced Neurodegeneration
Anesthetic-Induced Neuroinflammation:
Revealed as a further possible mechanism for cognitive impairment in newborn mice
Anesthesiology 2013; 118:
Recently, anesthetic-induced neuroinflammation has been revealed as a further possible mechanism for cognitive impairment in newborn mice

13. Possible Mechanisms of Anesthetic-Induced Neurotoxicity in the Immature Brain
Anaesthesia 2014; 69:
BDNF; brain-derived neurotrophic factor

14. Translating Animal Data to Clinical Settings
Areas of uncertainty in translation to humans:
The exact period of vulnerability
The dose required to cause injury
The clinical outcome likely to be seen
The role of anesthesia among the other factors
Dismissing the laboratory findings: no more logical than blinding accepting them
Changes seen in the laboratory are real
It is particularly difficult to link the laboratory findings to clinical practice
Areas of uncertainty in translation to humans are:
-the exact period of vulnerability
-the dose required to cause injury (as we saw, animals require high doses of IV anesthetics, and most studies have exposed animals to long periods of anesthesia
-the clinical outcome likely to be seen
-the role of anesthesia among the other factors
But, totally dismissing the laboratory findings because of theoretical limitations in translation is no more logical than blindly accepting them
The changes seen in laboratory work are indeed real. There is no reason to think that some degree of neuronal apoptosis would not be triggered by exposure to anesthetics in humans.

15. Clinical Data
Essentially based on retrospective and observational studies
The retrospective methodology presents limitations:
Initially sampled for different purposes and entities
Anesthetic agents may be obsolete
Study samples: representative of a fraction of the population
No way to control the indications for surgery
No way to control outcomes from the surgery itself
Available evidence remains mixed
As of today, clinical evidence regarding negative long-term cognitive and behavioral effects of anesthetics in young children is based on retrospective and observational studies.
Despite very careful analysis, the retrospective methodology with several potential confounders presents limitations:
Specific concerns include that the analyzed data were initially sampled for different purposes and entities (such as school administration, health insurance,…), the anesthetic agents at the time of exposure may not be in use today, the study samples are representative of only a fraction of the population (e.g. Medicare database), or that there is no way to control for factors leading to the need of surgery or outcomes from the surgery itself.
The currently available evidence remains mixed. Some studies suggest significant cognitive and behavioral deficits even with a single anesthesia exposure, whereas others saw no such effects after a single short exposure. In contrast, multiple or longer exposures may be injurious enough such that a long-term functional deficit results.

16. Clinical Retrospective Studies Ing C, et al
Clinical Retrospective Studies Ing C, et al. Pediatrics 2012; 130:e
Originally, investigation of long-term effects of perinatal U/S exposure
2868 children
11% exposed to anesthesia before the age of 3
Close early follow-up:1781 children
206: single exposure
52: multiple exposures
1523: unexposed children
Increased risk for disabilities in receptive language (tested at age 10)
75% increased risk of disability in abstract reasoning
Behavioral and motor testing did not differ between groups
Now, I would like to review with you the key findings of recent retrospective clinical studies.
The first observation was published in 2012 in the journal Pediatrics by a team of collaborators from Australia and the U.S.A.
They analyzed data that were prospectively obtained originally to investigate long-term effects of perinatal U/S exposure in Perth, Australia.
In this database, there were 2868 children born from 1989 to % of these children were exposed to anesthesia at a young age (<3). They also identified single versus multiple exposures before age 3 in a subset of patients with very close early follow up. It included 1781 children:
-206 single exposure
-52 multiple exposures
-1523 unexposed children
They found an increased risk for disabilities in receptive language, when tested at age 10 that was 2.4 fold (single exposure) or 3.5 fold (multiple exposures) higher when compared with unexposed children, even after adjusting for confounders.
Additionally, they detected a 75% increased risk of disability in abstract reasoning in single-exposed versus non exposed children
Behavioral and motor testing did not differ between groups.

17. Clinical Retrospective Studies Sprung J, et al
Clinical Retrospective Studies Sprung J, et al. Mayo Clin Proc 2012; 87:
Birth-cohort from Minnesota
2-fold higher risk for ADHD by the age of 19
If child exposed to 2 or more anesthetics before age 2
No correlation between ADHD and a single exposure
Association between anesthesia exposure and cognitive or behavioral issues: likely
Effects: dose-related
The second one was published by J Sprung from the Mayo Clinic in Rochester.
This study analyzed a birth-cohort from Minnesota. They observed a nearly 2-fold higher risk for a diagnosis of Attention Deficit Hyperactivity Disorder (ADHD) by age 19 when the child was exposed to 2 or more anesthetics before age 2.
In contrast, their data suggested no correlation between ADHD diagnosis and single exposure.
On the basis of these reports, it is reasonable to consider that an association between anesthesia exposure and cognitive or behavioral issues is likely, and that the effects are dose-related.
However, other retrospective studies were unable to find such an association…

18. Clinical Retrospective Studies Hansen TG, et al
Clinical Retrospective Studies Hansen TG, et al. Anesthesiology 2011; 114:
National cohort of Danish adolescents
Inguinal hernia repair at the age of 1 or less. N=2689
Compared to 14,575 matched controls
Same academic achievement scores
Confounders such as gender, birth weight, parental age and education were controlled for
The academic performance of a national cohort of Danish 15 to 16 yr-old adolescents (n=2689) who had undergone inguinal hernia repair between 1986 and 1990 at the age of 1 year or younger was compared with a random sample of 14,575 age-matched controls.
There was no evidence that the relatively brief general anesthetic (30-60 minutes) had affected academic achievement scores.
This when important confounder such as gender, birth weight, and parental age and education were controlled for.

19. Clinical Retrospective Studies Barteis M, et al
Clinical Retrospective Studies Barteis M, et al. Twin Res Hum Genet 2009; 12:
Data taken from the Young Netherland Twin Register
1143 pairs of monozygotic twins
Most pairs: both exposed or both not exposed to anesthesia
71 twin pairs discordant
Nationwide standardized test at age 12:
Academic performance similar
Teacher questionnaire:
Similar incidence of cognitive problems
Another trial was designed to test whether there was a causality between anesthesia administered at younger than 3 years and between 3 and 12 yrs and cognitive performance.
The data was taken from the Young Netherland Twin Register.
The authors studied 1143 pairs of monozygotic twins.
Most pairs of twins in this study consisted of twins who were either both exposed or both not exposed to anesthesia.
However, 71 twin pairs (15%) were discordant: one twin exposed, the other not exposed to anesthesia.
Academic performance on a nationwide standardized tests at age 12 was not different when comparing twins exposed to anesthesia to their unexposed twin sibling.
There was a similar incidence of cognitive problems, as assessed by a teacher questionnaire

20. Clinical Retrospective Studies
Cardiovascular, central nervous and respiratory systems: extremely sensitive and vulnerable to hemodynamic and metabolic changes
Outcome not chosen by the investigator
Do not provide the most meaningful measure of the cognitive or behavioral effect
Studies with negative results: broad measures of academic performance
Studies with positive results: individual tests of cognitive performance
Interpreting retrospective studies is also difficult.
It is well known that the cardiovascular, central nervous and respiratory systems are extremely sensitive and vulnerable to hemodynamic and metabolic changes.
Among the many methodological problems associated with human studies are the outcome measures available to the investigators. In retrospective studies, the outcome is not chosen by the investigators, and therefore may not provide the most meaningful measure of the cognitive or behavioral effect.
The majority of studies with negative results use broad measures of academic performance such as group tests of achievement and teacher-parent rating scales.
Studies using individual tests of cognitive performance have been uniformly positive, commonly in areas of speech and language

21. Conclusions Parents are aware!!
No available scientific evidence to change pediatric anesthesia practice
Not always possibility to postpone surgery or diagnostic test
New organization: “Strategies Mitigating Anesthesia-Related Neurotoxicity in Tots” (SmartTots)
Web site:
Supports several prospective clinical trials
The increasing public exposure to research in this area is making parents aware that anesthetic exposure could affect neurodevelopment in children.
As of today, NO scientific evidence is available to support a change in pediatric anesthesia practice.
Moreover, many diagnostic tests or surgical interventions that require anesthesia frequently cannot be postponed until a later development state.
An important step forward was the foundation of a new organization in 2010 that is solely dedicated to the cause of funding research to determine whether anesthesia impairs brain function in humans, what the specific deficit is, and how it can prevented and/or treated.
The SmartTots website ( remains an excellent resource for up-to-date information on this subject both for providers and families.
SmartTots currently supports several prospective clinical trials, including:

22. GAS Study (General Anesthesia and Spinal)
Children < 6 months
Inguinal hernia repair
General anesthesia or spinal anesthesia
Neurodevelopmental outcome and apnea
Preliminary results: 2015
This study compares the effects of general anesthesia or spinal anesthesia on neurodevelopmental outcome and apnea in infants undergoing inguinal hernia repair up to the age of 6 months.
Preliminary results are expected next year in 2015.

23. PANDA Study (Pediatric Anesthesia and NeuroDevelopmental Assessment)
Multicenter study
Age: up to 36 months
Inguinal hernia repair
Long-tem effects of anesthesia on cognitive function
This is a multicenter study that examines the long-term effects of anesthesia on cognitive function in children exposed to anesthesia for inguinal hernia up to the age of 36 months.

24. MASK Study (Mayo Safety in Kids)
Cohort study
Children in Rochester
Children: <3 years
One or more anesthetics
This is a collaborative cohort study involving researchers from the Mayo Clinic and the National Center for Toxicological Research in the USA.
Children in Rochester (Minnesota) who receive one or more anesthetics before the age of 3 years are being compared with children having no anesthetic exposure.
Results from these ongoing prospective clinical trials will help clarify whether anesthesia associated neurotoxicity affects the developing human brain and what the clinical relevance is.