**1. Introduction**

Studies performed in a variety of animal models including mice and rhesus monkeys have found that prolonged exposure of developing animals to most inhalational and intravenous anesthetic agents including sevoflurane, isoflurane, ketamine, propofol and anesthetic combinations induce neuroapoptosis [1–3]. The period of rapid synaptogenesis has been linked to the time of greatest vulnerability in developing brains to anesthetic-induced neurotoxicity [4–6]. The period of rapid brain growth in mice peaks at roughly 7 days after birth while this time frame in humans ranges from late in pregnancy until the 2nd or 3rd year of life [7, 8]. The neuroapoptosis observed in the growing brains of the young animals was also found to be coupled to long-term memory and learning deficits. For example, Shen and colleagues noted significant deficiencies in both memory and spatial learning in postnatal day 3 Sprague–Dawley rats following exposure to 1% sevoflurane. Their studies relied on the Morris water maze test to assess spatial learning/memory in the animals. Additionally, they found that these effects were both exposure number and dose dependent. They also observed that 7-week-old (adult) rats were insensitive to sevoflurane exposure with both the control and sevoflurane exposed animals displaying comparable results in the Water maze test [3]. The results of this study confirm that vulnerability to anesthetics is confined to an early period in development.

While many anesthetic agents are used clinically in the pediatric population, we choose to study the effects of the widely used anesthetic ketamine on our model of hESC-derived neurons. Ketamine is an N-methyl-D-aspartate-receptor antagonist and provides pain relief and sedation in children undergoing a variety of procedures. Additionally, ketamine is a drug that is often abused and abuse of ketamine during pregnancy is of concern for the developing brain of the fetus [19]. Despite the many studies implicating the negative effects of anesthetic agents on developing brains, the mechanisms behind the toxicity remain largely unknown. Possible roles of neuroinflammation, reactive oxygen species production, epigenetic changes, and calcium signaling in the mechanism of anesthetic-induced neurotoxicity have all been suggested [20–23]. However, considerable work remains to fully elucidate the mechanisms behind the neuronal toxicity. In this study, we investigated the effect of ketamine on the cell viability, intracellular calcium level, mitochondrial signaling, and microRNA profile using

Ketamine Induces Neuroapoptosis in Stem Cell–Derived Developing Human Neurons Possibly…

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Proper maintenance of intracellular calcium levels is critical for nearly every cellular process. In neurons, calcium is involved in the regulation of electrical activity, cell growth, metabolic activity, and many other processes [24]. Several studies have suggested that aberrant intracellular calcium level in neurons plays a critical role in the neuronal degeneration observed in many different neurological disorders [25]. The dysregulated intracellular calcium might induce the neurotoxicity through mitochondrial signaling. Mitochondria are highly dynamic organelles that undergo continuous cycles of fusion and fission in order to maintain cellular homeostasis. Mitochondrial fusion and fission result in a change in mitochondrial shape: either elongated, tubular, interconnected mitochondrial networks, or fragmented and discontinuous mitochondria, respectively. Unbalanced fission-fusion can lead to various pathological processes including neurodegeneration [26–29]. Shifting the balance towards fission has been associated with neuronal death in age-related neurodegenerative disease and brain injury [30, 31]. Inhibition of mitochondrial fission attenuated glutamate-induced neuronal death [31], translating to an increase in cell survival in the presence of oxidative stress [32]. Recent studies from Dr. Jevtovic's groups have shown that mitochondrial fission plays an important role in anesthetic neurotoxicity. The increased mitochondrial fission was found in neonatal rat brains after a sedative dose of midazolam followed by combined nitrous oxide

MicroRNAs are small, non-coding RNAs that function as negative regulators of gene expression. MicroRNAs are transcribed in hairpin structures in the nucleus by RNA polymerase II and the pri-miRNA produced is processed by the enzyme Drosha to cleave off a single hairpin loop. This forms the pre-miRNA which is exported to the cytoplasm. Once in the cytoplasm, the pre-miRNA is further processed to remove the hairpin loop and forms the mature microRNA strand. The mature microRNA strand then incorporates into an RNA-induced silencing complex where it can act to promote silencing of its target genes [34, 35]. Over 1000 microRNAs have been discovered in humans and they have been shown to play an important role in nearly every cellular process. Dysregulation of microRNAs has also been linked to several diseases including certain cancers [36]. The role of microRNAs in anesthetic-induced

the hESC-derived developing human neuron model.

and isoflurane anesthesia for 6 h [33].

neurotoxicity is just beginning to be studied.

The many animal studies have raised concerns regarding the applicability of the models to humans. Of particular concern are the many rodent studies in which hemodynamic properties were not properly controlled. While extensive studies in rhesus macaques, a non-human primate model have been performed [9–14], a suitable human model in the study of anesthetic-induced neurotoxicity is currently lacking and exposure of young children to anesthetics for study purposes would be unethical. Several human epidemiologic studies have been performed and many have suggested a link between anesthetic exposure in young children and learning and behavioral abnormalities when compared to their unexposed counterparts [15–17]. However, the human epidemiologic studies face many confounding variables that are often difficult to properly account for, leaving the results of these studies uncertain. While many of these studies are still ongoing, it is imperative to develop a proper human model to assess the effects of anesthetics in young children. Our study aimed to identify an appropriate human model to assess the effects of anesthetics on the developing human brain and also to dissect out the mechanisms behind the toxicity.

Human embryonic stem cells (hESCs) are derived from the inner cell mass of a human blastocyst. In 1998 James Thompson and colleagues at the University of Wisconsin-Madison developed a technique to isolate and culture hESCs *in vitro* [18]. The formative work of this group opened up the possibility for mechanistic-based studies using a human cell line, effectively eliminating the potential concerns regarding the relevancy of studies using animal models to humans. hESCs are immature, divide indefinitely and are capable of generating cells from all three germ layers which makes these cells an ideal model of early human neurons once differentiated. Using hESC-derived neurons has provided us with a reasonable human model by which to study anesthetic-induced developmental neurotoxicity.

While many anesthetic agents are used clinically in the pediatric population, we choose to study the effects of the widely used anesthetic ketamine on our model of hESC-derived neurons. Ketamine is an N-methyl-D-aspartate-receptor antagonist and provides pain relief and sedation in children undergoing a variety of procedures. Additionally, ketamine is a drug that is often abused and abuse of ketamine during pregnancy is of concern for the developing brain of the fetus [19]. Despite the many studies implicating the negative effects of anesthetic agents on developing brains, the mechanisms behind the toxicity remain largely unknown. Possible roles of neuroinflammation, reactive oxygen species production, epigenetic changes, and calcium signaling in the mechanism of anesthetic-induced neurotoxicity have all been suggested [20–23]. However, considerable work remains to fully elucidate the mechanisms behind the neuronal toxicity. In this study, we investigated the effect of ketamine on the cell viability, intracellular calcium level, mitochondrial signaling, and microRNA profile using the hESC-derived developing human neuron model.

**1. Introduction**

422 Mitochondrial Diseases

in development.

dissect out the mechanisms behind the toxicity.

which to study anesthetic-induced developmental neurotoxicity.

Studies performed in a variety of animal models including mice and rhesus monkeys have found that prolonged exposure of developing animals to most inhalational and intravenous anesthetic agents including sevoflurane, isoflurane, ketamine, propofol and anesthetic combinations induce neuroapoptosis [1–3]. The period of rapid synaptogenesis has been linked to the time of greatest vulnerability in developing brains to anesthetic-induced neurotoxicity [4–6]. The period of rapid brain growth in mice peaks at roughly 7 days after birth while this time frame in humans ranges from late in pregnancy until the 2nd or 3rd year of life [7, 8]. The neuroapoptosis observed in the growing brains of the young animals was also found to be coupled to long-term memory and learning deficits. For example, Shen and colleagues noted significant deficiencies in both memory and spatial learning in postnatal day 3 Sprague–Dawley rats following exposure to 1% sevoflurane. Their studies relied on the Morris water maze test to assess spatial learning/memory in the animals. Additionally, they found that these effects were both exposure number and dose dependent. They also observed that 7-week-old (adult) rats were insensitive to sevoflurane exposure with both the control and sevoflurane exposed animals displaying comparable results in the Water maze test [3]. The results of this study confirm that vulnerability to anesthetics is confined to an early period

The many animal studies have raised concerns regarding the applicability of the models to humans. Of particular concern are the many rodent studies in which hemodynamic properties were not properly controlled. While extensive studies in rhesus macaques, a non-human primate model have been performed [9–14], a suitable human model in the study of anesthetic-induced neurotoxicity is currently lacking and exposure of young children to anesthetics for study purposes would be unethical. Several human epidemiologic studies have been performed and many have suggested a link between anesthetic exposure in young children and learning and behavioral abnormalities when compared to their unexposed counterparts [15–17]. However, the human epidemiologic studies face many confounding variables that are often difficult to properly account for, leaving the results of these studies uncertain. While many of these studies are still ongoing, it is imperative to develop a proper human model to assess the effects of anesthetics in young children. Our study aimed to identify an appropriate human model to assess the effects of anesthetics on the developing human brain and also to

Human embryonic stem cells (hESCs) are derived from the inner cell mass of a human blastocyst. In 1998 James Thompson and colleagues at the University of Wisconsin-Madison developed a technique to isolate and culture hESCs *in vitro* [18]. The formative work of this group opened up the possibility for mechanistic-based studies using a human cell line, effectively eliminating the potential concerns regarding the relevancy of studies using animal models to humans. hESCs are immature, divide indefinitely and are capable of generating cells from all three germ layers which makes these cells an ideal model of early human neurons once differentiated. Using hESC-derived neurons has provided us with a reasonable human model by Proper maintenance of intracellular calcium levels is critical for nearly every cellular process. In neurons, calcium is involved in the regulation of electrical activity, cell growth, metabolic activity, and many other processes [24]. Several studies have suggested that aberrant intracellular calcium level in neurons plays a critical role in the neuronal degeneration observed in many different neurological disorders [25]. The dysregulated intracellular calcium might induce the neurotoxicity through mitochondrial signaling. Mitochondria are highly dynamic organelles that undergo continuous cycles of fusion and fission in order to maintain cellular homeostasis. Mitochondrial fusion and fission result in a change in mitochondrial shape: either elongated, tubular, interconnected mitochondrial networks, or fragmented and discontinuous mitochondria, respectively. Unbalanced fission-fusion can lead to various pathological processes including neurodegeneration [26–29]. Shifting the balance towards fission has been associated with neuronal death in age-related neurodegenerative disease and brain injury [30, 31]. Inhibition of mitochondrial fission attenuated glutamate-induced neuronal death [31], translating to an increase in cell survival in the presence of oxidative stress [32]. Recent studies from Dr. Jevtovic's groups have shown that mitochondrial fission plays an important role in anesthetic neurotoxicity. The increased mitochondrial fission was found in neonatal rat brains after a sedative dose of midazolam followed by combined nitrous oxide and isoflurane anesthesia for 6 h [33].

MicroRNAs are small, non-coding RNAs that function as negative regulators of gene expression. MicroRNAs are transcribed in hairpin structures in the nucleus by RNA polymerase II and the pri-miRNA produced is processed by the enzyme Drosha to cleave off a single hairpin loop. This forms the pre-miRNA which is exported to the cytoplasm. Once in the cytoplasm, the pre-miRNA is further processed to remove the hairpin loop and forms the mature microRNA strand. The mature microRNA strand then incorporates into an RNA-induced silencing complex where it can act to promote silencing of its target genes [34, 35]. Over 1000 microRNAs have been discovered in humans and they have been shown to play an important role in nearly every cellular process. Dysregulation of microRNAs has also been linked to several diseases including certain cancers [36]. The role of microRNAs in anesthetic-induced neurotoxicity is just beginning to be studied.

This chapter will outline: (1) the use of hESCs as a model system to study anesthetic-induced neurotoxicity in the developing human brain, (2) the protocols involved in dissecting the mechanisms behind anesthetic-induced developmental neurotoxicity. We have focused on the role of deregulated intracellular calcium, mitochondrial signaling (e.g., mitochondrial membrane potential, cytochrome c release from mitochondria into cytosol, and mitochondrial fission), and microR-NAs, and (3) a brief discussion on the possible role of intracellular calcium levels, mitochondrial signaling, and altered microRNA profiles in ketamine-induced developmental neurotoxicity.

**2.2. Neuronal characterization by immunofluorescence staining**

ketamine or neurobasal media as a vehicle-control in a 5% CO2

**2.4. Assessment of cell death by TUNEL staining**

Melville, NY).

incubator.

in a field.

**2.5. Calcium imaging**

**2.3. Ketamine exposure**

hESC-derived neurons cultured on matrigel-coated, glass coverslips for 2 weeks were fixed for 30 min in 1% paraformaldehyde at room temperature. The cells were then washed with phosphate-buffered saline (PBS). Next, the cells were incubated for 15 min in 0.5% Triton X-100 (Sigma-Aldrich) in PBS. The cells were washed once again with PBS and blocked for 20 min with 10% donkey serum at room temperature. The cells were then incubated with the primary antibodies [microtubule-associated protein 2 (MAP2), β-tubulin III or doublecortin (Abcam, Cambridge, MA)] for 1 h in a humidified, 37°C incubator. The cells were washed with PBS and incubated for 45 min with Alexa Fluor 488 or 594 donkey anti-mouse or rabbit immunoglobulin G (Invitrogen) secondary antibodies at 37°C. The cell nuclei were stained with Hoechst 33342 (Invitrogen). The coverslips were then mounted onto glass slides and imaged using a laser-scanning confocal microscope (Nikon Eclipse TE2000-U, Nikon Inc.,

Ketamine Induces Neuroapoptosis in Stem Cell–Derived Developing Human Neurons Possibly…

While the brain concentration of ketamine in humans during the induction and maintenance of anesthesia is not well understood, reports have found that the peak blood levels of ketamine could be as high as 103 μM. The levels required to maintain anesthesia are typically in the range of 10–20 μM. hESC-derived neurons were exposed to 6 h of 20 μM

conditions only for the microRNA studies. For all remaining studies, the cells were exposed to 24 h of 20 or 100 μM ketamine or neurobasal media as the vehicle control in the same

Apoptosis of the hESC-derived neurons was assessed using a cell death detection kit (Roche Applied Bio Sciences, Indianapolis, IN) based on terminal deoxynucleotidyl transferasemediated deoxyuridine triphosphate in situ nick end labeling (TUNEL) following directions provided by the manufacturer. This kit identifies single or double-stranded DNA breaks by labeling the free 3′-OH termini with altered nucleotides in a reaction with terminal deoxynucleotidyl transferase (TdT). Cells cultured on glass coverslips were exposed to ketamine or control conditions, rinsed and fixed with 1% paraformaldehyde. DNA fragmentation was analyzed using TdT, which incorporates into sites of DNA breaks. The nuclei were stained with Hoechst 33342 and the cells were imaged using the confocal microscope. Apoptosis/ necrosis was quantified by assessing the ratio of TUNEL-positive nuclei to total cell nuclei

Neurons were plated on matrigel-coated glass coverslips for the calcium imaging studies. Intracellular calcium was assayed using Fluo-4AM (Thermo Fisher Scientific). Neurons were

incubator with normoxic

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