**3.2. Ketamine-induced apoptosis**

TUNEL staining was used to assess cell death in hESC-derived neurons following ketamine exposure by labeling breaks in the DNA. The cells were exposed to 100 μM ketamine or control conditions for 24 h. The number of TUNEL-positive cells was significantly increased when compared to control treated cells following ketamine exposure (**Figure 2**). These findings confirm many of the previously published animal studies which have shown increased neuronal cell death following exposure of the developing brain to ketamine [39]. We focused on the investigation of the effect of ketamine on the intracellular calcium level, mitochondrial signaling, and microRNA expression in order to understand the mechanisms governing ketamine-induced cell death.

#### **3.3. Alterations in intracellular calcium levels**

exhibited a characteristic neuronal morphology with small cell bodies and long projections. The neurons also formed extensive, interconnected networks over time. The cells displayed a very distinct morphology at each stage of the differentiation protocol. The hESCs formed tight colonies when cultured on a feeder layer and the EBs formed three-dimensional aggregates when suspended in culture media. At the neural rosette stage, the NSCs formed tightly packed arrangements with a characteristic design and were bordered by several additional cell types. Once mechanically separated from the surrounding non-NSCs and digested, the NSCs separated from one another and spread out on the matrigel-coated dishes. At this point,

**Figure 1.** Differentiation protocol and confocal images of immunolabeled human embryonic stem cell (hESC)-derived neurons. (A) Neurons were derived from hESCs through a four step differentiation protocol. (B) Neurons were stained with antibodies against the neuron-specific proteins, microtubule-associated protein 2 (MAP2) and β-tubulin III to assess

the differentiation efficiency, and doublecortin to confirm the immaturity of the neurons [37].

The neurons were immunostained after 2 weeks in differentiation media and expressed the neuron-specific markers MAP2 and β-tubulin III and formed synapses as assessed by the positive staining of Synapsin I. Based upon the immunostaining, the differentiation protocol was 90–95% efficient in the generation of neurons. In an attempt to better gauge the maturity level of the hESC-derived neurons, the cells were also immunostained for doublecortin, a marker of immature/migrating neurons. In assessing the staining results, most of the neurons in culture (90–95%) were positive for this marker of immature neurons (**Figure 1B**) which suggests that this model system is a valuable representation of developing human

the cells proliferated extensively.

neurons.

428 Mitochondrial Diseases

Following exposure of 2-week-old hESC-derived neurons to 100 μM ketamine or control conditions for 24 h, the intracellular calcium levels in the cells were assessed using Fluo-4AM fluorescence. The intracellular calcium levels were significantly elevated in the ketaminetreated cells when compared to control treatment (**Figure 3**). Calcium is a critical ion in the body and is fundamental in proper neuronal functioning. In neurons, calcium is crucial in synaptic activity and plasticity, cell signaling, neurotransmitter release and is involved in nearly every aspect of the cell cycle [24]. The careful balance of intracellular calcium levels is crucial to cell survival. Calcium homeostasis dysregulation, in particular calcium overload in the cell, has been linked to many different neurodegenerative diseases [25]. The findings from this study suggest that disrupted intracellular calcium homeostasis may also be linked to ketamine-induced cell death in developing neurons which could prove to be a novel therapeutic target.

#### **3.4. Ketamine induces neuronal apoptosis via mitochondrial pathway**

The mitochondria are extremely important organelles involved in many cellular processes including energy production, cell signaling, and apoptosis [40–46]. Given that ketamine may

**Figure 2.** Exposure to 100 μM ketamine for 24 h induced significant cell death in the hESC-derived neurons. Ketamine induced an increase in the number of TUNEL-positive cells indicating significant cell death when compared to controltreated cells. \*P < 0.05 vs. control. n = 3 for each group.

**Figure 3.** Ketamine exposure for 24 h induced elevated cytosolic Ca2+ [(Ca2+) c ] of neurons. (A) Ketamine increased (Ca2+) c . Neurons were loaded with the Ca2+ indicator Fluo-4AM. The fluorescence images of free cytosolic Ca2+ in the 2-week-old differentiated neurons are shown. (B) Ketamine (100 μM, 24 h) significantly increased (Ca2+)c (\*\*P < 0.01, n = 3).

cause mitochondrial damage, we measured ΔΨm and distribution of cytochrome c in the cells. Treatment of hESC-derived neurons with 100 μM ketamine for 24 h significantly decreased ΔΨm (**Figure 4A**). In order to investigate the distribution of cytochrome c within the cells, the neurons were transduced with the virus CellLight™ mitochondria-GFP (green). GFP and TMRE signals were colocalized in the cells (**Figure 4B**), confirming that successfully labeling of mitochondria with GFP. GFP-positive cells reached 40% (**Figure 4C**). The distribution of cytochrome c in mitochondria and cytosol was examined using immunofluorescence staining. The results showed that cytochrome c was located within mitochondria in the control culture (**Figure 4D**). However, in the ketamine-treated cells, cytochrome c was released from the mitochondria into cytosol.

Neuroapoptosis is a commonly recognized harmful effect by anesthetics with mechanisms that are not fully understood. Apoptosis is a programmed cell death. Cytochrome c release from mitochondria precededing the loss of ∆Ψm is a key event in initiating mitochondria-involved apoptosis [47], eventually leading to the typical alterations related to apoptosis such as DNA fragmentation in cell nuclei [48, 49]. In this study, following ketamine exposure, there was a significantly increase in the TUNEL-positive apoptotic cells (**Figure 2**). Ketamine-induced cell death was accompanied by the significant decrease in ∆Ψm and the increased cytochrome c release from mitochondria into cytosol (**Figure 4**). These data were in agreement with those previously reported by others [48, 50–52], suggesting that ketamine induces human neuron to undergo mitochondria-mediated apoptosis pathway.

To maintain proper cellular function, the mitochondria continuously undergo cycles of fusion and fission. Unbalanced fusion/fission, particularly excessive fission/fragmentation can lead to various pathological conditions including neurodegeneration [53]. To assess mitochondrial fission shape in the hESC-derived neurons following exposure to ketamine, electron microscopy was used. The neurons were exposed to either 20 μM or 100 μM ketamine or control conditions for 24 h. The cells were then prepared and imaged on an electron microscope. The mitochondria appeared considerably fragmented in the ketamine- treated neurons when compared to control cells (**Figure 5**). Increased mitochondrial fission has been linked to the propofol-induced cell death in hESC-derived neurons as previously published by our group [54]. Our findings suggest that ketamine exposure results in an increase of mitochondrial fission within the developing neurons, which may contribute to the increased cell death observed in the ketamine-treated group.

**Figure4.** Ketamine decreases mitochondrial membrane potential (ΔΨm) and increases cytochrome c release from mitochondria into cytosol. (A) ΔΨm assay. Ketamine (100 Μm) treatment for 24 h decreased ΔΨm (\*P < 0.05 vs. control, n = 3). (B) Labeling mitochondria of neurons with CellLight™ mitochondria-green fluorescence protein (GFP) reagent. The GFP-positive cells were loaded with TMRE. GFP expression in mitochondria was confirmed by the colocalization of tetramethylrhodamine ethyl ester (TMRE/mitochondrial probe) and GFP signals within the cells. (C) Representative fluorescent images of the neurons transduced with CellLight™ mitochondria-GFP reagent. Blue are cell nuclei. 40% cells were GFP positive. (D) the effect of ketamine on the distribution of cytochrome c in neurons. Cells were labeled with CellLight™ mitochondria-GFP reagent expressed GFP in mitochondria and then treated with ketamine (100 μM, 24 h). The distribution of cytochrome c in cells was analyzed by immunofluorescence staining. Column 1 is the image of mitochondria; column 2 is the image of cytochrome c; and column 3 is the merged image. The inset in the top corner of each image is the magnified box indicated by white arrows. The orange signals in the merged images indicate the existence of cytochrome c inside the mitochondria, and the signals in the merged images indicate the existence of cytochrome c outside the mitochondria. Ketamine treatment (100 μM, 24 h) increased cytochrome c release from mitochondria into cytosol. *Note: The cytochrome c signals (indicated by blue* 

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*arrows) that do not overlap with GFP florescence were from non-transduced cells*. Scale bar = 10 μm [20].

Ketamine Induces Neuroapoptosis in Stem Cell–Derived Developing Human Neurons Possibly… http://dx.doi.org/10.5772/intechopen.72939 431

cause mitochondrial damage, we measured ΔΨm and distribution of cytochrome c in the cells. Treatment of hESC-derived neurons with 100 μM ketamine for 24 h significantly decreased ΔΨm (**Figure 4A**). In order to investigate the distribution of cytochrome c within the cells, the neurons were transduced with the virus CellLight™ mitochondria-GFP (green). GFP and TMRE signals were colocalized in the cells (**Figure 4B**), confirming that successfully labeling of mitochondria with GFP. GFP-positive cells reached 40% (**Figure 4C**). The distribution of cytochrome c in mitochondria and cytosol was examined using immunofluorescence staining. The results showed that cytochrome c was located within mitochondria in the control culture (**Figure 4D**). However, in the ketamine-treated cells, cytochrome c was released from

Neurons were loaded with the Ca2+ indicator Fluo-4AM. The fluorescence images of free cytosolic Ca2+ in the 2-week-old

c

] of neurons. (A) Ketamine increased (Ca2+)

(\*\*P < 0.01, n = 3).

c .

**Figure 3.** Ketamine exposure for 24 h induced elevated cytosolic Ca2+ [(Ca2+)

differentiated neurons are shown. (B) Ketamine (100 μM, 24 h) significantly increased (Ca2+)c

Neuroapoptosis is a commonly recognized harmful effect by anesthetics with mechanisms that are not fully understood. Apoptosis is a programmed cell death. Cytochrome c release from mitochondria precededing the loss of ∆Ψm is a key event in initiating mitochondria-involved apoptosis [47], eventually leading to the typical alterations related to apoptosis such as DNA fragmentation in cell nuclei [48, 49]. In this study, following ketamine exposure, there was a significantly increase in the TUNEL-positive apoptotic cells (**Figure 2**). Ketamine-induced cell death was accompanied by the significant decrease in ∆Ψm and the increased cytochrome c release from mitochondria into cytosol (**Figure 4**). These data were in agreement with those previously reported by others [48, 50–52], suggesting that ketamine induces human neuron to undergo mitochondria-mediated apop-

To maintain proper cellular function, the mitochondria continuously undergo cycles of fusion and fission. Unbalanced fusion/fission, particularly excessive fission/fragmentation can lead to various pathological conditions including neurodegeneration [53]. To assess mitochondrial fission shape in the hESC-derived neurons following exposure to ketamine, electron microscopy was used. The neurons were exposed to either 20 μM or 100 μM ketamine or control conditions for 24 h. The cells were then prepared and imaged on an electron microscope. The mitochondria appeared considerably fragmented in the ketamine- treated neurons when compared to control cells (**Figure 5**). Increased mitochondrial fission has been linked to the propofol-induced cell death in hESC-derived neurons as previously published by our group [54]. Our findings suggest that ketamine exposure results in an increase of mitochondrial fission within the developing neurons, which may contribute to the increased cell death

the mitochondria into cytosol.

observed in the ketamine-treated group.

tosis pathway.

430 Mitochondrial Diseases

**Figure4.** Ketamine decreases mitochondrial membrane potential (ΔΨm) and increases cytochrome c release from mitochondria into cytosol. (A) ΔΨm assay. Ketamine (100 Μm) treatment for 24 h decreased ΔΨm (\*P < 0.05 vs. control, n = 3). (B) Labeling mitochondria of neurons with CellLight™ mitochondria-green fluorescence protein (GFP) reagent. The GFP-positive cells were loaded with TMRE. GFP expression in mitochondria was confirmed by the colocalization of tetramethylrhodamine ethyl ester (TMRE/mitochondrial probe) and GFP signals within the cells. (C) Representative fluorescent images of the neurons transduced with CellLight™ mitochondria-GFP reagent. Blue are cell nuclei. 40% cells were GFP positive. (D) the effect of ketamine on the distribution of cytochrome c in neurons. Cells were labeled with CellLight™ mitochondria-GFP reagent expressed GFP in mitochondria and then treated with ketamine (100 μM, 24 h). The distribution of cytochrome c in cells was analyzed by immunofluorescence staining. Column 1 is the image of mitochondria; column 2 is the image of cytochrome c; and column 3 is the merged image. The inset in the top corner of each image is the magnified box indicated by white arrows. The orange signals in the merged images indicate the existence of cytochrome c inside the mitochondria, and the signals in the merged images indicate the existence of cytochrome c outside the mitochondria. Ketamine treatment (100 μM, 24 h) increased cytochrome c release from mitochondria into cytosol. *Note: The cytochrome c signals (indicated by blue arrows) that do not overlap with GFP florescence were from non-transduced cells*. Scale bar = 10 μm [20].

**Figure 5.** Ketamine increases mitochondrial fission as evidenced by the electron microscope images of differentiated neurons treated with the indicated concentrations (20 and 100 μM) of ketamine for 24 h. Scale bars = 500 nm.

expression changes following exposure to 24 h of ketamine along with additional cell death and mitochondrial signaling assays following 6 h of exposure to ketamine which will allow

**Figure 6.** The expression of several microRNAs was significantly altered following exposure to ketamine. Among 88 microRNAs investigated, exposure to ketamine decreased the expression of six microRNAs and increased the expression

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The mechanisms governing anesthetic-induced neurotoxicity in the developing brain are currently not well understood and this research has been limited by the lack of an appropriate human model. Collectively, the findings from this study indicate that (1) hESC-derived neurons represent a promising model to investigate the effects of anesthetic exposure in the developing human brain, (2) ketamine induces neuroapoptosis via mitochondrial pathway, and (3) dysregulation of intracellular calcium, increased mitochondrial fission, and altered microRNA expression might play important roles in ketamine-induced neuroapoptosis. The role of microRNAs in anesthetic-induced neurotoxicity is just beginning to be understood. Functional studies including knockdown and overexpression of microR-NAs of interest will further establish their role in the ketamine-induced neurotoxicity. Additionally, much work remains to establish a functional link between dysregulation of intracellular calcium, increased mitochondrial fission, decreased mitochondrial membrane potential, altered microRNA expression, and the observed neuroapoptosis. These studies are very promising and may reveal novel mechanisms of ketamine-induced developmental

The following grants supported this work: R01GM112696 from the NIH (to Dr. Xiaowen Bai)

and P01GM066730 from the NIH (to Dr. Zeljko J. Bosnjak).

for proper interpretation of the results.

of one microRNA. n = 3–4 for each group.

**3.6. Summary**

neurotoxicity.

**Acknowledgements**

#### **3.5. qRT-PCR analysis of microRNA expression**

Our lab was the first to show the role of microRNAs, specifically miR-21 in propofol-induced neurotoxicity in hESC-derived neurons [37]. Using a similar approach, we have identified several microRNAs with altered expression profiles in ketamine-treated hESC-derived neurons when compared to control-treated cells suggesting a role of microRNAs in ketamineinduced neurotoxicity. A total of 84 of the most abundantly expressed microRNAs were analyzed using the miFinder miRNA PCR arrays. A fold change of 1.3 between the control and ketamine-treated cells was considered significant. The data are expressed as percent of control with the control group set to 100%. The expression of seven microRNAs was found to be significantly altered following exposure to 6 h of ketamine when compared to controltreated cells. Of these seven, only miR-96 was found to be significantly upregulated with ketamine treatment. The remaining microRNAs (miRs-Let 7A, Let 7E, 21, 23b, 28-5p, 423-5p) were significantly downregulated with ketamine exposure compared to control-treated cells (**Figure 6**). Of these microRNAs, the downregulation of miR-21 was of particular interest since miR-21 is protective against ischemic injuries [55]. Downregulation of a protective microRNA may provide a mechanism by which ketamine is inducing neuronal toxicity in the hESCderived neurons. Interestingly, we also reported a significant downregulation of miR-21 in hESC-derived neurons following exposure to the anesthetic propofol and went on to confirm a functional role of this expression change in the propofol-induced neurotoxicity [37]. This research suggests that the mechanism of anesthetic-induced neurotoxicity among multiple anesthetic agents might converge on altered expression of microRNAs such as miR-21. While the other 6 microRNAs identified through these array studies have not been implicated previously in neuronal diseases, this approach has the potential to uncover novel roles of these microRNAs in ketamine-induced neurotoxicity. An aim of future studies will include functional studies to further elucidate the role of these microRNAs and the signaling components connecting these altered microRNAs and attenuated mitochondrial function in ketamineinduced neurotoxicity. Additionally, the current microRNA studies were done using a different time point (6 h) than the cell death and mitochondrial studies previously mentioned which used a time point of 24 h. This was done to ensure any potentially transient changes in microRNA expression were observed. Future studies will include the addition of microRNA

Ketamine Induces Neuroapoptosis in Stem Cell–Derived Developing Human Neurons Possibly… http://dx.doi.org/10.5772/intechopen.72939 433

**Figure 6.** The expression of several microRNAs was significantly altered following exposure to ketamine. Among 88 microRNAs investigated, exposure to ketamine decreased the expression of six microRNAs and increased the expression of one microRNA. n = 3–4 for each group.

expression changes following exposure to 24 h of ketamine along with additional cell death and mitochondrial signaling assays following 6 h of exposure to ketamine which will allow for proper interpretation of the results.

#### **3.6. Summary**

**3.5. qRT-PCR analysis of microRNA expression**

432 Mitochondrial Diseases

Our lab was the first to show the role of microRNAs, specifically miR-21 in propofol-induced neurotoxicity in hESC-derived neurons [37]. Using a similar approach, we have identified several microRNAs with altered expression profiles in ketamine-treated hESC-derived neurons when compared to control-treated cells suggesting a role of microRNAs in ketamineinduced neurotoxicity. A total of 84 of the most abundantly expressed microRNAs were analyzed using the miFinder miRNA PCR arrays. A fold change of 1.3 between the control and ketamine-treated cells was considered significant. The data are expressed as percent of control with the control group set to 100%. The expression of seven microRNAs was found to be significantly altered following exposure to 6 h of ketamine when compared to controltreated cells. Of these seven, only miR-96 was found to be significantly upregulated with ketamine treatment. The remaining microRNAs (miRs-Let 7A, Let 7E, 21, 23b, 28-5p, 423-5p) were significantly downregulated with ketamine exposure compared to control-treated cells (**Figure 6**). Of these microRNAs, the downregulation of miR-21 was of particular interest since miR-21 is protective against ischemic injuries [55]. Downregulation of a protective microRNA may provide a mechanism by which ketamine is inducing neuronal toxicity in the hESCderived neurons. Interestingly, we also reported a significant downregulation of miR-21 in hESC-derived neurons following exposure to the anesthetic propofol and went on to confirm a functional role of this expression change in the propofol-induced neurotoxicity [37]. This research suggests that the mechanism of anesthetic-induced neurotoxicity among multiple anesthetic agents might converge on altered expression of microRNAs such as miR-21. While the other 6 microRNAs identified through these array studies have not been implicated previously in neuronal diseases, this approach has the potential to uncover novel roles of these microRNAs in ketamine-induced neurotoxicity. An aim of future studies will include functional studies to further elucidate the role of these microRNAs and the signaling components connecting these altered microRNAs and attenuated mitochondrial function in ketamineinduced neurotoxicity. Additionally, the current microRNA studies were done using a different time point (6 h) than the cell death and mitochondrial studies previously mentioned which used a time point of 24 h. This was done to ensure any potentially transient changes in microRNA expression were observed. Future studies will include the addition of microRNA

**Figure 5.** Ketamine increases mitochondrial fission as evidenced by the electron microscope images of differentiated

neurons treated with the indicated concentrations (20 and 100 μM) of ketamine for 24 h. Scale bars = 500 nm.

The mechanisms governing anesthetic-induced neurotoxicity in the developing brain are currently not well understood and this research has been limited by the lack of an appropriate human model. Collectively, the findings from this study indicate that (1) hESC-derived neurons represent a promising model to investigate the effects of anesthetic exposure in the developing human brain, (2) ketamine induces neuroapoptosis via mitochondrial pathway, and (3) dysregulation of intracellular calcium, increased mitochondrial fission, and altered microRNA expression might play important roles in ketamine-induced neuroapoptosis. The role of microRNAs in anesthetic-induced neurotoxicity is just beginning to be understood. Functional studies including knockdown and overexpression of microR-NAs of interest will further establish their role in the ketamine-induced neurotoxicity. Additionally, much work remains to establish a functional link between dysregulation of intracellular calcium, increased mitochondrial fission, decreased mitochondrial membrane potential, altered microRNA expression, and the observed neuroapoptosis. These studies are very promising and may reveal novel mechanisms of ketamine-induced developmental neurotoxicity.

#### **Acknowledgements**

The following grants supported this work: R01GM112696 from the NIH (to Dr. Xiaowen Bai) and P01GM066730 from the NIH (to Dr. Zeljko J. Bosnjak).
