new_pdfs/10.1007_s12640-016-9615-7.txt

Neurotox Res (2016) 30:185–198 DOI 10.1007/s12640-016-9615-7

O R I G I N A L A R T I C L E

Ketamine Affects the Neurogenesis of the Hippocampal Dentate Gyrus in 7-Day-Old Rats

He Huang1 Dan Wang2

Cun-Ming Liu1 • Yu-Qing Wu2

Jie Sun1

Ting Hao2

Chun-Mei Xu2



Received: 15 December 2015 / Revised: 22 February 2016 / Accepted: 1 March 2016 / Published online: 10 March 2016 (cid:2) Springer Science+Business Media New York 2016

Abstract Ketamine has been reported to cause neonatal neurotoxicity via a neuronal apoptosis mechanism; how- ever, no in vivo research has reported whether ketamine could affect postnatal neurogenesis in the hippocampal dentate gyrus (DG). A growing number of experiments suggest that postnatal hippocampal neurogenesis is the foundation of maintaining normal hippocampus function into adulthood. Therefore, this study investigated the effect of ketamine on hippocampal neurogenesis. Male Sprague– Dawley rats were divided into two groups: the control group (equal volume of normal saline), and the ketamine- anesthesia group (40 mg/kg ketamine in four injections at 1 h intervals). The S-phase marker 5-bromodeoxyuridine (BrdU) was administered after ketamine exposure to postnatal day 7 (PND-7) rats, and the neurogenesis in the hippocampal DG was assessed using single- or double- immunofluorescence staining. The expression of GFAP in the hippocampal DG was measured by western blot anal- ysis. Spatial reference memory was tested by Morris water maze at 2 months after PND-7 rats exposed to ketamine treatment. The present results showed that neonatal keta- mine exposure significantly inhibited neural stem cell (NSC) proliferation, decreased astrocytic differentiation, and markedly enhanced neuronal differentiation. The dis- ruptive effect of ketamine on the proliferation and differ- entiation of NSCs lasted at least 1 week and disappeared the by 2 weeks after ketamine exposure. Moreover,

migration of newborn neurons in the granule cell layer and the growth of astrocytes in the hippocampal DG were inhibited by ketamine on PND-37 and PND-44. Finally, ketamine caused a deficit in hippocampal-dependent spatial reference memory tasks at 2 months old. Our results sug- gested that ketamine may interfere with hippocampal neurogenesis and long-term neurocognitive function in PND-7 rats. These findings may provide a new perspective to explain the adult neurocognitive dysfunction induced by neonatal ketamine exposure.

Keywords Ketamine (cid:2) Neonatal (cid:2) Neurogenesis (cid:2) Hippocampal dentate gyrus (cid:2) Morris water maze test

Ketamine, an N-methyl-D-aspartate (NMDA) receptor antagonist, is widely used in anesthesia, analgesia, and sedation during the neonatal period (Asadi et al. 2013; Guerra et al. 2011). However, ketamine has been reported short-term and long-term neurotoxicities, to induce including neuronal apoptosis and neurocognitive dysfunc- tion in the adult stage (Ikonomidou et al. 1999; Liu et al. 2011; Paule et al. 2011; Zou et al. 2009; Pfenninger et al. 2002; Wilder et al. 2009). As a commonly used anesthetic the safety of during pediatric anesthesia and sedation, ketamine has been the subject of concern for anesthesiol- ogists and the public.

& Yu-Qing Wu

xymzyqwu@126.com

1 Department of Anesthesiology, The First Affiliated Hospital

of Nanjing Medical University, Nanjing, China

2

Jiangsu Province Key Laboratory of Anesthesiology, Xuzhou Medical College, Xuzhou, China

The causal link between neuronal death in the hip- pocampus of the developing brain and neurocognitive dysfunction in the adult stage has not been investigated in detail. Curiously, a previous study showed that 4 h of hypercapnia exposure caused a similar degree of hip- pocampal neuronal death as 4 h of isoflurane in PND-7 rats, but only 4 h of isoflurane caused a long-term neu- rocognitive deficit (Stratmann et al. 2009). This raises

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suspicion regarding whether ketamine-induced hippocam- pal neuronal death in PND-7 rats can fully account for the neurocognitive dysfunction observed in the adult stage. Hence, it is worthwhile to study whether there is any other mechanism to explain the cognitive deficit in the adult stage after neonatal ketamine exposure other than anes- thesia-induced neuronal death.

The developing central nervous system (CNS) has a critical period called brain growth spurt (BGS), which lasts from the end of pregnancy to the first 2–3 weeks after birth in rodents; in humans, the corresponding period begins in the last trimester of pregnancy and continues until 2 years after birth (Byrnes et al. 2001). During this period, the brain exhibits a high degree of plasticity, and substantial neuro- genesis occurs rapidly and lays the foundation for the normal structure and function of the brain. The hippocampal dentate gyrus (DG) is one of only two restricted regions other than the subventricular zone (SVZ) where neurogenesis occurs during development and continues, at a slower rate, into adulthood (Lledo et al. 2006; Mongiat and Schinder 2011; Luskin 1993; Vadodaria and Jessberger 2014). The neuro- genesis of the hippocampal DG plays a critical role in the formation of hippocampal-dependent spatial learning and memory function (Dupret et al. 2008; Stone et al. 2011).

Neurogenesis is a complicated process that includes neural stem cell (NSC) proliferation, neuronal or astrocytic differ- entiation, and migration of newborn neurons. NSCs, which are located in the hilus/subgranular zone (SGZ) of hippocampal DG, partially begin to differentiate into neurons or astrocytes, while others retain the ability to divide. Some of the newly generated granule neurons can migrate into the granule cell layer (GCL) and functionally incorporate into the hip- pocampal circuit (granule neurons–CA3–CA1 loop) (Vado- daria and Jessberger 2014; van Praag et al. 2002).

Postnatal neurogenesis in the DG may be sensitive to outside stimulation, such as hypoxia–ischemia, hyperoxia, and stress (Bartley et al. 2005; Belnoue et al. 2013; Porzionato et al. 2013). In recent years, there has been increasing research into the effect of anesthetics on post- natal neurogenesis in the DG (Fang et al. 2012; Erasso et al. 2013; Nie et al. 2013; Stratmann et al. 2009). How- ever, the effects of ketamine on neonatal hippocampal neurogenesis in vivo have not been reported. The present study aims to explore the effects of ketamine on postnatal neurogenesis in the hippocampal DG in vivo.

Materials and Methods

Animal Treatment

All animal experiments were approved by the Institutional Animal Care and Use Committee of Nanjing Medical

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University. The timed-pregnant Sprague–Dawley rats were housed in a temperature-controlled (22–23 (cid:3)C) room on a 12 h:12 h light:dark cycle (light on at 8:00 AM) with free access to food and water. The PND-7 male rat pups (11–14 g) were randomly assigned to ketamine-treated and control groups. In the treated group, ketamine was diluted in 0.9 % normal saline, and PND-7 rats were intraperi- toneally administered with 40 mg/kg doses of ketamine in four injections at 1 h intervals (40 mg/kg 9 4 injections). Control rats received an equal volume of normal saline. Temperature probes were used to facilitate control of temperature at 36.5 ± 1 (cid:3)C using computer-controlled heater/cooler plates integrated into the floor of the cham- ber. Between each injection, animals were returned to their chamber to help maintain body temperature and reduce stress.

BrdU Injections

All animals received an intraperitoneal injection of BrdU (5-bromo-2-deoxyuridine; Sigma) at a dosage of 100 mg/ kg after ketamine anesthesia according to the following experimental schedule.

Experiment 1: To evaluate the effect of ketamine on the proliferation and differentiation of NSCs in the DG during the BGS, the PND-7 rats received a single intraperitoneal injection of BrdU on PND-7, 13, and 20 after ketamine treatment. The animals were then anesthetized and fixed by perfusion at 24 h after each BrdU injection. The experi- mental protocol is described in Tables 1a and 2.

Experiment 2: To exclude the GFAP/BrdU double- positive cells that were proliferative astrocytes, the PND-7 rats received a single intraperitoneal injection of BrdU on PND-7, 13, and 20 after exposure to treatment. The animals were then perfused at 3 h after each BrdU injection. The experimental protocol is detailed in Tables 1b and 2.

Experiment 3: To determine the effect of ketamine on the migration of newborn granule neurons in the DG, the PND-7 rats received three consecutive BrdU injections on PND-7, 8, and 9 after exposure to treatment. At 28 and 35 days after the last BrdU injection, the animals were anesthetized and fixed by perfusion. The experimental protocol is described in Table 1c and 2.

Cell Apoptotic Assays

Nestin/caspase-3 and GFAP/caspase-3 double-immunoflu- orescence staining was utilized to detect whether ketamine could induce the apoptosis of NSCs or astrocytes. At 12 h after the end of control and ketamine-anesthesia treatment, the neonatal rats were anesthetized and fixed by perfusion (n = 5 per group).

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Table 1 Experimental design

Total no. of BrdU injections

Postnatal day on which BrdU was administered

Survival (day) after the last BrdU injection

a. Experiment 1 (n = 5)

Effect of ketamine on the proliferation and differentiation

1

7

1

of NSCs in the DG of PND-7 rats

1

13

1

1

20

1

b. Experiment 2 (n = 5)

To exclude the GFAP/BrdU double-positive cells that were

1

7

3

proliferative astrocytes

1 1

13 20

3 3

c. Experiment 3 (n = 5)

Effect of ketamine on the migration of newborn granule

3

7–9

28

neurons in the DG of PND-7 rats

3

7–9

35

Table 2 Immunolabeling

Targeted process

NSC proliferation

Neuronal differentiation

Astrocytic differentiation

Astrocytic proliferation

IF stain

Nestin/BrdU b-tubulin III/BrdU

GFAP/BrdU

GFAP/BrdU

these steps. Blocking of nonspecific epitopes with 10 % donkey serum in PBS with 0.3 % Triton-X for 2 h at room temperature preceded incubation overnight at 4 (cid:3)C with the primary antibodies listed in Table 3 in PBS with 0.3 % Triton-X. On the next day, the sections were incubated with the appropriate secondary fluorescent antibodies (In- vitrogen Carlsbad, CA) for 2 h at room temperature.

Migration of newborn granule neurons

IF immunofluorescence

Tissue Preparation and Immunofluorescence

NeuN/BrdU

Astrocytic development was detected by using GFAP single-labeled staining. The sections were incubated over- night at 4 (cid:3)C with a fluorescent antibody for the GFAP (Table 3). After three washes in PBS, sections were incu- bated with secondary fluorescent antibody (Invitrogen) for 2 h at room temperature.

At the indicated time point, animals were deeply anes- thetized and then transcardially perfused with 0.9 % nor- mal saline followed by 4 % paraformaldehyde. The brains were removed, postfixed overnight in 4 % paraformalde- hyde, and placed in 30 % sucrose until sunk. The coronal sections of brain were cut consecutively at a thickness of 30 lm when the hippocampus was initially exposed. The fifteenth section was taken and stored in PBS. According to the Atlas of the Developing Rat Brain and previous reports (Ashwell and Paxinos 2008; Paxinos and Watson 1986), the positions of hippocampus coronal sections selected in to the our study were about 2.20–2.25 mm posterior bregma at PND-8 rats, about 2.35–2.40 mm posterior to the bregma at PND-14 rats, about 2.50–2.55 mm posterior to the bregma at PND-21 rats, and about 2.75–2.85 mm posterior to the bregma at PND-37 and PND-44 rats, respectively.

For Nestin/BrdU, b-tubulin III/BrdU, GFAP/BrdU, and NeuN/BrdU double-immunofluorescence the BrdU antigen was exposed by incubating the sections in 2-normal hydrochloric acid for 30 min at 37 (cid:3)C and then washed three times with PBS for 5 min between each of

staining,

To characterize the phenotype of cell apoptosis, brain sections were analyzed by double-labeled staining. The sections were incubated overnight at 4 (cid:3)C with the appro- priate primary antibodies listed in Table 3. After three washes with PBS, the sections were incubated with the suitable secondary fluorescent antibodies (Invitrogen) for 2 h at room temperature.

A skilled pathologist blinded to the study conditions examined the labeled sections using a laser scanning con- focal microscope (Fluoview 1000, Olympus). The number of single- or double-positive cells in the hippocampal DG was quantified using Image-Pro Plus software.

Western Blot Analysis

Thirty and thirty-seven days after the control or ketamine- anesthesia treatment, the animals were decapitated, and the hippocampal DG tissue was dissected carefully with ana- tomic microscope (leica EZ4HD). The harvested hip- pocampal tissues were homogenized on ice using lysate buffer plus protease inhibitors. The lysates were cen- trifuged at 14,000 rpm for 15 min at 4 (cid:3)C and were

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Table 3 Primary antibodies

Antibody name

Specificity

Host species

Dilution rates

Company

Nestin b-tubulin III

Neural stem cells

Newborn neurons

Rabbit

Rabbit

1:100

1:200

Abcam

Abcam

GFAP

Astrocytes

Rabbit

1:200

Millipore

NeuN

Mature neurons

Mouse

1:400

Millipore

BrdU

Newly generated cells

Mouse

1:1000

Sigma

BrdU

Newly generated cells

Rabbit

1:500

Abcam

Caspase-3

Cell apoptosis

Mouse

1:100

Santa Cruz

resolved by 12 % polyacrylamide gel electrophoresis, and the target proteins were transferred to nitrocellulose membranes. The blots were incubated with blocking buffer for 2 h at room temperature and then incubated for 24 h at 4 (cid:3)C with the primary antibodies: rabbit anti-GFAP anti- body (1:1000, Millipore) and GAPDH. The membranes were then incubated with appropriate secondary alkaline antibody donkey phosphatase-conjugated (1:10,000, Abcam) for 1 h. The band intensity was quan- tified using Image J software (n = 5 per group).

anti-rabbit

Morris Water Maze Test

Province Key Laboratory of Anesthesiology, Xuzhou Medical College, Xuzhou, China).

Statistical Analysis

The statistical analysis was conducted using SPSS 13.0, and the graphs were created using GraphPad Prism 5. The data were analyzed using Mann-Whitney U test. The interaction between time and group factors in a two-way ANOVA was used to analyze the difference of escape latency between rats in the control group and rats treated with ketamine in the MWM. The data are presented as the mean ± SD, and p \ 0.05 was considered statistically significant.

The hippocampal-dependent spatial memory abilities were tested by using the Morris water maze (MWM). Different set of rats were tested 2 months after administration of ketamine on PND-7. A circular, black painted pool (180 cm diameter, 50 cm deep) was filled with water to a depth of 30 cm. The water temperature was maintained at 25 ± 1 (cid:3)C. An invisible platform (10 cm diameter) was submerged 1 cm below the water surface and placed in the center of the III quadrant which was determined with four starting locations called I, II, III, and IV at equal distance on the edge of the pool. During five consecutive days, the experiments were conducted in a dark and quiet laboratory, all the rats were trained four times per day, the starting positions were random for each rat. When the rat found the platform, the rat was allowed to stay on it for 30 s. If a rat did not find the platform within 120 s, the rat would be guided gently to the place and allowed to stay on it for 30 s, and the latency time to find the hidden platform was recorded as 120 s. The average time from four trials rep- resented as the daily result for the rat. On the sixth day, the hidden platform was removed, and the rat was placed in the opposite quadrant. Rats were allowed to swim freely for 120 s. The numbers the rat swam to cross the previous platform area, and the times the rat stayed in the target quadrant within 120 s were recorded. Each animal’s path was tracked by a computerizing video system. After every trial, each rat was placed in a heater plates for 1 to 2 min until dry before being returned to its chamber. The data were analyzed using software for the MWM (Jiangsu

Results

Ketamine Anesthesia in Postnatal Rats Induced Inhibition of NSC Proliferation in the Hippocampal DG

As shown in Fig. 1d, e, the percentage (8 ± 1.04 %) and density (11 ± 1.08 lm2) of Nestin?/BrdU? cells in the ketamine-treated group were significantly decreased compared to those in the control group (14 ± 1.30 %; 20 ± 2.22 lm2) 1 day (PND-8) after exposure to keta- mine. This suppressive effect of ketamine on NSC pro- liferation was also found at 7 days (PND-14: 10 ± 0.77 vs. 14 ± 1.45 %; 15 ± 1.29 vs. 19 ± 2.08 lm2) after (PND-21: anesthesia 18 ± vs. 11 ± 1.32 1.75 lm2) after anesthesia. Typical immunofluorescence pictures are shown in Fig. 1a, b, c. In addition, we found that there were no significant differences in NSC prolif- eration at different time points (PND-8, 14, and 21) in either the control groups or the ketamine-treated groups. These data indicated that ketamine could significantly inhibit the proliferation of NSCs in the hippocampal DG of neonatal rats for at least 1 week and that the ability of NSC proliferation could recover after anesthesia.

but vs.

disappeared 13 ± 1.13 %;

at

14 days 17 ± 2.44

at 2 weeks

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Fig. 1 Effect of ketamine on the proliferation of NSCs in the hippocampal dentate gyrus (DG) of neonatal rats. The NSCs were labeled with primary antibodies against Nestin (green) and BrdU (red). The immunoreactive cells were visualized using a laser scanning confocal microscope (a, b and c; magnification: 9200); the scale bar is 100 lm. The filled arrows point to

Ketamine Anesthesia in Postnatal Rats Promoted the Neuronal Differentiation of NSCs in the Hippocampal DG

Figure 2d, e show that the percentage (18 ± 2.27 %) and density (23 ± 3.74 lm2) of b-tubulin III?/BrdU? cells were significantly increased in the ketamine-treated group com- pared to those in the control group (12 ± 2.07 %;

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Nestin/BrdU double-labeled cells. The ratio of Nestin?/BrdU? cells to Nestin? cells was calculated (d). The Y axis ‘‘(lm2)’’ represents the density of Nestin?/BrdU? cells in the DG (e). Data are presented as the mean ± SD (n = 5). **p\0.01 versus control group. GCL granule cell layer; ML molecular layer; PCL polymorphic cell layer (Color figure online)

17 ± 3.25/lm2) 1 day (PND-8) after exposure to ketamine. This stimulating effect of ketamine on neuronal differenti- ation of NSCs was also assessed at 7 days (PND-14: 16 ± 2.24 vs. 11 ± 1.25 %; 21 ± 3.01 vs. 16 ± 2.05 lm2) after anesthesia but was not detected at 14 days (PND-21: 12 ± 1.90 vs. 12 ± 2.34 %; 18 ± 2.28 vs. 17 ± 2.92 lm2) after anesthesia. Typical immunofluorescence pictures are shown in Fig. 2a, b, c. Moreover, we did not observe

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significant differences in the neuronal differentiation of NSCs at different time points (PND-8, 14 and 21) in either the control groups or the ketamine-treated groups. These data indicated that ketamine could significantly promote the neuronal differentiation of NSCs in the hippocampal DG of neonatal rats for at least 1 week, and this stimulating effect finally disappeared by 2 weeks after anesthesia.

Fig. 2 Effect of ketamine on the neuronal differentiation of NSCs in the hippocampal dentate gyrus (DG) of neonatal rats. The newborn neurons were labeled with primary antibodies against b-tubulin III (green) and BrdU (red). The immunoreactive cells were visualized using a laser scanning confocal microscope (a, b and c; magnification: 9200); the scale bar is 100 lm. The filled arrows point to b-tubulin

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Ketamine Anesthesia in Postnatal Rats Attenuated the Astrocytic Differentiation of NSCs in the Hippocampal DG

Similar to the effect of ketamine on NSC proliferation, the percentage (11 ± 0.89 %) and density (10 ± 1.46 lm2) of GFAP?/BrdU? cells were significantly decreased in the

III/BrdU double-labeled cells. The ratio of b-tubulin III?/BrdU? cells to BrdU? cells was calculated (d). The Y axis ‘‘(lm2)’’ represents the density of b-tubulin III?/BrdU? cells in the DG (e). Data are presented as the mean ± SD (n = 5). *p\0.05, **p\0.01 versus control group. GCL granule cell layer, ML molecular layer, PCL polymorphic cell layer (Color figure online)

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Fig. 3 Effect of ketamine on the astrocytic differentiation of NSCs in the hippocampal dentate gyrus (DG) of neonatal rats. The newborn astrocytes were labeled with primary antibodies against GFAP (green) and BrdU (red). The immunoreactive cells were visualized using a laser scanning confocal microscope (a, b and c; magnification: 9200); the scale bar is 100 lm. The filled arrows point to GFAP/ BrdU double-labeled cells. The ratio of GFAP?/BrdU? cells to BrdU? cells was calculated (d). The Y axis ‘‘(lm2)’’ represents the density of GFAP?/BrdU? cells in the DG (e). To exclude that GFAP/

ketamine-treated group compared to those in the control group (15 ± 1.30 %; 17 ± 2.07 lm2) 1 day (PND-8) after exposure to ketamine. This inhibitory effect of ketamine on astrocytic differentiation of NSCs was also detected at

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BrdU double-positive cells were the proliferative astrocytes, another set of animals was perfused and sacrificed at 3 h after BrdU injection on PND-7, 13, and 20. The proliferative astrocytes were also stained with primary antibodies against GFAP and BrdU, and the density of GFAP/BrdU double-labeled cells was calculated (f). Data are presented as the mean ± SD (n = 5). *p\0.05, **p\0.01 vs. control group. GCL granule cell layer, ML molecular layer, PCL polymorphic cell layer (Color figure online)

7 days (PND-14: 13 ± 1.40 vs. 15 ± 0.97 %; 12 ± 2.11 vs. 16 ± 1.88 lm2) after anesthesia but disappeared at 14 days (PND-21: 14 ± 1.57 vs. 14 ± 1.39 %; 13 ± 0.91 vs. 14 ± 0.82 lm2) after anesthesia (Fig. 3d, e). Typical

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immunofluorescence pictures are shown in Fig. 3a, b, c. Moreover, we did not observe significant differences in the astrocytic differentiation of NSCs at different time points (PND-8, 14 and 21) in either the control groups or the ketamine-treated groups. These data suggested that keta- mine could also inhibit the astrocytic differentiation of NSCs in the hippocampal DG of neonatal rats for at least 1 week, and this inhibitory effect finally disappeared by 2 weeks after anesthesia.

To exclude the GFAP/BrdU double-positive cells that were proliferative astrocytes, the animals were perfused at 3 h after BrdU injection on PND-7, 13, and 20. We found only a small amount of GFAP/BrdU double-positive cells in both the control and ketamine groups at the three time points, and there were no significant differences in the proliferation of matured astrocytes between the ketamine and control groups (Fig. 3f).

The NSCs and Astrocytes in the Hippocampal DG of Neonatal Rats are Resistant to Ketamine-Induced Cell Apoptosis

To investigate the effects of ketamine on the apoptosis of NSCs and astrocytes in the hippocampal DG of neonatal rats, we analyzed the nestin/caspase-3 and GFAP/caspase-3 double-positive cells in the hippocampal DG using double- immunofluorescence staining 12 h after the end of keta- mine anesthesia. The results showed that there were no significant changes in the numbers of nestin/caspase-3 or GFAP/caspase-3 double-positive cells in either the control or ketamine groups. These results suggested that the dosage and duration of ketamine used in our experiment could not induce the apoptosis of NSCs and astrocytes in the hip- pocampal DG (Fig. 4a, b).

Ketamine Anesthesia in Postnatal Rats Inhibited the Migration of Newborn Neurons in the GCL of the Hippocampal DG

According to previous research (Kempermann et al. 2003; Esposito et al. 2005), the hippocampal GCL can be divided into four zones (SGZ, GCL1, GCL2, and GCL3) from the inside to outside of the GCL. The newborn neurons dif- ferentiated from NSCs could migrate from the SGZ to different locations throughout the GCL. The experimental is shown in Fig. 5a. To better visualize the protocol migration of the newly generated neurons in the GCL, we examined the NeuN?/BrdU? cells in the GCL 28 days (PND-37) and 35 days (PND-44) after the last BrdU injection using double-immunofluorescence staining.

According to our findings, ketamine could significantly increase the rate of BrdU-positive neurons in the SGZ compared to the total BrdU-positive neurons in the GCL

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Fig. 4 Effect of ketamine on the apoptosis of NSCs and astrocytes in the hippocampal dentate gyrus of neonatal rats. The PND-7 rats were perfused and sacrificed at 12 h after four injections of 40 mg/kg ketamine at 1-h intervals. The apoptosis of NSCs and astrocytes is shown with nestin/caspase-3 (a) and GFAP/caspase-3 (b) double- staining (magnification: 9400; the scale bar immunofluorescence layer, PCL is 50 lm). GCL granule cell polymorphic cell layer

layer, ML molecular

PND-44: vs. (PND-37: 74 ± 6.11 vs. 26 ± 10.28 %) (Fig. 5d); the rate of BrdU- positive neurons in GCL1 compared to the total BrdU- positive neurons in the GCL was significantly decreased in the ketamine group compared to that in control group on PND-37 (24 ± 10.61 vs. 46 ± 12.57 %) and PND-44 (21 ± 9.65 vs. 51 ± 4.65 %) (Fig. 5e). The rate of BrdU- positive neurons in GCL2 compared to the total BrdU- positive cells in the GCL was significantly decreased in the ketamine group compared to that in control group on PND- 37 (5 ± 7.36 vs. 16 ± 5.06 %) and PND-44 (5 ± 6.85 vs. 23 ± 7.45 %) (Fig. 5f). The rate of BrdU-positive neurons in GCL3 compared to the total BrdU-positive neurons in the GCL showed no significant difference between the control and ketamine groups on PND-37 and PND-44. Typical immunofluorescence pictures are shown in Fig. 5b, c. Taken together, that neonatal ketamine exposure could inhibit the migration of postna- tally generated neurons in the GCL of the hippocampal DG and restrict them inside the GCL.

70 ± 16.73

36 ± 9.31 %;

these results suggest

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Fig. 5 Effect of ketamine on the migration of newborn neurons in the hippocampal dentate gyrus (DG) of neonatal rats. Experimental protocol (a). Representative photographs from a laser scanning confocal microscope are shown (b, c; magnification: a, e 9200; b– d and f–h 9400); the scale bar is 50 lm (a, b). The NeuN (green)/ BrdU (red) double-positive cells distributed in the GCL. The filled

Ketamine Anesthesia in Postnatal Rats Inhibited the Growth of Astrocytes in the Hippocampal DG

The normal migration of newborn neurons in the hip- pocampal DG is dependent on the development of

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arrows point to the double-positive cells. The percentage of BrdU- positive cells expressing NeuN in each cell layers of GCL was calculated (d, e, f). Data are presented as the mean ± SD (n = 5). *p\0.05, **p\0.01 versus control group. SGZ subgranular zone, GCL granule cell layer (Color figure online)

astrocytes in this area, which play a supporting role in the migration of newborn neurons. The experimental protocol is presented in Fig. 6a. Our results showed that ketamine could restrain the growth of radial glial cells in the hip- pocampal DG on PND-37 and PND-44, and the density of

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Fig. 6 Effect of ketamine on the development of radial glia in the hippocampal dentate gyrus (DG). Experimental protocol (a). Repre- sentative photographs from a laser scanning confocal microscope are shown (b; magnification: a, c, e and g 9200; b, d, f and h 9400); the scale bars are 100 lm (a) and 50 lm (b). The density of GFAP

GFAP-positive cells in the hippocampal DG was signifi- cantly reduced in the ketamine-treated group compared to that in the control group (PND-37: 176 ± 9.96 vs. 230 ± 9.95 lm2; PND-44: 193 ± 12.62 vs. 244 ± 10.97 lm2)

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positive cells in the DG was calculated (c). The expression level of GFAP in the DG was measured by Western blot analysis at the same time points (d, e). Data are presented as the mean ± SD (n = 5). **p\0.01 versus control group. GCL granule cell layer, ML molecular layer, PCL polymorphic cell layer (Color figure online)

(Fig. 6c). Typical immunofluorescence pictures are shown in Fig. 6b.

The hippocampus tissues of rats on PND-37 and PND- 44 were used for Western blot analysis. Quantification of

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the Western blot showed that the ketamine anesthesia induced less visible bands representing GFAP compared with the control group at these two time points (PND-37: 57 ± 4.06 vs. 89 ± 3.44 %; PND-44: 76 ± 6.88 vs. 93 ± 3.41 %; Fig. 6d, e).

that neonatal ketamine exposure had a significantly inhibitory effect on the growth of radial glial cells, which may be an important reason for the inhibition of migration of newborn neurons.

Together,

these data suggest

Neonatal Ketamine Exposure Caused Spatial Memory Impairment in the Adult Stage

Figure 7a–d showed the memory and learning performance of rats in 2 months old. The latency to find the hidden platform of two groups rats had a reduced tendency as training progressed, which indicated that the animals were learning from practice of everyday. However, during the five training days, the latency to locate the hidden platform in ketamine group was significantly longer than that in control group (p \ 0.05; Fig. 7a), indicating neonatal

Fig. 7 Anesthesia with ketamine in neonatal rats at postnatal day 7 (PND-7) induces learning and memory impairment in the adult stage. Ketamine anesthesia significantly increased the latency time of rat swimming in the Morris water maze (MWM) as compared with the control group (a). The times that the rats stayed in the target quadrant within 120 s was significantly reduced in ketamine group than that in

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ketamine exposure could induce significantly impairment in learning and memory functions during the adult stage. In the memory retrieval tests, the times that the rats stayed in the target quadrant within 120 s was significantly reduced in ketamine group than that in control group (28 ± 9.02 vs. 44 ± 7.80 %; Fig. 7b). Also, the numbers of crossing over the previous platform site within 120 s was significantly in control group reduced in ketamine group than that (2 ± 0.75 vs. 5 ± 1.41; Fig. 7c). The typical track chart were shown in Fig. 7d. These data suggested that exposing ketamine (40 mg/kg 9 4 injections) to PND-7 rats could cause hippocampal-dependent neurocognitive impairment in the adult stage.

Discussion

Widespread and growing research has reported that keta- mine has neurotoxic effects on the developing animal brain (Ikonomidou et al. 1999; Liu et al. 2011; Paule et al. 2011; Zou et al. 2009), and its safety in pediatric anesthesia has

control group (b). The numbers of crossing over the previous platform site within 120 s was significantly reduced in ketamine group than that in control group (c). Typical path chart of space exploration were exhibited (d). Data are presented as mean ± SD (n = 6). *p\0.05, **p\0.01 versus control group (Color figure online)

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been the subject of extensive concern for anesthesiologists and the public, based on the evidence that ketamine may have an association with neurocognitive impairment in children (Pfenninger et al. 2002; Wilder et al. 2009). However, the causal link between neuron death in the developing animal brain induced by anesthetics and long- term hippocampal-dependent neurocognitive deficits has not been elucidated. It is therefore of interest for us to explore the other mechanisms that can explain the hip- pocampal-dependent neurocognitive dysfunction caused by neonatal ketamine exposure.

The substantial neurogenesis in the hippocampal DG lasts for life in animals and humans (Abrous et al. 2005). the production of During the process of neurogenesis, granule cells may change dynamically with age. In the rat, the granule cells in the DG are generated from the 14th day of gestation until the adult stage, and approximately 80 % of the granule cells are produced postnatally with a peak around seven days after birth (Altman and Bayer 1990). The accumulated results have demonstrated that the factors interfering with neuron production (e.g., postnatal or adult) may have a significant impact on hippocampus-dependent function (Young et al. 1999; Kempermann and Gage 2002). However, only some newborn neurons can be selected by the DG and allowed to migrate into the normal position of the GCL to meet the functional demand (Dupret et al. 2007; Kee et al. 2007). Hence, neurogenesis in the DG plays a crucial role in the normal of structure and function of the hippocampus.

Numerous studies have suggested that NMDA-R plays an important role in regulating the neurogenesis of the hippocampal DG (Joo et al. 2007; Kitayama et al. 2004; Luk et al. 2003). However, the effects of blocking NMDA- R on the neurogenesis of the hippocampal DG are con- troversial (Nacher et al. 2001; Nacher and McEwen 2006; Arvidsson et al. 2001). Ketamine, as an NMDA-R inhi- bitor, was reported to inhibit the proliferation of NSCs isolated from the SVZ in the rat fetal cortex and enhance its neuronal differentiation in a previous in vitro study (Dong et al. 2012); however, its effect on postnatal neurogenesis in the hippocampal DG has not been studied in vivo. Hence, it might provide a new perspective to study the neonatal neurotoxicity of ketamine.

BrdU, a classical tool for the detection of cell fate, was used to test neurogenesis. The scheme and dose of BrdU administration in our tests was based on previous experi- ments (Guidi et al. 2005; Zhang et al. 2014). We first observed the change in NSC proliferation and differentia- tion in the DG within two weeks after ketamine anesthesia. Our results showed that ketamine could significantly inhi- bit the proliferation of NSCs with decreased numbers of Nestin/BrdU double-positive cells. It was also found that the astrocytic differentiation of NSCs was markedly

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attenuated with a decreased number of GFAP/BrdU dou- ble-positive cells, while the neuronal differentiation of NSCs was obviously promoted with an increased number of b-tubulin III/BrdU double-positive cells. Our present results are partially consistent with the reports of Dong et al. (Dong et al. 2012). In addition, these effects of ketamine on the proliferation and differentiation of NSCs could last at least 1 week but disappeared 2 weeks after neonatal ketamine exposure. It is known that mature astrocytes can proliferate after exposure to some types of stimulation, such as stroke (Barreto et al. 2011). To exclude the proliferative mature astrocytes from the newly differentiated astrocytes, GFAP/BrdU double-labeling immunostaining was performed 3 h after the BrdU injec- tion, by which time BrdU had been adequately incorpo- rated newly differentiated astrocytes had not been generated. It was found that only a small number of mature astrocytes were capable of proliferating in the hippocampus of neonatal rats, and ketamine did not significantly promote or suppress the proliferation of mature astrocytes. There was no sig- nificant difference in the number of GFAP/BrdU double- positive cells between the control and ketamine groups. Therefore, it was determined that the GFAP/BrdU double- positive cells detected at 24 h after the BrdU injection could represent the newborn astrocytes differentiated from NSCs. In addition, to observe the effect of ketamine on the apoptosis of NSCs and astrocytes in the DG of neonatal rats, the nestin?/caspase-3? and GFAP?/caspase-3? cells were measured using double-labeled immunofluorescence. The results showed that neither nestin/caspase-3 nor GFAP/caspase-3 double-positive cells were found in the control or ketamine groups. Although neuron apoptosis has been demonstrated to be induced by neonatal exposure to ketamine, the present dosage and duration of ketamine were unable to induce the apoptosis of NSCs and astrocytes in the DG of neonatal rats. Thus, it is suggested that the reduced numbers of nestin/BrdU double-positive cells and GFAP/BrdU double-positive cells were not caused by cell death after ketamine exposure.

into

the

proliferative

cells,

but

the

It is necessary for the newly differentiated neurons to migrate into the GCL of hippocampal DG to exert normal function. The abnormal migration of newborn granule neurons in the hippocampal DG is associated with hip- pocampal-specific cognitive deficits (Manning et al. 2012). The present study showed that ketamine could markedly inhibit the migration of newborn neurons with a decreased percentage of NeuN/BrdU double-positive cells in each layer of the GCL in the hippocampal DG both at PND-37 and PND-44. Further study indicated that the number of GFAP- positive cells and the expression of GFAP in hippocampal DG were significantly reduced in the ketamine group com- pared to the control group. Our findings suggest that the

Neurotox Res (2016) 30:185–198

reduced expression of GFAP may be caused by suppressing the astrocytic differentiation of NSCs after neonatal keta- mine exposure. The inhibitory effect of ketamine on the growth of astrocytes may result in abnormally positioned newborn neurons within the GCL during neuronal migration because astrocytes play a support role in the migration of newborn neurons (Sibbe et al. 2009).

A previous study reported that colchicine injection into the DG caused the impairment in hippocampal-dependent spatial memory, but the lesion was limited to the DG rather than other hippocampal regions (Keith et al. 2007). This result suggested that the DG damage alone could produce a hippocampal-type neurocognitive dysfunction. According to the present study, neonatal ketamine exposure induced a significant alteration of neurogenesis in the hippocampal DG, which may be an important reason leading to abnor- malities in the structure of the hippocampus. It might have a close association with the ketamine-induced neurocog- nitive impairment.

The mechanisms by which ketamine induce the inter- ference of neurogenesis in the hippocampal DG remain to be determined. In our previous in vitro study, suppressing Ca2?-PKCa-ERK1/2 signaling pathway may be involved in this inhibitory effect of ketamine on hippocampal NSCs proliferation (Yu-Qing et al. 2014). Thus, our future studies will include exploring whether ketamine exposure affects the hippocampal neurogenesis process through interfering with the calcium signaling pathway in vivo study.

In summary, neonatal ketamine exposure could interfere the hippocampal DG, with postnatal neurogenesis of including the inhibition of NSC proliferation and astrocytic differentiation, the promotion of neuronal differentiation, the inhibition of astrocytic growth, and neuronal migration in the GCL. These findings may account for the adult hippocampal-dependent dysfunction induced by neonatal ketamine exposure.

neurocognitive

Acknowledgments This work was supported by the National Nat- ural Science Foundation of China (81171013), the Key Subject of Colleges and Universities Natural Science Foundation of Jiangsu Province (10KJA320052).

Compliance with Ethical Standards

Conflict of interest The authors have declared that no competing interests exist.

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