Neuroprotective effects of dexmedetomidine against the ketamine-induced disturbance of proliferation and differentiation of developing neural stem cells in the subventricular zone

Background: Recent years, the number of neonatal patients receiving surgery under general anesthesia is increasing. Previous studies have indicated that ketamine can disturb the proliferation and differentiation of developing neural stem cells (NSCs). Therefore, the safe use of ketamine in pediatric anesthesia has drawn great concern among anesthesiologists and children’s parents. Dexmedetomidine (DEX) is widely used in sedation, antianxiety and analgesia. Recent studies have shown that DEX could provide neuroprotection against anesthetic-induced neurotoxicity in the developing brain. The aim of this in vivo study was to investigate whether DEX had neuroprotective effects on the proliferation and differentiation of NSCs in the subventricular zone (SVZ) following neonatal ketamine exposure. Methods: Postnatal day 7 (PND-7) male Sprague-Dawley rats were equally divided into the following 5 groups: Control group (n=8), ketamine group (n=8), 1 μg/kg DEX+ketamine group (n=8), 5 μg/kg DEX+ketamine group (n=8) and 10 μg/kg DEX+ketamine group (n=8). The proliferation and differentiation of NSCs in the SVZ were assessed by immunostaining with BrdU incorporation. Results: Neonatal ketamine exposure significantly inhibited NSC proliferation and astrocytic differentiation in the SVZ, and neuronal differentiation was markedly promoted. Furthermore, DEX pretreatment reversed the ketamine-induced disturbances in the proliferation and differentiation of NSCs at moderate (5 μg/kg) or high doses (10 μg/kg). Conclusion: Our findings demonstrate that DEX may have neuroprotective effects on NSCs in the SVZ of neonatal rats in a repeated ketamine anesthesia model.


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Ketamine is widely used in analgesia and sedative processes during pediatric examination and surgical operation [1][2]. Recently, an increasing number of research have suggested that neonatal ketamine exposure could cause neuroapoptosis and disturb normal neurogenesis in the developing brain and increase the risk of delayed neurocognitive dysfunction [3][4][5][6]. The evidence from clinical studies also supported that ketamine had long-term adverse effects on the neurocognitive function in children and infants [7][8].
Relevant research conclusions have prompted anesthesiologists to reevaluate the safety of using ketamine in pediatric anesthesia and to look for possible protective measures.
Dexmedetomidine (DEX), a highly selective α 2 -adrenoceptor agonist, is widely used for anxiolytic, sedative and analgesic in clinical pediatric anesthesia and intensive care [9][10]. DEX has shown protective effects on vital organs, including the decrease in lung and kidney damage and the reduction in neural apoptosis [11][12]. In anesthesia models of neonatal animal, DEX has been proven to produce protective effects on inhalation anesthetics-induced neurotoxicity in the developing brain [13][14]. In the clinical pediatric anesthesia, a medication strategy with DEX is increasingly being accepted as a way to reduce the adverse effects of ketamine [15][16]. However, the potential neuroprotective pathway of DEX needs to be further investigated.
Neurogenesis in the hippocampus and subventricular zone (SVZ) are important processes in the developing brain [17][18]. Early disruption of these regions has an adverse potential to alter the formation of neural circuits [19]. At present, little is known about whether DEX has protective effects on the ketamine-induced neurotoxicity of NSCs in SVZ. Therefore, the aim of this study was to investigate the impact of DEX pretreatment on neurogenesis in the SVZ in a repeated ketamine exposure model of neonatal rats, including detecting the proliferation capacity of NSCs and neuronal and astrocytic differentiation.

Animals and drug administration
All animal procedures were approved by the Institutional Animal Care and Use Committee of NanJing Medical University. Timed-pregnant Sprague-Dawley rats were housed at 24 °C on a 12-h light/dark cycle with free access to food and water. The postnatal day 7 (PND-7) male rats (11-14 g) selected from all the pups were used in the experiments. To ensure that hypoxia did not occur during anaesthesia in all newborn animals, arterial blood (0.25 ml) gas determination was performed by cardiac puncture immediately after the end of anesthesia through a 30-gauge needle (6 animals per group). Arterial blood gases were measured with a portable clinical analyzer (GEM Premier 3000 USA).

BrdU injections
Immediately after the treatment of each group, the rat received a single intraperitoneal injection of BrdU (5-bromo-2-deoxyuridine; Sigma, 100 mg/kg) in 0.9% NaCl solution. The animals were anesthetized with an overdose of chloral hydrate and fixed by perfusion at 24 h after BrdU injection. The detailed experimental protocol is listed in Table 1. Tissue preparation and immunofluorescence The brain of rats was isolated, removed and fifixed using 4% paraformaldehyde fixative for 6 h. Then, the brain was embedded using medium on ice and stored at -80 °C. The coronal brain sections were cut on a microtome at a thickness of 30 µm. When the SVZ was initially exposed in the slice, we selected three successive brain slices after each cut and a total nine brain slices were collected in each rat. Immunofluorescence procedures were conducted as described in our previously reported methods [5]. The sections were incubated with 50% formamide in PBS for 2 h at 65 °C preceded 2 N hydrochloric acid incubation for 30 min at 45 °C and 3 washes with PBS for 10 min. Blocking of nonspecific epitopes with 10% donkey serum in PBS with 0.3% Triton-X for 2 h at RT preceded incubation overnight at 4 °C with the appropriate primary antibody (Table 2)   List of primary antibodies, their suppliers, the animal used to raise the antibodies in, and the working dilution.

Immunofluorescence area quantifification
The sections were observed by a skilled pathologist blinded to this research using image stacks on a laser scanning confocal microscope (Fluoview 1000, Olympus). For each sample, we selected three sections for immunofluorescence of BrdU, three sections for immunofluorescence of β-tubulin III/BrdU and three sections for immunofluorescence of GFAP/BrdU respectively. The fluorescence area was measured using Image J (from the National Institutes of Health, Bethesda, MD, USA).

Statistical analysis
The data about proliferation and differentiation of NSCs were expressed as the medians and interquartile ranges (IQRs). The results about blood gas were expressed as mean ± SD. The statistical analysis and the graphs were generated using GraphPad Prism 5.
Significant differences between the groups were analyzed by one-way ANOVA. P < 0.05 was considered statistically significant.

Results
The arterial blood gas analyses In the experiment, all rats in each group survived until the end of the experiment. We observed that the animals' skin was ruddy and that the respiration was smooth. There were no significant changes in the pH, pCO 2 and pO 2 in any groups (Table 3). Table 3 The arterial blood gases analyse ( Dexmedetomidine protects NSCs from ketamine-induced injury Next, we investigated the impact of dexmedetomidine (DEX) on the ketamine-induced disturbance of the proliferation and differentiation of NSCs in the SVZ. As shown in Fig. 1D and 1E, our findings suggested that pretreated with 5 µg/kg DEX or 10 µg/kg DEX before  Fig. 3A and 3B). However, 1 µg/kg DEX pretreatment had no protective effect on the disturbance of proliferation and differentiation of NSCs induced by ketamine (p > 0.05, Fig. 1F, Fig. 2B and 3B).
In summary, these results suggest that moderate and high-dose DEX pretreatment may alleviate the disturbance in the proliferation and differentiation of NSCs in the SVZ induced by ketamine.

Discussion
Ketamine is a dissociative anesthetic and commonly used in pediatric anesthesia. An increasing preclinical evidence suggested that ketamine may causes neurodegeneration and neuroapoptosis in the developing brain and precipitate significant long-term cognitive sequelae in rodents and nonhuman primates. Epidemiological evidence has indicated that long-term neurotoxicity may ensue following prolonged and/or repeated exposure to ketamine in early life. The U.S. Food and Drug Administration (FDA) had issue the warning about prolonged and/or repeated exposure to general anesthetics may have a negative impact on developing children's brain [20].
Neurogenesis is an important process that occurs in multiple brain regions during the embryonic to adult stages, especially in the hippocampal dentate gyrus (DG) and subventricular zone (SVZ) [17][18]. The normal proliferation and differentiation of NSCs maintain a balance between the types and numbers of cells in the brain, which are the origin of neurons and glial cells. In rodents, the large number of neurons were established during gestation and extends for the first 21 postnatal days, which play a critical role in the formation of neural networks and neurocognitive functions. Our previous findings suggested that neonatal ketamine exposure disrupted the proliferation and differentiation of developmental NSCs in the hippocampal DG and caused hippocampus-dependent spatial memory dysfunction during the adult stage [5].
During the developmental neurogenesis in the SVZ, newly generated neurons migrate along the rostral migratory stream (RMS) to the olfactory bulb (OB), where the neurons were incorporated into the existing neural circuits and played a crucial role in long-term olfactory recognition memory [21][22]. Previous studies had suggested that early disruption of SVZ neurogenesis had the potential to alter the formation of neural circuit [23] and olfactory cognitive function during the adult stage [24]. This study demonstrated that ketamine exposure (40 mg/kg × 4) during the developmental stage significantly inhibited NSCs proliferation and astrocytic differentiation and promoted neuronal differentiation in the SVZ, which were consistent with our previous findings about fates of NSCs in the hippocampal DG and SVZ [5,25].
During clinical anesthesia procedure, the safe anesthetics including neuroprotective have been identified based on pharmacological and molecular biological probes [26].
Evidences had shown that α 2A subtype had a close relevance regarding sedation, analgesia, neuroprotection and sympatholysis [27]. The role of DEX in neonatal intensive care medicine and pediatric anesthesia has been an interesting topic of research in recent years. In preclinical studies, DEX has caught the attention of researchers and clinicians because it could produce neuroprotective properties on hippocampal neurogenesis and neuronal plasticity in the model of neonatal brain injury [28].
In the study regarding anesthetic-induced developmental neurotoxicity, DEX has been proven to produce neuroprotective effects on anesthetic-induced neurodegeneration and neuroapoptosis in the developing brain [13,14,[29][30][31]. A recent in vitro study demonstrated that DEX could protect embryonic cortex NSCs from ketamine-induced injury [32]. Although DEX has been extensively studied as a clinical anesthetic adjuvant DRUG, its neuroprotective effects on the proliferation and differentiation of NSCs exposed to ketamine are poorly understood at the animal level. In this study, the dose of DEX was based on clinical concentrations that had been used in children (1 µg/kg) [33]    Data are shown as the mean ± SD (8 tissue sections per group). **p<0.01 vs.