Compelling evidence in animal models have suggested that general anesthesia has unequivocal link to the change of developmental brain and persistent impairment in neurocognitive function. Recent large scale randomized controlled clinical trials confirm that the healthy infants received general anesthesia for less than 1 h is not associated with neurocognitive impairments in later childhood[33–34]. However, there is a lack of studies to clarify the potential impacts of longer exposure or multiple exposure to general anesthetics. The U.S. Food and Drug Administration (FDA) had issued that developmental neurotoxicity induced by multiple exposure or exposure several hours to anesthetics remains incompletely understood and the underlying mechanism required to be further investigated[35].
Neurogenesis occurs on a large scale of brain from a critical period of embryonic to postnatal stage, which includes NSC proliferation and differentiation. Neurogenesis in the certain brain is especially active throughout life in two distinct areas: the SVZ and hippocampal DG[16]. The disturbence of postnatal neurogenesis in the DG may lead to persistent impairments in learning, memory, and behaviors[36–37]. Previous findings have revealed that inhalation/intravenous anesthetics could affect the proliferation and differentiation of developmental hippocampal NSCs and lead to the long-term neurocognitive dysfunction[18–21]. On the basis of our precedent findings, the purpose of present study was to explore the mechanism of Notch signalling pathway in the role of ketamine exposure interfering with the neurogenesis of hippocampal dentate gyrus and damaging the learning and memory function in adulthood, and to provide a strong experimental basis for further understanding the neurotoxicity of ketamine in the neonatal period.
Based on Western blot analysis, 40 mg/kg ketamine anesthesia for 4 times in neonatal rats specifically inhibited the Notch signalling pathway, accompanied by a significant reduction in Jagged1, Notch1, NICD1 and Hes1 levels 24 h after the anesthesia in the hippocampal DG tissues. In addition, the same ketamine anesthesia in neonatal rats induced the long-term neurocognitive dysfunction at 2 months old. Given that Notch signalling pathway is closely relevant to the regulation of normal neurogenesis, we further investigated whether the ketamine-induced impairment of neurogenesis and neurocognitive function could be ameliorated by activating Notch1 signalling pathway.
The Notch1 receptor is a transmembrane protein, which can be activated by its ligand Jagged1. After interaction with ligand of the Jagged1, Notch1 receptor is cleaved by presenilin-1 and the γ-secretase enzyme complex, then a series of cleavages release the Notch1 intracellular domain (NICD1), which translocates into the nucleus where it induces transcription of Notch1 downstream target genes such as hairy enhancer of split (Hes) family member. In the present study, Jagged1 and LV-NICD1 were micro-injected into the bilateral hippocampal DG. Jagged1 can activate Notch1 signalling through extracellular pathway[38] and overexpression of NICD1 can activate Notch1 signalling through intracellular pathway[39]. Therefore, we choose two methods (Jagged1 or LV-NICD1) to activate Notch1 signalling through extracellular and intracellular pathway respectively. It was showed that the decreased expression of NICD1 and Hes1 induced by ketamine could be reversed by both Jagged1 and LV-NICD1 pretreatment.
The vast majority of NSCs are in a mitotically active condition in the developing brain, therefore, the balance between mitosis and quiescence of NSCs is crucial to maintain the neuron production and regeneration[40–41]. In recent years, postnatal neurogenesis is a particularly interesting filed in neuroscience to study the anesthetic induced neurotoxicity. Our previous study has revealed that neonatal ketamine exposure significantly inhibits NSC proliferation and astrocytic differentiation, meanwhile markedly enhances neuronal differentiation[22]. However, the potential mechanism has not been clarified. Notch signalling pathway is regarded as a ‘‘switch’’ to determine the NSC fate[25]. Although it has been demonstrated that up-regulation of NICD1 promotes the proliferation of NSCs in the postnatal hippocampal DG, and γ-secretase inhibitor or genetic ablation of Notch1 promotes cell cycle exit and the neuronal differentiation[42], the role of Notch1 signalling pathway in the disturbence of NSC proliferation and differentiation caused by neonatal ketamine exposure has not been clearly investigated. According to the methodology in our previous study[22], we performed the double staining to analyze the ratio of NSC proliferation and differentiation and the density of double positive cells in the hippocampal DG, and discovered the increase of NSC proliferation and astrocytic differentiation combined with the inhibition of neuronal differentiation in rats receiving Jagged1 or LV-NICD1 treatment compared with those in control groups. These results were mainly consistent with the findings in the previous research. The Hes family genes are essential effectors of Notch signalling pathway and negative regulators of neuronal differentiation, and upregulation of Hes1 could contribute to an decrease of neuronal population[43–44]. This study confirmed that the down expression of Notch targeted gene Hes1 induced by ketamine could be reversed by the Jagged1 or LV-NICD1 treatment. In addition, the disturbence of NSC proliferation and differentiation caused by ketamine could be specifically reversed by pre-activating the Notch1 signalling pathway with Jagged1 or overexpression of NICD1 in the hippocampal DG. All of the above findings suggested that neonatal ketamine exposure interfered with the proliferation and differentiation of NSCs in the hippocampal DG by inhibiting the Notch1-Hes1 signalling pathway.
A growing body of evidence strongly suggested that postnatal neurogenesis in the hippocampal DG may be highly susceptible to various physiological and pathological stimulation[37]. The damage of postnatal neurogenesis in the hippocampal DG has been associated with cognitive dysfunction in rodent models, such as the impairment in spatial learning, memory retention, memory retrieval. Our previous study had revealed that multiple exposure to clinically relevant dose of ketamine may interfere with hippocampal neurogenesis and long-term neurocognitive function in PND-7 rats. In this study, Jagged1 or LV-NICD1 was confirmed to ameliorate ketamine-induced long-term neurocognitive dysfunction, indicating that the proliferation and differentiation of neural stem cells regulated by Notch signaling pathway played a critical role in maintaining normal cognitive function.
Neurogenesis is a complicated process that includes not only NSC proliferation, neuronal and astrocytic differentiation but also the migration of newborn neurons and the formation of neuronal circuit. The appropriate number of newborn neurons in the developing brain is crucial for their migration and establishment of synaptic function[45]. Increased differentiation of NSCs into neurons during development stage may be detrimental to the formation of normal neural circuits. Our previous study indicated that ketamine interfered with the neurogenesis in hippocampal DG in PND-7 rats. Although ketamine promoted the neuronal differentiation of NSCs, the migration of newborn neurons in the GCL of hippocampal DG was markedly inhibited by ketamine[22]. In the present study, although neonatal exposure to ketamine markedly increased the differentiation of NSCs into neurons, the neurocognitive performance was significantly impaired in adulthood.
The Notch signalling pathway has been proved to be activated in the regulation of adult neurogenesis in the animal stroke model, including the promotion of NSC proliferation and acceleration of neuronal differentiation[46]. The present conclusions are not completely consistent with the previous findings partly because that the NSCs in the neonatal hippocampal DG have different biological characteristics compared with that in adult hippocampal neurogenesis[47]. During brain growth spurt (BGS), the peak levels of dentate gyrus neurogenesis in rodents last from the end of pregnancy to the first two weeks after birth, then with a change to a lower rate during adult stage[48]. The present study revealed that multiple ketamine exposure during neonatal stage influenced the balance between the activity and quiescence of NSCs, and also influenced the quantity and percentage of neurons/astrocytes in the developing hippocampal DG by inhibiting Notch1-Hes1 signalling pathway. However, the majority of NSCs in the adult hippocampal DG were kept in an inactive state. The promotion effects on the proliferation and neuronal differentiation of NSCs induced by stroke through activating the Notch1 signalling pathway should be taken as a protective mechanism to compensate the loss of neurons.
There are several limitations in this study. Although the present results showed neonatal anesthesia by ketamine in rats interfered with the proliferation and differentiation of hippocampal NSCs via inhibition of notch1 signalling pathway, it will be better to obtain the additional results in vitro. In addition, the effects of neonatal ketamine anesthesia on the migration of newborn neurons and the formation of neuronal circuit remain to be further investigated. Finally, although the present study demonstrated that neonatal exposure to ketamine could impair neurocognitive function in adulthood, its effect on hippocampal synaptic plasticity such as the growth of dendrites and dendritic spines as well as long-term potentiation (LTP) remain to be clarified.
In conclusion, the present study indicates that neonatal exposure to ketamine in rats interferes with the proliferation and differentiation of hippocampal NSCs and impairs neurocognitive function in adulthood via inhibition of Notch1 signalling pathway. These findings contribute to further understanding of the neonatal neurotoxicity induced by general anesthesia and its underlying mechanisms.