Bioscience, USA) and Neuroexplorer (ver.4.50.4; MBF. Bioscience), and the density and length of dendritic spines were determined. Statistical analysis. All data ...
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Original article Effects of ketamine and midazolam on morphology of dendritic spines in hippocampal CA1 region of neonatal mice TAN Hong, REN Rong-rong, XIONG Zhi-qi and WANG Ying-wei Keywords: dendritic spines; ketamine; midazolam; confocal Background It is a common phenomenon that children experience multiple general anesthesias in clinical practice, which raises the question whether repeated exposure to general anesthetics would interfere with the development of the central nervous system of children. The present study was designed to evaluate the effects of repeated treatment with ketamine or midazolam on postnatal dendrite development by examining the morphology of the dendritic spines of the pyramidal neurons in the hippocampal CA1 region in mice. Methods The transgenic green fluorescent protein-M line (GFP-M) mice were used in this study. Ketamine (100 mg/kg), midazolam (50 mg/kg) or saline (10 ml/kg) was administered intraperitoneally once a day on consecutive days from postnatal day 8 (P8) to postnatal day 12 (P12). At postnatal day 13 (P13) and postnatal day 30 (P30), the density and length of the apical dendritic spines of the pyramidal neurons in the hippocampal CA1 region were examined under a confocal microscope. Results At P13, for both the ketamine group and the midazolam group, the dendritic spines were found with a comparatively lower density and longer average length than in the control group. At P30, no significant difference in the density or average length of dendritic spines was found between the anesthetic group and control group. Conclusions This study indicated that repeated exposure to ketamine or midazolam in neonatal mice impaired dendritic spine maturation immediately afterwards, but this influence seemed to disappear during further postnatal development. Chin Med J 2009;122(4):455-459
D
uration and complexity of anesthesia procedures for newborns have increased as the consequence of the rapid advances in pediatric surgery and other procedures in clinical practice. Clinical anesthetists often have to explain to anxious parents about their concerns as to whether the anesthetics will interfere with the development of their child′s intelligence. To answer these questions, it is necessary to understand the effects of anesthetics on the developing nervous system, and this is especially critical for those who have to undergo a long period of anesthesia in their medical treatment.1 It is well known that the central nervous system (CNS) is not fully developed at birth and it takes a period of time for further maturation; so called synaptogenesis. For example, in mice rapid and intense synaptogenesis occurs within the first 2–3 postnatal weeks.2,3 The maturation of dendritic spines, the tiny protrusions on dendrites which receive excitatory synaptic input,4 plays a major role in the process of synaptogenesis. Against this background, we studied the density and length of dendritic spines to investigate the influence of anesthetics on the development of the CNS. Currently used anesthetics in clinical practice mainly work by two principal mechanisms:5 Gamma-Amino Butyric Acid A (GABAA) receptor agonists (e.g. midazolam, propofol and isoflurance) and N-methyl-Daspartate (NMDA) receptor antagonists (e.g. ketamine, nitrous oxide and xenon). These anesthetics are
considered comparably safe6 as the FDA certifies their legal application. Recent findings, however, indicated that these drugs could induce widespread neuronal apoptosis in immature rat brain if they were administered during synaptogenesis.7 This raised the questions regarding the potential risk of anesthetics to brain development. While the effects of anesthetics on the morphology of dendrite development has been studied in vitro,8 the effects in vivo are not clear. In this experiment, we studied the effects on dendrite development of repeated exposure to ketamine (NMDA antagonist) or midazolam (GABAA agonist), two general anesthetics commonly used in pediatric anesthesia. After the treatment we examined the morphology of dendritic spines of the hippocampus as it is a key brain region for high brain functions (e.g. learning and memory). Green DOI: 10.3760/cma.j.issn.0366-6999.2009.04.0018 Department of Anesthesiology, Xinhua Hospital, Shanghai Jiao Tong University, College of Medicine, Shanghai 200092, China (Tan H, Ren RR and Wang YW) Institute of Neuroscience and Key Laboratory of Neurobiology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China (Xiong ZQ) Correspondence to: Prof. WANG Ying-wei, Department of Anesthesiology, Xinhua Hospital, Shanghai Jiao Tong University, College of Medicine, Shanghai 200092, China (Tel: 86-2165790000 ext 7951. Fax: 86-21-65153984. Email: wangyingwei@ yahoo.com) This study was supported by a grant from Shuguang Program (08SG18) of Shanghai Municipal Education of Commission.
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Figure 1. Confocal microscopy images showing the morphology of hippocampal CA1 pyramidal neurons after repeated exposure to ketamine and midazolam in GFP-M mice at P13. Ketamine group (A,D,G), midazolam group (B,E,H), control group (C,F,I). Only a few pyramidal neurons are labeled by GFP and whole dendrites and their spines are clearly visible. Scale bars=100 µm (A-C) and 10 µm (D-F) and 3.3 µm (G-I).
fluorescent protein-M line (GFP-M) mice were used in the research because the dendritic spines of the hippocampal CA1 pyramidal neurons can be clearly visualized.9 In order to study the morphology of dendritic spines so as to demonstrate the effects of anesthetics during synaptogenesis, we developed an in vivo experiment to observe the density and length of spines in the CA1 area under a confocal laser scanning microscope. METHODS Animals GFP-M male and female mice were used in the present study. We chose GFP-M transgenic mice since some of the pyramidal neurons of hippocampal CA1 region can be marked by enhanced green fluorescent protein (EGFP) under a Thy1 promoter. These GFP-expressing pyramidal neurons take on Golgi-like appearance (Figure 1). As previous research demonstrated,9 the expression of GFP for up to 9 months has no discernible effect on synaptic structure. Thus, it would not interfere with the natural development of the mice and they could be a good model to study. The mice were housed in cages containing paddy husk as bedding material which was changed on alternate days. They were kept in a well-aerated room and
given a 12-hour light/dark cycle. The room temperature was maintained at 23°C. The experiments were approved by the Committee of Use of Laboratory Animals and Common Facility, Institute of Neuroscience, Chinese Academy of Sciences. Anesthetic treatment Eight-day-old GFP-M mice were used for all experiments. We gave intraperitoneal injection of anesthetics consecutively from postnatal day 8 (P8) to P12 during synaptogenesis, as the skin was too weak to tolerate intraperitoneal injection before P8. The effective anesthesia dose of intraperitoneal injection we adopted for maintaining a surgical plane of anesthesia was ketamine (Hrs, China, 100 mg/kg body weight), and midazolam (Npc, China, 50 mg/kg body weight). Totally 24 GFP-M mice were involved in the experiment. Those P8 mouse pups were randomly divided into three groups and received a single intraperitoneal injection per day of ketamine 100 mg/kg; midazolam 50 mg/kg and saline (Scrc, China) 10 ml/kg for five days. Then eight mice of each group were randomly divided into two groups. One was used for image studies at P13 for short-term post-anesthesia effects. The other was used for image studies at P30 for prolonged post-anesthesia effects.
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Slices preparation Mice were perfused with 4% paraformaldehyde (Sigma, USA) in 0.1 mol/L phosphate buffer (PB; pH 7.4) and brains were removed. Then the brains were transversely cut into 50 µm-thick sections after cryoprotection with 20% sucrose in 0.01 mol/L PBS. The slices were mounted onto gelatin (Sigma) glass slides and covered by 75% glycerin (Sigma) in PBS. Confocal microscopy Since many previous studies indicated that the hippocampus is closely related to learning and memory10,11 and in hippocampus of GFP-M mice, GFP is expressed more stably in CA1 region; we chose the hippocampal CA1 region as the target of the observation. GFP positive dendritic spines were imaged using a Zeiss 510 Meta confocal laser scanning microscope (Carl Zeiss MicroImaging GmbH, Germany). It produces a series of stacked images covering three-dimensional depths of dendrites and dendritic spines, allowing a more sensitive detection of dendritic spines as compared with Golgi staining by scrolling forward and backward through the entire series of stack images. Three to five GFP positive pyramidal neurons located in the hippocampal CA1 region were chosen at random in each brain. A 488 nm argon laser was used to detect GFP. The dendritic spines on the second and third order apical dendrites of pyramidal neurons were photographed. Dendrites were found using a 10× objective lens (zoom=1) (Figure 1A, B and C), then, a stack of spine images at 0.5 µm intervals was collected using a 63× objective lens (zoom=2) (Figure1 D, E and F). All images from three groups were handed over to a computer operator who was blinded as to the treatment of each group. These images were processed with Neurolucida software (ver.7.50.2; MBF Bioscience, USA) and Neuroexplorer (ver.4.50.4; MBF Bioscience), and the density and length of dendritic spines were determined. Statistical analysis All data were expressed by mean±standard error (SE) and were performed by the SPSS 15.0 statistical package. The Student′s t test, analysis of variance and q test were used and a difference between groups of P < 0.05 was considered significant. RESULTS Effects of ketamine or midazolam to the density and length of dendritic spines observed at P13 The density of dendritic spines can be taken as a measure of the complexity of the neuronal network and the higher density reflects increased connectivity between neurons,4 so we calculated the density of dendritic spines from the hippocampal CA1 region during synaptogenesis at P13 following the injection of anesthetics from P8 to P12. The density of dendritic spines was (0.751±0.041) spines/µm in the ketamine group, (0.701±0.033) spines/µm in the midazolam group and (1.002±0.043) spines/µm in the control group. These results demonstrated that repeated
injection of ketamine or midazolam would result in lower density of dendritic spines compared with the control group at P13 (P
