new_pdfs/10.1002_cbin.10349.txt

Cell Biology International ISSN 1065-6995 doi: 10.1002/cbin.10349

RESEARCH ARTICLE

Role of miR-34c in ketamine-induced neurotoxicity in neonatal mice hippocampus

Shu-e Cao*, Jianmin Tian, Shengyang Chen, Xiaoran Zhang and Yongqiang Zhang

Department of Anesthesiology, The First Affiliated Hospital of XinXiang Medical College, WeiHui, HeNan Province 453100, China

Abstract

Ketamine is a commonly used pediatric anesthetic, but it might affect development, or even induce neurotoxicity in the neonatal brain. We have used an in vivo neonatal mouse model to induce ketamine-related neurotoxicity in the hippocampus, and found that miR-34c, a microRNA associated with pathogenesis of Alzheimer’s disease, was significantly upregulated during ketamine-induced hippocampal neurodegeneration. Functional assay of silencing miR-34c demonstrated that downregulation of miR-34c activated PKC-ERK pathway, upregulated anti-apoptotic protein BCL2, and ameliorated ketamine-induced apoptosis in the hippocampus. Cognitive examination with the Morris water maze test showed that ketamine-induced memory impairment was significantly improved by miR-34c downregulation. Thus, miR-34c is important in regulating ketamine-induced neurotoxicity in hippocampus.

Keywords: hippocampus; ketamine; miR-34c; neurotoxicity

Introduction

Ketamine, synthesized in 1962, has been widely used in clinic anesthesia due to its rapid onset and minimal side-effects (Domino, 2010). Pharmacologically, the major mechanism of ketamine working as anesthetic is to inhibit the glutamate neuro-transmission through N-methyl-D-aspartate (NMDA) receptors (Ikonomidou et al., 1999 Olney et al., 1991), and a ketamine overdose could severely affect the development of neonatal brain and induce cortical neurotoxicity in both animals and humans (McGowan and Davis, 2008; Brambrink et al., 2012 Dong and Anand, 2013). In hippocampus, the major component of brain associated with memory and learning, new evidence had revealed through both in vitro and in vivo animal models, that repetitive or high dose adminis- tration of ketamine suppressed neural excitability, induced apoptosis impair learning and memory functions (Huang et al., 2012 Huang et al., 2013). Little is known about the underlying mechanisms or the associated signaling pathways during the process of hippocampal or memory neurodegeneration induced by anesthesia.

significantly

in hippocampus,

thus

MicroRNAs are endogenously expressed noncoding short RNAs regulating gene silencing by suppressing the transla- tion or degrading targeted messenger RNAs (Kim, 2005). They are abundantly expressed in various components in the brain, being involved in modulating embryogenesis, neural development, and maturation (Kosik and Krichevsky, 2005; Darnell et al., 2006 Kosik, 2006; Lau and Hudson, 2010). Among them, microRNA 34 c (miR-34c) is a member of miR-34 family, which includes three homologous miRNAs expressed at two different loci of chromosome (miR-34a, miR-34b, and miR-34c), and are involved in various aspects of neural development or degeneration (Agostini et al., 2011; Casci, 2012; Liu et al., 2012). miR-34c is a newly discovered modulator associated with pathogenesis of neurodegenera- tive disease (Zovoilis et al., 2011). Thus, we have determined whether miR-34c may regulate anesthesia-related neurotox- icity in hippocampus. Ketamine was introduced into an in vivo neonatal mouse model to induce anesthesia-related hippocampal neurotoxicity, and the effect of ketamine- induced neurodegeneration on the expression level of miR- 34c in hippocampus was measured. A lentiviral vector was used to downregulate miR-34c to investigate its functional

(cid:1)Corresponding author: e-mail: yongqiang.zhang@aol.com

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role in modulating anesthesia-induced neurotoxicity in hippocampus in vivo.

Materials and methods

Animals

C57BL/6 mice were purchased from Shanghai Laboratory Animal Center, Chinese Academy of Sciences (Shanghai, China). The in vivo induction of ketamine-related hippo- campal neurotoxicity was done at 2 weeks. Quantitative real time PCR of miR-34 family was used at 3 weeks, as was hippocampal injection of lentivirual vector of miR-34c. For analyses of TUNEL staining and Western blotting, 2-month old mice were used. All experimental procedures were reviewed and approved by the Animal Care Committee at the first affiliated Hospital of XinXiang Medical College.

Induction of ketamine-related hippocampal neurotoxicity

The in vivo protocol to induce ketamine-related hippocam- pal neurotoxicity was done as before with slight modifica- tions (Hayashi et al., 2002; Huang et al., 2012, Liu et al., 2012). Young C57BL/6 mice, postnatal 14 days, were intraperitoneally administrated with repeated dosage of 75 mg/kg ketamine per day for six consecutive days (n ¼ 28). Normal saline was injected in the control group of mice (n ¼ 25).

RNA isolation and reverse transcription

Hippocampal RNA was isolated with Trizol reagent (In Vitrogen, Carlsbad, CA, USA). Briefly, mice were anesthe- tized and decapitated. Hippocampal samples were retrieved and homogenized at 1 mL Trizol/0.1 g tissue. The quantity of RNA was assessed by spectrophotometry followed by 1% agarose gel electrophoresis. Total RNA was treated with 10 U of RNase free DNase I, and reverse transcription (RT) was done in a total volume of 20 mL with random hexamer primers using a High-Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA, USA). cDNA was stored at (cid:3)20(cid:4)C until further use.

Quantitative RT-PCR

Expression of miR-34a, miR-34b, miR-34c, and house- keeping gene GAPDH were measured by TaqMan micro- RNA RT-PCR on the ABI 7900 Real-time PCR System (Applied Biosystems, Foster City, CA, USA). Expression profiles of each gene were quantified using corresponding standard curves. End-point RT-PCR of miR-34a, miR-34b, miR-34c, and GAPDH used 50 ng of total RNA with a mirVana RT-PCR miRNA Detection Kit (Ambion, Austin,

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Texas, USA). PCR products were separated and visualized on a 4% agarose gel. Each sample was run in triplicate and a mean value of each Ct triplicate was used.

Lentivirus production and transduction

To downregulate miR-34c, the coding sequence for a 2’-O- methyl oligonucleotide of miR-34c inhibitor was UCCGU- CACAUCAAUCGACUAACG, and the non-specific control antisense sequence was UACUCUUUCUAGGAGGUU- GUUAUU (Yu et al., 2012). These two sequences were amplified and cloned into pCDH-CMV-MCS-EF1-coGFP for in vivo gene transfer, resulting in a miR-34c inhibitor vector (lenti-miR34c-I) and miR-34c non-specific control vector (lenti-miR34c-C) (System Biosciences, Mountain View, CA, USA). The lentivirual expression vectors and pPACK packaging vector were co-transfected into 293T cells, and viral particles were collected and concentrated to high titer.

Hippocampal injection

One day after the 6-day ketamine treatment, the injections of lent viruses were performed on the right side of the cortex. A tiny hole was drilled above hippocampus and a Hamilton syringe was used to inject 2 mL of lentivirus of miR-34c inhibitor (lenti-miR34c-I, 20 mM, n ¼ 17) or non- specific control (lenti-miR34c-C, 20 mM, n ¼ 14) at the coordinates assessed from bregma and skull surface: lateral þ1.5 mm, and vertical anteroposterior (cid:3)2.0 mm, (cid:3)1.5 mm. After injection, the incision was quickly sealed with dental cement.

Western blotting

Western blotting analysis was conducted at 2 months. Four mice with Lenti-miR34c-I injection and four mice with Lenti-miR34c-C injection were included in this analysis. Forty micrograms of hippocampal protein were collected and separated on an 8% NuPage Gel with MES buffer (Invitrogen, Carlsbad, CA, USA) and transferred to a polyvinylidene difluoride membrane. Primary antibody dilutions included 1:500 BCL2 (Santa Cruz, USA), 1:100 phosphorylated-PKC (p-PKC) (Sant Cruz Biotechnologies, Santa Cruz, CA, USA), 1:100 phosphorylated-ERK (p-ERK) (Sant Cruz Biotechnologies, Santa Cruz, CA, USA), and 1:1,000 b-actin (Cell Signaling, Danvers, MA, USA). Membranes were then incubated in primary antibody in Odyssey Blocking Buffer at 4(cid:4)C for 24 h, followed by three washes in 0.1% PBS-T and 1 h incubation at RT with 1:1,000 secondary antibodies. The films were visualized and quantified on the Odyssey Infrared Imaging Center (Li-Cor, Lincoln, NE, USA).

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TUNEL staining for hippocampal apoptosis

Hippocampal slices (350 mm) were prepared for terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) staining to detect the apoptosis, using an In Situ Cell Death Detection Kit according to manufacturer’s protocol (Roche, Branchburg, NJ, USA). Five mice with Lenti-miR34c-I injection and 5 mice with Lenti- miR34c-C injection were included in the analysis. Hippo- campal CA1 region was examined under a fluorescent scope. The apoptotic CA1 neurons were identified based on their size, location and immuno-reaction to TUNEL staining. The average number of the apoptotic neurons per 0.01 mm2 was measured and compared between control hippocampi and hippocampi treated with miR-34c inhibitor.

Morris water maze (MWM) testing

The MWM testing was carried out 1 month after hippocampal transfection of miR-34c knockdown. Eight mice with Lenti-miR34c-I injection and 5 mice with Lenti- miR34c-C injection were included in this analysis. In a large circular tank with a transparent platform (10 cm (cid:5) 10 cm), warm water at 26(cid:4)C was added to submerge the platform 1 cm below the surface. Visual cues of color paints were used to aid mice in locating the platform. The mice were given training sessions four times per day for one week before final testing. In each training session, the mice were put in the maze to locate the platform in 2 min followed by resting on the platform for 30 s. If mice did not locate the platform in 2 min, they were aided with flashing lights to the platform with 30 s of rest on top of the platform. On the final day of examination, the average swimming time and swimming distance were compared between control mice and the mice with miR-34c knockdown.

Statistical analysis

Statistic analysis was conducted with SPSS software (version 11.0). The measured data were presented as mean (cid:6) stan- dard deviations. The statistical differences were measured with a Student’s t-test, and the significance set at P < 0.05.

Results

miR-34c is upregulated in hippocampus by excessive ketamine administration

We examined whether the expression profiles of the miRNAs in the mirR-34 family would be modulated by excessive ketamine application that induces neurodegener- (Hayashi et al., 2002, Huang ation in hippocampus et al., 2012; Liu et al., 2012). After injecting C57BL/6 mice at 2 weeks with 75 mg/kg ketamine for 6 days, they were

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killed on the 7th day. Hippocampal mRNAs of miR-34 family, including miR-34a, miR-34b, and miR-34c, were measured by quantitative real-time PCR (qPCR). miR-34c was particularly upregulated, whereas miR-34a or miR-34b was relatively unchanged (Figure 1).

Knocking down miR-34c ameliorates ketamine-induced hippocampal neurotoxicity

To see if miR-34c has a role in modulating the neurotoxicity in hippocampus induced by excessive administration of keta- mine, we gave repeated daily administration of ketamine through systemic injection in young mice (P14) for six consecutive days. In hippocampus, the neurons in CA1 regions underwent significant apoptotic neurodegeneration, as previously reported (Hayashi et al., 2002, Huang et al., 2012; Liu et al., 2012). However, after injection of lentivirus containing miR-34c inhibitor (lenti-miR34-I) into mouse hippocampus on P21, TUNEL staining on 2-month old mouse hippocampal CA1 region showed that the number of apoptotic neurons was significantly reduced (Figure 2A), being about half of the number of apoptotic neurons in mice injected with control lentivirus (lenti-miR34c-C) (P < 0.05). Thus, inhibition of miR-34c helps ameliorate anesthesia- induced neurotoxicity in the hippocampus.

Knocking down miR-34c upregulates anti-apoptotic pathways during ketamine-induced hippocampal neurotoxicity

After systemic ketamine administration and hippocampal miR-34c lentivirus injection, Western blotting analysis was used on the 2-month old mice (Figure 2B). Anti-apoptotic protein Bcl2 was significantly upregulated by knocking

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Figure 1 Hippocampal miR-34c expression is upregulated by ketamine. Mice were treated with repetitive IP administration of ketamine, or normal saline (control) for 6 days. Expression of miR34a/b/c mRNAs (normalized to GAPDH) in hippocampus were examined by q-PCR. (*, P < 0.05, n ¼ 5).

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Figure 2 Knocking down miR-34c reduces apoptosis, upregulated Bcl2 and PKC-ERK signaling pathway after ketamine-induced hippocampal neurotoxicity. (A) TUNEL staining of hippocampal CA1 region in 2-month-old mice induced with ketamine-related neurotoxicity. Hippocampal injection of control vector (lenti-miR34c-C), or vector of miR-34c inhibitor (lenti-miR34c-I) was performed on P21. (B) Western blotting was also used to compare Bcl2 protein, phosphorylated PKC (p- PKC), and phosphorylated ERK (p-ERK), between miR-34c inhibitor treated mice and control mice after ketamine-induced hippocampal neurotoxicity. (Scale bar: 50 mM).

down miR-34c in the hippocampus. It was previously demonstrated that ketamine downregulated PKC pathway in the hippocampus to induce neuronal apoptosis (Huang et al., 2012; Liu et al., 2012). Here, we demonstrate that PKC pathway is indeed activated or strengthened, as more phosphorylated PKC and phosphorylated ERK were induced by genetically knocking down miR-34c after ketamine treatment.

Knocking down miR-34c increases memory performance after ketamine-induced hippocampal neurotoxicity

The question remained as to whether knocking down miR- 34c could rescue the memory loss after ketamine-induced neurotoxicity in hippocampus. Two-month-old mice were examined by the MWM test. For the control mice, lentivirus containing the non-specific control vector was injected in the hippocampus. Mice with hippocampal downregulation of miR-34c following ketamine-induced memory im- pairment, the averaged swimming time and swimming distance were both markedly reduced compared to the mice without hippocampal miR-34c downregulation (Figure 3). Thus, our result suggests that downregulation of miR-34c could increase memory.

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Figure 3 Knocking down miR-34c increases memory performance after ketamine-induced hippocampal neurotoxicity. Mice were initially induced with hippocampal neurotoxicity by repeated administra- tion of ketamine, and then followed by hippocampal injection of control vector (lenti-miR34c-C, left), or miR-34c inhibitor (lenti-miR34c-I, right). Morris water maze was used at 2 months to compare memory performance. Both swimming time (A) and swimming distance (B) were shortened in the mice receiving miR-34c inhibitor after ketamine- induced memory dysfunction in hippocampus. *, P < 0.05 (n ¼ 5).

Discussion

Ketamine is commonly used in pediatric anesthesia, but evidence suggests that excessive or repetitive usage of ketamine hinders or even damages the normal development of neonatal brain in both animal and human. We need to understand the underlying molecular mechanisms of this cortical neurotoxic event, as well as identify therapeutic targets to reduce or inhibit anesthesia-induced neuro- degeneration in the brain.

miR-34c had an important role in anesthesia-induced hippocampal neurodegeneration. Though all three members of the miR-34 family, miR-34a, miR-34b, and miR-34c, are expressed in hippocampus, little is known about their exact roles in modulating hippocampal maturation or develop- ment (Juhila et al., 2011). miR-34c is upregulated in neurodegenerative diseases or under stress condition, and targeted inhibition of miR-34c markedly improved learning capability in mice (Haramati et al., 2011; Zovoilis et al., 2011). After introducing hippocampal neurodegen- in vivo eration in mouse through ketamine induction,

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inhibition of miR-34c in hippocampus significantly im- proved MWM performance, with shortened swimming time and distance to locate platforms. Thus, along with previous findings, the results point to a critical role of miR-34c in regulating memory function through hippocampus in the brain.

It is noteworthy that anti-apoptotic protein Bcl2 was upregulated by miR-34c being knocked down after keta- mine-induced neurotoxicity in hippocampus. The expres- sion, or induced overexpression of Bcl2 protein by tumor necrosis factor (Tamatani et al., 1999) or estrogen receptors (Zhao et al., 2004), proved protective against neuronal apoptosis in the hippocampus. And miR-34a is inversely associated with Bcl2 expression in cortex in Alzheimer’s disease (Wang et al., 2009), but there has been no report identifying any of the miR-34 family in the regulation of Bcl2 expression in hippocampus. Thus, our finding showing that knocking down miR-34c upregulated Bcl2 expression level in hippocampus after ketamine-induced neurotoxicity is novel, and also indicates that miR-34 family microRNA might be directly involved in the regulation of neuronal apoptosis in the hippocampus. Thus, our data could further our understanding on identifying the underlying mecha- nisms of anesthesia-induce neurotoxicity, as well as developing targeted clinic therapies to treat anesthesia- induced neurotoxicity in neonatal brains.

Acknowledgements and funding

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Received 25 February 2014; accepted 5 June 2014. Final version published online January 2015.

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