new_pdfs/10.1016_j.biopha.2016.01.034.txt

Biomedicine & Pharmacotherapy 78 (2016) 322–328

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Original article

Neuronal apoptosis may not contribute to the long-term cognitive dysfunction induced by a brief exposure to 2% sevoflurane in developing rats

Yi Lu, Yan Huang, Jue Jiang, Rong Hu, Yaqiong Yang, Hong Jiang*, Jia Yan*

Department of Anesthesiology, Shanghai Ninth People's Hospital Affiliated to Shanghai Jiao Tong University, School of Medicine, 639 Zhi Zao Ju Road, Shanghai 200011, China

A R T I C L E

I N F O

A B S T R A C T

Article history: Received 7 November 2015 Accepted 26 January 2016

Keywords: Anesthesia Sevoflurane Neurotoxicity Neurodevelopment Apoptosis Developing brain

Background: Sevoflurane is an inhaled anesthetic commonly used in the pediatric. Recent animal studies suggest that early exposure to high concentration of sevoflurane for a long duration can induce neuroapoptosis and later cognitive dysfunction. However, the neurodevelopmental impact induced by lower concentration and shorter exposure duration of sevoflurane is unclear. To investigate whether early exposure to 2% concentration of sevoflurane for a short duration (clinically relevant usage of sevoflurane) can also induce neuroapoptosis and later cognitive dysfunction. Methods: Rat pups were subjected to control group, 2% sevoflurane for 3 h and 3% sevoflurane for 6 h. TUNEL assay and apoptotic enzyme cleaved caspase-3 measured by western blot were used for detection of neuronal apoptosis in frontal cortex and CA1 region of hippocampus 24 after sevoflurane treatment. Long-term cognitive function was evaluated by Morris water maze and passive avoidance test as the rats grew up. Results: The apoptotic levels in frontal cortex and CA1 region were significantly increased after rats exposed to 3% sevoflurane for 6 h (P 0.05), but not 2% sevoflurane for 3 h (P > 0.05). Exposure to both 2% sevoflurane for 3 h and 3% sevoflurane for 6 h could cause long-term cognitive dysfunction and animals exposed to 3% sevoflurane for 6 h exhibited worse neurodevelopmental outcomes (P Conclusion: It was suggested that neuronal apoptosis might not contribute to long-term cognitive dysfunction induced by 2% concentration and short exposure time of sevoflurane. Our findings also suggested that the mechanisms of sevoflurane-induced neurodevelopmental impact might be various, depending on the concentration and exposure duration. ã

<

<

0.05).

2016 Elsevier Masson SAS. All rights reserved.

1. Introduction

Over the past two decades, a series of animal experiments have indicated that various general anesthetics may be neurotoxic to the developing brain [1–5]. Thus, the potential for anesthetics-induced developmental neurotoxicity is of concern to the anesthesiologists, surgeons and parents of children undergoing surgery. The risks of childhood anesthesia have recently emerged as a public health concern [6,7]. In 2009, the U.S. Food and Drug Administration (FDA) established a partnership with the International Anesthesia Research Society (IARS) SmartTot to offer funds for preclinical and clinical studies concerning anesthetics-related neurodevelopmen- tal and

issues

[8]. Moreover,

the Pediatric Anesthesia

NeuroDevelopment Assessment (PANDA) study team holds a biennial scientific symposia to review recent preclinical and clinical data related to anesthetic neurotoxicity [9,10].

inhaled anesthetics [3]. Sevoflurane is a commonly used inhaled anaes- thetic for the induction and maintenance of general anesthesia during surgery. Because it has the advantage of a low blood–gas partition coefficient and pungency, sevoflurane is widely used as a pediatric anesthetic. Recently, several animal studies reported that early-life long (6–9 h) exposure to high concentrations (3–4%) of sevoflurane could cause neuronal apoptosis and subsequent long- term cognitive impairment [11–15]. However, 3 h exposure to the lower concentrations (1–2%) of sevoflurane more closely approx- imates typical general pediatric anesthetic episodes for anesthesia maintenance [16]. Whether lower concentrations and a shorter duration of exposure to sevoflurane can induce neuronal apoptosis and later cognitive impairment is unclear.

Every year, millions of children are exposed to

Corresponding authors.

E-mail addresses: mzkyanj@163.com (J. Yan), dr_hongjiang@163.com (H. Jiang).

http://dx.doi.org/10.1016/j.biopha.2016.01.034 0753-3322/ ã

2016 Elsevier Masson SAS. All rights reserved.

Y. Lu et al. / Biomedicine & Pharmacotherapy 78 (2016) 322–328

323

The immature human brain is most vulnerable to neurotoxic agents during the (BGS), which begins at mid- gestation and continues for 2–3 years after birth [17–19]. In rodents, the window of vulnerability to neurotoxic agents occurs first 2–3 weeks after birth [20,21]. In this primarily during the study, we aimed to investigate whether 3 h of exposure to 2% sevoflurane, as used in clinical practice, has pro-apoptotic effects on the developing brain and impairs the long-term cognitive function in rats in the same manner as prolonged exposure to high concentrations.

“brain growth spurt”

2. Materials and methods

2.1. Animals

in rodents occurs on postnatal day (PND) 7 [22], Sprague-Dawley (SD) PND7 rats weighing 14–18 g, provided by the Animal Center of Shanghai Jiao Tong University School of Medicine (Shanghai, China) were used in this study. The housing and treatment of the animals were in accordance with the National Institutes of Health guidelines for animal experimentation and approved by the institutional animal care and use committee. The animals were kept on a 12-h light/dark cycle (light from 7 am to 7 pm) with room temperature (23

Because peak

anesthesia-induced neurodegeneration

(cid:1)

1 (cid:3)C).

Animals were killed by lethal injection of pentobarbital at the time of blood sampling.

2.4. Analysis of apoptotic levels

2.4.1. TUNEL assay of brain

Twenty-four hours after sevoflurane exposure, six rats from each group (n = 6) were anesthetized with sodium pentobarbital fixed, dehydrated and made into and the brains were perfused, paraffin sections (5 mm), as described previously [24]. Apoptotic cells in the brain sections were detected by TUNEL Assay using the FragELTM DNA Fragmentation Detection Kit (Merck, Darmstadt, Germany), according to the manufacturer’s protocol. Briefly, brain sections were permeabilized with proteinase K (20 mg/ml) at room temperature for 20 min. Endogenous peroxidase was inactivated by 3% H2O2. Specimens were incubated for 1.5 h with terminal deoxynucleotidyl transferase (TdT) labelling reaction mixture, and apoptotic cells were visualized with 3,30-diaminobenzidine (DAB), and normal nuclei were counterstained with methyl green. Because to anesthetics at PND7 and the hippocampus is closely related to learning and memory [25], the number of apoptotic neurons in the frontal cortex and the CA1 region of the hippocampus was quantified. We selected two random viewing fields (400(cid:5)) per region (frontal cortex and CA1) from one brain section per animal for analysis in a double blinded manner.

the cerebral cortex

reaches peak vulnerability

2.2. Sevoflurane exposure

Rat pups were separated from their mothers for acclimatization prior to sevoflurane exposure. Pups from the same litter were randomly allocated to three different groups. Totally, ninety PND7 rats were included in this study (n = 30 for each group). Rats in the control group received 100% oxygen for 6 h in a chamber at 37 (cid:3)C. Rats in the other two groups were exposed to either 2% sevoflurane (SEVOFRANE1, Osaka, (Sevo1 group) or 3% sevoflurane for 6 h (Sevo2 group) under 100% oxygen in the same chamber at 37 (cid:3)C as described previously [13]. The concentration of sevoflurane in the chamber was monitored and maintained by a flow to the vaporizer as we described previously [23]. The gas chamber was 2 l/min. We chose these treatments because 3 h exposure to 2% seveflurane more closely approximates typical general pediatric anesthetic episodes for anesthesia maintenance [16] and 6 h exposure to 3% sevoflurane can cause neuronal apoptosis in developing animals [11,12,14,15].

Japan)

for 3 h

2.3. Arterial blood gas analysis

2.4.2. Western blot

Apoptosis was also assessed using western blot to quantify cleaved caspase-3 (Cl-Csp3) in all groups (n = 6). Briefly, tissue samples of the frontal cortex and CA1 region were collected from three groups twenty-four hours after sevoflurane exposure. Tissues were lysed in a buffer containing a protease inhibitor cocktail (Calbiochem, San Diego, CA, USA) and homogenated. The homogenate was centrifuged and the supernatant was collected for further analysis. Protein concentrations were measured by BCA Protein Assay Kit (Novagen, San Diego, CA, USA). Equal amounts of protein were boiled in loading buffer (Beyotime, Beijing, China) and separated by 10% polyacrylamide gel electrophoresis. Proteins were transferred to nitrocellulose, and the blots were probed overnight with anti-cleaved caspase-3 (1:200, Millipore, Darm- stadt, Germany) and b-actin antibodies (1:500, internal standard, Santa Cruz, San Diego, CA, USA) at 4 (cid:3)C. Primary antibodies were visualized using secondary antibodies conjugated to horseradish peroxidase (Santa Cruz, San Diego, CA, USA) and ECL reagent (Pierce, Rockford, IL, USA). Quantitative analysis of Cl-Csp3 was normalized to b-actin using the Quantity One software.

To determine adequacy of ventilation and oxygenation, arterial blood samples (n = 6) were obtained from the left cardiac ventricle in each group at the end of anesthesia, and the samples were immediately analyzed by a blood gas analyzer (Radiometer, ABL800, Denmark). We compared the pH, pO2, pCO2, oxygen saturation (sO2), and the concentrations of blood glucose (Glu), (cid:4)) among the groups. lactic acid (Lac) and bicarbonate (HCO3

2.5. Neurologic assessment

2.5.1. Morris water maze

To assess neurodevelopmental outcomes, particularly the learning and memory functions of juveniles, rats from all groups

Table 1 Arterial blood gas analysis for the three groups.

pH

pCO2 (mmHg)

pO2 (mmHg)

sO2 (%)

Lac (mmol/l)

HCO3

(cid:4) (mmol/l)

Glu (mmol/l)

Control 2%Sevo 3h 3%Sevo 6h

7.463 7.417 7.404

(cid:1) (cid:1) (cid:1)

0.030 0.025 0.045

29.4 30.6 32.3

(cid:1) (cid:1) (cid:1)

1.2 1.1 1.5

117.5 113.9 108.0

(cid:1) (cid:1) (cid:1)

4.8 6.9 5.4

99.6 99.5 99.3

(cid:1) (cid:1) (cid:1)

0.1 0.1 0.2

1.7 1.8 1.9

(cid:1) (cid:1) (cid:1)

0.2 0.2 0.2

20.2 21.2 21.0

(cid:1) (cid:1) (cid:1)

0.5 0.7 0.8

5.5 5.5 5.9

(cid:1) (cid:1) (cid:1)

0.3 0.4 0.4

aP

<

0.05 compared to the control group.

324

Y. Lu et al. / Biomedicine & Pharmacotherapy 78 (2016) 322–328

Fig. 1. TUNEL assay for apoptosis of frontal cortex and CA1 region of hippocampus in control and treatment groups. (A) Representative images of frontal cortex and CA1 region. TUNEL-positive apoptotic nuclei were stained by brown and normal nuclei were stained by cyan. Scale bar = 50 mm. (B) Quantification of apoptotic nuclei in the frontal cortex and CA1 region. *P

<

0.05 compared to control group, #P

<

0.05 compared to Sevo1 group.

Fig. 2. Western blot analysis of apoptotic enzyme Cl-Csp3 from frontal cortex and CA1 region of hippocampus in control and treatment groups. (A) Representative blots of Cl- Csp3. b-actin was used as the internal standard. (B) Quantification of the target protein expression levels. *P 0.05 compared to Sevo1 group.

<

0.05 compared to control group, #P

<

Y. Lu et al. / Biomedicine & Pharmacotherapy 78 (2016) 322–328

Fig. 3. Morris water maze test for the evaluation of learning and memory when the rats grew up to adolescence in control and treatment groups. (A) Statistical analysis of finding the former platform. (C) Representative traces of the paths latency in swum by the rats after the end of probe trials. The white circle in left lower quadrant represents the removed platform. (D) Statistical analysis of the number of times the former platform was crossed by the rats. (E) Statistical analysis of the percentage of time spent by the rats in the target quadrant. *P 0.05 compared to control group, #P

finding the hidden platform on trial days. (B) Statistical analysis of swimming distance before

<

<

0.05 compared to Sevo1 group.

were subjected to Morris water maze after reaching 6 weeks of age (n = 12), as previously described [24]. Briefly, a circular pool (1.6 m diameter, 60 cm height) was used for the water maze, and a submerged platform (10 cm diameter, 2 cm below the surface of fixed position in the pool. The water the water) was located at a 1 (cid:3)C. Probe trials were conducted twice (cid:1) temperature was set at 23 five consecutive days. In the trials, rats were trained to a day for finding swim to and locate the hidden platform. The time spent in the hidden platform and the swimming distance before reaching the platform were recorded. After the probe trials, the platform was removed, and the rats were allowed to swim freely for 120 s: the number of times that the former platform was crossed and the percentage of time spent in the target quadrant were determined. The entire behavioral test was recorded and analyzed using a MS-

type Water Maze Video analysis system (Chengdu Instrument Ltd., Chengdu, China). Finally, to investigate cognitive function during development, the passive avoidance test was performed at 3 months.

2.5.2. Passive avoidance test

The passive avoidance test was performed as previously described [26]. The apparatus used for the passive avoidance test included a behavioral stimulation controller and a video shuttle box (Chengdu Instrument Ltd., Chengdu, China). The test relies the natural preference of rats for darkness. Briefly, on the first trial day, the rats were placed in the illuminated compartment after 2 min of habituation to the dark compartment and allowed to re-enter the dark compartment. On the following day, an electric foot shock was

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Y. Lu et al. / Biomedicine & Pharmacotherapy 78 (2016) 322–328

floor of the dark compartment after the delivered through the grid rats entered. Twenty-four hours later, the retention of passive avoidance was determined by comparing the time elapsed prior to re-entry into the dark compartment with the arbitrary maximum time of 180 s.

2.6. Statistical analysis

(cid:1)

SEM. SAS 9.2 (SAS Institute Inc., Cary, North Carolina, USA) was used for statistical analysis. One-way ANOVA was used to determine statistically significant differences between the three groups, and Tukey’s post hoc analysis was performed to correct for multiple comparisons when applicable. Statistical significance was accepted as P

All data are expressed as the mean

<

0.05.

3. Results

3.1. Neonatal sevoflurane exposure does not induce metabolic or respiratory distress

indicate any signs of metabolic or respiratory distress in animals exposed to 2% sevoflurane for 3 h or 3% sevoflurane for 6 h, and the pH, pO2, (cid:4) did not differ significantly among pCO2, sO2, Glu, Lac and HCO3 the three groups (Table 1). Thus, there is little probability of brain injury induced by metabolic or respiratory distress during sevoflurane anesthesia.

Arterial blood gas analysis did not

3.2. Exposure to 2% sevoflurane for 3 h did not induce neuroapoptosis in the developing brain

0.05, Fig. 3C–E). These parameters were further decreased in 0.05, Fig. 3C–E). Long-term cognitive function of the three groups was also evaluated by the passive avoidance test when these rats became adults (3 months old). The results showed that the latency to re- enter the dark compartment was significantly decreased in both the Sevo1 and Sevo2 groups, with the Sevo2 rats displaying the findings demonstrated a shortest latency (P long-term following early exposure to both 2% sevoflurane for 3 h and 3% sevoflurane for 6 h.

<

(P the Sevo2 group, compared to the Sevo1 group (P

<

<

0.05, Fig. 4). These learning and memory deficiencies

4. Discussion

Sevoflurane is a volatile anesthetic that is frequently used in children as a sole agent or intravenous anesthetics. It is believed to be a g-aminobutyrate acid agonist (GABAA) [8,27]. Recently, animal experiments have suggested that by promoting neuronal apoptosis, sevoflurane is neurotoxic to the immature brain [3,11,12,14,28]. However, the experimental animals in these studies generally underwent a prolonged exposure to high concentration of sevoflurane by inhalation, which is unusual for clinical practice. In this study, we provided in vivo evidence that a clinically-relevant concentration and exposure duration of sevo- flurane (2% for 3 h) did not enhance neuronal apoptosis in two specific regions of the developing brain, whereas long-term cognitive deficiency still existed. This finding is important because the results indicated that enhanced neuronal apoptosis may not contribute to the long-term cognitive dysfunction induced by sevoflurane in normal clinical applications. Other potential mechanisms of sevoflurane-induced long-term cognitive deficien- cy should be studied to address the possible neurotoxicity.

in conjunction with

To study whether the clinically-relevant usage of sevoflurane (2% for 3 h) can induce neuroapoptosis similar to prolonged (6 h) exposure to high concentration in the developing brain, we assayed the apoptotic levels of the frontal cortex and CA1 region of the hippocampus after different treatments by using TUNEL assay and detection of the apoptotic enzyme Cl-Csp3 through western blotting. The TUNEL assay demonstrated no significant differences in apoptotic neuronal cells in either the frontal cortex or CA1 region of rats exposed to 2% sevoflurane (P > 0.05). However, exposure to 3% sevoflurane for 6 h increased the numbers of apoptotic cells in both the frontal cortex and CA1 region, compared with the control and Sevo1 groups (P

(3%) of sevoflurane

for 3 h versus

the controls

<

0.05, Fig. 1).

In addition, there was no significant difference between the Sevo1 and control groups in Cl-Csp3 protein expression analyzed by western blot (P > 0.05). However, Cl-Csp3 expression was significantly increased in the Sevo2 group (P

<

0.05, Fig. 2).

To study apoptotic effects, it is important to obtain results from more than one independent method (preferably several methods) before drawing a conclusion [29]. Thus, to assess apoptosis, we performed not only TUNEL assays but also detection of the apoptotic enzyme Cl-Csp3. Moreover, two independent behavioral tests were conducted to evaluate long-term cognitive outcomes after the rats reached adulthood. The results of the two assays were equivalent.

Early exposure to sevoflurane in rats results in significant concentration and exposure duration-dependent impairment in adulthood memory [13]. In this study, we found that early exposure to 3% sevoflurane for 6 h induced worse neurodevelop- mental outcomes than 2% sevoflurane for 3 h. Our results also confirmed the dose- and time-dependent neurotoxic effect of sevoflurane on the developing brain. Moreover, we demonstrated that a lower inhaled concentration and shorter exposure duration to sevoflurane long-term cognitive dysfunction, although no enhancement of neuronal apoptosis

in early

life could cause

3.3. Exposure to both 2% sevoflurane for 3 h and 3% sevoflurane for 6 h early in life can cause long-term cognitive dysfunction

To investigate the effects of early sevoflurane exposure on long- first subjected the rats from the control term cognitive function, we and treatment groups to the Morris water maze test when they were 6 weeks old. The result showed that the latency and swimming distance were significantly increased in rats from the Sevo1 and Sevo2 groups on trial day 4 and 5, compared to control group (P 0.05, Fig. 3A and B). Moreover, it took the Sevo2 group find the platform on trial day 4 and 5, compared to the more time to Sevo1 group (P 0.05, Fig. 3A). The rats from Sevo2 group also swam further than did the Sevo1 group on trial day 4 and 5 (P 0.05, Fig. 3B). The number of times that the rats crossed the platform and percentage of time spent in the target quadrant were significantly decreased in both the Sevo1 and Sevo2 groups

<

<

<

Fig. 4. Passive avoidance test for the cognitive function the rats grew up to adulthood in control and treatment groups. *P 0.05 compared to control group, #P

<

<

0.05 compared to Sevo1 group.

Y. Lu et al. / Biomedicine & Pharmacotherapy 78 (2016) 322–328

was found. There may be different mechanisms responsible for the neurotoxicity induced by the varying treatment conditions of sevoflurane.

The mechanism of neurodevelopmental impairment mediated by a brief exposure to lower concentration of sevoflurane is still not clear. Wu et al. suggested that physiological disturbance may contribute to sevoflurane-induced long-term learning and memo- ry dysfunction immature rats [30]. However, under our sevoflurane treatment conditions, no metabolic or respiratory distress was found in PND7 rats via arterial blood gas analysis. Therefore, physiological disturbance may be excluded in this study. Although the mechanism of neurotoxicity caused by sevoflurane is not fully understood, several recent studies provide clues to possible novel mechanisms of sevoflurane-induced neurotoxicity. First, Hu et al. suggested that by increasing tau protein expression, sevoflurane could change the arrangement of the microtubule cytoskeleton in the hippocampus of the developing rat brain [31]. Second, several researchers have found that early sevoflurane exposure had an impact on rat neuronal ultrastructure compo- nents, such as dendritic spines, synapses and mitochondria [28,32]. However, these studies did not demonstrate whether these mechanisms were associated with the long-term neuro- developmental impact induced by sevoflurane. Therefore, further studies are needed to explore the relationship between neuronal injury mechanisms and neurodevelopmental outcomes.

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In this study, we have shown that early exposure to both 2% sevoflurane impair adulthood learning and memory function but only exposure to 3% sevoflurane for 6 h could reinforce neuronal apoptosis. We therefore concluded that neuronal apoptosis might not contribute to the long-term cognitive dysfunction induced by a brief exposure to a in developing rats. Moreover, the mechanisms of sevoflurane-induced neurodevelopmental impact might be vari- ous, depending on the concentration and exposure duration. Further studies regarding the mechanism for the sevoflurane- induced neurodevelopmental impact are urgently needed to develop methods to protect the immature brain.

for 3 h and 3% sevoflurane

for 6 h may

lower concentration of sevoflurane

findings suggested

the

that

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Conflict of interest

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No conflicts of interest declared.

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Acknowledgment

can induce hippocampal neurodegeneration and long-term cognitive impairment via the ROS/HIF-1alpha pathway in developing rats, Cell. Physiol. Biochem. 33 (2014) 1715–1732.

This work was supported by research funds from Shanghai Municipal Commission of Health and Family Planning (grant number: 201440356).

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