new_pdfs/10.1007_s10072-014-1726-4.txt

Neurol Sci (2014) 35:1401–1404 DOI 10.1007/s10072-014-1726-4

O R I G I N A L A R T I C L E

Sevoflurane postconditioning provides neuroprotection against brain hypoxia–ischemia in neonatal rats

Xiaoyan Ren • Zhi Wang • Hong Ma • Zhiyi Zuo

Received: 25 February 2014 / Accepted: 15 March 2014 / Published online: 5 April 2014 (cid:2) Springer-Verlag Italia 2014

Abstract Application of volatile anesthetics after brain ischemia provides neuroprotection in adult animals (anes- thetic postconditioning). We tested whether postcondi- tioning with sevoflurane, the most commonly used general anesthetic in pediatric anesthesia, reduced neonatal brain injury in rats. Seven-day-old Sprague–Dawley rats were subjected to brain hypoxia–ischemia (HI). They were postconditioned with sevoflurane in the presence or absence of 5-hydroxydecanoic acid, a mitochondrial KATP channel inhibitor. Sevoflurane postconditioning dose- dependently reduced brain tissue loss observed 7 days after brain HI. This effect was induced by clinically relevant concentrations and abolished by 5-hydroxydecanoic acid. These results suggest that sevoflurane postconditioning protects neonatal brain against brain HI via mitochondrial KATP channels.

Abbreviations 5-HD KATP channels HI

5-Hydroxydecanoic acid ATP sensitive potassium channels Hypoxia–ischemia

Introduction

Neonatal brain injury occurs at about one in every 4,000 live births [1]. Most of them survive to adulthood and can long-term neurological and cognitive have significant impairment, such as cerebral palsy and epilepsy [2–4]. Thus, neonatal brain injury has a major impact on patients, their families and our society. However, there are no effective and practical interventions available for use in clinical practice to reduce neonatal brain injury, indicating an urgent need to identify these interventions.

Keywords Brain hypoxia–ischemia (cid:2) Neonates (cid:2) Postconditioning (cid:2) Sevoflurane

X. Ren (cid:2) H. Ma (&) Department of Anesthesiology, The First Hospital of China Medical University, Shenyang 110001, People’s Republic of China e-mail: mahong5466@yahoo.com

X. Ren (cid:2) Z. Wang (cid:2) Z. Zuo (&) Department of Anesthesiology, University of Virginia Health System, 1 Hospital Drive, PO Box 800710, Charlottesville, VA 22908-0710, USA e-mail: zz3c@virginia.edu

Neonatal brain injury is usually caused by brain hypoxia, ischemia or the combination of hypoxia and ischemia [1]. This situation is often modeled in rodents by inducing brain hypoxia–ischemia (HI) [5]. It has been shown that applica- tion of isoflurane, a volatile anesthetic used clinically, after focal brain ischemia provides neuroprotection in adult rodents [6]. This isoflurane postconditioning effect also protects brain against neonatal brain HI in rats [7]. However, isoflurane is now not commonly used in pediatric anesthesia in the USA. Sevoflurane is the most commonly used general anesthetic in pediatric anesthesia and also more commonly used in adults than isoflurane in current clinical practice in the USA and many other developed countries [8]. However, it is not known yet whether sevoflurane can induce a post- conditioning effect against neonatal brain HI.

Z. Wang Department of Anesthesiology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou 510120, China

Based on the information of isoflurane postconditioning, we hypothesize that sevoflurane postconditioning reduces

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neonatal brain injury. To test this hypothesis, we applied sevoflurane after brain HI in neonatal rats. Because mito- chondrial ATP-sensitive potassium (KATP) channels may be involved in neuroprotection and cardioprotection induced by volatile anesthetics [6, 9], we also determined whether mitochondrial KATP channels played a role in the sevoflurane postconditioning effects on neonatal rats using 5-hydroxydecanoic acid (5-HD), a specific inhibitor of mitochondrial KATP channels.

Methods

All experimental protocols were approved by the Institu- tional Animal Care and Use Committee of the University of Virginia (Charlottesville, VA). All surgical and experi- mental procedures were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH publications number 80-23) revised in 2011. Efforts were made to minimize the number of animals used and their suffering.

Neonatal brain hypoxia–ischemia modal

Brain HI was performed in 7-day-old male and female Sprague–Dawley rats as described previously [10, 11]. In brief, neonates were anesthetized by isoflurane and their left common carotid arteries were permanently ligated with a double 7-0 surgical silk. The procedure lasted \5 min. After surgery, neonates were returned to the cages with their mothers for 3 h. The neonates were then placed in a chamber filled with humidified 8 % oxygen–92 % nitrogen for 2 h at 37 (cid:3)C. The oxygen concentration and tempera- ture in the chamber were continuously monitored.

Drug application

The neonates were randomly divided into the following groups: (1) control, (2) brain HI, (3) brain HI and post- conditioning with 1, 2 and 3 % sevoflurane, (4) brain HI and 5-HD treatment (10 mg/kg) and (5) brain HI, 5-HD treatment and postconditioning with 2 % sevoflurane. Sevoflurane postconditioning was performed by exposing neonates to various concentrations of sevoflurane in 30 % O2 for 1 h immediately after brain HI. Neonates of brain HI alone group were placed in a chamber flushed with 30 % O2 for 1 h. The mitochondrial KATP channel inhibitor 5-HD was dissolved in normal saline and administered intraperitoneally just before the start of brain HI. The dose of 5-HD was based on a previous study in which intra- peritoneal injection of 10 mg/kg 5-HD blocked ischemic preconditioning-induced protection [12].

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Fig. 1 Neuroprotective effects of sevoflurane. Seven-day-old rats were subjected to or were not subjected to the brain HI and then postconditioned with various concentrations of sevoflurane. Brain was harvested at 7 days after the brain HI. The results are mean ± SD (n = 8–10). *P \ 0.05 compared with control rats. ^P \ 0.05 com- pared with rats that had brain HI only

Brain injury/tissue loss quantification

After 7 days of the brain HI, rats were sacrificed under deep isoflurane anesthesia and then their brains were har- vested as described previously [11, 13]. The hindbrain was removed from cerebral hemispheres and bilateral hemi- spheres were weighed separately. The weight ratio of left to right hemispheres was calculated.

Statistical analysis

The results are presented as mean ± SD (n C 6). Statisti- cal analysis was performed by one-way analysis of vari- ance followed by the Tukey’s test. A P B 0.05 was considered statistically significant.

Results

The control rats had similar right and left hemisphere weights. The left brain HI significantly reduced left cere- bral hemisphere weight examined 7 days after brain HI (weight to right cerebral hemispheres: 0.987 ± 0.012 of control group vs. 0.740 ± 0.049 of brain HI group, n = 8–10, P \ 0.001), suggesting brain tissue loss in this side of brain. This tissue loss was dose- dependently reduced by sevoflurane postconditioning, an effect that was already significant even after being post- conditioned with 1 % sevoflurane (Fig. 1) (weight ratio of left to right cerebral hemispheres: 0.740 ± 0.049 of brain HI group vs. 0.820 ± 0.051 of brain HI plus 1 % sevo- flurane group, n = 8–10, P = 0.003). The sevoflurane

ratio of

left

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Fig. 2 Inhibition of sevoflurane postconditioning-induced neuropro- tection by a mitochondrial KATP inhibitor. Seven-day-old rats were subjected to or were not subjected to the brain HI and then postconditioned with 2 % sevoflurane in the presence or absence of 10 mg/kg 5-HD. a Representative brain images. The inserted length marker = 2 mm; b Quantification results. The results are mean ± SD (n = 6–10). *P \ 0.05 compared with control rats, ^P \ 0.05 com- pared with rats that had brain HI only, #P \ 0.05 compared with rats that had brain HI and sevoflurane postconditioning. HD 5-HD, Sevo sevoflurane

postconditioning effects were abolished by 5-HD (weight ratio of left to right cerebral hemispheres: 0.884 ± 0.049 of brain HI plus 2 % sevoflurane group vs. 0.759 ± 0.044 of brain HI plus 2 % sevoflurane plus 5-HD group, n = 8–10, P \ 0.001), although 5-HD alone did not affect the brain tissue loss caused by brain HI (Fig. 2).

Discussion

Our results clearly showed that postconditioning with sevoflurane-induced neuroprotection against neonatal brain HI. These results extend the previous finding of isoflurane postconditioning effects in neonatal rats [7] to sevoflurane,

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the most commonly used general anesthetic in pediatric anesthesia. To test the effects of sevoflurane, we used the classical Rice–Vannucci model, a widely used and well- characterized animal model to study neonatal brain injury. This model incorporates both brain ischemia and hypoxia, factors that contribute to neonatal brain injury in humans [1, 5]. We monitored brain tissue loss that signifies brain structure damage as showed in our previous studies [11, 13].

Our results suggest that the sevoflurane postconditioning effects may be mediated by mitochondrial KATP channels because 5-HD, a mitochondrial KATP channel inhibitor, abolished sevoflurane effects and 5-HD alone did not alter brain tissue loss after brain HI. Consistent with this finding, our previous study has implicated the involvement of mitochondrial KATP channels in isoflurane postcondition- ing-induced neuroprotection in adult rats after focal brain ischemia [6]. These channels are inhibited by ATP and activated under energy-depleted conditions. They also can be activated by volatile anesthetics [14]. The opening of these channels produces an outward current. This current can maintain the mitochondrial membrane potential and reduce the opening of mitochondrial permeability transi- tion pore to inhibit cell injury and death [15, 16]. Of note, previous studies have shown that application of sevoflurane before brain ischemia (sevoflurane preconditioning) also provides neuroprotection. This effect may be mediated by inhibition of brain inflammation and activation of gluta- mate transporters in the brain [17–19]. Although these mechanisms play a role in the sevoflurane postcondition- ing-induced neuroprotection and whether there is a rela- tionship between these mechanisms and the mitochondrial KATP channel pathway require further investigation.

We used cerebral weight ratio to quantify the brain tissue loss after brain HI. The method is used by many studies before and the brain injury quantified by this method is highly correlated with that measured by bio- chemical, electrophysiological and morphometric methods [20, 21]. In addition, the cerebral weight ratio method is simple to perform and very objective.

Our finding may have clinical translational implications. Sevoflurane is the most commonly used general anesthetic in pediatric population [8]. It is often used in patients for Cesarean section if general anesthesia is used. One mini- mum alveolar concentration (the concentration in the lungs to prevent movement in 50 % subjects in response to sur- gical stimuli) of sevoflurane is *2 % in human [22] and 2.9 % in rats younger than 30 days [23]. Our results showed that sevoflurane at a concentration as low as 1 % was effective to induce neuroprotection, suggesting that a subclinical concentration for anesthesia can induce the postconditioning effect. In addition, we have demonstrated the postconditioning effect, which may be easy to apply

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clinically because its application does not require the predication of ischemia occurrence. However, exposure of neonatal mice to 3 % sevoflurane for 6 h can lead to brain cell apoptosis and increase in brain tumor necrosis factor a [24]. Thus, it is judicious to avoid exposure to a high concentration of sevoflurane for a long time so that the potential sevoflurane-induced neurotoxicity will not occur. In summary, we have shown that sevoflurane at clini- cally relevant concentrations can induce a postconditioning effect against neonatal brain injury. This effect may be mediated by mitochondrial KATP channels.

Acknowledgments This study was supported by Grants (R01 GM065211 and R01 GM098308 to Z. Zuo) from the National Insti- tutes of Health, Bethesda, Maryland, by a Grant from the International Anesthesia Research Society (2007 Frontiers in Anesthesia Research Award to Z. Zuo), Cleveland, Ohio, by a Grant-in-Aid from the American Affiliate (10GRNT3900019 to Z. Zuo), Baltimore, Maryland, and the Robert M. Epstein Professorship Endowment, University of Virginia.

Heart

Association

Mid-Atlantic

Conflict of interest None.

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