new_pdfs/10.1016_j.brainres.2015.10.050.txt

b r a i n r e s e a r c h 1 6 3 0 ( 2 0 1 6 ) 2 5 – 3 7

Available online at www.sciencedirect.com

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Research Report Sevoflurane postconditioning improves long-term learning and memory of neonatal hypoxia-ischemia brain damage rats via the PI3K/Akt-mPTP pathway

Zhongmeng Laia, Liangcheng Zhanga,n Qingxiu Xua

, Jiansheng Sua, Dongmiao Caib,

aDeparment of Anesthesiology, Fujian Medical University Union Hospital, 29 Xin-Quan Road, Fuzhou 350001, PR China bDeparment of Anesthesiology, The First Affiliated Hospital of Xiamen University, 55 Zhen-Hai Road, Xiamen 3610003, PR China

a r t i c l e i n f o

a b s t r a c t

Article history:

Accepted 16 October 2015

Available online 2 November 2015

Background: Volatile anesthetic postconditioning has been documented to provide neuro- protection in adult animals. Our aim was to investigate whether sevoflurane postcondi- tioning improves long-term learning and memory of neonatal hypoxia-ischemia brain

Keywords: Sevoflurane postconditioning Neonatal rat

Hypoxic-ischemic brain damage

Long-term learning and memory

PI3K/Akt pathway

Mitochondrial permeability

transition pore

damage (HIBD) rats, and whether the PI3K/Akt pathway and mitochondrial permeability

transition pore (mPTP) opening participate in the effect.

Methods: Seven-day-old Sprague-Dawley rats were subjected to brain HI and randomly allocated to 10 groups (n ¼24 each group) and treated as follows: (1) Sham, without hypoxia-ischemia; (2) HI/Control, received cerebral hypoxia-ischemia; (3) HIþAtractylo- side (Atr), (4) HIþCyclosporin A (CsA), (5) HIþsevoflurane (Sev), (6) HIþSevþ LY294002 (9) HIþSevþAtr, and (LY), (10) HIþSevþCsA. Twelve rats in each group underwent behavioral testing and their brains were harvested for hippocampus neuron count and morphology study.

(7) HIþSevþ L-NAME (L-N),

(8) HIþSevþ SB216763 (SB),

Brains of the other 12 animals were harvested 24 h after intervention to examine the expression of Akt, p-Akt, eNOS, p-eNOS, GSK-3β, p-GSK-3β by Western bolting and mPTP opening. Results: Sevoflurane postconditioning significantly improved the long-term cognitive performance of the rats, increased the number of surviving neurons in CA1 and CA3

hippocampal regions, and protected the histomorphology of the left hippocampus. These effects were abolished by inhibitors of PI3K/eNOS/GSK-3β. Although blocking mPTP opening simulated sevoflurane postconditioning-induced neuroprotection, it failed to enhance it.

Abbreviations: HIBD, hypoxia-ischemia brain damage; mPTP, mitochondrial permeability transition pore; eNOS, endothelial nitric

oxide synthase; mPTP, mitochondrial permeability transition pore; Atr, Atractyloside; CsA, Cyclosporin A;

TTC,

triphenyltertrazolium chloride; Sev,

sevoflurane; EL, Escape latency; HE, hematoxylin–eosin; BSA, ovine serum albumin;

least significant difference; PCA, principal components analysis.

SD, n Corresponding author. Fax: þ86 59183346181. E-mail addresses: guodh1981@163.com (Z. Lai), zhanglc6@163.com (L. Zhang), sjs2028@163.com (J. Su),

standard deviation; LSD,

caidongmiao@hotmail.com (D. Cai), 18201019@qq.com (Q. Xu).

http://dx.doi.org/10.1016/j.brainres.2015.10.050 0006-8993/& 2015 Elsevier B.V. All rights reserved.

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Conclusions: Sevoflurane postconditioning exerts a neuroprotective effect against HIBD in neonatal rats via PI3K/Akt/eNOS and PI3K/Akt/GSK-3β pathways, and blockage of mPTP opening may be involved in attenuation of histomorphological injury.

& 2015 Elsevier B.V. All rights reserved.

1.

Introduction

As a key part in learning and memory adjustment, the hippocampus, including the CA1, CA3, and DG regions, participates in integrating transferred outside information into the nerve center (Chan et al., 2010; Morris et al., 2012). The CA1 region in particular, is highly sensitive to hypoxia- ischemia brain damage (HIBD) (Hopkins and Haaland, 2004). In neonates HIBD may result from perinatal asphyxia, birth injury, and neonatal cardiac surgery, and is a common cause of neonatal death and neurobehavioral impairments (Fan et al., 2005). HIBD may cause apoptosis or necrosis of hippocampal neurons by a cascade of damaging reactions, thus impairing learning and memory (Zhang et al., 2002; Vannucci RC1 and Vannucci, 2001). Therefore, protection of hippocampal neurons is of great significance in prevention and treatment of HIBD.

of cerebral impairment, Akt activation after cerebral ischemia is part of the endogenous protection process and the PI3K/Akt pathway participates in sevoflurane protection against cere- bral ischemia-reperfusion injury, (Zitta et al., 2010; Ye et al., 2012) while neuronal mitochondria undergo permeability transition (Nieminen et al., 1996). Emerging evidence has demonstrated that sevoflurane postconditioning reduces cer- ebral HI-induced brain tissue loss via mitochondrial KATP channels, (Ren et al., 2014) and improves short-term learning and memory after focal cerebral ischemia-reperfusion injury in adult rats via inhibition of neuronal apoptosis through the PI3K/Akt pathway (Wang et al., 2010). However, studies have not examined the long-term learning and memory of neona- tal HIBD rats that received sevoflurane postconditioning, the role of PI3K/Akt-mPTP pathway in the process, and related signal-regulating downstream Akt kinases.

A method to provide this protective effective is ischemic or pharmacological postconditioning, which has been docu- mented to protect tissues and organs by making cells more tolerant of oxygen deficit (Peng et al., 2012; Danielisová et al., 2008; Hu et al., 2013). In cardiac studies, this postconditioning reduces calcium overload, the oxidative stress response, and ATP consumption in cells and mitochondria, thus protecting the cardiac muscle (Tsang et al., 2004; Lim et al., 2007; Lemoine et al., 2010; Fang et al., 2010). The mechanism involves activation of the Phosphatidylinositol-3 kinase (PI3K)/protein kinase B (Akt) pathway, possible activation of kinases including the downstream proteins of Akt, such as glycogen synthase kinase 3 (GSK-3β), P70S6 kinase (P70S6K), and endothelial nitric oxide synthase (eNOS), suppression of mitochondrial permeability transition pore (mPTP) opening, and opening potassium channels (Tsang et al., 2004; Lim et al., 2007; Lemoine et al., 2010; Fang et al., 2010). In studies

Therefore, the purposes of current study were to examine the potential protective effect of sevoflurane postcondition- ing on long-term cognitive performance in neonatal HIBD rats, the related underlying mechanism, and the role of mPTP opening in this process.

2.

Results

Sevoflurane postconditioning improves non-spatial

2.1. and spatial learning and memory

Compared with the HI/Control group, sevoflurane postcondi- tioning increased the DIs of the postconditioning groups (Po0.01), which were reduced by the LY294002 (a PI3K inhibitor), L-NAME (an eNOS inhibitor), and SB216763 (a GSK-3β inhibitor) (Po0.05). Atr did not worsening the DIs of the HI/Control group (P40.05), but it decreased the DIs of

Fig. 1 – Experimental protocol and animal grouping. Atr, atractyloside; CsA, Cyclosporin A; HI, hypoxia-ischemia; L-N, L- NAME; LY, LY294002; SB, SB216763; Sev, sevoflurane. P indicates postnatal age in days.

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Fig. 2 – Results of the novel object recognition test. Atr, atractyloside; CsA, Cyclosporin A; HI, hypoxia-ischemia; L-N, L-NAME; LY, LY294002; SB, SB216763; Sev, sevoflurane. Comparisons between groups were using one-way ANOVA with Fisher's LSD as post-hoc procedure. The detailed sample size, mean7SD are listed in Supplementary table S1.

Table 1 – Results of the place navigation study.

Day 1

Day 2

Day 3

Day 4

Day 5

Strategy (%)

Sham HI/Control HIþSev HIþSevþLY HIþSevþL-N HIþSevþSB HIþAtr HIþSevþAtr HIþCsA HIþSevþCsA

46.0078.04 74.1578.72 55.5277.45 70.2078.26 67.8377.40 67.9079.67 74.5079.24 74.17710.28 ▲ 55.9178.71 54.8778.64

▲n

n

▲

n

n

n

n

n

35.0976.20 68.3879.01 42.3877.11 66.66710.72 n 64.7878.02 63.02710.15 n 69.3079.56 68.7979.69 39.5675.57 39.8874.93

▲n

n

▲

n

n

n

▲

27.3875.60 56.3376.69 34.1075.69 53.7176.48 49.3876.61 49.2777.29 58.5679.34 56.5278.29 33.9276.83 32.7676.97

▲n

n

▲

n

▲n

▲n

n

n

▲

16.7873.86 47.3475.87 19.5473.22 45.4675.50 39.5875.33 38.5475.49 49.7475.05 48.5074.94 18.6673.18 18.6073.91

▲

n

▲

n

▲n

▲n

n

n

▲

14.3872.22 38.2273.93 16.2672.90 37.2573.43 32.3572.30 36.8673.59 39.2573.52 39.3173.35 15.8172.56 15.3772.52

▲

n

▲

n

▲n

n

n

n

▲

223(92.92) 164(74.55) 208(86.67) 175(79.55) 187(77.92) 171(77.73) 159(72.27) 162(73.64) 214(89.17) 217(90.42)

▲n

n

▲

n

n

n

n

n

▲

▲

▲

▲

▲

▲

▲

Atr, atractyloside; CsA, Cyclosporin A; HI, hypoxia-ischemia; L-N, L-NAME; LY, LY294002; SB, SB216763; Sev, sevoflurane. Data are presented as mean7standard deviation. ▲ Po0.05 when compared with HI/Control group. n Po0.05 when compared with HIþSev group.

HIþSev group (Po0.01). Cyclosporine A itself improved the DIs of HI/Control group (Po0.01), but produced no significant improvement in the HIþSev group (P40.05) (Fig. 2).

Results of the Morris water maze test are shown in Table 1

and Fig. 3. When compared with that of the HI/Control group

and of the groups receiving inhibitors (LY294002, L-NAME,

groups, while marginal strategy and random strategy were dominant in the HI/Control, HIþSevþLY/L-N/SB, HIþAtr, and In space exploration, no significant HIþSevþAtr groups. difference in swimming speed was found between the groups (Fig. 3B; F ¼ 0.504, P ¼0.869). Principal component analysis, including cross number (X1), probe time (X2), and probe

SB216763), the EL of the groups receiving Sev or CsA or both was significantly shortened (Po0.05). Atr eliminated the protective effect of Sev (Po0.05), although it did not worsen HI injury itself (P40.05). CsA alone shortened the EL, but did not enhance the protective effect of Sev (P40.05). Straight strategy and tendency strategy were the primary movement strategies in the Sham, HIþSev, HIþCsA, and HIþSevþCsA

length (X3) on the original platform quadrant, showed that the initial eigenvalues λ 1 was 2.469, and cumulative percent ¼ so the first principal was 82.296%, þ0.376X3 can be extracted to represent the 0.360X1 comprehensive index of rat's spatial memory (Fig. 3A). The result showed that sevoflurane postconditioning significantly improved the score of HI/Control group (Po0.01), which was

component Z1

þ0.367X2

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Fig. 3 – Results of the space exploration test. Atr, atractyloside; CsA, Cyclosporin A; HI, hypoxia-ischemia; L-N, L-NAME; LY, LY294002; SB, SB216763; Sev, sevoflurane. Comparisons between groups were using one-way ANOVA with Fisher’s LSD as post-hoc procedure. The detailed sample size, mean7SD are listed in Supplementary table S2.

respectively decreased by LY294002, L-NAME, and SB216763 (Po0.01). Atr did not reduce the score of HI/Control group (P ¼0.870), but decreased that of HIþSev group (Po0.01). The combined treatment (HIþSevþCsA) did not increase the score of HIþSev group (P¼ 0.414), but CsA alone increased the score of HI/Control group (Po0.01).

Sevoflurane postconditioning alleviates neuronal

2.2. damage and loss

2.4.

Ca2þ

induces mPTP opening

A decreasing tendency in optical density at 540 nm (OD540) was seen in the CaCl2-induced mPTP opening time in all groups. mPTP opening was expressed as a reduction in OD540 during a 5 min period (△OD540/min). In the HIþSev, HIþCsA, and HIþSevþCsA groups the decreases were lower than in the HI/Control, HIþAtr, and HIþSevþAtr groups, respectively (Po0.01). In the HIþSevþLY, HIþSevþL-N, and HIþSevþSB groups, the decreases were higher than in HIþSev group (Po0.05) (Fig. 8).

In the Sham group, neuronal degeneration and neuronal In the HI/Control, apoptosis were occasionally observed. HIþAtr, and HIþSevþAtr groups obvious pyramidal layer thinning (only 1–2 layers), cell body shrinkage and deformity, disordered cell arrangement, gliocyte proliferation, capillary edema, disconnected neighboring neurons, obvious neuronal apoptosis and loss, and low density were observed. In the HIþSev, HIþCsA, and HIþSevþCsA groups a denser pyramidal layer distribution (3–4 aligned layers), hyperchromatic cyto- plasm, regular cell arrangement, clear structure, only mini- mal few discrete neuronal loss, and remarkably reduced cell apoptosis and cellular atrophy were observed. In the groups given the inhibitors (LY294002, L-NAME, and SB216763), 2–3 layer derangement, cell body shrinkage and deformity, glial cell proliferation, cell connection loss, and obvious neuron apoptosis and neuron absence were seen (Figs. 4 and 5). The surviving neuron density of the CA1 and CA3 regions are shown in Fig. 6.

Sevoflurane postconditioning increases the expression

2.3. of p-Akt, p-eNOS, and p-GSK-3β in the left hippocampus

No significant difference in t-Akt/eNOS/GSK-3β expression was found in all groups (Fig. 7). The expressions of p-Akt/ eNOS/GSK-3β were higher in the HIþSev than in the HI/ Control group (Po0.05), and significantly decreased in the groups receiving inhibitors when compared with the HIþSev group (Po0.05).

3.

Discussion

The results of this study using the classical Rice–Vannucci sevoflurane postconditioning model improves long-term learning and memory in neonatal HIBD rats by reducing hippocampal neurons loss; (2) activation of the PI3K/Akt/eNOS and PI3K/Akt/GSK-3β pathways is involved in the neuroprotection provided by sevoflurane postcondi- tioning; and (3) blocking mPTP opening plays an important role in this effect.

showed that:

(1)

Morphological changes of brain tissues are direct indexes to measure HI-induced brain damage. The key brain devel- opment periods of rats are from 1 to 2 days before birth to 2 weeks after birth (Schousboe et al., 2004). Therefore, we intervened on postnatal day 7 and observed structural changes on day 42. The result showed that neuronal degen- eration and neuronal apoptosis were occasionally observed in CA1 and CA3 region in the Sham group, while HI/Control group experienced obvious damage. This finding is consistent with that of other study, (Kumral et al., 2004) and indicates that neonatal HIBD may cause ongoing damage to the hippocampus of rats, which lasts to puberty (postnatal day 35). The CA1 and CA3 regions protected by sevoflurane postconditioning showed denser pyramidal layer distribu- tion, hyperchromatic cytoplasm, regular cell arrangement, clear structure, only minimal neuronal loss, and remarkably reduced cell apoptosis and cellular atrophy, indicating that

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Fig. 4 – Hematoxylin and eosin staining of left hippocampal CA1 region neurons in 42 day old rats (magnification, 200 (cid:2) , 400 (cid:2) ; scale bars¼10 lm). Atr, atractyloside; CsA, Cyclosporin A; HI, hypoxia-ischemia; L-N, L-NAME; LY, LY294002; SB, SB216763; Sev, sevoflurane.

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Fig. 5 – Hematoxylin and eosin staining of left hippocampal CA3 region neurons in 42 day old rats (magnification, 200 (cid:2) ,400 (cid:2) ; scale bars¼10 lm). Atr, atractyloside; CsA, Cyclosporin A; HI, hypoxia-ischemia; L-N, L-NAME; LY, LY294002; SB, SB216763; Sev, sevoflurane.

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Fig. 6 – Number of surviving neurons in the left hippocampal CA1 and CA3 regions. Atr, atractyloside; CsA, Cyclosporin A; HI, hypoxia-ischemia; L-N, L-NAME; LY, LY294002; SB, SB216763; Sev, sevoflurane. Comparisons between groups were using one-way ANOVA with Fisher’s LSD as post-hoc procedure. The detailed sample size, mean7SD are listed in Supplementary table S3.

sevoflurane postconditioning is capable of protecting the hippocampus from HIBD.

is a hippocampus-dependent cognition (Ennaceur and Delacour, 1988). The novel object recognition test reflects non-spatial learning and memory ability, and can test instant, short- term, and long-term memory retention ability. Rats in the HI/ Control group showed much lower new object recognition than rats in the Sham group at all time points. Presumably, the instant, short-term, and long-term non-spatial memory abilities of HIBD rats were damaged. Sevoflurane postcondi- tioning, however, improved their non-spatial memory ability but the indexes did not reach the levels of the Sham group indicating that sevoflurane postconditioning alone is incap- able of completely eliminating damage to non-spatial mem- ory ability caused by cerebral HI damage.

In rats,

the habit of exploring new things

opening, of mPTPs are key factors that determine whether injury is reversible or irreversible. Mitochondrial permeability transition is a common pathway shared by death and apoptosis of (Kroemer and Reed, 2000; Juhaszova et al., 2009). Sevoflurane and isoflurane postcondi- tioning against cerebral ischemia-reperfusion injury involve inhibition of mPTP opening (Feng et al., 2005; Pagel et al., 2006). Interestingly, neuronal mitochondria undergo perme- ability transition as well, (Nieminen et al., 1996) and inhibi- tion of mPTP activity has become a novel neuroprotection strategy (Kristal et al., 2004). Crucial factors for mPTP opening are mitochondrial calcium overload, ATP depletion, and oxidative stress, and these are exactly what occur in the brains of neonatal HIBD rats during hypoxia-reoxygenation. Thus, sevoflurane postconditioning-conferred neuroprotec- tion against HIBD may also act on mPTP opening.

injured cells

The Morris water maze is a classic method testing hippocampus-related spatial learning and memory ability (Rodríguez et al., 2003). The Morris water maze test showed that sevoflurane postconditioning improved learning effi- ciency, learning strategy, and long-term spatial memory as compared to the HI/Control group. However, sevoflurane postconditioning did not improve these measures to the level of the Sham group.

Research has proven that ischemic injury is a dynamic process, and that rodents continue to loose neurons weeks after cerebral ischemia (Li et al., 1995; Du et al., 1996). This loss is especially characterized by brain damage during the growth period (Hu et al., 2000). Our research showed that compared with the Sham group, rats in the HI/Control group had obvious hippocampal neuron loss and more serious apoptosis, which is consistent with the findings of the aforementioned studies. Neurons almost completely depend on mitochondria-supplied ATP to maintain their function, so the state of mitochondria is an important factor that deter- mines whether injured neurons survive (Kann et al., 2005; Mancuso et al., 2007). mPTPs are highly conductive nonspe- cific channels across the outer mitochondrial and inner mitochondrial membrane. Opening, and the degree of

In the current study, the HIþSev group showed signifi- cantly lower mPTP activity, reduced long-term hippocampal pathological injuries, and improved behaviors as compared with the HI/Control group. Atr, a mPTP-specific opener, did not worsen hippocampal neuron damage or harm long-term learning and memory by itself, but appeared to reverse the neuroprotection of sevoflurane postconditioning. Although CsA, a mPTP-specific blocker, mimicked the neuroprotection provided by sevoflurane postconditioning, failed to enhance the effect of sevoflurane postconditioning. These results suggest that sevoflurane postconditioning possibly protects the brain by blocking the mitochondrial permeability transition and reducing metabolic energy disorder and neu- ron damage in the hippocampus and other tissues of HIBD rats.

it

Endothelial nitric oxide synthase (eNOS), a downstream kinase of Akt, can activate the release of eNO and inhibit platelet aggregation and generation of superoxides in vessels so as to protect endothelial function and promote heman- giectasis and neovascularization, (Rikitake et al., 2002) effi- ciently preventing secondary lower perfusion and the damage to vascular endothelial cells caused by oxygen- derived free radicals during reperfusion period after HIBD.

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Fig. 7 – Western blot analysis. Protein expression of p-Akt/p-eNOS/p-GSK-3β and t-Akt/t-eNOS/ t-GSK-3β (A) in the left hippocampus in the Sham, HI/Control, HIþSev, and HIþSevþLY groups were determined 24 hours after the intervention. (B and C) Relative density of p-Akt and t-Akt, respectively. β-Actin served as the internal control. Error bars represent standard error of the mean. Statistical comparisons were performed with the Student's t-test. #Po0.05 compared with HI/Control. *Po0.05 compared with HIþSev group. Atr, atractyloside; CsA, Cyclosporin A; HI, hypoxia-ischemia; L-N, L-NAME; LY, LY294002; SB, SB216763; Sev, sevoflurane.

The latter 2 are key processes in the occurrence and devel- opment of HIBD. Some researchers believe that NO is involved in mediating the preconditioning and postcondi- tioning effects of volatile anesthetics like sevoflurane and isoflurane, and it remarkably reduces myocardial infarction size in ischemia/reperfusion models (Tessier-Vetzel et al., 2006; Lamberts et al., 2009). Rastaldo et al. (2007) have shown that endogenous NO may activate PKG via cGMP, and ulti- mately affect the human body by inhibiting mPTP opening. The NO/cGMP pathway is one of the important molecular mechanisms by which volatile anesthetics like sevoflurane exert their effects (Johns, 1996). Other studies have shown that GSK-3β takes part in the transduction of several intra- cellular signaling pathways, genetic transcription and trans- lation, embryogenesis, and neuronal death and apoptosis Jope and Johnson, 2004; Chong (Balaraman et al., 2006;

et al., 2007). Furthermore, the PI3K/Akt/GSK-3β pathway provides negative feedback and promotes cell survival by phosphorylating ser9 of GSK-3β, and mediating GSK-3β activ- ity inhibition (Pap and Cooper, 1998; Duarte et al., 2008).

Ischemic/pharmacological preconditioning and postcondi- tioning produce cardioprotective effects through inhibiting mPTP activity and reducing apoptosis via the PI3K/Akt/GSK- 3β signaling pathway (Juhaszova et al., 2009; Feng et al., 2005; Gomez et al., 2008; Zhu et al., 2010). The concentration of GSK-3β in the central nervous system is extremely high, especially in the hippocampus (Hidenori et al., 2006). The expression level of GSK-3β reaches a peak during late preg- nancy and early after birth, and is closely related to develop- ment and reconstruction of neurons (Takahashi et al., 1994; Leroy and Brion, 1999). We found that the P13K pathway was activated to induce more phosphorylation of Akt, eNOS, and

b r a i n r e s e a r c h 1 6 3 0 ( 2 0 1 6 ) 2 5 – 3 7

of p90rsk2 under SB216763 treatment is necessary to further elucidate the mechanism in the future.

Fig. 8 – Alterations of mitochondrial permeability transition pore opening. Atr, atractyloside; CsA, Cyclosporin A; HI, hypoxia-ischemia; L-N, L-NAME; LY, LY294002; SB, SB216763; Sev, sevoflurane. Comparisons between groups were using one-way ANOVA with Fisher's LSD as post-hoc procedure. The detailed sample size, mean7SD are listed in Supplementary table S4.

GSK-3β in the HIþSev group than in the HI/Control, specific inhibitors LY294002, L-NAME, and SB216763 blocked sevoflur- ane postconditioning-induced expression of p-Akt, p-eNOS, and p-GSK-3β, respectively, and mPTP activity was greatly increased in the corresponding groups, neutralizing the effect of sevoflurane postconditioning on HIBD. These findings indicate that sevoflurane postconditioning may confer neu- roprotection against HIBD by inhibiting mitochondrial per- meability transition via the PI3K/Akt/eNOS and PI3K/Akt/ GSK-3β pathways. Although sevoflurane-induced activation of PI3K/Akt has been confirmed to provide heart and cerebral protections in several studies (Ye et al., 2012; Zhang et al., 2014), however, the underlying mechanism of sevoflurane- induced activation of PI3K/Akt is still unknown. It will be interesting to indentify the upstream components of the signaling pathway(s) that exert sevoflurane-induced activa- tion of PI3K/Akt. Previous studies have suggested that PI3K/ AKT/eNOS and PI3K/Akt/GSK-3β pathways may regulate mPTP through PKC-epsilon, reactive oxygen species, Ca2þ and mitochondrial ATP-dependent Kþ channels (Ren et al., 2014; Juhaszova et al., 2004; Pravdic et al., 2009). But the detailed mechanism is still not fully understood and is worthy of further investigation.

There are some limitations to this study. Arterial blood gas analysis was not performed during cerebral HI. Some researchers believe that cerebral HI itself, to a certain degree, can promote proliferation of cortical neurons and hippocam- pal neural precursor cells in neonatal rats, (Bartley et al., 2005) while sevoflurane lowers the cerebral metabolic rate, so both play a role in the recovery of neurological function. The result that Art neutralized the neuroprotective effect of sevoflurane postconditioning may partially be due to its effect of inhibiting energy generation rather than blocking mPTP. CsA may be incapable of blocking mPTP opening when mitochondria are seriously injured, and its neuroprotective effect may be dose-dependent. Whether combining different concentrations of sevoflurane and different doses of CsA can protect against HIBD to different degrees requires future study. We did not perform 2,3,5-triphenyltertrazolium chlor- ide (TTC) staining to determine brain infarct volume. Lastly, other pathways such as PI3K/Akt/mTOR or ERK1/2 were not studied.

In conclusion, sevoflurane postconditioning may improve long-term learning and memory of neonatal HIBD rats pos- sibly by blocking mPTP opening and reducing neuron death and apoptosis in the hippocampus via PI3K/Akt/eNOS and PI3K/Akt/GSK-3β pathways.

4.

Experimental procedures

4.1.

Animals

A total of 240 male and female clean Sprague-Dawley rats, 7 days of age and weighting 12–16 g, (Shanghai Slac Labora- tory Animal Co., Ltd., China) were used in this study. They were housed and treated in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 80-23, revised in 2011). All rats were maintained under standard laboratory temperature and humidity and a 12 day/night cycle (8 am/8 pm), and were allowed free access to food and water. The study was approved by the Experimental Animal Care Committee of the Fujian Medical University Union Hospital, and efforts were made to minimize the number of animals used and their suffering.

4.2.

Experimental protocol

It is noteworthy that sevoflurane postconditionin caused phosphorylation of GSK-3β at Ser9 and its inhibition, while SB216763 blocked sevoflurane-induced phosphorylation of GSK-3β and neuroprotection in our study. These two results seem conflicted since both Sev postconditioning and SB216763 treatment inhibits GSK-3β. However, previous stu- dies have consistently reported that SB216763 reduced the phosphorylation levels of GSK-3β Ser9 (possibly via inhibition of p90rsk2 (Zhang et al., 2003; Lochhead et al., 2001; Liang and Chuang, 2007), a kinase of GSK-3β Ser9), indicating the inhibition effect of SB216763 on GSK-3β has a complicated mechanism which eventually blocked the sevoflurane- induced protection in this study. Examining kinase activity

The animals were randomly allocated into 10 groups (n¼ 24 per group; Fig. 1): (1) Sham, without hypoxia-ischemia; (2) HI/ Control, received cerebral hypoxia-ischemia; (3) HIþAtractylo- side (Atr), (4) HIþCyclosporin A (CsA), treated like the control and respectively injected with Atr (10 mg/kg) and CsA (5 mg/ kg); (5) HIþsevoflurane (Sev), treated like the control and (6) HIþSevþLY, received sevoflurane postconditioning; (7) HIþSevþL-N, (10) HIþSevþCsA, treated like the HIþSev group and respectively injected with LY294002 (0.3 mg/kg), L-NAME (10 mg/kg), SB216763 (0.2 mg/kg), Atr (10 mg/kg), and CsA (5 mg/kg). LY294002, L-NAME, and SB216763 are specific blockers of

(8) HIþSevþSB,

(9) HIþSevþAtr,

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Akt, eNOS, and GSK-3β, respectively. Atr and CsA open and close, respectively, mPTPs. In each group, the brains of the rats that received behavioral testing (from 32-days-old to 42- days-old; n¼ 12 per group) were harvested for determination of hippocampal neuron count and morphology study, and the brains of the other 12 rats were harvested 24 h after the intervention for Western blot analysis, and study of mito- chondrial permeability transition pore opening.

4.3.

Cerebral HI model and sevoflurane postconditioning

The cerebral HI model was adapted from a procedure described previously (Ren et al., 2014). Briefly, the rats were anesthetized with pentobarbital sodium (0.5–1%, 40–50 mg/ kg, intraperitoneal), and their left common carotid arteries were permanently ligated with a double 7–0 surgical silk; the arteries in the Sham group, however, were not ligated. A dose of 5 mL of 0.1% DMSO or drug (LY294002, L-NAME, SB216763, Atr, CsA) with 0.1% DMSO was injected into the left lateral ventricle immediately after the surgery as previously described (Satoh and Onoue, 2005). After waking, the rats were returned to their cages with the mothers for 1.5–2.5 h, – and then placed in a chamber containing humidified 8% O2 92% N2 for 2 h. The air temperature in the chamber was maintained at 36.571 1C. The chamber was then exposed to room air for 15–20 min. For sevoflurane postconditioning, the animals were placed in a chamber containing 2.5% sevoflur- –70% N2 for 30 min after cerebral HI injury. After ane in 30% O2 waking, the neonates were cleaned with 75% alcohol and returned to their mothers.

4.4.

Novel object recognition test

The rats were evaluated with a nonspatial object recognition memory task 25 days after the intervention as described by Ennaceur and Delacour (1988) and Bruel-Jungerman et al. (2005). Briefly, for the first 3 days, after being comforted and stroked, each animal was put into an open chamber made of black plexiglas (80 (cid:2) 80 (cid:2) 60 cm3) for a 5 min acclimation and the test was conducted on the fourth day. Before the test, the animals received a 5 min training in the chamber containing 2 different objects (a white cube and a red cylinder) fixed at adjacent angles with a spacing of 10 cm from the field wall. Rats were put into the chamber with their backs turned towards the objects and allowed to explore the chamber freely for 5 minutes. Exploratory behavior can be identified when rats touch the objects with their noses or put their noses at places within 2 cm of the objects. To test memory storage, the white cube was kept in the chamber and the red cylinder was replaced by a blue semisphere. Exploratory time of new (T2) and old (T1) objects within 5 min was recorded and memorization ability of the rats was assessed by discrimina- þT2). The blue semisphere was replaced tion index: DI¼ T2/(T1 by a green prism 3 h after training, and the green prism was replaced by a yellow irregular shape 24 h after the training. The time each rat used to explore new and old objects was recorded for calculating DI. The DIs at 5 min, 3 h, and 24 h after the training (DI0 h, DI3 h, DI24 h) represent the instant, short-term, and long-term memory, respectively. Data with total exploration time less than 20 s were excluded from

statistic analysis. The field was always provided with even light, and the objects and fields were cleaned with 75% ethanol after each testing.

4.5.

Morris water maze test

After the novel object recognition test, the Morris water maze was used to test spatial learning and memory (Peng et al., 2012; Jiang et al., 2004). Briefly, a black circular pool (120 cm in diameter, 50 cm in height) was filled with water (2571 1C) to a depth of 25 cm and located in a quiet room. Chinese ink was added to make the water opaque. The water maze was conceptually divided into 4 quadrants, and a hyaline platform (10 cm in diameter) was submerged 1 cm below the surface of the water at the midpoint of the third quadrant. In the place navigation trial, each rat underwent 4 successive trials a day for 5 days for memory acquisition training, with a 15 min interval between trials for the rat to recover physically. The sequence of water-entry points differed each day, but the location of the platform was constant. Escape latency (EL) to find the platform was measured up to a maximum of 120 s. On locating the platform, the rat was left there for 15 s before the next trial. If the rats failed to locate the platform within 120 s, it was guided to the platform and allowed to stay there for 15 s. Latency and the search strategies, including straight strategy, tendency strategy, marginal strategy, and random strategy, were recorded for each trial. Twenty-four hours after the last training session, a space exploration trial was performed. The platform was removed from the pool and rats were allowed to swim freely for 60 s. Four indexes were calculated: (1) the time spent by the rats in the third quadrant in which the platform was hidden during acquisition trials; (2) the number of rats crossing exactly over the original position of the platform; (3) the search path in the target quadrant; (4) the total movement distance. Search speed was calculated by total movement distance divided by 120 cm/s. All trials were videotaped by a camera located 2 m above the water surface and computer analyzed.

4.6.

Histology of left hippocampal neurons

After the behavioral studies, rats were anesthetized with pentobarbital, transcardialy perfused with 200 mL of 4 1C heparin saline solution and then with 300 mL of 4% paraf- ormaldehyde. Left hippocampus was made into a wax block according to Paxinos–Waston methods. Continual coronal sections (4 mm in thickness) at approximately 3.3 mm caudal to bregma were obtained, and subjected to hematoxylin– eosin (HE) staining. The sections were examined by an observer blinded to the rat group assignment. Neurons microscopically showed a clear boundary, a round or an oval shape, a smooth cell membrane, basophilic cytoplasm (Nissl body), a large and round nucleus, a clear nuclear membrane and a large and round nucleolus will be defined as surviving neurons. Apoptotic neurons will not be regarded as surviving ones. Surviving neurons in pyramidal cell layer of the CA1 and CA3 regions were counted (n/mm) by two investigators blind to experimental conditions, and a count was deter- mined by averaging the total of 5 sections.

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4.7. Western blot analysis

Acknowledgments

Proteins were separated on a 12% SDS-PAGE gel, and then transferred to a nitrocellulose membrane (Bio-Rad, Hercules, USA). The membrane was blocked using 5% nonfat milk and incubated with a mouse anti-p-Akt, t-Akt, p-eNOS, t-eNOS, p-GSK-3β, and t-GSK-3β monoclonal antibody (mAb) (Cell Signaling Technology, Beverly, MA, USA) or a mouse anti-β- actin mAb (Sigma, USA). The proteins were visualized and quantified using ECL reagents (Pierce, IL, USA).

This work was supported by grants from Department of Edu- cation, Fujian Province (type A) (Grant number: JA12159) in part, and the Science Foundation of the Fujian Province, China (Grant No. 2015J01465).

Appendix A.

Supplementary material

4.8.

mPTP opening assay

Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.brainres. 2015.10.050

Preparation of mitochondria was adapted from a procedure described previously (Wu et al., 2006). All procedures were carried out in the cold (0–4 1C). Hippocampal pieces were placed in isolation buffer (250 mmol/L sucrose, 210 mmol/L mannitol, 1 mmol/L K-EDTA, 10 mmol/L Tris–HCl, pH 7.4) and homogenized (10 mL buffer/g). The homogenate was imme- diately centrifuged at 2000g for 3 min. The supernatant was centrifuged again at 2000g for 3 min, the second supernatant was decanted and centrifuged at 12,000g for 8 min, and the resulting supernatant was decanted and resuspended in isolation buffer without K-EDTA. The suspension was cen- trifuged at 12,000g for 10 min and the resulting mitochondrial pellet was resuspended in the same buffer. Mitochondrial protein concentration was quantified according to the Brad- ford's method using 1 g/mL bovine serum albumin (BSA) as standard. Purity and integrity of isolated mitochondria were confirmed by neutral red-Janus green B staining (Sigma, USA). Isolated mitochondria from the hippocampus (0.5 mg protein) was resuspended in swelling buffer (71 mmol/L sucrose, 215 mmol/L mannitol, and 10 mmol/L sodium succinate in 5 mmol/L HEPES, pH 7.4) to a final volume of 2 mL, and incubated at 25 1C for 2 min. mPTP-induced mitochondrial swelling was confirmed by 5 min incubation with the strong mPTP inhibitor CsA before addition of CaCl2, and was mea- sured with a spectrophotometer (Beckman DU800, USA) as a reduction in optical density at 540 nm (OD540) (Kristal and Brown, 1999; Baines et al., 2003).

4.9.

Statistical analysis

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