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. Author manuscript; available in PMC: 2011 Jul 1.
Published in final edited form as: Stroke. 2010 May 27;41(7):1521–1527. doi: 10.1161/STROKEAHA.110.583757

Isoflurane post-treatment reduces neonatal hypoxic-ischemic brain injury in rats by S1P/PI3K/Akt pathway

Yilin Zhou 1, Tim Lekic 1, Nancy Fathali 1, Robert P Ostrowski 1, Robert D Martin 1, Jiping Tang 1, John H Zhang 1
PMCID: PMC2917259  NIHMSID: NIHMS213421  PMID: 20508187

Abstract

Background and Purpose

Isoflurane, administered before or during cerebral ischemia, has been shown to exhibit neuroprotection in animal models of ischemic stroke. However, the underlying mechanism remains to be elucidated. In the present study we determined whether isoflurane post-treatment provides neuroprotection after neonatal hypoxia-ischemia (HI) in rats, and evaluated the role of the sphingosine-1-phosphate (S1P)/phosphatidylinositol-3-kinase (PI3K)/Akt pathway in this volatile anesthetic-mediated neuroprotection.

Methods

HI was induced in P10 rat pups by unilateral carotid ligation and two hours of hypoxia. For treatment, 2% isoflurane was administered immediately after HI for 1 hour. As pharmacological interventions, the S1P antagonist VPC23019 or PI3K inhibitor wortmannin or opioid antagonist naloxone was administered before HI. Isoflurane post-treatment was evaluated for effects on infarct volume at 48 hours after HI, brain atrophy and neurological outcomes at 4 weeks after HI. The expression of phosphorylated Akt (pAkt) and cleaved caspase 3 was determined by western blotting and immunofluorescence analysis.

Results

Isoflurane post-treatment significantly reduced infarct volume at 48 hours after HI. VPC23019 or wortmannin abrogated the neuroprotective effect of isoflurane, while naloxone did not inhibit the isoflurane-induced neuroprotection. Isoflurane post-treatment significantly preserved pAkt expression, and decreased cleaved caspase 3 levels. These effects were reversed by VPC23019 and wortmannin respectively. Isoflurane also confers long-term neuroprotective effects against brain atrophy and neurological deficits at 4 weeks after HI.

Conclusions

Isoflurane post-treatment provides lasting neuroprotection against hypoxic-ischemic brain injury in neonatal rats. Activation of S1P/PI3K/Akt pathway may play a key role in isoflurane post-treatment-induced neuroprotection.

Keywords: neonatal hypoxia-ischemia, isoflurane, Sphingosine 1-phosphate, Akt, apoptosis

Introduction

Perinatal hypoxia-ischemia (HI) continues to be a major contributor to mortality and life-long neurological impairments in infants and children.1,2 The incidence is as high as 1 in 4000 live births.2 Although the underlying pathophysiology of neonatal HI, including oxidative stress, excitotoxicity, inflammation and apoptosis,2 has been intensively studied, successful treatments for neonatal HI are still lacking.

Isoflurane, a volatile anesthetic, has been commonly and safely used in surgical procedures for decades. Accumulating evidence indicates that isoflurane, administered before or during experimental cerebral ischemia, provides neuroprotection in both in vivo and in vitro models.3,4 Isoflurane has also been reported to reduce ischemia-reperfusion injury in the myocardium5 and kidney6. However whether isoflurane, administered after hypoxic-ischemic insult, confers neuroprotection in neonates has not been studied yet. The molecular pathways underlying isoflurane-induced protection have been incompletely mapped and require to be further elucidated.

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid metabolite, and has been shown to bind to specific G protein-coupled receptors (the S1P receptors) and regulate multiple cellular events, including promoting cell proliferation, survival, migration and inhibiting apoptosis.7 The prosurvival PI3K/Akt have been shown to be downstream molecules regulated by the S1P1 receptor signaling.7 A recent study reported that isoflurane ameliorates renal ischemia-reperfusion injury by initiating S1P/S1P receptor signaling pathway.6 However, it is unknown whether the activation of S1P/ S1P receptor signaling transduction is vital for isoflurane-mediated neuroprotection in the setting of experimental cerebral hypoxia-ischemia. In the present study, we tested the effect of isoflurane as a post-treatment in a rat neonatal HI model. We hypothesized that isoflurane post-treatment will result in decreased brain injury and improved neurobehavioral outcomes by activating S1P/PI3K/Akt signaling pathway.

Materials and Methods

Animals

All experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Loma Linda University. Timed pregnant Sprague-Dawley rat were purchased from Harlan Laboratories, Indianapolis, IN. One hundred seventy seven P10 rat pups were used in this study and randomly divided into the following groups: sham-operated (n=18), HI group (n=31), HI groups treated with isoflurane (n=30), VPC23019+isoflurane (n=29), VPC23019 alone (n=10), wortmannin+isoflurane (n=29), wortmannin alone (n=10), naloxone+isoflurane (n=10) and naloxone alone (n=10). Rat pups of both genders were subjected to right common carotid artery ligation under isoflurane anesthesia. Surgery time for each pup did not exceed 5 minutes. After recovery for 1 hour, the pups were placed in a hypoxia chamber, which was submerged in a water bath at a stable temperature of 37 °C, and subjected to 8%O2 in N2for 2 hours. Sham-operated animals underwent anesthesia and incision only.

Isoflurane Post-treatment and Pharmacological Interventions

In the treatment group, rat pups received isoflurane post-treatment for 1 hour immediately after hypoxia by transferring them to 60 ml syringes continually flushed with 2% isoflurane, carried by 30%O2 and 70% medical air. In the HI group, pups were placed in the syringes flushed with 30%O2 and 70% medical air for 1 hour.

For pharmacological interventions, the specific S1P1/S1P3 antagonist VPC23019 (0.5mg/kg, intracerebroventricularly; VPC+HI+Iso group)8 or the PI3K inhibitor wortmannin (86ng/pup, intracerebroventricularly; WM+HI+Iso group)9 was administered just before the HI surgery. The nonselective opioid antagonist naloxone (5mg/kg, intraperitoneally; Naloxone+HI+Iso group)10 was given immediately before and after the hypoxia.

Infarct Volume and Brain Atrophy Quantification

As previously11, 2,3,5-triphenyltetrazolium chloride monohydrate (TTC) staining was performed to determine the infarct volume at 48 hours after HI. The infarct volume was traced and analyzed by Image J software (version 1.40; National Institutes of Health, Bethesda, MD). Brain atrophy was assessed at 4 weeks after HI. Brain tissue loss was expressed as the mass ratio of the ipsilateral hemisphere compared to the contralateral hemisphere.

Neurobehavioral Tests

All neurobehavioral tests were performed in a blinded set-up. At 4 weeks after HI, animals underwent the following three neurobehavioral tests:

T-maze test for spontaneous alternation has been used to examine exploratory behavior and working memory by hippocampus dysfunction. As previously11, rats were placed in the stem of a T-shaped maze and allowed to freely explore the two arms of the maze, throughout a 10-trial continuous alternation session. The spontaneous alternation rate was expressed as the ratio of the alternating choices to the total number of the choice.

In the foot-fault test,12 the rats were placed on a horizontal grid floor for 2 minute. The foot-fault was defined as when the rat inaccurately placed a fore or hind limb, and it fell through one of the openings in the grid. Number of foot-faults was recorded. The left/right side difference of foot-faults was used for the statistic analysis.

The modified grip-traction test has been used to test the muscle strength of the rat by hanging the rat to a horizontal rope by its forepaws.12 Time to falling (maximum 60 second) was recorded.

Western Blotting

The brain samples were collected at 24 hours after HI (n=6, respectively). Proteins of the ipsilateral hemisphere were extracted by homogenizing in RIPA buffer (Santa Cruz Biotechnology, Santa Cruz, CA). Western blotting was performed as described previously8 using anti-phospho-Akt (Ser473) and anti-cleaved caspase 3 (Cell Signaling Technology, Danvers, MA) antibodies.

Immunofluorescence staining

Triple–fluorescence staining of the ipsilateral hippocampus and peri-infarct region was performed at 24 hours after HI as described previously.8 The following primary antibodies were used:

1) anti-Akt1,2,3 (phospho-Ser473) antibody (Assay designs, Ann Arbor, MI), 2) anti-cleaved caspase 3 antibody (Cell Signaling Technology, Danvers, MA), and 3) anti-MAP2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA).

Statistical Analysis

Data were expressed as the mean±SEM. Statistical differences among groups were analyzed by using one-way ANOVA followed by Holm-Sidak method. P<0.05 was considered statistically significant.

Results

Mortality

The mortality rate in the HI group and isoflurane post-treatment group was as follows: 9.8% (3 of 31 pups) in the HI group and 6.7% (2 of 30 rats) in the isoflurane-treated group.

Isoflurane-Mediated Reduction in Infarct Volume Depends on S1P/PI3K/Akt Signaling Pathway

48 hours after neonatal HI brain injury, we determined the effect of isoflurane post-treatment on infarct volume. Isoflurane post-treatment resulted in a significant reduction in infarct volume compared to the HI group (21.9±3.4% and 31.4±2.1%, respectively; Fig. 1). There was no difference in isoflurane post-treatment-mediated reduction in infarct volume between genders (data not shown). To test if this isoflurane-induced neuroprotection depends on the S1P/PI3K/Akt signaling pathway or is via opioid receptor signal transduction, pups in the pharmacological intervention groups were pretreated with the S1P receptor antagonist VPC23019, the PI3K inhibitor wortmannin, or the nonselective opioid antagonist naloxone, respectively. Pretreatment with VPC23019 and wortmannin completely abrogated the reduction in infarct volume provided by isoflurane post-treatment (30.9±2.1% and 30.4±1.7%, respectively; Fig. 1), while naloxone did not inhibit the isoflurane-mediated neuroprotection (23.1±2.4%; Fig. 1). The compounds did not affect the infarct volume when administered alone (n=10, data not shown). These findings are consistent with the hypothesis that the isoflurane-induced neuroprotection requires activation of the S1P/PI3K/Akt signaling pathway.

Figure 1. Infarct Volume at 48 Hours After HI.

Figure 1

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Representative pictures of TTC-stained coronal brain sections (A) and quantitative analysis of infarct volume (B) in the HI group and groups treated with isoflurane (HI+Iso), VPC23019+isoflurane (VPC+HI+Iso), wortmannin+isoflurane (WM+HI+Iso), and naloxone+isoflurane (Naloxone+HI+Iso) at 48 hours after HI, n=9-10 in each group. Isoflurane post-treatment significantly reduced infarct volume. Pretreatment with VPC23019 or wortmannin abolished the reduction in infarct volume induced by isoflurane, whereas naloxone did not block the isoflurane-induced protection. Values are the mean±SEM; *P<0.05 vs. HI group, #P<0.05 vs. Isoflurane-treated group.

Isoflurane Confers Long-term Neuroprotection against Brain Atrophy and Neurological Deficits

Isoflurane post-treatment provided short-term protection against neonatal HI brain injury, we then sought to determine whether this beneficial effect is long-lasting. At 4 weeks after HI, extensive atrophy of ipsilateral brain tissue was observed in the HI group. Isoflurane post-treatment prevented the brain tissue loss (Fig. 2). Administration of VPC23019 and wortmannin abolished the isoflurane-induced protective effect against brain atrophy (Fig.2).

Figure 2. Brain Weight at 4 Weeks After HI.

Figure 2

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Representative pictures of brain (A) and quantitative analysis of brain weight (B) in groups of sham-operation (sham), HI or treated with isoflurane (HI+Iso), VPC23019+isoflurane (VPC+HI+Iso), wortmannin+isoflurane (WM+HI+Iso), n=7-10 in each group. At 4 weeks after HI, there was extensive brain tissue loss of ipsilateral hemisphere in the HI group. Isoflurane post-treatment attenuated the brain atrophy. Administration of VPC23019 or wortmannin blocked isoflurane-mediated protection. Values are the mean±;SEM; *P<0.05 vs. HI group, #P<0.05 vs. sham-operated group.

We also tested the effect of isoflurane post-treatment on the functional outcomes. Animals in the sham-operated group performed normally in all three neurobehavioral tests at 4 weeks after HI. T-maze testing for spontaneous alternation demonstrated a significantly worse performance in the HI group, compared to the sham-operated and isoflurane-treated groups (Fig. 3A). Animals in the HI group had significantly more foot-faults with their left-site fore and hind limb, which is contralateral to the brain injury site, compared to the sham-operated and isoflurane-treated groups (Fig. 3B). In the modified grip-traction test, rats in the HI group hung on to the rope about 15.5±2.8 seconds, compared to 37.6±0.9 seconds in the sham-operated group and 26.8±2.2 seconds in the treatment group (Fig. 3C). Likewise, pretreatment with VPC23019 or wortmannin reversed the isoflurane-mediated improvement in functional outcomes (Fig.3A-C). Taken together, isoflurane post-treatment significantly improved the neurobehavioral outcomes at 4 weeks after HI. This long-lasting isoflurane induced-neuroprotection also depends on the S1P/PI3K/Akt signaling pathway.

Figure 3. Neurobehavioral Tests at 4 Weeks After HI.

Figure 3

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Evaluation of neurological outcomes in groups of sham-operation (sham), HI or treated with isoflurane (HI+Iso), VPC23019+isoflurane (VPC+HI+Iso), wortmannin+isoflurane (WM+HI+Iso), n=7-10 in each group. Animals in the HI group exhibited severe neurobehavioral impairments in the T-maze test (A), foot-fault test (B) and modified grip-traction test (C). Isoflurane post-treatment significantly improved neurological outcomes at 4 weeks after HI. These beneficial effects were abrogated by administation of VPC23019 or wortmannin. Values are the mean±;SEM; *P<0.05 vs. HI group, #P<0.05 vs. sham-operated group.

Isoflurane Increases Phosphorylation of Akt and Decreases Expression of Cleaved Caspase-3 after HI

To further confirm that the S1P/PI3K/Akt signaling pathway underlies isoflurane-induced neuroprotection in the neonatal HI model, we measured phosphorylated Akt and cleaved caspase 3 levels in the ispilateral hemisphere (Fig. 4). The phosphorylation of Akt significantly increased after isoflurane post-treatment (Fig. 4A and 4B), whereas cleaved caspase 3 levels drastically decreased (Fig. 4C and 4D). These effects were reversed upon the pretreatment with VPC23019 or wortmannin, respectively (Fig. 4A-4D).

Figure 4. pAkt and Cleaved Caspase 3 Expression in Ipsilateral Hemisphere at 24 Hours After HI.

Figure 4

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Representative Western blots (A and C) and quantitative analysis of phosphorylated Akt (pAkt) and cleaved caspase 3 expression (B and D) in groups of sham-operation (sham; n=6), HI (HI; n=6) or treated with isoflurane (HI+Iso; n=6), VPC23019+isoflurane (VPC+HI+Iso; n=6), and wortmannin+isoflurane (WM+HI+Iso; n=6) at 24 hours after HI. The HI-induced decrease in pAkt level was prevented by isoflurane post-treatment. Pretreatment with VCP23019 or wortmannin reversed the isoflurane-mediated increase in pAkt expression (A and B). The cleaved caspase 3 level was increased in the HI group, which was decreased by isoflurane post-treatment. This isoflurane-induced reduction in the cleaved caspase 3 expression was blocked by administration of VPC23019 or wortmannin, respectively (C and D). Values are the mean±;SEM;*P<0.05 vs. HI group, #P<0.05 vs. sham-operated group.

Consistent with the western blotting results, the immunofluorescence analysis revealed that the expression of phosphorylated Akt was decreased in neurons in ipsilateral hippocampus and peri-infarct region of the HI group (Fig. 5G and 6F), which was restored by isoflurane post-treatment (Fig. 5L and 6J). VPC23019 or wortmannin prevented the isoflurane-induced increase in phosphorylated Akt level, respectively (Fig. 5Q, 5V, 6N and 6R). On the other hand, the cleaved caspase 3 level was increased in neurons in the HI group (Fig. 5H and 6G), but was reduced by isoflurane post-treatment (Fig. 5M and 6K). Once more, VPC23019 and wortmannin blocked the isoflurane-induced decrease in cleaved caspase 3 level, respectively (Fig. 5R, 5W, 6O and 6S). These results support the hypothesis that the activation of S1P/PI3K/Akt pathway contributes to the isoflurane-induced protection against HI brain injury.

Fig. 5. Representative Immunofluorescence: Colocalization of pAkt, Cleaved Caspase 3 With MAP2 Positive Cells in Ipsilateral Hippocampus at 24 Hours After HI.

Fig. 5

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Representative immunofluorescence images showing the colocalization of phosphorylated Akt (pAkt; green), and cleaved caspase 3 (red) with MAP2 (blue) positive cells in the ipsilateral hippocampus in groups of sham-operation (sham; A-D), HI (HI; F-I) or treated with isoflurane (HI+Iso, K-N), wortmannin+isoflurane (WM+HI+Iso; P-S), and VPC23019+isoflurane (VPC+HI+Iso; U-X) at 24 hours after HI. Cells in the ipsilateral hippocampus were fixed and stained with DAPI (E, J, O, T, Y). Arrows indicate apoptotic cells with pyknotic nuclei. The number of nuclei with an apoptotic morphology was much lower in the isoflurane-treated group, compared to the HI group and the groups pretreated with VPC23019 or wortmannin. Scale bar of images A-D, F-I, K-N, P-S, and U-X: 50 μm; scale bar of images E, J, O, T and Y: 20 μm.

Fig. 6. Representative Immunofluorescence: Colocalization of pAkt, Cleaved Caspase 3 With MAP2 Positive Cells in Ipsilateral Peri-infarct Region at 24 Hours After HI.

Fig. 6

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Representative immunofluorescence colocalization of phosphorylated Akt (pAkt; green), and cleaved caspase 3 (red) with MAP2 (blue) positive cells in the ipsilateral peri-infarct region in groups of sham-operation (sham; A-D), HI (HI; E-H) or treated with isoflurane (HI+Iso, I-L), wortmannin+isoflurane (WM+HI+Iso; M-P), and VPC23019+isoflurane (VPC+HI+Iso; Q-T) at 24 hours after HI. Scale bar: 50 μm.

Discussion

Isoflurane is used in clinical practice as a volatile anesthetic in the United States. In this study we tested 2 hypotheses: (1) Isoflurane post-treatment protects against HI brain injury in neonatal rats. (2) This protective effect is via the sphingosine 1-phosphate (S1P)/PI3K/Akt signaling pathway. We found that 2% isoflurane, given immediately after HI, reduced infarct volume in short-term, as well as brain atrophy and neurobehavioral deficits in long-term. Moreover, blocking the S1P receptor with VCP23019 or inhibiting PI3K by wortmannin abolished the isoflurane-mediated beneficial effects. In addition, we showed that administration of VCP23019 and wortmannin also blocked the isoflurane-induced recovery of Akt activity and decrease in cleaved caspase 3 expression, a marker of apoptotic cell death. These findings suggest that isoflurane-mediated neuroprotection against neonatal HI depends on S1P/PI3K/Akt signaling.

Volatile anesthetics, including isoflurane, have been demonstrated to provide protection against ischemic injury in various organ.3,5,6 Different mechanisms underlying this beneficial effect have been proposed. Isoflurane preconditioning has been shown to exhibit neuroprotection by upregulating inducible nitric oxide synthase (iNOS) in a neonatal HI model.3 Chiari et al. reported that PI3K pathway was involved in isoflurane-mediated protection against myocardial infarction.5 However, the upstream signaling pathway involved in volatile anesthetics-mediated protection is unclear. Weihrauch et al. reported that isoflurane mediates cardioprotection in a rabbit myocardial ischemia model via opioid receptor.13 However, we found that naloxone, an nonselective opioid antagonist, could not abolish the isoflurane post-treatment-induced neuroprotection in the neonatal HI model. The discrepancy between our finding and that of Weihrauch et al. is probably due to different species and ischemic models (organs) studied in these two experiments. Most volatile anesthetics are lipophilic and have been reported to activate sphingomyelin hydrolysis in brain tissue by increasing membrane fluidity and allowing the sphingomyelinase to increase its hydrolytic effect.14,15 Sphingosine is the sphingomyelin breakdown product. It is then phosphorylated by the sphingosine kinase to form sphingosine-1-phosphate (S1P). Likewise, isoflurane has been shown to enhance the production of S1P, the sphingolipid metabolite in the renal cortex in vivo16 and in human proximal tubule cells (HK-2)6. In addition, a recent study showed that isoflurane activates sphingosine kinase (SK) activity and synthesis of S1P in renal tubule cells to afford renal protection via S1P signaling pathway in the setting of renal ischemia-reperfusion injury.6 Thereby, we sought to examine whether there is a role for S1P signaling pathway in mediating isoflurane post-treatment-induced neuroprotection after neonatal HI. Since neurons and astrocytes express mainly S1P1- and S1P3- receptors,17 we employed VPC23019, a competitive antagonist at both the S1P1 and S1P3 receptors,18 as a pharmacological intervention. We found that administration of VPC23019 blocked the isoflurane-mediated reduction in infarct volume and apoptotic cell death, as indicated by the reversal of isoflurane-induced decrease in the cleaved caspase 3 levels in ipsilateral hemisphere. Furthermore, VCP23019 reversed isoflurane-induced activation of Akt. These data implicates that the activation of the S1P/PI3K/Akt signaling contributes to the isoflurane-mediated neuroprotective effects in neonatal HI.

S1P is an important lipid mediator, and ligand for a family of five G-protein-coupled receptors (S1P1-S1P5). Upon binding to its specific receptors, S1P has been implicated in diverse biological processes, including cell growth, differentiation, survival and migration.7 PI3K/Akt signaling cascade has been shown to play a key role in preventing apoptosis under hypoxic or ischemic conditions.5,8 Morales-Ruiz et al. demonstrated that S1P activates PI3K/Akt signaling through the pertussis toxin (PTx) sensitive G-protein in endothelial cells.19 In our current study, we firstly confirmed that wortmannin, a PI3K inhibitor, given intracerebroventricularly inhibited the increase in phosphorylated Akt levels seen after isoflurane post-treatment. We then demonstrated that pretreatment with wortmannin abolished the isoflurane-induced neuroprotection, as showing by a reversal of the reduction in infarct volume and cleaved caspase 3 expression provided by isoflurane post-treatment. Taken together, our findings indicate that the S1P/PI3K/Akt signaling pathway underlies isoflurane-mediated neuroprotection in neonatal HI.

Despite our findings, the reversal of isoflurane-mediated neuroprotection with the nonspecific inhibitor wortmannin cannot completely exclude involvement of other pathways.20 Studies using Akt siRNA would be more specific to confirm the contribution of the PI3K/Akt signaling cascades. In this study, we utilized VCP23019, a competitive antagonist at the S1P1 and S1P3 receptors,18 as pharmacological approach. Involvement of other S1P receptor subtypes cannot be ruled out. In this regard, further studies are needed. In contrast to its beneficial effect, isoflurane has been shown to induce apoptotic neurodegeneration in P7 rat pups when administered for 6 hours.21 However, Zhao et al. showed that isoflurane pretreatment (1.5% for 30 minutes) does not induce neuronal loss in P7 rat pups after HI.3 Also, Wise-Faberowski et al. reported that isoflurane treatment (30, 60, and 90 minutes) reduces oxygen and glucose deprivation (OGD)-induced neuronal apoptosis in a concentration-dependent manner (1.13%, 2.3%, or 3.3%) in neuronal cell culture prepared from newborn rats.4 Therefore, we believe that it is safe to administer isoflurane for 1 hour as a post-treatment after neonatal HI. Isoflurane appears to confer a dose-dependent dual action. Excessive exposure causes neurodegeneration, possibly through alteration of NMDA and GABAA receptors,21 which overwhelms the isoflurane-mediated protective pathways. Further experiments are warranted with the goal to enhance isoflurane neuroprotection and avoid its neurotoxicity.

Overall, we conclude that isoflurane post-treatment reduced infarct volume, brain atrophy and improved neurobehavioral outcomes in neonatal HI in rats. The isoflurane-induced neuroprotection is S1P/PI3K/Akt signaling pathway dependent. Since isoflurane has been introduced into clinical practice over 3 decades, it may represent a new and effective treatment against neonatal HI brain injury.

Acknowledgements and Funding

This study was supported partially by an Anesthesiology Research Fund in the Loma Linda University and by grants HD43120 and NS54685 from the National Institutes of Health to J.H.Z.

Footnotes

The authors report no conflicts of interest.

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