• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Stroke. Author manuscript; available in PMC Sep 1, 2013.
Published in final edited form as:
PMCID: PMC3429639

Isoflurane Attenuates blood-brain barrier disruption in Ipsilateral Hemisphere after subarachnoid hemorrhage in mice


Background and Purpose

We examined effects of isoflurane, volatile anesthetics, on blood-brain barrier (BBB) disruption in the endovascular perforation model of subarachnoid hemorrhage (SAH) in mice.


Animals were assigned to sham-operated, SAH+vehicle-air, SAH+1% or 2% isoflurane groups. Neurobehavioral function, brain water content, Evans blue dye extravasation and Western blotting for sphingosine kinases (SphKs), occludin, claudin-5, junctional adhesion molecule and vascular endothelial cadherin were evaluated at 24 hours post-SAH. Effects of SphK (DMS) or sphingosine-1-phosphate receptor-1/3 (S1P1/3) inhibitors (VPC23019) on isoflurane's action were also examined.


SAH aggravated neurological scores, brain edema and BBB permeability, which were prevented by 2% but not 1% isoflurane posttreatment. 2% isoflurane increased SphK1 expression and prevented a post-SAH decrease in expressions of the BBB-related proteins. Both DMS and VPC23019 abolished the beneficial effects of isoflurane.


2% isoflurane can suppress post-SAH BBB disruption, which may be mediated by SphK1 expression and S1P1/3 activation.

Keywords: subarachnoid hemorrhage, early brain injury, isoflurane, blood-brain barrier, sphingosine kinase-1, sphingosine-1-phosphate receptor


Blood-brain barrier (BBB) disruption has been an important prognostic factor after aneurysmal subarachnoid hemorrhage (SAH).1 The BBB is critical for brain homeostasis and is located at the cerebral microvessel endothelial cells, which maintain their barrier characteristics via cell-cell contacts made up of tight and adherens junctions.2 Stabilization of tight junctions involves a complex network of occludin, claudin-5 and junctional adhesion molecule (JAM).2 Adherens junctions consist of vascular endothelial (VE) cadherins.2

Recently, we reported that 2% isoflurane, a volatile anesthetic, prevented post-SAH neuronal apoptosis through sphingosine-related pathway activation.3 Sphingosine-1-phosphate (S1P) is generated from sphingomyelin by sphingosine kinase-1 (SphK1) and SphK2,4 and was reported to enhance endothelial barrier integrity.5 However, it remains undetermined whether isoflurane prevents BBB disruption. This study is the first to demonstrate that isoflurane posttreatment prevents BBB disruption after SAH in mice, and that the mechanism involves SphK1 expression and S1P receptor-1/3 (S1P1/3) activation.

Methods (expanded methods; please see http://stroke.ahajournals.org)

The Loma Linda University animal care committee approved all protocols.

In study 1, male CD-1 mice (30-38g; Charles River, Wilmington, MA) were randomly divided into sham-operated+vehicle-air (n=17), SAH+vehicle-air (n=25), SAH+1% isoflurane (n=9), and SAH+2% isoflurane (n=22) groups. SAH endovascular perforation model was produced and sham-operated mice underwent identical procedures except that the suture was withdrawn without puncture.3 One hour post-SAH, 1% or 2% isoflurane (Baxter, Deerfield, IL) was continuously administered for 1 hour with vehicle air (30% O2 and 70% medical air). All evaluations were blindly performed at 24 hours postsurgery. Eighteen-point SAH grading and eighteen-point neurological scores were evaluated in all surviving animals as previously described.3 Brain water content (n=6 per group) and Evans blue dye extravasation (n=5 per group) were measured as previously described.3 Western blot (n=6 per group) was performed on the left cerebral hemisphere (perforation side) using anti-SphK1 (Abgent, San Diego, CA), anti-SphK2 (Lifespan Biosciences, Seattle, CA), anti-occludin, anti-claudin-5, anti-JAM-A, and anti-VE-cadherin (Santa Cruz Biotechnology, Santa Cruz, CA) antibodies as previously described.3

In study 2, animals were randomly divided into dimethyl sulfoxide (DMSO, a vehicle)+sham-operated+vehicle-air (n=11), DMSO+SAH+2% isoflurane (n=15), N, N-dimethylsphingosine (DMS, a SphK antagonist; Enzo Life Sciences Inc., Plymouth Meeting, PA)+SAH+2% isoflurane (n=18), and VPC23019 (a S1P1/3-receptor antagonist; Avanti Polar Lipids Inc., Alabaster, Alabama)+SAH+2% isoflurane (n=18) groups. DMS (0.17μg/0.5μL) or VPC23019 (0.26μg/0.5μL) was infused into the right lateral ventricle at a rate of 0.1μL/minute 1 hour before surgery.3 The vehicle groups were given the same volume (0.5μL) of DMSO (1.1g/mL/kg) diluted in phosphate-buffered saline. Isoflurane was administered as study 1. SAH grading, neurological scores (all surviving animals), brain water content (n=6 per group) and Western blotting for SphK1, claudin-5 and VE-cadherin (n=5 per group) were performed at 24 hours postsurgery as described above.

Data were expressed as median±25th to 75th percentiles or mean±SD, and were analyzed using Kruskal-Wallis test followed by Steel-Dwass multiple comparisons, one-way analysis of variance (ANOVA) with Tukey-Kramer post hoc tests, Fisher's exact or chi-square tests as appropriate. P<0.05 was considered statistically significant.


Isoflurane Prevents Post-SAH BBB Disruption (Study 1)

The mortality was not different among the SAH groups (vehicle-air, 32.0% [8 of 25 mice]; 1% isoflurane, 33.3% [3 of 9]; and 2% isoflurane, 22.7% [5 of 22]) at 24 hours. No sham-operated mice died. SAH grade was similar among the groups (Figure 1A).

Figure 1
SAH grade (A), neurological score (B), brain water content (C) and Evans blue dye extravasation (D) at 24 hours post-SAH (study 1). Vehicle-air, SAH+vehicle-air group; 1% or 2% ISO, SAH+1% or 2% isoflurane group; left or right, left or right cerebral ...

Although 1% isoflurane had no significant effects, 2% isoflurane improved post-SAH neurological impairments (P=0.010), brain water content (P=0.003) and Evans blue dye extravasation (P=0.005) in the left cerebral hemisphere compared with the vehicle group (Figure 1B-D). Western blots showed that 2% isoflurane significantly increased SphK1, but not SphK2, in the left cerebral hemisphere compared with the sham (P=0.002) and vehicle (P<0.001) groups (Figure S1; http://stroke.ahajournals.org). In addition, 2% isoflurane increased expressions of occludin, JAM-A and VE-cadherin compared with the sham (P=0.001, respectively) and vehicle (P<0.001, respectively) groups, and claudin-5 expression compared with the vehicle group (P=0.010; Figure 2).

Figure 2
Representative Western blots and quantitative analysis of occludin (A), JAM-A (B), claudin-5 (C) and VE-cadherin (D) expressions in the left cerebral hemisphere at 24 hours post-SAH (study 1). The protein band density values are calculated as a ratio ...

SphK and S1P1/3-receptor Antagonists Inhibit Isoflurane's Effects (Study 2)

No sham-operated mice died. The mortality was not different among the DMSO+SAH+2% isoflurane (26.7%, 4 of 15 mice), DMS+SAH+2% isoflurane (38.9%, 7 of 18) and VPC23019+SAH+2% isoflurane (38.9%, 7 of 18) groups. SAH grade was similar among the groups (Figure S2A; http://stroke.ahajournals.org).

Both DMS and VPC23019 significantly aggravated neurological scores (P<0.001, P=0.003, respectively; Figure S2B; http://stroke.ahajournals.org) and brain edema in the left cerebral hemisphere (P<0.002, P=0.05, respectively; Figure 3A), and decreased expressions of SphK1, claudin-5 and VE-cadherin in 2% isoflurane-treated SAH mice (Figure 3B-D).

Figure 3
Brain water content (A), representative Western blots and quantitative analysis of SphK1 (B), claudin-5 (C) and VE-cadherin (D) expressions in the left cerebral hemisphere at 24 hours post-SAH (study 2). The protein band density values are calculated ...


A key pathologic manifestation of post-SAH early brain injury is BBB disruption.1 BBB dysfunction may allow greater influx of blood-borne cells and substances into brain parenchyma, thus amplifying inflammation, leading to further parenchymal damage and edema formation. In this study, one-hour 2% isoflurane administration at 1 hour post-SAH improved neurological score, brain edema and BBB permeability, associated with increased SphK1 expression and S1P1/3 activation. Isoflurane also prevented a post-SAH decrease in expressions of tight junction (occludin, JAM-A and claudin-5) and adherens junction (VE-cadherin) proteins.

S1P is well-known to decrease endothelial permeability.5-7 Vascular endothelial cells produce S1P, while express S1P1, S1P2 and S1P3 with S1P1>S1P2≈S1P3.7 S1P was reported to induce the formation of tight junctions through S1P1.5 FTY720, a S1P-receptor modulator, accompanied by an increase in S1P1/5 and a decrease in S1P3/4, reversed BBB disruption in a rat model of encephalomyelitis.6 This study suggested that isoflurane induced SphK1 to synthesize S1P, and that the activation of S1P1/3 was required for maintenance of post-SAH BBB function in mice. However, nothing is known on effects of S1P3 on endothelial permeability.7 Taken together, SphK1 and S1P1 activation may be key factors for isoflurane to induce S1P-mediated protection of post-SAH BBB.

Isoflurane anesthesia dose-dependently increases cerebral blood flow while decreases metabolism,8 and significantly increased BBB permeability associated with capillary dilatation at 3% in normal animals.9 Isoflurane inhibited neuronal injury dose-dependently, which was maximal at 2%.10 In this study, we tested 2 concentrations (1% and 2%) of isoflurane treatment since it is clinically relevant, and demonstrated that only 2% isoflurane is protective against post-SAH BBB disruption in mice. Our data suggest that 2% isoflurane may work not only for anesthesia but also for prevention of post-SAH BBB disruption through the sphingosine-related pathway.

This study has some limitations including no studies of effects of isoflurane or the inhibitors on cerebral blood flow and sham-operated animals, comparisons of isoflurane's neuroprotective effects with other anesthetics, as well as the detailed mechanisms how isoflurane protects or enhances BBB-related proteins. Thus, further studies are needed.

Supplementary Material




This study is partially supported by NIH NS060936 to JT and NS053407 to JHZ.




Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.


1. Doczi T. The pathogenetic and prognostic significance of blood-brain barrier damage at the acute stage of aneurysmal subarachnoid haemorrhage. Clinical and experimental studies. Acta Neurochir (Wien) 1985;77:110–132. [PubMed]
2. Brown RC, Davis TP. Calcium modulation of adherens and tight junction function: a potential mechanism for blood-brain barrier disruption after stroke. Stroke. 2002;33:1706–1711. [PubMed]
3. Altay O, Hasegawa Y, Sherchan P, Suzuki H, Khatibi NH, Tang J, et al. Isoflurane delays the development of early brain injury after subarachnoid hemorrhage through sphingosine-related pathway activation in mice. Crit Care Med. 2012 In press. [PMC free article] [PubMed]
4. Maceyka M, Payne SG, Milstein S, Spiegel S. Sphingosine kinase, sphingosine-1-phosphate, and apoptosis. Biochim Biophys Acta. 2002;1585:193–201. [PubMed]
5. Lee JF, Zeng Q, Ozaki H, Hand AR, Hla T, Wang E, et al. Dual roles of tight junction-associated protein, zonula occludens-1, in sphingosine 1-phosphate-mediated endothelial chemotaxis and barrier integrity. J Biol Chem. 2006;281:29190–29200. [PubMed]
6. Foster CA, Mechtcheriakova D, Storch MK, Balatoni B, Howard LM, Bornancin F, et al. FTY720 rescue therapy in the dark agouti rat model of experimental autoimmune encephalomyelitis: expression of central nervous system genes and reversal of blood-brain-barrier damage. Brain Pathol. 2009;19:254–266. [PubMed]
7. Lucke S, Levkau B. Endothelial functions of sphingosine-1-phosphate. Cell Physiol Biochem. 2010;26:87–96. [PubMed]
8. Maekawa T, Tommasino C, Shapiro HM, Keifer-Goodman J, Kohlenberger RW. Local cerebral blood flow and glucose utilization during isoflurane anesthesia in the rat. Anesthesiology. 1986;65:144–151. [PubMed]
9. Tétrault S, Chever O, Sik A, Amzica F. Opening of the blood-brain barrier during isoflurane anaesthesia. Eur J Neurosci. 2008;28:1330–1341. [PubMed]
10. Lee JJ, Li L, Jung HH, Zuo Z. Postconditioning with isoflurane reduced ischemia-induced brain injury in rats. Anesthesiology. 2008;108:1055–1062. [PMC free article] [PubMed]
PubReader format: click here to try


Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


Recent Activity

    Your browsing activity is empty.

    Activity recording is turned off.

    Turn recording back on

    See more...