Isoflurane delays the development of early brain injury after subarachnoid hemorrhage through sphingosine-related pathway activation in mice

Abstract

Objective: Isoflurane, a volatile anesthetic agent, has been recognized for its potential neuroprotective properties and has antiapoptotic effects. We examined whether isoflurane posttreatment is protective against early brain injury after subarachnoid hemorrhage and determined whether this effect needs sphingosine-related pathway activation.

Design: Controlled in vivo laboratory study.

Setting: Animal research laboratory.

Subjects: One hundred seventy-nine 8-wk-old male CD-1 mice weighing 30-38 g.

Interventions: Subarachnoid hemorrhage was induced in mice by endovascular perforation. Animals were randomly assigned to sham-operated, subarachnoid hemorrhage-vehicle, and subarachnoid hemorrhage+2% isoflurane. Neurobehavioral function and brain edema were evaluated at 24 and 72 hrs. The expression of sphingosine kinase, phosphorylated Akt, and cleaved caspase-3 was determined by Western blotting and immunofluorescence. Neuronal cell death was examined by terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end-labeling staining. Effects of a sphingosine kinase inhibitor N, N-dimethylsphingosine or a sphingosine 1 phosphate receptor inhibitor VPC23019 on isoflurane's protective action against postsubarachnoid hemorrhage early brain injury were also examined.

Measurements and main results: Isoflurane significantly improved neurobehavioral function and brain edema at 24 hrs but not 72 hrs after subarachnoid hemorrhage. At 24 hrs, isoflurane attenuated neuronal cell death in the cortex, associated with an increase in sphingosine kinase 1 and phosphorylated Akt, and a decrease in cleaved caspase-3. The beneficial effects of isoflurane were abolished by N, N-dimethylsphingosine and VPC23019.

Conclusions: Isoflurane posttreatment delays the development of postsubarachnoid hemorrhage early brain injury through antiapoptotic mechanisms including sphingosine-related pathway activation, implying its use for anesthesia during acute aneurysm surgery or intervention.

Figure 1

SAH grade (A, B) and neurological scores (C, D) in studies 1 and 2, respectively. Study 1 was evaluated at 24 and 72 hours, and study 2 was evaluated at 24 hours after SAH. Values are mean±SD; *P<0.05, ANOVA.

Figure 2

Brain water content. A, study 1, 24 hours after SAH; B, study 1, 72 hours after SAH; C, study 2, 24 hours after SAH. Values are mean±SD; *P<0.05, ANOVA.

Figure 3

Representative Western blots and quantitative analysis of SphK1 (A), SphK2 (B), phosphorylated Akt (Ser473) (p-Akt; C), and cleaved caspase-3 (D) in the left cerebral hemisphere at 24 hours after SAH (study 1). The p-Akt band density values are calculated as a ratio of that of total Akt, and the other proteins band density values are calculated as a ratio of that of β-actin. Values are mean±SD; *P<0.05, ANOVA.

Figure 4

Evaluation of TUNEL-positive cells in the ipsilateral basal cortex at 24 hours after SAH (study 1). A, representative brain section and immunofluorescence images showing the colocalization of NeuN (blue) with TUNEL (green)-positive cells; B, quantitative analysis of TUNEL-positive neurons. Scale bar, 50μm; values, mean±SD; *P<0.05, ANOVA.

Figure 5

Representative brain section and immunofluorescence images showing the relationship between TUNEL (green)-positive cells and SphK1 (red) expression (A) or phosphorylated Akt (Ser473) (p-Akt; red) expression (B) in the ipsilateral basal cortex at 24 hours after SAH (study 1). Scale bar: 50μm.

Figure 6

Representative Western blots and quantitative analysis of SphK1 (A), phosphorylated Akt (Ser473) (p-Akt; B), and cleaved caspase-3 (C) in the left cerebral hemisphere at 24 hours after SAH (study 2). The p-Akt band density values are calculated as a ratio of that of total Akt, and the other proteins band density values are calculated as a ratio of that of β-actin. Values are mean±SD; *P<0.05, ANOVA.