Isoflurane on brain inflammation

Abstract

Brain inflammation may play an important role in the pathophysiology of early brain injury after subarachnoid hemorrhage (SAH). Our aim was to demonstrate brain inflammation development and to determine whether isoflurane, a clinically available volatile anesthetic agent, prevents brain inflammation after SAH. This study used 162 8-week-old male CD-1 mice. We induced SAH with endovascular perforation in mice and randomly assigned animals to sham-operated (n=21), SAH+vehicle-air (n=35) and SAH+2% isoflurane (n=31). In addition to the evaluation of brain injury (neurological scores, brain edema and Evans blue dye extravasation), brain inflammation was evaluated by means of expression changes in markers of inflammatory cells (ionized calcium binding adaptor molecule-1, myeloperoxidase), cytokines (tumor necrosis factor [TNF]-α, interleukin-1β), adhesion molecules (intercellular adhesion molecule [ICAM]-1, P-selectin), inducers of inflammation (cyclooxygenase-2, phosphorylated c-Jun N-terminal kinase [p-JNK]) and endothelial cell activation (von Willebrand factor) at 24h post-SAH. Sphingosine kinase inhibitor (N, N-dimethylsphingosine [DMS]) and sphingosine-1-phosphate receptor-1/3 antagonist (VPC23019) were used to block isoflurane's effects (n=22, each). SAH caused early brain injury, which was associated with inflammation so that all evaluated markers of inflammation were increased. Isoflurane significantly inhibited both brain injury (P<0.001, respectively) and inflammation (myeloperoxidase, P=0.022; interleukin-1β, P=0.002; TNF-α, P=0.015; P-selectin, P=0.010; ICAM-1, P=0.016; p-JNK, P<0.001; cyclooxygenase-2, P=0.003, respectively). This beneficial effect of isoflurane was abolished with DMS and VPC23019. Isoflurane may suppress post-SAH brain inflammation possibly via the sphingosine-related pathway.

Keywords: BWC; Brain water content; COX-2; Cyclooxygenase-2; DMS; EBI; Early brain injury; ICAM-1; IL-1β; Inflammation; Intercellular adhesion molecule-1; Interleukin-1beta; Ionized calcium binding adaptor molecule-1; Isoflurane; MPO; Myeloperoxidase; N, N-dimethylsphingosine; Phosphorylated c-Jun N-terminal kinase; S1P; S1P1/3; SAH; SphK; Sphingosine 1-phosphate; Sphingosine kinase; Sphingosine-1-phosphate receptor-1/3; Subarachnoid hemorrhage; TNF-α; Tumor necrosis factor-alpha; iba-1; p-JNK; vWF; von Willebrand factor.

Figure 1

Representative Western blots, quantitative analysis of MPO expression (n=5 per group) (A), representative brain section and immunofluorescence for iba-1 (n=3 per group) (B) in the left cerebral hemisphere at 24 hours after SAH. The protein band density values are calculated as a ratio of that of β-actin. Vehicle, SAH+vehicle-air group; 2% ISO, SAH+2% isoflurane group; values, mean±SD; *P<0.05, ANOVA.

Figure 2

Representative Western blots and quantitative analysis of IL-1β (A) and TNF-α (B) expressions in the left cerebral hemisphere at 24 hours after SAH. The protein band density values are calculated as a ratio of that of β-actin. Vehicle, SAH+vehicle-air group; 2% ISO, SAH+2% isoflurane group; n=5 per group; values, mean±SD; *P<0.05, ANOVA.

Figure 3

Representative Western blots, quantitative analysis of P-selectin (n=5 per group) (A) and ICAM-1 (n=5 per group) (B) expressions, representative brain section and double immunofluorescence images showing the colocalization of P-selectin (red) with vWF (green) (n=3 per group) (C) in the left cerebral hemisphere at 24 hours after SAH. The protein band density values are calculated as a ratio of that of β-actin. Vehicle, SAH+vehicle-air group; 2% ISO, SAH+2% isoflurane group; values, mean±SD; *P<0.05, ANOVA.

Figure 4

Representative Western blots and quantitative analysis of p-JNK (A) and COX-2 (B) expressions in the left cerebral hemisphere at 24 hours after SAH. The protein band density values are calculated as a ratio of that of JNK or β-actin. Vehicle, SAH+vehicle-air group; 2% ISO, SAH+2% isoflurane group; n=5 per group; values, mean±SD; *P<0.05, ANOVA.

Figure 5

Representative Western blots and quantitative analysis of ICAM-1 (A) and IL-1β (B) expressions in the left cerebral hemisphere at 24 hours after SAH. The protein band density values are calculated as a ratio of that of β-actin. 2% ISO, 2% isoflurane group; n=6 per group; values, mean±SD; *P<0.05, ANOVA.