Isoflurane postconditioning reduces ischemia-induced nuclear factor-κB activation and interleukin 1β production to provide neuroprotection in rats and mice

Abstract

Application of isoflurane, a volatile anesthetic, after brain ischemia can reduce ischemic brain injury in rodents (isoflurane postconditioning). This study is designed to determine whether isoflurane postconditioning improves long-term neurological outcome after focal brain ischemia and whether this protection is mediated by attenuating neuroinflammation. Adult male Sprague-Dawley rats were subjected to a 90-min middle cerebral arterial occlusion (MCAO). Isoflurane postconditioning was performed by exposing rats to 2% isoflurane for 60min immediately after the MCAO. Isoflurane postconditioning reduced brain infarct volumes, apoptotic cells in the ischemic penumbral brain tissues and neurological deficits of rats at 4weeks after the MCAO. Isoflurane postconditioning reduced brain ischemia/reperfusion-induced nuclear transcription factor (NF)-κB (NF-κB) activation as well as interleukin 1β (IL-1β) and interleukin-6 production in the ischemic penumbral brain tissues at 24h after the MCAO. IL-1β deficient mice had smaller brain infarct volumes and better neurological functions than wild-type mice at 24h after a 90-min focal brain ischemia. Isoflurane posttreatment failed to induce neuroprotection in the IL-1β deficient mice. Our results suggest that isoflurane postconditioning improved long-term neurological outcome after transient focal brain ischemia. This protection may be mediated by inhibiting NF-κB activation and the production of the proinflammatory cytokine IL-1β.

Fig. 1. Diagrammatic presentation of the experimental protocol

Fig. 2. Isoflurane postconditioning improved histological and neurological function outcome assessed 4 weeks after focal brain ischemia in adult rats

A: Brain sections after Nissl staining from representative rats subjected to MCAO only or MCAO plus isoflurane postconditioning. B: Percentage of brain infarct volume in ipsilateral hemisphere volume. Results are the means ± S.E.M. (n = 17 – 18). C: Neurological deficit scores evaluated immediately before the animals were euthanized for the assessment of infarct sizes (data are presented in panel B) or assigned 7 to the animals that died before this end time point for observation. Results are presented in a box plot format (n = 20 – 25). ●: lowest or highest score (the score will not show up if it falls in the 95% interval); between lines: 95% interval of the data; inside boxes: 25 – 75% interval including the median of the data. D: The performance on rotarod. Rats were tested before and 4 weeks after the MCAO and the speed-latency index ratio of these two tests are presented. Results are the means ± S.E.M. (n = 17 – 18). ^ P < 0.05 compared with the corresponding MCAO only group. Iso: isoflurane postconditioning.

Fig. 3. Isoflurane postconditioning reduced dying cells evaluated at 4 weeks after the 90-min right middle cerebral arterial occlusion (MCAO) in rats

Cells in the penumbral cerebral cortex were examined after terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick-end labeling (TUNEL). Panels A, B and C are representatives of the sections from control, MCAO only and MCAO plus isoflurane postconditioning. Panel D: quantitative data are presented as the means ± S.E.M. (n = 5 for the control group, = 17 for MCAO, and = 18 for iso + MCAO groups). * P < 0.05 compared with the control group. ^ P < 0.05 compared with the MCAO only group. Iso: isoflurane postconditioning.

Fig. 4. Attenuation of ischemia-reperfusion-induced interleukin (IL)-1β (panel A), IL-6 (panel B) and IL-10 (panel C) expression by isoflurane postconditioning in rats

The frontal cerebral cortex area 1 (Fr1) and hippocampus ipisilateral or contralateral to the side of middle cerebral arterial occlusion (MCAO) were harvested at 24 h after the MCAO. Results are the means ± S.E.M. (n = 7 – 17). * P < 0.05 compared to sham-operated control. ^ P < 0.05 compared to MCAO only. Iso: isoflurane postconditioning.

Fig. 5. Isoflurane postconditioning reduced translocation of p65 to nuclei after the middle cerebral arterial occlusion (MCAO) in rats

The frontal cerebral cortex area 1 ipsilateral to the ischemia side was harvested at 24 h after the MCAO. Nuclear proteins were prepared for Western blotting. A representative Western blot is shown on the top panel and the graphic presentation of the p65 protein abundance quantified by integrating the volume of autoradiograms from 4 rats for each experimental condition is shown in the bottom panel. Values in graphs are the means ± S.E.M. * P < 0.05 compared with MCAO only. Iso: isoflurane postconditioning, PDTC: pyrrolidine dithiocarbamate.

Fig. 6. Isoflurane posttreatment reduced lipopolysaccharid (LPS)-induced interleukin (IL)-1β increase in mice

The frontal cerebral cortex was harvested at 6 h after intraperitoneal LPS injection for Western blotting. A representative Western blot is shown on the top panel and the graphic presentation of the IL-1β protein abundance quantified by integrating the volume of autoradiograms from 4 – 5 mice for each experimental condition is shown in the bottom panel. Values in graphs are the means ± S.E.M. * P < 0.05 compared with control mice. Iso: isoflurane postconditioning.

Fig. 7. Interleukin (IL)-1β deficient mice had better histological and neurological function outcome than wild-type mice after middle cerebral arterial occlusion (MCAO)

The results were evaluated at 24 h after the MCAO. A: Brain slices stained with 2,3,5-triphenyltetrazolium chloride from representative mice. B: Percentage of infarct volume in ipsilateral hemisphere volume. Results are the means ± S.E.M. (n = 6 – 8). C: Neurological deficit scores evaluated immediately before the animals were euthanized for the assessment of infarct sizes (data are presented in panel B) or assigned 7 to the animals that died before the end time point for observation. Results are presented in a box plot format (n = 6 – 8). ●: lowest or highest score (the score will not show up if it falls in the 95% interval); between lines: 95% interval of the data; inside boxes: 25–75% interval including the median of the data. D: The performance on rotarod. Mice were tested before and 24 h after the MCAO and the speed-latency index ratio of these two tests are presented. Results are the means ± S.E.M. (n = 6 – 8). * P < 0.05 compared with the wild-type mice.

Fig. 8. Isoflurane post-treatment failed to induce neuroprotection in interleukin (IL)-1β deficient mice after middle cerebral arterial occlusion (MCAO)

The results were evaluated at 24 h after the MCAO. A: Brain slices stained with 2,3,5-triphenyltetrazolium chloride from representative mice. B: Percentage of infarct volume in ipsilateral hemisphere volume. Results are the means ± S.E.M. (n = 6). C: Neurological deficit scores evaluated immediately before the animals were euthanized for the assessment of infarct sizes (data are presented in panel B) or assigned 7 to the animals that died before the end time point for observation. Results are presented in a box plot format (n = 6). ●: lowest or highest score (the score will not show up if it falls in the 95% interval); between lines: 95% interval of the data; inside boxes: 25–75% interval including the median of the data. D: The performance on rotarod. Mice were tested before and 24 h after the MCAO and the speed-latency index ratio of these two tests are presented. Results are the means ± S.E.M. (n = 6). Iso: isoflurane postconditioning.