Postconditioning with isoflurane reduced ischemia-induced brain injury in rats

Abstract

Background: Preexposure of brain to isoflurane, a commonly used anesthetic, induces ischemic tolerance. This phenomenon is called isoflurane preconditioning. However, it is not known whether isoflurane application after ischemia provides neuroprotection.

Methods: Corticostriatal slices (400 microm) freshly prepared from adult male Sprague-Dawley rats were subjected to a 15-min oxygen-glucose deprivation (OGD; to simulate ischemia in vitro). Isoflurane was applied after OGD. Brain slices were harvested 2 h after OGD for measuring 2,3,5-triphenyltetrazolium chloride (TTC) conversion to quantify cell injury. Adult male Sprague-Dawley rats were also subjected to middle cerebral arterial occlusion for 90 min and then treated with or without 2% isoflurane for 60 min started at the onset of reperfusion. The infarct volumes, neurologic deficit scores, and performance on rotarod were evaluated at 24 h after the onset of reperfusion.

Results: Isoflurane applied immediately after the 15-min OGD for 30 min dose-dependently reversed the OGD-induced decrease of TTC conversion. The TTC conversion was 34 +/- 16% and 58 +/- 28% of the control, respectively, for OGD alone and OGD plus 2% isoflurane (P < 0.05, n = 12). Application of 2% isoflurane for 30 min started at 10 min after the OGD also reduced the OGD-decreased TTC conversion. The presence of 0.3 microm glibenclamide, a general adenosine 5'-triphosphate-sensitive potassium channel blocker, or 500 microm 5-hydroxydecanoic acid, a mitochondrial adenosine 5'-triphosphate-sensitive potassium channel blocker, during the application of 2% isoflurane abolished the isoflurane preservation of TTC conversion. Application of isoflurane during reperfusion also improved neurologic outcome after brain ischemia.

Conclusions: The results suggest that isoflurane administrated after OGD or brain ischemia provides neuroprotection. Mitochondrial adenosine 5'-triphosphate-sensitive potassium channels may be involved in this protection.

Fig. 1. Time-course of oxygen-glucose deprivation (OGD)-induced cell injury

Corticostriatal slices from adult male rats were subjected to various lengths of OGD. Cell injury was quantified by 2, 3, 5-triphenyltetrazolium chloride (TTC) conversion 2 h after OGD. Results are means ± S.D. (n = 8 per time point). Statistical analysis was performed by one-way repeated measures analysis of variance on ranks followed by the Dunn's method. * P < 0.05 compared with control.

Fig. 2. Representative sections of corticostriatal slices stained with hematoxylin and eosin

The glucose-oxygen deprivation (OGD) was for 15 min at 37°C. Injured cells are presented with one of the following characteristics: cell swelling, vacuolization (commonly associated with light-staining cytoplasm) or presence of shrunken, darkened nuclei. Arrows in the OGD panel indicated neurons with condensed nuclei. The bar chart presents the quantification data. Results are means ± S.D. (n = 15). Statistical analysis was performed by one-way repeated measures analysis of variance followed by the Dunn's method. * P < 0.05 compared with control. ^ P < 0.05 compared with OGD only. Con: control; Iso: 2% isoflurane applied for 30 min at the onset of simulated reperfusion after the OGD. Scale bar: 20 μm.

Fig. 3. Time-course and dose-response of isoflurane postconditioning

Corticostriatal slices were postconditioned with 2% isoflurane for various durations after a 15-min oxygen-glucose deprivation (OGD) (Panel A) or with various concentrations of isoflurane for 30 min after a 15-min OGD (Panel B). Isoflurane was applied at the onset of simulated reperfusion after the OGD. Cell injury was quantified by 2,3,5-triphenyltetrazolium chloride (TTC) conversion 2 h after the OGD. Results are means ± S.D. (n = 11 for panel A and = 12 for panel B). Statistical analysis was performed by oneway repeated measures analysis of variance on ranks followed by the Dunn's method. * P < 0.05 compared with OGD only.

Fig. 4. Time-window of isoflurane postconditioning

Corticostriatal slices were postconditioned with 2% isoflurane for 30 min at various time points after the end of a 15-min oxygen-glucose deprivation (OGD). Cell injury was quantified by 2,3,5-triphenyltetrazolium chloride (TTC) conversion 2 h after OGD. Results are means ± S.D. (n = 9). Statistical analysis was performed by one-way repeated measures analysis of variance on ranks followed by the Dunn's method. * P < 0.05 compared with OGD only. Iso-post: isoflurane postconditioning.

Fig. 5. The inhibition of adenosine 5′-triphosphate-sensitive potassium channel blockers on isoflurane postconditioning-induced neuroprotection

Corticostriatal slices were subjected to a 15-min oxygen-glucose deprivation (OGD) and then were immediately postconditioned with or without 2% isoflurane for 30 min in the presence or absence of 0.3 μM glybenclamide or 500 μM 5-hydroxydecanoic acid (5-HD). Cell injury was quantified by 2,3,5-triphenyltetrazolium chloride (TTC) conversion 2 h after OGD. Results are means ± S.D. (n = 10). Statistical analysis was performed by one-way repeated measures analysis of variance followed by the Dunn's method. * P < 0.05 compared with OGD only. Gly: glybenclamide; Iso-post: isoflurane postconditioning.

Fig. 6

Improvement of neurological outcome by isoflurane postconditioning after transient right middle cerebral arterial occlusion (MCAO). MCAO was for 90 min and isoflurane postconditioning was for 60 min started at the onset of reperfusion. A: Percentage of infarct volume in ipsilateral hemisphere volume. Results are the means ± S.D. (n = 7 for the MCAO only group and n = 8 for the MCAO plus isoflurane postconditioning group). B: The performance on rotarod. Rats were tested before and 24 hr after the MCAO and the speed-latency index ratio of these two tests are presented. Results are the means ± S.D. (n = 7 for the MCAO only group and n = 8 for the MCAO plus isoflurane postconditioning group). C: Neurological deficit scores that were evaluated immediately before the animals were sacrificed for the assessment of infarct sizes (data are presented in Fig. 6A) are shown. Each circle represents the score for a single rat. A horizontal bar indicates the mean value for each group. * P < 0.05 compared with the corresponding MCAO only group. Iso: isoflurane postconditioning.