Glutamate transporter type 3 mediates isoflurane preconditioning-induced acute phase of neuroprotection in mice

Abstract

A pre-exposure to isoflurane reduces ischemic brain injury in rodents (isoflurane preconditioning). This neuroprotection has acute and delayed phases. Our previous in vitro studies suggest that the acute phase may involve excitatory amino acid transporters (EAATs). We determine whether this protection involves EAAT3, the major neuronal EAAT. Adult male EAAT3 knockout mice and their wild-type littermates were exposed or were not exposed to 1.5% isoflurane for 30 min. Sixty minutes later, they were subjected to a 90- or 60-min middle cerebral arterial occlusion (MCAO). Their neurological outcomes were evaluated 24h after the MCAO. In another experiment, cerebral cortex was harvested for Western blotting at 30 min after animals were exposed to 1.5% isoflurane for 30 min. Here, we showed that isoflurane reduced brain infarct volumes and improved neurological functions of wild-type mice after a 90-min MCAO. However, isoflurane pre-exposure did not change the neurological outcome of EAAT3 knockout mice no matter whether the MCAO was for 90 min or 60 min. Isoflurane increased phospho-Akt, a survival-promoting protein, in the wild-type mice but not in the EAAT3 knockout mice. The isoflurane-induced neuroprotection in the wild-type mice was abolished by LY294004, an Akt activation inhibitor. LY294004 alone did not affect the neurological outcome of the wild-type or EAAT3 knockout mice after focal brain ischemia. These results suggest that the isoflurane preconditioning-induced acute phase of neuroprotection involves EAAT3. The downstream event includes Akt activation.

Keywords: Akt; EAAT; ERK1/2; GAPDH; GSK3β; Glutamate transporter; Isoflurane; MCAO; Neuroprotection; Preconditioning; SpO(2); excitatory amino acid transporter; extracellular signal-regulated kinase 1/2; glyceraldehyde 3-phosphate dehydrogenase; glycogen synthase kinase 3β; middle cerebral arterial occlusion; pulse oximeter oxygen saturation.

Fig. 1. Isoflurane preconditioning-induced neuroprotection

Mice were pretreated with or without 1.5% isoflurane for 30 min at 60 min before a 90-min 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, C and D: Percentage of total, cortical and subcortical infarct volume in ipsilateral hemisphere volume. Results are the means ± S.D. (n = 10 – 11). E: Neurological deficit scores evaluated immediately before the animals were euthanized for the assessment of infarct sizes (data are presented in panels B, C and D) or assigned 7 to the animals that died before the end of observation period. Results are presented in a box plot format (n = 10 – 11). ●: 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. F: The performance on rotarod. Rats 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.D. (n = 10 – 11). * P < 0.05 compared with the wildtype mice subjected to MCAO only. ^ P < 0.05 compared with wild-type mice subjected to isoflurane preconditioning plus MCAO. Iso: isoflurane; LY: LY294004; EAAT3^−/−: EAAT3 knockout.

Fig. 2. Failure for isoflurane to induce neuroprotection in EAAT3 knockout mice

Wild-type mice had a 90-min middle cerebral arterial occlusion (MCAO). EAAT3 knockout mice were pre-treated with or without 1.5% isoflurane for 30 min at 60 min before a 60-min 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 total infarct volume in ipsilateral hemisphere volume. Results are the means ± S.D. (n = 7 – 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 of observation period. Results are presented in a box plot format (n = 7 – 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. Rats 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.D. (n = 7 – 8). Iso: isoflurane; LY: LY294004; EAAT3^−/−: EAAT3 knockout.

Fig. 3. Isoflurane-induced phosphorylation of protein kinases

The cerebral cortex was harvested at 30 min after mice were exposed to 1.5% isoflurane for 30 min. Total cell lysates were prepared for Western blotting. A: Representative Western blots. B – E: The graphic presentation of the phospho-Akt, phospho-ERK and Phospho-GSK3β protein abundance quantified by integrating the volume of autoradiograms from 6 – 8 mice for each experimental condition is shown. Values in graphs are the means ± S.D. * P < 0.05 compared with wild-type mice under control condition. EAAT3^−/−: EAAT3 knockout.

Fig. 4. No effect of isoflurane on the expression of protein kinases in wild-type mice

The cerebral cortex was harvested at 30 min after mice were exposed to 1.5% isoflurane for 30 min. Total cell lysates were prepared for Western blotting of total Akt, ERK and GSK3β. Representative Western blots are presented in the top panels. The graphic presentation of the Akt, ERK and GSK3β protein abundance quantified by integrating the volume of autoradiograms from 4 – 8 mice for each experimental condition is shown in the bottom pane. Values in graphs are the means ± S.D. Con: control; Iso: isoflurane.