Antisense knockdown of the glial glutamate transporter GLT-1, but not the neuronal glutamate transporter EAAC1, exacerbates transient focal cerebral ischemia-induced neuronal damage in rat brain

Abstract

Transient focal cerebral ischemia leads to extensive neuronal damage in cerebral cortex and striatum. Normal functioning of glutamate transporters clears the synaptically released glutamate to prevent excitotoxic neuronal death. This study evaluated the functional role of the glial (GLT-1) and neuronal (EAAC1) glutamate transporters in mediating ischemic neuronal damage after transient middle cerebral artery occlusion (MCAO). Transient MCAO in rats infused with GLT-1 antisense oligodeoxynucleotides (ODNs) led to increased infarct volume (45 +/- 8%; p < 0.05), worsened neurological status, and increased mortality rate, compared with GLT-1 sense/random ODN-infused controls. Transient MCAO in rats infused with EAAC1 antisense ODNs had no significant effect on any of these parameters. This study suggests that GLT-1, but not EAAC1, knockdown exacerbates the neuronal death and thus neurological deficit after stroke.

Fig. 1.

Western blot analysis of GLT-1 and EAAC1 protein levels in rats infused with GLT-1 and EAAC1 antisense, sense, and random ODNs. The amount of protein loaded per lane was 2 μg in the GLT-1 gels and 15 μg in the EAAC1 gels. Figure shows representative samples from each group. CC, Cerebral cortex;ST, striatum; CSF, artificial CSF;S, sense ODN; R, random ODN; andAS, antisense ODN. GLT-1 antisense infusion resulted in a significant decrease in GLT-1 protein levels compared with GLT-1 sense/random-infused controls. Similarly, EAAC1 antisense infusion significantly decreased EAAC1 protein levels compared with EAAC1 sense/random-infused controls. No significant changes were observed in the β-tubulin protein levels after the infusion of GLT-1 or EAAC1 sense/random/antisense.

Fig. 2.

Thionine-stained serial coronal sections from the brains of GLT-1 antisense and sense ODN-infused rats that underwent transient MCAO (1 hr) or sham operation. This figure shows only the GLT-1 sense-infused control, because there were no observable differences between the GLT-1 sense- and random-infused groups. Transient MCAO in GLT-1 antisense-infused rats resulted in significantly bigger infarcts compared with GLT-1 sense-infused controls.

Fig. 3.

Thionine-stained serial coronal sections from the brains of EAAC1 antisense and sense ODN-infused rats that underwent transient MCAO (1 hr) or sham operation. EAAC1 knockdown had no significant effect on the infarct size.

Fig. 4.

Effect of GLT-1 and EAAC1 antisense ODN infusion on rCBF after transient MCAO. The rCBF was measured using a laser Doppler flowmeter probe placed on the surface of the ipsilateral cortex (ischemic area) through a craniectomy over the MCA territory. Changes are expressed as percentage of the baseline. Values are mean ± SD. There were no statistically significant differences between the groups. Infusion of GLT-1 and EAAC1 sense and random led to no significant alterations in the rCBF after MCAO (data not shown).

Fig. 5.

End-point rCBF rates at 1 hr of MCAO as measured by autoradiography using [¹⁴C]AIP in the GLT-1 sense (n = 5), GLT-1 antisense (n = 5), EAAC1 sense (n = 4), and EAAC1 antisense (n = 4)-infused rats. Flow rates in the representative areas of cerebral cortex and striatum were averaged across the contralateral and ipsilateral sides of the brain. No significant differences were observed between GLT-1 sense- and antisense-infused groups and EAAC1 sense- and antisense-infused groups. The inset shows autoradiographs generated using the coronal brain sections (+0.5, −0.9, −2.1, and −3.9 mm from bregma) of [¹⁴C]AIP-administered GLT-1 sense- and antisense-infused groups.

Fig. 6.

Microscopic evaluation of the cerebral cortex (top panels) and striatum (bottom panels) from GLT-1 sense and antisense ODN-infused rats subjected to either sham operation or transient MCAO. The cortical neuronal layers (top panels) are indicated byI–VI. Severe neuronal loss in all cortical layers with a nearly total loss of the large pyramidal neurons from layer V and evident glial infiltration can be seen in the brains of GLT-1 antisense-infused/MCAO group. The striatum (bottom panels) of the GLT-1 antisense-infused/MCAO group also showed severe loss of the medium-sized striatal neurons (arrowheads), whereas the large striatal neurons (arrows) survived. The Figure shows only the sense-infused control, because there was no observable difference between sense- and random-infused groups.