Article Figures & Data
Figures
- 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.
Tables
ICV infusion n Cortex (mm3) Striatum (mm3) Total (mm3) None 6 172 ± 27 31 ± 5 203 ± 28 aCSF 5 175 ± 36 37 ± 7 208 ± 39 GLT-1 sense 5 167 ± 34 34 ± 9 204 ± 41 GLT-1 random 4 164 ± 33 32 ± 6 199 ± 47 GLT-1 antisense 8 246 ± 501-a,1-b 48 ± 71-a,1-b 294 ± 521-a,1-b EAAC1 sense 4 186 ± 44 33 ± 10 222 ± 47 EAAC1 random 4 175 ± 49 36 ± 9 210 ± 52 EAAC1 antisense 6 201 ± 46 39 ± 7 240 ± 50 Values are mean ± SD. The MCAO duration was 1 hr in all cases. Ischemic injury volumes were computed by volumetric analysis of the thionine-stained coronal sections using the NIH Image program. aCSF, Artificial CSF; ICV, intracerebroventricular.
↵F1-a p < 0.05, compared with the None or aCSF group (one-way ANOVA followed by Dunnet's multiple comparisons post test).
↵F1-b p < 0.05, compared with the respective sense or random group (ANOVA followed by Tukey-Kramer multiple comparisons post test).
- Table 2.
Physiological parameters in GLT-1 and EAAC1 sense, random, and antisense ODN-infused rats undergoing transient MCAO
ICV infusion n pH PaCO2 (mm Hg) PaO2 (mm Hg) Gl (mg/dl) Hb (gm/dl) Temperature (°C) Rectal Muscle None 6 7.41 ± 0.04 34.2 ± 3.3 136 ± 22 90 ± 6 13 ± 1.3 36.9 ± 0.4 36.5 ± 0.4 aCSF 5 7.38 ± 0.03 32.4 ± 2.6 133 ± 30 91 ± 5 14 ± 1.6 37.2 ± 0.4 36.3 ± 0.5 GLT-1 sense 5 7.42 ± 0.05 33.7 ± 3.6 142 ± 34 91 ± 8 13 ± 2.1 37.1 ± 0.5 36.5 ± 0.4 GLT-1 random 4 7.40 ± 0.04 33.3 ± 5.1 132 ± 31 88 ± 7 14 ± 1.7 37.4 ± 0.6 36.4 ± 0.3 GLT-1 antisense 8 7.38 ± 0.05 31.6 ± 4.8 138 ± 37 92 ± 8 14 ± 1.5 37.0 ± 0.5 36.7 ± 0.4 EAAC1 sense 4 7.39 ± 0.03 32.2 ± 2.1 133 ± 25 92 ± 8 13 ± 1.2 36.9 ± 0.6 36.7 ± 0.3 EAAC1 random 4 7.42 ± 0.04 34.8 ± 4.5 140 ± 38 93 ± 5 14 ± 1.3 37.2 ± 0.6 36.8 ± 0.2 EAAC1 antisense 6 7.43 ± 0.05 32.9 ± 3.8 137 ± 29 89 ± 6 13 ± 1.9 37.3 ± 0.5 36.5 ± 0.5 Values are mean ± SD. There were no statistically significant differences between the groups in any of the parameters. Gl, Glucose; Hb, hemoglobin; aCSF, artificial CSF; ICV, intracerebroventricular. The mean arterial blood pressure was observed to be in the ranges of 118–130 (pre-ischemia), 116–129 (ischemia), and 126–134 (post-ischemia) in all groups.
- Table 3.
Mortality rates and neuroscores after sham operation and transient MCAO in GLT-1 and EAAC1 sense, random, and antisense ODN-infused rats
ICV infusion Total rats (alive/dead) % mortality Neuroscores Neurological deficit Individual Median Sham-operated None 6 (6/0) 0 0, 0, 0, 0, 0, 0 0 None aCSF 5 (5/0) 0 0, 0, 0, 0, 0 0 None GLT-1 sense 5 (5/0) 0 0, 0, 0, 1, 1 0.40 None GLT-1 random 4 (4/0) 0 0, 0, 0, 1 0.25 None GLT-1 antisense 8 (7/1) 12 0, 0, 0, 1, 1, 1, 1 0.57 None EAAC1 sense 4 (4/0) 0 0, 0, 0, 0 0 None EAAC1 random 4 (4/0) 0 0, 0, 0, 0 0 None EAAC1 antisense 6 (5/1) 16 0, 0, 0, 0, 1 0.20 None Transient MCAO None 7 (6/1) 14 1, 2, 2, 2, 2, 2 1.832-a Mild–moderate aCSF 6 (5/1) 16 1, 2, 2, 2, 2 1.802-a Mild–moderate GLT-1 sense 5 (5/0) 0 1, 2, 2, 2, 3 2.002-a Mild–moderate GLT-1 random 5 (4/1) 20 1, 2, 2, 2 1.752-a Mild–moderate GLT-1 antisense 12 (8/4) 332-a,2-b 3, 3, 3, 3, 3, 4, 4, 4 3.382-a,2-b Severe–very severe EAAC1 sense 4 (4/0) 0 1, 2, 2, 2 1.752-a Mild–moderate EAAC1 random 4 (4/0) 0 1, 2, 2, 2 1.752-a Mild–moderate EAAC1 antisense 7 (6/1) 14 1, 1, 2, 2, 2, 3 1.832-a Mild–moderate In all groups, the MCAO duration was 1 hr. Neurological evaluation was conducted at 24 hr of reperfusion, and neuroscores were not given for the rats that failed to survive up to this period. The following scale was used for neurological scoring: 0, no neurological deficit; 1, mild neurological deficit; 2, moderate neurological deficit; 3, severe neurological deficit; and 4, very severe neurological deficit. ICV, Intracerebroventricular; aCSF, artificial CSF.
↵F2-a p < 0.05, compared with the corresponding sham control (one-way ANOVA followed by Tukey-Kramer multiple comparisons post test).
↵F2-b p < 0.05, compared with the respective sense/MCAO or random/MCAO control (one-way ANOVA followed by Tukey-Kramer multiple comparisons post test).
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