Overexpression of thioredoxin in transgenic mice attenuates focal ischemic brain damage

Abstract

Thioredoxin (TRX) plays important biological roles both in intra- and extracellular compartments, including in regulation of various intracellular molecules via thiol redox control. We produced TRX overexpressing mice and confirmed that there were no anatomical and physiological differences between wild-type (WT) mice and TRX transgenic (Tg) mice. In the present study we subjected mice to focal brain ischemia to shed light on the role of TRX in brain ischemic injury. At 24 hr after middle cerebral artery occlusion, infarct areas and volume were significantly smaller in Tg mice than in WT mice. Moreover neurological deficit was ameliorated in Tg mice compared with WT mice. Protein carbonyl content, a marker of cellular protein oxidation, in Tg mice showed less increase than did that of WT mice after the ischemic insult. Furthermore, c-fos expression in Tg mice was stronger than in WT mice 1 hr after ischemia. Our results suggest that transgene expression of TRX decreased ischemic neuronal injury and that TRX and the redox state modified by TRX play a crucial role in brain damage during stroke.

Figure 1

Expression of hTRX in various tissues of transgenic mice. Results of Western blot analysis of various tissues are shown. Protein content was analyzed in comparison with rTRX.

Figure 2

Western blot analysis of hTRX expression in Tg and WT brain. The hTRX mAb recognized hTRX as a single band of 13 kDa. In Tg mice, a strong hTRX signal was shown. However, no signal for hTRX was shown in WT mice.

Figure 3

Dorsal (A) and ventral (B) views of brains from WT and Tg mice. No gross anatomical difference was detected between the two sets of mice (n = 4 each). Intracardiac 5% India black injection revealed no anatomical abnormalities of the circle of Willis between the two groups (B, n = 4 each). Magnification: ×4.

Figure 4

Immunohistochemical study of hTRX in Tg mice. Immunoreactivity for hTRX was observed in cortex (A and C) and hippocampus (B and D). High-power view shows that hTRX exists in neurons, endothelial cells, and glial cells (C and D). hTRX immunoreactivity was not shown in WT mice (E and F). Magnification: A, B, E, and F, ×40; C and D ×200.

Figure 5

rCBF by laser-Doppler flowmetry during MCA occlusion in Tg and WT mice. Similarity in the relative changes in rCBF after MCA occlusion in Tg and WT mice in brain regions in the peri-infarct zone (penumbra) and the MCA core territory were shown. The rCBF level was determined simultaneously in the two regions by laser-Doppler flowmetry in six animals from Tg and WT. Time zero represents the point of MCA occlusion. Modest decreases was shown in the peri-infarct zone in both groups. More severe reduction in rCBF was present in a more deeply ischemic territory; no differences were detected between the two groups.

Figure 6

Infarct volumes in Tg and WT mice. Infarct size was analyzed 24 hr after MCA occlusion using 2,3,5-triphenyltetrazolium chloride staining. (A) Infarct areas for three coronal sections from rostral to caudal are shown. Significant differences were found in Tg and WT mice (n = 9 each). (B) Infarct volume was smaller in Tg mice than in WT mice (n = 9 each). Data are expressed as means ± SD. Statistical analysis is performed by using Student’s t test; ∗, P < 0.01, ∗∗, P < 0.05)

Figure 7

Changes in protein carbonyl content after MCA occlusion in Tg and WT mice. The DNP-derivatized protein samples prepared from brains subjected to MCA occlusion were separated on a 15% SDS/PAGE followed by Western blotting with primary antibody, specific to the DNP moiety of the proteins. In sham control animals, only a small amount of DNP moieties was detected, and no difference was detected between the two groups. One hour after ischemia, more DNP moieties were detected in WT mice than in Tg mice (I; ischemia, S; sham).

Figure 8

Expression of c-fos in Tg and WT mice during MCA occlusion. Densitometric analysis: (A) In sham-operated animals, c-fos expression was not different between the two groups. (B) After ischemic insult, c-fos expression was higher in Tg mice than in WT mice (data are expressed as means ± SD, statistical analysis performed by using Student’s t test; ∗, P < 0.05).