Astaxanthin reduces ischemic brain injury in adult rats

Abstract

Astaxanthin (ATX) is a dietary carotenoid of crustaceans and fish that contributes to their coloration. Dietary ATX is important for development and survival of salmonids and crustaceans and has been shown to reduce cardiac ischemic injury in rodents. The purpose of this study was to examine whether ATX can protect against ischemic injury in the mammalian brain. Adult rats were injected intracerebroventricularly with ATX or vehicle prior to a 60-min middle cerebral artery occlusion (MCAo). ATX was present in the infarction area at 70-75 min after onset of MCAo. Treatment with ATX, compared to vehicle, increased locomotor activity in stroke rats and reduced cerebral infarction at 2 d after MCAo. To evaluate the protective mechanisms of ATX against stroke, brain tissues were assayed for free radical damage, apoptosis, and excitoxicity. ATX antagonized ischemia-mediated loss of aconitase activity and reduced glutamate release, lipid peroxidation, translocation of cytochrome c, and TUNEL labeling in the ischemic cortex. ATX did not alter physiological parameters, such as body temperature, brain temperature, cerebral blood flow, blood gases, blood pressure, and pH. Collectively, our data suggest that ATX can reduce ischemia-related injury in brain tissue through the inhibition of oxidative stress, reduction of glutamate release, and antiapoptosis. ATX may be clinically useful for patients vulnerable or prone to ischemic events.

Figures

Figure 1.

ATX is a carotenoid and a primary pigment in crustaceans. A) Chemical structures of carotenoids ATX, β-carotene, lutein, and the retinoid, retinoic acid. B) Juvenile lobsters (Homarus americanus) fed an ATX-depleted diet exhibit minimal pigmentation (bottom) compared to an older lobster fed an ATX-containing diet (top). Size difference in the two lobsters is a consequence of age, not the lack of ATX. Photo courtesy of Dr. Michael Tlusty, New England Aquarium, Boston, MA, USA. Scale bar = 2 cm.

Figure 2.

Distribution of ATX in stroke brain. Animals received vehicle (veh) or ATX at 10-15 min before a 60-min MCAo. Tissue was collected at 10-15 min after reperfusion began for ATX analysis using MALDI-TOFMS. Distribution of a glycerophosphocholine species (P32:0+H) was used as an anatomical marker for veh (A) and ATX (B) treatments. Same sections were imaged in the mass range of m/z 616 Da, [ATX+K+−H2O]+ or 596 + 39 − 18, for veh (C) and ATX (D) treatments. The 616-Da signal (ATX) was evident at injection site (small red arrow, D) and cortical surface (large arrow, D) in the ATX-treated group. The average mass spectrum for a region of interest in the future infarction area (red circle, C, D) demonstrated detectable average ion counts for 616 Da in ATX-treated group (F) but not the vehicle-treated group (E). Scale bar = signal intensity (ion count) for all peaks in m/z range of 614-620 (C, D).

Figure 3.

Locomotor activity in animals treated with ATX or vehicle at 1 and 2 d after MCAo. Animals received vehicle (veh) or ATX followed by a 60-min MCAo. Locomotor activity was measured in 30-min sessions at 1 and 2 d after MCAo. Animals receiving ATX had a significant increase in horizontal (A–D) and vertical (E–G) activity compared to vehicle controls. There was also less rest time in stroke animals treated with ATX (H). HACTV, horizontal activity; TOTDIST, total distance; MOVNO, movement number; MOVTIME, movement time; VACTV, vertical activity; VMOVNO, vertical movement number; Vtime, vertical movement time; RESTIME, rest time. Data are presented as percentage of prestroke activity. *P < 0.05; 2-way ANOVA.

Figure 4.

Effects of ATX on cortical infarction induced by MCAo and reperfusion. Right middle cerebral artery was occluded for 60 min after bilateral common carotid ligation. Two days after MCAo, tissue was collected in 2-mm sections and stained with TTC. A) Volume of infarction = sum of infarction area (mm2) × 2-mm slice thickness for the 7 infarcted slices. B, C) Area of largest infarction per slice (B) and number of infarcted slices from each rat (C) were all significantly decreased in animals receiving ATX. *P < 0.05; Student’s t test. D) Representative TTC-stained sections of brains from vehicle (veh)-treated and ATX-treated animals.

Figure 5.

Aconitase enzymatic activity and lipid peroxidation in ATX-treated ischemic brain tissue. All animals received 60-min MCAo and reperfusion for 8-h (aconitase assay, A) or 24-h (MDA assay, B). Mitochondrial aconitase activity and MDA were normalized to total protein content. A) Aconitase data are presented as percentage of normalized aconitase activity in the ischemic hemisphere (R) compared to the normalized nonischemic contralateral hemisphere (L). *P < 0.05; 2-way ANOVA. B) MDA was also reduced after ATX treatment. Dotted line indicates MDA level in naive rat brain. *P < 0.05; Student’s t test.

Figure 6.

ATX alters ischemia and H2O2-induced glutamate release in the cortex. A) ATX suppresses ischemia-induced glutamate release in cerebral cortex. Glutamate levels were measured from the dialysates from 60 min before to 60 min after the onset of MCAo. B) ATX suppresses H2O2-induced glutamate release in cerebral cortex. Glutamate levels were measured from the dialysates for 60 min before and after H2O2 infusion. Pretreatment with ATX significantly reduced H2O2-mediated glutamate release. *P < 0.05; 2-way ANOVA.

Figure 7.

Mitochondrial cytochrome c levels are higher in ischemic cortex treated with ATX compared to vehicle. Animals pretreated with vehicle (veh) or ATX were sacrificed at 8 h after MCAo. A) Mitochondrial protein fractions from cortical tissue of the ischemic and nonischemic brain hemispheres were analyzed by Western blot. Ischemia/reperfusion decreases the level of mitochondrial cytochrome c protein but not cytochrome c oxidase protein. B) Densitometric analysis of changes in cytochrome c protein levels normalized to cytochrome c oxidase protein levels. *P < 0.05; 1-way ANOVA.

Figure 8.

Density of TUNEL(+) cells is decreased in stroke animals receiving ATX compared to vehicle. A, B) Animals were pretreated with vehicle or ATX before MCAo. Two days after MCAo, density of TUNEL(+) cells was quantified in penumbra (A) and core (B) of the infarction at three locations relative to bregma. There was a significant reduction in density of TUNEL-positive cells in both core and penumbra of animals receiving ATX pretreatment. *P < 0.05; 2-way ANOVA. C–F) Representative fields of TUNEL(+) cells in −0.8-mm bregma sections for vehicle-penumbra (C), ATX-penumbra (D), vehicle-core (E) and ATX-core (F). Scale bar = 200 μm

Figure 9.

CBF was not altered by ATX during ischemia. A) CBF was normalized (% control) by comparison to the mean blood flow before administration of MCAo in each animal. There was no difference in the decrease of CBF during MCAo between animals treated with vehicle or ATX (P=0.55; Student’s t test). B) Representative recording from a stroke animal treated with ATX.