Tocotrienol vitamin E protects against preclinical canine ischemic stroke by inducing arteriogenesis

Abstract

Vitamin E consists of tocopherols and tocotrienols, in which α-tocotrienol is the most potent neuroprotective form that is also effective in protecting against stroke in rodents. As neuroprotective agents alone are insufficient to protect against stroke, we sought to test the effects of tocotrienol on the cerebrovascular circulation during ischemic stroke using a preclinical model that enables fluoroscopy-guided angiography. Mongrel canines (mean weight=26.3±3.2 kg) were supplemented with tocotrienol-enriched (TE) supplement (200 mg b.i.d, n=11) or vehicle placebo (n=9) for 10 weeks before inducing transient middle cerebral artery (MCA) occlusion. Magnetic resonance imaging was performed 1 hour and 24 hours post reperfusion to assess stroke-induced lesion volume. Tocotrienol-enriched supplementation significantly attenuated ischemic stroke-induced lesion volume (P<0.005). Furthermore, TE prevented loss of white matter fiber tract connectivity after stroke as evident by probabilistic tractography. Post hoc analysis of cerebral angiograms during MCA occlusion revealed that TE-supplemented canines had improved cerebrovascular collateral circulation to the ischemic MCA territory (P<0.05). Tocotrienol-enriched supplementation induced arteriogenic tissue inhibitor of metalloprotease 1 and subsequently attenuated the activity of matrix metalloproteinase-2. Outcomes of the current preclinical trial set the stage for a clinical trial testing the effects of TE in patients who have suffered from transient ischemic attack and are therefore at a high risk for stroke.

Figures

Figure 1

Tocotrienol-enriched (TE) natural vitamin E protects against stroke-induced brain injury. (A and B) Effect of 10-week oral supplementation on cerebral cortex concentration of tocotrienols and tocopherols. (A) No tocotrienols were detected in brain of PBO-supplemented canines. Tocotrienol-enriched supplementation significantly increased α-, γ-, and δ- tocotrienol isomers in cerebral cortex. (B) A moderate, but significant (*P=0.047) increase in brain α-tocopherol level was observed as each TE gel capsule contains 61.5 mg of α-tocopherol. (C) Stroke-induced infarct volume in response to stroke. MD, mean diffusivity map taken at 1 hour; FLAIR, fluid attenuated inversion recovery taken at 24 hours. Representative coronal slice MR images of canine brain at (D) 1 hour demonstrating cytotoxic edema (*P<0.05) and (E) 24 hours demonstrating cytotoxic and vasogenic edema following reperfusion (*P<0.005). ND, not detected; PBO, placebo. Three dimensional volumetric reconstruction in color available online as Supplementary Figure S2.

Figure 2

Tocotrienol-enriched (TE) attenuates white matter injury following acute ischemic stroke. (A) Streamline tractography of placebo (PBO) and TE white matter fiber tracts was performed with two regions of interest (ROI) masks to visualize tracts connecting the corona radiata to the internal capsule at 24 hours. Fiber tracts were overlaid on T2-weighted structural scan (512 × 512 matrix) to visualize in context of contralateral (right) and ipsilateral (left) hemispheres in the coronal orientation. Sagittal views of contralateral and ipsilateral hemispheres demonstrate the protective effect of TE supplementation. (B) Probabilistic tractography reveals connectivity of white matter fiber tracts projecting from the seed region of the internal capsule to the corona radiata in representative canines. Color shift from black → red → yellow → white denotes a higher degree of relative connectivity between regions in the stroke-affected hemisphere of PBO- and TE-supplemented canines. (C) Variance of probabilistic tracts as a function of the distance from the internal capsule seed region. Contra, contralateral; ipsi, ipsilateral. Three dimensional color video available online as Supplementary Figure S3.

Figure 3

Tocotrienol-enriched (TE) supplement improves cerebrovascular collateral circulation during acute ischemic stroke. Cerebrovascular collaterals were identified by digital subtraction angiography (DSA) in placebo (PBO)- (A–E) and TE- (F–J) treated canines. To visualize collaterals of the stroke-affected MCA territory (green lines), pre-stroke arterial (A, F) and venous (B, G) DSA of left internal carotid artery (L-ICA) were compared with post-stroke arterial (D, I) and venous (E, J) DSA of right internal carotid artery (R-ICA). Post-stroke L-ICA DSA during the arterial phase (C, H) demonstrates effective MCA occlusion by embolic coil (marked by red oval). During the post-stroke arterial phase, greater collateral perfusion (black arrow) was observed in MCA territory of TE-supplemented canines as compared with PBO controls (I versus D). Likewise, more venous flow and contrast ‘blush' (black triangle) was observed in stroke-affected hemisphere of TE-supplemented canines (J versus E). Mean collateral score for PBO- and TE-supplemented canines was determined according to an 11-point scale (methods). (K) Collateral score during stroke was significantly higher in TE-supplemented canines as compared with PBO controls. *P<0.05. (L) Collateral score correlation with infarct volume (coefficient of determination, r2=0.821), open diamonds represent PBO, closed diamonds represent TE canines.

Figure 4

Tocotrienol-enriched (TE) supplement increases expression of arteriogenic markers in laser capture isolated cortex arterioles. (A–C) Arterioles (arrows, mean diameter 6.6±2.9 μm) were selectively captured from contralateral control and ipsilateral stroke-affected cerebral cortex 24 hours after stroke onset. (D) To verify specificity of captured elements, gene expression of vessel marker (VWF), neuron marker (NF-H), and glial marker (GFAP) was checked with real-time PCR. *P<0.05 VWF versus NF-H, ND, not detected. (E–H) Expression of arteriogenic genes was validated using real-time PCR in contralateral (white) and ipsilateral (black) arterioles. *P<0.05 in TE-supplemented control versus stroke. †P<0.05 in placebo (PBO) versus TE control tissue. (E) Chloride intracellular channel 1 (CLIC1). (F) Chloride intracellular channel 4 (CLIC4). (G) Tissue inhibitor of metalloproteinase 1 (TIMP1). (H) Vascular endothelial growth factor (VEGF).

Figure 5

Tocotrienol-enriched (TE) supplement inhibits matrix metalloproteinase-2 (MMP2) activity in stroke-affected cerebral cortex. No difference in MMP2 protein expression was observed by western blot (A) and densitometric analysis (B) of contralateral (contra) and ipsilateral (ipsi) somatosensory cortex of placebo (PBO)- and TE-supplemented canines 24 hours after stroke. Gelatin zymography (C) and densitometry (D) demonstrates significantly higher MMP-2 activity in stroke-affected hemisphere of PBO, not TE canines. *P<0.05 PBO cont versus stroke, †P<0.05 PBO stroke versus TE stroke.