## 4 Discussion
The effects might be attributed to arteriogenesis, a process that takes place in an early phase due to enhanced blood flow in the existing collateral blood vessels [28]. This improvement in collateral circulation might be attributed to the regulation of the extracellular matrix, as evidenced by the increase in TIMP-1 and reductions in both MMP-2 and MMP-9 [17; 20]. MMPs, together with plasmin, are essential for the degradation of the external elastic lamina and elastin, forming a space for expanding blood vessels [28]. Excessive MMP activity during the I/R event causes damage to the basal lamina, promoting blood-brain barrier disruption and inflammation [29; 30]. TIMP-1, the inhibitor of MMP-9, was significantly elevated in the locotrienol-treated group [20]. The downregulation of MMP-9 and the elevation of TIMP-1 are essential mechanisms that promote neuroprotection [30]. On the contrary, the level of vascular endothelial growth factor (VEGF) is not significantly changed by locotrienol-enriched treatment [20]. VEGF is an essential factor for angiogenesis, the process of sprouting of new blood vessels [31]. However, several studies had reported that the induction of angiogenesis in the early phase contributes to the aggravation of stroke, due to disruption of the blood-brain barrier and increased vascular permeability, resulting in worsening vasogenic edema and infarct size [31]. Thus, the downregulation of angiogenic factors, including VEGF by locotrienols, promotes beneficial outcomes in ischemic stroke. Another interesting finding is the increase in CLIC1 but not CLIC4 expression in the locotrienol-enriched group [20]. A previous study by [PERSON] et al. [32] reported that CLIC4 was an essential factor for collateral circulation and demonstrated that mice that were deficient in CLIC4 genes (homozygous _Clic4-\({}^{/-}\)_) had a fourfold-larger infarct size compared to the homozygous _Clic4\({}^{+/+}\)_. However, they did not extend their study regarding CLIC1, as negligible changes were observed in the collateral density and diameter between the postnatal and adult period in _Clic1\({}^{-/-}\)_ mice [32]. The exact reason for the contrasting expression of CLIC1 and CLIC4 remains to be elucidated, particularly with locotrienol treatment. In terms of myocardial I/R injury, locotrienols, particularly \(\gamma\)-tocotrienol, have been reported to protect the heart, as evidenced by the significant improvement of various functional parameters, including aortic flow and output recovery, the left ventricular developed pressure, and the first derivative of developed pressure [11; 12; 13; 14; 15]. These changes are often accompanied by a reduction in infarct size [11; 14; 15]. Mechanisms of locotrienol-induced cardioprotective effects are numerous, including regulation of PI3K/Akt/mTOR and MAPK pathways. The activation of PI3K promotes the phosphorylation of Akt, which in turn leads to the activation of mTOR [33]. Myocardial I/R injury has been shown to reduce the phosphorylation of Akt and mTOR [33]. [PERSON] et al. [14] had confirmed the role of locotrienol in modulating the PI3K/Akt/mTOR-signaling pathway since the use of wortmannin, the inhibitor of PI3K, reversed the inhibition of this pathway. Interestingly, they also found the potential role of locotrienols in modulating autophagy via the PI3K/Akt/mTOR pathway. Autophagy is an essential process that degrades damaged organelles and proteins and is frequently activated during stress conditions, such as myocardial I/R injury [34]. Tocotrienol has been reported to enhance the Beclin-1 level and LCII/LCI ratio [14]. The former component is required to form phosphatidylinositol 3-kinase class III (PI3K-III), a complex that is required for the initiation of autophagy in the presence of another complex, namely, an autophagy-related 1 (ATG1)/Unc-51 such as autophagy-activating kinase-1 (ULK1) [35].