2.1.2. Oxidative Stress

Oxidative stress is involved with the production of reactive oxygen species (ROS), which activates apoptosis, necrosis, and autophagy pathways during ischemic stroke [18]. Oxidative stress also reduces the bioavailability of nitric oxide (NO) in endothelial cells. Nitric oxide is a potent chemical that inhibits platelet aggregation and leukocyte adhesion [19]. The loss of NO will exacerbate the coagulation, which aggravates the ischemic insult, leading to more blood flow reduction in the brain. In addition, cells under ischemia can increase the levels of calcium, sodium, and adenosine diphosphate (ADP) that produce oxygen species in mitochondria. Reperfusion is a commonly used method to treat ischemic stroke, but this also produces more ROS to cause the secondary damage in the brain. Furthermore, ROS damages lipids, proteins, and nucleic acid via lipid peroxidation, protein oxidation, and DNA fragmentation and leads to further cell death [20].

#### 2.1.3. Inflammation Response

Inflammation response is an important event in ischemic stroke and the subsequent reperfusion. Ischemia and reperfusion activate immune system to respond to tissue injury including activation of endothelium and leukocytes. Specifically, the immune response is involved with the intravascular and parenchymal damage triggered by the interruption of blood supply. In other words, the immune system plays a central role in regulating the outcomes of stroke patients [21]. For the immune response, adhesion molecules on endothelial cells are upregulated during ischemia. For example, P-selectin was highly expressed when the mouse brain was damaged. P-selectin is a major adhesion molecule for the binding of platelets to endothelial cells, and this interaction causes proinflammatory signals [22]. ICAM-1 is another intercellular adhesion molecule and is upregulated during inflammation response. A study showed that knockdown of intercellular adhesion molecule-1 (ICAM-1) gene in mice reduced the infarction of the brain, suggesting that inflammation response plays a major role in pathogenesis of stroke [23].

Innate immune cells are major components during inflammation response because they sense endogenous danger signals through several receptors such as toll-like receptors (TLRs), retinoic acid-inducible gene (RIG)-1-like receptors, nucleotide oligomerization domain (NOD)-like receptors, C-type lectin receptors, and absent in melanoma (AIM2)-like receptors [24]. These receptors activate downstream signaling pathways, such as nuclear factor-κB (NF-κB), mitogen-activated protein kinase, and type 1 interferon pathways. These pathways upregulate proinflammatory cytokines, chemokines, and oxidative metabolites. The products including tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), IL-6, IL-18, and NO activate endothelial cells and astrocytes, and also act on leukocytes for their tissue infiltration in the brain, thus causing more tissue damage. Clinical studies found that in stroke patients, the increase of cytokines appeared in the cerebrospinal fluid and blood and this feature is correlated to patient survival [25]. In addition, the increased infiltration of leukocytes caused by the formation of fibrin and cytokines contributes to further tissue damage. Ischemia and reperfusion activate macrophages in the perivascular space. Activated macrophages release proinflammatory cytokines, which contribute to the expression of adhesion molecules in endothelium, promoting more leukocyte infiltration (especially neutrophils) to cause the BBB damage [26]. The large infiltration of neutrophils in a short period causes the release of proteases that can further destroy the BBB integrity and exacerbate oxidative stress, leading to severe brain damage.
