3.2.3. Neutrophils

Leukocytosis has been found to be a marker of inflammation in response to ischemic stroke. Leukocytosis is associated with a high degree of disability, impairment, and increased mortality [74]. Neutrophils are the first blood-derived immune cells to invade ischemic brain tissue, followed by monocytes. After brain ischemia, neutrophils undergo conformational changes due to the presence of numerous adhesive molecules, which facilitates their migration across the vessel wall into the brain tissue. In addition to bloodderived microglia and macrophages, neutrophils are among the most important leukocytes that infiltrate the post-ischemic brain. A high number of post-ischemic neutrophils in the brain come from the peripheral circulation. Later, neutrophils are attracted to the ischemic region by chemokines and then cause secondary damage to the ischemic tissue by releasing pro-inflammatory mediators, proteases, ROS, and MMPs [75]. These toxic mediators weaken the endothelial cell membrane and the basal lamina leading to permeability of the blood–brain barrier and the development of post-ischemic brain edema. Their onset is fairly early, reaching the brain within half an hour to several hours after ischemia, peaking over the next 3 days, and gradually decreasing over 15 days [8,76]. After five hours of recirculation, neutrophils enter the ischemically-damaged area of the brain [77,78]. Following neutrophil invasion, monocytes then adhere to the vessel wall and migrate towards the ischemic area with maximum involvement within 3–7 days after ischemia [76]. It has been noted that infiltrating neutrophils remain for more than a month in the ischemic areas of the brain and their presence is masked after 3 days by overactivation of microglia/macrophages in the inflammatory area [79]. These cells activate molecules capable of contact with the endothelial cells as early as 15 minutes after brain ischemia, and within 6–8 h they surround the brain's blood vessels and penetrate the brain [80–82]. Neutrophils are believed to block microcirculation in the brain either mechanically or by secreting vasoconstrictors, releasing pro-inflammatory factors, ROS, and enzymes with hydrolytic activities [83–85]. In addition, neutrophils produce MMP-9, which is a protease that damages the blood–brain barrier, enhancing brain edema and causing hemorrhagic transformation of acute ischemic stroke [86]. The size of ischemic infarction and level of neurological deficits positively correlate with an increase in the number and activity of neutrophils, which in turn leads to an increased risk of death [76,87]. In contrast to neutrophils, after brain ischemia, the number of lymphocytes decreases, and thus the neutrophil/lymphocyte ratio increases. This ratio is closely related to the size of the infarct and mortality [87]. Leukocytes, which include neutrophils and T lymphocytes, intensify ischemic brain damage in many ways. First, the neutrophils adhere to the endothelium, which blocks the flow of erythrocytes through the microcirculation, which leads to the noreflow phenomenon in the brain. Second, on the endothelial surface, activated neutrophils produce proteases, MMPs, and ROS, which significantly damage blood vessels and brain tissue. The consequence of the above phenomena is vasoconstriction and platelet aggregation inside the brain vessels [37,88]. Finally, infiltrating leukocytes further aggravate neuronal damage by activating pro-inflammatory mediators in and around the penumbra and in the core of the infarct [89].

#### 3.2.4. Lymphocytes

T lymphocytes become involved in the later stages of post-ischemic neurodegeneration of the brain. Lymphocytes surround the periphery of the ischemic lesion, and their number increases after 3 days, reaches a maximum after one week, and then decreases after another week [90]. The effect of various T lymphocytes on inflammation and thrombosis, with the consequence of increased brain damage and worsening of neurological deficits, has been demonstrated in studies in mice deficient in T lymphocytes [91–93]. In addition, immunodepletion of CD4<sup>+</sup> lymphocyte cells in mice increased neuronal loss and was associated with more severe neurological deficits 7 days after focal brain ischemia [94]. The study showed that mice without γδ T lymphocyte cells had reduced infarct volume postischemia and the same phenomenon was reported in mice after administration of antibodies

against the receptors of these cells [95]. Another study found an increase in CD4+CD28 null lymphocyte cells in stroke survivors or those who died from ischemic stroke [96]. This study revealed an association between the high probability of death after a stroke or new ischemic episodes and the CD4+CD28 null lymphocyte cell count, and proposed that the number of these lymphocytes could serve as a warning biomarker against recurrence of an ischemic event or death. Another study found that peripheral frequency of CD4+ lymphocyte cells and CD4+CD28 null lymphocyte cells was significantly higher in patients with acute ischemic stroke than in the control group [97]. The results of this investigation indicate that in acute ischemic stroke, a higher percentage of peripheral CD4+CD28 null lymphocyte cells may be associated with more massive brain damage. Analysis of this study also showed that the percentage of CD+CD28 null lymphocyte cells may be useful for distinguishing between subtypes of stroke. In addition, genetic study has found an increased expression of activating "pro-inflammatory" killer cell immunoglobulin-like receptor (KIR) genes in people with ischemic stroke, which likely explains the massive development of inflammation in the acute phase of stroke [98]. Accumulating evidence shows that both innate and adaptive immune cells penetrate the brain after ischemia. Up to one month after an experimental ischemic stroke, T lymphocyte cell invasion into the ischemic area has been observed and has been shown to persist for years in post-stroke patients [99]. Up to one month after focal brain ischemia, a significant increase in the number of different subtypes of T lymphocytes was observed in the peri-infarct zone [99]. T lymphocytes entering the brain after ischemia had a close interaction with activated astrocytes and a progressively developed pro-inflammatory phenotype as evidenced by markers of increased lymphocyte activation, pro-inflammatory cytokines TNF-α, INF-γ, IL-10, IL-17, and perforin, with appropriate T-bet and RORc transcription factors [99]. Treg immunodepletion using a specific CD-25 antibody aggravated tissue injury and impaired neurological deficits on day 7 after local brain ischemia in mice [94].
