The term “stroke” refers to a sudden loss of neurological function resulting from acute vascular disruption of cerebral blood flow. Approximately 80% of strokes are clinically manifested as cerebral infarction. Stroke is the second leading cause of death and the primary cause of physical disabilities in adults worldwide. In Bulgaria, there are 35,311 registered cases annually, of which 7175 result in fatalities. The number of survivors with varying degrees of disability is 28,136, with 25–50% experiencing speech and language disorders ^[1][189].

There are two main pathomorphological forms of cerebral infarction. The so-called anemic or pale infarction is characterized by a disruption of arterial blood flow in the corresponding area. The brain tissue in this region remains poorly perfused and becomes pale. After hemolysis of erythrocytes in the affected area (after 48 h), the infarct becomes yellowish-white. In the case of hemorrhagic infarction or red brain softening, there is initially an obstruction of an arterial vessel by a thrombus of sufficient duration, leading to the development of anemic infarction. Subsequently, the thrombus lyses or partially fragments, blocking distal, smaller branches of the respective artery. Blood flow is restored in the ischemic area, resulting in the seepage of blood into the zone. As a result, the necrotic area takes on a dark red-to-black color. The middle cerebral artery or its branch is most commonly affected. Hemorrhagic infarction of brain tissue can also occur with extensive thrombosis of the sagittal venous sinus ^[2][190]. Thrombosis of small arteries and arterioles leads to so-called lacunar infarcts, which are multiple and involve the simultaneous blood supply of several brain territories. Multifocal infarction is observed, often with hemorrhage ^[3][191].

Global cerebral ischemia develops in the context of severe hypotension or increased intracranial pressure. Although cerebral infarction is most commonly caused by ischemia (ischemic hypoxia or stagnant hypoxia), it can also result from a reduced amount of oxygen in the blood without a reduction in cerebral blood flow (hypoxia or anemic hypoxia), or it can be the result of toxins that prevent cells from using oxygen for oxidative processes (histotoxic hypoxia). In addition to oxygen deficiency, the brain tissue is also highly sensitive to glucose deficiency (hypoglycemia) ^[3][191].

The causes of cerebral infarction are numerous and can generally be represented as vasculopathies and vasculitis of large and small blood vessels. These conditions can manifest clinically in several ways, but the most common manifestation is infarction of the brain tissue.

Atherosclerosis of large blood vessels most commonly occurs extracranially and can be a source of emboli in small cerebral arteries, such as thrombus or atheromatous material. Less frequently, thromboembolism is a complication of arteritis or vascular aneurysms. Infectious vasculitis in the context of septicemia or in patients with immunodeficiency most commonly affects small arteries, arterioles, and venules, leading to thrombosis with infarction. Other rarer causes of embolism include fat embolism, air embolism, tumor embolism in malignant diseases, or embolism from an intervertebral disc following trauma. Disorders in the coagulant-anticoagulant system, various etiologies of vascular malformations, and vascular tumors are also among the causes of cerebral tissue infarction.

Saccular berry aneurysms, which affect smaller branches of the major cerebral arteries, can cause cerebral hemorrhage. Very rarely, small intracranial arteries can be affected following the dissection of a large blood vessel, such as the carotid artery. Depending on the affected blood vessel and the presence or absence of collateral vessels, the infarction can be limited beneath the pia mater, be deeper, or involve the entire zone supplied by the affected vessel ^[3][4][5][191,192,193]. Macroscopically, within the first 24 h, the infarcted gray brain matter becomes reddish-brown due to pronounced vascular congestion. Focal petechial hemorrhages may be present. The white brain matter also darkens in color and is congested, but the changes are more discrete. The boundaries of the infarcted area are difficult to determine earlier than 2 days after the incident. After this period, the affected tissue is significantly softer compared with the surrounding healthy parenchyma. After several days and weeks, the affected tissue undergoes liquefactive necrosis, and a cyst forms.

Histologically, at 4–6 h after the incident, neurons in the affected area become distorted and have pyknotic nuclei, perineuronal glia appears swollen with pale cytoplasm, and microvacuolization is observed among the neuropil. Between 6–48 h, the cytoplasm of neurons becomes increasingly eosinophilic, and the nuclei become amphophilic or also eosinophilic. After 2–3 days, it is difficult to identify neuronal nuclei, although their cytoplasmic boundaries are clearly visible. Glial cells, especially perifascicular and satellite OLs, undergo apoptosis and can be identified as apoptotic bodies within the infarcted tissue. Neutrophilic leukocyte infiltration is observed at the periphery of the infarct at 6–12 h. Between 1 and 2 days, the inflammatory reaction is abundant, but mononuclear cells predominate. Foamy macrophages accumulate in the infarcted area and around blood vessels. Neovascularization of the necrotic tissue occurs as a result of endothelial proliferation. After several months, the necrotic tissue is resorbed, and a pseudocyst lined with glial cells remains at the site of the incident, with blood vessels in the wall and residual foamy macrophages. Some of the foamy macrophages may contain hemosiderin. Numerous reactive astrocytes are observed in the surrounding parenchyma ^[3][4][5][191,192,193].

“Reactive astrogliosis” is a term used to describe cellular, functional, and molecular changes that astrocytes undergo following injury. When morphological changes have occurred, they can vary from hypertrophy of cell bodies and processes to alterations in protein profiles and/or proliferative activity ^[6][7][194,195]. The extent of observed changes depends on the severity of the injury. Therefore, astrocytic reactivity can be characterized as mild, moderate, diffuse, or severe. This quantitative analysis is based on the degree of GFAP expression. The more pronounced the astrocytic transformation and consequently the severity of the injury, the stronger the intensity of GFAP expression ^[6][8][194,196]. Several intercellular and intracellular signaling molecules regulate this process. The effect can be both protective and exacerbating the damage. Reactive astrocytes provide neuroprotection in the acute stage of ischemic brain injury while simultaneously interacting with immune cells from the blood, endothelial cells, and microglia, leading to the development of brain edema and potentiation of the neuroinflammatory response. In the chronic stage, reactive astrocytes are responsible for the formation of a glial scar, which aims to limit the damage zone, tissue remodeling, and restoration of neuronal functions ^[4][5][9][192,193,197].

Two subtypes of reactive astrocytes have been described: A1 and A2 reactive astrocytes. The proliferation of the A1 subtype is stimulated by secreted IL-1α, TNFα, and C1q from activated microglia, leading to neuronal and OL death. A2 reactive astrocytes have a neuroprotective effect and secrete trophic factors for neurons ^[10][198]. Among the markers expressed in reactive astrocytes are Lcn2, GFAP, Vimentin, and Timp1. Transcriptional analysis of reactive astrocytes in experimental animals following ischemia confirms the claim that this cell type, similar to microglia, has both pro-inflammatory and neuroprotective functions ^[11][12][199,200].

High-tech imaging methods have been used to study the ischemic penumbra in the human cerebral cortex ^[13][201]. Changes in astrocyte morphology can be described according to the stage of cerebral infarction. In the acute phase (1st to 4th day after ischemia), astrocytes exhibit increased proliferative activity and increased GFAP expression. In the subacute phase (4th to 8th day after ischemia), astrocytes with elongated processes and depolarized cell membranes are described, gradually forming a glial scar until the onset of the chronic stage (8th to 14th day after ischemia). Differences in astrocyte reactivity are associated with their sensitivity to ischemia, their location relative to the lesion core, and their subtypes ^[14][202]. Some authors believe that the different reactivity is also due to differences in the protein profile ^[15][203].

In ischemic brain injury, the lack of glucose is compensated by reactive astrocytes, which can initiate the process of glycolysis to produce lactate, serving as an energy source for neurons and transporting it to them through specialized transporters. Data regarding this function of reactive astrocytes and their role in the survival or degeneration of neurons are contradictory ^[16][204].

By establishing the connection between neurons and blood vessels within the neurovascular units in the CNS, astrocytes play a key role along with individual cells and pericytes in the aftermath of cerebral tissue infarction. Reactive astrocytes secrete VEGF (vascular endothelial growth factor) and MMPs, which increase vascular permeability and exacerbate ischemic damage in the acute phase of the incident. In the chronic phase, when the process of neuroregeneration prevails, the same bioactive molecules stimulate angiogenesis and the restoration of the BBB ^[17][18][205,206].

Excessive glutamate in the extracellular space of brain tissue has neurotoxic effects. This may be due to the inability of astrocytes to eliminate it through reuptake and convert it into glutamine. Therefore, by participating in the glutamate–glutamine cycle, reactive astrocytes have a protective effect on neurons ^[19][20][67,207]. Reactive astrocytes also secrete synaptic molecules, such as cholesterol-associated apolipoprotein E (APOE) and thrombospondin ^[21][22][208,209].

Astrocytes exert their antioxidant action through the production of glutathione, which is important for limiting the damage during ischemia in the cerebral cortex ^[23][210]. However, reactive astrocytes can also release ROS and nitric oxide, leading to oxidative stress ^[24][176]. Recent studies have shown that astrocytes can produce extracellular vesicles containing proteins, lipids, and nucleic acids and thus interact with other cell types. There is evidence that under ischemic conditions, reactive astrocytes can increase the survival of neurons through such vesicular activity ^[25][26][27][211,212,213]. However, studies on human astrocytes have demonstrated that these types of vesicles can be perceived by neurons and have adverse effects on their functioning and differentiation ^[28][214].

A significant portion of the mitochondria in the axons of retinal ganglion cells is normally degraded by astrocytes in the optic nerve head (ONH). This transcellular process of mitochondrial degradation, known as transmitophagy, likely occurs in other regions of the CNS as well, as structurally similar accumulations of degrading mitochondria are observed along neurites in the superficial layers of the cerebral cortex ^[29][215]. The introduction of astrocyte mitochondria into neighboring neurons has a protective effect during temporary focal cerebral ischemia in mice ^[30][216]. This raises the question of whether astrocytes in adult brain tissue can transfer mitochondria into affected neurons after ischemic injury ^[31][217]. During ischemic brain injury, astrocytes exert neuroprotective and anti-inflammatory effects, attributed to the secretion of erythropoietin, VEGF, GDNF (glial cell line-derived neurotrophic factor), and estrogen (17β-estradiol), which limit neuronal damage ^[32][33][34][218,219,220].

Gap junctions between astrocytes remain open during in vivo ischemia and in vitro hypoxia. This allows for the passage and rapid spread of pro-apoptotic factors, contributing to an increase in the size of the necrotic area. However, there is evidence from experiments on animals suggesting that astrocytic gap junctions can limit the zone of necrosis. The exact role of these contacts is still contradictory, and further research is needed to clarify their significance ^[18][35][206,221].

There is a hypothesis that reactive astrocytes forming a scar are actually astrocyte-like neural stem cells that differentiate into astrocytes. This transformation is thought to be modulated by specific genes activated after ischemia. Reactive astrocytes isolated from the peri-infarct cortex following ischemia can de-differentiate into neural-sphere-producing cells (NSPCs), which are multipotent cells capable of self-renewal. However, when transplanted, these cells have been shown to differentiate into astrocytes and OLs, but not neurons. Nevertheless, this demonstrates the high plasticity of reactive astrocytes. Recent studies indicate that reactive astrocytic glial cells after ischemia can be reprogrammed into functioning neurons, leading to a reduction in gliosis and restoration of synaptic contacts. There is also evidence that a combination of transcription factors can transform reactive astrocytes not only into neurons but also into neuroblasts. This highlights once again the plasticity of reactive glia and the potential for this property to find applications in targeted therapies following ischemic brain injury ^[18][36][206,222].

OLs are highly susceptible to ischemia, and a significant number of them die within three hours of an acute incident ^[37][223]. However, they play a crucial role in the chronic stage of ischemia as the main cellular population responsible for remyelinating affected axons ^[38][224]. After ischemia, mature OLs accumulate along the border of the infarct zone to participate in tissue recovery ^[39][225].

It should be noted that OLs do not have the capacity for self-renewal. In fact, ischemia stimulates the proliferation and differentiation of OPCs into myelinating Ols ^[40][226]. The number of OPCs increases in the penumbra (the surrounding region of the infarct) following ischemic brain injury but decreases in the center of the lesion. They undergo morphological transformation characterized by hypertrophy of cell bodies as well as molecular and genetic changes that stimulate their migration, proliferation, and differentiation ^{[41][42][43][44][45]}[227,228,229,230,231].

During ischemic brain injury, OLs undergo apoptosis induced by the complement system and the toxic effects of released glutamate and ATP. Additionally, OLs are influenced by inflammatory cytokines primarily released by microglia in the infarct area. For example, IFN-γ induces apoptosis, delays remyelination, and inhibits the proliferation and differentiation of OPCs. TNF-α also induces apoptosis and delays remyelination. IL-6, IL-11, and IL-17 have a beneficial effect by promoting the survival of OLs. IL-1β has contradictory effects: it promotes the survival of OLs on the one hand and contributes to their necrotization on the other hand ^[46][232]. Numerous studies indicate that interactions between microglia and OLs can have both favorable and unfavorable effects, depending on the stage of OL development ^[47][233].

NG2-glia rapidly respond to ischemic damage in brain tissue. Together with macrophages, these glial cells are identified within the first day after the onset of injury ^[7][195]. The observed changes in NG2-glia are morphological, including hypertrophy of the cell body, shortening and thickening of cellular processes, and increased intensity of NG2 expression ^[48][234]. Their proliferative activity is also enhanced, and NG2+ cells migrate to the periphery of the ischemic lesion ^[7][195]. After this stage, the number of NG2-glia gradually decreases, reaching optimal levels within 28 days after the injury. This suggests that these glial cells perform a regulatory function and maintain homeostatic balance following the injury ^[49][158].

Experimental studies conducted on mouse brains after focal cerebral ischemia reveal the presence of cells containing genes characteristic of both NG2-glia and reactive astrocytes. Immunohistochemical analysis of their protein profile confirms these characteristics. Consequently, they are referred to as astrocyte-like NG2-glia cells. Their profile resembles that of astrocytes in the cortical gray matter. They are localized in the postischemic glial scar and are likely related to its formation following ischemia ^[49][50][51][158,235,236]. Additionally, another study demonstrates the involvement of NG2-glia in the early stages of tissue recovery after brain injury ^[52][237].

Upon the appearance of a damaging factor, microglia undergo a transformation from a resting state to what is known as activated or amoeboid microglia ^[53][180]. These cells have undergone the following morphological changes: hypertrophy of the cell body and retraction and thickening of cellular processes ^[54][238]. Several markers are predominantly expressed in activated microglia, including CD45, MHCII, and CD68 ^[55][239]. Iba1, IB4, F4/80, and CD68 markers are also used for their study ^[56][57][181,240].

Following focal cerebral ischemia, different microglial phenotypes are observed depending on the location of microglia relative to the site of injury and the specific expression of surface cell proteins. In the periphery of the infarct, microglial cells are positive for Iba1 and negative for CD68. In the center of the infarct zone, cells are positive for both Iba1 and CD68, but they also show increased expression of CD11b ^[56][57][181,240]. While the activated microglia in the infarct zone exhibit phagocytic activity and primarily express MHCI ^[58][241], microglia in regions remote from the lesion express MHCII and are associated with neuronal degeneration ^[59][60][242,243]. These diverse morphological subtypes of microglial cells are presumed to perform different functions depending on their distance from the ischemia and the time elapsed since the incident ^{[57][61][62][63][64]}[240,244,245,246,247].

Microglia may participate in the reconstruction of blood vessels following ischemia. They carry out this function through the phagocytosis of endothelial cells and the release of the pro-angiogenic VEGF ^[65][248]. Endothelial proliferation, which represents the initial step of angiogenesis, is influenced by various pro- and anti-inflammatory cytokines, with TGF-α stimulating it and TGF-β inhibiting it ^[66][249]. Microglia with an anti-inflammatory effect, referred to as the M2 subtype, secrete TGF-β and are predominantly located in the ischemic area during the acute and subacute phases of cerebral infarction ^[67][250]. M2 glia also secrete pro-angiogenic factors such as VEGF and matrix metalloproteinase-9 (MMP-9) ^[68][251]. It has been demonstrated that following a stroke, the integrity of the BBB is maintained by pro-inflammatory factors such as TNFα, IL1-β, and IL-6, released by the so-called M1 microglia ^[69][252]. While resting microglia create an environment that inhibits endothelial proliferation, activated microglia have the opposite effect ^[66][249].

According to several studies, ischemia stimulates neurogenesis in both rodents ^[70][253] and primates ^[71][254]. In the ipsilateral SVZ, activated microglia with cellular processes facilitate the migration of neuronal precursor cells, while amoeboid microglia in the peri-infarct zone may have an adverse effect on neurogenesis ^[72][255].

Neuronal damage in ischemic brain injury enhances the phagocytic activity of microglia. Activated microglia have a dual role: on the one hand, they support neuronal survival ^[72][255], but on the other hand, uncontrolled microglial activation can lead to the release of cytotoxic factors such as superoxides ^[73][74][75][256,257,258], nitrogen oxide ^[76][259] and TNF-α ^[77][260]. NADPH oxidase is a mediator with neurotoxic effects and is released during ischemic brain injury. It stimulates microglia, which in turn causes long-term inhibition of synapses ^[78][261]. This means that the function of neuronal circuits after ischemia can be influenced by microglia.

Advances in vivo imaging techniques make the study of microglia in humans accessible. Activated microglia can be observed as early as 24–48 h after ischemic stroke, mainly localized in the center of the injury and gradually spreading to the peripheral ^[79][80][262,263]. Therefore, this cell type performs its function in both the acute ^[79][80][262,263] and subacute phases of brain infarction ^[81][82][264,265]. Clinical studies involving six patients within a period of 3 to 150 days after diagnosis of ischemic stroke confirm these findings: activated microglia can be observed in the early days of ischemia, distributed in the infarct area and gradually spreading toward the periphery. Reactive microglial cells are also present in the contralateral hemisphere at a later stage ^[83][266].

According to another study involving four patients, activated microglia are present in both the infarct zone and its surroundings during the first week after the incident, but the number of these cells gradually decreases over time. After 14 weeks, it has been observed that the increased amount of reactive microglia persists in the peri-infarct region, while it is lower in the center of the lesion ^[82][265]. In 18 patients with a first ischemic stroke in the subcortical region, different clinical outcomes were observed depending on whether activated microglia were mainly localized in the infarct zone or its surroundings. The effect of activated microglia in the core of the lesion is negative, while the activation of microglia in the peri-infarct region correlates with a positive clinical outcome ^[84][267]. The available contradictory data from human studies show that the question of whether reactive microglia have a positive or negative effect on recovery after ischemic stroke is still debatable.

During cerebral infarction, the integrity of the BBB is disrupted, which critically affects the extent of brain damage. Bioactive substances released by endothelial cells, brain glial cells, and immune cells provoke and sustain the inflammatory reaction in this area. Among the brain cell populations, microglial cells are the first to respond to the injury, activating through a series of molecular mechanisms and transforming into different functional subtypes. This is followed by infiltration of immune cells and a similar reaction by astrocytes. Microglia have the ability to modulate astrogliosis following brain ischemia, as reactive astrocytes possess a range of receptors for signaling molecules secreted by microglia. It can both stimulate and limit astrogliosis depending on specific conditions. On the other hand, microglia influence neurotransmission controlled by astrocytes by stimulating the release of glutamate into the extracellular space ^[18][85][86][206,268,269].

Astrocytes, in turn, can actively modulate microglial activity, both locally through paracrine mechanisms and at distant sites through mediators such as IL1β, the calcium signaling pathway, ATP, and the cytoplasmic calcium-binding S100β protein, and through the production of gliotransmitters, inflammatory cytokines, and RNA molecules. There is evidence that astrocytes can also influence gene expression in microglial cells ^[18][86][206,269].

The elimination of degenerated neurons resulting from ischemia is a process in which microglia and astrocytes interact once again. Experiments with mice modeling chronic cerebral hypoperfusion demonstrate characteristic cell interactions. The bodies of damaged neurons are infiltrated by processes of both astrocytes and microglia, forming a complex structure known as a triad, which exacerbates ischemic damage and contributes to an increased degree of neurodegeneration ^[87][270].

Reactive astrocytes secrete a range of bioactive molecules that are relevant to the functioning of OL cell lines ^[18][206]. Additional information is presented on Table 1.

The integrity of astrocyte-oligodendrocyte contacts is relevant to the proper functioning of OLs. Defects in gap junction contacts and the proteins that mediate them, both in astrocytes and OLs, primarily have a negative effect and lead to disruptions in axonal myelination ^[18][206]. CX43 is an astrocytic protein from the connexin family that participates in the formation of gap junction contacts. An interesting discovery is that in cell cultures of astrocytes and OLs under hypoxic conditions, where CX43 is blocked, OPC differentiation is facilitated ^[88][271].

Activated microglia are harmful to OL progenitor cells but improve the survival of mature Ols ^[47][233]. A recent study demonstrates that Iba1+ microglia have a beneficial effect on the OPC subpopulation in the early stages following an acute ischemic incident but lose this pro-regenerative potential in later stages. Immediately after ischemia, they contribute to increased OPC density and limit the demyelination process. In vitro, experiments have demonstrated the direct beneficial effect of microglial vesicles on the same OPC subpopulation ^[64][247].