, Ioana Adriana Ardelean 3,41 Psycho-neurosciences and Rehabilitation Department, Faculty of Medicine and Pharmacy, University of Oradea, 410087 Oradea, Bihor, Romania
2 Neurology Department, Clinical Municipal Hospital Oradea, 410154 Oradea, Bihor, Romania
3 Physiopathology Department, Faculty of Medicine and Pharmacy, University of Oradea, 410087 Oradea, Bihor, Romania
4 Cardiology Department, Clinical Emergency County Hospital Oradea, 410169 Oradea, Bihor, Romania
Abstract
With the larger variety of methods employed, recanalization therapy is increasingly used to treat acute ischemic stroke resulting in about one-third of patients undergoing early neurological deterioration, in which ischemia/reperfusion injuries are the main cause, leading to increases in the infarcted area, the no-reflow phenomenon, or hemorrhagic transformation. Efficient prevention or treatment of these injuries depends on extensive knowledge of the involved mechanisms. These pathways have dual, damaging, and neuroprotective effects, depending on the timing or protein subtype involved. The current article reviews the main mechanisms contributing to the pathophysiology of these injuries, such as mitochondrial dysfunction, cellular calcium overload, excitotoxicity, oxidative stress, apoptosis, and neuroinflammation.
Keywords
- Reperfusion injury
- Excitotoxicity
- Mitochondria
- Oxidative stress
- Apoptosis
- Neuroinflammation
The concept of ischemia/reperfusion injury emerged over 50 years ago when
Jennings and coworkers showed that in hearts subjected to coronary ligation,
reperfusion accelerated the development of necrosis [1]. Paradoxically, restoring
blood supply to an organ subjected to temporary glucose and oxygen deprivation
can injure the tissue [2, 3], as described in the kidneys, intestines, skeletal
muscles, liver, and cerebral tissue [4]. Cellular and molecular mechanisms
contribute to ischemia/reperfusion injuries, involving reactive oxygen species
(ROS), innate and adaptive immune systems, and dysfunction of cellular metabolism
and vascular and parenchymal cellular demise [5, 6]. Although much of the
research has been performed in animal models, with abrupt reperfusion after
transient ischemia [7] increasing the size of the infarction by as much as 70%
[8], in human patients, the increase in infarct size in the first 24 hours is
more limited [9]. However, hyperperfusion (defined as
A large amount of research has focused on unraveling the complex mechanisms of ischemia/reperfusion (I/R) injuries which are caused by a complex interplay between mitochondrial dysfunction, oxidative and nitrosative stress, calcium overload and excitotoxicity, activation of apoptosis, and inflammation [4]. This knowledge can open novel therapeutic opportunities for preventing them and extend the therapeutic windows for recanalization procedures.
Mitochondria are intracellular organelles with a double membrane that have a crucial role in energy generation, regulation of cell cycle, and apoptosis induction [13]. The inner membrane contains a series of enzyme complexes responsible for oxidative phosphorylation (complexes I–V) and the generation of adenosine triphosphate (ATP) [14].
Complex I or proton-pumping nicotinamide adenine dinucleotide (NAD) H
dehydrogenase oxidizes NADH by pumping 4 protons per 2 electrons passed to
ubiquinone, resulting in ubiquinol (QH
The lack of oxygen during ischemia inhibits the electron flow through the respiratory chain, preventing ATP synthase from generating ATP [17]. The rate of entry of electrons into complex I exceed the rate of transit through complex IV, causing them to build up at complexes I and III and slowing down the electron transport chain and the pumping of protons across the inner mitochondrial membrane, leading to a reduction of the mitochondrial membrane potential [16, 18].
Following the restoration of blood flow, the mitochondrial membrane potential is restored within 1 minute [19]. Still, the increased oxidative phosphorylation leads to mitochondrial hyperpolarization with dramatic consequences on the mitochondrial function and the increased generation of reactive oxygen species (ROS), which will further impair the normal mitochondrial function [20]. Indeed, after 30 minutes following reperfusion, mitochondrial function is significantly decreased in cells that will die [21].
Mitochondria are organelles whose dynamics, regulated by fission and fusion, have an important role in neuronal injury and recovery following ischemia [14]. Fission manifested as constriction and cleavage of mitochondria is regulated by dynamin-related protein 1 (Drp1), a mitochondrial-binding GTPase. It has been shown that global cerebral ischemia transiently increases phosphorylation of Drp1 [22] while Drp1 inhibitors reduced the infarct volume in a model of focal cerebral ischemia [23]. Mitochondrial fission also can initiate extrinsic apoptotic cell death, and fragmentation of these organelles in endothelial cells leads to endothelial dysfunction in postischemic tissues [4, 24].
During ischemia-hypoxia, the brain cells switch to anaerobic glycolysis to
supply the necessary ATP, which leads to the accumulation of lactate, NAD
High intracellular calcium promotes calcium from the endoplasmic reticulum via
activated ryanodine receptors [28]. It proves toxic by activating a series of
enzymes, such as the family of cysteine proteases known as calpains, which
degrade cytoskeletal, mitochondrial proteins and the endoplasmic reticulum [29].
Research has shown that pharmacological inhibition of calpains can protect the
brain against reperfusion injuries [30]. Another important pathway triggered by
increased cytosolic calcium levels is the activation of
Ca
High intracellular calcium levels also lead to the generation of danger signals, such as calcium pyrophosphate complexes and uric acid, which bind to the inflammasomes (intracellular protein complexes) and lead to the increased production of cytokines initiating inflammation [4].
Mitochondria act as a calcium buffer, attempting to normalize the cytosolic
calcium levels. The ion moves through the outer mitochondrial membrane through
the voltage-dependent anion-selective calcium channel and further into the
mitochondrial matrix mediated by the mitochondrial calcium uniporter [32, 33].
However, excessive mitochondrial Ca
As already mentioned, excess excitatory neuromediator (glutamate) release
significantly increases cellular calcium overload. Glutamate binds mainly to 2
ionotropic, ligand-gated ion channels: NMDARs and AMPARs. In the resting state,
magnesium blocks the channel pores of NMDARs. Glutamate binding to AMPARs causes
a partial depolarization of the postsynaptic membrane, which removes Mg
Under physiological conditions, presynaptic axonal terminals release quanta of
glutamate into the synaptic cleft to activate receptors on the postsynaptic
membrane [48]. Astrocytes clear glutamate from the synaptic cleft through
specific transporters (excitatory amino acid transporters—EAATs) and transform
it into glutamine or use it for their metabolism, thereby maintaining glutamate
homeostasis [49, 50]. However, this is a highly energy-consuming process, which
fails in oxygen and glucose deprivation conditions, as happens in ischemic
conditions. Glutamate uptake via EAATs occurs with co-transport of 3Na
Fig. 1.The dual role of NMDA receptors in determining the fate of
neurons: binding of glutamate to extrasynaptic NMDARs dephosphorylates
cAMP-responsive element-binding protein (CREB), inactivates the extracellular
signal-regulated kinase (ERK) pathway and promotes cell death, while binding of
glutamate to synaptic NMDARs promotes cell survival through activation of the
phosphoinositide-3-kinase (PI3K)/Akt pathway, which inactivates glycogen synthase
kinase 3
Reperfusion of ischemic tissue with oxygenated blood, although necessary for aerobic ATP production, leads to increased production of ROS, which can oxidize almost every biomolecule and induce cell dysfunction (oxygen paradox) [4]. Oxidative stress, defined as an imbalance between ROS production and the ability of the biological system to clear these highly reactive molecules, has been shown to significantly contribute to the pathophysiology of I/R injuries [52].
Three distinct phases of increased ROS generation have been identified in cell cultures [18, 53]: (i) during glucose and oxygen deprivation, due to mitochondrial depolarization and inhibition of complex IV leading to upstream accumulation of reduced compounds which enable leakage of electrons; (ii) 25–35 minutes after the oxygen and glucose deprivation, caused by activation of xanthine oxidase; (iii) after reperfusion.
The brain is particularly vulnerable to oxidative stress due to a series of characteristics: (i) it has the highest metabolic activity per unit weight, consuming 20–25% of the total body oxygen despite weighing only 2% of the total body weight [6, 13]; (ii) compared to other organs, such as the heart, kidney, or liver, it has significantly lower activities of antioxidants such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase, or heme-oxygenase-1 [54, 55]; (iii) it has lower activities of Cytochrome c oxidase, resulting in higher superoxide release from the mitochondrial electron transport chain during ATP production, which is also reduced [54]; (iv) the plasma membranes of brain cells are rich in polyunsaturated fatty acids, highly vulnerable to oxidative damage [56]; (v) it has a high ratio of membrane surface area compared to the cytoplasmic volume [57]; (vi) damaged cerebral parenchyma releases iron ions which can catalyze free radical reactions [57]; (vii) excessive neurotransmitter release during ischemia/reperfusion, such as glutamate and dopamine, resulting in cellular calcium overload, which impairs mitochondrial function and leads to excitotoxicity [4, 6].
The main ROS include superoxide anion (O
Cerebral ischemia inhibits the activity of complex I, leading to the accumulation of succinate through the reversed activity of succinate dehydrogenase, which reduces fumarate to succinate, and to a lesser extent, the activity of complex IV (cytochrome C oxidase) [61, 62]. The reduced activity of the final electron acceptor in the mitochondrial electron transport chain causes increased ROS production of upstream complexes, dramatically increased after oxygen delivery is restored by reperfusion [62, 63]. In addition, upon reperfusion, succinate dehydrogenase oxidizes the accumulated succinate and drives reverse electron transport through complex I, which is why complex I is regarded as the main ROS-generating mitochondrial site [61, 64]. However, at least 7 sites in the mitochondria can partially reduce oxygen and produce ROS [65, 66].
One class of enzymes mitigating the effects of ROS are the superoxide dismutases (SODs), with manganese SOD (MnSOD) being mainly a mitochondrial enzyme and copper-zinc SOD (Cu-ZnSOD) a cytosolic one. Complex I dysfunction after reperfusion influences MnSOD expression [62].
Reperfusion is associated with large increases in intracellular and
mitochondrial Ca
The outer mitochondrial membrane is associated with 2 monoamine-oxidases, monoamine oxidase-A and -B, which deaminate neurotransmitters at the expense of generating hydrogen peroxide [67].
Another highly reactive molecule produced by mitochondrial is nitric oxide (NO), which, at physiological concentrations, reversibly inhibits Cytochrome c oxidase and modulates oxygen consumption [68]. More recently, research has shown the involvement of another protein, p66Shc, located between the 2 mitochondrial membranes and forms molecular complexes with cytochrome c, thereby transferring electrons between itself and cytochrome c. It appears that this protein also contributes to increased ROS production, mitochondrial depolarization, and cytochrome c release [69, 70].
The mitochondrial permeability transition pore (MPTP) is a key player in I/R
injury. It is inhibited by low pH, so it is quiescent during ischemia, but the
increases in mitochondrial Ca
Fig. 2.During ischemia, oxygen and glucose deprivation leads to failure
of Na
NADPH oxidase (NOX) is a membrane enzymatic complex that generates superoxide while transferring electrons from NADPH to oxygen molecules across the cell membrane [59]. However, NOX is the main defense mechanism of macrophages and neutrophils. Exposure to microorganisms or inflammatory mediators can increase 50- to 100-fold the production of oxidative species [4]; NOX2 and NOX4 isoforms have been localized in the hippocampal CA1 region and cortex [68]. Experimentally, NOX2 knockout animals and NOX2 inhibitor-treated animals showed significantly reduced infarct sizes, demonstrating the role of NOX2 in oxidative stress-induced ischemic neuronal death [75]. The vascular NOX isoforms usually have lower activity levels, the ROS generated by them being more likely involved in signaling cascades. However, after ischemia-reperfusion, vascular NOXs can produce increased levels of ROS and produce oxidative stress [76].
Xanthine oxidase (XO) is a molybdo-flavin enzyme that exists in 2 forms: a
NAD-dependent dehydrogenase (xanthine dehydrogenase) and an oxygen-dependent
oxidase (xanthine oxidase) with a higher affinity for oxygen than NAD
The central nervous system expresses 3 kinds of nitric oxide synthases (NOS):
endothelial NOS (eNOS), which regulates cerebral blood flow, neuronal NOS (nNOS),
and inducible NOS (iNOS). Nitric oxide (NO) produced by eNOS after brain ischemia
promotes vasodilation and inhibits microvascular adhesion and aggregation, thus
exerting a protective effect [68]. However, ischemia activates nNOS through the
high intracellular Ca
ROS can also result from the activity of other intracellular enzymes, such as cytochrome P450 enzymes, cyclooxygenases, or lipoxygenases [59].
Cytochrome P450 enzymes (CYPs) are membrane-bound oxidases that use oxygen or NADPH to catalyze oxidation or reduction of lipids, steroids, cholesterol or other lipids, such as arachidonic acid [4]. They have a crucial role in vasoregulation, forming both vasoconstrictive compounds, such as 20-hydroxyeicosatetraenoic acid (20-HETE) and vasodilator epoxyeicosatrienoic acids [85]. The role of CYPs in I/R injury is complex, but research has suggested that 20-HETE may be significantly involved in the pathophysiology of these injuries, at least in neonatal brains [86]. Moreover, cerebral ischemia induces CYP expression [87].
Lipoxygenases (LOXs) catalyzes the synthesis of eicosanoids, such as leucotrienes and hydroxyeicosatetraenoic acids. Following cerebral ischemia, there is a massive release of free fatty acids from membrane stores [88]. 12/15 LOX oxidizes these lipids, leading to the generation of 12- and 15-HETE [89], and can damage the mitochondrial membrane, leading to increased ROS production and initiating apoptosis [90]. Experimentally, inhibition of 12/15 LOX with baicalein resulted in reduced infarct volume, similar to infarctions of animals in which ALOX15, the gene encoding for LOX12/15, was knocked out [91].
Another key enzyme in the generation of prostaglandins from arachidonic acid is cyclooxygenase (COX) [92, 93]. Both COX-1 and COX-2 isoforms cleave arachidonic acid, and upregulation of COX-2 is a hallmark of ischemia/reperfusion injuries [94], especially in the inflammatory cells, which invade the cerebral tissue after an ischemic injury [95]. Pharmacological inhibition or genetic inactivation of COX-2 resulted in the reduced magnitude of cerebral injury after focal or global cerebral ischemia [96, 97], although COX’s radical species have never been identified [98]. The reports on increased incidence of cardiovascular events, including stroke, after long-term treatment with COX-2 inhibitors, have challenged these agents’ therapeutic potential [98, 99].
Under normal conditions, the small amounts of ROS can be removed by the brain’s antioxidant enzymatic and non-enzymatic defenses. The antioxidant enzymes include superoxide dismutase (SOD), glutathione peroxidase (GPX), and catalase (CAT) [59]. Non-enzymatic antioxidant molecules are present mainly in extracellular spaces and include glutathione, vitamins C and A, N-acetylcysteine and melatonin [59, 100]. However, following cerebral ischemia, and especially after reperfusion, the production of ROS increases [93, 101], which, together with the downregulation of the enzymatic antioxidant defenses by ischemia [102], leads to significantly increased oxidative stress and oxidative species-induced cellular injury.
ROS has a series of detrimental effects, initiating several cell signaling cascades and altering the functions of enzymes and ion channels.
ROS can activate p53, a transcription factor controlling the gene expression of Bax, Bid and p53 upregulated modulator of apoptosis (PUMA). P53 opens the mitochondrial permeability transition pore and increases the mitochondrial membrane permeability (also caused by ROS), leading to cytochrome c release [60, 103]. This is pivotal in initiating apoptosis because released cytochrome c forms a complex with apoptotic protease activating factor-1 (APAF-1), pro-caspase-9 and ATP, and activates caspases [104]. In addition, p53 upregulates apoptosis signal-regulating kinase 1 (ASK1), which together with PUMA is involved in executing apoptotic cell death [105, 106].
Mitogen-activated protein kinases (MAPKs) are a family of serine/threonine kinases with substantial cell growth, survival, proliferation, and death. The 3 main MAPKs are extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs), and the p38 MAPKs [4]. ERKs are protective against ischemia-reperfusion injuries [4], the role of JNKs is controversial [107, 108]. At the same time, p38 MAPK is activated in response to I/R [109] but, depending on the isoform activated, can be either protective or harmful: it appears that activation of p38A is lethal to the cell [110]. In contrast, activation of the B isoform of p38 is cytoprotective and involved mainly in preconditioning [111].
ROS can interact with a variety of biological molecules. They can react with proteins, leading to their oxidation, degradation, or peptide bond cleavage, resulting in protein aggregation, enzyme inactivation, or modifications in the activity of ion channels [112, 113]. For example, oxidation and inactivation of glutamine synthetase in astrocytes prevent glutamate’s conversion into glutamine and contribute to ischemia-induced neurotoxicity in the gerbil brain [114].
Lipid peroxidation (ROS attacking the carbon-carbon bonds of polyunsaturated
fatty acids) is even more damaging than protein oxidation, being self-propagated
because lipid radicals are unstable and react with oxygen to form lipid peroxyl
radicals [59, 115]. These can react with other fatty acids to generate aldehydes,
such as malondialdehyde and 4-hydroxynonenal, the latter being a second messenger
which regulates several transcription factors including NF-
Finally, ROS can attack the DNA causing double-strand breaks, protein-DNA crosslinks, structural changes, or DNA mutations [117, 118], leading to increased poly (ADP-ribose) polymerase (PARP) activity in an attempt to repair DNA damage but which depletes the cells of the already reduced energy supplies [119].
This mechanism of cell death, with distinct features from necrosis, can be initiated by 2 main pathways: the extrinsic pathway, related to binding of specific molecules to the death receptors of the cell membrane, and the intrinsic pathway [120, 121].
A group of proteins, known as the Bcl-2 family, tightly regulate cell death and survival [14]. This family of proteins includes anti-apoptotic proteins, such as Bcl-2, Bcl-XL, Bcl-W, and pro-apoptotic proteins like Bax, Bad, Bid, Bim, Noxa, or PUMA [122]. The intrinsic pathways of apoptosis can be caspase-dependent or caspase-independent.
The ischemia-induced mitochondrial dysfunction and opening of the mitochondrial permeability transition pore (MPTP) lead to the release of cytochrome c and other pro-apoptotic factors such as apoptosis-inducing factor (AIF), high-temperature requirement protein A (HtrA2/OMI) [14], or second mitochondrion-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pI (SMAC/DIABLO [14]. Cytochrome c interacts with the cytosolic apoptotic-protease-activating factor-1 (Apaf-1) to form the apoptosome and, together with deoxyadenosine triphosphate, activates pro-caspase-9, which will cleave and activate caspase-3 [123]. Caspase-3 is a key mediator of apoptosis in animal models of stroke, its mRNA being upregulated 1 hour after the onset of focal ischemia [124]. It cleaves many proteins, among them PARP.
Aside from upregulation of the pro-apoptotic Bcl-2 protein subfamily by ischemia [125], the mitochondrial dysfunction and opening of the MPTP lead to the release of AIF, which translocates to the nucleus fragments DNA and inhibits PARP, thereby accelerating cellular damage and destruction [126]. SMAC/DIABLO binds to X chromosome-linked inhibitor-of-apoptosis protein (XIAP) and triggers apoptosis by suppressing the anti-apoptotic activity of XIAP [127]. In addition, increased cytosolic calcium activates calpains and caspase-8, leading to cleavage and activation of Bcl-2 interacting domain (BID) [128], which translocates to mitochondria when the cell receives a death signal. Activated BID induces conformational changes in other pro-apoptotic proteins, such as Bax, Bad, Bcl-XS, and inactivate anti-apoptotic proteins like Bcl-2 or Bcl-XL [129].
The cells also have anti-apoptotic pathways, but these are overwhelmed after an
ischemic insult. For example, inhibitor-of-apoptosis (IAP) proteins, including
XIAP, NIAP, and others, bind and suppress the activity of caspases -3, -7, and -9
[130]. Various members of the Bcl-2 family (Bcl-2, Bcl-XL, Bcl-w) also try to
suppress the apoptotic process [131]. CREB and NF-
This pathway also contributes to cell death after cerebral ischemia, being upregulated within 12 hours after the onset of focal cerebral ischemia and peaking 24 to 48 hours after the ischemic insult [121]. The binding of certain molecules initiates it on the surface receptors of the cells. These surface cell death receptors belong to the tumor necrosis factor receptor (TNFR) superfamily and include TNFR-1 and Fas. Forkhead 1, a transcription factor, stimulates the expression of several genes, such as Fas ligand (FasL), which binds to the Fas receptor and triggers recruitment of the cytoplasmic adaptor protein Fas-associated death domain protein (FADD) [121, 134]. FADD can bind to pro-caspase-8. The whole complex (FasL–Fas–FADD–procaspase-8) is also known as the death-inducing signaling complex (DISC). It is assembled within seconds of FasL binding to Fas, leading to activation of pro-caspase-8 and generation of caspase-8 [128]. Further, caspase-8 is released from the DISC complex and activates caspase-3 [121], leading to the execution phase of apoptosis. Fig. 3 (Ref. [135]) presents these pathways leading to apoptosis.
Fig. 3.Main pathways are leading to apoptosis. Opening of the mitochondrial permeability transition pore (MPTP) leads to the release of cytochrome C (cyt C), which together with the cytosolic apoptotic-protease-activating factor-1 (Apaf-1) activates procaspase-9. Active caspase-9 further activates caspases-3 and 7, leading to the execution phase of caspase-dependent apoptosis. The mitochondria also release apoptosis-inducing factor (AIF), high-temperature requirement protein A (HtrA2/OMI), as well as a second mitochondrion-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pI (SMAC/DIABLO), which inhibits the antiapoptotic X chromosome-linked inhibitor-of-apoptosis protein (XIAP), thereby leading to apoptosis. Various factors, such as increased cytosolic calcium-induced activation of calpains and caspase-8, cleave and activate Bcl-2 interacting domain (BID), which activates other pro-apoptotic proteins (Bax, Bad, Bcl-XS) and inactivates antiapoptotic proteins like Bcl-2 or Bcl-XL (caspase-independent apoptosis). The binding of Fas ligands (FasL) to Fas receptors recruits the cytoplasmic Fas-associated death domain (FADD) and pro-caspase-8, forming together the death-inducing signaling complex (DISC), which leads to activation of pro-caspase-8 and triggering of the extrinsic pathway of apoptosis. Adapted from [135].
The brain is an immune-privileged organ that is not readily accessible to immune cells due to the blood-brain barrier (BBB) [136]. The BBB has a layer of endothelial cells interconnected by tight junctions, placed on a basal membrane. Many pericytes are embedded [137] and are ensheathed on the abluminal aspect by astroglial endfeet [138].
Microglia is the primary immune cell of the central nervous system (CNS). The
resting-state has a small cell soma and numerous processes that monitor the CNS’s
microenvironment [139]. Upon activation, microglia retract their processes and
take on an amoeboid shape [140]. The main pathway for microglia activation is the
NF-
Transient middle cerebral artery occlusion as short as 15 minutes in
spontaneously hypertensive stroke-prone rats leads to microglial activation
[144], after which these cells migrate toward the ischemic lesion and remain
close to the neurons in a process called “capping”, which helps quick removal
of damaged neurons [145, 146]. The production of ROS via NADPH oxidase, of matrix
metalloproteinases and cytokines, as well as activation of CD14 receptors by iNOS
followed by the expression of toll-like receptor 4 (TLR4) in activated microglia
increase its neurotoxic effects in the infarcted core as well as in the penumbra
[147, 148, 149, 150, 151]. After transient middle cerebral artery occlusion in mice, microglial
and macrophage infiltration peaks 48–72 hours after the ischemic insult [152].
Once arrived at the site of injury, microglia produce a series of
pro-inflammatory cytokines, such as interleukin (IL)-1
However, microglia play a dual role after ischemic stroke, secreting pro-inflammatory as well as anti-inflammatory factors [147]. Research has shown that impaired microglial activation increased infarct size and potentiated neuronal apoptosis following ischemia [155]. Depletion of microglia with PLX3397, a dual colony-stimulating factor-1 inhibitor, increased infarct size and worsened the neurological deficits [156]. In addition, microglia produce a variety of neurotrophic factors which promote neuroplasticity and neurogenesis [157]. Thus, it appears that different subsets of microglial cells have different roles following cerebral ischemia [145].
Leukocytes are among the first blood-derived immune cells entering the brain
after cerebral ischemia, peaking at 48–72 hours and rapidly declining afterward
[143]. After minutes to hours after the ischemic insult, ROS, cytokines and
chemokines released by the damaged tissue induce the expression of adhesion
molecules on leukocytes and cerebral endothelial cells [145, 158]. Cytokines such
as tumor necrosis factor (TNF)-
Fig. 4.Leucocyte diapedesis. Leucocytes interact with endothelial cells expressing P selectins through P-selectin glycoprotein 1 (PSGL-1), leading to their “rolling” on the endothelial surface. Interaction of leucocyte integrins CD11a/CD18 and CD11b/CD18 with intercellular adhesion molecule 1 (ICAM-1) leads to firm adherence and aggregation of leucocytes. Diapedesis of leucocytes is facilitated by platelet-endothelial cell adhesion molecule 1 (PECAM-1). Adapted from [163].
Lymphocytes have a less important contribution in cerebral ischemic injury; the mechanisms are mainly related to the innate T-cell functions [164]. IL-17 secreting T cells aggravate ischemic injury [165], as do natural killer T cells, while IL-10-secreting regulatory lymphocytes (Tregs) have neuroprotective activity by downregulating postischemic inflammation [166, 167].
Cytokines are small polypeptides (8–26 kDa), normally expressed at very low levels, regulating immune responses [168].
IL-1
Tumor necrosis factor (TNF)-
IL-10 is an anti-inflammatory cytokine upregulated in ischemic stroke, peaking 3 days after the onset [178]. Animal research with intraventricular administration of IL-10 or adenoviral delivery of the IL-10 gene confirmed the neuroprotective effect of this cytokine [179, 180].
Another neuroprotective cytokine is interferon-
Chemokines are low molecular weight proteins (8–10 kDa) involved in cellular activation and leukocyte recruitment.
Monocyte chemoattractant protein-1 (MCP-1) directly increases the permeability of the BBB by causing tight-junction proteins to redistribute in endothelial cells [184] and recruits monocytes and activated lymphocytes into the brain after an ischemic insult [185].
Other chemokines upregulated in the first 3 hours after stroke are microglial response factor-1 (MRF-1), fractalkine, and macrophage inflammatory protein 1 (MIP-1), which all contribute to infiltration of the injured tissue with inflammatory cells and weaken the BBB [145].
However, stromal cell-derived factor 1 (SDF1), also known as C-X-C motif chemokine 12 (CXCL12), maybe neuroprotective, being found increased in the ischemic penumbra and facilitating homing of bone marrow stromal cells to the tissue injured by ischemia [186], thereby reducing the size of infarction and enhancing neural plasticity [187].
Although this family of enzymes is not a part of neuroinflammation, due to their significant involvement in BBB disruption, their course in acute ischemic stroke will be briefly discussed. MMPs are constitutive enzymes, such as MMP-2 and MMP-14, or inducible ones, like MMP-3 and MMP-9 [145]. The expression of MMP-9 increases significantly within 24 hours from the onset of ischemia in rats [188] and, together with tissue plasminogen activator, disrupts the BBB leading to hemorrhagic transformation [189]. Experimentally, MMP inhibition alleviates hemorrhage and brain edema and can also reduce infarct size [190]. On the other hand, plasma levels of MMP-3 were found to increase in patients with better functional and motor recovery [191], highlighting the dual role of these enzymes in stroke pathogenesis and recovery.
Despite the large amount of research focusing on the molecular pathophysiological mechanisms of ischemia/reperfusion injuries, the translation of these findings into clinically applicable therapies has been disappointing. As revascularization therapies continue to improve, gain popularity, and increase their therapeutic time window, reperfusion injuries are expected to increase in frequency. Clinical therapeutic advances have been hampered by coexisting risk factors that can prevent activation of cell survival programs and the dual nature of many of the described pathophysiological cascades, making correct timing an issue in their application. It is more likely that combined approaches, with concomitant employment of revascularization treatment, antioxidant, neuroprotective, and vasoprotective agents, will yield satisfactory results and extend the time window for efficient ischemic stroke treatment for the benefit of an expanding proportion of the aging population at risk for stroke.
ADP, adenosine diphosphate; AIF, apoptosis-inducing factor; Akt, protein kinase
B; AMPARs,
AJ conceptualized the study; AJ and IAA analyzed the literature and synthesized it; AJ wrote the first draft of the paper. Both authors agreed on the submitted material.
Not applicable.
We thank the two anonymous reviewers for their observations and suggestions which helped improve the manuscript.
This research received no external funding.
The authors declare no conflict of interest.
Epidemiology of early neurologic deterioration (END)
Worsening of the neurological status early in ischemic stroke is a common finding with serious short- and long-term consequences for the patient. Initially termed “stroke in progression”, or “stroke in evolution”, the accurate definition of the term in various trials depends on the neurological scale used to quantify the neurological deficit:
The incidence of END varies in different trials, ranging between 13% and one-third of patients [195, 196, 197, 198] but has been reported by some researchers to be as high as 43% [199].
Predisposing factors for END are:
Mechanisms contributing to END may include the following, summarized in Table 1.
| Conditions leading to END | Frequency | Mechanisms leading to END |
| Hemorrhagic transformation | 10% | Oxidative stress, neuroinflammation, matrix metalloproteinases – leading to weakening of the blood brain barrier and destruction of the vascular tissue, allowing blood to spill into the infarcted tissue |
| Cerebral edema | 19% | Anaerobic metabolism leading to tissue acidosis, failure of ionic pumps, leading to cytotoxic edema; compression of the vasculature with further impairment of glucose and oxygen supply, propagating in a cascade, leading to increase in intracranial pressure, shifting of brain tissue, herniation syndromes |
| Failure of collaterals | Most frequent cause | Extended area of tissue with oxygen and glucose deprivation, increased oxidative stress, ischemic and neuroinflammatory cascades leading to tissue infarction |
| Arterial reocclusion | 34% | Reignition of the ischemic cascade, increased oxidative stress, neuroinflammation |
| Recurrent stroke | 11% | New areas of infarction, cerebral edema, oxidative stress |
| Prolonged seizures | 5% | Excitotoxicity, oxidative stress |
| General conditions, infections | Variable, depending on the cause |
However, as already mentioned, most clinicians ascribe END to ischemia/reperfusion injuries, which can lead to additional cerebral injury, weaken the blood-brain barrier (BBB) and cause hemorrhagic transformation, potentiate cerebral edema, and contribute to the “no-reflow phenomenon” in which despite clot removal, efficient microvascular perfusion is not achieved. Angiographic studies in ischemic stroke patients confirmed the lack of reperfusion, although the patency of large vessels was restored [227].