Ischemic Stroke: An Underestimated Complication of COVID-19

The coronavirus disease 2019 (COVID-19) has spread rapidly as a pandemic around the world. In addition to severe acute respiratory syndrome, more and more studies have focused on the complication of COVID-19, especially ischemic stroke. Here, we propose several pathophysiological processes and possible mechanisms underlying ischemic stroke after COVID-19 for early prevention and better treatment of COVID-19-related stroke.

diagnosis exhibited neurological manifestations, and 4 in 88 cases (4.5%) with severe COVID-19 were affected by ischemic stroke, which was a higher frequency than that in mild cases (1 in 126 cases, 0.8%) [5]. Similarly, a higher proportion(67%) of cases with COVID-19 suffered from neurological feature in France, and this group revealed the association between disease severity and ischemic stroke incidence: 3 of 13 patients (23%) with severe COVID-19 had ischemic strokes [6].

Younger patients are also victims
Five cases of large-vessel stroke in patients younger than 50 years of age following SARS-CoV-2 infection have been reported in Mount Sinai Health System in New York City [7]. Increasing evidence suggests that ischemic stroke may occur after SARS-CoV-2 infection, even in young patients [8][9][10][11].

Acute ischemic stroke is the most common type of cerebrovascular diseases after COVID-19
Recently, Nannoni et al. reviewed published articles on acute cerebrovascular diseases (CVD) after COVID-19 (December 2019-September 2020) and found that among 108,571 patients with COVID-19, 1.4% of patients suffered from acute CVD. The most common manifestation in CVD was acute ischemic stroke (87.4%) [9,12]. Ischemic stroke could be the first manifestation after SARS-CoV-2 infection [8]. The incidence of COVID-19-related hemorrhagic stroke did not differ from that of non-COVID-19-hemorrhagic stroke [13].

COVID-19-related stroke has low incidence but poor outcome
As mentioned above, although the incidence of acute ischemic stroke was relatively low during hospitalization due to COVID-19, ranging from 1% to 6%, the mortality associated with it is substantially high, reaching as high as 38% [14,15]. Multivariate analysis demonstrated that patients with COVID-19 have more severe strokes and poorer outcomes compared with non-COVID-19 stroke [16]. Without other risk factors, ischemic stroke was an uncommon complication, exclusive of patients with a severe pulmonary injury. The presence of COVID-19 in patients who underwent EVT was an independent predictor of in-hospital mortality [14]. The presence of COVID-19 has been proved as an independent predictor of in-hospital mortality in ischemic stroke patients who underwent endovascular treatment [14]. These cases confirmed the connection between COVID-19 and ischemic stroke. Therefore, we conducted a commentary to investigate the possible pathophysiology of ischemic stroke after SARS-CoV-2 infection.

Pathways of SARS-CoV-2 to invade the brain
The SARS-CoV-2 nucleotide sequence is 82% identical with that of human SARS-CoV and 50% identical with that of MERS-CoV [17][18][19]. SARS-CoV-2 has been identified in cerebrospinal fluid by polymerase chain reaction [20]. Several studies have suggested that SARS-CoV-2, like most coronaviruses, is neurotropic [21]. SARS-CoV-2 can plausibly invade the brain via several routes. First, SARS-CoV-2 binds to the angiotensin converting enzyme 2 (ACE2) receptor on the endothelial cells, which comprise the main component of the bloodbrain barrier (BBB). SARS-CoV-2 transport across the vascular endothelium impairs the BBB and further enables virus invasion into the brain, where SARS-CoV-2 interacts with ACE2 on the surface of neurons, causing damage to the nervous system [22]. Second, SARS-CoV-2 passes across the BBB through adhesion to and subsequent infection of ACE2-expressing leukocytes, termed the Trojan horse mechanism [23]. Third, SARS-CoV-2 binds to the ACE2 receptor on the olfactory epithelium in the nasal cavity and invades the brain through the sieve plate near the olfactory bulb. This route is supported by observations of isolated anosmia and ageusia with or without respiratory symptoms [24,25].

ACE2, the door for SARS-CoV-2 to enter the body
Recently, ACE2 was identified as the receptor for SARS-CoV-2 that caused the COVID-19 pandemic. SARS-CoV and SARS-CoV-2, which both can bind to ACE2 receptors, have 76% homology in amino acid sequence [26], but the affinity of ACE2 for SARS-CoV-2 is 10-20 times higher than that of SARS-CoV, which explains why SARS-CoV-2 is more infectious. In the brain, ACE2 is expressed in several cell types, especially in cerebral vascular endothelial cells [27]. As shown in Figure 1, Following SARS-CoV-2 binds to ACE2, the membrane receptor ACE2 is functionally removed from the outer membrane, resulting in the downregulation of ACE2 surface expression [28]. Therefore, the ACE2-Ang-(1-7)-Mas receptor axis is substantially weakened, whereas the ACE-angiotensin II (Ang II)-Ang II receptor 1 (AT1R) axis mediating vasoconstriction, neuroinflammation, oxidative stress, apoptosis, and cell proliferation functions is relatively strengthened. In addition to protecting the brain from inflammation, apoptosis, and oxidative stress, Ang-(1-7) and the Mas receptor can also reduce platelet proliferation and glycoprotein VI activation by increasing nitric oxide (NO) and prostacyclin, thereby inhibiting thrombosis [29].
Taken together, SARS-CoV-2-mediated loss of ACE2 impairs endothelial cell function, leading to the occurrence or worsening of acute ischemic stroke [30]. Angiotensin converting enzyme 2 (ACE2), a dipeptidyl carboxypeptidase and a homolog of ACE, is an essential negative regulator of the renin-angiotensin system. Renin cleaves angiotensinogen (AGT) into angiotensin I (Ang I), which is hydrolyzed by ACE to angiotensin II (Ang II). Ang II has a high affinity to Ang II receptor 1 (AT1R) and plays a major physiological role in mediating vasoconstriction, neuroinflammation, oxidative stress, apoptosis, and cell proliferation, known as the classic ACE-Ang II-AT1R axis. ACE2 cleaves Ang II into Ang-(1-7), which effectively binds to the Mas receptor and counteracts adverse effects of the ACE-Ang II-AT1R axis, known as the ACE2-Ang-(1-7)-Mas receptor axis.  [31,32] and H1N1 influenza [33,34]. The thrombotic incidence appears higher in COVID-19, even with the utilization of thromboprophylaxis [35]. A study assessed hospitalized patients with COVID-19 in a New York city health system demonstrated that thrombotic events occurred in 16.0% patients [36].

COVID-19 and thrombosis
The incidence of venous thrombotic events is higher than arterial. A pooled analysis including thirty-five observational studies, showed that the pooled incidence of VTE was up to 41.9% and the pooled incidence of arterial thrombosis was 11.3% [37]. Klok et al. studied 184 ICU patients with COVID-19 and showed that PE was the most frequent thrombotic complication (81%) [38]. As to arterial thrombotic events, Modin et al found that 44 and 17 in 5119 COVID-19 patients suffered from ischemic stroke and AMI, respectively [39].
Higher incidence of thrombotic events was observed in COVID-19 patients with a severe condition [36,40]. Bilaloglu et al reported 244 in 829 ICU patients (29.4%) and 289 in 2505 non-ICU patients (11.5%) underwent a thrombotic event [36]. COVID-19 patients diagnosed with thrombotic complications were at higher risk of allcause death (HR 5.4; 95% CI 2.4-12) [41]. Some researchers pointed out that PE was the direct cause of death behind COVID-19 [42]. In severe COVID-19 patients, the significant increase of D-dimer is a good indicator for identifying high-risk group of thrombotic complications [43], especially for PE [44]. Increased Ddimer concentrations of greater than 1.0 μg/ml effectively predict the risk of VTE in COVID-19 patients [45]. Ddimer level-guided aggressive thromboprophylaxis treatments in patients with COVID-19 has essential clinical value [46]. COVID-19 is an endothelial disease, in the end. Small fibrinous thrombi were observed in small pulmonary vessels in areas of both damaged and more preserved lung parenchyma from severe COVID-19 patients [47][48][49]. Two studies involved 10 and 11 decedents both found thrombosis and microangiopathy in the small vessels and capillaries of the lungs, which may lead to death [50,51]. The high incidence of thrombotic events suggests an important role of SARS-CoV-2induced endothelial injury.
There are three triggers for thrombosis: hypercoagulability, endothelial damage, and abnormal hemodynamics.
SARS-CoV-2 infection and inflammatory reactions lead to vascular damage, hypercoagulability, thrombin activation, platelet aggregation, as well as plaque shedding due to hemodynamic changes, thereby promoting the occurrence of ischemic stroke.
The potential thrombosis pathogenesis behind COVID-19-related stroke As described above, many papers have reported that patients with COVID-19 are at increased risk of thrombosis, which results in COVID-19-related stroke. We summarize several pathogeneses and propose the potential mechanisms.

Coagulation dysfunction
Most patients with COVID-19, especially severe cases and critically ill patients, have varying degrees of coagulation dysfunction. Abnormal coagulation parameters are predictors of poor prognosis in COVID-19 patients [52]. Patients with COVID-19-related stroke have significantly higher D-dimer levels and blood viscosity than those with stroke alone [53]. A retrospective study of 191 COVID-19 patients in Wuhan demonstrated that 42% of patients with COVID-19 and 81% of deceased patients with COVID-19 had D-dimer levels higher than 1.0 μg/mL [54]. Chen et al. reported that 36 of 99 (36%) non-severe COVID-19 patients exhibited elevated D-dimer levels [55]. Tang et al. reported that non-survivors had significantly higher Ddimer and fibrin degradation products (FDP) than survivors. In the late stage of the disease, 71.4% of nonsurvivors and 0.6% of survivors had different degrees of disseminated intravascular coagulation, due to the excessive consumption of coagulation factors [56]. These findings suggest that coagulation disorders in COVID-19 patients are closely related to the severity of the disease.
Coagulation and anticoagulation systems include platelet thrombus formation, fibrin formation, and fibrinolysis. Under physiological conditions, anticoagulation is the main factor that ensures normal blood flow. Upon vascular endothelium damage, the subendothelial collagen is exposed to blood, thereby activating the coagulation system [57], resulting in a large amount of thrombin generation and fibrin deposition, which is the key factor of hypercoagulability. We suspect five mechanisms behind SARS-CoV-2-mediated coagulopathy (Fig. 2).
SARS-CoV-2 directly damages endothelial cells in various organs [58]. Autopsies have shown that SARS-CoV-2 virus particles are present in capillary endothelial cells, resulting in the disruption of their tight junctions, cell swelling, and loss of contact with the basement membrane, and endotheliitis [58,59]. After a vascular endothelial injury, the subendothelial collagens are exposed and release tissue factor (TF), triggering an exogenous coagulation cascade that leads to thrombin production and fibrin deposition [60].

Figure 2. Mechanisms of coagulation dysfunction triggered by SARS-CoV-2.
(2) Excessive cytokines activate coagulation. Excessive production and release of cytokines such as tumor necrosis factor (TNF), interleukin (IL)-1, and IL-6 are characteristic of COVID-19. TNF and IL-1 promote blood coagulation by inhibiting the expression of thromboregulatory protein (TM) and reducing the production of activated protein C (APC), thus promoting the coagulation reaction [61,62]. TM is expressed on the surface of endothelial cells and forms a thrombinthrombomodulin complex, inhibiting the activity of thrombin. Moreover, the complex can also activate APC. APC is a strong anticoagulant that promotes the release of tissue-type plasminogen activator, thereby activating the fibrin dissolution system and inactivating tissue plasminogen activator inhibitor [62].
IL-6 stimulates fibrinogen synthesis in the liver and induces megakaryocytes to produce a large number of platelets, both of which can promote the coagulation reaction. IL-6 can also stimulate monocytes to release TF, thereby initiating the exogenous coagulation cascade [63].
Thrombosis is one of the ways in which innate immunity strives to reduce the spread of infection [64]. After infecting the human body, SARS-CoV-2 is recognized by pathogen-associated molecular patterns (PAMP) receptors like Toll-like receptors (TLRs), initiating innate immune system. In addition, the complement system is activated after SARS-CoV-2 infection. C3a and C5 cleavage products (C5a and C5b) collectively influence many aspects of coagulation, including the activation of TF, the release of von Willebrand factor (vWF) from endothelial cells, and enhancement of P-selectin exposure [65].
Peripheral lymphocyte counts (mainly CD4+ and CD8+ T cells) were consistently and significantly reduced in COVID-19 patients, especially in severe cases, suggesting that the adaptive immunity is also weakened [54,66]. The degree of lymphopenia is correlated with the severity of COVID-19 [67]. SARS-CoV-2 can directly invade lymphocytes, particularly T cells, resulting in a compromised antiviral response due to a reduction in the number of lymphocytes [66]. However, it was demonstrated that although the counts of peripheral T cells were substantially reduced, their status was highly pro-inflammatory, manifested by increased numbers of Th17 cells and high cytotoxicity of CD8 T cells [48]. Dysregulation of T cell subsets can also increase the secretion of negative hematopoietic regulators (IL-2, interferon [IFN]-γ, and TNF), leading to coagulation dysfunction.
(4) Neutrophils are involved in the activation of the coagulation cascade.
Stimulated by inflammatory cytokines or infectious pathogens, neutrophil extracellular traps (NETs) are released from neutrophils to capture pathogens [68]. This fibrous network provides a scaffold for platelets to adhere to and capture erythrocytes and leukocytes, leading to platelet aggregation and thrombosis. Autopsies of deceased COVID-19 patients revealed neutrophil infiltration in pulmonary capillaries [68] and the level of NETs in the blood of COVID-19 patients is increased [69]. NETs can directly activate factor XII and TF and initiate an exogenous coagulation cascade. Moreover, histones in NETs also function as ligands for platelet TLRs to promote platelet activation and thrombosis [70].
Extramedullary megakaryocytes are present in the microvessels of most organs. The number of pulmonary megakaryocytes is increased by infection, impairing the respiratory system and the circulatory system [71]. Approximately 90% of lung-derived megakaryocytes are present in the pulmonary microcirculation. However, in cases of severe infection, a large proportion of megakaryocytes leave the lungs and enter the arterial circulation [72]. Megakaryocytes in the peripheral circulation can produce platelets, resulting in microvascular thrombosis in COVID-19 patients.
In summary, coagulation dysfunction increases the risk of thrombosis in COVID-19 patient.
The cytokine storm, also called hypercytokinemia, is the phenomenon of the aggressive release of proinflammatory cytokines and insufficient control of antiinflammatory responses due to immune dysfunction [75]. Under normal circumstances, the immune system can respond to external stimuli by secreting cytokines, helping the body to overcome the attack by pathogens. Following SARS-CoV-2 invasion, the virus is first recognized by innate immune system and activates immune cells, such as macrophages and dendritic cells (DCs), which can phagocytose and hydrolyse the virus [76]. Macrophages can release TNF-α in an endocrine manner, and dendritic cells secrete IL-12 and IL-6 in a paracrine manner [77]. Once the virus escapes recognition by the innate immune system, it is recognized by cell surface RNA pattern recognition receptors and form a protein complex, which promotes the translocation of transcription factors and upregulates the expression of pro-inflammatory factors. The release of cytokines facilitates the recruitment of more immune cells and secretion of more cytokines, thereby forming a positive feedback loop to continuously amplify the inflammatory response. This positive feedback results in two outcomes: 1) Pro-inflammatory cytokines are massively produced, disrupting the balance between pro-and anti-inflammatory cytokines. 2) Due to excessive response by the immune system, the cytokines start to attack healthy tissues rather than the virus, causing a systemic inflammatory response.
We hypothesize three possible mechanisms underlying cytokine storm-induced strokes after SARS-CoV-2 infection (Fig. 3). 1) Endothelial cell dysfunction leads to a disruption of the BBB. Brain endothelial cells express TNF-α and IL-6 receptors that may mediate local cell dysfunction [78], inducing the rupture of the BBB. Therefore, the integrity of the BBB is altered with increased permeability, leading to an elevated concentration of pro-inflammatory cytokines in the brain parenchyma [79]. 2) Excessive cytokines directly cause local neuronal necrosis. Enhanced permeability of the BBB enables excessive cytokine levels in the brain parenchyma, directly cause local neuronal necrosis. Necrosis may also recruit more infiltrating inflammatory cells around neurons to induce a stroke. 3) Injured endothelial cells induce coagulation and fibrinolysis imbalance. The injured endothelial cells induced by cytokines, in turn, release inflammatory factors in excess. This positive feedback promotes platelet activation and fibrinogen deposition, as well as inhibit fibrinolysis and thrombomodulin activity, leading to the imbalance of coagulation and fibrinolysis. The endothelial cells transform from an anti-to a procoagulant state, thereby facilitating thrombosis.
Among aPLs, LAC, the complex of aPL and plasma proteins (mainly β2-glycoproteins), is the greatest risk factor for arterial and venous thrombosis [83] and β2GPⅠ is the main binding cofactor for these antibodies [84]. Patients positive for LAC, aCL, and anti-β2GPⅠ antibodies, called triple-positive patients, have incidence rates for VTE of 9.8% (after 2 years) and 37.1% (after 10 years) during the follow-up period [85].
Following ROS stimulation, free thiols of β2GPⅠ form disulfide bonds, and the ring conformation of β2GPⅠ unfolds, exposing the normally shielded epitopes, thereby inducing autoantibody formation [88]. Thus, a positive feedback is formed to further promote oxidative stress. Increased levels of oxidative stress mediate damage to cell structures and serve as a trigger for ischemic stroke [89]. 2) Changes in coagulation factors: aPLs upregulate the release of TF, which is the key promoters of the exogenous coagulation cascade, from monocytes [86], neutrophils [87] and endothelial cells [90]. Furthermore, aPLs increase the level of coagulation factor Ⅺ containing free thiols (reduced factor Ⅺ), which is more prone to be activated by thrombin, factor Ⅻa, or factor Ⅺa compared to its unreduced form [91], participating in the endogenous coagulation pathway. LAC activates thrombin by promoting the binding of prothrombin to surface phospholipids and affecting its affinity [92]. 3) Platelet activation: Anti-β2GPⅠ cross-links vWF receptor glycoprotein Ⅰbα and ApoE receptor 2 to enhance platelet activation, promote thromboxane A2 (TXA2) release, and increase platelet adhesion [93]. Platelet activation initiates thrombus formation [94]. 4) Complement activation: Complement activation is involved in antiphospholipid antibody-induced thrombosis. Activation of complement by aPL generates C3a and C5a, which mediate leukocyte adhesion and thrombus formation, [95,96]. 5) Inhibition of endothelial nitric oxide synthase (eNOS): By inhibiting eNOS activity and reducing bioavailable NO, aPL promotes leukocyte-endothelial cell adhesion and thrombosis [97]. Autopsy reports of patients with COVID-19 revealed infiltration of leukocytes in small pulmonary vessels; therefore, we hypothesized that aPL also promotes leukocyte adhesion in cerebral vessels [58]. 6) Inhibition of annexin A5: AnnexinV is a calciumdependent protein that binds to phosphatidylserine residues, which form a shield that inhibits the formation of procoagulant complexes, including the TF-Ⅶa complex, the Ⅸa-Ⅷa complex, and the Ⅹa-Ⅴa complex [98]. The complex of anti-β2GPⅠ and β2GPⅠ can disrupt this shield, exposing procoagulant phosphatidylserines and, hence, promote thrombosis [99].
SARS-CoV-2 triggers the production of aPL by molecular mimicry and increased levels of cytokines [100]. It is reasonable to speculate that aPL serves as a potential mediator of cerebrovascular events in patients with COVID-19.

Abnormal ferritin metabolism
Ferritin was proven to be a predictor of mortality among 150 COVID-19 patients in Wuhan, since its level is significantly higher in non-survivors than in survivors [101]. Zhou et al. found that COVID-19 patients with elevated serum ferritin levels (>300 µg/L) had a 9-fold increase in pre-discharge mortality [54]. Subsequently, a growing number of researchers have recognized serum ferritin as a powerful index for COVID-19 severity, helping to identify cases with dismal prognosis [102,103]. The first reported case of COVID-19-related stroke showed that the patient's serum ferritin level was markedly elevated [104], which has attracted researchers' attention to the role of serum ferritin in COVID-19-related stroke.
Ferritin is an iron-binding molecule that stores major intracellular iron in all organisms [104]. A higher ferritin level is associated with an increased risk of ischemic stroke [105,106]. Ruddell et al. proposed the role of serum ferritin (mainly composed of L-ferritin subunits) as a proinflammatory signaling molecule in hepatic stellate cells [107]. Ferritin is involved in inflammatory/fibrotic states associated with the infection of various organs, such as the heart, lung, brain, kidney, and pancreas, all of which have cell types similar to hepatic stellate cells to mediate the fibrotic response to injury [107,108]. Serum ferritin can initiate the production of thrombus-like fibers through a variety of pathways and induce inflammation, thereby damaging cerebral blood vessels and causing neurological symptoms.

Treatment of COVID-19-related stroke
Multidisciplinary treatment of COVID-19-related stroke, including antiviral drugs, supportive therapy, and stroke treatment, has shown cheerful clinical effects. Alharthy et al. have shown that in life-threatening COVID-19, especially with immune dysregulation features such as antiphospholipid antibodies, therapeutic plasma exchange could be an effective rescue therapy [109]. Similar to stroke alone, treatment for ischemic stroke in COVID-19 patients is individualized and complicated, including thrombolysis [110,111] and thrombectomy [112,113] for acute interventions and antiplatelets and anticoagulants for secondary prevention. It should be noted that many patients with acute ischemic stroke lose the opportunity for acute intervention because of isolation and reluctance to present to the hospital [114,115]. The treatment efficacy for ischemic stroke following COVID-19 needs further evaluation. Ischemic stroke therapy in COVID-19 patients should not only be based on traditional guidelines, but on the experience and new insights from healthcare workers who are combating COVID-19 worldwide [116].

Conclusions
Despite the characteristic symptoms of respiratory distress, less than 2% of hospitalized patients with COVID-19 have an ischemic stroke [9,14,15,117]. The exact stroke pathophysiology following SARS-CoV-2 infection remains to be established by autopsies and pathology reports. Based on current knowledge, several possible mechanisms exist, including hypercoagulability, activation of the cytokine storm, excessive levels of antiphospholipid antibodies, and abnormal ferritin levels. During the lockdown period, factors such as lifestyle changes and sedentary lifestyle must be taken into consideration when assessing stroke risks [118]. It is unclear whether the patients who recover from COVID-19-related stroke experience any transient or long-lasting cerebrovascular sequelae. Clinical physicians should be aware that severe COVID-19 can lead to ischemic stroke, and close monitoring of the neurological status of COVID-19 patients is imperative.