SARS-CoV-2 Spike Protein Exacerbates Thromboembolic Cerebrovascular Complications in Humanized ACE2 Mouse Model

SARS-CoV-2 Spike Protein Exacerbates Thromboembolic Cerebrovascular Complications in Humanized ACE2 Mouse Model | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article SARS-CoV-2 Spike Protein Exacerbates Thromboembolic Cerebrovascular Complications in Humanized ACE2 Mouse Model Stan P. Heath, Veronica C. Hermanns, Maha Coucha, Mohammed Abdelsaid This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4649614/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 Oct, 2024 Read the published version in Translational Stroke Research → Version 1 posted 7 You are reading this latest preprint version Abstract COVID-19 increases the risk for acute ischemic stroke, yet the molecular mechanisms are unclear and remain unresolved medical challenges. We hypothesize that the SARS-CoV-2 spike protein exacerbates stroke and cerebrovascular complications by increasing coagulation and decreasing fibrinolysis by disrupting the renin-angiotensin-aldosterone system (RAAS). A thromboembolic model was induced in humanized ACE2 knock-in mice after one week of SARS-CoV-2 spike protein injection. hACE2 mice were treated with Losartan, an angiotensin receptor (AT 1 R) blocker, immediately after spike protein injection. Cerebral blood flow and infarct size were compared between groups. Vascular-contributes to cognitive impairments and dementia was assessed using a Novel object recognition test. Tissue factor-III and plasminogen activator inhibitor-1 were measured using immunoblotting to assess coagulation and fibrinolysis. Human brain microvascular endothelial cells (HBMEC) were exposed to hypoxia with/without SARS-CoV-2 spike protein to mimic ischemic conditions and assessed for inflammation, RAAS balance, coagulation, and fibrinolysis. Our results showed that the SARS-CoV-2 spike protein caused an imbalance in the RAAS that increased the inflammatory signal and decreased the RAAS protective arm. SARS-CoV-2 spike protein increased coagulation and decreased fibrinolysis when coincident with ischemic insult, which was accompanied by a decrease in cerebral blood flow, an increase in neuronal death, and a decline in cognitive function. Losartan treatment restored RAAS balance and reduced spike protein-induced effects. SARS-CoV-2 spike protein exacerbates inflammation and hypercoagulation, leading to increased neurovascular damage and cognitive dysfunction. However, the AT 1 R blocker, Losartan, restored the RAAS balance and reduced COVID-19-induced thromboembolic cerebrovascular complications. SARS-CoV-2 spike protein RAAS balance COVID-19-induced thromboembolic complications Vascular-contributes to cognitive impairments and dementia Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The Severe Acute Respiratory Syndrome Corona virus-2 (SARS-CoV-2) is a single-stranded RNA virus responsible for the COVID-19 pandemic. As of 2024, SARS-CoV-2 had killed over a million Americans. COVID-19 causes acute respiratory distress syndrome (ARDS) [ 1 , 2 ]. ARDS symptoms include severe pneumonia, hypoxemia, and elevated cytokines, known as a cytokine storm. Together, these can result in higher mortality rates, especially in patients with a concomitant cardiovascular disorder such as hypertension, diabetes, or obesity. Many studies reported that COVID-19 causes neurological symptoms [ 3 , 4 ]. COVID-19-induced neurological symptoms range from headaches and loss of taste and smell to encephalopathy, cognitive impairments, and stroke [ 5 ]. The mechanisms of these neurovascular disorders are unclear and represent a knowledge gap. COVID-19 caused life-threatening coagulopathies such as stroke and deep vein thrombosis [ 6 ]. Several reports indicate that these negative thrombotic events are associated with up to one-third of COVID-19 patients [ 7 – 9 ]. The molecular mechanisms behind increased hyper-coagulopathies and impaired fibrinolysis are unclear and require further investigation [ 10 ]. The development of vaccination against the SARS-CoV-2 virus has decreased mortality rates. However, long-term COVID-19-induced hyper-coagulopathy and cognitive dysfunction represent a significant medical challenge that requires intensive studies [ 11 ]. Angiotensin-converting enzyme-2 (ACE-2) is an enzyme that plays a crucial role in the RAAS balance. ACE-2 degrades angiotensin II, the bioactive form, which can bind to the angiotensin type receptor 1 (AT 1 R) or the angiotensin type receptor 2 (AT 2 R). AT 1 R is more abundant than AT 2 R, and its activation leads to physiological effects such as vasoconstriction, proinflammatory, thrombosis, and generation of reactive oxygen species [ 12 – 14 ]. Activation of the Ang II/AT 1 R axis pathway increases hyper-coagulations via upregulation of the tissue factor (TF), thrombin activation, and platelet aggregation [ 15 , 16 ]. In contrast, Ang II/AT 2 R axis opposes the effects of AT 1 R activation through vasodilation and anti-inflammatory responses and maintains hemostasis [ 17 – 19 ]. ACE-2 binds to the SARS-CoV-2 spike protein and acts as a functional receptor for the spike protein to internalize the viral particle [ 20 ]. SARS-CoV-2 downregulates the ACE-2 receptor [ 4 ], which decreases Ang II degradation, therefore increasing the RAAS destructive arm with increased activation of the abundant AT 1 R over AT 2 R [ 21 , 22 ]. This study aims to investigate one of the possible mechanisms by which SARS-CoV-2 Spike protein alters the coagulation and fibrinolysis hemostasis, leading to stroke and neurovascular complications. We hypothesize that the SARS-CoV-2 spike protein exacerbates stroke and neurovascular complications by increasing coagulation and decreasing fibrinolysis via disrupting the RAAS balance. We examined the effect of SARS-CoV-2 spike protein in a mouse model of humanized ACE-2 knock-in (hACE2 KI) mice. Spike protein was injected for seven days before the induction stroke. Stroke is induced through distal middle cerebral artery (MCA) thromboembolism using ferric chloride (FeCl 3 ). We examined cerebral blood flow, infarct-sized and vascular contribution to cognitive impairments, and dementia (VCID) in stroked animals after one week of SARS-CoV-2 spike protein injection. Our previous studies showed that SARS-CoV-2 spike protein disrupts RAAS balance and increases brain inflammation [ 4 ]. Here, our results provide novel evidence that SARS-CoV-2 Spike protein increased coagulation and decreased fibrinolysis in hACE2 KI mice. These effects were accompanied by decreased cerebral blood flow, increased neuronal death, and increased cognitive dysfunctions. Our results showed that using the AT 1 R blocker, Losartan restored the RAAS balance and reduced COVID-19-induced thromboembolic cerebrovascular complications. Materials and Methods Animals Transgenic humanized ACE2 KI mice (hACE2 KI), B6.129S2 (Cg)-Ace2 tm1(ACE2)Dwnt /J mice were purchased from Jackson Laboratory (Jax lab: stock no. 035000, Ellsworth, Maine, USA). hACE2 KI mice have human ACE2 cDNA replacing the endogenous mouse Ace2 sequences. The endogenous Ace2 regulatory elements direct expression of human ACE2, the receptor used for cellular entry by several coronaviruses, including severe acute respiratory syndrome coronavirus-1 (SARS-CoV) and − 2 (SARS-CoV-2) [ 4 , 23 ]. ARRIVE guidelines 2.0 were followed in conducting animal studies in accordance with the ethical guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Mercer University Institutional Animal Care and Use Committee (IACUC) is accredited by the American Association for Accreditation of Laboratory Animal Care, and has approved the animal protocols. A standard mice chow diet was provided to the animals, as was unlimited access to tap water. Twelve-hour cycles of light and dark were used for the mice. Animal Treatment hACE2 mice were randomly assigned to four groups: 1) sham, 2) stroke, 3) stroke + SARSCoV-2 spike protein injection, and 4) stroke + SARSCoV-2 spike protein injection + Losartan. A recombinant protein for SARS-CoV-2 nucleoprotein/spike protein (4ug/animal, Invitrogen, USA, Cat. No. RP-87706) was injected intravenously via jugular vein injection 7 days prior to MCA/FeCl 3 thromboembolic surgery. Sham animals were exposed to the MCA/FeCl 3 thromboembolic surgery without FeCl 3 treatment. Losartan (10 mg/kg body weight, Tokyo Chemical Industry, Tokyo, Japan, Cat. No. L0232) was added to the water supply of the animal cages. Losartan was first administered immediately after the injection of the recombinant spike protein for SARS-CoV-2. MCA/FeCl 3 Induced Thromboembolic Model Eight-week-old male hACE2 KI mice weighing 20-25g were randomly assigned for surgery. Mice were anesthetized using a mixture of Ketamine and Xylazine intraperitoneal (IP) injection (100mg/kg ketamine and 10 mg/kg Xylazine). Long-acting Buprenorphine (0.1 mg/kg) IP injection was also given before surgery to decrease postoperative pain. A vertical incision was made between the eye and ear to locate the temporal muscle. A horizontal incision is then made on the lateral ridge of the frontal bone, and a vertical incision from the rostral-most point of the parietal bone is made to expose the skull. The distal trunk of the middle cerebral artery is then located and exposed by drilling through the mouse skull. The meninges were removed to expose the artery, and the transient focal ischemia model was initiated by using fresh FeCl 3 (20%) soaked filter paper for five minutes. After removal, sterile warm saline was applied to the area for 10 minutes and then dried with sterile gauze. The temporal muscle was then placed back, the mouse was sutured, and the surgical area was disinfected with a povidone-iodine solution. The mouse was then monitored under a heat lamp until recovery. Assessment of Cerebral Blood Flow Cerebral blood flow was measured with the RFLSI Ⅲ Laser Speckle Imaging System (RWD, San Diego, CA, USA). Mice were anesthetized using isoflurane, and a vertical incision was made from the sagittal suture to the interferential suture on the mouse’s skull. RWD laser speckle imaging was used to record the cerebral blood flow. LSCI software (Version 5.0) was used to measure the perfusion per area at hours 1, 2, 3, 6, and 24. Perfusion was represented as a percentage ratio of the stroke to the non-stroke hemisphere of the brain. Assessment of Infarct Volume hACE2 mice brains were collected 24 hours after MCA/FeCl 3 thromboembolic model surgery. These brains were sectioned into 5 coronal 2mm thick slices using an acrylic brain matrix. Coronal brain slices were examined by staining with 2% 2,3,5-triphenyl tetrazolium chloride (TTC) and incubating for 15 min in a humidified incubator (37%C, 5% CO2). Sections were photographed with units of measure to be quantified blindly using Image-J software. Assessment of Cognitive Functioning Memory and learning functions were assessed at baseline, before SARS-CoV-2 spike protein injection, and 24 hours after MCA/FeCl 3 thromboembolic surgery using Novel Object Recognition testing (NOR) [ 24 , 25 ]. The NOR test is a 3-stage test used to examine recognition memory. The first stage involves habituation, with the mouse being exposed to its testing environment. The second is the familiarization stage, which involves the mouse being exposed to two identical objects. The final phase is the testing phase, which involves the replacement of one of the familiar objects with a novel object. The testing phase is recorded using ANY-maze software. Time spent around the novel object, the ratio of time spent investigating the novel object to the familiar object, and the total distance traveled were measured using the ANY-maze software recording and analyzed blindly. Cell Culture Primary human brain microvascular endothelial cells (HBMECs, from Angio-Proteomie, Boston, MA, USA, Cat. No. cAP-0002) were grown to confluency in complete media (MCDB-131 Complete, VEC Technologies, Inc., Rensselaer, NY, USA). HBMECs were switched to serum-reduced media and treated with SARS-CoV-2 recombinant spike protein (100 µM) with or without losartan (100 µM) for 24 hrs. The Next day, HBMECs were either kept under normoxia conditions (room air 21% oxygen) or placed in a hypoxia chamber (0.1% oxygen, BioSpherix, ProOx Model 110) for 6 hours. Cells were collected immediately once removed from the hypoxia chamber. Polymerase Chain Reaction RT-PCR was performed as described previously [ 4 ]. RNA was isolated using Triazole (Thermo-Fisher, USA, Cat. No. AC345480250) and quantified using Thermo Scientific NanoDrop 2000C Spectrophotometer (Thermo Scientific, USA). cDNA was prepared from isolated RNA using SuperScript IV VILO Master mix with an ezDNase kit (Invitrogen, USA, Cat. No. 11766050). The Quant Studio™ 3 Real-Time PCR System (Applied Biosystems, Thermo Scientific, USA) was utilized to run qRT-PCR using PowerUp SyBR Green Master mix (Applied Biosystems, Thermo Scientific, USA, Cat No. A25742). All primer sequences used are detailed in (Table 1 ). GAPDH was consistently used as the reference gene for normalization. Table 1 PCR primers. Primer Species FWD sequence REV sequence ACE2 Human 5’-TCC ATT GGT CTT CTG TCA CCC G-3’ 5’-AGA CCA TCC ACC TCC ACT TCT C-3’ Mouse 5’-TCC ATT GGT CTT CTG CCA TCC G-3’ 5’-AGA CCA TCC ACC TCC ACT TCT C-3’ AT 1 R Human 5’-CAG CGT CAG TTT CAA CTT GTA CG-3’ 5’-GCA GGT GAC TTT GGC TAC AAG C-3’ Mouse 5’-GCC ATT GTC CAC CCG ATG AAG T-3’ 5’-ACA CAT TTC GGT GGA TGA CGG C-3’ AT 2 R Human 5’-CCA TGT TCT GAC CTT CCT GGA TG-3’ 5’-CGG ATT AAC GCA GCT GTT GGT G-3’ Mouse 5’-CGT GAC CAA GTC CTG AAG ATG G-3’ 5’-GGA AGT GCC AGG TCA ATG ATG ACT G-3’ GAPDH Human 5’-GTC TCC TCT GAC TTC AAC AGC G-3’ 5’-ACC ACC CTG TTG CTG TAG CCA A-3’ Mouse 5’-CAT CAC TGC CAC CCA GAA GAC TG-3’ 5’-ATG CCA GTG AGC TTC CCG TTC AG-3’ Il-1β Human 5’-CCA CAG ACC TTC CAG GAG AAT G-3’ 5’-GTG CAG TTC AGT GAT CGT ACA GG-3’ Mouse 5’-TGG ACC TTC CAG GAT GAG GAC A-3’ 5’-GTT CAT CTC GGA GCC TGT AGT G-3’ I-6 Human 5’-AGA CAG CCA CTC ACC CTCT TAC G-3’ 5’-TTC TGC CAG TGC CTC TTT GCT G-3’ Mouse 5’-TAC CAC TTC ACA AGT CGG AGG C-3’ 5’-CTG CAA GTG CAT CAT CGT TGT TC-3’ MASR Human 5’-CAG CAC CAT CTT GGT CGT GAA G-3’ 5’-CAG CAG GTA AAG GAG TCT CAT GG-3’ Mouse 5’-CTG ACA GCC ATC AGT GTG GAG A-3’ 5’-GTG GTC ACC AAG CAC GAA AGT G-3’ NFκB Mouse 5’-GCT GCC AAA GAA GGA CAC GAC A-3’ 5’-GGC AGG CTA TTG CTC ATC ACA G-3’ TNFα Human 5’-CTC TTC TGC CTG CTG CAC TTT G-3’ 5’-ATG GGC TAC AGG CTT GTC ACT C-3’ Mouse 5’-GGT GCC TAT GTC TCA GCC TCT T-3’ 5’-GCC ATA GAA CTG ATG AGA GGG AG-3’ Immunoblotting RIPA buffer (Millipore, Billerica, MA, USA, Cat# 3P 20188) was used to extract protein from the hACE2 brain and HBMECs. Equal protein loads were separated on 10% SDS-polyacrylamide gel utilizing the Mini PROTEAN Tetra Cell SDS-PAGE Gel electrophoresis kit (Biorad Laboratories Inc, Hercules, CA). Gels were transferred onto nitrocellulose membranes using the Bio-Rad Trans-Blot Turbo (Biorad Laboratories Inc, Hercules, CA, USA). Membranes were blocked with 5% milk and incubated overnight with primary antibodies. Membranes were washed and incubated with an appropriate horseradish peroxidase-conjugated secondary antibody. Membranes were reacted with Western chemiluminescent HRP Substrate (Millipore, USA) and imaged using the iBright Imaging system (Invitrogen Thermo Fisher Scientific, USA, Model FL1500). Image-J software (Version 1.54g) was used to measure band intensity. β-actin was used for normalization. All antibodies used are listed in (Table 2 ). Table 2 Antibodies Primary Antibody Company CAT/ Lot/ Prod Recommended Concentration Dilution Immunoblot Secondary Antibody AT 1 R Invitrogen PA5-20812 1 mg/mL 1:1000 Rabbit AT 2 R Novus NBP1-60097 1 mg/mL 1:1000 Rabbit Β-actin R&D MAB8929 1 mg/mL 1:1000 Mouse PAI-1 Biotechne MAB-17861-100 1 mg/mL 1:1000 Rabbit TNFα NOVUS NBP1-19532 1 mg/mL 1:500 Rabbit TF-III R&D MAB-3178 1 mg/mL 1:1000 Mouse Statistical Analysis GraphPad prism version 10 or higher was employed for all data analysis. For animal studies, the sample size was determined from our previous work [ 4 ]. One-way ANOVA was utilized to assess mean differences between 1) sham, 2) stroke, 3) stroke + SARS-CoV-2 spike protein injection, and 4) stroke + SARS-CoV-2 spike protein injection + Losartan. Significance was determined at P < 0.05. Data is presented as mean ± standard deviation. A Tukey’s post-hoc test was used to adjust for the multiple comparisons to assess significant interaction effects from all analyses. Results SARS-CoV-2 spike protein intensifies RAAS imbalance after ischemic insult. We assessed the effect of SARS-CoV-2 spike protein on the gene expression of both RAAS arms in hACE2 KI mice brain and human microvascular endothelial cells (HBMECs) subjected to ischemic insult. We analyzed the gene expression of ACE2 in brain homogenate. Moreover, we measured AT 1 R, AT 2 R, and MASR in both the contralateral and ipsilateral brain homogenate using qRT-PCR. Singh et al. showed that a transient MCA occlusion stroke model increases ACE2 expression in the lung of mice [ 26 ]. Here, we showed that the MCA/FeCl 3 transient stroke model increases the ACE2 expression in the brain of hACE2 KI mice (Fig. 1 .A). We previously showed that SARS-CoV-2 spike protein downregulated ACE2 expression in hACE2 KI mice brains [ 4 ]. In the current study, a similar finding was reported. SARS-CoV-2 spike protein downregulates ACE2 gene expression in hACE2 KI brains after the thromboembolic occlusion model (Fig. 1 .A). Losartan treatment restored ACE2 gene expression after SARS-CoV-2 spike protein injection with the thromboembolic occlusion model. Next, we evaluated the AT 1 R, AT 2 R, and MASR in both stroke and non-stroke hemispheres. Our results showed that ischemic insult increases the expression of AT 1 R by 2-fold. Pre-injection of SARS-CoV-2 spike protein before the ischemic insult further increases the expression of AT 1 R to 4-fold in the brain of hACE2 KI mice (Fig. 1 .B). In contrast, SARS-CoV-2 spike protein decreased the gene expression of the RAAS protective arms AT 2 R and MASR (Fig. 1 .C-D). These results were confirmed in HBMECs that were treated with SARS-CoV-2 spike protein and subjected to hypoxia to mimic the ischemic insult. Our results showed that SARS-CoV-2 spike protein increases AT 1 R expression and decreases AT 2 R expression under normoxia control conditions. The addition of ischemic insult further augments SARS-CoV-2 spike protein-induced effects. Losartan treatment reduced spike protein-induced effects. (Fig. 1 .E-F). Increased inflammation after a double hit, SARS-CoV-2 spike protein followed by ischemic insult exacerbate inflammation in hACE2 brains. Stroke is known to increase brain inflammation [ 27 ]. We have previously shown that intravenous injection of SARS-CoV-2 spike protein increases inflammation in the brain of hACE2 KI mice [ 4 ]. Here, we examined the effect of SARS-CoV-2 spike protein injection coincident with ischemic insult on brain inflammation. Our results showed that SARS-CoV-2 spike protein pre-injection significantly intensifies inflammation after induction of thromboembolic occlusion model. Ischemic insults increased inflammation in both ipsilateral and contralateral brain hemispheres. Pre-injection of SARS-CoV-2 spike protein significantly increased NF Ϗ B, TNF-α, Il-1β, and Il-6 gene expression in the brains of hACE2 KI mice. Losartan showed a modest but significant effect in reducing the SARS-CoV-2 spike protein-induced effect (Fig. 2 .A-D). Our results showed similar findings when we examined the impact of SARS-CoV-2 spike protein on HBMCE subjected to hypoxia. SARS-CoV-2 spike protein significantly increased the expression of TNF-α under normoxia conditions. Pretreatment of HBMECs with SARS-CoV-2 spike protein prior to hypoxia further significantly increased endothelial cell inflammation (Fig. 2 .E). SARS-CoV-2 spike protein disrupts coagulation homeostasis We examined the effect of SARS-CoV-2 spike protein on the prothrombotic state. The disruption of the endothelial matrix leads to the release of tissue factor III (TF-III), which activates the coagulation cascade, resulting in thrombin activation. Our results showed that the thromboembolic stroke model exacerbated TF-III protein expression in hACE2 KI mice brain homogenate. The injection of spike protein before stroke further escalated TF-III expression. (Fig. 3 .A-C). PAI-1 inhibits the conversion of plasminogen to plasmin by urokinase-plasminogen activator and tissue-type plasminogen activator (tPA), thereby hindering fibrin degradation. Our results showed that the thromboembolic stroke model increased PAI-1, and the expression of PAI-1 was further increased by the injection of the spike protein seven days before the stroke. (Fig. 3 .A-C). Similar findings were observed in the HBMECs when exposed to hypoxic conditions with and without SARS-CoV-2 spike protein. Spike protein favors clot formation by increasing coagulation and decreasing fibrinolysis. Losartan treatment decreased spike protein-induced hypercoagulation. (Fig. 3 . D-E). SARS-CoV-2 spike protein decreases cerebral blood flow following stroke. The thromboembolic occlusion was induced in hACE2 KI mice using the MCA/FeCl 3 model. SARS-CoV-2 spike protein was injected intravenously one week before stroke induction. Laser speckle imaging was employed to measure cerebral blood flow at 1, 2, 3, 6, and 24 hours post-surgery. Our results showed that the thromboembolic model decreased cerebral blood flow in the stroke hemisphere compared to the contralateral hemisphere. The spontaneous recanalization occurred, and cerebral blood flow was restored within six hours of the embolic model. The pre-treatment with SARS-COV-2 spike protein showed a significant decrease in cerebral blood flow and increased vascular recanalization time to over 6 hrs. Treatment with Losartan helped in faster restoration of cerebral blood flow and decreased recanalization time compared to spike protein. (Fig. 4 .A-B). SARS-CoV-2 spike protein increases infarction volume following stroke. ACE2 KI mice were injected with SARS-CoV-2 spike protein one week before thromboembolic model induction. Brains were isolated and stained with TTC stain to detect dead infarct volume. Our results showed that pre-treatment with SARS-CoV-2 spike protein significantly increased infarct volume compared to stroke. Treatment with the AT 1 R blocker, Losartan, significantly reduced infarct volume (Fig. 5 , A-B). SARS-CoV-2 spike protein aggravates cognitive dysfunction after thromboembolic occlusion model. We have previously shown that SARS-CoV-2 causes cognitive dysfunction in hACE2 KI mice [ 4 ]. Here, we assessed the effect of SARS-CoV-2 spike protein on cognitive function after induction of the thromboembolic model. hACE2 KI mice were injected with SARS-CoV-2 spike protein one week before thromboembolic model induction. The Novel Object Recognition test was used to assess memory and learning in hACE2 KI mice. hACE2 KI mice were familiarized with two identical objects. On test day, one of the objects was replaced with a novel object. Time spent investigating the novel object indicates memory and learning cognition in the mouse. We assessed the total distance traveled for each animal to exclude motor dysfunction. Our results showed no significant changes between groups in the total distance traveled (Fig. 6 .A). There was a significant decrease in both the number of entries into the novel object zone and the time spent interacting with the novel object in the mice subjected to stroke. A similar decrease in the number of entries was observed in the group that received a prior spike protein injection before stroke. However, the spike protein pre-injection further exacerbated the reduction in time spent with the novel object. Treatment with Losartan significantly improved hACE2 KI mice’s cognitive function, as evidenced by the increase in both the time spent with the novel object and the number of entries into the novel object zone. (Fig. 6 .B-D) Discussion The COVID-19 pandemic caused over one million American deaths. Survivors suffered a wide range of cerebrovascular complications, including stroke and cognitive impairments. The potential mechanisms underlying these disorders are not fully understood. Our study tests one of the possible mechanisms by which SARS-CoV-2 disrupts coagulation hemostasis and increases cerebrovascular thromboembolic complications. The main finding of our study is that the SARS-CoV-2 spike protein disrupts the renin-angiotensin-aldosterone system (RAAS) balance in the brain vasculature. The SARS-CoV-2 spike protein increases Ang II/AT 1 R signaling in the brain’s endothelial cells at the expense of the Ang II/AT 2 R protective arm, which increases brain inflammation. Moreover, our results showed that RAAS imbalance contributes to increased coagulation and decreased fibrinolysis, exacerbating stroke, and vascular contribution to cognitive impairments and dementia (VCID). Lastly, restoration of RAAS balance using AT 1 R blocker, Losartan, decreased SARS-CoV-2 spike protein-induced thromboembolic cerebrovascular complications. With the development of effective COVID-19 vaccines and the reduction of COVID-19 mortality, many COVID-19-induced neurovascular complications are more clinically visible. COVID-19 causes a wide range of neurological disorders [ 28 – 31 ]. These neurological disorders ranged from headaches, loss of smell, and altered mental status to encephalitis and ischemic stroke. COVID-19 not only increased the ischemic stroke rates in the general population but also increased mortality and severity in stroke patients. COVID-19 infections worsen stroke outcomes, especially in patients with a prevalence of vascular risk factors, including age, male gender, hypertension, hyperlipidemia, ischemic heart disease, and diabetes mellitus [ 4 , 11 ]. There are multiple hypotheses that account for COVID-19’s increased thromboembolic events in patients, including increased vascular inflammation and cytokine storms, endothelial dysfunction, pericyte loss, blood-brain barrier dysfunction, and neuroinflammation [ 32 – 36 ]. Here, we hypothesized that SARS-CoV-2 spike protein exacerbates stroke and cerebrovascular complications by increasing coagulation and decreasing fibrinolysis via disrupting the RAAS balance. SARS-CoV-2 spike protein binds with the ACE-2 receptor as one of the binding sites to achieve cell entry. ACE-2 plays a crucial role in the degradation of Ang II, the bioactive form of the RAAS, to Ang 1–7. We have previously shown that SARS-CoV-2 spike protein decreases ACE-2 expression and increases Ang II/AT 1 R downstream inflammatory signaling and endothelial cell apoptosis in the brain of humanized ACE2 knock-in mice [ 4 ]. Our study also showed that spike protein significantly downregulated the RAAS protective arm with decreased AT 2 R and MAS receptor expression 3 . Singh et al. showed that transient MCA occlusion increases ACE-2 expression in mice, which might increase the binding affinity to SARS-CoV-2 spike protein [ 26 ]. In the present study, we provide novel evidence that the SARS-CoV-2 spike protein-induced RAAS imbalance increases coagulation and decreases fibrinolysis, which worsens ischemic stroke outcomes in a distal middle cerebral artery (MCA) thromboembolic model. Our results showed that SARS-CoV-2 spike protein increases coagulation via increased Tissue Factor III (TF-III) expression in brain endothelial cells. TF-III activates the extrinsic coagulation pathway that activates factor VII, which in turn catalyzes the conversion of the inactive factor X into the active factor Xa. In addition, SARS-CoV-2 spike protein increased the expression of Plasminogen activator inhibitor-1 (PAI-1), a serine protease inhibitor that inhibits endogenous tissue-type plasminogen activator (tPA) activation and hence prevents fibrinolysis. Elevated PAI-1 is associated with thrombosis and atherosclerosis [ 37 ]. These effects were reversed with the use of Losartan, an AT 1 R blocker. These findings were confirmed in human brain microvascular endothelial cells (HBMECs), in which spike protein increased TF-III and PAI-1 following exposure to hypoxic conditions in HBMECs exposed to hypoxia. We used a mild chemically induced distal transient MCA thromboembolic model where a clot forms and spontaneously recanalizes within a few hours. However, the model outcomes were significantly changed when animals were pre-injected with SARS-CoV-2 spike protein. Our study showed that pre-injection of SARS-CoV-2 spike protein intensified the decrease in cerebral blood flow and delayed recanalization in the thromboembolic model. These effects may be the result of increased clot formation and reduction in fibrinolysis. Our results showed that SARS-CoV-2 spike protein increased TF-III and PAI expression. Moreover, increased clot formation and delayed recanalization were associated with significant neurological damage, as seen with increased brain infarct size in hACE2 KI mice preinjected with spike protein compared to stroke. Restoration of RAAS balance using AT 1 R blocker, Losartan prevented SARS-CoV-2 spike protein-induced thromboembolic cerebrovascular complications. Finally, we reported that vascular dysfunction contributes to cognitive impairment and dementia associated with SARS-CoV-2 spike protein-induced thromboembolic stroke. These results agree with Ahmed et al., who showed that the restoration of RAAS via AT 2 R activation contributes to the improvement of cognitive impairments after stroke [ 38 ]. We showed that Losartan significantly improved cognitive functions after SARS-CoV-2 spike protein-induced thromboembolic ischemic stroke. In conclusion, our study provides new evidence that SARS-CoV-2 spike protein increased coagulation and decreased fibrinolysis in hACE2 KI mice. These effects were accompanied by decreased cerebral blood flow, increased neuronal death, and increased cognitive dysfunctions. Our results showed that restoring RAAS balance using the AT 1 R blocker, Losartan, restored the RAAS balance and reduced COVID-19-induced thromboembolic cerebrovascular complications. Declarations Funding: This study was funded by the American Heart Association grant number 23AIREA1045073 to MA. Author Contribution M.A. and SP.H. wrote the main manuscript text and SP.H and VH. prepared figures. All authors reviewed the manuscript. References Kempuraj D, et al. COVID-19, Mast Cells, Cytokine Storm, Psychological Stress, and Neuroinflammation. Neuroscientist. 2020;26(5–6):402–14. Panagiotakopoulos L, et al. Use of an Additional Updated 2023–2024 COVID-19 Vaccine Dose for Adults Aged >/=65 Years: Recommendations of the Advisory Committee on Immunization Practices - United States, 2024. MMWR Morb Mortal Wkly Rep. 2024;73(16):377–81. Hess DC, Eldahshan W, Rutkowski E. COVID-19-Related Stroke. Transl Stroke Res. 2020;11(3):322–5. Burnett FN et al. SARS-CoV-2 Spike Protein Intensifies Cerebrovascular Complications in Diabetic hACE2 Mice through RAAS and TLR Signaling Activation. Int J Mol Sci, 2023. 24(22). Cagnazzo F, et al. 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Angiotensin II receptor type 1 - An update on structure, expression and pathology. Biochem Pharmacol. 2021;192:114673. Levi M, et al. Inhibition of plasminogen activator inhibitor-1 activity results in promotion of endogenous thrombolysis and inhibition of thrombus extension in models of experimental thrombosis. Circulation. 1992;85(1):305–12. Shirbhate E, et al. Understanding the role of ACE-2 receptor in pathogenesis of COVID-19 disease: a potential approach for therapeutic intervention. Pharmacol Rep. 2021;73(6):1539–50. Dutsch A, Schunkert H. RAAS inhibition and beyond-cardiovascular medications in patients at risk of or affected by COVID-19. Herz. 2023;48(3):206–11. Kuster GM et al. SARS-CoV2: should inhibitors of the renin-angiotensin system be withdrawn in patients with COVID-19? Eur Heart J, 2020. 41(19): pp. 1801–1803. Zhou B, et al. SARS-CoV-2 spike D614G change enhances replication and transmission. Nature. 2021;592(7852):122–7. Coucha M, et al. Inhibition of Ephrin-B2 in brain pericytes decreases cerebral pathological neovascularization in diabetic rats. PLoS ONE. 2019;14(1):e0210523. Hardigan T, et al. Linagliptin treatment improves cerebrovascular function and remodeling and restores reduced cerebral perfusion in Type 2 diabetes. Am J Physiol Regul Integr Comp Physiol. 2016;311(3):R466–77. Singh V, et al. Stroke increases the expression of ACE2, the SARS-CoV-2 binding receptor, in murine lungs. Brain Behav Immun. 2021;94:458–62. Anrather J, Iadecola C. Inflammation and Stroke: An Overview. Neurotherapeutics. 2016;13(4):661–70. Sonneville R, Dangayach NS, Newcombe V. Neurological complications of critically ill COVID-19 patients. Curr Opin Crit Care. 2023;29(2):61–7. Lau VI, et al. Non-COVID outcomes associated with the coronavirus disease-2019 (COVID-19) pandemic effects study (COPES): A systematic review and meta-analysis. PLoS ONE. 2022;17(6):e0269871. Pavlovic T, et al. Predicting attitudinal and behavioral responses to COVID-19 pandemic using machine learning. PNAS Nexus. 2022;1(3):pgac093. Molaverdi G, et al. Neurological complications after COVID-19: A narrative review. eNeurologicalSci. 2023;33:100485. Jung JM, et al. New Directions in Infection-Associated Ischemic Stroke. J Clin Neurol; 2024. Marshall M. COVID and the brain: researchers zero in on how damage occurs. Nature. 2021;595(7868):484–5. Miners S, Kehoe PG, Love S. Cognitive impact of COVID-19: looking beyond the short term. Alzheimers Res Ther. 2020;12(1):170. Brinjikji W et al. Endotheliitis and cytokine storm as a mechanism of clot formation in COVID-19 ischemic stroke patients: A histopathologic study of retrieved clots. Interv Neuroradiol, 2023: p. 15910199231185804. Amruta N et al. Mouse Adapted SARS-CoV-2 (MA10) Viral Infection Induces Neuroinflammation in Standard Laboratory Mice. Viruses, 2022. 15(1). Juneja GK, et al. Biomarkers of coagulation, endothelial function, and fibrinolysis in critically ill patients with COVID-19: A single-center prospective longitudinal study. J Thromb Haemost. 2021;19(6):1546–57. Ahmed HA, et al. RAS modulation prevents progressive cognitive impairment after experimental stroke: a randomized, blinded preclinical trial. J Neuroinflammation. 2018;15(1):229. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 02 Oct, 2024 Read the published version in Translational Stroke Research → Version 1 posted Editorial decision: Revision requested 29 Jul, 2024 Reviews received at journal 27 Jul, 2024 Reviewers agreed at journal 17 Jul, 2024 Reviewers invited by journal 11 Jul, 2024 Editor assigned by journal 02 Jul, 2024 Submission checks completed at journal 30 Jun, 2024 First submitted to journal 27 Jun, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4649614","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":329993348,"identity":"5518e1bc-438c-407e-be7f-28832268baf3","order_by":0,"name":"Stan P. Heath","email":"","orcid":"","institution":"Mercer University","correspondingAuthor":false,"prefix":"","firstName":"Stan","middleName":"P.","lastName":"Heath","suffix":""},{"id":329993349,"identity":"d65d45f0-64b3-4440-ad73-91184190e910","order_by":1,"name":"Veronica C. Hermanns","email":"","orcid":"","institution":"Mercer University","correspondingAuthor":false,"prefix":"","firstName":"Veronica","middleName":"C.","lastName":"Hermanns","suffix":""},{"id":329993350,"identity":"a09a349c-d714-4803-8d38-442cc387ff47","order_by":2,"name":"Maha Coucha","email":"","orcid":"","institution":"South University","correspondingAuthor":false,"prefix":"","firstName":"Maha","middleName":"","lastName":"Coucha","suffix":""},{"id":329993352,"identity":"04791091-72c2-42ab-8160-7ad752389cb6","order_by":3,"name":"Mohammed Abdelsaid","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/0lEQVRIiWNgGAWjYDACCQYDhgcMDDwMB4CcD3BhNgJaEqBaGGeQooUBpIWZhxgt/LObNz5I+GMnw3eAO/GzbY5NPn977wOGD2WHcVty51ixQWJbMo/kAd7N0rnb0ixnnDluwDjjHG4tDDdyzCQSG5h5DA7wbgBqOWxgIJHGwMzbhluL/I0c8x8Jf+pBWjb/ttz238BA/hkD8188WgyAtjAksB0GadkmzbjtANAWNgZmRjxaDIF+kUhsOw7yyzbL3m3JBhJn0hgO9pxLx6lF7nbzxg8f/lTb8wEdduPnNjsD/vZjjA9+lFnj9j7CVw8Q7ANEqB8Fo2AUjIJRgAcAAOyTVtqrVUoNAAAAAElFTkSuQmCC","orcid":"","institution":"Mercer University","correspondingAuthor":true,"prefix":"","firstName":"Mohammed","middleName":"","lastName":"Abdelsaid","suffix":""}],"badges":[],"createdAt":"2024-06-27 15:35:59","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4649614/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4649614/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12975-024-01301-5","type":"published","date":"2024-10-02T15:57:50+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":61084066,"identity":"cfe139dc-d8fc-44b1-992b-036b0aef8274","added_by":"auto","created_at":"2024-07-25 11:25:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":191682,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eSARS-CoV-2 spike protein intensifies RAAS imbalance after ischemic insult\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e. We assessed the effect of SARS-CoV-2 spike protein on RAAS balance in ACE2 KI mice brain and HBMECs after ischemic insult. ACE2 KI mice were injected SARS-CoV-2 spike protein (SP) one week before thromboembolic model induction. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eA)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e RT-PCR analysis for\u0026nbsp;ACE2 gene expression in brain homogenate. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eB)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e RT-PCR analysis for\u0026nbsp;AT\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e\u003cem\u003eR\u0026nbsp;gene expression in brain homogenate. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eC)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e RT-PCR analysis for\u0026nbsp;AT\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e\u003cem\u003eR\u0026nbsp;gene expression in brain homogenate. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eD)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e RT-PCR analysis for\u0026nbsp;MAS\u0026nbsp;receptor gene expression in brain homogenate. (A-D, One-way ANOVA,\u0026nbsp;*p\u0026nbsp;\u0026lt;0.05 vs sham, \u003c/em\u003e\u003csup\u003e\u003cem\u003e#\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u0026lt;0.05 vs stroke (Stroke side),\u0026nbsp;n\u0026nbsp;= 4).\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eHBMECs were treated with SARS-CoV-2 spike protein for 24 hrs before exposure to hypoxia.\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e E)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Western blot representative and analysis for AT\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e\u003cem\u003eR\u0026nbsp;expression in HBMECs. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eF)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Western blot representative and analysis for AT\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e\u003cem\u003eR\u0026nbsp;expression in HBMECs. (E-F, One-way ANOVA, *\u0026nbsp;p\u0026lt;0.05 vs control, \u003c/em\u003e\u003csup\u003e\u003cem\u003e#\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u0026lt;0.05 vs hypoxia, n\u0026nbsp;= 4). Our results showed that pretreatment with SARS-CoV-2 spike protein intensifies RAAS imbalance after ischemic insult. (C: Control, H: Hypoxia, SP: Spike protein, SP+L: Spike protein +Losartan)\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-4649614/v1/4435dc84843de9b4ccb6a6fa.png"},{"id":61084067,"identity":"9a4a3fe0-f33d-4cc4-9404-739e7032b577","added_by":"auto","created_at":"2024-07-25 11:25:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":175845,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIncreased inflammation after a double hit, SARS-CoV-2 spike protein followed by ischemic insult exacerbate inflammation in hACE2 brains.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e We assessed the effect of pre-injecting SARS-CoV-2 spike protein on brain inflammation before applying ischemic insult. ACE2 KI mice were injected with SARS-CoV-2 spike protein (SP) one week before thromboembolic model induction. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eA)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e RT-PCR analysis for\u0026nbsp;NF\u003c/em\u003e\u003csub\u003e\u003cem\u003eϏ\u003c/em\u003e\u003c/sub\u003e\u003cem\u003eB gene expression in brain homogenate. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eB)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e RT-PCR analysis for\u0026nbsp;TNF-α\u0026nbsp;gene expression in brain homogenate. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eC)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e RT-PCR analysis for\u0026nbsp;Il-6\u0026nbsp;gene expression in brain homogenate. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eD)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e RT-PCR analysis for, Il-1β gene expression in brain homogenate. (A-D, One-way ANOVA,\u0026nbsp;*p\u0026nbsp;\u0026lt;0.05 vs. sham, #P\u0026lt;0.05 vs. stroke (Stroke side),\u0026nbsp;n\u0026nbsp;= 4-5).\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eHBMECs were treated with SARS-CoV-2 for 24 hrs before exposure to hypoxia.\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e E)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Western blot representative and analysis for TNF-α expression in HBMECs. (One-way ANOVA, *\u0026nbsp;p\u0026lt;0.05 vs. normoxia, #P\u0026lt;0.05 vs. hypoxia, n\u0026nbsp;= 3-4). Our results showed that SARS-CoV-2 intensifies inflammation when coincident with ischemic insult. (C: Control, H: Hypoxia, SP: spike protein, SP+L spike protein +Losartan)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-4649614/v1/b77e09350178874806d32dc2.png"},{"id":61084694,"identity":"2c274d9f-927f-46dd-ba73-dc2701e5fca2","added_by":"auto","created_at":"2024-07-25 11:33:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":215723,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eSARS-CoV-2 spike protein disrupts coagulation homeostasis.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e We assessed the effect of pre-injecting SARS-CoV-2 spike protein (SP) on coagulation homeostasis prior to applying ischemic insult. ACE2 KI mice were injected with SARS-CoV-2 spike protein (SP) one week before thromboembolic model induction. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eA)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Western blot representative for TF-III and PAI-1. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eB)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Western Blot analysis for PAI-1 expression in brain homogenate. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eC)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Western Blot analysis for TF-III expression in brain homogenate. (B-C, One-way ANOVA, *p \u0026lt;0.05 vs. sham, #P\u0026lt;0.05 vs. stroke (Stroke side), n = 4-5).\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eHBMECs were treated with SARS-CoV-2 spike protein for 24 hrs before exposure to hypoxia.\u003c/em\u003e\u003cem\u003e\u003cstrong\u003eD) \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eWestern blot representative and analysis for PAI-1. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eE) \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eWestern blot representative and analysis for TF-III (One-way ANOVA, * p\u0026lt;0.05 vs. control, #P\u0026lt;0.05 vs. hypoxia, n = 3-4). Our results showed that SARS-CoV-2 increases coagulation and decreases fibrinolysis. Losartan treatment decreased spike protein-induced hypercoagulation. (C: Control, H: Hypoxia, SP: spike protein, SP+L spike protein +Losartan)\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-4649614/v1/d8e3700ae653bca0cd151d83.png"},{"id":61084065,"identity":"753496bc-8fc9-4b98-9c65-1fbe8797b07f","added_by":"auto","created_at":"2024-07-25 11:25:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1101784,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eSARS-CoV-2 spike protein decreases cerebral blood flow following stroke.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e We assessed the effect of pre-injecting SARS-CoV-2 spike protein (SP) on cerebral blood flow and vascular recanalization following a thromboembolic model. ACE2 KI mice were injected with SARS-CoV-2 spike protein (SP) one week before thromboembolic model induction. \u003c/em\u003eLaser speckle imaging was employed to measure cerebral blood flow at various intervals of 1, 2, 3, 6, and 24 hours post-surgery. \u003cem\u003e\u003cstrong\u003eA)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Cerebral blood flow representative images. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eB)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Cerebral Blood flow and recanalization analysis. (A-B, One-way ANOVA, *p \u0026lt;0.05 vs. stroke, n = 6).\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eOur results showed that pre-treatment with SARS-COV-2 spike protein significantly decreased cerebral blood flow and increased vascular recanalization time. Treatment with Losartan reduced spike protein effects. (Stroke, SSP : Stroke + Spike protein, SSPL: Stroke + Spike protein+ Losartan)\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-4649614/v1/ccedc586ec608bfd2416f9f9.png"},{"id":61084064,"identity":"a5305112-d3ad-4008-bbc2-ec14eb9c7ca8","added_by":"auto","created_at":"2024-07-25 11:25:27","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":658583,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eSARS-CoV-2 spike protein decreases infarct volume following stroke.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e We assessed the effect of pre-injecting SARS-CoV-2 spike protein (SP) on infarct volume following a thromboembolic stroke model. ACE2 KI mice were injected with SARS-CoV-2 spike protein (SP) one week before thromboembolic model induction. \u003c/em\u003eBrains were isolated and stained with TTC stain to detect infarct volume. \u003cem\u003e\u003cstrong\u003eA)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Representative images. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eB)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Infarct volume analysis. (A-B, One-way ANOVA,\u0026nbsp;*p\u0026nbsp;\u0026lt;0.05 vs. stroke, \u003c/em\u003e\u003csup\u003e\u003cem\u003e#\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u0026lt;0.05 vs. Stroke + Spike protein,\u0026nbsp;n\u0026nbsp;= 5-8).\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eOur results showed that pre-treatment with SARS-CoV-2 spike protein significantly increased infarct volume compared to stroke. Treatment with Losartan reduced infarct volume. (Stroke +SP : Stroke + Spike protein, Stroke+ SP+ Losartan : Stroke + Spike protein+ Losartan)\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-4649614/v1/d11a127d2c78e69a06dd8af9.png"},{"id":61084068,"identity":"0ac933d0-f62f-403b-b22e-9dee77ce92ec","added_by":"auto","created_at":"2024-07-25 11:25:27","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":92282,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eSARS-CoV-2 spike protein aggravates cognitive dysfunction after thromboembolic occlusion model. \u003c/strong\u003e\u003c/em\u003e\u003cem\u003e\u0026nbsp;We assessed the effect of pre-injecting SARS-CoV-2 spike protein (SP) on cognitive dysfunction following a thromboembolic stroke model. ACE2 KI mice were injected with SARS-CoV-2 spike protein (SP) one week before thromboembolic model induction. \u003c/em\u003eA novel object recognition test was used to assess cognitive dysfunction. \u003cem\u003e\u003cstrong\u003eA)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Total distance traveled. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eB)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Time spent investigating the new object. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eC) \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eNumber of entries to novel object zone.\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e D) \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eTime spent on novel object to total time spent in object recognition. (A-D, One-way ANOVA,\u0026nbsp;*p\u0026nbsp;\u0026lt;0.05 vs. Sham, \u003c/em\u003e\u003csup\u003e\u003cem\u003e#\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u0026lt;0.05 vs. Stroke,\u0026nbsp;n\u0026nbsp;= 4-6).\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eOur results showed that pre-treatment with SARS-CoV-2 spike protein significantly increased cognitive dysfunction compared to stroke. Treatment with Losartan reduced cognitive impairments. (SP: Spike protein)\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-4649614/v1/9774d452e8d0873a2bb538da.png"},{"id":66096924,"identity":"fbd2162e-5a7d-48ec-9984-bc6b9e778584","added_by":"auto","created_at":"2024-10-07 16:11:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3527034,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4649614/v1/e1c2e475-ce27-4ff2-829c-4d28fd90981d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"SARS-CoV-2 Spike Protein Exacerbates Thromboembolic Cerebrovascular Complications in Humanized ACE2 Mouse Model","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe Severe Acute Respiratory Syndrome Corona virus-2 (SARS-CoV-2) is a single-stranded RNA virus responsible for the COVID-19 pandemic. As of 2024, SARS-CoV-2 had killed over a million Americans. COVID-19 causes acute respiratory distress syndrome (ARDS) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. ARDS symptoms include severe pneumonia, hypoxemia, and elevated cytokines, known as a cytokine storm. Together, these can result in higher mortality rates, especially in patients with a concomitant cardiovascular disorder such as hypertension, diabetes, or obesity.\u003c/p\u003e \u003cp\u003eMany studies reported that COVID-19 causes neurological symptoms [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. COVID-19-induced neurological symptoms range from headaches and loss of taste and smell to encephalopathy, cognitive impairments, and stroke [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The mechanisms of these neurovascular disorders are unclear and represent a knowledge gap. COVID-19 caused life-threatening coagulopathies such as stroke and deep vein thrombosis [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Several reports indicate that these negative thrombotic events are associated with up to one-third of COVID-19 patients [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The molecular mechanisms behind increased hyper-coagulopathies and impaired fibrinolysis are unclear and require further investigation [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The development of vaccination against the SARS-CoV-2 virus has decreased mortality rates. However, long-term COVID-19-induced hyper-coagulopathy and cognitive dysfunction represent a significant medical challenge that requires intensive studies [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAngiotensin-converting enzyme-2 (ACE-2) is an enzyme that plays a crucial role in the RAAS balance. ACE-2 degrades angiotensin II, the bioactive form, which can bind to the angiotensin type receptor 1 (AT\u003csub\u003e1\u003c/sub\u003eR) or the angiotensin type receptor 2 (AT\u003csub\u003e2\u003c/sub\u003eR). AT\u003csub\u003e1\u003c/sub\u003eR is more abundant than AT\u003csub\u003e2\u003c/sub\u003eR, and its activation leads to physiological effects such as vasoconstriction, proinflammatory, thrombosis, and generation of reactive oxygen species [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Activation of the Ang II/AT\u003csub\u003e1\u003c/sub\u003eR axis pathway increases hyper-coagulations via upregulation of the tissue factor (TF), thrombin activation, and platelet aggregation [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In contrast, Ang II/AT\u003csub\u003e2\u003c/sub\u003eR axis opposes the effects of AT\u003csub\u003e1\u003c/sub\u003eR activation through vasodilation and anti-inflammatory responses and maintains hemostasis [\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. ACE-2 binds to the SARS-CoV-2 spike protein and acts as a functional receptor for the spike protein to internalize the viral particle [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. SARS-CoV-2 downregulates the ACE-2 receptor [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], which decreases Ang II degradation, therefore increasing the RAAS destructive arm with increased activation of the abundant AT\u003csub\u003e1\u003c/sub\u003eR over AT\u003csub\u003e2\u003c/sub\u003eR [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis study aims to investigate one of the possible mechanisms by which SARS-CoV-2 Spike protein alters the coagulation and fibrinolysis hemostasis, leading to stroke and neurovascular complications. We hypothesize that the SARS-CoV-2 spike protein exacerbates stroke and neurovascular complications by increasing coagulation and decreasing fibrinolysis via disrupting the RAAS balance. We examined the effect of SARS-CoV-2 spike protein in a mouse model of humanized ACE-2 knock-in (hACE2 KI) mice. Spike protein was injected for seven days before the induction stroke. Stroke is induced through distal middle cerebral artery (MCA) thromboembolism using ferric chloride (FeCl\u003csub\u003e3\u003c/sub\u003e). We examined cerebral blood flow, infarct-sized and vascular contribution to cognitive impairments, and dementia (VCID) in stroked animals after one week of SARS-CoV-2 spike protein injection.\u003c/p\u003e \u003cp\u003eOur previous studies showed that SARS-CoV-2 spike protein disrupts RAAS balance and increases brain inflammation [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Here, our results provide novel evidence that SARS-CoV-2 Spike protein increased coagulation and decreased fibrinolysis in hACE2 KI mice. These effects were accompanied by decreased cerebral blood flow, increased neuronal death, and increased cognitive dysfunctions. Our results showed that using the AT\u003csub\u003e1\u003c/sub\u003eR blocker, Losartan restored the RAAS balance and reduced COVID-19-induced thromboembolic cerebrovascular complications.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eTransgenic humanized ACE2 KI mice (hACE2 KI), B6.129S2 (Cg)-Ace2\u003csup\u003etm1(ACE2)Dwnt\u003c/sup\u003e/J mice were purchased from Jackson Laboratory (Jax lab: stock no. 035000, Ellsworth, Maine, USA). hACE2 KI mice have human ACE2 cDNA replacing the endogenous mouse Ace2 sequences. The endogenous Ace2 regulatory elements direct expression of human ACE2, the receptor used for cellular entry by several coronaviruses, including severe acute respiratory syndrome coronavirus-1 (SARS-CoV) and \u0026minus;\u0026thinsp;2 (SARS-CoV-2) [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. ARRIVE guidelines 2.0 were followed in conducting animal studies in accordance with the ethical guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Mercer University Institutional Animal Care and Use Committee (IACUC) is accredited by the American Association for Accreditation of Laboratory Animal Care, and has approved the animal protocols. A standard mice chow diet was provided to the animals, as was unlimited access to tap water. Twelve-hour cycles of light and dark were used for the mice.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eAnimal Treatment\u003c/h2\u003e \u003cp\u003ehACE2 mice were randomly assigned to four groups: 1) sham, 2) stroke, 3) stroke\u0026thinsp;+\u0026thinsp;SARSCoV-2 spike protein injection, and 4) stroke\u0026thinsp;+\u0026thinsp;SARSCoV-2 spike protein injection\u0026thinsp;+\u0026thinsp;Losartan. A recombinant protein for SARS-CoV-2 nucleoprotein/spike protein (4ug/animal, Invitrogen, USA, Cat. No. RP-87706) was injected intravenously via jugular vein injection 7 days prior to MCA/FeCl\u003csub\u003e3\u003c/sub\u003e thromboembolic surgery. Sham animals were exposed to the MCA/FeCl\u003csub\u003e3\u003c/sub\u003e thromboembolic surgery without FeCl\u003csub\u003e3\u003c/sub\u003e treatment.\u003c/p\u003e \u003cp\u003eLosartan (10 mg/kg body weight, Tokyo Chemical Industry, Tokyo, Japan, Cat. No. L0232) was added to the water supply of the animal cages. Losartan was first administered immediately after the injection of the recombinant spike protein for SARS-CoV-2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eMCA/FeCl\u003csub\u003e3\u003c/sub\u003e Induced Thromboembolic Model\u003c/h2\u003e \u003cp\u003eEight-week-old male hACE2 KI mice weighing 20-25g were randomly assigned for surgery. Mice were anesthetized using a mixture of Ketamine and Xylazine intraperitoneal (IP) injection (100mg/kg ketamine and 10 mg/kg Xylazine). Long-acting Buprenorphine (0.1 mg/kg) IP injection was also given before surgery to decrease postoperative pain. A vertical incision was made between the eye and ear to locate the temporal muscle. A horizontal incision is then made on the lateral ridge of the frontal bone, and a vertical incision from the rostral-most point of the parietal bone is made to expose the skull. The distal trunk of the middle cerebral artery is then located and exposed by drilling through the mouse skull. The meninges were removed to expose the artery, and the transient focal ischemia model was initiated by using fresh FeCl\u003csub\u003e3\u003c/sub\u003e (20%) soaked filter paper for five minutes. After removal, sterile warm saline was applied to the area for 10 minutes and then dried with sterile gauze. The temporal muscle was then placed back, the mouse was sutured, and the surgical area was disinfected with a povidone-iodine solution. The mouse was then monitored under a heat lamp until recovery.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eAssessment of Cerebral Blood Flow\u003c/h2\u003e \u003cp\u003eCerebral blood flow was measured with the RFLSI Ⅲ Laser Speckle Imaging System (RWD, San Diego, CA, USA). Mice were anesthetized using isoflurane, and a vertical incision was made from the sagittal suture to the interferential suture on the mouse\u0026rsquo;s skull. RWD laser speckle imaging was used to record the cerebral blood flow. LSCI software (Version 5.0) was used to measure the perfusion per area at hours 1, 2, 3, 6, and 24. Perfusion was represented as a percentage ratio of the stroke to the non-stroke hemisphere of the brain.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eAssessment of Infarct Volume\u003c/h2\u003e \u003cp\u003ehACE2 mice brains were collected 24 hours after MCA/FeCl\u003csub\u003e3\u003c/sub\u003e thromboembolic model surgery. These brains were sectioned into 5 coronal 2mm thick slices using an acrylic brain matrix. Coronal brain slices were examined by staining with 2% 2,3,5-triphenyl tetrazolium chloride (TTC) and incubating for 15 min in a humidified incubator (37%C, 5% CO2). Sections were photographed with units of measure to be quantified blindly using Image-J software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAssessment of Cognitive Functioning\u003c/h2\u003e \u003cp\u003eMemory and learning functions were assessed at baseline, before SARS-CoV-2 spike protein injection, and 24 hours after MCA/FeCl\u003csub\u003e3\u003c/sub\u003e thromboembolic surgery using Novel Object Recognition testing (NOR) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe NOR test is a 3-stage test used to examine recognition memory. The first stage involves habituation, with the mouse being exposed to its testing environment. The second is the familiarization stage, which involves the mouse being exposed to two identical objects. The final phase is the testing phase, which involves the replacement of one of the familiar objects with a novel object. The testing phase is recorded using ANY-maze software. Time spent around the novel object, the ratio of time spent investigating the novel object to the familiar object, and the total distance traveled were measured using the ANY-maze software recording and analyzed blindly.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eCell Culture\u003c/h2\u003e \u003cp\u003ePrimary human brain microvascular endothelial cells (HBMECs, from Angio-Proteomie, Boston, MA, USA, Cat. No. cAP-0002) were grown to confluency in complete media (MCDB-131 Complete, VEC Technologies, Inc., Rensselaer, NY, USA). HBMECs were switched to serum-reduced media and treated with SARS-CoV-2 recombinant spike protein (100 \u0026micro;M) with or without losartan (100 \u0026micro;M) for 24 hrs. The Next day, HBMECs were either kept under normoxia conditions (room air 21% oxygen) or placed in a hypoxia chamber (0.1% oxygen, BioSpherix, ProOx Model 110) for 6 hours. Cells were collected immediately once removed from the hypoxia chamber.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003ePolymerase Chain Reaction\u003c/h2\u003e \u003cp\u003eRT-PCR was performed as described previously [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. RNA was isolated using Triazole (Thermo-Fisher, USA, Cat. No. AC345480250) and quantified using Thermo Scientific NanoDrop 2000C Spectrophotometer (Thermo Scientific, USA). cDNA was prepared from isolated RNA using SuperScript IV VILO Master mix with an ezDNase kit (Invitrogen, USA, Cat. No. 11766050). The Quant Studio\u0026trade; 3 Real-Time PCR System (Applied Biosystems, Thermo Scientific, USA) was utilized to run qRT-PCR using PowerUp SyBR Green Master mix (Applied Biosystems, Thermo Scientific, USA, Cat No. A25742). All primer sequences used are detailed in (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). GAPDH was consistently used as the reference gene for normalization.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePCR primers.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePrimer\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFWD sequence\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eREV sequence\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eACE2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHuman\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-TCC ATT GGT CTT CTG TCA CCC G-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u0026rsquo;-AGA CCA TCC ACC TCC ACT TCT C-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-TCC ATT GGT CTT CTG CCA TCC G-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u0026rsquo;-AGA CCA TCC ACC TCC ACT TCT C-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAT\u003csub\u003e1\u003c/sub\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHuman\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-CAG CGT CAG TTT CAA CTT GTA CG-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u0026rsquo;-GCA GGT GAC TTT GGC TAC AAG C-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-GCC ATT GTC CAC CCG ATG AAG T-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u0026rsquo;-ACA CAT TTC GGT GGA TGA CGG C-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAT\u003csub\u003e2\u003c/sub\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHuman\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-CCA TGT TCT GAC CTT CCT GGA TG-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u0026rsquo;-CGG ATT AAC GCA GCT GTT GGT G-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-CGT GAC CAA GTC CTG AAG ATG G-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u0026rsquo;-GGA AGT GCC AGG TCA ATG ATG ACT G-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eGAPDH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHuman\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-GTC TCC TCT GAC TTC AAC AGC G-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u0026rsquo;-ACC ACC CTG TTG CTG TAG CCA A-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-CAT CAC TGC CAC CCA GAA GAC TG-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u0026rsquo;-ATG CCA GTG AGC TTC CCG TTC AG-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eIl-1β\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHuman\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-CCA CAG ACC TTC CAG GAG AAT G-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u0026rsquo;-GTG CAG TTC AGT GAT CGT ACA GG-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-TGG ACC TTC CAG GAT GAG GAC A-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u0026rsquo;-GTT CAT CTC GGA GCC TGT AGT G-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eI-6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHuman\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-AGA CAG CCA CTC ACC CTCT TAC G-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u0026rsquo;-TTC TGC CAG TGC CTC TTT GCT G-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-TAC CAC TTC ACA AGT CGG AGG C-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u0026rsquo;-CTG CAA GTG CAT CAT CGT TGT TC-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eMASR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHuman\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-CAG CAC CAT CTT GGT CGT GAA G-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u0026rsquo;-CAG CAG GTA AAG GAG TCT CAT GG-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-CTG ACA GCC ATC AGT GTG GAG A-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u0026rsquo;-GTG GTC ACC AAG CAC GAA AGT G-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNFκB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-GCT GCC AAA GAA GGA CAC GAC A-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u0026rsquo;-GGC AGG CTA TTG CTC ATC ACA G-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTNFα\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHuman\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-CTC TTC TGC CTG CTG CAC TTT G-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u0026rsquo;-ATG GGC TAC AGG CTT GTC ACT C-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-GGT GCC TAT GTC TCA GCC TCT T-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u0026rsquo;-GCC ATA GAA CTG ATG AGA GGG AG-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eImmunoblotting\u003c/h2\u003e \u003cp\u003eRIPA buffer (Millipore, Billerica, MA, USA, Cat# 3P 20188) was used to extract protein from the hACE2 brain and HBMECs. Equal protein loads were separated on 10% SDS-polyacrylamide gel utilizing the Mini PROTEAN Tetra Cell SDS-PAGE Gel electrophoresis kit (Biorad Laboratories Inc, Hercules, CA). Gels were transferred onto nitrocellulose membranes using the Bio-Rad Trans-Blot Turbo (Biorad Laboratories Inc, Hercules, CA, USA). Membranes were blocked with 5% milk and incubated overnight with primary antibodies. Membranes were washed and incubated with an appropriate horseradish peroxidase-conjugated secondary antibody. Membranes were reacted with Western chemiluminescent HRP Substrate (Millipore, USA) and imaged using the iBright Imaging system (Invitrogen Thermo Fisher Scientific, USA, Model FL1500). Image-J software (Version 1.54g) was used to measure band intensity. β-actin was used for normalization. All antibodies used are listed in (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAntibodies\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePrimary Antibody\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCompany\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCAT/ Lot/ Prod\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRecommended Concentration\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDilution Immunoblot\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSecondary Antibody\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAT\u003csub\u003e1\u003c/sub\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInvitrogen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePA5-20812\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1 mg/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRabbit\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAT\u003csub\u003e2\u003c/sub\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNovus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNBP1-60097\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1 mg/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRabbit\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eΒ-actin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR\u0026amp;D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMAB8929\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1 mg/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMouse\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePAI-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBiotechne\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMAB-17861-100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1 mg/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRabbit\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTNFα\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNOVUS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNBP1-19532\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1 mg/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1:500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRabbit\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTF-III\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR\u0026amp;D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMAB-3178\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1 mg/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMouse\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eGraphPad prism version 10 or higher was employed for all data analysis. For animal studies, the sample size was determined from our previous work [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. One-way ANOVA was utilized to assess mean differences between 1) sham, 2) stroke, 3) stroke\u0026thinsp;+\u0026thinsp;SARS-CoV-2 spike protein injection, and 4) stroke\u0026thinsp;+\u0026thinsp;SARS-CoV-2 spike protein injection\u0026thinsp;+\u0026thinsp;Losartan. Significance was determined at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Data is presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. A Tukey\u0026rsquo;s post-hoc test was used to adjust for the multiple comparisons to assess significant interaction effects from all analyses.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cem\u003eSARS-CoV-2 spike protein intensifies RAAS imbalance after ischemic insult.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eWe assessed the effect of SARS-CoV-2 spike protein on the gene expression of both RAAS arms in hACE2 KI mice brain and human microvascular endothelial cells (HBMECs) subjected to ischemic insult. We analyzed the gene expression of ACE2 in brain homogenate. Moreover, we measured AT\u003csub\u003e1\u003c/sub\u003eR, AT\u003csub\u003e2\u003c/sub\u003eR, and MASR in both the contralateral and ipsilateral brain homogenate using qRT-PCR. Singh et al. showed that a transient MCA occlusion stroke model increases ACE2 expression in the lung of mice [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Here, we showed that the MCA/FeCl\u003csub\u003e3\u003c/sub\u003e transient stroke model increases the ACE2 expression in the brain of hACE2 KI mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.A). We previously showed that SARS-CoV-2 spike protein downregulated ACE2 expression in hACE2 KI mice brains [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In the current study, a similar finding was reported. SARS-CoV-2 spike protein downregulates ACE2 gene expression in hACE2 KI brains after the thromboembolic occlusion model (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.A). Losartan treatment restored ACE2 gene expression after SARS-CoV-2 spike protein injection with the thromboembolic occlusion model. Next, we evaluated the AT\u003csub\u003e1\u003c/sub\u003eR, AT\u003csub\u003e2\u003c/sub\u003eR, and MASR in both stroke and non-stroke hemispheres. Our results showed that ischemic insult increases the expression of AT\u003csub\u003e1\u003c/sub\u003eR by 2-fold. Pre-injection of SARS-CoV-2 spike protein before the ischemic insult further increases the expression of AT\u003csub\u003e1\u003c/sub\u003eR to 4-fold in the brain of hACE2 KI mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.B). In contrast, SARS-CoV-2 spike protein decreased the gene expression of the RAAS protective arms AT\u003csub\u003e2\u003c/sub\u003eR and MASR (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.C-D). These results were confirmed in HBMECs that were treated with SARS-CoV-2 spike protein and subjected to hypoxia to mimic the ischemic insult. Our results showed that SARS-CoV-2 spike protein increases AT\u003csub\u003e1\u003c/sub\u003eR expression and decreases AT\u003csub\u003e2\u003c/sub\u003eR expression under normoxia control conditions. The addition of ischemic insult further augments SARS-CoV-2 spike protein-induced effects. Losartan treatment reduced spike protein-induced effects. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.E-F).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eIncreased inflammation after a double hit, SARS-CoV-2 spike protein followed by ischemic insult exacerbate inflammation in hACE2 brains.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eStroke is known to increase brain inflammation [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. We have previously shown that intravenous injection of SARS-CoV-2 spike protein increases inflammation in the brain of hACE2 KI mice [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Here, we examined the effect of SARS-CoV-2 spike protein injection coincident with ischemic insult on brain inflammation. Our results showed that SARS-CoV-2 spike protein pre-injection significantly intensifies inflammation after induction of thromboembolic occlusion model. Ischemic insults increased inflammation in both ipsilateral and contralateral brain hemispheres. Pre-injection of SARS-CoV-2 spike protein significantly increased NF\u003csub\u003eϏ\u003c/sub\u003eB, TNF-α, Il-1β, and Il-6 gene expression in the brains of hACE2 KI mice. Losartan showed a modest but significant effect in reducing the SARS-CoV-2 spike protein-induced effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.A-D). Our results showed similar findings when we examined the impact of SARS-CoV-2 spike protein on HBMCE subjected to hypoxia. SARS-CoV-2 spike protein significantly increased the expression of TNF-α under normoxia conditions. Pretreatment of HBMECs with SARS-CoV-2 spike protein prior to hypoxia further significantly increased endothelial cell inflammation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.E).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eSARS-CoV-2 spike protein disrupts coagulation homeostasis\u003c/h2\u003e \u003cp\u003eWe examined the effect of SARS-CoV-2 spike protein on the prothrombotic state. The disruption of the endothelial matrix leads to the release of tissue factor III (TF-III), which activates the coagulation cascade, resulting in thrombin activation. Our results showed that the thromboembolic stroke model exacerbated TF-III protein expression in hACE2 KI mice brain homogenate. The injection of spike protein before stroke further escalated TF-III expression. (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.A-C). PAI-1 inhibits the conversion of plasminogen to plasmin by urokinase-plasminogen activator and tissue-type plasminogen activator (tPA), thereby hindering fibrin degradation. Our results showed that the thromboembolic stroke model increased PAI-1, and the expression of PAI-1 was further increased by the injection of the spike protein seven days before the stroke. (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.A-C). Similar findings were observed in the HBMECs when exposed to hypoxic conditions with and without SARS-CoV-2 spike protein. Spike protein favors clot formation by increasing coagulation and decreasing fibrinolysis. Losartan treatment decreased spike protein-induced hypercoagulation. (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. D-E).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eSARS-CoV-2 spike protein decreases cerebral blood flow following stroke.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe thromboembolic occlusion was induced in hACE2 KI mice using the MCA/FeCl\u003csub\u003e3\u003c/sub\u003e model. SARS-CoV-2 spike protein was injected intravenously one week before stroke induction. Laser speckle imaging was employed to measure cerebral blood flow at 1, 2, 3, 6, and 24 hours post-surgery. Our results showed that the thromboembolic model decreased cerebral blood flow in the stroke hemisphere compared to the contralateral hemisphere. The spontaneous recanalization occurred, and cerebral blood flow was restored within six hours of the embolic model. The pre-treatment with SARS-COV-2 spike protein showed a significant decrease in cerebral blood flow and increased vascular recanalization time to over 6 hrs. Treatment with Losartan helped in faster restoration of cerebral blood flow and decreased recanalization time compared to spike protein. (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.A-B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eSARS-CoV-2 spike protein increases infarction volume following stroke.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eACE2 KI mice were injected with SARS-CoV-2 spike protein one week before thromboembolic model induction. Brains were isolated and stained with TTC stain to detect dead infarct volume. Our results showed that pre-treatment with SARS-CoV-2 spike protein significantly increased infarct volume compared to stroke. Treatment with the AT\u003csub\u003e1\u003c/sub\u003eR blocker, Losartan, significantly reduced infarct volume (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, A-B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eSARS-CoV-2 spike protein aggravates cognitive dysfunction after thromboembolic occlusion model.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eWe have previously shown that SARS-CoV-2 causes cognitive dysfunction in hACE2 KI mice [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Here, we assessed the effect of SARS-CoV-2 spike protein on cognitive function after induction of the thromboembolic model. hACE2 KI mice were injected with SARS-CoV-2 spike protein one week before thromboembolic model induction. The Novel Object Recognition test was used to assess memory and learning in hACE2 KI mice. hACE2 KI mice were familiarized with two identical objects. On test day, one of the objects was replaced with a novel object. Time spent investigating the novel object indicates memory and learning cognition in the mouse. We assessed the total distance traveled for each animal to exclude motor dysfunction. Our results showed no significant changes between groups in the total distance traveled (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e.A). There was a significant decrease in both the number of entries into the novel object zone and the time spent interacting with the novel object in the mice subjected to stroke. A similar decrease in the number of entries was observed in the group that received a prior spike protein injection before stroke. However, the spike protein pre-injection further exacerbated the reduction in time spent with the novel object. Treatment with Losartan significantly improved hACE2 KI mice\u0026rsquo;s cognitive function, as evidenced by the increase in both the time spent with the novel object and the number of entries into the novel object zone. (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e.B-D)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe COVID-19 pandemic caused over one million American deaths. Survivors suffered a wide range of cerebrovascular complications, including stroke and cognitive impairments. The potential mechanisms underlying these disorders are not fully understood. Our study tests one of the possible mechanisms by which SARS-CoV-2 disrupts coagulation hemostasis and increases cerebrovascular thromboembolic complications. The main finding of our study is that the SARS-CoV-2 spike protein disrupts the renin-angiotensin-aldosterone system (RAAS) balance in the brain vasculature. The SARS-CoV-2 spike protein increases Ang II/AT\u003csub\u003e1\u003c/sub\u003eR signaling in the brain\u0026rsquo;s endothelial cells at the expense of the Ang II/AT\u003csub\u003e2\u003c/sub\u003eR protective arm, which increases brain inflammation. Moreover, our results showed that RAAS imbalance contributes to increased coagulation and decreased fibrinolysis, exacerbating stroke, and vascular contribution to cognitive impairments and dementia (VCID). Lastly, restoration of RAAS balance using AT\u003csub\u003e1\u003c/sub\u003eR blocker, Losartan, decreased SARS-CoV-2 spike protein-induced thromboembolic cerebrovascular complications.\u003c/p\u003e \u003cp\u003eWith the development of effective COVID-19 vaccines and the reduction of COVID-19 mortality, many COVID-19-induced neurovascular complications are more clinically visible. COVID-19 causes a wide range of neurological disorders [\u003cspan additionalcitationids=\"CR29 CR30\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. These neurological disorders ranged from headaches, loss of smell, and altered mental status to encephalitis and ischemic stroke. COVID-19 not only increased the ischemic stroke rates in the general population but also increased mortality and severity in stroke patients. COVID-19 infections worsen stroke outcomes, especially in patients with a prevalence of vascular risk factors, including age, male gender, hypertension, hyperlipidemia, ischemic heart disease, and diabetes mellitus [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. There are multiple hypotheses that account for COVID-19\u0026rsquo;s increased thromboembolic events in patients, including increased vascular inflammation and cytokine storms, endothelial dysfunction, pericyte loss, blood-brain barrier dysfunction, and neuroinflammation [\u003cspan additionalcitationids=\"CR33 CR34 CR35\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Here, we hypothesized that SARS-CoV-2 spike protein exacerbates stroke and cerebrovascular complications by increasing coagulation and decreasing fibrinolysis via disrupting the RAAS balance.\u003c/p\u003e \u003cp\u003eSARS-CoV-2 spike protein binds with the ACE-2 receptor as one of the binding sites to achieve cell entry. ACE-2 plays a crucial role in the degradation of Ang II, the bioactive form of the RAAS, to Ang 1\u0026ndash;7. We have previously shown that SARS-CoV-2 spike protein decreases ACE-2 expression and increases Ang II/AT\u003csub\u003e1\u003c/sub\u003eR downstream inflammatory signaling and endothelial cell apoptosis in the brain of humanized ACE2 knock-in mice [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Our study also showed that spike protein significantly downregulated the RAAS protective arm with decreased AT\u003csub\u003e2\u003c/sub\u003eR and MAS receptor expression \u003csup\u003e3\u003c/sup\u003e. Singh et al. showed that transient MCA occlusion increases ACE-2 expression in mice, which might increase the binding affinity to SARS-CoV-2 spike protein [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In the present study, we provide novel evidence that the SARS-CoV-2 spike protein-induced RAAS imbalance increases coagulation and decreases fibrinolysis, which worsens ischemic stroke outcomes in a distal middle cerebral artery (MCA) thromboembolic model.\u003c/p\u003e \u003cp\u003eOur results showed that SARS-CoV-2 spike protein increases coagulation via increased Tissue Factor III (TF-III) expression in brain endothelial cells. TF-III activates the extrinsic coagulation pathway that activates factor VII, which in turn catalyzes the conversion of the inactive factor X into the active factor Xa. In addition, SARS-CoV-2 spike protein increased the expression of Plasminogen activator inhibitor-1 (PAI-1), a serine protease inhibitor that inhibits endogenous tissue-type plasminogen activator (tPA) activation and hence prevents fibrinolysis. Elevated PAI-1 is associated with thrombosis and atherosclerosis [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. These effects were reversed with the use of Losartan, an AT\u003csub\u003e1\u003c/sub\u003eR blocker. These findings were confirmed in human brain microvascular endothelial cells (HBMECs), in which spike protein increased TF-III and PAI-1 following exposure to hypoxic conditions in HBMECs exposed to hypoxia.\u003c/p\u003e \u003cp\u003eWe used a mild chemically induced distal transient MCA thromboembolic model where a clot forms and spontaneously recanalizes within a few hours. However, the model outcomes were significantly changed when animals were pre-injected with SARS-CoV-2 spike protein. Our study showed that pre-injection of SARS-CoV-2 spike protein intensified the decrease in cerebral blood flow and delayed recanalization in the thromboembolic model. These effects may be the result of increased clot formation and reduction in fibrinolysis. Our results showed that SARS-CoV-2 spike protein increased TF-III and PAI expression. Moreover, increased clot formation and delayed recanalization were associated with significant neurological damage, as seen with increased brain infarct size in hACE2 KI mice preinjected with spike protein compared to stroke. Restoration of RAAS balance using AT\u003csub\u003e1\u003c/sub\u003eR blocker, Losartan prevented SARS-CoV-2 spike protein-induced thromboembolic cerebrovascular complications.\u003c/p\u003e \u003cp\u003eFinally, we reported that vascular dysfunction contributes to cognitive impairment and dementia associated with SARS-CoV-2 spike protein-induced thromboembolic stroke. These results agree with Ahmed et al., who showed that the restoration of RAAS via AT\u003csub\u003e2\u003c/sub\u003eR activation contributes to the improvement of cognitive impairments after stroke [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. We showed that Losartan significantly improved cognitive functions after SARS-CoV-2 spike protein-induced thromboembolic ischemic stroke.\u003c/p\u003e \u003cp\u003eIn conclusion, our study provides new evidence that SARS-CoV-2 spike protein increased coagulation and decreased fibrinolysis in hACE2 KI mice. These effects were accompanied by decreased cerebral blood flow, increased neuronal death, and increased cognitive dysfunctions. Our results showed that restoring RAAS balance using the AT\u003csub\u003e1\u003c/sub\u003eR blocker, Losartan, restored the RAAS balance and reduced COVID-19-induced thromboembolic cerebrovascular complications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThis study was funded by the American Heart Association grant number 23AIREA1045073 to MA.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eM.A. and SP.H. wrote the main manuscript text and SP.H and VH. prepared figures. All authors reviewed the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKempuraj D, et al. COVID-19, Mast Cells, Cytokine Storm, Psychological Stress, and Neuroinflammation. Neuroscientist. 2020;26(5\u0026ndash;6):402\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePanagiotakopoulos L, et al. Use of an Additional Updated 2023\u0026ndash;2024 COVID-19 Vaccine Dose for Adults Aged \u0026gt;/=65 Years: Recommendations of the Advisory Committee on Immunization Practices - United States, 2024. MMWR Morb Mortal Wkly Rep. 2024;73(16):377\u0026ndash;81.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHess DC, Eldahshan W, Rutkowski E. COVID-19-Related Stroke. Transl Stroke Res. 2020;11(3):322\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBurnett FN et al. 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RAS modulation prevents progressive cognitive impairment after experimental stroke: a randomized, blinded preclinical trial. J Neuroinflammation. 2018;15(1):229.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"translational-stroke-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"trsr","sideBox":"Learn more about [Translational Stroke Research](http://jcmr-online.biomedcentral.com)","snPcode":"12975","submissionUrl":"https://submission.nature.com/new-submission/12975/3","title":"Translational Stroke Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"SARS-CoV-2 spike protein, RAAS balance, COVID-19-induced thromboembolic complications, Vascular-contributes to cognitive impairments and dementia","lastPublishedDoi":"10.21203/rs.3.rs-4649614/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4649614/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCOVID-19 increases the risk for acute ischemic stroke, yet the molecular mechanisms are unclear and remain unresolved medical challenges. We hypothesize that the SARS-CoV-2 spike protein exacerbates stroke and cerebrovascular complications by increasing coagulation and decreasing fibrinolysis by disrupting the renin-angiotensin-aldosterone system (RAAS). A thromboembolic model was induced in humanized ACE2 knock-in mice after one week of SARS-CoV-2 spike protein injection. hACE2 mice were treated with Losartan, an angiotensin receptor (AT\u003csub\u003e1\u003c/sub\u003eR) blocker, immediately after spike protein injection. Cerebral blood flow and infarct size were compared between groups. Vascular-contributes to cognitive impairments and dementia was assessed using a Novel object recognition test. Tissue factor-III and plasminogen activator inhibitor-1 were measured using immunoblotting to assess coagulation and fibrinolysis. Human brain microvascular endothelial cells (HBMEC) were exposed to hypoxia with/without SARS-CoV-2 spike protein to mimic ischemic conditions and assessed for inflammation, RAAS balance, coagulation, and fibrinolysis. Our results showed that the SARS-CoV-2 spike protein caused an imbalance in the RAAS that increased the inflammatory signal and decreased the RAAS protective arm. SARS-CoV-2 spike protein increased coagulation and decreased fibrinolysis when coincident with ischemic insult, which was accompanied by a decrease in cerebral blood flow, an increase in neuronal death, and a decline in cognitive function. Losartan treatment restored RAAS balance and reduced spike protein-induced effects. SARS-CoV-2 spike protein exacerbates inflammation and hypercoagulation, leading to increased neurovascular damage and cognitive dysfunction. However, the AT\u003csub\u003e1\u003c/sub\u003eR blocker, Losartan, restored the RAAS balance and reduced COVID-19-induced thromboembolic cerebrovascular complications.\u003c/p\u003e","manuscriptTitle":"SARS-CoV-2 Spike Protein Exacerbates Thromboembolic Cerebrovascular Complications in Humanized ACE2 Mouse Model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-25 11:25:22","doi":"10.21203/rs.3.rs-4649614/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-29T13:22:31+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-27T11:23:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"197816421892990688496203951870594667609","date":"2024-07-17T07:23:34+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-11T14:41:41+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-03T00:21:09+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-01T01:36:45+00:00","index":"","fulltext":""},{"type":"submitted","content":"Translational Stroke Research","date":"2024-06-27T15:33:45+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"translational-stroke-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"trsr","sideBox":"Learn more about [Translational Stroke Research](http://jcmr-online.biomedcentral.com)","snPcode":"12975","submissionUrl":"https://submission.nature.com/new-submission/12975/3","title":"Translational Stroke Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"47493689-c92f-463f-9bbc-dde0d854e227","owner":[],"postedDate":"July 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-10-07T16:03:19+00:00","versionOfRecord":{"articleIdentity":"rs-4649614","link":"https://doi.org/10.1007/s12975-024-01301-5","journal":{"identity":"translational-stroke-research","isVorOnly":false,"title":"Translational Stroke Research"},"publishedOn":"2024-10-02 15:57:50","publishedOnDateReadable":"October 2nd, 2024"},"versionCreatedAt":"2024-07-25 11:25:22","video":"","vorDoi":"10.1007/s12975-024-01301-5","vorDoiUrl":"https://doi.org/10.1007/s12975-024-01301-5","workflowStages":[]},"version":"v1","identity":"rs-4649614","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4649614","identity":"rs-4649614","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}