Endothelial -targeted CD39 is protective in a mouse model of global forebrain ischaemia

doi:10.21203/rs.3.rs-4840216/v1

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Endothelial -targeted CD39 is protective in a mouse model of global forebrain ischaemia

https://doi.org/10.21203/rs.3.rs-4840216/v1

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Global ischemic brain injury occurs after cardiac arrest or prolonged hypotensive episodes following surgery or trauma. It causes significant neurological deficits even after successful re-establishment of blood flow. It is the primary cause of death in 68% of inpatient and 23% of out-of-hospital cardiac arrest cases, but there are currently no treatments. Endothelial activation and dysfunction impairing small vessel blood flow is the cause of brain damage. Purinergic signaling is an endogenous molecular pathway, where CD39 and CD73 catabolize extracellular adenosine triphosphate (eATP) to adenosine. After ischemia, eATP is released, triggering thrombosis and inflammation. In contrast, adenosine is anti-thrombotic, protects against oxidative stress, and suppresses the immune response.

Our group developed a bifunctional compound – anti-VCAM-CD39 that targets dysregulated endothelium and promotes adenosine generation at the infarct site, localising antithrombotic and anti-inflammatory effects of CD39. We investigated whether anti-VCAM-CD39 could improve outcome in a murine model of global ischaemia caused by dual carotid artery ligation (DCAL). Test drugs anti-VCAM-CD39 and controls were given 3h after 30min ischaemia. Assessments at 24h included neurological function, infarct volume, perfusion, albumin extravasation to assess blood-brain barrier (BBB) permeability.

We showed that there was an overall improvement in neurological deficit in αVCAM-CD39-treated mice after DCAL. MRI revealed that these mice had significantly smaller infarcts and reduced apoptotic activity on the side of permanent occlusion, compared to saline treated mice. There was reduced albumin extravasation in treated mice after DCAL, suggesting anti-VCAM-CD39 conferred neuroprotection in the brain through preservation of blood brain barrier (BBB) permeability. In vitro findings confirmed that αVCAM-CD39-mediated adenosine protected against hypoxia-induced endothelial cell death.

anti-VCAM-CD39 is a novel therapeutic that can promote neuroprotection, reduce tissue damage and inflammation in the brain after hypoxic brain injury in mice. These findings suggest that anti-VCAM-CD39 could be a new avenue of cardiac arrest therapy and could potentially be used in other cerebrovascular diseases where endothelial dysfunction is a constant underlying pathology.

ischaemia

dual carotid artery ligation

global hypoxia

CD39

VCAM-1

Hypoxic–ischemic encephalopathy or global hypoxic ischaemic brain injury (HIBI) is a consequence of global cerebral ischaemia due to cardiac arrest or after prolonged hypotensive episodes, such as hanging, strangulation or drowning[1]. It is a leading cause of mortality and is associated with significant long-term neurological disability in survivors[2]. The brain has a high metabolic need and is entirely dependent on a continuous supply of oxygen and glucose from the blood for proper functioning. HIBI can only be managed by establishing adequate oxygen delivery, thereby limiting secondary brain injury but there is currently no conclusive treatment[3]. Therefore, there is an unmet clinical need for new therapeutics.

As little as 5 minutes of hypoxic insult can cause widespread brain damage[4], including neuronal cell death, blood-brain barrier (BBB) permeability and tissue necrosis[5]. Two pathological processes mediate brain injury after HIBI. The first phase is the initial injury due to oxygen and glucose deprivation when blood flow is disrupted. The second phase is the ischaemia reperfusion injury (IRI) that follows re-establishment of blood flow after resuscitation following cardiac arrest[6].

IRI leads to endothelial cell (EC) injury and release of extracellular adenosine triphosphate (eATP). eATP signals via purinergic P2X_1 − 7 receptors on macrophages and T-cells to boost pro-inflammatory cytokines, tumour necrosis factor (TNF-α), IL-6 and IL-8, IL-1β and IL-18 production[7, 8]. EC activation during IRI leads to increased expression of vascular cell adhesion molecule-1 (VCAM-1)[9]. Leukocytes use VCAM-1 as one of the receptors to adhere to ECs and infiltrate tissues, amplifying inflammation. P- and E-selectins and von Willebrand factor (vWF) are also released, leading to a local prothrombotic environment[10].

CD39 is an ecto-nucleoside triphosphate diphosphohydrolase (NTPDase) expressed by ECs and is the dominant regulator of purinergic signalling in the vasculature [11]. eATP and eADP form substrates for CD39 enzymatic activity that converts a pro-inflammatory and pro-thrombotic vascular-EC environment to an anti-inflammatory and anti-thrombotic phenotype by the generation of adenosine. Adenosine production catalysed by CD39/CD73 is critical in protecting tissue against hypoxic and ischaemic insults[12]; CD39 reduces intravascular thrombosis[13] and CD39^–/– mice have larger infarct volumes after stroke[14]. We developed a new molecule, anti-VCAM-CD39 which consists of recombinant CD39 fused to a single chain fragment variable of VCAM-1 antibody. A unique construct that confers the anti-inflammatory and antithrombotic effect of CD39 to areas of endothelial dysfunction characterised by upregulation of VCAM-1, we recently showed that anti-VCAM-CD39 was protective in a mouse model of ischaemic stroke administered at a dose that was well below the threshold that caused haemostatic defect [15]. Given the overlapping pathophysiology of ischaemic stroke and global HIBI, we considered whether targeted delivery of CD39 to the activated endothelium would improve neurological outcome in a model of global forebrain IRI that we previously developed[16].

anti-VCAM-CD39 ameliorates brain damage in mice subjected to DCAL while reducing blood brain barrier permeability and limiting endothelial activation.

We found that after 0.125mg/kg of anti-VCAM-CD39 treatment, there was a significant decrease in the total infarct volume assessed by MRI-based T2* analysis (Fig. 1A), with distinct bilateral lesions evidenced by the representative MR images (Fig. 1B) that were smaller in the anti-VCAM-CD39 treated group. Treatment with non-targeted VCAM-CD39 did not significantly reduce infarct while there was a protective trend seen with VCAM blockade, when compared to saline. In fact, the non-targeted VCAM-CD39 yielded a significantly larger infarct on the side of permanent occlusion, when compared to the saline, anti-VCAM-CD39, and anti-VCAM-inactive CD39 treated groups. (p = 0.0219, p < 0.0001, p = 0.0001 respectively) (Fig. 1A and 1B). Corresponding Cresyl Violet-stained sections taken from the same brains clearly delineated the infarcts and absence of Cresyl violet positive cells in the same region, confirming cell death (Fig. 1B).

Apoptotic activity is normally increased due to ischaemic injury and corresponds to lesion volume as an indicator of cell death, which we have also previously demonstrated[17]. Post-treatment, apoptotic activity was significantly decreased in the permanent occlusion side of the brain after 24 hours after treatment with anti-VCAM-CD39 (Fig. 1C). On the side of transient occlusion, there seemed to be lower apoptotic activity overall, and similar levels of cell death in both saline and anti-VCAM-CD39 treated groups. Interestingly, on both sides of the brain, apoptotic activity seemed to be unchanged after non-targeted VCAM-CD39 when compared to saline treated animals. However, after anti-VCAM-inactive CD39 treatment, apoptotic activity was significantly increased from saline on both sides of the brain (p < 0.0001), suggesting that VCAM-blockade alone was not adequate for protection against cell death.

We found a significant reduction in extravasated albumin post anti-VCAM-CD39 treatment as compared to saline (Fig. 1D), suggesting a reduction in BBB permeability. Non-targeted VCAM-CD39 was not able to reduce albumin extravasation, while the trend was to increase BBB permeability with Anti-VCAM- inactive CD39 (p = 0.052). anti-VCAM-CD39 also minimised secretion of vWF into the plasma, consistent with reduced endothelial injury (Supplementary Fig. 1), further supporting the role of this construct in restoring BBB integrity.

As we have shown before[17], saline-treated mice post-DCAL had significantly impaired neurological function compared to sham (p = 0.001). These mice had a median Bederson score of 4, but mice treated with anti-VCAM-CD39 treatment had a median of 1 (Fig. 1E). Non-targeted VCAM-CD39 and Anti-VCAM- inactive CD39 were unable to reduce neurological deficit, confirming the synergistic effect seen of both CD39 activity and VCAM-blockade with treatment with anti-VCAM-CD39 .

anti-VCAM-CD39 significantly reduced inflammatory cytokines post-DCAL

We further analysed both the circulating plasma levels and tissue expression of cytokines that we have previously shown to be significantly increased following global forebrain ischaemia with DCAL[17] to gain an insight into the protective mechanism of anti-VCAM-CD39. Interestingly, it seems that it was the circulating plasma levels and not the gene expression of inflammatory cytokines systemic 1L1α (Fig. 2A and B) and TNFα (Fig. 2E and F) that were significantly decreased post treatment, while IL-6 (Fig. 2C and D) was only significantly decreased within the brain tissue.

We also analysed the gene expression of adenosine receptors (Fig. 3B) to understand the involvement of adenosine receptors. We found when anti-VCAM-CD39 was administered post-DCAL, there was a significant increase in gene expression of A_2B receptor (Fig. 3Aii) paired with a significant decrease in the gene expression of HIF-1α (Fig. 3Ai), suggests that adenosine generation was likely involved in the overall improvement of I/R injury found post-DCAL. No change in A_2A receptor expression was detected (not shown).

To confirm this, we investigated the ratio of adenosine and ATP in the plasma 24-hours post treatment. The increase in the plasma adenosine/ATP ratio confirms that αVCAM-cCD39 created a more protective, adenosine-rich milieu (Fig. 3B).

To confirm whether anti-VCAM-CD39 confers protection through adenosine signalling, we exposed brain endothelial cells to CGS-15943, a specific adenosine receptor antagonist, to oxygen glucose deprivation (OGD). We confirmed that OGD caused significant necrotic cell death (Fig. 3C), and treatment with anti-VCAM-CD39 was capable of significantly reducing LDH levels. When we treated the endothelial cells with CGS-15943 in addition to anti-VCAM-CD39, we observed a loss of the protective effect seen with anti-VCAM-CD39 treatment, suggesting that blocking adenosine signalling could limit the protective effect of anti-VCAM-CD39 .

Global hypoxic ischaemic brain injury (HIBI) is a condition that induces widespread brain damage due to oligemia and IRI[18]. We previously characterised a novel model of global HIBI[17] and confirmed that following injury, there were bilateral lesions in the forebrain, as well as increased BBB permeability; these are common indicators of IRI and oligemia in similar global ischaemia models[19–21]. Given the demonstrated efficacy of anti-VCAM-CD39 in an ischemic stroke model[15], we hypothesised that anti-VCAM-CD39 vwould be able to protect the brain against global HIBI due to the known effects of suppression of inflammatory eATP and generation of protective adenosine in improving hypoxia induced endothelial damage[22]. The neuroprotective effect of anti-VCAM-CD39 in reducing infarct volume reflected our data from the ischaemic stroke model[15]. This effect was not observed with control constructs (non-targeted VCAM-CD39 and anti-VCAM-inactive CD39 demonstrating the targeted effect of low-dose CD39 for neuroprotection. The reduction in plasma adenosine/eATP ratio confirms the dual hydrolytic effects of anti-VCAM-CD39 on both ATP and ADP. The functional effect of a reduction in infarct volume was evident with a reduction in Bederson score following anti-VCAM-CD39 treatment. Bederson scoring is a semi-quantitative test to assess neurological function in rodents, including forelimb flexion, circling behaviour, and balance beam tests[23]. While reduced caspase-3 activity in the permanently occluded side of the brain following anti-VCAM-CD39 correlates with a decrease in lesion volume in this cohort, the anti-VCAM component appeared to promote apoptotic activity. That there was no corresponding increase in infarct volume suggests that neuronal death was not increased in this cohort relative to vehicle administration. Therefore, this is potentially due to an accumulation of apoptotic T-cells that occurs when the interaction between VCAM-1 and its ligand VLA-4 is blocked[24]. With anti-VCAM-CD39 injection, the localised adenosine generation would likely inhibit T-cell infiltration[25], and there would not be a corresponding increase in T-cell apoptosis, although this remains to be confirmed.

The endothelium is a critical component of the BBB and endothelial permeability increases significantly in response to ischaemia and hypoxia, and extracellular adenosine is known to reduce hypoxia-associated vascular leakage[26]. We have previously confirmed that anti-VCAM-CD39 binds to VCAM-1 and minimises transendothelial migration of leukocytes[15]. Although a recent manuscript reported that inhibition of VCAM1 activation protects against BBB breakdown in a mouse model of chronic cerebral hypoxia, in our experiments VCAM-1 blockade alone did not reduce albumin extravasation[27]. Reduced capacity for adenosine generation (due to gene CD39 or CD73 gene deletion) results in significant increases in vascular leakage following exposure to ambient hypoxia. Release of vWF is a feature of endothelial activation[28] and consistent with induction of IRI, plasma vWF increased post-DCAL when compared to sham, while treatment with anti-VCAM-CD39 (Supplementary Fig. 1) significantly reduced plasma vWF, thereby confirming reduced endothelial activation and injury. This coincided with significant BBB protection, evidenced by a reduction in albumin extravasation after anti-VCAM-CD39 treatment. Further, adenosine-mediated activation of A_2B receptors has been found to decrease endothelial paracellular permeability during leukocyte transmigration[12].

That combined antithrombotic and anti-inflammatory effects of anti-VCAM-CD39 in the cerebrovascular circulation reduced hypoxia in the brain, which is likely reflected by a reduction in HIF-1α expression and correlated with a reduction in IL-6 expression and a reduction in plasma levels of IL-1α and TNFα. Both cytokines regulate HIF-1α activity in brain endothelial cells promoting hypoxia and inflammation as well as increasing BBB permeability and angiogenesis[29]. Here we showed an increase in gene expression of the A_2B receptor together with a correlative reduction in HIF-1α expression in the brains of mice after anti-VCAM-CD39 treatment. A_2B receptor signalling is known to provide neuroprotection and reduce pro-inflammatory cytokines TNF-α, IL-1, and IL-6[30], which aligns with our data. Dettori et al showed that chronic administration of an A_2B receptor agonist was able to significantly reduce infarct volume, BBB disruption and neurologic deficit after focal ischaemia in rats by preventing loss astrocyte loss in the striatum and reducing microglial activation[31].

In this work we have not identified which cell types were involved in the reduction of HIF-1α -IL1\(\:{\alpha\:}\)-IL6 and TNFα gene expression, and further immunolocalization experiments are needed to identify the contribution of astrocytes, pericytes and microglia which are all known to have a role in neuroprotection after ischaemia[32]. We showed that anti-VCAM-CD39 promoted a targeted increase in adenosine generation, and via A_2B receptor signalling, reduced neuroinflammation and promoted endothelial protection. Interestingly, while the mechanism of protection of anti-VCAM-CD39 appears to be adenosine-driven in this model, it differs to that seen in the ischaemic stroke model, where much of the protection appears to be due to anti-VCAM-CD39 mediated catabolism of eATP[15]. This further highlights the importance of purinergic signalling in brain injury and the need to further explore CD39-based therapeutics for these conditions.

Mice

Experiments were performed in male C57BL/6 mice (age, 8 ± 0.2 weeks; weight, 28.2 ± 2 g) obtained from an in-house colony (Monash Animal Research Platform, Clayton Australia) where they were placed on a 12h light-dark cycle with ad libitum access to food and water.

DCAL

Transient global forebrain hypoxia (30 minutes) was induced using DCAL, as described previously[17]. Briefly, mice are anaesthetised and left carotid artery is exposed and a calibrated flow probe is placed around the artery to measure blood flow velocity. After 10 min, the right carotid artery is permanently sutured to block blood flow in the artery. The left carotid artery is then transiently clamped for a period of 30 min (and cessation of blood flow is confirmed), and reperfusion is triggered.

Treatment Administration

We had previously determined that VCAM-1 was significantly upregulated 3 hours[17]. We tested anti-VCAM-CD39 at various doses and determined that the optimum therapeutic dose was 0.125mg/kg, which we administered intravenously 3 hours post-surgery. The corresponding dose of the controls (non-targeted CD39; equi-active- 1.5 mg/kg; and anti-VCAM-inactive CD39- 0.5 mg/kg) was similarly administered. Mice were assigned a random sample number, and all mouse groups were blinded during subsequent assessment. All animals were left to recover on the heat pad for at least 4 hours after procedure. Animals were separated and given supportive treatment and left on the heat rack overnight[17].

Euthanasia and tissue harvesting

At 24 hours post-surgery, mice were anaesthetized with Lethabarb (Pentobarbitone, 90mg/kg, Australia), and transcardially perfused with phosphate buffered saline (PBS) pH 7.4. Unless otherwise stated, a 6mm section of the infarcted brains was dissected and homogenised to 300mg wet weight of tissue per 1ml of Lysis Buffer (10mM Hepes pH 7.4, 10% Sucrose, 2mM EDTA, 0.1% CHAPS, 5mM DTT, 10ug/ml Aprotinin).

Functional Assessment

Neurological assessment was done through Bederson scoring as described previously[17, 33] 20–24 hours post-surgery.

Magnetic resonance imaging

At 24 hours post-stroke, in-vivo MRI Imaging was performed using a 9.4 T/20 cm Bruker MRI with actively decoupled volume transmit and surface-receive coils as described previously[34]. Refer to supplementary methods for further information.

Caspase-3/7 activity assay

The fluorogenic substrate (N-Acetyl-Asp-Glu-Val-Asp-7-amido-4-trifluoromethylcoumarin (Ac-DEVD-AFC) was used to measure caspase-3/7 activity as previously described[17]. The injured (ipsilateral) and uninjured (contralateral) cortices were dissected and homogenized to 300mg wet weight of tissue per ml of Caspase lysis Buffer (10mM HEPES pH 7.4, 10% Sucrose, 2mM EDTA, 0.1% CHAPS, 5mM DTT, 10µg/ml Aprotinin). 10µL of lysate was diluted in 90µl of Caspase reaction Buffer (40mM HEPES pH 7.4, 200mM NaCl, 2mM EDTA, 0.2% CHAPS, 0.1% Sucrose, 3mM DTT) along with Fluorogenic Caspase-3 Substrate Ac-DEVD-AFC (Final concentration 13µM) in a black 96 well plate (Perkin Elmer, Australia). The reaction was allowed to take place at 37°C and the fluorescence generated by the release of the fluorogenic group AFC on cleavage by caspase 3 was measured kinetically every 2 min for 200 minutes (Ex _{400 nm} and Em_{505 nm}) using a BMG FLUOstar Omega Microplate Reader (BMG Labtech, Australia).

Blood brain barrier permeability assay

Albumin content in the brain was determined using the Mouse Albumin ELISA Quantitation Set (Bethyl Laboratories, USA) according to the manufacturer’s instructions and as described[17, 35]. Albumin in the brain parenchyma is reflective of the extent of BBB damage as published[35]. Total protein was quantitated using the BCA protein assay (Pierce). The amount of albumin (ng) per microgram protein in each sample was calculated from the standard curve. Albumin extravasation was calculated as difference in albumin in the ipsilateral and contralateral cortex for each animal for mice.

Determination of plasma ATP and Adenosine levels

ATP and Adenosine levels in the plasma was determined as previously described[15].

Real-Time (RT)-PCR

Sections of ischemic brain tissue were dissected and kept in RNAlater (Thermo Fisher Scientific, Australia) for 24 hours before being stored at -80°c until use. For RNA extraction, tissues were homogenised in RLT lysis buffer (Qiagen, Hilden, Germany) and DNA-free RNA was prepared using RNeasy Mini Kit and DNase I set (Qiagen, Hilden, Germany) according to manufacturer’s protocols. Identical amounts of total RNA (1µg per sample) were reverse transcribed to generate cDNA and real-time PCR was performed as previously described[17].

Measurement of cytokine levels in plasma

25µL of plasma was analysed in duplicates using the LEGENDplex™ Mouse Inflammation Panel (BioLegend, United States) according to manufacturer’s instructions.

Oxygen Glucose deprivation (OGD) of Cells

To simulate HIBI, confluent monocultures of immortalised mouse brain endothelial cells (bEND3; ATCC, BSL1) were subjected to OGD. Confluent bEND3 cells seeded on a 12-well plate (7.5 x 10⁴ cells per well) were washed once and equilibrated at 37°C before cells were subjected to oxygen glucose deprivation (OGD) in Stimulation media (DMEM in 0.5%FBS) for 6 hours before collection. anti-VCAM-CD39 (10µg/mL), ±adenosine receptor antagonist CGS-15943 (200nM) (Sigma Aldrich, Australia), was then added. Next, cells were transferred to a hypoxic chamber (Modular Incubator Chamber; Billups-Rotheburg, Del Mar, CA, USA) which was flushed (6 min) with 100% nitrogen. The hypoxic chamber was kept humidified at 37°C for the duration of the experiment (6 hours). Anaerobic conditions were confirmed with the Anaerotest strip (Merck Millipore, Germany).

Cell death assay

Necrotic cell death was assessed by the lactate dehydrogenase (LDH) assay. Media and lysates of cells were collected and the LDH assay was run according to manufacturer protocol (Roche, Australia).

Statistical analysis

was performed using Prism 9 software (GraphPad, US). Normality tests were run to determine subsequent statistical test. Confirming normality, comparison of experimental datasets was performed by one-way ANOVA with Dunnett’s post-hoc correction or two-way ANOVA with Sidak’s or Dunnett’s post-hoc correction as stated. Non-normal datasets were compared with Kruskal-Wallis test with Dunn’s multiple comparisons test. Differences between two groups were assessed by two-tailed student t-tests (unpaired or unpaired with Welch’s correction for parametric data and Mann-Whitney test for non-parametric data). P < 0.05 was considered significant.

ADP	Adenosine diphosphate
ANOVA	Analysis of Variance
ATP	Adenosine triphosphate
BBB	Blood brain barrier
BCA	Bicinchoninic acid
bEND3	Brain endothelial cells
cDNA	Complementary dna
DCAL	Double carotid artery ligation
DMEM	Dulbecco's modified eagle medium
DMSO	Dimethyl sulfoxide
eATP	Extracellular adenosine triphosphate
HIBI	Hypoxic ischaemic brain injury
HIF-1	Hypoxia-inducible factor
HRP	Horseradish peroxidase
IRI	Ischaemia-reperfusion injury
IL	Interleukin
LDH	Lactate dehydrogenase
MRI	Magnetic resonance imaging
OGD	Oxygen glucose deprivation
PBS	Phosphate-buffered saline
PFA	Paraformaldehyde
RT-PCR	Real time polymerase chain reaction
ScFv	Single chain fragment variable
TBS	Tris-buffered saline
TNF-α	Tumour necrosis factor-α
VCAM-1	Vascular cell adhesion molecule 1
VLA-4	Very late antigen-4
vWF	Von willebrand factor

Ethics approval- All experiments were approved by Alfred Research Alliance Ethics Committee (ethics applications E/1683/18/M) in accordance with Australian code for the care and use of animals for scientific purposes 8^th Ed 2013 and in compliance with ARRIVE guidelines for reporting animal experiments.

Availability of data and materials- All data generated or analysed during this study are included in this published article and its supplementary information files.

Competing interests-The authors have declared that no conflict of interest exists.

Funding- NHMRC Project grant APP1141046 (HN) and Bethlehem Griffiths Research Foundation Grant (#1708; HN and MS).

Author Contributions-

NL: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Roles/Writing - original draft, Writing - review & editing.

IS, CS, IC, JC: Data curation, Investigation.

DKW, RB: MRI- Data curation, Software, Investigation, Methodology

SCR: Manuscript review and editing

HN: Conceptualization, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Writing - review & editing.

MS: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Validation, Roles/Writing - original draft, Writing - review & editing.

Acknowledgements -We acknowledge the Monash Histology Platform and the Monash Microimaging Platform, Monash University, for the provision of instrumentation, training and technical support. The authors acknowledge the facilities and scientific and technical assistance of the National Imaging Facility (NIF), a National Collaborative Research Infrastructure Strategy (NCRIS) capability at Monash Biomedical Imaging (MBI), a Technology Research Platform at Monash University.

Authors' information-

Corresponding author:Dr Maithili Sashindranath, Australian Centre for Blood Diseases, Central Clinical School, Monash University

Present address: Monash AMREP building, Level 1, Walkway, via The Alfred Centre, 99 Commercial Road, Melbourne, VIC 3004, Australia.

Email: [email protected]

Phone: +61 3 9903 0155

Fax: +61 3 9903 0228

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Endothelial -targeted CD39 is protective in a mouse model of global forebrain ischaemia

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