Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) was originally identified in immune cells as playing an important microbicidal role. In stroke and cerebrovascular disease, inflammation is increasingly being recognized as contributing negatively to neurological outcome, with NOX as an important source of superoxide. Several labs have now shown that blocking or deleting NOX in the experimental stroke models protects from brain ischemia. Recent work has implicated glucose as an important NOX substrate leading to reperfusion injury, and that NOX inhibition can improve the detrimental effects of hyperglycemia on stroke. NOX inhibition also appears to ameliorate complications of thrombolytic therapy by reducing blood-brain barrier disruption, edema formation, and hemorrhage. Further, NOX from circulating inflammatory cells seems to contribute more to ischemic injury more than NOX generated from endogenous brain residential cells. Several pharmacological inhibitors of NOX are now available. Thus, blocking NOX activation may prove to be a promising treatment for stroke as well as an adjunctive agent to prevent its secondary complications.

Therapeutic hypothermia has shown neuroprotective promise, but whether it can be used to improve outcome in stroke has yet to be determined in patients. Recombinant tissue plasminogen activator (rt-PA) is only given to a minority of patients with acute ischemic stroke, and is not without risk, namely significant brain hemorrhage.We explored whether mild hypothermia, in combination with rt-PA, influences the safety of rt-PA. Mice were subjected to middle cerebral artery occlusion (MCAO) using a filament model, followed by 24 hours reperfusion.Two paradigms were studied. In the first paradigm, cooling and rt-PA treatment began at the same time upon reperfusion, whereas in the second paradigm, cooling began soon after ischemia onset, and rt-PA began after rewarming and upon reperfusion. Experimental groups included: tPA treatment at normothermia (37°C), rt-PA treatment at hypothermia (33°C), no rt-PA at normothermia, and no rt-PA treatment at hypothermia. Infarct size, neurological deficit scores, blood brain barrier (BBB) permeability, brain hemorrhage, and expression of endogenous tissue plasminogen activator (tPA) and its inhibitor, plasminogen activator inhibitor (PAI-1) were assessed. For both paradigms, hypothermia reduced infarct size and neurological deficits compared to normothermia, regardless of whether rt-PA was given. rt-PA treatment increased brain hemorrhage and BBB disruption compared to normothermia, and this was prevented by cooling. However, mortality was higher when rt-PA and cooling were administered at the same time, beginning 1–2 hours post MCAO. Endogenous tPA expression was reduced in hypothermic mice, whereas PAI-1 levels were unchanged by cooling. In the setting of rt-PA treatment, hypothermia reduces brain hemorrhage, and BBB disruption, suggesting that combination therapy with mild hypothermia and rt-PA appears safe.

Objective

Microglia are among the first immune cells to respond to ischemic insults. Triggering of this inflammatory response may involve the microglial purinergic GPCR, P2Y12, activation via extracellular release of nucleotides from injured cells. It is also the inhibitory target of the widely used antiplatelet drug, clopidogrel. Thus, inhibiting this GPCR in microglia should inhibit microglial mediated neurotoxicity following ischemic brain injury.

Methods

Experimental cerebral ischemia was induced, in vitro with oxygen-glucose deprivation (OGD), or in vivo via bilateral common carotid artery occlusion (BCCAO). Genetic knock-down in vitro via siRNA, or in vivo P2Y12 transgenic mice (P2Y12-/- or P2Y12+/-), or in vivo treatment with clopidogrel, were used to manipulate the receptor. Neuron death, microglial activation, and microglial migration were assessed.

Results

The addition of microglia to neuron-astrocyte cultures increases neurotoxicity following OGD, which is mitigated by microglial P2Y12 deficiency (P<0.05). Wildtype microglia form clusters around these neurons following injury, which is also prevented in P2Y12 deficient microglia (P<0.01). P2Y12 knock-out microglia migrated less than WT controls in response to OGD-conditioned neuronal supernatant. P2Y12 (+/-) or clopidogrel treated mice subjected to global cerebral ischemia suffered less neuronal injury (P<0.01, P<0.001) compared to wild-type littermates or placebo treated controls. There were also fewer microglia surrounding areas of injury, and less activation of the pro-inflammatory transcription factor, nuclear factor Kappa B (NFkB).

Interpretation

P2Y12 participates in ischemia related inflammation by mediating microglial migration and potentiation of neurotoxicity. These data also suggest an additional anti-inflammatory, neuroprotective benefit of clopidogrel.

Background and purpose

Shelter-in-place (SIP) orders implemented to mitigate severe acute respiratory syndrome coronavirus 2 spread may inadvertently discourage patient care-seeking behavior for critical conditions like acute ischemic stroke. We aimed to compare temporal trends in volume of acute stroke alerts, patient characteristics, telestroke care, and short-term outcomes pre- and post-SIP orders.

Methods

We conducted a cohort study in 21 stroke centers of an integrated healthcare system serving 4.4+ million members across Northern California. We included adult patients who presented with suspected acute stroke and were evaluated by telestroke between January 1, 2019, and May 9, 2020. SIP orders announced the week of March 15, 2020, created pre (January 1, 2019, to March 14, 2020) and post (March 15, 2020, to May 9, 2020) cohort for comparison. Main outcomes were stroke alert volumes and inpatient mortality for stroke.

Results

Stroke alert weekly volume post-SIP (mean, 98 [95% CI, 92-104]) decreased significantly compared with pre-SIP (mean, 132 [95% CI, 130-136]; P<0.001). Stroke discharges also dropped, in concordance with acute stroke alerts decrease. In total, 9120 patients were included: 8337 in pre- and 783 in post-SIP cohorts. There were no differences in patient demographics. Compared with pre-SIP, post-SIP patients had higher National Institutes of Health Stroke Scale scores (P=0.003), lower comorbidity score (P<0.001), and arrived more often by ambulance (P<0.001). Post-SIP, more patients had large vessel occlusions (P=0.03), and there were fewer stroke mimics (P=0.001). Discharge outcomes were similar for post-SIP and pre-SIP cohorts.

Conclusions

In this cohort study, regional stroke alert and ischemic stroke discharge volumes decreased significantly in the early COVID-19 pandemic. Compared with pre-SIP, the post-SIP population showed no significant demographic differences but had lower comorbidity scores, more severe strokes, and more large vessel occlusions. The inpatient mortality was similar in both cohorts. Further studies are needed to understand the causes and implications of care avoidance to patients and healthcare systems.