Acute Stroke Presentation, Care, and Outcomes in Community Hospitals in Northern California During the COVID-19 Pandemic.

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.

Hyperglycemia promotes tissue plasminogen activator-induced hemorrhage by Increasing superoxide production.

OBJECTIVE: Risk of intracerebral hemorrhage is the primary factor limiting use of tissue plasminogen activator (tPA) for stroke. Clinical studies have established an association between admission hyperglycemia and the risk of hemorrhage with tPA use, independent of prior diabetes. Here we used an animal model of tPA-induced reperfusion hemorrhage to determine if this clinical association reflects a true causal relationship. METHODS: Rats underwent 90 minutes of focal ischemia, and tPA infusion was begun 10 minutes prior to vessel reperfusion. Glucose was administered during ischemia to generate blood levels ranging from 5.9 ± 1.8mM (normoglycemia) to 21 ± 2.3mM. In some studies, apocynin was administered to block superoxide production by nicotinamide adenine dinucleotide phosphate (NADPH). Brains were harvested 1 hour or 3 days after reperfusion to evaluate the effects of hyperglycemia and apocynin on oxidative stress, blood-brain barrier breakdown, infarct volume, and hemorrhage volume. RESULTS: Rats that were hyperglycemic during tPA infusion had diffusely increased blood-brain barrier permeability in the postischemic territory, and a 3- to 5-fold increase in intracerebral hemorrhage volumes. The hyperglycemic rats also showed increased superoxide formation in the brain parenchyma and vasculature during reperfusion. The effects of hyperglycemia on superoxide production, blood-brain barrier disruption, infarct size, and hemorrhage were all attenuated by apocynin. INTERPRETATION: These findings demonstrate a causal relationship between hyperglycemia and hemorrhage in an animal model of tPA stroke treatment, and suggest that this effect of hyperglycemia is mediated through an increase in superoxide production by NADPH oxidase.

Pyruvate protects against experimental stroke via an anti-inflammatory mechanism.

Pyruvate, a key intermediate in glucose metabolism, was explored as a potential treatment in models of experimental stroke and inflammation. Pyruvate was administered to rodents after the onset of middle cerebral artery occlusion (MCAO). Since the extent of inflammation is often proportional to the size of the infarct, we also studied a group of animals given lipopolysaccharide (LPS) to cause brain inflammation without cell death. Following MCAO, pyruvate did not affect physiological parameters but significantly reduced infarct volume, improved behavioral tests and reduced numbers of neutrophils, microglial and NFkappaB activation. Animals given LPS showed increased microglial and NFkappaB activation which was almost completely abolished by pyruvate. Lactate, a major metabolite of pyruvate, was increased after pyruvate administration. However, administration of lactate itself did not have any anti-inflammatory effects. Pyruvate protects against ischemia possibly by blocking inflammation, but lactate itself does not appear to explain pyruvate's anti-inflammatory properties.

FasL shedding is reduced by hypothermia in experimental stroke.

Protection by mild hypothermia has previously been associated with better mitochondrial preservation and suppression of the intrinsic apoptotic pathway. It is also known that the brain may undergo apoptotic death via extrinsic, or receptor-mediated pathways, such as that triggered by Fas/FasL. Male Sprague-Dawley rats subjected to 2 h middle cerebral artery occlusion with 2 h intraischemic mild hypothermia (33 degrees C) were assayed for Fas, FasL and caspase-8 expression. Ischemia increased Fas, but decreased FasL by approximately 50-60% at 6 and 24 h post-insult. Mild hypothermia significantly reduced expression of Fas and processed caspase-8 both by approximately 50%, but prevented ischemia-induced FasL decreases. Fractionation revealed that soluble/shed FasL (sFasL) was decreased by hypothermia, while membrane-bound FasL (mFasL) increased. To more directly assess the significance of the Fas/FasL pathway in ischemic stroke, primary neuron cultures were exposed to oxygen glucose deprivation. Since FasL is cleaved by matrix metalloproteinases (MMPs), and mild hypothermia decreases MMP expression, treatment with a pan-MMP inhibitor also decreased sFasL. Thus, mild hypothermia is associated with reduced Fas expression and caspase-8 activation. Hypothermia prevented total FasL decreases, and most of it remained membrane-bound. These findings reveal new observations regarding the effect of mild hypothermia on the Fas/FasL and MMP systems.

The inflammatory response in stroke.

Recent works in the area of stroke and brain ischemia has demonstrated the significance of the inflammatory response accompanying necrotic brain injury. Acutely, this response appears to contribute to ischemic pathology, and anti-inflammatory strategies have become popular. This chapter will discuss the current knowledge of the contribution of systemic and local inflammation in experimental stroke. It will review the role of specific cell types including leukocytes, endothelium, glia, microglia, the extracellular matrix and neurons. Intracellular inflammatory signaling pathways such as nuclear factor kappa beta and mitogen-activated protein kinases, and mediators produced by inflammatory cells such as cytokines, chemokines, reactive oxygen species and arachidonic acid metabolites will be reviewed as well as the potential for therapy in stroke and hypoxic-ischemic injury.

Monitoring the protective effects of minocycline treatment with radiolabeled annexin V in an experimental model of focal cerebral ischemia.

UNLABELLED: Minocycline is an antibiotic now recognized to have antiapoptotic and antiinflammatory properties. Because of these properties, minocycline may be of benefit in reducing neuronal apoptosis from ischemia and subsequent postischemic inflammation if administered soon after a stroke. We now explore the feasibility of using (99m)Tc-annexin V, an in vivo marker of apoptosis, with SPECT to monitor the antiapoptotic effects of minocycline therapy. METHODS: CB6/F1 adult male mice underwent unilateral distal middle cerebral artery occlusion (dMCA) occlusion and were imaged and sacrificed at 1, 3, 7, or 30 d after injury. Animals were given minocycline (or vehicle) 30 min and 12 h after dMCA occlusion and then given 22.5 mg/kg twice daily for up to 7 d. Before imaging, behavioral tests were performed to evaluate the neurologic function. After imaging, brains were collected for histology and assessed for the degree of apoptosis and microglial activation. RESULTS: (99m)Tc-Annexin V uptake in injured hemispheres was significantly decreased 2- to 3-fold by minocycline at all time points. Minocyline reduced infarct size as seen histologically and improved behavioral indices as late as 30 d. Infarct volume as seen histologically correlated with radiolabeled annexin V uptake seen by SPECT. In situ fluorescent microscopy demonstrated that annexin V bound primarily to neurons at 1 and 3 d, with a shift toward microglia by 7 and 30 d. CONCLUSION: We found that minocycline significantly reduces neuronal apoptosis and infarct size and improves neurologic outcome in mice after acute focal cortical ischemia.

Microglia potentiate damage to blood-brain barrier constituents: improvement by minocycline in vivo and in vitro.

BACKGROUND: Blood-brain barrier (BBB) disruption after stroke can worsen ischemic injury by increasing edema and causing hemorrhage. We determined the effect of microglia on the BBB and its primary constituents, endothelial cells (ECs) and astrocytes, after ischemia using in vivo and in vitro models. METHODS AND RESULTS: Primary astrocytes, ECs, or cocultures were prepared with or without added microglia. Primary ECs were more resistant to oxygen-glucose deprivation/reperfusion than astrocytes. ECs plus astrocytes showed intermediate vulnerability. Microglia added to cocultures nearly doubled cell death. This increase was prevented by minocycline and apocynin. In vivo, minocycline reduced infarct volume and neurological deficits and markedly reduced BBB disruption and hemorrhage in mice after experimental stroke. CONCLUSIONS: Inhibition of microglial activation may protect the brain after ischemic stroke by improving BBB viability and integrity. Microglial inhibitors may prove to be an important treatment adjunct to fibrinolysis.

Effects of high dose of simvastatin on levels of dopamine and its reuptake in prefrontal cortex and striatum among SD rats.

Statins are increasingly being used for the treatment of a variety of conditions beyond their original indication for cholesterol lowering. We previously reported that simvastatin increased dopamine receptors in the rat prefrontal cortex [Q. Wang, W.L. Ting, H. Yang, P.T. Wong, High doses of simvastatin upregulate dopamine D(1) and D(2) receptor expression in the rat prefrontal cortex: possible involvement of endothelial nitric oxide synthase, Br. J. Pharmacol. 144 (2005) 933-939] and restored its downregulation in a model of Parkinson's disease (PD) [Q. Wang, P.H. Wang, C. McLachlan, P.T. Wong, Simvastatin reverses the downregulation of dopamine D1 and D2 receptor expression in the prefrontal cortex of 6-hydroxydopamine-induced Parkinsonian rats, Brain Res. 1045 (2005) 229-233]. Here we explore the effects of simvastatin treatment on tissue dopamine content and reuptake. Sprague-Dawley rats were given simvastatin (1 and 10 mg kg(-1)day(-1), p.o.) for 4 weeks. Brain tissue from prefrontal cortex and striatum were taken out for dopamine content and its reuptake. Using high-performance liquid chromatographic-mass spectrometer (HPLC-MS), simvastatin (10 mg kg(-1)day(-1)) was found to increase dopamine content by 110% in the striatum but decreased by 76% in the prefrontal cortex compared with the saline treated group. Dopamine (DA) reuptake was unchanged in both brain regions. These results suggest that chronic treatment with high dose of simvastatin may affect DA tissue level in prefrontal cortex and striatum without changing on DA reuptake. This may have important clinical implications in psychiatric and striatal dopaminergic disorders.

Inflammation in adult and neonatal stroke.

This chapter will discuss the current knowledge of the contribution of systemic and local inflammation in acute and sub-chronic stages of experimental stroke in both the adult and neonate. It will review the role of specific cell types and interactions among blood cells, endothelium, glia, microglia, the extracellular matrix and neurons - cumulatively called "neurovascular unit" - in stroke induction and evolution. Intracellular inflammatory signaling pathways such as nuclear factor kappa beta and mitogen-activated protein kinases, and mediators produced by inflammatory cells such as cytokines, chemokines, reactive oxygen species and arachidonic acid metabolites, as well as the modifying role of age on these mechanisms, will be reviewed as well as the potential for therapy in stroke and hypoxic-ischemic injury.

Mild hypothermia reduces tissue plasminogen activator-related hemorrhage and blood brain barrier disruption after experimental stroke.

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 1­2 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.

Microglial P2Y12 deficiency/inhibition protects against brain ischemia.

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.

Bone marrow chimeras in the study of experimental stroke.

Inflammation is known to contribute to stroke evolution, and poststroke immune responses have been documented to emanate from the brain via microglia. However, circulating immune cells are increasingly recognized to play a significant role as well. Recent work has demonstrated the importance of the peripheral circulation and stroke pathogenesis. Understanding how the peripheral circulation contributes to ischemic brain injury may reveal important therapeutic targets and strategies. The use of bone marrow chimeras can be a useful tool in understanding the relative contributions of brain resident and peripheral inflammatory responses.

NOX inhibitors as a therapeutic strategy for stroke and neurodegenerative disease.

NADPH oxidase was originally identified in immune cells as playing an important microbicidal role. In neurodegenerative and cerebrovascular diseases, inflammation is increasingly being recognized as contributing negatively to neurological outcome, with NADPH-oxidase as an important source of superoxide. Recently, several forms of this oxidase have been found in a variety of non-immune cells. Neuronal NADPH oxidase is thought to participate in longterm potentiation and intercellular signaling. However, excessive superoxide production is damaging and has been shown to play an important role in the progression of brain injury. NADPH oxidase is a multisubunit complex composed of membrane-associated gp91(phox) and p22(phox) subunits and cytosolic subunits, p47(phox), p67(phox), and p40(phox) and Rac. When NADPH oxidase is activated through phosphorylatoin of p47(phox), cytosolic subunits translocate to the cell membrane and fuse with the catalytic subunit, gp91(phox). The activated enzyme complex transports electrons to oxygen, thus producing the superoxide anion (O2˙⁻), a precursor of reactive oxygen species. The advantage of a targeted NADPH oxidase inhibitor that would inhibit the production of superoxide non-phagocytic cells is clear. To date no such therapeutically viable inhibitor exists but recent research using current inhibitors has enhanced our knowledge of the role of NADPH oxidase in CNS diseases and provides impetus to develop a very specific, potent and safe NADPH oxidase inhibitor.

NADPH oxidase in stroke and cerebrovascular disease.

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.

Digitally quantifying cerebral hemorrhage using Photoshop and Image J.

A spectrophotometric hemoglobin assay is widely used to estimate the extent of brain hemorrhage by measuring the amount of hemoglobin in the brain. However, this method requires using the entire brain sample, leaving none for histology or other assays. Other widely used measures of gross brain hemorrhage are generally semi-quantitative and can miss subtle differences. Semi-quantitative brain hemorrhage scales may also be subject to bias. Here, we present a method to digitally quantify brain hemorrhage using Photoshop and Image J, and compared this method to the spectrophotometric hemoglobin assay. Male Sprague-Dawley rats received varying amounts of autologous blood injected into the cerebral hemispheres in order to generate different sized hematomas. 24h later, the brains were harvested, sectioned, photographed then prepared for the hemoglobin assay. From the brain section photographs, pixels containing hemorrhage were identified by Photoshop and the optical intensity was measured by Image J. Identification of hemorrhage size using optical intensities strongly correlated to the hemoglobin assay (R=0.94). We conclude that our method can accurately quantify the extent of hemorrhage. An advantage of this technique is that brain tissue can be used for additional studies.