American Heart Association

basic sciences

Matrix Metalloprotease 3 Exacerbates Hemorrhagic Transformation and Worsens Functional Outcomes in Hyperglycemic Stroke

Peggy Nguyen, MD

Hafez S, Abdelsaid M, El-Shafey S, Johnson MH, Fagan SC, Ergul A. Matrix Metalloprotease 3 Exacerbates Hemorrhagic Transformation and Worsens Functional Outcomes in Hyperglycemic Stroke. Stroke. 2016

Hyperglycemia in the setting of acute ischemic stroke has been associated with worsened outcomes, with an association seen with worsened vascular injury, increased infarct size, and hemorrhagic transformation; however, the mechanisms through which this occurs is not well elucidated. Here, matrix metalloprotease 3 (MMP3), which has previously been shown to contribute to tPA induced hemorrhagic transformation, was identified as a possible mediator of injury in hyperglycemic stroke.

Using animal models, researchers identified increased MMP3 activity n the cerebral vasculature with induced hyperglycemia, and found that the use of MMP3 inhibitor at reperfusion reduced MMP3 activity in the brain. When MMP3 was inhibited in the hyperglycemic animal stroke model, hemorrhagic transformation and edema, albeit not infarct size, were reduced. Early inhibition did not lead to a mortality benefit, but did reduce hyperglycemic induced neurobehavioral deficits and improved functional outcomes at days 3 and 7 after stroke. A similar association was seen in MMP3 knockdown mice in terms of reduced brain edema, without effect on infarct size, as well as increase in neurobehavioral composite score and grip strength.

Within this animal model, then, MMP3 does seem to be a mediator of injury in hyperglycemic stroke and may, at least partially, account for some of the poorer outcomes seen in hyperglycemic stroke. More importantly, the model is suggestive of MMP3 as a potential target for neuroprotection, not only in acute ischemic stroke, but possibly in hemorrhagic stroke, given its protective effect in hemorrhagic transformation, which is hugely important for hyperacute clinical research of neuroportection in the pre-emergency room setting.

D-4F is a promising neuroprotectant for stroke recovery

Peggy Nguyen, MD

Cui X, Chopp M, Zacharek A, Cui C, Yan T, Ning R, and Chen J. D-4F Decreases White Matter Damage After Stroke in Mice. Stroke. 2016

D-4F, an apolipoprotein analogue, has been studied as a potential therapy in the treatment of atherosclerosis. Prior studies suggest that D-4F may enhance HDL function, which could be potential target for neuroprotection in stroke. Here, the authors investigated the in vivo effect of D-4F on serum biomarkers, stroke outcome, inflammation and white matter recovery in a mouse stroke mode. Significantly, they found:

  • D-4F treatment at all dosages (2, 4, 8, 16 and 32 mg/kg daily for 7 days) had no significant effect on serum levels of HDL, total cholesterol, triglyceride and infarction volume but treatment at 16 mg/kg daily for 7 days did improve functional outcome after stroke as measured by the modified Neurological Severity Score (mNSS) at 7 days and as measured by the foot-fault test at 3 and 7 days after stroke.
  • D-4F treatment decreased white matter damage and increased oligodencrocyte progenitor cells after stroke. Accordingly, a better functional outcome (lower mNSS) significantly correlated with higher axonal density.
  • D-4F treatment decreased inflammatory markers TLR4 and TNFα but increased IGF1, which promotes neuronal and oligodendrocyte differentiation, proliferation, myelination and may serve as the pathway for via which D-4F treatment exerts its protective effects.

Both D-4F and IGF1 treatment of primary cortical neuron cultures in vitro increased neurite outgrowth significantly.

So far, none of the investigated neuroprotectants have been proven to be beneficial for stroke outcomes. Although this study is obviously limited by its subjects (being mouse models), the results show promise in identification of a possible neuroprotectant for stroke.

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IV tPA has an important role even in the presence of endovascular therapy.

Russell Mitesh Cerejo, MD

Desilles JP, Loyau S, Syvannarath V, Gonzalez-Valcarcel J, Cantier M, Louedec L, et al. Alteplase Reduces Downstream Microvascular Thrombosis and Improves the Benefit of Large Artery Recanalization in Stroke. Stroke. 2015

Dr. Jean-Philippe Desilles and colleagues studied the effects of IV tPA in downstream microvascular thrombosis. In their animal model they used rats with middle cerebral artery (MCA) occlusion for 60 minutes and then randomized them to IV tPA vs saline. They used Intravital fluorescence microscopy to visualize the circulating blood cells and fibrinogen. They also assessed for infarct volumes and neurological deficit. After sacrifice of the rats, they also assessed for patency of the microvasculature and plasma fibrinogen levels.

The authors found that immediately after occlusion of the MCA, there was early and pronounced adhesion of leukocytes and platelets that occurred almost exclusively in the venous compartment and persisted up to 1 hour post recanalization. There also was microthrombosis characterized by intraluminal deposition of fibrinogen developed in post-capillary micro vessels at sites of leukocyte and platelet accumulation, which caused complete cessation of blood flow.

Intravenous alteplase injection at 10 mg/kg 30 minutes after initiating MCA occlusion significantly reduced infarct size and also improved neurological deficits, evaluated 24 hours. In a subgroup of rats that were sacrificed before recanalization of the MCA occlusion showed that early administration of alteplase improves microvascular patency and stroke outcome indicating that alteplase exerts beneficial effects independently of its action on proximal arterial recanalization. Alteplase fibrinolytic activity induces a rapid and profound hypofibrinogenemia that prevents platelet aggregation and promotes disaggregation of freshly formed platelet aggregates.

These finding suggest that the use of IV alteplase treatment in eligible patients supplement endovascular therapy to improve the benefit of large artery recanalization, through targeting of downstream micro vascular thrombosis.

PD-L1 monoclonal antibody for prevention of inflammation after acute ischemic stroke

Alexander E. Merkler, MD

Bodhankar S, Chen Y, Lapato A, Dotson AL, Wang J, Vandenbark AA, et al. PD-L1 Monoclonal Antibody Treats Ischemic Stroke by Controlling Central Nervous System Inflammation. Stroke. 2015
Treatment options for acute ischemic stroke are limited. Although tPA and endovascular clot retrieval are beneficial, they are time-limited treatments which are not available to the majority of patients with stroke. Although targeting post-stroke inflammation is not a new concept, new advances in immunotherapy may lead to huge advances in post-stroke therapy.

Post-stroke inflammation begins with release of reactive oxygen species, which may trigger a cascade of activating complement, platelets, and endothelial cells leading to further neurological injury. Reperfusion, one of the goals of early acute ischemic stroke treatment, may also enhance the inflammatory process and lead to additional injury to brain tissue.

Dr. Bodhankar et al assess a new immunotherapy aimed at reducing the inflammation related to post-stroke reperfusion injury. The current study builds on their prior research, which showed that Programmed Death 1 (PD-1) receptor and its two ligands (PD-L1 and PD-L2) regulate the function of inflammatory immune cells and that mice deficient in the PD-L1 ligand had smaller infarct volumes when exposed to middle cerebral artery occlusion (MCAO). Based on the potentially pathogenic role of PD-L1 ligand, in the current study, the authors assess the effectiveness of blocking PD-L1 using a monoclonal antibody and assess outcomes via measuring stroke infarct volume and neurologic function.

Mice were exposed to transient focal ischemia via 1 hour of MCAO in the right brain hemisphere followed by 96h of reperfusion. Mice were given either monoclonal anti-PD-L1 antibody or an isotype matched control 4 hours following MCAO. First, the results support the theory that stroke and/or reperfusion leads to inflammation as both groups of mice had elevated leukocyte counts in the affected hemisphere, but not in the unaffected hemisphere. Second, as compared to mice treated with the control drug, mice treated with the monoclonal anti-PD-L1 antibody had a reduced number of pro-inflammatory cells in the ischemic hemisphere, supporting the anti-inflammatory effect of the antibody. Third, and most importantly, at 96 hours, infarct volume and neurological deficit were significantly reduced in mice that received the monocloncal antibody as compared to the matched controls. Noteworthy, however, is that 5 of the 73 monocloncal antibody treated mice developed severe hemorrhage and were excluded from the analysis while not of the control mice developed this complication.

Overall, the study provides exciting new vigor to post-stroke immunotherapy treatment. Blocking PD-L1 may be a viable treatment strategy in reducing post-stroke inflammation and thereby stroke infarct volume and neurological injury.

Targeting WNK3 in experimental stroke

Michelle Christina Johansen, MD

Begum G, Yuan H, Kahle KT, Li L, Wang S, Shi Y, et al. Inhibition of WNK3 Kinase Signaling Reduces Brain Damage and Accelerates Neurological Recovery After Stroke. Stroke. 2015 

Research targeting experimental stroke provides the foundation for future initiatives that lead to clinical implementation. Begum et al in their study investigate the WNK3-SPAK/OSR1 pathway in order to determine the role it plays in regulation of the bumetanide-sensitive Na-K-Cl cotransporter (NKCC1). This cotransporter has been shown to play an important role in the pathophysiology of experimental stroke but its exact mechanism has remained yet unexplained. By way of brief review, WNK3 (with no lysine) is a kinase that activates SPAK/OSR1 which in turn stimulates NKCC1. A similar pathway has been well researched in renal epithelial homeostasis but not in the brain. In vitro, inactivated WNK3 is a potent inhibitor of NKCC1 thus making it an attractive target for research. 

In their experiment, WNK3 wild-type (WT) and knockout (KO) mice were subjected to ischemic stroke via MCA occlusion for 60 minutes. Sensorimotor neurological deficit in the mice were evaluated post stroke. Infarct volume and hemispheric swelling was calculated postmortem and wet weight of the tissue was measured. A total of 61 mice were used in the study. All measurements were performed by investigators who were blinded to the study.

The researchers found that there was no difference in regional cerebral blood flow in the WNK3 KO and WNK3 WT littermates after MCA occlusion but after 24h of reperfusion, KO brains exhibited a marked reduction in cortical infarct volume relative to the wild type. Smaller infarcts were also noted in KO at 3 days post MCA occlusion by MRI and exhibited significantly less demyelination of the external capsule white matter tracks. This was shows to have ramifications functionally as KO mice exhibited accelerated recovery. There was decreased hemispheric swelling and albumin infiltration in KO mice. The investigators founds that focal cerebral ischemia activates NKCC1 by the WNK and SPAK/OSRI kinases and that this signaling pathway was regulated differently depending on brain region and cell type. No ischemic cell death was evident in untreated WNK3 KO neurons or in those treated with SPAK/OSR1 siRNAs (silencing RNAs).

The authors conclude that genetic inactivation of WNK3 or siRNA-mediated knockdown of SPAK/OSRI prevents demyelination and reduces primary oligodendrocyte death highlighting that this signaling complex serves as a potential target for neuroprotection and preservation of myelination following stroke. The team should be commended for working to elucidate the WNK3 pathway in the cerebrum. While this work is in its infantile state, one can only hope that WNK3 inhibition in the clinical setting would yield similar results.

Mitochondria Play an Important Role in Blood-Brain Barrier Permeability

Rizwan Kalani, MD

Doll DN, Hu H, Sun J, Lewis SE, Simpkins JW, and Ren X. Mitochondrial Crisis in Cerebrovascular Endothelial Cells Opens the Blood–Brain Barrier. Stroke. 2015 

Blood-brain barrier (BBB) dysfunction is seen in many neurological conditions, including cerebrovascular and neurodegenerative disorders. Infection after stroke is known to worsen clinical outcomes and prior preclinical studies have demonstrated that bacterial infection has effects on the BBB. In this report, Doll et al aimed to evaluate the molecular and cellular mechanisms by which this occurs.

They tested the effects of bacterial infection by mimicking it in a mouse model of stroke with intraperitoneal lipopolysaccharide (LPS) injection prior to transient middle cerebral artery occlusion (tMCAO). BBB permeability was tested by assessing the penetration of Evans blue, a dye that has high affinity for albumin, which enters brain tissue when the BBB is compromised. They found that LPS exposure significantly increased infarct volume and cerebrovascular permeability after tMCAO compared to saline-injected controls. There was higher cerebral neutrophil accumulation, measured by flow cytometry 6 hours after tMCAO, in the LPS-treated mice compared to controls. Along with this, the neutrophil to lymphocyte (NLR) ratio was significantly decreased in the blood and spleen of LPS-treated mice. In a cultured cerebrovascular endothelial cell model, they found that LPS-induced signaling through toll-like receptor 4 (TLR4) compromised oxidative phosphorylation without causing cell death. This occurred through reduction in the expression of multiple mitochondrial respiratory chain proteins. Blocking the respiratory chain pharmacologically with rotenone or oligomycin (inhibitors of respiratory chain complex I and V) in this in vitro model led to increased cell permeability and disruption of cell-cell tight junctions. This was then confirmed in vivo – the authors applied rotenone to the epidural surface of mice and showed that led to increased BBB permeability compared to controls. Finally, they then were able to demonstrate that treating mice with rotenone prior to tMCAO led to increased BBB permeability, higher stroke volumes, and worsened neurologic deficits.

This is the first study suggesting that bacterial infection may worsen the injury resulting from stroke by affecting mitochondrial metabolism in the cerebrovascular endothelium. It gives further insight into pathophysiologic mechanisms that, if also occur in humans, may provide novel pharmacologic targets for prevention/reduction in cerebral edema and potentially improving overall outcomes after stroke. Continual improvements in neuroimaging capability and molecular imaging techniques may allow for improved evaluation of BBB permeability in human subjects. This report should have implications for future translational research.

Effects of Post-Infarct MAG Antibody Treatment

Daniel Korya, MD

Barbay S, Plautz EJ, Zoubina E, Frost SB, Cramer SC, and Nudo RJ. Effects of Postinfarct Myelin-Associated Glycoprotein Antibody Treatment on Motor Recovery and Motor Map Plasticity in Squirrel Monkeys. Stroke. 2015

The regeneration of infarcted brain tissue has been a sought after feat in the neuroscience field since the conceptualization of stroke. This challenging and often elusive goal has been likened to a “holy grail” for those most concerned with this endeavor. Hundreds of molecules have been potential solutions to this puzzle, but only few have proven worthy of pursuit. One such molecule is actually an antibody called GSK249320. This antibody blocks the axon outgrowth inhibition molecule known as myelin-associated glycoprotein (MAG). So, it’s an anti-MAG antibody. MAG is a good target since it has been shown to be up-regulated in peri-infarct tissue after ischemic damage. 


In 2013, Cramer and colleagues published a study in Stroke proclaiming the safety and tolerability of escalating doses of GSK249230 in 42 post-stroke patients. In the present study, Barbay et al set out to move toward proving efficacy of GSK249320 by showing its ability to enhance recovery of skilled use of the forelimb in a non-human primate model of focal cortical ischemia. Essentially, the goal was to find out if the effects are evident in the organization of movement representations in spared cortical areas.

The study involved 9 monkeys that were either assigned to receiving the drug or placebo. First the monkeys were all taught a specific task that required the use of their distalThey were all taken to the OR and given a stroke in the distal forelimb representations (DFLs) in primary motor cortex. Afterward, they either got the drug or placebo at 24-hours post-stroke, and then weekly for seven weeks. At the end of this time period, the two groups were compared to evaluate for their ability to complete the previously learned task. Then, the monkey brains were analyzed to see if there were anatomic and histologic correlations.

This study found that the experimental group had almost immediate improvements compared with the control group. Their functional outcome was significantly improved at day 3 and day 9 (second treatment). There were some discrepancies in the analysis of the cortical areas that were explained by the length of the study. But, overall there were also significant changes neurophysiologically in the experimental groups stroke sites.