American Heart Association

basic sciences

Exendin-4: A Novel Candidate to Reduce Infarct Volume in Acute Ischemic Stroke with Hyperglycemia

Alexander E. Merkler, MD

Kuroki T, Tanaka R, Shimada Y, Yamashiro K, Ueno Y, Shimura H, et al. Exendin-4 Inhibits Matrix Metalloproteinase-9 Activation and Reduces Infarct Growth After Focal Cerebral Ischemia in Hyperglycemic Mice. Stroke. 2016

Hyperglycemia exacerbates acute brain injury and leads to worse outcomes in patients with ischemic stroke. In animal models of acute ischemic stroke, hyperglycemia is associated with increased infarct volume, increased blood-brain–barrier permeability, and hemorrhagic transformation. In order to avoid hyperlgycemia-induced brain injury, normoglycemia is recommended, and typically attained via use of insulin. Unfortunately, up to now, insulin has failed to show improvement in short-term outcomes in human studies and hypoglycemia, a not uncommon consequence of exogenous insulin is associated with further brain injury.[1] Exendin-4 is an agonist of Glucagon-like peptide-1 (a hormone secreted by the small intestines) that mitigates hyperglycemia in diabetes and has a low risk of hypoglycemia. In addition, exendin-4 has shown been shown to reduce oxidative stress and inflammation.

In the current article by Dr. Kuroki et al, the authors assess the protective effect of exendin-4 in a murine model of transient hyperglycemia in acute ischemic stroke using middle cerebral artery occlusion (MCAO). All mice underwent a 60 minute MCAO and were randomly assigned to four groups: 1) Transient hyperglycemia, 2) Transient hyperglycemia treated with insulin, 3) Transient hyperglycemia treated with exendin-4, or 4) control (no hyperglycemia). Histopathological evaluation was performed at 24 hours and 7 days after ischemic stroke.

Consistent with prior data, mice with induced hyperglycemia had significantly increased infarct volume, brain edema, and hemorrhagic transformation as compared with the control. In addition, hyperglycemia was associated with an increase in blood-brain-barrier disruption, more activation of matrix metalloproteinase-9, and a higher degree of neutrophilic infiltration in infracted tissue. Mice treated with Exendin-4, but not insulin, had attenuated levels of matrix metalloproteinase-9, decreased levels of TNF-α, and decreased neutrophilic infiltration. Furthermore, mice treated with Exendin-4 had significantly less total infarct volume at 24h and at 7 days after ischemic injury as compared to not only the control group, but also the insulin treated group. Finally, hyperglycemia decreased 7-day survival and the mice treated with Exendin-4, but not insulin had an improved survival rate.
Hyperglycemia is common in patients with ischemic stroke and leads to increased blood-brain-barrier disruption, increased inflammation, increased stroke volume, increased hemorrhagic transformation, and overall worse outcomes. Treatment of hyperglycemia in acute stroke is paramount, but by how much and by what mechanism is yet to be determined. Exendin-4 shows promise as a neuroprotective agent that can lower glucose levels and improve outcomes in acute ischemic stroke. 

Sumoylation of NCX3 a Possible Mechanism of Neuroprotection in Ischemic Preconditioning

Peggy Nguyen, MD

Cuomo O, Pignataro G, Sirabella R, Molinaro P, Anzilotti S, Scorziello A, et al. Sumoylation of LYS590 of NCX3 f-Loop by SUMO1Participates in Brain Neuroprotection Induced byIschemic Preconditioning. Stroke. 2016

Small ubiquitin-like modifier (SUMO) conjugation, or sumoylation, is a post-translational modification of various proteins similar to ubiquination, and has been noted in stress conditions including anoxia, hypothermia, and hypoxia. Changes in sumolyation patterns have been reported after brain ischemia, where it is thought to be possibly protective. To this end, the authors here attempt to further elucidate a possible mechanism underlying the role of sumoylation of the transmembrane protein NCX3, which is thought to be an effector of neuroprotection in ischemic mouse models.

Using mouse models, the authors identified 3 significant findings:
  • First, that SUMO1 conjugation does increase at various times points following induced ischemia via transient middle cerebral artery occlusion (tMCAO) (at 5 and 24 hours), after preconditioning (at 3, 5, 24, and 72 hours) and when preconditioning was combined with tMCAO (at 5 hours).
  • Second, using immunohistochemical stains, the authors identified NCX and SUMO1 colocalization to the neuronal cell bodies in the primary cortical neurons, with a probable sumoylation site in the NCX f-loop of the antiporter. 
  • Third, in SUMO1 knockdown mouse models, NCX3 expression decreased 72 hours after tMCAO  and after preconditioning + tMCAO and displayed a significant increase in ischemic volume after tMCAO at 24 and 72 hours after tMCAO induction.

Identifying targets for neuroprotection seems to be the next frontier in the world of stroke research. This takes us one step closer to characterizing the mechanisms underlying the possible neuroprotectant effect of ischemic preconditioning, whereby targeting either sumoylation of NCX, or regulation of NCX itself, may lead to the development of better neuroprotectants.

Vascular Cell Senescence Contributes To Blood-Brain Barrier Breakdown

Ilana Spokoyny, MD 

Accumulating senescent cells in tissues is known to contribute to age-related systemic organ dysfunction. The authors investigated whether a similar process happens in the cerebral vasculature, leading to compromised bloodbrain barrier and potentially contributing to neurodegenerative and cerebrovascular diseases. The bloodbrain barrier (BBB) is kept intact by endothelial cells (ECs) forming tight junctions, and these ECs are covered by pericytes, astrocyte end-feet, and the capillary basement membrane. BBB integrity is compromised by aging, but the exact process by which this occurs is still unknown. We know that senescent cells limit the regeneration potential of tissues and that there are more senescent vascular smooth muscle cells and endothelial cells found in aged peripheral vessels and atherosclerotic lesions, but the effects of increased senescent cells on the BBB have not been well studied. 

The authors used both an in vitro model made of endothelial cells, pericytes, and astrocytes; and an in vivo model in mice with accelerated aging phenotypes. In the in vitro model using senescent ECs and PCs, tight junction structure and barrier integrity were significantly impaired compared with the model using young ECs and PCs. The authors also determined that the reduced BBB integrity is due to altered TJ structure and distribution in the EC layer, rather than with decreased TJ protein expression.
The in vivo model also demonstrated this, with an exacerbation of senescence and compromised BBB integrity. Specifically, the coverage of cortical microvessels by tight junction proteins was impaired, but the coverage of microvessels by astrocytuc end-feet was not altered.

Limitations are noted, especially in the in vitro model, due to needing to keep the three cell types in different media (ECs in one, PCs and astrocytes in another); so  the possibility exists that if they were mixed we would see different effects. This is compensated by the in vivo model, however. Overall, this is an important study demonstrating a critical link and setting the foundation for future diagnostic and therapeutic advances in cerebrovascular and neurodegenerative disorders. 

Combined G-CSF and BMC Treatment Increases Hemorrhage Transformation Following Ischemia in Mice

Jay Shah, MD

Strecker J-K, Olk J, Hoppen M, Gess B, Diederich K, Schmidt A, et al. Combining Growth Factor and Bone Marrow Cell Therapy Induces Bleeding and Alters Immune Response After Stroke in Mice. Stroke. 2016

Cell-based therapies, such as transplantation of exogenous cells or stimulation of endogenous cells, for stroke are lacking. Bone marrow-derived cells (BMCs) are a viable option as they allow autologous transplantation and can be mobilized by granulocyte colony-stimulating factor (G-CSF). Cytokines released by BMCs contain neuroprotective properties and stimulate endogenous repair thereby potentially improving outcomes following ischemia. In this animal study, the authors’ hypothesis is that the combination of G-CSF and BMC is more effective than either single treatment in mice subjected to focal ischemia. There were 4 randomly assigned groups:  placebo, G-CSF, BMC, and G-CSF and BMC. Ischemia was induced by occlusion of the middle cerebral artery for 30 minutes. Treatment occurred 90 minutes after ischemia induction. Rotarod and cylinder test were used to assess motor performance and forelimb activity, respectively. At 1 or 7 days after ischemia, brains were harvested to assess for ischemic damage by various techniques.

Results revealed that mice treated with G-CSF alone showed increased running time compared to G-CSF and BMC group. Infarct volumes were reduced in G-CSF group compared to placebo. Parenchymal bleeding was only seen in mice treated with BMCs or combination therapy. There was evidence of increased astrogliosis in mice treated with G-CSF and BMC as evident by increased GFAP-signal intensity and increased mean blood vessel diameter.  

An unexpected result of this study was the detrimental effects of combination therapy, namely due to hemorrhagic transformation. Potential mechanisms to account for this complication include altered immune cell polarization, excessive astrogliosis, increased number of dilated blood vessels and blood brain barrier loss. There are several confounding variables. Foremost, time is certainly a crucial element in ischemia. In this study, treatment was conducted at a single time point. Treatment with BMC at a different time, perhaps earlier, may potentially alter results. It’s well established that molecular changes occur within minutes of ischemia and early intervention may be crucial to consistently affect outcome before irreversible molecular changes occur. Secondly, there is an assumption that all mice were clinically equal following ischemia but this may not be accurate. Lastly, additional studies will need to be conducted to further evaluate G-CSF and its impact on blood brain barrier. Cell-based treatment in animal models may not translate into human studies given the complex heterogeneity and multitude of variables that cannot be controlled. 

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.