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NLRP3 Inflammasome as a Therapeutic Target for Ischaemic Stroke: Are We Really There Yet?

Melissa Trotman-Lucas, PhD

Lemarchand E, Barrington J, Chenery A, Haley M, Coutts G, Allen JE, et al. Extent of Ischemic Brain Injury After Thrombotic Stroke Is Independent of the NLRP3 (NACHT, LRR and PYD Domains-Containing Protein 3) Inflammasome. Stroke. 2019;50:1232-1239.

Inflammation plays a key role in the fight against infection. However, following ischaemic brain injury, inflammation can play a very different role, exacerbating the severity of damage. Inflammation results in long lasting, ongoing damage from the onset of vessel blockage through to and during reperfusion of the ischaemic brain area. One possible player within the inflammation related post-stroke damage is the NLR family pyrin domain containing 3 (NLRP3) inflammasome. During ischaemic brain injury, NLRP3 senses multiple stroke-induced stimuli leading to the recruitment of the adaptor protein ASC (the apoptosis-associated speck-like pro-caspase-1) resulting in caspase 1 production leading to downstream IL-1β and IL-18 production and release. IL-1β is well-reported to have significant pro-inflammatory and pro-apoptotic effects during acute ischaemic stroke.   

A recent study by Lemarchand et al., published in Stroke, sought to determine the importance of NLRP3 to the damage occurring following ischaemic brain damage. Previous studies have reported associations between NLRP3 and an increase in the severity of ischaemic brain injury, leading to the suggestion that targeting NLRP3 could be a potential therapeutic avenue. These previous studies report NLRP3 inhibition to be protective during ischaemia, alongside data showing that mice deficient in NLRP3 show decreased damage when compared to WT counterparts. However, contrary to this, the group responsible for the paper discussed here have previously reported that ischaemic brain injury develops independent of the NLRP3 inflammasome in a rodent model of stroke, suggesting instead that the NLRC4 (NLR family, CARD containing 4) and AIM2 (absent in melanoma 2) inflammasomes contribute to the resulting brain injury, independent of NLRP3. Lemarchand et al. sought to categorically determine the role of NLRP3 in ischaemic stroke damage, using genetic and pharmacological inhibition of NLRP3. Furthermore, to increase the robustness of the data, the group utilized the FeCl3 (ferric chloride induced thrombosis) model of preclinical ischaemic stroke, where FeCl3 soaked strips are applied to the middle cerebral artery causing localized and immediate thrombus formation, a model that may have considerable clinical relevance.   

Reconsidering Role of Immune System in Neuropathophysiology After Stroke

Lin Kooi Ong, PhD

Perego C, Fumagalli S, Miteva K, Kallikourdis M, De Simoni M-G. Combined Genetic Deletion of IL (Interleukin)-4, IL-5, IL-9, and IL-13 Does Not Affect Ischemic Brain Injury in Mice. Stroke. 2019;50:2207–2215

Primary brain injury occurs immediately after the onset of stroke, and triggers a cascade of immune responses including glial activation, recruitment of peripheral immune cells and release of cytokines and chemokines. These inflammation responses may aggravate brain injury by enhancing oxidative stress, production of neurotoxic proteins and disruption of neurovascular unit. On the other hand, inflammation may also participate in waste clearance, production of neurotropic factors and support the survivor of neurons. The recognition of the crucial role of inflammation after stroke has motivated stroke researchers to investigate novel interventions to target brain inflammation processes, leading to improve neurological outcome.

Plasma, but Not Endothelial Fn-EDA, Promotes Ischemic Thrombo-Inflammation

Kristina Shkirkova, BSc

Dhanesha N, Chorawala MR, Jain M, Bhalla A, Thedens D, Nayak M, et al. Fn-EDA (Fibronectin Containing Extra Domain A) in the Plasma, but Not Endothelial Cells, Exacerbates Stroke Outcome by Promoting Thrombo-Inflammation. Stroke. 2019;50:1201–1209.

Reperfusion with mechanical thrombectomy and recombinant tissue plasminogen activator is a standard of care for patients with ischemic stroke. However, there is evidence from animal and clinical studies that the process of reperfusion contributes to vascular inflammation, secondary thrombosis and oxidative stress. The molecular mechanisms of this thrombo-inflammatory injury are not well understood.

The recent study by Dhanesha et al. published in Stroke examined the role of cellular fibronectin containing extra domain A (Fn-EDA) in thrombo-inflammatory injury. Fn-EDA is a glycoprotein that is present in a cellular form on the endothelial cells of the arteries and in a non-cellular form in the blood plasma. Previous studies have shown a significant elevation in the levels of plasma Fn-EDA in patients with cardiovascular disease. Additionally, severe vascular dysfunction in stroke is associated with increased expression of cellular Fn-EDA in activated endothelial cells. The aim of this study was to investigate contribution of plasma versus cellular Fn-EDA on stroke injury.

Phosphodiesterase-3 Inhibitors: The New Kid on the Block

Victor J. Del Brutto, MD

Bieber M, Schuhmann MK, Volz J, Kumar GJ, Vaidya JR, Nieswandt, et al. Description of a Novel Phosphodiesterase (PDE)-3 Inhibitor Protecting Mice From Ischemic Stroke Independent From Platelet Function. Stroke. 2018;50:478–486.

Inhibition of phosphodiesterase-3 (PDE-3) in platelets increases intracellular cAMP levels resulting in blockage of platelet aggregation induced by collagen, adenosine diphosphate, arachidonic acid, and epinephrine. In addition, PDE-3 inhibitors have a pleiotropic effect over blood vessels, which include arteriolar vasodilation, endothelial repair, smooth muscle anti-proliferative effect, and reduction of endothelial inflammatory response.

Although considered to have a central antiplatelet mechanism of action, PDE-3 inhibitors exert its vascular protective effect through the diverse therapeutic targets listed above. Cilostazol, a PDE-3 inhibitor prototype, is often used chronically in patients with peripheral vascular disease, as well as for coronary artery disease and stroke secondary prevention, particularly in Asian countries. There is growing evidence on the long-term efficacy and safety of cilostazol used among patients with non-cardioembolic stroke, especially when used in combination with aspirin or clopidogrel. However, little is known about its neuroprotective effects during acute ischemic injury.

Diabetic Condition Worsens Functional Deficits After Stroke

Lin Kooi Ong, PhD

Ma S, Wang J, Wang Y, Dai X, Xu F, Gao X, et al. Diabetes Mellitus Impairs White Matter Repair and Long-Term Functional Deficits After Cerebral Ischemia. Stroke. 2018

This article by Wang and colleagues aimed to investigate the impact of diabetes on brain recovery after stroke using a preclinical model, comparing wild type male mice to diabetes (db/db) mice. The team observed a significant decrease in sensorimotor performance in diabetes mice after stroke. It should be noted that the team did not observe deficit in memory function using the Morris water maze. There was an exacerbated white matter damage at both structural and functional levels. Further, there was an enhanced inflammatory response at the white matter in diabetes mice after stroke, such as a shift of microglia/macrophage to pro-inflammatory phenotype and higher levels of IL-1β and IL-6 expression. The inflammatory environment inhibited oligodendrogenesis, a brain repair mechanism to generate new myelinating oligodendrocytes. These findings provide compelling preclinical evidence that diabetic condition exacerbates functional deficits after stroke.

Article Commentary: “Synergistic Effects of Enriched Environment and Task-Specific Reach Training on Poststroke Recovery of Motor Function”

Kate Hayward, PhD, PT

Jeffers MS, Corbett D. Synergistic Effects of Enriched Environment and Task-Specific Reach Training on Poststroke Recovery of Motor Function. Stroke. 2018

Studies have previously demonstrated the efficacy of environmental enrichment and task-specific training to promote post-stroke recovery. The premise is that enrichment creates a neuroplastic milieu that is permissive for recovery, and task-specific training capitalizes on this environment to induce neuroplastic changes and promote motor recovery. Despite the efficacy of this synergistic approach, the respective contribution of each of these components had not been directly compared until this paper by Jeffers and Corbett (1).

This study demonstrated that the combination of environmental enrichment plus task-specific reach training (environment+reach) resulted in significant improvements in reaching (single-pellet retrieval) at both 4- and 9- weeks post-stroke compared to reach training alone or enrichment alone. Further, the enrichment+reach group was the only group that did not differ significantly from the sham group (no stroke) at 4- and 9-weeks post stroke. This indicates significant functional recovery had occurred; all groups were significantly impaired compared to sham at initial post-stroke assessment.

Investigation of Iron Overloaded Mice Administered Tissue Plasminogen Activator for Acute Ischemic Stroke

Kara Jo Swafford, MD

García-Yébenes I, García-Culebras A, Peña-Martínez C, Fernández-López D, Díaz-Guzmán J, Negredo P, et al. Iron Overload Exacerbates the Risk of Hemorrhagic Transformation After tPA (Tissue-Type Plasminogen Activator) Administration in Thromboembolic Stroke Mice. Stroke. 2018

Oxidative stress, activation of proteases and infiltration of circulating white cells are involved in short-term blood brain barrier (BBB) damage and hemorrhagic transformation (HT) after acute ischemic stroke. Iron can generate toxic reactive oxygen species associated with injury to the BBB after cerebral ischemia that may also increase HT. Some clinical studies found an increased risk of HT in the setting of iron overload. García-Yébenes, et al investigated whether iron overload increases the risk of HT with tPA in a murine model of ischemic stroke and sought to elucidate the mechanisms involved.

By |September 24th, 2018|basic sciences|0 Comments

Interview: Authors of “Future of Animal Modeling for Poststroke Tissue Repair”

A conversation with Prof. Johannes Boltze, MD, PhD, from the University of Lübeck, Germany, along with co-authors Michel M. Modo, PhD; Jukka Jolkkonen, PhD; and Marietta Zille, PhD, regarding the future of animal modeling for poststroke tissue repair.

From left, Johannes Boltze, Michel M. Modo, Jukka Jolkkonen, and Marietta Zille.

From left, Johannes Boltze, Michel M. Modo, Jukka Jolkkonen, and Marietta Zille.

Interviewed by Shashank Shekhar, MD, MS, Vascular Neurology Fellow, University of Mississippi Medical Center.

They will be discussing the paper “Future of Animal Modeling for Poststroke Tissue Repair,” published in the May issue of Stroke. The article is part of a Focused Update in Cerebrovascular Disease centered on stem cells and cell-based therapies.

Dr. Shekhar: First of all, I would like to thank Prof. Boltze and his co-authors for agreeing to do this interview. This is a very interesting paper where you have not only summarized the current animal research in tissue restoration and future trajectories in animal research for post-stroke repair, but also provided important strategies to overcome the hurdles in implementing successful and clinically relevant animal models.

Could you tell the readers why studying pre-clinical animal models for post-stroke tissue repair is important?

Dr. Boltze: True tissue repair, if it was achieved, will be a highly complicated endeavor that presumably requires numerous individual steps and the targeted modification of processes in the lesioned brain. Some of these processes may be currently unknown. Sophisticated in vitro systems, such as brain organoids, may be used to design intervention strategies towards a known mechanism on a cellular level, but the entire complexity of physiological and pathophysiological processes can only be studied in vivo so far.

Bone Marrow Derived Mononuclear Cells Improve Functional Outcomes in Animal Models of Ischemic Stroke

Mark R. Etherton, MD, PhD

Despite the advent of efficacious treatments for acute ischemic stroke, in the form of intravenous tPA and endovascular thrombectomy, post stroke disability is frequent. The prevalence of post stroke disability has served as the impetus for significant research into modalities to augment post stroke recovery. One promising approach is cellular therapy; including bone marrow derived mono-nuclear cells (BMMNCs), which have shown beneficial effects in animal models of ischemic stroke.

In this study, the authors conducted a systematic review of manuscripts using intravenous BMMNCs in animal models of ischemic stroke and performed a meta-analysis of histological and behavioral outcomes. They identified 22 studies in which the majority had assessments of common variables pertaining to infarct size and motor/functional outcomes.
While there was obvious heterogeneity among the individual studies with regards to methodologies and outcomes assessed. The pooled analysis was possible, in part, because the authors identified important shared approaches in the selection of specific animal models, timing of BMMNC injection, and outcome variables assessed (e.g. reduction in infarct size, cylinder test). BMMNC treated animals had significantly reduced infarct size (standardized mean difference -3.3, 95% CI: -4.3, -2.3) and enhanced performance on tests of sensorimotor function (cylinder test SMD -2.4, 95%CI: -3.1, -1.6).

This meta-analysis serves as an important summary of the pre-clinical data for one subtype of cellular therapy in ischemic stroke. BMMNCs have beneficial effects on infarct size and behavioral outcomes in animal models of ischemic stroke. Ideally, this study will serve as a platform on which future studies can build to target clinical trials for cellular therapies in human post stroke recovery.

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.