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basic sciences

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. 

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.