Michael Maniskas

Stroke neuroprotection revisited: Intra-arterial verapamil is profoundly neuroprotective in experimental acute ischemic stroke.

While clinical trials have now solidified the role of thrombectomy in emergent large vessel occlusive stroke, additional therapies are needed to optimize patient outcome. Using our previously described experimental ischemic stroke model for evaluating adjunctive intra-arterial drug therapy after vessel recanalization, we studied the potential neuroprotective effects of verapamil. A calcium channel blocker, verapamil is often infused intra-arterially by neurointerventionalists to treat cerebral vasospasm. Such a direct route of administration allows for both focused targeting of stroke-impacted brain tissue and minimizes potential systemic side effects. Intra-arterial administration of verapamil at a flow rate of 2.5 µl/min and injection volume of 10 µl immediately after middle cerebral artery recanalization in C57/Bl6 mice was shown to be profoundly neuroprotective as compared to intra-arterial vehicle-treated stroke controls. Specifically, we noted a significant (P ≤ 0.05) decrease in infarct volume, astrogliosis, and cellular apoptosis as well as a significant increase in neuronal survival and functional outcome over seven days. Furthermore, intra-arterial administration of verapamil was well tolerated with no hemorrhage, systemic side effects, or increased mortality. Thus, verapamil administered intra-arterially immediately following recanalization in experimental ischemic stroke is both safe and neuroprotective and merits further study as a potential therapeutic adjunct to thrombectomy.

Intra-arterial verapamil post-thrombectomy is feasible, safe, and neuroprotective in stroke:

Large vessel ischemic stroke represents the most disabling subtype. While t-PA and endovascular thrombectomy can recanalize the occluded vessel, good clinical outcomes are not uniformly achieved. We propose that supplementing endovascular thrombectomy with superselective intra-arterial (IA) verapamil immediately following recanalization could be safe and effective. Verapamil, a calcium channel blocker, has been shown to be an effective IA adjunct in a pre-clinical mouse focal ischemia model. To demonstrate translational efficacy, mechanism, feasibility, and safety, we conducted a group of translational experiments. We performed in vivo IA dose–response evaluation in our animal stroke model with C57/Bl6 mice. We evaluated neuroprotective mechanism through in vitro primary cortical neuron (PCN) cultures. Finally, we performed a Phase I trial, SAVER-I, to evaluate feasibility and safety of administration in the human condition. IA verapamil has a likely plateau or inverted-U dose–response with a defined toxicity level in mice (LD50 16–17.5 mg/kg). Verapamil significantly prevented PCN death and deleterious ischemic effects. Finally, the SAVER-I clinical trial showed no evidence that IA verapamil increased the risk of intracranial hemorrhage or other adverse effect/procedural complication in human subjects. We conclude that superselective IA verapamil administration immediately following thrombectomy is safe and feasible, and has direct, dose–response-related benefits in ischemia.

Selective intra-arterial drug administration in a model of large vessel ischemia.

With continuing disconnect between laboratory stroke treatment models and clinical stroke therapy, we propose a novel experimental model to study stroke and vessel recanalization that mirrors acute management of large vessel stroke, with concomitant directed pharmacotherapy. Using the tandem transient ipsilateral common carotid/middle cerebral artery occlusion (MCAO) model to induce stroke in mice we then added selective intra-arterial (IA) drug administration for directed pharmacotherapy. The IA model uses micro-angio tubing placed at the bifurcation of the CCA to selectively administer the drug to the internal carotid distribution. We have shown that delivery of pharmacotherapy agents selectively through an IA injection is feasible in a mouse model, which will permit studies involving pharmacotherapy, transgenic modification, and/or a combination. Our IA model has similarities to previously published models of IA injection but differs in that we do not leave an indwelling micro-port or catheter in our animals, which is not clinically relevant as it does not reflect the human condition or current clinical management. Furthermore, we optimized our model to selectively direct therapy to the ipsilateral, stroke affected hemisphere. By developing an IA drug delivery model that mirrors clinical conditions, we are bridging the gap between basic stroke research and what is standard practice in acute ischemic stroke intervention. The IA model of drug delivery can target agents directly to the site of injury while blunting systemic effects, dose penetration issues, and administration delay that have plagued the intraperitoneal and oral drug administration models.

Intra-arterial nitroglycerin as directed acute treatment in experimental ischemic stroke.

Background Nitroglycerin (also known as glyceryl trinitrate (GTN)), a vasodilator best known for treatment of ischemic heart disease, has also been investigated for its potential therapeutic benefit in ischemic stroke. The completed Efficacy of Nitric Oxide in Stroke trial suggested that GTN has therapeutic benefit with acute (within 6 hours) transdermal systemic sustained release therapy. Objective To examine an alternative use of GTN as an acute therapy for ischemic stroke following successful recanalization. Methods We administered GTN IA following transient middle cerebral artery occlusion in mice. Because no standard dose of GTN is available following emergent large vessel occlusion, we performed a dose–response (3.12, 6.25, 12.5, and 25 µg/µL) analysis. Next, we looked at blood perfusion (flow) through the middle cerebral artery using laser Doppler flowmetry. Functional outcomes, including forced motor movement rotor rod, were assessed in the 3.12, 6.25, and 12.5 µg/µL groups. Histological analysis was performed using cresyl violet for infarct volume, and glial fibrillary activating protein (GFAP) and NeuN immunohistochemistry for astrocyte activation and mature neuron survival, respectively. Results Overall, we found that acute post-stroke IA GTN had little effect on vessel dilatation after 15 min. Functional analysis showed a significant difference between GTN (3.12 and 6.25 µg/µL) and control at post-stroke day 1. Histological measures showed a significant reduction in infarct volume and GFAP immunoreactivity and a significant increase in NeuN. Conclusions These results demonstrate that acute IA GTN is neuroprotective in experimental ischemic stroke and warrants further study as a potentially new stroke therapy.

Internal carotid artery stenosis: A novel surgical model for moyamoya syndrome

Moyamoya is a cerebrovascular disorder characterized by progressive stenosis of the intracranial internal carotid arteries. There are two forms: Disease and Syndrome, with each characterized by the sub-population it affects. Moyamoya syndrome (MMS) is more prominent in adults in their 20’s-40’s, and is often associated with autoimmune diseases. Currently, there are no surgical models for inducing moyamoya syndrome, so our aim was to develop a new animal model to study this relatively unknown cerebrovascular disease. Here, we demonstrate a new surgical technique termed internal carotid artery stenosis (ICAS), to mimic MMS using micro-coils on the proximal ICA. We tested for Moyamoya-like vasculopathies by fluorescently labelling the mouse cerebrovasculature with Di I for visualization and analysis of vessel diameter at the distal ICA and anastomoses on the cortical surface. Results show a significant narrowing of the distal ICA and anterior cerebral artery (ACA) in the Circle of Willis, as observed in humans. There is also a significant decrease in the number of anastomoses between the middle cerebral artery (MCA) and the ACA in the watershed region of the cortex. While further characterization is needed, this ICAS model can be applied to transgenic mice displaying co-morbidities as observed within the Moyamoya syndrome population, allowing a better understanding of the disease and development of novel treatments.

Bilateral carotid artery stenosis causes unexpected early changes in brain extracellular matrix and blood-brain barrier integrity in mice

Bilateral carotid artery stenosis (BCAS) is one experimental model of vascular dementia thought to preferentially impact brain white matter. Indeed, few studies report hippocampal and cortical pathology prior to 30 days post-stenosis; though it is unclear whether those studies examined regions outside the white matter. Since changes in the blood-brain barrier (BBB) permeability precede more overt brain pathology in various diseases, we hypothesized that changes within the BBB and/or BBB-associated extracellular matrix (ECM) could occur earlier after BCAS in the hippocampus, cortex and striatum and be a precursor of longer term pathology. Here, C57Bl/6 mice underwent BCAS or sham surgeries and changes in the BBB and ECM were analyzed by collagen IV (vascular basement membrane component), α5 integrin (marker of endothelial activation), claudin-5 and occludin (tight junction proteins), Evans blue (permeability marker), Ki-67 (cell proliferation marker), and GFAP and CD11b (glial cell markers) immunohistochemistry after 14 days. Significant changes in markers of cerebrovascular integrity and glial activation were detected, not only in the striatum, but also in the hippocampus and cortex. In conclusion, this study demonstrates for the first time that changes in the BBB/ECM occur shortly after BCAS and within multiple brain regions and suggests such changes might underlie the gradual development of BCAS non-white matter pathology.

O-031 Combinational therapy following elvo in a mouse model of stroke demonstrates new frontiers in neuroprotection: the mavaric trial

Stroke remains a leading cause of morbidity and mortality in the United States. Potential therapies have encountered significant barriers in attempts to translate bench to bedside research. Because of this, we have evaluated novel roles for FDA approved drugs, repurposed for treating stroke. Two such drugs, verapamil and magnesium, represent drug classes with a long history in neuroprotection trials with mixed results. Using an intra-arterial (IA) model developed in our lab, we selectively delivered our agents of interest to the stroke affected region following experimental stroke and successful recanalization. In addition, we studied the effect of the drugs on primary cortical neurons (PCN) exposed to oxygen glucose deprivation (OGD) in vitro. In vivo experiments used 16 week old C57/Bl6 male mice that underwent a tandem transient common carotid/middle cerebral artery occlusion for 60 min. Following successful recanalization, verapamil (1.76 mg/kg) and magnesium (176 mg/kg) were administered IA through the internal carotid artery using a previously determined flow rate and injection volume. Physiological measurements (heart rate and blood pressure) were monitored for potential deleterious effects during and ten minutes following IA drug administration. A neurological score was used to determine differences in functional outcome on post-stroke days (PSD) 1 and 7. On PSD 7, animals were euthanized and brains flash frozen for immunohistochemistry analysis of infarct volume, mature neurons, and apoptosis. For comparison, single doses of verapamil, magnesium and saline were also administered IA. In vitro PCNs from C57/Bl6 embryonic day 18 mice were exposed to OGD (“stroke in a dish”) for 30 min in a sealed chamber. Following 30 min OGD, glucose deficient media was replaced with normal PCN media combined with verapamil (250, 325, and 500 ng) and magnesium (0.5, 1 and 2 mM) for 24 hours. Analysis was performed using Hoechst assay for cell viability, MAP2 for neurite extension and Bcl-2 for apoptosis. Control plates did not undergo OGD but did receive drug treatment. Both in vivo and in vitro results demonstrated that combinational therapy is safe and provides neuroprotection. In vivo results demonstrated drug administration did not have a negative effect on physiological measures during and after IA administration. Infarct volume shows a significant reduction in size when compared to control animals at PSD 7. Immunohistochemistry for mature neurons showed a greater population of healthy neurons in the treated group when compared to the control group. This correlated with measurements for apoptosis, as fewer number of apoptotic cells were found in the treated infarcted region when compared to control animals. In vitro results following OGD demonstrated direct neuroprotection of combinational drug therapy using Hoechst assay analysis compared to control. Further, neurite outgrowth measurements using MAP2 showed a greater number of primary, secondary and tertiary neurites compared to control. Lastly, Bcl-2 a measure of apoptosis showed combinational therapy had decreased apoptosis when compared to control. Combinational IA therapy with verapamil and magnesium is safe and effective for treatment of experimental ischemic stroke and may be an effective “stroke therapy cocktail” worthy of clinical study. Disclosures M. Maniskas: None. A. Trout: None. J. Goodman: None. G. Bix: None. J. Fraser: None.