Human Stroke Patients Research Articles

Low-Frequency Brain Oscillations Track Motor Recovery in Human Stroke.

The majority of patients with stroke survive the acute episode and live with enduring disability. Effective therapies to support recovery of motor function after stroke are yet to be developed. Key to this development is the identification of neurophysiologic signals that mark recovery and are suitable and susceptible to interventional therapies. Movement preparatory low-frequency oscillations (LFOs) play a key role in cortical control of movement. Recent animal data point to a mechanistic role of motor cortical LFOs in stroke motor deficits and demonstrate neuromodulation intervention with therapeutic benefit. Their relevance in human stroke pathophysiology is unknown. We studied the relationship between movement-preparatory LFOs during the performance of a visuomotor grip task and motor function in a longitudinal (<5 days, 1 and 3 months) cohort study of 33 patients with motor stroke and in 19 healthy volunteers. Acute stroke-lesioned brains fail to generate the LFO signal. Whereas in healthy humans, a transient occurrence of LFOs preceded movement onset at predominantly contralateral frontoparietal motor regions, recordings in patients revealed that movement-preparatory LFOs were substantially diminished to a level of 38% after acute stroke. LFOs progressively increased at 1 and 3 months. This re-emergence closely tracked the recovery of motor function across several movement qualities including grip strength, fine motor skills, and synergies and was frequency band specific. Our results provide the first human evidence for a link between movement-preparatory LFOs and functional recovery after stroke, promoting their relevance for movement control. These results suggest that it may be interesting to explore targeted, LFOs-restorative brain stimulation therapy in human stroke patients. ANN NEUROL 2019;86:853-865.

Neuroprotective therapy both for als and acute ischemic stroke

Although the underlying mechanism of amyotrophic lateral sclerosis (ALS) has not yet been fully clarified, several reports have implicated the involvement of oxidative stress under selective death of motor neurons in both ALS patients and animal models. Edaravone is a free radical scavenger, which is the first clinical drug for neuroprotection in the world which has been approved from 2001 in most ischemic stroke patients in Japan, and some of China and India. A recent multicenter prospective double-blind placebo-control clinical trial with edaravone for ALS patients conducted in Japan showed a positive effect for delaying the clinical score (ALS FRS-R) during the 24 weeks of examination. This clinical benefit of edaravone was shown as an add-on therapy after anti-glutamatergic riluzole. These data strongly suggest a potential underlying mechanism of oxidative stress in ALS and a clinical delay by a free radical scavenger. These translational studies on a free radical scavenger Edaravone allowed governmental permissions both for acute ischemic stroke after 2001 and for ALS after 2015. Edaravone was approved for ALS at 2015 in Japan, 2016 in Korea, and 2017 in USA. Use of Edaravone greatly reduced hemorrhagic transformation accompanied by tissue plasminogen activator (tPA) treatment, and may also extend therapeutic time window with tPA therapy for more than 4.5 h in human stroke patients for preserving neurovascular unit (NVU). An intensive Edaravine therapy for 3 days now showed a favorite recovery in 3 European countries.

Pharmacological Modulation of Neutrophil Extracellular Traps Reverses Thrombotic Stroke tPA (Tissue-Type Plasminogen Activator) Resistance.

Background and Purpose- Recanalization of the occluded artery is a primary goal in stroke treatment. Unfortunately, endovascular treatment is not always available, and tPA (tissue-type plasminogen activator) therapy is limited by its narrow therapeutic window; importantly, the rate of early arterial recanalization after tPA administration is low, especially for platelet-rich thrombi. The mechanisms for this tPA resistance are not well known. Since neutrophil extracellular traps (NETs) have been implicated in this setting, our aim was to study whether NET pharmacological modulation can reverse tPA resistance and the role of TLR4 (Toll-like receptor 4), previously related to NET formation, in thrombosis. Methods- To this goal, we have used a mouse photothrombotic stroke model, which produces a fibrin-free thrombus composed primarily of aggregated platelets and thrombi obtained from human stroke patients. Results- Our results demonstrate that (1) administration of DNase-I, which promotes NETs lysis, but not of tPA, recanalizes the occluded vessel improving photothrombotic stroke outcome; (2) a preventive treatment with Cl-amidine, impeding NET formation, completely precludes thrombotic occlusion; (3) platelet TLR4 mediates NET formation after photothrombotic stroke; and (4) ex vivo fresh platelet-rich thrombi from ischemic stroke patients are effectively lysed by DNase-I. Conclusions- Hence, our data open new avenues for recanalization of platelet-rich thrombi after stroke, especially to overcome tPA resistance.

Neuroprotection by Exogenous and Endogenous Neuregulin-1 in Mouse Models of Focal Ischemic Stroke.

Identifying novel neuroprotectants that can halt or reverse the neurological effects of stroke is of interest to both clinicians and scientists. We and others previously showed the pre-clinical neuroprotective efficacy of neuregulin-1 (NRG-1) in rats following focal brain ischemia. In this study, we examined neuroprotection by exogenous and endogenous NRG-1 using a mouse model of ischemic stroke. C57BL6 mice were subjected to middle cerebral artery occlusion (MCAO) followed by reperfusion. NRG-1 or vehicle was infused intra-arterially (i.a.) or intravenously (i.v.) after MCAO and before the onset of reperfusion. NRG-1 treatment (16μg/kg; i.a.) reduced cerebral cortical infarct volume by 72% in mice when delivered post-ischemia. NRG-1 also inhibited neuronal injury as measured by Fluoro Jade B labeling and rescued NeuN immunoreactivity in neurons. Neuroprotection by NRG-1 was also observed in mice when administered i.v. (100μg/kg) in both male and female mice. We investigated whether endogenous NRG-1 was neuroprotective using male and female heterozygous NRG-1 knockout mice (NRG-1+/-) compared with wild-type mice (WT) littermates. NRG-1+/- and WT mice were subjected to MCAO for 45min, and infarct size was measured 24h following MCAO. NRG-1+/- mice displayed a sixfold increase in cortical infarct size compared with WT mice. These results demonstrate that NRG-1 treatment mitigates neuronal damage following cerebral ischemia. We further showed that reduced endogenous NRG-1 results in exacerbated neuronal injury in vivo. These findings suggest that NRG-1 represents a promising therapy to treat stroke in human patients.

A co-ultramicronized palmitoylethanolamide/luteolin composite mitigates clinical score and disease-relevant molecular markers in a mouse model of experimental autoimmune encephalomyelitis

BackgroundPersistent and/or recurrent inflammatory processes are the main factor leading to multiple sclerosis (MS) lesions. The composite ultramicronized palmitoylethanolamide, an endogenous N-acylethanolamine, combined with the flavonoid luteolin, PEALut, have been found to exert neuroprotective activities in experimental models of spinal and brain injury and Alzheimer disease, as well as a clinical improvement in human stroke patients. Furthermore, PEALut enhances the expression of different myelin proteins in oligodendrocyte progenitor cells suggesting that this composite might have protective effects in MS experimental models.MethodsThe mouse model of experimental autoimmune encephalomyelitis (EAE) based on active immunization with a fragment of myelin oligodendrocyte glycoprotein (MOG35-55) was used. The daily assessment of clinical score and the expression of serum amyloid A (SAA1), proinflammatory cytokines TNF-α, IL-1β, IFN-γ, and NLRP3 inflammasome, as well as TLR2, Fpr2, CD137, CD3-γ, and TCR-ζ chain, heterodimers that form T cell surface glycoprotein (TCR), and cannabinoid receptors CB1, CB2, and MBP, were evaluated in the brainstem and cerebellum at different postimmunization days (PIDs).ResultsVehicle-MOG35-55-immunized (MOG35-55) mice developed ascending paralysis which peaked several days later and persisted until the end of the experiment. PEALut, given intraperitoneally daily starting on day 11 post-immunization, dose-dependently improved clinical score over the range 0.1–5 mg/kg. The mRNA expression of SAA1, TNF-α, IL-1β, IFN-γ, and NLRP3 were significantly increased in MOG35-55 mice at 14 PID. In MOG35-55 mice treated with 5 mg /kg PEALut, the increase of SAA1, TNF- α, IL-1β, and IFN-γ transcripts at 14 PID was statistically downregulated as compared to vehicle-MOG35-55 mice (p < 0.05).The expression of TLR2, Fpr2, CD137, CD3-γ, TCR-ζ chain, and CB2 receptors showed a significant upregulation in vehicle-MOG35-55 mice at 14 PID. Instead, CB1 and MBP transcripts have not changed in expression at any time. In MOG/PEALut-treated mice, TLR2, Fpr2, CD137, CD3-γ, TCR-ζ chain, and CB2 mRNAs were significantly downregulated as compared to vehicle MOG35-55 mice.ConclusionsThe present results demonstrate that the intraperitoneal administration of the composite PEALut significantly reduces the development of clinical signs in the MOG35-55 model of EAE. The dose-dependent improvement of clinical score induced by PEALut was associated with a reduction in transcript expression of the acute-phase protein SAA1, TNF-α, IL-1β, IFN-γ, and NLRP3 proinflammatory proteins and TLR2, Fpr2, CD137, CD3-γ, TCR-ζ chain, and CB2 receptors.

Polymorphonuclear Neutrophils Play a Decisive Role for Brain Injury and Neurological Recovery Poststroke.

Polymorphonuclear Neutrophils Play a Decisive Role for Brain Injury and Neurological Recovery Poststroke.

Abstract WMP45: Gene Expression Levels in Acute Large Vessel Occlusion Stroke in Humans

Background and Purpose: The Blood and Clot Thrombectomy Registry and Collaboration (BACTRAC) tissue bank collects blood proximal and distal to the intracranial thrombus during the process of mechanical thrombectomy in human stroke patients. These samples provide a unique resource in evaluating acute gene expression changes at the time of ischemic stroke. The purpose of this study was to evaluate gene expression changes occurring within the thrombus and across the occlusion in the intravascular space in acute ischemic stroke patients. Methods: We developed the BACTRAC protocol: an IRB-approved tissue banking approach for ELVO (clinicaltrials.gov NCT03153683). We evaluated relative concentrations of gene expression in 84 inflammatory molecules in thrombi removed from adults who received thrombectomy for ischemic stroke, in static blood distal to thrombus, and in peripheral circulation. Results: We analyzed the first 46 subjects (age = 67 ± 14.23, 20 males) in the BACTRAC registry. Results from qPCR gene expression analyses demonstrate that 21 genes (CCL1, CCL11, CCL13, CCL17, CCL26, CCL8, CSF3, CX3CL1, CXCL1, CXCL9, IFNA2, IL13, IL17C, IL17F, IL1A, IL27, IL3, IL33, LTA, TNFSF11, and TNFSF13) had at least 25 mean fold change in the distal blood compared to the peripheral blood. Fourteen genes (AIMP1, CCL11, CCL13, CCL15, CCL16, CCL23, CCR2, CCR4, CCR8, CD40LG, CXCL10, IL10RA, IL15, and TNFSF13B) had at least 10 mean fold change in the thrombus compared to the peripheral blood. Overall, these genes are associated with chemo-attraction and cell proliferation of monocytes, neutrophils, and T cells. Conclusions: These findings provide a novel insight into the initial pathology of large vessel stroke in humans, particularly in regard to identifying acute gene expression changes that occur during stroke.

Abstract 132: The Mammalian Target of Rapamycin Regulates Platelet Integrin Activation, Aggregation, and Ischemic Stroke

Rationale: Platelets are critical cellular effectors in the development of ischemic stroke. Translational control pathways, such as the mammalian target of rapamycin (mTOR), are active in platelets, and can be targeted therapeutically, but have not been examined in stroke. Objective: To determine if activation of the mTOR pathway regulates platelet activation and the development of ischemic stroke following stroke in vivo . Methods: We generated mice lacking mTOR specifically in platelets (KO). We assessed mTOR activation, platelet aggregation, integrin activation, and clot retraction in KO and wild-type control mice (WT). In both KO and WT mice, we also performed a transient middle cerebral artery occlusion (tMCAO) stroke model (t=1h followed with reperfusion for 24hrs). Following tMACO, blood flow in the MCA and stroke infarct size were determined. We also examined mTOR activation in human platelets from individuals with ischemic stroke (n=5) and healthy controls (n=4). Results: We confirmed the absence of mTOR in KO platelets. In platelets from WT mice, the mTOR pathway is activatable, triggering platelet activation and clot retraction in vitro . In comparison, indices of mTOR activation, including pAKT and pS6K, were significantly (p&lt;0.05) reduced in platelets from KO mice. Platelets from KO mice also had significant reductions in platelet aggregation (50% less), integrin αIIbβ3 activation (40% less), and clot retraction compared to WT mice. These differences were not due to alterations in platelet count. In the tMCAO model, KO mice had significantly smaller infarct sizes compared to WT mice (36.8±9.4mm 3 , n=7 vs. 80.3±8.1mm 3 , n=8; p&lt;0.004). Smaller infarct sizes in KO mice were not due to differences in blood flow during the ischemia or reperfusion phase, which were similar to WT mice. In platelets from human ischemic stroke patients, the mTOR pathway was activated as p4EBP1, a downstream target of mTOR, was 1.72-fold higher compared to healthy controls. Conclusion: These data demonstrate that inhibition of the mTOR pathway in platelets significantly reduces platelet activation and protects from ischemic stroke . Therapeutic targeting of the mTOR pathway in platelets may provide clinical benefit in the setting of ischemic stroke.

Abstract TMP26: Age Exacerbates the Pathogenicity of Neutrophils in Ischemic Stroke

Introduction: Ischemic stroke is a major cause of disability and mortality worldwide. Neutrophils may exacerbate stroke-related injury via the release of reactive oxygen species (ROS) and granule proteins. As age is the strongest risk factor for poor outcomes after ischemic stroke, we hypothesized that age exacerbates the deleterious functions of neutrophils after brain ischemia. Methods and Results: Young (3 month) and aged (20-22 month) mice were subjected to middle cerebral artery (MCAO) stroke. Aged mice had higher mortality than young mice at 7 days (0% young, 60% aged, p&lt;0.05, n=5/group). Aged mice also had greater neurodeficits than young mice (p&lt;0.05). Flow cytometry showed that aged animals display an increase in neutrophil proportions in the blood, spleen, lung and bone marrow (p&lt;0.001) after ischemic stroke compared to young. Furthermore, flow cytometry demonstrated that neutrophils from the blood of aged animals produced ROS at higher levels than neutrophils from young animals (p&lt;0.05). Aged stroke animals had higher serum IL-6 (p&lt;0.0001) and CXCL1 (p&lt;0.01), two powerful neutrophil activating cytokines, at 24 hours after stroke. Follow-up serum measurements in human ischemic stroke patients (n=143) demonstrated that increased age was associated with higher levels of IL-6 (p=0.014) and IL-8 (p&lt;0.0001) at 24 hours. We then used anti-Ly6G, a neutrophil specific monoclonal antibody, to deplete circulating neutrophils as a potential treatment for ischemic stroke. Mice were randomized to receive 500ug anti-Ly6G IP or control at 4, 24 and 48 hours after stroke (n=6-7/group). Treatment conferred no benefit to young mice, but resulted in a significant improvement in behavioral recovery in aged mice as measured by adhesive test at 7 days (p&lt;0.01), corner test at 14 days (p&lt;0.001) and hangwire test at 14 days (p&lt;0.05). Conclusions: Neutrophils from aged animals exhibit altered organ trafficking and a higher reactive oxygen species generation. Elevated levels of neutrophil-activating cytokines were seen in both aged animals and aged ischemic stroke patients at 24 hours. Finally, anti-neutrophil depletion therapy had no effect in young animals, but resulted in significantly enhanced behavioral recovery in aged animals.

Dietary salt promotes ischemic brain injury and is associated with parenchymal migrasome formation

Sodium chloride promotes vascular fibrosis, arterial hypertension, pro-inflammatory immune cell polarization and endothelial dysfunction, all of which might influence outcomes following stroke. But despite enormous translational relevance, the functional importance of sodium chloride in the pathophysiology of acute ischemic stroke is still unclear. In the current study, we show that high-salt diet leads to significantly worse functional outcomes, increased infarct volumes, and a loss of astrocytes and cortical neurons in acute ischemic stroke. While analyzing the underlying pathologic processes, we identified the migrasome as a novel, sodium chloride-driven pathomechanism in acute ischemic stroke. The migrasome was previously described in vitro as a migrating organelle, which incorporates and dispatches cytosol of surrounding cells and plays a role in intercellular signaling, whereas a pathophysiological meaning has not been elaborated. We here confirm previously reported characteristics of the migrasome in vivo. Immunohistochemistry, electron microscopy and proteomic analyses further demonstrate that the migrasome incorporates and dispatches cytosol of surrounding neurons following stroke. The clinical relevance of these findings is emphasized by neuropathological examinations, which detected migrasome formation in infarcted brain parenchyma of human stroke patients. In summary, we demonstrate that high-salt diet aggravates stroke outcomes, and we characterize the migrasome as a novel mechanism in acute stroke pathophysiology.

The protease‐activated receptor 4 Ala120Thr variant alters platelet responsiveness to low‐dose thrombin and protease‐activated receptor 4 desensitization, and is blocked by non‐competitive P2Y12 inhibition

Essentials The rs773902 SNP results in differences in platelet protease-activated receptor (PAR4) function. The functional consequences of rs773902 were analyzed in human platelets and stroke patients. rs773902 affects thrombin-induced platelet function, PAR4 desensitization, stroke association. Enhanced PAR4 Thr120 effects on platelet function are blocked by ticagrelor. SUMMARY: Background F2RL3 encodes protease-activated receptor (PAR)4 and harbors an A/G single-nucleotide polymorphism (SNP) (rs773902) with racially dimorphic allelic frequencies. This SNP mediates an alanine to threonine substitution at residue120 that alters platelet PAR4 activation by the artificial PAR4-activation peptide (PAR4-AP) AYPGKF. Objectives To determine the functional effects of rs773902 on stimulation by a physiological agonist, thrombin, and on antiplatelet antagonist activity. Methods Healthy human donors were screened and genotyped for rs773902. Platelet function in response to thrombin was assessed without and with antiplatelet antagonists. The association of rs773902 alleles with stroke was assessed in the Stroke Genetics Network study. Results As compared with rs773902 GG donors, platelets from rs773902 AA donors had increased aggregation in response to subnanomolar concentrations of thrombin, increased granule secretion, and decreased sensitivity to PAR4 desensitization. In the presence of PAR1 blockade, this genotype effect was abolished by higher concentrations of or longer exposure to thrombin. We were unable to detect a genotype effect on thrombin-induced PAR4 cleavage, dimerization, and lipid raft localization; however, rs773902 AA platelets required a three-fold higher level of PAR4-AP for receptor desensitization. Ticagrelor, but not vorapaxar, abolished the PAR4 variant effect on thrombin-induced platelet aggregation. A significant association of modest effect was detected between the rs773902 A allele and stroke. Conclusion The F2RL3 rs773902 SNP alters platelet reactivity to thrombin; the allelic effect requires P2Y12 , and is not affected by gender. Ticagrelor blocks the enhanced reactivity of rs773902 A platelets. PAR4 encoded by the rs773902 A allele is relatively resistant to desensitization and may contribute to stroke risk.

Protective effects of the angiotensin II AT2 receptor agonist compound 21 in ischemic stroke: a nose-to-brain delivery approach

Significant neuroprotective effects of angiotensin II type 2 (AT2) receptor (AT2 receptor) agonists in ischemic stroke have been previously demonstrated in multiple studies. However, the routes of agonist application used in these pre-clinical studies, direct intracerebroventricular (ICV) and systemic administration, are unsuitable for translation into humans; in the latter case because AT2 receptor agonists are blood-brain barrier (BBB) impermeable. To circumvent this problem, in the current study we utilized the nose-to-brain (N2B) route of administration to bypass the BBB and deliver the selective AT2 receptor agonist Compound 21 (C21) to naïve rats or rats that had undergone endothelin 1 (ET-1)-induced ischemic stroke. The results obtained from the present study indicated that C21 applied N2B entered the cerebral cortex and striatum within 30 min in amounts that are therapeutically relevant (8.4-9 nM), regardless of whether BBB was intact or disintegrated. C21 was first applied N2B at 1.5 h after stroke indeed provided neuroprotection, as evidenced by a highly significant, 57% reduction in cerebral infarct size and significant improvements in Bederson and Garcia neurological scores. N2B-administered C21 did not affect blood pressure or heart rate. Thus, these data provide proof-of-principle for the idea that N2B application of an AT2 receptor agonist can exert neuroprotective actions when administered following ischemic stroke. Since N2B delivery of other agents has been shown to be effective in certain human central nervous system diseases, the N2B application of AT2 receptor agonists may become a viable mode of delivering these neuroprotective agents for human ischemic stroke patients.

Treatment with Uric Acid Reduces Infarct and Improves Neurologic Function in Female Mice After Transient Cerebral Ischemia

Exogenous administration of uric acid, a naturally occurring antioxidant that scavenges reactive oxygen species in vasculature, has shown protective efficacy in both rodent models of stroke and human stroke patients in Spain as an adjuvant treatment to mechanical thrombectomy. Before clinical trials can be initiated in the United States, however, confirmation of efficacy in alternative preclinical models is required in accordance with stroke therapy academic industry roundtable-RIGOR criteria. To date, preclinical efficacy has only been established in the acute setting in male rodents. To address this need, we subjected 7- to 9-week old ovariectomized female mice to filament-induced right middle cerebral artery ischemia and reperfusion, an established preclinical model of mechanical thrombectomy. Fidelity of the procedure was monitored by laser Doppler flowmetry. A separate lab randomly assigned animals to vehicle versus uric acid infusion, which was initiated immediately after 45 minutes of reperfusion. Poststroke analysis of infarction size and neurologic function were conducted by investigators blind to treatment group, with a 7-day primary endpoint and a 3-day intermediary analysis at 1and. Infarct size and neurologic function at 7 days poststroke were significantly improved in uric acid-treated animals, relative to vehicle. Efficacy of uric acid in preclinical models of stroke is now expanded to include female mice analyzed at a later time point than has been investigated previously. These results support stroke therapy academic industry roundtable-RIGOR driven determination of the suitability of acute administration of uric acid as an adjuvant to mechanical thrombectomy in clinical trials for patients with stroke.

Abstract WP260: Uric Acid Reduces Infarct and Improves Neurological Function in Female Mice One Week After Transient Cerebral Ischemia

Background &amp; Purpose: Exogenous administration of uric acid, a naturally occurring antioxidant that scavenges reactive oxygen species in vasculature, has shown protective efficacy in both rodent models of stroke and in human stroke patients in Spain as an adjuvant treatment to mechanical thrombectomy. In accordance with STAIR-RIGOR criteria, confirmation of efficacy in alternative preclinical models is required before clinical trials can be initiated in the United States. To date, preclinical efficacy has only been established in the acute setting in male rodents. Methods: Seven to nine week old ovariectomized female mice were subjected to filament-induced right middle cerebral artery ischemia and reperfusion, an established preclinical model of mechanical thrombectomy. Fidelity of the procedure was monitored by laser Doppler flowmetry in all animals. A separate lab randomly assigned animals to vehicle vs uric acid infusion, which was initiated immediately after 45 minutes of reperfusion. Post-stroke analysis of infarction size and neurological function were conducted by investigators blind to treatment group, with a 7 day primary endpoint and intermediary analysis at 1and 3 days. Results: Infarct size and neurological function at 7 days post-stroke was significantly improved in uric acid-treated animals, relative to vehicle. Conclusions: Efficacy of uric acid in preclinical models of stroke is now expanded to include female mice analyzed at a more chronic time point than has been investigated previously. These results supports STAIR-RIGOR driven determination of the suitability of acute administration of uric acid as an adjuvant to mechanical thrombectomy in clinical trials for patients with stroke.

Abstract TMP107: Blood and Clot Thrombectomy Registry and Collaboration (BACTRAC) Protocol: Novel Method for Evaluating Human Stroke

Background: Research in ischemic stroke struggles from failures to translate stroke conditions between animal models and human patients. With mechanical thrombectomy, we have for the first time developed a protocol to facilitate tissue banking of intracranial thrombi as well as the blood immediately proximal and distal to it. These tissues provide a unique resource to correlate changes in the human condition to animal models. Our aim was to develop a reproducible protocol to bank these specimens for future gene expression and proteomic analysis. Methods: We developed an IRB-approved protocol for tissue processing during thrombectomy (www.clinicaltrials.gov NCT03153683). The protocol was a joint clinical/basic science effort among multiple laboratories and the Neurointerventional Radiology Service Line. We placed laboratory supplies in the angio suite, and developed a step-by-step protocol for specimen retrieval and processing. Results: Our protocol successfully yielded samples for analysis in all but one case. In our preliminary dataset, the protocol yielded adequate amounts of tissue for distal blood, proximal blood, and thrombus. We present the tissue banking protocol for dissemination, and highlight the training protocol and mechanics of on-call research staffing for 24/7 tissue processing. In addition, preliminary integrity analyses demonstrated high quality yields for RNA and protein. Conclusion: We have developed a novel tissue banking protocol to capture thrombus, and both proximal and distal blood to the intracranial thrombus in human stroke patients. The specimens are captured during the thrombectomy procedure, and allow us to study the initial molecular response to ischemic stroke in the human condition in ways previously unavailable.

Abstract 71: Circulating Inter-alpha Inhibitor Protein Levels Decline After Stroke in Humans: Exogenous Supplementation is Protective in Multiple Animal Models of Ischemic Stroke

Introduction: Inter-alpha Inhibitor Proteins (IAIP) are a family of endogenous plasma and extracellular matrix molecules. IAIPs have been shown to down-regulate inflammation, inhibit destructive serine proteases, and bind extracellular histones to neutralize cytotoxicity. Interestingly, many of these factors also regulate neuroprotection after stroke. In our initial studies, we found that IAIP treatment was neuroprotective in the middle cerebral artery occlusion model. Here, we analyzed if stroke alters IAIP using plasma samples from both mice and human stroke patients, and we investigated whether IAIP could provide neuroprotection using multiple stroke models. Methods: C57BL/6 mice were subjected to stroke with an autologous thromboembolic clot followed by reperfusion using t-PA. A separate cohort underwent the permanent occlusion model. Mice were randomized to receive IAIP or vehicle at 6h and 18h after stroke onset. Infarct was assessed 48h after reperfusion with t-PA, or 72h after permanent occlusion. Plasma samples from stroke patients and vascular risk factor matched controls were used for changes in IAIP levels using ELISA. Statistics were performed by two-sample t-test or Wilcoxon rank-sum test. P &lt;0.05 considered significant. Results: IAIP treatment significantly reduced total injury size (t-PA alone 22±2% vs. t-PA+IAIP 13±3%; n=4-5/grp). Protection was also seen after permanent occlusion (vehicle 59.4±3.9% vs IAIP 39.5±5.9%; n=6-8/gp, p&lt;.05), showing efficacy of IAIP in two different models. IAIP treatment improved survival rates (82% with IAIP vs 63% with vehicle) and neurological scores compared to vehicle group (p&lt;0.05). Mean IAIP levels were significantly lower (stroke patients 134.4 ug/ml; n=51 vs controls 157.5 ug/ml; n=40), in parallel, increased IAIP-Histone complexes were seen in these patients. Similar changes were identified in mouse samples. Conclusions: Our results show that delayed IAIP treatment can significantly reduce cell death in both permanent ischemia and/or in combination with t-PA. IAIP could potentially represent an important, novel treatment for stroke. Our studies in combination with t-PA further strengths this clinical translatability and to design personalized dosing based on IAIP levels.

Generation and characterization of tissue-type plasminogen activator transgenic rats

To address a species difference in the responsiveness to human recombinant tissue-type plasminogen activator (rt-PA) between rats and humans, tPA transgenic (Tg) rats were generated and characterized. In the rats, transcriptional regulation of tPA was designed under the control of the endogenous tPA promoter. There were no significant differences in hematological parameters between the tPA Tg and non Tg rats. Plasma tPA concentration was significantly increased and serum free PAI-1 was significantly decreased in the tPA Tg rats. Significant overexpression of tPA mRNA in five major organs was also confirmed in the tPA Tg rats. In contrast, the extent of tPA mRNA induction by pathophysiological stimuli (focal cerebral ischemia) was comparable in the two strains. Earlier increase in the plasma D-Dimer level was observed in the tPA Tg rats in a model of thromboembolism compared with the non Tg rats. On the other hand, there was no statistically significant prolongation of bleeding time in a rat model of bleeding between the two strains. rt-PA showed dose-related blood flow restoration in a rat model of thromboembolic stroke in the tPA Tg rats from a dose (1 mg/kg, i.v.) similar to clinical doses for human stroke patients. In conclusion, tPA Tg rats, in which tPA is overexpressed and endogenous fibrinolytic activity is enhanced without hemostatic abnormality, were generated. tPA Tg rats would be beneficial for the pharmacological and the toxicological evaluation of rt-PA and other various fibrinolytic enhancers.

Induced Pluripotent Stem Cell-Derived Neural Stem Cell Therapy Enhances Recovery in an Ischemic Stroke Pig Model

Induced pluripotent stem cell-derived neural stem cells (iNSCs) have significant potential as an autologous, multifunctional cell therapy for stroke, which is the primary cause of long term disability in the United States and the second leading cause of death worldwide. Here we show that iNSC transplantation improves recovery through neuroprotective, regenerative, and cell replacement mechanisms in a novel ischemic pig stroke model. Longitudinal multiparametric magnetic resonance imaging (MRI) following iNSC therapy demonstrated reduced changes in white matter integrity, cerebral blood perfusion, and brain metabolism in the infarcted tissue. The observed tissue level recovery strongly correlated with decreased immune response, enhanced neuronal protection, and increased neurogenesis. iNSCs differentiated into neurons and oligodendrocytes with indication of long term integration. The robust recovery response to iNSC therapy in a translational pig stroke model with increased predictive potential strongly supports that iNSCs may be the critically needed therapeutic for human stroke patients.

Phosphodiesterase 5 inhibition as a therapeutic target for ischemic stroke: A systematic review of preclinical studies

Phosphodiesterase 5 inhibitors (PDE5i), such as sildenafil (Viagra®) are widely used for erectile dysfunction and pulmonary hypertension. Preclinical studies suggest that PDE5i may improve functional outcome following ischemic stroke. In this systematic review we aimed to evaluate the effects of selective PDE5i in animal models of brain ischaemia. A systematic search in Medline, Embase, and The Cochrane Library was performed including studies in English assessing the effects of selective PDE5i. 32 publications were included describing outcome in 3646 animals. Neuroprotective effects of PDE5i were dependent on the NO-cGMP-PKG-pathway. These included reduced neuronal apoptosis (n=3 studies), oxidative stress (n=5), and neuroinflammation (n=2). PDE5i increased angiogenesis and elevated regional cerebral blood flow in the ischemic penumbra, and improved functional recovery. Some studies found that PDE5i treatment reduced lesion volume (n=9), others found no effect (n=9). Treatment was effective when administered within 24h post-ischemia, though treatment delayed to seven days improved outcome in one study. This review demonstrates both neuroprotective and neurorestorative effects of PDE5i in animal models of stroke, though the specific underlying signaling pathways relating to PDE5 inhibition and cGMP may remain serendipitous in some studies. There is currently limited evidence on the effects of selective PDE5i in human stroke patients, hence translation of preclinical results into clinical trials may be warranted.

Spontaneous ischaemic stroke lesions in a dog brain: neuropathological characterisation and comparison to human ischaemic stroke

BackgroundDogs develop spontaneous ischaemic stroke with a clinical picture closely resembling human ischaemic stroke patients. Animal stroke models have been developed, but it has proved difficult to translate results obtained from such models into successful therapeutic strategies in human stroke patients. In order to face this apparent translational gap within stroke research, dogs with ischaemic stroke constitute an opportunity to study the neuropathology of ischaemic stroke in an animal species.Case presentationA 7 years and 8 months old female neutered Rottweiler dog suffered a middle cerebral artery infarct and was euthanized 3 days after onset of neurological signs. The brain was subjected to histopathology and immunohistochemistry. Neuropathological changes were characterised by a pan-necrotic infarct surrounded by peri-infarct injured neurons and reactive microglia/macrophages and astrocytes.ConclusionsThe neuropathological changes reported in the present study were similar to findings in human patients with ischaemic stroke. The dog with spontaneous ischaemic stroke is of interest as a complementary spontaneous animal model for further neuropathological studies.