Transcranial photoacoustic characterization of neurovascular physiology during early-stage photothrombotic stroke in neonatal piglets in vivo

Perinatal ischemic stroke is estimated to occur in 1/2300–1/5000 live births, but early differential diagnosis from global hypoxia-ischemia is often difficult. In this study, we tested the ability of a hand-held transcranial photoacoustic (PA) imaging probe to non-invasively detect a focal photothrombotic stroke (PTS) within 2 hours of stroke onset in a gyrencephalic piglet brain. 17 stroke lesions of approximately 1-cm2 area were introduced randomly in anterior or posterior cortex via the light/dye PTS technique in anesthetized neonatal piglets (n = 11). The contralateral non-ischemic region served as control tissue for discrimination contrast for the PA hemoglobin metrics: oxygen saturation, total hemoglobin (tHb), and individual quantities of oxygenated and deoxygenated hemoglobin (HbO2 and HbR). The PA-derived tissue oxygen saturation at 2 hours yielded a significant separation between control and affected regions-of-interest (p < 0.0001), which were well matched with 24-hr post-stroke cerebral infarction confirmed in the triphenyltetrazolium chloride-stained image. The quantity of HbO2 also displayed a significant contrast (p = 0.021), whereas tHb and HbR did not. The analysis on receiver operating characteristic curves and multivariate data analysis also agreed with the results above. This study shows that a hand-held transcranial PA neuroimaging device can detect a regional thrombotic stroke in the cerebral cortex of a neonatal piglet. In particular, we conclude that the oxygen saturation metric can be used alone to identify regional stroke lesions. The lack of change in tHb may be related to arbitrary hand-held imaging configuration and/or entrapment of red blood cells within the thrombotic stroke.

Abstract 1122‐000198: Failure of Tenecteplase in Maintaining Collateral Circulation in Large Vessel Occlusion Strokes.

Introduction : Acute administration of alteplase with collateral patency has been systematically evaluated in acute ischemic stroke (AIS) patients. Large studies evaluating alteplase demonstrate a significant association of successful recanalization (TICI score) and good clinical outcome (mRS) with ASITN/SIR collateral grade greater than 2. However there is paucity of data looking at the association between IV tenecteplase (TNK) and acute collateralization. Our objective was to investigate early TNK use association for the degree of collateralization in subjects with AIS secondary to large vessel occlusion (LVO). Methods : Collateralization was assessed on digital subtraction angiography using the American Society of Intervention and Therapeutic Neuroradiology/Society of Interventional Radiology (ASTIN/SIR) scale. Grades were defined by the following: 0 no collaterals to ischemic region; 1 slow collaterals peripherally; 2 rapid collaterals peripherally; 3 slow collaterals within ischemic region; 4 complete retrograde perfusion to ischemic region. Subjects with LVO undergoing mechanical thrombectomy status post TNK as part of the pilot early clinical use of TNK within 4.5 hours of last known well were assigned a grade. Mean ASITN/SIR collateral grade was determined. Spearman’s rho was used to measure association of collateral grade with thrombolysis in cerebral infarction (TICI) score. Patients with TNK‐associated recanalization at time of cerebral angiogram were excluded from study. Social Science Statistics was used for data analysis. Results : From October 2020 to April 2021, 16 subjects (6 females; age, 63.25 95% CI [54.9207, 71.5793]) received TNK and underwent mechanical thrombectomy. From those subjects, 25 % (n = 4) had IV TNK‐associated recanalization with normalization of collateral blood flow and were excluded. Of the rest (n = 12, 75%) had a mean ASITN/SIR collateral grade of 1.08 (95% CI [0.5762, 1.5838]). Association between collateral grade and final TICI score was not statistically significant (rs = ‐0.33576, p = 0.28598) suggesting inability of TNK to result in/maintain a robust collateral flow. Conclusions : Poor correlation of collateral grade and final TICI score may have implications of faster progression in patients with ischemic stroke receiving TNK in the setting of LVO if immediate recanalization is not achieved. Larger prospective studies are needed to evaluate the effect of TNK on collateralization when compared to Alteplase.

Transcranial photoacoustic characterization of neurovascular physiology during early-stage photothrombotic stroke in neonatal piglets in vivo

Perinatal ischemic stroke is estimated to occur in 1/2300-1/5000 live births, but early differential diagnosis from global hypoxia-ischemia is often difficult. In this study, we tested the ability of a hand-held transcranial photoacoustic (PA) imaging to non-invasively detect a focal photothrombotic stroke (PTS) within 2 hours of stroke onset in a gyrencephalic piglet brain. 17 stroke lesions of approximately 1-cm2 area were introduced randomly in anterior or posterior cortex via the light/dye PTS technique in anesthetized neonatal piglets (n = 11). The contralateral non-ischemic region served as control tissue for discrimination contrast for the PA hemoglobin metrics: HbO2 saturation, total hemoglobin (tHb), and individual quantities of oxygenated and deoxygenated hemoglobin (HbO2 and HbR). The PA-derived tissue HbO2 saturation at 2 hours yielded a significant separation between control and affected regions-of-interest (p < 0.0001), which were well matched with 24-hr post-stroke cerebral infarction confirmed in the triphenyltetrazolium chloride (TTC)-stained image. The quantity of HbO2 also displayed a significant contrast (p = 0.021), whereas tHb and HbR did not. The analysis on receiver operating characteristic curves and multivariate data analysis also agreed with the results above. This study shows that a hand-held transcranial PA neuroimaging can detect a regional thrombotic stroke in cerebral cortex of a neonatal piglet. In particular, we conclude that the HbO2 saturation metric can be used alone to identify regional stroke lesions. The lack of change in tHb may be related to arbitrary hand-held imaging configuration and/or entrapment of red blood cells within the thrombotic stroke.

Role of Neutrophils in Cardiac Injury and Repair Following Myocardial Infarction

Neutrophils are first-line responders of the innate immune system. Following myocardial infarction (MI), neutrophils are quickly recruited to the ischemic region, where they initiate the inflammatory response, aiming at cleaning up dead cell debris. However, excessive accumulation and/or delayed removal of neutrophils are deleterious. Neutrophils can promote myocardial injury by releasing reactive oxygen species, granular components, and pro-inflammatory mediators. More recent studies have revealed that neutrophils are able to form extracellular traps (NETs) and produce extracellular vesicles (EVs) to aggravate inflammation and cardiac injury. On the contrary, there is growing evidence showing that neutrophils also exert anti-inflammatory, pro-angiogenic, and pro-reparative effects, thus facilitating inflammation resolution and cardiac repair. In this review, we summarize the current knowledge on neutrophils’ detrimental roles, highlighting the role of recently recognized NETs and EVs, followed by a discussion of their beneficial effects and molecular mechanisms in post-MI cardiac remodeling. In addition, emerging concepts about neutrophil diversity and their modulation of adaptive immunity are discussed.

PACAP-38 in Acute ST-Segment Elevation Myocardial Infarction in Humans and Pigs: A Translational Study

Acute myocardial infarction (MI) is one of the most common causes of death worldwide. Pituitary adenylate cyclase activating polypeptide (PACAP) is a cardioprotective neuropeptide expressing its receptors in the cardiovascular system. The aim of our study was to examine tissue PACAP-38 in a translational porcine MI model and plasma PACAP-38 levels in patients with ST-segment elevation myocardial infarction (STEMI). Significantly lower PACAP-38 levels were detected in the non-ischemic region of the left ventricle (LV) in MI heart compared to the ischemic region of MI-LV and also to the Sham-operated LV in porcine MI model. In STEMI patients, plasma PACAP-38 level was significantly higher before percutaneous coronary intervention (PCI) compared to controls, and decreased after PCI. Significant negative correlation was found between plasma PACAP-38 and troponin levels. Furthermore, a significant effect was revealed between plasma PACAP-38, hypertension and HbA1c levels. This was the first study showing significant changes in cardiac tissue PACAP levels in a porcine MI model and plasma PACAP levels in STEMI patients. These results suggest that PACAP, due to its cardioprotective effects, may play a regulatory role in MI and could be a potential biomarker or drug target in MI.

Detecting early myocardial ischemia in rat heart by MALDI imaging mass spectrometry

Diagnostics of myocardial infarction in human post-mortem hearts can be achieved only if ischemia persisted for at least 6–12 h when certain morphological changes appear in myocardium. The initial 4 h of ischemia is difficult to diagnose due to lack of a standardized method. Developing a panel of molecular tissue markers is a promising approach and can be accelerated by characterization of molecular changes. This study is the first untargeted metabolomic profiling of ischemic myocardium during the initial 4 h directly from tissue section. Ischemic hearts from an ex-vivo Langendorff model were analysed using matrix assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) at 15 min, 30 min, 1 h, 2 h, and 4 h. Region-specific molecular changes were identified even in absence of evident histological lesions and were segregated by unsupervised cluster analysis. Significantly differentially expressed features were detected by multivariate analysis starting at 15 min while their number increased with prolonged ischemia. The biggest significant increase at 15 min was observed for m/z 682.1294 (likely corresponding to S-NADHX—a damage product of nicotinamide adenine dinucleotide (NADH)). Based on the previously reported role of NAD+/NADH ratio in regulating localization of the sodium channel (Nav1.5) at the plasma membrane, Nav1.5 was evaluated by immunofluorescence. As expected, a fainter signal was observed at the plasma membrane in the predicted ischemic region starting 30 min of ischemia and the change became the most pronounced by 4 h. Metabolomic changes occur early during ischemia, can assist in identifying markers for post-mortem diagnostics and improve understanding of molecular mechanisms.

Abstract P217: Direct Myosin-2 Inhibition Restores Cerebral Perfusion in Cortical and Subcortical Regions Resulting in Functional Improvement After Ischemic Stroke

Inefficient cerebral reperfusion is a major unresolved problem in ischemic stroke treatment because hypoxia-induced constriction of capillaries remains persistent even after recanalization by thrombolysis or thrombectomy. Capillary constriction is mediated by actomyosin contraction in precapillary smooth muscle cells (SMCs). We have developed and tested a formulation and administration technique to target the ischemic brain region and developed a promising small-molecule myosin-2 inhibitor (para-aminoblebbistatin (AmBleb) that mainly exerts its effect through direct inhibition of smooth muscle myosin-2 (SMM) in SMCs. The efficacy of SMM inhibition was tested in a rodent transient Middle Cerebral Artery Occlusion (tMCAO) stroke model. SMM was targeted by the direct administration of AmBleb into the ischemic region. AmBleb significantly accelerated the improvement of neurological deficits. Regional cerebral blood flow (rCBF) in the most important cortical and subcortical regions (e.g. motor- and somatosensory cortices, optic pathways, striatum, corpus callosum) showed drastic improvement in the AmBleb treated animals in line with the significant reduction of neurological deficits. We further optimized AmBleb and developed our lead compound MPH-222, which possesses significantly improved pharmacological properties than those of AmBleb. As observed with AmBleb, MPH-222 also fully relaxed isolated human and rat cerebral arterioles, and improved neurological functions of tMCAO operated rats characterized by significantly improved neurological deficits as well as enhanced locomotor symmetry. Moreover, as opposed to AmBleb, markedly lower MPH-222 dose was enough to achieve efficient concentrations in the ischemic region when catheter-based direct intra-arterial administration was applied. This result suggests that direct myosin inhibitor administration may be an optimal add-on therapy to thrombectomy. Funded by the Hungarian National Research, Development and Innovation Office (NVKP 16-1-2016-0051 and PIACI-KFI-2019-00488).

Abstract 65: Targeted Drug Delivery via Acid-Triggered Release Antioxidant Nanoparticles Improves Efficacy of Na1 for Stroke Treatment

Introduction: Recently, NA1, a peptide designed to protect neurons against NMDA receptor-mediated excitotoxicity, demonstrated promise for acute stroke treatment. Enhanced efficiency of peptide delivery may result in improved efficacy. Hypothesis: The bioactive fragment of NA1 is nine amino acids of the carboxyl tail of GluN2B. Delivery of NR2B9c to cells in the brain is achieved through fusion with TAT, a peptide that binds to biological membranes without selectivity. We hypothesize that enhancing targeted delivery of NA1 to the ischemic brain improve the efficacy of NA1. Methods: We previously identified betulinic acid (BA) forming nanoparticles (NPs) as a novel formulation of stroke treatment delivery. After intravenous administration, BA NP penetrate the ischemic brain, reduce ischemia-induced infarction as an antioxidant agent, and allow for encapsulation and delivery of drugs. In this study, we engineered BA NPs for improved drug delivery preferentially to the ischemic brain through surface conjugation of AMD3100, and for acid-triggered drug release to selectively in the ischemic region, which is known to be acidic, through chemical modification. The resulting acid responsive, stroke- targeting antioxidant NPs, or ARSTA NPs, were evaluated for stroke treatment in the mouse transient middle cerebral artery occlusion (MCAO) model either alone or in combination with NA1, based on changes of infarct volumes, edema, mortality, and outcomes. Results: We found that ARSTA NPs enabled targeted delivery of NA1 preferentially to the ischemic brain and allowed for prompt drug release. We showed that intravenous delivery of 50 ug NA1 (or 1nM/g) encapsulated in 1 mg ARSTAs effectively reduced infarct volumes by 69.8%, edema by 60.3%, mortality (median survival >14 days). In contrast, delivery of ARSTA NPs alone (52.2%, 30.2%, 10) or NA1 (0.7%, 3.3%, 6 days) alone at a comparable dose exhibited significantly less efficacy. Consistently, treatment with NA1-loaded ARSTAs improved neurological outcomes in a degree significantly greater than that by either ARSTA NPs or NA1 alone. Conclusion: BA-NP encapsulating NA-1 may represent a novel platform for improved delivery to the ischemic brain. Improved delivery may result in improved efficacy.

A Soluble Epoxide Hydrolase Inhibitor Upregulated KCNJ12 and KCNIP2 by Downregulating MicroRNA-29 in a Mouse Model of Myocardial Infarction

Background: Soluble epoxide hydrolase inhibitors (sEHi) have anti-arrhythmic effects, and we previously found that the novel sEHi t-AUCB (trans-4[-4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid) significantly inhibited ventricular arrhythmias after myocardial infarction (MI). However, the mechanism is unknown. It’s known that microRNA-29 (miR-29) participates in the occurrence of arrhythmias. In this study, we investigated whether sEHi t-AUCB was protective against ischemic arrhythmias by modulating miR-29 and its target genes KCNJ12 and KCNIP2. Methods: Male 8-week-old C57BL/6 mice were divided into five groups and fed distilled water only or distilled water with t-AUCB of different dosages for seven days. Then, the mice underwent MI or sham surgery. The ischemic region of the myocardium was obtained 24 hours after MI to detect miR-29, KCNJ12, and KCNIP2 mRNA expression levels via real-time PCR and KCNJ12 and KCNIP2 protein expression levels via western blotting. Results: MiR-29 expression levels were significantly increased in the ischemic region of MI mouse hearts and the mRNA and protein expression levels of its target genes KCNJ12 and KCNIP2 were significantly decreased. T-AUCB prevented these changes dose-dependently. Conclusion: The sEHi t-AUCB regulates the expression levels of miR-29 and its target genes KCNJ12 and KCNIP2, suggesting a possible mechanism for its potential therapeutic application in ischemic arrhythmia.