Melatonin and calpeptin synergistically protect against post-reperfusion injury in a rat middle cerebral artery occlusion stroke model

Cerebral ischemia induces oxidative injury and increases the intracellular calcium ion concentration to activate several calcium-dependent proteases such as calpains. Calpain activation leads to various necrotic and apoptotic processes. Calpeptin is a potent, cell-permeable calpain inhibitor. As a strong antioxidant and free radical scavenger, melatonin shows beneficial effect in rodent models of focal cerebral ischemia when given prior to ischemia or reperfusion. This study was focused on the neuroprotective effects of melatonin and/or calpeptin given after onset of reperfusion. For this purpose, right-sided middle cerebral artery occlusion (MCAO) for 90 minutes followed by 24 or 72 hours of reperfusion was performed in male Sprague Dawley rats, then, melatonin 50 or 150 µg/kg, calpeptin 10, 15 or 50 µg/kg or a combination of melatonin 50 µg/kg plus calpeptin 15 or 50 µg/kg were injected via an intracerebroventricular route at 15 minutes after onset of reperfusion. Melatonin or calpeptin tended to reduce the relative infarct volume and significantly decreased the neurological deficit at 24 hours. The combination achieved a greater protection than each of them alone. Melatonin, calpeptin or the combination all decreased Fluoro-Jade B (FJB)+ degenerative neurons and cleaved/total caspase-3 ratio at 24 hours. These treatments did not significantly impact the density of surviving neurons and ED-1+ macrophage/activated microglia. At the 72-hour-reperfusion, melatonin or the combination decreased the relative infarct volume and neurological deficit. Nevertheless, only the combination reduced FJB+ degenerating neurons at 72 hours. In conclusion, a combination of melatonin and calpeptin exerted synergistic protection against post-reperfusion injury in a rat MCAO stroke model.

A Comparative Study of Koizumi and Longa Methods of Intraluminal Filament Middle Cerebral Artery Occlusion in Rats: Early Corticosterone and Inflammatory Response in the Hippocampus and Frontal Cortex

Two classical surgical approaches for intraluminal filament middle cerebral artery occlusion (MCAO), the Longa et al. (LM) and Koizumi et al. methods (KM), are used as alternatives in preclinical studies to induce stroke in rodents. Comparisons of these MCAO models in mice showed critical differences between them along with similarities (Smith et al. 2015; Morris et al. 2016). In this study, a direct comparison of MCAO-KM and MCAO-LM in rats was performed. Three days after MCAO, infarct volume, mortality rate, neurological deficit, and weight loss were similar in these models. MCAO-LM rats showed an increase in ACTH levels, while MCAO-KM rats demonstrated elevated corticosterone and interleukin-1β in blood serum. Corticosterone accumulation was detected in the frontal cortex (FC) and the hippocampus of the MCAO-KM group. IL1β beta increased in the ipsilateral hippocampus in the MCAO-KM group and decreased in the contralateral FC of MCAO-LM rats. Differences revealed between MCAO-KM and MCAO-LM suggest that corticosterone and interleukin-1β release as well as hippocampal accumulation is more expressed in MCAO-KM rats, predisposing them to corticosterone-dependent distant neuroinflammatory hippocampal damage. The differences between two models, particularly, malfunction of the hypothalamic–pituitary–adrenal axis, should be considered in the interpretation, comparison, and translation of pre-clinical experimental results.

Effect of early Sanguinate (PEGylated carboxyhemoglobin bovine) infusion on cerebral blood flow to the ischemic core in experimental middle cerebral artery occlusion

BackgroundSanguinate, a bovine PEGylated carboxyhemoglobin-based oxygen carrier with vasodilatory, oncotic and anti-inflammatory properties designed to release oxygen in hypoxic tissue, was tested to determine if it improves infarct volume, collateral recruitment and blood flow to the ischemic core in hyperacute middle cerebral artery occlusion (MCAO).MethodsUnder an IACUC approved protocol, 14 mongrel dogs underwent endovascular permanent MCAO. Seven received Sanguinate (8 mL/kg) intravenously over 10 min starting 30 min following MCAO and seven received a similar volume of normal saline. Relative cerebral blood flow (rCBF) was assessed using neutron-activated microspheres prior to MCAO, 30 min following MCAO and 30 min following intervention. Pial collateral recruitment was scored and measured by arterial arrival time (AAT) immediately prior to post-MCAO microsphere injection. Diffusion-weighted 3T MRI was used to assess infarct volume approximately 2 hours after MCAO.ResultsMean infarct volumes for control and Sanguinate-treated subjects were 4739 mm3 and 2585 mm3 (p=0.0443; r2=0.687), respectively. Following intervention, rCBF values were 0.340 for controls and 0.715 in the Sanguinate group (r2=0.536; p=0.0064). Pial collateral scores improved only in Sanguinate-treated subjects and AAT decreased by a mean of 0.314 s in treated subjects and increased by a mean of 0.438 s in controls (p<0.0276).ConclusionPreliminary results indicate that topload bolus administration of Sanguinate in hyperacute ischemic stroke significantly improves infarct volume, pial collateral recruitment and CBF in experimental MCAO immediately following its administration.

Adenosine A1R/A3R (Adenosine A1 and A3 Receptor) Agonist AST-004 Reduces Brain Infarction in a Nonhuman Primate Model of Stroke

Background and Purpose: Treatment with A1R/A3R (adenosine A1 and A3 receptor) agonists in rodent models of acute ischemic stroke results in significantly reduced lesion volume, indicating activation of adenosine A1R or A3R is cerebroprotective. However, dosing and timing required for cerebroprotection has yet to be established, and whether adenosine A1R/A3R activation will lead to cerebroprotection in a gyrencephalic species has yet to be determined. Methods: The current study used clinical study intervention timelines in a nonhuman primate model of transient, 4-hour middle cerebral artery occlusion to investigate a potential cerebroprotective effect of the dual adenosine A1R/A3R agonist AST-004. Bolus and then 22 hours intravenous infusion of AST-004 was initiated 2 hours after transient middle cerebral artery occlusion. Primary outcome measures included lesion volume, lesion growth kinetics, penumbra volume as well as initial pharmacokinetic-pharmacodynamic relationships measured up to 5 days after transient middle cerebral artery occlusion. Secondary outcome measures included physiological parameters and neurological function. Results: Administration of AST-004 resulted in rapid and statistically significant decreases in lesion growth rate and total lesion volume. In addition, penumbra volume decline over time was significantly less under AST-004 treatment compared with vehicle treatment. These changes correlated with unbound AST-004 concentrations in the plasma and cerebrospinal fluid as well as estimated brain A1R and A3R occupancy. No relevant changes in physiological parameters were observed during AST-004 treatment. Conclusions: These findings suggest that administration of AST-004 and combined A1R/A3R agonism in the brain are efficacious pharmacological interventions in acute ischemic stroke and warrant further clinical evaluation.

Histone Methyltransferase Dot1L Contributes to RIPK1 Kinase‐Dependent Apoptosis in Cerebral Ischemia/Reperfusion

Background Neuron apoptosis is a pivotal process for brain damage in cerebral ischemia. Dot1L (disruptor of telomeric silencing 1‐like) is only known histone H3K79 methyltransferase. It is not clear whether the role and mechanism of Dot1L on cerebral ischemia is related to regulate neuron apoptosis. Methods and Results We use a combination of mice middle cerebral artery occlusion stroke and neurons exposed to oxygen‐glucose deprivation followed by reoxygenation to investigate the role and mechanism of Dot1L on cerebral ischemia. We find knockdown or inhibition of Dot1L reversed ischemia‐induced neuronal apoptosis and attenuated the neurons injury treated by oxygen‐glucose deprivation followed by reoxygenation. Further, blockade of Dot1L prevents RIPK1 (receptor‐interacting protein kinase 1)‐dependent apoptosis through increased RIPK1 K63‐ubiquitylation and decreased formation of RIPK1/Caspase 8 complexes. In line with this, H3K79me3 enrichment in the promoter region of deubiquitin‐modifying enzyme A20 and deubiquitinase cylindromatosis gene promotes the increasing expression in oxygen‐glucose deprivation followed by reoxygenation ‐induced neuronal cells, on the contrary, oxygen‐glucose deprivation followed by reoxygenation decreases H3K79me3 level in the promoter region of ubiquitin‐modifying enzyme cIAP1 (cellular inhibitors of apoptosis proteins), and both these factors ultimately cause K63‐deubiquitination of RIPK1. Importantly, knockdown or inhibition of Dot1L in vivo attenuates apoptosis in middle cerebral artery occlusion mice and reduces the extent of middle cerebral artery occlusion ‐induced brain injury. Conclusions These data support for the first time, to our knowledge, that Dot1L regulating RIPK1 to the apoptotic death trigger contributes to cerebral ischemia injury. Therefore, targeting Dot1L serves as a new therapeutic strategy for ischemia stroke.

Effects of Magnetically Targeted Iron Oxide@Polydopamine-Labeled Human Umbilical Cord Mesenchymal Stem Cells In Cerebral Infarction In Mice

Mesenchymal stem cells are a potential therapeutic candidate for cerebral infarction due to their anti-inflammatory proprieties. However, ensuring the engraftment of sufficient cells into the affected brain area remains a challenge. Herein, magnetic targeting techniques were used for the noninvasive transplantation of a large number of cells noninvasively. Mice subjected to permanent middle cerebral artery occlusion surgery were administered mesenchymal stem cells labeled or not with iron oxide@polydopamine nanoparticles by tail vein injection. Iron oxide@polydopamine particles were characterized by transmission electron microscopy, and labeled mesenchymal stem cells were characterized by flow cytometry and their differentiation potential was assessed in vitro . Following the systemic injection of iron oxide@polydopamine-labeled mesenchymal stem cells into permanent transient middle cerebral artery occlusion-induced mice, magnetic navigation increased the MSCs localization to the brain lesion site and reduced the lesion volume. Treatment with iron oxide@polydopamine-labeled mesenchymal stem cells also significantly inhibited M1 microglia polarization and increased M2 microglia cell infiltration. Furthermore, western blotting and immunohistochemical analysis demonstrated that microtubule-associated protein 2 and NeuN levels were upregulated the brain tissue of mice treated with iron oxide@polydopamine-labeled mesenchymal stem cells. Thus, iron oxide@polydopamine-labeled mesenchymal stem cells attenuated brain injury and protected neurons by preventing pro-inflammatory microglia activation. Overall, the proposed iron oxide@polydopamine-labeled mesenchymal stem cells approach may overcome the major drawback of the conventional MSCs therapy for the treatment of cerebral infarction.

Mesenchymal stromal cell-derived small extracellular vesicles promote neurological recovery and brain remodeling after distal middle cerebral artery occlusion in aged rats

Small extracellular vesicles (sEVs) obtained from mesenchymal stromal cells (MSCs) promote neurological recovery after middle cerebral artery occlusion (MCAO) in young rodents. Ischemic stroke mainly affects aged humans. MSC-sEV effects on stroke recovery in aged rodents had not been assessed. In a head-to-head comparison, we exposed young (4–5 months) and aged (19–20 months) male Sprague–Dawley rats to permanent distal MCAO. At 24 h, 3 and 7 days post-stroke, vehicle or MSC-sEVs (2 × 106 or 2 × 107 MSC equivalents/kg) were intravenously administered. Neurological deficits, ischemic injury, brain inflammatory responses, post-ischemic angiogenesis, and endogenous neurogenesis were evaluated over 28 days. Post-MCAO, aged vehicle-treated rats exhibited more severe motor-coordination deficits evaluated by rotating pole and cylinder tests and larger brain infarcts than young vehicle-treated rats. Although infarct volume was not influenced by MSC-sEVs, sEVs at both doses effectively reduced motor-coordination deficits in young and aged rats. Brain macrophage infiltrates in periinfarct tissue, which were evaluated as marker of a recovery-aversive inflammatory environment, were significantly stronger in aged than young vehicle-treated rats. sEVs reduced brain macrophage infiltrates in aged, but not young rats. The tolerogenic shift in immune balance paved the way for structural brain tissue remodeling. Hence, sEVs at both doses increased periinfarct angiogenesis evaluated by CD31/BrdU immunohistochemistry in young and aged rats, and low-dose sEVs increased neurogenesis in the subventricular zone examined by DCX/BrdU immunohistochemistry. Our study provides robust evidence that MSC-sEVs promote functional neurological recovery and brain tissue remodeling in aged rats post-stroke. This study encourages further proof-of-concept studies in clinic-relevant stroke settings.

Nanoliposomes Reduce Stroke Injury Following Middle Cerebral Artery Occlusion in Mice

Background and Purpose: Neuroprotective strategies for stroke remain inadequate. Nanoliposomes comprised of phosphatidylcholine, cholesterol, and monosialogangliosides (nanoliposomes) induced an antioxidant protective response in endothelial cells exposed to amyloid insults. We tested the hypotheses that nanoliposomes will preserve human neuroblastoma (SH-SY5Y) and human brain microvascular endothelial cells viability following oxygen-glucose deprivation (OGD)–reoxygenation and will reduce injury in mice following middle cerebral artery occlusion. Methods: SH-SY5Y and human brain microvascular endothelial cells were exposed to oxygen-glucose deprivation–reoxygenation (3 hours 0.5%–1% oxygen and glucose-free media followed by 20-hour ambient air/regular media) without or with nanoliposomes (300 µg/mL). Viability was measured (calcein-acetoxymethyl fluorescence) and protein expression of antioxidant proteins HO-1 (heme oxygenase-1), NQO1 (NAD[P]H quinone dehydrogenase 1), and SOD1 (superoxide dismutase 1) were measured by Western blot. C57BL/6J mice were treated with saline (n=8) or nanoliposomes (10 mg/mL lipid, 200 µL, n=7) while undergoing 60-minute middle cerebral artery occlusion followed by reperfusion. Day 2 postinjury neurological impairment score and infarction size were compared. Results: SH-SY5Y and human brain microvascular endothelial cells showed reduced viability post–oxygen-glucose deprivation–reoxygenation that was reversed by nanoliposomes. Nanoliposomes increased protein expressions of HO-1, NQO1 in both cell types and SOD1 in human brain microvascular endothelial cells. Nanoliposomes-treated mice showed reduced neurological impairment and brain infarct size (18.8±2% versus 27.3±2.3%, P =0.017) versus controls. Conclusions: Nanoliposomes reduced stroke injury in mice subjected to middle cerebral artery occlusion likely through induction of an antioxidant protective response. Nanoliposome is a candidate novel agent for stroke.