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Dimethylarginine dimethylaminohydrolase 1 (DDAH1) protects against cerebral ischemia injury via regulating the level of asymmetric dimethylarginine (ADMA). This study is aimed at exploring the effect of adiponectin resistance on ADMA-induced neuronal loss in ischemic stroke (IS) and the underlying mechanism. DDAH1 knockout (DDAH1 -/- ) and wild-type (DDAH1 +/+ ) rats underwent middle cerebral artery occlusion/reperfusion (MCAO/R). Plasma and brain adiponectin levels and the expressions of adiponectin receptor 1 (APR1), adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper 1 (APPL1), adenosine monophosphate-activated protein kinase (AMPK), and phosphorylated AMPK were determined after 24 h, 3 days, and 7 days. Neurological behavior, infarct volume, and adiponectin signaling were evaluated using adiponectin peptide or AdipoRon. The levels of reactive oxygen species (ROS) and Forkhead box O1 (FOXO1) (a transcription factor for APR1) were also assessed. An oxygen-glucose deprivation/reoxygenation (OGD/R) model was established in primary neurons. DDAH1 was overexpressed in neurons, after which FOXO1 expression, ROS production, adiponectin resistance, and cell viability were detected. DDAH1 -/- rats showed no significant difference in adiponectin level in either plasma or brain after MCAO/R in DDAH1 +/+ rats, but downregulated APR1 expression and suppressed adiponectin signaling were observed. AdipoRon, but not adiponectin peptide, attenuated the neurological deficits and adiponectin resistance in DDAH1 -/- rats. ROS accumulation and phosphorylated FOXO1 expression also increased with DDAH1 depletion. Following DDAH1 overexpression, decreased cell viability and inhibited adiponectin signaling induced by OGD/R were alleviated in primary neurons, accompanied by reduced ROS production and phosphorylated FOXO1 expression. Our study elucidated that in IS, DDAH1 protected against adiponectin resistance in IS via the ROS/FOXO1/APR1 pathway., Competing Interests: The authors declare no conflicts of interest regarding the publication of this manuscript., (Copyright © 2022 Yichen Zhao et al.)

Dimethylarginine dimethylamino hydrolase-1 (DDAH-1) is an important regulator of nitric oxide (NO) metabolism that has been implicated in the pathogenesis of cardiovascular diseases. Nevertheless, its role in cerebral ischemia still needs to be elucidated. Herein, we examined the expression of DDAH-1 in the brain of rat by double-label immunofluorescence staining. DDAH-1 knock-out (DDAH-1 -/- ) and wild-type rats underwent middle cerebral artery occlusion/reperfusion (MCAO/R). After 24 h, neurological scores, TTC staining and TUNEL assay were used to evaluate neurological damages. 3 and 7-days infarct outcomes were also shown. Blood-brain-barrier (BBB) permeability was examined via Evans blue extravasation and tight junction (TJ) proteins expression and mRNA levels by western blot and RT-qPCR. The levels of plasma asymmetric dimethylarginine (ADMA), NO and ADMA in brain tissue were also assessed. In addition, supplementation of L-arginine to DDAH-1 -/- rats was used to explore its role in regulating NO. DDAH-1 was abundantly distributed in cerebral cortex and basal nuclei, and mainly expressed in neurons and endothelial cells. DDAH-1 -/- rats showed aggravated neurological damage and BBB disruption, including decrease of TJ proteins expression but indistinguishable mRNA levels after MCAO/R. DDAH-1 depletion and neurological damages were accompanied with increased ADMA levels and decreased NO concentrations. The supplementation with L-arginine partly restored the neurological damages and BBB disruption. To sum up, DDAH-1 revealed to have a protective role in ischemia stroke (IS) and IS-induced leakage of BBB via decreasing ADMA level and possibly via preventing TJ proteins degradation.

Mechanical thrombectomy is not only effective for managing patients with acute ischemic stroke (AIS), but it also enables a valuable histological analysis of thrombi. Previous studies indicated that regulatory T cells (Treg) adoptive transfer might alleviate the hemorrhagic transformation. However, whether Treg in intracranial thrombi correlates with hemorrhagic transformation after mechanical thrombectomy remains unclear. This study mainly analyzed the colocation of Treg markers in serial thrombus sections stained serially for CD4 and CD25 in groups of hemorrhagic or nonhemorrhagic transformation. Second, to investigate whether these immunohistochemical parameters could provide any additional information beyond hemorrhagic transformation, we compared the overlap between Treg markers among other groups, such as functional outcomes, stroke subtypes, and gender. Our results showed that the number of CD4 + CD25 + Treg cells was lower in the hemorrhagic transformation thrombi than in the nonhemorrhagic group ( p < 0.001) but there were no significant differences otherwise. The present finding of CD4 + CD25 + Treg cell reductions in thrombi associated with hemorrhagic transformation provides the histological evidence supporting that thromboinflammation might involve in the pathological process of an acute stroke after mechanical thrombectomy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Li Gong et al.)

Dimethylarginine dimethylamino hydrolase-1 (DDAH-1) as an indirect regulator of nitric oxide (NO) metabolism, its role in hypoxic preconditioning (HPC) and ischemic tolerance (IT) of ischemic stroke has still been unknown and needs to be elucidated. Herein, DDAH-1 knock-out (KO) and wild-type (WT) rats underwent HPC and middle cerebral artery occlusion/reperfusion (MCAO/R) model. After 24 h, neurological severity scores, TTC staining and TUNEL assay were used to evaluate neurological damages. To explore the mechanism, the expression of hypoxia inducible factor (HIF-1α) and its target genes were assessed by Western blot and RT-qPCR. NO and ADMA contents were also tested. In addition, supplementation of l-arginine to DDAH-1 KO rats was used to explore the role of DDAH-1 in regulating NO. After HPC the ischemic outcome improved in both KO and WT rats, while KO rats showed attenuated IT exhibiting less expression of HIF-1α and its target genes, lower NO but higher ADMA content. The supplement of l-arginine to KO rats partly alleviated neurological damages accompanied with higher expression of HIF-1α. To sum up, DDAH-1 could regulate the level of NO and enhance IT following HPC and MCAO model via activating the expression of HIF-1α and its target genes., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Manipulating the immune inflammatory response after cerebral ischemia has been a novel therapeutic strategy for ischemic stroke. This study attempted to investigate the effects of the transplantation of lymphocytes co-cultured with human cord blood-derived multipotent stem cells (HCB-SCs) on the inflammatory response in transient middle cerebral occlusion (tMCAO) rats., Methods: The tMCAO rats were subjected to the transplantation of lymphocytes co-cultured with HCB-SCs through tail vein injection. Infarct size and neurological deficits were measured at 48 hrs after stroke. Neurological deficits were assessed using Bederson's scoring system and tape removal test. Blood T cell flow cytometry was performed to measure the differentiation of regulatory T cells (Tregs). Western blot was used to detect the protein levels of inflammation-related molecules, apoptosis-related molecule, and signaling molecules in ischemic brain. TUNEL staining was performed to analyze cell apoptosis in ischemic cerebral cortex., Results: The transplantation of lymphocytes co-cultured with HCB-SCs significantly improved the neurological defects, reduced ischemic brain damage, and increased the proportion of peripheral CD4 + CD25 + Foxp3 + Tregs. Meanwhile, the transplantation of co-cultured cells decreased the expression of NLRP3 inflammasome and associated factors, such as caspase-1 and IL-1β, and inhibited the activation of NF-κB, ERK and caspase-3 in ischemic brain. The co-cultured cells significantly decreased the number of tMCAO-induced cell apoptosis., Conclusion: Lymphocytes co-cultured with HCB-SCs exhibit a neuroprotective effect after ischemic stroke by promoting Tregs differentiation and suppressing NLRP3 inflammasome activation and neuron apoptosis, and might be a promising therapeutic strategy for ischemic stroke., Competing Interests: The authors report no conflicts of interest in this work., (© 2019 Zhao et al.)

Background: Post-stroke depression (PSD) seriously affects the rehabilitation of nerve function and quality of life. However, the pathogenesis of PSD is still not clear. This study aimed to investigate the demographic, clinical, and biochemical factors in patients with PSD., Methods: Patients with an acute ischemic stroke, who met the inclusion criteria at Shanghai Tenth People's Hospital from April 2016 to September 2016, were recruited for this study. The stroke severity was assessed using the National Institutes of Health Stroke Scale (NIHSS), and the mental state was assessed using Mini-Mental State Examination (MMSE), Hamilton Depression Scale (HAMD), and Hamilton Anxiety Scale (HAMA) at 1 week of admission. The patients were divided into PSD and non-PSD groups. The demographic and clinical characteristics, as well as the biochemical factors, were compared between the two groups. A logistic regression analysis was performed to identify the risk factors for depression following stroke., Results: A total of 83 patients with acute ischemic stroke were recruited. Of these, 36 (43.4%) developed depression. The multivariate logistic regression analysis indicated that high NIHSS [odds ratio (OR): 1.84, 95% confidence interval (CI): 1.09-3.12, P = 0.023] and high HAMD scores (OR: 2.38, 95% CI: 1.61-3.50, P < 0.001) were independent risk predictors for PSD and so were lower dopamine level (OR: 0.64, 95% CI: 0.45-0.91, P = 0.014), lower 5-hydroxytryptamine level (OR: 0.99, 95% CI: 0.98-1.00, P = 0.046), higher tumor necrosis factor-α level (OR: 1.05, 95% CI: 1.00-1.09, P = 0.044), and lower nerve growth factor level (OR: 0.06, 95% CI: 0.01-0.67, P = 0.022)., Conclusions: The identification of higher NIHSS scores, higher HAMD scores, lower dopamine level, lower 5-hydroxytryptamine level, higher tumor necrosis factor-α level, and lower nerve growth factor level might be useful for clinicians in recognizing and treating depression in patients after a stroke.

Ischemic tolerance reduces brain damage and neurological dysfunction after brain ischemia. A ketogenic diet (KD) has disease-modifying effects in several neurodegenerative disorders. In this study, we fed mice with a KD for three weeks and performed reversible middle cerebral artery occlusion (MCAO) in the animals. KD-fed mice had a significantly reduced infarct volume, increased regional cerebral blood flow (rCBF) and extracellular adenosine levels in both the ischemic and the reperfusion phases. In vitro and in vivo experiments revealed that the KD-induced neuroprotection was mediated through the adenosine A1 receptor. The KD increased Akt and ERK1/2 phosphorylation via A1R activation. Besides, the KD also upregulated robustly HIF-1α/HIF-2α and HIF regulated genes, such as VEGF and EPO. A three-week preconditioning period with a KD improved ischemic tolerance in mice with MCAO. The underlying mechanisms might include elevated extracellular adenosine levels, and increased Akt and ERK1/2 phosphorylation via A1 adenosine receptor activation, together with upregulated HIFs and HIF-regulated genes., (Copyright © 2017 Elsevier B.V. All rights reserved.)

This study aimed to examine whether the neuroprotective effects of Mdivi-1 are attributable to extracellular ATP and adenosine. Mdivi-1 was administered prior to or post middle cerebral artery occlusion (MCAO). The extracellular adenosine was measured by in vivo microdialysis and high-pressure liquid chromatography (HPLC) in MCAO mouse model. Western blot was done to determine the influence of Mdivi-1 on the expression of CD39 and CREB phosphorylation both in vivo and in the cultured astrocytes. Intracellular cAMP and protein kinase A (PKA) activity were detected in primary astrocytes. Results showed that Mdivi-1 significantly reduced infarct volume and neurological scores when administered either prior to or post MCAO. Interestingly, pretreatment with Mdivi-1 resulted in marked increase of extracellular adenosine and concomitant decrease in ATP. The expression of CD39, but not CD73, was upregulated by Mdivi-1, which was associated with the elevated phosphorylated cAMP response element-binding protein (CREB), a transcription factor potentially regulating CD39 expression. In primary astrocytes, Mdivi-1 treatment induced increases in intracellular cAMP, PKA activity and CREB phosphorylation, and PKA-specific inhibitor completely reversed Mdivi-1-induced CD39 expression. Our results demonstrate that Mdivi-1 protects against ischemic brain injury through increasing extracellular adenosine, a process involving elevated CD39 expression that is likely modulated by cAMP/PKA/CREB cascade. Figure Potential mechanisms by which Mdivi-1 mediates the neuroprotection on cerebral ischemic stroke. Results from the present study indicate that Mdivi-1 protects against ischemic brain injury through increasing extracellular adenosine, a process involving elevated CD39 expression that is likely modulated by the cAMP/PKA/CREB cascades.

Our previous study has shown that PTEN-induced novel kinase 1 (PINK1) knocking down significantly induced mitochondrial fragmentation. Although PINK1 is proved to be associated with autosomal recessive parkinsonism and its function in this chronic pathological process is widely studied, its role in acute energy crisis such as ischemic stroke is poorly known. In this study by employing an oxygen-glucose deprivation (OGD) neuronal model, we explored the function of PINK1 in cerebral ischemia. Human PINK1, two PINK1 mutants W437X and K219M, or Pink1 shRNA were transduced before OGD using lentiviral delivery. Our results showed that over-expression of wild-type PINK1 significantly ameliorated OGD induced cell death and energy disturbance including reduced ATP generation and collapse of mitochondrial membrane potential. PINK1 over-expression also reversed OGD increased mitochondrial fragmentation, and suppressed the translocation of the mitochondrial fission protein dynamin-related protein 1 (Drp1) from the cytosol to the mitochondria. Transduction of the mutant PINK1 failed to provide any protective effect, while knockdown of Pink1 significantly increased the severity of OGD-induced neuronal damage. Importantly, inhibition of Drp1 reversed the effects of knocking down Pink1 on neuronal death and ATP production in response to OGD. This study demonstrates that PINK1 prevents ischemic damage in neurons by attenuating mitochondrial translocation of Drp1, which maintains mitochondrial function and inhibits ischemia-induced mitochondrial fission. These novel findings implicate a pivotal role of PINK1 regulated mitochondrial dynamics in the pathology of ischemic stroke. In this study by employing an oxygen-glucose deprivation (OGD) neuronal model, we explored the function of PINK1 in cerebral ischemia. We indicated that PINK1 significantly ameliorated OGD induced cell death and energy disturbance including reduced ATP generation and collapse of mitochondrial membrane potential by attenuating mitochondrial translocation of Drp1, which maintains mitochondrial function and inhibits ischemia-induced mitochondrial fission., (© 2013 International Society for Neurochemistry.)

Calcium toxicity remains the central focus of ischemic brain injury. Calcium channel antagonists have been reported to be neuroprotective in ischemic animal models but have failed in clinical trials. Rather than block the calcium channels, calbindin proteins can buffer excessive intracellular Ca2+, and as a result, maintain the calcium homeostasis. In the present study, we investigated the effect of calbindin D 28k (CaBD) in ischemic brain using the novel technique protein transduction domain (PTD)-mediated protein transduction. We generated PTD-CaBD in Escherichia coli, tested its biologic activity in N-methyl-D-aspartate (NMDA)- and oxygen-glucose deprivation (OGD)-induced hippocampal injury models, and examined the protection of the fusion protein using a rat brain focal ischemia model. Infarct volume was determined using 2,3,5-triphenyl-tetrazolium chloride staining; neuronal injury was examined using terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate-biotin nick end labeling (TUNEL) staining and cleaved caspase-3 assay. The results showed that the PTD-CaBD was efficiently delivered into Cos7 cells, hippocampal slice cells, and brain tissue. Pretreatment with PTD-CaBD decreased intracellular free calcium concentration and reduced cell death in NMDA- or OGD-exposed hippocampal slices (P<0.05). Intraperitoneal administration of PTD-CaBD before transient middle cerebral artery occlusion decreased brain infarct volume (280+/-47 versus 166+/-70 mm3, P<0.05), and improved neurologic outcomes compared with the control. Further studies showed that, compared with the control animals, PTD-CaBD decreased TUNEL (58%+/-7% versus 29%+/-3%, P<0.05)- and cleaved caspase-3 (62+/-4/field versus 31+/-6/field, P<0.05)-positive cells in the ischemic boundary zone. These results indicate that systemic administration of PTD-CaBD could attenuate ischemic brain injury, suggesting that PTD-mediated protein transduction might provide a promising and effective approach for the therapies of brain diseases, including cerebral ischemia.