Conditions Of Cerebral Ischemia Research Articles

ACTH-like Peptides Compensate Rat Brain Gene Expression Profile Disrupted by Ischemia a Day After Experimental Stroke.

Background: Ischemic stroke results from a disruption of cerebral blood flow. Adrenocorticotropic hormone (ACTH) serves as the basis for the creation of synthetic peptides as neuroprotective agents for stroke therapy. Previously, using RNA-Seq we first revealed differential expressed genes (DEGs) associated with ACTH(4-7)PGP (Semax) and ACTH(6-9)PGP peptides under cerebral ischemia conditions. Analysis was carried out at 4.5 h after transient middle cerebral artery occlusion (tMCAO) model in the ipsilateral frontal cortex of a rat brain. Methods: Here, we analyzed the penumbra-associated frontal cortex of rats and actions under the same peptides at 24 h after tMCAO using RNA-Seq. Results: 3774 DEGs (fold change > 1.5 and Padj < 0.05) were identified under ischemia conditions, whereas 1539 and 2066 DEGs were revealed under Semax and ACTH(6-9)PGP peptides at 24 h after tMCAO. Furthermore, both peptides significantly reduced expression distortions caused by ischemia for 1171 genes associated with immune and neurosignaling pathways. Concomitantly, there were 32 DEGs under ACTH(6-9)PGP versus Semax administration at 24 h after tMCAO. Besides, neurogenesis-, angiogenesis-, protein kinase- and growth factor-related DEGs were revealed under peptides action. Previously, we observed the neuroprotective effect of peptides at the histological level in rat brains at 24 h after tMCAO. Thus, here we demonstrate the transcriptome manifestation of this histological effect. Furthermore, comparison with previous data at the 4.5 h post-tMCAO time point showed that the pattern of peptide action on the transcriptome depends on the time elapsed after tMCAO. Conclusions: We revealed that the effect of ACTH(6-9)PGP was more similar to Semax than different from it a day after tMCAO. At this time point, ACTH-like peptides compensated rat brain gene expression profiles disrupted by ischemia. Thus, our results may be useful for selecting more effective structures for future anti-stroke drugs and appropriate post-stroke time points for their testing.

Melanocortin Derivatives Induced Vascularization and Neuroglial Proliferation in the Rat Brain under Conditions of Cerebral Ischemia.

Stroke remains the second leading cause of death worldwide. The development of new therapeutic agents focused on restoring vascular function and neuroprotection of viable tissues is required. In this study the neuroprotective activity of melanocortin-like ACTH(4-7)PGP and ACTH(6-9)PGP peptides was investigated in rat brain at 24 h after transient middle cerebral artery occlusion (tMCAO). The severity of ischemic damage, changes in the proliferative activity of neuroglial cells and vascularization of rat brain tissue were analyzed. The administration of peptides resulted in a significant increase in the volume density of neurons in the perifocal zone of infarction compared to rats subjected to ischemia and receiving saline. Immunohistochemical analysis of the proliferative activity of neuroglia cells using PCNA antibodies showed a significant increase in the number of proliferating cells in the penumbra and in the intact cerebral cortex of rats receiving peptide treatment. The effect of peptides on vascularization was examined using CD31 antibodies under tMCAO conditions, revealing a significant increase in the volume density of vessels and their sizes in the penumbra after administration of ACTH(4-7)PGP and ACTH(6-9)PGP. These findings confirm the neuroprotective effect of peptides due to the activation of neuroglia proliferation and the enhancement of collateral blood flow.

Identifying Qingkailing ingredients-dependent mesenchymal-epithelial transition factor-axiation "π" structuring module with angiogenesis and neurogenesis effects.

To explore the functional role of the drug-dependent mesenchymal-epithelial transition (Met)-axiation "π" structural module of neurogenesis after processing by three components of Qingkailing injection in neurogenesis and angiogenesis in cerebral ischemia. We used a Glutathione S-transferase (GST)-pull down assay, isothermal titration calorimetry assay, and other related methods to identify the relationships among Met, inositol polyphosphate phosphatase like 1 (Inppl1), and death associated protein kinase 3 (Dapk3) in this allosteric module. The biological effects of the modules of neurons generation composed of Met, Inppl1, and Dapk3 were measured through Western blot, apoptosis analysis, and double immunofluorescence labeling. The GST-pull down assay revealed that proline-serine-threonine rich domain of Met binds to the Src homology domain of Inppl1 to form a protein-protein complex; Dapk3 with a C-terminal domain interacts weakly with the protein kinase C domain of Met in the intracellular region. Thus, we obtained a "π" structuring module considered a neural regeneration module. The biological effects of angiogenesis and neurogenesis modules composed of Met, Inppl1, and Dapk3 were also verified. The study suggested that understanding the functional modules that contribute to pharmaceutics might provide novel signatures that can be used as endpoints to define disease processes under stroke or cerebral ischemia conditions.

DRP1 Regulation as a Potential Target in Hypoxia-Induced Cerebral Pathology

The following review considers current concepts concerning the characteristics of DRP1-related mitochondrial division in brain cells during hypoxic-ischemic pathology. The functional role of DRP1 in neurons and astroglia in cerebral ischemia conditions was analyzed. We discuss the potential for regulating DRP1 activity through the selective inhibitor of mitochondrial fission, mdivi-1. The article also presents data on DRP1 involvement in astro- and microglia-mediated intercellular mitochondrial transport. Understanding of the molecular mechanisms responsible for mitochondrial fission during hypoxic-ischemic exposure will allow us to consider DRP1 as an effective therapeutic target for treating conditions with a hypoxic component.

Evidence that enolase-phosphatase 1 exacerbates early cerebral ischemia injury and blood–brain barrier breakdown by enhancing extracellular matrix destruction and inhibiting the interaction between ADI1 and MT1-MMP

Enolase-phosphatase 1 (ENOPH1) is a newly identified enzyme associated with stress responses and cell proliferation. Our previous study found that ENOPH1 mediates cerebral microvascular endothelial cell apoptosis under cerebral ischemia conditions. In this study, we systematically provide mechanistic insights into the regulation of ENOPH1 in blood–brain barrier (BBB) dysfuction induced by early ischemia. ENOPH1 knockout mice (ENOPH1 KO) and wild type (WT) mice were exposed to transient middle cerebral artery occlusion (tMCAO) for 90 min followed by 3 h of reperfusion in vivo, and brain microvascular endothelial cell lines (bEnd.3 cells) were exposed to oxygen-glucose deprivation (OGD) in vitro. BEnd.3 cells were transfected with ENOPH1 shRNA to knockdown ENOPH1 expression. Brain ischemic damage and nerve function was assessed with 2, 3, 5-triphenyltetrazolium chloride (TTC) staining and neurological scores. BBB permeability and tight junction (TJ) protein and adherens junction (AJ) proteins expression were analyzed by FITC-dextran staining, western blotting and coimmunofluorescence. The MMP-2/9 activity was analyzed by gelatin zymography. Differential protein expression was assessed by quantitative proteomics. The interaction between ADI1 and MT1-MMP was measured by coimmunoprecipitation assay and coimmunofluorescence. Knockout of ENOPH1 ameliorated cerebral ischemic injury, decreased BBB permeability, inhibited the activity of MMP-2/9, upregulated the expression of TJ/AJ proteins and reversed extracellular matrix destruction after ischemia in vivo. Mechanistic studies have shown that ENOPH1 silencing enhanced the interaction between ADI1 and MT1-MMP by promoting the nuclear translocation of ADI1 to inhibit MT1-MMP in bEnd.3 cells after OGD and decreasing the expression of Tnc and Fn1 to inhibit ECM degradation. Our results reveal that ENOPH1 increases the activity of MMP-2/9, then promotes TJ protein and extracellular matrix degradation, and eventually destroys the stability of the BBB. Therefore, ENOPH1 is a new therapeutic target for ischemic stroke.

Effects of Neuroglobin in Cerebral Ischemia and Introduction of Omega-3 Polyunsaturated Fatty Acids

The aim of the work was to evaluate the content of neuroglobin in the brain of rats with cerebral ischemia and under the conditions of administration of omega-3 polyunsaturated fatty acids. The content of neuroglobin in the cytoplasm of neurons in the parietal cortex and hippocampus of rats under conditions of cerebral ischemia and administration of omega-3 polyunsaturated fatty acids was studied. Against the background of the introduction of the drug ω-3 PUFAs, there was an improvement in the indicators of antioxidant protection of brain neurons, which was manifested by an increase in the content of neuroglobin in hippocampal neurons.

Scutellarin acts on the AR-NOX axis to remediate oxidative stress injury in a mouse model of cerebral ischemia/reperfusion injury

BackgroundOxidative stress plays an important role in the pathology of ischemic stroke. Studies have confirmedthat scutellarin has antioxidant effects against ischemic injury, and we also reported that the involvement of Aldose reductase (AR) in oxidative stress and cerebral ischemic injury, in this study we furtherly explicit whether the antioxidant effect of scutellarin on cerebral ischemia injury is related to AR gene regulation and its specific mechanism. MethodsC57BL/6N mice (Wild-type, WT) and AR knockout (AR−/−) mice suffered from transient middle cerebral artery occlusion (tMCAO) injury (1 h occlusion followed by 3 days reperfusion), and scutellarin was administered from 2 h before surgery to 3 days after surgery. Subsequently, neurological function was assessed by the modified Longa score method, the histopathological morphology observed with 2,3,5-triphenyltetrazolium chloride (TTC) and hematoxylin-eosin (HE) staining. Enzyme-linked immunosorbent assay (Elisa) was used to detect the levels of ROS, 4-hydroxynonenal (4-HNE), 8-hydroxydeoxyguanosine (8-OHDG), Neurotrophin-3 (NT-3), poly ADP-ribose polymerase-1 (PARP1) and 3-nitrotyrosine (3-NT) in the ischemic penumbra regions. Quantitative proteomics profiling using quantitative nano-HPLC-MS/MS were performed to compare the protein expression difference between AR−/− and WT mice with or without tMCAO injury. The expression of AR, nicotinamide adenine dinucleotide phosphate oxidases (NOX1, NOX2 and NOX4) in the ipsilateral side of ischemic brain were detected by qRT-PCR, Western blot and immunofluorescence co-staining with NeuN. ResultsScutellarin treatment alleviated brain damage in tMCAO stroke model such as improved neurological function deficit, brain infarct area and neuronal injury and reduced the expression of oxidation-related products, moreover, also down-regulated tMCAO induced AR mRNA and protein expression. In addition, the therapeutic effect of scutellarin on the reduction of cerebral infarction area and neurological function deficits abolished in AR−/− mice under ischemia cerebral injury, which indicated that the effect of scutellarin treatment on tMCAO injury is through regulating AR gene. Proteomic analysis of AR−/− and WT mice indicated AR knockout would affect oxidation reaction even as NADPH related process and activity in mice under cerebral ischemia conditions. Moreover, NOX isoforms (NOX1, NOX2 and NOX4) mRNA and protein expression were significant decreased in neurons of penumbra region in AR−/− mice compared with that in WT mice at 3d after tMCAO injury, which indicated that AR should be the upstream protein regulating NOX after cerebral ischemia. ConclusionsWe first reported that AR directly regulates NOX subtypes (not only NOX2 but also NOX1 and NOX4) after cerebral ischaemic injury. Scutellarin specifically targets the AR-NOX axis and has antioxidant effects in mice with cerebral ischaemic injury, providing a theoretical basis and accurate molecular targets for the clinical application of scutellarin.

Study of the Neuroprotective Activity of a New Fumaric Acid Derivative

In a narrowing beam-walking test, on the 21st day after the occlusion of the middle cerebral artery, it was found that the succinic salt of diethylaminoethanol fumaric ester (PDES), at doses of 10 and 75 mg/kg, led to a statistically signifi cant decrease in the severity of sensorimotor defi cit of the anterior and posterior contralateral limbs of male rats under the conditions of cerebral ischemia. The test substance at a dose of 10 mg/kg reduced the volume of ischemic damage to the cerebral cortex of rats by 1.5 times. In terms of neuroprotective activity, the effect of the succinic salt of diethylaminoethanol fumaric ester at a dose of 10 mg/kg is comparable to that of Citicoline at a dose of 500 mg/kg.

Correction of mitochondrial dysfunction by succinic acid derivatives under experimental cerebral ischemia conditions

Abstract The aim of the study. To evaluate the effect of succinic acid derivatives on changes of mitochondrial function in rats under cerebral ischemia conditions. Materials and methods. In this work, the effect of succinic acid, ethylmethylhydroxypyridine succinate, and acetylaminosuccinic acid at doses of 50 mg/kg, 100 mg/kg, and 200 mg/kg (per os) on the change of the neuronal mitochondria function was studied. Cerebral ischemia was reproduced by the Tamura method. The following parameters were evaluated: changes in aerobic/anaerobic metabolism, mitochondrial membrane potential, the opening rate of the mitochondrial pore of transitional permeability and the activity of apoptotic systems. Results. During the study, it was found that the use of the test-compounds at doses of 100 mg/kg and 200 mg/kg contributed to an increase in ATP-generating activity, as well as the maximum respiration level and respiratory capacity, while accompanied by a decrease in the intensity of anaerobic metabolism reactions. Also, upon administration of the test succinic acid derivatives, an increase in the mitochondrial membrane potential and latent opening time of the mitochondrial pore transitional permeability were observed. Moreover, the activity of caspase-3 and apoptosis-inducing factor on groups treated by test objects at doses of 100 mg/kg and 200 mg/kg was significantly lower than that in untreated animals. Conclusion. The studied succinic acid derivatives contribute to the restoration of mitochondrial function in cerebral ischemia conditions, while the most effective dose can be considered to be 100 mg/kg.

Changes in the biogenesis of mitochondria in brain tissue in animals under conditions of cerebral ischemia

Changes in the biogenesis of mitochondria in brain tissue in animals under conditions of cerebral ischemia

Influence of some cinnamic acid derivatives on changes of the tricarboxylic acid cycle enzymes activity in rats under brain ischemia conditions

The aim of the study was to assess the effect of certain derivatives of cinnamic acids on changes of the tricarboxylic acid cycle enzymes activity under experimental cerebral ischemia.&#x0D; Materials and methods. Brain ischemia was modeled by irreversible right-sided coagulation of the middle cerebral artery. Test compounds: 4-hydroxy-3,5-ditretbutyl cinnamic acid, coumaric, coffee, synapic, cinnamic, 4-hydroxycinnamic and ferulic acids, as well as a reference drug succinic acid was administered at a dose of 100 mg / kg per os for 3 days after the reproduction of ischemia. Then, changes in the activity of aconitase, citrate synthase, and -ketoglutarate dehydrogenase were evaluated in the supernatant of the brain.&#x0D; Results. The use of all the studied compounds and the reference drug helped to restore the activity of enzymes of the tricarboxylic acid cycle. The most pronounced results were obtained when animals were treated by 4-hydroxy-3,5-ditretbutyl cinnamic acid, against the background of which the activity of citrate synthase was higher than in animals treated by succinic, coumaric, coffee, synapic and ferulic acids by 1.53 (p 0.05), 1.41 (p 0.05), 1.4 (p 0.05), 1.46 (p 0.05) and 1.41 (p 0.05) times, respectively. Also, with the administration of 4-hydroxy-3,5-ditretbutyl cinnamic acid, the activity of aconitase was higher compared to rats that were administered with succinic, coumaric, coffee, synapic and ferulic acids by 2.47 (p 0.05), 2.49 (p 0.05), 3.44 (p 0.05), 2.59 (p 0.05) and 1.9 (p 0.05) times, respectively.&#x0D; Conclusion. The administration of the studied in this work cinnamic acid derivatives helps to restore the activity of citrate synthase, aconitase, and -ketoglutarate dehydrogenase in rats under conditions of cerebral ischemia. The most pronounced changes in the activity of enzymes were obtained with the iadministration of 4-hydroxy-3,5-ditretbutyl cinnamic acid.

Hyperexpression of TLR2 and TLR4 in patients with ischemic stroke in acute period of the disease

Pathogenesis of ischemic stroke is actively involved in the system of innate immunity. Under conditions of cerebral ischemia, a number of biologically active substances are released that interact with innate immunity receptors, in particular TLR2 and TLR4, which exacerbate inflammation in brain tissue. Identification of predictor markers at the level of the innate immunity system may foresee the clinical course of ischemic stroke and ensure timely treatment. Our objective was to study expression of TLR2 and TLR4 receptors in peripheral blood leukocytes in patients with ischemic stroke in the dynamics of the disease. 27 people were included in the study. The main group consisted of patients with ischemic stroke of varying severity (n = 19). Patients of the main group were divided into two subgroups: with an NIHSS index value of &lt; 10 (n = 10) and &gt; 10 (n = 9). The control group included healthy donors with no history of acute and chronic inflammatory diseases (n = 8). Peripheral blood leukocytes were used as the test material. To determine expression of the TLR2 and TLR4 genes, RT-PCR in real time was used. Surface expression of TLRs was determined by flow cytometry. A study of the TLR2 and TLR4 gene expression showed that on the 1st, 3rd and 7th day post-stroke, the TLR4 gene expression in patients was significantly increased, when compared to the control group (p &lt; 0.01), whereas TLR2 gene expression on the 3rd day of the disease was not statistically different from the control group. A study of surface expression of receptors showed that the average TLR2 fluorescence intensity on the patients’ peripheral blood monocytes was significantly increased on the 1st and 3rd day of disease when compared to the control group. The surface expression of TLR4 on monocytes has a statistically significant increase only on day 7. Assessment of surface expression of TLRs in subgroups with different severity values by NIHSS showed that patients with a NIHSS index &gt; 10 had a significantly higher level of surface of TLR2 expression over the observation period, while the largest difference in TLR4 expression in the subgroups was observed on the 1st day of the disease (p &lt; 0.05). Patients with ischemic stroke showed an increase in TLR2 and TLR4 expression at the gene and protein level, compared to healthy donors. These indices can be considered possible predictors for clinical prognosis of ischemic stroke.

Ethylmethylhydroxypyridine succinate, acetylcysteine and choline alphoscerate improve mitochondrial function under condition of cerebral ischemia in rat

The study was devoted to see the effect of ethylmethylhydroxypyridine succinate, acetylcysteine and choline alphoscerate on the mitochondrial function under condition of experimental cerebral ischemia in rat. An integrated approach was used with the assessment of changes in the respirometric and antiapoptotic functions of mitochondria. It was found that the application of the objects under study contributed to a decrease in the intensity of glycolysis and the restoration of aerobic glucose metabolism with an increase in ATP synthesis, as well as a decrease in the dissociation of reactions in mitochondrial complexes I, II, IV and V. In addition, the introduction of the studied drugs reduced the intensity of caspase-dependent and caspase-independent apoptosis reactions, which, together with the restoration of mitochondrial respiratory function, contributed to the decrease in the size of the necrosis zone of the brain.&#x0D; Video Clip of Methodology:&#x0D; Rat middle cerebral artery occlusion: 8 min 7 sec Full Screen Alternate

EVALUATION OF THE MITOCHONDRIA RESPIROMETRIC FUNCTION IN THE CONDITIONS OF PATHOLOGIES OF VARIOUS GENESES

The aim of the paper is to assess the change in the mitochondrial respirometric function under conditions of various pathologies.Materials and methods. The study was performed on male Wistar rats. Experimental focal cerebral ischemia, traumatic brain injury, coronary occlusive myocardial infarction and muscle dysfunction were used as pathological models. Focal ischemia was reproduced by the method of irreversible thermocoagulation of the middle cerebral artery. Traumatic brain injury was modeled by the method of free fall of the load. Experimental myocardial infarction was reproduced by ligating the descending branch of the left coronary artery. Muscle dysfunction was modeled by the method of «forced swimming with a 20% burden». The respiratory function of mitochondria was assessed by the method of respirometry by the change in oxygen consumption when introducing mitochondrial respiration into the medium: Oligomycin, Rotenone and FCCP. Additionally, we evaluated the intensity of the glycolysis process and the activity of respiratory complexes I, II, IV and V. In order to comprehensively assess the respiratory function, an ELISA study was conducted to determine the concentration of ATP, mitochondrial ATP synthetase, cytochrome C oxidase and NADP-Oxidase 4.Results. In the course of the study it was established that under conditions of experimental cerebral ischemia, traumatic brain injury, myocardial infarction and muscle dysfunction, the ATP-generating ability of mitochondria the maximum breathing and respiratory capacity deteriorated, herby the decrease in overall respiratory function was accompanied by an increase in glycolysis, which was uncompensated, as well as dysfunction of mitochondrial complexes I, II, IV and V, confirmed by an increase in NADPH oxidase 4 activity and a decrease in cytochrome C oxidases and ATP synthetase. As a result, the observed changes in mitochondrial respiration function contributed to a decrease in ATP concentration under conditions of cerebral ischemia - by 3.2 times (p &lt;0.05), traumatic brain injury – by 2.6 times (p &lt;0.05), myocardial infarction – by 1.8 times (p &lt;0.05) and muscle dysfunction – by 4 times (p &lt;0.05).Conclusion. Basing on the data obtained, we can assume that in conditions of cerebral ischemia, traumatic brain injury, myocardial infarction and muscle dysfunction, there is deterioration of the mitochondrial respirometric function with inhibition of ATP synthesis and increased glycolysis.

Endothelotropic activity of 4-hydroxy-3,5-di-tret-butylcinnamic acid in the conditions of experimental cerebral ischemia

Introduction: The aim of the study was to evaluate the endothelioprotective activity of 4-hydroxy-3,5-di-tret-butylcinnamic acid in conditions of experimental cerebral ischemia.&#x0D; Materials and methods: The brain ischemia was reproduced by the method of irreversible right-sided thermocoagulation of the middle cerebral artery. As comparative drugs, mexidol (30 mg/kg) and sulodexide (30 U/kg) were used. The vasodilating function of the vascular endothelium was assessed by the change in the rate of cerebral blood flow when the synthesis of nitric oxide was modified. Antithrombotic function was assessed by changes in the concentration of thromboxane A2, fibrinogen, von Willebrand factor activity and platelet aggregation activity. Serum concentration of C-reactive protein served as a marker of the state of anti-inflammatory endothelial function. To determine the potential mechanism of endothelioprotective activity of 4-4-hydroxy-3,5-di-tret-butylcinnamic acid, the anti-radical activity of this compound toward superoxide and nitrosy-radicals was assessed; and the effect of the compound on the mitochondrial function was studied, by evaluating the functional activity of mitochondrial ATP synthetase and cytochrome-c-oxidase by ELISA.&#x0D; Results and discussion: In the course of the study, a positive effect of 4-hydroxy-3,5-di-tret-butylcinnamic acid on the state of endothelial function in cerebral ischemia was established, which was expressed in the preservation of vasodilating (restoring the vascular reaction to acetylcholine, nitro-L-arginine methyl ether, L-arginine), antithrombotic (a decrease in the concentration of thromboxane A2, fibrinogen and von Willebrand factor activity by 241.9% (p &lt;0.05), 73.5% (p &lt;0.05), 20.4% (p &lt;0.05), respectively, a decrease in the degree of aggregation and platelet aggregation rate by 56.7 % (p &lt;0.05) and 52.8% (p &lt;0.05), respectively, and anti-inflammatory vascular endothelial function (99.1% C-reactive protein reduction (p &lt;0.05)). The 4-hydroxy-3,5-di-tret-butylcinnamic acid compound in vitro tests suppressed generation of superoxide (IC50 = 1.99 mg/ml) and nitrosyl radical (IC50 = 1.92 mg/ml), eliminated NO-synthase uncoupling, and restored the mitochondrial function (increase in mitochondrial ATP synthase and cytochrome-c-oxidase activity by 23.5% (p &lt;0.05) and 110.8% (p &lt;0.05), respectively).&#x0D; Conclusion: The study demonstrated the presence of endotheliotropic activity of 4-hydroxy-3,5-di-tret-butylcinnamic acid, which is expressed in the preservation of vasodilating, antithrombotic and anti-inflammatory functions of the vascular endothelium in conditions of cerebral ischemia. At the same time, the anti-radical properties of this compound, as well as the direct effect on the functional activity of the NO-synthase system and the improvement of the mitochondrial function, may underlie the endotheliotropic effects of 4-hydroxy-3,5-di-tret-butylcinnamic acid.

The Role of ATP-Sensitive Potassium Channels and Nitric Oxide in the Protective Effect of Preconditioning of the Brain

Objective. The role of ATP-dependent potassium (K+ ATP) channels in the neuroprotective effect of ischemic (IPre) and pharmacological (PPre) preconditioning and changes in blood levels of nitric oxide (NO) metabolites were studied in conditions of cerebral ischemia. Materials and methods. Ischemic stroke (IS) was modeled in male rats (n = 86) by electrocoagulation of a branch of the middle cerebral artery (MCA). The nonselective K+ ATP channel blocker glibenclamide and the K+ ATP channel activator diazoxide were used. IPre and PPre were performed one day before MCA occlusion. Blood concentrations of NO, nitrates (NO3 –) and nitrites (NO2 –) were determined in experimental animals at 5, 24, and 72 h after MCA occlusion. Results. IPre decreased the lesion zone by 37% (p < 0.05), while prior administration of glibenclamide countered the action of IPre. The protective effect of PPre was analogous to that of IPre. Decreases in blood levels of oxygenated R-conformers of hemoglobin-bound NO (Hb-NO) were seen 5 h after MCA occlusion, with an inversely proportional increase in the concentration of nonoxygenated T-conformers; there were also increases in NO3 – and NO2 – concentrations. NO3 – and NO2 – levels showed normalization by one day after MCA occlusion, along with changes in the concentrations of Hb-NO complexes – R-conformers dominated, while the blood level of T-conformers reached a minimum. Furthermore, by 24 h there was a correlation between blockade of K+ ATP channels and decreases in serum NO3 – and NO2 – levels (p < 0.03). Conclusions. The neuroprotective effect of preconditioning was due to activation of K+ ATP channels. Analysis of blood levels of NO metabolites in rats with IS showed that Hb-NO complexes in the R-conformation stored and carried NO to the tissues, releasing NO on occurrence of the R → T transition in ischemic conditions.

Transient middle cerebral artery occlusion in mice induces neuronal expression of RNF213, a susceptibility gene for moyamoya disease

Although recent genome-wide and locus-specific association studies revealed that the RING finger protein 213 (RNF213) gene is an important susceptibility gene for moyamoya disease (MMD), the exact mechanism by which the genetic alteration of RNF213 contributes to the development of MMD has not yet been elucidated. A quantitative reverse transcription polymerase chain reaction (PCR) analysis revealed that the constitutive expression of the RNF213 gene was very low in adult and embryonic brain tissue. However, information regarding the temporal and spatial expression patterns of the RNF213 gene under the condition of cerebral ischemia, which is one of characteristic pathologies associated with MMD, is currently limited. In order to address this critical issue, Rnf213 mRNA expression was investigated in mouse brains subjected to 60min of transient middle cerebral artery occlusion (tMCAO). Male C57BL6/j mice underwent tMCAO through the intraluminal blockade of MCA. Expression of the Rnf213 gene in the tMCAO brain was investigated with in situ RNA hybridization and a real-time PCR analysis from 1 to 72h after tMCAO. In situ RNA hybridization revealed a significant increase in Rnf213 mRNA levels in the cerebral cortex supplied by the affected MCA, especially at the penumbra area, as early as 6h after tMCAO, and these levels had increased further by 24h. Rnf213 gene expression remained unchanged in the non-ischemic hemisphere or control specimens. Double staining of Rnf213 mRNA with NeuN immunohistochemistry revealed Rnf213 hybridization signal expression mostly in neurons. The real-time PCR analysis confirmed induction of the Rnf213 gene after tMCAO. Therefore, the Rnf213 gene was up-regulated in the ischemic brain, especially at the penumbra area, 6h after tMCAO. Early increases in RNF213 gene expression in neurons after tMCAO indicate its involvement in cerebral ischemia, which is an underlying pathology of MMD. Further investigation is required to clarify its exact role in the pathophysiology of MMD.

Treatment of stroke with liposomal neuroprotective agents under cerebral ischemia conditions

Since the proportion of patients given thrombolytic therapy with tissue plasminogen activator (t-PA) is very limited because of the narrow therapeutic window, the development of new therapies for ischemic stroke has been desired. We previously reported that liposomes injected intravenously accumulate in the ischemic region of the brain via disruption of the blood–brain barrier that occurs under cerebral ischemia. In the present study, we investigated the efficacy of a liposomal neuroprotective agent in middle cerebral artery occlusion (MCAO) rats to develop ischemic stroke therapy prior to the recovery of cerebral blood flow. For this purpose, PEGylated liposomes encapsulating FK506 (FK506-liposomes) were prepared and injected intravenously into MCAO rats after a 1-h occlusion. This treatment significantly suppressed the expansion of oxidative stress and brain cell damage. In addition, administration of FK506-liposomes before reperfusion significantly ameliorated motor function deficits of the rats caused by ischemia/reperfusion injury. These findings suggest that FK506-liposomes effectively exerted a neuroprotective effect during ischemic conditions, and that combination therapy with a liposomal neuroprotectant plus t-PA could be a promising therapeutic strategy for ischemic stroke.

Stress-induced neuroprotective effects of epiregulin and amphiregulin.

Members of the epidermal growth factor family play important roles in the regulation of cell growth, proliferation, and survival. However, the specific roles of each epidermal growth factor family member with respect to brain injury are not well understood. Gene chip assay screens have revealed drastic increases in the expression of the epidermal growth factor family members amphiregulin and epiregulin following lipopolysaccharide stimulation, which activates an immune response. Both immune activity and endoplasmic reticulum stress are activated during cerebral ischemia. We found that the expression levels of amphiregulin and epiregulin were significantly increased under conditions of cerebral ischemia. Because endoplasmic reticulum stress increased the expression of amphiregulin and epiregulin in glial cells, endoplasmic reticulum stress may be a key mediatory factor of pathophysiological activity. Recombinant epiregulin and amphiregulin proteins effectively inhibited endoplasmic reticulum stress and the subsequent induction of neuronal cell death. Therefore, the upregulation of the epidermal growth factor family members epiregulin and amphiregulin may play a critical role in preventing endoplasmic reticulum stress-induced cell death, thus providing a potential therapy for brain injury.

Magnesium treatment for neuroprotection in ischemic diseases of the brain.

This article reviews experimental and clinical data on the use of magnesium as a neuroprotective agent in various conditions of cerebral ischemia. Whereas magnesium has shown neuroprotective properties in animal models of global and focal cerebral ischemia, this effect could not be reproduced in a large human stroke trial. These conflicting results may be explained by the timing of treatment. While treatment can be started before or early after ischemia in experimental studies, there is an inevitable delay of treatment in human stroke. Magnesium administration to women at risk for preterm birth has been investigated in several randomized controlled trials and was found to reduce the risk of neurological deficits for the premature infant. Postnatal administration of magnesium to babies after perinatal asphyxia has been studied in a number of controlled clinical trials. The results are promising but the trials have, so far, been underpowered. In aneurysmal subarachnoid hemorrhage (SAH), cerebral ischemia arises with the onset of delayed cerebral vasospasm several days after aneurysm rupture. Similar to perinatal asphyxia in impending preterm delivery, treatment can be started prior to ischemia. The results of clinical trials are conflicting. Several clinical trials did not show an additive effect of magnesium with nimodipine, another calcium antagonist which is routinely administered to SAH patients in many centers. Other trials found a protective effect after magnesium therapy. Thus, it may still be a promising substance in the treatment of secondary cerebral ischemia after aneurysmal SAH. Future prospects of magnesium therapy are discussed.