A Sphingosine-1-Phosphate Receptor Modulator Attenuated Secondary Brain Injury and Improved Neurological Functions of Mice after ICH.

BACKGROUND: Stroke activates the immune system and induces brain infiltration by immune cells, aggravating brain injury. Poststroke immunomodulation via (S1P-)receptor modulation is beneficial; however, the S1P-modulator in clinical use (FTY-720) is unspecific, and undesirable side effects have been reported. Previously, we tested effects of a novel selective S1P-receptor modulator, Siponimod, on ICH-induced brain injury in acute stage of the disease. In the current study, we investigated whether protective effects of Siponimod, evaluated in a short-term study, will protect the brain of ICH animals at long term as well. METHODS: 134 C57BL/6N mice were divided into sham and ICH-operated groups. Collagenase model of ICH was employed. ICH animals were divided into Siponimod treated and nontreated. Dose- and time-dependent effects of Siponimod were investigated. Contraplay between development of brain injury and the number of lymphocytes infiltrating the brain was investigated by forelimb placing, T-Maze test, brain water content calculation, MRI scanning, and immunostaining. RESULTS: Depending on the therapeutic strategy, Siponimod attenuated the development of brain edema, decreased ICH-induced ventriculomegaly and improved neurological functions of animals after ICH. It was associated with less lymphocytes in the brain of ICH animals. CONCLUSION: Siponimod is able to decrease the brain injury and improves neurological functions of animals after ICH.

Serelaxin activates eNOS, suppresses inflammation, attenuates developmental delay and improves cognitive functions of neonatal rats after germinal matrix hemorrhage.

Germinal matrix hemorrhage (GMH) is a detrimental form of neonatal CNS injury. Following GMH-mediated eNOS inhibition, inflammation arises, contributing to GMH-induced brain injury. We investigated the beneficial effects of Serelaxin, a clinical tested recombinant Relaxin-2 protein, on brain injury after GMH in rats. We investigated whether effects of Serelaxin are mediated by its ability to activate the GMH-suppressed eNOS pathway resulting in attenuation of inflammatory marker overproduction. GMH was induced by intraparenchymal injection of bacterial collagenase (0.3U). Seven day old Sprague-Dawley rat pups (P7) were used (n = 63). GMH animals were divided in vehicle or serelaxin treated (3 µg once, 30 µg once, 30 µg multiple, i.p., starting 30 after GMH and then daily). Sham operated animals were used. We monitored the developmental profile working memory and spatial function (T-maze and open field test respectively). At day 28, all rats underwent MRI-scans for assessment of changes in cortical thickness and white matter loss. Effects of Serelaxin on eNOS pathway activation and post-GMH inflammation were evaluated. We demonstrated that Serelaxin dose-dependently attenuated GMH-induced developmental delay, protected brain and improved cognitive functions of rats after GMH. That was associated with the decreased post-GMH inflammation, mediated at least partly by amelioration of GMH-induced eNOS inhibition.

Hydrogen gas reduced acute hyperglycemia-enhanced hemorrhagic transformation in a focal ischemia rat model.

Hyperglycemia is one of the major factors for hemorrhagic transformation after ischemic stroke. In this study, we tested the effect of hydrogen gas on hemorrhagic transformation in a rat focal cerebral ischemia model. Sprague-Dawley rats (n=72) were divided into the following groups: sham; sham treated with hydrogen gas (H(2)); Middle Cerebral Artery Occlusion (MCAO); and MCAO treated with H(2) (MCAO+H(2)). All rats received an injection of 50% dextrose (6 ml/kg i.p.) and underwent MCAO 15 min later. Following a 90 min ischemic period, hydrogen was inhaled for 2 h during reperfusion. We measured the level of blood glucose at 0 h, 0.5 h, 4 h, and 6 h after dextrose injection. Infarct and hemorrhagic volumes, neurologic score, oxidative stress (evaluated by measuring the level of 8 Hydroxyguanosine (8OHG), 4-Hydroxy-2-Nonenal (HNE) and nitrotyrosine), and matrix metalloproteinase (MMP)-2/MMP-9 activity were measured at 24 h after ischemia. We found that hydrogen inhalation for 2 h reduced infarct and hemorrhagic volumes and improved neurological functions. This effect of hydrogen was accompanied by a reduction of the expression of 8OHG, HNE, and nitrotyrosine and the activity of MMP-9. Furthermore, a reduction of the blood glucose level from 500+/-32.51 to 366+/-68.22 mg/dl at 4 h after dextrose injection was observed in hydrogen treated animals. However, the treatment had no significant effect on the expression of ZO-1, occludin, collagen IV or aquaporin4 (AQP4). In conclusion, hydrogen gas reduced brain infarction, hemorrhagic transformation, and improved neurological function in rats. The potential mechanisms of decreased oxidative stress and glucose levels after hydrogen treatment warrant further investigation.

Hyperbaric and normobaric reoxygenation of hypoxic rat brain slices--impact on purine nucleotides and cell viability.

Hyperbaric oxygen treatment has been suggested as able to reduce hypoxia induced neuronal damage. The aim of the study was to compare the impact of different reoxygenation strategies on early metabolical (purine nucleotide content determined by HPLC) and morphological changes (index of cell injury after celestine blue/acid fuchsin staining) of hypoxically damaged rat neocortical brain slices. For this purpose slices (300 microm and 900 microm) were subjected to either 5 or 30 min of hypoxia by gassing the incubation medium with nitrogen. During the following reoxygenation period treatment groups were administered either 100% oxygen (O) or room air (A) at normobaric (1 atm absolute, NB-O; NB-A) or hyperbaric (2.5 atm absolute, HB-O; HB-A) conditions. After 5 min of hypoxia, both HB-O and NB-O led to a complete nucleotide status restoration (ATP/ADP; GTP/GDP) in 300 microm slices. However, reoxygenation after 30 min of hypoxia was less effective, irrespective of the oxygen pressure. Furthermore, administering hyperbaric room air resulted in no significant posthypoxic nucleotide recovery. In 900 microm slices, both control incubation as well as 30 min of hypoxia resulted in significantly lower trinucleotide and higher dinucleotide levels compared to 300 microm slices. While there was no significant difference between HB-O and NB-O on the nucleotide status, morphological evaluation revealed a better recovery of the index of cell injury (profoundly injured/intact cell-ratio) in the HB-O group. Conclusively, the posthypoxic recovery of metabolical characteristics was dependent on the duration of hypoxia and slice thickness, but not on the reoxygenation pressure. A clear restorative effect on purine nucleotides was found only in early-administered HB-O as well as NB-O in contrast to room air treated slices. However, these pressure independent metabolic changes were morphologically accompanied by a significantly improved index of cell injury, indicating a possible neuroprotective role of HB-O in early posthypoxic reoxygenation.

Early biochemical and histological alterations in rat corticoencephalic cell cultures following metabolic damage and treatment with modulators of mitochondrial ATP-sensitive potassium channels.

The present study was aimed at characterizing alterations of the nucleotide content and morphological state of rat corticoencephalic cell cultures subjected to metabolic damage and treatment with modulators of mitochondrial ATP-dependent potassium channels (mitoK(ATP)). In a first series of experiments, in vitro ischemic changes of the contents of purine and pyrimidine nucleoside diphosphates and triphosphates were measured by high performance liquid chromatography (HPLC) and the corresponding histological alterations were determined by celestine blue/acid fuchsin staining. As an ischemic stimulus, incubation with a glucose-free medium saturated with argon was used. Ischemia decreased the levels of adenosine, guanine and uridine triphosphate (ATP, GTP, UTP) and increased the levels of the respective dinucleotides ADP and UDP, whereas the GDP content was not changed. Both 5-hydroxydecanoate (5-HD) and diazoxide failed to alter the contents of nucleoside diphosphates and triphosphates, when applied under normoxic conditions. 5-HD (30 microM) prevented the ischemia-induced changes of nucleotide and nucleoside levels. Diazoxide (300 microM), either alone or in combination with 5-hydroxydecanoate (30 microM) was ineffective. Pyruvate (5 mM) partially reversed the effects of ischemia or ischemia plus 2-deoxyglucose (20mM) in the incubation medium. Diazoxide (300 microM) and 5-HD (30 microM) had no effect in the presence of pyruvate (5mM) and 2-deoxyglucose (20mM). Staining the cells with celestine blue/acid fuchsin in order to classify them as intact, reversibly or profoundly injured, revealed a protective effect of 5-HD. When compared with 5-HD, diazoxide, pyruvate and 2-deoxyglucose had similar but less pronounced effects. In conclusion, these results suggest a protective role of 5-hydroxydecanoate on early corticoencephalic nucleotide and cell viability alterations during ischemia.

Early biochemical and histological changes during hyperbaric or normobaric reoxygenation after in vitro ischaemia in primary corticoencephalic cell cultures of rats.

In a first series of experiments, the morphological changes of corticoencephalic cells by ischaemia were determined by staining with celestine blue-acid fuchsin in order to classify cells as intact, dark basophilic (supposedly reversibly injured) and preacidophilic or acidophilic (profoundly injured). Hypoxia and glucose-deprivation (in vitro ischaemia) markedly decreased the number of intact cells and correspondingly increased the number of both reversibly and profoundly damaged cells. The morphological characteristics indicated a partial recovery during reoxygenation either in the absence or presence of glucose and irrespective of whether normobaric or hyperbaric oxygen was used. In a second series of experiments, nucleoside triphosphate and diphosphate levels were determined in corticoencephalic cultures by high-performance liquid chromatography. Hypoxia in combination with glucose-deficiency markedly decreased the ATP:ADP, GTP:GDP and UTP:UDP ratios. A still larger fall of these ratios was observed both after normobaric and hyperbaric reoxygenation. In contrast, both normobaric and hyperbaric reoxygenation in the presence of glucose led to an almost complete recovery near the control normoxic values. In conclusion, the histological changes were not adequately reflected by changes in the nucleoside triphosphate:diphosphate ratios and, in addition, hyperbaric oxygen had neither favourable nor unfavourable effects on the early morphological and functional restitution of ischaemically damaged cells under the conditions of the present study.

Alterations of purine and pyrimidine nucleotide contents in rat corticoencephalic cell cultures following metabolic damage and treatment with openers and blockers of ATP-sensitive potassium channels.

Rat corticoencephalic cell cultures were investigated by high performance liquid chromatography for changes in the levels of adenosine 5'-triphosphate (ATP), guanosine 5'-triphosphate (GTP), uridine 5'-triphosphate (UTP), cytidine 5'-triphosphate (CTP), and the respective nucleoside diphosphates. Hypoxia was induced by gassing the incubation medium for 30 min with 100% argon. Removal of glucose was caused by washing the cultures in glucose-free medium at the beginning of the 30 min incubation period. Whereas hypoxia or glucose-deficiency alone failed to alter the nucleotide levels, the combination of these two manipulations was clearly inhibitory. Diazoxide (300 microM) an opener of ATP-dependent potassium channels (K(ATP)) did not alter the nucleotide contents either in a normoxic and glucose-containing medium, or a hypoxic and glucose-free medium. By contrast, the K(ATP) channel antagonist tolbutamide (300 microM) aggravated the hypoxic decrease of nucleotide levels in a glucose-free medium, although it was ineffective in a normoxic and glucose-containing medium. Hypoxia and glucose-deficiency decreased the ATP/ADP and UTP/UDP ratios, but failed to change the GTP/GDP ratio. Diazoxide and tolbutamide (300 microM each) had no effect on the nucleoside triphosphate/diphosphate ratios either during normoxic or during hypoxic conditions. In conclusion, corticoencephalic cultures are rather resistant to in vitro ischemia. Although they clearly respond to the blockade of plasmalemmal K(ATP) channels (plasmaK(ATP)) by tolbutamide, these channels appear to be maximally open as a consequence of the fall in intracellular nucleotides and, therefore, diazoxide has no further effect.

Rapid assay for one-run determination of purine and pyrimidine nucleotide contents in neocortical slices and cell cultures.

The present study describes the measurement of endogenous nucleoside di- and triphosphate contents (ATP, GTP, UTP, CTP, ADP, GDP and UDP) in rat neocortical brain slices and mixed neuronal/astrocytic corticoencephalic cultures. Determination was by means of anion-exchange HPLC using a binary gradient of 0.3 M ammonium carbonate and water. In addition, a new method is described for the identification of nucleoside triphosphates, using digestion of the nucleotides by phosphoglycerate kinase and partial splitting of nucleoside diphosphates to shift the equilibrium of the phosphoglycerate kinase reaction in direction of breakdown of nucleoside triphosphates. Finally, the determination of the sum of creatine and creatine phosphate is suggested as an alternative reference value instead of protein under conditions when cells are cultured in protein-containing medium.

[Diastereomers of nonionic oligonucleotide analogs. VI. Isolation of diastereomers of ethyl phosphotriesters of the octanucleotide d(GCCAAACA) by high performance affinity chromatography]. / Issedovanie diastereomerov neionnykh analogov oligonukleotidov. VI. Razdelenie diastereomerov étilovykh fosfotriéfirov oktanukleotida d(GCCAAACA) metodom vysokoéffektivnoi affinnoi khromatografii.

Diastereomers of oligonucleotide ethyl phosphotriesters were separated by high-performance complementary (affinity) chromatography on a column with the immobilized complementary oligonucleotide. The elution buffer contained 0.18 M K2HPO4, pH 7.5, and 30% acetonitrile. The temperature of the separation was a few degrees lower than Tm of corresponding oligonucleotide complexes. The diastereomers separated completely or partially were: d[GCC(Et)AAACA], d[GCCA(Et)AACA], d[GCAA(Et)ACA], d[GCC(Et)A(Et)AACA], d[GCC(Et)AA(Et)ACA], d[GCCA(Et)A(Et)ACA], d[GCC(Et)A(Et)A(Et)ACA].