Ghrelin reduces cerebral ischemic injury in rats by reducing M1 microglia/macrophages

The purpose of this study was to investigate the effect of Ghrelin on the polarization of microglia/ macrophages after cerebral ischemia (CI) in rats. 60 wild-type SD rats were randomly divided into sham group, CI group, CI+Ghrelin group, 20 rats in each group. The modified Longa suture method was used to establish the middle cerebral artery occlusion (MCAO) model in rats. Before surgery, Ghrelin was injected subcutaneously (100μg/kg, twice a day) for 4 consecutive weeks. After modeling, neurological function scores were performed with three behavioral experiments: mNSS score, Corner test, and Rotarod test, to evaluate the recovery of neurological function after Ghrelin treatment. At the same time, the brain tissues were collected and stained with 2,3,5-triphenyltetrazolium chloride (TTC) to detect the cerebral infarct volume. RT-qPCR was used to detect the expression of TNF-α and IL-1β in the ischemic brain tissue, and the TUNEL staining was used to detect the apoptosis of brain tissue. Flow cytometry was used to detect the percentage of M1 type microglia/macrophages which were isolated by trypsin digestion of fresh cerebral cortex. Then, the Western blotting and immunofluorescence method were used to detect the phosphorylation level of AKT (P-AKT) and AKT. Compared with the CI group, the neurological function of the rats in the CI+Ghrelin group was dramatically improved, and the cerebral infarction area was dramatically reduced. At the same time, the expression of TNF-α and IL-1β in the ischemic brain tissue of rats in the CI+Ghrelin group decreased, and the apoptotic cells in the brain tissue also decreased. Compared with the CI treatment group, the activation of M1 microglia/macrophages in the cortex of the ischemic side of the infarct and the peri-infarct area in the CI+Ghrelin group was dramatically inhibited. At the same time, the ratio of P-AKT/AKT of the brain tissue in the CI+Ghrelin group was dramatically higher than that of the CI group. In the rat cerebral ischemia model, Ghrelin can promote the repair of brain damage and the recovery of neurological function after ischemia. Its mechanism may be related to activating AKT to selectively reduce M1 microglia/macrophages, reducing inflammation and cell apoptosis in brain tissue.

Influence of Low Concentrations of Carbon Monoxide on Metabolism of Isolated Heart under Conditions of Ischemia-Reperfusion

The purpose of the study was to determine the effect of different concentrations of carbon monoxide on the metabolism of isolated mice hearts. Materials and methods. To elucidate the effect of low concentrations of carbon monoxide on the myocardium, we performed retrograde perfusion of isolated hearts of laboratory mice with Krebs-Henseleit solution, which was saturated with carbon monoxide for 5, 10, and 30 minutes. We then determined how different concentrations of carbon monoxide affected coronary volumetric flow rate, myocardial glucose and calcium uptake, creatinine release, and aspartate aminotransferase release. During perfusion, R-wave amplitude and R-R interval were measured using an electrocardiograph. To determine the effect of ischemia on the heart muscle during perfusion with solutions of different concentrations, we measured the area of the affected myocardium after staining with 2,3,5-triphenyltetrazolium chloride. Results and discussion. After these studies, it was found that different concentrations of carbon monoxide had a dose-dependent effect on the isolated mouse heart. However, the dependence of the effects does not follow the pattern «lowest concentration – lowest effect». At the same time, an increase in concentration did not mean an increase in adverse effects on the myocardium. Even on the contrary, the smallest concentration led to increased signs of ischemic myocardial damage. In particular, the use of the solution, through which carbon monoxide was passed for 5 minutes, caused vasoconstrictor effect during perfusion. At the end of reperfusion, vasoconstrictor effect was observed after using a solution through which carbon monoxide was passed for 10 minutes. Increased glucose uptake was observed in the group with 30-minute carbon monoxide permeation against the background of the minimal myocardial creatinine release. In this group there was also a decrease in Ca2+ loss at the beginning of reperfusion (immediately after ischemia). The above phenomenon explains the least degree of ischemic myocardial damage in the isolated mouse heart. The obtained data should be expanded. Since it is difficult to accurately determine the dose of carbon monoxide, then the use of donor compounds is promising. Such compounds include CORM-2 and CORM-3. Under physiological conditions, they decompose in a controlled manner, releasing a specific amount of carbon monoxide. Conclusion. The obtained results indicate that at different concentrations of carbon monoxide can differently influence different structures of cardiomyocyte: at one concentration it binds to calcium channels, other concentrations influence ion channels of plasma membrane, which can explain all these dependencies

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.

Methodology of asystolic donor heart’s condition study in an experiment using small laboratory animals

In transplantation, there has always been an acute problem of the discrepancy between the number of donor organs and the number of recipients, including donor hearts. There are various ways to increase the pool of donor organs, one of them is the use of asystolic or non-heart-beating donors. Due to poor myocardial tolerance of ischemia during the asystole period, as well as because of the difficulties in diagnosing cardiac diseases of the asystolic donor, which can be contraindication to transplantation. Therefore, an in-depth study of the state of the myocardium in asystolic donors is required. Currently, there is no generally accepted protocol for working with asystolic heart donors. This protocol should include methods of heart conditioning and assessing of myocardium state. For its development we need more experimental and preclinical studies. A protocol for such a study is proposed. The modeling of an asystolic donor using rats is described on the basis of experimental work carried out by a team of authors. The article describes the following technical aspects: anesthetic guidance, asystole detection criterion, maintaining the rat body temperature in accordance with the human body temperature during cardiac arrest, surgical aspects of performing the main experimental model. The Langendorff model of isolated cardiac perfusion was chosen as the main model for assessing the state of the myocardium of a small laboratory animal. Intra-left ventricular pressure, volume of coronary blood flow, heart rate and the presence of post-reperfusion arrhythmias were selected as criteria for assessing the state of donor hearts. Assessment of the volume of damage to the donor heart is carried out using triphenyltetrazolium chloride staining of the donor organ.

Seed viability of common ragweed (Ambrosia artemisiifolia L.) is affected by seed origin and age, but also by testing method and laboratory

Common ragweed (Ambrosia artemisiifolia L.) is an annual Asteraceae species native to North America which is highly invasive across Europe and has harmful impacts, especially on human health and agriculture. Besides its wide ecological range, particularly its high reproductive power by seeds is promoting its spread to various habitats and regions. To prevent further spread and to control the plant, the European Commission funded projects and COST-Actions involving scientists from all over Europe. A joint trial was set up comprising eight different laboratories from Europe to study seed viability variation in different seed samples. Three different testing methods (viability test with 2,3,5-triphenyltetrazolium chloride (TTC), a germination test combined with a subsequent TTC test and a crush test) were tested within the EU-COST-Action SMARTER network to four different seed origins. The viability test results from different laboratories were compared for variation amongst tests and laboratories. The main aim was to optimise the reliability of testing procedures, but results revealed not only significant effects of seed origin and seed age on seed viability, but also considerable differences between the output of the individual testing methods and furthermore between laboratories. Due to these significant differences in the results of the testing labs, additionally a second test was set up. Twelve Austrian ragweed populations were used for TTC testing to obtain a precise adjustment of the testing method as well as a tight guideline for interpreting the results, particularly for the TTC state “intermediate” since a proper classification of TTC-intermediate coloured seeds is still a challenge when determining viability rates.

Inhibiting YAP in Endothelial Cells From Entering the Nucleus Attenuates Blood-Brain Barrier Damage During Ischemia-Reperfusion Injury

Blood-brain barrier (BBB) damage is a critical event in ischemic stroke, contributing to aggravated brain damage. Endothelial cell form a major component of the BBB, but its regulation in stroke has yet to be clarified. We investigated the function of Yes-associated protein 1 (YAP) in the endothelium on BBB breakdown during cerebral ischemia/reperfusion (I/R) injury. The effects of YAP on BBB dysfunction were explored in middle cerebral artery occlusion/reperfusion (MCAO/R)-injury model mice and using brain microvascular endothelial cells (BMEC) exposed to oxygen-glucose deprivation/reoxygenation (OGD/R) injury. The degree of brain injury was estimated using staining (2,3,5-Triphenyltetrazolium chloride, hematoxylin and eosin) and the detection of cerebral blood flow. BBB breakdown was investigated by examining the leakage of Evans Blue dye and evaluating the expression of tight junction (TJ)-associated proteins and matrix metallopeptidase (MMP) 2 and 9. YAP expression was up-regulated in the nucleus of BMEC after cerebral I/R injury. Verteporfin (YAP inhibitor) down-regulated YAP expression in the nucleus and improved BBB hyperpermeability and TJ integrity disruption stimulated by cerebral I/R. YAP-targeted small interfering RNA (siRNA) exerted the same effects in BMEC cells exposed to OGD/R injury. Our findings provide new insights into the contributions made by YAP to the maintenance of BBB integrity and highlight the potential for YAP to serve as a therapeutic target to modulate BBB integrity following ischemic stroke and related cerebrovascular diseases.

Shenxiong Drop Pill exerts neuroprotective effect against focal cerebral ischemia partly via regulation of the expressions of ICAM-1 and caspase-3

Purpose: To investigate the effect of Shenxiong Drop Pill (SXDP) on cerebral infarction (CI) in rats, and the involvement of anti-inflammatory response in the process.Methods: Rats were sacrificed at three different time points, viz, 24, 48 and 72 h after establishment of CI model. Neurological deficit score (NDS) was determined using Bederson’s neurological behavioral scoring method, whereas triphenyltetrazolium chloride (TTC) staining was used to show brain injury. The integrated optical density (IOD) of Nissl bodies and caspase-3-positive nerve cells were measured with Nissl staining and SP kit, respectively. The mRNA expression of intercellular adhesion molecule 1(ICAM-1) was determined using reverse transcription-polymerase chain reaction (RT-PCR).Results: SXDP produced neuroprotective effect at high, medium, and low doses. The infarct volumes in the high-, medium- and low-dose SXDP, and cyclophosphamide groups were significantly reduced at each time point. Different doses of SXDP significantly reduced the mRNA expression of ICAM-1 and the IOD of caspase-3.Conclusion: These results indicate that SXDP exerts neuroprotective effects against ischemic injury by negatively regulating ICAM-1/caspase-3 downstream of inflammatory and apoptosis pathways.

The Neuroprotective Effect of l-Borneolum on Cerebral Ischemic Stroke by Regulating Dll4/Notch1 Pathway

Aiml-Borneolum is a monoterpene compound witch deserved from Blumea balsamifera (L.) DC, this study aimed to investigate the potential mechanism of l-borneolum on cerebral ischemic stroke (CIS) rats and provide evidence for the development of l-borneolum in CIS.MethodsPermanent middle cerebral artery occlusion (pMCAO) model rats were applied to this study. Neurological function was assessed by modified neurological severity scores (mNSS) and Longa neurological function scoring methods. The pathological changes of cerebral tissue were evaluated by 2,3,5-triphenyltetrazolium chloride (TTC) and hematoxylin-eosin (HE) staining. Ultrastructure of blood brain barrier (BBB) was observed by transmission electron microscopy. Additionally, the expression of Notch1, Dll4, Hey1, Hes1, Hes5, VEGFA and p65 in the cortex were determined by Western blotting (WB) while expression of caspase 3 were determined by immunohistochemical method (IHC). Resultsl-Borneolum improved neurological function in a dose-dependently. l-Borneolum significantly alleviated brainstem edema and inflammation, as well as improved the ultrastructure of capillary and BBB in cortex. Moreover, 0.2 g/kg l-borneolum substantially decreased the protein expressions of Dll4, Notch1, Hes1, Hes5, and VEGFA in the cortex while decreased the level of Caspase-3 in the cortex of rats. Conclusionsl-Borneolum could repair neurological function by regulating Dll4/Notch1 signaling pathway.

Invitro Toxicity of Binary Mixtures of Glyphosate and 2, 2 Dichlorovinyl Dimethyl Phosphate on Bacterial Isolates

The in vitro toxicity of glyphosate (Gly) and 2, 2 Dichlorovinyl dimethyl phosphate (DDVP) single compound and binary mixtures was assessed against Pseudomonas sp. and Bacillus sp. isolated from Otamiri River, Imo state, Nigeria was investigated. The toxicity response was assessed using the inhibitory effect of the single and binary mixtures on isolates dehydrogenase activity; and 2,3,5 triphenyltetrazolium chloride (TTC) was used as the artificial electron acceptor. The binary mixtures were composed using fixed ratios of glyphosate and 2, 2 Dichlorovinyl dimethyl phosphate in ratios of 20% Gly:80% DDVP, 40% Gly: 60% DDVP, 50% Gly: 50% DDVP, 60% Gly: 40% DDVP and 80% Gly: 20% DDVP. Results obtained showed that the isolates exhibited different degrees of logistic and sigmoidal toxicity trends with areas of hormesis at low concentrations of the toxicants. Furthermore, isobolographic analysis on the toxic interaction of the mixtures presented both synergism and antagonism, based on the relative ratio of the component mixtures. Increasing concentration of glyphosate in the binary mixture caused a shift in the interaction effect from antagonism to synergism. Our findings showed that isolates exhibited tolerance to glyphosate and 2,2 dichlorovinyl dimethyl phosphate and their binary mixtures exposure at concentration range of 0-1000mg/L; above which has deleterious effects on the aquatic organisms. It is evident that there are considerable differences in pesticide sensitivity among the bacterial species and that the presence of glyphosate and 2, 2-dichlorovinyl dimethyl phosphate in the aquatic environment may present toxicological risk to microbial diversity.

Evaluation of electroporated area using 2,3,5-triphenyltetrazolium chloride in a potato model

Irreversible electroporation (IRE) is a tissue ablation method, uses short high electric pulses and results in cell death in target tissue by irreversibly permeabilizing the cell membrane. Potato is commonly used as a tissue model for electroporation experiments. The blackened area that forms 12 h after electric pulsing is regarded as an IRE-ablated area caused by melanin accumulation. Here, the 2,3,5-triphenyltetrazolium chloride (TTC) was used as a dye to assess the IRE-ablated area 3 h after potato model ablation. Comparison between the blackened area and TTC-unstained white area in various voltage conditions showed that TTC staining well delineated the IRE-ablated area. Moreover, whether the ablated area was consistent over time and at different staining times was investigated. In addition, the presumed reversible electroporation (RE) area was formed surrounding the IRE-ablated area. Overall, TTC staining can provide a more rapid and accurate electroporated area evaluation.