The effect of erythropoietin on endometrial edema during ischemia–reperfusion injury in rats

This experimental study examined the effect of erythropoietin (Epo) in a rat model and particularly in an ischemia–reperfusion protocol. The potential beneficial effect of Epo was studied pathologically using endometrial edema (EE) lesions. Endometrial edema was evaluated 60 min after reperfusion (Groups A and C) and 120 min after reperfusion (Groups B and D) in rats. Epo was administered only in Groups C and D. Epo administration non-significantly increased the EE scores by 0.05 (p = 0.9315). Reperfusion non-significantly increased the EE scores by 0.15 (p = 0.6508). Epo administration and reperfusion together non-significantly increased the EE scores by 0.027 (p = 0.8898). Epo administration, reperfusion, and their interaction reduced the EE scores from significant to non-significant levels. Perhaps a study time longer than 2 h or a higher Epo dose could result in complete resolution of the endometrial edema formed as a result of the ischemia–reperfusion injury in this rat model.     

Hyperbaric oxygen preconditioning reduces ischemia–reperfusion injury by inhibition of apoptosis via mitochondrial pathway in rat brain

This study examined the hypothesis that apoptotic inhibition via mitochondrial pathway was involved in hyperbaric oxygen preconditioning (HBO-PC)–induced neuroprotection on ischemia–reperfusion injury in rat brain. Male Sprague–Dawley rats (250∼280 g, n=144) were divided into control, middle cerebral artery occlusion (MCAO) for 90 min, and HBO-PC plus MCAO groups. HBO-PC was conducted four times by giving 100% oxygen at 2.5 atm absolute (ATA), for 1 h at 12 h intervals for 2 days. At 24 h after the last HBO-PC, MCAO was performed and at 24 h after MCAO, neurological function, brain water content, infarct volume, and cell death were evaluated. Enzymatic activity of capase-3 and −9, and expression of cytochrome c, Bcl-2 and Bax proteins were performed in the samples from hippocampus, ischemic penumbra and core of the brain cortex, respectively. HBO-PC reduced brain edema, decreased infarction volume, and improved neurological recovery. HBO-PC reduced cytoplasm cytochrome c levels, decreased caspase enzyme activity, upregulated the ratio of Bcl-2 and Bax expression, and abated the apoptosis of ischemic tissue. HBO-PC protects brain tissues from ischemia–reperfusion injury by suppressing mitochondrial apoptotic pathways.     

Role of mitochondrial ATP-sensitive potassium channel-mediated PKC-ε in delayed protection against myocardial ischemia/reperfusion injury in isolated hearts of sevoflurane-preconditioned rats

This study aimed to determine the role of mitochondrial adenosine triphosphate-sensitive potassium (mitoK_ATP) channels and protein kinase C (PKC)-ε in the delayed protective effects of sevoflurane preconditioning using Langendorff isolated heart perfusion models. Fifty-four isolated perfused rat hearts were randomly divided into 6 groups (n=9). The rats were exposed for 60 min to 2.5% sevoflurane (the second window of protection group, SWOP group) or 33% oxygen inhalation (I/R group) 24 h before coronary occlusion. The control group (CON) and the sevoflurane group (SEVO) group were exposed to 33% oxygen and 2.5% sevoflurane for 60 min, respectively, without coronary occlusion. The mitoK_ATP channel inhibitor 5-hydroxydecanoate (5-HD) was given 30 min before sevoflurane preconditioning (5-HD+SWOP group). Cardiac function indices, infarct sizes, serum cardiac troponin I (cTnI) concentrations, and the expression levels of phosphorylated PKC-ε (p-PKC-ε) and caspase-8 were measured. Cardiac function was unchanged, p-PKC-ε expression was upregulated, caspase-8 expression was downregulated, cTnI concentrations were decreased, and the infarcts were significantly smaller (P<0.05) in the SWOP group compared with the I/R group. Cardiac function was worse, p-PKC-ε expression was downregulated, caspase-8 expression was upregulated, cTnI concentration was increased and infarcts were larger in the 5-HD+SWOP group (P<0.05) compared with the SWOP group. The results suggest that mitoK_ATP channels are involved in the myocardial protective effects of sevoflurane in preconditioning against I/R injury, by regulating PKC-ε phosphorylation before ischemia, and by downregulating caspase-8 during reperfusion.     

The opposite roles of nNOS in cardiac ischemia–reperfusion-induced injury and in ischemia preconditioning-induced cardioprotection in mice

The role of neuronal nitric oxide synthase (nNOS) in cardiac ischemia–reperfusion (IR) and ischemia preconditioning (IP) is still controversial. Here, we focused on the possible roles of nNOS in cardiac IR and IP. Wild type C57BL/6 (WT) mice were subjected to coronary artery occlusion for 30 min followed by 24-h reperfusion (IR). Cardiac injury (infarct size and apoptotic cell number) was increased, associated with elevation of oxidative stress (lipid peroxidation) and nitrative stress (nitrotyrosine formation). A potent nNOS inhibitor, L-VNIO, and a superoxide dismutase mimetic and peroxynitrite scavenger, MnTBAP, significantly reduced IR-induced increases of oxidative/nitrative stress and cardiac injury. IR-induced cardiac injury in nNOS^−/− (KO) mice was significantly lower than that in WT mice. MnTBAP markedly reduced IR-induced cardiac injury by suppression of oxidative/nitrative stress in KO mice. Cardiac IP was performed by three cycles of 5-min IR before 30-min ischemia followed by 24-h reperfusion. IP attenuated IR-induced cardiac injury in WT mice associated with reductions of oxidative/nitrative stress. IP-induced reduction of cardiac injury and oxidative/nitrative stress were eliminated by pretreatment with L-VNIO. In contrast with WT mice, IP had no protective effects in nNOS KO mice. In conclusion, nNOS played a dual role during cardiac IR and IP; nNOS exacerbated IR-induced injury by increasing oxidative/nitrative stress and contributed to IP-induced protection by inhibition of oxidative/nitrative stress.     

Seizures are associated with brain injury severity in a neonatal model of hypoxia–ischemia

Hypoxia–ischemia is a significant cause of brain damage in the human newborn and can result in long-term neurodevelopmental disability. The loss of oxygen and glucose supply to the developing brain leads to excitotoxic neuronal cell damage and death; such over-excitation of nerve cells can also manifest as seizures. The newborn brain is highly susceptible to seizures although it is unclear what role they have in hypoxic-ischemic (H/I) injury. The aim of this study was to determine an association between seizures and severity of brain injury in a piglet model of perinatal H/I and, whether injury severity was related to type of seizure, i.e. sub-clinical (electrographic seizures only) or clinical (electrographic seizures+physical signs). Hypoxia (4% O₂) was induced in anaesthetised newborn piglets for 30 min with a final 10 min period of hypotension; animals were recovered and survived to 72 h. Animals were monitored daily for seizures both visually and with electroencephalogram (EEG) recordings. Brain injury was assessed with magnetic resonance imaging (MRI), ¹H-MR spectroscopy (¹H-MRS), EEG and by histology (haematoxylin and eosin). EEG seizures were observed in 75% of all H/I animals, 46% displayed clinical seizures and 29% sub-clinical seizures. Seizure animals showed significantly lower background amplitude EEG across all post-insult days. Presence of seizures was associated with lower cortical apparent diffusion coefficient (ADC) scores and changes in ¹H-MRS metabolite ratios at both 24 and 72 h post-insult. On post-mortem examination animals with seizures showed the greatest degree of neuropathological injury compared to animals without seizures. Furthermore, clinical seizure animals had significantly greater histological injury compared with sub-clinical seizure animals; this difference was not apparent on MRI or ¹H-MRS measures. In conclusion we report that both sub-clinical and clinical seizures are associated with increased severity of H/I injury in a term model of neonatal H/I.     

The interaction between ischemia–reperfusion and immune responses in the kidney

Kidney ischemia–reperfusion injury (IRI) engages both the innate and adaptive immune responses. Cellular mediators of immunity, such as dendritic cells, neutrophils, macrophages, natural killer T, T, and B cells, contribute to the pathogenesis of renal injury after IRI. Postischemic kidneys express increased levels of adhesion molecules on endothelial cells and toll-like receptors on tubular epithelial cells. Soluble components of the immune system, such as complement activation proteins and cytokines, also participate in injury/repair of postischemic kidneys. Experimental studies on the immune response in kidney IRI have resulted in better understanding of the mechanisms underlying IRI and led to the discovery of novel therapeutic and diagnostic targets.     

The rise of [Na+]i during ischemia and reperfusion in the rat heart—underlying mechanisms

Intracellular Na⁺ concentration ([Na⁺]_i) rises in the heart during ischemia, and on reperfusion, there is a transient rise followed by a return toward control. These changes in [Na⁺]_i contribute to ischemic and reperfusion damage through their effects on Ca²⁺ overload. Part of the rise of [Na⁺]_i during ischemia may be caused by increased activity of the cardiac Na⁺/H⁺ exchanger (NHE1), activated by the ischemic rise in [H⁺]_i. In support of this view, NHE1 inhibitors reduce the [Na⁺]_i rise during ischemia. Another possibility is that the rise of [Na⁺]_i during ischemia is caused by Na⁺ influx through channels. We have reexamined these issues by use of two different NHE1 inhibitors, amiloride, and zoniporide, in addition to tetrodotoxin (TTX), which blocks voltage-sensitive Na⁺ channels. All three drugs produced cardioprotection after ischemia, but amiloride (100 μM) and TTX (300 nM) prevented the rise in [Na⁺]_i during ischemia, whereas zoniporide (100 nM) did not. Both amiloride and zoniporide prevented the rise of [Na⁺]_i on reperfusion, whereas TTX was without effect. In an attempt to explain these differences, we measured the ability of the three drugs to block Na⁺ currents. At the concentrations used, TTX reduced the transient Na⁺ current (I _Na) by 11 ± 2% while amiloride and zoniporide were without effect. In contrast, TTX largely eliminated the persistent Na⁺ current (I _Na,P) and amiloride was equally effective, whereas zoniporide had a substantially smaller effect reducing I _Na,P to 41 ± 8%. These results suggest that part of the effect of NHE1 inhibitors on the [Na⁺]_i during ischemia is by blockade of I _Na,P. The fact that a low concentration of TTX eliminated the rise of [Na⁺]_i during ischemia suggests that I _Na,P is a major source of Na⁺ influx in this model of ischemia.     

Role of PKCβII and PKCδ in blood–brain barrier permeability during aglycemic hypoxia

Blood–brain barrier (BBB) dysfunction contributes to the pathophysiology of cerebrovascular diseases such as stroke. In the present study, we investigated the role of PKC isoforms in aglycemic hypoxia-induced hyperpermeability using an in vitro model of the BBB consisting of mouse bEnd.3 cells. PKCβII and PKCδ isoforms were activated during aglycemic hypoxia. CGP53353, a specific PKCβII inhibitor, significantly attenuated aglycemic hypoxia-induced BBB hyperpermeability and disruption of occludin and zonula occludens-1 (ZO-1), indicating a deleterious role of PKCβII in the regulation of BBB permeability during aglycemic hypoxia. Conversely, rottlerin, a specific PKCδ inhibitor, exacerbated BBB hyperpermeability and tight junction (TJ) disruption during aglycemic hypoxia, indicating a protective role of PKCδ against aglycemic hypoxia-induced BBB hyperpermeability. Furthermore, disruption of TJ proteins during aglycemic hypoxia was attenuated by PKCβII DN and PKCδ WT overexpression, and aggravated by PKCβII WT and PKCδ DN overexpression. These results suggest that PKCβII and PKCδ counter-regulate BBB permeability during aglycemic hypoxia.     

Comparison of cardioprotective and anti-inflammatory effects of ischemia pre- and postconditioning in rats with myocardial ischemia–reperfusion injury

Objective  

To compare cardioprotective and anti-inflammatory effects of ischemia preconditioning (IPC) and ischemia postconditioning (IPOC) in a rat myocardial ischemia–reperfusion injury (IRI) model.     

Expression of ether à go-go potassium channels in human gliomas

Ether à go-go (EAG) K(+) channels have been shown to be involved in tumor generation and malignant growth. Gliomas have not been investigated thus far. Using RT-PCR we investigated healthy human brain and human gliomas of different subtypes and malignancy grades for the expression of human EAG1 and eag-related gene (ERG) 1 channels. mRNA of both channels was detected in all tissues. Expression was strong in normal brain, moderate in high-grade and high in low-grade gliomas. Our findings suggest a differential expression of hEAG1 and hERG1 in gliomas depending on the malignancy grade and nature of the tumor cells. However, the hypothesis that EAG channels are related to the oncogenic process itself is only partly supported by this study.     

ATP-sensitive potassium channels modulate in vitro tocolytic effects of β2-AR agonists on uterine muscle rings in rats in early but not in late pregnancy

Aim

To investigate whether ATP-sensitive potassium (K_ATP) channels modulate the tocolytic effect of β₂-AR agonists (ritodrine and salmeterol) in early-pregnant (day 6) and late-pregnant (day 22) rat uterus in vitro, in order to examine the relation between the K_ATP channel sulphonylurea-binding regulatory subunit (SUR) expression and pharmacological reactivity of β₂-AR agonists.

Methods

The tocolytic effects of ritodrine and salmeterol (10^-10-10^-5 M) on spontaneous rhythmic contractions were investigated cumulatively, alone, or in the presence of the K_ATP channel blocker glibenclamide (10^-6 M) and the K_ATP channel opener pinacidil (10^-9-10^-7 M) after 5-min preincubation.

Results

β₂-AR agonist induced myometrial relaxation was inhibited by glibenclamide and enhanced by pinacidil on day 6, when SUR1 expression levels were high. Neither glibenclamide nor pinacidil mediated tocolytic effect was measured on day 22.

Conclusion

Low expression of the K_ATP channels at the end of gestation may facilitate enhanced excitability and contractility in the rat myometrium. The combination of a betamimetic and a K_ATP channel opener will therefore not be of therapeutic relevance in the treatment of preterm delivery.A number of agents have tocolytic effect, including β₂-adrenergic receptor (β₂-AR) agonists, magnesium sulfate, prostaglandin synthesis inhibitors, Ca²⁺-channel blockers, nitrogen monoxide donors, and oxytocin receptor antagonists (1). β₂-AR agonists (such as salmeterol, terbutaline, fenoterol, hexoprenaline, and ritodrine) delay preterm labor for at least 48 hours, which is why they are the drugs of choice in the treatment of preterm labor (2). Of all tocolytics in use, β-mimetics have the most undesirable side-effect profile. The most serious reported side-effects associated with the administration of β₂-AR agonists are pulmonary edema, hypotension, and tachycardia (3,4). Promising new therapeutic approach for the treatment of preterm delivery is the combination β₂-AR agonists and 17alpha-hydroxyprogesterone (5) or Ca²⁺-channel blocker nifedipine (6).Adenosine triphosphate (ATP)-sensitive potassium channels (K_ATP channels) are involved in β-AR agonists-induced smooth muscle relaxation in pulmonary vasorelaxation in the rat (7), vasodilatation in the rat diaphragmatic microcirculation (8), vasorelaxation in the rat mesenteric artery (9), detrusor muscle relaxation in the rat (10), and myometrial relaxation in non-pregnant buffaloes (11). K_ATP channels are formed by a combination of two types of subunits, the pore-forming inwardly rectifying subunit (Kir_6x) and the sulphonylurea-binding regulatory subunit (SUR) (12). We earlier reported (13) that SUR subunits, SUR1 and SUR2, are both expressed in the rat uterus during gestation. SUR1 expression was elevated in the early pregnancy (day 6) and then dramatically decreased from day 8 to term, while the level of SUR2 subunit remained unchanged.The aim of the present study was to investigate the role of the K_ATP channel in β₂-AR agonist-induced myometrial relaxation. We studied the tocolytic effects of β₂-AR agonists (salmeterol, ritodrine) in the presence of glibenclamide (K_ATP channel blocker) and pinacidil (K_ATP channel opener) in early pregnant (day 6) and late pregnant (day 22) rats in vitro, in order to clarify the relation between SUR1 expression and pharmacological reactivity of β₂-AR agonists.     

Contribution of nitric oxide synthase (NOS) in blood–brain barrier disruption during acute focal cerebral ischemia in normal rat

Endogenous level of nitric oxide (NO) is increased in the brain following the stroke, and deactivation of NO synthase has been shown to attenuate its destructive actions in animal stroke models using middle cerebral artery occlusion (MCAO) procedures. However, little is known about the effects of NO in cerebral vascular integrity and edema during acute cerebral ischemia. Here we investigated whether NO plays any role in the progression of blood–brain barrier (BBB) disruption and edema formation in ischemia/reperfusion injury. Intraperitoneal administration of NO substrate l-arginine (300 mg/kg), or NOS inhibitor (l-NAME, 1 mg/kg), was done in normal rats at 20 min before a 60-min MCAO. Mean arterial blood pressures (MAP) and regional cerebral blood flow (rCBF) were continuously recorded during experiment. Neurological deficit score (NDS) was evaluated 12 h after termination of MCAO followed with evaluations of cerebral infarction volume (CIV), edema formation and cerebral vascular permeability (CVP), as determined by the Evans blue dye extravasations (EBE) technique. No significant changes were observed in the values of MAP and rCBF with l-arginine or l-NAME during ischemia or reperfusion periods. There was a 75–85% reduction in rCBF in during MCAO which returned back to its pre-occlusion level during reperfusion. Acute cerebral ischemia with or without l-arginine augmented NDS (4.00 ± 0.44 and 3.00 ± 0.30), in conjunction with increased CIV (518 ± 57 mm³ and 461 ± 65 mm³), provoked edema (3.09 ± 0.45% and 3.30 ± 0.49%), and elevated EBE (8.28 ± 2.04 μg/g and 5.09 ± 1.41 μg/g). Inhibition of NO production by l-NAME significantly improved NDS (1.50 ± 0.22), diminished CIV (248 ± 56 mm³), edema (1.18 ± 0.58%) and EBE (1.37 ± 0.12 μg/g). This study reconfirms the cerebroprotective properties of reduced tissue NO during acute ischemic stroke, and it also validates the deleterious actions of increased NOS activity on the disruption of cerebral microvascular integrity and edema formation of ischemia/reperfusion injuries in normal rat, without changing arterial blood pressure or blood flows to ischemic regions.     

The roles of CRISPR–Cas systems in adaptive immunity and beyond

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Modulation of ATP-sensitive potassium channel activity by flash-photolysis of ‘caged-ATP’ in rat heart cells

We have used caged-ATP to investigate the kinetic behavior of K_ATP channels in ventricular cells from rat heart. In whole cells, loaded with caged-ATP, an increase of intracellular [ATP] following a UV light flash produced a decrease of K_ATP channel current that was too slow ( 300 ms) to be explained by the expected timecourse of ATP release ( 3 ms) and the time-course of channel blockade by ATP ( 20 ms). In isolated membrane patches, caged-ATP itself caused partial blockade of K_ATP channels. Under these conditions, photorelease of ATP caused channel activity to decline further. The results suggest that caged-ATP can bind to the K_ATP channel but, on binding, decreases the open probability to a lesser extent than does ATP. Additionally, the observations indicate that for photolytically-generated ATP to bind to the channel, caged-ATP must first unbind (slowly) from the channel. We conclude that caged-ATP is not fully caged with respect to its allosteric action on the K_ATP channel.     

Emulsified isoflurane postconditioning produces cardioprotection against myocardial ischemia–reperfusion injury in rats

Emulsified isoflurane (EIso) preconditioning can induce cardioprotection. We investigated whether EIso application after ischemia protects hearts against reperfusion injury and whether it is mediated by the inhibition of apoptosis. Rats were subjected to 30-min coronary occlusion followed by 180-min reperfusion. At the onset of reperfusion, rats were intravenously administered saline (sham, control group), 30 % intralipid (IL group) or 2 ml kg^?1 EIso (EIso group) for 30 min. After reperfusion, infarct sizes, myocardial apoptosis and expression of Bcl-2, Bax and caspase-3 proteins were determined. Hemodynamic parameters were not different among groups. Compared with control and intralipid group, EIso limited infarct size, inhibited apoptosis, increased the expression of Bcl-2, decreased the expression of Bax, cleaved caspase-3, and enhanced Bcl-2/Bax ratio. EIso protects hearts against reperfusion injury when administered at the onset of reperfusion, which may be mediated by the inhibition of apoptosis via modulation of the expression of pro- and anti-apoptotic proteins.     

The dynamic roles of TGF-β in cancer

The transforming growth factor-β (TGF-β) signalling pathway plays a critical and dual role in the progression of human cancer. During the early phase of tumour progression, TGF-β acts as a tumour suppressor, exemplified by deletions or mutations in the core components of the TGF-β signalling pathway. On the contrary, TGF-β also promotes processes that support tumour progression such as tumour cell invasion, dissemination, and immune evasion. Consequently, the functional outcome of the TGF-β response is strongly context-dependent including cell, tissue, and cancer type. In this review, we describe the molecular signalling pathways employed by TGF-β in cancer and how these, when perturbed, may lead to the development of cancer. Concomitantly with our increased appreciation of the molecular mechanisms that govern TGF-β signalling, the potential to therapeutically target specific oncogenic sub-arms of the TGF-β pathway increases. Indeed, clinical trials with systemic TGF-β signalling inhibitors for treatment of cancer patients have been initiated. However, considering the important role of TGF-β in cardiovascular and many other tissues, careful screening of patients is warranted to minimize unwanted on-target side effects.