The Effects of Lipopolysaccharide Derivatives in Rodent Models of Cardiac Arrhythmia.

BACKGROUND: Several previous studies have suggested that sublethal doses of Escherichia coli lipopolysaccharide (endotoxin) and monophosphoryl lipid A Re595, a nonpyrogenic derivative of Salmonella minnesota lipopolysaccharide, exhibit antiarrhythmic effects in the rat model of ischemia-reperfusion arrhythmias. METHODS: In this study, the protective effect of lipopolysaccharide derivatives was also further investigated in drug (aconitine or ouabain)-induced arrhythmia models, and conclusions were drawn with particular emphasis on the molecular characteristics of different types of lipopolysaccharide. RESULTS: The importance of the molecular structure for the antiarrhythmic effect of monophosphoryl lipid A and E. coli lipopolysaccharide was tested in the ischemia-reperfusion arrhythmia model. In contrast to monophosphoryl lipid A from Salmonella typhimurium SL 684 which has only monophosphoryl residue in its structure, monophosphoryl lipid A Re595, obtained from S. minnesota, and E. coli lipopolysaccharide which have both mono and diphosphoryl residue reduced the duration of ventricular tachycardia (e.g., during reperfusion: vehicle: 176 ± 22.8; monophosphoryl lipid A Re595: 132.83 ± 12.1, as second, n=8-10, P < .05) and the incidence of ventricular fibrillation. The antiarrhythmic effects of E. coli lipopolysaccharide and monophosphoryl lipid A Re595 in ischemia-reperfusion arrhythmia model were absent in either aconitine- (e.g., onset time for ventricular ectopic beats: saline 25.3 5.0, E. coli lipopolysaccharide 24.3 ± 7.1; vehicle: 24.0 ± 4.5, monophosphoryl lipid A SL684 23.8 ± 4.3, as second, n=6, P > .05) or ouabain-induced arrhythmia models in mice. CONCLUSION: Therefore, we conclude that lipopolysaccharide derivatives exhibit antiarrhythmic effect only in ischemia-reperfusion arrhythmias, and lipopolysaccharide should possess diphosphoryl groups in its subcomponent composition for this antiarrhythmic effect.

Depressive-like behavior accompanies neuroinflammation in an animal model of bipolar disorder symptoms induced by ouabain.

INTRODUCTION: A previous study from our Laboratory showed no alteration in inflammatory parameters seven days after ouabain (OUA) administration, a Na+K+ATPase inhibitor, which was previously considered only a mania model. However, the administration of OUA in rats was recently validated as a model of bipolar disorder (BD) symptoms, demonstrating that 14 days after single intracerebroventricular (ICV) administration, OUA also induces depressive-like behavior. Therefore, it is important to investigate the long-term effect of OUA on inflammatory parameters since this mechanism seems to play a key role in BD physiopathology. METHODS: Adult male Wistar rats received a single ICV administration of OUA or artificial cerebrospinal fluid (aCSF). From the fourth day after the ICV infusion, the rats received saline or Lithium (Li) for 14 days. The open-field test was performed on the 7th day after OUA. On the 14th day, locomotion was re-evaluated, and the forced swimming test (FST) was used to evaluate depressive-like behavior. Inflammatory parameters were assessed in the frontal cortex and hippocampus. RESULTS: OUA increased the locomotion of rats after seven days, considered a mania-like behavior. In the FST, OUA increased the time of immobility on the 14th day, considered a depressive-like behavior. Li reversed the mania-like behavior and partially reversed the depressive-like behavior. Furthermore, OUA increased the levels of interleukin (IL)-1ß, IL-6, IL-10, TNF-α, and CINC-1 in the frontal cortex and hippocampus. Li treatment reverses all these inflammatory alterations. CONCLUSION: This study suggests that the long-term Na+K+ATPase inhibition effects induce depressive-like behavior, which was accompanied by inflammation in the BD symptoms model.

HDAC inhibitors reverse mania-like behavior and modulate epigenetic regulatory enzymes in an animal model of mania induced by Ouabain.

BACKGROUND: The etiology of bipolar disorder (BD) is multifactorial, involving both environmental and genetic factors. Current pharmacological treatment is associated with several side effects, which are the main reason patients discontinue treatment. Epigenetic alterations have been studied for their role in the pathophysiology of BD, as they bridge the gap between gene and environment. OBJECTIVE: Evaluate the effects of histone deacetylase inhibitors on behavior and epigenetic enzymes activity in a rat model of mania induced by ouabain. METHODS: Adult male rats were subjected to a single intracerebroventricular injection of ouabain (10-3 M) followed by 7 days of valproate (200 mg/kg) or sodium butyrate (600 mg/kg) administration. Locomotor and exploratory activities were evaluated in the open-field test. Histone deacetylase, DNA methyltransferase, and histone acetyltransferase activity were assessed in the frontal cortex, hippocampus, and striatum. RESULTS: Ouabain induced hyperactivity in rats, which was reversed by valproate and sodium butyrate treatment. Ouabain did not alter the activity of any of the enzymes evaluated. However, valproate and sodium butyrate decreased the activity of histone deacetylase and DNA methyltransferase. Moreover, there was a positive correlation between these two enzymes. CONCLUSION: These results suggest that targeting epigenetic mechanisms may play an important role in mania-like behavior management.

Intracerebroventricular injection of ouabain causes mania-like behavior in mice through D2 receptor activation.

Intracerebroventricular (ICV) administration of ouabain, an inhibitor of the Na, K-ATPase, is an approach used to study the physiological functions of the Na, K-ATPase and cardiotonic steroids in the central nervous system, known to cause mania-like hyperactivity in rats. We describe a mouse model of ouabain-induced mania-like behavior. ICV administration of 0.5 µl of 50 µM (25 pmol, 14.6 ng) ouabain into each lateral brain ventricle results in increased locomotor activity, stereotypical behavior, and decreased anxiety level an hour at minimum. Fast-scan cyclic voltammetry showed that administration of 50 µM ouabain causes a drastic drop in dopamine uptake rate, confirmed by elevated concentrations of dopamine metabolites detected in the striatum 1 h after administration. Ouabain administration also caused activation of Akt, deactivation of GSK3ß and activation of ERK1/2 in the striatum of ouabain-treated mice. All of the abovementioned effects are attenuated by haloperidol (70 µg/kg intraperitoneally). Observed effects were not associated with neurotoxicity, since no dystrophic neuron changes in brain structures were demonstrated by histological analysis. This newly developed mouse model of ouabain-induced mania-like behavior could provide a perspective tool for studying the interactions between the Na,K-ATPase and the dopaminergic system.

Effect of photobiomodulation therapy on neuronal injuries by ouabain: the regulation of Na, K-ATPase; Src; and mitogen-activated protein kinase signaling pathway.

BACKGROUND: To determine whether photobiomodulation (PBM) rescued the disruption of Na+/Ca2+ homeostasis and mitochondrial membrane potential by ouabain; the Na, K-ATPase inhibitor. For PBM in this study, a 660 nm LED array was used at energy densities of 0.78, 1.56, 3.12, 6.24, and 9.36 J/cm2. RESULTS: HCN-2 neuronal cells treated with ouabain showed loss of cell polarity, disrupted cell morphology, and decreased cell viability, which were improved after PBM treatment. We found that ouabain-induced Na, K-ATPase inhibition promoted activation of downstream signaling through Src, Ras, and mitogen-activated protein kinase (MAPK), which were suppressed after PBM treatment. This provided evidence of Na, K-ATPase α-subunit inactivation and intracellular Ca2+ increase. In response to ouabain, we observed activation of Src and MAPK by Na, K-ATPase, decreased mitochondrial membrane potential, and Na+-dependent Ca2+ increases, which were restored by PBM treatment. CONCLUSIONS: This study demonstrated that Na+/K+ imbalance could be regulated by PBM treatment in neuronal cells, and we suggest that PBM is a potential therapeutic tool for Na, K-ATPase targeted neuronal diseases.

The potassium channel blocker, dalfampridine diminishes ouabain-induced arrhythmia in isolated rat atria.

The aim of the present experiment was to investigate the possible antiarrhythmic effects of dalfampridine in ouabain-induced arrhythmia in rats. Twenty-four male rats including the control and dalfampridine-incubated (100 µM to 10 mM) ouabain-stimulated (40 µM) groups were used. After induction of anesthesia, the atria were isolated and the time of onset of arrhythmia and asystole were recorded. The contractile force of atria was also measured. Dalfampridine at concentration of 1 mM significantly postponed the onset of arrhythmia and asystole compared to control group (p ≤ .05). Ouabain significantly increased the atrial beating rate in control group (p ≤ .05), while pretreatment of isolated atria with dalfampridine reversed this effect. Incubation of isolated atria with ouabain did not alter the contractile force in both control- and dalfampridine-treated groups (p > .05). It is concluded that dalfampridine might possess antiarrhythmic properties in reducing the atrial arrhythmias.

Non-ß-Blocking Carvedilol Analog, VK-II-86, Prevents Ouabain-Induced Cardiotoxicity.

BACKGROUND: It has been shown that carvedilol and its non ß-blocking analog, VK-II-86, inhibit spontaneous Ca2+ release from the sarcoplasmic reticulum (SR). The aim of this study is to determine whether carvedilol and VK-II-86 suppress ouabain-induced arrhythmogenic Ca2+ waves and apoptosis in cardiac myocytes. Methods and Results: Rat cardiac myocytes were exposed to toxic doses of ouabain (50 µmol/L). Cell length (contraction) was monitored in electrically stimulated and non-stimulated conditions. Ouabain treatment increased contractility, frequency of spontaneous contractions and apoptosis compared to control cells. Carvedilol (1 µmol/L) or VK-II-86 (1 µmol/L) did not affect ouabain-induced inotropy, but significantly reduced the frequency of Ca2+ waves, spontaneous contractions and cell death evoked by ouabain treatment. This antiarrhythmic effect was not associated with a reduction in Ca2+ calmodulin-dependent protein kinase II (CaMKII) activity, phospholamban and ryanodine receptor phosphorylation or SR Ca2+ load. Similar results could be replicated in human cardiomyocytes derived from stem cells and in a mathematical model of human myocytes. CONCLUSIONS: Carvedilol and VK-II-86 are effective to prevent ouabain-induced apoptosis and spontaneous contractions indicative of arrhythmogenic activity without affecting inotropy and demonstrated to be effective in human models, thus emerging as a therapeutic tool for the prevention of digitalis-induced arrhythmias and cardiac toxicity.

Involvement of IL-1ß and IL-6 in antiarrhythmic properties of atorvastatin in ouabain-induced arrhythmia in rats.

PURPOSE: Evidence show that statins possess wide beneficial cardioprotective and anti-inflammatory effects; therefore, in the present experiment, we investigated the antiarrhythmic properties of atorvastatin in ouabain-induced arrhythmia in isolated rat atria and the role of several inflammatory cytokines in this effect. MATERIALS AND METHODS: Male rats were pretreated with either of atorvastatin (10 mg/kg) or vehicle, orally once daily for 6 weeks. After induction of anesthesia, we isolated the atria and after incubation with ouabain, time of onset of arrhythmia and asystole as well as atrial beating rate and contractile force were recorded. We also measured the atrial levels of IL-1ß, IL-6, and TNF-α after the injection of ouabain to animals. RESULTS: Pretreatment with atorvastatin significantly delayed the onset of arrhythmia and asystole compared with vehicle-treated group (p < .01, p < .001, respectively). Incubation of ouabain boosted both atrial beating rate and contractile force in vehicle-treated group (p < .05), while these responses in atorvastatin-treated group were not significant (p > .05). Injection of ouabain elevated the atrial levels of IL-1ß, IL-6, and TNF-α, while pretreatment of animals with atorvastatin could reverse the ouabain-induced increase in atrial IL-1ß and IL-6 (p < .01 and p < .05, respectively). CONCLUSIONS: It is concluded that observed antiarrhythmic effects of atorvastatin might be attributed to modulation of some inflammatory cytokines, at least IL-1ß and IL-6.

Ouabagenin is a naturally occurring LXR ligand without causing hepatic steatosis as a side effect.

Ouabagenin (OBG) is an aglycone of the cardiotonic steroid ouabain and until now was considered a biologically inactive biosynthetic precursor. Herein, we revealed that OBG functions as a novel class of ligand for the liver X receptor (LXR). Luciferase reporter assays and in silico docking studies suggested that OBG has LXR-selective agonistic activity. In addition, OBG repressed the expression of epithelial sodium channel (ENaC), a LXR target gene, without causing hepatic steatosis, a typical side effect of conventional LXR ligands. This remarkable biological activity can be attributed to a unique mode of action; the LXR agonist activity mainly proceeds through the LXRß subtype without affecting LXRα, unlike conventional LXR ligands. Thus, OBG is a novel class of LXR ligand that does not cause severe side effects, with potential for use as an antihypertensive diuretic or a tool compound for exploring LXR subtype-specific biological functions.

The cardiac glycoside ouabain activates NLRP3 inflammasomes and promotes cardiac inflammation and dysfunction.

Cardiac glycosides such as digoxin are Na+/K+-ATPase inhibitors that are widely used for the treatment of chronic heart failure and cardiac arrhythmias; however, recent epidemiological studies have suggested a relationship between digoxin treatment and increased mortality. We previously showed that nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasomes, which regulate caspase-1-dependent interleukin (IL)-1ß release, mediate the sterile cardiovascular inflammation. Because the Na+/K+-ATPase is involved in inflammatory responses, we investigated the role of NLRP3 inflammasomes in the pathophysiology of cardiac glycoside-induced cardiac inflammation and dysfunction. The cardiac glycoside ouabain induced cardiac dysfunction and injury in wild-type mice primed with a low dose of lipopolysaccharide (LPS), although no cardiac dysfunction was observed in mice treated with either ouabain or LPS alone. Ouabain also induced cardiac inflammatory responses, such as macrophage infiltration and IL-1ß release, when mice were primed with LPS. These cardiac manifestations were all significantly attenuated in mice deficient in IL-1ß. Furthermore, deficiency of NLRP3 inflammasome components, NLRP3 and caspase-1, also attenuated ouabain-induced cardiac dysfunction and inflammation. In vitro experiments revealed that ouabain induced NLRP3 inflammasome activation as well as subsequent IL-1ß release from macrophages, and this activation was mediated by K+ efflux. Our findings demonstrate that cardiac glycosides promote cardiac inflammation and dysfunction through NLRP3 inflammasomes and provide new insights into the mechanisms underlying the adverse effects of cardiac glycosides.

Contributions of Mouse and Human Hematopoietic Cells to Remodeling of the Adult Auditory Nerve After Neuron Loss.

The peripheral auditory nerve (AN) carries sound information from sensory hair cells to the brain. The present study investigated the contribution of mouse and human hematopoietic stem cells (HSCs) to cellular diversity in the AN following the destruction of neuron cell bodies, also known as spiral ganglion neurons (SGNs). Exposure of the adult mouse cochlea to ouabain selectively killed type I SGNs and disrupted the blood-labyrinth barrier. This procedure also resulted in the upregulation of genes associated with hematopoietic cell homing and differentiation, and provided an environment conducive to the tissue engraftment of circulating stem/progenitor cells into the AN. Experiments were performed using both a mouse-mouse bone marrow transplantation model and a severely immune-incompetent mouse model transplanted with human CD34+ cord blood cells. Quantitative immunohistochemical analysis of recipient mice demonstrated that ouabain injury promoted an increase in the number of both HSC-derived macrophages and HSC-derived nonmacrophages in the AN. Although rare, a few HSC-derived cells in the injured AN exhibited glial-like qualities. These results suggest that human hematopoietic cells participate in remodeling of the AN after neuron cell body loss and that hematopoietic cells can be an important resource for promoting AN repair/regeneration in the adult inner ear.

Cariprazine delays ouabain-evoked epileptiform spikes and loss of activity in rat hippocampal slices.

In the only bipolar cycling in vitro model, rat hippocampal slices are treated with the sodium pump inhibitor ouabain, which induces epileptiform activity, followed by refractory activity loss that recovers and cycles back to epileptiform activity. Thus, clinical cycling seen in patients with bipolar disorder is modeled on a cellular level as alternating hyperactivity and hypoactivity interspersed with normal activity. In this study, we tested the ability of cariprazine a new antipsychotic candidate to block ouabain-induced changes in rat hippocampal slices. Cycling of population spikes and epileptiform bursts was evoked using an extracellular stimulation electrode located in the Schaeffer collaterals of 400-µm-thick rat hippocampal slices treated with ouabain (3.3µM) alone or in combination with cariprazine (1, 5, 25, and 50µM). Responses were recorded using an extracellular electrode placed in the cell body layer of the CA1 region. Cariprazine 25 and 50µM delayed ouabain-induced epileptiform burst onset and subsequent activity loss. Lower cariprazine concentrations were ineffective. Cariprazine delays the onset of ouabain-induced epileptiform bursts and the loss of spiking activity similarly to that previously demonstrated with the mood stabilizer lithium. These results suggest that cariprazine may have therapeutic potential for treatment of bipolar disorder.

Rostafuroxin protects from podocyte injury and proteinuria induced by adducin genetic variants and ouabain.

Glomerulopathies are important causes of morbidity and mortality. Selective therapies that address the underlying mechanisms are still lacking. Recently, two mechanisms, mutant ß-adducin and ouabain, have been found to be involved in glomerular podocytopathies and proteinuria through nephrin downregulation. The main purpose of the present study was to investigate whether rostafuroxin, a novel antihypertensive agent developed as a selective inhibitor of Src-SH2 interaction with mutant adducin- and ouabain-activated Na,K-ATPase, may protect podocytes from adducin- and ouabain-induced effects, thus representing a novel pharmacologic approach for the therapy of podocytopathies and proteinuria caused by the aforementioned mechanisms. To study the effect of rostafuroxin on podocyte protein changes and proteinuria, mice carrying mutant ß-adducin and ouabain hypertensive rats were orally treated with 100 µg/kg per day rostafuroxin. Primary podocytes from congenic rats carrying mutant α-adducin or ß-adducin (NB) from Milan hypertensive rats and normal rat podocytes incubated with 10(-9) M ouabain were cultured with 10(-9) M rostafuroxin. The results indicated that mutant ß-adducin and ouabain caused podocyte nephrin loss and proteinuria in animal models. These alterations were reproduced in primary podocytes from NB rats and normal rats incubated with ouabain. Treatment of animals, or incubation of cultured podocytes with rostafuroxin, reverted mutant ß-adducin- and ouabain-induced effects on nephrin protein expression and proteinuria. We conclude that rostafuroxin prevented podocyte lesions and proteinuria due to mutant ß-adducin and ouabain in animal models. This suggests a potential therapeutic effect of rostafuroxin in patients with glomerular disease progression associated with these two mechanisms.

Ouabain inhibits placental sFlt1 production by repressing HSP27-dependent HIF-1α pathway.

Up-regulation of placental soluble fms-like tyrosine kinase 1 (sFlt1) contributes to the pathogenesis of preeclampsia. To evaluate novel upstream pathways that regulate placental sFlt1 production, we screened a library of natural compounds (n=502) in human placental cell lines. Here, we report 3 compounds in the cardiac glycoside family, ouabain, gitoxigenin, and digitoxin, that inhibit placental sFlt1 production at nanomolar concentrations in vitro. We further characterized ouabain and demonstrated that it inhibits sFlt1 mRNA and protein expression in human placental cytotrophoblasts and explant cultures in a dose- and time-dependent manner. Ouabain down-regulated sFlt1 production by inhibiting hypoxia-inducible factor 1 (HIF-1α) protein expression in the placenta. Furthermore, we found that phosphorylation of heat-shock protein 27 (HSP27) was necessary for ouabain to inhibit HIF-1α translation. In a rat model of pregnancy-induced hypertension, ouabain reduced mean arterial pressure and enhanced placental HSP27 phosphorylation without any adverse effects on pups. Further studies are needed to explore the usefulness of targeting HIF-1α/HSP27 pathway in preeclampsia.

Ouabain-induced cochlear nerve degeneration: synaptic loss and plasticity in a mouse model of auditory neuropathy.

Ouabain application to the round window can selectively destroy type-I spiral ganglion cells, producing an animal model of auditory neuropathy. To assess the long-term effects of this deafferentation on synaptic organization in the organ of Corti and cochlear nucleus, and to ask whether surviving cochlear neurons show any post-injury plasticity in the adult, we quantified the peripheral and central synapses of type-I neurons at posttreatment times ranging from 1 to 3 months. Measures of normal DPOAEs and greatly reduced auditory brainstem responses (ABRs) confirmed the neuropathy phenotype. Counts of presynaptic ribbons and postsynaptic glutamate receptor patches in the inner hair cell area decreased with post-exposure time, as did counts of cochlear nerve terminals in the cochlear nucleus. Although these counts provided no evidence of new synapse formation via branching from surviving neurons, the regular appearance of ectopic neurons in the inner hair cell area suggested that neurite extension is not uncommon. Correlations between pathophysiology and histopathology showed that ABR thresholds are very insensitive to even massive neural degeneration, whereas the amplitude of ABR wave 1 is a better metric of synaptic degeneration.

Discovery of aroyl piperazine derivatives as IKr & IKs dual inhibitors for cardiac arrhythmia treatment.

Combined blockade of IKr and IKs potassium channels is considered to be a promising therapeutic strategy for arrhythmia. In this study, we designed and synthesized 15 derivatives through modifying the hit compound 7 that was discovered by screening in-house database by whole-patch clamp technique. All of the compounds were evaluated on CHO and HEK 293 cell lines stably expressing hERG (IKr) and hKCNQ1/KCNE1 (IKs) potassium channels, and half of them exhibited improved dual IKr and IKs inhibitory effects compared to the hit compound. Compounds 7a and 7b with potent dual inhibitory activities were selected for further in vivo evaluations. Due to the preferable pharmacological behaviors, compound 7a deserved further optimization as a promising lead compound.

Ouabain-induced apoptosis in cochlear hair cells and spiral ganglion neurons in vitro.

Ouabain is a common tool to explore the pathophysiological changes in adult mammalian cochlea in vivo. In prior studies, locally administering ouabain via round window membrane demonstrated that the ototoxic effects of ouabain in vivo varied among mammalian species. Little is known about the ototoxic effects in vitro. Thus, we prepared cochlear organotypic cultures from postnatal day-3 rats and treated these cultures with ouabain at 50, 500, and 1000 µM for different time to elucidate the ototoxic effects of ouabain in vitro and to provide insights that could explain the comparative ototoxic effects of ouabain in vivo. Degeneration of cochlear hair cells and spiral ganglion neurons was evaluated by hair-cell staining and neurofilament labeling, respectively. Annexin V staining was used to detect apoptotic cells. A quantitative RT-PCR apoptosis-focused gene array determined changes in apoptosis-related genes. The results showed that ouabain-induced damage in vitro was dose and time dependent. 500 µM ouabain and 1000 µM ouabain were destructively traumatic to both spiral ganglion neurons and cochlear hair cells in an apoptotic signal-dependent pathway. The major apoptotic pathways in ouabain-induced spiral ganglion neuron apoptosis culminated in the stimulation of the p53 pathway and triggering of apoptosis by a network of proapoptotic signaling pathways.

Different methods of immunosuppresion do not prolong the survival of human cord blood-derived neural stem cells transplanted into focal brain-injured immunocompetent rats.

Cerebrovascular diseases are the leading cause of severe disability worldwide, with an enormous financial burden for society. There is growing evidence that stem cell-based therapy may positively influence recovery from stroke. Cord blood is an attractive source of ontogenetically young, yet safe, stem cells. Conceptually, preclinical studies in which donor cells were of human origin have been the most valuable, and thus, it is likely that these cells will be used in clinical trials. Unfortunately, immunological barriers impede discordant xenotransplantations. We have previously observed acute rejection of cord blood derived neural stem cells (HUCB-NSC) after transplantation to the brains of intact animals. Since it was reported recently that a brain lesion may actually improve the chances of graft survival, in this study, we used infarcted animals as graft recipients. In ongoing studies, we tested three immunosuppressive regimes: group I received cyclosporine A (CsA: 10 mg per kg i.p.); group II received a triple-drug therapy (CsA: 10 mg per kg i.p., azathioprine: 5 mg per kg i.p., and methylprednisolone: 1.5 mg per kg i.m.); group III included rats that were formerly desensitized with HUCB, group IV had not undergone immunosuppression. Animals were sacrificed at five time-points: 1, 3, 7, 14, and 21 days post-transplantation to evaluate graft survival and the time-course of immunological response. We observed a gradual decrease in the number of transplanted cells, with complete disappearance by day 14, surprisingly, with no difference among the experimental groups. The involvement of the innate immune system in the process of graft rejection dominated over an adaptive immunoresponse, with the highest activity on day 3, and subsequent fading of immune cell infiltration. In this work, we have shown that none of our immunosuppressive strategies proved adequate to prevent rejection of human stem cell grafts after transplantation into immunocompetent animals.

Increased constrictor tone induced by ouabain treatment in rats.

Ouabain (Oua)-induced hypertension in rodents provides a model to study cardiovascular changes associated with human hypertension. We examined vascular function in rats after a long-term treatment with Oua. Systolic blood pressure was measured by tail-cuff plethysmography in male Sprague-Dawley rats treated with Oua (≈ 25 µg/d) or placebo for 8 weeks. Blood pressure increased in Oua-treated animals, reaching 30% above baseline systolic blood pressure after 7 weeks. At the end of treatment, vascular responses were studied in mesenteric resistance arteries (MRAs) by wire myography. Contraction to potassium chloride in intact and denuded arteries showed greater sensitivity in Oua-treated animals. Contraction to phenylephrine and relaxation to acetylcholine were similar between groups with a lower response to sodium nitroprusside in Oua-treated arteries. Sensitivity to endothelin-1 was higher in Oua-treated arteries. Na⁺-K⁺ ATPase activity was decreased in MRAs from Oua-treated animals, whereas protein expression of the Na⁺-K⁺ ATPase α2 isoform was increased in heart and unchanged in mesenteric artery. Preincubation with indomethacin (10⁻5 M) or Nω-nitro-L-arginine methyl ester (10⁻4 M) abolished the differences in potassium chloride response and Na⁺-K⁺ ATPase activity. Changes in MRAs are consistent with enhanced vascular smooth muscle cell reactivity, a contributor to the increased vascular tone observed in this model of hypertension.

Inhalation of hydrogen gas attenuates ouabain-induced auditory neuropathy in gerbils.

AIM: Auditory neuropathy (AN) is a hearing disorder characterized by abnormal auditory nerve function with preservation of normal cochlear hair cells. This study was designed to investigate whether treatment with molecular hydrogen (H(2)), which can remedy damage in various organs via reducing oxidative stress, inflammation and apoptosis, is beneficial to ouabain-induced AN in gerbils. METHODS: AN model was made by local application of ouabain (1 mmol/L, 20 mL) to the round window membrane in male Mongolian gerbils. H(2) treatment was given twice by exposing the animals to H(2) (1%, 2%, and 4%) for 60 min at 1 h and 6 h after ouabain application. Before and 7 d after ouabain application, the hearing status of the animals was evaluated using the auditory brainstem response (ABR) approach, the hear cell function was evaluated with distortion product otoacoustic emissions (DPOAE). Seven days after ouabain application, the changes in the cochleae, especially the spiral ganglion neurons (SGNs), were morphologically studied. TUNEL staining and immunofluorescent staining for activated caspase-3 were used to assess the apoptosis of SGNs. RESULTS: Treatment with H(2) (2% and 4%) markedly attenuated the click and tone burst-evoked ABR threshold shift at 4, 8, and 16 kHz in ouabain-exposed animals. Neither local ouabain application, nor H(2) treatment changed the amplitude of DPOAE at 4, 8, and 16 kHz. Morphological study showed that treatment with H(2) (2%) significantly alleviated SGN damage and attenuated the loss of SGN density for each turn of cochlea in ouabain-exposed animals. Furthermore, ouabain caused significantly higher numbers of apoptotic SGNs in the cochlea, which was significantly attenuated by the H(2) treatment. However, ouabain did not change the morphology of cochlear hair cells. CONCLUSION: The results demonstrate that H(2) treatment is beneficial to ouabain-induced AN via reducing apoptosis. Thus, H(2) might be a potential agent for treating hearing impairment in AN patients.