Journal articles on the topic 'Alpha attenuation test'

To test the hypothesis that alpha 2-adrenoceptor activity exerts a dual control of coronary blood flow, i.e., vasoconstriction and augmentation of the vasodilatory effect of adenosine, four doses of adenosine were infused into left anterior descending coronary artery before and during alpha 2-adrenoceptor stimulation or attenuation in anesthetized open-chest dogs. During a moderate alpha 2-adrenoceptor attenuation (yohimbine or rauwolscine, ic), which did not alter coronary blood flow (CBF) at the base-line condition, the hyperemic response of CBF to infused adenosine was markedly reduced, whereas during the potent attenuation both base-line CBF and adenosine-induced hyperemic CBF were significantly increased. Inversely, the moderate alpha 2-stimulation (0.03 microgram.kg-1.min-1 norepinephrine with prazosin, ic, or 0.04 microgram.kg-1.min-1 clonidine ic, under propranolol pretreatment) augmented the adenosine-induced coronary vasodilation, but the potent alpha 2-stimulation (0.3 microgram.kg-1.min-1 norepinephrine with prazosin ic, or 0.3 microgram.kg-1.min-1 clonidine ic) reduced both base-line CBF hyperemic CBF. In contrast, alpha 2-adrenoceptor activity did not affect papaverine-induced coronary vasodilation. Moreover, the reactive hyperemic flow after a brief coronary occlusion was reduced significantly during the moderate alpha 2-adrenergic attenuation, but it was augmented during the potent one. These results indicate that the moderate activation of the alpha 2-adrenoceptor augments the hyperemic response of CBF to both exogenous and endogenous adenosine, whereas the potent alpha 2-activation may mask this vasodilatory effect through the coronary vasoconstrictive effect.

Our goals were to establish that an alpha 2-adrenergic agonist (clonidine) inhibits ACh release from airway nerve endings and to test effects of iberiotoxin (IBTX), an inhibitor of fast-conductance, Ca(2+)-activated K+ channels on alpha 2-adrenergic and muscarinic attenuation of ACh release. Guinea pig tracheas were mounted between electrodes in buffer containing indomethacin and neostigmine, and high-performance liquid chromatography with electrochemical detection was used to measure ACh release during electrical field stimulation. Clonidine inhibited ACh release in a concentration-dependent fashion [maximum reduction: 48 +/- 3%; 50% inhibitory constant (IC50): 0.1 microM], and idazoxan, alpha 2-adrenergic antagonist, reversed the effect. However, IBTX failed to alter clonidine-induced attenuation of ACh release. In contrast, IBTX did cause an increase in tracheal tension. In addition, IBTX failed to reverse any of the potent autoinhibitory effects of endogenous ACh. Our results confirm the presence of inhibitory alpha 2-adrenergic receptors. However, activation of IBTX-sensitive K+ channels does not appear necessary for either alpha 2-adrenergic or muscarinic cholinergic inhibition of ACh release.

α-Pinene, an organic terpene compound found in coniferous trees, is used as a safe food additive and is contained in many essential oils. Moreover, some studies have shown that α-pinene suppresses neuronal activity. In this study, we investigated whether inhalation of α-pinene suppresses dizocilpine (MK-801-) induced schizophrenia-like behavioural abnormalities in mice. Mice inhaled α-pinene 1 h before the first MK-801 injection. Thirty minutes after MK-801 injection, the open field, spontaneous locomotor activity, elevated plus maze, Y-maze, tail suspension, hot plate, and grip strength tests were conducted as behavioural experiments. Inhalation of α-pinene suppressed the activity of mice in the spontaneous locomotor activity test and although it did not suppress the MK-801-induced increased locomotor activity in the open field test, it remarkably decreased the time that the mice remained in the central area. Inhalation of α-pinene suppressed the MK-801-induced increased total distance travelled in the Y-maze test, whereas it did not alter the MK-801-induced reduced threshold of antinociception in the hot plate test. In the tail suspension and grip strength tests, there was no effect on mouse behaviour by administration of MK-801 and inhalation of α-pinene. These results suggest that α-pinene acts to reduce MK-801-induced behavioural abnormalities resembling those seen in neuropsychiatric disorders. Therefore, both medicinal plants and essential oils containing α-pinene may have potential for therapeutic treatment of schizophrenia.

Introduction. Decrease of daily alertness is a common cause of accidents in the work place, especially traffic accidents. Therefore, an increasing interest exists to determine reliable indicators of a tendency to fall asleep involuntarily. Objective. To determine an optimal electroencephalographic (EEG) indicator of an involuntary tendency to fall asleep, we performed a study on neurologically healthy subjects, after one night of sleep deprivation. Total sleep deprivation was aimed at increasing daily sleepiness in healthy subjects, providing us with an opportunity to test different methods of evaluation. Methods. We applied a visual analogue scale for sleepiness (VASS), EEG registration with the specific test of alpha activity attenuation (TAA) in 87 healthy subjects. The test was performed in a standard way (sTAA) as well as in accordance with new modifications related to changes of EEG filter width in the range from 5 to 32 Hz (mTAA). Results. After sleep deprivation, we observed involuntary falling asleep in 54 subjects. The comparison of VASS results showed no differences, contrary to a more objective TAA. Between two variants of TAA, the modified test provided us with a better prediction for subjects who would fall asleep involuntarily. Conclusion. The application of a more objective EEG test in evaluation of daily alertness represents the optimal method of testing. Modified TAA attracts special attention, offering a simple solution for reliable testing of decreased daily alertness in medical services related to professional aircraft personnel.

IntroductionAlthough schizophrenia was previously associated with affected spatial neuronal synchronization, surprisingly little is known about the temporal dynamics of neuronal oscillations in schizophrenia. However, since coordination of neuronal process in time represents an essential aspect of practically all cognitive processes, it might be strongly affected in schizophrenia patients.ObjectivesTo test the hypothesis of abnormal temporal neuronal dynamics in patients with schizophrenia.AimsWe aimed at quantification and comparisons of long-range temporal correlations (LRTCs) in patients and normal subjects.MethodsWe measured 21 patients with schizophrenia (n = 18) or schizoaffective disorder (n = 3) and 28 age and gender matched controls. Ongoing neuronal oscillations were recorded with multi-channel EEG at rest condition. EEG was analyzed with spectral analysis and with the detrended fluctuation analysis allowing quantification of LRTCs.ResultsThe amplitude of neuronal oscillations in alpha and beta frequency ranges did not differ between the patients and controls. However, LRTCs were strongly attenuated in schizophrenia patients: in centro-parietal areas and fronto-central areas for alpha and beta oscillations, respectively. In addition we observed a negative correlation between the strength of the negative symptoms and LRTCs.ConclusionsSmall values of LRTCs and their correlation with the negative symptoms in schizophrenia patients demonstrate that the temporal dynamics of neuronal oscillations are essential for normal brain functioning. Attenuated LRTCs might indicate a more intermittent neuronal dynamics possibly allowing for more random associations between neuronal activations, which in turn might relate to the occurrence of positive symptoms in schizophrenia.

AbstractBackground:Peripheral neuropathy is the dose limiting side effect of many anticancer drugs. Flavonoids exhibit good antinociceptive effect in animal models. Their efficacy against different types of nociception has been documented. The present study investigated the effect of flavonol (3-hydroxy flavone), 3′,4′-dimethoxy flavonol, 6,3′-dimethoxy flavonol, 7,2′-dimethoxy flavonol and 7,3′-dimethoxy flavonol against paclitaxel-induced peripheral neuropathy in mice.Methods:A single dose of paclitaxel (10 mg/kg, i.p.) was administered to induce peripheral neuropathy in mice and the manifestations of peripheral neuropathy such as tactile allodynia, cold allodynia and thermal hyperalgesia were assessed 24 h later by employing Von Frey hair aesthesiometer test, acetone bubble test and hot water tail immersion test, respectively. The test compounds were prepared as a suspension in 0.5% carboxymethyl cellulose and were administered s.c. in various doses (25, 50, 100 and 200 mg/kg). The above behavioral responses were assessed prior to and 30 min after drug treatment. In addition, the effect of test compounds on proinflammatory cytokines like tumor necrosis factor-alpha (TNF-α), interleukin-1-beta (IL-1β) and free radicals was investigated by using suitablein vitroassays.Results:A dose-dependent attenuation of tactile allodynia, cold allodynia and thermal hyperalgesia was evidenced in mice treated with flavonol derivatives. The test compounds inhibited TNF-α, IL-1β and free radicals in a concentration-dependent manner.Conclusions:These results revealed that flavonol and its dimethoxy derivatives ameliorated the manifestations of paclitaxel-induced peripheral neuropathy in mice. The inhibition of proinflammatory cytokines and free radicals could contribute to this beneficial effect.

ABSTRACT The herpes simplex virus (HSV) virion host shutoff (vhs) protein, the product of the UL41 (vhs) gene, is an important determinant of HSV virulence. vhs has been implicated in HSV interference with host antiviral immune responses, down-regulating expression of major histocompatibility complex molecules to help HSV evade host adaptive immunity. The severe attenuation of vhs-deficient viruses in vivo could reflect their inability to escape immune detection. To test this hypothesis, BALB/c or congenic SCID mice were infected intravaginally (i.vag.) with the HSV type 2 (HSV-2) vhs null mutant 333d41 or the vhs rescue virus 333d41R. vhs-deficient virus remained severely attenuated in SCID mice compared with rescue virus, indicating that vhs regulation of adaptive immune responses does not influence HSV pathogenesis during acute infection. Innate antiviral effectors remain intact in SCID mice; prominent among these is alpha/beta interferon (IFN-α/β). The attenuation of HSV-2 vhs mutants could reflect their failure to suppress IFN-α/β-mediated antiviral activity. To test this hypothesis, 129 and congenic IFN-α/β receptor-deficient (IFN-α/βR−/−) mice were infected i.vag. with wild-type virus, vhs null mutants 333-vhsB or 333d41, or the vhs rescue virus 333d41R. Whereas vhs-deficient viruses showed greatly reduced replication in the genital mucosa of 129 mice compared with wild-type or vhs rescue viruses, they were restored to nearly wild-type levels of replication in IFN-α/βR−/− mice over the first 2 days postinfection. Only wild-type and vhs rescue viruses caused severe genital disease and hind limb paralysis in 129 mice, but infection of IFN-α/βR−/− mice restored the virulence of vhs-deficient viruses. vhs-deficient viruses replicated as vigorously as wild-type and rescue viruses in the nervous systems of IFN-α/βR−/− mice. Restoration was specific for the vhs mutation, because thymidine kinase-deficient HSV-2 did not regain virulence or the capacity to replicate in the nervous systems of IFN-α/βR−/− mice. Furthermore, the defect in the IFN-α/β response was required for restoration of vhs-deficient virus replication and virulence, but the IFN-α/β-stimulated protein kinase R pathway was not involved. Finally, vhs of HSV-2 has a unique capacity to interfere with the IFN-α/β response in vivo, because an HSV-1 vhs null mutant did not recover replication and virulence after i.vag. inoculation into IFN-α/βR−/− mice. These results indicate that vhs plays an important role early in HSV-2 pathogenesis in vivo by interfering with the IFN-α/β-mediated antiviral response.

Left–right (L–R) differences in mammographic parenchymal patterns are an early predictor of breast cancer risk; however, the basis for this asymmetry is unknown. Here, we use retinoid X receptor alpha heterozygous null (RXRα +/− ) mice to propose a developmental origin: perturbation of coordinated anterior–posterior (A–P) and L–R axial body patterning. We hypothesized that by analogy to somitogenesis—in which retinoic acid (RA) attenuation causes anterior somite pairs to develop L–R asynchronously—that RA pathway perturbation would likewise result in asymmetric mammary development. To test this, mammary glands of RXRα +/− mice were quantitatively assessed to compare left- versus right-side ductal epithelial networks. Unlike wild-type controls, half of the RXRα +/− thoracic mammary gland (TMG) pairs exhibited significant L–R asymmetry, with left-side reduction in network size. In RXRα +/− TMGs in which symmetry was maintained, networks had bilaterally increased size, with left networks showing greater variability in area and pattern. Reminiscent of posterior somites, whose bilateral symmetry is refractory to RA attenuation, inguinal mammary glands (IMGs) also had bilaterally increased network size, but no loss of symmetry. Together, these results demonstrate that mammary glands exhibit differential A–P sensitivity to RXRα heterozygosity, with ductal network symmetry markedly compromised in anterior but not posterior glands. As TMGs more closely model human breast development than IMGs, these findings raise the possibility that for some women, breast cancer risk may initiate with subtle axial patterning defects that result in L–R asymmetric growth and pattern of the mammary ductal epithelium. This article is part of the themed issue ‘Provocative questions in left–right asymmetry’.

Microglia-mediated neuroinflammation is one of the key mechanisms involved in acute brain injury and chronic neurodegeneration. This study investigated the inhibitory effects of 2-hydroxy-4-methylbenzoic anhydride (HMA), a novel synthetic derivative of HTB (3-hydroxy-4-trifluoromethylbenzoic acid) on neuroinflammation and underlying mechanisms in activated microglia in vitro and an in vivo mouse model of Parkinson’s disease (PD). In vitro studies revealed that HMA significantly inhibited lipopolysaccharide (LPS)-stimulated excessive release of nitric oxide (NO) in a concentration dependent manner. In addition, HMA significantly suppressed both inducible NO synthase and cyclooxygenase-2 (COX-2) at the mRNA and protein levels in LPS-stimulated BV-2 microglia cells. Moreover, HMA significantly inhibited the proinflammatory cytokines such as interleukin (IL)-1beta, IL-6, and tumor necrosis factor-alpha in LPS-stimulated BV-2 microglial cells. Furthermore, mechanistic studies ensured that the potent anti-neuroinflammatory effects of HMA (0.1, 1.0, and 10 μM) were mediated by phosphorylation of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) in LPS-stimulated BV-2 cells. In vivo evaluations revealed that intraperitoneal administration of potent neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, 20 mg/kg, four times a 1 day) in mice resulted in activation of microglia in the brain in association with severe behavioral deficits as assessed using a pole test. However, prevention of microglial activation and attenuation of Parkinson’s disease (PD)-like behavioral changes was obtained by oral administration of HMA (30 mg/kg) for 14 days. Considering the overall results, our study showed that HMA exhibited strong anti-neuroinflammatory effects at lower concentrations than its parent compound. Further work is warranted in other animal and genetic models of PD for evaluating the efficacy of HMA to develop a potential therapeutic agent in the treatment of microglia-mediated neuroinflammatory disorders, including PD.

ABSTRACT Several Shigella flexneri mutants with defects in aromatic amino acid and/or purine biosynthesis have been evaluated as vaccines in humans or in animal models. To be suitable as a vaccine, a mutant has to show virulence attenuation, minimal reactogenicity, and a good immunogenic potential in animal models. With this aim, we have constructed five S. flexneri 5 (wild-type strain M90T) mutants with inactivation of one or two of the loci purEK, purHD, and guaBA, governing early or late steps of purine biosynthesis. The mutants have been analyzed in vitro in cell cultures and in vivo in the Sereny test and in the murine pulmonary model of shigellosis. M90T guaBA, M90T guaBA purEK, M90T guaBA purHD, and M90T purHD purEK gave a negative result in the Sereny test. In contrast, in the murine pulmonary model all of the strains had the same 50% lethal dose as the wild type, except M90T guaBA purHD, which did not result in death of the animals. Nevertheless, bacterial counts in infected lungs, immunohistochemistry, and reverse transcription-PCR analysis of mRNAs for tumor necrosis factor alpha (TNF-α), gamma interferon (IFN-γ), interleukin-1β (IL-1β), IL-6, IL-12, and inducible nitric oxide synthase (iNOS) revealed significant differences among the strains. At 72 h postinfection, M90T guaBA purHD still induced proinflammatory cytokines and factors such as IL-1β, IL-6, TNF-α, and iNOS, along with cytokines such as IL-12 and IFN-γ. Moreover, in the absence of evident lesions in murine tissues, this mutant highly stimulated major histocompatibility complex class II expression, showing a significant ability to activate the innate immunity of the host.

ABSTRACT Surfactant protein A (SP-A), a member of the collectin family, selectively binds to Pneumocystis carinii and mediates interactions between pathogen and host alveolar macrophages in vitro. To test the hypothesis that mice lacking SP-A have delayed clearance of Pneumocystis organisms and enhanced lung injury, wild-type C57BL/6 (WT) and SP-A-deficient mice (SP-A−/−) with or without selective CD4+-T-cell depletion were intratracheally inoculated with Pneumocystis organisms. Four weeks later, CD4-depleted SP-A-deficient mice had developed a more severe Pneumocystis infection than CD4-depleted WT (P. carinii pneumonia [PCP] scores of 3 versus 2, respectively). Whereas all non-CD4-depleted WT mice were free of PCP, intact SP-A−/− mice also had evidence of increased organism burden. Pneumocystis infection in SP-A-deficient mice was associated histologically with enhanced peribronchial and/or perivascular cellularity (score of 4 versus 2, SP-A−/− versus C57BL/6 mice, respectively) and a corresponding increase in bronchoalveolar lavage (BAL) cell counts. Increases in SP-D content, gamma interferon, interleukin-4, interleukin-5, and tumor necrosis factor alpha in BAL fluid occurred but were attenuated in PCP-infected SP-A−/− mice compared to WT mice. There were increases in total BAL NO levels in both infected groups, but nitrite levels were higher in SP-A−/− mice, indicating a reduction in production of higher oxides of nitrogen that was also reflected in lower levels of 3-nitrotyrosine staining in the SP-A−/− group. We conclude that despite increases in inflammatory cells, SP-A-deficient mice infected with P. carinii exhibit an enhanced susceptibility to the organism and attenuated production of proinflammatory cytokines and reactive oxygen-nitrogen species. These data support the concept that SP-A is a local effector molecule in the lung host defense against P. carinii in vivo.

Severe pruritus is a characteristic feature of atopic dermatitis (AD) and is closely related to its activity. Recent studies have shown that IL-31 is a key determinant of pruritus in AD. Anti-IL-31 receptor alpha (IL-31RA) antibody treatment has also been reported to improve pruritus clinically, subsequently contributing to the attenuation of AD disease activity. Therefore, IL-31 has been thought to be an important cytokine for regulating pruritus and AD disease activity; however, how IL-31 is involved in the immune response in AD has remained largely unknown. Epidermal Langerhans cells (LCs) and dermal dendritic cells (DCs) derived from bone marrow cells have been reported to play a critical role in AD pathogenesis. LCs and DCs produce Ccl 17 and Ccl 22, which chemoattract Th2 cells, leading to AD development. Therefore, we aimed to clarify how IL-31/IL-31RA interaction affects Ccl 17 and Ccl 22 production. To test this, we analyzed murine bone marrow-derived DCs (BMDCs) stimulated with IL-4, an important cytokine in AD development. We found that IL-31RA expression was upregulated by IL-4 stimulation in a dose-dependent manner in BMDCs. Furthermore, IL-31 upregulates Ccl 17 and Ccl 22 production in the presence of IL-4, whereas IL-31 stimulation alone did not produce Ccl 17 and Ccl 22. These findings suggest that IL-4 mediates IL-31RA expression and IL-31/IL-31RA interaction augments Ccl 17 and Ccl 22 production in BMDCs, which promotes Th2-deviated immune response in AD. Since we previously reported that soybean tar Glyteer, an aryl hydrocarbon receptor (AHR) ligand, impairs IL-4/Stat 6 signaling in BMDCs, we examined whether Glyteer affects IL-31RA expression induced by IL-4 stimulation. Glyteer inhibited upregulation of IL-31RA expression induced by IL-4 stimulation in a dose-dependent manner. Glyteer also inhibited Ccl 17 and Ccl 22 production induced by IL-4 and IL-31 stimulation. Taken together, these findings suggest that Glyteer treatment may improve AD disease activity by impairing IL-31/IL-31RA interaction in DCs.

ABSTRACT Mycobacterium tuberculosis is associated with the activation of cytokine circuits both at sites of active tuberculosis in vivo and in cultures of mononuclear cells stimulated by M. tuberculosis or its components in vitro. Interactive stimulatory and/or inhibitory pathways are established between cytokines, which may result in potentiation or attenuation of the effects of each molecule on T-cell responses. Here we examined the interaction of transforming growth factor β1 (TGF-β1) and interleukin-10 (IL-10) in purified protein derivative (PPD)-stimulated human mononuclear cell cultures in vitro. TGF-β1 induced monocyte IL-10 (but not tumor necrosis factor alpha) production (by 70-fold, P < 0.02) and mRNA expression in the absence but not in the presence of PPD. Both exogenous recombinant (r) IL-10 and rTGF-β1 independently suppressed the production of PPD-induced gamma interferon (IFN-γ) in mononuclear cells from PPD skin test-positive individuals. Synergistic suppression of IFN-γ in cultures containing both rTGF-β1 and rIL-10 was only seen when the responder cell population were peripheral blood mononuclear cells (PBMC) and not monocyte-depleted mononuclear cells and when PBMC were pretreated with rTGF-β1 but not with rIL-10. Suppression of PPD-induced IFN-γ in PBMC containing both rTGF-β1 (1 ng/ml) and rIL-10 (100 pg/ml) was 1.5-fold higher (P< 0.05) than cultures containing TGF-β1 alone and 5.7-fold higher (P < 0.004) than cultures containing IL-10 alone. Also, neutralization of endogenous TGF-β1 and IL-10 together enhanced PPD-induced IFN-γ in PBMC in a synergistic manner. Thus, TGF-β1 and IL-10 together potentiate the downmodulatory effect on M. tuberculosis-induced T-cell production of IFN-γ, and TGF-β1 alone enhances IL-10 production. At sites of active M. tuberculosis infection, these interactions may be conducive to the suppression of mononuclear cell functions.

ABSTRACT We present our measurements of the H α, [O iii], and [O ii] luminosity functions as part of the Lyman Alpha Galaxies at Epoch of Reionization (LAGER) survey using our samples of 1577 z = 0.47 H α-, 3933 z = 0.93 [O iii]-, and 5367 z = 1.59 [O ii]-selected emission line galaxies in a 3 deg2 single, CTIO/Blanco DECam pointing of the COSMOS field. Our observations reach 5σ depths of 8.2 × 10−18 erg s−1 cm−2 and comoving volumes of (1−7) × 105 Mpc3 making our survey one of the deepest narrow-band surveys. We select our emission line galaxies via spectroscopic confirmation, photometric redshifts, and colour–colour selections. We measure the observed luminosity functions for each sample and find best fits of $\phi ^\star = 10^{-3.16^{+0.09}_{-0.09}}$ Mpc−3 and $L^\star = 10^{41.72^{+0.09}_{-0.09}}$ erg s−1 for H α, $\phi ^\star = 10^{-2.16^{+0.10}_{-0.12}}$ Mpc−3 and $L^\star = 10^{41.38^{+0.07}_{-0.06}}$ erg s−1 for [O iii], and $\phi ^\star = 10^{-1.97^{+0.07}_{-0.07}}$ Mpc−3 and $L^\star = 10^{41.66^{+0.03}_{-0.03}}$ erg s−1 for [O ii], with α fixed to −1.75, −1.6, and −1.3, respectively. An excess of bright &gt;1042 erg s−1 [O iii] emitters is observed and may be due to active galactic nucleus (AGN) contamination. Corrections for dust attenuation are applied assuming AHα = 1 mag. We also design our own empirical rest-frame g − r calibration using SDSS DR12 data, test it against our z = 0.47 H α emitters with zCOSMOS 1D spectra, and calibrate it for (g − r) between −0.8 and 1.3 mag. Dust and AGN-corrected star formation rate densities (SFRDs) are measured as log10ρSFR/(M⊙ yr−1 Mpc−3) = −1.63 ± 0.04, −1.07 ± 0.06, and −0.90 ± 0.10 for H α, [O iii], and [O ii], respectively. We find our [O iii] and [O ii] samples fully trace cosmic star formation activity at their respective redshifts in comparison to multiwavelength SFRDs, while the H α sample traces ∼70 per cent of the total z = 0.47 SFRD.

Abstract Abstract 4982 Lenalidomide is an immunomodulatory agent that stimulates T-cell activation, enhances natural killer cells and inhibits cytokines, as tumor necrosis factor-alpha. It is FDA-approved for use in both transfusion-dependent myelodysplastic syndrome, especially 5q deletion syndrome and refractory and relapsed multiple myeloma. The known side effects are myelosuppression, diarrhea/constipation, fever, fatigue, rash and thromboembolism, while pulmonary complications are rarely reported. We present a patient with myelodysplastic syndrome on lenalidomide with acute pulmonary toxicity. A 63-year-old Caucasian man was diagnosed with low-intermediate risk myelodysplastic syndrome with 5q deletion after presenting with anemia and thrombocytopenia. He was started on lenalidomide at 21mg daily dose but after 5 days began to develop dyspnea on exertion, cough and fever. He was admitted for treatment of community acquired pneumonia. Physical examination revealed a middle aged man in respiratory distress. Oxygen saturation on room air was 86%. Chest examination revealed dry crackles. Heart sounds were regular without murmurs or gallops. Laboratory tests revealed hemoglobin of 8.6g/dl, normal white count at 10,000 and platelets of 7,000/uL. BNP and echocardiogram were normal and chest radiography was initially unremarkable. He was started on antibiotics but developed respiratory failure requiring endotracheal intubation and mechanical ventilation. CT scan of the chest showed diffuse interstitial reticulonodular opacities most prominent at the apices with scattered ground glass attenuation. Cultures from blood, sputum, urine and stool and brochoalveolar lavage did not reveal an infectious source. Broad spectrum antibiotics and antifungals were withheld. Clinical and radiologic improvement was noted after withdrawal of lenalidomide and initiation of steroids. He was persistently transfusion-dependent hence lenalidomide was reintroduced. Within 6 days of lenalidomide, respiratory symptoms recurred. He later improved after withdrawal of the offending drug. Our myelodysplastic patient developed acute respiratory failure after lenalidomide introduction which improved with steroids and drug withdrawal. He had symptom recurrence after reintroduction of lenalidomide. The exposure history, clinical and radiological examination, positive challenge test with exclusion of other etiologies support the diagnosis of lenalidomide-induced hypersensitivity pneumonitis. This is the third reported case of lenalidomide-induced hypersensitivity pneumonitis and it is likely that this entity is underdiagnosed due to concurrent steroid use especially in myeloma patients. Clinicians should be aware of this potential complication in patients who present with fever, hypoxemia and diffuse pulmonary infiltrates unresponsive to broad spectrum antibiotics as this disease is reversible if diagnosed early. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 3210 5-HMF (5-hydroxymethyl-2-furfural; Aes-103) is a breakdown product of glucose that has potent anti-sickling properties in vitro and in vivo (transgenic sickle cell mice) and produces concentration-dependent left shifts in p50 values of the oxygen equilibrium curve (OEC), indicating increased oxygen affinity. These pharmacological properties are likely based on the binding of 5-HMF to the valine residue of alpha globin chains of HbS and possibly to lysine. Increasing the oxygen affinity of HbS is known to reduce sickling. Aes-103 is being developed as a potential treatment for sickle cell disease (SCD). The current first in man study was a double-blind, placebo-controlled, ascending single dose evaluation of the safety, pharmacokinetics and pharmacodynamics of 5-HMF given as an oral solution at 300 mg, 1000 mg, 2000 mg and 4000 mg to healthy normal subjects. In each dosing cohort, 6 subjects received 5-HMF and 2 received placebo. A total of 20 adult subjects of African descent with normal hemoglobin (Hb), received a single dose of study drug or placebo on up to 2 occasions separated by 2–4 weeks. The mean age of the subjects was 28 years, mean BMI was 25.8, and 13 were males, 7 were females. Written informed consent was obtained. Safety measures consisted of adverse events (AEs), vital signs, ECG, clinical chemistry, hematology, urinalysis and physical exams. No subjects were discontinued from the study due to an AE. During the day of dosing a total of 14 AEs occurred. All were mild, with 3 occurring in the placebo arm (constipation, dry mouth, dizziness), none at 300 mg, 2 at 1000 mg (diarrhea, headache), 3 at 2000 mg (feeling hot, somnolence, dyspnea—during hypoxia testing) and 6 at 4000 mg (nausea (2), abdominal pain, dizziness, somnolence (2)). Following dosing, there were no clinically significant differences between drug and placebo treated subjects in respect to heart rate, blood pressure, ECG, physical exams, clinical chemistry, hematology and urinalysis results. The pharmacokinetic profile of 5-HMF in plasma showed dose-proportional kinetics with an upward trend towards higher Cmax and AUC at higher doses. Mean plasma Cmax concentrations ranged from 34 to 699 ng/ml, Tmax ranged from 0.42 to 0.67 h, AUC ranged from 33 to 875 ng.ml/hr and t1/2 ranged from 0.38–0.76 h. 5-HMF levels in RBC hemolysate were typically 5–15 times that of corresponding plasma levels with Cmax ranging from 152–3640 ng/ml, Tmax ranging from 0.71–0.83 h, AUC ranging from 473–10103 ng/ml/h and half-life ranging from 1.61–2.13 h. Measurements of RBC hemolysate levels did not include the 5-HMF bound to hemoglobin. The elevated levels and longer half-life of 5-HMF in RBCs relative to plasma probably reflects the binding affinity of Hb for 5-HMF and the equilibrium between Hb bound 5-HMF, free 5-HMF in the RBC and in plasma. The main pharmacodynamic endpoint was the change in blood oxygen level (SpO2) during a 5-minute hypoxia challenge test in which 12% O2 was inhaled. The hypoxia challenge was administered prior to dosing and then at 0.75, 2, 4 and 8 h post-dose. Results showed an appreciable attenuation of the drop in SpO2 values due to hypoxia. For example, at 2 hours post-dose for the placebo treated subjects the mean SpO2 values declined by an average of 12.3% after 5 minutes of hypoxia. In contrast, the 2000 mg 5-HMF treated subjects had a mean decline in SpO2 of 8.7%. The attenuation of hypoxia effects was dose-dependent (minimal effect at 300 mg of 5-HMF) and was time-dependent following 5-HMF dosing (largest protection seen at 2–4 h post-dose, no protection at 8 h post dose). At 2 hours after 5-HMF doses of 1000–4000 mg, the SpO2 values from 18 hypoxia test sessions showed significantly (p<0.05, t-test) smaller decrements than what occurred in the same time point in the pooled placebo treated subjects. In summary, single oral doses of 300–4000 mg of 5-HMF given to healthy normal volunteers were well tolerated, rapidly absorbed and preferentially taken up into RBCs relative to plasma, had a dose-proportional pharmacokinetic profile and showed a pharmacodynamic change (protection against desaturation during hypoxia) consistent with the expected increase in oxygen affinity and with the compound's proposed mechanism of action in SCD patients. A similar ascending single dose, placebo controlled, double-blind study in patients with SCD at steady state is currently ongoing at the NIH (see www.clinicaltrials.gov). Disclosures: Stern: AesRx, LLC: Employment, Equity Ownership.

The spin trap N-tert-butyl-alpha-phenylnitrone (PBN) has a high avidity for free radical species and hence functions as an antioxidant in many biological systems. As such, we hypothesized that PBN would have powerful antioxidant effects on muscle function. We examined the effects of PBN on directly stimulated in vitro (37 degrees C) rat diaphragm. First, a dose-response curve for the effects of PBN on force frequency (n = 8) was established by comparing PBN-treated muscle strips (0.01-10 mM) with time- and stimulus-matched control strips. Second, the effect of 1.0 mM PBN on muscle endurance (n = 8) was established. Our findings were as follows. 1) Compared with baseline, peak twitch and low-frequency muscle tensions increased in a dose-dependent fashion, with peak effects at 1.0 mM PBN. 2) Muscle function at all stimulation frequencies was depressed at doses above 1.0 mM PBN. 3) Complete inhibition at 10 mM PBN was reversed with caffeine administration or washout. 4) During early fatigue, 1.0 mM PBN facilitated force. However, endurance time decreased in the PBN-treated group. We conclude that PBN has direct reversible dose-dependent effects on diaphragm function. However, facilitation of low-frequency forces and the lack of fatigue-attenuating properties suggest that PBN has atypical antioxidant effects on muscle function.

Background In the current study, the potency and spread of the antinociception induced by MPV-2426, a novel alpha2-adrenoceptor agonist, was characterized in neuropathic and non-neuropathic animals. Methods Neuropathy was induced by unilateral ligation of two spinal nerves in the rat. After lumbar intrathecal or systemic administration of MPV-2426, thermally and mechanically evoked responses of nociceptive neurons of the rostroventromedial medulla were recorded during pentobarbitone anesthesia. To obtain a behavioral correlate of neurophysiologic findings, nocifensor reflex responses evoked by thermal and mechanical stimuli were assessed in unanesthetized neuropathic and control animals. Results After intrathecal administration, MPV-2426 and dexmedetomidine produced a dose-related antinociceptive effect, independent of the submodality of the noxious test stimulus or the pathophysiologic condition. This antinociceptive effect was spatially restricted to the inputs from the lower half of the body, and it was reversed by atipamezole, an alpha2-adrenoceptor antagonist. After systemic administration in non-neuropathic animals, MPV-2426 had no antinociceptive effect on responses to rostroventromedial medulla neurons, whereas systemically administered dexmedetomidine produced a dose-related suppression of nociceptive signals to the rostroventromedial medulla, independent of the site of test stimulation. In a behavioral study, intrathecal MPV-2426 produced a dose-dependent suppression of nocifensor responses evoked by noxious mechanical or heat stimuli, whereas systemic administration of MPV-2426 had no effects. Conclusions Intrathecal MPV-2426 has spatially limited antinociceptive properties in neuropathic and non-neuropathic conditions because of its action on spinal alpha2-adrenoceptors. These properties may be advantageous when designing therapy for spatially restricted pain problems.

Abstract Administration of agonistic monoclonal antibodies or recombinant cytokines is a potential approach to enhance antitumor immunity in bone marrow (BM) transplant recipients, but is complicated by toxicity due to proinflammatory cytokine-mediated vital organ damage. We used a murine syngeneic bone marrow transplant (BMT) model, in which administration of anti-CD40 antibody early after BMT results in overproduction of interleukin-12 (IL-12) and interferon-γ (IFN-γ), and lethal gut toxicity to examine the protective effect of the spin trap inhibitor, alpha phenyl-tert-butyl nitrone (PBN). Administration of PBN protected transplant recipients from mortality by significantly attenuating gut toxicity, but did not effect a reduction in the levels of proinflammatory cytokines (IL-12, IFN-γ, tumor necrosis factor α [TNF-α], or nitrate/nitrite). Moreover, PBN did not compromise anti-CD40 antibody-mediated antitumor effects in a nontransplantation lymphoma model. Collectively, these data suggest that PBN administration may represent a novel approach for reduction of toxicity without compromise of antitumor effects resulting from administration of therapeutic antibodies in both transplantation and nontransplantation settings. (Blood. 2005;105:428-431)

Ferroptosis and inflammation induced by cerebral hemorrhage result in an excessive inflammatory response and irreversible neuronal injury. Alleviating ferroptosis might be an effective way to prevent neuroinflammatory injury and promote neural functional recovery. Pyridoxal isonicotinoyl hydrazine (PIH), a lipophilic iron-chelating agent, has been reported to reduce excess iron-induced cytotoxicity. However, whether PIH could ameliorate the effects of hemorrhagic stroke is not completely understood. In the present study, the preventive effects of PIH in an intracerebral hemorrhage (ICH) mouse model were investigated. Neurological score, rotarod test, and immunofluorescence around the hematoma were assessed to evaluate the effects of PIH on hemorrhagic injury. The involvement of ferroptosis and inflammation was also examined in vitro to explore the underlying mechanism. Results showed that administration of PIH prevented neuronal cell death and reduced lipid peroxidation in Erastin-treated PC-12 cells. In vivo, mice treated with PIH after ICH attenuated neurological deficit scores. Additionally, we found PIH reduced ROS production, iron accumulation, and lipid peroxidation around the hematoma peripheral tissue. Meanwhile, ICH mice treated with PIH showed an upregulation of the key ferroptosis enzyme, glutathione peroxidase 4, and downregulation of cyclooxygenase-2. Moreover, PIH administration inhibited proinflammatory polarization and reduced interleukin-1 beta and tumor necrosis factor alpha in ICH mice. Collectively, these results demonstrated that PIH protects mice against hemorrhage stroke, which was associated with mitigation of inflammation and ferroptosis.

In response to high-altitude long-term hypoxemia, the cerebral arteries of fetal and adult sheep show decreased contractile responses to norepinephrine (NE) and other agonists. To test the hypothesis that hypoxia-induced developmental and vessel specific cerebral artery contractility changes are mediated, in part, by changes in alpha1-adrenergic receptor (alpha1-AR) density and/or NE-induced inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] responses, we performed the following study. In common carotid (Com) and main branch cerebral (MBC) arteries from normoxic adult ewes and near-term fetuses and those acclimatized to high altitude (3,820 m), we quantified alpha1-AR density (maximal binding in fmol/mg protein) and affinity (dissociation constant in nM) with the alpha1-AR antagonist [3H]prazosin. In addition, we quantified NE-induced Ins(1,4,5)P3 responses in these arteries. With long-term hypoxemia, alpha1-AR density in fetal and adult Com decreased 75% (from 113 +/- 18 to 28 +/- 5 fmol/mg protein) and 66% (from 54 +/- 3 to 18 +/- 4 fmol/mg protein), respectively, from normoxic control values. alpha1-AR density of the fetal and adult MBC decreased 76% (from 47 +/- 4 to 11 +/- 1 fmol/mg protein) and 61% (from 23 +/- 3 to 9 +/- 3 fmol/mg protein), respectively, from controls. In hypoxemic adult Com, the NE-induced Ins(1,4,5)P3 response decreased 51% (from 309 +/- 38 to 151 +/- 24%) from the control value. In fetal and adult MBC, long-term hypoxemia was associated with decreases of 35% (from 345 +/- 40 to 225 +/- 30%) and 44% (from 355 +/- 55 to 199 +/- 16%), respectively, from control values. We conclude that in the adult Com and MBC vessels, acclimatization to high-altitude, long-term hypoxemia was associated with significant decreases in both alpha1-AR density values and Ins(1,4,5)P3 responses to NE. Similarly, in the fetal MBC arteries, high-altitude hypoxemia was associated with marked attenuation of both alpha1-AR density and NE-induced Ins(1,4,5)P3 responses. The magnitude of decreases in NE-induced Ins(1,4,5)P3 responses in these vessels correlated fairly well with the decreases in alpha1-AR density. These findings suggest that changes in noradrenergic receptor-second messenger coupling may play a role in altered cerebrovascular tone in association with high-altitude acclimatization and other forms of long-term hypoxia in both fetus and adult.

Recent evidence suggests that postischemic myocardial dysfunction ("stunning") is mediated by iron-catalyzed free radical reactions, but the exact time window during which the critical iron-mediated damage develops remains unknown. Furthermore, the evidence that iron promotes free radical reactions in vivo is indirect. Thus open-chest dogs undergoing a 15-min coronary occlusion and 4 h of reperfusion were given one of the following intracoronary infusions: desferrioxamine (DF) beginning 2 min before reperfusion (group I), DF beginning 1 min after reperfusion (group II), iron-loaded DF in dosage identical to group I (group III), or vehicle (controls, group IV). Recovery of contractile function was substantially greater in group I than in controls, whereas in groups II and III it was indistinguishable from controls. To determine whether the protection afforded by DF was due to inhibition of free radical reactions, myocardial production of free radicals was directly assessed by intracoronary infusion of the spin trap alpha-phenyl N-tert-butyl nitrone (PBN). In controls (group VI), radical adducts of PBN were released in the coronary venous blood after reperfusion. DF given as in group I (group V) markedly suppressed myocardial production of PBN adducts. These results strongly suggest that a substantial portion of the damage responsible for myocardial stunning is caused by iron-catalyzed free radical reactions that develop in the initial seconds of reperfusion and can be prevented by administration of iron chelators started just before reflow. Furthermore, the results demonstrate that attenuation of postischemic dysfunction by DF is associated with attenuation of free radical reactions in vivo, thereby providing direct evidence for a pathogenetic role of iron-catalyzed free radical reactions in myocardial stunning in the intact animal.

Background and objectives: Ascorbic acid, alpha lipoic acid (ALA) and silymarin are well-known antioxidants that have hepatoprotective effects. This study aims to investigate the effects of these three compounds combined with attenuating drug-induced oxidative stress and cellular damage, taking acetaminophen (APAP)-induced toxicity in rats as a model both in vivo and in vitro. Materials and Methods: Freshly cultured primary rat hepatocytes were treated with ascorbic acid, ALA, silymarin and their combination, both with and without the addition of APAP to evaluate their in vitro impact on cell proliferation and mitochondrial activity. In vivo study was performed on rats supplemented with the test compounds or their combination for one week followed by two toxic doses of APAP. Results: Selected liver function tests and oxidative stress markers including superoxide dismutase (SOD), malondialdehyde (MDA) and oxidized glutathione (GSSG) were detected. The in vivo results showed that all three pretreatment compounds and their combination prevented elevation of SOD and GSSG serum levels indicating a diminished burden of oxidative stress. Moreover, ascorbic acid, ALA and silymarin in combination reduced serum levels of liver enzymes; however, silymarin markedly maintained levels of all parameters to normal ranges. Silymarin either alone or combined with ascorbic acid and ALA protected cultured rat hepatocytes and increased cellular metabolic activity. The subjected agents were capable of significantly inhibiting the presence of oxidative stress induced by APAP toxicity and the best result for protection was seen with the use of silymarin. Conclusions: The measured liver function tests may suggest an augmented hepatoprotection of the combination preparation than when compared individually.

Objective. The present study aims at evaluating the beneficial effect of Nigella sativa (NS) oil mouth rinse in the management of chemotherapy- (CT-) induced oral mucositis (OM) in patients with acute myeloid leukemia (AML). Methods. Fifty-four AML patients were participated in this study and randomly allocated to either the test group or a control group. The patients of the test group received NS oil mouth rinse during 28-day CT, while the participants of the control group received a “magic mouthwash” formula. The primary outcome of this study was the incidence and severity of CT-induced OM in terms of erythema and ulcer. The secondary outcomes were the pain severity score, swallowing function, and the salivary concentrations of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α). Results. NS oil mouth rinse attenuated the progression of CT-induced OM compared with the control formula (AUC = 5.9 vs. 38.4, P<0.05) and significantly decreased the erythema and ulceration scores (AUC of total OMAS = 11.4 vs. 85.9, P<0.001) compared with the magic mouthwash formula. It also reduced the pain score and enabled all the participants of this group to consume normal food during treatment. It significantly decreased salivary IL-6 (AUC = 7376 vs. 16599, P<0.001), while the changes of TNF-α levels were not significant (AUC = 676.9 vs. 885.2, P>0.05). Conclusions. NS oil mouth rinse is effective in attenuating the severity of CT-induced OM and improves the pain and swallowing function in AML patients.

Doxorubicin (DOX) is a broad-spectrum antitumor drug while its use is limited due to its neurobiological side effects associated with depression. We investigated the neuroprotective efficacy of dl-3-n-butylphthalide (dl-NBP) against DOX-induced anxiety- and depression-like behaviors in rats. dl-NBP was given (30 mg/kg) daily by gavage over three weeks starting seven days before DOX administration. Elevated plus maze (EPM) test, forced swimming test (FST), and sucrose preference test (SPT) were performed to assess anxiety- and depression-like behaviors. Our study showed that the supplementation of dl-NBP significantly mitigated the behavioral changes induced by DOX. To further explore the mechanism of neuroprotection induced by dl-NBP, several biomarkers including oxidative stress markers, endoplasmic reticulum (ER) stress markers, and neuroinflammatory cytokines in the hippocampus were quantified. The results showed that dl-NBP treatment alleviated DOX-induced neural apoptosis. Meanwhile, DOX-induced oxidative stress and ER stress in the hippocampus were significantly ameliorated in dl-NBP pretreatment group. Our study found that dl-NBP alleviated the upregulation of malondialdehyde (MDA), nitric oxide (NO), CHOP, glucose-regulated protein 78 kD (GRP-78), and caspase-12 and increased the levels of reduced glutathione (GSH) and activities of catalase (CAT), glutathione reductase (GR), and glutathione peroxidase (GPx) in the hippocampus of rats exposed to DOX. Additionally, the gene expression of interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-alpha (TNF-α) and protein levels of inducible nitric oxide synthase (iNOS) were significantly increased in DOX-treated group, whereas DOX-induced neuroinflammation was significantly attenuated in dl-NBP supplementation group. In conclusion, dl-NBP could alleviate DOX-induced anxiety- and depression-like behaviors via attenuating oxidative stress, ER stress, inflammatory reaction, and neural apoptosis, providing a basis as a therapeutic potential against DOX-induced neurotoxicity.

Abstract Very premature infants are at risk of bleeding complications and are frequently given plasma because of a perception that coagulation test results are abnormal. However, “abnormal” clotting times may simply be due to physiological immaturity. We hypothesized that prospective characterization of coagulation tests alongside assessment of thrombin generation would provide information on overall haemostatic balance in this vulnerable patient cohort. In a prospective observational study, blood was drawn into citrated tubes from cord blood of neonates <30/40 and subsequently on day 1, 3 and fortnightly until 30/40 corrected gestational age from non-heparinised lines. Exclusion criteria included antenatal intraventricular haemorrhage and lack of informed consent. Platelet poor plasma was obtained by centrifugation of whole blood. Prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen, procoagulant and anticoagulant factor activity were measured and tissue factor (TF)-stimulated thrombin generation was characterized in a calibrated automated thrombography assay with a 1pM TF stimulus using Thrombinoscope™ software. Control plasma was obtained from cord blood of term neonates. Between April 2013 and June 2014, 92 patients <30/40 were admitted. Six infants were excluded and 86 recruited. Mean gestational age and birth weight were 27 (23.7-29.9) weeks and 1019 (530-1590) g respectively. Median (5th-95thpercentile) Day 1 PT, APTT, and fibrinogen were 17.5 (12.9-26.7) s, 82.9 (53.4-139.8) s and 1.4 (0.7-3.7) g/L respectively which were significantly prolonged compared with published results of term infants; p<0.001. Serial analysis revealed correction of PT, APTT and fibrinogen with increasing postnatal age; p<0.001. Further analysis of procoagulant and anticoagulant mechanisms was performed in a subset of infants (N=16) where sufficient plasma was available from umbilical cord samples. Despite apparent “prolongations” in clotting times, we found that preterm peak thrombin generated was similar to term infants (94.3 (33.4-149.8) nM vs. 93.5 (74.3-133.8) nM; p=0.94). Minor, non-significant differences in the area under the thombin generation curve (endogenous thrombin potential) were observed (88 (37.1-126.3) nm/min vs. 101.3 (76.8-139.7) nm/min; p=0.09). Alpha-2-macroglobulin (α2M)-bound thrombin cleaves the fluorogenic thrombin substrate utilized in this assay and contributes to measured thrombin generation. However, in keeping with published data in a more mature cohort, α2M activity was lower in preterm vs. term infants. Consequently, we speculate that true ETP in preterm infants may be higher, undermining the current perception that these infants are at risk of bleeding due to “abnormal” clotting times. In keeping with published data in a more mature cohort, mean activity of procoagulant factors II, VII, IX and X were reduced in preterm vs. term infants (0.33 (0.18-0.5) IU/ml vs. 0.45 (0.35-0.6) IU/ml, p=0.003; 0.38 (0.14-0.57) IU/ml vs. 0.42 (0.31-0.59) IU/ml, p=0.29; 0.19 (0.09-0.50) IU/ml vs. 0.28 (0.19-0.37) IU/ml, p=0.004 and 0.33 (0.13-0.52) IU/ml vs. 0.43 (0.32-0.58) IU/ml, p=0.02) respectively. Conversely, activity of anticoagulant factors Protein C (0.14U/ml (0.06-0.24) IU/ml vs. 0.27 (0.18-0.39) IU/ml; p=0.002), free protein S (0.39 (0.28-0.55) IU/ml vs. 0.47 (0.36-0.59) IU/ml; p=0.02), antithrombin (0.22 (0.06-0.36) IU/ml vs. 0.52 (0.38-0.69) IU/ml; p=0.0001) and tissue factor pathway inhibitor (7.7 (2.6-16.9) vs. 10 (4.2-15.6) ng/ml; p=0.14) was lower in preterm vs. term infants. No differences in attenuation in thrombin generation in response to the anticoagulant activated protein C were observed in preterm vs. term infants, p=0.92. In conclusion, in the largest prospective study to date of very preterm infants, we describe typical ranges for widely available coagulation tests. We also demonstrate differences in both procoagulant and anticoagulant pathways, to which standard clotting tests may have limited sensitivity. Finally, we show for first time that thrombin generation is similar in very preterm and term infants. These findings call into question the current practice of exposing infants to plasma products based on standard laboratory parameters, a practice which is associated with enormous potential harm and is not evidence based. These findings have the potential to impact on neonatal practice worldwide. Disclosures Ní Áinle: LEO Pharma: Research Funding; Bayer Healthcare Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees.

Abstract Anxiety leads to a global decline in quality of life and increase in social burden. However, treatments are limited, because the molecular mechanisms underlying complex emotional disorders are poorly understood. We explored the anxiolytic effects of 8-O-acetyl shanzhiside methylester (8-OaS), an active component in Lamiophlomis rotata (L. rotata; Benth.) or Kudo, a traditional herb that has been shown to be effective in the clinical treatment of chronic pain syndromes in China. Two mouse anxiety models were used: forced swimming stress (FSS)–induced anxiety and complete Freund’s adjuvant (CFA)–induced chronic inflammatory pain. All animal behaviors were analyzed on the elevated plus maze and in the open-field test. 8-OaS significantly ameliorated anxiety-like behaviors in both anxiety models and inhibited the translation enhancement of GluN2A, GluN2B, and PSD95. Moreover, a reduction in GABA receptors disrupted the excitatory/inhibitory (E/I) balance in the basolateral amygdala (BLA), indicated by increased excitatory and decreased inhibitory presynaptic release. 8-OaS also blocked microglia activation and reduced the phosphorylation of p38, c-Jun N-terminal kinase (JNK), NF-κB p65, and tumor necrosis factor alpha (TNF-α) in the BLA of anxiety mice. 8-OaS exhibits obvious anxiolytic effects by regulating the excitatory/inhibitory (E/I) synaptic transmission and attenuating inflammatory responses in the BLA.

The aim of this study is to assess whether overexpressing neuregulin 4 (Nrg4), a growth factor known to attenuate hepatic lipogenesis, in mesenchymal stem cells (MSCs) could enhance their ability to ameliorate insulin resistance (IR) and improve lipid metabolism in high-fat diet (HFD)-fed mice. Six-week-old C57BL/6 mice were fed a HFD for 12 weeks and then were given intravenous transplantation of adipose tissue-derived MSCs (ADSCs) or ADSCs overexpressing Nrg4 (Nrg4-ADSCs). Assessment of body weight and blood glucose and insulin levels as well as glucose tolerance test and insulin tolerance test was performed four and eight weeks after cell injection. Triglyceride (TG) and total cholesterol (TC) levels in the plasma and liver were also measured. The mRNA levels of glucose transporter 4 (GLUT4), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) in muscle and adipose tissues were assessed by Real-time Polymerase Chain Reaction (RT-PCR) analysis. Expression of genes related to lipid metabolism, including sterol regulatory element binding protein-1c (SREBP-1c) and fatty acid synthase, was evaluated at the mRNA and protein levels by RT-PCR and western blotting, respectively. The HFD-fed mice receiving ADSCs or Nrg4-ADSCs showed reduced blood glucose levels and enhanced insulin sensitivity, with the Nrg4-ADSC group exhibiting increased improvement in these aspects. HFD-induced changes in the expression of GLUT4 and IL-6 and TNF-α in skeletal muscle and adipose tissues were partially reversed by ADSC or Nrg4-ADSC infusion; however, no difference was observed between these two groups. Nrg4-ADSC-treated mice showed less fat cell deposition and lower TG and TC levels in the serum and liver with decreased expression of SREBP-1c and fatty acid synthase compared with the ADSC group. ADSC transplantation can reduce blood glucose level and ameliorate IR induced by HFD. The protective effects of ADSC can be attributed to suppression of inflammation and augmentation of glucose uptake in skeletal muscle and adipose tissues. More importantly, Nrg4 overexpression in ADSCs could strengthen this efficacy by attenuating hepatic lipogenesis. Impact statement Due to high-fat and high-sugar diets accompanied by sedentary lifestyles, diabetes has become a global epidemic. Literature findings suggest a potential therapeutic effect of Nrg4 on treating obesity-related metabolic disorders including type 2 diabetes (T2D). Adipose tissue-derived MSCs (ADSCs) were used in our study as they are abundant and can be harvested with minimally invasive procedures. In the end, our study reveals that ADSC transplantation improves glucose tolerance and metabolic balance in HFD-fed mice by multiple mechanisms, including upregulating GLUT4 expression and suppressing inflammation. More importantly, our study shows that Nrg4 overexpression could improve the efficacy of ADSCs in ameliorating insulin resistance (IR) and other obesity-related metabolic disorders, given the function of Nrg4 in attenuating hepatic lipogenesis. It would provide a new therapeutic strategy for the treatment of obesity, IR, and T2D.

Introduction: Benign Prostatic Hyperplasia (BPH) is the most common health problem of male elderly population resulting in lower urinary tract symptoms (LUTS), characterized by frequency, urgency, hesitancy, nocturia, dysuria and incomplete voiding. Aims & Objectives: To compare the different doses of petroleum ether extracts of Sphaeranthus indicus Linn (SiP) against Dutasteride, on hormonal parameters (serum testosterone and PSA) in testosterone induced BPH in albino mice. Place and Duration of study: This experimental study was conducted at the Animal House of PGMI, Lahore for 6 months. Material & Methods: Thirty-six healthy adult male mice, divided into 6 groups, were administered testosterone, different doses of petroleum ether extract SiL extracts (SIP) and dutasteride. Animals in group 1, taken as control, were given 0.1ml corn oil daily subcutaneously for 28 days. The animals in the remaining 5 groups (# 2-6) were given 3 mg/kg body weight testosterone dissolved in corn oil subcutaneously for 28 days, to induce BPH. The animals in group 3 were given 20 mg/kg/day Dutasteride orally for 28 days. Animals in groups 4-6 were given 25,50mg and 75mg/ kg body weight petroleum ether extract (SIP) orally for 28 days. Blood samples were drawn on day 0, 14 and 28 for the estimation of serum testosterone and serum PSA, by ELISA technique. Data was analyzed using SPSS Version 20.0. Comparison (between groups at baseline, day14 and day 28)was done by using one-way ANOVA and Post Hoc Tukey’s test Results: Decrease in PSA level and increase in serum testosterone supports the alpha reductase inhibiting activity of SiP, in a dose dependent manner, in testosterone induced BPH. SI petroleum ether extract 75mg/kg exhibited efficacy similar to that of dutasteride. Conclusion: Sphaeranthus indicus Linn (SiP) has shown efficacy equal to that of dutasteride in attenuating the testosterone induced BPH, in albino mice.

Background: Tumor necrosis factor alpha (TNFα) is a cytokine that may mediate inflammatory histopathology of the dorsal root ganglion following lumbar disc herniation.1 Soluble TNF receptor II (sTNFRII) competitively binds TNFa with clinical value for painful radiculopathy.2 Bioactive peptides expressed with elastin-like polypeptides (ELP) fusion partners gain a thermally responsive domain, by which they can undergo hydrophobic collapse and separate from solution to aggregate at physiological temperatures.3 Protein release from such a depot may locally sustain drug presence, an effect demonstrated for non-fusion ELP after intra-articular injection.4 Methods: We expressed sTNFRII fused to ELP to demonstrate potential bidomain functionality. Protein Expression. A gene encoding ELP-(VPGVG)60 was subcloned adjacent to the sTNFRII and transformed into E.coli for expression.5 Protein Safety. Endotoxin content of purified fusion protein was evaluated using a limulus amebocyte lysate endpoint assay and compared to non-fusion ELP using a two-tailed Student’s t-test. Thermal Responsiveness. Dynamic light scattering evaluated the inverse thermal phase transition behaviour of ELP-sTNFRII, and absorbance spectrophotometry quantified the in vitro depot release at 37°C. Fusion Domain Function. Anti-TNFα bioactivity was assessed by the in vitro inhibition of TNFα-induced glutamate production by microglia. Single-factor ANOVA analyzed treatment differences for ELP-sTNFRII, commercial sTNFRII (positive control), and non-fusion ELP (negative control). A 44 kDa recombinant fusion protein was expressed from E. coli and purified by inverse transition cycling. Results: Measured endotoxin content for ELP-sTNFRII was comparable to ELP alone (p < 0.01), well below FDA levels for biomedical implants. The fusion protein underwent a thermally-induced phase transition and formed observable aggregates of ~240 nm upon heating to physiological temperatures (Tt = 32°C). Slow release was observed from this depot with a time constant of 21 ± 3 hours. The fusion protein demonstrated anti-TNFα activity in vitro by attenuating TNFα-induced microglial glutamate production, albeit requiring a greater concentration than the free antagonist to achieve the same effect.(p < 0.01). Conclusion: Fusion of a sTNFRII protein to an ELP can serve to generate a thermally-induced drug depot that may sustain anti-cytokine activity of agents delivered locally to a nerve region. Further directions may involve studying in vivo biodistribution after perineural delivery of ELP and in vivo disease modifying activity of this agent.

Abstract Objectives Neuroinflammation and oxidative stress (OS) are implicated as mediators of cognitive decline and neurodegenerative diseases. Microglia, the primary immune cells of the central nervous system (CNS), are key regulators of inflammation and OS in the CNS. Blueberries (BB) are rich in anti-inflammatory and antioxidant polyphenols and reduce inflammation and OS when administered to microglia before pathogenic stimuli such as lipopolysaccharide (LPS). However, the therapeutic value of BBs administered to microglial cells after activation by LPS on inflammation and OS has not been examined. This study investigated the potential differential effects of pre-, post-, and pre-/post-BB on LPS-induced inflammation and OS in rat microglia. Methods Rat microglia were pre-treated with freeze-dried BB diluted in media (0.5 mg/mL) or control media (C) for 24 h, incubated overnight with LPS (0 or 100 ng/mL), and post-treated with BB or C for 24 h. Standard immunochemical techniques were used to measure biomarkers of inflammation and OS, including nitrite, tumor necrosis factor alpha (TNFɑ), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2). Data were analyzed using two-way ANOVAs with treatment group (pre-/post-BB) and LPS exposure as experimental factors. Post-hoc testing was performed using Tukey's test to determine differences among groups. Results Preliminary results showed that all BB treatments (pre-, post- and pre-/post-BB) reduced LPS-induced nitrite and TNFɑ relative to no BB (P &lt; 0.01). Pre-BB was more effective at reducing nitrite than post-BB (P &lt; 0.01), although neither was significantly different than pre-/post-BB. In contrast, the attenuating effects of pre-BB and post-BB on TNFɑ were not different from each other, although both treatments were more beneficial than pre-/post-BB (P &lt; 0.001). Compared to no BB, both pre-BB and post-BB, although not pre-/post-BB reduced LPS-induced iNOS expression (P &lt; 0.05), and only pre-BB attenuated COX2 expression (P &lt; 0.01). Conclusions Results suggest that BBs can target the downstream effects of microglial activation in addition to preventing stressor-induced neuroinflammation and OS. Therefore, although potentially more effective, BBs may not need to be present prior to microglial activation for beneficial effects. Funding Sources Supported by USDA intramural funding.

Video gaming, specifically action video gaming, seems to improve a range of cognitive functions. The basis for these improvements may be attentional control in conjunction with reward-related learning to amplify the execution of goal-relevant actions while suppressing goal-irrelevant actions. Given that EEG alpha power reflects inhibitory processing, a core component of attentional control, it might represent the electrophysiological substrate of cognitive improvement in video gaming. The aim of this study was to test whether non-video gamers (NVGs), non-action video gamers (NAVGs) and action video gamers (AVGs) exhibit differences in EEG alpha power, and whether this might account for differences in visual information processing as operationalized by the theory of visual attention (TVA). Forty male volunteers performed a visual short-term memory paradigm where they memorized shape stimuli depicted on circular stimulus displays at six different exposure durations while their EEGs were recorded. Accuracy data was analyzed using TVA-algorithms. There was a positive correlation between the extent of post-stimulus EEG alpha power attenuation (10–12 Hz) and speed of information processing across all participants. Moreover, both EEG alpha power attenuation and speed of information processing were modulated by an interaction between group affiliation and time on task, indicating that video gamers showed larger EEG alpha power attenuations and faster information processing over time than NVGs – with AVGs displaying the largest increase. An additional regression analysis affirmed this observation. From this we concluded that EEG alpha power might be a promising neural substrate for explaining cognitive improvement in video gaming.

ABSTRACT Human papillomaviruses (HPVs) are small, double-stranded DNA viruses that are significant risk factors in the development of cancer, and HPV accounts for approximately 5% of all worldwide cancers. Recent studies using data from The Cancer Genome Atlas (TCGA) have demonstrated that elevated levels of estrogen receptor alpha (ERα) are associated with improved survival in oropharyngeal cancers, and these elevated receptor levels were linked with human papillomavirus-positive cancers (HPV+cancers). There has been a dramatic increase in HPV-related head and neck squamous cell carcinomas (HPV+HNSCCs) over the last 2 decades, and therapeutic options for this ongoing health crisis are a priority; currently, there are no antiviral therapeutics available for combatting HPV+cancers. During our TGCA studies on head and neck cancer, we had also discovered the overexpression of ERα in HPV+cancers. Here, we demonstrate that 17β-estradiol (estrogen) attenuates the growth/cell viability of HPV+cancers in vitro, but not HPV-negative cancer cells. In addition, N/Tert-1 cells (foreskin keratinocytes immortalized with human telomerase reverse transcriptase [hTERT]) containing human papillomavirus 16 (HPV16) have elevated levels of ERα and growth sensitivity after estrogen treatment compared with parental N/Tert-1 cells. Finally, we demonstrate that there are potentially two mechanisms contributing to the attenuation of HPV+ cell growth following estrogen treatment. First, estrogen represses the viral transcriptional long control region (LCR) downregulating early gene expression, including E6/E7. Second, expression of E6 and E7 by themselves sensitizes cells to estrogen. Overall, our results support the recent proposal that estrogen could be exploited therapeutically for the treatment of HPV-positive oral cancers. IMPORTANCE Human papillomaviruses cause around 5% of all human cancers, yet there are no specific antiviral therapeutic approaches available for combatting these cancers. These cancers are currently treated with standard chemoradiation therapy (CRT). Specific antiviral reagents are desperately required, particularly for HPV+HNSCC whose incidence is increasing and for which there are no diagnostic tools available for combatting this disease. Using data from The Cancer Genome Atlas (TCGA), we and others determined that the estrogen receptor alpha (ERα) is overexpressed in HPV+HNSCC and that elevated levels are associated with an improved disease outcome. This has led to the proposal that estrogen treatment could be a novel therapeutic approach for combatting HPV+cancers. Here, we demonstrate that estrogen attenuates the growth of HPV+epithelial cells using multiple mechanisms, supporting the idea that estrogen has potential as a therapeutic agent for the treatment of HPV+HNSCC.

The nitric oxide-cGMP-dependent protein kinase pathway is a central regulator of cardiovascular physiology. Protein Kinase G I (PKGI), a principle mediator of this pathway, has been implicated as a negative regulator of cardiac hypertrophy, but the specific mechanisms involved are unknown. To test the hypothesis that PKGIα regulates cardiac hypertrophy, we characterized the cardiac phenotype of mice homozygous for a mutant form of PKGIα in which critical amino acids in the N-terminal leucine zipper (LZ) motif have been substituted to disrupt PKGIα LZ binding to specific downstream effector proteins. In the unstressed state, male PKG Iα leucine zipper mutant ( LZM) mice develop progressive LV hypertrophy compared with wild type (WT) littermates with LV mass/tibia length 12.3% greater at 30 weeks of age (p=0.05), and 27% greater at 60 weeks (p=0.001). Compared with WT littermates, the hearts of 30 week old PKGIα mutants are hypercontractile with decreased end systolic diameter (p=0.02) and increased fractional shortening on echocardiography (p=0.03). Invasive hemodynamics demonstrate that LZM mice also have increased LV systolic pressure (p=0.04), developed pressure (p=0.05), and LV dP/dt max (p=0.13). To evaluate the response to hemodynamic stress, cardiac hypertrophy was induced by transaortic constriction (TAC) in male WT and LZM mice. TAC resulted in early mortality in the LZM mice (60%) compared to the WT (19%) mice at 21 days post procedure (p=0.008), with evidence of accelerated LV hypertrophy in the mice that died early (LV mass/tibia length 9.8 mg/mm in the early LZM deaths vs 7.2 mg/mm in WT survivors, p<0.001). Additionally, the LZM early deaths had evidence of congestive heart failure with increased RV mass (RV mass/ tibia length 1.8 mg/mm in LZM early deaths vs 1.2 mg/mm in WT survivors, p<0.05), and increased lung mass (23.1 mg/mm LZM early deaths vs 12.5 mg/mm in WT survivors, p<0.005). These findings support that the N-terminal LZ domain of PKGIα is required for suppression of cardiac hypertrophy. The early mortality following TAC in the LZM mice also suggests a critical role for PKGIα in attenuating pathologic cardiac remodeling, identifying PKGIα as an attractive candidate drug target for prevention of cardiac hypertrophy and failure.

Previous studies demonstrate that Mycobacterium vaccae NCTC 11659 (M. vaccae), a soil-derived bacterium with anti-inflammatory and immunoregulatory properties, is a potentially useful countermeasure against negative outcomes to stressors. Here we used male C57BL/6NCrl mice to determine if repeated immunization with M. vaccae is an effective countermeasure in a “two hit” stress exposure model of chronic disruption of rhythms (CDR) followed by acute social defeat (SD). On day –28, mice received implants of biotelemetric recording devices to monitor 24-h rhythms of locomotor activity. Mice were subsequently treated with a heat-killed preparation of M. vaccae (0.1 mg, administered subcutaneously on days –21, –14, –7, and 27) or borate-buffered saline vehicle. Mice were then exposed to 8 consecutive weeks of either stable normal 12:12 h light:dark (LD) conditions or CDR, consisting of 12-h reversals of the LD cycle every 7 days (days 0–56). Finally, mice were exposed to either a 10-min SD or a home cage control condition on day 54. All mice were exposed to object location memory testing 24 h following SD. The gut microbiome and metabolome were assessed in fecal samples collected on days –1, 48, and 62 using 16S rRNA gene sequence and LC-MS/MS spectral data, respectively; the plasma metabolome was additionally measured on day 64. Among mice exposed to normal LD conditions, immunization with M. vaccae induced a shift toward a more proactive behavioral coping response to SD as measured by increases in scouting and avoiding an approaching male CD-1 aggressor, and decreases in submissive upright defensive postures. In the object location memory test, exposure to SD increased cognitive function in CDR mice previously immunized with M. vaccae. Immunization with M. vaccae stabilized the gut microbiome, attenuating CDR-induced reductions in alpha diversity and decreasing within-group measures of beta diversity. Immunization with M. vaccae also increased the relative abundance of 1-heptadecanoyl-sn-glycero-3-phosphocholine, a lysophospholipid, in plasma. Together, these data support the hypothesis that immunization with M. vaccae stabilizes the gut microbiome, induces a shift toward a more proactive response to stress exposure, and promotes stress resilience.

Background: Reperfusion following ischemia can lead to more injuries than ischemia itself especially in diabetic patients. The aim of this study was to evaluate the effect of dexmedetomidine on ischemia-reperfusion injury (IRI) in rats with have hepatic IRI and diabetes mellitus. Methodology: Twenty-eight Wistar Albino rats were randomised into four groups as control (C), diabetic (DC), diabetic with hepatic ischemia-reperfusion injury (DIR), and diabetic but administered dexmedetomidine followed by hepatic IRI (DIRD) groups. Hepatic tissue samples were evaluated histopathologically by semiquantitative methods. Malondialdehyde (MDA), superoxide dismutase (SOD), glutathion s-transpherase (GST), and catalase (CAT) enzyme levels were investigated in liver and kidney tissues as oxidative state parameters. Results: In Group DIR; hepatocyte degeneration, sinusoidal dilatation, pycnotic nucleus, and necrotic cells were found to be more in rat hepatic tissue; while mononuclear cell infiltration was higher in the parenchyme. MDA levels were significantly lower; but SOD levels were significantly higher in Group DIRD with regard to Group DIR. In the IRI induced diabetic rats’ hepatic and nephrotic tissues MDA levels, showing oxidative injury, were found to be lower. SOD levels, showing early antioxidant activity, were higher. Conclusion: The enzymatic findings of our study together with the hepatic histopathology indicate that dexmedetomidine has a potential role to decrease IRI. Key words: Hepatic ischemia reperfusion injury; Diabetes mellitus; Dexmedetomidine; Rat; MDA; SOD Citation: Sezen SC, Işık B, Bilge M, Arslan M, Çomu FM, Öztürk L, Kesimci E, Kavutçu M. Effect of dexmedetomidine on ischemia-reperfusion injury of liver and kidney tissues in experimental diabetes and hepatic ischemia-reperfusion injury induced rats. Anaesth Pain & Intensive Care 2016;20(2):143-149 Received: 21 November 2015; Reviewed: 10, 24 December 2015, 9, 10 June 2016; Corrected: 12 December 2015; Accepted: 10 June 2016 INTRODUCTİON Perioperative acute tissue injury induced by ischemia-reperfusion is a comman clinical event caused by reduced blood supply to the tissue being compromised during major surgery. Ischemia leads to cellular injury by depleting cellular energy deposits and resulting in accumulation of toxic metabolites. The reperfusion of tissues that have remained in ischemic conditions causes even more damage.1 Furthermore hepatic ischemia-reperfusion injury (IRI) demonstrates a strong relationship with peri-operative acute kidney injury.2 The etiology of diabetic complications is strongly associated with increased oxidative stress (OS). Diabetic patients are known to have a high risk of developing OS or IRI which results with tissue failure.3 The most important role in ischemia and reperfusion is played by free oxygen radicals.1 In diabetes, characterized by hyperglycemia, even more free oxygen radicals are produced due to oxidation of glucose and glycosylation of proteins.3 The structures which are most sensitive to free oxygen radicals in the cells are membrane lipids, proteins, nucleic acids and deoxyribonucleic acids.1 It has been reported that endogenous antioxidant enzymes [superoxide dismutase (SOD), glutathion s-transpherase (GST), catalase (CAT)] play an important role to alleviate IRI.4-8 Also some pharmacological agents have certain effects on IRI.1 The anesthetic agents influence endogenous antioxidant systems and free oxygen radical formation.9-12 Dexmedetomidine is a selective α-2 adrenoceptor agonist agent. It has been described as a useful and safe adjunct in many clinical applications. It has been found that it may increase urine output by considerably redistributing cardiac output, inhibiting vasopressin secretion and maintaining renal blood flow and glomerular filtration. Previous studies demonstrated that dexmedetomidine provides protection against renal, focal cerebral, cardiac, testicular, and tourniquet-induced IRI.13-18 Arslan et al observed that dexmedetomidine protected against lipid peroxidation and cellular membrane alterations in hepatic IRI, when given before induction of ischemia.17 Si et al18 demonstrated that dexmedetomidine treatment results in a partial but significant attenuation of renal demage induced by IRI through the inactivation of JAK/STAT signaling pathway in an in vivo model. The efficacy of the dexmedetomidine for IRI in diabetic patient is not resarched yet. The purpose of this experimental study was to evaluate the biochemical and histological effects of dexmedetomidine on hepatic IRI in diabetic rat’s hepatic and renal tissue. METHODOLOGY Animals and Experimental Protocol: This study was conducted in the Physiology Laboratory of Kirikkale University upon the consent of the Experimental Animals Ethics Committee of Kirikkale University. All of the procedures were performed according to the accepted standards of the Guide for the Care and Use of Laboratory Animals. In the study, 28 male Wistar Albino rats, weighing between 250 and 300 g, raised under the same environmental conditions, were used. The rats were kept under 20-21 oC at cycles of 12-hour daylight and 12-hour darkness and had free access to food until 2 hours before the anesthesia procedure. The animals were randomly separated into four groups, each containing 7 rats. Diabetes was induced by a single intraperitoneal injection of streptozotocin (Sigma Chemical, St. Louis, MO, USA) at a dose of 65 mg/kg body weight. The blood glucose levels were measured at 72 hrs following this injection. Rats were classified as diabetic if their fasting blood glucose (FBG) levels exceeded 250 mg/dl, and only animals with FBGs of > 250 mg/dl were included in the diabetic groups (dia­betes only, diabetes plus ischemia-reperfusion and diabetes plus dexmedetomidine-ischemia-reperfusion). The rats were kept alive 4 weeks after streptozotocin injection to allow development of chronic dia­betes before they were exposed to ischemia-reperfusion.(19) The rats were weighed before the study. Rats were anesthetized with intraperitoneal ketamine 100 mg/kg. The chest and abdomen were shaved and each animal was fixed in a supine position on the operating table. The abdomen was cleaned with 1% polyvinyl iodine and when dry, the operating field was covered with a sterile drape and median laparotomy was performed. There were four experimental groups (Group C (sham-control; n = 7), (Group DC (diabetes-sham-control; n = 7), Group DIR (diabetes-ischemia-reperfusion; n = 7), and Group DIRD (diabetes-ischemia-reperfusion-dexmedetomidine; n = 7). Sham operation was performed on the rats in Group C and Group DC. The sham operation consisted of mobilization of the hepatic pedicle only. The rats in this group were sacrificed 90 min after the procedure. Hepatic I/R injury was induced in Groups DIR and DIRD respectively with hepatic pedicle clamping using a vascular clamp as in the previous study of Arslan et al.(17) After an ischemic period of 45 min, the vascular clamp was removed. A reperfusion period was maintained for 45 min. In Group DIRD, dexmedetomidine hydrochloride 100 μg/kg, (Precedex 100 μg/2 ml, Abbott®, Abbott Laboratory, North Chicago, Illinois, USA) was administrated via intraperitoneal route 30 minutes before surgery. All the rats were given ketamine 100 mg/kg intraperitoneally and intracardiac blood samples were obtained. Preserving the tissue integrity by avoiding trauma, liver and renal biopsy samples were obtained. Biochemical Analysis: The liver and renal tissues were first washed with cold deionized water to discard blood contamination and then homogenized in a homogenizer. Measurements on cell contest require an initial preparation of the tissues. The preparation procedure may involve grinding of the tissue in a ground glass tissue blender using a rotor driven by a simple electric motor. The homogenizer as a tissue blender similar to the typical kitchen blender is used to emulsify and pulverize the tissue (Heidolph Instruments GMBH & CO KGDiax 900 Germany®) at 1000 U for about 3 min. After centrifugation at 10,000 g for about 60 min, the upper clear layer was taken. MDA levels were determined using the method of Van Ye et al,(20) based on the reaction of MDA with thiobarbituric acid (TBA). In the TBA test reaction, MDA and TBA react in acid pH to form a pink pigment with an absorption maximum at 532 nm. Arbitrary values obtained were compared with a series of standard solutions (1,1,3,3-tetraethoxypropane). Results were expressed as nmol/mg.protein. Part of the homogenate was extracted in ethanol/chloroform mixture (5/3 v/v) to discard the lipid fraction, which caused interferences in the activity measurements of T-SOD, CAT and GST activities. After centrifugation at 10.000 x g for 60 min, the upper clear layer was removed and used for the T-SOD, CAT, GST enzyme activity measurement by methods as described by Durak et al21, Aebi22 and Habig et al23 respectively. One unit of SOD activity was defined as the enzyme protein amount causing 50% inhibition in NBTH2 reduction rate and result were expressed in U/mg protein. The CAT activity method is based on the measurement of absorbance decrease due to H2O2 consumption at 240 nm. The GST activity method is based on the measurement of absorbance changes at 340 nm due to formation of GSH-CDNB complex. Histological determinations: Semiquantitative evaluation technique used by Abdel-Wahhab et al(24) was applied for interpreting the structural changes investigated in hepatic tissues of control and research groups. According to this, (-) (negative point) represents no structural change, while (+) (one positive point) represents mild, (++) (two positive points) medium and (+++) (three positive points) represents severe structural changes. Statistical analysis: The Statistical Package for the Social Sciences (SPSS, Chicago, IL, USA) 20.0 softwre was used for the statistical analysis. Variations in oxidative state parameters, and histopathological examination between study groups were assessed using the Kruskal-Wallis test. The Bonferroni-adjusted Mann-Whitney U-test was used after significant Kruskal-Wallis to determine which groups differed from the others. Results were expressed as mean ± standard deviation (Mean ± SD). Statistical significance was set at a p value < 0.05 for all analyses. RESULTS There was statistically significant difference observed between the groups with respect to findings from the histological changes in the rat liver tissue (hepatocyte degeneration, sinüsoidal dilatation, pycnotic nucleus, prenecrotic cell) determined by light microscopy according to semiquantitative evaluation techniques (p < 0.0001). In Group DIR, hepatocyte degeneration was significantly high compared to Group C, Group DC and Group DIRD (p < 0.0001, p < 0.0001, p = 0.002, respectively), (Table 1, Figure 1-4). Similarly, sinüsoidal dilatation was significantly higher in Group DIR (p < 0.0001, p = 0.004, p = 0.015, respectively). Although, pcynotic nucleus was decreased in Group DIRD, it did not make a significant difference in comparison to Group DIR (p = 0.053), (Table 1, Figure 1-4). The prenecrotic cells were significantly increased in Group DIR, with respect to Group C, Group DC and Group DIRD (p < 0.0001, p = 0.004, p < 0.0001, respectively), (Table 1, Figure 1-4). Table 1. The comparison of histological changes in rat hepatic tissue [Mean ± SD)] p**: Statistical significance was set at a p value < 0.05 for Kruskal-Wallis test *p < 0.05: When compared with Group DIR Figure 1: Light microscopic view of hepatic tissue of Group C (control). VC: vena centralis, *: sinusoids. ®: hepatocytes, k: Kupffer cells, G: glycogen granules, mc: minimal cellular changes, Hematoxilen & Eosin x 40 Figure 2: Light-microscopic view of hepatic tissue of Group DC (diabetes mellitus control) (G: Glycogen granules increased in number, (VC: vena centralis, *:sinusoids. ®:hepatocytes, k:Kupffer cells, G: glycogen granules, mc: minimal cellular changes; Hematoxylin & Eosin x 40) Figure 3: Light-microscopic view of hepatic tissue of Group DIR (Diabetes Mellitus and ischemia-reperfusion) (VC: vena centralis, (H) degenerative and hydrophic hepatocytes, (dej) vena centralis degeneration (centrolobar injury) (*): sinusoid dilatation. (←) pycnotic and hyperchromatic nuclei, MNL: mononuclear cell infiltration, (¯) congestion, K: Kupffer cell hyperplasia, (­) vacuolar degeneration (Hematoxylin & Eosin x 40) Figure 4: Light-microscopic view of hepatic tissue of Group DIRD (Diabetes Mellitus and ischemia-reperfusion together with dexmedetomidine applied group) (VC: vena centralis, (MNL) mononuclear cell infiltration, (dej) hydrophilic degeneration in hepatocytes around vena centralis, (conj) congestion, G: glycogen granules, (←) pycnotic and hyperchromatic nuclei, sinusoid dilatation (*) (Hematoxylin & Eosin x 40) Besides, in liver tissue parenchyma, MN cellular infiltration was a light microscopic finding; and showed significant changes among the groups (p < 0.0001). This was significantly higher in Group DIR, compared to Group C, DC, and DIRD (p < 0.0001, p=0.007, p = 0.007, respectively), (Table 1, Figure 1-4). The enzymatic activity of MDA, SOD and GST in hepatic tissues showed significant differences among the groups [(p = 0.019), (p = 0.034). (p = 0.008) respectively]. MDA enzyme activity was significantly incresed in Group DIR, according to Group C and Group DIRD (p = 0.011, p = 0.016, respectively), (Table 2). In Group DIR SOD enzyme activity was lower with respect to Group C and Group DIRD (p = 0.010, p = 0.038, respectively), (Table 2). The GST enzyme activity was significantly higher in Group DIR, when compared to Group C, DC and DIRD (p = 0.007, p = 0.038, p = 0.039, respectively), (Table 2). Table 2. Oxidative state parameters in rat hepatic tissue [Mean ± SD] p**: Statistical significance was set at a p value < 0.05 for Kruskal-Wallis test *p < 0.05: When compared with Group DIR The enzymatic activity of MDA, SOD in renal tissues, showed significant differences among the groups [(p < 0.0001), (p = 0.008) respectively ]. MDA enzyme activity was significantly incresed in Group DIR, according to Group C and Group DIRD (p < 0.0001, p < 0.0001, respectively). Also MDA enzyme activity level was significantly increased in Group DC, in comparison to Group C and Group DIRD (p = 0.003, p = 0.001, respectively), (Table 3). In Group DIR SOD enzyme activity was lower with respect to Group C and Group DIRD (p = 0.032, p = 0.013, respectively), (Table 3). The GST enzyme activity was significantly higher in Group DIR than the other three groups, however; CAT levels were similar among the groups (Table 3). Table 3: Oxidative state parameters in rat nephrotic tissue [Mean ± SD)] p**: Statistical significance was set at a p value < 0.05 for Kruskal-Wallis test *p < 0.05: When compared with Group DIR DISCUSSION In this study, we have reported the protective effect of dexmedetomidine in experimental hepatic and renal IRI model in the rat by investigating the MDA and SOD levels biochemically. Besides, hepatic histopathological findings also supported our report. Ischemic damage may occur with trauma, hemorrhagic shock, and some surgical interventions, mainly hepatic and renal resections. Reperfusion following ischemia results in even more injury than ischemia itself. IRI is an inflammatory response accompanied by free radical formation, leucocyte migration and activation, sinusoidal endothelial cellular damage, deteoriated microcirculation and coagulation and complement system activation.1 We also detected injury in hepatic and renal tissue caused by reperfusion following ischemia in liver. Experimental and clinical evidence indicates that OS is involved in both the pathogenesis and the complications of diabetes mellitus.25,26 Diabetes mellitus is a serious risk factor for the development of renal and cardiovascular disease. It is also related to fatty changes in the liver.27 Diabetes-related organ damage seems to be the result of multiple mechanisms. Diabetes has been associated with increased free radical reactions and oxidant tissue damage in STZ-induced diabetic rats and also in patients.26Oxidative stress has been implicated in the destruction of pancreatic β-cells28 and could largely contribute to the oxidant tissue damage associated with chronic hyperglycemia.29 A number of reports have shown that antioxidants can attenuate the complications of diabetes in patients30 and in experimental models.28,31 This study demonstrated that diabetes causes a tendency to increase the IRI. There is a lot of investigations related to the pharmacological agents or food supplements applied for decreasing OS and IRI. Antioxidant agents paly an important role in IRI by effecting antioxidant system or lessening the formation of ROS. It has been reported that anesthetic agents too, are effective in oxidative stress.1 During surgical interventions, it seems rational to get benefit from anesthetic agents in prevention of OS caused by IRI instead of using other agents. It has been declared that; dexmedetomidine; as an α-2 agonist with sedative, hypnotic properties; is important in prevention of renal, focal, cerebral, cardiac, testicular and tourniquet-induced IRI.13-18 On the other hand Bostankolu et al. concluded that dexmedetomidine did not have an additional protective role for tournique induced IRI during routine general anesthesia.32 In this study; we have shown that dexmedetomidine has a reducing effect in IRI in diabetic rats. Some biochemical tests and histopathological evaluations are applied for bringing up oxidative stress and IRI in the tissues. Reactive oxygen species (ROS) that appear with reperfusion injury damage cellular structures through the process of the lipid peroxidation of cellular membranes and yield toxic metabolites such as MDA.33 As an important intermidiate product in lipid peroxidation, MDA is used as a sensitive marker of IRI.34 ROS-induced tissue injury is triggered by various defense mechanisms.35 The first defence mechanisms include the antioxidant enzymes of SOD, CAT, and GPx. These endogenous antioxidants are the first lines of defence against oxidative stres and act by scavenging potentially damaging free radical moieties.36 There is a balance between ROS and the scavenging capacity of antioxidant enzymes.1-8 In this study, for evaluation of oxidative damage and antioxidant activity, MDS, SOD, GST and CAT levels were determined in liver and kidney tissues. MDA levels in hepatic and renal tissues were higher in Group DIR compared to Group C and Group DIRD. GST levels were higher in Group DIR compared to all the other three groups. When the groups were arranged from highest to lowest order, with respect to CAT levels, the order was; Group DIR, Group DIRD, Group DC and Group C. However, the difference was not significant. The acute phase reactant MDA, as a marker of OS, was found to be high in Group DIR and low in Group DIRD. This could be interpreted as the presence of protective effect of dexmedetomidine in IRI. IRI developing in splanchnic area causes injury also in the other organs.35 Leithead et al showed that clinically significant hepatic IRI demonstrates a strong relationship with peri-operative acute kidney injury.2 In our experimental research that showed correlation to that of research by Leithead et al. After hepatic IRI in diabetic rats renal OS marker MDA levels were significantly more in Group DIR than Group DIRD. In our study, we observed histopathological changes in the ischemic liver tissue and alterations in the level of MDA, SOD, GST and CAT levels which are OS markers. Histopathological changes of the liver tissues are hepatocyt degeneration, sinusoidal dilatation, nuclear picnosis, celluler necrosis, mononuclear cell infiltrationat paranchimal tissue. These histopathological injury scores were significantly lower in the Group DIRD than those in group DIR. LIMITATION Study limitation is there was no negative control group, as this type of surgical intervention is not possible in rats without anesthesia. CONCLUSION The enzymatic findings of our study together with the hepatic histopathology indicate that dexmedetomidine has a potential role to decrease ischemia-reperfusion injury. Conflict of interest and funding: The authors have not received any funding or benefits from industry or elsewhere to conduct this study. 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