Acute Pretreatment Research Articles

The Histamine H3 Receptor Antagonist E159 Reverses Memory Deficits Induced by Dizocilpine in Passive Avoidance and Novel Object Recognition Paradigm in Rats.

The involvement of histamine H3 receptors (H3Rs) in memory is well known, and the potential of H3R antagonists in therapeutic management of neuropsychiatric diseases, e.g., Alzheimer disease (AD) is well established. Therefore, the effects of histamine H3 receptor (H3R) antagonist E159 (2.5–10 mg/kg, i.p.) in adult male rats on dizocilpine (DIZ)-induced memory deficits were studied in passive avoidance paradigm (PAP) and in novel object recognition (NOR) using pitolisant (PIT) and donepezil (DOZ) as standard drugs. Upon acute systemic pretreatment of E159 at three different doses, namely 2.5, 5, and 10 mg/kg, i.p., 2.5 and 5 but not 10 mg/kg of E159 counteracted the DIZ (0.1 mg)-induced memory deficits, and this E159 (2.5 mg)-elicited memory-improving effects in DIZ-induced amnesic model were moderately abrogated after acute systemic administration of scopolamine (SCO), H2R antagonist zolantidine (ZOL), but not with H1R antagonist pyrilamine to the animals. Moreover, the observed memory-enhancing effects of E159 (2.5 mg/kg, i.p.) were strongly abrogated when animals were administered with a combination of SCO and ZOL. Furthermore, the E159 (2.5 mg)-provided significant memory-improving effect of in DIZ-induced short-term memory (STM) impairment in NOR was comparable to the DOZ-provided memory-enhancing effect, and was abolished when animals were injected with the CNS-penetrant histamine H3R agonist R-(α)-methylhistamine (RAMH). However, E159 at a dose of 2.5 mg/kg failed to exhibit procognitive effect on DIZ-induced long-term memory (LTM) in NOR. Furthermore, the results observed revealed that E159 (2.5 mg/kg) did not alter anxiety levels and locomotor activity of animals naive to elevated-plus maze (EPM), demonstrating that improved performances with E159 (2.5 mg/kg) in PAP or NOR are unrelated to changes in emotional responding or in spontaneous locomotor activity. These results provide evidence for the potential of drugs targeting H3Rs for the treatment of neuropsychiatric disorders, e.g., AD.

Effects of lorcaserin (Belviq®) on nicotine- and food-maintained responding in non-human primates

BackgroundAccumulating evidence suggests that the FDA-approved serotonin 5-HT2C receptor agonist, lorcaserin (Belviq®), may be a promising candidate for the management of substance use disorders, including nicotine addiction. The present study was conducted to determine the efficacy and selectivity of acute or continuous lorcaserin treatment for decreasing the reinforcing effects of nicotine in a primate species. MethodsAdult rhesus monkeys (n=4) with a history of nicotine self-administration (>2years) responded for injections of nicotine (0.32–100μg/kg IV) or food pellets under a fixed-ratio schedule of reinforcement during daily 100-min sessions. When responding was stable, lorcaserin was administered either as an acute pretreatment (0.1–1.0mg/kg, IM) or by continuous infusion (0.1mg/kg/hr, SC for 3–5days). Daily activity patterns were also monitored immediately following experimental sessions. ResultsResults indicate that acute lorcaserin pretreatment produced significant and dose-dependent decreases in nicotine-maintained responding across a >100-fold range of self-administered nicotine doses. Continuous lorcaserin treatment decreased intake of 10μg/kg/inj nicotine to about 50% of baseline values. Food-maintained responding was only moderately decreased in 3 of 4 subjects after acute administration and unaffected in all subjects during continuous treatment. Daily activity also was significantly decreased—to ≤50% of control values—following experimental sessions in which acute lorcaserin was administered. ConclusionsThese data indicate that lorcaserin reduces IV self-administration of nicotine at a dose that decreases motoric activity but less consistently disrupts food-maintained responding. Further research into lorcaserin’s potential utility for the management of nicotine dependence is warranted.

Anti-inflammatory, Antinociceptive, and Antioxidant Activities of Methanol and Aqueous Extracts of Anacyclus pyrethrum Roots.

Anacyclus pyrethrum (L.) is a plant widely used in Moroccan traditional medicine to treat inflammatory and painful diseases. The objective of the present study was to evaluate the antinociceptive, anti-inflammatory and antioxidant activities of aqueous and methanol extracts of Anacyclus pyrethrum roots (AEAPR and MEAPR). The anti-inflammatory effect of AEAPR and MEAPR was determined in xylene–induced ear edema and Complete Freund’s Adjuvant (CFA)-induced paw edema. The antinociceptive activity of AEAPR and MEAPR (125, 250, and 500 mg/kg) administered by gavage was examined in mice by using acetic acid-induced writhing, hot plate, and formalin tests, and the mechanical allodynia were assessed in CFA-induced paw edema. In addition, the in vitro antioxidant activities of the extracts were determined by using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging method, ferric reducing power and β-carotene-linoleic acid assay systems. AEAPR and MEAPR produced significant reductions in CFA-induced paw edema and xylene-induced ear edema. A single oral administration of these extracts at 250 and 500 mg/kg significantly reduced mechanical hypersensitivity induced by CFA, which had begun 1 h 30 after the treatment, and was maintained till 7 h. Chronic treatment with both extracts significantly reduced mechanical hypersensitivity in persistent pain conditions induced by CFA. Acute pretreatment with AEAPR or MEAPR at high dose caused a significant decrease in the number of abdominal writhes induced by acetic acid injection (52.2 and 56.7%, respectively), a marked increase of the paw withdrawal latency in the hot plate test, and also a significant inhibition of both phases of the formalin test. This antinociceptive effect was partially reversed by naloxone pretreatment in the hot plate and formalin tests. Additionally, a significant scavenging activity in DPPH, reducing power and protection capacity of β-carotene was observed in testing antioxidant assays. The present study suggests that AEAPR and MEAPR possess potent anti-inflammatory, antinociceptive and antioxidant effects which could be related to the presence of alkaloids and phenols in the plant. In addition, the antinociceptive effect of APR extracts seems to partly involve the opioid system. Taken together, these results suggest that Anacylcus pyrethrum may indeed be useful in the treatment of pain and inflammatory disorders in humans.

Blockade of alcohol escalation and "relapse" drinking by pharmacological FAAH inhibition in male and female C57BL/6J mice.

Anandamide (AEA)-dependent signaling is regulated by the catabolic enzyme fatty acid amide hydrolase (FAAH). Several lines of evidence have demonstrated that FAAH and AEA are involved in the behavioral effects of alcohol. Therefore, we investigated whether a selective FAAH inhibitor, URB597 (cyclohexylcarbamic acid 3'-[aminocarbonyl]-[1,1'-biphenyl]-3-yl ester), altered alcohol intake in mice in a voluntary alcohol drinking model. Mice, subjected to 3weeks of chronic intermittent access (IA) in a two-bottle choice paradigm with 24-h access every other day, developed rapid escalation of alcohol intake and high preference. We evaluated the pharmacological effects of URB597 after both acute (1-day) withdrawal from chronic IA and 1-week withdrawal using the alcohol deprivation effect (ADE) model. AEA and N-acyl ethanolamide (NAE) abundances were determined after chronic IA, acute (1-day), or long-term (1 and 2weeks) withdrawal in four brain regions. Acute pretreatment with URB597 reduced alcohol intake and preference after acute withdrawal. This effect was blocked by pretreatment with a selective type 1 cannabinoid receptor (CB1) antagonist, suggesting a CB1-mediated mechanism. Both single- and multiple-dosing regimens with an effective dose of URB597 prevented the ADE, with no tolerance development after the multi-dosing regimen. AEA and NAE levels were transiently increased in all brain regions measured after acute withdrawal, indicating that the endocannabinoid system is involved in acute alcohol withdrawal stress response. FAAH inhibitors reduce alcohol escalation and "relapse" drinking in mice.

GABAA receptor positive allosteric modulators modify the abuse-related behavioral and neurochemical effects of methamphetamine in rhesus monkeys

GABAA receptor positive allosteric modulators (GABAA receptor modulators) are commonly used for the treatment of insomnia. Nevertheless, the effects of these compounds on psychostimulant-induced sleep impairment are poorly understood. Because GABAA receptor modulators have been shown to decrease the abuse-related effects of psychostimulants, the aim of the present study was to evaluate the effects of temazepam (0.3, 1.0 or 3.0 mg/kg) and eszopiclone (0.3, 1.0 or 3.0 mg/kg), two GABAA receptor modulators, on the behavioral neuropharmacology of methamphetamine in adult rhesus macaques (n = 5). Sleep-like measures and general daytime activity were evaluated with Actiwatch monitors. Methamphetamine self-administration (0.03 mg/kg/inf) was evaluated during morning sessions. Methamphetamine-induced dopamine overflow was assessed through in vivo microdialysis targeting the nucleus accumbens. Nighttime treatment with either temazepam or eszopiclone was ineffective in improving sleep-like measures disrupted by methamphetamine self-administration. Acute pretreatment with a low dose of temazepam before self-administration sessions increased methamphetamine self-administration without affecting normal daytime home-cage activity. At a high dose, acute temazepam pretreatment decreased methamphetamine self-administration and attenuated methamphetamine-induced increases in dopamine in the nucleus accumbens, without decreasing general daytime activity. Acute eszopiclone treatment exerted no effects on methamphetamine intake or drug-induced increases in dopamine. Our study suggests that treatments based on GABAA receptor modulators are not effective for the treatment of sleep disruption in the context of psychostimulant use. In addition, distinct GABAA receptor modulators differentially modulated the abuse-related effects of methamphetamine, with acute treatment with the high efficacy GABAA receptor modulator temazepam decreasing the behavioral and neurochemical effects of methamphetamine.

Effects of acute pre-treatment with ethanolamine on isoprenalineinduced myocardial infarction in adult Wistar rats

Ethanolamine (Etn), a biogenic amine, protects isolated hearts against MI, but its effects in vivo are unknown. In this study, we investigated the effects of Etn pretreatment on isoprenaline (ISO)-induced MI in vivo. Wistar rats were treated with either Etn (10mg.kg-1, i.p.) or saline, prior to either ISO (67mg.kg-1, s.c.) or saline. Haemodynamic- and electrocardiographic parameters were recorded under anaesthesia, 24 hours post-treatment with ISO. Infarct sizes were determined by triphenyltetrazolium chloride staining. Oxidative stress plasma parameters, conjugated dienes (CD) and thio-barbituric acid reactive substances (TBARS), were measured by spectrophotometric analysis. Etn decreased ISO-induced pathological Q-waves, but had no effect on ISO-induced low-voltage electrocardiogram. In addition, Etn decreased overall mortality induced by ISO treatment. However, Etn did not prevent ISO-induced infarction or the systolic- and diastolic hypotensive effects. Etn also enhanced the ISO-induced increase in heart-to-body weight ratio, and reversed the decrease in lungs-to-body weight ratio. Neither ISO nor Etn altered CD or TBARS. These results suggest that, despite a tendency to modulate cardiac electrical activity, Etn did not prevent ISO-induced MI or myocardial dysfunction in vivo.

Unbiased Screening of Kawasaki Disease Sera for Viral Antigen Exposure

BackgroundKawasaki disease(KD) is a medium-vessel vasculitis with a predilection for coronary arteries and is of unknown etiology. KD is responsible for the majority of acquired pediatric cardiovascular disease in the industrialized world, and is associated with development of coronary artery aneurysms in approximately 25% of untreated patients. Epidemiologic, pathologic, and clinical characteristics of KD display notable overlap with common pediatric viral illnesses, leading some to hypothesize that a viral infection is the inciting agent for KD.MethodsWe investigated viral exposure history in KD patients by utilizing a recently developed technique to profile sera against the known human virome in an unbiased manner. We collected sera during the acute (pretreatment) and subacute phases of illness from 35 patients meeting clinical diagnostic criteria for KD, preferentially selecting patients with coronary involvement and/or late presentation. Control samples included healthy children and patients with known viral infections. Using phage immunoprecipitation sequencing(PhIP-seq), the sera were screened against a phage display library expressing epitopes that cover the complete reference protein sequences of the known 206 viruses with human tropism.ResultsThe mean patient age was 4.6 years(range 0.4–16.9) and mean day of illness at acute sample collection was 14.5 days(range 5 to 32). A majority of patients demonstrated coronary artery changes during the course of their illness(22/35, 62%). Sera from patients with KD demonstrated patterns of viral infection to common pediatric viruses with similar signal intensity and distribution to healthy control children. Interestingly, one patient demonstrated a strong signal to parvovirus B19. She presented on Illness Day 29 with periungual peeling and severe hip arthritis, and her initial KD course with 14 days of fever and 4/5 clinical criteria was missed.ConclusionAlthough sera obtained early in the disease course could have missed a titer rise, we conclude that patients with KD do not exhibit unique serologic evidence of infection to known viruses or a viral exposure history that differs from age-similar healthy children.Disclosures All authors: No reported disclosures.

Phytochemical screening and analgesic profile of the lyophilized aqueous extract obtained from Chrysobalanus icaco leaves in experimental protocols

Context: Chrysobalanus icaco L. (Chrysobalanaceae) has been used for the treatment of abdominal pain and cramps.Objective: Assess the chemical and pharmacological profile of the lyophilized aqueous extract from C. icaco leaves (AEC).Materials and methods: Chromatographic methods were used to assess compounds from AEC. Mice were treated with vehicle (control group) or AEC (100, 200 or 400 mg/kg, p.o.) (group with 7–8 mice) and the analgesic profile was assessed employing the acetic acid-induced writhing, formalin, hot plate tests and hyperalgesia induced by carrageenan (CG) or tumour necrosis factor-alpha. The animal motor performance was assessed using rota-rod and grip strength tests.Results: The chromatographic profile of AEC demonstrated the presence of terpenoid compounds. The acute pretreatment with AEC, at all doses, produced a significant (p < 0.01) inhibition of painful bahaviour (11.4 ± 3.6; 10.3 ± 2.8; 11.3 ± 2.2) when compared to the control group (24.7 ± 4.7) in acetic acid-induced writhing test. In the formalin test, AEC were effective in the second phase (p < 0.01) (57.2 ± 10.3; 56.3 ± 9.2; 54.7 ± 8.9) when compared to control group (121.9 ± 18.5). No response was observed in the hot plate test. The higher dose of AEC produced a significant (p < 0.01 or p < 0.05) inhibitory effect on the mechanical hyperalgesia test. AEC did not affect the motor performance of the mice.Discussion: The terpenoids from AEC are known for its analgesic and anti-inflammatory properties. So, these results corroborate the experiments using the AEC in inflammatory pain protocols.Conclusion: Our results suggest that AEC act against inflammatory pain.

Neuroprotective effect of lercanidipine in middle cerebral artery occlusion model of stroke in rats

Oxidative stress, inflammation and apoptotic neuronal cell death are cardinal mechanisms involved in the cascade of acute ischemic stroke. Lercanidipine apart from calcium channel blocking activity possesses anti-oxidant, anti-inflammatory and anti-apoptotic properties. In the present study, we investigated neuroprotective efficacy and therapeutic time window of lercanidipine in a 2h middle cerebral artery occlusion (MCAo) model in male Wistar rats. The study design included: acute (pre-treatment and post-treatment) and sub-acute studies. In acute studies (pre-treatment) lercanidipine (0.25, 0.5 and 1mg/kg, i.p.) was administered 60min prior MCAo. The rats were assessed 24h post-MCAo for neurological deficit score (NDS), motor deficit paradigms (grip test and rota rod) and cerebral infarction via 2,3,5-triphenyltetrazolium chloride (TTC) staining. The most effective dose was found to be at 0.5mg/kg, i.p., which was considered for further studies. Regional cerebral blood flow (rCBF) was monitored till 120min post-reperfusion to assess vasodilatory property of lercanidipine (0.5mg/kg, i.p.) administered at two different time points: 60min post-MCAo and 15min post-reperfusion. In acute studies (post-treatment) lercanidipine (0.5mg/kg, i.p.) was administered 15min, 120min and 240min post-reperfusion. Based on NDS and cerebral infarction via TTC staining assessed 24h post-MCAo, effectiveness was evident upto 120min. For sub-acute studies same dose/vehicle was repeated for next 3days and magnetic resonance imaging (MRI) was performed 96h after the last dose. Biochemical markers estimated in rat brain cortex 24h post-MCAo were oxidative stress (malondialdehyde, reduced glutathione, nitric oxide, superoxide dismutase), blood brain barrier damage (matrix metalloproteinases-2 and -9) and apoptotic (caspase-3 and -9). Lercanidipine significantly reduced NDS, motor deficits and cerebral infarction volume as compared to the control group. Lercanidipine (60min post-MCAo) significantly increased rCBF (86%) as compared to vehicle treated MCAo group (64%) 120min post-reperfusion, but failed to show vasodilatation with 15min post-reperfusion group. Lercanidipine (13.78±2.78%) significantly attenuated percentage infarct volume as evident from diffusion-weighted (DWI) and T2-weighted images as compared to vehicle treated MCAo group (25.90±2.44%) investigated 96h post-MCAo. The apparent diffusion coefficient (ADC) was also significantly improved in lercanidipine group as compared to control group. Biochemical alterations were significantly ameliorated by lercanidipine till 120min post-reperfusion group and MMP-9 inhibition observed even with 240min group. Thus, lercanidipine revealed significant neuroprotective effect mediated through attenuation of oxidative stress, inflammation and apoptosis.

Effects of repeated morphine on ultrasonic vocalizations in adult rats: increased 50-kHz call rate and altered subtype profile.

Adult rat 50-kHz vocalizations have been proposed to indicate a positive affective state, putatively revealed by a predominance of trill calls over flat calls. However, short-term exposure to non-sedative doses of the euphorigen morphine suppresses calling, with no discernible shift in trill or flat call prevalence. This study aimed to determine whether morphine acutely increases 50-kHz call rates or alters the relative prevalence of trill or flat calls, after long-term morphine exposure or acute pharmacological pretreatment. Experiment 1 comprised 10 once-daily tests, alternating between saline and morphine, 1mg/kg SC, followed by dose-response testing (0, 0.3, 1, and 3mg/kg). Experiment 2 was similar but included additional testing with morphine in combination with the antinausea drug ondansetron or the peripheral opioid antagonist methylnaltrexone. In experiment 3, morphine was again combined with ondansetron or methylnaltrexone but in rats that were initially drug naïve. In animals that were initially drug naïve, morphine tended to suppress calling and did not alter the 50-kHz call subtype profile. In morphine-experienced rats, morphine acutely increased the 50-kHz call rate and promoted trills over flat calls; short calls were also inhibited. Neither ondansetron nor methylnaltrexone detectably altered any effect of morphine on calling, nor did these two drugs affect 50-kHz calling when given alone. With chronic exposure, morphine acutely enhances 50-kHz calling and differentially promotes trill calls, mainly at the expense of flat calls. These effects appear consistent with a positive affect interpretation of 50-kHz vocalizations.

Effects of the GLP-1 Agonist Exendin-4 on Intravenous Ethanol Self-Administration in Mice.

Glucagon-like peptide 1 (GLP-1) receptor agonists have been shown to decrease ethanol (EtOH) drinking in rodent assays. The GLP-1 system also powerfully modulates food and fluid intake, gastrointestinal functions, and metabolism. To begin to understand the neurobiological mechanisms by which GLP-1 receptor ligands may be able to control EtOH intake, it is important to ascertain whether they can modulate the direct reinforcing effects of EtOH, without the confound of effects on ingestive behaviors generally. We trained experimentally naïve, free-fed C57BL/6J mice to self-administer EtOH intravenously. Once stable EtOH intake was acquired, we tested the effect of acute pretreatment with the GLP-1 receptor agonist Exendin-4. Effect of Exendin-4 on operant behavior reinforced by a palatable liquid food was similarly evaluated as a control. Intravenous EtOH functioned as a positive reinforcer in over half the mice tested. In mice that acquired self-administration, EtOH intake was high, indeed, reaching toxic doses; 3.2μg/kg Exendin-4 decreased intravenous EtOH intake by at least 70%, but had no significant effect on food-maintained operant responding. This experiment produced 2 main conclusions. First, although technically challenging and yielding only moderate throughput, the intravenous self-administration procedure in mice is feasible, and sensitive to pharmacological manipulations. Second, GLP-1 receptor agonists can powerfully attenuate voluntary EtOH intake by directly modulating the reinforcing effects of EtOH. These findings support the potential usefulness of GLP-1 receptor ligands in the treatment of alcohol use disorder.

Chromosome abnormalities at onset of complete remission are associated with worse outcome in patients with acute myeloid leukemia and an abnormal karyotype at diagnosis: CALGB 8461 (Alliance).

Achievement of complete remission is essential for long-term survival of acute myeloid leukemia patients. We evaluated the prognostic significance of cytogenetics at complete remission in 258 adults with de novo acute myeloid leukemia and abnormal pre-treatment karyotypes, treated on Cancer and Leukemia Group B front-line studies, with cytogenetic data at onset of morphological complete remission. Thirty-two patients had abnormal karyotypes at time of initial complete remission. Of these, 28 had at least 1 abnormality identified pre-treatment, and 4 acute myeloid leukemia-related abnormalities not detected pre-treatment. Two hundred and twenty-six patients had normal remission karyotypes. Patients with abnormal remission karyotypes were older (P<0.001), had lower pre-treatment white blood counts (P=0.002) and blood blast percentages (P=0.004), were less often classified as Favorable and more often as Adverse among European LeukemiaNet Genetic Groups (P<0.001), and had shorter disease-free survival (median 0.6 vs. 0.9 years; P<0.001) and overall survival (median 1.2 vs. 2.2 years; P<0.001) than patients with normal remission karyotypes. Sixteen patients with normal remission karyotypes also harbored non-clonal abnormalities unrelated to pre-treatment karyotypes. They had shorter overall survival than 210 patients with only normal metaphases (P=0.04). Forty-eight patients with any clonal or non-clonal chromosome abnormality at complete remission had worse disease-free survival (median 0.6 vs. 1.0 years; P<0.001) and overall survival (median 1.2 vs. 2.5 years; P<0.001) than 210 patients with exclusively normal metaphases. In multivariable analyses, after adjustment for age, the presence of any remission abnormality was associated with shorter disease-free survival (P=0.03) and overall survival (P=0.01). We conclude that detection of any abnormality at complete remission is an adverse prognostic factor. (clinicaltrials.gov identifier: 00048958).

Separate and Combined Effects of Naltrexone and Extended-Release Alprazolam on the Reinforcing, Subject-Rated, and Cardiovascular Effects of Methamphetamine.

Opioid antagonists (eg, naltrexone) and positive modulators of γ-aminobutyric acid type A receptors (eg, alprazolam) each modestly attenuate the abuse-related effects of stimulants. A previous study demonstrated that acute pretreatment with the combination of naltrexone and alprazolam attenuated a greater number of the subject-rated effects of D-amphetamine than the constituent drugs alone. This study tested the hypothesis that maintenance on the combination of naltrexone and alprazolam XR would attenuate the reinforcing and "positive" subject-rated effects of methamphetamine to a greater extent than the constituent drugs alone.Eight non-treatment-seeking, stimulant-using individuals completed a placebo-controlled, crossover, double-blind inpatient protocol. Participants were maintained on naltrexone (0 and 50 mg), alprazolam XR (0 and 1 mg), and the combination of naltrexone and alprazolam XR (50 mg and 1 mg, respectively) for 6 to 7 days. Under each maintenance condition, participants sampled intranasal doses of methamphetamine (0, 10, and 30 mg), and were then offered the opportunity to work for the sampled dose on a modified progressive-ratio procedure. Subject-rated drug effect questionnaires, psychomotor, and physiology assessments were collected.Intranasal methamphetamine functioned as a reinforcer and produced prototypical stimulant-like "positive" subject-rated and physiological effects. Maintenance on naltrexone significantly decreased the reinforcing, but not subject-rated drug effects of 10-mg methamphetamine. Alprazolam XR and the combination of naltrexone and alprazolam XR did not impact methamphetamine self-administration or subject-rated drug effects. The results support the continued evaluation of naltrexone for methamphetamine dependence, as well as the identification of other drugs that enhance its ability to reduce drug-taking behavior.

Phorbol Dibutyrate (PDB) Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) May Involve Down‐Regulation of MicroRNAs (miRs)

In colonic epithelia, the concerted action of apical membrane Cl− channels and basolateral membrane Na+/K+‐ATPase, Na+‐K+‐2Cl− cotransporters and K+ channels, account for net Cl− secretion. CFTR, the major apical Cl− channel, is regulated by protein kinases (PK) A and C. We had previously showed in human colonic T84 cells that the cAMP activator, prostaglandin E1 (PGE1,10nM), stimulated Cl− secretion via CFTR, measured as short circuit current (ΔISc, μA/cm2) and this was attenuated by both acute and prolonged pretreatment with the PKC activator, phorbol dibutyrate (PDB, 100nM). Further, 2h of pretreatment with PDB significantly reduced CFTR mRNA. Since microRNAs (miRs) are known to modulate proteins by either degrading mRNA, or by inhibiting translation, we initially tested whether 6 specific CFTR related miRs: −145, −331, −384, −494, −1246 and 1290 were regulated by PDB. However, the expressions of these miRs were not significantly altered by PDB (FASEB 29:855.5,’15). In this study, we further explored the molecular basis of the attenuation in Cl− secretion. We first confirmed, by Western blotting, that CFTR protein expression is down regulated at 2h PDB treatment (n=2). In addition, the mRNA (60%, n=3) and protein expression for NKCC was also decreased. Since CFTR 3′ UTR contains almost 500 recognized miR target sites, we next undertook a detailed algorithm screening process involving TargetScan, miRDB, EMBL‐EBI and Diana to identify further possible CFTR miR regulators. We hypothesized that the miRs identified through algorithmic screening would be up‐regulated after PDB treatment, leading to a decrease in CFTR expression. We specifically screened miRs: −362, −433, −509, −600, −607, −619, −631, and −944, which were identified by at least 3 algorithms. After treating T84 cells with ±100nM PDB for 2h, 6h, and 24h, RNA was isolated using TriZol Reagent and cDNA was synthesized using an Invitrogen cDNA synthesis kit. We compared the expression levels of these miRs using an Invitrogen miRNA qRT‐PCR Kit with U6 snRNA as an internal control. Based on three separate experiments each (p&lt;0.05), the results show that miR −607 (Control: 1.37±0.18, 2h: 0.51±0.08, 6h: 0.77±0.09, 24h: 0.55±0.18), miR‐619 (Control: 2.04±0.92, 2h: 0.58±0.05, 6h: 0.42±0.06, 24h: 0.44±0.04), and miR‐944 (Control: 1.50±0.30, 2h: 0.46±0.07, 6h: 0.36±0.08, 24h: 0.40±0.13) expressions were significantly down‐regulated at all measured time‐points. miR‐631 (Control: 1.24±0.18) expression was significantly down‐regulated at 2h (0.60±0.05) and 6h (0.58±0.04) but not at 24h (0.78±0.22). The results with miR‐607 are in contrast to studies in Caco2 cells, where an increase in miR‐607 leads to a decrease in CFTR expression (Biochem J. 438, p.25–32 2011). The other miRs tested did not show significant alteration of expression at any measured time‐point. Thus, PDB treatment reduces CFTR and NKCC expression and down regulates specific miRs. We speculate that perhaps there is an intermediary protein, the target of miRs: −607, −619, and −944, that alters CFTR/NKCC expression.Support or Funding InformationAmerican Physiological Society Undergraduate Research Excellence Fellowship (UGREF)

Acute and Chronic Pretreatment With Atenolol Attenuates Intestinal Ischemia and Reperfusion Injury in Hypercholesterolemic Rats.

To evaluate the protective effects of preinjury atenolol (acute v chronic) on apoptosis, contractility, oxidative stress, and inflammatory markers in hypercholesterolemic rats undergoing intestinal ischemia-reperfusion (I/R) injury. Prospective, experimental animal study. University laboratory. Male Wistar rats (n = 32). Rats were divided into the following 4 groups: 1 group was fed a normal diet (ND) (group ND+NoAT [no atenolol]), and the other 3 groups were fed a high-cholesterol diet (HCD)-group HCD+NoAT, group HCD+ChAT (chronic atenolol, 3 mg/kg/day for 8 weeks), and group HCD+AcAT (acute atenolol, 1.5 mg/kg, given 5 minutes before intestinal clamping). All rats underwent I/R injury. The superior mesenteric artery was clamped for 60 minutes, then opened for 120 minutes (reperfusion). Apoptotic cells and stimulated contractions of ileal segments were examined. Tissue markers of intestinal I/R injury were examined. Intestinal malondialdehyde, superoxide dismutase, and nitrate/nitrite levels were measured. The chronic atenolol group had fewer apoptotic cells and higher superoxide dismutase activity compared with the other groups. Intestinal contraction was higher in both atenolol pretreatment groups compared with the NoAT groups. Chronic and acute atenolol resulted in lower ileal levels of malondialdehyde and immunolabeling-positive cells (intestinal inducible nitric oxide synthase, endothelial nitric oxide synthase, interleukin-1, and interleukin-8) after I/R injury compared with the no atenolol groups. Both chronic and acute pre-I/R injury treatment with atenolol attenuated I/R injury in this hypercholesterolemic rat model. These findings should encourage future studies of atenolol in hypercholesterolemic patients undergoing procedures with a high risk of intestinal ischemia.

Ethanol Withdrawal-Induced Impaired Recognition Is Reversed by Chronic Exposure to Stress and the Acute Administration of Corticosterone in Mice.

The present study was designed to ascertain the effects of repeated exposure to stress and the acute administration of corticosterone (1, 3, 10 mg/kg, intraperitoneally (i.p.)) on the ethanol withdrawal-induced impairment of novel object recognition in mice. Mice were chronically treated with 3% ethanol for 7 d, with or without exposure to restraint stress for 1 h/d. A significant decrease in cognitive function was observed in the ethanol plus no stress group at 48 h after the discontinuation of ethanol treatment. This impaired recognition was recovered in the ethanol plus stress group. Moreover, we investigated the effects of acute pretreatment with corticosterone, which is a corticosteroid-type hormone produced in the cortex of the adrenal glands, on the impaired recognition after the discontinuation of ethanol treatment in mice. The impaired recognition in the 3% ethanol alone-treated group at 48 h after the discontinuation of ethanol treatment was recovered by treatment with the middle dose (3 mg/kg) of corticosterone, but not with the low or high doses (1, 10 mg/kg). These results suggest that chronic stress during the development of ethanol dependence may reduce the impaired recognition after the discontinuation of ethanol treatment. Moreover, acute pretreatment with the middle dose of corticosterone also recovered the impaired recognition after the discontinuation of ethanol treatment in mice. Adequate regulation of the hypothalamic-pituitary-adrenal (HPA) axis by corticosterone may improve the impaired recognition after the discontinuation of ethanol treatment.

Mechanisms of tau and Aβ-induced excitotoxicity

Excitotoxicity was originally postulated to be a late stage side effect of Alzheimer׳s disease (AD)-related neurodegeneration, however more recent studies indicate that it may occur early in AD and contribute to the neurodegenerative process. Tau and amyloid beta (Aβ), the main components of neurofibrillary tangles (NFTs) and amyloid plaques, have been implicated in cooperatively and independently facilitating excitotoxicity. Our study investigated the roles of tau and Aβ in AD-related excitotoxicity. In vivo studies showed that tau knockout (tau−/−) mice were significantly protected from seizures and hippocampal superoxide production induced with the glutamate analog, kainic acid (KA). We hypothesized that tau accomplished this by facilitating KA-induced Ca2+ influx into neurons, however lentiviral tau knockdown failed to ameliorate KA-induced Ca2+ influx into primary rat cortical neurons. We further investigated if tau cooperated with Aβ to facilitate KA-induced Ca2+ influx. While Aβ biphasically modulated the KA-induced Cacyt2+ responses, tau knockdown continued to have no effect. Therefore, tau facilitates KA-induced seizures and superoxide production in a manner that does not involve facilitation of Ca2+ influx through KA receptors (KAR). On the other hand, acute pretreatment with Aβ (10min) enhanced KA-induced Ca2+ influx, while chronic Aβ (24h) significantly reduced it, regardless of tau knockdown. Given previously published connections between Aβ, group 1 metabotropic glutamate receptors (mGluRs), and KAR regulation, we hypothesized that Aβ modulates KAR via a G-protein coupled receptor pathway mediated by group 1mGluRs. We found that Aβ did not activate group 1mGluRs and inhibition of these receptors did not reverse Aβ modulation of KA-induced Ca2+ influx. Therefore, Aβ biphasically regulates KAR via a mechanism that does not involve group 1mGluR activation.

Chlorella vulgaris reduces the impact of stress on hypothalamic–pituitary–adrenal axis and brain c-fos expression

Predominantly emotional stressors activate a wide range of brain areas, as revealed by the expression of immediate early genes, such as c-fos. Chlorella vulgaris (CV) is considered a biological response modifier, as demonstrated by its protective activities against infections, tumors and stress. We evaluated the effect of acute pretreatment with CV on the peripheral and central responses to forced swimming stress in adult male rats. Pretreatment with CV produced a significant reduction of stress-related hypothalamic–pituitary–adrenal activation, demonstrated by decreased corticotrophin releasing factor gene expression in the hypothalamic paraventricular nucleus (PVN) and lower ACTH response. Hyperglycemia induced by the stressor was similarly reduced. This attenuated neuroendocrine response to stress occurred in parallel with a diminished c-fos expression in most evaluated areas, including the PVN. The data presented in this study reinforce the usefulness of CV to diminish the impact of stressors, by reducing the HPA response. Although our results suggest a central effect of CV, further studies are necessary to understand the precise mechanisms underpinning this effect.

Abstract 10246: Hydroxychloroquine Pretreatment Reduces Myocardial Ischemia-Reperfusion Injury: Role of Cardiac Extracellular-Signal-Regulated Kinase 5 and Autophagy

Background: Hydroxychloroquine (HCQ) is an antimalarial drug, which is also widely used to treat chronic rheumatologic diseases. Since HCQ was reported to inhibit cell autophagy and to activate extracellular-signal-regulated kinase 5 (ERK5) in vascular endothelial cells, we designed the current study to determine the effects of HCQ on cardiac ischemia-reperfusion (I-R) injury and post-I-R expression of ERK5 and autophagy marker proteins. Methods: Adult C57BL/6J mice of both genders were pretreated with HCQ (50 mg/kg, i.p.) 1 hour prior to isolation of the hearts, which were subjected to 30 min of no-flow global ischemia followed by 60 min of reperfusion in Langendorff mode. Ventricular function was continuously assessed and myocardial infarct size was determined at the end of I-R. Heart samples were collected following normoxic perfusion (no-ischemic controls), I-R, or I-R with HCQ for assessing ERK5 and autophagy-related proteins with Western blots. Results: HCQ pretreatment reduced infarct size significantly in the female hearts (P&lt;0.05) as compared with the male hearts (Fig. A). Post-I-R cardiac function was better in HCQ-treated males (Fig. B). I-R resulted in a robust increase in total ERK5 (Fig. C) and phosphorylated ERK5 (Thr218/Tyr220) in both genders, which was abolished in HCQ-treated groups. Conversely, either I-R or HCQ did not affect the post-I-R cardiac expression of autophagy-related proteins (e.g., Atg5, Beclin-1, LC3II/LC3I ratio), except Beclin-1 phosphorylation was inhibited in HCQ-treated male hearts, but not females (Fig. D). Conclusions: Acute HCQ pretreatment affords cardioprotection against I-R injury in both genders. Interestingly, cardioprotective effects of HCQ are associated with a strong inhibitory effect on the induction of ERK5 following I-R in the heart, indicating a novel molecular mechanism underlying the HCQ-induced cardioprotection. However, the cardioprotective dose of HCQ has no major impact on cardiac autophagy.

Blockade of cholinergic transmission elicits somatic signs in nicotine-naïve adolescent rats.

High doses of the nicotinic acetylcholine receptor (nAChR) antagonist mecamylamine can elicit somatic signs resembling those associated with nicotine withdrawal in nicotine-naïve adult rats. Understanding this phenomenon, and its possible modulation by acute nicotine and age, could inform the use of mecamylamine as both an experimental tool and potential pharmacotherapy for tobacco dependence and other disorders. This study evaluated the ability of high-dose mecamylamine to elicit somatic signs in adolescent rats, and the potential for acute nicotine pretreatment to potentiate this effect as previously reported in adults. Single or repeated injections of mecamylamine (1.5 or 3.0 mg/kg, s.c.) elicited somatic signs in nicotine-naïve adolescents, but this effect was not influenced by 2 h pretreatment with acute nicotine (0.5 mg/kg, s.c.). In an initial evaluation of the effects of age in this model, mecamylamine (2.25 mg/kg, s.c.) elicited somatic signs in nicotine-naïve adolescents and adults. This effect was modestly enhanced following acute nicotine injections in adults but not in adolescents, even when a higher nicotine dose (1.0 rather than 0.5 mg/kg, s.c.) was used in adolescents to account for age differences in nicotine pharmacokinetics. These studies are the first to show that mecamylamine elicits somatic signs in nicotine-naïve adolescent rats, an effect that should be considered when designing and interpreting studies examining effects of high doses of mecamylamine in adolescents. Our findings also provide preliminary evidence that these signs may be differentially modulated by acute nicotine pretreatment in adolescents versus adults.