scholarly journals Involvement of N-Methyl-D-Aspartate Receptors in the Anticonvulsive Effects of Licofelone on Pentylenetetrazole-Induced Clonic Seizure in Mice

2021 ◽  
Vol 11 (1) ◽  
pp. 14-21
Author(s):  
Ramtin Gholizadeh ◽  
Zohreh Abdolmaleki ◽  
Taraneh Bahremand ◽  
Mehdi Ghasemi ◽  
Mehdi Gharghabi ◽  
...  
Keyword(s):  
Combined Treatment ◽  
Clonic Seizure ◽  
In Vivo Model ◽  
Anticonvulsant Effect ◽  
No Production ◽  
Acute Administration ◽  
Seizure Models ◽  
Clonic Seizures ◽  
Anticonvulsant Effects

Background and Purpose: Licofelone is a dual 5-lipoxygenase/cyclooxygenase inhibitor, with well-documented anti-inflammatory and analgesic effects, which is used for treatment of osteoarthritis. Recent preclinical studies have also suggested neuroprotective and anti-oxidative properties of this drug in some neurological conditions such as seizure and epilepsy. We have recently demonstrated a role for nitric oxide (NO) signaling in the anti-epileptic activity of licofelone in two seizure models in rodents. Given the important role of N-methyl-D-aspartate receptors (NMDARs) activation in the NO production and its function in the nervous system, in the present study, we further investigated the involvement of NMDAR in the effects of licofelone (1, 3, 5, 10, and 20 mg/kg, intraperitoneal [i.p.]) in an in vivo model of seizure in mice.Methods: Clonic seizures were induced in male NMRI mice by intravenous administration of pentylenetetrazol (PTZ).Results: Acute administration of licofelone exerted anticonvulsant effects at 10 (p<0.01) and 20 mg/kg (p<0.001). A combined treatment with sub-effective doses of the selective NMDAR antagonist MK-801 (0.05 mg/kg, i.p.) and licofelone (5 mg/kg, i.p.) significantly (p<0.001) exerted an anticonvulsant effect on the PTZ-induced clonic seizures in mice. Notably, pre-treatment with the NMDAR co-agonist D-serine (30 mg/kg, i.p.) partially hindered the anticonvulsant effects of licofelone (20 mg/kg).Conclusions: Our data suggest a possible role for the NMDAR in the anticonvulsant effects of licofelone on the clonic seizures induced by PTZ in mice.

scholarly journals Studies on Anticonvulsant Effects of Novel Histamine H3R Antagonists in Electrically and Chemically Induced Seizures in Rats

2018 ◽  
Vol 19 (11) ◽  
pp. 3386 ◽  
Author(s):  
Alaa Alachkar ◽  
Dorota Łażewska ◽  
Gniewomir Latacz ◽  
Annika Frank ◽  
Agata Siwek ◽  
...  
Keyword(s):  
Clonic Seizure ◽  
Maximal Electroshock ◽  
Protective Effects ◽  
Chemically Induced ◽  
Seizure Models ◽  
Dose Dependent Fashion ◽  
Full Protection ◽  
Anticonvulsant Effects

A newly developed series of non-imidazole histamine H3 receptor (H3R) antagonists (1–16) was evaluated in vivo for anticonvulsant effects in three different seizure models in Wistar rats. Among the novel H3R antagonists examined, H3R antagonist 4 shortened the duration of tonic hind limb extension (THLE) in a dose-dependent fashion in the maximal electroshock (MES)-induced seizure and offered full protection against pentylenetetrazole (PTZ)-induced generalized tonic-clonic seizure (GTCS), following acute systemic administration (2.5, 5, 10, and 15 mg/kg, i.p.). However, only H3R antagonist 13, without appreciable protective effects in MES- and PTZ-induced seizure, fully protected animals in the strychnine (STR)-induced GTCS following acute systemic pretreatment (10 mg/kg, i.p.). Moreover, the protective effect observed with H3R antagonist 4 in MES-induced seizure was completely abolished when animals were co-administered with the H3R agonist (R)-α-methylhistamine (RAMH, 10 mg/kg, i.p.). However, RAMH failed to abolish the full protection provided by the H3R antagonist 4 in PTZ-induced seizure and H3R antagonist 13 in STR-induced seizure. Furthermore, in vitro antiproliferative effects or possible metabolic interactions could not be observed for compound 4. Additionally, the predictive in silico, as well as in vitro, metabolic stability for the most promising H3R antagonist 4 was assessed. The obtained results show prospective effects of non-imidazole H3R antagonists as innovative antiepileptic drugs (AEDs) for potential single use against epilepsy.

Zearalenone-induced changes in biochemical parameters, oxidative stress and apoptosis in cardiac tissue

2015 ◽  
Vol 35 (6) ◽  
pp. 623-634 ◽  
Author(s):  
I Ben Salem ◽  
M Boussabbeh ◽  
F Neffati ◽  
MF Najjar ◽  
S Abid-Essefi ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cardiac Tissue ◽  
Fusarium Species ◽  
Combined Treatment ◽  
In Vivo Studies ◽  
Protein Carbonyls ◽  
Acute Administration ◽  
Dependent Manner ◽  
Immunological Parameters

Zearalenone (ZEN) is a mycotoxin from Fusarium species commonly found in food commodities and is known to cause reproductive disorders. Several in vivo studies have shown that ZEN is haematotoxic and hepatotoxic and causes several alterations of immunological parameters. Meantime, the available information on the cardiotoxic effects of ZEN is very much limited. In the present study, we investigated the toxic effects of ZEN in heart tissues of Balb/c mice. We demonstrated that ZEN (40 mg kg−1 body weight (b.w.)) increased creatine phosphokinase, lactate dehydrogenase, aspartate transaminase, alanine transaminase, total cholesterol and triglyceride levels and induced oxidative stress as monitored by measuring the malondialdehyde level, the generation of protein carbonyls, the catalase and superoxide dismutase activity and the expression of the heat shock proteins (Hsp 70). We also demonstrated that acute administration of ZEN triggers apoptosis in cardiac tissue. Furthermore, we aimed to evaluate the safety and efficacy of crocin (CRO), a natural carotenoid, to prevent ZEN-induced cardiotoxicity in mice. In fact, combined treatment of ZEN with different doses of CRO (50, 100, and 250 mg kg−1 b.w.) showed a significant reduction of ZEN-induced toxicity for all tested markers in a dose-dependent manner. It could be concluded that CRO was effective in the protection against ZEN-induced toxicity in cardiac tissue.

scholarly journals Indolent course of tubulointerstitial disease in a mouse model of subpressor, low-dose nitric oxide synthase inhibition

2008 ◽  
Vol 295 (3) ◽  
pp. F717-F725 ◽  
Author(s):  
Adelina Stoessel ◽  
Alexander Paliege ◽  
Franziska Theilig ◽  
Francesco Addabbo ◽  
Brian Ratliff ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Dysfunction ◽  
Basement Membrane ◽  
Mouse Model ◽  
Asymmetric Dimethylarginine ◽  
In Vivo Model ◽  
No Production ◽  
Collagen Xviii

Deficiency of nitric oxide (NO) represents a consistent manifestation of endothelial dysfunction (ECD), and the accumulation of asymmetric dimethylarginine occurs early in renal disease. Here, we confirmed in vitro and in vivo the previous finding that a fragment of collagen XVIII, endostatin, was upregulated by chronic inhibition of NO production and sought to support a hypothesis that primary ECD contributes to nephrosclerosis in the absence of other profibrotic factors. To emulate more closely the indolent course of ECD, the study was expanded to an in vivo model with NG-monomethyl-l-arginine(l-NMMA; mimics effects of asymmetric dimethylarginine) administered to mice in the drinking water at subpressor doses of 0.3 and 0.8 mg/ml for 3–6 mo. This resulted in subtle but significant morphological alterations detected in kidneys of mice chronically treated with l-NMMA: 1) consistent perivascular expansion of interstitial matrix components at the inner stripe of the outer medulla and 2) collagen XVIII/endostatin abundance. Ultrastructural abnormalities were detected in l-NMMA-treated mice: 1) increased activity of the interstitial fibroblasts; 2) occasional detachment of endothelial cells from the basement membrane; 3) splitting of the vascular basement membrane; 4) focal fibrosis; and 5) accumulation of lipofuscin by interstitial fibroblasts. Preembedding labeling of microvasculature with anti-CD31 antibodies showed infiltrating leukocytes and agglomerating platelets attaching to the visibly intact or denuded capillaries. Collectively, the data indicate that the mouse model of subpressor chronic administration of l-NMMA is not a robust one (endothelial pathology visible only ultrastructurally), and yet it closely resembles the natural progression of endothelial dysfunction, microvascular abnormalities, and associated tubulointerstitial scarring.

Contributions of endothelium-derived relaxing factors to control of hindlimb blood flow in the mouse in vivo

2007 ◽  
Vol 293 (2) ◽  
pp. H1072-H1082 ◽  
Author(s):  
Sharyn M. Fitzgerald ◽  
Homaira Bashari ◽  
Jessica A. Cox ◽  
Helena C. Parkington ◽  
Roger G. Evans
Keyword(s):  
Nitric Oxide ◽  
Blood Flow ◽  
Vascular Tone ◽  
Jugular Vein ◽  
Combined Treatment ◽  
Additional Treatment ◽  
Tonic Inhibition ◽  
No Production ◽  
Hyperemic Response

We determined the contributions of various endothelium-derived relaxing factors to control of basal vascular tone and endothelium-dependent vasodilation in the mouse hindlimb in vivo. Under anesthesia, catheters were placed in a carotid artery, jugular vein, and femoral artery (for local hindlimb circulation injections). Hindlimb blood flow (HBF) was measured by transit-time ultrasound flowmetry. Nω-nitro-l-arginine methyl ester (l-NAME, 50 mg/kg plus 10 mg·kg−1·h−1), to block nitric oxide (NO) production, altered basal hemodynamics, increasing mean arterial pressure (30 ± 3%) and reducing HBF (−30 ± 12%). Basal hemodynamics were not significantly altered by indomethacin (10 mg·kg−1·h−1), charybdotoxin (ChTx, 3 × 10−8 mol/l), apamin (2.5 × 10−7 mol/l), or ChTx plus apamin (to block endothelium-derived hyperpolarizing factor; EDHF). Hyperemic responses to local injection of acetylcholine (2.4 μg/kg) were reproducible in vehicle-treated mice and were not significantly attenuated by l-NAME alone, indomethacin alone, l-NAME plus indomethacin with or without co-infusion of diethlyamine NONOate to restore resting NO levels, ChTx alone, or apamin alone. Hyperemic responses evoked by acetylcholine were reduced by 29 ± 11% after combined treatment with apamin plus charybdotoxin, and the remainder was virtually abolished by additional treatment with l-NAME but not indomethacin. None of the treatments altered the hyperemic response to sodium nitroprusside (5 μg/kg). We conclude that endothelium-dependent vasodilation in the mouse hindlimb in vivo is mediated by both NO and EDHF. EDHF can fully compensate for the loss of NO, but this cannot be explained by tonic inhibition of EDHF by NO. Control of basal vasodilator tone in the mouse hindlimb is dominated by NO.

Possible Involvement of Endogenous Opioids and Nitric Oxide in the Anticonvulsant Effect of Acute Chloroquine Treatment in Mice

2017 ◽  
Vol 41 (S1) ◽  
pp. S630-S630
Author(s):  
A. Abkhoo
Keyword(s):  
Nitric Oxide ◽  
Effective Dose ◽  
Clonic Seizure ◽  
Endogenous Opioids ◽  
Seizure Threshold ◽  
Anticonvulsant Effect ◽  
Nitric Oxide Signaling ◽  
Nos Inhibitor ◽  
Neuronal Nos ◽  
Anticonvulsant Effects

IntroductionChloroquine, a 4-aminoquinoline derivative, has long been used for the treatment of malaria and rheumatological disorders, including rheumatoid arthritis and systemic lupus erythematosus. Accumulating evidence now suggests potential use of chloroquine as a neuroprotectant. Studies have shown that nitric oxide (NO) pathway is involved in the chloroquine actions. Considering the fact that nitrergic neurotransmission plays a crucial role in the central nervous system functioning, in the present study we evaluated whether nitrergic system is involved in the anticonvulsant effects of chloroquine in a model of clonicseizure in mice.MethodsClonic seizure threshold was determined by infusion of pentylenetetrazole (PTZ, 0.5%) at a constant rate of 1 mL/min into the tail vein of male Swiss mice (23–29 g). Minimal dose of PTZ (mg/kg of mice weight) needed to induce clonicseizure was considered as an index of seizure threshold.ResultsChloroquine (5 mg/kg, acutely 30 min before test, intraperitoneally), i.p significantly increased the seizure threshold. Acute co-administration of a non-effective dose of the non-selective NO synthase (NOS) inhibitor, L-NAME (L-NG-Nitro-L-arginine methyl ester hydrochloride,5 mg/kg, i.p.) or the selective inhibitor of neuronal NOS, 7-NI (7-nitroindazole, 40 mg/kg, i.p.) with an effective dose of chloroquine (5 mg/kg) inhibited its anticonvulsant effects. Co-administration of a non-effective dose the selective inducible NOS inhibitor, aminoguanidine (100 mg/kg, i.p.) with chloroquine 5 mg/kg did not alter its anticonvulsant effects.ConclusionChloroquine increases the PTZ-induced clonic seizure threshold in mice. We demonstrated for the first time that nitric oxide signaling probably through neuronal NOS could be involved in the anticonvulsant effects of chloroquine in this model of seizure in mice.Disclosure of interestThe author has not supplied his/her declaration of competing interest.

Altered l-arginine metabolism results in increased nitric oxide release from uraemic endothelial cells

2002 ◽  
Vol 103 (1) ◽  
pp. 31-41 ◽  
Author(s):  
Raj C. THURAISINGHAM ◽  
Norman B. ROBERTS ◽  
Mark WILKES ◽  
David I. NEW ◽  
A. Claudio MENDES-RIBEIRO ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
In Vivo Model ◽  
No Production ◽  
Arginine Metabolism ◽  
Enos Expression ◽  
Nitric Oxide Release ◽  
Excessive Consumption ◽  
And Control

Results regarding the nitric oxide (NO) system in uraemia are contradictory. l-Arginine, the precursor of NO, is also metabolized by arginase to form ornithine and urea. In the present study, endothelial NO production and arginine metabolism in uraemia were assessed. In addition an in vivo model was used to examine excess consumption of NO in uraemia. NO and amino acid measurements were made from basal and stimulated (by bradykinin) uraemic and control endothelial cells. Reverse-transcriptase PCR was used to assess endothelial NO synthase (eNOS) and inducible NOS (iNOS) expression. Finally, aortae of uraemic rats were stained for nitrotyrosine (a marker of peroxynitrite). Basal uraemic cells produced more NO than the control cells. l-Arginine levels were greater in uraemic (supernatants/cells), but ornithine levels were higher in control (supernatants/cells). Following stimulation, NO levels in supernatants were similar, but the rise in NO production was greater in control compared with uraemic cells; l-arginine levels still remained higher in uraemic supernatants/cells. Differences in ornithine concentration (supernatants/cells) disappeared following bradykinin stimulation, due to a rise in ornithine levels in the uraemic group. There was no difference in eNOS expression, nor was iNOS detected in either group. Only aortae from uraemic rats showed evidence for nitrotyrosine staining. These studies demonstrated increased basal NO release in uraemic endothelial cells, perhaps by inhibition of arginase and hence diversion of arginine to the NO pathway. The increased NO produced under basal conditions may be inactive due to excessive consumption, resulting in peroxynitrite formation. Interestingly, bradykinin appears to restore arginase activity in uraemia, resulting in normalization of NO production.

scholarly journals Interactions among Lacosamide and Second-Generation Antiepileptic Drugs in the Tonic-Clonic Seizure Model in Mice

2021 ◽  
Vol 22 (11) ◽  
pp. 5537
Author(s):  
Katarzyna Załuska-Ogryzek ◽  
Paweł Marzęda ◽  
Paula Wróblewska-Łuczka ◽  
Magdalena Florek-Łuszczki ◽  
Zbigniew Plewa ◽  
...  
Keyword(s):  
Second Generation ◽  
Screen Test ◽  
Clonic Seizure ◽  
Tonic Clonic Seizure ◽  
Maximal Electroshock ◽  
Isobolographic Analysis ◽  
Clonic Seizures ◽  
Seizure Model

Combination therapy with two or three antiseizure medications (ASMs) is sometimes a preferred method of treatment in epilepsy patients. (1) Background: To detect the most beneficial combination among three ASMs, a screen test evaluating in vivo interactions with respect to their anticonvulsant properties, was conducted on albino Swiss mice; (2) Methods: Classification of interactions among lacosamide (LCM) and selected second-generation ASMs (lamotrigine (LTG), pregabalin (PGB), oxcarbazepine (OXC), and topiramate (TPM)) was based on the isobolographic analysis in the mouse maximal electroshock-induced seizure (MES) model. Interactions among LCM and second-generation ASMs were visualized using a polygonogram; (3) Results: In the mouse MES model, synergy was observed for the combinations of LCM + TPM + PGB and LCM + OXC + PGB. Additivity was reported for the other combinations tested i.e., LCM + LTG + TPM, LCM + LTG + PGB, LCM + LTG + OXC, and LCM + OXC + TPM in this seizure model. No adverse effects associated with triple ASM combinations, containing LCM and second-generation ASMs were observed in mice; (4) Conclusions: The combination of LCM + TPM + PGB was the most beneficial combination among the tested in this study, offering synergistic suppression of tonic-clonic seizures in mice subjected to the MES model. Both the isobolographic analysis and polygonogram method can be recommended for experimental epileptology when classifying interactions among the ASMs.

scholarly journals NAD(P)H oxidase-derived peroxide mediates elevated basal and impaired flow-induced NO production in SHR mesenteric arteries in vivo

2008 ◽  
Vol 295 (3) ◽  
pp. H1008-H1016 ◽  
Author(s):  
Xiaosun Zhou ◽  
H. Glenn Bohlen ◽  
Steven J. Miller ◽  
Joseph L. Unthank
Keyword(s):  
Arterial Disease ◽  
Mesenteric Arteries ◽  
No Production ◽  
Acute Administration ◽  
Spontaneously Hypertensive ◽  
Direct Measurements ◽  
No Signaling ◽  
Wistar Kyoto ◽  
Selective Placement

Nitric oxide (NO) and reactive oxygen species (ROS) have fundamentally important roles in the regulation of vascular tone and remodeling. Although arterial disease and endothelial dysfunction alter NO and ROS levels to impact vasodilation and vascular structure, direct measurements of these reactive species under in vivo conditions with flow alterations are unavailable. In this study, in vivo measurements of NO and H2O2 were made on mesenteric arteries to determine whether antioxidant therapies could restore normal NO production in spontaneously hypertensive rats (SHR). Flow was altered from ∼50–200% of control in anesthetized Wistar-Kyoto rats (WKY) and SHR by selective placement of microvascular clamps on adjacent arteries while NO and H2O2 were directly measured with microelectrodes. Relative to WKY, SHR had significantly increased baseline NO and H2O2 concentrations (2,572 ± 241 vs. 1,059 ± 160 nM, P < 0.01; and 26 ± 7 vs. 7 ± 1 μM, P < 0.05, respectively). With flow elevation, H2O2 but not NO increased in SHR; NO but not H2O2 was elevated in WKY. Apocynin and polyethylene-glycolated catalase decreased baseline SHR NO and H2O2 to WKY levels and restored flow-mediated NO production. Suppression of NAD(P)H oxidase with gp91ds-tat decreased SHR H2O2 to WKY levels. Addition of topical H2O2 to increase peroxide to the basal concentration measured in SHR elevated WKY NO to levels observed in SHR. The results support the hypothesis that increased vascular peroxide in SHR is primarily derived from NAD(P)H oxidase and increases NO concentration to levels that cannot be further elevated with increased flow. Short-term and even acute administration of antioxidants are able to restore normal flow-mediated NO signaling in young SHR.

scholarly journals Involvement of N-methyl-D-aspartate receptors and nitric oxide in the anticonvulsant effects of dantrolene against pentylenetetrazole-induced seizures in mice

2017 ◽  
Vol 04 (02) ◽  
pp. 112-118
Author(s):  
Akbarzadeh Samad ◽  
Heidary Fatemeh ◽  
Keshavarz Mojtaba
Keyword(s):  
Nitric Oxide ◽  
Nmda Receptors ◽  
Clonic Seizure ◽  
Negative Control ◽  
No Synthase ◽  
Tonic Clonic Seizure ◽  
Control Group ◽  
No Donor ◽  
Clonic Seizures ◽  
Anticonvulsant Effects

Abstract Objective N-methyl-d-aspartate (NMDA) receptors and nitric oxide (NO) have important roles in the pathology and treatment of pentylenetetrazole (PTZ)-induced seizures. We aimed to show the involvement of these two systems in the anticonvulsant effects of dantrolene against PTZ-induced seizures. Methods The male albino Swiss strain of mice (N = 56) randomly allocated to the seven separate groups and treated with dantrolene (40 mg/kg), dantrolene (40 mg/kg) + L-arginine (100 mg/kg, a NO donor), dantrolene (40 mg/kg) + N-Nitroarginine methyl ester (L-NAME) (100 mg/kg, a NO synthase inhibitor), dantrolene (40 mg/kg) + NMDA (50 mg/kg), dantrolene (40 mg/kg) + MK801 (1 mg/kg, a selective NMDA antagonist), Diazepam (5 mg/kg, the positive control) and saline (the negative control). Seizures were induced by intraperitonial injection of PTZ (90 mg/kg). The onsets of clonic and tonic-clonic seizures, as well as the death of animals, were recorded. Results Dantrolene significantly increased the onset of clonic, tonic-clonic seizures and death of animals challenged with PTZ. The onset of tonic-clonic seizure in animals treated with dantrolene alone and dantrolene + L-NAME was higher than the control group. In contrast, the onset of tonic-clonic seizure in the animals treated with dantrolene + L-arginine was significantly lower than the dantrolene-treated group. The onset of clonic and tonic-clonic seizures in animals treated with dantrolene + MK801 were significantly higher than the control and dantrolene + NMDA groups. Conclusion Dantrolene protected animals against PTZ-induced seizures and mortality. The inhibition of NO synthase and NMDA receptors may contribute to the dantrolene anticonvulsant effects on the PTZ-induced seizure.

scholarly journals COMPARATIVE STUDY OF ANTICONVULSANT EFFECT OF THE LEAVES OF SAPINDUS EMARGINATUS AND ACORUS CALAMUS IN EXPERIMENTALLY INDUCED ANIMAL MODELS OF EPILEPSY

2021 ◽  
pp. 36-39
Author(s):  
PRIYADARSHINI SHOUGRAKPAM ◽  
ABHISHEK BHATTACHARJEE ◽  
NGANGOM GUNINDRO ◽  
SANJENBAM RITA
Keyword(s):  
Anticonvulsant Activity ◽  
Clonic Seizure ◽  
Acorus Calamus ◽  
Anticonvulsant Effect ◽  
Maximal Electroshock ◽  
Group V ◽  
Gum Acacia ◽  
Group I ◽  
Seizure Models ◽  
Animal Models Of Epilepsy

Objective: To compare anticonvulsant activity of methanol extracts of Sapindus emarginatus (MESE) and Acorus calamus (MEAC) in experimental seizure models in mice. Methods: Hind limb tonic extension (HLTE) in Maximal electroshock (MES) seizure and clonic seizure in Pentylenetetrazol (PTZ) seizure models were assessed. Group I (control) mice received 1% gum acacia in distilled water (1 ml/100 g). Topiramate (50 mg/kg) was administered in group II (standard) animals. Group III and IV mice were treated with 200 and 400 mg/kg of MESE, respectively. Mice in group V and VI were given MEAC at the dose of 200 and 400 mg/kg, respectively. Drugs were given orally suspended in 1% gum acacia suspension (1 ml/100 g) for 7 d. Next day after 1 h of drug administration, the seizure was induced for evaluation. Results: Anticonvulsant property of both extracts was confirmed by reduction (p<0.001) in HLTE phase in MES model; delayed onset of the clonic seizure (p<0.001) and its shortened phase (p<0.001) in PTZ model when compared with the control. MESE-200 mg/kg produced significantly longer (p<0.001) HLTE phase with lower protection (40.34%) among the different doses of the extracts. Clonic seizure onsets and durations in PTZ model were comparable among the different extract-treated groups; however, mortality was higher (66.6%) with MESE-200 mg/kg. Conclusion: Anticonvulsant activity of MESE and MEAC was evident; however, MESE at the dose of 200 mg/kg was less effective.

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