Mice were injected intraperitoneally on different days, following a rotational design, with: (i) 1 ml/kg saline solution (0.9% NaCl; control condition); (ii) 0.1 mg/kg methylscopolamine (muscarinic receptor antagonist, Sigma-Aldrich); (iii) 1 mg/kg atenolol (β1-adrenergic receptor antagonist, Sigma-Aldrich); (iv) methylscopolamine + atenolol (at the same above-indicated doses; for both vagal and sympathetic blockade). Each injection was separated by at least a 2-day washout period. Drug doses were selected on the basis of previous studies (Statello et al., 2017 (link)). ECG recordings were performed during the hour that preceded (baseline condition) and the 2 h that followed each injection. All injections were done between 14.00 and 15.00 h.
Methylscopolamine
Manufactured by Merck Group
Sourced in United States, Macao, France
Methylscopolamine is a pharmaceutical compound used as a laboratory reagent. It functions as a muscarinic acetylcholine receptor antagonist.
Automatically generated - may contain errors
Lab products found in correlation
Pilocarpine, by Merck Group (25 mentions)
Lithium chloride (licl), by Merck Group (9 mentions)
Pilocarpine hydrochloride, by Merck Group (6 mentions)
Diazepam, by Merck Group (6 mentions)
Terbutaline, by Merck Group (3 mentions)
Diazepam, by Pfizer (3 mentions)
Medetomidine hydrochloride, by Bio-Techne (2 mentions)
Water for injection, by Baxter (2 mentions)
Isoflurane, by Baxter (2 mentions)
Kainic acid, by Bio-Techne (1 mentions)
Sc 58125, by Bio-Techne (1 mentions)
Pentobarbital sodium, by Merck Group (1 mentions)
Lithium carbonate, by Merck Group (1 mentions)
Pilocarpine hcl, by Merck Group (1 mentions)
β actin antibody, by Cell Signaling Technology (1 mentions)
Psc833, by Novartis (1 mentions)
L9650, by Merck Group (1 mentions)
Atenolol, by Merck Group (1 mentions)
Pilocarpine, by MP Biomedicals (1 mentions)
Sprague dawley rats, by Inotiv (1 mentions)
40 protocols using methylscopolamine
1
Impact of Autonomic Blockade on ECG
2
Synthesis and Characterization of Compound TG6-10-1
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Methylscopolamine, terbutaline, pilocarpine, and sodium pentobarbital were purchased from Sigma‐Aldrich. SC‐58125 and kainic acid were purchased from Tocris Bioscience. 16,16‐dimethyl prostaglandin E2 (dmPGE2) was purchased from Cayman Chemical. Diazepam was purchased from Henry Schein. Compound TG6‐10‐1 was synthesized accordingly,22 and the purity was confirmed by LC/MS and NMR in the Medicinal Chemistry Core at the University of Tennessee Health Science Center.
3
Lithium-Pilocarpine Seizure Model in Rats
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The procedure was essentially as described previously [26 (link),29 (link)] with minor modifications. At postnatal day 23 (P23), male Sprague Dawley rats (the animal colony of the Mossakowski Medical Research Institute, Polish Academy of Sciences in Warsaw) were injected intraperitoneally (i.p.) with lithium carbonate (222 mg/kg; Sigma-Aldrich, Steinheim, Germany) dissolved in saline (pH equalized to 7.4). At P24, 18–20 h after Li+ treatment, animals were injected i.p. with methyl-scopolamine (1 mg/kg; Sigma-Aldrich, Steinheim, Germany) and thirty minutes later with pilocarpine (40 mg/kg; Sigma-Aldrich, Steinheim, Germany). From then on, the animal’s behavior was continuously monitored utilizing five-stage Racine scale [72 (link)]: 1: mouth and facial movement, 2: head nodding, 3: forelimb clonus, 4: rearing with forelimb clonus, 5: rearing and falling with forelimb clonus, considering stages 1–3 as a focal and 4–5 as a generalized seizure. Rats were decapitated either 15′ (short period) or 60′ (long period) after Pilo administration (see also Section 4.4 ). Control rats that did not receive Pilo were given equal volumes of saline at the same time.
4
Pilocarpine-Induced Epilepsy Model in Rats
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The pilocarpine model of epilepsy represents a suitable paradigm for studying the pathophysiological mechanisms of TLE and has been used in several studies (28 (link)). In summary, male and female Wistar rats were pre-treated with methyl scopolamine (Sigma Co.) at a dose of 1 mg/kg subcutaneously to inhibit the peripheral cholinergic effects of pilocarpine (12 (link)). Thirty minutes later, pilocarpine HCl (Sigma Co.) at a dose of 300 mg/kg was injected intraperitoneally with the aim of inducing long-standing SE (12 (link), 29 (link)). After 5 h of SE, diazepam (Merck) at a dose of 10 mg/kg was administered subcutaneously to limit motor seizures (30 (link)). The surviving animals (7 males and 9 females) were fed a special fractionated diet until complete recovery, which ends the acute period of the model. After this period, the animals were transferred to video surveillance. Control male and female rats were injected with methyl scopolamine (1 mg/kg, s.c.) followed 30 min later by a saline injection (1 ml/kg, i.p.) as a substitute for pilocarpine.
5
Pilocarpine-induced Status Epilepticus Model
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The PILO-induced SE model was established according to our previous description [14 (link)]. Briefly, ICR mice (9–10 weeks old) were injected with methyl scopolamine (1 mg/kg, Sigma-Aldrich, St. Louis, MO, USA) intraperitoneally (i.p.) 30 min prior to PILO (290 mg/kg, i.p., Sigma-Aldrich, St. Louis, MO, USA) administration. The animals were placed in a plastic chamber (10 × 15 × 30 cm), and their behavior was observed before and after PILO administration. The control mice were injected with saline (0.1 mL/10 g i.p.) instead of PILO. SE was defined by the occurrence of five generalized convulsive seizures. To terminate SE, all mice were injected with diazepam (DZP; Cercine®, 10 mg/kg, i.p.; Takeda Pharmaceutical Ltd. Osaka, Japan), once or more as needed to suppress convulsive seizures. LEV (LKT Labs, Inc., St. Paul, MN, USA) was orally administered at a dose of 360 mg/kg within 30 min after DZP injection and thereafter twice a day (at 8:30 and 17:30). As a result, the SE + LEV 6 h group was given LEV once, and the SE + LEV 2 days group was given LEV 5 times.
6
Chronic Epilepsy Model in C57BL/6 Mice
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A total of 118 five-week-old male C57BL/6 mice (18–22 g) were used for the generation of chronic epilepsy models. A single systemic injection of pilocarpine (400 mg/kg, intraperitoneal, Sigma, St. Louis, MO, USA) was given to each mouse for the induction of SE, as described previously [17 (link)–19 ]. To reduce peripheral muscarinic effects, methylscopolamine (1 mg/kg, intraperitoneal, Sigma, St. Louis, MO, USA) was administered to the mice 30 min before the pilocarpine injection. To interrupt the prolonged seizure, diazepam (5 mg/kg, intraperitoneal) was injected at 40 min after the onset of SE. SE was defined as continuous tonic-clonic seizures following several discontinuous convulsive seizures (stage ≥ 4) [20 (link)].
7
Investigating P-glycoprotein Modulators
8
Pilocarpine-Induced Seizures in Mice
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To determine the seizure threshold, 8-week-old mice were pre-treated with 423 mg/kg lithium chloride (intraperitoneally (i.p.), Sigma, L9650). 18 h later we injected 1 mg/kg Methylscopolamine (Sigma, S8502). Seizures were induced 30 min later by intraperitoneal injection of 80 mg/kg pilocarpine (Sigma, P6503). Seizures were scored for 3 h according to a modified Racine scale: 0 (no changes, normal behavior), 1 (behavioral arrest, motionless starring, orofacial automatism), 2 (head nodding), 3 (lordotic posture, chewing), 4 (rearing), 5 (falling, loss of postural control), 6 (generalized tonic-clonic activity, complete loss of control). Surviving animals were sacrificed 4 weeks later (Racine, 1972 (link)).
9
Lithium chloride-pilocarpine rat model of status epilepticus
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All procedures were carried out in accordance with University of Nizhny Novgorod regulations. 3–6 weeks old male Sprague-Dawley (Wistar for LTP experiments) rats were injected with lithium chloride (127 mg/kg, Sigma Aldrich) 20–24 h prior to pilocarpine and methylscopolamine (1 mg/kg, Sigma Aldrich) 20 min prior to pilocarpine. Then pilocarpine (Tocris), 10 mg/kg was injected every 30 min (but no more than 60 mg/kg) to induce SE which characterized with generalized seizures lasting for at least 20 min (Supplementary Figure 1 ). To reduce mortality, phenazepam 1 mg/kg was injected every 10 min for 30–40 min after 20 min of generalized seizures.
10
Lithium Chloride-Induced Pilocarpine Epilepsy Model
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