LCX001 was provided by the Laboratory of Computer-Aided Drug Design & Discovery, Beijing Institute of Pharmacology and Toxicology (Xiao et al., 2016 (link)). Fentanyl (Sigma, Beijing, China) and TH-030418 were administered to generate opioid-induced respiratory depression models. TH-030418 (N-methyl-7a-[(R)-1-hydroxy-1-methyl-3-(thien-3-yl)-propyl]-6,14-endo-ethanotetrahydronororipavine) was a thienorphine derivative, synthesized by our institute. Compared with morphine or dihydroetorphine, TH-030418 displayed non-selective and high binding affinity to all subtypes of opioid receptors (Wen et al., 2011 (link)). TH-030418 also exerted a strong and long-acting analgesic effect. Ampakines, LCX001 and CX614 (Tocris, Bristol, United Kingdom), were dissolved in a 20% hydroxypropyl-β-cyclodextrin (HPCD; Sigma-Aldrich, Beijing, China) 0.45% saline solution. Propofol was purchased from AstraZeneca (Viale Dell’industria, Italy). Pentobarbital (Nembutal; Merck, Beijing, China), Fentanyl, TH-030418, and morphine hydrochloride (Qinghai Pharmaceutical Factory, Xining, China) were dissolved in 0.45% saline solution at room temperature and used at different doses. The volume of each dose was 1 ml/kg for SD rats and 10 ml/kg for KM mice.
Fentanyl
Manufactured by Merck Group
Sourced in United States
Description not available
Automatically generated - may contain errors
Lab products found in correlation
Diazepam, by Merck Group (4 mentions)
Tramadol, by Merck Group (2 mentions)
Forskolin, by Merck Group (2 mentions)
3 isobutyl 1 methyl xanthine (ibmx), by Merck Group (2 mentions)
Methadone, by Merck Group (2 mentions)
Imipramine, by Merck Group (2 mentions)
Desipramine, by Merck Group (2 mentions)
Superfrost plus slide, by Thermo Fisher Scientific (2 mentions)
Hydroxypropyl β cyclodextrin hpcd, by Merck Group (1 mentions)
Propofol, by AstraZeneca (1 mentions)
Antibiotic mixture, by Thermo Fisher Scientific (1 mentions)
Droperidol, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
D ala2, by Merck Group (1 mentions)
N me phe4, by Merck Group (1 mentions)
Fentanyl, by Cayman Chemical (1 mentions)
Codeine, by Merck Group (1 mentions)
Amiloride, by Merck Group (1 mentions)
Glibenclamide, by Merck Group (1 mentions)
25 protocols using fentanyl
1
Opioid-Induced Respiratory Depression Models
2
Chronic Opioid Administration Protocol
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Morphine, methadone, oxycodone (Francopia, France), and fentanyl (Sigma Aldrich, France), all hydrochloride, were dissolved in saline solution (0.9% NaCl) and injected via i.p. route. All animals received 0.1mL/10g of bodyweight. Chronic treatment consisted of 2 daily injections (at 9:00 am and 5:00 pm ) with morphine (10mg/kg), methadone (2.5mg/kg), fentanyl (0.25mg/kg), or oxycodone (1mg/kg). These doses are equally effective in the locomotor activity assay (supplementary Figure 1 ). This test was chosen as it has a larger range of dose response than the hot-plate, where 10mg/kg of morphine results in 100% of analgesia (Figure 1 ). Control groups received saline under the same conditions.
3
Histological Analysis of Carotid Artery Endothelial Cells
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Histological analysis was performed as described previously (27 (link)). At 4 weeks after HCHFD treatment and MCP interventions, all rats were sacrificed following anesthesia with Fentanyl (0.4 mg/kg; Sigma-Aldrich; Merck KGaA) and carotid artery vascular endothelial cells (CAVECs) were collected. CAVECs were fixed in 4% phosphate-buffered saline-buffered formalin for >24 h, followed by processing for conventional paraffin embedding. Sections (5-um thickness) were mounted on glass slides, dewaxed, rehydrated with distilled water and stained with hematoxylin and eosin. The CAVECs were examined using a light microscope to identify pathological changes.
4
Immunohistochemistry of Lymphatic and Vascular Markers
5
Opioid receptor agonist evaluation
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[D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) and fentanyl were obtained from Sigma, St. Louis, MO, USA. Morphine and oxycodone were obtained from the NIDA Drug Supply Program. NKTR-181 was supplied by Nektar Therapeutics (San Francisco, CA, USA). For cellular experiments, all compounds were made up as stock solutions in water and diluted and used on the day of the experiment. The same stocks were used for all cellular experiments. For behavioral outcomes, oxycodone and NKTR-181 were dissolved in sterile saline immediately prior to use. The protocols for the use and disposal of Schedule II compounds followed the guidelines outlined by the UCLA Department of Environmental Health and Safety.
6
Examining Opioid Addiction and IRAK4 Inhibitor
7
Fentanyl Self-Administration and Abstinence Tests
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After a week of recovery following surgery, we trained rats to self-administer fentanyl for 10 days as described above. For the first cohort of animals (n = 12) we trained rats to self-administer fentanyl (Sigma Aldrich) at 2.5 μg/kg/infusion. For the second cohort (n = 16), fentanyl was not available from our original source. fentanyl sourced from Cayman Chemical visibly occluded rats from responding at 2.5 μg/kg/infusion. Thus, for cohort 2 we reduced the concentration of fentanyl (Cayman Chemical) to 2.0 μg/kg/infusion for days 3 and 4 of self-administration and to 1.5 μg/kg/infusion for the rest of the self-administration phase. Since the terminal levels of responding for both cohorts were not significantly different (see section “Supplementary Results ”), we pooled the data together. During training, Active Lever presses resulted in fentanyl infusion paired with a compound 5s tone-light cue located above the Active Lever.
Incubation extinction tests: After 10 days of training, we injected vehicle intracranially into the BNST of all rats 15 minutes prior to Day 1 of forced abstinence extinction test. After 30 days of forced abstinence (Day 30), we retested the same rats and injected either intra-BNST vehicle in n = 13 rats or intra-BNST R121919 in n = 15 rats in a mixed within-between subject design.
Incubation extinction tests: After 10 days of training, we injected vehicle intracranially into the BNST of all rats 15 minutes prior to Day 1 of forced abstinence extinction test. After 30 days of forced abstinence (Day 30), we retested the same rats and injected either intra-BNST vehicle in n = 13 rats or intra-BNST R121919 in n = 15 rats in a mixed within-between subject design.
8
Electrophysiological Effects of Opioid Drugs
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Tramadol, fentanyl, and codeine (purity ≥98%), purchased from Sigma-Aldrich, were freshly dissolved in the bath solution to the desired concentration just before use. Nav1.5 current was measured at baseline conditions (no drug) and after 5–8 min wash-in of these drugs at various concentrations that are close to the lethal concentrations of these drugs.33–35 (link) Tramadol was measured at 1, 10, 30, 100, 300, or 1000 µm . fentanyl was measured at 1 µm . codeine was measured at 100 µm .
9
Comprehensive Inflammatory Modulation Protocol
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Carrageenan, lipopolysaccharide (LPS) E. coli 0111:B4, indomethacin, BK, prostaglandin E2 (PGE2), dopamine, forskolin, dibutyryl cyclic adenosine monophosphate (cAMP), glibenclamide, dipyrone, fentanyl, diazepam, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), ruthenium red, camphor, amiloride, capsaicin, cinnamaldehyde, and menthol were purchased from Sigma (St. Louis, MO, USA). Tumor necrosis factor- α (TNF-α), interleukin-1β (IL-1β) and cytokine-induced neutrophil chemoattractant 1 (CINC-1) were purchased from R&D Systems (Pittsburgh, PA, USA). Ketamine and xylazine were obtained from Syntec Laboratory (Cotia, SP, Brazil). Oxytetracycline hydrochloride was purchased from Pfizer (São Paulo, SP, Brazil). Dexamethasone was obtained from Química Santa Marina (Rio de Janeiro, RJ, Brazil).
10
Opioid Vaccine Formulation and Administration
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Fentanyl was acquired from Sigma–Aldrich, and heroin was provided
by NIDA. Heroin is soluble in bacteriostatic 0.9% NaCl solution; however,
the Fentanyl solubility in aqueous solution is limited (0.2 mg/mL
in water). Therefore, Fentanyl was dissolved in 10% dimethyl sulfoxide,
10% Tween 80, and saline for drug administration. Heroin24 (link) and Fentanyl haptens11 (link) were synthesized per our literature procedure, and protein conjugation
proceeded as previously described.23 (link) Vaccines
were formulated with 50% (v/v) glycerol and 50 μg of CpG/dose
per mouse and stored at −80 °C. Immediately prior to administration,
the vaccines were thawed and mixed with 0.8 mg of alum/dose per mouse
for at least half an hour.
by NIDA. Heroin is soluble in bacteriostatic 0.9% NaCl solution; however,
the Fentanyl solubility in aqueous solution is limited (0.2 mg/mL
in water). Therefore, Fentanyl was dissolved in 10% dimethyl sulfoxide,
10% Tween 80, and saline for drug administration. Heroin24 (link) and Fentanyl haptens11 (link) were synthesized per our literature procedure, and protein conjugation
proceeded as previously described.23 (link) Vaccines
were formulated with 50% (v/v) glycerol and 50 μg of CpG/dose
per mouse and stored at −80 °C. Immediately prior to administration,
the vaccines were thawed and mixed with 0.8 mg of alum/dose per mouse
for at least half an hour.
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