CIA was induced as describe above. Two weeks after the first immunization, rats were randomly assigned to the following groups: (1) normal group, model group, curcumin (100 mg/kg) group, mecamylamine (1 mg/kg, Sigma) group, curcumin (100 mg/kg) + mecamylamine (1 mg/kg) group, hexamethonium (4 mg/kg, Sigma) group, curcumin (100 mg/kg) + hexamethonium (4 mg/kg) group, and leflunomide (2 mg/kg) group; (2) normal group, model group, curcumin (100 mg/kg) group, α-bungarotoxin (1 μg/kg, Abcam Inc., Cambridge, UK) group, curcumin (100 mg/kg) + α-bungarotoxin (1 μg/kg) group, and nicotine (300 μg/kg) group. Curcumin and leflunomide were suspended in 0.5% CMC-Na and orally administered daily 2 weeks. Antagonists were dissolved in 0.9% NaCl solution and intraperitoneally injected 10 min before curcumin administration. Nicotine was dissolved in 0.9% NaCl solution and intraperitoneally injected. Normal and model group rats were orally administered 0.5% CMC-Na in the same schedule.
α bungarotoxin
Manufactured by Abcam
Sourced in United Kingdom, United States
α-bungarotoxin is a neurotoxin that binds specifically and with high affinity to nicotinic acetylcholine receptors. It is commonly used as a research tool to study the structure, function, and distribution of nicotinic acetylcholine receptors in various biological systems.
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Lab products found in correlation
Mecamylamine, by Merck Group (1 mentions)
Hexamethonium, by Merck Group (1 mentions)
Gts 21, by Abcam (1 mentions)
Antibodies against β actin, by Merck Group (1 mentions)
Antibodies against iba1, by Fujifilm (1 mentions)
Pparγ, by Cell Signaling Technology (1 mentions)
Antibodies against bcl 2, by Abcam (1 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (1 mentions)
Sp8 inverted scanning confocal microscope, by Leica (1 mentions)
C9891, by Merck Group (1 mentions)
P 97 puller, by Sutter Instruments (1 mentions)
Histamine, by Merck Group (1 mentions)
Prostaglandin e2, by Merck Group (1 mentions)
6 protocols using α bungarotoxin
1
Curcumin and Nicotinic Receptor Modulators in CIA
2
Neuroinflammation and Oxidative Stress
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GTS-21, α-bungarotoxin, and antibodies against Bcl2 and PGC-1α were purchased from Abcam (Cambridge, UK). LPS (Escherichia coli serotype 055:B5), methyllycaconitine, and antibodies against β-actin and BDNF were purchased from Sigma-Aldrich (St. Louis, MO, USA). MPTP was obtained from Tokyo Chemical Industry Co. (Tokyo, Japan). Antibodies against Iba-1 were purchased from Wako (Osaka, Japan). Antibodies against phospho-/total forms of AMPK, Akt, MAP kinases, CREB, and antibodies for IL-6, TGF-β, NQO1, PPAR-γ, and TH were obtained from Cell Signaling Technology (Beverley, CA, USA). While antibodies against phospho-p47phox were provided by Assaybiotech (Sunnyvale, CA, USA), those against TNF-α, Nrf2, HO-1, catalase, lamin A, and COX-2 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against iNOS and IL-1β were purchased from BD Biosciences (San Jose, CA, USA), and an antibody for 4-hydroxy-2E-nonenal (HNE) was purchased from Alpha Diagnostic International (San Antonio, TX, USA).
3
Immunostaining of Neuromuscular Junctions
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Animals were fixed in 4% paraformaldehyde for 3 h at room temperature. After fixation, the embryos were rinsed several times with PBS and then incubated in PBS containing 1 mg/mL collagenase (20 min, C9891, Sigma-Aldrich, Saint-Quentin Fallavier, France) to remove skin. The collagenase was washed off with PBS Triton X-100 (PBST; 1 h) and heads were cut away. After an incubation of 30 min in blocking solution (1% BSA, 1% triton, PBS, 2% goat serum), the embryos were incubated overnight at 4 °C in synaptic vesicle 2 (sv2, 1:200; Developmental Studies Hybridoma Bank; University of Iowa, Iowa USA) antibody diluted in blocking solution. The embryos were then washed and incubated for 30 min in PBST containing α-bungarotoxin conjugated to Alexa 488 (αBTX, 1:1000; Abcam). The embryos were rinsed several times with PBST and then incubated in freshly prepared block solution containing a secondary antibody (Alexa Fluor 568, 1:1000; Life Technologies, Saint-Aubin, France) for 3h at RT before mounting on glass slide in 50% glycerol. The NMJs were visualized using a Leica SP8 Inverted scanning confocal microscope. All captured images of stained embryos were processed using Imaris Image Analysis software and ImageJ.
4
Investigating a7nAChR in VNS-Induced Neuroprotection
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To further investigate the function of a7nAChR in the VNS-induced neuroprotective response, the specific a7nAChR antagonist α-bungarotoxin was used to inhibit a7nAChR expression level. The rats were deeply anesthetized with 10% hydration chlorine aldehyde (0.35 ml/kg; intraperitoneal injection) and were placed in a stereotaxic frame with a head holder. The α-bungarotoxin (0.5 ug/kg; Abcam, USA) and equivalent volume of saline (antagonist placebo control) were injected into the left lateral ventricle of rats in each group. We repeated the lateral cerebral ventricle injection test alone in advance to finally determine the injection stereotaxic coordinates: 1 mm before the anterior fontanel, 1.5 mm to the left, and a depth of 4.5 mm, and marked the location on the skull surface with ink. We used a dental drill to drill the point to the dura mater, then punctured the dura mater with a needle tip, using sterile cotton swabs to stop bleeding and dry the surgical field. A catheter was inserted 4.5 mm vertically, and then we slowly injected the drugs. The catheter was removed 1 min after administration of drugs, then we sutured the scalp incision. After 30 min, rats were subjected to PMCAO surgery [26 ]. However, the specific agonist of a7nAChR (PHA543613; 1.0 mg/kg; R&D System, USA) was injected intraperitoneally as the reagent can pass through the blood-brain barrier.
5
Extracellular Recording of Lateral Line Neurons
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Our recording procedures were previously described in detail [28] (link). Briefly, larvae were anesthetized, mounted, and microinjected in the heart with 125 µM α-bungarotoxin to block muscle activity (Abcam, Cambridge, Massachusetts). Larvae were then rinsed and returned to normal extracellular solution (in mM: 130 NaCl, 2 KCl, 2 CaCl2, 1 MgCl2 and 10 HEPES, pH 7.8, 290 mOsm). Extracellular recordings were performed at room temperature with borosilicate glass recording electrodes (Sutter Instruments, Novato, CA) fabricated with long tapers and resistances between 5 and 15 MΩ in extracellular solution (P-97 Puller; Sutter Instruments, Novato, CA). Extracellular action currents were recorded from an individual lateral line afferent neuron in the loose-patch configuration (seal resistances ranged from 20 to 80 MΩ). Recordings were done in voltage-clamp mode, sampled at 50 µs/pt, and filtered at 1 kHz with an EPC 10 amplifier and Patchmaster software (Heka Electronic, Bellmore, New York).
6
Modulation of Neuroinflammation in Rats
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Rats were randomly divided into 4 groups: the control, CM, PUN-282987, and α-bungarotoxin groups. In the control group, 2 μL of PBS (0.1 M, pH 7.4) was slowly infused through the cannula. In the other 3 groups, 2 μL of IS was infused. The IS contained 1 mM histamine, serotonin, and bradykinin and 0.1 mM prostaglandin E2 (all from Sigma, St. Louis, MO, USA). After 7 days of exposure by infusion, the vehicle (PBS, 0.1 M, pH 7.4) was administered by intracerebroventricular (I.C.V) injection to the control and CM groups, and PNU-282987 (2.5 μmol/animal, P6499-10MG; Sigma) and α-bungarotoxin (1.0 μg/animal, ab120542; Abcam, Cambridge, MA, USA) were administered to the PNU-282987 and α-bungarotoxin groups, respectively (on the eighth day; Figure 1 ). The optimal doses were based on previous studies in rats.49 (link),50 (link)
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