One week after surgery, kainic acid (5.3 mg/ml PBS; Cayman Chemical, Ann Arbor, MI, USA) was injected intraperitoneally at a dose of 25–100 mg kainic acid/kg mouse body weight (in increments of 10–25 mg/kg) until the mouse went into status epilepticus, defined as persistent epileptiform activity on the EEG accompanied by recurrent clinical seizures for at least 12 hours (the minimum time we determined in pilot experiments to induce epilepsy). Some mice received diazepam i.p. (5 mg/kg) (Hospira, Lake Forest, IL, USA) if clinically evident convulsions were persistent and severe. Mice were injected with PBS (i.p.) 4–8 hr after induction of status epilepticus to maintain adequate hydration.
Kainic acid
Manufactured by Cayman Chemical
Sourced in United States
Kainic acid is a naturally occurring amino acid that acts as a potent agonist of kainate receptors, a class of ionotropic glutamate receptors in the central nervous system. It is commonly used as a research tool in the study of excitotoxicity and neurodegenerative processes.
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8 protocols using kainic acid
1
Kainic Acid-Induced Epilepsy Model
2
Kainic Acid-Induced Seizures in Scn8a Mouse
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Kainic acid (Cayman Chemical) was dissolved in phosphate-buffered saline at 2.5 mg/ml. Adult Scn8aR1872W/+,CAG-Cre-ER+/− mice were first treated with a tamoxifen dose of 1.0 mg/20 g body weight to activate the conditional allele. Six weeks after tamoxifen treatment, Scn8aR1872W/+,CAG-Cre-ER+/− mice and littermate controls lacking Cre were randomly selected and injected intraperitoneally with 20 mg/kg Kainic acid. Mice were monitored for 2 h. Seizure severity was scored by an experimenter blinded to genotype. As no sex differences were observed, data from both sexes were combined.
3
Kainic Acid-induced Neurodegeneration Model
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Kainic acid (KA, 78050, Cayman Chemicals, Ann Arbor, MI), indomethacin (IND, 19233-51, Nacalai tesque, Tokyo, Japan), liraglutide (2499410G1021, Novo Nordisk, Bagsværd, Danmark), and sitagliptin phosphate monohydrate (SPM, A4036, ApexBio, Boston, MA, USA) were used for animal treatments. Fluoro Jade C (FJC, TR-100-FJ, Biosensis, CA) was used for staining of degenerating neurons. Pre-stained Protein Marker (02525-35, Nacalai tesque, Tokyo, Japan) was used for Western blots. MK-0524 (DP1 antagonist, 1480, Axon MEDCHEM, Groningen, Netherlands), OC000459 (DP2 antagonist, 1913, Axon MEDCHEM, Groningen, Netherlands) were used as DP selective inhibitors.
4
Neuronal ER Stress Modulation Assay
5
Characterizing Neuronal Responses in Rat Primary Cortex
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Rat primary cortical neurons were prepared from Sprague-Dawley embryos as previously described31 (link) and in accordance with approved procedures by the NIH ACUC. Isolated cells were plated at 6 × 104 cells/well in 96-well polyethyleneimine-coated plates. Fifty percent media exchanges were performed on days in vitro (DIV) 4, 6, 8, 11, and 13. On DIV6, transductions were performed with the following viruses: AAV1-CMV’-GLuc (5.8 × 109 vg/mL), AAV1-SYN1-GLuc (5.8 × 109 vg/mL), AAV1-EF1α-GLuc (5.8 × 109 vg/mL), AAV1-EF1α-iCre (1.0 × 1011 vg/mL), AAV1-Syn1-Dio-hM3D(Gq)-mCherry (1.0 × 1011 vg/mL), and AAV1-Syn1-DIO-hM4D(Gi)-mCherry (1.0 × 1011 vg/mL). On DIV13, cells were treated with kainic acid (100 μM; Cayman Chemical Company, Ann Arbor, MI, USA), glutamate (100 μM; Sigma-Aldrich), or CNO (0.03, 0.3, 3 μM; Enzo Life Sciences, Farmingdale, NY, USA) via 50% media exchange. Extracellular media samples were collected 24 h post-treatment. Subsequently, a subset of cells was rinsed twice with PBS and incubated with lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% Nonidet P-40 [NP-40], and protease inhibitors; Sigma-Aldrich) for 20 min at 4°C. Cell viability was assessed 24 h post-treatment via CellTiter96 AQueous One Solution Cell Proliferation Assay (MTS assay; Promega).
6
Modeling Kainic Acid-Induced Status Epilepticus
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The SE model was established in SE group and MINO group by intraperitoneal (i.p.) injection of kainic acid (1.5 mg/kg, Cayman Chemical Company). Diazepam (0.5 mg/kg, i.p., Kingyork Group) was administered 30 min after the beginning of seizures of score 3 to terminate the SE. After successful modeling, the mice in the MINO group were administered intraperitoneal injections of minocycline (10 mg/kg, Sigma–Aldrich) once a day for 3 consecutive days. The Ctl group mice received only 0.9% sodium chloride solution and diazepam. Mice were excluded from the study due to failure to induce SE or death after SE. The ratio of failure to induce SE and death after SE were 21% and 14%, respectively.
7
Viral Modulation of Kainic Acid-Induced Seizures
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Five days before inducing SC, the mice were anesthetized via intraperitoneal (i.p.) injection of pentobarbital sodium (7.5 mg/mL, 0.05 mL/10 g) and fixed in a stereotactic frame (RWD Company, Shenzhen, China). A 5-μL Hamilton syringe was placed 0.4 mm posterior and 1 mm lateral to the bregma at a depth of 2 mm relative to the parietal bone and located in the right lateral cerebral ventricle. Each group was injected with 1 μL (0.5 μL/min) virus at the following titers: AV group, 5.21 × 1010 PFU/mL; OAV group, 9.95 × 1010 PFU/mL; and NC group, 1 × 1011PFU/mL. 5 days after the ICV injection, SC was induced in the mice of all SC subgroups by i.p. injection of kainic acid (2 mg/kg, Cayman Chemical Company, Ann Arbor, United States). After 30 min, all mice received a dose of pentobarbital sodium (7.5 mg/mL, 0.05 mL/10 g i.p.) to terminate SC. Mice were excluded from the study due to failure to induce SC failure or death after SC.
8
Subconvulsive Kainic Acid Model for Autism
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To develop a reliable subconvulsive model for autism in development, male and female mixed and same sex litter rat pups were randomly assigned to receive three subconvulsive doses of KA for 15 days beginning on P6 with three 5-day step-up intervals. Kainic acid (Cayman Chemicals) was dissolved in phosphate buffered saline (PBS) to a stock solution of 10 mg/ml adjusted to pH 7.4 and stored at 4 o C until use. A second stock solution was prepared (0.25 mg/ml) then diluted to three final concentrations (25 µg 0.5 mg/kg, 50 µg, 1 mg/kg, 100 µg, 2 mg/kg). The lowest dose was administered by subcutaneous (s.c.) injections until P10, followed by intraperitoneal (ip) injections of the two higher doses with maturation. A schematic diagram of our subconvulsive treatment regimen is illustrated in Figure 1. Open handling was tested daily before the next dosing. Additional behavioral tests were performed on P21 and P22 and KA was re-injected at the end of behavioral testing to keep a 24 h interval between tests. All animals remained with their lactating mother and then sacrificed on P23, at 24 h after the last subconvulsive injection. There were 11 litters in this study, with approximately half treated with low doses of KA (n=58) and the other with PBS (n=54).
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