Inflammatory Cytokines as Targets for Epilepsy Drug Therapy

Abstract

Seizures and epilepsy are associated with a neuroinflammatory process. Proinflammatory cytokines, such as IL-1β, IL-6, and TNF-α, are acutely expressed after seizures in experimental models and, in some cases, still overexpressed later during the chronic stage. Increased levels of these cytokines have been found in the serum from epileptic patients. Pharmacological approaches in rodents support that neuroinflammation is involved in modulating seizure susceptibility, neuronal damage, and other neurological disorders associated with epilepsy. This chapter review studies suggesting that proinflammatory cytokine systems are potential therapeutic targets for the pharmacological treatment of epilepsy.

Appendices

Appendix 1: Intracerebroventricular (i.c.v) Injection of Cytokines in 14-Day-Old Rat Pups

1.1 Materials

Anesthesia system, isoflurane anesthesia system, hair clippers, scalpel, forceps, fine scissors, 25-gauge needle, stereotaxic frame adapted for neonatal rodents, heating blanket, handheld drill for skull openings, microinfusion pump, bone wax, sterile suture, and dry glass bead sterilizer.

1.2 Solutions

Picric acid saturated solution, sterile saline.

1.3 Protocol

• 1. Anesthetize the rat with isoflurane (3 %, for induction) and shave the top of the head.

• 2. Place the anesthetized rat in a stereotaxic frame adapted for neonatal rodents and maintain the rat’s body temperature with a heating blanket. Anesthetized rats with isoflurane (1.5–2 %, for maintenance) during the surgery.

Note: Surgery must be performed in aseptic conditions; surgical instruments must be sterilized.

• 3. Clean the skin with iodine solution to reduce the risk of contamination.

• 4. Make a small incision in the skin to expose the skull.

• 5. Make a small burr hole at the following coordinates: −4.0 mm posterior to bregma, −1.6 mm lateral to the midline (right side).

• 6. Insert a 25-gauge needle (0.5 mm) into the hole and lower it until it is at the depth of 3.5 mm below the surface of the skull.

• 7. Infuse drug over a period of 5 min (0.2 μl/min) using a microinfusion pump.

Note: Prepare stock solution of lyophilized drug by dissolving them in 1 μl of sterile saline solution.

• 8. Seal the hole with bone wax and close the skin with a single suture.

• 9. Apply the saturated picric acid solution to the wound to prevent maternal cannibalism.

• 10. After recovery, the pup is transferred to its mother.

Appendix 2: Determination of IL-1β and IL-1RI mRNA Expression by qRT-PCR

2.1 Materials

mirVana^TM PARIS^TM kit (AM1556, Applied Biosystems, USA), agarose gel for electrophoresis, M-MLV reverse transcriptase (Invitrogen, USA), cDNA, primers (forward and reverse), Maxima SYBR Green/ROX qPCR Master Mix (Fermentas, Thermo Scientific, USA), nuclease-free water, spectrophotometer, electrophoresis system, and thermal cycler.

2.2 Protocol

• 1. Isolate RNA from brain samples with mirVana^TM PARIS^TM kit (AM1556) following the manufacturer’s recommendations (Applied Biosystems, USA).

• 2. Determine RNA concentration with a spectrophotometer by measuring the absorbance at 260 nm.

• 3. Assess RNA integrity by agarose gel electrophoresis.

• 4. Perform reverse transcription reactions with 1 μg of total RNA in a final volume of 20 μl, using M-MLV reverse transcriptase (Invitrogen, USA), according to the manufacturer’s recommendations.

• 5. Perform real-time PCR reactions in triplicate as follows: each sample containing 1 μl of cDNA, 5 pmoles of each primer (forward and reverse), 10 μl of Maxima SYBR Green/ROX qPCR Master Mix (Fermentas, Thermo Scientific, USA), and nuclease-free water up to 20 μl.

Note: Include triplicates for each sample containing primers for β-actin to normalize the data.

• 6. Run reactions in a thermal cycler, according to the following protocol: 2 min at 50 °C, 10 min at 95 °C, 40 cycles of 30 s at 95 °C, and 1 min at 60 °C, followed by a melting curve analysis.

Note: Primers were designed using the Primer 3 software [96], and sequences are shown in Table 1.

Table 1 Sequence of primers used to amplify IL-1β, IL-1RI, and β-actin

• 7. Calculate amplification efficiencies by using lineal regression and determine changes in relative gene expression using β-actin as internal control.

Appendix 3: Localization of IL-1β and IL-1RI by Immunohistochemistry

3.1 Materials

Peristaltic pump, microtome, humidity chamber, histology jars, hydrophobic pen, aqueous mounting media, microwave, timer, and light microscope.

3.2 Solutions

4 % phosphate-buffered paraformaldehyde, 0.1 M phosphate buffer (0.1 M PB), saline solution, 10 mM citrate solution (pH 6.0), ethanol solutions: 70, 80, 95, and 100 %, blocking buffer (0.5 % goat serum in 0.1 M PB).

3.3 Protocol

3.3.1 Tissue Processing

• 1. Anesthetize rats with an overdose of pentobarbital.

• 2. Perform transcardiac perfusion with saline solution followed by 4 % phosphate-buffered (PB) paraformaldehyde.

• 3. Keep brain in situ at 4 °C overnight.

• 4. After removal of the brain, dehydrate in graded series of ethanol solutions (70 %, 8 h; 80 %, 2 × 3 h; 95 %, 2 × 1.5 h; 100 %, 2 × 1 h) and xylene (2 × 40 min) and then embed it in paraffin.

• 5. Section brains into 10-μm-thick coronal sections at the level of dorsal hippocampus and mount sections on gelatin-coated glass slides; keep slides at room temperature.

• 6. Deparaffinize (in xylene, 2 × 10 min) and rehydrate sections in a graded series of ethanol (100 %, 2 × 10 min; 95 %, 2 × 5 min; and 70 %, 1 × 5 min) and dH₂O (2 × 10 min) with a final wash in 0.1 M PB (1 × 10 min).

3.3.2 Immunodetection of IL-1β and IL-1RI

• 1. Perform antigen retrieval procedure by heating brain sections in a 10 mM citrate solution (pH 6.0) using a microwave oven [2 min at high power and 10 min at power 20]; then allow the slides to slowly cool to room temperature for 45 min.

• 2. Rinse slides in dH₂O (1 × 10 min), wash them in 0.1 M PB (2 × 10 min), and incubate in a blocking buffer (0.5 % goat serum in 0.1 M PB) at room temperature for 1 h.

• 3. Incubate slides overnight at 4 °C in a humidified chamber with primary antibody [rabbit polyclonal anti-IL-1β (ab9787, Abcam) diluted 1:500 or rabbit polyclonal anti-IL-1RI (sc 25775, Santa Cruz) diluted 1:200 with blocking buffer].

• 4. Wash sections in 0.1 M PB (3 × 10 min) and then expose them to biotinylated goat anti-rabbit (BA 1000, Vector Labs) diluted 1:1000 with the blocking solution for 2 h at room temperature.

• 5. Wash sections in 0.1 M PB (3 × 10 min) and then incubate with ABC-peroxidase complex 1:1000 (Vectastain ABC kit elite DK-6100 standard, Vector Labs) during 90 min.

• 6. Wash sections in 0.1 M PB (3 × 10 min) and reveal staining after incubation with AB peroxidase substrate kit, 3,3′-diaminobenzidine (SK-4100, Vector Labs).

• 7. Wash sections in 0.1 M PB (3 × 10 min), dehydrate it in graded series of ethanol solutions (70 %, 1 min; 95 %, 1 min; 100 %, 1 min) and xylene (20 dips), and then mount with nonaqueous mounting medium.

• 8. Examine brain sections with a light microscope.