Pentylenetetrazole-Induced Kindling Mouse Model

요약

This protocol describes a method of chemical kindling with pentylenetetrazole and provides a mouse model of epilepsy. This protocol can also be used to investigate vulnerability to seizure induction and pathogenesis after epileptic seizures in mice.

초록

Pentylenetetrazole (PTZ) is a GABA-A receptor antagonist. An intraperitoneal injection of PTZ into an animal induces an acute, severe seizure at a high dose, whereas sequential injections of a subconvulsive dose have been used for the development of chemical kindling, an epilepsy model. A single low-dose injection of PTZ induces a mild seizure without convulsion. However, repetitive low-dose injections of PTZ decrease the threshold to evoke a convulsive seizure. Finally, continuous low-dose administration of PTZ induces a severe tonic-clonic seizure. This method is simple and widely applicable to investigate the pathophysiology of epilepsy, which is defined as a chronic disease that involves repetitive seizures. This chemical kindling protocol causes repetitive seizures in animals. With this method, vulnerability to PTZ-mediated seizures or the degree of aggravation of epileptic seizures was estimated. These advantages have led to the use of this method for screening anti-epileptic drugs and epilepsy-related genes. In addition, this method has been used to investigate neuronal damage after epileptic seizures because the histological changes observed in the brains of epileptic patients also appear in the brains of chemical-kindled animals. Thus, this protocol is useful for conveniently producing animal models of epilepsy.

서문

Epilepsy is a chronic neurological disorder that is characterized by recurrent seizures and affects approximately 1% of people. The underlying mechanisms of epileptogenesis and seizure generation in epilepsy patients cannot be fully clarified in clinical studies. Therefore, an appropriate animal model is required for the study of epilepsy¹.

A variety of animal models of epilepsy have been used to investigate the physiology of epilepsy and to identify anti-epileptic drugs^2,3. Among these models, pharmacological seizure induction is a common method used to generate an animal model for the investigation of the pathology of epilepsy⁴. This method is inexpensive and simple. Electrode-mediated kindling is also a commonly used method, but the costs of this procedure are higher, and the method requires surgical and electrical skills to induce repetitive seizures⁵.

Pharmacological induction is also advantageous because the timing and number of seizures are easily controlled. Genetic mouse models that exhibit spontaneous seizures are also used in the study of epilepsy. However, predicting when and how often the seizures arise in these genetic models may be impossible⁶. A monitoring system is required to observe the epileptic behavior of genetically modified mice⁶.

Kainic acid, pilocarpine and pentylenetetrazole (PTZ) are widely used as seizure-inducing drugs⁷. Kainic acid is an agonist for glutamate receptors, and pilocarpine activates cholinergic receptors. PTZ is a gamma aminobutyric acid (GABA)-A receptor antagonist⁸. PTZ suppresses the function of inhibitory synapses, leading to increased neuronal activity. This regulation causes generalized seizures in animals⁹. A single injection of kainic acid and pilocarpine can induce acute seizures, especially status epilepticus (SE)^10,11 and kainic acid- or pilocarpine-mediated SE promotes chronic spontaneous and recurrent seizures^12,13. Electroencephalographic (EEG) recordings and behavior analysis have indicated that spontaneous recurrent seizures are observed a month after a single injection^12,13. A single injection of a convulsive dose of PTZ also induces acute seizure. However, chronic spontaneous seizures after a single injection of PTZ are difficult to promote. Chronic administration of PTZ is required to induce repetitive seizures¹⁴. In either method, the generation of repetitive seizures is able to induce a pathology more similar to that of human epilepsy than the generation of acute seizures. In the case of PTZ, each injection evokes a seizure, and seizure severity becomes more severe in a stepwise manner with each injection. Finally, a single low-dose PTZ injection induces a severe tonic-clonic seizure. In this phase, each injection evokes severe seizures. In addition, the seizure latency and duration also change over the course of the injections. The latency to tonic seizure often becomes shorter in the latter phase of kindling¹⁵. Furthermore, seizure aggravation is accompanied by a prolonged seizure duration¹⁶. Investigating the molecular mechanism regulating the seizure severity, latency, and duration is useful for screening anti-epileptic drugs^17,18,19.

Seizures are commonly induced by a single systemic administration of PTZ, and the recovery is very fast, within 30 min^4,5. Thus, the number of seizures is more controllable in the PTZ-kindling model. However, EEG monitoring has indicated that generalized spikes may be seen up to 12 h after PTZ-mediated seizure²⁰. Therefore, animals should preferably remain under observation for 24 h after the myoclonic or tonic seizure²¹ for more precise analysis of the kindling mechanisms.

The administration of anti-epileptic drugs, such as ethosuximide, valproate, phenobarbital, vigabatrin, and retigabine³, before or after PTZ injection mitigates the aggravation of the seizure severity^3,22,23. Similarly, knockout mice that lack genes involved in seizure exacerbation, such as matrix metalloproteinase-9²⁴, FGF-22²⁵ and neuritin²⁶, have been shown to exhibit reduced seizure severity after multiple PTZ injections. In addition, observing histopathological alterations after epileptic seizures is possible with this method. In patients with temporal lobe epilepsy, there are typical histological changes in the brain, such as mossy fiber sprouting^27,28, abnormal granule neuron migration²⁹, astrogliosis³⁰, neuronal cell death in the hippocampus^31,32, and hippocampal sclerosis³³. Similar changes are observed in epileptic model animals. Among the available methods, PTZ-mediated chemical kindling is a good, reproducible and inexpensive method to produce an animal model of epilepsy. In a pilocarpine-mediated SE model, seizure control is difficult and many mice die or fail to develop SE³⁴. In contrast, mortality and seizure severity are more controllable in the PTZ model. Additionally, PTZ is less expensive than kainic acid, and skills in mouse brain surgery are not required for drug administration.

프로토콜

All experimental procedures were approved by the Animal Care and Use Committee of the Tokyo Metropolitan Institute of Medical Science. Postnatal 8 - 16-week-old mice are recommended. Any inbred strain is acceptable for the experiment. C57BL/6 mice are more resistant to PTZ, whereas BALB/c and Swiss albino mice are more sensitive to PTZ. C57BL/6 were used in this study. Vulnerability to PTZ also depends on the age of the mouse. Compared to younger mice, older mice are more refractory to PTZ³⁵. The number of animals used for this method can vary, but at least 6 - 10 animals are required for each condition.

1. Preparation of PTZ

1. Dissolve 2 mg/mL PTZ in sterile 0.9% (w/v) NaCl. Prepare PTZ on the day of use.

2. Injection of PTZ

1. Perform all experiments between 9:00 a.m. and 12:00 p.m. (noon).
2. Measure the animal's body weight.
3. Place the animal in an observation chamber for habituation.
4. During the habituation period (3 min), calculate the volume of PTZ solution for the injection based on the body weight of the animal and the previously determined injection dose.
   NOTE: For example, when 35 mg/kg PTZ is used, 0.875 mg of PTZ is required for a mouse weighing 25 g (35 mg/kg x 0.025 kg = 0.875 mg). Therefore, 437.5 μL of a 2 mg/mL PTZ solution should be injected (0.875 mg / 2 mg/mL = 0.4375 mL). The injection dose of PTZ depends on the mouse genotype and strain. For wild-type C57BL/6 mice, a dose of 30-35 mg PTZ/kg body weight is recommended for the first trial. Sensitivity to PTZ depends on the mouse strain used^36,37. The injection dose varies according to the purpose of the experiment.
5. Inject PTZ intraperitoneally with a 1-mL syringe attached to a 27-gauge needle into the left or right quadrant of the abdomen of the animal. Avoid injecting at the midline.
   1. Avoid repeated injections at the same position.
6. Observe animal behaviors for 30 min after PTZ administration (Figure 1A), and classify and score the abnormal behavior as shown below. If possible, keep the animals under observation for 24 h, or at least an additional 6-10 h post-injection, especially once the seizure severity score reaches 3 or above.
   1. Note any mild seizures or behavioral changes in the animals beyond the 30-min observation period. This prolonged observation period may address a particularly important and complete effect of a subconvulsive dose of PTZ in the generation of chronic unprovoked seizures in epilepsy.
   2. In addition, measure the seizure duration of each observed seizure as changes in seizure duration relate to the seizure severity. Moreover, the latency to the first seizure after the PTZ injection is another important measure to collect. Monitoring the seizure frequency, duration, and latency is critical for any post-seizure molecular studies.
7. Inject PTZ every other day (Figure 1A). The number of injections depends on the objective of the experiment. Representative examples are as follows.
   1. To generate completely kindled animals, once an animal experiences a seizure with a score of 5 (tonic-clonic seizure), finish the injection within additional three administrations. In this case, increase the injection dose if the seizure score does not increase for three consecutive administrations. Each animal can receive a different number and dose of PTZ injections.
   2. Fix the number and dose of PTZ injection in every condition to evaluate the vulnerability to PTZ, including in assessments of anti-epilepsy drugs and studies of the phenotype of genetically modified mice. Give all animals the same number of PTZ injections; a total of 8 to 12 injections is recommended. If an animal dies, the seizure score should be denoted as 6.
   3. Determine the injection dose necessary to maintain a high seizure score (4 or 5) for 10 or more injections to study the histopathological changes that occur epileptic seizures. In this case, the total injection number should range from 25 to 30. Decrease the injection dose if the seizure score reaches 5. If the seizure score decreases to 3 or lower, increase the injection dose.

3. Seizure Score

1. Observe the animal behaviors and record the scores.
   1. Classify and score the epileptic behaviors as follows^38,39 (Figure 1B):
      0: normal behavior, no abnormality.
      1: immobilization, lying on belly.
      2: head nodding, facial, forelimb, or hindlimb myoclonus.
      3: continuous whole-body myoclonus, myoclonic jerks, tail held up stiffly.
      4: rearing, tonic seizure, falling down on its side.
      5: tonic-clonic seizure, falling down on its back, wild rushing and jumping.
      6: death.
2. Change the behavioral criteria based on the Racine score⁴⁰, depending on the experimental conditions.

4. Post-Seizure Analysis

1. Immunohistopathological analysis
   1. Perfusion fixation
      1. Preparation
         1. Dissolve 4% (w/v) paraformaldehyde (PFA) in 0.1 M phosphate buffer (pH 7.4). Heat the solution to approximately 70 °C with the addition of a drop of 1 M NaOH (approximately 1 µL of 1 M NaOH for 50 mL PFA solution).
         2. Prepare ice-cold 4% PFA and phosphate-buffered saline (PBS).
         3. Set up a peristaltic pump, with a tube and needle. Run approximately 20 mL of water through the tube to clear out residue. Then, place the open end of the tube in ice-cold PBS.
      2. Transcardial perfusion and preparation of the fixed brain
         1. Deeply anesthetize the mouse with 50% isoflurane/50% ethanol in a small jar.
         2. Keep the mouse in a supine position by pinning the tips of their forelimbs and hindlimbs.
         3. Cut the ventral midline of the abdomen and expose the diaphragm.
         4. Cut the diaphragm along the costal margin. Then, cut both lateral sides of the ribs and body. Pinch the xiphoid process by the locking forceps and evert the rib cage. Maintain the position of the xiphoid process by pinning it with a needle or pinching it with forceps. Expose the heart.
         5. Insert the needle into the left ventricle and make a cut in the right atrium using scissors. Be aware not to push the needle too far into the heart, as it can pierce an interior wall.
         6. Begin a steady flow of ice-cold PBS through the needle to wash out the blood (approximately 15 - 20 mL/min).
         7. When the blood has been cleared from the body, move the tube to a 4% paraformaldehyde solution (approximately 60 mL) without the addition of bubbles. Then, stop the perfusion.
         8. Cut the rear neck and spine and peel off the head skin and the dorsal part of the skull with dissecting scissors.
         9. Cut the premaxillary bone between the eye orbits and completely remove the dorsal portion of the skull.
         10. Create a gap between the brain and jugal bone from the posterior side and cut the trigeminal nerves and optic chiasm underneath the brain. Carefully remove the brain and place it in a vial containing 4% PFA. Post-fix the brain at 4°C for 12-16 h.
   2. Brain slice preparation
      1. Transfer the fixed brain to 20% sucrose/PBS until the brain sinks into the solution to allow for cryoprotection.
      2. Cut the fixed brain based on the required slice orientation and required part of the brain.
      3. Set the cryomicrotome. Set the blade in position and maintain the microtome stage at a temperature under -20 °C.
      4. Place the pre-cut brain on the stage and coat the brain with 20% sucrose/PBS. The sucrose/PBS solution will gradually freeze, covering the brain on the stage, until the brain is completely embedded in frozen 20% sucrose/PBS.
      5. Slice the brain (30 µm thickness) by sliding the blade through the tissue. Transfer the slices into PBS and keep them at 4 °C.
   3. Fluorescent immunohistochemistry
      1. Wash the necessary slices in 0.1% Triton X-100/PBS (PBST) for 10 min, 3 times at room temperature (RT).
      2. Block the slices by incubating them in PBST with 2% goat serum or 5% bovine serum albumin (BSA) for 1 - 2 h at RT.
      3. Incubate the slices in an antibody solution with the suitable concentration of primary antibody in PBST with 2% goat serum or 5% BSA for 1 to 7 overnights at 4 °C.
      4. Wash the slices in PBST for 10 min, 3 times at RT.
      5. Incubate the slices in a secondary antibody solution with the suitable concentration of secondary antibody in PBST for 1 - 2 h at RT, light-protected.
      6. Mount the slices onto glass slides and cover with mounting medium and cover glass.
      7. Observe the slices with fluorescence microscopy.
2. Three-chamber test
   1. Preparation
      1. Prepare at least 2 2 - 6-month-old 129/Sv mice of the same sex as the subject mice to be the "Stranger mouse." Habituate all mice in the cage before the analysis. For each training session, place the mouse in the cage and leave the mouse for 15 min.
      2. Before each habituation phase with the subject mouse, clean the entire apparatus and both cages by wiping the surface with 70% ethanol.
      3. Place the empty cages in the two side chambers of the 3-chamber apparatus and open the doors between the chambers.
      4. To habituate the subject mouse, place a subject mouse in the center chamber and allow the mouse to freely move throughout the apparatus until the mouse has investigated both cages, plus an additional 5 min. Subject animals that may be fearful of the cage do not investigate the cage for 20 min and should not be used for this analysis. Such animals avoid the cage and rarely come close to the cage.
      5. After the animal moves into the center chamber, close the doors and let the mouse move freely in the center chamber for 5 min. During this period, put one of the 129/Sv mice in a cage to habituate to the cage.
   2. Sociality analysis
      1. Carry out this analysis immediately after the habituation period of the subject mouse.
      2. Place the cage containing a 129/Sv mouse, termed the "Stranger 1 cage", in one of the side chambers and place the empty cage, termed the "Object cage", in the other side chamber.
      3. Open the door and monitor the behavior of the subject mouse.
      4. Allow the subject mouse to move freely for 10 min and record the following behavioral parameters. If the subject mouse is afraid of the mouse in the Stranger 1 cage, as indicated by the mouse avoiding the Stranger 1 cage and remaining in the corner of one of the chambers, the animal should not be used for the analysis.
         a) Time spent in each chamber, the Stranger 1 chamber, the Object chamber, and center chamber.
         b) Time spent investigating each cage, the Stranger 1 cage and the Object cage. (The mouse is classified as investigating the cage if the mouse is sniffing or toughing the mouse or cage)
         c) The number of entrances into each chamber (optional)
         d) Total distance travelled (optional)
      5. After the mouse moves into the center chamber, close the doors.
   3. Social novelty analysis
      1. Carry out this analysis immediately after the sociality analysis.
      2. Wipe both cages with 70% ethanol.
      3. Place another 129/Sv mouse in one of the cages, termed the "Stranger 2 cage" and place the Stranger 2 cage in one of the chambers. Place the Stranger 1 mouse in the other cage, termed the "Stranger 1 cage", and place the Stranger 1 cage in the other chamber.
      4. Open the door and monitor the behavior of the subject mouse.
      5. Allow the subject mouse to move freely for 10 min, and measure the same behavioral parameters described for the sociality analysis.
         NOTE: The Stranger 1 and Stranger 2 mouse should be chosen randomly. The Stranger 1 chamber and Object chamber as well as the Stranger 1 chamber and Stranger 2 chamber should be randomly determined.
3. Contextual fear discrimination
   1. Conditioning
      1. Before each conditioning trial, clean the experimental apparatus by wiping the surface with 70% ethanol.
      2. Place a mouse in a conditioning apparatus with specific conditions (apparatus shape, wall color, floor material, odor, lighting, and background noise volume should be pre-determined). For example, the conditioning apparatus used here was a square apparatus with clear plexiglass walls, a metal grid floor, an ethanol odor, 100 lux brightness, and 65-dB background white noise.
      3. Condition the mouse by foot-shock with pseudo-random timing of 0.1-mA electrical shocks for 2 s x 3 times over the course of 5 min.
      4. Return the mouse to the home cage after conditioning.
   2. Memory assessment
      1. Before each assessment, clean the experimental apparatus by wiping the surface with 70% ethanol.
      2. The next day following conditioning, place the mouse into the same apparatus as shock condition and measure the freezing time over the course of 5 min.
      3. Later the same day, place the mouse in a novel apparatus and measure the freezing time during a 5-min assessment. A triangular apparatus with white plexiglass walls, plate floor, no odor, 30 lux brightness, and 70-dB white noise was used here.
      4. Compare the freezing time between the same condition and novel condition. The mice with a normal memory ability will freeze more in the same condition than in the novel condition. Freezing is defined immobility of the animal for more than 2 s.

결과

Repetitive injection of PTZ induces an increase in seizure severity. Six C57BL/6 mice were treated with PTZ, and another 6 mice were treated with saline as a control group. The PTZ dose was 35 mg/kg, and 10 injections were administered. The seizure score gradually increased with PTZ injections, whereas no seizures or abnormal behaviors were evoked by saline injections (Figure 2). ANOVA followed by Bonferroni test showed a significant difference between the PT...

토론

Here, we present a widely accessible protocol for the establishment of a pharmacological animal model of epilepsy. PTZ-mediated chemical kindling has a long history and is a commonly accepted model for the study of the histopathology and cellular pathology of epilepsy⁴¹. The chemical kindling model of epilepsy has been reviewed previously by Suzdak and Jansen, 1995⁴². Pharmacological seizure induction, especially with PTZ, is an easy and simple method for evoking severe sei...

자료

Name	Company	Catalog Number	Comments
Pentylenetetrazole	Sigma-Aldrich	P6500
Sodium chloride	MANAC	7647-14-5
Mouse	CLEA Japan		C57Bl/6NJcl, postnatal 8 week, male
Syringe (1mL)	Terumo	SS-01T
Needle(27G x 3/4") (0.40 x 19 mm)	Terumo	NN-2719S
Weighing scale	Mettler	PE2000	This item is a discontinued product. Almost equivalent to FX-2000i with FXi-12-JA from A&D company.
Paraformaldehyde	Sigma-Aldrich	P6148
Sodium hydroxide	nacalai tesque	31511-05
Peristatic pump	ATTO	SJ1211
Sucrose	nacalai tesque	30404-45
Microtome	Yamato	REM-700	This item is a discontinued product. Almost equivalent to REM-710
Microtome blade	Feather	S35
Triton X-100	Sigma-Aldrich	X-100
anti-synaptoporin antibody	Synaptic systems	102 002
anti-ZnT3 antibody	Synaptic systems	197 002
anti-doublecortin	Santa Cruz	sc-8066	This item is a discontinued product. We did not test equivalent product (sc-271390).
Contextual fear discrimination test apparatus	O'hara
Three chamber test apparatus	Muromachi