Name: pentylenetetrazole-induced kindling mouse model
Uploaded: 2018-06-12T12:30:00
Description: Watch this Scientific Journal Video about Pentylenetetrazole-Induced Kindling Mouse Model at JoVE.com

This work was partly supported by JSPS KAKENHI grant numbers 24700349, 24659093, 25293239, JP18H02536, and 17K07086, MEXT KAKENHI grant numbers 25110737 and 23110525, AMED Grant Number JP18ek0109311, and the SENSHIN Medical Research Foundation and the Japan Epilepsy Research Foundation.

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 ...

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...

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 drugs2,3. 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 seizures12,13. Electroencephalographic (EEG) recordings and behavior analysis have indicated that spontaneous recurrent seizures are observed a month after a single injection12,13. 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 drugs17,18,19.
Seizures are commonly induced by a single systemic administration of PTZ, and the recovery is very fast, within 30 min4,5. 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 severity3,22,23. 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 sprouting27,28, abnormal granule neuron migration29, astrogliosis30, neuronal cell death in the hippocampus31,32, 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.

<table>
	<tbody>
		<tr>
			<td>Pentylenetetrazole</td>
			<td>Sigma-Aldrich</td>
			<td>P6500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>MANAC</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse</td>
			<td>CLEA Japan</td>
			<td></td>
			<td>C57Bl/6NJcl, postnatal 8 week, male</td>
		</tr>
		<tr>
			<td>Syringe (1mL)</td>
			<td>Terumo</td>
			<td>SS-01T</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle(27G x 3/4&#34;) (0.40 x 19 mm)</td>
			<td>Terumo</td>
			<td>NN-2719S</td>
			<td></td>
		</tr>
		<tr>
			<td>Weighing scale</td>
			<td>Mettler</td>
			<td>PE2000</td>
			<td>This item is a discontinued product. Almost equivalent to FX-2000i with FXi-12-JA from A&#38;D company.</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>nacalai tesque</td>
			<td>31511-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Peristatic pump</td>
			<td>ATTO</td>
			<td>SJ1211</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>nacalai tesque</td>
			<td>30404-45</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Yamato</td>
			<td>REM-700</td>
			<td>This item is a discontinued product. Almost equivalent to REM-710</td>
		</tr>
		<tr>
			<td>Microtome blade</td>
			<td>Feather</td>
			<td>S35</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>X-100</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-synaptoporin antibody</td>
			<td>Synaptic systems</td>
			<td>102 002</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-ZnT3 antibody</td>
			<td>Synaptic systems</td>
			<td>197 002</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-doublecortin</td>
			<td>Santa Cruz</td>
			<td>sc-8066</td>
			<td>This item is a discontinued product. We did not test equivalent product (sc-271390).</td>
		</tr>
		<tr>
			<td>Contextual fear discrimination test apparatus</td>
			<td>O&#39;hara</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Three chamber test apparatus</td>
			<td>Muromachi</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

pentylenetetrazole-induced kindling mouse model

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.

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...

Watch this Scientific Journal Video about Pentylenetetrazole-Induced Kindling Mouse Model at JoVE.com

Pentylenetetrazole-Induced Kindling Mouse Model

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.

Pentylenetetrazole (PTZ) is a GABA-A receptor antagonist. An intraperitoneal injection of PTZ into an animal induces an acute, severe seizure at a high ...

This method can help the deep analysis of epilepsy. Seizure will induce histological changes in the brain which may be involved in the neuropsychiatric developmental disorders. The main advantage with this technique is that self-conversive dose of PTZ can induce controllable seizures better than the other current methods.Begin preparing the injection by dissolving 2 milligrams per milliliter PTZ in sterile 0.9 percent sodium chloride. Weigh the animal, then place it in an observation chamber for a 3-minute habituation period. Next, inject PTZ interperitoneally with a 1 milliliter syringe attached to a 27 gauge needle into the left or right quadrant of the abdomen of the animal.Finally, observe the animal after PTZ injection for abnormal behavior. Obtain at least two two-to-six month old 129 SV mice who are of the same sex as the subject mice to be the stranger mouse. Each stranger mouse must be kept separately.For each training session, leave the mouse in the cage for habituation for 15 minutes. Clean the entire apparatus in both cages by wiping the surface with 70%ethanol. Then, place the empty cages in the two side chambers of the three-chamber apparatus and open the doors between the chambers.To habituate the subject mouse, place the subject mouse in the center chamber and allow the mouse to move freely throughout the apparatus until the mouse has investigated both cages, plus an additional five minutes. After the animal moves into the center chamber, close the doors and let the mouse move freely in the center chamber for five minutes. During this period, put one of the 129 SV mice in a cage to habituate to the cage.Immediately after the habituation period of the subject mouse, place the cage containing the 129 SV mouse, termed the Stranger One cage, in one of the side chambers and place the empty cage, termed the Object cage, in the other side chamber. Open the door and monitor the behavior of the subject mouse. Allow the subject mouse to move freely for 10 minutes and record four behavioral parameters.Number one, the time spent in each chamber, the Stranger One chamber, the Object chamber and the center chamber. Number two, time spent investigating each cage, the Stranger One cage and the Object cage. Number three, the number of entrances into each chamber and Number four, the total distance traveled.After the mouse moves into the center chamber, close the doors. After wiping the cages, begin the social novelty analysis by placing another 129 SV mouse in one of the cages, termed the Stranger Two cage, and place the Stranger Two cage in one of the chambers. Place the Stranger One mouse in the other cage, termed the Stranger One cage and place the Stranger One cage in the other chamber.Finally, open the door and monitor the behavior of the subject mouse. Allow the subject mouse to move freely for 10 minutes and measure the same behavioral parameters described for the sociality analysis. Begin by placing a mouse in a conditioning apparatus with specific conditions.For example, a square apparatus with clear plexiglass walls, a metal grid floor, an ethanol odor, 100 lux brightness and 65 decibel background white noise. Condition the mouse by foot shock with pseudo-random timing of a 0.1 milliamp electrical shocks for two seconds three times over the course of five minutes. Then, return the mouse to the home cage after conditioning.The day after conditioning, perform a memory assessment by placing the mouse into the same apparatus as the shock condition and measuring the freezing time over the course of five minutes. Later the same day, place the mouse in a novel apparatus and measure the freezing time during a five-minute assessment. Finally, compare the freezing time between the same condition and novel condition.Results indicated that the seizure score gradually increased with PTZ injections, whereas no seizures or abnormal behaviors were evoked by saline injections. Further, repetitive seizure promoted aberrant axonal branch formation or mossy fiber sprouting and abnormal migration of granule cells in the hippocampus. PTZ-treated mice showed normal sociality.Mice spent more time in the Stranger One chamber than in the Object chamber and investigated the Stranger One cage more than they investigated the Object cage. PTZ-treated mice showed abnormal social novelty, indicative of impaired social memory. PTZ-treated mice also showed impaired memory in the contextual fear test.Following this procedure, other methods like Morris water maze or the other memory tests can be performed to answer the additional questions about the impaired memory ability affected by epileptic seizure. After its development, this technique paves the way for the researchers in the field of epilepsy to explore as a mechanism of seizure-induced neuropsychiatric developmental disorders by using model animals.

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