Pentylenetetrazole-Induced Kindling Mouse Model

Summary

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.

Abstract

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.

Introduction

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 gene....

Protocol

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....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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