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 number of animals used for this method can vary, but at least 6 - 10 animals are required for each condition.
1. Preparation of PTZ
<ol>
	<li>Dissolve 2 mg/mL PTZ in sterile 0.9% (w/v) NaCl. Prepare PTZ on the day of use.</li>
</ol>
2. Injection of PTZ
<ol>
	<li>Perform all experiments between 9:00 a.m. and 12:00 p.m. (noon).</li>
	<li>Measure the animal&#39;s body weight.</li>
	<li>Place the animal in an observation chamber for habituation.</li>
	<li>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 &#956;L 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 used36,37. The injection dose varies according to the purpose of the experiment.</li>
	<li>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.
	<ol>
		<li>Avoid repeated injections at the same position.</li>
	</ol>
	</li>
	<li>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.
	<ol>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Inject PTZ every other day (Figure 1A). The number of injections depends on the objective of the experiment. Representative examples are as follows.
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
3. Seizure Score
<ol>
	<li>Observe the animal behaviors and record the scores.
	<ol>
		<li>Classify and score the epileptic behaviors as follows38,39 (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.</li>
	</ol>
	</li>
	<li>Change the behavioral criteria based on the Racine score40, depending on the experimental conditions.</li>
</ol>
4. Post-Seizure Analysis
<ol>
	<li>Immunohistopathological analysis
	<ol>
		<li>Perfusion fixation
		<ol>
			<li>Preparation
			<ol>
				<li>Dissolve 4% (w/v) paraformaldehyde (PFA) in 0.1 M phosphate buffer (pH 7.4). Heat the solution to approximately 70 &#176;C with the addition of a drop of 1 M NaOH (approximately 1 &#181;L of 1 M NaOH for 50 mL PFA solution).</li>
				<li>Prepare ice-cold 4% PFA and phosphate-buffered saline (PBS).</li>
				<li>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.</li>
			</ol>
			</li>
			<li>Transcardial perfusion and preparation of the fixed brain
			<ol>
				<li>Deeply anesthetize the mouse with 50% isoflurane/50% ethanol in a small jar.</li>
				<li>Keep the mouse in a supine position by pinning the tips of their forelimbs and hindlimbs.</li>
				<li>Cut the ventral midline of the abdomen and expose the diaphragm.</li>
				<li>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.</li>
				<li>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.</li>
				<li>Begin a steady flow of ice-cold PBS through the needle to wash out the blood (approximately 15 - 20 mL/min).</li>
				<li>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.</li>
				<li>Cut the rear neck and spine and peel off the head skin and the dorsal part of the skull with dissecting scissors.</li>
				<li>Cut the premaxillary bone between the eye orbits and completely remove the dorsal portion of the skull.</li>
				<li>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&#176;C for 12-16 h.</li>
			</ol>
			</li>
		</ol>
		</li>
		<li>Brain slice preparation
		<ol>
			<li>Transfer the fixed brain to 20% sucrose/PBS until the brain sinks into the solution to allow for cryoprotection.</li>
			<li>Cut the fixed brain based on the required slice orientation and required part of the brain.</li>
			<li>Set the cryomicrotome. Set the blade in position and maintain the microtome stage at a temperature under -20 &#176;C.</li>
			<li>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.</li>
			<li>Slice the brain (30 &#181;m thickness) by sliding the blade through the tissue. Transfer the slices into PBS and keep them at 4 &#176;C.</li>
		</ol>
		</li>
		<li>Fluorescent immunohistochemistry
		<ol>
			<li>Wash the necessary slices in 0.1% Triton X-100/PBS (PBST) for 10 min, 3 times at room temperature (RT).</li>
			<li>Block the slices by incubating them in PBST with 2% goat serum or 5% bovine serum albumin (BSA) for 1 - 2 h at RT.</li>
			<li>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 &#176;C.</li>
			<li>Wash the slices in PBST for 10 min, 3 times at RT.</li>
			<li>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.</li>
			<li>Mount the slices onto glass slides and cover with mounting medium and cover glass.</li>
			<li>Observe the slices with fluorescence microscopy.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Three-chamber test
	<ol>
		<li>Preparation
		<ol>
			<li>Prepare at least 2 2 - 6-month-old 129/Sv mice of the same sex as the subject mice to be the &#34;Stranger mouse.&#34; 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.</li>
			<li>Before each habituation phase with the subject mouse, clean the entire apparatus and both cages by wiping the surface with 70% ethanol.</li>
			<li>Place the empty cages in the two side chambers of the 3-chamber apparatus and open the doors between the chambers.</li>
			<li>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.</li>
			<li>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.</li>
		</ol>
		</li>
		<li>Sociality analysis
		<ol>
			<li>Carry out this analysis immediately after the habituation period of the subject mouse.</li>
			<li>Place the cage containing a 129/Sv mouse, termed the &#34;Stranger 1 cage&#34;, in one of the side chambers and place the empty cage, termed the &#34;Object cage&#34;, in the other side chamber.</li>
			<li>Open the door and monitor the behavior of the subject mouse.</li>
			<li>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)</li>
			<li>After the mouse moves into the center chamber, close the doors.</li>
		</ol>
		</li>
		<li>Social novelty analysis
		<ol>
			<li>Carry out this analysis immediately after the sociality analysis.</li>
			<li>Wipe both cages with 70% ethanol.</li>
			<li>Place another 129/Sv mouse in one of the cages, termed the &#34;Stranger 2 cage&#34; and place the Stranger 2 cage in one of the chambers. Place the Stranger 1 mouse in the other cage, termed the &#34;Stranger 1 cage&#34;, and place the Stranger 1 cage in the other chamber.</li>
			<li>Open the door and monitor the behavior of the subject mouse.</li>
			<li>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.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Contextual fear discrimination
	<ol>
		<li>Conditioning
		<ol>
			<li>Before each conditioning trial, clean the experimental apparatus by wiping the surface with 70% ethanol.</li>
			<li>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.</li>
			<li>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.</li>
			<li>Return the mouse to the home cage after conditioning.</li>
		</ol>
		</li>
		<li>Memory assessment
		<ol>
			<li>Before each assessment, clean the experimental apparatus by wiping the surface with 70% ethanol.</li>
			<li>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.</li>
			<li>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.</li>
			<li>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.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

The authors declare no conflicts of interest.

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 ...

pentylenetetrazole-induced-kindling-mouse-model

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32.3K Views.  Tokyo Metropolitan Institute of Medical Science. 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.

Video: Pentylenetetrazole-Induced Kindling Mouse Model

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

Detection of Neuritic Plaques in Alzheimer's Disease Mouse Model

Alzheimer's disease (AD) is the most common neurodegenerative disorder leading to dementia. Neuritic plaque formation is one of the pathological hallmarks of Alzheimer's disease. The central component of neuritic plaques is a small filamentous protein called amyloid &beta; protein (A&beta;)1, which is derived from sequential proteolytic cleavage of the beta-amyloid precursor protein (APP) by &beta;-secretase and &gamma;-secretase. The amyloid hypothesis entails that A&gamma;-containing plaques as the underlying toxic mechanism in AD pathology2. The postmortem analysis of the presence of neuritic plaque confirms the diagnosis of AD. To further our understanding of A&gamma; neurobiology in AD pathogenesis, various mouse strains expressing AD-related mutations in the human APP genes were generated. Depending on the severity of the disease, these mice will develop neuritic plaques at different ages. These mice serve as invaluable tools for studying the pathogenesis and drug development that could affect the APP processing pathway and neuritic plaque formation. In this protocol, we employ an immunohistochemical method for specific detection of neuritic plaques in AD model mice. We will specifically discuss the preparation from extracting the half brain, paraformaldehyde fixation, cryosectioning, and two methods to detect neurotic plaques in AD transgenic mice: immunohistochemical detection using the ABC and DAB method and fluorescent detection using thiofalvin S staining method.

Detection of Neuritic Plaques in Alzheimer&#039;s Disease Mouse Model

One of the pathological characteristics of AD is the formation of Amyloid &#x03B2; protein positive neuritic plaques. In this protocol we describe two methods to detect neuritic plaques in transgenic AD model mice: immunohistochemical detection using the ABC and DAB method and fluorescent detection using thioflavin S staining method.

Alzheimer's disease (AD) is the most common neurodegenerative disorder leading to dementia. Neuritic plaque formation is one of the pathological hallmarks ...

Methods for Intravenous Self Administration in a Mouse Model

Animal models have been developed to study the reinforcing effects of drugs, including the intravenous self-administration (IVSA) paradigm. The advantages of using an IVSA paradigm to study the reinforcing properties of drugs of abuse such as cocaine include the fact that the drug is self-administered instead of experimenter-administered, the schedule of reinforcement can be altered, and accurate measurement of the quantities of drug consumed as well as the timing and pattern of IV injections can be obtained. Furthermore, the intravenous route of administration avoids potential confounds related to first pass metabolism or taste, and produces rapid increases in blood and brain drug levels. As outlined in this video, intravenous self-administration can be obtained without prior food restriction or prior drug training following careful catheter placement during surgery and meticulous daily catheter flushing and maintenance. Experimental procedures outlined in this paper include a description of animal housing and acclimation methods, operant training using sweetened milk solutions, and catheter implantation surgery.

The intravenous self-administration (IVSA) paradigm is considered to be the gold standard in examining the reinforcing properties of drugs of abuse in rodents. This manuscript outlines the experimental procedures and surgical techniques necessary to obtain reliable IVSA data. In particular, meticulous catheter implantation and maintenance are highlighted.

Animal models have been developed to study the reinforcing effects of drugs, including the intravenous self-administration (IVSA) paradigm. The advantages ...

Trypsin Digest Protocol to Analyze the Retinal Vasculature of a Mouse Model

Trypsin digest is the gold standard method to analyze the retinal vasculature 1-5. It allows visualization of the entire network of complex three-dimensional retinal blood vessels and capillaries by creating a two-dimensional flat-mount of the interconnected vascular channels after digestion of the non-vascular components of the retina. This allows one to study various pathologic vascular changes, such as microaneurysms, capillary degeneration, and abnormal endothelial to pericyte ratios. However, the method is technically challenging, especially in mice, which have become the most widely available animal model to study the retina because of the ease of genetic manipulations 6,7. In the mouse eye, it is particularly difficult to completely remove the non-vascular components while maintaining the overall architecture of the retinal blood vessels. To date, there is a dearth of literature that describes the trypsin digest technique in detail in the mouse. This manuscript provides a detailed step-by-step methodology of the trypsin digest in mouse retina, while also providing tips on troubleshooting difficult steps.

Trypsin digest is one of the most commonly used methods to analyze retinal vasculature. This manuscript describes the method in detail, including key alterations to optimize the technique and remove the non-vascular tissue while preserving the overall architecture of the vessels.

Trypsin digest is the gold standard method to analyze the retinal vasculature 1-5. It allows visualization of the entire network of complex ...

The Corneal Micropocket Assay: A Model of Angiogenesis in the Mouse Eye

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets containing specific growth factors that trigger blood vessel growth throughout the naturally avascular cornea, angiogenesis can be measured and quantified. In this assay the angiogenic response is generated over the course of several days, depending on the type and dose of growth factor used. The induction of neovascularization is commonly triggered by either basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF). By combining these growth factors with sucralfate and hydron (poly-HEMA (poly(2-hydroxyethyl methacrylate))) and casting the mixture into pellets, they can be surgically implanted in the mouse eye. These uniform pellets slowly-release the growth factors over five or six days (bFGF or VEGF respectively) enabling sufficient angiogenic response required for vessel area quantification using a slit lamp. This assay can be used for different applications, including the evaluation of angiogenic modulator drugs or treatments as well as comparison between different genetic backgrounds affecting angiogenesis. A skilled investigator after practicing this assay can implant a pellet in less than 5 min per eye.

The protocol describes the corneal micropocket assay as developed in mice.

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets ...

Measuring Progressive Neurological Disability in a Mouse Model of Multiple Sclerosis

After intracerebral infection with the Theiler's Murine Encephalomyelitis Virus (TMEV), susceptible SJL mice develop a chronic-progressive demyelinating disease, with clinical features similar to the progressive forms of multiple sclerosis (MS). The mice show progressive disability with loss of motor and sensory functions, which can be assessed with multiple apparatuses and protocols. Among them, the Rotarod performance test is a very common behavioral test, its advantage being that it provides objective measurements, but it is often used assuming that it is straightforward and simple. In contrast to visual scoring systems used in some models of MS, which are highly subjective, the Rotarod test generates an objective, measurable, continuous variable (i.e., length of time), allowing almost perfect inter-rater concordances. However, inter-laboratory reliability is only achieved if the various testing parameters are replicated. In this manuscript, recommendations of specific testing parameters, such as size, speed, and acceleration of the rod; amount of training given to the animals; and data processing, are presented for the Rotarod test.

An optimized testing protocol is presented in this paper for the Rotarod performance test, used for measuring progressive neurological disability in TMEV-infected mice.

After intracerebral infection with the Theiler's Murine Encephalomyelitis Virus (TMEV), susceptible SJL mice develop a chronic-progressive demyelinating ...

Neurobehavioral Assessments in a Mouse Model of Neonatal Hypoxic-ischemic Brain Injury

We performed unilateral carotid artery occlusion on CD-1 mice to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of neonatal HI brain injury by studying neurobehavioral functions in these mice compared to non-operated (i.e., normal) mice. During the study, Rice-Vannucci&#39;s method was used to induce neonatal HI brain damage in postnatal day 7-10 (P7-10) mice. The HI operation was performed on the pups by unilateral carotid artery ligation and exposure to hypoxia (8% O2 and 92% N2 for 90 min). One week after the operation, the damaged brains were evaluated with the naked eye through the semi-transparent skull and were categorized into subgroups based on the absence (&#34;no cortical injury&#34; group) or presence (&#34;cortical injury&#34; group) of cortical injury, such as a lesion in the right hemisphere. On week 6, the following neurobehavioral tests were performed to evaluate the cognitive and motor functions: passive avoidance task (PAT), ladder walking test, and grip strength test. These behavioral tests are helpful in determining the effects of neonatal HI brain injury and are used in other mouse models of neurodegenerative diseases. In this study, neonatal HI brain injury mice showed motor deficits that corresponded to right hemisphere damage. The behavioral test results are relevant to the deficits observed in human neonatal HI patients, such as cerebral palsy or neonatal stroke patients. In this study, a mouse model of neonatal HI brain injury was established and showed different degrees of motor deficits and cognitive impairment compared to non-operated mice. This work provides basic information on the HI mouse model. MRI images demonstrate the different phenotypes, separated according to the severity of brain damage by motor and cognitive tests.

We performed unilateral carotid artery occlusion on postnatal day 7-10 CD-1 mouse pups to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of HI brain injury. We studied neurobehavioral functions in these mice compared to non-operated normal mice.

We performed unilateral carotid artery occlusion on CD-1 mice to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of neonatal HI ...

The Mouse Hindbrain As a Model for Studying Embryonic Neurogenesis

The mouse embryo forebrain is the most commonly employed system for studying mammalian neurogenesis during development. However, the highly folded forebrain neuroepithelium is not amenable to wholemount analysis to examine organ-wide neurogenesis patterns. Moreover, defining the mechanisms of forebrain neurogenesis is not necessarily predictive of neurogenesis in other parts of the brain; for example, due to the presence of forebrain-specific progenitor subtypes. The mouse hindbrain provides an alternative model for studying embryonic neurogenesis that is amenable to wholemount analysis, as well as tissue sections to observe the spatiotemporal distribution and behavior of neural progenitors. Moreover, it is easily dissected for other downstream applications, such as cell isolation or molecular biology analysis. As the mouse hindbrain can be readily analyzed in the vast number of cell lineage reporter and mutant mouse strains that have become available, it offers a powerful model for studying the cellular and molecular mechanisms of developmental neurogenesis in a mammalian organism. Here, we present a simple and quick method to use the mouse embryo hindbrain for analyzing mammalian neural progenitor cell (NPC) behavior in wholemount preparations and tissue sections.

This article demonstrates how the mouse embryonic hindbrain can be used as a model for studying developmental neurogenesis in both whole organ and tissue section preparations.

The mouse embryo forebrain is the most commonly employed system for studying mammalian neurogenesis during development. However, the highly folded ...

Preparation and Implantation of Electrodes for Electrically Kindling VGAT-Cre Mice to Generate a Model for Temporal Lobe Epilepsy

It was discovered that electrical kindling of VGAT-Cre mice led to the spontaneous motor and electrographic seizures. A recent paper focused on how unique VGAT-Cre mice were used in developing spontaneous recurring seizures (SRS) after kindling and a likely mechanism - insertion of Cre into the VGAT gene - disrupted its expression and reduced GABAergic tone. The present study extends these observations to a larger cohort of mice, focusing on key issues such as how long the SRS continues after kindling and the effect of the animal's sex and age. This report describes the protocols for the following key steps: making headsets with hippocampal depth electrodes for electrical stimulation and for reading the electroencephalogram; surgery to affix the headset securely on the mouse's skull so that it does not fall off; and key details of the electrical kindling protocol such as duration of the pulse, frequency of train, duration of train, and amount of current injected. The kindling protocol is robust in that it reliably leads to epilepsy in most VGAT-Cre mice, providing a new model to test for novel antiepileptogenic drugs.

This report describes the methods to generate a model of temporal lobe epilepsy based on the electrical kindling of transgenic VGAT-Cre mice. Kindled VGAT-Cre mice may be useful in determining what causes epilepsy and for screening novel therapies.

It was discovered that electrical kindling of VGAT-Cre mice led to the spontaneous motor and electrographic seizures. A recent paper focused on how unique ...

Optimization of the Retinal Vein Occlusion Mouse Model to Limit Variability

Mouse models of retinal vein occlusion (RVO) are often used in ophthalmology to study hypoxic-ischemic injury in the neural retina. In this report, a detailed method pointing out critical steps is provided with recommendations for optimization to achieve consistently successful occlusion rates across different genetically modified mouse strains. The RVO mouse model consists primarily of the intravenous administration of a photosensitizer dye followed by laser photocoagulation using a retinal imaging microscope attached to an ophthalmic guided laser. Three variables were identified as determinants of occlusion consistency. By adjusting the wait time after rose bengal administration and balancing the baseline and experimental laser output, the variability across experiments can be limited and a higher success rate of occlusions achieved. This method can be used to study retinal diseases that are characterized by retinal edema and hypoxic-ischemic injury. Additionally, as this model induces vascular injury, it can also be applied to study the neurovasculature, neuronal death, and inflammation.

Here, we describe an optimized protocol for retinal vein occlusion using rose bengal and a laser-guided retinal imaging microscope system with recommendations to maximize its reproducibility in genetically modified strains.

Mouse models of retinal vein occlusion (RVO) are often used in ophthalmology to study hypoxic-ischemic injury in the neural retina. In this report, a ...