Experimental modeling of epilepsy in lithium-pilocarpine rats

Summary

Source: Practical Experimental Techniques in Neurobiology, Fourth Military Medical University Press

Operation method

basic program

Principle

Epilepsy (epilepsy) is one of the most common neurological disorders. It is characterized by recurrent convulsive seizures accompanied by different clinical and electroencephalographic manifestations. Animal experiments are the most important means of studying the pathogenesis of epilepsy in humans, and dozens of animal models have been used in epilepsy research, which are classified as hereditary and artificially induced according to the mode of generation. The choice of animal model depends on the problem to be solved, the type of epilepsy and the purpose of the study. This section focuses on the cone-pilocarpine ignition rat seizure model, which was first reported by Honchar et al. in 1983. It has similar characteristics to human status epilepticus (SE) or temporal lobe epilepsy, and is suitable for exploring the formation mechanism of temporal lobe epilepsy and screening antiepileptic drugs, and is used in the molecular biology and electrophysiology of SE and complex partial seizures. The modeling mechanism is as follows: acetylcholine (ACh) plays an important role in epileptogenesis, and systemic administration of the ACh receptor agonist pilocarpine can cause seizures; lithium chloride can effectively increase the body's sensitivity to pilocarpine, resulting in a reduction of pilocarpine dosage and a significant reduction in the mortality rate of animals due to the toxic effects of pilocarpine; bromomethylscopolamine is mainly used to attenuate the pilocarpine-induced peripheral nervous system reactions (hematemesis, salivation, etc.).

Materials and Instruments

Adult SD rats (200-250g)
Lithium chloride Pilocarpine Bromomethylscopolamine Sodium phenobarbital injection 0.9% saline
Syringe

Move

1. Estimate the total requirement of various drugs according to the expected number of experimental animals, weigh the total requirement of scopolamine bromide, lithium chloride and pilocarpine, and dissolve the experimental drugs to the working concentration with 0.9% saline (need to be filtered).

2 1d before modeling, lithium chloride 3mmol/kg was injected intraperitoneally.

3. 18-20h after lithium chloride injection,1mg/kg of scopolamine bromomethyl was injected subcutaneously and the time of administration was recorded.

4. The first dose of pilocarpine 30mg/kg was given intraperitoneally after 30min to observe the degree of epileptiform seizures in rats. Thereafter, an additional dose of 10 mg/kg was given intraperitoneally at 30 min intervals until the occurrence of SE-like seizures of grade IV or above without significant intervals (see interpretation of results). The extreme dose was 60 mg/kg.

5. 1h after SE, terminate the seizure with an injection of phenobarbital sodium 40mg/kg.

6. 30 min after the injection of sodium phenobarbital, 10 ml of saline was given subcutaneously to replace the fluid loss caused by the modeling process.

7. Interpretation of results.

(1) Model grading standard: Racine grading standard was adopted for seizure symptoms in rats:

① Grade 0 without any response; ② Grade I facial clonus, including blinking, whisker movement, rhythmic chewing, etc.; ③ Grade II: Grade I plus rhythmic head nodding; ④ Grade III: Grade II plus myoclonus of the forelimbs, but no hind limbs in upright position; ⑤ Grade IV: Grade III plus hind limbs in upright position; ⑥ Grade V full-blown tonic-clonic seizure with loss of body position control. Modeling was judged to be successful when the experimental animals showed 1h of consecutive seizures of grade IV or higher degree. :

(2) Epilepsy model staging: this epilepsy model is temporally divided into three main periods:

①Acute periodSE state that occurs within minutes to 1h after Pilocar-pine injection and lasts for about 24h, this state is characterized by tonic-clonic generalized seizures;

② Latent phase After SE, seizures are moderated and almost no seizures occur, also known as silent phase, which can last from 1 week to several weeks;

③ Chronic epileptic phase After the latent phase, characterized by recurrent spontaneous seizures (SRSs), with an average of 2-3 seizures per week. Electroencephalographic manifestations: ① In the acute phase, significant 0-rhythm and cortical low-voltage fast electrical activity is seen in the hippocampus, which develops into high-voltage fast electrical activity with spikes in the hippocampus; ② In the latent phase, normal EEG activity is shown; ③ In the chronic phase, initial SRSs are often characterized by paroxysmal hippocampal emissions with no cortical recording alterations, and then evolve gradually into graded cortical and hippocampal synchronized emissions.

Caveat

1. Pilocarpine should be freshly prepared before modeling and stored away from light during use to ensure the efficacy of the drug.

2. The procedure for mice is the same as that for rats, and the weight range of the selected animals is 20-25 g. Taking C57BL/6 mice as an example, the dosage of the modeling drugs is lithium chloride 10 mmol/kg, and pilocarpine 100 mg/kg.

3. If the pilocarpine injection has reached the extreme maximum, and the rats still do not have seizures of grade IV or above, the modeling can be considered as a failure.

Common Problems

1. During the modeling process, if rats were seen to have intermittent grade IV seizures accompanied by persistent grade III seizures, and there was no clear autonomous crawling and groping movements during the seizures, the modeling criteria could also be judged to be met.

2. The author had applied sodium phenobarbital to terminate the seizure in time when the rat had a grade III seizure to establish a mild seizure model, and the histological staining confirmed that the hippocampus of the rats showed pathological changes different from those after SE.

3. The rats had a high mortality rate due to frequent seizures within 1 week after successful modeling. 5% dextrose sodium chloride 3-5ml intraperitoneal injection can be given daily, 2/d, to strengthen the energy supply.

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