For years, researchers around the world are using animals as models for inducing human diseases to better understand their pathophysiology and for evaluation followed by development of safe and effective therapeutic interventions. Animal models are primarily employed to predict a response similar to that of humans, towards newly developed therapeutic agents. For this reason, the continuous interest to develop better models for post traumatic epilepsy has been widely acknowledged, as traumatic brain injury represents one of the apparent factors that can be retrospectively linked to epilepsy.
Although, the precise mechanisms that underlie the pathogenesis of post-traumatic epilepsy are still poorly understood, the iron-induced epileptic rat model is of interest because it may serve to mimic clinical epilepsy appearing in persons who have suffered head trauma. Production of epileptiform electrographic discharges in iron-induced experimental epilepsy is believed to be mediated by membrane lipid peroxidation initiated by the action of oxygen radicals such as superoxide anion, hydrogen peroxide and hydroxyl radicals. The iron chloride (FeCl3) model of post-traumatic epilepsy involves the injection of iron salts into the cortex of rats (simulating red cell extravasation after traumatic brain injury), which leads to edema, necrosis with subsequent gliosis and focal recurrent epileptiform discharges. There is significant evidence from both in-vivo and in-vitro studies that iron-containing compounds induce the production of oxidative species. For example, there is increased production of malonaldehyde (a product of lipid peroxidation) after injection of ferric chloride into rat isocortex. Similarly, superoxide radicals are increased in brain tissue adjacent to the site of iron salt injection. Also, in the dorsal hippocampus, markers of lipid peroxidation are significantly elevated when measured in the dissected organ. In vivo levels of lipid peroxides and the activities of the antioxidant enzymes such as superoxide dismutase, Glutathione peroxidase, Glutathione reductase, catalase and Glucose-6-phosphate dehydrogenase were also found to be altered in the epileptogenic focus after intracortical FeCl3 injection. So, this article is intended to provide a methodology for the induction of post-traumatic epilepsy in rat model.
Rats of either sex preferably male of age 4-6 months, weighing 300-450g are used for experimental studies. Animals are housed in standard polypropylene cages under controlled hygienic environmental conditions. The rats are maintained at temperature (23 ± 4ºC) under a 12-hour light - dark cycle.
1. Rats to be anaesthetized either with gas anesthesia (e.g., isofluorane) or intraperitoneal injection of standard dose of ketamine (80mg/kg body weight) and xylazine (10mg/kg body weight).
2. Surgery to be performed under deep anaesthesia as mentioned below.
3. First, hair is removed from the skull region with the help of scissors or electric shaver to expose the skin.
4. The rat is fixed in the stereotaxic apparatus.
5. Betadiene solution is to be applied on the exposed skin of the rat.
6. Slight deep incision is to be made on the skin parallel to the central portion of the skull and expose that area with the help of surgical blade.
7. Mark the coordinates on the skull anteroposterior = 1mm, lateral =1 mm and ventral (depth) = 1.5 mm to induce epilepsy.
8. Drill holes of 0.5mm diameter into the skull.
9. Inject 5μl of 100mM ferric chloride intracortically with the help of injector cannulas to induce experimental epilepsy.
10. Rate of ferric chloride injection is 1μl/minute.
11. Place four stainless steel epidural screw electrodes bilaterally over the parietal cortex with the help of stereotaxic apparatus following the atlas.
12. Coordinates will be 2 mm posterior and anterior to bregma and 2 mm lateral surrounding the somatosensory region.
13. Also place one screw upon the frontal sinus to serve as animal ground.
14. Connect free ends of each electrode wire to a 9-pin connector.
15. Affix the connector to the surface of the skull with the help of dental acrylic cement to make a robust platform.
16. Proper post-operative care will be done and animals were allowed to recover and habituate for 1 week before electroencephalographic (EEG) recordings.
17. Spread nebasulf powder over the surgical area to avoid any infection and to ensure proper healing.
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