Proposals involving animal models of status epilepticus (SE) must be designed and performed with due consideration of minimizing animal discomfort, distress, pain, and mortality consistent with the experimental goals. Studies must be designed to include the least amount of seizure activity for the shortest amount of time to still produce meaningful data. Major considerations for seizure studies should include the method of induction, the provision of appropriate supportive care (commensurate with the number, duration, and severity of seizures), and criteria for either treatment of the seizure by drug administration (such as diazepam) or euthanasia of the affected animal. Protocols should clearly state the goals of the study, and clearly justify the duration of SE required to achieve those goals.
Method of Induction
Brain damage producing epileptiform activity may be induced by a number of different methods: electrical stimulation, percussion, or exposure to excitotoxic or neurotoxic conditions or substances.
Many different and varied protocols exist for induction of SE; a list of some relevant references may be found at the end of this policy. Regardless of the method chosen, it is understood that the procedures required to investigate anatomical, electrophysiological, and molecular consequences of SE may cause distress and injury, and even require loss of some animals in order to achieve the aims of the research. The goal should be to provide for the welfare of animal subjects and reduce pain, distress and mortality whenever possible when compatible with experimental objectives.
Monitoring / Premature Euthanasia
During the acute phase of SE, animals must be continuously monitored. If they are in a cage during this time, they should be individually caged to prevent inadvertent injury between animals. Cages should also not contain bedding, which might asphyxiate an animal that cannot raise its head; lining of the cage with paper toweling or disposable blue hospital underpads (chux) is recommended. To better describe the level of seizure activity, it is recommended that the activity be staged every 30 minutes, using the scale of Racine (1972) - stage 1, mouth and facial twitches; stage 2, clonic head movements; stage 3, unilateral forelimb clonus followed by contralateral clonus; stage 4, clonic rearing; stage 5, loss of postural control. If stage 3 or greater seizures are observed for 2 or more hours, an anticonvulsant should be used (see below) if it would not interfere with the study. Withholding of anticonvulsant medication in this instance must be scientifically justified and described in the animal use protocol. Prolonged seizures that cannot be controlled with the use of drugs should be considered as a basis for premature euthanasia of the animal. Also, animals which develop excessive seizures, accompanied by tonic flexion and/or extension of the limbs, should be euthanized; such animals, if they are allowed to survive, often develop hindlimb paralysis of variable severity.
Following the acute phase, animals should be monitored at least once daily, including weekends or holidays. Criteria for premature euthanasia during this period should include weight loss of 10% or greater. (If transient weight loss exceeding 10% might be expected in the first few days following induction, this should be detailed in the animal use protocol and justified.) Any unusual frequency and/or duration of seizure activity during the chronic phase should also be considered as criteria for premature euthanasia.
Supportive Care
Anticonvulsants
Benzodiazepines (such as diazepam) and barbiturates (such as pentobarbital) have proved to efficiently abort or attenuate SE, thus preventing the relatively high mortality associated with prolonged seizures in several models. For example, Gibbs et al. (1998) recommended administration of diazepam at 4 mg/kg IP one hour after excitotoxic injection, repeated at 3 and 5 hours, while Buckmaster (2004) and Han et al. (2009) administered diazepam at 10 mg/kg two hours after onset of stage 3 or greater seizures. In models that may induce SE lasting longer than 2 hours or that may be expected to induce seizures of a high stage, 20-30 mg/kg pentobarbital (i.p.) at an interval greater than 30 minutes following injection can protect animals from lethal effects of convulsants, while allowing the production of sufficient damage to generate the pathological effects required for experimental study (Han 2009).
Fluid Therapy
As early as one hour following status development, pharmaceutical-grade isotonic saline or lactated Ringer’s solution (at 0.01 to 0.05 ml/gram body weight) should be administered every 2-3 hours in order to maintain adequate hydration. Fluid administration should continue until the animal is able to drink on its own.
Special Feeding
Because animals are sometimes unable to eat pelleted rodent chow during the recovery phase, provision of liquid or soft food may be essential for some models. Once the animal is able to drink, supportive beverages such as Ensure (rats seem to prefer the chocolate flavor) or Gatorade (especially the red, fruit juice flavors) may be useful as supplements. Animals may also be offered baby food, or other soft food supplements (such as pelleted diet that has been moistened and mashed with Napa nectar). Oral gavaging of these should not be necessary but may be attempted if the animal is unable to drink normally, or consume the softened food. Supplemental feeding must continue twice daily until the animal is able to eat regular pelleted food. Consider the caloric requirements of the animal when planning the feeding schedule (for a mouse, approx. 175 kcal /BWkg0.75, for an adult rat, 114 kcal/BWkg0.75, although this can vary with strain, age, etc) (ILAR, 1995).
At any time, investigators must contact the DLAM veterinary staff if they have any questions about the condition of their animals or their special care.
References
- Andre V, Ferrandon A, Marescaux C, Nehlig A. (2000) The lesional and epileptogenic consequences of lithium- pilocarpine induced status epilepticus are affected by previous exposure to isolated seizures: Effects of amygdala kindling and maximal electroshocks. Neuroscience 99:469-481.
- Buckmaster PS. (2004) Laboratory animal models of temporal lobe epilepsy. Comp Med. 54:473-85.
- Curia G. (2008). The pilocarpine model of temporal lobe epilepsy (invited review). J Neurosci Methods. 172:143-57.
- Gibbs JW, Shumate MD, Coulter DA. (1997) Differential epilepsy-associated alterations in postsynaptic circuit rearrangements accompanying development of TLE. J. Neurophysiol. 77:1924–1938.
- Glien M, Brandt C, Potschka H, Voight H, Ebert U, Loscher W. (2001) Repeated low dose treatment of rats with pilocarpine: Low mortality but high proportion of rats developing epilepsy. Epilepsy Res. 46:111-119.
- Han SR et al. (2009) Differential expression of activating transcription factor-2 and c-Jun in the immature and adult rat hippocampus following lithium-pilocarpine induced status epilepticus. Yonsei Med J. 30:200-5.
- Hellier J, Patrylo P, Buckmaster P, Dudek FE. (1998) Recurrent spontaneous motor seizures after repeated low-dose systemic treatment with kainate: Assessment of a rat model of temporal lobe epilepsy. Epilepsy Res. 31: 73-84.
- Leite JP, Garcia-Cairasco NA, Cavalheiro EA. (2002) New insights from the use of pilocarpine and kainate models. Epilepsy Res. 50:93-103.
- Nutrient Requirements of Laboratory Animals, Fourth Revised Edition, 1995 (1995) Institute for Laboratory Animal Research (ILAR).
- Racine, RJ. (1972) Modification of seizure activity by electrical stimulation. II. Motor seizure. Electroencephalogr Clin Neurophysiol. 32:281-94.
Approved 11/24/03; Revised 12/14/09