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
