Animals and Drug Treatments
All animal procedures were conducted in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) in accordance with protocols approved by the Institutional Animal Care and Use Committee and the principles outlined in the NIH Guide for the Care and Use of Laboratory Animals. Male C57BL/6 mice (Charles River, Tacoma, WA, weight, 25–30 g) were habituated to the testing environment 4 days prior to behavioral testing.
Mice were treated with combinations of the following drugs by intraperitoneal injection in a volume of 0.1 ml 0.9% saline / 10 g body weight: (i) erythro-9-(2-hydroxy-3-nonyl)-adenine hydrochloride (EHNA, Sigma-Aldrich): 10 mg/kg; (ii) 5-iodotubercidin (ITU, RBI): 3.1 mg/kg; (iii) kainic acid (KA, Sigma-Aldrich): 35 or 40 mg/kg; (iv) caffeine (Sigma-Aldrich): 40 mg/kg.
Mouse model for SUDEP
Inhibition of the two major adenosine removing enzymes adenosine deaminase (ADA) and adenosine kinase (ADK) with their respective inhibitors EHNA (10mg/kg, i.p.) and ITU (3.1mg/kg, i.p.) was performed 15 min prior to induction of acute seizures with KA (35mg/kg, i.p., ). Mice were randomly divided into two groups, (i) EI-K (pretreatment with EHNA and ITU followed by kainic acid), and (ii) SAL-K (pretreatment with 0.9% saline followed by kainic acid).
Injection paradigm and seizure scores of SUDEP model
To explore the effects of adenosine receptor blockade in our SUDEP model, two groups of animals (SUDEP+CAF and SUDEP+SAL) were subjected to treatment with caffeine immediately after seizure onset (). Mice received pretreatment with EHNA (10 mg/kg, i.p.) and 5-ITU (3.1 mg/kg, i.p.), 15 min prior to injection of kainic acid (40 mg/kg, i.p.). 30 sec after seizure onset (at first appearance of a seizure score of 5) animals from the SUDEP+CAF group received caffeine (40mg/kg, i.p.) whereas animals from the SUDEP+SAL group received a saline injection. The survival time following kainic acid injection was recorded in each animal.
Caffeine prolongs survival time of SUDEP mice
Assessment of seizure activity
Following kainic acid administration, seizure activity was scored according to the Racine scale in 5-min bins: stage 1, mouth and facial twitches; stage 2, clonic head movements; stage 3, unilateral forelimb clonus followed by contralateral clonus; stage 4, clonic rearing; and stage 5, loss of postural control and falling.
Differences among groups were analyzed using one-way ANOVA followed by Bonferroni post hoc test. P < 0.05 was determined as significant. Values are expressed as means ± standard deviation.