doi:10.1016/j.expneurol.2004.12.012 
Copyright © 2004 Elsevier Inc. All rights reserved.
Seizure suppression and lack of adenosine A1 receptor desensitization after focal long-term delivery of adenosine by encapsulated myoblasts
Martin Güttingera, Vivianne Padrunb, William F. Pralongb and Detlev Boisona, c, 
, 
aInstitute of Pharmacology and Toxicology, University of Zurich, CH-8057 Zurich, Switzerland
bInstitute of Neurosciences, Federal Institute of Technology (EPF), CH-1015 Lausanne, Switzerland
cInstitute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, Federal Institute of Technology (ETH), CH-8057 Zurich, Switzerland
Received 29 October 2004;
revised 25 November 2004;
accepted 10 December 2004.
Available online 12 February 2005.
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Abstract
Adenosine is an important inhibitory modulator of brain activity. In a previous ex vivo gene therapy approach, local release of adenosine by encapsulated fibroblasts implanted into the vicinity of an epileptic focus, was sufficient to provide transient protection from seizures (Huber, A., Padrun, V., Deglon, N., Aebischer, P., Mohler, H., Boison, D., 2001. Grafts of adenosine-releasing cells suppress seizures in kindling epilepsy. Proc. Natl. Acad. Sci. U. S. A. 98, 7611-7616). Long-term seizure suppression beyond 2 weeks was precluded by limited life expectancy of the encapsulated fibroblasts. To study the feasibility for long-term seizure suppression by adenosine releasing brain implants, in the present contribution, mouse C2C12 myoblasts were engineered to release adenosine by genetic inactivation of adenosine kinase. After encapsulation, the myoblasts were grafted into the lateral brain ventricles of epileptic rats kindled in the hippocampus. While seizure activity in animals with wild-type implants remained unaltered, 1 week after grafting all rats with adenosine-releasing implants (n = 25) displayed complete protection from convulsive seizures and a corresponding reduction of afterdischarges in EEG-recordings. The duration of seizure suppression was maintained for a period of 3 weeks in 50% of the animals ranging to a maximum of 8 weeks in one animal. During the course of these experiments, adenosine A1 receptors remained responsive to selective agonists and antagonists indicating a lack of desensitization of A1 receptors after local long-term exposure to adenosine. Furthermore, local release of adenosine did not affect locomotor activity, whereas systemic application of the A1 agonist 2-chloro-N6-cyclopentyladenosine caused strong sedation. Thus, the local release of adenosine by cellular implants provides a feasible option for a potential side-effect free approach for the long-term treatment of focal epilepsies.
Keywords: Adenosine; Adenosine kinase; A1 receptor; Epilepsy; Kindling; Seizure suppression; Cell therapy; Myoblasts; Cell encapsulation; Sedation
Fig. 1. Characterization of adenosine-releasing C2C12-M1 myoblasts. (A) After mutagenesis and selection for ADK deficiency the C2C12 cell clone M1 (C2C12-M1) was analyzed by Western blot analysis. Aqueous cell protein extracts from C2C12-WT and C2C12-M1 myoblasts corresponding to 20 and 40 μg of total protein were probed with a polyclonal antiserum raised against recombinant mouse ADK. C2C12-M1 myoblasts were devoid of ADK immunoreactivity. (B) The amount of adenosine in supernatants from 105 adherent C2C12-M1 and C2C12-WT myoblasts was measured 2, 4, 8, and 24 h after incubation using an enzyme-coupled bioluminescent assay. Data are represented as means (n = 5) ± SDs.
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Fig. 2. Behavioral seizure suppression in kindled rats. Seizure activity of kindled rats was analyzed after application of a test stimulus at the indicated time points, before and after implantation of an adenosine-releasing cell capsule. One and 2 weeks before grafting of encapsulated C2C12-M1 myoblasts, all rats (n = 25) displayed convulsive grade 5 seizures. One week after implantation of C2C12-M1 myoblast capsules into the lateral brain ventricle of kindled rats (n = 25), behavioral seizures were completely suppressed in all rats. Two animals lost their pedestal after 1 week and were excluded from the experiment (n = 23 from week two onward). At day 31 after implantation, three rats were tested with the selective A1 receptor antagonist DPCPX. After this pharmacological control experiment, two animals were killed to retrieve capsules from animals, which were still protected by the implant (n = 21 from week 5 onward). Five weeks after implantation of the C2C12-M1 myoblast capsules, five rats were still protected by the grafts. One animal was completely protected from seizures 8 weeks after implantation, when the experiment was terminated. *To provide a control for the lack of desensitization of A1 receptors, animals with recurrence of seizure activity after 2 to 6 weeks were tested on the following day with the A1 receptor agonist CCPA (3 mg/kg, i.p.). All animals (n = 20) were again protected from seizure activity 20 min after drug injection.
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Fig. 3. Representative EEG recordings after application of test stimulations in kindled rats. Bilateral intrahippocampal EEGs were recorded after application of a test stimulus in kindled rats that had received (A) a control C2C12-WT capsule or (B) an adenosine-releasing C2C12-M1 capsule. (A) Kindled rats were tested once weekly after implantation of a wild-type C2C12 myoblast capsule. Epileptic afterdischarges were associated with grade 5 seizures and remained unchanged 1 to 8 weeks after implantation of the control capsule. Representative traces are shown 1, 2, and 4 weeks after implantation. (B) Kindled rats grafted with an adenosine-releasing C2C12-M1 capsule were tested once weekly. In the animal shown, 1 to 4 weeks after implantation, epileptic afterdischarges were dramatically reduced and associated with grade 0 behavioral seizures. A recording from week one is shown. Systemic application of the selective A1 receptor antagonist DPCPX at day 31 after implantation of the capsule resulted in a complete restoration of epileptic afterdischarges including a convulsive grade 5 seizure, when tested 30 min after DPCPX injection. Four days after DPCPX injection (day 35), the same rat was again protected from seizures (grade 0). (C) EEG recordings from a different animal with a C2C12-M1 implant, which was protected for 8 weeks. DPCPX injection at day 49 transiently restored grade 5 seizures and epileptic afterdischarges (left), while 56 days (8 weeks) after implantation the animal was again protected from convulsive seizures and afterdischarges (right). After this recording, the animal was killed to retrieve a capsule from a long-term protected animal. In each EEG, the upper trace represents the recording of the ipsilateral (i) hippocampus (kindling stimulation and capsule implantation side), while the lower trace represents the recording from the contralateral (c) hippocampus.
Fig. 4. Photomicrographs of encapsulated myoblasts after hematoxylin-eosin staining. (A) C2C12-M1 myoblast capsule retrieved at 8 weeks from a kindled rat, which was protected from seizures at the time point of retrieval. Nuclei of viable myoblasts are stained by hematoxylin. (B) C2C12-M1 myoblast capsule retrieved at 5 weeks from a rat with recurrence of seizure activity. No hematoxylin staining of cell nuclei is evident. Scale bars: (in A) A, B, 20 μm.
Fig. 5. Locomotor activity of non-kindled sham-operated control rats and kindled rats in the open-field. Total locomotor activity was assessed in the open-field. Sham-operated control rats (n = 6) and kindled rats (n = 14) were analyzed for changes in locomotor activity 1 week before (“untreated”) and after implantation of an adenosine-releasing C2C12-M1 myoblast capsule (“intraventricular adenosine”). Locomotor activity was not altered by the local release of adenosine (F(1,26) = 0.006, not significant). In contrast, another group of sham-operated control (n = 6) and kindled (n = 6) rats displayed significantly reduced total locomotor activity 20 min after i.p. injection of CCPA (1.5 and 3.0 mg/kg), as compared to control vehicle injection (P < 0.01, Dunnett). Results are expressed as means ± SEMs.
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