As outlined above, AATs constitute a neurochemical rationale for the suppression or prevention of seizures in epilepsy. To circumvent side effects of systemic AATs, focal AATs were tested by transplantation of adenosine-releasing cells into the vicinity of an epileptogenic focus.11
The first generation of therapeutic implants was based on rodent fibroblasts engineered to release adenosine. These cells were encapsulated into semi-permeable polymer membranes to prevent immune-rejection and to prevent network interactions. Intraventricular implants of these devices provided nearly complete protection from seizures in kindled rats, which was limited to 2 to 4 weeks due to poor long-term survival of the encapsulated cells.38
These results also demonstrated that local paracrine release of adenosine is sufficient to prevent seizures; thus, functional integration of therapeutic cells into hippocampal networks is not necessary.
generation of adenosine-releasing therapeutic cells was generated in our laboratory by bi-allelic genetic disruption of the Adk
-gene in mouse embryonic stem cells (mESCs).39
The cells were subjected to a neural differentiation protocol40 in vitro.
Resulting neural precursor cells (NPs) released adenosine and were transplanted into the infrahippocampal fissure of rats prior to hippocampal kindling. When analyzed 26 days after grafting, we found dense clusters of graft-derived cells within the infrahippocampal fissure that likely formed a reservoir for the paracrine release of adenosine. In addition, graft derived cells migrated into the ipsilateral CA1, stained positive for NeuN, and assumed a neuronal morphology with long, branching processes.14
These data demonstrate that adenosine-releasing stem cell-derived brain-implants display improved survival characteristics compared to encapsulated cell grafts. One week after grafting kindling was initiated and the subsequent increase in seizure activity was compared to recipients of corresponding wild-type (wt) cells and to sham-operated animals. Strikingly, kindling in recipients of adenosine releasing ES-derived NPs was strongly retarded.14
Thus, 22 days after grafting and after 48 kindling stimulations, recipients of adenosine releasing NPs failed to display generalized (stage 4 and 5) seizures; instead, these animals displayed more immature kindling parameters. This delay in the progressive development of behavioral seizures was observed in the presence of electrographic afterdischarges elicited by each kindling-stimulation. These findings suggest a novel antiepileptogenic or disease modifying function of stem cell-mediated adenosine delivery; using this approach however, true antiepileptogenic effects are difficult to assess due to overlapping anti-ictogenic effects of adenosine. In addition, this study left open the question whether ADK-deficient stem cell-derived brain implants would be equally effective in epileptogenesis models that involve astrogliosis and spontaneous recurrent seizures.
To assess potential antiepileptogenic effects of adenosine-releasing mESC-derived NPs in a model that involves astrogliosis and the development of spontaneous seizures we chose a mouse model of CA3-selective epileptogenesis.21
Twenty-four hours after unilateral intraamygdaloid injection of KA (initial epileptogenesis-precipitating injury, IPI) ADK-deficient NPs were injected into the infrahippocampal fissure ipsilateral to the KA-injection. Controls received respective wt cells or a corresponding sham procedure. When analyzed three weeks later, all graft recipients had dense clusters of graft-derived cells located within the infrahippocampal fissure. In addition, individual cells had migrated into the ipsilateral CA1 and assumed a neuronal morphology. In all animals, the CA3-selective IPI was confirmed by histological analysis. Most importantly, recipients of adenosine releasing Adk−/−
NPs were characterized by a significant reduction in astrogliosis and by almost normal ADK levels in the ipsilateral CA3, whereas prominent astrogliosis and upregulation of ADK was found in the CA3 of control animals. These findings indicate that in recipients of adenosine-releasing stem cell derived brain implants two important features of epileptogenesis – astrogliosis and upregulation of ADK –were significantly reduced. In concordance with normal ADK levels, all recipients of adenosine-releasing cells were completely protected from any seizure activity, whereas respective control animals displayed >4 electrographic seizures per hour. Thus, adenosine releasing stem cell-derived brain implants prevented the expression of seizures in a spontaneous seizure model.21
Reduced astrogliosis and lack of ADK-upregulation in the therapeutic group suggest that adenosine releasing stem cell-derived brain implants exert at least some antiepileptogenic effects. Indeed, adenosine acting on astrocytic adenosine A1
receptors was shown to inhibit reactive astrogliosis.41
Reduced astrogliosis in turn would limit epileptogenic upregulation of ADK, thus ameliorating the seizure-triggering adenosine-deficiency.