Suppression of kindling epileptogenesis by adenosine releasing stem cell-derived brain implants
1RS Dow Neurobiology Laboratories, Legacy Research, Portland, OR, USA and 2Institute of Reconstructive Neurobiology, LIFE & BRAIN Center, University of Bonn and Hertie Foundation, Bonn, Germany
Correspondence to: Detlev Boison, PhD, R.S. Dow Neurobiology Laboratories, Legacy Research, 1225 NE 2nd Ave, Portland, OR 97232, USA E-mail: dboison{at}downeurobiology.org
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Summary |
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Epilepsy therapy is largely symptomatic and no effective therapy is available to prevent epileptogenesis. We therefore analysed the potential of stem cell-derived brain implants and of paracrine adenosine release to suppress the progressive development of seizures in the rat kindling-model. Embryonic stem (ES) cells, engineered to release the inhibitory neuromodulator adenosine by biallelic genetic disruption of the adenosine kinase gene (Adk–/–), and respective wild-type (wt) cells, were differentiated into neural precursor cells (NPs) and injected into the hippocampus of rats prior to kindling. Therapeutic effects of NP-derived brain implants were compared with those of wt baby hamster kidney cells (BHK) and adenosine releasing BHK cell implants (BHK-AK2), which were previously shown to suppress seizures by paracrine adenosine release. Wild-type NP-graft recipients were characterized by an initial delay of seizure development, while recipients of adenosine releasing NPs displayed sustained protection from developing generalized seizures. In contrast, recipients of wt BHK cells failed to display any effects on kindling development, while recipients of BHK-AK2 cells were only moderately protected from seizure development. The therapeutic effect of Adk–/–-NPs was due to graft-mediated adenosine release, since seizures could transiently be provoked after blocking adenosine A1 receptors. Histological analysis of NP-implants at day 26 revealed cell clusters within the infrahippocampal cleft as well as intrahippocampal location of graft-derived cells expressing mature neuronal markers. In contrast, BHK and BHK-AK2 cell implants only formed cell clusters within the infrahippocampal cleft. We conclude that ES cell-derived adenosine releasing brain implants are superior to paracrine adenosine release from BHK-AK2 cell implants in suppressing seizure progression in the rat kindling-model. These findings may indicate a potential antiepileptogenic function of stem cell-mediated adenosine delivery.
Key Words: adenosine; adenosine kinase; epileptogenesis; stem cells; cell therapy
Abbreviations: ADD, afterdischarge duration; ADK, adenosine kinase; BHK cells, baby hamster kidney cells; DPCPX, 8-cyclopentyl-1,3-dipropylxanthine; EGFP, enhanced green fluorescent protein; ES cell, embryonic stem cell; FGF-2, basic fibroblast growth factor; NP, neural precursor.
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Received July 13, 2006. Revised January 25, 2007. Accepted March 5, 2007.
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Introduction |
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Adenosine is thought to act as an endogenous anticonvulsant (Lee et al., 1984
; Dragunow et al., 1985; Boison, 2005; Fredholm et al., 2005a). Indeed, deficits in adenosinergic neuromodulation contribute to epileptogenesis and seizure activity (Young and Dragunow, 1994; Rebola et al., 2003; Gouder et al., 2004; Dulla et al., 2005; Fedele et al., 2005). In particular, increased levels of adenosine kinase (ADK), the major adenosine metabolizing enzyme, can be a direct cause for seizures (Fedele et al., 2005) or increased sensitivity to cell death (Pignataro et al., 2006), and pharmacological inhibition of ADK or activation of adenosine A1 receptors are effective in the suppression of pharmacoresistant seizures (Kowaluk and Jarvis, 2000; Gouder et al., 2003; Gouder et al., 2004). In addition, adenosine, by activation of adenosine A1 receptors, is thought to keep an epileptic focus localized (Fedele et al., 2006) and to prevent status epilepticus development (Young and Dragunow, 1994). Thus therapies, which are aimed to augment the adenosine system during epileptogenesis, should constitute a rational antiepileptogenic approach. Unfortunately, systemic manipulation of the adenosine system is associated with significant peripheral side effects (Dunwiddie and Masino, 2001). However, focal treatment strategies for partial epilepsies may circumvent these side effects (Nilsen and Cock, 2004). Thus, seizure suppression by adenosine can be exploited by intracerebral implants of cells engineered to release adenosine. This has transiently been accomplished by encapsulated fibroblasts or myoblasts engineered to lack ADK (Huber et al., 2001; Güttinger et al., 2005b). In contrast to encapsulated adenosine releasing cells, which exert their therapeutic effects exclusively by a paracrine mode of action (Güttinger et al., 2005a), stem cell-derived brain implants may integrate into the adult hippocampus (Ruschenschmidt et al., 2005) and may therefore directly influence the progression of epileptogenesis. Embryonic stem (ES) cells are amenable to genetic modification and to proliferation in an undifferentiated state and are thus able to provide unlimited numbers of engineered cells for transplantation. To avoid teratoma formation by undifferentiated ES cells (Lindvall et al., 2004), a protocol has been established, which allows the directed differentiation of ES cells into defined populations of neural precursor (NP) cells (Okabe et al., 1996; Brüstle et al., 1997).
We recently disrupted both alleles of ADK in mouse ES cells (Adk–/–) (Fedele et al., 2004) to induce therapeutic adenosine release. Intraventricular transplantation of encapsulated Adk–/– stem cell-derived cells provided transient but complete seizure suppression in kindled rats (Güttinger et al., 2005a). Long-term studies were precluded by the low survival rate of these encapsulated implants.
In these previous studies, encapsulated cells provided suppression of fully kindled seizures by paracrine release of adenosine. However, possible therapeutic interactions of paracrine cell-mediated adenosine release and of stem cell-mediated effects on epileptogenesis have not been addressed previously. Neuronal brain implants may have different effects on epileptogenesis depending on their origin and integration into pre-existing networks (Buzsaki et al., 1988). To address the question whether Adk–/– ES cell-derived NPs combine adenosine-mediated and endogenous antiepileptogenic activity, we compared epileptogenesis in rats, which either received Adk–/– or wild-type (wt) ES cell-derived NPs, with therapeutic effects from paracrine adenosine release mediated by adenosine releasing BHK-AK2 cell implants.
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Methods |
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Cell culture
Wild-type and genetically altered ES cells (Adk+/+ and Adk–/–, respectively) and baby hamster kidney (BHK) cells (Adk+/+ BHK and Adk–/– BHK-AK2, respectively) used in these experiments have been described previously (Huber et al., 2001
; Fedele et al., 2004). NP cells were generated from Adk–/– and Adk+/+ ES cells using a well-established step-wise differentiation protocol (Okabe et al., 1996; Brüstle et al., 1997). NPs were routinely cultured on poly-ornithine-coated dishes in N3 medium, which is based on a 1:1 mixture of DMEM with Ham's F12 supplemented with insulin (25 µg/ml), human apo-transferrin (100 µg/ml), progesterone (20 nM), putrescine (100 µM), sodium selenite (30 nM), penicillin (100 U/ml) and streptomycin (100 µg/ml). In addition, laminin (1 µg/ml) was added when plating the cells. To keep the cells in a proliferative state, the growth factor FGF-2 (basic fibroblast growth factor) (10 ng/ml) was added daily. Prior to transplantation, proliferating neural precursors were transduced with a pMOWS-EGFP (enhanced green fluorescent protein) retrovirus, which does not affect the cellular differentiation potential of transduced cells (Ketteler et al., 2002). For transduction of NPs, subconfluent producer cells GP-E-86 (Markowitz et al., 1988) were maintained in N3 medium for 24 h. Subsequently, virus-containing supernatant was filtered, supplemented with FGF-2 and polybrene (4 µg/ml) and allowed to transduce NPs. Transduction efficacies of NPs were about 25%. For transplantation, the cells were harvested 2–4 days later and resuspended at a concentration of 2 x 105 cells per µl HBSS containing 0.1% DNAse. The analysis of the amount of adenosine released from ES cell-derived NPs was performed after growing the cells under neurogenic conditions as described previously (Fedele et al., 2004).
Animals and surgery
All animal procedures were conducted in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care 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 Sprague–Dawley rats were used at a body weight of 300 g. All rats were acclimatized for at least 1 week before being used in the experiments. The rats were housed under 12 h light/dark cycle (lights on from 8:00 AM) with food and water provided ad libitum. Two days before cell implantation or sham treatment, immunosuppression with cyclosporine was initiated and maintained daily during the course of the experiment.
Anesthesia was induced with 3% isoflurane, 67% N2O, 30% O2 and maintained with 1.5% isoflurane, 68.5% N2O, 30% O2, while rats (n = 26) were placed in a Kopf stereotactic frame. First, the cell injection was performed using a glass capillary (inner diameter of tip: 70–90 µm). 2 x 106 cells were injected in a volume of 10 µl using a drill hole above the left brain hemisphere 2 mm rostral to bregma and 1.6 mm lateral to the midline. Using an identical procedure, sham-treated control animals received Hank's buffered saline solution instead of cells. Using this drill hole the glass capillary was inserted into the brain using an angle of 45° from vertical and an angle of 45° from the midline. Thus, a diagonal injection tract was created aiming at a coordinate of 5.5 mm caudal to bregma, 5.5 mm to the right of the midline and 7.5 mm below the dura. Cells were injected during retraction of the cannula at a rate of 
1 µl per mm per min. Thus, the injected cells were deposited over a relatively long injection tract within the right hippocampus and adjacent to the electrode implantation site (Fig. 4). The diagonal cell injection approach pursued here was characterized by a number of important advantages compared with ‘traditional’ vertical intrahippocampal injection approaches: (i) coverage of most of the dorso-ventral extent of the hippocampus by placement of the grafted cells into the infra-hippocampal cleft; (ii) minimization of damage to the ipsilateral hippocampus; (iii) compatibility with the electrode containing pedestal of the animals.
Next, a bipolar, coated, stainless steel electrode (0.20 mm in diameter, Plastics One, Roanoke, VA, USA) was implanted into the right hippocampus and fixed with a pedestal of dental acrylate. Coordinates for the hippocampal electrodes were (tooth bar at 0), 5.5 mm caudal to bregma, 5.5 mm lateral to midline and 7.5 mm ventral to dura (Fig. 4).
Kindling
One week after electrode implantation, the animals were stimulated unilaterally six times every second day with a Grass S-88 stimulator (1-ms square-wave pulses of 5 V at 50-Hz frequency for 10 s, 30-min interval between stimulations). Behavioural seizures were scored according to the scale of Racine (Racine, 1978). Each animal was stimulated for a total of 48 stimulations, which was equivalent to 8 test days. The electroencephalogram (EEG) was recorded for periods of 1 min before and 5 min after application of the stimulating pulse using a Grass Electroencephalogram Model 8–16. The EEGs recordings were scanned and assembled to consecutive sequences.
Drug treatment
DPCPX (8-cyclopentyl-1,3-dipropylxanthine) was purchased from Sigma and dissolved in DMSO (1 mg/ml). After the completion of 48 kindling stimulations, all animals were tested again with the same stimulus 2 days later to determine the baseline seizure response. Four hours after this test all animals were injected with DPCPX (1 mg/kg, i.p.) and retested after 30 min. The next stimulus was delivered 24 h after the DPCPX injection. During the course of the whole experiment all animals, including the sham controls, received daily injections of the immunosuppressant cyclosporine A (12.5 mg/kg, i.p.) (Sandimmune, Novartis, Basel, Switzerland).
Histology
One day after completion of the experiments the rats were transcardially perfused with 4% paraformaldehyde in phosphate buffer (0.15M, pH 7.4). Brains were then post-fixed in the same fixative for 6 h and cryoprotected in 10% dimethyl sulfoxide (DMSO) in PBS (v/v) before being cut into a total of 72 coronal sections (40 µm thickness) covering a range of 3.5–6.5 mm caudal to bregma thus covering the full extent of the lateral hippocampus. Histological analysis was performed in series with every sixth section treated equally as follows (n = 10 to 12 per treatment): (i) to characterize the location of graft-derived cell clusters a Cresyl violet stain was performed; (ii) to further characterize the intrahippocampal implants, graft-based EGFP fluorescence was determined. (iii–vi) In additional sets of sections from each animal the EGFP fluorescence was correlated with immunofluorescence stainings. To characterize the implanted cells, the following antibodies were used; (iii) primary ADK antibody (Gouder et al., 2004), (iv) a monoclonal mouse antibody against the astrocytic marker, glial fibrillary acidic protein (GFAP) (MAB360; Chemicon International, Temecula, CA, USA); (v) a monoclonal mouse antibody against the neuron-specific nuclear protein, NeuN (MAB377; Chemicon International), and (vi) a monoclonal mouse nestin antibody with specificity for neuronal stem and endothelial cells (MAB5326, Chemicon International). Brain sections, were washed in PBS for 30 min changing the solution three times, and were then incubated overnight at 4°C in a solution with the primary antibodies diluted 1:1000 (NeuN), 1:4000 (ADK), 1:1000 (nestin) or 1:15000 (GFAP) in Tris–Triton, pH 7.4 with 2% normal serum and 0.2% Triton X-100. Sections were then washed three times for 10 min in TBS plus 0.05% Triton X-100, pH 7.4, followed by a 30 min incubation at room temperature in a solution containing the secondary antibodies donkey antimouse, conjugated to Cy3 (1:300) (Jackson Immuno Research, West Grove, PA, USA). Sections were washed a further three times, mounted on gelatin-coated slides and coverslipped with Dako fluorescent mounting medium (Carpentaria, CA, USA). To verify the specificity of the monoclonal antibodies, control stainings were performed with secondary antibody only. These control stainings were devoid of localized immunofluorescence (data not shown). To determine the proportion of NeuN positive cells, the total number of NeuN and GFP positive cells was counted within the CA1 region of four brain sections each from 5 Adk–/– and 4 wt NP graft recipients.
Confocal imaging
Confocal images were acquired using a Leica TCS-SP2 confocal system with a Leica DM-R upright microscope fitted with a Plan APO 40.0 x 0.75 objective. EGFP was excited at 488 nm with an Argon laser and emission filtered at 499–535 nm. Cy-3 was excited at 543 nm with a GrNe laser and emission filtered at 551–678 nm. For each image in a stack, the EGFP and Cy3 images were acquired sequentially, with two frames averaged per image.
Statistics
Unless stated otherwise, errors are given as ± SD and data were analysed using one-way ANOVA with Student–Newman–Keuls Test. *P < 0.05, **P < 0.01, ***P < 0.001.
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Results |
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Suppression of kindling epileptogenesis by intrahippocampal implants of ADK deficient NPs
The direct intrahippocampal transplantation of a stem cell-derived delivery system for adenosine may combine beneficial stem cell-dependent therapeutic effects with paracrine adenosine release. Adk+/+ and Adk–/– ES cells were differentiated to NPs as described previously (Fedele et al., 2004
). One week after growth in neurogenic conditions, Adk–/– NPs released 9.00 ± 4.55 ng adenosine/105 cells in 2 h, while the amount of adenosine released from wt cells, 0.33 ± 0.20 ng adenosine/105 cells in 2 h, was minimal. Three groups of rats received intrahippocampal implants of Adk+/+ (n = 4) or Adk–/– (n = 5) NPs, or sham treatments (n = 4). One week after surgery hippocampal kindling was initiated. Each rat received a total of eight kindling sessions every second day, each session comprised of six stimulations delivered every 30 min. Thus, each animal received a total of 48 stimulations (Fig. 1A). After each stimulation, the seizure score was determined according to the scale of Racine (1978), with stages 1–3 being partial seizures of growing intensity and stages 4 and 5 being generalized seizures with clonic and tonic components. During the first 24 stimulations (= session 1–4), acquisition of kindling was markedly delayed in recipients of stem cell-derived implants compared with sham controls (P < 0.001) (Fig. 1A and B). This effect was independent of genotype. Thus, intrahippocampal NP implants initially retard epileptogenesis. During the next 24 stimulations kindling acquisition continued in sham controls but also in wt cell recipients, with both groups reaching kindling criteria (= stage 4 and 5 seizures) by session 7. In contrast, recipients of adenosine releasing Adk–/– NPs continued to display significant protection from further epileptogenesis (P < 0.001) and failed to reach generalized stage 4 and 5 seizures after 48 stimulations (Fig. 1A and B). Thus, animals treated with Adk–/– NPs did not develop generalized seizures even 18 stimulations or 3 test days after their first occurrence in sham-treated controls (Fig. 1A). These results indicate a powerful suppression of kindling development by adenosine releasing stem cell-derived brain implants. In line with our previous demonstration that intraventricular adenosine releasing brain implants do not cause any sedative side effects (Güttinger et al., 2005b), in the present study we did not see any obvious signs for sedative or other side effects.
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Intrahippocampal implants of Adk–/– NPs reduce afterdischarge duration in EEG recordings
To further quantify the antiepileptogenic effect of adenosine releasing intrahippocampal NP implants, we determined the duration of electroencephalographic afterdischarges (ADD) elicited by each test stimulus during the kindling acquisition phase (Fig. 2A). The ADD was averaged according to session number (n = 6 stimulations) and treatment group (sham, wt and Adk–/– graft recipients). During the first four sessions, the ADD of cell recipients was significantly reduced compared with sham-treated controls. By session number 5, the ADD of wt graft recipients had approached those of sham-treated controls (Fig. 2B), whereas the ADD of Adk–/– NP-treated animals remained reduced until termination of the experiment after session 8 (i.e. 48 stimulations) (Fig. 2A). At that time the ADD of sham-treated animals or wt graft recipients had reached a plateau with average values exceeding 70 s, while the ADD of Adk–/– graft-treated animals remained reduced with values of 53.5 ± 19 s. Thus, the behavioural seizure response as observed in Fig. 1 was closely paralleled by the duration of afterdischarges in respective EEGs.
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Therapeutic effect of Adk–/– NPs is due to released adenosine
Unfortunately, direct in vivo measurements of adenosine are subject to large variations depending on the methods used (Delaney and Geiger, 1996
). The use of microdialysis probes to measure synaptic adenosine is compromised by the fact that the devices are usually engulfed by astrocytic processes (Benveniste et al., 1989), which can efficiently metabolize extracellular purines (Gu et al., 1996). To demonstrate that the therapeutic effect of Adk–/– NP implants was due to released adenosine, at the end of the experiment (i.e. 2 days after test session 8, Fig. 1B), all rats were injected with the A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) (1 mg/kg, i.p.). Adk–/– graft recipients displayed an average seizure stage of 2.8 ± 0.8 1 h before and of 3.0 ± 0.7 1 day after DPCPX administration (Fig. 3A, ADK–/–). However, when stimulated 30 min after DPCPX injection, all rats reacted with generalized stage 4–5 seizures. In contrast, both sham-treated controls and Adk+/+ NP recipients uniformly displayed generalized stage 4 or 5 seizures irrespective of DPCPX treatment (Fig. 3A, WT and SHAM). It is important to note that DPCPX had no effect in non-kindled control animals, which displayed only wet-dog (wd) shakes (Fig. 3A, CON). The pharmacological control experiments described here demonstrate (i) increased seizure thresholds in Adk–/– graft recipients 26 days after grafting, (ii) that the therapeutic effect of the grafts is due the release of adenosine, and (iii) that Adk–/– graft recipients are principally capable to express generalized seizures 26 days after transplantation.
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The graft-dependent seizure response at the end of the experiment was analysed in more detail in respective EEG recordings taken after test stimuli delivered either before, 30 min after, or 1 day after DPCPX application. The duration of ADD was determined and averaged for each treatment group (Fig. 3B). At the end of the kindling paradigm, the stimulus-elicited average ADD in sham controls and in wt graft recipients was around 80 s irrespective of DPCPX treatment. However, after completion of 48 kindling stimulations, animals with adenosine releasing implants were characterized by a profound suppression of the ADD to 55.0 ± 16.5 s before DPCPX, or to 57.0 ± 16.5 s 1 day after DPCPX (P < 0.05), values that corresponded to the lack of generalized seizures (Fig. 3B). The ADD suppression was adenosine-dependent, since seizure activity was transiently restored to an ADD of 81.0 ± 11.4 s, being comparable with wt or sham controls, when assessed 30 min after DPCPX (1 mg/kg, i.p.) injection. In contrast, DPCPX, which is known to be neither convulsive nor proconvulsive in vivo (Chesi and Stone, 1997
), failed to elicit any stimulus-elicited afterdischarge in non-kindled control animals. We conclude that the reduction of the ADD in ADK-deficient graft recipients at the end of the kindling phase is due to graft-mediated adenosine release.
ES cell-derived NPs are located within the ipsilateral hippocampus
Since the suppression of kindling progression by Adk–/– ES cell-derived brain implants was maintained for a period of more than 3 weeks, we expected to find viable grafts in the treated animals. In coronal brain sections from animals sacrificed 26 days after grafting we detected dense cellular transplants in the vicinity of the surgical injection tract, located in the infrahippocampal cleft (Fig. 4). Apart from these cell clusters, in all animals we detected individual graft-derived cells in the CA1 region of the ipsilateral hippocampus (Figs 4C and 5A1–A3). Many of these graft-derived cells showed a morphology and orientation comparable with mature hippocampal neurons displaying an elaborate dendritic tree and a principal axon. In these studies, we did not find any differences in cell numbers [49.9 ± 9.9 Adk–/– graft derived cells per CA1 per slice (n = 4 slices from 5 animals, each) versus 46.1 ± 6.3 graft derived cells per CA1 per slice (n = 4 slices from 4 animals, each), P > 0.1] or graft location between Adk+/+ (Fig. 5A3) and Adk–/– (Fig. 5A1 and A2) graft recipients. In all animals (n = 9), the location of the graft was confined to the ipsilateral hippocampus while the contralateral hippocampus (Fig. 5A4) as well as all other brain areas analysed were devoid of graft-derived cells. As expected, Adk–/– cells did not express ADK, while ADK colocalized with EGFP fluorescence in Adk+/+ transplants (Fig. 5D1 and D2). As revealed by confocal imaging, the differentiation of NP-derived brain implants did not appear to be influenced by the genotype of the graft. In Adk–/– graft recipients, 56% of these cells (556 out of 998 cells counted) expressed the neuronal marker NeuN (Fig. 5B1). Wild-type graft recipients displayed a similar degree of neuronal marker expression (53%, 391 out of 738 cells counted) (Fig. 5B2). Since every sixth of the 40-µm brain sections was stained for NeuN immunoreactivity and GFP/NeuN positive cells were found in a total of four brain sections per animal, we thus analysed a tissue block spanning 19 brain slices and 760 µm [= (4 x 40 µm) + (3 x 5 x 40 µm)]. By extrapolation of cell numbers (46.1–49.9 cells per slice) and efficacy of GFP transduction (25%), we estimate the presence of > 3500 [= 46.1 x 19 x 4] Adk–/– or wt graft-derived cells located within the CA1 region. Although unlabelled graft-derived cells were not further analysed in this in vivo study, neural differentiation of GFP positive and GFP negative cells in vitro was comparable (data not shown). Thus, the cellular fate of GFP-labelled cells in vivo is likely to be representative for the fate of the non-labelled cells. Further analysis revealed that intrahippocampal graft-derived cells did neither express GFAP (Fig. 5C1) nor nestin (Fig. 5C2). No tumours were detected within a post-operative follow-up of 26 days, although longer survival times will be required to address the safety of this approach.
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Therapeutic effects of Adk–/– NPs are superior to paracrine adenosine release from BHK cells
To investigate whether suppression of kindling development by Adk–/– NPs is due to paracrine adenosine release or due to additional stem cell-specific effects, we performed a control experiment transplanting either wt Adk+/+ BHK or engineered Adk–/– BHK-AK2 cells releasing 19.5 ± 1.3 ng adenosine per 105 cells per hour (Huber et al., 2001
) into rat hippocampi 1 week prior to the onset of kindling. In our previous experiments we were able to demonstrate that encapsulated BHK-AK2 but not wt BHK cells provided transient but almost complete seizure suppression in fully kindled rats (Huber et al., 2001). In these types of experiments, seizure suppression was entirely due to paracrine adenosine release (Güttinger et al., 2005a, b). Here the direct transplantation of BHK cells into the hippocampus of rats allowed us to evaluate the paracrine adenosine-mediated effects kindling development. Since BHK cells are not able to integrate functionally into the brain, they constitute a valid control to selectively study paracrine effects of adenosine. Two groups of rats received intrahippocampal implants of Adk+/+ BHK cells (n = 6) or Adk–/– BHK-AK2 cells (n = 5). Injection procedure, cell numbers and immunosuppression were identical to the NP cell transplantations (Fig. 1). One week after surgery hippocampal kindling was initiated and maintained exactly as described for the NP graft recipients for a total of six stimulation sessions (i.e. 36 stimulations), before the animals were sacrificed for histological analysis. Wt graft recipients developed kindling progression (Fig. 6), which was comparable with sham controls (P > 0.05). Thus, in contrast to NP cells, BHK cells do not have an endogenous potential to reduce kindling progression. However, adenosine releasing BHK-AK2 cells led to a significant reduction of kindling development (P < 0.001) compared with wt BHK cells, a protective effect that was maintained during the six kindling sessions (Fig. 6). During the first four kindling sessions, the therapeutic effect of BHK-AK2 cells was comparable with the antiepileptic efficacy of Adk–/– NP cells (P > 0.05). However, during kindling session 5 and 6, the therapeutic effect of BHK-AK2 cells was significantly reduced (P < 0.05) compared with the Adk–/– NP-mediated effect. Seizure activity in BHK-AK2 recipients in session 6 was comparable with seizure activity in Adk–/– graft recipients in session 8 (P > 0.05). We therefore suggest that Adk–/– NP cells initially suppress kindling development by endogenous stem cell-mediated effects and paracrine adenosine release, whereas during later stages of kindling development (i.e. beyond session 5) Adk–/– NPs are likely to display therapeutic effects, which (i) go beyond paracrine adenosine release (i.e. diminished protection by BHK-AK2 cells, Fig. 6) and (ii) go beyond endogenous stem cell mediated effects (i.e. diminished protection by Adk+/+ NPs, Fig. 1). Thus, the location of NP-derived NeuN positive cells within the CA1 region of the hippocampus appears to be critical in maintaining the therapeutic effect of ES cell-derived adenosine releasing brain implants.
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The experiment was terminated at a timepoint (i.e. after session 6, = 18 days after transplantation), when seizure activity in BHK and wt NP cell recipients was comparable. Histological analysis revealed clusters of viable BHK and BHK-AK2 cells within the infrahippocampal cleft (Fig. 6C and D), which were comparable in size and location with the NP cell implants (Fig. 4). However, in contrast to the NP cell implants, we did not find any BHK cells at other locations within the brain.
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Discussion |
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Since epilepsy has many aetiologies that all lead to an imbalance in neurotransmission and neuromodulation, there is no identifiable deficit, such as a lack of dopamine in Parkinson's disease, to be restored by cell therapy. However, in temporal lobe epilepsy (TLE), there is one common lesion: hippocampal sclerosis (Blumcke et al., 1999a
, b), which in a status epilepticus model has recently been demonstrated to lead to a deficit in adenosinergic neuromodulation (Gouder et al., 2004). Indeed, overexpression of ADK, as observed in astrogliotic tissue, can—by reducing levels of protective adenosine—contribute to epileptogenesis and be the cause for seizures (Gouder et al., 2004; Fedele et al., 2005). Therefore, the local and direct implantation of cell, which release adenosine into an area that is prone to adenosine dysfunction (i.e. the hippocampus) should retard or prevent epileptogenesis.
This hypothesis is supported by the potent antiseizure and neuroprotective effects of adenosine and its analogues (Lee et al., 1984; Dragunow and Faull, 1988; Fredholm, 1997; Boison, 2005; Fredholm et al., 2005b), which have exhibited efficacy in pharmacoresistant epilepsy (Gouder et al., 2003). In the present contribution, we provide a novel adenosine-based approach to retard kindling development and to suppress generalized seizures in seizure susceptible animals. This therapeutic effect was achieved by local cell-mediated adenosine release from direct intrahippocampal transplants of stem cell-derived NPs. We used a well-characterized protocol (Okabe et al., 1996) to generate NPs from either wt (Simpson et al., 1997) or ADK deficient (Fedele et al., 2004) ES cells. In our previous studies, we were able to demonstrate that local intracerebral implants of encapsulated adenosine releasing cells can suppress fully kindled seizures in the rat by a paracrine mode of action (Huber et al., 2001; Güttinger et al., 2005a, b). In these experiments we demonstrated that a unilateral brain implant of adenosine releasing cells suppresses epileptic activity in bilateral EEG recordings, suggesting that adenosine is able to prevent the spread of seizure activity (Huber et al., 2001; Boison et al., 2002). The ability of endogenous adenosine to prevent the spread of seizures and epilepsy-associated cell loss has recently been demonstrated in a status epilepticus model (Fedele et al., 2006). In our previous cell transplantation experiments, however, the therapeutic efficacy was temporal due to the limited long-term viability of encapsulated cells. Our present work goes far beyond these initial studies for several reasons:
- Potential antiepileptogenic activity: currently, epilepsy therapy is largely symptomatic and no effective therapy is available to prevent epileptogenesis. We therefore analysed stem cell-derived brain implants in an experimental paradigm, in which we could study the progression of seizure development. We chose the rat hippocampal kindling-model of epilepsy, in which the progression of seizure development is well characterized. By repetitive administration of subconvulsive stimuli permanent alterations of neuronal circuitry are achieved that render the brain more susceptible to seizures, which leads to a gradual progression in seizure severity. Thus, the rat kindling model is widely considered to be a model of epileptogenesis and therapies, which suppress kindling-development have predictive value for antiepileptogenic effects (Racine, 1978; McNamara et al., 1985; Lothman and Williamson, 1994). However, it has to be noted that the rat kindling-model does not reflect all histopathological changes observed in human temporal lobe epileptogenesis. Also, the time frame of seizure development is different, with seizures developing during the course of days to weeks in kindling, but years in human temporal lobe epileptogenesis. In summary, the rat kindling model is of great value for the preclinical evaluation of potentially disease modifying therapies, but, as with all animal models, caution has to be taken when extrapolating results to the clinical situation. Our results (Figs 1 and 2) clearly demonstrate that intrahippocampal implants of ES-derived NPs initially retard the progression of kindling development. This effect is even more pronounced and most importantly long-lasting, when the cells are engineered to release adenosine. We therefore suggest that adenosine releasing stem cell-derived brain implants may have a pronounced antiepileptogenic effect. Since epileptogenesis in patients, once seizures initially occur, is not a completed pathophysiological state, but rather a dynamic process with most severe consequences like pharmacoresistant seizures and cognitive deterioration still to develop (Blumcke et al., 2002; Engel, 2002; Elger et al., 2004), the time course of disease aggravation might provide a sufficient time window for therapeutic cell transplantation to prevent the progression to chronic epilepsy.
- Stem cell-mediated effects and local paracrine adenosine release: stem cell-mediated effects on epileptogenesis and therapeutic effects mediated by cellular release of adenosine have not yet been compared directly before. While it is beyond the scope of our study to elucidate the potential mechanisms of endogenous stem cell-mediated protective effects, a large body of evidence exists that endogenous stem cells and their neuronal and glial progenitor derivatives, as well as intracerebral implants of these cells, exert powerful protective effects on the brain (Tai and Svendsen, 2004; Kurozumi et al., 2005; Trendelenburg and Dirnagl, 2005). Most transplantation studies are aimed at replacing damaged neurons and glia. However, in many cases, functional improvements are considered a result of stem cell-induced self-repair and neuroprotection rather than cell replacement (Tai and Svendsen, 2004). Indeed, endogenous and exogenous stem cells are thought to provide neuroprotection by trophic support (Nomura et al., 2005), immunomodulation (Nomura et al., 2005), scavenging of reactive oxygen species, or by secretion of the neuroprotectant erythropoietin (Trendelenburg and Dirnagl, 2005). Thus, stem cells might be used as ‘chaperones’ for injured nervous tissues. Finally, these stem cell-mediated effects may be combined with the targeted delivery of therapeutic agents as described here in our attempt to establish an adenosine-based stem cell pharmacology. Neuronal brain implants may have different effects on epileptogenesis depending on their integration into pre-existing networks. Thus, neuronal brain implants can either suppress seizure development or be epileptogenic depending on their origin (Buzsaki et al., 1988). Here we describe potential antiepileptogenic effects mediated by intrahippocampal brain implants with the following order of therapeutic efficacy: Adk–/– NP > Adk–/– BHK-AK2 > Adk+/+ NP > Adk+/+ BHK > sham (Fig. 6B). In addition, we demonstrate the location of NP derived NeuN positive cells within the CA1 region of the hippocampal formation and associate prolonged antiepileptogenesis and seizure suppression with the continued presence of Adk–/– graft-derived cells. We have previously characterized the functional properties of ES cell-derived neurons engrafted into the normal adult or epileptic hippocampus (Ruschenschmidt et al., 2005) as well as into the embryonic brain (Wernig et al., 2004) and into organotypic hippocampal slice cultures (Benninger et al., 2003). Thus, although not tested directly here, the presence of graft-derived cells expressing a mature neuronal marker (Fig. 5) lends support to the possibility of direct interactions of the implanted cells when analysed histologically at day 26. Since in our study wt ES cell-derived NPs suppress kindling development initially—a beneficial effect, which is prolonged, when the cells release adenosine—we can conclude that endogenous NP-mediated effects and paracrine adenosine release from these cells combine to retard epileptogenesis and to suppress seizures. The importance of endogenous NP-mediated effects is further supported by our findings that adenosine releasing BHK-AK2 cells, which are not able to integrate functionally into brain and exert therapeutic effects exclusively by a paracrine mode of action (Huber et al., 2001) retard kindling development to a lesser degree than comparable Adk–/– NP cell transplants (Figs 1 and 6). In addition, our finding that suppression of kindling development by BHK-AK2 cells, which release approximately eight times more adenosine than Adk–/– NP cells, is less pronounced than the therapeutic effect of a comparable number of Adk–/– NP cells lends support to the notion that the location of Adk–/– NP-derived cells with neuronal appearance within the CA1 region (as early as 1 week after transplantation in mice; unpublished observations) contributes to a more specific suppression of seizure development, possibly by more localized adenosine-based effects requiring much lower concentrations of adenosine to be effective. In conclusion, stem-cell mediated intrahippocampal delivery of adenosine provides clear benefits compared with paracrine adenosine release from fibroblasts, which is an important finding. However, the mechanisms how stem-cell derived brain implants provide these beneficial effects, e.g. by trophic effects or by functional integration, and how these effects interact with paracrine adenosine delivery, have yet to be determined and will provide the basis for future studies.
- Prospect for long-term therapeutic efficacy: previous adenosine-based treatment approaches relied on the use of encapsulated cells. Such intraventricular brain implants—although very effective in seizure suppression—were characterized by limited life-expectancy of the encapsulated cells. In these studies, the therapeutic effect started to decline during the second week after grafting (Huber et al., 2001; Güttinger et al., 2005b). In the experiments described here, DPCPX control experiments initiated 24 days after cell implantation demonstrated complete functionality of the implanted cells. The experiment was terminated at a time point, when the implants were functional. As demonstrated histologically, we found clusters of graft-derived cells in the infrahippocampal cleft and individual cells, which were located within the ipsilateral CA1 region and which were mostly positive for the neuronal marker NeuN (54–58%), but negative for the astrocyte marker GFAP. Although not further investigated here, the NeuN negative graft-derived cells with neuronal morphology are most likely to constitute cells at an earlier stage of differentiation. The mature neuronal morphology and CA1 location of our implants suggests excellent graft acceptance and survival of the implants. Therefore, we speculate that the anticonvulsant effect of the grafts might be extended far beyond the 26-day time span described here. Thus, direct intrahippocampal implants of adenosine releasing ES cell-derived NPs might display an enhanced potential for long-term seizure suppression compared with hitherto used encapsulated cell grafts.
The work described here provides the first proof-of-principal-application of stem cells engineered to release adenosine as direct intrahippocampal implants. Intrahippocampal implants of ADK-deficient ES cell-derived NPs led to a retardation of epileptogenesis during kindling development and prevented the occurrence of generalized seizures. Thus, ADK deficient stem cells combine two important prerequisites for the development of future antiepileptogenic therapies.
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* These authors contributed equally to this work.
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The pMOWS-EGFP plasmid was a generous gift from Dr U. Klingmüller (DKFZ, Heidelberg, Germany). We thank Dr D. Fedele (University of Zurich, Switzerland) for performing the adenosine assays. This work was supported by grant R01 NS047622-01A2 from the National Institutes of Health, the Good Samaritan Hospital Foundation, the Epilepsy Research Foundation through the generous support of Arlene & Arnold Goldstein Family Foundation, the Deutsche Forschungsgemeinschaft (SFB TR3, D2) and the Hertie Foundation.
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