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. 2009 Sep;219(1):126-35.
doi: 10.1016/j.expneurol.2009.05.018. Epub 2009 May 19.

Antiepileptic effects of silk-polymer based adenosine release in kindled rats

Cory Szybala  1 , Eleanor M Pritchard, Theresa A Lusardi, Tianfu Li, Andrew Wilz, David L Kaplan, Detlev Boison
Affiliations

Antiepileptic effects of silk-polymer based adenosine release in kindled rats

Cory Szybala et al. Exp Neurol. 2009 Sep.

Abstract

Pharmacotherapy for epilepsy is limited by high incidence of pharmacoresistance and failure to prevent development and progression of epilepsy. Using the rat hippocampal kindling model, we report on the therapeutic potential of novel silk-based polymers engineered to release the anticonvulsant adenosine. Polymers were designed to release 1000 ng adenosine per day during a time span of ten days. In the first experiment rats were kindled by hippocampal electrical stimulation until all animals reacted with stage 5 seizures. Adenosine-releasing or control polymers were then implanted into the infrahippocampal fissure ipsilateral to the site of stimulation. Subsequently, only recipients of adenosine-releasing implants were completely protected from generalized seizures over a period of ten days corresponding to the duration of sustained adenosine release. To monitor seizure development in the presence of adenosine, adenosine-releasing or control polymers were implanted prior to kindling. After 30 stimulations--delivered from days 4 to 8 after implantation--control animals had developed convulsive stage 5 seizures, whereas recipients of adenosine-releasing implants were still protected from convulsive seizures. Kindling was resumed after nine days to allow expiration of adenosine release. During additional 30 stimulations, recipients of adenosine-releasing implants gradually resumed kindling development at seizure stages corresponding to those when kindling was initially suspended, while control rats resumed kindling development at convulsive seizure stages. Blockade of adenosine A1 receptors did not exacerbate seizures in protected animals. We conclude that silk-based adenosine delivery exerts potent anti-ictogenic effects, but might also have at least partial anti-epileptogenic effects. Thus, silk-based adenosine augmentation holds promise for the treatment of epilepsy.

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Figures

Figure 1
Figure 1
Daily release of adenosine from silk-based polymers. (A) Adenosine release in vitro was determined for each day shown, based on averaged values from N = 3 polymers. Note the stable release rate of around 1000 ng adenosine per day from day 4 to 10, corresponding to the pre-designed target release rate. Errors are given as ± SD. (B) Assessment of antiepileptogenesis in the rat kindling model according to Silver et al., 1991. Kindling is initiated during drug delivery followed by a washout period of the drug; subsequently, kindling is resumed in the absence of the drug. Five potential kindling outcomes are shown: red, normal kindling development of an untreated or sham-treated animal; dark blue, kindling in the presence of a drug with no effects on seizure expression and epileptogenesis; light blue, kindling development under the influence of a drug that suppresses seizures, but not epileptogenesis; violet, kindling development under the influence of a drug that exerts partial antiepileptogenic effects; orange, kindling under the influence of a drug that completely suppresses epileptogenesis. Note that the criterion for complete suppression of epileptogenesis is a shift of the kindling curve to the right; the number of drug-free kindling stimulations needed to trigger a specific seizure stage should be the same as in control animals.
Figure 2
Figure 2
Suppression of fully kindled seizures by implant-derived adenosine. Fully kindled rats (criterion: at least three consecutive stage 5 seizures) received infrahippocampal implants of silk-based polymers with a daily target release rate for adenosine of 0 ng (N = 4, red), or 1000 ng (N = 5, blue). Individual test stimulations were delivered at days 4, 6, 10, 14, 18, and 21. Seizure stages, averaged across animals from each group, are shown for every stimulus. Note that recipients of a target dose of 1000 ng adenosine per day are completely protected from any seizures during the 10 days after implantation corresponding to sustained release of adenosine during that time period. Errors are given as ± SD. Data were analyzed by two way ANOVA followed by a Bonferroni test; the significance of interaction between groups was determined as F=2.390; P<0.05; significance levels of individual tests is indicated: * P<0.05, ** P<0.01.
Figure 3
Figure 3
Influence on epileptogenesis by adenosine releasing polymers. (A) Experiment 2A: Four days after infrahippocampal implantation of silk-based polymers with daily target release rates for adenosine of 0 ng (N = 5, red), or 1000 ng (N = 8, blue) kindling stimulations were delivered at a rate of 6 stimulations per day on days 4, 6, 8, and 11 following implantation. A total of 24 kindling stimulations were delivered. On day 12 DPCPX (1 mg/kg, i.p.) was injected 30 min prior to stimulation. Each animal was tested again on day 13 (no DPCPX). Seizure stages were averaged across animals from each group for each individual stimulus. Note that recipients of a target dose of 1000 ng adenosine per display significant protection from kindling development, while DPCPX does not increase the seizure score. Errors are given as ± SD. Data were analyzed by two way ANOVA followed by a Bonferroni test; the significance of interaction between groups was determined as F=6.704, P<0.0001; significance levels of individual tests are indicated: * P<0.05, ** P<0.01, *** P<0.001. (B) Experiment 2B: Four days after infrahippocampal implantation of silk-based polymers with daily target release rates for adenosine of 0 ng (N = 7, red), or 1000 ng (N = 5, blue) kindling stimulations were delivered at a rate of 6 stimulations per day on days 4, 5, 6, 7, and 8 following implantation. A total of 30 kindling stimulations were delivered. Please note the increased kindling frequency compared to (A). After the 30th kindling stimulation, kindling was discontinued for 9 days. Kindling stimulations were resumed at day 18. Seizure stages were averaged across animals from each group for each individual stimulus. Note that recipients of a target dose of 1000 ng adenosine per day resumed kindling at day 18 at a level at which kindling was discontinued at day 8. After 7 consecutive stage 5 seizures kindling was discontinued in control animals due to animal welfare considerations. Errors are given as ± SD. Data were analyzed by two way ANOVA followed by a Bonferroni test; the significance of interaction between groups was determined as F=19.36, P<0.0001; significance levels of individual tests are indicated: ** P<0.01, *** P<0.001.
Figure 4
Figure 4
Afterdischarges during kindling acquisition. Representative EEG recordings are shown from control polymer recipients (0 ng) and adenosine implant recipients (1000 ng) on the first and fifth day of kindling (corresponding to day 4 and day 8 after polymer implantation). The scale bar represents the 10 second stimulation interval. Note the presence of electrographic afterdischarges (2 – 3 Hz frequency) in the adenosine implant recipients. The afterdischarge duration (ADD) on day 1 and 5 of kindling was determined after each kindling-stimulation by analyzing the respective EEG recordings. ADDs were averaged for each day (n = 6 stimulations) and treatment type: implants releasing target doses of 0 ng adenosine (N=7) or 1000 ng (N=5) adenosine per day. Errors are given as ± SD. Data were analyzed by ANOVA; ADDs were not statistically different, P > 0.05.
Figure 5
Figure 5
Characterization of implants before and after implantation. (A, B) Example of Image J degradation analysis (pre-implantation sample #1, Table 1): The total surface area (yellow line) in pixels is measured (A), then the sum of the surface areas of all the pores (blue lines) is measured (B). The percentage porosity is calculated by taking the ratio of pore surface area over total implant area. Scale bars in images A and B = 300 μm. (C) Cresyl violet stain of a representative sagittal rat brain section 4 weeks post implantation. The polymer implant is dark blue and indicated with arrow. Scale bar = 3 mm. (D) Hematoxylin & eosin stain showing the morphology of representative infrahippocampal aqueous-derived adenosine-loaded silk fibroin implant after 4 weeks. Scale bar = 300 μm. Solid arrow = remaining scaffold.
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