Fig 1.
Chemical structure of the four investigated compounds.
Fig 2.
Concentration-dependent inhibition of ATP-induced [Ca2+]i response.
The studies were carried out in HEK293 cells, stably transfected with the mouse P2X7R (a), rat P2X7R (b), and human P2X7R (c), respectively (fluo-4 microfluorometry, cell suspension, microplate reader). Concentration-response curves are shown for the four tested compounds tanshinone IIA-SO3Na (TIIAS, red), Brilliant Blue G (BBG, blue), AFC-5128 (AFC, green), JNJ-47965567 (JNJ, yellow) as well as for the two P2X7R antagonists A-438079 (A43, orange) and AZ-10606120 (AZ, pink) for comparison. The curves were fitted by using a 4-parameter logistic function (SigmaPlot). Data points are expressed as mean ± SEM (n = 5–7 independent experiments performed as duplicates). ATP-induced Ca2+ responses are calculated in percentage of the respective controls with solvent alone.
Table 1.
Potency (IC50 ± SEM in μM) of the four investigated compounds in comparison with A-438079 and AZ-10606120 (fluorometric Ca2+ assay).
Fig 3.
Maximal Electroshock Seizure threshold test (MES-T) in mice.
(a) Effects of tanshinone IIA SO3Na (TIIAS), Brilliant Blue G (BBG), AFC-5128 (AFC) and JNJ-47965567 (JNJ) on the threshold for tonic (hindlimb extension) electroshock seizures. The columns represent the CC50 values with confidence limits for 95% probability of compounds (20 animals per group), expressed in percent of the parallel estimated control thresholds (C = 100%, open columns). Controls received the corresponding vehicle i.p.; doses of compounds (mg/kg i.p.) are indicated below the columns. TIIAS and BBG were administered 45 min before and AFC and JNJ 30 min before threshold determination. Mean of control thresholds: 4.6 to 5.4 mA. There is not a statistically significant difference (Probit analysis). (b) Influence of AFC-5128 (AFC) and JNJ-47965567 (JNJ) on the anticonvulsant efficacy of carbamazepine (CBZ) in the MES-T (representation as in (a), CC50 values and confidence limits, expressed in percent of the parallel estimated control thresholds (C = 100%, open columns). AFC (25 or 50 mg/kg) and JNJ (15 or 30 mg/kg) were administered s.c. 30 min before threshold determination (additional injection of ethylcellulose [EC] i.p. 30 min instead of CBZ 7 mg/kg); the antiepileptic drug was given i.p. 30 min before (injection of vehicle s.c. 30 min instead of AFC or JNJ). Doses of compounds are indicated below the columns. Mean of control thresholds (injection of vehicle s.c. and ethylcellulose i.p., 30 min before each): 5.34 (AFC 25 mg/kg), 4.89 (AFC 50 mg/kg), 5.44 (JNJ 15 mg/kg), and 4.52 mA (JNJ 30 mg/kg), respectively. Significance level: **P < 0.01 (Probit analysis).
Table 2.
Pentylenetetrazol seizure threshold test (PTZ-T) in mice.
Table 3.
Influence of AFC-5128 as well as JNJ-47965567 upon the protective action of ethosuximide against PTZ-induced clonic seizures (PTZ-T) in mice.
Fig 4.
PTZ-induced kindling in rats. Influence of the four investigated compounds on the development of PTZ-kindling.
(a) First series: Tanshinone IIA SO3Na (TIIAS, 30 mg/kg) and Brilliant Blue G (BBG, 50 mg/kg) were administered i.p. 45 min before PTZ with vehicle (20% PEG 400). (b) Second series: AFC-5128 (AFC, 30 mg/kg) was given i.p. 45 min before PTZ with vehicle (DMA/β-CD). (c) Third series: JNJ-47965567 (JNJ, 15 mg/kg) was given s.c. 30 min before PTZ with vehicle (SBE-β-CD). The control groups (n = 12) received the corresponding vehicle alone. In addition, in the third series a further control group pre-treated with 0.9% NaCl-solution was included. PTZ (35 mg/kg i.p.) was injected once every 48 h (three times a week on Monday, Wednesday and Friday) for 20 successive sessions. After 20 PTZ-injections and an 8-day interruption of the kindling procedure, the rats were further kindled with only vehicle pre-treatment instead of the compounds (21st to 25th PTZ-injection). Values shown represent mean seizure stages. In order to keep the curves clear from the others, error bars (± SEM) were indicated only in Fig 4b. (SEM in all line graphs was not higher than ± 0.4.) Significance level: *P <0.05 (two-way repeated measures ANOVA, Holm-Sidak post hoc test).
Fig 5.
(a) Mean number of PTZ-injections needed to produce the first generalized seizure in the three series of PTZ-kindling (see Fig 4). Left part: impact of Tanshinone IIA SO3Na (TIIAS) and Brilliant Blue G (BBG); Middle: impact of AFC-5128 (AFC); Right part: impact of JNJ-47965567 (JNJ) in comparison with the corresponding vehicle controls (n = 12). Animals, which showed no generalized seizure (< stage 3) up to the 20th PTZ-injection, were calculated with “20”. Significance level: *P <0.05, ***P < 0.001 (Mann-Whitney rank sum test). (b) Effects of tanshinone IIA-SO3Na (TIIAS), Brilliant Blue G (BBG), AFC-5128 (AFC) and JNJ-47965567 (JNJ) on PTZ-induced seizures in fully PTZ-kindled rats in comparison with carbamazepine (CBZ) and Ca-valproate (VP). Each pair of columns represents the mean seizure stage (+ SEM) of a tested group, after vehicle treatment (vehicle seizure stage) and after compound treatment (compound seizure stage), indicated as white and coloured column, respectively). The number of animals within the group is shown on the top of the columns, the doses of compounds and the time of pre-medication before PTZ-injection are shown below the columns. Significance level: *P < 0.05, **P < 0.01, ***P < 0.001 (paired t-test).
Fig 6.
Representative photomicrographs of celestine blue/acid fuchsin (CB/AF) and Fluoro-Jade B (FJB)-stained coronal sections from the right dorsal anterior hippocampus of rats.
(a) CB/AF staining, control animal, 24h after the 25th vehicle injection (20% PEG 400, vehicle-only group), higher magnification view of CA3 pyramidal cell layer below. (b) CB/AF staining, PTZ-treated rat, 24h after the 25th PTZ injections (vehicle/PTZ group), higher magnification view below. Damaged cells (dark red-violet, black arrow) are rarely observed, however, in some cases cell-free gaps (blue arrow) can be seen indicating cell loss (see magnification of CA3). (c) FJB staining, control animal. (d) FJB staining, PTZ-treated rat. Bright green-fluorescent FJB-positive cells (white arrows) are rarely observed.
Fig 7.
Representative light microscopy photomicrographs showing Iba 1-immunopositive microglial cells from the right dorsal anterior hippocampus of the rat.
(a) Control animal, 24h after the 25th vehicle injection (20% PEG 400, vehicle-only group); higher magnification view of CA3 subfield (outlined area) on the right. (b) PTZ-kindled rat, 24h after the 25th PTZ injections (vehicle/PTZ group). (c) AFC-5128 pre-treated rat 24h after the 25th PTZ injections (compound/PTZ group). (d): Elongated microglia with heavy immunoprecipitation (same magnification, vehicle/PTZ group). Photomicrographs were made by using a light microscope (Axioskop) equipped with a CCD digital camera (AxioCam, ICc 1).
Fig 8.
Representative confocal images of triple immunofluorescence showing the localization of P2X7R in the dorsal hippocampus of the rat (CA3 subfield).
(a) Control animal, 24h after the 25th vehicle injection (20% PEG 400, vehicle-only group), (b) PTZ-treated rat, 24h after the 25th PTZ injections (vehicle/PTZ group). Immunofluorescence for P2X7R (Cy3, red), Iba 1 (Cy2, green), GFAP (Alexa Fluor 647-fluorescence, color coded in blue), and the corresponding overlays are presented. Strong P2X7R immunoreactivity was found only on Iba 1-positive microglial cells and their fine elaborated processes (arrows). Photomicrographs were made by using a confocal laser scanning microscope (LSM 510 Meta).