Taken together, while P2X7R antagonism seems to have no effect on acute PTX-induced epileptiform activity, treatment with P2X7R antagonists reduced seizure-like events under chronic inflammatory conditions.
3.3. Synergist effect of P2X7R antagonism in carbamazepine-resistant iPSC-derived neuronal network model of epileptiform-like events
Recent studies showed that blocking the P2X7R attenuates pharmacoresistant status epilepticus in a mouse model (Beamer et al., 2022). We, therefore, hypothesized that P2X7R antagonism may also enhance the effects of conventional ASMs in our in vitro hiPSC-derived neural network model treated with neuroinflammatory agents.
As before, hiPSC-derived neural networks were treated with PTX and the same cocktail of neuroinflammatory agents as before (i.e., TNF-ɑ, IL-1ɑ, IL-1β and C1q) for 7-12 days. Loose patch-clamp experiments were performed in baseline conditions and in the presence of the common ASM carbamazepine (50 µM) (Figure 5a, b). Neurons were categorised as unresponsive to carbamazepine if carbamazepine application failed to reduce firing frequency by more than 30%, leading to 68.8% carbamazepine-unresponsive neurons when co-treated with PTX and neuroinflammatory agents (Figure 5c). Next, we wanted to explore if the P2X7R antagonist AFC-5128 attenuates carbamazepine-resistant epileptiform-like burst firing. Representative traces depicting burst firing indicated attenuation of epileptiform-like activity when the neural network was co-treated with carbamazepine and AFC-5128 (Figure 5d). While the burst frequency did not change significantly in the presence of carbamazepine, this was reduced to a significant level via the co-application of carbamazepine and AFC-5128 (PTX vs. PTX and CBZ (carbamazepine): 0.1022 ± 0.0156 vs. 0.1187 ± 0.0264 Hz; p = 0.8666; PTX and CBZ vs. PTX, CBZ and AFC-5128: 0.1187 ± 0.02645 Hz vs. 0.04713 ± 0.01130 Hz; p = 0.0442) (Figure 5e). Interestingly, co-application of carbamazepine and AFC-5128 also led to a reduction in the number of spikes in burst (PTX vs. PTX and CBZ: 27.94 ± 3.583 vs. 15.73 ± 3.678; p = 0.0173; PTX and CBZ vs. PTX and CBZ and AFC-5128: 15.73 ± 3.678 vs. 12.71 ± 3.239; p = 0.0017) (Figure 5f). Similarly, a reduction in the burst duration was observed after carbamazepine application that was further reduced in the presence of AFC-5128, which was statistically significant compared to the burst duration in the presence of carbamazepine alone ([F(2,11) = 8.6; p = 0.0039]; PTX vs. PTX and CBZ: 1857 ± 227.1 vs. 1321 ± 241.5 ms; p = 0.1390; PTX and CBZ vs. PTX, CBZ and AFC-5128: 1321 ± 241.5 vs. 888.5 ± 125.6 ms; p = 0.0585; PTX vs. PTX, CBZ and AFC-5128: 1857 ± 227.1 vs. 888.5 ± 125.6 ms;p = 0.0098) (Figure 5g). Moreover, while the IBI was only slightly increased in the presence of carbamazepine, the mean IBI was further increased when carbamazepine and AFC-5128 were co-applied, although not statistically significant ([F(2,11) = 1.199, p = 0.2970]; PTX vs. PTX and CBZ: 11.06 ± 2.493 vs. 15.19 ± 5.057 s;p = 0.5748; PTX and CBZ vs. PTX, CBZ and AFC: 15.19 ± 5.057 vs. 106.4 ± 84.75 s; p = 0.5526) (Figure 5h).
To further quantify these observations, histograms were made using log-transformed IBI distributions. The data shows a right-shift in the IBI distribution indicating longer IBI after the co-application of carbamazepine and AFC-5128 (Figure 5i-k). Kernel density estimation (KDE) also reveals a shift towards a longer IBI distribution after co-application of carbamazepine and AFC-5128 (Figure 5l). The shape parameters of the distribution of the IBIs were measured by fitting a gamma distribution to the histogram. The distribution of the IBIs and corresponding gamma-distribution fits are shown in Figure 5m-o. The shape and scale parameter as a measurement of the IBI distribution for PTX or PTX and CBZ or for co-application of PTX, CBZ and AFC-5128 is shown in Table 2. The shape parameter as a measure of the spread of the IBI distribution increased form 10.418 in the presence of PTX and CBZ to 17.847 during the co-application of PTX, carbamazepine and AFC-5128.