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.