Week 4

Our astrocyte experimental results are in!

Quick recap on last week: Our astrocytes have matured and are ready for experimental work. We tested the impact of BX559 – a potent activator of glial cells that induces a distinct ALS phenotype – on astrocyte mediated cytokine release. We wanted to determine the optimum dose and time for BX559 to mediate an inflammatory response in our astrocytes. We added BX559 at 9 different doses and tested the media at 24hr, 48hr and 72hr post-addition for cytokine release. We were looking for the expression of 4 cytokines – TNFα, IL-1β, IL-8 and IL-6.

This week we have the results, but they are not as straightforward as you may think…

24 hours:

We found a dose-dependent increase of all 4 cytokines up to a certain point at which we observed a plateau. For IL-1β and IL-6, the plateau occurs at around 0.8ng/mL of BX559, IL-8 at around 1.6ng/ml, and there is no obvious plateau for TNF. See Figure 1 below.

Figure 1. Graphs illustrating cytokine release in astrocytes 24 hours after introduction of BX559 at different concentrations.

48 hours:

The results are less clear. We do see a dose-dependent increase in IL-1β and TNF until they hit an apparent plateau at around 0.8ng/ml. However, for IL-6 and IL-8, we cannot say their expression levels are increasing in conjunction with increased BX559 dosage. Please see Figure 2.

Figure 2. Graphs illustrating cytokine release in astrocytes 48 hours after introduction of BX559 at different concentrations.

72 hours:

The 72-hour results are inconclusive. We cannot deduce that there is a dose dependent increase in the release of any cytokines. We also see extremely high IL-6 and IL-8 expression levels at dose 0.0ng/ml, which is difficult to explain. We hypothesise that the cells might have suffered from confluence related stress, without any media changing across the 72hr time period. Please see Figure 3 below.

Figure 3. Graphs illustrating cytokine release in astrocytes 72 hours after introduction of BX559 at different concentrations.

From the data we can see that the longer the cells are incubated with BX559, the less conclusive the results are. We suspect that we should have tested earlier timepoints on cytokine expression – 8 and 16 hours. Therefore, we believe that we haven’t yet found the optimal dose and time for BX559 to induce an inflammatory response. Our findings infer that the growth of the cells negatively impacts their cytokine release.

These results weren’t necessarily what we were hoping for, but they offer highly valuable insight into what a cell/molecular biological study looks like in practice – they can throw curveballs, and you must respond promptly!

In the coming weeks, we will be testing BX559 induced cytokine expression in astrocytes at earlier time points, to try and definitively determine the optimal dose and time for BX559 to mediate an inflammatory response. Make sure you stay tuned to find out how we will correct our experimental results.

#12wdc #ALS #MND #drugdiscovery #Ulysses