Two ways a cell can die

Cells die in two broad ways. One is messy: the cell bursts, spills its contents and sets off inflammation around it. The other is orderly: the cell takes itself apart in a controlled sequence, packs up the pieces and lets neighbouring cells clear them away.

The orderly kind is called apoptosis. It is useful in a healthy body. It is how you get rid of worn-out or damaged cells without fuss. The trouble after a brain injury is that apoptosis gets switched on in neurons that might have survived, and once a neuron dismantles itself it is gone.

The Hainan team wanted to know whether SS-31 reduced this kind of cell death after the bleed, and if so, how.

Counting the dead

They used a stain called TUNEL. When a cell goes through apoptosis, it chops up its DNA into pieces. TUNEL attaches a marker to the cut ends, so dying cells light up under the microscope.

They paired it with a second marker, NeuN, which only lights up in neurons. A cell glowing with both markers is a neuron that is dying.

Two investigators who did not know which group each sample came from counted these cells in six random fields under high magnification, in the temporal cortex at 24 hours after the bleed.

The results:

  • Sham rats had only a few dying cells.
  • Untreated and vehicle-treated bleed rats had many.
  • SS-31 rats had significantly fewer.

Following the death signal upstream

Counting dead cells tells you that something worked. It does not tell you how. So the team went further, and looked at the chain of signals that tells a cell to die.

Here is the chain in plain terms, as the paper describes it.

Step one: Bax moves. Bax is a protein that normally drifts around the main body of the cell. When a cell is under severe stress, Bax moves onto the outer surface of the mitochondria.

Step two: the mitochondria leak. Once Bax gathers there, the outer membrane of the mitochondria becomes permeable. A protein called cytochrome c, which normally sits inside the mitochondria helping make energy, escapes into the rest of the cell.

Step three: the executioners switch on. Cytochrome c outside the mitochondria is an alarm. It triggers a cascade of enzymes called caspases. The one at the end of the line, caspase-3, does the actual dismantling.

So if SS-31 is stopping apoptosis at the mitochondria, you would expect to see less Bax on the mitochondria, less cytochrome c escaping, and less active caspase-3.

What they found

The team separated each brain sample into two parts, the mitochondria and everything else, and measured the proteins in each.

After the bleed:

  • Bax went up in the mitochondria and down in the rest of the cell. It had moved.
  • Cytochrome c went down in the mitochondria and up in the rest of the cell. It had escaped.

In SS-31 treated rats, both of those shifts were reversed. Less Bax moved to the mitochondria, and less cytochrome c leaked out.

Then caspase-3. Using two separate methods, a tissue stain and a protein measurement for the active, cut form of the enzyme, the team found more caspase-3 after the bleed and less in SS-31 treated rats.

Why this chain is convincing

What I like about this part of the study is that each link supports the next. Fewer dying neurons. Less of the protein that starts the process. Less of the alarm signal. Less of the enzyme that finishes the job.

That is the kind of layered evidence that makes a mechanism believable. It points to SS-31 stepping in at the mitochondria, before the cell commits to dying.

The authors conclude that SS-31 protects neurons in this model by inhibiting the mitochondrial pathway of apoptosis. That fits everything else they measured.

A note for later in the series

The Bax finding is one I want you to remember, because it gets interesting when we look at other labs.

In this paper, Bax moving onto the mitochondria was reduced by SS-31, measured in brain tissue taken at a single time point. A later study by a different group, which we cover in another series, watched Bax move in living cells in real time and reached a rather different conclusion about what SS-31 does to it. Both can be true in their own settings. Understanding why is one of the more useful lessons in reading this field.

For now, in this model, the chain is consistent: SS-31 kept the death signal from reaching the point of no return.

Next, we look at the other half of the story, oxidative stress, and what it means to measure "rust" inside a cell.

This series discusses a peer-reviewed laboratory study in rats. The results have not been shown in people. Nothing here is medical advice, and SS-31 is discussed for research purposes only.