Where They Disagree

A paper that cites the paper it questions

Here is something you do not often see. One of the four studies in this collection directly tests a claim made by another, cites it by name, and reaches a different conclusion.

The Wisconsin BAX study lists the Hainan brain bleed study in its references. It describes its own findings as inconsistent with that earlier report. Then it sets out to work out why.

That is science working the way it should, and it is worth looking at closely.

The claim

The Hainan team gave rats a brain bleed, treated them with SS-31 thirty minutes later, and measured proteins in the brain 24 hours after the bleed.

They split each tissue sample into mitochondria and the rest of the cell. After the bleed:

• BAX, a protein that triggers cell death, had moved onto the mitochondria.
• Cytochrome c, which normally stays inside mitochondria, had moved out.

With SS-31, both shifts were reduced. Less BAX on the mitochondria, less cytochrome c escaping.

Their reading: SS-31 suppresses BAX moving onto mitochondria, and so blocks the cell's self-destruct pathway.

The test

The Wisconsin team asked whether that reading holds up when you watch BAX directly.

They used human retinal support cells, tagged BAX, cytochrome c and the mitochondria with different coloured fluorescent markers, and filmed individual cells every two minutes as they went through self-destruction triggered by a chemical called staurosporine.

Here is what they found with SS-31:

• BAX still gathered on the mitochondria in every cell.
• BAX did not start gathering any later.
• BAX gathered slightly more slowly, but for slightly longer.
• The total amount of BAX that gathered was the same.
• Cytochrome c escaped in every cell where BAX gathered.
• The mitochondria broke apart just as much.

Their conclusion: SS-31's protection is not at the level of BAX once apoptosis has started.

Can both be right?

At first glance, these results contradict each other. One says SS-31 reduces BAX on mitochondria. The other says it does not.

But look at what each one actually measured.

The brain study took a snapshot. It measured BAX in a whole piece of tissue at one moment, 24 hours after the injury. That tells you how much BAX was on mitochondria across many cells at that time.

The Wisconsin study made a film. It followed individual cells from before BAX moved until after the process finished.

Here is the key point. A snapshot showing less BAX on mitochondria could mean SS-31 blocked BAX in each cell. Or it could mean fewer cells ever reached the stage where BAX moves at all.

The Wisconsin team themselves raised this possibility, wondering whether a delay might explain the difference. They tested for a delay in their cells and did not find one. But their cells were not facing the same kind of stress as a rat brain after a bleed.

A reading that fits both

Here is one way to reconcile them, and it fits the pattern from Part 2.

In a living brain after a bleed, the stress builds over hours. SS-31 might protect mitochondria early, reducing the oxidative damage and keeping cells further from the edge. Fewer cells then cross into the self-destruct process. A snapshot at 24 hours would show less BAX on mitochondria overall, not because BAX was blocked in each cell, but because fewer cells got that far.

In the Wisconsin experiment, staurosporine is a strong, direct trigger. It pushes cells straight into the death pathway. Once a cell is committed, SS-31 does not stop it.

Both results can be true in their own settings. What the Wisconsin study rules out is the simple version of the claim: that SS-31 stands in BAX's way once the trigger has been pulled.

Other differences that matter

It is also worth remembering how different the two set-ups were:

Any of these could contribute to the difference. That is the honest answer: we do not know exactly why the results differ, but there are plenty of reasonable explanations that do not require either lab to be wrong.

What the muscle study adds

The 2011 muscle abstract fits with the Wisconsin side of this. SS-31 raised the protective protein HSP70 but was not reported to switch off Bad, another protein from the same family as BAX.

So two of the three studies that looked at this family of death proteins did not find SS-31 acting on them directly. The one that did measured a snapshot in tissue, where an earlier protective effect could explain the result.

Why this disagreement is good news

It would be easy to see this disagreement as a problem. I think it is one of the most useful things in the whole collection.

It narrows down how SS-31 probably works. It replaces a dramatic claim, "SS-31 blocks cell death", with a more careful one, "SS-31 helps keep stressed cells away from the point of no return". And it shows independent labs testing each other's claims instead of just repeating them.

When you see SS-31 described online as something that "stops apoptosis", you now know that at least one carefully designed study tested that claim and found it does not hold once the process has begun.

Next, the dose puzzle: how the amounts used in these four studies compare, and why none of them translate into a human dose.

This series discusses four laboratory studies in animals and cultured cells. None tested SS-31 in people. Nothing here is medical advice, and SS-31 is discussed for research purposes only.