Four approaches for long-term fixation-based brain banking
When I was a freshman at Vassar College, I was taking Biology 105: Ecology of Birds, when our professor, Jason Jones, told us something that has stuck with me. He told us that biology, and life more generally, is all about trade-offs.
Close to two decades later (I’m getting older!), I love thinking about the world in terms of trade-offs. So naturally, I think about brain banking from this perspective too.
Just as in the initial fixation procedure in brain banking, there are clear trade-offs in one’s choice of long-term storage method.
Cryopreservation with cryoprotectant agents to prevent ice formation is sometimes used to maintain morphology in fixed brain sections until they are processed for further studies. For example, here is the method described by Insausti 1995 for storage:
The brain slabs were then equilibrated in solutions of 10% and 20% glycerol in 0.1 M phosphate buffer and 2% dimethylsulfoxide, and kept frozen at -80°C until processing.
It’s well-established that this cryopreservation-based preservation method prevents ice damage in brain tissue. It’s also well-established that very low-temperature preservation allows the preservation of tissue for many decades.
But freezers are expensive. The financial costs add up and have to be paid by someone. There are also other costs of low-temperature storage, such as environmental ones.
So most brain banks end up using cheaper storage solutions. Many simply store the fixed brain tissue in the liquid state at room temperature.
Building off of my previous post describing trade-offs in the initial fixation procedure, we can imagine a 2x2 matrix of different procedures, based on the initial preservation method and the long-term storage method:
Quadrant #1 is expected to have higher preservation quality but will be limited in access to the labs that can perform this procedure and substantially more expensive over time. On the other hand, Quadrant #4 will be scalable and cheap.
While I described this in terms of categorical quadrants, in reality, the axes represent spectrums. For example, the maintenance of neural structures can be improved when using liquid preservation, for example by refrigeration and/or by strategies to maintain the solution at a neutral pH.
As an aside, these four approaches obviously aren’t the only ways to do brain banking. For example, one could eschew fixatives altogether and opt for pure cryopreservation approaches. Alternatively, the fixed tissue could be embedded, for example in paraffin, instead of being stored in the liquid state.
How good is the preservation in Quadrant #4? I think that’s an important open question. Putting aside the question of perfusion vs immersion, as discussed in my previous post, it’s not clear to me that connectivity cannot still be traced even after decades of liquid-state storage, if the right imaging methods are used.
One of the best examples of this can be found in the work of Andrew Dwork’s lab at New York State Psychiatric Institute and Columbia University. For example, Rosoklija 2013 studied brain tissue with the following characteristics:
We used human and monkey necropsy tissues, from cerebral cortex and hippocampus. Human tissue was fixed in 10% formalin for different periods of time, from 15 months up to 55 years. The interval between death and autopsy varied from 6 to 30 h. Although the human brain specimens had been placed in buffered formalin, over time, the solution lost its buffering capacity and acidified to pH 4–5 during fixation…
And here are their results, using the Golgi–Kopsch method of staining:
With tissue fixed by immersion in formalin for 15 months or longer, the Golgi–Kopsch method was superior to rapid Golgi. Tissue fixed for 5–20 years in formalin yielded some well-impregnated neurons with the rapid Golgi method, but considerably more with the Golgi–Kopsch method (Fig. 2). However, for tissue with 50 years of fixation, rapid Golgi staining was clearly inadequate, while the Golgi–Kopsch method gave excellent results, comparable to tissue fixed for a few years (Fig. 3). When fixation ranged from 15 months to 20 years, virtually all cases yielded large numbers of apparently well-impregnated neurons. Whether extended fixation produces quantitative changes in extent of impregnation remains to be determined, but qualitative changes are not noted.
You can see in the image that neuronal morphology and dendritic spines look as expected.
Now, the Golgi method stains only 1-5% of neurons and one could argue that the neurons that are not stained might be degraded for some reason. But it’s not clear to me why some cells would degrade over time and others would remain so well preserved while they are all sitting in the same formalin solution. After all, they’re made of the same things — gels of crosslinked proteins and other biomolecules.
Based on this data, I think it is probably fair to say that neuron morphology seems to be retained even after 50 years of storage in formalin at room temperature. And if it’s maintained at 50 years, why not 100 or more?
At the beginning of Carl Sagan’s book “Broca’s Brain”, written in 1979, he muses on what kind of information might be present in brains stored for a long period of time in formalin:
And here was Broca's brain floating, in formalin and in fragments, before me. I could make out the limbic region which Broca had studied in others. I could see the convolutions on the neocortex. I could even make out the grey-white left frontal lobe in which Broca's own Broca's area resided, decaying and unnoticed, in a musty corner of a collection that Broca had himself begun.
It was difficult to hold Broca's brain without wondering whether in some sense Broca was still in there - his wit, his sceptical mien, his abrupt gesticulations when he talked, his quiet and sentimental moments. Might there be preserved in the configuration of neurons before me a recollection of the triumphant moment when he argued before the combined medical faculties (and his father, overflowing with pride) on the origins of aphasia? A dinner with his friend Victor Hugo? A stroll on a moonlit autumn evening, his wife holding a pretty parasol, along the Quai Voltaire and the Pont Royal? Where do we go when we die? Is Paul Broca still there in his formalin-filled bottle? Perhaps the memory traces have decayed, although there is good evidence from modern brain investigations that a given memory is redundantly stored in many different places in the brain. Might it be possible at some future time, when neurophysiology has advanced substantially, to reconstruct the memories or insights of someone long dead? And would that be a good thing? It would be the ultimate breach of privacy. But it would also be a kind of practical immortality, because, especially for a man like Broca, our minds are clearly a major aspect of who we are.
I think these are still open questions. If anything, data from the past 40 years have made Sagan’s speculations more plausible.
As a historical aside, it’s not entirely clear to me that Paul Broca's brain is actually preserved. Upon a visit to Paris, Leonard LaPointe was not able to find it. One speculation I have is that the brain was labeled as such because it was one of the brains in the collection that Broca helped to preserve and study. Also, formaldehyde wasn’t yet used in pathology when Broca died (1880), so at a minimum, there would have been a period of time when his brain was preserved in some other liquid preservative, likely an alcohol-based one, which seem to have substantially worse conservation quality than aldehydes.
Reference
LaPointe, Leonard L. "Broca's brain: brother, wherefore art thou?" Journal of Medical Speech - Language Pathology, vol. 15, no. 1, Mar. 2007, pp. vii+. Gale Academic OneFile, link.gale.com/apps/doc/A161132215/AONE?u=nysl_oweb&sid=googleScholar&xid=424e7599.