> Stepped Glycerol Concentrations Up To 75%

January 2001

We have another set of reports on our sheep brain samples from the independent Canadian service laboratory.

For readers in a hurry, the main feature of this report was that stepped concentration of glycerol up to a maximum of 75% was possibly a smidgen better than one pass at 75%.

Previous reports showed clearly that use of 75% glycerol perfusate, one pass, produced results much better than with untreated controls, and just as good as using stepped glycerol concentrations of 10%, 20%, and 30%. But we had not previously tried stepped concentration all the way to 75%, which was done this time. The steps were 10%, 20%, 40%, and 75%.

We have not yet tried continuous ramping rather than stepped ramping. However, a look at previous literature, starting with Farrant's pioneering work with stepped concentrations, does not suggest that any remarkable improvement is likely with continuously ramped rather than stepped concentrations.

One of the stepped samples in this series included 5% DMSO in the perfusate, and while overall results were about the same as without the DMSO, there were differences correlated with different brain sites, which may repay further study. It is worth remembering here that some reports indicate that addition of DMSO can reduce toxicity of some CPAs, so that there could be an advantage not discernible from photos. Photos do not show chemistry, only histology. (We plan later work to include chemistry and electrophysiology.)

This set included four sheep brain samples. The lab did not know which samples were which, or even the nature of the treatment of any of them, so there was no possibility of reports being biased.

#1 was with stepped glycerol up to 75%, as noted above. #2 was the same, but with 5% DMSO included. #3 was prepared in the same way as #2, but did not show naked-eye shrinkage, suggesting impaired circulation in that brain during washout or perfusion. (This reminds us of individual variability, especially when dealing with real-life or real-death situations, i.e. with patients who may have impaired vasculature from age, disease, or trauma.) #4 was another control, frozen in the same way but not washed out or perfused.

This set did not include a one-pass 75% sample, but the lab had results of that on file and made the comparison with the stepped sample (#1 of set 3). The results were very nearly the same. The scientist in charge reports that, "My impression of [stepped] and [one pass] is that the middle regions of the brain look the same. I think I could argue that [stepped] is subjectively slightly better on the inner-most and outer-most brain regions. Although the gaps in the intercellular regions are quite similar, there are fewer peri-nuclear gaps in the [stepped]."

These reports also tell us that stepping all the way up to 75% is a little better than stepping with a maximum of 30%.

Again, the control brain (not washed out or perfused but frozen in the same way as the others) showed much worse results in all respects. This doesn't mean that straight freezing leaves the situation hopeless-just that it puts a much heavier burden on future technology.

Since the micrograms do not print out well, and are similar to those previously published, we omit them here, although we plan to put some on our web site. However, a nice feature of the latest report is inclusion of graphs comparing gaps (ice holes and disjunctures) by percentage area of gaps and by mean gap area; and also by brain location--outer region, mid region, or inner region. This may tell us something about heat transfer or/and penetration of perfusate in different regions of the brain.

The bar chart above shows percentage area of gaps for four different brains. #1 (left) was treated by stepped concentration of glycerol-10%, 20%, 40%, 75%. #2 was the same, except it also contained 5% DMSO. #3 was handled the same as #2, except that the results were different because of individual variation-this brain did not show as much shrinkage to the naked eye, presumably because of impaired circulation. #4 was a control, not perfused but frozen in the same way.

In each case, the left or blue bar represents a sample from the outer layer, the middle or red bar the middle layer, and the right or yellow bar the outer layer.

The only obvious conclusion from this chart is that the perfused brains suffered much less disruption (gaps in the tissues including ice holes) than the unperfused controls. We already knew this, but a quantitative visualization is helpful.

It might be appropriate to insert a remark here about shrinkage, edema, and osmotic shock. Our sheep brains (and heads) always show shrinkage, because the glycerol circulating in the vasculature draws water out of the tissues. This is "bad" to a certain extent, and "osmotic shock" may result, and there may be some "denaturing" by concentration of solutes. But edema (swelling) is worse, and is often encountered with other methods. Less water left in the tissues also means less ice after freezing. We also recall that some organisms can revive after preservation by drying alone, even many years later, which tends to strengthen the case that--other things equal--less water is better.

-- Robert Ettinger

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