Rapatz, et al. also found cracking to occur in frog hearts cooled to below Tg after impregnation with multimolar concentrations of ethylene glycol. For this reason valves are stored in liquid nitrogen vapor. I believe there have been published reports about this problem and I will do a literature search to see what I come up with. Visible fracturing does not occur in my experience in brains poorly loaded with cryoprotectant. We then tried cutting them up with various types of saws.
Fractures do occur in straight frozen animals, but are smaller.
Anyone who has looked at an ice cube in a tray will understand about fracturing in ice, even at relatively high subzero temperatures. So, one reason fracturing may not have occured is that the glycerol concentration in the tissue may not have been very high. Again, without data on arterial vs. venous glycerol concentration as a minimum, it is hard to evaluate the results. On the other hand, CI observed shrinkage of the brains in response to glycerolization and that is a reasonably good indicator of gross adequacy of distribution of the agent. Isolated brains freed from contact with a container during cooling CAN be cooled down to -196C without fracturing or with minimal fracturing.