Cryofixation and Chemopreservation

The most common modern protocol for imaging brain structure at high magnification is to chemically fix the brain with aldehydes (formaldehyde, glutaraldehyde) and heavy metals like osmium and then prepare it for electron microscopy imaging. Using this method, a tremendous amount of detailed anatomical information about the structure of the brain in its healthy and pathological state has been obtained, including the effects of (prolonged) ischemia.

Almost from its inception, however, the limitations of this method have been recognized. In particular, when fixatives are introduced to the brain through the process of perfusion a number of distinct artifacts are produced, notably shrinking of the brain and a reduction of the extracellular space. While different solutions and protocols have been developed to reduce these artifacts, the gold standard for ultrastructural analysis is a method that does not use aldehydes at all; cryofixation.

In cryofixation small tissue samples are rapidly cooled (without freezing) and then prepared for electron microscopy. This method produces the most realistic images of the ultrastructure of the brain, as evidenced by papers that compared this method with aldehyde fixation or used advanced tools to understand the properties of the brain without doing electron microscopy.

Although the word “vitrification” is rarely used in the context of cryofixation, the pristine images in this method can only be achieved when ice formation is avoided through ultra-rapid cooling. Vitrification without the use of high concentrations of (toxic) cryoprotectants would be quite attractive if it could be scaled to the size of organs (or even humans!) but unfortunately this method can only be used on very small tissue samples.

The pristine images obtained from cryofixation raise some important issues. Does conventional aldehyde fixation produce only predictable distortions or is identity-specific information irreversibly lost? What are the ultrstructural effects of the heavy metal exposure when cryofixed samples are prepared for electron microscopy? In a more general sense, to what degree can we be confident that a technology can produce a completely realistic image of the ultrastructure of the brain?

Will computer simulations of scanned fixed brains need extensive correction if they are to serve as a simulation of the brain? One clear advantage of using viability assays in addition to electron microscopy is that we can test brain slices or whole brains for resumption of function (or retention of memory) after subjecting them to experimental protocols. This is a clear advantage of the use of cryopreservation technologies over chemical fixation. In a cryonics case we can monitor the patient from the start of our procedures to the point of long term care and collect data and viability information. In the case of chemopreservation no such feedback is possible and taking brain biopsies for electron microscopy is all we can do to assess the effects of our cryopreservation procedures.

It is tempting for a cryonics organization to choose the method of preservation that produces the most crisp electron micrographs. In reality, however, there are challenges and unknown issues. Cryofixation cannot be scaled to work for cryonics. What is the effect of conventional aldehyde perfusion in ischemic brains? How do aldehyde fixed brains look on the molecular level compared to cryopreserved brains? How can we know that identity-critical information is not irreversibly altered? And, last but not least, any preservation technology that renders tissue dead by conventional criteria cannot be considered as a means for achieving true human suspended animation.

Originally published as a column (Quod incepimus conficiemus) in Cryonics magazine, September, 2015