In situ chemical fixation of whale brains
As discussed by R. Michael Perry in his recent contribution to Cryonics Magazine, “Alternatives to Cryonics: A Very Preliminary Study,” (3rd Quarter 2007) chemical fixation of the brain may be a substitute for cryopreservation in circumstances where cryonics is not feasible or affordable. Several issues come into play when attempting to determine whether chemical fixation results in acceptable preservation of ultrastructure. An important question is whether chemical fixatives are uniformly distributed throughout the brain, preventing the occurrence of islands of tissue decomposition due to inadequate fixation.
In their 2002 paper, Knudsen et al. provide some preliminary answers to this question. Due to the difficulty in obtaining fresh brains for study from large aquatic mammals, the researchers developed a novel method for in situ fixation of (minke) whale brains. The procedure involved cutting a triangular opening in skull and pouring 8% formalin solution into the epidural and subdural space until it leaked out through the foramen magnum. The foramen magnum was then plugged, the triangular bone piece replaced to reduce loss of fixative, and the fixative level was checked and refilled every 4 hours, if necessary. Pilot studies indicated that a diffusion period of at least 60 hours is required for adequate fixation of large volume (average = 2201 g) minke whale brains, so the researchers set the in situ fixation period at 72 hours “to ensure that even the largest brains were sufficiently fixed prior to excision.” The brains were then excised and stored in formalin for at least 2 months prior to gross and microscopic examination.
Gross examination revealed that “there were no cases where the brain tissue was liquefied or smelled sour due to post mortem bacterial growth and the occurrence of artifacts and autolytic changes due to incomplete fixation was generally low.” Microscopic examination showed well-preserved cells and myelin in all parts of the brain. Specifically, histological evaluation categorized 97.3 to 100% of samples taken from different sites (brain stem and spinal cord, cerebellum, and cerebrum) as ‘good’ fixation (occurrence of mild autolytic changes) or ‘excellent’ fixation (without autolytic changes). “Swiss cheese” artifacts, caused by the invasion of gas-forming anaerobic bacteria, were observed in restricted (central) parts of the brain in 22 of 38 brains, especially in the thalamus, brain stem, and cerebellar vermis. White matter vacuolation was also observed in some of the brains, again in the thalamus and cerebellar vermis. However, in every case, the vacuoles were few (1 to 5) and small (1 to 3 mm).
The authors conclude that “the subsequent histological examination showed that these brains were, in many ways, better preserved than the routine autopsy brains of human and veterinary medicine. We regard the time span from death to start of fixation as the most decisive or crucial factor for this successful result.” They indicate that, although ≈75% of the fixations began within 2 hours post mortem, there were some instances where fixation started later (up to 6 hours), and that variations in fixation quality are likely due to the occurrence of autolytic changes. Importantly, it was noted that even careful handling of fresh brains always results in compression damage, and that fixation of the brain in situ was an excellent remedy for this problem.
In situ whale brain diffusion fixation appears to produce good preservation of the structure of the whole brain, especially in cases where fixation is begun soon after death. If such results can be achieved by passive diffusion of vastly larger brains than the human brain, investigation of the feasibility of reproducable uniform chemical fixation of complete humans brains as a method of biopreservation is warranted.