Setting criteria for monitoring autolysis using pig brain samples.

Session

LSA.2 - Dynamic interactions in cells, organoids, tissue and entire organisms

Authors

Dr. Deniz Tafrali (2), M.Sc Mariella Sele (3), Priv. Doz. Dr. Dagmar Kolb (1), Assoc. Prof. Dr. Gerd Leitinger (2)

Affiliations

1. Core Facility Ultrastructure Analysis, Center for Medical Research, Medical University of Graz
2. Gottfried Schatz Research Center, Medical University of Graz
3. Gottfried Schatz Research Center, Medical Univesity of Graz

Keywords

autolysis, frontal lobe, pig brain

Abstract text

Pigs (Sus scrofa domesticus) can be valuable for translational brain research. The advantage of electron microscopic studies with pig brains over human brain samples is that the pig brain samples can be obtained directly after slaughtering. In contrast, for ethical and practical reasons, there is a long post-mortem interval for human brain samples. During that interval, autolyses changes the appearance of the tissue and cells. 

Thus, publications on early post-mortem changes and autolysis in human brain tissue seem hardly possible, and in general studies of early autolysis in the mammalian frontal lobe are scarce. To set reliable criteria for post-mortem autolysis in the mammalian frontal lobe, we obtained samples of a frontal lobe of a pig during slauthtering. We prepared tissue samples from its frontal lobe in post-mortem intervals of 0, 3, 6 and 12 h. Tissue was stored at 4°C during that time and at every time point, we chemically fixed the tissue and processed it for producing TEM thin sections. 

Ultrastructural alterations of 51 frontal lobe neurons, astrocytes and oligodendrocytes were investigated. We found that neurons and glial cells responded differently to this lack of nutrients and oxygen: after 12 h the diameter of neuronal cells tended to decrease (neurons: 12,61 μm to 11,25 μm; pyramid cells: 23,15 μm to 14,24 μm) while glial cells gained in diameter (astrocytes: 8,9 μm to 12,4 μm; oligodendrocytes: 9,73 μm to 10,41 μm). Chromatin condensation occurred in all frontal lobe cells beginning at 3 h. Complete ribosome detachment from the rER was not found in nearly any investigated cell (1:51 overall). Vacuolization of the cytoplasm was first seen at 3 h. The occurrence of lysosomes was high throughout the PMIs in all cells (84.6 % at 0 h to 100 % at 12 h). 

This work confirms several autolytic ultrastructural changes in neural and glial cells described in other study animals (eg.references [1-4]). Chromatin condensation, neurons shrinking, glial cells swelling, intracytoplasmic vacuolization and the occurrence of lysosomes are valid indicators for autolysis. In the pig frontal lobe, the occurrence of rER without any ribosomes and structural disintegration of all mitochondria are comparatively unreliable markers for autolysis[5].

References

[1] J George, AJ Van Wettere, BB Michaels, D Crain, GA Lewbart, J PeerJ 4 (2016) e1943.

[2] H Hirsch, HA Müller, Pflüg. Arch. für Gesamte Physiol. Menschen Tiere 275 (1962) p. 277.

[3] SV Sheleg, JR LoBello, H Hixon, SW Coons, D Lowry, Int J Clin Exp Pathol. 1 (2008) p. 291.

[4] GR Stinnett, S Lin, AV Korotcov, L Korotcova, PD Morton, SD Ramachandra, A Pham, S Kumar, K Agematsu, D Zurakowski, PC Wang, RA Jonas, N Ishibashi, J. Am. Heart Assoc. 6.(2017) e005997.

[5] This study was funded by the Autrian Science Foundation FW, grant number P 29370.