Biochemical and molecular studies using human autopsy brain tissue

Abstract

The use of human brain tissue obtained at autopsy for neurochemical, pharmacological and physiological analyses is reviewed. RNA and protein samples have been found suitable for expression profiling by techniques that include RT-PCR, cDNA microarrays, western blotting, immunohistochemistry and proteomics. The rapid development of molecular biological techniques has increased the impetus for this work to be applied to studies of brain disease. It has been shown that most nucleic acids and proteins are reasonably stable postmortem. However, their abundance and integrity can exhibit marked intra-and intercase variability, making comparisons between case-groups difficult. Variability can reveal important functional and biochemical information. The correct interpretation of neurochemical data must take into account such factors as age, gender, ethnicity, medicative history, immediate ante-mortem status, agonal state and post-mortem and post-autopsy intervals. Here we consider issues associated with the sampling of DNA, RNA and proteins using human autopsy brain tissue in relation to various ante-and postmortem factors. We conclude that valid and practical measures of a variety of parameters may be made in human brain tissue, provided that specific factors are controlled.

Human postmortem tissue: What quality markers matter?

“…Traditionally, a low postmortem interval (Barton et al, 1993;Ferrer-Alcon et al, 2003;Harrison et al, 1995;Lewis, 2002;Trotter et al, 2002) has been the hallmark of high tissue quality. More recently tissue pH (Hynd et al, 2003;Kingsbury et al, 1995;Li et al, 2004) has been used, and even more recently, specific markers of RNA quality have been introduced (Imbeaud et al, 2005;Johnston et al, 1997;Miller et al, 2004). In a cohort of human postmortem cases, we have determined these standard tissue quality measures, checked their collection correlates, and tested them for an association with RNA quality and with quantification of representative proteins.…”

Expression profile of Bag 1 in the postmortem brain

“…Similarly, postmortem variables such as tissue pH, storage temperature and postmortem intervals can also modify overall neurochemical integrity. In this regard, experimental brains are normally cooled to 4 °C to minimize degradation of certain species of mRNA in individual tissue samples (Bahn et al, 2001;Hynd et al, 2003). Here we have found an apparent effect of a postmortem variable (e.g., storage temperature) on Bag 1 expression.…”

“…The correct interpretation of neurochemical data must take into account postmortem intervals and storage temperatures (Torres et al, 1992;Bahn et al, 2001;Hynd et al, 2003;Torres et al, 2004). The approach outlined here provides a means with which to examine these two factors in the context of profile expression and cellular stability of an anti-apoptotic protein.…”

“…It is generally thought that both agonal (i.e., premortem) and postmortem states can yield a strong influence on proteins subsequently measured in autopsy tissue (Barton et al, 1993;Hynd et al, 2003). For instance, rapidity of death, nature of the agonal state and disease phenotype are particularly relevant premortem factors affecting the stability of specific proteins.…”

“…Under this lethal drug regimen, death ensues within 3-5 min (Torres et al, 1992). As death occurs over a time-course of less than 24 hr, there is minimal evidence of cerebral hypoxia as described by Hynd et al (2003). After the 4 hr interval, brains were removed from calvaria and immediately fixed in a 4% paraformaldehyde solution (dissolved in 0.1 M sodium phosphate buffer, pH 7.2) for 5 consecutive days at 4 °C.…”