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Factors Affecting Ultrastructural Quality in the Prefrontal Cortex of the Postmortem Human Brain.

Author(s): Glausier JR, Konanur A, Lewis DA

Publication: J Histochem Cytochem, 2019, Vol. 67, Page 185-202

PubMed ID: 30562121 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effects of PMI; specimen pH; storage time; and patient age, sex, psychiatric diagnosis, and cause of death on neuronal profiles in brain tissue. The effects of PMI, pH, and cause of death on secondary structures was also examined. Dorsolateral prefrontal cortex tissue was obtained from 30 subjects at autopsy (23 males, 7 females, aged 22-64 y, median age 48.8 ± 10.7 years, PMI range 5.9–24.3 h) and classified into three groups based on the cause of death (trauma, cardiac-related, or substance use). Brain pH was measured with an electrode in a tissue specimen that was snap-frozen within 1 hr of excision and homogenized in distilled water. An approximately 1 cm-thick coronal block was immersed in a solution of 4% paraformaldehyde and 0.2% glutaraldehyde for 24 h at room temperature, followed by 24 h at 4°C, rinsed with phosphate buffer saline (PBS), and sectioned at 50 μm thickness using a vibratome. Tissue sections were stored in a 30% ethylene glycol and 30% glycerol cryoprotectant solution at -30°C until histology processing (3–773 days, average 246.0 ± 296.1 days). Sections were rinsed in PBS, postfixed in 1% osmium tetroxide for 60 min, and then dehydrated in ascending ethanol concentrations. Tissue sections were then embedded in resin for 3 h, mounted on glass slides, and incubated for 2 days at 60°C. Five electron micrographs at the same approximate tissue depth were digitally imaged and captured at 25,000X on a transmission electron microscope. Investigators determined the total number of identifiable neuronal profiles per 160 μm2 analyzed per subject and secondary measures of ultrastructural preservation included counts of postsynaptic densities and mitochondria, as well as postsynaptic density length.

   Summary of Findings:

   Brain pH showed a modest positive correlation with the number of neuronal profiles observed (r= 0.5, P=0.003) and while female subjects had an average 38% fewer identifiable neuronal profiles than male subjects (P=0.04), this appeared to be due to differences in pH as mean pH was significantly lower (0.4 pH units) in females than males (P=0.003). Cause of death had a significant effect on the number of identifiable neuronal profiles (P=0.01) with patients who died of trauma averaging 38% more neuronal profiles than those with cardiac-related deaths (P=0.03) and 55% more than in patients whose death was attributed to substance use (P=0.02). PMI and specimen storage duration (≤200 days or ≥500 days) were weakly negatively correlation with number of neuronal profiles observed but differences did not reach statistical significance (r= −0.3, P=0.09). Neither age at time of death nor a history of a psychiatric diagnosis were correlated with number of neuronal profiles. PMI was significantly negatively correlated with the number of postsynaptic densities (r= −0.4, P=0.03) but was not correlated with the postsynaptic density length or number of mitochondria. In contrast, brain pH (r=0.004, P=1.0) was not correlated with the number of postsynaptic densities or number of mitochondria and showed a negative but not statistically significant correlation with postsynaptic densities length (r= −0.4, P=0.06). Cause of death had no effect on the number of postsynaptic densities or mitochondria but there was an effect on postsynaptic density length (P=0.03) with statistically nonsignificant differences between specimens obtained from patients with cause of death attributed to trauma and cardiac (P=0.05) or substance use (P=0.07).

   Biospecimens

   • Tissue - Brain

   Preservative Types

   • Frozen
   • Formalin

   Diagnoses:

   • Autopsy
   • Other diagnoses
   • Cardiovascular Disease

   Platform:

   Analyte	Technology Platform
   Morphology	Electron microscopy

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Cause of death	Cardiac Trauma Substance use
   Preaquisition	Patient age	22-64 y, median age 48.8 ± 10.7 y
   Preaquisition	Patient gender	Female Male
   Preaquisition	Postmortem interval	5.9–24.3 h
   Preaquisition	Diagnosis/ patient condition	History of psychiatric diagnosis
   Storage	Storage duration	≤200 days ≥500 days
   Biospecimen Aliquots and Components	pH	A range of values investigated

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