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Correction: Individual Case Analysis of Postmortem Interval Time on Brain Tissue Preservation.

Author(s): Blair JA, Wang C, Hernandez D, Siedlak SL, Rodgers MS, Achar RK, Fahmy LM, Torres SL, Petersen RB, Zhu X, Casadesus G, Lee HG

Publication: PLoS One, 2016, Vol. 11, Page e0157209

PubMed ID: 27258001 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This paper explored the effects of postmortem interval (PMI) on morphological, protein, and RNA endpoints in case-matched hippocampal specimens subjected to different PMI durations (4.5 – 53.5 h) at 4°C. To minimize inter-individual variability, hippocampal specimens from five postmortem donors (4 males, 1 female; 42-78 y) were dissected and a 2-4 mm thick section was preserved as soon as possible (4.5 – 23 h), while the remaining specimen was stored in a closed plastic container at 4°C and used to assess one or two additional PMI timepoints (6.5 – 53.5 h total PMI). Although one donor (42 y, male) lacked histopathologic abnormalities, the remaining donors were diagnosed with Alzheimer’s disease (2 donors) and moderate or severe atherosclerosis of intracranial arteries/vessels (1 donor each). Tissues destined for histological and immunohistochemical evaluation were preserved in 10% neutral buffered formaldehyde, paraffin-embedded, and sectioned at a thickness of 6 µm; additional details on tissue fixation, processing, and storage were not provided. Tissues targeted for Western blot and qRT-PCR analysis were frozen at -80°C; the duration of frozen storage was not specified. Nissl stained FFPE tissue sections were used to assess morphological integrity. Immunohistochemistry included targeted proteins routinely included in anatomical and neuropathological assessment: collagen IV, SMI-34 (phosphoneurofilament marker), HNE (a lipid peroxidation marker), α-tublin, cytochrome oxidase 1 (COX-1), double-stranded DNA, NeuN, GFAP (an astrocyte marker), rRNA, and markers of phosphorylated tau (phosphorylated tau Ser396/404, phosphorylated tau Ser202/Thr205).  Western blot analysis and qRT-PCR analysis were limited to two cases (initial PMIs of 4.5 and 5.0 h).  For Western blot analysis, frozen samples were homogenized in lysis buffer containing protease and phosphatase inhibitors, centrifuged, and the protein concentration of lysates was determined using the bicinchoninic acid assay (BCA). For each sample, 10 or 30 µg of protein was run per well on a 10% SDS PAG gel with coomassie blue staining, and protein was transferred to immobilon membranes, blocked, and incubated with primary antibodies against α-tubulin, β-actin, GAPDH, PHF1, phosphorylated tau, NeuN, 4-hydroxynonenal, and tau-5. For qRT-PCR analysis of GAPDH and B2M, total RNA was extracted, purified, and reverse transcribed according to previously reported methods.  Isolated RNA was also compared to a mouse RNA standard on an ethidium bromide gel.

   Summary of Findings:

   Neuronal morphology, visualized by Nissl-stained neurons in FFPE specimens, was not appreciably different in the case-matched hippocampal specimens that experienced different PMIs at 4°C (4.5-5 h versus 17.25 versus 42.75 h).

   While protein integrity in frozen specimens was generally similar among the PMIs evaluated in the two cases examined (5-49.5 h and 4.5-53.5 h), some smearing on the coomassie blue-stained SDS-PAGE gel was observed in one case after a PMI of 24 h, while weaker bands were noted in the other.  Western blot analysis revealed a slight decrease in NeuN levels in one frozen case after a PMI of 53.5 h (relative to a 4.5 h PMI) but stable levels in the other case (5.0 versus 49.5 h PMI) and no change in GAPDH or actin in either case.  α-tubulin was significantly and negatively correlated to PMI in frozen specimens based on data from Western blot analysis of 2 cases (p<0.02), with levels decreased substantially after a PMI ≥22 h compared to shorter PMIs (4.5-10 h). Conversely, immunohistochemical staining for α-tubulin in FFPE specimens remained stable after a PMI of 48 h for three of the six cases examined.  COX-1 and 4-hydroxynonenal immunostaining of two FFPE cases remained stable among the PMIs evaluated (22 vs 41 h, 23 vs 46 h PMI).  In the two donors diagnosed with Alzheimer’s disease, all phosphorylated tau protein markers remained stable at the PMIs investigated (≤53 h) when analyzed by immunohistochemistry of FFPE specimens or Western blot analysis of frozen specimens, including stable immunohistochemical staining of phosphorylated tau in neurofibrillary tangles.  GFAP, an astrocytosis marker, and? collagen IV both displayed stable immunohistochemical staining in one case across the PMIs examined (23 versus 46 h).

   Degradation of RNA isolated from frozen postmortem hippocampus specimens was apparent in ethidium bromide-stained gels at all PMIs examined in a single case (5, 25.5, 49.5 h PMI). While cDNA was successfully transcribed, a slight increase in qRT-PCR Ct values of GAPDH and B2M was observed with longer PMIs (5 h versus 25.5-49.5 h) in one of the two cases examined, and RNA yields were too low in specimens with a PMI of 30.5 and 53.5 h in the remaining case.  Immunohistochemical staining using an antibody against rRNA also displayed a reduction in staining intensity when PMIs of 22 and 41 h were compared in a single case, while immunopositive staining for DNA remained stable in specimens with a PMI of 23 and 46 h.

   Biospecimens

   • Tissue - Brain

   Preservative Types

   • Formalin
   • Frozen

   Diagnoses:

   • Alzheimer's Disease
   • Autopsy
   • Normal

   Platform:

   Analyte	Technology Platform
   Protein	Immunohistochemistry
   Protein	Western blot
   Protein	1D/2D gels
   Protein	Colorimetric assay
   RNA	Electrophoresis
   RNA	Real-time qRT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Postmortem interval	4.5 – 53.5 h
   Immunohistochemistry Specific	Targeted peptide/protein	Collagen IV SMI-34 Alpha tubulin COX-1 Double-stranded DNA phosphorylated tau Ser396/404 phosphorylated tau Ser202/Thr205 GFAP rRNA
   Western blot Specific	Targeted peptide/protein	Beta actin GAPDH phosphorylated tau NeuN tau-5 4-hydroxynonenal
   Real-time qRT-PCR Specific	Targeted nucleic acid	GAPDH B2M

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