Embedding Brain Tissue for Routine Histopathology: A Processing Step Worthy of Consideration in the Digital Pathology Era.

The importance of technical quality for histopathologic examination has only increased in recent years with the expanding use of digital pathology. The University of Kentucky Alzheimer's Disease Center (UK-ADC) Neuropathology Core has decades of experience with brain histopathology and has emphasized the importance of quantitative assessments of histopathologic hallmarks. Technical artifacts and nonuniform samples are challenging for high-throughput digital analyses after the slides have been scanned, so that methodological optimization may be helpful. We do not know of published literature that systematically reviews how different procedures at the various stages of tissue processing can impact the quality of the histopathologic preparations in human brain samples. We wanted to pass along our experience in the hope that it will help others to improve their results. Here we describe the UK-ADC method of embedding for neuropathologic evaluation and provide specific examples (with a comparison to another processing workflow) that help support the idea that the methods and tools used in the embedding process can alter the quality of the formalin-fixed paraffin-embedded histopathologic results. The process used at the UK-ADC has been successful for us, but results may vary in relation to each embedding machine and with other factors.

Alzheimer Disease Pathology-Associated Polymorphism in a Complex Variable Number of Tandem Repeat Region Within the MUC6 Gene, Near the AP2A2 Gene.

We found evidence of late-onset Alzheimer disease (LOAD)-associated genetic polymorphism within an exon of Mucin 6 (MUC6) and immediately downstream from another gene: Adaptor Related Protein Complex 2 Subunit Alpha 2 (AP2A2). PCR analyses on genomic DNA samples confirmed that the size of the MUC6 variable number tandem repeat (VNTR) region was highly polymorphic. In a cohort of autopsied subjects with quantitative digital pathology data (n = 119), the size of the polymorphic region was associated with the severity of pTau pathology in neocortex. In a separate replication cohort of autopsied subjects (n = 173), more pTau pathology was again observed in subjects with longer VNTR regions (p = 0.031). Unlike MUC6, AP2A2 is highly expressed in human brain. AP2A2 expression was lower in a subset analysis of brain samples from persons with longer versus shorter VNTR regions (p = 0.014 normalizing with AP2B1 expression). Double-label immunofluorescence studies showed that AP2A2 protein often colocalized with neurofibrillary tangles in LOAD but was not colocalized with pTau proteinopathy in progressive supranuclear palsy, or with TDP-43 proteinopathy. In summary, polymorphism in a repeat-rich region near AP2A2 was associated with neocortical pTau proteinopathy (because of the unique repeats, prior genome-wide association studies were probably unable to detect this association), and AP2A2 was often colocalized with neurofibrillary tangles in LOAD.

The Amygdala as a Locus of Pathologic Misfolding in Neurodegenerative Diseases.

Over the course of most common neurodegenerative diseases the amygdala accumulates pathologically misfolded proteins. Misfolding of 1 protein in aged brains often is accompanied by the misfolding of other proteins, suggesting synergistic mechanisms. The multiplicity of pathogenic processes in human amygdalae has potentially important implications for the pathogenesis of Alzheimer disease, Lewy body diseases, chronic traumatic encephalopathy, primary age-related tauopathy, and hippocampal sclerosis, and for the biomarkers used to diagnose those diseases. Converging data indicate that the amygdala may represent a preferential locus for a pivotal transition from a relatively benign clinical condition to a more aggressive disease wherein multiple protein species are misfolded. Thus, understanding of amygdalar pathobiology may yield insights relevant to diagnoses and therapies; it is, however, a complex and imperfectly defined brain region. Here, we review aspects of amygdalar anatomy, connectivity, vasculature, and pathologic involvement in neurodegenerative diseases with supporting data from the University of Kentucky Alzheimer's Disease Center autopsy cohort. Immunohistochemical staining of amygdalae for Aß, Tau, α-synuclein, and TDP-43 highlight the often-coexisting pathologies. We suggest that the amygdala may represent an "incubator" for misfolded proteins and that it is possible that misfolded amygdalar protein species are yet to be discovered.