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The coronavirus SARS-CoV-2 causes COVID-19, a predominantly respiratory disease that has been reported to be associated with numerous neurological signs, symptoms and syndromes. More than 20 published studies have used RT-PCR methods to determine viral SARS-CoV-2 genomic presence in postmortem brain tissue and the overall impression is that viral brain invasion is relatively uncommon and occurs in low copy numbers, supporting indirect mechanisms as the cause of most neurological phenomena. Hypoxic-ischemic brain injury and stroke are one such possible indirect mechanism, as acute ischemia or stroke concurrence with COVID-19 has been reported as being 0.5% to 20%. Immunohistochemical stains for β-amyloid precursor protein (APP) have been suggested to be a “signature” change of hypoxic leukoencephalopathy or COVID-19 brain disease, although prior reports have not had a non-COVID-19 control group. We therefore compared the prevalence and intensity of white matter APP staining in the brains of subjects dying with and without COVID-19. Clinical and neuropathological results, including semi-quantitative assessment of the density of white matter APP staining, were compared between 20 COVID-19 cases and 20 pre-COVID-19 autopsy cases, including 10 cases with autopsy-proven non-COVID-19 pneumonia and 10 cases without pneumonia. Positive APP white matter staining in at least one of the two brain regions (precentral gyrus and cingulate gyrus) studied was not significantly more common in COVID-19 vs controls (14/20 vs 12/20). Comparing density scores from both brain regions combined, the mean scores for COVID-19 cases were higher than those for controls of both types together but not significantly different when restricting to controls with pneumonia. Among control cases, cases with pneumonia had significantly higher scores. The presence or absence of a major neuropathologically-defined neurodegenerative disorder did not significantly affect the APP scores. The major finding is that while APP white matter staining cannot be regarded as a specific marker of COVID-19, as it does not occur with significantly greater probability in in COVID-19 brains as compared to non-COVID-19 brains, it is possible that white matter APP staining, representing acute or subacute axonal damage, may be a common occurrence in the perimortem period, and that it may be more intense in subjects dying with pneumonia, regardless of cause.

Stroke is one of the most serious complications of Covid-19 disease but it is still unclear whether stroke is more common with Covid-19 pneumonia as compared to non-Covid-19 pneumonia. We investigated the concurrence rate of autopsy-confirmed acute brain ischemia, acute brain infarction and acute brain hemorrhage with autopsy-proven acute non-Covid pneumonia in consecutive autopsies in the Arizona Study of Aging and Neurodegenerative Disorders (AZSAND), a longitudinal clinicopathological study of normal aging and neurodegenerative diseases. Of 691 subjects with a mean age of 83.4 years, acute pneumonia was histopathologically diagnosed in 343 (49.6%); the concurrence rates for histopathologically-confirmed acute ischemia, acute infarction or subacute infarction was 14% and did not differ between pneumonia and non-pneumonia groups while the rates of acute brain hemorrhage were 1.4% and 2.0% of those with or without acute pneumonia, respectively. In comparison, in reviews of Covid-19 publications, reported clinically-determined rates of acute brain infarction range from 0.5% to 20% while rates of acute brain hemorrhage range from 0.13% to 2%. In reviews of Covid-19 autopsy studies, concurrence rates for both acute brain infarction and acute brain hemorrhage average about 10%. Covid-19 pneumonia and non-Covid-19 pneumonia may have similar risks tor concurrent acute brain infarction and acute brain hemorrhage when pneumonia is severe enough to cause death. Additionally, acute brain ischemia, infarction or hemorrhage may not be more common in subjects dying of acute pneumonia than in subjects dying without acute pneumonia.

Biochemical analysis of human brain tissue is typically done by homogenizing whole pieces of brain and separately characterizing the proteins, RNA, DNA, and other macromolecules within. While this has been sufficient to identify substantial changes, there is little ability to identify small changes or alterations that may occur in subsets of cells. To effectively investigate the biochemistry of disease in the brain, with its different cell types, we must first separate the cells and study them as phenotypically defined populations or even as individuals. In this project, we developed a new method for the generation of whole-cell-dissociated-suspensions (WCDS) in fresh human brain tissue that could be shared as a resource with scientists to study single human cells or populations. Characterization of WCDS was done in paraffin-embedded sections stained with H&E, and by phenotyping with antibodies using immunohistochemistry and fluorescence-activated cell sorting (FACS). Additionally, we compared extracted RNA from WCDS with RNA from adjacent intact cortical tissue, using RT-qPCR for cell-type-specific RNA for the same markers as well as whole transcriptome sequencing. More than 11,626 gene transcripts were successfully sequenced and classified using an external database either as being mainly expressed in neurons, astrocytes, microglia, oligodendrocytes, endothelial cells, or mixed (in two or more cell types). This demonstrates that we are currently capable of producing WCDS with a full representation of different brain cell types combined with RNA quality suitable for use in biochemical analysis.

Due to the absence of core clinical features, many subjects with neuropathologically-confirmed dementia with Lewy bodies (DLB) are never diagnosed as such during life. Most of these are diagnosed with Alzheimer’s disease dementia (ADD) or unspecified dementia. Unrecognized DLB therefore is a critical impediment to clinical studies and treatment trials of both ADD and DLB. There are numerous published studies that suggest that olfactory function tests may be able to differentiate some neurodegenerative conditions from each other and from normal subjects, but there are very few studies with neuropathological confirmation of diagnosis. We compared University of Pennsylvania Smell Identification Test (UPSIT) results in 209 subjects: 1) 29 concurrently meeting intermediate or high consensus clinicopathological criteria for both DLB and ADD 2) 96 meeting criteria for ADD without DLB 3) 84 control subjects that were non-demented and without parkinsonism at death. The DLB subjects had significantly lower (one-way ANOVA p < 0.0001, pairwise Bonferroni p < 0.05) first and mean UPSIT scores (13.7 and 13.2) than ADD (23.3 and 22.2) or controls (29.6 and 28.9). For subjects with first and mean UPSIT scores less than 20 and 17, respectively, Firth logistic regression analysis, adjusted for age, gender and mean MMSE score, conferred statistically significant odds ratios of 17.5 and 18.0 for predicting a DLB vs ADD diagnosis, as compared to 3.3 for the presence or absence of visual hallucinations throughout the clinical observation period. To our knowledge, this is the largest study to date comparing olfactory function in subjects with neuropathologically confirmed DLB and ADD. Olfactory function testing may be a convenient and inexpensive strategy for enriching dementia studies or clinical trials with DLB subjects, or conversely, reducing the inclusion of DLB subjects in ADD studies or trials.

The Bielschowsky silver method has been the most commonly-used histological stain for demonstrating the neuritic plaques and neurofibrillary tangles that define Alzheimer’s disease (AD). The stain has been critical to providing a common measure allowing large clinicopathological correlation studies that have demonstrated statistically significant and independent contributions of both lesion types to cognitive impairment and dementia. The continuing relevance of neuritic plaques, and the Bielschowsky stain as the method of choice for their demonstration, is also indicated by its use as the US Food and Drug Administration (FDA) “standard of truth” for autopsy confirmation of the validity of PET amyloid imaging agents. Many modifications of the Bielschowsky stain have been published but it is still known to frequently generate artifactual staining that at times mimics the appearance of amyloid plaques. In this study we found that pretreatment with oxidizing agents prior to the initial incubation in silver nitrate eliminates these artifacts and reduces the commonly observed high background staining. This new method may be valuable for Alzheimer’s disease (AD) researchers and neuropathologists.