Young COVID-19 Patients Show a Higher Degree of Microglial Activation When Compared to Controls

Abstract

The severe acute respiratory syndrome-corona virus type 2 (SARS-CoV-2) is the cause of human coronavirus disease 2019 (COVID-19). Since its identification in late 2019 SARS-CoV-2 has spread rapidly around the world creating a global pandemic. Although considered mainly a respiratory disease, COVID-19 also encompasses a variety of neuropsychiatric symptoms. How infection with SARS-CoV-2 leads to brain damage has remained largely elusive so far. In particular, it has remained unclear, whether signs of immune cell and / or innate immune and reactive astrogliosis are due to direct effects of the virus or may be an expression of a non-specific reaction of the brain to a severe life-threatening disease with a considerable proportion of patients requiring intensive care and invasive ventilation activation. Therefore, we designed a case-control-study of ten patients who died of COVID-19 and ten age-matched non-COVID-19-controls to quantitatively assess microglial and astroglial response. To minimize possible effects of severe systemic inflammation and / or invasive therapeutic measures we included only patients without any clinical or pathomorphological indication of sepsis and who had not been subjected to invasive intensive care treatment. Our results show a significantly higher degree of microglia activation in younger COVID-19 patients, while the difference was less and not significant for older COVID-19 patients. The difference in the degree of reactive gliosis increased with age but was not influenced by COVID-19. These preliminary data warrants further investigation of larger patient cohorts using additional immunohistochemical markers for different microglial phenotypes.

Keywords: COVID-19; SARS-CoV-2; astrocytes; microglia; nervous system; neuroinflammation; neuropathology.

Figure 1

Schematic procedure for digitally supported quantification of immunostaining intensities. Digitalized slides of immunostained brain tissue (A) were separately deconvoluted for DAB [(B), upper panel] and converted to grayscale [(B), lower panel]. Both acquired binary images were globally thresholded (C) with distinct value cut-offs. Pixel quantities were measured within manually assigned anatomical regions (D). Assessment of immunostaining intensities (E) was based on ratios calculated from pixels with above-threshold values of the deconvoluted DAB image [(C), upper panel, DAB] over pixels with above-threshold value of the grayscale image [(C), lower panel, overall tissue] within their respective anatomical regions (D).

Figure 2

Comparison of microglial marker HLA immunostaining in COVID-19- and control cerebral tissue stratified according to patient age at death. (A–F) Representative images of cerebral HLA immunostaining (A), comprising cortex (C) and white matter (E). The colorized overlay image (B) indicates categorized pixel intensities in the cortex [(D), brown: DAB, indigo: immunonegative tissue] and white matter [(F), magenta: DAB, sky blue: immunonegative tissue]. Scale bar is 250 μm. (G–I) Dot plots showing staining intensity ratios calculated from cerebral tissue in young (<60y) vs. old (≥90y) patients in total (G), and separately in the cortex (H) and white matter (I). *p < 0.05, ns, not significant; n = 5 per group.

Figure 3

Representative microscopic images for GFAP and HLA immunostaining in COVID-19 patients and controls. GFAP staining in cortex (A,B) and white matter (C,D) of old COVID-19 patients and controls; and GFAP staining in cortex (E,F) and white matter (G,H) of young COVID-19 patients and controls. HLA staining in cortex (I,J) and white matter (K,L) of old COVID-19 patients and controls; and HLA staining in cortex (M,N) and white matter (O,P) of young COVID-19 patients and controls. Scale bar = 100 μm.

Figure 4

Comparison of GFAP immunostaining in COVID-19 and control cerebral tissue stratified according to patient age. (A–F) Representative images of cerebral GFAP immunostaining (A), comprising cortex (C) and white matter (E). The colorized overlay image (B) indicates categorized pixel intensities in the cortex [(D), gold: DAB, blue: immunonegative tissue] and white matter [(F), orange: DAB, sky blue: immunonegative tissue]. Scale bar is 250 μm. (G–I) Dot plots showing staining intensity ratios calculated from cerebral tissue in young (<60y) vs. old (≥90y) patients in total (G), and separately in the cortex (H) and white matter (I). (J–O) Representative image of cerebral GFAP immunostaining (J), comprising cortex (L) and white matter (N). The colorized overlay image (K) indicates categorized pixel intensities in perivascular regions of the cortex [(M), red: DAB, turquoise: immunonegative tissue] and parenchymal regions of the cortex [(M), yellow: DAB, blue: immunonegative tissue] as well as perivascular regions of the white matter [(O), mocha: DAB, light green: immunonegative tissue] and parenchymal regions of the white matter [(O), orange: DAB, sky blue: immunonegative tissue]. Scale bar is 250 μm. (P–S) Dot plots showing staining intensity ratios in young (<60y) vs. old (≥90y) patients in cortical perivascular (P) and cortical parenchymal (Q) as well as perivascular white matter (R) and parenchymal white matter (S) regions. *p < 0.05, ns, not significant; n = 5 per group.

Figure 5

Anti-GFAP and anti-vimentin immunoreactivity of human cortical gray matter in COVID-19 patients. Three-dimensional projection of vimentin (orange) and GFAP (cyan) antibody staining of 250 μm thick sections of perivascular (A) and parenchymal (B) gray matter of frontal cortex of a young patient with COVID-19 (age 54). Perivascular (C) and parenchymal (D) staining of an elderly patient (age 99). Squares indicate regions depicted in the following images. (E–H) magnified views of the stainings shown in (A–D). Scale bars: (A–D), 100 μm; (E,F), 30 μm.