Subtle vascular and astrocytic changes in the brain of coronavirus disease 2019 (COVID-19) patients

Abstract

Background and purpose: In the central nervous system, a multitude of changes have been described associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, such as microglial activation, perivascular lymphocyte cuffing, hypoxic-ischaemic changes, microthrombosis, infarcts or haemorrhages. It was sought here to assess the vascular basement membranes (vBMs) and surrounding perivascular astrocytes for any morphological changes in acute respiratory syndrome (coronavirus disease 2019, COVID-19) patients.

Methods: The light microscopy morphology of the vBMs and perivascular astrocytes from brains of 14 patients with confirmed SARS-CoV-2 infection was analysed and compared to four control patients utilizing fluorescent immunohistochemistry for collagen IV and astrocytes (GFAP), endothelia (CD31), tight junction 1 (TJ1) adhesion protein, as well as the aquaporin 4 (AQP4) water channel. On 2D and 3D deconvoluted images from the cortex and white matter, vessel densities, diameters, degree of gliosis, collagen IV/GFAP and GFAP/AQP4 colocalizations were calculated, as well as the fractal dimension of astrocytes and vBMs viewed in tangential planes.

Results: Fractal dimension analysis of the GFAP-stained astrocytes revealed lower branching complexities and decreased GFAP/collagen IV colocalization for COVID-19 patients. Interestingly, vBMs showed significantly increased irregularities (fractal dimension values) compared to controls. Vessel diameters were increased in COVID-19 cases, especially for the white matter, TJ1 protein decreased its colocalization with the endothelia, and AQP4 reduced its co-expression in astrocytes.

Conclusions: Our data on the irregularity of the basement membranes, loss of endothelial tight junction, reduction of the astrocyte end-feet and decrease of AQP4 suggest subtle morphological changes of the blood-brain barrier in COVID-19 brains that could be linked with indirect inflammatory signalling or hypoxia/hypercapnia.

Keywords: COVID-19; aquaporin 4; astrocytes; brain blood vessels; fractal dimension; vascular basement membranes.

FIGURE 1

A variety of histopathology changes have been identified in the brains of COVID‐19 patients: (a) small extravasated groups of red blood cells; (b), (c) microthrombosis; (d), (e) stasis in both white and grey matter; (f) perivascular and (g) perineuronal accumulation of lymphocytes; (h) hypoxic neurons; (i) chromatolysis; (j) microinfarctions; (k) smooth muscle cell proliferation; and (l) occasional leptomeningeal haemorrhage. Arrows indicate the respective pathological denominations

FIGURE 2

Exemplary images of various degrees of gliosis seen together with the blood vessel basement membranes (GFAP/collagen IV). In the grey matter of both COVID‐19 (a), (a1) and control patients (b), (b1), as well as in the white matter (c), (c1), (d), (d1) of the two groups, there were regions with low and high GFAP reactivity

FIGURE 3

Evaluation of gliosis and blood vessel morphological features. (a) GFAP expression does not show significant differences between COVID‐19 and control patients, whilst astrocyte morphology (b) assessed by fractal dimension (FD) showed a clear‐cut decrease in COVID‐19 patients. (c) Both groups had the same vascular densities, lower for the white matter compared to the grey matter, whilst vessel diameters (d) increased significantly in the white matter of the COVID‐19 group. GFAP coverage (overlapping coefficient) of the blood vessel basement membranes showed a significant drop in COVID‐19 cases, as both overall values (e) or as individual values for grey and white matter regions (f). Significance is shown for Student's t testing. Data are expressed as mean ± SD. 40× image field corresponds to an area of 12569.26 μm²

FIGURE 4

Evaluation of collagen IV irregularities on tangential images, as stained for collagen IV. Exemplary images of a control group blood vessel (a), (a1) and a COVID‐19 group irregular blood vessel (b), (b1). (c) The COVID‐19 group has a clear‐cut higher average fractal dimension (FD) of the basement membrane asperities compared to control cases, and (d) the area percentage of the blood vessel lumens covered by irregularities also tends to increase for the disease group. (e) Only capillaries (i.e., <9 μm diameter vessels) have been considered in this analysis, and within this sector there was a clear‐cut enlargement of the vessels for COVID‐19 patients. (f) There is no clear‐cut correlation between the vessel diameters and the FD of their basement membranes. Significance is shown for Student's t testing (c)–(e) and Pearson (r) score (f). Data are expressed as mean ± SD

FIGURE 5

Evaluation of tight junction 1 (TJ1) adhesion molecule colocalization degree with the endothelia (CD31). Exemplary double stained and deconvoluted TJ1/CD31 images for the COVID‐19 (a)–(c) and control groups (d)–(f). TJ1/CD31 colocalization clearly decreases in COVID‐19 cases as an overall evaluation (g) or segmented for white and grey matter regions (h). Whilst there is a weak but significant direct correlation between the colocalization coefficient with the diameter of the blood vessel for control cases (i), this becomes an inverse correlation for COVID‐19 patients (j). Significance is shown for Student's t testing (g), (h) and Pearson (r) score (i), (j). Data are expressed as mean ± SD in (g), (h)

FIGURE 6

Aquaporin 4 (AQP4) expression in the GFAP‐positive astrocytes decreases for COVID‐19 patients. (a)–(c), (a₁); (d)–(f), (d₁) AQP4 was expressed mostly at the levels of the cell processes, but also on the cell bodies, for both control and COVID‐19 cases. (g)–(i), (g₁) Occasional activated‐like astrocytes with eccentric nuclei and retracted processes also reveal co‐staining on the cell body. (j) Overall AQP4‐GFAP colocalization drops for COVID‐19 cases compared to control patients, and if visualized for grey and white matter (k) the highest difference occurs in the white matter. Significance is shown for Student's t testing. Data are expressed as mean ± SD