Fetal Brain Damage in Human Fetuses with Congenital Cytomegalovirus Infection: Histological Features and Viral Tropism

Abstract

Human cytomegalovirus (HCMV) causes congenital neurological lifelong disabilities. To date, the neuropathogenesis of brain injury related to congenital HCMV (cCMV) infection is poorly understood. This study evaluates the characteristics and pathogenetic mechanisms of encephalic damage in cCMV infection. Ten HCMV-infected human fetuses at 21 weeks of gestation were examined. Specifically, tissues from different brain areas were analyzed by: (i) immunohistochemistry (IHC) to detect HCMV-infected cell distribution, (ii) hematoxylin-eosin staining to evaluate histological damage and (iii) real-time PCR to quantify tissue viral load (HCMV-DNA). The differentiation stage of HCMV-infected neural/neuronal cells was assessed by double IHC to detect simultaneously HCMV-antigens and neural/neuronal markers: nestin (a marker of neural stem/progenitor cells), doublecortin (DCX, marker of cells committed to the neuronal lineage) and neuronal nuclei (NeuN, identifying mature neurons). HCMV-positive cells and viral DNA were found in the brain of 8/10 (80%) fetuses. For these cases, brain damage was classified as mild (n = 4, 50%), moderate (n = 3, 37.5%) and severe (n = 1, 12.5%) based on presence and frequency of pathological findings (necrosis, microglial nodules, microglial activation, astrocytosis, and vascular changes). The highest median HCMV-DNA level was found in the hippocampus (212 copies/5 ng of human DNA [hDNA], range: 10-7,505) as well as the highest mean HCMV-infected cell value (2.9 cells, range: 0-23), followed by that detected in subventricular zone (1.7 cells, range: 0-19). These findings suggested a preferential viral tropism for both neural stem/progenitor cells and neuronal committed cells, residing in these regions, confirmed by the expression of DCX and nestin in 94% and 63.3% of HCMV-positive cells, respectively. NeuN was not found among HCMV-positive cells and was nearly absent in the brain with severe damage, suggesting HCMV does not infect mature neurons and immature neural/neuronal cells do not differentiate into neurons. This could lead to known structural and functional brain defects from cCMV infection.

Keywords: Brain damage; Congenital infection; Cytomegalovirus; Human fetuses; Neural/neuronal cells; Viral tropism.

Fig. 1

Histological findings in brain injury. Diffuse microglial activation (increased number of rod-shaped cells, arrows) (a), astrocytosis (cell with clear nuclei, named Alzheimer type II, arrow) (b) and vascular changes (plump endothelial cell protruding into the vessel lumen, arrow) (c) detected in all brain regions (hematoxylin–eosin staining 40 HPF, scale bar 50 µm; frontal lobe, case number 3)

Fig. 2

Microglial nodules. Microglial nodules (arrow) composed of histiocytes (grooved reniform nucleus), lymphocytes (round nucleus), and microglial cells (slender nucleus) (a) were occasional (< 3/brain region) in mild brain damage (b) (hematoxylin–eosin staining, 20 and 10 HPF, scale bar 50 µm; white matter temporal lobe, case number 8). Microglial nodules are multiple (≥ 3/brain region) in moderate and severe brain injury (c) (hematoxylin–eosin staining, 10 HPF, scale bar 50 µm; white matter temporal lobe, case number 7). Microglial nodules were absent in control cases (d) (hematoxylin–eosin staining, 10 HPF, scale bar 50 µm; white matter temporal lobe)

Fig. 3

Cortical necrosis and polymicrogyria. Necrosis in cortical layer III (star) and polymicrogyria (arrow) (a) were found in the brain with severe injury (hematoxylin–eosin staining, 4 HPF, scale bar 500 µm; parietal lobe, case number 9). Necrosis area at higher magnification (between arrows) (b) showed stromal lysis, fewer cortical cells, and numerous macrophages (hematoxylin–eosin staining, 20 HPF, scale bar 100 µm; parietal lobe, case number 9). Polymicrogyria was evident as abundant cortical folds compared to normal cortex with a smooth surface (arrow) and regular neuronal migration (c) (hematoxylin–eosin staining, 4 HPF, scale bar 500 µm; parietal lobe, control case)

Fig. 4

HCMV-DNA levels and HCMV-positive cell distribution in the brain. Tissue viral load (A) and distribution of mean HCMV-infected cell values (B) in different brain areas. Total subventricular zone refers to both periventricular areas and ganglionic eminence. Mean HCMV-infected cell values on four lobes were calculated considering only the positive cells detected on the cortex and underlying white matter. Tissue viral load in the subventricular zone was not evaluated due to the difficulties to anatomically dissect the thin layer of the periventricular region

Fig. 5

HCMV-infected cells along the migration pathway. HCMV-infected cells (brown-stained cells) from subventricular zone (filled arrow) to cortex (dashed arrow) (a). Most of these positive cells, located along the migration pathway, show slender extension typical of radial glia (b), as more highlighted in (c) (HCMV immunohistochemistry, 4 HPF, scale bar 500 µm; 10 HPF, scale bar 50 µm; 40 HPF scale bar 50 µm; respectively. Temporal lobe, case number 7)

Fig. 6

HCMV-antigen and neural/neuronal cell marker expression. HCMV-positive cell expressing nestin identified by both brown and red staining (filled arrow) in a cerebral area where nestin (red staining) was expressed by not infected cells (dashed arrow) (a) (double immunohistochemistry, 40 HPF, scale bar 25 µm, hippocampus, case number 7). HCMV-positive cell expressing nestin (filled arrow) in a cerebral area where this marker was almost absent by uninfected cells (dashed arrow) (b) (double immunohistochemistry, 40 HPF, scale bar 25 µm, cortex temporal lobe, case number 7). HCMV-positive cell expressing DCX (filled arrow) in a cerebral area where DCX (red staining) was expressed by not infected cells (dashed arrow) (c) (double immunohistochemistry, 40 HPF, scale bar 25 µm, cortex temporal lobe, case number 7). HCMV-positive cell not expressing NeuN (brown staining, filled arrow) in the cortex where this marker was expressed by uninfected cells (dashed arrow) (d) (double immunohistochemistry, 40 HPF, scale bar 25 µm, cortex temporal lobe, case number 7)

Fig. 7

NeuN expression and brain damage. Expression of NeuN (red cells) in cortical area of temporal lobe in case with severe (a) moderate (b) and mild (c) encephalic damage (double immunohistochemistry, 4 HPF, scale bar 500 µm, case number 9, 7, 8)

Fig. 8

Expression of neural/neuronal cell markers in control cases. Expression of nestin in subventricular zone (a), white matter (b) and cortical area (c); expression of DCX in cortical area (d) hippocampus (e) subventricular zone (f) and white matter (g); expression of NeuN in cortical area (h) and subventricular zone (i) (immunohistochemistry, 4 HPF, scale bar 500 µm)