Chronological brain lesions after SARS-CoV-2 infection in hACE2-transgenic mice

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes respiratory disease, but it can also affect other organs including the central nervous system. Several animal models have been developed to address different key questions related to Coronavirus Disease 2019 (COVID-19). Wild-type mice are minimally susceptible to certain SARS-CoV-2 lineages (beta and gamma variants), whereas hACE2-transgenic mice succumb to SARS-CoV-2 and develop a fatal neurological disease. In this article, we aimed to chronologically characterize SARS-CoV-2 neuroinvasion and neuropathology. Necropsies were performed at different time points, and the brain and olfactory mucosa were processed for histopathological analysis. SARS-CoV-2 virological assays including immunohistochemistry were performed along with a panel of antibodies to assess neuroinflammation. At 6 to 7 days post inoculation (dpi), brain lesions were characterized by nonsuppurative meningoencephalitis and diffuse astrogliosis and microgliosis. Vasculitis and thrombosis were also present and associated with occasional microhemorrhages and spongiosis. Moreover, there was vacuolar degeneration of virus-infected neurons. At 2 dpi, SARS-CoV-2 immunolabeling was only found in the olfactory mucosa, but at 4 dpi intraneuronal virus immunolabeling had already reached most of the brain areas. Maximal distribution of the virus was observed throughout the brain at 6 to 7 dpi except for the cerebellum, which was mostly spared. Our results suggest an early entry of the virus through the olfactory mucosa and a rapid interneuronal spread of the virus leading to acute encephalitis and neuronal damage in this mouse model.

Keywords: Coronavirus Disease 2019; SARS-CoV-2; animal model; brain; hACE2-transgenic mice; meningoencephalitis; neuropathology.

Conflict of interest statement

Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Figures

Figure 1.

Progression of weight loss after inoculation of hACE2-transgenic mice with either 103 or 104 SARS-CoV-2 TCID50 per animal. Significant differences between groups are shown as asterisks above each time point. Red asterisks show differences between the 104 TCID50-inoculated group and noninfected animals. Blue asterisks show differences between 103 TCID50-inoculated group and noninfected animals. Green asterisks show differences between 104 and 103 TCID50-inoculated groups. ns, P > .05; *, P ≤ .05; **, P ≤ .01; ***, P ≤ .001; ****, P ≤ .0001. The bars show the mean ± standard deviation. Figure 2. Survival curves of hACE2-transgenic mice inoculated with either 103 or 104 TCID50 per animal of SARS-CoV-2 and the negative controls.

Figures 3–5.

Virological studies. No significant differences in viral loads in brain were observed between the 2 dose groups at 4 and 7 days postinoculation (dpi). In the high-dose group, viral genomic DNA was found in the brain as early as 2 dpi. Figure 3. Viral load in the brain of SARS-CoV-2-inoculated hACE2-transgenic mice measured by means of viral genomic RNA. Figure 4. Viral load in the brain of SARS-CoV-2-inoculated hACE2-transgenic mice measured by means of viral subgenomic RNA. Figure 5. Viral load in the brain of SARS-CoV-2-inoculated hACE2-transgenic mice measured by means of infectious virus (in cell culture). Each dot represents a single animal. ns, nonsignificant differences. The bars show the mean ± standard deviation.

Figures 6–11.

SARS-CoV-2 infection, brain, hACE2-transgenic mice, at 7 days postinoculation, 104 TCID50 dose, hematoxylin and eosin. Figure 6. Occipital cortex. Mononuclear cell infiltrate in the leptomeninges with a thrombus in a blood vessel (arrow). Figure 7. Mesencephalon. A perivascular cuff of mononuclear cells, and a thrombus within a blood vessel. Figure 8. Occipital cortex, gray to white matter transition. There is vasculitis (arrow) and small hemorrhages (bottom left). Figure 9. Thalamus. Intravascular thrombus (arrow) in a small blood capillary. Figure 10. Hippocampal formation. Vacuolar degeneration of neurons in the dorsal end of the subiculum. Figure 11. Medulla oblongata. Myelin sheath vacuolation, limited to the root of the facial nerve.

Figure 12.

SARS-CoV2 immunolabeling in the nervous system of hACE2-transgenic mice. Mice were inoculated with 103 (blue; euthanized at 4 and 7 dpi) or 104 (pink; euthanized at 2, 4, 6, and 7 dpi) TCID50 of SARS-CoV-2. Different areas of the brain and nasal cavity were semiquantitatively scored from 0 (no immunolabeling) to 3 (maximum immunolabeling) and the mean scores in each group are shown. Levels from 0 to 3 are shown by concentric circles, with the outside circle being 3. At 2 dpi, virus was only found in the olfactory mucosa, but at 4 dpi the virus had reached most brain areas (for both doses of virus). Virus throughout the brain was maximal at 6 and 7 dpi, with the exception of the cerebellum which is almost spared. At 7 dpi, the virus is no longer observed in the olfactory mucosa. See individual scores in Supplemental File S2. Each axis of the star-graph represents a studied region (Om, olfactory mucosa; Cc, cerebellar cortex; Mob, medulla oblongata; P, pons; M, mesencephalon; HT, hypothalamus; T, thalamus; S, striatum; Oc, occipital cortex; Tc, temporal cortex; Pc, parietal cortex; Fc, frontal cortex; H, hippocampus; Pfc, piriform cortex; Ob, olfactory bulb).

Figures 13–18.

SARS-CoV-2 infection, olfactory mucosa (Fig. 13), and brain (Figs. 14–18), hACE2-transgenic mice, immunohistochemistry for SARS-CoV-2. Figure 13. Small clusters of immunolabeled cells in the olfactory epithelium (2 dpi, 104 TCID50 dose). Figure 14. Widespread intracytoplasmic neuronal immunolabeling in all layers of the occipital cortex (7 dpi, 104 TCID50 dose). Inset: immunolabeled cytoplasmic inclusion bodies in a neuron (arrowheads). Figure 15. The early distribution of SARS-CoV-2 antigen immunolabeling with patchy involvement of thalamus, CA1, CA2, CA3, and subiculum of the hippocampus (the lateral portion of CA1 and the dentate gyrus are mostly spared) and the parietal cortex (4 dpi, 104 TCID50 dose). Figure 16. The cerebellar cortex is mostly spared, and only the perikaryon and dendrites of a few piriform (Purkinje) cells are immunolabeled (7 dpi, 104 TCID50 dose). Figure 17. Parietal cortex. All degenerating neurons with cytoplasmic vacuoles are immunolabeled (7 dpi, 104 TCID50 dose). Figure 18. Mesencephalon. There is immunolabeling of neurons but not of the endothelium in a vessel that has a cuff of mononuclear cells (the same blood vessel depicted in Fig. 7; 7 dpi, 104 TCID50 dose).

Figures 19–20.

Quantification of the neuroinflammatory response in brains of hACE2-transgenic SARS-CoV-2-infected mice (103 TCID50 dose) at 4 dpi and 7 dpi, compared to mock-inoculated negative controls. Different areas of the brain were semiquantitatively scored from 0 (no immunolabeling) to 4 (maximum immunolabeling) and the mean scores in each group are shown. Levels from 1 to 4 are shown by concentric circles, with the outside circle being 4. Each axis of the star-graph represents a brain region; the value given is the average score for all the animals in that group in that particular area (Pfc, piriform cortex; Cc, cerebellar cortex; Mob, medulla oblongata; P, pons; M, mesencephalon; HT, hypothalamus; T, thalamus; S, striatum; Oc, occipital cortex; Tc, temporal cortex; Fc, frontal cortex; Pc, parietal cortex; H, hippocampus). Figure 19. Scoring of GFAP immunolabeling. Astrogliosis (hypertrophy and hyperplasia of astrocytes) is most evident in the brainstem (thalamus, hypothalamus, mesencephalon, pons, and medulla oblongata), piriform cortex, and occipital cortex, and is evident at 4 dpi (green squares) but more intense at 7 dpi (yellow triangles). The hippocampus, parietal cortex, striatum, and cerebellar cortex show small or no differences compared to uninoculated negative controls (gray circles). Figure 20. Scoring of the IBA1 immunolabeling. Widespread microgliosis is most severe at 7 dpi (yellow triangles), but was similar at 4 dpi (green squares) as in the negative controls (gray circles). Microglial spares only the cerebellar cortex. See individual scores in Supplemental Table S2.

Figures 21–23.

SARS-CoV-2 infection (103 TCID50 dose), brain (mesencephalon), hACE2-transgenic mice. (a) Hematoxylin and eosin (HE), (b) immunohistochemistry (IHC) for GFAP, and (c) IHC for IBA1. Figure 21. Negative control mouse (mock-inoculated). Figure 22. SARS-CoV-2-infected at 4 dpi. (a) The increase in cellularity is not striking in HE-stained sections. (b) Moderate increase in number and hypertrophy of astroglial cells. (c) Moderate increase in number and activation of microglial cells. Figure 23. SARS-CoV-2-infected at 7 dpi. (a) Markedly increased cellularity. (b) Marked increase in number and size of astrocytes near vessels and in neuropil. (c) Marked increase in number and size of microglial cells.

Figure 24.

SARS-CoV-2 infection (104 TCID50 dose, 7 dpi), brain (mesencephalon), hACE2-transgenic mice. Immunohistochemistry. (a) Several CD3-positive cells (T cells) are in most perivascular cuffs (black arrows) and occasional T cells infiltrate the neuropil (white arrows). (b) Comparatively, there are low numbers of CD20-positive cells near vessels and in neuropil (B cells, arrows). (c) Numerous IBA1-positive cells (macrophages) are observed in perivascular cuffs (arrows). IBA1-positive activated microglial cells are present in the neuropil.