Fig. 2: SARS-CoV-2 viral antigens, S and N protein, were detected in the dorsal medulla and in the substantia nigra in five COVID-19 subjects.

Topographical localization of SARS-CoV-2 viral protein immunoreactivities (triangles, right half) and microglial nodules (asterisks, left half) throughout the brainstem. a, b N and S protein IHC, real-time RT-PCR cycle thresholds for SARS-CoV-2 N gene and RNAseP quality control in our COVID-19 cohort at the level of the medulla (L) and midbrain (M). c At the level of the midbrain, immunoreactivities are found mainly within the boundaries of the substantia nigra, with the exception of Subject #3, which also presented immunoreactive neurons within the interstitial nucleus of Cajal; microglial nodules were confined mainly within the boundaries of the tegmentum, and were not detected neither within the pes nor the tectum. c1 SARS-CoV-2 spike protein IHC at the level of the substantia nigra reveals immunoreactive neurons (mean of two immunoreactivities per mm²) with well-marked processes (black arrows); negative neurons can also be found nearby (white arrows). c2 SARS-CoV-2 Nucleocapsid Protein IHC reveals a similar pattern of immunoreactive neurons and axons throughout the substantia nigra. d At the level of the pons, Subject #3 presented immunoreactive neurons (mean of five immunoreactivities per mm²) within the basilary nuclei, while microglial nodules were found both within the basis, as well as the dorsal pons in proximity to the facial nucleus. d1 SARS-CoV-2 spike protein IHC at the level of the pons in Subject #3, displaying immunoreactive neurons (black arrows) within the basilary nuclei of the pons; non-reactive cells can also be appreciated (white arrows). e At the level of the upper medulla oblongata, immunoreactivities were found at the level of the dorsal motor nucleus of the vagus, solitary tract nucleus and nucleus ambiguus; microglial nodules were prominent within the Vagal Trigone and Area Postrema, but were also found within the reticular formation and the inferior olivary complex. e1–2 SARS-CoV-2 spike protein IHC at the level of the solitary tract nucleus and nucleus ambiguus; immunoreactive neurons can be seen within the anatomical boundaries of these nuclei (black arrows), along with non-reactive cells (white arrows). Inset of a single reactive neuron within the solitary tract nucleus, spike protein immunohistochemistry. f At the level of the Lower Medulla Oblongata, immunoreactivities were found at the level of the spinal trigeminal nucleus and medullary reticular formation. Microglial nodules were found within the medullary reticular formation. f1 SARS-CoV-2 Spike Protein IHC at the level of the medullary reticular formation in the lower medulla (black arrows); non-reactive cells are indicated with a white arrow. Double label N and S protein (red) and Beta-III Tubulin (green) fluorescent immunohistochemistry in COVID-19 subjects (g–j) and controls (k). Distinct immunoreactive neurons and neurites can be appreciated in both the medulla (dorsal motor nucleus of the vagus) (h) and midbrain (substantia nigra) (g, i, j). Juxtavascular immunoreactive neurons in proximity to an immunoreactive vessel in the midbrain of COVID-19 subject #9 (j, inset). Control subjects present no viral protein immunoreactivity (k). l Double label ACE2R (green) and Beta-III Tubulin (red) fluorescent immunohistochemistry reveals ACE2R expression of midbrain neurons.