Fig 2: Axioscan fluorescent montages (a) and confocal fluorescent micrographs (b–f) of GFAP (red), P75NTR (green) and SOX10 (magenta) immunoreactivity in sagittal cryosections of a–d 17 pcw and f 12 pcw human foetal olfactory system. Scale bars a1, a2 500 µm, a2i 200 µm, a2ii 100 µm, b–e 40 µm. f 20 µm. Dashed white lines roughly denote inner and outer boundaries of the olfactory nerve layer ONL, olfactory bulb OB, olfactory epithelium OE, glomerular layer GL, olfactory nerves ON asterisk. Boxes in a2 show position of magnified areas in a2i and a2ii. a GFAP immunoreactivity in the ONL and ONs is sparse. Yellow arrowhead a1 shows GFAP+ astrocytes, a2i shows the majority of ONs asterisks do not express significant GFAP, white arrowhead a2ii shows GFAP+ cells in ONL, blue arrows show sporadic GFAP in olfactory nerves asterisk. b, c GFAP in the ONL and GL, white and blue arrowheads show GFAP+ cells in ONL and glomerular layer, respectively. Yellow arrowheads show GFAP+ presumptive astrocytes in the deeper layers of the OB. d Sporadic GFAP in olfactory nerves which are surrounded by P75NTR+ perineurial cells. Co-labelling of GFAP & P75NTR is not observed. Pattern of GFAP and P75NTR co-labelling in peripheral facial nerves e is similar to that seen in occasional small nerve bundles in nasal stroma f and a2ii white arrow

Fig 3: Fluorescent micrographs of sagittal cryosections from 17 pcw human foetal olfactory system immunolabelled with antibodies towards P75NTR (green), S100 (red), SOX10 (magenta), and DAPI nuclear stain (blue). a Axioscan micrograph montages of the olfactory bulb (OB) and olfactory nerves (ON, asterisk). Scale bar 500 µm. S100 labels the entire olfactory nerve layer (ONL) and ONs, whereas P75NTR surrounds the outside of the OB and ONs. P75NTR labelling was also observed surrounding blood vessels (blue arrowhead); on cells within the nasal stroma (white arrowheads) and intensely expressed in meningeal tissue (yellow arrowhead). b Cross-section of olfactory nerves showing P75NTR+ processes (white arrows) surrounding S100+ OECs. b1 Scale bar 100 µm. b2–4 Scale bar 20 µm, shows magnified images of area within white box in b1. c2–7 Serial optical sections through a 13 µm z-stack (c1) showing DAPI labelled nuclei and P75NTR+ processes of perineurial cells (white arrows) surrounding S100+ OECs within a cross-section of an olfactory nerve (asterisk). Scale bar 20 µm. d1–3 In the outer ONL, P75NTR is expressed on cells (white arrowheads) surrounding ONs as they enter the ONL, but not on SOX10/S100+ OECs. Dashed white lines show rough boundary of ONL, scale bar 20 µm. e P75NTR+ cells surround presumptive arterioles in the ONL, scale bar 20 µm

Fig 4: The lamina propria of the olfactory mucosaConfocal images of sections through the olfactory mucosa of COVID #105. No viral puncta or nucleocapsid-IR signal were detected in this non-informative case.(A) OSNs harbor OMP puncta, S100B-IR signal identifies OECs, and p75-IR signal identifies pONFs.(B) Sustentacular cells in the olfactory epithelium and Bowman’s gland cells in the lamina propria harbor UGT2A1 puncta. OSN axons contain TUBB3-IR signal and OECs contain S100B-IR signal.(C) Layers of p75-IR pONFs surround olfactory axon fascicles. OMP puncta occur in an intraepithelial cyst or invagination of the olfactory epithelium.(D) OMP puncta occur in the olfactory epithelium. In the deeper but not in the superficial lamina propria, OECs are enwrapped by pONFs.(E) Axon fascicles are enwrapped by pONFs.(F and G) OMP-IR olfactory axon fascicles are enwrapped by pONFs. The dashed square in (F) is imaged at higher magnification in (G).

Fig 5: Perineurial olfactory nerve fibroblasts form an anatomical barrierConfocal images of sections through olfactory mucosa of COVID #68 (Alpha), #87 (Delta), #89 (Delta), #94 (Delta), and #108 (Omicron BA.1).(A) N puncta do not invade a fascicle of TUBB3-IR OSN axons.(B) A swarm of N puncta stays separate from fascicles containing OSN axons and GFAP-IR OECs.(C) In a section close to (B), N puncta remain outside the enwrapment by p75-IR pONFs.(D) A longitudinally sectioned olfactory axon fascicle containing S100B-IR OECs and p75-IR pONFs is devoid of N puncta. The blood vessel at the left emits autofluorescence.(E) The area marked with an asterisk in (D) imaged at higher magnification.(F and G) Arrays of N puncta do not invade axon fascicles containing OECs and enwrapped by pONFs.(H) Fascicles of OMP-IR and TUBB3-IR OSN axons remain free of N puncta. The blood vessel at the center emits autofluorescence.(I) N puncta stay clear of a fascicle of OSN axons.(J) An ant-like column of N puncta escorts a fascicle of OSN axons. See also Figure S4.