Fig 1.
Steps of herpetic infection and monitoring in the active storage machine (ASM).
(A) Two-week epithelial restoration step. (B) Inoculation with HSV-1 suspension. The ASM epithelial lid was opened under a laminar flow hood. (C) Daily monitoring of the ASM using a macro zoom microscope. Asterisk: corneal support machined in polyether ether ketone; black arrowhead: storage medium tank; white arrowhead: pressure sensor; black arrow: peristaltic pump; white arrow: solenoid valve.
Fig 2.
Observation with a slit lamp and cobalt blue filter of dendritic herpetic keratitis in a patient (A, personal database of GT) and of a dendritic epithelial lesion obtained in the ASM at day 4 post infection (B).
Fig 3.
Ex vivo models of herpetic lesions on corneas.
(A) Chronology of the development of herpetic lesions on the corneas stored in the active storage machine (ASM) versus organ culture (OC). Bright field macroscopic observation (x1 and x6.3 zooms) after scratching (D0) and infection at D3, D4 and D5. In the OC cornea, the red arrows show the initial scratch; and the yellow arrows, the "buds" that appeared at the edges of the infected area. Note that the OC cornea presented a characteristic tessellated pattern (highlighted by retro illumination) corresponding to the numerous deep posterior folds caused by stromal edema. On the contrary, the ASM-stored cornea was thinner, presented no folds and thus appeared smooth. (B) Anti HSV-gB immunolabeling on unfixed whole human corneas. Observation with a fluorescence macro-zoom microscope (x1 and x6.3 zooms) of the corneal epithelium infected with HSV-1 in the ASM or OC and labeled with anti-HSV-gB and Hoechst after 5 days' incubation in the ASM or OC. Yellow asterisks show full-thickness epithelial ulcers where nuclei of Hoechst-positive keratocytes were visible through transparency. Yellow arrows show the rounded infected cells demonstrating the cytopathogenic effect (CPE). Note that the corneal limbus and the scleral rim were strongly autofluorescent on this wavelength, but this did not prevent corneal imaging. Scale bar: 1000 μm for low magnification images and 200 μm for high magnification images.
Fig 4.
En face view of the corneal epithelium after HSV-1 infection.
Confocal microscopy of corneal epithelium infected in the active storage machine or in organ culture and labelled with anti-gB/E-cadherin after methanol fixation or K3 or DSC2/3 after 0.5% PFA fixation, a mean 5 days after HSV-1 inoculation. The white arrows show the rounded infected cells exhibiting the HSV-1 induced cytopathogenic effect with a higher nuclear-cytoplasmic ratio. No gB staining was observed in negative controls. Nuclei were stained with To-PRO 3 Iodide. Scale bar: 50 μm.
Fig 5.
Cross-section of the corneal epithelium and anterior stroma after HSV-1 infection.
Only infected areas are shown. Histology (hematoxylin, eosin and saffron, scale bar: 50 μm) and immunohistochemistry of paired corneas infected in the active storage machine (ASM) or in organ culture (OC) (scale bar: 20μm) were compared with uninfected corneas (the ASM control group is presented here). Cells infected by HSV-1 expressed HSV-1 gB. Epithelial organization and maturation were labeled by the three epithelial markers (K3, E-Cadherin, DSC2/3), and the epithelial basement membrane integrity by expression of laminin-5. Yellow arrows mark gB+ keratocytes and white arrows marked the cytopathogenic effect (CPE) in epithelial cells. No gB staining was observed in uninfected controls. The thickness of the corneal epithelium in the ASM was greater than in passive OC because the ASM can regenerate an epithelium with more layers than in passive OC [30] and, in this thicker epithelium, the CPE further increased the difference between the ASM and OC. Nuclei were stained with To-PRO 3 Iodide.
Fig 6.
Ultrastructure of the corneal epithelium after HSV-1 infection.
Corneas were stored either in the active storage machine (ASM) (A to C) or in organ culture (D to G). The microvilli (green arrows) present only on the superficial cells of corneas in the ASM demonstrated normal epithelial maturation. (A and B) Corneas with dendritic or geographic ulcers. Enveloped viral particles in the intercellular spaces or in cytoplasmic vacuoles. Viral capsids in the nucleus. (C) Cornea without macroscopic lesion. Viral capsids in the nuclei of epithelial cells. Desmosomes were intact (blue arrows). (D) Enveloped viral particles in the intercellular spaces and viral capsids in the nuclei of epithelial cells. (E) Enveloped viral particles on Bowman's layer in the absence of epithelial cells. (F) Infected keratocyte with viral capsids in the nucleus and enveloped viral particles in the disorganized cytoplasm. (G) Reconstruction of an infected keratocyte. BL: Bowman's layer, ER: Endoplasmic reticulum, N: Nucleus. Yellow arrow: enveloped viral particle. Red arrow: Viral capsid. Scale bar: 2 μm.
Fig 7.
Disappearance of intraepithelial nerve structures after organ culture and storage in the active storage machine.
Nerves were revealed by immunostaining of the Neurofilament-L (NF in red). One cornea was studied fresh, immediately after retrieval (A) and its paired cornea was studied after 14 days' organ culture storage and 14 days' additional storage in the active storage machine (C). (A and C) Nerve map of the whole flat-mounted cornea with its limbus and proximal conjunctiva. Scale bar: 2 mm. (B and D). Close-up of the nerves, and of epithelial cell nuclei labeled with DAPI (in blue) (x10 objective). The epithelium of the stored cornea was very disorganized and had far fewer cells. Scale bar: 200 μm.