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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online April 10, 2002 as doi:10.1096/fj.01-0803fje. |
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Institut für Medizinische Mikrobiologie, Abteilung Klinische Virologie, Universität Münster, Münster, Germany; and
* Klinik und Poliklinik für Augenheilkunde, Abteilung für Experimentelle Ophthalmologie, Universität Münster, Münster, Germany
2Correspondence: Institut für Medizinische Mikrobiologie, Abteilung Klinische Virologie, Von-Stauffenberg-Strasse 36, 48151 Münster, Germany. E-mail: kuehnj{at}uni-muenster.de
SPECIFIC AIMS
Reciprocal transmission between epithelial cells and neurons of the PNS in direct contact with the epithelium is a crucial step in the life cycle of HSV and other alphaherpesviruses. Using a dual-chamber organ model based on embryonic chick tissues, our aim was to investigate HSV-1 transfer between corneal epithelia and trigeminal ganglia in vitro.
PRINCIPAL FINDINGS
1. Embryonic chick cornea explants are efficiently reinnervated by outgrowing trigeminal (TG) neurites in the dual-chamber model
As an in vitro alternative to animal models, we developed a dual-chamber model consisting of embryonic chick TG explants and embryonic chick cornea slices separated by a diffusion barrier. When TG explants were placed in the dual-chamber system, a fraction of neurites spread preferentially toward the notch of the ring separating the two compartments of the system. After penetrating the agarose diffusion barrier and contacting the front of the outgrowing multilayered epithelial cell carpet, the ingrowth of trigeminal axons into the cornea was followed by formation of elaborate axonal terminal branches. The reinnervation process was complete in 
6 days. The number of neurites crossing the agarose diffusion barrier ranged between 20 and 100 in each experiment.
2. Embryonic chick corneal cells are susceptible to lytic replication of HSV-1
The susceptibility of embryonic chick corneal cells to lytic HSV-1 infection was tested by inoculation of epithelial cell layers with 106 PFU HSV-1 strain 17. Entry of HSV-1 was allowed to proceed for 1 h at 37°C. Remaining unpenetrated virions were inactivated by exposing cells to low pH buffer. Progeny virus could be detected in the supernatants as early as 12–14 h postinfection (p.i.), with maximum levels reached at 36 h p.i. Analysis of infected cultures by immunofluorescence showed that infection started at the outer margin of the corneal cell layer and spread from cell to cell toward the inner parts of the epithelial layer.
3. HSV-1 replicates in chick trigeminal explants but is not secreted into culture supernatants
No infectious progeny virus could be detected in the supernatants of TG explants infected with 106 PFU HSV-1 within a 72 h follow-up. However, when TG explants were dispersed at 48 h p.i. and cocultivated with Vero cells, 30 HSV-replicating foci per TG explant were produced. Infection of in vitro cultured cells derived from dispersed TG explants resulted in the expression of late HSV-1 antigens 48 h p.i. in ganglion and satellite cells, respectively.
4. The dual-chamber system supports anterograde axonal transport of HSV-1 in chick TG neurites and spread of infection to reinnervated chick corneal cells
The anterograde axonal transport of HSV from infected TG to the corneal epithelium was analyzed by infection of the TG compartment with 106 PFU HSV-1. After 24, 48 and 72 h p.i., TG and corneal cells were fixed in situ and HSV-infected cells were visualized by immunoperoxidase staining with an anti-HSV-1 rabbit serum. At 24 h p.i., expression of HSV proteins was restricted to the portions of neurites and non-neuronal cells located in the trigeminal compartment of the system. As shown in Fig. 1
, at 48 h p.i. most of the neurites reaching through the diffusion barrier and corneal cells in direct contact with nerve terminals were intensively stained. HSV-positive corneal epithelial cells showed a cytopathic effect similar to that observed after direct infection.
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5. The dual-chamber system supports retrograde axonal spread of HSV-1 in chick neurites
To demonstrate retrograde axonal transport, the corneal compartment of the dual-chamber system was inoculated with 106 PFU HSV-1. When TG were dispersed and cocultivated with Vero cells 48 h p.i., infectious centers could be detected as soon as 24 h after cocultivation.
6. Effect of virus dose on retrograde axonal transport
The effect of virus dose on retrograde axonal transport was studied by infecting corneal epithelia with 101–106 PFU HSV-1, harvesting, and dispersing the TG explants at various intervals p.i., as indicated in Fig. 2
A, and detecting expression of HSV antigens by immunofluorescence. As controls, TG were directly infected accordingly. As displayed in Fig. 2B
, expression of HSV proteins in directly infected TG began at 12 h p.i. (1–3 positive cells per TG), reached a maximum at 36–48 h p.i. and later (
100 positive cells), and exhibited a dose dependency in the early phase (see the graphs for 104-106 PFU in Fig. 2B
). In contrast, retrogradely infected TG showed a low and fairly constant number of HSV-positive cells within the first 36 h p.i. irrespective of the inoculum, followed by a dose-dependent increase in the number of HSV-positive cells after 36 h p.i.
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CONCLUSIONS
In searching for an easy and generally applicable experimental approach that enables direct analysis of virus transfer between primary epithelial cells and sensory neurites, we investigated the spread of HSV-1 in a dual-chamber organ model using chick embryonic tissues. Here we present a novel organ model based on corneal epithelia and TG explants that supports anterograde axonal transport of HSV to epithelial cells as well as retrograde axonal transport to sensory neurons (Fig. 3
). Due to the efficient reinnervation of corneal tissue, our model appears to be well suited for the detailed analysis of viral retrograde axonal transport. Our data show that HSV-1 can reach the trigeminal ganglion by retrograde axonal transport after entering free nerve terminals without the need of prior replication in the corneal epithelium. These results suggest that the organ model presented in this study holds particular promise for the direct observation and molecular analysis of HSV spread between epithelia and sensory neurons and may be an attractive alternative to current experimental approaches based on laboratory animals or human fetal tissues.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0803fje; to cite this article, use FASEB J. (April 10, 2002) 10.1096/fj.01-0803fje. 
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