Tracking the spread of a lacZ-tagged herpes simplex virus type 1 between the eye and the nervous system of the mouse: comparison of primary and recurrent infection

Abstract

The spread of herpes simplex virus type 1 (HSV-1) during primary ocular infection and after reactivation of latent infection in the trigeminal ganglion (TG) was examined in the mouse using a genetically modified virus containing the lacZ reporter gene under the control of the immediate-early 110 promoter. Whole tissue mounts of the eye and lids, their sensory nerves, and TG with the attached dorsal root entry zone (DRE) into the central nervous system (CNS) were stained for beta-galactosidase. Sixteen hours after inoculation of the cornea by scarification, staining was found in the scarified epithelium of the cornea and in the unscarified conjunctiva. By 24 h, staining was also seen in a few TG neurons and by 96 h their number had greatly increased and their distribution was more widespread. Stained cells (identified as Schwann cells by their staining for glial fibrillary acidic protein [GFAP] or S-100) in the TG were first seen close to stained neurons at 40 h, and by 48 h lines of such cells extended partway toward the periphery and toward the DRE. By 72 h, these lines had reached the periphery and the DRE where the adjacent CNS was also stained. In the cornea, stained cells with the morphology and arrangement of Schwann cells were seen from 40 to 120 h. After reactivation of latent infection, 10 of 22 samples had positively stained neurons. In eight samples, corneal and lid epithelial cells were stained. No stained Schwann cells were seen in the TG; however, branched networks of such cells were present in the cornea and the lids. This detailed sequential analysis has provided new information on the involvement of Schwann cells in the pathogenesis of primary and recurrent HSV-1 disease in the TG and the cornea.

FIG. 1

Diagrammatic representation of β-Gal staining in whole tissue mounts (ocular structures, their trigeminal nerve supply, the trigeminal ganglion, and its root in the brain stem) at different times after inoculation of the cornea with HSV-1 SC16 IE110 lacZ. (A) Key to diagram. In the mounted specimen, the part containing the conjunctiva, lids, and skin (lower right in panel A) remains attached to the ophthalmic nerve, but for clarity this is drawn separately. Similarly, in the specimen, the iris (lower center in panel A) is a part of the anterior segment but again, for clarity, this is drawn separately. TG1, ophthalmic part of the trigeminal ganglion; TG2, maxillary part; TG3, mandibular part; oph. n, ophthalmic nerve; max. n, maxillary nerve; man. n, mandibular nerve; B.S, brainstem; d.r.e, dorsal root entry zone; cor, cornea; ir, iris; p.m., pupillary margin; sk, skin; l.m, lid margin; i.p. con, inferior palpebral conjunctiva; s.p. con, superior palpebral conjunctiva. In panels B through F, the colored areas indicate cells or regions with β-Gal staining: red, individual neuronal cell bodies (where such cells were too numerous to count, the region involved is represented as a larger red area); blue dashed lines, Schwann cells; yellow, areas of staining in epithelial tissues (cornea, conjunctiva, skin) and iris; blue-green, staining in the CNS.

FIG. 2

(A) Histochemical detection of β-galactosidase and immunohistochemical detection of GFAP in mouse ophthalmic nerve 120 h after inoculation of the cornea with HSV-1 SC16 110 lacZ. Panel 1 shows a β-galactosidase (blue) and GFAP (brown) double-positive cell; panel 2 shows a β-galactosidase-positive cell stained as for panel 1, except that the anti-GFAP antibody was replaced by normal rabbit serum. (B) Histochemical detection of β-galactosidase in mouse tissues at different timepoints after similar inoculations (1 through 8). Expression was detected in the corneal epithelium (1) and the superior palpebral fornix of the conjunctiva (2) at 16 h after inoculation (the arrow marks nonspecific staining of meibomian glands), isolated neurons in the TG 24 h after infection (3), small groups of neurons, satellite cells, and Schwann cells in the TG 48 h after inoculation (4), large numbers of neurons, satellite cells and Schwann cells in the TG 72 h after infection (5), Schwann cells of the ophthalmic nerves 96 h after infection (6), the CNS at the DRE and Schwann cells in the PNS 120 h after infection (7) and the iris 120 h after inoculation (8) (arrow marks pupillary margin). (C) Histochemical detection of β-galactosidase in mouse tissues at different timepoints after reactivation of latent virus in the TG by UV irradiation of the cornea: a reactivating neuron in TG1 2 days after irradiation of the cornea (the arrow marks axonal staining) (1), a network of corneal nerve Schwann cells 2 days after UV irradiation (the inset shows apparent nuclear staining in Schwann cells [arrows], at higher magnification) (2), a dendritic lesion of the corneal epithelium 3 days after irradiation (3), and a lesion of the eyelid margin 3 days after irradiation (4).

FIG. 3

Ophthalmic nerve 120 h after inoculation of HSV-1 SC16 IE110 lacZ. N, uninfected nucleus of myelinating Schwann cell; A, myelinated axon; IN, nucleus of unmyelinated Schwann cell with marginated chromatin. The inset shows area of infected nucleus at higher magnification. Arrows indicate virus capsids.