Abstract

The conjunctiva forms the mucosal lining of all mammalian eyelids and serves to protect and support the anterior portion of the eye and surrounding orbit. Recently, we have observed its close relationship with-associated lymphoid tissue (CALT) in the Florida manatee as an integral part of the immune system in this species.¹ Recent studies utilizing ultrastructural methodology have been used to understand and characterize CALT and EALT more fully.^2-4 The CALT/EALT of marine mammals have yet to be examined by electron microscopy. The present study examines ultrastructurally the conjunctiva and CALT in the Florida manatee, Trichechus manatus latirostris and represents the first examination of its kind on any marine mammal.

For this study, portions of the upper and lower eyelids (superior and inferior palpebrae) and third eyelid (nictitating membrane) from 3 animals were collected from the Marine Mammal Pathobiology Laboratory in St. Petersburg, Florida and were placed in either: 2% glutaraldehyde with 0.1M sodium cacodylate buffer, embedded in an epoxy resin, sectioned at 1 µm for semi-thin sections; or 70 nm for ultrathin sections; or 10% buffered formalin, embedded in paraffin, and sectioned at 5 µm. Ultrathin sections were stained by lead citrate and uranyl acetate. Paraffin sections were stained using either hematoxylin and eosin or Masson's trichrome. The animals in this study had died from various causes including acute and chronic complications from boat strikes, red tide exposure, and cold stress.

The conjunctiva and CALT were found to be identical in both the upper and lower eyelids. The conjunctiva was most pronounced in thickness towards the external margins of these eyelids possessing a well developed stratified squamous epithelium of 7–15 layers. Melanocytes infiltrated throughout much of the basal cell layer and became fewer in number towards the conjunctival fornix. Sporadically, the epithelium thinned to a few layers at locations where lymphoid tissue protruded towards the external surface. At these places, cells were observed with more electron-lucent cytoplasm than surrounding epithelial cells and had numerous endocytotic vesicles. We believe these cells to be M cells, similar to those described in the rabbit and dog.^1,2 The organization of lymphatic follicles was similar to that described in humans and rabbits.^2,4

With regard to the third eyelid, neither protrusions of follicular regions toward the ocular surface nor the similar presence of M cells within the epithelial lining were observed. As in the upper and lower eyelids, the epithelium became reduced to few layers having transitioned into pseudostratified to stratified columnar epithelium as it became confluent with the principal ducts of the nictitating gland. The epithelium along these largest ducts and those of the accessory glands of the upper and lower eyelids had prominent intercellular spaces, which appeared similar to examples of terrestrial spp that had associated inflammation with or without infection.⁴ While lymphoid tissue existed primarily in a diffuse manner, occasional clusters of lymphocytes were observed, having lied mostly next to secretory cells. Small lymph vessels were observed in these regions. Other solitary cells of defense, including macrophages, plasma cells, heterophils and eosinophils were encountered.

Acknowledgements

Thanks to the Marine Mammal Pathobiology Laboratory staff, especially Drs. Martine De Wit and Chris Torno for sample collection. This work was supported by a grant from the Florida Fish and Wildlife Conservation Commission. This study was conducted under Federal Fish and Wildlife Permit #MA067116-1 and IACUC protocol #200801803.

References

1.  McGee JL, Samuelson DA, Lewis PA, Lore D, Schwarz E, Greenbarg E, Goldbach K. Morphological and histochemical description of conjunctiva-associated lymphoid tissue (CALT) in the Florida manatee, Trichechus manatus latirostris. Intern Assoc Aquat Anim Med 39th Ann Conf 2008; 19–20.

2.  Giuliano EA, Moore CP, Phillips TE. Morphological evidence of M cells in healthy canine conjunctiva-associated lymphoid tissue. Graefes Arch Clin Exp Ophthalmol 2002; 240: 220–226.

3.  Knop N, Knop E. Ultrastructural anatomy of CALT follicles in the rabbit reveals characteristics of M-cells, germinal centres and high endothelial venules. J Anat 2005; 207: 409–426.

4.  Nichols B. Conjunctiva. Microsc Res Tech 1996; 33(4): 296–319. Review.