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Neuropeptides and the Innervation of the Avian Lacrimal Gland

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Neuropeptides and the Innervation of the Avian Lacrimal Gland Investigative Ophthalmology & Visual Science, Vol. 30, No. 7, July 1989 Copyright © Association for Research in Vision and Ophthalmology Neuropeptides and the Innervation of the Avian Lacrimal Gland Denjomin Wolcorr,* Patrick A. 5ibony,j- and Kent T. Keyser^: The chicken Harderian gland, the major lacrimal gland, has two major cell populations: a cortical secretory epithelium and a medullary interstitial cell population of lymphoid cells. There is an exten- sive acetylcholinesterase (AChE) network throughout the gland, as well as catecholamine positive fibers among the interstitial cells. There are substance P-like (SPLI) and vasoactive intestinal poly- peptide-like (VIPLI) immunoreactivite fibers throughout the gland. These fibers are particularly dense and varicose among the interstitial cells. The adjacent pterygopalatine ganglion complex has neuronal somata that exhibit VIPLI and were AChE-positive. This ganglion complex also contains SPLI and catecholamine-positive fibers. In regions of the ganglion, the somata appear surrounded by SPLI varicosities. Surgical ablation of the ganglion eliminated or reduced the VIPLI, AChE and catecholamine staining in the gland. The SPLI was reduced only in some regions. Ablation of the superior cervical ganglion or severance of the radix autonomica resulted in the loss of catecholamine staining in the pterygopalatine ganglion and the gland. Severance of the ophthalmic or infraorbital nerves had no effect on the VIPLI or the SPLI staining pattern in the gland. Invest Ophthalmol Vis Sci 30:1666-1674, 1989 The avian Harderian gland is the major source of further investigation. Using immunohistochemical serous fluid1 and immunoglobulins2 in tears. Like the techniques and surgical ablations, the source, pat- mammalian lacrimal gland, it is innervated by both terns and regional distribution of cholinergic, adren- sympathetic and parasympathetic nerve fibers.34 The ergic and neuropeptide innervation of the chicken avian gland differs from the mammalian lacrimal Harderian gland were studied. gland in the number and distribution of its lympho- cytes. Where lymphocytes are scattered throughout Materials and Methods the interstitial connective tissue of the mammalian gland, the avian gland has a larger concentrated pop- Adult white leghorn chickens weighing 2-4 kg were ulation of lymphoid cells located in the central re- used in all the experiments and treated in accordance gions or medulla of the gland.4"6 In pigeons, the me- with the ARVO Resolution on the Use of Animals in dulla also contains a dense network of amine-positive Research. All surgical manipulations were carried out and acetylcholinesterase (AChE)-positive nerve under deep anesthesia induced by intramuscular in- fibers.4 The occurrence of axon terminals in proxim- jection of 2-3 ml of a 1:1 mixture of ketamine and ity to lymphocytes within this region has led to the xylazine. suggestion that these autonomic nerve fibers may The Harderian gland was exposed by making a modulate lymphocyte activity and thus influence the vertical incision through the rostral third of the lower secretory immune system. However, before studies eyelid and an inferior conjunctival peritomy. In order confirming this hypothesis can be undertaken, the to facilitate the exposure, the vitreous was aspirated, innervation of the chicken Harderian gland requires the globe was gently retracted superiorly and with blunt dissection, using an operating microscope, the Harderian gland was exposed along the inferomedial From the Departments of *Anatomical Sciences, fNeurology orbital wall. Ablation of the various structures de- and "("Ophthalmology, School of Medicine, State University of New York, Stony Brook, Long Island, New York, and the ^Department scribed below was accomplished by surgical transec- of Neuroscience, School of Medicine, University of California at tion or cautery. The radix autonomica is found along San Diego, La Jolla, California. the superior margin of the gland and the pterygopala- Supported by NIH grant NS-19350. tine ganglion is a small bulbous enlargement at the Submitted for publication: December 2, 1985; accepted De- rostral end of the radix autonomica. While ablation cember 27, 1988. Reprint requests: Dr. Benjamin Walcott, Department of Ana- of the radix autonomica was easily visualized with the tomical Sciences, School of Medicine, H.S.C., State University of operating microscope, destruction of the pterygopal- New York, Stony Brook, NY 11794-8081. atine ganglion required histological confirmation be- 1666 Downloaded from iovs.arvojournals.org on 09/23/2021 No. 7 AVIAN LACPJMAL GLAND / Walcorr er ol 1667 cause of its small size. The ophthalmic nerve and the nuclear Corp., Stillwater, MN) were diluted 1:1000 infraorbital nerve were exposed and destroyed proxi- with phosphate-buffered saline (PBS) containing mal to the anterior margin of the gland. The animals 0.25% Triton X-100. Twelve micron frozen sections were sacrificed 8 to 9 days following ablation proce- were thaw mounted on gelatine coated slides and in- dures and in all instances the gland, ganglion and cubated with the primary antisera overnight at room radix autonomica were studied. The contralateral temperature. After washing with 1% BSA in PBS for gland, radix autonomica and ganglion were processed 30 min the sections were exposed for 1 hr to the in parallel and served as controls. The source of appropriate secondary antiserum conjugated to ei- amine-positive fibers was studied by surgically re- ther fluorescein isothiocyanate (FITC) or rhodamine. moving the left superior cervical ganglia in six (1-5- After washing with Millonig's buffer, the slides were day-old) chicks. Controls included four additional coverslipped using a glycerol/buffer media. chicks who underwent sham surgery; the superior Controls for specificity consisted of "blocks" in cervical ganglion was exposed but not removed. Fol- which the primary antiserum was reacted overnight lowing this procedure, the sympathectomized chicks with an excess of the pure peptide (10~6 M) before its exhibited a persistent ipsilateral ptosis. These chicks application to tissue sections. Nonspecific primary were sacrificed and studied 150 days later. antisera or the omission of the primary antisera fol- The normal histology of the gland and the pterygo- lowed by the secondary antisera were used as addi- palatine ganglion were examined using 3-4 nm thick tional controls. Controls were processed simulta- sections of plastic embedded tissue after perfusion neously with the experimental tissue sections. The through the ascending aorta with 4% paraformalde- sections were examined and photographed with a hyde or 2.5% glutaraldehyde in 0.1 M phosphate Zeiss epifluorescence compound microscope buffer (pH 7.4). Tissue was post fixed in the same equipped with a 50 W mercury burner. fixative for 2-4 hr. After dehydration with an alcohol series, the tissue was embedded in glycol methacry- Results late, JB-4 plastic (DuPont-Sorvall, Wilmington, DE). Harderian Gland Sections were stained with hematoxylin and eosin and then coverslipped. The chicken Harderian gland is a lingual-shaped merocrine gland located on the ventro-medial surface Acetylcholinesterase of the orbit. It extends from the orbital apex near the optic nerve to the rostral angle of the orbit where a The distribution of acetylcholinesterase in the solitary duct drains into the medial fornix of the nic- gland and pterygopalatine ganglion was studied using 7 8 titating membrane. It is bounded laterally by the a modification of the Coupland-Holmes technique. globe and medially by periorbita and bone. The gland For controls a specific AChE inhibitor, BW284C51 (5 X 10"5 mM) (Sigma Chemical Co., St. Louis, MO) is surrounded by a thin connective tissue capsule; was added to the "preincubation" and incubation so- fibrous septae separate it into many tubulo-acinar lution of one half of the slides. Extraocular muscle containing lobules (Fig. 1). The secretory acini, con- served as a positive control. sisting of columnar epithelium, are located in the pe- ripheral or cortical regions of the gland. The central region of the gland or the medulla consists of smaller Histofluorescence and Immunocytochemical tubules that drain into a large central duct. The more Methods distal tubules and central duct are surrounded by a To localize the catecholamine-containing fibers, 12 large number of plasma cells and lymphocytes. Nu- nm frozen sections of unfixed tissue were treated with merous capillaries and small numbers of fibroblasts the glyoxylic acid method.9 can also be found in this region of the gland. Arteri- Substance P-like immunoreactivity (SPLI) and va- oles are restricted to the surface of the gland and the soactive intestinal polypeptide-like immunoreactiv- interlobular septae. ity (VIPLI) were studied in Harderian gland frag- AChE-positive nerve fibers were present through- ments that were immersion-fixed in a solution con- out the gland, particularly in the interstitial connec- sisting of 4% paraformaldehyde, 0.1 M phosphate tive tissue along the small tubules draining each lob- buffer (pH 7.4), 0.01 M sodium periodate and 0.1 M ule (Fig. 2). These fiber bundles extend from the cap- D, L-Lysine for 4 hr and then cryoprotected over- sule to form a network of fibers in the medulla of the night in buffered 30% sucrose. Both monoclonal rat gland. AChE-positive fibers surrounding epithelial antiserum to substance P (SP) (Accurate Biochemi- acini were uncommon. Pretreatment with a specific
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