Macaque Bulbar Conjunctiva Lymphatic Dimensional Distribution, Functional Structure And The Relationship With The Drainage Of Aqueous Humor

Purpose: To investigate three dimension distribution of the conjunctival lymphatic vessels and its functional structure and the relationship to aqueous humor drainage ways.Materials and Method: Eight adult Chinese rhesus monkeys, 4-10kg in weight, four ofthem are female.Part I(1) Visualization by trypan blue0.1-0.2 mL of 1% trypan blue was injected by a 30-gauge needle at two depths of the conjunctiva in every different quadrant: just under the conjunctival epithelium (the superficial layer) and under the Tenon's capsule (the deep layer). The courses of injections were recorded by video-tapes under slitlamp.(2) Visualization by fluorescein sodium5% fluorescein sodium was injuected into the anterior chamber by a 30-gauge needle. The courses of injecting under slitlamp were recorded by video-tapes.(3) Double visualization by trypan blue and fluorescein sodiumSuccessively inject 1% trypan blue beneath the conjunctiva and 5% fluorescein sodium in anterior chamber thus to investigate the relationship of two drainage channels. Courses were recorded by video-tapes. Part II (1)Enzyme-histochemistry staining for whole-mount preparationsMonkeys were sacrificed by an overdosed injection of anesthetic and the bulbar conjunctivas were dissected along the limbus and the cul-de-sac in fornix, divided into 4 pieces according to quandrants, fully streched and fixed by 4%paraformaldehyde in 4℃ for about 24 hours. Redundant Tenon' capsule tissue was pruned off before processed to 5'-Nase-ALPase double staining.(2) Enzyme-histochemistry staining for conjunctiva sectionsMonkeys were sacrificed by an overdosed anesthetic and were fixed by perfusion. Full thickness pieces of globe wall were excised, including limbus, conjunctiva, sub-conjunctiva tissues and sclera, into 4 trapezia-shaped pieces according to quadrants. Cryosections for these tissuses were made and processed by 5'-Nase-ALP double staining.(3) Immunohistochemistry staining for conjunctiva sectionsCryosections made as enzyme-histochemistry staining were stained for VEGFR-3 by the ABC way, and counterstained by hematoxylin, mounted, observed under light microscope.All images for these staining were taken under Leica DFC 300FX, and analysed by Leica QWin V3.1 image processing system. Results:1. The width of rhesus monkeys' bulbar conjunctiva: 10.22 ± 0.80mm(superior), 8.0 ± 1.06mm(nasal), 9.0 ± 0.75mm(infenor), 9.94 ± 1.00mm(temple).2. The two-dimensional distribution of rhesus monkeys' bulbar conjunctival lymphatic vessels: Teichmann lymphatic ring can be seen parallel to the limbus. Fine vessles radiate from the limbus pass towards the peripheral lymphatic ring. The limbus area was manifest by crowded fine vessels, while typical large rings dominated the middle area and irregular vessels in the peripheral area (near the fornix).3. The direction of conjunctival lymphatic drainage: the superior injection dyes finally disappear in the superonasal or the superior ocular tissues; the temple injection dyes go separately from superior to the superonasal and from inferior to the inferonasal; the inferior inhection dyes terminated in inferonasal or inferior orbital tissues, the nasal injection dyes go separately from superior to superonasal and from inferior to the inferonasal.4. The three-dimensional distribution of conjunctival lymphatics: the bulbar conjunctival lymphatic vessels can be divided into the superficial layer and the deep layer, both of which lie in the conjunctival stroma, but not in the Tenon' capsule and the episclera.5. The functional structure of conjunctival lymphatics: (1) the blind ends— initial parts of the lymphatic vessels: can be seen everywhere, and less, thiner in the peripheral than the limbus and middle areas. (2) valve-like structure — can be found both in large vessels as the middle rings and fine vessels, including some blind ends.6. The fluorescein sodium pass from the anterior chamber and the trypan blue injected beneath the bulbar conjunctiva have no overlapped drainage vessles.Conclusions:1. The rhesus monkeys' conjunctiva contains ample lymphatic vessles, including Teichmann ring, radiating vessels near the limbus, the annular lymphatic vessels in the middle, and irregular network in peripheral. The conjunctival vessels are divided into two layers— fine lines in the superficial layer and typical large vessels in the deep layer, both lie in the stroma of conjuntival tissue. And no evidence shows the deeper tissues - Tenon'scapsule and the episcleral - have any lymphatioc vessles. And most of the conjunctival lymphatics were finally directed toward the nasal in to the deep orbital tissue while part of the superior vessels went down to the inferior and disappear in the deep orbital tissues. Blind ends and valves can be seen in every quadrant, from the limbus to the peripheral area. In normal, the conjunctival lymphatic vessels have no relationship to the aqueous drainage.2. Whole-mount preperations of conjunctiva precessed by 5'-Nase-ALP double specific enzyme staining is an optimal and useful method to study the conjunctival lymphatic vessels.3. The width of normal rhesus monkeys' bulbar conjunctiva decrease as follows: the superior, the temple, the inferior and the nasal.