rat limbal vasculature

7/30/2019 Rat Limbal Vasculature

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Limbal Microvasculature of the Rat EyeJohn C. Morrison*^ F. W . Fraunfelder,* Scott T. Milne* and Chester G. Moore* f

Purpose. To define the microvascular anatomy of anterior segment blood supply and aqueousdrainage in the rat eye.Methods. External limbal blood vessels were studied by direct inspection of normal, living rateyes and eyes injected intracamerally with fluorescein dye. Intraocular connections of thesevessels were then docum ented with scanning electron microscopy of methylmethacrylate m i-crovascular luminal castings.Results. The rat limbus possesses a circumferential vascular ring consisting of a single arteryand a venous plexus. The limbal artery communicates with radial anterior ciliary arteries andwith perforating arterioles arising from the long posterior ciliary arteries. The venous plexus isconnected to a circumferential Schlem m's canal by num erous transcleral , aqueous-containingcollector channels and drains into multiple radial veins located within die episclera.Conclusions. These findings illustrate anatomic similarities between rats and primates, both inanterior segment blood supply and aqueous humor drainage. The rat l imbal artery providescollateral perfusion of the anterior segment from anterior and long posterior ciliary systems.The direct commun ications between identifiable external aqueous-containing veins, a circum-ferential episcleral venous plexus, and an internal Schlemm's canal provides the anatomicbasis for producing chronically elevated intraocular pressure in rats using retrograde injectionof mild sclerosing agents into the aqueous humor outflow pathways, and for administeringdrugs and other agents to the entire trabecular meshwork and Schlemm's canal. Invest Oph-thalmol Vis Sci. 1995;36:751-756.

M Co nti nu ed progress in understanding glaucomatousJJ optic nerve damage relies on the development of ap-propr iate and cost-effective animal models. AlthoughJ, analyses of enucleated huma n glaucomatous eyes haveprovided much information,1'2 studies in live monkeyswith experimentally elevated intraocular pressure


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75 2 Investigative Ophthalmology & Visual Science, March 1995, Vol. 36, No. 3

FIGURE l. In vivo photograph of rat l imbus showing anteriorciliary artery (asterisk) and aqueous-containing veins (arrows)supplying limbal artery and venous plexus, respectively.Original magnification, X60.

drainage in the rat eye, two major determinants ofaqueous humor dynamics and intraocular pressure.MATERIALS AND METHODSAll experiments were performed in accordance withthe ARVO Statement for the Use of Animals in Oph-thalmic and Vision Research. Twenty adult brown Nor-way and albino Lewis rats were anesthetized by intra-peritoneal injection of a 1.5 ml/kg solution con-taining 5 ml ketamine (100 mg/ml), 2.5 ml xylazine(20 mg /ml), 1 ml acepromazine (10 mg/ml), and 1.5ml sterile water. All animals were examined with aWild M3Z dissecting microscope (Wild Leitz; Heer-brugg, Switzerland) to assess general features of theexternal limbal vasculature. The anterior chambersof four animals were injected with a 1:20 dilution offluorescein dye using a 32-gauge needle. The patternof dye drainage from the anterior chamber was thendocumented using a slit lamp camera equipped witha medium SE-40 blue excitor filter.

For microcorrosion casting studies, methylmeth-acrylate casting medium was prepared by diluting Bat-son's #17 base (Polysciences, Warrington, PA) with anequal volume of methylmethacrylate monomer (Aid-rich, Milwaukee, Wl). This mixture was divided intotwo separate containers, adding catalyst to one andpromoter to the other as previously described.24 Theabdominal aortas of 16 rats were isolated and cannu-lated retrogradely with 20-gauge catheters securedwith 5-0 silk sutures. The separate catalyst and pro-moter mixtures were then combined, mixed for 30seconds, and aspirated into plastic 60-ml syringes.Casting medium was injected by moderate thumb

FIGURE 2. Rat limbus after injection of fluorescein dye intothe anterior chamber. Unfilled l imbal artery stands outagainst the dye-filled limbal venous plexus (arrowheads). Dye-filled, radial aqueous-containing veins are also visible(arrows). Original magnification, X15.

pressure into the abdominal aorta, and the right auri-cle was punctured to allow efflux of blood and plasticfrom the head. The corneas were also incised beforeinjection of casting medium to reduce intraocularpressure an d maximize filling of intraocu lar bloo d ves-sels. Incisions did not exceed 2.5 mm. Injection wascontinued until a noticeable increase in viscosity oc-curred.After allowing 1 hou r for polymerization, the eyeswere enucleated and placed in 4% paraformaldehydefor 24 hours. Th e tissue was then digested in 6 M KOHsolution overnight at 60C, and the resultant castingswere gently washed with distilled water and allowedto air dry. Castings were then mounted, coated withgold palladium (40 nm), and examined under anAMR 1000 scanning electron microscope (Amray;Bedford, MA). Scanning electron microscopy and se-quential microdissection2' were employed to docu-ment the details of the limbal blood vessels and theirintraocular connections. Casts of arteries and veins

were identified by their distinctive endothelial nuclearimprint pattern, the former appearing more fusiformand linear.RESULTSDirect observations revealed a perilimbal vascular ringcomposed of a single artery and a venous plexus in allanimals (Fig. 1). The limbal artery was supplied exter-nally by anterior ciliary arteries located in the episclera.The venous plexus was drained posteriorly by severalradial veins within the episclera and conjunctiva. Fluores-cein dye injected into the anterior chamber escaped


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Limbal Microvasculature of the Rat Eye 75 3

FIGURE 3. External view of microvascular casting demonstra-ting limbal vascular ring composed of single artery (arrow)and venous plexus (arrowhead), the latter connecting toaqueous-containing v i i s (AV). Asterisk indicates capillarybed, which is connected to the limbal venous plexus. Castingorientation conforms to orientation of Figures 1 and 2. Orig-inal magnification, X90.

rapidly into the limbal and episcleral veins, illustratingthe importance of these vessels as a route for aqueoushumor outflow (Fig. 2). The limbal artery did not fillwith fluorescein and appeared in silhouette against thedye-filled limbal veins.Scann ing electron microscopy of methylmethacry-late luminal cast ings confirmed the pre sence of a sin-

FIGURE4. Limbal artery (arrow) connects internally with thelong posterior ciliary artery (asterisk) through a perforatingarteriole near the origin of the major arterial circle. Thispartially dissected specimen also shows the relationship ofSchlemm's canal (SC) and limbal venous plexus (arrow-heads) to limbal and long posterior ciliary arteries. Originalmagnification, X90.

gle limbal artery with th e venous plexus (Fig. 3). Care-ful microdissection revealed intraocular connectionsof the limbal artery to the long posterior ciliary arter-ies in the form of limbal perforating arterioles (Fig.4) . These perforators, two per eye, were consistentlyfound nasally and temporally, just before bifurcationof the long posterior ciliary arteries into the majorarterial circle.Externally, the multiple channels of the limbalvenous plexus were found adjacent to the limbal artery(Fig. 3). Internally, careful microdissection revealed acast of Schlemm 's canal, which appea red as a flattenedchann el lying deep to the limbal plexus but external tothe iris vessels (Fig. 4). Numerous transcleral collectorchannels were found connecting Schlemm's canal tothe limbal venous plexus (Fig. 5), providing an ana-tomic pathway for intracameral fluid to gain access tothe episcleral limbal veins.All castings without obviousfillingdefects revealedthat the venous plexus was a complete ring aroundthe entire limbus (Fig. 6A). The extremely delicatenature of the casting prevented the successful isolationof the Schlemm's canal in its entirety, although cir-cumferentially oriented portions, often several clockhours in length, were frequently noted (Fig. 6B).No major differences from these overall patterns,summarized in Figure 7, were noted among any of dieanimals and castings studied. No differences in eitherblood supply or aqueou s humo r outflow patfiways wereseen between pigmented and nonpigmented animals.DISCUSSIONAlthough specific descriptions of rat ciliary processcapillary beds and the trabecular meshwork exist, this

FIGURE 5. Anterior view of casting demonstrates Schlemm'scanal (SC) communicating with the limbal plexus (asterisk)through a transcleral collector channel (arrow). Originalmagnification, X375.


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75 4 Investigative Ophthalmology & Visual Science, March 1995, Vol. 36, No. 3is the first overall study of the major blood vesselsresponsible for serving the anterio r segment of the rateye. We have defined a circumferential limbal vascularplexus that has arterial and venous components andoffers the potential for affecting aqueous humor for-mation and drainage.Although prepared under nonphysiologic condi-tions, the casts illustrated here do not show significantartifacts offillingor plastic extravasation. As such, theyshow the potential for blood and aqueous flow, andthe anatomic possibilities represented allow specula-tion on their significance.Although previous work has dem onstrated severalsimilarities in the microvasculature of rat and primateciliary processes/1' our observations indicate that rats

FIGURE 6. (A) Montage of a single, complete, circumferentiallimbal venous plexus with numerous aqueous-containingveins (arrows). Original magnification, xlf>. (B) Magnifiedview of lower right-hand corner of montage in 6A, demon-strating a segment of'Schlemm's canal (arrows), lying inter-nal and parallel to the limbal plexus (arrowheads). Originalmagnification, X55.

LPCAAV

ACAFIGURE 7. Diagram of the rat limbal microvasculature, illus-trating the association of arteries (light outline) and veins(heavy outline). Limbal artery (arrows) is supplied by ante riorciliary arteries (ACA) and long posterior ciliary arteries(LPCA), providing collateral blood supply for the iris (I)and ciliary processes (CP). Venous plexus (asterisk) is con-nected to Schlemm's canal (SC) through multiple collectorchannels (arrowheads) and drains into radial aqueous-con-taining veins (AV).possess further similarities to primates with regard tooverall anterior segment blood supply. In rats, thearterial component of the limbal vascular plexus, thelimbal artery, is supplied externally by anterior ciliaryarteries. In addition, the limbal artery communicateswith the intraocular long p osterior ciliary arteries justbefore bifurcation into the major arterial circle. Be-cause arterioles for die iris and ciliary processes arisefrom the major arterial circle, the rat anterior uveahas the potential, thro ugh the limbal artery, to receivecollateral blood supply from the anterio r and the longposterior ciliary arteries. This is analogous to pri-mates27 and reflects the importance of maintainingadequate ciliary body perfusion under varying physio-logic and pathologic conditions.28"32

Conventional light and electron microscopic stud-ies have previously demonstrated that rats possess anidentifiable Schlemm's canal and trabecular mesh-work with an ultrastructure similar to that of pri-mates.33"35 Our methylmethacrylate castings not onlyconfirm the presence of a Schlemm's canal in rats,they illustrate the overall anatomy of aqueous humoraccess to the general circulation.Lowering eye pressure before injecting methyl-methacrylate, a techniqu e successfully used to dem on-strate aqueous humor outflow pathways in other spe-cies,36 allowed successful retrograde filling ofSchlemm's canal, which appears to be an extensiveand apparently complete channel. The connection ofSchlemm's canal, through transcleral collector chan-nels, to the episcleral venous plexus and then to radialepiscleral veins confirms tha t the rat trabecular mesh-work and Schlemm's canal are responsible for at leasta portion of aqueous humor drainage, analogous to


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Limbal Microvasculature of the Rat Eye 75 5the "conventional" route of aqueous humor outflowin primates.37The concentric, interconnected Schlemm's canaland limbal venous plexus shown in this study presenta double, circular drainage system for aqueous hum orinto the general circulation. This offers the potentialof introducing agents to the entire trabecular mesh-work through a retrograde injection into a singleaqueous-containing vein. Using a specially designedmicroneedle, we recently dem onstrated the feasibilityof such an injection using hypertonic saline.38 Thismethod produces scarring of aqueous humor outflowpathways, resulting in prolonged elevation of IOP anda new potential model of chronic IOP-induced opticnerve damage in rats.Key Wordstrabecular meshwork, animal glaucoma model, rats,Schlemm's canal, major arterial circleReferences

1. Quigley HA, Green WR. The histology of human glau-coma cupping and optic nerve damage: Clinicopatho-logic correlation in 21 eyes. Ophthalmology. 1979;86:1803-1827.2. Quigley HA, Addicks EM, Green WR, Maumenee AE.Optic nerve damage in human glaucoma: II: The siteof injury and susceptibility to damage. Arch Ophthal-mol. 1981;99:635-649.3. Anderson DR, Hendrickson AH. Effect of intraocularpressure on rapid axoplasmic transport in monkeyoptic nerve. Invest O phthalmol Vis Sci. 1974;13:77l -783.4. Minckler DS, Bunt AH, Klock IB. Radioautographicand cytochemical ultrastructu ral studies of axoplasmictransport in the monkey optic nerve head. Invest Oph-thalmol Vis Sci. 1978; 17:33-50.5. deKater AW, Smyth JRJr, Rosenquist RC, Epstein DZ.The slate turkey: A model for secondary angle closureglaucoma. Invest Oph thalmol Vis Sci. 1986; 27:1751-1754.6. Samuelson DA, Gun n GG, Gelatt KN. Ultrastructuralchanges in the aqueous outflow apparatus of beagleswith inherited glaucoma. Invest Ophthalmol Vis Sci.1989:30:550-561.7. Wyman M, Ketring K. Congenital glaucoma in thebasset hound: A biologic model. Trans Am Acad Oph-thalmol Otolaryngol. 1976;81:OP645-OP651.8. Bunt-Milan AH, Dennis MB Jr, Bensug er RE. Opticnerve head axonal transport in rabbits with hereditaryglaucoma. Exp Eye Res. 1987;44:537-551.9. Podos SM. Animal models of human glauco ma. TransAm Acad Ophthalmol Otolaryngol. 1976;81:OP632-OP635.10. Shields BM. A Study Guide for Glaucoma. Baltimore:Williams & Wilkins; 1982:373-375.

11 . Quigley HA, Hoh man RM. Laser energy levels for tra-becular meshwork damage in the primate eye. InvestOphthalmol Vis Sci. 1983;24:1305-1306.

12. Gherezghiher T, March WF, Nordquist RE, Koss MC.Laser-induced glaucoma in rabbits. Exp Eye Res.1986; 43:885-894.13. Quigley HA, Addicks EM. Chronic experimental glau-coma in primates: I: Production of elevated intraocu-lar pressure by anterior chamber injection of autolo-gous red blood cells. Invest Ophthalmol Vis Sci.1980;19:126-136.14. Knep per PA, Breen M , Weinstein HG , Black LJ. In-traocular pressure and glycosaminoglycan distribu-tion in the rabbit eye: Effect of age and dexametha-sone. Exp Eye Res. 1978; 27:567-575 .15. Levene RZ, Rothberger M, Rosenberg S. Corticoste-roid glaucoma in the rabbit. Am J Ophthalmol.1974; 78:505-510.16. Bonomi L, Perfetti S, Noya E, Bellucci R, TomazzoliL. Experimental corticosteroid ocular hypertension in

the rabbit. Graefe's Arch Clin Exp Ophthalmol. 1978;209:73-82.17. Francois J. Corticosteroid glaucoma. Ophthalmologica.1984; 188:76-81.18. Zhan GI, Miranda OC, Bito LZ. Steroid glaucoma:Corticosteroid-induced ocular hypertension in cats.Exp Eye. Res. 1992; 54:211-218 .19. Thorpe RM, Kolker AE. A tonographic study of waterloading in rabbits. Arch Ophthalmol. 1967; 77:238.20. Bonomi L, Tomazzoli L, Jaria D. An improved m odelof experimentally induced ocular hypertension in therabbit. Invest Ophthalmol. 1976;15:781-784.21 . Kinnear A, Lauber JK, Boyd TAS. Genesis of light-induced avian glaucoma. Invest Ophthalmol. 1974;13:872-875.22. Moore CG, Milne ST, Morrison JC . Non-invasive mea-surement of rat intraocular pressure with the tono-pen. Invest Ophthalmol Vis Sci. 1993;34:363-369.23. Mermoud A, Baerveldt G, Minckler DS, Lee MB, RaoNA. Intraocular pressure in Lewis rats. Invest Ophlhal-mo l Vis Sci. 1994;35:2455-2460.24. Fahrenbach WH, Bacon DR, Morrison JC , Van BuskirkEM. Controlled vascular corrosion casting of the rab-bi t eye. J Electron Microsc Technique. 1989; 10:15-26.25. Morrison JC, Van Buskirk EM. Sequential microdissec-tion and scanning electron microscopy of ciliary mi-crovascular castings. Scanning Electron Microsc. 1984;2:857-865.26. Morrison JC, DeFrank MP, Van Buskirk EM. Comp ara-tive microvascular anatomy of m amma lian ciliary pro-cesses. Invest Ophthalmol Vis Sci. 1987; 12:1325-1340.27. Morrison JC, Van Buskirk EM. Anter ior collateral cir-culation in the primate eye. Ophthalmology. 1983;90:707-715.28. OlverJM , Lee JP. Recovery of anterio r segm ent circu-lation after strabismus surgery in adult patients. Oph-thalmology. 1992;99:305-315.29. OlverJM, LeeJP. The effects of strabismus surgery onanterior segm ent circulation. Eye. 1989;3:318-326.30. Hayreh SS, Scott WE. Fluorescein iris angiography: II:Disturbances in iris circulation following strabismusoperation of the various recti. Arch Ophthalmol.1978; 96:1390-140 0.31. Fishman PH, Repka MX , Green WR, D'Anna SA, Guy-


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75 6 Investigative Ophthalmology & Visual Science, March 1995, Vol. 36 , No. 3ton DL. A primate model of anterior segment isch-emia after strabismus surgery: The ro le of the conjunc-tival circulation. Ophthalmology. 1990;97:456-461.

32. Talusan ED, Schwartz B. Fluorescein angiography:Demo nstration of flow pattern of anterior ciliary arter-ies. Arch Ophthalmol. 1981:99:1074-1080.

33 . Tripathi RC. Ultrastructure of the exit pathway of theaqueous in lower mammals. Exp Eye Res. 1972; 12:311 -314.

34. van Der Zypen E. Experimental morphological studyon structure and function of the filtration angle ofthe rat eye. Ophthalmologica. 1977; 174:285-298.35 . McMenamin PG, Al-Shakarchi. The effect of various

levels of intraocular pressure on the rat aqueous out-flow system. J Anat. 1989; 162:67-82.36. Van Buskirk EM. The canine eye: The vessels of aque-ous drainage. Invest Ophthalmol Vis Sri. 1979; 18:223-

290.37 . Shields MB. Aqueous humor dynamics: I: Anatomyand physiology. In: Shields MB. Textbook of Glaucoma.

3rd ed. Baltimore: Williams & W ilkins; 1992:15-36 .38. Moore CG, Milne S, Johnson EC, Morrison JC. A ratmodel of pressure-induece optic nerve damage.ARVO Abstracts. Invest Ophthalmol Vis Sri. 1993;34:1141.

Announcement

Call for N ominations for the Jin H . Kinoshita LectureshipThe National Foundat ion for Eye Research, an organizat ion dedicated to the sup-port of cataract research, is pleased to ann ou nce a call for no min at ion s for th e th irdJin H. Kinoshi ta Lectureship ( hon orar ium , $15,000) to be given at the Intern at ion alCooperat ive Cataract Research Group meeting in Kallua-Rona, Hawaii , November1 5 - 1 9 , 1995. Nominat ions should include a curriculum vi tae and a support inglet ter. The Nominat ion Committee consists of Suguru Fukushi , MD; Joseph Hor-witz , PhD; Venkat N. Reddy, PhD; and Abraham Spector, PhD, Chair.

The deadl ine for nomina t ions is June 1, 1995. Please send nominat ions to:Dr. Abraham SpectorDepar tment of Ophtha lmologyCollege of Physicians & Surgeons of Columbia University630 West 158th StreetNew YorkNY 10032

rat limbal vasculature

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