The mechanism of aqueous outflow in birds

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(1973) 15, 425-431

The Mechanism of Aqueous Outflow in Birds II. An Ultrastructural

Study of Perfused Eyes

.In order to detect the route of flow of the large molecules of the aqueous humour, a rolloidally suspended electron-dense tracer (Thorotrast-particle size approx. 10 nm) was injected in vivo into the ant.erior chambers of chicken eyes at a controlled intraocular pressure. After about. 20 min. the eyes mere fixed and processed for electron microscopy. It M-as observed that almost all vacuoles in the endothelial lining of the aqueous sinus were filled with the tracer element from their basal openings but a leakage of this material into the lumen of the aqueous sinus was seen only from a small proportion of vacuoles whirl] have openings on the luminal aspert. Xo leaka,ge of thr tracser element occurred, however. across the endothelial cell junctions and the majorit’!of micropinocytotic vesicles were devoid of tracer part’icles. Vsriahle concentrations of the tracer particles were also seen in the lumen of the aqueous sinus and the intraseleral collector channels. It is concluded that the porosity of the endothelial lining of the aqueous sinus is dependent upon the dynamic proc’ess of endothelial vacuolation and t’hat the bulk transfer of the aqueous humour takes place via the transient t,ranscellular channels created during this vacuolation cycle. Reference has heen made to the factors which could possibly influence this process. A close similarit,y of this mechanism of aqueous outflow in the eyes of birds to that of primates (Tripathi, 1969a,b, 1971a) and lower mammals (Tripathi and Tripathi. 1972) indicates that the dynamic process of endothelial vacuolation is a fundamental biological process, probabl! common to the eyes of many vertebrates.

1. Introduction the previous paper (Tripathi and Tripathi, 1973) we have described the structural and ultrastructural morphology of the angular ayneous sinus/plexus in the eyes of chicken. pigeon and snow goose. It is concluded that the endothelial lining of t,hrr angular aqueoussinusis a continuous singlelayered membrane and the giant vacuoles. especially those at the transcellular stage. may normally have an important role to play in the bulk transfer of the aqueous humour. The ainl of the present study is to provide experimental evidence a’sto the morphogenesisand functional significanccb of giant vacuoles and also to evaluate whether or not large moleculesof the aqueous humour can gain accessinto the lumen of the aqueous sinus by micropinocytotic transport or by seepageacrosst,he intercellular junctions of the endothelial cells. In this study, w-e have investigated the fatz of an cllectron-dense tracer of colloi~lal In

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ways through complex epithelial and endothelial layers) following an intracamer;rl injection at a physiological level of intraocular prwsuw. 2. Materials and Methods I:n(ler Sembutal slowlv over a period stahiiized colloidal using a narrow bore thus aclueons hntnour

anaesthesia. the anterior chambers of six chicken eyes were inject,etI of 5 tnin with ()*I:5 ml of an rlrc.tr.on-dense tracer (Thorotrast ~dT,“,, thorium dioxide in aqueous Dcxtrin. particle size approx. II I tmi ! needle. To avoid any rise of intraocular pressure, the same volume of was sinllllt~;l~~eo~1sl~ \vithclr;rwu front t#he anterior cha,mher by ;I

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syringe attached to a. second nwrrow how neetlle. .After wIlout 20 ftlilh the anini;il b;b killed with an overdose of Nernbutwl. The eyes were enucleated and fixed irnnrrtliatcl! ill

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fixation, dehydration and embedding powsses. ilyitation was ;~voiflrd 11~careful ha wlii~ly a.nd changing of reagents. The tissue hlorks were prwessecl anti scc+iorrrtl for f.lc.c~tr~I~l nlicroscopp as described in t,he prrvious pitper (Tripathi a.1~1Tripathi. 1973). Onlv swticrllh from the central region of each block were weepted for analysis of the results. ‘Elrc.trcll)nlicrographs were taken using all ;\EI E.\It; electrcw micwscope. 3. Results Semi-thin sections of the angular region stained with toluidine blue and examined Ijy light microscopy reveal that the morphological organization of the angular aqueous sinus, its endothelial lining and t.he giant vacuoles is closely comparahlu to that seen in normal eyes (F’g. I). lJltmstru.cture

Thin sections examined by electron microscops reveal a morphological organization similar to that present in normal eves. It’ is most, remarkable to note, however, t,hat almost all electron optically empty spaces in and around the a,ngular aqueous sinus seen in normal eyes are now filled with a variable concentration of the tracer element,. The concentration is heavy in t,he supporting tissue zone of t,he inner aspect

FIG. 1. Photomicrograph of the aqueous sinus (.4S) of a chicken eye following intracameral fusion with an electron dense tracer; arrows point to prominent vacuoles in the endothelial lining. the normal morphological organization of this region. TM, Trabecular meshwork; A, artery; acleral leaf; C, Crampton’s muscle. ( : 665)

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PK. 2. tiurvey el~,ctronmicrograp~l of the inner wall perfusion in chicken eye. Note the concent)ration of the of t,hv entlothelial meshwork and the giant vacuoles (C) cytic irwlusiona of the tracer vlemmt ill a mwrophitye.

of the squeous sinus (AS) fullowing intrecamwal el&ron-dense tracer element in t,he open spacer: of the cndothelial lining. Arrow point to ph;lg,,. ( . MOIII

of the aqueous sinus (Fig. 2). In further contrast8 to normal eyes, the majority of thta vacuoles in the endothelial cells lining the inner wall of the aqueous sinus are filled with tracer element (Figs 2-5). In some instances, the pseudopodia of wandering cells are ,seen lodged within a vacuole (Fig. 6) or partially or completely occluding the basal or luminal openings of the vacuoles (Fig. 7). It, is rare to see vacuoles with luminal openings only and in such cases the vacuoles present a somewhat collapsed profile and appear relatively empty (Fig. 8). Variable concentrations of tracer element are seen in t,he septae bridging the sinus and in the connective tissue surrounding the artery located within the septum; no particles of tracer element are; however, seen within bhe lumen of the artery. Small concentrations of the tracer element are also seen in some regions of the scleral leaf and supporting connective tissue zone of the scleral wall of the sinus. There is, however, an increased concentration in some regions especially where occasional giant vacuoles are to be seen. Rarely, a macrophage is also seen partly trapped within a vacuole (Figs 9 and 10). A relatively small but variable concentration of the tracer element8 is seen in the lumen of the aqueous sinus (Figs 3-10) and some collector channels (Fig. 11). In comparison with the giant vacuoles, the micropinocytotic vesicles in the endothelial cells lining the aqueous sinus rarely contain tracer particles (Figs 3-5 and 7). The occluding zonules between adjacent endothelial cells are intact and no leakage of the tracer element is seen across hhe intercellular junction (Figs 6 and 7). 3t

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Fra. 6. Electronmicrograph of the inner wall of the aqueous sinus (AR) following intracameral per. fusion in chicken eye. The gia& vacuole (V) in the endothelial crll lining is filled with the tracer element and contains part of a macrophage with phaqocytic inclusions of the tracer element. OZ, Occluding zcrnult~. ( ,,: 10,000)

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FIGS !I and 10. Serial electronmicropraphs of the outer wall of the aqueoux sinus (AS) following intracamwnl perfksiotr in chicken eye. The giant vacuole (V) in thv rndothelial cell lining of the outer wall contains trecer element and is occupied by pseudopodia (Ps) of a macrophagr (MP). Note in Fig. 9 tht, vacuolr appears wholly intracellular. whereas in Fig. 10 it has a basal opening (arrows) occupied by t,he mecrophagc. There we wme particles of tracw element in the v~mpnct connertiw t,iww zone. ( 13.5001

4. Discussion The distinctive anatomical disposition of the angular aqueous sinus ill avim t’vt+ has impressed a number of workers (SW Tripathi and Tripathi. 1973). Itut the fu\~:t,ional nature of this structure has remained unelucitlated. It. has been rcmarketl that this structure is generally devoid of hlootl in the enucleated eye. but it is not knower what, it contains in life (Rochoa-l)uvignearltl. 1943). injection cxperimrnts havr heen undertaken in the past. to clarify whether or not t,here arf communicat,ions between the angular aqueous sinus a,nd the anterior chamber. By injecting Prussian t)lue into the carotid and t’hr lunlen of the aqueoli, c: sinus. .Kochon-Duvigneaucl the episcleral (1893) concluded that while there is a direct communication hetween veins and the lumen of the aqueous sinus. thr inner wall of the lat,ter formetl a, barricl for the leakage of the dye. By injecting the tlyt~ tlirect,ly into the anterior chamber. however. he observed a passage of t,hc blue mass into the episcleral veins hut on the basis of his histological study he contended that this leakage of the dye from tht, anterior chamber probably resulted from an artefactual rupture of the inner wall of wit,h no open Comtnunicathe sinus and thatJ the anterior chamber is a closrtl cavity tion with lumen of t,hc angular nqu~us &ils. In our present stu(lv on the fate of thcb colloidall~ suspended tracer elerrrt#nt. wc have paid special care’in tissue handling so a,s to avoid or minimize possible sources of artefact,s. It’ is realized t,hat some dislodg,rrment and ext)raction of the bracrr materia,l

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taspeci;tllv front large open tissue spaces can occur clurinp the tissue processing. ‘I’hc~ results of our st,udy, however, suggest the following conclusions. ‘I’htx cntlothelial lining of the aqueous sinus forms a barrier to the outflow of tltta t cw,cer elt~tnent. That, the intercellular junctions are not permeable to the tzacet elettlettt is apparent since no leakage of this ntatt,rial was st~l across the occluding zonult~s. This experiment. howrIver. does not negxte the possil)ility that tttolecult+ ,~ttta~llt~rthan tht, tracer element used in our invcstigatiott coultl seep across the ~11 ,junc‘tions. The I)ulk transfer of the tracer eletttent hy micropinocytotic vesicles also finds littIc support, from this experiment, since for the tluratiott of thts perfusion. only ~~+.,~ional caveolae cont,ainetl particles of the trac’ct eletnent. I IL caontrast to the above findings, following intracatneral perfbion with a colloitiall\ ~t~spt~trtietl tttattbrial. almost all vacuole,5 are readily tillrtl with the t,racer rlettit~nt. This c~lt~;trl>~indicates thxt the c~ntlothelial vwcuo1e.s trortttally taontain aqueo~ hu~nour I’~OI~I I tic* anterior chatnl~er. Leakage of t,tte tracer c4etut~ut frottt some vacuoles into t tiv I~ittlen of t tie aqueous siiiib suggest> that ttttav represent (hither trauscellul~t ~‘tliltltl~lh. i.e. vacuoles having communication Ott I)oth I~sal ant1 lunrinal surface<. or tttt)\p art’ vacuoles only having luruinal opening-. It i * interc&ig to note t,liat in I ttf+.(* t~xpt~rintentally perfused dyes. the wandering cells often contained phagocytosclti partic*les of the tracer element and appeartat to htl pasqitig through the transcrllulat t*ttiiut& ah xeeit in tiortrtal eye-. ( Iti the basis of t,hese ohst!rva,tions. the out’flow of thr> cohithlly suspended part icu. 1. of t ftt> ,supportinp connect,ive tissue zone of the scleral \\-a11of the sinus suggests that ttorttlall~ SOIIIC amount of the a.queous humour tltust ftave access to these regionc. It woultl appear: however, that’ eventually a proportion of this aqueous humour is t~liriiitiatt~tl into the aqueou s sinus via t& occaGtta1 vacuoles present in hhe rnrfot ttelbl lining of the outer wall and the septae. iis itidicatrtl I)\* the presence of t,racer t~lettrrnt in tlie\r vacuoles in perfused eyei. Ttlt> passage of t.he tracer element front the lun~cn of t.he sinus into the intraseleral

Our sittcere thanks are due to Professor Norrl~ II dsht,ori. F.R.S. for his continuetl interest ittltl help in this work. Enthusiastic, ~~ssistatwe was provided hv Mr R. Howrs ;I.II~ 11~ 4. Ahmetl md secretarial help by Miss 13. A. Streek. It is a pleasure t,o ackttowledpe the fill;ttt(,i;tl support of the Medical Kese;trc.h (‘outtc.il title Grant CT971/127/C. REPERESCES (‘ale, D. F. and Rorhon-Duvigneaud, Kochon-Duvigneaud, Trip&hi, R. C. Tripathi, R. C. of London. Tripathi, R. C. Tripathi, R. C. Tripathi, R. C. Tripathi, R. C. Tripathi, R. C. Tripathi, R. C. vol. 5 (Ed. Tripathi, R. C. Tripathi, R. C.

Tripathi, R. C. (1971). Exp. b’ye Res. 12, 25. A. (1893). Arch.. O~~hthnlmol. (Prrris) 13, 20. 41. (1943). Les I’euz et In Vision drs I~~rtdbrCs. Paris: Masson (1968). Ezp. Eye Res. 7, 335. (1969a). Ultrastructure of the exit, pathway of the aqueous. Ph.D.

et (‘ie. Thesis.

lyniv.

(1969b). Tmns. Ophthnlmol. Sot. U.K. 89, 449. (1970). Proc. ,Olst Int. Ophfhnl~mol. Congr. Merico, 8-14 March, p. 267. (1971a). Exp. Eye Res. 11, 116. (1971b). Exp, Eye Res. 12, 311. (1972). R&f. J. Op~t~~Z?~~oZ. 56, 157. (1973). Comparative physiology and anatonly of the outflow pathway. In The: Eye, Davson, H.). London and Sew York: Xcadernic Press. In press. and Tripathi, B. J. (1972). h’xp. Eye Res. 14, 73. and Tripathi, B. J. (1973.1. Exp. Eye Res. 15, 40%