The fine structure of the trabecular meshwork at graded levels of intraocular pressure

Exy. Eyp Res. (1975) 20: 505-521

The Fine Structure of the Trabecular Meshwork at Graded Levels of Intraocular Pressure (1) Pressure Effects Within the Near-Physiological Range (8-30 mmHg) TAN

GRIERSON

ASII

KILLIAM

R.

LEE

‘l’h(. outflow apparatus from rhesus monkeys which had been subjected to graded levels of intraocnlar pressure (8-30 mmHg) wax studied by transmission electron microscopy. At the higher pressures marked distension of the outer corneoscleral and the endothelial layers of the meshwork occurred, but cell-to-cell contact across the intertrabecular spaces and in the cndothelial meshwork was maintained by extended endot’helial cell processes. The elasticity of the native cells was attributed to the presenre of abundant intracytoplasmic tni?rofilaments. “Button” junctions were observed at the sites of process apposition and these were considered to be an important factor in the cellular adhesion. The endothelium lining the trabecular aspect of Schlemm’s canal remained in close contact with the underlying meshwork throughout the pressure range and cellular rather than extracellular contacts were thought to be the major anchoring mechanism. With increase in intraocular pressure, there was an apparent decrease in extracellular material in the endothelial meshwork. A statistical analysis demonstrated a significant increase in the counts of lysosomes. lysosome complexes, multivesicular bodies and lipid vesicles in the meshwork cells at 20 mntHp when compared to t-he counts at 15 mmHg (the control pressure).

1. Introduction In a recent publication, it was shown by light and scanning electron microscopy: that increases in intraocular pressure, from 0 to 50 mmHg. produced progressivr distension of the corneoscleral and endothelial layers of the tSrabecular meshwork (Grierson and Lee. 1974). The tissue was subsequently studied by transmission electron microscopy in an attempt to determine those properties of bhe trabecular meshwork which permit, distension without disruption. Particular attention was paid to the anchorage mechanismsof the endothelium lining the trabecular aspect of Schlemm’s canal ant1 to the inter-process attachments of the endothelial cells within the meshwork. The studv also includes an assessmentof the changes induced in the cellular and estracellular components of the endothelial meshwork. Thr results are presented in t’wo sections. The first section describesthe changes observed in the near physiological range of 8-30 nunHg and the second the changes at 0 and 50 mmHg, becauseai the latter pressures,there were pathological changes which were not apparent in the near physiolo&cal range. A description of the effect< of graded intraocular pressureson the mechanismsof fluid flow through the lining rntlot~helium of Schlemm’s camalwill be t,he subject of a further communication. ~r~led

2. Materials and Methods The outflow exalninerl

apparatus by transmission

from the epes of electron microscopy. 36

14 rhesus

monkeys

(Macnca

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Kefore the pressure esperinieuts \vere performe~l, it. was ttecessilr\. to esclucle tltc. possibility that the artificial aqueous used as an infusion fluid had MI; sigttifi(.ant effect, on the ultrastructure of the meshwork. -1 prelirninarr experiment TVS therefore perfot~tttt~~l (111three monkeys which \vere anaesthet,izerl with Seruylan attd Pentothal. The right evr in each animal was cannulaterl with two d&gauge tteedles and its aqueous repl;hcptl by, thtb slow infusiou of BdrBny’s solutiotI (BArAny, 1964). The infusion fluid was itItrotluce(l kJ) the 11u~Jua1 manipulation of a perisbaltic pump which caused t,he fluid t.o How iuto ;Itttl out of the anterior chamber via t.lIe itttracameral needles. When the imterior ctt;tnIher contents had been replaced, the infusion wan halted and the eye ws left iu situ for I hr. After t,his period the left’ eye was cannulated in a similar manner to the right eye attrl t,hr fluid in both eyes was replaced by 2-4” ,, $utaraldehvde i11 phosphate buffer by ttte;ttts of t,tte peristaltic pump. Ten minut,es Inter the animals were s:u,rificed by an ovrrtlose of’ EUthtill: t,he eyes mere enucleatetl and itumersetl in fixative of the same concentr;ttic,tt. Nine monkeys mere used in l”essure-cotitrolletl experinieuts iii which fixatitrtt wits ;tchievetl by itltri~callleral itifusion [see Grierson antI Lee (l!U-i) f or it tletailetl desc~riptioti of the apparatus and techniques]. III three groups of three animals, t,he ititriiowliw pressure was m;~itit;iined at either 8, 2-L r)r 3( t InmHg by a manometer system wtttainittg Blir;iny’s solution (B;ir;iny 1964). In each esperitttent,, the control eye was maint;tined ;Lt 1.5 1111nIig. After 1 hr the eves were fisetl in viva bv clamping t’he reservoir cotttaiuing mock aqueous and allowing 2- 4” o phosphate buffered glutaraldehyde (a flurore~ceitt tlve Gldit~ive confirmed rapid distribution) to enter t,he anterior chamber from ;I sccwttl reservoir. The fixative reservoir wts set at the same height ;LSthe mock ;~queous rwervoir, 60 tltxt the intraocular pressure ~-as considered t,o be constitnt~ throughout the esperitnent. An exception was made in the three eyes nt:tint~titIetl at, 8 mtnH,n, bewuse fixtttivr etlt,r! wits slo\v, so infusion n-as twisted l)y getitl\- with~lrirwing it strtall quautit,v elf fluit f’roitl the attt,erior chamber through the mock a.qieous infusiun needle: after this procetlure t,lie jIttr;It~?ul;tr pressure ~v;I.,sttlloweri t)o retww to 8 JIIII~H~. 111 ettrh l’ressurr-experimrllt, J t) tttiti \VilS allowed for fisat,ive peiietr;ttic,ti before the itttiniiil. ‘: \vere kiltell ;tnrl 30 tt1i11 ;&et, tlwtjll the irtlterior segments were csciwtl ittttl stotwl in fis;ttive. ttt 30 ttntIH,g itttritocttli~t 111 two niotike~s the esperitrtetit~al eves were trr;tittt;linetl pressure and the controls at, 15 rrtntHg ‘ittt.rztocular pressure. The regime WIS similar t.o ttt;tt described above, except that’ fiuatiott 1~;~s achievetl by in viva c:wot,icL perfusirpn ilt, a r;tte and pressure consistent with t,he animal’s carotid t&w1 flow. The anterior segments for ilt lf3iWtm 24 hr. Meridi~.~tial were store11 itt 2--4’fC, phosphate I~utYewtl ~llltitr;ll~lellvll~~ blocks of littibal tissue from eiwli qua(lrattt ‘crere I\-astied itr buffer atlcl post-fixctl it1 I”,, trcl~~iutn tetrositle for 1 hr. The ntat.eri;tl uxs ;tgain ~~;tsh~cl itl buRer. tlelt~tlrated in grdetl alrc~hols, cleared in propylene osicle :I 1111embedtlerl ill Ar&ite. UltrtLt,ltitt (St N-&N t K) sections were cut in the meridional plane on tltt LKl? ITttrotome III atId were examittetl in t,he Phillips 30~1 nrul the Siemetls Elmiskop Id electron microscoprn. For rout,itIo examination, the thin sections were double stainetl iu ur;tnyl acetate alld lead I,itrate. However, for more det:iiIed investig;ttioit of cell-to-cell junct~iow, some I~loclcs of littiM tissue \vere t,reated \\;it)li uranyl :tcetate prior to tleti~dratiott accordiug t,o the prow lure etnployetl by Farquar and Pal& (1M.i) : secticjtts from these blocks were es~ttrtiuetl without further staining. Preliminary quantitative examination of the meshwork endotlIelia1 cells suggested that the cytoplasmic content of lysosomes, lysososomal complexes, multivesicular botlies iLtI(l lipid vesicles was increased at the higher pressures. Therefore a quantitative analysis; WAS undertakeu bo compare the incidence of t,hese particular organeltes at the pressures of 13 illltl 30 mmHg. Rince the area of the individual meshwork cells is non-uniform in rittt(lonI ultrat8hin sections, the analysis could hare heen influenced bv observer selection ;tttd regiottal variation. To achieve compaaable measurements, 10 of the largest entlothelial cells were chosen from good quality coded sections cut from each of 20 coded blocks (five from each quadrant) and for this purpose five experimental eyes and five control eyes provided the tissue.

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The organelle counts were made directly in the electron microscope and distinction was made between the cells of the endothelial meshwork and the cells in the remainder of the meshwork (i.e. cells lining trabeculae). The results were expressed as organelle counts per 100 cells examined and paired t-tests were used for the statistical analysis.

3. Results The initial experiment showed that the exposure of the outflow apparatus to the mock aqueous solution for one hour had no recognizable effect on cell ultrastructure. Although no significant differences in tissue appearances were found when the two fixation techniques of carotid perfusion and anterior chamber infusion were comparetl. the ultrastructural preservation was better with the lat,ter technique.

FIG. 1. Short Schlemm’s cane1 material, e.g. the in (a) shows part “en bloc” staining plssma membranes.

stumpy processes from the endothelinl cells (En’) linilcg the trabecular aspect. of insert into the underlying meshwork and come il1t.o association with extracellula~ elastic-like clumps (E) shown in (a) or with meshwork uells shown in (b). The insert of the intercellular cleft between two endothalial cells lining the trabecular wall. 13~ the intercellular junction (arrow) is seen as a region of fitsion between the apposing Fifteen mmHg intraocular pressure. [(a nncl b) 20 OOII: iusert .,: 160 000]

Control tissue F$tem mnHg intraocular p~~sure. The 15 nunHg intraocular pressure control group conformed to a general pattern and the examination of the ultrastructure was concentrated on the outer regions of the meshwork where the most significant pressure induced changes have been shown to occur (Grierson and Lee. 1974). The endothelium lining the trabecular aspect of Xchlemm’s canal was a continuous monolayer of cells whose lateral borders met either in a narrow zone of opposition

prrssurc. Intertrabeculiu FN. 3. Part of the outer corneoscloral meshwork at 15 mm H, u intraocular spaces (ITS) ere crossed by short processes and are narrowed to ;t 20%400-a pap where the lining cndothelial cells on neighbowing trabeculae come into contact (arrcbws). Membritnr-bound prcdilcs are occnsionally observed in the intertrebocular spaces. ( .: 10 000)

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or in a more complex zone of opposition formed by cellular overlap or tongue-ingroove insertion. tn each intercellular cleft, from one to four junctional modifications were identified and from “en bloc” staining these were shown to represent limited regions of fusion between opposing plasma membranes [Fig. l(a)]. The endothelial cells contained cytoplasmic microfilaments which were found throughout the cell cytoplasm. hut were prominent in the. short stumpy processes which projected through the‘ patchy hascment membranr. These processes calne into contact with either ~~xt~r;~~;~~llular materials. endothclial meshwork cells or nleshwork cell processes (Figs 1 antI

2).

Thcx c~ndotlrelial meshwork was considered t*o be the region M,wcen the basal aslject of t!~r: t~ntlothr~liun~ lining Schletnm’s canal and the first layer of corncoscleral tral)t~culae and as such was a variable, though narrow region (8 -16 pm thick). The tlati\,(t ~11s were usually oricntatetl with their long axes parAle to the endotheliutn lining the canal and formed a network by process contacks Mwren neighbourirq cell\ (Fig. 2). Th e 1)rocesscs were usually short, l)ut coriltl extentl up t’o 2 pm in length. ESt 1.~l~(X’111&11ltliaterial was idcnt~ificd in the spar15 between tlrc native cells and printilril~ it consisted of fibrils. ground sut)&ancP>VI)ll:~geufi I)ro< and “elasfic’-like ni;lttGl (Figs 1 and 2).

staining, between two BIG:.4. (a) A junotion,which resembles a macula occludens by conventional processes in the corneoscleral meshwork. The junction is recognized as an apparent obliteration of the int~ereellular space between two opposing plasma,membranes. ( i 120 000) (h) KYth uranyl acetate “en bloc” treatment &l no subsequent section staining, the type of junction shown in (a) is seen to be septilaminar, i.e. there is no fusion of t.he opposing plasma membranes which are separated by a 30-40-8 gap. ( x 160 000) (c) At the region of opposition (arrows) between t,wo processe, F. and the endothelial meshwork cell (E&ICI), adhering junctions can be identified. From the high magnification insert (d) it is apparent that tht intcrcrllular space is narrowed but not occluded, at the site of the macula adhaerens. Microfilaments can bc identified in the meshwork cell cytoplasm and tend to be orient,ated along the long axis of the cell. ( 27 5UlJ: insrrt ;:, 44 000) Fifteen mmHg intraocular pr~surr. R

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In the outer corncoscleral ndlwork. the adjacent trabeculae ww separatfal I II? narrow intertrabecular spacrs and cellular contact across the spaces was maintainc~c I tither by process connecbions 01 11v opposition between the perinuclear t)ulyc+ ot mdothelial cells on adjacent trat,eAlae (Fig. 3). In both the corneoscleral aud endothelial parts of the meshwork the cells tl;ul a tlistinctive arrangement of cytoplasmic microfilaments similar to t~liat seei) in t!lcb The microfilaments wer’c of intletern~inat~~ cndothelial cells lining Schlenm’s canal. length. less than 100 A in diameter ant1 ran in tnmdles which trntled to be orientatctl ol’ jU1ic~8iOll were itlrntifietl ~~f!tw?en in parallel to the long axes of t,he cell s. Two types L the regions of process and cellular opposition (Fig. 4). Roth were macular or huttou junctions; the first was a simple atlhering junction ant! thr: secontl. from convcrttional staining, appeared to hc a region of fusion hetwecn the opposing plasma inciiihrilne~. However, when the second type of junction was exminctl after many1 acetatcb “en IAoc” staining, it was evident, that t,tte opposing plasma mcint)rancs were not fuml. of this ty)e havf~ Iwf?n tmt, were separated by a 3OMO A gal) ; Illemtnxne moflifications called “ gap junctions” t)v ItCVc~l R:trnovsk,v ( 1965). ilntl

Eight 1))111 Hg iratrcroculfxr prrs.vtrf~. The cells lining the trat)ecular wall of Hchlett~lu’s canal were narrow in cross-section and the nuclei were often deeply infolded. so that the cells appeared to be more compressed than in the control tissue (Fig. 5). Junctional modifications were apparent in the tortuous zone of contact between neighboaring cells (Figs 5 and 6).

FIG. 5. At 8 mmHg intraocular pressure many of the individual cells in the endothelium lining the trabecular aspect of Schlemm’s canal have a squat appearance, invagineted lateral borders (arrows) and prominent infoldings in their nuclei. Under the lining endothelium there is a wide diffuse basement membrane material. ( x 13 500)

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FIG. 6. The endnthelial meshwork (EMW) at 8 mmHg is a compact, region iu which the nativcx rell~ WC arr~rgetl wit,h their long axes pardlel to the lining mtl~~thelium of Schlrmm‘s canal (EN). Short, process connections between the endot~helial meshwork cells are evident (BI.I.OU.S) and the narrow ertraplasmoid cellular spaces are packed with extracdlular material some of which rrsvmhlcs thP granular material in the lumen of Schlemm’s carrd. ( I 13 000)

Yru. 7. Part of the endothelial meshwork at 22 mmHg. Wide ~~lectron empty extracellular spaces are evident. Extracellular material, whew present, ia focally aggregated near the lining cdothelium of Schlcmm’s canal (EN). The native eclls tend t.u bo oricntatecl at 90” to the lining rndotheli~m~ itnd cellto-cd1 contects we maintained h,v lotq t.hin pcesn~s. ( ‘~‘8000)

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The compact endothelial meshwork was reduced to less than 10 pm in thickness and the native cells were arranged in two or three irregular layers usually lying parallel to the endothelium lining Schlemm’s canal. The narrow and tortuous extracellular spaces were filled with extracellular material similar to that described in the control tissue. In addition, the extracellular spaces in some regions contained granular plasmoid material identical to that present in the lumen of Schlemm’s canal (Pig. 6). In the outer corncoscleral meshwork the intert,rabecular spaces were constricted and on occasion almost eliminated. ‘I’wnty-two m,m Hg intraodar pressure. At ‘J‘2mmHg. the endothelium lining the t,sa,\lecularwall of Schlemm’s canal was usually in close contact with the underlying rntlotht~lial meshwork, but limited regions of separation were occasionally identified.

The rndothelial meshwork was distended (in excessof 20 pm) and the distension produced a reorientation of the endothelial cells so that their long axes tended t*o lie at) right, angles rather than parallel to the endotheliurn lining the trabecular aspect of Schletnm’s canal. Although the cells were more separateand hence the extracellular spaceswere larger, cell-to-cell contacts were still present and the integrity of the tissue was maintained. The extracellular material was focally aggregated and was usually found in limited regions close to the endothelium lining Schlemm’s canal; elsewherewide electron-lucent extracellular spaceswere a prominent feature (Fig. 7).

514

1. (:ItIEI~SoS

Axu

IV. II.

LEE

The distension. however. was not rmiform anti focal Imlges in the endotheliuni lining the t,rabecular aspect of Schlenlrn’s canal IWIY a f’eaturp of note. By comparison with the tissue at, lower pressures. the intertral~ecular spaces in the outclr corncoscleral meshwork werta wide, but, process attachmtmts remained and junctions were still obvious and intact at the region of contact. The JX’OCCWW wertl thin and extended (l--7 ~111in length) and the longitutlinal nl icrofilament) organization was pronounced (Fig. 8). Tkirty wm Hy i~~trnocultr~ pw.ssu~‘~~. l_)esp&b t fir fa,ct that t’tic cells lining t,tir were attenriatett and bhe hasetnent nieriihran~~ trabecular aspect of Schlemni’s CNlid material was absent. there was no estrrlsive loss of cell-to-cell adhesion.

(b)

Distension of the endothelial meshwork was striking and the thickness of this layer varied between 15 and 35 pm. The most advanced distension was evident in the posterior part, where loss of cell-to-cell contact was prevalent in the most severely affected areas [Fig. 9(a)]. On the other hand process contacts persisted elsewhere, not only between native endothelial meshwork cells: but also between the meshwork cells and processes from the endothelium lining Schlemm’s canal [Fig. 9(b)]. Extracellular material was reduced in quantity throughout the endothelial meshwork (Fig. 9) and in some areas it could not be detected in the wide extracellular spaces. The outer corneoscleral meshwork was more distended at this pressure than at

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22 mmHg. The widely separated traheculae were bridged by delicattl cell progress which often reached a length of IO pm. IntracytoplasmL uiicrofilan~ent~s \verc orientated along the long axes of the cell processes and the junctional modifications were apparently unaltered. There WLS (Dvidence of cellular separatiou from some of the: trahecular cores and at these foci. the basement membrane was diminished or al)sentS and the underlying collagen matrix was either disorganized or diminished. By contrast, where cellular attachment per&cd, focal segment H of basement membrane were thickened (Fig. 10). ,4 general qualitative assessment of t,he effects of change in intraocular pressure 011 the fine structure of the trabecular meshwork cells showed no ohviolls variation in either the number of mitochondria or the amount of rough endoplastnic reticulum. Similarl~y significant variations in Inicropinocytosis could not be detected. By contrast there appeared to be an increase in the number of lysosomes, lpsosonial complexes, multivesicular bodies and lipid vesicles (Fi,.(I 11) in the meshwork cells at 30 tntnHg

when compared with the 15 mmHg controls. The results of the quantitative analysis of the tissue at these two pressures are summarized in Figs 12-15 and they shop. for each animal, an increase in the organ& counts at 30 mmHg in both the endothelial meshwork cells and the cells lining the tr,ibeculae. The increase was variable. however, and. for example, was about IOO”,, in t,he lip&l vesicle count and al)out 500”{, for secondary lysosomes. Wh en tAr total counts for the five animals was compared statistically, the differences in organelk counts were significant.

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Lysosomes

TE.C

Lyosomal

I

I

15

30

complexes

EMW

100

05 I

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I 30

515

Multtveslculor

bodies

EMW

i-EC

75

25

i 0

I

I

15

30

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Lipid

vesicles

E.M.W.

T.E.C

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I

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30

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PIGS 16-15. Show the results of the lysosome. lysosome complru. multiresicular body aud lipid vesicle counts from 100 cell sections in the endothclial meshwork (E,MW) and also from traheculur cndothelial cells (TEC) elsewhere in the meshwork. A line cormccts the control (1.5 mmHg) and the experimental (30 mmHp) counts for each animal and the animal number for each individual is the same in all the figures (in animals 1-3. fixation was achieved by anterior chamber infusion and in animals 1 and 5 by carotid perfusion). When the organelle counts in the control and experimental eves were compared statistically by the paired t-test, the difference was significant (P~041).

4. Discussion It is evident from the present investigation that progressive tlistension of the trabecular meshwork can occur without causing extensive cellular damage and loss of cell-to-cell contacts in either the endothelium lining the trahecular aspect of Schlemm’s canal or the underlying meshwork. The native cells can adapt their

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morphology partly because of inherent elasticity and partly because of effective intercellular adhesion. The elasticity probably depends upon the presence of cytoplasmic microfilaments which have previously been described in trabecular meshwork cells by Spelsberg and Chapman (1962), Vegge (1963), Holmberg (1965) and Inomata: Bill and Stnelser (1972) and th etr similarity to contractile elements in smooth muscle cells has been emphasized (Tripathi, 1974). It is of interest to note that when cellular attenuation involves the whole cell. as in the endothelium lining Schletnm’s canal. (rat,her than the cell processes. as in the trabecular meshwork cells) changes arca ohser\-tld in nuclear outline which are remarkably similar to those described by hlajno. Shea ;1nt1 l,eventhal (1969) in contract,ile ~a~scular endot~helium which also has it significant microfilament content. Thcb *mall junctions present in the intercrllular rlr:fts between the endothelial cells lining the trabecular aspect of Schlemm’s canal provide sufficient8 cell-to-cell adhesion to nlaintain tltcb integrity of the entlotttelial monolayer up to. at least. 30 tnntHg intra( bcula,r pressure. The junctions have 1~~1 considered to IW zonulae occludentrs t),v *(~v~~ral aut,Itors (Holtnberg. 1965; Tripathi. 1968; Inomata rt al.. 1972). t)ut recently Shal)o. Reese> and Gaasterlantl (1973) 1.i,lve stated that, the junctions arca nlacular occlutl~ntes based on their findings usinK fhr, uratryl acetate “en Moe” staitiittg techtiiclue. Our prelitninarv studies lia\c confirrnt~d that’ tnentt~rane fusion OCC’I~I~~in t8titl ,junctional region. Ll~t~ cottclusivc idc&ficatioii of non-wrtefactiial discotltitiitities. its drmonst~ratecl hy Shah0 et al., has not t teen achievetl. III tlltl t~rdl~ecular meshwork. the anchorage provitlctl I>? the gap junctions and ttttj tttit(‘lll>tt’ atlttat~rentes is the protjable exI)lattat8ion for the aljilitr of the meshwork cel!~ to n~aintain intercellular contact in spite of deformation in the distended tissur. .\lt’hc 1r1y11the cndotheliutn lining the trat)ccular aspect of Schlt~rn~‘s canal does not ha\-rs :I clistinctivr hasetnent ni(~tnbratie. it is maintainetl against, pressure gradient antI t hctrefore requires sotne form of anchorage to prevent tie.tachment from ttttl untlt~t~l~ing meshwork. Extracellular material. e.g. tine fihrillar material. ground sul~stancc~ ant1 cxlastic-like concentrations. lies closr to t,ttc lining r~ndotheliutn in ttttb 1:s 111 IIIH~ control tissue and this tnaterial may Ilavtl atlhrsivt~ propert,ies. Also tlttb small wII proc(‘ss projections into the ground substance. dcscrihed in this paper ant1 t)v It\otttata et al. (1972) anti ‘fripathi (1974): would provide stability. However at, 30 111n~Hg int,raocular pressure’. when presumably tttr pressure gradient is increasrtl ant1 t hta forces acting on the endothelial ntonola,ver are greater. the endotheliunl remains in close contact with the underlying tncxsltn-ork ~cn though much of tht> (~xtr;rAlular material is diminished in this a,rca. Sinccl endothelial tneshwork cell procths;:: att8achtnents to the processes from t,he entlothelium lining the trabecular u-all of S~~hletntii’s canal are intact. it is cotisitleretl that cellular and not extracellular c+itlc~iit,s play I hr. major part in maintaining the posit’ioti of the trabecular wall lining il

t’llclottlc’lill~~~.

In +v(xraI investigations, attention has hcen I& to the material present in the extr;lc~rllular sljaces of the endothelial meshwork (Vegge and Ringvold. 19f0; Inotllata et al. 1972; Lutjen-Drecoll, 1973; Tripathi. 1974). This material was consitlerecl to be of importance because changes in the nature. distribution and densit,r of t81ttbrxtracellolar substances may influence the rat,e of aqueous flow through the extractblhtlar spaces to the endothelial lining the trahccular wall of Schlemtn’s canal. In thr present study it ltas been shown that, as intraocular pressure increases. tht> ext~racrllular spaces become dilated and. particularly at 30 mtnHp, extracellular tttat,ckri;rls are lost (presumably t,hey are washed through the endotheliutn lining the

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canal). The changes may be of importance to the determinat,ion of facility in physiological experiments and the smallest practical stepwise increment!s in pressure antI flow rates would be desirable to minimize the loss of extracellular material. (iross tissue or cellular damage is not apparent within the range of 8 ~30 mrnHg intraocular pressure employed in this experimental model. although at the upper limit there wr(’ signs of loss of contact between some cells covering the traheculae. The increase in the counts of lysosomes, lysosornal complexes. multivesicular bodies and lipid vesicles at 30 mmHg. when compared with 15 mmHg, may be t’he result of two factors (a) an increase in the size of the organelles and (h) an increase in organelle numbers. As the lysosomal complexes often appeared to be larger a,t the higher pressure, both factors probably contribute to the increased count in the case of this organelle type, whereas with the other three organelles there were no obvious size changes. so the increasrll count represents a true increase in incidence.

Number

of vesicles

It has tIeen shown that, like lysosomes and lysosomal complexes, multivcsiculat Mies contain acid phosphatases (Smith and Paryuar, 1966). An increase in t,hese t,hrec cellular organelles may therefore be an indication of early autophagic degeneration and is probably a representation of a cell-response to a less favourahle environment. Similarly, if the residue of the autolytic processes was rich in lipid, this would explain the increase in lipid vesicles. In addition to the increase in the numher~ of multivesicular bodies there appears to be a decrease in the mean number of vesicles wit,hin the organelles as shown in Fig. 16. If the vesicles themselves contain hytlrolvtic enzymes then the decrease in vesicle content could be explained by increased enzvnle demand by cells involved in autophapic tligestion.

This work was supported by the Scott,ish Hospital Endowment Research Trust (Grimt No. 395) and this help is gratefully acknowledged. Facilities for the experimental work were generously provided by Dr Murray Harper of the Wellcome Surgical Research Institute. We also wish to thank Dr John Paul of the Royal Heatson Memorial Hospital, Gbsgow, for kindly permitting the use of the electron microscope.

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