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British Journal of Ophthalmology, 1979, 63, 9-16
The trabecular wall of Schlemm's canal: a study of theeffects of pilocarpine by scanning electron microscopyI. GRIERSON, W. R. LEE, H. MOSELEY,' AND S. ABRAHAMFrom the University Departments of Ophthalmology and Pathology, The University, Glasgow, and'West of Scotland Health Boards Department of Clinical Physics and Bio-Engineering, Glasgow
SUMMARY The scanning electron microscope was used to study the endothelium lining the trabecularwall of Schlemm's canal in 10 human eyes enucleated in treatment of choroidal melanomas. Theeyes of 5 patients had been treated before enucleation with pilocarpine drops, and the remaining 5were untreated and served as controls. In the pilocarpine-treated tissue there was an increasedprominence of cellular bulges in the endothelial monolayer, and the endothelial pores were bothlarger and more frequent than in the untreated eyes. By the use of Poiseuille's formula it wascalculated that the resistance offered by the pore system to the drainage of aqueous in thepilocarpine-treated group was approximately one-ninth of that in the control series.
In a previous investigation the present authorsshowed by light microscopy and transmissionelectron microscopy (TEM) that the incidence ofgiant vacuoles and transcellular pathways in theendothelium of Schlemm's canal was greater inhuman eyes which had been treated with topicalpilocarpine than in a suitable control group of asimilar age range (Grierson et al., 1978). This findingwas in disagreement with the results of a previousquantitative TEM investigation (Holmberg andBa'rany, 1966) in which it was shown that vacuolecounts in the endothelium of Schlemm's canal werelower after the topical application of pilocarpine.The experiment of Holmberg and Barany wasconducted on a group of cynomolgus monkeys, andboth gross facility and net outflow were increasedby the action of the drug. In an attempt to explaintheir findings Holmberg and Barany proposed thatthe vacuolar flow pathways had become lesstortuous and that straightening out of the vacuolarchannels would account both for the decrease inincidence in single sections and for the increasedrate of drainage.
Svedbergh (1976), however, has criticised the useof TEM in the assessment of changes in flow path-ways through the lining endothelium of Schlemm'scanal. This criticism is largely based on the fact thatonly a small proportion of giant vacuoles aretranscellular channels (Kayes, 1967; Tripathi, 1968,1971, 1974; Inomata et al., 1972; Grierson and Lee,
Address for reprints: Dr W. R. Lee, Tennent Institute ofOphthalmology, University of Glasgow, 38 Church Street,Glasgow Gl 1 6NT
9
1975; Grierson et al., 1978), and it is possible tofind transcellular channels which are not associatedwith a vacuolar swelling (Inomata et al., 1972;Grierson and Lee, 1975).
Quantitative data on the luminal openings ofvacuolar and nonvacuolar transcellular channelscan be obtained by examining the endotheliumlining the trabecular aspect of Schlemm's canalwith the scanning electron microscope (SEM). Thistechnique provides a useful means of examiningrelatively large areas of exposed canal endotheliumand overcomes the sampling problem associatedwith TEM. It was therefore considered appropriateto apply scanning electron microscopy to the studyof the material which was used in the initial lightmicroscopy and TEM investigation of the effects ofpilocarpine in the human outflow system. Inparticular, the analysis was directed towards thevariation in incidence and size of the luminal poresin the endothelium of the treated and untreatedtissue.
Materials and methods
The tissue for this investigation came from 10patients with an ostensibly normal anterior segment,and each eye was enucleated in treatment of achoroidal melanoma. With 5 patients the eye to beenucleated was subjected to a topical application of2 to 4% pilocarpine on 4 separate occasions at6-hourly intervals before surgery. The remaining 5patients were untreated, and the tissue served as acontrol. On enucleation the eyes were immersed in2 to 4% glutaraldehyde in phosphate buffer. There-
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0L Grierson, W. R. Lee, H. Moseley, and S. Abrahant
after blocks of limbal tissue were removed, washedin buffer, postfixed in 1 % buffered osmium tetroxide,and washed again in phosphate buffer.The technique for dissection of the trabecular
wall of Schlemm's canal has been described pre-viously (Lee, 1971). With a ground razor blade acut was made from the anterior limit of the canalacross the peripheral cornea and into the supraciliaryspace from the posterior portion. The dissectionleaves 2 pieces of tissue, one which shows thesurface of the scleral sulcus and the other theendothelium of the trabecular wall.The tissue was freeze-dried in a Balzars Micro
BA3 unit which maintained a vacuum of 10-5 Torr.Before water extraction the tissue was passedthrough liquid Arcton12 and frozen in liquid nitro-gen. The dried tissue was coated with gold in aPolaron Sputter Coater E5000 and examined in aCambridge Stereoscan S600.For the quantitative analysis of endothelial
porosity coded pieces of tissue from pilocarpinetreated and untreated eyes were processed in parallelto minimise variation in preparation. Mappingphotographs of the endothelial monolayer weretaken at a screen magnification of x 3000 andenlarged to a print size which corresponded to anarea of 2500 1Im2. The number of cellular bulgesand surface openings were recorded on codedphotomicrographs, and the total area examined ineach eye was between 250 000 and 400 000 yiM2.Maximum pore widths were measured from sets ofprints which corresponded to a surface area of100 000 yiM2 with the use of a x 7 magnifier whichincorporated a calibrated graticule.
Results
Examination of the dissected portions of Schlemm'scanal from both the treated and untreated eyesshowed that the general topography of the endo-thelium lining the trabecular aspect of the canalwas similar to previously published findings(Hoffmann and Dumitrescue, 1971; Bill andSvedbergh, 1972; Segawa, 1973). In some regionsthe endothelium was flat, whereas in others therewere distinct ridges, humps, and areas of disruptionwhere septa had been torn. The individual cells ofthe endothelial monolayer were elongated andspindle shaped, and each had a prominent centralovoid bulge (Fig. 1).Each bulge contained the endothelial cell nucleus
and possibly a giant vacuole, but, in adequatelyprepared tissue, identification of the giant vacuoleswas speculative except where the delicate vacuolarshell was mechanically disrupted during dissectionto reveal its hollow interior. If this disruption was
Fig. I A dissection of the endothelium (EN) lining thetrabecular aspect of Schlemm's canal in an untreated eye.The trabecular meshwork is indicated by an arrow. Theiris (I) is seen beneath the trabecular meshwork (arrow)(x 120)
sufficiently extensive the meshwork pore at the baseof the cystic cavity was exposed (Fig. 2).
Openings with a smooth outline were found bothon and off the bulges. As in previous reports ofBill (1970), Bill and Svedbergh (1972), Segawa (1973),and Lee and Grierson (1975), the former wereconsidered to be the lumen pores of giant vacuolesand the latter to be the openings of nonvacuolartranscellular channels (Fig. 2). The distributions ofboth types of pore were not uniform, and areas ofparticular abundance and also areas of deficiencywere common to both the eyes treated with pilocar-pine and the untreated tissue. Surface pitting wasalso found to a greater or lesser extent, both on andoff the bulges. This was an artifact which may havebeen produced by the crystallisation of surfacedeposits (plasma) during the freeze-drying, and thiswas a troublesome feature because the largest pitscould have been mistaken for pores. Therefore theidentification of true pores was not always clear-cut,but with care and critical evaluation most of theartifacts could be eliminated. In addition the studywas essentially relative, and it was assumed that thepreparation hazards were equally applicable to thetreated and the untreated tissues.There were qualitative differences in the endo-
thelial bulges which protruded from the endothelialcells of the monolayer between the treated anduntreated tissue. In the pilocarpine treated tissuethe cellular bulges occupied a larger portion of themonolayer. They protruded further into the canaland were often seen as smooth-walled spherical
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The trabecular wall of Schlemm's canal: a study of the effects ofpilocarpine
Fig. 2 (a) Pores in endothelial bulges (arrows) fromthe trabecular wall ofSchlemm's canal in an untreated eye( x 3800). (b) A bulge (B), which has a large artifactualopening, in the trabecular wall endothelium of Schlemm'scanal in an untreated eye. The bulge appears to be hollowand a meshwork pore (arrow) is evident at the base ofthe cavity. Note the non-bulge pore elsewhere in theendothelium (small arrow) (x 3800)
grape-like structures, whereas the bulges in theuntreated tissue were usually less obtrusive, more
ovoid, and sometimes had a rough crenated surface(Fig. 3). The bulges in the treated tissue were moreprone to total or partial collapse (the action ofsurface tension forces during the drying process)and to artifactual tearing than in the untreatedtissue. Presumably this was due to the presence ofparticularly large giant vacuoles with delicate andtherefore vulnerable cytoplasmic shells.
It was our impression that pores both on and offthe bulges were more in evidence in the pilocarpine-treated series than in the untreated group. However,there was considerable variation within both groups,
and a quantitative analysis was undertaken todetermine in as precise a manner possible the effectsof pilocarpine on the canal pores of the endothelialflow pathways.
QUANTITATIVE ANALYSISThree structural features were counted in theendothelial monolayer: (a) bulges (since somecontained giant vacuoles), (b) openings on the bulges,and (c) openings elsewhere on the endothelial cells.
There were three major sources of error orinaccuracy in the quantitative analysis. (a) Truepores were defined as openings with a regular andsmooth outline to distinguish them from raggeddeficits which resulted from the preparation.However, the distinction was not always clear-cutand therefore an overcount was possible. Conversely,true pores may have been damaged and were thuseliminated from the count. (b) Particular difficultywas encountered with pores outside the openingswhich could have been obscured by grooves andridges in the monolayer. (c) The analysis was basedon viewing from one angle only, so that inevitablya proportion of the openings would have beenmissed.
In view of these problems in analysis a check wascarried out on duplicate photographic prints toassess interobserver differences. The difference wasfound to be 8% for counts of bulges and 15% forcounts of endothelial pores. Table 1 shows thatthere was no significant difference in the numbersof cellular bulges between the treated and untreatedgroups. However, there were substantially greaternumbers of endothelial pores both on and off thebulges to the extent that there were nearly threetimes as many in the pilocarpine treated as in theuntreated group.Measurements of maximum pore diameters were
made without stereoscopic correction. Withoutcorrection a pore will appear erroneously smallerthan its 'true' size, the error depending on the tiltof the pore relative to the viewing angle and thedirection of its long axis relative to the viewingdirection. Since this error was common to themeasurements of pore dimensions in both groups,comparison between the 2 groups could be justified.There was a much smaller population of pores inthe untreated than in the pilocarpine-treated series,and the histograms (Fig. 4) showed that in bothgroups the populations were not normally distri-buted but had a distinct skew, with smaller porespredominating. The mean pore diameters in thepilocarpine-treated eyes were slightly larger than inthe untreated eyes. The average diameter of thepores in the control group was 0 9 [±m and in thepilocarpine-treated series was 1-3 ,um.
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2L Grierson, W. R. Lee, H. Moseley, and S. Abraham
Fig. 3 The endothelium liningthe trabecular aspect ofSchlemm's canal in (a) apilocarpine treated and (b) an
untreated eye. In the pilocarpinetreated tissue the endothelialbulges are smooth walledspherical grape-like structures,whereas the bulges in the controlare less obtrusive and more ovoid( x 700)
Table 1 Results (expressed per square millimetre) obtained from the morphological analysis of surlace structuresin the lining endothelium of the trabecular wall of Schlemm's canal
Patientno. Age Sex Bulges
Treated1 46
2 50
3 60
4 61
5 75
Mean ± SD
Untreated1 312 633 63
4 68
5 75
Mean + SD
M
F
FF
M
F
M
F
FF
5690
4730
4630
4670
4970
4970 ±450
5740
4550
4130
3340
4690
4490±880
Pores onbulges
370
9001050
760
460
710 290*
380
200
220
150
280
250±90
Percentage bulgeswith pores
7
19
23
16
915+7
7
45
6
5±1
Pores outsidebulges
130
380
340
210
210
26041 00t
160
60
9075
120
100±40
Total poresin endolelium
500
1280
1390
970670
960+ 380*
540
260
310
225
400
350+130
Levels of significance: *P < 0'001. tP < 0-01.
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The trabecular wall of Schlemm's canal: a study of the effects ofpilocarpine
When the pore distributions were pooled andexpressed as percentage incidences it was found thatthere was a distinct difference in the pore distributionprofile between the treated and the untreated groups(Fig. 5). Only 3% of the pore population in thepilocarpine-treated series had a diameter greaterthan 3 0 ,um, which was the maximum size foundin the control group. However, in the untreatedgroup small pores predominated, and approximately70% of the population had a diameter of 1 F±m orless compared with 45% of the pores in the pilocar-
Treated
50
50
L0O
30
20
10
a
0
z
N =163x=1 2,um
30
20
Untreated
N 25R=0-8,m
N= 23
= %Um
N=40x 0-09,um
10
30
20
10
20
10
0- 06-1-1-1-6-21- 26-31-36-41-0-06-1-1-16-21- 26-31-05 1-0 15 20 25 3-0 354*0 4 5 15 20 25 30 35
Pore diameters jum1 Pore diameters (um
Fig. 4 The distribution ofpores on the basis of theirdiameters, from a sample area of 100 000 [sm2 for eachof the 10 eyes under investigation. N is the number ofpores and x is the mean diameter
35 .E UntreatedE Treated
'a.2Q
21-25 26-30 31Pore diameters (,um)
Fig. 5 The percentage incidence ofpores in each of thevarious size categories from the grouped data from theS treated and S untreated eyes
pine population. On the other hand less than 9%of the pores from the control population were1 6 ,um or greater, which can be compared with avalue of 28% from the pore population in thepilocarpine-treated tissue.
Discussion
The instantaneous 3-dimensional image of the SEMhelped to highlight topographical differences in theendothelial monolayer on the trabecular aspect ofSchlemm's canal between the pilocarpine treatedand the untreated group. In particular it was shownthat the endothelial bulges became more pronounced,and the porosity of the endothelium was increasedby the action of the drug. The increased prominenceof the cellular bulges was probably the result of theincrease in size and incidence of giant endothelialvacuoles in the treated eyes (Grierson et al., 1978).Similar findings are associated with increase inintraocular pressure (Grierson and Lee, 1975).
It was of interest to plot the bulge pore incidenceobtained in the present study against the appropriatevacuolar counts published in the previous paper(Grierson et al., 1978). From Fig. 6 it can be seenthat high counts of bulge pores are associated withhigh incidences of giant vacuoles. Although the dataare insufficient for meaningful statistical analysis,the trend encourages the assumption that the poresin the bulges are the luminal openings of giantvacuoles.
In both groups it was found that the distributionof endothelial pores was nonuniform, there beingareas of relative abundance and other regions wherepores could not be found. This observation wasconsidered to be further evidence for the proposalthat there are preferential flow pathways throughthe endothelial meshwork to the overlying endo-thelial monolayer (Rohen et al., 1967; Bill and
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I. Grierson, W. R. Lee, H. Moseley, and S. Abraham
Svedbergh, 1972; Grierson and Lee, IS1976; Lee and Grierson, 1974, 197'1976).SEM is particularly suited to the
relatively rare and sporadically distithelial pores because of the large awhich can be examined by this faciliwas possible to demonstrate quantita
Giant Vocuolesper Section
25s
20 -
15-
10-
5-
o
0
x
*.
0
x
500
Pores on Bulges per mm
Fig. 6 A scatter diagram which shows thgiant vacuoles per section as derived by lig(Grierson et al., 1978) plotted against thepore count for the corresponding eye
)74; Grierson, pores were greater in both numbers and size in the5; Svedbergh, pilocarpine-treated series than in the untreated
group. Thus the findings of the present investigationstudy of the do not support the suggestion by Holmberg and
ributed endo- Barany (1 966) that the effect of pilocarpine essentiallyreas of tissue is to change the shape of the flow pathways ratherity. Indeed, it than to produce alterations in their numbers.tively that the However, our results must be interpreted with
some caution because pore analysis by SEM is notwithout hazard. Preparation artifacts, like icecrystal formation, tend to produce holes in theendothelial monolayer and raise the pore count,whereas protein masking, produced by the reflux of
x blood into segments of Schlemm's canal during thetrauma of enucleation, tends to cover pores and
x x depress the pore count. Both factors will influencethe absolute values obtained in the analysis, but as
they were equally likely to occur in treated anduntreated tissue they are not considered to invalidatecomparison between the 2 groups. In the light ofthe problems associated with all aspects of prepara-tion of the tissue for SEM it is of value to makecomparison between the data obtained from the
x= TREATED human control tissue with that obtained by otherauthors for normotensive humans and monkeys(Table 2).The incidence of endothelial bulges found from
the present study fits reasonably well with the valuespreviously reported. If it is considered that the totalsurface area of the trabecular wall of Schlemm'scanal is 11 mm2 (McEwen, 1958), then this wouldgive a value from our data of 50 000 for the numbersof bulges, and therefore the cells, which make upthe monolayer. Bill and Svedbergh (1972) had a
12 much lower estimate of 23 000 cells in man, bute incidence of our figure is very close to the 55 000 estimated by,ht microscopy Segawa (1973).endothelial The size range of the cellular pores is also in
fairly close agreement with the values previously
Table 2 A comparison of data obtained in quantitative SEM studies of the endothelium lining the trabecular wallof Schlemm's canal
PoreAuthor Species IOP Bulgesl Jirequency Total poresl Pore size
mm2 on bulges mm2 in plmBill (1970) Vervet and Rhesus monkey Normal 4000 29% 1200 0-3-2 0
Lee (1971) Rhesus monkey 18-20 mmHg 6000 - - 0 2-1 0
Hoffman and Dumitrescu (1971) Human Normal _ _ up to 2 0
Bill and Svedbergh (1972) Human Normal 2000 - 1800 0 15-5-0Segawa (1973) Human Normal 5000 20% 1000 0 15-1-5
Lee and Grierson (1975),Grierson (1976) Rhesus monkey 15 mmHg 3800 15% 950 0-14-0
Svedbergh (1976) Cynomologus monkey 12 mmHg - - 1640 0-25-5 0
Present study Human Normal 4500 520 350 02- 3 0
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The trabecular wall of Schlemm's canal: a study of the effects ofpilocarpine
reported (Table 2), but our values for the total poreincidence and the frequency of pores on bulges areextremely low. To some extent the difference maybe accounted for by the quantitative techniqueadopted in the present study. The pore incidenceswere determined from micrographs similar to themethod adopted by Lee and Grierson (1975) andGrierson (1976) for the study of pressure effects onpore incidence in the rhesus monkey. Bill andSvedbergh (1972) and Svedbergh (1976), who arethe only other investigators to use a detailedquantitative approach to the investigation of endo-thelial pores, obtained particularly high values forpore incidence, and their counts were made directlyfrom the display screen on the SEM. The use ofphotomicrographs rather than the display screenhas several advantages because coding and 'blind'assessment, repeatability testing, and determinationof interobserver error are much more easilyachieved. However, an undercount is inevitablebecause 'true' pores are often more difficult toidentify in electron micrographs and viewing wasfrom one angle only, so that pores hidden behindthe bulges are missed. On the other hand overcount-ing due to counting from the same area twice wouldbe less likely from photomicrographs than from thedisplay screen.With the possibility of overcounting in the
technique adopted by Bill and Svedbergh (1972)and undercounting by the present approach itwould seem reasonable that a value somewherebetween 350 and 1800 pores per mm2 (4000 and20 000 respectively for the whole canal) will approachthe true incidence in the older human eye. To someextent the large discrepancy is probably also dueto the fact that the tissue in both investigations wasfixed by immersion without pressure maintenance.It has been reported that immersion fixation is theleast satisfactory technique for the study of tempor-ary pressure-sensitive pathways, which may beradically altered in the time interval betweenenucleation and penetration of the appropriatelayers of tissue by fixative (Grierson, 1976;Svedbergh, 1976).
It is of interest to consider the present results interms of fluid conductance in order to assess thecapacity of the canal endothelium to facilitate thedrainage of aqueous humour. The fluid conductance,C (,ul min-1 mmHg-') of a pore is the flow, Q(,ul min-') of fluid through the pore per unit dropin pressure, AP (mmHg) across the pore.
C Q (1)
The mathematical approaches to problems of
conductance in such a complex biological system as
in the canal endothelium are to be considered inmore detail in a later paper. However, the modelproposed by Bill and Svedbergh (1972) is suitablefor a general estimation of the properties of conduct-ance in the lining endothelium. Bill and Svedberghrepresented the pore as an opening 0 3 ,um longand of various diameters as measured.
Fluid conductance through a tube is given byPoiseuille's formula:
where Q =AP =
r =
L =V =
AP r4Q =8 Lv
fluid flow (cm3 sec-1)pressure drop (dynes cm-2)radius of tube (cm)length of tube (cm)fluid viscosity (poise)
(2)
It may be shown that, after rearranging the termsand assuming a viscosity of 0 007 poise, which is theviscosity of water, formula (2) may be expressed as:
C lx 3563 (3)
where C = conductance (,ul min-' mmHg-')d = diameter of tube (,um)1 = length of tube (,um)
Flow through an aperture is given by the equation(Happel and Brenner, 1965):
AP r3
3v (4)
It is also possible to rearrange formula (4) andexpress it more conveniently as:
=2099 (5)
According to Bill and Svedbergh the resistance,which is the inverse of the conductance, of each poremay be taken to be the resistance as calculated byformula (3) plus the resistance derived from formula(5). The values obtained for pore conductance andpore resistance by using this approach are shown inTable 3. Because of the small numbers involved,the pore counts in the 5 eyes in both the untreatedgroup and the pilocarpine-treated group have beensummed together to produce results for an equiva-lent area of 500 000 pLm2 and extrapolated to 11 mm2in each group.The results are presented when all the pores are
included and also when those pores above a diameterof 3 p.m have been omitted. Since the large poreshave a considerable effect on the conductance, theinclusion of a potential artifact such as a large-diameter pore will greatly increase the calculatedconductance. Thus, while the pores of diameter
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I. Grierson, W. R. Lee, H. Moseley, and S. Abraham
Table 3 Calculated values for the conductance andresistanice in the pore system in the lining endothelium ofthe trabecular wall of Schlemm's canal. The data wereobtained from the grouped treated and untreated eyesand were calulated for an area of 500 000 tIm2. Thefigures in parentheses are conversions to an area of 11 mm2
Pore conductance Pore resistance(pl min-. niinHg-g) (mmHg mw. 1l 1)
All pores Pores <3 mIn All pores Pores <3 m
Treated 0-985 0-837 1-02 1-20(21.7) (18-4) (0 0462) (0 0543)
Untreated 0104 9-61(2-29) - (0-437) -
greater than 3 ,um represent only 3% of the totalnumber in the pilocarpine-treated group, theycontribute 15% to the fluid conductance. The ratiobetween the pore resistance of the control groupand the pore resistance of the pilocarpine-treatedgroup is 9 47 when all the pores counted are includedand 8-04 when only those less than 3 t,m are con-sidered. Therefore according to this mathematicalapproach the effect of pilocarpine was to decreasethe resistance in the pores by a probable factor of 9.
In addition it can be seen from Table 3 that theresistance offered by the pore system in the un-treated human eyes is 0 44 mmHg min-' 1I-1. Thisvalue represents only one-sixth of the total trabecularresistance (3 mmHg min-' VIl-') as estimated byGrant (1958). These observations are in agreementwith those of Bill and Svedbergh (1972), whoconsidered that the bulk of the trabecular resistanceis outside the canal endothelium and is probablyin the underlying endothelial meshwork.
This work was supported by the Ross Foundation and theScottish Hospital Endowments Research Trust (grant 442),and this help is gratefully acknowledged. We are indebtedto the Anatomy and Engineering Departments of GlasgowUniversity for the use of their scanning electron microscopes.It is also a pleasure to acknowledge the helpful co-operationof our clinical colleagues in the Scottish Western Region.
References
Bill, A. (1970). Scanning electron microscopic studies of thecanal of Schlemm. Experimental Eye Research, 10, 214-218.
Bill, A., and Svedbergh, B. (1972). Scanning electron micro-scopic studies of the trabecular meshwork and the canalof Schlemm-an attempt to localise the main resistanceto outflow of aqueous humor in man. Acta Ophthalmolo-gica, 50, 295-320.
Grant, M. W. (1958). Further studies of facility of flowthrough the trabecular meshwork. Archives of Ophthal-mology, 60, 523-533.
Grierson, I. (1976). The morphology of the outflow apparatusof the eye with particular reference to its structuralappearance at various levels of intraocular pressure. Ph.D.Thesis. University of Glasgow.
Grierson, 1., and Lee, W. R. (1974). Changes in the monkeyoutflow apparatus at graded levels of intraocular pressure:a qualitative analysis by light microscopy and scanningelectron microscopy. Experimental Eve Research, 19, 21-33.
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