Exp. Eye Res. (1975) 21, 433-449
A Light and Transmission Electron-microscopic Study of the Rat Iris in Pupillary Dilation and Constriction
Department of Anntvmy, McGill
liniversity,
P.0. Rex 6070, &!oatrenl, Quebec
AXD WILLIAMS
Department of Anatomy,
Uniwrsity
8.
WEBBER*
of Bhtish
(Received 7 April
Colrw~hin. T’omwwer
B.C., Camdn
197,5, Boston)
Adult rat pupils were dilated with a mixture of 5” ,(, phenylephrine hydrochloride and 0.5”,, cyclopentolate, or constricted with 0.125y0 echothiophate iodide. The iris tissues were then prepared for and examined by light and electron microscopy. In pupillary dilation the iris is short as seen in meridional sections. The posterior epithelial cells are high columnar and are separated from the adjacent cells thus giving a scalloped appearance to the posterior surface of the iris. The nuclei have irregular outlines. They are oriented with their long axis along the long axis of the cells. Bundles of intracellular fila&ent,s, often associated witkthe nuclei, may f&n a hammock around the nucleus. The dilator muscle laver is relativelv thick. The nuclei are highlv indented. The most prominent structures present are the series of dilator hillocks and Khe& associated profusely branching dilator processes seen all along the boundary zone between the dilator and the stroma. They protrude int.o the stroma. The stromal cells and blood vessels appear to bc disposed in vertical columns perpendicular to the posterior epithelial and dilator layers. [n pupillaq constriction the iris is long and thin as seen in meridional sections. Most of the posterior surface of the iris is smooth. Both the posterior epit,helial and dilator layers are thin. The nuclei of the posterior epit,helial cells and of t,he dilator mnscle cells have smooth nuclear outlines and thev are oriented along the length of the cells. The intracellular filaments, mainly situatei in the posterior portions of the posterior epithelial cells, are seen as longitudinal bundles parallel to the length of the cells. Dilator hillocks and dilator processes. so prominent in pupillary dilation. are absent. The stromal components are oriented I~~rallel to the posberior surface of the iris.
1. Introduction The irip is a dynamic stru&ure capable of highly precise and rapidly occurring changes in pupillary diameter both in response to changing light conditions and in response to rniotics and mydriatics. In extreme pupillary constriction, the iris presents a large surface area with a small pupil. In pupillary dilation the pupil is much enlargad and the iris appears as a thin rim of tissue. Obviously this must entail structural changes within the iris tissue to accommodate for the great changes in total surface area from pupillary constriction to pupillary dilation: Also the iris architecture must be structurally adapt,ed to facilitate these movements so that they occur as smoothly as possible. Scanning electron microscopic studies of the constricted and dilated rat iris have revealed dramatic changes both on the posterior and anterior surfaces of the iris (Lim and Webber, 1975). To corroborate and amplify these findings, the present investigation is carried out using both the light and transmission electron microscopes. Changes * Please direct
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A. Webber. 433
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in the shape and orientation of the posterior epitheliutn. the tlilator and thtr st,rom;lI elements, as well as the relationships of these iris components to each other. are hettrl examined with the light microscope. The rat iris is also examined with the trallsmissioll electron microscope to see what ultrastructural changes. if any. OCWT. cspccially iu the posterior epithelium and the dilator.
2. Methods Adult rats of the Wistar straiu were used. Their pupils were dilated with a mixture of 59; phenylephrine hydrochloride aud 0~5~;~ cyalopeutolate, or constrict,ed with 0.1250,, echothiophate iodide. These drugs were allowed to act. for 15-20 miu, zt which time t,he pupils were widely dilated or much constricted, as the case may be. For the light microscopic studies, the rats were auesthetized with ether and the eyes removed immediately and immersed in the fixative of 4: O glutaraldehyde in 0.1 >I-sodium cacodylate buffer (Sabatini, Bensch and Baruett, 1963) at pH 7.2 at room temperature. For the transmission electron microscopic studies, the rats were anesthetized 1)~ au iutraperitoneal injection of sodium peutobarbitol and a subcutaneous injection of sodiunl pheuobarbitone. The left common carotid artery was exposed and cannulated. Thr cannula was connected to a ‘20 ml syringe containing the fixative of 4% glutaraldehyde iu 0.1 Iw-sodium cacodylate buffer. The inferior vena cava was also exposed. The fixative was perfused through the carotid artery at a pressure of 100 nmHg. As soon as perfusion started, the inferior vena cava was cut just below the rem1 vessels for drainage to take . place. The perfused eyes were removed and immersed in fresh fixative. Au antero-posterior slit was made through the corueal~scleral junctiou extending front the anterior and posterior chambers into the vitreous humor to facilitate the peuetratiou of the fixative into the iris tissue. After about 30 miu in the fixation, the posterior halves of the eyes and the lenses were removed and discarded. The eyes were fixed for a total of 4 hr, washed with and stored in 0.1 wsodium cacodylate buffer at pH 7.2 at 4°C. The anterior portions of the eyes, with the &tached iris, were trimmed into smaller pieces while in the buffer wash. The tissues were post-fixed for 1 hr in 19; osmium tetroxide in 0.1 >I-sodium eacodylate buffer, stained en bloc iu urarlyl acetate for 1 hr, dehydrated through a graded series of alcohols, infiltrated ant1 embedded in epou Araldite. Thick sections for the light microscopic st)udies were stained with toluidine blue aud examine(l on >l Zeiss Photomicroscope II. For the transmission electrou microscopic studies, thin sections of well-preserved tissues, ils determined light microscopically on thick section, were stained with lead citrate for 2.11~2..5 mill and exanlinwl on a Philips EN 200 operatitlp at 60 kV.
3. Results Light microscopicstudy (a) The iris in papillary dilation. When the pupil is well dilated the iris is short in terms of its dimensions from the root to the tip of the iris, as seen in meridoneal sections. The iris tends to be disposed in a slight convex arch towards the surface of the cornea due to the fact that the stroma in the middle third of the iris is thicker than that at the pupillary or peripheral region [Fig. l(a)]. In meridional sections. the posterior epithelial cells form a layer covering the posterior surface of the iris [Fig. l(a)]. Th e major portion of each cell may be discretely separated from its neighbouring cell or cells thus giving the posterior surface a deeply fringed appearance (Figs 1 and 2). Most probably cytoplasmic processes from neighbouring cells interdigitate with each other. Sometimes the posterior epithelial cells
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may be so closely abutted on each other t.hat there are no gaps in between the cells. The posterior epithelial cells are most often large, columnar in shape and they have a highly irregular cell outline [Figs l(b), 2(a) and (b)]. The height of the cells varies with the degree of pupillary dilation. The anterior border of these cells is in apposition with the anterior epithelium and dilator muscle cells. In the center of each cell is an elongated nucleus wioh a very irregular outline, oriented with the long axis of the
FIIJ. 1. (a) The iris in pupillary dilation (LM). The iris is short. Both the posterior epithelium (p) and dilator (d) are distinctly visible. The st,roma is thick especially in t.hc middle one third of the iris so that the iris appears to buckle anteriorly. Dilator muscle spurs (arrows) are seen in the pupillary half of the iris. The sphincter (s) is a mass of small cells at the pupillary tip ( i 90). (b) The iris in pupillarp dilat’ion {IX). The posterior epithelial cells (p) are peg-like girin g the posterior surface a deeply fringed appearance. The nuclei are large and are situated in the middle of the cells. The dilator (d) is a relatively thick layer. Fine dark-staining processes from the dilator are seen all along the dilator-stroma boundary (arr(ws). The strorna is filled with blood vessels having round or oval lumens. Some of the blood vessels appear to have only an endothelial lining (bv 1) while others possess in addition a layer of pericytes (br 2). The stromal cells are arranged in columns extending from the anterior surface of the iris to t,he dilatorPstroma boundary. The stromal cell nuclei are kept at, a distance from the dilator. The anterior surface of the iris is highly scalloped in outline. This is mainly due to the bulging outwards of the iris Idoud vessels ( ,: 240).
Fro. 2. (a) The iris in pupillary dilation (LM). processes devoid of nuclei. Groups of dilat,or cells spurs may also show fine dilator processrs (arrow). undulating outline ( x 380). (b) The iris in pupillarg large, high columnar with an irregular CPII out,line. the long axes of the cells. The dilator layer (d) is densely staining hillocks of dilator material (arrows) dilator. From these hillocks arise numerws highly fan-like manner. The stromal cells, the intercellular disposed perpendicular to the posterior surface of
The posterior epithelial cells (p) show numerous rell or muscle spurs encroach on the Aroma. The musrle The anterior surface of thr iris shows a wlati\x4,v dilation (LM). The posterior epithelial cells (p) aw The irregularly shaped nuclei are elongated along quit:: thick. The nuclei are irregular in shape. Small, are distributed all alon the stromal surfxe of tho branched dilator processes which spread out in a spaces and the lumens of the blood vessels aw all. the iris ( Y 1401)).
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nucleus along the long axis of the cell. At the junction of the dilator and sphincter there is an abrupt change from a columnar to a squamous epithelium lining the whole posterior aspect of the sphincter region up to the pupillary border. The dilator muscle layer is relatively thick and quite readily discernible with the light microscope (Figs 1 and 2). The nuclei. placed more or less in the center of the cells. va.ry both in size and shape. They are not as regularly placed within the entirety of the muscle layer as in the posterior epithelium [Fig. 2(b)]. These observations are probably a result of the plane of sectioning of the material. The nuclei are irregular in shape, sometimes somewhat rounded, wit,h grooves and indentations so that thev may even appear lobulated. At the boundary between the dilator and the stroma: the cytoplasm stains much more intensely, probably owing to an accumulation of myofilaments in this region [Fig. 2(b)]. At intervals all along this boundary zone, the dilat,or tnuscle cells send out a series of arhorescent processes [Pigs l(b) and 2(b)]. These cell processes, simple or complex, also stain darkly. Large or major processes with their many minor processe;i which radiate ant1 spread out in a fan-like manner, protrude into the stroma [Fig. 2(b)]. At times it seems that not only dilator cell processes but whole grcups of muscle cells encroach on the stroma [Figs l(a) and 2(a)]. The sphincter muscle is a distinct, but not, a compact. bundle at the pupillary region of thr iris [Fig. l(a)]. The s;troma extends from the root. to the pupillary margin of the iris. No attempt is rnwtl~ to stringently identify the various cell tvpes within the stroma. This has been adequately done (Hogan, Alvarado and Weddell, 1971). The main concern within this st.udv is to examine the overall arrangement of the cellular elements within the stroma rather than the inter-relationships of individual cell types. The stroma over the sphincter region is not very cellular and there are large intercellular spaces present. In the middle of the iris the stromal cells are very closely packed together. It i> impossiblt~ to trace the continuity of each cell. However. the numerous cyt,oplaxrnic cell processes tieem to be disposed perpendicularly with respect to the posterior layers of the iris. The spaces in between the cells and their processes give the impression of vertical linearity [Figs l(b) and 2(h)]. The stromal ct>lls then appear to he arrangetl in vcbrtical columns from the anterior to the posterior surface of t,he iris. Also, t.he nucifbi of the stromal cells seem to be kept away at a dist’ance from the dilator cell processes [Figs l(b) and 2(b)]. Only the stromal cell cytoplasmic processes appear to come into close proximity to the dilator muscle cell layer. The nuclei, having more bulk and perhaps being less deformable, seem to be held aloof from the dilator almost so as not to impede the l’ostulated folding up of the dilator during pupillary dilation. Most. of the blood vessels are large with open lumina filled with erybhrocytes. It is not certain if the blood vessels are kept patent as a result of the drugs employed. The lumina may be round or oval in shape (Figs 1 and 2). When the lumina are oval. the length of the oval is oriented similarly to the cells, that is, perpendicular to the post,erior surface of the iris. These blood vessels thus appear to be squeezed in frown side t,o side but not sufficiently to occlude the lumina. This is perhaps important for the maintenance of vital cell functions even in extreme pupillary dilation. The walls of the iridial vessels are not very thick in relationship to t.heir size. This is probably for greater flexibility during movements of the iris in pupillary dilation and constriction. The anterior surface of the iris may show a scalloped or uneven contour (Fig. 1) due to colutnns of cells protruding outwards or dipping inwards, or due to the bulging outwards of large blood vessels.
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iv. (‘. LIJI ASI) I\‘. A. \Vlc:Hl observed in the dilator and sphincter musclescorroborating the observations of Kellv and Arnold (1972). In this study, attention is focused only on specific aspects of the posterior epithelial and dilator layers, as it is here that ultrastructural changes are ohserved between pupillary dilation and constriction. (~6)The iris in pzq%blar~ tiilntion. The bulk of each posterior epithelial cell is $epatated from that of its neighbour hy deep and narrow grooves (Figs 4, 5. 6). There is a basement lamina following the contours of the posterior surface of the iris (Figs At 5; 6). The basal cell membranesalong the posterior and lateral walls of the posterior epit helial cells show numerous compl.icated infoldings which extend relatively deepl!F into t.he cell. The cell infoldings interdigitate with each other in a three-dimensional fashion so that they are always sectioned in different planes. The basement larnina follows the general contours of the posterior epithelial cells but it doesnot follow the outlines of the basal cell infoldings. In addition to the usual organellesin the cytoplasm of the posterior epithelial cells there are massesof fine filaments which have not been observed before (Figs 4, 5, 6). The filaments are sometimesvery clearly seenin bundles. They seemto cascadedown to surround the nucleus of the posterior epithelial cells (Pig. 5). These filaments are also found in the more apical part of the cell (Fig. 4). They form bundles which curve around the cell organelles. Very rarely is a bundle of filaments cut in cross-section (Fig. 6). Here, it is clearly seen that the filaments do form a distinct bundle devoid of cell organelles.
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The nuclei of the posterior epithelial cells assume different shapes hut in aLl instanceh they arc highly indented. Tongue-like cytoplasmic processes. devoid of cell organrll~~S~. occupy the indentations of the nuclear envelope (Figs 4. 5).
PIG. 4. The iris in pupillary dilatiou (TEM). The major portion of each posterior ppithrlial cell is separated from the next by a deep groove. The cell membrane is deeply and complexly infolded. The cell infoldings (ci) interdigitat,e in various planes. The basement lamiua (bm) follows the overall contours of the cells but not the contours of the cell infoldings. In the anterior cytoplasm there are bundles of intracellular filaments (if) cascading down. The nucleus is indentrd and the indentation is occupied by cell cytoplasm (*) ( i: 16 700).
It is impossible to delineate the boundaries of one post,erior epithelial cell from the next, nor the boundaries between the posterior epithelium and the dilat,or due to the large number of interdigitations of the processes of individual cells with each other or with those of neighbouring cells. Much like the posterior epithelial cells, the nuclei of the dilator muscle cells have a highly convoluted outline. The most striking feature, however, is seen along the stromal poles of the cells (Fig. 7). The sarcoplasm is very dense so that the myoflamentous nature of this layer is not always distinguishable but sometimes apparent [Fig. 7(b)]. There is one constant and interesting feature of the dilator that is always
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observed in irises in pupillary dilation. Numerous arborescent protrusions are regularly found all along the length of the dilator-stroma boundary [Fig. 7(a)]. These dilator processes may be relatively simple in configuration where they consist of single projections of the dilator into the stroma [Fig. 7(b)]. More often, the dilator
FIG. 5. The iris in pupillary dilation (TEM). The basement lamina (bm) of the posterior epithelial cell does not follow the contours of the cell infoldings (ci). A large bundle of intracellular filaments (if) forms a hammock around the nucleus. The indentations of the nucleus are filled with tongues of cytoplasm (*) ( x 23 500).
processes are quite complex and branch profusely (Fig. 7). Small hillocks of dilator muscle are disposed along the length of the dilator-stroma boundary. From these hillocks arise long, delicate dilator processes which seem to branch extensively in all planes (Fig. ‘7). All of the stromal surface of the dilator including the dilator hillocks and dilator processes is covered by a basement lamina. (b) The iris in pupillary constriction. In pupillary constriction, the posterior epithelial and dilator layers are much thinned out. Certain changes from that present in pupillary dilation are observed.
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The posterior surface of the iris is relatively smooth (Fig. 8). Thr~ :~rc no groovt’s in Iletween the posterior epithelial cells so that one riclye of cells cannot he s(~l)a~‘ate(l from the next. The basal cell membranes appear fo show a~ 111any itlfoldin,ns 2~s in pupillary dilation. The basement; la,nlina forms a straight covering for tht, I~asal surfaces of the posterior epithelial cells (Figs 8. 9). The intracellul:~r filanrt~nts.
FIG. 6. The iris in pupillary dilation (TEY). A large irregularly shaped nucleus is situated in the middle of the posterior epithelial cell. Intracellular filaments sectioned longitudinally (if I) and in cross-section (if 2) are present. The intracellular filaments form a discrete bundle (if 2). A basement lamiua (hm) is present (X 18 600).
longitudinally sectioned and usually located in the posterior portions of the cells appear to be aligned parallel to the length of the cells (Figs 8, 9 and 10). They run in bundles which may branch [Figs 8 and 10(a)] and criss-cross [Fig. 10(a)]. These filaments are quite prominent,ly seen in some cells and less so in others. Sometimes
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Fro. ‘7. (a) The iris in pupillary dilation (TEM). The nucleus of the dilator cell has a highly irregular and indented outline. The cytoplasm is very electron-dense especially in the region of the dilator hillocks (dh) and dilator processes (dp). Hillocks of dilator material are seen all along the stromal boundary of the dilator. From these hillocks arise a complex and profusely branching series of dilator processes which protrude int,o the stroma. The basement lamina (bm) of the dilator follows the contour of the dilator processes. The iris stroma consists of cells, blood vessels and an extensive network of collagen fibera which are sectioned in all planes ( x 8600). (b) The iris in pupillary dilation (TEM). The dilator cytoplasm is very electron-dense. There are few organelles in the stromal poles of the cells. The dilator processes may be simple protrusions of the dilat,or into the stroma (dp I) or they may arise from a dilator hillock (dh) and branch (dp 2). The basement lamins (bm) follows the outlinru of the dilator processes ( ,: 18 600).
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den,cities of the cell nlem branes a,rt~seen and occasionally a bundle of filan~ents appears< to attach to the densities of the ccl1 nlenlhrane [Fig. IO(h)]. Remarkable changes are seen with respect to the shapes of the nuclei of the posttriot epithelium and dilator when compared to that’ observed during pupillary dilation. The nuclei of the posterior epithelial cells are oval or elongated a& have a relatively smooth outline (Figs 9 and 11). The length of the nuclei are orientrtl parallel to th length of t,he cells and the posterior surface of thr iris.
E'IG. 8. The iris in pupillary constriction (TEM). Both the posterior epithelial (p) and dilator (d) layers are thin. The posterior surface of the iris is smooth and is covered by a basement lamina (bm). Intracellular filaments (if) are present in the cytoplasm of the posterior epithelial cell. The posterior epithelium and anterior epithelium are joined by apparent fusions of the cell membranes (double arrows). Occasionally the membranes are close together and there is Borne modification of the immediately adjacent cytoplasm (single arrow). Almost all the the dilator cell is occupied by a cigar-shaped nucleus oriented parallel to the posterior surface of the iris. The anterior surface of the dilator is smoot,h ( x 20 500).
A cursory glance at the boundary between the posterior epithelium and the dilator suggests that there are a number of cell junctions. This study is by no means intended to be an intensive or extensive study, The cell membranes may only come close together (Fig. 9) or the membranes may appear to fuse (Fig. 8) without any specialization of the adjacent cytoplasm. Occasionally the cell membranes are in close apposition and there is an apparent increased density of the surrounding cytoplasm (Fig. 8). Also, microvillous cytoplasmic processes from both the posterior epithelial and dilator
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layers interdigitate with each other, seemingly in a plane parallel to the posterior surface of the iris (Fig. 9). The dilator is thin (Figs 8,9 and 11). There is little cytoplasm in comparison to the size of the nucleus. The length of the long and thin dilator nucleus is oriented parallel to the length of the cell. The nuclear out,line is usuaUy smooth (Fig. 8) although rarely the nuclear envelope may be indented and a spit of cytoplasm is seen occupying the space (Fig. 9).
FIG. 9. The iris in pupillary constriction (TEN). There is an occasional bulge of the posterior surface of the iris. Cell infoldings (ci) are numerous. Intracellular filaments (if) are found in the posterior portion of the cells and are closely associated with the nucleus. The nucleus of the posterior epithelium is oval in shape and has a smooth outline. The nucleus of the dilator shows a deep indentation parallel to the length of the iris. Microvillous cytoplasmic processes from both layers interdigitate with each other (*). The contractile filamentous portion of the dilator is only a thin strip at the stromal pole of the cell. The basement lamina is perceptible as a straight line (bm). The stromal surface of t,he dilator is smooth (xlS600).
The muscular portion of the dilator cells is confined to a small region to the basal poles of the cells (Figs 8, 9 and 11). The sarcoplasm is not as dense as in pupiuary dilation. The myofilaments are seen running along the length of the stromal poles of the cells (Figs 8, 9 and 11). The anterior stromal surface of the dilator is normally smooth (Figs 8 and 9). Arborescent dilator processes are rarely seen (Fig. 11). They
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arc only present in isolated spots and are very simple in configuration. Small and simple dilator processes jut into t.he stroma (Fig. 1I). They do not show the complex branching that is observed in ln~pillary dilation. 4. Discussion in this investigation the iris is maintained in a fixed state of miosis or mydriasi.s with the aid of chemical mediators. A mixture of phenylephrine hydrochloride, a syniI’athomimetic drug, and cyclopentolate, an anti-muscarinic agent. is used to dilate the pupil. while echothiophatc iodide. a powerful anticholinest-rase. is used t,ct conytNrict~ the pupil (Goodman and Gilman. 1967). The question arises as to whether thtb histological and ultrastructural changes of the iris tissue that are seen in ohis study. as induced b;v the local administration of drugs. are truly indicative of the changes that, normally occur in response t’o changing light conditions without the intervent,ion of tin c.xternal agent. From the present studies this question remains unresolvetl although there is some indication from our preliminary unpublished data that there is no difference that can be perceived. Without, the aid of drugs it is difficult to obtain the iris with t,he desired pupillary diameter. The most striking differences between the histology and ultrastructure of the iris when it is in pupillary dilation from when it is in pupillary constriction are primarily noted in the posterior epithelium and the dilator muscle layer. From miosis to mydriasis there is an apparent drastic change in the total exposed area of the iris, as is clearly shown in scanning ele.ctron microscopic studies (Lim and Webber. 1975). The shapes of the posterior epithelial cells and the dilator cells. and their relationships to each other alter in response to changes in pupillarv size. In pupillary dilation the post,erior surface of the iris’is most. often deeply fringed a;; a result, of the rows of peg-like epithelial cells. Thel- do not appear to be arranged in arcades as in the monkey iris where each arch is n&c up of eight to 10 cells (Alphen. 1963) although probably each ridge of cells in the rat iris map be made up of a staggered series of cells whose cytoplasmic processes int?rtligitate with each other to some extent. In pupillary constriction the configuration of the posterior epithelial cells is markedly changed. They flatten out and appear as a thin layer. There s?ems to be a remarkable fluidity and malleability to the epithrlial cells and to their relationships wit.11 each other and with the dilator muscle cells. Although the thickness of the basement, lamina was not measured, it appears qualitatively t,o have been t(hinnetl out in pupillary constriction and is less readily perceptible. The nuclei of the posterior epithelial cells also appear to be highly plastic and change both in their nuclear outline and in their orientation within the cells. Masses of intSracellular filaments, often associated with the nuclei. change in their orientation FII:. 10. (a) The iris in pupillary constriction (TEN). Cell infoldings (ci) occupy most of the posterlr,r aspect of the posterior epithelium. There are also large buudles of intracellular filaments (if) which run parallvl to the posterior surface of the iris. The filaments may branch, or they may come together and intermesh ( Y 25 000). (b) The iris in pupillary constrict,ion (TEM). The intracellular filaments (if) run in bun~lh~s parallel to t.he posterior surface of the iris. Sometimes they appear to attach to dense areas on the wll membrane of the posterior epithelium (arrow) ( IX 600). Flu. 11. The iris in pupillacy constriction (TEN). A few dilator processes (dp) are seeu along the anterior surface of the dilator. These are relatively simple protrusions of the dilator into the stroma. The myofilaments of the dilator are seen as longitudinal bundles running along the length of the cells. They are found in the stromal as well as the deeper park of the cells. The basement lamina (hm) is readily yisiblr (x20 500).
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within the cell in n1iosi.q a,nd rnydriasis as well. \Ve can only speculate as to thtb pos~il)lt* nature and function of t,hrse filanlrlnts. Perhaps. thcay arc only a structursl conq)orlrnt of the epithelial cells. ()r. thev. UIRV have a. certain elast,icitv t,o thclnr. Rut ZN\. calasticity WOUI~ seem to I)e fen&onal onlr if the filatrlents are anchorc~tl in position ;It certain point’s. There is only a hint that s;nle of the filaments are :~t~tachcd to ttrcb crll membranes. Or. perhaps the filaments mav havta contractile properties. Thcv rc1r111l possibly be actin filaments but? this aspect‘was not explored further. (.‘hanges are also ohserved in the dilator layer from pupillary dilation to pupillary constrichion. The dilator nuclei are also able to change both in their shape ant1 orientation within the cell. The> most striking changes t,hough are seen along thfa boundary zone between the dilat,or cells and the stroma. In pupillarp dilation numerous simple or complex arhorescent dilator processes a.re ol)servcd all along the bounttarv zone. They stain verv darkly wit,11 toluidine blue and are also very elect,ron dense. The dilator processes apiear ~0 6, wpll-st,ructured cornponcnts. Since the dilator isattachetl by iti; epithelial portion to the posterior c~pithelium. which is itself a Imlky st:.ucturr. in pupillary dilation there seems to I)e not enough room to accommodate all of the dilator cells. Parts of’ the cells appear to be squeezed ~JU~WardS and this is appartJnt,l\, ~nost possible anteriorly into the relatively loose stroma. It would I)e inbercsting Tao speculate that there a,re certain Tvell-defined regions all along the strotnal surfaor of the dilator muscle cells where dilator processes preferentially protrude outwards. They do not seem to have been formed haphazardly. Perhaps. the ctilator cells art’ also arranged in contractile units. .4t least. in the sphincter, there is some indication t,hat the muscle cells are arranged in functional groups (Hogan. Alvarado and Weddell, 1971). This may be the case with the dilator as well. The force of contraction for each functional unit of cells may 1)e eoncsntrated at certain points. Herth the dilator material would l-bulge outward as a hillock from which would arise a seriiss of profusely branched dilator processes. In pupillary constriction the dilator layer is harely visihle light microscopically. With the transmission electron microscope the dilator cells are thin. The stromal surfaces of the dilator, unlike in pupillary dilation. is usually smooth. Very rarely there may be a few short dilator processes jutting into the stroma. The whole anterior surface of the dilator has been flattened. It appears that all of the dilator cell can he accommodated within one layer and there is no necessity for protrusions of the dilator to occur. In the stroma the main concern is in the overall arrangement of the cells and their changes in orientation from pupillary dilat,ion to pupillary constriction. In mydriasis, the stromal cells and their processes appear to be disposed in vert.ical columns extending from the anterior surface of the iris to the boundary zone with the dilator. The spaces in between the cells accentuate this impression of vertical linearity. In miosis? the stromal cells and the associated intercellular spaces are no longer perpendicular but’ parallel to the posterior surface of the iris. It has been previously shown that the collagen network of the iris stroma follows closely the changes in orientabion of the stromal cells (Lim and Webber, 1972). Thus, between the two extremes of pupillary size, the stromal components can apparently go through a shift in position. ACKNOWLEDGMENTS We greatly appreciate the assistance of Mrs Pam Gill in performing the perfusiorlti aud that of Mrs Leny Volkov in the printing of the micrographs. This research was supported by a Medical Research Council Studentship to Wan C. Lim.
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REFERENCES Alphen. G. W. H. 51. (1963). The st.ructural changes in miosis and myclriasis of the monkey eye. Arch.. Ophthdmol. 69, 802-14. Hogan. M. J., Alvarado, J. A. and \Veddell, J. E. (1971). Histology qf thr Humnn Eye-n Atlna nnrl Text. W. B. Saunders Co.. Toronto. Kelly. R,. E. and Arnold, J. W. (1972). Nyofilaments of the pupillary muscles of the iris fixed ill situ. J. Ultrastruet. Res. 40, 53%45. Lim. W. C. and Webber, W. A. (197’7). The collagen network in the stroma of the rat iris. Proc. Con. Fed. Biol. Sot.? p. 181. Lim, IV. C. and Wcbber, W. A. (1975). A scanning electron microscopic study of the posterior and anterior surfaces of the rat iris in pupillary dilation and constriction. Exp. Eye Res. 20,44561. Saba.tini. D. D., Bansch. K. and Barrnett, R. J. (1963). Cytochemistry and electron microscopy. The preservation of cellular ultrastructure and enzymatic activity hy aldehyde fixation. J. Cdl Biol. 17, 19-58.
A Light and Transmission Electron-microscopic Study of the Rat Iris in Pupillary Dilation and Constriction
Department of Anntvmy, McGill
liniversity,
P.0. Rex 6070, &!oatrenl, Quebec
AXD WILLIAMS
Department of Anatomy,
Uniwrsity
8.
WEBBER*
of Bhtish
(Received 7 April
Colrw~hin. T’omwwer
B.C., Camdn
197,5, Boston)
Adult rat pupils were dilated with a mixture of 5” ,(, phenylephrine hydrochloride and 0.5”,, cyclopentolate, or constricted with 0.125y0 echothiophate iodide. The iris tissues were then prepared for and examined by light and electron microscopy. In pupillary dilation the iris is short as seen in meridional sections. The posterior epithelial cells are high columnar and are separated from the adjacent cells thus giving a scalloped appearance to the posterior surface of the iris. The nuclei have irregular outlines. They are oriented with their long axis along the long axis of the cells. Bundles of intracellular fila&ent,s, often associated witkthe nuclei, may f&n a hammock around the nucleus. The dilator muscle laver is relativelv thick. The nuclei are highlv indented. The most prominent structures present are the series of dilator hillocks and Khe& associated profusely branching dilator processes seen all along the boundary zone between the dilator and the stroma. They protrude int.o the stroma. The stromal cells and blood vessels appear to bc disposed in vertical columns perpendicular to the posterior epithelial and dilator layers. [n pupillaq constriction the iris is long and thin as seen in meridional sections. Most of the posterior surface of the iris is smooth. Both the posterior epit,helial and dilator layers are thin. The nuclei of the posterior epit,helial cells and of t,he dilator mnscle cells have smooth nuclear outlines and thev are oriented along the length of the cells. The intracellular filaments, mainly situatei in the posterior portions of the posterior epithelial cells, are seen as longitudinal bundles parallel to the length of the cells. Dilator hillocks and dilator processes. so prominent in pupillary dilation. are absent. The stromal components are oriented I~~rallel to the posberior surface of the iris.
1. Introduction The irip is a dynamic stru&ure capable of highly precise and rapidly occurring changes in pupillary diameter both in response to changing light conditions and in response to rniotics and mydriatics. In extreme pupillary constriction, the iris presents a large surface area with a small pupil. In pupillary dilation the pupil is much enlargad and the iris appears as a thin rim of tissue. Obviously this must entail structural changes within the iris tissue to accommodate for the great changes in total surface area from pupillary constriction to pupillary dilation: Also the iris architecture must be structurally adapt,ed to facilitate these movements so that they occur as smoothly as possible. Scanning electron microscopic studies of the constricted and dilated rat iris have revealed dramatic changes both on the posterior and anterior surfaces of the iris (Lim and Webber, 1975). To corroborate and amplify these findings, the present investigation is carried out using both the light and transmission electron microscopes. Changes * Please direct
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in the shape and orientation of the posterior epitheliutn. the tlilator and thtr st,rom;lI elements, as well as the relationships of these iris components to each other. are hettrl examined with the light microscope. The rat iris is also examined with the trallsmissioll electron microscope to see what ultrastructural changes. if any. OCWT. cspccially iu the posterior epithelium and the dilator.
2. Methods Adult rats of the Wistar straiu were used. Their pupils were dilated with a mixture of 59; phenylephrine hydrochloride aud 0~5~;~ cyalopeutolate, or constrict,ed with 0.1250,, echothiophate iodide. These drugs were allowed to act. for 15-20 miu, zt which time t,he pupils were widely dilated or much constricted, as the case may be. For the light microscopic studies, the rats were auesthetized with ether and the eyes removed immediately and immersed in the fixative of 4: O glutaraldehyde in 0.1 >I-sodium cacodylate buffer (Sabatini, Bensch and Baruett, 1963) at pH 7.2 at room temperature. For the transmission electron microscopic studies, the rats were anesthetized 1)~ au iutraperitoneal injection of sodium peutobarbitol and a subcutaneous injection of sodiunl pheuobarbitone. The left common carotid artery was exposed and cannulated. Thr cannula was connected to a ‘20 ml syringe containing the fixative of 4% glutaraldehyde iu 0.1 Iw-sodium cacodylate buffer. The inferior vena cava was also exposed. The fixative was perfused through the carotid artery at a pressure of 100 nmHg. As soon as perfusion started, the inferior vena cava was cut just below the rem1 vessels for drainage to take . place. The perfused eyes were removed and immersed in fresh fixative. Au antero-posterior slit was made through the corueal~scleral junctiou extending front the anterior and posterior chambers into the vitreous humor to facilitate the peuetratiou of the fixative into the iris tissue. After about 30 miu in the fixation, the posterior halves of the eyes and the lenses were removed and discarded. The eyes were fixed for a total of 4 hr, washed with and stored in 0.1 wsodium cacodylate buffer at pH 7.2 at 4°C. The anterior portions of the eyes, with the &tached iris, were trimmed into smaller pieces while in the buffer wash. The tissues were post-fixed for 1 hr in 19; osmium tetroxide in 0.1 >I-sodium eacodylate buffer, stained en bloc iu urarlyl acetate for 1 hr, dehydrated through a graded series of alcohols, infiltrated ant1 embedded in epou Araldite. Thick sections for the light microscopic st)udies were stained with toluidine blue aud examine(l on >l Zeiss Photomicroscope II. For the transmission electrou microscopic studies, thin sections of well-preserved tissues, ils determined light microscopically on thick section, were stained with lead citrate for 2.11~2..5 mill and exanlinwl on a Philips EN 200 operatitlp at 60 kV.
3. Results Light microscopicstudy (a) The iris in papillary dilation. When the pupil is well dilated the iris is short in terms of its dimensions from the root to the tip of the iris, as seen in meridoneal sections. The iris tends to be disposed in a slight convex arch towards the surface of the cornea due to the fact that the stroma in the middle third of the iris is thicker than that at the pupillary or peripheral region [Fig. l(a)]. In meridional sections. the posterior epithelial cells form a layer covering the posterior surface of the iris [Fig. l(a)]. Th e major portion of each cell may be discretely separated from its neighbouring cell or cells thus giving the posterior surface a deeply fringed appearance (Figs 1 and 2). Most probably cytoplasmic processes from neighbouring cells interdigitate with each other. Sometimes the posterior epithelial cells
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may be so closely abutted on each other t.hat there are no gaps in between the cells. The posterior epithelial cells are most often large, columnar in shape and they have a highly irregular cell outline [Figs l(b), 2(a) and (b)]. The height of the cells varies with the degree of pupillary dilation. The anterior border of these cells is in apposition with the anterior epithelium and dilator muscle cells. In the center of each cell is an elongated nucleus wioh a very irregular outline, oriented with the long axis of the
FIIJ. 1. (a) The iris in pupillary dilation (LM). The iris is short. Both the posterior epithelium (p) and dilator (d) are distinctly visible. The st,roma is thick especially in t.hc middle one third of the iris so that the iris appears to buckle anteriorly. Dilator muscle spurs (arrows) are seen in the pupillary half of the iris. The sphincter (s) is a mass of small cells at the pupillary tip ( i 90). (b) The iris in pupillarp dilat’ion {IX). The posterior epithelial cells (p) are peg-like girin g the posterior surface a deeply fringed appearance. The nuclei are large and are situated in the middle of the cells. The dilator (d) is a relatively thick layer. Fine dark-staining processes from the dilator are seen all along the dilator-stroma boundary (arr(ws). The strorna is filled with blood vessels having round or oval lumens. Some of the blood vessels appear to have only an endothelial lining (bv 1) while others possess in addition a layer of pericytes (br 2). The stromal cells are arranged in columns extending from the anterior surface of the iris to t,he dilatorPstroma boundary. The stromal cell nuclei are kept at, a distance from the dilator. The anterior surface of the iris is highly scalloped in outline. This is mainly due to the bulging outwards of the iris Idoud vessels ( ,: 240).
Fro. 2. (a) The iris in pupillary dilation (LM). processes devoid of nuclei. Groups of dilat,or cells spurs may also show fine dilator processrs (arrow). undulating outline ( x 380). (b) The iris in pupillarg large, high columnar with an irregular CPII out,line. the long axes of the cells. The dilator layer (d) is densely staining hillocks of dilator material (arrows) dilator. From these hillocks arise numerws highly fan-like manner. The stromal cells, the intercellular disposed perpendicular to the posterior surface of
The posterior epithelial cells (p) show numerous rell or muscle spurs encroach on the Aroma. The musrle The anterior surface of thr iris shows a wlati\x4,v dilation (LM). The posterior epithelial cells (p) aw The irregularly shaped nuclei are elongated along quit:: thick. The nuclei are irregular in shape. Small, are distributed all alon the stromal surfxe of tho branched dilator processes which spread out in a spaces and the lumens of the blood vessels aw all. the iris ( Y 1401)).
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nucleus along the long axis of the cell. At the junction of the dilator and sphincter there is an abrupt change from a columnar to a squamous epithelium lining the whole posterior aspect of the sphincter region up to the pupillary border. The dilator muscle layer is relatively thick and quite readily discernible with the light microscope (Figs 1 and 2). The nuclei. placed more or less in the center of the cells. va.ry both in size and shape. They are not as regularly placed within the entirety of the muscle layer as in the posterior epithelium [Fig. 2(b)]. These observations are probably a result of the plane of sectioning of the material. The nuclei are irregular in shape, sometimes somewhat rounded, wit,h grooves and indentations so that thev may even appear lobulated. At the boundary between the dilator and the stroma: the cytoplasm stains much more intensely, probably owing to an accumulation of myofilaments in this region [Fig. 2(b)]. At intervals all along this boundary zone, the dilat,or tnuscle cells send out a series of arhorescent processes [Pigs l(b) and 2(b)]. These cell processes, simple or complex, also stain darkly. Large or major processes with their many minor processe;i which radiate ant1 spread out in a fan-like manner, protrude into the stroma [Fig. 2(b)]. At times it seems that not only dilator cell processes but whole grcups of muscle cells encroach on the stroma [Figs l(a) and 2(a)]. The sphincter muscle is a distinct, but not, a compact. bundle at the pupillary region of thr iris [Fig. l(a)]. The s;troma extends from the root. to the pupillary margin of the iris. No attempt is rnwtl~ to stringently identify the various cell tvpes within the stroma. This has been adequately done (Hogan, Alvarado and Weddell, 1971). The main concern within this st.udv is to examine the overall arrangement of the cellular elements within the stroma rather than the inter-relationships of individual cell types. The stroma over the sphincter region is not very cellular and there are large intercellular spaces present. In the middle of the iris the stromal cells are very closely packed together. It i> impossiblt~ to trace the continuity of each cell. However. the numerous cyt,oplaxrnic cell processes tieem to be disposed perpendicularly with respect to the posterior layers of the iris. The spaces in between the cells and their processes give the impression of vertical linearity [Figs l(b) and 2(h)]. The stromal ct>lls then appear to he arrangetl in vcbrtical columns from the anterior to the posterior surface of t,he iris. Also, t.he nucifbi of the stromal cells seem to be kept away at a dist’ance from the dilator cell processes [Figs l(b) and 2(b)]. Only the stromal cell cytoplasmic processes appear to come into close proximity to the dilator muscle cell layer. The nuclei, having more bulk and perhaps being less deformable, seem to be held aloof from the dilator almost so as not to impede the l’ostulated folding up of the dilator during pupillary dilation. Most. of the blood vessels are large with open lumina filled with erybhrocytes. It is not certain if the blood vessels are kept patent as a result of the drugs employed. The lumina may be round or oval in shape (Figs 1 and 2). When the lumina are oval. the length of the oval is oriented similarly to the cells, that is, perpendicular to the post,erior surface of the iris. These blood vessels thus appear to be squeezed in frown side t,o side but not sufficiently to occlude the lumina. This is perhaps important for the maintenance of vital cell functions even in extreme pupillary dilation. The walls of the iridial vessels are not very thick in relationship to t.heir size. This is probably for greater flexibility during movements of the iris in pupillary dilation and constriction. The anterior surface of the iris may show a scalloped or uneven contour (Fig. 1) due to colutnns of cells protruding outwards or dipping inwards, or due to the bulging outwards of large blood vessels.
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iv. (‘. LIJI ASI) I\‘. A. \Vlc:Hl observed in the dilator and sphincter musclescorroborating the observations of Kellv and Arnold (1972). In this study, attention is focused only on specific aspects of the posterior epithelial and dilator layers, as it is here that ultrastructural changes are ohserved between pupillary dilation and constriction. (~6)The iris in pzq%blar~ tiilntion. The bulk of each posterior epithelial cell is $epatated from that of its neighbour hy deep and narrow grooves (Figs 4, 5. 6). There is a basement lamina following the contours of the posterior surface of the iris (Figs At 5; 6). The basal cell membranesalong the posterior and lateral walls of the posterior epit helial cells show numerous compl.icated infoldings which extend relatively deepl!F into t.he cell. The cell infoldings interdigitate with each other in a three-dimensional fashion so that they are always sectioned in different planes. The basement larnina follows the general contours of the posterior epithelial cells but it doesnot follow the outlines of the basal cell infoldings. In addition to the usual organellesin the cytoplasm of the posterior epithelial cells there are massesof fine filaments which have not been observed before (Figs 4, 5, 6). The filaments are sometimesvery clearly seenin bundles. They seemto cascadedown to surround the nucleus of the posterior epithelial cells (Pig. 5). These filaments are also found in the more apical part of the cell (Fig. 4). They form bundles which curve around the cell organelles. Very rarely is a bundle of filaments cut in cross-section (Fig. 6). Here, it is clearly seen that the filaments do form a distinct bundle devoid of cell organelles.
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The nuclei of the posterior epithelial cells assume different shapes hut in aLl instanceh they arc highly indented. Tongue-like cytoplasmic processes. devoid of cell organrll~~S~. occupy the indentations of the nuclear envelope (Figs 4. 5).
PIG. 4. The iris in pupillary dilatiou (TEM). The major portion of each posterior ppithrlial cell is separated from the next by a deep groove. The cell membrane is deeply and complexly infolded. The cell infoldings (ci) interdigitat,e in various planes. The basement lamiua (bm) follows the overall contours of the cells but not the contours of the cell infoldings. In the anterior cytoplasm there are bundles of intracellular filaments (if) cascading down. The nucleus is indentrd and the indentation is occupied by cell cytoplasm (*) ( i: 16 700).
It is impossible to delineate the boundaries of one post,erior epithelial cell from the next, nor the boundaries between the posterior epithelium and the dilat,or due to the large number of interdigitations of the processes of individual cells with each other or with those of neighbouring cells. Much like the posterior epithelial cells, the nuclei of the dilator muscle cells have a highly convoluted outline. The most striking feature, however, is seen along the stromal poles of the cells (Fig. 7). The sarcoplasm is very dense so that the myoflamentous nature of this layer is not always distinguishable but sometimes apparent [Fig. 7(b)]. There is one constant and interesting feature of the dilator that is always
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observed in irises in pupillary dilation. Numerous arborescent protrusions are regularly found all along the length of the dilator-stroma boundary [Fig. 7(a)]. These dilator processes may be relatively simple in configuration where they consist of single projections of the dilator into the stroma [Fig. 7(b)]. More often, the dilator
FIG. 5. The iris in pupillary dilation (TEM). The basement lamina (bm) of the posterior epithelial cell does not follow the contours of the cell infoldings (ci). A large bundle of intracellular filaments (if) forms a hammock around the nucleus. The indentations of the nucleus are filled with tongues of cytoplasm (*) ( x 23 500).
processes are quite complex and branch profusely (Fig. 7). Small hillocks of dilator muscle are disposed along the length of the dilator-stroma boundary. From these hillocks arise long, delicate dilator processes which seem to branch extensively in all planes (Fig. ‘7). All of the stromal surface of the dilator including the dilator hillocks and dilator processes is covered by a basement lamina. (b) The iris in pupillary constriction. In pupillary constriction, the posterior epithelial and dilator layers are much thinned out. Certain changes from that present in pupillary dilation are observed.
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The posterior surface of the iris is relatively smooth (Fig. 8). Thr~ :~rc no groovt’s in Iletween the posterior epithelial cells so that one riclye of cells cannot he s(~l)a~‘ate(l from the next. The basal cell membranes appear fo show a~ 111any itlfoldin,ns 2~s in pupillary dilation. The basement; la,nlina forms a straight covering for tht, I~asal surfaces of the posterior epithelial cells (Figs 8. 9). The intracellul:~r filanrt~nts.
FIG. 6. The iris in pupillary dilation (TEY). A large irregularly shaped nucleus is situated in the middle of the posterior epithelial cell. Intracellular filaments sectioned longitudinally (if I) and in cross-section (if 2) are present. The intracellular filaments form a discrete bundle (if 2). A basement lamiua (hm) is present (X 18 600).
longitudinally sectioned and usually located in the posterior portions of the cells appear to be aligned parallel to the length of the cells (Figs 8, 9 and 10). They run in bundles which may branch [Figs 8 and 10(a)] and criss-cross [Fig. 10(a)]. These filaments are quite prominent,ly seen in some cells and less so in others. Sometimes
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Fro. ‘7. (a) The iris in pupillary dilation (TEM). The nucleus of the dilator cell has a highly irregular and indented outline. The cytoplasm is very electron-dense especially in the region of the dilator hillocks (dh) and dilator processes (dp). Hillocks of dilator material are seen all along the stromal boundary of the dilator. From these hillocks arise a complex and profusely branching series of dilator processes which protrude int,o the stroma. The basement lamina (bm) of the dilator follows the contour of the dilator processes. The iris stroma consists of cells, blood vessels and an extensive network of collagen fibera which are sectioned in all planes ( x 8600). (b) The iris in pupillary dilation (TEM). The dilator cytoplasm is very electron-dense. There are few organelles in the stromal poles of the cells. The dilator processes may be simple protrusions of the dilat,or into the stroma (dp I) or they may arise from a dilator hillock (dh) and branch (dp 2). The basement lamins (bm) follows the outlinru of the dilator processes ( ,: 18 600).
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den,cities of the cell nlem branes a,rt~seen and occasionally a bundle of filan~ents appears< to attach to the densities of the ccl1 nlenlhrane [Fig. IO(h)]. Remarkable changes are seen with respect to the shapes of the nuclei of the posttriot epithelium and dilator when compared to that’ observed during pupillary dilation. The nuclei of the posterior epithelial cells are oval or elongated a& have a relatively smooth outline (Figs 9 and 11). The length of the nuclei are orientrtl parallel to th length of t,he cells and the posterior surface of thr iris.
E'IG. 8. The iris in pupillary constriction (TEM). Both the posterior epithelial (p) and dilator (d) layers are thin. The posterior surface of the iris is smooth and is covered by a basement lamina (bm). Intracellular filaments (if) are present in the cytoplasm of the posterior epithelial cell. The posterior epithelium and anterior epithelium are joined by apparent fusions of the cell membranes (double arrows). Occasionally the membranes are close together and there is Borne modification of the immediately adjacent cytoplasm (single arrow). Almost all the the dilator cell is occupied by a cigar-shaped nucleus oriented parallel to the posterior surface of the iris. The anterior surface of the dilator is smoot,h ( x 20 500).
A cursory glance at the boundary between the posterior epithelium and the dilator suggests that there are a number of cell junctions. This study is by no means intended to be an intensive or extensive study, The cell membranes may only come close together (Fig. 9) or the membranes may appear to fuse (Fig. 8) without any specialization of the adjacent cytoplasm. Occasionally the cell membranes are in close apposition and there is an apparent increased density of the surrounding cytoplasm (Fig. 8). Also, microvillous cytoplasmic processes from both the posterior epithelial and dilator
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layers interdigitate with each other, seemingly in a plane parallel to the posterior surface of the iris (Fig. 9). The dilator is thin (Figs 8,9 and 11). There is little cytoplasm in comparison to the size of the nucleus. The length of the long and thin dilator nucleus is oriented parallel to the length of the cell. The nuclear out,line is usuaUy smooth (Fig. 8) although rarely the nuclear envelope may be indented and a spit of cytoplasm is seen occupying the space (Fig. 9).
FIG. 9. The iris in pupillary constriction (TEN). There is an occasional bulge of the posterior surface of the iris. Cell infoldings (ci) are numerous. Intracellular filaments (if) are found in the posterior portion of the cells and are closely associated with the nucleus. The nucleus of the posterior epithelium is oval in shape and has a smooth outline. The nucleus of the dilator shows a deep indentation parallel to the length of the iris. Microvillous cytoplasmic processes from both layers interdigitate with each other (*). The contractile filamentous portion of the dilator is only a thin strip at the stromal pole of the cell. The basement lamina is perceptible as a straight line (bm). The stromal surface of t,he dilator is smooth (xlS600).
The muscular portion of the dilator cells is confined to a small region to the basal poles of the cells (Figs 8, 9 and 11). The sarcoplasm is not as dense as in pupiuary dilation. The myofilaments are seen running along the length of the stromal poles of the cells (Figs 8, 9 and 11). The anterior stromal surface of the dilator is normally smooth (Figs 8 and 9). Arborescent dilator processes are rarely seen (Fig. 11). They
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arc only present in isolated spots and are very simple in configuration. Small and simple dilator processes jut into t.he stroma (Fig. 1I). They do not show the complex branching that is observed in ln~pillary dilation. 4. Discussion in this investigation the iris is maintained in a fixed state of miosis or mydriasi.s with the aid of chemical mediators. A mixture of phenylephrine hydrochloride, a syniI’athomimetic drug, and cyclopentolate, an anti-muscarinic agent. is used to dilate the pupil. while echothiophatc iodide. a powerful anticholinest-rase. is used t,ct conytNrict~ the pupil (Goodman and Gilman. 1967). The question arises as to whether thtb histological and ultrastructural changes of the iris tissue that are seen in ohis study. as induced b;v the local administration of drugs. are truly indicative of the changes that, normally occur in response t’o changing light conditions without the intervent,ion of tin c.xternal agent. From the present studies this question remains unresolvetl although there is some indication from our preliminary unpublished data that there is no difference that can be perceived. Without, the aid of drugs it is difficult to obtain the iris with t,he desired pupillary diameter. The most striking differences between the histology and ultrastructure of the iris when it is in pupillary dilation from when it is in pupillary constriction are primarily noted in the posterior epithelium and the dilator muscle layer. From miosis to mydriasis there is an apparent drastic change in the total exposed area of the iris, as is clearly shown in scanning ele.ctron microscopic studies (Lim and Webber. 1975). The shapes of the posterior epithelial cells and the dilator cells. and their relationships to each other alter in response to changes in pupillarv size. In pupillary dilation the post,erior surface of the iris’is most. often deeply fringed a;; a result, of the rows of peg-like epithelial cells. Thel- do not appear to be arranged in arcades as in the monkey iris where each arch is n&c up of eight to 10 cells (Alphen. 1963) although probably each ridge of cells in the rat iris map be made up of a staggered series of cells whose cytoplasmic processes int?rtligitate with each other to some extent. In pupillary constriction the configuration of the posterior epithelial cells is markedly changed. They flatten out and appear as a thin layer. There s?ems to be a remarkable fluidity and malleability to the epithrlial cells and to their relationships wit.11 each other and with the dilator muscle cells. Although the thickness of the basement, lamina was not measured, it appears qualitatively t,o have been t(hinnetl out in pupillary constriction and is less readily perceptible. The nuclei of the posterior epithelial cells also appear to be highly plastic and change both in their nuclear outline and in their orientation within the cells. Masses of intSracellular filaments, often associated with the nuclei. change in their orientation FII:. 10. (a) The iris in pupillary constriction (TEN). Cell infoldings (ci) occupy most of the posterlr,r aspect of the posterior epithelium. There are also large buudles of intracellular filaments (if) which run parallvl to the posterior surface of the iris. The filaments may branch, or they may come together and intermesh ( Y 25 000). (b) The iris in pupillary constrict,ion (TEM). The intracellular filaments (if) run in bun~lh~s parallel to t.he posterior surface of the iris. Sometimes they appear to attach to dense areas on the wll membrane of the posterior epithelium (arrow) ( IX 600). Flu. 11. The iris in pupillacy constriction (TEN). A few dilator processes (dp) are seeu along the anterior surface of the dilator. These are relatively simple protrusions of the dilator into the stroma. The myofilaments of the dilator are seen as longitudinal bundles running along the length of the cells. They are found in the stromal as well as the deeper park of the cells. The basement lamina (hm) is readily yisiblr (x20 500).
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within the cell in n1iosi.q a,nd rnydriasis as well. \Ve can only speculate as to thtb pos~il)lt* nature and function of t,hrse filanlrlnts. Perhaps. thcay arc only a structursl conq)orlrnt of the epithelial cells. ()r. thev. UIRV have a. certain elast,icitv t,o thclnr. Rut ZN\. calasticity WOUI~ seem to I)e fen&onal onlr if the filatrlents are anchorc~tl in position ;It certain point’s. There is only a hint that s;nle of the filaments are :~t~tachcd to ttrcb crll membranes. Or. perhaps the filaments mav havta contractile properties. Thcv rc1r111l possibly be actin filaments but? this aspect‘was not explored further. (.‘hanges are also ohserved in the dilator layer from pupillary dilation to pupillary constrichion. The dilator nuclei are also able to change both in their shape ant1 orientation within the cell. The> most striking changes t,hough are seen along thfa boundary zone between the dilat,or cells and the stroma. In pupillarp dilation numerous simple or complex arhorescent dilator processes a.re ol)servcd all along the bounttarv zone. They stain verv darkly wit,11 toluidine blue and are also very elect,ron dense. The dilator processes apiear ~0 6, wpll-st,ructured cornponcnts. Since the dilator isattachetl by iti; epithelial portion to the posterior c~pithelium. which is itself a Imlky st:.ucturr. in pupillary dilation there seems to I)e not enough room to accommodate all of the dilator cells. Parts of’ the cells appear to be squeezed ~JU~WardS and this is appartJnt,l\, ~nost possible anteriorly into the relatively loose stroma. It would I)e inbercsting Tao speculate that there a,re certain Tvell-defined regions all along the strotnal surfaor of the dilator muscle cells where dilator processes preferentially protrude outwards. They do not seem to have been formed haphazardly. Perhaps. the ctilator cells art’ also arranged in contractile units. .4t least. in the sphincter, there is some indication t,hat the muscle cells are arranged in functional groups (Hogan. Alvarado and Weddell, 1971). This may be the case with the dilator as well. The force of contraction for each functional unit of cells may 1)e eoncsntrated at certain points. Herth the dilator material would l-bulge outward as a hillock from which would arise a seriiss of profusely branched dilator processes. In pupillary constriction the dilator layer is harely visihle light microscopically. With the transmission electron microscope the dilator cells are thin. The stromal surfaces of the dilator, unlike in pupillary dilation. is usually smooth. Very rarely there may be a few short dilator processes jutting into the stroma. The whole anterior surface of the dilator has been flattened. It appears that all of the dilator cell can he accommodated within one layer and there is no necessity for protrusions of the dilator to occur. In the stroma the main concern is in the overall arrangement of the cells and their changes in orientation from pupillary dilat,ion to pupillary constriction. In mydriasis, the stromal cells and their processes appear to be disposed in vert.ical columns extending from the anterior surface of the iris to the boundary zone with the dilator. The spaces in between the cells accentuate this impression of vertical linearity. In miosis? the stromal cells and the associated intercellular spaces are no longer perpendicular but’ parallel to the posterior surface of the iris. It has been previously shown that the collagen network of the iris stroma follows closely the changes in orientabion of the stromal cells (Lim and Webber, 1972). Thus, between the two extremes of pupillary size, the stromal components can apparently go through a shift in position. ACKNOWLEDGMENTS We greatly appreciate the assistance of Mrs Pam Gill in performing the perfusiorlti aud that of Mrs Leny Volkov in the printing of the micrographs. This research was supported by a Medical Research Council Studentship to Wan C. Lim.
LX1 $ND
TEhI-DILATED.
CONSTRICTED
RST
IRIS
449
REFERENCES Alphen. G. W. H. 51. (1963). The st.ructural changes in miosis and myclriasis of the monkey eye. Arch.. Ophthdmol. 69, 802-14. Hogan. M. J., Alvarado, J. A. and \Veddell, J. E. (1971). Histology qf thr Humnn Eye-n Atlna nnrl Text. W. B. Saunders Co.. Toronto. Kelly. R,. E. and Arnold, J. W. (1972). Nyofilaments of the pupillary muscles of the iris fixed ill situ. J. Ultrastruet. Res. 40, 53%45. Lim. W. C. and Webber, W. A. (197’7). The collagen network in the stroma of the rat iris. Proc. Con. Fed. Biol. Sot.? p. 181. Lim, IV. C. and Wcbber, W. A. (1975). A scanning electron microscopic study of the posterior and anterior surfaces of the rat iris in pupillary dilation and constriction. Exp. Eye Res. 20,44561. Saba.tini. D. D., Bansch. K. and Barrnett, R. J. (1963). Cytochemistry and electron microscopy. The preservation of cellular ultrastructure and enzymatic activity hy aldehyde fixation. J. Cdl Biol. 17, 19-58.
