histochemical and electron microscopic studies of the iridic pigment epithelium in the albino rabbit

Z. Zellforsch. 115, 1-16 (1971) �9 by Springer-Verlag 1971

Histochemical and Electron Microscopic Studies of the Iridic Pigment Epithelium in the Albino Rabbit

J. HVIDBERG-HANSEN Department of Anatomy, University of Odense, Odense, Denmark

Received October 5, 1970

Summary. On the basis of the occurrence, at the light microscopic level, of alkaline and acid phosphatases, the pigment epithelium covering the posterior surface of the iris in the albino rabbit can be divided into two zones not previously described, viz. a central zone close to the pupil, approximately corresponding to the area occupied by the iridic sphincter muscle, and a peripheral zone extending to the ciliary body. The central zone which is in intimate relation with the lens was found to have a high content of both phosphatases. At the fine structural level it exhibits a marked pinoeytotic activity in the epithelium at the interdigitations between adjacent cells. Electron microscopy revealed that acid phosphatase is localized to the walls of the pinocytotic vesicles. Alkaline phosphatase is in evidence at the surface membrane folds and at microvillous processes between the epithelial cells and the adjoining muscle cells. Unlike the distribution of the acid phosphatase, that of the alkaline phosphatase does not differ fundamentally in the two zones at the fine structural level.

In a series of dehydrogenases studied, staining with a view to succinic-, isocitric- and glucose-6-phosphate dehydrogenases revealed an evenly distributed content of enzyme throughout the epithelium. As to the lactic- and fl-hydroxybutyric dehydrogenases, contents seem to be lower in the pupillary than in the peripheral zone.

Key-Words: Iris--Rabbit--Pinocytosis--Enzymes--Electron microscopy.

Introduction

The epithelium covering the posterior surface of the iris is composed of two layers of cells. The posterior layer is characterized by its high content of pigment. I n the albino rabbit, p igment granules are colourless, probably because thy- rosinase is lacking (Danneel and Schaumann, 1938 ; Kuki ta and Fitzpatrick, 1955). The anterior layer, facing the s t roma and continuous with the pigment epithelium of the ciliary body, is par t ly t ransformed into myo-epithelial cells, viz. the iridic sphincter muscle and the iridic dilatator muscle. The fine structure of the pigment epithelium covering the posterior surface of the iris has been studied in various mammals , for instance by Tousimis and Fine (1959) in man and Rhesus monkey, by Richardson (1964) in the albino rabbit, and by Shively and Epling (1969) in the dog. In addition, the cell s tructure in the eat has been briefly discussed by Geltzer (1969) and tha t in the mouse by Rhodin (1963). The cells, the shapes of which depend on the size of the pupil (van Alphen, 1963), present a ra ther uniform structure in the various species studied. The nuclei are usually indented ; the large ovoid pigment granules which according to Tousimis (1963) are very uniform in the various species represent the most conspicuous elements in the cytoplasm.

So far, the histochemistry of iridic epithelial cells has not been an object of special s tudy, but comments on the enzymology of the iridic cells have appeared in several publications concerning the histochemistry of the ciliary body.

1 Z. Zellforsch., Bd. 115

2 J. Hvidberg-Hansen:

One impor t an t funct ion of the epithelial p igment cells is to produce and store

large amounts of p igment granules which, together with the s t romal p igment ,

makes iris impervious to l ight and prevents reflect ion of l ight in the bulb. The

p igment epi thel ium in albino rabbi ts has been studied with a view to eva lua t ing

whe ther morphological and his tochemical factors might suggest the i nvo lvemen t

of o ther functions. Owing to the staining methods used in l ight microscopy, eyes

from albino rabbi ts were chosen as exper imenta l material .

Material and Methods

Twenty-one albino rabbits of either sex, each weighing about 3.5 kg, were used in the experiments. The eyes were enucleated immediately after the rabbits had been killed by a blow to the skull.

Prior to examination by light microscopy, the anterior 1/3 Of the bulb was cut off, the lens excised, and sectors of the cornea, the ciliary body and the iris were either fixed in for- malin-calcium or they were frozen in fresh condition. Thin sections, 8-16 ~, of the chamber angle and the entire iris were cut in a cryostat. One or several sectors of the iris were cut together with rabbit liver and rabbit kidney in order to stabilize the tissue during the pro- cedure and also to serve as control in the enzyme-histochemical staining process.

Unspecific alkaline and acid phosphatases were determined according to Burstonc's simultaneous diazo salt technique (1958) while dehydrogenases were determined according to Thomas and Pearse's method (1961) as modified by Andersen (1965). Control experiments included heat inhibition and omission of substrate. The interval of time between start of incubation and appearance of the first demonstrable traces of colour served as standard of the strength of reactions. Incubation proceeded along a logarithmic scale arranged round about the average, predetermined, intervals of time.

The eyes from a total of 11 rabbits were studied in the electron microscope. The iris was fixed immediately in 6% glutaric aldehyde in cacodylate buffer, pH 7.4. The presence of alkaline phosphatases was examined in accordance with Hugon and Borgers' method (1967) which seemed to be more suitable than the one suggested by Mizutani and Barrnett (1965); still, whole sectors of the iris, not cut in the cryostat, were incubated. Incubation covered periods of from 5 to 30 minutes at 20 ~ C. Barka's method (1964) was used for the demonstration of acid phosphatase, also without preceding freeze-microtomy. Incubation for 20-30 minutes at 37 ~ C was found to be optimal.

Upon embedding in Epon 812, the ultrathin sections were cut on a LKB microtome. The sections were studied in a JEM T 7 electron microscope. Uncontrasted sections were used. Contrasting using lead hydroxyde-uranyl acetate, usually in accordance with Reynold's method (1963), served for illustrative purposes. By way of control, osmium-inactivated tissue specimens were examined.

Results

Light Microscopy Using the occurrence of phosphatases in the epi thel ium as basis, the iris can

be d iv ided into two zones. One zone is ar ranged concentr ical ly round about the pupil, i ts ex ten t a lmost equall ing tha t of the iridic sphincter muscle, the other

being ar ranged per ipheral ly to the l a t t e r ; this second zone is also of annular shape and extends to the ciliary body; in rabbits , the borderl ine be tween the ciliary body and the iris is serrated at the so-called iridic processes (Kozart ,

1968). Demarca t ion be tween the two zones is not distinct. The epi the l ium in the central zone was found to contain unspecific alkaline

phosphatase in an amount a lmost equal to t ha t in l iver tissue and in k idney

tissue, de te rmined af ter adequa te periods of incubat ion (table). Alkaline phos-

Iridic Epithelium in the Albino Rabbit 3

Fig. 1. Iris seen from the posterior aspect after staining for alkaline phosphatases. Enzymatic activity is visible in the epithelium in the region of the sphincter (S). Reactions are positive

also in the ciliary body (C) and in the iridic processes (IP)

phatases were present only in minor quantities in the epithelial cells facing the iridic dilatator muscle. Upon incubat ion of an intact iris, the division into zones was visible, not merely in freeze sections, but also to the gross view, cf. Fig. 1. Alkaline phosphatase was in evidence in the two epithelial cell layers of the ciliary body. Furthermore, staining was observed in the vascular endothelium of the ciliary body and the iridic vessels as well as the iridic nerves stained red. The lat ter feature has been observed also by Frangois and Rabaey (1951).

Distr ibution of the acid phosphatases was almost the same as t ha t of the alkaline phosphatases, viz. the content was high in the zone close to the pupil whereas staining in the peripheral zone required incubat ion over longer periods. Apar t f rom the epithelium, also cells scattered in the s t roma close to the anterior surface of the iris stained rapidly and distinctly (Fig. 2).

1"

J. Hvidberg-Hansen:

Table

Enzyme a Liver Ciliary Iridic epithelium Limbic Kidney tissue epithelium conjunetival tissue

dilata- sphincter epithelium tor area area

A lkP + + + + b + + + + § + + + § + + + + AeidP + + + + + + + + + + + + + + § + + + + + + + + + + fl-0HDH + + + + § + + + + ~ + LDH + + + + + + + + § + § + + + § SDH § + + + + + § + + + + § 2 4 7 2 4 7 ICDH + + + + + + + + + + + § + + + G-6-PDH + + + + + + + + § + + + + + + + + + +

a The following abbreviations are used in the table: Alk P: unspecific alkaline phosphatase; Acid P : unspecific acid phosphatase; fl-OHDH : fl-hydroxybutyrie dehydrogenase; LDH : lactic dehydrogenase; SDH: succinic dehydrogenase; ICDH: isocitrie dehydrogenase; G-6-PDH: glucose-6-phosphate dehydrogenase.

bFi rs t sign of staining: + + + + + = v e r y fast; + + + + ~ f a s t ; + + + - - m o d e r a t e ; + + = slow; + ~ very slow. Liver tissue with which the other tissues are compared is arbitrarily denoted: + + + +.

Dehydrogenases were found to be r a the r evenly d i s t r i bu t ed in the i r idic ep i the l ium (table). The ac t i v i t y of f i -hydroxybu ty r i e dehydrogenase and lact ic dehydrogenase a t the si te of the ir idic sphinc te r muscle was a l i t t le weaker t han t h a t a t the i r idic d f la ta to r muscle.

I n the i r idic ep i the l ium close to the pupil , the con ten t of f l -hyd roxybu ty r i e dehydrogenase was a p p a r e n t l y equal to t h a t in the sphinc te r muscle cells and in s t roma cells. I n con t rad i s t inc t ion to the i r idic sptf inctcr muscle, the d f l a ta to r muscle was found to conta in surpr is ingly high amount s of the enzyme, a l though no t qui te as high, appa ren t ly , as amoun t s in the epi the l ia l cells facing the muscle. The content of lact ic acid dehydrogenase was analogous to t h a t of f i -hydroxybu ty r i c dehydrogenase (table).

Succinic dehydrogenase as well as isocitr ic dehydrogenase were demons t rab le , as a charac ter i s t ic feature , in the form of granular s ta in ing in the ep i the l ium in which the granules p robab ly were commensura te wi th the mi tochondr ia . Re- p r e sen t a t i on in the two epi thel ia l zones was equal ly high and the muscle cells s t a ined dis t inct ly . The same appl ied to glueose-6-phosphate dehydrogenase which, however , was presen t to minor degree in the uns t r i a t ed muscle cells.

The enzymat i c fea tures discussed in the above were not r e la ted to the size of pupi ls of the irises examined.

Electron Microscopy The fine s t ruc ture of the epi thel ia l p igmen t ceils is i l lus t ra ted in Fig. 3.

Throughou t the pos ter ior surface of the iris, the cells are covered b y a basement membrane , the th ickness of 120-140 nm, and cont inuous with the m e m b r a n e covering the ci l iary body. The p lasma m e m b r a n e facing the pos ter ior chamber is m a r k e d b y infoldings (Figs. 3, 5, 7). According to the au tho r ' s opinion, the


Fig. 2. Radial cross section (somewhat bent) of the iris stained for acid phosphatases. Activity is visible in the epithelium covering the iridic sphincter muscle (S), in the epithelium of the ciliary body (C), and in the iridic processes (IP). Activity in the epithelium of the peripheral zone is very discrete (cf. text). Acid phosphatase positive cellular elements are in evidence in

the iridic stroma, especially near the anterior surface (arrows)

infoldings are comparable with the ones composing the deep intercellular interdigitations which represent a highly conspicuous feature of the plasma membranes of adjacent, non-pigmented epithelial cells observed by Tormey (1963) in serial sections from parts of the ciliary body. The intercellular space between the epithelial cells and the myoepithelial cells is occasionally seen to be expanded and microvilli originating in either layer of cells are present. Comparable spaces - - t h e ciliary canals-- in the ciliary body have been described by Tormey (1963). They are not quite as distinct in the iris, but microvilli are demonstrable even at the pupil lary margin, cf. Fig. 7 c.

Fig. 3a-c. Fine structure of the pigment epithelium of the iris. a The epithelium covering the di latator muscle cells. The plasma membrane of adjacent cells is marked by several, deep intercellular interdigitations (ID). A muscle cell wi th nucleus (ND), Golgi zone (G), and cen- trioles are seen (C). PC the posterior chamber; N the indented nucleus of the pigment epithelium, b Pigment epithelium of iris in the sphincter region; vesicles (V) are in evidence together with broad intercellular interdigitations (ID) with micropinocytosis (arrows).

Pinocytotic vesicles appear more distinctly in the inset c

Fig. 4a-c. Alkaline phosphatases in the epithelium of the anterior portion of the ciliary body, visualized by means of Hugon and Borgers' technique (1967) (incubation for 15 minutes at 20 ~ C). Deposits of lead phosphate indicate activity in the intercellular interdigitations (ID) (cf. inset b) and at the borderline between the two epithelial layers (arrows). Notice the difference from cell to cell in activity of unspecific alkaline phosphatase content in the non-pigmented epithelium. Inset c demonstrates activity on the surface of the villi in the ciliary canals (C). PC posterior chamber; N nucleus of non-pigmented epithelium of ciliary body;

N P nucleus of pigmented epithelial cell

Fig. 5 a-c. Iris stained for alkaline phosphatases, a Pigment epithelium cell of the peripheral zone with sparse act ivi ty of unspecific alkaline phosphatase, visualized by means of Hugon and Borgers' technique (incubation for 15 minutes at 20 ~ C). Lead deposits in the infoldings (ID) and in a non-identified body (B). The posterior chamber (PC) is filled out with a fibrillar material , probably vitreous substance left there at dissection, b The epithelium in the central zone corresponding to the iridic sphincter muscle. Signs of alkaline phosphatase act ivi ty in the broad infoldings (ID) are observed together with deposits in the micropinocytotic vesicles;

details be t te r seen in the inset e. PC posterior chamber, N nucleus of the epithelium

Fig. 6a and b. Acid phosphatase in the epithelium of the anterior portion of the ciliary body, visualized by means of Barka 's technique (1964)( incubation for 15 minutes a t 37 ~ C). Lead deposits are seen in the lysosomes (L) and a t the junction of the two epithelial layers (arrows), cf. also inset b. In the nucleus of the pigmented epithelium (NP), lead particles with relation to the chromatin are visible, which is in contrast to features in the nucleus of non-pigmented

epithelium (/V). PC posterior chamber


:Fig. 7 a-c. Acid phosphatase in the pigment epithelium of the iris (incubation for 20-30 minutes at 37 ~ C). a An over-all view including the iridic dilatator muscle (D). The epithelium near the posterior chamber (PC) contains only discrete lead deposits. A fibrocyte-like cell close to an iridic vessel displays a certain activity in the Golgi zone (G), cf. text. b Activity in

Iridic Epithelium in the Albino Rabbit l l

The nuclei in the epithelial layer are of ovoid shapes and indentures are often deep; nuclei containing one or several nucleoles represent a common finding. The nuclei are centrally located. The mitochondria contain a dark matrix. Golgi zones are not conspicuous and secretory granules are not demonstrable. On the other hand, some albinotic pigment granules and bodies reminding of lysosomes are in evidence together with vesicles of varying sizes. The endoplasmatic reticulum is mainly of the smooth type. "Vesicle-bounded vacuoles" (Wegner, 1967) represented an occasional, although not dominating, feature in the region of the iridie dilatator muscle. Such organelles were not observed in the zone close to the pupil, whereas the cytoplasm is most electron dense in cells from the central zone. In addition to these dissimilarities of the two zones in the pigment epithelium, the plasma membrane folds in the epithelium of the central zone presented a rather different configuration, often being broader and more irregular. The numerous micropinocytotic vesicles near the above mentioned broad infoldings presented a characteristic finding, Fig. 3e. Such pinocytotie activity was not observed in the ciliary body or in the epithelium of the peripheral zone. The micropinocytotic vesicles had not the character of intracellular, coated vesicles and their walls were rather smooth. Finally, this zone comprised several larger vesicles (cf. Fig. 3b), whereas lysosomes were sparse.

Only hydrolases were exposed to enzyme histoehemical examinations using the electron microscope. The epithelium of the ciliary body was selected as tissue of reference because the fine structure of this epithelium repeatedly has been an object of similar studies (Cameron and Cole, 1965; Tarkkanen et al., 1965; Shiose and Sears, 1965; Frangois and Wainstein, 1969). The phosphatase activity is assumed to occur at sites where lead deposits are in evidence; it should be accen- tuated, however, that uncontrasted sections were used in the examinations and, whenever possible, localizations were controlled by comparison with neighbouring sections.

Activity of unspecific phosphatases occurred throughout the iris and the ciliary body, but in general, the phosphatase content seemed to conform with that observed by light microscopy as previously described. Moreover it should be added that contents of phosphatases differed greatly from cell to cell, el. Fig. 4a, a finding which could be corroborated by observations under the light microscope.

Two main cellular localizations could be pointed out: Firstly, phosphatase was localized to the intercellular interdigitations where it was seen as fine granules in the intercellular spaces; occasionally they might adhere to the plasma membranes {Fig. 4b). Secondly, alkaline phosphatase was in evidence in the afore mentioned ciliary canals and in the corresponding processes in the iris, often in connection with microvilii (Fig. 4c). The previously mentioned difference in

the epithelium of the central zone corresponding to the iridic sphincter muscle. At this site, the lead phosphate deposits are localized to the wall of the pinocytotic vesicles (arrows). c Acid phosphatase activity in relation to villi (V) in a canal-like structure is observed between the pigment epithelium with albinotic pigment granules (AP) and a sphincter muscle cell (SM).

Albinotic pigment granules may occasionally display acid phosphatase activity (arrowhead)


morphology of interdigitations in the iris and in the ciliary body was brought out more distinctly by the lead deposits, cf. Figs. 4 and 5. The pinocytotic vesicles in the central zone contained occasionally lead particles, either in the vesicles or in invaginations in the plasma membrane.

Alkaline phosphatases were not demonstrable in the endothelium of vessels. On the other hand, axons of nerve cells in the iris were active at the surface of membranes, particularly at Ranvier 's nodes; this finding is commensurate with those observed in the light microscope.

The occurrence of acid phosphatase in the epithelium of the ciliary body differed markedly from that in the two zones of the iridic pigment epithelium. Deposits in the ciliary body were most conspicuous at the transition between the two layers of epithelial cells and at the surface of the microvilli (Fig. 6) which is commensurate with features observed in the case of alkaline phosphatase. Furthermore, distinct activity was localized to lysosomes in that part of the cells which was farthest away from the chamber. Finally, lead granules were demonstrable in the chromatin of nuclei in the pigment epithelium covering the ciliary body ; the latter observation is commensurate with findings by Franqois and Wainstein (1969) who in the light microscope observed acid phosphatase act ivi ty in these nuclei. Such deposits had not the appearance of an "unspecific nuclear phosphatasc ac t iv i ty" as the one to occur after incubation over long periods of time, in particular at high pH. In part of the material, some delicate lead deposits were observed at the infoldings whereas acitivity at the Golgi zones was moderate.

Acid phosphatase activity was very discrete in the iridic pigment epithelium of the peripheral zone; if present, it was localized mainly to lysosomes.

Findings in the iridic epithelium in the central zone were quite different in tha t numerous pinocytotic vesicles were marked by minor lead deposits (Fig. 7). At anterior sites of the cells, corresponding to the microvilli, lead deposits were also in evidence. A few lysosomes presenting activity were observed in the epithelium of the central as well as the peripheral zones; lead deposits in a few of the albinotic pigment granules were noted but they did not represent a typical finding.

In the extreme front of the iridic stroma it was possible to demonstrate cells in which activity at the Golgi zone was distinct. Lead deposits in a few lysosomes were also demonstrable in these cells; according to their structure they might be fibrocytcs ; they were assumed to correspond to those observed by light microscopy and described in the above. Fig. 7a illustrates a fibrocyte in which the acid phosphatase activity is of the same order; it is seen to be lying in relation to the characteristically composed iridic vessels.

Discussion

The concept according to which the iris may be supposed to have functions other than tha t of a shutter, for instance, producer of chamber water, has been advocated before (Masuda et al., 1961) and indeed, the ultrastructural composition of the epithelium (Richardson, 1964) seems also to indicate tha t the cells are something more than just pigment containers. Apart from the possible function

Iridic Epithelium in the Albino l~abbit 13

as a producer of chamber water, other, so far ignored, functions may also be conceivable; in this conjunction it seems relevant to direct the attention to its close contact with the lens. Thus the capillaries behind the sphincter represent the most intimate interrelation between the lens and the vascular bed; it is separated from the latter merely by the iridie epithelium which at this site generally is resting against the anterior surface of the lens.

As regards the conclusions previously drawn by other investigators concerning the content of enzymes in pigment epithelium, it deserves notice that Frangois and Rabaey (1951) as well as Allen and Friedenwald (1953) failed to discover alkaline phosphatase in the iridic epithelium in rabbits whereas Lessel and Kuwabara (1964) successfully demonstrated the presence of acid as well as of alkaline phosphatases; also Shanthaveerappa and Bourne (1964) ascertained the presence of a strong acid phosphatase in the epithelium, but they did not mention alkahne phosphatase. The latter authors declared that parts of the iridie epithelium had been found to react strongly to suceinic acid dehydrogenase, mode- rately to cytochromoxidase and monoamine oxidase. According to Mustakallio (1967), however, the latter enzyme had not been demonstrable. This discrepancy between findings reported by the individual investigators in matters concerning the content of hydrolytic enzymes in the iridic epithelium may find its explanation in the fact that the pigment epithelium at this site is inhomogeneous; this has been demonstrated in the present study in which the iridie pigment epithelium has been divided into zones, a feature not previously mentioned by other authors.

The finding of unspecific alkaline phosphatase in the ciliary body is in agree- ment with observations made by Shiose and Sears (1965) and by Frangois and Wainstein (1969). In the present study, the marked activity on the surface of mierovilli in the vicinity of ciliary canals and similar iridic processes represented a characteristic feature. In considerations concerning the functions of the ciliary body and the iris, a certain weight should be attached to this finding (Shiose, 1965). The fact that unspecific phosphatases were in evidence also near the infoldings makes it more difficult to draw any inferences with a view to the specific phosphatases (Novikoff et al., 1961). Hence, the latter have not been studied.

That part of the present study which is concerned with the demonstration of dehydrogenases was instituted with a view to obtaining a picture, if possible, of the metabolic possibilities of epithelial cells; in this conjunction it should be mentioned that the oxygenic tension of the chamber water in albino rabbits ranges at a rather low level, about 30-35 Hg (Wegener and M~ller, 1970). The occurrence of lactic acid dehydrogenase might be suggestive of an anaerobic glycolysis, but enzymes from Krebs' cycle: succinic acid- and isoeitric acid dehydrogenase were present actually in rather high concentrations, thus indi- cating an aerobic metabolism. The demonstrated differences in the iridic pigment epithelium as regards contents of fi-OH butyric acid dehydrogenase and lactic acid dehydrogenase are not sufficiently marked to allow any conclusions to be drawn in matters concerning the metabolism.

Such cells in the anterior marginal layers in the iridic stroma as present acid phosphatase activity may be either macrophages or, more likely, mast cells like those observed in the stroma of the ciliary body which, according to Francois and Wainstein (1969), were found to have acid phosphatase activity. In the


present study, electron microscopy disclosed that the connective tissue of s troma contained some cells much reminding of fibrocytcs; these cells produced an intense acid phosphatase activity in the Golgi zone and in lysosomes. Mast cells presenting acid phosphatase activity were not demonstrable and maerophages were sparsely represented.

A characteristic finding in the present study was the pinocytotic activity in the iridic epithelium at the site of the sphincter. According to Kaye et al. (1962), such pinocytotic activity might occur in the intercellular spaces in the corneal epithelium. Using colloid particles as tracers, these authors found signs suggestive of an active transportation between chamber water and corneal stroma. In this s tudy acid phosphatasc has been found to occur in connection to micropinoeytotie vesicles; a similar activity in pinocytotic vesicles in amoebae has been demonstrated by Novikoff (1960) and by Birns (1960). According to Novikoff (1961) such activity might be linked up with pinocytosis also in other cells. So far, it has been rather difficult to have this postulation confirmed and, by way of explanation, Novikoff declared that it might be a mat ter of only minor amounts of acid phosphatase and that the methods hitherto used for the demonstration of enzymes may not have been sufficiently subtile. When it has been possible in the present s tudy to demonstrate such activity, the reason may be that freeze-microtomy during preparation was omitted. The iris is a thin, sheet formed process, measuring 90-270 ~ (Prince, 1964) with a loose stroma which is permeable even for molecules as large as dextran (Gregersen, 1960). In the case of sub-microscopic examinations of enzymes, it is generally considered contra-indicated to use tissue tha t has not been cut in the cryostat although some authors admittedly have been successful when they used techniques not including freeze-mierotomy (Mishima, 1966). As it is the difficulties involved in penetration tha t argue primarily in favour of freeze-microtomy prior to examinations of the specimens, I have taken special efforts to s tudy the edges of the specimens. The enzyme activity in this location was not found to be particularly intense or in principle different. The acid phosphatase reaction in Golgi zones in cells at sites below the epithelium (Fig. 7a) indicate that penetration is satisfactory in this particular tissue.

The fact tha t electron microscopy, in contrast to light microscopy, failed to disclose alkaline phosphatase in the endothelium of vessels is explicable when the sensitivity of this phosphatase towards glutaric aldehyde fixation is taken into consideration (Goldfischer et al., 1964; Shiose, 1965).

The findings in this study, in particular that of a pinocytotic activity, seem to encourage a continuation of studies concerning the postulation that the epithelium covering the posterior surface of the iris may have some functional share in the fields of transportation; it might be of advantage to include a s tudy of the route of migration of colloidal particles.

References

Allen, R.A., Friedenwald, J .S.: Distribution of substrate-specific alkaline phosphatases in the ocular tissues. Arch. Ophthal. 50, 671-684 (1953).

Alphen, G. W. H. M. van: The structural changes in miosis and mydri~sis of the monkey eye. Arch. Ophthal. 69, 802-814 (1963).


Andersen, H. : Sulphydryl "nothingdehydrogenase" aetivityand lacticdehydrogenase-NADH 2 cytoehrome C reduetase reactions in tissues of the human foetus. Acta histochem. (Jena) 21, 120-134 (1965).

Barka, T.: Electron hist,)chemical localization of acid phosphatase activity in the small intestine of the mouse. J. ttistochem. Cytochem. 12, 229-238 (1964).

Birns, M.: The localization of acid phosphatase activity in the ameba, Chaos chaos. Exp. Cell Res. 20, 202-205 (1960).

Burstone, M. S.: The relationship between fixation and techniques for the histochemical localization of hydrolytic enzymes. J. Histochem. Cytoehem. 6, 322-339 (1958).

Cameron, E., Cole, D. F. : Some studies on enzyme histochemistry of the ciliary epithelium. In : Eye structure I I (J. W. l~ohen, ed.). Stuttgart: Schattauer 1965.

Danneel, R., Schaumann, K. : Zur Physiologie der K~lteschw~rzung beim Russenkanin. Biol. Zbl. 58, 242-260 (1938).

Francois, J., Rabaey, M. : Localisation histochimique de la phosphatase alcaline dans l'oeil de certains mammif~res. Ann. Oculist. (Paris) 184, 481497 (1951).

- - Wainstein, 1~.: Phosphatases in ocular tissues. Ophtha]mologica (Basel) 157, 231-247 (1969).

Geltzer, A. I.: Autonomic innervation of the cat iris. Arch. Ophthal. 81, 70-83 (1969). Goldfischer, S., Essner, E., Novikoff, A. B. : The localization of phosphatase activities at the

level of ultrastructure. J. ttistochem. Cytochem. 12, 72-95 (1964). Gregersen, E. : Studies on the spongy structure of the human iris and its imbibition with the

aqueous humor. Copenhagen: Munksgaard 1960. Hugon, J., Borgers, M. : Submicroscopic localization of the alkaline phosphatase activity in

the duodenum of the rat. Exp. Cell Res. 45, 698-702 (1967). Kaye, G. I., Pappas, G. D., Donn, A., Mallett, N. : Studies on the cornea I I : The uptake and

transport of colloidal particles by the living rabbit cornea in vitro. J. Cell Biol. 12,481-501 (1962).

Kozart, D. M. : Light and electron microscopic study of regional morphologic differences in the processes of the ciliary body in the rabbit. Invest. 0phthal. 7, 15-33 (1968).

Kukita, A., Fitzpatrick, T. B. : Demonstration of tyrosinase in melanocytes of the human hair matrix by autoradiography. Science 121, 893-894 (1955).

Lessell, S., Kuwabara, T. : Phosphatase histochemistry of the eye. Arch. Ophthal. 71,851-860 (1964).

Masuda, S., Ogino, F., Yoshikawa, T., Ryu, G., Nojika, H., Komichi, S., Mitsukawa, K., Takee, T. : Studies on the function of the iris and the ciliary body. I. The amount of chamber water produced in the iris and the ciliary bodies. Acta Soc. ophthal, jap. 65, 1291-1295 (1961).

Mishima, Y. : Cellular and subcellular differentiation of melanin phagocytesis and synthesis by the lysosomal and melanosomal activity. J. invest. Derm. 46, 70-75 (1966).

Mizut~ni, A., Barrnett, 1~. J . : Fine structural demonstration of phosphatase activity at ptI 9. Nature (Lond.) 206, 1001-1003 (1965).

Mustakallio, A. : Monoamine oxidase activity in various structures of the mammalian eye. Acta ophthal. (Kbh.) Suppl. 93, 1--62 (1967).

Novikoff, A. B. : Biochemical and staining reactions of cytoplasmic constituents. In : Devel- oping cell system and their control (D. Rudnick, ed.). New York: Ronald Press Co. 1960.

- - Lysosomes and related particles. In : The cell, vol. I I (J. Brachet and A. E. Mirsky, eds.). New York and London: Academic Press 1961.

- - Drucker, J., Shin, W.-Y., Goldfischer, S.: Further studies of the apparent adenosine- triphosphatase activity of cell membranes in formot-ealcium-fixed tissues. J. Histoehem. Cytochem. 9, 434-451 (1961).

Prince, J. H. : The rabbit in eye research. Springfield: Charles C. Thomas 1964. Reynolds, E. S. : The use of lead citrate at high pH as an electron-opaque stain in electron

microscopy. J. Cell Biol. 17, 208-212 (1963). Rhodin, J. A. G.: An atlas of ultrastructure. Philadelphia: W. B. Saunders Co. 1963. Richardson, K. C. : The fine structure of the albino rabbit iris with special reference to the

identification of adrenergic and cholinergic nerves and nerve endings in its intrinsic muscles. Amer. J. Anat. 114, 173-184 (1964).

16 J. Hvidberg-Hansen: Iridic Epithelium in the Albino Rabbit

Shanthaveerappa, T. R., Bourne, G. H. : Histochemical studies on the distribution of oxidative and dephosphorylating enzymes of the rabbit eye. Acta anat. (Basel) 57, 192-219 (1964).

Shiose, Y. : Localization of nucleoside phosphatase activity in the ciliary epithelium of the albino rabbit. In: Eye structure I I (J. W. Roben, ed.). Stuttgart: Schattauer 1965.

- - Sears, M. : Localization and other aspects of the histochemistry of nucleoside phosphatases in the ciliary epithelium of albino rabbits. Invest. Ophthal. 4, 64-75 (1965).

Shively, J. N., Epling, G. P.: Fine structure of the canine eye: Iris. Amer. J. vet. Res. 80, 13-25 (1969).

Tarkkanen, A., Pajari, S., Niemi, M. : Enzyme histochemistry of the human ciliary epithelium. In: Eye structure I I (J. W. Rohen, ed.). Stuttgart: Schattauer 1965.

Thomas, E., Pearse, A. G. E. : The fine localization of dehydrogenase in the nervous system. Histochemie 2, 266-282 (1961).

Tormey, J. McD. : Fine structure of the ciliary epithelium of the rabbit, with particular reference to "infolded membranes", "vesicles ", and the effects of diamox. J. Cell Biol. 17, 641~659 (1963).

Tousimis, A. J. : Pigment cells of the mammalian iris. Ann. N. u Acad. Sci. 160, 447-466 (1963).

- - Fine, B. S. : Ultrastructure of the iris: An electron microscopic study. Amer. J. Ophthal. 48, (5) pt. 2, 397417 (1959).

Wegener, J. , M~ller, P .M. : Personal communication (1970). Wegner, K. : Regional differences in ultrastructure of the rabbit ciliary processes: The effect

of anesthetics and fixation procedures. Invest. Ophthal. 6, 177-191 (1967).

J . Hvidberg-Hansen, M. D. Department of Anatomy University of Odense DK-500O Odense, Denmark

histochemical and electron microscopic studies of the iridic pigment epithelium in the albino rabbit

Documents