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Cytochemical study of macular dystrophy of the cornea (Groenouw II): an ultrastructural study
Exp. Eye Res. (1974) 18, 163-169
Cytochemical Study of Macular Dystrophy of the Cornea (Groenouw II) : an Ultrastructural Study BEATRICE GRAF, TYTVESPOULIQUEN,* MARIE-ALICE JEAN-PIERRE FAURE AND GUY OFFRET
FROUIN,
(Received 3 July 1972, and in revised form 14 November 1973) A cytochemical study of a case of macular dystrophy (Groenouw II) by means of optical and electron microscopy is reported. The epithelial lesions are slighter than the stromal lesions. The kerat,ocytes are the site of important degeneration. The substance secreted by these cells is PAS-positive under both the optical and electron microscope. It is also alcianophil. This substance may be a glycoprotein rather than an acid mucopolysaccharide. The simultaneous positivity of the PAS reaction and the Alcian blue stain argues in favour of the hypothesis, which should be confirmed by a biochemical study.
1. Introduction Macular dystrophy of the cornea represents one of the principal, but rare, forms of recessive hereditary parenchymatous dystrophy of the cornea (Morgan, 1969), so that a light- and electron-microscope study of the cornea in this condition is of some general interest. In the present work a perforating graft was performed on a women aged 66 years suffering from bilateral dystrophy, and the excised cornea1 disc was examined. 2. Methods Immediately after keratoplasty the sample of cornea was subdivided into two, one half being destined for light microscopy and the other for electron microscopy. Light microscopy Tissue was fixed in Bouin’s fluid, dehydrated
and embedded in paraffin.
Stains
(a) Haematoxylin, eosin, safranin. (b) PAS. (MacManus).
Oxidation
by periodic acid for 30 min.
(c) Alcian blue at pH = 2.5 and at pH = 1.0. (d) Alcian blue with MgCl, = 0.1 M. (e) Alcian blue with MgCl, = 1 M.
(f) Thioflavin. (g) Toluidine blue. Electron microscopy Half of the sample was immediately
fixed in a 5% solution of glutaraldehyde
in phosphate
buffer for 30 min, rinsed in buffer solution, and post-6xed by treating with 1% osmium tetroxide in phosphate buffer for 24 hr. It was rinsed in buffer solution, dehydrated and embedded in an aralditelepon mixture. Ultra-thin sections were cut on a microtome (Reichert OM.UII) and examined in a Phillips EM 300 electron microscope (Ete. Frer. Lissac) . * Requests for reprints to Dr Y. Pouliquen, Laboratoire de la Clinique Ophtalmologique de I’HotelDieu, Place de Parvis Notre-Dame, Paris IVe, France. 163
H. (:ltAF.
I64 Stain,ing
Y. I’OI~I,IQl-ES.
11.-A.
FROI.IS.
.I..l’.
FAVRE,
c:. OFFKE’I
of the sectims
(a) Standard staining by many1 acetate, lead citrate. (b) Cytochemical stain to show t,he presence of polysaccharides ---periodic acid, thiocarbohydrazide, silver proteinate~~~according to the method described by Thiery (1947). (i) Periodic acid, 30 min; thiocarbohydrazide, 60 hr; silver proteinate, 20 min. (ii) Controls were prepared in the same way, but without oxidation by periodic acid-thiocarbohydrazide, 60 hr ; silver proteinate, 20 nun; or with oxidation by periodic acid, 30 min, without thiocarbohydrazide; silver proteinate, 20 min.
FIG . 1. (a) Aspect of the epithelial region and adjacent stroma. There is abundant Alcian blue-1 )osit,ive depos it below the basal membrane. Light microscope. Stain: AI&n blue. pH = 2.6 MgCl, 0.1 31. (b) Sin& II deposit below the basal membrane. Light microscope. Stain: Alcian blue. pH = 2.6 Mg( :I, 1 M.
CORNEAL
MACULAR
DYSTROPHY
16.5
3. Results Light nlicroscopy (stain: haernatoxylin,
eosin, safmnin)
1 e1’mm is irregular, sometimes thickened, consisting of six to eight (a) Th e ep’th layers of cells, whilst in other regions it is normal. The basement membrane follows the irregularities of the layer of basal cells. In some regions the epithelium is lacking. (b) Bowman’s membrane is normal in appearance ; however, in the central area, where t*here is an accumulation of substance in the sub-epithelial region, it is lacking. (c) The stroma. The bundles of fibrils of collagen, at this magnification, seem to have a normal arrangement; the keratocytes seem to be globular and sometimes vacuolated. There is an accumulation of material in the sub-epithelial region, in the central area of the sample, in the region corresponding to the break in Bowman’s membrane and at the thickening of the epithelium.
5%~. 3. Aspect of an epithelial cell. Dilatation of the ergastoplasm oont,aining micro-granulofilamentous material. Abundance of free ribosomes. Presence of dense inclusions outside. Persistence of tonofilaments. Stain: wanyl acetate-lead citrate Gx. ( x 31 350)
A deposit of substance is also observed throughout, the whole of the stroma but which is nevertheless predominant in the anterior region. This deposit is localized in the neighbourhood of the keratocytes, between the layers of the stroma. Unfortunately we were unable to examine, in this sample, the extreme posterior region corresponding to Descemet’s membrane and the endothelium.
166
H. GRAF,
Y. POULIQUEX,
M.-A.
FROl’lS.
*J.-P.
FAURE,
G. OFFRET
Light microscopy: cytochemical ataills The substance accumulated in the sub-epithelial region and the deposits situated in the stroma have the same staining affinities. (a) PAS-positive following 30 min of oxidation by periodic acid. However the intensity of the reaction is weak. (b) Alcian blue: positive at pH ‘3.6, at pH 1.0: with MgCl, O-1 M [Fig. l(a)] and with MgCl, 1 M [Fig. l(b)]. (c) Thioflavin T-negat,ive reaction.
FIG. 3. Keratocyte in a state of severe degeneration. Only a voluminous oval-shaped formation is recognizable consisting of a microfibrillar substance. Irregular arrangement of these microfibrils. Stain : uranyl acetate-lead citrate Gx. ( x 40 000)
Electron microscopy: standard examination Epithelium. Certain epithelial cells show alterations, the most important of which are an extreme dilatation of the ergastoplasmic cisternae containing a less dense microgranulo-filamentous material, and certain cisternae contain very dense inclusions, of hazy outline. These inclusions are also observed outside the cisternae. We do not know their significance. There is also an abundance of free ribosomes in the cytoplasm, isolated or in groups. (Fig. 2.) Stroma. The keratocytes: (a) these are in a considerably degenerate condition. At this stage, it is difficult to recognize the different organelles contained in these cells: the nucleus is generally lacking, the cytoplasmic membrane is ruptured. In the cyto-
CORNEAL
MACULAR
DYSTROPHY
167
plasm, there are clear vacuoles of varying sizes, dense bodies, resembling a blackberry, consisting of small dense granules that may correspond to free ribosomes. The mitochondria are no longer recognizable, but voluminous formations, sometimes polycyclic, seem to be the elements that are most consistently found in the cytoplasm of these keratocytes. These formations consist of a microfibrillar substance that is less dense in all directions at high magnifications (Fig. 3). Formations of this type can be seen in the neighbourhood of the cells (Fig. 4).
Fro. 4. Keratocyte in a state of degeneration in which the cytoplasm contains formations consisting of a microfibrillar material. Long period fiber (Arrow). Stain: uranyl acetate-lead citrate Gx. ( x 26 000)
(b) Extracellular space : in the neighbourhood of the cells this is profoundly modified; the collagen fibrils losing their regular arrangement, and normal spacing. The periodic striation is often visible. Apart from the previously described formations. there are patches of granular material, microfibrils and fibrils of varying diameter. A few long-period fibres are also observed (Fig. 4). Electron microscopy: cytochemical stak The microtlbrillar substance showed positive reactions to periodie acid stain---thiocarbohydrazide, silver proteinate. It did not react to a short staining time of 30 min with thiocarbohydrazide (T.C.H.). It, showed a positive reaction with a long staining time of T.C.H. of 60 hr. This reaction resulted in the presence of fine particles of 20 A arranged irregularly in the fibrillar network (Fig. 5). On the other hand, no positive reaction is observed in the
168
R. (:K,ZF.
Y. I’OITI,IQUF:N.
JI.-A.
FKOI:lS.
.I.-I’.
FAURF 1. t’. . CIPF’HE’I
other structures apart from certain particles which correspond to a glycogen (positive stain with a short staining time of T.(!.H. of 30 min). The collagen fibrils show slight, staining. The reaction is negative if eit,her oxidation by periodic acid or the action ot thiocarbohydrazide is omitted.
PIG. 5. Aspect of intra-cytoplasmic formations: presence of fine particles in an irregular arrangement, corresponding to silver precipitates. Stain: periodic acid, thiocarbohydrazide (T.C.H.), silver proteinatr (T.C.H. = 60 hr) Gx. ( :i 40 000)
4. Discussion In general, our observations agree with those of Teng (1966) both with respect to the alterations in the keratocytes and the epithelium, which consist largely of the accumulation of material in the agranular endoplasmic reticulum, the changes in both stroma and epithelium running parallel, but those in the epithelium being less severe. The cytochemical studies reported here revealed the presence of both PASpositive and Alcian blue-positive material, the latter corresponding to keratin sulphate. As to the nature of the PAS-positive material, we may consider two hypotheses : (a) The tissue contains both keratin sulphate, responsible for the positive Alcian blue reaction, and another compound, containing several glycol groups, responsible for PAS-positivity ; (b) a single compound, responding positively to both reaction procedures, is present. The latter hypothesis conforms with the finding of Robert and Dische (1965) of a glycoprotein linked to collagen from normal cornea ; the compound was a sulphated sialofucoglucosaminogalactomannoglycan; the positive reaction to
CORNEAL
MACULAR
DYSTROPHY
169
Alcian blue would be due to the sulphate group and the PAS-positivity to one of several glycol or hexose groups in the molecule, as suggested by Garner (1969). ACKNOWLEDGMENTS
The authors wish to thank Madame Colette Favard-Sereno for all the advice she has given us. We also wish t.o thank Madame J. Biseon for her efficient technical assistance. This research was carried out in collaboration with M. A. Girard. Research Group associated with the National Centre for Scientific Research 273 (Inflammation and Immunopathology of the eye). Professor G. Offret. Group 86 of INSERM : Professor P. Payrau. Grant 7122193. REFERENCES Garner, A. (1969). Histochemistry of cornea1 macular dystrophy. Innvest. Ophthdmol. 8, 5. Morgan, 0. (1969). Histochemistry of cornea1 macular dystrophy. Invest. OphthuZmoE. 8, 5. Robert, L. and Dische, Z. (1965). Analysis of sulfated sialofucoglucosaminogalactomarmosidoglycan from cornea1 stroma. Biockm. Biophys. Res. Commun. 10, 209. Teng, C. C. (1966). Macular dystrophy of the cornea. A hi&chemical and electron microscopic study. Amer. J. OphthuZmoZ. 62,3. Thiery, J. P. (1967). Mise en evidence des polysaccharides sur coupes fines en microscopic Blectronique. .I. Micmsc. 6, 987.
Cytochemical Study of Macular Dystrophy of the Cornea (Groenouw II) : an Ultrastructural Study BEATRICE GRAF, TYTVESPOULIQUEN,* MARIE-ALICE JEAN-PIERRE FAURE AND GUY OFFRET
FROUIN,
(Received 3 July 1972, and in revised form 14 November 1973) A cytochemical study of a case of macular dystrophy (Groenouw II) by means of optical and electron microscopy is reported. The epithelial lesions are slighter than the stromal lesions. The kerat,ocytes are the site of important degeneration. The substance secreted by these cells is PAS-positive under both the optical and electron microscope. It is also alcianophil. This substance may be a glycoprotein rather than an acid mucopolysaccharide. The simultaneous positivity of the PAS reaction and the Alcian blue stain argues in favour of the hypothesis, which should be confirmed by a biochemical study.
1. Introduction Macular dystrophy of the cornea represents one of the principal, but rare, forms of recessive hereditary parenchymatous dystrophy of the cornea (Morgan, 1969), so that a light- and electron-microscope study of the cornea in this condition is of some general interest. In the present work a perforating graft was performed on a women aged 66 years suffering from bilateral dystrophy, and the excised cornea1 disc was examined. 2. Methods Immediately after keratoplasty the sample of cornea was subdivided into two, one half being destined for light microscopy and the other for electron microscopy. Light microscopy Tissue was fixed in Bouin’s fluid, dehydrated
and embedded in paraffin.
Stains
(a) Haematoxylin, eosin, safranin. (b) PAS. (MacManus).
Oxidation
by periodic acid for 30 min.
(c) Alcian blue at pH = 2.5 and at pH = 1.0. (d) Alcian blue with MgCl, = 0.1 M. (e) Alcian blue with MgCl, = 1 M.
(f) Thioflavin. (g) Toluidine blue. Electron microscopy Half of the sample was immediately
fixed in a 5% solution of glutaraldehyde
in phosphate
buffer for 30 min, rinsed in buffer solution, and post-6xed by treating with 1% osmium tetroxide in phosphate buffer for 24 hr. It was rinsed in buffer solution, dehydrated and embedded in an aralditelepon mixture. Ultra-thin sections were cut on a microtome (Reichert OM.UII) and examined in a Phillips EM 300 electron microscope (Ete. Frer. Lissac) . * Requests for reprints to Dr Y. Pouliquen, Laboratoire de la Clinique Ophtalmologique de I’HotelDieu, Place de Parvis Notre-Dame, Paris IVe, France. 163
H. (:ltAF.
I64 Stain,ing
Y. I’OI~I,IQl-ES.
11.-A.
FROI.IS.
.I..l’.
FAVRE,
c:. OFFKE’I
of the sectims
(a) Standard staining by many1 acetate, lead citrate. (b) Cytochemical stain to show t,he presence of polysaccharides ---periodic acid, thiocarbohydrazide, silver proteinate~~~according to the method described by Thiery (1947). (i) Periodic acid, 30 min; thiocarbohydrazide, 60 hr; silver proteinate, 20 min. (ii) Controls were prepared in the same way, but without oxidation by periodic acid-thiocarbohydrazide, 60 hr ; silver proteinate, 20 nun; or with oxidation by periodic acid, 30 min, without thiocarbohydrazide; silver proteinate, 20 min.
FIG . 1. (a) Aspect of the epithelial region and adjacent stroma. There is abundant Alcian blue-1 )osit,ive depos it below the basal membrane. Light microscope. Stain: AI&n blue. pH = 2.6 MgCl, 0.1 31. (b) Sin& II deposit below the basal membrane. Light microscope. Stain: Alcian blue. pH = 2.6 Mg( :I, 1 M.
CORNEAL
MACULAR
DYSTROPHY
16.5
3. Results Light nlicroscopy (stain: haernatoxylin,
eosin, safmnin)
1 e1’mm is irregular, sometimes thickened, consisting of six to eight (a) Th e ep’th layers of cells, whilst in other regions it is normal. The basement membrane follows the irregularities of the layer of basal cells. In some regions the epithelium is lacking. (b) Bowman’s membrane is normal in appearance ; however, in the central area, where t*here is an accumulation of substance in the sub-epithelial region, it is lacking. (c) The stroma. The bundles of fibrils of collagen, at this magnification, seem to have a normal arrangement; the keratocytes seem to be globular and sometimes vacuolated. There is an accumulation of material in the sub-epithelial region, in the central area of the sample, in the region corresponding to the break in Bowman’s membrane and at the thickening of the epithelium.
5%~. 3. Aspect of an epithelial cell. Dilatation of the ergastoplasm oont,aining micro-granulofilamentous material. Abundance of free ribosomes. Presence of dense inclusions outside. Persistence of tonofilaments. Stain: wanyl acetate-lead citrate Gx. ( x 31 350)
A deposit of substance is also observed throughout, the whole of the stroma but which is nevertheless predominant in the anterior region. This deposit is localized in the neighbourhood of the keratocytes, between the layers of the stroma. Unfortunately we were unable to examine, in this sample, the extreme posterior region corresponding to Descemet’s membrane and the endothelium.
166
H. GRAF,
Y. POULIQUEX,
M.-A.
FROl’lS.
*J.-P.
FAURE,
G. OFFRET
Light microscopy: cytochemical ataills The substance accumulated in the sub-epithelial region and the deposits situated in the stroma have the same staining affinities. (a) PAS-positive following 30 min of oxidation by periodic acid. However the intensity of the reaction is weak. (b) Alcian blue: positive at pH ‘3.6, at pH 1.0: with MgCl, O-1 M [Fig. l(a)] and with MgCl, 1 M [Fig. l(b)]. (c) Thioflavin T-negat,ive reaction.
FIG. 3. Keratocyte in a state of severe degeneration. Only a voluminous oval-shaped formation is recognizable consisting of a microfibrillar substance. Irregular arrangement of these microfibrils. Stain : uranyl acetate-lead citrate Gx. ( x 40 000)
Electron microscopy: standard examination Epithelium. Certain epithelial cells show alterations, the most important of which are an extreme dilatation of the ergastoplasmic cisternae containing a less dense microgranulo-filamentous material, and certain cisternae contain very dense inclusions, of hazy outline. These inclusions are also observed outside the cisternae. We do not know their significance. There is also an abundance of free ribosomes in the cytoplasm, isolated or in groups. (Fig. 2.) Stroma. The keratocytes: (a) these are in a considerably degenerate condition. At this stage, it is difficult to recognize the different organelles contained in these cells: the nucleus is generally lacking, the cytoplasmic membrane is ruptured. In the cyto-
CORNEAL
MACULAR
DYSTROPHY
167
plasm, there are clear vacuoles of varying sizes, dense bodies, resembling a blackberry, consisting of small dense granules that may correspond to free ribosomes. The mitochondria are no longer recognizable, but voluminous formations, sometimes polycyclic, seem to be the elements that are most consistently found in the cytoplasm of these keratocytes. These formations consist of a microfibrillar substance that is less dense in all directions at high magnifications (Fig. 3). Formations of this type can be seen in the neighbourhood of the cells (Fig. 4).
Fro. 4. Keratocyte in a state of degeneration in which the cytoplasm contains formations consisting of a microfibrillar material. Long period fiber (Arrow). Stain: uranyl acetate-lead citrate Gx. ( x 26 000)
(b) Extracellular space : in the neighbourhood of the cells this is profoundly modified; the collagen fibrils losing their regular arrangement, and normal spacing. The periodic striation is often visible. Apart from the previously described formations. there are patches of granular material, microfibrils and fibrils of varying diameter. A few long-period fibres are also observed (Fig. 4). Electron microscopy: cytochemical stak The microtlbrillar substance showed positive reactions to periodie acid stain---thiocarbohydrazide, silver proteinate. It did not react to a short staining time of 30 min with thiocarbohydrazide (T.C.H.). It, showed a positive reaction with a long staining time of T.C.H. of 60 hr. This reaction resulted in the presence of fine particles of 20 A arranged irregularly in the fibrillar network (Fig. 5). On the other hand, no positive reaction is observed in the
168
R. (:K,ZF.
Y. I’OITI,IQUF:N.
JI.-A.
FKOI:lS.
.I.-I’.
FAURF 1. t’. . CIPF’HE’I
other structures apart from certain particles which correspond to a glycogen (positive stain with a short staining time of T.(!.H. of 30 min). The collagen fibrils show slight, staining. The reaction is negative if eit,her oxidation by periodic acid or the action ot thiocarbohydrazide is omitted.
PIG. 5. Aspect of intra-cytoplasmic formations: presence of fine particles in an irregular arrangement, corresponding to silver precipitates. Stain: periodic acid, thiocarbohydrazide (T.C.H.), silver proteinatr (T.C.H. = 60 hr) Gx. ( :i 40 000)
4. Discussion In general, our observations agree with those of Teng (1966) both with respect to the alterations in the keratocytes and the epithelium, which consist largely of the accumulation of material in the agranular endoplasmic reticulum, the changes in both stroma and epithelium running parallel, but those in the epithelium being less severe. The cytochemical studies reported here revealed the presence of both PASpositive and Alcian blue-positive material, the latter corresponding to keratin sulphate. As to the nature of the PAS-positive material, we may consider two hypotheses : (a) The tissue contains both keratin sulphate, responsible for the positive Alcian blue reaction, and another compound, containing several glycol groups, responsible for PAS-positivity ; (b) a single compound, responding positively to both reaction procedures, is present. The latter hypothesis conforms with the finding of Robert and Dische (1965) of a glycoprotein linked to collagen from normal cornea ; the compound was a sulphated sialofucoglucosaminogalactomannoglycan; the positive reaction to
CORNEAL
MACULAR
DYSTROPHY
169
Alcian blue would be due to the sulphate group and the PAS-positivity to one of several glycol or hexose groups in the molecule, as suggested by Garner (1969). ACKNOWLEDGMENTS
The authors wish to thank Madame Colette Favard-Sereno for all the advice she has given us. We also wish t.o thank Madame J. Biseon for her efficient technical assistance. This research was carried out in collaboration with M. A. Girard. Research Group associated with the National Centre for Scientific Research 273 (Inflammation and Immunopathology of the eye). Professor G. Offret. Group 86 of INSERM : Professor P. Payrau. Grant 7122193. REFERENCES Garner, A. (1969). Histochemistry of cornea1 macular dystrophy. Innvest. Ophthdmol. 8, 5. Morgan, 0. (1969). Histochemistry of cornea1 macular dystrophy. Invest. OphthuZmoE. 8, 5. Robert, L. and Dische, Z. (1965). Analysis of sulfated sialofucoglucosaminogalactomarmosidoglycan from cornea1 stroma. Biockm. Biophys. Res. Commun. 10, 209. Teng, C. C. (1966). Macular dystrophy of the cornea. A hi&chemical and electron microscopic study. Amer. J. OphthuZmoZ. 62,3. Thiery, J. P. (1967). Mise en evidence des polysaccharides sur coupes fines en microscopic Blectronique. .I. Micmsc. 6, 987.