Download Phagocytosis of polystyrene spheres in the rabbit corneal

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Phogocytosis of Polystyrene Spheres in the Robbit Corneol Endothelium:
Contribution of Lysosomoi Enzymes to the Endothelioi Degenerotion
Saroshi Hara, Sei-ichi Ishiguro, and Karsuyoshi Mizuno
solution (BSS), pH 7.6, according to the method of
Hollyfield and Ward.7 The concentration of polystyrene was adjusted to 10" particles per milliliter.
Injection of polystyrene spheres: New Zealand white
rabbits, weighing 2 to 3 kg each, were anesthesized with
an intramuscular injection of ketamine hydrochloride.
A 27-gauge needle inserted at the limbus was used to
aspirate 200 ^1 of aqueous humor; the same amount
of polystyrene sphere suspension was injected into the
anterior chamber of the left eye at the point. The anterior chamber of the right eye received 200 ix\ of BSS
as a control. This investigation adhered to the ARVO
Resolution on the Use of Animals in Research.
Tissue preparation: At 20 min, 1 hr, 4 hr, and 24 hr
after the injection of polystyrene spheres, the animals
were killed by an injection of ketamine into the marginal ear vein, and the eyes were enucleated. All of the
following procedures were carried out at about 4°C.
For electron microscopic study, the excised cornea was
fixed in 2.5% glutaraldehyde containing 0.1 M phosphate buffer, pH 7.4, for 2 hr. For biochemical study,
the Descemet's membrane-endothelium complex of 8
mm in diameter was peeled from the stroma with a
sharpened jeweler's forceps. The complex was put in
0.5 ml of 0.25 M sucrose solution and the homogenate
was made using the same Potter-Elvehjem homogenizer for each experiment. A 0.1 ml aliquot was used
to determine the DNA content. The remaining 0.4 ml
was centrifuged at 20,000 g for 20 min; the supernatant
was the unsedimentable fraction. The pellet was resuspended in 0.5 ml of 0.25 M sucrose solution, and
that was the sedimented fraction, which contained most
of the intact lysosomes.8 These unsedimentable and
sedimented fractions were subjected to the lysosomal
enzyme assays after being frozen and thawed six times.
Total lysosomal enzyme activities of the homogenate
were, therefore, obtained by adding the activities in the
sedimented fraction and in the unsedimentable fraction.
The rabbit corneal endothelium phagocytized polystyrene
spheres 0.5 Mm in diameter. After phagocytizing spheres, the
endothelium degenerated, and lost from the Descemet's
membrane. Lysosomal enzyme activities of the endotheliumDescemet's membrane complex, such as acid phosphatase,
/?-glucuronidase and N-acetyl-/?-D-glucosaminidase, were
assayed and the total activities per microgram DNA were
almost constant. The unsedimentable activities in the complex,
however, increased by phagocytosis of polystyrene spheres,
which indicated an extralysosomal release of lysosomal enzymes. Released lysosomal enzymes probably would have accounted for the degeneration of the corneal endothelium. Invest Ophthalmol Vis Sci 26:1631-1634, 1985
Although the cultured corneal endothelium has been
reported to phagocytized polystyrene beads' and take
up low density lipoprotein,2 few reports describe endocytosis of the corneal endothelium. Saccharated iron3
and colloidal particles4 have been taken up by the endothelium of living rabbit corneas after the irrigation
of those substances into the anterior chamber. Pigment
granules have been degraded in the human corneal endothelium, 5 and copper has been found in the corneal
endothelium in Wilson's disease.6
The corneal endothelium sometimes is degenerated
by the uptake of saccharated iron3 or copper.6 Lysosomal enzymes, which have very high activities in the
corneal endothelium (unpublished data), digest
phagocytized substance. It is, therefore, important to
understand the changes of lysosomal enzyme activities
and the intracellular distribution of those enzymes
during phagocytosis to clarify the pathogenesis of corneal endothelial degeneration. The purpose of this paper is to describe phagocytosis of polystyrene beads by
the rabbit corneal endothelium by electron microscopy
and changes in the intracellular distribution of the lysosomal enzymes biochemically.
Materials and Methods. Polystyrene spheres were
washed in three changes of the sterile balanced salt
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INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / November 1985
Vol. 26
Fig. I. Transmission electron micrograph of the corneal endothelium at 20 min after the injection of polystyrene sphere suspension into the
anterior chamber. Polystyrene spheres (arrows) were phagocytized and surrounded by a membrane, indicating a phagosome. Vacuoles and
swollen mitochondria were seen in the cytoplasm (X27,000).
1.5
1.0
0.5
0
Untreated 0
1
4
24
Time after Injection of Polystyrene Beads (hr)
Fig. 2. DNA content in the 8-mm diameter endothelium-Descemet's membrane complex. At each time period after injection of
polystyrene sphere suspension into the anterior chamber, one eye
was enucleated, and the 8-mm diameter endothelium-Descemet's
complex was peeled and prepared for biochemical analysis as described in the text. DNA content was measured by the method of
Kissane and Robins.10 Means and standard deviations of four separate
experiments are shown.
Lysosomal enzyme assay: Acid phosphatase [E.C.
3.1.3.2] 0-gIucuronidase [E.C. 3.2.1.31] and N-acetyl/3-D-glucosaminidase [E.C. 3.2.1.30] were assayed in
the manner described by Hayasaka and Shiono,9 using
p-nitrophenyl derivatives as substrate.
Determination of DNA content: The DNA of Descemet's membrane-endothelium complex was fluorometrically assayed by the method of Kissane and Robins10 using diaminobenzoic acid. Calf thymus DNA
was used as the standard. Specific lysosomal enzyme
activities of the corneal endothelium-Descemet's
membrane complex were expressed as total activities
of the complex per microgram of DNA.
Histologic examination: The excised cornea was also
examined with transmission electron microscopy
(TEM). The tissue was postfixed in 1% osmium tetroxide and dehydrated in a series of graded ethanol.
The tissue was embedded in epoxy resin (EPON 812),
thin sectioned, stained with lead citrate-uranyl acetate,
and examined by a JEOL-lOOc transmission electron
microscope.
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Reports
Measurement of the intraocular pressure: At 20, 40,
60, 120, 240 min and one day after the injection of
spheres, intraocular tension was measured in the rabbits
using the Perkins hand-held applanation tonometer,
following local anesthesia with a drop of 4% oxybuprocaine hydrochloride. No restraint was used on the
rabbits during the measurement.
Results. All values were determined as the means
plus standard deviations of four different experiments.
The rabbit corneal endothelium showed no remarkable
changes after the injection of BSS. After 20-min exposure to polystyrene sphere suspension, the rabbit
corneal endothelium phagocytized the spheres (Fig. 1).
The sphere was surrounded by a membrane, which
disclosed a phagosome. A few endothelial cells were
degenerated and contained numerous vacuoles and
swollen mitochondria. Cell membranes were disrupted
and a large number of spheres penetrated into the cell
after 60-min exposure to polystyrene sphere suspension. As the corneal endothelium was exposed to the
spheres for a longer period, the extent of degenerated
cells increased, resulting in direct exposure of Descemet's membrane to the anterior chamber, caused by
the large number of cells lost. Figure 2 shows the DNA
content in the 8-mm diameter corneal endotheliumDescemet's complex. The decrease of DNA indicated
a loss of endothelium from the complex.
Specific activities of acid phosphatase, 0-glucuronidase, and N-acetyl-/3-D-glucosaminidase in the complex did not change during experiments (Fig. 3). These
three lysosomal enzyme activities in the unsedimentable fraction, however, increased significantly (Fig. 4).
The intraocular pressure, which rose to 13 Perkins tonometer readings 20 min after the injection of spheres,
decreased to 10 Perkins tonometer readings 60 min
after the injection.
Discussion. Under certain clinical situations the
corneal endothelium has been induced to take pigment
granules5 and copper.6 It is, therefore, clear that endocytosis occurs in the corneal endothelium in pathologic conditions, although the role of endocytosis in
this tissue is not known in physiologic situation.
The corneal endothelial cells that take up copper6
or iron3 sometimes show vacuolation of their cytoplasm. In this study, the corneal endothelium also degenerated after the phagocytosis of polystyrene spheres.
Although the intraocular pressure temporarily increased to 13 during the experiments, the pressure rise
could not have caused the endothelial degeneration,
because no changes were noticeable in the control corneal endothelium irrigated with BSS after maintaining
the ocular pressure at 15 for 20 min.
Possibly, the degeneration of the corneal endothe-
0
1
4
24
Untreated
Time after Injection of Polystyrene Beads (hr)
Fig. 3. Lysosomal enzyme activities in the endothelium-Descemef s
complex per microgram of DNA. Lysosomal enzymes such as acid
phosphatase (O
O), /3-glucuronidase ( •
• ) , and N-acetyljS-D-glucosaminidase (A
A) were assayed in the complex which
had been treated by irrigating polystyrene sphere suspension in the
anterior chamber. Means and standard deviations of four separate
experiments are shown.
Hum under the present conditions occurred as a result
of changes in the intracellular distribution of the lysosomal enzyme though the mechanism of the extralysosomal release of lysosomal enzymes is still unclear.
Unsedimentable activities of lysosomal enzymes in-
80
70
60
E
50
40
>.
30
20
10
Untreated 0
1
4
24
Time after Injection of Polystyrene Beads (hr)
Fig. 4. Proportion of lysosomal enzyme activity of unsedimentable
fraction in thcendothelium-Descemet's membrane complex. Symbols
are the same as those used in Fig. 3. Unsedimentable fraction was
prepared as described in the text.
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INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / November 1985
creased after the injection of polystyrene spheres, which
indicated an extralysosomal release of lysosomal enzymes. Released lysosomal enzymes probably would
have accounted for the degeneration of the corneal endothelium. Disturbances of the phagolysosomal system
might account for the degeneration of the endothelial
cells in some pathologic conditions.
Key words: cornea, endothelium, phagocytosis, polystyrene
spheres, lysosomal enzymes
3.
4.
5.
From the Department of Ophthalmology, Tohoku University
School of Medicine, Miyagi, Japan. Submitted for publication: September 6, 1984. Reprint requests: Satoshi Hara, MD, Department
of Ophthalmology, Tohoku University School of Medicine, 1-1
Seiryo-cho, Sendai, Miyagi 980 Japan.
6.
References
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1. Tripathi RC and Tripathi BJ: Human trabecular endothelium,
corneal endothelium, keratocytes and scleral fibroblasts in primary cell culture: a comparative study of growth characteristics,
morphology, and phagocytic activity by light and scanning electron microscopy. Exp Eye Res 35:611, 1982.
2. Goldminz D, Vlodavsky I, Johnson K, and Gospodarowicz D:
Contact inhibition and the regulation of endocytosis in the corneal
endothelium: correlation with a restricted surface receptor lateral
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Vol. 26
mobility and the appearance of a fibronectin meshwork. Exp
Eye Res 29:331, 1979.
Cibis PA and Yamashita T: Experimental aspects of ocular siderosis and hemosiderosis. Am J Ophthalmol 48:465, 1969.
Iwamoto T and Smelser G: Electron microscopy of the human
corneal endothelium with reference to transport mechanisms.
Invest Ophthalmol 4:270, 1965.
Kampik A, Patrinely JR, and Green RW: Morphologic and clinical features of retrocorneal melanin pigmentation and pigmented
pupillary membrane: review of 225 cases. Surv Ophthalmol 27(3):
161, 1982.
Tso MOM, Fine BS, and Thorpe HE: Kayser-Fleischer ring and
associated cataract in Wilson's disease. Am J Ophthalmol 79:
479, 1975.
Hollyfield JG and Ward A: Phagocytic activity in the retinal
pigment epithelium of the frog Rana pipiens 1. Uptake of polystyrene spheres. J Ultrastruct Res 46:327, 1974.
Hayasaka S, Hara S, and Mizuno K: Distribution and some
properties of cathepsin D in the retinal pigment epithelium. Exp
Eye Res 21:307, 1974.
Hayasaka S and Shiono T: a-Fucosidase, a-mannosidase and /3N-acetylglucosaminidase of the bovine retinal pigment epithelium. Exp Eye Res 34:565, 1982.
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Hydration Stability of Intracomeal Hydrogel Implants
W. Houdijn Beekhuis* and Bernard E. McCorey
A hydrogel intracorneal lens for refractive keratoplasty must
have predictable and stable optics when implanted in the corneal stroma. A series of experiments was performed to evaluate the hydrogel hydration stability when in the corneal
stromal environment. Hydrogel ICLs of 54%, 63%, 66% and
71% water content showed no loss of hydration (by weight)
after one week in the rabbit corneal stroma. In vitro experiments with hydrogel discs of 56%, 65%, 69.5% and 75% water
content were subjected to swelling pressures ranging from 55
to 150 mmHg in a suction chamber. Only the hydrogel of
75% water content showed a significant loss of hydration at
the physiologic swelling pressure of 55 mmHg. This study
shows that hydrogel materials with up to 69.5% water content
can be expected to be dimensionally stable when used in keratorefractive surgery. Invest Ophthalmol Vis Sci 26:16341636, 1985
Refractive keratoplasty with hydrogel lenticules has
the potential advantage of utilizing predictable preoperative designed lenticules. Yet, McCarey and Andrews,1 observed that Permalens lenticules, when measured by pachymetry, were 28 ± 2% (n = 13) thinner
in vivo than the preoperative measurement. This was
further confirmed in preliminary investigations in
monkeys. The thinning was considerable and thought
to possibly influence the amount of refractive change
by the hydrogel intracorneal lens (ICL). KJyce et al2
observed high water content hydrogel materials (93%
and 95% water content) to have, respectively, a 26%
and 29% thinning of their original thickness when exposed to the physiologic stromal swelling pressure. In
this study we examined changes in the hydration of
hydrogel ICLs of various water contents (55%—75%
water) when subjected to normal stromal swelling
pressure. The objective of this investigation was to establish if there is dimensional stability of the hydrogel
lenticules used in experimental alloplastic keratorefractive surgery.
Materials and Methods. Water content and diameter
of hydrogel intracorneal implants in rabbits: Hydrogel
ICLs (Vistamarc®; Jacksonville, FL) with water contents of approximately 55%, 63%, 66% and 70% were
individually measured preoperatively for their wet
weights and diameters. The wet weight was determined
after the hydrogel had equilibrated with a phosphate
buffered saline solution (pH 7.4 .and 306 mOsmol).
The preoperative diameter was determined with the
wet ICL supported by a steel ball of approximately the
same radius of curvature, using a calibrated eye piece
in the slitlamp. After these measurements had been
taken, the hydrogel ICLs were implanted in rabbit corneas via a lamellar pocket incision. After one postoperative wk, the lenses were removed from the cornea
and their wet weights immediately determined. The
ICL dry weights were determined after the lenses were
dehydrated at 60°C in a vacuum desiccator for 48 hr.
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