Abstract
purpose. To evaluate corneal endothelial morphology in mice without secreted
extracellular superoxide dismutase (SOD) in normal ageing and in a
lipopolysaccharide (LPS)-induced inflammation model and to measure the
contents of SOD isoenzymes in the mouse cornea and the superoxide
radical concentrations in corneas with and without extracellular SOD.
methods. The central corneal endothelium of wild-type and extracellular
SOD–null mice were studied in micrographs at eight different ages and
after a unilateral intravitreal injection of LPS, with the
contralateral eye serving as the control. The activities of the SOD
isoenzymes in the mouse cornea were determined with a direct assay, the
superoxide radical concentration was assessed by lucigenin-induced
chemiluminescence, and the extracellular SOD distribution was mapped
with immunohistochemistry.
results. The activities of the cytosolic Cu- and Zn-containing SOD, the
mitochondrial Mn-containing SOD and extracellular SOD were 4300, 15,
and 340 U/g wet weight, respectively. Extracellular SOD was found in
the epithelium, stroma, and endothelium. The concentration of
extracellular superoxide radicals was doubled in extracellular SOD-null
corneas, and the endothelial cell density decreased more with age in
extracellular SOD-null than in wild-type control corneas. In the
LPS-induced inflammation model, the cell density decreased more, and
the cells became more irregular in extracellular SOD-null than in
wild-type corneas.
conclusions. In the mouse cornea, absence of extracellular SOD leads to a higher
concentration of extracellular superoxide radicals, an enhancement in
the spontaneous age-related loss of endothelial cells, and an increased
susceptibility to acute inflammatory endothelial damage. Extracellular
SOD is likely to have a protective role in the corneal
endothelium.
The continuous monolayer of hexagonal cells forming the
corneal endothelium has an important function in maintaining corneal
deturgescence and thereby corneal transparency.
1 During the life span of an individual, corneal endothelial cell density
decreases due to a continuous cell loss.
2 In higher
mammals, the loss of endothelial cells is compensated for by sliding
and thinning of adjacent cells to cover the defect,
1 3 but
when these compensatory mechanisms are insufficient, corneal edema
occurs—a major clinical problem and a leading cause of corneal
transplantation.
1 In lower mammals, mitosis may also
compensate for lost corneal endothelial cells,
4 5 but
because these cells are essentially amitotic in all vertebrates in the
resting condition,
4 a gradual reduction of cell density
with age is seen in many species, including the mouse.
6 There are many indices supporting an involvement of reactive oxygen
species in the mechanisms underlying corneal endothelial cell
loss,
7 8 and specifically, the superoxide anion radical
has been ascribed a possible role in age-related cell
loss.
9 10
The loss of corneal endothelial cells can be accelerated in
various stress conditions, such as endothelial wounds,
4 5 intraocular surgery,
11 and systemic
12 or
ocular diseases, including uveitis.
13 Under such
conditions, surviving cells stretch and slide to cover areas of lost
cells, which makes them more irregular and elongated, and the degree of
irregularity in the shape of cells reflects the degree of ongoing
repair of the endothelium.
11 The superoxide anion radical
has been ascribed a role also in the accelerated loss of corneal
endothelial cells seen in inflammations.
14 15 16 In
experimental settings, endotoxin-induced uveitis (EIU), with
administration of lipopolysaccharide (LPS) systemically or
intravitreally,
17 has been used to study endothelial
integrity and regenerative capacity in vivo.
18 Oxygen free
radicals
15 and their reaction products with nitric oxide
(NO)
18 19 are involved in the formation of EIU, and
scavengers of oxygen free radicals, including derivatized superoxide
dismutase (SOD)
20 21 have been demonstrated to reduce the
harmful effects of EIU, indicating an involvement of superoxide
radicals also in the formation of EIU.
To protect tissues from superoxide radicals, there are three SOD
isoenzymes in mammals: the cytosolic Cu- and Zn-containing SOD
(CuZn-SOD),
22 the mitochondrial Mn-containing SOD
(Mn-SOD)
23 and the secreted, interstitially located
extracellular SOD (EC-SOD).
24 The latter has a high
affinity to sulfated glycosaminoglycans and exists mainly anchored to
proteoglycans in the connective tissue matrix and on cell
surfaces.
25 26 Because the substrate of the SOD
isoenzymes, the superoxide anion radical, penetrates biological
membranes poorly, the three SOD isoenzymes exert separate protective
roles. We have found that the human cornea has a very high content of
EC-SOD,
27 located around the epithelial cells, in the
stroma, and in the endothelial layer.
28
To summarize, several investigations link oxygen free radicals to
corneal endothelial cell loss, and the superoxide scavenger EC-SOD is
present in the corneal endothelial layer. Therefore, the present study
was undertaken to elucidate a possible protective role for EC-SOD in
the corneal endothelium by investigating the effect of the absence of
this isoenzyme on corneal endothelial morphology in normal ageing and
in EIU in a murine model. In addition, EC-SOD distribution was mapped
with immunohistochemistry, and the contents of EC-SOD and the other SOD
isoenzymes were measured in the mouse cornea. Finally, the
concentration of superoxide radicals in the presence and absence of
EC-SOD was determined.
The ARVO Statement for the Use of Animals in Ophthalmic and
Vision Research was followed in this investigation. The research ethics
committee of Umeå University, Umeå, Sweden, approved the
investigation. EC-SOD–null mice (initial background C57BL/6 x 129/SV)
were obtained from a breeding colony established at Umeå
University.
29 The EC-SOD–null mice were backcrossed 10
times into C57BL-6J, and C57BL-6J mice were used throughout as the
wild-type control, except for the 12- and 24-month groups, in which the
EC-SOD–null mice were backcrossed five times into C57BL-6J, and
wild-type littermates were used as the control. The EC-SOD–null
genotype is completely without EC-SOD activity but has unaltered
activities of CuZn-SOD, Mn-SOD, catalase, glutathione peroxidase,
glutathione reductase, and glucose-6-phosphate dehydrogenase in all
tissues examined.
29 All animals were kept under identical
conditions in a 12-hour light–dark environment.
Forty-six corneas from wild-type mice were dissected, pooled,
and extracted as previously described.
27 The SOD enzymatic
activity was determined by the direct spectrophotometric method, using
KO
2,
30 31 as previously
detailed.
27 Cyanide (3 mM) was used to distinguish between
the cyanide-sensitive isoenzymes EC-SOD and CuZn-SOD and the resistant
Mn-SOD. To separate EC-SOD from the other two SOD isoenzymes,
chromatography on a Sepharose column (Con A-Sepharose; Pharmacia
Biotech, Uppsala, Sweden) was used as detailed
previously.
32 Finally, the CuZn-SOD activity was
calculated as the total SOD activity minus the Mn-SOD and EC-SOD
activities.
For protein analysis, Coomassie brilliant blue (G-250; Bio-Rad
Laboratories, Inc., Hercules, CA) was used,
33 standardized
with human serum albumin. The DNA concentration was determined with
fluorometry as a complex with bisbenzimidazole (Hoechst 33258; Sigma
Chemical Co., St. Louis, MO)
34 using calf thymus DNA as a
standard.
Two freshly enucleated eye globes from wild-type mice and
two from EC-SOD–null mice, aged 4 months, were fixed in buffered
formaldehyde solution. The samples were processed for standard
immunohistochemical staining according to the peroxidase-antiperoxidase
(PAP) technique,
35 and 4-μm-thick, serial sagittal
sections were photographed in a photomicroscope (Carl Zeiss,
Oberkochen, Germany) with a digital microscope camera (DKC-5000; Sony
Corp., Tokyo, Japan). Polyclonal rabbit antibodies were used, raised
against a synthetic polypeptide corresponding to amino acids 21-42 in
the mouse EC-SOD sequence,
29 and purified on protein A
Sepharose column followed by immobilization of the peptide on a
coupling gel (SulfoLink Pierce, Inc., Rockford, IL). The EC-SOD–null
globes were used as the negative control. The photographs were
digitally stored and processed on computer with image-processing
software (Photoshop; Adobe Systems, Mountain View, CA).
Analysis of the Superoxide Anion Radical with Lucigenin-Derived
Chemiluminescence
Twelve corneas from six EC-SOD–null mice and 12 corneas from
six wild-type mice, ages 13 to 23 weeks, were dissected and kept in
RPMI 1640 medium without phenol red (Gibco/BRL, Life Technologies,
Inc., Gaithersburg, MD) at 37°C in a humidified atmosphere of 95%
air-5% CO
2 for 2 to 5 hours. For analysis, the
corneas were transferred to vials containing 0.5 ml RPMI with 25 μM
lucigenin
36 (Sigma Chemical Co.) and placed in a
luminometer (TD 20/20; Turner Designs, Inc., Sunnyvale, CA).
Luminescence was measured after a 60-second delay during three
consecutive 60-second periods. Thereafter, 20 μg bovine CuZn-SOD was
added to the vials to remove the extracellular superoxide
radicals,
37 and the luminescence was again measured after
a 60-second delay during three consecutive 60-second periods. The means
of the three measurements were used for calculations. The corneas were
then blotted, placed on small stainless steel plates, and weighed
before and after drying at 60°C for 24 hours, for calculation of dry
and wet weights.
The cornea of one eye from each animal was dissected and mounted
between two steel plates with a round hole with a diameter of 1.2 mm,
and the endothelium was stained with alizarin red S and trypan blue, as
proposed by Sperling.
38 No alcohol fixation was used.
Specimens were placed on a glass slide, and the central part of the
endothelium was photographed in a light microscope, at ×400
magnification. Photographs were scanned and analyzed on a computer
(Macintosh, Apple Computer, Cupertino, CA) using NIH Image (developed
at the National Institutes of Health and available in the public domain
at http://rsb.info.nih.gov/nih-image/).
Cell area (
A), perimeter (
P), maximal inertia
moment (
I max), and minimal inertia
moment (
I min) were determined for a
central cluster of 50 cells in each specimen by marking the cell
corners with a digitizer pen. Because the cell clusters consisted of a
continuous monolayer of cells in all specimens investigated (including
the LPS investigation, described later), the corneal endothelial cell
density in cells per square millimeter (
D) could be
calculated as 10
6/
A. The degree of
elongation (
DE) of cells
5 was calculated as
(
I max −
I min)/(
I max+
I min), and the deviation from the
ideal hexagonal shape (hexagon shape factor, HSF) of each cell as abs
(
P 2/
A − 13.856), a
modification of the shape factor formula suggested by Collin and
Grabsch.
39 The value of this modified formula is zero for
a hexagon, and deviations in shape (increased pleomorphism) render
higher values. In some groups, one or two specimens were excluded
because of preparation or staining artifacts. Eight of 10 specimens in
the 2.25-month group and all seven specimens in the
4-month group (
Fig. 1 ,
Table 1 ) were control samples from the LPS investigation (see following
section).
To exclude a difference between the genotypes in the size of the whole
eye globe as an explanation for differences in corneal endothelial cell
density, 8 eyes from four wild-type mice and 10 eyes from five
EC-SOD–null mice, aged 9 months, were dissected, weighed, and
photographed in a dissection microscope with a sagittal projection with
the same equipment as used for the corneas, and the limbus-to-limbus
and limbus-to-vertex distances were measured on the photographs.
Three different sets of 10 EC-SOD–null and 10 wild-type mice
were injected with 25 μg Salmonella abortus LPS
in the vitreous of the right eye. The ages of the three groups of
animals were 4, 2.25, and 2.25 months, respectively. After 4, 7, and 10
days, respectively, the animals were killed and the corneas examined as
described earlier, with the left eye as a control.
Student’s two-tailed t-test was generally used for
statistical analysis, except in the LPS model, for which Student’s
paired t-test was used. P < 0.05 was
considered statistically significant.
The total SOD activity of the mouse cornea was 4700 U/g wet
weight, of which CuZn-, EC-, and Mn-SOD accounted for 4300, 340, and 15
U/g wet weight, respectively. The DNA content of mouse cornea was 3.1
mg/g wet weight, and the protein content was 15 mg/g wet weight.
Immunohistochemistry showed staining for EC-SOD both in the
corneal epithelial and endothelial layers and weaker staining in the
stroma
(Fig. 1A) . Staining of the EC-SOD–null corneas was negligible
(Fig. 1B) .
The total lucigenin-derived luminescence of the corneas did not
differ significantly between EC-SOD–null and wild-type mice (6.4 ± 1.9 relative light units (RLU)/μg wet weight and 5.4 ± 2.6 RLU/μg wet weight, respectively,
P = 0.30).
After addition of bovine CuZn-SOD, however, the luminescence was
significantly more reduced in the EC-SOD–null than in the wild-type
group (2.3 ± 1.0 RLU/μg, compared with 0.96 ± 1.2
RLU/μg,
P = 0.0068). Because it is unlikely that the
SOD added entered the corneal cells during the few minutes of
incubation, the SOD-inhibitable luminescence was interpreted as
deriving from extracellular superoxide radicals.
37 The wet
and dry weights for the specimens did not differ between the two groups
(data not shown).
The mean cell density decreased continuously with age in mice of
both genotypes, but the decrease was more extensive in the EC-SOD–null
mice—the differences in density reaching statistical significance from
2 months
(Table 1) . No differences in the weights of the whole eye and
the limbus-to-limbus or limbus-to-vertex distance were found between
wild-type and EC-SOD–null mice, showing that differences in eye size
did not explain the differences in corneal endothelial cell density
(data not shown).
After LPS injection, all animals showed signs of severe uveitis in
the treated eye within 48 hours, including photophobia, epiphora,
posterior synechiae, hypopyon, and corneal edema
(Fig. 2A) . Some variations in the inflammatory response were noted between
animals, but no obvious difference was seen between the two genotypes.
No signs of inflammation were seen in the untreated contralateral eyes.
At day 4, the corneal edema had resolved in all cases. At this stage,
the endothelial cells were more elongated and showed increased
pleomorphism in the EC-SOD–null but not in the wild-type specimens. No
decrease in cell densities was seen in the groups. At day 7, the cell
densities were reduced in both groups, compared with the untreated
contralateral eyes, although more in the EC-SOD–null group than in the
wild-type group, and the cells showed increased pleomorphism in the
EC-SOD–null group. At day 10, no changes were seen in the wild-type
specimens, compared with the contralateral eyes, whereas the
EC-SOD–null specimens still showed 27% fewer cells and increased
pleomorphism
(Figs. 2B 2C 2D 2E) .
In this study, EC-SOD was present in the murine corneal
endothelium, and absence of this isoenzyme resulted in increased
extracellular superoxide levels in the murine cornea and a decrease in
corneal endothelial cell density, both in normal aging and in the
presence of acute inflammation. These findings suggest that EC-SOD has
a role in protecting the corneal endothelium against oxidative stress.
In humans, accelerated age-dependent endothelial cell loss with
increased apoptosis of corneal endothelial cells is seen in
Fuchs endothelial dystrophy.
40 Photo-oxidative
injury has been shown to induce corneal endothelial cell apoptosis in
animal models.
7 8 It could be speculated that the
accelerated age-related endothelial cell loss in EC-SOD–null mice is
caused by an increased extracellular superoxide concentration, both
from the basal metabolism and from photochemical reactions.
In an acute injury, altered morphology, cell elongation, and
pleomorphism are more pronounced features,
11 as found in
the current study in the LPS inflammation model. A prominent feature of
this model is invasion of polymorphonuclear leukocytes,
15 in our animals, appearing as hypopyon formations
(Fig. 2A) . Superoxide
radicals produced by these are likely to contribute to endothelial
damage. Another important inflammatory effector substance in EIU is
NO.
16 18 19 NO is normally formed by the corneal
endothelium and is of importance in its function.
41 In the
presence of excessive superoxide radicals, however, the toxic
peroxynitrite (ONOO
−)
19 42 can be
formed from NO, and superoxide and is probably an important route
leading to inflammatory damage in the corneal endothelium and other
ocular tissues.
16 42 Suppression of
ONOO
− toxicity may be one of the mechanisms by
which EC-SOD protects the corneal endothelium in this model. The
present results are in accordance with previous findings in the
EC-SOD–null mice of a marked enhancement of inflammatory lung damage
induced by high oxygen tension.
29
The activity of CuZn-SOD, related to wet weight, is higher in the mouse
cornea than in the human cornea.
27 When comparing tissues
with a low cellularity, such as the cornea, intracellular enzymes are
better related to the DNA content, and the CuZn-SOD activity per
milligram DNA is found to be rather lower in the mouse cornea than in
the human cornea. The activity of EC-SOD in the human cornea is related
to wet weight or protein content or is approximately four times higher
than that which we found in the present study in the murine cornea.
Compared with other mouse organs, corneal EC-SOD content is moderately
high.
29 Absence of EC-SOD in the mouse cornea results in
an increased basal formation of extracellular superoxide radicals and
an endothelium more susceptible to age-related and inflammatory damage.
The findings suggest that EC-SOD may also be of importance in the
integrity of the human corneal endothelium.