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.
Analysis of the Superoxide Anion Radical with Lucigenin-Derived
Chemiluminescence
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.
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.