Corneal Endothelial Integrity in Mice Lacking Extracellular Superoxide Dismutase

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. 

Materials and Methods

Animals

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. 

SOD Analysis

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 KO₂, ^{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. 

Immunohistochemistry

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% CO₂ 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. 

Corneal Endothelial Morphology at Different Ages

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 10⁶/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. 

LPS Inflammation Model

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. 

Statistics

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. 

Results

SOD Isoenzyme Activities in the Mouse Cornea

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

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) . 

Superoxide Radical Concentration

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). 

Age-Related Changes in Corneal Endothelial Morphology

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). 

LPS Inflammation Model

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) . 

Discussion

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. 

 Submitted for publication November 14, 2000; revised May 24, 2001; accepted July 11, 2001.

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Figure 1.

 

Serial transverse formaldehyde-fixed sections of wild-type and an EC-SOD–null mouse corneas, stained with the PAP technique and counterstained with hematoxylin. A polyclonal rabbit antibody against a murine EC-SOD polypeptide was used as the primary antibody. Both the epithelial and endothelial layers clearly showed staining, whereas the stroma exhibited weaker staining (A). Staining of the EC-SOD–null cornea was negligible (B). Scale bars, 10μ m.

Table 1.

 

Corneal Endothelial Cell Morphology in Mice at Different Ages

	6 Days	1 Month	2 Months	2.25 Months	4 Months	9 Months	12 Months	24 Months
Wild Type
D (cells/mm2)	6984 ± 1050	4172 ± 179	3884 ± 292	3359 ± 314	2737 ± 388	2449 ± 179	2345 ± 178	1944 ± 141
P (μm)	47.7 ± 3.8	59.9 ± 1.3	61.7 ± 2.3	66.4 ± 2.9	73.5 ± 4.7	76.7 ± 2.7	78.9 ± 3.1	86.3 ± 3.0
DE	0.16 ± 0.022	0.14 ± 0.01	0.13 ± 0.01	0.13 ± 0.02	0.12 ± 0.01	0.12 ± 0.01	0.11 ± 0.02	0.13 ± 0.01
HSF	2.04 ± 0.37	1.28 ± 0.08	1.06 ± 0.17	1.06 ± 0.26	0.96 ± 0.08	0.95 ± 0.09	0.87 ± 0.13	1.06 ± 0.13
n	8	10	10	10	7	9	10	8
EC-SOD null
D (cells/mm2)	6965 ± 984	4191 ± 365	3401 ± 170	2812 ± 161	2183 ± 295	2023 ± 197	1940 ± 140	1540 ± 98
P (μm)	48.1 ± 3.2	59.9 ± 2.6	65.9 ± 1.6	72.5 ± 1.8	82.3 ± 5.5	84.7 ± 3.9	86.8 ± 3.3	98.0 ± 3.2
DE	0.17 ± 0.010	0.14 ± 0.02	0.13 ± 0.01	0.13 ± 0.02	0.11 ± 0.01	0.12 ± 0.01	0.11 ± 0.01	0.13 ± 0.01
HSF	2.30 ± 0.31	1.31 ± 0.27	1.00 ± 0.17	1.02 ± 0.23	0.91 ± 0.10	0.90 ± 0.18	0.85 ± 0.16	1.14 ± 0.14
n	8	10	10	10	9	9	8	8
Probabilities
D	NS	NS	0.00045	0.00026	0.0095	0.00020	0.000058	0.000019
P	NS	NS	0.00027	0.000052	0.0041	0.00016	0.00014	0.0000029
DE	NS	NS	NS	NS	NS	NS	NS	NS
HSF	NS	NS	NS	NS	NS	NS	NS	NS

 Factors expressing the degree of pleomorphism, of wild type and EC-SOD–null mice at different ages. The data are expressed as mean ± SD. A continuous cluster of 50 central cells was measured in each specimen. Student’s two-tailed t-test was used for calculation of probabilities, comparing the wild type with the EC-SOD–null group.

Figure 2.

 

(A) Uveitis in an EC-SOD–null mouse 48 hours after intravitreal injection of LPS, showing posterior synechiae (white arrows), hypopyon (short black arrow), and corneal edema (long black arrow). (B–E) Murine corneal endothelium at day 10 after a unilateral intravitreal LPS injection, stained with alizarin red S and trypan blue. (B) EC-SOD–null mouse, untreated control eye; (C) EC-SOD–null mouse, LPS-treated eye, showing enlarged and irregular cells; (D) wild-type mouse, untreated control eye; and (E) wild-type mouse, LPS-treated eye.

Table 2.

 

Corneal Endothelial Cell Morphology after LPS Injection

	4 Days					7 Days					10 Days
		LPS	Contralateral	P	LPS	Contralateral	P	LPS	Contralateral	P
Wild type
D (cells/mm2)	2600 ± 385	2737 ± 388	NS	2837 ± 202	3356 ± 274	0.0027	3120 ± 167	3263 ± 168	NS
P (μm)	76.0 ± 5.5	73.5 ± 4.7	NS	72.6 ± 2.2	66.7 ± 2.7	0.0015	68.5 ± 1.7	67.3 ± 1.6	NS
DE	0.13 ± 0.01	0.12 ± 0.01	NS	0.12 ± 0.01	0.13 ± 0.02	NS	0.12 ± 0.01	0.11 ± 0.01	NS
HSF	0.99 ± 0.07	0.96 ± 0.07	NS	1.19 ± 0.21	1.16 ± 0.34	NS	1.02 ± 0.17	0.97 ± 0.10	NS
n	7	7		8	8		9	9
EC-SOD null
D (cells/mm2)	2150 ± 142	2183 ± 295	NS	2098 ± 260	2790 ± 174	0.0016	2002 ± 191	2720 ± 213	0.00018
P (μm)	83.1 ± 2.8	82.3 ± 5.5	NS	84.3 ± 5.8	72.7 ± 2.0	0.0023	86.8 ± 4.5	73.4 ± 2.9	0.00010
DE	0.14 ± 0.02	0.11 ± 0.01	0.0059	0.12 ± 0.01	0.13 ± 0.03	NS	0.14 ± 0.02	0.12 ± 0.01	NS
HSF	1.25 ± 0.26	0.91 ± 0.10	0.014	1.08 ± 0.20	0.95 ± 0.21	0.015	1.37 ± 0.50	0.92 ± 0.09	0.050
n	9	9		8	8		8	8

 Factors expressing the degree of pleomorphism, of wild type and EC-SOD null mice 4, 7, and 10 days after a unilateral intravitreal LPS injection. The data are expressed as mean ± SD. The ages of the three groups of mice were 4, 2.25, and 2.25 months, respectively. A continuous cluster of the central cells was measured in each specimen. Student’s paired t-test was used for calculation of probabilities, comparing the treated eyes with the untreated contralateral eyes.