# Change in Long-Spacing Collagen in

Authors: Yoshihiro Akimoto; Hajime Sawada; Mica Ohara-Imaizumi; Shinya Nagamatsu; Hayato Kawakami
Journal: Experimental Diabetes Research
 (2008)

---

## Abstract

We  examined changes in the ultrastructure and localization of major extracellular matrix components, including 5 types of collagen (type I, III, IV, VI, and VIII), laminin, fibronectin, and heparan sulfate proteoglycan in Descemet's membrane of the cornea of diabetic GK rats. In the cornea of diabetic GK rats, more long-spacing collagen fibrils were observed in Descemet's membrane than in the membrane of the nondiabetic Wistar rats. Both GK and Wistar rats showed an age-dependent increase in the density of the long-spacing collagen. Immunoelectron microscopy showed that type VIII collagen was localized in the internodal region of the long-spacing collagen, which was not labelled by any of the other antibodies used. The antidiabetic agents nateglinide and glibenclamide significantly suppressed the formation of the long-spacing collagen in the diabetic rats. Long-spacing collagen would thus be a useful indicator for studying diabetic changes in the cornea and the effect of antidiabetic agents.


---

## Body

## 1. Introduction

Corneal keratopathy is one of the
diabetic complications. Clinically, the diabetic cornea often shows superficial
punctate keratopathy and persistent epithelial defects and recurrent epithelial
erosion that are considered to be a form of diabetic keratoepitheliopathy.
Various degrees of epithelial disturbance take place in the diabetic cornea.
Thickening of epithelial basement membrane in the diabetic cornea has been
extensively studied [1, 2].
In a previous study, we showed that hemidesmosomes in the epithelial basal
cells were decreased in number in the diabetic rats and that the basement
membrane detached from the epithelial basal cells [3]. Also, the
content of O-GlcNAc-modified proteins was found to be increased in the corneal
epithelium as well as in the nerves, kidneys, and pancreas of diabetic rats [3–5].The diabetic
corneal endothelium has been shown by speculum-aided microscopy to have morphological
abnormalities such as polymorphism [6]. The endothelial cells
vary in cell shape and in cell area in the diabetic rat and human cornea [7, 8]. At the posterior side of the cornea, a thick
basement membrane called Descemet’s membrane is located adjacent to the
endothelium. In the normal human cornea, long-spacing collagen, which is
cross-striated fiber bundle, is located only in the anterior-banded zone of
Descemet's membrane [9, 10]. However, in the cornea of normal
Sprague-Dawley rats, there is no such collagen in Descemet's membrane [11]. Although the normal rat
and human Descemet's membranes differ in this regard, in the corneas of both diabetic
human patients and Streptozotocin-induced diabetic rats, unusual long-spacing
collagen was observed scattered in Descemet’s membrane [11, 12].The spontaneously
diabetic Goto-Kakizaki (GK) rat is a nonobese model of type 2 diabetes that was
developed by the selective breeding of glucose-intolerant Wistar rats [13–15]. In the eyes of GK rats, various
abnormalities have been reported, including decreased retinal microcirculation [15], elevated levels of vascular endothelial growth factor [16], nitric oxide synthase activity in the retina [17], delayed
wound closure, as well as phenotypic changes in the corneal epithelium [18]. However,  little attention
has been paid to Descemet’s membrane in GK rats. To investigate Descemet’s
membrane in terms of the pathogenesis of diabetes mellitus, in the present
study we examined the ultrastructural morphology, and immunohistochemically
determined the composition of Descemet’s membrane in the cornea of diabetic GK
rats in comparison with normal Wistar rats. Furthermore, we examined if the
morphological change detected could be prevented by antidiabetic agents. Our
findings revealed that unusual long-spacing collagen appeared and increased in
content rapidly with aging in the Descemet's membrane of the diabetic rat cornea,
and that its appearance could be suppressed by the antidiabetic agents.


## 2. Materials and Methods

### 2.1. Animals and Tissues

All experimental procedures using
laboratory animals were approved by the Animal Care and Use Committee of Kyorin
University School of Medicine. The corneas of 15-, 33-, and 62-week-old male
(n=6 for each age) Goto-Kakizaki rats and Wistar rats (as normal controls),
obtained from Kurea (Tokyo, Japan), were
used in the present study. Rats were
housed under 12-hour light: 12-hour dark cycle and given free access to food and water. Serum
glucose levels in Wistar and GK rats, which were measured after an overnight
fast, were, respectively, 158.0±12.0 and 375.9±11.6 (mean±SEM) mg/dL at 15 weeks, 118.5±10.5 and 333.8±22.4 mg/dL at 33 weeks, and
167.0±28.6 and 314.9±48.7 mg/dL at 62 weeks. As
reported previously [5], serum insulin levels were also
higher in the GK rats.


### 2.2. Antibodies

A monoclonal antibody (clone
9H3) against Type VIII collagen and a monoclonal antibody (clone 15B6) against
Type VI collagen were prepared and characterized as described previously [19, 20]. Polyclonal antibody against laminin and type IV collagen were purchased from EY laboratory (San Meteo, Calif, USA) and LSL (Tokyo, Japan),
respectively. Monoclonal antibody against heparan sulfate proteoglycan and
fibronectin were purchased from Upstate Biotechnology (Lake Placid, NY, USA) and Chemicon International (Temecula, Calif, USA), respectively. Alexa568-conjugated donkey anti-rabbit or mouse IgG and
SYBR-Green I were obtained from Molecular Probes (Eugene, Ore, USA).


### 2.3. Immunohistochemical Localization

Immunofluorescence
observation for localization of components of extracellular matrix was
performed as described earlier [21]. Corneas were fixed in 4%
paraformaldehyde in 0.1 M phosphate buffer (pH7.3) for 1 hour at 4°C. After
having been washed with PBS, the specimens were embedded in OCT compound
(Miles; Elkhart, Ill, USA).
Frozen sections (4 μm thick) were made, washed with PBS, and incubated for 10
minutes in 5% BSA in PBS. The sections were then incubated with
anti-extra-cellular matrix component antibody for 1 hour at room temperature,
washed with PBS, and subsequently incubated with Alexa 568-conjugated donkey
anti-rabbit or mouse IgG antibody (1:200). Nuclei were stained with SYBR-Green
I (1:500). After a final wash with PBS, the specimens were mounted in 90%
glycerol-0.1 M Tris-HCl buffer (pH8.5) containing 0.5 mM p-phenylene diamine,
and observed under a laser scanning confocal microscope (LSM510, Zeiss, Mass, USA). For a control
experiment, the specimens were incubated with normal rabbit or mouse IgG or
with 0.1% BSA-PBS alone instead of the primary antibodies. No positive staining
was observed in the control experiment (data not shown).


### 2.4. Electron Microscopy

Corneas were fixed in
phosphate-buffered 2.5% glutaraldehyde (pH7.4). Strips of cornea were taken
from the central part of the cornea, and were postfixed in 1%OsO4 in 0.1 M phosphate buffer (pH7.4), and dehydrated with graded
alcohols. After immersion in propylene
oxide, the specimens were embedded in Epon 812.
Ultrathin sections were cut perpendicular to the epithelium, doubly
stained with uranyl acetate and lead citrate, and examined with a transmission
electron microscope, TEM-1010 (JEOL, Tokyo, Japan).


### 2.5. Immunoelectron Microscopy

Immunoelectron microscopic
observation for localization of components of extracellular matrix was
performed as described earlier [22]. Fixation was carried out in the same way as for light
microscopy. Ultrathin frozen-sections were cut at −90 to −100°C. The sections were washed with PBS and
pretreated with 1% BSA in PBS for 10 minutes. After a PBS rinse, they were
incubated with the desired antibodies for 1 hour, washed with PBS, and
incubated with colloidal gold-conjugated goat anti-rabbit or mouse IgG
antibodies for 1 hour. After another wash with PBS, the sections were refixed
in 2% glutaraldehyde-0.1 M phosphate buffer, pH7.4, and embedded in a mixture
of methylcellulose, polyethyleneglycol, and uranyl acetate.


### 2.6. Antidiabetic Agents and Experimental Design

The administration of antidiabetic drugs
was started at 8 weeks old. Nateglinide (50 mg/kg) or glibenclamide (2 mg/kg)
was suspended in 0.5% methylcellulose and administered to GK rats via a stomach
tube in volume of 10 mL/kg [23–25]. These doses of the antidiabetic
agents were chosen from the data on their suppressive effects on the peak blood
glucose levels after oral sucrose or glucose loading of fasted normal rats for
15 weeks [23, 26]. GK rats were fed twice daily (9:00 and 16:00)
for 1 hour and were given nateglinide or glibenclamide orally just before each
meal. Control rats were treated with 0.5% methylcellulose alone (the vehicle).


### 2.7. Statistical Analysis

Results were expressed as themean±standard  deviation,  and the statistical analysis was done by the use of the unpaired
Student’s t-test. Differences were defined as significant at P<.05.


## 2.1. Animals and Tissues

All experimental procedures using
laboratory animals were approved by the Animal Care and Use Committee of Kyorin
University School of Medicine. The corneas of 15-, 33-, and 62-week-old male
(n=6 for each age) Goto-Kakizaki rats and Wistar rats (as normal controls),
obtained from Kurea (Tokyo, Japan), were
used in the present study. Rats were
housed under 12-hour light: 12-hour dark cycle and given free access to food and water. Serum
glucose levels in Wistar and GK rats, which were measured after an overnight
fast, were, respectively, 158.0±12.0 and 375.9±11.6 (mean±SEM) mg/dL at 15 weeks, 118.5±10.5 and 333.8±22.4 mg/dL at 33 weeks, and
167.0±28.6 and 314.9±48.7 mg/dL at 62 weeks. As
reported previously [5], serum insulin levels were also
higher in the GK rats.


## 2.2. Antibodies

A monoclonal antibody (clone
9H3) against Type VIII collagen and a monoclonal antibody (clone 15B6) against
Type VI collagen were prepared and characterized as described previously [19, 20]. Polyclonal antibody against laminin and type IV collagen were purchased from EY laboratory (San Meteo, Calif, USA) and LSL (Tokyo, Japan),
respectively. Monoclonal antibody against heparan sulfate proteoglycan and
fibronectin were purchased from Upstate Biotechnology (Lake Placid, NY, USA) and Chemicon International (Temecula, Calif, USA), respectively. Alexa568-conjugated donkey anti-rabbit or mouse IgG and
SYBR-Green I were obtained from Molecular Probes (Eugene, Ore, USA).


## 2.3. Immunohistochemical Localization

Immunofluorescence
observation for localization of components of extracellular matrix was
performed as described earlier [21]. Corneas were fixed in 4%
paraformaldehyde in 0.1 M phosphate buffer (pH7.3) for 1 hour at 4°C. After
having been washed with PBS, the specimens were embedded in OCT compound
(Miles; Elkhart, Ill, USA).
Frozen sections (4 μm thick) were made, washed with PBS, and incubated for 10
minutes in 5% BSA in PBS. The sections were then incubated with
anti-extra-cellular matrix component antibody for 1 hour at room temperature,
washed with PBS, and subsequently incubated with Alexa 568-conjugated donkey
anti-rabbit or mouse IgG antibody (1:200). Nuclei were stained with SYBR-Green
I (1:500). After a final wash with PBS, the specimens were mounted in 90%
glycerol-0.1 M Tris-HCl buffer (pH8.5) containing 0.5 mM p-phenylene diamine,
and observed under a laser scanning confocal microscope (LSM510, Zeiss, Mass, USA). For a control
experiment, the specimens were incubated with normal rabbit or mouse IgG or
with 0.1% BSA-PBS alone instead of the primary antibodies. No positive staining
was observed in the control experiment (data not shown).


## 2.4. Electron Microscopy

Corneas were fixed in
phosphate-buffered 2.5% glutaraldehyde (pH7.4). Strips of cornea were taken
from the central part of the cornea, and were postfixed in 1%OsO4 in 0.1 M phosphate buffer (pH7.4), and dehydrated with graded
alcohols. After immersion in propylene
oxide, the specimens were embedded in Epon 812.
Ultrathin sections were cut perpendicular to the epithelium, doubly
stained with uranyl acetate and lead citrate, and examined with a transmission
electron microscope, TEM-1010 (JEOL, Tokyo, Japan).


## 2.5. Immunoelectron Microscopy

Immunoelectron microscopic
observation for localization of components of extracellular matrix was
performed as described earlier [22]. Fixation was carried out in the same way as for light
microscopy. Ultrathin frozen-sections were cut at −90 to −100°C. The sections were washed with PBS and
pretreated with 1% BSA in PBS for 10 minutes. After a PBS rinse, they were
incubated with the desired antibodies for 1 hour, washed with PBS, and
incubated with colloidal gold-conjugated goat anti-rabbit or mouse IgG
antibodies for 1 hour. After another wash with PBS, the sections were refixed
in 2% glutaraldehyde-0.1 M phosphate buffer, pH7.4, and embedded in a mixture
of methylcellulose, polyethyleneglycol, and uranyl acetate.


## 2.6. Antidiabetic Agents and Experimental Design

The administration of antidiabetic drugs
was started at 8 weeks old. Nateglinide (50 mg/kg) or glibenclamide (2 mg/kg)
was suspended in 0.5% methylcellulose and administered to GK rats via a stomach
tube in volume of 10 mL/kg [23–25]. These doses of the antidiabetic
agents were chosen from the data on their suppressive effects on the peak blood
glucose levels after oral sucrose or glucose loading of fasted normal rats for
15 weeks [23, 26]. GK rats were fed twice daily (9:00 and 16:00)
for 1 hour and were given nateglinide or glibenclamide orally just before each
meal. Control rats were treated with 0.5% methylcellulose alone (the vehicle).


## 2.7. Statistical Analysis

Results were expressed as themean±standard  deviation,  and the statistical analysis was done by the use of the unpaired
Student’s t-test. Differences were defined as significant at P<.05.


## 3. Results

### 3.1. Changes in Ultrastructural Morphology of Descemet’s Membrane in the Cornea of Diabetic and Normal Rats with Aging

The ultrastructure of Descemet’s membrane
in the cornea in 15-, 33-, and 62-week-old diabetic and nondiabetic rat corneas
was examined by electron microscopy. The thickness of the membrane remained
unchanged in both groups. In the 15-week-old rat cornea, abnormal collagen
fibril bundles (long-spacing collagen) were frequently observed in Descemet’s
membrane of diabetic rats, whereas they were observed less so in that of the
normal rats. Figure1 shows electron microscopic images of the long-spacing
collagen. The banding pattern was wide, averaging 110–120 nm. A
rod-like structure was observed in the internodal region of typical long-spacing
collagen (Figure 1). The size and number of long-spacing collagen molecules increased with aging in both the normal and diabetic Descemet's membrane (Figures 2 and 3). At
62 weeks, the average length of the long-spacing collagen was 0.25–1.0 μm in
normal rats and 0.5–2.15 μm in the diabetic ones. In the diabetic cornea, the
number of long-spacing collagen fibrils in the membrane increased more than in
the normal cornea with aging. The
density of these collagen fibrils was higher adjacent to the endothelium and
lower toward the stroma (Figure 2).Figure 1
Electron microscopic image of long-spacing collagen present in Descemet's
membrane of a diabetic GK rat. Arrow indicates a rod-like structure in the
internodal region. Scale bar, 100 nm.Electron micrographs of Descemet’s
membrane of Wistar (a), (b), (c) and GK (d), (e), (f) rat corneas at 15 (a), (d), 33 (b),
(e), and 62 (c), (f) weeks. Long-spacing collagen (arrowheads) accumulates with
increasing age. Dm: Descemet’s membrane; En: endothelium; S: stroma. Scale bar:
500 nm.
(a)(b)(c)(d)(e)(f)Figure 3
Number of long-spacing collagen
fibrils per10μm2 of Descemet’s membrane in nondiabetic Wistar rat (▪)
and diabetic GK rat (□) corneas. In the diabetic cornea, the
long-spacing collagen in Descemet’s increases more rapidly than in the normal
cornea with aging. Data are presented as the mean±SD (n=6).  P*<.05,  P**<.01.


### 3.2. Immunohistochemical Localization  of Type VIII Collagen

It was earlier shown that type VIII
collagen is localized in Descemet's membrane [19]. So we
examined the localization of type VIII collagen in the rat cornea by laser
confocal scanning microscopy (Figure 4). Whereas weak staining was observed in Descemet's
membrane of the normal cornea, more intense punctate staining was observed in that
of the diabetic one (Figure 4).Immunofluorescence localization
of type VIII collagen (red color) in Descemet’s membrane of the cornea from
62-week-old rats (a), (b) and Nomarsky microscopic images of the same optical
fields (c), (d). (a), (c) Wistar rat. (b), (d) GK rat. Green color (SYBR-Green I) indicates the nuclei
of endothelial cells. Dm: Descemet’s membrane; En: endothelial cell; S: stroma.
Scale bar: 10μm.
(a)(b)(c)(d)


### 3.3. Immunoelectron Microscopic Localization of Type VIII Collagen

Localization of type VIII collagen in
Descemet’s membrane was examined immunoelectron-microscopically by using the
colloidal-gold labeling method (Figure5).
In the normal cornea, the colloidal gold was detected diffusely in
Descemet’s membrane (Figure 5(a)). In the diabetic cornea, however, it was found in
the region between bands of the long-spacing collagen (Figure 5(b)). This
localization of type VIII collagen in the diabetic rat is consistent with that
reported previously for the diseased human Descemet’s membrane [9, 10].Immunoelectron-microscopic
detection of type VIII collagen in Descemet’s membrane by the colloidal gold labeling
method. (a) In the cornea of a 15-week-old Wistar rat, the colloidal gold label
is distributed diffusely in the membrane. (b) In the diabetic cornea of a
15-week-old GK rat, the gold marker is mainly observed in the internodal region
of the long-spacing collagen. Scale bar, 100 nm.
(a)(b)


### 3.4. Localization of other Components of Extracellular Matrix

Next we examined the localization of the
other components of the extracellular matrix, that is, laminin and type I, III,
IV, and VI collagens. There is a report that type VI collagen becomes localized
in the long-spacing collagen in the corneoscleral meshwork of human eyes [27]. So we examined immunoelectron-microscopically whether type VI
collagen could also be detected in the long-spacing collagen of Descemet’s
membrane. In both the normal and diabetic cornea, the colloidal gold label was
mostly seen in the corneal stroma; but none was detected in either Descemet’s
membrane or in the long-spacing collagen of this membrane (Figure 6). Collagens
type I and III showed the same distribution as the type VI (data not shown). Laminin
(Figure 7) and type IV collagen (data not shown) were localized in the
amorphous material of Descemet’s membrane, but were not present in the
long-spacing collagen in neither
the normal nor
diabetic cornea.Immunoelectron-microscopic localization of type VI collagen in
Descemet’s membrane by the colloidal gold labeling method. In both normal and
diabetic corneas, the label was detected mostly in the stroma, with little
found in Descemet’s membrane (a), (b) or in the long-spacing
collagen (c), (d). Dm:
Descemet’s membrane; S: stroma. Scale bar: 100 nm.
(a)(b)(c)(d)Immunoelectron-microscopic localization of laminin in Descemet’s
membrane by the colloidal-gold labeling method. In both the normal (a) and
diabetic (b) cornea, the label was diffusely distributed in the Descemet’s
membrane but did not attach to the long-spacing collagen. Scale bar: 100 nm.
(a)(b)


### 3.5. Effect of Antidiabetic Agents on the Formation of Long-Spacing Collagen

The morphological change in long-spacing
collagen is thought to be one of the complications of diabetes. Thus we
examined whether the abnormal formation of long-spacing collagen could be suppressed
by the antidiabetic agents nateglinide and glibenclamide. As shown in our
previous study [25], during the period of antidiabetic agent
treatments, there was no difference in fasting blood glucose levels among
vehicle-treated, nateglinide-treated, and glibenclamide-treated GK rat. Nateglinide administration reduced blood
glucose levels 1 hour after feeding, whereas glibenclamide reduced blood
glucose levels 2 and 3 hours after feeding, but not 1 hour after feeding [25]. Although the degree of the antidiabetic effect was different
between the 2 agents, both of them significantly (P<.05) inhibited the formation of the long-spacing collagen in
the diabetic GK rats (Figure 8). Glibenclamide treatment was more effective than
nateglinide treatment (Figure 8).Figure 8
Inhibition by antidiabetic
agents of the formation of long-spacing collagen in 15-week-old GK rats. The
rats were administered methylcellulose alone (GK-Control), nateglinide
(GK-Nateglinide) or glibenclamide (GK-Glibenclamide) for 15 weeks, which agents
were suspended in methylcellulose. The number of long-spacing collagen fibrils
in the Descemet’s membrane was examined by electron microscopy. Data are
presented as themean±SD (n=4). GK, GK rats. * P<.05, ** P<.01.


## 3.1. Changes in Ultrastructural Morphology of Descemet’s Membrane in the Cornea of Diabetic and Normal Rats with Aging

The ultrastructure of Descemet’s membrane
in the cornea in 15-, 33-, and 62-week-old diabetic and nondiabetic rat corneas
was examined by electron microscopy. The thickness of the membrane remained
unchanged in both groups. In the 15-week-old rat cornea, abnormal collagen
fibril bundles (long-spacing collagen) were frequently observed in Descemet’s
membrane of diabetic rats, whereas they were observed less so in that of the
normal rats. Figure1 shows electron microscopic images of the long-spacing
collagen. The banding pattern was wide, averaging 110–120 nm. A
rod-like structure was observed in the internodal region of typical long-spacing
collagen (Figure 1). The size and number of long-spacing collagen molecules increased with aging in both the normal and diabetic Descemet's membrane (Figures 2 and 3). At
62 weeks, the average length of the long-spacing collagen was 0.25–1.0 μm in
normal rats and 0.5–2.15 μm in the diabetic ones. In the diabetic cornea, the
number of long-spacing collagen fibrils in the membrane increased more than in
the normal cornea with aging. The
density of these collagen fibrils was higher adjacent to the endothelium and
lower toward the stroma (Figure 2).Figure 1
Electron microscopic image of long-spacing collagen present in Descemet's
membrane of a diabetic GK rat. Arrow indicates a rod-like structure in the
internodal region. Scale bar, 100 nm.Electron micrographs of Descemet’s
membrane of Wistar (a), (b), (c) and GK (d), (e), (f) rat corneas at 15 (a), (d), 33 (b),
(e), and 62 (c), (f) weeks. Long-spacing collagen (arrowheads) accumulates with
increasing age. Dm: Descemet’s membrane; En: endothelium; S: stroma. Scale bar:
500 nm.
(a)(b)(c)(d)(e)(f)Figure 3
Number of long-spacing collagen
fibrils per10μm2 of Descemet’s membrane in nondiabetic Wistar rat (▪)
and diabetic GK rat (□) corneas. In the diabetic cornea, the
long-spacing collagen in Descemet’s increases more rapidly than in the normal
cornea with aging. Data are presented as the mean±SD (n=6).  P*<.05,  P**<.01.


## 3.2. Immunohistochemical Localization  of Type VIII Collagen

It was earlier shown that type VIII
collagen is localized in Descemet's membrane [19]. So we
examined the localization of type VIII collagen in the rat cornea by laser
confocal scanning microscopy (Figure 4). Whereas weak staining was observed in Descemet's
membrane of the normal cornea, more intense punctate staining was observed in that
of the diabetic one (Figure 4).Immunofluorescence localization
of type VIII collagen (red color) in Descemet’s membrane of the cornea from
62-week-old rats (a), (b) and Nomarsky microscopic images of the same optical
fields (c), (d). (a), (c) Wistar rat. (b), (d) GK rat. Green color (SYBR-Green I) indicates the nuclei
of endothelial cells. Dm: Descemet’s membrane; En: endothelial cell; S: stroma.
Scale bar: 10μm.
(a)(b)(c)(d)


## 3.3. Immunoelectron Microscopic Localization of Type VIII Collagen

Localization of type VIII collagen in
Descemet’s membrane was examined immunoelectron-microscopically by using the
colloidal-gold labeling method (Figure5).
In the normal cornea, the colloidal gold was detected diffusely in
Descemet’s membrane (Figure 5(a)). In the diabetic cornea, however, it was found in
the region between bands of the long-spacing collagen (Figure 5(b)). This
localization of type VIII collagen in the diabetic rat is consistent with that
reported previously for the diseased human Descemet’s membrane [9, 10].Immunoelectron-microscopic
detection of type VIII collagen in Descemet’s membrane by the colloidal gold labeling
method. (a) In the cornea of a 15-week-old Wistar rat, the colloidal gold label
is distributed diffusely in the membrane. (b) In the diabetic cornea of a
15-week-old GK rat, the gold marker is mainly observed in the internodal region
of the long-spacing collagen. Scale bar, 100 nm.
(a)(b)


## 3.4. Localization of other Components of Extracellular Matrix

Next we examined the localization of the
other components of the extracellular matrix, that is, laminin and type I, III,
IV, and VI collagens. There is a report that type VI collagen becomes localized
in the long-spacing collagen in the corneoscleral meshwork of human eyes [27]. So we examined immunoelectron-microscopically whether type VI
collagen could also be detected in the long-spacing collagen of Descemet’s
membrane. In both the normal and diabetic cornea, the colloidal gold label was
mostly seen in the corneal stroma; but none was detected in either Descemet’s
membrane or in the long-spacing collagen of this membrane (Figure 6). Collagens
type I and III showed the same distribution as the type VI (data not shown). Laminin
(Figure 7) and type IV collagen (data not shown) were localized in the
amorphous material of Descemet’s membrane, but were not present in the
long-spacing collagen in neither
the normal nor
diabetic cornea.Immunoelectron-microscopic localization of type VI collagen in
Descemet’s membrane by the colloidal gold labeling method. In both normal and
diabetic corneas, the label was detected mostly in the stroma, with little
found in Descemet’s membrane (a), (b) or in the long-spacing
collagen (c), (d). Dm:
Descemet’s membrane; S: stroma. Scale bar: 100 nm.
(a)(b)(c)(d)Immunoelectron-microscopic localization of laminin in Descemet’s
membrane by the colloidal-gold labeling method. In both the normal (a) and
diabetic (b) cornea, the label was diffusely distributed in the Descemet’s
membrane but did not attach to the long-spacing collagen. Scale bar: 100 nm.
(a)(b)


## 3.5. Effect of Antidiabetic Agents on the Formation of Long-Spacing Collagen

The morphological change in long-spacing
collagen is thought to be one of the complications of diabetes. Thus we
examined whether the abnormal formation of long-spacing collagen could be suppressed
by the antidiabetic agents nateglinide and glibenclamide. As shown in our
previous study [25], during the period of antidiabetic agent
treatments, there was no difference in fasting blood glucose levels among
vehicle-treated, nateglinide-treated, and glibenclamide-treated GK rat. Nateglinide administration reduced blood
glucose levels 1 hour after feeding, whereas glibenclamide reduced blood
glucose levels 2 and 3 hours after feeding, but not 1 hour after feeding [25]. Although the degree of the antidiabetic effect was different
between the 2 agents, both of them significantly (P<.05) inhibited the formation of the long-spacing collagen in
the diabetic GK rats (Figure 8). Glibenclamide treatment was more effective than
nateglinide treatment (Figure 8).Figure 8
Inhibition by antidiabetic
agents of the formation of long-spacing collagen in 15-week-old GK rats. The
rats were administered methylcellulose alone (GK-Control), nateglinide
(GK-Nateglinide) or glibenclamide (GK-Glibenclamide) for 15 weeks, which agents
were suspended in methylcellulose. The number of long-spacing collagen fibrils
in the Descemet’s membrane was examined by electron microscopy. Data are
presented as themean±SD (n=4). GK, GK rats. * P<.05, ** P<.01.


## 4. Discussion

Our
present study revealed that a diabetes-associated increase in the number of
long-spacing collagen fibrils occurred in Descemet’s membrane in the cornea of
type II diabetes model GK rats. This result is consistent with the findings
made in the corneas of diabetic humans and type I diabetes model Streptozotocin-induced
diabetic rats [11, 12].
The long-spacing collagen was much more abundant in the diabetic cornea than in
the nondiabetic one, and it increased with aging in both the nondiabetic Wistar
and diabetic GK rats (Figures 2 and 3). These results reveal that the age-associated
morphological change in Descemet's membrane was accelerated by diabetes.Immunoelectron
microscopy showed that the long-spacing collagen contained type VIII collagen
molecules (Figure5). However, antibodies against
type I, III, IV, or VI collagen did not bind to the long-spacing collagen.
These results are consistent with those obtained from human and bovine Descemet’s
membrane [28, 29]. Type VIII collagen
was found as a product of rabbit corneal and bovine aortic endothelial cells [30, 31]. Type VIII collagen is a major
constituent of the hexagonal lattice of Descemet’s membrane [19]. Descemet’s lattice collagen can assemble into other long-spacing fibrils
with a longer periodicity [19]. It is thought that the function
of type VIII collagen is to provide an open, porous structure that can
withstand compressive force [19]. Under some special condition of
the diabetic state, type VIII collagen may contribute to the assembly of these
unusual long-spacing collagen fibrils. It was earlier postulated that the
aggregates of wide-spacing collagen fibrils may reflect an excessive
glycosylation in diabetes [11, 12].Long-spacing
collagen is also observed in the eyes from patients with
iridocorneal-endothelial syndrome, primary open-angle glaucoma, age-related
macular degeneration, and Fuchs’ endothelial corneal dystrophy [9, 10, 32–34]. In
Fuchs’ corneal dystrophy, Descemet’s membrane thickens abnormally; and many
long-spacing collagen fibrils
are formed in its posterior layer [10, 35]. A recent
study showed that Fuchs’ corneal dystrophy results from a mutation in the gene
encoding alpha 2 chain of type VIII collagen [36, 37]. Lack of
type VIII collagen results in dysgenesis of the anterior segment of the eye, in
which Descemet’s membrane is markedly thinned [38]. Whereas
human and mouse corneas have an anterior banded layer and a posterior unbanded
layer in their Descemet’s membrane, in the present study these 2 layers could
not be distinguished clearly in the rat cornea.
The long-spacing collagen tended to be localized in the posterior side
of Descemet’s membrane in the diabetic  rat cornea (Figure 2). This localization
is consistent with that observed in Fuch's corneal dystrophy.Diabetes
induces the dysfunction of corneal endothelium [39–42]. High glucose levels in
diabetes cause the increase in sorbitol accumulation [43],
advanced glycation end products [44], and O-GlcNAc-modified
proteins [3] in the cornea. These changes may cause
dysfunction of corneal endothelial cells and induce the morphological change of
Descemet’s membrane as shown in the present study. Kaji et al. [44] reported
that advanced glycation end products in Descemet’s membrane may be responsible
for the corneal endothelial abnormalities in diabetes.D-Phenylalanine derivative drug nateglinide and sulfonylurea drug
glibenclamide are antidiabetic agents that increase insulin secretion. When we
examined the effect of nateglinide and glibenclamide on the morphological
changes in Descemet’s membrane of GK rats, we found that the abnormal formation
of the long-spacing collagen was significantly suppressed by the administration
of either nateglinide or glibenclamide (Figure8). These results suggest that
control of postprandial hyperglycemia is essential to prevent the abnormal
formation of long-spacing collagen in type-2 diabetes.In the present study, glibenclamide treatment was more effective
than nategelinide treatment (Figure8). The different effects of these two
antidiabetic agents may be due to their different action mechanisms.
Nateglinide and glibenclamide display different effects on insulin secretion in
beta cell. Our previous study showed that decreased first-phase insulin release
was partially recovered when GK rats were treated with nateglinide, whereas no
first-phase release occurred with glibenclamide treatment [25].
Nateglinide administration reduced blood glucose 1 hour after feeding, whereas
glibenclamide administration reduced the blood glucose level 2 and 3 hours
after feeding [25]. Glibenclamide treatment is more effective
than nateglinide treatment in the dysfunction of second-phase insulin release.
The present study suggests that glibenclamide treatment might be more effective
in the inhibition of long-spacing collagen formation by recovering second-phase
insulin release.In
summary, more long-spacing collagen fibrils were observed in Descemet’s
membrane of diabetic GK rats than in the membrane of the nondiabetic Wistar
rats. Type VIII collagen was localized
in the internodal region of the long-spacing collagen. Further studies are needed to elucidate the
role of type VIII collagen in the formation of long-spacing collagen. Antidiabetic
agents nateglinide and glibenclamide significantly suppressed the formation of
the long-spacing collagen in the diabetic rats. The
long-spacing collagen of the cornea would appear to be a useful indicator for
studying diabetic changes in the cornea and the effect of antidiabetic agents.



---

*Source: 818341-2008-09-02.xml*