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(Investigative Ophthalmology and Visual Science. 2005;46:4281-4287.)
© 2005 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.04-1361
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Vascular Damage in a Mouse Model of Diabetic Retinopathy: Relation to Neuronal and Glial Changes

Rachel A. Feit-Leichman,1 Reiko Kinouchi,1,2 Masumi Takeda,1,2 Zhigang Fan,1 Susanne Mohr,3 Timothy S. Kern,3,4 and Dong Feng Chen1,4

1From the Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts; the 2Department of Ophthalmology, Asahikawa Medical College, Asahikawa, Japan; and the 3Department of Medicine and Ophthalmology, Center for Diabetes Research, Case Western Reserve University, Cleveland, Ohio.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. Lack of information about the development of diabetic retinopathy in mice has greatly hindered the use of genetic mouse models for the study of disease mechanisms and the development of therapeutic strategies. The objective of this study was to characterize the occurrence and pathologic progression of diabetic retinopathy in C57Bl/6J mice.

METHODS. Diabetes was induced with five consecutive injections of streptozotocin (STZ). The retinas were collected at different time points (2 weeks to 22 months) after the induction of diabetes and examined by using molecular, histologic, and immunohistochemical techniques and morphometric analysis.

RESULTS. There was transient induction of cell apoptosis and caspase-3 activation in retinal neurons of C57Bl/6 mice within days of diabetes induction. Glial fibrillary acidic protein (GFAP), a marker of glial activation, likewise was transiently upregulated, seemingly in astrocytes but not in Müller cells. These abnormalities quickly returned to normal; ultimately, no detectable loss of retinal ganglion cells (RGCs) was noted by any of three independent methods (number of cells in ganglion cell layer of retinal cross-sections, retrograde labeling of retinal ganglion cells with fluorescent dye, or TUNEL staining) after up to a 1-year duration of diabetes. Despite this apparent lack of evidence for progressive damage in neurons and glial cells, diabetic mice developed vascular disease characteristic of the early stage of diabetic retinopathy beginning at 6 months after the onset of disease. The vascular damage—formation of acellular capillaries and pericyte ghosts—continued to increase through the 18 months examined.

CONCLUSIONS. Diabetic C57Bl/6J mice develop capillary lesion that are characteristic of the early stages of diabetic retinopathy in patients. The data suggest that diabetes-induced degeneration of retinal capillaries can develop independent of neuronal loss or chronic GFAP upregulation in glial cells.

Diabetic retinopathy is a leading cause of adult blindness and is the most common complication of diabetes. It affects more than 90% of people with diabetes, ultimately leading to retinal edema, neovascularization, and vision loss in some patients.1 2 Vascular changes characteristic of diabetic retinopathy in humans, including breakdown of the blood–retinal barrier, thickening of the capillary basement membrane (BM), reduction in the number of pericytes, and an increase in the number of acellular capillaries, have been widely documented in diabetic rats, dogs, and cats.3 4 5 However, mice as a model of diabetic retinopathy have been less studied.

Recently, it has been reported that diabetes also induces damage in nonvascular retinal neurons and Müller glial cells.6 7 Impaired retinal electrophysiology and neurodegeneration have been detected in at least some diabetic patients.8 9 10 In diabetic rats, changes in retinal physiology and biochemistry have been reported at as early as 1 to 2 months of hyperglycemia.8 11 12 In addition, retinal glial cells, primarily Müller glia change from quiescent to an injury-associated phenotype and express high levels of GFAP—a hallmark of glial cell activation—in the human retina during early diabetes.13 In rat Müller glia, alterations in GFAP expression patterns7 13 14 15 and translocation of GAPDH to the nucleus (a proapoptotic change)16 have been observed after 2 to 5 months of experimental diabetes. In contrast, capillary obliteration first becomes significantly greater than normal after years of diabetes in humans or after approximately 6 months of diabetes in rats. Thus, diabetes-induced nonvascular abnormalities precede the development of the vascular cell changes in rats and may contribute to the pathogenesis of the vascular disease.6 To date, the relation of the nonvascular abnormalities to the development of the vascular lesion characteristic of diabetic retinopathy is only beginning to be explored.

Because of its great potential for genetic manipulation, the mouse offers a unique opportunity to study the molecular pathways involved in disease development; however, descriptions of the retinal disease that develops in the diabetic mouse are incomplete and often contradictory. In some studies, STZ-induced diabetic mice showed no distinct vascular retinopathy other than thickening of the retinal capillary BM for up to 20 months,17 whereas other reports have demonstrated that the early stages of vascular diabetic retinopathy develops18 and are inhibited in diabetic or galactosemic mice deficient in intercellular adhesion molecule (ICAM) or CD18.18 19 Other studies that focused on diabetes-induced changes in endothelial cells and pericytes yielded mixed results.20 21 22 Reports on effects of diabetes on retinal neurons in mice also have been contradictory. Increased frequency of apoptotic retinal neurons has been reported in some18 23 24 but not all25 studies of diabetic mice. To clarify these contradictions and provide systematic characterization of retinal disease after induction of diabetes, we investigated the occurrence and time course of diabetes-induced changes in neurons, glia, and vasculature in the retina of C57Bl/6J mice, a commonly used mouse strain.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Experimental Animals
All experiments were performed in accordance with the protocol approved by the Animal Care and Use Committee of The Schepens Eye Research Institute (SERI) and Case Western Reserve University and the tenets of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. C57Bl/6J mice (Jackson Laboratory, Bar Harbor, ME) aged 7 to 10 weeks were rendered diabetic with five consecutive daily intraperitoneal injections of STZ (55 mg/kg) freshly dissolved in citrate buffer (pH 4.5). Development of diabetes (defined by blood glucose greater than 250 mg/dL) was verified 1 week after the first STZ injection (Glucometer Elite XL; Bayer Corp., Elkhart, IN). Mice were housed in the SERI animal facility with 12-hour light/dark cycle and allowed free access to food and water. Body weight was recorded fortnightly. Glycemic control was estimated on multiple occasions from the measurement of glycohemoglobin (GHb), using either a GHb assay (Glyc-Affin; PerkinElmer, Norton, OH) or a glycohemoglobin assay (Helena Glyco Tek Laboratory, Beaumont, TX).

Immunohistochemical Staining
The eyes or fresh retinal wholemounts were fixed in 4% paraformaldehyde for 1 hour. For staining of retinal sections, the eye was then immersed in 30% sucrose for 4 hours, embedded in optimal cutting temperature (OCT) compound (Tissue-Tek; Sakura Finetek, Torrance, CA), and sectioned at 30 µm. Retinal wholemounts or sections were then incubated in 1% Triton X-100 in 0.1% citrate for 1 hour and stained with GFAP antibody conjugated to Cy-3 (1:5000; Sigma-Aldrich, St. Louis, MO) at 4°C overnight. Immunofluorescence labeling was then observed under a microscope equipped with fluorescence illumination (TE300; Nikon, Tokyo, Japan).

Western Blot Analysis
Freshly isolated mouse retinas were frozen in liquid nitrogen. They were then homogenized with 1x lysis buffer (Reporter Lysis 5x Buffer; Promega, Madison, WI) and kept at 4°C for 1 hour. The protein concentration was determined by a BCA kit (Micro BCA Assay Reagent Kit; Pierce Chemical, Rockford, IL). Ten micrograms of protein from each sample were electrophoresed in 10% SDS-PAGE with protein markers (BMA ProSieve Color Protein Markers; BioWhittaker Molecular Applications, Rockland, ME). After electrophoresis, proteins were transferred to nitrocellulose membrane. The blot was then incubated in blocking solution (1% bovine serum albumin, 2.5% milk protein, 0.2% Tween-20 in PBS) for 1 hour, followed by reactions with primary antibodies against GFAP (1:5000; Sigma-Aldrich) or Ras-GAP as a control (1:2000; Sigma-Aldrich) in blocking buffer at 4°C overnight. The membrane was incubated with goat anti-rabbit conjugated with horseradish peroxidase (1:5000; Chemicon International, Temecula, CA) and goat anti-mouse conjugated with horseradish peroxidase (1:5000; Chemicon International) in blocking buffer for 1 hour at room temperature. Antibody detection was performed with enhanced chemiluminescence (Chemiluminescence System and SuperSignal West Pico Chemiluminescent Substrate; Pierce Chemical). Densitometric analysis was performed with Image J software (available by ftp at zippy.nimh.nih.gov/ or at http://rsb.info.nih.gov/nih-image; developed by Wayne Rasband, National Institutes of Health, Bethesda, MD).

Caspase Activity Assay
Caspase-3 activity was measured as described previously.26 Briefly, equal amounts of retinal protein extracts (usually 15 µg) were incubated in a lysate buffer containing the fluorogenic caspase-3 substrate (DEVD-AFC; 2.5 µM) at a total volume of 100 µL at 32°C for 1 hour. Cleavage of the substrate emitted a fluorescence signal that was quantified by a fluorescence plate reader (excitation: 400 nm, emission: 505 nm; Spectra FluorPlus; Tecan, Durham, NC).

Preparation of Trypsin-Digested Retinal Vasculature
The enucleated eye was placed in 10% buffered formalin for 4 to 5 days. The retina was dissected and incubated in a 3% crude trypsin solution (DIFCO, Detroit, MI) containing 0.2 M sodium fluoride at 37°C for 2 hours, as described elsewhere.27 The neuroretinal tissue was gently brushed away, and the resultant isolated vascular tree was air dried onto a glass microscope slide.

Tdt-dUTP Terminal Nick-End Labeling
To evaluate apoptosis in retinal vascular cells, the slide-mounted retinal vascular digests were incubated with 1% Triton X-100 in 0.1% sodium citrate for 1 hour, to enhance penetration of reagents into cells, followed by staining with TUNEL. TUNEL-positive capillary nuclei were then counted, and the data were expressed per retina. To evaluate cell apoptosis in the neuroretina, the mouse eye was fixed in 4% paraformaldehyde for 15 minutes. The retina was then isolated and placed in 2% Triton X-100 in PBS for 1 hour at room temperature. Retinas treated with DNase were used as a positive control for TUNEL. The retinas were flatmounted, and all samples for TUNEL were incubated with terminal deoxynucleotidyl transferase conjugated to fluorescein (In Situ Cell Death Detection Kit; Fluorescein Roche Diagnostics, Indianapolis, IN), which labels free 3'OH DNA, at 37°C for 1 hour. TUNEL-positive cells were counted within the entire retina of each mouse.

Quantitation of Acellular Capillaries and Pericyte Ghosts
After TUNEL-positive cells were counted, coverslips were soaked off, and the retinal vasculature then was stained with hematoxylin-periodic acid-Schiff. Acellular capillaries were counted in four to seven field areas in the mid retina. Acellular capillaries were identified as capillary-sized vessel tubes (not <20% the diameter of surrounding healthy capillaries) having no nuclei anywhere along their length. Pericyte ghosts were estimated from the prevalence of spaces in the capillary BMs from which pericytes had disappeared. Although counting the ghosts, at least 1000 capillary cells (endothelial cells and pericytes) were counted in five mid-retinal field areas. Ghosts on any acellular vessel were excluded. All measurements were made in a masked manner.

Electron Microscopy
After enucleation, the eye was incubated in half-strength Karnovsky overnight. The anterior segment and vitreous were removed, and the eyecup was washed in 0.1 M cacodylate buffer for 5 to 10 minutes. Tissue was postfixed in 2% osmium tetroxide (Sigma-Aldrich), dehydrated, and embedded in Spurr’s resin. Capillaries were selected at random from the inner nuclear layer and photographed under a transmission electron microscope (model 410; Philips, Eindhoven, The Netherlands). Approximately 20 electron micrographs were taken of each retinal sample. The area and perimeter of the outer and inner BM were measured with Image J software (NIH). BM thickness was calculated by dividing its areas by its length; results are reported in square micrometers per 1000 micrometers.

Retrograde Labeling of Retinal Ganglion Cells
Mice were anesthetized by intraperitoneal administration of a mixture of xylazine (2.5 mg/mL) and ketamine (12.5mg/mL). The skin over the cranium was incised to expose the scalp. A hole (2 x 1 mm) was cut with a scalpel, 4 mm posterior to the bregma and 1 mm lateral to the midline on both sides of the midline raphe. The superior colliculi were exposed by gentle aspiration of the overlying occipital cortex. A piece of Gelfoam (Pharmacia & Upjohn; Kalamazoo, MI) soaked in a 5% solution of fluorescent gold tracer (Fluorogold; Fluorochrome, Denver, CO) was directly applied to the superior colliculus. The skull was replaced, and the overlying skin was sutured and covered with topical antibiotic ointment. Seven days after application of the tracer (time to allow retrograde uptake of dye and labeling of the RGC somata) mice were euthanatized in a CO2 gas chamber and the eyes enucleated. Retinas were isolated and placed in 4% paraformaldehyde for 10 minutes (Sigma-Aldrich). They were then flattened as wholemounts on microscope slides (VWR, West Chester, PA). Ten fields at 20x magnification (area of 0.09 mm2) were photographed randomly from each retina with a fluorescence microscope. The total number of RGC bodies was counted in each field using Image J software (NIH).

Cell Counts in the Ganglion Cell Layer
Formalin-fixed whole eyes were embedded in paraffin and sectioned sagittally (4 µm), so that each section passed through or next to the optic nerve and the center of the cornea were collected. Sections were stained with hematoxylin and eosin (H&E), and the number of cells in the ganglion cell layer (GCL) was counted for a 250-µm linear distance on each side of the optic nerve (adjacent to the optic nerve). The counts from the two sides were averaged and reported per unit length of retina.

Statistical Analysis
Results are presented as mean ± SD. The Mann-Whitney test or two-paired Student’s t-test was used to assess significance between two groups. One-way ANOVA followed by the post hoc Tukey (Fisher’s protected least significant difference) test was used to assess statistical significance between multiple groups. P ≤ 0.05 was regarded as statistically significant.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Induction of Diabetes in C57Bl/6J Mice
To induce diabetes in C57Bl/6J mice, we administered intraperitoneal STZ at 55 mg/kg for five consecutive days. For most animals, the levels of blood glucose levels reached maximum elevation at 4 weeks after STZ injection and remained elevated throughout the time course of study. GHb levels in diabetic mice were significantly higher (>3-fold) than control animals (3.7 ± 0.6%; P < 0.01), and all diabetic mice evaluated retained elevated GHb until death. As male mice consistently developed and maintained higher blood sugar levels than female mice (Chen DF, unpublished observation, 2000), male mice were selected for the studies. Some animals maintained body weight and survived without insulin supplements, whereas others that tended to lose weight were given small amounts (0.2 U, 0–3 times per week) of insulin to prevent weight loss. A small number of animals did not respond to STZ and were used as the control for effects of the drug.

Neural Apoptosis in the Early Diabetic Retina
TUNEL was performed in wholemount retinas at 2 weeks and 1, 2, and 6 months after induction of diabetes (Fig. 1A) . A significant increase in TUNEL-positive cells, primarily localized in the GCL was observed in retinas of mice at 2 weeks of diabetes (P < 0.05). However, at 1 month, the number of TUNEL-positive cells in diabetic mice declined toward normal, although remaining slightly increased compared with the control group (P = 0.17). At 2 and 6 months of diabetes, no significant differences in neuronal apoptosis were observed between groups. To preclude that the transient induction of neuronal apoptosis is caused by a possible toxic effect of STZ, we performed TUNEL in retinas of mice in which diabetes did not develop after STZ injection. We found no significant difference of the number of TUNEL-positive cells in STZ-treated mice in which diabetes did not develop in comparison with the age-matched control, even at 2 weeks after STZ injection (4.0 ± 1.3; P > 0.1 compared with age-matched control group). Thus, the results indicate that the mouse neuroretina undergoes a transient and small amount of cell apoptosis immediately after induction of diabetes.



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  		FIGURE 1. Lack of evidence for significant neuronal loss in the mouse retina in the early stage of diabetes. (A) Counts of TUNEL-positive cells in the STZ-treated and control mouse retinas at various time points after diabetes (n = 5/group). (B) Photomicrograph of representative retinal sections stained with H&E. (C) Quantification of neuronal loss in the GCL, assessed by number of cells counted in the GCL at 6 and 12 months after induction of diabetes (C). (D) Photomicrograph of retrogradely labeled RGCs (arrowhead) in a retinal wholemount. (E) Counts of retrogradely labeled RGCs in the diabetic and control mouse retinas (n = 4/group). No observable difference in RGC loss was noted between the control and diabetic groups.

 
As the TUNEL-positive cells were localized primarily in the GCL, we further assessed diabetes-induced neuronal loss by counting cells in the GCL at 6 and 12 months of diabetes, using retinal sections taken from diabetic and age-matched control mice (Figs. 1B 1C) . After these long durations of diabetes, we found no significant difference in the number of cells in the GCL between the diabetic and nondiabetic mice (Fig. 1C) . To corroborate this result, we also performed retrograde labeling of RGCs in diabetic (n = 4) and control (n = 4) mice at 3 months of diabetes (Figs. 1D 1E) . Labeled RGCs from both eyes were counted. Again, we observed that the retinas of diabetic mice contained a number of RGCs (4980 ± 1663 RGC/mm2) similar to that of the control group (4942 ± 1445 RGC/mm2; P > 0.5; Fig. 1E ).

Because of the unexpected transient increase in the number of TUNEL-positive neuronal cells in the mouse retina, we examined the activity of caspase-3, a downstream inducer of apoptosis. Coinciding with the transient induction of neural apoptosis, we noted a significant increase in caspase-3 activity in diabetic mouse retinas compared with age-matched control group over the first month of diabetes, which likewise diminished during the ensuing 2 to 4 months after induction of diabetes (Fig. 2) . Caspase-3 activity increased again after 5 months, which appeared to correlate with the induction of retinal vascular cell apoptosis at the later stage, as indicated later in this study. Thus, these results suggest that retinal neurons undergo a small and transient apoptosis soon after the development of diabetes in mice, but this abnormality is not maintained at longer durations of diabetes, nor does it result in a significant loss of RGCs in diabetic C57Bl/6J mice.



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  		FIGURE 2. Activity of caspase-3 measured from retinal homogenates prepared at various time points after diabetes induction (n = 5/group). Caspase-3 activity was significantly greater than normal at 1 month after induction of diabetes, then diminished to normal, and began to increase again after approximately 6 months. *P < 0.05.

 
Glial Cell Changes in the Diabetic Retina
Activation of Müller glial cells, characterized by upregulation of GFAP, is another component of early diabetic retinopathy in diabetic rats.13 To evaluate retinal glial cell changes, we examined expression of GFAP in mice from 2 weeks to 12 months after diabetes induction. With Western blot analysis, the largest increase of GFAP expression was observed at 1 month after the induction of diabetes (Fig. 3) , and this abnormality remained significant at 2 months but quickly returned toward normal with longer durations of diabetes. To determine further which cell type was responsible for the increase in GFAP level after the onset of diabetes, we performed immunofluorescence staining in retinal sections, at 1, 2, and 6 month of diabetes. Increased GFAP expression at 1 and 2 months of diabetes was consistent with the labeling pattern of astrocytes in the GCL (Fig. 4) . In agreement with the quantification of GFAP levels with Western blot, after 6 months of diabetes, the intensity of GFAP labeling in the diabetic retinas returned to normal. At any time point examined, no upregulation of GFAP expression was observed in Müller glial cell bodies or other retinal layers that were consistent with Müller glial cell labeling.



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  		FIGURE 3. Diabetes induced transient upregulation of GFAP expression in the mouse retina. (A) Western blot analysis of GFAP levels in the retinas of diabetic (D) and age-matched control (C) mice at various time points after STZ injection. Anti-RAS-GAP was used as a control. (B) Densitometric measurement of band densities in Western blot analysis. For each time point, the band-density level of control mouse retinas was set to 100%, and the band-density level of diabetic mouse retinas is presented as a percentage of the age-matched control retinas (n ≥ 3/group). Note that the level of GFAP expression increased in the diabetic mouse retinas beginning at 1 month after induction of diabetes and continued through 2 months, but returned to normal at 6 months.

 


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  		FIGURE 4. Absence of apparent Müller glia activation in the diabetic mouse retina. Representative photomicrographs of retinal sections that were prepared from diabetic (E–H) and age-matched control (A–D) mice at 1, 2, and 6 months after STZ-injection (n = 3/group). GFAP upregulation was observed in the GCL, with a pattern resembling that of astrocytes (arrowheads), in the diabetic retinas at 1 and 2 months of diabetes. However, GFAP labeling was not detected in Müller glial cell bodies that normally appear in the inner nuclear layer or their processes in other retinal layers at any age examined.

 
Development of Vascular Features Characteristic of Diabetic Retinopathy in the Diabetic Mouse Retina
Next, we studied the vascular disease in the mouse retina at various time points, from 6 to 19 months, after STZ-injection. Features of early vascular damage associated with diabetic retinopathy include the formation of acellular capillaries and pericyte ghosts and induction of vascular cell apoptosis. At 6 months after diabetes induction, a statistically significant increase in the number of acellular capillaries was noted in the diabetic mice compared with the age-matched control animals (P < 0.01; Fig. 5 ). With increasing duration of diabetes, the frequency of acellular capillaries increased in diabetic animals but not in nondiabetic control subjects. A significant increase of capillary cell apoptosis and prevalence of pericyte ghosts were also observed in diabetic mice after 6 to 9 months of diabetes, and the frequency of the lesion likewise increased with increasing duration of diabetes (Fig. 6) . These results indicate that the mouse exhibits vascular abnormalities characteristic of early diabetic retinopathy in rats and humans.



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  		FIGURE 5. STZ-treated mice exhibited acellular capillaries, beginning at 6 months after induction of diabetes. (A, B) Low- and high-magnification photomicrographs of trypsin-digested retinas stained with periodic acid-Schiff hematoxylin. Arrowhead: an acellular capillary. (C) Quantitative analysis of acellular capillaries counted per mm2 trypsin-digested retinas indicating a significant increase in the number of acellular capillaries compared to control mice beginning at 6 months after STZ-injection (n > 6/group). *P < 0.001; **P < 0.0001 with ANOVA test.

 


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  		FIGURE 6. Induction of capillary apoptosis and formation of pericyte ghosts in the diabetic mouse retina. (C) The merged image of light microscopy (A) and TUNEL (B) of trypsin-digested retina. Arrows: TUNEL+ cells. (D, E) Counts of apoptotic capillary cells (D) and pericyte ghosts (E) in trypsin-digested retinas prepared from STZ-treated and age-matched control mice (n > 5/group). *P < 0.05, **P < 0.01 (Mann-Whitney test).

 
BM Thickening
Thickening of the capillary BM is a phenomenon that is often observed in diabetic retinopathy. To evaluate whether retinal capillaries from diabetic mice undergo BM thickening, we examined by transmission electron microscopy the mouse retina after 6, 12, and 15 months of induced diabetes. To minimize variability, the examination was conducted only in capillaries located in the outer nuclear layer of the retina. We found that capillary BM thickness tended to increase at longer durations of diabetes compared with the age-matched control, but the results did not achieve statistical significance (Fig. 7) .



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  		FIGURE 7. Absence of capillary BM thickening in the retinas of diabetic mice. Quantification of BM thickness at various time points after STZ-injection, using transmission electron microscopy (n ≥ 6/group). No significant difference was detected between the control and STZ-treated groups at 6 to 15 months after induction of diabetes. P > 0.1 (Student’s t-test).

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Several important findings emerged from the present study. First, diabetic C57Bl/6J mice exhibit development of vascular disease characteristic of early diabetic retinopathy in rats and humans,4 notably induction of acellular capillaries and vascular cell apoptosis and formation of pericyte ghosts, as early as 6 months after induction of diabetes. The severity of these lesions is progressive, becoming more severe with longer duration of diabetes. Second, vascular lesions develop despite only mild and transient abnormalities in the neural retina, unlike the degeneration of RGCs and Müller glial changes that have been reported in humans and rats.6 9 13 14 15

The mouse has not been studied extensively as a model of diabetic retinopathy. The small size of the mouse eye presents unique challenges in determining capillary lesions in the mouse retina. For example, pericyte ghosts are much more difficult to detect in mice than they are in rats or larger species. Although the mouse brings special problems, the ability to use genetically modified mice to study the pathogenesis of diabetic retinopathy gives a major advantage over the use of any other animal species, offsetting the potential hardships. Herein, we report a systematic study of the occurrence and progression of retinal disease of diabetic retinopathy in the mouse. Our results and those of others28 indicate that the C57Bl/6J mouse has vascular lesions consistent with the early stages of diabetic retinopathy, similar to findings in diabetic rats and human. Likewise, the Akita mouse shows development of capillary lesions in the early stages of diabetic retinopathy.18 Of note, we found only slight thickening of retinal capillary BMs in our mice, even as capillary degeneration was developing. This suggests that processes leading to capillary BM thickening are in some way different from those leading to hyperglycemia-mediated death of retinal capillary cells.

In the present study, we quantified neuronal cell death in the mouse retina at various times after the induction of diabetes, and the results were corroborated using three methods: (1) TUNEL, (2) counting the number of cells in the GCL in H&E-stained retinal sections, and (3) counting surviving RGCs in retinal wholemounts using a retrograde tracer (Fluorogold; Fluorochrome). In each case, we did not detect significant loss of retinal neurons in diabetic mice. Our inability to detect RGC loss in the diabetic mouse seems not to be due to a methodological problem, because quantifying ganglion cells by the same method (in cross sections) revealed a significant decrease in the number of RGCs in diabetic rats after 8 months of diabetes (Kern TS, unpublished results, 2004). Altogether, our studies indicated a transient activation of proapoptotic processes in the mouse retina after induction of diabetes, but it did not lead to significant loss of neurons in long-term diabetes.

These findings disagree with a much shorter study by Martin et al.23 who found a progressive neuronal loss in the closely related mouse strain (C57Bl/6) used in our study, which became significantly greater than normal at 10 weeks after induction of diabetes. Differences between these studies that could account for the different conclusions seemed to be few. First, we provided low doses of insulin to the mice to prevent diabetes-induced weight loss and failure to grow, whereas Martin et al.23 administered no insulin to any of the experimental mice. Second, in our procedure, diabetes was induced in mice at the age of 7 to 10 weeks, by using five consecutive injections of low-dose STZ (55 mg/mL), whereas Martin et al. induced diabetes in 3-week-old mice with three injections of a higher dose (75 mg/mL). Finally, the minor differences in the genetic background of the mice used (C57Bl/6J versus C57Bl/6) might also result in the different observations. In a study of retinal sections of Akita mice, significantly fewer cells were also reported in the GCL than in nondiabetic control animals.18

Neuronal lesions commonly evoke a response of glial cells, which become activated and undergo reactive gliosis, a process characterized by proliferation and changes in intermediate filament (e.g., GFAP) production.29 In rat retina, GFAP upregulation in Müller glia (but not astrocytes) develops soon after diabetes, and continues for many months.14 In the present study, we observed a transient response (caspase-3) in RGCs of the diabetic mouse retina at 1 to 2 months of diabetes, which correlated with the transient presence of TUNEL-positive neuronal cells. Likewise, GFAP upregulation in diabetic mice was transient and limited. Moreover, the upregulation of GFAP in the mouse retina seemed to occur in astrocytes, but not Müller glia, unlike what happens in diabetic rats.15 These data suggest that although diabetes transiently triggers death signals in a small population of neurons of the mouse retina, the damage is probably not strong enough to evoke the response of Müller glial cells in the mice,6 or GFAP upregulation is not a sufficient marker of glial activation in this model. Müller glial cells do develop proapoptotic changes in retinas of diabetic rats and mice, as evidenced by translocation of GAPDH to the nucleus16 (and unpublished data, 2004), a change that has been closely identified with apoptosis in other cell types.30 Whether these abnormalities detected in neurons and glia were sufficient to contribute to the vascular lesions that developed at longer durations of diabetes cannot be positively determined at present.

Recent reports comparing biochemical sequelae of hyperglycemia in retinas and other tissues between mice and rats indicated several similarities31 and differences.25 32 33 At the similar glucose levels, mice had much lower polyol pathway activity in the retina than did rats.25 31 34 35 Activation of the polyol pathway has been shown to account for the induction of early neuronal apoptosis, GFAP upregulation in Müller cells, and capillary BM thickening in the retina of diabetic rats.25 36 37 38 Thus, lower activity of the polyol pathway in the diabetic mouse retina might be a factor in the modest evidence of diabetes-induced damage of retinal neurons, Müller glial cells, and BM thickening.22

It is intriguing that diabetes-induced microvascular lesions develop in the mouse retina, despite the absence of significant neuronal loss (as assessed by three independent methods) and persistent glial cell activation (as assessed by GFAP induction). These observations provide no support to a postulate that diabetes-induced early changes in neurons and Müller glia contribute critically to the later development of vascular lesions in diabetic retinopathy.6 9


   Acknowledgements
 
The authors thank Mara Lorenzi for critical comments, Debra Shaumberg for statistical analysis, Patricia Pearson for electron microscopy, and Casey Miller and Todd Hoehn for supervision and maintenance of diabetic animals.


   Footnotes
 
4 Contributed equally to the work and therefore should be considered equivalent authors. Back

Supported by National Eye Institute Grants EY012983 (DFC), EY00300 (TSK), and EY014380 (SM); the Massachusetts Lion’s Eye Research Fund, the Kristin C. Dietrich Diabetes Research Award; the Animal Models of Diabetes Complications Consortium (AMDCC; funded by the National Institute of Health); Juvenile Diabetes Research Foundation Grant CDA-2-2000-390 (SM); and the Juvenile Diabetes Research Foundation Center for Diabetic Retinopathy at the Schepens Eye Research Institute.

Submitted for publication November 22, 2004; revised June 22, 2005; accepted September 16, 2005.

Disclosure: R.A. Feit-Leichman, None; R. Kinouchi, None; M. Takeda, None; Z. Fan, None; S. Mohr, None; T.S. Kern, None; D.F. Chen, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Dong Feng Chen, Schepens Eye Research Institute, Harvard Medical School, 20 Staniford Street, Boston, MA 02114; dfchen{at}vision.eri.harvard.edu.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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