Corneal Complications in Streptozocin-Induced Type I Diabetic Rats

Materials

Phospho (p)-EGFR (Tyr1068), AKT, and pAKT (Ser473) antibodies were from Cell Signaling (Danvers, MA). P-ERK1/2 (p42/p44), ERK2 (p42 MAPK), EGFR, and actin antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). β-catenin, ZO-1 and occludin antibodies were from BD Biosciences (San Jose, CA), Invitrogen (Carlsbad, CA), and Zymed Laboratory (South San Francisco, CA), respectively. Horseradish peroxidase conjugate secondary antibodies were from Bio-Rad (Hercules, CA). FITC conjugate secondary antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA). Contour glucometer was from Bayer HealthCare (Mishawaka, IN). All other reagents and chemicals were purchased from Sigma-Aldrich (St. Louis, MO).

Animals

Six-week-old male Sprague-Dawley (SD) rats, weighing approximately 150 g, were purchased from Charles River Laboratories (Wilmington, MA) and housed under standard conditions with continuously available water and chow (LabDiet 5001 Rodent Chow, Dexter, MI). All investigations conformed to the regulations of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, the National Institutes of Health, and the guidelines of the Animal Investigation Committee of Wayne State University.

Induction of Diabetes and Characterization of Physical Properties in SD Rats

SD rats were randomly divided into the DM and age-matched normal control (NL) groups. Type I DM was induced with an intraperitoneal injection of 40 mg/kg of STZ in ice-cold 0.5 M citrate buffer, pH 4.5, and a second dose of 40 mg/kg STZ 24 hours later.³³ The NL group received 2 doses of intraperitoneal injection of the citrate buffer only. Serum glucose levels were monitored from rat tail vein using a glucometer. All STZ rats developed glucose levels higher than 400 mg/dL 3 days postinjection. Glucose levels and body weight were monitored weekly. At 4 and 8 weeks postinjection, body length without tails was measured and corneal diameter was measured (Castroviejo Calipers; V. Mueller, Wurmlingen, Germany).

Slit Lamp Examination

Rats were anesthetized with an intraperitoneal injection of rodent anesthesia cocktail containing 70 mg/kg ketamine and 7 mg/kg xylazine. Animals were examined with a slit lamp (Zeiss, Oberkochen, Germany) at 8 weeks post-STZ injection for corneal irregularity, defects, edema, infiltrations, and cataract while the pupils were dilated with 1% ophthalmic solution (Tropicamide Ophthalmic Solution; Akron, Buffalo Grove, IL). Corneal surface was further examined with 1% fluorescein sodium and 1% Rose Bengal staining, and photographed with a digital camera (Pentax; Golden, CO) attached to the slit lamp under Cobalt blue or red free filters, respectively. To quantitate Rose Bengal staining pattern, each cornea is divided into four quadrants and each quadrant is scored based on the following scale: 0, no staining; 1, punctate staining; 2, continuous staining covering <50% area; 3, continuous staining covering >50% area but not confluent; and 4, confluent staining. The final score for each cornea is the total of scores from all quadrants.

Evaluation of Tear Secretion and Corneal Sensitivity

Tear secretion was determined with phenol red–impregnated cotton threads (Zone-Quick, Tokyo, Japan). The threads were placed in the medial canthus for 1 minute and the length of the wetted part, turning red on soaking tears, was measured. Corneal sensation was measured with an esthesiometer (Cochet-Bonnet; Luneau, France) in unanesthetized rats. The testing began with the nylon filament fully extended to its maximal length of 60 mm and shortened by 5 mm each time a negative response was observed until a positive response was obtained. A positive blinking response was recorded by two observers and each testing was repeated three times.

Confocal Microscopic Examination

Starting at Week 2 after STZ injection, rat corneas were scanned with a digital confocal scanning microscope (ConfoScan-4; Nidek Technologies, Gamagori, Japan). Topical proparacaine 0.5% (Akron) was applied to the cornea of anesthetized rats. A drop of GenTeal gel was applied to the tip of a 40× objective lens and the central cornea was scanned using the Auto Full mode at 25 frames per second with scan step of 5 μm and total of 350 images. Each cornea was scanned three times and the corneal thickness was calculated as the peak to peak distance in the z-scan profile and averaged among three scans. To analyze corneal innervation, three representative images of the sub-basal nerve plexus from each rat were selected and nerve fiber length was traced and measured (NeuronJ plug-in of ImageJ software developed by Wayne Rasband, National Institues of Health, Bethesda, MD; available at http://rsb.info.nih.gov/ij/index.html). The final length for each animal is the mean of three scans and expressed in an arbitrary unit.

Determination of Corneal Epithelial Wound Healing

Topical proparacaine 0.5% was applied to NL and DM rat corneas 8 weeks after STZ injection. A 5-mm circular wound was first demarcated with a trephine in the central cornea and the epithelium was then removed with a blunt scalpel blade under a dissecting microscope (Zeiss). Bacitracin ophthalmic ointment (Fougera, Melville, NY) was applied to the cornea after surgery to prevent infection. To document the healing process, the remaining denuded area was visualized with fluorescein staining, photographed, and quantitated (Photoshop, version 6; Adobe, San Jose, CA). Healing rate was calculated as (original wound area − current wound area)/original wound area in percentage.

Determination of Protein Expression by Western Blot Analysis

The epithelium removed during corneal debridement was lysed with radioimmunoprecipitation assay buffer (RIPA). Protein concentrations were determined using a protein assay kit (Micro BCA; Pierce, Rockford, IL). Levels of various proteins were determined using specific antibodies. Densitometry was measured with image analysis software (Kodak 1 D).

Histopathology and Immunohistochemistry

To determine histologic changes, unwounded corneas from DM or NL rats 8 weeks after STZ injection were fixed in 4% paraformaldehyde, paraffin-embedded, stained with hematoxylin and eosin, and photographed. Corneas at various time points postwounding were embedded (OCT; Ted Pella, Inc., Redding, CA), snap frozen, and cryostat-sectioned for immunofluorescence staining. The slides were fixed in 4% paraformaldehyde, blocked with bovine serum albumin, incubated with primary antibody or mouse or rabbit IgG isotype controls overnight at 4°C, followed by incubation with FITC-conjugated secondary antibody. The slides were then washed in PBS and mounted with mounting medium (Vectashield; Vector Labs, Burlingame, CA) with DAPI for nuclear staining and examined under a fluorescence microscope (Carl Zeiss Axioplan 2 equipped with an ApoTome digital camera). Negative controls (mouse or rabbit IgG isotypes) exhibited no fluorescence (data not shown).

Statistical Analysis

Each experiment used n = 8–10 rats in each group, NL or DM, unless otherwise noted in the figure legends. Results are mean ± SEM. Statistical parameters were ascertained (SigmaStat; Systat Software, San Jose, CA) with Student's t-test between two groups. P < 0.05 indicates a significant difference.

Maintenance of Hyperglycemia and Attenuation of Growth

The blood sugar levels were approximately 100 mg/dL in vehicle-injected normal control (NL) and higher than 500 mg/dL in STZ diabetic (DM) rats (Fig. 1A). While NL rats maintained a steady weight gain, 2.7- and 3.4-fold increases over the initial weight at 4 and 8 weeks postinjection; the body weight of STZ-rats increased only 2.1- and 2.2-fold and were 23% and 36% lower than that of the controls. The body length was also decreased, but to a lesser extent, 15% and 12% at these two time points (Fig. 1C). The DM rats also exhibited adipose and muscle tissue wasting, polyuria, polydypsia, and some exhibited diarrhea and intestinal bloating.

Alterations in Ocular Properties

To characterize the ocular changes caused by hyperglycemia, DM and NL rats were subjected to a battery of ophthalmic examinations and measurements. Unlike the body weight and length, the corneal size of DM rats was only slightly smaller than that of the controls, 5.50 mm versus 5.79 mm, a 5% decrease in diameter at 8 weeks of DM (Fig. 2A). No gross difference in histology was observed (Fig. 2B). Slit lamp examination revealed no significant ocular irregularity or epithelial morphologic changes (Fig. 2C). Fluorescein staining of the cornea revealed no visible defects (Fig. 2D). DM rats started showing lens opacity around 3 weeks of hyperglycemia and by 8 weeks nearly 100% of the DM rats developed cataract (Fig. 2C); while the lenses of the NL rats remained clear.

Rat corneas were also examined with confocal microscopy (ConfoScan; Nidek Technologies; Fig. 2E). The morphology of corneal epithelium, stroma (data not shown), and endothelium was compared between the two groups, and no significant differences were noted. Corneal thickness (Fig. 2F) was similar between NL and DM rats, averaging approximately 170 μm.

Attenuation of Tear Film Protection and Tear Secretion

The rat corneas of DM and NL rats were stained with Rose Bengal, a dye commonly used to visualize conjunctiva but also useful in highlighting tear film impairment.³⁴ Unlike ocular surface fluorescein staining that showed no difference between NL and DM rats, Rose Bengal staining was much stronger in the DM rats than that in the controls (Fig. 3A). The staining was quantitated as described in the Methods section and DM rats scored 7.5, higher than the 1.75 score of NL rats, indicating a deficiency of tear film protection (Fig. 3B). Tear secretion was measured with cotton threads. Consistent with Rose Bengal staining deficiency, DM rats had significantly reduced amount of tears than that of the NL rats (Fig. 3C).

Impairment of Corneal Sensitivity and Innervation

Corneal sensitivity was determined with an esthesiometer and the values were expressed as the length of the esthesiometer thread in centimeters with 6 being the longest or most sensitive (Fig. 4A). Seventy-five percent of NL rats and 58% of DM rats showed a positive blinking response at 6 cm and on average DM rats exhibited a slight yet significant decrease in sensitivity. To visualize corneal innervation, the subbasal plexus of nerve fibers was assessed with confocal microscopy (ConfoScan; Nidek Technologies). Compared with the NL rats, the nerve fibers of the DM rats are thinner and have fewer branches (Fig. 4B). The length of the nerves in each frame was calculated; and Figure 4C shows a significant reduction from 13 arbitrary units of the NL to four in the DM.

Delay in Epithelial Wound Healing

A 5-mm wound was made in the center of the cornea, leaving the limbal region intact. The healing progress was monitored with fluorescein staining to highlight denuded area and photographed (Fig. 5A). The healing rate was calculated and shown in Figure 5B. The NL rats healed 66%, 90%, 94%, and 100% at 24, 32, 40, and 48 hours postwounding (hpw), while the DM rats healed 51%, 66%, 83%, and 90%. More than one third of NL corneas completely closed the wound by 40 hpw and 100% by 48 hpw; while only one third of DM corneas healed completely by 48 hpw.

Attenuation of EGFR Signaling

We previously demonstrated the critical role of EGFR signaling in corneal epithelial wound healing and homeostasis. Using Western blot analysis, we confirmed these observations and showed that the phosphorylation (activation) of EGFR, and its two downstream effectors AKT and ERK, was significantly diminished in the corneal epithelia collected from rats of 8 weeks DM, compared with the controls (Figs. 6A and 6B). The expression of total proteins of EGFR, AKT, and ERK was also slightly lower with no statistical differences. We further extended our study to AKT activation and distribution via immunohistochemistry to 53 days postwounding (dpw) (Fig. 6C). Unwounded NL cornea exhibited stronger staining of phosphor-AKT compared with the DM. At 2, 4, and 11 dpw, more layers of cells positive of p-AKT staining were seen in the NL than in the DM, indicating a stronger AKT activation. After 23 dpw, the staining pattern of p-AKT was similar between the DM and NL corneas.

Disruption of Cell-Cell Junction

We also examined cell-cell junctions in the corneal epithelia. The expression of tight junction proteins ZO-1 and occludin, and adherens junction protein β-catenin was detected with Western blot analysis (Fig. 7A). The expression levels for these proteins were similar between the NL and DM corneas (Fig. 7B). The distribution of β-catenin (Fig. 7C) and ZO-1 (Fig. 7D) was then evaluated with immunohistochemistry, and no difference was observed between the unwounded NL and DM corneas. At 2 dpw, NL corneas exhibited continuous β-catenin staining in all epithelial layers and continuous ZO-1 staining in the apical layers in the central cornea; while the DM corneas showed sparse β-catenin staining in the apical layers and minimal ZO-1 staining in the central cornea. The staining pattern of β-catenin and ZO-1 was similar between the two groups in the limbal region at 2 dpw (data not shown). No significant difference in the distribution of β-catenin and ZO-1 was noticed at 7 dpw, indicative of a delayed but not absent formation of cell-cell junctions in the DM corneas.