April 2017
Volume 58, Issue 4
Open Access
Cornea  |   April 2017
Mitochondrial and Morphologic Alterations in Native Human Corneal Endothelial Cells Associated With Diabetes Mellitus
Author Affiliations & Notes
  • Benjamin T. Aldrich
    University of Iowa Carver College of Medicine, Department of Ophthalmology and Visual Sciences, Iowa City, Iowa, United States
    Iowa Lions Eye Bank, Coralville, Iowa, United States
    Cornea Research Center, University of Iowa, Iowa City, Iowa, United States
  • Ursula Schlötzer-Schrehardt
    Department of Ophthalmology, University of Erlangen-Nürnberg, Erlangen, Germany
  • Jessica M. Skeie
    University of Iowa Carver College of Medicine, Department of Ophthalmology and Visual Sciences, Iowa City, Iowa, United States
    Iowa Lions Eye Bank, Coralville, Iowa, United States
    Cornea Research Center, University of Iowa, Iowa City, Iowa, United States
  • Kimberlee A. Burckart
    Iowa Lions Eye Bank, Coralville, Iowa, United States
    Cornea Research Center, University of Iowa, Iowa City, Iowa, United States
  • Gregory A. Schmidt
    Iowa Lions Eye Bank, Coralville, Iowa, United States
    Cornea Research Center, University of Iowa, Iowa City, Iowa, United States
  • Cynthia R. Reed
    Iowa Lions Eye Bank, Coralville, Iowa, United States
    Cornea Research Center, University of Iowa, Iowa City, Iowa, United States
  • M. Bridget Zimmerman
    University of Iowa College of Public Health, Department of Biostatistics, Iowa City, Iowa, United States
  • Friedrich E. Kruse
    Department of Ophthalmology, University of Erlangen-Nürnberg, Erlangen, Germany
  • Mark A. Greiner
    University of Iowa Carver College of Medicine, Department of Ophthalmology and Visual Sciences, Iowa City, Iowa, United States
    Iowa Lions Eye Bank, Coralville, Iowa, United States
    Cornea Research Center, University of Iowa, Iowa City, Iowa, United States
  • Correspondence: Mark A. Greiner, Cornea and External Diseases, Department of Ophthalmology and Visual Sciences, University of Iowa Carver College of Medicine, 200 Hawkins Drive, Iowa City, IA 52242, USA; mark-greiner@uiowa.edu
  • Friedrich E. Kruse, Department of Ophthalmology, University of Erlangen-Nuremberg, Schwabachanlage 6, 91054 Erlangen, Germany; friedrich.kruse@uk-erlangen.de
Investigative Ophthalmology & Visual Science April 2017, Vol.58, 2130-2138. doi:10.1167/iovs.16-21094
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      Benjamin T. Aldrich, Ursula Schlötzer-Schrehardt, Jessica M. Skeie, Kimberlee A. Burckart, Gregory A. Schmidt, Cynthia R. Reed, M. Bridget Zimmerman, Friedrich E. Kruse, Mark A. Greiner; Mitochondrial and Morphologic Alterations in Native Human Corneal Endothelial Cells Associated With Diabetes Mellitus. Invest. Ophthalmol. Vis. Sci. 2017;58(4):2130-2138. doi: 10.1167/iovs.16-21094.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: To characterize changes in the energy-producing metabolic activity and morphologic ultrastructure of corneal endothelial cells associated with diabetes mellitus.

Methods: Transplant suitable corneoscleral tissue was obtained from donors aged 50 to 75 years. We assayed 3-mm punches of endothelium-Descemet membrane for mitochondrial respiration and glycolysis activity using extracellular flux analysis of oxygen and pH, respectively. Transmission electron microscopy was used to assess qualitative and quantitative ultrastructural changes in corneal endothelial cells and associated Descemet membrane. For purposes of analysis, samples were divided into four groups based on a medical history of diabetes regardless of type: (1) nondiabetic, (2) noninsulin-dependent diabetic, (3) insulin-dependent diabetic, and (4) insulin-dependent diabetic with specified complications due to diabetes (advanced diabetic).

Results: In total, 229 corneas from 159 donors were analyzed. Insulin-dependent diabetic samples with complications due to diabetes displayed the lowest spare respiratory values compared to all other groups (P ≤ 0.002). The remaining mitochondrial respiration and glycolysis metrics did not differ significantly among groups. Compared to nondiabetic controls, the endothelium from advanced diabetic samples had alterations in mitochondrial morphology, pronounced Golgi bodies associated with abundant vesicles, accumulation of lysosomal bodies/autophagosomes, and focal production of abnormal long-spacing collagen.

Conclusions: Extracellular flux analysis suggests that corneal endothelial cells of donors with advanced diabetes have impaired mitochondrial function. Metabolic findings are supported by observed differences in mitochondrial morphology of advanced diabetic samples but not controls. Additional studies are needed to determine the precise mechanism(s) by which mitochondria become impaired in diabetic corneal endothelial cells.

Diabetes mellitus is well known to be associated with excess glycosylation of blood proteins and vascular tissue,13 and organ damage due to small vessel disease.46 However, because the cornea is avascular, diabetes is not investigated traditionally as a risk factor for corneal endothelial cell dysfunction. Numerous studies of cataract surgery have been conducted to compare outcomes between patients with and without diabetes. Compared to nondiabetics, diabetic individuals show differences in corneal endothelial cell morphology, corneal thickness, recovery in clarity, and greater endothelial cell loss following routine cataract surgery.710 Diabetes also slows the recovery of the cornea after vitreoretinal surgery, with corneal edema persisting longer after vitrectomy for patients with diabetes than in nondiabetic patients.1114 The observed differences in endothelial cell loss and morphology, combined with the apparent impairment of endothelial pump function,1518 suggest that diabetes increases the risk of damage and dysfunction in the corneal endothelium following surgical stress. 
A wide base of evidence supports the assertion that corneal endothelial cells are impaired functionally,7,8,11,12,1924 morphologically,25,26 and biochemically2731 by diabetes mellitus. Yet, only two major studies have investigated the impact of diabetes on cornea transplant survival. Price et al.32 studied 3640 consecutive penetrating keratoplasty (PK) cases and found that diabetes mellitus specifically increased the risk of endothelial decompensation and graft failure compared to nondiabetic cornea transplant recipients. The 5-year risk of graft failure was significantly greater in recipients with diabetes compared to recipients without diabetes (5.7% vs. 2.4%). Additionally, the risk of endothelial failure was twice as high in patients with diabetes as in patients without diabetes 5 years postoperatively. In contrast, the Cornea Donor Study (CDS), a multicenter prospective randomized study of over 1000 PK cases, found no significant impact of donor diabetes history on graft survival at 5 and 10 years.3335 However, the CDS was neither powered nor purposed to assess the impact of diabetes on graft survival or endothelial cell morphology. Unfortunately, basic studies that investigate the relationship between diabetes and corneal endothelial cell health and provide clarity to this issue are lacking. Moreover, few corneal studies have considered diabetes from a standpoint that accounts for the significant variability in severity inherent to this complex systemic disease. With the increasing prevalence of diabetes in the cornea donor and transplant recipient pool,32,3437 there is a growing need to examine the influence of diabetes on corneal endothelial cell health in greater detail. 
Recently, our group described a method capable of quantifying mitochondrial respiration of native corneal endothelial cells from cornea donors using extracellular flux analysis of oxygen consumption.38 Recognizing that metabolic dysfunction in diabetes is associated with changes in mitochondrial respiration activity3941 and morphology,42,43 we conducted an investigation to examine the relationship between diabetes and mitochondrial changes in endothelial cells of donor corneas. In this initial study, we used extracellular flux analysis of oxygen consumption and pH to quantify several metrics of mitochondrial respiration and glycolysis for native human donor corneal endothelial cells. The resulting values were then compared between cells from nondiabetic and diabetic donors, with special attention given to donor medical history to account for disease severity. In addition, we performed qualitative and quantitative analyses of cellular and mitochondrial morphology in nondiabetic and diabetic corneal endothelium samples using transmission electron microscopy imaging. Results from these experiments provide a preliminary assessment of the health impact to corneal endothelial cells in individuals with diabetes mellitus, and a basis for future investigations into the potential mechanism(s) that contribute to cellular dysfunction in this disease. 
Methods
All experimental procedures conformed to the tenets of the Declaration of Helsinki. The institutional review boards at the University of Iowa and University of Erlangen-Nürnberg determined that approval was not required for this study and research consent was obtained for all donor corneas. 
Donor Corneas
Corneoscleral tissues included for analysis were obtained, inspected, and stored in medium (Optisol GS; Bausch & Lomb, Irvine, CA, USA) at 4°C in accordance with Eye Bank Association of America (EBAA) and Iowa Lions Eye Bank (ILEB)–compliant protocols. All tissues included for analysis were from donors aged between 50 and 75 years and determined to be suitable for corneal transplantation according to standard ILEB protocol. All experimental testing was performed within 14 days of procurement. Prior to assays, all tissues were analyzed via noncontact specular microscopy (KeratoAnalyzer EKA-10; Konan Medical USA, Irvine, CA, USA) at ILEB to assess endothelial cell density (ECD), hexagonality (hex), and coefficient of variation (CV) from the average of three independent images obtained using a 100-cell, center count method. All tissues were also analyzed by slit-lamp examination (BQ-900 LED; Haag-Streit Diagnostics, Mason, OH, USA) to assess for tissue health according to standard protocol at ILEB. Corneas analyzed in this study had a minimum ECD of 2000 cells/mm2. Corneas were excluded from analysis if the review of medical records or postmortem serology results conducted by ILEB technicians revealed evidence of sepsis or infectious disease posing a risk for disease transmission via cornea transplantation. 
Donor tissue characteristics tracked for this study include donor age, sex, death to preservation time (D/P), preservation to assay time (P/A), and prior intraocular surgery. Tissues with central corneal endothelial guttae in both corneas from a single donor on slit-lamp examination were defined as displaying bilateral guttae. 
Donor Diabetes Status and Group Classification
To gain a detailed understanding of the influence of diabetes on corneal endothelial cells, a donor history of diabetes mellitus and information about medical care related to this diagnosis were ascertained by a review of donor medical records and donor family interviews performed at the time of tissue donation using standard ILEB screening protocols. A manual review of donor records was performed first to identify donors with a history of diabetes mellitus with or without home insulin use (search terms: “diabetic,” “diabetes,” “DM,” “NIDDM,” “IDDM,” “insulin dependent,” “insulin dependency”). Subsequently, we performed a manual review of diabetic donor records to identify prototypic microvascular complications of diabetes mellitus including neuropathy, nephropathy, retinopathy, ulceration of the extremities, amputation of the extremities, renal failure, and ophthalmic treatment for diabetic retinopathy. Microvascular complications were counted as evidence of end-organ damage due to diabetes only if the records indicated specifically that the complication occurred as a result of diabetes (e.g., diabetic neuropathy, diabetic retinopathy, renal failure due to diabetes, amputations due to diabetes). A history of myocardial infarction was not counted as evidence of end-organ damage due to diabetes, given the high prevalence of nondiabetic coronary artery disease in the US population. 
Donor tissues were classified into four groups according to the absence or presence of a donor's diagnosis of diabetes mellitus and historic markers of disease severity. The classification system used for this investigation allowed stratification of diabetic donors based on disease severity using information readily available in the medical record at the time of donation and related specifically to diabetes progression (treatment with home insulin, medical complications from diabetes).4449 Tissues from donors lacking a diagnosis of diabetes mellitus or complications due to diabetes in their medical history were defined as the nondiabetic (ND) sample group. Tissues from diabetic donors with no history of home insulin use (with or without notation of medical complications secondary to diabetes) were classified as the nonadvanced diabetes noninsulin-dependent (NAD-ni) sample group. Tissues from diabetic donors with a history of home insulin use but without notation of medical complications secondary to diabetes were classified as the nonadvanced diabetes insulin-dependent (NAD-i) sample group. Finally, tissues from diabetic donors with a history of home insulin use and end-organ damage specifically noted in the medical history as occurring as a result of diabetes were classified as the advanced diabetes (AD) sample group. 
Ancillary diabetes care data such as disease type, duration of disease, and recent hemoglobin A1c values were not available routinely at the time of tissue procurement and thus could not be used to classify donor tissues. 
Metabolic Assays
Tissue preparation and extracellular flux assays of mitochondrial respiration and glycolysis were performed as described previously.38 In brief, after prestripping the endothelium-Descemet membrane complex (EDM), 3-mm diameter EDM punches were mounted in wells of a XF24 cell culture microplate (Agilent Technologies, Santa Clara, CA, USA). After acclimation for 1 hour in nonbuffered assay medium, metabolic activity of the EDM samples was quantified using a commercial kit (XF Cell Mito or Glycolysis Stress Test Kits; Agilent Technologies) on a Seahorse XFe24 extracellular flux analyzer (Agilent Technologies). Following extracellular flux analysis, tissues were labeled fluorescently using nucleic acid stain (Sytox Green; Life Technologies, Grand Island, NY, USA) and cell counts used to compute the oxygen consumption rate per cell (OCR; pmole/min/cell) and extracellular acidification rate per cell (ECAR; mpH/min/cell). 
Raw OCR values were used to calculate several different key parameters of mitochondrial function per manufacturer's directions (Agilent Technologies; Supplementary Fig. S1). To assess the steady state oxygen consumption, basal respiration was calculated using the third measurement prior to drug injections (baseline OCR) minus the minimum measurement obtained after the addition of electron transport chain (ETC) complex I inhibitors, rotenone (1 μM) and antimycin A (1 μM). To evaluate the proportion of basal respiration contributing directly to ATP synthesis, ATP-associated oxygen consumption was calculated using the third baseline measurement minus the minimum of three measurements obtained after the addition of an ATP synthase inhibitor, oligomycin (1 μM). The remaining portion of basal respiration not contributing to ATP production, proton [H+] leak, was calculated using the minimum of three measurements obtained after the addition of oligomycin (1 μM) minus the minimum measurement obtained after the addition of rotenone (1 μM) and antimycin A (1 μM). The maximum level to which endothelial cells were capable of increasing electron transport chain activity was assessed by quantifying the maximal respiration, which was calculated using the maximum of three measurements obtained after the addition of an ETC uncoupler, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP, 0.5 μM) minus the minimum measurement obtained after the addition of rotenone (1 μM) and antimycin A (1 μM). The ability of the endothelial cells to increase respiratory activity above basal respiration to meet energy demand was assessed by quantifying the spare respiratory capacity, which was calculated using the maximum of three measurements obtained after the addition of FCCP (0.5 μM) minus the third baseline measurement. Lastly, the proportion of oxygen consumption due to nonmitochondrial cellular enzymatic activity, nonmitochondrial respiration, was calculated using the minimum measurement obtained after the addition of rotenone (1 μM) and antimycin A (1 μM). 
Raw ECAR values were used to calculate several different key parameters of glycolytic activity per manufacturer's directions (Agilent Technologies; Supplementary Fig. S2). To assess the steady-state glycolysis activity, glycolysis was calculated using the maximum of the three measurements obtained after the addition of glucose (10 mM) minus the third measurement prior to glucose injection (baseline ECAR). The maximum level to which the endothelial cells were capable of increasing glycolysis activity was assessed by quantifying the glycolytic capacity, which was calculated using the maximum of three measurements obtained after addition of the ATP synthase inhibitor, oligomycin (1 μM), minus the third measurement prior to glucose injection. The ability of the endothelial cells to increase glycolysis activity above basal activity levels to meet energy demand was assessed by quantifying the glycolytic reserve, which was calculated using the maximum of three measurements obtained after the addition of oligomycin (1 μM) minus the maximum of the three measurements obtained after the addition of glucose. Lastly, the proportion of extracellular acidification due to nonglycolytic activity, nonglycolytic acidification, was calculated using the third measurement prior to glucose injection. 
Transmission Electron Microscopy (TEM) Imaging
Tissue preparation and TEM analyses were performed as described previously.50,51 Briefly, corneal specimens were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer, postfixed in 2% buffered osmium tetroxide, dehydrated in graded alcohol concentrations, and embedded in epoxy resin according to standard protocols. We stained 1-μm semithin sections for orientation with toluidine blue. Ultrathin sections (80 nm) were stained with uranyl acetate and lead citrate and examined with transmission electron microscopes (EM 906E; Carl Zeiss Microscopy, Oberkochen, Germany). For qualitative TEM analysis, we analyzed 10 entire sections per specimen, which were cut from two different planes of the tissue block. For quantitative analysis of mitochondrial counts and surface area, average surface area per mitochondria, and total mitochondrial surface area per field, an automated image-processing system (DigiVision; Zeiss Microscopy) with an integrated software package (analySIS; Soft Imaging Systems, Münster, Germany) was used. The measurements were performed according to a defined random sampling procedure, using the bars of the supporting grid square as points of reference, by which 10 consecutive endothelial cells adjacent to the right side of a grid bar were analyzed at a magnification of ×7000 (field of view = 20 μm2). Cellular areas containing cell nuclei were avoided. 
Statistical Analysis
Linear mixed model analysis accounting for two eyes from a single donor was used to examine the relationship between donor diabetic grouping and the parameters obtained from extracellular flux analysis of oxygen and pH (e.g., spare respiratory capacity and glycolytic capacity). Additional variables incorporated into the model included age, ECD, hex, CV, D/P, P/A, bilateral guttae, and prior intraocular surgery. Since parameter measures were not normally distributed, natural log transformation was applied to the parameter values to normalize the data distribution for use in the linear mixed model analysis. Differences between groups from this analysis were expressed as ratios of group means. Statistical analyses were performed using statistical software (SAS version 9.4; SAS Institute, Inc., Cary, NC, USA). A value of P ≤ 0.05 was considered statistically significant. 
Due to a limited number of samples, all NAD corneas (NAD-ni and NAD-i) were treated as a single group when analyzing and comparing data obtained from TEM images. From these samples, the estimate of the mean difference with 95% confidence intervals (CI) in the number of mitochondria per μm2, surface area per mitochondria in μm2, and total mitochondrial surface area per 20 μm2 field of view between the diabetic groups (AD and NAD) and control (ND) were calculated. 
Results
In total, 154 corneas (102 donors; 52 diabetic corneas) were assayed for mitochondrial respiration; 60 corneas (42 donors; 26 diabetic corneas) assayed for glycolysis activity; and 15 corneas (15 donors; 10 diabetic corneas) used for imaging analysis. A mean of 2.8 punches per cornea assayed (range: 1–5, SEM 0.09) and 2.9 punches per cornea assayed (range: 1–5, SEM 0.14) were included for analyses of mitochondrial respiration and glycolysis, respectively. Summary statistics for donor tissue variables included in each assay are shown in Table 1. Donor age, sex, D/P, P/A, ECD, hex, and CV did not differ significantly between assays. 
Table 1
 
Donor Characteristics of Corneal Tissue by Experimental Assay*
Table 1
 
Donor Characteristics of Corneal Tissue by Experimental Assay*
Diabetic Status and Mitochondrial Respiration
Raw OCR data displayed a trend toward increased basal respiration and reduced response to FCCP in AD samples compared to the other groups (Fig. 1). From the raw OCR plot profiles (Fig. 1), NAD-i samples appeared to display a slight increase in mean basal respiration over NAD-ni and ND samples and a larger mean FCCP response relative to all other groups. However, the regression analysis of derived metrics uncovered a significant difference only in spare respiratory capacity among the groups analyzed (Table 2). Specifically, the AD group had significantly lower spare respiratory capacity values compared to each of the other groups analyzed (P < 0.002) with an average mean ratio value of 0.22 (78% reduction) among the three comparisons. Although it did not reach the level of significance (P ≥ 0.28), AD samples did maintain an increase in basal respiration compared to all other samples with an average mean ratio value of 1.39 among the three comparisons. In contrast, the apparent relative increases in basal respiration and FCCP response for NAD-i compared to NAD-ni and ND samples (Fig. 1) were nullified almost completely by including donor history variables in the model with average mean ratio values of 1.00 and 1.05 for basal respiration and spare respiratory capacity comparisons, respectively (Table 2). 
Figure 1
 
Mitochondrial respiration activity in the endothelium of nondiabetic and diabetic donor corneas (n = 154 corneas; 102 donors). Mitochondrial stress assay oxygen consumption rate plot profiles (OCR, pmole/min/cell) are presented for ND (solid blue line, n = 102); NAD-ni (dashed red line, n = 24); NAD-i (dashed purple line, n = 16); and AD (solid green line, n = 12) corneas. Vertical dashed lines indicate assay drug injections and abbreviations above them represent specific compounds injected. O, oligomycin; F, FCCP; R/A, rotenone/antimycin A. Error bars represent SEM.
Figure 1
 
Mitochondrial respiration activity in the endothelium of nondiabetic and diabetic donor corneas (n = 154 corneas; 102 donors). Mitochondrial stress assay oxygen consumption rate plot profiles (OCR, pmole/min/cell) are presented for ND (solid blue line, n = 102); NAD-ni (dashed red line, n = 24); NAD-i (dashed purple line, n = 16); and AD (solid green line, n = 12) corneas. Vertical dashed lines indicate assay drug injections and abbreviations above them represent specific compounds injected. O, oligomycin; F, FCCP; R/A, rotenone/antimycin A. Error bars represent SEM.
Table 2
 
Regression Analysis Comparing Mitochondrial Respiration Parameters Among Nondiabetic and Diabetic Cornea Samples
Table 2
 
Regression Analysis Comparing Mitochondrial Respiration Parameters Among Nondiabetic and Diabetic Cornea Samples
Diabetic Status and Glycolysis
Unlike mitochondrial respiration, raw ECAR plot profiles were largely overlapping among the groups analyzed and diabetes did not appear to be associated with an appreciable amount of variation in glycolytic activity (Fig. 2). Linear mixed model analysis did uncover a slightly reduced glycolytic reserve in AD samples compared to the other groups with an average mean ratio value of 0.58 among the three comparisons (P ≥ 0.51; Table 3). However, the differences observed in glycolytic reserve and all other glycolysis metrics analyzed did not differ significantly among groups (Table 3). 
Figure 2
 
Glycolysis activity in the endothelium of nondiabetic and diabetic donor corneas (n = 60 corneas; 42 donors). Glycolysis stress assay extracellular acidification rate plot profiles (ECAR, mpH/min/cell) are presented for ND (solid blue line, n = 34); NAD-ni (dashed red line, n = 10); NAD-i (dashed purple line, n = 9); and AD (solid green line, n = 7) corneas. Vertical dashed lines indicate assay drug injections and abbreviations above them represent specific compounds injected. G, glucose; O, oligomycin; 2-DG, 2-deoxy-d-glucose. Error bars represent SEM.
Figure 2
 
Glycolysis activity in the endothelium of nondiabetic and diabetic donor corneas (n = 60 corneas; 42 donors). Glycolysis stress assay extracellular acidification rate plot profiles (ECAR, mpH/min/cell) are presented for ND (solid blue line, n = 34); NAD-ni (dashed red line, n = 10); NAD-i (dashed purple line, n = 9); and AD (solid green line, n = 7) corneas. Vertical dashed lines indicate assay drug injections and abbreviations above them represent specific compounds injected. G, glucose; O, oligomycin; 2-DG, 2-deoxy-d-glucose. Error bars represent SEM.
Table 3
 
Regression Analysis Comparing Glycolysis Parameters Among Nondiabetic and Diabetic Cornea Samples
Table 3
 
Regression Analysis Comparing Glycolysis Parameters Among Nondiabetic and Diabetic Cornea Samples
Diabetic Status and Corneal Endothelial Cell Ultrastructure
Several phenotypic changes in cellular ultrastructure were observed in diabetic samples (n = 10, 5 AD, and 5 NAD) compared to ND controls (n = 5). In the qualitative analysis, samples from donors with diabetes appeared to have distinct alterations in mitochondrial morphology compared to control samples (Fig. 3), including dense packing (NAD-ni, NAD-i, and AD), loss of cristae (AD), and a tendency toward larger and more distended mitochondria (NAD-ni, NAD-i, and AD). Cells from all three diabetic groups appeared to have prominent and inflated Golgi bodies associated with abundant vesicles (Fig. 4B). Samples from the AD group were observed to have intracellular accumulations of lysosomal bodies or autophagosomes compared to their nondiabetic counterparts (Fig. 4C). Two AD samples analyzed displayed a focal production of abnormal extracellular matrix protein resembling long-spacing collagen extending from the cell surface into the Descemet membrane (Fig. 4D) and AD endothelial cells appeared to have more frequent overlap between neighboring cells (Figs. 4E, 4F). In the quantitative analysis, the mean number of mitochondria per μm2 was nearly indistinguishable among the ND (3.05; CI 2.29, 3.82), NAD (2.98; CI: 2.24, 3.72), and AD (2.76; CI: 1.60, 3.93) groups. However, the average surface area of the mitochondria increased with diabetes severity, with mean mitochondrial size larger by 54% (0.047 μm2; CI: −0.026, 0.120) in the NAD group (0.133 μm2), and by 67% (0.058 μm2; CI: −0.015, 0.130) in the AD group (0.144 μm2) compared to controls (0.086 μm2). The net result was an increase in total mitochondrial area by 43% (2.26 μm2; CI: −1.45, 5.97) in the NAD group (7.49 μm2), and by 39% (2.02 μm2; CI: –1.69, 5.73) in the AD group (7.25 μm2) compared to controls (5.23 μm2). Overall, when combining both the AD and NAD groups together, diabetic samples demonstrated 60% greater mean surface area per mitochondria (0.052 mm2; CI: −0.005, 0.105; P = 0.052) and 41% greater mean total mitochondrial surface area (2.14 mm2; CI: −0.53, 4.81; P = 0.11) compared to the ND group. 
Figure 3
 
Transmission electron micrographs showing endothelial cell mitochondrial morphology in (A) nondiabetic and (BD) advanced diabetic corneas. Mitochondria in advanced diabetic corneas frequently displayed a loss of cristae (B, D; asterisks) with electron-dense inclusion bodies (B; arrows). Compared to control samples (A), mitochondria in diabetic corneas (NAD and AD) were densely packed and displayed a greater variation in size with many large, distended mitochondria (BD). Arrows in (C) highlight lysosomal bodies/autophagosomes observed in diabetic corneas. Magnification bars equal 1 μm.
Figure 3
 
Transmission electron micrographs showing endothelial cell mitochondrial morphology in (A) nondiabetic and (BD) advanced diabetic corneas. Mitochondria in advanced diabetic corneas frequently displayed a loss of cristae (B, D; asterisks) with electron-dense inclusion bodies (B; arrows). Compared to control samples (A), mitochondria in diabetic corneas (NAD and AD) were densely packed and displayed a greater variation in size with many large, distended mitochondria (BD). Arrows in (C) highlight lysosomal bodies/autophagosomes observed in diabetic corneas. Magnification bars equal 1 μm.
Figure 4
 
Transmission electron micrographs showing endothelial cell and organelle morphology in (A) nondiabetic and (BF) advanced diabetic corneas. Arrows highlighting features unique to diabetic samples include: (B) prominent and inflated Golgi bodies associated with abundant vesicles (NAD and AD corneas); (C) accumulation of lysosomal bodies/autophagosomes (AD corneas); (D) focal production of abnormal extracellular matrix (long-spacing collagen; AD corneas); and (EF) overlap of neighboring cells (AD corneas). Asterisks in (E) highlight abnormal mitochondria with loss of cristae. Arrowhead in (F) highlights an area with prominent Golgi vesicles. Magnification bars equal 1 μm, except in (D) where it equals 0.5 μm.
Figure 4
 
Transmission electron micrographs showing endothelial cell and organelle morphology in (A) nondiabetic and (BF) advanced diabetic corneas. Arrows highlighting features unique to diabetic samples include: (B) prominent and inflated Golgi bodies associated with abundant vesicles (NAD and AD corneas); (C) accumulation of lysosomal bodies/autophagosomes (AD corneas); (D) focal production of abnormal extracellular matrix (long-spacing collagen; AD corneas); and (EF) overlap of neighboring cells (AD corneas). Asterisks in (E) highlight abnormal mitochondria with loss of cristae. Arrowhead in (F) highlights an area with prominent Golgi vesicles. Magnification bars equal 1 μm, except in (D) where it equals 0.5 μm.
Discussion
Corneal endothelial cells from donors with evidence of advanced diabetes mellitus, specifically a medical history of home insulin use and prototypic comorbidities due to this microvascular disease, had mitochondrial respiration parameters that deviated from the remaining diabetic and nondiabetic donor tissue studied in this investigation. The endothelium of corneas with AD displayed a significant reduction in spare respiratory capacity (average among comparisons, 78%) and a moderate but insignificant increase in basal respiration (average among comparisons, 39%). However, there was no evidence of a compensatory shift toward glycolysis associated with disease. Consistent with our findings of impaired mitochondrial respiration in the corneal endothelium of AD samples, increased basal respiration and decreased spare respiratory capacity are observed in peripheral blood mononuclear cells collected from diabetic patients.40,41 The metabolic changes observed in AD corneal endothelium are also consistent with cells experiencing stress and susceptible to cell death.5257 Additional studies are needed to define more clearly the relationship between spare respiratory capacity and the health of the corneal endothelium, but our results confirm that the mitochondria of corneal endothelial cells can be impacted by diabetes despite the avascular nature of this tissue. 
Mitochondrial respiration assay results were further supported by ultrastructural analyses that, in addition to other cellular changes, revealed morphologic changes in the mitochondria of AD corneal endothelium. Endothelial cells from cornea donors with a history of advanced diabetes appeared to have large, distended, densely packed mitochondria with abnormally staining or absent cristae. These observations are consistent with shifts in mitochondrial dynamics and also a possible disruption of the normal mitophagy process.40 Mitochondrial dynamics include the balance of fission and fusion.58,59 Under normal resting conditions, mammalian cells have a balanced rate of fission and fusion events, occurring constantly and independent of changes occurring in their mitochondrial microtubule networks. An imbalance in mitochondrial dynamics is demonstrated in some pathologic conditions, including diabetes.60,61 Fission events lead to mitochondrial products that include healthy and unhealthy mitochondria, the latter of which are tagged for mitophagy.62 In diabetes, it has been observed that impaired mitophagy results in dysfunctional mitochondria detectable in the cell cytoplasm.40 Reported changes in mitochondrial dynamics of hyperglycemic tissues from diabetic patient samples, like the changes in diabetic donor corneal endothelium we observed with our respirometry assays and ultrastructural analyses, result in dysfunctional mitochondria.40 
Our investigation also provides evidence of increased cellular injury with progression of systemic diabetic disease. Diabetes has been shown in some studies to increase corneal endothelial cell loss with or without intraocular surgery, suggesting that diabetic hyperglycemia contributes to cell death,710,19 while other studies have found no difference in endothelial cell density with or without intraocular surgery between diabetics and nondiabetics.63 The same tension has been demonstrated surrounding keratoplasty, where some studies have shown a deleterious impact of diabetes on donor tissue36 but not outcomes33,64 using diabetic tissue, and other studies have shown negative impact32,65 or no impact33 on keratoplasty outcomes in diabetic recipients. Considering the increasing prevalence of this disease,66 it is imperative to obtain greater clarity on the issue of diabetes and endothelial cell health. Results of our study highlight the importance of stratifying diabetic samples or cases on the basis of disease severity when trying to account for the influence of diabetes on endothelial health following cornea transplant surgery. Although we cannot discount the possibility that additional comorbidities (e.g., hypertension) influence endothelial health, the metabolic data presented here indicate that insulin dependence and comorbidities secondary to diabetes need to be considered as covariables when analyzing transplant outcomes. We acknowledge that medical histories can be incomplete, even though we have found several meaningful correlations with classifications based on a medical history of diabetes complementary to the work presented in this manuscript67 and Liaboe et al.68 We aim in future studies to further quantitate and classify the differences in diabetes severity among these groups by measuring advanced glycation end products, mitochondrial pathogenic gene mutation associations (e.g., m.3243A>G), and mitochondrial protein expression alterations. These categorizations will be used to help stratify tissues without having to rely solely on medical history reports. 
In conclusion, results of this study demonstrate that the mitochondria of corneal endothelial cells from diabetic donors suffer from mitochondrial dysfunction indicative of disease severity. Two features that became apparent during this study were a significant reduction in spare respiratory capacity and corresponding changes in mitochondrial morphology. Both features are tied intimately to cellular health and highlight the need to further investigate the molecular mechanism(s) by which mitochondria become altered in the corneal endothelium of diabetic individuals. However, it is important to emphasize that the metabolic and morphologic abnormalities observed in this study were only significant for a small proportion of diabetics (i.e., AD samples only) and underscore the need to stratify diabetics into meaningful groups when studying this broad, complex disease. From a transplant perspective, our data indicate that a general diagnosis of diabetes mellitus may not necessarily represent a significant risk factor for poor endothelial cell health and that caution appears warranted, particularly for individuals with a history of insulin dependence and comorbidities resulting directly from this disease. 
Acknowledgments
The authors thank the donors and their families who, by providing research consent, made this study possible. 
Supported by Beulah and Florence Chair in Cornea/External Disease and Refractive Surgery; Lloyd and Betty Schermer; and the M. D. Wagoner and M. A. Greiner Cornea Excellence Fund. 
Disclosure: B.T. Aldrich, None; U. Schlötzer-Schrehardt, None; J.M. Skeie, None; K.A. Burckart, None; G.A. Schmidt, None; C.R. Reed, None; M.B. Zimmerman, None; F.E. Kruse, None; M.A. Greiner, Eye Bank Association of America (S) 
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Figure 1
 
Mitochondrial respiration activity in the endothelium of nondiabetic and diabetic donor corneas (n = 154 corneas; 102 donors). Mitochondrial stress assay oxygen consumption rate plot profiles (OCR, pmole/min/cell) are presented for ND (solid blue line, n = 102); NAD-ni (dashed red line, n = 24); NAD-i (dashed purple line, n = 16); and AD (solid green line, n = 12) corneas. Vertical dashed lines indicate assay drug injections and abbreviations above them represent specific compounds injected. O, oligomycin; F, FCCP; R/A, rotenone/antimycin A. Error bars represent SEM.
Figure 1
 
Mitochondrial respiration activity in the endothelium of nondiabetic and diabetic donor corneas (n = 154 corneas; 102 donors). Mitochondrial stress assay oxygen consumption rate plot profiles (OCR, pmole/min/cell) are presented for ND (solid blue line, n = 102); NAD-ni (dashed red line, n = 24); NAD-i (dashed purple line, n = 16); and AD (solid green line, n = 12) corneas. Vertical dashed lines indicate assay drug injections and abbreviations above them represent specific compounds injected. O, oligomycin; F, FCCP; R/A, rotenone/antimycin A. Error bars represent SEM.
Figure 2
 
Glycolysis activity in the endothelium of nondiabetic and diabetic donor corneas (n = 60 corneas; 42 donors). Glycolysis stress assay extracellular acidification rate plot profiles (ECAR, mpH/min/cell) are presented for ND (solid blue line, n = 34); NAD-ni (dashed red line, n = 10); NAD-i (dashed purple line, n = 9); and AD (solid green line, n = 7) corneas. Vertical dashed lines indicate assay drug injections and abbreviations above them represent specific compounds injected. G, glucose; O, oligomycin; 2-DG, 2-deoxy-d-glucose. Error bars represent SEM.
Figure 2
 
Glycolysis activity in the endothelium of nondiabetic and diabetic donor corneas (n = 60 corneas; 42 donors). Glycolysis stress assay extracellular acidification rate plot profiles (ECAR, mpH/min/cell) are presented for ND (solid blue line, n = 34); NAD-ni (dashed red line, n = 10); NAD-i (dashed purple line, n = 9); and AD (solid green line, n = 7) corneas. Vertical dashed lines indicate assay drug injections and abbreviations above them represent specific compounds injected. G, glucose; O, oligomycin; 2-DG, 2-deoxy-d-glucose. Error bars represent SEM.
Figure 3
 
Transmission electron micrographs showing endothelial cell mitochondrial morphology in (A) nondiabetic and (BD) advanced diabetic corneas. Mitochondria in advanced diabetic corneas frequently displayed a loss of cristae (B, D; asterisks) with electron-dense inclusion bodies (B; arrows). Compared to control samples (A), mitochondria in diabetic corneas (NAD and AD) were densely packed and displayed a greater variation in size with many large, distended mitochondria (BD). Arrows in (C) highlight lysosomal bodies/autophagosomes observed in diabetic corneas. Magnification bars equal 1 μm.
Figure 3
 
Transmission electron micrographs showing endothelial cell mitochondrial morphology in (A) nondiabetic and (BD) advanced diabetic corneas. Mitochondria in advanced diabetic corneas frequently displayed a loss of cristae (B, D; asterisks) with electron-dense inclusion bodies (B; arrows). Compared to control samples (A), mitochondria in diabetic corneas (NAD and AD) were densely packed and displayed a greater variation in size with many large, distended mitochondria (BD). Arrows in (C) highlight lysosomal bodies/autophagosomes observed in diabetic corneas. Magnification bars equal 1 μm.
Figure 4
 
Transmission electron micrographs showing endothelial cell and organelle morphology in (A) nondiabetic and (BF) advanced diabetic corneas. Arrows highlighting features unique to diabetic samples include: (B) prominent and inflated Golgi bodies associated with abundant vesicles (NAD and AD corneas); (C) accumulation of lysosomal bodies/autophagosomes (AD corneas); (D) focal production of abnormal extracellular matrix (long-spacing collagen; AD corneas); and (EF) overlap of neighboring cells (AD corneas). Asterisks in (E) highlight abnormal mitochondria with loss of cristae. Arrowhead in (F) highlights an area with prominent Golgi vesicles. Magnification bars equal 1 μm, except in (D) where it equals 0.5 μm.
Figure 4
 
Transmission electron micrographs showing endothelial cell and organelle morphology in (A) nondiabetic and (BF) advanced diabetic corneas. Arrows highlighting features unique to diabetic samples include: (B) prominent and inflated Golgi bodies associated with abundant vesicles (NAD and AD corneas); (C) accumulation of lysosomal bodies/autophagosomes (AD corneas); (D) focal production of abnormal extracellular matrix (long-spacing collagen; AD corneas); and (EF) overlap of neighboring cells (AD corneas). Asterisks in (E) highlight abnormal mitochondria with loss of cristae. Arrowhead in (F) highlights an area with prominent Golgi vesicles. Magnification bars equal 1 μm, except in (D) where it equals 0.5 μm.
Table 1
 
Donor Characteristics of Corneal Tissue by Experimental Assay*
Table 1
 
Donor Characteristics of Corneal Tissue by Experimental Assay*
Table 2
 
Regression Analysis Comparing Mitochondrial Respiration Parameters Among Nondiabetic and Diabetic Cornea Samples
Table 2
 
Regression Analysis Comparing Mitochondrial Respiration Parameters Among Nondiabetic and Diabetic Cornea Samples
Table 3
 
Regression Analysis Comparing Glycolysis Parameters Among Nondiabetic and Diabetic Cornea Samples
Table 3
 
Regression Analysis Comparing Glycolysis Parameters Among Nondiabetic and Diabetic Cornea Samples
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