The Pennsylvania State University

The Graduate School

College of Medicine

HISTOLOGIC AND ELECTRORETINOGRAPHIC EVIDENCE OF

DIABETES-INDUCED RETINAL NEURODEGENERATION

A Dissertation in

Anatomy

by

William F. Robinson, Jr.

© 2010 William F. Robinson, Jr.

Submitted in Partial Fulfillment

of the Requirements

for the Degree of

Doctor of Philosophy

December 2010

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The dissertation of William F. Robinson, Jr. was reviewed and approved* by the following:

Alistair J. Barber

Associate Professor of Ophthalmology and Cellular and Molecular Physiology

Dissertation Advisor

Chair of Committee

Patricia J. McLaughlin

Professor of Neural and Behavioral Sciences; Director, Graduate Program in Anatomy

Shao-Min Zhang

Assistant Professor of Neural and Behavioral Sciences

Steve Abcouwer

Associate Professor of Surgery and Cellular and Molecular Physiology

*Signatures are on file in the Graduate School

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ABSTRACT

Diabetic retinopathy is a vision-threatening condition, currently affecting more than 5 million Americans. It is the leading cause of new cases of blindness in working-aged adults. The pathogenesis of the disease is poorly understood, but the earliest symptoms include subtle visual disturbances such as color and contrast sensitivity changes, delayed dark adaptation, and difficulty seeing in low-light environments. The earliest clinical signs of diabetic retinopathy are vascular abnormalities such as microaneurysms and edema. There is considerable evidence suggesting that retinal neurodegeneration takes place before the onset of such vascular changes.

Previous research in our lab has established that retinal neurodegeneration is an apoptotic event, leading to retinal thinning. Animal models of experimental diabetes have been used to determine that retinal thinning takes place preferentially in the inner retina. The retinal ganglion cells are especially vulnerable, demonstrating the greatest loss in peripheral regions of the retina. The current study seeks to further explore the effects of diabetes on the neuronal components of retinas from human donors and from diabetic mice. The studies compiled here seek to test the hypotheses that early stages of diabetes lead to histologic evidence of neurodegeneration in human donor tissue, and that diabetes in the Ins2Akita mouse leads to alterations in rhodopsin content and electroretinographic output.

Donor retinas from humans who had had diabetes for less than 10 years were compared with retinas from age-matched nondiabetic donors. Comparisons were made on the basis of retinal thickness and cell counts. Ins2Akita mice, which spontaneously develop diabetes at 4 weeks of age, were tested after being diabetic for 7 weeks. Electroretinograms were recorded and the following waveforms were analyzed for amplitude and implicit time: scotopic threshold response, a- and b- waves, and oscillatory potentials.

Results of the human histopathology study indicate that, while there are no differences between retinal thickness measurements from diabetic vs. nondiabetic donors, the retinas from donors with short-term diabetes had fewer cells in the peripheral regions of the outer nuclear layer. Cell counts from the cone photoreceptors were unchanged, indicating rod loss. Studies of rod photoreceptor protein content demonstrated a decrease in rhodopsin content in diabetic mice, without a change in the protein content of other phototransduction proteins (transducin and phosphodiesterase). ERG results from diabetic mice demonstrate reduced scotopic threshold response and delayed oscillatory potentials, compared to age-matched controls.

The findings presented here confirm that neurodegeneration takes place in the peripheral retina early in the progression of diabetes in humans. In contrast to animal models in which the inner retina is implicated, it may be that the photoreceptors of the outer retina are the cells most vulnerable to neurodegeneration in humans with diabetes. Diabetic mice, however, demonstrate loss of rhodopsin without concomitant photoreceptor loss. ERG results in the Ins2Akita diabetic mice show deficits in scotopic vision and changes in oscillatory potentials similar to those seen in humans with diabetes.

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TABLE OF CONTENTS PAGE

List of Figures v List of Tables vi List of Abbreviations vii Acknowledgments viii Chapter One 1

Introduction 2

Chapter Two 10 Effect of Short-Duration Diabetes on Human Donor Retinas 10

Introduction 11 Experimental Procedures 13 Results 18 Discussion 27

Chapter Three 30 Effect of Diabetes on Photoreceptor Protein Expression 30

in Diabetic Mouse Retinas

Introduction 31 Experimental Procedures 35 Results 41 Discussion 49

Chapter Four 52

Electroretinographic Response to Diabetes in Ins2Akita Mice 52

Introduction 53 Experimental Procedures 60 Results 63 Discussion 84

Chapter Five 88

Concluding Discussion 88

References 95

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LIST OF FIGURES PAGE 1. Cross section of human retina demonstrates layered cellular structure 20

2. Cell layer thickness in central and peripheral regions of human retinas 21

3. Cell counts indicate fewer ONL cells in peripheral retinas from diabetic donors 23

4. Immunolabeled cones enable differential cone counts in human retinas 25

5. TUNEL labeling in donor retinas indicates frequency of apoptotic cells 26

6. Diabetes reduces rhodopsin protein content in 11 week Ins2Akita mice 41

7. Quantification of oligomers indicates that rhodopsin protein content in oligomer 43 form is not changed in diabetes

8. Transducin protein content is not altered by diabetes in 11 week old mice 44

9. Phosphodiesterase protein content is not altered by diabetes in 11 week old mice 45

10. Photoreceptor mRNA expression was unchanged in mice diabetic for 10 weeks 46

11. ONL cell counts are unaltered by diabetes in 11 week old mice 47

12. Immunofluorescent image analysis to compare rhodopsin content 48

13. ERG output demonstrates a- and b-wave in 11 week old mice 54

14. Oscillatory potentials recorded in Ins2Akita mice 56

15. Scotopic Threshold Response is recorded in dark-adapted animals 58

16. Positive STR amplitude is reduced in diabetic Ins2Akita mice 65

17. Negative STR is unchanged in 11 week old diabetic mice 67

18. ERG a-wave recordings for 11 week old Ins2Akita mice are largely unchanged 69 with diabetes

19. ERG b-wave response is largely unchanged by 11weeks age 71

20. Oscillatory potentials are significantly delayed in 11 week old diabetic mice 73

21. There is no difference in pSTR between controls, treated-, or untreated- 75 diabetic mice at 14 weeks

22. There is no difference in nSTR between controls, treated-, or untreated- 77 diabetic mice at 14 weeks

23. There is no difference in a-wave amplitude or implicit time measurements, 79 when comparing 14 week old controls, treated-, and untreated- diabetic mice

24. Untreated 14 week old diabetic mice demonstrate reduced b-wave implicit times 81

25. ERG oscillatory potentials for 14 week old Ins2Akita mice do not demonstrate 83 differences between controls, and treated, or untreated diabetic animals

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LIST OF TABLES PAGE

Table 1 Summary of donor demographics 14

Table 2 Detailed description of donor demographics 19

Table 3 Mass and blood glucose levels measured in diabetic and control mice 64

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LIST OF ABBREVIATIONS

µV Microvolt

AU Arbitrary Unit (of fluorescence)

DM Diabetes Mellitus

DR Diabetic Retinopathy

ERG Electroretinogram

INL Inner Nuclear Layer

IPL Inner Plexiform Layer

L-cone Long Wavelength-Sensitive Cone

M-cone Middle-Wavelength Sensitive Cone

ms Millisecond

NDRI National Disease Research Interchange

ONL Outer Nuclear Layer

OP Oscillatory Potential

OPL Outer Plexiform Layer

PDE Phosphodiesterase

pSTR Positive Scotopic Threshold Response

RGC Retinal Ganglion Cell

S-cone Short-Wavelength Sensitive Cone

STR Scotopic Threshold Response

STZ Streptozotocin

TUNEL Terminal deoxynucleotidyl transferase dUTP nick-end labeling

VEGF Vascular Endothelial Growth Factor

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ACKNOWLEDGMENTS

The author would like to thank Mrs. Rhona Ellis (deceased) for collecting and archiving the

human tissue; Dr. Kang Li (Penn State Hershey Histology Core facility) for paraffin

embedding, sectioning, and H&E staining; Mr. Wade Edris (Penn State Hershey Imaging

Core facility) for help with confocal microscopy; Ms. Ann Banko (Penn State Hershey

Department of Surgery) for performing the qRT-PCR work; and Dr. Peter Macleish

(Morehouse School of Medicine, Atlanta, GA) for the kind gift of mAb 7G6 anti-cone

arrestin antibody.

  

CHAPTER ONE

Introduction

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Diabetes mellitus affects approximately 17.7 million Americans and is predicted to double by

the year 2030 1. The incidence of diabetic retinopathy correlates with the length of time the

patient has the disease, reaching as high as 97% prevalence by the 15th year after the onset of

diabetes 2. The serious and debilitating effects of diabetic retinopathy encompass several

facets of vision loss, including decreases in contrast sensitivity, latency in adaptation to

darkened environments, and loss of visual acuity. Diabetic retinopathy usually affects both

eyes, though the severity may vary. Vision changes may begin subtly, but frequently

progress to severe vision deficits, making diabetic retinopathy the leading cause of legal

blindness in working-aged adults 2, 3.

The clinical diagnosis and staging of diabetic retinopathy are made on the basis of observed

changes in the retinal vasculature. The fundus may be evaluated by ophthalmoscopic

examination. The first observable stage is referred to as nonproliferative diabetic

retinopathy, and may include microaneurysms, small vessel occlusion, and ischemia in the

retina. Nonproliferative diabetic retinopathy may be subdivided into mild, moderate, and

severe stages. As the vascular pathologies develop, one may progress to proliferative

diabetic retinopathy, characterized by proliferation of new vasculature (neovascularization).

The new vessels tend to be more permeable, allowing for lipid exudates and hemorrhages.

Macular edema involves the accumulation of plasma and other fluids in the macular region of

the retina. 10% of the total diabetic population has macular edema, though prevalence tends

to correlate with severity of diabetic retinopathy. Macular edema may be due to increased

vessel permeability and/or breakdown of the blood-retinal barrier 4.

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The earliest symptoms of diabetic retinopathy, as experienced by those who suffer from the

disease, are often subtle and may be difficult for the patient to describe. Vision abnormalities

range from difficulty with hue discrimination to areas of diminished vision within the visual

field known as scotomas. One of the earliest symptoms of visual disturbance is likely to be

loss of contrast sensitivity. Brinchmann-Hansen and colleagues studied 51 patients with

diabetes, finding that deficits in contrast sensitivity correlate with the degree of diabetic

retinopathy 5. Other authors report that abnormalities in contrast sensitivity occur before the

onset of vascular changes associated with diabetic retinopathy 6, 7.

Hue discrimination and color-contrast sensitivity are also symptoms associated with early

stages of diabetic retinopathy. Fristöm studied human subjects with and without diabetes,

subdividing the diabetic sample into groups based on presence of microaneurysms and hard

exudates. He found significant differences between non-diabetics and those with diabetes,

and that color contrast sensitivity deficits correlate with the degree of diabetic retinopathy 8.

Greenstein and colleagues came to similar conclusions and also found that the short-

wavelength sensitive (S-cone) pathway sensitivity was diminished in 70% of patients with

nonproliferative diabetes while the middle-wavelength sensitive (M-cone) pathway

sensitivity remained largely unchanged 9. Additional studies have indicated that color

contrast sensitivity is impaired before the onset of vascular changes in diabetic retinopathy

patients 10, 11.

Many patients with diabetes complain of difficulty with driving at night and have trouble

seeing when entering a darkened room due to delayed dark adaptation 12. Clinical testing

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supports these anecdotal reports. In 1979 Henson and North used a dark adaptometer with 35

diabetic patients to measure the length of time required to recognize a stimulus after exposure

to bright light. Results indicated a significant delay in dark adaptation in diabetes 13.

A growing number of studies demonstrate the impact of diabetes on the neural portions of the

retina. In 1998, Barber and coworkers first suggested that apoptotic changes in the eyes of

diabetic animals indicated retinal neurodegeneration. Samples taken from streptozotocin

(STZ)-induced diabetic rats after 1, 3, 6, and 12 months of diabetes demonstrated increased

retinal apoptosis coupled with retinal thinning compared with age-matched control rats.

Specifically, there was a 9-10 fold increase in the number of apoptotic cells in the retinas of

diabetic rats. Furthermore, there was a 22% reduction in the thickness of the inner plexiform

layer of the retina 7.5 months after the onset of diabetes in STZ rats 14. Studies performed in

vitro using R28 neuronal cells demonstrated the protective effects of insulin in reducing

neural apoptosis, 15 and that such pro-survival effects could be blocked by high glucose and

glucosamine 16.

Continued research has shown apoptosis of specific cell types within the inner retina. Retinal

ganglion cells (RGCs) are especially vulnerable to apoptosis. Abu-El-Asrar and colleagues

used immunohistochemistry to demonstrate cleaved caspase-3 immunolabeling in the

ganglion cells of retinas from diabetic subjects. Other pro-apoptotic factors, Bax and Fas,

were also expressed in the ganglion cells and identified by immunohistochemistry 17.

5  

Martin and coworkers came to the same conclusion regarding neurodegeneration of ganglion

cells in STZ diabetic mice, using TUNEL labeling techniques to detect apoptosis. Further

verification was performed, noting morphologic changes indicative of apoptosis via electron

microscopy 18. In another morphology-based study of ganglion cells, Gastinger and

colleagues studied Ins2Akita mice, which have a point mutation in the insulin 2 gene,

predisposing the heterozygotes to spontaneous loss of pancreatic beta cells with subsequent

loss of insulin production and hyperglycemia. Their study demonstrated that within 3

months, Ins2Akita diabetic mice lost 16.4% of retinal ganglion cells peripherally, and those

that survived underwent morphologic changes, including abnormal swellings throughout the

cell and increases in ON-type RGC dendrite length, density, and number of dendritic

terminals 19.

Gastinger and coworkers used TUNEL labeling and caspase-3 immunoreactivity in rat

retinas to identify a significant increase in apoptosis 2 weeks after streptozotocin-induced

diabetes, compared with age-matched controls. After 12 weeks, Ins2Akita diabetic mice

showed a 72% increase in apoptotic cells, compared to control littermates 20. Other retinal

layers and cell types have also been implicated in neurodegeneration. Amacrine cells, which

have cell bodies in the inner nuclear and ganglion cell layers, with synapses in the inner

plexiform layer, were identified undergoing apoptosis in diabetic animal models. After 6

months of diabetes, Ins2Akita mice demonstrated a 16-20% reduction in amacrine cell

numbers, compared to nondiabetic littermates 20.

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While studying neuronal nitric oxide synthase (nNOS) expression in diabetic mice, Park and

colleagues described a significant reduction in the outer nuclear layer after 12 weeks of

diabetes and a 30% reduction after 24 weeks. They suggest that, in diabetes, nNOS-

expressing bipolar cells in the inner nuclear layer lead to the increased synthesis of nitric

oxide, which may be responsible for photoreceptor degeneration in the outer retina via

glutamate excitotoxicity 21. This would support work carried out by the Smith lab, which

studied H&E stained sections in diabetic and age-matched control mice and found that, in

addition to inner retina degeneration, the thickness of the outer nuclear layer was also

significantly decreased by 10 weeks after the induction of diabetes 18.

While an accumulating body of information has documented the changes to the various cell

types involved in diabetic retinopathy, it is important to remember that these structural

changes are likely indicative of functional changes. Due to the organized, stratified anatomy

of the mammalian retina, these structural changes in retinal ganglion cell layers and nuclear

layers indicate the loss of specific cell types which have particular functions within the visual

pathway. Additionally, decreases in inner- and outer plexiform layers may indicate

degeneration of synaptic transmission within the neural retina. Animal models demonstrate

that diabetes has a neurodegenerative effect on the retina, and suggest that such neuronal

dysfunction might be related to a decrease in synaptic protein levels 22, 23. Given the

detrimental effects of diabetes on neuronal cells and synaptic proteins in the retina, it is

important to proceed with animal research and translational research that can lead to greater

understanding and improved vision in humans with diabetes.

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The electroretinogram (ERG) is frequently used to measure the electrical responses

originating from the retina due to light stimulus. The response, recorded by an electrode

placed on the cornea, produces a waveform with several components, provided by different

cell types from the neural retina. Immediately following light stimulus, the a-wave is a

negative deflection produced by the photoreceptors. The post-receptor b-wave response is a

large positive deflection originating primarily from the ON-center bipolar cells 24, 25,

modified by input from OFF-center bipolar and horizontal cells 26. The oscillatory potentials

(OPs) are small, higher frequency wavelets on the ascending portion of the b-wave, and are

thought to represent the modulation of interactions between bipolar, amacrine, and ganglion

cells 27, 28, and are often analyzed in clinical and research studies of diabetic retinopathy.

The scotopic threshold response (STR) is another component of the ERG, considered to be

an indicator of retinal ganglion cell function 28. While this response is often not measured,

because it can only be determined in response to very low intensity flashes of light after

prolonged dark adaptation, there is good evidence that it is reduced in both humans and rats

with diabetes 29-31. Furthermore, electrophysiological ganglion cell function was found to be

compromised even in children with diabetes 32. These data provide functional evidence of

diabetes-induced retinal ganglion cell degeneration.

The negative consequences of vascular and neuronal pathologies are apparent. Treatment

options for patients suffering with diabetic retinopathy, however, are limited. The current

standard of care for treatment of diabetic retinopathy is laser photocoagulation. This

technique, developed in 1968, utilizes laser energy focused on the retina, to treat the vascular

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pathologies associated with diabetic retinopathy 3, 33. Photocoagulation treatment serves to

ablate peripheral blood vessels, and is largely successful at halting neovascularization; but in

so doing, peripheral regions of the retina are destroyed in an effort to salvage central vision.

Photocoagulation treatment is unlikely to improve vision after the procedure but rather serves

to prevent further vision loss. Unfortunately, repeated treatments of retinal photocoagulation

are sometimes required 34.

Other therapeutic options for treating diabetic retinopathy have been designed to treat the

vascular effects of disease progression with angiotensin converting enzyme inhibitors 35, 36 or

circulating endothelial progenitor cells 37. Neuroprotective treatments proposed include

Exendin-4, a glucagon-like peptide-1 (GLP-1) receptor agonist 38; lipoic acid 39 and lutein 40.

Adrenomedullin and erythropoietin therapies are reported to have both vasculoprotective and

neuroprotective benefits 41, 42. Clinical trials have been conducted to test the effectiveness of

PKC inhibitors, and while these have demonstrated visual improvements in patients,

compared with placebo groups 43, the benefits did not meet endpoints for FDA approval.

Another promising therapy in diabetic retinopathy is the use of compounds that block

vascular endothelial growth factor (VEGF) and thereby prevent angiogenesis. These anti-

VEGF drugs demonstrate the ability to reverse neovascularization 44 and improve visual

acuity 45. Unfortunately, the administration of these drugs is by repeated intravitreal

injection, which understandably diminishes patient appeal.

Maintaining tight glycemic control and preventing hypertension are logical preventative

measures that can be taken to reduce the risks of diabetic retinopathy 46-48. However, the

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limitations of the drug therapies and the destructive nature of laser photocoagulation therapy

serve to underscore the importance of a more complete understanding of the pathogenesis in

diabetic retinopathy.

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CHAPTER TWO

Effect of Short-Duration Diabetes on Human Donor Retinas

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INTRODUCTION

Diabetic retinopathy is a disease process that takes its toll on the vascular and neuronal

components of the retina. The majority of persons with diabetes will progress to clinically

detectable diabetic retinopathy within 15 years of diagnosis 48, 49. Diabetic retinopathy often

causes appreciable vision loss and is the leading cause of new cases of blindness in working

aged adults 50. Clinically observable indicators of vascular disease (microaneurysms,

neovascularization, macular edema) are accompanied by functional vision loss, which may

be due to neurodegenerative changes 14, 51-54 that are more difficult to detect in vivo 55, 56.

Postmortem specimens, however, allow for histological analysis of retinal pathology in

diabetes, providing direct access to the tissue to perform immunolabeling, layer-specific cell

counts, and selective imaging from whole-mount and cross-sectional perspectives.

Once thought to be a strictly microvascular pathology, diabetic retinopathy is now

characterized as having a neurodegenerative component as well 14, 36, 57-61. Indicators of

neurodegeneration in animal models of diabetes include histologic evidence such as loss of

retinal thickness 14, 18, 21, 38, 42, 51, 52, 60-62, increased neural apoptosis 14, 18, 42, 52, 62, increased

reactivity of glial cells 53, 62-64, and decreased neuronal cell counts 14, 18, 38, 42, 51, 52, 60, 62.

Clinical studies also indicate functional alterations in color and contrast sensitivity 5, 8, 65-69,

dark adaptation, 13, 70-72, and electrophysiological changes 38, 51. Therefore, the evidence

strongly suggests that loss of visual function and neurodegeneration are prevailing

consequences of diabetes in both humans and animals. The present study seeks to test the

hypothesis that histologic evidence of neurodegeneration can be demonstrated in human

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retinal tissue from donors with diabetes and that the results will indicate findings similar to

those seen in experimental diabetes animal models.

Animal models have been used to study many of the complications associated with diabetes.

The most frequently utilized model involves the use of streptozotocin (STZ), a chemical that

is toxic to pancreatic beta cells. When STZ is injected into rats or mice, the animals quickly

lose the ability to produce insulin and become hyperglycemic. STZ-induced diabetes in

animals leads to many of the same alterations observed in humans with diabetes, including

vascular pathology 42, 73, glial reactivity 53, 63, and alterations in ERG response 35, 38, 74-78.

Barber and colleagues used the STZ rat model to identify neural apoptosis and retinal

thinning in diabetes 14, and went on to elaborate on this retinal neurodegeneration by

studying the Ins2Akita mouse model. The Ins2Akita mouse has a point mutation in the insulin 2

gene which leads to insulin protein misfolding in pancreatic beta cells, and by four weeks of

age, beta cell death with hypoinsulinemia and hyperglycemia. Morphometric analysis

enabled Barber’s lab to discover an overall thinning of the diabetic retinas at 22 weeks of

hyperglycemia, and more severe thinning in the inner retina peripherally 62.

In the following study the thickness of cell layers and the number of cell bodies were

quantified in post mortem human retinas from 3 groups of donors; those with no diabetes

(ND), diabetes and no insulin treatment ((-)INS), and diabetes with insulin treatment

((+)INS). The donors were carefully age-matched and their duration of diabetes was reported

as less than ten years. The results indicate that peripheral regions of retinas had a lower

number of outer nuclear layer cells compared to age-matched non-diabetic retinas. The

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number of cones was unaffected by diabetes, suggesting that only rod photoreceptors were

lost in donors with this relatively short duration of diabetes.

EXPERIMENTAL PROCEDURES

Human Donor Tissue

Postmortem human eyes were obtained from the National Disease Research Interchange

(NDRI) in Philadelphia, PA. Eyes were accepted only if they met the following criteria:

donor was < 68 years of age; no history of infection, ischemia (no ventilator), chemotherapy,

or radiation treatment to the head. Enucleation was performed within 12 hours of time of

death, and eyes were immediately placed in formalin fixation and delivered within 48 hours.

Donor records collected by NDRI were limited to basic information including the donor’s

gender, age, race, cause of death, time of death, time of enucleation, and a brief medical

history, including diabetic status and duration, and whether or not the donor was taking

insulin. Donor statistics are summarized in Table 1. Upon receipt from NDRI, the eyes were

sagittally hemisected, using a dissecting microscope, and the temporal and nasal hemispheres

were embedded in paraffin blocks for subsequent sectioning. Paraffin-embedded tissue was

sectioned at 5µm thickness and stained with hematoxylin and eosin (H & E) or labeled by

immunohistochemistry. Care was taken to select well-preserved sections close to the optic

nerve with minimal tissue artifact such as folding.

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Table 1. Summary of donor demographics

Donor Group

n Gender (M/F)

Average age Diabetes duration

ND 7 6/1 55±10 yrs. -

(-)INS 6 4/2 54±10 yrs. 3±2 yrs.

(+)INS 6 4/2 56±12 yrs. 5±3 yrs.

ND, non-diabetic; (-)INS, diabetic donors not using insulin; (+)INS, diabetic donors using

insulin.

Tissue Staining and Image Acquisition

H & E staining was performed in the Histology Core Facility at Penn State Hershey Medical

Center. Slides were then photographed with a Spot RT KE diagnostic camera (Diagnostic

instruments, Sterling Heights, MI) mounted on an Olympus BH-2 microscope (Lake Success,

NY), using a 20× dry objective lens. Sixteen images were acquired from each retina: four

regions from four serial sections: two centrally and two peripherally (one 8 mm superior to

the central region and one 8 mm inferior to the central region).

TUNEL Labeling

Additional retina cross sections were labeled using Terminal deoxynucleotidyl transferase

dUTP nick-end labeling (TUNEL) to detect cells undergoing apoptosis at the time of death.

TUNEL labeling was performed using the ApopTag Peroxidase In Situ Apoptosis detection

kit (Millipore, Billerica, MA), in accordance with the kit instructions, with the addition of

using 3-amino-9ethylcarbazole (Sigma, St. Louis, MO) as the peroxidase substrate. Some

retina sections were pretreated with DNase (Sigma, St. Louis, MO) to provide a positive

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control. Other DNase-treated sections were processed without terminal deoxynucleotidyl

transferase (TdT) enzyme, serving as negative control. Masked TUNEL-labeled cross

sections were viewed with an Olympus BH2 microscope (Lake Success, NY) using the 20×

and 40× dry objective lenses. Three sections from each donor were quantified with regard to

frequency of TUNEL-positive cells. Positive cells were identified by cell layer and location

(central or peripheral) within the sagittal plane. Images were captured using an attached

Sony 3CCD video camera (Sony, Tokyo, Japan).

Morphometric and Morphologic Analyses

H & E images were analyzed using Image J software (W. S. Rasband, public domain via U.S.

National Institutes of Health, Bethesda, Maryland, http://rsb.info.nih.gov/ij/, 1997-2010).

Image J was used to measure the thickness of the following layers: ganglion cell layer + inner

plexiform layer (GCL + IPL), inner nuclear layer (INL), outer plexiform layer (OPL), and

outer nuclear layer (ONL). Cell counts were also performed on each of the images of H & E-

stained retina sections. Using Image J to randomly outline 100 micron regions of central and

peripheral retina, cell counts were carried out for the GCL, INL, and ONL. Eight separate

cell counts from the central retina of each donor were averaged to determine the number of

cells per 100 microns of retina. The procedure was repeated for the peripheral retina in each

of the same layers. The identity of each slide was masked during cell counts and

morphometric analysis.

Cone subpopulations were quantified using a Leica DM RXA2 fluorescent microscope

(Leica Microsystems, Bannockburn, IL) with a 1.2 mm diameter field using the 20×

16  

objective oil-immersion lens. Cell bodies and nuclear layers were detected using Hoechst

nuclear stain. Immunofluorescence with the monoclonal mouse antibody (mAb) 7G6

(generously provided by Dr. Peter Macleish, Morehouse School of Medicine, Atlanta, GA),

which recognizes cone arrestin, enabled quantification of the total number of cone

photoreceptors within the field. As before, two central and two peripheral regions were

sampled from each of the four sections from each of the donor retinas. After data collection,

the cell numbers from each region of each donor sample were averaged and expressed as

cells/mm. Immunofluorescence to red/green opsin enabled quantification of middle- (MWS)

and long-wavelength sensitive (LWS) cones (also known as green and red cones,

respectively). The number of anti- red/green opsin-positive cells was subtracted from the

total number of mAb 7G6-positive cells to estimate the number of short-wavelength sensitive

(SWS), or blue cones.

Immunohistochemistry

Sagittally hemisected, paraffin-embedded, postmortem human donor eyes were sectioned (5µ

thick) and mounted on Colorfrost/Plus microscope slides (Fisher Scientific, Fair Lawn, NJ).

Retina tissue sections were deparaffinized in xylene (Fisher Scientific) with gentle agitation

followed by rehydration in graduated alcohol. Antigen retrieval was performed by

incubating sections in 10mM citrate buffer (pH 6.0) at 80ºC for 30 minutes. After cooling,

the retina sections were incubated at room temperature for 1.5 hours in a blocking agent of

10% donkey serum (Jackson ImmunoResearch, West Grove, PA) and 0.1% Triton X-100 in

phosphate-buffered saline (PBST). Tissue sections were then incubated in primary antibody

diluted in blocking agent at 4ºC overnight. Retina sections were labeled with rabbit Anti-

17  

Opsin, red/green polyclonal antibody (1:100, Millipore, Billerica, MA), and mAb 7G6 for all

cones79 (1:1, Peter Macleish, Atlanta, GA). After overnight incubation in the primary

antibody, the sections were washed in PBST, counterstained with Hoechst (0.5 µg/mL,

Sigma-Aldrich, St. Louis, MO) and incubated in the following immunofluorescent affinity

purified secondary antibodies diluted in blocking agent: Cy2-conjugated donkey anti-rabbit

(1:1000, Jackson ImmunoResearch, West Grove, PA) and Cy3-conjugated donkey anti-

mouse (1:1000, Jackson ImmunoResearch, West Grove, PA). Secondary antibodies were

incubated for 1 hour in the dark at room temperature. Slides were then washed in PBST, and

coverslipped using Aqua Polymount (Polysciences, Warrington, PA) mounting medium.

Confocal Microscopy

Fluorescently labeled slides were imaged with a laser confocal microscope (TCS SP2 AOBS,

Leica Microsystems, Manheim, Germany), utilizing a 405 nm laser for Hoechst (detection

range 410-480 nm), a 488 nm laser for Cy2 (detection range 493-530 nm), and a 543 nm

laser for Cy3 (detection range 548-630 nm). Z-section stacks of optical slices were taken

using either a 40× or 63× inverted oil objective lens and reconstructed, using the Leica

software, as a maximum projection, and saved in 512 × 512 pixel format.

Statistical Analysis

Statistical analyses were performed using GraphPad Prism software (version 5.01, GraphPad

Software, Inc., La Jolla, CA). Values from the 3 donor groups were analyzed using 1-way

analysis of variance (ANOVA) with the Newman-Keuls Multiple Comparison post hoc test

to determine differences between groups. Donor ages are expressed as mean ± standard

18  

deviation. Error bars in all graphs are expressed as mean ± standard error of the mean

(S.E.M.). An asterisk (*) denotes significance at p < 0.05.

RESULTS

Donor Demographics

30 eyes (19 right, 11 left) were used in this study, from 19 donors. All 12 diabetic donors

can be considered to have had Type II, or adult onset diabetes, given their age and short

duration of diabetes. A complete list of information is shown in Table 2, including the cause

of death. The most common cause of death was cardiac arrest. There were 14 males and 5

females. The average age of donors was 55 (±10.66) years. There was no statistically

significant difference in mean age between the three donor groups.

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Table 2. Detailed description of donor demographics

Donor Sex Age Cause of death

ND M 68 Multi-system organ failure

ND M 48 Respiratory arrest

ND M 60 Cardiac arrest

ND M 40 Cardiac arrest

ND M 60 Cardiac arrest

ND M 47 Motorcycle accident

ND F 65 Unknown

(-)INS M 52 Cardiac arrest

(-)INS F 65 Cardiac arrest

(-)INS F 55 Respiratory arrest

(-)INS M 66 Lymphoma

(-)INS M 49 Cerebral hemorrhage

(-)INS M 38 Cardiac arrest

(+)INS M 63 Cardiac arrest

(+)INS M 53 Cardiac arrest

(+)INS F 62 Chronic Pulmonary Obstructive Disorder

(+)INS F 33 Drug overdose

(+)INS M 57 Cardiac arrest

(+)INS M 66 Respiratory arrest

ND, non-diabetic; (-)INS, diabetic donors not using insulin; (+)INS, diabetic donors using

insulin.

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Quantification of Retinal Layer Thickness

Measurements of retinal thickness were made in H&E sections from paraffin-embedded

retinas from 6 (-)INS diabetic, 6 (+)INS diabetic, and 7 ND donors (Figure 1), in central and

peripheral regions. Results indicate that there were no significant differences in thickness for

any of the cell layers in sections from diabetic donors, compared to ND donors, in both the

central (Figure 2 A) and peripheral (Figure 2 B) regions. When the diabetic groups were

combined (data not shown), the average ONL thickness was 36.99 (±3.87) microns centrally,

and 26.41 (±2.90) microns peripherally; compared with 42.75 (±2.33) microns centrally and

29.86 (±1.68) microns peripherally in the ND donors, which was not significantly different

from the age-matched ND measurements.

Fig. 1. Cross section of human retina demonstrates layered cellular structure. Retinal thickness

measurments were taken at each of the following layers: GCL + IPL, INL, OPL, ONL. GCL,

ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer;

ONL, outer nuclear layer.

21  

Fig. 2. Cell layer thickness in central and peripheral regions of human retinas. The thickness of

cell layers was measured in paraffin sections from human donors. A) There were no significant

differences in retinal layer thickness in the central retina. B) There were also no significant

differences between groups in the peripheral region of retinas, despite a trend towards reduction in the

thickness of the ONL in both diabetic groups. Data were analyzed by 1-way ANOVA and expressed

as mean ± standard error of the mean. ND, non-diabetic; (-)INS, diabetic donors not using insulin;

(+)INS, diabetic donors using insulin.

22  

Quantification of Retinal Layer Cell Number

Cell bodies in the GCL, INL, and ONL were quantified in each of the samples, in both

central and peripheral regions of the donor retinas. Cell body counts were compared between

the ND, (-)INS diabetic and (+)INS diabetic groups. There was no significant difference

between the groups in the number of cell bodies in the GCL or the INL (Figure 3A and 3B).

In contrast, the ONL in the peripheral regions of the retina contained significantly fewer cells

in (-)INS diabetic and (+)INS diabetic groups compared to ND donors (p < 0.05) (Figure

3B).

23  

Fig. 3. Cell counts indicate fewer ONL cells in peripheral retinas from diabetic donors. Cell

bodies were counted in 1.2 mm regions of H&E paraffin sections from human donor tissue, in central

and peripheral retina. A) There were no significant differences between donor groups in the number

of cell bodies in each of the retinal layers GCL, INL, and ONL in the central regions. B) In the

peripheral regions of retinas there were no significant differences in cell body numbers in GCL and

INL, however there were significantly fewer cell bodies in the peripheral ONL of both the (-)INS

group and (+)INS group compared to the ND group. Data were analyzed by 1-way ANOVA and

expressed as mean ± standard error of the mean. * p < 0.05

A 

B 

24  

Quantification of Photoreceptor Populations

The loss of ONL cell bodies suggested photoreceptor drop-out. Immunofluorescence was

used to further identify the subtypes of cells in the photoreceptor layer. The monoclonal

antibody (mAb) 7G6 anti-cone arrestin, was used to label the cone photoreceptors in the

peripheral retina (Figure 4A). Sections were also labeled with polyclonal anti-red/green

opsin, which is specific for the outer segments of medium/long wavelength sensitive

(red/green) cones. Co-localization of these two antibodies was taken to indicate the presence

of medium wavelength sensitive (MWS) cones or long wavelength sensitive (LWS) cones.

Cells that were mAb 7G6 positive, but negative for anti-red/green opsin were assumed to be

short wavelength sensitive (SWS), or blue, cones (Figure 4A, arrow).

After immunolabeling the cones from all three groups of donor retinas, a fluorescent

microscope was used to visualize the cone cells. Two peripheral regions were selected for

quantification, each 8 mm from the posterior aspect of the tissue sample. At 20×

magnification, all of the mAb 7G6-positive cells were counted, representing the total cone

count for each 1.2 mm span of retina. Similarly, all of the cells labeled with anti-red/green

opsin were counted in the same region of retina. The difference between the two counts was

taken as the number of SWS (blue) cones. As expected, the number of SWS cones was

dramatically outnumbered by the MWS and LWS cones, with the SWS cones comprising

about 15% of the total cone population. There was no significant difference between donor

groups with regard to the numbers of cones found (Figure 4B).

25  

Fig. 4. Immunolabeled cones enable differential cone counts in human retinas. Paraffin sections

of human donor retina were labeled with anti-red/green opsin (green) and Mab7G6 for all cones (red).

Hoechst dye was used to label all nuclei (blue). A) Confocal image of retina from a non-insulin-

dependent diabetic donor: positive 7G6 labeling was observed throughout the cell bodies and outer

segments of all cone photoreceptors, while red/green opsin immunoreactivity occurred only in the

outer segments of long- and middle- wavelength cone receptors. Co-localization of both antibodies at

the outer segment (yellow) indicated long- or middle-wavelength cones, while the outer segments of

short-wavelength cones were red (arrow). Nuclear material of cell bodies is labeled with Hoechst

(blue). B) Cone counts indicate that there was no significant difference between donor groups with

regard to total cones or Short-wavelength cones and Middle-/Long-wavelength cones.

Quantification of TUNEL-positive Cells

Data from TUNEL-positive cell counts in cross sections from human donor retinas

demonstrate a higher frequency of apoptotic cells in the ganglion cell layers. This was the

case in both central and peripheral regions of retinas. The greatest number of TUNEL-

positive cells was found in the GCLs of retinas from diabetic donors who were not using

insulin. There was great variability in results across the different groups and even within

samples from the same donor. Due to the fact that the frequency of TUNEL-positive cells

A B

26  

did not fall into a normal bell-shaped distribution of data points, it was not appropriate to

perform parametric statistics in these cell counts (Figure 5).

Fig. 5. TUNEL labeling in donor retinas indicates frequency of apoptotic cells. TUNEL-positive

cells were quantified in retinal cross sections from human donors. A) Sections from human donors

were TUNEL-labeled (reddish-brown) to detect cells undergoing apoptosis. B) TUNEL labeling in

central retinas demonstrated higher frequency of apoptotic cells in retinas from non-insulin using

diabetic donors, particularly in the retinal ganglion cell layer. C) Peripheral retinas also showed

higher frequencies of apoptotic cells in the retinal ganglion cell layer. TUNEL, Terminal

deoxynucleotidyl transferase dUTP nick-end labeling.

A

27  

DISCUSSION

Retinal neurodegeneration is thought to be responsible for a cumulative loss of neurons and

photoreceptors in diabetes. This has become a well-established concept in animal models of

diabetes 57, 80. Studies using human postmortem tissue have also shown evidence of

increased apoptosis in both the vascular and neural tissues of the retina 14, 81. Most

postmortem studies, however, have examined retinas from donors with advanced stages of

diabetic retinopathy. The present study examined the possibility of cell loss in retinas from

human donors with shorter durations of diabetes, and little evidence of vascular retinopathy,

testing the hypothesis that such cell loss could be quantified in human donor tissue. The

donors in this study were reported to have had adult onset diabetes for an average of 4.3

(±2.5) years, which can be considered a relatively short duration in humans.

This study found that the thickness of retinal layers was unaffected by diabetes, but that the

cell bodies in the peripheral ONL were depleted by 19.2% in the (-)INS group and 21.6% in

the (+)INS group of diabetic donors, suggesting that a small but significant loss of

photoreceptors had occurred. Quantification of subgroups of cells using

immunohistochemistry determined no change in the cone density, implying that the loss of

cells in the peripheral ONL can be attributed to depletion of rod photoreceptors. This finding

differs from animal studies that measured reductions in the thickness of the IPL and INL in

mice and rats and after 3.5 to 7 months of experimental diabetes 14, 18, 62. It is possible that

larger changes in these animals with uncontrolled experimental diabetes may have masked

any subtle changes in the ONL. It is also the case that the changes in the ONL of the human

retinas were only revealed by counting cell bodies, which was not an approach used in the

28  

animal studies. Some investigators have noted photoreceptor apoptosis 20, 52 and delayed a-

waves in the ERG 76, suggesting the possibility that the neurodegeneration in animals occurs

at an accelerated rate compared to humans and more rapidly affects many, if not all, cellular

components of the retina. In contrast, cell loss in human retinas may occur more gradually,

preferentially affecting a subpopulation of cells beginning with the peripheral rod

photoreceptors.

The results from the quantification of TUNEL-positive cells support previous findings in

which it has been demonstrated that ganglion cells are vulnerable to degeneration by

apoptosis 14, 18. The high frequency of TUNEL-positive RGCs does not support the data

collected in the morphometric study and cell count study presented here. These studies did

not show any differences in RGC + IPL layer thickness or in RGC counts with diabetes. It

must be noted, however, that the TUNEL study portrays a transient “snapshot” of apoptosis

that was under way at the time of the donor’s death, while the morphometry and cell count

studies demonstrate retinal layer thickness and number of cells remaining after years of

diabetes. It may be the case that photoreceptor degeneration took place prior to this

“snapshot” or that photoreceptors undergo a form of cell death other than apoptosis and

therefore do to get labeled with TUNEL staining.

Clinical studies have noted retinal thinning in diabetes 61, 82, mostly using optical coherence

tomography (OCT) techniques to image the retina. The advantage of OCT technology is that

it can be performed in vivo in patients for diagnostic purposes, although these studies were

limited to a region with a radius of 3 mm from the fovea 61, 82-84 or the optic nerve head 85. It

29  

is conceivable that larger changes in retinal thickness may occur in the peripheral regions but

could not be observed in these OCT studies.

One possible explanation for a preferential loss of peripheral rod photoreceptors is that these

cells may be impacted more by the metabolic stress caused by diabetes. It has been

suggested that the rods have such a high metabolic demand that these cells are preferentially

impacted by changes in oxygen tension or blood flow associated with dark adaptation in

diabetes 71, 86. This is a difficult hypothesis to test, but there is strong evidence that diabetes

impacts the speed of dark adaptation in humans 13, even in patients with no clinical evidence

of vascular pathology, suggesting that the rod photoreceptors may undergo functional deficits

after shorter durations of diabetes.

30  

CHAPTER THREE

Effect of Diabetes on Photoreceptor Protein Expression

in Diabetic Mouse Retinas

31  

INTRODUCTION

The interpretation of a visual image is dependent on light as a stimulus, and the presence of

an intact cellular and synaptic pathway from the retina to the visual cortex. Photoreceptors

serve as the interface where light energy is converted into signals that are modulated by the

different cell types of the retina before leaving the eye via the optic nerve. The

phototransduction cascade is a complex process with separate pathways for cone-dominated

color vision and rod-dominated scotopic vision. Much of the research into diabetic

retinopathy has investigated changes in vasculature that occur in this disease process. More

recent studies have explored the role of neurodegeneration in the diabetic retina, discovering

pathology and apoptosis localized to the inner retina. Despite the fact that the

phototransduction cascade has been studied extensively, little research has been undertaken

to investigate the role of diabetes in the outer retina where the photoreceptors are located.

Rod and cone photoreceptors are not equally distributed across the retina. Although not

found in the fovea of humans, rods account for 95% of all photoreceptors, and are the

dominant cell type of the peripheral outer nuclear layer 87, 88. Humans have 3 different cone

cell types, those sensitive to light of short-, medium-, and long-wavelengths; allowing for

perception of blue, green, and red colors respectively. Cone photoreceptors dominate the

fovea of the retina and their population falls off dramatically outside of the parafoveal

regions. The previous chapter provides evidence that humans with diabetes for a relatively

short period of time demonstrated an increased loss of rod photoreceptors.

32  

Animal studies of photoreceptor degeneration in diabetes tend to focus on structural or

functional changes to the outer retina. The structure of the retina is organized in a stratified

architecture, allowing for histologic identification of photoreceptor cell bodies, which occupy

the outer nuclear layer of the retina. Park and coworkers described a remarkable loss of

photoreceptors in diabetic rats by 12 weeks after streptozotocin (STZ) injection with

subsequent ONL thinning by 24 weeks after the onset of diabetes 52. Structural changes that

are seen in the vasculature of patients with diabetic retinopathy are not as evident in animal

models. X-ray irradiation was utilized in an attempt to model neovascularization in diabetic

rats. Findings indicated that the combined effects of diabetes and radiation lead to outer

nuclear layer (ONL) thinning followed by loss of photoreceptor cells along with RPE

hypertrophy and inner retina atrophy 89. A study which investigated the structural and

functional effects of diabetes, performed by Aizu and colleagues, found an 18.9% reduction

in photoreceptor layer thickness in 1-month diabetic Sprague Dawley rats. The same study

demonstrated a significant reduction in a-wave amplitude, implicating photoreceptor

dysfunction in 1-month diabetic Brown Norway rats 51.

A small number of histological studies of postmortem donor eyes from humans with diabetes

have demonstrated photoreceptor loss. While both of the following studies were performed

using retina tissue from donors who had had laser photocoagulation treatment for diabetic

retinopathy, Shah and coworkers documented photoreceptor loss in areas removed from the

treatment sites 90. In a separate study destruction of photoreceptors and RPE cells was noted

at the sites of laser burns, with RPE hypertrophy at the edges of the scars and the presence of

inner nuclear layer cells lying directly on the intact Bruch’s membrane 91.

33  

Specific electroretinogram (ERG) waveforms have been analyzed to understand alterations in

photoreceptor function in humans. The a-wave originates from photoreceptor activity, and

the slope of the leading edge of the a-wave has been used in a computational photopigment

transduction model to determine log S, identified as an indicator of photoreceptor sensitivity

92. Using this model, humans with diabetes demonstrate diminished photoreceptor sensitivity

compared to age-matched controls 74.

Using a paired-flash ERG protocol, it is possible to extract the rod contribution to the

photoreceptor response. Diabetic rats demonstrated a reduction in the amplitude of the rod

response as early as 2 days post STZ injection. It has been suggested that this very early

dysfunction may be due to STZ toxicity, as the rod response returns to normal within 4

weeks. By 12 weeks, however, the diabetic rats again showed rod dysfunction compared

with age-matched controls. The authors attribute this alteration to a decrease in the length of

rod photoreceptor outer segments and the loss of rhodopsin 75, 93.

Mice are nocturnal animals, and as such, have a much greater preponderance of rods, which

account for 97% of photoreceptors 94. Furthermore mice are protanopic dichromats, unable

to perceive red, since they have only short- and medium-wavelength sensitive cones 95. The

rod structure and function in mice, however, are very similar to human, and the predominant

protein, rhodopsin, is highly conserved between the two species 96. Rhodopsin is a G-protein

coupled receptor with 7 transmembrane domains, located primarily within the disc

membranes of the rod photoreceptor outer segments. While there are a great many published

34  

reports that have used rodents to study photoreceptor structure and function, there is less

information regarding the effects of diabetes on rhodopsin expression, in rodents or humans.

Rhodopsin is the most abundant protein of the outer retina, accounting for >90% of the

protein content of rod outer segment membrane 97. When rhodopsin absorbs a photon of

light, it undergoes a conformational change, leading to the activation of the rhodopsin

molecule. When the activated rhodopsin comes into contact with the heterotrimeric G-

protein transducin, the α-subunit of transducin is released, which allows for the exchange of

GDP for GTP. The activated α-transducin/GTP subunits dissociate from the β/γ subunits

then bind to and activate phosphodiesterase 6 (PDE 6). As the activated PDE 6 hydrolyzes

the cyclic GMP, the cytoplasmic concentration of GMP is reduced to the point that GMP is

no longer able to hold open the sodium (Na+) ion channels. The Na+ channel closure leads

to membrane hyperpolarization 98. While activated, rhodopsin catalyzes the nucleotide

exchange of GDP for GTP in many transducin α-subunits, allowing for signal amplification.

The phototransduction cascade is halted by phosphorylation of the activated rhodopsin which

leads to binding by the protein arrestin 99.

Due to the fact that rhodopsin is a rod-specific protein, it is a useful marker for western

blotting and immunohistochemistry assays, used to identify the presence of rod

photoreceptors. Evidence suggests that rhodopsin can form dimers 100, 101, and higher order

oligomers that may improve the efficiency of transducin activation 102. The present study

sought to test the hypothesis that there is a loss of rod photoreceptors and thus, a subsequent

reduction in rhodopsin protein content in Ins2Akita mice after 7 weeks of diabetes, compared

35  

to age-matched controls. To test this hypothesis, diabetic and control mouse retinas were

assayed by immunoblotting, quantitative real-time polymerase chain reaction (qRT-PCR),

and immunohistochemistry for rhodopsin protein content. Additionally, the outer nuclear

layer cell bodies of photoreceptors were counted on slides taken from cross-sections of

diabetic and control mouse retinas.

EXPERIMENTAL PROCEDURES

Animals

Heterozygous C57BL/6J Ins2Akita mice were bred and maintained at the Penn State College

of Medicine Juvenile Diabetes Research Foundation Diabetic Retinopathy Center Animal

Facilities Core. Ins2Akita mice have a spontaneous mutation in the Ins2 gene that prevents

disulfide bonds from forming, leading to protein misfolding, endoplasmic reticulum stress,

and eventual pancreatic beta cell death, rendering the animals hypoinsulinemic and

hyperglycemic at 4 weeks of age. In the present study, data was collected from 11 week old

mice that had been diabetic for 7 weeks. Female Ins2Akita mice demonstrate later onset and a

lesser degree of hyperglycemia 103; for this reason, only male mice were used in the present

study. Animals were housed in the Penn State College of Medicine animal facility in a

standard 12 hour light/dark cycle with littermates in plastic cages, with ad libitum access to

food and water in accordance with the Institutional Animal Care and Use Committee

guidelines, standards set forth in the ARVO Statement of the Use of Animals in Ophthalmic

and Vision Research, and the NIH Guidelines for the Care and Use of Laboratory Animals.

36  

Immunoblotting

Mice were anesthetized with an intraperitoneal injection of ketamine/xylazine (0.136 mg

ketamine + 0.0096 mg xylazine / g body weight) prior to sacrifice. Immediately following

decapitation by guillotine, retinas were removed, placed in centrifuge tubes, flash frozen in

liquid nitrogen, and transferred to a -70 degree freezer. Frozen retina samples were sonicated

in a detergent based lysis buffer (100mM NaCl, 1.0% Triton X-100, 0.05% sodium

deoxycholate, 0.2% SDS, 2 mM EDTA, 10 mM HEPES, 1 mM sodium orthovanadate, 10

mM sodium fluoride, 10 mM sodium pyrophosphate, 1 mM benzamidine, 10 mM

microcystin, and 1 Complete™ Mini EDTA-free Protease Inhibitor Cocktail Tablet (Roche

Molecular Biochemicals, Indianapolis, IN) for every 10 mL) on ice and centrifuged at 12,500

rpm. The supernatant was removed for protein quantification assay and electrophoresis using

NuPAGE apparatus and gels (Invitrogen, Carlsbad, CA). Overnight transfer to nitrocellulose

membrane was performed and the following day the membranes were blocked with 5% milk

in Tris-buffered saline with 0.05% Tween-20, incubated in primary antibodies against

rhodopsin (mouse monoclonal antibody 1D4 at 1:200, Abcam, Cambridge, MA) or rod

transducin α (rabbit polyclonal antibody 3504 at 1:500, Abcam, Cambridge, MA) or PDE 6 α

(rabbit polyclonal antibody 5659 at 1:500, Abcam, Cambridge, MA) and the appropriate

secondary antibodies (anti- mouse, anti-rabbit IgG alkaline phosphatase linked, GE

Healthcare UK Limited, Little Chalfont Buckinhamshire, UK) and developed by incubating

on ECF substrate (GE Healthcare UK Limited) before reading blots on Typhoon 9400

Variable Mode Imager (Amerisham Biosciences Corp, Piscataway, NJ). Blots were

quantified using ImageQuant 5.0 software (Molecular Dynamics, Inc., Sunnyvale, CA).

37  

RNA Isolation and qRT-PCR Analysis

Total RNA was isolated from mouse retinas using the RNeasy Mini Kit (QIAGEN 74104,

Gaithersburg, MD) following the manufacturer’s instructions with some modification. The

“Protocol: Purification of Total RNA from Animal Tissues” supplied with the kit was

followed except for the following steps: Homogenization of tissue was done by pipetting

each retina in 700ul RLT buffer containing 1%β-mercaptoethanol until it disintegrated. The

homogenate was then placed in a QIAshredder column (QIAGEN 79656, Gaithersburg, MD)

and subjected to centrifugation at 13,200xg in a Labnet Spectrafuge™ 24D microcentrifuge

(Labnet International, Woodbridge, NJ) at room temperature for 2 min. The resulting lysate

was subjected to centrifugation for 3 min at 13,200xg. The supernatant, avoiding genomic

DNA and any precipitate at the bottom of the tube, was transferred to another tube. To this

sample was added 1 volume of 70% ethanol, and the manufacturer’s protocol was then

followed.

The RNA was quantitated using a Nanodrop 1000 spectrophotometer (Thermo Fisher

Scientific, Wilmington, DE). 350 ng of RNA was used as a template for reverse transcription

with the Omniscript RT Kit plus RNase inhibitor (0.5U/µl), oligo-dT primers (0.825µM) and

random hexamers (11µM) (all reagents purchased from QIAGEN, Gaithersburg, MD) by

incubation at 37°C for 90 min in a Techne TC-512 thermal cycler (Burlington, NJ). cDNA

products were diluted six-fold in water. 2µl of this dilution was used as template for PCR

with the TaqMan Gene Expression Assay (Applied Biosystems, Carlsbad, CA) for 18s rRNA

(Hs99999901_s1) and 5µl of the dilution was used as template for PCR with the TaqMan

Gene Expression Assays for β-actin (Mm00607939_s1), rhodopsin (Mm00520345_m1) and

38  

Gnat1 (Mm00492388_g1) along with the TaqMan Universal PCR 2X Master Mix (Roche,

Indianapolis, IN).

PCR reactions were run in an ABI 7900HT sequence detection system (Applied Biosystems,

Carlsbad, CA) located in the Functional Genomics Core Facility of the Section of Research

Resources, Penn State College of Medicine using the following parameters: 50°C for 2 min,

95°C for 10 min then 40 cycles of 95°C for 15 s and 60°C for 1 min. Data was analyzed

using the ddCt method. Expression of each target gene was normalized to that of 18s rRNA.

Immunohistochemistry

Mice were anesthetized with an intraperitoneal injection of ketamine/xylazine (0.136 mg

ketamine + 0.0096 mg xylazine / g body weight) prior to sacrifice. Immediately following

decapitation by guillotine, globes were removed and placed in 2% paraformaldehyde for 10

minutes. Eyes were then blotted dry, submerged in Optimal Cutting Temperature Compound

(Tissue-Tek, Redding, CA) and lowered into isopentane on dry ice until frozen.

Paraformaldehyde-fixed, frozen mouse eyes were sectioned (10µ thick) and mounted on

Colorfrost/Plus microscope slides (Fisher Scientific, Fair Lawn, NJ). Antigen retrieval was

performed by incubating sections in 10mM citrate buffer (pH 6.0) at 80ºC for 30 minutes.

After cooling, the retina sections were incubated at room temperature for 1.5 hours in a

blocking agent of 10% donkey serum (Jackson ImmunoResearch, West Grove, PA) and 0.1%

Triton X-100 in phosphate-buffered saline (PBST). Tissue sections were then incubated in

primary antibody diluted in blocking agent at 4ºC overnight. Retina sections were labeled

with rabbit anti-cone arrestin polyclonal antibody (1:1000, Millipore, Billerica, MA), and

39  

mouse monoclonal antibody RET-P1 (1:500, Millipore, Billerica, MA). After overnight

incubation in the primary antibody, the sections were washed in PBST, counterstained with

Hoechst (0.5 µg/mL, Sigma-Aldrich, St. Louis, MO) and incubated in the following

immunofluorescent affinity purified secondary antibodies diluted in blocking agent: Cy2-

conjugated donkey anti-mouse (1:1000, Jackson ImmunoResearch, West Grove, PA) and

Cy3-conjugated donkey anti-rabbit (1:1000, Jackson ImmunoResearch, West Grove, PA).

Secondary antibodies were incubated for 1 hour in the dark at room temperature. Slides were

then washed in PBST, and coverslipped using Aqua Polymount (Polysciences, Warrington,

PA) mounting medium for confocal microscopy.

Confocal Microscopy

Fluorescently labeled slides were imaged with a laser confocal microscope (TCS SP2 AOBS,

Leica Microsystems, Manheim, Germany), utilizing a 405 nm laser for Hoechst (detection

range 410-480 nm), a 488 nm laser for Cy2 (detection range 493-530 nm), and a 543 nm

laser for Cy3 (detection range 548-630 nm). Z-section stacks of optical slices were taken

using either a 40× or 63× inverted oil objective lens and reconstructed, using the Leica

software, as average- and maximum projections, and saved in 512 × 512 pixel format (Figure

12).

Quantification of Immunofluorescence and Outer Nuclear Layer Cells

Quantification of relative rhodopsin and cone arrestin immunoreactivity was performed using

image analysis software (Image J, W. S. Rasband, public domain via U.S. National Institutes

of Health, Bethesda, MD, http://rsb.info.nih.gov/ij/, 1997-2010). Using average projections

40  

generated by confocal microscopy, a region of interest encompassing the area of

photoreceptor outer segments was selected with Image J, and average intensity recorded.

Background measurements from each sample were subtracted to obtain a measurement of

fluorescence, recorded in arbitrary units (AUs).

The cell bodies of the outer nuclear layer (ONL) were counted from confocal maximum

projection images. Using Image J to randomly outline 100 micron regions of central and

peripheral retina, cell counts were carried out in the ONL. 4 separate cell counts from the

central retina of each animal were averaged to determine the number of cells per 100 microns

of retina. The procedure was repeated for the peripheral retina in the ONL. The identity of

each slide was masked during cell counts.

Statistical Analysis

Statistical analyses were performed using Excel software (Microsoft Office 2010, Redmond,

Washington). Values from the 2 groups (diabetic and control) were analyzed using Student’s

two-tailed t-test to determine differences between groups. Error bars in all graphs are

expressed as mean ± standard error of the mean (S.E.M.). An asterisk (*) denotes

significance at p < 0.05.

41  

RESULTS

Immunoblot Analysis of Rod Outer Segment Proteins Rhodopsin, Transducin and

Phosphodiesterase

After normalizing to actin, the rhodopsin content was found to be reduced in the retinas from

11 week old diabetic mice, compared to age-matched control mice. Quantification of the blot

fluorescence indicated that rhodopsin content was decreased by 27 % in the Ins2Akita mice,

which had been hyperglycemic for 7 weeks (Figure 6).

Fig. 6. Diabetes reduces rhodopsin protein concentration in 11 week Ins2Akita mice. A) Retinas

from 11 week old Ins2Akita and control mice were immunoblotted for rhodopsin. B) Rhodopsin

protein content was significantly diminshed in diabetic mice, compared to age-matched controls. *p

< 0.05.

A

B

42  

An alternative method of calculating rhodopsin content involved summing the volumes of

bands representing the monomeric, dimeric, and trimeric forms of rhodopsin taken from

another set of 11 week old mice and then normalizing to actin. The results of this

quantification revealed a different outcome in which there was not a difference in summed

rhodopsin oligomer content in the diabetic mice, compared to controls (Figure 7).

43  

 

Fig. 7. Quantification of oligomers indicates that rhodopsin protein content in oligomer form is

not changed in diabetes. A) Retinas from 11 week old Ins2Akita and control mice were

immunoblotted for rhodopsin. Monomers, dimers, and trimers were summed and then normalized to

actin. B) Oligomeric rhodopsin protein content was unchanged in diabetic mice, compared to age-

matched controls.

A

B

44  

Transducin and phosphodiesterase, photoreceptor proteins involved in the phototransduction

cascade were also detected in retinas from 11 week old mice. Subsequent quantification of

these blots demonstrated that there was no difference between the diabetic and control

animals, with respect to transducin α (Figure 8) or phosphodiesterase 6 α (Figure 9) protein

content.

Fig. 8. Transducin protein content is not altered by diabetes in 11 week old mice. A) Retinas

from 11 week old Ins2Akita and control mice were immunoblotted for transducin α subunit. B) There

is no difference in transducin protein content in diabetic mice, compared to age-matched controls.

A

B

45  

Fig. 9. Phosphodiesterase protein content is not altered by diabetes in 11 week old mice.

A) Retinas from 11 week old Ins2Akita mice, and control mice were immunoblotted for the

phosphodiesterase 6 α subunit. B) There was no difference in phosphodiesterase protein content in

diabetic mice, compared to age-matched controls. PDE, phosphodiesterase.

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)

Messenger RNA (mRNA) expression in retinas from 14 week old mice was quantified by

qRT-PCR. β-actin transcript was used as an endogenous control, and remained unchanged

in samples from diabetic (-)INS, diabetic (+)INS, and controls (data not shown). Retinas

harvested from 14 week old mice demonstrated no significant difference in rhodopsin mRNA

expression levels in insulin-treated or untreated diabetic mice, relative to control levels.

There was also no difference in transducin mRNA expression measured in the two groups of

diabetic mice, compared to controls (Figure 10).

A

46  

    

Fig. 10. Photoreceptor mRNA expression was unchanged in mice that were diabetic for 10

weeks. A) mRNA expression levels for rhodopsin were not different in diabetic mice, compared to

age-matched controls, regardless of whether or not they received insulin treatment. B)

Quantification of mRNA levels of transducin reavealed no difference in mRNA expression in diabetic

animals, compared to controls. n=9 control, n=5 untreated diabetic, n=6 insulin-treated diabetic.

Quantification of Outer Nuclear Layer Cells

Quantification of cell bodies for photoreceptor cells found in the central and peripheral outer

nuclear layer yielded no difference between diabetic animals and age-matched controls

(Figure 11).

47  

Fig. 11. ONL cell counts are unaltered by diabetes in 11 week old mice. A) Cell counts from the

outer nuclear layer of 11 week old mice indicated that there was no difference between the numbers

of photoreceptor cell bodies due to diabetes in the central retina. B) Likewise, peripheral retinas from

diabetic mice were not different from controls with regard to ONL cell counts.

Rhodopsin Immunofluorescence

Another measure of protein content included in this study involved measuring the

fluorescence of cy-2 conjugated rhodopsin in images taken using confocal microscopy. The

fluorescence of all of the pixels in the region of the image corresponding to the outer

segments was averaged to gain an understanding of the amount of rhodopsin present in the

photoreceptor outer segments. In this small study, there was no difference in measured

fluorescence between the diabetic and control mice at this 11 week time point (Figure 12).

48  

A

Fig. 12. Immunofluorescent confocal image analysis to compare rhodopsin content in 11 week-

old Ins2Akita mouse retinas. Cy-2 conjugated rhodopsin is labeled in green, Cy-2 conjugated cone

arrestin is labeled in red, and nuclear DNA is counterstained with Hoechst, in blue. A) Control retina.

B) Diabetic retina. There was no difference in the level of fluorescence in Cy-2 conjugated rhodopsin

due to diabetes in either C) central retina or D) peripheral retina.

A B

49  

DISCUSSION

Neurodegeneration taking place due to diabetic retinopathy has been considered to be an

event that occurs primarily in the inner retina. Retinal thinning, indicative of neuronal loss,

has been identified in patients with diabetes 61, 104 and in animal models of diabetes 14, 60, 62.

Apoptotic markers have been identified in the ganglion cells of diabetic animals 18, 20, and

humans 17. Fewer investigations have explored the neurodegenerative effects of diabetes on

the outer retina. Postmortem human histological analysis of the outer retina has been limited

to donors with longstanding diabetes or patients who received laser photocoagulation

treatment 90, 91, 105. Park and colleagues published a descriptive account of photoreceptor loss

in STZ-induced diabetic rats 52, but included no statistical comparison to indicate the extent

of cell loss, nor did the histology indicate the area of retinas sampled. The present study

examined 11 week old diabetic Ins2Akita, and age-matched control mice, to determine the

effects of diabetes on the photoreceptor layer. Outer nuclear layer cell counts were

performed centrally and peripherally in order to determine possible photoreceptor

degeneration. Immunoblotting, immunohistochemistry, and qRT-PCR were used to measure

rhodopsin expression in order to estimate potential loss within the rod photoreceptor

population.

In this study, western blot analysis demonstrated that rhodopsin content was diminished in

Ins2Akita mouse retinas after 7 weeks of diabetes, compared to age-matched controls. This

finding was not able to be confirmed by immunohistochemistry, since fluorescence levels of

immunolabeled rhodopsin in diabetic samples were not different from control samples. This

discrepancy may be due to a very small sample size in the immunohistochemistry study (n =

50  

3), along with variability in fluorescence values. There appears to be a trend toward

decreased rhodopsin fluorescence in the peripheral diabetic samples, although this did not

reach statistical significance.

Transducin immunoblots indicate that transducin content in the diabetic retinas was not

different from levels seen in control samples, even as rhodopsin levels in the diabetic mice

were decreased. While the author was unable to find any other studies that used

immunoblotting to measure rhodopsin content in diabetic retinas, similar findings were

reported in BETA2/NeuroD1 Null mice. These mice serve as a model for Bardet-Biedl

syndrome, a genetic disorder characterized by rod-cone dystrophy and increased incidence of

diabetes. The BETA2/NeuroD1 Null mice also demonstrate a loss of rhodopsin in the

absence of transducin changes 106. In chapter 4 of this dissertation, ERG results demonstrate

that, at this same 11-week time point, there was no difference between diabetic and control

mice with regards to the rod-dominated a-wave. One might expect the a-wave to be

diminished in situations where there is a significant loss of rhodopsin. The fact that the a-

wave is unchanged in the rhodopsin-deficient mice can be explained by the amplification

properties of the phototransduction cascade. Since each rhodopsin molecule is able to

activate many transducin molecules, it is possible to lose a portion of the rhodopsin pool and

still maintain phototransduction, as long as transducin and PDE levels remain optimal.

ONL cell counts in 11 week-old diabetic mice were not different from values measured in

age-matched control mice. Other animal studies examining the ONL have had conflicting

results. The most frequently used method of detecting histologic change in the ONL is by

51  

thickness measurements. Some studies have measured loss in ONL thickness in diabetic

animals 18, 52, while others have found thinning of alternate retinal layers without ONL

change 14, 62.

The aim of this study was to test the hypothesis that diabetes leads to rod photoreceptor

degeneration. The original intent was to measure rhodopsin as an indicator of photoreceptor

number. It must be noted that the reduction in rhodopsin content seen in the diabetic mice

occurred after 7 weeks of diabetes while, during the same time period, there was no apparent

loss of photoreceptor cell bodies. Nor was there a loss of transducin or phosphodiesterase, so

the original hypothesis must be rejected. It appears that there is a loss of monomeric

rhodopsin content in diabetes, but this does not correlate with a change in mRNA expression

or a loss of photoreceptors. It is possible that this loss of rhodopsin is due to diabetes-

induced shortening of the outer segments, where the majority of rhodopsin is localized.

Outer segment shortening has been documented in diabetic rats 51 and in humans with

diabetic macular edema 107.

52  

CHAPTER FOUR

Electroretinographic Response to Diabetes in Ins2Akita Mice

53  

INTRODUCTION

Electroretinography (ERG) involves the use of a recording electrode placed on the cornea of

a subject, which measures the electrophysiologic output originating from the retina. The

signal that is recorded is the response to a light stimulus; usually a flash of light of short

duration and variable intensity. The resultant waveforms have been identified and the

component parts of the output have been assigned to the retinal cell types from which they

originate. Holmgren first demonstrated that there was a change in the electrical potential of

an eye due to light stimulation in 1865 108. Following the work of Ragnar Granit, who

performed ERG studies in anesthetized cats 108, further studies have been able to determine

the cellular layers responsible for the separate components of the ERG waveform. Modern

electroretinography is now widely used in clinical and research settings to identify

pathologies in many vision disorders. Patients with diabetes, for instance, frequently exhibit

reduced or delayed ERG responses. Animal research in ERG studies, particularly in the use

of mammalian models of disease, is important in the furthering of our understanding of

disease pathology, progression, and treatment options. Research related to the study of

diabetic retinopathy has largely been performed using the rat as a model. In these studies,

rats are often injected with streptozotocin (STZ), which is toxic to pancreatic beta cells.

Fewer studies have been performed in STZ- or alloxan- injected mice. Another mouse model

of diabetes is the Ins2Akita mouse, which has a spontaneous mutation leading to the

destruction of beta cells and subsequent diabetes. To date, there are no published reports of

ERG data from this Ins2Akita diabetic mouse model.

54  

The basic component parts of the ERG waveform are most frequently referred to as the a-

wave, which is a negative deflection from baseline, and a positively directed b-wave (Figure

13). Granit developed an alternate naming system, using Roman numerals to label the

cellular responses in the order in which they were suppressed by deeper levels of anesthesia.

Some researchers continue to refer to the P-I, P-II, and P-III nomenclature, but the majority

of published reports use the convention of a- and b-waves. The a-wave is the earliest

response to light stimulus and originates from the outer retina; specifically, the

photoreceptors. Following the a-wave, the post-photoreceptoral b-wave is produced by the

depolarization of bipolar cells, with contributions from amacrine cells and ganglion cells. Xu

and colleagues proposed that there is also a Muller cell contribution to the b-wave 109, but

studies by Karwoski and colleagues and Green and coworkers refute this claim 24, 25.

Fig. 13. ERG output demonstrates a- and b-wave in 11 week old mice. The photoreceptor-

dominated a-wave is seen as a negative trough, while the bipolar dominant b-wave exhibits a positive

peak. Data collected included amplitude, measured in microvolts (µV) and implicit time, measured in

milliseconds (ms).

b-wave

a-wave

55  

Oscillatory potentials (OPs) consist of a series of higher frequency wavelets that are

superimposed upon the rising slope of the b-wave. They are thought to originate from the

interactions of bipolar cells, amacrine cells, and ganglion cells 27, 28. Any disease process that

impacts these cell types is likely to affect the shape of the OP wavelets. In order to measure

the amplitude and implicit times (time from the onset of stimulus to the peak of each OP),

bandpass filters are used to isolate the OPs from the b-wave (Figure 14). Changes in OP

parameters are the most commonly cited alterations in ERG studies involving patients with

diabetes. Early clinical studies of patients with diabetes were concerned with OP changes

associated with diabetic retinopathy. In 1962, Yonemura and colleagues reported

deterioration of oscillatory potentials in patients with diabetes, most of whom had been

diagnosed with retinopathy, but also in a smaller number of patients without

ophthalmoscopic evidence of retinopathy 110. Additional clinical trials followed, establishing

that humans with diabetic retinopathy have specific alterations in ERG response, including

reduced OP amplitudes 111-113 and increased OP implicit times 112, 114. Juen and Kieselbach

noted that patients ages 18 – 33 had significant loss of OP amplitude after being diagnosed

with diabetes for an average of only 7 years, prior to the advent of any notable vascular

changes associated with diabetic retinopathy 111. Alterations in OPs have been correlated

with loss of visual acuity, hue discrimination, 112 and contrast sensitivity 113. Other early

studies showing that the electroretinogram was altered within a few years of the onset of

juvenile diabetes suggested that this could be used as an early diagnostic approach 115, 116.

56  

Fig. 14. Oscillatory potentials recorded in Ins2Akita mice are filtered using a bandpass filter to

obtain amplitude and implicit time measurements. A) Unfiltered waveform: note wavelets on

rising slope of the b-wave. B) OPs after filtration with a bandpass filter.

Alterations in a- and b- waves have been reported in patients with diabetes, but the results are

not as dramatic as those seen in OP changes. Bresnick and Palta recorded ERGs from 72

patients with diabetes and compared the data to recordings from 29 control subjects. They

found a significant delay in the implicit time of a-waves recorded from the patients with

diabetes. The same study also determined the effects of photocoagulation treatment on ERG

response and found that the eyes that received pan-retinal photocoagulation showed a

significant delay in the b-wave implicit time, compared to the untreated contralateral eye 114.

Chung and coworkers measured ERG responses in diabetic patients with and without

vascular signs of retinopathy. They reported decreased b-wave amplitudes at lower flash

intensities and delayed b-wave implicit times in all groups, even in patients without

retinopathy 117.

A B

57  

Additional waveforms have been described in the ERG field of study, such as the c-wave, the

d-wave, and the scotopic threshold response. The c-wave originates from the retinal

pigmented epithelium (RPE) with input from photoreceptors 118. The d-wave, a second

positive response following the b-wave, can only be elicited under special conditions with

longer flash exposures. The d-wave is a response to activation of the off-bipolar pathway 119.

While the c- and d- waves have not been addressed to a great extent in diabetic literature,

there have been a small number of studies published that utilize the scotopic threshold

response (STR) as a measure. The STR is a proximal retina waveform that is more sensitive

to low-level illumination than the b-wave and saturates at a lower light intensity 120. It can

only be elicited in subjects who have been dark adapted. In order to record the STR, dark-

adapted subjects are exposed to flashes of very low intensity, producing an inner retina

response (Figure 15). As flash stimuli increase in intensity, bipolar cell depolarization will

eventually lead to b-wave encroachment. Saszik and colleagues performed intravitreal γ-

aminobutyric acid (GABA) injections in mice to eliminate the STR in order to determine that

the b-wave encroachment is initiated at a flash intensity of -4.3 log sc td s, equivalent to

0.00005 c.s/m2 120. The positive component of the STR (pSTR) denotes ganglion cell

activity while the negative deflection (nSTR) is a combined response from ganglion cell and

amacrine cell activity 28, 121. Aylward recorded ERGs in 50 diabetic patients and 10

nondiabetic subjects, finding a correlation between severity of diabetic retinopathy and STR

abnormalities 30.

58  

Fig. 15. Scotopic Threshold Response is recorded in dark-adapted animals. The positive STR is

followed by the negative STR. Note that as the flash intensity grows brighter (0.0001 and 0.0005

c.s/m2), the pSTR is masked by the emerging b-wave.

Animal models provide useful insight into the normal and abnormal functions and

physiological processes that are common across species. ERG studies have been performed

in such mammalian species as primates, cats, guinea pigs, rats, and mice. Sieving and

colleagues performed pioneering work in isolating and understanding the STR by recording

ERGs in dark-adapted cats 122. Saszik and coworkers further defined our understanding of

the STR waveform using C57/BL6 mice 120. Mice have been utilized for years in

electroretinography studies as a way to learn more about the electrophysiology of vision 120,

123, 124. For instance, Lei and colleagues studied rhodopsin knockout (rho-/-) mice and cone

photoreceptor function loss 1 (cpfl1) mice to determine differences in OP results based on

pure cone and pure rod pathways 125.

59  

ERG studies in diabetic animal models have been carried out largely in streptozotocin-

induced diabetic rats 31, 35, 38, 75-78. Several studies reported a reduction of the a-wave

amplitude by 12 weeks after the onset of diabetes, suggesting loss of photoreceptor function

35, 75, 93. Additionally, Hancock and Kraft found a delay in the a-wave implicit time 76, a

result which had also been reported in humans with diabetes 114, 126. Animal studies and

clinical trials show differing results with regards to b-wave response to diabetes. Animal

studies consistently demonstrate a decrease in b-wave amplitude by 12 weeks after the onset

of diabetes 38, 76, 77. Phipps and coworkers recorded decreased b-wave amplitudes as early as

2 days after STZ injection 75. The same study also determined that there was no change in b-

wave latency in the diabetic rat model, a finding that was replicated in another animal study

31. In clinical trials of humans with diabetes, however, it is an increase in b-wave latency that

has been shown to change with diabetic retinopathy 41, 117. Taken together the results of many

ERG studies provide evidence of loss of function in photoreceptors, amacrine cells, bipolar

and horizontal cells. Potential explanations for this include reduction in the actual cell

numbers, or functional deficits in neurotransmission between neurons.

In addition to the diabetic rat studies, there have been a small number of published reports

that used diabetic mice to determine the effects of diabetes on retinal electrophysiology.

Muriach and colleagues found a decrease in b-wave amplitude in alloxan-induced diabetic

mice 40. In a report by Johnsen-Soriano and coworkers, the b-wave amplitude was measured

as a way to assess the effectiveness of a proposed treatment. The b-wave amplitude was

restored to normal values in diabetic mice after 3 weeks of treatment using lipoic acid 39.

While these are important discoveries, there is another mouse model of diabetes that has not

60  

been represented in the ERG literature. The Ins2Akita mouse does not require the introduction

of toxins to induce a diabetic state and has been validated as a useful animal in modeling the

characteristics of diabetic retinopathy 19, 20, 60, 62. The Ins2Akita mouse contains a point

mutation in the Ins2 gene that prevents disulfide bonding of the proinsulin A- and B- chains.

Without the formation of these disulfide bonds, the subsequent misfolded proteins

accumulate in the pancreas, leading to eventual beta cell apoptosis. Beta cell death results in

hypoinsulinemia and hyperglycemia, making the Ins2Akita mouse a useful model for the

spontaneous onset of diabetes in humans 62, 103, but there are, as yet no published reports of

ERG data from Ins2Akita mice. The aim of this study was to test the hypothesis that Ins2Akita

mice exhibit ERG abnormalities similar to those seen in human diabetic retinopathy.

EXPERIMENTAL PROCEDURES

Animals

Heterozygous C57BL/6J Ins2Akita mice were bred and maintained at the Penn State College

of Medicine Juvenile Diabetes Research Foundation Diabetic Retinopathy Center Animal

Facilities Core. Ins2Akita mice have a spontaneous mutation in the Ins2 gene that prevents

disulfide bonds from forming, leading to protein misfolding, endoplasmic reticulum stress,

and eventual pancreatic beta cell death, rendering the animals hypoinsulinemic and

hyperglycemic at 4 weeks of age. In the present study, data was collected from 11- and 14-

week old mice that had been diabetic for 7 and 10 weeks, respectively. Female Ins2Akita mice

demonstrate later onset and a lesser degree of hyperglycemia. 103. For this reason, only male

61  

mice were used in the present study. Animals were housed in the Penn State College of

Medicine animal facility in a standard 12 hour light/dark cycle with littermates in plastic

cages, with ad libitum access to food and water in accordance with the Institutional Animal

Care and Use Committee guidelines, standards set forth in the ARVO Statement of the Use

of Animals in Ophthalmic and Vision Research, and the NIH Guidelines for the Care and

Use of Laboratory Animals.

Diabetic animals receiving insulin treatment were anesthetized with an intraperitoneal

injection of ketamine/xylazine (0.072 mg ketamine, (Phoenix Pharmaceuticals, St. Joseph,

MO), and 0.005 mg xylazine, (Vedco, St. Joseph, MO) /g body weight). While under

anesthesia, the external surface of the animal’s abdomen was prepared with povidone- iodine

antiseptic solution (Betadine, Purdue Pharma, Stamford, CT). Two 13 mg insulin implants

(LinBit for Mice, Sustained Release Insulin Implants; LinShin Canada, Inc., Toronto,

Ontario) were inserted subcutaneously into the flank region, using a12-gauge trocar. These

insulin pellets have a release rate of 0.1 U/day/implant over a period of 30 days.

Mice were dark adapted overnight in a ventilated, light-proof chamber with unlimited access

to food and water. All subsequent work involved in preparation for ERG testing was

performed under red L.E.D. illumination, as mice do not have long wavelength sensitive

cones capable of detecting red light. ERGs were recorded using the Espion2 system

(Diagnosys LLC, Lowell, MA). The mice were anesthetized with an intraperitoneal injection

of ketamine/xylazine (0.072 mg ketamine, (Phoenix Pharmaceuticals, St. Joseph, MO), and

0.005 mg xylazine (Vedco, St. Joseph, MO) /g body weight). Whiskers were trimmed to

62  

enable free access to the animal’s cornea and both eyes were dilated with Atropine sulfate

1% (Bauch & Lomb, Tampa, FL) and Tropicamide 1% (Wilson Ophthalmic, Mustang, OK).

The mouse was then placed on Espion2’s warming platform which maintains the animal’s

body temperature during set-up and recording. A platinum needle ground electrode (Grass

Technologies, West Warwick, RI) was placed subcutaneously between the animal’s scapulae.

A negative electrode was placed either medial to the eye being tested using a platinum

needle, or in the ipsilateral cheek, using a copper-plated clip electrode. A contact lens-style

recording electrode (LKC, Gaithersburg, MD) was filled with conductive lubricating gel

(GenTeal, Novartis, East Hanover, NJ) and placed over the eye to be tested. In all cases save

one, the left eye was tested. The exception involved injury to the left eye and the right eye

was used for recordings. The contralateral eye was treated with the same lubricating gel to

prevent corneal drying during the time the animal was under anesthesia. The animal was

positioned on the warming platform, on his side with the recording electrode oriented upward

toward the Ganzfeld stimulator, which was lowered into place over the animal.

The ERG flash protocol consisted of a stepwise progression of full field flashes, beginning

with very dim (subthreshold) stimuli, and progressing to greater flash energies. All stimuli

presented to the animals were composed of broad spectrum white light. Lower intensity

flashes were produced by L.E.D., while brighter flash intensities were produced by xenon

bulb. Following each of the brightest steps, there was a dark adaptation period of 1 to 5

minutes, to allow the animal to recover from photobleaching in preparation for the next

stimulus. Oscillatory potentials were filtered with a bandpass filter of 10-1000 Hz.

Responses to the stimuli were recorded from a number of consecutive sweeps, (50 sweeps at

63  

the lowest flash intensities, fewer at the higher intensities) and averaged for a final output.

Amplitudes and implicit times were collected for each of the following ERG waveform

components: positive scotopic threshold (pSTR), negative scotopic threshold (nSTR), a-

wave, b-wave, and oscillatory potentials (OPs). OP results were summed in a manner

previously described by Hancock and Kraft 76.

RESULTS

Animal Data

The control animals continued to gain mass throughout the study. Diabetic animals failed to

gain mass at the same rate as controls, and weighed significantly less that controls at 11- and

14 weeks of age. Blood glucose averages in control mice remained below 250 mg/dL

throughout testing. Untreated diabetic mice averaged 795 mg/dL at 11 weeks and 823 mg/dL

at 14 weeks. Animals that received insulin implants began to gain weight, but did not attain

masses seen in controls. Insulin restored blood glucose levels to near-euglycemic ranges,

significantly lower than those recorded in the untreated animals (Table 3).

 

64  

 

11 weeks 14 weeks

n= Mass (g) Blood glucose (mg/dL)

n= Mass (g) Blood glucose (mg/dL)

Control 20 28±2.6a 227±76 b 10 31±2.9c,d 209±49e

Diabetic (-) insulin

19 25±2.2a 795±144b 6 24±3.4c 823±108e

Diabetic (+) insulin

- - - 6 26±1.5d 297±122

Table 3. Mass and blood glucose levels measured in diabetic and control mice at 11 weeks and

14 weeks of age. Half of the diabetic mice in the 14 week-old group received insulin implants to

lower their blood glucose levels. ap < .0001, bp < .0001, cp < .005, dp < .005, ep < .0001

Positive Scotopic Threshold Response

11 week old dark adapted Ins2Akita mice exposed to very dim (scotopic) flash intensities

exhibited a significantly diminished amplitude response, compared to age-matched control

mice. At flash intensities of 0.00001 c.s/m2 and 0.00005 c.s/m2, the average amplitudes were

5.28(±1.00) µv and 16.52(±2.05) µv, respectively, in diabetic mice; compared to 9.64(±0.87)

µv and 26.08(±2.59) µv in controls. There were significant differences in the results at

0.0001 c.s/m2 and 0.0005 c.s/m2 as well, although at these brighter flash intensities, the

bipolar cell response was encroaching so that the ganglion cell dominant pSTR became lost

in the emerging b-wave (Figure 16 A). The pSTR implicit times for the diabetic mice,

however, were not different from those of the control mice (Figure 16 B).

65  

 

Fig. 16 Positive scotopic threshold response for 11 week old Ins2Akita mice. A) pSTR amplitude

was diminished at all scotopic flash intensities in diabetic mice compared to age-matched control.

Flash intensities of 0.00001 and 0.00005 c.s/m2 are considered to be in the scotopic range. The higher

flash intensities presented here mark the first positive peak, but will also include bipolar cell signal

from the encroaching b-wave. B) There was no difference between groups with regard to pSTR

implicit times. * p < 0.05

66  

Negative Scotopic Threshold Response

The nSTR measured in 11 week old mice showed no difference in amplitude or implicit time

(Figures 17 A and 17 B). At higher flash intensities, above the scotopic range, there was a

significant difference in amplitude, with diabetic responses diminished in comparison with

age-matched controls (Figure 17 A)

67  

Fig. 17. Negative STR is unchanged in diabetic mice. A) nSTR amplitude measurements were not

different through the scotopic stimulus range. There was a significantly reduced amplitude in the

diabetic mice at a flash intensity of 0.0005 c.s/m2, but this was outside the scotopic range. B) There

was no difference between the diabetic and control groups with regard to nSTR implicit times across

any of the tested flash intensities.

68  

a-Wave

11 week old diabetic mice showed no difference in a-wave amplitudes, with any of the tested

flash intensities, compared to age-matched controls (Figure 18 A). While the lower intensity

flashes trended toward delayed a-wave implicit times in the diabetic animals, this difference

did not reach significance until the flash intensity reached 1.0 c.s/m2. Interestingly, at 10

c.s/m2, the a-wave trough was significantly delayed in the control animals (Figure 18 B).

69  

 

Fig. 18. ERG a-wave recordings for 11 week old Ins2Akita mice are largely unchanged with diabetes. A) There were no differences between diabetic and control mice with regards to a-wave amplitude. B) At two flash intensities, 1.0 and 10.0 c.s/m2, there were statistically significant differences in implicit time. At the lower flash intensity, recordings from diabetic animals were delayed, but at the next higher flash intensity, the control mice exhibited delayed a-waves.

70  

b-Wave

At 11 weeks of age, the b-waves recorded from diabetic mice were not different in amplitude

from the control mice (Figure 19 A). Furthermore, the b-wave implicit times were not

significantly different in the diabetic and control mice, except at one flash intensity: 1.0

c.s/m2 (Figure 19 B).

71  

 

Fig. 19. ERG b-wave response is largely unchanged by 11weeks age. A) b-wave amplitude was

unchanged at all flash intensities tested. B) The control mice demonstrated a significant b-wave delay

at a single flash intensity, 1.0 c.s/m2.

B

72  

Oscillatory Potentials

OPs were filtered with a bandpass filter and OPs 1 through 4 were recorded and summed to

determine both amplitude and implicit times. The summed OP amplitudes were not

significantly different between the two groups. At all flash intensities, the diabetic mice had

lower average summed amplitudes, but this trend did not reach significance. (Figure 20 A).

The lower flash intensities resulted in a delay in the implicit times of summed OPs in the

diabetic mice compared to the age-matched controls. Significant delays were recorded at

0.001, 0.01, and 0.1 c.s/m2 (Figure 20 B).

73  

 

Fig. 20. Oscillatory potentials are significantly delayed in 11 week old diabetic mice.

A) Summed oscillatory potential amplitudes were not significantly reduced in diabetic mice. B) At

the lower flash intensities, however, summed oscillatory potentials were significantly delayed in the

diabetic animals, compared to age-matched controls. * p < 0.05

74  

A subset of the diabetic Ins2Akita mice were further divided into untreated (DM – insulin) and

insulin treated (DM + insulin). The insulin treated group received subcutaneous sustained-

release insulin implants in order to normalize their blood glucose values. When the animals

reached 14 weeks of age (diabetic for 10 weeks), ERG protocols were repeated.

Positive Scotopic Threshold Response

One week after the subcutaneous insulin was implanted into the diabetic mice, dark adapted

animals were tested using the previously outlined ERG protocol. ERGs were recorded using

the dimmest flash stimuli capable of eliciting responses, resulting in scotopic threshold

responses. The averaged pSTR responses at scotopic flash intensities failed to demonstrate

any differences between the three age-matched groups: control, untreated diabetic mice, and

insulin-treated diabetic mice, with regard to pSTR amplitude (Figure 21 A), or implicit time

(Figure 21 B).

75  

 

Fig. 21. There is no difference in pSTR between controls, treated-, or untreated- diabetic mice

at 14 weeks. ERGs recorded were 1 week after 6 of the diabetic mice received subcutaneous insulin

implants. A) There was not a significant difference between the groups with regard to pSTR

amplitude. B) pSTR implicit time was also not different between the three groups.

76  

Negative Scotopic Threshold Response

Stimulus flashes of scotopic intensity yielded highly variable nSTR amplitude results. The

amplitudes recorded for the control mice were not significantly different from either the

treated or untreated diabetic groups (Figure 22 A). There was also no difference between the

three groups’ nSTR implicit times (Figure 22 B).

77  

 

Fig. 22. There is no difference in nSTR between controls, treated-, or untreated- diabetic mice

at 14 weeks. A) There was not a significant difference between the groups with regard to pSTR

amplitude. B) pSTR implicit time was also not different between the three groups.

A

B

78  

a-Wave

Similar to the results from the 11 week-old mice, there were no differences in average a-

wave amplitudes between diabetic and control mice at 14 weeks of age. Furthermore, the a-

wave amplitude did not vary significantly due to the presence or absence of insulin in the

diabetic animals (Figure 23 A). The averaged implicit times of the a-waves at each flash

intensity also demonstrated no difference between groups, although there is an interesting

trend in the results recorded at 10 c.s/m2, wherein the untreated diabetic animals exhibited an

earlier a-wave trough than controls (though this did not reach significance as it did at the

same flash intensity in the 11 week-old mice), and the insulin-treated diabetic animals had a-

wave implicit values almost identical to control values (Figure 23 B).

79  

 

Fig. 23. There is no difference in a-wave amplitude or implicit time measurements, when

comparing 14 week old controls, treated-, and untreated- diabetic mice. A) a-wave amplitudes

are not different across the three groups. B) a-wave implicit times are not different across the three

groups.

80  

b-Wave

The b-wave amplitude values for 14 week-old mice were largely unchanged compared to the

11 week-old values, and there was no difference in amplitude averages when compared

across the 3 groups (Figure 24 A). Implicit times recorded in the 14 week-old mice reveal

that, at all flash intensities, the untreated animals exhibited a decrease in average implicit

time in the untreated diabetic group, compared to age-matched controls. This difference was

significant at all flash intensities except for 0.5 and 1 c.s/m2. At all flash intensities, the

average implicit times for the treated diabetic mice were statistically similar to the control

values (Figure 24 B).

81  

Fig. 24. Untreated 14 week old diabetic mice demonstrate reduced b-wave implicit times.

A) There was no difference in amplitude between groups at any of the flash intensities tested. B) The

uncontrolled diabetic mice had significantly reduced implicit times compared to age-matched controls

at stimulus intensities of 0.001, 0.005, 0.01, 0.05, 0.1, and 10 c.s/m2. The insulin-treated diabetic

mice had implicit times that were not different from age-matched controls at all flash intensities.

B

82  

Oscillatory Potentials

The OPs recorded from 14 week-old mice did not result in a significant difference between

untreated diabetic mice, insulin-treated diabetic, or control mice. There were no differences

in summed OP amplitudes (Figure 25 A), or summed implicit times (Figure 25 B).

83  

 

Fig. 25. ERG oscillatory potentials for 14 week old Ins2Akita mice do not demonstrate

differences between controls, and treated, or untreated diabetic animals. A) Summed oscillatory

potential amplitudes are not significantly different from each other in the three groups. B) Summed

oscillatory potential implicit times are also not significantly different from each other in the three

groups.

84  

DISCUSSION

Electroretinography is an important research tool in the field of vision research. The

component parts of the resulting waveform can be useful in determining the cellular origin of

electrophysiological dysfunction. Neurodegeneration, such as that seen in diabetic

retinopathy, can be correlated with changes in specific aspects of ERG waveforms. This

study used the Ins2Akita diabetic mouse as a model for diabetic retinopathy in an effort to

study the early effects of diabetes on the retina and the role that insulin plays in changes to

ERG responses. In this study, the author tested the hypothesis that ERG alterations measured

in humans with diabetes can be demonstrated in similar results from Ins2Akita mice.

The present study found disturbances in the ERG results from 11 week-old diabetic mice

(duration of diabetes, 7 weeks) compared to age-matched nondiabetic counterparts. ERG

results from diabetic mice demonstrated delayed OPs and diminished pSTR compared to

control values. The delay in OP implicit time that was noted in the diabetic mice indicates

pathology localized to the inner retina. At this early time point, the mice did not show any

significant differences in a- or b-wave responses. At 14 weeks of age (10 weeks diabetic),

untreated diabetic mice demonstrated a significantly reduced b-wave implicit time, compared

to age-matched controls and diabetic mice that had received insulin implants. At this later

time point, the differences in OP latency and pSTR amplitude were lost. This is likely due to

a smaller number of animal subjects leading to the study being underpowered. Power

analyses were performed to determine the number of mice that would be necessary to

determine a difference between the groups. Results indicated that the required sample size

85  

would have needed to be 23 to 27 mice for the STR experiments and 8 to 21 mice for the OP

study, to demonstrate a difference (Chang Bioscience calculator, Castro Valley, CA).

The Ins2Akita mouse has been used previously as a valuable model of diabetic retinopathy, but

this is the first time, to the author’s knowledge, that this mouse has been used to study the

effects of diabetes on ERG results. While this is an important first step, there are some

limitations to the present study. As this was the first ERG study performed using Ins2Akita

mice, the protocol was designed to study all of the major waveforms across a broad range of

flash intensities. Responses were recorded under scotopic environments, with pauses built

into the protocol, after flash stimuli, to allow the animal to readapt to a darkened

environment. Time constraints limited the number of steps of increasing flash intensities that

could be included in the protocol, in order to finish testing within a period of time wherein

the animals remained under anesthesia. This made it necessary to increase the luminance of

the flash stimuli fairly quickly over a limited number of steps. This was most problematic

during the STR testing. The results indicate that in recordings resulting from flash stimuli of

0.0001 c.s/m2 and greater, the emerging b-wave was interfering with the STR. It would have

been preferable to have recorded STRs at a greater number of the intervening flash intensities

between 0.00001 and 0.0001 c.s/m2 in order to more narrowly define the differences in STRs

from diabetic and control animals. Another limitation of this study is the small number of

subjects used in the insulin treatment experiment. By dividing the number of diabetic

animals into two groups (untreated and insulin treated), the author hoped to determine

whether or not insulin could rescue diminished or delayed ERG results seen in diabetic

86  

animals. Unfortunately, halving the number of diabetic animals left the study underpowered

and did not provide an adequate sample size to offset the variation in the results.

Clinical studies in humans with diabetes have demonstrated findings similar to those shown

here, including changes in OP implicit time and diminished STR amplitude. The Pardue lab

found OP results very similar to those seen in this study. Specifically, they found that

dimmer flash intensities were more sensitive at detecting OP changes in diabetes. At lower

flash energies (below current standards set by the International Society for Clinical

Electrophysiology of Vision), ERG responses were able to uncover evidence of pathology

not discernable at higher flash intensities 127. These findings are paralleled in the current

study, in which OP delays were evident in the diabetic mice at flash intensities of 0.001,

0.01, and 0.1 c.s/m2 after being diabetic for only 7 weeks. Brighter stimuli, however, failed

to show a difference in OP implicit time values.

There have not been a large number of clinical studies focusing on the scotopic threshold

response in humans with diabetes. Aylward noted a correlation between diminished STR

amplitude and severity of diabetic retinopathy 30. Graham and Vaegan reported similar

findings in the ERGs derived from diabetic subjects 121. Kaneko and colleagues studied

patients with type II diabetes who had maintained tight glycemic control and who

demonstrated no vascular retinal abnormalities. These patients had STR values that were not

different from age-matched control subjects 128. It is interesting to note that these studies in

human scotopic ERG responses only measured the negative portion of the STR (nSTR). A

more recent study in STZ-induced diabetic rats was able to uncover the smaller positive

87  

deflection (pSTR) that precedes the nSTR. Similar to the findings presented here, Kohzaki

and coworkers found that the pSTR amplitude was reduced in rats 4, 8, and 11 weeks after

onset of diabetes. These changes in pSTR were evident before the onset of any nSTR

changes and before the onset of changes in STR implicit times 31. The fact that the ganglion

cell-dominated pSTR is affected prior to the onset of nSTR changes in the work by Kohzaki

and coworkers, and in the present study, may indicate pathology specific to the ganglion

cells. The nSTR is thought to be the response from not only ganglion cells, but also includes

amacrine cell function. While it is known that amacrine cells are lost by apoptosis in

diabetes 20, and that this occurs within the same timeframe as ganglion cell loss 19, the

magnitude of this loss may not have been sufficient at this early time point to affect the

nSTR.

88  

CHAPTER FIVE

Concluding Discussion

89  

This dissertation serves as a means to explore the neurodegenerative effects of diabetes in the

retina, with an emphasis on learning more about the earliest alterations that take place in the

disease progression. To that end, the author made use of laboratory techniques involving

immunohistochemistry, western blotting, histologic staining, microscopy, and

electroretinography. These assays yielded results which uncovered alterations in protein

expression, cell population, and electrophysiology, all of which occur in response to early

diabetic changes in the retina. The human retinas that were studied came from donors who

had had type II diabetes for less than 10 years. These were compared to retinas from non-

diabetic humans of similar ages. The animal model used was the Ins2Akita diabetic mouse.

These mice were 11-14 weeks old, which means that they had hyperglycemia for only 7 to 10

weeks. This is an important distinction, as human studies frequently examine subjects with

advanced stages of proliferative diabetic retinopathy; and animal studies are often conducted

in diabetic animals that have had diabetes for an extended period of time. The use of

younger mice not only models the earliest diabetes-induced changes, but also eliminates any

age-related confounding variables.

The human histopathology study presented in chapter 2 establishes the loss of peripheral rod

photoreceptors from donors with a short duration of diabetes, compared with age-matched

non-diabetic donors. While this photoreceptor degeneration is supported by others, no

definitive mechanism has been confirmed by which photoreceptor loss takes place. It is well

established that photoreceptors are responsible for the extremely high metabolic demands of

the retina 129. In cases wherein the retina is compromised, such as ischemic conditions,

retinal tissue may not be able to meet those metabolic demands, leading to eventual neuronal

90  

loss 130. Furthermore, hypoxic conditions lead to increased expression of VEGF which

signals neovascularization 131. Dr. Geoffrey Arden has proposed the possibility that the

metabolic demands of the rods in the diabetic retina are the cause of diabetic retinopathy; that

the oxygen demands of supporting a full complement of photoreceptors will, in certain

situations (such as dark adaptation), create a hypoxic situation in the retina. Hypoxia then

induces VEGF-induced neovascularization. He surveyed a small population of patients with

both retinitis pigmentosa, a rod-degenerating disease, and diabetes, and found that these

patients did not develop the vascular signs of diabetic retinopathy. A disease process like

retinitis pigmentosa would lead to progressive destruction and removal of rod photoreceptors.

Photoreceptor outer segment ion channels are normally held open as their default position in

the dark. The ion pumps, located in the inner segment, require considerable energy to

maintain this so-called “dark current.” As the photoreceptor population decreases in retinitis

pigmentosa, so would the oxygen demands, thereby preventing diabetic retinopathy 86. The

assumptions of Dr. Arden’s ideas as to the causative factors in diabetic retinopathy have not

been rigorously tested, but it is interesting to consider the possibility that the loss of rod

photoreceptors in diabetes may serve a protective role by lessening the metabolic demands of

the retina.

The rhodopsin content study, presented in chapter 3 demonstrates, by immunoblot analysis,

diabetes-induced reduction of rhodopsin in retinas harvested from Ins2Akita mice, compared to

age-matched controls. This represents a 27% decline in rhodopsin content, within a span of 7

weeks of hyperglycemia. While the reasons for this loss of rhodopsin are unknown,

possibilities include the loss of photoreceptor number or a decrease in outer segment length.

91  

ONL cell quantification (Figure 11) did not find a difference in the number of ONL cell

bodies with diabetes. This may represent a case in which there is a difference in disease

progression in the animal model compared to human pathology, or these results may reflect

the fact that this data was collected from a small sample of mice. Photoreceptor outer

segment length was not measured in this study, but diminished outer segment length has been

demonstrated previously in diabetes 51, 107.

The ERG evidence presented in chapter 4 establishes the Ins2Akita mouse as useful in

modeling certain aspects of diabetic electrophysiology. After only 7 weeks of

hyperglycemia, these diabetic mice demonstrate defects in ERG responses to stimuli

compared to their age-matched counterparts. ERG recordings from dark adapted diabetic

mice had a significantly diminished scotopic threshold response. Oscillatory potentials were

delayed in these Ins2Akita mice as well. These changes are similar to some of the early

changes seen in ERG recordings from humans with diabetes. It will be important to repeat

the insulin treatment studies in the future, with an increased sample size, in order to

determine if insulin is able to rescue the ERG waveforms that were reduced or delayed in this

study.

The reduction in the positive scotopic threshold response in the Ins2Akita mice indicates

pathology in the retinal ganglion cell population. This further supports the many

neurodegeneration studies that have found increased ganglion cell apoptosis in diabetes. The

present ERG study also found delays in the oscillatory potentials of diabetic animals, which

may be the result of the loss of synaptic connections within the inner plexiform layer. Taken

92  

together, the results of the ERG study point to inner retinal neurodegeneration in diabetes.

This is in contrast to the findings in the rhodopsin content study, which demonstrate a

reduction of rhodopsin, an outer retina protein, in diabetic mice. The rhodopsin content was

diminished without a concomitant loss of transducin or phosphodiesterase, two other

photoreceptor proteins. This suggests that rhodopsin content may be reduced without the

loss of the photoreceptor cells that contain these proteins. In humans, however, the rod

photoreceptor is the very cell type which was implicated in chapter 2. There, it was

determined that, in humans with a short duration of diabetes, rod photoreceptors were

reduced in number compared with cell counts from age-matched donors. There are obvious

similarities and differences in the results from these studies, emphasizing the importance of

animal models in research as a way to study certain aspects of a disease. Care must be taken,

however, to refrain from interpreting the results from one model and imposing those

interpretations too broadly. There are findings that are presented here, that may be useful to

gain a better understanding of diabetic retinopathy. The histologic evidence from human

donors that rod photoreceptors are preferentially lost early in diabetes is an important aspect

of diabetes that has not been explored in the literature.

While the experimental results presented in this dissertation address some of the current gaps

in knowledge regarding diabetic retinopathy, they also point to other unanswered questions.

Future experiments need to be designed to better understand the photoreceptor loss that was

measured in diabetic human donor tissue. Such work would benefit from the use of

additional donor eyes, and may concentrate on donors with different durations of diabetes.

Comparisons also need to include eyes from donors with type I diabetes. It would also be

93  

helpful if donor eyes could be matched with more complete donor information such as

HbA1c levels and clinical notes regarding retinopathy status. Future histologic studies of

human retinas might also benefit from horizontal sectioning, parallel to a line through the

fovea and optic disc, in order to better localize photoreceptor loss. Improved fixation and

camera technology may enable accurate rod photoreceptor counts.

Additional experiments need to be designed in order to further explore the diabetes-induced

changes that were documented in the Ins2Akita mice. Why is the protein content of the

monomeric form of rhodopsin diminished in diabetic mice, while the total protein content

and mRNA expression of rhodopsin and transducin are unaltered? Electroretinographic

responses in the current study also leave unanswered questions. Future ERG studies will

require an increased number of animal subjects to detect the effects of insulin on the various

recorded waveforms. Paired-flash ERGs will provide more insight into dark-adaptation

deficiencies. Additional scotopic flash intensities need to be added to the ERG protocol to

collect important information related to the scotopic threshold response. These and other

experiments will add to the understanding of the neurodegenerative changes that take place

in diabetic retinopathy.

This dissertation reports experimental results that establish and confirm diabetes-induced

neurodegeneration in the retinas of humans and spontaneously diabetic animals. The

collection of these data may improve our understanding of early retinal responses to diabetes.

Such understanding will enable clinicians to have a more complete picture of the pathology

and progression of diabetic retinopathy, allowing for them to better educate and treat their

94  

patients. It may prove therapeutic, based on the results presented here, to develop treatment

options that target the outer retina in an effort to reduce or delay the loss of rod

photoreceptors in patients with diabetes. The rhodopsin results reported here may serve to

offer some good news to patients with diabetes, as the data suggest that there is redundancy

built into the phototransduction cascade. Although rhodopsin content is diminished in the

diabetic mice, the nature of signal amplification is such that, downstream signaling

compensates for this loss, demonstrated by the fact that transducin and phosphodiesterase

levels remained unchanged as did the photoreceptor-dominant a-wave. The ERG evidence of

diabetes-induced neurodegeneration provides further evidence of the usefulness of the

Ins2Akita mouse as a model for diabetic retinopathy. The alterations in waveforms are very

similar to changes seen in humans with diabetes, and thus may prove useful in developing

treatment options targeted to the inner retina cell types responsible for the abnormal ERG

results demonstrated here.

95  

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VITA

William F. Robinson, Jr. Education 2010 Ph.D. candidate in Anatomy

Pennsylvania State University, College of Medicine, Hershey, PA 2001 Doctor of Physical Therapy; graduation 2000, DPT awarded 2001

Belmont University, Nashville, TN Thesis: The Effects of Cryotherapy on Balance Strategy Selection

1992 Bachelor of Science in Biology; cum laude

Belmont University, Nashville, TN Awards and Honors 1. Student-Fellow Research Award from Penn State Hershey Eye Center Vision Research

Day; ($500 prize), June 2010. 2. Sigma-Aldrich 2009 Histotechnology Stain Contest Winner; 1st Place ($200 prize),

October 2009. http://www.sigmaaldrich.com/life-science/cell-biology/hematology-and-histology/learning-center/2009-stain-winners.html

3. Recipient of Life Care Scholarship, Belmont University Physical Therapy Program,

1999. 4. Graduated Cum Laude, Belmont University, 1992. 5. Recipient of Schering-Plough Scholarship, Belmont University, 1991. Publications in Preparation 1. Robinson, W.F., Mohr, S., Barber, A.J. (2010) Peripheral rod photoreceptors are

depleted in postmortem human retinas after a short duration of diabetes. In preparation.

2. Stevenson, J., Lam, J., Robinson, W.F., Hauck, R. (2010) Capitate morphology: radiographic evaluation with routine wrist films. In preparation.

3. Barber, A.J., Robinson, W.F., Jackson, G.R. (2010) Neurodegeneration in Diabetic

Retinopathy. In: Visual Dysfunction in Diabetes: The Science of Patient Impairment and Improvement. Humana Press. In press.