the PI3kinase-Akt pathway in STZ-diabetic rat retinas, accompanied by reduced insulin receptor kinase activity [126]. Systemic administration of IGF-1 was found to reduce the amount of apoptosis measured by TUNEL and caspase-3 activity in STZ rats, suggesting that increased growth factor signaling may protect the retina [127].

A less widely considered explanation of neuronal cell death and dysfunction is a change in the way intracellular calcium concentration is regulated. Calcium is an especially potent signal in neurons, responsible for initiating many metabolic events, including plastic changes at the synapse [128, 129]. Some in vitro studies indicate that elevated levels of glucose augmented the intracellular calcium response to membrane depolarization [130] (Fig. 6).

SUMMARY AND CONCLUSIONS

Diabetic retinopathy is considered to be a vascular disease of the retina, because clinically identifiable signs of the disease include vascular lesions such as microaneurysms and loss of the blood-retinal barrier leading to macular edema (nonproliferative stage). Later in the disease, there can be vascular proliferation and ischemia (proliferative stage) resulting in profound vision loss, although progression to this stage is less common [131, 132]. Clinical detection of diabetic retinopathy is almost exclusively through recognition of the vascular indications of the disease. These symptoms are accompanied by loss of visual acuity [133], and the patient usually recognizes the effects of the disease as a reduction in quality of life due to gradual deterioration of functional vision [134]. There is little doubt that diabetes reduces the ability of the retina to function correctly, but retinal function is difficult to measure in the clinic, so the fundus examination is regarded as the standard method to diagnose and map the progress of diabetic retinopathy. The gradual loss of retinal structure and function can, however, be interpreted as the most basic indication that neurodegeneration of the retina, leading to compromised visual function, is a prevalent component of diabetic retinopathy. Future advances in diagnosis and treatment of diabetic retinopathy will likely include consideration of this important aspect of the disease.

Fig. 6. (continued) calcium. The live cells were imaged by confocal microscopy during membrane depolarization by addition of 20 mM KCl. (A) Five seconds of baseline images of cells were recorded, followed by depolarization with 20 mM KCl. (B) In control cells with 5 mM glucose, the intracellular fluorescence increased transiently and returned almost to baseline within 65 s. (C) Cells grown with 20 mM glucose had baseline fluorescence similar to control cells. (D) Cells grown with 20 mM glucose displayed a more dramatic increase in fluorescence in response to KCl, and this did not return to baseline within 65 s. (E) Relative quantification of whole cell fluorescence (cytoplasmic and nuclear), by digital image analysis, indicated that there was a significant increase in calcium-induced fluorescence in the cells grown with 20 mM glucose compared to those grown with 5 mM glucose. Addition of mannitol did not alter the calcium response compared to the control cells, indicating that the effect was not due to osmotic changes in the media (*p < 0.05). Similar results were obtained from primary cultures of retinal cells. Taken from Santiago et al. [130].

REFERENCES

1. Przedborski S, Vila M, et al. Neurodegeneration: what is it and where are we? J Clin Invest. 2003;111(1):3–10.

2. Wolter JR. Diabetic retinopathy. Am J Ophthalmol. 1961;51:1123–39. 3. Bloodworth Jr JM. Diabetic retinopathy. Diabetes. 1962;11:1–22.

4. Bresnick GH. Diabetic retinopathy viewed as a neurosensory disorder. Arch Ophthalmol. 1986;104:989–90.

5. Schmidt D. The mystery of cotton-wool spots—a review of recent and historical descriptions. Eur J Med Res. 2008;13(6):231–66.

6. Roy MS, Rick ME, et al. Retinal cotton-wool spots: an early finding in diabetic retinopathy? Br J Ophthalmol. 1986;70(10):772–8.

7. Kern TS, Barber AJ. Retinal ganglion cells in diabetes. J Physiol. 2008;586(Pt 18):4401–8. 8. Iseki S. DNA strand breaks in rat tissues as detected by in situ nick translation. Exp Cell

Res. 1986;167(2):311–26.

9. Gavrieli Y, Sherman Y, et al. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol. 1992;119(3):493–501.

10. Wijsman JH, Jonker RR, et al. A new method to detect apoptosis in paraffin sections: in situ end-labeling of fragmented DNA. J Histochem Cytochem. 1993;41(1):7–12.

11. Hammes HP, Federoff HJ, et al. Nerve growth factor prevents both neuroretinal programmed cell death and capillary pathology in experimental diabetes. Mol Med. 1995;1(5):527–34.

12. Mizutani M, Kern TS, et al. Accelerated death of retinal microvascular cells in human and experimental diabetic retinopathy. J Clin Invest. 1996;97(12):2883–90.

13. Kern TS, Tang J, et al. Response of capillary cell death to aminoguanidine predicts the development of retinopathy: comparison of diabetes and galactosemia. Invest Ophthalmol Vis Sci. 2000;41(12):3972–8.

14. Kowluru RA, Odenbach S. Role of interleukin-1beta in the development of retinopathy in rats: effect of antioxidants. Invest Ophthalmol Vis Sci. 2004;45(11):4161–6.

15. Sugiyama T, Kobayashi M, et al. Enhancement of P2X(7)-induced pore formation and apoptosis: an early effect of diabetes on the retinal microvasculature. Invest Ophthalmol Vis Sci. 2004;45(3):1026–32.

16. Barber AJ, Lieth E, et al. Neural apoptosis in the retina during experimental and human diabetes. Early onset and effect of insulin. J Clin Invest. 1998;102(4):783–91.

17. Martin PM, Roon P, et al. Death of retinal neurons in streptozotocin-induced diabetic mice. Invest Ophthalmol Vis Sci. 2004;45(9):3330–6.

18. Ning X, Baoyu Q, et al. Neuro-optic cell apoptosis and microangiopathy in KKAY mouse retina. Int J Mol Med. 2004;13(1):87–92.

19. Gastinger MJ, Singh RS, et al. Loss of cholinergic and dopaminergic amacrine cells in streptozotocin-diabetic rat and Ins2Akita-diabetic mouse retinas. Invest Ophthalmol Vis Sci. 2006;47(7):3143–50.

20. Park SH, Park JW, et al. Apoptotic death of photoreceptors in the streptozotocin-induced diabetic rat retina. Diabetologia. 2003;46(9):1260–8.

21. Feit-Leichman RA, Kinouchi R, et al. Vascular damage in a mouse model of diabetic retinopathy: relation to neuronal and glial changes. Invest Ophthalmol Vis Sci. 2005;46(11): 4281–7.

22. Kanamori A, Nakamura M, et al. Diabetes has an additive effect on neural apoptosis in rat retina with chronically elevated intraocular pressure. Curr Eye Res. 2004;28(1):47–54.

23. Srinivasan A, Roth KA, et al. In situ immunodetection of activated caspase-3 in apoptotic neurons in the developing nervous system. Cell Death Differ. 1998;5(12):1004–16.

46. Lopes de Faria JM, Russ H, et al. Retinal nerve fibre layer loss in patients with type 1 diabetes mellitus without retinopathy. Br J Ophthalmol. 2002;86(7):725–8.

47. Takahashi H, Goto T, et al. Diabetes-associated retinal nerve fiber damage evaluated with scanning laser polarimetry [see comment]. Am J Ophthalmol. 2006;142(1):88–94.

48. Lim MC, Tanimoto SA, et al. Effect of diabetic retinopathy and panretinal photocoagulation on retinal nerve fiber layer and optic nerve appearance. Arch Ophthalmol. 2009;127(7):857–62.

49. Scott TM, Foote J, et al. Vascular and neural changes in the rat optic nerve following induction of diabetes with streptozotocin. J Anat. 1986;144:145–52.

50. Mizutani M, Gerhardinger C, et al. Muller cell changes in human diabetic retinopathy. Diabetes. 1998;47(3):445–9.

51. Mohr S, Xi X, et al. Caspase activation in retinas of diabetic and galactosemic mice and diabetic patients. Diabetes. 2002;51(4):1172–9.

52. Kowluru RA, Koppolu P. Diabetes-induced activation of caspase-3 in retina: effect of antioxidant therapy. Free Radic Res. 2002;36(9):993–9.

53. Krady JK, Basu A, et al. Minocycline reduces proinflammatory cytokine expression, microglial activation, and caspase-3 activation in a rodent model of diabetic retinopathy. Diabetes. 2005;54(5):1559–65.

54. Vincent JA, Mohr S. Inhibition of caspase-1/interleukin-1beta signaling prevents degeneration of retinal capillaries in diabetes and galactosemia. Diabetes. 2007;56(1):224–30.

55. Kern TS, Miller CM, et al. Topical administration of nepafenac inhibits diabetes-induced retinal microvascular disease and underlying abnormalities of retinal metabolism and physiology. Diabetes. 2007;56(2):373–9.

56. Vanguilder HD, Brucklacher RM, et al. Diabetes downregulates presynaptic proteins and reduces basal synapsin I phosphorylation in rat retina. Eur J Neurosci. 2008;28(1):1–11.

57. Kurihara T, Ozawa Y, et al. Angiotensin II type 1 receptor signaling contributes to synaptophysin degradation and neuronal dysfunction in the diabetic retina. Diabetes. 2008;57(8):2191–8.

58. do Carmo A, Lopes C, et al. Nitric oxide synthase activity and L-arginine metabolism in the retinas from streptozotocin-induced diabetic rats. Gen Pharmacol. 1998;30(3):319–24.

59. Roufail E, Soulis T, et al. Depletion of nitric oxide synthase-containing neurons in the diabetic retina: reversal by aminoguanidine. Diabetologia. 1998;41(12):1419–25.

60. Giove TJ, Deshpande MM, et al. Increased neuronal nitric oxide synthase activity in retinal neurons in early diabetic retinopathy. Mol Vis. 2009;15:2249–58.

61. Yu HM, Xu J, et al. Coupling between neuronal nitric oxide synthase and glutamate receptor 6-mediated c-Jun N-terminal kinase signaling pathway via S-nitrosylation contributes to ischemia neuronal death. Neuroscience. 2008;155(4):1120–32.

62. Leist M, Nicotera P. Apoptosis, excitotoxicity, and neuropathology. Exp Cell Res. 1998;239(2):183–201.

63. Cogen J, Cohen-Cory S. Nitric oxide modulates retinal ganglion cell axon arbor remodeling in vivo. J Neurobiol. 2000;45(2):120–33.

64. Green DG, Kapousta-Bruneau NV. A dissection of the electroretinogram from the isolated rat retina with microelectrodes and drugs. Vis Neurosci. 1999;16(4):727–41.

65. Karwoski CJ, Xu X. Current source-density analysis of light-evoked field potentials in rabbit retina. Vis Neurosci. 1999;16(2):369–77.

66. Sieving PA, Murayama K, et al. Push-pull model of the primate photopic electroretinogram: a role for hyperpolarizing neurons in shaping the b-wave. Vis Neurosci. 1994;11(3):519–32.

67. Wachtmeister L. Oscillatory potentials in the retina: what do they reveal. Prog Retin Eye Res. 1998;17(4):485–521.

208 Barber et al.

91. Zhang L, Inoue M, et al. Alterations in retrograde axonal transport in optic nerve of type I and type II diabetic rats. Kobe J Med Sci. 1998;44(5–6):205–15.

92. Zhang LX, Ino-ue M, et al. Retrograde axonal transport impairment of largeand mediumsized retinal ganglion cells in diabetic rat. Curr Eye Res. 2000;20(2):131–6.

93. Ino-Ue M, Zhang L, et al. Polyol metabolism of retrograde axonal transport in diabetic rat large optic nerve fiber. Invest Ophthalmol Vis Sci. 2000;41(13):4055–8.

94. Sima AA, Zhang WX, et al. Impaired visual evoked potential and primary axonopathy of the optic nerve in the diabetic BB/W-rat. Diabetologia. 1992;35(7):602–7.

95. Papakostopoulos D, Hart JC, et al. The scotopic electroretinogram to blue flashes and pattern reversal visual evoked potentials in insulin dependent diabetes. Int J Psychophysiol. 1996;21(1):33–43.

96. Lopes de Faria JM, Katsumi O, et al. Neurovisual abnormalities preceding the retinopathy in patients with long-term type 1 diabetes mellitus. Graefes Arch Clin Exp Ophthalmol. 2001;239(9):643–8.

97. Klein R. Age-related eye disease, visual impairment, and driving in the elderly. Hum Factors. 1991;33(5):521–5.

98. Bailey CC, Sparrow JM. Visual symptomatology in patients with sight-threatening diabetic retinopathy. Diabet Med. 2001;18(11):883–8.

99. Sokol S, Moskowitz A, et al. Contrast sensitivity in diabetics with and without background retinopathy. Arch Ophthalmol. 1985;103(1):51–4.

100. Di Leo MA, Caputo S, et al. Nonselective loss of contrast sensitivity in visual system testing in early type I diabetes. Diabetes Care. 1992;15(5):620–5.

101. Bangstad HJ, Brinchmann-Hansen O, et al. Impaired contrast sensitivity in adolescents and young type 1 (insulin-dependent) diabetic patients with microalbuminuria. Acta Ophthalmol. 1994;72(6):668–73.

102. Dosso AA, Yenice-Ustun F, et al. Contrast sensitivity in obese dyslipidemic patients with insulin resistance. Arch Ophthalmol. 1998;116(10):1316–20.

103. Roy MS, Gunkel RD, et al. Color vision defects in early diabetic retinopathy. Arch Ophthalmol. 1986;104(2):225–8.

104. Cho NC, Poulsen GL, et al. Selective loss of S-cones in diabetic retinopathy. Arch Ophthalmol. 2000;118(10):1393–400.

105. Daley ML, Watzke RC, et al. Early loss of blue-sensitive color vision in patients with type I diabetes. Diabetes Care. 1987;10(6):777–81.

106. Rockett M, Anderle D, et al. Blue-yellow vision deficits in patients with diabetes. West J Med. 1987;146(4):431–3.

107. Ong GL, Ripley LG, et al. Assessment of colour vision as a screening test for sight threatening diabetic retinopathy before loss of vision. Br J Ophthalmol. 2003;87(6):747–52.

108. Moss SE, Klein R, et al. The 14-year incidence of visual loss in a diabetic population. Ophthalmology. 1998;105(6):998–1003.

109. Arend O, Remky A, et al. Contrast sensitivity loss is coupled with capillary dropout in patients with diabetes. Invest Ophthalmol Vis Sci. 1997;38(9):1819–24.

110. Arden GB. The absence of diabetic retinopathy in patients with retinitis pigmentosa: implications for pathophysiology and possible treatment. Br J Ophthalmol. 2001;85(3):366–70.

111. de Gooyer TE, Stevenson KA, et al. Retinopathy is reduced during experimental diabetes in a mouse model of outer retinal degeneration. Invest Ophthalmol Vis Sci. 2006;47(12): 5561–8.

112. Barber AJ. A new view of diabetic retinopathy: a neurodegenerative disease of the eye. Prog Neuropsychopharmacol Biol Psychiatry. 2003;27(2):283–90.

113. Ambati J, Chalam KV, et al. Elevated gamma-aminobutyric acid, glutamate, and vascular endothelial growth factor levels in the vitreous of patients with proliferative diabetic retinopathy. Arch Ophthalmol. 1997;115(9):1161–6.

114. Lieth E, Barber AJ, et al. Glial reactivity and impaired glutamate metabolism in short-term experimental diabetic retinopathy. Diabetes. 1998;47(5):815–20.

115. Kowluru RA, Engerman RL, et al. Retinal glutamate in diabetes and effect of antioxidants. Neurochem Int. 2001;38(5):385–90.

116. Ng YK, Zeng XX, et al. Expression of glutamate receptors and calcium-binding proteins in the retina of streptozotocin-induced diabetic rats. Brain Res. 2004;1018(1):66–72.

117. Lieth E, LaNoue KF, et al. Diabetes reduces glutamate oxidation and glutamine synthesis in the retina. Exp Eye Res. 2000;70(6):723–30.

118. Puro DG. Diabetes-induced dysfunction of retinal Muller cells. Trans Am Ophthalmol Soc. 2002;100:339–52.

119. Ward MM, Jobling AI, et al. Glutamate uptake in retinal glial cells during diabetes. Diabetologia. 2005;48(2):351–60.

120. Santiago AR, Hughes JM, et al. Diabetes changes ionotropic glutamate receptor subunit expression level in the human retina. Brain Res. 2008;1198:153–9.

121. Santiago AR, Gaspar JM, et al. Diabetes changes the levels of ionotropic glutamate receptors in the rat retina. Mol Vis. 2009;15:1620–30.

122. Schmidt AM, Yan SD, et al. Activation of receptor for advanced glycation end products: a mechanism for chronic vascular dysfunction in diabetic vasculopathy and atherosclerosis [review] [89 refs]. Circ Res. 1999;84(5):489–97.

123. Tilton RG, Chang K, et al. Prevention of diabetic vascular dysfunction by guanidines. Inhibition of nitric oxide synthase versus advanced glycation end-product formation. Diabetes. 1993;42(2):221–32.

124. Kern TS, Engerman RL. Pharmacological inhibition of diabetic retinopathy: aminoguanidine and aspirin. Diabetes. 2001;50(7):1636–42.

125. Nitta A, Murai R, et al. Diabetic neuropathies in brain are induced by deficiency of BDNF. Neurotoxicol Teratol. 2002;24(5):695–701.

126. Reiter CE, Wu X, et al. Diabetes reduces basal retinal insulin receptor signaling: reversal with systemic and local insulin. Diabetes. 2006;55(4):1148–56.

127. Seigel GM, Lupien SB, et al. Systemic IGF-I treatment inhibits cell death in diabetic rat retina. J Diabetes Complications. 2006;20(3):196–204.

128. Verkhratsky A. Physiology and pathophysiology of the calcium store in the endoplasmic reticulum of neurons. Physiol Rev. 2005;85(1):201–79.

129. Verkhratsky A, Shmigol A. Calcium-induced calcium release in neurones. Cell Calcium. 1996;19(1):1–14.

130. Santiago AR, Rosa SC, et al. Elevated glucose changes the expression of ionotropic glutamate receptor subunits and impairs calcium homeostasis in retinal neural cells. Invest Ophthalmol Vis Sci. 2006;47(9):4130–7.

131. Bloodworth Jr JM, Molitor DL. Ultrastructural aspects of human and canine diabetic retinopathy. Invest Ophthalmol. 1965;4(6):1037–48.

132. Aiello LP, Gardner TW, et al. Diabetic retinopathy. Diabetes Care. 1998;21(1):143–56.

133.Moss SE, Klein R, et al. The incidence of vision loss in a diabetic population. Ophthalmology. 1988;95(10):1340–8.

134. Association AD. Economic costs of diabetes in the U.S. in 2007. Diabetes Care. 2008;31(3):596–615.