activity was increased in the retinas of alloxan-diabetic rats after 14 months, but not 2 months, of hyperglycemia [52]. In rats after 3 months of STZ diabetes, the increased caspase-3 activity was reversed by the anti-inflammatory drug, minocycline, suggesting the possibility that caspase-3 dependent apoptosis is due to an inflammatory signal [53]. Minocycline also reduced caspase-1 activity in STZ-diabetic mouse retinas [54]. Further evidence of a link between inflammatory signaling and caspase enzyme activation is provided by a study with nepafenac, a COX-1/-2 inhibitor, given topically to the eye. In this study, the anti-inflammatory treatment inhibited the increase in caspases-3 and -6 after 9 months of diabetes [55].

While the evidence for increases in apoptosis-associated enzymes is compelling, the cell types in which these changes take place are not easily determined. It is arguable that these changes occur in vascular cells as well as, or to the exclusion of, neurons. Other biochemical evidence for changes in neurons comes from measurements of synapse-specific proteins such as postsynaptic density 95 (PSD95), and synaptic vesicleassociated proteins such as synaptophysin. The retinal content of several synaptic proteins was found to be decreased after the first month of hyperglycemia in STZ-diabetic rats [56] (Fig. 5). These changes were accompanied by a further depletion in the content of phosphorylated synapsin 1, suggesting a reduction in the mobilization of neurotransmitter vesicles. Interestingly, the content reduction in synaptophysin was reversed by angiotensin II receptor blockers [57].

Other biochemical changes that could be associated with neurodegeneration include increases in nNOS, which increased in both protein content and activity in retinas from STZ rats [58]. It was proposed that nNOS provided a regulatory link between neurons and vascular blood flow and that the number of nNOS-positive neurons was depleted by diabetes [59]. A similar study confirmed the increase in nNOS expression and identified multiple subtypes of nNOS-containing neurons, including amacrine, bipolar, and horizontal cells, that were damaged by diabetes [28, 32]. Elevated levels of nNOS are accompanied by increases in the production of nitric oxide, especially in the plexiform layers, measured by a novel in situ immunohistochemical imaging technique [60]. Elevated levels of nitric oxide could have a dramatic influence on neuronal function, including altered glutamate receptor signaling [61], increased peroxynitrite production associated with excitotoxicity [62], and altering RGC axon morphology [63].

FUNCTIONAL EVIDENCE OF NEURODEGENERATIVE CHANGES

There is an abundant variety of electrophysiological studies indicating that diabetes induces functional changes in the retina. Many of these studies will be reviewed elsewhere in this volume; however, some electrophysiological studies specifically indicate a neurodegenerative mechanism.

Electrophysiological Evidence for Neurodegeneration

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

Fig. 5. Diabetes decreased the content of synaptic proteins in rat retinas. Synaptic proteins were quantified by western blot in the retinal homogenates from STZ-diabetic and control rats after 1 and 3 months of hyperglycemia. (A) Protein bands were apparent at the predicted molecular weight for each synaptic protein, and band densities were standardized to b-actin in the same sample. (B) Relative protein content was obtained as % control. There was a significant reduction in each of the proteins measured in the retinas from STZ-diabetic rats (n = 8 per group, *p < 0.05, **p < 0.01, *p < 0.001). Taken from Vanguilder et al. [56].

different cell types from the neural retina. Immediately following light stimulus, the a-wave is a negative deflection produced by the photoreceptors. The postreceptor b-wave response is a large positive deflection originating primarily from the ON-center bipolar cells [64, 65] modified by input from OFF-center bipolar and horizontal cells [66]. The oscillatory potentials (OPs) are small, higher frequency wavelets on the ascending portion of the b-wave, are thought to represent the modulation of interactions between bipolar, amacrine, and ganglion cells [67, 68], and are often analyzed in clinical and research studies of diabetic retinopathy.

Clinical studies of patients with diabetes were concerned with OP changes associated with diabetic retinopathy. In 1962, Yonemura et al. reported deterioration of oscillatory potentials not only 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 [69]. Additional clinical studies followed, establishing that humans with diabetic retinopathy have specific alterations in ERG response, including reduced OP amplitude [70–72] and increased OP latency [71]. Juen and Kieselbach noted that patients of 18–33 years old 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 [72]. Alterations in OPs have been correlated with loss of visual acuity, hue discrimination [71, 73], and contrast sensitivity [70]. Other studies showing that the ERG was altered within a few years of the onset of juvenile diabetes suggested that this could be used as an early diagnostic approach [74, 75].

Much of the research into the effects of diabetes on the a-wave has been performed in the STZ-diabetic rat model. Several studies reported a reduction of the a-wave amplitude by 12 weeks after the onset of diabetes, suggesting loss of photoreceptor function [76–78]. Hancock and Kraft also found a delay in the a-wave implicit time [79], a result which had also been reported in humans with diabetes [71, 80]. Animal studies consistently demonstrate a decrease in b-wave amplitude by 12 weeks after the onset of diabetes [79, 81, 82]. Phipps et al. recorded decreased b-wave amplitudes as early as 2 days after STZ injection [77]. 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 [83]. The b-wave latency is increased in patients with diabetic retinopathy [84, 85]. Taken together, the results of many ERG studies provide evidence of loss of function in photoreceptors, amacrine cells, bipolar and horizontal cells. The mechanism for these electrophysiological changes is unclear. The small amounts of cell death are unlikely to give rise to these large changes in the electrophysiological output of the retina. A more likely possibility is that changes in the amount of neurotransmitter release, or transmembrane ionic currents could account for the electrophysiological deficits.

The scotopic threshold response (STR) is another component of the ERG, considered to be an indicator of RGC function [68, 86, 87]. While this response is often not measured, because it can only be determined in response to very low intensity flashes of light, there is good evidence that it is reduced in both humans and rats with diabetes [83, 88, 89]. Furthermore, electrophysiological ganglion cell function was found to be compromised even in children with diabetes [90]. These data provide functional evidence of diabetes-induced RGC degeneration.

Optic Nerve Retrograde Transport

Several studies have determined that diabetes causes functional reductions in retrograde transport along the optic nerve. Retrograde axonal transport of fluorogold into medium and large RGCs was reduced in STZ-diabetic rats (but not a type II animal model) [91, 92]. The effect was reduced by treating rats with an aldose reductase inhibitor, suggesting that the loss of function in diabetes may be due to activation of the polyol pathway [93]. Loss of the visually evoked potential, accompanied by optic nerve pathology, in spontaneously diabetic BB/W rats is a further indication that optic nerve function is compromised by diabetes [94].

Other Changes in Visual Function

There have been a variety of studies applying psychophysical testing on humans with diabetes, recording a number of deficits that could be explained by altered neural function or neurodegeneration. A study of visual evoked potential, a measure of the visual cortex response to a flash of light, found that the evoked response was reduced and delayed in juvenile patients with diabetes [95]. Furthermore, the evoked response to stimuli with low contrast sensitivity was reduced more than stimuli with greater contrast sensitivity in patients with type 1 diabetes but no evidence of vascular retinopathy [96].

Reduced night vision is often associated with the early stages on diabetic retinopathy [97]. Loss of night vision may be associated with reduced contrast sensitivity and prolonged dark adaptation, and patients with maculopathy are often aware of peripheral field defects and color vision abnormalities [98].

Contrast sensitivity has also been studied extensively in diabetic patients. In a larger study, a group of non-insulin-dependent diabetic subjects with minimal visible fundus signs of diabetic retinopathy had abnormal contrast sensitivity at one or more spatial frequencies [99]. In another study of type 1 diabetic subjects with no retinopathy, there was a reduction in contrast sensitivity at multiple spatial frequencies between 1.0 and 9.6 cycles/degree [100]. A similar study indicated that presence of microalbuminuria predicted a reduction in contrast sensitivity in type 1 patients [101]. Subjects with insulin resistance and dyslipidemia also have significant reductions in mesopic and low photopic contrast sensitivity, suggesting that this loss of function is not limited to those with severe insulin-dependent diabetes [102].

Color vision defects can also occur in humans with diabetes [103]. A histological study on postmortem retinas found a selective reduction in the number of S-cones in samples from donors with diabetes, possibly by apoptosis [104], which could explain the tritan color confusion and loss of sensitivity to blue light that is known to occur in diabetic retinopathy [105–107].

The studies on visual function in humans with diabetes indicate that there are specific deficits that are measurable early on in the course of the disease, often in the absence of gross vascular defects evident by fundus examination. These data suggest that changes in neural function begin early in diabetes. It is important that we examine the cellular substrate for various elements of vision, such as contrast sensitivity, dark adaptation, and color contrast, in order to develop better ways to protect vision in diabetes. In order to develop better treatments for neurodegenerative changes in the retina, a number of theories for the causative mechanisms have evolved, which we will attempt to summarize next.

POTENTIAL MECHANISMS OF RETINAL

NEURODEGENERATION IN DIABETES

The relationship between vascular permeability and retinal neurodegeneration in diabetes is still unclear. It is reasonable to assume, however, that a breach in the blood-retinal barrier will give rise to local changes in neural function that could result in necrosis or apoptosis of neurons. There is a clinical link between macular edema and loss of visual

acuity, although correlations with more sensitive measures of visual function have not been attempted [108]. Equally, reductions in contrast sensitivity have been correlated with reductions in capillary density, as an index of ischemia in the retina [109].

Related to the concept that the neural retina is compromised by ischemia is the proposal that the retina becomes hypoxic in diabetes. One proposed mechanism for hypoxia is that poor blood flow to the inner retina, in concert with the heavy metabolic demand from photoreceptors under dark-adapted conditions, leads to tissue oxygen depletion. This is based on observations that diabetic retinopathy is limited in situations where the photoreceptors are lost, like retinitis pigmentosa or in animal models such as the rhodopsin knockout mouse (Rho−/−) [110, 111].

Glutamate excitotoxicity is a commonly considered mechanism for many diseases involving neurodegeneration and has been suggested to occur in diabetes [112]. GABA and glutamate levels were increased in vitreous of 22 patients with proliferative diabetic retinopathy, compared to a similar set of nondiabetic patients who had pars plana vitrectomy [113]. Similar increases have been measured in rats [114, 115]. Elevated concentrations of glutamate and GABA increased immunoreactivity for glutamate receptors NMDA and GluR2/3, accompanied by increased expression of calcium-binding proteins calbindin and parvalbumin in ganglion, amacrine, and bipolar cells [116]. Furthermore, glutamate oxidation was 62% less than controls in retina explants from STZ-diabetic rats, related to the reduction in the activity and content of glutamine synthetase, suggesting a reduced ability to process glutamate in the retina [117]. Reductions in the uptake rate of glutamate into Müller cells have also been measured [118, 119], along with alterations in the expression of some glutamate receptor subunits [120, 121]. The weak NMDA receptor antagonist has been reported to correct electroretinographic changes, prevent loss of RGCs, and reduce the amount of retinal vascular permeability in diabetic Brown-Norway rats, suggesting that this class of drugs may represent a useful therapeutic to prevent loss of function in diabetic retinopathy [37]. There is an intimate relationship between oxidative stress, nitric oxide toxicity, and glutamate excitotoxicity, and diabetes may induce all these biochemical processes in the retina [115].

The role of advanced glycation end-products (AGEs) in diabetic complications and retinopathy in particular has been discussed widely, especially since the AGE receptor was discovered [122]. The specific effect of AGEs on neurons in the retina has not been as well defined. Many studies have shown that treatment with aminoguanidine, an inhibitor of AGE formation, can rescue the vascular changes in diabetes [13, 123, 124]. This drug also reduced the loss of nNOS-containing neurons in STZ rats [59]; however, it failed to improve the abnormal ERG response in diabetic rats [76]. It may be that the effect of AGEs in neurons is indirect, acting by inducing an inflammatory response in glial cells and the vasculature [29].

Another mechanism that may be responsible for pathological changes to neurons in diabetes is loss of growth factor signaling, either through reduction in abundance of the growth factors or through loss of receptor sensitivity and second messenger signaling. BDNF was depleted from both brain and retina of diabetic rats [31, 125]. Loss of tyrosine hydroxylase-positive amacrine cells was prevented by injection of exogenous BDNF in rats [31]. There are also reductions in the kinase activity of components of