Ординатура / Офтальмология / Учебные материалы / Retinal Vascular Disease Joussen Springer

6.2The Phenotype of the Retinal Vasculature Is Determined by the Tissue It Serves

It is generally accepted that the development and maintenance of blood vessels are directed by local signals from the tissues they perfuse. This is particularly the case of the central nervous system, in which the vascular endothelial cells express proteins that form specialized tight junctions, which render the blood vessels impermeable to water and solutes in the lumen [93]. This is achieved by the expression of tight junction proteins such as occludin, claudins, zonula occludins, and several others, which make a tight seal between vascular endothelial cells [10, 25, 103]. (For more information on tight junctions see Chapter 8.1, this volume.) The vascular endothelial cells also express many unique transporters that regulate the entry of other substances, such as amino acids, into the sensitive neural parenchyma, and express unique basement membrane characteristics [14, 75] (Fig. 6.1). Together these properties establish physiological boundaries, termed the blood-brain and blood-retina barrier [26, 27].

The unique phenotype of the vasculature in the brain and retina is induced by the neural tissue. In a classic study on blood-brain barrier development, Stewart and Wiley demonstrated that transplanting avascular tissue from neonatal quail brain into the coelomic cavity of chick embryos causes the endothelial cells that grow into the quail neural tissue and adopt blood-brain barrier properties, becoming less

permeable to circulating dye. Conversely, chick brain vessels lose their barrier properties after growing I 6 into quail coelomic tissue grafts [94]. It is now widely accepted that the signals required to induce the bar-

rier phenotype in vascular endothelial cells of the central nervous system are derived from the glial cells, although the specific identity of these signals is still unclear [49, 89, 101].

The retinal vasculature has a similar phenotype to that of the brain [9]. The components comprising the vascular endothelial basement membrane in the retina and brain are also similar [14], as is the neural source of the barrier phenotype. For example, retinal Müller cells injected into the iris of rat eyes causes the vasculature in that region to adopt barrier properties [100]. Also, culture media from astrocytes increase the expression of the tight junction protein, ZO-1, in primary bovine retinal endothelial cells [35, 36]. These data suggest that glia manufacture a soluble factor that induces the expression of the features of the blood-retina barrier in vascular endothelial cells.

6.3Diabetes Causes a Loss of the Blood-Retina Barrier Phenotype

It is well established that diabetes increases the permeability of the vasculature in the retina [28]. The most direct method that has been used to measure retinal vascular permeability is by imaging fluorescein leaking from the blood vessels into the vitreous and retinal tissue [31, 51, 60]. Other approaches that have been used to study this phenomenon in animal models include measuring the leak of radiolabeled tracers, Evans blue dye, and albumin [83, 106, 116]. Immunohistochemical approaches in specimens from both humans and rats with diabetes have also identified regions around major blood vessels that contain more albumin, suggesting increased permeability to protein as well as water and small molecules [107, 108, 109]. This increase occurs soon after the onset of diabetes, beginning in the larger superficial vessels and spreading to the capillary bed [13, 80]. This suggests that diabetes induces a progressive loss of barrier properties in the retinal vasculature.

Fig. 6.1. Basement membrane components contribute to a unique phenotype of vascular barriers. Sections from rat brain and retina were labeled by immunohistochemistry for von Willebrand factor and agrin. a von Willebrand factor is abundant in all blood vessels and appears as a punctuate cytoplasmic antigen. b The same vessel was also labeled for agrin, a basement membrane glycoprotein that is only expressed on vessels in tissue that have a blood-tissue barrier. The unique properties of vessels that form blood-tissue barriers are induced by signals from the tissue that they serve. (From Fig. 1 in [14]. Reproduced with permission of Developmental Dynamics)

6.4The Increase in Vascular Permeability Is Likely Due to an Increase in the Expression of VEGF

The evidence currently suggests that the primary factor increasing blood-retina barrier permeability in diabetes is the potent growth and proliferation protein, vascular endothelial growth factor (VEGF). VEGF-induced increases in retinal vascular permeability occur through regulation of vascular endothelial tight junction proteins [9]. VEGF content is

increased in the vitreous of patients with prolifera- 6 I tive diabetic retinopathy [4, 6]. Similarly, diabetes increases the expression of both VEGF mRNA and protein in STZ-diabetic rat retina within as little as 1 week of the onset of diabetes, correlating with increases in vascular permeability that can be prevented by treatment with VEGF Trap, a high-affinity VEGF receptor blocker [67]. This suggests that VEGF is a primary inducer of retina vascular permeability

in diabetes.

6.5VEGF Originates from the Neural Tissue of the Retina in Diabetes

The primary source of retinal VEGF in diabetes remains to be identified, but recent research utilizing immunofluorescent microscopy indicates that this growth factor originates from specific cell types within the neural retina. The ganglion cells and amacrine cells appear to be potent sources of VEGF, which has also been detected in the nerve fiber layer, the retinal pigment epithelial cell layer, the outer plexiform layer, the photoreceptor layer and the inner retina [34]. VEGF content is known to be increased in diabetic retinopathy and in many co-morbid complications including ischemia and oxidative stress [32, 72]. Immunohistochemical studies indicate that neurons and glial cells are the main source of VEGF in diabetes in both humans and rodent models [7, 110]. VEGF protein detected by immunohistochemistry appears to accumulate at the vascular endothelial cell walls [61]. mRNA transcript encoding VEGF protein, however, is localized to discrete neuronal populations, but not endothelial cells, suggesting that VEGF protein is synthesized in neurons and migrates to the blood vessel walls [40, 67]. These data indicate that the neural tissue of the retina is the most likely source of VEGF, and that diabetes-induced increases in VEGF expression may be caused by the response of neurons and glia to physiological stresses imposed by diabetes.

6.6Injury and Neurodegeneration in the Brain Are Accompanied by Increased VEGF Expression and Vascular Abnormalities

Neurodegenerative diseases often involve both neural and vascular pathology. Growing evidence suggests that changes within the neural tissue and vasculature are intimately linked. While VEGF is identified as playing a central role in diabetic retinopathy, it is also well recognized in studies of acute neurodegeneration stemming from cerebral ischemia and stroke [53]. VEGF mRNA expression is increased in

an animal model of brain ischemia induced by experimental heart attack, increasing between 24 and 48 h after the ischemic event and returning to normal about 7 days later [78]. VEGF expression is also known to increase in humans following transient stroke, and similar results have been obtained after transient forebrain ischemia in rats, as well as in retinal ischemia [57, 88]. Cerebral ischemia-reperfusion is followed by an acute increase in vascular permeability with a distinctive time course [48, 77, 112]. It is likely that VEGF plays a role in this transient loss of blood-brain barrier integrity [57, 78], but may also be responsible for some degree of neuroprotection, because blocking VEGF increases the infarct volume in a rat model of brain ischemia [117]. Topical application of VEGF after cerebral ischemia also reduces infarct volume, suggesting that the growth factor may have dual roles, including beneficial effects on neuronal cell survival [44].

VEGF also appears to be involved in chronic neurodegenerative diseases. Increases in VEGF expression have been noted in brain tissue from patients with Alzheimer’s disease, with the immunoreactivity being most prominent within brain astrocytes and the walls of large blood vessels [53]. VEGF and the inflammatory cytokine, TGF-beta, are also found to be increased in the cerebrospinal fluid of patients with Alzheimer’s disease as well as those with vascular dementia [98]. Immunoreactivity for VEGF colocalizes with plaques containing beta-amyloid in Alzheimer’s brains, again suggesting that it may have an important role in the neuropathogenesis of the disease [118]. One possible outcome of elevated VEGF in these brains is the reduction of neuronal apoptosis, because it can be neuroprotective in some circumstances [50, 73, 91, 95 – 97, 113, 117]. A genetic study demonstrated that two single-nucleotide polymorphisms within the promoter region of the VEGF gene have a significantly higher incidence in a population with Alzheimer’s disease, compared to a healthy control group, suggesting that some VEGF promoter polymorphisms are either more toxic to the brain, or less able to prevent neurodegeneration [29]. These data support a role for VEGF in other neurodegenerative diseases.

It is also becoming increasingly clear that vascular pathology occurs in Alzheimer’s disease. Vascular lesions and breakdown of the blood-brain barrier may contribute to cerebral degeneration [52]. The vascular lesions include loss of endothelial cell markers, increased vascular inflammatory markers, basement membrane thickening and intracerebral hemorrhages and microinfarcts [79]. It is possible that these changes are accompanied by transient increases in cerebrovascular permeability, because serum albumin and IgG immunoreactivity are also

evident in amyloid plaques [114]. The vascular lesions of Alzheimer’s disease are less well established than the neuronal pathology, presumably because of the difficulties in examining the threedimensional structure of the vascular structure. These abnormalities are, however, reminiscent of the vascular lesions well established in the retinas as a result of diabetes, suggesting that similar mechanisms may be at work in the two diseases.

In central nervous system degenerations like hypoxic ischemia and Alzheimer’s disease, the loss of neurons by apoptosis is well established. Data suggest that important vascular complications may contribute to these diseases, some of which may be mediated by VEGF. A similar relationship between the neural tissue and the blood vessels of the retina likely exists in diabetic retinopathy.

6.7Diabetes Increases Neural Apoptosis, Leading to a Cumulative Reduction in the Thickness of the Inner Layers of the Retina

Neurodegeneration of the retina can provide a direct explanation for vision loss in diabetic retinopathy. Numerous studies now show that diabetes increases retinal apoptosis, reduces the number of retinal ganglion cells, and causes atrophy of the inner plexiform layer, which is composed entirely of neuronal and glial projections. Diabetes also reduces survival of retinal neurons [15, 43], induces reactive changes typical of pathological insults in retinal glial cells [16, 64, 83], and causes the appearance of abnormal swellings on centrifugal axons [37], giving rise to the concept that neurodegeneration occurs in diabetic retinopathy [12].

Recent studies of retinal cell loss in diabetes have quantified apoptosis by the number of TUNEL-posi- tive cells in whole and sectioned retinas from STZdiabetic rats, compared to age-matched controls. Increased numbers of apoptosis-positive cells are consistently counted in whole rat retinas after 1, 3, 6 and 12 months of STZ diabetes (Fig. 6.2). Similar data have been obtained from two postmortem retinas of humans with diabetes [15]. Despite several reports of increased apoptosis in the diabetic retina, the specific identities of apoptotic cells are less well established. Studies have reported TUNEL-positive labeling in trypsin-digested retinas, in which only the vasculature remains, implying that the cells are of vascular origin [54, 63]. Interestingly, many apoptotic cells detected by TUNEL staining and active caspase-3 immunoreactivity in STZ-diabetic rat retina are distinct from the vasculature, indicating a neural identity [33, 39] (Fig. 6.3). Although TUNELpositive cells have been detected in the outer nuclear

6 The Neuronal Influence on Retinal Vascular Pathology 111

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Fig. 6.2. Diabetes increases apoptosis in the retina. Apoptosis was quantified in whole retinas of STZ rats after 1, 3, 6 and 12 months of diabetes. The number of apoptotic cells, identified by TUNEL, was standardized to the surface area of each whole-mount retina, calculated by image analysis. The retinas from STZ-diabetic rats contained significantly more TUNEL-positive cells at all time points studied, compared to the age-matched control rats (*p < 0.01, **p < 0.001). (From Fig. 4 in [15]. Reproduced with permission of the American Society for Clinical Investigation)

Fig. 6.3. Apoptosis in the neural retina. A whole-mounted rat retina was labeled for the vascular basement membrane antigen, agrin (green) and the active form of the apoptosis enzyme, caspase-3 (red). The majority of cells with positive immunoreactivity for active caspase-3 were spatially separated from the vasculature, indicating that they were not vascular cells, and more likely to be part of the neural tissue

layer, the most prevalent diabetes-induced cell loss appears to occur in the inner retina, where ganglion, bipolar and amacrine cells are depleted [33, 38, 76, 86]. For example, in rats after 7.5 months of STZ diabetes there is a 10 % reduction in large cell bodies in

thickness of the inner plexiform and inner nuclear layers was diminished by 22 % and 14 % respectively. Together these data suggest a loss of neurons and their processes from the inner part of the retina (Fig. 6.4). Additional studies demonstrate that 1 month of STZ diabetes decreases the thickness of the inner plexiform layer by 10 % in Sprague-Daw- ley rats and by nearly 16 % in Brown Norway rats [5]. Apoptosis indicated by TUNEL-positive and active caspase-3-immunoreactive cells in the ganglion cell layer has been further confirmed by the appearance of fragmented DNA in electron micrographs. These data are consistent with the observation that the number of axons in the optic nerve decreases in STZ-diabetic rats, implying a loss of retinal ganglion cells [85]. Collectively, these data suggest that there is a predominant loss of retinal ganglion, bipolar and amacrine neurons, and the projections needed to communicate between these cells in the retinas of diabetic rats.

Diabetes also increases apoptosis and reduces the thickness of the inner plexiform and inner nuclear layers in the retinas of Ins2Akita diabetic mice [18]. This mouse is spontaneously diabetic due to a point mutation on the second insulin gene, causing degeneration of pancreatic beta cells. The mice develop significant hyperglycemia 4 – 5 weeks after birth [119]. After 4 weeks of diabetes in these mice, the number of cells detected and quantified by counting cells labeled with an antibody to the active form of caspase-3 is significantly higher in retinas from diabetic mice compared to control littermates, suggesting an increase in apoptosis [18].

Morphological changes in the Ins2Akita mouse retinas are also similar to those observed in STZ-diabet- ic rats, including reduced inner plexiform layer thickness. In mice that had been diabetic for 22 weeks there was a 16.7 % reduction in the thickness of the central part of the inner plexiform layer and a 27 % reduction in the peripheral part of this layer compared to non-diabetic littermates in both the central and peripheral retina [18] (Fig. 6.4B, C). A similar study on STZ-diabetic mice also reported a loss of 20 – 25 % of the cell bodies in the ganglion cell layer after 14 weeks of diabetes [62].

These studies demonstrate that retinal degeneration is an effect of diabetic pathology rather than the method of diabetes induction, as they occur in multiple species and in both drug-induced and transgenic models. Collectively, these data suggest that diabetes leads to loss of neurons in the inner retina, as well as the projections necessary for communication between these cells. Importantly, the increase in apo-

Fig. 6.4. Degeneration of the inner retina. The layers of the inner retina become less thick over long periods of diabetes in both rats and mice. a Retinal morphology was measured in sections from streptozotocin-diabetic rats after 7.5 months. The thickness of the inner plexiform and inner nuclear layers was significantly less in STZ rats compared to controls (*p < 0.001), while there was no difference in the outer layers (OPL+ONL). b In Ins2Akita mice, after 5.5 months of hyperglycemia, the thickness of IPL and INL was significantly less in the peripheral retina compared to littermate controls (*p < 0.05). c The thickness of the IPL in the central part of the retina was also significantly less in the Ins2Akita diabetic mice, compared to controls (*p < 0.05), but the thickness of the central INL was unchanged by diabetes. IPL inner plexiform layer, INL inner nuclear layer, OPL outer plexiform layer, ONL outer nuclear layer, RET total retinal thickness, STZ strep- tozotocin-diabetic rats, Ins2 Ins2Akita diabetic mice. (a from Fig. 7 in [15]. Reproduced with permission of the American Society for Clinical Investigation. b, c From Fig. 8 in [18]. Reproduced with permission of Investigative Ophthalmology and Visual Science)

ptosis occurs soon after the onset of experimental diabetes, corresponding to the earliest changes in vascular permeability [9, 13].

6.8Specific Neurons Are Lost by Apoptosis in Diabetes

Several studies have reported that apoptosis in the retina increases with diabetes, but the identity of the dying cells is less well established. Some studies have reported TUNEL positive labeling in trypsine digest retinas, implying that the cells were vascular [54, 63]. Other work, however, has focused on identifying specific subsets of retinal neurons undergoing apoptosis in diabetes. Gastinger et al. [38] recently demonstrated that active caspase-3 immunoreactivity colocalizes with NeuN, a nuclear antigen found in the nuclei of most types of mature neurons (Fig. 6.5). This study also measured a significant depletion of amacrine cell numbers in whole retinas from Ins2Akita mice after 6 months of diabetes, including both dopaminergic and cholinergic cells, identified by immunoreactivity to tyrosine hydroxylase and choline acetyltransferase, respectively. Diabetes has been shown to cause degeneration of a third type of amacrine cells that produce nitric oxide (NO), decreasing the number of NO-immunoreactive cells and attenuating the neuroprotective effect of NO [41a]. This is particularly interesting in light of the regulatory role of NO in vasodilation and ocular blood flow. Data from other histology studies demonstrate that several subsets of neurons are affected adversely by diabetes, including NO-pro- ducing bipolar cells, photoreceptors, horizontal cells and retinal ganglion cells [1, 62, 76]. Therefore, diabetes increases the attrition of many types of neuron in the retina, leading to chronic neurodegeneration.

Fig. 6.5. Retinal neurons undergo apoptosis in diabetes. Whole retinas from STZ rats after 1 month of diabetes were labeled by immunohistochemistry for the active form of the apoptosis enzyme, caspase-3, and nuclear antigen unique to neurons, Neu-N. A Cells undergoing apoptosis were positive for caspase- 3 immunoreactivity (red). B The same cell also contained immunoreactivity for Neu-N (green), identifying it as a neuron. C The two antigens colocalized (yellow) and the apoptotic cell was surrounded by Neu-N positive cells that were not undergoing apoptosis. (From Fig. 2 in [39]. Reproduced with permission of Investigative Ophthalmology and Visual Science)

of Neurodegeneration

Retinas from diabetic rats may exhibit axonal degeneration similar to that observed in other neurodegenerative diseases. A histological study of crosssections of optic nerve from STZ-diabetic rats found that there was a reduction in the number of axons, while the density of glial cells was increased [85]. Functional deficits in retrograde transport may also occur within the optic nerve, indicated by a decreased accumulation of Fluorogold in large and medium sized retinal ganglion cells when injected into the dorsal lateral geniculate nucleus of STZ-dia- betic rats [121, 122]. In a similar study, inhibition of glucose disposal through the polyol pathway with an aldose reductase inhibitor normalized the rate of retrograde transport of Fluorogold through the optic nerve, suggesting that the reduction of retrograde transport is due to metabolic effects of hyperglycemia [47].

Abnormalities such as axoplasmic swellings have also been noted on centrifugal axons labeled by immunohistochemistry for phosphorylated heavy neurofilament protein, which forms part of the cytoskeleton structure in many axons [37]. A preliminary study reported the presence of abnormal swellings and constrictions on retinal ganglion cell axons in Ins2Akita mice crossbred with Thy1.YFP mice, in which yellow fluorescent protein was expressed under the control of the Thy1 promoter [38]. In the retina, Thy1 is expressed exclusively by ganglion cells, and this cross results in a subpopulation of endogenously fluorescent retinal ganglion cells expressing YFP throughout all their neuronal projections, including the axon. These histological characteristics are reminiscent of those noted in degeneration of the sciatic nerve induced by transection and diabetes [41, 99], and suggest that diabetes causes neurodegeneration and abnormalities in the retinal ganglion cell axons.

Another preliminary report suggests that a 1- month duration of diabetes leads to a reduction in the expression of synaptic vesicle-associated proteins such as VAMP2, SNAP-25, synaptophysin, and synapsin 1 [105]. These data are in concordance with other studies demonstrating that synaptic proteins, such as synaptophysin, are depleted from the brain during diabetes and in Alzheimer’s disease, and suggest that the mechanisms of functional communication between neurons in the retina are compromised soon after the onset of diabetes [23, 70]. Taken together these observations suggest that diabetes induces neurodegenerative characteristics in the retina, including loss of synaptic integrity and axonal

6.10The Glial Cells of the Retina React to Diabetes As if an Injury Has Occurred

The glial cells of the retina and brain provide metabolic support for neurons. The highly specialized Müller cells span the retina from inner to outer limiting membranes while the astrocytes form a monolayer close to the inner limiting membrane. The very high metabolic demand and abundant neurotransmitter activity of the retina makes the glial cells extremely important to retinal function, because they regulate potassium, calcium and proton currents, as well as neurotransmitters such as glutamate [68, 81].

In diabetes the expression pattern of the glial cell intermediate filament, glial fibrillary acidic protein (GFAP), is altered in both the astrocytes and Müller cells of the rat retina. Diabetes reduces GFAP content in astrocytes, which normally exhibit high levels of GFAP in the retina, while markedly upregulating GFAP expression in Müller cells, which do not express this protein under normal conditions (Fig. 6.6). These

observations are significant because GFAP expression is an established response to several types of stress and injury in the brain and retina [45, 71, 74]. Other studies have also shown that diabetes alters GFAP expression in both human and rat retina [11, 43, 58, 64, 83].

Other metabolic functions of glial cells are also affected by diabetes. The rate of conversion of glutamate into glutamine is reduced by diabetes [58], as are glutamate oxidation and glutamine synthesis [59]. Together these data show that major metabolic processes supporting glutamate neurotransmission are compromised by diabetes, which could lead to inappropriate neuronal communication and possibly to excitotoxicity due to excess stimulation of neuronal ionotropic glutamate receptors.

Like the brain, the retina contains microglial cells, which are normally quiescent but adopt a reactive state during infection and injury. The microglial cells also become reactive in the retinas of mice and rats with diabetes [18, 83, 120] (Fig. 6.7). When quiescent these cells have long fine processes that appear to interact with other cells including neurons. When the retina is stressed by neuroinflammation, ischemia, or some other types of damage, however, microglia adopt a reactive morphology in which their processes become contracted and swollen. The activation of microglia may in part be due to increased expression of cytokines, but this process may be blocked by anti-inflammatory drugs such as minocycline [56]. Taken together these data suggest that the microglia and macroglia of the retina respond to diabetes in a manner that suggests an underlying stress or metabolic insult.

Fig. 6.6. Retinal macroglia express a stress reaction in diabetes. The stress reaction of astrocytes and Müller cells was visualized by immunohistochemistry for glial fibrillary acidic protein (GFAP) in whole-mount retinas from control and STZ-injected rats after 4 months of diabetes. A In control rats the astrocytes, which form a monolayer at the superficial surface of the retina, express high levels of GFAP. B In STZ rats GFAP expression in the astrocytes is reduced. C Focusing deeper into the retina reveals that the Müller cells do not express GFAP in the control rats. D Focusing at a similar level in the retinas of STZ diabetic rats reveals that GFAP expression is high in the Müller cell processes. (From Fig. 2 in [16]. Reproduced with permission of

Investigative Ophthalmology and Visual Science)

Fig. 6.7. Retinal microglia become reactive in diabetes. Whole retinas from Ins2Akita diabetic mice, after 2 months of hyperglycemia, were labeled by immunohistochemistry for the calcium binding protein called Iba1, which is unique to microglia in the retina. A In non-diabetic littermates the microglia had a quiescent morphology, with many long, fine processes. B In diabetic mice the microglia of some regions had a reactive morphology with short swollen processes. (From Fig. 6 in [18]. Reproduced with permission of Investigative Ophthalmology and Visual Science)

6.11There is a Loss of Function in Diabetic Retinopathy That Begins Soon After the Onset of Diabetes

The onset of vision loss often precedes the clinically measurable changes in the vasculature, and likely stems from disruptions of the neural retina. Diabe- tes-induced neuronal degeneration has been observed in postmortem human retinas as well as in rodent models of diabetes [3, 15, 18, 20, 62, 65, 76, 87, 115]. Such retinal neurodegeneration is a fundamental pathological feature of other high-incidence retinal diseases, including glaucoma and age-related macular degeneration, that involve functional vision loss [2, 55]. Normal vision is dependent on intact signal transduction pathways through the network of neurons in the retina, which is further dependent on undisrupted cell-cell interactions between neuronal, glial, vascular and epithelial cells. Due to the interdependent nature of these cells, the degeneration of one subpopulation has the potential to impact the function and overall health of the entire retina in a feedforward manner. This is very likely one contributing mechanism to the progressive nature of diabetic retinopathy, emphasizing the need for early intervention.

Deficits in vision occurring among people with diabetes include decreases in night vision, contour and contrast sensitivity, detail discrimination and visual acuity [46, 66, 90, 102]. Although normal daytime vision is the product of complex neuronal processing, various aspects of vision can be attributed to inputs from distinct neuronal types in the retina. Clinical studies have detected a loss of neuronal function with basic vision examinations and psychophysical testing, correlated with underlying neurophysiological disruptions using electroretinography. The electroretinogram (ERG) provides an assessment of neuronal electrical responses to visual stimuli and can be used in humans as well as animal models of diabetes. Studies of humans with diabetes using this technology have demonstrated retinal dysfunction that precedes visible vascular changes characteristic of diabetic retinopathy [24, 30, 42]. Data from these studies suggest that the function of the inner retina is most affected by diabetes, with reduced amplitude and frequency of amacrine cell responses to visual stimuli, indicated by aberrant oscillatory potentials [19, 104]. In conclusion, data showing changes in the electrophysiological response of the retina suggest that diabetes rapidly induces a loss of function that may precede the gross vascular pathology [21].

6.12 Conclusions

Neurodegeneration in the retina is accompanied by loss of neural function, which may begin soon after

the onset of diabetes. These early changes may be reflected in the increase in expression of VEGF, I 6 which occurs within the neurons and glial cells, presumably as a response to stresses induced by diabe-

tes. VEGF, and possibly other vasoactive factors, causes the increases in vascular permeability and proliferation that are more widely recognized as diabetic retinopathy. VEGF is currently being studied as a potential target for a variety of novel therapies [8, 69, 84, 92]. Since retinal function may not be reversible, however, other current approaches should involve direct targeting of survival of the neural retina with therapeutic agents such as ACE inhibitors [22], cannabidiol [33], and growth factors such as brain derived neurotrophic factor [87], insulin and IGF-I [17, 82, 86], which may exert a neuroprotective effect, reducing the numbers of cells lost to diabetes and perhaps limiting the increased expression of VEGF that leads to angiogenesis and vascular permeability.

6.13 Methodology

Here we summarize two of the methodologies that were developed to collect the apoptosis and immunohistochemical data discussed earlier.

A. Terminal dUTP nick end labeling (TUNEL) on whole retina

1.Retinas are dissected on ice immediately and fixed for 20 min in 10 % normal buffered formalin and rinsed before mounting flat onto coated microscope slides, by four radial cuts. Samples are placed at 4 °C overnight and allowed to dehydrate.

2.At the time of staining, retinas are rehydrates in PBS, fixed further in formalin or 4 % paraformaldehyde, 10 min @r.t., rinsed in PBS (twice for 5 min each) and

dehydrated in 50 %, 75 , 95 %, 100 % ethanol, 20 min each.

3.The retinas are cleared in xylene (2 times for 10 min) and then stored in xylene overnight at 4 °C. This step removes much of the lipid in the tissue.

4.The next day the samples are allowed to come to room temperature, placed in fresh xylene, and incubated at

60 °C for 30 min, then rehydrated in ethanol, 100 %, 95 %, 75 %, 10 min each, then in 50 % ethanol for 5 min, before two 5 min rinses in water.

5.The retinas are permeabilized in 0.3 % Triton X-100 (15 min), washed in TRIS/NaCl buffer (100 mM TRIS, 150 mM NaCl, pH 8.0) and then digested with 20 μg/ml

proteinase K in TRIS/NaCl buffer, for 30 min. This step is stopped by rinsing in water.

6.Endogenous peroxidase activity is quenched with 3 % H2O2 for 10 min.

7.After further rinsing in saline the TUNEL protocol is carried out using an in situ terminal transferase labeling kit. Terminal transferase is stored at –20 °C and prepared fresh in incubation buffer containing digoxigenin labeled nucleotides. The tissue is covered with plastic coverslips and incubated at 37 °C for 1 h. Then the enzyme reaction is washed off.

B. Immunohistochemistry for confocal microscopy on whole retina

1.Dissect whole retinas carefully using a dissection microscope and fine scissors and forceps. Keep the retina as intact as possible with no cuts or tears. Handle retinas gently by edges using very fine pointed forceps.

2.Fix each whole retina in paraformaldehyde, and rinse in PBS. The optimum concentration of fixative and duration of this step must be empirically determined for every antigen and primary antibody. Optimum fixation can vary between 10 min and several hours in 2 % or 4 % paraformaldehyde.

3.Retinas are transferred to 200 μl of 10 % serum containing 0.3 % Triton X-100, in 96-well microtiter plate wells, for 1 – 2 h.

4.Transfer to separate wells containing 200 μl of primary antibody and incubate at 4 °C for 3 – 4 days. The optimal concentration for each antibody must be empirically determined.

5.Transfer retinas to six-well culture plates and wash repeatedly in 10 ml volumes of wash buffer (PBS with 0.3 % Triton X-100). Each wash should last 1 h, and then incubate in wash buffer overnight.

6.Transfer retinas back to a 96-well plate for the secondary antibody incubation. Fluorescent conjugates for each secondary antibody should be chosen carefully to be compatible with the available microscopes. The concentrations of secondary antibodies must also be optimized empirically.

7.Repeat the washing step as described in step 5. Then mount the tissue on microscope slides by making four radial cuts to flatten each retina, using fine scissors, pointed forceps and a soft brush. This step is best done with a dissection microscope. Tissue should be mounted in a non-fluorescent mounting media. The volume of media should be adjusted to account for the thickness of the retinal tissue.

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