August 2004
Volume 45, Issue 8
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Retina  |   August 2004
Expression of Apoptosis Markers in the Retinas of Human Subjects with Diabetes
Author Affiliations
  • Ahmed M. Abu El-Asrar
    From the Department of Ophthalmology, College of Medicine, King Saud University, Riyadh, Saudi Arabia; the
  • Lieve Dralands
    Department of Ophthalmology and the
  • Luc Missotten
    Department of Ophthalmology and the
  • Ibrahim A. Al-Jadaan
    King Khaled Eye Specialist Hospital, Riyadh, Saudi Arabia.
  • Karel Geboes
    Laboratory of Histochemistry and Cytochemistry, University of Leuven, Belgium; and the
Investigative Ophthalmology & Visual Science August 2004, Vol.45, 2760-2766. doi:10.1167/iovs.03-1392
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      Ahmed M. Abu El-Asrar, Lieve Dralands, Luc Missotten, Ibrahim A. Al-Jadaan, Karel Geboes; Expression of Apoptosis Markers in the Retinas of Human Subjects with Diabetes. Invest. Ophthalmol. Vis. Sci. 2004;45(8):2760-2766. doi: 10.1167/iovs.03-1392.

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      © 2016 Association for Research in Vision and Ophthalmology.

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Abstract

purpose. To investigate the expression of the apoptotic mediators in the retinas from human subjects with diabetes mellitus.

methods. Ten donor eyes from five subjects with diabetes mellitus, and eight eyes from four nondiabetic subjects without known ocular disease serving as control subjects were examined. Immunohistochemical techniques were used with antibodies directed against glial fibrillary acidic protein (GFAP), caspase-3, Fas, Fas ligand (FasL), Bax, Bcl-2, survivin, p53, extracellular signal-regulated kinases (ERK1/2), and p38.

results. In retinas from all subjects without diabetes, weak Bcl-2 immunoreactivity was confined to GFAP-positive glial cells in the nerve fiber layer. Weak immunoreactivity for ERK1/2 was noted in a few nuclei in the inner nuclear layer and in a few Müller cell processes. Cytoplasmic immunostaining for survivin was noted in the retinal pigment epithelial cells. There was no immunoreactivity for the other antibodies tested. All diabetic retinas showed cytoplasmic immunoreactivity for caspase-3, Fas, and Bax in ganglion cells. FasL immunoreactivity was detected in GFAP-positive cells. Upregulation of Bcl-2 immunoreactivity was noted in GFAP-positive cells in nerve fiber and ganglion cell layers, and Bcl-2 induction was noted in Müller cell processes. Strong immunoreactivity for ERK1/2 was observed in many nuclei in the inner nuclear layer in GFAP-positive cells in the nerve fiber and ganglion cell layers and numerous Müller cell processes. Survivin immunoreactivity was not altered in the diabetic retinas. There was no immunoreactivity for p53 and p38.

conclusions. Ganglion cells in diabetic retinas express several proapoptosis molecules, suggesting that these cells are the most vulnerable population. Glial cells in diabetic retinas are activated and express several antiapoptosis molecules in addition to the cytotoxic effector molecule FasL, suggesting a possible role of glial cells in induction of apoptosis in ganglion cells.

Increased apoptosis of neural retinal cells in experimental diabetes in rats and diabetes mellitus in humans was recently documented. At particular risk are retinal ganglion cells, which demonstrated a 10% decrease in cell number in diabetic rats compared with control eyes. Apoptotic cells did not colocalize with endothelial cells. These data confirmed that nonvascular cells, most likely ganglion cells, become apoptotic in the diabetic rat retinas. 1 Apoptotic death requires synthesis of new proteins by degenerating cells and can be prevented by protein synthesis inhibitors. 2 3  
The molecular basis for the apoptogenic effects of diabetes in the retina is not yet identified. The molecular events regulating apoptosis are complex and involve genes that are both proapoptotic and antiapoptotic. 4 A number of mediators are involved in apoptosis, including caspases, Fas/Fas ligand (FasL), Bax/Bcl-2, survivin, and p53. 
Central to the implementation of apoptosis is a class of cysteine aspartate-specific proteases of the interleukin-1β–converting enzyme family known as caspases. They exist as proenzymes that are proteolytically processed to their active forms in response to an apoptosis-inducing stimulus. Activated caspases cleave each other’s precursors into mature, active enzymes in a proteolytic cascade. Activated caspases kill cells by degrading structural elements and DNA repair enzymes and by indirect activation of chromosomal endonucleases. 5 6 Fas (CD95) is a type I transmembrane glycoprotein belonging to the tumor necrosis factor-α receptor superfamily. 7 On ligation with agonistic antibody or the natural FasL, Fas trimerizes and recruits several proteins that share a death domain that leads to the formation of a specific death-inducing signaling complex at the intracellular region of the Fas receptor. 8 The recruitment of caspase-8 to the death-inducing signaling complex results in proteolytic activation of the enzyme, which, in turn activates a series of other caspase members. 9 FasL is a type II transmembrane glycoprotein that induces apoptosis in target cells in both the membrane-bound form and the soluble form. 10 Cell survival and apoptotic death have been shown to be regulated by genes of the bcl-2 family. Thus, several studies demonstrated a protective, antiapoptotic effect of Bcl-2 protein in neural cells both in vitro and in vivo. 11 12 Several proteins have been identified that share homology with Bcl-2. Some of these, such as Bax, render cells more susceptible to apoptotic stimuli. 13 14 Survivin is a protein that inhibits apoptosis and regulates cell division. It inhibits apoptosis by either directly or indirectly interfering with the function of caspases. 15 The nuclear phosphoprotein p53 is a key determinant in the process of apoptosis in many cell types, acting to promote apoptosis. 16 p53 is a DNA-binding transcription factor originally recognized as a tumor suppressor, and mutations in this gene are found in approximately half of all human tumors. 17  
Transfer of information for cell death or survival programs is organized by the cascades of kinases, by which several adaptive and protective or pathogenic proteins are functionally activated by phosphorylation. Among the signal transduction pathways involved in cell fate, mitogen-activated protein kinases (MAPKs) occupy a central place. Members of the MAPK family include extracellular signal-regulated kinases (ERK, p44 MAPK/ERK1, and p42 MAPK/ERK2), c-Jun NH2-terminal protein kinases (JNK), and p38 MAPK. JNK and p38 MAPKs are strongly activated by stress signals such as inflammatory cytokines, bacterial lipopolysaccharide, heat shock, ultraviolet light, osmotic shock, and ischemia and have been suggested to contribute to cell death. ERK, in contrast, is activated in response to mitogens and survival factors, subsequently modulating the activity of many transcriptional factors, leading to cell growth, and differentiation. In general, ERK1/2 activation has been shown to promote survival in a number of situations. 18 19 20 21  
Because retinal neural cell death in diabetes is thought to be due to an apoptotic mechanism, it is important to know which apoptotic mediators are specifically expressed during apoptosis of retinal cells in diabetes. Therefore, we used immunohistochemical techniques to study the expression of the apoptotic markers caspase-3, Fas, FasL, Bax, Bcl-2, survivin, p53, ERK1/2, and p38 in the retinas from diabetic patients and in the retinas from subjects without diabetes. 
Methods
We obtained 10 human donor eyes postmortem from five subjects with diabetes mellitus. Donor eyes were obtained and used in the study in accordance with the provisions of the Declaration of Helsinki for research involving human tissue. No subject had a history of retinal photocoagulation. We also obtained eight eyes from four persons with no history of diabetes or of ocular disease, as determined by gross pathologic examination. Immediately after the specimens arrived in the laboratory, an incision was made 3 mm posterior to the limbus, and the cornea, iris, lens, and vitreous were gently removed. The retina and uveal tissue were dissected from the surrounding tissue, fixed in 4% paraformaldehyde, and embedded in paraffin. 
Immunohistochemical techniques were used. After deparaffinization, endogenous peroxidase was abolished with 2% hydrogen peroxide in methanol for 3 minutes, and nonspecific background staining was blocked by incubating the sections for 5 minutes in normal swine serum. For caspase-3, Fas, Bcl-2, survivin, ERK1/2, and p38 detection, the sections underwent heat-induced antigen retrieval with a microwave oven (three 5-minute cycles in 10 mM Tris-EDTA buffer [pH 9] at 650 W). For p53 immunohistochemistry, the sections were microwaved (three 5-minute cycles in 10 mM citrate buffer [pH 6] at 650 W). Subsequently, the sections were incubated with the monoclonal and polyclonal antibodies listed in Table 1 . The specificity of the antibodies used is indicated in Table 2 . Optimum working concentration and incubation time for the antibodies were determined earlier in pilot experiments. For caspase-3, Fas, Bcl-2, p53, ERK1/2, and p38 immunohistochemistry, the sections were incubated for 30 minutes with goat anti-rabbit or anti-mouse immunoglobulins conjugated to peroxidase-labeled dextran polymer (EnVision+; Dako, Carpinteria, CA). For FasL, Bax, and survivin immunohistochemistry, the sections were incubated for 30 minutes with the biotinylated secondary antibody and reacted with the avidin-biotinylated peroxidase complex (Dako). The reaction product was visualized by incubation for 10 minutes in 0.05 M acetate buffer at pH 4.9, containing 0.05% 3-amino-9-ethylcarbazole (Sigma-Aldrich, Bornem, Belgium) and 0.01% hydrogen peroxide, resulting in bright-red immunoreactive sites. The slides were faintly counterstained with Harris hematoxylin. Finally, the sections were rinsed with distilled water and coverslipped with glycerol. 
Omission or substitution of the primary antibody with an irrelevant antibody of the same species and staining with chromogen alone were used as negative controls. Sections from patients with colorectal carcinoma were used as positive controls for survivin, caspase-3, Fas, and FasL; sections from patients with Barrett esophagus and adenocarcinoma were used as positive controls for p53; sections from patients treated for lymphoma were used as positive controls for Bcl-2 and Bax; sections from patients with Crohn’s disease were used as positive controls for ERK1/2 and p38; and sections from peripheral nerves were used as positive controls for glial fibrillary acidic protein (GFAP). The sections from the control patients were obtained from patients treated at the University Hospital (University of Leuven, Belgium), in full compliance with the tenets of the Declaration of Helsinki. 
All sections were examined by two independent observers (AMA, KG). One of them (KG) was unaware of the origin of the specimens. In case of disagreement, the results obtained by the blinded observer were used. The staining was graded on the basis of the presence or absence of immunoreactivity, intensity of immunoreactivity, thickness of the staining, and the homogeneous or heterogeneous character of staining. 
Results
Donor age and sex, type of diabetes, duration of diabetes, presence or absence of arterial hypertension, retinopathy status, cause of death, and death-to-enucleation times are summarized in Table 3 . Nonproliferative diabetic retinopathy was documented to be present in two cases (cases 1 and 2). The status of the retina was unknown in two cases (cases 3 and 4). One case (case 5) had no retinopathy. 
There was no staining in the negative control slides (Fig. 1) and when the chromogen alone was applied. A summary of the results is given in Table 4 . Similar findings were noted in retinas from all subjects without diabetes. Retinas from subjects without diabetes showed weak immunoreactivity for GFAP very close to internal limiting membrane in nerve fiber layer, and ganglion cell layer (data not shown). Weak Bcl-2 immunoreactivity was observed very close to internal limiting membrane in nerve fiber layer in GFAP-positive cells (Fig. 2A) . Weak immunoreactivity for ERK1/2 was noted in a few scattered nuclei in the inner nuclear layer and in a few processes consistent with the morphologic appearance of Müller cells (Fig. 2B) . Cytoplasmic immunostaining for survivin was noted in the retinal pigment epithelial cells (Fig. 2C) and in the pigmented and nonpigmented layers of the ciliary body epithelium (data not shown). There was no immunoreactivity for caspase-3, Fas, FasL, Bax, p53, and p38. 
Similar findings were noted in all diabetic retinas. Strong immunoreactivity for GFAP was noted in nerve fiber layer and ganglion cell layer. Müller cell process that extend radially throughout the retina showed moderate GFAP immunoreactivity (data not shown). Granular cytoplasmic immunoreactivity for caspase-3 (Fig. 3A) , Fas, and Bax was noted in ganglion cells. These cells had large cell bodies characteristic of ganglion cells. Figure 3A shows a representative result of the expression of these mediators in ganglion cells. In addition, few cells in inner nuclear layer showed immunoreactivity for Fas. Cytoplasmic granular immunoreactivity for FasL was detected in the nerve fiber layer, the ganglion cell layer, and numerous cell processes that extend radially through the entire width of the retina (Fig. 3B) . The pattern of immunoreactivity for FasL was similar to the pattern of cells that stained for GFAP. Upregulation of Bcl-2 immunoreactivity was noted in nerve fiber layer and ganglion cell layer and Bcl-2 induction was noted in cell processes in inner plexiform layer which was similar to the pattern of cells that stained for GFAP (Fig. 3C) . Strong immunoreactivity for ERK1/2 was observed in many nuclei in inner nuclear layer, in nerve fiber and ganglion cell layers, and in numerous cell processes that extend radially through all the retinal layers (Fig. 3D) . Immunostaining in the nuclei in the inner nuclear layer was continuous, with immunostaining in cell processes. Based on the morphologic assessment of cell types, increased immunostaining for ERK1/2 in diabetic retinas was associated with glial cells. The distribution and intensity of survivin immunoreactivity was not altered in the diabetic retinas compared with retinas from subjects without diabetes. There was no immunoreactivity for p53 and p38. 
Discussion
There are three important findings of the present study: (1) Ganglion cells in diabetic retinas expressed the apoptosis-promoting factors caspase-3, Fas, and Bax; (2) glial activation in diabetic retinas was associated with upregulation of ERK1/2 signaling pathway that has a protective effect on apoptotic signaling from death receptors. In addition, glial cells in diabetic retinas showed upregulation of the antiapoptotic marker Bcl-2 and expressed the cytotoxic effector molecule FasL; and (3) retinal pigment epithelial cells in diabetic and nondiabetic retinas expressed the antiapoptotic protein survivin. 
Distinct members of the caspase family are involved in both the initiation and execution phases of apoptosis, with the initiator caspases coupling cellular signaling pathways to caspase activation and the downstream effector caspases being responsible for the cleavage of cellular substrates. Among them, caspase-3 is the executioner caspase known to play a central role in the proteolytic cascade during apoptosis. 22 23 The detection of activated caspase-3 is a very reliable way to identify cells destined to die by apoptosis, even before many of the morphologic characteristics (e.g., DNA fragmentation) are present. 24 In the present study, the executioner caspase-3 immunoreactivity was observed in ganglion cells in diabetic retinas. Our observations are consistent with previous reports showing caspase-3 activation in the retinas of diabetic animals and diabetic patients. 25 26 Several studies demonstrated that caspase-3 is involved in the apoptotic death of retinal ganglion cells induced by ischemia, 27 28 excitotoxicity, 28 29 axotomy, 30 and chronic ocular hypertension. 31 Inhibition of caspase-3 activity reduced apoptotic cell death induced in retinal cells by excitotoxicity and ischemia. 27 28 29  
In diabetic retinas, Fas immunoreactivity was observed in ganglion cells, and FasL immunoreactivity was localized to glial cells. These findings indicate that both Fas death system components are available for interaction, possible Fas-mediated ganglion cell death, and a possible role of glial cells in the induction of apoptosis in ganglion cells. Our results are in agreement with a previous report showing that levels of Fas and FasL were elevated in the retinas of diabetic rats. 32 In vitro studies showed that cultured astrocytes express FasL and that FasL expressed in astrocytes was functional, as astrocytes induced apoptosis in neuronal cells through FasL. 33 The crucial role of the Fas/FasL death receptor system in apoptotic neuronal cell death after ischemia has recently been shown. Rat cerebral ischemia resulted in upregulation of Fas and FasL in the apoptotic areas of the postischemic brain. 34 35 In pontosubicular neuron necrosis, an age-specific response to severe hypoxic–ischemic injury occurring in neonates, neuronal Fas expression was significantly increased and FasL expression was found mainly on astrocytes and microglial cells. 36 Moreover, mice deficient in the Fas/FasL pathway were highly resistant to ischemia-induced neuronal damage. 34 In vitro studies demonstrated that oxidative stress and hypoxia and reoxygenation induce FasL expression in microglial cells. 37  
In the present study, Bax, a proapoptotic member of the genetic program that governs apoptosis, was expressed in ganglion cells in diabetic retinas. These observations are consistent with a previous report showing that Bax levels are increased in human diabetic retinas and that Bax immunoreactivity is limited to the inner retina and is prominent in ganglion cells. 38 Several studies demonstrated that Bax is a major effector of apoptotic ganglion cell death in the retina after ischemia, excitotoxicity, axotomy, and in retinal degeneration. 28 39 40 41 42 Bax antisense oligonucleotides reduced axotomy-induced retinal ganglion cell death by reduction of Bax protein expression, indicating that Bax induction is a prerequisite for the execution of retinal ganglion cell apoptosis. 40  
It is noteworthy that the sensory retina from diabetic subjects with or without retinopathy showed immunoreactivity for the apoptosis-promoting factors in ganglion cells. This observation suggests that retinal ganglion cells in subjects with diabetes express proapoptosis molecules and undergo apoptosis independent of the occurrence of retinopathy. There is increasing evidence that retinal ganglion cell death occurs early in diabetes and that neurodegeneration is an important component of diabetic retinopathy. 1 Diabetes results in various metabolic and biochemical abnormalities in the retina such as increased oxidative stress, 43 accumulation of advanced glycation end products, 44 and increased levels of glutamate, 45 which have been shown to induce the expression of the proapoptosis molecules and lead to apoptosis in neuronal cells. In vivo and in vitro studies demonstrated that oxidative stress induced by high glucose leads to activation of caspase-3 and apoptosis in dorsal root ganglion neurons. 46 47 The apoptosis induced by high glucose could be effectively inhibited by pretreatment with caspase-3 inhibitors. 25 In vitro studies showed that advanced glycation end products induce the increase of the proapoptosis markers caspase-3 and Bax and increase apoptosis in cultured rat retinas. 48 Glutamate treatment of cultured rat retinas induces caspase-3 expression and apoptotic cell death in ganglion cells, and an intervention to caspase-3 provides effective protection to retinal neurons against glutamate excitotoxicity. 29 These observations establish that a metabolic abnormality characteristic of diabetes is sufficient to upregulate the expression of the apoptosis-promoting factors and stimulate a death pathway in retinal ganglion cells. 
In the present study, diabetic retinas showed ERK1/2 upregulation in glial cells. Similarly, upregulation of ERK1/2 expression was also detected in glial cells in retinas of glaucomatous human eyes, 49 and in retinas of experimental endotoxin-induced uveitis. 50 Our observations further document glial activation in retinas of human subjects with diabetes. The glial cells and the ERK pathway may be chronically activated in diabetic retinas because of the continuous presence of extracellular stimulatory factors, such as ischemia, 51 oxidative stress, 43 or glutamate excitotoxicity, 45 which are implicated in diabetic retinopathy. Ischemia, 52 glutamate excitotoxicity, 53 and oxidative stress 54 have been associated with the activation of ERK1/2 in brain glial cells. Recently reported studies have established the concept that activation of ERK1/2 confers survival advantages to cells in the face of activation of apoptotic pathways. 19 55 Our observation of activated ERK signaling in glial cells in diabetic retinas suggests that the activity of this kinase pathway may account, in part, for the relative protection of glial cells against damage, whereas retinal ganglion cells undergo apoptosis. In vitro studies demonstrated that ischemia and elevated hydrostatic pressure induce apoptosis in retinal ganglion cells, whereas cocultured glial cells survive the same stress conditions. 56  
Several studies demonstrated that the ERK1/2 signaling pathway is involved in the pathogenesis of diabetic retinopathy. ERK activity increased in the retina of diabetic animals compared with normal control animals. 57 Poulaki et al. 58 demonstrated that hyperglycemia-induced retinal vascular endothelial growth factor expression requires ERK1/2 and that inhibition of the ERK1/2 pathway reduces retinal hypoxia-inducible factor-1α levels, and suppresses retinal vascular endothelial growth factor upregulation, and blood–retinal barrier breakdown in diabetic animals. Moreover, advanced glycation end products were found to stimulate vascular endothelial growth factor expression and activate the transcription factor hypoxia-inducible factor-1α in an ERK-dependent pathway. 59  
In the nondiabetic retinas, weak Bcl-2 immunoreactivity was confined to GFAP-positive cells in the innermost retinal layers. Previous reports demonstrated that Bcl-2 immunoreactivity was detected in Müller cells in human, 60 rabbit, 61 and rat 62 retinas. Diabetic retinas showed Bcl-2 upregulation in glial cells in nerve fiber and ganglion cell layers and Bcl-2 induction in Müller cell processes. This result is in contrast to a previous study, in which Mizutani et al. 60 demonstrated that in the adult human retina, Bcl-2 levels are not modified by diabetes. The differences in technique, and the antibodies may account for this discrepancy. Upregulation of Bcl-2 immunoreactivity in retinal Müller cells has also been observed in a mice model of neurotoxin-induced retinal neuronal degeneration, 41 after optic nerve transection in rats, 62 and in rats with inherited retinal dystrophy. 63 The increased expression of the antiapoptotic marker Bcl-2 in glial cells in diabetic retinas may contribute to preventing glial cells from undergoing apoptosis. 
In our study, we showed that survivin, a protein that inhibits apoptosis, 15 was expressed by retinal pigment epithelial cells in diabetic and nondiabetic retinas. The distribution and intensity of survivin immunoreactivity was not modified by diabetes. Similarly, αB-crystallin, a member of the small heat shock proteins that exerts an antiapoptotic effect was reported to be constitutively expressed by retinal pigment epithelial cells. 64 The expression of these antiapoptotic proteins in retinal pigment epithelial cells may reflect the fact that retinal pigment epithelial cells have minimal mitotic capacity, are in a postmitotic state, and have to survive for the lifetime of the organism. 65  
In conclusion, these observations suggest that diabetes induces an apoptogenic environment in the retina, that retinal ganglion cells are the most vulnerable population, and that the Fas death system and glial cells may be involved in the induction of cell death by apoptosis in ganglion cells. In addition, our results suggest that glial cells and retinal pigment epithelial cells are prevented from undergoing apoptosis. The use of antiapoptotic agents could play a role in the treatment of diabetic injury in the retina. 
 
Table 1.
 
Monoclonal and Polyclonal Antibodies Used in the Study
Table 1.
 
Monoclonal and Polyclonal Antibodies Used in the Study
Primary Antibody Dilution Incubation Time Source*
Anti-cleaved caspase-3 (Asp175) (pc) 1:25 120 minutes Cell Signaling Technology
Anti-Fas (B-10) (mc) 1:50 Overnight Santa Cruz Biotechnology, Inc.
Anti-FasL (N-20) (pc) 1:50 30 minutes Santa Cruz Biotechnology, Inc.
Anti-Bax (B-9) (mc) 1:50 30 minutes Santa Cruz Biotechnology, Inc.
Anti-Bcl-2 (clone 124) (mc) 1:10 30 minutes Dako
Anti-survivin (D-8) (mc) 1:20 30 minutes Santa Cruz Biotechnology, Inc.
Anti-p53 (clone DO-7 (mc) 1:10 30 minutes Dako
Anti-phospho-ERK1/ERK2 MAPK (E10) (mc) 1:30 Overnight Cell Signaling Technology
Anti-p38 MAPK (pc) 1:30 Overnight Cell Signaling Technology
Anti-GFAP (pc) 1:300 30 minutes Dako
Table 2.
 
Specificity of the Antibodies Used in the Study
Table 2.
 
Specificity of the Antibodies Used in the Study
Primary Antibody Specificity
Anti-cleaved caspase-3 Detects the large fragment of activated caspace-3 (17–20 kDa)
Anti-Fas Detects a peptide mapping at the carboxyl terminus of Fas of human origin
Anti-FasL Detects a peptide mapping at the amino terminus of FasL of rat origin
Anti-Bax Maps at amino acids 1-171 representing all but the carboxyl terminal 21 amino acids of Bax of mouse origin
Anti-Bcl-2 Reacts with the Bcl-2 oncoprotein
Anti-survivin Raised against a recombinant protein corresponding to amino acids 1-142 of survivin of human origin
Anti-p53 Reacts with a 53-kDa protein. The epitope recognized is located between the N terminal amino acids 1 and 45 and possibly between amino acids 37 and 45 of the human p53 protein
Anti-phospho-ERK1/ERK2 MAPK Detects endogenous levels of p44 and p42 MAP kinase (ERK1 and ERK2) dually phosphorylated at threonine 202 and tyrosine 204
Anti-p38 MAPK Detects total p38 MAP kinase (phosphorylation-state–independent) levels.
Anti-GFAP Reacts strongly with human GFAP.
Table 3.
 
Subject Characteristics
Table 3.
 
Subject Characteristics
Case Number Age/Sex Type of Diabetes Duration of Diabetes (y) Arterial Hypertension Retinopathy Status Cause of Death Death to Enucleation Time (h)
Subjects with diabetes
 1 75 F NIDDM 14 + NPDR Coronary artery disease 17
 2 70 M NIDDM 20 NPDR Renal failure 9
 3 58 M NIDDM 16 Unknown Intracranial bleeding 10
 4 67 F NIDDM 18 Unknown Coronary artery disease 21
 5 73 F NIDDM 15 + Absent Cerebrovascular accident 9
Subjects without diabetes
 5 52 M Colon cancer metastasis 17
 6 42 M Unknown 15
 7 79 M Coronary artery disease 17
 8 64 F Coronary artery disease 18
Figure 1.
 
Photomicrograph of a retina from a diabetic subject. This is a negative control slide that was treated identically with the omission of the primary antibody showing no staining. NFL, nerve fiber layer; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer. Scale bar, 10 μm.
Figure 1.
 
Photomicrograph of a retina from a diabetic subject. This is a negative control slide that was treated identically with the omission of the primary antibody showing no staining. NFL, nerve fiber layer; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer. Scale bar, 10 μm.
Table 4.
 
Summary of Staining Results
Table 4.
 
Summary of Staining Results
Antibody Retinas from Four Subjects without Diabetes* Retinas from Five Subjects with Diabetes*
Anti-cleaved caspase-3 − (8/8) + (10/10)
Anti-Fas − (8/8) + (10/10)
Anti-FasL − (8/8) +++ (10/10)
Anti-Bax − (8/8) + (10/10)
Anti-Bcl-2 + (8/8) ++ (10/10)
Anti-survivin ++ (8/8) ++ (10/10)
Anti-p53 − (8/8) − (10/10)
Anti-phospho-ERK1/ERK2 MAPK + (8/8) +++ (10/10)
Anti-p38 MAPK − (8/8) − (10/10)
Anti-GFAP + (8/8) +++ (10/10)
Figure 2.
 
Photomicrographs of retinas from nondiabetic subjects. (A) Immunohistochemical staining for Bcl-2 showing weak immunoreactivity confined to the innermost retina in the nerve fiber layer (NFL; arrow). (B) Immunohistochemical staining for ERK1/2 showing weak immunoreactivity in a few nuclei in the inner nuclear layer (INL; arrow) and in a few Müller cell processes (arrowhead). (C) Immunohistochemical staining for survivin showing cytoplasmic immunoreactivity for survivin in retinal pigment epithelial cells (RPE; arrow). Abbreviations are defined in Figure 1 . Scale bar, 10 μm.
Figure 2.
 
Photomicrographs of retinas from nondiabetic subjects. (A) Immunohistochemical staining for Bcl-2 showing weak immunoreactivity confined to the innermost retina in the nerve fiber layer (NFL; arrow). (B) Immunohistochemical staining for ERK1/2 showing weak immunoreactivity in a few nuclei in the inner nuclear layer (INL; arrow) and in a few Müller cell processes (arrowhead). (C) Immunohistochemical staining for survivin showing cytoplasmic immunoreactivity for survivin in retinal pigment epithelial cells (RPE; arrow). Abbreviations are defined in Figure 1 . Scale bar, 10 μm.
Figure 3.
 
Photomicrographs of retinas from diabetic subjects. (A) Immunohistochemical staining for caspase-3 showing immunoreactivity in ganglion cells (arrows). (B) Immunohistochemical staining for FasL showing immunoreactivity in numerous cell processes (arrows). (C) Immunohistochemical staining for Bcl-2 showing strong immunoreactivity in nerve fiber (NFL) and ganglion cell (GCL) layers and in cell processes in the inner plexiform layer (IPL; arrow). (D) Immunohistochemical staining for ERK1/2 showing strong immunoreactivity in nuclei in the inner nuclear layer (INL; arrow) and in numerous cell processes (arrowheads). Abbreviations are defined in Figure 1 . Scale bar, 10 μm.
Figure 3.
 
Photomicrographs of retinas from diabetic subjects. (A) Immunohistochemical staining for caspase-3 showing immunoreactivity in ganglion cells (arrows). (B) Immunohistochemical staining for FasL showing immunoreactivity in numerous cell processes (arrows). (C) Immunohistochemical staining for Bcl-2 showing strong immunoreactivity in nerve fiber (NFL) and ganglion cell (GCL) layers and in cell processes in the inner plexiform layer (IPL; arrow). (D) Immunohistochemical staining for ERK1/2 showing strong immunoreactivity in nuclei in the inner nuclear layer (INL; arrow) and in numerous cell processes (arrowheads). Abbreviations are defined in Figure 1 . Scale bar, 10 μm.
The authors thank Christel Van den Broeck for technical assistance. 
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Figure 1.
 
Photomicrograph of a retina from a diabetic subject. This is a negative control slide that was treated identically with the omission of the primary antibody showing no staining. NFL, nerve fiber layer; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer. Scale bar, 10 μm.
Figure 1.
 
Photomicrograph of a retina from a diabetic subject. This is a negative control slide that was treated identically with the omission of the primary antibody showing no staining. NFL, nerve fiber layer; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer. Scale bar, 10 μm.
Figure 2.
 
Photomicrographs of retinas from nondiabetic subjects. (A) Immunohistochemical staining for Bcl-2 showing weak immunoreactivity confined to the innermost retina in the nerve fiber layer (NFL; arrow). (B) Immunohistochemical staining for ERK1/2 showing weak immunoreactivity in a few nuclei in the inner nuclear layer (INL; arrow) and in a few Müller cell processes (arrowhead). (C) Immunohistochemical staining for survivin showing cytoplasmic immunoreactivity for survivin in retinal pigment epithelial cells (RPE; arrow). Abbreviations are defined in Figure 1 . Scale bar, 10 μm.
Figure 2.
 
Photomicrographs of retinas from nondiabetic subjects. (A) Immunohistochemical staining for Bcl-2 showing weak immunoreactivity confined to the innermost retina in the nerve fiber layer (NFL; arrow). (B) Immunohistochemical staining for ERK1/2 showing weak immunoreactivity in a few nuclei in the inner nuclear layer (INL; arrow) and in a few Müller cell processes (arrowhead). (C) Immunohistochemical staining for survivin showing cytoplasmic immunoreactivity for survivin in retinal pigment epithelial cells (RPE; arrow). Abbreviations are defined in Figure 1 . Scale bar, 10 μm.
Figure 3.
 
Photomicrographs of retinas from diabetic subjects. (A) Immunohistochemical staining for caspase-3 showing immunoreactivity in ganglion cells (arrows). (B) Immunohistochemical staining for FasL showing immunoreactivity in numerous cell processes (arrows). (C) Immunohistochemical staining for Bcl-2 showing strong immunoreactivity in nerve fiber (NFL) and ganglion cell (GCL) layers and in cell processes in the inner plexiform layer (IPL; arrow). (D) Immunohistochemical staining for ERK1/2 showing strong immunoreactivity in nuclei in the inner nuclear layer (INL; arrow) and in numerous cell processes (arrowheads). Abbreviations are defined in Figure 1 . Scale bar, 10 μm.
Figure 3.
 
Photomicrographs of retinas from diabetic subjects. (A) Immunohistochemical staining for caspase-3 showing immunoreactivity in ganglion cells (arrows). (B) Immunohistochemical staining for FasL showing immunoreactivity in numerous cell processes (arrows). (C) Immunohistochemical staining for Bcl-2 showing strong immunoreactivity in nerve fiber (NFL) and ganglion cell (GCL) layers and in cell processes in the inner plexiform layer (IPL; arrow). (D) Immunohistochemical staining for ERK1/2 showing strong immunoreactivity in nuclei in the inner nuclear layer (INL; arrow) and in numerous cell processes (arrowheads). Abbreviations are defined in Figure 1 . Scale bar, 10 μm.
Table 1.
 
Monoclonal and Polyclonal Antibodies Used in the Study
Table 1.
 
Monoclonal and Polyclonal Antibodies Used in the Study
Primary Antibody Dilution Incubation Time Source*
Anti-cleaved caspase-3 (Asp175) (pc) 1:25 120 minutes Cell Signaling Technology
Anti-Fas (B-10) (mc) 1:50 Overnight Santa Cruz Biotechnology, Inc.
Anti-FasL (N-20) (pc) 1:50 30 minutes Santa Cruz Biotechnology, Inc.
Anti-Bax (B-9) (mc) 1:50 30 minutes Santa Cruz Biotechnology, Inc.
Anti-Bcl-2 (clone 124) (mc) 1:10 30 minutes Dako
Anti-survivin (D-8) (mc) 1:20 30 minutes Santa Cruz Biotechnology, Inc.
Anti-p53 (clone DO-7 (mc) 1:10 30 minutes Dako
Anti-phospho-ERK1/ERK2 MAPK (E10) (mc) 1:30 Overnight Cell Signaling Technology
Anti-p38 MAPK (pc) 1:30 Overnight Cell Signaling Technology
Anti-GFAP (pc) 1:300 30 minutes Dako
Table 2.
 
Specificity of the Antibodies Used in the Study
Table 2.
 
Specificity of the Antibodies Used in the Study
Primary Antibody Specificity
Anti-cleaved caspase-3 Detects the large fragment of activated caspace-3 (17–20 kDa)
Anti-Fas Detects a peptide mapping at the carboxyl terminus of Fas of human origin
Anti-FasL Detects a peptide mapping at the amino terminus of FasL of rat origin
Anti-Bax Maps at amino acids 1-171 representing all but the carboxyl terminal 21 amino acids of Bax of mouse origin
Anti-Bcl-2 Reacts with the Bcl-2 oncoprotein
Anti-survivin Raised against a recombinant protein corresponding to amino acids 1-142 of survivin of human origin
Anti-p53 Reacts with a 53-kDa protein. The epitope recognized is located between the N terminal amino acids 1 and 45 and possibly between amino acids 37 and 45 of the human p53 protein
Anti-phospho-ERK1/ERK2 MAPK Detects endogenous levels of p44 and p42 MAP kinase (ERK1 and ERK2) dually phosphorylated at threonine 202 and tyrosine 204
Anti-p38 MAPK Detects total p38 MAP kinase (phosphorylation-state–independent) levels.
Anti-GFAP Reacts strongly with human GFAP.
Table 3.
 
Subject Characteristics
Table 3.
 
Subject Characteristics
Case Number Age/Sex Type of Diabetes Duration of Diabetes (y) Arterial Hypertension Retinopathy Status Cause of Death Death to Enucleation Time (h)
Subjects with diabetes
 1 75 F NIDDM 14 + NPDR Coronary artery disease 17
 2 70 M NIDDM 20 NPDR Renal failure 9
 3 58 M NIDDM 16 Unknown Intracranial bleeding 10
 4 67 F NIDDM 18 Unknown Coronary artery disease 21
 5 73 F NIDDM 15 + Absent Cerebrovascular accident 9
Subjects without diabetes
 5 52 M Colon cancer metastasis 17
 6 42 M Unknown 15
 7 79 M Coronary artery disease 17
 8 64 F Coronary artery disease 18
Table 4.
 
Summary of Staining Results
Table 4.
 
Summary of Staining Results
Antibody Retinas from Four Subjects without Diabetes* Retinas from Five Subjects with Diabetes*
Anti-cleaved caspase-3 − (8/8) + (10/10)
Anti-Fas − (8/8) + (10/10)
Anti-FasL − (8/8) +++ (10/10)
Anti-Bax − (8/8) + (10/10)
Anti-Bcl-2 + (8/8) ++ (10/10)
Anti-survivin ++ (8/8) ++ (10/10)
Anti-p53 − (8/8) − (10/10)
Anti-phospho-ERK1/ERK2 MAPK + (8/8) +++ (10/10)
Anti-p38 MAPK − (8/8) − (10/10)
Anti-GFAP + (8/8) +++ (10/10)
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