Abstract
purpose. This study aimed to determine the association between advanced glycation end products (AGEs) and glaucoma based on the known synergism between oxidative stress with AGEs and the evidence of oxidative stress during glaucomatous neurodegeneration.
methods. The extent and cellular localization of immunolabeling for AGEs and their receptor, RAGE, were determined in histologic sections of the retina and optic nerve head obtained from 38 donor eyes with glaucoma and 30 eyes from age-matched donors without glaucoma.
results. The extent of AGE and RAGE immunolabeling was greater in older than in younger donor eyes. However, compared with age-matched controls, an enhanced accumulation of AGEs and an up-regulation of RAGE were detectable in the glaucomatous retina and optic nerve head. Although some retinal ganglion cells (RGCs) and glia exhibited intracellular immunolabeling for AGEs, increased AGE immunolabeling in glaucomatous eyes was predominantly extracellular and included laminar cribriform plates in the optic nerve head. Some RAGE immunolabeling was detectable on RGCs; however, increased RAGE immunolabeling in glaucomatous eyes was predominant on glial cells, primarily Müller cells.
conclusions. Given that the generation of AGEs is an age-dependent event, increased AGE accumulation in glaucomatous tissues supports that an accelerated aging process accompanies neurodegeneration in glaucomatous eyes. One of the potential consequences of AGE accumulation in glaucomatous eyes appears to be its contribution to increased rigidity of the lamina cribrosa. The presence of RAGE on RGCs and glia also makes them susceptible to AGE-mediated events through receptor-mediated signaling, which may promote cell death or dysfunction during glaucomatous neurodegeneration.
Extended exposure of proteins to reducing sugars leads to the nonenzymatic glycation of amino groups by Maillard reaction, which alters the biological activity and degradation processes of proteins. In the early stage of this posttranslational protein modification, the synthesis of intermediates leads to the formation of Amadori compounds. In the late stage, advanced glycation end products (AGEs) irreversibly form through cross-linking after a complex cascade of chemical modifications, including “oxidation.” These reactive products, formed on intracellular and extracellular proteins, lipids, and nucleic acids, have complex structures that exhibit pigmentation and fluorescence properties. Several AGEs have been chemically characterized, whereas new compounds remain to be identified. Studies of the contribution of protein glycation to diseases have been primarily focused on its relationship to diabetes and diabetes-related complications, which in the eye include retinopathy, optic neuropathy, and cataract.
1 However, it has become clear that glycation-associated damage is not limited to patients with diabetes. Although it does not cause rapid or remarkable cell damage, glycation advances slowly and, in conjunction with oxidation, accompanies every fundamental process of cellular metabolism. Such alterations affect the physiological aging process because AGEs accumulate in various tissues in the course of aging.
2 3 Mostly because of their association with oxidative stress, AGEs have also been implicated in many neurodegenerative diseases, such as Alzheimer disease, amyotrophic lateral sclerosis, and Huntington disease.
4 5 As in many other age-dependent neurodegenerative diseases of the brain, oxidative stress-associated age-dependent pathogenic processes are not unexpected in glaucoma because this disease is also more common in the elderly.
6
AGEs are commonly thought to exacerbate disease progression through two general mechanisms. First, these modified proteins form detergent-insoluble and protease-resistant nondegradable aggregates and impair normal cellular/tissue functions. In neurons, such aggregates may interfere with axonal transport and intracellular protein traffic.
7 Second, AGEs modulate cellular function through binding to specific receptors. AGE-binding receptors, which are mostly found on monocytes, macrophages, endothelial cells, pericytes, microglia, and astrocytes, include receptor for AGE (RAGE). RAGE is a multiligand signal transduction receptor of the immunoglobulin superfamily.
2 8 Binding of AGEs to receptors such as RAGE induces the release of profibrotic cytokines, such as TGF-β, and proinflammatory cytokines, such as TNF-α and IL-6. Cell activation in response to AGE-modified proteins has also been associated with increased expression of extracellular matrix proteins, vascular adhesion molecules, and growth factors. However, depending on the cell type and concurrent signaling, RAGE-mediated events result not only in cell activation/proliferation, chemotaxis, and angiogenesis, but also in the generation of reactive oxygen species and apoptotic cell death.
2 3 4 8 Pharmacologic inhibition of RAGE-mediated cell activation with specific antagonists has been proposed as a therapeutic intervention in diseases in which AGE accumulation is a suspected etiological factor for vascular complications of diabetes, renal insufficiency, atherosclerosis, and neurodegeneration.
9 10
Thus, AGEs lead to the generation of reactive oxygen species, whereas AGE production is promoted by oxidative stress. Based on such a synergism between AGEs and oxidative stress, along with the growing evidence of oxidative stress during glaucomatous neurodegeneration,
11 this study aimed to determine the association of AGEs and their receptor, RAGE, with glaucoma, which is also an age-dependent disease.
6 Through immunohistochemical analysis, the extent and cellular localization of AGE and RAGE were determined in the retina and optic nerve head of eyes of donors with glaucoma compared with control eyes from age-matched donors. These revealed an enhanced accumulation of AGEs and an up-regulation of RAGE in the glaucomatous retina and optic nerve head, which support that an accelerated aging process accompanies neurodegeneration in glaucomatous eyes. Findings of this study suggest that one of the consequences of AGE accumulation in glaucomatous eyes may be the contribution of these aggregates to increased rigidity of the lamina cribrosa in the glaucomatous optic nerve head. The presence of RAGE on RGCs and glia, including mainly Müller cells, also makes them susceptible to AGE-mediated events through receptor-mediated signaling, which may promote cell death or dysfunction in patients with glaucoma.
Histologic sections were incubated with a mixture of anti–mouse and anti–rabbit primary antibodies for 1 hour at room temperature. Primary antibodies to AGEs and RAGE were the same as described. In addition, a rabbit antibody against glial fibrillary acidic protein (GFAP) was used as a marker of astrocytes, and an antivimentin antibody (1:200; Santa Cruz Biotechnology, Santa Cruz, CA) was used to recognize Müller cells. To identify RGCs, a rabbit antibody to brn-3 (1:200; Santa Cruz Biotechnology) was used. After incubation with primary antibody and a washing step, slides were incubated with a mixture of Alexa Fluor 488– or 568–conjugated anti–mouse, anti–goat, or anti–rabbit IgGs (1:400; Molecular Probes, Eugene, OR) for another hour at room temperature. Negative controls were performed by replacing the primary antibody with serum, or sections were incubated with each primary antibody followed by the inappropriate secondary antibody to determine that each secondary antibody was specific to the species against which it was made. After washing and mounting, slides were examined and images were recorded under a fluorescence microscope equipped with a digital camera (Carl Zeiss).
To determine whether AGE generation is associated with protein oxidation in glaucomatous eyes, double-immunofluorescence labeling was also performed for AGEs and protein carbonyls, as previously described.
11 Briefly, additional sections were incubated with 0.01% 2,4-dinitrophenylhydrazine (DNPH; Sigma-Aldrich) in 2 N HCl for 1 hour at room temperature. After washing and blocking steps, slides were incubated with a polyclonal goat antibody recognizing DNP (1:100; Biomeda, Foster City, CA) and the anti-AGE antibody described. Secondary antibodies included Alexa Fluor 568–conjugated anti–goat IgG and Alexa Fluor 488–conjugated anti–mouse IgG (1:400; Molecular Probes). Anti–DNP antibody or the DNPH treatment was omitted, or the primary antibody was replaced with serum, to confirm the specificity of immunolabeling.
Similar to the control retina, immunolabeling of the optic nerve head for AGEs or RAGE was faint in control eyes
(Figs. 4A 4B) . However, optic nerve head sections from glaucomatous eyes exhibited increased immunolabeling for AGEs and RAGE
(Figs. 4C 4D)in all histologic slides examined. AGE and RAGE immunolabeling of the glaucomatous optic nerve head were qualitatively graded as moderate or strong. Digital image analysis revealed that the extent of AGE immunolabeling was 36% ± 5% in the glaucomatous optic nerve head but less than 6% in age-matched controls
(Fig. 1) . The extent of RAGE immunolabeling in the optic nerve head was similarly greater in glaucomatous eyes (9% ± 1%) than in control eyes (less than 3%).
Based on double-immunofluorescence labeling, increased AGE immunolabeling in the glaucomatous optic nerve head was most detectable extracellularly in cribriform plates of the lamina cribrosa. Some GFAP-positive astrocytes and nerve bundles were also positive for AGE immunolabeling in these tissues
(Fig. 4E) . Although AGE immunolabeling of the optic nerve head was predominantly extracellular, increased RAGE immunolabeling in glaucomatous eyes was mainly localized to GFAP-positive astrocytes located at the prelaminar and laminar regions of the optic nerve head
(Fig. 4F) . Blood vessels also exhibited immunolabeling for AGEs and RAGE in the glaucomatous optic nerve head.
Control slides in which the primary antibody was omitted or replaced with serum were all negative for specific immunolabeling for AGEs or RAGE. Although the intensity and extent of immunolabeling and the number of immunolabeled cells exhibited individual or regional differences, increased immunolabeling of glaucomatous tissues for AGEs and RAGE was widespread and detectable in all slides examined. To determine the retinal orientation of histologic sections, 12 freshly obtained donor eyes were marked for nasal, temporal, superior, and inferior quadrants before processing. The correlation between immunolabeling and functional damage was estimated in these glaucomatous eyes. However, no correlation was detected between the location of visual field defects and AGE or RAGE immunolabeling in corresponding retinal quadrants of these individual eyes. In addition, it was considered that, because of the retrospective nature of data collection, assessment of a relationship between AGE or RAGE immunolabeling with other clinical variables would not be precisely informative.
This immunohistochemical study detected that in comparison with control eyes from age-matched donors, there was an increase in immunolabeling of the glaucomatous retina and optic nerve head for different AGE structures, including N(epsilon)-(carboxymethyl)lysine. Because the generation of AGEs is an age-dependent event, findings of this study support that an accelerated aging process accompanies neurodegeneration in glaucomatous eyes. Findings of this study also demonstrate that enhanced accumulation of AGEs in glaucomatous eyes is associated with oxidative stress, as assessed by protein carbonyl immunoreactivity. Protein carbonyl formation is an important marker for protein oxidation, which can arise from direct free radical attack on amino acid side chains. The distribution of protein carbonyls in the glaucomatous human retina was similar to that detected in the eyes of rats with ocular hypertension, which exhibit oxidative modification of important proteins present in RGCs and glia, including Müller cells.
11 The colocalization of AGEs and protein carbonyls in the glaucomatous retina and optic nerve head supports the known synergism between AGEs and oxidative stress, in which AGEs lead to the generation of reactive oxygen species and AGE production is promoted by oxidation.
20
AGEs can initiate a wide range of abnormal responses with serious consequences for macromolecular function. These include inappropriate expression of important structural proteins, enzymes, and growth factors, induction of TNF-α and nitric oxide synthase, alterations in cellular growth dynamics and migration, glial activation, accumulation of extracellular matrix molecules, promotion of vasoregulatory dysfunction, induction of oxidative cascades, and initiation of cell death pathways. Many of these are receptor-mediated events through the binding of RAGE and can be significantly reversed by pharmacologic inhibitors.
21 It is now clear that AGEs are neurotoxic,
22 and they may act as mediators not only of diabetic complications but also of many age-related abnormalities, including age-related neurodegenerative diseases.
1
Increasing numbers of reports confirm widespread AGE accumulation at sites of different ocular abnormalities, and various in vitro and in vivo studies highlight the putative pathophysiological role of AGEs in retinal cell dysfunction. For example, AGEs have been shown to accumulate in the diabetic retina
23 and optic nerve head
24 and have been associated with retinopathy.
25 26 Because of the colocalization of AGEs with RAGE at sites of diabetic microvascular injury, it has also been suggested that this ligand–receptor interaction represents an important mechanism in the pathogenesis of diabetic complications.
27 28 Furthermore, inhibitors of AGE formation
29 30 31 32 or RAGE
28 have ameliorated neuronal dysfunction and vascular disease in diabetic eyes. In addition, it has been suggested that the accumulation of AGEs contributes to the progression of age-related macular degeneration in human eyes because they induce receptor-mediated activation of retinal pigment epithelial cells.
33
Similar to other diseases, AGEs detected in glaucomatous tissues may be directly cytotoxic or may initiate receptor-mediated signaling. Increased AGE immunolabeling in glaucomatous eyes was detectable in RGCs, their axons, and glia. These intracellular aggregates may interfere with normal cellular functions, including axonal transport and intracellular protein traffic.
7 However, accelerated accumulation of AGEs in glaucomatous tissues was predominant in the extracellular matrix of the retina and optic nerve head. Because AGEs accumulate with age on many long-lived macromolecules such as collagen, it is not surprising that these products were prominently detectable within the extracellular matrix. Accumulation of AGEs in the extracellular matrix may elicit several alterations, including decreased solubility, decreased susceptibility to enzymes, and changes in thermal stability, mechanical strength, and stiffness. It has been suggested that such alterations in the physicochemical properties of the extracellular matrix contribute to the development of various age-related abnormalities.
34 35 36 37 38 Extracellular accumulation of AGEs in the glaucomatous optic nerve head may be particularly important because extracellular matrix sheets of the lamina cribrosa provide mechanical support for RGC axons.
The ability of the optic nerve head to withstand elevations in intraocular pressure decreases with increasing age because of age-related alterations in the proportion of various components of the extracellular matrix in the lamina cribrosa.
39 40 In addition to these alterations, a linear increase in AGE accumulation has been observed in the aging lamina cribrosa, possibly associated with decreased elasticity of the lamina cribrosa in the elderly.
41 Extensive evidence supports that the content and distribution of age-dependent alterations of the extracellular matrix are even more severe in the glaucomatous optic nerve head.
42 43 Clinical observations are consistent with histopathologic findings in glaucomatous eyes.
44 Current findings support that the accelerated accumulation of AGEs in laminar cribriform plates accompanies other extracellular matrix alterations in the glaucomatous optic nerve head, which influence the susceptibility of stressed axons to sustain neuronal damage in glaucomatous eyes.
45 46 Increased accumulation of AGEs in laminar cribriform plates and blood vessels of the glaucomatous optic nerve head may facilitate axonal damage by compromising the ability of lamina cribrosa to bear the strain caused by elevated intraocular pressure or by impairing the microcirculation.
The AGE receptor RAGE was also up-regulated in the glaucomatous retina and optic nerve head. Some astrocytes and RGCs exhibited immunolabeling for RAGE in glaucomatous tissues; however, increased RAGE immunolabeling was predominantly localized to Müller cells in the glaucomatous retina. The up-regulation of RAGE in neurons and glia is consistent with observations in patients with brain injury.
4 5 47 48 RAGE up-regulation in glaucomatous eyes suggests that in addition to direct cytotoxic effects of intracellular or extracellular AGEs, these reactive products may initiate specific receptor-mediated signaling that can promote cell death and dysfunction. The presence of RAGE on RGCs makes them susceptible targets of AGE-mediated events. Increased RAGE immunolabeling of glial cells in glaucomatous tissues similarly indicates that AGEs can modulate glial functions through receptor-mediated signaling.
A predominant up-regulation of RAGE on glial cells raises exciting possibilities. First, based on known outcomes of RAGE-mediated signaling, glial up-regulation of RAGE in glaucomatous eyes may be associated with glial activation
14 and activated glial migration.
49 Despite the preferential susceptibility of RGCs and their axons to primary or secondary degeneration, glial cells survive the widespread and chronic tissue stress present in the glaucomatous optic nerve head and retina.
12 15 Although glial cells are relatively protected against glaucomatous injury,
50 they significantly respond to glaucomatous stressors by persistently exhibiting an activated phenotype in glaucomatous human eyes.
14 Chronic signaling through RAGE may explain, in part, how glial cells persistently remain activated in these eyes, even after elevated intraocular pressure is lowered.
Second, RAGE-mediated signaling in glial cells may be associated with alterations in their neurosupportive functions during glaucomatous neurodegeneration. Despite the well-known role of glial cells in supporting RGCs, considerable evidence suggests that under glaucomatous stress conditions, their neu rosupportive ability may diminish and glial cells may become neurodestructive by the release of increased amounts of neurotoxic substances
50 or by the activation of an aberrant immune response.
51 This is supported by increased glial production of TNF-α,
13 nitric oxide synthase,
52 and antigen-presenting ability of glial cells
53 in glaucomatous human eyes. Based on known consequences of AGE/RAGE signaling, it seems reasonable to hypothesize that age-dependent and oxidative stress–induced events mediated through AGE/RAGE signaling play a role in the decreased ability of glial cells to protect RGCs from glaucomatous injury. For example, one of the numerous neurosupportive functions of glial cells is associated with their ability to control extracellular levels of excitotoxic amino acids such as glutamate. Müller cells are known to be the primary cell type responsible for the removal of excess glutamate from the extracellular space in the retina.
54 55 56 However, not only are glutamate transporters significantly reduced in human and experimental glaucoma,
57 58 glutamine synthetase, which modulates the glutamate–glutamine cycle, is oxidatively modified during glaucomatous neurodegeneration.
11 These findings lend support to the notion that besides other dysfunctions, depression of the glutamate balancing function of Müller cells caused by oxidative inactivation can facilitate RGC death in glaucoma. However, whether a specific RAGE-dependent mechanism is associated with glial cell activation or oxidative stress–associated glial dysfunction in glaucoma must be proven in future studies. It is also tempting to determine whether AGE/RAGE signaling in glial cells could be associated with their immune regulatory function during aberrant activation of the immune system in patients with glaucoma.
51 59
It seems confusing that advanced glycation processes occur in nondiabetic glaucomatous eyes. However, based on the known process of AGE generation, glucose and its degradation products can participate in the aberrant glycation of proteins
60 61 and the generation of AGEs.
62 Energy supply for neurons in the retina is generated from glucose through the glycolytic pathway. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a major glycolytic enzyme in this pathway. If GAPDH activity were to decline, the two glycolytic intermediates, glyceraldehyde-3-phosphate and dihydroxyacetone phosphate, would likely accumulate, leading to the production of methylglyoxal. Methylglyoxal has been identified as a precursor of AGEs in the lens
63 and retina
64 and has been associated with increased stiffness of the human lamina cribrosa and peripapillary sclera during the physiological aging process.
65 What links such alternative metabolic routes to AGE generation in glaucoma is the evidence of oxidative modification of GAPDH in ocular hypertensive retinas.
11 In fact, GAPDH is easily affected by oxidants, resulting in the loss of dehydrogenase activity.
66 Therefore, it seems highly possible that a similar oxidation-induced decline in GAPDH activity during glaucomatous neurodegeneration may be associated with accelerated AGE formation in glaucomatous tissues, even in the presence of normal blood glucose levels. Such facilitated AGE formation by oxidative stress is consistent with the known synergism between reactive oxygen species and AGEs. Our more recent in vivo study using a proteomic approach has demonstrated aberrant protein glycation in ocular hypertensive and diabetic retinas and has identified common targets of this posttranslational protein modification in two different disease models (Atmaca-Sonmez P, et al.
IOVS 2006;47:ARVO E-Abstract 197). Increased retinal protein glycation in rats with ocular hypertension and AGE accumulation by physiological aging through lifelong exposure to normoglycemia are consistent with the findings of increased AGE accumulation in ocular tissues of nondiabetic donors with glaucoma in the present study.
In summary, findings of this study demonstrate an enhanced accumulation of AGEs and an up-regulation of RAGE in the glaucomatous retina and optic nerve head. Known consequences of AGE/RAGE signaling suggest that key cellular events associated with glaucomatous neurodegeneration, such as oxidative stress, glial activation and dysfunction (including activated glial migration, increased glial production of TNF-α and nitric oxide synthase, and activated immunoregulatory function), inappropriate activation of signaling molecules (including mitogen-activated protein kinases and nuclear factor-κB), activated immune response, and neuronal apoptosis, may all have important links to advanced glycation processes in glaucoma. These warrant further study for a better understanding of the pathogenic importance of AGE/RAGE-mediated cytotoxicity in glaucomatous neurodegeneration.
Supported in part by National Eye Institute Grants R01 EY013813 and R24 EY015636 and by an unrestricted grant to the University of Louisville Department of Ophthalmology and Visual Sciences from Research to Prevent Blindness Inc. GT is a recipient of the Research to Prevent Blindness Sybil B. Harrington Special Scholar Award.
Submitted for publication June 30, 2006; revised August 14, September 28, and October 17, 2006; accepted January 3, 2007.
Disclosure:
G. Tezel, None;
C. Luo, None;
X. Yang, None
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “
advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Corresponding author: Gülgün Tezel, Department of Ophthalmology and Visual Sciences, University of Louisville School of Medicine, Kentucky Lions Eye Center, 301 E. Muhammad Ali Boulevard, Louisville, KY 40202;
[email protected].
Table 1. Demographic and Clinical Data of Donor Eyes
Table 1. Demographic and Clinical Data of Donor Eyes
| Age (y) | Sex | Diagnosis | Average IOP | C/D | VF Damage |
| Donors with glaucoma | | | | | | |
| 1 | 54 | M | POAG | 19 | 0.7 | Moderate |
| 2 | 54 | M | POAG | 20 | 0.7 | Moderate |
| 3 | 56 | F | POAG | 18 | 0.9 | Advanced |
| 4 | 59 | M | POAG | 18 | 0.6 | Moderate |
| 5 | 59 | M | POAG | 18 | 0.8 | Moderate |
| 6 | 68 | F | NPG | 16 | 0.8 | Moderate |
| 7 | 69 | F | POAG | 16 | 0.5 | Moderate |
| 8 | 69 | F | POAG | 17 | 0.5 | Moderate |
| 9 | 70 | M | POAG | 19 | 0.6 | Moderate |
| 10 | 70 | M | POAG | 19 | 0.7 | Moderate |
| 11 | 72 | F | POAG | 21 | 0.8 | Moderate |
| 12 | 72 | F | POAG | 18 | 0.7 | Moderate |
| 13 | 74 | F | POAG | 20 | 0.7 | Moderate |
| 14 | 74 | F | NPG | 16 | 0.8 | Moderate |
| 15 | 74 | F | NPG | 17 | 0.9 | Advanced |
| 16 | 75 | F | NPG | 16 | 0.85 | Moderate |
| 17 | 75 | F | NPG | 15 | 0.8 | Mild |
| 18 | 76 | F | POAG | 18 | 0.9 | Advanced |
| 19 | 76 | M | POAG | 18 | 1.0 | Advanced |
| 20 | 76 | M | POAG | 25 | 1.0 | Advanced |
| 21 | 78 | M | POAG | 22 | N/A | N/A |
| 22 | 79 | F | POAG | 20 | 0.7 | Moderate |
| 23 | 79 | F | POAG | 13 | 0.9 | Advanced |
| 24 | 82 | M | POAG | 23 | N/A | N/A |
| 25 | 82 | F | POAG | 24 | 0.8 | Moderate |
| 26 | 82 | F | NPG | 15 | 0.8 | Moderate |
| 27 | 82 | F | NPG | 15 | 0.8 | Moderate |
| 28 | 84 | F | NPG | 13 | 0.95 | Advanced |
| 29 | 84 | F | NPG | 12 | 0.95 | Advanced |
| 30 | 85 | M | POAG | 17 | 1.0 | Advanced |
| 31 | 85 | M | POAG | 22 | 0.5 | Moderate |
| 32 | 91 | F | POAG | 21 | 0.9 | Advanced |
| 33 | 91 | F | POAG | 20 | 0.8 | Moderate |
| 34 | 91 | F | POAG | 16 | 0.7 | Moderate |
| 35 | 91 | F | POAG | 16 | 0.9 | Advanced |
| 36 | 94 | F | POAG | 16 | 0.7 | Moderate |
| 37 | 94 | M | POAG | 22 | 0.8 | Moderate |
| 38 | 94 | M | POAG | 20 | 0.5 | Moderate |
| Control donors | | | | | | |
| 1 | 44 | M | — | — | — | — |
| 2 | 44 | M | — | — | — | — |
| 3 | 49 | F | — | — | — | — |
| 4 | 49 | F | — | — | — | — |
| 5 | 55 | M | — | — | — | — |
| 6 | 55 | M | — | — | — | — |
| 7 | 56 | M | — | — | — | — |
| 8 | 56 | M | — | — | — | — |
| 9 | 59 | F | — | — | — | — |
| 10 | 59 | F | — | — | — | — |
| 11 | 68 | F | — | — | — | — |
| 12 | 68 | F | — | — | — | — |
| 13 | 70 | M | — | — | — | — |
| 14 | 70 | M | — | — | — | — |
| 15 | 73 | F | — | — | — | — |
| 16 | 73 | F | — | — | — | — |
| 17 | 76 | F | — | — | — | — |
| 18 | 76 | F | — | — | — | — |
| 19 | 79 | M | — | — | — | — |
| 20 | 79 | M | — | — | — | — |
| 21 | 82 | F | — | — | — | — |
| 22 | 82 | F | — | — | — | — |
| 23 | 84 | F | — | — | — | — |
| 24 | 84 | F | — | — | — | — |
| 25 | 85 | M | — | — | — | — |
| 26 | 85 | M | — | — | — | — |
| 27 | 91 | F | — | — | — | — |
| 28 | 91 | F | — | — | — | — |
| 29 | 94 | F | — | — | — | — |
| 30 | 94 | F | — | — | — | — |
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