AIMS To localise advanced glycation end products (AGEs) in human optic nerve head.
METHODS Optic nerve samples from 13 elderly individuals (seven diabetics and six non-diabetics) were obtained at necropsy. Pyrraline, an advanced glycation end product, was immunohistochemically localised in the optic nerve heads.
RESULTS In the diabetic subjects, moderate to intense immunoreactivity for pyrraline was detected in sclera, pia mater, cribriform plates, connective tissues in the optic nerve, and around vessels in the optic nerve and pia mater. Immunoreactivity for pyrraline was also detected around retinal vessels. In the non-diabetic subjects, slight or no immunoreactivity for pyrraline was found in cribriform plates and around the optic nerve vessels.
CONCLUSION Accumulation of AGEs in cribriform plates and around vessels in the optic nerve may contribute to the development of optic neuropathy in diabetic patients.
- optic nerve head
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Age is a major risk factor for chronic open angle glaucoma (COAG). Virtually every study agrees that the prevalence of COAG increases with age of the population being considered.Diabetes is also reported to be a risk factor for COAG in several studies, although further information from population based studies is necessary to definitely prove the role of diabetes in the development of COAG. While direct mechanical damage to the optic nerve and/or microangiopathy in the optic nerve head is a possible contributing mechanism in the pathogenesis of glaucoma, the exact nature of age or diabetes related changes that contribute to the development of COAG is not known.
Recently, advanced glycation end products (AGEs) have been revealed to have an important role in age related changes and diabetic complications in several organs. AGEs have also been reported to accumulate in diabetic or ageing ocular tissues including cornea, lens,vitreous, and retina. Several AGE structures have been identified including ε-2-(formyl-5-hydroxymethyl-pyrrol-l-yl)-l-norleucine (pyrraline), pentosidine,N[ε]-(carboxymethyl) lysine (CML), and crossline. Among them, pyrraline is one of the major Maillard compounds resulting from long time incubation of glucose and proteins. Pyrraline is known to increase in lens, sclerosed glomeruli, and perivascular extracellular matrix (ECM) in elderly subjects and diabetic patients.
In the present study, we performed immunohistochemistry for pyrraline in the human specimens to localise AGEs in the optic nerve head.
Materials and methods
Thirteen optic nerve samples of 13 individuals (seven diabetics and six non-diabetics) were obtained at the time of necropsy at the division of pathology, University of Tokyo Hospital. The clinical characteristics of the subjects are summarised in Table 1. None of these subjects had suffered from any diseases affecting the optic nerve.
Polyclonal antibody to pyrraline was produced as previously reported. To prepare immunogen, 6 mg of caproyl pyrraline was incubated with 6 mg of keyhole limpet haemocyanin (KLH) in the presence of 50 mM of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (Pierce, Rockford, IL, USA) hydrochloride and 2.5 mM of N-hydroxysulphosuccinimide (Pierce, Rockford, IL, USA) at room temperature for 1 hour. The reaction was terminated by 20 mM of 2-mercaptoethanol and dialysed against phosphate buffered saline (PBS) for 24 hours. To raise the polyclonal antibody, 0.5 mg of caproyl pyrraline conjugated KLH in 50% Freund's complete adjuvant was injected intradermally into a rabbit at 20 skin sites, followed by four booster injections with the same amount of hapten conjugated KLH in 50% Freund's incomplete adjuvant. The serum was taken 10 days after the final immunisation for further affinity purification. The antiserum against hapten conjugated KLH was passed over 5 ml of protein G immobilised sepharose gel (Amersham Pharmacia Biotech, Uppsala, Sweden). The column was extensively washed with PBS and eluted with 0.1 M citric acid buffer (pH 3.0). The pooled antibody fraction was neutralised, concentrated, and dialysed against PBS and used as affinity purified polyclonal antibody in the present study.
Immunohistochemical staining was performed by the streptavidin-biotin-complex method. After fixation with 4% paraformaldehyde, optic nerve samples were embedded in paraffin. At least, 10 longitudinal sections 4 μm thick were obtained in each optic nerve sample. The sections were preincubated in 1.0% hydrogen peroxide solution in PBS for 20 minutes at room temperature to inhibit the endogenous peroxidase activity. After washing with PBS three times for 10 minutes at 4°C, the sections were incubated with 10% normal goat serum (Histofine, Nichirei, Tokyo) for 2 hours at room temperature to inhibit non-specific binding. The sections were then exposed to primary antibody (rabbit polyclonal antibody for pyrraline) diluted to 1.0 mg/ml with PBS containing 1% bovine serum albumin and 0.04% sodium azide for 24 hours at 4°C. After washing with PBS three times for 20 minutes at room temperature, secondary antibody (goat biotinylated anti-rabbit IgG: Histofine) was added for 2 hours at room temperature. The sections were then incubated with peroxidase conjugated streptavidin for 2 hours at room temperature. To visualise the immunoreactions, sections were incubated with diaminobenzidine (DAB) in PBS. Finally, sections were counterstained with haematoxylin. For negative control, we replaced the primary antibody with normal rabbit serum IgG (1.0 mg/ml) diluted with PBS containing 1% bovine serum albumin and 0.04% sodium azide. Following these preparations, the sections were processed to immunostaining as described above. The intensity of immunoreactivity was graded by a masked observer qualitatively as negative, weak, moderate, or intense.
Since the sample number was small for statistical analysis, the intensity of immunoreactivity was reclassified into two groups: negative (negative or weak immunoreactivity) and positive (moderate or intense). Then, the immunoreactivity for pyrraline was compared between diabetics and non-diabetics using a two tailed Fisher's exact test, with p<0.05 deemed significant.
The results of immunohistochemistry are summarised in Table 2. In the diabetic subjects, moderate to intense immunoreactivity for pyrraline was detected in sclera, pia mater, cribriform plates, connective tissues in optic nerve, and around vessels in optic nerve and pia mater (Fig 1A and B). Immunoreactivity for pyrraline was also detected around retinal vessels (Fig 1C). There was no immunoreactivity in optic nerve axons. In the negative controls in which normal rabbit serum IgG was used instead of primary polyclonal antibody to pyrraline, there was no immunoreactivity in optic nerve heads (data not shown).
In the non-diabetic subjects, slight or no immunoreactivity for pyrraline was found in cribriform plates and around optic nerve vessels (Fig 2A and B). There was no immunoreactivity in optic nerve axons (Fig2B) and retinas (Fig 2C).
The differences in the intensity of pyrraline immunoreactivity between diabetics and non-diabetics were statistically significant in cribriform plates, sclera, pia mater, and vessels in each tissue (p<0.01).
AGEs have been shown to accumulate in diabetic and ageing organs, including ocular tissues (cornea, lens,vitreous, and retina). Accumulation of AGEs in ECM was shown to elicit several changes of ECM including decreased solubility, decreased susceptibility to enzymes, and changes in such properties as thermal stability, mechanical strength, and stiffness. These changes in the physicochemical properties of ECM are believed to contribute, in part, to the development of age related changes and diabetic complications, including cardiovascular disease, retinopathy, nephropathy, indurated skin and joint stiffness. Albonet al reported a linear age related increase of pentocidine, an advanced glycation end product, in lamina cribrosa. Accumulation of AGEs in ECM of optic nerve heads in the elderly may decrease elasticity of lamina cribrosa and compromise the ability of cribriform plates to bear the strain caused by elevated intraocular pressure. The results of the current study showed that accumulation of pyrraline in cribriform plates was accelerated in diabetics compared with non-diabetics, suggesting that the mechanical properties of the lamina cribrosa change more severely in diabetics than in non-diabetics. Such alterations in cribriform plates may contribute to more frequent development of glaucoma in diabetics.
Owing to the scarcity of samples, we could not examine the accumulation of pyrraline in younger subjects. We obtained one sample of a fetus of 34 weeks, in which immunoreactivity for pyrraline was completely absent in ECM and vessels in the optic nerve head (data not shown). A previous study demonstrated a linear age related increase of pentosidine in the lamina cribrosa in subjects ranging from 3 weeks to 92 years old. Thus, it is probable that pyrraline accumulates in lamina cribrosa with age.
Dramatic changes in ECM of lamina cribrosa in COAG have been reported including a decrease of collagen density and an appearance of masses of elastin. These changes in ECM may alter the mechanical properties of the tissue early in the disease. In the current series, optic nerve heads from eyes with glaucoma were not available. Examination of AGEs in glaucomatous subjects may further clarify the involvement of AGEs in the development of glaucoma.
AGEs have been shown to accumulate in myelin and tubulin of peripheral nerves. Non-enzymatic glycation of such neural proteins is thought to impair axonal transport, which may induce diabetic neuropathy. In the present study, immunoreactivity for pyrraline was not detected in neural component of optic nerves, suggesting that diabetic optic neuropathy is different from diabetic neuropathy in other peripheral nerves in that it is not induced by the accumulation of AGEs in the neural proteins.
In conclusion, advanced accumulation of AGEs in cribriform plates and around optic nerve vessels in diabetics may contribute to the development of optic neuropathy by compromising the ability of cribriform plates to bear the strain caused by elevated intraocular pressure and/or impairing the microcirculation.
The authors have no commercial or proprietary interest in the product or company described in this paper.
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