Ординатура / Офтальмология / Английские материалы / Aging and Age Related Ocular Diseases_Lutjen-Drecoll_2000

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Key Words

Immunohistochemistry W Anterior eye segment W Optic nerve W Heart muscle W Dot blot W ·B-Crystallin

Abstract

·B-Crystallin is constitutively expressed in a variety of tissues including the nervous system, the eye, heart and striated muscles and the kidney. The functional significance of the protein in the different cell populations is not yet known. Experimental data indicate that mechanical stress to the cells might play a role but that there is also a close correlation with markers of oxidative activity. Increased expression of ·B-crystallin is seen in a number of age-related degenerative diseases. Whether aging per se induces expression of the protein has not been investigated yet. In this study tissue samples of the anterior eye segment, optic nerve, heart muscle and thyroid gland from mouse, rat, pig, cow and human donors of different age groups were investigated with immunohistochemical methods. ·B-Crystallin levels in heart muscle and optic nerve samples from different species and different age groups were investigated using protein immunoblotting (dot blot) and the mRNA levels using semiquantitative PCR methods. The results showed that neither in heart muscle known to show constitutively high

amounts of the protein nor in nonlenticular eye tissues with variations in staining intensity of different cell populations or in glandular cells studied for the first time, there were significant age-related staining differences. Dot blot methods as a quantitative evaluation method gave similar results. There were, however, species differences. In the eye these differences could be due to functional differences related to the development of a fovea centralis and an accommodative system in primates. In addition, in all mouse tissues there was less protein expression than in the other species. Differences in the absolute life span might be a factor involved in ·B-crys- tallin expression. In summary the findings show that an increase in ·B-crystallin with age may occur but is not a general phenomenon in tissues constitutively expressing this protein.

Copyright © 2000 S. Karger AG, Basel

Introduction

Until recently it has been assumed that ·B-crystallin occurs exclusively in ocular lenses forming part of the structural ·-crystallins [1]. However, in the last decade the presence of the protein has also been demonstrated in a variety of nonlenticular cells in the body. Immunoand

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Northern blotting have been used to identify the protein and its corresponding mRNA in heart and skeletal muscle, in Schwann cells of the peripheral nervous system, in skin, kidney, lung and inner ear (table 1). The functional significance of the protein in these different cell populations is not yet known.

In vitro ·B-crystallin gene expression can be induced by heat, chemical and osmotic stress, consistent with the idea that ·B-crystallin is a functional heat shock protein [2±4]. Horwitz [5] was the first to demonstrate that ·B- crystallin has chaperone-like properties protecting other proteins against heat aggregation and aid chemically denatured proteins to renature properly [for a review, see 6].

In vivo, ·B-crystallin was mainly found in desmin preparations from heart muscle. Immunohistochemical staining indicates that it might be involved in the organization of cytoskeletal filaments of the Z lines [7]. Previous studies have already revealed a high concentration of ·B- crystallin in both fetal and adult hearts with similar amounts of the protein [8, 9]. This finding may be related to the early functional maturation of this tissue. In adult rats, passive tension increased ·B-crystallin expression in fast and slow skeletal muscles, whereas denervation increased expression of the protein in fast but decreased it in slow muscles [10]. These data indicate that mechanical stress to the cells might be one of the factors explaining the

constitutive presence of the stress protein. However, other factors may also play a role. In muscle cells and in some of the epithelial cells which constitutively express ·B-crys- tallin, Iwaki et al. [11] found a close correlation with markers of oxidative activity. During early development, in chicken ·B-crystallin one of the proteins is selectively present in cells undergoing cytomorphological reorganization [12]. Hence it might be involved in cellular morphogenesis [13]. On the other hand, increased expression of ·B-crystallin is also seen in age-related degenerative diseases. Rosenthal fibers of astrocytes of patients with Alexander's disease express ·B-crystallin [14, 15]. The protein has also been found in glial cells in a number of other neurodegenerative diseases [16±19]. In fibroblasts, which normally do not express the protein, ·B-crystallin has been detected in patients with Werner's syndrome, a disease characterized by inherited premature aging [20]. In senescence-accelerated (SAMP) mice, ·B-crystallin expression in the brain cortex and cerebellum was higher than in the control group [21]. Whether aging per se induces expression of the protein has not been investigated yet. We therefore studied the distribution of ·B- crystallin in the anterior eye segment, in the retrolaminar optic nerve and the heart muscle in various age groups of rats and mice, as well as in some age groups of pigs, cows and primates. In addition, quantitative immunoblotting and semiquantitative PCR methods were used to study

Table 2. Number of tissues in different age groups investigated by immunohistochemistry

the concentration and mRNA levels of ·B-crystallin in the retrolaminar optic nerve and heart muscle in different age groups of the same species.

Material and Methods

Tissue samples of the anterior eye segment, retrolaminar optic nerve, heart muscle and thyroid gland were collected from mouse, rat, pig, cow and human donors. From our own breedings, C57/ Black6 mice (3 weeks to 23 months of age), SAMP accelerated-aging mice (11±12 months of age), Wistar rats (3 weeks to 12 months of age) and pink-eyed rdy+ Royal College of Surgeons (RCS) rats (9 days to 22 months of age) were used. From the local slaughterhouse tissue samples of pigs (5, 6 months and 6 years of age) and cows (7 months and 5, 6 years of age) were obtained (tables 2, 3). Human tissue was obtained from 19to 87-year-old donors (postmortem time listed in table 5).

Immunohistochemistry

Anterior eye segments, optic nerve, heart muscle and gland tissue samples were fixed in 4% paraformaldehyde for 4±6 h, then rinsed in Tris-buffered saline (TBS, pH 7.4). Frozen, 14-Ìm-thick sections through the heart and glands, sagittal and tangential sections through the retrolaminar optic nerve and the anterior eye segment were mounted on poly-L-lysine-covered glass slides. In addition, whole mounts of the anterior eye segment of rat eyes were stained free floating. After preincubation with Blotto's dry milk solution [22] for 30 min to reduce nonspecific background staining, incubation with a polyclonal rabbit anti-·B-crystallin antibody (dilution 1:400; antibody described in Flügel et al. [23]) was performed overnight in a moist chamber at room temperature. Sagittal sections through the optic nerve were also stained with a polyclonal rabbit anti-GFAP antibody (1:200; Bio Genex Laboratories, San Ramon, Calif., USA). After rinsing in TBS, a fluorescein-conjugated goat antirabbit Cy3

antibody was added (dilution 1:800; Dianova, Hamburg, Germany) for 1 h. The slides were rinsed again in TBS and mounted with Kaiser's glycerin gelatine. An Aristoplan fluorescence microscope (Leitz, Wetzlar, Germany) was used to examine the staining reaction.

Immunoelectron Microscopy

Paraformaldehyde-perfused small heart muscle tissue samples of 3 Wistar rats were immersion fixed in a mixture of 4% paraformaldehyde and 30% dimethylformamide (DMF) for 1 h on ice. Cryoprotection was performed by washing in 30% DMF twice for 30 min on ice. The samples were then incubated in methylbutane for 30 min and rapidly frozen in liquid nitrogen using an EM-CPC workstation (Leica). Low-temperature embedding was performed using Lowicryl HM20 according to the instruction manual of the manufacturer (Polysciences Europe, Eppelheim, Germany). After UV polymerization, ultrathin sections were mounted on Ni-grids, air dried and preincubated with 2% bovine serum albumin in phosphate-buffered saline (PBS, pH 7.4) for 20 min. Incubation with the primary antibody (rabbit anti-·B-crystallin, 1:100) followed for 60 min. The grids were then washed and labeled using goat antirabbit IgG and 10-nm gold particles. Uranyl-stained sections were viewed with a Zeiss EM 902 (Zeiss, Oberkochen, Germany).

Protein Immunoblotting (Dot Blot)

Frozen unfixed heart muscle and retrolaminar optic nerve tissue samples were homogenized with 2 ! sample buffer (20% glycerol, 6% sodium dodecyl sulfate in 0.12 M Tris, pH 6.8) and five times freeze-and-thaw lysed, using liquid nitrogen and a 37 ° C waterbath, respectively. Following spinning at 13,000 g for 10 min, the protein content of the supernatant was measured using BCA protein assay reagent (Pierce, Rockford, Ill., USA). The samples were diluted with 2 ! sample buffer to equalize protein concentration and further diluted with 20% methanol to a working dilution of 10 Ìg protein/ 100 Ìl. A standard of purified ·B-crystallin was prepared with bovine serum albumin to reach the same protein concentration as the tissue samples. Methanol-presoaked nitrocellulose transfer mem-

Table 3. Number of tissues in different age groups investigated by protein immunoblotting (dot blot) and polymerase chain reaction (PCR)

branes (Schleicher and Schuell, Dassel, Germany) were fixed in a 96well dot blot apparatus (Minifold I SRC 96 D, Schleicher and Schuell) and 100 Ìl of the standard and probes were added. After vacuum filtering and washing in 20% methanol the transfer membrane was air dried.

For immunostaining, the membrane was incubated with PBS containing 0.05% Tween 20 and 1% milk powder for 1 h. The primary antibody (·B-crystallin diluted 1:20,000) was then added and allowed to react overnight at room temperature. After washing twice in PBS + Tween and once in alkaline phosphate buffer (0.1 M NaCl, 5 mM MgCl, 0.1 M Tris; pH 9.5), the membrane was incubated with an alkaline-phosphatase-conjugated goat antirabbit IgG antibody (dilution 1:10,000; Promega, Madison, Wisc., USA) for 2 h. Visualization was achieved using fast NBT/BCIP buffered substrate tablets (Sigma, St. Louis, Mo., USA). The reaction was stopped after 10 min with 20 mM EDTA in PBS and the membrane was air dried.

For densitometric quantitation of ·B-crystallin the membranes were scanned with a Hewlett Packard Scanner (Scan Jet IIcx) and analyzed using TINA (ray test) and EXCEL software.

Semiquantitative Polymerase Chain Reaction

Total RNA of rat and human optic nerve specimens and rat heart muscle was isolated by the guanidinium thiocyanate phenol-chloro- form single-step extraction (RNA isolation kit; Stratagene, La Jolla, Calif., USA). The structural integrity of the total RNA samples was analyzed by 1.2% agarose gel electrophoresis with ethidium bromide staining. To remove traces of contaminating genomic DNA, all RNA samples were treated with 3 units of RQ RNase-free DNase (Promega) for 35 min at 37°C. Samples were then extracted with phenolchloroform (1:1) and precipitated by addition of ethanol. First-strand complementary DNA preparation and polymerase chain reaction (PCR) amplification were performed with the same methods and conditions described previously [24] using a specific primer for glyc- eraldehyde-3-phosphate dehydrogenase, which served as an internal standard. The primers were purchased from Roth (Karlsruhe, Germany). Semiquantitative evaluation was performed using a LumiImager workstation (Boehringer Mannheim, Mannheim, Germany).

Results

Immunohistochemistry

Anterior Eye Segment. Staining for ·B-crystallin in the anterior eye segment showed clear differences between the various species investigated.

Mouse anterior eye segments revealed a bright ·B-crys- tallin staining only in the corneal endothelium and epithelium. A small number of nonpigmented ciliary epithelial (NPE) cells were stained particularly in the apical region of the most anterior pars plicata; the epithelial cells of the pars plana remained unstained. No positive staining for ·B-crystallin was found in the trabecular meshwork and outflow pathways, iris or ciliary muscle.

In the rat, the corneal endothelial cells showed slight ·B-crystallin staining that continued into the anterior portion of the trabecular meshwork. Intense staining for ·B- crystallin was seen in iris, ciliary and retinal pigmented epithelial (RPE) cells but with regional differences. In the iris, the entire layer of the pigmented epithelium stained for ·B-crystallin. In the anterior part of the pars plicata, some NPE cells were intensely stained, whereas in the posterior pars plicata only slight ·B-crystallin staining was seen in some pigmented epithelial cells (PE). Staining intensity increased in the pars plana region. The RPE cells stained for ·B-crystallin in a scattered way, showing unstained RPE cells next to intensely stained ones (fig. 1a).

Porcine eyes showed an intense ·B-crystallin staining of the corneal endothelium and clear staining in the trabecular meshwork. The scleral spur cells and the ciliary muscle cells remained unstained. The iris epithelium and almost all epithelial cells of the pars plicata showed positive staining for ·B-crystallin with pronounced labeling in the apical region. In the pars plana, staining was present in NPE and PE cells but was much less distinct.

Staining of the anterior eye segment of bovine and primate eyes confirmed our results of previous studies. In bovine eyes the corneal endothelium, iris and ciliary body NPE, PE and RPE cells were stained positively for ·B- crystallin.

In primate eyes, intense staining was seen in the corneal endothelium in the iris and ciliary epithelium, especially at the tips of the pars plicata. In addition, staining for ·B-crystallin was found in the ciliary muscle and in the trabecular meshwork but was mainly restricted to the outer portion adjacent to Schlemm's canal.

In none of the five species investigated were age-relat- ed differences seen in staining intensity or principal distribution of stained cells (table 4). The individual variations were greater than the age-related differences.

a

b

Fig. 1. Immunohistochemical demonstration of ·B-crystallin in the eye. a Whole mount of the anterior retinal pigmented epithelium of a rat eye. The RPE cells show scattered staining for ·B-crystallin. !140. b Sagittal section through the rat retrolaminar optic nerve. Intense staining is present in glial cells within the individual nerve fiber bundles, whereas the astrocyte processes separating the bundles from the connective tissue septa remain unstained. !180.

Optic Nerve. In the mouse and rat optic nerve, only glial cells stained for ·B-crystallin (fig. 1b). Astrocyte processes forming a rim separating the nerve fiber bundles from the connective tissue septa appeared unstained. Considerably more staining was found in optic nerve of the pig and cow. Numerous glial cells with their cytoplasmic processes appeared ·B-crystallin positive, including the astrocyte processes of the nerve fiber bundle rim. The staining intensity was more pronounced in the peripheral part of the optic nerve and less intense in the central part. The distribution of ·B-crystallin in the primate optic nerve was comparable to that described for the porcine and bovine optic nerve. The slightly larger variability in staining intensity of human optic nerve specimens might be explained by the greater variability of the postmortal time. In none of the species studied were age-related differences found in staining intensity or distribution of staining.

a

b

Fig. 2. Ultrastructural demonstration of ·B-crystallin in the heart muscle. Gold particles showing localization of ·B-crystallin are scattered in the cytoplasm adjacent to the Z bands. a !13,300. b !48,000.

Heart Muscle. The distribution of ·B-crystallin within the heart muscle was similar in all animals studied. Using immunohistochemistry, the antibody against ·B-crystal- lin labeled almost exclusively the region of the Z band of cardiac myocytes (fig. 2a). In addition, Schwann cells also stained positive for ·B-crystallin. Ultrastructural studies of the rat heart muscle revealed that most of the gold particles were located in the Z band region, but a number of gold particles was also scattered in the adjacent cytoplasm (fig. 2b). These particles were not adhering to cytoskeletal filaments or cell organelles. The staining intensity varied greatly between the different species studied. Weak staining was found in heart muscle cells of the mouse, clear staining in that of rats and intense staining in the pig, cow and human heart muscle. Investigation of animals in different age groups did not show clear-cut differences in staining intensity.

Thyroid Gland. In the thyroid gland of all species studied (mouse, rat, pig, human), follicular epithelial cells showed bright staining for ·B-crystallin (fig. 3a). The staining was not evenly distributed within the circumference of the follicles and varied in different parts of the gland (fig. 3b). The colloid of the follicle remained unstained as did the parafollicular cells of the stroma. The intracellular distribution of ·B-crystallin was regular and showed no preference for any compartment. The mouse thyroid gland revealed less intense staining than the thyroid of the other three species investigated.

No age-related staining differences were found.

Protein Immunoblotting (Dot Blot) and PCR

Optic Nerve. In rat eyes, quantitative densitometric evaluation of ·B-crystallin in the dot blots (fig. 4a) showed that there were no differences in the protein expression with increasing age (fig. 5a). The interindivid-

Table 4. Distribution of ·B-crystallin in the anterior eye segment of various animals

Fig. 3. Immunohistochemical demonstration of ·B-crystallin in human (a) and rat (b) thyroid gland. The thyroidal follicular cells are intensely stained throughout the entire cytoplasm. There are, however, staining differences in cells within single follicles. The endocrine cells of the parathyroid gland (right half of b) are unstained. Arrows indicate positively stained Schwann cells. a !80. b !200.

Fig. 4. Dot blot analysis of ·B-crystallin in retrolaminar optic nerve (a) and heart muscle (b) of different species. The standard for quantitative evaluation is shown in lanes 1 and 2. a Optic nerve samples show different amounts of the protein when comparing mouse (lane 3), rat (lanes 4 and 5), pig (lane 6), cow (lanes 7 and 8) and human samples (lanes 9 and 10). b ·B-Crystallin in heart muscle of mouse (lane 10), rat (lanes 6±9), pig (lane 5, bottom), cow (lane 5, top) and human samples (lanes 2, bottom, 3 and 4).

ual differences were predominant with ·B-crystallin levels varying between 90 and 310 ng/mg protein.

In mouse eyes of different age groups, ·B-crystallin levels varied between 210 ng/mg protein in a 3-week-old mouse to 100 ng/mg protein in a 21-month-old animal (fig. 6a). In 2 SAMP mice, 11 and 12 months old, only 70 ng/mg protein were detected.

The bovine optic nerves were derived from animals 7 months, 2, 5 and 6 years old (maximal life span of cows is about 15 years). Individual differences ranged between 790 and 2,250 ng/mg protein (fig. 7a). In porcine optic nerves only 5- to 6-month-old animals and one 6-year-old animal could be investigated (maximal life span of pigs is about 10 years). In the younger group, 2 animals had lev-

aB-Crystallin (ng/mg protein)

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Fig. 5. Densitometric quantitation of ·B-crystallin (ng/mg total protein) in the dot blots of rat optic nerve (a) and heart muscle (b). There are individual but no age-related differences in the amount of ·B- crystallin in both tissues.

Fig. 6. Densitometric quantitation of ·B-crystallin (ng/mg total protein) in the dot blots of mouse optic nerve (a) and heart muscle (b). The amount of ·B-crystallin in the optic nerve is lower in the old animals compared to the young mouse. The protein expression in the heart muscle shows no age-related differences.

els of around 500 ng/mg protein, whereas 2 other animals had ·B-crystallin levels of approximately 2,000 ng/mg protein. The old animal had 550 ng/mg protein ·B-crys- tallin (fig. 8).

In human eyes there was no age-related decrease or increase in the amount of ·B-crystallin in the optic nerve either, but individual differences ranging between 470 and 2,450 ng/mg protein were observed (table 5). These data are in the range of those obtained from porcine and bovine optic nerves, but the postmortem time of the human tissues was between 4 h and 3 days, compared to the immediately obtained tissue of the animals.

PCR analysis was performed with optic nerve tissues derived from rat and human donors. In the 5 human optic nerves (donor age 41±87 years) and in the rat optic nerves, derived from 8 animals (1.5±15 months old), no obvious differences in ·B-crystallin mRNA were seen (fig. 9).

Heart. In the rat heart muscle the mean concentration of ·B-crystallin was 1,570 ng/mg protein with no changes in protein expression during aging (fig. 5b). In the heart muscle of mice protein concentrations of ·B-crystallin were between 260 and 580 ng/mg protein (fig. 6b). The amount of ·B-crystallin remained nearly at the same level in all age groups.

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Fig. 7. Densitometric quantitation of ·B-crystallin (ng/mg total protein) in the dot blots of bovine optic nerve (a) and heart muscle (b). The amount of ·B-crystallin in the optic nerve does not change with age. In the heart muscle, levels of the protein in 5- and 6-year-old animals are higher than in the 2-year-old animals.

In the bovine heart muscle higher values of ·B-crystal- lin (mean concentration of 3,210 ng/mg protein) were measured. In 2-year-old animals the values varied between 370 and 2,530 ng/mg protein, in the 5- and 6-year- old animals values increased towards 2,040±8,540 ng/mg protein (fig. 7b). In pigs only heart muscle tissue from three 5-month-old animals was available. ·B-Crystallin levels were 820, 970 and 1,000 ng/mg protein, respectively. The human heart, even if obtained more than 12 h postmortem, still showed concentrations of up to 10,650

Fig. 8. Densitometric quantitation of ·B-crystallin (ng/mg total protein) in the dot blot of porcine optic nerve. The amount of ·B-crystal- lin in the optic nerve shows individual but no age-related differences.

Fig. 9. Agarose gel electrophoresis and ethidium bromide staining of PCR products amplified from human (a) and rat (b) optic nerve tissue with primers specific for ·B-crystallin. Glyceraldehyde-3-phos- phate dehydrogenase (white arrow) shows equal amounts of total cDNA preparation. All human samples show similar amounts of ·B- crystallin mRNA (black arrow). In the rat, an increase in ·B-crystal- lin mRNA is seen with increasing age of the animals.

ng/mg protein (mean concentration 2,040 ng/mg protein; table 5).

The ·B-crystallin mRNA levels obtained from 11 rat heart muscles of different age groups showed no age-relat- ed differences (data not shown).