Muller Cell Expression of Gliol Fibrillory Acidic Protein offer Genetic ...

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1322 INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / November 1984 Vol. 25 each containing two 15-watt fluorescent bulbs were positioned 18 cm above the bottom of the cage. These conditions resulted in an incident luminance of approximately 200-ft candles at the floor of the cage. The temperature in the cage was 24 ± 1 °C. Age-matched Sprague-Dawley rats maintained in a 12-hr overhead room light/ 12-hr dark environment, were used as controls. All rats were enucleated under ether anesthesia between 1:00 and 2:30 PM. After a slit was made in the cornea, the lens and vitreous were removed and the globe was immersed in 4% formalin in 0.13 M phosphate buffer (pH 7.4). Immunofluorescence After 6 hr in fixative at room temperature, the eyes were bisected, transferred to 30% sucrose in 0.13 M phosphate buffer and stored at 4°C overnight. Sections were cut at a thickness of 20 nm using a cryostat at —20°C. The sections were mounted on chrome alum-gelatin coated slides and air-dried overnight at room temperature. Plastic rings (0.75-cm diameter) were mounted with fingernail polish around the sections to form incubation wells. Each well contained an experimental section of retina (RCS or CL-damaged) and a control section. The sections were treated for 10 min at room temperature with 1% goat serum and 4% bovine serum albumin (BSA) in phosphate buffered saline (PBS) followed by overnight incubation at 4°C in GFAP antiserum diluted 1:100 in PBS containing 0.3% Triton X-100. The GFAP antiserum, provided by Dr. Larry Eng (Veterans Medical Center; Palo Alto, CA), was raised in rabbits against GFAP obtained from multiple sclerosis plaques. 8 Control sections were treated identically with an IgG fraction from preimmune rabbit serum. Sections were washed twice (15 min each) with PBS at room temperature and incubated for 30 min at room temperature in the dark in sheep anti-rabbit IgG-fluorescein isothiocyanate (Cappel Laboratories), diluted 1:50 in PBS with 0.3% Triton X-100. After two 10-min washes in phosphate buffer, the plastic rings were removed and the sections were coverslipped with 80% glycerol in 0.13 M phosphate buffer containing 5% n-propyl gallate. 9 The sections were examined with a Zeiss microscope equipped for epifluorescence. Measurement of Outer Nuclear Layer After enucleation, eyes were stored in fixative at 4°C overnight. They were bisected vertically just temporal to the optic nerve head. The bisected eyes were washed for several hours in phosphate buffer and then dehydrated through a graded series of ethanol. The half of the eye including the optic nerve head was embedded in plastic (Sorvall Embedding Medium), with the cut edge of the eyecup placed flat to allow for sectioning along the inferior-superior plane, including the optic nerve head. In some cases the eyes were bisected after 6 hr in fixative, and the half without the optic nerve head was processed for immunofluorescence, while the remaining half was stored in fixative overnight. Sections were cut at a thickness of 2.5 ^m on a Sorvall JB-4 microtome and stained for 30 sec with 10% Richardson's stain. A section through the optic nerve head from each eye was chosen and the thickness of the outer nuclear layer was measured 250 ixm, 500 fim, and 750 /xm from the optic nerve head in the superior and inferior hemispheres. The mean and standard error of the mean of these six measurements were calculated. Preparation of Triton X-100 Insoluble Proteins Triton-insoluble proteins were obtained from retina according to Pruss et al. 10 Four to six retinas were homogenized in ice-cold PBS containing the protease inhibitors p-chloromercuribenzoate, phenyl methane sulfonyl fluoride and o-phenanthraline, each at 1 raM concentration. The homogenate was centrifuged at 8000 g for 10 min at 4°C. The pellet was extracted with PBS containing 0.6 M KC1, 0.5% Triton X-100 and the protease inhibitors, and centrifuged at 8000 g for 10 min at 4°C. After a second extraction with Triton X-100, the pellet was washed four times in ice-cold PBS and solubilized by boiling in SDSpolyacrylamide sample buffer." In the light damage experiments, Sprague-Dawley rats had been exposed to constant light for 3 or 7 days. Control animals had been kept in 12-hr light/12-hr dark cycle. RCS rats were 45 and 70 days old. Congenic, 45-day-old ^y" 1 " rats were used as controls. Polyacrylamide Gel Electrophoresis and Electroblotting to Nitrocellulose One dimensional SDS-polyacrylamide gel electrophoresis (PAGE) was performed according to the procedure of Fairbanks et al" using protein standards ranging in molecular weight from 15-94,000. Proteins were transferred from PAGE gels to nitrocellulose membranes (BIORAD) in a Hoefer Transphor electrophoresis apparatus at 0.8 mV for 1 hr at room temperature. 12 After 1 hr blocking in Tris-buffered saline (TBS) containing 3% BSA, the blots were treated overnight in anti-GFAP diluted in TBS and 1% BSA. After several washes, the blots were incubated with goat anti-rabbit IgG (Cappel) for 1 hr (1:1000 dilution in TBS and 1% BSA). Following a 30-min exposure to the peroxidase-antiperoxidase complex
No. 11 GFAP IN MULLER CELLS / Eisenfeld er al. 1023 Fig, 1. Light micrographs of normal, constant light-damaged and RCS rat retinas. A, Normal Sprague-Dawley rat retina. O, outer segments; ON, outer nuclear layer; IN, inner nuclear layer; 1, inner plexiform layer; G, ganglion cell layer. B, Sprague-Dawley rat retina after 3 days in constant light. C, 38-day-old RCS rat. Note the decreased thickness of the ON. The inner retina appears normal. (1:500 in PBS and 1% BSA), the blots were stained in Tris-saline containing 4-chloronaphthol (0.5 mg/ ml) and hydrogen peroxide (0.025%). 13 Results Thickness of Outer Nuclear Layer Examples of normal, CL-damaged and RCS retinas (Figs. 1A-C) illustrate the extent of photoreceptor degeneration. The amount of damage caused by a 3-day exposure of CL was somewhat variable from animal to animal. The thickness of the outer nuclear layer of a control Sprague-Dawley rat was 46.0 ± 1.0 /xm (mean ± SEM, n = 3) while for rats kept in CL for 3 days it ranged from 22-47 nm with a mean of 37.7 ± 2.7 nm (n = 11), representing a 20% loss of photoreceptors (Fig. IB). In the 38-day-old RCS-rdy + rats without inherited retinal degeneration, the thickness of the outer nuclear layer was 45.6 ± 1.0 jum (n = 6). In 38-day-old RCS retinas with inherited retinal degeneration, the outer nuclear layer thickness was only 17.8 ± 0.6 ^m (n = 7), representing an average reduction by 61% (Fig. 1C). No abnormalities were apparent in the inner retina in either condition of photoreceptor degeneration (Fig. IB, C). Immunofluorescence Sections treated with preimmune serum showed only autofluorescence that was pale green for the neural retina and yellow for erythrocytes (Figs. 2A> 3A). In control Sprague-Dawley rats, GFAP staining was confined to filamentous structures in the innermost retina, including the nerve fiber and ganglion cell layers and encircling blood vessels (Fig. 2B). GFAP positive cells were also abundant in the optic nerve head. From their location and morphology, these GFAP positive cells were interpreted as astrocytes. In some cases, there was a light, finely particulate staining in the outer segment layer. After 1 day in CL, the GFAP staining did not differ from that in control retinas. After 3 days in CL, variable numbers of radially oriented processes were stained, some more strongly than others. These processes extended from the inner limiting membrane through the inner plexiform and inner nuclear layers, with occasional positive fibers in the outer nuclear layer (Fig. 2C). This pattern of staining closely matched the distribution of Muller cell processes and appeared to represent the appearance of GFAP reactivity in Muller cells. Staining was most intense
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