A direct contact between astrocyte and vitreous body is possible in the rabbit eye due to discontinuities in the basement membrane of the retinal inner limiting membrane.

Different from most mammalian species, the optic nerve of the rabbit eye is initially formed inside the retina where myelination of the axons of the ganglion cells starts and vascularization occurs. Astrocytes are confined to these regions. The aforementioned nerve fibers known as medullated nerve fibers form two bundles that may be identified with the naked eye. The blood vessels run on the inner surface of these nerve fiber bundles (epivascularization) and, accordingly, the accompanying astrocytes lie mostly facing the vitreous body from which they are separated only by the inner limiting membrane of the retina. The arrangement of the astrocytes around blood vessels leads to the formation of structures known as glial tufts. Fragments (N = 3) or whole pieces (N = 3) of the medullated nerve fiber region of three-month-old male rabbits (Orictolagus cuniculus) were fixed in glutaraldehyde followed by osmium tetroxide, and their thin sections were examined with a transmission electron microscope. Randomly located discontinuities (up to a few micrometers long) of the basement membrane of the inner limiting membrane of the retina were observed in the glial tufts. As a consequence, a direct contact between the astrocyte plasma membrane and vitreous elements was demonstrated, making possible functional interactions such as macromolecular exchanges between this glial cell type and the components of the vitreous body.

A direct contact between astrocyte and vitreous body is possible in the rabbit eye due to discontinuities in the basement membrane of the retinal inner limiting membrane

Different from most mammalian species, the optic nerve of the rabbit eye is initially formed inside the retina where myelination of the axons of the ganglion cells starts and vascularization occurs. Astrocytes are confined to these regions. The aforementioned nerve fibers known as medullated nerve fibers form two bundles that may be identified with the naked eye. The blood vessels run on the inner surface of these nerve fiber bundles (epivascularization) and, accordingly, the accompanying astrocytes lie mostly facing the vitreous body from which they are separated only by the inner limiting membrane of the retina. The arrangement of the astrocytes around blood vessels leads to the formation of structures known as glial tufts. Fragments (N = 3) or whole pieces (N = 3) of the medullated nerve fiber region of three-month-old male rabbits (Orictolagus cuniculus) were fixed in glutaraldehyde followed by osmium tetroxide, and their thin sections were examined with a transmission electron microscope. Randomly located discontinuities (up to a few micrometers long) of the basement membrane of the inner limiting membrane of the retina were observed in the glial tufts. As a consequence, a direct contact between the astrocyte plasma membrane and vitreous elements was demonstrated, making possible functional interactions such as macromolecular exchanges between this glial cell type and the components of the vitreous body

A practical method for the radioisotope labeling of rabbit astrocytes with (3)H-thymidine.

A practical method for the radioisotope labeling aimed at the study of the proliferative behavior of astrocytes was described. It consisted in injecting 20 microCi of (3)H-thymidine into the vitreous body and tracing by autoradiography labeled astrocytes located both inside and outside the retina, e.g. optic nerve and neighboring parts of the central nervous system. The paraffin sections were immunostained for glial fibrillary acidic protein (GFAP) previous to autoradiographic processing. The semiquantitative analysis of labeled astrocytes was carried out on autoradiographs of semithin sections of rabbits killed as early as 6 h and as late as 3 months after the single intravitreal injection of (3)H-thymidine. Compared with the technique of labeling astrocytes by systemic administration (single injection or continuous infusion) of (3)H-thymidine into small animals, the method described herein has the following outstanding features: (i) it is much more economical in terms of the amount of labeled precursor used per animal; (ii) the labeling of the astrocytes is obtained as early as 6 h and remains up to 3 months after injection; (iii) the immunolabeling of the astrocytes is compatible with autoradiography; (iv) it is less risky to the experimental animal and to the environment; (v) it can be used in animals much larger than rats or mice.

Immunohistochemistry in association with scanning electron microscopy for the morphological characterization and location of astrocytes of the rabbit retina.

The purpose of the present investigation was to establish a method for the morphological characterization and location of the several types of astrocytes in the rabbit retina. Whole retinas were incubated with unlabeled antibody to glial fibrillary acidic protein (GFAP) and, afterwards, treated with secondary antibody labeled according to the requirements for the visualization of the antigen-antibody reaction either with the confocal or transmission electron microscope. Specimens treated similarly to the latter were osmium enhanced and analyzed with scanning electron microscopy (SEM). The different immunohistochemical approaches led to the conclusion that the cells selectively visualized with the SEM are astrocytes. The higher resolution and depth of focus of this instrument allowed a better morphological characterization and a more precise location of the astrocytes in the several levels of the inner portion of the rabbit retina. The method described herein, in which pre-embedding immunohistochemistry for GFAP on rabbit retinas was associated with osmium enhancement and examination with SEM, proved to be reliable and efficient for the morphological characterization and location of astrocytes.