A to H. Tissue culture of the prelaminar region, JC-1 staining.

<p>A sequence showing the transformation from normal mitochondria into vesicular, inflated mitochondria. Healthy mitochondria are small compared to the nucleus (blue fluorescence). The normal metabolic activity of the mitochondrial membrane concentrates the dye (JC-1) on the inside and favors the formation of J-aggregates that fluoresce in bright red (A). As the ionic activity of the membrane decreases, the mitochondrion swells, the dye is less concentrated, and the hue changes from red to orange and finally to green (<b>B</b> to <b>H</b>) before mitochondrial disruption. This effect indicates mitochondrial-activated apoptosis as a main path for cell loss in prelaminar tissue culture. Here, the inflation of the mitochondria results from the decay of ionic pump function, which manifests as an increase in mitochondrial size and dilution of the dye from bright red to orange, yellow and green when increasingly diluted.</p

Schematic drawing of the experimental arrangement.

<p>The prelaminar region of the optic nerve head is excised and cultured in shallow medium to allow an adequate supply of oxygen to reach the tissue. As reported elsewhere, the axons are destroyed, leaving a matrix of astrocytes and capillaries that adapt to culture conditions. Microscopy (LM, TEM, and CLSM, including immunolabeling) is used to show that the tissue retains normal characteristics, including the important zonula adherens, and contains the glucose transport molecules GLUT1 and GLUT3. A comparison between the freshly excised and the cultured tissues by western blot shows the proportional quantities of the coupled molecules under study. This approach was followed in a previous report for MCTs and was performed here for GLUTs. (LM: light microscopy; TEM: transmission electron microscopy; CLSM: confocal laser scanning microscopy).</p

The levels of LDH in culture.

<p>The levels decrease from the first day of culture and stabilize at approximately day 6. Necrosis in the tissue is caused by the sectioning and manipulation process and is induced minimally or not at all by the culture conditions. The values are the means, with the standard errors represented by vertical bars. *The mean values were significantly different from those of the Day 2 group (P < 0.05).</p

Histology of the prelaminar explant LM and TEM.

<p><b>a</b>) Semithin section of a full-length prelaminar explant following excision. The thickness varies between 100 and 300 microns, being thinner in the center. The central thickness in this case is approximately 280 microns. Toluidine blue inclusion, epoxy resin; magnification in the figure. <b>b</b>) Central zone (green square) in a). The arrangement in columns of astrocytes is highlighted by the dotted line in part of the section. Axonal bundles pass between the astrocytic columns. The area of the orange pentagon includes a capillary as part of a column and a portion of an axonal bundle. This type of section allows for GLUT-1 labeling in the endothelial vascular cells to serve as a positive control for axonal labeling of GLUT-1. <b>c</b>) The area enclosed in the red square is formed exclusively by axons wrapped in an extremely tight web of astrocytic expansions. The complexity of the arrangement is reminiscent of the neuropil in the central nervous system. The TEM micrograph shows three axons (Ax) and numerous astrocytic expansions. This type of section allows for the identification of the involved cells even in the absence of membrane counterstaining, which is required for immunolabeling with gold micelles. Cell identification is aided by the presence of GFAP and Neurofilament staining. The magnification is indicated in the figures.</p

List of antibodies for western blot.

<p>Only the final dilutions used are listed.</p><p>List of antibodies for western blot.</p

Explant of the prelaminar region of the optic nerve viewed via LM and CLSM.

<p>As explants are thinner than 300 microns, the penetration of culture medium is sufficient for the survival of the explant for more than a week. <b>A</b>) LM photography of an original 2 x magnification of an explant after 5 days of culture; toluidine blue staining of semithin section in epoxy resin. <b>B</b>) An explant after 6 days of culture at 40x; toluidine blue staining of semithin section in epoxy resin. <b>C)</b> CLSM micrograph of a prelaminar explant after 8 days of culture labeled for GFAP. GFAP is abundant, indicating the preservation of the main astrocytic characteristics, including rounded nuclei with a healthy shape stained with DAPI. A GFAP-negative capillary is present in the upper left corner and is delimited by astrocytic expansions.</p

TEM micrographs.

<p><b>A</b>, <b>B</b>, <b>C</b> and <b>D</b>: Colloidal gold immunolabeling of GLUT3. The gold particles (arrows) delimit the boundaries between the clear areas (axons) and the GFAP-rich darker areas of astrocytes. The magnification is given in the figures.</p

List of procedures used to detect apoptosis.

<p>List of procedures used to detect apoptosis.</p

CLSM micrographs of the prelaminar region labeled for Glial Fibrillary Acidic Protein (A, B) and Neurofilament.

<p>A, <b>B</b>) An axon-free perivascular glial sheet surrounding the trunks of the central vessels can be distinguished (PVG) based on the distribution of the axons in the prelaminar region. The remaining prelaminar tissue contains axons that are wrapped in astrocytic expansions (PAA). The somas of the astrocytes are loosely distributed in the superficial fiber layer and pile up in columns on top of the beams of the <i>lamina cribrosa</i> (dark areas at the bottom of <b>B</b>). <b>C, D</b>) Labeling with neurofilament delimits the superficial fiber layer, with a branch of a central vessel with the unlabeled perivascular glial sheet on top (PVG). The axons form bundles that pass between the astrocytic columns and formed by the piles of astrocytic somas (unstained dark area at the bottom). The magnification is given in the figures.</p

CLSM.

<p>Astrocyte nuclei are shown in blue (DAPI). Positive staining of the target (GLUT1) shows green fluorescence. <b>A, B</b>: GLUT1, long thin arrows point to positive green fluorescence in the surface astrocytes (sa) near the central vessels (cv), the astrocytes surrounding the capillaries (v), as well as in the columnar astrocytes (ca). The short thick arrows point towards their axonal expansions (ae). <b>C</b> Specific staining of GLUT-1 at the level of the columnar astrocytes (ca, short arrow) as well as in the axonal bundles (long arrow). <b>D</b> GLUT1 labeled by a chain with a secondary anti-idiotypic antibody. The long arrow points towards an astrocytic expansion that enters into the axonal bundle.</p