Maurice H. Bernstein

Phagocytosis by retinal pigment epithelium explants in culture.

Phagocytosis of rod outer segments and of latex beads was demonstrated in retinal pigment epithelium explants from adult albino rats. Uptake of latex particles increased steadily over a 24 hr period while ingestion of rod outer segments peaked after 2 hr of incubation, then decreased. Postnatal changes in phagocytic capabilities of retinal epithelium were examined in 10- to 35-day-old rats. Retinal epithelium from 10–15 day neonatal animals ingested lastex particles but few rod outer segments. The number of outer segment packets ingested increased with increasing age of the explant donor. The results suggest that the mechanisms involved in phagocytosis of nonspecific particles (latex beads) appear earlier in development than the mechanisms necessary for phagocytosis of photoreceptor outer segments.

Malachite Green: Applications in Electron Microscopy

Incorporation of malachite green into a glutaraldehyde fixative results in enhanced staining of a number of cellular elements. Ribosomes and myofilaments exhibit increased electron density, but cell membranes generally are not stained. In certain tissues, lipid inclusions are uniformly and heavily stained. Other populations of lipid droplets exhibit differential affinity for malachite green, facilitating their division into subclasses. In addition to its function as a dye, malachite green has previously been reported to stabilize lipid elements soluble in aqueous glutaraldehyde. Such a component was observed in the stroma of uterine endometrium. The variety of cell components which exhibit increased contrast after preparation with malachite green suggests that this technique may find widespread application in fine structure studies.

MANNOSE-SENSITIVE HRP ENDOCYTOSIS BY THE RETINAL PIGMENT EPITHELIUM

Mannose-sensitive endocytosis by rat retinal pigment epithelium (RPE) explants was characterized using the mannose-rich glycoprotein horseradish peroxidase (HRP). The number of HRP-containing endosomes in the RPE was morphologically quantitated by light microscopy while the amount of HRP ingested was biochemically quantitated by enzyme assay. HRP internalized via a mannose-sensitive receptor was differentiated from fluid-phase uptake in competitive inhibition studies using D-mannose. Morphological results showed that most HRP-containing endosomes formed within the first 15 min of incubation and showed little increase in number during 4 hr of continued incubation with HRP. In contrast, the biochemical assay showed a steady increase in the amount of HRP in RPE endosomes measured over time. The addition of 10 mM D-mannose to the incubation medium was associated with a significant decrease in both the number of HRP-containing endosomes and the amount of HRP ingested by RPE explants. Values indicate that half of the total uptake of HRP is mediated by a mannose-sensitive receptor while the balance is ingested via non-specific fluid-phase endocytosis.

Daily patterns of the retinal pigment epithelium. Microperoxisomes and phagosomes

Retinal tissue was taken from adult albino rats maintained on a regular light-dark (14–10) cycle. Animals were killed at specific time intervals throughout the day. All tissues were incubated in an alkaline diaminobenzidine reagent for cytochemical demonstration of the peroxidatic activity of catalase. Enzymatic activity was seen in the retinal epithelium in microperoxisomes and in small phagosomes (secondary lysosomes). Microperoxisomes have membrane-limited coarsely granular contents and measure from 100 to 300 nm. Quantitative studies involved counting the numbers of microperoxisomes, large phagosomes, and small phagosomes in a given length of retinal epithelium. The numbers of large phagosomes reflect the synchronous burst of rod outer segment phagocytosis initiated with the onset of light. Microperoxisomes, having accumulated in the dark, show a precipitous drop with the onset of light, then rebound to a peak number three hours into the light period, then decline to a low level after four hours of the light period and remain at this low level for the balance of the light period. A small-scale repeat of this pattern is seen relative to the beginning of the dark period. Small phagosomes, or secondary lysosomes, accumulate slowly but progressively through the light period. With the end of the light period there is a sharp drop in the number of small phagosomes.

Localization of (3H2)vitamin A in mouse retina

Abstract Following intraperitoneal injection of [ 3 H 2 ]vitamin A acetate, the accumulation of labeled material in mouse retinas was studied by autoradiographic techniques. Significant accumulations of label were observed in the pigment epithelium and photoreceptor cells. Isotope continued to accumulate for 2 days, then achieved a steady state which was maintained through 9 days. The major localization within the photoreceptor cells was in the outer segments; inner segments and nuclei showed lesser amounts of label but were significantly above background level. Although the concentration of label varied markedly from one region of the photoreceptor cells to another, the distribution within each region was homogeneous. Photoreceptor outer segments gave no evidence of localized banding of labeled material. A metabolic pathway for vitamin A within mouse photoreceptor cells involving inner segments and nuclei is suggested.

Effect of Estrogen on the Affinity of Malachite Green for Staining Cardiac Lipid Inclusions in Mice

The effect of estradiol-17-beta on lipids of the ventricular myocardium of mice has been studied with a cytochemical technique in which malachite green was added to glutaraldehyde. This malachite green-glutaraldehyde fixative enhances the visualization of certain phospholipid-related elements. Estrogen induces an affinity of ventricular cardiac lipid inclusions for the cationic dye malachite green. The staining affinity is evidenced only in the estrous female, not in diestrus. In oophorectomized animals, malachite green staining is seen only following estradiol injection, but this effect is blocked by progesterone. In the male, ventricular lipids do not stain, nor do they develop malachite green affinity with estrogen stimulation. These results imply a blockade of the estradiol-mediated dye affinity by progesterone and testosterone. This reinforces the concept of the heart as a target organ for sex steroids and expands the previously described estrogen effects on myocardium.