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A quantitative study of intramembrane changes during cell junctional breakdown in the dystrophic rat retinal pigment
Insights
Intercellular junctions in the retinal pigment epithelium (RPE) of Royal College of Surgeons (RCS) rats with inherited retinal dystrophy break down, leading to significant changes in tight and gap junctions and increased particle density.
Area of Science:
- Ophthalmology
- Cell Biology
- Neuroscience
Background:
- Inherited retinal dystrophy in Royal College of Surgeons (RCS) rats is characterized by breakdown of intercellular junctions in the retinal pigment epithelium (RPE).
- Previous studies indicate junctional breakdown occurs between three and six postnatal weeks.
Purpose of the Study:
- To quantitatively analyze freeze-fracture changes in the dystrophic RPE using computer techniques.
- To measure specific parameters of tight junctions, gap junctions, and intramembrane particles.
Main Methods:
- Quantitative computer analysis of freeze-fracture electron microscopy images.
- Measurement of tight junctional strand length and anastomoses per square micron.
- Quantification of gap junctional aggregates (number and area) and background particle density per square micron.
Main Results:
- At 10 weeks and later, dystrophic RPE shows decreased tight junctional strand length and anastomoses, and reduced number and area of gap junctions.
- A significant increase in background intramembrane particle density was observed in dystrophic RPE.
- Correlational analysis revealed significant relationships between changes in tight junctions, gap junctions, and background particle density.
Conclusions:
- Breakdown of intercellular junctions in dystrophic RPE involves significant alterations in tight and gap junction structures.
- Increased background particle density is associated with junctional breakdown, with contributions from both junctional and other sources.
- Despite junctional breakdown, differences in particle density between apical and basal membranes are maintained, suggesting partial integrity of tight junctions.
Abstract:
Previous electron microscope freeze-fracture and tracer studies have revealed that intercellular junctions in the retinal pigment epithelium (RPE) of Royal College of Surgeons (RCS) rats with inherited retinal dystrophy [5] break down between three and six postnatal weeks [6, 7]. In this study quantitative computer techniques were used to analyze the freeze-fracture changes in the dystrophic RPE. The following parameters were measured: length of tight junctional strands/micron2; number of tight junctional strand anastomoses/micron2; number of gap junctional aggregates/micron2; area of gap junctional aggregates/micron2; and density of background intramembrane particles/micron2. At three postnatal weeks, the dystrophic junctional complex membrane is similar to normal, but at 10 weeks and later there are dramatic decreases in tight junctional strand length/micron2 and number of anastomoses/micron2, as well as in the number/micron2 and area of gap junctions/micron2, while the density of background particles/micron2 is dramatically increased. Correlational analysis revealed that changes in gap and tight junctions were significantly related to each other and to the increase in background particle density. The diameter of background particles within the normal and post-breakdown dystrophic junctions was measured in order to see whether the dispersal of gap and tight junctional particles (8-10 nm) into the surrounding membrane contributes to the increased particle density. These measures showed that background particles in all size ranges were more numerous in the dystrophic RPE, but that the largest increase was in the smallest diameter particles (6-7 nm). Thus, while gap and tight junctional sized particles contribute to the increase, particles from other sources may also be involved. Particle density of apical and basal membranes in the normal and in the 10 week and older dystrophic RPE was analyzed to study the effects of tight junctional breakdown on the distribution of intramembrane particles. These measures showed that particle density was greater basally than apically in the normal RPE and that particle density in both membranes decreased slightly in the dystrophic RPE, but that their ratio remained unchanged. It has been shown previously that even a single intact tight junctional strand is sufficient to maintain differences in particle density between apical and basal surfaces [14, 15] and in the majority of abnormal dystrophic junctional complexes at least one tight junctional strand remains intact.(ABSTRACT TRUNCATED AT 400 WORDS)