Abstract
Purpose::
To determine, using anatomical and biochemical methods, the porosity of the structural scaffold of ageing human Bruch’s membrane. Disulphide bridging leads to fibre cross-linking and stabilisation of proteinaceous debris and its role in the ageing of Bruch’s was assessed by measurement of free thiols. Finally, the reversal potential for reducing disulphide links and subsequent effects on membrane transport were quantified.
Methods::
Inter-fibrillar spaces in the inner/outer and elastin layers of Bruch’s membrane were approximated to pores and the relative distribution of pore radii was obtained by electron microscopy. A plot of pore radii versus age was used to designate changes in porosity in individual layers of Bruch’s membrane from donors aged 7-81 years. The porosity of the resistance barrier of Bruch’s was examined biochemically by determining the size exclusion limit for transport of dextran molecules. Free thiols were quantified using the fluorescent probe 7-diethylamino-3-(4-maleimidylphenyl)-4-methylcoumarin (CPM) and an age-profile constructed. Improvements in porosity in response to disulphide reduction by dithiothreitol was assessed by monitoring changes in fluid transport.
Results::
Pore radii in the inner collagenous layer of Bruch’s membrane showed an age-dependent reduction (P<0.05) but changes in the elastic and outer collagenous layer were not significant. Biochemically, the molecular weight exclusion limit to movement of dextran molecules was determined to be 75 ± 10 kDa (n=8; age range 4-92 years) and remained independent of age. However, the flux of a much smaller dextran (21.2kDa) was reduced with ageing (n = 18, age range 4-90 years; p<0.001). The level of free thiols in Bruch’s-choroid preparations from 28 donors, aged 9-86 years declined linearly (p<0.005), a reduction of 62% between the youngest and oldest donor. Incubations with dithiothreitol markedly improved the fluid dynamics of Bruch’s membrane (p<0.05).
Conclusions::
Anatomically, the spacing within the structural scaffold of Bruch’s showed little age-related variation but the diminished flux of dextran molecules signified a marked reduction in the porosity of the membrane. The age-related reduction in free thiols implied greater disulphide bridge formation and may be one of the mechanisms for trapping and stabilizing debris within the confines of the extracellular matrix. The plasticity of the debris and potential for destabilisation of debris within ageing Bruch’s was inferred from the improvement in fluid dynamics after treatment with dithiothreitol.
Keywords: age-related macular degeneration • aging • Bruch's membrane