Biochemical studies of the tear film in humans and rabbits

Abstract

Clinically, a decrease in tear film stability has been associated with both dry eye and contact lens discontinuation. Rabbits are known to have a significantly longer inter-blink time compared to humans, perhaps due to their very stable tear film. The reason for this, however, has never been studied at a detailed biochemical level. This research project therefore sets out to examine the tear film biochemistry and molecular interactions in humans and rabbits and determine how these affect the tear stability. The research outcomes could lead to the development of novel ways to increase tear film stability and hence ocular comfort. Initially, tear break-up time (TBUT) of rabbit tears was measured with a slit-lamp biomicroscope and tearscope. The average TBUT of rabbit tears was 30 minutes, which is in line with their long inter-blink time reported in the literature. The tear osmolality and surfactant properties were measured using the vapour pressure osmometer, and the Langmuir trough, respectively. Compared to human tears, rabbit tears had a higher osmolality and demonstrated different tear surfactant properties, which indicates the different biochemical composition of tears from the two species. In this thesis, the concentrations of metal cations were tested using the inductively coupled plasma mass spectrometer (ICP-MS) and inductively coupled plasma atomic emission spectrometer (ICP-AES). A higher concentration of divalent cations was found in rabbit tears. Their influence on the tear film stability was analysed in vitro by measuring surface tension using the Langmuir trough and in vivo by grading the tear break-up with a slit-lamp biomicroscope. In rabbit tears, reducing the divalent cations concentration significantly decreased the maximum surface pressure from 37 mN/m to 30 mN/m in vitro and resulted in more extensive tear disturbance in vivo. In humans however, changed concentrations of divalent cations did not show any change. Tear protein and lipid composition as well as protein-protein interactions were analysed using a range of proteomic and lipidomic techniques. A clear difference in composition was found between human and rabbit tears. The components present only or more abundantly in rabbit tears, along with divalent cations, were further assessed to determine their influence on human tear surfactant properties using the Langmuir trough. The predominant proteins in rabbits, lipophilins and prolactin inducible protein (PIP), exhibited very strong surface activity and bringing human tear surfactant properties closer to that of rabbit tears. Other components, including dihydrolanosterol, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), which are more abundant in rabbit tears, demonstrated no change to human tear surfactant properties. This thesis demonstrated that the predominant proteins and divalent cations found in rabbit tears could be potentially used to improve human tear film stability. Ultimately, outcomes of this research may lead to the development of novel ways to increase tear film stability to provide lasting relief from debilitating dry eye symptoms and increase comfort during contact lens wear.