The development, evaluation and use of freshly isolated renal proxinal tubule systems in the fischer rat

The investigation of renal pathophysiology and toxicology has traditionally been advanced by the development of increasingly defined and refined in vitro preparations. This study has sought to develop and evaluate various methods of producing pure samples of renal proximal tubules (PTs) from the Fischer rat. The introduction summarised the most common in vitro preparations together with the parameters used to monitor viability - particularly with regard to toxic events. The most prevalent isolation methods have involved the use of collagenase to produce dissociation of the cortex. However, the present study has shown that even the mildest collagenase treatment caused significant structural damage which resulted in a longevity of only 3hr in suspension. An alternative mechanical isolation technique has been developed in this study that consists of perfusion loading the renal glomeruli with Fe304 followed by disruption of the cortex by homogenisation and sequential sieving. The glomeruli are removed magnetically and the PTs then harvested by a 64μM sieve. PTs isolated in this way showed a vastly superior structural preservation over their collagenase isolated counterparts; also oxygen consumption and enzyme leakage measurements showed a longevity in excess of 6hr when incubated in a very basic medium. Attempts were then made to measure the cytosolic calcium levels in both mechanical and collagenase isolated PTs using the fluorescent calcium indicator Fura. However results were inconclusive due to significant binding of the Fura to the external PT surfaces. In conclusion, PTs prepared by the present mechanical isolation technique exhibit superior preservation and longevity compared with even the mildest collagenase isolation technique and hence appear to offer potential advantages over collagenase isolation as an in vitro renal system.

Contact lenses and sport

Contact lenses seem to be the ideal method of vision correction for ametropic people who participate in sporting activities. This thesis sets out to evaluate the viewpoint of the optometric professional and that of the patient on the use of contact lenses in sport and to establish if education is needed within this area. It also aims to provide some scientific evidence on the effect of exercise on the physiology of the cornea with and without contact lenses. Silicone hydrogel contact lenses have previously been suggested to impede heat dissipation from the cornea compared to mid water hydrogels. This was further demonstrated with exercise. The physiological integrity of the cornea is dependant on the amount of oxygen available to its surfaces. Contact lenses can disrupt the diffusion of oxygen to the cornea. Previous methods of measuring the oxygen consumption of the cornea have been limited by their invasive nature and assessment of only a small surface area of the cornea. They are not suitable to measure corneal oxygen consumption during exercise with and without contact lenses. A new method needed to be established. This was achieved by designing a novel method by the use of an oxygen sensor inside an airtight goggle using dynamic quenching of luminescence method. This established a non-contact way of measuring the effect oxygen uptake with and without contact lenses in vivo, allowing the contact lens to be undisturbed in their natural environment. The new method differentiated between the closed-eye and the open-eye condition with a good within-visit repeatability. It also illustrated that the cornea utilises oxygen at a faster rate during controlled aerobic exercise at moderate intensity. New contact lenses are available specifically for sport, these claim to reduce glare and increase contrast for daylight outdoor sports. However, visual benefits of these types of contact lenses cannot be measured easily in an indoor clinical environment, such as the optometric practice. To demonstrate any potential benefits of these lenses emulation of them should be conducted outdoors.