Longitudinal in vivo monitoring of the neuropathology in ovine neuronal ceroid lipofuscinoses

Abstract

Batten disease or neuronal ceroid lipofuscinoses (NCLs) is one of the most common reasons for childhood dementia, characterized by brain atrophy, blindness, mental decline and premature death. Although animal model neurological research is traditionally conducted using small rodents, large animal models are becoming more popular for several reasons. Longitudinal monitoring of disease progression and individual variations are essential in the development of translational medicine and whereas small rodents have relatively short life spans and different brain structures compared with humans, sheep provide an excellent model for long-term studies of diseases and their treatment. Three naturally occurring ovine models are currently being utilised to increase knowledge about different types of NCL and to aid in development of treatment strategies for translation to humans. These sheep models include a CLN5 Borderdale, a CLN6 South Hampshire and a CLN6 Merino model. This thesis contains investigations of techniques for longitudinal in vivo monitoring that can be correlated with the progression of neuropathological events in all three ovine NCL models, both in the natural progression of the diseases and in trials of potential therapeutic regimes. Previous studies have described the neuropathological development of disease in the CLN5 Borderdale and the CLN6 South Hampshire models. In the present study similar investigations were conducted with the CLN6 Merino model, and the three ovine models compared. The general development of neuropathology in CLN6 Merino sheep follows that of the other two models. Common themes are neuronal loss from the cerebral cortex that proceeds in a regional pattern, with neuroinflammation, neurogenesis and ubiquitous accumulation of storage material within the cells. Immunohistochemistry indicated possible cross-regulation between the CLN5 and CLN6 proteins. In vivo techniques are essential in the monitoring of progressive diseases such as the NCLs. Neuroimaging through computed tomography (CT) and magnetic resonance imaging (MRI) are suitable tools to describe neuropathological changes without the need to sacrifice animals. Here, current techniques and protocols for CT scanning in ovine NCL research were reviewed and improved. The congruence of brain size and intracranial volume (ICV) in ovine NCL was established and techniques for the monitoring of brain size in therapeutic trials were validated and correlated with neuropathological changes. Furthermore, 3-dimensional reconstruction was used to monitor regionality of the cortical changes and this could be correlated with findings from post examination. CT based surgery planning was used to improve accuracy of intracereboventricular gene therapy delivery and reduce duration of the surgical procedure. Pilot studies on the use of MRI as a longitudinal in vivo monitoring modality in ovine NCL research were conducted and these indicated that MRI will yield enhanced detail compared with CT and that MRI measurements can be closely correlated with the neuropathological changes observed at post mortem examination. However, some specific technical issues were identified and will need to be addressed in the future. These include image quality, fit of templates and an important need to establish ovine-specific image acquisition protocols. Blindness in ovine NCL results from atrophy of photoreceptor cells in the retina as well as changes in the central visual pathways. Treatment should aim not only to treat the atrophy of the brain, but also to prevent or halt the development of blindness. Previous trials have indicated the need for ocular delivery of therapy to achieve this. As for neurodegeneration, in vivo monitoring of retinal pathology is essential for the longitudinal assessment of treatment efficacy. Here, protocols were established for the use of repeated electroretinography (ERG) to monitor the development of blindness in the CLN5 Borderdale and the CLN6 South Hampshire models. The results show that ERG is an invaluable tool for monitoring the progression of retinal degeneration in ovine NCL. Furthermore, it was established that the course of development of retinal changes differs between the two ovine models that were investigated. The measurements enabled some degree of differentiation between the retinal and central components of the blindness in ovine NCL. Finally, it was shown that the in vivo monitoring tools established throughout the studies described in this thesis are invaluable for the assessment of treatment efficacy in gene therapy trials for ovine NCL. The measures evaluated here were shown to correspond with other clinical measures of treatment efficacy, such as behavioural observations, maze-testing and neuropathology. Key words: Neuronal ceroid lipofuscinosis, Batten disease, lysosomal storage disease, large animal models, sheep, neuroimaging, neurodegeneration, neuroinflammation, gene therapy, computed tomography, magnetic resonance imaging, electroretinography, in vivo monitoring.