title: Investigating Cell Proliferation in the Nervous System
Creators: name: Cavanagh, Brenton
Publisher: name: Griffith University
Year: 2017
Subjects: subject: Cell proliferation
subject: Nervous system
subject: Neural tissue

description: Cell proliferation is a strictly regulated process which is preceded by DNA synthesis and results in an increase in the number of new cells. It is essential for the development, regeneration and is upregulated in tumours. Whilst the study of cell proliferation is fundamental for many arms of biomedical investigation, the techniques used in its study have remained unchanged for decades. Neuroscience is one such field where cell proliferation in the adult can be a rare event and where molecular biology techniques are accelerating discoveries. Unfortunately, the limitations of studying cell proliferation have become a constraint in the field. This thesis examines the development of techniques to identify, characterise, quantify and profile proliferative cells in neural tissue. The techniques are provided in the context of a collection of studies that applied the techniques to secure data in a testable framework. The nervous system of the brain and olfactory mucosa were used to develop techniques to investigate cells on a histological and molecular scale. Initial investigation developed and improved tissue processing workflow and techniques to provide the optimal samples for histological analysis. The optical properties of the sample were improved, allowing deeper imaging in thick tissue sections. The embedding of frozen samples was altered to make use of gradients of OCT embedding media that minimised cell lysis. Embedment in the wax polyethelene glycol was used for sectioning at room temperature. Both techniques preserved tissue cytoarchitecture and antigenicity, allowing fluorescent labelling with multiple markers simultaneously in tissues with well conserved ultrastructure. These optimally prepared thick tissue samples provided a means of imaging multiple phenotypes and cell states in a single specimen (multiplexed), rather than multiple replicates of tissue sections, with different markers. The high optical clarity of the section facilitated high resolution 3D image acquisition, using optimised imaging techniques, further increasing the amount of information obtained from a sample set. These histological techniques were applied to the olfactory mucosa and substantia nigra to quantify cell proliferation and neurogenesis. EdU a thymidine analogue was used to label cells during the S-phase of the cell cycle, identifying cells that had undergone proliferation during exposure. It was shown that although neurogenesis was present in the olfactory mucosa, cell proliferation that occurred in the substantia nigra rarely gave rise to cells of a neural lineage. These techniques enabled the development of novel cell quantification methods, using stereology principles. Due to the ease and significantly less fragile nature, the substantia nigra as opposed to the olfactory system, was chosen to develop this technique. By quantifying subtypes of dopaminergic neurons in the substantia nira pars compacta of both the mouse and rat, an unbiased and accurate method of cell estimation was developed. The embedding method, multiple labelling immunofluorescence and serial optical sections obtained from thick specimens enabled significant improvements to stereological assessment. Thus, multiplexed cellular data was accurately quantified in brain tissue. These techniques were used to label multiple cell phenotypes during a defined period of exposure to EdU. This provided a powerful tool to investigate tissue where data on cell division and development was required together with cell – cell interaction of specific cell phenotypes that were labelled fluorescently. Specific tissue regions were quantified accurately and unbiasedly employing the advanced cell estimation technique. Expanding on the ability to effectively label and analyse cellular structures in tissue sections, in-situ, the capability to isolate and extract biomolecules from proliferating cells for analysis was developed. Thus, providing insight into the cellular differences that occur in proliferating cells. The basis of the technique involved the dissociation and fluorescent labelling of samples pre-labelled with EdU. These labelled cells were then isolated using FACS and RNA was then extracted for assessment of quality and analysis. The optimisation of each step was required to conserve the cell integrity and RNA quality. An enzyme cocktail that provided a gentle dissociation of neuronal tissue, decreased copper in the EdU labelling reaction, and minimising cell disruption during FACS was developed. Thereby, single proliferating cells and their RNA content was effectively extracted from neural tissues. The RNA extracted from the dividing cells showed significant expression differences of several key RNA products when compared to the non-dividing cells of the same tissue. These techniques provide a powerful tool kit to investigate proliferating cells of neural origin. Their use is not limited to the tissues and applications outlined in this thesis but are translatable to other tissue and biomolecules.