General Anaesthetic Modulation of Memory-Related Gene Expression Using an In vitro Brain Slice Model
Stayte, L. T. O. (2016). General Anaesthetic Modulation of Memory-Related Gene Expression Using an In vitro Brain Slice Model (Thesis, Master of Science (Research) (MSc(Research))). University of Waikato, Hamilton, New Zealand. Retrieved from http://hdl.handle.net/10289/11487
Permanent Research Commons link: https://hdl.handle.net/10289/11487
General anaesthetics cause widespread neurochemical and physiological changes in the brain. However, the precise mechanism of amnesic action is largely unknown. Gene expression changes in the hippocampus have been a focal point for investigation in this area, while effects on the cerebral cortex have been largely underreported even though the cerebral cortex has been shown to play a large role in memory consolidation and storage. Amnesia is likely due to the change in expression of memory-related genes within the neocortex or hippocampus of the mammalian brain. The first aim of this research was to investigate the in vitro cortical gene expression pattern of two memory-related genes; activity-regulated cytoskeleton-associated protein (Arc) and brain-derived neurotrophic factor (Bdnf), after a t=4 hour exposure to propofol- or sevoflurane- induced anaesthesia using an adult mouse brain slice model and real-time quantitative PCR. Five animals were used for each anaesthetic and a t=0 hour control, t=4 hour control and t=4 hour treated 400 μm slice were taken from each animal. Seizure-like activity was recorded from the brain slices to ensure viability of the tissue before carrying out the anaesthetic exposure. RNA was extracted, DNAse-treated, and then converted to cDNA. Quantitative PCR was then carried out to analyse Bdnf and Arc expression differences between the t=4 hour control and t=4 hour treated samples using Gapdh, β2m, Actb and HRPT1 as reference genes. The second research aim was to determine the Bdnf protein expression level and localisation after a t=4 hour exposure to propofol using western blot and immunohistochemistry methodologies. Our research demonstrated that Arc was significantly down-regulated after exposure to sevoflurane for t=4 hours (p<0.05). Arc was also shown to be up-regulated after a t=4 hour exposure to propofol while Bdnf showed a downregulation to sevoflurane but an upregulation to propofol, however this data was not statistically significant. Western blot data showed that the rabbit polyclonal Bdnf antibody was binding to an off-target epitope at 55 kDa with mouse brain, heart, lung, liver, spleen and kidney whole tissue lysate. A newly sourced commercial Bdnf antibody was validated and western blot data showed recognition of the Bdnf epitope at the correct predicted size of 28 kDa in the mouse brain, heart and kidney. Immunohistochemistry of frozen mouse brain sections failed to produce a positive signal for two different Bdnf antibodies due to encountering technical issues. Hematoxylin and Eosin staining showed the tissue was still intact after the sectioning procedure. These issues could be resolved in the future by improving the tissue fixation length and optimising the antigen retrieval step. Eleven recommendations have been made to provide further insight into the gene expression levels of memory-related genes in the mouse brain. This includes varying the induction and maintenance of the two anaesthetic drugs, role of epigenetic modification, evaluating the mental state of the mice (depression), investigating Bdnf knockout, differential expression between areas of the brain and brain electrical activity. In addition, the gene expression pattern after a period of anaesthetic exposure should also be analysed. This may help elucidate the causes of postoperative cognitive dysfunction. Whole transcriptome analysis using RNA-Seq could be used to determine the expression levels of other known memory-related genes after anaesthetic exposure.
University of Waikato
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