title: General Anaesthetic Modulation of Memory-Related Gene Expression Using an In vitro Brain Slice Model

authors: Stayte, Liam Thomas Oliver

subjects: General Anaesthetic

abstract: 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.

text: General Anaesthetic Modulation of Memory-Related Gene Expression Using an In vitro Brain Slice Model 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.

year: 2016

title: Book Review: A Brief History of Theology: From the New Testament to Feminist Theology – By Derek Johnston

authors: Pratt, Douglas

subjects: book review

abstract: This article reviews the book: “A Brief History of Theology: From the New Testament to Feminist Theology”, by Derek Johnston.

text: Book Review: A Brief History of Theology: From the New Testament to Feminist Theology – By Derek Johnston This article reviews the book: “A Brief History of Theology: From the New Testament to Feminist Theology”, by Derek Johnston.

year: 2010

title: The Hospital Transfers Project: Supporting whanau engagement during hospitalisations

authors: Masters-Awatere, Bridgette

abstract: For Māori, as the Indigenous peoples of Aotearoa New Zealand, the care of those who are unwell has always been the concern of whānau (family) and community. Māori have established knowledge systems relating to health and wellbeing, and long-standing practices for both promoting good health and responding to illness (Taskforce on Whānau Centred Initiatives [hereafter referred to as the Taskforce], 2009; Waitangi Tribunal, 2001). These systems recognise the importance of relationships between peoples and broader environments to health and wellbeing (Mark & Lyons, 2010; Taskforce, 2009), something which has been more recently acknowledged in Māori health strategy and policy by government (Ministry of Health, 2014b).

text: The Hospital Transfers Project: Supporting whanau engagement during hospitalisations For Māori, as the Indigenous peoples of Aotearoa New Zealand, the care of those who are unwell has always been the concern of whānau (family) and community. Māori have established knowledge systems relating to health and wellbeing, and long-standing practices for both promoting good health and responding to illness (Taskforce on Whānau Centred Initiatives [hereafter referred to as the Taskforce], 2009; Waitangi Tribunal, 2001). These systems recognise the importance of relationships between peoples and broader environments to health and wellbeing (Mark & Lyons, 2010; Taskforce, 2009), something which has been more recently acknowledged in Māori health strategy and policy by government (Ministry of Health, 2014b).

year: 2019