ItemA microbiota-targeted strategy to attenuate antipsychotic-induced weight gain(University College Cork, 2023) Lipuma, Timothy; Schellekens, Harriet; English, JaneBackground: Atypical antipsychotics such as olanzapine are an essential treatment for psychotic-spectrum disorders, but their use is associated with significant weight gain and increased cardiometabolic disease risk. Attenuating these side effects could improve the tolerability and adherence to antipsychotic medications. Evidence suggests that the microbiome plays a role in antipsychotic-induced weight gain, thus targeting the microbiome may be a viable therapeutic strategy to attenuate the side effect profile of antipsychotics like olanzapine. Furthermore, metabolomics approaches are being increasingly employed to elucidate disease pathophysiology and potential therapeutic targets, but these strategies have not yet been applied to the problem of antipsychotic-induced obesity and hyperphagia. Aims: The primary aims of this study are to (1) investigate if combined microbiome-targeted treatments (probiotic [APC1472], prebiotic [xanthohumol], and their combination) with olanzapine attenuate antipsychotic-induced obesity, metabolic dysfunction, and hyperphagia in female Sprague-Dawley rats, and (2) analyse blood plasma using discovery metabolomics to generate potential mechanistic and therapeutic targets related to the side effects of olanzapine. Methods: Animals were treated with olanzapine (2 mg/kg body weight) alone (n=12), olanzapine with probiotic (n=11), olanzapine with prebiotic (n=11), olanzapine with probiotic and prebiotic (n=12) or control vehicle (n=12) twice a day via intraperitoneal injection for 31 days. Changes in body weight, adiposity, glucose metabolism, dietary intake, anxiety-like behaviour, plasma biomarkers (corticosterone, insulin, ghrelin), and hypothalamic and hepatic gene expression were examined. Ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS) and subsequent metabolomic analysis using Progensis QI and Metaboanalyst were used to characterise plasma differences between the olanzapine treatment group and controls. Results: After the study conclusion, quality control issues with the probiotic formulation were discovered, limiting the interpretability of the data from those treatment groups. However, the olanzapine treatment displayed increased weight gain, dietary intake, and hypothalamic genes related to ghrelinergic signalling. Olanzapine did not increase adiposity, change hepatic gene expression, plasma biomarkers, or hypothalamic genes related to anorexigenic signalling. No treatments attenuated olanzapine-induced weight gain. There were no observed differences in anxiety-like behaviour between any groups. Lastly, the metabolomics investigation revealed several highly differentially expressed metabolites; two androstanoids and one endocannabinoid (oleamide). Conclusion: These findings indicate that olanzapine-associated increases in hypothalamic ghrelinergic signalling can occur before or without the onset of peripheral changes in metabolic health. Although the attenuation of olanzapine-associated increases in hypothalamic ghrelinergic signalling could not be assessed due to the quality control issues with the probiotic, targeting ghrelinergic signalling via microbiome-targeted approaches warrants further research. Additionally, the metabolomics analyses highlight oleamide as a novel metabolite that is potentially at the intersection of the endocannabinoid system, the microbiota, and olanzapine treatment, but further research is needed to clarify if the observed increase in oleamide is due to changes in host and/or microbial metabolism. ItemLong term potential of a saturated sodium chloride solution for the anatomical preservation of human cadavers(University College Cork, 2022-03-23) O'Flynn, Carrie; Toulouse, AndréThe anatomical world has relied heavily on formaldehyde as an embalming agent since its use began in the 1890s. Efforts to move away from formaldehyde have intensified in recent years, largely in response to health concerns. Another important motivation is to seek out ways to an improved anatomical cadaver. Several new techniques have been investigated for their abilities to provide cadavers with both life-like features and longevity of preservation. A simple saturated salt solution (saturated NaCl solution) was used to embalm 4 cadavers in two phases of study, without the addition of formalin. As “soft-fix” methods are generally viewed as short-term preservatives, the long-term preservative action of the saturated NaCl solution method was assessed. The suitability of this cadaver type for teaching and training was considered; specifically, its utility as a training model for ultrasound-guided regional anaesthesia (USGRA). The saturated NaCl solution method conferred long-lasting preservation of structures with retention of tissue colour and pliability; however, the rapid onset of deterioration occurred when gross dissection began. The cadavers proved to have some utility as simulation models for USGRA training, but lack of vascular circulation limited this suitability. ItemThe role of retinoic acid in glioma growth control(University College Cork, 2021-07-22) Flynn, Patricia Margaret; Toulouse, André; Hand, Collette; Bermingham, Niamh; Jansen, MichaelTumours of the central nervous system are known as gliomas, arising from the astrocytes, oligodendrocytes, ependymal cells or from glial progenitor cells. Although a relatively rare diagnosis, there is disproportionate morbidity associated with a glioma diagnosis, owing to its diffuse and infiltrative nature and partly, in the restricted accessibility of the tumour to treatment. Several obstacles prevent effective treatment, namely, the blood brain barrier, peripheral inactivation of systemic treatment, outward tumoural convection pressures, and cancer stem cell resistance. Despite therapeutic advances, the relapse rate and the mortality linked to glioma remains high, with most patients surviving less than 2 years following diagnosis. The terminal differentiation of malignant cells using a differentiation agent such as retinoic acid (RA) could be a promising scientific advance in the treatment of this disease. Endogenous retinoic acid is the primary active metabolite of vitamin A. It is a small, lipophilic differentiation agent that acts as a ligand for a family of nuclear receptors (RARs) to regulate the expression of target genes. The main family of nuclear receptors comprises three genes, RARα, RARβ and RARγ, each coding for multiple isoforms. The selective stimulation of these isoforms with RA has been shown to mainly inhibit cellular proliferation but is also known in some cases to promote such proliferation. The use of all-trans retinoic acid as an agent of differentiation has also been highly successful in the treatment of acute promyelocytic leukaemia. The canonical retinoic acid pathway involves proteins that are responsible for the conversion of precursors of RA, their transport and the transcription of genes downstream of the RARs. In this thesis, gene expression of components of the canonical RA signalling pathway was analysed from existing microarray data for a panel of 1100 gliomas of various histological grades. The analysis was performed on the R2 Genomics Analysis and Visualization Platform. Expression of individual genes was extracted from the datasets and analysed according to WHO grade. The results showed that the expression of key components of the pathway was altered in high grade gliomas compared to the lower grades. The expression levels of RBP1, RBP2, and RBP3 (involved in the transport of retinol) were significantly altered in high grade glioma, with an increase in the expression levels of RBP1 and RPB2 and the decrease in expression level of RBP3. The genes involved in the oxidation of retinol to retinal, ADH1A, ADH1B, ADH1C, ADH4, ADH7, RDH5 RDH10, RDH11 and RDH16 were all significantly lower in high grade glioma. The expression levels of the genes involved in the oxidation of retinal to retinoic acid, ALDH8A1, and ALDH1A1 (RALDH1) are also significantly reduced in high grade glioma. The genes involved in the intranuclear transcription of the RA pathway are affected by high grade glioma. Retinoic acid receptor genes RARA, RARB, RARG, retinoid X receptor genes RXRA, RXRB, RXRG, transcriptional co-activator genes EP300, NCOA1, NCOA2, transcriptional co-repressor genes HDAC2, NCOR1, and NRPI1 are all significantly lower in high grade glioma with an increase in the expression of transcriptional repressor HDAC1. There is also significantly reduced expression of CYP26B1, involved in the metabolism of RA. Together, lower expression of the enzymes responsible for the intracellular formation of RA from its precursors and its intranuclear transcriptional machinery could potentially lead to a reduction in RA signalling. This suggests that the targeted activation of the RA pathways in gliomas with reduced signalling capacity could be used to regulate cancerous growth. Using two established glioblastoma cell lines, the growth altering properties of retinoic acid, some of its synthetic derivatives and specific retinoic acid receptor isoforms was assessed. Results show that manipulation of the retinoic acid signalling pathway by selectively stimulating different isoforms of the RARs can lead to changes in growth patterns that differ depending on the cellular context. Opposing growth patterns were produced in the two glioblastoma cell lines, A172 and U87-MG in response to ATRA, with growth suppression of the A172 cell line and proliferation of the U87-MG cell line. More detailed insight into the growth responses mediated by isoform specific retinoids and overexpression of isoforms highlighted the potential for suppressing the growth of the cell lines by targeting individual receptor isoforms. While further research is needed, these results show that targeting specific receptors in cell lines can lead to growth reduction and may pave the way to the use of isoform selective retinoids in the treatment of glioma. ItemDeciphering the role of microbially-derived metabolites on the microbiota-gut-brain axis(University College Cork, 2021-01-25) Spichak, Simon; Cryan, John; Dinan, Timothy G.; Science Foundation Ireland; Irish Research CouncilThe trillions of microbial organisms residing in the gut, microbiota, are now recognized as major modulators of physiology and health, quickly becoming one of the most exciting emerging areas in neuroscience. Preclinical and clinical research alike suggests that the metabolites produced by these gut microbes modulate brain, behavior and disease. Short-chain fatty acids, tryptophan metabolites and bile acids are promising targets for new microbiome-based therapies. But, little is known about their mechanisms. To this end, the second chapter of the thesis collates 278 studies relating to the human microbiota-gut-brain axis, identifying trends and technical/bioinformatics limitations. These studies across different disorders of the brain as well as healthy human behavioral functions. Then a 35 of these studies was reanalyzed with an up-to-date bioinformatics pipeline. New tools, mainly the gut-brain modules provide a predictive framework for identifying whether these gut microbial metabolic pathways are dysregulated in brain diseases and disorders. We uncovered evidence of disease-related alterations in microbial metabolic pathways in Alzheimer’s Disease, schizophrenia, anxiety and depression. Previous human studies suggest that astrocyte immunity and metabolism is affected by short-chain fatty acids. Thus we grew primary male and female mouse astrocyte cultures, treating them with acetate, butyrate and propionate. Butyrate treatment (0 – 25μM) increased gene expression of Bdnf and Pgc1-α expression, implicating histone-deacetylase inhibitor pathways only in female cells. Acetate (0 – 1500 μM) positively correlated with Ahr and Gfap expression in males, suggesting an immune modulatory role. These findings show a novel sex-dependent impact of acetate and butyrate, but not propionate on astrocyte gene expression. These studies increase understanding of microbial metabolites and how they might impact the brain. It also provides guidance to improve and direct future investigations aimed at identifying the mechanisms of other metabolites. ItemThe effects of stressors during early life on hippocampal neurogenesis and microglial activation in the male and female brain(University College Cork, 2021-04-05) McGovern, Andrew J.; Nolan, Yvonne M.; O'Leary, OliviaStress during critical periods of brain development and maturation such as adolescence is associated with an increased risk of developing stress-related psychiatric disorders which are more common in women than men. Early life stress such as maternal separation (MS), juvenile stress (JS) and inflammatory insults like lipopolysaccharide (LPS), have been found to induce anxiety and depressive-like behaviours and decrease adult hippocampal neurogenesis in rodents. However, the effects of early life stress on adult hippocampal neurogenesis and associated function have been mostly assessed in male rodents. The impact of early life stress on microglia, which are involved in the regulation of adult hippocampal neurogenesis and dendritic remodelling, has also been predominantly examined in male rodents. Thus, in this study we assessed adult hippocampal neurogenesis and hippocampal microglia following LPS administration in MS juvenile female Sprague-Dawley rats and following JS in male and female Sprague-Dawley rats in adulthood. MS increased the number of newly born hippocampal neurons in the ventral hippocampus, reduced the dendritic complexity of newly born neurons in the whole hippocampus and increased the soma size of microglia, indicating activation. LPS reduced newly born hippocampal dendritic complexity and increased the number of microglia in the dorsal hippocampus. Conversely, LPS administration in MS rats reduced the number of microglia in the dorsal hippocampus and MS attenuated microglial activation in response to LPS. LPS administration in MS increased dendritic complexity in the granule cell layer (GCL) and further reduced dendritic complexity in the ventral but not dorsal hippocampus of juvenile female rats. JS did not affect hippocampal neurogenesis in adult male or female rats but reduced the cell soma size of microglia in the GCL in the dorsal hippocampus of females. We observed significant sex differences in adult rats; females had fewer newly born neurons with less dendritic complexity in the dorsal hippocampus than males. There were also fewer microglia in the molecular layer (ML) of the hippocampus in adult female than male rats. Together the data here shows that the effect of early life stressors differentially affects hippocampal neurogenesis and hippocampal microglia dependent on age, sex and subregion of the hippocampus analysed.