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The Role of PACAP Neurons in the Control of Metabolism and Fertility by Leptin
Get PDFThe reproductive or Hypothalamic-Pituitary-Gonadal (HPG) axis is tightly regulated by its own internal negative feedback system, as well as by external factors such as the body’s stress responses. One key regulatory factor is the energy status of the body. Several lines of evidence present the reproductive system as metabolically gated, as sufficient energy stores are required for organism go through puberty and be fertile.
Several hormones are used by the body to signal energy status. Leptin is a major metabolic hormone released from adipose tissue. It travels into the hypothalamus to signal satiety, but also serves as a permissive signal for the HPG axis. Leptin deficiency or leptin receptor knockout results in an obese phenotype and infertility. As the GnRH neurons which drive the HPG axis do not express leptin receptors, several intermediary neurons have been identified as communicating leptin’s fertility actions. A neuronal population recently suggested to fulfil this role is the Pituitary Adenylate Cyclase Activating Polypeptide or PACAP neurons. PACAP is a neurotrophic factor expressed across many different tissues which serves a wide number of functions including as a satiety signalling peptide and regulating fertility. A recent study found the PACAP neurons of the ventral premammillary nucleus (PMV) region synapse with the kisspeptin neurons (which stimulate reproductive activity), and when PACAP is knocked out from the PMV, it causes significant delays in puberty onset and impaired female fertility. As these neurons also express leptin receptors, perhaps these neurons act as conduits for leptin’s actions on the reproductive axis.
The aim of the experiment was to investigate the role of PACAP in leptin’s regulation of fertility, with body weight and adiposity measured as secondary endpoints. We used the Cre-lox system to create two genetic mouse models to manipulate leptin receptor expression. To test whether PACAP is necessary for leptin’s metabolic and reproductive actions, leptin receptors were knockout out selectively from PACAP neurons. These animals had no changes in body weight compared to controls, but females showed a moderate but significant ~2-day delay in puberty onset. To test whether PACAP is sufficient for leptin’s metabolic and reproductive actions, PACAP-Cre animals were crossed with animals containing a loxP-flanked stop sequence within the leptin receptor. In Cre-expressing neurons the stop condon was excised, allowing the leptin receptor to be expressed. Thus, only PACAP neurons had functional leptin receptors. These animals exhibited slightly reduced body weights compared to total leptin receptor knockout animals, but puberty and fertility were not restored. An additional experiment was performed to map hypothalamic PACAP neuron populations from birth to adulthood. Using PACAP-Cre mice crossed to animals with floxed GFP, several regions such as the SCN showed increased PACAP expression at later development stages. These results show PACAP neurons are necessary for normal functional fertility and timing, but they are not sufficient for mediating leptin’s fertility role. PACAP expression may also be linked to development
Investigating the roles of epicardial fat and long-chain acylcarnitines in cardio-metabolic disease
Get PDFHeart fat, termed epicardial adipose tissue (EAT), expands in cardio-metabolic disease; however, whether hypertrophy of the comprising adipocytes underlies this expansion is unknown. Part one of my thesis aimed to examine the relationship between EAT adipocyte size, macroscopic EAT thickness, and obesity level. I also investigated the role of EAT adipocytokine release in cardiovascular pathogenesis by determining the effect of the EAT-derived factor, resistin, on human myocardium function.
EAT adipocytes were measured in histological sections of EAT procured from post-mortem case (N=43) and surgery patient (N=49) cohorts. In each cohort, EAT adipocyte size did not correlate with the obesity measure, body mass index (BMI) (post-mortem cohort: r=0.1, P=0.4; surgery cohort: r=0.1, P=0.5), despite finding a positive correlation for paired subcutaneous adipocytes (r=0.6, P<0.0001). Furthermore, in the surgery cohort, adipocyte size did not correlate with EAT thickness (r=-0.1, P=0.6). Exploratory analysis of adipocyte size frequency distributions indicated that EAT from obese cases contains a greater proportion of ‘smaller’ adipocytes. Enzyme-linked immunosorbent assay revealed that human EAT (N=36) releases resistin. When applied to human atrial trabeculae (N=10), exogenous resistin (7, 12, 20 ng/mL) did not change the propensity for spontaneous muscle contractions. Resistin did, however, dose-dependently increase trabeculae contractility (maximal 2.9-fold increase, P<0.0001), while also reducing post-pause contraction potentiation (maximal 2-fold decrease, P=0.002).
These data suggest that expansion of EAT in obesity might be associated with hyperplastic, and not hypertrophic, remodelling of EAT adipocytes. Additionally, the findings suggest that resistin released from EAT exerts a direct inotropic, but not an arrhythmogenic, effect on human myocardium.
Circulating levels of the metabolites, long-chain acylcarnitines (LCACs), are associated with CVD risk. Part two of my thesis sought to determine if LCACs also directly alter cellular Ca2+ handling and human myocardial function.
Human atrial trabeculae were stimulated at 60 contractions per minute and treated with LCAC species 18:1 (1-25 µM, n=8 per concentration). LCAC 18:1 dose-dependently increased contractility (maximal 1.5-fold increase, P<0.01) and increased arrhythmic contraction propensity (maximal 50% increase, P<0.05). To investigate LCAC effects on Ca2+ handling, human embryonic kidney cells with inducible ryanodine receptor (RyR2) expression were loaded with the cytosolic Ca2+ indicator, fluo-4-AM, or the intra-endoplasmic reticulum Ca2+ indicator, D1ER. All LCAC 18:1 concentrations tested (0.1-25 µM) promoted spontaneous Ca2+ release events by reducing the threshold for RyR2-mediated Ca2+ release (89% of control, P<0.0001). In addition to promoting spontaneous Ca2+ release, higher LCAC 18:1 concentrations also markedly increased the rates of Ca2+ and Zn2+ influx (maximal 3- to 7-fold increases, P<0.0001) from the extracellular environment. Treatment with a different LCAC, LCAC 16:0, induced analogous effects on Ca2+ release, Ca2+ influx, and Zn2+ influx, but to a lesser extent than that induced by LCAC 18:1, suggesting that fatty acyl chain and likely membrane disruption underlies the effects of LCACs.
Circulating LCACs directly alter human myocardium function. In vitro, this is linked to LCAC-induced membrane perturbation that promotes Ca2+ influx and enhances spontaneous RyR2 Ca2+ release. This suggests that high LCACs levels might contribute to cardiovascular pathogenesis in addition to offering diagnostic utility
The crisis of representation in the refugee resettlement sector in New Zealand
Get PDFResettlement practitioners are powerful advocates for resettling individuals but are often accused of relying on representational practices that promote inaccurate assumptions of psychopathology. The preoccupation with post-traumatic stress disorder and vicarious traumatisation that characterises such representations can result in resettling communities being subjected to two levels of stigmatisation within society. They are thereby simultaneously at risk and a risk in their new society of settlement and their resilience and opportunities for vicarious resilience in the sector are potentially silenced.
Informed by the transformative paradigm, this thesis recognises that positivist psychological research reinforcing assumptions of psychopathology has historically been promoted throughout the resettlement sector. The transformation anticipated in this research was to raise awareness of the range of responses to trauma and trauma work by sharing critical reflections from resettlement practitioners, obtained through socially constructed semi-structured interviews, together with recently published positivist psychiatric research. This approach to research was chosen to explore identified concerns of local resettling communities and challenge practitioners to reconsider how they represent resettling individuals and resettlement work.
The primary aim of this research was to explore how psychopathological representations are resisted and/or reproduced by practitioners working within the resettlement sector in Wellington, New Zealand. A total of 25 interviews with a cross section of resettlement practitioners (psychiatrists, psychologists, social workers, interpreters and volunteers) were conducted. Six interconnected themes were identified; “They’re people”, “This is not paradise”, “Psychotherapy”, “Pretty damaged people”, “Oh, those poor people” and “People have no idea”. The first three themes resisted the three assumptions of psychopathology which imply that the vast majority of resettling individuals suffer from PTSD, caused by their pre-displacement trauma, and require specialist psychological intervention. The remaining three themes corresponded with the implications of these assumptions, such as, promoting the ‘at risk’ status of resettling individuals and resettlement practitioners and reliance on the assumptions of psychopathology in advocacy. The conclusion of the analysis was that practitioners are potentially caught in a crisis of representation. Central to this crisis is the way in which resettling communities’ psychological wellbeing was represented and the assumptions made about the type of assistance they should receive. Subsequently, practitioners felt compelled to continue to rely on psychopathological representations of resettling communities and resettlement work, in order to obtain recognition and resources for the services they provided. Importantly, in some cases, they continued to do this, knowing that these representations did not accurately reflect the resilience of resettling communities or the realities of supporting them to settle in New Zealand. In addition, they acknowledged that such representations could compromise successful settlement outcomes by perpetuating stigma, societal prejudice and service provision that reinforces passive styles of resettlement. Such critical reflections corroborate the concerns of representatives of resettling communities as well as clinical research published during the course of this research.
The secondary aim of this thesis was to raise awareness of the assumptions of psychopathology that resettlement practitioners tend to rely on and to promote the resilience of resettling communities and realities of supporting them to successfully settle in New Zealand. My approach to sharing my research resulted in a constructive collaboration with the New Zealand Red Cross. As part of this collaboration, I conducted a regional training tour that enabled me to sensitise approximately 500 practitioners to the assumptions of psychopathology being promoted in the resettlement sector and encourage them to critically reflect on the ways in which they represent their work and resettling clients. While the research resonated with most practitioners across the country, a critical incident with one specialist mental health service, revealed contrasting perspectives consistent with the crisis of representation I had conceptualised. It also highlighted the significance of the relational context in the reception of critical research.
In light of the pervasiveness of the assumptions of psychopathology that inform service provision and pragmatism required of practitioners, the recommendation from this research is that practitioners receive ongoing professional development in order to be as critically reflexive and culturally responsive as they are required to be by their professional associations. This research also recommends future participatory research initiatives in collaboration with local resettling communities to identify alternative interventions that acknowledge their resilience and respond to their priorities for resettlement and recovery.
The first contribution of this research has been to identify and illustrate the implications of the crisis of representation within the New Zealand resettlement context during the period 2014 - 2018. The second contribution of this research has been to go beyond simply recommending initiatives to increase critical reflexivity and to actually create opportunities to do so throughout the sector. The third contribution of this research has been to conduct reciprocal research informed by the transformational paradigm – a first in the Department of Psychological Medicine at the University of Otago, Wellington
Novel glycosaminoglycan mimetic's show therapeutic potential in post-stroke recovery
No full textEach year, millions of people are affected by stroke resulting in death or long-term disabilities. Due to improvements in stroke treatment resulting in improved survival following having a stroke, more stroke survivors are living with long-lasting disabilities in sensorimotor and cognitive functions. Due to this, it is critical to find new therapeutic interventions to improve mechanisms of recovery and improve functional outcomes in the field of stroke research. An interesting class of biological molecules for stroke recovery are glycosaminoglycans (GAGs), as they can be expressed in hundreds of different configurations, playing many critical roles in biological processes, whist acting on a large variety of cell types. Due to recent advances in the field, glycomimetics can be efficiently synthesised. The overall aim of this thesis was to explore the potential therapeutic effects of glycomimetic compounds on recovery following stroke. Specifically, we aimed to assess the effects of glycomimetic compounds on inflammation and immune cell activation, functional recovery and axonal sprouting patterns and networks following stroke.
A panel of glycomimetic compounds of different sizes and sulfation patterns were first screened in vitro to investigate whether they altered inflammation of immune cell maturation under conditions of LPS-stimulation. All compounds successfully reduced the inflammatory response following LPS-stimulation, whilst many also modulate the maturation of dendritic cells (DCs) by altering the expression of co-stimulatory molecules CD86 and MCHII. The therapeutic potential of two glycomimetic compounds showing similar success in vitro, were then tested in vivo to assess changes in immune cell signalling and populations, axonal sprouting and behavioural recovery following photothrombotic stroke to the motor cortex (MC) in mice. Glycomimetic compounds were given at a delay of 3-days to promote recovery during the sub-acute phase of stroke. Interestingly, we saw a decrease in the stroke-induced infiltration of B-lymphocytes into the brain following treatment with compound A, but not G. In addition, both glycomimetic compounds, A and G improved gross motor impairments on the grid-walking task and changed the distribution of axonal sprouting by increasing connections to the premotor and prefrontal cortex, whilst dramatically decreasing the sprouting response into the direction of somatosensory cortex. Consistent with this loss in sprouting to the somatosensory cortex, we also observed impaired recovery in the cylinder task as a measure of forepaw asymmetry, which corroborates prior publications assessing some glycomimetic compounds for stroke recovery.
As rehabilitation clinically overlaps with our intervention window, we thought that it would be imperative to train a cohort of mice on the pasta matrix reaching task (PMRT) in order to try and maximise the extent of recovery. A second rational for this is that rehabilitation may also interfere with potential pharmacological treatments. Assessment of the rehabilitation intervention alone, using the pasta matrix reaching task (PMRT), was able to abolish gross motor impairments and forepaw asymmetries observed at 4-weeks post-stroke in all stroke groups. In addition, a combinational approach of rehabilitation and treatment with glycomimetic compounds changed the distribution of axonal sprouting to support functional recovery by the rescue of axonal sprouting to the somatosensory cortex. There is also potential to limit any aberrant axonal sprouting using a combination approach, as we observed a reduction in the sum of axons coordinates compared to rehabilitation alone. Together, this provides evidence that glycomimetic compounds have the potential to improve recovery following stroke, however, a combinational approach with rehabilitation was more successful. Additionally, these studies highlight that a component of rehabilitation should be undertaken in preclinical experiments to support the accuracy and translatability of mechanisms involved in therapeutic agents.
Normal cognitive processing is the result of dynamic connections between multiple brain regions. Previously, the Clarkson group has observed delayed impairments in spatial working memory following photothrombotic stroke to the prefrontal cortex (PFC) in mice. The mechanisms underpinning this impairment remain poorly unknown, but may involve loss of connections to other brain regions. Therefore, we assessed neural projections by injecting retrograde tracers into two regions associated with cognition, the prelimbic cortex (PLC) and ventral hippocampus (vHPC). A global loss of projections into the PLC was observed, in particular from the midline thalamic nuclei mediodorsal (MD and nucleus reuniens (Re). To confirm the involvement of these thalamic nuclei in spatial working memory function, a “silent stroke” induced by L-NIO injection targeting either midline thalamic nuclei was used and which resulted in the same delayed spatial memory impairment. These data support our hypothesis that a loss in connections and in particular, a loss of connections between the PLC and MD and Re, likely underpins the development of delayed memory impairments we have observed following stroke to the PFC and which is similar to reports also observed in humans. In addition to the changes in PLC connections, we also observed altered projections into the vHPC that may be a compensatory mechanism due to global loss of inputs into the PLC. The current thesis provides evidence that functional recovery is, in part, reliant on intact and correct connections being maintained or produced between multiple brain regions, which are likely compromised by the chronic changes in inflammatory signalling pathways. In addition, this thesis offers further support for the use of glycomimetics as a therapeutic option. However, much needed work is still required, especially around understanding differences in sulfation as this can greatly affect the different areas of functional recovery, inflammation, sprouting, angiogensisis, and growth factor support to name just a few
Investigating the inflammatory response within the ageing oviduct
Get PDFOne in four people attempting to conceive will experience infertility. The current theory is that infertility is caused by a decreased number and quality of eggs in the ovary. However, ovarian aging cannot be the sole cause of infertility. In natural conception, the first days of embryonic growth occur within the oviduct. In IVF, this growth period occurs in the lab, eliminating the oviduct's effect on the embryo. Previously the role of the oviduct has not been questioned. The difference of embryos reaching the blastocyst stage between young and aged mice was quantified to establish the extent of the preimplantation loss. To investigate the level of expression of growth promoters and inhibitors in the oviducts of aged and young mice during pregnancy, RT-qPCR was completed. Additionally, spectral cytometry was used to investigate if a change in the Treg cell population occurred with advancing maternal age. This study showed that the number of embryos surviving to the blastocyst stage was significantly decreased in aged mice. No difference was identified between young and aged mice and the expression of embryo growth-promoting or inhibiting factors within the oviduct. This study concluded that there were no changes in the presence of T regulatory cells with advancing maternal age. Our results showed that preimplantation loss is increased in aged mice compared to young mice due to a post-ovulatory effect. We confirmed that the increase in preimplantation loss is not due to a change in the expression of growth promoters or inhibitors within the oviduct. Nor is it due to a change in the response of the maternal inflammatory system to pregnancy. We postulate that an increase in abnormal eggs within the ovary is occurring with advancing maternal age, and is resulting in an increased number of aneuploidy embryos degrading before implantation
Dietary fat intakes and cardiovascular disease risk in adults with type 2 diabetes: a systematic review and meta-analysis
No full textDefining Leptin-Responsive nNOS Neurons for Puberty and Fertility
Get PDFReproduction is a resource intensive process by which animals propagate their genes, hence it must be coordinated carefully with the body’s availability of energy stores to produce viable offspring. The adipocyte-derived hormone leptin relays the peripheral metabolic status of the body to the gonadotropin-releasing hormone (GnRH) neurons to control the hypothalamic- pituitary-gonadal (HPG) axis. This communication is primarily achieved in the hypothalamus, via hormone-responsive neurons operating upstream of GnRH neurons, since GnRH cells do not express leptin receptors (LepR). Nitric oxide synthase-1 (nNOS) neurons co-expressing LepR are suggested to be a direct conduit whereby leptin signals are relayed to the HPG axis. Inhibition of nNOS activity has been shown to cause sporadic ovulation, blunted pre-ovulatory luteinising hormone (LH) levels, and consequent hypothalamic hypogonadism in female mice.
This project initially aimed to elucidate whether nNOS neurons are sufficient to mediate leptin’s permissive actions on reproductive function in vivo, in the absence of leptin signals from any other neurons. Cre-lox technology was adopted to produce transgenic mice that would express LepR in only the nNOS neurons, referred to as the LepR-rescue group. Age at puberty onset, estrous cyclicity, insulin tolerance, and body weights were analysed. Unexpectedly, no significant differences were found between the LepR-rescues and wildtype controls.This appeared to result from Cre-mediated germline excision of the target STOP-flox sequence, which led to global expression of the intact LepR.
Since prior gamma-aminobutyric acid (GABA) neuron-specific LepR knockout experiments suggested that leptin can act via the GABA-ergic neurons but not the glutamatergic neurons to signal GnRH release, this study mapped co-localisation of nNOS neurons that are both GABAergic and leptin-responsive, to raise functional significance for specific hypothalamic regions. Immunohistochemistry (IHC) of both nNOS and phosphorylated signal transducer and activator of transcription 3 (pSTAT3, a marker for leptin signalling) was performed on brain sections of GABA neuron reporter mice (Vgat-Cre X tdTomato). The percentage co-localisation of GABA and leptin-induced pSTAT3 in nNOS neurons was 13% in the arcuate nucleus, 3% in the dorsomedial hypothalamic nuclei and in the medial preoptic area. It is anatomically evident that nNOS neurons form a part of the neuroendocrine network in controlling puberty and fertility
Medication safety in New Zealand general practice
Get PDFBackground
General practice has been traditionally considered a low-risk healthcare setting, however high patient volumes and high prescribing rates elevate the risk of harm. Māori and Pasifika patients are at greater risk of healthcare harm. The extent of medication-related harm, and harm by ethnicity, is unknown in New Zealand general practice.
Determining the nature and extent of these harms is a first step to addressing these problems. Strategies to reduce harm increasingly involve patients. Providing risk information at an appropriate health literacy level can improve patient engagement.
Aims
1) To identify problems associated with medication use in New Zealand general practice
a) To evaluate the extent of medication-related harm arising from prescribing in NZ general practice
b) To evaluate an automated clinician alert system to see whether there were any inequities in clinician action taken based on patient ethnicity or other demographic factors
2) To explore strategies to improve medication safety in New Zealand general practice
a) To explore what patients and prescribers would like in a decision support and communication tool
b) To explore whether a tailored information package for patients can improve knowledge of NSAIDs and reduce self-reported use of NSAIDs
Methods
This thesis used a pragmatic mixed-methods approach in four linked studies. The first two studies are analyses of general practice record review data. They outlined problems associated with medication use, by estimating the amount of medication-related harm, and by determining whether clinician action varies by patient ethnicity when notified of the harm. Studies three and four explored strategies to improve medication safety.
Patients and prescribers were interviewed in the third study to understand what they would like from a medication decision support and communication tool. The fourth study considered the feasibility of conducting a randomised controlled trial to test whether a tailored information package is acceptable and effective in improving knowledge of non- steroidal anti-inflammatory drugs (NSAIDs) and reducing self-reported NSAID use in patients at risk of renal damage.
Results
1. The estimated incidence rate of all medication-related harms in New Zealand general practice was 73.9 harms per 1000 patient-years, the estimated incidence of preventable or potentially preventable medication-related harms was 15.6 per 1000 patient-years, and the estimated hospitalisation rate was 1.1 per 1000 patient-years. Most harms were minor (1390/1762, 78.9%), but one in five harms were moderate or severe (373/1762, 21.1%); three patients died (3/9076, 0.03%). Most medication-related harms were not preventable (n=1432, 81.3%); the remainder were considered preventable or potentially preventable (n=330, 18.7%).
2. Analysis of whether clinicians took action following a risk alert found no evidence of a difference in the odds of having action taken by patient ethnicity, however the estimated odds for Māori and Pasifika patients were lower compared with Europeans (Māori OR 0.88, 95 %CI 0.63–1.22; Pasifika OR 0.88, 95 %CI 0.52–1.49). Females had significantly lower odds of having action taken compared with males (OR 0.76, 95 %CI 0.59–0.96).
3. Patients want as much information as possible about their medications and risk, but doctors find it difficult to communicate that information. Participants were cautiously optimistic about a prescribing risk assessment and communication tool, but worried about potential harm arising from its use. They also identified requirements for the tool and features to avoid. Culturally safe and trustworthy doctor-patient relationships are required before successful implementation of any tool.
4. Patients at risk of renal damage are willing to participate in a study via email recruitment, and engage with an interactive learning activity about non-steroidal anti-inflammatory drugs online. This randomized feasibility trial demonstrated that this research method is feasible for the purposes of recruiting patients and testing the effects of providing this targeted information package.
Conclusion
Medication-related harm in general practice is common. Patient and prescriber perspectives are needed to inform harm-reduction strategies. Use of a targeted alert system has potential to mitigate risk from medication-related harm. Clinicians typically take action on alerts arising from a general practice electronic alert system, but our study suggested that they appear to take less action for women and Māori and Pasifika patients. Recognising clinician biases may improve the equitability of health care provision; respectful, culturally safe doctor-patient relationships are critical to the successful implementation of any risk-mitigation tool. Delivering medication-risk information to targeted patients online is feasible, and further studies are required to determine determine whether that improves knowledge or changes behaviour
Open Access at the University of Otago: what do we know?
No full textThis infographic is the Otago-specific version of a national research project to take a snapshot of the proportion of research by New Zealand authors that is open access. This project was the of the Concul of New Zealand University Librarians (CONZUL). See more here: https://www.universitiesnz.ac.nz/about-universities-new-zealand/unz-committees-and-working-groups/council-new-zealand-universityThree out of five research outputs by Otago authors in 2017 are behind paywalls as of June 2019. Of the 868 articles that are behind a paywall, 84% of these could be deposited legally in a non-commercial repository to make them free-to-access. This is despite a citation advantage of 93% for research that is openly accessible. Where open access was achieved by a paid method (Article Processing Charges) this was estimated to have cost NZ$735,000 for 2017