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The thermoneutral zone (TNZ) curve lies between the ambient temperatures (Ta) where an endothermic animal uses the least amount of energy to maintain a balance between the heat production from the animal’s own metabolism and the heat lost to the environment. If the animal is exposed to Ta’s over the upper critical temperature (UCT), which is the highest temperature that is still in the TNZ, the animals have to use energy to cool down. If they are exposed to temperatures lower than the lower critical temperature (LCT), which is the lowest temperature that is still in the TNZ, the animal have to use energy to warm up. In the present study oxygen consumption was measured at different Ta’s to determine the TNZ in two and three week old broiler chickens (Gallus gallus domesticus). Two different protocols were used and compared, a pseudorandom protocol in which chickens were exposed to seven temperatures in two hour periods for each run and a more typical progressive protocol in which Ta decreased gradually, one or two degrees per hour. The TNZ in two weeks old chickens was between 30.7 ˚C- 36.4 ˚C and between 28.8 ˚C- 32.7 ˚C in the three weeks old chickens. In chickens the TNZ shifts remarkably during the first few weeks of life towards lower temperatures as the animals acquire thermoregulatory competence. The method with a pseudorandom protocol takes more factors, like activity, into consideration than a typical progressive protocol.

Liver injury instigates a proinflammatory response in tissue-resident macrophages, called Kupffer cells (KCs), resulting in the recruitment of monocytes and neutrophils. The high energy demand required for a rapid proinflammatory response in macrophages like KCs is achieved through metabolic reprogramming. This is supported by increased glycolysis. On the other hand, injury resolution requires hepatic macrophages to undergo an anti-inflammatory polarization, which relies on oxidative phosphorylation (OXPHOS). In addition to shifts in mechanisms of adenosine triphosphate (ATP) production, lipid metabolic reprogramming supplies metabolic intermediates and lipids for membrane remodeling and the production of inflammatory mediators. AMP-activated protein kinase (AMPK) is a master metabolic regulator that influences the metabolic reprogramming of macrophages. While AMPK activation promotes an anti-inflammatory polarization, disruption of activity exacerbates proinflammatory signaling. For this thesis work, we addressed whether macrophage AMPK is protective against liver injury by altering immunometabolism. Specifically, we investigated this question in the context of chronic (nonalcoholic steatohepatitis (NASH)) and acute (acetaminophen (APAP) overdose) liver injury. While APAP overdose is a robust and directly translational model of acute injury, models of NASH-induced hepatic fibrosis rely on nutrient-deficient diets like the choline-deficient high-fat diet (CDAHFD) or genetic manipulation. Despite the utility of these models, they seldom mirror the pathogenesis of human NASH, with diets like CDAHFD being completely dissociated from metabolic syndrome. Moreover, models are required to address the divergence between male and female mice. Recently, there has been a shift towards addressing other variables that drive inflammation and metabolism. At room temperature (RT) (22 °C), mice experience cold stress that alters various biological functions. Cold stress drives brown adipose tissue (BAT) activation and upregulates corticosterone production and immunosuppression, all processes that blunt NASH progression. Giles et al. (2016) demonstrated that housing mice at thermoneutrality (TN) (30 °C) exacerbated metabolic-dysfunction associated fatty liver disease (MAFLD) progression toward NASH in both male and female mice. Since then, we and others have implemented TN housing with different dietary interventions and mice strains. We determined that 16-week Western diet (WD) feeding of male and female mice at 29 °C was insufficient to drive hepatic fibrosis, however alterations in glucose tolerance and elevated liver injury enzymes as well as profibrotic gene expression in male mice may indicate that a longer timeline is necessary (24 weeks). Given that our TN NASH model did not produce hepatic fibrosis, we implemented the CDAHFD to investigate macrophage AMPK in chronic liver injury. Male and female AMPK Flox (Prkaa1 fl/fl/Prkaa2 fl/fl) and MacKO (Flox-LysM-Cre+) mice were fed CDAHFD for 8 weeks. In this time frame, CDAHFD produces a lean euglycemic phenotype with hepatic steatosis, inflammation, and fibrosis, to which AMPK MacKO had no influence. Moreover, intervention with a low dose of metformin had no effect, contrary to the reduction in hepatic steatosis observed in HFD-fed mice. Although macrophage AMPK is dispensable in the CDAHFD model of chronic liver injury, acute liver injury needed to be addressed. We found that priming with systemic activation of a direct AMPK activator MK-8722 did not influence hepatic injury and necrosis in our model of APAP-induced liver injury (AILI). Moreover, deletion of hepatocellular AMPK (Flox-Alb-Cre+) or AMPK MacKO did not influence injury at 24 hours post overdose. Despite the lack of effect of systemic AMPK activation, we were interested in a nanoparticle-based targeting of direct AMPK activator MK-8722 (NP-MK8722) delivery. We determined that PLGA-PEG nanoparticles (NPs) accumulated in hepatic macrophages as early as 2 hours post-injection, but NP-MK8722 did not alter hepatic necrosis, injury, or immune infiltration. Overall, my thesis work has advanced our knowledge of the effects of housing temperatures on NASH pathogenesis. Moreover, we are the first to address the effects of macrophage AMPK signaling in NASH and AILI. This is especially true for assessing how AMPK deficiency and targeted activation influences KC immunometabolism during injury.

Previous data have shown that the most prevalent, indirect plasma volume (PV) measurement technique, which utilises changes in haematocrit (Hct) and haemoglobin concentration ([Hb]), underestimates actual PV changes during immersion, when compared to a direct tracer-dilution method. An increase in the F-cell ratio (whole-body haematocrit (Hctw) to large-vessel haematocrit (Hctv) ratio) has been purported as a possible explanation, probably due to hydrostatic and thermally-mediated changes during water immersion. Previous investigators have not quantified the F-cell ratio during immersion. Therefore, this study sought to determine the effect of the F-cell ratio on the indirect method during both, thermoneutral and cold-water immersions. Seven healthy males were tested three times, seated upright in air (control: 21.2°C SD ±1.1), and during thermoneutral (34.5oC SD ±0.2) and cold-water immersion (18.6oC SD ±0.2), immersed to the third intercostal space for 60 min. Measurements during the immersion tests included PV (Evans blue dye column elution, Evans blue dye computer programme, and Hct [Hb]), red cell volume (RCV; sodium radiochromate), cardiac frequency (fc) and rectal temperature (Tre). Plasma volume during the control trial remained stable, and equivalent across the three tests. There was a hydrostatically-induced increase in PV during thermoneutral immersion, when determined by the Evans blue dye method (16.2%). However, the Hct/[Hb] calculation did not adequately reflect this change, and underestimated the relative PV change by 43%. In contrast, PV decreased during cold immersion when determined using the Evans blue dye method by 17.9% and the Hct/[Hb] calculation by 8.0%, respectively, representing a 52% underestimation by the latter method. There was a non-significant decline in RCV during both immersions. Furthermore, an increase (8.6%) and decrease (-14.4%) in blood volume (BV) was observed during thermoneutral and cold-water immersions, respectively. The decline in RCV during thermoneutral immersion attenuated the BV expansion. Despite the disparity between the PV methods, there was no increase in the F-cell ratio during either immersion. In contrast, there was a significant decline in the F-cell ratio during the control: air and thermoneutral immersion, which may indicate that other, undefined variables may impact on the stability of the red cell compartment. The current study is the first to show that the Hct/[Hb] method clearly underestimates PV changes during both thermoneutral and cold-water immersion. Furthermore, RCV was shown, for the first time, to decline during both immersions. However, the changes in the F-cell ratio during this study, did not account for the underestimation of PV change using the Hct/[Hb] method.

Volume overload is a common feature in patients on haemodialysis (HD). This contributes significantly to the cardiovascular disease burden seen in these patients. Clinical assessments of the volume state are often inaccurate. Techniques such as interdialytic blood pressure, relative blood volume monitoring, bioimpedance are available to improve clinical effectives. However all these techniques exhibit significant shortcomings in their accuracy, reliability and applicability at the bed side. We evaluated the usefulness of a dual compartment monitoring technique using Continuous Segmental Bioimpedance Spectroscopy (CSBIS) and Relative Blood Volume (RBV) as a tool to assess hydration status and determine dry weight. We also sought to evaluate the role of Atrial Natriuretic Peptide (ANP) and B-type Natriuretic Peptide (BNP) as a volume marker in dialysis patients. The Retrospective analysis of a historical cohort (n = 376, 55 Diabetic) showed a significant reduction in post-dialysis weights in the first three months of dialysis (72.5 to 70kg, p<0.027) with a non-significant increase in weight between months 6-12. The use of anti-hypertensive agents reduced insignificantly in the first 3 months, increased marginally between months 3-6 and significantly increased over the subsequent 6 months. The residual urea clearance (KRU) fell and dialysis times increased. The cohort was very different to that dialysing at Tassin and showed a dissociation between weight reduction and BP control. This may relate to occult volume overload. CSBIS-RBV monitoring in 9 patients with pulse ultrafiltration (pulse UF) showed distinct reproducible patterns relating to extra cellular fluid (ECF) and RBV rebound. An empirical Refill Ratio was then used to define the patterns of change and this was related to the state of their hydration. A value closer to unity was consistent with the attainment of best achievable target weight. The refill ratio fell significantly between the first (earlier) and third (last) rebound phase (1.97 ± 0.92 vs 1.32 ± 0.2). CSBIS monitoring was then carried out in 31 subjects, whilst varying dialysate composition, temperature and patient posture to analyse the effects of these changes on the ECF trace and to ascertain whether any of these interventions can trigger a change in the slope of the ECF trace distinct to that caused by UF. Only, isovolemic HD caused a change in both RBV and ECF in some patients that was explained by volume re-distribution due to gravitational shifts, poor vascular reactivity, sodium gradient between plasma and dialysate and the use of vasodilating antihypertensive agents. This has not been described previously. These will need to be explored further. The study did demonstrate a significant lack of comparability of absolute values of RECF between dialysis sessions even in the same patient. This too has not been described previously. This is likely to be due to subtle changes in fluid distribution between compartments. Therefore a relative changes must be studied. This sensitivity to subtle changes may increase the usefulness of the technique for ECF tracking through dialysis. The potential of dual compartment monitoring to track volume changes in real time was further explored in 29 patients of whom 21 achieved weight reductions and were able to be restudied. The Refill Ratio decreased significantly in the 21 patients who had their dry weights reduced by 0.95 ± 1.13 kg (1.41 ± 0.25 vs 1.25 ± 0.31). Blood pressure changes did not reach statistical significance. The technique was then used to examine differences in vascular refill between a 36oC and isothermic dialysis session in 20 stable prevalent patients. Pulse UF was carried out in both these sessions. There were no significant differences in Refill Ratios, energy removed and blood pressure response between the two sessions. The core temperature (CT) of these patients was close to 36oC and administering isothermic HD did not confer any additional benefit. Mean BNP levels in 12 patients during isovolemic HD and HD with UF did not relate to volume changes. ANP concentrations fell during a dialysis session in 11 patients from a mean 249 ± 143 pg/ml (mean ± SD) at the start of dialysis to 77 ± 65 pg/ml at the end of the session (p<0.001). During isolated UF levels did not change but fell in the ensuing sham phase indicating a time lag between volume loss and decreased generation. (136±99 pg/ml to 101±77.2 pg/ml; p<0.02) In a subsequent study ANP concentrations were measured throughout dialysis and in the post-HD period for 2 hours. A rebound in ANP concentration was observed occurring at around 90 min post-HD. The degree of this rebound may reflect the prevailing fluid state and merit further study. We have shown the utility of dual compartment monitoring with CSBIS-RBV technique and its potential in assessing volume changes in real time in haemodialysis patients. We have also shown the potential of ANP as an independent marker of volume status in the same setting. Both these techniques merit further study.