Investigation Of Hypoxia And Mitochondrial Dysfunction In The Central Nervous System Resulting From Focal And Systemic Inflammation

Inflammation is an important feature of several seemingly disparate neurological disorders, including multiple sclerosis, Parkinson’s disease and sepsis-related brain dysfunction. Inadequate oxygenation and mitochondrial dysfunction have been implicated in these and other CNS pathologies in which inflammation is found. Indeed, inflammation can have direct or indirect effects on mitochondrial function, for example, via reactive oxygen/nitrogen species, or through compromised perfusion respectively. However, the study of oxygenation and mitochondrial function in the CNS has been limited as tissues are typically excised for study in vitro, invariably exposing cells and their mitochondria to non-physiological environments. To overcome these limitations, the work described in this thesis involved the study of mitochondrial dysfunction and tissue oxygenation in the CNS during local and systemic inflammation in whole-animal preparations under physiological and pathophysiological conditions. The experiments include development of in vivo optical imaging techniques to assess the redox potential of mitochondria, without the application of dyes, and with an intact blood supply. Using this technique in conjunction with established methods we investigated mitochondrial function and tissue oxygen concentrations in cortical and retinal models of local and systemic inflammation. Our findings reveal that mitochondrial flavoprotein autofluorescence imaged in the cortex of anaesthetised mice can be used to assess an aspect of mitochondrial function (redox potential) in the CNS in vivo. Additionally, we show that certain types of inflammation are associated with tissue hypoxia in the brain and retina, and that this can have profound functional consequences for cerebral mitochondria during systemic inflammation. Hypothermia was also explored as a potential therapeutic strategy to attenuate inflammation-induced functional deficits. Collectively, these findings further our understanding of the mechanisms underlying neurological deficits associated with inflammation, and reveal mitochondrial redox state imbalances in certain inflammatory conditions with potential implications for the treatment of CNS disorders in which inflammation plays a role

The impact of bone cancer on the peripheral encoding of mechanical pressure stimuli

Skeletal metastases are frequently accompanied by chronic pain that is mechanoceptive in nature. Mechanistically, cancer-induced bone pain (CIBP) is mediated by peripheral sensory neurons innervating the cancerous site, the cell bodies of which are housed in the dorsal root ganglia (DRG). How these somatosensory neurons encode sensory information in CIBP remains only partly explained. Using a validated rat model, we first confirmed cortical bone destruction in CIBP but not sham-operated rats (day 14 after surgery, designated “late”-stage bone cancer). This occurred with behavioural mechanical hypersensitivity (Kruskal–Wallis H for independent samples; CIBP vs sham-operated, day 14; P < 0.0001). Next, hypothesising that the proportion and phenotype of primary afferents would be altered in the disease state, dorsal root ganglia in vivo imaging of genetically encoded calcium indicators and Markov Cluster Analysis were used to analyse 1748 late-stage CIBP (n = 10) and 757 sham-operated (n = 9), neurons. Distinct clusters of responses to peripheral stimuli were revealed. In CIBP rats, upon knee compression of the leg ipsilateral to the tumour, (1) 3 times as many sensory afferents responded (repeated-measures analysis of variance: P < 0.0001 [vs sham]); (2) there were significantly more small neurons responding (Kruskal–Wallis for independent samples (vs sham): P < 0.0001); and (3) approximately 13% of traced tibial cavity afferents responded (no difference observed between CIBP and sham-operated animals). We conclude that an increased sensory afferent response is present in CIBP rats, and this is likely to reflect afferent recruitment from outside of the bone rather than increased intraosseous afferent activity

Protection of cerebral microcirculation, mitochondrial function and electrocortical activity by small-volume resuscitation with terlipressin in a model of haemorrhagic shock

Background During early treatment of haemorrhagic shock, cerebral perfusion pressure can be restored by small-volume resuscitation with vasopressors. Whether this therapy is improved with additional fluid remains unknown. We assessed the value of terlipressin and lactated Ringer’s solution (LR) on the early recovery of the microcirculation, tissue oxygenation, and mitochondrial and electrophysiological function in the rat cerebral cortex. Methods Animals treated with LR replacing three times (3x) the volume bled (n=26), terlipressin (n=27), terlipressin plus LR of 1x (n=26), 2x (n=16), or 3x (n=15) were compared with untreated (n=36) and sham-operated rats (n=17). In vivo confocal microscopy was used to assess cortical capillary perfusion, changes in tissue oxygen concentration, and mitochondrial membrane potential and redox state. Electrophysiological function was assessed by cortical somatosensory evoked potentials (SEPs), spinal cord dorsum potential, and peripheral electromyography. Results Compared with sham, the mean (SD) area of perfused vessels was lower in rats subjected to haemorrhagic shock: 82 (10)% vs. 38 (12)%; P<0.001) and impaired oxygen concentration, mitochondrial redox state (99±4 vs. 59±15 % of baseline; P<0.001), and SEPs (97±13% vs. 27±19% of baseline). Adminstration of terlipressin plus 1X or 2X LR was able to recover these measures, but terlipressin+3LR or 3LR alone were not as effective. Spinal cord dorsum potential was preserved in all groups, but no therapy protected electromyographic function. Conclusion Resuscitation from haemorrhagic shock using terlipressin with small-volume LR was superior to high-volume LR, with regard to cerebral microcirculation, and mitochondrial and electrophysiological function

Hypothermia protects brain mitochondrial function from hypoxemia in a murine model of sepsis

Sepsis is commonly associated with brain dysfunction, but the underlying mechanisms remain unclear, although mitochondrial dysfunction and microvascular abnormalities have been implicated. We therefore assessed whether cerebral mitochondrial dysfunction during systemic endotoxemia in mice increased mitochondrial sensitivity to a further bioenergetic insult (hyoxemia), and whether hypothermia could improve outcome. Mice (C57bl/6) were injected intraperitoneally with lipopolysaccharide (LPS) (5 mg/kg; n = 85) or saline (0.01 ml/g; n = 47). Six, 24 and 48h later, we used confocal imaging in vivo to assess cerebral mitochondrial redox potential and cortical oxygenation in response to changes in inspired oxygen. The fraction of inspired oxygen (FiO2) at which the cortical redox potential changed was compared between groups. In a subset of animals, spontaneous hypothermia was maintained or controlled hypothermia induced during imaging. Decreasing FiO2 resulted in a more reduced cerebral redox state around veins but preserved oxidation around arteries. This pattern appeared at a higher FiO2 in LPS-injected animals, suggesting an increased sensitivity of cortical mitochondria to hypoxemia. This increased sensitivity was accompanied by a decrease in cortical oxygenation, but was attenuated by hypothermia. These results suggest that systemic endotoxemia influences cortical oxygenation and mitochondrial function, and that therapeutic hypothermia can be protective

Immune or Genetic-Mediated Disruption of CASPR2 Causes Pain Hypersensitivity Due to Enhanced Primary Afferent Excitability

Human autoantibodies to contactin-associated protein-like 2 (CASPR2) are often associated with neuropathic pain, and CASPR2 mutations have been linked to autism spectrum disorders, in which sensory dysfunction is increasingly recognized. Human CASPR2 autoantibodies, when injected into mice, were peripherally restricted and resulted in mechanical pain-related hypersensitivity in the absence of neural injury. We therefore investigated the mechanism by which CASPR2 modulates nociceptive function. Mice lacking CASPR2 (Cntnap2-/-) demonstrated enhanced pain-related hypersensitivity to noxious mechanical stimuli, heat, and algogens. Both primary afferent excitability and subsequent nociceptive transmission within the dorsal horn were increased in Cntnap2-/-mice. Either immune or genetic-mediated ablation of CASPR2 enhanced the excitability of DRG neurons in a cell-autonomous fashion through regulation of Kv1 channel expression at the soma membrane. This is the first example of passive transfer of an autoimmune peripheral neuropathic pain disorder and demonstrates that CASPR2 has a key role in regulating cell-intrinsic dorsal root ganglion (DRG) neuron excitability

Height-diameter allometry and above ground biomass in tropical montane forests: Insights from the Albertine Rift in Africa

Tropical montane forests provide an important natural laboratory to test ecological theory. While it is well-known that some aspects of forest structure change with altitude, little is known on the effects of altitude on above ground biomass (AGB), particularly with regard to changing height-diameter allometry. To address this we investigate (1) the effects of altitude on height-diameter allometry, (2) how different height-diameter allometric models affect above ground biomass estimates; and (3) how other forest structural, taxonomic and environmental attributes affect above ground biomass using 30 permanent sample plots (1-ha; all trees ≥ 10 cm diameter measured) established between 1250 and 2600 m asl in Kahuzi Biega National Park in eastern Democratic Republic of Congo. Forest structure and species composition differed with increasing altitude, with four forest types identified. Different height-diameter allometric models performed better with the different forest types, as trees got smaller with increasing altitude. Above ground biomass ranged from 168 to 290 Mg ha-1, but there were no significant differences in AGB between forests types, as tree size decreased but stem density increased with increasing altitude. Forest structure had greater effects on above ground biomass than forest diversity. Soil attributes (K and acidity, pH) also significantly affected above ground biomass. Results show how forest structural, taxonomic and environmental attributes affect above ground biomass in African tropical montane forests. They particularly highlight that the use of regional height-diameter models introduces significant biases in above ground biomass estimates, and that different height-diameter models might be preferred for different forest types, and these should be considered in future studies

Search for dark matter produced in association with bottom or top quarks in √s = 13 TeV pp collisions with the ATLAS detector

A search for weakly interacting massive particle dark matter produced in association with bottom or top quarks is presented. Final states containing third-generation quarks and miss- ing transverse momentum are considered. The analysis uses 36.1 fb−1 of proton–proton collision data recorded by the ATLAS experiment at √s = 13 TeV in 2015 and 2016. No significant excess of events above the estimated backgrounds is observed. The results are in- terpreted in the framework of simplified models of spin-0 dark-matter mediators. For colour- neutral spin-0 mediators produced in association with top quarks and decaying into a pair of dark-matter particles, mediator masses below 50 GeV are excluded assuming a dark-matter candidate mass of 1 GeV and unitary couplings. For scalar and pseudoscalar mediators produced in association with bottom quarks, the search sets limits on the production cross- section of 300 times the predicted rate for mediators with masses between 10 and 50 GeV and assuming a dark-matter mass of 1 GeV and unitary coupling. Constraints on colour- charged scalar simplified models are also presented. Assuming a dark-matter particle mass of 35 GeV, mediator particles with mass below 1.1 TeV are excluded for couplings yielding a dark-matter relic density consistent with measurements