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The protocol was carried out as follows. After an initial 2 min period of normoxia, the O2 was turned off, allowing the superfusate to become saturated with N2, thus making it hypoxic. After approximately 100 s of hypoxia ( LEE011 at this level for a further 2 min. This sequence was then carried out in the presence of increasing concentrations of cytokine, either IL-1��, TNF-�� or IL-6 (0.1, 0.3 and 1 ng ml?1) or LPS (10 and 100 ng ml?1), with a 3 min normoxic interval of normal Tyrode superfusion between each hypoxic challenge. A further hypoxic challenge was carried out at the end of each protocol to ensure that the chemosensitive unit being tested was still active, giving a total of five hypoxic challenges in the cytokine experiments and four in the LPS experiments. Only units that still responded to hypoxia at the end were included in the analysis. All data were digitized learn more using an A/D converter (1401 micro; Cambridge Electronic Design, Cambridge, UK), and captured and analysed using Spike2 v6.02 (Cambridge Electronic Design), which allowed discrimination of individual units from multi-unit nerve recordings. The unitary nature of discriminated recordings was determined by noting the relative constancy of spike shape and amplitude by spike superimposition. The Kolmogorov�CSmirnov test was applied to test for normality of data distribution. For normally distributed data, a two-way repeated-measures ANOVA (with Bonferroni correction to control the family wise type I error) was applied to test for significance of the effect of treatment (concentration of cytokines or LPS) and time. For non-parametric data, Friedman's test was applied with Dunn's post hoc test. The criterion for statistical significance was P Resiquimod animal was tested (n= 27), and values are expressed as means �� SEM. All statistical analyses were carried out using Prism 4 v4.03 (GraphPad Software, Inc., San Diego, CA, USA). The results of these experiments confirmed previous findings (O��Leary et al. 2004; Mac Grory et al. 2010) that these cells respond strongly to changes in , significantly increasing their discharge rate in response to hypoxia (from 0.71 �� 0.23 to 10.95 �� 1.74 Hz; P