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Inflammation status of rabbit carotid artery model endothelium during intermittent hypoxia exposure and its relationship with leptin

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Abstract

Objectives

To develop an intermittent hypoxia/reoxygenation (IH/ROX) rabbit carotid artery model and then investigate the inflammation status of rabbit carotid artery endothelium after IH exposure and its relationship with leptin.

Materials and methods

After anesthetization, rabbit’s right common carotid artery was cleared of surrounding tissue with anatomic microscope, cannulated to its distal part and the proximal part was ligated. Preparations were challenged by changing the PO2 of the gas mixture equilibrating the perfusate. Alternate perfusing (2 mL/min) of equilibrated perfusate bubbled with normoxia or hypoxia gas mixtures formed IH/ROX cycles in the right carotid common artery, simulating the pattern of hypoxic episodes seen in obstructive sleep apnea (OSA), or continuous perfusing of hypoxia perfusate to form continuous hypoxia (CH) modes. Sixty adult male New Zealand White rabbits (2.5–3.0 kg) were separated into six groups, ten per group. Groups were: A, intermittent normoxia (IN) group, perfused with perfusion equilibrated with 21% O2 [PO2 about 141 ± 2.87 mmHg] for 15 s and 21% O2 for 1 min 45 s, 60 cycles; B, severe IH group, 5% O2 [PO2 about 35.2 ± 1.27 mmHg] 15 s and 21% O2 1 min 45 s, 60 cycles; C, mild IH group, 10% O2 [PO2 about 54.3 ± 3.31 mmHg] 15 s and 21% O2 1 min 45 s, 60 cycles; D, severe IH+Lep group, protocol was the same with severe IH group; E, CH group, IN for 1 h 45 min and then 5% O2 for 15 min; and F, Lep group, the same with IN group. Right common carotid artery parts distal to the cannula were harvested after exposure, and endothelial cell layers were gotten from longitudinal outspread vessels. Nuclear factor κB (NFκB) DNA binding activities of partial cell layers were measured with electrophoretic mobility shift assay in the IN group, severe IH group, mild IH group, and CH group nuclear extracts. The other part of the cell layers in the IN group, severe IH group, severe IH+Lep group, and Lep group were cultured for 2 h, and during the culture procedure, recombinated human leptin solutions were added to culture dishes of severe IH+Lep group and Lep group (resulted concentration, 10 ng/mL). Enzyme-linked immunosorbent assay was used to analyze medium interleukin-6 (IL-6) concentrations, reverse transcription polymerase chain reaction was used to analyze endothelial cell Ras homology A (RhoA) mRNA expression levels. Statistical analysis was done with SPSS 11.5 software package.

Results

NFκB DNA binding activities were significantly different between groups (F = 112.428, P < 0.001). This activity in the severe IH group (4.27 ± 0.64) was higher than that in the mild IH group (2.33 ± 0.45, P < 0.001), IN group (1.00 ± 0.26, P < 0.001), and CH group (1.15 ± 0.36, P < 0.001). RhoA mRNA expression levels were different in groups (F = 26.634, P < 0.001).This level in the severe IH+Lep group (2.54 ± 0.53) was higher than that in the severe IH group (1.57 ± 0.44, P = 0.002), IN group (1.00 ± 0.31, P < 0.001), and Lep group (1.31 ± 0.30, P < 0.001). IL-6 concentrations were different in groups (F = 79.922, P < 0.001). IL-6 concentration in the severe IH+Lep group (1591.50 ± 179.57 pg/mL) was higher than that in the severe IH group (1217.20 ± 320.62 pg/mL, P = 0.036), IN group (325.40 ± 85.26 pg/mL, P < 0.001), and Lep group (517.40 ± 183.09 pg/mL, P < 0.001).

Conclusions

IH/ROX activated the inflammation pathway significantly in the endothelium, which was more intensive than CH and intensity-dependent. When exposed to both IH/ROX and leptin, inflammation occurs more dramatically. It means that synergic activating roles were performed by IH/ROX and leptin. This study may have a clinical implication that IH can cause endothelial damage through activated inflammation in OSA patients, and if the OSA patients have obesity at the same time, the endothelial damage or the inflammation would be more significant because of elevated leptin level as a synergic factor.

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Acknowledgements

This study was supported by grants from the National Natural Science Foundation of China (no. 30800507).

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Authors and Affiliations

  1. Respiratory Department of Tianjin Medical University General Hospital, Tianjin, 300052, China

    Jing Feng, Bao-yuan Chen, Chun-xia Liu, Pan-feng Chen, Mei-nan Guo, Li-xia Dong & Shuo Li

  2. Tianjin Neurology Research Institute, Tianjin, 300052, China

    Lin-yang Cui

  3. Endocrinology Research Institute, Tianjin Medical University, Tianjin, 300052, China

    Bao-li Wang

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  1. Jing Feng
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Correspondence to Bao-yuan Chen.

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Feng, J., Chen, By., Cui, Ly. et al. Inflammation status of rabbit carotid artery model endothelium during intermittent hypoxia exposure and its relationship with leptin. Sleep Breath 13, 277–283 (2009). https://doi.org/10.1007/s11325-009-0246-6

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  • DOI: https://doi.org/10.1007/s11325-009-0246-6

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