Contribution of inflammation on carotid body chemosensory potentiation induced by intermittent hypoxia
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(2) 200. R. Del Rio et al.. et al. 2009), but it is matter of debate if ROS per se may increase the CB chemosensory discharges (Gonzalez et al. 2007). Thus, it is likely that molecules downstream of the ROS signals may mediate the CIH-induced effects of ROS on CB chemoreception. Among other molecules upregulated in the CB by CIH, such as ET-1 and iNOS (Iturriaga et al. 2009; Del Rio et al. 2010, 2011), pro-inflammatory cytokines has been proposed as mediators of the CB chemosensory potentiation induced by CIH (Iturriaga et al. 2009; Del Rio et al. 2011). Recently, we found that CIH induced a ROS-dependent increases of TNF-a and IL-1b within the CB, suggesting that these pro-inflammatory cytokines may mediate the ROS-induced potentiation (Del Rio et al. 2011). To test this hypothesis, we study the effects of the anti-inflammatory drug ibuprofen on the increased immunoreactive levels of TNF-a and IL-1b in the rat CB, and the potentiation of the carotid chemosensory responses to hypoxia.. 28.2 28.2.1. Methods Exposure to Intermittent Hypoxia and Subcutaneous Ibuprofen Therapy. Unrestrained, freely moving rats housed in individual chambers were exposed to hypoxic cycles of 5% O2 for 20 s, followed by room air for 280 s, applied 12 times/h, 8 h/day during 21 days (Del Rio et al. 2010). In the Sham condition, the hypoxic exposure was replaced by means of flushing equal flow of compressed air into the chambers. The room temperature was kept at 23–25°C. The experimental procedures were approved by the Bio-Ethical Committee of the Biological Sciences Faculty, P. Universidad Católica de Chile, and were performed according to the NIH Guide for the Care and Use of Laboratory Animals. Two days before the onset of CIH or sham exposures, animals were anesthetized with isofluorane 3% in oxygen and an osmotic minipump (Alzet Scientific Products, USA) implanted subcutaneously in the back of each animal. The pumps delivered 2.5 ml/h and were filled with 400 mg ibuprofen (40 mg/kg/day) in 2 ml NaCl 0.9% or NaCl 0.9% alone.. 28.2.2. Recording of Carotid Body Chemosensory Discharges. Rats were anesthetized with sodium pentobarbitone (40 mg/kg i.p), placed in supine position and the body temperature was maintained at 38.0 ± 0.5 C. One carotid sinus nerve was dissected, placed on a pair of platinum electrodes and covered with warm mineral oil. The neural signal was pre-amplified, filtered and fed to an electronic spike-amplitude discriminator, allowing the selection of action potentials of given amplitude above the noise to be counted with a frequency meter for measuring the frequency of carotid chemosensory discharge expressed in Hz. Barosensory fibers were eliminated by crushing the common carotid artery wall between the carotid sinus and the carotid body. The other carotid sinus nerve was cut to prevent cardioventilatory reflexes evoked by the activation of the CB. The chemosensory discharge was measured at several isocapnic levels of PO2. At the end of the experiments rats were killed by an overdose of sodium pentobarbitone (100 mg/kg i.p.).. 28.2.3. Western Blot for p65 Subunit of the NF-kB Transcription Factor. The CB tissue was rapidly harvested from anesthetized rats, and the CBs were frozen and stored at −80°C until analyzed. The CBs from 4 to 5 rats were pooled and the protein was extracted by.
(3) 28 Contribution of Inflammation on Carotid Body Chemosensory Potentiation…. 201. sonication in lysing buffer plus 2% protease inhibitor cocktail. Following a centrifugation at 12,000 g for 20 min at 4°C, protein concentration in the supernatant was measured using a Lowry assay kit. Samples were adjusted to the same concentrations of protein using equal volumes of loading buffer containing b-mercaptoethanol. For electrophoresis, 25 mg of protein were fractionated in a 10% polyacrylamide gel. Proteins were transferred onto membranes and treated overnight at 4°C with mouse anti-p65 primary antibody (1:500, Cell Signaling, USA) followed by one hour incubation with the secondary antibody conjugated with horseradish peroxidase (rabbit anti-mouse IgG, 1:2,500, Sigma, USA). Immune complexes on the membrane were visualized using enhanced chemiluminescence and the specific bands according to the expected molecular weight were analyzed with the ImageJ software (NIH, USA).. 28.2.4. Immunohistochemical Detection of Cytokines in the Carotid Body. Quantitative immunohistochemistry was used to measure the levels of TNF-a and IL-1b in the rat CB exposed to CIH for 21 days. Anesthetized rats were perfused intracardially with phosphate saline buffer followed by buffered 4% paraformaldehyde, and post-fixed at 4°C for 12 h. Samples were dehydrated in ethanol, included in paraffin, cut in 5 mm sections and mounted on silanized slides. Deparaffinized samples were submitted to microwave based antigen retrieval protocol. Samples were incubated with 0.3% H2O2 to inhibit endogenous peroxidase and then in blocking normal horse serum solution. Slides were incubated with specific antibodies overnight at 4°C in humidity chambers for detection of TNF-a (1:20, goat anti- TNF-a, Santa Cruz Biotech., USA) and IL-1b (1:100, rabbit anti- IL-1b, Santa Cruz Biotech., USA). Negative controls were performed by omission of the primary antibody. After rinse in cold PBS, samples were incubated with secondary antibodies conjugated to biotin and revealed at 37°C in a dark chamber with 3,3-diaminobenzidine tetrahydrochloride. Finally, samples were counterstained with Harris Hematoxylin and permanently mounted. Photomicrographs of the CB tissue were taken at 100x with a CCD-camera coupled to a microscope, digitized and analyzed using a color deconvolution algorithm with the ImageJ software (NIH, USA).. 28.2.5. Statistical Data Analysis. Data are expressed as means ± SEM. Differences between two groups were assessed by Student T-test comparisons. Differences between more groups were assessed with one or two-way ANOVA tests, followed by appropriated posthoc comparisons.. 28.3 28.3.1. Results Intermittent Hypoxia Increased the Expression of the p65 Subunit of the NF-kB Transcription Factor. Since an enhanced production of ROS induced by hypoxia-reoxygenation increased the expression and the translocation of NF-kB to the nucleus, inducing the expression of IL-1b and TNF-a (JanseenHeininger et al. 2000), we studied if intermittent hypoxia increased the levels of the p65 subunit of the.
(4) 202. R. Del Rio et al.. Fig. 28.1 CIH-induced accumulation of NF-kB in the rat CB. Upper panel, immunoblot for the p65 subunit of the NF-kB transcription factor. Lower panel, band densitometry quantification for sham and CIH rat CBs. *, p < 0.05 Student T-test, n = 4–5 CBs per group. NF-kB transcription factor in the CBs from rats exposed to CIH. Figure 28.1 shows an immunoblot of the p65 subunit of the NF-kB transcription factor. Clearly, CIH produced a significant increase in the p65 levels in the CBs from rats exposed to CIH for 21 days.. 28.3.2. Effect of CIH on TNF-a and IL-1b Immunoreactivity in the Rat CB. The exposure to CIH for 21 days increased the immunoreactive levels of TNF-a and IL-1b in the rat CB, as is illustrated in Fig. 28.2. Note that TNF-a and IL-1b were mainly confined to the glomus cells, but it is worth noting that not all glomus cells showed positive immunoreactive staining for the cytokines.. 28.3.3. Effects of Ibuprofen on TNF-a and IL-1b Immunoreactivity in the Rat CB. We found a significant increased of TNF- and IL-1b immunoreactivity in the CB from rats exposed to CIH for 21 days, which was prevent by ibuprofen treatment (see Table 28.1). Ibuprofen prevented the increased TNF-a and IL-1b levels, compared with the optical integrated intensity measured in the CBs of CIH-treated rats..
(5) 28 Contribution of Inflammation on Carotid Body Chemosensory Potentiation…. 203. Fig. 28.2 Micrographs showing positive immunoreactivity for TNF-a and IL-1b in the CB from a Sham rat and CIH-treated rat. Note that positive staining is mainly confined to round to ovoid cells sharing histological morphology with CB chemoreceptor cells. Scale bars 20 mm. Table 28.1 Effect of ibuprofen on CIH-induced enhanced of TNF-a and IL-1b in the CB Sham CIH TNF-a –ir (a.u.) 11.8 ± 2.3 49.3 ± 1.9** IL-1b –ir (a.u.) 16.5 ± 1.5 36.8 ± 2.6**. expression CIH + IB 9.8 ± 1.8 17.2 ± 1.1. Data are presented as mean ± SEM. Sham: control animals; CIH: rats exposed to chronic intermittent hypoxia; CIH + IB: rats treated with ibuprofen during the exposure to chronic intermittent hypoxia; TNF-a –ir: tumor necrosis factor alpha immunoreactivity; IL-1b –ir: interleukin 1 beta immunoreactivity **p < 0.01 compared to Sham. 28.3.4. Effects of Ibuprofen on CB Chemosensory Potentiation Induced by CIH. Rats exposed to CIH showed enhanced CB chemosensory responses to acute hypoxia as compared with Sham, and Sham-IB rats (Fig. 28.3). However, the CIH-induced potentiation of the CB chemosensory response to acute hypoxia was not prevented by ibuprofen. The two-way ANOVA analysis showed that the overall curve for CIH and CIH-IB treated groups were not different..
(6) 204. R. Del Rio et al.. Fig. 28.3 Anti-inflammatory treatment failed to prevent CB chemosensory potentiation induced by CIH. Summary of the CB chemosensory responses induced by several levels of inspired PO2 in 4 Sham rats (□), 4 CIH-treated rats (●), 4 Sham + IB rats (♦) and 4 CIH + IB (▼). ƒCSN, frequency of chemosensory discharges in Hz. **, p < 0.01; *, p < 0.05 CIH compared to sham; #, p < 0.05 CIH + IB compared to sham, Bonferroni test after 2-way ANOVA. 28.4. Discussion. Present results showed that ibuprofen, which prevented the CIH-increased TNF-a and IL-1b expression in the CB, did not prevent the potentiation of the CB chemosensory responses to acute hypoxia. Thus, the CIH-induced potentiation of the CB chemosensory responses does not depend on the increased TNF-a and IL-1b levels in the CB. The inhibitory effect of ibuprofen on the cytokine accumulation induced by CIH in the CB is consistent with its known anti-inflammatory effect. Although, ibuprofen is considered a non-selective inhibitor of cyclooxygenases 1 and 2, is a well known inhibitor of the nuclear translocation of the transcription factor, NF-kB, which mediates the TNFa and IL-1b production (Jenseen-Heininger et al. 2000). Some studies have shown that the pro-inflammatory cytokines IL-1b, IL-6, and TNF-a, are excitatory modulators of CB oxygen transduction process in the rat (Lam et al. 2008; Liu et al. 2009; Shu et al. 2007). It is known that the rat glomus cells express these cytokines along with their functional receptors, IL-1R1 and TNF-R1 (Lam et al. 2008; Wang et al. 2002). In addition, Shu et al. (2007) found that the application of IL-1b to glomus cells of the rat CB inhibit the O2-dependent voltage-gated K+ currents and the increase of intracellular Ca2+. Intermittent hypoxia increases the levels of ROS in the rat CB (Pawar et al. 2009; Del Rio et al. 2010). In response to oxidative stress, NF-kB induces the expression of pro-inflammatory cytokines such as IL-1b and TNF-a in the CB, suggesting that chemoreceptor cells can synthesize and release cytokines (Del Rio et al. 2011). Inflammatory processes are also involved in the enhanced CB chemosensory response to hypoxia in rats exposed to sustained hypoxia (Lam et al. 2008; Liu et al. 2009). In fact, Lam et al. (2008) found that sustained hypobaric hypoxia increased the mRNA expression of IL-1b and TNF-a, and the IL-1R1 and TNF-R1 receptors. Besides that, Liu et al. (2009) found that the concurrent administration of ibuprofen and dexamethasone to rats exposed to sustained hypoxia reduced the potentiation of the CB chemosensory response to acute hypoxia and reduced the cytokine mRNA expression. We found that intermittent hypoxia produces a progressive increase of the TNF-a and IL-1b levels in the rat CB (Del Rio et al. 2011). Present results showed that the enhanced CB chemosensory response to hypoxia induced by CIH was not prevented by ibuprofen, while the increased levels of IL-1b and TNF-a in the CB were blocked by the anti-inflammatory treatment. Thus, our results suggest that the mechanisms underlying the hypoxic CB chemosensory potentiation induced by sustained and intermittent hypoxia are different..
(7) 28. Contribution of Inflammation on Carotid Body Chemosensory Potentiation…. 205. In summary, ibuprofen prevented the increased CB levels of TNF-a and IL-1b induced by CIH, but did not prevent the enhanced CB chemosensory responses to acute hypoxia induced by CIH. Thus, our results suggest that IL-1b and TNF-a do not mediate the potentiation of the CB chemosensory response to hypoxia induced by CIH. Acknowledgements This work was supported by grant 1100405 from the National Fund for Scientific and Technological Development of Chile (FONDECYT).. References Del Rio R, Moya EA, Iturriaga R (2010) Carotid body and cardiorespiratory alterations in intermittent hypoxia: the oxidative link. Eur Respir J 36:143–150 Del Rio R, Moya EA, Iturriaga R (2011) Differential expression of pro-inflammatory cytokines, endothelin-1 and nitric oxide synthases in the rat carotid body exposed to intermittent hypoxia. Brain Res 1395:74–85 Garvey JF, Taylor CT, McNicholas WT (2009) Cardiovascular disease in obstructive sleep apnoea syndrome: the role of intermittent hypoxia and inflammation. Eur Respir J 33:1195–1205 González C, Agapito MT, Rocher A, González-Martin MC, Vega-Agapito V, Gomez-Niño A, Rigual R, Castañeda J, Obeso A (2007) Chemoreception in the context of the general biology of ROS. Respir Physiol Neurobiol 157:30–44 Iturriaga R, Moya EA, Del Rio R (2009) Carotid body potentiation induced by intermittent hypoxia: implications for cardiorespiratory changes induced by sleep apnoea. Clin Exp Pharmacol Physiol 36:1197–1204 Janssen-Heininger YM, Poynter ME, Baeuerle PA (2000) Recent advances towards understanding redox mechanisms in the activation of nuclear factor kappaB. Free Radic Biol Med 28:1317–1327 Lam SY, Tipoe GL, Liong EC, Fung ML (2008) Chronic hypoxia upregulates the expression and function of proinflammatory cytokines in the rat carotid body. Histochem Cell Biol 130:549–559 Liu X, He L, Stensaas L, Dinger B, Fidone S (2009) Adaptation to chronic hypoxia involves immune cell invasion and increased expression of inflammatory cytokines in rat carotid body. Am J Physiol Lung Cell Mol Physiol 296:L158–L166 Pawar A, Nanduri J, Yuan G, Khan SA, Wang N, Kumar GK, Prabhakar NR (2009) Reactive oxygen species-dependent endothelin signaling is required for augmented hypoxic sensory response of the neonatal carotid body by intermittent hypoxia. Am J Physiol Regul Integr Comp Physiol 296:R735–R742 Peng YJ, Overholt JL, Kline D, Kumar GK, Prabhakar NR (2003) Induction of sensory long-term facilitation in the carotid body by intermittent hypoxia: implications for recurrent apneas. Proc Natl Acad Sci U S A 100:10073–10078 Rey S, Del Rio R, Alcayaga J, Iturriaga R (2004) Chronic intermittent hypoxia enhances cat chemosensory and ventilatory responses to hypoxia. J Physiol 560:577–586 Shu HF, Wang BR, Wang SR, Yao W, Huang HP, Zhou Z, Wang X, Fan J, Wang T, Ju G (2007) IL-1beta inhibits IK and increases [Ca2+]i in the carotid body glomus cells and increases carotid sinus nerve firings in the rat. Eur J Neurosci 25:3638–3647 Somers VK, White DP, Amin R, Abraham WT, Costa F, Culebras A, Daniels S, Floras JS, Hunt CE, Olson LJ, Pickering TG, Russell R, Woo M, Young T (2008) Sleep apnea and cardiovascular disease. J Am Coll Cardiol 52:686–717 Wang X, Wang BR, Duan XL, Zhang P, Ding YQ, Jia Y, Jiao XY, Ju G (2002) Strong expression of interleukin-1 receptor type I in the rat carotid body. J Histochem Cytochem 50:1677–1684.
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