Respiratory Physiology & Neurobiology 221 (2016) 11–18

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Morphological changes in the rat carotid body following acute sodium nitrite treatment Dimitrinka Y. Atanasova a,b , Nikolai E. Lazarov a,c,∗ a b c

Institute of Neurobiology, Bulgarian Academy of Sciences, Sofia, Bulgaria Institute of Experimental Morphology, Pathology and Anthropology with Museum, Bulgarian Academy of Sciences, Sofia, Bulgaria Department of Anatomy and Histology, Medical University of Sofia, Sofia, Bulgaria

a r t i c l e

i n f o

Article history: Received 30 June 2015 Received in revised form 21 October 2015 Accepted 23 October 2015 Available online 31 October 2015 Keywords: Carotid body Hemic hypoxia Reactive oxygen species Sodium nitrite Structural plasticity Vasodilation

a b s t r a c t The carotid body (CB) is a small neural crest-derived chemosensory organ that detects the chemical composition of the arterial blood and responds to its changes by regulating breathing. The effects of acute nitrite treatment on the CB morphology in rats were examined by morphometry. We found that 1 h after administrating a single dose of sodium nitrite, the CB underwent structural changes characterized by a prominent increase in its size with a marked, several-fold dilation of the blood vessels. The obvious CB enlargement mostly due to apparent vasodilation and glomus cell hypertrophy was at its highest one day later and persisted until the fifth day. 20 days after the treatment, the CB regained its size to the normoxic control state. Morphometric analysis revealed that the CB size increase in treated animals is statistically significant when compared to that of untreated controls. It can be inferred that the nitrite-exposed CB displays remarkable structural plasticity and enlarges its size mostly through vascular expansion. © 2015 Elsevier B.V. All rights reserved.

1. Introduction The carotid body (CB) is a polymodal peripheral chemoreceptor that registers the levels of pO2, pCO2 and pH in the arterial blood (Gonzalez et al., 1994; Prabhakar and Joyner, 2015). It plays an essential role in initiating an appropriate respiratory and cardiovascular response to hypoxia, hypercapnia and acidosis, leading to the restoration of blood gas homeostasis (reviewed in Gonzalez et al., 1994 and recently by Kumar and Prabhakar, 2012). The organ consists of cell clusters of two cell types, i.e. neuron-like glomus (or type I) cells and glial like sustentacular (or type II) cells, intermingled with a dense network of fenestrated capillaries and nerve bundles, and separated by connective tissue (Gonzalez et al., 1994; Verna, 1997; Atanasova et al., 2011; Kumar and Prabhakar, 2012). It is well-known that chronic hypoxia induces gene expression, leading to profound morphological changes at a cellular level in the CB (Wang and Bisgard, 2002; Kusakabe et al., 2005). A number of previous studies have described the structural alterations in the rat CB upon exposure to sustained hypoxia (Laidler and Kay, 1975;

Abbreviations: CB, carotid body; ET-1, endothelin-1; NaNO2 , sodium nitrite; NO, nitric oxide; NOS, nitric oxide synthase; ROS, reactive oxygen species. ∗ Corresponding author at: Department of Anatomy and Histology, Medical University of Sofia, 2, Zdrave Street, BG-1431 Sofia, Bulgaria. Fax: +359 2 8518 783. E-mail address: [email protected] (N.E. Lazarov). http://dx.doi.org/10.1016/j.resp.2015.10.015 1569-9048/© 2015 Elsevier B.V. All rights reserved.

McGregor et al., 1984; Lahiri et al., 2000; Kusakabe et al., 2005; Matsuda et al., 2006; Pardal et al., 2007). It has been revealed that in humans the long-term hypoxia caused a several-fold increase of the CB size mostly due to marked vasodilation and hyperplasia of the glomus cells (Heath et al., 1985), or their hypertrophy in rats (Wang and Bisgard, 2002; Wang et al., 2008). Such a morphological adaptive response to prolonged hypoxia occurs during acclimatization to high altitudes (Arias-Stella and Valcarcel, 1976; Wang and Bisgard, 2002). Conversely, no definite evidence concerning a volume increase of glomus cells in the rats exposed to short-term (for up to 24 h) hypoxia has been provided so far (see Kato et al., 2010 and references therein). On the other hand, the mechanisms of the origin of hypoxia are exceptionally diverse and their exact effects on the CB morphology remain to be established. It has recently been proposed that one putative hypoxia-sensing mechanism is the production of oxygen radicals (López-Barneo et al., 2008). It is also established that the repeated episodes of hypoxia-reoxygenation produce local oxidative stress in the CB due to accumulation of reactive oxygen species (ROS), and their increased levels result in enhanced chemosensory response to hypoxia and cellular damage (Del Rio et al., 2010; Iturriaga and Del Rio, 2012). Nonetheless, since no chemosensory excitatory effects of ROS have been registered, the direct involvement of ROS in the transduction of oxygen levels in the CB has been questioned (Gonzalez et al., 2007). Furthermore, the morphological

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Fig. 1. Conventional hematoxylin and eosin (H&E) stained sections showing the morphological characteristics of the carotid body (CB) in normoxic and hypoxic rats. (A) A section from the control normoxic CB. High-power view of the area inside the rectangle in (H). (B) CB morphology 1 h after acute sodium nitrite-induced hypoxia. Note the slight enlargement of its size. The glomic clusters (G) are compact and surrounded by slightly distended blood vessels (BV). (C) 5 h following hypoxic exposure, a marked vasodilation in the CB is observed without apparent glomus cell hypertrophy. (D) Morphological changes of the CB 1 day later. Note the increased CB size and dilated blood vessels. (E) Representative photomicrograph showing the persistent vasodilation 5 days following hypoxic exposure. (F) H&E-stained sections from the hypoxic CB 20 days after hypoxic termination. Note that the hypoxic CB has almost the same size as its normoxic state (H) but the parenchyma is more compact. (G) High power view of the boxed area in the previous figure illustrating the morphological features of recovering CB. Note the compact glomeruli and hypertrophic glomus cells. The blood vessels have relatively narrower lumens and the extracellular matrix is somewhat expanded. Scale bar = 100 ␮m (F and H); 50 ␮m (B–E); 25 ␮m (G).

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alterations of the CB after exposure to hypoxic chemicals such as sodium nitrite have not been studied yet. Sodium nitrite (NaNO2 ), an oxidative metabolite of nitric oxide (NO), is commonly used for inducing chemical hypoxia in experimental animal models (Naik et al., 2006). Current research has provided convincing evidence that sodium nitrite reduces the oxygen-carrying capacity of blood by oxidizing hemoglobin to methemoglobin, and thus causes hemic hypoxia (May et al., 2000). Moreover, time- and dose-dependent relationships between exposure to nitrite and its plasma and methemoglobin levels have been reported (Kohn et al., 2002). A more recent study, however, has demonstrated that methemoglobinemia is not likely to affect the ventilatory response of the CB to hypoxia (Haouzi et al., 2011). Besides, it has been shown that nitrite infusions are also associated with formation of NO, decreased arterial pressure and increased blood flow (Cosby et al., 2003). The present study aimed to make an animal model for investigating the effects of acute nitrite treatment on the CB morphology in rats. Since generated ROS and methemoglobin are potential mediators of CB structural alterations, we attempted to elucidate the mechanisms underlying the CB chemosensory potentiation and, in particular, the contribution of local oxidative stress, endothelial dysfunction and some vasoactive substances to the enhanced CB responsiveness to sodium nitrite administration.

2. Materials and methods 2.1. Animals, experimental design and tissue preparation The experiments were carried out on adult male Wistar rats, weighing 250–300 g. The animals were kept under standard laboratory conditions (12-h light/dark cycle, temperature 23 ± 2 ◦ C, 50% relative humidity) with food and water available ad libitum. The procedures for animal handling were conducted in accordance with the ethical guidelines of the EU Directive 2010/63/EU for the protection of animals used for scientific purposes and the experimental protocol was approved by the Bioethical Commission of the Biomedical Research at the Institute of Experimental Morphology, Pathology and Anthropology with Museum of the Bulgarian Academy of Sciences. All efforts were made to minimize the number of animals used and their suffering. They were divided into a group of treated rats that received sodium nitrite with seven recovery periods (n = 5 in each group) and an age-matched control group (n = 5) of untreated rats injected with the same volume of normal saline. Chemical hypoxia was induced by a single intraperitoneal injection of sodium nitrite (50 mg/kg body weight). The animals were deeply anesthetized with Nembutal (50 mg/kg, i.p., Abbott Laboratories, North Chicago, IL) and transcardially perfused at different time intervals (1 h, 5 h, 24 h, 48 h and 5, 10 and 20 days) after the administration of sodium nitrite, first with 0.05 M phosphate buffered saline (PBS), followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.2–7.4. This neutral buffered fixative penetrates rapidly and does not cause excessive tissue shrinking or distortion of the cellular structure (Thavarajah et al., 2012). After perfusion, the carotid bifurcations were quickly removed, both CBs were immediately dissected out and specimens were postfixed in the same fixative overnight at 4 ◦ C. Thereafter, the tissue blocks were washed in tap water and then in distilled water, dehydrated, embedded in paraffin and cut into 7 ␮m thick sections. The sections were routinely stained with hematoxylin and eosin to examine the CB morphology. After staining, the specimens were examined and photographed with a Nikon research microscope equipped with a DXM1200c digital camera. The digital images were saved in TIF

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format and processed using Adobe Photoshop CS3 software (Adobe Systems, Inc., San Jose, CA). 2.2. Digital image analysis and statistics For calculation of the total surface area of the CB and the diameter of blood vessels, hematoxylin and eosin-stained sections from the CB of sodium nitrite-treated and control untreated animals were selected and measured by the Nikon NIS Element Digital Imaging software. The CB in each section was delineated, and the transectional area was measured. To precisely evaluate the expanded vasculature in treated animals and to compare the diameter changes with those in chronically hypoxic rats, different size ranges of the vessels were elected according to Kusakabe et al. (2004). The number of blood vessels of different calibres—less than 5 ␮m, 6–10 ␮m, 11–15 ␮m, 16–20 ␮m, 21–25 ␮m, 26–30 ␮m and 31–35 ␮m in the normoxic controls, and, additionally, 35–40 ␮m, 41–50 ␮m, 51–60 ␮m, 61–70 ␮m and 71–90 ␮m in the hypoxic CB was expressed as a percentage of the total number of blood vessels. Fifty images taken from ten CBs of five rats from each group, both control and experimental, provided an adequate sample size. The obtained values were given as mean ± S.E.M. Additionally, using the same software, cell size of the glomus cells was directly measured. At least 250 glomus cells from each group and time interval, whose boundaries were clearly visible at a higher magnification of the light microscope, were randomly selected, outlined and measured. Since the glomus cells are not perfectly round, we calculated at least two diameters and, thus 500 measurements for each group were further used for statistical analysis. To illustrate the distribution of the data, we have displayed them as box-and-whisker plots. The central line in each box plot represents the median, whereas the lower and upper halves of the box are the 25th and 75th percentiles, respectively. The whiskers on the box are the minimum and maximum scores for the group. Statistical analysis was performed using SigmaStat® 11.0 software package (Systat Software Inc). Experimental data (CB total surface area, the surface area occupied by blood vessels, their mean diameter and percentage of different blood vessel diameter ranges) were evaluated by Student’s t-test or Mann–Whitney U test, to parametric and nonparametric data, respectively, and further analyzed by multiple comparisons between the control and experimental groups using two-way ANOVA. Differences were considered statistically significant if p-values were 0.05) increased between the 5th h (5h–H) and 5th day (5d–H) of hypoxic exposure in comparison with that of the normoxic control Wistar rats (Wis-C). Data are means ± S.E.M. (n = 5 per each group); analysis of data by two-way ANOVA indicating a main effect of Hypoxia [F6,89 = 8.669, p < 0.001]; *p < 0.05 vs Wis-C.

Fig. 3. Statistical comparison of the surface area occupied by the blood vessels in normoxic control and hypoxic rats. Total area of blood vessels in 5 h (5h–H) hypoxic CB (0.000295 ± 0.000039) mm2 is significantly (p < 0.001) enhanced compared with the value (0.000115 ± 0.000005 mm2 ) in normoxic CB (Wis-C). Values are mean ± S.E.M. (n = 5); analysis of data by two-way ANOVA demonstrating a main Hypoxic treatment [F6,3827 = 22.609, p < 0.001]; * p < 0.05 vs Wis-C; 0 p < 0.05 vs 20d-H; & p < 0.05 vs 5d-H; # p < 0.05 vs 2d–H.

Fig. 4. Statistical comparison of the mean diameter of blood vessels in normoxic and hypoxic CB. Representative histograms demonstrating that the CB vessel diameter is significantly (p < 0.05) increased at 1 hour, 5 h and 1–5 days after hypoxic exposure in comparison with that of the normoxic controls (Wis-C). Data are means ± S.E.M. (n = 5 per each group); analysis of data by two-way ANOVA indicating a main effect of hypoxia [F6,3816 = 26.228, p < 0.001]; *p < 0.05 vs Wis-C; 0 p < 0.05 vs 20d-H; & p < 0.05 vs 5d-H.

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Fig. 5. The percentage of different blood vessel diameter ranges in normoxic and hypoxic CB. The histograms represent the estimated proportion of blood vessels of different caliber in normoxic control group (A) and those after 1 h (B), 5 h (C), 1 day (D), 2 days (E), 5 days (F) and 20 days (G) of hypoxic exposure. Morphometric data are presented as mean ± S.E.M.

tor antagonist bosentan considerably reduced the elevation of ROS level and the increased release of ET-1 in hypoxic animals, but has no effect on the CB chemosensory activity in control animals (Rey et al., 2006), indicating that ET-1 actions on heightened chemosen-

sory responses to acute hypoxia are mediated by ET-A receptors. However, the enhanced expression of ET-1 has been observed during the first week of hypoxia (Del Rio et al., 2011), thus suggesting that a ROS induced increase of ET-1 release could be involved in the

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review, see Kumar and Prabhakar, 2012) and ventilatory control (Powell, 2007) during hypoxia, it might be concluded that acute nitrite treatment is associated with striking structural changes that appear shortly after its application and persist up to 20 days later. These alterations are consistent with morphological changes in the rat CB during the course of hypoxic adaptation and recovery reported by Kusakabe et al. (2005). 4.1. Summary

Fig. 6. Statistical comparison of the mean diameter of the glomus cells in normoxic and hypoxic CB. Morphometric data are presented as box plots, where the upper and lower boundaries of the color boxes represent the 75th and 25th percentiles, respectively. The black line within the box represents the median and the whiskers represent the minimum and maximum values that lie within 1.5x the interquartile range from the end of the box. Values outside this range are represented by color circles. Data are means ± S.E.M. Statistical differences between groups are compared using the Mann–Whitney’s U test. *p < 0.05 vs Wis-C; ◦ p < 0.05 vs 20d-H; & p < 0.05 vs 5d-H; # p < 0.05 vs 2d-H, $ p < 0.05 vs 1d-H.

early CB response to hypoxia (see Iturriaga and Del Rio, 2012 and references therein). It is well-known that under hypoxic stress, regardless of the cause, peripheral chemoreceptors show remarkable plasticity; they will both swell the size of chemosensory cells and increase their number (Kumar and Prabhakar, 2012). Under such conditions, the level of ROS drops, thereby inducing K+ channel inhibition, the ATP level declines, a failure of ion-motive ATPase occurs and together with the closure of K+ channels, leading to membrane depolarization and Ca2+ influx through voltage-gated Ca2+ channels, and subsequent activation of calcium-dependent phospholipases and proteases. These events result in uncontrolled cell swelling, hydrolysis of main cellular components, and eventually to cell necrosis (reviewed by Michiels, 2004). In this context, the herein observed increase in glomus cell size may indicate an alteration in their metabolism, in particular a switch from aerobic to anaerobic metabolism. Sodium nitrite is a strong oxidant which can cause increased cellular oxidative stress (May et al., 2000), and thus evokes significant swelling in stressed cells. Accordingly, this may explain the nitrite-induced increase in the CB size 20 days after its administration. It is also worth noting that cell swelling is the earliest and most universal indicator of potentially reversible cellular injury which may become irreversible unless oxygenation is restored. To our knowledge, this is the first study describing sodium nitrite-induced morphological alterations in the CB during the course of its recovery. In this regard, it has been described that the oxidative effects of nitrite on hemoglobin are time- and dose-dependent (Doyle et al., 1981; Kohn et al., 2002), which could explain the temporal profile of the observed morphological changes. Our results showed that these started shortly after acute nitrite application and 5 h later the rat CB was enlarged and showed obvious vasodilation. Subsequently vasodilation diminished but the size of the CB still remained larger 20 days after the administration. Since the half-time for recovery from methemoglobinemia is estimated to be 60–120 min (Kohn et al., 2002), this could determine the time course of concordant early structural alterations. However, the hemoglobin-mediated vasodilation may also increase the blood flow, hemoglobin saturation and tissue oxygenation (Cosby et al., 2003). Based on the known role of CB in time-dependent changes in arterial blood gases (for a recent

In conclusion, we demonstrate that acute treatment with sodium nitrite causes statistically significant enlargement of the CB and a pronounced vasodilation. Although the initial nitrite-induced expansion of blood vessels decreases within five days after the nitrite application, the CB size still remains larger. We also find that 20 days following administration, CB morphology recovers to that of untreated controls. Further studies are required to elucidate the mechanisms by which the observed structural changes affect the sensitivity of CB to hypoxic chemicals such as sodium nitrite. Competing interests The authors declare no conflicts of interest. Authors’ contribution N.L. initiated the study, planned and designed the experiments and wrote the paper. D.A. performed the experiments, image analysis and did the photographic work. Both authors were involved in the process of analysis and interpreting the data, read and approved the final version of the manuscript. Acknowledgements The authors wish to thank Dr. Emilia Petrova (Institute of Experimental Morphology, Pathology and Anthropology with Museum of the Bulgarian Academy of Sciences) for providing the necessary facility to carry out this research and for her help with the hypoxic animals, Dr. Zlatina Nenchovska (Institute of Neurobiology of the Bulgarian Academy of Sciences) for her assistance with the image analysis and Dr. Angel Dandov (Medical University of Sofia) for critical reading of the manuscript. References Arias-Stella, J., Valcarcel, J., 1976. Chief cell hyperplasia in the human carotid body at high altitudes; physiologic and pathologic significance. Hum. Pathol. 7, 361–373. Atanasova, D.Y., Iliev, M.E., Lazarov, N.E., 2011. Morphology of the rat carotid body. Biomed. Rev. 22, 41–55. Bisgard, G.E., 2000. Carotid body mechanisms in acclimatization to hypoxia. Respir. Physiol. 121, 237–246. Chen, J., Dinger, B., Stensaas, L., Fidone, S., 2001. Involvement of vascular endothelial growth factor (VEGF) in carotid body vascular remodeling induced by chronic hypoxia. FASEB J. 15, A153. Chen, J., He, L., Dinger, B., Stensaas, L., Fidone, S., 2002. Role of endothelin and endothelin A-type receptor in adaptation of the carotid body to chronic hypoxia. Am. J. Physiol. Lung Cell Mol. Physiol. 282, L1314–L1323. Cosby, K., Partovi, K.S., Crawford, J.H., Patel, R.P., Reiter, C.D., Martyr, S., Yang, B.K., Waclawiw, M.A., Zalos, G., Xu, X., Huang, K.T., Shields, H., Kim-Shapiro, D.B., Schechter, A.N., Cannon 3rd, R.O., Gladwin, M.T., 2003. Nitrite reduction to nitric oxide by deoxyhemoglobin vasodilates the human circulation. Nat. Med. 9, 1498–1505. Del Rio, R., Moya, E.A., Iturriaga, R., 2010. Carotid body and cardiorespiratory alterations in intermittent hypoxia: the oxidative link. Eur. Respir. J. 36, 143–150. Del Rio, R., Moya, E.A., 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. Doyle, M.P., Pickering, R.A., DeWeert, T.M., Hoekstra, J.W., Pater, D., 1981. Kinetics and mechanism of the oxidation of human deoxyhemoglobin by nitrites. J. Biol. Chem. 256, 12393–12398.

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Morphological changes in the rat carotid body following acute sodium nitrite treatment.

The carotid body (CB) is a small neural crest-derived chemosensory organ that detects the chemical composition of the arterial blood and responds to i...
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