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Histochemical and immunohistochemical localization of nitrergic structures in the carotid body of spontaneously hypertensive rats
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Histochemical and immunohistochemical localization of nitrergic structures in the carotid body of spontaneously hypertensive rats Dimitrinka Y. Atanasovaa,b, Angel D. Dandovc, Nikolay D. Dimitrovb, Nikolai E. Lazarova,c,* a
Institute of Neurobiology, Bulgarian Academy of Sciences, Sofia, Bulgaria Department of Anatomy, Faculty of Medicine, Trakia University, Stara Zagora, Bulgaria c Department of Anatomy and Histology, Medical University of Sofia, Sofia, Bulgaria b
A R T I C LE I N FO
A B S T R A C T
Keywords: Carotid body Hypertension NADPH-d histochemistry NOS immunohistochemistry Spontaneously hypertensive rats
The carotid body (CB) is a multipurpose metabolic sensor that acts to initiate cardiorespiratory reflex adjustments to maintain homeostasis of blood-borne chemicals. Emerging evidence suggests that nitric oxide increases the CB chemosensory activity and this enhanced peripheral chemoreflex sensitivity contributes to sympathoexcitation and consequent pathology. The aim of this study was to examine by means of NADPH-diaphorase histochemistry and nitric oxide synthase (NOS) immunohistochemistry the presence and distribution of nitrergic structures in the CB of spontaneously hypertensive rats (SHRs) and to compare their expression patterns to that of age-matched normotensive Wistar rats (NWRs). Histochemistry revealed that the chemosensory glomus cells were NADPH-d-negative but were encircled by fine positive varicosities, which were also dispersed in the stroma around the glomeruli. The NADPH-d-reactive fibers showed the same distributional pattern in the CB of SHRs, however their staining activity was weaker when compared with NWRs. Thin periglomerular, intraglomerular and perivascular varicose fibers, but not glomus or sustentacular cells in the hypertensive CB, constitutively expressed two isoforms of NOS, nNOS and eNOS. In addition, clusters of glomus cells and blood vessels in the CB of SHRs exhibited moderate immunoreactivity for the third known NOS isoenzyme, iNOS. The present study demonstrates that in the hypertensive CB nNOS and eNOS protein expression shows statistically significant down-regulation whereas iNOS expression is up-regulated in the glomic tissue compared to normotensive controls. Our results suggest that impaired NO synthesis could contribute to elevated blood pressure in rats via an increase in chemoexcitation and sympathetic nerve activity in the CB.
1. Introduction The carotid body (CB) is a polymodal chemosensory organ that registers levels of arterial blood gases such as oxygen and carbon dioxide as well as the hydrogen ion concentration (pH), and responds to their changes by initiating corrective respiratory and cardiovascular reflexes to restore homeostasis (Gonzalez et al., 1994; Kumar and Prabhakar, 2012). The detection of chemosensory stimuli is mediated by specialized receptor cells in the CB, the neuron-like glomus (or type I) cells which depolarize and release one (or more) neurotransmitter(s) that stimulate apposed chemoafferent nerve fibers (Atanasova et al., 2011). These sensory nerve fibers, running through the carotid sinus nerve, a branch of the glossopharyngeal nerve, send signals to brain centers to initiate cardiorespiratory adjustments that correct the condition (Nurse, 2005, 2014; Leonard et al., 2018). In addition to sensory
input, the CB receives a rich autonomic innervation by postganglionic fibers of sympathetic origins from the closely located superior cervical ganglion via the ganglioglomerular nerve and from parasympathetic vasomotor fibers of ganglion cells located inside or near the CB, respectively (McDonald and Mitchell, 1975; Kondo, 1976; Ichikawa, 2002). The latter serve as a source of the putative excitatory transmitter acetylcholine (Gauda et al., 2004). These autonomic fibers mainly contribute to the CB vascular innervation and some of them may also innervate glomus cells (McDonald and Mitchell, 1975). The chemosensory glomus cells are typically aggregated in clusters called glomeruli, and are incompletely invested by the extended processes of neighboring glial-like, sustentacular (type II) cells. There is recent evidence that the latter may actively participate in paracrine signaling during chemotransduction by the release of certain “gliotransmitters” such as ATP (Nurse, 2014; Leonard et al., 2018). Being a key excitatory
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Corresponding author at: Department of Anatomy and Histology, Medical University of Sofia, 2, Zdrave Street, BG-1431, Sofia, Bulgaria. E-mail addresses: [email protected] (D.Y. Atanasova), [email protected]fia.bg (A.D. Dandov), [email protected] (N.D. Dimitrov), [email protected]fia.bg (N.E. Lazarov). https://doi.org/10.1016/j.acthis.2019.151500 Received 4 September 2019; Received in revised form 25 November 2019; Accepted 12 December 2019 0065-1281/ © 2020 Elsevier GmbH. All rights reserved.
Please cite this article as: Dimitrinka Y. Atanasova, et al., Acta Histochemica, https://doi.org/10.1016/j.acthis.2019.151500
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For immunohistochemical analysis, after perfusion the carotid bifurcations were removed, the CBs were dissected out and samples were postfixed in the same fixative of 4 % paraformaldehyde in 0.1 M PB, pH 7.4 overnight at 4 °C. Afterwards, the tissue samples were washed in tap water, then in distilled water, dehydrated, embedded in paraffin and cut into 5 μm thick sections. Thereafter, cryostat or paraffin sections were processed for either NADPH-d histochemistry or NOS immunohistochemistry, respectively.
CB neurotransmitter ATP mediates chemosensory function and is involved via P2X signaling in efferent inhibition of chemoreceptors during hypoxia (Nurse, 2005; Campanucci et al., 2006). In addition to these excitatory transmitters, other chemical mediators produced within the CB can regulate the chemosensory process. Indeed, previous research has revealed that the gaseous messenger nitric oxide (NO) exerts a peripheral autonomic control of blood flow, neuronal activity and chemoreception in the rat CB via actions on both the receptor elements and their associated blood vessels (Wang et al., 1993, 1994; Höhler et al., 1994; Prabhakar, 1999; Atanasova et al., 2016). It has been found that under normoxic conditions NO increases the CB chemosensory activity, while during hypoxia it is primarily an inhibitory modulator of chemoreception (Iturriaga, 2001). On the other hand, there is emerging experimental evidence that an abnormal enhanced CB chemosensory discharge contributes to the autonomic dysfunction and elicits sympathetic hyperactivity, which is a common hallmark of sympathetic-related diseases including systemic hypertension (for recent reviews, see Iturriaga et al., 2016; Iturriaga, 2018). Importantly, it has been reported that the bilateral CB ablation in some hypertensive patients causes an immediate and sustained fall in blood pressure (Paton et al., 2013) and attenuates the elevated blood pressure in animal models of hypertension (McBryde et al., 2013). These results suggest that hypertension is strongly dependent on the CB input (Abdala et al., 2012), although the mechanisms underlying the CB chemosensory potentiation elicited by this disease are not completely known. Therefore, considering the involvement of the CB and the putative role of NO in the pathogenesis of hypertension, we examined the presence and distribution of the nitrergic structures in the CB of spontaneously hypertensive rats (SHRs) by means of NADPH-diaphorase (NADPH-d) histochemistry and nitric oxide synthase (NOS) immunohistochemistry, and the alterations in their expression patterns compared to these in the CB of normotensive Wistar rats (NWRs).
2.3. NADPH-d histochemistry NADPH-d histochemical technique was applied according to Scherer-Singler et al. (1983). Briefly, the sections were incubated for 30−60 min at 37 °C in a staining solution consisting of 1 mg/ml reduced β-NADPH, 0.25 mg/ml nitroblue tetrazolium (both from Sigma, St. Louis, MO, USA), and 0.3 % Triton X-100 dissolved in Tris-buffered saline (TBS), pH 7.4. This mixture was freshly prepared prior to use. Following incubation, the slides were rinsed in TBS, washed with distilled water (3 × 15 min) and coverslipped in Kaiser′s glycerol jelly (Merck, Darmstadt, Germany). Control reactions were performed with the omission of the substrate from the incubation medium or the addition of 0.1 mM dichlorophenolindophenol (DPIP), an inhibitor of NADPH-d activity. No final reaction product was observed in any of the control sections under these conditions. 2.4. NOS immunohistochemistry For NOS immunostaining, adjacent sections were deparaffinized and processed by the avidin-biotin-peroxidase complex (ABC) technique of Hsu et al. (1981). They were initially rinsed in PBS several times, treated with 1.2 % H2O2 in absolute methanol to block endogenous peroxidase activity and preincubated in 5 % normal serum in 0.01 M PBS for 1 h at room temperature. Subsequently, they were incubated for 24 h at 4 °C with monoclonal primary antibodies against the three known isoforms of NOS as follows: neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS). The mouse monoclonal antibodies [anti-nNOS (N31020), anti-eNOS (N30020) and antiiNOS (N39120)] were from BD Biosciences Pharmingen (San Diego, CA) and used at a dilution of 1:100 in PBS containing 0.25 % bovine serum albumin and 0.1 % sodium azide. After washing in PBS, sections were consecutively treated for 2 h each at room temperature with biotinylated horse anti-mouse IgG (diluted 1:250) and avidin-biotin complex (Vectastain ABC Kit; both from Vector Labs, Burlingame, CA, USA). The peroxidase activity was developed with 3,3I-diaminobenzidine tetrahydrochloride (DAB; Sigma) and 0.01 % H2O2 in 0.05 M TrisHCl buffer, pH 7.54. Finally, the slides were first washed in running tap water and then in distilled water, dehydrated in a graded series of alcohols, cleared in xylene and coverslipped with Entellan (Merck). Specificity of immunostaining was controlled by either preabsorption of the primary antibody with its native antigen or by substitution of the primary and/or secondary antiserum with normal serum. These methods entirely abolished immunoreactivity.
2. Materials and methods 2.1. Animals The experiments were carried out on adult (12-week-old; n=8) SHRs and age-matched NWRs (n=8) of either sex (250–300 g body weight). The animals were obtained from the Experimental and Breeding Base for Laboratory Animals in Slivnitsa, Bulgaria. All experimental procedures were consistent with the European Communities Council Directive 2010/63 /EU. The experimental design was in accordance with national rules on animal experiments and was approved by the Ethics Committee of the Institute of Neurobiology, Bulgarian Academy of Sciences. The experimental procedures were approved by the Research Ethics Commission of the Medical University of Sofia and the experimental studies were conducted in keeping with the EU Directive 2010/63/EU for animal experiments. 2.2. Tissue preparation For histochemical and immunohistochemical experiments, the rats were deeply anesthetized with sodium pentobarbital (Nembutal; 50 mg/kg, i.p.) and transcardially perfused, first with heparinized phosphate buffered saline (PBS) followed by ice-cold 4 % paraformaldehyde in 0.01 M phosphate buffer (PB), pH 7.4. For the purpose of histochemical experiments, after perfusion the carotid bifurcations were excised and postfixed in the same fixative overnight at 4 °C and then placed into ice-cold cryoprotectant solution of 30 % sucrose in PB until they sank. Trimmed tissues were subsequently embedded in Tissue-Tek O.C.T. compound, sectioned on a cryostat at 10 μm thickness; the sections were thaw-mounted onto glass slides precoated with chrome gelatin and air dried at room temperature.
2.5. Photodocumentation and image processing After histochemical and immunohistochemical reactions, the stained and processed sections were digitalized using a Nikon DXM 1200c research microscope (Nikon Inc., Tokyo, Japan) equipped with a DMX 1200 digital camera. The digitized images were precisely captured with an objective lens 40x and a total magnification 400×. As an initial step, the system underwent an appropriate calibration to correct the captured images. The light source and camera settings were kept the same for every single image. The images were recorded in TIF format and processed for removal of artifacts using Adobe Photoshop CC software (Adobe Systems Inc., San Jose, CA). 2
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2.6. Densitometric analysis and statistics NADPH-d and NOS staining intensity were quantified in binaryconverted pictures using the semi-automated densitometrical evaluation after a threshold setting by means of the open source program ImageJ 1.48v (NIH, Bethesda, MD, USA). First we outlined the region of interest with an area of 50 μm2. Relative staining intensities were then semi-quantified and the results were presented as percentage areas. Two blinded assessors performed the evaluations and the obtained results were averaged. The statistical analyses between groups were performed using the GraphPad Prism software package (GraphPad Software Inc., San Diego, CA, USA; version 5.04 for Windows). The data are presented as means ± standard error of the mean (SEM). We have implemented unpaired t-test with Welch’s correction for Gaussian distributed nNOS and iNOS and Mann-Whitney U-test for non-Gaussian distributed eNOS and NADPH-d. The results were considered statistically significant where p < 0.05. To indicate the level of significance the following symbols were used: *p < 0.05 and ***p < 0.001.
Fig. 2. Densitometrical analysis of NADPH-d staining intensity in normotensive Wistar rats (NWR; n = 3; blue column) and spontaneously hypertensive rats (SHR; n = 3; red column). The NADPH-d staining intensity was statistically significantly reduced in the CB of hypertensive compared to normotensive animals. The level of significance: * p < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
3. Results 3.1. NADPH-d staining
running between the cell clusters in NWRs. In the CB of age-matched SHRs, we found an almost identical staining pattern, i.e. the NADPH-d negative glomus cells were surrounded by arborizations of reactive beaded nerve fibers (Fig. 1c, d). In addition, NADPH-d-stained varicosities passed between the CB glomeruli and were also observed perivascularly. However, the NADPH-d-
In the normotensive CB, light microscopic NADPH-d histochemistry revealed that the chemosensory glomus cells typically aggregated in cell clusters were negative but were enveloped by fine positive fibers in a basket-like manner (Fig. 1a and b). Such scattered NADPH-d-stained varicosities were also seen around the blood vessels in the CB. In addition, NADPH-d activity was observed in single varicose nerve fibers
Fig. 1. Histochemical demonstration of NADPH-d activity in the carotid body (CB) of normotensive Wistar rats (NWR) and spontaneously hypertensive rats (SHR). (A) Overview of the CB in NWR showing NADPH-d staining of NOS. (B) Higher magnification of the boxed area in (A). Note that NADPH-d reactive nerve fiber arborizations (arrows) are associated with the glomus cells and also surround the blood vessels (BV). Low- (C) and high (D)-power photomicrographs of the CB in SHR demonstrating fine periglomerular (arrows) and perivascular (arrowheads) NADPH-d varicose fibers that are less intensely stained than in NWR. Scale bars =50 μm. 3
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Fig. 3. Immunohistochemical localization of NOS isoenzymes in the carotid body (CB) of normotensive Wistar rats (NWR) and spontaneously hypertensive rats (SHR). Light photomicrographs showing the localization of nNOS in the CB of NWR and SHR (A and D). Sparse beaded immunoreactive nerve fibers are observed in close proximity to clusters of glomus cells (arrows) and are also associated with CB vasculature (arrowheads). Note the weaker staining intensity of immunoreactive structures in the SHR compared to control NWR. eNOS-immunopositive varicosities (arrows) and dot-like structures (arrowheads), presumably nerve endings, are visible in the vicinity of glomus and endothelial cells of the CB blood vessels (BV) in both NWR (B) and SHR (E). In the latter the intensity of the immunoreaction is distinctly weaker and the number of immunostained fibers and their terminals is obviously reduced. The CB structures are iNOS-immunonegative in the normotensive CB (C) while a subset of glomus cells (arrows) and the walls of blood vessels (arrowheads) show moderate-to-high intensity staining for iNOS in the hypertensive CB (F). Scale bars =50 μm.
reactive fibers showed statistically significant weaker blue-staining intensity in the hypertensive than in the normotensive CB (SHR 43.45 ± 3.75 %, NWR 55.95 ± 3.52 %, p < 0.05) (Fig. 2).
compared to the control NWR group (SHR 83.93 ± 3.03 %, NWR 13.24 ± 2.85 %, p < 0.001). The analysis of the nNOS (SHR 39.30 ± 6.71 %, NWR 86.38 ± 1.84 %, p < 0.001) and eNOS (SHR 6.67 ± 2.99 %, NWR 53.49 ± 4.01 %, p < 0.001) staining intensity yielded opposing results in the hypertensive CB whereas it was statistically decreased in SHRs compared to normotensive animals.
3.2. NOS immunostaining Overally, immunohistochemistry demonstrated that in the CB of NWRs both parenchymal cell types, glomus and sustentacular cells, were immunonegative for all three isoforms of the NOS. Immunostained sections for nNOS in the normotensive CB showed virtually the same pattern of immunoreactivity when compared to that seen following NADPH-d histochemistry. Specifically, moderate to strong nNOS and eNOS immunostaining was observed in varicose nerve fibers and their endings found in close proximity to clusters of glomus cells, and also in varicosities that were intimately associated with CB vasculature (Fig. 3a and b). Sparse beaded immunoreactive nerve fibers were visible in the stroma around the glomic lobules as well. Conversely, no iNOS-immunopositive staining was observed in the CB structures of NWRs (Fig. 3c). In the CB of SHRs, a weak immunopositive reaction for nNOS and eNOS was restricted to pericellular and perivascular thin varicosities while glomus and sustentacular cells were devoid of immunoreactivity (Fig. 3d and e). Interestingly, iNOS immunostaining showing moderate to high intensity was detected in clusters of glomus cells in the CB of SHRs (Fig. 3f). The immunopositive reaction was also seen in the walls of blood vessels.
4. Discussion The results of the present study show that NOS is not found at detectable levels in the chemosensory glomus cells of normotensive animals. In contrast, the nerve fibers and their endings which are associated with glomus cells and blood vessels in both the normotensive and hypertensive CB are positive for NADPH-d activity, and express nNOS and eNOS. Furthermore, iNOS protein expression is induced in clusters of glomus cells and the blood vessels of the CB in SHRs, but not in NWRs. It is generally accepted that essential hypertension is associated with endothelial dysfunction, increased oxidative stress in blood vessels and activation of the inflammatory mediators (Schulz et al., 2011). Observations in certain brain regions show that in SHRs NO synthesis is different from that of control normotensive strains (see in Zanzinger, 1999) and that these alterations might be associated with the enhancement of vascular resistance, which could account for high blood pressure (Marín and Rodríguez-Martínez, 1997). In this regard, it has been proposed that eNOS itself can be a superoxide source and thereby it contributes to endothelial dysfunction (Forstermann and Munzel, 2006). Such a transformation of eNOS from a protective enzyme in the vasculature to a contributor of oxidative stress has already been observed in animal models of hypertension (Forstermann and Munzel, 2006). On the other hand, earlier studies have shown that the superoxide anion may enhance iNOS expression by activating nuclear factorkappa B (Blackwell and Christman, 1997). Here, we provide evidence
3.3. Densitometrical analysis of NOS immunoreactivity The densitometric analysis revealed that the ratio of glomus cells immunoreactive for nNOS, eNOS and iNOS was significantly different between NWR and SHR groups (Fig. 4). Specifically, a prominent increase in the iNOS immunostaining was observed in the SHRs, 4
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Fig. 4. Densitometrical analysis of (A) anti-nNOS, (B) anti-eNOS and (C) anti-iNOS staining intensity in normotensive Wistar rats (NWR; n = 5; blue column) and spontaneously hypertensive rats (SHR; n = 5; red column). The nNOS (A) and eNOS (B) staining intensity was statistically decreased in the CB of hypertensive compared to normotensive animals. (C) Prominent increase in the iNOS immunostaining was observed in the CB of SHR. The level of significance: *** p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
sensitivity to hypoxia is associated with the absence of eNOS expression in CB blood vessels (Prabhakar and Semenza, 2012). In keeping with its role as a circulatory chemical sensor, the CB is considered a highly vascular organ (Kumar and Prabhakar, 2012). NO derived from eNOS is a potent vasodilator and regulates the blood flow to the CB (Prabhakar and Semenza, 2012). Consistent with this statement, the current results suggest that a reduced eNOS expression observed in the CB of SHRs might elicit vasoconstriction, enhanced vascular dysfunction and consequent blood pressure elevation. In conclusion, the results of the present study provide histochemical and immunohistochemical evidence that in the hypertensive state the expression of nNOS and eNOS is statistically down-regulated while the levels of iNOS are up-regulated in the CB. The altered NO production modulates the chemosensory processing in hypertensive animals by its direct action on glomus cells and indirectly on the CB vasculature. Our findings further support and extend the new concept that a heightened chemosensory discharge causes sympathetic neural activation which, along with the elevated production of neurotrophic factors (cf. Atanasova and Lazarov, 2014), contributes to the development of hypertension. Thus, it is tempting to speculate that the CB and NOS may represent potential therapeutic targets for the treatment of hypertensive patients.
for a decreased expression of nNOS and eNOS, and a de novo expression of iNOS in the CB of SHRs. It is likely that reactive oxygen species directly alter endothelial function or may cause changes in the vascular tone by several mechanisms including decreased NO production. Previous research has revealed that the endothelial dysfunction in hypertension may not be associated only with an impaired NO metabolism but also with an increased production of angiotensin II (Ang II) and Ang II-induced superoxide anion generation (reviewed in Consolim-Colombo and Bortolotto, 2018). Accordingly, our recent research has demonstrated that the glomus cells are richly endowed with angiotensin AT1 receptors (Atanasova et al., 2018) and confirms prior findings that a local renin-angiotensin system exists in the rat CB (Lam and Leung, 2002). Simultaneous activation of this system and the interaction of NO with Ang II may further potentiate the direct effects of NO on blood vessels. Besides such an effect, NO has an important role in the regulation of cardiovascular autonomic control (reviewed by Zanzinger, 1999) and carotid chemoreception (Iturriaga, 2001). Taken together, these findings suggest that the altered expression of NOS isoenzymes elicits chemosensory potentiation via an increase in oxidative stress and Ang II levels and a decrease in NO production in the CB. A growing body of evidence suggests that the chemosensory reflex is a major regulator of sympathetic tone (reviewed in Kumar and Prabhakar, 2012). It is also well established that that oxidative stress and Ang II are potential mediators of both chemosensory and cardiorespiratory alterations via activation of the sympathetic nervous system (see, Zanzinger, 1999). In turn, CB activity is under a tonic inhibitory influence from NO generated by the nerve endings and blood vessels (Prabhakar, 1999). There is convincing evidence that nNOS-expressing nerve fibers in the CB originate from neurons located in the petrosal ganglion and in paraganglia along the glossopharyngeal and carotid sinus nerves (Wang et al., 1993; Prabhakar, 1999; Campanucci and Nurse, 2007). Given that nNOS immunoreactivity is present in both sensory and autonomic (parasympathetic) ganglion cells innervating the CB, it is believed that afferent fibers act on glomus cells, thus controlling chemoreception while autonomic fibers control the blood flow by acting on vasculature (Campanucci et al., 2012). Further studies reveal that the latter underlie the basis for NO-mediated efferent inhibition of the CB sensory discharge (Wang et al., 1993, 1994; Prabhakar, 1999). In particular, it has been proposed that NO released from efferent nerves during hypoxia regulates the excitability of glomus cells, resulting in decreased afferent nerve activity in the CB (Iturriaga, 2001; Campanucci and Nurse, 2007). However, under hypertensive conditions, NOS activity is inhibited and NO production is reduced, which contributes to chemoreflex activation leading to elevated sympathetic activity and high blood pressure. Therefore, a decrease in the level of NO by inhibition of NOS could be associated with hypertension in rats. Furthermore, the available data indicate that such decreased
Author contributions Study concept and design: NL. Performed experiments: DA, AD, and ND, Image analysis, photographic work and illustrations: DA and ND. Wrote paper: NL and DA. Edited paper: NL and AD. All authors were involved in the process of analysis and interpreting the data, have read and approved the final version of the manuscript. Funding This work was supported by the Medical Faculty of Trakia University, Stara Zagora, Bulgaria, contract No. 10/2018. Declaration of Competing Interest The authors declare that they have no conflict of interests. Acknowledgement The authors would like to thank Sabina Mitova for her excellent technical assistance. References Abdala, A.P., McBryde, F.D., Marina, N., Hendy, E.B., Engelman, Z.J., Fudim, M., Sobotka, P.A., Gourine, A.V., Paton, J.F., 2012. Hypertension is critically dependent
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Histochemical and immunohistochemical localization of nitrergic structures in the carotid body of spontaneously hypertensive rats Dimitrinka Y. Atanasovaa,b, Angel D. Dandovc, Nikolay D. Dimitrovb, Nikolai E. Lazarova,c,* a
Institute of Neurobiology, Bulgarian Academy of Sciences, Sofia, Bulgaria Department of Anatomy, Faculty of Medicine, Trakia University, Stara Zagora, Bulgaria c Department of Anatomy and Histology, Medical University of Sofia, Sofia, Bulgaria b
A R T I C LE I N FO
A B S T R A C T
Keywords: Carotid body Hypertension NADPH-d histochemistry NOS immunohistochemistry Spontaneously hypertensive rats
The carotid body (CB) is a multipurpose metabolic sensor that acts to initiate cardiorespiratory reflex adjustments to maintain homeostasis of blood-borne chemicals. Emerging evidence suggests that nitric oxide increases the CB chemosensory activity and this enhanced peripheral chemoreflex sensitivity contributes to sympathoexcitation and consequent pathology. The aim of this study was to examine by means of NADPH-diaphorase histochemistry and nitric oxide synthase (NOS) immunohistochemistry the presence and distribution of nitrergic structures in the CB of spontaneously hypertensive rats (SHRs) and to compare their expression patterns to that of age-matched normotensive Wistar rats (NWRs). Histochemistry revealed that the chemosensory glomus cells were NADPH-d-negative but were encircled by fine positive varicosities, which were also dispersed in the stroma around the glomeruli. The NADPH-d-reactive fibers showed the same distributional pattern in the CB of SHRs, however their staining activity was weaker when compared with NWRs. Thin periglomerular, intraglomerular and perivascular varicose fibers, but not glomus or sustentacular cells in the hypertensive CB, constitutively expressed two isoforms of NOS, nNOS and eNOS. In addition, clusters of glomus cells and blood vessels in the CB of SHRs exhibited moderate immunoreactivity for the third known NOS isoenzyme, iNOS. The present study demonstrates that in the hypertensive CB nNOS and eNOS protein expression shows statistically significant down-regulation whereas iNOS expression is up-regulated in the glomic tissue compared to normotensive controls. Our results suggest that impaired NO synthesis could contribute to elevated blood pressure in rats via an increase in chemoexcitation and sympathetic nerve activity in the CB.
1. Introduction The carotid body (CB) is a polymodal chemosensory organ that registers levels of arterial blood gases such as oxygen and carbon dioxide as well as the hydrogen ion concentration (pH), and responds to their changes by initiating corrective respiratory and cardiovascular reflexes to restore homeostasis (Gonzalez et al., 1994; Kumar and Prabhakar, 2012). The detection of chemosensory stimuli is mediated by specialized receptor cells in the CB, the neuron-like glomus (or type I) cells which depolarize and release one (or more) neurotransmitter(s) that stimulate apposed chemoafferent nerve fibers (Atanasova et al., 2011). These sensory nerve fibers, running through the carotid sinus nerve, a branch of the glossopharyngeal nerve, send signals to brain centers to initiate cardiorespiratory adjustments that correct the condition (Nurse, 2005, 2014; Leonard et al., 2018). In addition to sensory
input, the CB receives a rich autonomic innervation by postganglionic fibers of sympathetic origins from the closely located superior cervical ganglion via the ganglioglomerular nerve and from parasympathetic vasomotor fibers of ganglion cells located inside or near the CB, respectively (McDonald and Mitchell, 1975; Kondo, 1976; Ichikawa, 2002). The latter serve as a source of the putative excitatory transmitter acetylcholine (Gauda et al., 2004). These autonomic fibers mainly contribute to the CB vascular innervation and some of them may also innervate glomus cells (McDonald and Mitchell, 1975). The chemosensory glomus cells are typically aggregated in clusters called glomeruli, and are incompletely invested by the extended processes of neighboring glial-like, sustentacular (type II) cells. There is recent evidence that the latter may actively participate in paracrine signaling during chemotransduction by the release of certain “gliotransmitters” such as ATP (Nurse, 2014; Leonard et al., 2018). Being a key excitatory
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Corresponding author at: Department of Anatomy and Histology, Medical University of Sofia, 2, Zdrave Street, BG-1431, Sofia, Bulgaria. E-mail addresses: [email protected] (D.Y. Atanasova), [email protected]fia.bg (A.D. Dandov), [email protected] (N.D. Dimitrov), [email protected]fia.bg (N.E. Lazarov). https://doi.org/10.1016/j.acthis.2019.151500 Received 4 September 2019; Received in revised form 25 November 2019; Accepted 12 December 2019 0065-1281/ © 2020 Elsevier GmbH. All rights reserved.
Please cite this article as: Dimitrinka Y. Atanasova, et al., Acta Histochemica, https://doi.org/10.1016/j.acthis.2019.151500
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For immunohistochemical analysis, after perfusion the carotid bifurcations were removed, the CBs were dissected out and samples were postfixed in the same fixative of 4 % paraformaldehyde in 0.1 M PB, pH 7.4 overnight at 4 °C. Afterwards, the tissue samples were washed in tap water, then in distilled water, dehydrated, embedded in paraffin and cut into 5 μm thick sections. Thereafter, cryostat or paraffin sections were processed for either NADPH-d histochemistry or NOS immunohistochemistry, respectively.
CB neurotransmitter ATP mediates chemosensory function and is involved via P2X signaling in efferent inhibition of chemoreceptors during hypoxia (Nurse, 2005; Campanucci et al., 2006). In addition to these excitatory transmitters, other chemical mediators produced within the CB can regulate the chemosensory process. Indeed, previous research has revealed that the gaseous messenger nitric oxide (NO) exerts a peripheral autonomic control of blood flow, neuronal activity and chemoreception in the rat CB via actions on both the receptor elements and their associated blood vessels (Wang et al., 1993, 1994; Höhler et al., 1994; Prabhakar, 1999; Atanasova et al., 2016). It has been found that under normoxic conditions NO increases the CB chemosensory activity, while during hypoxia it is primarily an inhibitory modulator of chemoreception (Iturriaga, 2001). On the other hand, there is emerging experimental evidence that an abnormal enhanced CB chemosensory discharge contributes to the autonomic dysfunction and elicits sympathetic hyperactivity, which is a common hallmark of sympathetic-related diseases including systemic hypertension (for recent reviews, see Iturriaga et al., 2016; Iturriaga, 2018). Importantly, it has been reported that the bilateral CB ablation in some hypertensive patients causes an immediate and sustained fall in blood pressure (Paton et al., 2013) and attenuates the elevated blood pressure in animal models of hypertension (McBryde et al., 2013). These results suggest that hypertension is strongly dependent on the CB input (Abdala et al., 2012), although the mechanisms underlying the CB chemosensory potentiation elicited by this disease are not completely known. Therefore, considering the involvement of the CB and the putative role of NO in the pathogenesis of hypertension, we examined the presence and distribution of the nitrergic structures in the CB of spontaneously hypertensive rats (SHRs) by means of NADPH-diaphorase (NADPH-d) histochemistry and nitric oxide synthase (NOS) immunohistochemistry, and the alterations in their expression patterns compared to these in the CB of normotensive Wistar rats (NWRs).
2.3. NADPH-d histochemistry NADPH-d histochemical technique was applied according to Scherer-Singler et al. (1983). Briefly, the sections were incubated for 30−60 min at 37 °C in a staining solution consisting of 1 mg/ml reduced β-NADPH, 0.25 mg/ml nitroblue tetrazolium (both from Sigma, St. Louis, MO, USA), and 0.3 % Triton X-100 dissolved in Tris-buffered saline (TBS), pH 7.4. This mixture was freshly prepared prior to use. Following incubation, the slides were rinsed in TBS, washed with distilled water (3 × 15 min) and coverslipped in Kaiser′s glycerol jelly (Merck, Darmstadt, Germany). Control reactions were performed with the omission of the substrate from the incubation medium or the addition of 0.1 mM dichlorophenolindophenol (DPIP), an inhibitor of NADPH-d activity. No final reaction product was observed in any of the control sections under these conditions. 2.4. NOS immunohistochemistry For NOS immunostaining, adjacent sections were deparaffinized and processed by the avidin-biotin-peroxidase complex (ABC) technique of Hsu et al. (1981). They were initially rinsed in PBS several times, treated with 1.2 % H2O2 in absolute methanol to block endogenous peroxidase activity and preincubated in 5 % normal serum in 0.01 M PBS for 1 h at room temperature. Subsequently, they were incubated for 24 h at 4 °C with monoclonal primary antibodies against the three known isoforms of NOS as follows: neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS). The mouse monoclonal antibodies [anti-nNOS (N31020), anti-eNOS (N30020) and antiiNOS (N39120)] were from BD Biosciences Pharmingen (San Diego, CA) and used at a dilution of 1:100 in PBS containing 0.25 % bovine serum albumin and 0.1 % sodium azide. After washing in PBS, sections were consecutively treated for 2 h each at room temperature with biotinylated horse anti-mouse IgG (diluted 1:250) and avidin-biotin complex (Vectastain ABC Kit; both from Vector Labs, Burlingame, CA, USA). The peroxidase activity was developed with 3,3I-diaminobenzidine tetrahydrochloride (DAB; Sigma) and 0.01 % H2O2 in 0.05 M TrisHCl buffer, pH 7.54. Finally, the slides were first washed in running tap water and then in distilled water, dehydrated in a graded series of alcohols, cleared in xylene and coverslipped with Entellan (Merck). Specificity of immunostaining was controlled by either preabsorption of the primary antibody with its native antigen or by substitution of the primary and/or secondary antiserum with normal serum. These methods entirely abolished immunoreactivity.
2. Materials and methods 2.1. Animals The experiments were carried out on adult (12-week-old; n=8) SHRs and age-matched NWRs (n=8) of either sex (250–300 g body weight). The animals were obtained from the Experimental and Breeding Base for Laboratory Animals in Slivnitsa, Bulgaria. All experimental procedures were consistent with the European Communities Council Directive 2010/63 /EU. The experimental design was in accordance with national rules on animal experiments and was approved by the Ethics Committee of the Institute of Neurobiology, Bulgarian Academy of Sciences. The experimental procedures were approved by the Research Ethics Commission of the Medical University of Sofia and the experimental studies were conducted in keeping with the EU Directive 2010/63/EU for animal experiments. 2.2. Tissue preparation For histochemical and immunohistochemical experiments, the rats were deeply anesthetized with sodium pentobarbital (Nembutal; 50 mg/kg, i.p.) and transcardially perfused, first with heparinized phosphate buffered saline (PBS) followed by ice-cold 4 % paraformaldehyde in 0.01 M phosphate buffer (PB), pH 7.4. For the purpose of histochemical experiments, after perfusion the carotid bifurcations were excised and postfixed in the same fixative overnight at 4 °C and then placed into ice-cold cryoprotectant solution of 30 % sucrose in PB until they sank. Trimmed tissues were subsequently embedded in Tissue-Tek O.C.T. compound, sectioned on a cryostat at 10 μm thickness; the sections were thaw-mounted onto glass slides precoated with chrome gelatin and air dried at room temperature.
2.5. Photodocumentation and image processing After histochemical and immunohistochemical reactions, the stained and processed sections were digitalized using a Nikon DXM 1200c research microscope (Nikon Inc., Tokyo, Japan) equipped with a DMX 1200 digital camera. The digitized images were precisely captured with an objective lens 40x and a total magnification 400×. As an initial step, the system underwent an appropriate calibration to correct the captured images. The light source and camera settings were kept the same for every single image. The images were recorded in TIF format and processed for removal of artifacts using Adobe Photoshop CC software (Adobe Systems Inc., San Jose, CA). 2
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2.6. Densitometric analysis and statistics NADPH-d and NOS staining intensity were quantified in binaryconverted pictures using the semi-automated densitometrical evaluation after a threshold setting by means of the open source program ImageJ 1.48v (NIH, Bethesda, MD, USA). First we outlined the region of interest with an area of 50 μm2. Relative staining intensities were then semi-quantified and the results were presented as percentage areas. Two blinded assessors performed the evaluations and the obtained results were averaged. The statistical analyses between groups were performed using the GraphPad Prism software package (GraphPad Software Inc., San Diego, CA, USA; version 5.04 for Windows). The data are presented as means ± standard error of the mean (SEM). We have implemented unpaired t-test with Welch’s correction for Gaussian distributed nNOS and iNOS and Mann-Whitney U-test for non-Gaussian distributed eNOS and NADPH-d. The results were considered statistically significant where p < 0.05. To indicate the level of significance the following symbols were used: *p < 0.05 and ***p < 0.001.
Fig. 2. Densitometrical analysis of NADPH-d staining intensity in normotensive Wistar rats (NWR; n = 3; blue column) and spontaneously hypertensive rats (SHR; n = 3; red column). The NADPH-d staining intensity was statistically significantly reduced in the CB of hypertensive compared to normotensive animals. The level of significance: * p < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
3. Results 3.1. NADPH-d staining
running between the cell clusters in NWRs. In the CB of age-matched SHRs, we found an almost identical staining pattern, i.e. the NADPH-d negative glomus cells were surrounded by arborizations of reactive beaded nerve fibers (Fig. 1c, d). In addition, NADPH-d-stained varicosities passed between the CB glomeruli and were also observed perivascularly. However, the NADPH-d-
In the normotensive CB, light microscopic NADPH-d histochemistry revealed that the chemosensory glomus cells typically aggregated in cell clusters were negative but were enveloped by fine positive fibers in a basket-like manner (Fig. 1a and b). Such scattered NADPH-d-stained varicosities were also seen around the blood vessels in the CB. In addition, NADPH-d activity was observed in single varicose nerve fibers
Fig. 1. Histochemical demonstration of NADPH-d activity in the carotid body (CB) of normotensive Wistar rats (NWR) and spontaneously hypertensive rats (SHR). (A) Overview of the CB in NWR showing NADPH-d staining of NOS. (B) Higher magnification of the boxed area in (A). Note that NADPH-d reactive nerve fiber arborizations (arrows) are associated with the glomus cells and also surround the blood vessels (BV). Low- (C) and high (D)-power photomicrographs of the CB in SHR demonstrating fine periglomerular (arrows) and perivascular (arrowheads) NADPH-d varicose fibers that are less intensely stained than in NWR. Scale bars =50 μm. 3
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Fig. 3. Immunohistochemical localization of NOS isoenzymes in the carotid body (CB) of normotensive Wistar rats (NWR) and spontaneously hypertensive rats (SHR). Light photomicrographs showing the localization of nNOS in the CB of NWR and SHR (A and D). Sparse beaded immunoreactive nerve fibers are observed in close proximity to clusters of glomus cells (arrows) and are also associated with CB vasculature (arrowheads). Note the weaker staining intensity of immunoreactive structures in the SHR compared to control NWR. eNOS-immunopositive varicosities (arrows) and dot-like structures (arrowheads), presumably nerve endings, are visible in the vicinity of glomus and endothelial cells of the CB blood vessels (BV) in both NWR (B) and SHR (E). In the latter the intensity of the immunoreaction is distinctly weaker and the number of immunostained fibers and their terminals is obviously reduced. The CB structures are iNOS-immunonegative in the normotensive CB (C) while a subset of glomus cells (arrows) and the walls of blood vessels (arrowheads) show moderate-to-high intensity staining for iNOS in the hypertensive CB (F). Scale bars =50 μm.
reactive fibers showed statistically significant weaker blue-staining intensity in the hypertensive than in the normotensive CB (SHR 43.45 ± 3.75 %, NWR 55.95 ± 3.52 %, p < 0.05) (Fig. 2).
compared to the control NWR group (SHR 83.93 ± 3.03 %, NWR 13.24 ± 2.85 %, p < 0.001). The analysis of the nNOS (SHR 39.30 ± 6.71 %, NWR 86.38 ± 1.84 %, p < 0.001) and eNOS (SHR 6.67 ± 2.99 %, NWR 53.49 ± 4.01 %, p < 0.001) staining intensity yielded opposing results in the hypertensive CB whereas it was statistically decreased in SHRs compared to normotensive animals.
3.2. NOS immunostaining Overally, immunohistochemistry demonstrated that in the CB of NWRs both parenchymal cell types, glomus and sustentacular cells, were immunonegative for all three isoforms of the NOS. Immunostained sections for nNOS in the normotensive CB showed virtually the same pattern of immunoreactivity when compared to that seen following NADPH-d histochemistry. Specifically, moderate to strong nNOS and eNOS immunostaining was observed in varicose nerve fibers and their endings found in close proximity to clusters of glomus cells, and also in varicosities that were intimately associated with CB vasculature (Fig. 3a and b). Sparse beaded immunoreactive nerve fibers were visible in the stroma around the glomic lobules as well. Conversely, no iNOS-immunopositive staining was observed in the CB structures of NWRs (Fig. 3c). In the CB of SHRs, a weak immunopositive reaction for nNOS and eNOS was restricted to pericellular and perivascular thin varicosities while glomus and sustentacular cells were devoid of immunoreactivity (Fig. 3d and e). Interestingly, iNOS immunostaining showing moderate to high intensity was detected in clusters of glomus cells in the CB of SHRs (Fig. 3f). The immunopositive reaction was also seen in the walls of blood vessels.
4. Discussion The results of the present study show that NOS is not found at detectable levels in the chemosensory glomus cells of normotensive animals. In contrast, the nerve fibers and their endings which are associated with glomus cells and blood vessels in both the normotensive and hypertensive CB are positive for NADPH-d activity, and express nNOS and eNOS. Furthermore, iNOS protein expression is induced in clusters of glomus cells and the blood vessels of the CB in SHRs, but not in NWRs. It is generally accepted that essential hypertension is associated with endothelial dysfunction, increased oxidative stress in blood vessels and activation of the inflammatory mediators (Schulz et al., 2011). Observations in certain brain regions show that in SHRs NO synthesis is different from that of control normotensive strains (see in Zanzinger, 1999) and that these alterations might be associated with the enhancement of vascular resistance, which could account for high blood pressure (Marín and Rodríguez-Martínez, 1997). In this regard, it has been proposed that eNOS itself can be a superoxide source and thereby it contributes to endothelial dysfunction (Forstermann and Munzel, 2006). Such a transformation of eNOS from a protective enzyme in the vasculature to a contributor of oxidative stress has already been observed in animal models of hypertension (Forstermann and Munzel, 2006). On the other hand, earlier studies have shown that the superoxide anion may enhance iNOS expression by activating nuclear factorkappa B (Blackwell and Christman, 1997). Here, we provide evidence
3.3. Densitometrical analysis of NOS immunoreactivity The densitometric analysis revealed that the ratio of glomus cells immunoreactive for nNOS, eNOS and iNOS was significantly different between NWR and SHR groups (Fig. 4). Specifically, a prominent increase in the iNOS immunostaining was observed in the SHRs, 4
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Fig. 4. Densitometrical analysis of (A) anti-nNOS, (B) anti-eNOS and (C) anti-iNOS staining intensity in normotensive Wistar rats (NWR; n = 5; blue column) and spontaneously hypertensive rats (SHR; n = 5; red column). The nNOS (A) and eNOS (B) staining intensity was statistically decreased in the CB of hypertensive compared to normotensive animals. (C) Prominent increase in the iNOS immunostaining was observed in the CB of SHR. The level of significance: *** p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
sensitivity to hypoxia is associated with the absence of eNOS expression in CB blood vessels (Prabhakar and Semenza, 2012). In keeping with its role as a circulatory chemical sensor, the CB is considered a highly vascular organ (Kumar and Prabhakar, 2012). NO derived from eNOS is a potent vasodilator and regulates the blood flow to the CB (Prabhakar and Semenza, 2012). Consistent with this statement, the current results suggest that a reduced eNOS expression observed in the CB of SHRs might elicit vasoconstriction, enhanced vascular dysfunction and consequent blood pressure elevation. In conclusion, the results of the present study provide histochemical and immunohistochemical evidence that in the hypertensive state the expression of nNOS and eNOS is statistically down-regulated while the levels of iNOS are up-regulated in the CB. The altered NO production modulates the chemosensory processing in hypertensive animals by its direct action on glomus cells and indirectly on the CB vasculature. Our findings further support and extend the new concept that a heightened chemosensory discharge causes sympathetic neural activation which, along with the elevated production of neurotrophic factors (cf. Atanasova and Lazarov, 2014), contributes to the development of hypertension. Thus, it is tempting to speculate that the CB and NOS may represent potential therapeutic targets for the treatment of hypertensive patients.
for a decreased expression of nNOS and eNOS, and a de novo expression of iNOS in the CB of SHRs. It is likely that reactive oxygen species directly alter endothelial function or may cause changes in the vascular tone by several mechanisms including decreased NO production. Previous research has revealed that the endothelial dysfunction in hypertension may not be associated only with an impaired NO metabolism but also with an increased production of angiotensin II (Ang II) and Ang II-induced superoxide anion generation (reviewed in Consolim-Colombo and Bortolotto, 2018). Accordingly, our recent research has demonstrated that the glomus cells are richly endowed with angiotensin AT1 receptors (Atanasova et al., 2018) and confirms prior findings that a local renin-angiotensin system exists in the rat CB (Lam and Leung, 2002). Simultaneous activation of this system and the interaction of NO with Ang II may further potentiate the direct effects of NO on blood vessels. Besides such an effect, NO has an important role in the regulation of cardiovascular autonomic control (reviewed by Zanzinger, 1999) and carotid chemoreception (Iturriaga, 2001). Taken together, these findings suggest that the altered expression of NOS isoenzymes elicits chemosensory potentiation via an increase in oxidative stress and Ang II levels and a decrease in NO production in the CB. A growing body of evidence suggests that the chemosensory reflex is a major regulator of sympathetic tone (reviewed in Kumar and Prabhakar, 2012). It is also well established that that oxidative stress and Ang II are potential mediators of both chemosensory and cardiorespiratory alterations via activation of the sympathetic nervous system (see, Zanzinger, 1999). In turn, CB activity is under a tonic inhibitory influence from NO generated by the nerve endings and blood vessels (Prabhakar, 1999). There is convincing evidence that nNOS-expressing nerve fibers in the CB originate from neurons located in the petrosal ganglion and in paraganglia along the glossopharyngeal and carotid sinus nerves (Wang et al., 1993; Prabhakar, 1999; Campanucci and Nurse, 2007). Given that nNOS immunoreactivity is present in both sensory and autonomic (parasympathetic) ganglion cells innervating the CB, it is believed that afferent fibers act on glomus cells, thus controlling chemoreception while autonomic fibers control the blood flow by acting on vasculature (Campanucci et al., 2012). Further studies reveal that the latter underlie the basis for NO-mediated efferent inhibition of the CB sensory discharge (Wang et al., 1993, 1994; Prabhakar, 1999). In particular, it has been proposed that NO released from efferent nerves during hypoxia regulates the excitability of glomus cells, resulting in decreased afferent nerve activity in the CB (Iturriaga, 2001; Campanucci and Nurse, 2007). However, under hypertensive conditions, NOS activity is inhibited and NO production is reduced, which contributes to chemoreflex activation leading to elevated sympathetic activity and high blood pressure. Therefore, a decrease in the level of NO by inhibition of NOS could be associated with hypertension in rats. Furthermore, the available data indicate that such decreased
Author contributions Study concept and design: NL. Performed experiments: DA, AD, and ND, Image analysis, photographic work and illustrations: DA and ND. Wrote paper: NL and DA. Edited paper: NL and AD. All authors were involved in the process of analysis and interpreting the data, have read and approved the final version of the manuscript. Funding This work was supported by the Medical Faculty of Trakia University, Stara Zagora, Bulgaria, contract No. 10/2018. Declaration of Competing Interest The authors declare that they have no conflict of interests. Acknowledgement The authors would like to thank Sabina Mitova for her excellent technical assistance. References Abdala, A.P., McBryde, F.D., Marina, N., Hendy, E.B., Engelman, Z.J., Fudim, M., Sobotka, P.A., Gourine, A.V., Paton, J.F., 2012. Hypertension is critically dependent
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