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Increased total volume and dopamine β-hydroxylase immunoreactivity of carotid body in spontaneously hypertensive rats
Autonomic Neuroscience: Basic and Clinical 169 (2012) 49–55
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Increased total volume and dopamine β-hydroxylase immunoreactivity of carotid body in spontaneously hypertensive rats Kouki Kato a, b, Jun Wakai a, b, Hideki Matsuda c, Tatsumi Kusakabe d, Yoshio Yamamoto a, b,⁎ a
Laboratory of Veterinary Biochemistry and Cell Biology, Faculty of Agriculture, Iwate University, Morioka, Japan Department of Basic Veterinary Science, United Graduate School of Veterinary Science, Gifu University, Gifu, Japan Department of Otolaryngology, Yokohama City University School of Medicine, Yokohama, Japan d Laboratory for Anatomy and Physiology, Department of Sport and Medical Science, Kokushikan University, Tokyo, Japan b c
a r t i c l e
i n f o
Article history: Received 5 March 2012 Received in revised form 21 March 2012 Accepted 25 March 2012 Keywords: Spontaneously hypertensive rat Carotid body Dopamine β-hydroxylase Noradrenaline Sympathetic nervous system
a b s t r a c t Under hypertension, it has been reported that the carotid body (CB) is enlarged and noradrenaline (NA) content in CB is increased. Therefore, it is hypothesized that morphological and neurochemical changes in CB are induced in hypertensive animal models. In the present study, we examined the morphological features and dopamine β-hydroxylase (DBH) immunoreactivity in CB of spontaneously hypertensive rats (SHR/Izm) and Wistar Kyoto rats (WKY/Izm). The CB of SHR/Izm was elongated in terms of the cross section of center and was enlarged in the reconstructed images compared with that of WKY/Izm, and the total volume of CB in SHR/Izm (0.048 ± 0.004 mm 3) was significantly (p b 0.05) increased compared with the value in WKY/Izm (0.032 ± 0.006 mm3). By immunohistochemistry, immunoreactivity for tyrosine hydroxylase in CB was mainly observed in glomus cells and the immunostaining properties were similar between WKY/Izm and SHR/Izm. On the other hand, DBH immunoreactivity was mainly observed in nerve fibers around blood vessels and observed in a few glomus cells in CB of WKY/Izm. The number of glomus cells with strong DBH immunoreactivity was increased in SHR/Izm compared with that in WKY/Izm. In conclusion, the present study exhibited the enlargement of CB as three-dimensional image and revealed the enhanced immunoreactivity for DBH of glomus cells in SHR/Izm. These results suggest that the morphology of CB is affected by the effect of sympathetic nerve and that the signal transduction from CB is regulated by NA in glomus cells under hypertensive conditions. © 2012 Elsevier B.V. All rights reserved.
1. Introduction The carotid body (CB) is located bilaterally at the bifurcation of the common carotid artery and is the peripheral chemoreceptor responsible for monitoring changes in PO2, PCO2 and pH in arterial blood (Nurse, 2005; Lahiri et al., 2006; Prabhakar, 2006). The decrease in PO2 is detected by glomus cells (type I cells) within CB, which are synaptically connected with carotid sinus nerve (CSN), leading to an increase in the afferent sensory discharge to the nucleus of the solitary tract (Gonzalez et al., 1994; Lahiri et al., 2006). As a result, appropriate autonomic changes including stimulation of breathing and a raise in blood pressure are caused under environmental hypoxia (Prabhakar, 2006). In essential hypertensive patients, it is known that the CB is enlarged (Habeck, 1986). In addition to morphological changes in the CB, hyperventilation was reported in essential hypertensive patients ⁎ Corresponding author at: Laboratory of Veterinary Biochemistry and Cell Biology, Faculty of Agriculture, Iwate University, 3-18-8 Ueda, Morioka, Iwate 020-8550, Japan. Tel./fax: + 81 19 621 6273. E-mail address: [email protected] (Y. Yamamoto). 1566-0702/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.autneu.2012.03.005
at resting conditions (Trzebski et al., 1982). Furthermore, CB is innervated by postganglionic sympathetic nerve from the superior cervical ganglion (Verna et al., 1984) and an elevated level of sympathetic nerve activity was observed in the patients (Anderson et al., 1989; Grassi et al., 1998, 2000). These previous studies imply that alteration in chemoreceptor reflex under hypertensive conditions is attributable to the changes in CB, including morphological changes. Spontaneously hypertensive rat (SHR) strains are animal models of essential hypertension and are used to study the pathophysiology of essential hypertension. It has been reported that the volume of CB in SHR strains is increased compared with that of age-matched genetically comparable Wistar Kyoto rats (WKY) (Alho et al., 1984). In addition to the morphological changes, it has also been reported that dopamine (DA) content was similar but noradrenaline (NA) content was increased by 50% in the CB of the New Zealand strain of hypertensive rat compared with those of normotensive rat (Pallot and Barer, 1985). It is generally known that DA and NA are present in glomus cells (Gonzalez et al., 1994) and are inhibitory neuromodulators in CB chemotransduction via dopaminergic D2 receptor and adrenergic α2 receptor, respectively (Nurse, 2005; Lahiri et al., 2006).
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Therefore, there is the possibility that the signal transduction from CB to the nucleus of the solitary tract is regulated by the inhibitory NA in SHR strains. Considering these previous studies in essential hypertensive human patients and hypertensive rats, it is hypothesized that morphological and neurochemical changes are induced in CB and these changes modulate chemosensitivity of CB under hypertensive conditions. In order to verify this hypothesis, it is necessary to investigate the histological features and NA expression in detail in CB under hypertensive conditions. In the present study, we reexamined the morphological features in CB of SHR/Izm. Then, to estimate the effect of hypertension on the NA biosynthesis pathway in CB, we examined the immunoreactivity for dopamine β-hydroxylase (DBH; EC 1.14.17.1), the enzyme catalyzing the synthesis of NA from DA, in addition to tyrosine hydroxylase (TH; EC 1.14.16.2), the rate-limiting enzyme of catecholamine biosynthesis, in CB of SHR/Izm. We also discussed the effect of the morphological and neurochemical changes occurring within CB of hypertensive animals on CB chemosensitivity. 2. Materials and methods 2.1. Animals Sixteen-week-old male rats of SHR/Izm (body weight: 310–360 g) were used in the present study, and age-matched male rats of WKY/ Izm (body weight: 330–380 g) were also used as controls (Japan SLC, Hamamatsu, Japan). A total of six rats (a total of twelve CBs) in each strain were used in the present study. All procedures for animal handling were performed in accordance with the guidelines of the local animal ethics committee of Iwate University (accession number: 201047). 2.2. General histological analysis Each rat was anesthetized by intraperitoneal injection of pentobarbital sodium (55.5 mg/kg) and transcardially perfused through the ascending aorta with Ringer's solution (300 ml) and then with 4% paraformaldehyde in 0.1 M phosphate buffer containing 0.5% picric acid (pH 7.4; 300 ml). The bifurcation of carotid arteries was removed and further fixed with the same fixative for 2–3 h at 4 °C. Then, the tissues were rinsed in PBS (pH 7.4), soaked in 30% sucrose in PBS, and frozen with O.C.T. compound medium (Sakura Finetek, Tokyo, Japan). The tissues were serially sectioned at a thickness of 10 μm using a cryostat (CM 1900, Leica, Wetzlar, Germany) and mounted on glass slides coated with chrome alum–gelatin. Every other section was processed for hematoxylin and eosin staining to examine the morphological features in CB of SHR/Izm. After the hematoxylin and eosin staining, the sections were examined using a light microscope (BX-50, Olympus, Tokyo, Japan). The remaining semi-serial sections were used for immunohistochemistry (ABC method) for TH as described later in order to calculate the total volume of CB and to reconstruct three-dimensional images of CB. 2.3. Immunohistochemistry for TH and DBH Another series of serial cryostat sections of the CB was prepared as mentioned earlier, and the serial sections were alternately used for immunohistochemistry for TH and DBH. The semi-serial cryostat sections were rinsed with PBS, soaked in methanol containing 0.3% H2O2 at room temperature, and rinsed with PBS. To prevent non-specific binding, the sections were incubated for 30 min with non-immune donkey serum (1:50 dilution) diluted with the dilution buffer (2 mM NaH2PO4, 5 mM Na2HPO4, 0.37 M NaCl, 0.5% Triton X-100) at room temperature. Then, the sections were rinsed with PBS and incubated overnight at 4 °C with monoclonal mouse antibody against TH (1:1000 dilution; MAB318, Chemicon, Temecula, CA,
USA) or monoclonal mouse antibody against DBH (1:4000 dilution; MAB308, Chemicon). After rinsing with PBS, the sections were incubated for 30 min with a biotinylated donkey anti-mouse IgG (1:500 dilution; Jackson Immunoresearch, West Grove, PA, USA) at room temperature. Then, the sections were further rinsed with PBS and incubated for 30 min with an avidin–biotin–peroxidase complex (Vector, Burlingame, CA, USA). After rinsing with PBS, the sections were incubated with 0.02% 33′-diaminobenzidine tetrahydrochloride in Tris–HCl buffer containing 0.006% H2O2 for 5–10 min, and washed with PBS and pure water. Finally, the sections were dehydrated in a graded series of ethanol, cleared with xylene, coverslipped, and examined using a light microscope (BX-50, Olympus, Tokyo, Japan). For further investigation of immunoreactivity for TH and DBH, the semi-serial cryostat sections were processed for immunofluorescent staining. The sections were rinsed with PBS, incubated for 30 min with non-immune donkey serum (1:50 dilution), and rinsed with PBS. Then, the sections were incubated overnight at 4 °C with either monoclonal mouse antibody against TH (1:1000 dilution; MAB318, Chemicon) together with polyclonal guinea pig antibody against synaptophysin (1:1000 dilution; Syn-GP-Af300-1, Frontier Science, Sapporo, Japan) as a marker protein for glomus cells or monoclonal mouse antibody against DBH (1:4000 dilution; MAB308, Chemicon International, Temecula, CA, USA) together with polyclonal guinea pig antibody against synaptophysin (1:1000 dilution; Syn-GP-Af300-1, Frontier). Then, the sections were rinsed with PBS and incubated for 90 min with either Alexa Fluor 488-labeled donkey anti-mouse IgG (1:200; A21202; Invitrogen, Tokyo, Japan) together with Cy3-labeled donkey anti-guinea pig IgG (1:100; 706-165-148; Jackson Immunoresearch) for TH and DBH immunostained sections at room temperature. After rinsing with PBS, the coverslips were mounted onto glass slides with mounting medium and the sections were examined using an epifluorescence microscope (E600, Nikon, Tokyo, Japan). 2.4. Morphometry For calculation of the total volume of the CB, TH immunostained semi-serial sections by the ABC method (see Immunohistochemistry for TH and DBH) were used because TH immunoreactivity was uniformly observed in glomus cells in the present study. In each TH immunostained section, the cross-sectional area was calculated using the ImageJ analysis program (National Institutes of Health, Bethesda, MD, USA; http://rsb.info.nih.gov/ij/). Then, the sum of cross-sectional area was multiplied by the thickness of the two sections (20 μm), and the value was considered as the total volume of the organ. In addition, three-dimensional images of CB were reconstructed on a personal computer using Delta Viewer software (http://delta.math.sci.osaka-u. ac.jp/DeltaViewer/) from the TH immunostained semi-serial sections. 2.5. Gray scale for DBH immunoreactivity To analyze the immunoreactivity for DBH in glomus cells, gray scale intensity (range 0–255: black = 0, white = 255) of DBH immunofluorescence was measured using the ImageJ analysis program (National Institutes of Health, Bethesda, MD, USA; http:// rsb.info.nih.gov/ij/). Cytoplasm of the glomus cells was identified on micrographs of the section stained for synaptophysin. The DBH immunostained images were converted into gray scale images (256 shades of gray), and then gray scale intensity for DBH immunofluorescence in cytoplasm of glomus cells was measured. At least 700 glomus cells were randomly measured for each rat. Additionally, to analyze the distribution of DBH immunoreactive nerve fibers within CB, the area occupied by DBH immunoreactive nerve fibers was measured. Micrographs of the DBH immunostained section were converted to binary images. Then, DBH immunoreactivity in glomus
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cells was eliminated and the ratio of the area occupied by DBH immunoreactive nerve fibers per unit area was measured. 2.6. Statistical analysis The measured values are given as mean ± S.D. Three CBs from SHR/Izm and four CBs from WKY/Izm were used for statistical analysis for total volume of CB, and total volume of CB between the two strains was analyzed by Student's t-test. The difference in the distribution of glomus cells based on gray scale intensity for DBH was analyzed by Kolmogorov–Smirnov tests between the SHR/Izm group and the WKY/Izm group, and all glomus cells measured in three CBs from SHR/Izm and all the cells measured in the same number of CBs from WKY/Izm were contained in the SHR/Izm group and the WKY/Izm group, respectively. Three CBs from SHR/Izm and the same number of CBs from WKY/Izm were used for statistical analysis for the ratio of area occupied by DBH immunoreactive nerve fibers, and Student's t-test was used for statistical analysis between the two strains. Differences with a value of p b 0.05 were considered to be statistically significant.
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Morphometrical analysis revealed that the total volume of CB was 0.032 ± 0.006 mm 3 in WKY/Izm and was 0.048 ± 0.004 mm 3 in SHR/ Izm, and the value of SHR/Izm significantly (p b 0.05) increased compared with that of WKY/Izm (Fig. 2). Thus, the present morphological study reconfirmed the enlargement of the CB in SHR/ Izm in comparison with that in WKY/Izm.
3.2. TH and DBH immunoreactivity The results of immunohistochemistry for TH and DBH in the CB of WKY/Izm and SHR/Izm are shown in Figs. 3 and 4. No differences in
3. Results 3.1. Morphological characteristics In WKY/Izm, the cross section of center of the CB was ellipsoidal in hematoxylin and eosin stained sections (Fig. 1A) and the shape of the organ was oval in the reconstructed images (Fig. 1C). In SHR/Izm, the cross section of center of the CB was elongated in the hematoxylin and eosin sections (Fig. 1B) and the size of the organ was enlarged in the reconstructed images compared with that in WKY/Izm (Fig. 1D). Numerous glomus cells were observed in CB of SHR/Izm as in the case of WKY/Izm (Fig. 1F). Numerous blood vessels were also observed in CB of SHR/Izm, and there were no distinct differences in the diameter of blood vessels between the two rat strains (Fig. 1E, F).
Fig. 2. Total volume of carotid body (CB) in WKY/Izm and SHR/Izm. Total volume of CB in SHR/Izm (0.048 ± 0.004 mm3) was significantly increased compared with the value in WKY/Izm (0.032 ± 0.006 mm3). Means ± S.D. values are shown (*: p b 0.05, in comparison to WKY/Izm).
Fig. 1. Hematoxylin and eosin-stained sections of carotid body (CB) and reconstructed images of CB. In CB of WKY/Izm, the cross section of center was ellipsoidal (A) and the shape was oval (C). In SHR/Izm, the CB was elongated in terms of the cross section of center (B) and the organ was enlarged compared with that of WKY/Izm (D). Numerous glomus cells and blood vessels were observed in the CB of SHR/Izm, and there were no distinct differences in the diameter of blood vessels between the two rat strains (E, F).
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Fig. 3. Immunoreactivity for tyrosine hydroxylase (TH) and dopamine β-hydroxylase (DBH) in carotid body (CB) of WKY/Izm and SHR/Izm. TH immunoreactivity was mainly observed in glomus cells in CB of both WKY/Izm (A) and SHR/Izm (B). In the CB of WKY/Izm, DBH immunoreactivity was mainly observed in varicosity of nerve fibers and there were few DBH immunoreactive glomus cells (C). In CB of SHR/Izm, DBH immunoreactivity was observed in varicosity of nerve fibers and also observed in glomus cells (D). DBH immunoreactive glomus cells appeared to be increased in number in SHR/Izm compared with that in WKY/Izm (D, arrows).
Fig. 4. Immunofluorescence images for tyrosine hydroxylase (TH) and dopamine β-hydroxylase (DBH) in carotid body (CB) of WKY/Izm and SHR/Izm. Green fluorescence indicates immunoreactivity for TH (A, B) or DBH (C, D) and red fluorescence indicates immunoreactivity for synaptophysin, as a marker for glomus cells. The TH immunoreactivity was observed in the cytoplasm of glomus cells in the CB of WKY/Izm (A) and SHR/Izm (B). The fluorescence intensity for TH in the glomus cells did not appear to be different between WKY/Izm and SHR/Izm. The DBH immunoreactivity was mainly observed in varicosity of nerve fibers associated with blood vessels in WKY/Izm (C). The DBH immunoreactivity was also observed in the cytoplasm of glomus cells but there were few DBH immunoreactive glomus cells in the CB of WKY/Izm (C, arrowhead). In the CB of SHR/Izm, the DBH immunoreactivity was observed in the cytoplasm of glomus cells in addition to varicosity of nerve fibers associated with blood vessels (D). In SHR/Izm, the DBH immunoreactive glomus cells existed in isolation (D, arrowheads) or formed clusters (D, arrow). The DBH immunoreactive glomus cells in SHR/Izm showed more intense DBH immunofluorescence than the cells in WKY/Izm (C, D).
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the staining properties were identified between the ABC method and the immunofluorescent staining method. TH immunoreactivity was mainly observed in glomus cells in CB of WKY/Izm (Fig. 3A). In glomus cells of WKY/Izm, TH immunoreactivity was observed in the cytoplasm (Fig. 4A). A few nerve fibers were also immunoreactive for TH in the CB of WKY/Izm (figure not shown). In the CB of SHR/Izm, TH immunoreactivity was mainly observed in glomus cells (Fig. 3B), and TH immunoreactivity was observed in the cytoplasm of glomus cells (Fig. 4B). There were no apparent differences in the immunofluorescence intensity for TH in glomus cells between SHR/Izm and WKY/Izm (Fig. 4A, B). In the CB of SHR/ Izm, a few nerve fibers were also immunoreactive for TH (figure not shown). In the CB of WKY/Izm, DBH immunoreactivity was mainly observed in varicosity of nerve fibers (Fig. 3C) and the DBH immunoreactive nerve fibers were associated with blood vessels (Fig. 4C). In the CB of
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WKY/Izm, although DBH immunoreactivity was also observed in glomus cells, there were few DBH immunoreactive glomus cells (Fig. 4C, arrowhead). In the CB of SHR/Izm, DBH immunoreactivity was observed in varicosity of nerve fibers around blood vessels (Figs. 3D and 4D). DBH immunoreactivity was also observed in glomus cells in the CB of SHR/Izm and the DBH immunoreactive glomus cells appeared to be increased in number compared with those in WKY/Izm (Fig. 3D, arrows). In glomus cells, DBH immunoreactivity was observed in the cytoplasm, and the DBH immunoreactive glomus cells existed in isolation (Fig. 4D, arrowheads) or formed clusters (Fig. 4D, arrow). The DBH immunoreactive glomus cells in SHR/Izm showed more intense DBH immunofluorescence than the cells in WKY/Izm (Fig. 4C, D). Immunoreactivity for synaptophysin was observed in the cytoplasm of glomus cells and the staining properties were similar between WKY/ Izm and SHR/Izm (Fig. 4). 3.3. Image analysis of DBH immunoreactivity
Fig. 5. Scatter graph of glomus cells based on immunoreactive intensity for dopamine β-hydroxylase (DBH) in WKY/Izm and SHR/Izm. The scatter graph shows that the number of glomus cells with strong DBH immunoreactivity was increased in SHR/Izm compared with that in WKY/Izm.
The scatter graph of glomus cells based on gray scale intensity for DBH immunoreactivity showed that the number of glomus cells with strong DBH immunoreactivity was increased in SHR/Izm compared with that in WKY/Izm (Fig. 5). The histogram of gray scale intensity for DBH immunoreactivity showed that the ratio of DBH negative glomus cells (gray scale intensity is 0) was 44% and the ratio of DBH nearly negative glomus cells (gray scale intensity is 10 or less) was 54% in WKY/Izm (Fig. 6). In SHR/Izm, the ratio of DBH negative glomus cells was 53% and the ratio of DBH nearly negative glomus cells was 37% and the total ratio of DBH negative glomus cells and DBH nearly negative glomus cells was decreased in SHR/Izm (90%) compared with WKY/Izm (98%) (Fig. 6). Moreover, the ratio of glomus cells with intense DBH immunoreactivity (gray scale intensity is greater than 10) was increased in SHR/Izm compared with that in WKY/Izm (Fig. 6). Additionally, by Kolmogorov–Smirnov tests, it was revealed that the distribution of glomus cells based on gray scale intensity for DBH was significantly (p b 0.05) different between WKY/ Izm and SHR/Izm, indicating that DBH immunoreactivity of glomus cells was statistically increased in SHR/Izm compared with that in WKY/Izm. The ratio of the area occupied by DBH immunoreactive nerve fibers per unit area was 6.9 ± 1.1% in WKY/Izm and 5.5 ± 0.9% in SHR/
Fig. 6. Histogram representing the ratio of glomus cells based on immunoreactive intensity for dopamine β-hydroxylase (DBH) in WKY/Izm (left panel) and SHR/Izm (right panel). The x-axis and y-axis represent gray scale intensity for DBH and ratio of glomus cells, respectively. The ratio of glomus cells with intense DBH immunoreactivity (gray scale intensity is 10 or more) was increased in SHR/Izm compared with that in WKY/Izm. The distribution was significantly (p b 0.05) different between WKY/Izm and SHR/Izm analyzed by Kolmogorov–Smirnov tests.
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Fig. 7. The ratio of area occupied by dopamine β-hydroxylase (DBH) immunoreactive nerve fibers per unit area. The ratio of area occupied by DBH immunoreactive nerve fibers per unit area was 6.9 ± 1.1% in WKY/Izm and 5.5 ± 0.9% in SHR/Izm, and there was no significant difference between WKY/Izm and SHR/Izm. Means± S.D. values are shown.
Izm (Fig. 7). There was no significant difference in the value between WKY/Izm and SHR/Izm. 4. Discussion In the present study, it was revealed that CB of SHR/Izm is enlarged in size as shown in the three-dimensional image. Moreover, apparent vasodilatation could not be observed in the CB of SHR/Izm. These results are supported by the report that the diameter of blood vessels within CB in SHR strains is similar to that in WKY strains (Takahashi et al., 2011). It is generally accepted that chronic hypoxia (10% O2) causes the enlargement of the CB with vasodilatation (Wang and Bisgard, 2002; Kusakabe et al., 2005). Interestingly, it was reported that chronic hypercapnic hypoxia (10% O2, 6–7% CO2) caused the enlargement of the rat CB without vasodilatation within the CB (Kusakabe et al., 2005). Thus, between SHR/Izm and hypercapnic hypoxic rats, there are similarities in the volume and the diameter of blood vessels in the CB. It was reported that renal sympathetic nerve activity is increased when rats are exposed to hypercapnic hypoxia (Hirakawa et al., 1997). Given this finding, it is suggested that sympathetic nerve input from superior cervical ganglion is increased, thereby inducing vasoconstriction within the CB of hypercapnic hypoxic rats. Therefore, in hypercapnic hypoxic rats, the enlargement of the CB would be caused by the effect of hypoxia and vasodilatation would be inhibited by the effect of increased sympathetic nerve activity. On the other hand, previous electrophysiological studies showed that sympathetic nerve activity was also increased in SHR strains compared with that in WKY strains (Judy and Farrell, 1979; Lundin et al., 1984; Sugimura et al., 2008). Given this finding, it is suggested that increased sympathetic activity causes vasoconstriction within the CB of SHR/Izm as in the case of the hypercapnic hypoxic rats, leading to reduction in blood supply to the CB. As a result of the decreased blood supply, the CB of SHR/Izm would be rendered regional hypoxia and become enlarged without vasodilatation. Therefore, the morphology of the CB would be similar between SHR/Izm and hypercapnic hypoxic rats due to the effect of hypoxia and the increased sympathetic nerve activity. It has been reported by immunohistochemistry that chronic hypoxia enhances the immunoreactivity for TH in the CB (Wang et al., 1998; Wang and Bisgard, 2002; Hui et al., 2003). Although it is suggested that the CB of SHR/Izm is rendered regional hypoxia as mentioned earlier, immunoreactivity for TH in CB was similar
between SHR/Izm and WKY/Izm in the present study. Therefore, it might be that the regional hypoxic condition in the CB under hypertension is slightly different from the systemic hypoxic condition under environmental hypoxia. On the other hand, the present immunohistochemical study showed that DBH immunoreactivity is enhanced in glomus cells of SHR/Izm. Considering the previous report that NA content was increased in the CB of the New Zealand strain of hypertensive rat (Pallot and Barer, 1985), it is suggested that NA biosynthesis is facilitated in glomus cells of CB in SHR/Izm. It has been suggested that NA in glomus cells, along with DA, is inhibitory to CB activity and is neuromodulator in signal transduction from CB to the nucleus of the solitary tract (Kou et al., 1991; Almaraz et al., 1997; Overholt and Prabhakar, 1999). Furthermore, it has been reported that CSN discharge of SHR strains under normoxic conditions is not different from that of normotensive rats (Fukuda et al., 1987). Therefore, in SHR/Izm, it is suggested that DBH is increased in glomus cells in order to synthesize NA, and furthermore to maintain the CSN discharge at a normal level by the inhibitory NA. Moreover, it is reported that minute ventilation under air-breathing conditions is not different between SHR strains and WKY rat strains (Grisk et al., 1996). Thus, in SHR/Izm, normal respiration under resting conditions would be attributable to the normal CSN activity modulated by the effect of NA in addition to DA. It is reported that sympathetic nerve activity is increased in the SHR strain compared with that in the WKY strain (Judy and Farrell, 1979; Lundin et al., 1984; Sugimura et al., 2008) and NA content is increased in the CB of the New Zealand strain of hypertensive rat compared with that in normotensive rat (Pallot and Barer, 1985). These findings suggest that NA release from sympathetic nerve fibers in addition to glomus cells within the CB is increased in SHR/Izm; therefore, it was expected that DBH immunoreactive nerve fiber within the CB is increased in SHR/Izm compared with that in WKY/ Izm. In contrast, DBH immunoreactive nerve fibers were at similar levels between the two rat strains in the present study. Because it is known that DBH is released from synaptic vesicles together with NA (Smith et al., 1970; Weinshilboum et al., 1971), it is suggested that DBH immunoreactive nerve fibers are not increased in SHR/Izm. In conclusion, the present study exhibited the enlargement of CB as three-dimensional image and revealed the enhanced immunoreactivity for DBH of glomus cells in SHR/Izm. These results suggest that the morphology of CB is affected by the effect of sympathetic nerve and that the signal transduction from CB is regulated by NA in glomus cells under hypertensive conditions. Acknowledgments The authors thank Associate Professor Misuzu Yamaguchi-Yamada (Laboratory of Veterinary Biochemistry and Cell Biology, Faculty of Agriculture, Iwate University) for valuable comments and suggestions. This study was supported by grants-in aid from the Japan Society for the Promotion of Science to TK (21500636), HM (21592202) and YY (22580330). References Alho, H., Partanen, M., Koistinaho, J., Vaalasti, A., Hervonen, A., 1984. Histochemically demonstrable catecholamines in sympathetic ganglia and carotid body of spontaneously hypertensive and normotensive rats. Histochemistry 80, 457–462. Almaraz, L., Pérez-García, M.T., Gómez-Nino, A., González, C., 1997. Mechanisms of α2adrenoceptor-mediated inhibition in rabbit carotid body. Am. J. Physiol. 272, 628–637. Anderson, E.A., Sinkey, C.A., Lawton, W.J., Mark, A.L., 1989. Elevated sympathetic nerve activity in borderline hypertensive humans. Evidence from direct intraneural recordings. Hypertension 14, 177–183. Fukuda, Y., Sato, A., Trzebski, A., 1987. Carotid chemoreceptor discharge responses to hypoxia and hypercapnia in normotensive and spontaneously hypertensive rats. J. Auton. Nerv. Syst. 19, 1–11. Gonzalez, C., Almaraz, L., Obeso, A., Rigual, R., 1994. Carotid body chemoreceptors: from natural stimuli to sensory discharges. Physiol. Rev. 74, 829–898.
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Autonomic Neuroscience: Basic and Clinical journal homepage: www.elsevier.com/locate/autneu
Increased total volume and dopamine β-hydroxylase immunoreactivity of carotid body in spontaneously hypertensive rats Kouki Kato a, b, Jun Wakai a, b, Hideki Matsuda c, Tatsumi Kusakabe d, Yoshio Yamamoto a, b,⁎ a
Laboratory of Veterinary Biochemistry and Cell Biology, Faculty of Agriculture, Iwate University, Morioka, Japan Department of Basic Veterinary Science, United Graduate School of Veterinary Science, Gifu University, Gifu, Japan Department of Otolaryngology, Yokohama City University School of Medicine, Yokohama, Japan d Laboratory for Anatomy and Physiology, Department of Sport and Medical Science, Kokushikan University, Tokyo, Japan b c
a r t i c l e
i n f o
Article history: Received 5 March 2012 Received in revised form 21 March 2012 Accepted 25 March 2012 Keywords: Spontaneously hypertensive rat Carotid body Dopamine β-hydroxylase Noradrenaline Sympathetic nervous system
a b s t r a c t Under hypertension, it has been reported that the carotid body (CB) is enlarged and noradrenaline (NA) content in CB is increased. Therefore, it is hypothesized that morphological and neurochemical changes in CB are induced in hypertensive animal models. In the present study, we examined the morphological features and dopamine β-hydroxylase (DBH) immunoreactivity in CB of spontaneously hypertensive rats (SHR/Izm) and Wistar Kyoto rats (WKY/Izm). The CB of SHR/Izm was elongated in terms of the cross section of center and was enlarged in the reconstructed images compared with that of WKY/Izm, and the total volume of CB in SHR/Izm (0.048 ± 0.004 mm 3) was significantly (p b 0.05) increased compared with the value in WKY/Izm (0.032 ± 0.006 mm3). By immunohistochemistry, immunoreactivity for tyrosine hydroxylase in CB was mainly observed in glomus cells and the immunostaining properties were similar between WKY/Izm and SHR/Izm. On the other hand, DBH immunoreactivity was mainly observed in nerve fibers around blood vessels and observed in a few glomus cells in CB of WKY/Izm. The number of glomus cells with strong DBH immunoreactivity was increased in SHR/Izm compared with that in WKY/Izm. In conclusion, the present study exhibited the enlargement of CB as three-dimensional image and revealed the enhanced immunoreactivity for DBH of glomus cells in SHR/Izm. These results suggest that the morphology of CB is affected by the effect of sympathetic nerve and that the signal transduction from CB is regulated by NA in glomus cells under hypertensive conditions. © 2012 Elsevier B.V. All rights reserved.
1. Introduction The carotid body (CB) is located bilaterally at the bifurcation of the common carotid artery and is the peripheral chemoreceptor responsible for monitoring changes in PO2, PCO2 and pH in arterial blood (Nurse, 2005; Lahiri et al., 2006; Prabhakar, 2006). The decrease in PO2 is detected by glomus cells (type I cells) within CB, which are synaptically connected with carotid sinus nerve (CSN), leading to an increase in the afferent sensory discharge to the nucleus of the solitary tract (Gonzalez et al., 1994; Lahiri et al., 2006). As a result, appropriate autonomic changes including stimulation of breathing and a raise in blood pressure are caused under environmental hypoxia (Prabhakar, 2006). In essential hypertensive patients, it is known that the CB is enlarged (Habeck, 1986). In addition to morphological changes in the CB, hyperventilation was reported in essential hypertensive patients ⁎ Corresponding author at: Laboratory of Veterinary Biochemistry and Cell Biology, Faculty of Agriculture, Iwate University, 3-18-8 Ueda, Morioka, Iwate 020-8550, Japan. Tel./fax: + 81 19 621 6273. E-mail address: [email protected] (Y. Yamamoto). 1566-0702/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.autneu.2012.03.005
at resting conditions (Trzebski et al., 1982). Furthermore, CB is innervated by postganglionic sympathetic nerve from the superior cervical ganglion (Verna et al., 1984) and an elevated level of sympathetic nerve activity was observed in the patients (Anderson et al., 1989; Grassi et al., 1998, 2000). These previous studies imply that alteration in chemoreceptor reflex under hypertensive conditions is attributable to the changes in CB, including morphological changes. Spontaneously hypertensive rat (SHR) strains are animal models of essential hypertension and are used to study the pathophysiology of essential hypertension. It has been reported that the volume of CB in SHR strains is increased compared with that of age-matched genetically comparable Wistar Kyoto rats (WKY) (Alho et al., 1984). In addition to the morphological changes, it has also been reported that dopamine (DA) content was similar but noradrenaline (NA) content was increased by 50% in the CB of the New Zealand strain of hypertensive rat compared with those of normotensive rat (Pallot and Barer, 1985). It is generally known that DA and NA are present in glomus cells (Gonzalez et al., 1994) and are inhibitory neuromodulators in CB chemotransduction via dopaminergic D2 receptor and adrenergic α2 receptor, respectively (Nurse, 2005; Lahiri et al., 2006).
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Therefore, there is the possibility that the signal transduction from CB to the nucleus of the solitary tract is regulated by the inhibitory NA in SHR strains. Considering these previous studies in essential hypertensive human patients and hypertensive rats, it is hypothesized that morphological and neurochemical changes are induced in CB and these changes modulate chemosensitivity of CB under hypertensive conditions. In order to verify this hypothesis, it is necessary to investigate the histological features and NA expression in detail in CB under hypertensive conditions. In the present study, we reexamined the morphological features in CB of SHR/Izm. Then, to estimate the effect of hypertension on the NA biosynthesis pathway in CB, we examined the immunoreactivity for dopamine β-hydroxylase (DBH; EC 1.14.17.1), the enzyme catalyzing the synthesis of NA from DA, in addition to tyrosine hydroxylase (TH; EC 1.14.16.2), the rate-limiting enzyme of catecholamine biosynthesis, in CB of SHR/Izm. We also discussed the effect of the morphological and neurochemical changes occurring within CB of hypertensive animals on CB chemosensitivity. 2. Materials and methods 2.1. Animals Sixteen-week-old male rats of SHR/Izm (body weight: 310–360 g) were used in the present study, and age-matched male rats of WKY/ Izm (body weight: 330–380 g) were also used as controls (Japan SLC, Hamamatsu, Japan). A total of six rats (a total of twelve CBs) in each strain were used in the present study. All procedures for animal handling were performed in accordance with the guidelines of the local animal ethics committee of Iwate University (accession number: 201047). 2.2. General histological analysis Each rat was anesthetized by intraperitoneal injection of pentobarbital sodium (55.5 mg/kg) and transcardially perfused through the ascending aorta with Ringer's solution (300 ml) and then with 4% paraformaldehyde in 0.1 M phosphate buffer containing 0.5% picric acid (pH 7.4; 300 ml). The bifurcation of carotid arteries was removed and further fixed with the same fixative for 2–3 h at 4 °C. Then, the tissues were rinsed in PBS (pH 7.4), soaked in 30% sucrose in PBS, and frozen with O.C.T. compound medium (Sakura Finetek, Tokyo, Japan). The tissues were serially sectioned at a thickness of 10 μm using a cryostat (CM 1900, Leica, Wetzlar, Germany) and mounted on glass slides coated with chrome alum–gelatin. Every other section was processed for hematoxylin and eosin staining to examine the morphological features in CB of SHR/Izm. After the hematoxylin and eosin staining, the sections were examined using a light microscope (BX-50, Olympus, Tokyo, Japan). The remaining semi-serial sections were used for immunohistochemistry (ABC method) for TH as described later in order to calculate the total volume of CB and to reconstruct three-dimensional images of CB. 2.3. Immunohistochemistry for TH and DBH Another series of serial cryostat sections of the CB was prepared as mentioned earlier, and the serial sections were alternately used for immunohistochemistry for TH and DBH. The semi-serial cryostat sections were rinsed with PBS, soaked in methanol containing 0.3% H2O2 at room temperature, and rinsed with PBS. To prevent non-specific binding, the sections were incubated for 30 min with non-immune donkey serum (1:50 dilution) diluted with the dilution buffer (2 mM NaH2PO4, 5 mM Na2HPO4, 0.37 M NaCl, 0.5% Triton X-100) at room temperature. Then, the sections were rinsed with PBS and incubated overnight at 4 °C with monoclonal mouse antibody against TH (1:1000 dilution; MAB318, Chemicon, Temecula, CA,
USA) or monoclonal mouse antibody against DBH (1:4000 dilution; MAB308, Chemicon). After rinsing with PBS, the sections were incubated for 30 min with a biotinylated donkey anti-mouse IgG (1:500 dilution; Jackson Immunoresearch, West Grove, PA, USA) at room temperature. Then, the sections were further rinsed with PBS and incubated for 30 min with an avidin–biotin–peroxidase complex (Vector, Burlingame, CA, USA). After rinsing with PBS, the sections were incubated with 0.02% 33′-diaminobenzidine tetrahydrochloride in Tris–HCl buffer containing 0.006% H2O2 for 5–10 min, and washed with PBS and pure water. Finally, the sections were dehydrated in a graded series of ethanol, cleared with xylene, coverslipped, and examined using a light microscope (BX-50, Olympus, Tokyo, Japan). For further investigation of immunoreactivity for TH and DBH, the semi-serial cryostat sections were processed for immunofluorescent staining. The sections were rinsed with PBS, incubated for 30 min with non-immune donkey serum (1:50 dilution), and rinsed with PBS. Then, the sections were incubated overnight at 4 °C with either monoclonal mouse antibody against TH (1:1000 dilution; MAB318, Chemicon) together with polyclonal guinea pig antibody against synaptophysin (1:1000 dilution; Syn-GP-Af300-1, Frontier Science, Sapporo, Japan) as a marker protein for glomus cells or monoclonal mouse antibody against DBH (1:4000 dilution; MAB308, Chemicon International, Temecula, CA, USA) together with polyclonal guinea pig antibody against synaptophysin (1:1000 dilution; Syn-GP-Af300-1, Frontier). Then, the sections were rinsed with PBS and incubated for 90 min with either Alexa Fluor 488-labeled donkey anti-mouse IgG (1:200; A21202; Invitrogen, Tokyo, Japan) together with Cy3-labeled donkey anti-guinea pig IgG (1:100; 706-165-148; Jackson Immunoresearch) for TH and DBH immunostained sections at room temperature. After rinsing with PBS, the coverslips were mounted onto glass slides with mounting medium and the sections were examined using an epifluorescence microscope (E600, Nikon, Tokyo, Japan). 2.4. Morphometry For calculation of the total volume of the CB, TH immunostained semi-serial sections by the ABC method (see Immunohistochemistry for TH and DBH) were used because TH immunoreactivity was uniformly observed in glomus cells in the present study. In each TH immunostained section, the cross-sectional area was calculated using the ImageJ analysis program (National Institutes of Health, Bethesda, MD, USA; http://rsb.info.nih.gov/ij/). Then, the sum of cross-sectional area was multiplied by the thickness of the two sections (20 μm), and the value was considered as the total volume of the organ. In addition, three-dimensional images of CB were reconstructed on a personal computer using Delta Viewer software (http://delta.math.sci.osaka-u. ac.jp/DeltaViewer/) from the TH immunostained semi-serial sections. 2.5. Gray scale for DBH immunoreactivity To analyze the immunoreactivity for DBH in glomus cells, gray scale intensity (range 0–255: black = 0, white = 255) of DBH immunofluorescence was measured using the ImageJ analysis program (National Institutes of Health, Bethesda, MD, USA; http:// rsb.info.nih.gov/ij/). Cytoplasm of the glomus cells was identified on micrographs of the section stained for synaptophysin. The DBH immunostained images were converted into gray scale images (256 shades of gray), and then gray scale intensity for DBH immunofluorescence in cytoplasm of glomus cells was measured. At least 700 glomus cells were randomly measured for each rat. Additionally, to analyze the distribution of DBH immunoreactive nerve fibers within CB, the area occupied by DBH immunoreactive nerve fibers was measured. Micrographs of the DBH immunostained section were converted to binary images. Then, DBH immunoreactivity in glomus
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cells was eliminated and the ratio of the area occupied by DBH immunoreactive nerve fibers per unit area was measured. 2.6. Statistical analysis The measured values are given as mean ± S.D. Three CBs from SHR/Izm and four CBs from WKY/Izm were used for statistical analysis for total volume of CB, and total volume of CB between the two strains was analyzed by Student's t-test. The difference in the distribution of glomus cells based on gray scale intensity for DBH was analyzed by Kolmogorov–Smirnov tests between the SHR/Izm group and the WKY/Izm group, and all glomus cells measured in three CBs from SHR/Izm and all the cells measured in the same number of CBs from WKY/Izm were contained in the SHR/Izm group and the WKY/Izm group, respectively. Three CBs from SHR/Izm and the same number of CBs from WKY/Izm were used for statistical analysis for the ratio of area occupied by DBH immunoreactive nerve fibers, and Student's t-test was used for statistical analysis between the two strains. Differences with a value of p b 0.05 were considered to be statistically significant.
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Morphometrical analysis revealed that the total volume of CB was 0.032 ± 0.006 mm 3 in WKY/Izm and was 0.048 ± 0.004 mm 3 in SHR/ Izm, and the value of SHR/Izm significantly (p b 0.05) increased compared with that of WKY/Izm (Fig. 2). Thus, the present morphological study reconfirmed the enlargement of the CB in SHR/ Izm in comparison with that in WKY/Izm.
3.2. TH and DBH immunoreactivity The results of immunohistochemistry for TH and DBH in the CB of WKY/Izm and SHR/Izm are shown in Figs. 3 and 4. No differences in
3. Results 3.1. Morphological characteristics In WKY/Izm, the cross section of center of the CB was ellipsoidal in hematoxylin and eosin stained sections (Fig. 1A) and the shape of the organ was oval in the reconstructed images (Fig. 1C). In SHR/Izm, the cross section of center of the CB was elongated in the hematoxylin and eosin sections (Fig. 1B) and the size of the organ was enlarged in the reconstructed images compared with that in WKY/Izm (Fig. 1D). Numerous glomus cells were observed in CB of SHR/Izm as in the case of WKY/Izm (Fig. 1F). Numerous blood vessels were also observed in CB of SHR/Izm, and there were no distinct differences in the diameter of blood vessels between the two rat strains (Fig. 1E, F).
Fig. 2. Total volume of carotid body (CB) in WKY/Izm and SHR/Izm. Total volume of CB in SHR/Izm (0.048 ± 0.004 mm3) was significantly increased compared with the value in WKY/Izm (0.032 ± 0.006 mm3). Means ± S.D. values are shown (*: p b 0.05, in comparison to WKY/Izm).
Fig. 1. Hematoxylin and eosin-stained sections of carotid body (CB) and reconstructed images of CB. In CB of WKY/Izm, the cross section of center was ellipsoidal (A) and the shape was oval (C). In SHR/Izm, the CB was elongated in terms of the cross section of center (B) and the organ was enlarged compared with that of WKY/Izm (D). Numerous glomus cells and blood vessels were observed in the CB of SHR/Izm, and there were no distinct differences in the diameter of blood vessels between the two rat strains (E, F).
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Fig. 3. Immunoreactivity for tyrosine hydroxylase (TH) and dopamine β-hydroxylase (DBH) in carotid body (CB) of WKY/Izm and SHR/Izm. TH immunoreactivity was mainly observed in glomus cells in CB of both WKY/Izm (A) and SHR/Izm (B). In the CB of WKY/Izm, DBH immunoreactivity was mainly observed in varicosity of nerve fibers and there were few DBH immunoreactive glomus cells (C). In CB of SHR/Izm, DBH immunoreactivity was observed in varicosity of nerve fibers and also observed in glomus cells (D). DBH immunoreactive glomus cells appeared to be increased in number in SHR/Izm compared with that in WKY/Izm (D, arrows).
Fig. 4. Immunofluorescence images for tyrosine hydroxylase (TH) and dopamine β-hydroxylase (DBH) in carotid body (CB) of WKY/Izm and SHR/Izm. Green fluorescence indicates immunoreactivity for TH (A, B) or DBH (C, D) and red fluorescence indicates immunoreactivity for synaptophysin, as a marker for glomus cells. The TH immunoreactivity was observed in the cytoplasm of glomus cells in the CB of WKY/Izm (A) and SHR/Izm (B). The fluorescence intensity for TH in the glomus cells did not appear to be different between WKY/Izm and SHR/Izm. The DBH immunoreactivity was mainly observed in varicosity of nerve fibers associated with blood vessels in WKY/Izm (C). The DBH immunoreactivity was also observed in the cytoplasm of glomus cells but there were few DBH immunoreactive glomus cells in the CB of WKY/Izm (C, arrowhead). In the CB of SHR/Izm, the DBH immunoreactivity was observed in the cytoplasm of glomus cells in addition to varicosity of nerve fibers associated with blood vessels (D). In SHR/Izm, the DBH immunoreactive glomus cells existed in isolation (D, arrowheads) or formed clusters (D, arrow). The DBH immunoreactive glomus cells in SHR/Izm showed more intense DBH immunofluorescence than the cells in WKY/Izm (C, D).
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the staining properties were identified between the ABC method and the immunofluorescent staining method. TH immunoreactivity was mainly observed in glomus cells in CB of WKY/Izm (Fig. 3A). In glomus cells of WKY/Izm, TH immunoreactivity was observed in the cytoplasm (Fig. 4A). A few nerve fibers were also immunoreactive for TH in the CB of WKY/Izm (figure not shown). In the CB of SHR/Izm, TH immunoreactivity was mainly observed in glomus cells (Fig. 3B), and TH immunoreactivity was observed in the cytoplasm of glomus cells (Fig. 4B). There were no apparent differences in the immunofluorescence intensity for TH in glomus cells between SHR/Izm and WKY/Izm (Fig. 4A, B). In the CB of SHR/ Izm, a few nerve fibers were also immunoreactive for TH (figure not shown). In the CB of WKY/Izm, DBH immunoreactivity was mainly observed in varicosity of nerve fibers (Fig. 3C) and the DBH immunoreactive nerve fibers were associated with blood vessels (Fig. 4C). In the CB of
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WKY/Izm, although DBH immunoreactivity was also observed in glomus cells, there were few DBH immunoreactive glomus cells (Fig. 4C, arrowhead). In the CB of SHR/Izm, DBH immunoreactivity was observed in varicosity of nerve fibers around blood vessels (Figs. 3D and 4D). DBH immunoreactivity was also observed in glomus cells in the CB of SHR/Izm and the DBH immunoreactive glomus cells appeared to be increased in number compared with those in WKY/Izm (Fig. 3D, arrows). In glomus cells, DBH immunoreactivity was observed in the cytoplasm, and the DBH immunoreactive glomus cells existed in isolation (Fig. 4D, arrowheads) or formed clusters (Fig. 4D, arrow). The DBH immunoreactive glomus cells in SHR/Izm showed more intense DBH immunofluorescence than the cells in WKY/Izm (Fig. 4C, D). Immunoreactivity for synaptophysin was observed in the cytoplasm of glomus cells and the staining properties were similar between WKY/ Izm and SHR/Izm (Fig. 4). 3.3. Image analysis of DBH immunoreactivity
Fig. 5. Scatter graph of glomus cells based on immunoreactive intensity for dopamine β-hydroxylase (DBH) in WKY/Izm and SHR/Izm. The scatter graph shows that the number of glomus cells with strong DBH immunoreactivity was increased in SHR/Izm compared with that in WKY/Izm.
The scatter graph of glomus cells based on gray scale intensity for DBH immunoreactivity showed that the number of glomus cells with strong DBH immunoreactivity was increased in SHR/Izm compared with that in WKY/Izm (Fig. 5). The histogram of gray scale intensity for DBH immunoreactivity showed that the ratio of DBH negative glomus cells (gray scale intensity is 0) was 44% and the ratio of DBH nearly negative glomus cells (gray scale intensity is 10 or less) was 54% in WKY/Izm (Fig. 6). In SHR/Izm, the ratio of DBH negative glomus cells was 53% and the ratio of DBH nearly negative glomus cells was 37% and the total ratio of DBH negative glomus cells and DBH nearly negative glomus cells was decreased in SHR/Izm (90%) compared with WKY/Izm (98%) (Fig. 6). Moreover, the ratio of glomus cells with intense DBH immunoreactivity (gray scale intensity is greater than 10) was increased in SHR/Izm compared with that in WKY/Izm (Fig. 6). Additionally, by Kolmogorov–Smirnov tests, it was revealed that the distribution of glomus cells based on gray scale intensity for DBH was significantly (p b 0.05) different between WKY/ Izm and SHR/Izm, indicating that DBH immunoreactivity of glomus cells was statistically increased in SHR/Izm compared with that in WKY/Izm. The ratio of the area occupied by DBH immunoreactive nerve fibers per unit area was 6.9 ± 1.1% in WKY/Izm and 5.5 ± 0.9% in SHR/
Fig. 6. Histogram representing the ratio of glomus cells based on immunoreactive intensity for dopamine β-hydroxylase (DBH) in WKY/Izm (left panel) and SHR/Izm (right panel). The x-axis and y-axis represent gray scale intensity for DBH and ratio of glomus cells, respectively. The ratio of glomus cells with intense DBH immunoreactivity (gray scale intensity is 10 or more) was increased in SHR/Izm compared with that in WKY/Izm. The distribution was significantly (p b 0.05) different between WKY/Izm and SHR/Izm analyzed by Kolmogorov–Smirnov tests.
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Fig. 7. The ratio of area occupied by dopamine β-hydroxylase (DBH) immunoreactive nerve fibers per unit area. The ratio of area occupied by DBH immunoreactive nerve fibers per unit area was 6.9 ± 1.1% in WKY/Izm and 5.5 ± 0.9% in SHR/Izm, and there was no significant difference between WKY/Izm and SHR/Izm. Means± S.D. values are shown.
Izm (Fig. 7). There was no significant difference in the value between WKY/Izm and SHR/Izm. 4. Discussion In the present study, it was revealed that CB of SHR/Izm is enlarged in size as shown in the three-dimensional image. Moreover, apparent vasodilatation could not be observed in the CB of SHR/Izm. These results are supported by the report that the diameter of blood vessels within CB in SHR strains is similar to that in WKY strains (Takahashi et al., 2011). It is generally accepted that chronic hypoxia (10% O2) causes the enlargement of the CB with vasodilatation (Wang and Bisgard, 2002; Kusakabe et al., 2005). Interestingly, it was reported that chronic hypercapnic hypoxia (10% O2, 6–7% CO2) caused the enlargement of the rat CB without vasodilatation within the CB (Kusakabe et al., 2005). Thus, between SHR/Izm and hypercapnic hypoxic rats, there are similarities in the volume and the diameter of blood vessels in the CB. It was reported that renal sympathetic nerve activity is increased when rats are exposed to hypercapnic hypoxia (Hirakawa et al., 1997). Given this finding, it is suggested that sympathetic nerve input from superior cervical ganglion is increased, thereby inducing vasoconstriction within the CB of hypercapnic hypoxic rats. Therefore, in hypercapnic hypoxic rats, the enlargement of the CB would be caused by the effect of hypoxia and vasodilatation would be inhibited by the effect of increased sympathetic nerve activity. On the other hand, previous electrophysiological studies showed that sympathetic nerve activity was also increased in SHR strains compared with that in WKY strains (Judy and Farrell, 1979; Lundin et al., 1984; Sugimura et al., 2008). Given this finding, it is suggested that increased sympathetic activity causes vasoconstriction within the CB of SHR/Izm as in the case of the hypercapnic hypoxic rats, leading to reduction in blood supply to the CB. As a result of the decreased blood supply, the CB of SHR/Izm would be rendered regional hypoxia and become enlarged without vasodilatation. Therefore, the morphology of the CB would be similar between SHR/Izm and hypercapnic hypoxic rats due to the effect of hypoxia and the increased sympathetic nerve activity. It has been reported by immunohistochemistry that chronic hypoxia enhances the immunoreactivity for TH in the CB (Wang et al., 1998; Wang and Bisgard, 2002; Hui et al., 2003). Although it is suggested that the CB of SHR/Izm is rendered regional hypoxia as mentioned earlier, immunoreactivity for TH in CB was similar
between SHR/Izm and WKY/Izm in the present study. Therefore, it might be that the regional hypoxic condition in the CB under hypertension is slightly different from the systemic hypoxic condition under environmental hypoxia. On the other hand, the present immunohistochemical study showed that DBH immunoreactivity is enhanced in glomus cells of SHR/Izm. Considering the previous report that NA content was increased in the CB of the New Zealand strain of hypertensive rat (Pallot and Barer, 1985), it is suggested that NA biosynthesis is facilitated in glomus cells of CB in SHR/Izm. It has been suggested that NA in glomus cells, along with DA, is inhibitory to CB activity and is neuromodulator in signal transduction from CB to the nucleus of the solitary tract (Kou et al., 1991; Almaraz et al., 1997; Overholt and Prabhakar, 1999). Furthermore, it has been reported that CSN discharge of SHR strains under normoxic conditions is not different from that of normotensive rats (Fukuda et al., 1987). Therefore, in SHR/Izm, it is suggested that DBH is increased in glomus cells in order to synthesize NA, and furthermore to maintain the CSN discharge at a normal level by the inhibitory NA. Moreover, it is reported that minute ventilation under air-breathing conditions is not different between SHR strains and WKY rat strains (Grisk et al., 1996). Thus, in SHR/Izm, normal respiration under resting conditions would be attributable to the normal CSN activity modulated by the effect of NA in addition to DA. It is reported that sympathetic nerve activity is increased in the SHR strain compared with that in the WKY strain (Judy and Farrell, 1979; Lundin et al., 1984; Sugimura et al., 2008) and NA content is increased in the CB of the New Zealand strain of hypertensive rat compared with that in normotensive rat (Pallot and Barer, 1985). These findings suggest that NA release from sympathetic nerve fibers in addition to glomus cells within the CB is increased in SHR/Izm; therefore, it was expected that DBH immunoreactive nerve fiber within the CB is increased in SHR/Izm compared with that in WKY/ Izm. In contrast, DBH immunoreactive nerve fibers were at similar levels between the two rat strains in the present study. Because it is known that DBH is released from synaptic vesicles together with NA (Smith et al., 1970; Weinshilboum et al., 1971), it is suggested that DBH immunoreactive nerve fibers are not increased in SHR/Izm. In conclusion, the present study exhibited the enlargement of CB as three-dimensional image and revealed the enhanced immunoreactivity for DBH of glomus cells in SHR/Izm. These results suggest that the morphology of CB is affected by the effect of sympathetic nerve and that the signal transduction from CB is regulated by NA in glomus cells under hypertensive conditions. Acknowledgments The authors thank Associate Professor Misuzu Yamaguchi-Yamada (Laboratory of Veterinary Biochemistry and Cell Biology, Faculty of Agriculture, Iwate University) for valuable comments and suggestions. This study was supported by grants-in aid from the Japan Society for the Promotion of Science to TK (21500636), HM (21592202) and YY (22580330). References Alho, H., Partanen, M., Koistinaho, J., Vaalasti, A., Hervonen, A., 1984. Histochemically demonstrable catecholamines in sympathetic ganglia and carotid body of spontaneously hypertensive and normotensive rats. Histochemistry 80, 457–462. Almaraz, L., Pérez-García, M.T., Gómez-Nino, A., González, C., 1997. Mechanisms of α2adrenoceptor-mediated inhibition in rabbit carotid body. Am. J. Physiol. 272, 628–637. Anderson, E.A., Sinkey, C.A., Lawton, W.J., Mark, A.L., 1989. Elevated sympathetic nerve activity in borderline hypertensive humans. Evidence from direct intraneural recordings. Hypertension 14, 177–183. Fukuda, Y., Sato, A., Trzebski, A., 1987. Carotid chemoreceptor discharge responses to hypoxia and hypercapnia in normotensive and spontaneously hypertensive rats. J. Auton. Nerv. Syst. 19, 1–11. Gonzalez, C., Almaraz, L., Obeso, A., Rigual, R., 1994. Carotid body chemoreceptors: from natural stimuli to sensory discharges. Physiol. Rev. 74, 829–898.
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