J Appl Physiol 104: 803-808, 2008.
First published December 20, 2007; doi:10.1152/japplphysiol.00393.2007
8750-7587/08 $8.00
Sustained hypoxia-induced proliferation of carotid body type I cells in rats
Z.-Y. Wang,1
E. B. Olson, Jr.,3
D. E. Bjorling,1
G. S. Mitchell,2 and
G. E. Bisgard2
Departments of 1Surgical Sciences, 2Comparative Biosciences and 3Population Health Sciences, University of Wisconsin-Madison, Madison, Wisconsin
Submitted 12 April 2007
; accepted in final form 14 December 2007
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ABSTRACT
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Sustained hypoxia (SH) has been shown to cause profound morphological and cellular changes in carotid body (CB). However, results regarding whether SH causes CB type I cell proliferation are conflicting. By using bromodeoxyuridine, a uridine analog that is stably incorporated into cells undergoing DNA synthesis, we have found that SH causes the type I cell proliferation in the CB; the proliferation occurs mainly during the first 1–3 days of hypoxic exposure. Moreover, the new cells survive for at least 1 mo after the return to normoxia. Also, SH does not cause any cell death in CB as examined by the terminal deoxynucleotidyl transferase-mediated dUTP-X nick-end labeling assay. Taken together, our results suggest that SH stimulates CB type I cell proliferation, which may produce long-lasting changes in CB morphology and function.
bromodeoxyuridine; terminal deoxynucleotidyl transferase-mediated dUTP-X nick end-labeling assay; chemoreceptor; mitosis
THE CAROTID BODIES (CB) are small sensory organs at the bifurcation of the common carotid artery. The CB is a special chemoreceptor organ that detects the levels of PO2, pH, and PCO2 in the arterial blood (11, 22). In response to the decrease of O2 level or the increase of CO2 level, the CB reflex rapidly induces hyperventilation (11, 22). The CB is highly vascularized, and it contains the specialized, chemosensitive type I cells. Type I cells, like sympathetic neurons and chromaffin cells of the adrenal medulla, originate from the neural crest (14, 24) and migrate toward the carotid bifurcation. In adult animals, type I cells retain some neuronal characteristics; specifically, they are excitable and secrete a number of neurochemicals, including dopamine, acetylcholine, norepinephrine, and several neuropeptides (11, 13, 22). Type I cells are stimulated by decreased arterial PO2 or pH or by increased PCO2, and they are thought to release neurotransmitters that elicit action potentials in chemoafferent neurons with axons in the carotid sinus nerve. Chemoafferent neurons project to the nucleus of the solitary tract in the medulla, thereby initiating chemoreflexes (10). CB chemoreceptors exhibit considerable plasticity when exposed to sustained hypoxia (SH), including increased sensitivity to hypoxia (4).
In association with functional changes in CB chemoreceptors exposed to SH, profound morphological and cellular changes are observed, including increased CB volume, type I cell hypertrophy, angiogenesis, vascular dilation, and alternations in neurochemical expression and nerve innervation (4, 11, 15, 24). Several studies also provide suggestive evidence that SH induces type I cell mitosis, as revealed by condensing chromatin with irregular patches in the nucleus of some type I cells (2, 8). Bee and Pallot (3) reported that many CB cells are labeled when rats that have been administered [3H]thymidine are exposed to 4 days of SH. However, the cell labeling was too dense to determine whether they were type I cells (3). On the other hand, other studies provide no evidence for type I cell mitosis in rats exposed to SH for up to 4 wk (15, 21). We reexamined whether SH causes type I cell proliferation using bromodeoxyuridine (BrdU), a uridine analog that is stably incorporated into cells undergoing DNA synthesis. We also examined the time course of hypoxia-induced type I cell proliferation and whether proliferation is accompanied by CB cell death (e.g., apoptosis or necrosis) during hypoxia.
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METHODS
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Experimental Design
All experimental procedures were approved by the Animal Care and Use Committee at the University of Wisconsin. Male Sprague-Dawley rats (250–300 g) were divided into six groups. In four groups treated with hypoxia, rats were exposed to hypoxia for 1, 2, 3, and 7 days, respectively. All rats in these four groups were treated with BrdU throughout the hypoxic exposure (see below) and then killed for analysis. Rats of group 5 were maintained in room air and served as a control. They were treated with BrdU for 7 days and then killed.
Rats in group 6 (n = 3) were exposed to hypoxia for 7 days (with BrdU treatment) and were then maintained in room air for another 4 wk (without BrdU treatment) before death to determine the longevity of new cells.
BrdU Treatment
Twelve hours before hypoxic exposure, rats were given drinking water containing BrdU (0.8 mg/ml, Sigma, St. Louis, MO) (23); this treatment continued for the duration of the designed hypoxic exposure. The control group was also treated with BrdU in the drinking water for 7 days.
Hypoxic Exposure
O2 was mixed with N2 to achieve a 12% O2 concentration; this mixture was flushed through the chamber at a rate sufficient to maintain the CO2 concentration below 0.5%. Both O2 and CO2 concentrations inside the chamber were monitored continuously. After the hypoxic or normoxic exposure (with the exception of group 6) the rats were killed and tissues were collected immediately (see below).
Histology and Immunohistochemistry
Rats were deeply anesthetized with pentobarbital sodium (50 mg/kg ip) and perfused through the heart with heparinized saline, followed with 2% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS; pH 7.4, 250 ml, 25–30 ml/min). The carotid bifurcations, with CB and superior cervical ganglion (SCG) were removed and postfixed in the same fixative for 4 h at 4°C. The tissues were cryoprotected with 30% sucrose in PBS overnight at 4°C, and were then embedded with optimal cutting temperature compound and sectioned serially at 10 µm using a cryostat. About 150 sections were made of each tissue sample. Every fifteenth section was selected, stained with hematoxylin and eosin (H & E) and examined microscopically to identify the sections containing the CB.
BrdU staining.
Six to eight sections that contained the middle part of the CB from each rat were used for double-immunofluorescence staining. After rinsing in PBS, the sections were incubated with 50% formaldehyde in 2x SSC (0.03 M sodium citrate buffer containing 0.3 M sodium chloride) at 65°C for 2 h to denature DNA. After rinsing in 2x SSC, the sections were treated with 2 M HCl at 37°C for 30 min and then incubated in 0.1 M borate buffer for 10 min (9). The sections were rinsed in PBS and blocked with 10% normal goat serum for 1 h. Sections were first incubated overnight at 4°C with a mouse anti-BrdU antibody (1:150; Becton Dickinson Immunocytometry Systems, San Jose, CA). The sections were rinsed in PBS and incubated with a biotinylated goat anti-mouse IgG (1:250; Sigma) for 1 h. Rhodamine red-X-conjugated streptavidin (1:500, Jackson ImmunoResearch, West Grove, PA) was used to reveal staining. After rinsing, the sections were further incubated with a rabbit anti-tyrosine hydroxylase (TH) antibody (1:500, Chemicon International, Temecula, CA) overnight at 4°C. The TH immunoreactivity was revealed with a fluorescent isothiocyanate (FITC)-conjugated goat anti-rabbit IgG antibody (1:500, Sigma) for 90 min. The slides were rinsed and coverslipped using an antifading solution (Vector Laboratories, Burlingame, CA). The sections were examined with a Nikon E600 microscope and digital images were imported to a computer using a Spot digital camera.
Quantification of type I cells.
Type I cells occur as clusters and have a large oval-shaped nucleus. Therefore, they could be easily identified. TH staining revealed the location of type I cell clusters and confirmed type I cell identification. The number of BrdU-positive type I cells was then counted. However, the cytoplasmic staining of TH is associated with all type I cells. Because of the "bleedthrough" of fluorescence, the type I cells in the cluster are not readily distinguished from each other. Therefore, after counting the number of BrdU-positive type I cells, the same slides were stained with H & E, and the total number of type I cells was counted. A ratio of the number of BrdU-positive type I cells to the total number of type I cells was calculated in the same tissue section, and this ratio was averaged for 6–8 tissue sections examined from each animal.
TUNEL assay.
Four to six sections that contained the middle part of the CB from each rat were used. The TUNEL (TdT-mediated dUTP-X nick end labeling) reaction was used to reveal DNA breaks, which are the hallmark of the late phase of apoptosis or cell necrosis. After rinsing in PBS, the TUNEL assay was performed using an ApopTag kit (Serologicals, Norcross, GA). Briefly, tissue sections were treated with proteinase K (20 µg/ml) for 15 min at room temperature. The slides were then incubated with a mixture of terminal deoxynucleotidyl transferase enzyme and digoxigenin-dNTP at 37°C for 1 h. After rinsing, staining was revealed using an affinity purified sheep anti-digoxigenin antibody, conjugated with FITC. The slides were rinsed and coverslipped using an anti-fading solution (Vector). For positive controls, some slides were treated with DNAse I for 30 min at 37°C to generate DNA breaks. TUNEL assay was also performed with rat mammary tissues included in the kit as a positive control. Sections were examined with a Nikon E600 microscope.
Calculation of CB volume.
H & E staining was performed with all tissue sections containing CB tissues, and digital photoimages were taken using a Spot camera and imported into a computer. The CB area was outlined and measured using Image-Pro plus software (Media Cybernetics, Carlsbad, CA). Calibration was established using a micrometer. Total CB volumes were calculated based on the area of the CB in each section, section thickness, and the total number of sections containing the CB.
Data Analysis
Data are presented as means ± SE. One-way ANOVA was performed to determine whether there was a significant difference among experimental groups. Individual groups were then compared using Tukey's multiple comparison test, and P values <0.05 were considered significant.
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RESULTS
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No overt changes in behavior and activity were observed in rats treated with BrdU. Histologically, SH caused vasodilation and enlargement of CB as reported previously (24). Furthermore, SH increased the CB volume (Fig. 1).
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Fig. 1. Sustained hypoxia (SH) increases the total volume of carotid body (CB). The increase of CB volume appears after the first day of SH, although significant enlargement is observed after 7 days of SH. Values are means ± S; n = 4. *P < 0.01 vs. control group (no hypoxic exposure). #P < 0.05 vs. 1-day SH group.
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CB type I cells were observed in clusters as identified by TH positive staining and by their large oval-shaped nuclei. Immunofluorescence indicating BrdU incorporation was limited to cell nuclei as anticipated. Generally, no BrdU-labeled type I cells were observed in the CBs of control animals, although one or two BrdU-labeled connective tissue cells could be occasionally observed. Following 1 day SH, a few BrdU-positive type I cells were observed (Fig. 2). However, on days 2 and 3, the prevalence of BrdU-positive type I cells increased dramatically compared with day 1 (P < 0.01) (Figs. 2, 3, and 4). By day 3 of SH, the increase in BrdU-labeled type I cells appeared to approach the maximum. Seven-day exposures numerically further increased the ratio of labeled cells, but this difference did not rise to a significant level compared with the value of day 3 (P > 0.05; Figs. 2 and 4).
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Fig. 2. Representative photoimages showing immunofluorescence staining of bromodeoxyuridine (BrdU), a uridine analog that is stably incorporated into cells undergoing DNA synthesis, in the nucleus of cells of CBs in control rats (A; no hypoxic exposure) and in rats exposed to 1 (B), 2 (C), 3 (D), and 7 (E) days SH. F: staining in CBs of rats exposed to SH for 7 days (with BrdU treatment) and were then maintained in room air for another 4 wk (without BrdU treatment) before death. Scale bar, 50 µm.
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Fig. 3. Double-immunofluorescence staining showing 3 days of SH-induced cell proliferation in the rat CB. Green fluorescence indicates the staining of tyrosine hydroxylase, a marker protein in type I cells. Orange-yellow fluorescence indicates the staining of BrdU in the nucleus of cells. I, type I cells; II, type II cells; E, endothelial cells of blood vessel. Note that there are more BrdU-positive type I cells than BrdU-positive type II and endothelial cells. Scale bar, 50 µm.
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Fig. 4. SH induces the proliferation of CB type I cells. The number of BrdU-positive type I cells is expressed as a ratio of the total number of type I cells in the same tissue section. Values are means ± SE; n = 4. *P < 0.01 vs. 1-day SH group. #P < 0.05 vs. 2 days SH group.
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In addition to type I cell proliferation, SH also increased BrdU labeling in connective tissue cells, endothelial cells and type II cells (15) within the CB. However, the majority of BrdU-labeled cells in CB were type I cells (Fig. 3).
We also examined cell proliferation in the SCG. The neurons in SCG, like CB type I cells, originate from the neural crest (25). No BrdU-labeled neurons were observed in this tissue. There were some labeled glia cells, but SH did not affect the proliferation of these cells (Fig. 5).
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Fig. 5. Representative photoimages showing immunofluorescence staining of BrdU in superior cervical ganglion of control rats (A; no hypoxic exposure) and of rats exposed to 7 days SH (B). No BrdU-labeled neurons are observed. Some glia cells are labeled with BrdU, but SH does not affect the proliferation of these cells. Scale bar, 50 µm.
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TUNEL analysis was used to determine whether SH causes cell death. No TUNEL positive cells were seen in the CBs from the control animals or from the rats treated with 1–7 days SH. Also, no TUNEL positive cells were observed in SCG (not shown). After treatment of CB sections with DNAse I to generate DNA breaks, a strong TUNEL signal was observed. Furthermore, in rat mammary tissue that contains apoptotic cells, positive results were observed, demonstrating that the TUNEL assay worked under our experimental conditions (Fig. 6).
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Fig. 6. The terminal deoxynucleotidyl transferase-mediated dUTP-X nick end-labeling (TUNEL) analysis is used to determine whether SH causes cell death. No TUNEL-positive cells are seen in the CBs from the control animals or from the rats treated with 1–7 days SH (A). After treatment of tissue slides with DNAse I to generate DNA breaks, a strong TUNEL signal is observed (B). demonstrating that the TUNEL assay works under our experimental conditions. Scale bar indicates 50 µm.
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In group 6, rats were exposed to SH (with BrdU treatment) for 7 days and then kept in room air for another 4 wk (without BrdU treatment) to determine the longevity of new cells. The ratio of BrdU-labeled type cells was 28.1 ± 2.2%, and it was not significantly different from rats killed immediately following 7 days SH (29.3 ± 4.7, P > 0.05) (Fig. 2). There was no evidence for CB cell death in this group of rats as indicated by the TUNEL assay (not shown).
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DISCUSSION
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Our results demonstrate that there is the proliferation of CB type I cells that occurs largely within the first 3 days of SH. Furthermore, the new type I cells are viable for at least 4 wk following sustained hypoxia. Conflicting literature reports concerning hypoxia-induced CB cell proliferation may be due to the different techniques used, and the time points investigated (2–3, 7–8, 15, 21). By using a convenient, noninvasive and relatively unambiguous treatment of adding BrdU into the drinking water (23), we have provided clear evidence that SH induces type I cell proliferation in the rat CB.
Type I cells started to proliferate after 
1 day of hypoxic exposure, increased dramatically at 2 days, and approached to a plateau after 3 days of SH. In a preliminary study, two rats were exposed to 2 wk of SH. Two days before death, the rats were given BrdU intraperitoneally every 12 h. In this study, very few BrdU-labeled type I cells were observed (4–6 cells/tissue section; Wang and Bisgard, unpublished observations). Thus it appears that type I cell proliferation mainly happens during the first few days of SH, perhaps up to 1 wk. The limited capacity of type I cell proliferation was also observed in culture (18). The mechanism of increased proliferation exclusively within the first few days of exposure remains to be explored. However, this observation may help to explain why there is no sign of type I cell mitosis in rats exposed to weeks or months of hypoxia (7, 15, 21).
Little is known concerning the cellular mechanisms underlying the hypoxia-induced type I cell proliferation. Studies with in vitro cultured type I cells have shown that basic fibroblast growth factor and endothelins promote type I cell division (17–20). Hypoxia can stimulate type I cell proliferation in a serum-free, chemically defined medium, suggesting that hypoxia may exert mitogenic action on type I cells independent of any exogenous hormonal or neural factors (18). Indeed, type I cells are capable of producing growth factors and neurotrophins, which may regulate cell proliferation by an autocrine mechanism (5, 20). Interestingly, SH did not promote any neuronal proliferation in SCG. SCG provides sympathetic innervation to blood vessels in CB, and the neurons in SCG, like CB type I cells, originate from neural crest (14, 25). This finding suggests that, although CB type I cells and SCG neurons share the same origin, only CB type I cells that are specialized as chemoreceptors are subject to proliferative effect of SH.
Ischemia or severe hypoxia may cause cell death in the central nervous system (1, 16). To our knowledge, no studies concerning possibility of SH-induced cell death in the CB have been reported. By doing TUNEL assay, which reveals DNA breaks (the hallmark of late phase of apoptosis or necrosis), we have no evidence of any cell death in the CB following 1, 2, 3, or 7 days SH. Although type I cells possess some neuron-like characteristics, they are specialized so that they may tolerate hypoxic stresses more effectively than neurons. On the other hand, the hypoxic exposure (12% O2) used in the present study was only moderate. It remains possible that more severe hypoxia and/or longer exposures would initiate CB type I cell death.
Chen et al. (6) studied the effects of SH on normoxic and hypoxia-evoked carotid sinus nerve activity and found that basal nerve activity progressively increases during SH. Significant changes are first observed only after 3 days of SH, and both basal nerve activity and the response to acute hypoxia continued to increase up to day 9 of SH. Further increases were not observed in preparations from animals exposed to SH for up to 16 days. The increase of nerve activity mirrors SH-induced type I cell proliferation in some respects, although the effect is delayed. Either CB type I cell proliferation is unnecessary for SH-enhanced carotid sinus nerve activity or it is necessary, but requires further maturation before the cells are able to contribute to the functional effects. Moreover, our study indicates that the majority of newly generated type I cells remain viable for at least 4 wk posthypoxia, and these new generated type I cells are likely to contribute to SH-induced alternations in CB function.
In conclusion, we have demonstrated that SH stimulates type I cell proliferation in the rat, that the proliferation occurs mainly within the first 1–3 days of hypoxic exposure, and that the new cells survive for at least 4 wk after the return to normoxia. Because no SH-induced cell death of type I cells is observed and majority of new generated type I cells persist at least 4 wk after termination of SH, our results suggest that SH induces long-lasting changes in CB morphology and possibly function.
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GRANTS
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-68255.
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ACKNOWLEDGMENTS
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We thank Laura Emrick for technical assistance.
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FOOTNOTES
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Address for reprint requests and other correspondence: Z.-Yi Wang, Dept. of Surgical Sciences, School of Veterinary Medicine, Univ. of Wisconsin-Madison, 2015 Linden Dr., Madison, WI 53706 (e-mail: wangz{at}svm.vetmed.wisc.edu)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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Copyright © 2008 by the American Physiological Society.
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ABSTRACT
METHODS
