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The CB constitute the first line of defense against hypoxia in mature mammals, triggering an increase in breathing and arousal whenever necessary (1). Before birth, the CB are relatively insensitive to hypoxia, being adapted to the chronically low Po2 in utero (2). The sudden rise in Po2 at the time of birth results in a progressive increase in CB sensitivity to hypoxia, a resetting process that takes approximately 2 wk to complete in rodents (3). If the normal postnatal rise in Po2 is delayed or accelerated, CB function can be significantly impaired (3–6).

Although the molecular mechanisms underlying this resetting of the CB are still unknown, catecholamines appear to play an important role in this process (7,8). The dopamine content and the level of mRNA encoding TH, the rate-limiting enzyme in catecholamine synthesis, are used as two principal indicators of catecholamine metabolism in the rat CB. Both of these parameters are relatively high in the fetus, but decrease rapidly after birth (7–10). Exposure of adult rats to hypoxia enhances the expression of TH mRNA in their CB and, therefore, it is proposed that fetal hypoxia results in a high level of TH mRNA in utero, which is reduced in response to the sudden rise in Po2 at birth (11,12).

The postnatal increase in Po2 and fall in the level of TH mRNA in the CB might involve hypoxia-inducible factors (HIF), members of the basic helix-loop-helix superfamily of eukaryotic transcription factors (13,14). The HIF-1 heterodimer consists of HIF-1α and HIF-1β (ARNT) (14), whereas HIF-2 contains HIF-2α and HIF-1β (15). The activities of these factors are regulated by the stability of their α subunits, which function as direct sensors of intracellular oxygen concentrations. Binding of HIF-1 or HIF-2 heterodimers to the hypoxia responsive element located in the promoter of the TH gene promoter (16,17) regulates transcription of this gene (18). Thus, there is strong evidence that HIF-1α plays a role in chemosensitivity in adult mice (19) and HIF-2α and TH are co-expressed in the CB of adult mice (20,21).

To determine whether HIF-1α or HIF-2α participate in the perinatal resetting of the CB, we first investigated here whether hypoxia in vivo induces expression of these proteins in the CB of adolescent rats [postnatal d 40 (P40)]. Thereafter, we analyzed developmental changes in the expression of these two factors in the CB of rats of different ages, ranging from embryonic d 20 (E20) to P14.

METHODS

Animals and treatment.

Male Sprague-Dawley rats (B & K Universal, Stockholm, Sweden) were studied on E20, at the time of birth (P0), and on P2, P7, P14, and P40, with birth occurring in all cases during the night following the E20. At least five to six pups of each age, randomly selected from different litters, were analyzed. All animals were housed at sea level in an air-conditioned room at 24°C, with a 12-h light-dark cycle and access to food and water ad libitum.

We also compared the CB parameters of five adolescent (P40) rats subjected to normobaric hypoxia (8% O2/92% N2) for 6 h or to normoxic conditions (five animals in each group). The CO2 level in the chamber was maintained at the same level as that of room air. The regional ethical committee for animal experimentation, which follows the regulations of the European Community, approved this study.

Immunostaining.

Pregnant rats were killed on E20 by cervical dislocation and casarean section was performed within 2 min thereafter. To minimize the time elapsed during removal of the pups and, thereby, the risk for alterations in the expression of HIF-1α or HIF-2α [both of which exhibit rapid turnover and short half-time decay (14)] due to an increase in O2 tension, only three pups from each mother were used. The carotid bifurcations of all rat pups were excised under deep anesthesia (50 mg pentobarbital/kg body weight) within 3 min following delivery, after which the animals were killed immediately by decapitation.

Adolescent rats (P40) exposed to hypoxia in vivo (6 h) were subsequently anesthetized directly with pentobarbital (50 mg/kg) and perfused transcardially with PBS (0.1 M, pH 7.4) containing 4% paraformaldehyde for 10 min (flow rate = 30 mL/min). To prevent reestablishment of normoxic conditions during this period, both anesthesia and fixation were performed under 8% O2. Subsequently, the carotid bifurcation was immediately fixed for 60 min in ice-cold 4% paraformaldehyde in PBS, followed by immersion in 30% sucrose overnight.

Cryosections with a thickness of 10–14 μm were prepared and allowed to adhere to SuperFrostPlus glass slides (Menzel-Glaser, Braunschweig, Germany). These sections were then permeabilized with 0.1% saponin and 0.1% TritonX, blocked with 7% normal goat (NGS) or donkey (NDS) serum, and incubated overnight at 4°C with the primary antibody in PBS containing 3.5% serum, 0.1% saponin, and 0.1% Triton X. Thereafter, the sections were washed, incubated with secondary antibody in the same PBS solution, washed again, and examined by confocal microscopy.

This procedure was used to examine a number of different parameters. TH immunoreactivity in the CB was used as a marker for glomus cells (22). The cellular hypoxic response and the localization of HIF-1α and HIF-2α at various stages of development were determined by double immunostaining (TH/HIF-1α or TH/HIF-2α). For this purpose, HIF-1α was probed with an affinity-purified mouse MAb (diluted 1:500) against human HIF-1α (Novus Biologicals, Inc., Littleton, CO) (23); HIF-2α with an affinity-purified goat polyclonal antibody (1:400) against human HIF-2α (sc-8712, Santa Cruz Biotechnology, Santa Cruz, CA) (24); and tyrosine hydroxylase with polyclonal sheep antibody (1:500) against rat tyrosine hydroxylase (Calbiochem-Novabiochem San Diego, CA) (25). Goat-anti-mouse Alexa 546 (1:200), rabbit-anti-goat Alexa 546 (1:200), and donkey-anti-sheep Alexa 488 (1:200) (all from Molecular Probes, Eugene, OR) were used as the secondary antibodies.

These same conditions were used to characterize the nuclear translocation of HIF-1α and HIF-2α following hypoxia in vivo in adolescent rats, except that in this case freshly prepared hydrogen peroxide (3%, 37°C, 10 min) was first used to block endogenous peroxidase activity. Exposure to the primary antibodies was followed by incubation with mouse anti-goat (Goat ExtrAvidin Peroxidase EXTRA-1 kit, Sigma Chemical Co., St. Louis, MO) or goat anti-mouse (Mouse ExtrAvidin Peroxidase EXTRA-2 kit; Sigma Chemical Co.) antibodies that had been biotinylated (1:15) for 1 h at room temperature. The tissue sections were incubated with ExtrAvidin-peroxidase (1:15) for 30 min at room temperature, after which sites of antigen-antibody interaction were visualized with diaminobenzidine tetrahydrochloride and 0.01% H2O2 in 2.5 mM Tris-Cl (pH 7.6) for 10 min (Fast DAB Kit; Sigma Chemical Co.). The slides were subsequently mounted in ProLong Antifade (Molecular Probes).

Controls in which the primary antibody was omitted were run for each immunocytochemical procedure to ensure that there was no unspecific immunoreactivity originating from the secondary antibodies. In the case of the double immunostaining, we carefully tested the level of cross-reactivity by performing two single immunostainings, switching the secondary antibodies, which clearly revealed that staining due to cross-reactivity was negligible (8% of the total staining). As an additional control, we omitted the secondary antibodies to determine the background level of autofluorescence.

Analysis.

The immunolabeled tissues were scanned with a Zeiss LSM410 or a Leica TCS SP inverted confocal scanning laser microscope using 63×/1.4 N.A. and 20×/0.75 N.A. objectives. Green fluorescence was produced by excitation at 488 nm and detected with a 515–540 nm band-pass filter. In the case of red fluorescence, excitation was at 543 nm and a 570 nm long-pass filter was used for detection.

Because of the small size of the CB, standard approaches to quantify the levels of different proteins in this organ are problematic. Therefore, we used immunoquantification of the HIF proteins. Densitometric analysis of the staining for HIF-1α and -2α was performed applying ImageJ software from the National Institutes of Health Internet site (http://rsb.info.nih.gov) to 15–30 TH+ cells in one image from each section. From each image, we also analyzed 15–30 TH− cells to determine the background level of staining, as well as staining for HIF-1α or HIF-2α in sustentacular cells (TH−). For each age and mode of treatment (normoxia/hypoxia), the mean level of staining for five or six sections from each of two or three CB was determined.

During perinatal maturation, the diameter and volume of the CB may increase, which would affect our calculations. However, our estimates of the diameter (122 ± 15.4 μm at E20 versus 146 ± 44.3 μm at P14), as well as of the volume (10.1 ± 1.25 × 104 mm3 at E20 versus 16.3 ± 2.56 × 104 mm3 at P14) of this organ indicated no significant alteration in these macroscopic parameters during the perinatal period investigated.

Statistical analysis.

To determine whether the responses varied with age or treatment (hypoxia/normoxia), the data were analyzed using one-way ANOVA. In cases where the difference was thus found to be significant, modified t tests with the Bonferroni correction were applied a priori for comparison of pairs of responses. A p value ≤0.05 was considered to be statistically significant.

RESULTS

HIF-1α and HIF-2α are induced in adolescent rats in response to hypoxia in vivo.

On P40, HIF-1α and HIF-2α were found to be expressed constitutively in the CB of rats maintained under normoxic conditions. When rats of the same age were exposed to 8% O2 for 6 h, immunostaining of the CB for HIF-1α was elevated 7-fold (Fig. 1). Intense nuclear and cytoplasmic immunostaining was observed, primarily in the glomus cells (as indicated by co-localization with the neural-specific marker TH) (Fig. 1). The level of HIF-1α staining in TH− cells was only half of that in TH+ cells. In addition, immunopositive, flat nuclei, most likely localized in endothelial cells, were observed in some of the blood vessels.

Figure 1
figure 1

Expression of HIF-1α in the carotid body of adolescent rats after exposure to hypoxia (6 h, 8% O2) in vivo. (A) These photomicrographs (×10) illustrate the immunolocalization of the HIF-1α protein in a whole carotid body of adolescent rats (P40) after sustained hypoxia. We observed staining for HIF-1α in cell clusters and flat nuclei associated with blood vessels, probably located in endothelial cells (circle). (B) The arrows indicate strong nuclear and cytoplasmic immunostaining for HIF-1α under hypoxic conditions (×100). (C) Immunostaining for HIF-1α in normoxic and hypoxic carotid bodies. The bar indicates a distance of 100 μm. (D) Densitometric quantitation of the immunostaining for HIF-1α in hypoxic (n = 5) and normoxic (n = 5) CB, revealing a higher level in the hypoxic organ. *Statistically significant difference. (E) Double immunolabeling for HIL-1α and TH in the CB of adolescent rats (P40) exposed to hypoxia (6 h at 8% O2) in vivo. The localization of TH is depicted in green and that of HIF-1α in red. In the overlaid image, HIF-1α-positive cells appear red, TH-positive cells green, and cells expressing both of these markers yellow. Hypoxia induces the expression of HIF-1α primarily in TH+ cells. Internal scale bars = 100 μm.

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In addition, expression of HIF-2α was strongly up-regulated in these hypoxic CB, as indicated by strong and specific nuclear and cytoplasmic immunostaining (Fig. 2). This immunoreactivity was observed in numerous cells arranged in clusters. No immunoreactivity was detected in CB sections processed in the same manner except for omission of the primary antibodies against HIF-1α, HIF-2α, or TH (not shown).

Figure 2
figure 2

Photomicrographs depicting the expression and localization of the HIF-2α protein in the carotid bodies of adolescent rats (P40) exposed to normoxia (n = 3) or sustained hypoxia (6 h at 8% O2; n = 5) in vivo. (A) Immunostaining for HIF-2α in the carotid bodies of rats maintained under normoxic conditions (×10). (B) After sustained hypoxia, the carotid body exhibits strong immunostaining for HIF-2α (×10). (C) The arrows indicate nuclear and cytoplasmic immunostaining for HIF-2α in response to hypoxia (×100).

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Expression of HIF-1α and HIF-2α in the rat CB at different stages of development.

TH immunoquantification was performed during the CB development (E20, 62.01 ± 10.6; P0, 71.97 ± 6.3; P2, 53.9 ± 4.21; P7, 55.68 ± 11.80; P14, 48.62 ± 8.30; P40, 45.8 ± 8.8). There was a general tendency (p = 0.07) for a general decrease in TH immunoquantification with age (regression: y = –0.4161x + 60.628). Immunostaining for HIF-1α was also present at all stages investigated, from E20 to P40. On E20, P0, and P2, this protein was shown using double-immunostaining (HIF-1α/TH) to be present in both glomus (TH+) and sustentacular cells (TH−). However, subsequent to the perinatal period (i.e. at P7 and P14), HIF-1α was still expressed in sustentacular cells, but deeply decreased from glomus cells (Fig. 3). Quantitation of these morphologic changes by densitometry confirmed that the levels of HIF-1α in TH+ and TH− cells were similar (with a ratio of approximately 1) at early developmental stages (E20, P0, and P2), but that this ratio was significantly lower (around 0.35) on P7 and P14 (Fig. 3).

Figure 3
figure 3

Immunostaining for HIF-1α and TH during maturation of the rat carotid body, i.e. at E20, birth (P0), and P2, P7, and P14 (A) and after hypoxia in vivo (P40) (B). In the overlaid images, cells that are only positive for HIF-1α appear red, TH-positive (TH+) cells appear green, and cells expressing both of these markers appear yellow. In early stages of development (i.e. E20, P0, and P7; n = 5 or 6), HIF-1α is expressed by both TH+ and TH− cells. At later stages (P7 and P14; n = 5 or 6), staining for HIF-1α and TH is present in different cells. (B) This graph depicts the ratio of HIF-1 staining (as quantified by densitometry) in TH+ vs TH− cells. At early stages (E20, P0, and P2; n = 5 or 6), HIF-1α staining in TH+ and TH− cells is of the same intensity; whereas at later stages [P7, P14, and P40 normoxic (NX) rats; n = 5 or 6], HIF-1α is expressed primarily in the TH− cells. After exposure of P40 rats (n = 5) to hypoxia (HX) in vivo, expression of HIF-1α was strongly enhanced in TH+ cells. *Statistically significant difference compared with the ratio at E20. †Statistically significant difference compared with P40 rats exposed to normoxic conditions.

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Immunoreactivity toward HIF-2α was present and of similar intensity in CB of all ages and was restricted to cells arranged in clusters. Double immunostaining (HIF-2α/TH) revealed that this protein was expressed in glomus cells (TH+) only and not in sustentacular cells (TH−) (Fig. 4). Indeed, all cells expressing HIF-2α also expressed TH.

Figure 4
figure 4

Immunostaining for HIF-2α and TH during maturation of the rat carotid body, i.e. at E20, birth (P0), and P2, P7, and P14. In the overlaid images, cells expressing both of these markers appear yellow. During this time period, expression of HIF-2α and TH is entirely co-localized.

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DISCUSSION

Our major finding in the present investigation is that immunoreactivity toward HIF-1α is relatively high in the fetal CB and decreases substantially several days after birth. Moreover, we show that hypoxia in vivo induces the expression of both HIF-1α and HIF-2α in the CB of adolescent rats.

The high level of HIF-1α in the fetal CB is remarkable, because HIF are not usually expressed under normal conditions in healthy organs (26–28). Although the fetus is not regarded as being hypoxic, it is well known that the Po2 in utero is relatively low, which may explain our finding that HIF are constitutively expressed during this period.

Here, we demonstrate that severe hypoxia (8% O2) induces expression of both HIF-1α and HIF-2α in the CB of adolescent rats, relative to their basal levels of expression. Exposure to this degree of hypoxia represents a strong but tolerable challenge, which can be observed under certain physiologic or pathophysiological situations. Furthermore, we found a significant reduction in the intensity of immunostaining for HIF-1α, but not in HIF-2α during the first week of postnatal life.

This decrease in HIF-1α immunoreactivity was restricted to glomus cells, without any change with respect to sustentacular cells. The glomus cells are situated in the vicinity of capillaries and are thereby particularly sensitive to changers in blood parameters such as Po2, pH, CO2, and endocrine factors. Moreover, increased mitochondrial production of reactive oxygen species is required for stabilization of HIF-1α and such production may vary in different cell types (29). Indeed, large mitochondria with numerous cristae and a matrix of relatively low density are typically seen in glomus cells, whereas the mitochondria in sustentacular cells appear to be denser and smaller and to contain fewer cristae (30,31). This difference might also contribute to the difference in the levels of HIF-1α immunostaining in these two cell types.

Immunoreactivity toward HIF-1α was seen to decrease relatively slowly after birth, in contradiction to the suggestion that this decrease is a response to the immediate increase in Po2 experienced after birth. However, other factors related to the stress of being born, e.g. changes in the circulating levels of hormones such as angiotensin and insulin (32–35), might play a role in this context. For example, the levels of angiotensin and IGF1 in the blood of rat and human babies are very high during the first two postnatal days, after which they fall markedly (36,37). We propose that these high levels may compensate for the sudden loss of hypoxic stimulus and sustain a high level of HIF-1α expression during the first 2 d of postnatal life. In contrast, endocrine factors that influence the expression of HIF-2α have yet to be identified.

In adolescent rats, we found that severe and prolonged hypoxia stimulates the expression of both HIF-1α and HIF-2α in the CB, resulting in pronounced nuclear staining. The reason why such a decrease in oxygen tension up-regulates both of these factors in adolescent rats, whereas only the level of HIF-2α expression is maintained during perinatal transition is not presently clear. The stage of maturation is the major difference between these experimental protocols and represents an obvious possible answer. We can also speculate that expression of HIF-1α may be more responsive to hormones (see also above) and moderate changes in Po2, whereas HIF-2α may be up-regulated in the CB only by severe hypoxia. Therefore, we hypothesize that adaptation of the CB to prolonged hypoxia may be mediated in part either by HIF-1α and/or HIF-2α. Indeed, it has been established that HIF-1α and HIF-2α activate the transcription of genes whose products mediate adaptive responses to hypoxia. To date, more than 30 such HIF-1α-responsive genes have been identified (38).

The perinatal decrease in the level of HIF-1α in the CB during the critical period of resetting, suggests a possible coupling between HIF and dopaminergic mechanisms. An HIF-1/O2-responsive element has been detected in the promoter of the TH gene. Indeed, hypoxia in vitro can regulate TH promoter activity through the binding of HIF-1α or HIF-2α (18). Furthermore, prolonged exposure of rats to hypoxia results in adaptation and up-regulation of the level of TH mRNA and dopamine content of the CB (11,39). Earlier studies have also demonstrated an inverse relationship between the decrease in dopamine metabolism (as reflected in the dopamine content and level of TH mRNA) and the increase in chemoreceptor activity during the first week of postnatal life (7–10).

In our investigation, the level of TH immunoreactivity tended to decrease during this critical period. However, we also observed a fall in the level of immunostaining for HIF-1α in glomus cells during the first postnatal week, i.e. during the same period as the decreases in dopamine content and TH mRNA reported by others (8,10). These results provide evidence that HIF-1α and, possibly, HIF-2α as well participate in activation of the catecholaminergic pathway.

Our present findings are also consistent with reports demonstrating that chronic hypoxia results in up-regulation of the HIF-1α protein in catecholaminergic neurons of the brainstem (40). Moreover, carotid body function in adult HIF-1α −/+ heterozygous mice is abnormal (19). However, several other putative targets for HIF-1α and HIF-2α could also be involved in the adaptation of the CB to hypoxia. For instance, in the CB of adult rats exposed to 4 wk of hypoxia, HIF-1α up-regulates the expression of VEGF and VEGFR-1 (41). Furthermore, HIF-1α might also up-regulate the expression of the inducible nitric oxide synthase and thus increase the local level of NO, which has been described as a key factor in the chemosensitive process (42,43). Finally, HIF-1α plays a key role(s) in the regulation of glucose and overall cell metabolism (44) and metabolic adaptation is a necessary part of the response of any tissue to chronic hypoxia. Thus, HIF-1α could play an important role in the metabolic adaptation of both glomus and sustentacular cells in the CB to chronic hypoxia.

In conclusion, the level of the HIF-1α protein in the CB is selectively regulated during the perinatal transition period. In contrast, sustained hypoxia induces both HIF-1α and HIF-2α in the CB of the adolescent rat. These processes might be associated with trophic adjustments and remodeling of the CB, which occur during acclimatization to hypoxia.