1Division of Neonatology, Rainbow Babies and Children's Hospital, Case Western Reserve University, Cleveland, Ohio; and 2Department of Physiology and Biophysics, Howard University, Washington, District of Columbia
Submitted 18 January 2005 ; accepted in final form 6 April 2005
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ABSTRACT |
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neurotrophins; airway smooth muscle; brain-derived neurotrophic factor
Increasing evidence from studies of airway hyperreactivity in asthmatic patients suggests that NTs, especially NGF, may participate in the immunological dysfunction of allergic airway diseases and serve as a link between airway inflammation and airway hyperresponsiveness (9, 13, 37, 43, 45). Recent studies by de Vries et al. (17) suggest that administration of the neurokinin-1 receptor antagonist completely blocks NGF-induced airway hyperresponsiveness, suggesting a strong association between NTs and tachykinin peptides in regulating airway activity. To identify the role of BDNF and NGF in hyperoxia-induced bronchopulmonary changes and resultant airway hyperresponsiveness, it is first necessary to document whether these NTs and their receptors are present in the lung tissue of developing mammals and whether hyperoxic stress affects their expression.
Hence, in the present study, we evaluated the effects of hyperoxic exposure on the production and expression of NGF and BDNF as well as their corresponding high-affinity receptors TrkA and TrkB, respectively, in the lung of developing rat pups. Our data demonstrate for the first time that hyperoxia induces BDNF production as well as expression of TrkB in the lung, specifically, in peribronchial smooth muscle. This enhanced BDNF production and TrkB expression were not accompanied by any changes in production and expression of NGF or TrkA. We speculate that BDNF may be directly involved in hyperreactivity of intrapulmonary airways following hyperoxic exposure in the developing lung.
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MATERIALS AND METHODS |
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Animal hyperoxic exposure. Pathogen-free 5-day-old Sprague-Dawley rat pups (Charles River, Wilmington, MA) were mixed between litters and randomized for continuous exposure to either hyperoxia (>95% oxygen) or normoxia (room air) over a 7-day period. The hyperoxic environment was achieved by continuous oxygen delivery (2 l/min) to a sealed Plexiglas chamber where rat pups were confined. Oxygen concentration was monitored continuously with an Oxygen Analyzer (TED 60T; VWR Scientific Products, Bridgeport, NJ). Litters assigned to normoxia were kept in open cages in room air and served as controls. Food and water were supplied ad libitum to both groups, and nursing mothers were rotated every 24 h between the two groups to avoid oxygen toxicity in the mothers and eliminate maternal effects. Rat pups were killed at the end of exposure, and lung and tracheae were collected. A total of 23 animals in each group were used from three different exposed cohorts for the following experiments: i.e., 10 animals from each group for RNA extraction and RT-PCR, 10 animals from each group for ELISA, and three animals from each group for histological studies including immunohistochemistry and in situ hybridization.
RNA extraction and RT-PCR.
Cellular RNA was isolated with Trizol reagent (Invitrogen Life Technologies, Carlsbad, CA) from individual rat pup lungs (n = 10 in each group), and tracheae were pooled from 10 animals in each group before RNA extraction. First-strand complementary DNA (cDNA) was synthesized using 5 µg of total RNA and oligo(dT) primers with SuperScript II RT-PCR 1st-strand DNA synthesis system (Invitrogen Life Technologies). The PCR was carried out with 0.2 µM gene-specific primers, 2 units Taq DNA polymerase, and 2 µl cDNA from the reverse transcription reaction. The primer sets for genes of interest were designed on the basis of sequence data from the National Center for Biotechnologies Information databases and purchased from Invitrogen Life Technologies. The primer sequences for BDNF were 5'-GCGGCAGATAAAAAGACTGC-3' (forward) and 5'-GCCAGCCAATTCTCTTTTTG-3' (reverse), generating a fragment of 238 bp. The primer sequences for NGF were 5'-CAACAGGACTCACAGGAGCA-3' (forward) and 5'-TCCAGTGCTTGGAGTCAATG-3' (reverse), generating a fragment of 254 bp. Used as a control for RNA input, the primer sequences for -actin were 5'-AGCCATGTACGTAGCCATCC-3' (forward) and 5'-CTCTCAGCTGTGGTGGTGAA-3' (reverse), generating a fragment of 228 bp. After initial denaturation at 94°C for 5 min, amplification was conducted in the linear range of 25 cycles for BDNF and
-actin and 35 cycles for NGF with the following cycling parameters: denaturation at 94°C for 45 s; annealing at 55°C for 30 s; extension at 72°C for 90 s; and final extension at 72°C for 5 min. End products were analyzed on a 2% agarose gel stained with ethidium bromide, and band fluorescent intensities were measured and analyzed with Image J software (NIH Image; National Institutes of Health, Bethesda, MD).
Enzyme-linked immunosorbent assays. To evaluate the production of BDNF and NGF in response to hyperoxic exposure in lung and trachea, enzyme-linked immunosorbent assays (ELISA) (BDNF and NGF ELISA Emax kits; Promega, Madison, WI) was performed as described (5). Protein extracted from homogenates of extrathoracic trachea and lung was used for ELISA assay (n = 10 in each group). In brief, immediately after animals were killed, trachea and lung were collected and immersed in liquid nitrogen. They were then homogenized in ice-cold lysis buffer (137 mM NaCl, 20 mM Tris·HCl, pH 8.0, 1% Triton X-100, 10% glycerol, 1 mM PMSF, 10 µg/ml aprotinin, 1 µg/ml leupeptin, and 0.5 mM sodium vanadate). Protein concentrations were measured by Bio-RAD Protein Assay kit (Bio-RAD, Hercules, CA). Experimental samples (200 µg protein/well) and standards were added as duplicated wells to the 96-well plate precoated with monoclonal primary capture antibody. After incubation with specific secondary antibody, horseradish peroxidase (HRP)-conjugated species-specific antibody was added; the reaction was developed with tetramethylbenzidine and stopped with addition of hydrochloric acid. The absorbance was then read at 450 nm using a kinetic microplate reader (Vmax; Molecular Devices, Sunnyvale, CA), and data were analyzed against the standard curve (Softmax, Molecular Devices). The sensitivity of this assay is 15.6 pg/ml and has <3% cross-reactivity with other related neurotrophic factors at 100 ng/ml.
Tissue harvesting and processing for in situ hybridization and immunohistochemistry. Three animals in each group were randomly selected, and tissue sections were obtained from their single-lobed left lung and processed for in situ hybridization and immunohistochemistry studies. Immediately after death, the heart and lung were exposed via a midline incision through the sternum. The pulmonary artery and vein were ligated. The tracheae were cannulated, and lungs were inflated with a 1:1 mixture of Tissue Tek OCT Compound (Sakura Finetek, Torrance, CA) and normal saline. The lungs were then removed and immediately immersed in liquid nitrogen. Cryosections were obtained from the single-lobed left lung and were cut into 5-µm slices. They were placed on positively charged slides (Super Frost Plus; Fisher Scientific, Hanover, IL) and air dried for 1 h before fixation in acetone at 20°C for 5 min. The slides were then processed for in situ hybridization and immunohistochemistry accordingly.
BDNF in situ hybridization.
In situ hybridization on sequential tissue sections was performed using digoxigenin-labeled cRNA probe to detect the BDNF mRNA expression in lung, as described previously (10). In brief, the plasmid template was linearized by digestion with BamH1 or HindIII, to generate the corresponding sense or antisense transcripts, under T3 or T7 promoters, respectively. Postrestriction digest DNA was treated with proteinase K and was phenol-chloroform extracted. The reaction mixture contained 40 mM Tris·HCl, pH 8.0, 6 mM MgCl2, 10 mM dithiothreitol (DTT), 2 mM spermidine, 10 mM NaCl, 1 unit/µl RNase inhibitor, and 1 mM ATP, GTP, CTP with 0.65 mM UTP and 3.5 mM digoxigenin-111-UTP. Product was treated with DNase and the labeled cRNA probe was recovered by ethanol precipitation. Tissue sections were briefly treated with protease (125 µg/ml; Sigma Chemical, St. Louis, MO) and acetylated with 0.25% (vol/vol) acetic anhydride in 0.1 M triethanolamine-HCl. Pretreated tissue sections were incubated with hybridization solution containing 20 mM Tris·HCl buffer (pH 7.4), 5 µg of cRNA probe, 0.5 mg/ml tRNA, 0.1 M DTT, 50% formamide, 0.3 M NaCl, 10 mM NaH2PO4 (pH 8.0), 5 mM EDTA, 10% dextran sulfate, and 1x Denhardt solution (Sigma). Sense or antisense probes were hybridized with tissue overnight on microslides at 55°C in a humidified chamber. After hybridization, the slides were washed for 30 min each at 55°C in 5x SSC (0.3 M NaCl/0.03 M sodium citrate, pH 7.0) and 2x SSC containing 10 mM DTT and treated with RNase. The slides were then washed in 2x SSC containing 50% formamide and 10 mM DTT at 65°C for 30 min and finally washed twice with 1x SSC containing 1% sodium pyrophosphate and 15 mM DTT at 55°C for 30 min. After being washed with PBS, the samples were blocked with 5% sheep serum for 30 min and incubated overnight in the presence of antidigoxigenin antibody conjugated with alkaline phosphatase. On the next day, the slides were washed with PBS, and color product was developed using nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate in 100 mM Tris·HCl buffer, pH 9.5, containing 100 mM NaCl, 50 mM MgCl2, and 10 mM levamisole. The reaction was stopped with 10 mM Tris and 1 mM EDTA, pH 8.0, following the development of optimal visible color.
Immunohistochemistry. To anatomically detect BDNF, NGF protein, and their corresponding receptors, we used the Vectastain Elite ABC kit (Vector Laboratory, Burlingame, CA) and the following primary antibodies from Santa Cruz Biotechnology: 1) anti-BDNF polyclonal IgG (SC-546), 2) anti-TrkB polyclonal IgG (SC-8316), 3) anti-NGF polyclonal IgG (SC-548), and 4) anti-TrkA polyclonal IgG (SC-118). Optimal primary antisera dilutions were assessed in a series of preliminary experiments, and they served as isotype controls for each other since all primary antibodies have the same isotype of rabbit IgG. The immunohistochemical specificity of the antibodies has been extensively studied in human tissues (42). Briefly, after quenching of endogenous peroxidase by using 0.3% H2O2 in PBS and blocking unspecific binding by blocking serum, we performed stepwise incubation. The primary antibodies (diluted to 1:800) were incubated at 4°C overnight, followed by application of biotinylated secondary antibody and ABC reagent. Diaminobenzidine chromogen (DAB substrate, Vector Laboratory) was used to visualize the antibody. Tissue sections stained with secondary antibody alone served as control.
Bright field microscopy. Sections were examined with a microscope (LEICA DMLB) equipped with the adequate filter systems. For each trait, the intensity of the signal for in situ and immunolabeled structures and the intensity of the background signal were measured using Sigma Scan Pro image analysis software (SPSS, Chicago, IL). Only sites expressing BDNF mRNA or immunolabeled specific protein signals at least two times above the background were considered to express the BDNF mRNA or BDNF, NGF, TrkA, and TrkB protein signals.
Statistical analysis. Results are presented as mean values ± SD, and Student's t-test was used to determine the difference between animal groups. P < 0.05 was regarded as a statistically significant difference.
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RESULTS |
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BDNF and NGF protein levels in lung and extrathoracic trachea. To further understand the responses of neurotrophins to hyperoxia, we evaluated the expression of BDNF and NGF protein in lung and extrathoracic trachea by ELISA (n = 10 in each group). BDNF protein level was substantially enhanced in hyperoxia-exposed lung; however, it was undetectable in the trachea in either hyperoxic or normoxic groups. Mean level of BDNF in the lungs of the normoxic group was 39.4 ± 6.9 pg/mg protein, compared with 147.6 ± 14.3 pg/mg protein in the hyperoxic group (P < 0.001, Fig. 3A). Compared with BDNF, substantial amounts of NGF were present in both lung parenchyma and extrathoracic trachea. NGF levels were similar between normoxic and hyperoxic groups in both lung and trachea. In normoxic vs. hyperoxic groups, the mean levels of NGF were 851.2 ± 42.2 pg/mg protein and 950.3 ± 37 pg/mg protein in the lung and 450 ± 19 pg/mg protein and 496 ± 15.2 pg/mg protein in the trachea, respectively (P > 0.1). Therefore, hyperoxic exposure enhanced BDNF expression in lung but had no obvious effect on NGF level (Fig. 3B).
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As shown in Fig. 5, in the normoxic rat pups, BDNF and TrkB were present in the bronchial epithelium, whereas in the hyperoxic rats, BDNF and TrkB protein were present not only in the bronchial epithelium, but also in peribronchial smooth muscle, forming a double layer appearance that is extremely similar to the BDNF mRNA distribution in the hyperoxia-exposed group. This result was consistent in all animals we randomly selected for histological study (n = 3 in each group). The data suggest that hyperoxic exposure upregulates expression of both BDNF and its high-affinity receptor TrkB in peribronchial smooth muscle, thus producing a multipotent effect.
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DISCUSSION |
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Expression of BDNF and TrkB receptors. In normoxia-exposed rat pups, low levels of BDNF mRNA and BDNF protein traits were present only in bronchial epithelial cells of the intrapulmonary airways. However, in neonatal rats exposed to hyperoxic stress, robust BDNF mRNA and protein expression occurred in peribronchial smooth muscle, indicating that these cells are capable of producing BDNF when challenged. Furthermore, hyperoxic exposure significantly elevated BDNF protein levels in lung tissue. These results indicate for the first time that synthesis of BDNF mRNA and protein, expressed by intrapulmonary airways, is upregulated following exposure to hyperoxia. Furthermore, our data suggest that peribronchial smooth muscle is the major source of the significant increase of BDNF levels in lungs after 7 days of exposure to hyperoxia, although we cannot totally exclude a contribution from resident macrophages that may be attracted to the lung by hyperoxic exposure (27).
Mechanisms involved in hyperoxia-induced BDNF upregulation are not well understood. Conceivably, high BDNF levels in neonatal rats exposed to oxygen could be linked to oxygen free radicals that activate redox-sensitive transcriptional factors (i.e., c-Fos, c-Jun, Egr-1, NF-B) (38). It has been shown that immediate-early genes such as c-Fos regulate the expression of BDNF, both in vivo and in vitro (50). Furthermore, activation of the NF-
B site in promoter 3 of the BDNF gene upregulates BDNF expression (39). Overexpression of BDNF may confer partial protection against hyperoxia-mediated cytotoxicity that can be explained partly by decreasing conversion from O2 to H2O2, a critical step for oxidant-mediated cell injury (49).
As already indicated, the increase in BDNF levels may represent a secondary event, a product of inflammatory cells attracted by hyperoxic lung injury. There are similarities between hyperoxic and allergic inflammation, since both activate a number of inflammatory mediators such as cytokines and adhesion molecules, some of which are found in high concentrations in airways of infants developing bronchopulmonary dysplasia (27). In an animal model of bronchial asthma, increased constitutive expression of BDNF mRNA was observed in the respiratory epithelium of sensitized mouse lungs (11). In addition, BDNF mRNA was detected in airway inflammatory infiltrates and bronchoalveolar lavage fluid (BALF) cells of ovalbumin-sensitized and aerosol-challenged mice. The highest BDNF protein levels were detected in BALF after long-term allergen aerosol exposure (11). Analysis of BDNF production by different cell types, including isolated proinflammatory cells, has revealed that fibroblasts, as well as activated lymphocytes and monocytes, are major cellular sources of BDNF (1, 30). Our present data suggest smooth muscle as a major source of increased BDNF production in response to hyperoxia. We have considered the possibility that BDNF may be anterogradely transported via axon terminals from airway-related vagal preganglionic neurons that innervate the airways (16, 2022) and taken up by the peribronchial smooth muscle cells that express TrkB receptors. However, in extrathoracic trachea, which is innervated by the same pool of vagal preganglionic neurons as intrapulmonary airways (2022), BDNF was undetectable, in contrast to relatively high levels of NGF. This lack of changes in BDNF in extrathoracic trachea excluded a contribution from anterograde transport to the elevation of BDNF protein in the lungs.
The effects of NTs are mediated through a family of tyrosine kinase receptors known as the Trk receptors (TrkA, TrkB, TrkC), and the low-affinity p75 neurotrophin receptor (p75NTR). The TrkB receptor is activated by BDNF, and it also responds to NT-4/5; the TrkC receptor is activated by NT-3, whereas NGF binds to TrkA receptors (15). The TrkB receptors are expressed in several subunits. Furthermore, truncated and catalytic isoforms of TrkB are coexpressed in neurons of rat and mouse (4). Although the significance of the "full-length" kinase-containing form is known, the role of truncated isoforms that lack the kinase domain is not well understood. In addition, BDNF and other NTs can bind with similar affinity to the nonselective p75NTR. Hence, the biological effects of BDNF can be mediated either by specific TrkB receptor signaling alone or by the conjoint activation of TrkB and p75NTR receptors by other NTs (15).
In the present study, we characterized the effect of hyperoxic stress on distribution and levels of TrkB receptor expression. As with BDNF protein expression in control neonatal rats, within the intrapulmonary airways, low levels of TrkB receptor protein signal were present only in bronchial epithelial cells. Exposure to hyperoxia caused redistribution of detectable TrkB receptors with a robust expression in peribronchial smooth muscle, indicating that these cells express the TrkB receptor gene that is silent in neonatal rats breathing room air. Activation of the TrkB gene encoding TrkB receptor protein by hyperoxia probably occurs via mechanisms that lead to BDNF upregulation. BDNF itself, when overexpressed and released, may activate this gene, leading to increased TrkB receptor protein expression. Together, these data indicate for the first time that the BDNF-TrkB-dependent system may play a significant role in regulating airway smooth muscle function following exposure to hyperoxic stress in neonatal rats.
Expression of NGF and TrkA receptors. In recent years, numerous studies have described the biological role of NGF (33), including its role in lung diseases (8). Investigations in humans showed that NGF concentrations in peripheral blood correlate well with the severity of allergic manifestations. The highest levels of NGF were measured in patients with asthma. Changes in NGF paralleled BDNF levels, indicating that factors that regulate expression of NGF including hormones, glucocorticosteroids, or inflammatory mediators also influence BDNF traits (8). Furthermore, it has been shown that NGF regulates the expression of BDNF mRNA in the peripheral nervous system (3). The results of the present study suggest that upregulation of BDNF and its receptors in response to hyperoxic stress is not necessarily associated with an increase in NGF and TrkA receptors, as hyperoxia had no effect on distribution and levels of NGF and TrkA receptors along the tracheobronchial tree. The differences in the response of NGF and BDNF expression to hyperoxic stress could be related to the differences in baseline levels of these NTs in neonatal rats breathing room air. Our findings in the developing respiratory system support the notion that the BDNF-TrkB receptor signaling pathway may respond to specific environmental changes without observed changes in NGF.
Role of BDNF-TrkB signaling pathway in hyperoxia-induced lung injury and altered airway function. Prolonged hyperoxic exposure could induce airway hyperreactivity by enhancing neuronal constrictive influences, increasing airway smooth muscle mass, and/or modulating airway inflammatory changes. It seems that in neonatal rats exposed to hyperoxic stress of 57 days in duration, mechanisms responsible for airway hyperreactivity are largely related to increase in contractile responses to cholinergic inputs. We and others (23) have shown that under in vivo (25) and in vitro conditions (2, 41), contractile responses of airway smooth muscle are disproportionally increased in hyperoxia-exposed neonatal rats compared with controls. Data have suggested that increased duration of the contraction could be related to a hyperoxia-induced decrease in relaxing mechanisms (7, 41). Although the contribution of BDNF to hyperoxia-induced changes in airway reactivity is not well understood, recent studies indicate that, in allergen-challenged mice, BDNF has no proinflammatory effects and does not affect inflammatory conditions or induce any inflammation by itself (12). Therefore, it seems unlikely that BDNF affects airway function indirectly via inflammatory effects on airway smooth muscle.
The possibility that hyperoxia alters neuronal control of the airways leading to airway hyperresponsiveness via the BDNF-TrkB receptor system is based on studies showing that BDNF enhances quantal neurotransmitter release (47), facilitates synaptic transmission (14), and unmasks silent synapses (26). In addition, BDNF may cause rapid excitation of neurons via activation of high-affinity TrkB receptors (28), inducing postsynaptic long-term potentiation (32). Airway-related vagal preganglionic neurons and the majority of airway intrinsic ganglionic cells express a cholinergic phenotype (19), defined by the production of choline acetyltransferase (ChAT), an enzyme involved in the biosynthesis of acetylcholine (ACh). Synthesis of ChAT, as well as production and release of Ach, may be facilitated by NTs. For example, NGF and BDNF increase the immunoreactivity of vesicular ACh transporter in cultured neurons from the embryonic rat septum (46). If a similar phenomenon occurs in the airways, then BDNF could upregulate cholinergic traits within the network controlling airway smooth muscle tone and consequently cause an increase in airway reactivity. In addition, hyperoxia acting through an increase in expression of BDNF may cause sensory hyperinnervation of the airways, similar to changes observed in transgenic mice overexpressing NGF (24). In hyperoxia-exposed neonatal rats, there is increased expression of the preprotachykinin gene that encodes substance P, as well as substance P levels (2). While substance P in neonatal rats induces relaxation of preconstricted airway smooth muscle (40), it potentiates the contractile effects of electrical field stimulation under in vitro conditions (2). Furthermore, previous studies have shown an association between NTs and tachykinins in regulating airway activity (17). Hence, BDNF may participate in hyperoxia-induced airway hyperresponsiveness by altering neuronal control of the airways.
Alternatively, the increase in BDNF-TrkB receptors might have some protective effects. Hyperoxia induces death of airway epithelial cells by both apoptosis and necrosis (6, 38). Increased BDNF levels and upregulation of TrkB receptors may contribute to reduced apoptotic processes, promoting survival and repair of injured lung structures. It has been shown that BDNF provides protection against apoptotic cell death and the Trk signaling cascade must be activated for this response to occur (31). BDNF may alternatively exert a protective effect on lung structures via increased expression and release of vasoactive intestinal peptide (VIP). VIP is present in intrinsic airway ganglionic neurons (18), as well as in airway-related vagal preganglionic neurons (29), and when released may exert protective effects on lung injury induced by free radicals as well as reducing bronchoconstrictive responses (44).
In summary, by using in situ hybridization and immunohistochemistry in addition to measuring BDNF mRNA and BDNF protein levels within the extrathoracic trachea and lungs, we have observed that exposure to hyperoxic stress upregulates BDNF mRNA and BDNF protein and increases the expression of the TrkB receptor signal within peribronchial smooth muscle. There was no corresponding effect on NGF and TrkA receptors. These data suggest that the BDNF-TrkB-dependent system may play a significant role in altered airway function following hyperoxic stress, and we speculate that it may contribute to hyperoxia-induced airway hyperresponsiveness in early postnatal life. Future in vitro and in vivo physiological studies employing exogenously administrated BDNF and blockade of endogenous BDNF in normoxic and hyperoxic rat pups will be needed to address this speculation.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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REFERENCES |
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