Persistent respiratory changes following intermittent hypoxic stimulation in cats and human beings

Respiratory Physiology & Neurobiology 140 (2004) 1–8

Persistent respiratory changes following intermittent hypoxic stimulation in cats and human beings Kendall F. Morris a,∗ , David Gozal b,1 a

b

Department of Physiology and Biophysics, University of South Florida Medical Center, 12901 Bruce B. Downs Blvd. MDC Box 8, Tampa, FL 33612-4799, USA Departments of Pediatrics and Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, KY 40202-1788, USA Accepted 17 December 2003

Abstract Repeated intermittent hypoxia or other stimulation of carotid chemoreceptors produces a consistent long-term increase in respiratory nerve activity in vagotomized, artificially ventilated anesthetized or decerebrate animals, but variable results have been reported in more intact preparations. We sought additional variables that could be measured to help gain an understanding of persistent respiratory responses to intermittent hypoxia. The variance of respiratory phases decreased in 10 of 11 recordings from vagotomized anesthetized cats during long-term facilitation induced by carotid chemoreceptor stimulation. The variance of expiratory time was reduced in 10 awake human beings exposed to repetitive, brief episodes of isocapnic hypoxia (6% O2 in N2 , 60 s). Respiratory frequency was increased in humans and tidal volume decreased so that minute ventilation remained unchanged. The results suggest that there are persistent changes in the output of the respiratory central pattern generator following intermittent peripheral chemoreceptor stimulation or hypoxia. © 2004 Elsevier B.V. All rights reserved. Keywords: Hypoxia, intermittent, nerve activity; Mammals, cat; Mammals, humans; Nerve, respiratory, hyposia; Timing, long-term facilitation

1. Introduction Repeated stimulation of carotid chemoreceptors produces a persistent increase in frequency and amplitude of respiratory efferent activity in vagotomized, paralyzed, artificially ventilated anesthetized or decerebrate animals (Millhorn et al., 1980a,b; Hayashi ∗ Corresponding author. Tel.: +1-813-974-1549; fax: +1-813-974-3079. E-mail address: [email protected] (K.F. Morris). 1 Present address: Department of Pediatrics, University of Louisville, 571 S. Preston St., Suite 321, Louisville, KY 402021788, USA.

et al., 1993; Bach and Mitchell, 1996; Morris et al., 1996a). Several studies in more intact preparations have reported the existence of long-term facilitation (Cao et al., 1992; Mateika and Fregosi, 1997; Turner and Mitchell, 1997). Additional research suggests that expression of long-term facilitation depends on age, gender, preconditioning and genetics (Gozal and Gozal, 1999; Ling et al., 2001; Behan et al., 2003; for reviews see: Johnson and Mitchell, 2002; Feldman et al., 2003; Mitchell and Johnson, 2003). A preliminary report investigating persistent effects of repeated hypoxia in awake humans (Stewart et al., 1994) reported an increase in ventilation. However,

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subsequent studies failed to detect a long-term change in the majority of healthy, awake human beings (McEvoy et al., 1996; Jordan et al., 2002), while it was detected in snorers during non-rapid eye movement sleep (Babcock and Badr, 1998; Aboubakr et al., 2001; Babcock et al., 2002). Our own preliminary studies of intermittent hypoxia in humans also initially failed to detect persistent changes in ventilation. A review of the literature suggested several limitations in previous analysis methods. Non-human studies focused on individual differences in breathing pre- versus post-intermittent hypoxia (McEvoy et al., 1996; Jordan et al., 2002). Human studies, including our own initially, grouped data (McEvoy et al., 1996; Babcock and Badr, 1998; Aboubakr et al., 2001; Babcock et al., 2002; Jordan et al., 2002), perhaps losing individual differences in group variation. The spinal activity of serotonin is necessary for the full expression of long-term facilitation (Ling et al., 2001; Johnson and Mitchell, 2002; for a review see: Mitchell and Johnson, 2003). Systemic administration of a non-specific serotonin receptor competitive inhibitor blocks both the induction and expression of long-term facilitation (Millhorn et al., 1980b; Bach and Mitchell, 1996). The major source of serotonin in the brainstem is the raphe nuclei (Palkovits et al., 1974; Jacobs and Azmitia, 1992). Electrical stimulation of raphe obscurus can induce long-term facilitation similar to that of carotid chemoreceptor stimulation (Millhorn, 1986). Neurons in raphe obscurus respond to carotid chemoreceptor stimulation with rate changes and show evidence in cross correlation histograms for connectivity to cells with respiratory modulated firing rates in brainstem regions implicated in respiratory control and with phrenic nerve (Morris et al., 1996a, 2001). Previous work has suggested that the caudal raphe nuclei are involved in motor gain control (Yates et al., 1992; Lindsey et al., 1998). Long-term facilitation of respiration may involve a resetting of this gain control (Morris et al., 1996a, 2001; for reviews see: Morris et al., 2000, 2003). Measurement of persistent changes in breathing after intermittent hypoxia tended to focus on the more obvious, gross changes in amplitude of motor output or ventilation. Bruce (1996) suggested that the contributions of various stimuli, including chemoreceptor stimulation, to the variability of breathing

might provide further insight into respiratory control mechanisms. Gain control resetting may be expressed as increased amplitude and frequency of motor output with less variability in a system with negative feedback removed. We hypothesized that while the increased motor output may not be expressed as increased pulmonary ventilation in some more intact systems, changes in pattern and decreased variability might remain. We re-examined the long-term changes in respiratory activity in a cat model previously studied in our laboratory in order to more carefully describe persistent changes in respiratory motor output for more accurate comparison with human responses. We sought evidence for the hypothesis that persistent changes in ventilatory pattern, including a decrease in the variance of respiratory phases, is a component of long-term facilitation of breathing.

2. Methods 2.1. Cat studies Materials and methods of cat experiments have been described elsewhere (Morris et al., 1996a, b). The data consisted of all recordings (n = 11) with a long-term facilitation of integrated phrenic amplitude following repeated carotid chemoreceptor stimulation in nine vagotomized, thoracotomized, artificially ventilated, adult cats of either sex (2.5–5.7 kg). Other results from these experiments have been reported (Morris et al., 1996a, b). Anesthesia was maintained with dial/urethane (allobarbital, CIBA, 60.0 mg kg−1 ; urethane, 240 mg kg−1 ). Neuromuscular blockade was accomplished with a bolus of gallamine triethiodide (2.2 mg kg−1 ) followed by constant infusion (0.4 mg kg−1 h−1 ). The animals were given additional dial/urethane if there was an increase in blood pressure or phrenic activity in response to periodic noxious stimuli (toe pinch). The C5 phrenic nerve rootlet was desheathed and cut peripherally for subsequent placement on bipolar, silver wire, hook electrodes in a pool of mineral oil. Control activity was recorded for at least 15 min prior to the onset of stimulation. Carotid chemoreceptors were stimulated in series of 3–6 stimuli at 3–5 min intervals with 200 ␮l of CO2 saturated, 0.9% saline solution, injected over a period

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of 30 s through a catheter inserted into the external carotid artery. Phrenic nerve multiunit activity was amplified and fed into a resistor-capacitor “leaky” integrator with a time constant of 200 ms. A moving, smoothed first derivative was used to detect the start and end of inspiration (Korten and Haddad, 1989) from the digitized, integrated phrenic nerve signal. The criterion for long-term facilitation was integrated phrenic nerve amplitude at least 2 standard deviations greater than pre-stimulus levels. Two samples of 15–21 respiratory cycles each were measured at the beginning and end of the control period prior to the first stimulation and a third beginning approximately 5 min after the last stimulus period, after the period of short-term potentiation. The samples were chosen to be long enough to provide robust statistical analysis, while minimizing potential effects that might be attributed to metabolism of anesthetic or deterioration of the preparation. The mean durations of the inspiratory and expiratory phases and the standard deviations of those means were calculated. While the means of random samples from a normal population display a t distribution, the standard deviations have an F distribution. The student’s t-test (P < 0.05) was used to detect changes in means and the F-test (P < 0.05) used to assess changes in variance. 2.2. Human studies Three female and seven male adults (ages: 31.0±3.7 years), were studied after signing an informed consent of the experimental protocol, which received approval from the Institutional Review Board. All subjects were healthy and had no history of cardiopulmonary disease. To confirm this, pulmonary function studies were performed in the laboratory, located near sea level (mean atmospheric pressure 761 mmHg). The best forced vital capacity (FVC), forced expiratory volume in 1 s (FEV1 ), mean forced expiratory flow during the middle half of forced vital capacity (FEF25–75% ) and maximal expiratory flow volume curves were obtained from forced expiration into a wedge spirometer (Collins, DSIIa, Braintree, MA), and corrected for body temperature, pressure saturated (BTPS). Individual test results were considered abnormal if they were >±2S.D. from available reference values (Crapo et al., 1981), and led to exclusion from the study. Mean FVC was 117 ± 8

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(±S.E.M.)% predicted, FEV1 was 97 ± 3% predicted, and FEF25–75% was 88 ± 5% of predicted. 2.2.1. Ventilatory measurements Subjects were studied awake, sitting comfortably, wearing nose clips, and spontaneously breathing through a mouthpiece. All subjects were visually monitored to ensure that they did not fall asleep. Oxygen saturation was continuously measured by pulse oximetry (Nellcor 3000, Hayward, CA). Subjects were connected via the mouthpiece to a Hans Rudolph pneumotachograph, and to a two-way non-rebreather valve (Hans Rudolph, Kansas City, MO). PCO2 was sampled continuously at the expiratory port of the two-way valve, and analyzed breath-by-breath using an infrared microcapnometer (Columbus Instruments, Columbus, OH). The gas monitor was calibrated with gas mixtures of known CO2 concentrations. The end-expiratory carbon dioxide tension (PETCO2 ) was held constant throughout the experiment at 45 mmHg. This was achieved by a custom-made microcomputer assembly (Lab-LC, National Instruments, Austin, TX) that provided control signals to the gas flow controllers, so that the CO2 composition of the inspired gas mixture could be adjusted to force PETCO2 at the desired concentration. The dead space of this system was approximately 85 ml. During each test, expiratory flow was measured with a heated pneumotach and a pressure transducer (Valydine, Northridge, CA). The signal was calibrated with a mechanically-driven pump yielding 1000 ml stroke volume at a frequency of 10 strokes/min. Corrections were made for changes in gas viscosity due to the changes in oxygen concentration in the inhaled gas mixture which was warmed and humidified immediately before the inspiratory port of the two-way non-rebreather respiratory valve. Breath-by-breath tidal volume (VT ) was obtained by analog integration of the flow signal. Analog output channels were continuously displayed on screen, and digitally acquired onto a MacIntosh Personal Computer System at 125 Hz sampling frequency, as dictated by the Nyquist theorem, using MacLab Digital Acquisition Software (ADInstruments, Castle Hill, Australia). During subsequent off-line analysis, VT , inspiratory time (TI ) and SaO2 were measured for each breath. Expiratory time (TE ), respiratory rate (RR; 60/TI + TE ), and expired ventilation (V˙ E ) were calculated.

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2.2.2. Hypoxic challenges Following an initial 2–4 min period of tidal breathing of an isocapnic normoxic gas mixture to establish baseline, subjects were surreptitiously switched to the designated hypoxic gas mixture by a silent, remote pneumatic valve switch mechanism (Hans Rudolph, Kansas City, MO). The hypoxic gas mixture consisted of either 6% O2 in N2 or 10% O2 in N2 , to which CO2 was added by the computer-controller to maintain PETCO2 at the pre-set level. The hypoxic gas was administered for 60 s, at which time subjects were switched back to isocapnic normoxia for the remainder of the test. At least three cycles as described above were performed by each subject with recovery periods of 15 min being allowed between trials. Runs in which PETCO2 differed by more than 4 mmHg from the individual resting PETCO2 levels at any given time during the experiment were discarded. Hypoxic challenges with 6 and 10% O2 were administered on different days, and in random order. 2.2.3. Data analysis Respiratory analog data for each run were analyzed on a breath-by-breath basis by a custom-designed peak-trough detection procedure (Wavemetrics, Lake Oswedo, OR). Ventilatory measures in isocapnic normoxia before the onset of the hypoxic stimulus (pre) and after subjects’ V˙ E returned to pre-hypoxic baseline values (post) were compared. Thus, two samples of at least 20 respiratory cycles each were measured for pre and post conditions. The sample sizes were chosen to provide robust statistical measures while minimizing effects of boredom and concomitant sleepiness. The mean durations of the inspiratory and expiratory phases and the standard deviations of those means were calculated. The student’s t-test was used to detect changes in means and the F-test was used to assess changes in variance. A P-value of <0.05 was considered to achieve statistical significance in either case.

3. Results 3.1. Cat experiments In agreement with previous reports (Millhorn et al., 1980a, b; Hayashi et al., 1993; Bach and Mitchell,

1996) there was an increase in respiratory frequency measured as a decrease in mean inspiratory or expiratory time in the recordings of anesthetized, vagotomized cats after carotid chemoreceptor stimulation. Ten of the 11 recordings had a decrease in variance of inspiratory or expiratory phase. Only one recording had a significant increase in inspiratory phase variance. None of these changes was evident between the two pre-stimulus control periods. The measurements of TI , TE and coefficients of variation are given in Table 1. The average percent decreases in TI and TE and percent decreases of the coefficients of variation (i.e. the average percent decreases of standard deviations normalized by expression as a percent of mean) before and after induction of long-term facilitation for all trials are summarized in the first row of Table 4.

Table 1 Measures of TI and TE and coefficients of variation (CV; normalized standard deviations, i.e. standard deviation expressed as a percent of the mean) before and after protocols Pre

CV

Post

CV

1.71 2.57 3.48 5.42 2.78 2.16 4.63 2.35 1.43 1.81 2.46

11.91 54.87 6.74 31.34 17.68 8.36 12.62 44.26 18.87 9.11 12.74

1.62 1.53∗ 2.41∗ 4.95 1.72∗ 1.75∗ 2.73∗ 2.26 1.28 1.95 1.45∗

5.10∗ 12.86∗ 8.07 15.78∗ 4.10∗ 5.27∗ 11.85∗ 23.99∗ 18.84 13.86∗ 13.33∗

TE 1.04 0.85 2.14 2.90 3.17 2.12 1.72 1.80 0.41 1.76 1.38

22.33 27.02 4.11 6.77 15.30 5.36 3.83 22.51 24.44 8.62 9.82

0.92 0.79 1.97∗ 2.49∗ 2.42∗ 1.68∗ 1.65∗ 1.55∗ 0.41 1.30∗ 0.99∗

5.45∗ 8.60∗ 4.15 7.39 2.49∗ 5.98 4.11 9.50∗ 10.65∗ 18.47∗ 18.55

TI

Eleven recordings were made with nine anesthetized, vagotomized cats; induction of long-term facilitation of respiration was by close arterial injection of CO2 saturated saline. Asterisks denote significant changes.

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Table 2 Mean ± standard deviation of ventilatory measurements in 10 subjects during isocapnic normoxia before and following at least three 1 min challenges of isocapnic hypoxia with 10 and 6% O2 10% O2

6% O2

Pre

Post

V˙ E (l min−1 ) VT (l) RR (br min−1 ) PETCO2 (Torr)

7.16 0.52 13.8 44.9

TI (s) TE (s)

1.56 ± 0.21 2.79 ± 0.66

± ± ± ±

1.84 0.09 2.9 1.5

7.30 0.46 15.9 44.8

Pre ± ± ± ±

1.14 0.06 1.7 1.1

7.03 0.58 13.1 44.9

1.40 ± 0.15 2.38 ± 0.36

Post ± ± ± ±

1.87 0.13 2.6 1.4

7.47 0.44 16.9 45.0

1.51 ± 0.22 3.07 ± 0.65

± ± ± ±

1.18 0.09 1.5 1.0

1.29 ± 0.13 2.26 ± 0.33

While each individual showed combinations of significant decreases in TI , TE or variance, as a group there was only a non-significant trend in those directions; individual changes were lost in relatively larger group variations.

3.2. Human experimental results During the baseline control periods, there were no significant trends in any measured variable. Table 2 gives group means and standard deviations of ventilatory measurements from before and after brief intermittent exposure to the two levels of hy-

poxia. While the average TI and TE decreased, there were no statistically significant changes in any variable; individual changes were submerged in group variance. When individual data were analyzed, increases in respiratory frequency were apparent for both post 10 and 6% O2 . The increases in frequency were due to

Table 3 TI , TE and coefficients of variation (CV) before and after protocols TE (6% O2 ) Pre 3.97 3.53 2.93 2.24 3.36 2.46 2.97 3.55 3.49 2.17 TE (10% O2 ) 3.96 2.60 2.23 2.14 2.96 2.17 2.79 3.51 3.48 2.05

TI (6% O2 ) CV

Post

9.82 9.63 10.24 14.29 10.12 10.57 8.42 10.14 12.89 12.90

2.96∗

CV

2.35∗ 1.99∗ 1.87∗ 2.43∗ 2.06∗ 2.57∗ 2.37∗ 2.25∗ 1.78∗

9.36∗ 9.55∗ 9.09∗ 10.70∗ 7.28∗ 5.45∗ 6.75∗ 10.22∗ 6.18∗

10.86 12.31 15.25 13.55 10.47 12.44 8.60 11.11 12.07 11.71

2.78∗ 2.38∗ 1.99∗ 2.03∗ 2.76 2.02 2.55∗ 3.02∗ 2.36∗ 1.92∗

8.99∗ 10.08∗ 10.55∗ 7.88∗ 10.14 7.92∗ 6.67∗ 5.96∗ 9.32∗ 6.77∗

7.77∗

Pre 1.48 1.51 1.30 1.50 1.76 1.52 1.42 1.51 1.93 1.20 TI (10% O2 ) 1.54 1.58 1.33 1.61 1.79 1.58 1.48 1.53 1.94 1.24

CV

Post

CV

16.22 17.88 16.92 16.00 15.34 17.11 18.31 19.21 16.06 15.83

1.30∗ 1.26∗ 1.19∗ 1.20∗ 1.35∗ 1.31∗ 1.22∗ 1.49∗ 1.52∗ 1.11∗

14.62 17.46 15.13 8.33∗ 13.33∗ 16.79 18.03 9.40∗ 16.45 14.41

14.29 16.46 13.53 15.53 15.08 15.19 18.24 18.30 16.49 14.52

1.36∗ 1.37∗ 1.15∗ 1.48∗ 1.56∗ 1.44∗ 1.29∗ 1.38∗ 1.77∗ 1.16∗

14.71 16.79 12.17 14.19 13.46 14.58 17.05 16.67 14.69 14.66

Results from 10 awake human beings were after at least three 1 min isocapnic challenges with 6 or 10% O2 in N2 . Asterisks denote significant changes.

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Table 4 Summary of the average ± standard deviation percent decreases in TI , TE and coefficients of variation (CV) before and after protocols Average percentage decrease in TI duration

Average percentage decrease of CV for TI

Average percentage decrease in TE duration

Cat

21.0 ± 17.9

24.8 ± 41.67

14.3 ± 9.2

9.4 ± 66.4

Human 6% Human 10%

13.8 ± 6.8 10.6 ± 2.5

14.5 ± 19.1 5.4 ± 5.5

25.2 ± 8.4 12.9 ± 9.8

23.4 ± 17.8 28.1 ± 13.7

decreases in both TI and TE . TI was significantly reduced in all 10 subjects after both protocols. TE was decreased post 10% in eight, unchanged in the remainder, and decreased post 6% in all 10. However, decreases in VT offset increases in frequency such that VE were not significantly changed. A reduced variance of respiratory frequency emerged after the challenges and was sustained for the duration of the measurements after the hypoxic challenges (ranging from 20 to 30 min). The decrease in frequency variability was primarily dependent on significant reductions in TE variance with lesser contributions from TI variance. Although there was an average decrease, only three subjects showed a significant decrease in inspiratory phase variance post 6% O2 , none after 10%, none had an increase. Nine of the 10 showed decreases in TE variance after 10% and all had decreased after 6% O2 . TI , TE and coefficients of variation are given in Table 3. The average decreases in durations and the coefficients of variation (normalized standard deviations) of TI and TE after both protocols are reported in Table 4.

4. Discussion The most significant result of this study is that all human subjects tested showed persistent, significant individual changes in respiratory pattern. Results from the experiments with anesthetized, vagotomized cats support the hypothesis that an increase in frequency and a decrease of respiratory phase variance accompany long-term facilitation of respiratory activity induced by carotid chemoreceptor stimulation. While we did not observe an increase in pulmonary minute ventilation in the experiments with human beings, those data also indicate persistent increased frequency and a decrease in variance.

Average percentage decrease of CV for TE

In a study of conscious dogs, Cao et al. (1992) reported a long-term facilitation that was expressed primarily as an increase in tidal volume, similar to the increased integrated phrenic amplitude in less intact models. In a series of elegant experiments with awake, vagally intact goats, Turner and Mitchell (1997) were able to unmask long-term facilitation of respiration following repeated hypoxia. The animals were kept normocapnic with supplementary inspired CO2 . They reported a mean 68% increase in minute ventilation. In contrast to the experiments with dogs an increase in frequency made the greatest contribution to long-term facilitation, similar to the present findings in human beings. Other experiments comparing vagotomized and non-vagotomized freely breathing anesthetized cats suggested that there was a masked persistent memory in the latter (Mateika and Fregosi, 1997). It was found that vagal mechanisms inhibited long-term facilitation in diaphragm and upper airway electromyographic activity but tidal volume was increased long-term. It was hypothesized that there was a long-term facilitation of accessory muscle activity. Vagal feedback may have also contributed to masking long-term facilitation in the present study. In human beings, a preliminary report found suggestions of long-term facilitation (Stewart et al., 1994). Others reported long-term facilitation during sleep, but only in snorers (Babcock and Badr, 1998; Aboubakr et al., 2001; Babcock et al., 2002). In agreement with other studies of males and females (McEvoy et al., 1996; Jordan et al., 2002), we found no change in minute ventilation in any subject after the hypoxic challenges; the changes in tidal volume offset the changes in frequency. Although the latter study reported that two subjects had a postintermittent hypoxia persistent increase in ventilation >10%, neither an analysis of individual changes in

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breathing pattern nor an analysis of variance was reported in either study. The differences in the results in other animals and human beings may relate to species differences or differences in the experimental paradigm. In addition, the previous studies in human beings grouped data, i.e. sought evidence that group ventilation was significantly greater than pre-stimulus means, whereas animal studies tended to measure individual changes in respiratory activity for differences pre- versus post-stimuli. Significant differences in respiratory patterns of individuals may be lost due to relatively large group variation. We controlled CO2 levels in the experiments with humans reported here during hypoxic hyperpnea but it is possible that small changes (<4.0 mmHg) affected the stimulus protocol. Since there was no change in alveolar ventilation in the post-stimulus period, no additional CO2 was necessary and there were no significant differences in pre- versus post-stimuli that may have contributed to the observed changes in breathing. Two different magnitudes of hypoxic stimulation were employed. Although significant increases in frequency and reductions in the coefficient of variation for expiratory time occurred following the hypoxic stimuli, there was no evidence of a dose-dependent effect. The reductions in variance were similar with either protocol. Previous reports have suggested that lack of expression of long-term facilitation in human beings is due to concurrent hypoxic stimulation and hypoxic depression (Powell et al., 1998; Jordan et al., 2002). Although in the present study the duration of each stimulus was constrained to 1 min with longer periods between hypoxic exposures than the protocols of other studies (McEvoy et al., 1996; Babcock and Badr, 1998; Aboubakr et al., 2001; Jordan et al., 2002; Babcock et al., 2002), this possibility still exists. The changes in frequency and variance noted here might be the result of these competing effects as well as vagal input noted above.

Acknowledgements This work was supported by N.I.H. Grants NS19814, HL69932, HL63912, and HL66358. The authors thank J. Gilliland, C. Orsini, D. Baekey, J.E.

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Torres, and K.D. Morris for excellent technical support and Roger Shannon and Bruce G. Lindsey for comments on the manuscript.

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