Edited by Tamas Bartfai, The Scripps Research Institute, La Jolla, CA, and accepted by the Editorial Board April 24, 2007 (received for review January 2, 2007)
IL-1β and anoxia rapidly induce brainstem mPGES-1. mPGES-1 activity in the microsomal fraction of cortex and brainstem, including endothelial cells of the BBB, was analyzed in 9-d-old mice (n = 33) treated with IL-1β or vehicle and subjected to normoxia or normoxia plus anoxia (100% N2, 5 min). (A) In wild-type mice, mPGES-1 activity was measured at 90 min after NaCl (control) or 90 min and 180 min after IL-1β treatment. Higher endogenous mPGES-1 activity was observed in the brainstem compared with cortex in control mPGES-1+/+ mice. In addition, IL-1β induced mPGES-1 activity in a time-dependent manner. (B) At 90 min, IL-1β-treated mice exhibited approximately 2-fold higher activity in the brainstem compared with saline-treated mice. Anoxia also significantly induced mPGES-1 activity. Moreover, the effects of IL-1β and transient anoxic exposure were additive. When IL-1β-treated mice were exposed to anoxia, 4-fold higher activity was observed in the brainstem compared with control mice. However, mice with genetic deletion of mPGES-1 gene displayed negligible activity in response to IL-1β and anoxia. Data are presented as mean ± SEM. ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001.
IL-1β depresses respiration through mPGES-1 activation. Using whole-body flow plethysmography, basal respiration and the ventilatory response to hyperoxia were examined in 9-d-old mPGES-1 wild-type mice (n = 66) and mPGES-1 knockout mice (n = 34) after i.p. administration of either IL-1β (n = 52) or NaCl (n = 48). (A) Plethysmograph recordings illustrate breathing during normoxia and hyperoxia in wild-type mice given NaCl or IL-1β (5-s period, breath amplitude 1 μl/s). (B and C) All mice responded to hyperoxia with a reduction in f R (breaths per min). IL-1β depressed f R to a greater extent than NaCl in mPGES-1 mice+/+, whereas IL-1β did not alter respiration during normoxia or hyperoxia in mPGES-1−/− mice. mPGES-1+/+ mice exhibited a greater respiratory depression during hyperoxia compared with mPGES-1−/− mice. Data are presented as mean ± SEM. ∗, P < 0.05 compared with mPGES-1+/+ mice given NaCl.
IL-1β reduces anoxic survival via mPGES-1. Nine-day-old mPGES-1+/+ mice (n = 37) and mPGES-1−/− mice (n = 20) were exposed to 5 min of anoxia (100% N2) at 80 min after peripheral administration of IL-1β (n = 29) or vehicle (n = 28). (A) Plethysmograph recording of mPGES-1+/+ mouse given NaCl depicting the initial hyperpnea and subsequent gasping response to anoxia. The mouse autoresuscitated after 100% O2 was administered. (B) Plethysmograph recording of mPGES-1+/+ mouse given IL-1β showing the brief hyperpnea period and subsequent gasping response to anoxia. The mouse failed to autoresuscitate after 100% O2 was administered. (C) The number of gasps tended to differ between groups (Wilcoxon χ2, P = 0.06). When comparing treatment effects within each genotype, IL-1β decreased the number of gasps in wild-type mice, whereas this effect was not observed in mice lacking mPGES-1. (D) IL-1β reduced the survival rate anoxic compared with NaCl in mPGES-1+/+ mice, but not in mPGES-1−/− mice. Data are presented as mean ± SEM. ∗, P < 0.05; ∗∗, P < 0.01.
PGE2 depresses brainstem respiratory activity and induces apnea via brainstem EP3Rs. Respiration was examined in EP3R+/+ (n = 13) and EP3R−/− (n = 25) neonatal mice after administration of PGE2 (n = 19) or NaCl (n = 19). (A) PGE2 was injected i.c.v. at 0 min followed by normoxia and a 1-min hyperoxic challenge in newborn EP3R+/+ (■) and EP3R−/− (□) mice. The EP3R+/+ mouse exhibited a lower f R (breaths per min) and an irregular respiratory rhythm with elevated coefficient of variation (C.V.) during normoxia and hyperoxia due to apneic breathing. In the EP3R−/− mouse, basal f R did not decrease after the postanesthesia period, and there was less variability in the respiratory pattern. No temperature difference or dependency was observed during the first 20 min after i.c.v. administration of PGE2. (B) Plethysmograph recordings (10-s periods with breath amplitude of 1 μl/s) demonstrate apnea episodes in response to PGE2 during normoxia in an EP3R+/+ mouse, but not in an EP3R−/− mouse. (C) In EP3R+/+ mice, PGE2 induced more apneas during normoxia and hyperoxia compared with vehicle. This effect of PGE2 was not observed in EP3R−/− mice. (D) In en bloc brainstem spinal–cord preparations from 2- to 3-d-old EP3R+/+ pups (■, n = 5), PGE2 (20μg/liter) reversibly depressed respiratory rhythm generation to 64 ± 5% of control frequency (f R) (ANOVA repeated measures design, P < 0.01). PGE2 did not affect respiratory activity in preparations from EP3R−/− mice (□, n = 6). (E) In transverse medullary sections, respiration-related neurons within the RVLM ventral to the nucleus ambiguus (NA) and including the preBötC coexpress NK1R (red) and EP3R (green). The arrows indicate EP3R and NK1R colocalization (yellow) in some RVLM respiration-related neurons. (F) NK1R, but no EP3R, expression was identified in an EP3R−/− mouse. (Scale bar, 100 μm.) Data are presented as mean ± SEM. ∗, P < 0.05 compared with EP3R+/+ mice given NaCl.
PGE2 in CSF is correlated to apnea index in neonates. CSF was collected from infants in the neonatal intensive care unit who had clinical indications for lumbar puncture (n = 12, mean postnatal age 16 ± 4 d; mean gestational age 32 ± 2 week). Infants then underwent a cardiorespiratory recording (duration 9.2 ± 2.4 h). PGE2 concentrations in the CSF were analyzed using a standardized enzyme immunoassay (EIA) protocol and correlated to the infectious marker CRP and apnea index (number of apneas per h). Central PGE2 concentrations were positively correlated to the CRP levels in blood (P = 0.01). Moreover, a striking association was observed between central PGE2 concentrations and apnea index (P < 0.05). Here, we distinguish between undetectable levels of PGE2 (0 ± 0 pg/ml) compared with high levels of PGE2 (52 ± 22 pg/ml). Data are presented as mean ± SEM.
Model for IL-1β-induced respiratory depression and autoresuscitation failure via a PGE2-mediated pathway. During a systemic immune response, the proinflammatory cytokine IL-1β is released into the peripheral blood stream. It binds to its receptor (IL-1R) located on endothelial cells of the BBB. Activation of IL-1R induces the synthesis of PGH2 from arachidonic acid (AA) via COX-2 and the synthesis of PGE2 from PGH2 via of the rate-limiting enzyme mPGES-1. PGE2 is released into the brain parenchyma and binds to its EP3R located in respiratory control regions of the brainstem, e.g., NTS and the RVLM. This results in depression of central respiration-related neurons and breathing, which may fatally decrease the ability to gasp and autoresuscitate during hypoxic events.
Respiration during normoxia, hyperoxia, and anoxia in EP3R mice after central PGE2 administration
| Genotype | Treatment | Normoxia | Hyperoxia | Hyperpnea | ||||
|---|---|---|---|---|---|---|---|---|
| fR | VT | VE | fR | VT | VE | fR | ||
| EP3R+/+ | NaCl (n = 7) | 281 ± 17 | 3.8 ± 0.4 | 1,065 ± 75 | 234 ± 19 | 7.0 ± 3.0 | 1,598 ± 642 | 327 ± 13 |
| PGE2 (n = 6) | 247 ± 13* | 3.7 ± 0.4 | 901 ± 154 | 190 ± 16 | 4.4 ± 1.1 | 745 ± 102 | 267 ± 11** | |
| EP3R−/− | NaCl (n = 12) | 247 ± 15 | 5.3 ± 0.6 | 1,322 ± 157 | 200 ± 23 | 5.4 ± 0.9 | 1,057 ± 213 | 288 ± 11 |
| PGE2 (n = 13) | 256 ± 10 | 5.2 ± 0.5 | 1,350 ± 129 | 229 ± 9 | 6.7 ± 1.3 | 1,509 ± 299 | 290 ± 9 | |
f R (breaths per min), tidal volume (V T) (μl per breath per g), and minute ventilation (V E)(μl·min·g) during normoxia, hyperoxia (100% O2), and anoxia (100% N2) were examined in 9-d-old EP3R+/+ mice (n = 13) and EP3R−/− mice (n = 25) after i.c.v. injection of PGE2 or vehicle. When comparing treatment effects within each genotype, PGE2 significantly depressed f R during normoxia and hyperpnea in EP3R+/+ mice, but not in EP3R−/− mice. PGE2 also tended to reduce f R during hyperoxia in EP3R+/+ mice (ANOVA, P = 0.11), but not in EP3R−/− mice. Data are presented as mean ± SEM.
↵*, P < 0.05;
↵**, P < 0.01.
|
Hofstetter et al. 10.1073/pnas.0611468104. |
Table 2. Respiration during normoxia and hyperoxia in mPGES-1 mice after peripheral IL-1β administration
|
Genotype |
Treatment |
Normoxia |
Hyperoxia | ||||
|
|
|
fR |
VT |
VE |
fR |
VT |
VE |
|
mPGES-1+/+ |
NaCl (n = 33) |
234 ± 6 |
3.2 ± 0.1 |
745 ± 30 |
181 ± 6 |
4.4 ± 0.4 |
791 ± 85 |
|
IL-1β (n = 33) |
224 ± 5** |
3.2 ± 0.2 |
730 ± 38 |
155 ± 7* |
3.9 ± 0.2 |
628 ± 43 | |
|
mPGES-1−/− |
NaCl (n = 15) |
247 ± 7 |
2.8 ± 0.2 |
684 ± 45 |
195 ± 14 |
3.9 ± 0.5 |
771 ± 142 |
|
IL-1β (n = 19) |
245 ± 7 |
2.7 ± 0.1 |
660 ± 41 |
206 ± 11 |
3.9 ± 0.3 |
795 ± 67 | |
fR (breaths per min), tidal volume (VT) (μl per breath per g), and minute ventilation (VE) (μl·min·g) during normoxia and hyperoxia (100% O2) were examined in 9-d-old mPGES-1+/+ and mPGES-1−/− mice after intraperitoneal injection of IL-1β or vehicle. When comparing treatment effects within each genotype, IL-1β tended to reduce basal fR in mPGES-1+/+ mice (Wilcoxon χ2, P = 0.17), but not in mPGES-1−/− mice. All mice responded to hyperoxia with a reduction in fR. IL-1β depressed fR during hyperoxia in mPGES-1+/+ mice, and this effect was not apparent in mPGES-1−/− mice. mPGES-1+/+ mice exhibited a greater extent of respiratory depression during hyperoxia compared to mPGES-1−/− mice. Data are presented as mean ± SEM. *, P < 0.05; **, P < 0.05 when normalized by weight.
Table 3. Biphasic ventilatory response to anoxia
|
Genotype |
Treatment |
Hyperpnea |
Gasping response | |||
|
|
|
fR |
Duration |
Gasp no. |
Gasp fR |
Duration |
|
mPGES-1+/+ |
NaCl (n = 20) |
368 ± 11 |
63 ± 2 |
38 ± 2 |
25 ± 1 |
94 ± 6 |
|
IL-1β (n = 17) |
390 ± 11 |
61 ± 2 |
30 ± 2** |
23 ± 1 |
82 ± 7 | |
|
mPGES-1−/− |
NaCl (n = 8) |
339 ± 25 |
55 ± 4 |
37 ± 3 |
23 ± 3 |
113 ± 18 |
|
IL-1β (n = 12) |
338 ± 24 |
57 ± 2 |
36 ± 3 |
18 ± 2 |
146 ± 23 | |
Newborn mice with variable expression of mPGES-1 were exposed to anoxia at 80 min after peripheral administration of IL-1β or vehicle. Mice exhibited an initial increase in fR, VT, and VE during hyperpnea followed by gasping response during hypoxic ventilatory depression. When comparing treatment effects within each genotype, IL-1β decreased the number of gasps in wild-type mice, whereas this effect was not observed in mice with reduced expression of mPGES-1. Data are presented as mean ± S.E.M.. **, P < 0.01.
SI Materials and Methods
Animals. All animals were killed via decapitation immediately following experimentation, and genotyping was performed using PCR and Southern blot analysis. Data from some of the wild-type DBA/1lacJ mice were included in the characterization of respiratory behavior in neonatal DBA/1lacJ mice (1). All mice were reared under standardized conditions with a 12-h light:12-h dark cycle. Food and water were provided ad libitum.
Human Subjects. Infants from the neonatal intensive care unit at Karolinska University Hospital were included in this study (postnatal age mean 16 ± 4 d). Infants were eligible for inclusion if they underwent a lumbar puncture for clinical indications such as suspected infection, neurological changes, and cardiorespiratory problems. Infants were excluded if they had intraventricular hemorrhage (grade ³2), white matter disease (PVL-periventricluar leukomalacia), seizures, posthemorrhagic hydrocephalus, or congenital abnormalities. Pertinent medical information was documented, including neonatal delivery data, medical conditions, infectious markers, respiratory therapy, and medications. Cardiorespiratory recordings were performed within 18 h after the lumbar puncture (mean: 4.8 ± 1.7 h).
Immunohistochemistry. Brainstems from 9-day-old wild-type and EP3R-knockout pups were rapidly dissected after decapitation, fixed in 4% paraformaldehyde, and cryoprotected overnight in 15% sucrose in PBS, pH 7.4. The brainstems were then rapidly frozen, and 14 mm transversal sections were serially collected in a cryostat (Leica CM3050 S, Leica Microsystems Nussloch GmbH). Sections were dried in air, rehydrated with PBS, and endogenous peroxidases were inhibited using 0.3% hydrogen peroxide for 10 min. After subsequent PBS washes, the sections were blocked and permeabilized in 5% goat serum (Jackson Immunoresearch Laboratories, West Grove, PA), 1% BSA (Sigma-Aldrich), and 0.3% Triton X-100 (Sigma-Aldrich) in PBS for 45 min followed by overnight incubation with a rabbit NK-1R antibody (1:20,000 dilution; Sigma-Aldrich). The sections were then washed in PBS and incubated with a biotinylated secondary antibody (goat anti-rabbit; Vector Laboratories, Burlingame, CA) at a 1:50 dilution. After 1-h incubation, the sections were rinsed and incubated with peroxidase-conjugated Vectastain ABC (1:100 dilution; Vector Laboratories) for 30 min followed by Cy3-conjugated Tyramide signal amplification (TSA, 1:50; PerkinElmer, Boston, MA) for 2 min. The reaction was stopped in PBS and blocked with 5% donkey serum (Jackson), 1% BSA (Sigma-Aldrich), and 0.3% Triton X-100 (Sigma-Aldrich) in PBS for 45 min. The sections were then incubated at 4C overnight with a rabbit EP3R antibody (Cayman Chemical, MI) at a 1:50 dilution. The following day, the sections were rinsed in PBS and incubated for 1h with Alexa 488-conjugated secondary antibody (donkey anti-rabbit; Molecular probes). After following PBS washes, the sections were mounted in Vectashield Hard Set mounting medium (Vector Laboratories). To rule out the risk of possible cross-reactions, primary antibodies were titrated to determine the optimal dilutions, and control slides were included with the respective primary antibody omitted. Moreover, brainstem slices from EP3R knockout mice (n = 4) were studied using the above protocols with normal NK1R staining, but no detectable EP3R. Images were processed using ImageJ software (NIH, Bethesda, MD).
Unrestricted Whole-Body Flow Plethysmography. As described previously, the chamber was calibrated by repeatedly injecting standardized volumes of air (5-200 ml) with preset precision syringes (Hamilton Bonaduz AG, Switzerland) (1). 95% of gas exchange occurred within 35 s of administration, which was verified by CO2 content analyses (Metek CD-3A and S-3A, PA, USA). A Plexiglas chamber (35 ml) was connected to a highly sensitive direct airflow sensor (0-200 ml/min; TRN3100, Kent Scientific Corporation, Litchfield, CT, USA). The flow signal was amplified by a four-channel amplifier (P/N 770 S/N 5; SENSElab, Somedic Sales, Hörby, Sweden), converted to digital signal, and recorded at 100 Hz by an online computer using DasyLab software (Datalog GmbH & Co. KG, Mönchengladbach, Germany). Respiratory frequency (fR, breaths/min), tidal volume (VT, μl/breath), and minute ventilation (VE, μl/min) were calculated. Chamber temperature was maintained at 30.1 ± 0.1°C in accordance with the documented thermoneutral range for neonatal mice by immersing the chamber in a thermostat-controlled water bath (2).
Brainstem-Spinal Cord Preparation. The brainstem was rostrally decerebrated between the cranial nerve VI roots and the lower border of the trapezoid body so that the pons was removed. The preparation was continuously perfused in a 1.5 ml chamber with artificial cerebrospinal fluid (aCSF): 130 mM NaCl, 3.3 mM KCl, 0.8 mM KH2PO4, 0.8 mM CaCl2, 1.0 mM MgCl2, 26 mM NaHCO3, and 30 mM D-glucose at 28°C (flow rate, 3-4 ml/min). The solution was continuously equilibrated with 95% O2 and 5% CO2 to pH 7.4 (3, 4).
Plethysmograph Data Analysis. As there is a variable response to anoxia based upon age (5), we attempted to perform all recordings at age P9; however, in an effort to minimize confounding age-related effects, weight was used as a correlate of age and only animals with weights within 1 SD of the population mean weight were included in the anoxia and survival analyses (1).
Animal Characteristics. In the plethysmography experiments following i.p. injection of IL-1b or NaCl, the mPGES-1+/+ mice possessed a lower weight than mPGES-1-/- mice (4.4 ± 0.1 g vs. 4.9 ± 0.1 g, respectively). There was no difference in animal gender. Animal skin temperature at baseline (34.7 ± 0.1°C) and 70 min after injection (34.8 ± 0.1°C) was similar between groups. After anoxia, mPGES-1+/+ mice possessed a higher skin temperature than mPGES-1-/- mice (32.2 ± 0.1°C vs. 31.4 ± 0.2°C, respectively). In the C57BL/6 mice, there was no difference in animal weight (4.5 ± 0.1 g), animal gender, baseline temperature (34.4 ± 0.2°C), temperature at 70 min (34.5 ± 0.5°C), or after anoxia (30.4 ± 0.1°C). In the plethysmography experiments following i.c.v. injection of PGE2 or vehicle, the C57BL/6 mice exhibited no difference in animal gender and postanesthesia temperature (31.0 ±0.2°C). However, EP3R+/+ mice weighed more than EP3R-/- mice (4.9 ± 0.1 g vs. 4.1 ± 0.1 g, respectively). Skin temperature was measured in 9 d-old EP3R+/+ mice (n = 13) and EP3R-/- mice (n = 26) at baseline and each min during normoxia, hyperoxia, and anoxia following i.c.v. injection of PGE2 or vehicle. No difference in temperature was apparent until anoxic exposure at 23 min after injection. At that time, the EP3R-/- mice possessed a lower skin temperature than EP3R+/+ mice (30.9 ± 0.3°C vs. 31.8 ± 0.3°C, respectively). The temperature similarly differed during the postanoxic period at 30 - 31 min (29.8 ± 0.2°C vs. 30.4 ± 0.1°C, respectively).
Drugs. Recombinant mouse IL-1b (Nordic Biosite, Täby, Sweden) was reconstituted in sterile NaCl to produce a 1 mg/ml working solution. PGE2 (Cayman Chemicals, Ann Arbor, MI) was diluted in aCSF to a concentration of 2 nmol/ml for in vivo experiments and 20 mg/liter (60 nM) for in vitro experiments.
Impedance Pneumography. Infant cardiorespiratory activity was measured noninvasively by using impedance pneumography and was recorded by an event monitoring system (KIDS; Hoffrichter, Schwerin, Germany). The monitor was programmed to record baseline respiratory rates and events exceeding the apnea threshold. Apnea was defined as a [mtequ]10-sec reduction of the mean impedance signal amplitude during the preceding 0.5 s to [lt]16% of the mean amplitude measured during the preceding 25 s. The 60-s periods before and after the event were also stored in the monitor's memory.
Plethysmography After i.p. Injection of IL-1b or NaCl. Skin temperature was recorded at baseline, at 70 min, and after removal from the chamber. Rectal temperature was not measured because rectal probe placement may alter respiratory behavior. The anogenital distance was measured to approximate gender.
Plethysmography After i.c.v. Injection of PGE2 or Vehicle. Respiration was examined using flow plethysmography in 9-d-old C57BL/6mice (n=38) with variable expression of EP3R. Animal skin temperature was recorded at baseline and at each subsequent minute by using a thermistor temperature probe.
Plethysmography Data Analysis. Regularity of breathing was quantified using the coefficient of variation (C.V.) (i.e., SD divided by mean of breath-by-breath interval during 60-s periods).
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