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Published ahead of print on June 30, 2004, doi:10.1164/rccm.200403-261OC
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American Journal of Respiratory and Critical Care Medicine Vol 170. pp. 665-672, (2004)
© 2004 American Thoracic Society

Original Article

Long-Term Intermittent Hypoxia

Reduced Excitatory Hypoglossal Nerve Output

Sigrid C. Veasey, Guanxia Zhan, Polina Fenik and Domenico Pratico

Center for Sleep and Respiratory Neurobiology, Division of Sleep Medicine, Department of Medicine, and Center for Experimental Therapeutics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania

Correspondence and requests for reprints should be addressed to Sigrid Carlen Veasey, M.D., 972 Maloney Bldg., 600 Spruce St., Philadelphia, PA 19104. E-mail: veasey{at}mail.med.upenn.edu


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Humans with long-standing sleep apnea show mixed responses to serotonergic therapies for obstructive sleep apnea. We hypothesize that long-term intermittent hypoxia may result in oxidative injury to upper airway motoneurons, thereby diminishing serotonergic motoneuronal excitation. Unilateral serotonin and glutamate agonist and antagonist microinjections into the hypoglossal motor nuclei in adult rats exposed to 3 weeks of intermittent hypoxia showed reduced hypoglossal nerve responsiveness (logEC50) for serotonin and N-methyl-D-aspartate. However, long-term intermittent hypoxia did not appear to alter hypoglossal response to {alpha}-amino-3-hydroxy-methylisoxazole-4-propionic acid injections. There was no reduction in hypoglossal motoneuron soma number or in serotonergic postsynaptic receptor mRNA copy numbers within single-cells; in contrast, there was an increase in isoprostanes in the dorsal medulla. Systemic 4-hydroxyl-2,2,6,6-tetramethylpiperidin-1-oxyl (tempol) throughout exposure to intermittent hypoxia improved the EC50 for serotonin to a larger extent than glutamate and normalized medullary isoprostanes. Protein kinase C activity within the hypoglossal nucleus was increased after long-term intermittent hypoxia. These results suggest that long-term intermittent hypoxia reduces serotonergic and N-methyl-D-aspartate excitatory output of hypoglossal nerves, and that reduced excitatory responsiveness and lipid peroxidation are largely prevented with superoxide dismutase treatment throughout hypoxia/reoxygenation. Similar alterations in neurochemical responsiveness may occur in select persons with obstructive sleep apnea.

Key Words: glutamate • microinjection • serotonin • signal transduction

Obstructive sleep apnea is a prevalent disorder (1) with significant cardiovascular and neurobehavioral morbidities (2–7). Persons with obstructive sleep apnea have narrowed or collapsible upper airways, such that sleep state–dependent reductions in upper airway dilator motoneuron activity result in repeated episodes of airway occlusion (8). Upper airway occlusion in many individuals manifests as intermittent episodes of hypoxia terminating with stimulus induced arousal, return of motoneuron excitation, restoration of a patent airway, and reoxygenation (8). Hypoxia/reoxygenation events may occur as frequently as once every minute or two of sleep (1).

Sleep-related reductions in serotonergic modulation of upper airway dilator nerves contribute to the sleep state–dependent reductions in upper airway motoneuron activity in normal animals (9–11). However, clinical trials of serotonergic drugs in persons with obstructive sleep apnea show small, if any, responses (12). One plausible explanation for the minimal responsiveness to serotonergic drugs is that the disease process itself may alter neurochemical responsiveness to serotonergic drugs. Serotonergic neuromodulation may be modified by both intermittent hypoxia (IH) and alterations in neurochemical input to dilator motoneurons (13–18). Depending upon the duration and severity of hypoxia/reoxygenation events, IH may cause either excitatory or inhibitory changes in nerve response (13–17). Short-term (< 24 hours) IH results in a serotonin-dependent increase in phrenic and hypoglossal nerve activity (13–17). Although the effects of long-term IH (LTIH) on control of motoneurons have not been explored, several other neuronal populations show reduced function with LTIH exposure (19–22). LTIH results in reduced excitability in CA1 hippocampal neurons, and induces nitrosative and oxidative neuronal injury in many brain regions (19, 20, 23). Nitrosylation, nitration, and oxidation have been shown to alter receptor function and signaling protein activities (24, 25), and humans with obstructive sleep apnea show evidence of oxidative stress before therapy (26, 27). We hypothesized that LTIH, modeling the oxygenation patterns alone of severe obstructive sleep apnea, would result in significant oxidative changes in excitability of upper airway dilator nerves. Thus, LTIH-altered responsiveness to serotonergic excitation may contribute to the variable responses to serotonergic medications in persons with sleep apnea, and this altered responsiveness could also contribute to disease progression in persons with this disorder.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
Experiments were performed on 156 adult male 300- to 400-g Sprague-Dawley rats (Charles River, Wilmington, MA), in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the University of Pennsylvania's animal care committee. Rats were housed in cages of three, maintained on a 12-hour light:dark cycle with lights on at 7:00 P.M., with food pellets and water ad libitum.

IH Exposure
During both LTIH and sham LTIH, each home cage of rats was placed within a plexiglass chamber (15" x 20" x 20"; Biospherix, Redfield, NY). Flow rates of 99% pure nitrogen gas and 99% pure oxygen into the chambers were varied with an automated oxygen profile system (Oxycycler model A84XOV; Biospherix), to result in episodic (90-second cycle length) reductions from an ambient oxygen level of 21 to 9%, where the 9% was maintained for just 5 seconds, using an established protocol (20, 21, 23). Sham LTIH was produced for the same cycle length time, with fluctuations from 21 to 19%. A third condition was Tempol-LTIH (with 2 mM 4-hydroxyl-2,2,6,6-tetramethylpiperidin-1-oxyl [tempol; Sigma-Aldrich, St. Louis, MO] in the drinking water throughout IH). LTIH and Sham LTIH conditions were produced throughout the light period of the 24-hour ambient light cycle for a total of 3 weeks. Ambient oxygen, carbon dioxide, and humidity levels and temperature were recorded throughout study.

Microinjection Agonist Studies
Hypoglossal microinjection studies were performed on rats 12 hours after removal from one of the three conditions: LTIH (n = 44), Tempol-LTIH (n = 20), or Sham LTIH (n = 42). General anesthesia, line placement, tracheostomy, hypoglossal cuff electrode placement, and microinjection procedures were performed as previously described (28, 29). One series of LTIH, Tempol-LTIH, and Sham LTIH rats received serotonin (5-HT; Sigma-Aldrich) injections as randomized series of 5-HT concentrations (0.0001–1,000 µM) injected (20 {eta}l) into one hypoglossal nucleus every 5 minutes using coordinates relative to the calamus scriptorius (0.2 mm lat, 1.05 ventral, and 0.6 mm rostral). A dose of 10,000 µM was injected (20 {eta}l) upon completion of the randomized doses, as this concentration has lasting effects on hypoglossal nerve activity (29). Hypoglossal nerve activity was quantified as the amplitude (µV) from peak respiratory activity to baseline end-expiratory activity using the rectified moving average of the whole nerve recording and methods as previously detailed (28, 29). Agonist effect was defined as the percentage of baseline nerve activity and was determined 30 seconds after completion of injection (maximum drug effect). Additional series of rats were used for glutamatergic agonists (Sigma-Aldrich), glutamate, N-methyl-D-aspartate (NMDA) or {alpha}-amino-3-hydroxy-methylisoxazole-4-propionic acid (AMPA), using randomized concentrations as above for serotonin.

Microinjection Antagonist Studies
Methysergide maleate, a broad spectrum 5-HT antagonist (Sigma-Aldrich), was dissolved in phosphate-buffered saline (PBS) and used to test local baseline 5-HT contribution to hypoglossal nerve output by determining the IC50 for methysergide, using the nonlinear regression fit and determining the dose for 50% reduction with GraphPad Prism 3.0 software (29). The NMDA antagonist used was MK-801 and the AMPA antagonist was 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide disodium (NBQX) (both from Sigma-Aldrich). Concentrations of all antagonists tested were 0.002 to 200 µM in PBS, and one antagonist was administered per animal. Drug effect was quantified as percentage of baseline nerve activity 5 minutes after injection, to allow for adequate diffusion.

Quantification of 5-HT Receptor Subtype mRNA Copies in Single Hypoglossal Motoneurons
Individual hypoglossal motoneurons were laser dissected (PixCell II; Arcturus, Mountain View, CA) from 10-µm coronal sections of the hypoglossal nuclei from the brains of 4°C PBS-perfused rats (n = 9 LTIH and 9 Sham LTIH), and from the single hypoglossal motoneuron, RNA was purified, reverse transcribed, and resultant cDNA was used as a template for PCR and real-time PCR amplification. The primer sets and molecular beacon sequences (30) were designed using Primer Express 1.0 software (Applied Biosystems, Foster City, CA) and synthesized by Integrated DNA Technologies, Inc. (Coralville, IA). Standard curves were generated for each primer/probe set using serial dilutions of known quantities of DNA: 100,000, 10,000, 1,000, 100, and 10 copies per well, using the methods of Medhurst and coworkers 2000 (30). Standard curves for each set plotted CT versus the log of the initial number of DNA copies. Absolute sensitivity was previously determined to be similar for all primer probe sets (31). Single-factor ANOVA with Neumans-Keuls was used with statistical significance at p < 0.05.

Serotonin Receptor Autoradiography in the Hypoglossal Nucleus
Autoradiography with [125I] 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) was used to compare intrinsic 5-HT2A/2C receptor binding site densities in the hypoglossal nucleus of LTIH and Sham LTIH rats. Methods were performed as previously described with the following modifications (32). Rats were decapitated, and brainstems were dissected and immediately frozen onto tissue pedestals with O.C.T. (Miles, Inc. Elkhart, IN). Sequential 12-µm coronal sections were cut at –20°C and thaw-mounted onto Superfrost Plus Slides (Fisher, Pittsburgh, PA). Air-dried slides were preincubated with 100 µl of 50 mM Tris-HCl buffer (pH 7.5) containing 10 mM MgSO4 and 0.1% (wt/vol) bovine serum albumin at room temperature for 10 min to remove endogenous serotonin, and then incubated with a similar mixture including 200 ppm [125I] DOI (Sigma Aldrich) for 60 min. Nonspecific binding was determined with 5 µM methysergide "blank" in the incubation mixture. Slides were dried in warm air and apposed to a cassette placed in a PhoshorImager SI system (Molecular Dynamics, Sunnyvale, CA), and image analysis was performed with ImageQuant Software V 5.2. In each cassette, we paired LTIH and Sham LTIH slides (matched for rostral-caudal area of the hypoglossal nucleus), along with nonspecific binding slides. Relative density was achieved by first measuring binding density within a circumferential area drawn over the hypoglossal nucleus. From this value, we subtracted the density from the same circumferential area density on the nonspecific binding slide and then compared values from the two conditions on the same cassette. A nonpaired t test for eight animals in each group was used to compare relative hypoglossal LTIH and Sham LTIH [3H] DOI baseline binding.

Measurement of Lipid Peroxidation in the Hypoglossal Nucleus
Levels of the isoprostane isomer most closely linked to neurodegeneration, d4-8,12-iso-iPF2{alpha}-VI (33), were measured (n = 6 LTIH, n = 6 tempol LTIH and n = 6 Sham LTIH rats). Rats were deeply anesthetized with 1.3 gm urethane intraperitoneally and then perfused intracardially with 4°C 0.9% PBS with 2mM EDTA and 20mM BHT, pH 7.4 (33). Brains were immediately removed, flash frozen in liquid nitrogen and then a block dissection of bliateral hypoglossal nuclei was performed. The hypoglossal tissue was homogenized, and total lipids were extracted using Folch solution (chloroform:methanol 2:1 vol) (34). Base hydrolysis was performed using 15% KOH at 45°C for 1 h. A fixed amount of internal standard of d4-8,12-iso-iPF2{alpha}-VI extracted on a C18 cartridge column was added to each sample. Thin layer chromatography was used for purification of the eluate, and negative ion chemical ionization gas chromatography-mass spectrometry was used to assay d4-8,12-iso-iPF2{alpha}-VI.

Measurement of Protein Kinase C Activity in the Hypoglossal Nucleus
To better localize the source of intermittent hypoxia effect on hypoglossal neuron responsiveness to 5-HT, the sum activity of Ca2+/phospholipid-dependent protein kinase C (PKC) isotypes was measured with a nonradioactive PKC assay measuring quantity of phosphorylated peptide substrate for two levels of substrate (PepTag; Promega, Madison, WI). Briefly, rats (n = 6 LTIH and 6 Sham LTIH) were decapitated and a block of the medulla was dissected and frozen onto a tissue pedestal as for autoradiography slice preparation. A block of medulla was dissected using boundaries, 1 mm rostral to calamus scriptorius (CS) to 0.3 mm caudal to CS. The lateral boundaries were 0.3 mm lateral to CS, and the dorso-ventral boundaries were 0.5 to 1.5 mm. Individual blocks were rapidly homogenized in 10 vols of homogenization buffer (20 mM Tris-HCl, pH 8; 1 mM EDTA, pH 8; 0.5 mM DTT). After 5 minutes of centrifugation at 500 x g (4°C), supernatant was collected, and 10 µl was sampled for protein quantification. The PKC assay was run using aliquots of 1 to 3 µg protein in 5 µl. PKC activator 5X was sonicated for 30 seconds. Samples were prepared with 5 µl PKC reaction buffer; 5 µg PepTag peptide; 5 µl PKC activator; 1 µl Peptide protector; and 5 µl of homogenized hypoglossal sample. Four microliters of PKC at 2.5 µg/ml was added to positive controls, whereas no tissue was placed in negative control vials. Reactions were terminated at 30 minutes by placing tubes in boiling water. Agarose gel electrophoresis was run with 80% glycerol (1 µl) at 100 V for 20 minutes and then photographed under ultraviolet light. Spectrophotometric quantitation of phosphorylation was performed on gel bands.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of Chronic Intermittent Hypoxia on the Responsiveness of Hypoglossal Nerve Output to Exogenous 5-HT
Serotonin dose–response microinjection studies were successfully completed in 15 of 17 LTIH rats and 13 of 17 Sham LTIH rats. Serotonin injected into the hypoglossal nucleus produced a dose-dependent response in both LTIH and Sham LTIH rats, coefficients of determination, r2 = 0.24, p < 0.001 and 0.38, p < 0.0001, respectively. The dose–response relationships are shown in Figure 1A. There was an overall effect on the dose–response relationship by ambient oxygen condition, F = 21.6, p < 0.0001. The maximal effect of 5-HT on hypoglossal nerve activity was significantly diminished under conditions of LTIH, 174 ± 1.9% of baseline nerve activity, compared with 225% ± 3 for Sham LTIH, p < 0.01. In addition, LTIH shifted the LOG EC50 (µM) to the right from Sham LTIH: 1.7± 0.1 (95% CI, 1.5–2.0) to LTIH: 2.6 ± 0.04 (95% CI, 2.4–2.8), p < 0.05. These results demonstrate that LTIH results in a rightward shift of the EC50 for 5-HT microinjected into the XII nucleus and a reduction in the maximum 5-HT nerve response.



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  		Figure 1. Effects of long-term intermittent hypoxia on serotonin responses and post-synaptic receptors in hypoglossal nuclei. (A) Dose-dependent effects of serotonin microinjected into the hypoglossal nucleus on hypoglossal nerve respiratory activity. Response curves for rats exposed to long-term intermittent hypoxia (LTIH) for 3 weeks (n = 15 adult rats, open circles) and for rats exposed to sham LTIH for 3 weeks (n = 13 adult rats, closed circles). EC50 is shifted rightwards in LTIH, p < 0.0001. (B) Dose-dependent effects of methysergide injected into the hypoglossal nucleus on hypoglossal respiratory activity (LTIH, n = 9, open circles; sham LTIH, n = 6, closed circles) N.S. (C) Mean ± SE mRNA copy numbers of 5-HT2A and 5-HT2C in individual laser-captured hypoglossal motoneurons (Sham LTIH, n = 8, light gray bars; LTIH, n = 8, dark gray bars, N.S.). (D) Representative autoradiograms for [125I] 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) in the medulla (sham LTIH, left panel; LTIH, right panel, N.S.). Hypoglossal nucleus (arrow) and pyramidal tract (PT) show increased 5-HT2A,2C binding in sham LTIH and LTIH.

 
Effects of Chronic Intermittent Hypoxia on Baseline Hypoglossal Nerve Activity and Intrinsic 5-HT Activity
A second set of microinjections in separate animals was performed to characterize intrinsic hypoglossal nerve activity and 5-HT intrinsic activity. For 15 rats in each group, there were no differences in either the amplitude of hypoglossal respiratory activity (LTIH, 1.2 ± 0.3 µV versus Sham LTIH, 1.0 µV ± 0.3) or the amplitude of end-expiratory nerve activity relative to electrical baseline (LTIH, 0.2 ± 0.01 µV versus Sham LTIH, 0.2 µV ± 0.02). Statistical power to detect a twofold difference in either tonic or phasic amplitude voltage was greater than 0.99. There were no differences in dose response curves for methysergide (5-HT2A and 2C receptor antagonist), F = 0.4, N.S. Under both conditions, LTIH, n = 9 and Sham LTIH, n = 6, a dose response was observed with methysergide, F = 5.4, p < 0.001 (Figure 1B). At a concentration of 200 µM, there was a significant reduction in hypoglossal nerve peak activity for both groups: 65% ± 13 of baseline activity for Sham LTIH and 59% ± 13 for LTIH, p < 0.05, yet no difference between conditions and no difference in the IC50 for LTIH and Sham LTIH: 1.9 ± 1.1 versus 1.9 ± 1.0, N.S. Thus, LTIH did not alter baseline 5-HT activity for the XII nerve, as determined with a methysergide dose–response curve for nerve activity.

Effects of LTIH on Hypoglossal Motoneuron 5-HT Receptor Subtype Transcription
5-HT2A and 5-HT2C receptor mRNA numbers for single XII moto-neurons were unchanged by LTIH. Single hypoglossal motoneuron cell bodies, identified by size as previously reported (31), were laser captured from 11 LTIH (158 soma) and 11 Sham LTIH (120 soma) rats and assayed individually for two of the selected six 5-HT postsynaptic receptor subtypes. Copy numbers for 5-HT receptor subtypes, 3, 4, 6, and 7 were < 100 copies/cell for both conditions. Copy numbers of 5-HT2A receptor mRNA were 1418 ± 400 for Sham LTIH (n = 8 rats) and 1388 ± 226 for LTIH (n = 8 rats), N.S., and copy numbers for 5-HT2C were 1505 ± 325 for Sham LTIH (n = 8 rats) and 2649 ± 346 for LTIH (n = 7 rats), N.S., shown in Figure1C. Therefore, 5-HT2A and 5-HT2C receptor mRNA numbers for single XII motoneurons were unchanged by LTIH.

Hypoglossal 5-HT2A/2C Receptor Binding Density
Serotonin receptor binding (hypoglossal:hypoglossal methysergide blank) for [3H] DOI were similar for both conditions, Sham LTIH (2.1 ± 0.1, n = 8 rats) and for LTIH (2.6 ± 0.3, n = 8 rats), t = 2.1, p = 0.06. Representative autoradiograms for Sham LTIH and LTIH are presented in Figure 1D. Thus, 5-HT receptor binding affinity in the XII nucleus was unchanged by LTIH.

Effects of LTIH on Hypoglossal Nerve Response to Glutamate
Glutamate dose–response microinjection studies were successfully completed in 13 LTIH rats and 15 Sham LTIH rats, showing a dose-dependent response in both LTIH and Sham LTIH rats, coefficients of determination, r2 = 0.09, p < 0.05 and 0.30, p < 0.01, respectively. The dose–response relationships are shown in Figure 2A. There was an overall effect on the dose–response relationship by ambient oxygen condition, F = 37.8, p < 0.0001. The maximal effect of glutamate on hypoglossal nerve activity was significantly diminished under conditions of LTIH, 138.7 ± 2.8% of baseline nerve activity, compared with 332.6% ± 12.5 for Sham LTIH, p < 0.01. In addition, LTIH shifted the LOG EC50 to the right from Sham LTIH: 0.2 ± 0.4 (95%CI, –0.98–1.4) to LTIH: 2.8 ± 0.04 (95% CI, 2.3–3.4), p < 0.001. NMDA dose responses were successful in 10 Sham LTIH and 9 LTIH rats, showing for both groups a dose responsiveness, as seen in Figure 2B. There was dose–response pattern evident for LTIH and Sham LTIH, r2 = 0.23, p < 0.01 and r2 = 0.36, p < 0.001, respectively. EC50 for Sham LTIH was 0.1 ± .2 and EC50 LTIH was 2.3 ± 0.1, p < 0.001. AMPA dose responses were successful in eight Sham LTIH and nine LTIH rats, showing for both groups a dose responsiveness, as seen in Figure 2C. There was no effect of ambient oxygen exposure on dose–response pattern. EC50 for Sham LTIH was 2.2 ± 1.5 and EC50 LTIH was 2.9 ± 4.1. The large variance in EC50 for this drug, would have required > 1,000 animals to detect a 30% shift in EC50. In summary, whole XII nerve responses to hypoglossal nucleus injections of glutamate and NMDA, but not AMPA, were significantly shifted rightward.



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  		Figure 2. Hypoglossal whole nerve responses to glutamate agonists and antagonists microinjected into the ipsilateral hypoglossal nucleus vary with exposure to LTIH. In each panel, LTIH is denoted by closed circles, dashed line; sham LTIH by open circles, solid line. (A) Glutamate (Glu) microinjection dose responses (LTIH, n = 13; sham LTIH, n = 15). LTIH reduces the logEmax (p < 0.05) and shifts the EC50 to the right (F = 37.8, p < 0.0001). (B) NMDA microinjection dose responses (LTIH, n = 10; sham LTIH, n = 9). LTIH effect on EC50, p < 0.01. (C) AMPA microinjection dose responses (LTIH, n = 9; sham LTIH, n = 8). LTIH effect on EC50, N.S. (D) Dose–response curves for MK-801 (NMDA antagonist) hypoglossal nerve activity. Dose response is evident for sham LTIH, n = 7, p < 0.001, and no dose response for LTIH, n = 7, N.S. The two curves varied significantly, F = 32.7, p < 0.001. (E) Dose–response curves for NBQX (AMPA antagonist) on hypoglossal nerve activity show no difference between sham LTIH, n = 7 and LTIH, n = 7. (F) Tempol (closed triangle, dotted line; n = 12 rats), shows an improvement in the logEmax, but no change in the logEC50.

 
Effects of LTIH on Intrinsic NMDA and AMPA Activity
For MK-801 (NMDA antagonist) a dose–response effect was present only for the Sham LTIH (n = 7) group, with an r2 = 0.68, p < 0.01. Significant reductions in hypoglossal nerve activity in Sham LTIH rats were observed with doses, 20 and 200 µM, p < 0.05. In LTIH rats, doses <= 200 µM did not reduce hypoglossal activity significantly (Figure 2D). There were differences in the curves for LTIH and Sham LTIH, F = 32.7, p < 0.0001. NBQX, an AMPA antagonist, was effective in both sham LTIH and LTIH rats in reducing nerve activity, p < 0.05. There were, however, no differences in NBQX dose–response curves for LTIH (n = 7) and Sham LTIH (n = 7, F = 0.6, N.S.) (Figure 2E). LTIH, therefore, resulted in a smaller reduction in XII nerve activity with NMDA antagonism, whereas effects of the AMPA antagonist on hypoglossal nerve activity were unchanged.

Baseline PKC Activity
PKC activity in homogenates of the dorsal medulla differed statistically for the two conditions (LTIH and Sham LTIH), t = 3.3, p < 0.05. For 2 µg C1 peptide, PKC activity was 31.2 ± 4.5 pmol/3 µg protein/minute in Sham LTIH rats (n = 5) and 45.6 ± 4.5 pmol/3 µg protein/minute in LTIH rats (n = 5), N.S. t = 3.3, p < 0.05, shown in Figure 3A. Thus, PKC activity in XII nucleus micropunches was elevated in LTIH rats.



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  		Figure 3. (A) Protein kinase C activity in dorsal medulla homogenates. (Top panel) Lane 1, negative control; lane 2, positive control with 10 µg PKC; lane 3, positive control with 20 µg PKC; lane 4, sham LTIH, 0.2 µg C1 peptide; lane 5, sham LTIH, 2 µg C1 peptide; lane 6, LTIH, 0.2 µg C1 peptide; lane 7, LTIH, 2 µg C1 peptide. (Bottom panel) Average PKC activity ± SE (sham LTIH, n = 5, striped bar; LTIH, n = 5, gray bar). *p < 0.05. (B) Effect of tempol throughout LTIH on hypoglossal nerve response to serotonin (LTIH, n = 15, open circles; sham LTIH, n = 13, closed circles). Systemic 4-hydroxyl-2,2,6,6-tetramethylpiperidin-1-oxyl (tempol, 5 mM) therapy significantly improved the 5-HT EC50, p < 0.01. (C) Effects of intermittent hypoxia on isoprostane (8,12–iso-iPF2{alpha}) levels in the cortex and medulla. Average isoprostane values ± SE for sham LTIH, n = 6, striped bars; LTIH, n = 6, light gray bars; tempol LTIH, n = 6, dark gray bars. *p < 0.05 LTIH versus sham LTIH or tempol LTIH.

 
Effects of a Superoxide Dismutase Mimetic on Hypoglossal Excitatory Responses
Dose–response curves differed significantly for Sham LTIH, LTIH, Tempol-LTIH, F = 19, p < 0.001 (Figure 3B). The variance in LOG EC50 for TEMPOL-LTIH was greater than the variance for either LTIH or Sham LTIH. The LOG EC50 for Tempol-LTIH was 2.1 ± 0.3 µM (n = 12). This was not significantly higher than the values obtains for rats exposed to normoxia was 1.7 ± 0.1 (n = 13). The Emax under conditions of Tempol-LTIH (215 ± 12%) trended toward higher than LTIH (174 ± 2%), N.S., and was close to the Emax under conditions of Sham LTIH (225 ± 5%). Glutamate dose–response was also changed significantly by tempol, F = 36.8, p < 0.0001. However, LOG EC50 for Tempol-LTIH was 2.8 ± 0.2 µM (n = 12), N.S. from LTIH (2.6 ± 0.2) (Figure 2E). In summary, systemic administration of a superoxide dismutase mimetic throughout LTIH prevented the LTIH effects on serotonergic XII nerve responsiveness and reduced LTIH effects on glutametergic nerve response.

Effects of Chronic Intermittent Hypoxia and Superoxide Dismutase Mimetic Treatment on Hypoglossal Isoprostane Levels
Isoprostane (d4-8,12-iso-iPF2{alpha}-VI) levels in homogenates of dorsal medulla were increased in LTIH rats compared with Sham LTIH, LTIH, and Tempol-LTIH rats, and results are present in Figure 3C. Isoprostanes were significantly elevated in both the hypoglossal nucleus and frontal cortex, p < 0.01. Isoprostane levels in Tempol-LTIH rats did not differ significantly from levels in Sham LTIH rats. Therefore, superoxide dismutase treatment throughout LTIH prevented an increase in brain isoprostane levels.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Exposure to LTIH, producing arterial oxyhemoglobin desaturation patterns similar to those observed in severe obstructive sleep apnea, shifted the dose–response curves for 5-HT– and NMDA-hypoglossal nerve output in adult rats significantly rightward. Neither 5-HT2A/2C binding density in the hypoglossal nucleus or 5-HT2A and 2C receptor mRNA copy numbers in individual hypoglossal motoneurons were affected by LTIH. Baseline nerve activity and 5-HT activation in the hypoglossal nucleus of hypoglossal whole nerve, as measured with whole hypoglossal nerve output response, were also unchanged by LTIH. In contrast, LTIH increased lipid peroxidation injury in the dorsal medial medulla. Further, systemic treatment with a superoxide dismutase mimetic throughout exposure to intermittent hypoxia prevented the reduced serotonergic responsiveness and largely averted intermittent hypoxia-induced lipid peroxidation within the dorsal region of the medulla. Together, these data suggest that LTIH impairs the responsiveness of hypoglossal nerves to 5-HT and NMDA in the motor nuclei, at least in part, through redox changes. The AMPA response and activity of protein kinase C, a major signaling enzyme for 5-HT2A and 2C and NMDA, were not affected by LTIH in this model.

Responsiveness of hypoglossal whole nerve activity to 5-HT and glutamate agonists was determined using microinjections into intact anesthetized, tracheostomized, ventilatory-controlled animals. In other neuronal groups, measures of neuronal excitability and plasticity may be achieved with either slice preparations or neuronal cultures. A major concern when using the latter two techniques for pharmacological trials of adult hypoglossal motoneurons, however, is the health, including oxidative stress, and integrity of membrane function and thus, the baseline excitatory capability of this population of neurons (35–37).

We have shown that treatment with a superoxide dismutase mimetic throughout LTIH largely prevents the marked rightward shift in 5-HT dose–response curve. This supports the hypothesis that redox alterations contribute to the reduced nerve responsiveness to 5-HT after LTIH. Although other brain regions are also affected by LTIH, including the cortex (20, 21, 23), hippocampus (19, 21), and sleep wake regions (23), there are several features of motoneurons that render this particular group of neurons at greater risk of oxidative injury (38–40). The large soma and long axonal projections require high metabolic activity and high mitochondrial function (38). A further burden is the high content of neurofilament protein susceptible to oxidative and nitrative changes leading to misfolding and aggregations and poor nerve conduction (39). Motoneurons, however, are not the only group of neurons to succumb to oxidative injury from LTIH (19–23, 41, 42), where drugs targeting reduction of proinflammatory mediators (22) or superoxide have been shown to largely prevent neural injury from LTIH (21, 41, 42). This raises the possibility that respiratory control neurons might also be affected by LTIH.

Our findings with LTIH resulting in reduced responsiveness contrast with results from recent studies with short-term IH that show largely excitatory effects after brief exposures to mild IH: increased 5-HT activity at respiratory motoneurons or increased 5-HT modulation of the AMPA response (13–18). In the present study, LTIH did not modify either baseline hypoglossal nerve activity or acute 5-HT activation of hypoglossal nerve output. Further, there was no difference in the short-term nerve response to AMPA or baseline AMPA activation in the hypoglossal nucleus. Thus, the effects of LTIH on respiratory nerve activity in our model differ substantially from the effects of shorter term and less frequent IH (13–18). Future studies are required to characterize the duration and severity effects on hypoglossal nerve responsiveness. Identification of duration and severity effects will provide windows through which to further explore mechanisms of the varied responses to intermittent hypoxia.

Hypoglossal nerve output response to NMDA was markedly shifted to the right in LTIH rats and was only partially corrected with superoxide dismutase therapy. Tempol has been shown to reduce both nitration and oxidation of signaling proteins and other key cellular proteins and lipids (43, 44), but may not impact upon nitrosylation. Nitrosative modulation of NMDA receptors is an important regulatory mechanism for neuronal responsiveness (45–50). Nitrosylation of Cys 399 on the NR2A NMDA subunit, and to a lesser extent nitrosylation of the Cys 744 and 798 on NR1, protects taxed neurons by reduced calcium influx (45). Whether the reduced NMDA effect could be prevented with nitric oxide synthase inhibitors should be studied. The change in NMDA dose–response Hill slope after LTIH raises the possibility that either multiple receptor subtypes are involved in the reduced responsiveness or that a G-protein involved in the NMDA response was altered by LTIH (51).

Under many conditions, serotonergic receptors, particularly the 5-HT2A receptors, undergo transcriptional changes (52, 53). Infants who succumb to Sudden Infant Death Syndrome have reduced 5-HT1/2A/2C binding in the medulla oblongata (54, 55). Our findings, however, show that the mechanisms of reduced serotonergic responsiveness involve neither reductions in transcription of postsynaptic 5-HT excitatory receptors nor reductions in 5-HT2A /2C binding densities.

The present study demonstrates that LTIH results in significantly increased isoprostane d4-8,12-iso-iPF2{alpha}-VI levels in the hypoglossal region of the medulla. To our knowledge, this is the first report of increased isoprostanes in the brainstem. LTIH induced brainstem isoprostanes may have relevance to certain neurodegenerative diseases. Lipid peroxidation has been implicated in the pathogenesis of several neurodegenerative disorders, including amyotropic lateral sclerosis (32, 56–59). This raises the possibility that LTIH, as in severe obstructive sleep apnea, could worsen select neurodegenerative processes. With excellent murine models of these diseases, this important area should be further investigated.

The results presented here have several important implications for the effects of sleep-related hypoxia/reoxygenation events on the neural function. The data demonstrate that severe LTIH reduces the responsiveness of hypoglossal nerve output to 5-HT and NMDA. The effects of LTIH appear distinct from the effects of short-term IH. Thus, severity and/or duration of intermittent hypoxia in persons with obstructive sleep apnea may have significant effects on responsiveness of upper airway dilator nerves to both serotonergic drugs and intrinsic neurochemical drive. Moreover, oxidative changes underlie the LTIH-reduced responsiveness. This would suggest that individual vulnerability to oxidative stress could contribute to varied responsiveness to pharmacotherapies and to the severity of altered nerve function. Similarly, the close relationship between LTIH-reduced nerve responses and oxidative changes raises the possibility that oxidative changes will have to be corrected before testing the effectiveness of serotonergic drugs in, at least a subset of, persons with sleep apnea.


   FOOTNOTES
 
Supported in part by NIH HLBI 60287.

Conflict of Interest Statement: S.C.V. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; G.Z. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; P.F. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; D.P. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form March 1, 2004; accepted in final form June 25, 2004


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 RESULTS
 DISCUSSION
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