Carotid chemoafferent activity is not necessary for all phrenic long-term facilitation following acute intermittent hypoxia

Phrenic long-term facilitation (pLTF) is a form of respiratory plasticity induced by acute intermittent hypoxia (AIH) or episodic carotid chemoafferent neuron activation. Surprisingly, residual pLTF is expressed in carotid denervated rats. However, since carotid denervation eliminates baroreceptor feedback and causes profound hypotension during hypoxia in anesthetized rats, potential contributions of these uncontrolled factors or residual chemoafferent neuron activity to residual pLTF cannot be ruled out. Since ATP is necessary for hypoxic carotid chemotransduction, we tested the hypothesis that functional peripheral chemoreceptor denervation (with intact baroreceptors) via systemic P2X receptor antagonism blocks hypoxic phrenic responses and AIH-induced pLTF in anesthetized rats. Pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS; 100 mg/kg i.v.), a non-selective P2X receptor antagonist, was administered to anesthetized, vagotomized, paralyzed and ventilated male Sprague-Dawley rats prior to AIH (3, 5 min episodes of 10% O(2); 5 min intervals). Although PPADS strongly attenuated the short-term hypoxic phrenic response (20 ± 4% vs. 113 ± 15% baseline; P < 0.001), pLTF was reduced but not eliminated 60 min post-AIH (25 ± 4% vs. 51 ± 11% baseline; n = 8 and 7, respectively; P < 0.002). Thus, AIH initiates residual pLTF out of proportion to the diminished hypoxic phrenic response and chemoafferent neuron activation. Although the mechanism of residual pLTF following functional chemo-denervation remains unclear, possible mechanisms involving direct effects of hypoxia on the CNS are discussed.     

Lipopolysaccharide attenuates phrenic long-term facilitation following acute intermittent hypoxia

Lipopolysaccharide (LPS) induces inflammatory responses, including microglial activation in the central nervous system. Since LPS impairs certain forms of hippocampal and spinal neuroplasticity, we hypothesized that LPS would impair phrenic long-term facilitation (pLTF) following acute intermittent hypoxia (AIH) in outbred Sprague-Dawley (SD) and inbred Lewis (L) rats. Approximately 3h following a single LPS injection (i.p.), the phrenic response during hypoxic episodes is reduced in both rat strains versus vehicle treated, control rats (SD: 84 ± 7% vs. 128 ± 14% baseline for control, p < 0.05; L: 62 ± 10% vs. 90 ± 9% baseline for control, p < 0.05). At 60 min post-AIH, pLTF is also diminished by LPS in both strains: (SD: 22 ± 5% vs. 73.5 ± 14% baseline for control, p < 0.05; L: 18 ± 15% vs. 56 ± 8% baseline for control, p < 0.05). LPS alone does not affect phrenic burst frequency in either rat strain, suggesting that acute LPS injection has minimal effect on brainstem respiratory rhythm generation. Thus, systemic LPS injections and (presumptive) inflammation impair pLTF, a form of spinal neuroplasticity in respiratory motor control. These results suggest that ongoing infection or inflammation must be carefully considered in studies of respiratory plasticity, or during attempts to harness spinal plasticity as a therapeutic tool in the treatment of respiratory insufficiency, such as spinal cord injury.     

Protein kinase C activity in the nucleus tractus solitarii is critically involved in the acute hypoxic ventilatory response, but is not required for intermittent hypoxia-induced phrenic long-term facilitation in adult rats

Protein kinase C (PKC) is a broadly expressed and critically important signalling protein with a wide range of functional roles, including central components of respiratory control. For example, systemic and targeted administration of PKC inhibitors within the nucleus of the solitary tract (nTS) markedly attenuates peak hypoxic ventilatory responses (HVR). Protein kinase C activation in phrenic motor nucleus has also been implicated in some forms of acute respiratory plasticity, such as phrenic long-term facilitation (pLTF), a persistent enhancement of phrenic motor output following acute intermittent hypoxia. To further examine the role of PKC within the nTS, the selective PKC antagonist bisindolylmaleimide I (BIM I) was microinjected in the area corresponding to the nTS via bilateral osmotic pumps in normoxic adult male Sprague-Dawley rats; control animals received bisindolylmaleimide V (BIM V, inactive analogue). In one series of experiments, hypoxic challenges (fractional inspired ) were conducted in unrestrained animals (n = 8 per group). No differences in baseline ventilation emerged; however, peak HVR was attenuated following BIM I (P < 0.01), primarily owing to reductions in respiratory frequency increases (P < 0.01). In a second series of experiments, integrated phrenic nerve activity was recorded in anaesthetized, vagotomized, paralysed and ventilated rats exposed to three 5 min hypoxic episodes separated by 5 min hyperoxia . During baseline conditions, no differences emerged in phrenic nerve output; however, phrenic nerve output measured during the initial hypoxic exposure was significantly attenuated in BIM I-treated rats (P < 0.01). In contrast, both groups of animals displayed significant pLTF (BIM I versus BIM V; n.s.). Thus, we conclude that PKC activation within the nTS is critically involved in the central response to acute hypoxia, but does not appear to play a role in either eliciting or maintaining pLTF.     

The role of NADPH oxidase in chronic intermittent hypoxia-induced pulmonary hypertension in mice

Obstructive sleep apnea, characterized by intermittent periods of hypoxemia, is an independent risk factor for the development of pulmonary hypertension. However, the exact mechanisms of this disorder remain to be defined. Enhanced NADPH oxidase expression and superoxide (O2(-).) generation in the pulmonary vasculature play a critical role in hypoxia-induced pulmonary hypertension. Therefore, the current study explores the hypothesis that chronic intermittent hypoxia (CIH) causes pulmonary hypertension, in part, by increasing NADPH oxidase-derived reactive oxygen species (ROS) that contribute to pulmonary vascular remodeling and hypertension. To test this hypothesis, male C57Bl/6 mice and gp91phox knockout mice were exposed to CIH for 8 hours per day, 5 days per week for 8 weeks. CIH mice were placed in a chamber where the oxygen concentration was cycled between 21% and 10% O2 45 times per hour. Exposure to CIH for 8 weeks increased right ventricular systolic pressure (RVSP), right ventricle (RV):left ventricle (LV) + septum (S) weight ratio, an index of RV hypertrophy, and thickness of the right ventricular anterior wall as measured by echocardiography. CIH exposure also caused pulmonary vascular remodeling as demonstrated by increased muscularization of the distal pulmonary vasculature. CIH-induced pulmonary hypertension was associated with increased lung levels of the NADPH oxidase subunits, Nox4 and p22phox, as well as increased activity of platelet-derived growth factor receptor beta and its associated downstream effector, Akt kinase. These CIH-induced derangements were attenuated in similarly treated gp91phox knockout mice. These findings demonstrate that NADPH oxidase-derived ROS contribute to the development of pulmonary vascular remodeling and hypertension caused by CIH.     

Hsp90 regulates NADPH oxidase activity and is necessary for superoxide but not hydrogen peroxide production

The goal of this study was to identify whether heat-shock protein 90 (Hsp90) regulates the production of superoxide and other reactive oxygen species from the NADPH oxidases (Nox). We found that pharmacological and genetic inhibition of Hsp90 directly reduced Nox5-derived superoxide without secondarily modifying signaling events. Coimmunoprecipitation and bioluminescence resonance energy transfer studies suggest that the C-terminus of Nox5 binds to Hsp90. Long-term Hsp90 inhibition reduced Nox5 expression and provides further evidence that Nox5 is an Hsp90 client protein. Inhibitors of Hsp90 also reduced superoxide from Nox1, Nox2 (neutrophils), and Nox3. However, Nox4, which emits only hydrogen peroxide, was unaffected by Hsp90 inhibitors. Hydrogen peroxide production from the other Nox enzymes was not affected by short-term inhibition of Hsp90, but long-term inhibition reduced production of all reactive oxygen species coincident with loss of enzyme expression. Expression of chimeric Nox enzymes consisting of N-terminal Nox1 or Nox3 and C-terminal Nox4 resulted in only hydrogen peroxide formation that was insensitive to Hsp90 inhibitors. We conclude that Hsp90 binds to the C-terminus of Noxes1-3 and 5 and is necessary for enzyme stability and superoxide production. Hsp90 does not bind to the C-terminus of Nox4 and is not required for hydrogen peroxide formation.     

Inspiratory activation is not required for episodic hypoxia-induced respiratory long-term facilitation in postnatal rats

Episodic hypoxia causes repetitive inspiratory activation that induces a form of respiratory plasticity termed long-term facilitation (LTF). While LTF is a function of the hypoxic exposures and inspiratory activation, their relative importance in evoking LTF is unknown. The aims of this study were to: (1) dissociate the relative roles played by episodic hypoxia and respiratory activation in LTF; and (2) determine whether the magnitude of LTF varies as a function of hypoxic intensity. We did this by examining the effects of episodic hypoxia in postnatal rats (15–25 days old), which unlike adult rats exhibit a prominent hypoxia-induced respiratory depression. We quantified inspiratory phrenic nerve activity generated by the in situ working-heart brainstem before, during and for 60 min after episodic hypoxia. We demonstrate that episodic hypoxia evokes LTF despite the fact that it potently suppresses inspiratory activity during individual hypoxic exposures ( P < 0.05). Specifically, we show that after episodic hypoxia (three 5 min periods of 10% O₂) respiratory frequency increased to 40 ± 3.3% above baseline values over the next 60 min ( P < 0.001). Continuous hypoxia (15 min of 10% O₂) had no lasting effects on respiratory frequency ( P > 0.05). To determine if LTF magnitude was affected by hypoxic intensity, the episodic hypoxia protocol was repeated under three different O₂ tensions. We demonstrate that the magnitude and time course of LTF depend on hypoxic severity, with more intense hypoxia inducing a more potent degree of LTF. We conclude that inspiratory activation is not required for LTF induction, and that hypoxia per se is the physiological stimulus for eliciting hypoxia-induced respiratory LTF.     

Propofol abolished the phrenic long-term facilitation in rats

The aim was to investigate the effect of propofol anesthesia on the phrenic long-term facilitation (pLTF) in rats. We hypothesized that pLTF would be abolished during propofol-compared with urethane anesthesia.     

Attenuated phrenic long-term facilitation in orexin neuron-ablated mice

We examined phrenic long-term facilitation (LTF) in urethane-anesthetized, vagotomized, paralyzed, and artificially ventilated orexin neuron-ablated mice and their wild-type littermates. Effect of isocapnic single hypoxic episode (SHE, for 45 s) and intermittent hypoxia (IH, 5 times of SHE separated by 5 min) on phrenic nerve activity (PNA) was measured for 1–2 h. In wild-type mice, amplitude of PNA gradually increased after cessation of IH and reached 55 ± 15% above the baseline (n = 7, p < 0.05) whereas the burst rate of PNA did not change. Qualitatively similar but significantly attenuated response (16 ± 8%) was observed in orexin neuron-ablated mice. SHE did not affect amplitude nor frequency in both animals. We conclude that orexin contributes to eliciting phrenic LTF at least in part in mice. This study also showed, for the first time, phrenic LTF following IH in WT mice. Characteristics of phrenic and ventilatory LTF in mice were similar to those in rats.     

5-HT evokes sensory long-term facilitation of rodent carotid body via activation of NADPH oxidase

5-Hydroxytryptamine (5-HT) evokes long-term activation of neuronal activity in the nervous system. Carotid bodies, the sensory organs for detecting arterial oxygen, express 5-HT. In the present study we examined whether 5-HT evokes sensory long-term facilitation (LTF) of the carotid body, and if so by what mechanism(s). Experiments were performed on anaesthetized adult rats and mice. Sensory activity was recorded from carotid bodies ex vivo . Spaced (3 × 15 s of 100 n m at 5 min intervals) but not mass (300 n m , 45 s) application of 5-HT elicited LTF, whereas both modes of 5-HT application evoked initial sensory excitation of the carotid bodies in rats. Ketanserin, a 5-HT₂ receptor antagonist prevented sensory LTF but not the initial sensory excitation. Spaced application of 5-HT activated protein kinase C (PKC) as evidenced by increased phosphorylations of PKC at Thr⁵¹⁴ and myristoylated alanine-rich C kinase substrate (MARCKS) and these effects were abolished by ketanserin as well as bisindolylmaleimide (Bis-1), an inhibitor of PKC. Bis-1 prevented 5-HT-evoked sensory LTF. 5-HT increased NADPH oxidase activity and PKC-dependent phosphorylation of p47^phox subunit of the oxidase complex. NADPH oxidase inhibitors (apocynin and diphenyl iodinium), as well as an anti-oxidant ( N -acetyl cysteine), prevented 5-HT-evoked sensory LTF. Mice deficient in gp91^phox, the membrane subunit of the NADPH oxidase complex, showed no sensory LTF, although responding to 5-HT with initial afferent nerve activation, whereas both LTF and initial excitation by 5-HT were seen in wild-type mice. These results demonstrate that spaced but not mass application of 5-HT elicits sensory LTF of the carotid body via activation of 5-HT₂ receptors, which involves a novel signalling mechanism coupled to PKC-dependent activation of NADPH oxidase.     

Reduced respiratory neural activity elicits phrenic motor facilitation

We hypothesized that reduced respiratory neural activity elicits compensatory mechanisms of plasticity that enhance respiratory motor output. In urethane-anesthetized and ventilated rats, we reversibly reduced respiratory neural activity for 25-30 min using: hypocapnia (end tidal CO(2)=30 mmHg), isoflurane (~1%) or high frequency ventilation (HFV; ~100 breaths/min). In all cases, increased phrenic burst amplitude was observed following restoration of respiratory neural activity (hypocapnia: 92±22%; isoflurane: 65±22%; HFV: 54±13% baseline), which was significantly greater than time controls receiving the same surgery, but no interruptions in respiratory neural activity (3±5% baseline, p<0.05). Hypocapnia also elicited transient increases in respiratory burst frequency (9±2 versus 1±1bursts/min, p<0.05). Our results suggest that reduced respiratory neural activity elicits a unique form of plasticity in respiratory motor control which we refer to as inactivity-induced phrenic motor facilitation (iPMF). iPMF may prevent catastrophic decreases in respiratory motor output during ventilatory control disorders associated with abnormal respiratory activity.     

Acute intermittent hypoxia induces phrenic long-term facilitation which is modulated by 5-HT1A receptor in the caudal raphe region of the rat

Obstructive sleep apnoea (OSA) is characterized by periods of upper airway collapse accompanied by repeated episodes of hypoxia. In experimental animals repeated bouts of hypoxia may evoke sustained augmentation of phrenic nerve activity, known as phrenic long‐term facilitation (pLTF). This form of physiological compensation might contribute to stable breathing, minimizing the occurrence of apnoeas and/or hypopnoeas during sleep in patients with OSA. Serotonin (5‐HT) has been shown to modulate respiratory neuronal activity, possibly via projections originating in the raphe nuclei. Our model focuses on the effects of 5‐HT_1A receptors blockade by selective antagonist WAY‐100635 into the caudal raphe region on phrenic long‐term facilitation after exposure to acute intermittent hypoxia (AIH) episodes. Adult, male, urethane‐anaesthetized, vagotomized, paralyzed and mechanically ventilated Sprague–Dawley rats were exposed to AIH protocol. Experimental group received microinjection of WAY‐100635 into the caudal raphe nucleus, whereas the control group received saline into the same site. Peak phrenic nerve activity and respiratory rhythm parameters were analysed during five hypoxic episodes, as well as at 15, 30 and 60 min after the end of hypoxias. In the control group, 1 h post‐hypoxia pLTF was developed. Microinjections of selective 5‐HT_1A receptor antagonist WAY‐100635 into the raphe nuclei prior to the AIH protocol prevented induction of pLTF. These results suggest that 5‐HT_1A receptor activation at supraspinal level is important for induction of pLTF, which is suggested to be an important respiratory neuroplasticity model in animal studies that possibly correlates with OSA in humans.     

Episodic spinal serotonin receptor activation elicits long-lasting phrenic motor facilitation by an NADPH oxidase-dependent mechanism

Phrenic long-term facilitation (pLTF) is a serotonin (5-HT)-dependent augmentation of phrenic motor output induced by acute intermittent hypoxia (AIH). AIH-induced pLTF requires spinal NADPH oxidase activity and reactive oxygen species (ROS) formation. Since 5-HT receptor activation stimulates NADPH oxidase activity in some cell types, we tested the hypothesis that episodic spinal 5-HT receptor activation (without AIH) is sufficient to elicit an NADPH oxidase-dependent facilitation of phrenic motor output (pMF). In anaesthetised, artificially ventilated adult male rats, episodic intrathecal 5-HT injections (3 × 6 μl injections at 5 min intervals) into the cerebrospinal fluid (CSF) near cervical spinal segments containing the phrenic motor nucleus elicited a progressive increase in integrated phrenic nerve burst amplitude (i.e. pMF) lasting at least 60 min post-5-HT administration. Hypoglossal (XII) nerve activity was unaffected, suggesting that effective doses of 5-HT did not reach the brainstem. A single 5-HT injection was without effect. 5-HT-induced pMF was dose dependent, but exhibited a bell-shaped dose–response curve. Activation of different 5-HT receptor subtypes, specifically 5-HT₂ versus 5-HT₇ receptors, may underlie the bell-shaped dose–response curve via a mechanism of 'cross-talk' inhibition. Pre-treatment with NADPH oxidase inhibitors, apocynin or diphenylenodium (DPI), blocked 5-HT induced pMF. Thus, episodic spinal 5-HT receptor activation is sufficient to elicit pMF by an NADPH oxidase-dependent mechanism, suggesting common mechanisms of ROS formation with AIH-induced pLTF. An understanding of the mechanisms giving rise to AIH-induced pLTF and 5-HT induced pMF may inspire novel therapeutic strategies for respiratory insufficiency in diverse conditions, such as sleep apnoea, cervical spinal injury or amyotrophic lateral sclerosis.     

Episodic phrenic-inhibitory vagus nerve stimulation paradoxically induces phrenic long-term facilitation in rats

All respiratory long-term facilitation (LTF) is induced by inspiratory-excitatory stimulation, suggesting that LTF needs inspiratory augmentation and is the result of a Hebbian mechanism (coincident pre- and post-synaptic activity strengthens synapses). The present study examined the long-term effects of episodic inspiratory-inhibitory vagus nerve stimulation (VNS) on phrenic nerve activity. We hypothesized that episodic VNS would induce phrenic long-term depression. The results are compared with those obtained following serotonin receptor antagonism or episodic carotid sinus nerve stimulation (CSNS). Integrated phrenic neurograms were measured before, during and after three episodes of 5 min VNS (50 Hz, 0.1 ms), each separated by a 5 min interval, at a low (˜50 μA), medium (˜200 μA) or high (˜500 μA) stimulus intensity in anaesthetized, vagotomized, neuromuscularly blocked and artificially ventilated rats. Medium- and high-intensity VNS eliminated rhythmic phrenic activity during VNS, while low-intensity VNS only reduced phrenic burst frequency. At 60 min post-VNS, phrenic amplitude was higher than baseline (35 ± 5 % above baseline, mean ± S.E.M., P < 0.05) in the high-intensity group but not in the low- (-4 ± 4 %) or medium-intensity groups (-10 ± 15 %), or in the high-intensity with methysergide group (4 mg kg⁻¹, I.P.) (-11 ± 5 %). These data, which are inconsistent with our hypothesis, indicate that phrenic-inhibitory VNS induces a serotonin-dependent phrenic LTF similar to that induced by phrenic-excitatory CSNS (33 ± 7 %) and may require activation of high-threshold afferent fibres. These data also suggest that the synapses on phrenic motoneurons do not use the Hebbian mechanism in this LTF, as these motoneurons were suppressed during VNS.     

NADPH oxidase is required for neutrophil-dependent autoantibody-induced tissue damage

The contribution of phagocyte-derived reactive oxygen species to tissue injury in autoimmune inflammatory diseases is unclear. Here we report that granulocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase crucially contributes to tissue injury in experimental models of the antibody-mediated autoimmune disease epidermolysis bullosa acquisita. Neutrophil cytosolic factor 1-deficient mice lacking functional NADPH oxidase were resistant to skin blistering by the passive transfer of antibodies against type VII collagen. Pharmacological inhibition or deficiency of human NADPH oxidase abolished dermal-epidermal separation caused by autoantibodies and granulocytes ex vivo. In addition, recruitment of granulocytes into the skin was required for tissue injury, as demonstrated by the resistance to experimental blistering of wild-type mice depleted of neutrophils and of CD18-deficient mice. Transfer of neutrophil cytosolic factor 1-sufficient granulocytes into neutrophil cytosolic factor 1-deficient mice demonstrated that granulocytes provide the NADPH oxidase required for tissue damage. Our findings identify granulocyte-derived NADPH oxidase as a key molecular effector engaged by pathogenic autoantibodies and provide relevant targets for prevention of tissue damage in granulocyte-mediated autoimmune diseases.     

Phrenic long-term facilitation requires NMDA receptors in the phrenic motonucleus in rats

Exposure to episodic hypoxia induces a persistent augmentation of respiratory activity, known as long-term facilitation (LTF). LTF of phrenic nerve activity has been reported to require serotonin receptor activation and protein syntheses. However, the underlying cellular mechanism still remains poorly understood. NMDA receptors play key roles in synaptic plasticity (e.g. some forms of hippocampal long-term potentiation). The present study was designed to examine the role of NMDA receptors in phrenic LTF and test if the relevant receptors are located in the phrenic motonucleus. Integrated phrenic nerve activity was measured in anaesthetized, vagotomized, neuromuscularly blocked and artificially ventilated rats before, during and after three episodes of 5 min isocapnic hypoxia ( P _a,O2= 30–45 mmHg), separated by 5 min hyperoxia (50% O₂). Either saline (as control) or the NMDA receptor antagonist MK-801 (0.2 mg kg⁻¹, i.p. ) was systemically injected ∼1 h before hypoxia. Phrenic LTF was eliminated by the MK-801 injection (vehicle, 32.8 ± 3.7% above baseline in phrenic amplitude at 60 min post-hypoxia; MK-801, −0.5 ± 4.1%, means ± s.e.m. ), with little change in both the CO₂-apnoeic threshold and the hypoxic phrenic response (HPR). Vehicle (saline, 5 × 100 nl) or MK-801 (10 μ m ; 5 × 100 nl) was also microinjected into the phrenic motonucleus region in other groups. Phrenic LTF was eliminated by the MK-801 microinjection (vehicle, 34.2 ± 3.4%; MK-801, −2.5 ± 2.8%), with minimal change in HPR. Collectively, these results suggest that the activation of NMDA receptors in the phrenic motonucleus is required for the episodic hypoxia-induced phrenic LTF.     

Microinjection of methysergide into the raphe nucleus attenuated phrenic long-term facilitation in rats

Exposure to acute intermittent hypoxia (AIH) evokes persistent increase in respiratory activity that lasts up to 60 min after hypoxic episodes have ceased. This persistent increase in phrenic nerve activity (PNA) is known as phrenic long-term facilitation (LTF). AIH-induced phrenic LTF in anesthetized rats is serotonin dependant. The present study was performed to determine whether microinjection of methysergide (4 mM, 20 ± 5 nl), a broad spectrum 5-HT receptor antagonist, into the caudal raphe nuclei influences phrenic LTF. Peak integrated PNA and respiratory frequency were recorded at 15, 30, and 60 min after five 3-min episodes of normocapnic hypoxia in urethane-anesthetized, vagotomized, paralyzed and ventilated male Sprague–Dawley rats. In control animals, phrenic nerve amplitude was elevated 66.7 ± 8.6% from baseline 1 h after episodic hypoxia, indicating phrenic LTF. Experimental microinjections of methysergide prior to AIH exposure attenuated phrenic LTF (amplitude increase 2.62 ± 2.9% over baseline). We conclude that methysergide microinjections into the caudal raphe region attenuated phrenic LTF induced by AIH, indicating involvement of 5-HT receptor activation at a supraspinal level.     

Serotonin receptor subtypes involved in vagus nerve stimulation-induced phrenic long-term facilitation in rats

Episodic vagus nerve stimulation (VNS) induces phrenic long-term facilitation (LTF, a persistent augmentation of phrenic nerve activity after the stimulation ends), sensitive to the serotonin 5-HT(1,2,5,6,7) receptor antagonist methysergide and similar to that elicited by episodic hypoxia or carotid sinus nerve stimulation. This study examined the effect of ketanserin (5-HT(2) antagonist) or clozapine (5-HT(2,6,7) antagonist) on VNS-induced LTF in anesthetized, vagotomized, paralyzed and ventilated rats to determine which receptor subtype(s) is involved. Three episodes of 5 min VNS (50 Hz, 0.1 ms, approximately 500 microA) with 5 min intervals elicited phrenic LTF in control (amplitude: 38% above baseline at 60 min post-VNS) and ketanserin (2 mg x kg(-1), i.p.) pre-treated rats (45%), but not clozapine (3 mg x kg(-1)) rats (8%). These data suggest that unlike hypoxia-induced LTF (5-HT(2) receptor-dependent), VNS-induced LTF requires non-5-HT(2) serotonin receptors, perhaps 5-HT(6) and/or 5-HT(7) subtype(s).     

低压氧舱慢性间断性缺氧诱导大鼠膈神经长时程易化

目的:长时程易化(long-term facilitation,LTF)是反映呼吸可塑性的重要电生理指标,与睡眠呼吸紊乱疾病密切相关。3~5个低氧周期的急性间断性缺氧可以诱导膈神经LTF,而持续一周以上的慢性间断性缺氧(chronic intermittent hypoxia,CIH)可以诱导更大的增强的LTF(enhanced LTF)。以往制备CIH大鼠LTF模型多用氧(10%)+氮(90%)混合气(5 min)、常氧(5 min)交替通气,每天12 h,连续7 d以上,实验需要大量混合气,费用较高。我们模拟高原缺氧制备了低压氧舱大鼠CIH模型,表达增强的膈神经LTF。方法:成年SD大鼠置于密闭容器内进行5 min低压缺氧、5 min常氧交替通气,每天12 h,持续7 d。通过空气抽提进行低压缺氧,使舱内气压逐渐下降到210~220 mmHg,相当于海拔约9000 m。第8 d,动物进行急性间断性缺氧,诱导膈神经LTF表达。对照组大鼠只进行急性间断性缺氧,统计学分析两组动物膈神经LTF的表达变化。结果:低压氧舱CIH大鼠较正常对照组对缺氧反应更加敏感,表现为缺氧期膈神经放电的频率和幅度快速增加。在急性间断性缺氧结束后30 min和60 min,CIH组大鼠膈神经放电幅度较基础水平分别增加了(116.3±6.5)%和(106.1±19.2)%,而对照组分别增加(60.4±7.8)%和(48.2±11.0)%,两组之间有显著性差别(P(0.01),表明CIH诱导了比对照组更加强大的LTF,形成增强的LTF。结论:我们建立了低压氧舱CIH大鼠膈神经LTF模型,为进一步研究LTF的发生机理、揭示与睡眠呼吸紊乱疾病的相关性提供了实验平台。     

Respiratory long-term facilitation following intermittent hypoxia requires reactive oxygen species formation

Acute intermittent hypoxia (AIH) elicits a form of respiratory plasticity known as long-term facilitation (LTF). LTF is a progressive and sustained increase in respiratory motor output as expressed in phrenic and hypoglossal (XII) nerve activity. Since reactive oxygen species (ROS) play important roles in several forms of neuroplasticity, and ROS production is increased by intermittent hypoxia, we tested the hypothesis that ROS are necessary for phrenic and XII LTF following AIH. Urethane-anesthetized, paralyzed, vagotomized and pump-ventilated Sprague-Dawley rats were exposed to AIH (11% O2, 3, 5 min episodes, 5 min intervals), and both phrenic and XII nerve activity were monitored for 60 min post-AIH. Although phrenic and XII LTF were observed in control rats, i.v. manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin pentachloride (MnTMPyP), a superoxide anion scavenger, attenuated both phrenic and XII LTF in a dose dependent manner. Localized application of MnTMPyP (5.5 mM; 10 microl) to the intrathecal space of the cervical spinal cord (C4) abolished phrenic, but not XII LTF. Thus, ROS are necessary for AIH-induced respiratory LTF, and the relevant ROS appear to be localized near respiratory motor nuclei since cervical MnTMPyP injections impaired phrenic (and not XII) LTF. Phrenic LTF is a novel form of ROS-dependent neuroplasticity since its ROS-dependence resides in the spinal cord.