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Low-dose (< 2 h/day), acute intermittent hypoxia (AIH) elicits multiple forms of serotonin-dependent phrenic motor plasticity and is emerging as a promising therapeutic strategy to restore respiratory and non-respiratory motor function after spinal cord injury (SCI). In contrast, high-dose (> 8 h/day), chronic intermittent hypoxia (CIH) undermines some forms of serotonin-dependent phrenic motor plasticity and elicits pathology. CIH is a hallmark of sleep disordered breathing, which is highly prevalent in individuals with cervical SCI. Interestingly, AIH and CIH preconditioning differentially impact phrenic motor plasticity. Although mechanisms of AIH-induced plasticity in the phrenic motor system are well-described in naïve rats, we know little concerning how these mechanisms are affected by chronic SCI or intermittent hypoxia preconditioning. Thus, in a rat model of chronic, incomplete cervical SCI (lateral spinal hemisection at C2 (C2Hx), we assessed serotonin type 2A, 2B and 7 receptor expression in and near phrenic motor neurons and compared: 1) intact vs. chronically injured rats; and 2) the impact of preconditioning with varied "doses" of intermittent hypoxia (IH). While there were no effects of chronic injury or intermittent hypoxia alone, CIH affected multiple receptors in rats with chronic C2Hx. Specifically, CIH preconditioning (8 h/day; 28 days) increased serotonin 2A and 7 receptor expression exclusively in rats with chronic C2Hx. Understanding the complex, context-specific interactions between chronic SCI and CIH and how this ultimately impacts phrenic motor plasticity is important as we leverage AIH-induced motor plasticity to restore breathing and other non-respiratory motor functions in people with chronic SCI.
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Hipoxia , Neuronas Motoras , Nervio Frénico , Receptores de Serotonina , Traumatismos de la Médula Espinal , Animales , Masculino , Ratas , Médula Cervical/lesiones , Médula Cervical/metabolismo , Vértebras Cervicales , Enfermedad Crónica , Hipoxia/metabolismo , Neuronas Motoras/metabolismo , Plasticidad Neuronal/fisiología , Nervio Frénico/metabolismo , Ratas Sprague-Dawley , Receptores de Serotonina/metabolismo , Receptores de Serotonina/biosíntesis , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/fisiopatologíaRESUMEN
Microglia are innate CNS immune cells that play key roles in supporting key CNS functions including brain plasticity. We now report a previously unknown role for microglia in regulating neuroplasticity within spinal phrenic motor neurons, the neurons driving diaphragm contractions and breathing. We demonstrate that microglia regulate phrenic long-term facilitation (pLTF), a form of respiratory memory lasting hours after repetitive exposures to brief periods of low oxygen (acute intermittent hypoxia; AIH) via neuronal/microglial fractalkine signaling. AIH-induced pLTF is regulated by the balance between competing intracellular signaling cascades initiated by serotonin vs adenosine, respectively. Although brainstem raphe neurons release the relevant serotonin, the cellular source of adenosine is unknown. We tested a model in which hypoxia initiates fractalkine signaling between phrenic motor neurons and nearby microglia that triggers extracellular adenosine accumulation. With moderate AIH, phrenic motor neuron adenosine 2A receptor activation undermines serotonin-dominant pLTF; in contrast, severe AIH drives pLTF by a unique, adenosine-dominant mechanism. Phrenic motor neuron fractalkine knockdown, cervical spinal fractalkine receptor inhibition on nearby microglia, and microglial depletion enhance serotonin-dominant pLTF with moderate AIH but suppress adenosine-dominant pLTF with severe AIH. Thus, microglia play novel functions in the healthy spinal cord, regulating hypoxia-induced neuroplasticity within the motor neurons responsible for breathing.
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Plasticity is a fundamental property of the neural system controlling breathing. One key example of respiratory motor plasticity is phrenic long-term facilitation (pLTF), a persistent increase in phrenic nerve activity elicited by acute intermittent hypoxia (AIH). pLTF can arise from distinct cell signaling cascades initiated by serotonin versus adenosine receptor activation, respectively, and interact via powerful cross-talk inhibition. Here, we demonstrate that the daily rest/active phase and the duration of hypoxic episodes within an AIH protocol have profound impact on the magnitude and mechanism of pLTF due to shifts in serotonin/adenosine balance. Using the historical "standard" AIH protocol (3, 5-min moderate hypoxic episodes), we demonstrate that pLTF magnitude is unaffected by exposure in the midactive versus midrest phase, yet the mechanism driving pLTF shifts from serotonin-dominant (midrest) to adenosine-dominant (midactive). This mechanistic "flip" results from combined influences of hypoxia-evoked adenosine release and daily fluctuations in basal spinal adenosine. Since AIH evokes less adenosine with shorter (15, 1-min) hypoxic episodes, midrest pLTF is amplified due to diminished adenosine constraint on serotonin-driven plasticity; in contrast, elevated background adenosine during the midactive phase suppresses serotonin-dominant pLTF. These findings demonstrate the importance of the serotonin/adenosine balance in regulating the amplitude and mechanism of AIH-induced pLTF. Since AIH is emerging as a promising therapeutic modality to restore respiratory and nonrespiratory movements in people with spinal cord injury or ALS, knowledge of how time-of-day and hypoxic episode duration impact the serotonin/adenosine balance and the magnitude and mechanism of pLTF has profound biological, experimental, and translational implications.
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Hipoxia , Serotonina , Ratas , Animales , Humanos , Ratas Sprague-Dawley , Transducción de Señal , AdenosinaRESUMEN
Carbon dots (CDs) from glucose were synthesized using two of the most common bottom-up methods, namely, microwave assisted (MW) and hydrothermal carbonization (HT). Synthetic parameters such as reaction time, temperature, and precursor concentration were changed to study the effects of each parameter on CD size, structure, surface functionalities, charge, photoluminescence behavior, quantum yield, cytotoxicity, blood-brain barrier (BBB) crossing ability and bioimaging. A detailed analysis is performed to compare the structure and properties of the CDs synthesized in ten different conditions. We show that the synthesis route drastically changes the structure, properties, and related functions of glucose-derived CDs yielding two different subtypes of CDs. Surprisingly, CDs that was synthesized via HT method showed specific anticancer activity against a neuroblastoma cell line while being non-toxic towards healthy cell lines, indicating significant potential for therapeutic applications. CDs synthesized via MW crosses the BBB in zebrafish and rat models, and accumulates in neurons. CDs synthesized via MW method showed high biocompatibility and a great potential to be used for bioimaging applications in vitro and in vivo targeting neurons. Finally, a formation mechanism of CDs is proposed for both HT and MW synthesis routes.
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Neuroblastoma , Puntos Cuánticos , Ratas , Animales , Carbono/química , Puntos Cuánticos/química , Microondas , Nitrógeno/química , Pez Cebra , Línea Celular , Neuroblastoma/tratamiento farmacológico , GlucosaRESUMEN
Cervical spinal cord injury (cSCI) impairs neural drive to the respiratory muscles, causing life- threatening complications such as respiratory insufficiency and diminished airway protection. Repetitive "low dose" acute intermittent hypoxia (AIH) is a promising strategy to restore motor function in people with chronic SCI. Conversely, "high dose" chronic intermittent hypoxia (CIH; â¼8 h/night), such as experienced during sleep apnea, causes pathology. Sleep apnea, spinal ischemia, hypoxia and neuroinflammation associated with cSCI increase extracellular adenosine concentrations and activate spinal adenosine receptors which in turn constrains the functional benefits of therapeutic AIH. Adenosine 1 and 2A receptors (A1, A2A) compete to determine net cAMP signaling and likely the tAIH efficacy with chronic cSCI. Since cSCI and intermittent hypoxia may regulate adenosine receptor expression in phrenic motor neurons, we tested the hypotheses that: 1) daily AIH (28 days) downregulates A2A and upregulates A1 receptor expression; 2) CIH (28 days) upregulates A2A and downregulates A1 receptor expression; and 3) cSCI alters the impact of CIH on adenosine receptor expression. Daily AIH had no effect on either adenosine receptor in intact or injured rats. However, CIH exerted complex effects depending on injury status. Whereas CIH increased A1 receptor expression in intact (not injured) rats, it increased A2A receptor expression in spinally injured (not intact) rats. The differential impact of CIH reinforces the concept that the injured spinal cord behaves in distinct ways from intact spinal cords, and that these differences should be considered in the design of experiments and/or new treatments for chronic cSCI.
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Síndromes de la Apnea del Sueño , Traumatismos de la Médula Espinal , Ratas , Animales , Neuronas Motoras , Receptores Purinérgicos P1 , Hipoxia , AdenosinaRESUMEN
Purpose: The mouthpiece is the standard interface for spirometry tests. Although the use of a mouthpiece can be challenging for patients with orofacial weakness, maintaining a proper seal with a facemask can be an issue for healthy individuals during forceful efforts. We compared respiratory muscle activity and tests using a mouthpiece and facemask in healthy adults to investigate whether they can be used interchangeably. Methods: In this observational study, subjects (n=12) completed forced vital capacity, maximal respiratory pressure, and peak cough flow with a mouthpiece and facemask. Root mean square values of the genioglossus, diaphragm, scalene, and sternocleidomastoid were compared between conditions. Results: When switching from a mouthpiece to a facemask, significantly higher values were seen for peak cough flow (average bias= -54.36 L/min, p<0.05) and the difference seen with MEP and MIP were clinically significant (average bias: MEP=27.33, MIP=-5.2). Additionally, submental activity was significantly greater when MIP was conducted with a mouthpiece. No significant differences were seen in respiratory muscle activity during resting breathing or spirometry. Conclusion: There are clinically significant differences with cough and MEP tests and neck muscles are activated differently based on interface. Considering the small sample size, our findings suggest a facemask may be used to complete some PFTs.
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Intermittent hypoxia elicits protocol-dependent effects on hypoglossal (XII) motor plasticity. Whereas low-dose, acute intermittent hypoxia (AIH) elicits serotonin-dependent plasticity in XII motor neurons, high-dose, chronic intermittent hypoxia (CIH) elicits neuroinflammation that undermines AIH-induced plasticity. Preconditioning with repeated AIH and mild CIH enhance AIH-induced XII motor plasticity. Since intermittent hypoxia pre-conditioning could enhance serotonin-dependent XII motor plasticity by increasing serotonergic innervation density of the XII motor nuclei, we tested the hypothesis that 3 distinct intermittent hypoxia protocols commonly studied to elicit plasticity (AIH) or simulate aspects of sleep apnea (CIH) differentially affect XII serotonergic innervation. Sleep apnea and associated CIH are common in people with cervical spinal injuries and, since repetitive AIH is emerging as a promising therapeutic strategy to improve respiratory and non-respiratory motor function after spinal injury, we also tested the hypotheses that XII serotonergic innervation is increased by repetitive AIH and/or CIH in rats with cervical C2 hemisections (C2Hx). Serotonergic innervation was assessed via immunofluorescence in male Sprague Dawley rats, with and without C2Hx (beginning 8 weeks post-injury) exposed to 28 days of: 1) normoxia; 2) daily AIH (10, 5-min 10.5% O2 episodes per day; 5-min normoxic intervals); 3) mild CIH (5-min 10.5% O2 episodes; 5-min intervals; 8 h/day); and 4) moderate CIH (2-min 10.5% O2 episodes; 2-min intervals; 8 h/day). Daily AIH, but neither CIH protocol, increased the area of serotonergic immunolabeling in the XII motor nuclei in both intact and injured rats. C2Hx per se had no effect on XII serotonergic innervation density. Thus, daily AIH may increases XII serotonergic innervation and function, enhancing the capacity for serotonin-dependent, AIH-induced plasticity in upper airway motor neurons. Such effects may preserve upper airway patency and/or swallowing ability in people with cervical spinal cord injuries and other clinical disorders that compromise breathing and airway defense.
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Vértebras Cervicales/lesiones , Nervio Hipogloso/metabolismo , Hipoxia/metabolismo , Neuronas Serotoninérgicas/metabolismo , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/terapia , Animales , Nervio Hipogloso/química , Hipoxia/patología , Masculino , Ratas , Ratas Sprague-Dawley , Neuronas Serotoninérgicas/química , Traumatismos de la Médula Espinal/patologíaRESUMEN
Respiratory failure is the main cause of death in amyotrophic lateral sclerosis (ALS). Since no effective treatments to preserve independent breathing are available, there is a critical need for new therapies to preserve or restore breathing ability. Since acute intermittent hypoxia (AIH) elicits spinal respiratory motor plasticity in rodent ALS models, and may restore breathing ability in people with ALS, we performed a proof-of-principle study to investigate this possibility in ALS patients. Quiet breathing, sniff nasal inspiratory pressure (SNIP) and maximal inspiratory pressure (MIP) were tested in 13 persons with ALS and 10 age-matched controls, before and 60 min post-AIH (15, 1 min episodes of 10% O2, 2 min normoxic intervals) or sham AIH (continuous normoxia). The root mean square (RMS) of the right and left diaphragm, 2nd parasternal, scalene and sternocleidomastoid muscles were monitored. A vector analysis was used to calculate summated vector magnitude (Mag) and similarity index (SI) of collective EMG activity during quiet breathing, SNIP and MIP maneuvers. AIH facilitated tidal volume and minute ventilation (treatment main effects: p < 0.05), and Mag (ie. collective respiratory muscle activity; p < 0.001) during quiet breathing in ALS and control subjects, but there was no effect on SI during quiet breathing. SNIP SI decreased in both groups post-AIH (p < 0.005), whereas Mag was unchanged (p = 0.09). No differences were observed in SNIP or MIP post AIH in either group. Discomfort was not reported during AIH by any subject, nor were adverse events observed. Thus, AIH may be a safe way to increase collective inspiratory muscle activity during quiet breathing in ALS patients, although a single AIH presentation was not sufficient to significantly increase peak inspiratory pressure generation. These preliminary results provide evidence that AIH may improve breathing function in people with ALS, and that future studies of prolonged, repetitive AIH protocols are warranted.
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Esclerosis Amiotrófica Lateral/terapia , Hipoxia , Músculos Respiratorios/fisiología , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mecánica Respiratoria/fisiologíaRESUMEN
The blood-brain barrier (BBB) is a major obstacle for drug delivery to the central nervous system (CNS) such that most therapeutics lack efficacy against brain tumors or neurological disorders due to their inability to cross the BBB. Therefore, developing new drug delivery platforms to facilitate drug transport to the CNS and understanding their mechanism of transport are crucial for the efficacy of therapeutics. Here, we report (i) carbon dots prepared from glucose and conjugated to fluorescein (GluCD-F) cross the BBB in zebrafish and rats without the need of an additional targeting ligand and (ii) uptake mechanism of GluCDs is glucose transporter-dependent in budding yeast. Glucose transporter-negative strain of yeast showed undetectable GluCD accumulation unlike the glucose transporter-positive yeast, suggesting glucose-transporter-dependent GluCD uptake. We tested GluCDs' ability to cross the BBB using both zebrafish and rat models. Following the injection to the heart, wild-type zebrafish showed GluCD-F accumulation in the central canal consistent with the transport of GluCD-F across the BBB. In rats, following intravenous administration, GluCD-F was observed in the CNS. GluCD-F was localized in the gray matter (e.g. ventral horn, dorsal horn, and middle grey) of the cervical spinal cord consistent with neuronal accumulation. Therefore, neuron targeting GluCDs hold tremendous potential as a drug delivery platform in neurodegenerative disease, traumatic injury, and malignancies of the CNS.
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KEY POINTS: While respiratory complications following opioid use are mainly mediated via activation of mu opioid receptors, long-latency off-target signalling via innate immune toll-like receptor 4 (TLR4) may impair other essential elements of breathing control such as respiratory motor plasticity. In adult rats, pre-treatment with a single dose of morphine blocked long-term facilitation (LTF) of phrenic motor output via a long-latency TLR4-dependent mechanism. In the phrenic motor nucleus, morphine triggered TLR4-dependent activation of microglial p38 MAPK - a key enzyme that orchestrates inflammatory signalling and is known to undermine phrenic LTF. Morphine-induced LTF loss may destabilize breathing, potentially contributing to respiratory side effects. Therefore, we suggest minimizing TLR-4 signalling may improve breathing stability during opioid therapy. ABSTRACT: Opioid-induced respiratory dysfunction is a significant public health burden. While respiratory effects are mediated via mu opioid receptors, long-latency off-target opioid signalling through innate immune toll-like receptor 4 (TLR4) may modulate essential elements of breathing control, particularly respiratory motor plasticity. Plasticity in respiratory motor circuits contributes to the preservation of breathing in the face of destabilizing influences. For example, respiratory long-term facilitation (LTF), a well-studied model of respiratory motor plasticity triggered by acute intermittent hypoxia, promotes breathing stability by increasing respiratory motor drive to breathing muscles. Some forms of respiratory LTF are exquisitely sensitive to inflammation and are abolished by even a mild inflammation triggered by TLR4 activation (e.g. via systemic lipopolysaccharides). Since opioids induce inflammation and TLR4 activation, we hypothesized that opioids would abolish LTF through a TLR4-dependent mechanism. In adult Sprague Dawley rats, pre-treatment with a single systemic injection of the prototypical opioid agonist morphine blocks LTF expression several hours later in the phrenic motor system - the motor pool driving diaphragm muscle contractions. Morphine blocked phrenic LTF via TLR4-dependent mechanisms because pre-treatment with (+)-naloxone - the opioid inactive stereoisomer and novel small molecule TLR4 inhibitor - prevented impairment of phrenic LTF in morphine-treated rats. Morphine triggered TLR4-dependent activation of microglial p38 MAPK within the phrenic motor system - a key enzyme that orchestrates inflammatory signalling and undermines phrenic LTF. Morphine-induced LTF loss may destabilize breathing, potentially contributing to respiratory side effects. We suggest minimizing TLR-4 signalling may improve breathing stability during opioid therapy by restoring endogenous mechanisms of plasticity within respiratory motor circuits.
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Morfina , Nervio Frénico , Receptor Toll-Like 4 , Animales , Hipoxia , Morfina/farmacología , Plasticidad Neuronal , Ratas , Ratas Sprague-Dawley , Médula EspinalRESUMEN
Although cervical spinal cord injury (cSCI) disrupts bulbo-spinal serotonergic projections, partial recovery of spinal serotonergic innervation below the injury site is observed after incomplete cSCI. Since serotonin contributes to functional recovery post-injury, treatments to restore or accelerate serotonergic reinnervation are of considerable interest. Intermittent hypoxia (IH) was reported to increase serotonin innervation near respiratory motor neurons in spinal intact rats, and to improve function after cSCI. Here, we tested the hypotheses that spontaneous serotonergic reinnervation of key respiratory (phrenic and intercostal) motor nuclei: 1) is partially restored 12 weeks post C2 hemisection (C2Hx); 2) is enhanced by IH; and 3) results from sprouting of spared crossed-spinal serotonergic projections below the site of injury. Serotonin was assessed via immunofluorescence in male Sprague Dawley rats with and without C2Hx (12 wks post-injury); individual groups were exposed to 28 days of: 1) normoxia; 2) daily acute IH (dAIH28: 10, 5 min 10.5% O2 episodes per day; 5 min normoxic intervals); 3) mild chronic IH (IH28-5/5: 5 min 10.5% O2 episodes; 5 min intervals; 8 h/day); or 4) moderate chronic IH (IH28-2/2: 2 min 10.5% O2 episodes; 2 min intervals; 8 h/day), simulating IH experienced during moderate sleep apnea. After C2Hx, the number of ipsilateral serotonergic structures was decreased in both motor nuclei, regardless of IH protocol. However, serotonergic structures were larger after C2Hx in both motor nuclei, and total serotonin immunolabeling area was increased in the phrenic motor nucleus but reduced in the intercostal motor nucleus. Both chronic IH protocols increased serotonin structure size and total area in the phrenic motor nuclei of uninjured rats, but had no detectable effects after C2Hx. Although the functional implications of fewer but larger serotonergic structures are unclear, we confirm that serotonergic reinnervation is substantial following injury, but IH does not affect the extent of reinnervation.
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Médula Cervical/fisiopatología , Hipoxia , Regeneración Nerviosa/fisiología , Serotonina/metabolismo , Traumatismos de la Médula Espinal/fisiopatología , Animales , Médula Cervical/metabolismo , Vértebras Cervicales , Nervios Intercostales/metabolismo , Nervios Intercostales/fisiopatología , Masculino , Neuronas Motoras/fisiología , Nervio Frénico/metabolismo , Nervio Frénico/fisiopatología , Ratas , Ratas Sprague-Dawley , Recuperación de la Función/fisiología , Traumatismos de la Médula Espinal/metabolismoRESUMEN
BACKGROUND: Inflammation undermines multiple forms of neuroplasticity. Although inflammation and its influence on plasticity in multiple neural systems has been extensively studied, its effects on plasticity of neural networks controlling vital life functions, such as breathing, are less understood. In this study, we investigated the signaling mechanisms whereby lipopolysaccharide (LPS)-induced systemic inflammation impairs plasticity within the phrenic motor system-a major spinal respiratory motor pool that drives contractions of the diaphragm muscle. Here, we tested the hypotheses that lipopolysaccharide-induced systemic inflammation (1) blocks phrenic motor plasticity by a mechanism that requires cervical spinal okadaic acid-sensitive serine/threonine protein phosphatase (PP) 1/2A activity and (2) prevents phosphorylation/activation of extracellular signal-regulated kinase 1/2 mitogen activated protein kinase (ERK1/2 MAPK)-a key enzyme necessary for the expression of phrenic motor plasticity. METHODS: To study phrenic motor plasticity, we utilized a well-characterized model for spinal respiratory plasticity called phrenic long-term facilitation (pLTF). pLTF is characterized by a long-lasting, progressive enhancement of inspiratory phrenic nerve motor drive following exposures to moderate acute intermittent hypoxia (mAIH). In anesthetized, vagotomized and mechanically ventilated adult Sprague Dawley rats, we examined the effect of inhibiting cervical spinal serine/threonine PP 1/2A activity on pLTF expression in sham-vehicle and LPS-treated rats. Using immunofluorescence optical density analysis, we compared mAIH-induced phosphorylation/activation of ERK 1/2 MAPK with and without LPS-induced inflammation in identified phrenic motor neurons. RESULTS: We confirmed that mAIH-induced pLTF is abolished 24 h following low-dose systemic LPS (100 µg/kg, i.p.). Cervical spinal delivery of the PP 1/2A inhibitor, okadaic acid, restored pLTF in LPS-treated rats. LPS also prevented mAIH-induced enhancement in phrenic motor neuron ERK1/2 MAPK phosphorylation. Thus, a likely target for the relevant okadaic acid-sensitive protein phosphatases is ERK1/2 MAPK or its upstream activators. CONCLUSIONS: This study increases our understanding of fundamental mechanisms whereby inflammation disrupts neuroplasticity in a critical population of motor neurons necessary for breathing, and highlights key roles for serine/threonine protein phosphatases and ERK1/2 MAPK kinase in the plasticity of mammalian spinal respiratory motor circuits.
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Inflamación/metabolismo , Neuronas Motoras/enzimología , Plasticidad Neuronal/fisiología , Fosfoproteínas Fosfatasas/metabolismo , Nervio Frénico/enzimología , Animales , Inflamación/inducido químicamente , Lipopolisacáridos/toxicidad , Potenciación a Largo Plazo/fisiología , Sistema de Señalización de MAP Quinasas , Masculino , Ratas , Ratas Sprague-Dawley , Fenómenos Fisiológicos RespiratoriosRESUMEN
Moderate acute intermittent hypoxia (AIH) elicits a persistent, serotonin-dependent increase in phrenic amplitude, known as phrenic long-term facilitation (pLTF). Although pLTF was originally demonstrated by carotid sinus nerve stimulation, AIH still elicits residual pLTF in carotid denervated (CBX) rats via a distinct, but unknown mechanism. We hypothesized that exaggerated hypoxia-induced hypotension after carotid denervation leads to greater spinal tissue hypoxia and extracellular adenosine accumulation, thereby triggering adenosine 2A receptor (A2A)-dependent pLTF. Phrenic activity, arterial pressure and spinal tissue oxygen pressure were measured in anesthetized CBX rats. Exaggerated hypoxia-induced hypotension after CBX was prevented via intravenous phenylephrine; without the hypotension, spinal tissue hypoxia during AIH was normalized, and residual pLTF was no longer observed. Spinal A2A (MSX-3), but not serotonin 2 receptor (5-HT2) inhibition (ketanserin), abolished residual pLTF in CBX rats. Thus, pLTF regulation may be altered in conditions impairing sympathetic activity and arterial pressure regulation, such as spinal cord injury.
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Cuerpo Carotídeo , Hipotensión/etiología , Hipotensión/metabolismo , Hipoxia/complicaciones , Potenciación a Largo Plazo , Nervio Frénico/fisiopatología , Adenosina/metabolismo , Animales , Presión Arterial , Análisis de los Gases de la Sangre , Desnervación , Ketanserina/farmacología , Potenciación a Largo Plazo/efectos de los fármacos , Masculino , Fenilefrina/farmacología , Ratas , Ratas Sprague-Dawley , Receptor de Adenosina A2A/metabolismo , Antagonistas del Receptor de Serotonina 5-HT2/farmacología , Médula Espinal/patologíaRESUMEN
Respiratory motor neuron survival is critical for maintenance of adequate ventilation and airway clearance, preventing dependence to mechanical ventilation and respiratory tract infections. Phrenic motor neurons are highly vulnerable in rodent models of motor neuron disease versus accessory inspiratory motor pools (e.g. intercostals, scalenus). Thus, strategies that promote phrenic motor neuron survival when faced with disease and/or toxic insults are needed to help preserve breathing ability, airway defense and ventilator independence. Adenosine 2A receptors (A2A) are emerging as a potential target to promote neuroprotection, although their activation can have both beneficial and pathogenic effects. Since the role of A2A receptors in the phrenic motor neuron survival/death is not known, we tested the hypothesis that A2A receptor antagonism promotes phrenic motor neuron survival and preserves diaphragm function when faced with toxic, neurodegenerative insults that lead to phrenic motor neuron death. We utilized a novel neurotoxic model of respiratory motor neuron death recently developed in our laboratory: intrapleural injections of cholera toxin B subunit (CtB) conjugated to the ribosomal toxin, saporin (CtB-Saporin). We demonstrate that intrapleural CtB-Saporin causes: 1) profound phrenic motor neuron death (~5% survival); 2) ~7-fold increase in phrenic motor neuron A2A receptor expression prior to cell death; and 3) diaphragm muscle paralysis (inactive in most rats; ~7% residual diaphragm EMG amplitude during room air breathing). The A2A receptor antagonist istradefylline given after CtB-Saporin: 1) reduced phrenic motor neuron death (~20% survival) and 2) preserved diaphragm EMG activity (~46%). Thus, A2A receptors contribute to neurotoxic phrenic motor neuron death, an effect mitigated by A2A receptor antagonism.
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Antagonistas del Receptor de Adenosina A2/farmacología , Toxina del Cólera/toxicidad , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/metabolismo , Nervio Frénico/efectos de los fármacos , Nervio Frénico/metabolismo , Saporinas/toxicidad , Animales , Apoptosis/efectos de los fármacos , Diafragma/inervación , Masculino , Purinas/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
Respiratory motor neuron death arises from multiple neurodegenerative and traumatic neuromuscular disorders. Despite motor neuron death, compensatory mechanisms minimize its functional impact by harnessing intrinsic mechanisms of compensatory respiratory plasticity. However, the capacity for compensation eventually reaches limits and pathology ensues. Initially, challenges to the system such as increased metabolic demand reveal sub-clinical pathology. With greater motor neuron loss, the eventual result is de-compensation, ventilatory failure, ventilator dependence and then death. In this brief review, we discuss recent advances in our understanding of mechanisms giving rise to compensatory respiratory plasticity in response to respiratory motor neuron death including: 1) increased central respiratory drive, 2) plasticity in synapses on spared phrenic motor neurons, 3) enhanced neuromuscular transmission and 4) shifts in respiratory muscle utilization from more affected to less affected motor pools. Some of these compensatory mechanisms may prolong breathing function, but hasten the demise of surviving motor neurons. Improved understanding of these mechanisms and their impact on survival of spared motor neurons will guide future efforts to develop therapeutic interventions that preserve respiratory function with neuromuscular injury/disease.
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Muerte Celular/fisiología , Neuronas Motoras/fisiología , Enfermedades Neuromusculares/fisiopatología , Plasticidad Neuronal/fisiología , Nervio Frénico/fisiología , Reclutamiento Neurofisiológico/fisiología , Respiración , Centro Respiratorio/fisiología , Músculos Respiratorios/fisiología , Animales , HumanosRESUMEN
Spinal chloride-dependent synaptic inhibition is critical in regulating breathing and requires neuronal chloride gradients established by cation-chloride cotransporters Na+-K+-2Cl- (NKCC1) and K+-Cl- (KCC2). Spinal transection disrupts NKCC1/KCC2 balance, diminishing chloride gradients in neurons below injury, contributing to spasticity and chronic pain. It is not known if similar disruptions in NKCC1/KCC2 balance occur in respiratory motor neurons after incomplete cervical contusion (C2SC). We hypothesized that C2SC disrupts NKCC1/KCC2 balance in phrenic motor neurons. NKCC1 and KCC2 immunoreactivity was assessed in CtB-positive phrenic motor neurons. Five weeks post-C2SC: 1) neither membrane-bound nor cytosolic NKCC1 expression were significantly changed, although the membrane/cytosolic ratio increased, consistent with net chloride influx; and 2) both membrane and cytosolic KCC2 expression increased, although the membrane/cytosolic ratio decreased, consistent with net chloride efflux. Thus, contrary to our original hypothesis, complex shifts in NKCC1/KCC2 balance occur post-C2SC. The functional significance of these changes remains unclear.
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Médula Cervical/lesiones , Contusiones/metabolismo , Neuronas Motoras/metabolismo , Nervio Frénico/metabolismo , Miembro 2 de la Familia de Transportadores de Soluto 12/metabolismo , Simportadores/metabolismo , Animales , Membrana Celular/metabolismo , Membrana Celular/patología , Médula Cervical/metabolismo , Médula Cervical/patología , Vértebras Cervicales , Contusiones/patología , Citosol/metabolismo , Citosol/patología , Modelos Animales de Enfermedad , Masculino , Neuronas Motoras/patología , Nervio Frénico/patología , Distribución Aleatoria , Ratas Endogámicas Lew , Traumatismos de la Médula Espinal/metabolismo , Cotransportadores de K ClRESUMEN
Loss-of-function mutations in ATP13A2 are associated with three neurodegenerative diseases: a rare form of Parkinson's disease termed Kufor-Rakeb syndrome (KRS), a lysosomal storage disorder termed neuronal ceroid lipofuscinosis (NCL), and a form of hereditary spastic paraplegia (HSP). Furthermore, recent data suggests that heterozygous carriers of mutations in ATP13A2 may confer risk for the development of Parkinson's disease, similar to the association of mutations in glucocerebrosidase (GBA) with both Parkinson's disease and Gaucher's disease, a lysosomal storage disorder. Mutations in ATP13A2 are generally thought to be loss of function; however, the lack of human autopsy tissue has prevented the field from determining the pathological consequences of losing functional ATP13A2. We and others have previously neuropathologically characterized mice completely lacking murine Atp13a2, demonstrating the presence of lipofuscinosis within the brain - a key feature of NCL, one of the diseases to which ATP13A2 mutations have been linked. To determine if loss of one functional Atp13a2 allele can serve as a risk factor for disease, we have now assessed heterozygous Atp13a2 knockout mice for key features of NCL. In this report, we demonstrate that loss of one functional Atp13a2 allele leads to both microgliosis and astrocytosis in multiple brain regions compared to age-matched controls; however, levels of lipofuscin were only modestly elevated in the cortex of heterozygous Atp13a2 knockout mice over controls. This data suggests the possibility that partial loss of ATP13A2 causes inflammatory changes within the brain which appear to be independent of robust lipofuscinosis. This study suggests that heterozygous loss-of-function mutations in ATP13A2 are likely harmful and indicates that glial involvement in the disease process may be an early event that positions the CNS for subsequent disease development.
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Adenosina Trifosfatasas/genética , Gliosis/genética , Proteínas de la Membrana/genética , Lipofuscinosis Ceroideas Neuronales/genética , Adenosina Trifosfatasas/metabolismo , Animales , Encéfalo/metabolismo , Encéfalo/patología , Gliosis/metabolismo , Heterocigoto , Lipofuscina/metabolismo , Mutación con Pérdida de Función , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Lipofuscinosis Ceroideas Neuronales/metabolismo , ATPasas de Translocación de ProtónRESUMEN
KEY POINTS: Although adenosine 2A (A2A ) receptor activation triggers specific cell signalling cascades, the ensuing physiological outcomes depend on the specific cell type expressing these receptors. Cervical spinal adenosine 2A (A2A ) receptor activation elicits a prolonged facilitation in phrenic nerve activity, which was nearly abolished following intrapleural A2A receptor siRNA injections. A2A receptor siRNA injections selectively knocked down A2A receptors in cholera toxin B-subunit-identified phrenic motor neurons, sparing cervical non-phrenic motor neurons. Collectively, our results support the hypothesis that phrenic motor neurons express the A2A receptors relevant to A2A receptor-induced phrenic motor facilitation. Upregulation of A2A receptor expression in the phrenic motor neurons per se may potentially be a useful approach to increase phrenic motor neuron excitability in conditions such as spinal cord injury. ABSTRACT: Cervical spinal adenosine 2A (A2A ) receptor activation elicits a prolonged increase in phrenic nerve activity, an effect known as phrenic motor facilitation (pMF). The specific cervical spinal cells expressing the relevant A2A receptors for pMF are unknown. This is an important question since the physiological outcome of A2A receptor activation is highly cell type specific. Thus, we tested the hypothesis that the relevant A2A receptors for pMF are expressed in phrenic motor neurons per se versus non-phrenic neurons of the cervical spinal cord. A2A receptor immunostaining significantly colocalized with NeuN-positive neurons (89 ± 2%). Intrapleural siRNA injections were used to selectively knock down A2A receptors in cholera toxin B-subunit-labelled phrenic motor neurons. A2A receptor knock-down was verified by a â¼45% decrease in A2A receptor immunoreactivity within phrenic motor neurons versus non-targeting siRNAs (siNT; P < 0.05). There was no evidence for knock-down in cervical non-phrenic motor neurons. In rats that were anaesthetized, subjected to neuromuscular blockade and ventilated, pMF induced by cervical (C3-4) intrathecal injections of the A2A receptor agonist CGS21680 was greatly attenuated in siA2A (21%) versus siNT treated rats (147%; P < 0.01). There were no significant effects of siA2A on phrenic burst frequency. Collectively, our results support the hypothesis that phrenic motor neurons express the A2A receptors relevant to A2A receptor-induced pMF.
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Neuronas Motoras/metabolismo , Nervio Frénico/metabolismo , Receptor de Adenosina A2A/metabolismo , Potenciales de Acción , Agonistas del Receptor de Adenosina A2/farmacología , Animales , Toxina del Cólera/farmacología , Masculino , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/fisiología , Nervio Frénico/citología , Nervio Frénico/fisiología , Ratas , Ratas Sprague-DawleyRESUMEN
In SOD1G93A transgenic rat model of ALS, breathing capacity is preserved until late in disease progression despite profound respiratory motor neuron (MN) cell death. To explore mechanisms preserving breathing capacity, we assessed inspiratory EMG activity in diaphragm and external intercostal T2 (EIC2) and T5 (EIC5) muscles in anesthetized SOD1G93A rats at disease end-stage (20% decrease in body mass). We hypothesized that despite significant phrenic motor neuron loss and decreased phrenic nerve activity, diaphragm electrical activity and trans-diaphragmatic pressure (Pdi) are maintained to sustain ventilation. We alternatively hypothesized that EIC activity is enhanced, compensating for impaired diaphragm function. Diaphragm, EIC2 and EIC5 muscle EMGs and Pdi were measured in urethane-anesthetized, spontaneously breathing female SOD1G93A rats versus wild-type littermates during normoxia (arterial PO2 ~90mmHg, PCO2 ~45mmHg), maximal chemoreceptor stimulation (MCS: 10.5% O2/7% CO2), spontaneous augmented breaths and sustained tracheal occlusion. Phrenic MNs were counted in C3-5; T2 and T5 ventrolateral MNs were counted. In end-stage SOD1G93A rats, 29% of phrenic MNs survived (vs. wild-type), yet integrated diaphragm EMG amplitude was normal. Nevertheless, maximal Pdi decreased ~30% vs. wild type (p<0.01) and increased esophageal to gastric pressure ratio (p<0.05), consistent with persistent diaphragm weakness. Despite major T2 and T5 MN death, integrated EIC2 (100% greater than wild type) and EIC5 (300%) EMG amplitudes were increased in mutant rats during normoxia (p<0.01), possibly compensating for decreased Pdi. Thus, despite significant phrenic MN loss, diaphragm EMG activity is maintained; in contrast, Pdi was not, suggesting diaphragm dysfunction. Presumably, increased EIC EMG activity compensated for persistent diaphragm weakness. These adjustments contribute to remarkable preservation of breathing ability despite major respiratory motor neuron death and diaphragm dysfunction.
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Esclerosis Amiotrófica Lateral/fisiopatología , Diafragma/fisiopatología , Músculos Intercostales/fisiopatología , Músculos Respiratorios/fisiopatología , Esclerosis Amiotrófica Lateral/genética , Animales , Electromiografía , Femenino , Neuronas Motoras/patología , Neuronas/patología , Nervio Frénico/patología , Nervio Frénico/fisiopatología , Ratas , Ratas Sprague-Dawley , Ratas Transgénicas , Respiración , Superóxido Dismutasa-1/genéticaRESUMEN
Phrenic motor neurons are recruited across a range of motor behaviors to generate varying levels of diaphragm muscle (DIAm) force. We hypothesized that DIAm motor units are recruited in a fixed order across a range of motor behaviors of varying force levels, consistent with the Henneman Size Principle. Single motor unit action potentials and compound DIAm EMG activities were recorded in anesthetized, neurally intact rats across different motor behaviors, i.e., eupnea, hypoxia-hypercapnia (10% O2 and 5% CO2), deep breaths, sustained airway occlusion, and sneezing. Central drive [estimated by root-mean-squared (RMS) EMG value 75 ms after the onset of EMG activity (RMS75)], recruitment delay, and onset discharge frequencies were similar during eupnea and hypoxia-hypercapnia. Compared with eupnea, central drive increased (â¼25%) during deep breaths, and motor units were recruited â¼12 ms earlier (P < 0.01). During airway occlusion, central drive was â¼3 times greater, motor units were recruited â¼30 ms earlier (P < 0.01), and motor unit onset discharge frequencies were significantly higher (P < 0.01). Recruitment order of motor unit pairs observed during eupnea was maintained for 98%, 87%, and 84% of the same pairs recorded during hypoxia-hypercapnia, deep breaths, and airway occlusion, respectively. Reversals in motor unit recruitment order were observed primarily if motor unit pairs were recruited <20 ms apart. These results are consistent with DIAm motor unit recruitment order being determined primarily by the intrinsic size-dependent electrophysiological properties of phrenic motor neurons.