RESUMEN
Cervical spinal cord hemisection at C2 leads to paralysis of the ipsilateral hemidiaphragm in rats. Respiratory function of the paralyzed hemidiaphragm can be restored by activating a latent respiratory motor pathway in adult rats. This pathway is called the crossed phrenic pathway and the restored activity in the paralyzed hemidiaphragm is referred to as crossed phrenic activity. The latent neural pathway is not latent in neonatal rats as shown by the spontaneous expression of crossed phrenic activity. However, the anatomy of the pathway in neonatal rats is still unknown. In the present study, we hypothesized that the crossed phrenic pathway may be different anatomically in neonatal and adult rats. To delineate this neural pathway in neonates, we injected wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP), a retrograde transsynaptic tracer, into the phrenic nerve ipsilateral to hemisection. We also injected cholera toxin subunit B-horseradish peroxidase (BHRP) into the ipsilateral hemidiaphragm following hemisection in other animals to determine if there are midline-crossing phrenic dendrites involved in the crossed phrenic pathway in neonatal rats. The WGA-HRP labeling was observed only in the ipsilateral phrenic nucleus and ipsilateral rostral ventral respiratory group (rVRG) in the postnatal day (P) 2, P7, and P28 hemisected rats. Bilateral labeling of rVRG neurons was shown in P35 rats. The BHRP study showed that many phrenic dendrites cross the midline in P2 neonatal rats at both rostral and caudal parts of the phrenic nucleus. There was a marked reduction of crossing dendrites observed in P7 and P28 animals and no crossing dendrites observed in P35 rats. The present results suggest that the crossed phrenic pathway in neonatal rats involves the parent axons from ipsilateral rVRG premotor neurons that cross at the level of obex as well as decussating axon collaterals that cross over the spinal cord midline to innervate ipsilateral phrenic motoneurons following C2 hemisection. In addition, midline-crossing dendrites of the ipsilateral phrenic motoneurons may also contribute to the crossed phrenic pathway in neonates.
Asunto(s)
Diafragma/patología , Parálisis/patología , Nervio Frénico/patología , Recuperación de la Función , Traumatismos de la Médula Espinal/patología , Médula Espinal/patología , Envejecimiento , Animales , Animales Recién Nacidos , Dendritas/patología , Dendritas/fisiología , Diafragma/crecimiento & desarrollo , Diafragma/fisiopatología , Femenino , Masculino , Vías Nerviosas/crecimiento & desarrollo , Vías Nerviosas/patología , Vías Nerviosas/fisiopatología , Trazadores del Tracto Neuronal , Neuronas/patología , Neuronas/fisiología , Parálisis/fisiopatología , Nervio Frénico/crecimiento & desarrollo , Nervio Frénico/fisiopatología , Ratas , Ratas Sprague-Dawley , Recuperación de la Función/fisiología , Médula Espinal/crecimiento & desarrollo , Médula Espinal/fisiopatología , Traumatismos de la Médula Espinal/fisiopatologíaRESUMEN
The present study investigated the involvement of the adenosine 3'5'-cyclic monophosphate-dependent protein kinase A (cAMP-PKA) pathway in the activation of the crossed-phrenic pathways after left C2 spinal cord hemisection. Experiments were conducted on left C2 spinal cord hemisected, anesthetized, vagotomized, pancuronium paralyzed, and artificially ventilated male Sprague-Dawley rats. One week post-injury, the ipsilateral phrenic nerve exhibited no respiratory-related activity indicating a functionally complete hemisection. Intrathecal spinal cord administration of the cAMP analog, 8-Br-cAMP at the level of the phrenic nucleus resulted in an enhancement of contralateral phrenic nerve output and a restoration of respiratory-related activity in the phrenic nerve ipsilateral to the hemisection. Furthermore, pre-treatment with Rp-8-Br-cAMP, a PKA inhibitor, abolished the effects of 8-Br-cAMP. These results suggest that PKA activation is necessary for the cAMP-mediated respiratory recovery following high cervical spinal cord injury and that activation of intracellular signaling cascades may represent an important strategy for improving respiratory function after spinal cord injury.
Asunto(s)
Vértebras Cervicales/lesiones , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , AMP Cíclico/fisiología , Fenómenos Fisiológicos Respiratorios , Transducción de Señal/fisiología , Traumatismos de la Médula Espinal/fisiopatología , Médula Espinal/fisiopatología , Animales , Apnea/inducido químicamente , Apnea/fisiopatología , Dióxido de Carbono/farmacología , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Interpretación Estadística de Datos , Electrofisiología , Inhibidores Enzimáticos/farmacología , Lateralidad Funcional/efectos de los fármacos , Lateralidad Funcional/fisiología , Masculino , Nervio Frénico/efectos de los fármacos , Nervio Frénico/fisiopatología , Ratas , Ratas Sprague-Dawley , Tionucleótidos/farmacologíaRESUMEN
High cervical spinal cord hemisection interrupts the descending respiratory drive from the medulla to the ipsilateral phrenic motoneurons, consequently leading to the paralysis of the ipsilateral hemidiaphragm. Previous studies have shown that chronic oral administration of theophylline, a phosphodiesterase inhibitor and an adenosine receptor antagonist, can restore function to the quiescent phrenic nerve and hemidiaphragm ipsilateral to hemisection. Both of these actions of theophylline result in an increase in 3'-5'-cyclic adenosine monophosphate (cAMP). Furthermore, the chronic theophylline-mediated respiratory recovery persists long after the animals have been weaned from the drug. To date, the precise cellular mechanisms underlying the recovery induced by theophylline are still not known. Since theophylline has two modes of action, in the present study we tested whether chronic administration of pentoxifylline, a non-selective phosphodiesterase inhibitor, rolipram, a phosphodiesterase-4 specific inhibitor, and 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), an adenosine A1 receptor antagonist, would induce recovery similar to that induced by theophylline in male Sprague-Dawley rats following a left C2 spinal cord lesion. Recovery of left phrenic nerve activity was assessed at 5 or 10 days after the last drug administrations to assess the persistent nature of the recovery. Pentoxifylline, rolipram and DPCPX, all capable of modulating 3',5'-cyclic monophosphate (cAMP) levels, brought about long-term respiratory recovery in the phrenic nerve ipsilateral to the left C2 lesion at 5 and 10 days after the last drug administration. Therefore, these results suggest that compounds capable of regulating cAMP levels may be therapeutically useful in promoting functional recovery following spinal cord injury.
Asunto(s)
Inhibidores de Fosfodiesterasa/uso terapéutico , Nervio Frénico/efectos de los fármacos , Recuperación de la Función/efectos de los fármacos , Traumatismos de la Médula Espinal/tratamiento farmacológico , Xantinas/uso terapéutico , Potenciales de Acción/efectos de los fármacos , Animales , Vértebras Cervicales/patología , Diafragma/efectos de los fármacos , Diafragma/inervación , Modelos Animales de Enfermedad , Electromiografía , Masculino , Pentoxifilina/uso terapéutico , Nervio Frénico/fisiopatología , Ratas , Ratas Sprague-Dawley , Respiración/efectos de los fármacos , Rolipram/uso terapéutico , Traumatismos de la Médula Espinal/patología , Factores de TiempoRESUMEN
1. Previous studies from our laboratory have established that a latent respiratory motor pathway can be activated to restore function to a hemidiaphragm paralysed by upper cervical (C2) spinal cord hemisection during a reflex known as the 'crossed phrenic phenomenon'. In addition, theophylline, a general adenosine A1 and A2 receptor antagonist, can activate the latent pathway by acting centrally through antagonism at adenosine receptors. 2. The present study was designed to assess the relative contributions of adenosine A1 and A2 receptors in inducing functional recovery in our model of spinal cord injury. Specific adenosine A1 and A2 agonists and antagonists were used in an electrophysiological study. 3. Our results demonstrate that, in hemisected rats, systemic administration of the adenosine A1 receptor-specific antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) restores, in a dose-dependent manner, phrenic nerve respiratory related output that is lost following hemisection. Furthermore, DPCPX augments respiratory activity in non-injured animals. The A2 receptor agonist CGS-21680 mediates its effects by predominantly acting on peripheral rather than central nervous system (CNS) receptors. CGS-21680 modulates respiratory related phrenic nerve activity in non-injured animals by enhancing tonic activity, but does not induce recovery of phrenic nerve activity in hemisected animals in the majority of cases. When CGS-21680 was administered prior to DPCPX in hemisected rats, the magnitude of recovery of respiratory function was significantly greater than that elicited by DPCPX alone. However, when the A2 receptor agonist was administered after DPCPX, the magnitude of recovery was virtually unchanged, whereas activity in the right phrenic nerve was significantly enhanced. The A1 receptor agonist N6-cyclohexyladenosine depressed respiratory activity in non-injured, as well as hemisected, rats. The A2 receptor antagonist 3,7-dimethyl-1-propargylxanthine did not affect respiratory activity. 4. We conclude that while antagonism at central adenosine A1 receptors mediates functional restitution in hemisected animals, activation of A2 receptors located outside of the CNS subserves the A1 receptor-mediated respiratory recovery.
Asunto(s)
Adenosina/análogos & derivados , Nervio Frénico/fisiología , Agonistas del Receptor Purinérgico P1 , Antagonistas de Receptores Purinérgicos P1 , Traumatismos de la Médula Espinal/fisiopatología , Adenosina/farmacología , Adenosina/uso terapéutico , Animales , Vértebras Cervicales/efectos de los fármacos , Vértebras Cervicales/lesiones , Vértebras Cervicales/fisiología , Femenino , Fenetilaminas/farmacología , Fenetilaminas/uso terapéutico , Nervio Frénico/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Receptores Purinérgicos P1/fisiología , Traumatismos de la Médula Espinal/tratamiento farmacológico , Xantinas/farmacología , Xantinas/uso terapéuticoRESUMEN
The aim of the present study was to specifically investigate the involvement of serotonin [5-hydroxytryptamine (5-HT(2))] receptors in 5-HT-mediated respiratory recovery after cervical hemisection. Experiments were conducted on C(2) spinal cord-hemisected, anesthetized (chloral hydrate, 400 mg/kg ip), vagotomized, pancuronium- paralyzed, and artificially ventilated female Sprague-Dawley rats in which CO(2) levels were monitored and maintained. Twenty-four hours after spinal hemisection, the ipsilateral phrenic nerve displayed no respiratory-related activity indicative of a functionally complete hemisection. Intravenous administration of the 5-HT(2A/2C)-receptor agonist (+/-)-2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI) induced respiratory-related activity in the phrenic nerve ipsilateral to hemisection under conditions in which CO(2) was maintained at constant levels and augmented the activity induced under conditions of hypercapnia. The effects of DOI were found to be dose dependent, and the recovery of activity could be maintained for up to 2 h after a single injection. DOI-induced recovery was attenuated by the 5-HT(2)-receptor antagonist ketanserin but not with the 5-HT(2C)-receptor antagonist RS-102221, suggesting that 5-HT(2A) and not necessarily 5-HT(2C) receptors may be involved in the induction of respiratory recovery after cervical spinal cord injury.
Asunto(s)
Receptores de Serotonina/fisiología , Fenómenos Fisiológicos Respiratorios , Anfetaminas/farmacología , Animales , Asfixia/fisiopatología , Vértebras Cervicales , Femenino , Ketanserina/farmacología , Nervio Frénico/efectos de los fármacos , Nervio Frénico/fisiopatología , Pirazinas/farmacología , Ratas , Ratas Sprague-Dawley , Receptores de Serotonina/efectos de los fármacos , Valores de Referencia , Antagonistas de la Serotonina/farmacología , Agonistas de Receptores de Serotonina/farmacología , Traumatismos de la Médula Espinal , Compuestos de Espiro/farmacología , Sulfonamidas/farmacologíaRESUMEN
Previous studies have demonstrated that during respiratory stress (hypercapnia and hypoxia), a latent crossed respiratory pathway can be activated to produce hemidiaphragm recovery following an ipsilateral C2 spinal cord hemisection. The present study investigates the effects of ventral medullary chemoreceptor area stimulation by microinjection of (1S,3R)-aminocyclopentanedicarboxylic acid (ACPD), a glutamate metabotropic receptor agonist, on activating the latent pathway following left C2 spinal cord hemisection in rats in which end-tidal CO2 was maintained at a constant level. Experiments were conducted on anesthetized, vagotomized, paralyzed, and artificially ventilated rats in which phrenic nerve activity was recorded bilaterally. Before drug injection, the phrenic nerve contralateral to hemisection showed vigorous respiratory-related activity, but the phrenic nerve ipsilateral to hemisection showed no discernible respiratory-related activity. ACPD (1-100 nl, 1 mM) was injected directly into the region of the retrotrapezoid nucleus (RTN), a known medullary chemoreceptor area. Microinjection of ACPD into the right RTN increased respiratory-related activity in the right phrenic nerve (contralateral to hemisection). ACPD (>5 nl, 1 mM) microinjection also significantly induced respiratory recovery in the phrenic nerve ipsilateral to hemisection in a dose-dependent manner. The present study indicates that respiratory recovery can be achieved by stimulation of respiratory circuitry without increasing CO2 levels.
Asunto(s)
Células Quimiorreceptoras/fisiología , Bulbo Raquídeo/fisiología , Vías Nerviosas/fisiología , Neuronas/fisiología , Fenómenos Fisiológicos Respiratorios , Animales , Dióxido de Carbono/fisiología , Células Quimiorreceptoras/efectos de los fármacos , Cicloleucina/análogos & derivados , Cicloleucina/farmacología , Relación Dosis-Respuesta a Droga , Electrofisiología , Femenino , Bulbo Raquídeo/citología , Bulbo Raquídeo/efectos de los fármacos , Microinyecciones , Vías Nerviosas/efectos de los fármacos , Neuronas/efectos de los fármacos , Nervio Frénico/efectos de los fármacos , Nervio Frénico/fisiología , Intercambio Gaseoso Pulmonar , Ratas , Ratas Sprague-Dawley , Receptores de Glutamato Metabotrópico/agonistas , Recuperación de la Función/efectos de los fármacos , Respiración Artificial , Fenómenos Fisiológicos Respiratorios/efectos de los fármacos , Médula Espinal/fisiología , Estimulación Química , Volumen de Ventilación Pulmonar/fisiología , VagotomíaRESUMEN
Cervical spinal cord injury leads to a disruption of bulbospinal innervation from medullary respiratory centers to phrenic motoneurons. Animal models utilizing cervical hemisection result in inhibition of ipsilateral phrenic nerve activity, leading to paralysis of the hemidiaphragm. We have previously demonstrated a role for serotonin (5-HT) as one potential modulator of respiratory recovery following cervical hemisection, a mechanism that likely occurs via 5-HT2A and/or 5-HT2C receptors. The present study was designed to specifically examine if 5-HT2A and/or 5-HT2C receptors are colocalized with phrenic motoneurons in both intact and spinal-hemisected rats. Adult female rats (250-350 g; n = 6 per group) received a left cervical (C2) hemisection and were injected with the fluorescent retrograde neuronal tracer Fluorogold into the left hemidiaphragm. Twenty-four hours later, animals were killed and spinal cords processed for in situ hybridization and immunohistochemistry. Using (35)S-labeled cRNA probes, cervical spinal cords were probed for 5-HT2A and 5-HT2C receptor mRNA expression and double-labeled using an antibody to Fluorogold to detect phrenic motoneurons. Expression of both 5-HT2A and 5-HT2C receptor mRNA was detected in motoneurons of the cervical ventral horn. Despite positive expression of both 5-HT2A and 5-HT2C receptor mRNA-hybridization signal over phrenic motoneurons, only 5-HT2A silver grains achieved a signal-to-noise ratio representative of colocalization. 5-HT2A mRNA levels in identified phrenic motoneurons were not significantly altered following cervical hemisection compared to sham-operated controls. Selective colocalization of 5-HT2A receptor mRNA with phrenic motoneurons may have implications for recently observed 5-HT2A receptor-mediated regulation of respiratory activity and/or recovery in both intact and injury-compromised states.
Asunto(s)
Células del Asta Anterior/metabolismo , Neuronas Motoras/metabolismo , Nervio Frénico/metabolismo , Receptores de Serotonina/genética , Traumatismos de la Médula Espinal/metabolismo , Transcripción Genética , Animales , Células del Asta Anterior/patología , Femenino , Regulación de la Expresión Génica , Inmunohistoquímica , Hibridación in Situ , Neuronas Motoras/patología , Nervio Frénico/patología , ARN Mensajero/análisis , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Receptor de Serotonina 5-HT2A , Receptor de Serotonina 5-HT2C , Receptores de Serotonina/análisis , Valores de Referencia , Traumatismos de la Médula Espinal/genética , Traumatismos de la Médula Espinal/patologíaRESUMEN
Cerebral ischemia studies demonstrating that stimulation of adenosine A1 receptors by either endogenously released adenosine or the administration of selective receptor agonists causes significant reductions in the morbidity and mortality associated with focal or global brain ischemias have triggered interest in the potential of purinergic therapies for the treatment of traumatic injuries to the brain and spinal cord. Preliminary findings indicate that activation of A1 adenosine receptors can ameliorate trauma-induced death of central neurons. Other avenues of approach include the administration of agents which elevate local concentrations of adenosine at injury sites by inhibiting its metabolism to inosine by adenosine deaminase, rephosphorylation to adenosine triphosphate by adenosine kinase; or re-uptake into adjacent cells. Amplification of the levels of endogenously released adenosine in such a 'site and event specific' fashion has the advantage of largely restricting the effect of such inhibitors to areas of injury-induced adenosine release. Another approach involving purinergic therapy has been applied to the problem of respiratory paralysis following high spinal cord injuries. In this instance, the adenosine antagonist theophylline has been used to enhance residual synaptic drive to spinal respiratory neurons by blocking adenosine A1 receptors. Theophylline induced, and maintained, hemidiaphragmatic recovery for prolonged periods after C2 spinal cord hemisection in rats and may prove to be beneficial in assisting respiration in spinal cord injury patients.
Asunto(s)
Adenosina/metabolismo , Lesiones Encefálicas/metabolismo , Lesiones Encefálicas/terapia , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/terapia , Lesiones Encefálicas/fisiopatología , Circulación Cerebrovascular , Humanos , Receptores Purinérgicos P1/metabolismo , Parálisis Respiratoria/metabolismo , Parálisis Respiratoria/fisiopatología , Parálisis Respiratoria/terapia , Traumatismos de la Médula Espinal/fisiopatologíaRESUMEN
Previous investigations from our laboratory have demonstrated qualitatively that a latent respiratory pathway can be activated by systemic theophylline administration to restore function to a hemidiaphragm paralyzed by an upper (C2) cervical spinal cord hemisection in adult rats. The present study seeks to extend the previous investigations by contrasting and quantitating the actions of theophylline, 8-phenyltheophylline, enprofylline, and 8(p-Sulfophenyl)theophylline in restoring function 24 h after hemidiaphragm paralysis. The alkylxanthines were selected based on their diverse pharmacologic profiles to elucidate the mechanisms that underlie functional recovery after spinal cord injury. To quantitatively assess the magnitude of recovery, electrophysiological experiments were conducted on pancuronium-paralyzed, hemisected animals under standardized recording conditions. The total absence of respiratory-related activity in the phrenic nerve ipsilateral to the hemisection and paralyzed hemidiaphragm was used as the index of a functionally complete hemisection. Thereafter, drug-induced recovered activity in the phrenic nerve ipsilateral to hemisection was quantified and expressed either as a percentage of contralateral phrenic nerve activity in the same animal prior to drug administration or as a percentage of predrug activity in the homolateral nerve in noninjured animals. With either approach, theophylline (5-15 mg/kg) and 8-phenyltheophylline (5-10 mg/kg) dose-dependently induced respiratory-related recovered activity. Enprofylline, a potent bronchodilator, and 8(p-Sulfophenyl)theophylline, an adenosine receptor antagonist with limited access to the central nervous system, were ineffective. Maximal recovery was attained with theophylline (15 mg/kg) and 8-phenyltheophylline (10 mg/kg). At these doses, theophylline and 8-phenyltheophylline induced recovery that was 70.0 +/- 2.5 and 69.3 +/- 4.1% of predrug contralateral nerve activity respectively. When expressed as a percentage of activity in the homolateral nerve in noninjured animals, the magnitude changed to 32.9 +/- 4.9 and 35.7 +/- 6.9%, respectively. Involvement of adenosine receptors in the alkylxanthine-induced actions was confirmed in experiments with the adenosine analog, N6 (l-2-phenylisopropyl) adenosine (L-PIA). It is concluded that central adenosine receptor-mediated mechanisms are implicated in the recovery of respiratory-related activity after spinal cord injury. Furthermore, our results suggest a potential for a new therapeutic approach in the rehabilitation of spinal cord patients with respiratory deficits.
Asunto(s)
Broncodilatadores/farmacología , Mecánica Respiratoria/efectos de los fármacos , Traumatismos de la Médula Espinal/tratamiento farmacológico , Traumatismos de la Médula Espinal/fisiopatología , Teofilina/análogos & derivados , Xantinas/farmacología , Animales , Vértebras Cervicales , Diafragma/efectos de los fármacos , Diafragma/inervación , Diafragma/fisiopatología , Relación Dosis-Respuesta a Droga , Femenino , Lateralidad Funcional , Nervio Frénico/fisiopatología , Ratas , Ratas Sprague-Dawley , Mecánica Respiratoria/fisiología , Teofilina/farmacologíaRESUMEN
The present study investigates the role of serotonin in respiratory recovery after spinal cord injury. Experiments were conducted on C(2) spinal cord hemisected, anesthetized, vagotomized, paralyzed, and artificially ventilated rats in which end-tidal CO(2) was monitored and maintained. Before drug administration, the phrenic nerve ipsilateral to hemisection showed no respiratory-related activity due to the disruption of the descending bulbospinal respiratory pathways by spinal cord hemisection. 5-Hydroxytryptophan (5-HTP), a serotonin precursor, was administrated intravenously. 5-HTP induced time- and dose-dependent increases in respiratory recovery in the phrenic nerve ipsilateral to hemisection. Although the 5-HTP-induced recovery was initially accompanied by an increase in activity in the contralateral phrenic nerve, suggesting an increase in descending respiratory drive, the recovery persisted well after activity in the contralateral nerve returned to predrug levels. 5-HTP-induced effects were reversed by a serotonin receptor antagonist, methysergide. Because experiments were conducted on animals subjected to C(2) spinal cord hemisection, the recovery was most likely mediated by the activation of a latent respiratory pathway spared by the spinal cord injury. The results suggest that serotonin is an important neuromodulator in the unmasking of the latent respiratory pathway after spinal cord injury. In addition, the results also suggest that the maintenance of 5-HTP-induced respiratory recovery may not require a continuous enhancement of central respiratory drive.
Asunto(s)
5-Hidroxitriptófano/farmacología , Nervio Frénico/fisiopatología , Mecánica Respiratoria/efectos de los fármacos , Traumatismos de la Médula Espinal/fisiopatología , Animales , Dióxido de Carbono/análisis , Relación Dosis-Respuesta a Droga , Femenino , Lateralidad Funcional , Metisergida/farmacología , Neuronas/efectos de los fármacos , Neuronas/fisiología , Nervio Frénico/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Respiración Artificial , VagotomíaRESUMEN
The present study was carried out to test the hypothesis that dividing microglia are responsible for the depression of crossed phrenic nerve activity documented at 2 weeks postphrenicotomy in an injury model which superimposes the effects of spinal cord injury on peripheral axotomy. Crossed phenic nerve activity is defined as the respiratory activity recorded from the phrenic nerve during the crossed phrenic phenomenon (CPP) which is a respiratory reflex induced by respiratory stress following an ispsilateral spinal cord hemisection. Young adult female Sprague-Dawley rats were subjected to left intrathoracic phrenicotomies. Cytarabine (Cyt-A, a powerful antimitotic drug) or saline-filled miniosmotic pumps were then implanted into the cisterna magna and 2 weeks were allowed to pass at which time the CPP was induced by a left C2 spinal cord hemisection and transection of the contralateral phrenic nerve. Control studies including bromodeoxyuridine labeling of mitotic cells and a triple immunofluorescent protocol were carried out to verify that microglial cells were the primary cell type undergoing mitosis in the current injury model and that Cyt-A completely inhibited cellular proliferation. Quantitative electrophysiological analysis of crossed phrenic nerve activity showed that there is a statistically significant depression of activity at 2 weeks postphrenicotomy when animals were infused with saline compared to controls. Crossed phrenic nerve activity levels were not significantly different, however, from control levels when 2-week postphrenicotomized rats were infused with Cyt-A. Immunofluorescent studies showed that the majority of cells dividing in response to phrenicotomy were microglia. Furthermore, there were no astrocytes seen dividing at any time. From the results, we conclude that activated microglial cells may be responsible for the depression in crossed phrenic activity normally seen 2 weeks postphrenicotomy. Further, the activation of microglia may be related to the astrocytic response to injury. The activated microglial cell may be acting as a coordinator of various aspects of the injury response. Alternatively, the activation of microglia may be a necessary step in the cascade of multiple events that take place in the spinal cord after injury.
Asunto(s)
Citarabina/farmacología , Mitosis/efectos de los fármacos , Neuroglía/efectos de los fármacos , Nervio Frénico/fisiopatología , Traumatismos de la Médula Espinal/fisiopatología , Médula Espinal/efectos de los fármacos , Animales , Axotomía , División Celular/efectos de los fármacos , Electrofisiología/métodos , Femenino , Lateralidad Funcional , Bombas de Infusión Implantables , Neuroglía/patología , Neuroglía/fisiología , Nervio Frénico/efectos de los fármacos , Nervio Frénico/patología , Ratas , Ratas Sprague-Dawley , Mecánica Respiratoria/efectos de los fármacos , Mecánica Respiratoria/fisiología , Médula Espinal/patología , Médula Espinal/fisiopatología , Traumatismos de la Médula Espinal/patologíaRESUMEN
Electrophysiological recordings taken from the whole phrenic nerve have been utilized previously to describe the gradual increase in functional recovery of a hemidiaphragm paralyzed by ipsilateral C2 hemisection during the crossed phrenic phenomenon (CPP). Although the increase in activity has been temporally correlated with hemisection-induced morphological alterations of the phrenic nucleus, suggesting an association of the increased activity with the morphological alterations, whole phrenic nerve recordings during the CPP can provide only limited information. The purpose of the present study, therefore, was to use phrenic single-axon recording techniques to better understand the mechanisms underlying the recovery of respiratory activity during the expression of the CPP. Recordings from the whole phrenic nerve on the right side and from small fascicles of the phrenic nerve that show only the activity of single phrenic axons (units) on the left side were made in the neck before left spinal hemisection and during the CPP. The results indicated that there were two types of units firing before and during the CPP: an early- and a late-firing unit based on the time of their firing onset in relation to whole phrenic nerve activity. Ten early units and 25 late units were identified according to the shape of their spikes before hemisection as well as during the CPP. In addition to these units, 20 new units were recruited during CPP activity. These new units were mainly of the late-onset type. The results also indicated that there was a significant increase in the frequency of firing of both early and late units. The results specifically indicate therefore that the increase in respiratory activity recorded previously in the whole phrenic nerve during the CPP is most likely due to: (i) an increase in firing frequency for both early- and late-firing units and (ii) a recruitment of predominantly late-firing units into the CPP response. These results are important in understanding more completely the mechanisms that can facilitate recovery of the diaphragm after spinal cord injury.
Asunto(s)
Axones , Diafragma/fisiopatología , Nervio Frénico/fisiopatología , Traumatismos de la Médula Espinal/fisiopatología , Animales , Electrofisiología , Femenino , Plasticidad Neuronal , Nervio Frénico/patología , Ratas , Ratas Sprague-Dawley , Mecánica RespiratoriaRESUMEN
The present study investigates the effect of 5-hydroxytryptophan (5-HTP), a serotonin precursor, on crossed phrenic nerve activity (CPNA) in rats subjected to a left C2 spinal cord hemisection. Electrophysiological experiments were conducted on anesthetized, vagotomized, paralyzed, and artificially ventilated rats to assess phrenic nerve activity. The left phrenic nerve lost rhythmic activity due to the disruption of the bulbospinal respiratory pathways following spinal cord hemisection. Activity was induced in the left phrenic nerve (CPNA) by temporary asphyxia. 5-HTP administration increased CPNA during asphyxia in the left phrenic nerve in a dose-dependent fashion. Specifically, in a group of eight animals, application of 5-HTP at 0.5, 1.0, and 2.0 mg/kg significantly increased CPNA by 102.2+/-18.5%, 200.8+/-58.1%, and 615.0+/-356.9% compared with predrug control values, respectively. 5-HTP-induced increases in CPNA were reversed by methysergide (2-6 mg/kg, i.v.), a serotonin receptor antagonist. The results suggest that serotonin is involved in the modulation of crossed phrenic nerve activity following spinal cord injury.
Asunto(s)
5-Hidroxitriptófano/farmacología , Neuronas/fisiología , Nervio Frénico/fisiopatología , Serotonina/fisiología , Traumatismos de la Médula Espinal/fisiopatología , Médula Espinal/fisiopatología , Animales , Asfixia , Vértebras Cervicales , Femenino , Lateralidad Funcional , Metisergida/farmacología , Neuronas/efectos de los fármacos , Parálisis , Pargilina/farmacología , Nervio Frénico/efectos de los fármacos , Nervio Frénico/fisiología , Ratas , Ratas Sprague-Dawley , Médula Espinal/fisiología , VagotomíaRESUMEN
C2 spinal cord hemisection results in synaptic and astroglial changes in the phrenic nucleus which have been associated with the recovery of the ipsilateral hemidiaphragm during expression of the crossed phrenic phenomenon. As part of our ongoing analysis of the neurotransmitters involved, the present study investigated the effects of systemic administration of para-chlorophenylalanine (p-CPA), a serotonin (5-HT) synthesis inhibitor, on plasticity in the rat phrenic nucleus 4 h following C2 hemisection. Hemisected control rats demonstrated typical morphological changes in the ipsilateral phrenic nucleus including: (1) an increased number and length of synaptic active zones and (2) an increased number and length of dendrodendritic membrane appositions. p-CPA treatment 3 days prior to hemisection reduced 5-HT levels and resulted in an attenuation of these changes in the ipsilateral phrenic nucleus 4 h following hemisection compared to hemisected controls. In addition, p-CPA treatment attenuated injury-induced alterations in immunohistochemical staining of glial fibrillary acidic protein (GFAP), although Western blot analysis demonstrated that overall levels of GFAP did not differ significantly between groups. The results suggest that inhibition of 5-HT synthesis by p-CPA attenuates hemisection-induced plasticity in the phrenic nucleus 4 h following an ipsilateral C2 hemisection.
Asunto(s)
Astrocitos/fisiología , Fenclonina/farmacología , Neuronas Motoras/fisiología , Neuroglía/fisiología , Plasticidad Neuronal , Nervio Frénico/fisiopatología , Antagonistas de la Serotonina/farmacología , Traumatismos de la Médula Espinal/fisiopatología , Animales , Astrocitos/efectos de los fármacos , Astrocitos/ultraestructura , Axones/fisiología , Axones/ultraestructura , Dendritas/fisiología , Dendritas/ultraestructura , Femenino , Proteína Ácida Fibrilar de la Glía/análisis , Inmunohistoquímica , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/ultraestructura , Neuroglía/efectos de los fármacos , Neuroglía/ultraestructura , Plasticidad Neuronal/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Médula Espinal/patología , Médula Espinal/fisiopatología , Médula Espinal/ultraestructura , Sinapsis/fisiología , Sinapsis/ultraestructura , Factores de TiempoRESUMEN
The present study assesses the effects of para-chlorophenylalanine (p-CPA), a serotonin-depleting drug, on the recovery of respiratory-related activity in the phrenic nerve induced by asphyxia 4 h following ipsilateral C2 hemisection in young adult rats. HPLC analysis was used to quantify levels of serotonin (5-HT), dopamine (DA), norepinephrine, and the 5-HT metabolite, 5-hydroxyindoleacetic acid, in the C4 segment of the spinal cord, all of which were significantly lower in p-CPA-treated hemisected rats compared to hemisected controls receiving saline. Hemisection alone was found to significantly increase 5-HT levels and significantly decrease DA levels compared to normal controls. Eight of eight saline-injected rats expressed recovery of respiratory-related activity in the ipsilateral phrenic nerve during asphyxia 4 h following hemisection, while only 4/8 rats in the p-CPA-treated group expressed recovery in the ipsilateral nerve. Quantification of integrated phrenic nerve wave-forms indicated that the mean amplitude of respiratory-related activity in the ipsilateral phrenic nerve was significantly lower in p-CPA-treated rats than in saline controls. In addition, saline controls demonstrated significant increases in mean respiratory frequency and mean amplitude of contralateral phrenic nerve activity during asphyxia, compared to normocapnia. However, p-CPA-treated rats did not express significant differences in either mean respiratory frequency or mean amplitude of integrated respiratory wave-forms during asphyxia, compared to normocapnia. The results suggest that p-CPA treatment attenuates the recovery of respiratory-related activity in the phrenic nerve 4 h following ipsilateral C2 hemisection and attenuates asphyxia-induced increases in respiratory frequency and respiratory burst amplitude recorded from the contralateral phrenic nerve.
Asunto(s)
Fenclonina/farmacología , Nervio Frénico/fisiopatología , Antagonistas de la Serotonina/farmacología , Serotonina/metabolismo , Traumatismos de la Médula Espinal/fisiopatología , Médula Espinal/fisiopatología , Animales , Asfixia , Cromatografía Líquida de Alta Presión , Dopamina/metabolismo , Femenino , Lateralidad Funcional , Ácido Hidroxiindolacético/metabolismo , Norepinefrina/metabolismo , Nervio Frénico/metabolismo , Ratas , Ratas Sprague-Dawley , Mecánica Respiratoria/efectos de los fármacos , Mecánica Respiratoria/fisiología , Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/metabolismo , Factores de TiempoRESUMEN
Previously, we demonstrated that a single intravenous injection of theophylline can induce recovery in a hemidiaphragm paralyzed by cervical (C2) spinal cord hemisection for up to 3 h. The present study contrasts the actions of enprofylline and theophylline on inducing hemidiaphragmatic recovery after cervical spinal cord hemisection. Both drugs are methylxanthines; however, theophylline is an adenosine receptor antagonist while enprofylline is not. To further test the involvement of adenosine receptors, N6 (L-2-phenylisopropyl) adenosine (L-PIA), an analogue of adenosine was used in conjunction with theophylline. Following a left C2 spinal cord hemisection, animals were injected with either enprofylline (2.5-20 mg/kg) or theophylline (15 mg/kg) alone or in combination. Theophylline-injected animals demonstrated robust respiratory-related activity in the previously quiescent left phrenic nerve and hemidiaphragm. No recovery was observed in any of the enprofylline-injected rats. When enprofylline injection was followed later with theophylline, recovery occurred. Prior L-PIA administration blocked theophylline-induced recovery. When given after theophylline, L-PIA attenuated and then blocked the induced activity in both the nerve and hemidiaphragm ipsilateral to spinal cord hemisection. We conclude that adenosine receptor antagonism is implicated in hemidiaphragmatic recovery after hemisection and theophylline may be useful in the treatment of spinal cord injured patients with respiratory deficits.
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Broncodilatadores/farmacología , Diafragma/efectos de los fármacos , Receptores Purinérgicos P1/efectos de los fármacos , Teofilina/farmacología , Xantinas/farmacología , Animales , Diafragma/fisiopatología , Electrofisiología , Femenino , Ratas , Ratas Sprague-Dawley , Receptores Purinérgicos P1/fisiología , Médula Espinal/cirugíaRESUMEN
Based on a previous demonstration that acutely administered theophylline induces respiratory-related recovery in an animal model of spinal cord injury, the influence of chronically administered theophylline on maintaining recovery was assessed. The absence of respiratory-related activity in the left phrenic nerve and hemidiaphragm of rats subjected to an ipsilateral C2 spinal cord hemisection was confirmed electrophysiologically 24 h after injury. Theophylline was then injected i.p. for 3-30 consecutive days. Recovery of respiratory-related activity was observed in the majority (29 out of 32) of the experimental animals. We conclude that theophylline not only induces, but also maintains recovery for prolonged periods after cervical spinal cord injury.
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Diafragma/fisiopatología , Traumatismos de la Médula Espinal/fisiopatología , Teofilina/farmacología , Animales , Electrofisiología , Femenino , Inyecciones Intraperitoneales , Cuello , Nervio Frénico/fisiopatología , Ratas , Ratas Endogámicas , Respiración/fisiologíaRESUMEN
In this study, we describe a new method for quantitative assessment of phrenic inspiratory motor activity in two models of cervical spinal cord contusion injury. Anesthetized rats received contusion injury either to the descending bulbospinal respiratory pathway on one side of the spinal cord alone (C2 lateralized contusion) or to both the descending pathway, as well as the phrenic motoneuron pool bilaterally (C4/C5 midline contusion). Following injury, respiratory-associated phrenic nerve motor activity was recorded under standardized and then asphyxic conditions. Phrenic nerve efferent activity was rectified, integrated, and quantitated by determining the mean area under the integrated neurograms. The mean integrated area of the four inspiratory bursts recorded just before turning off the ventilator (to induce asphyxia) was determined and divided by the integrated area under the single largest respiratory burst recorded during asphyxia. This latter value was taken as the maximal inspiratory motor response that the rat was capable of generating during respiratory stress. Thus, a percentage of the maximal inspiratory motor drive was established for breathing in control and injured rats under standardized conditions. The results indicate that noninjured rats use 52 +/- 1.8% of maximal inspiratory motor drive under standardized conditions. In C2-contused rats, the results showed that while the percentage of maximal inspiratory motor drive on the noncontused side was similar to the control (55 +/- 4.1%), it was increased on the contused side (78 +/- 2.6%). In C4/5 lesions, the results indicate that this percentage was increased on both sides (77 +/- 4.4%). The results show the feasibility for performing quantitative evaluation of respiratory dysfunction in an animal model of cervical contusion injury. These findings lend to further development of this model for investigations of neuroplasticity and/or therapeutic interventions directed at ameliorating respiratory compromise following cervical spinal cord trauma.
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Nervio Frénico/fisiopatología , Pruebas de Función Respiratoria , Parálisis Respiratoria/fisiopatología , Traumatismos de la Médula Espinal/fisiopatología , Animales , Vértebras Cervicales , Contusiones/complicaciones , Contusiones/fisiopatología , Femenino , Neuronas Motoras/fisiología , Ratas , Ratas Sprague-Dawley , Reproducibilidad de los Resultados , Parálisis Respiratoria/etiología , Traumatismos de la Médula Espinal/complicacionesRESUMEN
The objective of the present study was to characterize the microglial and astroglial reaction in the phrenic nucleus following either an ipsilateral C2 spinal cord hemisection, a peripheral phrenicotomy, or a combination of the two injuries in the same adult rat. The present study used three different fluorescent markers and a confocal laser image analysis system to study glial cells and phrenic motoneurons at the light microscopic level. Young adult female rats were divided into one combined injury group (left phrenicotomy and left C2 spinal hemisection with periods of 1 to 4 weeks between injuries, N = 12) and three other groups consisting of noninjured animals (N = 3), animals that received C2 hemisection only (N = 3), and animals with phrenicotomy only (survival periods of 2 (N = 3) and 4 (N = 3) weeks after phrenicotomy). Fluorogold was injected into the diaphragm to label phrenic motoneurons in all animals. Microglia and astrocytes were labeled with Texas red and fluorescein, respectively, and were visualized simultaneously along with phrenic motoneurons. The results suggest that the microglial and astrocytic response in the superimposed injury model are similar to the glial reactions characteristically seen in a peripheral axotomy alone model. These reactions include proliferation and migration of microglial cells along the perineuronal surface (peaking at 2 weeks) and the hypertrophy of astrocytes (peaking at 4 weeks). In addition, the increase in astrocytic tissue, which is characteristically seen in response to axotomy alone, is significantly enhanced in the superimposed injury model. Also, there is a large and rapid increase in GFAP-positive astrocytes within 24 hours after hemisection alone. The information gained from the present study will aid in determining, predicting, and eventually manipulating central nervous system responses to multiple injuries with the objective of reestablishing function in the damaged CNS.
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Astrocitos/patología , Microglía/patología , Neuronas Motoras/patología , Nervio Frénico/lesiones , Degeneración Retrógrada , Traumatismos de la Médula Espinal/patología , Médula Espinal/patología , Animales , División Celular , Movimiento Celular , Cordotomía , Diafragma/inervación , Femenino , Colorantes Fluorescentes , Hipertrofia , Procesamiento de Imagen Asistido por Computador , Microscopía Confocal , Desnervación Muscular , Ratas , Ratas Sprague-Dawley , Traumatismos de la Médula Espinal/complicaciones , Cicatrización de HeridasRESUMEN
Interruption of the main descending respiratory drive to phrenic motoneurons by cold block or spinal cord hemisection results in morphological modifications of the ipsilateral phrenic nucleus in the rat. The modifications consist of an increase in the number of multiple synapses and dendrodendritic appositions and elongation of the asymmetric and symmetric synaptic active zones. Hemisection and hemispinalization by cold block cause not only "functional deafferentation" of the ipsilateral phrenic neurons (i.e., a loss of ipsilateral descending respiratory drive), but also an increase in the remaining contralateral descending respiratory drive. The contralateral respiratory pathways connect with phrenic motoneurons ipsilateral to cold block or hemisection by decussating collateral axons which cross the spinal cord midline below the hemisection/cold block site. Thus, the phrenic nucleus synaptic plasticity could possibly be induced by functional deafferentation or by an increase of the descending respiratory drive. To differentiate between these two possible inducers of the plasticity, we assessed the synaptic morphology of the phrenic nucleus of nonoperated rats exposed to 48 h of hypoxia in an atmosphere chamber. The hypoxia exposure produces an increased descending respiratory drive without functional deafferentation. The quantitative data extracted from electron micrographs of the phrenic nucleus from four experimental rats were compared with the data from four normal breathing animals. Phrenic nucleus morphometric analysis showed that there was no significant difference in the mean number of single synapses between the samples from control animals (141 +/- 12.12) and the experimental animals (156 +/- 26.73). Similarly, no significant difference was detected in the total number of synaptic active zones of control animals (178.25 +/- 11.13) and experimental animals (195.05 +/- 5.35). Furthermore, the length of synaptic active zones of asymmetrical synapses (0.21 +/- 0.024 micron) or symmetrical synapses (0.22 +/- 0.022 micron) did not change significantly compared to the synaptic active zone length in control animals (0.21 +/- 0.018 micron for asymmetrical and 0.21 +/- 0.010 micron for symmetrical). We conclude that no synaptic plasticity occurs in the phrenic nucleus without functional deafferentation in spite of an increase in descending respiratory drive. Therefore functional deafferentation may be the primary inducer of phrenic nucleus synaptic plasticity occurring after hemisection or cold block.