Mechanisms of ventilatory adaptations to chronic hypercapnia

 描述(由申请人提供) 由于肺部或神经肌肉疾病或医源性干预引起的慢性高碳酸血症在退伍军人事务部患者中很常见。低容量机械通气量的允许性高碳酸血症是防止肺部气压创伤的标准护理，但慢性高碳酸血症对大脑的影响在很大程度上是未知的。我们的目标是了解中枢神经系统对慢性高碳酸血症反应的时间进程和分子机制，以延长和改善慢性二氧化碳滞留VA患者的生活质量。我们的中心假设是慢性高碳酸血症在呼吸控制网络中引起适应性和/或适应性不良的神经可塑性。海马区的代偿过程依赖于谷氨酸受体亚单位(GluA，Glun)表达的变化或改变突触强度/网络功能的活动状态。其他形式的神经可塑性需要改变5-羟色胺(5-HT)系统(TPH，SERT)。本研究以颈动脉体失神经(CBD)山羊为研究对象，研究了低通气性高碳酸血症对神经化学物质的影响。CBD后30d以上，呼吸相关核团和非呼吸性脑干核团谷氨酸受体亚单位和5-羟色胺系统标志物的表达均发生改变，提示高碳酸血症本身参与了这些变化。因此，我们将通过在成年山羊的清醒和NREM睡眠中完成三个特定目标(SA)来验证我们的中心假设。SA-1测试5-羟色胺系统谷氨酸受体亚单位和标志物的变化是否与慢性高碳酸血症期间呼吸控制的适应性或非适应性变化相关。我们假设，在吸入二氧化碳(InCO2)增加6%的30天内，将会出现：a)多相呼吸变化，这将与脑干呼吸核团内GluA或Glun亚单位的激活状态的多相变化呈正相关；b)随着时间的推移，通气性二氧化碳化学反射减少，这将与5-羟色胺系统标志物的下降呈正相关；c)清醒和NREM睡眠之间的PaCO2、通气量和呼吸肌活动的差异大于正常。通气量、动脉血气和呼吸肌活动(清醒和睡眠)，以及二氧化碳化学反射(清醒)将在长期接触6%二氧化碳或30天室内空气呼吸之前和期间进行测量。在慢性高碳酸血症5天、15天和30天、慢性正常碳酸血症30天和未手术对照组中，山羊将被安乐死，并从脑干核团中提取组织块用于谷氨酸受体亚单位、5-羟色胺标记物的表达和启动神经可塑性的分子的蛋白质印迹分析。Sa 2功能测试慢性高碳酸血症期间呼吸控制的适应性/非适应性变化是否与关键部位谷氨酸受体反应性的变化有关，而谷氨酸受体反应性有助于二氧化碳的化学接收和呼吸节律的产生。我们假设，在清醒的山羊体内，通过向斜方后核(RTN)或Prebötzinger复合体(PrebötC)注射谷氨酸受体拮抗剂，可以缓解慢性增加的INCO_2引起的高呼吸，并且这种减弱将与注射前的通气量呈正相关。在慢性高碳酸血症30天或慢性正常碳酸血症30天(对照组)前、中、后2-5天，在二氧化碳浓度升高期间，将长期植入微管(MT)，用于注射谷氨酸受体拮抗剂犬尿酸(Kyna)。Sa 3功能测试慢性高碳酸血症时呼吸控制的变化是否与RTN或PrebötC内神经调节剂的变化相关，和/或依赖于5-HT2A受体的神经调节。我们假设高二氧化碳血症会降低兴奋性神经调节剂和对透析到RTN和PrebötC中的5-HT2a受体拮抗剂的通气性反应。通过植入MT，在高碳酸血症或正常碳酸血症30天前、中、后分别用或不用拮抗剂透析模拟脑脊液。将对流出的透析液进行神经调节剂含量分析。

 DESCRIPTION (provided by applicant) Chronic hypercapnia due to lung or neuromuscular disease or iatrogenic intervention is common in Veteran's Affairs patients. Permissive hypercapnia from low volume mechanical ventilation is standard care for protection against barotrauma in the lung, but the effects of chronic hypercapnia on the brain are largely unknown. Our goal is to understand the time course and molecular mechanisms of the central nervous system's response to chronic hypercapnia in order to extend and improve the quality of life in VA patients with chronic CO2 retention. Our central hypothesis is that chronic hypercapnia elicits adaptive and/or maladaptive neuroplasticity within the respiratory control network. Compensatory processes in the hippocampus depend on changes in glutamate receptor subunit (GluA, GluN) expression or activity state altering synaptic strength/network function. Other forms of neuroplasticity require changes in the serotonin (5-HT) system (TPH, SERT). Our studies on carotid body denervated (CBD) goats characterized effects of hypoventilation-induced hypercapnia on neurochemicals. Over 30 days after CBD, the expression of glutamate receptor subunits and markers of the 5-HT system were altered in respiratory-related and non-respiratory brainstem nuclei, indicating that hypercapnia per se contributed to these changes. Thus, we will test our central hypothesis by completing three Specific Aims (SA) on adult goats during both wakefulness and NREM sleep. SA 1 tests whether changes in glutamate receptor subunits and markers of the 5-HT system correlate with adaptive or maladaptive changes in ventilatory control during chronic hypercapnia. We hypothesize that over 30 days of increased inspired CO2 (InCO2) of 6% there will be: a) multiphasic changes in ventilation which will positively correlate with multiphasic changes in the activation state of GluA or GluN subunits within brainstem respiratory nuclei, b) a decrease over days in the ventilatory CO2 chemoreflex which will positively correlate with a decrease in markers of the 5-HT system, and c) a greater than normal difference in PaCO2, ventilation, and respiratory muscle activity between wakefulness and NREM sleep. Ventilation, arterial blood gases, and respiratory muscle activity (awake and asleep), and the CO2 chemoreflex (awake) will be measured before and during up to 30 days of chronic 6% CO2 exposure or 30 days of room air breathing. At 5, 15, and 30 days of chronic hypercapnia, 30 days of chronic normocapnia, and in an unoperated control group, goats will be euthanized and tissue punches from brainstem nuclei will be extracted for Western blot analyses of glutamate receptor subunits, 5-HT marker expression, and molecules that initiate neuroplasticity. SA 2 functionally tests if the adaptive/maladaptive changes in ventilatory control during chronic hypercapnia correlate with changes in glutamate receptor responsiveness within key sites that contribute to CO2 chemoreception and respiratory rhythm generation. We hypothesize that the hyperpnea during chronic increased InCO2, in awake goats will be attenuated by injection of a glutamate receptor antagonist into the retrotrapezoid nucleus (RTN) or preBötzinger Complex (preBötC), and the attenuation will positively correlate with the ventilation prior to injections. Microtubule (MT's) will be chronically implanted for injections of the glutamate receptor antagonist kynurenic acid (KynA) during increased InCO2 before, during, and 2-5 days after 30 days of chronic hypercapnia or 30 days of chronic normocapnia (control). SA 3 functionally tests if changes in ventilatory control during chronic hypercapnia correlate with changes in neuromodulators within the RTN or preBötC and/or are dependent upon 5-HT2A receptor neuromdoulation. We hypothesize InCO2 hypercapnia will decrease excitatory neuromodulators and the ventilatory response to a 5-HT2A receptor antagonist dialyzed into the RTN and preBötC. Through implanted MT's, mock cerebrospinal fluid will be dialyzed with or without antagonist before, during, and after 30 days of hypercapnia or normocapnia. Effluent dialyzed fluid will be analyzed for neuromodulator content.