Catherine A. Mayer

BDNF Is a Target-Derived Survival Factor for Arterial Baroreceptor and Chemoafferent Primary Sensory Neurons

Brain-derived neurotrophic factor (BDNF) supports survival of 50% of visceral afferent neurons in the nodose/petrosal sensory ganglion complex (NPG; Ernfors et al., 1994a; Jones et al., 1994; Conover et al., 1995; Liu et al., 1995; Erickson et al., 1996), including arterial chemoafferents that innervate the carotid body and are required for development of normal breathing (Erickson et al., 1996). However, the relationship between BDNF dependence of visceral afferents and the location and timing of BDNF expression in visceral tissues is unknown. The present study demonstrates that BDNF mRNA and protein are transiently expressed in NPG targets in the fetal cardiac outflow tract, including baroreceptor regions in the aortic arch, carotid sinus, and right subclavian artery, as well as in the carotid body. The period of BDNF expression corresponds to the onset of sensory innervation and to the time at which fetal NPG neurons are BDNF-dependent in vitro. Moreover, baroreceptor innervation is absent in newborn mice lacking BDNF. In addition to vascular targets, vascular afferents themselves express high levels of BDNF, both during and after the time they are BDNF-dependent. However, endogenous BDNF supports survival of fetal NPG neurons in vitro only under depolarizing conditions. Together, these data indicate two roles for BDNF during vascular afferent pathway development; initially, as a target-derived survival factor, and subsequently, as a signaling molecule produced by the afferents themselves. Furthermore, the fact that BDNF is required for survival of functionally distinct populations of vascular afferents demonstrates that trophic requirements of NPG neurons are not modality-specific but may instead be associated with innervation of particular organ systems.

Chemoafferent degeneration and carotid body hypoplasia following chronic hyperoxia in newborn rats

1 To define the role of environmental oxygen in regulating postnatal maturation of the carotid body afferent pathway, light and electron microscopic methods were used to compare chemoafferent neurone survival and carotid body development in newborn rats reared from birth in normoxia (21 % O2) or chronic hyperoxia (60 % O2). 2 Four weeks of chronic hyperoxia resulted in a significant 41 % decrease in the number of unmyelinated axons in the carotid sinus nerve, compared with age‐matched normoxic controls. In contrast, the number of myelinated axons was unaffected by hyperoxic exposure. 3 Chemoafferent neurones, located in the glossopharyngeal petrosal ganglion, already exhibited degenerative changes following 1 week of hyperoxia from birth, indicating that even a relatively short hyperoxic exposure was sufficient to derange normal chemoafferent development. In contrast, no such changes were observed in the vagal nodose ganglion, demonstrating that the effect of high oxygen levels was specific to sensory neurones in the carotid body afferent pathway. Moreover, petrosal ganglion neurones were sensitive to hyperoxic exposure only during the early postnatal period. 4 Chemoafferent degeneration in chronically hyperoxic animals was accompanied by marked hypoplasia of the carotid body. In view of previous findings from our laboratory that chemoafferent neurones require trophic support from the carotid body for survival after birth, we propose that chemoafferent degeneration following chronic hyperoxia is due specifically to the loss of target tissue in the carotid body.

Vagal afferents modulate cytokine-mediated respiratory control at the neonatal medulla oblongata

Perinatal sepsis and inflammation trigger lung and brain injury in preterm infants, and associated apnea of prematurity. We hypothesized that endotoxin exposure in the immature lung would upregulate proinflammatory cytokine mRNA expression in the medulla oblongata and be associated with impaired respiratory control. Lipopolysaccharide (LPS, 0.1mg/kg) or saline was administered intratracheally to rat pups and medulla oblongatas were harvested for quantifying expression of mRNA for proinflammatory cytokines. LPS-exposure significantly increased medullary mRNA for IL-1β and IL-6, and vagotomy blunted this increase in IL-1β, but not IL-6. Whole-body flow plethysmography revealed that LPS-exposed pups had an attenuated ventilatory response to hypoxia both before and after carotid sinus nerve transection. Immunochemical expression of IL-1β within the nucleus of the solitary tract and area postrema was increased after LPS-exposure. In summary, intratracheal endotoxin-exposure in rat pups is associated with upregulation of proinflammatory cytokines in the medulla oblongata that is vagally mediated for IL-1β and associated with an impaired hypoxic ventilatory response.

Effect of supplemental oxygen on reinitiation of breathing after neonatal resuscitation in rat pups

To test our hypothesis that resuscitation with 21% and 40% oxygen (O2) would shorten time to onset of respiratory activity when compared with resuscitation with 100% O2, diaphragmatic electromyogram (EMG) electrodes were inserted in Sprague-Dawley rat pups, age 8–10 d before intubation and mechanical ventilation with 5% O2 to induce cessation of respiratory activity. Each animal was then resuscitated with 100% and 21% O2 (n = 10) or 100% and 40% O2 (n = 11) for 30 s before the ventilator was disconnected. Recovery of diaphragm activity was compared between resuscitation groups. Blood gas status and heart rate data were characterized in additional rat pups. Time to first respiratory effort was 36 ± 21 s (mean ± SD) for room air resuscitation and 72 ± 22 s for 100% O2, (p = 0.002). In contrast, there was no difference in time to onset of diaphragm activity when resuscitation with 40% O2 was compared with 100% O2: 84 ± 27 s versus 76 ± 23 s, respectively (p > 0.05). Resuscitation with 100% and 40% O2 both resulted in hyperoxia and hypocapnia when compared with room air, without effect on heart rate. Our findings indicate that even modest hyperoxic resuscitation will result in a delayed onset of respiration compared with normoxic gas, via a mechanism that may involve both hyperoxemic and hypocapnic inhibition of chemoreceptors.

Airway inflammation and central respiratory control: results from in vivo and in vitro neonatal rat.

In infants, respiratory infection elicits tachypnea. To begin to evaluate the role of brainstem cytokine expression in modulation of breathing pattern changes, we compared the pattern generated after endotracheal instillation of lipopolysaccharide (LPS) in in vivo rat pups to local pro-inflammatory cytokine injection in the nucleus tractus solitarius (nTS) in an in vitro en bloc brainstem spinal cord preparation. We hypothesized that both challenges would elicit similar changes in patterning of respiration. In anesthetized, spontaneously breathing rat pups, lipopolysaccharide (LPS) or saline was instilled in the airway of urethane-anesthetized rats (postnatal days 10-11). We recorded diaphragm EMG over the subsequent 2h and saw a 20-30% decrease in interburst interval (Te) at 20-80min post-injection in LPS-instilled animals with no significant change in Ti. In contrast, IL-1β injections into the nTS of en bloc in vitro brainstem-spinal cord preparations from 0 to 5 day-old pups maintained Ti and caused an increase in Te as early as 20min later, decreasing frequency for 80-120min after injection. Our results suggest that the neonatal respiratory response to the cytokine IL-1β mediated inflammatory response depends on the site of the inflammatory stimulus and that the direct effect of IL-1β in the nTS is to slow rather than increase rate.

Hyperbilirubinemia diminishes respiratory drive in a rat pup model

Although apnea is common in premature babies, there is a paucity of information concerning the pathophysiologic basis of these episodes and their relationship to other perinatal conditions such as hyperbilirubinemia. Unconjugated hyperbilirubinemia in premature infants, even in moderately high levels, may cause encephalopathy affecting brainstem functions and has been linked to increased incidence of apnea in these infants. Thus, there is a need to clarify mechanisms by which bilirubin may alter respiratory control and induce apnea of prematurity. In this study, bilirubin or placebo was infused i.v. in 9-d-old rat pups (n = 36). Serum hyperbilirubinemia peaked in the first hours after bilirubin infusion. Twenty-four hours after bilirubin infusion, respiration was recorded by plethysmography at rest and under hypercapnic and hypoxic conditions. In treated pups, minute ventilation in room air was significantly reduced, hyperventilatory response to CO2 was blunted, and hypoxic ventilatory depression was increased, compared with placebo-injected rat pups. Brainstem bilirubin deposition and immunoreactivity to bilirubin was detected in the brainstem on histologic analysis. We speculate that high serum bilirubin levels may cause prolonged inhibition of brainstem autonomic function and that this could underlie the exacerbation of apnea noted in premature babies who have experienced jaundice.

Microglia modulate brainstem serotonergic expression following neonatal sustained hypoxia exposure: implications for sudden infant death syndrome.

Neonatal sustained hypoxia exposure modifies brainstem microglia and serotonin expression. The altered brainstem neurochemistry is associated with impaired ventilatory responses to acute hypoxia and mortality. The deleterious effects of sustained hypoxia exposure can be prevented by an inhibitor of activated microglia. These observations demonstrate a potential cause of the brainstem serotonin abnormalities thought to be involved in sudden infant death syndrome.

Vulnerability of neonatal respiratory neural control to sustained hypoxia during a uniquely sensitive window of development

The first postnatal weeks represent a period of development in the rat during which the respiratory neural control system may be vulnerable to aberrant environmental stressors. In the present study, we investigated whether sustained hypoxia (SH; 11% O2) exposure starting at different postnatal ages differentially modifies the acute hypoxic (HVR) and hypercapnic ventilatory response (HCVR). Three different groups of rat pups were exposed to 5 days of SH, starting at either postnatal age 1 (SH1-5), 11 (SH11-15), or 21 (SH21-25) days. Whole body plethysmography was used to assess the HVR and HCVR the day after SH exposure ended. The primary results indicated that 1) the HVR and HCVR of SH11-15 rats were absent or attenuated (respectively) compared with age-matched rats raised in normoxia; 2) there was a profoundly high (∼84% of pups) incidence of unexplained mortality in the SH11-15 rats; and 3) these phenomena were unique to the SH11-15 group with no comparable effect of the SH exposure on the HVR, HCVR, or mortality in the younger (SH1-5) or older (SH21-25) rats. These results share several commonalities with the risk factors thought to underlie the etiology of sudden infant death syndrome, including 1) a vulnerable neonate; 2) a critical period of development; and 3) an environmental stressor.

Lung and brainstem cytokine levels are associated with breathing pattern changes in a rodent model of acute lung injury.

Acute lung injury evokes a pulmonary inflammatory response and changes in the breathing pattern. The inflammatory response has a centrally mediated component which depends on the vagi. We hypothesize that the central inflammatory response, complimentary to the pulmonary inflammatory response, is expressed in the nuclei tractus solitarii (nTS) and that the expression of cytokines in the nTS is associated with breathing pattern changes. Adult, male Sprague-Dawley rats (n=12) received intratracheal instillation of either bleomycin (3units in 120μl of saline) or saline (120μl). Respiratory pattern changed by 24h. At 48h, bronchoalveolar lavage fluid and lung tissue had increased IL-1β and TNF-α levels, but not IL-6. No changes in these cytokines were noted in serum. Immunocytochemical analysis of the brainstem indicated increased expression of IL-1β in the nTS commissural subnucleus that was localized to neurons. We conclude that breathing pattern changes in acute lung injury were associated with increased levels of IL-1β in brainstem areas which integrate cardio-respiratory sensory input.

Impaired hypoxic ventilatory response following neonatal sustained and subsequent chronic intermittent hypoxia in rats

Neonatal chronic intermittent hypoxia (CIH) enhances the ventilatory sensitivity to acute hypoxia (acute hypoxic ventilatory response, HVR), whereas sustained hypoxia (SH) can have the opposite effect. Therefore, we investigated whether neonatal rats pre-treated with SH prior to CIH exhibit a modified HVR. Rat pups were exposed to CIH (5% O2/5min, 8h/day) between 6 and 15 days of postnatal age (P6-15) after pre-treatment with either normoxia or SH (11% O2; P1-5). Using whole-body plethysmography, the acute (5min, 10% O2) HVR at P16 (1 day post-CIH) was unchanged following CIH (67.9±6.7% above baseline) and also SH (58.8±10.5%) compared to age-matched normoxic rats (54.7±6.3%). In contrast, the HVR was attenuated (16.5±6.0%) in CIH exposed rats pre-treated with SH. These data suggest that while neonatal SH and CIH alone have little effect on the magnitude of the acute HVR, their combined effects impose a synergistic disturbance to postnatal development of the HVR. These data could provide important insight into the consequences of not maintaining adequate levels of oxygen saturation during the early neonatal period, especially in vulnerable preterm infants susceptible to frequent bouts of hypoxemic events (CIH) that are commonly associated with apnea of prematurity.