Cerebral Erythropoietin Prevents Sex-Dependent Disruption of Respiratory Control Induced by Early Life Stress.

Injuries that occur early in life are often at the root of adult illness. Neonatal maternal separation (NMS) is a form of early life stress that has persistent and sex-specific effects on the development of neural networks, including those that regulate breathing. The release of stress hormones during a critical period of development contributes to the deleterious consequences of NMS, but the role of increased corticosterone (CORT) in NMS-induced respiratory disturbance is unknown. Because erythropoietin (EPO) is a potent neuroprotectant that prevents conditions associated with hyperactivation of the stress neuroaxis in a sex-specific manner, we hypothesized that EPO reduces the sex-specific alteration of respiratory regulation induced by NMS in adult mice. Animals were either raised under standard conditions (controls) or exposed to NMS 3 h/day from postnatal days 3-12. We tested the efficacy of EPO in preventing the effects of NMS by comparing wild-type mice with transgenic mice that overexpress EPO only in the brain (Tg21). In 7-days-old pups, NMS augmented CORT levels ~2.5-fold by comparison with controls but only in males; this response was reduced in Tg21 mice. Respiratory function was assessed using whole-body plethysmography. Apneas were detected during sleep; the responsiveness to stimuli was measured by exposing mice to hypoxia (10% O2; 15 min) and hypercapnia (5% CO2; 10 min). In wild-type, NMS increased the number of apneas and the hypercapnic ventilatory response (HcVR) only in males; with no effect on Tg21. In wild-type males, the incidence of apneas was positively correlated with HcVR and inversely related to the tachypneic response to hypoxia. We conclude that neural EPO reduces early life stress-induced respiratory disturbances observed in males.

Developmental expression patterns of erythropoietin and its receptor in mouse brainstem respiratory regions.

Erythropoietin (EPO) is a hypoxia-inducible hormone, classically known to enhance red blood cell production upon binding its receptor (EPOR) present on the surface of the erythroid progenitor cells. EPO and its receptor are also expressed in the central nervous system (CNS), exerting several non-hematopoietic actions. EPO also plays an important role in the control of breathing. In this review, we summarize the known physiological actions of EPO in the neural control of ventilation during postnatal development and at adulthood in rodents under normoxic and hypoxic conditions. Furthermore, we present the developmental expression patterns of EPO and EPORs in the brainstem, and with the use of in situ hybridization (ISH) and immunofluorescence techniques we provide original data showing that EPOR is abundantly present in specific brainstem nuclei associated with central chemosensitivity and control of ventilation in the ventrolateral medulla, mainly on somatostatin negative cells. Thus, we conclude that EPO signaling may act through glutamatergic neuron populations that are the primary source of rhythmic inspiratory excitatory drive. This work underlies the importance of EPO signaling in the central control of ventilation across development and adulthood and provides new insights on the expression of EPOR at the cellular level.

Brain-derived erythropoietin protects from intermittent hypoxia-induced cardiorespiratory dysfunction and oxidative stress in mice.

Study Objectives: Based on the fact that erythropoietin (Epo) administration in rodents protects against spatial learning and cognitive deficits induced by chronic intermittent hypoxia (CIH)-mediated oxidative damage, here we tested the hypothesis that Epo in the brain protects against cardiorespiratory disorders and oxidative stress induced by CIH in adult mice. Methods: Adult control and transgenic mice overexpressing Epo in the brain only (Tg21) were exposed to CIH (21%-10% O2-10 cycles/hour-8 hours/day-7 days) or room air. After CIH exposure, we used the tail cuff method to measure arterial pressure, and whole-body plethysmography to assess the frequency of apneic episodes at rest, minute ventilation, and ventilatory responses to hypoxia and hypercapnia. Finally, the activity of pro-oxidant (XO-xanthine oxidase, and NADPH) and antioxidant (super oxide dismutase) enzymes was evaluated in the cerebral cortex and brainstem. Results: Exposure of control mice to CIH significantly increased the heart rate and arterial pressure, the number of apneic events, and the ventilatory response to hypoxia and hypercapnia. Furthermore, CIH increased the ratio of pro-oxidant to antioxidant enzymes in cortex and brainstem tissues. Both physiological and molecular changes induced by CIH were prevented in transgenic Tg21 mice. Conclusions: We conclude that the neuroprotective effect of Epo prevents oxidative damage in the brain and cardiorespiratory disorders induced by CIH. Considering that Epo is used in clinics to treat chronic kidney disease and stroke, our data show convincing evidence suggesting that Epo may be a promising alternative drug to treat sleep-disorder breathing.

Pharmacological, but not genetic, alteration of neural Epo modifies the CO2/H+ central chemosensitivity in postnatal mice.

Cerebral erythropoietin (Epo) plays a crucial role for respiratory control in newborn rodents. We showed previously that soluble Epo receptor (sEpoR: an Epo antagonist) reduces basal ventilation and hypoxic hyperventilation at postnatal day 10 (P10) and in adult mice. However, at these ages (P10 and adulthood), Epo had no effect on central chemosensitivity. Nevertheless, it is known that the sensitivity to CO2/H+ during the mammalian respiratory network maturation process is age-dependent. Accordingly, in this study we wanted to test the hypothesis that cerebral Epo is involved in the breathing stimulation induced by the activation of central CO2/H+ chemoreceptors at earlier postnatal ages. To this end, en bloc brainstem-spinal cord preparations were obtained from P4 mice and the fictive breathing response to CO2-induced acidosis or metabolic acidosis was analyzed. This age (P4) was chosen because previous research from our laboratory showed that Epo altered (in a dose- and time-dependent manner) the fictive ventilation elicited in brainstem-spinal cord preparations. Moreover, as it was observed that peripheral chemoreceptors determined the respiratory sensitivity of central chemoreceptors to CO2, the use of this technique restricts our observations to central modulation. Our results did not show differences between preparations from control and transgenic animals (Tg21: overexpressing cerebral Epo; Epo-TAgh: cerebral Epo deficient mice). However, when Tg21 brainstem preparations were incubated for 1h with sEpoR, or with inhibitors of ERK/Akt (thus blocking the activation of the Epo molecular pathway), the fictive breathing response to CO2-induced acidosis was blunted. Our data suggest that variation of the Epo/sEpoR ratio is central to breathing modulation during CO2 challenges, and calls attention to clinical perspectives based on the use of Epo drugs at birth in hypoventilation cases.

Hypercapnic ventilatory response is decreased in a mouse model of excessive erythrocytosis.

The impact of cerebral erythropoietin (Epo) in the regulation of the hypercapnic ventilatory response (HcVR) is controversial. While we reported that cerebral Epo does not affect the central chemosensitivity in C57Bl6 mice receiving an intracisternal injection of sEpoR (the endogenous antagonist of Epo), a recent study in transgenic mice with constitutive high levels of human Epo in brain and circulation (Tg6) and in brain only (Tg21), showed that Epo blunts the HcVR, maybe by interacting with central and peripheral chemoreceptors. High Epo serum levels in Tg6 mice lead to excessive erythrocytosis (hematocrit ~80-90%), the main symptom of chronic mountain sickness (CMS). These latter results support the hypothesis that reduced central chemosensitivity accounts for the hypoventilation observed in CMS patients. To solve this intriguing divergence, we reevaluate HcVR in Tg6 and Tg21 mouse lines, by assessing the metabolic rate [O consumption (V̇) and CO production (V̇)], a key factor modulating ventilation, the effect of which was not considered in the previous study. Our results showed that the decreased HcVR observed in Tg6 mice (~70% reduction; < 0.01) was due to a significant decrease in the metabolism (~40%; < 0.0001) rather than Epo's effect on CO chemosensitivity. Additional analysis in Tg21 mice did not reveal differences of HcVR or metabolism. We concluded that cerebral Epo does not modulate the central chemosensitivity system, and that a metabolic effect upon CO inhalation is responsible for decreased HcVR observed in Tg6 animals. As CMS patients also show decreased HcVR, our findings might help to better understand respiratory disorders at high altitude.

The central chemosensitivity is not altered by cerebral erythropoietin.

The stimulation of central chemoreceptors by CO2 is considered essential for breathing. The supporting evidence include the fact that central apnea in neonates correlates with immaturity of the CO2-sensing mechanism, and that congenital central hypoventilation syndrome (CCHS) is characterized by the absence of a ventilatory response to elevated PCO2. We reported previously that cerebral erythropoietin (Epo) is a potent respiratory stimulant upon normoxia and hypoxia. The injection of soluble Epo receptor (sEpoR; the natural EpoR competitor to bind Epo) via the cisterna magna (ICI: intra-cisternal injection) decreases basal ventilation in adult and newborn mice. Moreover, sEpoR induces respiratory depression in adult and newborn mice exposed to hypoxia. In this study we tested the hypothesis that endogenous brain Epo also modulates the respiratory stimulation induced by the activation of central CO2 chemoreceptors. Adult and newborn male and female mice received an injection of sEpoR or vehicle via the cisterna magna. Twenty-four hours later basal minute ventilation and the ventilatory response to hypercapnia (5% CO2) were evaluated by plethysmography. Our results did not show a difference in the hypercapnic response between sEpoR and vehicle-injected male or female mice at postnatal or adult ages. We concluded that endogenous brain Epo does not contribute to modulating the PCO2-mediated central activation of breathing.