Jorge Soliz

Endogenous brain erythropoietin is a potent sex-specific respiratory stimulant in adult and newborn mice

We tested the hypothesis that endogenous brain Epo is a respiratory stimulant. Adult (3 mo) and newborn (10 days) male and female mice received an intracisternal (cisterna magna) injection of soluble Epo receptor (sEpoR; competes with EpoR to bind Epo; 50 μg/ml) or vehicle (0.1% BSA in PBS). Twenty-four hours after injection, we used whole body plethysmography to record minute ventilation (V̇e) tidal volume (VT), respiratory frequency (fR), O2 consumption (V̇o2), and CO2 production (V̇co2) under normoxia and progressive exposure to hypoxia (12-10-6% O2; 10 min each). In adult male and female mice sEpoR decreased normoxic V̇e (-25%), due to a decrease of VT in males and fR in females. Moreover, sEpoR injection decreased the ventilatory response to 12% O2, assessed as V̇e/V̇o2 or V̇e/V̇co2, in male but not in female mice. In newborn male and female mice sEpoR decreased V̇e (-37% in males, -59% in females) and VT (-38% in males, -47% in females) in normoxia and fR in females. During hypoxia, sEpoR decreased V̇e/V̇o2 and V̇e/V̇co2 in mice of both sexes. Upon extreme hypoxia (6% O2), the newborn mice treated with sEpoR showed respiratory depression, signs of asphyxia (gasping) and a high mortality rate in males and females. We concluded that endogenous brain Epo is a potent respiratory stimulant under normoxia and hypoxia in adult and newborn mice. Because sex-specific effects are different in newborn male and female, sex steroids secreted at different ages mice appear to modulate the effects of Epo on respiratory regulation in normoxia and in response to hypoxia.

Erythropoietin and respiratory control at adulthood and during early postnatal life

Erythropoietin (Epo) was originally discovered as a cytokine able to increase the production of red blood cells upon conditions of reduced oxygen availability. Now we know that Epo does far more than only augmenting the number of erythrocytes. Since the demonstration that Epo (and its receptor) is expressed in the mammalian brain, several elegant experiments were performed to reveal the function of this molecule in the neuronal tissue. Accordingly to its anti-apoptotic, neurotrophic and proliferative effects in the bone marrow, it was suitably suggested that upon pathological conditions Epo exerts neuroprotective functions (i.e. reducing the infarct volume of stroke, thus allowing better and faster recovery). We considered however, that Epo in brain might also exert a physiological function. Indeed, we found that Epo is an important modulator of the respiratory control system. By using adult mice we showed that Epo increases the hypoxic ventilatory response by interacting with both the central respiratory network (brainstem) as well as the main peripheral sensory organs detecting systemic hypoxia, the carotid bodies. More recently, our research turned to examine the exciting hypothesis that Epo is also implicated in the regulation of the neuronal control of ventilation during the postnatal development. The objective of this review is to summarize the role and mode of action of Epo on respiratory control in adult mammals and highlight the potential pathways by which this cytokine achieve this function. Additionally, we review recent evidences showing that Epo play a crucial role in setting the respiratory motor output (measured on the isolated brainstem spinal cord preparation, en bloc technique) during the early postnatal life.

Erythropoietin and its antagonist regulate hypoxic fictive breathing in newborn mice.

Clinical use of erythropoietin in adult and newborn patients has revealed its involvement in neuroprotection, neurogenesis, and angiogenesis. More recently, we showed in adult mouse, that brain erythropoietin interacts with the major brainstem centers associated with respiration to enhance the ventilatory response to acute and chronic conditions of physiological hypoxia (e.g., as occurring at high altitude). However, whether brain erythropoietin is involved in breathing regulation in newborns remains unknown. In this study, en bloc brainstem-spinal cord preparations were obtained from mice at postnatal day 4. After various periods (30, 60, or 90 min) of incubation with 0, 25, or 250 U of erythropoietin, preparations were superfused with artificial cerebrospinal fluid bubbled with normoxic or hypoxic gas mixtures. The electrophysiological fictive breathing produced by axons at the C4 ventral root was next recorded. Our results show that erythropoietin attenuates the hypoxia-mediated decrease of the central respiratory activity and improves post-hypoxic recovery. Additional analysis revealed that the soluble erythropoietin receptor (the endogenous erythropoietin antagonist) dramatically decreases neural hypoxic respiratory activity, confirming the specific erythropoietin effect on respiratory drive. These results imply that erythropoietin exerts main modulation and maintenance of respiratory motor output during hypoxic and post-hypoxic challenges in 4-days old mice.

PI3K and MEK1/2 molecular pathways are involved in the erythropoietin-mediated regulation of the central respiratory command.

Erythropoietin stimulation modulates the central respiratory command in newborn mice. Specifically, the central respiratory depression induced by hypoxia is attenuated by acute (1h) or abolished by chronic erythropoietin stimulation. However, the underlying mechanisms remain unknown. As MEK and PI3K pathways are commonly involved in Epo-mediated effects of neuroprotection and erythropoiesis, we investigated here the implication of PI3K and MEK1/2 in the Epo-mediated regulation of the central respiratory command. To this end, in vitro brainstem-spinal cord preparations from 3 days old transgenic (Tg21; constitutively overexpressing erythropoietin in the brain specifically) and control mice were used. Our results show that blockade of PI3K or MEK1/2 stimulates normoxic bursts frequency in Tg21 preparations and abolish hypoxia-induced frequency depression in control preparations. These results show that MEK1/2 and PI3K pathways are involved in the Epo-mediated regulation of the central respiratory command. Moreover, this is the first demonstration that MEK1/2 and PI3K are involved in the brainstem central respiratory command.

Chronic overexpression of cerebral Epo improves the ventilatory response to acute hypoxia during the postnatal development

Clinicians observed that the treatment of premature human newborns for anemia with erythropoietin (Epo) also improved their respiratory autonomy. This observation is in line with our previous in vitro studies showing that acute and chronic Epo stimulation enhances fictive breathing of brainstem‐spinal cord preparations of postnatal day 3–4 mice during hypoxia. Furthermore, we recently reported that the antagonization of the cerebral Epo (by using the soluble Epo receptor; sEpoR) significantly reduced the basal ventilation and the hypoxic ventilatory response of 10 days old mice. In this study, we used transgenic (Tg21) mice to investigate the effect of the chronic cerebral Epo overexpression on the modulation of the normoxic and hypoxic ventilatory drive during the post‐natal development. Ventilation was evaluated by whole body plethysmography at postnatal ages 3 (P3), 7 (P7), 15 (P15) and 21 (P21). In addition Epo quantification was performed by RIA and mRNA EpoR was evaluated by qRT‐PCR. Our results showed that compared to control animals the chronic Epo overexpression stimulates the hypoxic (but not the normoxic) ventilation assessed as V˙E/V˙O2 at the ages of P3 and P21. More interestingly, we observed that at P7 and P15 the chronic Epo stimulation of ventilation was attenuated by the down regulation of the Epo receptor in brainstem areas. We conclude that Epo, by stimulating ventilation in brainstem areas crucially helps tolerating physiological (e.g., high altitude) and/or pathological (e.g., respiratory disorders, prematurity, etc.) oxygen deprivation at postnatal ages.

Post-natal hypoxic activity of the central respiratory command is improved in transgenic mice overexpressing Epo in the brain.

Previous studies indicated that erythropoietin modulates central respiratory command in mice. Specifically, a one-hour incubation of the brainstems with erythropoietin attenuates hypoxia-induced central respiratory depression. Here, using transgenic mice constitutively overexpressing erythropoietin specifically in the brain (Tg21), we investigated the effect of chronic erythropoietin stimulation on central respiratory command activity during post-natal development. In vitro brainstem-spinal cord preparations from mice at 0 (P0) or 3 days of age (P3) were used to record the fictive inspiratory activity from the C4 ventral root. Our results show that erythropoietin already stimulates the hypoxic burst frequency at P0, and at P3, erythropoietin effectively stimulates the hypoxic burst frequency and amplitude. Because the maturation of the central respiratory command in mice is characterized by a decrease in the burst frequency with age, our results also suggest that erythropoietin accelerates the maturation of the newborn respiratory network and its response to hypoxia.

Brain‐derived neurotrophic factor interacts with astrocytes and neurons to control respiration

Respiratory rhythm is generated and modulated in the brainstem. Neuronal involvement in respiratory control and rhythmogenesis is now clearly established. However, glial cells have also been shown to modulate the activity of brainstem respiratory groups. Although the potential involvement of other glial cell type(s) cannot be excluded, astrocytes are clearly involved in this modulation. In parallel, brain‐derived neurotrophic factor (BDNF) also modulates respiratory rhythm. The currently available data on the respective roles of astrocytes and BDNF in respiratory control and rhythmogenesis lead us to hypothesize that there is BDNF‐mediated control of the communication between neurons and astrocytes in the maintenance of a proper neuronal network capable of generating a stable respiratory rhythm. According to this hypothesis, progression of Rett syndrome, an autism spectrum disease with disordered breathing, can be stabilized in mouse models by re‐expressing the normal gene pattern in astrocytes or microglia, as well as by stimulating the BDNF signaling pathway. These results illustrate how the signaling mechanisms by which glia exerts its effects in brainstem respiratory groups is of great interest for pathologies associated with neurological respiratory disorders.

Enhanced erythropoietin expression in the brainstem of newborn rats at high altitude

In addition to its role in elevating red blood cell number, erythropoietin (Epo) exerts protective functions against acute and delayed degenerative diseases of the brain. Moreover, we have recently demonstrated that endogenously synthesized Epo and soluble Epo receptor (a negative regulator of Epo binding to the Epo receptor) in the central nervous system play a crucial role in facilitating the ventilatory response and acclimatization to hypoxia. Here we hypothesized that cerebral Epo in the brainstem is implicated in the process that allows cardiorespiratory acclimatization to high altitude hypoxia during the postnatal period. Thus, we evaluated the postnatal ontogeny of cerebral Epo concentration of Sprague-Dawley rats living and reproducing at high altitude for longer than 19 years (3600 m in La Paz, Bolivia). Our results show that postnatal Epo concentration in high-altitude rats is higher in the brainstem than in the forebrain. Moreover, although Epo concentration in the forebrain of high-altitude rats is similar to sea-level controls, Epo level in the brainstem is surprisingly 2-fold higher in high-altitude rats than in sea-level controls. These findings strongly suggest that brainstem Epo plays an important role in tolerance to high altitude hypoxia after birth. From a clinical perspective, a better understanding of the role of Epo in the postnatal development of cardiorespiratory responses in neonates exposed to acute or chronic hypoxia might be useful.

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.

Erythropoietin and the sex-dimorphic chemoreflex pathway.

During hypoxic or hypoxemic conditions, tissue oxygenation and arterial O(2) carrying capacity are upregulated by two complementary systems, namely the neural respiratory network (central and peripheral) that leads to increased minute ventilation thereby increasing tissue oxygenation, and erythropoietin (Epo) release by the kidney that activates erythropoiesis in bone marrow to augment arterial blood O(2) carrying capacity. Despite the fact that both neural respiratory control and Epo-mediated elevation of red blood cells are responsible for keeping arterial O(2) content optimal, no interaction between these systems has been described so far. Here we review data obtained in our laboratory demonstrating that ventilatory and erythropoietic systems are tightly connected. We found Epo is the key factor mediating this relationship through modulation of the chemoreflex pathway. Moreover, we showed that this interaction occurs in a sex-dependent manner.